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+# Apoptosis: A Janus Bifrons In Tcell **Immunotherapy**
+
+Yong Gu Lee,1,2,3 Nicholas Yang,1,2 Inkook Chun,1,2 Patrizia Porazzi,1,2 Alberto Carturan,1,2 Luca Paruzzo,1,2,4 Christopher Tor Sauter,1,2 Puneeth Guruprasad,1,2 Raymone Pajarillo ,1,2 Marco Ruella 1,2,5 To cite: Lee YG, Yang N, Chun I, 
+et al. Apoptosis: a *Janus bifrons* in T-cell immunotherapy. *Journal* for ImmunoTherapy of Cancer 2023;11:e005967. doi:10.1136/
+jitc-2022-005967
+► Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi.org/10. 1136/jitc-2022-005967). Accepted 04 February 2023 © Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
+
+1Division of Hematology and Oncology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA
+2Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA
+3College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do, Republic of Korea 4Department of Oncology, University of Turin, Torino, Piemonte, Italy 5Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania, USA Correspondence to Dr Marco Ruella; mruella@upenn.edu
+
+## Abstract
+
+Immunotherapy has revolutionized the treatment of cancer. In particular, immune checkpoint blockade, bispecific antibodies, and adoptive T-cell transfer have yielded unprecedented clinical results in hematological malignancies and solid cancers. While T cell-based immunotherapies have multiple mechanisms of action, their ultimate goal is achieving apoptosis of cancer cells. Unsurprisingly, apoptosis evasion is a key feature of cancer biology. Therefore, enhancing cancer cells' sensitivity to apoptosis represents a key strategy to improve clinical outcomes in cancer immunotherapy. Indeed, cancer cells are characterized by several intrinsic mechanisms to resist apoptosis, in addition to features to promote apoptosis in T cells and evade therapy. However, apoptosis is doublefaced: when it occurs in T cells, it represents a critical mechanism of failure for immunotherapies. This review will summarize the recent efforts to enhance T cell-based immunotherapies by increasing apoptosis susceptibility in cancer cells and discuss the role of apoptosis in modulating the survival of cytotoxic T lymphocytes in the tumor microenvironment and potential strategies to overcome this issue.
+
+## The Dual Role Of Apoptosis In Cancer Immunotherapy
+
+Cancer immunotherapies exploit the immune system to combat cancers and thus have revolutionized the field of immuno-oncology, leading to unprecedented outcomes in relapsed and refractory patients.1 Especially, modulation of T cell's anticancer activity through immune checkpoint blockade (ICB) (eg, anti-programmed cell death protein-1 
+(PD-1)/programmed death ligand-1 or anticytotoxic T-lymphocytes-associated protein 4 (CTLA-4) antibodies, online supplemental box 1) showed a significant clinical response in a subset of solid and hematologic malignancies. Bispecific antibodies (online supplemental box 1) represent another strategy triggering cancer recognition by T 
+cells.2 The anti-CD19/CD3 bispecific T-cell engager blinatumomab was approved in 2014 for B-acute lymphoblastic leukemia (B-ALL) and several anti-CD20/CD3, and anti-BCMA/CD3 antibodies are in advanced clinical development (online supplemental box 1).3–6 Although checkpoint inhibitors are currently the treatment backbone for several cancer types, many patients eventually develop secondary resistance and progressive disease in the end.7 Chimeric antigen receptor T-cell (CAR-T) therapy, a form of adoptive cell transfer (ACT),8 has also demonstrated substantial anticancer efficacy in treating relapsed or refractory B-cell leukemias, lymphomas, and multiple myeloma, which resulted in the approval of multiple CAR-T products by the US Food and Drug Administration (FDA) (online supplemental box 1).9–14 Nevertheless, approximately 50% of pediatric B-ALL and up to 70% of patients with B-cell lymphoma still do not respond or eventually relapse to the CAR-T 
+therapy.10 12 13 15 Therefore, improving the potency of T cell-based immunotherapies is critical for improving the clinical outcomes of patients with cancer.
+
+The ultimate goal of anticancer therapy, including T cell-based immunotherapies, is to eliminate cancer cells, mainly by efficiently inducing apoptosis in cancer cells. Apoptosis, a programmed cellular mechanism leading to cell death, is a complex biological process involving a vast array of tightly controlled cellular components.16 The acquisition of resistance to programmed cellular death (eg, apoptosis) is a key feature of cancer progression.17 For instance, genetic alteration of the anti-apoptotic regulator (eg, translocation and/or gain of B-cell lymphoma 2 (BCL2)) has been well characterized as a key biological marker in multiple lymphomas, including follicular B-cell non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, and B-cell chronic lymphocytic leukemia 
+(CLL).18 High levels of BCL-2 expression protect these fast-growing lymphomas against apoptosis, allowing malignant B cells to survive under various stress factors, such as cytokine deprivation. The critical role of apoptosis in cancer development has been further identified during the transformation of premalignant cells into 
+
+![1_image_0.png](1_image_0.png)
+
+malignant cells. While MYC expression in premalignant cells increases sensitivity to apoptosis, a similar expression of MYC in malignant cells provides a strong proliferative advantage without inducing apoptosis. This proliferative advantage of MYC expression can be attributed to the co-expression of anti-apoptotic regulators (ie, BCL-2) in malignant cells, indicating that acquiring resistance to apoptosis by increasing expression of anti-apoptotic regulator (ie, BCL-2) during malignant transformation is an important checkpoint in cancer development.18 19 Considering the critical role of apoptotic resistance in cancer development, this resistance may also provide a strong protective mechanism against T cell-based immunotherapies. Therefore, it is essential to understand not only the general molecular mechanisms of apoptosis but also the evasion mechanism of cancer cells to enhance the anticancer activity of T cell-based immunotherapies.
+
+Multiple cellular insults and external stimuli, broadly categorized as intrinsic or extrinsic, can promote apoptosis. Intrinsic apoptosis is triggered by DNA damage, excessive reactive oxygen species (ROS), hypoxia, or cellular/metabolic stress.20 In contrast, extrinsic apoptosis is initiated by the so-called 'death ligands,' such as Fas ligand (FasL or CD95L), TRAIL (TNF-related apoptosis-inducing ligand), and tumor necrosis factors 
+(TNFs).21 Immune cells, particularly T cells, use both pathways to activate apoptosis in cancer cells (figure 1). On T-cell receptor (TCR) engagement, T cells release cytolytic granules containing granzymes and perforin in the immune synaptic space to initiate the intrinsic apoptotic pathway. Perforins are pore-forming proteins that diffuse across immunological synapses and oligomerize to form pores on the target cell membrane, facilitating the entry of granzymes into the target cell.22 Granzymes generally enter target cells through pores formed by perforin; it has also been described that granzymes can pass the cell membrane of target cells without requiring perforin, via pinocytosis.23 Four subsets of granzymes 
+(granzyme A, B, K, and M) have been identified in human T cells; particularly granzyme B plays an essential role in promoting T cell-induced apoptosis in target cells.24 On entry into the target cell, granzyme B cleaves BH3 Interacting Domain Death Agonist (BID), a BCL-2 homology 
+(BH3)-only pro-apoptotic protein that plays an essential role in promoting apoptosis. Cleavage of BID results in the generation of the active form of BID (truncated BID/tBID). Subsequently, tBID activates pro-apoptotic effector proteins, such as BAX and BAK, by interfering with their interaction with BCL-2. This activation of effector proteins leads to the induction of instability of the mitochondrial membrane potential and the release of cytochrome c from the mitochondria to the cytoplasm. Cytochrome c leads to the formation of apoptosome complexes (cytochrome c:APAF-1:Caspase-9), which activate effectors, Caspase-3 and Caspase-7, promoting downstream apoptotic signaling cascades.25 However, to trigger the extrinsic apoptotic pathway, the engagement of T cell-derived death ligands and their associated receptors in target cells is necessary. When T cells are activated, it leads to an increase in the expression of death ligands, such as FasL, TNF-α, and TRAIL. These death ligands bind to their respective death receptors (eg, FasL-Fas 
+(CD95), TNF-α-TNFRSF1A (TNFR1), and TRAILTNFRSF10A (DR4)) on target cells, triggering the formation of death-inducing signaling complexes (DISCs), 
+comprizing adaptor proteins (eg, Fas-associated death domains (FADD) or TNF receptor type 1-associated death protein (TRADD)) and initiator Caspase-8. DISCs eventually initiate downstream apoptotic signaling cascades to induce cellular apoptosis.25 Cancers have developed several strategies to evade immune cell-mediated apoptosis. For instance, mutation of TP53, a key tumor suppressor gene that confers resistance to apoptosis is strongly associated with decreased immune function genes (eg, granzymes and perforin) in patients with gastric cancer.26 27 This observation suggests that aberrant TP53 activity could affect the anticancer immune response. Modulation of anti-apoptotic (eg, BCL-2, CFLAR, and BIRC2) and pro-apoptotic proteins (FAS, FADD, TNFRSF10B (death receptor 5—DR5), BID, and Caspase-8) is another important mechanism used by cancer cells to blunt cancer immunotherapy's anticancer efficacy.28–33 Maruyama *et al* reported that >40% of patients with metastatic renal cell cancer with no response or progressive disease were positive for immunohistochemical staining of BCL-2, while patients with complete or partial responses were negative for BCL-2 during the treatment course of immune-stimulatory treatments (eg, interferon (IFN)-α, IFN-γ, and interleukin-2).28 Furthermore, we performed a retrospective analysis of the clinical response of patients with lymphoma treated with anti-CD19 CAR-T therapy and showed that patients with genetic alterations in BCL-2 (ie, gain or translocation of BCL-2) show significantly lower response and overall survival to CAR-T treatments than patients without genetic alterations of BCL-2,33 implicating that genetic alteration of BCL-2 plays a crucial role in the anticancer efficacy of CAR-T therapy. In addition to the effect of altered intrinsic regulators of apoptosis on cancer immunotherapy, our group also demonstrated that CAR-T cells' anticancer efficacy is significantly reduced when leukemic cells display decreased expression of positive regulators of apoptosis, especially in the death receptor pathway (FasL, 
+TRAIL, and TNF-α).34 By using unbiased genome-wide CRISPR knock-out (KO) screening, we identified that the deletion of anti-apoptotic regulators (eg, BIRC2, CFLAR, 
+and TRAF2) in the B-ALL cell line NALM-6 led to significant enhancement of the anticancer activity of CAR-T 
+cells, while KO of pro-apoptotic regulators (eg, FADD, Caspase-8, BID, and TNFRSF10B) resulted in a decrease of anticancer activity of CAR-T cells. Further validation with clinical data revealed that the downregulation of proapoptotic regulators was significantly associated with a poor clinical response in patients with B-ALL treated with anti-CD19 CAR-T cells. Likewise, Upadhyay *et al* showed that Fas-FasL-mediated cancer killing plays a crucial role in T cell-based immunotherapy, and the expression of Fas in cancer strongly correlates with the clinical outcome of CAR-T therapy.29 Importantly, these studies suggest that mechanisms conferring resistance to apoptosis in some cancer cells can also drive T-cell dysfunction, leading to poor clinical outcomes of T cell-based immunotherapy.
+
+Given that resistance to apoptosis in cancers could be a critical factor associated with poor clinical outcomes of T-cell mediated immunotherapy by causing dysfunction of T cells, this review will highlight several novel therapeutic strategies designed to augment T cell-mediated cancer apoptosis. Furthermore, it discusses tumor-derived or tumor microenvironment (TME)-derived factors that govern T-cell apoptosis and rational strategies to prevent it.
+
+## Strategies To Enhance T-Cell Mediated Cancer Apoptosis
+
+Despite remarkable clinical outcomes of T cell-based immunotherapies, a substantial number of patients do not benefit from these approaches.9–15 Considering the importance of cancer apoptosis susceptibility in the cytolytic activity of T-cell therapy, several interesting therapeutic approaches have been investigated to overcome apoptosis resistance in cancer cells (figure 2 and table 1).
+
+First, researchers have tested whether conventional anticancer therapeutics, such as chemotherapy or radiotherapy, can enhance cancer apoptosis during immunotherapy. Both, chemotherapeutic agents (eg, alkylating agents, anthracyclines, vinca alkaloids, and antimetabolites) and radiation (eg, X-ray), potently promote intrinsic 
+
+![3_image_0.png](3_image_0.png)
+
+apoptosis in cancer cells by inducing DNA damage and/
+or inhibiting cell cycle.35 36 Moreover, treatment with selected chemotherapies (eg, etoposide, doxorubicin, and 5-fluorouracil) and radiation can also modulate death receptor-mediated extrinsic apoptosis in cancer cells by affecting the transcriptional activity of death receptors 
+(eg, Fas and TNFRSF10 families).37–41 On exposure to etoposide, DR5 and Fas expression increased in human lung, colorectal, prostate, bladder, and breast cancer cell lines, leading to improved immune cell (natural killer T cell-mediated killing).37 Similarly, sublethal doses of doxorubicin upregulate TRAIL receptors on cancer cells (eg, MAR and JOHW colorectal carcinoma cell lines), 
+promoting natural killer and tumor-infiltrating lymphocyte (TIL) cytotoxicity.39 Interestingly, the authors also observed that doxorubicin treatment reduced the expression of intracellular FLICE inhibitory protein (c-FLIP), a key anti-apoptotic inhibitor of death receptor-mediated apoptosis. This finding suggests that doxorubicin can sensitize cancer cells to immune cell-mediated extrinsic apoptosis by modulating both pro-apoptotic and anti-apoptotic regulators. 
+
+| Table 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | Summary of the preclinical combinations of pro-apoptotic drugs and T cell-based immunotherapies   |                                            |                    |                           |                      |     |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------|--------------------------------------------|--------------------|---------------------------|----------------------|-----|
+| Class                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Drug                                                                                              | Target                                     | Combination        | Cancer type               | Effect               | Ref |
+| Chemo                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Cisplatin,                                                                                        | DNA damage                                 | NKT cells          | NSCLC cell lines          |                      |     |
+| therapy                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | etoposide                                                                                         | CRC cell lines PC cell lines BC cell lines | Sensitization to TRAILmediated and FasLmediated apoptosis                    | 37 38                     |                      |     |
+| Chemo                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Doxorubicin,                                                                                      | DNA damage                                 | Vγ9Vδ2 T cells     | CRC cell lines            | Sensitization to TRAILmediated apoptosis                      |     |
+| therapy                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | 5-fluorouracyl                                                                                    |                                            |                    |                           |                      |     |
+| Chemo                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Doxorubicin                                                                                       | DNA damage                                 | NK or T cells      | Melanoma and bladder cancer  Sensitization to TRAILmediated apoptosis                           | 39                   |     |
+| therapy                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | cell lines                                                                                        |                                            |                    |                           |                      |     |
+| Radiation                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Sublethal                                                                                         | -                                          | antitumor CTLs and | CRC cell lines            | Sensitization to TRAILmediated and FasLmediated apoptosis                      | 40  |
+| irradiation                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | NK cells                                                                                          |                                            |                    |                           |                      |     |
+| Radiation                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Sublethal                                                                                         | -                                          | CEA-specific HLAA2-restricted CD8(+)                    |                           |                      |     |
+| irradiation                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | CTLs                                                                                              | 23 human carcinoma cell lines  (12 colons, 7 lungs, and 4  prostate) Sensitization to FasLmediated apoptosis                                            | 41                 |                           |                      |     |
+| SMAC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Birinapant                                                                                        | Inhibition of XIAP                         | anti-CD19 CAR T    | B-ALL                     | Increase of CAR T cellmediated apoptosis                      | 34  |
+| mimetic                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | and cIAP1/2                                                                                       | cells                                      |                    |                           |                      |     |
+| SMAC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Birinapant                                                                                        | Inhibition of IAPs                         | anti-HER2 CAR T    | HER2+patient-derived      | Sensitization to TNF-αmediated apoptosis                      | 56  |
+| mimetic                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | cells                                                                                             | colorectal tumoroids                       |                    |                           |                      |     |
+| SMAC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Birinapant                                                                                        | Inhibition of IAPs                         | anti-CD19 CAR T    | B-ALL                     | Sensitization to TNF-αmediated apoptosis                      | 32  |
+| mimetic                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | cells                                                                                             |                                            |                    |                           |                      |     |
+| SMAC                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | ASTX660                                                                                           | Inhibition of IAPs                         | cytotoxic TIL      | HNSCC                     | Enhanced immunogenic | 59  |
+| mimetic                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | cell death                                                                                        | 65                                         |                    |                           |                      |     |
+| BH3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | ABT737                                                                                            | Bcl-2 family antiapoptotic proteins                                            | anti-CD19 CAR T    | Patients with childhood   | Increase of CAR T cellmediated apoptosis                      |     |
+| mimetic                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | cells                                                                                             | precursor-B ALL                            |                    |                           |                      |     |
+| BH3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | Venetoclax                                                                                        | Bcl-2 family antiapoptotic proteins                                            | anti-CD19 CAR T    | B-ALL and B-lymphoma cell | Increase of CAR T cellmediated apoptosis                      | 67  |
+| mimetic                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | cells                                                                                             | lines                                      |                    |                           |                      |     |
+| B-ALL, B-cell acute lymphoblastic leukemia; BC, breast cancer; BCL-2, B-cell lymphoma 2 ; CAR, chimeric antigen receptor; CRC,  colorectal cancer; CTL, cytotoxic T cell; FasL, Fas ligand; HER2, human epidermal growth factor receptor-2; HNSCC, head and neck  cancer; IAP, inhibitor of apoptosis proteins; NK, natural killer; NKT, natural killer T; NSCLC, non-small cell lung cancer; PC, prostate  cancer; SMAC, second mitochondria-derived activator of caspase; TIL, tumor-infiltrating lymphocyte; TNF, tumor necrosis factor; TRAIL, |                                                                                                   |                                            |                    |                           |                      |     |
+
+Another evidence of chemotherapy-induced immune killing reported that 5-fluorouracil treatment upregulates the expression of DR5 in colon cancer-initiating cells in vitro, leading to enhancement of T cell-mediated cytotoxicity.38 In addition to chemotherapeutic agents, irradiation, which causes DNA damage and intracellular stress, can also lead to the induction of death receptor-mediated cancer cell apoptosis. For instance, a sublethal dose of irradiation can upregulate the expression of Fas and DR5 in colorectal carcinoma cell lines, making them susceptible to TRAIL-induced and Fas-induced apoptosis.40 Further investigation identified that a sublethal dose of irradiation can increase Fas expression in over 40% of the cancer cell lines, including colon, lung, and prostate cancers.
+
+41 This suggests that radiation may be an effective strategy in combination with T cell-based immunotherapy to enhance the sensitivity of extrinsic apoptosis in cancer cells. In addition to the modulation of cytolytic activity of endogenous T cells in radiation therapy and chemotherapy, the use of recombinant anti-Fas and anti-DR4/5 agonists along with radiation and chemotherapeutic agents has been investigated. Treatment with recombinant TRAIL combined with bortezomib, vorinostat 
+(SAHA), and valproic acid significantly induced cancer cell apoptosis by sensitizing cancer cells to extrinsic apoptosis signal.42–45 Finally, with ample preclinical evidence that chemotherapy and radiation therapy can increase the sensitivity of cancer cells to T cell-mediated apoptosis, various clinical trials exploiting the combination of immunotherapy and chemo/radiation have been registered and conducted 
+(see online supplemental table 1). First, the combination of pembrolizumab (online supplemental box 1) 
+and chemotherapy (ie, carboplatin and either paclitaxel or nanoparticle albumin-bound-paclitaxel) significantly improved overall survival and progression-free survival in patients with metastatic squamous non-small cell lung cancer (NSCLC) as compared with chemotherapy only treated patients.46 Another clinical investigation using an immunotherapeutic combination (nivolumab and ipilimumab, online supplemental box 1) with chemotherapy 
+(carboplatin, paclitaxel, pemetrexed, and cisplatin) 
+showed similar results.47 These two independent clinical trials eventually led to FDA approval of chemotherapy in combination with checkpoint blockade for first-line metastatic squamous NSCLC treatment. Despite the substantial synergy between immune checkpoint inhibitors and chemo/radiation therapy, one caveat remains: apoptosis of T cells can also be increased by chemotherapy and radiation due to the lack of ability of chemotherapy and radiation to distinguish target cells (ie, cancer cells) and effector cells (ie, T cells). The resistance mechanisms of T cells to chemotherapy-induced and radiation-induced apoptosis remain largely unknown. Several studies have highlighted that memory T cells can escape apoptosis triggered by chemotherapy and irradiation, implicating the potential role of memory T cells in synergy strategies.48 49 Considering the resistance of memory T cells to apoptosis, one possible explanation is that a high level of BCL-250 and low level of Bcl-2-like 11 (BIM)51 expression in memory T cells may increase the threshold of apoptotic sensitivity, allowing them to evade chemotherapy-induced and radiation-induced apoptosis. However, further investigations are required to fully understand survival mechanisms of T cells during chemotherapy and radiation.
+
+While chemotherapy and/or radiation increase the sensitivity of cancer cells to apoptosis, direct inhibition of apoptotic regulators has also been observed in combination with T cell-based immunotherapy. One example is the inhibitor of apoptosis proteins (IAPs) that are overexpressed in many cancers52 and include several members, such as cellular IAP1 (cIAP1), cIAP2, X-linked IAP (xIAP), neuronal apoptosis inhibitory protein (NAIP), 
+livin, and survivin.53 cIAP1 plays a critical role in inhibiting TNF-α-mediated apoptosis by preventing the formation of the apoptotic complex (FADD/RIPK1/Caspase-8). 
+
+Moreover, xIAP inhibits apoptosis by blocking Caspase-3 and Caspase-7 by directly binding to them.54 Given the importance of IAPs in inhibiting apoptosis in cancer, multiple agents have been investigated to promote IAP 
+degradation, thereby sensitizing cancer cells to apoptosis. Second mitochondria-derived activator of caspase 
+(SMAC) mimetics are small synthetic molecules whose structural and functional features are similar to SMAC, which are endogenous antagonists of IAPs. Several SMAC mimetics (eg, birinapant, LCL-161, ASTX660, Debio1143, BV-6, GDC-0152, CUCD-427, HGS1029, and AT-406) have been developed (online supplemental box 2).55 Particularly, birinapant has been extensively evaluated for its anticancer properties, including in combination with T cell-based immunotherapies such as immune checkpoint inhibitors, such as anti-PD-1 and anti-CTLA-4 antibodies, driving significantly enhanced TNF-α-mediated cancer cell apoptosis and increase in survival in a murine glioblastoma (GBM) model.32 34 56 57 Birinapant also improved the anticancer efficacy of CAR-T therapy in murine models. Treatment with birinapant enhanced anti-human epidermal growth factor receptor-2 CAR-T cells-mediated cancer killing by sensitizing cancer cells to TNF-mediated apoptosis.56 Likewise, Song *et al* also found that treatment of birinapant enhances CAR-T cell-mediated tumor killing in GBM model.58 Using a CRISPR Cas9 KO library, key mediators of synergy between CAR-T cells and birinapant such as RIPK1, FADD, and TNFRSF10B in cancer cells are identified.32 Our group also demonstrated that birinapant treatment improved CAR-T cells ability to eliminate B-ALL, which otherwise lacks sensitivity to extrinsic apoptosis34; however, these results were obtained in vitro and in vivo validation is required to exclude toxicity on CAR T cells. In addition to birinapant, Ye *et al* studied the combinatorial efficacy of cytotoxic TILs and ASTX660, another antagonist of cIAP1/2 and xIAP, in a preclinical model with head and neck squamous cell carcinoma 
+(HNSCC).59 The authors found that ASTX660 treatment induced—in the presence of TNF-α—calreticulin 
+(CRT) expression, heat shock proteins 70/90, and high mobility group protein on the surface of human HNSCC cell lines (eg, UMSCC-46 and UMSCC-47). These are key molecular signatures for immunogenic cell death (ICD), suggesting that ASTX660 and TNF-α promoted ICD in HNSCC cell lines. ASTX660-mediated induction of ICD was further confirmed in syngeneic murine cancer models (HNSCC) when combined with radiation. Interestingly, the authors identified that ASTX660 treatment plus TNF-α led to clonal expansion of antigen-specific T-cell clones. This might be due to the enhancement of the antigen-processing machinery in cancer cells, as evidenced by the upregulation of critical components of the antigen-processing machinery (eg, human leukocyte antigen (HLA)-A, HLA-B, HLA-C, ERp57, CRT (intracellular), Transporter associated with antigen processing 1 (TAP1), and TAP2) in human HNSCC cell lines after exposure to ASTX660 and TNF-α.
+
+Members of the BCL-2 family, such as BCL-2, BCL-XL, 
+MCL-1, BAX, and BAK, play a critical role in regulating intrinsic apoptosis by modulating the permeabilization of the mitochondrial membrane.60 As previously discussed, upregulation of BCL-2 activity via overexpression or translocation is one of the key features of various cancers.61 Several BCL-2 inhibitors have been developed, including obatoclax, AT101, ABT737, S-055746, S65487, PNT-2258, navitoclax, and venetoclax (online supplemental box 2).62 In particular, venetoclax, an orally available smallmolecule inhibitor with high specificity to BCL-2, has demonstrated substantial anticancer efficacy in treating CLL, other lymphomas, and acute myeloid leukemia, leading to its FDA approval in these settings.63 Recently, Kohlhapp *et al* reported that venetoclax could enhance the anticancer efficacy of anti-PD-1 antibody (MDX-1106) 
+treatment.64 Interestingly, the authors found that venetoclax treatment increased tumor infiltrating effector memory T cells, which could also explain the potential role of memory T cells in synergy with pro-apoptotic drugs. The beneficial effects of venetoclax in T cellbased immunotherapy were further identified by our group. Using a combination of venetoclax and CAR-T therapy, we demonstrated a significant improvement in CAR-T cells' anticancer activity against various lymphoma and leukemia xenograft models (eg, OCI-Ly18, MINO, 
+NALM6, KG-1, and MOLM-14).33 In addition to venetoclax, the combinatory effect of different BCL-2 inhibitors 
+(ie, ABT737) with CAR-T therapy was tested, and it was found that adding ABT737 to CART19 resulted in an increase in Caspase 3/7 activity in cancer cells, leading to cancer killing enhancement.65 While, increasing cancer cell sensitivity to apoptosis using aforementioned pro-apoptotic molecules results in the enhancement of the anticancer response of T cellbased immunotherapies in some models, one potential concern of this approach is the unintended toxicity of these agents on effector immune cells such as T cells, which could be critical for the long-term efficacy of combination immunotherapy. A study showed that SMAC mimetic (ie, LBW242) treatment significantly inhibited virus-specific CD8+ T-cell expansion in vivo by inducing T-cell apoptosis, ultimately leading to the failure of virus replication.66 Moreover, although Lee *et al* and Kohlhapp et al demonstrated that venetoclax augmented the anticancer response of CAR-T therapy and anti-PD-1 treatment, they also found that co-culture of venetoclax with genetically non-modified T cells and CAR-T cells potently reduced their viability.33 64 These observations strongly suggest that careful design of combination therapies and the sequence of administration are required to avoid T-cell toxicity and ensure long-term therapeutic efficacy. One possible administration strategy to avoid bystander effects on T cells is to pretreat the cancer cells with cytotoxic drugs. Recently we reported that patients with lymphoma receiving venetoclax during bridging therapy prior CAR-T cell infusion achieved significant improvement in clinical response compared with patients treated with no venetoclax-included bridging therapy.33 In line with our clinical observations, pretreatment of cancer cells with venetoclax enhanced CAR-T cell-mediated anticancer activity in vitro.67 These preclinical and clinical data strongly suggest that pre-sensitizing cancer cells with anti-apoptotic inhibitors could enhance the anticancer effect of T cell-based immunotherapy while reducing toxicity to T cells.
+
+## T-Cell Apoptosis Limits Anticancer Immunity In The Tme
+
+Apoptosis in cancer therapy could induce both cancer cells to die and result in T-cell death. T-cell apoptosis is an *indirect* result of multiple immunosuppressive mechanisms in cancer genesis. For example, T-cell dysfunction, such as exhaustion, is a physiological state in which T cells lose their effector functions while maintaining viability. Prolonged exhaustion ultimately leads to T 
+cells undergoing cellular apoptosis.68 Furthermore, the immunosuppressive TME, including immunosuppressive immune cells (eg, T regulatory cells, tumor-associated macrophages, and myeloid-derived suppressor cells)69 70 and lack of key nutrients (ie, low arginine and changes in available metabolites)71–73 also have substantial effects on the proliferation and survival of cytotoxic T cells in the TME. Because there are already extensive revisions of the literature on T-cell exhaustion74–76 and other immunosuppressive factors,77–79 we focused on the mechanisms of immune evasion that *directly* trigger apoptosis in T-cells.
+
+On activation, T cells enhance the expression of proapoptotic proteins (eg, FasL, TRAILs, and TNF), potentially promoting the death of target cells as well as death receptors on their surface (Fas, TRAIL receptors, TNF receptor). This upregulation of death receptors increases the susceptibility of activated T cells to apoptosis.80 81 This process is called activation-induced cell death (AICD) and plays a vital role in maintaining peripheral immune tolerance and preventing autoimmune disease development.82 Cancer cells can take advantage of this T-cell liability by using it as a potential immunoevasion strategy. 
+
+Reports in the late 1990s demonstrated that FasL expression in several malignancies (melanoma, colon, head/
+neck, liver, and lung) serves as a mechanism of cancer evasion.83–86 This phenomenon, coupled with evidence that T cells increase the expression of Fas on activation, highlights that cancers can induce apoptosis in T cells via the extrinsic pathway to evade immune surveillance. There has also been evidence of upregulation of TRAIL in a few malignancies (melanoma, liver, breast, and lung), although its correlation with the clinical outcome has been controversial.87–90 While Bron *et al* found no correlation with prognosis in patients with melanoma,87 Cross et al observed a negative association between TRAIL expression in breast cancers and the clinical outcome of patients with breast cancer.88 Moreover, heterogeneity exists in the ubiquity of FasL and TRAIL expression across cancers. Another captivating aspect of this mechanism is the observation that cancer can secrete exosomes expressing FasL and independently induce T-cell apoptosis.91–93 Such observations amplify the potency of FasL-mediated apoptosis of T cells directed by cancer cells, which may cause peripheral T-cell dysfunction.94 This cancer-induced, death receptor-mediated T-cell apoptosis has been proven to directly hinder responses to immunotherapy.95–99 Zhu *et al* used a novel autochthonous melanoma mouse model to demonstrate that FasL-mediated T-cell apoptosis facilitates cancer resistance to anti-CTLA-4 antibody, anti-PD-1 antibody, and ACT.98 Similar to TCR-mediated activation, CAR-driven T-cell activation also increases the susceptibility of CAR-T cells to apoptosis by upregulating death receptors and associated ligands on their surface. Hyperstimulation of CAR-T cells by incorporating two co-stimulatory domains (CD28 and 4-1BB) also increases Fas and DR5 expression and promoted CAR-T cells apoptosis.96 97 Lastly, tonic signaling of 4-1BB co-stimulation due to greater antiCD19 CAR expression driven by a strong promoter, such as retroviral long terminal repeat, increases levels of FasL, 
+leading to apoptosis of CAR-T cells on activation.99 While introducing multiple co-stimulatory domains into CAR 
+construct was intended to enhance activation,95 these data present a potential concern of overstimulation suggesting a need of 'modulating' CAR-activation in T cells rather than just boosting it.
+
+Cancer and TME cells also secrete factors that can directly trigger T-cell apoptosis.100 101 Galectins are a family of proteins produced and secreted by various cells, including cancer and immune cells.102 Galectins bind to β-galactosides on glycoproteins and glycolipids via a conserved carbohydrate recognition domain, thereby regulating miscellaneous biological events, including apoptosis.100 101 Many studies have demonstrated that cancer-secreted galectin-3 (Gal-3) can induce T-cell apoptosis in various cancers, including melanoma, lung, and colorectal cancer, on binding to their target TCRs, such as CD7, CD29, CD45, and CD71.103–108 Mechanistically, cancer-secreted Gal-3 binds to CD45, activating independent pathways involving protein kinase C and ROS, resulting in sustained ERK 1/2 phosphorylation, Caspase-9 activation, cytochrome c release, and Caspase-3 activation to induce apoptosis.109 Besides the function of Gal-3, secreted Gal-1 in the TME also correlated with increased cancer progression (following ICB therapy), which could be due to T-cell apoptosis, likely mediated through a CD45-binding dependent mechanism.110–116 However, this correlation is not consistent across cancers. While elevated Gal-1 correlates with T-cell apoptosis in pancreatic117 and lung110 cancer cell lines, it was not confirmed in a melanoma cell line108 or in vitro against activated primary T cells,118 suggesting that its effects may be heterogeneous across malignancies.
+
+Similarly, gangliosides and sialic acid-containing glycosphingolipids found on outer plasma membranes are overexpressed in cancers and shed into the TME.119 Although the apoptotic effects of gangliosides and their expression in different cancers have not been investigated as extensively as galectins, Finke and Tannenbaum have elucidated their general effect on T-cell apoptosis through a series of studies. Finke *et al* demonstrate in both a GBM 
+and a renal cell carcinoma model that cancer gangliosides are responsible for inducing T-cell apoptosis.120 121 Moreover, Bharti and Singh show the induction of bone marrow cell apoptosis through T-cell lymphoma-derived gangliosides.122 Regarding the mechanism of gangliosidemediated T-cell apoptosis, gangliosides have been shown to be internalized by activated T cells, resulting in ROS production, cytochrome c release, and Caspases-8 and Caspase-9 activation.123 This implies that gangliosides may promote both intrinsic and extrinsic apoptosis. Notably, gangliosides facilitate the intrinsic pathway of apoptosis, as evidenced by the induction of ROS, cytochrome c release, Caspase-9 activation, and downregulation of antiapoptotic BCL genes, such as BCL-XL and BCL-2.124 Metabolic pathways and associated enzymes may also play important roles in T-cell apoptosis. For instance, glucose deprivation can reduce the proliferation of Jurkat cells and primary human T cells in vitro.125 This reduction might be linked to the increase in intrinsic apoptosis since the knockdown of pro-apoptotic BH-3-only protein (ie, Noxa) improves the survival of T cells when limited glucose is available. Considering that T cells encounter significant competition in the uptake of glucose by cancer cells in TME,126 the lack of glucose in T cells may increase the susceptibility of T cells to apoptosis, leading to impairment of the anticancer activity of T cells. In addition to the glycolytic pathway, fatty acid metabolism is critical for T cell-mediated anticancer activity. While T cells use fatty acid oxidation to form and maintain the memory phenotype,127 inhibiting fatty acid synthase potentially reduces the expression of FasLs, preventing T cells from restimulation-induced cell death.128 In addition to the intrinsic alteration of T-cell metabolism in inducing apoptosis, metabolites from cancer cells may also promote apoptosis in T cells. For example, kynurenine, a metabolite of tryptophan by indoleamine 2,3-dioxygenase in cancer cells, can induce apoptosis in thymocytes and terminally differentiated T helper cells.129 The last well-documented secretion-based methods of direct cancer-induced T-cell apoptosis are the acidic and hypoxic stress found in the TME. Acidity is caused by the 'Warburg effect', whereby cancer cells preferentially engage in aerobic glycolysis rather than oxidative phosphorylation metabolism of glucose.130 Consequently, they increase their glucose intake to meet their energy demands, producing excess lactate acid, which is secreted into the microenvironment, causing acidification of the extracellular space.131 Long-term exposure (>3 days) to acidic pH in the TME (pH 6.5) caused permanent damage and T-cell apoptosis in C57BL-murine B16-melanoma TILs.132 Under extreme conditions, acidic stress (pH 
+3.3 for 25 min at 37°C) induces intrinsic apoptosis in Jurkat T cells by increasing cell cycle arrest.133 Although in vitro studies demonstrated that acidic stress can alter apoptosis in T cells, the effect of acidic conditions in vivo remains unknown and requires careful validation. Along with increased acidity, hypoxia in the TME can also be a critical factor affecting T-cell apoptosis. Kiang *et al* found hypoxia-induced apoptosis in the Jurkat cell line.134 The authors attributed apoptosis to increase NO production due to the upregulation of NO synthase, subsequently increasing Caspase-9 activation, cytochrome c levels, and Caspase-3 activation. In addition, hypoxia (1% O2
+) 
+induces apoptosis in primary T cells from healthy donors, hypothesizing it to result from a buildup of endogenous adenosine in the extracellular medium. The authors found that T cells had an upregulation of the adenosine receptor A2aR. The downstream effects of these receptors in inducing apoptosis have not been characterized.135
+
+## Strategies To Avoid T-Cell Apoptosis In The Tme
+
+Long-term survival and functionality of T cells are critical to ensure the anticancer efficacy of cancer immunotherapies.136 Several strategies have been developed to prevent T-cell apoptosis. Yamamoto *et al* established a novel CAR-T cell that inhibits FasL-mediated T-cell apoptosis by truncating the intracellular death domain of Fas or introducing a point mutation (I246N) in the Fas death domain.137 These modifications allow CAR-T cells to become resistant to cancer-induced FasL-mediated enhancement of CAR-T cells and venetoclax combination effects.
+
+apoptosis by inhibiting the recruitment of FADD into the apoptotic complex and preventing DISC formation. 
+
+The failure of DISC formation enhanced CAR-T cell persistence and anticancer activity in a murine B16 melanoma cancer model. Importantly, Fas-engineered T cells did not show uncontrolled proliferation, at least in in-vivo models, suggesting that modulation of T-cell extrinsic apoptosis may be a safe and feasible strategy.137 Similarly, another study reported that CRISPR-mediated KO of Fas reduced the AICD of anti-CD19 CAR-T cells during chronic exposure to target cells, which led to increased T-cell expansion.138 In addition to modifying extrinsic apoptosis in T cells, Charo *et al* generated murine T cells that overexpress BCL-2 and tested whether this modification leads to enhanced anticancer activity of cytolytic T cells by preventing apoptosis.35 The authors identified that BCL-2 overexpressing T cells show superior anticancer activity compared with wild-type T cells by improving long-term survival in the absence of a survival signal. Recently, another study revealed that constitutive overexpression of BCL-2 in CAR-T cells improves CAR-T cells proliferation and reduces AICD in CAR-T 
+cells.139 Our group further demonstrates that higher levels of BCL-2 expression in CAR-T cells of patients with lymphoma significantly correlate with enhanced clinical response (ie, CAR-T persistence and overall survival) of CAR-T therapy, suggesting that modulating intrinsic apoptosis in T cells is an important strategy to enhance CAR-T therapy.33 In addition to BCL-2, there are other critical anti-apoptotic regulators (ie, MCL-1 and BCL-xL) affecting T-cell survival and differentiation. Studies using transgenic expression of these anti-apoptotic regulators have suggested their potential implications in T cell-based immunotherapy. For instance, constitutive expression of BCL-xL rescued activation-induced cell death of CD8+
+T cells in a viral infectious model.140 Enhanced expression of MCL-1 promotes long-term memory formation in the acute phase of vaccinia virus infections.141 Despite the beneficial effect of BCL-2 family overexpression in T 
+cells, altering the BCL-2 signal in T cells requires additional attention, as the constitutive expression of BCL-2 in murine T cells promoted T-cell lymphoma development (ie, 18 of 68 BCL-2 transgenic mice developed T-cell lymphoma).142 Finally, developing strategies to avoid T-cell apoptosis would be beneficial for preventing potential apoptosis of T cells when combining immunotherapies with proapoptotic drugs. Our group recently reported a novel strategy to overcome venetoclax-mediated CAR-T cell toxicity by developing venetoclax-resistant CAR-T cells 
+(ven-CAR-T).33 In ven-CAR-T, we introduced a mutant form of BCL-2 containing a point mutation at the 104 amino acid residue (Phe104Leu or F104L) located in the binding pocket of venetoclax. Accordingly, venetoclax cannot bind to BCL-2(F104L) and loses its inhibitory function.143 144 Therefore, by overexpressing BCL-2(F104L) in ven-CAR-T, ven-CAR-T showed strong resistance to venetoclax, leading to a significant 
+
+## Conclusions
+
+As immunotherapy is ready to make its next steps and advances as a line of therapy for patients, a critical factor is the development of strategies to overcome the current limitations that preclude responses in a significant subset of patients. This review discussed the dual role of apoptosis in T cell-based immunotherapy, from cancer (ie, resistance to apoptosis) as well as a T-cell side (ie, apoptotic death). Most cancers are characterized by resistance to apoptosis through several mechanisms, including neutralizing pro-apoptotic signals by either increasing expression of anti-apoptotic molecules such as IAPs and BCL-2 or decreasing positive regulators of the death receptormediated apoptosis. Therefore, increasing the sensitivity of cancer cells to apoptosis should be considered a vital strategy to improve the anticancer activity of T cell-based immunotherapies. Combining pro-apoptotic drugs may be an appealing approach for sensitizing cancer cells to T cell-mediated death; for instance, inhibiting key antiapoptotic regulators (IAPs and BCL-2) by targeted small molecules (SMAC mimetics and ABT737) enhanced CAR-T cell-mediated anticancer activities. However, because such drugs may also induce T-cell apoptosis, careful consideration of the administration timing/dose of pro-apoptotic drugs or apoptosis-sensitizing treatments must be made to determine the optimal therapeutic regimens.
+
+Regarding T-cell apoptosis, cancer evades immunotherapy by secreting pro-apoptotic inducers against cytolytic T cells and developing a hostile TME. Thus, there is a clear need for combinations that can prevent these evasion mechanisms. CAR-T cell therapy presents a versatile option not only for combination strategies but also for the possibility of performing genetic engineering (eg, Fas KO, mutant Fas, or constitutive overexpression of BCL-2). However, as a consequence of enhancing T-cell survival/expansion by aforementioned modulations, safety concerns such as abnormal lymphoproliferation and tumorigenesis of modified T cells appear. Therefore, it is critical to include safety switches in these models to maximize safety in clinical use (eg, the anti-inducible Caspase-9 system and antibody-mediated cellular cytotoxicity using a truncated epidermal growth factor receptor/
+anti-epidermal growth factor receptor antibody).
+
+In conclusion, apoptosis is a crucial player in T cellbased immunotherapy. Deep knowledge of mechanisms of apoptosis resistance in cancer and T-cell biology is necessary to promote cancer cell apoptosis and prevent T-cell death. Several novel agents being developed together with the most recent advances in bioengineering will pave the way for the success of next-generation therapeutic combinations.
+
+## 
+
+Twitter Puneeth Guruprasad @PuneethGuru_ and Marco Ruella @MarcoRuella Contributors YGL and MR wrote and revised the manuscript. NY, IC, PP, AC, LP, 
+CTS, PG, and RP revised the manuscript. MR and YGL funded the project. MR supervised the project. All authors reviewed and approved the manuscript. Funding This research was supported by the Mark Foundation (MR), the Upenn TAPITMAT grant (MR), the Lymphoma Research Foundation (YGL), Basic Science Research Program through the national Research Foundatio of Korea 
+(2022R1C1C1011603, YGL), NCI 1K99CA212302 and NCI R00CA212302 (MR), 
+Laffey McHugh Foundation (MR), Parker Institute for Cancer Immunotherapy (MR), and Berman and Maguire Funds for Lymphoma Research at Penn (MR). Competing interests M. Ruella reports grants from the Mark Foundation, Upenn TAPITMAT, the Lymphoma Research Foundation, the NCI (1K99CA212302 and R00CA212302), the Parker Institute for Cancer Immunotherapy, and the Berman and Maguire Funds for Lymphoma Research at the University of Pennsylvania during the conduct of the study; grants from Novartis outside the submitted work; a patent for BCL-2 and CART pending; is listed as an inventor of CART technologies, University of Pennsylvania, partly licensed to Novartis, Tmunity, and viTToria Biotherapeutics; research funding from AbClon, Beckman Coulter, Lumicks, and ONI; consultancy for/honoraria from NanoString Technologies Inc. and GLG; advisory boards for AbClon, Bayer, Sana, Bristol Myers Squibb, GSK, and viTToria Biotherapeutics; and is a scientific founder of viTToria Biotherapeutics. No disclosures were reported by the other authors.
+
+## Patient Consent For Publication Not Applicable.
+
+Provenance and peer review Commissioned; externally peer reviewed. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/. ORCID iDs Raymone Pajarillo http://orcid.org/0000-0003-3299-0929 Marco Ruella http://orcid.org/0000-0003-4301-5811
+
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medical/md/PMC10202116.md

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+# Open Access Full Text Article O R I G I N A L R E S E A R C H Predictors For The Longevity Of People With Diabetes In Buno Bedele And Illubabor Zones, South-West Ethiopia
+
+Melaku Tadege 1, Azmeraw Misganaw2, Zemenay Truneh1, Awoke Seyoum Tegegne 3 1Department of Statistics, Injibara University, Injibara, Amhara, Ethiopia; 2Department of Statistics, Mettu University, Mettu, Oromia, Ethiopia; 3Department of Bio-Statistics, Bahir Dar University, Bahir Dar, Ethiopia Correspondence: Awoke Seyoum Tegegne, Email bisrategebrail@yahoo.com Introduction: Currently, diabetes is a global health problem and it affects many people, especially in the developing continents. As patients' living conditions improve and the science of medicine advances, the longevity of such patients has increased greatly. Therefore, the purpose of this study was to identify predictors for the association of the longevity of people with diabetes in Buno Bedele and Illubabor Zones, South-west Ethiopia.
+
+Methods: The study applied a retrospective cohort study design approach. In particular, long rank tests for longevity experience and Cox semi-parametric regression were implemented to compare and investigate the predictors associated with the longevity of patients with diabetes.
+
+Results: Among all the patients who participated in this study, 56.9% were females and the rest were males. From the Cox regression result, age (AHR = 1.0550, 95% CI: (1.0250, 1.0860), p-value = 0.001), female patients (AHR = 0.2200, 95% CI: (0.0390, 0.5290)), rural patients (AHR = 0.2200, 95% CI: (0.1000, 0.4890), p-value = 0.001), the existence of fasting blood glucose complication (AHR = 1.2040, 95% CI: (1.0930, 1.4460), p-value = 0.001), the existence of blood pressure (AHR = 1.2480, 95% CI: (1.1390, 1.5999), p-value = 0.0180), treatment type, Sulfonylureas (AHR = 4.9970, 95% CI: (1.4140, 17.6550), p-value = 0.0120), treatment type, Sulfonylurea and Metformin (AHR = 5.7200, 95% CI: (1.7780, 18.3990), p-value = 0.0030) were significantly affected the longevity of people with diabetes.
+
+Conclusion: The findings of the current study showed that the patient's age, sex of patients, residence area, the existence of complications, existence of pressure, and treatment type were major risk factors related to the longevity of people with diabetes. Hence, health-related education should be given to patients who come to take treatment to have better longevity for people with diabetes. More attention should be given to aged patients, male and urban patients, patients under complication treatment, and patients under treatment with single-treatment medication.
+
+Keywords: DMD, mortality, Log rank, Cox proportional hazards model, longevity
+
+## Introduction
+
+Non-communicable diseases (NCDs) are the leading cause of death worldwide and present a huge threat to health and development, particularly in developing continents. The diseases kill about 41 million people and account for 74% of all deaths each year, and about 17 million people die from an NCD before age 70 which leads to 86% of premature deaths occurring in developing continents. The diseases consist of cardiovascular diseases, cancers, chronic respiratory diseases, and diabetes. All these four groups account for over 80% of all premature non-communicable deaths.
+
+Diabetes consists of three major groups, namely, T1DM, T2DM, and gestational diabetes mellitus.1 In T2DM, the pancreas makes less insulin than it used to, and the body becomes resistant to insulin while in T1DM, the body's system damages the cells that discharge insulin, eventually removing insulin fabrication from the body.2 On the other hand, gestational diabetes is well defined as the amount of glucose intolerance with onset or first gratitude throughout pregnancy.3 T1DM is a hereditary complaint that existed in individuals at a young age life. On the other hand, T2DM 
+is mainly developed over time.4 Hence, an individual with high blood glucose levels can lead to serious health complications, whether an individual has T1DM or T2DM.5 So if an individual has either condition, he/she needs to take the right steps to manage it.
+
+Diabetes is the most serious metabolic disease in the world as many people with diabetes are unaware that they are living with diabetes or not may be for up to a decade before diagnosis.6 Hence, hyperglycemia over a long period of time is strongly associated with the development of diabetic complications.7 Diabetic complications lead to damage of tiny vessels like neuropathy, nephropathy, retinopathy, and enormous blood vessels as in cardiovascular diseases.8 It is well known and established that in diabetes, long-term complications result from abnormal regulation of glucose metabolism, and all manifestations of cardiovascular disease, coronary heart disease, stroke, and peripheral vascular disease are substantially more common in patients with diabetes than in normal people.9 Patients with diabetes have a two-to-four-fold increased risk of fatal and nonfatal coronary events.10 Previous studies indicate that there are various mechanisms for preventing and treating diabetes. Screening of a person with pre-diabetes reveals a chance to detect those individuals who are at high risk for emerging diabetes and at high risk of developing cardiovascular disease (CVD).11 Identifying people with pre-diabetes to condemn the progression at an early stage has several advantages.12 Such interventions may be categorized as primordial, primary, secondary, or tertiary-level strategies, based on the pathophysiological stage at which they are being targeted.13 Primordial interventions are activities of an individual such as reduction of fat or salt intake, increased physical activity, and weight loss and belong to the whole population.14 The primary intervention is a strategy that belongs to the prevention of type 2 diabetes progressions.15 This would include the use of pharmacological agents and lifestyle modification.16 Prevention for the development of diabetic complications belongs to secondary prevention, and tertiary prevention includes the handling of specific diabetic complications, to prevent excess amount of morbidity and mortality.17 Therefore, diabetes is highly prevalent with an increasing incidence globally.18 World Health Organization (WHO) 
+declared that about 90% of the people in the world who suffered from diabetes belong to T2DM.19 It is an epidemic in many low- and middle-income countries with many people in younger age groups.20 The latest International Diabetes Federation (IDF) report declared that the global prevalence of diabetes mellitus was estimated to be 3.3% of adults, with total cases of diabetes in adults as 1,920,000 and this will be increased to 55 million by 2045.21 Some of the African countries with the highest number of people living with diabetes are Nigeria (3.9 million), South Africa (2.6 million), Ethiopia (1.9 million), and Tanzania (1.7 million).22 One of the previous systematic reviews of diabetes conducted in 2011 in sub-Saharan Africa (SSA) indicates that about 12.1 million people are living with diabetes in Africa and if the condition continues in this trend, by 2030, about 23.9 million people will be expected to live with diabetes.23 International Diabetes Federation (IDF) indicates that Ethiopia is one of the 48 countries in African regions where 24 million people are living with diabetes.24 Another study indicates that Ethiopia is one of the top five countries with the highest number of people living with diabetes in sub-Saharan Africa.25 Some hospital-based statistical data evidence showed that the prevalence of the occurrence of diabetes and associated death are increasing,26 and this is why the authors are initiated to investigate the situation to identify the risk factor associated with the longevity of people with diabetes. There is a scarcity of such research or investigations associated with risk factors for the longevity of people with diabetes in the study area. Longevity is the long waiting time of people with diabetes or people with diabetes that cannot be cured in a short time. It is the hazard/risk of people living with diabetes.
+
+## Materials And Methods Study Area
+
+The current study was done in Buno Bedele and Illu Aba Bora zones, Oromia Region, Ethiopia. Buno Bedele and Illu Aba Bora zones are located in the south-west part of Ethiopia.
+
+## Data
+
+An institution-based cohort study design was conducted in the current study. The data collection was conducted by health professionals and the investigators strictly followed the data quality at Buno, Bedele, and Illubabor zones. Data were analyzed by using SAS software version 9.2.
+
+## Sample Size And Selection Method
+
+The study was conducted in two senior hospitals and five health centers. The two hospitals were Illubabor General Hospital and Buno Bedele Specialized Hospitals, selected based on the service they gave and the number of people served in the health institution. Health centers were selected randomly. The systematic random sample selection technique was applied to select patients, considering their id number as code. The study was conducted in a period from September 2008 to January 2018. All people with diabetes who were under treatment in the study period were considered the target population. The sample size was determined using single population proportions' sample size calculation, considering a 95% confidence level, 4% degree of precision, and 33% proportion of TB/HIV co-infection.27 A stratified random sampling method was used for selecting a representative sample from each of the two hospitals in the study area. Hence, the sample size was allocated proportionally based on the number of patients followed up at each hospital. Patients were selected randomly using their ART unique identification number. The study considered all people living with diabetes and under treatment fulfilled inclusion criteria, regardless of their treatment category during the study period. Hence, a total of 444 randomly selected patients were considered for the current investigation.
+
+## Study Variables
+
+The response variable for this study was longevity/long waiting time for people with diabetes. The predictor variables included in the study were gender (male, female), age of the patients, residence area (rural, urban), marital status, fasting blood sugar, the existence of complications (yes, no), patients with hypertension (yes, no), type of medication (treatment type), and type of diabetes mellitus.
+
+## Data Collection Procedures And Their Quality
+
+All the relevant information about the patient's health-related issue was taken from the patient's chart in the hospital records, repeated questioning of the patient at different visits, and the relationship of the onset to well-known historical events. During data collection, all participants had completed the physical examination. Laboratory examination results like patients' blood glucose, weight, and blood pressure were recorded at each visiting time.
+
+The long-term complications of diabetes were also categorized based on WHO Report.28 Blood glucose was usually measured by the glucose oxidase method with Dextrostix and an Eyestone reflectance meter. Urine was examined for protein content by precipitation with 20% Sulfosalicylic acid.29
+
+## Methods Of Data Analysis Non-Parametric Methods
+
+The Kaplan–Meier, Nelson-Aalen, and Life Tables were used to estimate the longevity of people living with diabetes and hazard functions.
+
+The Product-Limit estimator with all information about uncensored and censored observations was used for estimates of the survival function.30 The observed data were used to estimate the conditional probability which is used to estimate the overall survival function.
+
+The Log rank test was used for comparing two or more survival curves (longevity of people with diabetes) in the case that the distribution is right-skewed and censored data sets. On the other hand, the Wilcoxon test was used when there is no censoring in the data set. Log rank test was computed at 5% level of significance to compare the survival experience between different categories to all categorical predictors.
+
+## Semi-Parametric Models
+
+The Cox-Proportional Hazard Model was used for estimating time-varying, time-independent, continuous, and discrete covariates. Kaplan–Meier estimate and Log rank test were also used for all categorical variables to know if there is a significant difference among the categories of each predictor variable.
+
+## Kaplan–Meier Estimation
+
+The well-known study method for the estimator of the survival function, proposed by (Kaplan and Meier, 1995) is the Product-Limit estimator. This estimator technique incorporates information from all observations included in the study, both uncensored (event) and censored estimates. This considers survival at any point in time as a series of steps defined at the observed and censored times. The observed data were used to estimate the conditional probability of established longevity at each observed time for people living with complications and then multiply them to obtain an estimate of the overall survival function.
+
+## Log Rank Test
+
+The most frequently used nonparametric test is a Log rank test, sometimes called the Co-Mantel or Mantel test. The Log rank statistic is approximately standard normal; it can be viewed as time-stratified. It is a nonparametric test for comparing two or more survival curves, and it is more powerful than Wilcoxon's other non-parametric tests. Because the Log rank test considers right skewed and censoring data sets, whereas the Wilcoxon test is used when there is no censoring in the data set.
+
+## Semi-Parametric Models The Cox-Proportional Hazard Model
+
+The usual statistical model used for survival data is the proportional hazards (PH) model, which is known as the Cox regression model. This model has certain assumptions to conduct a given research but with no assumption on particular form of a probability distribution for the survival times. The Cox proportional hazard model is popular because of its flexibility in selecting covariates. The two other issues that make it popular are it does not make any assumption about the underlying survival distribution and does not require estimation of the baseline hazard rate, to estimate the regression parameters. It is a multiple regression method and is used to evaluate the effect of multiple covariates on the hazard.25 It is a semi-parametric model for the hazard function that allows the addition of covariates while keeping the baseline hazards unspecified, and we can take only positive values.25
+
+## Results
+
+There were 444 patients in the cohort study, of which 61 (13.7%) were dead. Of the total number of people with diabetes, 206 (46.4%) of them were females and 238 (53.6%) were males. The majority of patients, 194 (47.7%) of them were unmarried. Among all the patients, 296 (66.7%) of them were urban patients, and others were rural patients. Of all the people with diabetes, about 288 (64.9%) of them were type 2, and 156 (35.1%) of them were type 1. About 159 (35.8%) of them were living with fasting glucose complications. Of all the patients, 186 (41.9%) were hypertensive (Refer to Table 1).
+
+## Longevity Of People With Diabetes/Log Rank Survival Estimate
+
+From the Log rank test, the survival practice of patients related to profession and diabetes mellitus type, marital status, residence area, treatment type, and hypertensive status had a significant difference in the longevity of people with diabetes under the complicated risk stage at a 5% statistically significant level (Refer to Table 2). Hence, Table 2 indicates that the expected waiting time (longevity) of married people to be cured of the disease was significantly shorter than that of separated and never married people with diabetes (p-value < 0.01). Similarly, the expected waiting time of people with diabetes tocure from the disease was significantly shorter than urban patients (p-value = 0.010). In the same way, complication status and hypertensive status had significant differences with their counterparts.
+
+| Table 1 Descriptive Statistical Analysis Variables Category   | n                   | %    |      |
+|---------------------------------------------------------------|---------------------|------|------|
+| Sex                                                           | Male                | 206  | 46.4 |
+| Female                                                        | 238                 | 53.6 |      |
+| Marital status                                                | Living with partner | 173  | 39   |
+| Living with partner                                           | 271                 | 61   |      |
+| Residence area                                                | Rural               | 148  | 33.3 |
+| Urban                                                         | 296                 | 66.7 |      |
+| Diabetes mellitus                                             | Type 1              | 156  | 35.1 |
+| Type 2                                                        | 288                 | 64.9 |      |
+| Longevity                                                     | Cured               | 61   | 13.7 |
+| Censored/under risk treatment                                 | 383                 | 86.3 |      |
+| Complication                                                  | Yes                 | 159  | 35.8 |
+| No                                                            | 285                 | 64.2 |      |
+| Treatment type                                                | Sulfonylurea        | 70   | 15.8 |
+| Metformin                                                     | 102                 | 23.0 |      |
+| Sulfonylurea and Metformin                                    | 99                  | 22.3 |      |
+| Insulin therapy                                               | 104                 | 23.4 |      |
+| Sulfonylurea and insulin therapy                              | 69                  | 15.5 |      |
+| Blood Pressure                                                | Yes                 | 186  | 41.9 |
+| No                                                            | 258                 | 58.1 |      |
+
+| Table 2 The Long Rank Test Result Variables Categories   | Mean (SD) Estimate   | p-value       |       |
+|----------------------------------------------------------|----------------------|---------------|-------|
+| Sex                                                      | Female               | 50.848 (5.23) | 0.653 |
+| Male                                                     | 60.038 (2.36)        |               |       |
+| Marital status                                           | Married              | 50.404 (1.29) | 0.001 |
+| Separated                                                | 58.977 (2.31)        |               |       |
+| Never married                                            | 62.501 (3.41)        |               |       |
+| Residence                                                | Rural                | 39.199 (2.35) | 0.010 |
+| Urban                                                    | 57.551 (2.41)        |               |       |
+| Diabetes mellitus type                                   | Type 1               | 62.842 (4.21) | 0.001 |
+| Type 2                                                   | 57.742 (2.91)        |               |       |
+| Treatment type                                           | Sulfonylurea         | 52.566 (3.18) | 0.029 |
+| Metformin                                                | 58.167 (1.45)        |               |       |
+| Sulfonylurea and Metformin                               | 50.474 (2.41)        |               |       |
+| Insulin therapy                                          | 61.839 (2.35)        |               |       |
+| Sulfonylurea and insulin therapy                         | 57.492 (3.17)        |               |       |
+| Complication status                                      | Yes                  | 60.440 (3.26) | 0.013 |
+| No                                                       | 56.147 (2.18)        |               |       |
+| Hypertensive status                                      | Yes                  | 56.889 (3.27) | 0.001 |
+| No                                                       | 41.640 (2.62)        |               |       |
+
+## Cox Proportional Hazard Model Results
+
+The Cox proportional hazard model in Table 3 indicates that as age increased by one year, the expected hazard/risk of the longevity of people living with diabetes was increased by 5.5% (AHR = 1.055, CI (1.025, 1.086), p-value ≤ 0.001), given that the other covariates are constant.
+
+Comparing rural patients with urban, the expected hazard of the longevity of people living with diabetes for rural patients was decreased by 78.1% as compared to urban patients (AHR = 0.2210, 95% CI: (0.1000, 0.4890), p-value < 0.01), given the other conditions constant.
+
+| Table 3 Multivariable Analysis of Cox Proportional Hazard Model Covariates P-value   | AHR    | 95.0% CI Lower   | Upper   |         |
+|--------------------------------------------------------------------------------------|--------|------------------|---------|---------|
+| Age                                                                                  | 0.001  | 1.0550           | 1.0250  | 1.0860  |
+| Sex (Ref.=Male) Female                                                               | 0.0480 | 0.2200           | 0.0390  | 0.5290  |
+| Residence (Ref.=Urban) Rural                                                         | 0.001  | 0.2210           | 0.1000  | 0.4890  |
+| Marital status (Ref.=Unmarried) Married                                              | 0.6410 | 0.7780           | 0.2700  | 2.2410  |
+| Separated                                                                            | 0.6680 | 0.7470           | 0.1970  | 2.8330  |
+| DM type (Ref. =Type two) Type one                                                    | 0.3280 | 0.5330           | 0.1510  | 1.8780  |
+| Fasting blood sugar                                                                  | 0.5480 | 0.9990           | 0.9970  | 1.0020  |
+| Complication (Ref.=no) Yes                                                           | 0.001  | 1.2040           | 1.0930  | 1.4460  |
+| Blood Pressure (Ref.=no) Yes                                                         | 0.0180 | 1.2480           | 1.1390  | 1.5999  |
+| Treatment type (Ref.= Sulfonylurea and insulin therapy (Ref)) Sulfonylurea           | 0.0120 | 4.9970           | 1.4140  | 17.6550 |
+| Metformin                                                                            | 0.3480 | 1.7680           | 0.5380  | 5.8050  |
+| Sulfonylurea and Metformin                                                           | 0.0030 | 5.7200           | 1.7780  | 2.3990  |
+| Insulin therapy                                                                      | 0.6560 | 1.3160           | 0.3920  | 4.4160  |
+| Abbreviations: Ref, Reference category; AHR, Adjusted hazard rate.                   |        |                  |         |         |
+
+The sex of patients was one of the other significant covariates affecting the longevity of people living with diabetes. 
+
+Hence, comparing female patients with males, the expected risk of the longevity of female people living with diabetes was decreased by 88% as compared to male patients (AHR = 0.2200, 95% CI: (0.0390, 0.5290), p-value= 0.0480), keep the other conditions constant.
+
+Fasting blood glucose complications also significantly affected the risk of longevity of people living with diabetes. 
+
+Comparing those people with diabetes who had also fasting blood glucose complications with people free from fasting blood glucose complications, the expected risk of longevity of people living with diabetes for those who had also fasting blood glucose complications was increased by 20.4% as compared to those without blood glucose complication (AHR = 1.2040, 95% CI: (1.0930, 1.4460), p-value < 0.01), considering the other covariates constant.
+
+Blood pressure also significantly affected the dependent variable. Hence, the expected risk of the longevity of people with diabetes who had also blood pressure was increased by 24.8% as compared to people with diabetes but with no blood pressure (AHR = 1.2480, 95% CI: (1.1390, 1.5990), p-value = 0.0180), given the other things constant.
+
+Treatment type also affected the study variables. Comparing those people with diabetes with treatment type Sulfonylurea and insulin therapy, the expected risk of people living a long time whose treatment type Sulfonylurea was 4.997 times that of people living with diabetes whose treatment type Sulfonylurea and insulin therapy (AHR = 4.9970, 95% CI: (1.4140, 17.6550), p-value= 0.0120), given the other conditions constant (Refer to Table 3).
+
+## Discussions
+
+The significant variables that affected the study variable are discussed as follows. The age of people with diabetes significantly affects the long life of people under the risk conditions. Hence, as age increased, the complication of the risk of treatment for diabetes also increased and this further leads to taking a long time to be cured of the disease. The potential reason for this might be the fact that aged people have a small number of white blood cells, used to fight diseases, and such people may be exposed to additional diseases. This result was consistent with another previously conducted study.27 The sex of people with diabetes also plays a significant role in the variation of the risk of longevity of such patients. 
+
+One of the mechanisms of preventing and treating diabetes is doing physical exercises. Most of the time females are busy at home managing children and cooking food and this leads to a reduction in blood glucose. Hence, females are less likely to be exposed to the risk of living with diabetes under treatment as compared to males. This result was similar to one of the other previously conducted research29 but contradicted another study.30 The potential reason for the contradiction might be the study area, sample size, and study period.
+
+Comparing rural patients with urban, rural people are always engaged in physical exercises for daily livelihood activities and this leads to reduction of fats and blood glucose. Hence, people with diabetes can cure from diabetes in a short time with simple treatment as compared to urban people with diabetes.31 Fasting blood glucose complications also significantly affected the risk of longevity of people living with diabetes. 
+
+Comparing those people with fasting blood glucose complications with people free from fasting blood glucose complications, the people without fasting blood complications need a short time to be free from the disease, assuming that the other conditions remain constant. This result is supported by one of the studies conducted previously.31 Blood pressure also affects the dependent variable. The duration of time for curing diabetes for people who have blood pressure is short as compared to people with blood pressure in addition to diabetes. The possible reason for this might be the interaction effect of the two treatments given for diabetes and blood pressure. This result is supported by other studies.32 Treatment type also affects the study variables. Comparing those people with diabetes with treatment type Sulfonylurea and insulin therapy, people with diabetes whose treatment type Sulfonylurea need a long time period to be free from as compared to those people with diabetes with treatment type Sulfonylurea and insulin therapy. This result is contradicted by another study conducted previously,33 and this needs further investigation for the result to be coherent and consistent.
+
+## Limitations
+
+This study is not without limitations. One of the limitations is that the study was done in only two zones and including that more health institutions may give additional information about the predictor variables. Since the data were secondary, some important variables such as doing physical exercise or not, job type, and others may also reveal additional information. Authors recommend this for future research including more variables and additional health institutions.
+
+## Conclusion
+
+Important significant predictors for the variable of interest were identified for proper intervention to the program. Hence, the age of patients, the sex of patients, the residence area of patients, the existence of complications, and the existence of blood pressure, diabetes type, and treatment type significantly affected the longevity of people with diabetes to wait a long time under risk treatment.
+
+The study recommends that zonal health institutions and the Ministry of Health should give more attention to those people who were in a long time under treatment to be cured of the disease. Health professionals should care for patients of older age, living with complications, and living in urban. The result obtained in the current study would be used as a baseline for future researchers, managers, and policymakers.
+
+## Abbreviations
+
+T1DM, Type one diabetes patients; T2DM, Type two diabetes patients; SSA, Sub-Saharan African; AHR, adjusted hazard rate; CI, confidence Interval; NCDs, Non-communicable diseases; DMD, Diabetes mellitus disease.
+
+## Data Sharing Statement
+
+The data used in the current study are available under the corresponding author and can be attached on request.
+
+## Ethics Approval And Consent To Participate
+
+The study was carried out after receiving an ethical clearance endorsement letter from Mettu University Research Technical and Ethical Review Board and permission was obtained from the health office of the Buno Bedele and Illubabor zone. This study used secondary data from medical case records and patients were not contacted by investigators, and consent to participate was not taken from respondents. During data analysis, the authors used only the id number of each patient to be confidential.
+
+## Consent For Publication
+
+This manuscript was not published or is not under consideration for publication by other journals.
+
+## Acknowledgments
+
+The authors need to forward their acknowledgement for all the health staffs at the two hospitals. Data collectors shall also owe our heartfelt thanks for their cooperation for the success of this research work.
+
+## Author Contributions
+
+All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.
+
+## Funding
+
+There is no agent/institution that funded this research.
+
+## Disclosure
+
+The authors report no conflicts of interest in this work.
+
+## References
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+a secondary analysis of the Look AHEAD randomised clinical trial. *Lancet Diabetes Endocrinol*. 2014;2(10):801–809.
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+(with or without sulfonylureas) in insulin-naive patients with type 2 diabetes (SUSTAIN 4): a randomised, open-label, parallel-group, multicentre, multinational, phase 3a trial. *Lancet Diabetes Endocrinol*. 2017;5(5):355–366.
+
+32. Derks M, Bastiaannet E, van de Water W, et al. Impact of age on breast cancer mortality and competing causes of death at 10 years follow-up in the adjuvant TEAM trial. *Eur J Cancer*. 2018;99:1–8. doi:10.1016/j.ejca.2018.04.009 33. Macedo AF, Douglas I, Smeeth L, et al. Statins and the risk of type 2 diabetes mellitus: cohort study using the UK clinical practice research datalink. *BMC Cardiovasc Disord*. 2014;14:1–12. doi:10.1186/1471-2261-14-85 Diabetes, Metabolic Syndrome and Obesity Dovepress Publish your work in this journal Diabetes, Metabolic Syndrome and Obesity is an international, peer-reviewed open-access journal committed to the rapid publication of the latest laboratory and clinical findings in the fields of diabetes, metabolic syndrome and obesity research. Original research, review, case reports, hypothesis formation, expert opinion and commentaries are all considered for publication. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. 
+
+Submit your manuscript here: https://www.dovepress.com/diabetes-metabolic-syndrome-and-obesity-journal

medical/md/PMC10484673.md

@@ -0,0 +1,535 @@
+# Genetically Encoded Rna-Based Sensors With **Pepper** Fluorogenic **Aptamer**
+
+## Zhenyin Chen1,2,3, Wei Chen1,4, Zhayila Reheman1,5, Haodong Jiang1,3, Jiahui Wu6 And Xing Li **1,2,7,8,***
+
+1Beijing Institute of Life Sciences, Chinese Academy of Sciences, Beijing 100101, China, 2Department of Pulmonary and Critical Care Medicine, Department of Inflammation and Clinical Allergology, the Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China, 3University of Chinese Academy of Sciences, Beijing 100049, China, 4Institute of Cytology and Genetics, the Hengyang Key Laboratory of Cellular Stress Biology, Hengyang Medical School, University of South China, Hengyang, Hunan 421001, China, 5School of Life Science, Hebei University, Baoding, Hebei 071000, China, 6Department of Chemistry, University of Massachusetts, Amherst, MA01003, USA, 7Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China and 8State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China Received January 24, 2023; Editorial Decision July 02, 2023; Accepted July 21, 2023
+
+## Abstract
+
+Sensors to measure the abundance and **signaling**
+of intracellular molecules are crucial for understanding their physiological functions. Although conventional fluorescent protein-based sensors have been designed, RNA-based sensors are promising imaging tools. Numerous RNA-based sensors have been developed. These sensors typically contain RNA Gquadruplex (RG4) motifs and thus may be suboptimal in living cells. Here we describe **RNA-based** sensors based on Pepper, a fluorogenic RNA without an RG4 motif. With Pepper, we engineered various sensors for metabolites, synthetic **compounds,**
+proteins and metal ions in vitro and in living **cells.**
+In addition, these sensors show high activation and selectivity, demonstrating their universality and robustness. In the case of sensors responding to Sadenosylmethionine (SAM), a metabolite **produced**
+by methionine adenosyltransferase **(MATase),** we showed that our sensors exhibited positively correlated fluorescence responding to different SAM levels. Importantly, we revealed the SAM biosynthesis pathway and monitored MATase activity and gene expression spatiotemporally in living individual human cells. Additionally, we constructed a **ratiometric**
+SAM sensor to determine the inhibition efficacy of a MATase inhibitor in living cells. Together, these sensors comprising Pepper provide a useful platform for imaging diverse cellular targets and their **signaling** pathway.
+
+![0_image_0.png](0_image_0.png)
+
+## Introduction
+
+Genetically encoded sensors are imaging tools that allow cellular small molecules, proteins and ions to be imaged over time in living individual cells. These sensors can characterize the spatial and temporal distributions of the cellular targets and provide insights into how various signaling pathways regulate intracellular molecules (1). The conventional genetically encoded sensors are composed of a fluorescent protein flanking a target-binding domain. Target binding to the sensor triggers conformational changes and foldsthe fluorescent protein, thusinducing fluorescence
+(2). However, the lack of a target-binding protein domain
+*To whom correspondence should be addressed. Tel: +86 10 84504251; Fax: +86 10 64807099; Email: lix@biols.ac.cn prevents the application of fluorescent protein-based sensors (3,4). Additionally, the low signal-to-noise ratio of fluorescent protein-based sensors limits the sensitive detection of cellular targets (1,2,5).
+
+In addition to fluorescent protein-based sensors, genetically encoded sensors can be composed of RNA (6). RNAbased sensors use a target-binding RNA to connect to a fluorogenic RNA aptamer, such as Spinach or Broccoli (7,8).
+
+In RNA-based sensors, the fluorophore-binding pocket of Spinach/Broccoli is connected to an RNA stem whose thermodynamic stability is regulated by target-binding RNA
+aptamers (6). Target binding induces a conformational change that regulates RNA folding, resulting in fluorescence (6). These genetically encoded RNA-based biosensors allow cellular targets to be imaged in real time.
+
+However, these RNA-based biosensors widely use fluorogenic RNA aptamers containing RNA G-quadruplex
+(RG4) motifs (6,9–20). RG4-containing aptamers or biosensors may be suboptimal in living cells as RG4 may be unfolded or depleted by the cellular machinery
+(Supplementary Figure S1) (21–24). It is therefore optimal to develop RNA-based biosensors with non-RG4 motifs and robust optical performance for cell-based study.
+
+Here, we describe an approach for generating RNAbased sensors with bright and stable fluorogenic RNA without RG4 motifs. These sensors are composed of Pepper, a recently reported fluorogenic RNA that activates the fluorescence of the otherwise non-fluorescent HBC fluorophore
+(25). Pepper exhibits several crucial merits. Pepper does not harbor the RG4 motif (26,27). Although there is no direct evidence or data to show that RG4-containing fluorogenic aptamers cannot function in cells, previous research suggests that RG4 motifs may be unfolded by cellular helicase
+(21,22) in living cells, which could inhibit the function of RG4-containing aptamers (22–24,28). Therefore, generating RNA-based sensors using Pepper or other RG4-free fluorogenic aptamers (29–33) would avoid this potential problem. Moreover, Pepper and HBC complexes showed an order of magnitude enhanced cellular fluorescence intensity and fluorescence turn-on ratio, one or two orders of magnitude enhanced affinity, ∼20◦C increased Tm, expanded pH
+tolerance and a broad spectral range available for live cell studies relative to other fluorogenic RNA aptamers (25).
+
+In addition, Pepper has high stability since Pepper has a monomeric structure, and magnesium independence, without needing scaffold RNA and little interference with RNA
+compartmental localization (25). However, Pepper has not been designed as the RNA-based sensor for tracking devise targets,such assmall molecules, proteins and ions, and their cellular signaling pathway.
+
+We thus constructed Pepper-based sensors and imaged various targets in living cells, including signaling molecules, metabolites,small molecular drugs, proteins and metal ions, as well asthe cellularsignaling pathways. We first fused Pepper into an S-adenosylmethionine (SAM)-binding aptamer, developing an RNA-based sensor that is highly activated upon binding SAM. Using SAM sensors, we readily detected endogenous SAM and its dynamics with cell-to-cell heterogeneity. Additionally, we imaged the metabolic origin of SAM synthesis, and the activity and gene expression of methionine adenosyltransferase (MATase) in single living human cells. Moreover, we constructed a ratiometric SAM
+sensor to measure the half-maximal inhibitory concentration (IC50) of the MATase inhibitor AG-270. Furthermore, we showed that other target-binding aptamers, including tetracycline-binding, guanine-binding, protein-binding and metal ion-binding aptamers, can be converted into Pepper for intracellular sensing, demonstrating the universality of the non-RG4 RNA sensortools.Importantly, Pepper-based sensors overcome the key defects of the previous Broccolibased sensors which cannot accurately detect the target in vitro and in cells. Overall, Pepper provides a bright, stable, multicolor and non-RG4 platform for generating universal and robust RNA-based sensor tools with minimal cellular perturbations in living bacteria and human cells.
+
+## Materials And **Methods** Reagents And **Equipment**
+
+All DNAs were ordered from Tsingke Biotechnology (Beijing, China). Fluorescence was measured on a FluoroMax + spectrofluorometer (Horiba Scientific) using FluorEssence software (v.3.9). Fluorescence imaging was acquired on an Olympus SpinSR10 microscope (Olympus). Data were plotted using Origin 2021 software. All RNA-binding fluorophores, including HBC, HBC620, BI,
+DFHO and tetramethyl rhodamine-dinitroaniline (TMRDN) used in this study were synthesized as described previously (25,34,35).
+
+## Cell Lines And **Transfection**
+
+HEK293T (ATCC-CRL-11268) and HeLa (ATCC-CCL-2)
+cells were cultured under standard tissue culture conditions
+(37◦C and 5% CO2) in Dulbecco's modified Eagle's medium
+(DMEM; Invitrogen C11995500BT) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were screened for mycoplasma contamination by a mycoplasma detection kit (Vazyme D101).
+
+To transfect cells with plasmids, Lipofectamine 3000 (Invitrogen 92008) was used as the transfection reagent, and transfection was performed according to the manufacturer's instructions. Cells were seeded onto 24-well plates (Nest 801006) 1 day prior to transfection.
+
+## Preparation Of Rna In **Vitro**
+
+For all Pepper-based sensor RNAs, double-stranded DNA
+(dsDNA) templates were designed to contain a 5 T7 promoter to be used for in *vitro* transcription. dsDNA templates were prepared from single-stranded DNA oligos (Tsingke Biotechnology). DNA templates were amplified by polymerase chain reaction (PCR) using Taq DNA polymerase (Vazyme P112) and checked for quality using 1.5%
+agarose gel electrophoresis. PCRs were purified with the PCR purification kit (Axygen AP-PCR-250).
+
+In *vitro* transcription reactions using the T7 High Yield RNA Transcription Kit (Vazyme DD4201) were carried out according to the manufacturer's instruction. Transcription reactions were terminated by treatment with RNase-free DNase I at 37◦C for at least 15 min. An 80 -l aliquot of RNase-free water was added followed by an equal volume of water-saturated phenol–chloroform, and this was then centrifuged at 4◦C, 12 000 rpm for 15 min. The supernatant liquid was aspirated with sodium acetate and 2 times the volume of anhydrous ethanol, and left to settle at –20◦C for 30 min. Afterwards, the precipitate was washed with 75%
+anhydrous ethanol prepared with enzyme-free water and centrifuged for 5 min, and then dissolved in enzyme-free water.
+
+## In Vitro Characterization Of The **Sensors**
+
+All concentrations quoted below are final concentrations in the measurement. In *vitro* purified RNA (1 -M) was mixed with HBC (10 -M) in the absence or presence of SAM
+(0.1 mM, Sigma-Aldrich A7007), tetracycline (0.1 mM, BBI
+A600504-0025), guanine (0.1 mM, Solarbio G8260) or silver acetate (CH3COOAg, 0.1 mM, Sigma-Aldrich 216674)
+in the buffer (40 mM HEPES, pH 7.4, 100 mM KCl and 1 mM MgCl2). After a 1 h incubation, the fluorescence of each sample was measured at 37◦C using a spectrofluorometer with 485 nm excitation, 530 nm emission, 5 nm slit widths and 0.1 s integration time. The buffer containing HBC (10 -M) was measured as a background signal. The background signal was subtracted from the signal obtained from each RNA sample measurement. Pepper- or Broccoli-based RNA sensors were measured with a cognate fluorophore (10 -M HBC or 10 -M BI) in 40 mM HEPES
+pH 7.4, 100 mM KCl and 1 mM MgCl2 buffer. Corn-based RNA sensors were measured with DFHO (10 -M) in 40 mM HEPES pH 7.4, 100 mM KCl and 5 mM MgCl2 buffer.
+
+For protein sensors, in *vitro* transcribed RNA (0.2 -M)
+was mixed with HBC (10 -M) in the absence or presence of streptavidin (100 -g/ml, ∼2 -M) in the buffer. After a 1 h incubation, the fluorescence signal of each sample was measured at room temperature (∼25◦C) using a spectrofluorometer with 485 nm excitation, 530 nm emission, 5 nm slit widths and 0.1 s integration time.
+
+The sequences of all Pepper-based sensors used in this study are shown in Supplementary Table S1.
+
+## Selectivity Measurement Of Pepper-Based **Sensors**
+
+To test the selectivity of the Pepper-based SAM sensors, Pepper-based SAM sensor RNA (1 -M) was mixed with HBC (10 -M) in the presence of the target (0.1 mM)
+or its analogs in the buffer. The samples were incubated at 37◦C for 1 h. The fluorescence signal of each sample was measured at 37◦C using a spectrofluorometer with 505 nm excitation, 545 nm emission, 5 nm slit widths and 0.1 s integration time. Analogs of SAM include SAH
+(Aladdin S139501), adenosine (Coolaber CA1241) or methionine (Coolaber CM7211). Analogs of tetracycline include doxycycline (Solarbio ID0670), neomycin (Solarbio N8090), tobramycin (Solarbio T8810), gentamycin (Solarbio G8170), ampicillin (Solarbio A1170) and kanamycin
+(Solarbio K1030). Analogs of guanine including guanosine (MACKLIN G810366) and adenine (BBI A6000130025) were used in guanine sensor selectivity measurement. Bovine serum albumin (BSA; Cusabio NP009501B),
+lysozyme (Cusabio NP004301C) and ovalbumin (Cusabio NP004201C) was used in streptavidin sensor selectivity measurement.
+
+## Dose–Response Curve **Measurements**
+
+Dose–response curves were determined by measuring fluorescence in the presence of fixed concentrations of RNA
+sensor (0.2 -M streptavidin sensor, 1 -M SAM sensor, tetracycline sensor, guanine sensor or Ag+ sensor) and fixed concentrations of HBC (10 -M) in relation to target concentration. Pepper-based sensor RNA was incubated for 1 h with a range of concentrations of target molecules in the buffer with HBC (10 -M) at 37◦C. After a 1 h incubation, the fluorescence signal of each sample was measured using a spectrofluorometer at 37◦C with an excitation wavelength of 485 nm, an emission wavelength of 530 nm, a slit width of 5 nm and an integration time of 0.1 s.
+
+## Microscopy And Image **Processing**
+
+To image Escherichia *coli* cells, we coated confocal dishes with poly-L-lysine (Sangon Biotech A600751) for at least 4 h and rinsed them once in water. We incubated isopropyl--
+D-thiogalactopyranoside (IPTG)-induced E. *coli* with M9 minimal medium (Gibco A1374401) containing HBC (10
+-M) on pre-treated confocal dishes. Live cell fluorescence images were acquired through a ×100 oil objective mounted on a microscope. Images were analyzed using Fiji software.
+
+During live cell imaging, conditions were maintained at 37◦C.
+
+To image mammalian cells, we coated 24-well confocal plates with poly-D-lysine (Sangon E607014) for at least 4 h and rinsed them once in water. One day after transfection, the cells were subcultured onto the pre-treated confocal plates. One hour before imaging, the cell medium was changed to phenol red-free DMEM (Biological Industries 06–1052-04–1ACS) supplemented with 10% FBS and HBC (10 -M). Live cell fluorescence images were acquired through a ×20 air objective (NA 0.75) mounted on the microscope. Images were analyzed using Fiji software. During live cell imaging, conditions were maintained at 37◦C and 5% CO2.
+
+## Flow Cytometry Of Pepper-Based Sensor Expressing **Cells**
+
+Two days after transfection of Pepper-based sensors, HEK293T cells were harvested and resuspended in phosphate-buffered saline (PBS) containing HBC (10 -M)
+and 4% FBS with or without cycloleucine (30 mM) or the cognate targets (tetracycline 0.1 mM, guanine 0.1 mM,
+CH3COOAg 20 -M) and kept on ice until analysis. Cells were analyzed using an LSRFortessa (BD Biosciences).
+
+Populations of cells were gated to avoid cell doublets detected by forward and side scattering, followed by gating for live cells by fluorescein isothiocyanate (FITC) fluorescence. Untransfected cells were used as a negative control for gating of fluorescence of Pepper-based sensors. Plots were generated using FlowJo software.
+
+## Determination Of The Ic50 Of Ag-270 With A Ratiometric Sam Sensor
+
+To determine the activity of MATase, we constructed a plasmid expressing an F30 scaffold with a Pepper-based SAM
+sensor in one arm and RhoBAST in the other (Supplementary Table S2). Then 24-well confocal plates were coated with poly-D-lysine (Sangon E607014) for at least 4 h and rinsed once in water. HEK293T cells were transfected with plasmid expressing the ratiometric SAM sensor or control RNA. One day after transfection, the cells were subcultured onto the pre-treated confocal plates. In the meantime, different concentrations of AG-270 (5000, 1666.67, 555.56, 185.18, 61.73, 20.58, 6.86, 2.29 and 0.76 nM) were added to the medium. After incubation for 24 h, HBC (10 -M) and TMR-DN (100 nM) were added to the medium forimaging.
+
+To process the image of the ratiometric SAM sensor, it is important to generate the green-to-red fluorescence ratio only for areas containing the cells. The red channel was used to create a binary mask to identify the cells. The region of interest (ROI) on this mask was regenerated in the images of both red and green channels. Then, areas outside the ROI
+were cleared. Finally, images containing a green-to-red fluorescence ratio were generated by dividing the masked green channel image by a masked red channel image. The ratio in the image is coded by 16-color look up tables.
+
+To calculate the IC50 of AG-270, we counted the greento-red fluorescence ratio signal of >20 cells at each pre-set concentration. The inhibition rate was calculated according to the following equation:
+
+$$Inhibition\;rate\;=\left(1-\frac{Signal_{sample}-Signal_{min}}{Signal_{max}-Signal_{min}}\right)\;\times100\%$$
+
+Subsequently, we plotted the logarithm of the concentration against the inhibition rate in Origin software and fitted it with a dose–response curve. The IC50 was acquired as the concentration corresponding to the 50% inhibition rate on the curve.
+
+## Detection Of Matase Expression By Real-Time Pcr
+
+Total RNA was isolated from HeLa cells using TRIzol reagent (Invitrogen 15596026) following the manufacturer's instructions. The quality and quantity of RNA were assessed using Nano drop (Thermo Fisher). Reverse transcription was performed using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme R312) with 1 -g of RNA in a 20 -l system.
+
+Real-time PCR analysis was conducted using Power SYBR Green PCR Master Mix (Applied Biosystems, 4309155) and carried out on a LightCycler 480 Real-Time PCR System (Roche Applied Science). Each target gene was amplified using the following primer sequences: glyceraldehyde phosphate dehydrogenase (GAPDH) forward primer 5-TGGGTGTGAAACCATGAGAAGT-3, reverse primer 5-TGAGTCCTTCCACGATACCAA-3; MAT1A
+forward primer 5-GTGTGACCACTCTCTAAGTG-3, reverse primer 5-TGCCGGTCTTGCACACTGTC-3.
+
+The amplification protocol included an initial denaturation step at 95◦C for 30 s, followed by 40 cycles of denaturation at 95◦C for 10 s, and annealing and extension at 60◦C
+for 15 s. A melt curve analysis was performed to verify the specificity of the amplification products.
+
+Gene expression levels were calculated using the 2(–-Ct)
+method (36), where the Ct values of the target gene were normalized to the Ct values of GAPDH and compared with a calibratorsample. Statistical analysis was performed using Origin 2021 software.
+
+For quality control, three technical replicates were included, and no-template controls (NTCs) were used to monitor potential contamination or primer-dimer formation.
+
+## Live-Cell Imaging Of Pepper-Based Sensors In E. **Coli**
+
+BL21 E. *coli* cells(Tsingke TSC-E01) were transformed with 40 ng of plasmid DNA expressing the Pepper-based sensor chimeras in pET28a under the control of a T7 promoter.
+
+Cells were plated, grown overnight and single colonies were picked forinoculation in Luria broth containing kanamycin
+(Solarbio K1030, 50 -g/ml). The broth was diluted to OD600 = 0.4, followed by the addition of 1 mM IPTG to the broth and shaking at 37◦C for 2 h. A 100 -l aliquot of culture was then removed, centrifuged and resuspended in 2 ml of pH 6.0 M9 minimal medium containing HBC (2
+-M), dispensed onto poly-L-lysine-coated confocal dishes
+(Nest 806001) and incubated at 37◦C for 45 min.
+
+To image SAM synthesis, cells were treated with 2 -l of 1000x methionine stock added to a final concentration of 50 mg/ml. Cells were imaged using the live-cell imaging conditions described above. Images were taken intermittently over a period of 3 h at 10 min intervals with an exposure time of 500 ms per image.
+
+For tetracycline, guanine or Ag+ imaging, the target (0.1 mM) was added to the M9 medium and incubated with cells at 37◦C for 45 min. Cells were imaged using the live-cell imaging conditions described above.
+
+To image the streptavidin level, BL21 E. *coli* cells(Tsingke TSC-E01) were co-transformed with pET28a plasmid (carrying a kanamycin resistance gene) expressing a streptavidin sensor and pET21a plasmid (carrying an ampicillin resistance gene) expressing the streptavidin protein. Cells were plated and grown overnight on solid Luria broth plates containing both ampicillin (Solarbio A1170, 100 -g/ml) and kanamycin (Solarbio K1030, 50 -g/ml). Single colonies were picked for inoculation in Luria broth containing both ampicillin (100 -g/ml) and kanamycin (50 -g/ml). The broth was diluted to OD600 = 0.4, followed by the addition of 1 mM IPTG to the broth and shaking at 37◦C for 2 h. A 100 -l aliquot of culture was then removed, centrifuged and resuspended in 2 ml of pH 6.0 M9 minimal medium containing HBC (2 -M), dispensed onto poly-Llysine-coated confocal dishes and incubated at 37◦C for 45 min. Cells were imaged using the live-cell imaging conditions described above.
+
+## Intracellular Imaging In Living Mammalian **Cells**
+
+For intracellular SAM imaging, HEK293T or HeLa cells were transfected with Tornado plasmids encoding SAM
+sensors or Pepper aptamers. After 1 day of transfection, the cells were passaged onto coated glass-bottomed plates. After 3 days of transfection, cells were imaged using the livecell imaging conditions described above.
+
+For imaging SAM synthesis and consumption, cells expressing the SAM sensor were incubated with HBCcontaining medium 1 h before imaging. DMEM lacking amino acids was supplemented with 1× minimal essential medium with non-essential amino acids (MEM NEAA)
+and other essential amino acids except methionine to generate the methionine-free medium. Immediately before imaging, we switched the medium to methionine-free DMEM.
+
+Cells were imaged for 4 h at 30 min intervals. Then, methionine (100 -M final) was reintroduced to the medium. Cells were imaged for another 4 h at 30 min intervals.
+
+To monitor the activity of MATase, we added cycloleucine (Ark Pharm AK-29341, 30 mM) to the medium.
+
+Cells were then imaged for 2 h at 10 min intervals. We then withdrew the cycloleucine by changing the cell medium to fresh medium and continued to image the cells at 10 min intervals for 3 h.
+
+To image the MATase transcription level in HeLa cells, we treated HeLa cells with SAHA (suberoylanilide hydroxamic acid; Yuanye Bio, S42929). Cells were imaged for 8 h at 30 min intervals.
+
+For tetracycline or guanine imaging, HEK293T cells were transfected with Tornado plasmids encoding a tetracycline sensor or a guanine sensor. After 1 day of transfection, the cells were passaged onto coated glass-bottomed plates. After 3 days of transfection, we replaced the cell culture medium with phenol red-free DMEM (Biological Industries 06–1052-04–1ACS) containing HBC (10 -M) and tetracycline (BBI A600504) or guanine (Solarbio G8260)
+and incubated it at 37◦C for 1 h. Cells were then imaged using the live-cell imaging conditions described above.
+
+## Quantification And Statistical **Analysis**
+
+All in *vitro* experiments were performed in three independent repeats. The data are represented as the mean ± standard deviation (SD) of three independent repeats in the plots (n = 3).
+
+For the trajectory plot in Figure 3, Figure 4 and Supplementary Figure S7, the fluorescence intensity of the cell was calculated using Fiji by measuring the total fluorescence signal in an ROI divided by ROI area (-m2) and subtracting the background based on the fluorescence intensity of an untransfected cell. The ROI was defined as the cell cytoplasm area.
+
+## Results Strategy For Designing Rna-Based Sensors With **Pepper**
+
+Prior strategies for the construction of RNA-based biosensors mainly took advantage of the formation of RG4containing pockets by an allosteric effect triggered with the fused targeting-binding aptamer (Figure 1A; Supplementary Figure S2). For example, Spinach/Broccoli have been extensively constructed as RNA-based sensors for various targets (4,6,9–20,37). These Spinach/Broccoli-based sensors all contain RG4 motifs that can be regulated by targetbinding RNA aptamers (Figure 1A; Supplementary Figure S2). This RG4-dependent design is also found in other fluorogenic RNA aptamer-based sensors. For example, our lab and the Jaffrey lab developed a series of Red Broccoli-, Spinach2- and Corn-based sensors (6,20,38,39). These sensors contain RG4 motifs in the fluorogenic aptamer domain.
+
+We wondered if fluorogenic aptamers without an RG4 motif could be reasonably constructed as an RNA-based sensor. Pepper is a recently reported RNA aptamer that does not harbor an RG4 motif (Figure 1A, B) (26,27). In addition, Pepper is bright, stable and colorful as an RNA
+tag that enables the imaging of diverse RNAs in living cells
+(25). However, Pepper has not been designed as an RNAbased sensor for various cellular targets, including intracellular small molecules, proteins and ions.
+
+We next sought to design Pepper-based sensors. Similar to protein-based sensors and other RNA-based sensors
+(1,6,40), Pepper-based sensors should comprise three domains: Pepper as the fluorescent indicator, an RNA aptamer for specific binding to targets and a transducer domain that transmits the targeting-binding event to a fluorescence signal. The target-binding aptamer and transducer need to be inserted into a structurally critical stem of Pepper.
+
+To identify the structurally critical stem, we studied the Systematic Evolution of Ligands by Exponential enrichment (SELEX) process and the crystal structure of Pepper
+(25,26). The SELEX library generated Pepper containing two 26 base random stretches separated by a 12 base fixed sequence (25). The 12 base fixed sequence with two bases forms a stable stem–loop structure (stem–loop 1) in Pepper (Figure 1C). Because the major part of stem–loop 1 was the fixed sequence in every library member, it may be less likely to make sequence-specific contacts with the HBC
+fluorophore. The crystal structure of Pepper supports that stem–loop 1 does not interact with HBC (26). We then assumed that stem–loop 1 in Pepper has a structural role in HBC binding that could be exploited in sensor design (26).
+
+To verify this idea, we mutated stem–loop 1 with different sequences. Mutagenesis revealed that stem–loop 1 has an essential structural role in Pepper fluorescence (Figure 1C, D). We thus used stem–loop 1 as an entry point for the insertion of targeting–binding aptamers, and designed various Pepper-based sensors for small molecules and proteins.
+
+## In Vitro Characterization Of The Pepper-Based **Sensor**
+
+We first asked if a small molecule-binding aptamer inserted into stem–loop 1 can modulate Pepper fluorescence intensity. To test this, we chose SAM-III riboswitch aptamer binding to SAM, a metabolite that acts as a methyl donor for nearly all cellular methylation reactions, and regulates the activities of DNA, RNA and proteins (41,42). We previously found that this SAM-III riboswitch can regulate RG4 folding in Spinach/Broccoli (Supplementary Figure S2), and induces fluorescence when fused via a single transducer domain (6,13,39). In this study, we reasoned that the SAM-III riboswitch could regulate Pepper folding and fluorescence.
+
+To generate a Pepper-based SAM sensor, we fused the SAM-binding aptamerto stem–loop 1 in Pepper via a transducer domain (Figure 2A). The transducer domain is an RNA duplex that is unhybridized in the absence of SAM,
+thus disrupting the Pepper structure. However, SAM addition causes the SAM aptamer to become stabilized, which then brings the strands of the transducer together, resulting
+
+![5_image_0.png](5_image_0.png)
+
+in the duplex formation and subsequent folding of Pepper.
+
+To optimize the SAM sensor, we tested different transducer domains, each with different thermodynamic stability (Figure 2B). Among these sensors, the SAM sensor containing transducer 2 exhibitsthe largest fold increase in fluorescence
+(19.3-fold), and high selectivity (Figure 2C, D). We therefore used transducer 2 for all subsequent experiments on the SAM sensor.
+
+We next asked whether SAM sensor fluorescence could reflect SAM concentration. SAM physiological concentrations within different cell types are heterogeneous, typically at the tens to hundreds of micromolar level (9–11). In some cases, the SAM physiological concentration has been reported to reach the millimolar level in cells (12). We then tested the SAM concentration–response fluorescence with the Pepper-based SAM sensor and the reported Broccolibased SAM sensors (13).
+
+Pepper-based sensors give gradual increases in fluorescence with increasing SAM concentrations ranging from 1 to 500 -M (Figure 2E). When the SAM concentration exceeds 500 -M, the fluorescence signal of Pepper-based sensors saturates. Similarly, Broccoli-based SAM sensors showed fluorescence increases with increasing SAM concentration, and a good linearity between 0.1 and 100 -M.
+
+However, when the SAM concentration is >500 -M,
+Broccoli-based SAM sensors showed an obvious artificial decrease in fluorescence (Figure 2E). This may be because SAM diminishes Broccoli fluorescence (Supplementary Figure S3). These data indicate that the Pepper-based sensor showed the corresponding response to SAM at a physiological concentration. In addition, unlike Broccolibased sensors, the Pepper-based sensor is less prone to inhibition by high concentrations of SAM.
+
+## Imaging Sam And Its Biosynthesis Pathway With **Cell-To-Cell** Heterogeneity
+
+We next imaged SAM in living human cells with the Pepperbased SAM sensor. To do so, we expressed the SAM sensor as a circular RNA via a Tornado (Twister-optimized RNA for durable overexpression) expressing system in living HEK293T cells (13). The Tornado system recently reported by the Jaffrey lab allows RNA-based sensors to be accumulated to micromolar levels in living mammalian cells, similar to protein-based sensors (13). After treating with HBC (10 -M), we observed strong fluorescence in SAM sensor-expressing HEK293T cells with confocal microscopy and flow cytometry (Figure 3B; Supplementary Figures S4A, E and S5). These data indicate that a Pepperbased SAM sensor can image endogenous SAM in living human cells (43).
+
+We next monitored the consumption and biosynthesis of SAM in living cells. Methionine is the substrate for SAM biosynthesis in cells, as methionine can be converted to SAM by MATase (Figure 3A; Supplementary Figures S6 and S7A) (44). We detected Pepper fluorescence easliy when SAM sensor-expressing cells were cultured in complete medium containing methionine. On switching cells to
+
+![6_image_0.png](6_image_0.png)
+
+a methionine-free medium, we observed a drop in fluorescence (Figure 3B, C).
+
+To further verify that the decrease in fluorescence was caused by SAM consumption when there was a lack of methionine, we then added methionine (100 -M) to the medium to see if the fluorescence would recover. By reintroducing methionine, cellular fluorescence was restored in 4 h (Figure 3B, C). With the SAM sensor, we found that HEK293T cells exhibit three patterns of SAM synthesis rates. For type I cells, SAM levels rapidly increase to the original level, and continue to rise gradually thereafter.
+
+Type I cells could have a weaker SAM consumption ability but a higher SAM synthesis ability, and the SAM level in Type I cells at 8 h is higher than that of the initial state (t = 0 h). Type II cells showed a moderate SAM consumption and synthesis ability. Type III cells exhibited a gradual increase in SAM level and reached a plateau, which is lower than the SAM level at the initialstate in fluorescence (t = 0 h) (Figure 3B, C). In addition, we also observed SAM biosynthesis in bacterial cells expressing the SAM sensor (Supplementary Figure S7). These data indicate that the SAM sensor enables real-time detection of SAM consumption and biosynthesis in living cells.
+
+## Monitoring Matase Activity And Gene **Expression**
+
+We next monitored MATase activity in living human cells.
+
+Cycloleucine is able to inhibit MATase activity and decrease the endogenous SAM level (Figure 4A) (45). We added cycloleucine (30 mM) to the medium of HEK293T cells expressing the SAM sensor. The cellular fluorescence decreased to a minimum within 2 h upon cycloleucine treatment (Figure 4B, C). We then removed the cycloleucine by replacing the medium with a fresh cycloleucine-free medium. Cellular fluorescence was recovered within 3 h after cycloleucine removal (Figure 4B, C). As a control, we
+
+![7_image_0.png](7_image_0.png)
+
+Type I cells **Type II cells**
+
+![7_image_1.png](7_image_1.png)
+
+Methionine Methionine
+
+![7_image_2.png](7_image_2.png)
+
+used the original Pepper aptamer, to which SAM was unable to bind and thus modulate fluorescence. The fluorescence of cells expressing Pepper was unaffected by cycloleucine treatment and removal (Figure 4B, C). Overall, these data show that the SAM sensor enables real-time monitoring of MATase activity in living individual human cells.
+
+We next asked if the SAM sensor can monitor the MATase gene expression level in HeLa cancer cells. MATase mRNA is expressed at a low level in cancer cells, due to the histone acetylation proximal to the promoter region of the MATase gene (46). Thus, inhibition of histone acetylation may induce the expression of MATase mRNA,
+leading to SAM accumulation. We expressed SAM sensor in HeLa cells and treated the cells with SAHA, a histone acetylation inhibitor, to up-regulate the expression of MATase mRNA (Figure 4A). A significant increase in cellular fluorescence was observed 8 h after the addition of SAHA (Figure 4D, E). Quantitative reverse transcription–
+PCR (RT–qPCR) verified that MATase mRNA expression was elevated after SAHA treatment (Figure 4F). These data suggest that the SAM sensor can indicate intracellular epigenetic regulation of MATase expression in cancer cells.
+
+Last, we wondered about Pepper-based sensors exhibiting multicolor modulation. The Pepper aptamer can bind different HBC analogs and produce fluorescence of varying colors (25). For example, Pepper binds HBC620 to produce red fluorescence at a wavelength of 620 nm (Supplementary Figure S8A) (25). We suspected that this multicolor property could be extended to the Pepper-based sensor. To test this, we imaged cells expressing the SAM sensor with HBC620. SAM-dependent red fluorescence emissions were observed under the red fluorescence filter (Supplementary Figure S8B, Ct). These data indicate that the Pepper-based sensor exhibits color modulation, which is especially crucial when used for ratiometric or multitarget sensing with other orthogonal aptamers or aptamer-based sensors.
+
+## Assessment Of Matase Inhibitor Efficacy Using A **Ratiometric** Sam **Sensor**
+
+To accurately investigate MATase activity, we next sought to design a ratiometric SAM sensor. The signal of the ratiometric sensor is independent of the RNA expression level, thus enabling quantitative assessment of MATase activity. We chose another non-RG4 fluorogenic RNA aptamer, RhoBAST (47), as an internal reference in the ratiometric SAM sensor. RhoBAST binds specifically TMR-DN to emitred fluorescence, making RhoBAST an orthogonal fluorogenic aptamer to the green fluorescent Pepper.
+
+We next developed a ratiometric SAM sensor with the Pepper-based SAM sensor and RhoBAST. To facilitate the proper folding of each aptamer, we inserted the Pepper-based SAM sensor and RhoBAST into two arms of an F30 scaffold (Figure 5A) (48). After incubation with
+
+![8_image_0.png](8_image_0.png)
+
+![9_image_0.png](9_image_0.png)
+
+SAM, the ratiometric SAM sensor showed a significant increase in green fluorescence, while the red fluorescence was not affected (Figure 5B). These results demonstrate that RhoBAST can be used as a normalizer in the ratiometric SAM sensor.
+
+We next asked if the ratiometric SAM sensor can be used for SAM imaging in living cells. We expressed the ratiometric SAM sensor in HEK293T cells using the Tornado expression system (13). After incubating the HEK293T cells with HBC and TMR-DN fluorophores, we readily detected the green fluorescence of the Pepper-based SAM sensor and the red fluorescence of RhoBAST (Figure 5C). The ratiometric signal can be acquired by calculating the green-tored fluorescence intensity (Figure 5C). Because RhoBAST
+fluorescence is not affected by SAM, the fluctuations of the ratiometric signal can therefore reflect the fluctuations of the cellular SAM level and MATase activity. These data showed that the ratiometric SAM sensor functions effectively in living cells.
+
+We next asked if the ratiometric sensor can quantitate the inhibition efficacy of MATase inhibitors. We added AG-270
+(49), a recently reported MATase inhibitor, to HEK293T
+cells expressing the ratiometric SAM sensor or a control RNA. After 24 h incubation, we assessed MATase activity with the ratiometric signal by measuring the green-tored fluorescence ratio. The ratiometric signal in cells expressing the ratiometric SAM sensor was significantly reduced with increasing concentrations of AG-270, while the control group was not affected by AG-270 (Figure 5D, E).
+
+The decrease of the ratiometric signal can reflect the inhibition rate of MATase. By analyzing the ratiometric signal at various concentrations of AG-270, we plotted a dose–
+response curve and determined the IC50 value to be ∼19.4 nM (Figure 5D), which is consistent with the previous report (IC50 = 14 nM) (49). These results show that the ratiometric SAM sensor can accurately detect the efficacy of MATase inhibitors in living cells.
+
+## Modular Generation For Detection Of Other Small **Molecules**
+
+To determine the versatility of the Pepper-based sensor, we incorporated other target-binding RNA sequences with Pepper and detected other small molecules. We generated the tetracycline sensor and the guanine sensor using Pepper (Figure 6A, E) (6,12,16,50). For the tetracycline sensor, an 83.7-fold fluorescence enhancement was observed in tetracycline sensors with the optimal transducer sequence after incubating with tetracycline (0.1 mM) (Figure 6B;
+Supplementary Figure S9A). Furthermore, the tetracycline sensor showed high selectivity with other antibiotics, including neomycin, tobramycin, gentamycin, ampicillin and kanamycin (Figure 6C). In addition, we found that doxycycline, a structural analog of tetracycline, was able to induce fluorescence of the tetracycline sensor, but this was significantly lower than tetracycline-induced fluorescence (Supplementary Figure S10). This is because doxycycline exhibits lower binding affinity (17.7 -M) toward the tetracycline aptamer compared with tetracycline (0.77 nM). For the guanine sensor, the optimal transducer gave a 5-fold signal-to-noise ratio and high specificity (Figure 6F, G; Supplementary Figure S9B). These data suggest that Pepperbased sensors can be applied to other small molecular metabolites and drugs.
+
+We next asked if the Pepper-based sensor can image tetracycline or guanine in living bacterial cells. We expressed the tetracycline sensor or the guanine sensor in BL21 E. *coli* cells incubated with HBC (2 -M). The E. *coli* cells expressing the sensor exhibit minimal fluorescence in the absence of the target. When incubated with a medium containing a tetracycline or guanine target (0.1 mM), cells expressing the tetracycline sensor or guanine target sensor showed significant fluorescence enhancement, respectively (Supplementary Figure S11A, B). These data suggest that the Pepperbased sensor can be designed to be a widely applicable small molecule imaging platform in bacteria.
+
+To determine if these Pepper-based sensors could be used in living human cells, we expressed the circular tetracycline sensor or guanine sensor in HEK293T cells using the Tornado expression system. Upon incubation with a medium containing guanine or tetracycline, the cells exhibited significant fluorescence enhancement (Figure 6D, H; Supplementary Figure S4B, C, E, and S5B, C). Thus, the Pepperbased sensor can be designed for detection of various small molecule targets in living human cells.
+
+## Pepper-Based Sensors Allow Sensing Of Metal Ions And **Proteins**
+
+We next asked if a Pepper-based sensor can detect metal ions. To test this, we designed a silver ions (Ag+) sensor using Pepper. Ag+ is harmful to human health; excess exposure to silver ions may lead to toxic symptoms including loss of organ function, growth retardation and mitochondrial damage through the promotion of oxidative stress
+(51). Therefore, imaging Ag+ helps to understand the mechanism of Ag+ damage to cells. However, there has been no report of imaging Ag+ in human cells with a genetically encoded fluorescent sensor. To detect Ag+ in living human cells, we designed a Pepper-based Ag+ sensor using cytosine–Ag+–cytosine (C–Ag+–C) metallo base pairs
+(52). We inserted C–C mismatches into Pepper stem–loop 1
+(Figure 7A). In the absence of Ag+, the C–C mismatches resulted in an unstable stem–loop 1 structure, leading to low fluorescence. In the presence of Ag+, the C–Ag+–C metallo base pair was formed to stabilize the stem–loop 1 structure
+(53), leading to enhanced fluorescence (Figure 7A). We optimized the Ag+ sensor by adjusting the position and number of C–C mismatch insertions. The optimized sensor design achieves a 3.7-fold fluorescence enhancement (Figure 7B;
+Supplementary Figure S9C). Other metal ions failed to induce fluorescence in the Ag+ sensor (Figure 7C). These data demonstrate that a Pepper-based sensor can be designed for metal ion detection.
+
+We next expressed the Ag+ sensor in HEK293T cells. Cell fluorescence was measured by confocal microscopy or flow cytometry after incubation with Ag+ (20 -M) for 1 h. Ag+-
+dependent cell fluorescence was clearly observed (Figure 7D; Supplementary Figures S4D, E and S5D). In addition, we observed Ag+-dependent cell fluorescence in living E.
+
+coli cells (Supplementary Figure S11C). These data demonstrate that an Ag+ sensorisforthe first time imaged in mammalian cells using a genetically encoded fluorescent sensor, and the Pepper-based sensor is a comprehensive imaging platform for this metal ion in living cells.
+
+We next asked if Pepper can generate sensors of proteins.
+
+To test this idea, we fused a streptavidin-binding aptamer to Pepper via different transducer stems with different thermodynamic stabilities (Figure 7E) (9). We next tested the fluorescence responsiveness of each RNA against streptavidin. The optimal sensor exhibited a 7.2-fold fluorescence increase following the addition of streptavidin (2 -M),
+and shows high selectivity (Figure 7F, G; Supplementary Figure S9D).
+
+We next imaged protein expression levels in living cells using a Pepper-based sensor. To test this, we expressed the streptavidin sensor alone or co-expressed it with streptavidin target in BL21 E. *coli* cells. Cells expressing the
+
+![11_image_0.png](11_image_0.png)
+
+sensor alone exhibit low background fluorescence. In contrast, cells co-expressing the sensor and streptavidin showed significantly enhanced fluorescence (Figure 7H). These data demonstrate that Pepper-based sensors provide a generalizable approach for generating bright and stable sensors for proteins.
+
+## Discussion
+
+Here we converted Pepper, an RG4-free, bright, stable and multiple-spectral fluorogenic RNA, into an RNA-based sensor for measuring the abundance and signaling of intracellular molecules. Pepper-based sensors show high brightness and stability (25). Importantly, Pepper does not harbor the RG4 motif to trigger the cellular RNA unfolding machinery, which enables it to be developed as various robust sensors in living cells. Crucially, Pepper exhibits other key merits for sensing study, including high brightness, high stability, high folding, high binding affinity and high pH tolerance, allowing Pepper to be constructed as robust RNAbased sensors in living cells.
+
+We designed sensors for metabolite detection in *vitro* and in *vivo*. We first rationally designed a Pepper-based sensor for SAM, a small molecular metabolite that is a methyl donor for nearly all cellular methylation reactions. The Pepper-based SAM sensor only exhibits fluorescence when SAM is present. We showed that the sensor works both in vitro and in *vivo* with high activation and high brightness.
+
+![12_image_0.png](12_image_0.png)
+
+Intracellular physiological SAM concentrations vary in different cell types. Therefore, the ideal SAM sensor allows high-range detection of SAM. We found thqt the previously designed Broccoli-based SAM sensor exhibits an artificial fluorescence decrease on SAM, when the SAM concentration gradually increases above 100 -M. In contrast, the Pepper-based SAM sensor exhibits the corresponding response to SAM at a physiological concentration, and is less prone to inhibition by high concentrations of SAM.
+
+Therefore, a Pepper-based sensoris more suitable for detecting physiologically relevant SAM concentrations compared with a Broccoli-based sensor.
+
+With a Pepper-based sensor, we revealed SAM signal function over time in living human cells, and tracked the biosynthesis pathway of SAM with cell-to-cell variation.
+
+Furthermore, we monitored the activity of MATase and determined the inhibition of MATase inhibitors in living individual human cells. Additionally, our SAM sensor detected the gene expression of MATase and verified the agonist drug to accumulate SAM in cancer cells, which has not been reported by other fluorogenic RNA-based or fluorescent protein-based sensors.
+
+We constructed a ratiometric RNA-based sensor to determine the IC50 of MATase inhibitors in live cells. To the best of our knowledge, this is the first time that the IC50 of a drug has been identified by an RNA-based sensor directly in live cells. Therefore, we expect that RNA-based sensors can be used to identify or screen drugs in living human cells.
+
+Furthermore, we demonstrated the versatility of the sensor. We designed Pepper-based sensors for various other targets, including metabolites, synthetic compounds, proteins and metal ions in living cells, demonstrating the universality of Pepper-based sensors. One key process is that this work is the first time that Ag+ has been observed with a genetically encoded sensor in living mammalian cells. The monitoring of intracellular levels of various heavy metal ions is of great significance. However, heavy metal ions cause denaturation of the protein (54), which may limit the application of fluorescent proteins to sense heavy metal ions. Our study demonstrated that RNA has the potential to bind metal ions selectively, and we designed an RNAbased sensor for Ag+ in living human cells for the first time, introducing a new approach for ionomics study.
+
+In addition to Pepper, other fluorogenic RNA aptamers without an RG4 motif have been reported, including Squash (43), RhoBAST (47) and Riboglow (32), but most aptamers have not been designed as RNA-based sensors.
+
+Recently reported Squash/Broccoli were designed as RNAbased ratiometric sensors (43). Although Squash alone does not contain an RG4 motif, Squash incorporates RG4containing Broccoli for ratiometric sensing, potentially preventing its application.
+
+Additionally, Pepper-based sensors show multicolor modulation, exhibiting the high flexibility of RNA-based sensors. Such flexibility is especially useful when used for ratiometric biosensors, orthogonal sensors or for creating stable cell lines or transgenic animals.
+
+## Data **Availability**
+
+All data are available in the main text or the supplementary data.
+
+## Supplementary **Data**
+
+Supplementary Data are available at NAR Online.
+
+## Acknowledgements
+
+We thank Professor Fangqing Zhao and Zhongxuan Zhang for their comments and suggestions.
+
+## Funding
+
+The National Science Foundation of China [32271515 and 32311530120 to X. Li]; Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project [TSBICIP-CXRC038 to X. Li]; the University of Massachusetts [start-up grant to J. Wu]; and Beijing Institutes of Life Science, Chinese Academy of Sciences [Talent research start-up fund to X. Li].
+
+Conflict of interest *statement.* X. Li and Z. Chen are authors of a Chinese patent application on MATase activity detection and MATase inhibitor identification, which are related to the technology described in this manuscript. The remaining authors declare no competing interests.
+
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medical/md/PMC10554762.md

@@ -0,0 +1,20 @@
+therapy, there is increased prevalence of endocrinopathies. Periodic hormonal assessment along with close monitoring of new symptoms results in early detection of endocrinopathies as the time of onset varies significantly with the type of ICI agent and the use of single vs combination therapy.
+
+Presentation: Saturday, June 17, 2023 Abstract citation ID: bvad114.1073
+
+## Endocrine Disrupting Chemicals Sat443
+
+Prevalence Of Immune Checkpoint Inhibitor-related Endocrinopathies In Patients With Cancers Shanti Pittampalli, MD, Sarah R. Lubin, MD Candidate, and Runa Acharya, MD
+
+Upstate Medical University, Syracuse, NY, USA
+
+## Disclosure: S. Pittampalli: None. S.R. Lubin: None. R. Acharya: None.
+
+Background: Immune checkpoint inhibitors (ICIs),
+emerged as the primary treatment modality for a wide range of cancers improving overall survival. Nevertheless, improved overall survival is complicated by endocrine dysfunctions including thyroid dysfunction, adrenalitis, diabetes mellitus, and hypophysitis. Such adverse events offset the benefits of ICI therapy and are associated with significant morbidity and mortality. Limited data exists concerning the burden of the ICI-related endocrinopathies in cancer treatment. Objective: To identify the prevalence and timing of onset of various endocrinopathies in patients with cancers receiving ICIs. Methods: A retrospective study was conducted at SUNY Upstate Medical University, NY
+comprising of adult patients. The study population consisted of all patients age 18 and above seen at Upstate Medical University between 1 January 2019 and 31 December 2021. Anonymized aggregate-level data was collected using an EPIC Slicer Dicer to identify all adult patients with cancer who underwent ICI therapy and a total of 199 patients were included. Demographic, clinical, histopathological, laboratory data were examined. The study was reviewed by IRB and deemed exempt.
+
+The following ICI-related endocrinopathies were considered: Hypothyroidism, hyperthyroidism, diabetes mellitus, adrenal insuffiency, and hypophysitis. ICI treatments included: anti-PD-1 (nivolumab, pembrolizumab, cemiplimab), anti-PD-L1 (atezolizumab, avelumab, durvalumab),
+and combination therapy (ipilimumab and nivolumab, or any other ICI combinations). Statistical analysis including descriptive statistics, ANOVA was conducted using IBM
+SPSS software. Results: A total of fifty patients (25%) developed ICI-related endocrinopathy (n=28 M, 22 F) with a mean age of onset 63.69 years. Majority of the patients were being treated for lung cancers (51%) and had hypothyroidism (88%) followed by adrenal insufficiency (9%) and diabetes (2%). There were no cases of hyperthyroidism or hypophysitis observed. The average time of onset of endocrinopathies after initiation of ICI therapy was 22 weeks
+(anti-PD-1=122 days, anti-PD-L1=180 days and combination therapy= 252 days) with a significantly later onset with combined therapy when compared to anti-PD-1 inhibitors (252 vs 122 days, p= 0.004). Conclusion: With the increasing use of immune checkpoint inhibitors in cancer J Endocrine Soc, Volume 7, Issue Supplement_1, October–November 2023 A572

medical/md/PMC10579731.md

@@ -0,0 +1,273 @@
+# The Integrative Feedback Tool: 
+
+![0_Image_1.Png](0_Image_1.Png) Assessing A Novel Feedback Tool Among Emergency Medicine Residents
+
+Katarzyna M. Gore, Jessen Schiebout, Gary D. Peksa, Sara Hock, Rahul Patwari, Michael Gottlieb Department of Emergency Medicine, Rush University Medical Center, Chicago, IL, USA
+Objective Feedback is critical to the growth of learners. However, feedback quality can be variable in practice. Most feedback tools are generic, with few targeting emergency medicine. We created a feedback tool designed for emergency medicine residents, and this study aimed to evaluate the effectiveness of this tool. Methods This was a single-center, prospective cohort study comparing feedback quality before and after introducing a novel feedback tool. Residents and faculty completed a survey after each shift assessing feedback quality, feedback time, and the number of feedback episodes. Feedback quality was assessed using a composite score from seven questions, which were each scored 1 to 5 points (minimum total score, 7 points; maximum, 35 points). Preintervention and postintervention data were analyzed using a mixed-effects model that took into account the correlation of random effects between study participants. Results Residents completed 182 surveys and faculty members completed 158 surveys. The use of the tool was associated with improved consistency in the summative score of effective feedback attributes as assessed by residents (P=0.040) but not by faculty (P=0.259). However, most of the individual scores for attributes of good feedback did not reach statistical significance. With the tool, residents perceived that faculty spent more time providing feedback (P=0.040) and that the delivery of feedback was more ongoing throughout the shift (P=0.020). Faculty felt that the tool allowed for more ongoing feedback (P=0.002), with no perceived increase in the time spent delivering feedback (P=0.833). 
+
+Conclusion The use of a dedicated tool may help educators provide more meaningful and frequent feedback without impacting the perceived required time needed to provide feedback.
+
+Keywords Feedback; Medical education; Resident education eISSN: 2383-4625 Original Article
+
+![0_image_0.png](0_image_0.png)
+
+Received: 5 November 2022 Revised: 19 January 2023 Accepted: 7 February 2023 Correspondence to: Katarzyna M. Gore Department of Emergency Medicine, Rush University Medical Center, 1750 West Harrison St, Suite 108 Kellogg, Chicago, IL 60612, USA Email: knosek2@gmail.com How to cite this article:
+
+![0_image_2.png](0_image_2.png) Gore KM, Schiebout J, Peksa GD, Hock S, Patwari R, Gottlieb M. The integrative feedback tool: assessing a novel feedback tool among emergency medicine residents. Clin Exp Emerg Med 2023;10(3):306-314. https://doi.org/10.15441/ceem.22.395 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https:// creativecommons.org/licenses/by-nc/4.0/).
+
+![1_image_1.png](1_image_1.png)
+
+What is already known Feedback is not always focused on or formally taught as part of graduate medical education, and, as a result, many clinical faculty may not have significant training in the matter. Furthermore, many faculty may not have the time to prioritize keeping up to date with evolving literature in medical education given their other clinical and administrative commitments. This can lead to significant variability in how feedback is delivered and result in learner dissatisfaction with the quality of feedback provided as well as missed opportunities for growth and development. What is new in the current study Overall, the use of an interactive feedback delivery tool improved consistency in attributes of effective feedback without impacting the perceived time to deliver feedback.
+
+## Introduction
+
+Feedback is important in all fields and is a critical aspect of medical training. In fact, the Accreditation Council of Graduate Medical Education (ACGME) declares feedback to be an essential and required component of resident training [1]. However, studies have demonstrated that current feedback quality can vary, leaving some learners and faculty dissatisfied with the adequacy of the feedback they receive [2–7]. This can be particularly challenging in the emergency department (ED) setting due to time constraints, frequent interruptions, high patient acuity, and learners at multiple stages of training [8,9].
+
+To be effective, feedback should be goal-oriented, constructive, based on observed activities, and timely [10]. It should also focus on specific elements of performance, address how the task was done, and provide guidance to help learners grow beyond their current competence [11]. It is important as well to consider the relationship between the feedback giver and receiver. Borrowing from the psychological concept of a therapeutic alliance, an "educational alliance" is a conceptual framework that incorporates a mutual understanding of educational goals with an agreement on how to work toward those goals [6]. Learners can engage in reflective conversations to relate their self-assessment with educator observations. An educational alliance is strengthened when these discussions are held regularly and often by individuals who exhibit trust and mutual respect. Learners engaged in these alliances are more likely to use the feedback they receive effectively 
+[8,11–16]. However, in the ED setting, feedback is more commonly delivered at the end of the shift in a summative format, frequently using a Milestones-based checklist [17]. This limits the ability to integrate the feedback or sustain the educational alliance since the learner's next ED shift is often with a different faculty member [8].
+
+Feedback is not always focused on or formally taught as part of graduate medical education, so clinical faculty may not have 
+
+![1_image_0.png](1_image_0.png) significant training in the matter. Furthermore, many faculty may not have the time to prioritize keeping up to date with evolving literature in medical education given their other clinical and administrative commitments [18–22]. This can lead to significant variability in how feedback is delivered and result both in learner dissatisfaction with the quality of feedback provided and missed opportunities for growth and development [23–25]. 
+
+To address this need, we developed a novel feedback tool (Fig. 
+
+1) to guide feedback delivery and allow opportunities for integration into the shift. Using a structured tool, residents could identify their specific learning objectives from a full list modeled after the Emergency Medicine (EM) Milestones [26]. Informed by Kolb's theory of experiential learning, the residents then receive realtime feedback on specific instances after a patient encounter, alter their practice, and see if any changes they made are effective 
+[8,27,28]. Having the learner choose the specific skills in an organized system, with clearly defined and achievable goals to work on during their shift, may prevent defensive reactions and better facilitate learning [5,23]. This could also allow the learner and faculty member to visually track improvements to enable a more comprehensive summative evaluation at the end of the shift. 
+
+Our primary goal was to evaluate the impact of a novel tool on the overall consistency in providing attributes of effective feedback in a cohort of EM residents and faculty. A subgroup analysis was planned to evaluate the consistency with regard to specific attributes of effective feedback. Secondary outcomes included differences in perceived feedback timing (i.e., how long feedback takes) and frequency.
+
+## Methods
+
+Ethics statement The study was approved by the Institutional Review Board of 
+
+![2_image_0.png](2_image_0.png)
+
+![2_image_1.png](2_image_1.png)
+
+Fig. 1. Sample milestone-based tool. The resident decides before the shift where they feel they fall on the scale (circle). The resident decides midway through the shift how they are doing (marked as "X"). The resident and supervising physician decide where on the scale the resident performed (square). CT, computed tomography.
+
+Rush University Medical Center (No. 19031105-IRB01). Informed consent was obtained from all interested participants, and all methodologies and procedures were conducted in line with the Declaration of Helsinki guidelines.
+
+## Study Setting
+
+This was a single-center, prospective cohort study comparing a composite feedback score before and after a novel feedback tool was introduced. The study was conducted at Rush University Medical Center, a 3-year EM residency program at an urban academic center in Chicago, Illinois, USA, and enrolled 36 EM residents and 38 EM faculty members. All EM resident and faculty physicians were eligible for inclusion in the study (with the exception of the authors), though survey completion was optional. 
+
+We excluded medical students and non-EM residents. All faculty are trained in EM. 
+
+## Study Design
+
+The preintervention phase occurred from August 24, 2020 to October 8, 2020. During this period, faculty gave residents feedback based on the existing end-of-shift evaluation model used in our department. This consisted of an electronic end-of-shift card, which was informed by the EM Milestones. Feedback was not standardized across faculty, and they had not received any specialized training. During the preintervention time period, residents and faculty completed a survey evaluating their feedback experience after each shift (Supplementary Materials 1, 2). Survey reminders were posted throughout the ED, and individualized emails were sent to resident and faculty physicians before each shift.
+
+We reviewed the literature to identify components of effective feedback and existing feedback-assessment tools. We identified a paucity of existing feedback-assessment tools appropriate for use in this study; therefore, a new tool was created. Based on a thorough review of existing literature, we determined that high-quality feedback should be tangible, goal-referenced, actionable, personalized, timely, ongoing, and consistent [8,10]. We drafted a survey to assess these specific components, with the cumulative summary score of all seven aforementioned elements serving as our primary outcome. 
+
+The survey was then piloted and iteratively refined by the authors. Content validity was determined by discussion among attending ED physicians, including an assistant program director, associate dean of the medical college, and core faculty members, which included two individuals with extensive experience publishing and presenting on feedback. Response process validity was determined by piloting the survey on two attending physicians, including one core faculty and one noncore faculty member. The survey included seven questions evaluating the feedback quality (Supplementary Material 1), which were assessed using a Likert scale of 1 (strongly disagree) to 5 points (strongly agree). The consistency in providing attributes of effective feedback (feedback quality) was assessed as a summative score, with a total minimum of 7 points and total maximum of 35 points. The survey also asked about the time spent on feedback (<1, 1–3, 3–5, 5–7, or >7 minutes) and the number of feedback instances per shift (0, 1, 2, 3, 4, or ≥5). Study data were collected and managed using Research Electronic Data Capture (REDCap) electronic data capture tools. REDCap is a secure, web-based software platform designed to support data capture for research studies that provides an intuitive interface for validated data capture, audit trails for tracking data manipulation and export procedures, automated export procedures for seamless data downloads to common statistical packages, and procedures for data integration and interoperability with external sources [29].
+
+From August 24, 2020 to October 8, 2020, we trained our residents and faculty on the new feedback tool. Training was 30 minutes in length and covered only the use of the feedback tool. We did not conduct specific training regarding feedback best practices or other faculty development during the entire study time period. Faculty were educated on the use of the feedback tool during a faculty meeting with most faculty present, while residents were educated during their conference day. Any absent resident or faculty member was sent both a video and verbal explanation of the feedback tool. After allowing time for training and uptake, we collected postintervention data from October 15, 2020 to March 19, 2021, using the same process described above for the preintervention period.
+
+## Feedback Tool
+
+The feedback tool was developed based on EM Milestones ver. 1.0 (Supplementary Material 3) [26]. All milestones were included, and each milestone was split into 10 strata based on the five levels and criteria described in the EM Milestones document. We chose 10 strata to provide a wide berth of options for faculty and residents to rate their skill level. Each milestone had its own separate form and was paper-based to facilitate ease of completion and collection. Because data suggest that feedback may be better received when the message is presented conceptually in a visual manner [30], we used a visual scale to track progress directly (Fig. 1).
+
+Prior to each shift, residents selected two milestones on which to focus for the shift. Blank feedback tool forms were stored in a folder near the resident and faculty workstations. Before seeing patients, residents would circle their self-perceived level for both milestones and have a conversation with the faculty about what they needed to do to get to the next level. Midway through the shift, the resident and faculty would revisit the document to see if progress had been made on each milestone. An "X" was placed on the visual scale to indicate where they thought they were at that point in time, prompting another conversation on opportunities for improvement. At the end of the shift, the resident marked the visual scale with a square to denote where they thought they had ended up. This response was independent of the end-of-shift evaluations completed by attendings, separating this feedback process from the formal evaluation process. 
+
+## Statistical Analysis 
+
+![3_Image_0.Png](3_Image_0.Png)
+
+A dependent means sample size calculation indicated 140 assessments were needed based on an alpha value of 0.05, power of 80%, and mean total score difference of 1 between the preintervention and postintervention arms. The normality of data was assessed by visual inspection of histogram plots. We report descriptive statistics for the participant responses using median with interquartile range (IQR) values. Preintervention and postintervention data were analyzed using a linear mixed-effects model that took into account the correlation of random effects between study participants and reported as mean estimates with standard deviations. An *a priori*, two-sided, P-value of <0.05 was considered statistically significant. Comparative data were reported as differences and 95% confidence interval values. A post hoc Bonferroni correction was completed for the subanalyses and set at P<0.005 given the use of one model per the 10 strata evaluated (i.e., original alpha value of 0.05 divided by 10). Analyses were performed using IBM SPSS ver. 22.0 (IBM Corp).
+
+## Results
+
+Thirty-one residents and 35 faculty participated in the study. In the preintervention period, residents completed 101 total surveys, with a median of four surveys (IQR, 2–6) per person, while faculty completed 94 surveys, with a median of three surveys (IQR, 1–5) per person. In the postintervention period, residents completed 81 total surveys, with a median of two surveys (IQR, 1–4) per person, while faculty completed 64 surveys, with a median of three surveys (IQR, 2–4) per person. Characteristics of the participant groups are noted in Table 1. 
+
+The resident data suggested that there was a significant improvement in the composite feedback score after the intervention 
+(linear mixed-model mean estimate, preintervention=26.6/35.0 vs. postintervention=28.2/35.0; P=0.041) (Table 2). Compared to 
+
+| Table 1. Characteristics of the study population  Presurvey   | Postsurvey No. of  surveys  (%)   |           |           |           |
+|---------------------------------------------------------------|-----------------------------------|-----------|-----------|-----------|
+| Resident                                                      | 24 (100)                          | 101 (100) | 25 (100)  | 81 (100)  |
+| Postgraduate year 1                                           | 8 (33.3)                          | 32 (31.7) | 7 (28.0)  | 18 (22.2) |
+| Postgraduate year 2                                           | 7 (29.2)                          | 28 (27.7) | 10 (40.0) | 29 (35.8) |
+| Postgraduate year 3                                           | 9 (37.5)                          | 41 (40.6) | 8 (32.0)  | 34 (42.0) |
+| Faculty (yr)                                                  | 28 (100)                          | 94 (100)  | 20 (100)  | 64 (100)  |
+| <5                                                            | 10 (35.7)                         | 29 (30.9) | 4 (20.0)  | 18 (28.1) |
+| 5–10                                                          | 6 (21.4)                          | 17 (18.1) | 5 (25.0)  | 13 (20.3) |
+| >10                                                           | 12 (42.9)                         | 48 (51.1) | 11 (55.0) | 33 (51.6) |
+| No. of                                                        |                                   |           |           |           |
+| participants  (%)                                             | No. of  surveys  (%)              | No. of    |           |           |
+| participants  (%)                                             |                                   |           |           |           |
+| Characteristic                                                |                                   |           |           |           |
+
+| Table 2. Linear mixed model for resident data comparing feedback received before and after feedback tool implementation Question Preintervention  Postintervention  (n=81)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | P-value   |          |       |
+|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------|----------|-------|
+| (n=101)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |           |          |       |
+| How many distinct instances did you receive feedback from a faculty member about your performance                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | 2.6±2.2   | 2.8±2.0  | 0.285 |
+| today?a) Approximately how much time did your faculty preceptor spend providing feedback?b)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | 3.1±1.9   | 3.4±1.6  | 0.036 |
+| Feedback quality evaluationc) My feedback was tangible (identified specific, positive or negative behaviors).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | 3.9±1.4   | 4.1±1.1  | 0.084 |
+| My feedback was goal-referenced (suggested a goal, addressed progress towards a goal).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | 3.7±1.5   | 4.0±1.2  | 0.063 |
+| My feedback was actionable (suggested something I can work to correct or should do more of).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | 3.8±1.4   | 4.0±1.1  | 0.125 |
+| My feedback was personalized (tailored to my performance).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | 4.1±1.3   | 4.3±1.0  | 0.193 |
+| My feedback was timely (offered in close proximity to the actions it addressed).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   | 3.9±1.4   | 4.0±1.1  | 0.217 |
+| My feedback was ongoing (offered throughout the shift versus only at the end).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | 3.5±1.8   | 3.9±1.4  | 0.023 |
+| My feedback was consistent (similar in content to other feedback I've received in similar situations).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | 3.8±1.4   | 3.9±1.1  | 0.367 |
+| My feedback addressed my progress towards the goal of residency graduation (helped evaluate my development towards independent practice).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | 4.0±1.3   | 4.1±1.0  | 0.446 |
+| Composite score                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | 26.6±7.7  | 28.2±6.2 | 0.041 |
+| Values are presented as estimate mean±standard deviation. a)Answer options were 0, 1, 2, 3, 4, or ≥5. b)Answer options were <1, 1–3, 3–5, 5–7, or >7 minutes. c)Composite score calculated from the below seven variables. Table 3. Linear mixed model for faculty data comparing feedback received before and after feedback tool implementation Question Preintervention  Postintervention  (n=64) P-value (n=94) On how many distinct instances did you give feedback to this resident about their performance today?a) 3.6±1.8 3.4±1.6 0.405 Approximately how much time did you spend providing feedback?b) 3.0±1.4 2.9±1.2 0.833 Feedback quality evaluationc) The feedback I gave was tangible (identified specific, positive or negative behaviors). 4.0±1.0 3.8±0.8 0.097 The feedback I gave was goal-referenced (suggested a goal, addressed progress towards a goal). 3.6±1.3 3.8±1.1 0.071 The feedback I gave was actionable (suggested something I can work to correct or should do more of). 3.7±1.2 3.8±1.0 0.451 The feedback I gave was personalized (tailored to my performance). 3.9±1.0 3.9±0.9 0.828 The feedback I gave was timely (offered in close proximity to the actions it addressed). 3.8±1.2 3.8±1.0 0.915 The feedback I gave was ongoing (offered throughout the shift versus only at the end). 3.3±1.5 3.8±1.4 0.002 The feedback I gave was consistent (similar in content to other feedback I've given in similar situations). 4.0±1.0 3.9±1.1 0.122 The feedback I gave addressed their progress towards the goal of residency graduation (helped evaluate  3.9±1.1 4.0±1.0 0.465 development towards independent practice) Composite score 26.2±6 26.9±5.8 0.259 Values are presented as estimate mean±standard deviation. a)Answer options were 0, 1, 2, 3, 4, or ≥5. b)Answer options were <1, 1–3, 3–5, 5–7, or >7 minutes. c)Composite score calculated from the below seven variables. |           |          |       |
+
+![4_image_0.png](4_image_0.png)
+
+before the implementation of the feedback tool, residents perceived that the faculty spent more time providing feedback (preintervention=3.1/5.0 vs. postintervention=3.4/5.0, P=0.036) and that feedback was more ongoing throughout the shift (preintervention=3.5/5.0 vs. postintervention=3.9/5.0, P=0.023). 
+
+In the faculty group, the difference in the overall composite feedback score was not statistically significant (preintervention= 26.2/35.0 vs. postintervention=26.9/35.0, P=0.259) (Table 3). Faculty felt that the tool led to more ongoing feedback over the course of the shift (preintervention=3.3/5.0 vs. postintervention=
+3.8/5.0, P=0.002) without a perceived increase in time spent delivering feedback (P=0.833). 
+
+## Discussion
+
+As medical education continues to advance and new generations of medical learners transform the ways in which they acquire knowledge, it is critical that the ways in which feedback is given to these learners also evolve [31,32]. Using our novel feedback tool, we found significantly increased consistency in the composite score of attributes of effective feedback (feedback quality) 
+without a significant change in time perceived by faculty devoted to delivering feedback. 
+
+Prior literature has focused primarily on faculty development sessions to improve feedback delivery, with fewer studies focusing on supporting tools. One study used a training session on feedback delivery paired with a reminder card and booklet for documentation of noted observations and found a modest improvement in written evaluations and improvement in residents' perception that feedback would impact their clinical practice [33]. 
+
+Another study used an extensive training session coupled with a skills checklist to be completed in observed encounters and found that these interventions improved how specific the content of feedback was and that direct observation was viewed by residents as a valuable aspect of their training [32]. These studies, however, required dedicated faculty coaching and time commitments for the observations, which may be more challenging to secure in the ED setting [33,34]. Other studies have focused on providing tools that can increase the ease with which resident evaluations can be completed, whether using app-based systems or QR codes; these studies have primarily focused on increasing the number of evaluations completed rather than on the feedback itself [35–38]. While increasing the quantity of feedback may be important, unintended consequences, such as degrading the process into one of "form filling" and "checking boxes," may occur [39]. Most importantly, many of the studies on feedback interventions and tools were conducted outside the ED environment and were limited by their retrospective or qualitative design, with few prospective case-control studies, further highlighting the need for an ED-specific tool. 
+
+We believe there are several unique benefits to our tool. One of the main individual attributes of effective feedback that did reach statistical significance in both the faculty and resident groups was "my feedback was ongoing." We believe that having an interactive, physical tool available throughout the shift may be a key to navigating the challenge of the busy ED with frequent interruptions. A visible feedback tool allows the learner and facilitator to be reminded of the need to have continued conversations related to resident performance. This tool also allows learners to choose their learning goals as well as to reflect on where they stand and how they are progressing, thereby moving the feedback session from a unilateral delivery of feedback to a bilateral discussion [6]. It also emphasized self-reflection and accountability to the process by using clear anchors and a visual tool. Finally, the tool standardizes the approach to giving feedback, is simple to use, and aligns with the existing Milestones framework while simultaneously necessitating only minimal formal training for residents and faculty. 
+
+Interestingly, most of the individual attributes of effective feedback did not reach statistical significance independently. As a subgroup analysis, this study was not powered for the analysis of the specific components; therefore, it is possible that it may have been underpowered to detect a difference in the individual feedback components. Alternatively, while having set goals chosen at 
+
+![5_image_0.png](5_image_0.png) the beginning of the shift in general can improve the ability to provide concrete feedback, it becomes challenging when the chosen goals are not addressed during the shift. In order to address this, residents were asked to pick a pair of milestones to discuss during the shift so there was a greater chance of having something relevant to provide feedback on. We did not, however, keep track of which milestones were more likely to be selected, if the milestones were applicable to the shift experience, or if residents were given feedback on the full breadth of milestones. This may have contributed to the lack of statistical significance in certain individual scores of effective feedback. For instance, the individual attribute "my feedback was tangible" relies on having instances during the shift that are applicable to the specific milestone chosen. In the future, it may be beneficial to assign several milestones to each shift to ensure residents have a greater chance of receiving feedback on clinically applicable milestones, which may lead to improvement in the scores of the individual attributes of effective feedback. Additionally, removing some milestones that are better assessed outside of the clinical setting from the pool of possible milestones to give feedback on may improve the relevance and effectiveness of the feedback given.
+
+Overall, the use of an interactive feedback delivery tool improved consistency in attributes of effective feedback without impacting the perceived time to deliver feedback. Many of the individual attributes of effective feedback did not reach clinical significance, and future research is needed to evaluate the validity of this tool in other settings and among different learner groups. 
+
+There are several important limitations to consider with this study. First, this was conducted at a single EM residency program, and future studies are needed to assess the external validity of the tool itself as well as the findings on its effects on the cumulative attributes of effective feedback. In the future, it would also be beneficial to directly measure the amount of time required to utilize the tool and provide feedback, as some of the responses to questions relied on the subjective assessment of time which is subject to recall bias. In addition, it may be helpful to ask direct questions regarding ease of use and perceived intrusions on workflow. Additionally, this study was conducted using a pre-post design. While there were no feedback interventions other than the tool performed during this time period and no new faculty hired, it is possible that faculty feedback may have improved over time. Another limitation is that this tool was derived using the prior iteration of the Milestones, which have recently been revised. However, as the intervention focused on the delivery model, rather than the specific Milestone categories, we do not anticipate this to significantly impact the findings. Moreover, responses were voluntary, and it is possible this may have led to selection 
+
+![6_image_0.png](6_image_0.png) bias. Finally, the outcomes assessed the impact on a cumulative feedback score but did not assess the impact on patient care or educational significance. While statistically significant, the clinical difference of a mean total score increase of 1 point is unclear, and future studies should ascertain the threshold of a clinically significant difference. Future studies should also assess this among non-EM specialties using specialty-specific Milestones. Studies should also assess this longitudinally, evaluating for the impact on resident performance and potential implications for remediation and competency-based advancement assessments.
+
+## Supplementary Materials
+
+Supplementary Material 1. Resident survey. Supplementary Material 2. Faculty survey. Supplementary Material 3. Sample feedback tool. Supplementary materials are available from https://doi.org/10. 15441/ceem.22.395.
+
+## Ethics Statement
+
+The study was approved by the Institutional Review Board of Rush University Medical Center (No. 19031105-IRB01). Informed consent was obtained from all interested participants.
+
+## Conflict Of Interest
+
+No potential conflict of interest relevant to this article was reported.
+
+## Funding
+
+None.
+
+## Acknowledgments
+
+The authors thank the emergency medicine residents and faculty at Rush University Medical Center (Chicago, IL, USA) for their assistance with the conduct of this study.
+
+## Author Contributions
+
+Conceptualization: all authors; Data curation: all authors; Formal analysis: GDP; Investigation: all authors; Methodology: all authors; Project administration: all authors; Supervision: KMG, MG; 
+Writing–original draft: all authors; Writing–review & editing: all authors. All authors read and approved the final manuscript.
+
+## Orcid
+
+Katarzyna M. Gore https://orcid.org/0000-0001-5310-3327 Jessen Schiebout https://orcid.org/0000-0002-9106-2957 Gary D. Peksa https://orcid.org/0000-0003-3625-8137 Sara Hock https://orcid.org/0000-0001-7495-2272 Rahul Patwari https://orcid.org/0000-0001-8040-992X Michael Gottlieb https://orcid.org/0000-0003-3276-8375
+
+## References
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+![8_image_0.png](8_image_0.png)
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+Available online at www.sciencedirect.com
+
+![0_image_0.png](0_image_0.png)
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+![0_image_2.png](0_image_2.png)
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+![0_image_3.png](0_image_3.png)
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+![0_image_4.png](0_image_4.png)
+
+Case **Report**
+
+![0_image_1.png](0_image_1.png)
+
+Iliac neck dilatation causes rupture of **abdominal**
+aortic aneurysm previously treated **with**
+endovascular aortic aneurysm **repair**✩
+Maki Fuchigami, MDa, Yukihisa Ogawa, MD, PhDa,c,∗, Kiyoshi Chiba, MD, PhDb, Masahide Komagamine, MD, PhDb, Shintaro Nawata, MD, PhDa, Satoshi Kinebuchi, MDb, Hidefumi Mimura, MD, PhDa, Takeshi Miyairi, MD, PhDb, Hiroshi Nishimaki, MD, *PhDb* a Department of Radiology, St. Marianna University School of Medicine, 2-16-1, Sugao, Miyamae, *Kawasaki,*
+Kanagawa, 216-8511, *Japan* b Department of Cardiovascular Surgery, St. Marianna University School of Medicine, 2-16-1, Sugao, *Miyamae,*
+Kawasaki, Kanagawa, 216-8511, *Japan* c Department of Radiology, Tokai University School of Medicine Hachioji Hospital, 1838 Ishikawa, Hachioji, *Tokyo,*
+192-0032, *Japan* a r t i c l e i n f o Article *history:* Received 29 June 2023 Revised 16 September 2023 Accepted 18 September 2023 Keywords: Abdominal aortic aneurysm Endoleak Endovascular aortic aneurysm repair Iliac neck dilation Rupture a b s t r a c t A 78-year-old male had undergone endovascular aortic aneurysm repair (EVAR) 7 years prior to presentation. Although the sac was stable 6 months ago,the patient presented with shock at arrival, and CT showed aortic rupture with rapid expansion due to type Ib endoleak caused by iliac neck dilatation (IND). The aneurysm sac was excluded using an endovascular strategy. Bell-bottom iliac limbs can cause IND associated with type Ib endoleak. Additionally, the risk of rupture is high when re-expansion of an aneurysm occurs after sac regression after EVAR. Therefore, close follow-up is mandatory for patients with IND after EVAR.
+
+© 2023 The Authors. Published by Elsevier Inc. on behalf of University of Washington.
+
+This is an open access article under the CC BY-NC-ND license
+(http://creativecommons.org/licenses/by-nc-nd/4.0/)
+
+## Introduction
+
+Endovascular abdominal aortic aneurysm repair (EVAR), along with open surgery, is the standard treatment for abdominal aortic aneurysms (AAA) in most patients with suitable anatomy and reasonable life expectancy. However, 25%-40% of patients with AAA treated with EVAR have common iliac artery aneurysms (CIAA) [1], and the management of these patients is still controversial [2]. Although commercially available  limbs have various sizes to accommodate a wide range of iliac morphologies, there is a potential risk of iliac neck dilation (IND) which may affect the long-term outcomes of EVAR [3]. In this report, we describe the rapid expansion of a previously excluded AAA resulting in a rupture due to a type Ib endoleak, which is defined as an inadequate seal at distal end of the stent-graft caused by IND [4].
+
+## Case **Report**
+
+A 78-year-old male had undergone EVAR on the right side using an Endurant device (Medtronic, Minneapolis, MN) with a bell-bottom iliac limb (24 mm) 7 years ago. His medical history includes postchemotherapy for lung cancer and surgical clipping for subarachnoid hemorrhage. The clinical course was uneventful with the sac stable 6 months prior to presentation. However, the patient was urgently admitted to our hospital and presented unconsciousness with a blood pressure of 68/40 mm Hg, a heart rate of 90 bpm, a respiratory rate of 30/min, and SpO2 of 90% (in room air). His hemoglobin level and platelet count were 8.4 g/dL and 104^103/μL, respectively, and he received 2 units of RCC and 2 units of FFP.
+
+Contrast-enhanced computed tomography (CT) revealed a massive hematoma surrounding the rapidly expanded AAA (Fig. 1). Retrospective evaluation of the CT scans showed that the size of the AAA decreased after EVAR (55 mm-37 mm). The AAA was dilated to 39 mm 6 months ago and the size at rupture was 45 mm. The size of the iliac neck on the right side gradually expanded over time. In the last follow-up, the legs were raised from 26 mm to 28 mm and the endograft retracted (Fig. 2). The rupture was determined to be a result of a type Ib endoleak from the leg float.
+
+Open surgery was deemed to be high risk due to his frailty, so emergency EVAR with iliac extension and internal iliac artery embolization was performed.
+
+Under general anesthesia, 2 Excluder extensions (W.L. Gore
+& Associates, Inc., Flagstaff, AZ) were placed in the right leg of the main body and the right external iliac artery in the hybrid operating room (Fig. 3). The right internal iliac artery was difficult to cannulate; therefore, it was embolized with 50% n-butyl-cyanoacrylate-lipiodol mixture after the leg was completed using a 4F-Cobra catheter (Medikit, Tokyo, Japan)
+placed near the origin of the internal iliac artery. Completion angiography showed sac exclusion without endoleaks.
+
+His hemoglobin level and platelet count were elevated to 11.2 g/dL, and 147^103/μL, respectively, 4 hours after the operation.
+
+The postoperative course was uneventful, and the patient is currently being followed up on as an outpatient.
+
+## Discussion
+
+The current case report highlights a late type Ib endoleak induced by IND after EVAR.The commercially available iliac limb has a diameter of 28 mm, allowing for placement in CIA up to 25 mm in diameter. However, bell-bottom iliac limbs can result in late type Ib endoleaks, causing IND. A comparative study consisting of 239 iliac limbs from 128 patients, 61 iliac limbs were ≥ 20 mm in diameter and 178 were < 20 mm in diameter, found that type Ib endoleaks were significantly higher in patients ≥ 20 mm diameter iliac limbs (18% vs 3.9%,
+P = .001) [5]. Another study showed that type Ib endoleak occurred in 16.1% of patients with the bell-bottom type, whereas it occurred in 7.7% without the bell-bottom type [6]. This may be due to the relatively strong radial force associated with the bell-bottom type. Therefore, EVAR with a bell-bottom type may not be preferable for patients with a long-life expectancy, and using iliac branch devices or external iliac extension may produce better outcomes if anatomically feasible [5]. A recent study found that patients whose AAA sac diameter regressed to ≤ 40 mm after EVAR presented a very low rate of re-expansion, reintervention, or rupture, and most of the sac re-expansion occurred at least 3 years after reaching ≤ 40 mm in sac diameter [7].In the current case,however, sac regression
+(37 mm) was observed 5 years ago, but a rapid AAA expansion occurred due to a secondary type Ib endoleak. This resulted in a rupture despite the aneurysmal sac diameter not
+
+![1_image_0.png](1_image_0.png)
+
+![2_image_0.png](2_image_0.png)
+
+![3_image_0.png](3_image_0.png)
+
+reaching the AAA diameter immediately after the EVAR. The lack of data regarding the timing of rapid expansion or rupture means that close follow-up is mandatory, and early reintervention may solve this issue. Although EVAR is the standard procedure for treating anatomically suitable infrarenal AAA,
+physicians should be aware of this potentially lethal clinical scenario.
+
+## Conclusion
+
+Bell-bottom iliac limbs can cause IND associated with type Ib endoleak. Additionally, the risk of rupture is high when re-expansion of an aneurysm occurs after sac regression after EVAR. Therefore, close follow-up is mandatory for patients with IND after EVAR.
+
+## Author **Contributions**
+
+MF: Data collection; MF, YO: Manuscript preparation; Critical review and revision: all authors; Final approval of article: all authors; Accountability for all aspects of the work: all authors.
+
+## Patient **Consent**
+
+Written informed consent was obtained from the patient for the publication of this case report and any accompanying images.
+
+## Ethics **Approval**
+
+All procedures were performed in accordance with the ethical standards of the institution and the 1964 Helsinki Declaration.
+
+REFERENCES
+[1] Brown CR, Greenberg RK, Wong S, Eagleton M, Mastracci T,
+Hernandez AV, et al. Family history of aortic disease predicts disease patterns and progression and is a significant influence on management strategies for patients and their relatives. J Vasc Surg 2013;58:573–81.
+
+[2] Pagliariccio G, Gatta E, Schiavon S, Cicilioni CG, Lattanzi S,
+Dimitri E, et al. Bell-bottom technique in iliac branch era:
+mid-term single stent graft performance. CVIR Endovasc 2020;3:57.
+
+[3] Gonçalves FB, Oliveira NF, van Rijn MJ, Ultee KFJ, Hoeks SE,
+Raa ST, et al. Iliac seal zone dynamics and clinical consequences after endovascular aneurysm repair. Eur J Vasc Endovasc Surg 2017;53:185–92.
+
+[4] Moll FL, Powell JT, Fraedrich G, Verzini F, Haulon S,
+Waltham M, et al. Management of abdominal aortic aneurysms clinical practice guidelines of the European society for vascular surgery. Eur J Vasc Endovasc Surg 2011;41:S1–S58.
+
+[5] Gray D, Shahverdyan R, Reifferscheid V, Gawenda M,
+Brunkwall JS. EVAR with flared iliac limbs has a high risk of late type 1b endoleak. Eur J Vasc Endovasc Surg 2017;54:170–6.
+
+[6] Rouby AF, Kuntz S, Delay C, Thaveau F, Georg Y, Lejay A,
+et al. Volume change after endovascular treatment of common iliac arteries ≥ 17 mm diameter: assessment of type 1b endoleak risk factors. Eur J Vasc Endovasc Surg 2020;59:51–8.
+
+[7] Andraska EA, Phillips AR, Reitz KM, Asaadi S, Dai Y, Tzeng E,
+et al. Longer follow-up intervals following endovascular aortic aneurysm repair are safe and appropriate after marked aneurysm sac regression. J Vasc Surg 2022;76:454–60.

medical/md/PMC10649656.md

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+# Ukraine Trauma Project: The Feasibility Of Introducing Advanced Trauma-Care Skills To Frontline Emergency Medical Services **Responders**
+
+Gerard Bury ,1 Christopher Fitzpatrick,2 Bernard Heron,2 Walter Cullen,3 Eithne Scully,2 Kateryna Kachurets,2 Lyudmyla Zacharchenko2 To cite: Bury G, Fitzpatrick C, 
+Heron B, *et al*. Ukraine Trauma Project: the feasibility of introducing advanced trauma-care skills to frontline emergency medical services responders. *BMJ Open* 2023;13:e077895. doi:10.1136/ bmjopen-2023-077895
+► Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2023-077895). Received 18 July 2023 Accepted 18 October 2023 © Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
+
+1General Practice, University College Dublin, Dublin, Ireland 2UCD Centre for Emergency Medical Science, University College Dublin, Dublin, Ireland 3School of Medicine, University College Dublin, Dublin, Ireland Correspondence to Dr Gerard Bury; gerard.bury@ucd.ie
+
+## Abstract
+
+Objectives To design, develop, deliver and assess a training initiative on haemorrhage control for emergency medical services (EMS) staff in Ukraine, in an active wartime setting.
+
+Design Using the Medical Research Council framework for complex interventions, a training programme was designed and developed in a collaboration between Irish and Ukrainian colleagues and delivered by experienced prehospital clinicians/educators. Feedback was gathered from participants. Setting The Russian invasion of Ukraine has caused large numbers of trauma patients with limited access to advanced prehospital emergency care. Ukrainian authorities requested support in delivering such care.
+
+Participants Ukrainian EMS nominated clinical staff as trainees, in partnership with an educational institution in Kyiv.
+
+Intervention One day provider and train-the-trainer courses were developed and delivered, focused on early delivery of tranexamic acid (TXA), using intraosseous access (IO) in victims of wartime trauma. Outcome measures Safe organisation and delivery of courses, assessed knowledge and skills competence and self-reported satisfaction and pre/
+post confidence/competence. Results Two provider and one train-the-trainer courses and four equipment supply exercises were delivered for 89 EMS staff (doctors, nurses, paramedics); none had prior experience of IO or prehospital delivery of TXA. All participants were assessed as competent as providers and/or trainers. High levels of satisfaction and significantly improved self-assessed confidence and competence were reported. Conclusion Rapid design and delivery of a training programme focused on an identified need for advanced care of trauma patients in a wartime setting has been possible. Training and immediate access to appropriate equipment was demonstrated. Evidence of frequency of use and safe, effective interventions has not been collected; such data are important for evaluation but difficult to collect in this setting. A high level of demand for this training now exists.
+
+## Strengths And Limitations Of This Study
+
+⇒ The Medical Research Council framework for complex interventions is a useful tool for urgent operational research.
+
+⇒ Ukraine has experienced serious systems problems in providing advanced prehospital care to the many victims of trauma since the Russian invasion. Experience and expertise from other European countries can be of value.
+
+⇒ Extracts from professional advanced prehospital emergency care courses can be adapted for presentation to frontline healthcare providers.
+
+⇒ This study reports some important indicators of feasibility but contains no data on clinical effectiveness. ⇒ Course based and self-assessment tools may provide useful feedback on training but cannot replace clinical evaluation of safe and effective delivery of therapeutic interventions.
+
+## Introduction
+
+The Russian invasion of Ukraine in February 2022 has led to very large numbers of military and civilian trauma casualties, with significant variability between sources. Recent published reports of US government estimates indicate that between 189500 and 223000 Russian soldiers have been killed (35000 to 43 000) or wounded; the equivalent figures for Ukraine's losses are between 124500 and 131000 soldiers killed or injured.1 As of 3 April 2023, the UN Commission for Human Rights recorded 22607 civilian casualties since the invasion with 8451 killed and 14156 injured.2 Evidence from battlefield studies strongly suggests that many deaths and serious injuries relate to massive bleeding immediately after injury and that most preventable deaths can be achieved with control of blood loss.3–5 Vigorous efforts to address the challenges of massive blood loss have been exemplified by the US military's Tactical Combat Casualty Care (TCCC) training and management programmes for all service members, widely adopted or reflected by similar programmes in many other countries, including Ukraine.6 From March 2022, large numbers of Ukrainian refugees began to arrive in Ireland (almost 80000 had arrived by April 2023) and many institutions around the country offered accommodation, logistic and educational supports. University College Dublin (UCD) School of Medicine engaged closely with arriving Ukrainian doctors and subsequently received requests from public representatives and emergency medical services (EMS) in Ukraine to offer training support for care of trauma patients. UCD Centre for Emergency Medical Science has a long background in training Advanced Paramedics in Irish emergency services and Combat Medical Technicians in the Irish Defence Forces; a full range of core and advanced trauma care skills is delivered by those services and practitioners.7 8 These requests were very positively received; this descriptive paper outlines the response to those requests, through the establishment of the Ukraine Trauma Project. The feasibility of an effective response had to be assessed by pilot training and supply exercises, in order to assess the potential for a larger programme of work.
+
+The objectives of the paper are to report on the design, development, delivery and assessment of the feasibility of a training initiative on haemorrhage control for EMS staff in Ukraine. A specific study objective was to assess the self-reported precourse and postcourse confidence and competence of trainees in the clinical and teaching skills included.
+
+## Methods
+
+The study describes the approach to the requests for assistance, outlining the principles adopted, the implementation of that response and brief outlines of the provider and training the trainer courses established. The 2008 Medical Research Council guidance on complex interventions provided an appropriate framework for this approach and for reporting on the intervention; the major components are development, feasibility/pilot testing, evaluation and implementation.9 The development and feasibility/pilot phases are most relevant to this paper.
+
+Feedback from course participants on the initial two courses was gathered. Participants were nominated by Ukrainian statutory and voluntary EMS agencies providing frontline prehospital trauma care. Feedback on both courses was limited to feasibility issues (access, positive issues, advice on changes); in course 2, brief data were collected from participants on training, relevant clinical skills and clinical teaching and the perceived impact of training. In course 2, precourse and postcourse self-completion Likert questionnaires were developed by the authors (online supplemental file 1) and administered anonymously, covering self-reported confidence and competence in a range of clinical and teaching issues. 
+
+SPSS V.27 was used for analysis and Wilcoxon signed rank test was used for pre/post grouped comparisons. No identifying or demographic information was requested other than duration of prior medical training.
+
+## Patient And Public Involvement
+
+It was not appropriate to involve patients or the public in the design of the study, given the setting.
+
+## Framework Of Response
+
+1. *Partnership approach*. Support offered from Ireland was made possible with the agreement and guidance of Ukrainian partner agencies and key stakeholders. 
+
+Sustainability, acceptability and relevance were identified as key elements of any successful initiative—any contribution had to address important unmet needs and to ideally be iterative. Ukrainian partner organisations include charitable fundraising bodies, educational partner agencies and state/military/EMS agencies which were the sources of invitations for the missions set out below and acted as operational supports. The Ukrainian embassy in Ireland also provided formal endorsement and support.
+
+2. Preliminary discussions between Irish and Ukrainian doctors on key challenges, with preparation of proposal, rationale and outline delivery plan. This early discussion identified the need for a focused response to early, severe blood loss as the key, addressable goal for a training package. That package would build on the widespread adoption of TCCC training across Ukrainian military and civilian EMS services.
+
+3. *Key interventions identified*. The early administration of tranexamic acid (TXA) significantly reduces mortality and morbidity following severe trauma, including battlefield injuries; however, early (preferably within onehour) intravenous or intraosseous (IO) administration is required for best effect. An important understanding to emerge from our discussions was that while a wide range of resuscitative measures for trauma (including TXA) were available in stabilisation centres, hours could elapse before trauma patients reached these centres. There were no established systems in place to provide TXA at or near the point of injury.
+
+A rapid intervention programme to train first responders to administer TXA by IO routes immediately after serious injury was identified. If the initial dose of TXA does not occur until later than 3 hours after injury, there is some evidence of increased harm.10 Additional measures addressed included use of pelvic binders (used also as ad-hoc junctional tourniquets) 
+and an introduction to the use of inhaled methoxyflurane for analgesia following resuscitation. The importance of early extremity tourniquet application was stressed.
+
+4. On-line, in-depth presentation/review of proposal to *in-country* Ukrainian decision makers. A secure online meeting was set up to present these proposals (with detail on the evidence base) to a key group of Ukrainian doctors, paramedics and trainers involved in the delivery of EMS services locally. Mediated by medically qualified interpreters, a detailed discussion led to agreement and strong support for the proposals.
+
+5. *Programme design/equipment purchase/fundraising*. A 
+oneday, pilot provider programme was designed (outlined below), based on teaching materials in Ukrainian, skills stations taught by experienced clinicians/trainers and an assessment of competence and confidence. 
+
+Individual treatment kits were designed, purchased and prepared; significant practical input was received from EMS and military medical experts on design of the kits. Fundraising was entirely from philanthropic and charitable donations, in partnership with UCD's registered charity, UCD Foundation.
+
+6. *Pilot mission*: with the support of experienced volunteer clinicians/trainers a pilot mission was planned for November 2022 to deliver a provider course to staff nominated by Ukrainian EMS services. In addition, the first batches of standardised kits would be distributed.
+
+7. *Constraints*: the reality of an ongoing war, the difficulties of reliable communication and planning with those at the frontline or close to it, the volunteerism needed for trainers to offer their services, language interpretation challenges, having units and services identify trainees, release them and transport them to and from the secure locations where training occurred and the uncertainty of whether resupply of kit could happen are among the many real-world issues which place this project in a very different setting to the mainstream of health services or medical education research. They also reflect the fact that the training and responsibilities offered are of a different standard, structure and acceptability to those used in peacetime conditions. 
+
+The risks and challenges inherent in all of these constraints were acknowledged by the individuals and organisations involved and accepted as worthwhile.
+
+## Implementation Of Response
+
+Mission 1: a four-person trainer team delivered pilot course 1 (provider/oneday) at a secure location in Ukraine. The course was attended by 29 staff from military medical units and civilian EMS services. Two trainers with extensive prehospital emergency care experience in Ireland and two Ukrainian doctors working in Ireland made up the team. The visit also supplied standardised IO/TXA kits × 30, multipurpose pelvic binders, combat tourniquets and inhaled methoxyflurane units x 30.
+
+Mission 2: at the request of the trainees from mission 1, delivery of further standardised TXA kits × 20 was made to the group about 4weeks later.
+
+Mission 3: a six-person trainer team delivered course 2 (provider and train the trainers course/3days) at a different secure location in Ukraine. The course was attended by 60 staff from military medical units and civilian EMS services and was delivered by five staff from Ireland with extensive prehospital emergency care/
+training experience and one Ukrainian doctor working in Ireland, with support from two local professional interpreters. In addition, participants were provided with standardised IO/TXA kits × 50.
+
+## Key Training Principles Provider Course
+
+1. Builds throughout on TCCC stage 1 (care under fire) 
+and 2 (care when safe) 'All Service Member' course, extensively taught as the basis of EMS care in Ukraine, using the 'MARCH (Massive bleeding, Airway, Respiration, Circulation, Hypothermia)' approach. Key TCCC skills include sequence of care, combat tourniquets, wound packing, haemostatic dressings and airway care. Immediate tourniquet use for massive bleeding is an essential, life-saving procedure highlighted by TCCC and emphasised by our training.11 12 2. Essential theory only: to include key indications, understanding the role of TXA and possible complications. Standard indications include gunshot wound above knee or elbow, amputation above hand or foot, head injury with altered level of consciousness, obvious severe blood loss/clinical shock.
+
+3. Focus on key skills, taught by experienced providers in small groups: IO access (humerus and tibia only, using simulation materials), preparation and administration of initial TXA dose (1g in 100 mL NaCl over 10min), use of pelvic binder (including as junctional binder).
+
+4. Other initiatives: a specific request was made from Ukrainian partners for a basic introduction to Chemical, Biological, Radiological, Nuclear and Explosive 
+(CBRNE) skills, particularly because of exposure to phosphorus burns; the issue was addressed by a certified CBRNE instructor. In earlier discussions about analgesia, it was accepted that the use of opiate analgesia was inappropriate in frontline/combat environments because of the potential for psychological effects; however, a proposal was made to trial the use of methoxyflurane, a self-administered, non-narcotic, inhaled analgesic for follow-up pain relief after administration of TXA and not as preparation for IO access. Both TXA and methoxyflurane are licensed for use in Ukraine.
+
+5. Assessment: core knowledge items and individual assessment of core skills.
+
+## Train The Trainer Course
+
+1. Initial successful completion of provider course. 2. Key teaching and learning principles introduced and demonstrated for both didactic and skills teaching. 
+
+The course comprised two full days of teaching, resourced by six experienced faculty members. More than half of the available time and teaching sessions were devoted to small group teaching sessions, focused on clinical skills acquisition and skills teaching.
+
+3. Participants assigned teaching tasks from provider course and were assessed for competence.
+
+4. Clear demonstration of effective interactions with colleagues.
+
+## Results Feedback From Participants
+
+Course 1 The initial provider course was attended by 29 staff from a range of military and civilian EMS agencies. Participants included four registered medical practitioners from a range of disciplines and 'medics' whose training and backgrounds ranged from professional paramedic training to those with very limited or informal training. At least 25 of the group were first responders to combat injuries and had provided early care which sometimes extended for hours before evacuation to a care facility.
+
+The focus was mainly on the feasibility of organising and delivering the course and structured feedback was kept to a minimum. The positives included the safe, on-time attendance of 29/30 expected candidates most of whom came from frontline units, the availability of good quality teaching facilities, the attention and interaction demonstrated by candidates and their complete attention to the content of the course. Individual skills stations assessed the knowledge base and ability of candidates to safely and accurately insert an IO needle and prepare and administer the correct dose of TXA. Each of the candidates was assessed as competent to carry out these tasks by direct observation by a faculty member of each candidate undertaking the specific clinical tasks of preparation of intravenous fluids and TXA for administration and IO insertion in tibial and humeral sites using training mannikins. The obvious limitations included a single day in which to carry out training, language challenges, variable background training and experience and the unfamiliarity of candidates with the procedures involved.
+
+## Course 2
+
+Of 60 participants, 55 returned feedback forms. Table 1 shows that more than half of the group selected to attend had at least a year's training in medical or prehospital medical care; four of these clinicians were registered medical practitioners and the remainder were emergency paramedics. Most of the group were first responders to combat injuries and had provided early care which sometimes extended for hours before evacuation to a care facility.
+
+Table 2 indicates that most clinical tasks were familiar to participants but confidence levels were limited; following the course, significant improvements in confidence were reported. While use of a combat tourniquet was not unexpectedly the most familiar task, there was some familiarity with parenteral access and administration of 
+
+| Table 1     | Completed months of medical training (n=55)   |
+|-------------|-----------------------------------------------|
+| >12 months  | 32 (58.2%)                                    |
+| 6–12 months | 13 (23.6%)                                    |
+| >3<6 months | 4 (7.3%)                                      |
+| >1<3 months | 5 (9.1%)                                      |
+| <1month     | 1 (1.8%)                                      |
+
+TXA. By contrast, the use of methoxyflurane or (basic) CBRN procedures were very unfamiliar but improved significantly.
+
+Table 3 reports similar significant changes in confidence levels at completion of the course. Candidates were provided with copies of the teaching materials and teaching models used including a Ukrainian language version of the TCCC ASM materials, Ukrainian language PowerPoint presentations on each of the clinical skills taught and a number of tibial and humeral models for IO access training. Each of the components formed the basis for the assessed teaching sessions.
+
+## Discussion
+
+The Ukraine Trauma Project was established as a real-time humanitarian effort to share evidence-based trauma interventions from a well-established civilian training and care system in Ireland to wartime environments in Ukraine where they were not available but could potentially have real impact. Ireland's EMS training and delivery systems are relatively small and could not aspire to provide widespread advanced EMS training or to duplicate existing basic training systems. However, the 'action-research' approach outlined here has identified key trauma interventions in which civilian expertise could be potentially cascaded to where it might have effect.
+
+| Table 2                                                                                                       | Confidence in clinical procedures: (1, no   |           |        |
+|---------------------------------------------------------------------------------------------------------------|---------------------------------------------|-----------|--------|
+| confidence, 4 completely confident) (n=55) Before the  After the  course course Median (range) Median (range) | P value                                     |           |        |
+| Intraosseous access                                                                                           | 2.0 (1–4)                                   | 4.0 (3–4) | <0.001 |
+| Intravenous access                                                                                            | 3.0 (1–4)                                   | 4.0 (2–4) | <0.001 |
+| Administration of TXA                                                                                         | 2.0 (1–4)                                   | 4.0 (3–4) | <0.001 |
+| Use of combat tourniquet                                                                                      | 4.0 (1–4)                                   | 4.0 (3–4) | 0.008  |
+| Use of pelvic binder                                                                                          | 3.0 (1–4)                                   | 4.0 (3–4) | <0.001 |
+| Use of PENTHROX                                                                                               | 1.0 (1–4)                                   | 4.0 (3–4) | <0.001 |
+| CBRN procedures                                                                                               | 1.0 (1–4)                                   | 4.0 (2–4) | <0.001 |
+| CBRN, Chemical, Biological, Radiological, Nuclear; TXA, tranexamic acid.                                      |                                             |           |        |
+
+| confidence, 4 completely confident) (n=55) Before the  course After the course Median (range) Median (range) P value Intraosseous access 2.0 (1–4) 4.0 (3–4) <0.001 Intravenous access 2.0 (1–4) 4.0 (2–4) <0.001 Administration of TXA 3.0 (1–4) 4.0 (3–4) <0.001 Use of combat tourniquet 4.0 (1–4) 4.0 (3–4) 0.006 Use of pelvic binder 3.0 (1–4) 4.0 (3–4) <0.001 Use of PENTHROX 1.0 (1–4) 4.0 (2–4) <0.001 CBRN procedures 2.0 (1–4) 4.0 (2–4) <0.001 CBRN, Chemical, Biological, Radiological, Nuclear; TXA, tranexamic acid.   |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+The initial steps outlined here demonstrate the feasibility of the key steps needed to implement such a response; the specific study objective to assess the self-reported precourse and postcourse confidence and competence of trainees demonstrated significant improvements in key clinical and teaching issues. Clearly, further data collection will be needed to establish the utility, availability and impact of the clinical interventions themselves and planning is underway to gather data to address these issues. Nonetheless, anecdotal case reports from participants in our first course indicate the intervention has been carried out in critically injured patients on a number of occasions, very close to and soon after battlefield injuries. Course candidates have returned to EMS services in which they provide care to both military and civilian patients.
+
+The process outlined here reflects aspects of familiar health services research such as the 2008 MRC framework for complex interventions, whose four phases are described as development or identification of an intervention, feasibility, evaluation and implementation. The collaborative approach taken rapidly identified a critical gap in the trauma systems in use in Ukraine (early availability of TXA), which could be addressed using well established clinical interventions from Irish EMS systems (IO 
+or intravenous administration of TXA) and which proved compatible with a rapid intervention cascade programme 
+(provider and training the trainer courses). The evaluation has necessarily been constrained by having to deliver the intervention in a wartime setting but has demonstrated that it is possible to do so, that participants demonstrate evidence of significant learning and satisfaction and that the local EMS agencies find the process sufficiently worthwhile to send their staff on the courses and to request the urgent delivery of more. TXA is licensed and available for use in Ukraine and is familiar within hospital but not prehospital systems. While our project has supplied TXA from Ireland for use by trainees, a system-wide adoption of early TXA use should not encounter significant supply issues. IO access kit is currently much less available in Ukraine but is used by some prehospital agencies; intravenous access/infusion kit is widely available. Again, systemwide adoption of early TXA use will need to examine the training/logistic options available.
+
+Important limitations include limited training time, language challenges, the variable backgrounds of candidates as well as the dangers and unpredictability of working in an active wartime setting. More specifically, the effectiveness, safety and accessibility of the interventions included have not been demonstrated in this setting and must be explored in future research.
+
+Those leading the project do not underestimate the challenges inherent in asking candidates to take responsibility for the interventions offered in this course. Candidates for the Advanced Paramedic Training Programme in Ireland must complete a minimum of 2years as registered Paramedics and then undertake a oneyear Master's level academic, clinical training and internship programme which is built around comprehensive advanced diagnostic and procedural training, including IO access and TXA administration. A course lasting some days cannot have the same objectives, content or outcomes, particularly for candidates who may have limited backgrounds in paramedic or medical practice. Nonetheless, the recognition of major combat injuries is a current responsibility of these medics; the additional use of a technical procedure may be achievable, clinically effective and requires continuing evaluation.
+
+Limited data have emerged from the war in Ukraine on the roles and effects of early care trauma systems. Our results align with those of Kivlehan *et al* who reported significant improvements in knowledge and confidence among Ukrainian participants in a 5-day WHO-ICRC 
+Basic Emergency Care course carried out in 2022.13 Epstein *et al* compared some of the lessons learnt from the US 'Global War on Terror' to the work of their group in supporting surgical care services in Ukraine.14 Riley has examined the potential lessons from the war for largescale NATO led medical operations15 and Zasiekina has explored the impact of Post Traumatic Stress Disorder at community level.16 However, we believe that this study is one of the first to describe an intervention aimed at directly improving the care of trauma patients through collaboration and dissemination of critical interventions. Further research to evaluate implementation of these procedures and to evaluate effectiveness (to the extent possible in this setting) is being planned.
+
+Further training and supply of appropriate items of equipment is planned, at the specific request of the Ukrainian EMS agencies involved, with the intention of complementing more generalised introductory TCCC training. That ongoing collaboration will provide the context for further research and development in relation to the initiatives described in this paper.
+
+Twitter Lyudmyla Zacharchenko @Lyudmyla Acknowledgements The authors thank all of those who contributed to fundraising and support and delivery of the work described in this report. Contributors GB and CF designed and developed the protocol, and all authors contributed to study implementation, data collection, analysis and interpretation of data. All authors confirm that they have contributed to the content of the paper and have approved the version of the paper submitted and agree to be accountable for resolution of any issues arising. GB acts as guarantor. Funding Fundraising was entirely from philanthropic and charitable donations, in partnership with UCD's registered charity, UCD Foundation. Competing interests None declared. Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. Patient consent for publication Not applicable. Ethics approval This study involves human participants and was approved by Institutional Review Board, Prytula Institution, Kiev VO-2023-320. Participants gave informed consent to participate in the study before taking part. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data sharing not applicable as no datasets generated and/or analysed for this study. All data collected are presented in the paper. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been 
+
+## 
+
+peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iD Gerard Bury http://orcid.org/0000-0002-4441-6724
+
+## References
+
+1 Washington post; April 6th, 2023. 2 Office of the UN Commission on Human Rights. UNHCR operational response, delivery updates; 2023.
+
+3 Kelly JF, Ritenour AE, McLaughlin DF, *et al*. Injury severity and causes of death from operation Iraqi freedom and operation enduring freedom: 2003–2004 versus 2006. *J Trauma* 2008;64(2 Suppl):S21–6; 4 Champion HR, Bellamy RF, Roberts CP, *et al*. Profile of combat injury. 
+
+J Trauma 2003;54(5 Suppl):S13–9. 
+
+5 Eastridge BJ, Mabry RL, Seguin P, *et al*. Death on the battlefield 
+(2001Y2011): implications for the future of combat casualty care. J Trauma Acute Care Surg 2012;73(6 Suppl 5):S431–7. 
+
+6 Tactical combat casualty care guidelines. Deployed Medicine, 2021. 
+
+Available: https://deployedmedicine.com/content/40 7 Clinical practice guidelines - advanced Paramedic. Naas: PreHospital Emergency Care Council, 2021.
+
+8 Loughman S, Berry C, Hickey P, *et al*. Irish defence forces combat medical technician training: experience of a novel university medical school-based programme. *BMJ Mil Health* 2022;168:128–31. 
+
+9 Craig P, Dieppe P, Macintyre S, *et al*. Developing and evaluating complex interventions: the new medical research Council guidance. 
+
+BMJ 2008;337. 
+
+10 Roberts I, Edwards P, Prieto D, *et al*. Tranexamic acid in bleeding trauma patients: an exploration of benefits and harms. *Trials* 2017;18:48. 
+
+11 Shlaifer A, Yitzhak A, Baruch EN, *et al*. Point of injury tourniquet application during operation protective edge-what do we learn? J Trauma Acute Care Surg 2017;83:278–83. 
+
+12 Yatsun V. Application of hemostatic tourniquet on wounded extremities in moderntrenchwarfare:the view of a vascular surgeon. Mil Med 2022. 
+
+13 Kivlehan SM, Allen A, Viun O, *et al*. Evaluation of change in emergency care knowledge and skills among front-line healthcare providers in Ukraine with the basic emergency care course: a pretest/ post-test study. *BMJ Open* 2022;12. 
+
+14 Epstein A, Lim R, Johannigman J, *et al*. Putting medical boots on the ground: lessons from the war in Ukraine and applications for future conflict with near-peer adversaries. *J Am Coll Surg* 2023;237:364–73. 
+
+15 Riley MR. How should the defence medical services prepare for an article 5 NATO collective defence operation with the prospect of high volumes of combat casualties? *BMJ Mil Health* 2023. 
+
+16 Zasiekina L, Zasiekin S, Kuperman V. Post-traumatic stress disorder and moral injury among Ukrainian civilians during the ongoing war. J Community Health 2023;48:784–92. 

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+Contents lists available at ScienceDirect 
+
+![0_image_0.png](0_image_0.png)
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+![0_image_2.png](0_image_2.png)
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+![0_image_3.png](0_image_3.png)
+
+journal homepage: www.cell.com/heliyon 
+
+![0_image_4.png](0_image_4.png)
+
+Validation of combined AFP, AFP-L3, and PIVKA II for diagnosis 
+
+![0_image_1.png](0_image_1.png)
+
+and monitoring of hepatocellular carcinoma in Chinese patients Tianying Ren a, Xu Hou b, Xin Zhang c, Dongliang Chen a, Juan Li a, Yingnan Zhu d, Zhiheng Liu b,**, Dawei Yang a,* 
+a Zhong Yuan Academy of Biological Medicine, Liaocheng People's Hospital, Liaocheng, 252000, PR China b Department of Hepatobiliary Surgery, Liaocheng People's Hospital, Liaocheng, 252000, PR China c Department of Clinical Laboratory, Zibo Central Hospital, Zibo, 255036, Shandong, PR China d *Department of Laboratory Medicine, Liaocheng People's Hospital, Liaocheng, 252000, PR China* ARTICLE INFO 
+
+| Keywords:  Hepatocellular carcinoma  Alpha-fetoprotein  Diagnosis  Prothrombin induced by vitamin K absence-II   |
+|------------------------------------------------------------------------------------------------------------------|
+
+ABSTRACT 
+Background: In this study, we aimed to investigate the performance of GALAD, GALAD-C, and GAAP models in Chinese population in comparison to our newly build statistical model. 
+
+Methods: In this study, we built the AALP model based on age, α-fetoprotein (AFP), AFP-L3, and prothrombin induced by vitamin K absence-II (PIVKA II) to differentiate between patients with HCC and patients with CLD. We then compared the serum levels of AFP-L3 and PIVKA II in patients with HCC who were defined as remission or progression and showed the prognostic value of combined biomarkers. 
+
+Results: The AUC value of the AALP model for HCC detection was 0.939 and AALP model exhibited a sensitivity of 81 % and a high specificity of 95 %. AALP model also exhibited good performance in the subgroups of patients with CLD. Furthermore, we demonstrated the consistency between imaging results and serum levels of AFP-L3 and PIVKA II. 
+
+Conclusions: The AALP model achieved a good diagnostic performance and a high sensitivity for predicting HCC patients. Our research also showed that AFP-L3 and PIVKA II are complementary to each other but irreplaceable in the clinical detection and monitoring of HCC. 
+
+## 1. **Introduction**
+
+Primary liver cancer is the fifth most common malignant tumor and the second leading cause of mortality in China [1–3]. Primary liver cancer includes hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular–cholangiocarcinoma. HCC accounts for 75–85 % of all liver cancer cases and ICC accounts for 10%–20 % [4]. The number of HCC cases and mortality will increase over the next 20 years [5]. Most patients with hepatocellular carcinoma (HCC) are diagnosed at an advanced disease stage and the 5-year survival rate is only 12 % [6]. It would be beneficial to identify effective molecular diagnostic markers that will allow the early detection of cancers and improve long-term survival and patient quality of life [7]. 
+
+AFP is the most utilized surveillance biomarker for screening HCC. However, the sensitivity and specificity values of AFP for HCC 
+are only 41%–65 % and 76%–94 %, respectively [8]. AFP levels are not significantly increased in nearly 40 % of patients with HCC, 
+* Corresponding author. Zhong Yuan Academy of Biological Medicine, Liaocheng People's Hospital, Liaocheng, 252000, PR China. 
+
+** Corresponding author. Department of Hepatobiliary Surgery, Liaocheng People's Hospital, Liaocheng, 252000, PR China. 
+
+E-mail addresses: lzh8623@163.com (Z. Liu), yangdawei775@163.com (D. Yang). 
+
+https://doi.org/10.1016/j.heliyon.2023.e21906 Received 4 August 2023; Received in revised form 31 October 2023; Accepted 31 October 2023 Available online 1 November 2023 2405-8440/© 2023 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
+(http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+whereas AFP levels are increased in patients with CLD [9,10]. Owing to the limited performance of AFP for the diagnosis of HCC in patients with CLD, AFP, and AFP-L3 combined with prothrombin induced by vitamin K absence-II (PIVKA II) were suggested to increase their diagnostic value for differentiating between patients with HCC patients and patients with CLD [11]. 
+
+AFP-L3 is a glycol-forms of AFP. The other two glycol-forms are AFP-L1 and AFP-L2 [12]. AFP-L3 is more specific for HCC, whereas AFP-L1 is correlated with hepatic inflammation and AFP-L2 is correlated with pregnancy [13]. AFP-L3 is calculated as the fraction of AFP-L3 to total AFP and is usually used as an HCC diagnostic biomarker [14]. Protein induced by the absence of vitamin K or antagonist-II (PIVKA II), also known as des-gamma-carboxy prothrombin (DCP), is undetectable in healthy patients and is used as a biomarker for diagnosing HCC [15]. PIVKA II is recommended as a surveillance biomarker by the Europe Association for the Study of Liver and the American Association for the Study of liver [16]. However, previous research has shown that PIVKA-II combined with AFP increased diagnostic accuracy in HCC patients with HCV [17]. To clarify whether combined three biomarker could increase diagnostic accuracy in HCC patients, we investigated the association of combined biomarkers with diagnostic accuracy. To go a step further, we continued to consider increasing diagnostic accuracy by designing diagnostic models based on Chinese patients. Johnson et al. developed the GALAD model which is based on gender, age, and three biomarkers AFP, AFP-L3, and PIVKA II. The performance of the GALAD model has been validated in Germany and Japan but has not been evaluated in the Chinese population. Liu et al. developed GALAD-C and GAAP models based on Chinese patients. They showed that GALAD-C is superior to GALAD and GAAP models in Chinese patients. However, the effectiveness of this scoring model should be further verified in different clinical cohorts. We aimed to construct a new model based on the Chinese population. We aimed to evaluate the prognostic value of AFP, AFP-L3, and PIVKA II for the diagnosis of HCC and compare the performance of GALAD, GALAD-C, and GAAP models with our new model. In addition, we aimed to compare the consistency between serum levels of AFP-L3 and PIVKA II and imaging results included magnetic resonance imaging 
+(MRI), computed tomography (CT), and angiography. 
+
+## 2. **Materials And Methods** 2.1. Study Design And Sample Collections
+
+Between 2018 and 2022, we enrolled 1241 patients from the Liaocheng People's Hospital. The patients were primarily diagnosed with HCC or had chronic disease (CLD), cirrhosis, or chronic hepatitis. There were a total of 395 patients with HCC and 846 with CLD. 
+
+The CLD patients were assigned to 3 subgroups in accordance with their clinical diagnosis—hepatitis carrier (HC) group, chronic hepatitis B (CHB) group, and liver cirrhosis (LC) group. The HC, CHB, and LC groups included 217, 206, and 423 patients, respectively. 
+
+All subjects were informed about the procedures, purpose, and study course, and every participant provided his or her signed written consent. The research protocol was approved by the Ethics Committee of the Liaocheng People's Hospital (201803007). This study was conducted in compliance with the principles of the Declaration of Helsinki and its subsequent amendments. 
+
+## 2.2. Inclusion Criteria
+
+The diagnostic standards for HCC issued by the National Health Commission of the People's Republic of China were as follows: A) 
+contrast-enhanced ultrasound, computed tomography (CT) or magnetic resonance imaging (MRI) results indicating typical imaging lesions of HCC, and the lesion site had typical blood flow changes or B) suspected small nodules found using contrast-enhanced ultrasound, CT, or MRI were confirmed by positron emission tomography examination; C) pathological examination results were consistent with the histopathological diagnosis of HCC. The diagnostic criteria for chronic liver diseases (hepatitis or CH) were as follows: (1) histopathological diagnosis or (2) in the absence of a histological diagnosis, ultrasound, CT, or MRI imaging results indicated splenomegaly without liver space-occupying lesions. The diagnostic criteria for cirrhosis were as follows: (1) the determination of liver fibrosis status was carried out utilizing Metavir scoring system. (2) in the case of non-cirrhotic portal hypertension, endoscopy revealed varices in the esophagogastric or digestive tract that were not in the usual location (3) the characteristics of liver cirrhosis or portal hypertension were evident in B-ultrasound or CT and other imaging examination such as the presence of an irregular liver outline, abnormal proportion of liver lobes, or uneven liver density. Indirect indications of liver cirrhosis encompass esophageal and gastric varices, ascites, and splenomegaly. (4) in patients lacking histological, endoscopic or imaging examinations, the presence of following abnormalities indicates liver cirrhosis, as suggest by 2 out of 4 following items): 1) platelet count (PLT) < 100 × 109/L, with no other evident cause; 2) serum albumin <35 g/L, excluding malnutrition or kidney disease as underlying causes; 3) international normalized ratio (INR) > 1.3 or prolonged prothrombin time (PT) (after discontinuation of thrombolysis or anticoagulants for more than 7 days). 4) AST/PLT ratio index (APRI): adult APRI score >2. 
+
+## 2.3. Exclusion Criteria
+
+The patients with the following diseases were excluded: (1) autoimmune liver disease; (2) ICC; (3) drug-induced hepatitis; (4) 
+warfarin or vitamin K treatment; (5) metabolic liver disease; (6) schistosomiasis. 
+
+## 2.4. Afp, Afp-L3, And Pivka Ii Tests
+
+Blood samples were collected in coagulation tubes and the plasma was separated via centrifugation at 3000 rpm for 10 min. The plasma was then used for further analyses. The biomarkers were measured using the μTASWako i30 automatic immunofluorescence analyzer (FUJIFILM Wako Pure Chemical Corporation, Japanese). The reagent was sourced from the μTASWako i30 Medical Company. 
+
+The minimum quantitative limit of AFP was 0.5 ng/mL, while that of AFP-L3% was 0.5 %. The minimum quantitative limit of PIVKA II 
+was 5 mAU/mL. In the logistic regression analysis, 0.5 % represented AFP-L3 <0.5 %, 99.5 % represented AFP-L3% >99.5 %, and 5 % represented PIVKA II < 5 mAU/mL. 
+
+## 2.5. Statistical Analysis
+
+SPSS version 24.0 software, GraphPad Prism version 5.0, and MedCalc statistical software were employed to perform the analyses. 
+
+p < 0.05 and < 0.01 were considered to indicate statistical significance. Correlations between HCC and sex were calculated by the Chisquare test. Correlations between HCC and age, TB, AST, ALP, and GGT were analyzed by Mann–Whitney U test. p < 0.05 and <0.01 were considered to indicate statistical significance. The feasibility of using AFP/AFP-L3/PIVKA II as a potential biomarker for distinguishing patients with HCC from CLD was assessed through receiver operating characteristic (ROC) curve analysis. The ROC curve analysis was performed by calculating the maximal area under the curve (AUC). Logistic regression analyses adjusted for age, AFP, 
+AFP-L3, PIVKAII, ALP, AST, GGT, and TB was applied to determine the significant independent predictors of HCC. MedCalc software was used to analyze the diagnostic performance of the respective tests between each diagnostic model. 
+
+## 3. **Results** 3.1. The Demographic And Clinical Characteristics Of Patients
+
+In this study, 1241 patients (395 patients with HCC and 846 with CLD) were enrolled and analyzed. The clinical characteristics of the patients are shown in Table 1. The inclusion and exclusion criteria of patients are described in the Materials and Methods section. 
+
+We found statistically significant differences between HCC and non-HCC groups in the clinical parameters and the patients with HCC 
+exhibited a higher level of AFP (p < 0.001), AFP-L3 (p < 0.001), PIVKA II (p < 0.001), TB (p < 0.001), DB, ALP, GGT, ALT (p < 0.001), and AST (p < 0.001) (Table 1). 
+
+## 3.2. Serum Levels Of Afp, Afp-L3, And Pivka Ii In Patients With Cld And Hcc
+
+To investigate the diagnosis value of AFP, AFP-L3, and PIVKA II in HCC, we compared the serum levels of AFP, AFP-L3, and PIVKA 
+II in the HCC group and CLD group. The median levels of AFP, AFP-L3, and PIVKA II in the HCC group were significantly higher than those in the CLD group (Fig. 1A–C). Subsequently, the CLD group was divided into the following three groups according to the clinical diagnosis: hepatitis carrier (HC) group, chronic hepatitis B (CHB) group, and liver cirrhosis (LC) group. HC, CHB, and LC groups contained 217, 206, and 423 patients, respectively. We compared the serum levels of AFP, AFP-L3, and PIVKA II in these three subgroups. We found that the serum levels of AFP, AFP-L3, and PIVKA II were significantly different in these three subgroups. The serum levels of AFP, AFP-L3, and PIVKA II in the CHB group were significantly higher than those in the HC group (p < 0.01) 
+(Fig. 2A–C). In the LC group, the serum levels of AFP and AFP-L3 were significantly higher than those in the CHB group (p < 0.01) 
+
+| Clinical characteristics of the patients included in the study.  Variables All patients (N = 1241)   | p-value*         |              |        |
+|------------------------------------------------------------------------------------------------------|------------------|--------------|--------|
+| HCC (N = 395)                                                                                        | CLD (N = 846)    |              |        |
+| Age year                                                                                             | 59 ± 0.6         | 51 ± 0.4     | <0.001 |
+| Gender male % (N) †                                                                                  | 81.0(320)        | 74.3 %(629)  | 0.01   |
+| ALT U/L                                                                                              | 85.9 ± 8.5       | 45.0 ± 3.4   | <0.001 |
+| AST U/L                                                                                              | 110.9 ± 9.0      | 42.0 ± 3.1   | <0.001 |
+| Albumin g/L                                                                                          | 33.5 ± 0.3       | 40.5 ± 0.2   | <0.001 |
+| Globulin g/L                                                                                         | 33.2 ± 0.4       | 34.9 ± 0.6   | 0.053  |
+| TB μmol/l                                                                                            | 54.5 ± 4.6       | 26.0 ± 1.2   | <0.001 |
+| DB μmol/l                                                                                            | 32.1 ± 3.8       | 8.8 ± 0.9    | <0.001 |
+| ALP IU/L                                                                                             | 145.2 ± 6.8      | 83.1 ± 2.1   | <0.001 |
+| GGT IU/L                                                                                             | 125.7 ± 7.3      | 43.2 ± 2.8   | <0.001 |
+| AFP ng/ml                                                                                            | 41318.4 ± 14,953 | 43.9 ± 44.1  | <0.001 |
+| AFP-L3 %                                                                                             | 40.5 ± 1.6       | 1.6 ± 0.1    | <0.001 |
+| PIVKA II mAU/ml                                                                                      | 14638.7 ± 2244.8 | 183.6 ± 83.6 | <0.001 |
+| TNM staging system % (N)  I                                                                          | 97(24.6)         |              |        |
+| II                                                                                                   | 62(15.7)         |              |        |
+| III                                                                                                  | 66(16.7)         |              |        |
+| IV                                                                                                   | 170(43.0)        |              |        |
+
+3
+
+![3_image_0.png](3_image_0.png)
+
+(Fig. 2A and B). In the LC group, the serum levels of PIVKA II were higher than those in the CHB group (p < 0.05) (Fig. 2C). The serum levels of AFP, AFP-L3, and PIVKA II in the CHB group were significantly higher than those in the HCC group (p < 0.01) (Fig. 2A–C). 
+
+Increased levels of AFP, AFP-L3, and PIVKA II indicate a high risk of liver disease progression. We also analyzed the association of AFP, AFP-L3, and PIVKA II with various TNM staging in patients with HCC. We observed that high content of AFP, AFP-L3, and PIVKA II were associated with advanced TNM stage (Supplementary Fig. 1). These results indicated that AFP, AFP-L3, and PIVKA II can be potential diagnostic markers for HCC. 
+
+A single marker as AFP has been reported in the diagnosis of HCC; however, its diagnostic value is limited by low specificity and positive predictive value. In this study, we compared the ROC curves based on the cutoff value of 20.0 ng/mL for AFP, AFP-L3 was 10 
+%, and PIVKAII 40 mAU/mL. The AUC value of AFP, AFP-L3, and PIVKA II was 0.73, 0.851, and 0.839 respectively. Meanwhile, the AUC value of combined biomarkers was 0.877 (Fig. 3). Compared to the limitation of single biomarker detection, the combined diagnosis of HCC markers can increase the accuracy of HCC diagnosis. 
+
+## 3.3. The Establishment And Validation Of The Aalp Model
+
+In this study, we found that AFP, AFP-L3, and PIVKA II were associated with clinical characteristics. To construct a diagnostic model for HCC, we summarized all the independent potential factors affecting the count of diagnostic models by performing binary logistic regression analysis. The independent diagnostic factors of patients with HCC were found and analyzed (Table 2). In this model, age, AFP, AFP-L3, and PIVKAII levels were risk factors for the diagnostic score of HCC. In this model, sex was not a risk factor for the diagnostic score of HCC (p > 0.05) (Supplementary Table 1). Thus, the diagnostic model was constructed based on age, AFP-L3, AFP, and PIVKA II. In this model, AFP-L3 scores were converted to 0 if AFP-L3 > 10 %, otherwise, AFP-L3 scores were converted to 1. The HCC diagnostic score was calculated by the formula as follows: Z = − 7.245 + 0.056 × age +0.431 log10 (AFP) + 3.112 × AFP-L3 (0 for AFP-L3 < 10 %, 1 for AFP-L3 ≥ 10 %) + 1.162 × log10 (PIVKA II). Table 3 presents 95 % prediction and confidence intervals. These results showed that the serum levels of AFP, AFP-L3, and PIVKAII and age could be effective risk factors for HCC with an AUC value of 0.939 using this diagnostic model (Fig. 4 and Table 3). In this model, the p-value of risk factors <0.05, and the p-value of the Hosmer–Lemeshow test was 0.782 (>0.05). Therefore, the predictive performance of the established model can be accepted. 
+
+## 3.4. Comparison Of Galad, Galad-C, Gaap, And Aalp Model
+
+The GALAD model was described using the following equation [18]: 
+
+![3_image_1.png](3_image_1.png)
+
+Z = − 10.08 + 0.09 × age+1.67 × sex+2.34log10 (AFP) +0.04 × AFP-L3+1.33 × log10 (PIVKA II), sex = 1 for males and = 0 for females. 
+
+The GALAD-C model was described using the following equation [19]: 
+Z = − 11.501 + 0.099 × age+0.073 × sex+0.84 log10 (AFP) +0.073 × AFP-L3+2.364 × log10 (PIVKA II), sex = 1 for males and =
+
+![4_image_0.png](4_image_0.png)
+
+| Significant independent factors affecting the diagnostic values in patients with HCC due to binary logistic regression analysis.  Multivariate analysis for Z score  β SE 95%CI   | p value   |       |               |       |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------|-------|---------------|-------|
+| Age                                                                                                                                                                               | 0.056     | 0.009 | 1.039–1.076   | 0.000 |
+| Log PIVKAII                                                                                                                                                                       | 1.162     | 0.135 | 2.453–4.166   | 0.000 |
+| Log AFP                                                                                                                                                                           | 0.43      | 0.131 | 1.19–1.988    | 0.001 |
+| AFP-L3                                                                                                                                                                            | 3.112     | 0.285 | 12.854–39.247 | 0.000 |
+| The p value of Hosmer-Lemeshow test was 0.782.                                                                                                                                    |           |       |               |       |
+
+| Table 3  Diagnostic performance of the GALAD, GALAD-C, GAAP, and AALP model for HCC.  Model AUC (95%CI) p value   | Standard error     |       |       |
+|-------------------------------------------------------------------------------------------------------------------|--------------------|-------|-------|
+| AALP                                                                                                              | 0.939(0.923–0.955) | 0.000 | 0.008 |
+| AFP                                                                                                               | 0.793(0.763–0.823) | 0.000 | 0.015 |
+| AFP-L3                                                                                                            | 0.851(0.824–0.867) | 0.000 | 0.014 |
+| PIVKA II                                                                                                          | 0.839(0.854–0.899) | 0.000 | 0.014 |
+| Combined                                                                                                          | 0.877(0.854–0.899) | 0.000 | 0.012 |
+| GALAD                                                                                                             | 0.930(0.913–0.947) | 0.000 | 0.009 |
+| GALAD-C                                                                                                           | 0.937(0.920–0.953) | 0.000 | 0.008 |
+| GAAP                                                                                                              | 0.916(0.897–0.934) | 0.000 | 0.010 |
+| 95%CI: 95 % Confidence Interval, AUC: area under the ROC curve, combined: combined AFP, AFP-L3 and PIVKA II.      |                    |       |       |
+
+0 for females. 
+
+The GAAP model was described using the following equation [19]: 
+Z = − 11.203 + 0.094 × age+0.699 × sex+1.076 log10 (AFP) +2.376 × log10 (PIVKA II), sex = 1 for males and = 0 for females. 
+
+The AALP model was described using the following equation: 
+Z = − 7.245 + 0.056 × age+0.431 log10(AFP)+3.112 × AFP-L3(0 for AFP-L3<10 %, 1 for AFP-L3≥10 %) +1.162 × log10(PIVKA 
+II). 
+
+The probability of HCC (P (HCC)) in an individual patient was calculated using the following formula: P (HCC) = exp (Z)/(1+ exp 
+(Z)) 
+The GALAD, GALAD-C, GAAP, and AALP models showed good prognostic value for patients with HBV/HCV having HCC (Fig. 4A). 
+
+The GALAD, GALAD-C, GAAP, and AALP models also showed good prognostic value for subgroups of CLD patients (Fig. 4B–D). In the 
+
+![5_image_0.png](5_image_0.png)
+
+GAAP model, the AUC of the ROC curves was 0.916 (95 % CI = 0.897–0.934, p < 0.001). In the GALAD model, the score was 0.930 (95
+%CI = 0.913–0.947, p < 0.001). In the AALP model, the score was 0.937 (95 %CI = 0.920–0.953, p < 0.001). In the GALAD-C model, the score was 0.939 (95 % CI = 0.923–0.955, p < 0.001) (Table 3 and Fig. 4A ). Furthermore, we used MedCalc software to compare these four models and found that the AALP model was superior to the GALAD-C model and GAAP model ( p < 0.01). GALAD-C model was superior to GALAD model ( p < 0.01). The AALP model was not superior to the GALAD model ( p > 0.05) (Supplementary Table 2).
+
+The sensitivity and specificity of the AALP model were 0.85 and 0.923 respectively. The difference between the GALAD-C model, GALAD model, and GAAP model was very less in terms of sensitivity and specificity ( Supplementary Table 3 ). These results indicated that the AALP model exhibited good sensitivity for predicting the likelihood of HCC.
+
+## 3.5. The Diagnostic Value And Monitoring Recurrence In Patients With Hcc
+
+The above studies showed that AFP-L3 and PIVKA II serum levels were better predictors of HCC diagnosis than the AFP serum level.
+
+Nevertheless, potential of AFP-L3 and PIVKA II for postoperative recurrence diagnosis has not been systematically studied yet. HCC
+recurrence is often detected by imaging examinations [ 19 ]. To assess AFP-L3 and PIVKA II levels to detect HCC postoperative recurrence, we included patients with HCC who underwent various treatment, included ablation, TACE and resection. First, we compared AFP-L3 and PIVKA II serum levels in patients with HCC showing progression. In most of the patients, either serum levels of AFP-L3 or PIVKA II increased according to the line charts ( Fig. 5A and B). The AFP-L3 value increased from 0.5 to 40.2 maximally. The PIVKA II value increased from 2480 to 51,302 maximally (Supplementary Table 4). Some exceptional changes were observed in patients showing HCC progression, and AFP-L3 and PIVKA II serum levels did not increase in these patients. 
+
+Similarly, we compared AFP-L3 and PIVKA II serum levels in patients with HCC remission. Either AFP-L3 or PIVKA II serum levels decreased in a majority of patients with HCC remission (Fig. 5C and D). The AFP-L3 value decreased from 99.5 to 0.5 maximally. The PIVKA II value decreased from 61,831 to 17,167 maximally. However, there were some exceptions. The AFP-L3 value increased from 0.5 to 13 in one patient with HCC who showed no progression, whereas the PIVKA II value increased from 13 to 178 in another patient showing no progression (Supplementary Table 5). 
+
+To verify whether AFP, AFP-L3, and PIVKA II levels could be used for monitoring dynamic disease statuses, we further selected four patients with HCC and tracked serum levels of these three biomarkers before and after diagnosis or treatment (Table 4). The imaging results of the patients (CT or MRI) were systematically analyzed. Then, we generated a timeline to assess the consistency between the imaging results and the serological results. The positive serological test criteria for these three biomarkers were AFP >20 ng/mL, AFPL3 > 10 %, and PIVKA II > 40 mAU/mL. 
+
+Case 1: Age, 58; Male. The person was diagnosed with HCC in November 2020 and multiple lesions were found (Fig. 6A). AFP-L3 and PIVKA II test results were positive at this point (Table 4). Subsequently, the patient received transhepatic arterial chemotherapy and embolization (TACE). Only one lesion was observed and the patient was not in remission according to CT (Fig. 6B). However, remission was observed in August 2021 (Fig. 6C). AFP-L3 and PIVKA II serological test results were negative at this point. During this process, serum AFP, AFP-L3, and PIVKA II levels decreased gradually. AFP-L3 levels decreased from 82.8 % to 71.3 % and from 71.3 % 
+to <0.5 %. PIVKA II levels decreased from 294 mAU/mL to 30 mAU/mL and from 30 mAU/mL to 6 mAU/mL (Table 4). AFP levels decreased from 127.9 ng/mL to 18.8 ng/mL and from 18.8 ng/mL to 3.1 ng/mL. 
+
+Case 2: Age, 53; Male. The person initially experienced recurrence after surgical treatment in January 2021 with positive AFP-L3 and PIVKA II results (86.9 % and 743 mAU/mL, respectively) (Table 4). However, liver cancer had progressed according to imaging results. The tumor size was 3.5 × 3.1 cm (Fig. 6D). In February 2021, the serological test results were 85.1 % and 212 mAU/mL with a tumor size of 3.2 × 3.0 cm (Fig. 6E). In April 2021, the serological results were 91.5 % and 1402 mAU/mL with a tumor size of 3.5 × 4.0 cm. In May 2021, the serological test results were 91.8 % and 24,565 mAU/mL with a tumor size of 4.9 × 6.1 cm (Fig. 6F). 
+
+Moreover, portal vein tumor thrombus (PVTT) was observed (Fig. 6G). Hence, AFP, AFP-L3, and PIVKA II levels increased as time progressed with an increased size of the lesion (Table 4). 
+
+Case 3: Age, 60; Female. The person was initially diagnosed with liver cirrhosis and showed negative results for AFP, AFP-L3, and PIVKA II in June 2020 (Fig. 6H and Table 4). The disease progressed into liver cancer with AFP-L3-positive and PIVKA II-negative results in September 2020(Fig. 6I). After receiving radiofrequency ablation, cancer remission was observed and the serum test turned negative (Fig. 6J). AFP-L3 serum levels increased significantly from <0.5 % to 38.6 %, whereas PIVKA II serum levels were less than 40 mAU/mL, showing a negative result in clinical testing. 
+
+Case 4: Age, 48; Male. The person presented multiple lesions after surgical resection (Fig. 6K). The serological results were PIVKA 
+II-positive (2480 mAU/mL) and AFP-L3-negative (9.1, <10 %) in January 2019 (Table 4). Later, MRI results showed enlarged multiple lesions as well as new lesions in December 2019. This case had progression of disease (Fig. 6L). The serological results showed were PIVKA II-positive (6801 mAU/mL) and AFP-L3-negative (8.4 %) at this time. 
+
+Based on the above comparison and analyses, we found that these two independent serum biomarkers showed similar performances 
+
+![6_image_0.png](6_image_0.png)
+
+| three biomarkers were AFP >20 ng/mL, AFP-L3 >10 %, and PIVKA II > 40 mAU/mL, respectively.  Date AFP (ng/mL) AFP-L3(%) PIVKA II (mAU/mL)   | Disease status   |       |        |                                                          |                              |
+|--------------------------------------------------------------------------------------------------------------------------------------------|------------------|-------|--------|----------------------------------------------------------|------------------------------|
+| Case 1                                                                                                                                     | 2020.11          | 127.9 | 82.8   | 294                                                      | HCC, multiple lesions        |
+| 2021.2                                                                                                                                     | 18.8             | 71.3  | 30     | Single lesions  Tumor size 10.5 × 9.5 cm                 |                              |
+| 2021.8                                                                                                                                     | 3.1              | <0.5  | 6      | Remission                                                |                              |
+| Case 2                                                                                                                                     | 2021.1           | 18.5  | 86.9   | 743                                                      | HCC  Tumor size 3.5 × 3.1 cm |
+| 2021.2                                                                                                                                     | 32.6             | 85.1  | 212    | HCC  Tumor size 3.2 × 3.0 cm                             |                              |
+| 2021.4                                                                                                                                     | 128              | 91.5  | 1402   | HCC  Tumor size 3.5 × 4.0 cm                             |                              |
+| 2021.5                                                                                                                                     | 465.5            | 91.8  | 24,565 | HCC  Tumor size 4.9 × 6.1 cm  Portal vein tumor thrombus |                              |
+| Case 3                                                                                                                                     | 2020.3           | 6.1   | <0.5   | 13                                                       | liver cirrhosis              |
+| 2020.12                                                                                                                                    | 7.9              | 38.6  | 15     | HCC, single lesions  Tumor size 2.1 × 2.1 × 1.7 cm       |                              |
+| 2021.4                                                                                                                                     | 3.5              | <0.5  | 27     | Remission                                                |                              |
+| Case 4                                                                                                                                     | 2019.1           | 6.7   | 9.1    | 2480                                                     | HCC, multiple lesions        |
+| 2019.12                                                                                                                                    | 8.2              | 8.4   | 6801   | progression                                              |                              |
+
+![7_image_0.png](7_image_0.png)
+
+Fig. 6. Dynamic imaging results from 4 clinical cases. (A–C) Case 1, this case became remission. (D–G) Case 2, this case progressed with enlarged tumor. (H–J) Case 3, this case progressed from liver cirrhosis to HCC and finally became remission. (K–L) Case 4, this case progressed with new lesions. (A)Case 1, initial imaging results with elevated AFP, AFP-L3, and PIVKA II. MRI showing two lesions of HCC (arrows). (B) Case 1, CT 
+showing a tumor with parenchymal iodized oil deposition (tumor size 10.5 × 9.5 cm) (arrow). (C) Case 1, CT showing an inactive lesion. (D) Case 2, MRI showing an active lesion, tumor size 3.5 × 3.1 cm (arrow). (E) Case 2, MRI showing this lesion size becoming 3.2 × 3.0 cm after radiofrequency therapy. (F) Case 2, a larger lesion size was observed (arrow), with tumor size 3.5 × 4.0 cm. (G) Case 2, this pre-present lesion became larger (4.9 ×
+6.1 cm) and PVTT appeared. (H) Case 3, MRI showing no HCC detection in this case. (I) Case 3, this case was diagnosed as HCC with a single lesion 
+(arrow). (J) Case 3, No active lesion was observed and this case went into remission. (K) Case 4, multiple lesions were located in the right liver lobe (arrows). (L) Case 4, a new lesion was detected in the right liver lobe relative to that before MRI imaging (arrows). PVTT, portal vein tumor thrombus. 
+
+and were complementary to the diagnosis and monitoring of HCC. Thus, AFP-L3 and PIVKA II can be considered potential prognostic biomarkers. 
+
+## 4. **Discussion**
+
+The majority of HCC arises from liver cirrhosis and chronic liver diseases (CLDs) [20]. The common risk factors for HCC included viral hepatitis (HBV/HCV), alcoholic abuse, and non-alcoholic fatty liver disease [5,21]. HCC diagnosis and staging are predominantly based on pathological and imaging studies. HCC should be screened routinely via abdominal ultrasound and evaluation of serum alpha-fetoprotein (AFP) levels every 6 months [22]. However, pathological analysis is invasive and has certain disadvantages, such as obtaining adequate tissues is difficult and surgical risks may exist. Serological testing has the advantages of being non-invasive and allowing real-time monitoring; thus, it can be a potential alternative to pathological testing. AFP is used to diagnose, monitor, and terminate HCC; however, it has limited sensitivity and specificity. To improve the prognosis of patients with HCC, identifying more effective biomarkers is essential. A systematic review suggested that AFP and AFP-L3 combined with PIVKA II could potentially be a valuable prognosis tool for patients with HCC [23]. 
+
+Here, we evaluated the diagnostic value of AFP-L3 and PIVKA II levels in patients with HCC. The diagnostic value of serum AFP-L3 and PIVKA II levels was found to be better than that of serum AFP levels. Moreover, we showed that the AUC value of the combination of AFP, AFP-L3, and PIVKA II was 0.877, the sensitivity was 81.0, and the specificity was 95.5. The combined diagnosis of the three markers greatly improved the early diagnosis accuracy of HCC. This finding is consistent with other study findings [24–26]. AFP 
+combined with AFP-L3 and PIVKA II can improve the sensitivity of HCC diagnosis, especially for patients with AFP-negative HCC. 
+
+To improve the diagnostic accuracy, an HCC prediction model, AALP, was established based on age and the three markers. In the binary logistic regression model, these three biomarkers were significantly related to patients with HCC. The identified signature was integrated with clinical features to establish a composite diagnostic model, which reliably demonstrated accurate diagnostic predictions for patients with CLD and HCC. The AUC value of the AALP model was 0.939 (95 % CI: 0.923–0.955), which indicated that AALP model performed good in predicting HCC accurately. The AALP model also showed good performance in differentiating patients with HCC from subgroups of patients with CLD. 
+
+In the AALP model, age and AFP, AFP-L3, and PIVKA II levels were risk factors and these results were consistent with previous published findings [19]. For example, the incidence of HCC increases with age, and older age is independently associated with HCC development [11,27,28]. AFP-L3 has been suggested as a biomarker for early HCC detection due to its higher specificity than AFP [11]. 
+
+PIVKA II exhibits higher specificity and sensitivity than AFP for HCC diagnostic [22]. Furthermore, increased AFP, AFP-L3, and PIVKA 
+II serum levels have been observed in patients with HCC than in patients with CLD [11,13,29]. 
+
+However, in this model, sex was not a risk factor unlike it was in the other three models. This finding is inconsistent with previous findings [11,18]. There may be several possible reasons for this result. One possible reason is differences in populations enrolled in the studies. Another possible reason is the different methodological approaches used in the studies. Herein, binary logistic regression was performed using the stepwise backward method. Furthermore, unlike other models, AFP-L3 was calculated as 0 or 1 in the AALP model. These reasons may explain why sex was not a risk factor in the present model. 
+
+To validate the performance of the AALP model, we compared it with the other three models. Liu et al. built the GALAD-C model for Chinese patients with HCC and validated that GALAD-C showed better performance than the GALAD model built for patients from the USA [19]. This finding is consistent with our findings and the hypothesis that enrolling different populations results in different diagnostic models has been confirmed. Another study also revealed that differences in demographic and biochemical characteristics led to different results. Further scientific studies based on a multicenter clinical trial are required to verify the present findings. 
+
+Further, to validate the performance of the combination of AFP, AFP-L3, and PIVKA II for diagnosing and monitoring patients with HCC, we collected the clinical samples and disease statuses of the patients. By comparing the serological results with the imaging results, we subsequently validated the diagnostic capabilities of the combination of AFP, AFP-L3, and PIVKA II for detecting and monitoring HCC. HCC always develops in response to chronic inflammation of the liver, and AFP levels are closely related to tumor size, stage, and pathological grade [30]. Patients undergoing surgical resection with recurrence often present increased AFP levels 
+[31]. Similar results could be found for AFP-L3 and PIVKAII [32,33]. Decreased AFP-L3 and/or PIVKA II levels generally indicate a good prognosis, whereas increased AFP-L3 and/or PIVKA II levels suggest a poor prognosis [34–36]. Here, patients with HCC were categorized into three subgroups: increased AFP-L3 and PIVKA II levels, increased AFP-L3 levels, and increased PIVKA II levels. In Case 1 and Case 2, when tumors were present, or tumor recurrence occurred, AFP-L3 and PIVKA II tests were positive. In Case 3, when the tumor was observed by imaging results, AFP-L3 was positive and PIVKA II was negative. After treatment, remission was observed in this patient with AFP-L3-negative results. In Case 4, when local tumor progression was observed, the serum AFP-L3 test was negative and PIVKA II was positive. AFP-L3 and PIVKA II levels can predict CLD progressing to HCC. These cases also showed the utility of AFP-L3 and PIVKA II levels in monitoring recurrence in patients with HCC who were clinically treated. Previous studies showed that the correlation between AFP-L3 and PIVKA II levels was low and they were two independent biomarkers [37,38]. AFP-L3 is particularly useful in predicting HCC recurrence after receiving radiofrequency ablation [39]. PIVKA II is particularly useful in predicting HCC with PVTT [40]. AFP-L3 and PIVKA II are useful in patients with low levels of AFP (<20 ng/mL). Overall, the present study shows that two biomarkers, AFP-L3 and PIVKA II, complement each other and are not irreplaceable. Further, both these biomarkers are recommended to use in clinical applications. Nevertheless, several limitations should be noted. First, this research was a single center research. further this new model should be validated in multicenter studies. Second, we were unable to analyze combined biomarkers according to pathological stage. 
+
+## 5. **Conclusion**
+
+In conclusion, we developed and validated the AALP model to predict CLD in patients with HCC. The proposed model yielded statistically significant results, which were better than those yielded by the current GALAD-C model. The present results suggest that AFP combined with AFP-L3 and PIVKA II can be used as a prognosis biomarker and can increase the accuracy of HCC diagnosis. Moreover, AFP-L3 and PIVKA II levels are complementary to each other but irreplaceable. These results provide a valuable theoretical basis for the utility of AFP-L3 and PIVKA II levels for diagnosing HCC and monitoring its recurrence. 
+
+## Data Availability Statement
+
+Data will be made available on request. 
+
+## Funding
+
+This research was funded by Natural Science Foundation of Shandong Province (No. ZR2021MH215),Liaocheng Key R&D Plan 
+(2022YDSF37), Medical and Health Technology Development Plan Project of Shandong Province (202204080720) and Scientific Research Fund of Liaocheng People's Hospital (LYQN201935). 
+
+## Credit Authorship Contribution Statement
+
+Tianying Ren: Writing - review & editing, Writing - original draft, Software, Methodology, Investigation, Data curation. **Xu Hou:** 
+Software, Methodology, Formal analysis, Data curation. **Xin Zhang:** Software, Methodology, Investigation, Formal analysis, Data curation. **Dongliang Chen:** Visualization, Methodology, Data curation. **Juan Li:** Validation, Data curation. **Yingnan Zhu:** Software, Formal analysis. **Zhiheng Liu:** Writing - review & editing, Writing - original draft, Visualization, Supervision, Project administration, Investigation, Conceptualization. **Dawei Yang:** Writing - review & editing, Writing - original draft, Supervision, Project administration, Investigation, Funding acquisition, Data curation, Conceptualization. 
+
+## Declaration Of Competing Interest
+
+The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 
+
+## Acknowledgements
+
+The authors would like to thank MJEditor (www.mjeditor.com) for its linguistic assistance during the preparation of this manuscript. 
+
+## Abbreviations
+
+AFP alpha-fetoprotein AFP-L3 lens culinaris agglutinin-reactive fraction of AFP PIVKA II protein induced by vitamin K absence or antagonist-II CLD chronic liver disease HCC hepatocellular carcinoma ROC receiver operating characteristics 
+
+AUC area under the curve 
+
+CI confidence interval 
+
+CT computed tomography MRI magnetic resonance imaging 
+
+PVTT portal vein tumor thrombus TACE transcatheter arterial chemoembolization and embolization RFA received radiofrequency ablation 
+
+## Appendix A. **Supplementary Data**
+
+Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2023.e21906. 
+
+## References
+
+| [10] D.-S. Bai, C. Zhang, P. Chen, et al., The prognostic correlation of afp level at diagnosis with pathological grade, progression, and survival of patients with  hepatocellular carcinoma, Sci. Rep. 7 (1) (2017), 12870.  [11] G.P. Caviglia, M. Ciruolo, M.L. Abate, et al., Alpha-fetoprotein, protein induced by vitamin k absence or antagonist ii and glypican-3 for the detection and  prediction of hepatocellular carcinoma in patients with cirrhosis of viral etiology, Cancers 12 (11) (2020).  [12] D. Li, T. Mallory, S. Satomura, Afp-l3: a new generation of tumor marker for hepatocellular carcinoma, Clin. Chim. Acta 313 (1) (2001) 15–19.  [13] J.Y. Choi, S.W. Jung, H.Y. Kim, et al., Diagnostic value of afp-l3 and pivka-ii in hepatocellular carcinoma according to total-afp, World J. Gastroenterol. 19 (3)  (2013) 339–346.  [14] H. Koga, H. Iwamoto, H. Suzuki, et al., Clinical practice guidelines and real-life practice in hepatocellular carcinoma: a Japanese perspective, Clin. Mol. 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Asahina, K. Tsuchiya, N. Tamaki, et al., Effect of aging on risk for hepatocellular carcinoma in chronic hepatitis c virus infection, Hepatology 52 (2) (2010)  518–527.  [29] H.A. Lee, Y.R. Lee, Y.S. Lee, et al., Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein improves diagnostic accuracy for hepatocellular carcinoma,  World J. Gastroenterol. 27 (28) (2021) 4687–4696.  [30] C. Liu, G.Q. Xiao, L.N. Yan, et al., Value of alpha-fetoprotein in association with clinicopathological features of hepatocellular carcinoma, World J.  Gastroenterol. 19 (11) (2013) 1811–1819.  [31] C.N. Yeh, M.F. Chen, Resection of peritoneal implantation of hepatocellular carcinoma after hepatic resection: risk factors and prognostic analysis, World J.  Surg. 28 (4) (2004) 382–386.  [32] S. Nakamura, K. Nouso, K. Sakaguchi, et al., Sensitivity and specificity of des-gamma-carboxy prothrombin for diagnosis of patients with hepatocellular  carcinomas varies according to tumor size, Am. J. Gastroenterol. 101 (9) (2006) 2038–2043.  [33] H.W. Lee, G.W. Song, S.G. Lee, et al., Patient selection by tumor markers in liver transplantation for advanced hepatocellular carcinoma, Liver Transplant. 24 (9)  (2018) 1243–1251.  [34] D.Y. Kim, B.N. Toan, C.K. Tan, et al., Utility of combining pivka-ii and afp in the surveillance and monitoring of hepatocellular carcinoma in the asia-pacific  region, Clin. Mol. Hepatol. 29 (2) (2023) 277–292.  [35] J. Cheng, W. Wang, Y. Zhang, et al., Prognostic role of pre-treatment serum afp-l3% in hepatocellular carcinoma: systematic review and meta-analysis, PLoS One  9 (1) (2014), e87011.  [36] K. Nouso, Y. Kobayashi, S. Nakamura, et al., Prognostic importance of fucosylated alpha-fetoprotein in hepatocellular carcinoma patients with low alphafetoprotein, J. Gastroenterol. Hepatol. 26 (7) (2011) 1195–1200.  [37] K.S. Ahn, D.R. O'Brien, Y.H. Kim, et al., Associations of serum tumor biomarkers with integrated genomic and clinical characteristics of hepatocellular  carcinoma, Liver Cancer 10 (6) (2021) 593–605.  [38] D. Zhou, G.F. Hu, W.C. Gao, et al., Hepatocellular carcinoma with tumor thrombus in bile duct: a proposal of new classification according to resectability of  primary lesion, World J. Gastroenterol. 26 (44) (2020) 7005–7021.  [39] M. Force, G. Park, D. Chalikonda, et al., Alpha-fetoprotein (afp) and afp-l3 is most useful in detection of recurrence of hepatocellular carcinoma in patients after  tumor ablation and with low afp level, Viruses 14 (4) (2022).  [40] T. Li, Y. Yu, J. Liu, et al., Pivka-ii level is correlated to development of portal vein tumor thrombus in patients with hbv-related hepatocellular carcinoma, Infect.  Agents Cancer 14 (2019) 13.   |
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medical/md/PMC10850765.md

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+# Open Access Full Text Article O R I G I N A L R E S E A R C H Pharmacogenomic Analysis Of Cyp3A5*3 And Tacrolimus Trough Concentrations In Vietnamese Renal Transplant Outcomes
+
+Thi Van Anh Nguyen1,*, Ba Hai Le 2,*, Minh Thanh Nguyen2,*, Viet Thang Le 3,*, Viet Tien Tran4,*, Dinh Tuan Le 5,*, Duong Anh Minh Vu 2,*, Quy Kien Truong 3,*, Trong Hieu Le 2,*, Huong Thi Lien Nguyen 2,*
+1Department of Pharmacy, 103 Military Hospital, Hanoi, Vietnam; 2Department of Clinical Pharmacy, Hanoi University of Pharmacy, Hanoi, Vietnam; 3Department of Nephrology and Dialysis, 103 Military Hospital, Hanoi, Vietnam; 4Department of Infectious Diseases, 103 Military Hospital, Hanoi, Vietnam; 5Department of Rheumatology and Endocrinology, 103 Military Hospital, Hanoi, Vietnam
+*These authors contributed equally to this work Correspondence: Huong Thi Lien Nguyen, Department of Clinical Pharmacy, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hoan Kiem, Hanoi, Vietnam, Tel +84904308406, Email huongntl@hup.edu.vn Purpose: CYP3A5 polymorphisms have been associated with variations in the pharmacokinetics of tacrolimus (Tac) in kidney transplant patients. Our study aims to quantify how the CYP3A5 genotype influences tacrolimus trough concentrations (C0) in a Vietnamese outpatient population by selecting an appropriate population pharmacokinetic model of Tac for our patients.
+
+Patients and Methods: The external dataset was obtained prospectively from 54 data of adult kidney transplant recipients treated at the 103 Military Hospital. All published Tac population pharmacokinetic models were systematically screened from PubMed and Scopus databases and were selected based on our patient's available characteristics. Mean absolute prediction error (MAPE), mean prediction error, and goodness-of-fit plots were used to identify the appropriate model for finding the formula that identifies the influence of CYP3A5 genotype on the pharmacokinetic data of Vietnamese patients.
+
+Results: The model of Zhu et al had a good predictive ability with MAPE of 19.29%. The influence of CYP3A5 genotype on tacrolimus clearance was expressed by the following formulas: CL=F ¼ 27; 2�½ð Þ WT=70 0; 75��½ð Þ HCT=0; 35 0; 501�
+�½ð Þ POD=180 0; 0306��CYP3A5 Lð Þ =h . The simulation result showed that Tac C0 was significantly higher in patients not expressing CYP3A5 (p< 0.001).
+
+Conclusion: The incorporation of the CYP3A5 phenotype into Zhu's structural model has significantly enhanced our ability to predict Tacrolimus trough levels in the Vietnamese population. This study's results underscore the valuable role of CYP3A5 phenotype in optimizing the forecast of Tac concentrations, offering a promising avenue to assist health-care practitioners in their clinical decisionmaking and ultimately advance patient care outcomes.
+
+Keywords: tacrolimus, population pharmacokinetic, CYP3A5, Vietnam
+
+## Introduction
+
+Tacrolimus (Tac) is the most used immunosuppressive medication to prevent acute rejection following renal transplantation. It has a narrow therapeutic index and its pharmacokinetics (PK) exhibits high inter- and intra-individual variability. Therefore, therapeutic drug monitoring (TDM) of Tac has been implemented regularly to maintain efficacy and minimize side effects. In Vietnam, the initial dose of Tac is adjusted exclusively based on the patient's weight. Subsequently, therapeutic drug monitoring for tacrolimus involves monitoring the trough concentration (C0), relying solely on the physician's experience due to the absence of any tools or software incorporating patient information. In our most recently published study, it was observed that utilizing this approach was associated with a significant occurrence of acute rejection following kidney transplantation, particularly in cases with lower C0 levels.1 The significant occurrence of acute rejection in our population can be attributed to the prevalence of various CYP3A5 gene polymorphisms, particularly the highly prevalent *3 allele, which has been consistently shown in previous pharmacogenetic studies to influence individual dosage requirements and the variability of tacrolimus levels.2–4 Some studies conducted in Vietnam have also examined the high prevalence of the *3 allele of the CYP3A5 gene and its impact on Tacrolimus trough concentration.5,6 However, a precise formula to accurately quantify the influence of CYP3A5 genotype on the pharmacokinetic data of Vietnamese patients for determining the Tacrolimus dose has not yet been identified.
+
+Pharmacist-led activities for individualized dosing have gained recognition worldwide as a valuable approach to optimizing medication therapy. For tacrolimus, by utilizing a model-informed precise dosing (MIPD) tool that incorporates covariates such as CYP3A5 genotype into a population pharmacokinetic (PopPK) model, pharmacists are able to provide the most effective approach for determining appropriate individual doses and facilitating TDM in routine practice.7 However, it is important to note that PopPK models used in these tools have not been specifically tested on Vietnamese kidney transplantation patients.
+
+Therefore, the objectives of the present study were as follows: (1) to identify the optimal population pharmacokinetic model of tacrolimus integrating the CYP3A5 phenotype specifically tailored for Vietnamese Renal Transplant Outpatients and 
+(2) to analyze the impact of the CYP3A5 phenotype on tacrolimus trough concentrations through the simulation of Tac C0 utilizing the selected pharmacokinetic model. The results of this study will provide robust evidence of the influence of the CYP3A5 genotype, enabling the development and implementation of MIPD software tools, which will assist in determining the optimal individual starting dose and facilitating TDM in Vietnamese kidney transplant patients.
+
+## Materials And Methods Patients And External Data Collection
+
+The external dataset was obtained prospectively from data of 54 outpatient renal transplant recipients treated at 103 Military Hospital, Vietnam. Patients between the ages of 18 and 75 years who received a single organ renal transplant from either a living donor or a deceased donor were eligible, and the patients received Tac as a primary immunosuppressant treatment. The exclusion criteria were retransplanted patients and non-Vietnamese renal transplant recipients.
+
+Tac C0 was collected before the morning dose when the patient who was on the outpatient drug monitoring schedule went back to the clinic. The administering time and the sampling time of patients were accurately recorded. Demographic and clinical information data were also collected.
+
+## Immunosuppression Regimen
+
+The immunosuppressive protocol at our institution consists of a triple-drug therapy consisting of tacrolimus, mycophenolate mofetil (MMF), and steroids. Induction therapy included basiliximab 20 mg (Simulect®, Novartis) at day 0 and day 4 after transplantation, 500 mg intravenous (IV) methylprednisolone (Solu Medrol®: Pfizer) pre and 12 h postoperation. Oral tacrolimus (Prograf®, Astellas Pharma) was started at night 1 day before transplantation with a dose of 0.1 mg/kg/day administered in two divided doses. Subsequent doses were adjusted based on clinical evaluation and Tac levels. Mycophenolate mofetil (Cellcept®, Roche) was started with tacrolimus at a dose of 1 g twice a day and adjusted to lower doses in the presence of diarrhea or prolonged fever. The next IV dose of steroid decreased by half in consecutive days to 40 mg/day within one to two weeks post-transplant. Oral prednisolone (15 mg/day) was initiated right after and was tapered every week to a stable period of 5 mg/day.
+
+## Bioassay
+
+Tac C0 was quantified by chemiluminescence immunoassay (CMIA, analyzed on the Architect system, Abbott Diagnostics, IL, USA). The detection limit was 1.5 ng/mL. The correlation coefficient of >0.90 for specimens between 2.0 and 30 ng/mL. The Precision of ≤10% total coefficient variation (CV).
+
+Pharmacogenetic analysis: The determination of CYP3A5 genotype was performed on blood samples from each patient. Two single nucleotide polymorphisms of CYP3A5, CYP3A5*1 and CYP3A5*3 were determined by direct sanger sequencing (ABI3500 Biosystem, Thermo Fisher Scientific, Waltham, MA, USA).8
+
+## Reviews Of The Published Tacrolimus Population Pharmacokinetic Models
+
+Published tacrolimus PopPK models were systematically searched from PubMed and Scopus databases from the date of their inception to May 2022. Additional studies were also screened from the reference lists of the identified articles. Search terms included ("kidney" OR "renal") AND "transplant" AND "Tacrolimus" AND ("population pharmacokinetic"). Studies published in English, twice-daily oral tacrolimus, and conducted in patients >18 years were included. Exclusion criteria for study selection were external validation studies. Based on screened Tac PopPK models in adult renal transplantation, we continued to choose models in which the covariates resemble ours such as PopPK models containing CYP3A5 alleles and studies that had model data.
+
+## External Predictability Evaluation
+
+The external evaluation was conducted by using MONOLIX software (version 2020 R1 Lixoft, France). Published models were re-modeled and the parameters were set to the published values in Monolix. The output was analyzed using the R package (version 3.1.1, http://www.r-project.org). The predictive performances of the model in the external dataset were assessed using two approaches: a priori and a posteriori (Bayesian approach).9 By using an a priori approach, the prediction value was estimated based on only typical parameters of the selected PopPK model and covariates such as hematocrit (HCT), weight, and genotypes. Meanwhile, the predictions that followed the Bayesian approach were estimated by integrating the observed concentrations in patients.
+
+The predictive performances of the model were evaluated through by Goodness of fit plot and two parameters that describe the accuracy and precision of model prediction (MPE and MAPE).
+
+$${\mathrm{MPE}}={\frac{\sum_{j=1}^{n}(p r e d j-o b s j)}{n}}$$
+
+- MPE (mean prediction error):
+
+$$(1)$$
+$$\text{MAPE}=\frac{\sum_{j=1}^{n}\left(\frac{\left|\textit{predj}-\textit{obsj}\right|}{\textit{obsj}}\right)\times100}{n}$$
+$$(2)$$
+
+- MAPE (mean absolute prediction error):
+where n is the number of concentration points; predj and obsj are population predicted and observed tacrolimus concentrations, respectively.
+
+Model performance based on the MAPE values was classified as acceptable prediction (MAPE < 50%), good prediction (10% < MAPE < 20%), and excellent prediction (MAPE < 10%).10
+
+## Impact Of The Cyp3A5 Phenotype On Tacrolimus Target Concentrations
+
+Using the appropriate model, simulations (N=1000) were conducted to examine the impact of the CYP3A5 phenotype on achieving target concentrations of tacrolimus. The simulation was performed using Simulix 2023R1 (Lixoft SAS, a Simulations Plus company, France).
+
+Simulated patient Tac C0 data were divided into two groups based on their allelic status for CYP3A5 - CYP3A5*3 
+(non-expressors) and CYP3A5*1 (expressors). The statistical significance of differences between the two groups was examined using the independent t-test. Statistical analyses were performed using RStudio; a p-value less than 0.05 was considered statistically significant.
+
+## Results Patients And External Validation Data
+
+A total of 54 kidney transplant patients with 167 Tac C0 samples were collected for external evaluation. The characteristics of these patients are summarized in Table 1.
+
+On average, the postoperative day of our patients was approximately 4 years. Approximately 60% of the patients expressed a genotype with a CYP3A5*3 variant. The mean tacrolimus concentration was 5.6 ± 1.5 ng/mL. Figure 1 illustrates the tacrolimus concentrations of patients over time.
+
+## Reviews Of The Published Tacrolimus Population Pharmacokinetic Models
+
+Following systematic research, a total of 256 articles were selected from PubMed and Scopus databases. After removing some duplications, 160 full texts were chosen when reviewing the titles and abstracts. Of 160 studies, 38 were chosen based on the inclusion and exclusion criteria. We then added articles from reference lists, and 44 Tac PopPK models in adult renal transplantation were collected. Finally, based on the available characteristics of our external cohort, five models were chosen for evaluating prediction. A PRISMA flow diagram of study selection is presented in Figure 2.
+
+Table 2 describes the characteristics of the patient population used to build PopPK models. Most studies (4/5) were undertaken in the Asian population. Although the study period varied greatly, from immediate up to a few years following transplantation, 3/5 studies were performed within the first year and two studies within 3 years. The median (range) number of patients was 102.5 (32–234). The mean age of subjects used for model building was between 38 and 55 years old. More than half of patients carried CYP3A5*3/*3 allele.
+
+Only one study was developed using intensive sampling data, while the remainder used Tac C0. 4/5 studies used immunoassays to measure Tac concentrations.
+
+The PopPK of Tac was described using a one-compartment model with first-order elimination in four studies (Table 3). 
+
+One model utilized the Erlang (transit) model to describe the absorption phase. Inter-patient variability was described using exponential error models in 4/5 studies. Additive, proportional, and combined (ie, additive, and proportional) error models were tested to describe the residual variability. Four covariates were identified as having a significant influence on Tac wholeblood CL/F: postoperative days, hematocrit (HCT), patient weight, and CYP3A5 phenotype. The influence of the CYP3A5 genotype on tacrolimus clearance was expressed by the following formulas.
+
+| Table 1 Characteristics of Our External Dataset Characteristics Results (N=54) Sex, n (%) Male 42 (77.8%) Female 12 (22.2%) Agea 36.0 ± 8.2 Weight (kg)b 58 (53.0–63.0) Hematocrit (%)a 43.5 ± 3.8 Postoperative period (days)a 1426.6 ± 74.2 Total daily tacrolimus dose (mg)b 6.0 (4.0–7.0) Tacrolimus trough concentration (ng/mL)a 5.6 ± 1.5 CYP3A5 genotype, n (%) *1*1/*1*3 23 (42.6%) *3*3 31 (57.4%) Notes: a Mean ± SD, b Median (quartile).   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+![4_image_0.png](4_image_0.png)
+
+| Table 2 Summary of Demographic Characteristics of the Included Published Studies Study Han et al  Zhang et al  Woillard et al   | Zhu et al   | Ling et al        |                  |              |                  |
+|---------------------------------------------------------------------------------------------------------------------------------|-------------|-------------------|------------------|--------------|------------------|
+| 2013.11                                                                                                                         | 2017.12     | 201113            | 2018.14          | 2020.15      |                  |
+| Mean age ± SD (range) (years)                                                                                                   | 38 (19–65)  | 39 (19–64)        | 55 (18–69)       | 38.26 ± 9.95 | 39 (18–68)       |
+| Mean weight ± SD (range) (kg)                                                                                                   | 59.0 ± 10.2 | 60.2 (44.0–107.0) | 65 (46–97)       | 57.02 ± 9.92 | 60 (34–95)       |
+| Mean Hematocrit ± SD (range) (%)                                                                                                | 35 ± 7.8    | 39.1 (18.4–60.7)  | 32.3 (20.9–46.6) | 33.77 ± 7.83 | 30.9 (18.8–49.6) |
+| CYP3A5 *1*1/ *1*3                                                                                                               | 32          | 39                | 1                | 57           | 213              |
+| CYP3A5 *3*3                                                                                                                     | 48          | 44                | 31               | 84           | 121              |
+| No. of patients                                                                                                                 | 80          | 83                | 32               | 141          | 234              |
+| No. of samples                                                                                                                  | 2788        | 2109              | 145              | 1232         | 936              |
+| No. of PK dots/ patients                                                                                                        | 34.85       | 25.41             | 4.53             | 8.74         | 4                |
+| Bioassay                                                                                                                        | MEIA        | EMIT              | LCMS             | CMIA         | EMIT             |
+| Abbreviations: CMIA, chemiluminescence immunoassay; EMIT, enzyme-multiplied immunoassay; MEIA, microparticle enzyme-linked immunoassay; LCMS, Liquid chromatography–mass spectrometry.                                                                                                                                 |             |                   |                  |              |                  |
+
+## Predictive Performance Assessment
+
+The predictive performance expressed by MPE and MAPE is presented in Figures 3 and 4. The model of Zhang et al and the model of Han et al resulted in an underprediction at MPE of −2.31 and −0.06, respectively. The rest of the models showed a higher estimation than the observed values. Using a Bayesian approach, the predictive results of PopPK models have been improved by reducing MPE values. The models of Woillard et al and Zhu et al showed good accuracy with MPE of −0.01 and −0.15, respectively. The model of Zhang et al still tends to predict a much smaller value than the observed value with an MPE of −2.18. The model of Ling et al resulted in overestimation. In contrast, the model of Han et al predicted lower concentrations than the observed values.
+
+When using the a priori approach, all models give poor predictive results with a high MAPE (37.68–80.07%). Threefifths of the models are acceptable with 20% < MAPE < 50%. The much lower MAPE value reflects the improved forecasting ability of the PopPK model using a Bayesian approach. Three models by Han et al, Woillard et al, and Zhu et al gave good estimation with a MAPE of 16.06%, 14.81%, and 19.29%, respectively. Ling et al's model indicated an acceptable prediction with a MAPE of 28.58%.
+
+Goodness-of-fit plots were generated to visually compare the observed concentrations with the predicted concentrations of the PopPK model, thereby enabling the validation of the models. Figure 5 shows how well the predicted concentration fits the observed concentration using a Bayesian approach.
+
+The models of Woillard et al, Zhu et al bring relatively good results exhibiting a good fit between the predicted and the observed concentration. Zhu's model had the same characteristics as ours with Asian patients, POD, age, weight, HCT, CYP3A5 polymorphisms, and Tac immunoassays. Models of Zhang et al, Ling et al, and Han et al have poor predictions.
+
+## Influence Of Cyp3A5 Genotype On Tacrolimus Trough Concentrations
+
+The model of Zhu was used to simulate the data from Vietnamese patients to evaluate the influence of the CYP3A5 genotype on Tac C0. The simulation results showed that Tac C0 were significantly higher in patients not expressing CYP3A5 (p<0.001, Figure 6).
+
+## Discussion
+
+Vietnam currently lacks a formula to calculate Tacrolimus dosage based on CYP3A5 genotype and pharmacokinetic data specific to Vietnamese patients. This study aims to fill that gap by evaluating published Tacrolimus population Pharmacogenomics and Personalized Medicine 2024:17 
+
+| Table 3 Summary of Structural, Statistical, and Covariate Models of the Included Published Studies Study Structural Model Pharmacokinetic Parameters and Covariate Relationships                                                                                                                                                                                                                                                                                                                                                                                                                         | Inter-Subject                                               | Residual         | Model     |     |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------|------------------|-----------|-----|
+| Variability                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Variability                                                 | Evaluation       |           |     |
+| Ka = 4.5 h−1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                             |                  |           |     |
+| 1-CMT                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                             |                  |           |     |
+| 2013.11                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | First-order absorption and  elimination                     |                  |           |     |
+| Han et al                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | Prop: 40.0%                                                 | RMSE,            |           |     |
+| CL=F Lð                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Þ¼ =h 22:9�eð0:17ðCYP3A5�1�3Þþ0:0525ðCYP3A5�3�3ÞÞ �eð0:297ð | Þþ               |           |     |
+| LowHct 0:117ð                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | Þ                                                           |                  |           |     |
+| NormalHct Þ �POD 0:00762                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | 49.8                                                        | Bootstrap        |           |     |
+| V=FðLÞ ¼ 716�e0:355�WTKG 59:025                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | 48.7                                                        |                  |           |     |
+| Ka = 4.5 h−1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                             |                  |           |     |
+| Zhang et al                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | 1-CMT                                                       |                  |           |     |
+| 2017.12                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | First-order absorption and  elimination                     | Add: 2.33 ng/mL  | GOF, VPC, |     |
+| CL=FðL=hÞ ¼ 22:4�eð 0:0526� 83 PODÞ � e 0:32�CYP3A5�ð39:1=HCTÞ 0:548                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | 18.8                                                        | Bootstrap        |           |     |
+| 43.7                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                             |                  |           |     |
+| V=FðLÞ¼ 179 � POD0:842  CYP3A5 = 0 if patients did not express CYP3A5, others CYP3A5 = 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |                                                             |                  |           |     |
+| Woillard                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | 2-CMT                                                       | Ktr = 5.11 h−1   | 24        | VPC |
+| Add: 0.71 ng/mL Prop: 11.3%                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                             |                  |           |     |
+| et al                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | Erlang's delay and absorption                               | Mtt = 0.78 h     | 32.6      |     |
+| 2011.13                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Ka = 7.31 h−1                                               |                  |           |     |
+| time                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | 28                                                          |                  |           |     |
+| First-order elimination                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | CL=F Lð                                                     | Þ¼ =h 21:2 HCT ð | Þ         |     |
+| =35  1:14                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |                                                             |                  |           |     |
+| h                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | ix 2CYP3A5  �                                               |                  |           |     |
+| V1/F = 140.94 (L)  Q/F (L/h) = 79  V2/F (L) = 271  CYP3A5 = 0 if patients did not express CYP3A5, others CYP3A5 = 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | 31  54  60                                                  |                  |           |     |
+| Ka=3.089 h−1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                             |                  |           |     |
+| Zhu et al                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | 1-CMT                                                       |                  |           |     |
+| 2018.14                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | First-order absorption and  elimination                     | Bootstrap        |           |     |
+| Add: 2.5 ng/mL Prop: 18.8%                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                             |                  |           |     |
+| CL=F ¼ 27:2� ð                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | Þ                                                           |                  |           |     |
+| WT=70 0:75 h i � ð                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | Þ                                                           |                  |           |     |
+| HCT=0:35  0:501 h i � ð                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Þ                                                           |                  |           |     |
+| POD=180 0:0306 h i �CYP3A5 Lð Þ =h                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                             |                  |           |     |
+| CYP3A5 = 0.753 if patients did not express CYP3A5, others CYP3A5 =1  V/F=240 L (constant)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | 28.8                                                        |                  |           |     |
+| 1-CMT                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                             |                  |           |     |
+| 2020.15                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | First-order absorption and  elimination                     |                  |           |     |
+| Ling et al                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Ka=4.5 h−1                                                  | GOF,             |           |     |
+| Add: 1.40 ng/mL                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Bootstrap                                                   |                  |           |     |
+| Prop: 24.4%                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                             |                  |           |     |
+| CL=F Lð                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Þ¼ =h 23:3 � ð                                              | Þ                |           |     |
+| HCT=0:309  0:445 � ½ð0:897 if POD >10Þ orð1 if POD � 10Þ� � 0:657CYP3A5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | 21.9                                                        |                  |           |     |
+| CYP3A5 = 0 if patients did not express CYP3A5, others CYP3A5 =1  V/F (L)=240                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                             |                  |           |     |
+| Abbreviations: Add, additive; CL/F, the clearance; CMT, compartment; CYP, cytochrome P450; CYP3A5*1*3 and CYP3A5*3*3, alleles of CYP3A5; 1GOF, goodness of fit; HCT, hematocrit; Ka, absorption rate constant; Ktr, the  absorption rate; Mtt, mean transit time; POD, post-operative day; Prop, proportional; Q/F, apparent intercompartmental oral clearance; RMSE, Root mean square error; V/F, apparent volume of distribution; V1/F, apparent volume of  distribution of the central; V2/F, volume of distribution of peripheral compartment; VPC, visual predictive check; WTKG, body weight (kg). |                                                             |                  |           |     |
+
+Nguyen et al
+
+![7_image_0.png](7_image_0.png)
+
+pharmacokinetic (PopPK) studies and selecting an appropriate model to determine the influence of CYP3A5 genotype on Tacrolimus concentration (Tac C0) in the Vietnamese population. This study is a crucial step towards developing a model-informed precision dosing approach, which will assist pharmacists in prescribing the optimal initial and adjusted doses, thereby enhancing patient outcomes globally.
+
+After a thorough systematic review, we carefully selected models that were applicable to our dataset, resulting in a collection of five final models for further analysis. When compared to our population, we observed similarities in terms of age, weight, and the prevalence of the CYP3A533 allele. Notably, the studies conducted by Woillard et al and Zhu et al showed promising predictive results that could be useful in clinical practice for predicting tacrolimus concentration (C0). Woillard's model utilized a two-compartment model with Erlang absorption and demonstrated the lowest mean absolute percentage error (MAPE) of 14.81%. However, it is worth mentioning that the study had a higher number of nonexpresser CYP3A5 *3/*3 carriers compared to only one expresser CYP3A5 *1/*3 carrier.
+
+![8_image_0.png](8_image_0.png)
+
+In contrast, Zhu's model exhibited numerous resemblances to our study, encompassing Asian demographics, postoperative day, age, weight, HCT, CYP3A5 polymorphisms, and Tacrolimus immunoassays. Consequently, Zhu's model was chosen for predicting target Tacrolimus concentrations. The impact of CYP3A5 genotype on tacrolimus clearance was identified in Zhu's equation CL/F = 27.2×[(WT/70)0.75]×[(HCT/0.35)−0.501]×[(POD/180)0.0306]×CYP3A5(L/h). Our simulation results revealed significantly higher Tacrolimus C0 levels in CYP3A5 non-expressors. The CYP3A5*3 allele causes alternative splicing and protein truncation, resulting in the absence of CYP3A5 enzyme activity. Therefore, Tac metabolism in renal transplant recipients carrying CYP3A5*3*3 is much slower than that of CYP3A5 expressors, which is supported by a number of studies and meta-analyses such as the finding of Chauhan et al. 16 Our findings are consistent with those of previous studies. 6,16,17 In addition to CYP3A5, the post-operative day (POD) serves as a crucial covariate included in
+
+Nguyen et al **Dovepress**
+
+![9_image_0.png](9_image_0.png)
+
+four out of the five examined models. However, there is some inconsistency in the effect of POD on tacrolimus clearance among these models. The studies by Han et al, Ling et al, and Staatz et al18 showed a decrease in tacrolimus clearance as PODs increased. Conversely, the models by Zhang et al and Zhu et al aligned with the findings of Antignac et al19 suggesting that an increase in tacrolimus clearance is associated with the recovery of gastrointestinal mobility and metabolism. In our study, most patients had a long average POD of 1426.6 days, leading to misestimations in the models of Zhang et al and Ling et al. Although the models of Han et al and Zhu et al did not exhibit consistent impacts of POD, both models demonstrated good predictive abilities. Hence, further research is needed to explore the influence of POD on tacrolimus clearance. Nevertheless, it is still regarded as one of the most significant covariates in tacrolimus population pharmacokinetic (PopPK) models for renal transplantation patients.
+
+Numerous studies have demonstrated the significance of hematocrit (HCT) in influencing population pharmacokinetic 
+(PopPK) models in kidney transplant patients. Specifically, tacrolimus has a strong binding affinity to erythrocytes in plasma, leading to higher HCT levels indicating an increased fraction of tacrolimus bound to erythrocytes and a decreased fraction in the plasma. In our study, all models considered HCT as a crucial covariate, and four models agreed that higher HCT levels were associated with a decrease in tacrolimus clearance. Moreover, the models developed by Han et al and Zhu et al incorporated patient weight as a covariate, which affected either the volume of distribution11 or the clearance of tacrolimus.14 Both studies concurred that increased body weight was associated with an increase in tacrolimus volume distribution, likely due to the drug's lipophilic properties.
+
+In comparison to the recent study conducted by Methaneethorn et al on Thai patients, which showcased effective predictive performance using Bayesian methods and two-compartment models, it is crucial to highlight that their research did not take into account the influence of CYP3A5 genotype as a covariate.10 Conversely, our study holds a distinct advantage over external investigations as we performed an external evaluation using both one- and two-compartment models, and notably, all of the models we assessed incorporated CYP3A5 as a covariate.
+
+On the other hand, our study had certain limitations. Firstly, we did not include other patient factors such as CYP3A4, BSA, and corticosteroid concentration. The fixed dose of corticosteroid, specifically prednisolone 5 mg, prevented us from investigating its effect in this analysis. Although we assessed the genetic polymorphism of the CYP3A4 gene in phase one, the frequency of the CYP3A422 allele, which is associated with decreased CYP3A4 activity, was below 2%. 
+
+In South Asia, the rate of CYP3A422 allele was even lower, at <0.01%.20 Hence, we made the decision not to analyze the CYP3A4 genotype. Additionally, there was variation in Tac bioassay methods across studies. This could introduce inconsistencies when applying population pharmacokinetic (PopPK) models to predict concentration scores in a patient population that is different from the original population used to construct the model.21 However, the CMIA method used in our study is currently the most commonly used method for quantitative analysis of Tacrolimus C0 levels.
+
+Based on the findings of this study, we can confidently assert that the application of population pharmacokinetic (PopPK) 
+models using the Bayesian approach enables the accurate prediction of individual tacrolimus concentration levels. This, in turn, allows for the optimization of anti-rejection therapy for kidney transplant patients. In conclusion, further evaluation of population pharmacokinetic models for tacrolimus is warranted, and the development of early dose adjustment strategies in clinical practice is essential to enhance treatment outcomes and minimize adverse effects in kidney transplant recipients.
+
+## Conclusion
+
+The incorporation of the CYP3A5 phenotype into Zhu's structural model has significantly enhanced our ability to predict Tacrolimus trough levels in the Vietnamese population. This study's results underscore the valuable role of CYP3A5 phenotype in optimizing the forecast of Tac concentrations, offering a promising avenue to assist health-care practitioners in their clinical decision-making and ultimately advance patient care outcomes.
+
+## Ethics Approval
+
+All methods were carried out in accordance with the Declaration of Helsinki. The study received ethical approval from the Ethics Committee in Biomedical Research 103 Military Hospital (No. 04/CNChT-HĐĐĐ). Participants were informed, and their consent was obtained for the research. All kidneys were donated voluntarily with written informed consent and these were conducted in accordance with the Declaration of Istanbul.
+
+## Acknowledgments
+
+The authors sincerely thank the staff at 103 Military Hospital, Vietnam, for the accommodating data.
+
+## Funding
+
+This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
+
+## Disclosure
+
+The authors declare no conflicts of interest in this work.
+
+## References
+
+8. Sarasamma S, Gracious N, Nair S, Radhakrishnan R. Pharmacogenomics of CYP3A5 polymorphism: predicting dose-adjusted trough levels of tacrolimus in south Indian renal transplant patients. *J Pharmacogenomics Pharmacoproteomics*. 2016;7(161):2.
+
+9. Owen JS, Fiedler-Kelly J. Introduction to population pharmacokinetic/pharmacodynamic analysis with nonlinear mixed effects models. In: 
+Chapter 8: Introduction to Model Evaluation. John Wiley & Sons, Inc; 2014:212–217.
+
+10. Methaneethorn J, Lohitnavy M, Onlamai K, Leelakanok N. Predictive Performance of Published Tacrolimus Population Pharmacokinetic Models in Thai Kidney Transplant Patients. *Eur J Drug Metab Pharmacokinet*. 2022;47(1):105–116. doi:10.1007/s13318-021-00735-8 11. Han N, Yun HY, Hong JY, et al. Prediction of the tacrolimus population pharmacokinetic parameters according to CYP3A5 genotype and clinical factors using NONMEM in adult kidney transplant recipients. *Eur J Clin Pharmacol*. 2013;69(1):53–63. doi:10.1007/s00228-012-1296-4 12. Zhang HJ, Li DY, Zhu HJ, Fang Y, Liu TS. Tacrolimus population pharmacokinetics according to CYP3A5 genotype and clinical factors in Chinese adult kidney transplant recipients. *J Clin Pharm Ther*. 2017;42(4):425–432. doi:10.1111/jcpt.12523 13. Woillard JB, de Winter BC, Kamar N, Marquet P, Rostaing L, Rousseau A. Population pharmacokinetic model and Bayesian estimator for two tacrolimus formulations--twice daily Prograf and once daily Advagraf. *Br J Clin Pharmacol*. 2011;71(3):391–402. doi:10.1111/j.1365-2125.2010.03837.x 14. Zhu W, Xue L, Peng H, et al. Tacrolimus population pharmacokinetic models according to CYP3A5/CYP3A4/POR genotypes in Chinese Han renal transplant patients. *Pharmacogenomics*. 2018;19(13):1013–1025. doi:10.2217/pgs-2017-0139 15. Ling J, Dong LL, Yang XP, et al. Effects of CYP3A5, ABCB1 and POR*28 polymorphisms on pharmacokinetics of tacrolimus in the early period after renal transplantation. *Xenobiotica*. 2020;50(12):1501–1509. doi:10.1080/00498254.2020.1774682 16. Chauhan PM, Hemani RJ, Solanki ND, et al. A systematic review and meta-analysis recite the efficacy of Tacrolimus treatment in renal transplant patients in association with genetic variants of CYP3A5 gene. *Am J Clin Exper Urol*. 2023;11(4):275–292.
+
+17. Khan AR, Raza A, Firasat S, Abid A. CYP3A5 gene polymorphisms and their impact on dosage and trough concentration of tacrolimus among kidney transplant patients: a systematic review and meta-analysis. *Pharmacogen J*. 2020;20(4):553–562. doi:10.1038/s41397-019-0144-7 18. Staatz CE, Willis C, Taylor PJ, Tett SE. Population pharmacokinetics of tacrolimus in adult kidney transplant recipients. *Clin Pharmacol Ther*. 
+
+2002;72(6):660–669. doi:10.1067/mcp.2002.129304 19. Antignac M, Barrou B, Farinotti R, Lechat P, Urien S. Population pharmacokinetics and bioavailability of tacrolimus in kidney transplant patients. 
+
+Br J Clin Pharmacol. 2007;64(6):750–757. doi:10.1111/j.1365-2125.2007.02888.x 20. Brunet M, van Gelder T, Åsberg A, et al. Therapeutic drug monitoring of tacrolimus-personalized therapy: second consensus report. Ther Drug Monit. 2019;41(3):261–307. doi:10.1097/FTD.0000000000000640 21. Zhao CY, Jiao Z, Mao JJ, Qiu XY. External evaluation of published population pharmacokinetic models of tacrolimus in adult renal transplant recipients. *Br J Clin Pharmacol*. 2016;81(5):891–907. doi:10.1111/bcp.12830 Pharmacogenomics and Personalized Medicine Dovepress Publish your work in this journal Pharmacogenomics and Personalized Medicine is an international, peer-reviewed, open access journal characterizing the influence of genotype on pharmacology leading to the development of personalized treatment programs and individualized drug selection for improved safety, efficacy and sustainability. This journal is indexed on the American Chemical Society's Chemical Abstracts Service (CAS). The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www. dovepress.com/testimonials.php to read real quotes from published authors. 
+
+Submit your manuscript here: https://www.dovepress.com/pharmacogenomics-and-personalized-medicine-journal

medical/md/PMC2808942.md

@@ -0,0 +1,170 @@
+# The Mouse Genome Database: Enhancements And Updates
+
+Carol J. Bult*, James A. Kadin, Joel E. Richardson, Judith A. Blake and Janan T. Eppig and the Mouse Genome Database Groupy The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609 USA
+Received September 15, 2009; Accepted October 1, 2009
+
+## Abstract
+
+The Mouse Genome Database (MGD) is a major component of the Mouse Genome Informatics
+(MGI, http://www.informatics.jax.org/) database resource and serves as the primary community model organism database for the laboratory mouse. MGD is the authoritative source for mouse gene, allele and strain nomenclature and for phenotype and functional annotations of mouse genes. MGD contains comprehensive data and information related to mouse genes and their functions, standardized descriptions of mouse phenotypes, extensive integration of DNA and protein sequence data, normalized representation of genome and genome variant information including comparative data on mammalian genes.
+
+Data for MGD are obtained from diverse sources including manual curation of the biomedical literature and direct contributions from individual investigator's laboratories and major informatics resource centers, such as Ensembl, UniProt and NCBI. MGD collaborates with the bioinformatics community on the development and use of biomedical ontologies such as the Gene Ontology and the Mammalian Phenotype Ontology. Recent improvements in MGD described here includes integration of mouse gene trap allele and sequence data, integration of gene targeting information from the International Knockout Mouse Consortium, deployment of an MGI Biomart, and enhancements to our batch query capability for customized data access and retrieval.
+
+## Introduction
+
+The Mouse Genome Database (MGD) is an integrated database of genetic, genomic and phenotypic data for the laboratory mouse (1–3). MGD is a central component of the Mouse Genome Informatics (MGI) database resource (http://www.informatics.jax.org), the community model organism database for the laboratory mouse. Other MGI data resources integrated with MGD includes the Gene Expression Database (GXD) (4), the Mouse Tumor Biology Database (MTB) (5),
+the Gene Ontology (GO) project (6) and the MouseCyc database of biochemical pathways (7). Data in MGD are updated daily. There are typically four to six major software releases per year to support access and display of new data types.
+
+The primary data types maintained in MGD include mouse genes and other genome features along with their function and phenotype annotations, associations of genome features with nucleotide and protein sequences, genetic and physical maps, gene families, mutant phenotypes, SNPs and other polymorphisms animal models of human disease, and mammalian homology. A recent summary of MGD content is shown in Table 1.
+
+MGD is the authoritative source for mouse gene, allele and strain nomenclature, Gene Ontology annotations for mouse gene function, and Mammalian Phenotype (MP)
+Ontology (8) annotations for phenotype associations.
+
+MGD contains the most comprehensive source of mouse phenotype information and associations between human diseases and mouse models. MGI curatorial staff acquire data by direct data loads from other databases, from direct submission from researchers and from published literature. To facilitate data integration, MGI employs recognized standards for genetic nomenclature and functional annotation to describe mouse sequence data, genes,
+
+| Table 1. Summary of MGD data content (10 September 2009) MGD data statistics 10 September 2009 Genes with nucleotide sequence data 28 891 Genes with protein sequence data 26 255 Genes (including uncloned mutations) 36 323 Genes with GO annotations 18 167 Mouse/human orthologs 17 787 Mouse/rat orthologs 16 768 Genes with one or more mutant allelesa 17 227 Genes with one or more phenotypic allelesb 8363 Total mutant allelesa 524 527 Phenotypic allelesb 22 666 Targeted alleles 13 721 Gene trapped alleles 501 232 Human diseases with one or more mouse models 964 QTLs 4248 Number of references 146 597 Mouse RefSNPs 10 089 692 a Mutant alleles include those occurring in mice and/or in ES cell lines. b Phenotypic alleles include only those mutant alleles present in mice.   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+strains, expression data, alleles and phenotypes. All data associations in MGD are supported with evidence and citations.
+
+Researchers can query MGD using keyword searches, vocabulary browsers and advanced web-based query forms. Keyword search supports the use of the wildcard characters (i.e.*) for broad searches and the use of quotation marks for specific phrases search. MGD also provides vocabulary browsers for GO annotations, MP annotations and Human Disease Term annotations to support browsing of the database content. The webbased query forms in MGD allow, users to construct queries of differing degrees of specificity. For example, using the Genes and Markers Query form in MGD, a researcher query broadly for all genes on mouse Chromosome 3 or specifically for genes on Chromosome 3 that are associated with specific phenotypes and/or functions (i.e. show me all genes on mouse Chromosome 3 that are associated with respiratory distress and that have been annotated functionally as being enzymes). The MGI MouseBLAST server allows users to interrogate the MGI database using nucleotide and/or protein sequences. Access to data in MGD is also facilitated by summary data files that are updated nightlyand available for download via FTP, and through direct SQL (Structured Query Language; user account is required).
+
+The staff of MGD collaborates with members of other large genome informatics resources including NCBI
+(http://www.informatics.jax.org), Ensembl (http://www
+.ensembl.org), UCSC Genome Browser (http://genome
+.ucsc.edu) and the Vertebrate Genome Annotation
+(Vega) group (http://vega.sanger.ac.uk/index.html), to maintain a comprehensive catalog of mouse genes and other genome features, and also to resolve inconsistencies in the representation of mouse genome features as needed. Biological annotations for mouse genes based on MGD curation are incorporated into scores of external informatics resources and software products.
+
+## New In 2009 Completing The Representation Of Mouse Gene Traps
+
+Release of 4.3 of MGD added over 500 000 mouse ES cell lines and sequences for gene traps from the NCBI Genome Survey Sequences Database (dbGSS), including those from the International Gene Trap Consortium (IGTC),
+and from Lexicon Genetics. Database records for genetrap alleles in MGD now include the following information:
+
+(i) sequence tag information, including genome coordinates,
+(ii) cell line IDs from organizations supplying gene trap sequence data to dbGSS,
+(iii) information about the parent stem cell line and the gene trap vector used to produce each mutant,
+(iv) information about whether a mouse has been produced from the gene trap mutant cell line,
+(v) phenotypic data for specific mouse genotypes carrying this gene trap allele,
+(vi) identification of any mouse models with phenotypic similarity to human diseases associated with the allele,
+(vii) links to the International Mouse Strain Resource
+(IMSR) for access to available mouse strains and cell lines, and
+(viii) official allele nomenclature.
+In addition to the rich annotation details for gene trap alleles (Figure 1), the location and structure of the gene traps in a genomic context are available from mouse GBrowse (Figure 2). GBrowse contains separate tracks for DNA and RNA-based gene traps. In addition, there is a summary track which displays the number of traps per gene. Since GBrowse includes the gene predictions from NCBI, Vega and Ensembl in individual tracks, it is straightforward to compare the location of the gene traps relative to multiple gene predictions.
+
+Gene trap data are easily accessed from gene detail pages via hypertext links in the Phenotypes section of the report. Direct queries for gene traps in MGD can be accomplished using the dbGSS sequence accession identifiers or by searching for specific parameters on the Phenotypes, Alleles and Disease Query form. Tabdelimited reports of gene traps in MGD can be viewed or downloaded from the MGI FTP site.
+
+## Incorporation Of International Knockout Mouse Consortium Data
+
+The International Knockout Mouse Consortium (IKMC) is a broad based international effort to generate knockout alleles for every mouse gene (9,10). As IKMC generates ES cell lines carrying new targeted mutant alleles, these are incorporated into MGD and provide official nomenclature and MGI identifiers. Thus, IKMC alleles are accessible with all other mouse mutant alleles. As IKMC mutant ES cell lines are used to produce mice and those mice are phenotyped, data will be available in MGD for comparative phenotyping with all other extant mouse mutant data.
+
+![2_image_0.png](2_image_0.png)
+
+![3_image_0.png](3_image_0.png)
+
+Primary access to IKMC progress and resources is available through a common web portal (http://www
+.knockoutmouse.org). To facilitate access to IKMC information and resources from within MGI, curated links to the IKMC web site are now available from MGI gene detail pages and also from tracks in mouse GBrowse (Figure 2).
+
+## Enhanced Batch Query Tool Capabilities
+
+The Batch Query Tool is particularly useful for researchers who use non-MGI mouse gene accession identifiers in their analyses but who want to connect those identifiers to the rich functional and phenotypic annotations for mouse genes contained in MGD. The initial release of the MGI Batch Query Tool
+(http://www.informatics.jax.org/javawi2/servlet/WIFetch?
+
+page=batchQF) provided the ability to access information about nomenclature, genome location, function, or phenotype associations for many genes/markers in a single query (2). Allowable input into the Batch Query Tool included current gene symbols, Ensembl gene ids, EntrezGene ids, VEGA gene ids, MGI ids, RefSeq ids and GenBank sequence accession ids. These data can be uploaded as a file or pasted into a text box on the query form. Users specified the desired output and output format (web or tab-delimited text). Recent additions to the Batch Query Tool include the ability to use Affymetrix microarray probe identifiers as inputs into the query tool and the ability to download phenotype and functional annotation terms, gene expression data from the Gene Expression Database (GXD), and human disease terms that are associated with the user supplied id lists. In addition, the Batch Query Tool will now accept mixed lists of identifiers as input, for example. MGI:96677, Pax6, 16590, OTTMUSG00000015949, Q3UFR6, which are, respectively, an MGI accession identifier, a gene symbol, an Entrez Gene identifier, a VEGA mouse gene identifier, and a UniProt protein record accession identifier.
+
+## Mgi Biomart
+
+To support cross database integration and data mining, MGI now supports a BioMart application (Figure 3). BioMart is a 'query-oriented data management system' that is designed to support a federated approach to data integration (11). The unique aspect of the MGI BioMart query tool relative to existing data access mechanisms for MGI is that the resources supports the ability of users to combine data and annotations from MGI with data from external databases, such as the gene annotation
+
+![4_image_0.png](4_image_0.png)
+
+data from Ensembl (http://www.ensembl.org), 'on the fly'.
+
+The BioMart also supports iterative query refinement and allows users to save query results in a variety of output formats.
+
+## Other Information Mouse Gene, Allele And Strain Nomenclature
+
+MGD is the authoritative source of symbols and names for mouse genes, alleles and strains. The nomenclature in MGD follows the guidelines set by the International Committee on Standardized Genetic Nomenclature for Mice (http://www.informatics.jax.org/nomen). This official nomenclature is widely disseminated through regular data exchange and curation of shared links between MGI and other bioinformatics resources. MGD
+staff members work with editors of journal publications to promote adherence to mouse nomenclature standards in publications.
+
+To support consistency of nomenclature across multiple mammalian species, members of the MGD nomenclature group coordinate gene names and symbols with nomenclature specialists from the Human Gene Nomenclature Committee (HGNC) (http://www.genenames.org/) and the rat genome database (RGD; http://rgd.mcw.edu).
+
+The mouse and human nomenclature committees collaborate with scientific experts in specific domain areas to represent the latest knowledge about gene families such as the NLR gene family (12). The MGD nomenclature coordinator can be contacted by email
+(nomen@informatics.jax.org).
+
+## Electronic Data Submission
+
+MGD accepts contributed data sets from individuals and organizations for any type of data maintained by the database. The most frequent types of contributed data are mutant and phenotypic allele information originating with the large mouse mutagenesis centers and repositories that contribute to the International Mouse Strain Resource [IMSR, http://www.imsr.org (13)]. Each electronic submission receives a permanent database accession ID. All data sets are associated with their source, either a publication or an electronic submission reference. Details about data submission procedures can be found at http://www.informatics.jax.org/mgihome/submissions/ submissions_menu.shtml.
+
+Suggestions and corrections to the representation of data and information in MGD can be submitted using the 'Your Input Welcome' link which appears in the upper right hand corner of gene and allele detail pages.
+
+## Community Outreach And User Support
+
+The MGD resource has full-time staff members who are dedicated to user support and training. Members of the User Support team can be contacted via email, web requests, phone or FAX.
+
+(i) World wide web: http://www.informatics.jax.
+
+org/mgihome/support/support.shtml
+(ii) Email access: mgi-help@informatics.jax.org
+(iii) Telephone access: +1-207-288-6445
+(iv) FAX access: +1-207-288-6132
+MGD User Support staff are available for on-site training on the use of MGD and other MGI data resources. The traveling tutorial program includes lectures, demos and hands-on tutorials that can be customized according to the research interests of the audience.
+
+Online training materials for MGD and other MGI
+data resources are available as FAQs and on-demand help documents. In addition, a freely available Mouse Genome Informatics tutorial is available via Open Helix (http://www.openhelix.com/mgi).
+
+## Other Outreach
+
+MGI-LIST (http://www.informatics.jax.org/mgihome/ lists/lists.shtml) is a moderated and active email bulletin board supported by the MGD User Support group. The MGI list serve has over 2100 subscribers. On an average there are three posts per day.
+
+## High-Level Overview Of The Main Components And Implementation
+
+MGD is implemented in the Sybase relational database management system with -180 tables within which the biological information is stored. BLAST-able databases and genome assembly files for sequence data are stored outside the relational database. An editing interface and automated load programs are used to input data into the MGD system. The editing interface (EI) is an interactive, graphical application used by curators. Automated load programs that integrate larger data sets from many sources into the database include quality control (QC) checks and processing algorithms that integrate the bulk of the data automatically and identify issues to be resolved by curators or the data provider. Thus, through EI and automated loads, we acquire and integrate large amounts of data into a high-quality, knowledgebase.
+
+Public data access to MGD is provided primarily through the web interface (WI) where users can interactively query and download our data through a web browser. MouseBLAST allows users to do sequence similarity searches against a variety of rodent sequence databases that are updated weekly from selected sequence databases from NCBI, UniProt and other providers. Mouse GBrowse allows users to visualize mouse data sets against the genome as a series of linear tracks. FTP reports are a major source for other data providers who link to or use MGD data in their products, and for computational biologists who use MGD data in their analyses. Programmatic access to MGD via web services (SOAP) is also supported (http://www.informatics.jax.org/mgihome/other/web_
+service.shtml). All MGD files and programs are openly and freely available.
+
+## Citing Mgd
+
+For a general citation of the MGI resource please cite this article. In addition, the following citation format is suggested when referring to datasets specific to the MGD
+component of MGI: Mouse Genome Database (MGD),
+Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Maine (URL: http://www.informatics .jax.org). [Type in date (month, year) when you retrieve the data cited.]
+
+## Funding
+
+National Institutes of Health National Human Genome Research Institute grant HG000330. Funding for open access charge: National Institutes of Health grant HG000330. Conflict of interest statement. None declared.
+
+## References
+
+1. Blake,J.A., Bult,C.J., Eppig,J.T., Kadin,J.A., Richardson,J.E. and the Mouse Genome Database Group. (2009) The Mouse Genome Database genotypes::phenotypes. Nucleic Acids Res., 37, D712–D719.
+
+2. Bult,C.J., Eppig,J.T., Kadin,J.A., Richardson,J.E., Blake,J.A. and the Mouse Genome Database Group. (2008) The Mouse Genome Database (MGD): mouse biology and model systems. Nucleic Acids Res., 36, D724–D728.
+
+3. Eppig,J.T., Blake,J.A., Bult,C.J., Kadin,J.A., Richardson,J.E. and the Mouse Genome Database Group. (2007) The Mouse Genome Database (MGD): new features facilitating a model system. Nucleic Acids Res., 35, D630–D637.
+
+4. Smith,C.M., Finger,J.H., Hayamizu,T.F., McCright,I.J., Eppig,J.T.,
+Kadin,J.A., Richardson,J.E. and Ringwald,M. (2007) The mouse Gene Expression Database (GXD): 2007 update. Nucleic Acids Res., 35, D618–D623.
+
+5. Krupke,D.M., Begley,D.A., Sundberg,J.P., Bult,C.J. and Eppig,J.T.
+
+(2008) The Mouse Tumor Biology database. Nat. Rev. Cancer, 8, 459–465.
+
+6. The Gene Ontology Consortium. (2008) The Gene Ontology (GO) project in 2008. Nucleic Acids Res., 36, D440–D444.
+
+7. Evsikov,A., Dolan,M., Genrich,M.J., Patek,E. and Bult,C.J. (2009)
+MouseCyc: a curated biochemical pathways database for the laboratory mouse. Genome Biol., 10, R84.
+8. Smith,C.L., Goldsmith,C.A. and Eppig,J.T. (2005)
+The Mammalian Phenotype Ontology as a tool for annotating, analyzing and comparing phenotypic information.
+
+Genome Biol., 6, R7.
+
+9. The International Mouse Knockout Consortium. (2007) A mouse for all reasons. Cell, 128, 9–13.
+
+10. Collins,F.S., Finnell,H., Rossant,J. and Wurst,W. (2007) A new partner for the international knockout mouse consortium. Cell, 129, 235.
+
+11. Smedley,D., Haider,S., Ballester,B., Holland,R., London,D.,
+Thorisson,G. and Kasprzyk,A. (2009) BioMart - biological queries made easy. BMC Genomics, 10, 22.
+
+12. Ting,J.P., Lovering,R.C., Alnemri,E.S., Bertin,J., Boss,J.M.,
+Davis,B.K., Flavell,R.A., Girardin,S.E., Godzik,A., Harton,J.A. et al. (2008) The NLR gene family: a standard nomenclature. Immunity, 28, 285–287.
+
+13. Strivens,M. and Eppig,J.T. (2004) Visualizing the laboratory mouse: capturing phenotype information. Genetica, 122, 89–97.

medical/md/PMC2862674.md

@@ -0,0 +1,221 @@
+# Clinical Science Effects Of Sildenafil On Autonomic Nervous Function During Sleep In Obstructive Sleep Apnea
+
+Christiane Neves,I,II Sérgio Tufik,I Felipe Chediek,I Dalva Poyares,I Fátima Cintra,I Marina Roizenblatt,I Fabiano Abrantes,I Marina Ariza Monteiro,I Suely RoizenblattI,II doi: 10.1590/S1807-59322010000400008 Neves C, Tufik S, Chediek F, Poyares D, Cintra F, Roizenblatt M, et al. Effects of sildenafil on autonomic nervous function during sleep in obstructive sleep apnea. Clinics. 2010;65(4):393-400.
+
+OBJECTIVE: To evaluate the effects of sildenafil on the autonomic nervous system in patients with severe obstructive sleep apnea. METHODS: Thirteen male patients with severe obstructive sleep apnea (mean age 43±10 years with a mean body mass index of 26.7±1.9 kg/m2) received a single 50-mg dose of sildenafil or a placebo at bedtime. All-night polysomnography and heart rate variability were recorded. Frequency domain analysis of heart rate variability was performed for the central five-minute sample of the longest uninterrupted interval of slow wave and rapid eye movement sleep, as well as for one-minute samples during apnea and during slow wave and rapid eye movement sleep after resumption of respiration. 
+
+RESULTS: Compared to the placebo, sildenafil was associated with an increase in the normalized high-frequency (HFnu) components and a decrease in the low/high-frequency components of the heart rate variability ratio (LF/HF) in slow wave sleep (p<0.01 for both). Differences in heart rate variability parameters between one-minute post-apnea and apnea samples (D= difference between resumption of respiration and apnea) were assessed. A trend toward a decreasing magnitude of DLF activity was observed during rapid eye movement sleep with sildenafil in comparison to placebo (p=0.046). Additionally, D LF/HF in SWS and rapid eye movement sleep was correlated with mean desaturation (sR= -0.72 and -0.51, respectively, p= 0.01 for both), and D HFnu in rapid eye movement sleep was correlated with mean desaturation (sR= 0.66, p= 0.02) and the desaturation index (sR= 0.58, p = 0.047). CONCLUSIONS: The decrease in arousal response to apnea/hypopnea events along with the increase in HFnu components and decrease in LH/HF components of the heart rate variability ratio during slow wave sleep suggest that, in addition to worsening sleep apnea, sildenafil has potentially immediate cardiac effects in patients with severe obstructive sleep apnea. KEYWORDS: Nitric oxide; Heart rate variability; Phosphodiesterase-5; Erectile dysfunction; Oxyhemoglobin.
+
+## Introduction
+
+Obstructive sleep apnea (OSA) is associated with a number of conditions prevalent among middle-aged men1-4 and is a major factor contributing to erectile dysfunction.5 Concerns have arisen about the increasing percentage of men using selective phosphodiesterase-5 (PDE-5) inhibitor drugs to treat erectile dysfunction.6 Our previous research showed I Department of Psychobiology, Universidade Federal de São Paulo - São Paulo/SP, Brazil. II Department of Internal Medicine, Universidade Federal de São Paulo - São Paulo/SP, Brazil. Email: christianen1@hotmail.com Tel: 55 16 3967.0768 Received for publication on December 04, 2009 First review completed on December 29, 2009 Accepted for publication on January 28, 2010 that this concern is justified; sildenafil has a magnifying effect on respiratory events in patients with severe OSA.7 Furthermore, OSA remains undiagnosed in 80% of patients.8 Our previous study found that an increase in the number and duration of obstructive respiratory events might be related to the nasal congestion that is frequently reported by sildenafil users.9 These events could also be linked to a ventilation-perfusion mismatch10 related to the nitric oxide (NO)-dependent vasodilatory effect of sildenafil in the absence of ventilation. Although we observed worsening apnea and oxyhemoglobin desaturation, our study found an interesting less-than-expected increase in arousal.7 The blunted arousability of OSA patients after sildenafil consumption may result from cerebral vasodilation due to increased cerebrovascular reactivity to hypercapnia.11 However, the blunted arousability may also reflect an impaired autonomic nervous system response. 
+
+Research has shown an association between cardiac autonomic function measures and the severity of sleepdisordered breathing.12 Heart rate variability (HRV) is not the most direct measure of autonomic activity but is widely used because of its non-invasiveness and ease of application.13 In patients with OSA, HRV differs in apnea and post-apnea periods. The increased negative intrathoracic pressure (the Muller maneuver) during respiratory effort arises from parasympathetic predominance.14 However, during apnea events, a progressive increase in sympathetic activity also occurs due to oxyhemoglobin desaturation.15 The maximum sympathetic activity occurs at the end of the apnea event; from then on, sympathetic activity decreases until the recovery of normal respiration.16 A bilateral relationship exists between arousal and sympathetic activity in OSA17, and both are related to sleep fragmentation. 
+
+To the best of our knowledge, the influence of sildenafil on autonomic nervous modulation in patients with severe OSA has not yet been reported. The primary goal of the present study is to examine this relationship during slow wave sleep (SWS) and rapid eye movement (REM). A secondary objective is to evaluate sildenafil-induced modifications of HRV during apnea and after the resumption of respiration. 
+
+## Patients And Methods
+
+Heart rate variability recordings were performed during polysomnography (PSG) for the thirteen subjects included in our previous study.7 These recordings were evaluated and compared with the patients' PSG respiratory parameters. 
+
+Patients included in the study were between the ages of 40 and 65 years and had a body mass index (weight in kilograms/height in meters squared) of less than 30, an apnea-hypopnea index (AHI) of more than 30 events/ hour of total sleep time (TST), and an oxygen desaturation (>4%) index of 10 or more/hour of TST, as evidenced by polysomnography performed less than six months earlier. 
+
+Exclusion criteria included daytime hypoxemia, concomitant significant debilitating illness, the use of nitrates or drugs that could influence sleep, current alcohol or drug abuse, and a current or previous habit of smoking more than 10 cigarettes a day. Further exclusion criteria included acute or chronic respiratory disease based on symptoms and respiratory function test results, systemic arterial hypertension, and evidence of previous or present cardiac disease based on symptoms, 12-lead electrocardiography, or Doppler echocardiography. Subjects who participated in trials with continuous positive airway pressure devices in the previous six months were also eliminated from the study.7 The study protocol was approved by the local Ethics Review Committee, and all participants signed written informed consent forms. 
+
+## Study Design
+
+After consenting to participate in the study, all of the patients were evaluated for the presence of vascular or metabolic disease (i.e., arterial hypertension, diabetes, coronary artery disease, cerebrovascular disease, hypercholesterolemia, and diabetes mellitus) and smoking habits. To exclude patients with cardiac and pulmonary hypertension (defined as a pulmonary arterial pressure above 20 mmHg) and other respiratory diseases, electrocardiography, Doppler echocardiography, and respiratory function tests were performed. 
+
+Polysomnography recordings were carried out after a night of adaptation to the sleep laboratory. Each participant received two coded envelopes, one containing the drug and the other containing the placebo, and was asked to randomly select one of the envelopes and take the pill that was inside at bedtime. The pill in the remaining envelope was administered on the next recording night. The codes were opened at the end of the study. 
+
+## Polysomnography
+
+Bedtime was based on each patient's habits. At least seven hours of recording time were obtained. The following measurements were collected: an electroencephalogram (at positions C3-A2, C4-A1, and O1-A2 of the International 10-20 System), a bilateral electrooculogram, a submental electromyogram, and an electrocardiogram (modified V2 lead). 
+
+Respiration was monitored as follows: airflow was measured with a nasal cannula/pressure transducer system (Pro-Tech Services Inc; Mukilteo, WA, USA) and a mouth thermocouple; chest and abdominal efforts were measured with uncalibrated, inductive, respiratory plethysmographic belts; arterial oxygen saturation (SaO2
+) was measured with pulse oximetry (Ohmeda Hatfield, Herts., England), and body position movements were measured with a mercury gauge. Body position was determined by a sensor. Data were collected using a 16-channel computerized sleep system (Harmonie 5.2; Stellate Systems Inc., Montreal, Quebec) with a sampling rate of 512 Hz. 
+
+An experienced researcher, blinded to the medication condition of the participants, performed the sleep scoring of each patient's three PSGs; the scoring was conducted according to previously established parameters.18 Total sleep time (TST) was defined as the time elapsed between the first and last recorded epoch of sleep, excluding wakefulness. Arousals lasting more than 3 seconds were scored according to the criteria established by the American Sleep Disorders Association.19 According to the parameters established by the Taskforce of the American Academy of Sleep Medicine,20 apnea was defined as a period of breathing cessation and hypopnea, as a 50% reduction in breathing, or as less than a 50% reduction in breathing associated with a 4% desaturation of oxyhemoglobin or arousal. The minimum duration of an event was ten seconds. The apnea hypopnea index was defined as the total number of apneas and hypopneas/hour of TST. Obstructive AHI was defined as the number of obstructive apneas plus hypopneas/hours of TST, mixed AHI was defined as mixed apneas plus hypopneas/hours of TST, and central AHI was defined as central apneas plus hypopneas/hours of TST. The percentage of TST spent in apnea/hypopnea events (%TST AH) was calculated to estimate the duration of respiratory events during sleep. The desaturation index (DI) corresponded to the number of arterial oxygen desaturations (with a drop greater than 4%) per hour of TST. The percentage of TST spent with less than 90% oxyhemoglobin saturation (Sat O2
+<90%) was also measured.
+
+## Heart Rate Variability
+
+Frequency domain analysis of HRV21 was performed for the central five-minute sample of the longest interval of SWS and REM sleep that was free of stage shifts, artifacts or arousals.22 Additional frequency domain analysis of HRV was carried out in one-minute samples during apnea and after the opening of the upper airway in SWS and REM. Samples during an apnea event were eligible only if they occurred during obstructive events and were followed by a post-apnea one-minute period that was free of further apnea or artifact events. The interval corresponding to the opening of the airway was excluded.23 The spectra of the selected samples were adjusted for respiration using the Welch method with 128-sample Hanning windowing.24 This study focused on the low frequency band (LF: 0.04 to 0.15 Hz), which is influenced by sympathetic activity, and on the high frequency component (HF: 0.15 to 0.40 Hz), which is under vagal control, is synchronous with respiratory frequency, and represents vagal heart activity.13 The LF/HF ratio reflects sympathovagal modulation.25 Normalized (nu) values and spectral analysis were used to quantify changes in the components of HRV. This method is more sensitive to fluctuations in cardiac autonomic influence than time domain indexes of HRV because the spectral measures are normalized (nu) in relation to total power 
+(e.g., LFnu = LF/total power and HFnu= HF/total power).26 The power density in the very low frequency range (VLF: 0.0033 to 0.04 Hz) was not analyzed because short-term HRV analysis cannot detect this component. Because the total frequency (TF) includes the sum of very low, low, and high frequency power, TF values were also not included in this analysis.21
+
+## Statistical Analysis
+
+A Shapiro-Wilk test was used to examine the normality of the distribution, and values were expressed as mean ± standard error (SE) (Table 1). The differences among groups were analyzed using a dependent sample t-test or Wilcoxon test. Differences in HRV parameters between one-minute post-apnea and apnea periods (D= difference between postapnea and apnea) were assessed. In addition, a Spearman test was used to determine correlations between DHRV 
+parameters and the following respiratory variables: mean desaturation of oxyhemoglobin, AHI, DI and respiratory arousal index. Statistical evaluations were conducted with Statistica (version 6.0) for Windows software (Statsoft, Inc., Tulsa, OK, 2004). Significance was established at p<0. 05. 
+
+## Results
+
+Table 1 shows the effects of sildenafil and the placebo on HRV and respiratory parameters during SWS and REM sleep. As described in our previous study,7 we observed an increase in mean desaturation (p= 0.02), in the apnea hypopnea index (p= 0.0006), and in the desaturation index (p=0.002) as well as a decrease in mean saturation (p=0.01) after sildenafil treatment in comparison to the placebo. An increase in HF (p=0.0001) and a decrease in LF/HF in SWS sleep (p=0.002) were also observed after sildenafil treatment. The respiratory arousal index did not significantly differ in relation to the treatments, but a decrease in SWS, in terms of both minutes and percentage of TST, was observed after sildenafil. 
+
+A decreased magnitude of the difference (D) in the LF component of HRV was observed in REM sleep after sildenafil treatment in comparison to the placebo (p=0.046). 
+
+A clear trend toward a decrease in D LF/HF was also observed in REM sleep after sildenafil (Figure 1).
+
+Examinations of the correlations between HRV and respiratory parameters showed that D LF/HF in SWS and REM sleep was correlated with mean desaturation (sR = 
+-0.72 and -0.51, respectively, p = 0.01 for both), whereas D HF in REM sleep was correlated with mean desaturation 
+(sR=0.66, p = 0.02) and the desaturation index (sR = 0.58, p 
+= 0.047).
+
+| Sleep parameter                                                                                                                                                                                                                                             | Placebo           | Sildenafil    | p           |       |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------|---------------|-------------|-------|
+| SWS                                                                                                                                                                                                                                                         | LF                | 0.74 (0.04) § | 0.72 (0.03) | 0.81  |
+| HF                                                                                                                                                                                                                                                          | 0.12 (0.02)       | 0.28 (0.03)   | 0.0001      |       |
+| LF/HF                                                                                                                                                                                                                                                       | 10.14 (2.29) §    | 3.57 (0.62)   | 0.002       |       |
+| SWS min                                                                                                                                                                                                                                                     | 77.0 (7.3)        | 53.3 (4.4)    | 0.001       |       |
+|                                                                                                                                                                                                                                                             | %TST              | 17.1 (1.4)    | 12.5 (0.9)  | 0.001 |
+| Respiratory arousal index                                                                                                                                                                                                                                   | 0.5 (0.2)         | 0.7 (0.2) §   | 0.16        |       |
+| REM                                                                                                                                                                                                                                                         | LF                | 0.75 (0.04)   | 0.74 (0.04) | 0.89  |
+| HF                                                                                                                                                                                                                                                          | 0.25 (0.04)       | 0.26 (0.04)   | 0.89        |       |
+| LF/HF                                                                                                                                                                                                                                                       | 5.33 (1.05)       | 5.09 (1.29) § | 0.92        |       |
+| REM min                                                                                                                                                                                                                                                     | 90.1 (8.4)        | 91.1 (5.8)    | 0.89        |       |
+|                                                                                                                                                                                                                                                             | %TST              | 20.3 (1.5)    | 21.3 (1.1)  | 0.38  |
+| Respiratory arousal index                                                                                                                                                                                                                                   | 17.3 (1.9)        | 18.2 (1.9) §  | 0.22        |       |
+| Respiratory data                                                                                                                                                                                                                                            | Saturation (mean) | 93.8 (0.4)    | 92.1 (0.5)  | 0.01  |
+| Desaturation (mean)                                                                                                                                                                                                                                         | 8 (1.3) §         | 9.9 (1.3) §   | 0.02        |       |
+| Apnea hypopnea index                                                                                                                                                                                                                                        | 32.3 (3.1)        | 48.0 (5.7) §  | 0.0006      |       |
+| Desaturation index                                                                                                                                                                                                                                          | 18.5 (2.5)        | 30.3 (4.0) §  | 0.002       |       |
+| Mean (standard error). §  Data with nonparametric distribution. Heart rate variability was analyzed in the central five-minute sample of the longest interval  of slow wave sleep (SWS) and REM sleep that was free of stage shifts, artifacts or arousals. |                   |               |             |       |
+
+Table 1 - Analysis of heart rate variability in slow wave sleep and REM sleep. Respiratory sleep parameters after placebo and sildenafil.
+
+## Discussion
+
+This double-blind, cross-over, placebo-controlled study builds on data from our previous research on the respiratory effects of sildenafil in 13 patients with severe OSA.7 The present study examined the impact of sildenafil on HRV during SWS and REM sleep as well as differences in HRV 
+between apnea and respiration resumption. We used shortterm frequency domain analysis throughout the sleeping period and paid special attention to high-frequency and low-frequency ranges. 
+
+Our previous study found that sildenafil was correlated with impaired respiratory parameters during sleep (e.g., mean values of saturation, desaturation, apnea hypopnea index and desaturation index) that were not followed by an increase in the respiratory arousal index.7 This study found that sildenafil was correlated with an increase in HFnu and a decrease in the LF/HF components of HRV during SWS but not during REM sleep. We examined the mean difference in HRV parameters (D) between post-apnea and apnea events and identified a trend toward a reduced modification of LFnu and of the LF/HF components of HRV during REM sleep with sildenafil in comparison to the placebo. 
+
+These findings suggest that, in patients with severe OSA, a single dose of sildenafil (50 mg) before sleep affects sleep HRV, with predominance of parasympathetic upon sympathetic tone. This was evident in SWS, but not in REM, in which greater sympathetic tone typically occurs.12 Furthermore, the trend toward less modification of LFnu and of the LF/HF components of HRV during REM sleep with sildenafil suggests that either the increased LF overshadows the HF increment in this sleep stage or that a ceiling effect occurs in the sympathetic tone that is related to the severity of respiratory events.12, 15, 16 In the evaluation of post-apnea and apnea modification in HRV, the first open breath was excluded because of the typical surge of sympathetic activity triggered by hypoxia or by the activation of carotid and/or aortic chemoreceptors.27 As found in previous studies (see Younes27 for reviews), the amplitude of the modification of HRV parameters had relatively little influence on the number of apnea events in these patients. 
+
+This research is clinically important because abnormal autonomic control is a key factor in the causal link between OSA and cardiovascular disease.14,16 Apnea is associated with increased circulating norepinephrine and with the loss of vagal tone, a combination that may underlie life-threatening arrhythmias.28 Long-term complications, i.e., hypertension, myocardial infarction, and stroke, may result from repeated temporary loss of NO in tissues. This loss of NO is produced by a lack of oxygen, one of the two essential substrates of NO.10 Manipulation of the NO/cyclic GMP system may therefore be a therapeutic option in this circumstance. Our finding that the HF component of HRV increases in severe OSA patients after the use of sildenafil shows that caution must be exercised when changes in HRV spectral indices are interpreted to represent changes in vagal or sympathetic activities.29 Sometimes, estimates of HF do reflect heart rate variability;30 instead, they reflect both non-respiratory sinus arrhythmia and respiratory instability. The emergence of hyperpneic breaths following the termination of apnea can increase estimates of HF, leading to the erroneous interpretation of increased vagal tone during these episodes.31
+
+![4_image_0.png](4_image_0.png)
+
+The influence of sleep stage on respiration and HRV 
+has been reviewed in detail.12 The shift from wakefulness to non-REM sleep is accompanied by a progressive increase in parasympathetic modulation and a decrease in sympathetic modulation of the heart rate. On the other hand, REM sleep, which typically occurs at 90-minute intervals, is associated with significantly greater sympathetic modulation than nonREM sleep. In addition, ventilation and breathing patterns vary according to sleep stage; non-uniform ventilatory patterns can substantially influence LF and HF, particularly in sleep-disordered breathing conditions. High-frequency power is closely associated with the respiratory modulation of heart rate, commonly known as respiratory sinus arrhythmia. Both tidal volume and breathing frequency compromise the reliability of the HF component of HRV as a measure of parasympathetic activity.31 Clinically, OSA is a REM-sleep related disease; obstructive events occur more frequently, for a longer duration of time, and with more desaturation of oxyhemoglobin during REM sleep than non-REM sleep.32 Variable surges in sympathetic activity are associated with a range of arousal events. Arousals can vary from increases in muscular and respiratory activity to sleep stage shifts and full awakening.33 Changes at the cortical level may not be visible in SWS because the magnitude of the autonomic activation is lower than in light and REM sleep. Previous studies reported that around 30% of apneas/hypopneas, particularly in NREM sleep, are not terminated by visible cortical arousals.34 The present study provides evidence that, after administration of sildenafil, either hypoxemia or increased upper airway resistance due to nasal congestion may lead to a lack of arousals during SWS as a response to apneas and hypopneas. The same was not observed in REM sleep due to eased sympatho-vagal balance and arousability.35 Exacerbation of obstruction and an increase in inspiratory effort make upper airway pressure even more negative. An ineffective pharyngeal dilator reflex may also impair arousal and lead to central apnea events in OSA.28 It is also possible that the profound change in respiratory pattern observed in the cases of severe OSA in this study may have had a direct influence on the excitatory drive of peripheral and central chemoreceptors after sildenafil administration. In addition to a direct effect on chemoreceptor afferent input, an indirect action at the level of central respiratory drive may profoundly influence vagal efferent activity.36 The predominant cholinergic control of airway smooth muscle at the level of the neuro-effector junction has recently been reviewed in detail by Jordan36 and the anatomical organization of airway pathways in the brainstem has been reviewed by Mazzone and Canning.37The data presented in this study also raise important questions about the role of NO in the responsiveness of the cerebral vasculature to changes in blood gas tension as well as to sildenafil. NO plays a direct role in obstructive apnea events; during these events, there is an intermittent failure to transport nasal NO to the lungs with each breath and a decrease in NO synthesis due to the lack of oxygen.38 In this context, sildenafil, a PDE-5 inhibitor, potentially impairs NO-dependent protective mechanisms of adaptation to intermittent hypoxia. For example, it may affect the compensatory mechanism that matches perfusion to ventilation and alter efferent pathways that control the activity of pharyngeal dilators and thoracic musculature.10, 39 Furthermore, neuronal and endothelial NO exerts an inhibitory nitrergic influence on both inotropic and chronotropic cardiac responses to adrenergic stimulation.40 NO acts to increase acetylcholine release in cholinergic neurons. Conversely, NO generated in the sympathetic ganglia reduces the release of noradrenaline.41 The results of our study are also supported by the findings of Chowdhary et al.,42 which demonstrated that enhancement of baroreflex gain by NO may underlie the modulation of cardiac vagal control. An increase in HF, but not in LF, was observed with NO-synthetase drug inhibitors and exogenous NO donors.
+
+In addition to the reported involvement of NO in sleep termination, other studies have reported that NO43 has a somnogenic effect in OSA patients (see Gautier-Sauvigné44 for a review of this literature). These studies support our finding that the use of sildenafil is associated with blunted arousal response to respiratory events during sleep. 
+
+Our data on the modification of HRV components between post-apnea and apnea events are heterogeneous; this may highlight the individual pattern of collapsibility of the upper airway and of ventilatory instability. The degree of airway collapsibility and the necessary effort required to open it are inversely proportional to and dependent on the degree of arousal.45 Several methodological considerations must be acknowledged. These include the application of HRV analysis to non-stationary conditions such as sleep in OSA patients. To overcome this difficulty, we used spectral analysis of central 5-minute samples of the longest interval of SWS and REM sleep that was free of stage shifts, artifacts or arousals.23 However, because AHI was high, particularly after the use of sildenafil, it was not possible to obtain fiveminute samples of SWS or REM sleep that were free of apnea or hypopnea events; adjustments for respiration were performed in the spectra of the selected samples.24 This study has several limitations. First, we estimated changes in heart frequency using HRV rather than baroreceptor reactivity. However, it was not feasible to perform recordings of baroreflex or vagal efferent activity which are supposedly impaired during sleep in severe OSA 
+patients. Second, overnight measures of CO2 and the number of central sleep apnea events would provide additional information regarding chemoreceptor influence on HRV parameters. Third, sildenafil is designed to influence penile erection for 2 to 5 hours; this is less time than a full night of sleep. However, little is known about the duration of the other effects of this drug. Further studies of long-term PDE-5 inhibitors such as vardenafil and of daily sildenafil doses are needed to improve our understanding of this group of drugs. Finally, the small sample size of this study raises questions about the applicability of results. The data from the 13 patients, however, show a consistently low ratio of arousals and apnea/hypopnea events during SWS and REM sleep, as well as increased desaturation and lower saturation after the use of sildenafil. Additionally, because all eligible recorded apneas/hypopneas were similarly analyzed, the large amount of data may be representative of the characteristics of the respiratory events.
+
+The lack of increase in the arousal index during SWS 
+may suggest a link between the worsening of OSA and HRV abnormalities after sildenafil. These findings provide evidence of a potential risk of sildenafil use by OSA patients with cardiovascular disease. Our data support Stein et al.'s suggestion that increases in HF estimates do not always reflect better heart rate variability.30 In conclusion, the decrease in arousal response to apnea/
+hypopnea events along with the increase in HF and decrease in LH/HF components of heart rate variability during SWS suggest that, in addition to worsening sleep apnea, sildenafil has potentially immediate cardiac effects in patients with severe obstructive sleep apnea.
+
+## Acknowlegdements
+
+This study was supported by grants of CEPID- FAPESP, 
+Sao Paulo, Brazil and Associação Fundo Incentivo a Psicofarmacologia.
+
+The authors would like to thank Francisca Veloso for her support in all steps of the study.
+
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medical/md/PMC3101051.md

@@ -0,0 +1,225 @@
+# C-Jun Nh2-Terminal Kinase Contributes To Dexmedetomidineinduced Contraction In Isolated Rat Aortic Smooth Muscle
+
+Seong-Ho Ok,1 Young Seok Jeong,1 Jae-Gak Kim,1 Seung-Min Lee,1 Hui-Jin Sung,1 Hye Jung Kim,4 Ki Churl Chang,4 Seong-Chun Kwon,5 and Ju-Tae Sohn2,3 1Department of Anesthesiology and Pain Medicine, Gyeongsang National University Hospital, Jinju; 2Department of Anesthesiology and Pain Medicine, 3Institute of Health Sciences, and 4Department of Pharmacology, Gyeongsang National University School of Medicine, Jinju; 5Department of Physiology, Kwandong University College of Medicine, Kangneung, Korea.
+
+Received: May 25, 2010 Revised: September 18, 2010 Accepted: September 20, 2010 Corresponding author: Dr. Ju-Tae Sohn, Department of Anesthesiology and Pain Medicine, Gyeongsang National University Hospital, Jinju 660-702, Korea. Tel: 82-55-750-8586, Fax: 82-55-750-8142 E-mail: jtsohn@nongae.gsnu.ac.kr ∙ The authors have no financial conflicts of interest. © Copyright: Yonsei University College of Medicine 2011 This is an Open Access article distributed under the terms of the Creative Commons Attribution NonCommercial License (http://creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
+
+Purpose: Dexmedetomidine, a full agonist of α2B-adrenoceptors, is used for analgesia and sedation in the intensive care units. Dexmedetomidine produces an initial 
+
+![0_image_0.png](0_image_0.png)
+
+transient hypertension due to the activation of post-junctional α2B-adrenoceptors on vascular smooth muscle cells (SMCs). The aims of this *in vitro* study were to identify mitogen-activated protein kinase (MAPK) isoforms that are primarily involved in full, α2B-adrenoceptor agonist, dexmedetomidine-induced contraction of isolated rat aortic SMCs. **Materials and Methods:** Rat thoracic aortic rings without endothelium were isolated and suspended for isometric tension recording. Cumulative dexmedetomidine (10-9 to 10-6 M) dose-response curves were generated in the presence or absence of extracellular signal-regulated kinase (ERK) inhibitor PD 98059, p38 MAPK inhibitor SB 203580, *c-Jun* NH2-terminal kinase (JNK) inhibitor SP 600125, L-type calcium channel blocker (verapamil and nifedipine), and α2-adrenoceptor inhibitor atipamezole. Dexmedetomidine-induced phosphorylation of ERK, JNK, and p38 MAPK in rat aortic SMCs was detected using Western blotting. **Results:** SP 
+600125 (10-6 to 10-5 M) attenuated dexmedetomidine-evoked contraction in a concentration-dependent manner, whereas PD 98059 had no effect on dexmedetomidine-induced contraction. SB 203580 (10-5 M) attenuated dexmedetomidine-induced contraction. Dexmedetomidine-evoked contractions were both abolished by atipamezole and attenuated by verapamil and nifedipine. Dexmedetomidine induced phosphorylation of JNK and p38 MAPK in rat aortic SMCs, but did not induce phosphorylation of ERK. **Conclusion:** Dexmedetomidine-induced contraction involves a JNK- and p38 MAPK-mediated pathway downstream of α2-adrenoceptor stimulation in rat aortic SMCs. In addition, dexmedetomidine-induced contractions are primarily dependent on calcium influx via L-type calcium channels.
+
+Key Words: Dexmedetomidine, mitogen-activated protein kinase, α2B-adrenoceptors, hypertension, rat aorta
+
+## Introduction
+
+The highly selective α2-adrenergic agonist dexmedetomidine has a relative α2/α1 selectivity ratio of 1,620, which is 5-10 times greater than that of other compounds 
+(e.g., α2/α1 selectivity ratio: clonidine, 220; UK 14304, 300).1 Dexmedetomidine is a full agonist of α2B-adrenoceptors and a partial agonist of α2A- and α2C-adrenoceptors.2 In humans and mammals, dexmedetomidine produces an initial transient increase in blood pressure after intravenous injection.3-8 In addition, intravenous doses of dexmedetomidine produce transient systemic and coronary vasoconstriction.6-8 Activation of post-junctional α2B-adrenoceptors on vascular smooth muscle is responsible for the initial transient hypertension.9,10 In response to receptor stimulation, Ca2+ is mobilized from intracellular stores and/or the extracellular space to increase cytosolic Ca2+ concentrations in vascular smooth muscle cells (SMCs), leading to the activation of the Ca2+-calmodulin-myosin light chain kinase (MLCK) pathway.11 In addition to increased cytosolic Ca2+-induced contraction, contraction mediated by protein kinases involving Rho-kinase, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and receptor tyrosine kinase is believed to play important roles in agonist-induced contraction.11,12 Such protein kinasemediated contraction leads to the phosphorylation of 20-kDa myosin regulatory light chain (MLC20) and inhibition of myosin light chain phosphatase (MLCP).11,12 MAPKs are serinethreonine protein kinases that consist of three isoforms: extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK).13 A growing body of evidence demonstrates that MAPK pathway activation via G 
+protein-coupled receptors is involved in the modulation of contraction in vascular smooth muscle.14,15 ERK contributes to contractions induced by the partial α2B-adrenoceptor agonist UK 14304 in blood vessles.16,17 As peripheral vasoconstriction in response to α2-agonists primarily involves the α2Badrenoceptor, MAPK isoforms activated by α2-adrenergic agonists may differ for each α2-adrenoceptor agonist, depending on the agonist's affinity for the α2B- adrenoceptor subtype.2,9,10 However, from the best knowledge available to the authors, the MAPK isoform principally involved in the dexmedetomidine-induced contraction in isolated rat aortic SMCs has not been previously identified. The goal of the current *in vitro* study was to identify the MAPK isoform that is mainly involved in the dexmedetomidine-induced contraction of isolated rat aortic SMCs.
+
+## Materials And Methods
+
+All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee at Gyeongsang National University. 
+
+## Preparation Of Aortic Rings For Tension Measurements
+
+Male Sprague Dawley rats weighing 250-350 g each were anesthetized by intraperitoneal administration of pentobarbital sodium (50 mg/kg). The aortic rings for tension measurement were prepared according to the method described in the previous studies.18,19 The descending thoracic aorta was dissected free, and surrounding connective tissues and fat were removed under microscopic guidance while aorta was bathed in Krebs solution composed of following components: 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.4 mM CaCl2, 25 mM NaHCO3, 11 mM 
+glucose, and 0.03 mM EDTA. The aorta was then cut into 2.5-mm rings and suspended on Grass isometric transducers (FT-03, Grass Instrument, Quincy, MA, USA) under a 3.0-g resting tension in a 10-mL Krebs bath at 37°C, aerated continuously with 95% O2 and 5% CO2 to maintain pH 
+values within the 7.35-7.45 range. The rings were equilibrated at a 3.0-g resting tension for 120 min, during which time bathing solution was changed every 30 min. In all aortic rings, the endothelium was intentionally removed from the aortic rings by inserting a 25-gauge needle into the lumen of the rings and gently rubbing the ring for a few seconds. When the contraction in response to phenylephrine 
+(10-7 M) had stabilized, endothelial removal was confirmed by the absence of a relaxation response to acetylcholine (10-5 M). The contractile response evoked by isotonic 30 mM 
+KCl was measured for all aortic rings and used as a reference value. The isotonic 30 mM KCl solution was prepared by replacing the NaCl in the Krebs solution with an equimolar amount of KCl. After washing out the KCl from the organ bath and allowing the isometric tension to return to baseline, a cumulative concentration-response curve to either dexmedetomidine or levomedetomidine was obtained as described below. A single ring was used for each concentration-response curve. The cyclooxygenase inhibitor indomethacin (10-5 M) and the nitric oxide synthase inhibitor NW-nitro-L-arginine methyl ester (L-NAME, 10-5 M) were also included in the Krebs solution to prevent the release of endogenous prostaglandins and nitric oxide, respectively, from any residual endothelium. 
+
+## Experimental Protocol
+
+The first series of experiments assessed the cumulative concentration-response curves to the medetomidine enantiomers dexmedetomidine or levomedetomidine (10-9 to 10-6 M) in the endothelium-denuded rings. A subsequent concentration of dexmedetomidine was added after the previous concentration elicited a sustained and stable contraction for 5 min. 
+
+The second series of experiments was designed to determine which isoform of MAPK is functionally important in mediating dexmedetomidine-induced contraction. The effect of MAPK inhibitors (ERK inhibitor: 10-5 M PD 98059, p38 MAPK inhibitor: 10-6 and 10-5 M SB 203580, JNK inhibitor: 
+10-6, 3×10-6, and 10-5 M SP 600125) on the dexmedetomidine concentration-response curve was assessed by comparing each dexmedetomidine-induced contraction in the presence or absence of each MAPK inhibitor. The incubation period for each MAPK inhibitor was 20 min prior to addition of dexmedetomidine. Inhibitor concentrations were chosen based on the reported 50% inhibitory concentration.12,20 The third series of experiments was designed to examine the effect of dexmedetomidine on the voltage-operated calcium channel-induced contraction. The initial baseline response induced by isotonic 30 mM KCl was recorded in the endothelium-denuded aorta. After washing out KCl from the organ bath and allowing it to return to the baseline resting tension, the second isotonic 30 mM KCl-induced contraction in the same aorta was determined by adding 30 mM 
+KCl solution containing dexmedetomidine (0, 3×10-8, 10-7, 3×10-7, or 10-6 M) to the organ bath.
+
+Finally, the effects of the L-type calcium channel blocker 
+(10-5 M verapamil and 5×10-6 M nifedipine) and the α2adrenoceptor antagonist atipamezole (10-4 M) on the dexmedetomidine concentration-response curve were assessed by comparing cumulative dexmedetomidine concentrationresponse curves in the presence and absence of each inhibitor. Each inhibitor was added to the organ bath 20 min prior to the addition of dexmedetomidine. 
+
+## Cell Culture
+
+The cells were isolated from the rat thoracic aorta by enzymatic dissociation and grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/
+mL penicillin, and 100 μg/mL streptomycin. The cells were subcultured twice a week by harvesting with trypsin/EDTA 
+and seeding in 7.5×105 /mm2 flasks. For experiments, the cells between passage numbers 2 and 10 were seeded into dishes (2 cells/mm2), fed every other day, and used at confluence (6-7 days). The cells were deprived of serum over-
+
+## Night Prior To Treatment. Western Blot Analysis
+
+Western blot analysis was performed according to the method described in the previous study.21 In brief, the cells were lysed in PRO-PREP protein extract solution for total cell lysates, and the lysates were centrifuged at 100,000×g for 20 min at 4°C. Protein concentration was determined using the Bradford method. For preparation of sample loading, equal volumes of 2×sodium dodecyl sulfate (SDS) sample buffer (0.1 mol/L Tris-HCI, 20% glycerol, 4% SDS, and 0.01% bromophenol blue) and supernatant fractions from the lysates were mixed. Proteins (60 microgram) were separated by 10% SDS-polyacrylamide gel electrophoresis for 90 min at 110 V. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes for 2 h at 20 mA using SD Semi-dry Transfer Cells (Bio-Rad Laboratories, Hercules, CA, USA). After blocking the membranes using 5% nonfat milk in Tris-buffed saline (TBS, pH 7.0), 
+the membranes were incubated overnight at 4°C with specific antibodies at a dilution of 1 : 500 in 5% skim milk in TBS-T. Bound antibody was detected by horseradish peroxidase (HRP)-conjugated anti-goat or anti-rabbit IgG. The membranes were washed and developed using a Western Blotting Luminol Reagent System (iNtRON Biotechnology, Houston, TX, USA) and autoradiography. 
+
+## Drugs
+
+All drugs were of the highest purity available commercially: 
+acetylcholine, L-NAME, indomethacin, verapamil, nifedipine, PD 98059, SB 203580, and SP 600125 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dexmedetomidine, levomedetomidine, and atipamezole were kind gifts from Orion Pharma (Turku, Finland). All concentrations are expressed as the final molar concentration in the organ bath. DMEM, FBS, penicillin, streptomycin, trypsin/EDTA, and glutamine were supplied by Gibco BRL (Rockville, MD, 
+USA). PRO-PREP protein extract solution and electrochemiluminescence (ECL) Western blotting detection reagents were supplied by iNtRON Biotechnology (Houston, TX, USA). Anti-phospho-ERK and anti-ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-JNK, anti-JNK, anti-phosphop38 MAPK, and anti-p38 MAPK antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Nifedipine, PD 98059, SB 203580, and SP 600125 were dissolved in dimethyl sulfoxide (DMSO) (final organ bath concentration: 0.1% DMSO) as was indomethacin. Unless stated otherwise, all other drugs were dissolved and diluted in distilled water.
+
+## Data Analysis
+
+Values are expressed as the mean±SD of n rats from which descending thoracic aortic rings were obtained. Contractile responses to dexmedetomidine and levomedetomidine are expressed as a percentage of the maximum contraction to 30 mM isotonic KCl. Maximal contraction was defined as the contractile response to 10-6 M dexmedetomidine. Statistical analysis for the comparison of maximal contractions between the no-drug and inhibitor-treated groups was performed using the Student's t-test for paired samples or a one-way analysis of variance (ANOVA) by Tukey's multiple comparison test. Band intensities from the Western blot analyses were assessed by scanning densitometry, and statistical analysis was performed using the Mann-Whitney U 
+test. Scanning densitometry was performed using an Image Master® VSD (Pharmacia Biotech Inc., San Francisco, CA, 
+USA). The effect of dexmedetomidine on the 30 mM KClinduced contraction was analyzed by an unpaired Student's t-test. Responses to each concentration of dexmedetomidine were compared using repeated measures ANOVA followed by a Tukey's multiple comparison test. A p value of less than 0.05 was considered statistically significant. 
+
+![3_image_0.png](3_image_0.png)
+
+## Results
+
+Dexmedetomidine produced concentration-dependent contractions in endothelium-denuded rings (p<0.001: 10-7 to 10-6 M versus 10-9 M), whereas levomedetomidine produced no contractile response (maximal contraction: p<0.001 versus dexmedetomidine) (Fig. 1). 
+
+The ERK inhibitor PD 98059 (10-5 M), p38 MAPK inhibitor SB 203580 (10-5 M), and JNK inhibitor SP 600125 (10-5 M) did not significantly affect the baseline resting tension. PD 
+98059 (10-5 M) had no effect on dexmedetomidine-induced contraction (Fig. 2A). SB 203580 (10-5 M) attenuated dexmedetomidine-induced maximal contractions (maximal contraction: p<0.001 versus no drug) (Fig. 2B). SP 600125 (10-6, 3×10-6, and 10-5 M) attenuated dexmedetomidine-induced maximal contractions in a concentration-dependent manner 
+(maximal contraction: p<0.001 versus no drug) (Fig. 2C). 
+
+Dexmedetomidine (3×10-8 to 10-6 M) increased 30 mM 
+KCl-induced contraction (3×10-8 and 3×10-7 M: p=0.002 versus no drug; 10-7 M: p=0.001 versus no drug; 10-6 M: 
+p=0.006 versus no drug) (Fig. 3A). The maximum contractile effect of dexmedetomidine in endothelium-denuded rings was markedly attenuated by verapamil (10-5 M) and nifedipine (5×10-6 M) relative to responses in the absence of each inhibitor (maximal contraction: p<0.001 versus no drug) (Fig. 3B, C). Dexmedetomidine-induced contractions were completely abolished by atipamezole (10-4 M) (maximal contraction: p<0.001 versus no drug) (Fig. 3B). 
+
+The above results (Fig. 2) indicate the involvement of JNK 
+and p38 MAPK in dexmedetomidine-induced contractions. 
+
+Thus, we examined whether dexmedetomidine (10-7 and 10-6 M) induces activation of JNK and p38 MAPK pathways in rat aortic SMCs (Fig. 4A). JNK phosphorylation was induced at both 5 minutes and 10 minutes by dexmedetomidine (10-6 M) (Fig. 4A). p38 MAPK phosphorylation was induced at 10, 30, and 60 minutes by dexmedetomidine (10-6 M) (Fig. 
+
+4A). As expected, the ERK1/2 pathway was not activated at 5, 10, 30 and 60 minutes by dexmedetomidine (10-7 or 10-6 M) (Fig. 4A). Fig. 4B shows that dexmedetomidine (10-6 M) 
+treatment induced phosphorylation of p38 and JNK, but not ERK1/2, at 10 minutes compared to control (0 minute). 
+
+## Discussion
+
+Despite the increased use of dexmedetomidine for analge-
+
+![4_image_0.png](4_image_0.png)
+
+![4_image_1.png](4_image_1.png)
+
+respectively). *p=0.002, †p=0.001, ‡p=0.002 and §p=0.006 compared with no drug. (B) Effects of verapamil and atipamezole on dexmedetomidine concentration-response curves in endothelium-denuded rings. Data represent the mean±SD and are expressed as the percentage of the maximal contraction induced by isotonic 30 mM KCl (isotonic 30 mM KCl-induced contraction: 100%=2.37±0.53 g [n=6], 100%=2.64±0.59 g [n=5] and 100%=1.47±0.24 g 
+[n=5] for endothelium-denuded rings with no drug, the verapamil (10-5 M)- 
+and atipamezole (10-4 M)-pretreated endothelium-denuded rings, respectively). Maximal contraction: *p<0.001 compared with no drug. (C) Effects of nifedipine on dexmedetomidine concentration-response curves in endothelium-denuded rings. Data represent the mean±SD and are expressed as the percentage of the maximal contraction induced by isotonic 30 mM KCl (isotonic 30 mM KCl-induced contraction: 100%=2.44±0.51 g [n=6] and 100%=2.45±0.48 g [n=6] for endothelium-denuded rings with no drug and the nifedipine (5×10-6 M)-pretreated endothelium-denuded rings, respectively). Maximal contraction: *p<0.001 compared with no drug. 
+
+Relative density Relative density Relative density
+
+![5_image_0.png](5_image_0.png)
+
+![5_image_1.png](5_image_1.png)
+
+sia and sedation in intensive care units, we believe that the present study is the first to investigate the specific MAPK 
+isoforms involved in dexmedetomidine-induced contractions of rat aortic SMCs.22 The major findings of the present study are as follows: 1) SP 600125 and SB 203580 attenuated dexmedetomidine-induced contractions, whereas PD 
+98059 had no effect on dexmedetomidine-induced contractions; 2) dexmedetomidine induced phosphorylation of JNK and p38 MAPK, whereas dexmedetomidine had no effect on ERK phosphorylation; 3) dexmedetomidine-induced contractions were attenuated by the L-type calcium channel antagonist (verapamil and nifedipine), indicating a role for extracellular Ca2+; these contractions were virtually abolished by a high concentration of the α2-adrenoceptor antagonist atipamezole.
+
+The L-isomer of medetomidine has no effect on hemodynamics.3 In agreement with a previous study by Schmeling, et al.,3 levomedetomidine failed to contract endothelium-denuded rings, suggesting that the effect of dexmedetomidine is stereoselective. 
+
+Protein kinases, including Rho-kinase, PKC, and MAPK, 
+mediate agonist-induced contractions.11,12 Three major MAPK 
+families have been identified (i.e., ERK, JNK, and p38 MAPK) in vascular SMCs.13 Several lines of evidence suggest that the ERK-dependent MAPK pathway is activated by receptor agonists such as phenylephrine, angiotensin II, 
+and 5-hydroxytryptamine, producing contraction of vascular smooth muscle.23-25 PD 98059 has been shown to attenuate contractions in response to UK 14304 in isolated endothelium-denuded rat aorta and porcine palmar lateral veins, suggesting that UK 14304-induced contractions involve ERK pathway activation.16,17 In contrast with previous reports, PD 98059 did not alter the dexmedetomidine concentration-response curve in this study, while dexmedetomidineinduced contractions were attenuated by SP 600125 and SB 
+203580, suggesting that the JNK and p38 MAPK pathways are involved in dexmedetomidine-induced contractions.16,17 In addition, both postsynaptic α2A- and α2B-adrenoceptors are present in rat aortas.26 This finding may be due to differences in α2-adrenoceptor subtype affinities, α2-adrenoceptor subtype distributions, incubation period and α2//α1 selectivity ratios, as well as the fact that the α2B-adrenoceptor is the main α2-adrenoceptor subtype involved in vascular smooth muscle contraction.1,2,9,10,27 However, as SP 600125 also nonspecifically inhibits MLCK, which is involved in vascular smooth muscle contraction, we cannot rule out the possibility that the inhibition of MLCK induced by SP 600125 may attenuate dexmedetomidine-induced contraction.28 Thus, we should be very cautious about interpreting data using SP 
+600125 as inhibitor of JNK. If a potent and specific inhibitor that is selective for JNK can be developed, further study using this selective inhibitor will be needed. 
+
+Dexmedetomidine and clonidine enhance high potassiuminduced contractions in isolated endothelium-denuded vessels.29,30 In accordance with previous studies, dexmedetomidine augmented 30 mM KCl-induced contractions in this in vitro study, suggesting that dexmedetomidine has a synergistic effect on voltage-operated calcium channel-induced contraction.29,30 Verapamil and nifedipine attenuated contractile responses to dexmedetomidine, suggesting that dexmedetomidine-induced contraction involves L-type calcium channels activation. These results indicate that the major signaling mechanism for dexmedetomidine-induced contractions is associated with the Ca2+-calmodulin-MLCK pathway. However, although verapamil attenuated dexmedetomidine-induced contraction, we could not rule out the possibility that the inhibition of α2-adrenoceptors induced by verapamil may attenuate dexmedetomidine-induced contraction because verapamil can also bind to α2-adrenoceptors.31 Another Ltype calcium channel blocker, nifedipine, which does not compete for [3H] yohimbine binding to alpha-2 adrenoceptors, attenuated dexmedetomidine-induced contraction, indicating a significant role for extracellular Ca2+ entry through L-type calcium channels in the calcium-dependent mechanism for dexmedetomidine-induced contraction.31 Dexmedetomidine-induced contractions of rat aortas involve activation of the lipoxygenase pathway.32 Metabolites generated by the lipoxygenase pathway greatly accelerate calcium entry, favoring smooth muscle contraction.33 Taken together, these results suggest that activation of the lipoxygenase pathway involved in dexmedetomidine-induced contractions may be associated with L-type calcium channel-induced contractions in aortic smooth muscle. 
+
+To confirm whether JNK and p38 MAPK mediate dexmedetomidine-induced contractions, Western blot analyses were performed. In agreement with results from isometric tension measurements in this *in vitro* study, dexmedetomidine was shown to induce phosphorylation of both JNK 
+and p38 MAPK in rat aortic SMCs, while dexmedetomidine did not induce phosphorylation of ERK. UK 14304-induced contraction increases ERK phosphorylation in porcine palmar lateral veins.17 Treatment of the renal tubular cell line LLC-PK1-α2B with dexmedetomidine activates ERK via a pathway involving arachidonic acid metabolism.34 Dexmedetomidine is a full agonist of α2B-adrenoceptors, whereas UK 14304 is a partial agonist of α2B-adrenoceptors.2 Thus, this finding may be due to differences in α2-adrenoceptor subtype affinities and species.2 As SP 600125 attenuated dexmedetomidine-induced maximal contractions in a concentration-dependent manner, these results suggest that dexmedetomidine-induced contraction is primarily mediated by activation of a JNK-mediated pathway and, in part, by activation of a p38 MAPK-mediated pathway. There are several cross talk points among the signal transduction pathways of protein kinase-mediated contraction.12 Therefore, it is very difficult to fully elucidate the signaling pathways involving JNK 
+and p38 MAPK that are responsible for dexmedetomidineinduced contractions. 
+
+Dexmedetomidine produces an initial transient increase in blood pressure, followed by a long-lasting reduction in blood pressure in both humans and animals.3-8,35 This initial, transient increase in blood pressure may be ascribed to the time differences between the direct binding of dexmedetomidine on α2B-adrenoceptors of vascular SMCs and the diffusion of dexmedetomidine into the central nervous system to decrease sympathetic activity; these time differences might be unavoidable effects of dexmedetomidine and may be associated with both dosage and intravenous injection speed.4,5 Oral and buccal administration of dexmedetomidine in humans did not produce initial transient hypertension, suggesting that these time differences were eliminated.36,37 In the current study, a dexmedetomidine concentration (10-8 M) that corresponds approximately to the plasma concentration (2×10-9 to 8×10-9 M) of dexmedetomidine used for sedation in humans did not produce any contractile responses, but supraclinical doses (10-7 to 10-6 M) of dexmedetomidine did so.38 However, any clinical implication of dexmedetomidine on regional hemodynamics must be tempered by the fact that the aorta was used in this *in vitro* experiment, whereas organ blood flow is controlled by changes in the diameters of arterioles with diameters less than 150 μm. Despite this limitation, taking into consideration results from previous studies, as well as the current results, these findings suggest that dexmedetomidine-induced JNKand p38 MAPK-mediated contraction may contribute to the initial transient increase in blood pressure after dexmedetomidine infusion.3-8,35 Dexmedetomidine-induced contractions in vascular smooth muscle are primarily attenuated by endothelial nitric oxide produced via stimulation of endothelial α2-adrenoceptors by dexmedetomidine.29,32,39 Taken together, these results suggest that patients, with compromised endothelial integrity, such as those with diabetes, hypertension, and atherosclerosis, may exhibit extremely high blood pressures due to exaggerated dexmedetomidine-induced vasoconstriction. 
+
+In conclusion, these results indicate that contraction induced by dexmedetomidine involves both JNK- and p38 MAPK-mediated pathways downstream of α2-adrenoceptor stimulation in rat aortic SMCs. In addition, dexmedetomidine-induced contractions are primarily dependent on calcium influx via L-type calcium channels.
+
+## Acknowledgements
+
+This paper was supported by Bumsuk Academic Research Fund in 2010.
+
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+30. Hamasaki J, Tsuneyoshi I, Katai R, Hidaka T, Boyle WA, Kanmura Y. Dual alpha (2)-adrenergic agonist and alpha (1)-adrenergic antagonist actions of dexmedetomidine on human isolated endothelium-denuded gastroepiploic arteries. Anesth Analg 2002;94: 
+1434-40.
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+31. Motulsky HJ, Snavely MD, Hughes RJ, Insel PA. Interaction of verapamil and other calcium channel blockers with alpha 1- and alpha 2-adrenergic receptors. Circ Res 1983;52:226-31.
+
+32. Kim HJ, Sohn JT, Jeong YS, Cho MS, Kim HJ, Chang KC, et al. 
+
+Direct effect of dexmedetomidine on rat isolated aorta involves endothelial nitric oxide synthesis and activation of the lipoxygenase pathway. Clin Exp Pharmacol Physiol 2009;36:406-12. 
+
+33. Vanhoutte PM. Calcium-entry blockers, vascular smooth muscle and systemic hypertension. Am J Cardiol 1985;55:17B-23B.
+
+34. Cussac D, Schaak S, Denis C, Paris H. alpha 2B-adrenergic receptor activates MAPK via a pathway involving arachidonic acid metabolism, matrix metalloproteinases, and epidermal growth factor receptor transactivation. J Biol Chem 2002;277:19882-8.
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+35. Shah S, Sangari T, Qasim M, Martin T. Severe hypertension and bradycardia after dexmedetomidine for radiology sedation in a patient with acute transverse myelitis. Paediatr Anaesth 2008;18: 
+681-2.
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+36. Zub D, Berkenbosch JW, Tobias JD. Preliminary experience with oral dexmedetomidine for procedural and anesthetic premedication. Paediatr Anaesth 2005;15:932-8.
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+37. Sakurai Y, Obata T, Odaka A, Terui K, Tamura M, Miyao H. Buccal administration of dexmedetomidine as a preanesthetic in children. J Anesth 2010;24:49-53. 
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medical/md/PMC3314821.md

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+# Statistical Characteristics Of Finger-Tapping Data In Huntington'S Disease
+
+Chrystalina A. Antoniades - Jan Ober •
+Stephen Hicks - Gill Siuda - R. H. S. Carpenter - Christopher Kennard - Andrea H. Nemeth Received: 14 July 2011 / Accepted: 3 January 2012 / Published online: 19 January 2012
+- The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Measuring the rate of finger tapping is a technique commonly used as an indicator of impairment in degenerative neurological conditions, such as Huntington's disease. The information it provides can be greatly enhanced by analysing not simply the overall tapping rate, but also the statistical characteristics of the individual times between each successive response. Recent technological improvements in the recording equipment allow the responses to be analysed extremely quickly, and permit modification of the task in the interest of greater clinical specificity. Here we illustrate its use with some pilot data from a group of manifest HD patients and age-matched controls. Even in this small cohort, differences in the responses are apparent that appear to relate to the severity of the disease as measured by conventional behavioural tests.
+
+C. A. Antoniades (&) - S. Hicks - C. Kennard - A. H. Nemeth Division of Clinical Neurology, Nuffield Department of Clinical Neurosciences, Level 6 West Wing, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK e-mail: Chrystalina.antoniades@clneuro.ox.ac.uk C. A. Antoniades - R. H. S. Carpenter Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK J. Ober Maciej Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland G. Siuda - A. H. Nemeth Dementia and Neurodegenerative Diseases Network, John Radcliffe Hospital, Oxford, UK A. H. Nemeth Department of Clinical Genetics, Churchill Hospital, Oxford, UK
+Keywords Finger tapping - Huntington's disease
+
+## 1 Introduction
+
+Huntington's disease (HD) is a devastating inherited neurodegenerative condition. It is characterised not only by the movement abnormalities, but also by cognitive impairment and abnormal behaviour, as well as weight problems and sleep disturbances [9]. HD patients experience a complex variety of movement problems, which include not only chorea, but also akinesia and bradykinesia. A number of studies have assessed impairment of simple motion sequences in HD patients as a way of quantifying the progression of symptoms [2, 7, 12, 15].
+
+Sequential hand movements in HD patients have been examined at various stages of the condition [17], with markedly slower execution of movements by the HD patients when compared with controls. As well as performing movements more slowly, HD patients are also slower in switching from one movement to the next [1]. Garcia Ruiz and colleagues [7] studied the degree of bradykinesia and timing in genetically confirmed HD individuals compared with controls, using the four CAPIT timed tasks previously used for PD [10]. There were no significant differences between patients with and without anti-dopaminergic drugs. These results have been reproduced in more recent studies that reported significantly reduced tapping rates in manifest HD patients as compared to controls, but not premanifest individuals; Unified Huntington's Disease Rating Scale (UHDRS) motor scores and duration of the disease were highly correlated with the tapping results, something which did not correlate with the CAG repeat lengths [3, 13–15].
+
+Longitudinal studies have shown a significant decline in tapping rate over a period of 3 years in manifest HD
+patients, and a strong correlation between UHDRS scores and the motor tests [2]. Furthermore, the variability of finger tapping (using target intervals of 600 and 1,200 ms) correlated with an index of the probability of motor onset, estimated from CAG length and age [8].
+
+Quick and easy-to-use hand tapping devices have enabled the number of taps in 30 s, and the variability in tapping rhythm and fatigue over the testing period, to be measured. Initial cross-sectional testing of HD patients using an early model of such a device showed that the tapping frequency correlated significantly with the motor UHDRS and independence scores [11]. Longitudinal data from a small cohort followed over 10 years revealed that this correlation was maintained over time, suggesting the technique may provide an objective measure of disease progression. Recently, a large study, TrackHD, used a force transducer to measure paced finger tapping in premanifest and manifest HD patients; significant differences between premanifest and manifest patients were found, again suggesting that finger-tapping measures are an important way of monitoring the progression of the disorder [16].
+
+Here we present a pilot study, using a new portable device in which the sensors are activated purely by contact and are independent of force; this facilitates the rapid collection of finger-tapping data, and allows the possibility of introducing modifications of the basic task that may have diagnostic utility. Because it provides information not merely about average tapping rates, but about the statistical distribution of the individual intervals between responses, it enables the behaviour to be characterised more specifically and quantitatively, sometimes revealing aberrant patterns of response that would not be detected by conventional average measures; this is likely to aid monitoring and diagnosis.
+
+## 2 Materials And Methods 2.1 Participants
+
+The present study was approved by the Local Regional Ethics Committee and was conducted in the John Radcliffe Hospital, Oxford, UK. Participants were recruited from the patients attending the regional HD clinic. All had a positive genetic test for HD, and were evaluated by an experienced clinician (AHN) using a standard neurological examination and the UHDRS to ascertain whether individuals had motor manifestation of Huntington's disease (Table 1). There were two experimental groups: eight in a manifest group (MG) with overt motor signs, and a small aged-matched group of three controls (C). All participants gave their informed consent after the procedures had been explained to them.
+
+| controls (C)                                                       | M (n = 8)     | C (n = 3)    |
+|--------------------------------------------------------------------|---------------|--------------|
+| Age: mean ± SD                                                     | 54.50 ± 10.08 | 54.33 ± 7.81 |
+| CAG repeats                                                        | 42.17 ± 1.80  | N/A          |
+| UHDRS (motor score)                                                | 31.75 (11–81) | N/A          |
+| (mean, range) Total functional capacity                            | 12.00 ± 0.71  | N/A          |
+| VF (mean, range)                                                   | 26.86 (17–45) | N/A          |
+| Hand tapping l (reciprocal median latency) mean ± SE (s-1 ) Single | 4.08 ± 0.60   | 4.95 ± 0.85  |
+| Alternating                                                        | 3.18 ± 0.41   | 4.06 ± 0.53  |
+| Pronating                                                          | 2.14 ± 0.30   | 3.49 ± 0.74  |
+| r (SD of main distribution) mean ± SE (s-1 ) Single                | 0.75 ± 0.14   | 0.35 ± 0.12  |
+| Alternating                                                        | 0.49 ± 0.06   | 0.44 ± 0.21  |
+| Pronating                                                          | 0.41 ± 0.03   | 0.41 ± 0.07  |
+| Except where stated, all figures are mean - 1SE                    |               |              |
+
+## 2.2 Recording The Finger Tapping
+
+The tapping pad (Fig. 1 left: Ober Consulting Poland) has two touch sensors each of 60-mm diameter, their centres 115-mm apart; each has an associated blue LED providing feedback whenever one of the sensor active fields is touched. The sensors are activated purely by contact, independent of force, as they work by monitoring electrical impedance, with a threshold that is automatically adjusted to the background noise level. Left and right taps are recorded independently, at a sampling frequency of 1 kHz
+(resolution 1 ms), for a fixed period of 30s. Participants sat in front of the pad with their forearm resting on the table. Three protocols were used, and in each case the subject used their dominant hand: (1) alternation: the instructions were to tap the left and right sensors alternately as rapidly as possible using the tip of the index finger; (2) pronation/supination: alternately as before and as rapidly as possible, but with the hand in the supine position when tapping medially side, but prone on when tapping laterally, using the finger nail rather than finger tip; (3) single: tapping the right sensor only, with the index finger. For each task, the successive intervals between taps were recorded, generating histograms of the distribution of intervals between consecutive taps. The statistics of these inter-tap intervals for hand tapping was then analysed using the computer application SPIC (saccadic programming and instrumentation computer
+[4]), calculating best-fit parameters (l and r) of the LATER
+(linear approach to threshold with ergodic rate) model [5].
+
+![2_image_0.png](2_image_0.png)
+
+## 3 Results 3.1 Distributions Of Inter-Tap Intervals
+
+Figure 1 (right) shows a typical distribution plot from one of the participants in the study. As usually found for saccadic and evoked manual reaction times, distributions of the intervals between taps in this task are skewed, with a long tail of longer latencies, and in general the reciprocal of reaction time, or promptness, follows a Gaussian distribution (and is therefore more amenable to statistical analysis).
+
+Fig. 2 Examples of unusual
+
+![2_image_1.png](2_image_1.png)
+
+latency distributions. a A subject showing marked bimodality in the alternation task, with one group of responses around 300 ms and another around 450 ms: responses in each direction are very similar. b A subject showing sporadic inattention in the pronation task: in both directions a small group of responses (top right) is markedly delayed relative to the main distribution. c Marked left/ right difference; responses to the right are about 40 ms faster (alternation task). d A manifest HD patient with exceptionally slow responses, affecting all three types of movement, but most marked for the pronation/
+supination task Consequently if inter-tap interval distributions are plotted cumulatively, on a probit scale, using a reciprocal abscissa
+(a reciprobit plot), they will be expected to generate a straight line, as seen on Fig. 1 (right). Such a distribution can be fully described by just two parameters: these are l, its mean (which is also the reciprocal of the median latency),
+and r, its standard deviation. A large value of l corresponds to increased promptness or speed of response, and thus a shorter interval; because of the reciprocal relationship, the units for these two parameters are s-1 or Hz. The best-fit values of the parameters can be determined automatically
+
+![3_image_0.png](3_image_0.png)
+
+by minimisation of the Kolmogorov–Smirnov one-sample statistic.
+
+For most subjects, the observed distribution of reciprocal inter-tap intervals has the expected normal distribution, but in some cases significant, idiosyncratic deviations are observed. Figure 2 shows examples of unusual distributions for individual participants in this study. Figure 2a demonstrates a bimodal pattern of response, possibly analogous to the population of 'express' saccades sometimes seen in saccadic latency distributions, first described by Fischer [6]. A different kinds of abnormality (Fig. 2b) is the existence of a distinct sub-set of aberrantly long intertap intervals (around 600–700 ms), probably due to inattention: in this case they represent only some 5% of all trials. Some participants show marked differences for rightward and leftward movements (Fig. 2c), implying some asymmetry of neurological function. Finally, Fig. 2d shows responses from one manifest HD patient with remarkably long inter-tap intervals, and very marked
+
+![3_image_1.png](3_image_1.png)
+
+differences between the three types of task. An important point to note is that many of these characteristic features would have been entirely missed by the conventional technique of simply counting the total number of taps in a fixed time-period: they are only revealed by quantitative analysis of the distribution of individual intervals.
+
+## 3.2 Values Of The Underlying Parameters
+
+Best-fit values of l and r were estimated for all participants and conditions. Figure 3 shows the average values of the parameters broken down by type of response and category of participant (controls and manifest). When comparing the three different tasks for the HD participants, l is significantly different between the alternation and pronating tasks (p = 0.0011) and between pronating and single
+(p = 0.0024), and r is significantly different for pronating versus alternating (p = 0.045), but no other comparisons are significant at p = 0.05. When comparing the patients with the controls, for all three tasks the patients show the expected reduction in l (equivalent to increased time between taps), but because of the very small group sizes in this pilot study, the only difference seen in Fig. 3, that is significant at p = 0.05 (unpaired t test), is for r with single tapping (p = 0.036).
+
+It is instructive to plot l and r as a scatter plot for the two groups (Fig. 4). What is then seen is an apparent segregation between the manifest patients (M) and the controls; once again, it would obviously be desirable to have more participants than was available for this preliminary evaluation.
+
+Finally, some idea of the test's predictive validity can be seen by looking at the correlation between the observed values of l and r and a more general measure of the patients' neurological status, the total motor score (TMS).
+
+Regression analysis reveals R values that are significant or near-significant for l (Pearson: p = 0.063, 0.008 and 0.065
+(Fig. 5) for alternation, pronation and single tapping, respectively), but not for r.
+
+Fig. 5 Relation between l and r in each of three types of test, and the total motor score, across all manifest HD subjects and controls (the latter are assigned a TMS score of zero)
+
+ 345
+
+![4_image_0.png](4_image_0.png)
+
+## 4 Discussion
+
+The aim of this study was to evaluate the potential use of a new device for quantitative assessments in disorders, such as HD in which hand-tapping tapping is abnormal. It has two key features: first, the force required to be exerted by the subject to register a response is very low (essentially zero); this makes it feasible to use variations on the basic alternation task that can pose a greater challenge to a patient with relatively mild impairment. Figure 2d provides a particularly clear example of this, when comparing ordinary alternation with the pronation task: the difference in l for the two types of task is equivalent to over 450 ms of latency difference. The second potential benefit is that by providing sequential information about individual responses in each direction, a great deal of data are generated in a short period of time, from which much more can be calculated than the average response time that has previously been conventionally used. For simple tapping, the parameter r seems to provide particularly clear discrimination between subject groups (Fig. 3, right), and it is possible that a combination of l and r together may be helpful in this respect (Fig. 4). Because responses in the two directions are not conflated, lateral asymmetries
+(Fig. 2c), suggesting a relatively one-sided functional impairment, become obvious and can be quantified. Other kinds of idiosyncrasies, not previously noted (such as the bimodality of Fig. 2a, or the sporadic inattention of Fig. 2b) are also revealed, and are equally capable of quantification. Another advantage of the device is that it is small, lightweight and portable and the software is easy-touse allowing its potential ease of introduction to a clinical environment.
+
+This is, in other words, a simple tool that has the potential to address a general problem in studying not only in HD, but also other neurodegenerative disorders: a lack of objective and genuinely quantitative neurological tests, by which disease progress can be monitored and treatments evaluated with respect to the particular needs of individual patients. Obviously much more data are needed to provide true validation, and to discover what aspects of the interval distributions will be most useful for this process, and this work is currently in hand.
+
+Acknowledgments This research was supported by a grant from the Wellcome Trust (073735), by the National Institute of Health Research (NIHR), by the Oxford Biomedical Research Centre, and by the Dementias and Neurodegenerative Diseases Research Network
+(DENDRON). Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
+
+## References
+
+1. Agostino R, Berardelli A, Formica A, Accornero N, Manfredi M
+(1992) Sequential arm movements in patients with Parkinson's disease. Huntington's disease and dystonia. Brain 115(Pt 5):
+1481–1495 2. Andrich J, Saft C, Ostholt N, Muller T (2007) Assessment of simple movements and progression of Huntington's disease. J Neurol Neurosurg Psychiatry 78(4):405–407 3. Biglan KM, Ross CA, Langbehn DR, Aylward EH, Stout JC,
+Queller S et al (2009) Motor abnormalities in premanifest persons with Huntington's disease: The PREDICT-HD study. Mov Disord 24(12):1763–1772 4. Carpenter RHS (1994) SPIC: a PC-based system for rapid measurement of saccadic responses. J Physiol 4P:480 5. Carpenter RH, Williams ML (1995) Neural computation of log likelihood in control of saccadic eye movements. Nature 377(6544):59–62 6. Fischer B, Ramsperger E (1984) Human express saccades:
+extremely short reaction times of goal directed eye movements.
+
+Exp Brain Res [Experimentelle Hirnforschung] 57(1):191–195 7. Garcia Ruiz PJ, Hernandez J, Cantarero S, Bartolome M, Sanchez Bernardos V, de Garcia Yebenez J (2002) Bradykinesia in Huntington's disease. A prospective, follow-up study. J Neurol 249(4):437–440 8. Hinton SC, Paulsen JS, Hoffmann RG, Reynolds NC, Zimbelman JL, Rao SM (2007) Motor timing variability increases in preclinical Huntington's disease patients as estimated onset of motor symptoms approaches. J Int Neuropsychol Soc 13(3):539–543 9. Huntington's Disease Collaborative Research Group (1993) A
+novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 72:971– 983 10. Kirkwood SC, Siemers E, Bond C, Conneally PM, Christian JC,
+Foroud T (2000) Confirmation of subtle motor changes among presymptomatic carriers of the Huntington disease gene. Arch Neurol 57(7):1040–1044 11. Michell AW, Goodman AO, Silva AH, Lazic SE, Morton AJ,
+Barker RA (2008) Hand tapping: a simple, reproducible, objective marker of motor dysfunction in Huntington's disease.
+
+J Neurol 225(8):1145–1152 12. Muller T, Schafer S, Kuhn W, Przuntek H (2000) Correlation between tapping and inserting of pegs in Parkinson's disease. Can J Neurol Sci 27(4):311–315 13. Paulsen JS, Langbehn DR, Stout JC, Aylward E, Ross CA, Nance M et al (2007) Detection of Huntington's disease decades before
+diagnosis: the Predict HD study. J Neurol Neurosurg Psychiatry 79(8):874–880 14. Paulsen JS, Hayden M, Stout JC, Langbehn DR, Aylward E, Ross CA et al (2006) Preparing for preventive clinical trials: the Predict-HD study. Arch Neurol 63(6):883–890 15. Saft C, Andrich J, Meisel NM, Przuntek H, Muller T (2006)
+Assessment of simple movements reflects impairment in Huntington's disease. Mov Disord 21(8):1208–1212 16. Tabrizi SJ, Langbehn DR, Leavitt BR, Roos RA, Durr A, Craufurd D et al (2009) Biological and clinical manifestations of Huntington's disease in the longitudinal TRACK-HD study: cross-sectional analysis of baseline data. Lancet Neurol 8(9):791–801 17. Thompson PD, Berardelli A, Rothwell JC, Day BL, Dick JP,
+Benecke R et al (1988) The coexistence of bradykinesia and chorea in Huntington's disease and its implications for theories of basal ganglia control of movement. Brain 111(Pt 2):223–244

medical/md/PMC4075911.md

@@ -0,0 +1,237 @@
+# Original Article Incidence Of Pulmonary Embolism During Copd Exacerbation*,**
+
+Incidência de embolia pulmonar durante exacerbação da DPOC
+
+## Abstract
+
+Objective: Because pulmonary embolism (PE) and COPD exacerbation have similar presentations and symptoms, PE can be overlooked in COPD patients. Our objective was to determine the prevalence of PE during COPD exacerbation and to describe the clinical aspects in COPD patients diagnosed with PE. **Methods:** This was a prospective study conducted at a university hospital in the city of Ankara, Turkey. We included all COPD 
+patients who were hospitalized due to acute exacerbation of COPD between May of 2011 and May of 2013. 
+
+All patients underwent clinical risk assessment, arterial blood gas analysis, chest CT angiography, and Doppler ultrasonography of the lower extremities. In addition, we measured D-dimer levels and N-terminal pro-brain natriuretic peptide (NT-pro-BNP) levels. **Results:** We included 172 patients with COPD. The prevalence of PE 
+was 29.1%. The patients with pleuritic chest pain, lower limb asymmetry, and high NT-pro-BNP levels were more likely to develop PE, as were those who were obese or immobile. Obesity and lower limb asymmetry were independent predictors of PE during COPD exacerbation (OR = 4.97; 95% CI, 1.775-13.931 and OR = 2.329; 95% CI, 1.127-7.105, respectively). **Conclusions:** The prevalence of PE in patients with COPD exacerbation was higher than expected. The association between PE and COPD exacerbation should be considered, especially in patients who are immobile or obese. 
+
+Keywords: Pulmonary disease, chronic obstructive; Pulmonary embolism; Risk factors.
+
+## Resumo
+
+Objetivo: Visto que a embolia pulmonar (EP) e a exacerbação da DPOC têm apresentação e sintomas comuns, o diagnóstico de EP pode ser negligenciado nesses pacientes. Nosso objetivo foi determinar a prevalência de EP durante a exacerbação da DPOC e descrever os aspectos clínicos em portadores de DPOC diagnosticados com EP. **Métodos:** Estudo prospectivo conduzido em um hospital universitário na cidade de Ancara, Turquia. Entre maio de 2011 e maio de 2013, todos os pacientes hospitalizados por exacerbação aguda da DPOC foram incluídos no estudo. Todos os pacientes foram submetidos a avaliação de risco clínico, gasometria arterial, angiotomografia de tórax e ultrassonografia Doppler de membros inferiores. Além disso, foram medidos os níveis de dímero-D e de N-terminal pro-brain natriuretic peptide (NT-pro-BNP). **Resultados:** Foram incluídos 172 pacientes com DPOC. A prevalência de EP foi de 29,1 %. Os pacientes com DPOC e dor torácica pleurítica, assimetria de membros inferiores e altos níveis de NT-pro-BNP, assim como aqueles que estavam obesos ou imobilizados, apresentavam maior probabilidade de desenvolver EP. Obesidade e assimetria de membros inferiores foram preditores independentes de EP nos pacientes com exacerbação da DPOC (OR = 4,97; IC95%, 1,77513,931 e OR = 2,329; IC95% CI, 1,127-7,105, respectivamente). **Conclusões:** A prevalência de EP em pacientes com exacerbação da DPOC foi maior que a esperada. A associação entre EP e exacerbação da DPOC deve ser considerada nesses pacientes, especialmente naqueles imobilizados ou obesos. 
+
+Descritores: Doença pulmonar obstrutiva crônica; Embolia pulmonar; Fatores de risco.
+
+## Introduction
+
+Not only is COPD a significant cause of morbidity worldwide, but it is also the fourth-leading cause of mortality today and is estimated to be the third-leading cause of death by 2020. 
+
+Exacerbations of COPD are the episodic periods of the disease, characterized by deterioration of respiratory function. Most deaths caused by COPD appear to occur during exacerbations. Respiratory infections are responsible for 50-70% of COPD 
+exacerbations, and environmental pollution causes another 10%. Nearly 30% of all COPD 
+exacerbations have unknown etiology.(1,2) Although a meta-analysis found that the prevalence of pulmonary embolism (PE) was 20% among patients who were in a period of exacerbation of COPD,(3)
+that prevalence was found to be 13.7% in a recent study.(4) The prevalence of PE in post-mortem studies ranges from 28% to 51%.(5,6)
+The presentation of common symptoms of COPD exacerbations, such as dyspnea and cough, might cause the diagnosis of acute PE 
+to be overlooked. Patients with COPD are at risk of developing PE due to various reasons, such as immobility, systemic inflammation, and polycythemia. In addition, COPD has been recently defined as an independent risk factor for PE.(7) Mortality and delay in diagnosis of PE are higher in patients with COPD.(8) The prevalence of PE in a highly specific group of patients who were hospitalized for severe exacerbation with unknown origin was reported to be 25%.(9) Gunen et al. reported that venous thromboembolism 
+(VTE) was three times more prevalent in patients with an exacerbation of unknown origin than in patients with an exacerbation of known origin.
+
+(4) The exact prevalence of PE in patients who are in a period of an acute exacerbation of COPD and the clinical features of those patients are as yet unclear. The objective of the present study was to determine the prevalence of PE 
+in patients during COPD exacerbation and to describe the clinical aspects in those patients diagnosed with PE.
+
+## Methods
+
+This was a prospective study conducted in a university hospital in the city of Ankara, Turkey. 
+
+All COPD patients who were hospitalized due to acute exacerbation of COPD between May of 2011 and May of 2013 were included in the study. The study protocol was approved by the Research Ethics Committee of Ufuk University, located in Ankara, Turkey. All participating patients gave written informed consent. Exclusion criteria were having a history of hypersensitivity after the injection of contrast material; having chronic renal disease, pneumonia, or congestive heart failure; having been under anticoagulant treatment; and being unable to give written informed consent because of confusion or dementia. Patients with an inconclusive diagnosis of COPD were also excluded. As a result, a total of 172 patients were included in the study. Figure 1 shows the design of the study.
+
+The diagnosis of COPD was confirmed by medical history and previous medical records 
+(chest X-rays and pulmonary function testing). The severity of COPD was determined by using the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria. Acute exacerbation was diagnosed when the patient with COPD had a worsening in respiratory symptoms beyond the normal day-to-day variations that led to a change in medication.(10)
+Detailed clinical evaluations were performed for all participants by medical history, physical examination, and chest X-rays. Blood samples were taken immediately for the evaluation of D-dimer, blood workup, arterial blood gas analysis, and N-terminal pro-brain natriuretic peptide (NT-pro-BNP) levels. D-dimer levels were measured with the Tina-quant® D-dimer assay system (Boehringer, Mannheim, Germany), which is a particle-enhanced immunoturbidimetric assay. 
+
+We used Roche Elecsys ProBNP assay (Roche Diagnostics, Mannheim, Germany) in order to determine NT-pro-BNP levels.
+
+Risk factors for developing VTE, such as surgery, malignancy, immobility (bed rest > 48 h), and previous VTE, were noted while recording the history of the patient. We used the simplified version of the revised Geneva score,(11) Well's score,(12) and the clinical classification proposed by Miniati et al.(13) in all patients who were included in the study.
+
+Chest CT angiography (CTA) was performed with a 16-section multidetector CT scanner 
+(GE Light Speed 16; GE Healthcare, Milwaukee, Wisconsin, USA) within 24 h of admission. The patients were injected 100 mL of non-ionic contrast media (Iohexol Omnipaque 300/100; GE Healthcare, Milwaukee, WI, USA) via an 18G 
+
+![2_image_0.png](2_image_0.png)
+
+needle into the antecubital vein at a rate of 4 mL/s using a power injector (Medrad Stellant Dual; Medrad, Indianola, PA, USA). Chest CTA was carried out using a dedicated workstation 
+(Advanced Workstation 4.0; GE Healthcare). The diagnosis of PE was reached when an intraluminal filling defect surrounded by intravascular contrast or total occlusion of the pulmonary arterial lumen was detected at any level of the pulmonary arteries. 
+
+The localization of the thrombi was noted.
+
+Doppler ultrasonography of the deep veins of the lower extremities was performed by an experienced radiologist who was blinded about the angiographic results. A standard method was used with a dedicated ultrasound unit (Logiq 7®; 
+GE Healthcare) and a 10L linear array transducer 
+(bandwidth, 6-10 MHz) in order to investigate the presence/absence of intravenous thrombi.
+
+Arterial blood gas analyses were performed with a GEM Premier™ 3000 blood gas/electrolyte analyzer (model 570; Instrumentation Laboratory, Lexington, MA, USA). Interpretation of the results was as follows: acidosis, pH < 7.35; alkalosis, pH > 7.45; hypercapnia, PaCO2
+ > 45 mmHg; hypocapnia, PaCO2
+ < 35 mmHg; and hypoxemia, PaO2
+ < 80 mmHg. Hypoxemia was further graded as mild (60-80 mmHg); moderate (40-59 mmHg); 
+or severe (< 40 mmHg).(14)
+Patients were considered obese with a body mass index (BMI) ≥ 30 kg/m²,(15) whereas a diagnosis of cachexia was given to male patients with a BMI < 16 kg/m2 and to female patients with a BMI < 15 kg/m2.
+
+(16)
+A diagnosis of hypertension was confirmed when blood pressure was ≥ 140/90 mmHg on three separate occasions after hospital admission.(17) A diagnosis of diabetes mellitus was based on the American Diabetes Association criteria.(18) The participants on antihypertensive or antidiabetic treatment were considered to have hypertension or diabetes mellitus. A diagnosis of coronary artery disease was based on previous medical records (echocardiogram and CTA) of the patients. 
+
+Anemia was diagnosed based on hemoglobin levels (≤ 13.5 g/dL in males ≥ 18 years of age and ≤ 12.0 g/dL in females ≥ 18 years of age).(19)
+The data was analyzed using the Statistical Package for the Social Sciences 11.5 pocket program (SPSS Inc., Chicago, IL, USA). Categorical variables were expressed as absolute and relative frequencies, whereas continuous variables were expressed as means, standard deviations, medians, minimum values, and maximum values. The chi-square test was used in order to compare two independent groups of categorical variables. 
+
+The Mann-Whitney U test was used in order to compare two independent groups of continuous variables. Statistical significance was set at a value of p < 0.05. Variables with p < 0.1 in the univariate analysis were evaluated by multiple logistic regression analysis in order to define independent risk factors of outcome variables.
+
+## Results
+
+A total of 172 patients were enrolled in the study. The mean age was 71.31 ± 9.62 years; 142 patients (82.6%) were male, and 30 patients 
+(17.4%) were female. The distribution of patients according to GOLD stages (GOLD I-IV) were as follows: 7.0%, 37.2%, 28.5%, and 27.3%, 
+respectively. Most of the patients (73.8%) had comorbidities accompanying COPD. Demographic properties and basic clinical characteristics of the COPD patients who were included in the study are shown in Table 1.
+
+The prevalence of PE was 29.1%, and all of the patients who had PE based on CTA results also had deep vein thrombosis. The prevalence of PE did not differ between GOLD stages (p > 
+0.05). The localization of thrombi in patients who had PE and the results of the Doppler ultrasonography are shown in Table 2.
+
+The ratios of low probability according to the revised Geneva score,(11) Well's score,(12) and the clinical classification by Miniati et al.(13) in patients who had a confirmed diagnosis of PE 
+was 18%, 24%, and 44%, respectively. The clinical probabilities of the patients who were diagnosed with PE (positive results on CTA) according to the three abovementioned scores are shown in Table 3.
+
+There was no statistically significant difference between the groups of patients with PE and without PE in terms of age, gender distribution, and presence/number of accompanying comorbidities (p > 0.05 for all). The prevalence of obesity was significantly higher among the patients with PE (p = 0.033). The prevalence of other comorbidities (cachexia, hypertension, diabetes mellitus, coronary artery disease, and anemia) was not different between the groups with or without PE (p > 0.05). However, the prevalence of immobility was significantly higher among those with PE (p = 0.024). Risk factors for VTE (trauma, malignancy, surgery, congestive heart failure, or previous history of VTE) did not significantly differ between the two groups (p 
+> 0.05). In addition, the presence of symptoms, such as cough, sputum, hemoptysis, dyspnea, tachycardia, fever, etc., did not significantly differ between the two groups (p > 0.05). Pleuritic chest pain and lower limb asymmetry were significantly more prevalent among those diagnosed with PE 
+(p = 0.038, and p = 0.002, respectively). 
+
+Levels of D-dimers and NT-pro-BNP were significantly higher among the patients with PE 
+than among those without (p < 0.001 vs. p = 
+0.006). Arterial blood gas analyses revealed that the patients with PE had higher pH values and 
+
+| Table 1 - Demographic properties and general clinical  characteristics of the study population.a Variable Result Age, yearsb 71.31 ± 9.62 Gender Male 142 (82.6) Female 30 (17.4) FEV1 , mLb 1,502.82 ± 359.86 FEV1 , % of predictedb 55.8 ± 19.4 mMRCb 1.39 ± 1.02 Exacerbations/yearb 1.26 ± 0.71 GOLD stage I 12 (7.0) II 64 (37.2) III 49 (28.5) IV 47 (27.3) Comorbidities Obesity 24 (13.9) Cachexia 6 (3.5) Hypertension 35 (20.3) Diabetes mellitus 26 (15.1) Coronary artery disease 30 (17.4) Anemia 17 (9.9) mMRC: modified Medical Research Council scale; and GOLD:  Global Initiative for Chronic Obstructive Lung Disease.  a Values expressed as n (%), except where otherwise indicated.  b Values expressed as mean ± SD.   | Table 2 - Localization of the thrombi on chest CT  angiography and Doppler ultrasonography of the  lower limbs in the 172 patients studied.a Localization Patients Chest CT angiographyb Main pulmonary artery 10 (5.8) Segmental 8 (4.7) Subsegmental 32 (18.6) Unilateral 45 (26.2) Bilateral 5 (2.9) Doppler ultrasonographyb Proximal deep vein 10 (5.8) Distal deep vein 18 (10.5) Distal superficial vein 22 (12.8) a Values expressed as n (%). b Thrombi detected in 50 patients  (29.1%).Table 3 - Clinical probabilities of the 50 patients  diagnosed with pulmonary embolism according to  the scoring systems used in the study.a Scoring system Probability Low Moderate High Revised Geneva(11) 9 (18) 32 (64) 9 (18) Well's(12) 12 (24) 34 (68) 4 (8) Miniati et al.(13) 22 (44) 6 (12) 22 (44) a Values expressed as n (%).   |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+lower PaCO2 levels than did those without (p < 
+0.01 and p < 0.05, respectively). The length of hospitalization was longer in the patients with PE than in those without (p = 0.001). However, the three-month mortality rate and the need for intensive care did not differ between the groups (p > 0.05). The clinical and laboratory findings in the patients with and without PE 
+are compared in Table 4.
+
+Multiple logistic regression analysis revealed that obesity and lower limb asymmetry were the independent variables that predicted the presence of PE in the patients with COPD (OR 
+= 4.97; 95% CI, 1.775-13.931 and OR = 2.329; 95% CI, 1.127-7.105, respectively).
+
+## Discussion
+
+The present study showed that PE was present in 29.1% of the patients who were hospitalized due to an exacerbation of COPD. Patients with pleuritic chest pain, lower limb asymmetry, and high NT-pro-BNP levels were more likely to develop PE, as were those who were obese or immobile. Obesity and lower limb asymmetry were independent predictors of PE in the patients with COPD exacerbation.
+
+Most deaths caused by COPD occur during the periods of exacerbation. The course of the disease can be worsened by PE. Because clinical features of PE are nonspecific (e.g., 
+dyspnea and pleuritic chest pain), it might be underdiagnosed in patients with COPD during periods of exacerbation. Visual confirmation of the clot with an imaging technique is required in order to warrant anticoagulation therapy in appropriate dose and duration. The prevalence of PE in COPD exacerbations is not precisely known, but a recent meta-analysis reported that the prevalence of PE in COPD exacerbation with an unknown cause was 20%. The prevalence was higher (24.7%) in four studies that included hospitalized COPD patients.(3) However, TillieLeblond et al. found the prevalence of PE to be 25% in COPD patients with severe exacerbation of 
+
+| Table 4 - Clinical and laboratory characteristics of the patients with and without pulmonary embolism.a Characteristic Pulmonary embolism p Yes No Age, yearsb 72.08 ± 10.89 71.00 ± 9.08 > 0.05 Gender Male 38 (76.0) 104 (85.2) > 0.05 Female 12 (24.0) 18 (14.8) Comorbidities Cachexia 11 (22.0) 12 (9.8) > 0.05 Obesity 5 (4.1) 1 (2.0) 0.033 Hypertension 18 (36.0) 42 (34.4) > 0.05 Diabetes mellitus 13 (26.0) 21 (17.2) > 0.05 Coronary artery disease 20 (40.0) 46 (37.7) > 0.05 Anemia 8 (16.0) 19 (15.6) > 0.05 D-dimer, µg/mLb 2.38 ± 2.80 1.06 ± 1.51 < 0.001 Hematocrit, %b 40.20 ± 6.77 41.70 ± 6.15 > 0.05 Pleuritic chest pain 12 (24.0) 14 (11.5) 0.038 Lower limb asymmetry 11 (22.0) 7 (5.7) 0.002 Hemoptysis 1 (2.0) 1 (0.8) > 0.05 NT-pro-BNP, pg/Lb 1,664 ± 3,247 1,188 ± 3,233 0.006 Arterial blood gasb pH 7.470 ± 0.072 7.400 ± 0.039 < 0.01 PaCO2 , mmHg 34.0 ± 20.0 37.5 ± 10.1 < 0.05 PaO2 , mmHg 57.0 ± 14.9 60.0 ± 13.3 > 0.05 Length of hospital stay, days 11.42 ± 5.69 8.89 ± 4.05 0.001 ICU need 5 (10.0) 5 (4.1) > 0.05 Three-month mortality rate, % 12.0 6.6 > 0.05 NT-pro-BNP: N-terminal pro-brain natriuretic peptide. a Values expressed as n (%), except where otherwise indicated. b Values expressed as mean ± SD.   |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+unknown origin.(9) In our study, we also evaluated the prevalence of PE among hospitalized patients with COPD exacerbation, and it was even higher 
+(29.1%).
+
+Gunen et al. found the prevalence of PE 
+among patients who were hospitalized due to COPD exacerbation to be 13.7%,(4) which was lower than that in our study. They found that being female, having chest pain, having hypotension, and having syncope to be predictors of PE in patients with COPD exacerbation. Our study did not reveal a significant relationship between the genders. Similarly, we found positive relationships of pleuritic chest pain and lower limb asymmetry with the occurrence of PE. 
+
+Gunen et al. found that none of the patients with low-risk determination and 20.7% of those with moderate-risk determination had PE. In contrast, our study showed that, among the patients with PE, 24% had a low clinical probability, and 68% 
+had a moderate clinical probability according to the Well's score. Fernández et al. reported that patients with PE and COPD had a lower pre-test probability for PE than the patients with PE but without COPD.(8) In the present study, a low probability according to all three scoring systems (revised Geneva, Well's and Miniati et al.)(11-13) could not rule out PE in the patients with COPD exacerbation. These results showed that the clinical risk assessment of patients with COPD exacerbation for PE can mislead clinicians. 
+
+The presence of common symptoms in COPD 
+exacerbation and PE might be the reason that causes a high incidence of low-risk probability in these patients. COPD has been recently defined as an independent risk factor for PE.(7) Further studies are necessary to evaluate the inclusion of COPD to the criteria for the clinical risk assessment for PE in order to overcome this problem.
+
+The variation in the prevalence of PE is thought to be the result of differences in study populations and study design. The prevalence of PE in COPD 
+patients who were admitted to the emergency department was reported to be 3.3% in a study.
+
+(20) The low prevalence in that study might result from the evaluation of patients admitted to the emergency department. Additionally, the authors did not further evaluate the COPD patients who did not have a clinical suspicion of PE and had low D-dimer levels (< 0.5 µg/mL). We evaluated all hospitalized patients with COPD exacerbation using imaging methods without considering their clinical probabilities for PE and D-dimer levels. In a recent study by Choi et al., the prevalence of PE among hospitalized patients with COPD 
+exacerbation was distinctly lower than that in our study (5% vs. 29.1%), despite the similarities in the selected population and the design of the two studies.(21) In contrast to their study, however, the location of PE in most of our patients was peripheral. In both studies, NT-pro-BNP levels were significantly higher in COPD patients with PE. Although the length of hospital stay was longer in the COPD patients with PE in our study, that study did not reveal a difference between the two groups in terms of length of hospital stay.
+
+In contrast to the study by Gunen et al., which reported no difference in PaCO2 levels in COPD 
+patients with or without PE,(4) the present study showed that respiratory alkalosis and hypocapnia were more common in patients with COPD 
+exacerbation accompanying PE. Tillie-Leblond et al. found lower PaCO2 levels in patients with COPD exacerbation and PE.(9) Similarly, previous reports showed that a decrease in PaCO2 during COPD exacerbation might indicate PE.(22,23)
+It is known that BNP is released from the heart into the circulation. Levels of BNP increase in patients with congestive heart failure and acute myocardial infarction; BNP is also a marker of right ventricular dysfunction in acute PE(24) and correlates with pulmonary arterial pressure.(25) Gunen et al. 
+
+showed that indicators of acute right heart failure on echocardiogram were more prevalent in COPD 
+patients with PE.(4) In our study, we excluded the patients with congestive heart failure on their initial evaluation. NT-pro-BNP levels were significantly higher in COPD patients with PE than in those without PE. Echocardiograms require expertise and cannot be performed at all medical centers. 
+
+However, NT-pro-BNP measurements might be more accessible for the evaluation of right heart failure and pulmonary arterial pressure in COPD 
+patients without congestive heart failure who are being investigated for PE.
+
+A few symptoms are closely related to the localization of the thrombus in patients with PE. 
+
+Hypotension and syncope were more common symptoms in the study by Gunen et al., which can be explained based on the location of the thrombi 
+(half of the patients with PE had a centrally located thrombus).(4) In the present study, the thrombi in patients with COPD exacerbation and PE were most commonly peripheral (segmental and subsegmental) in 80% of the patients, and pleuritic chest pain was the most common symptom. In our study population, the peripheral location of most of the thrombi might be the reason of the similar mortality rates in the COPD patients with and without PE. However, it is important to detect and properly treat a peripherally located thrombus in order to prevent recurrence, which might cause death.
+
+Our study had some limitations. Although this was the largest study to evaluate the prevalence and the characteristics of patients with COPD 
+exacerbation associated with PE, it was a single center study. In addition, the present study only investigated the prevalence of PE and the clinical conditions in which PE was suspected in the patients with COPD exacerbation.
+
+In conclusion, the prevalence of PE in the patients with COPD exacerbation was higher than expected in our study. Exacerbation of COPD and PE can be associated. Because of that, PE should be considered in patients with COPD exacerbation, especially in those who are immobile, have pleuritic chest pain, and present high D-dimer, NT-pro-BNP, and pH levels, as well as presenting low PaCO2 levels. Obesity and lower limb asymmetry were independent predictors of the presence of PE in our patients. Larger studies are necessary in order to evaluate the prevalence of PE in patients with COPD 
+exacerbation in a more precise fashion and to provide understanding about the factors and mechanisms that influence the development of PE in this population.
+
+## References
+
+1. Sapey E, Stockley RA. COPD exacerbations . 2: Aetiology. 
+
+Thorax. 2006;61(3):250-8. http://dx.doi.org/10.1136/
+thx.2005.041822 2. Seneff MG, Wagner DP, Wagner RP, Zimmerman JE, 
+Knaus WA. Hospital and 1-year survival of patients admitted to intensive care units with acute exacerbation of chronic obstructive pulmonary disease. JAMA. 1995;274(23):1852-7. http://dx.doi.org/10.1001/
+jama.1995.03530230038027 3. Rizkallah J, Man SF, Sin DD. Prevalence of pulmonary embolism in acute exacerbations of COPD: A systematic review and metanalysis. Chest. 2009;135(3):786-93. 
+
+http://dx.doi.org/10.1378/chest.08-1516 4. Gunen H, Gulbas G, In E, Yetkin O, Hacievliyagil SS. 
+
+Venous thromboemboli and exacerbations of COPD. 
+
+Eur Respir J. 2010;35(6):1243-8. http://dx.doi.
+
+org/10.1183/09031936.00120909 5. Baum GL, Fisher FD. The relationship of fatal pulmonary insufficiency with cor pulmonale, rightsided mural thrombi and pulmonary emboli: a preliminary report. Am J Med Sci. 1960;240:609-12.
+
+6. Mitchell RS, Silvers GW, Dart GA, Petty TL, Vincent TN, 
+Ryan SF, et al. Clinical and morphologic correlations in chronic airway obstruction. Aspen Emphysema Conf. 
+
+1968;9:109-23.
+
+7. Poulsen SH, Noer I, Møller JE, Knudsen TE, Frandsen JL. 
+
+Clinical outcome of patients with suspected pulmonary embolism. A follow-up study of 588 consecutive patients. 
+
+J Intern Med. 2001;250(2):137-43. http://dx.doi.
+
+org/10.1046/j.1365-2796.2001.00866.x 8. Fernández C, Jiménez D, De Miguel J, Martí D, Díaz G, 
+Sueiro A. Chronic obstructive pulmonary disease in patients with acute symptomatic pulmonary embolism [Article in Spanish]. Arch Bronconeumol. 2009;45(6):286-90. http://dx.doi.org/10.1016/j.arbres.2008.10.008 9. Tillie-Leblond I, Marquette CH, Perez T, Scherpereel A, Zanetti C, Tonnel AB, et al. Pulmonary embolism in patients with unexplained exacerbation of chronic obstructive pulmonary disease: prevalence and risk factors. Ann Intern Med. 2006;144(6):390-6. http://
+dx.doi.org/10.7326/0003-4819-144-6-200603210-00005 10. Global Initiative for Chronic Obstructive Lung Disease 
+- GOLD [homepage on the Internet]. Bethesda: Global Initiative for Chronic Obstructive Lung Disease [cited 2013 Aug 26]. Global Strategy for the Diagnosis, Management, and Prevention of COPD - Revised 2011. [Adobe Acrobat document, 90p.]. Available from: http://www.goldcopd.org/.
+
+11. Klok FA, Mos IC, Nijkeuter M, Righini M, Perrier A, Le Gal G, et al. Simplification of the revised Geneva score for assessing clinical probability of pulmonary embolism. 
+
+Arch Intern Med. 2008;168(19):2131-6. http://dx.doi. org/10.1001/archinte.168.19.2131 12. Wells PS, Owen C, Doucette S, Ferguson D, Tran H. 
+
+Does this patient have deep vein thrombosis? JAMA. 
+
+2006;295(2):199-207. http://dx.doi.org/10.1001/
+jama.295.2.199 13. Miniati M, Prediletto R, Formichi B, Marini C, Di Ricco G, Tonelli L, et al. Accuracy of clinical assessment in the diagnosis of pulmonary embolism. Am J Respir Crit Care Med. 1999;159(3):864-71. http://dx.doi.org/10.1164/ ajrccm.159.3.9806130 14. Rose BD, TW Post. Clinical physiology of acid-base and electrolyte disorders. 5th ed. New York: McGraw Hill; 2001.
+
+15. NHLBI Obesity Education Initiative Expert Panel on the Identification, Evaluation, and Treatment of Obesity in Adults. Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults: 
+The Evidence Report. Bethesda: National Heart, Lung, and Blood Institute; 1998.
+
+16. Schols AM, Broekhuizen R, Weling-Scheepers CA, Wouters EF. Body composition and mortality in chronic obstructive pulmonary disease. Am J Clin Nutr. 2005;82(1):53-9.
+
+17. Carretero OA, Oparil S. Essential hypertension. Part I: 
+definition and etiology. Circulation. 2000; 101(3):32935. http://dx.doi.org/10.1161/01.CIR.101.3.329 18. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2010;33 Suppl 1:S62-9. 
+
+http://dx.doi.org/10.2337/dc10-S062 19. White CT, Barrett BJ, Madore F, Moist LM, Klarenbach SW, Foley RN, et al. Clinical practice guidelines for evaluation of anemia. Kidney Int Suppl. 2008;(110):S4-6. 
+
+http://dx.doi.org/10.1038/ki.2008.268
+
+20. Rutschmann OT, Cornuz J, Poletti PA, Bridevaux PO, 
+Hugli OW, Qanadli SD, et al. Should pulmonary embolism be suspected in exacerbation of chronic obstructive pulmonary disease? Thorax. 2007;62(2):121-5. http:// dx.doi.org/10.1136/thx.2006.065557 21. Choi KJ, Cha SI, Shin KM, Lee J, Hwangbo Y, Yoo SS, et al. Prevalence and predictors of pulmonary embolism in Korean patients with exacerbation of chronic obstructive pulmonary disease. Respiration. 2013;85(3):203-9. http://
+dx.doi.org/10.1159/000335904 22. Lippmann M, Fein A. Pulmonary embolism in the patient with chronic obstructive pulmonary disease. A 
+diagnostic dilemma. Chest. 1981;79(1):39-42. http://
+dx.doi.org/10.1378/chest.79.1.39 23. Rodger MA, Jones G, Rasuli P, Raymond F, Djunaedi H, 
+Bredeson CN, et al. Steady-state end-tidal alveolar dead space fraction and D-dimer: bedside tests to exclude pulmonary embolism. Chest. 2001;120(1):115-9. http://
+dx.doi.org/10.1378/chest.120.1.115 24. Klok FA, Mos IC, Huisman MV. Brain-type natriuretic peptide levels in the prediction of adverse outcome in patients with pulmonary embolism: a systematic review and meta-analysis. Am J Respir Crit Care Med. 
+
+2008;178(4):425-30. http://dx.doi.org/10.1164/
+rccm.200803-459OC
+25. Yetkin Ö, In E, Aksoy Y, Hacıevliyagil SS, Günen H. Brain natriuretic peptide in acute pulmonary embolism: its association with pulmonary artery pressure and oxygen saturations. Turkish Resp J. 2006;7(3):105-8.
+
+## About The Authors
+
+Evrim Eylem Akpınar MD. Department of Chest Diseases, Ufuk University School of Medicine, Ankara, Turkey.
+
+Derya Hosgün MD. Department of Chest Diseases, Ufuk University School of Medicine, Ankara, Turkey.
+
+Serdar Akpınar MD. Atatürk Chest Diseases and Chest Surgery Training Hospital, Ankara, Turkey.
+
+Gökçe Kaan Ataç MD. Department of Radiology, Ufuk University School of Medicine, Ankara, Turkey.
+
+Beyza Doganay PhD. Department of Biostatistics, Ankara University School of Medicine, Ankara, Turkey.
+
+Meral Gülhan Chief. Department of Chest Diseases School of Medicine, Ufuk University, Ankara, Turkey.

medical/md/PMC4859320.md

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+
+## Depression And Hiv Risk Among Men Who Have Sex With Men In Tanzania
+
+Hycienth Ahanekua, Michael W. Rossb, Joyce E. Nyonic, Beatrice Selwyna, Catherine Troisid, Jessie Mbwamboe, Adeniyi Adeboyef and Sheryl McCurdyf aDepartment of Epidemiology, Human Genetics and Environmental Sciences, The University of Texas School of Public Health, Houston, TX, USA; bDepartment of Family Medicine and Community Health, University of Minnesota, Minneapolis, MN, USA; cDepartment of Sociology and Anthropology, University of Dar es Salaam, Dar es Salaam, Tanzania; dDepartment of Management, Policy and Community Health, The University of Texas School of Public Health, Houston, TX, USA; eDepartment of Psychiatry, Muhimbili University of Health Sciences, Dar es Salaam, Tanzania; fDepartment of Health Promotion and Behavioral Sciences, The University of Texas School of Public Health, Houston, TX, USA
+ABSTRACT
+Studies have shown high rates of depression among men who have sex with men (MSM) in developed countries. Studies have also shown association between depression and HIV risk among MSM. However, very little research has been done on depression among African MSM. We assessed depression and HIV risk among a sample of MSM in Tanzania. We reviewed data on 205 MSM who were recruited from two Tanzanian cities using the respondent driven sampling method. Demographic and behavioral data were collected using a structured questionnaire. HIV and sexually transmitted infections data were determined from biological tests. Depression scores were assessed using the Patient Health Questionnaire (PHQ-9). For the analysis, depression scores were dichotomized as depressed (PHQ > 4) and not depressed (PHQ ≤ 4).
+
+Bivariate and multivariable Poisson regression analyses were conducted to assess factors associated with depression. The prevalence of depression in the sample was 46.3%. The mean (±SD) age of the sample was 25 (±5) years. In bivariate analysis, depression was associated with self-identifying as gay (p = .001), being HIV positive (p < .001: <8% of MSM knew they were HIV infected) and having a high number of sexual partners in the last 6 months (p = .001). Depression was also associated with sexual (p = .007), physical (p = .003) and verbal (p < .001) abuse. In the Poisson regression analysis, depression was associated with verbal abuse (APR =
+1.91, CI = 1.30–2.81). Depression rates were high among MSM in Tanzania. It is also associated with abuse, HIV and HIV risk behaviors. Thus, reducing the risk of depression may be helpful in reducing the risk of HIV among MSM in Africa. We recommend the colocation of mental health and HIV preventive services as a cost-effective means of addressing both depression and HIV risk among MSM in Africa.
+
+## Introduction
+
+Depression is a leading cause of morbidity and disability in the world (Moussavi et al., 2007; Murray, Lopez, Mathers, & Stein, 2001). The prevalence of depression in the general population ranges from 0.3% in Czech Republic to 10% in the USA (Andrade, Caraveo-Anduaga, Berglund, Bijl, & Dragomericka, 2003). Research, particularly in high income countries, has shown higher rates of depression and mental health disorders among men who have sex with men (MSM). In the Netherlands, MSM were three times more likely to be depressed compared to heterosexual men (Sandfort, de Graaf, Bijl, &
+Schnabel, 2001). In the USA, several studies report high rates of depression among MSM - 33% in Massachusetts
+(Reisner et al., 2009), 23.1% in Chicago (Fendrich, Avci, Johnson, & Mackesy-Amiti, 2013) and 22.8% in Seattle (Perdue, Hagan, Thiede, & Valleroy, 2003). Reviews of other studies showed rates of depression that were three to five times higher among MSM compared to the general adult male population (Cochran & Mays, 2000; Lhomond & Saurel-Cubizolles, 2009; Pratt & Brody, 2008).
+
+Depression among MSM has further public health implications because of its association with risky sexual behavior and HIV infection. In one study, MSM who engage in unprotected anal intercourse were 10 times more likely to be depressed compared to those who do not engage in unprotected anal intercourse (Reisner et al., 2009). In another study, MSM with high depression scores were five times more likely to be depressed than those with low depression scores. In their study of MSM in Seattle, CONTACT Hycienth Ahaneku hycpet@yahoo.com Department of Epidemiology, Human Genetics and Environmental Sciences, The University of Texas School of Public Health, Houston, TX, USA
+ARTICLE HISTORY
+Received 10 September 2015 Accepted 20 January 2016 KEYWORDS
+Men who have sex with men; HIV; depression; Africa; Tanzania Perdue et al. (2003) reported a significant association between high depression scores and multiple sexual partners (≥3) in the preceding six months. Given the high rates of depression among MSM, the association with risky sexual behavior is cause for concern (Fendrich et al., 2013; Perdue et al., 2003; Reisner et al., 2009).
+
+While there has been considerable research on depression among MSM in high income countries, there is little research on depression among MSM in Africa. To our knowledge only two studies (both in South Africa) have researched depression among African MSM (Stoloff et al., 2013; Tucker et al., 2013). Furthermore, MSM in Africa face both cultural and civil hostility as a result of their sexual orientation and identity (Baral et al., 2009; Semugoma, Nemande, & Baral, 2012). Furthermore, over 30 countries in Africa, including Tanzania have civil or criminal laws against MSM behavior
+(Itaborahy, 2012). This situation may contribute to the risk of HIV infection, through a cycle of internalized hostility, depression and HIV risk behavior. Thus, there is need for more research to assess depression among MSM in Africa. The objectives of our study are: (1) to determine the prevalence of depression among a sample of MSM in Tanzania; (2) to determine the association between depression and HIV in the sample; (3) to determine other factors associated with depression in the sample. We hypothesize that depression will be higher among HIV-positive men than HIV-negative men.
+
+## Methods Study Design And Sampling
+
+Using a cross-sectional epidemiologic design, data were collected between 2012 and 2013 from two cities in Tanzania: a large metropolitan city (Dar es Salaam) and a small provincial city (Tanga). Dar es Salaam (DES) is the capital city of Tanzania and is a large metropolitan city with over four million inhabitants. Tanga is a smaller city in the northeastern part of the country with a population of about 275,000 people. Two hundred MSM and 100 MSM were recruited from DES and Tanga, respectively. Before recruitment we mapped several MSM sites in each city including hotels, parks, clubs and bars
+(Ross, Nyoni, Bowen, Williams, & Kashiha, 2012). Starting with initial "seeds" of five people in each city, the respondent driven sampling (RDS) method was used to recruit subjects into the study (Heckathorn, 1997, 2002). RDS is a chain-referral sampling method that starts with initial seeds of recruiters who recruit subjects into a study. These newly recruited subjects then recruits a limited number of other MSM into the study. This process continues in recruitment "waves" until the desired sample size is reached. MSM behavior is both illegal and a social stigma in Tanzania (Itaborahy, 2012). The RDS method allows us to reach such a closed community with less difficulty. Subjects were eligible for the study if they were male, 18 years or older, could provide informed consent for the study and indicated they have had sex with another male within six months preceding data collection. Seeds were chosen from three different regions of each city, and covering three decades of age
+(20s–40s). A more detailed account of the recruitment process is referenced elsewhere (Ross et al., 2014).
+
+## Data Collection And Procedure And Study Approval
+
+All research activities were managed by research supervisors and assistants with experience and training in MSM
+field research. The participant self-administered the questionnaire using a pencil and paper form. If the participant had any difficulty with any question or item, the research assistant explained the item/question in a manner consistent with the item's meaning. After the participant was done, the research assistant checked the questionnaire for completeness and consistency of the data while the participant was still present. Any inconsistencies and missing data were clarified with the respondents. Meetings with each participant were conducted in private store fronts/offices rented for the project.
+
+Our study is a secondary study of data collected on a primary study to assess HIV, risk behavior and stigma among MSM in Tanzania (Ross et al., 2014). The study was approved by the Institutional Review Boards of the University of Texas Health Science Center at Houston and the Tanzanian National Institute for Medical Research.
+
+## Dependent (Outcome) Variable
+
+Depression, our main outcome variable, was measured using the Patient Health Questionnaire - 9 scale
+(PHQ-9). The PHQ-9 is a validated nine- item instrument that scores each of the nine DSM-IV depressive symptoms between 0 (not present at all) and 3 (present nearly every day) (Adewuya, Ola, & Afolabi, 2006; Kroenke, Spitzer, & Williams, 2001; Monahan et al., 2009; Ola et al., 2006). Thus, scores ranges between 0 and 27. For our study, depression was dichotomized into: No depression (0–4) and depression (>4)
+(Monahan et al., 2009). A cut of point of four was used because this is the threshold for at least mild depression (Kroenke et al., 2001; Kroenke & Spitzer, 2009). Additionally, since this is a population-based study, using a cut-off point of four increases the sensitivity of the tool to capture all respondents with any level of depression irrespective of the severity. The PHQ-9 has been validated in East African populations (Monahan et al., 2009).
+
+## Independent Variables
+
+The independent variables for this study fall into five main domains: (1) Demographics; (2) Sexual behavior;
+(3) MSM network size; (4) Violence/abuse and Internalized homonegativity (IH); (5) Sexual Diseases (HIV status, diagnosis of sexually transmitted infection (STI),
+ever been tested for HIV). See Table 1 for variables in each domain.
+
+IH is the acceptance and internalization of negative attitudes toward one's own sexual orientation (Shidlo, 1994).
+
+| Table 1. Results of bivariate analysis between depression and variables in each domain. Not depressed Depressed                                                                                                    | Total   |         |        |        |           |           |             |         |
+|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------|---------|--------|--------|-----------|-----------|-------------|---------|
+| Demographics                                                                                                                                                                                                       | PRa     | 95% CIb |        |        |           |           |             |         |
+| N                                                                                                                                                                                                                  | %       | N       | %      | N      | %         | P-value   |             |         |
+| City                                                                                                                                                                                                               | 110     | 95      | 205    | 1.23   | 0.86–1.73 | 0.215     |             |         |
+| Dar es Salaam (DES)                                                                                                                                                                                                | 71      | 50.7%   | 69     | 49.3%  | 140       | 68.3%     |             |         |
+| Tangac                                                                                                                                                                                                             | 39      | 60.0%   | 26     | 40.0%  | 65        | 31.7%     |             |         |
+| Currently employed                                                                                                                                                                                                 | 110     | 95      | 205    | 1.01   | 0.69–1.49 | 0.957     |             |         |
+| Yes                                                                                                                                                                                                                | 90      | 53.6%   | 78     | 46.4%  | 168       | 82.0%     |             |         |
+| Noc                                                                                                                                                                                                                | 20      | 54.1%   | 17     | 45.9%  | 37        | 18.0%     |             |         |
+| Self-identified sexual orientation                                                                                                                                                                                 | 106     | 94      | 200    | 1.90   | 1.24–2.91 | 0.001     |             |         |
+| Gay/homosexual                                                                                                                                                                                                     | 64      | 45.4%   | 77     | 54.6%  | 141       | 70.5%     |             |         |
+| Bisexualc                                                                                                                                                                                                          | 42      | 71.2%   | 17     | 28.8%  | 59        | 29.5%     |             |         |
+| Education                                                                                                                                                                                                          | 110     | 95      | 205    | 0.82   | 0.59–1.15 | 0.239     |             |         |
+| Primary school or less                                                                                                                                                                                             | 41      | 59.4%   | 28     | 40.6%  | 69        | 33.7%     |             |         |
+| Sec school or morec                                                                                                                                                                                                | 69      | 50.7%   | 67     | 49.3%  | 136       | 66.3%     |             |         |
+| Ever had a meaningful sexual relationship with a female                                                                                                                                                            | 110     | 95      | 205    | 0.73   | 0.55–0.97 | 0.038     |             |         |
+| Yes                                                                                                                                                                                                                | 81      | 58.7%   | 57     | 41.3%  | 138       | 67.3%     |             |         |
+| Noc                                                                                                                                                                                                                | 29      | 43.3%   | 38     | 56.7%  | 67        | 32.7%     |             |         |
+| Age                                                                                                                                                                                                                | 23.99d  | 5.27e   | 24.74d | 5.3e   | 24.71d    | 5.21e     | 24.00–25.42 | 0.314   |
+| Abuse and stigma Ever been a victim of sexual abuse                                                                                                                                                                | 110     | 95      | 205    | 1.53   | 1.16–2.04 | 0.007     |             |         |
+| Yes                                                                                                                                                                                                                | 19      | 37.3%   | 32     | 62.7%  | 51        | 24.9%     |             |         |
+| Noc                                                                                                                                                                                                                | 91      | 59.1%   | 63     | 40.9%  | 154       | 75.1%     |             |         |
+| Ever been a victim of physical abuse                                                                                                                                                                               | 110     | 95      | 205    | 1.58   | 1.20–2.10 | 0.003     |             |         |
+| Yes                                                                                                                                                                                                                | 21      | 36.8%   | 36     | 63.2%  | 57        | 27.8%     |             |         |
+| Noc                                                                                                                                                                                                                | 89      | 60.1%   | 59     | 39.9%  | 148       | 72.2%     |             |         |
+| Ever been a victim of verbal abuse                                                                                                                                                                                 | 110     | 95      | 205    | 1.95   | 1.44–2.63 | <0.001    |             |         |
+| Yes                                                                                                                                                                                                                | 31      | 35.6%   | 56     | 64.4%  | 87        | 42.4%     |             |         |
+| Noc                                                                                                                                                                                                                | 79      | 66.9%   | 39     | 33.1%  | 118       | 57.6%     |             |         |
+| IH                                                                                                                                                                                                                 | 23.25d  | 8.19e   | 21.77d | 7.52e  | 23.03d    | 7.87e     | 21.95–24.12 | 0.184   |
+| Sexual behavior Used condom with most recent transactional sex                                                                                                                                                     | 97      | 89      | 186    | 1.35   | 0.95–1.93 | 0.069     |             |         |
+| Did not use condom                                                                                                                                                                                                 | 59      | 47.6%   | 65     | 52.4%  | 124       | 66.7%     |             |         |
+| Used condomc                                                                                                                                                                                                       | 38      | 61.3%   | 24     | 38.7%  | 62        | 33.3%     |             |         |
+| Condom use in receptive anal sex with last three partners                                                                                                                                                          | 110     | 95      | 205    | 100.0% | 2.31      | 1.68–3.18 | <0.001      |         |
+| Did not use condom                                                                                                                                                                                                 | 30      | 32.6%   | 62     | 67.4%  | 92        | 44.9%     |             |         |
+| Used condomc                                                                                                                                                                                                       | 80      | 70.8%   | 33     | 29.2%  | 113       | 55.1%     |             |         |
+| Condom use in insertive anal sex with last three partners                                                                                                                                                          | 110     | 95      | 205    | 100.0% | 0.61      | 0.39–0.98 | 0.02        |         |
+| Did not use condom                                                                                                                                                                                                 | 31      | 68.9%   | 14     | 31.1%  | 45        | 22.0%     |             |         |
+| Used condomc                                                                                                                                                                                                       | 79      | 49.4%   | 81     | 50.6%  | 160       | 78.0%     |             |         |
+| Number of people you have had sex with in past 6 months                                                                                                                                                            | 4.97d   | 5.18e   | 10.68d | 16.27e | 7.86d     | 11.02e    | 6.59–9.13   | 0.001   |
+| Sexual diseases HIV status                                                                                                                                                                                         | 97      | 79      | 176    | 1.84   | 1.36–2.48 | <0.001    |             |         |
+| Positive                                                                                                                                                                                                           | 14      | 31.8%   | 30     | 68.2%  | 44        | 25.0%     |             |         |
+| Negativec                                                                                                                                                                                                          | 83      | 62.9%   | 49     | 37.1%  | 132       | 75.0%     |             |         |
+| Current diagnosis of STI                                                                                                                                                                                           | 104     | 86      | 190    | 1.26   | 0.88–1.79 | 0.235     |             |         |
+| Positive                                                                                                                                                                                                           | 16      | 45.7%   | 19     | 54.3%  | 35        | 18.4%     |             |         |
+| Negativec                                                                                                                                                                                                          | 88      | 56.8%   | 67     | 43.2%  | 155       | 81.6%     |             |         |
+| Ever been tested for HIV                                                                                                                                                                                           | 108     | 93      | 201    | 0.93   | 0.66–1.33 | 0.703     |             |         |
+| Yes                                                                                                                                                                                                                | 86      | 54.4%   | 72     | 45.6%  | 158       | 78.6%     |             |         |
+| Noc                                                                                                                                                                                                                | 22      | 51.2%   | 21     | 48.8%  | 43        | 21.4%     |             |         |
+| MSM network size                                                                                                                                                                                                   | Mean    | SD      | Mean   | SD     | Mean      | SD        | 95% CIb     | P-value |
+| How many gay men over 15 years whom you know by name                                                                                                                                                               | 13.66   | 15.99   | 19.33  | 21.31  | 15.45     | 17.73     | 13.02–17.88 | 0.039   |
+| How many of these have you seen in the past one month                                                                                                                                                              | 7.53    | 9.81    | 14.38  | 17.47  | 10.54     | 13.63     | 8.67–12.41  | 0.001   |
+| How many gays do you consider as close friends                                                                                                                                                                     | 3.41    | 3.62    | 4.36   | 7.00   | 3.68      | 4.86      | 3.01–4.35   | 0.236   |
+| a PR, prevalence ratio. b 95% confidence interval. c The reference category for computing the prevalence ratio. The prevalence ratio is computed by dividing the prevalence of depression in the main group by the |         |         |        |        |           |           |             |         |
+
+IH was measured using an 8-item short version of the 28-item Reactions to Homosexuality Scale (Ross & Rosser, 1996; Smolenski, Diamond, Ross, & Simon Rosser, 2010). The 8-item short version is formatted for a 6-point Likert type response from 0 (strongly disagree) to 5 (strongly agree). Thus, the scores ranged between 0 and 40.
+
+HIV status was based on result of tests carried out on blood samples provided by the subjects. HIV status was assessed using two methods: Determine, Abbot Laboratories, USA for initial testing; and Unigold, Trinity Biotech PLC, Ireland for confirmation of the initial test.
+
+These tests require minimal infrastructure and allows for quick turnaround time for results. Tests were initially done using the Abbot Determine test and confirmed with the Unigold test. In the event of any discordant results, the result of the Unigold test was adopted.
+
+Validation studies done in five African countries showed greater than 98% sensitivity and specificity for these tests (Piwowar-Manning et al., 2010). Voluntary counseling and testing for HIV was offered to all participants (12% of the participants declined being tested)
+and all HIV-positive men were referred to the HIV center in Dar es Salaam or the Bombo regional Hospital in Tanga.
+
+STI was diagnosed as positive if the participant tested positive for syphilis, Chlamydia or gonorrhea. The respondents provided blood samplesfor the syphilis test and urine and anal swab samples for the Chlamydia and gonorrhea tests. Syphilis antibody was tested using DetermineTP Rapid Syphilis Assay (Inverness Medical Innovations, MA, USA). Chlamydia and gonorrhea were tested using APTIMA Combo2 (Hologic Gen-Probe, CA, USA).
+
+## Power And Sample Size Estimation
+
+Based on past research studies, a priori estimates of depression prevalence were: 36% among HIV-positive MSM and 16% among HIV-negative MSM (Kelly et al., 1998; Rosenberger et al., 1993; Stoloff et al., 2013; Tucker et al., 2013).Thus, assuming an 80%
+power and a two-sided significance level of .05, we needed a minimum of 150 people to detect a difference of 20% points in depression rates between HIV-positive and HIV-negative participants. However, considering the RDS method, we factored in a design effect of 1.5 for our study (Johnston et al., 2010; Salganik, 2006).
+
+Thus, the final minimum sample size for this study was 225 subjects. However, due to missing data, we only had 205 subjects with complete data for depression. With this number of subjects, we had 80% power to detect a 21% point difference in depression prevalence between HIV-positive and HIV-negative subjects.
+
+## Data Analysis
+
+Simple descriptive statistics were conducted for each variable. Bivariate analysis (chi-square tests and t-tests) were performed to assess relationship between depression and each independent variable. Variables with p-values ≤.200 were subsequently used for a Poisson multivariable regression analysis.
+
+A Poisson regression model with robust variance estimation was used to assess association between depression and the independent variables (Barros & Hirakata, 2003). Poisson regression is commonly used to assess discrete outcomes in longitudinal studies (Singer & Willett, 2003). To adapt this regression method to our cross-sectional data, we assigned a follow-up time of one year to every participant. Thus, the offset variable was set at log (1) which is zero. The effect sizes from this Poisson model will thus yield prevalence ratios which appropriately reflect the cross-sectional design of our study. Using a robust estimation method will yield confidence intervals that are more precise than using standard variance estimation methods (Barros & Hirakata, 2003).
+
+All analyses were done using SPSS 21 and were based on two-sided significance level of .05.
+
+## Results
+
+A total of 300 MSM participated in the study. However, we only had depression data on 205 participants. Thus, results presented here are based on the 205 participants with depression data. The prevalence of depression among our sample was 46.3% (95/205).
+
+The mean age and standard deviation of the sample was 24.7 years and 5.2 years, respectively. Most of the participants indicated they were gay/homosexual (71%) and had at least a secondary school education (66%).
+
+Those who identified as gay/homosexual were twice as likely to be depressed compared to bisexuals (PR = 1.9, CI = 1.24–2.91). However, those who have had a meaningful sexual relationship with a female were significantly less likely to be depressed than those who have never had a meaningful sexual relationship with a female (PR =
+0.73, CI = 0.54–0.97). Most respondents reported not experiencing any form of sexual abuse (75%), physical abuse (72%) or verbal abuse (58%). Depression was significantly more prevalent in those who have experienced sexual abuse (PR = 1.53, CI = 1.16–2.04), physical abuse (PR = 1.58, CI = 1.20–2.10) and verbal abuse (PR = 1.95, CI = 1.44–2.63) compared to those who have not experienced each of these, respectively. Depression was significantly more prevalent among those who did not use a condom in receptive anal sex with all of their last
+
+| Table 2. Multiple poisson regression showing factors associated with depression as the dependent variable. Poisson regression with robust estimators APRa LCLb                                                                                                                                                                                                                   | UCLc   | P-value   |      |       |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------|-----------|------|-------|
+| How many gay men that you know have you seen in the past one month                                                                                                                                                                                                                                                                                                               | 1.01   | 1.00      | 1.02 | 0.06  |
+| No of different men you have had sex with in the past 6 months                                                                                                                                                                                                                                                                                                                   | 1.01   | 1.00      | 1.01 | 0.141 |
+| IH                                                                                                                                                                                                                                                                                                                                                                               | 1.01   | 1.00      | 1.04 | 0.32  |
+| Ever had a meaningful sexual relationship with a woman (Yes vs. Nod )                                                                                                                                                                                                                                                                                                            | 1.00   | 0.67      | 1.25 | 0.579 |
+| Ever been a victim of verbal abuse (Yes vs. Nod )                                                                                                                                                                                                                                                                                                                                | 1.91   | 1.30      | 2.81 | 0.001 |
+| Ever been a victim of sexual abuse (Yes vs. Nod )                                                                                                                                                                                                                                                                                                                                | 1.11   | 0.82      | 1.52 | 0.501 |
+| HIV status (Positive vs. Negatived )                                                                                                                                                                                                                                                                                                                                             | 1.33   | 1.00      | 1.86 | 0.102 |
+| Self-identified sexual relationship (Homosexual vs. bisexuald )                                                                                                                                                                                                                                                                                                                  | 1.15   | 0.67      | 1.97 | 0.623 |
+| Used condom with most recent transactional partner (Did not use condom vs. Used condomd )                                                                                                                                                                                                                                                                                        | 1.34   | 0.91      | 1.96 | 0.134 |
+| Used condom in receptive anal sex with last three partners (Did not use condom vs. Used condomd )                                                                                                                                                                                                                                                                                | 1.33   | 0.88      | 2.01 | 0.177 |
+| Used condom in insertive anal sex with last three partners (Did not use condom vs. Used condomd )                                                                                                                                                                                                                                                                                | 1.05   | 0.61      | 1.81 | 0.86  |
+| a APR = adjusted Prevalence Ratio. Each variable was adjusted for all the other variables in the table. b LCL = 95% lower confidence level. c UCL = 95% upper confidence level. d Reference category for each variable. For example, those who have ever experienced verbal abuse were 1.9 times more likely to be depressed than those who have never experienced verbal abuse. |        |           |      |       |
+
+three partners (PR = 2.31, CI = 1.68–3.18) compared to those who used. The mean number of sexual partners within the last 6 months was significantly higher for the depressed participants than non-depressed participants (10.68 vs. 4.97, p = .001). With regard to MSM network size, those who were depressed had higher indices of network size than those who were not depressed.
+
+Those who were depressed were significantly more likely to have seen more gay people who they know within the last one month (14.38 vs. 7.53, p = .001).
+
+Further analysis on MSM network size (not shown in table) reveal that gay/homosexual men had significantly larger network size compared to bisexual men (13 vs. 7, p < .001). Furthermore, those who did not use condom in receptive anal sex with their last three partners had significantly larger network size (15 vs. 7, p < .001) than those who used condom. Network size was significantly associated with number of sexual partners in the last six months (rho = 0.260, p < .001).
+
+Depression was significantly more prevalent among HIV-positive MSM compared to HIV-negative MSM
+(PR = 1.84, CI = 1.36–2.48).
+
+Of all the variables in our Poisson regression model
+(Table 2), only one variable was significantly associated with depression. Those who have ever experienced verbal abuse had about twice the prevalence of depression compared to those that have never had verbal abuse (APR =
+1.91, CI: 1.29–2.81).
+
+## Discussion
+
+Our study is one of very few studies that have assessed depression among MSM in Africa. We found a 46.3% prevalence of depression among our study sample. Our result is similar to the 44% and 56% prevalence reported in two other studies in South Africa (Stoloff et al., 2013; Tucker et al., 2013). We note that these three studies report depression prevalence that are higher than those reported for MSM in high income countries. Studies among MSM in the USA and Australia report prevalence of depression ranging from 22.8% to 33% (Fendrich et al., 2013; Mao et al., 2009; Perdue et al., 2003; Reisner et al., 2009). We also note that some of the non-African studies used instruments that were different from our study. It is unlikely that the large difference in depression prevalence could have been entirely due to differences in instruments used. In fact, one of the South African studies used instruments similar to the studies in the USA and reported prevalence that were higher than those from the USA.
+
+So what could be responsible for the high prevalence of depression among African MSM? Anti-gay laws, cultural stigma and human right abuses toward MSM may be contributory factors here (Itaborahy, 2012; Mayer et al. 2013). In the multivariate analysis, those who have ever experienced verbal abuse had significantly higher prevalence of depression compared to those who have never experienced verbal abuse (see Table 2).
+
+Additionally, 25%, 28% and 42% of the participants reported ever experiencing sexual, physical and verbal abuse, respectively. These abuses are likely related to their sexual orientation (Anderson, Ross, Nyoni, & McCurdy, 2015).Other African studies have reported similarly high prevalence of human right abuses ranging from 26% in Botswana to 60% in Lesotho (Baral et al., 2009, 2011, 2013).
+
+Depression among African MSM presents a major concern for two reasons. One, MSM represents a high risk population for HIV. Two, Africa carries a greater burden of HIV compared to other regions of the world. In the bivariate analysis, being HIV positive, having a high number of sexual partners and engaging in unprotected receptive anal intercourse with the last three partners were associated with depression (see Table 1). Our data support findings from other studies that have found associations between depression and risky sexual behavior or HIV infection (Fendrich et al., 2013; Perdue et al., 2003; Reisner et al., 2009; Tucker et al., 2013, 2014). Knowledge of HIV status cannot entirely explain the association between HIV and depression in our data since only five of the 64 HIV-positive men (7.8%) were aware of their status. The association may be mediated by risky sexual behavior. Further exploration of risky sexual behavior as a mediating factor between depression and HIV may be needed.
+
+In regard to MSM Network size, bivariate analysis showed that network size was associated with self-identifying as homosexual (as opposed to bisexual), nonuse of condom in receptive anal sex and having a large number of sexual partners in the last six months. These results suggest that a large network size may be fostering an environment for more casual relationships among MSM and engaging in risky sexual behavior which is associated with depression (see discussion above). Thus, those MSM with larger networks may be viable targets for public health interventions aimed at delivering HIV prevention and mental health services.
+
+## Limitations
+
+Our study has a few limitations including large number of MSM with no depression data (although further analysis did not show any significant difference between those with depression data and those missing depression data with respect to age, education, HIV status or city of residence), inability to make causal inferences on depression (because of the cross-sectional design) and reporting and recall bias. We also note that our findings may not be generalizable to older "closeted" MSM who were outside the primary network and cannot be reached by the recruitment process (RDS). Additionally, information bias could have resulted from research assistants influencing response of the research participants in their bid to clarify questions on the questionnaire. However, we do not envisage these limitations would have any significant impact our conclusions since we used structured and well validated instruments for our study (Adewuya et al., 2006; Monahan et al., 2009; Ross et al., 2010). Moreover, the research assistants received training on research techniques on preventing undue influence on participants' responses.
+
+## Conclusion
+
+In summary, the study showed a high prevalence of depression and its association with abuse, sexual risk behaviors and HIV infection. The high prevalence of depression as well as its positive association with HIV risk behaviors among our study sample demonstrates the need for assessing and addressing mental health needs among MSM. Researchers have acknowledged this complex interaction between HIV infection, high risk sexual behavior, stigma/abuse and depression and have suggested the co-location of mental health services with HIV services as a cost-effective means of addressing mental health among MSM (Fendrich et al., 2013; Stoloff et al., 2013).
+
+## Disclosure Statement
+
+No potential conflict of interest was reported by the authors.
+
+## Funding
+
+This work was supported by US National Institute of Mental Health [grant number 5R21MH090908] (M. W. R.).
+
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+1097/QAI.0b013e318298700e Monahan, P. O., Shacham, E., Reece, M., Kroenke, K., Ong'or, W. O., Omollo, O., … Ojwang, C. (2009). Validity/reliability of PHQ-9 and PHQ-2 depression scales among adults living with HIV/AIDS in Western Kenya. Journal of General Internal Medicine, 24(2), 189–197. doi:10.1007/s11606008-0846-z Moussavi, S., Chatterji, S., Verdes, E., Tandon, A., Patel, V., &
+Ustun, B. (2007). Depression, chronic diseases, and decrements in health: Results from the World Health Surveys. The Lancet, 370, 851–858. doi:10.1016/S01406736(07)61415-9 Murray, C. J. L., Lopez, A. D., Mathers, C. D., & Stein, C.
+
+(2001). The global burden of disease 2000 project: Aims, methods and data sources. Geneva: WHO. Retrieved from: http://www.who.int/healthinfo/paper36.pdf Ola, B. A., Adewuya, A. O., Ajayi, O. E., Akintomide, A. O.,
+Oginni, O. O., & Ologun, Y. A. (2006). Relationship between depression and quality of life in Nigerian outpatients with heart failure. Journal of Psychosomatic Research, 61, 797–800. doi:10.1016/j.jpsychores.2006.04.022 Perdue, T., Hagan, H., Thiede, H., & Valleroy, L. (2003).
+
+Depression and HIV risk behavior among Seattle-area injection drug users and young men who have sex with men.
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+AIDS Education and Prevention, 15 (1), 81–92. doi:10.
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+1521/aeap.15.1.81.23842 Piwowar-Manning, E., Tustin, N., Sikateyo, P., Kamwendo, D.,
+Chipungu, C., Maharaj, R., … Jackson, J. B. (2010).
+
+Validation of rapid HIV antibody tests in five African countries. Journal of the International Association of Physicians in AIDS Care (Chicago, Ill. : 2002), 9(3), 170–
+172. doi:10.1177/1545109710368151 Pratt, L. A., & Brody, D. J. (2008). Depression in the United States household population, 2005–2006. NCHS Data Brief, (7): 1–8. Retrieved from http://www.cdc.gov/nchs/
+data/databriefs/db07.pdf Reisner, S. L., Mimiaga, M. J., Skeer, M., Bright, D., Cranston, K., Isenberg, D., … Mayer, K. H. (2009). Clinically significant depressive symptoms as a risk factor for HIV infection among black MSM in Massachusetts. AIDS and Behavior, 13(4), 798–810. doi:10.1007/s10461-009-9571-9 Rosenberger, P. H., Bornstein, R. A., Nasrallah, H. A., Para, M.
+
+F., Whitaker, C. C., Fass, R. J., & Rice, R. R. (1993).
+
+Psychopathology in human immunodeficiency virus infection: Lifetime and current assessment. Comprehensive Psychiatry, 34 (3), 150–158. doi:10.1016/0010-440X(93)
+90041-2 Ross, M. W., Nyoni, J., Ahaneku, H. O., Mbwambo, J.,
+McClelland, R. S., & McCurdy, S. A. (2014). High HIV seroprevalence, rectal STIs and risky sexual behaviour in men who have sex with men in Dar es Salaam and Tanga, Tanzania. BMJ Open, 4(8), e006175. doi:10.1136/bmjopen2014-006175 Ross, M. W., Nyoni, J., Bowen, A. M., Williams, M. L., &
+Kashiha, J. J. (2012). Sexual and geographic organisation of men who have sex with men in a large East African city: Opportunities for outreach. BMJ Open, 2(6), e001813. doi:10.1136/bmjopen-2012-001813 Ross, M. W., & Rosser, B. R. S. (1996). Measurement and correlates of internalized homophobia: A factor analytic study.
+
+Journal of Clinical Psychology, 52, 15–21. doi:10.1002/(SICI)
+1097-4679(199601)52:1 < 15::AID-JCLP2>3.0.CO;2-V
+Ross, M. W., Smolenski, D. J., Kajubi, P., Mandel, J. S.,
+McFarland, W., & Raymond, H. F. (2010). Measurement of internalized homonegativity in gay and bisexual men in Uganda: Cross-cultural properties of the Internalized Homonegativity Scale. Psychology, Health and Medicine, 15, 159–165. doi:10.1080/13548500903527746 Salganik, M. J. (2006). Variance estimation, design effects, and sample size calculations for respondent-driven sampling. Journal of Urban Health : Bulletin of the New York Academy of Medicine, 83(Suppl. 1), 98–112. doi:10.1007/
+s11524-006-9106-x Sandfort, T. G., de Graaf, R., Bijl, R. V., & Schnabel, P. (2001).
+
+Same-sex sexual behavior and psychiatric disorders: Findings from the Netherlands mental health survey and incidence study (NEMESIS). Archives of General Psychiatry, 58 (1): 85–91. doi:10.1001/archpsyc.58.1.85 Semugoma, P., Nemande, S., & Baral, S. D. (2012). The irony of homophobia in Africa. The Lancet, 380(9839), 312–314.
+
+doi:10.1016/S0140-6736(12)60901-5 Shidlo, A. (1994). Internalized homophobia: Conceptual and empirical issues in measurement. In B. Greene & G. M. Herek (Eds.), Lesbian and gay psychology: Theory, research, and clinical practice (Vol. 1, pp. 176–205). Thousand Oaks, CA: Sage.
+
+Singer, J. D., & Willett, J. B. (2003). Applied longitudinal data analysis: Modeling change and event occurrence. New York, NY: Oxford University Press.
+
+Smolenski, D. J., Diamond, P. M., Ross, M. W., & Simon Rosser, B. R. (2010). Revision, criterion validity, and multi-group assessment of the reactions to homosexuality scale. Journal of Personality Assessment, 92(6), 568–576.
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+doi:10.1080/00223891.2010.513300 Stoloff, K., Joska, J. A., Feast, D., De Swardt, G., Hugo, J.,
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+09540121.2013.764383 Tucker, A., Liht, J., de Swardt, G., Jobson, G., Rebe, K., McIntyre, J., & Struthers, H. (2014). Homophobic stigma, depression, self-efficacy and unprotected anal intercourse for peri-urban township men who have sex with men in Cape Town, South Africa: A cross-sectional association model. AIDS Care, 26
+(7), 882–889. doi:10.1080/09540121.2013.859652

medical/md/PMC5266882.md

@@ -0,0 +1,135 @@
+II.- -THE LATEST WORK ON DYSPEPSIA. 
+
+By A. Lockhart Gillespie, M.B., M.R.C.P.E., Medical Registrar, Edinburgh Royal Infirmary. 
+
+Science is rapidly replacing empiricism in the treatment of gastric disorders, and every month sees some new procedure for gaining new and more accurate knowledge of the processes which go on in that viscus in health or disease. Not only are new methods advertised, but clinical evidence also accumulates with regard to those which have reached, what we might call, the age of puberty. 
+
+The different tests devised by Ewald and others to demonstrate both the motility and digestive power of the stomach have lately attracted a good deal of attention, both in this country and abroad. 
+
+As salol splits into phenol and salicylic acid only in alkaline or neutral media, and as it is not absorbed until this action has taken place, Ewald and Sievus proposed to make use of the drug as an indicator of the length of time during which food remained in the stomach. Under normal circumstances, they found that salicylic acid could be found in the urine in from three-quarters of an hour to one hour and a quarter. They concluded, however, that to perform the test properly, the salol should only be given when the stomach contents have become markedly acid,?that is, an hour or an hour and a half after meals. It was soon found clinically, however ? Kullmann (l),1 Brunner (2), Decker (3), Leo (4), 
+Pal (5), and Kodczjewski (6)?that although in cases of dilated stomach, especially where the pylorus was much contracted, the salicylic acid did not appear for a considerable time, yet the varia^ 
+tions in health were so great as to preclude any useful deductions. 
+
+More particular statements as to the reactions in health and disease were made by Wolff (12), who found the normal limit-to be an hour and a half; and by Hiiberlin (13), who found the reaction delayed in 87 per cent, of all cancer cases examined by him. 
+
+But others?Huber (7), Bourget (8), Silberstein (9), Ochmen (10) 
+?in working at the subject, found that all traces of the salol disappear from the urine in health in twenty-four to twenty-seven hours, while in cases of enlargement of the stomach or enfeeble-ment of its walls, forty to sixty-six hours may elapse before the disappearance occurs. 
+
+These clinical observations, curiously enough, are in direcf antagonism to some experimental work lately chronicled. In th last edition of his great work on Digestion, Ewald (11) mentior that there is a very considerable mass of evidence to show that th contents of the small intestine are acid throughout, and this h&c been corroborated by Macfadyen in some observations made on the 1 Figures within parentheses refer to bibliography at end of article. 
+
+contents of the ileum in man. The acidity is due to acetic and lactic acids; and it can easily be proved that salol is prevented from splitting into carbolic and salicylic acids by a solution of acetic acid. 
+
+Klemperer's oil method may just be mentioned to be dismissed, but several well-known observers have recommended the use of iodide of potassium in the same way as that in which Ewald advised the use of salol. 
+
+Known generally as G-iinzberg's test, it has apparently been independently worked out by Sahli (14) in Berne, and by Margan in France. The main idea of the process is, to judge by the length of time which elapses between the ingestion of iodide of potash and its appearance in the saliva. The salt is enclosed in a caoutchouc tube stoppered with fibrin, each writer on the subject having a favourite form. The time taken in digesting the fibrin determines the appearance of the iodide in the saliva, dependent, of course, on the absorptive power of the stomach. 
+
+Penzoldt and Faber (15) also used iodide of potash enclosed in gelatine, to test the absorptive power simply. Glinzberg's test has recently been investigated by Drs Symons Eccles (16) and Tolcher Eccles (17),?indeed, their investigations are not yet completed, but, as far as they have gone, they consider the test of value. 
+
+They first examine the activity of absorptio7i of the stomach by neans of tincture of rhubarb, which, as has been shown by Lauder 3runton, soon appears in the urine, and may be detected in that uid by its reaction when liquor potass? is added. Then they use iinzberg's capsule to estimate the digestive power. The normal active period for the latter they give as seventy-eight minutes. 
+
+)me practical observations on these methods will also be found 
+>ted in some cases of dilated stomach narrated by Dr Cutler (18). 
+
+In the hope of getting rid of some of the inconveniences which ise from the use of the stomach-tube, Dr Einhorn, in 1890, introced a 
+" stomach-bucket," which is, in fact, a silver bucket, hollow, the shape of the bulb of a probang, and with an open top. This icket is swallowed and recovered again by means of a silk thread Cached to it. It generally contains sufficient of the stomach intents to test for free HC1. A good description of it will be mnd in the Dublin Journal for February last by Dr H. C. 
+
+weedy (19). The advantages claimed for it are, that it is more mple and easy to use than the tube, that it is a great advantage 
+-> the general practitioner, and that there is no danger attendant i its use. 
+
+It does not appear that this method has any advantages over the ube, for if a tube with a very soft rubber end or nozzle, however tiff the rest of it may be, be used, cases suffering from cancer or leer may be examined with ease and without danger. 
+
+Dr Soltan Fenwick, writing on this subject, imputes all sorts f dangers to the process of washing out the stomach?convulsions EDIN. MED. JOUHN. XXXVIII.?2. p and tetany, syncope and sudden death, perforation and haemorrhage, injury and poisoning, may in his opinion result from it. The con-vulsions, he says, it is true, may only be set up by the irritation of the passage of the tube, the existing dilated stomach accounting for the tendency. But he is on surer ground when he speaks of the dangers which may occur in cases of gastric ulcer, if strong expulsive efforts are made by the patient during the passage of the tube. He recommends a more discriminating employment of lavage. 
+
+On the other hand, Dr Harvey Attfield (20) reports most excellent results in the treatment of chronic gastric affections by lavage; while Prof. Chas. G. Stockton (21), and H. M. Fisher, M.D. 
+
+(28), also recommend it. 
+
+Dr Einhorn (22) has invented an electrode for use in the stomach of much the same form as his " stomach-bucket," the sill* thread in this case being replaced by a soft wire clothed in indiarubber. The circuit, is as usual completed by introducing somt water into the stomach, and placing the other electrode on the epigastric region. The author claims to have increased the acidity of the contents by this means, and to have diminished the absorp-tion period for the chemical tests, as Ewald has claimed before him. Wolff (24), however, categorically denies this, observing, however, that the current may be of use in dilating oesophageal strictures (!), and in restoring tone to the walls of the stomach ir atonic ectasis; while Dr Stockton (23) speaks enthusiastically c its use in all cases in which the motility of the stomach walls weakened. Cases with marked gastric catarrh do best with t! 
+
+continuous current, the anode being used within. 
+
+Gastric ulcer has, as usual, come in for a considerable amount discussion. Dreschfeld (25), in the Medical Chronicle for last ye gave a full description of our later knowledge, while Henry (2 last February, in a paper on this subject, pointed out that f hyperacidity found in such cases is a relative term. In one cl of patients, for instance in chlorotic women, a normal acidity is oft hyperacid to the stomach walls. He points out well, too, tl injury to the stomach walls, if the acidity present be relative normal, will not produce an ulcer; but if hydrochloric acid solutic 5 per cent, say, be injected into the viscus, " the loss of substanc instead of rapidly healing, would degenerate into a typical rom ulcer." 
+The latest note on this subject is that of Weir's (27), in t" New International Medical Magazine, who discusses some cases laparotomy for perforating round ulcer of the stomach, and givv some history of the operation. 
+
+The effect which the presence of albuminoids has on the reaction and properties of free HC1 is attracting more attention. Hayej and Winter's (29, 30) method, as published in their book, late, reviewed in this Journal, has provoked considerable criticism. 
+
+Bouveret and Magnien (31), and Wagner (32) of St Petersburg, have published notes on the process. The first two named think it cumbrous and little suited for its purpose; the last has a better opinion of it, but thinks it needs further corroboration. 
+
+Rosenheim (33) has written fully on the effect albuminoids and amido-acids have on HC1, and took occasion to doubt the results obtained by Salkowski and Kumagawa (34). This led Salkowski 
+(35) to repeat his experiments with ainido-acids, and to modify his opinions concerning them. His latest research shows that the presence of amido-acids during peptic digestion only exerts a slight malign influence. 
+
+The micro-organisms of the stomach have not been troubled much lately; the only note on the subject is a paper by E. Hirschfeld (36), on the effect of an artificial gastric juice on acetic and actic fermentation, in which he shows that HC1 alone is a more powerful germicide than HC1 plus pepsine. 
+
+The only other thing of note is a pretty little quarrel which has een going on in the pages of Pfluger's Archiv between Prof. 
+
+Wagner on the one hand, and C. Friedheim and H. Leo on the 
+.her, about the merits of Leo's calcium carbonate test for HC1. ? 
+
+1. Kullmann.?Inaugural Dissertation, Giessen, 1888. 
+
+2. Brunner.?Deutsche Medicinische Wochenschrift, No. 7, 1889. 
+
+3. Decker.?Berliner Klin. Wochensch., No. 45, 1889. 
+
+4. Leo.?Diagnost. der Krankheiten der Verdanungsorgane, s. 81. 
+
+5. Pal.?Berliner Klin. Wochensch., No. 48, 1889. 
+
+5. Rodczjewski.?Maly's J'ahresbericlit der Tliierchemie, 1887. 
+
+;. Huber.?Miinchencr Med. Wochensch., No. 19, 1889. 
+
+'. Bourget.?Bee. Med. de la Suisse Bomande, pp. 103-106, 1888. 
+
+. Silberstein.?Deutsche Med. Wochensch., No. 9, 1891. 
+
+. Oceimen.?Inaugural Dissertation, G-iessen, 1891. 
+
+Ewald.?Klinik der Verdarningskrankheiten, vol. i., p. 156 in the 1886 edition. (Page 172 in Dr Saundby's translation of the 1891 edition.) 
+J. Wolff.?Zeitschrift fur Klin. Med., 1883, Bd. vi. s. 112. 
+
+Haberlin.?Deutsches Archiv fur Klin. Med., Bd. xlv. s. 337. 
+
+?. Sahli.?Correspondenz-blatt fur Schiveitzer Aerzte, 3, 1891. 
+
+5. Penzoldt und Faber.?Berliner Klin. Wochensch., s. 313-315, 1882. 
+
+). Symons Eccles.?The Practitioner, April 1892. 
+
+7. Tolcher Eccles.?The Practitioner, March 1892. 
+
+>. Cutler.?The Boston Medical and Surgical Journal, Dec. 3, 1891. 
+
+). Tweedy.?The Dublin Journal of Medical Science, February 1892, p. 104. 
+
+1. Attfield.?The Practitioner, February 1892. 
+
+1. Stockton.?The Medical and Surgical Beporter, January 16, 1892. 
+
+no ) The Journal of the American Medical Association, Sl?:} Feb. 13,1892. 
+
+25. Dreschfeld.? The Medical Chronicle, vol. xiv. pp. 81, 249. 
+
+26. Henry.?The New York Medical Journal, February 27, 1892. 
+
+27. Weir.?The International Medical Magazine, vol. i. No. 1. 
+
+28. Fishkr.?The Medical and Surgical Reporter, Nov. 14, 1891. 
+
+29. Hayem and Winter.?Du Chimisme Stomcical, 1891. 
+
+30. Hayem.?Revue Mid. de la Suisse romancle, xi. 3, 1891. 
+
+31. Bouveret et Magnien.?Lyon Mdd., xxiii. 31, 32. 
+
+32. Wagner.?Arch, de Physiologic, xxiii. 3, 1891. 
+
+33. Rosenheim?Centralblatt f. Klin. Med., September 1891. 
+
+34. Salkowski und Kumagawa.? Virchow's Archiv, Bd. cxxii. 
+
+s. 235. 
+
+35. Salkowski.? Virch. Archiv, Bd. cxxvii. s. 501. 
+
+36. Hirschfeld.?Arch. f. d. gesammt. Phys., xlvii. 9 und 10 s. 510. 

medical/md/PMC5665032.md

@@ -0,0 +1,286 @@
+Ivyspring International Publisher Journal of *Cancer* 2017; 8(16): 3166-3172. doi: 10.7150/jca.19060 Research Paper
+
+# Programmed Death Ligand 1 (Pd-L1) Expression In Primary Angiosarcoma
+
+Gerardo Botti1*, Giosuè Scognamiglio1*, Laura Marra1, Antonio Pizzolorusso2, Maurizio Di Bonito1, Rossella De Cecio1, Monica Cantile1, Annarosaria De Chiara1 1. Pathology Unit, Istituto Nazionale Tumori Fondazione "G. Pascale", via Mariano Semmola, 80131 Napoli, Italy; 2. Department of Muscle-skeletal Oncology, Istituto Nazionale Tumori Fondazione "G. Pascale", via Mariano Semmola, 80131 Napoli, Italy.
+
+* Equal contributors Corresponding author: Monica Cantile: Pathology Unit, Istituto Nazionale Tumori Fondazione "G. Pascale", via Mariano Semmola, 80131 Napoli, Italy TEL.+390815903745; FAX. +390815903718; e-mail: m.cantile@istitutotumori.na.it
+© Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license 
+(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
+
+Received: 2017.01.05; Accepted: 2017.03.31; Published: 2017.09.15
+
+## Abstract
+
+Angiosarcomas are rare malignant endothelial-cell tumors of vascular or lymphatic origin, and are among the most aggressive subtypes of soft-tissue sarcomas. The prognosis is poor and treatment is challenging in many cases. PD-1/PD-L1 pathway plays a critical role in immune escape of tumor cells. Recent studies described that PD-L1 is widely expressed in various types of cancer, providing the basis for the development of PD1/PD-L1 antibodies as anti-cancer immunotherapy. Despite the well-known potential of PD-L1 as prognostic and predictive biomarker, only few studies described its IHC expression in cancer subtypes for the extreme difficulty in developing standard protocol with the different antibody clones available. We analyzed the IHC expression of PD-L1 on a series of angiosarcomas at different body location, showing its aberrant expression in about 66% of samples with no relation with prognosis. Our study allowed us to correctly define PD-L1 staining in angiosarcoma tumor tissues with final purpose to stratify patients for immune checkpoint inhibitors therapies.
+
+Key words: angiosarcoma, PD-L1, IHC evaluation.
+
+## Introduction
+
+Angiosarcomas are rare malignant vascular tumors, which account for approximately 2% of all soft tissue sarcomas. It is characterized by rapidly proliferating, extensively infiltrating atypical cells derived from blood vessels and lining irregular blood-filled spaces [1].
+
+Angiosarcomas can arise at different sites and in different organs with distinct features, more frequently in skin and soft tissue, but also in the liver, breast, spleen, bone, or heart.
+
+The most common form of angiosarcoma is cutaneous angiosarcoma, with a higher incidence in elderly patients [2], followed by soft tissue angiosarcomas with a very aggressive clinical behaviour [1], while primary mammary angiosarcoma, affecting a mainly younger females is very uncommon [3]. 
+
+Several studies suggested that angiosarcomas had a very poor prognosis, with 5-year survival of only 10–15%. This highly aggressive tumour spreads widely, recurs locally and metastasizes early [1]. 
+
+Surgery is the mainstay of treatment, but the high frequency of local recurrence of this strategy is discouraging. Radiotherapy was generally performed in cases of widely spread and unresectable tumours, but the outcomes were usually unsatisfactory [4, 5]. Therefore, further adjuvant or supportive therapy is necessary for the treatment of angiosarcoma. 
+
+Recently, chemotherapy and immunotherapy using recombinant interleukin-2 (rIL-2) have been studied as potential treatments [6]. However, the optimal treatment for these tumours has not been clearly established.
+
+PD-L1 is a 40kDa transmembrane protein that is expressed on a wide variety of normal tissues including natural killer cells, macrophages, myeloid dendritic cells, B cells, epithelial cells, and vascular endothelial cells [7]. Recently, several studies showed that PD-1/PD-L1 pathway may have a key role in the interaction of tumor cells with host immune response, and tumor cells PD-L1 expression may serve as a mechanism of adaptive immune resistance [8]. Many human cancers have been shown to express PD-L1 in most of cases, and its expression was also correlated with a poor prognosis, highlighting that it can represents a potential prognostic and predictive biomarker in human solid tumors [9-13]. In support of these experimental evidences, early-phase trials using monoclonal antibodies targeting PD-1 or PD-L1 revealed a real efficacy in patients with refractory tumors [14].
+
+However, recent reports revealed that the expression of the PD-L1 on tumor cells is not uniform, because of different antibodies clones, with variable specificity, often doubtful topographical localization and not well defined score.
+
+The purpose of this study was to analyze the immunohistochemical expression of PD-L1 to define adequate evaluation criteria, and investigate its prognostic role in angiosarcomas.
+
+## Materials And Methods Patient Selection
+
+From 2006 to 2015, 24 Formalin Fixed Paraffin Embedded (FFPE) samples from angiosarcoma patients were retrieved from the Department of Pathology, at the National Cancer Institute "Giovanni Pascale Foundation" of Naples, Italy. All selected cases were primary angiosarcoma. We excluded secondary angiosarcoma originating as consequences of breast radiotherapy.
+
+Medical records for all cases were reviewed for clinical information. The following clinical and pathological parameters were evaluated for each tumor included in the study: patient age at initial diagnosis, tumour size, histologic features, tumour differentiation, type of surgery (for tumour removal) 
+and clinical outcome. 
+
+In addition to H&E evaluation, all specimens were characterized for all routinely diagnostic immunophenotypic parameters.
+
+## Immunohistochemical Analysis
+
+Immunohistochemical staining for PD-L1 was performed on 5-μm-thick sections obtained from formalin-fixed, paraffin-embedded tissue of the selected cases. Prepared slides were incubated for 12 minutes at 110°C in Cell Conditioning Solution 
+(Ventana medical Systems [Cat \#: 950-124], Tucson, Arizona, USA), using a commercial steamer as the heat source (Biocare Medical, Decloaking Chamber DC12, Pike Lane Concord CA, USA). After cooling for 15 minutes, manual staining was performed.
+
+Following a 5 min incubation with a peroxidase blocking reagent (DAKO \#SM801, Glostrup, Denmark, UE) followed by two, 5 min rinses in Bond Wash Solution (Leica, \#AR9590). Protein blocking reagents were applied for 5 min with a protein serum block (1% goat serum (Abcam Cambridge Science Park Milton Road, Cambridge, UK), 4% BSA in PBS. 
+
+Blocking reagents were removed without rinsing before adding primary antibody. Primary anti-PDL-1 antibody clone SP-142 (Spring Bioscience [\#M4420] 
+Koll Center Pkwy, Pleasanton, CA, USA) was diluted in one commercial primary antibody diluent (Bond primary antibody diluent, \# AR9352, Leica Biosystems, Newcastle, UK) at a concentration of 3,75 μg/ml. Slides were incubated with the antibody for 1 hour at room temperature, followed by two steps of 5 min in Bond Wash Solution. The Goat Anti-Rabbit + 
+HRP (horseradish peroxidase) visualization reagent (DAKO, Cat \# SK001, Glostrup, Denmark, UE), which is biotin-independent and reduces the potential for background or nonspecific staining from endogenous biotin, was used for primary antibody detection. After two, 5 min rinses in Bond Wash Solution the secondary antibody was incubate for 35-minute. 
+
+Follow incubation with steady plus DAB (3,3'-diaminobenzidine) kit (ABCAM, \#AB103723,). 
+
+The slides were rinses for 5 in distillated water. The slides were counterstained with hematoxylin for 30 second, rinsed in distilled water, dehydrated in an ethanol series (30%, 70%, 100%) and four changes of 100% xylene, and permanently sealed with coverslips in automatic (DAKO \#CS100, Glostrup, Denmark, UE).
+
+## Immunohistochemistry **Evaluation**
+
+Antigen expression was evaluated independently by two pathologists (ADC and MDB) 
+using a light microscopy. For each sample, at least five fields (inside the tumor and in the peripheral areas) 
+and >500 cells were analysed. Using a semi-quantitative scoring system microscopically and referring to each antigen scoring method in other studies, each observer evaluated the intensity, extent and subcellular distribution.
+
+For TIL detection, lymphocytes were quantified by manual expression scoring, considering as tumor infiltrating (TIL) if they were located within 1 high-power field (HPF; ×40) of carcinoma cells. An immunohistochemistry characterization of immune cells was performed by CD3 (for T-cells), CD20 (for B-cells) and CD163 (for macrophages).
+
+For PD-L1 assessment we considered both a qualitative and a quantitative parameter. For the qualitative criteria we considered i) the immunoreactivity of membrane: "absent" (negative samples), "incomplete" or "complete" (positive samples); and ii) the intensity of the reaction at the membrane level: "mild", "moderate" and "intense". For the quantitative criteria we considered positive samples when PD-L1 staining is detected in ≥ 5% of tumor cells.
+
+## Statistical Analysis
+
+The association between PD-L1 expression with clinical-pathological parameters was conducted using Fisher's Exact test determining whether a relationship exists between the different variables included in the study. The level of significance was defined as P<0.05. 
+
+Overall survival (OS) and disease-free survival (DFS) 
+curves were calculated using the Kaplan-Meier method. All the statistical analyses were carried out using the Statistical Package for Social Science v. 20 software (SPSS Inc., Chicago, IL, USA).
+
+OS was defined as the time from diagnosis (first biopsy) to death by any cause or until the most recent follow-up. DFS was measured as the time from diagnosis to the occurrence of progression, relapse after complete remission, or death from any cause.
+
+## Results Clinical-Pathological Features Of Angiosarcoma Patients
+
+In our cohort, we have included 24 primary angiosarcoma samples from different body locations.
+
+The age of patients ranged from 23–91 years, with an average age of 58 years. Females were 14/24 while males were 10/24. The prevalent sites of primary lesion were breast (7/24), followed by soft tissues (5/24), bone (4/24), skin (4/24), and visceral 
+(4/24). Regarding tumor grading and histological differentiation, 12/24 were classified as low grade/well differentiated tumors, and 12/24 as high grade/poor differentiated tumors. All information is summarized in Table 1.
+
+## Pdl1 **Expression In Primary Angiosarcoma** Samples And Relation With Clinical- **Pathological** Features And Prognosis Of **Patients**
+
+PD-L1 was detected in 16/24 (66%) of angiorsarcoma samples, with a heterogeneous expression of PD-L1 on tumor cells. 
+
+The incomplete (Figure 1a, 1b) and complete 
+(Figure 1c, 1d) immunoreactivity of membrane was equally distributed in our case series, with a prevalent mild intensity of reaction, and with or without cytoplasmic positivity (Figure 1e, 1f) associated with a membrane staining.
+
+In most of the analyzed samples the presence of infiltrating lymphocyte (TIL) was poor, only in 3/20 
+(15%) it was consistent. TIL component showed a PD-L1 positive staining also in negative tumor samples (Figure 2a, 2b).
+
+Based on statistical elaboration of PD-L1 protein expression analysis with the clinical-pathological parameters in angiosarcomas, we showed no statistically significance with age, gender and site location while a strong statistical association was detected with tumor differentiation (p-value= 0.027). 
+
+PD-L1 appeared overexpressed in high grade/poor differentiated tumors, and variably expressed in low grade/well differentiated angiosarcomas. All data are schematized in Table 1.
+
+Regarding the relation with patients survival, we showed only a weak trend of statistical association with DFS (0.118) (metastatic patients have a prevalent expression of PD-L1), while no statistically significance was detected with Overall survival (p-value= 0.726). 
+
+Kaplan-Meier curves relative to DFS and OS are illustrated in Figure 3. 
+
+| Tot pz             | PD-L1 (-)   | (+)        | P-value   |       |       |
+|--------------------|-------------|------------|-----------|-------|-------|
+| Age                | <57         | 12         | 6         | 6     | 0,193 |
+| (50%)              | (50%)       | (50%)      |           |       |       |
+| 58+                | 12          | 2          | 10        |       |       |
+| (50%)              | (16,7%)     | (83,3%)    |           |       |       |
+| Gender             | M           | 14 (58,3%) | 4         | 10    | 0,673 |
+| (28,6%)            | (71,4%)     |            |           |       |       |
+| F                  | 10 (41,7%)  | 4          | 6         |       |       |
+| (40%)              | (60%)       |            |           |       |       |
+| Grade              | Low         | 12         | 7         | 5     | 0,027 |
+| (50%)              | (58,3%)     | (41,7%)    |           |       |       |
+| High               | 12          | 1          | 11        |       |       |
+| (50%)              | (8,3%)      | (91,7%)    |           |       |       |
+| Site location Bone | 4           | 0          | 4         | 0,258 |       |
+| (16,6%)            | (0%)        | (100%)     |           |       |       |
+| Skin               | 4           | 1          | 3         |       |       |
+| (16,6%)            | (25%)       | (75%)      |           |       |       |
+| Breast             | 7           | 3          | 4         |       |       |
+| (29,2%)            | (42,9%)     | (57,1%)    |           |       |       |
+| Soft tissue 5      | 1           | 4          |           |       |       |
+| (20,8%)            | (20%)       | (80%)      |           |       |       |
+| Visceral           | 4           | 3          | 1         |       |       |
+| (16,6%)            | (75%)       | (25%)      |           |       |       |
+
+![3_image_0.png](3_image_0.png)
+
+![3_image_1.png](3_image_1.png)
+
+![4_image_0.png](4_image_0.png)
+
+## Discussion
+
+Angiosarcomas are rare malignant mesenchymal tumours, showing a wide range of differentiation from highly differentiated tumours, which resemble hemangiomas, to poorly differentiated anaplastic lesions [1].
+
+Primary Angiosarcoma had a very poor prognosis with a high rate of metastasis involving most commonly lungs, liver, regional lymph nodes and bone.
+
+Cutaneous angiosarcoma is the most common form of angiosarcoma [2], with a higher incidence in elderly patients as well as Soft Tissue Angiosarcomas 
+[1]. Primary angiosarcoma of the Breast is the most common mesenchymal tumour of the breast while angiosarcoma of the Gastrointestinal Tract is quite rare. Both lesions, in the most of cases, are followed to a radiation therapy for other malignant and benign diseases [15]. 
+
+Angiosarcoma is a tumor with a very poor prognosis, also because of known molecular alterations and consequently of specific therapeutic targets. Therefore, it is treated mainly with surgery followed by chemotherapy and/or radiation therapy.
+
+Several drugs directed against PD-1/PD-L1 axis are now undergoing clinical trials in various tumor diseases [16], but PD-L1 role as prognostic and predictive biomarker, among various studies, is very moot [17-23] also because the different assays for IHC staining and different cutoff values assigned.
+
+In this study we assessed the immunohistochemical expression of PD-L1 mainly to clarify the potential role in the progression of angiosarcoma, but also to define a correct IHC 
+staining.
+
+A case series of 24 angiosarcomas from different body localization were enrolled into this study. Our results showed a heterogeneous expression of PD-L1 on tumor cells. 
+
+For its assessment and evaluation, not yet described in the literature, we defined both qualitative, considering the immunoreactivity of membrane and the intensity of the reaction at the membrane level, and quantitative parameters, considering the percentage of positive tumor cells ≥ 5%.
+
+PD-L1 was detected in about 66% of angiosarcoma samples with a prevalent immunoreactivity of membrane. Only in few cases a mild cytoplasmic associated with membrane staining was present.
+
+The data available in the literature related to the expression of PD-L1 are not uniform, because of the different antibodies clones used, with variable specificity, often doubtful topographical localization and not well defined score. Moreover, in the most of studies in which PD-L1 expression was realized by immunohistochemistry, a consistent cytoplasmic expression was detected, without clarify and define the real value of this positivity in tumor cells [24].
+
+Whereas for other malignancies the prognostic value of PD-L1 is abundantly described in the literature, also for the therapeutic framing of the patients, for the soft tissue tumors this role has not been yet well defined. In a single study PD-L1 IHC expression was described in several histological subtypes of sarcomas [25]. However, the limitation of this study was that PD-L1 expression was analyzed on 20 different histological types, with a few number of specimens for each tumor types without defining its prognostic value. In this study D'Angelo SP et al. 
+
+described the PDL-1 expression in the all three angiosarcoma cases and also in TIL inside tumors [25].
+
+In our series, PD-L1 showed only a strong statistical association with tumor differentiation. 
+
+PD-L1 was overexpressed in high grade/poor differentiated tumors, and variably expressed in low grade/well differentiated angiosarcomas. No significant statistical association was detected with DFS and OS, highlighting that PD-L1 would not seem to be a prognostic marker in this tumor disease.
+
+Actually the prognostic value of PD-L1 in solid tumors is much debated and often conflicting data were described in the literature.
+
+Although most of the findings associated PD-L1 expression with a poor prognosis [26, 27], several studies assigned an opposite value to its expression.
+
+Schalper KA *et al.* quantized *in situ* PD-L1 mRNA 
+levels in a large case series of stage I-III breast carcinomas showing that its expression was associated with longer recurrence-free survival [17].
+
+Moreover, another immunohistochemical study showed that PD-L1 positive patients had a significantly greater disease-control rate, in association with longer progression-free survival after EGFR-tyrosine kinase inhibitor therapy and overall survival in EGFR mutation positive Lung adenocarcinoma [22].
+
+More recently Yang *et al.* described that tumor PD-L1 expression and increased CD4+T cell infiltrations in the tumor stroma were independent predictors of better overall survival in stage I 
+pulmonary squamous cell carcinoma [23]. 
+
+However, also tumours where PD-L1 does not appear to be a prognostic marker [28], the use of the anti-PD1/PD-L1 drugs produces significant therapeutic effects [29]. In fact the only expression of the PD-L1 protein on tumor cells has been thought to be a good predictor of a patient's response.
+
+In conclusion, in clinical practice, the most important purpose is to predict the treatment response from PD-1/PD-L1 blockade, by a correct standardization of operative protocols for the PD-L1 determination in tumor cells.
+
+In our study, the absence of strong correlations between PD-L1 expression and survival of angiosarcoma patients could in part due to the small number of specimens, but also to the extreme aggressiveness of this tumor for which the prognosis is poor in the most of cases.
+
+In angiosarcomas, despite PD-L1 is not related to the prognosis, its detection could be very useful in the therapeutic stratification of patients to enroll for specific therapies associated to PD-1/PD-L1 blockade.
+
+## Abbreviations
+
+PD-1: Programmed cell death protein 1; PD-L1: 
+Programmed death-ligand 1; IHC: immunohistochemistry; FFPE: Formalin-fixed, paraffin-embedded; H&E: Hematossilin and eosin; OS: Overall survival; DFS: disease-free survival; TIL: 
+Tumor infiltrating lymphocytes.
+
+## Acknowledgements
+
+The authors thank all of the study participants for their great effort.
+
+## Authors' Contributions
+
+ADC and MC were responsible for the conception and design of the study. AP was responsible for provision of patients clinical information. GS and LM collected samples for immunohistochemical analysis. MDB, RDC and GB 
+were responsible for immunohistochemical evaluation. All authors were involved in manuscript writing and provided final approval of the manuscript.
+
+## Availability Of Data And Materials
+
+All relevant data of the study were already included in the manuscript, including the survival curves although not significant.
+
+## Ethics Approval And Consent
+
+This study was approved by the Ethics Committee of INT Fondazione Pascale.
+
+Written informed consent was obtained from the patient for publication of this manuscript and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
+
+## Competing **Interests**
+
+The authors have declared that no competing interest exists.
+
+## References
+
+1. Young RJ, Brown NJ, Reed MW, et al. Angiosarcoma. Lancet Oncol. 2010; 11:983-91.
+
+2. Costigan DC, Doyle LA. Advances in the Clinicopathologic and Molecular Classification of Cutaneous Mesenchymal Neoplasms. Histopathology. 2016; 68:776-95.
+
+3. Arora TK, Terracina KP, Soong J, et al. Primary and secondary angiosarcoma of the breast. Gland Surg. 2014; 3:28-34.
+
+4. D'Angelo SP, Munhoz RR, Kuk D, et al. Outcomes of Systemic Therapy for Patients with Metastatic Angiosarcoma. Oncology. 2015; 89:205-14.
+
+5. Ravo V, Falivene S, De Chiara A, et al. Treatment of cutaneous angiosarcoma of the face: efficacy of combined chemotherapy and radiotherapy. Tumori. 2013; 99:e211-5.
+
+6. Ohguri T, Imada H, Nomoto S, et al. Angiosarcoma of the scalp treated with curative radiotherapy plus recombinant interleukin-2 immunotherapy. Int J Radiat Oncol Biol Phys. 2005; 61:1446-53.
+
+7. Butte MJ, Peña-Cruz V, Kim MJ, et al. Interaction of human PD-L1 and B7-1. 
+
+Mol Immunol. 2008; 45:3567-72.
+
+8. Akbay EA, Koyama S, Carretero J, et al. Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer Discov. 
+
+2013; 3:1355-63.
+
+9. Zhang Y, Wang L, Li Y et al. Protein expression of programmed death 1 ligand 1 and ligand 2 independently predict poor prognosis in surgically resected lung adenocarcinoma. Onco Targets Ther. 2014; 7:567-73.
+
+10. Choueiri TK, Fay AP, Gray KP et al. PD-L1 expression in non clear-cell renal cell carcinoma. Ann Oncol. 2014; 25:2178-84. 
+
+11. Taube JM, Klein A, Brahmer JR et al. Association of PD-1, PD-1 Ligands, and Other Features of the Tumor Immune Microenvironment with Response to Anti–PD-1 Therapy. Clin Cancer Res. 2014; 20:5064-74. 
+
+12. Harshman LC, Choueiri TK, Drake C. Subverting the B7-H1/PD-1 Pathway in Advanced Melanoma and Kidney Cancer. Cancer J. 2014; 20:272-80.
+
+13. Soliman H, Khalil F, Antonia S. PD-L1 Expression Is Increased in a Subset of Basal Type Breast Cancer Cells. PLoS One. 2014; 9:e88557.
+
+14. Topalian SL, Hodi FS, Brahmer JR. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012; 366:2443–54.
+
+15. Policarpio-Nicolas ML, Nicolas MM, Keh P, Laskin WB. Postradiation angiosarcoma of the small intestine: a case report and review of literature. Ann Diagn Pathol. 2006;10:301-5.
+
+16. Ohaegbulam KC, Assal A, Lazar-Molnar E, et al. Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway. Trends Mol Med. 2015; 21: 24–33.
+
+17. Schalper KA, Velcheti V, Carvajal D, et al. In situ tumor PD-L1 mRNA 
+expression is associated with increased TILs and better outcome in breast carcinomas. Clin Cancer Res. 2014; 20:2773-82. 
+
+18. Chen YB, Mu CY, Huang JA. Clinical significance of programmed death-1 ligand-1 expression in patients with non-small cell lung cancer: a 5-year-follow-up study. Tumori. 2012; 98 :751-5.
+
+19. Wang L, Ren F, Wang Q, et al. Significance of Programmed Death Ligand 1 
+(PD-L1) Immunohistochemical Expression in Colorectal Cancer. Mol Diagn Ther. 2016;20:175-81.
+
+20. Jacquelot N, Roberti MP, Enot DP, et al. Immunophenotyping of stage III 
+melanoma reveals parameters associated with patient prognosis. J Invest Dermatol. 2016; 136:994-1.
+
+21. Xu F, Xu L, Wang Q, et al. Clinicopathological and prognostic value of programmed death ligand-1 (PD-L1) in renal cell carcinoma: a meta-analysis. Int J Clin Exp Med. 2015; 8:14595-603.
+
+22. Lin C, Chen X, Li M, et al. Programmed Death-Ligand 1 Expression Predicts Tyrosine Kinase Inhibitor Response and Better Prognosis in a Cohort of Patients With Epidermal Growth Factor Receptor Mutation-Positive Lung Adenocarcinoma. Clin Lung Cancer. 2015; 16:e25-35.
+
+23. Yang CY, Lin MW, Chang YL, et al. Programmed cell death-ligand 1 expression is associated with a favourable immune microenvironment and better overall survival in stage I pulmonary squamous cell carcinoma. Eur J Cancer. 2016; 57:91-103.
+
+24. Muenst S, Schaerli AR, Gao F, et al. Expression of programmed death ligand 1 
+(PD-L1) is associated with poor prognosis in human breast cancer. Breast Cancer Res Treat. 2014; 146:15-24.
+
+25. D'Angelo SP, Shoushtari AN, Agaram NP, et al. Prevalence of tumor-infiltrating lymphocytes and PD-L1 expression in the soft tissue sarcoma microenvironment. Hum Pathol. 2015; 46:357-65.
+
+26. Leite KR, Reis ST, Junior JP, et al. PD-L1 expression in renal cell carcinoma clear cell type is related to unfavorable prognosis. Diagn Pathol. 2015; 10:189. 
+
+27. Muenst S, Soysal SD, Gao F, et al. The presence of programmed death 1 
+(PD-1)-positive tumor-infiltrating lymphocytes is associated with poor prognosis in human breast cancer. Breast Cancer Res Treat. 2013; 139:667-76.
+
+28. Ukpo OC, Thorstad WL, Lewis JS. B7–H1 expression model for immune evasion in human papillomavirus-related oropharyngeal squamous cell carcinoma. Head Neck Pathol. 2013; 7:113-21.
+
+29. Sorensen SF, Zhou W, Dolled-Filhart M, et al. PD-L1 Expression and Survival among Patients with Advanced Non–Small Cell Lung Cancer Treated with Chemotherapy. Translational Oncology. 2016, 9:64-69.

medical/md/PMC5787966.md

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+# Barriers And Enablers To Adoption Of Intrauterine Device As A Contraceptive Method: A Multi‑Stakeholder Perspective
+
+## Namita Mishra1, Meely Panda2, Souvik Pyne3**, Nallala Srinivas3**, Sandipana Pati4**, Sanghamitra Pati5**
+
+1Department of Health and Family Welfare, Sub‑Divisional Hospital, Cuttack, Government of Odisha, 2Department of Community Medicine, Kalinga Institute of Medical Sciences, 3*Indian Institute of Public Health Bhubaneswar,* 
+Public Health Foundation of India, Bhubaneswar, 4*Disease Surveillance Medical Officer, Directorate of Public Health,* 
+Government of Odisha, 5*Regional Medical Research Centre, Indian Council of Medical Research, Chandrasekharpur,* 
+Bhubaneswar, Odisha, India
+
+## Abstract
+
+Background: Promoting family planning practices aid considerably in attaining Millennium Development Goals by various mechanisms. Despite concerted health system efforts, adoption of especially reversible contraceptive methods such as intrauterine devices (IUDs) has remained negatively skewed in India, which is the pioneer country to implement Family Planning programme way back in 1952. Although few studies in India have looked into the reasons for its nonacceptance, literature from Odisha was scant and hence the study was undertaken. **Methodology:** A cross‑sectional study using qualitative methods was done in the Mahanga Tehsil of Cuttack district. In‑depth interviews were conducted with women of reproductive age (WRA) and focused group discussions (FGDs) among health workers and health professionals were held separately. Data analysis was done using thematic framework approach supported by Atlas Ti software. **Results:** There were 31 in‑depth interviews with WRA, two FGDs with health workers, and one FGD with health professionals. Availability of IUD services was low and wherever available, being located far away affected its physical accessibility. Most women were reluctant to ask health workers about services owing to their shyness while many women felt using IUDs breached their autonomy and privacy. The existence of fear and misconceptions regarding its use rooting from lack of knowledge and poor service quality also impeded its adoption by women. **Conclusion:** There is a pressing need to enhance the demand of IUDs by dispelling the myths among women through effective information, education, and communication and also to improve the availability of IUDs.
+
+Keywords: Contraceptive, health, intrauterine device, qualitative research, women
+
+## Introduction
+
+Family planning helps women prevent unintended pregnancies, delay early childbearing, and space births at least 2 years apart.[1] Fostering family planning practice alleviates poverty, Address for correspondence: Dr. Sanghamitra Pati, ICMR - Regional Medical Research Centre, Department of Health Research, Govt. of India, Chandrasekharpur, Bhubaneswar, Odisha 751023, India.
+
+E‑mail: drsanghamitra12@gmail.com
+
+Access this article online
+
+![0_image_0.png](0_image_0.png)
+
+www.jfmpc.com
+
+| Access this article online Website: www.jfmpc.com DOI:  10.4103/2249-4863.222028   |
+|------------------------------------------------------------------------------------|
+
+accelerates socioeconomic development, increases child schooling, promotes gender equality, and decreases maternal and infant mortality.[2] As per the United Nations Population Fund estimates, widespread use of family planning could lower Maternal Mortality Ratio (MMR) by 20% and Infant Mortality Rate by as much as 25–30% in developing countries. Thus, there is a growing consensus that a good approach to family planning would help in achieving the Millennium Development Goals (MDGs). An important cause for these This is an open access article distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as the author is credited and the new creations are licensed under the identical terms.
+
+For reprints contact: reprints@medknow.com How to cite this article: Mishra N, Panda M, Pyne S, Srinivas N, Pati S, 
+Pati S. Barriers and enablers to adoption of intrauterine device as a contraceptive method: A multi-stakeholder perspective. J Family Med Prim Care 2017;6:616-21.
+
+© 2017 Journal of Family Medicine and Primary Care | Published by Wolters Kluwer - Medknow 616 high fertility rates is the low availability and use of family planning services.[3,4]
+there is no study done in Odisha. Thus, this study aimed to explore the prevalent perspectives regarding IUD (CuT) from a multistakeholder perspective.
+
+## Methodology Study Design
+
+We did a cross‑sectional study using qualitative methods among three groups of people who were recruited using a purposive sampling.
+
+## Study Setting
+
+Mahanga tehsil of Cuttack district was chosen since it is the largest with 192 villages, 29 subcenters, and a population of 186,930 as per census 2011.
+
+## Data Collection
+
+India is the second most populous country in the world and also was the pioneer to launch the Family Planning Programme as early as in 1952. Subsequently, its name got changed to Family Welfare Programme and then to Reproductive and Child Health Programme. This change was made to emphasize and increase the acceptance of contraceptive methods. Since its inception in 1951, the National Family Planning Programme has been dominated by demographic goals by focusing primarily on sterilization, largely obviating client choice, and limiting availability to a narrow range of services. In April 1996, the Indian government abolished method‑specific family planning targets throughout the country. In October 1997, India reoriented the national program's approach to more broadly address health and family limitation needs. The new approach involved a more comprehensive set of reproductive and child health services and a focus on client choice, service quality, gender issues and underserved groups, including adolescents, men, and postmenopausal women. The Family Welfare Programme in India explains that small differences in the family size will have a tremendous impact on the population growth. Hence, the objective of Family Welfare Programme is that the people should adopt the small family norm to stabilize the country's population at the level of some 1533 million by the year 2050.[5]
+Despite concerted efforts by the health system in promotion of family planning services, the unmet need for contraception still remains a problem. As per District Level Household and Facility Survey (DLHS)‑3 estimates the Contraceptive Prevalence Rate (CPR) for India is 47.1 percent while the unmet need of contraception is 21.3%, with 7.9% for spacing, and 13.4 percent for limiting births.[2,6,7] The intrauterine device (IUD) is the most frequently used reversible family planning method worldwide. However, its acceptability and usage are low in many developing countries with a majority of women choosing female sterilization or oral contraceptive pills (OCP) for birth control.[8] According to Health and Family Welfare Statistics 2014–2015, there were 5,277,460 IUD insertions in India compared to 5,602,284°CP 
+users.[9]
+Odisha is the eleventh populous state in India having a MMR of 235 (SRS 2010–2012) which is much above the national average and obviously far away from the MDG‑5 target.[10] The CPR in Odisha is 46.8% for any modern method according to Annual Health Survey 2011–2012 while according to DLHS‑3, the unmet need for contraception in Odisha is about 19.8%, of which 6.5% 
+is for spacing and 13.3% for limiting.[2,6,7] Again, according to Health and Family Welfare Statistics 2014–2015, in Odisha, there were 84,608 IUD insertions compared to 154,929 OCP users showing a very stark disparity.[9]
+There are few studies in India examining the reasons for nonacceptance of IUDs and perceptions of women, but The study was conducted from June to August 2014. All study tools were used in Odia language. First, women of reproductive age group (WRA), i.e., belonging to age group of 15–49 years from different villages under Mahanga Community Health Centre (CHC) were interviewed to find out the perception of the beneficiaries about the contraceptive services being provided to them. In‑depth interviews were done for 31 beneficiaries selected through purposive sampling so as to have uniform representation of tribal and general population. We used an in‑depth interview guideline including six structured questions for capturing the sociodemographic details. Second, health workers (Auxiliary Nurse Midwives or ANMs) were interviewed to know about the enablers and barriers of the contraceptive services, especially IUD being provided as experienced by them. Two focused group discussions (FGDs) comprising six members each were done with the health workers (ANMs) using an FGD guideline. Third, health professionals from different fields were interviewed to explore the challenges contraceptive services and come up with possible solutions. An FGD was conducted with 10 health professionals using a FGD guideline.
+
+## Data Analysis
+
+Grounded theory approach was used to sum‑up the collected data. In‑depth interviews and FGDs were transcribed and translated to English. Coding was done, and they were successively condensed into relevant subthemes and themes. Atlas Ti (Version 7 [Computer sofware], Berlin, Scientific Sofware Development)software was used, and ultimately representation was done using the thematic framework approach.
+
+## Ethical Considerations
+
+Informed consent was obtained from all the participants and thus the participation was purely voluntary. Adequate privacy was ensured while conducting interviews. Confidentiality and anonymity were maintained during the procedure. Necessary ethical approval was obtained from the Institutional Ethics Committee of the Indian Institute of Public Health‑Bhubaneswar.
+
+## Results
+
+There were total 31 participants for in‑depth interviews having median age of 26 years while the lowest was 20 years and highest being 33 years. All were homemakers and majority had low educational level. Sixteen women were of Hindu general caste; five women were of Hindu scheduled caste while ten women belonged to Muslim religion. The majority of women's household monthly income was below Rs. 10,000. Their detailed individual sociodemographic profiles are depicted in Annexure 1. Key issues emerging from in‑depth interviews, and FGDs were identified and then condensed into the following thematic areas.
+
+## Accessibility
+
+Women felt the CHC was far away, and so services offered there were physically inaccessible to them. ANMs added that women had to arrange their transportation out of their pocket raising economic concerns. Women felt embarrassed to enquire and discuss the methods of contraception and sex‑related topics freely with them leading to lack of information access. "I had heard the word CuT but thought that it was an operative procedure, and as Accredited Social Health Activist (ASHA) is senior to me in age, I felt shy to ask her in detail about CuT" (Respondent MMn6). "I feel bad to talk about this bad issue with others" (Respondent MS1). Health professionals believed that the beneficiaries should be comfortable with the person who is distributing the contraceptives and outlets for distribution should be opened up at convenient places for improving access issues.
+
+## Availability
+
+The beneficiaries said that the nearby subcenters though equipped for IUD services did not had trained staffs to deliver them. Most ANMs also reiterated the fact. Health professionals felt availability and accessibility were interrelated. They suggested that contraceptive services should be made universally available as a basket of choice so that people could access it easily.
+
+## Autonomy And Privacy
+
+Few women said that they enjoyed the freedom of getting oral pills from shops by not depending on ASHAs or ANMs, and they felt that in taking oral pills their privacy was not hampered where as in IUD service their privacy was hampered. "I know CuT but my husband does not recommend CuT, so I cannot use" (Respondent MG5). ANMs too reported that women liked to preserve their autonomy and privacy and not depend on anyone while using contraceptive methods. Health professionals suggested, if the mother‑in‑law or the husband was motivated, they could help the woman in reaching out for the IUDs as well as maintain adequate privacy.
+
+## Misconceptions
+
+The interviewed women harbored varying misconceptions regarding CuT like "it may or may not suit," "it will melt inside body," "it will go inside chest," "it will go inside abdominal cavity," "it will cause cancer," "it will stick to the wall permanently." ANMs further added that women felt that it results in white badly smelling discharge, permanently stay in abdomen, cause secondary infertility, swelling of abdomen, anemia, headache, head reeling, weakness, and irregular menses. Health professionals opined that although IUDs were very effective, wrong notions among women about it were major hurdles for their uptake and the service providers were yet to clarify the misconceptions effectively.
+
+## Fear
+
+Women reported that they were afraid of instrumentation and also the side effects of IUDs postinsertion such as bleeding, abdominal cramps, and apprehensive of unusual reactions. ANMs seconded the women's fear. Health professionals discerned that fear and misconceptions were obvious to crop up, but these could be alleviated by the positive teachings of the health workers. They emphasized the importance of knowledge imparting periodically.
+
+## Awareness And Knowledge
+
+Many women lacked knowledge about IUDs because they said there was inadequate promotion of the method by health‑care providers as well as their illiteracy. They felt partial dissemination of information by health‑care providers often led to wrong perceptions in their minds. ANMs propounded that women were not aware of the contraceptive methods, and there were no information education and communication (IEC) and Behaviour Change Communication (BCC) programs running from government side for raising awareness. Health professionals felt that both the beneficiaries as well as the providers were often unaware of the government policies, and there must be initiatives to improve methods so as to impart more knowledge and awareness among all sections to reduce disparity.
+
+## Quality
+
+Women raised concerns about service quality. They expected motivation from doctors and not just from ANMs or ASHAs. They also felt that Village Health and Nutrition Day (VHND) spots were not suitable places for them to gather and avail the service. Some women had an idea that IUD services were expensive. Most women said that ASHAs did not say much about CuT but promoted more about sterilization and oral pills. ANMs reiterated the lack of user‑friendliness of VHND spots for women to interact with health providers and said beneficiaries took them less seriously and thus relied less on them. Health professionals highlighted that people preferred things 
+
+## Et Al
+
+which were beneficial, low cost, easily available as well as highly effective. They suggested that if all these could be possible only if government gave prior attention to the quality of services rather than forcing on things or simply trying to explain the benefits. Thus, analyses brought forward various perspectives across multiple stakeholders, namely, WRA, health workers, and health professionals shrouding the issue of contraception, especially IUDs.
+
+## Discussion
+
+According to the Health Belief Model (HBM), four factors influence an individual's protective health behaviors: perception of susceptibility to negative health outcomes, perception of the severity of the negative outcome, benefits of preventive behavior in relation to the potential negative outcome, barriers, and perceived barriers to implementing the protective behavior. Although some find the HBM to be outdated, the original constructs and concepts can still be considered in gaining insight into decision‑making regarding sexual behaviors and risk‑taking.[11,12]
+In rural areas, many women were unaware of the contraceptive methods leading to low demand of this service from health‑care providers. Most of the women thought contraception meant pills. Out of 31 women, 12 had never heard the word CuT. Eight out of 19 women, who had heard about CuT, knew no more than that. An operational research study done in Gujarat stressed the importance of IEC which improved client performance after the intervention.[13]
+From among the 11 women who knew about IUDs in details, all had fear of side effects. They had less perceived benefits of CuT over oral pills. This might had been due to the differential dissemination of information by health‑care providers. Moreover, the women narrated that the ASHAs motivated them more about sterilization and pills. This may be because there is no incentive for promoting IUDs while that for sterilization is incentivized. 
+
+The studies by Rati et al. and Murarkar et al. mentioned that scattered knowledge of women regarding contraceptives led to myths and misconceptions resulting in nonacceptance of IUD 
+as a spacing method.[14,15]
+Loads of misconceptions existed in the minds of women. Discussing sex‑related issues was a forbidden issue for them, and it had been a social norm to maintain secrecy regarding this matter. Thus, most women could not replace their myths with correct information. A hospital‑based study in Bhopal also reiterated the fact of the presence of deep‑rooted misconceptions dissuading them from accepting IUDs.[16]
+Variation of autonomy in the selection of contraceptive methods arose due to the ignorance of the women, especially housewives, dominance of husbands and mother‑in‑laws, associated stigma, and lack of privacy. Similar results were found in a study by Yadav et al. on agreement and concordance regarding reproductive intentions and contraception between husbands and wives in rural Ballabgarh, India.[17]
+Almost all ANMs admitted that many women did not take their advice seriously. VHND spots were neither user‑friendly nor had privacy, which were essential for providing services. Women mostly of low income and low literacy status thought it to be a credit to manage spacing between pregnancies without using any method. These women either used calendar method or withdrawal method. Few bitter experiences from peer groups became propaganda, and even ASHAs were unable to persuade them in the right direction with proper information. Most Muslim women did not use contraceptive methods except some who used withdrawal technique and oral pills. Thus, we can understand that the issue of family planning is deeply rooted in an array of individual and social factors making it relevant to family physicians since their practice involves considering the person as a whole.
+
+## Conclusion And Recommendations
+
+This study focused on the exploration of factors influencing the acceptability of contraceptives, especially IUDs among WRA. Most of the rural women were not aware of the necessity of an effective contraception to avoid unwanted pregnancy. More than two‑thirds of the interviewed women in rural settings did not know about the CuT method. Among those who knew about the method, most did not use it either due to fear of side effects or some prevalent myths. Some were reluctant about the method because they felt lack of autonomy and privacy. Some were unable to avail the services because of restrictions by their husbands and in‑laws. In addition, such services were inaccessible and were not up to an acceptable quality. Because of the above facts, there was low demand from the acceptors' side. The findings that had emerged from this study thus provide some recommendations to increase the demand. Effective IEC, BCC should be promoted continuously with the help of health workers for better acceptance of CuT. VHND sessions should be improved in respect of site, logistics, and trained health providers. At facility level, i.e., at subcenters, quality services should be made available. Doctors and health workers should be trained in large numbers for providing quality services. Majority women recommended that interactive sessions between doctors and women should be undertaken at community level, maybe conducting "swasthya‑melas" frequently. Improving education level of rural women might modify the sociocultural aspects of extreme shyness and maintaining social distances.
+
+Thus, a concerted effort with collaboration and cooperation of various sectors and departments are warranted to fulfill the vision and mission more accurately.
+
+## Acknowledgments
+
+We gratefully thank all the study participants comprising WRA, health workers, and health professionals. We also sincerely thank the Government of Odisha for nominating the candidate for the course.
+
+## Financial Support And Sponsorship
+
+This study was a part of Postgraduate Diploma in Public Health Management dissertation work.
+
+## Conflicts Of Interest
+
+There are no conflicts of interest.
+
+## References
+
+1. National Institute Health and Family Welfare. Population and Development: A Discourse on Family Planning in Odisha. New Delhi: Policy Unit, NIHFW; 2014.
+
+2. Kols A. Reducing unmet need for family planning: 
+Evidence‑based strategies and approaches. Outlook 2008;25:1.
+
+3. Ringheim K, Gribble J, Foreman M. Integrating family planning and maternal and child health care: Saving lives, money, and time. Int Fam Plan Perspect 2007;33:6‑12.
+
+4. Abrejo FG, Shaikh BT, Saleem S. ICPD to MDGs: Missing links and common grounds. Reprod Health 2008;5:4.
+
+5. Visaria L, Jejeebhoy S, Merrick T. From family planning to reproductive health: Challenges facing India. Int Fam Plan Perspect 1999;1:S44‑9.
+
+6. International Institute for Population Sciences (IIPS). 
+
+National Family Health Survey (NFHS‑3), 2005‑06. Mumbai: International Institute for Population Sciences; 2007.
+
+7. International Institute for Population Sciences (IIPS). 
+
+District Level Household and family Survey (DLHS‑3), 
+2007‑08. Mumbai: International Institute for Population Sciences; 2010.
+
+8. Ambadekar NN, Rathod KZ, Zodpey SP. Study of cu T 
+utilization status and some of the factors associated with discontinuation of cu T in rural part of yavatmal district. 
+
+Indian J Community Med 2011;36:54‑6.
+
+9. Ministry of Health and Family Welfare. Family Welfare Statistics in India 2015. New Delhi: Ministry of Health and Family Welfare; 2016.
+
+10. National Health Mission. Health Statistics. Department of Health and Family Welfare, Government of Odisha. Available from: http://www.nrhmorissa.gov.in/frmhealthstatistics. aspx. [Last cited on 2016 Sep 13].
+
+11. Carpenter CJ. A meta‑analysis of the effectiveness of health belief model variables in predicting behavior. Health Commun 2010;25:661‑9.
+
+12. Downing‑Matibag TM, Geisinger B. Hooking up and sexual risk taking among college students: A health belief model perspective. Qual Health Res 2009;19:1196‑209.
+
+13. Khan ME, Kar SS, Desai VK, Patel P, Itare BP, Barge S. 
+
+Increasing the Accessibility, Acceptability and Use of the IUD in Gujarat, India. Frontiers Final Report; May, 2008.
+
+14. Rati MS, Jawadagi MS, Pujari MJ. A study to assess the factors affecting acceptance of intrauterine device (IUD) among rural women of Hirebagewadi, Belgaum. IOSR J Nurs Health Sci 2014;3:37‑52.
+
+15. Murarkar SK, Soundale SG, Lakade RN. Study of contraceptive practices and reasons for not accepting contraceptives in rural India: Chanai village as a case study. Indian J Sci Technol 2011;4:915‑6.
+
+16. Gadre SS, Ahirwar R. Level of acceptance of IUCD insertion in Indian women‑a cross‑sectional mixed research from central India. Int J Reprod Contracept Obstet Gynecol 2015;4:1079‑85.
+
+17. Yadav K, Singh B, Goswami K. Agreement and concordance regarding reproductive intentions and contraception between husbands and wives in rural Ballabgarh, India. Indian J Community Med 2010;35:19‑23.
+
+| Annexure 1: Sociodemographic profile of in‑depth interviews participants   |             |           |            |                           |          |           |
+|----------------------------------------------------------------------------|-------------|-----------|------------|---------------------------|----------|-----------|
+| Subject code                                                               | Age (years) | Education | Occupation | Income per month (in Rs.) | Religion | Ethnicity |
+| MS1                                                                        | 25          | 10th fail | HW         | 5500                      | Hindu    | Gen       |
+| MS2                                                                        | 30          | 10th fail | HW         | 7000                      | Hindu    | Gen       |
+| MS3                                                                        | 31          | 10th      | HW         | 5000                      | Hindu    | Gen       |
+| MK4                                                                        | 30          | 10th fail | HW         | 9000                      | Hindu    | Gen       |
+| MJ5                                                                        | 28          | 10th fail | HW         | 3500                      | Hindu    | Gen       |
+| MB1                                                                        | 25          | 8th       | HW         | 4000                      | Hindu    | Gen       |
+| MG1                                                                        | 28          | 7th       | HW         | 5000                      | Hindu    | SC        |
+| MG2                                                                        | 23          | 4th       | HW         | 4000                      | Hindu    | Gen       |
+| MG3                                                                        | 25          | 3rd       | HW         | 6000                      | Hindu    | SC        |
+| MG4                                                                        | 26          | 12th      | HW         | 6000                      | Hindu    | Gen       |
+| MG5                                                                        | 28          | 7th       | HW         | 10,000                    | Hindu    | SC        |
+| MG6                                                                        | 22          | 4th       | HW         | 5000                      | Hindu    | Gen       |
+| MG7                                                                        | 21          | 9th       | HW         | 9000                      | Hindu    | SC        |
+| MG8                                                                        | 24          | 8th       | HW         | 5000                      | Hindu    | SC        |
+| MJe1                                                                       | 22          | 7th       | HW         | 5000                      | Muslim   |           |
+| Mm2                                                                        | 23          | 10th pass | HW         | 15,000                    | Muslim   |           |
+| MM3                                                                        | 30          | 5th       | HW         | 5000                      | Muslim   |           |
+| MM4                                                                        | 27          | 7th       | HW         | 5000                      | Muslim   |           |
+| MM5                                                                        | 30          | 10th      | HW         | 4500                      | Hindu    | Gen       |
+| MM6                                                                        | 24          | 9th       | HW         | 20,000                    | Muslim   |           |
+| MMn1                                                                       | 28          | 10th      | HW         | 6000                      | Hindu    | Gen       |
+| MMn2                                                                       | 28          | 9th       | HW         | 10,000                    | Hindu    | Gen       |
+| MGn3                                                                       | 33          | 3rd       | HW         | 5000                      | Hindu    | Gen       |
+| MGn4                                                                       | 21          | 10th      | HW         | 3000                      | Hindu    | Gen       |
+| MGn5                                                                       | 24          | 7th       | HW         | 7000                      | Hindu    | Gen       |
+| MMn6                                                                       | 23          | 10th      | HW         | 12,000                    | Hindu    | Gen       |
+| MN7                                                                        | 26          | 7th       | HW         | 10,000                    | Muslim   |           |
+| MN8                                                                        | 30          | 7th       | HW         | 6000                      | Muslim   |           |
+| MN9                                                                        | 20          | 9th       | HW         | 20,000                    | Muslim   |           |
+| MN10                                                                       | 27          | 10th      | HW         | 10,000                    | Muslim   |           |
+| MN11                                                                       | 22          | 8th       | HW         | 6000                      | Muslim   |           |
+| HW: House wife                                                             |             |           |            |                           |          |           |

medical/md/PMC6614857.md

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+Open Access Full Text Article
+
+# Original Research Integrative Analysis Of The Lncrna-Associated Cerna Network Reveals Lncrnas As Potential Prognostic Biomarkers In Human Muscle-Invasive Bladder Cancer
+
+| Lei Lyu Wei Xiang Jin-Yan Zhu Tao Huang Jing-Dong Yuan Chuan-Hua Zhang Department of Urology, Wuhan No. 1 Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, People's Republic of China   |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+Correspondence: Chuan-Hua Zhang Department of Urology, Wuhan No. 1 Hospital, Huazhong University of Science and Technology, No. 215 zhongshan Road, Wuhan 430030, Hubei Province, People's Republic of China Tel +86 1 808 648 5857 Email CH_Zhang07@163.com This article was published in the following Dove Press journal: Cancer Management and Research
+
+| and Genomes (KEGG) pathway enrichment analyses were performed to determine the principal functions of significantly dysregulated mRNAs. The dysregulated lncRNA-associated ceRNA network of MIBC was constructed based on the bioinformatics data, and the correlations between lncRNA expression and clinical features were analyzed using a weighted gene coexpression network analysis (WGCNA). Six cancer specific lncRNAs from the ceRNA network were randomly selected to detect their expression in 32 paired MIBC tissue samples and 5 bladder cancer cell lines using quantitative real-time polymerase chain reaction (qRT-PCR). Results: The ceRNA network was constructed with 30 lncRNAs, 13 miRNAs and 32 mRNAs. Seventeen lncRNAs in the ceRNA network correlated with certain clinical features, and only 1 lncRNA (MIR137HG) correlated with the overall survival (OS) of patients with MIBC (log-rank test P<0.05). GO and KEGG analyses revealed roles for the potential mRNA targets of MIR137HG in epithelial cell differentiation and the peroxisome proliferator-activated receptor (PPAR) and tumor necrosis factor (TNF) signaling pathways. The expression data from TCGA were highly consistent with the verification results of the MIBC tissue samples and bladder cancer cell lines. Conclusion: These findings improve our understanding of the regulatory mechanism of the lncRNA-miRNA-mRNA ceRNA network and reveal potential lncRNAs as prognostic biomarkers of MIBC. Keywords: muscle-invasive bladder cancer (MIBC), long noncoding RNA (lncRNA), competing endogenous RNA (ceRNA), bioinformatics analysis   |
+|---|
+
+## Introduction
+
+Bladder cancer is the most common urological malignancy and a major cause of mortality in the elderly population in China.1 Although the mortality rate of bladder cancer has decreased, approximately 30% of patients have muscle-invasive bladder cancer (MIBC, pathologically classified as ≥T2 using the American Joint Committee on Cancer TNM staging system) at the time of the initial diagnosis, and the tumor may rapidly progress and metastasize, resulting in a poor prognosis.2 Currently, radical cystectomy (RC) and chemoradiotherapy are considered the main treatments for MIBC.3 However, many patients with MIBC develop a metastatic disease within 2 years of diagnosis and usually succumb to their disease.4 Therefore, investigations of the mechanism that promotes MIBC progression and the identification of new molecular prognostic biomarkers are crucial.
+
+With the development of high-throughput sequencing technology, numerous studies have reported key genes associated with cancer progression. However, the identification of genes associated with the progression of MIBC remains challenging. Interest in the biological roles of long noncoding RNAs (lncRNAs) in cancer progression and development has increased.5 The lncRNAs, ranging from 200 nucleotides to 100 kb in length, can regulate gene expression at the transcriptional, posttranscriptional and epigenetic levels.6 Aberrantly expressed lncRNAs are associated with various human tumors, such as squamous cell lung carcinoma, renal cell carcinoma, breast cancer and glioblastoma.7–10 In bladder cancer, lncRNAs may function as oncogenes or tumor suppressors and potentially affect the prognosis of patients. For example, upregulated expression of the lncRNA UCA1 promotes the proliferation and invasion of bladder cancer, and lncRNA MALAT-1 might contribute to the epithelial to mesenchymal transition (EMT) and metastasis of bladder cancer in vitro by activating the Wnt/β-catenin signaling pathway.11,12 However, the molecular mechanisms by which lncRNAs regulate the progression and development of MIBC remain largely unknown.
+
+The formation and development of tumors are complex processes regulated by gene networks, and lncRNAs may interact with miRNAs, mRNAs or other molecules with regulatory capacities. The competing endogenous RNA (ceRNA) hypothesis was proposed as a novel regulatory mechanism between noncoding RNAs and coding RNAs.13 Notably, lncRNAs may act as ceRNAs to interact with miRNAs through miRNA response elements (MREs) and thereby regulate mRNA expression. Accumulating data have confirmed this complex crosstalk of the ceRNA network in many diseases, particularly in cancer.
+
+According to Zheng et al14, the lncRNA HOTAIR promotes cell migration and invasion by regulating MKL1 expression through the inhibition of miR206 expression in HeLa cells. Wu et al15 showed that the lncRNA MEG3 inhibited the progression of prostate cancer by modulating miR-9-5p expression. However, studies employing large samples to investigate the ceRNA mechanisms of lncRNAs in MIBC are still rare, and the relationships between lncRNAs and the clinical features of MIBC are unclear.
+
+In this study, we obtained RNA sequencing data from 373 MIBC tissue samples and 19 adjacent nontumor bladder tissue samples from The Cancer Genome Atlas
+(TCGA) database. Subsequently, we constructed an lncRNA-miRNA-mRNA ceRNA regulatory network and discovered specific lncRNA markers in the MIBC tissue samples. Moreover, we analyzed the correlations between these specific lncRNAs and clinical features of MIBC.
+
+More importantly, we used tissue samples from our patients and bladder cancer cells to verify the bioinformatics analysis results using quantitative real-time polymerase chain reaction (qRT-PCR). The findings from the present study not only improve our understanding of the function of lncRNAs in the ceRNA network but also provide potential diagnostic biomarkers for MIBC.
+
+## Methods Patients And Samples
+
+Information from 430 patients was obtained from TCGA
+(https://portal.gdc.cancer.gov/). Additionally, RNA expression profiles and clinical data (age, gender, neoplasm histologic grade, tumor stage and pathologic TNM stage information) were also extracted from TCGA. The exclusion criteria were: (1) a pathologic diagnosis (Tis and T1)
+of a condition other than MIBC, (2) preoperative radiochemotherapy, (3) a lack of complete data for analysis, and
+(4) the presence of other malignancies. As a result, 373 tumor tissue samples and 19 adjacent nontumor bladder tissue samples were obtained for further analysis (Table 1). The study followed the publication guidelines provided by TCGA (http://cancergenome.nih.gov/publications/publica tionguidelines). In addition, 32 pairs of MIBC and paracarcinoma tissue samples were collected from patients without other malignancies who did not receive preoperative radiotherapy or chemotherapy at the Wuhan No. 1 Hospital, Tongji Medical College, Huazhong University of Science and Technology. The tissues were quickly frozen in liquid nitrogen immediately after surgery and stored until RNA extraction. Written informed consent was obtained from all patients, and was in accordance with
+
+| Table 1 Clinical features of the patients with MIBC from TCGA Characteristics Patients (n=373) n % Gender Male 274 73.5 Female 99 26.5 Age (years) ≤60 96 25.7 >60 277 74.3 Tumor stage Stage II 103 27.6 Stage III 140 37.5 Stage IV 130 34.9 Neoplasm histological grade Low 19 5.1 High 352 94.4 Unknown 2 0.5 TNM staging system T2 119 31.9 T3 195 52.3 T4 59 15.8 TNM staging system N0 222 59.5 N1-3 127 34.1 NX 24 6.4 TNM staging system M0 176 47.2 M1 8 2.1 MX 189 50.7 Abbreviations: MIBC, muscle-invasive bladder cancer; TCGA, The Cancer Genome Atlas; TNM, tumor nodes metastasis.   |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+the Declaration of Helsinki. This study protocol was approved by the institutional ethics committee of Wuhan No. 1 Hospital, Tongji Medical College, Huazhong University of Science and Technology.
+
+## Acquisition And Analysis Of Rna (Lncrna, Mirna And Mrna) Expression Profiles
+
+RNA sequencing data (RNA-Seq2 level 3 data) from human MIBC (pathologic stage T2~T4) samples were collected from the TCGA data portal (https://portal.gdc.cancer.
+
+gov/, platform: Illumina HiSeq 2,000 RNA Sequencing, through Dec, 2018). In addition, the level 3 miRNA sequencing data, which were downloaded from TCGA, were obtained from Illumina HiSeq 2,000 miRNAseq platforms. No further normalization was needed, as TCGA had already normalized the RNA (lncRNA, mRNA and miRNA)
+sequencing expression profile data. Then, we divided the samples into 4 groups (adjacent nontumor; and tumor stages
+Ⅱ, Ⅲ and Ⅳ) and used Empirical Analysis of Digital Gene Expression Data in R (edgeR) to compare differences in the expression levels of RNAs between stage II tumor tissues and adjacent nontumor bladder tissues, between stage III
+tumor tissues and adjacent nontumor bladder tissues, between stage IV tumor tissues and adjacent nontumor bladder tissues and between all tumor tissues and adjacent nontumor bladder tissues (P≤0.05, log2 fold change (logFC)
+>2.0, false discovery rate (FDR)<0.01). Next, we identified intersections of differentially expressed lncRNAs, miRNAs and mRNAs from the 4 comparison groups for hierarchical clustering and further bioinformatics analyses. The flow chart below shows the framework used in this study (Figure 1).
+
+## Functional Enrichment Analysis
+
+A Gene Ontology (GO) analysis contains three domains: molecular functions (MFs), cellular components (CCs) and biological processes (BPs). The GO database (http://www. geneontology.org) was used to analyze differentially expressed intersecting mRNAs. The MFs, CCs and BPs of up- and downregulated mRNAs were identified. The potential functions of the aberrantly expressed intersecting mRNAs involved in signaling pathways were analyzed using the Kyoto Encyclopedia of Genes and Genomes
+(KEGG) (http://www.kegg.jp). In addition, the relationships among the enriched clusters from the GO and KEGG pathway analyses were visualized using Metascape (http://www.metascape.org/).
+
+## Construction Of The Lncrna-Mirnamrna Cerna Network
+
+The lncRNA-miRNA-mRNA ceRNA network was constructed based on the ceRNA hypothesis that lncRNAs regulate the activity of mRNAs by sequestering and binding miRNAs, thereby acting as miRNA sponges. The lncRNA-miRNA interactions were predicted by miRcode databases (http://www.mircode.org/), and the Starbase (http://starbase.sysu.edu.cn/), miRDB (http://mirdb.org/)
+and TargetScan (http://www.targetscan.org/vert_72/) databases were used to identify the target mRNAs to establish the ceRNA network. Then, the differentially expressed intersecting lncRNAs, miRNAs and mRNAs were retained to construct a ceRNA network. The ceRNA network was visualized using Cytoscape v3.6.
+
+![3_image_0.png](3_image_0.png)
+
+## Weighted Gene Coexpression Network Analysis (Wgcna)
+
+The scale-free coexpression network was constructed using the "WGCNA"16 package in R to reveal the correlated gene modules and clinical features. First, aberrantly expressed lncRNA matrices from 373 MIBC samples and complete clinical data were evaluated to determine whether the samples and genes were of sufficient quality.
+
+Outlier samples were removed to ensure that the results of the constructed coexpression network were reliable. Next, the coexpression similarity matrix was transformed into an adjacency matrix by choosing an appropriate soft-thresholding power β value. Then, a topological matrix was established using the topological overlap measures (TOM), and average linkage hierarchical clustering was performed according to the TOM-based dissimilarity measure. Finally, the coexpression gene modules and moduletrait relationships were identified using the dynamic hybrid cut method (a bottom-up algorithm). Moreover, gene significance (GS) was calculated to measure the correlation between gene expression and sample traits (age, gender, neoplasm histologic grade, tumor stage and pathologic TNM stage), and module significance (MS) was calculated to identify the correlations between modules and sample traits. Statistically significant modules were identified with P<0.05.
+
+## Survival Analysis
+
+Kaplan-Meier survival curves were generated to evaluate the prognostic value of the lncRNAs in the ceRNA network using the R package "survival" (https://CRAN.Rproject.org/package=survival). For the overall survival
+(OS) analysis, the 373 patients were divided into 2 groups according to median expression of the lncRNAs (high vs low). The survival curves of samples with low expression of the lncRNAs and high expression of the lncRNAs were compared using the log-rank test. P<0.05 indicated statistically significant differences.
+
+## Cell Culture
+
+Five human bladder cancer cell lines (T24, J82, UM-UC3, TCCSUP and SW780) and a human immortalized uroepithelial cell line (SV-HUC-1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, US). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Thermo Scientific HyClone, Logan, UT, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C and 5% CO2.
+
+## Qrt-Pcr
+
+Total RNA was extracted using the Trizol Reagent kit (Invitrogen, Carlsbad, CA, USA) and was reverse transcribed into cDNAs by using PrimeScript RT-polymerase (Takara, Dalian, China). Real-time PCR was performed on the cDNA
+templates using specific primers (Sangon, Shanghai, China)
+and the SYBR master mix (Takara, Dalian, China). The relative expression levels of the lncRNAs were calculated as a ratio normalized to GAPDH expression. Comparative quantification was performed using the 2−ΔΔCt method. The sequences of the specific primers used in the present study are listed below. LINC00518 (ENSG00000183674): F, 5ʹGCTGTATAGAAGG TGCTAATG-3ʹ; and R, 5ʹ-AGTAGAG
+GCTAGAACTGGA A-3ʹ. ADAMTS9-AS1 (ENSG0000024 1158): F, 5ʹ-ACTTCTTGCCATTCCTCTG-3ʹ; and R, 5ʹCTGTCTT CTTAGTGCTCTGT-3ʹ. MIR137HG (ENSG000 00225206): F, 5ʹ-GGTGGATAATAC GGATTACG-3ʹ; and R, 5ʹ-GCTGTGGTGAGTCAAGAT-3ʹ. LINC00525 (ENSG000 00146666): F, 5ʹ-TACATCCACGAAGCAGATT-3ʹ; and R, 5ʹ-CAACGCA AGAGTTAGTCAG-3ʹ. AC110491.1 (ENSG 00000261292): F, 5ʹ-ACATACAAGGC TATCAGACA-3ʹ; and R, 5ʹ-TGAGACA GAGGACAGAAGA-3. GAPDH (ENSG00000111640): F, 5ʹ-TGAAGGTCGGAGT CAACG G-3ʹ; and R, 5ʹ-CCTGGA AGATGGTGATGGG-3ʹ.
+
+## Statistical Analysis
+
+Statistical analyses were performed by R Studio (R version 3.5.1) and SPSS 19.0 (SPSS Inc., Chicago, IL, USA).
+
+Differentially expressed RNAs were identified using edgeR from R Studio. Two-tailed unpaired Student's ttest was used to compare the differences between two groups. Differences were considered statistically significant when P<0.05.
+
+## Results Differentially Expressed Mrnas And Their Functions In Mibc
+
+A total of 1,683 mRNAs (logFC >2, FDR<0.01) were differentially expressed between MIBC tissue samples and adjacent nontumor bladder tissue samples from the TCGA
+database. Notably, 1,746 mRNAs were differentially expressed between stage II MIBC samples and adjacent nontumor bladder tissues, 1,645 mRNAs were differentially expressed between stage III MIBC samples and adjacent nontumor bladder tissues, and 1,678 mRNAs were differentially expressed between stage IV MIBC samples and adjacent nontumor bladder tissues. Next, we identified 1,136 intersecting mRNAs (Figure 2A) from these mRNAs (493 upregulated and 643 downregulated). We performed GO and KEGG pathway enrichment analyses using Metascape to determine the potential functions of these dysregulated mRNAs. The GO terms of in which the upregulated genes were enriched were mainly DNA packaging complexes, mitotic nuclear division and transcription regulation in the mitotic cell cycle (Figure 3A), while the main functions of the downregulated mRNAs involved muscle system processes, the regulation of membrane potential and the extracellular matrix (Figure 3B). The significant KEGG pathways in which the upregulated genes were enriched mainly included the cell cycle, transcriptional misregulation in cancer, the IL-17 signaling pathway and the peroxisome proliferator-activated receptor (PPAR) signaling pathway (Figure 4A). The pathways associated with the downregulated mRNAs included pathways associated with cancer, the MAPK signaling pathway, proteoglycans in cancer and cell adhesion molecules (Figure 4B). Based on the results of the functional enrichment analyses, these dysregulated mRNAs participated in carcinogenesis and the development of MIBC by regulating related BPs and key pathways.
+
+## Prediction Of Mirna Targets And Construction Of The Cerna Network
+
+A total of 263 differentially expressed lncRNAs (181 upregulated and 82 downregulated) were identified between MIBC tissues and adjacent nontumor tissues, and the tumor tissues from patients with stage II, III and IV MIBC were compared to adjacent nontumor tissues (Figure 2B).
+
+Subsequently, we identified 115 aberrantly expressed miRNAs (96 upregulated and 19 downregulated) by selecting the intersections of specific miRNAs across the 4 comparison groups (Figure 2C). Then, we focused on the target
+
+![5_image_0.png](5_image_0.png)
+
+![5_image_1.png](5_image_1.png)
+
+relationships between these 115 miRNAs and 263 specific lncRNAs identified above. Finally, 13 specific miRNAs were predicted to target 30 specific lncRNAs (Tables 2 and 3),
+based on the analysis using miRcode.
+
+In the next step, we chose the 13 miRNAs mentioned above to predict targeted mRNAs using the TargetScan, Starbase and miRDB databases. We then compared the predicted mRNAs to the 1136 intersecting specific mRNAs and obtained the mRNAs present in both databases. Finally, 11 specific miRNAs (hsa-mir-301b and hsa-mir-143 did not target mRNAs from the intersecting specific mRNAs) were identi-fied to interact with the 32 intersecting mRNAs (Table 4).
+
+Some of these targeted mRNAs are cancer-associated genes, such as AKAP12, EPHA7, ZEB1, TMEM100, FGF2, E2F7 and BTG2. Based on the aforementioned data, the lncRNAmiRNA-mRNA ceRNA network was established and visualized using Cytoscape 3.6. As shown in Figure 5A, 30 lncRNAs, 13 miRNAs and 32 mRNAs were involved in the
+
+![6_image_0.png](6_image_0.png) 
+proposed network. Regression analyses were performed between the expression levels of the key lncRNAs and mRNAs in the ceRNA network to further validate the prediction. The expression levels of the lncRNAs AC110491.1 and ADAMTS9-AS1 were also positively correlated with the mRNAs AKAP12, EPHA7 or TMEM100, suggesting that AC110491.1 and ADAMTS9-AS1 competitively bound to MREs in hsa-mir-141-3p, hsa-mir-183-5p or hsa-mir-195-5p to prevent the miRNA-mediated inhibition of target gene expression (Figure 5B).
+
+## The Correlations Between Mibc-Specific Lncrnas And Clinical Features
+
+We performed a WGCNA to construct a gene coexpression network, identify the correlations between the specific lncRNAs and clinical features, including age, gender, neoplasm histologic grade, tumor stage and pathologic TNM stage, and further study the 30 key lncRNAs in the ceRNA network. Finally, 9 gene modules were identified using the dynamic tree cutting method. We then analyzed the relationship between the gene modules and the clinical features of the sample (Figure 6). The lncRNAs from the modules with the highest positive or negative scores that correlated with clinical features were selected to intersect with lncRNAs from the ceRNA network. As shown in Table 5, the aberrant expression of 3 lncRNAs
+(MIR137HG, LINC00460 and TM4SF19-AS1) significantly correlated with the tumor stage and pathologic T
+stage, 9 lncRNAs (C2orf48, C20orf197, AP002478.1, LINC00518, AC073352.1, C9orf163, IGF2-AS, DLEU7-
+AS1 and AC078778.1) significantly correlated with the neoplasm histologic grade, the expression of 3 lncRNAs Table 2 Key lncRNAs, miRNAs and mRNAs involved in the ceRNA networklncRNAs FDR miRNAs Log2 (FC) 
+FDR mRNAs Log2 (FC) 
+FDR
+3.46E-04 7.94E-04 5.97E-05 1.39E-03 1.67E-14 2.91E-10 3.23E-08 2.94E-18 5.47E-19 8.29E-20 6.29E-28 6.93E-20 3.42E-09 2.76E-47 7.50E-20 1.81E-22 1.98E-59 9.55E-25 3.32E-05 1.37E-46 1.21E-12 8.09E-25 2.69E-42 6.37E-30 4.62E-15 1.09E-20 1.66E-60 1.31E-20 3.97E-24 6.31E-41 3.93E-58 2.51E-13
+
+| PRICKLE2 −2.34 LRRK2 −2.44                                                                                                                                                                                                                                                                    | ITPR1 −2.87 SHISA6 −2.87                                                                                                                        | CFL2 −3.24 MKX −3.30                                                                     | FAM129A −3.70                                                          |             |              |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------|------------------------------------------------------------------------|-------------|--------------|
+| 3.22 2.82 2.75 2.75                                                                                                                                                                                                                                                                           | 2.25 2.18 TGFBR3 −2.21 CYBRD1 −2.21 BTG2 −2.23                                                                                                  | SELE −2.46 NACC2 −2.51 RUNX1T1 −2.65 AKAP12 −2.66 SLC25A25 −2.69 ZFPM2 −2.73 SALL3 −2.78 | FGF2 −2.92 RAB23 −2.95 ZEB1 −2.96 TPM2 −3.03 GREM2 −3.09 TMEM100 −3.13 | EPHA7 −3.39 | SERTM1 −3.99 |
+| 2.31                                                                                                                                                                                                                                                                                          |                                                                                                                                                 |                                                                                          |                                                                        |             |              |
+| WNT7A                                                                                                                                                                                                                                                                                         | HOXC13                                                                                                                                          |                                                                                          |                                                                        |             |              |
+| ELAVL2                                                                                                                                                                                                                                                                                        | CCNE1                                                                                                                                           |                                                                                          |                                                                        |             |              |
+| POLQ                                                                                                                                                                                                                                                                                          |                                                                                                                                                 |                                                                                          |                                                                        |             |              |
+| DIO1                                                                                                                                                                                                                                                                                          | E2F7                                                                                                                                            |                                                                                          |                                                                        |             |              |
+| 2.43E-04 1.66E-16 4.39E-12 2.25E-16 1.03E-12 1.39E-08 5.30E-05                                                                                                                                                                                                                                | 5.27E-09 9.40E-06 1.06E-35                                                                                                                      | 2.06E-42                                                                                 |                                                                        |             |              |
+| 5.79E-11                                                                                                                                                                                                                                                                                      | 1.15E-11                                                                                                                                        |                                                                                          |                                                                        |             |              |
+| hsa-mir-195-5p −2.52 hsa-mir-383-5p −3.18 hsa-mir-143-3p −3.58                                                                                                                                                                                                                                |                                                                                                                                                 |                                                                                          |                                                                        |             |              |
+| 6.77 4.83 3.86 3.71 3.00 2.70 2.67 2.57 2.35 2.14 hsa-mir-210-3p hsa-mir-96-5p hsa-mir-183-5p hsa-mir-31-5p hsa-mir-141-3p hsa-mir-503-5p hsa-mir-205-5p hsa-mir-519d hsa-mir-301b hsa-mir-429 8.81E-08 1.36E-05 1.15E-04 5.94E-05 2.57E-06 6.42E-05 3.11E-06 1.72E-05 3.85E-09               | 1.87E-05 3.35E-10 9.36E-05 2.46E-12 1.28E-05 1.95E-05 1.66E-06 5.20E-12 1.82E-06 1.47E-08 3.07E-17 6.07E-14 2.74E-09 1.79E-25 1.19E-14 2.50E-45 | 1.96E-85                                                                                 |                                                                        |             |              |
+| 1.98E-03 5.62E-03                                                                                                                                                                                                                                                                             | 2.28E-03                                                                                                                                        | 3.32E-51                                                                                 | Abbreviations: FC, fold change; FDR, false discovery rate.             |             |              |
+| LINC00472 −2.11 LINC00402 −2.29 NALCN-AS1 −2.45 AC008676.1 −2.50 PART1 −2.74 LINC00163 −2.87 JAZF1-AS1 −3.10 AC110491.1 −3.63 ADAMTS9-AS2 −4.02 ADAMTS9-AS1 −4.95 HCG22 −7.22                                                                                                                 |                                                                                                                                                 |                                                                                          |                                                                        |             |              |
+| 7.11 6.14 5.07 5.07 4.45 4.14 3.40 3.13 2.89 2.70 2.67 2.64 2.51 2.48 2.35 2.30 2.24 2.24 2.23 TM4SF19-AS1 LINC00460 LINC00392 LINC00518 AC011453.1 AL513123.1 AP000525.1 AP002478.1 AC073352.1 LINC00525 LINC00487 AC078778.1 DLEU7-AS1 ERVH48-1 MIR137HG C20orf197 IGF2-AS C9orf163 C2orf48 |                                                                                                                                                 |                                                                                          |                                                                        |             |              |
+
+Log2 (FC) 
+submit your manuscript | www.dovepress.com 6068 Cancer Management and Research 2019:11 Lyu et al Dovepress
+
+| Table 3 Specific miRNAs that target specific lncRNAs lncRNAs miRNAs IGF2-AS hsa-mir-503-5p, hsa-mir-519d-3p LINC00525 hsa-mir-301b, hsa-mir-96-5p, hsa-mir-141-3p, hsa-mir-31-5p, hsa-mir-383-5p PART1 hsa-mir-301b, hsa-mir-141-3p, hsa-mir-143-3p, hsa-mir-195-5p, hsa-mir-429, hsa-mir-205-5p, hsa-mir-31-5p C2orf48 hsa-mir-143-3p, hsa-mir-195-5p, hsa-mir-519d-3p, hsa-mir-183-5p C20orf197 hsa-mir-143-3p, hsa-mir-519d-3p, hsa-mir-383-5p AP002478.1 hsa-mir-503-5p, hsa-mir-195-5p, hsa-mir-519d-3p, hsa-mir-205-5p LINC00518 hsa-mir-141-3p, hsa-mir-143-3p C9orf163 hsa-mir-143-3p, hsa-mir-195-5p, hsa-mir-205-5p AC008676.1 hsa-mir-301b, hsa-mir-141-3p, hsa-mir-143-3p, hsa-mir-519d-3p, hsa-mir-31-5p LINC00487 hsa-mir-143-3p, hsa-mir-183-5p, hsa-mir-205-5p, hsa-mir-31-5p AP000525.1 hsa-mir-503-5p, hsa-mir-31-5p AL513123.1 hsa-mir-141-3p, hsa-mir-183-5p LINC00392 hsa-mir-183-5p HCG22 hsa-mir-96-5p, hsa-mir-195-5p, hsa-mir-31-5p, hsa-mir-383-5p MIR137HG hsa-mir-31-5p NALCN-AS1 hsa-mir-195-5p, hsa-mir-205-5p, hsa-mir-31-5p, hsa-mir-383-5p ERVH48-1 hsa-mir-301b, hsa-mir-96-5p, hsa-mir-141-3p LINC00472 hsa-mir-503-5p, hsa-mir-141-3p, hsa-mir-143-3p, hsa-mir-195-5p, hsa-mir-383-5p AC110491.1 hsa-mir-141-3p, hsa-mir-143-3p, hsa-mir-429, hsa-mir-205-5p LINC00460 hsa-mir-503-5p, hsa-mir-143-3p, hsa-mir-429 JAZF1-AS1 hsa-mir-143-3p, hsa-mir-519d-3p, hsa-mir-205-5p LINC00163 hsa-mir-143-3p, hsa-mir-183-5p, hsa-mir-205-5p, hsa-mir-31-5p, hsa-mir-210-3p LINC00402 hsa-mir-141-3p, hsa-mir-143-3p, hsa-mir-519d-3p, hsa-mir-429, hsa-mir-383-5p TM4SF19-AS1 hsa-mir-141-3p, hsa-mir-205-5p DLEU7-AS1 hsa-mir-96-5p, hsa-mir-195-5p ADAMTS9-AS1 hsa-mir-301b, hsa-mir-96-5p, hsa-mir-31-5p ADAMTS9-AS2 hsa-mir-301b, hsa-mir-96-5p, hsa-mir-141-3p, hsa-mir-143-3p, hsa-mir-183-5p, hsa-mir-205-5p, hsa-mir-31-5p AC078778.1 hsa-mir-301b AC073352.1 hsa-mir-96-5p AC011453.1 hsa-mir-143-3p, hsa-mir-205-5p   |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+Table 4 Specific mRNAs that target specific miRNAs
+
+| miRNAs          | mRNAs                                                                                       |
+|-----------------|---------------------------------------------------------------------------------------------|
+| hsa-mir-141-3p  | ZEB1, ELAVL2, EPHA7                                                                         |
+| hsa-mir-183-5p  | ZEB1, AKAP12                                                                                |
+| hsa-mir-195-5p  | TGFBR3, WNT7A, FGF2, RAB23, TMEM100, CCNE1, TPM2, MKX, PRICKLE2, E2F7, RUNX1T1, BTG2, ITPR1 |
+| hsa-mir-205-5p  | LRRK2, ZEB1, SHISA6                                                                         |
+| hsa-mir-210-3p  | SERTM1                                                                                      |
+| hsa-mir-31-5p   | HOXC13, SELE                                                                                |
+| hsa-mir-383-5p  | DIO1                                                                                        |
+| hsa-mir-429     | ZEB1, ZFPM2                                                                                 |
+| hsa-mir-503-5p  | GREM2                                                                                       |
+| hsa-mir-519d-3p | CYBRD1, POLQ, FAM129A, ELAVL2, SALL3, CFL2, NACC2                                           |
+| hsa-mir-96-5p   | ZEB1, SLC25A25                                                                              |
+
+(LINC00525, LINC00392 and AC011453.1) was dysregulated in tumors with the pathologic N stage, and 2 lncRNAs (ERVH48-1 and NALCN-AS1) were markedly differentially expressed in tumors with the pathologic M stage. More importantly, the results of the Kaplan-Meier survival curves revealed a significant negative correlation
+
+![9_image_0.png](9_image_0.png)
+
+(Figure 7A). We constructed a coexpression network based on the Pearson correlation coefficient (|cor|>0.4, P<0.01) between MIR137HG and the mRNAs to predict the potential targets of MIR137HG. Three hundred mRNAs potentially targeted MIR137HG, and the nt your ipt 
+
+![10_image_0.png](10_image_0.png)
+
+expression levels of the top 20 mRNAs that were potentially positively or negatively correlated with MIR137HG
+are shown as a heatmap in Figure 7B. Subsequently, enrichment analyses based on GO and KEGG pathways were performed to predict the potential functions of all mRNAs targeted by MIR137HG. The results from the GO and KEGG analyses showed that MIR137HG may be involved in epithelial cell differentiation and the PPAR signaling pathway (Table 6).
+
+## Qrt-Pcr Verification Of Representative Lncrnas In Mibc Tissue Samples And Bladder Cancer Cell Lines
+
+We randomly chose 6 lncRNAs (MIR137HG, LINC00525, AC110491.1, ERVH48-1, LINC00518 and ADAMTS9-
+AS1) from the ceRNA network to investigate their expression patterns in 32 paired samples from patients with MIBC and 5 bladder cancer cell lines (T24, J82, UMUC-3,TCCSUP and SW780) using qRT-PCR to verify the reliability and validity of the above bioinformatics analysis results. As shown in Figure 8A and B, the expression of 4 lncRNAs (MIR137HG, LINC00525, ERVH48-1 and LINC00518) was upregulated, while the expression of AC110491.1 and ADAMTS9-AS1 was downregulated in MIBC tissues and bladder cancer cells (T24, J82, UM-UC3, TCCSUP and SW780) compared with the expression in adjacent nontumor bladder tissues and the immortalized SV-HUC-1 uroepithelial cells, respectively. The results were consistent with the results of the bioinformatics analysis described above. A subsequent analysis was performed to correlate the expression of these 6 lncRNAs with clinical features. As shown in Figure 8C–E, LINC00518 significantly correlated with the neoplasm histologic grade, MIR137HG significantly correlated with the pathologic T stage, LINC00525 significantly correlated
+
+| Table 5 The correlations between key lncRNAs from the ceRNA network and clinical features of MIBC Clinical feature Upregulated (fold change) Downregulated (fold change) Tumor stage (III+IV vs II) MIR137HG (2.21), LINC00460 (1.34), TM4SF19-AS1 (1.12) Neoplasm histological grade (High vs Low) C2orf48 (5.13), C20orf197 (1.18), AP002478.1 (1.46) C9orf163 (0.63), IGF2-AS (0.32) LINC00518 (10.48), AC073352.1 (1.32) DLEU7-AS1 (0.76), AC078778.1 (0.91) TNM staging system T3+T4 vs T2 MIR137HG (1.34), LINC00460 (1.45), TM4SF19-AS1 (1.22) N1-3 vs N0 LINC00525 (1.31), LINC00392 (1.38) AC011453.1 (0.91) M1 vs M0 ERVH48-1 (1.76) NALCN-AS1 (0.11) Abbreviations: MIBC, muscle-invasive bladder cancer; TNM, tumor nodes metastasis.   |
+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+![11_image_0.png](11_image_0.png)
+
+with the pathologic N stage and ERVH48-1 significantly correlated with the pathologic M stage. Thus, bioinformatics analysis described above was reliable and valid.
+
+## Discussion
+
+Based on accumulating evidence, lncRNAs play central roles in cancer development and progression. Notably, lncRNAs can regulate various BPs, including cell proliferation, apoptosis, invasion, immune response and development.17–20 Although the exact mechanism is unclear, an increasing number of researchers have focused on ceRNA coexpression networks in which lncRNAs may affect the expression of proteincoding genes by interacting with miRNAs. Recent studies have confirmed several potential ceRNAs in various human tumors, such as renal cell carcinoma, lung adenocarcinoma, hepatocellular carcinoma and gastric cancer.21–24 Zhang et al25 revealed significant correlations between the expression of 3 key lncRNAs (LINC00355, HULC and IGF2-AS) and clinical features of human colon adenocarcinoma. Sui et al26 revealed correlations between the expression of 5 cancer-specific lncRNAs (BCRP3, LINC00472, CHIAP2, BMS1P20 and UNQ6494) and OS in patients with lung adenocarcinoma and confirmed that the 5 lncRNAs were independent predictors of prognosis using a univariate Cox regression analysis.
+
+However, the potential function of the ceRNA network in MIBC is still poorly understood.
+
+In the present study, we first identified 263 lncRNAs, 115 miRNAs and 1,136 mRNAs that were aberrantly expressed in tumor stages II~IV of MIBC according to data from TCGA. Using GO and KEGG analyses, we determined the functions and signaling pathways in which these aberrantly expressed mRNAs were involved. Subsequently, we used bioinformatics tools to establish a ceRNA coexpression network with 30 lncRNAs, 13
+
+Table 6 GO and KEGG analyses of the mRNA targets of MIR137HG
+
+| MIR137HG Items                                                                   | Category ID   | Log10P   |
+|----------------------------------------------------------------------------------|---------------|----------|
+| GO Epithelial cell differentiation                                               | GO:0030855    | −4.92    |
+| Regulation of cytokine production                                                | GO:0001817    | −4.49    |
+| Focal adhesion                                                                   | GO:0005925    | −4.31    |
+| Connective tissue development                                                    | GO:0061448    | −4.52    |
+| KEGG PPAR signaling pathway                                                      | hsa03320      | −2.71    |
+| ECM-receptor interaction                                                         | hsa04512      | −1.73    |
+| PI3K-Akt signaling pathway                                                       | hsa04151      | −1.57    |
+| TNF signaling pathway                                                            | hsa04668      | −1.35    |
+| Abbreviations: GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes. |               |          |
+
+miRNAs and 32 mRNAs. We further analyzed the correlations between lncRNAs and mRNAs in the ceRNA network. We also investigated the relationships between the lncRNAs that were involved in the ceRNA network and clinical features and identified 1 key lncRNA from the ceRNA network that was related to OS. These results were verified in MIBC tissue samples from 32 patients and 5 bladder cancer cell lines (T24, J82, UM-UC-3, TCCSUP and SW780) using qRT-PCR.
+
+The GO and KEGG pathway analyses of the 1,136 intersecting mRNAs identified the important BPs and pathways in MIBC, most of which were classic pathways and BPs that play critical roles in bladder cancer, such as transcriptional misregulation in cancer, the MAPK signaling pathway, and the cell cycle. Interestingly, some of the other novel BPs and pathways involved in MIBC progression and development, such as muscle system processes, DNA packaging complexes, calcium signaling pathways and protein digestion, supplemented the findings of previous studies.
+
+Based on the ceRNA hypothesis, we selected the lncRNAs, miRNAs and mRNAs that were aberrantly expressed in MIBC to construct a MIBC ceRNA network using bioinformatics tools (miRcode, TargetScan, Starbase and miRDB). This ceRNA network provided considerable information to improve our understanding of the key role of lncRNAmediated gene regulatory networks in MIBC development.
+
+From the ceRNA network, we ultimately identified 30 lncRNAs, 13 miRNAs and 32 mRNAs that formed a complex network. For example, mRNAs such as ZEB1, TGFBR3, E2F7 and HOXC13 were regulated by multiple lncRNAs (LINC00525, MIR137HG and HCG22) through a pathway mediated by miRNAs (miR-141-3p, miR-31-5p and miR-
+195-5p). These results were consistent with the GO and KEGG enrichment analyses, suggesting that transcriptional misregulation in cancer is involved in MIBC progression. Moreover, the results from the ceRNA network also revealed that 2 downregulated lncRNAs (AC110491.1 and ADAMTS9-AS1) were positively correlated with the expression of 3 tumor suppressor protein-coding genes (AKAP12, EPHA7 and TMEM100). A-kinase anchoring protein 12 (AKAP12) is a kinase scaffold protein with known tumor suppressor activity that is involved in the development and progression of a variety of tumors.27 AKAP12 expression is downregulated in prostate cancer, colon cancer and hepatocellular carcinoma (HCC), and the downregulated expression of AKAP12 is associated with a poor prognosis and high recurrence rates after treatment.28 EPHA7 is a member of the tyrosine kinase receptor family that exerts negative effects on cancer cell proliferation, migration, and adhesion, depending on the presence of membrane-bound ephrin ligands and coreceptors.29 According to Li et al30, the downregulation of EPHA7 suppresses prostate cancer malignancy by targeting the PI3K/Akt signaling pathway. TMEM100 is a novel gene that was first identified as a transcript from the mouse genome in 2001.31 TMEM100 was recently shown to inhibit the proliferation of lung cancer cells, and TMEM100 may function as a novel tumor suppressor gene in HCC proliferation and metastasis.32,33 According to the aforementioned conclusion, we speculated that AC110491.1 and ADAMTS9-AS1 might function as tumor suppressors in MIBC, as AKAP12, EPHA7 and TMEM100 were coexpressed with AC110491.1 and ADAMTS9-AS1, and this coexpression was mediated by miRNAs.
+
+We then used a recently developed methodology to construct a weighted gene coexpression network and analyze the correlations between the expression of specific lncRNAs from the ceRNA network and several clinical features, such as age, gender, neoplasm histologic grade, tumor stage and pathologic TNM stage. Seventeen of 30 lncRNAs from the ceRNA network were significantly associated with clinical features.
+
+Twelve of these 17 lncRNAs were reported to be associated with cancer. For example, LINC00460, which is upregulated by CBP/P300, promotes carcinogenesis in esophageal squamous cell carcinoma.34 LINC00518 contributes to multidrug resistance in breast cancer.35 However, no report has described the correlation between MIBC and the expression of the aforementioned lncRNAs. Therefore, we analyzed the association between the expression of 17 clinical cancer featurespecific lncRNAs and OS, and we found that only MIR137HG
+was significantly associated with the OS of patients with
+
+![13_image_0.png](13_image_0.png)
+
+MIBC. MIR137HG has been reported to be associated with the genetic basis of schizophrenia. 36 Since previous studies have rarely reported the biological functions of MIR137HG in cancer, we further performed GO and KEGG enrichment analyses of the mRNAs that were coexpressed with MIR137HG to explore the functions of MIR137HG.
+
+ript I v MIR137HG may function as an oncogene in MIBC by regulating epithelial cell differentiation, cytokine production, the PPAR signaling pathway and TNF signaling pathway. In our study, we predicted that miR-31-5p was targeted by MIR137HG based on the analysis using miRcode. Meanwhile, the results from the TargetScan, Starbase and miRDB databases further revealed that miR-31-5p targeted the 3ʹ untranslated regions (3ʹUTR) of HOXC13 (Figure 5).
+
+Based on this findings, MIR137HG/miR-31-5p/HOXC13 may represent a ceRNA network that is involved in the tumorigenesis of MIBC. Recently, miR-31 was shown to play dual roles in tumor development. Lv et al37 reported that miR-31 functions as a oncomiR to promote breast cancer formation by regulating the TGFβ and Wnt/β-catenin signaling pathways.
+
+However, Creighton et al38 observed anti-proliferative and pro-apoptosis effects of miR-31 on prostate cancer, osteosarcoma and ovarian cancer. Thus, miR-31 might regulate different biological processes in different cancers, depending on the cellular origin of the tumor. HOXC13, a member of the homeobox HOXC gene family, has been reported to correlate with the development of cancers such as colon cancer,39 esophageal squamous cell carcinoma40 and skin tumors.41 HOXC13 promotes proliferation and inhibits apoptosis in cancers by regulating numerous biological processes and signaling pathways, including the EMT42 and TNF signaling pathway.43 Based on this indirect evidence, MIR137HG functions as an lncRNA-associated ceRNA to promote the progression of MIBC by regulating the expression of miR-31-5p and HOXC13. Because the development of cancer is a complex biological process correlated with multiple genes, the functions of MIR137HG will be further verified in vivo and in vitro in our future research.
+
+Finally, we randomly selected 6 lncRNAs from the ceRNA network (MIR137HG, LINC00525, AC110491.1, ERVH48-1, LINC00518 and ADAMTS9-AS1) and detected their expression in 32 paired MIBC tissue samples and 5 bladder cancer cell lines using qRT-PCR to validate the findings from our bioinformatics analysis. We also analyzed the correlations between the expression of the 6 lncRNAs and clinical features. The expression data from TCGA were highly consistent with the results of the verification experiment using separate MIBC tissue samples and bladder cancer cell lines. These results further support the conclusions based on our bioinformatics analysis.
+
+## Conclusions
+
+In the present study, we successfully identified numerous potential prognostic genes and determined their functions and the signaling pathways in which they participate in MIBC. Moreover, we constructed an lncRNA-miRNAmRNA ceRNA regulatory network to explore the potential roles of lncRNAs in MIBC. Importantly, we further studied the correlation between these key lncRNAs and clinical features, which might serve as a reference for future research. Our findings provide novel insights into and a better understanding of the lncRNA-associated ceRNA network in MIBC. Further studies are needed to completely elucidate the effects of these genes on the development of bladder cancer.
+
+## Acknowledgments
+
+This study was supported by grants from the National Natural Science Foundation of China (81502204), the Hubei Province Nature Science Foundation of China (2014CFB399) and Wuhan Clinical Medical Research of China (WX14A01).
+
+## Disclosure
+
+The authors report no conflicts of interest in this work.
+
+## References
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+
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medical/md/PMC6698310.md

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+www.transonc.com
+
+![0_image_1.png](0_image_1.png)
+
+ORIGINAL ARTICLE
+
+![0_image_0.png](0_image_0.png)
+
+Gene Alterations in TripleNegative Breast Cancer Patients in a Phase I/II Study of Eribulin and Olaparib Combination Therapy Akihiko Shimomura1,*, Kan Yonemori1, Masayuki Yoshida2, Teruhiko Yoshida3, Hiroyuki Yasojima4, Norikazu Masuda4, Kenjiro Aogi5, Masato Takahashi6, Yoichi Naito7, Satoru Shimizu8, Rikiya Nakamura9, Akinobu Hamada10, Hirofumi Michimae11, Jun Hashimoto1,12, Harukaze Yamamoto1, 13, Asuka Kawachi1, Chikako Shimizu1, 14, Yasuhiro Fujiwara1 and Kenji Tamura1 1Department of Breast and Medical Oncology, National Cancer Center Hospital, Tokyo, Japan 2Department of Pathology, National Cancer Center Hospital, Tokyo, Japan 3Department of Genetic Medicine, National Cancer Center Hospital, Tokyo, Japan 4Department of Breast Surgery, National Hospital Organization Osaka National Hospital, Osaka, Japan 5Department of Breast Surgery, National Hospital Organization Shikoku Cancer Center, Matsuyama, Japan 6Department of Breast Surgery, National Hospital Organization Hokkaido Cancer Center, Sapporo, Japan 7Department of Breast and Medical Oncology, National Cancer Center Hospital East, Kashiwa, Japan 8Department of Breast and Endocrine Surgery, Kanagawa, Cancer Center, Yokohama, Japan 9Department of Breast Surgery, Chiba, Cancer Center, Chiba, Japan 10Department of Molecular Pharmacology, National Cancer Center Research Institute, Tokyo, Japan 11Department of Biostatistics, Kitasato University School of Pharmacy, Tokyo, Japan 12Department of Medical Oncology, St. Luke's International Hospital, Tokyo, Japan 13Department of Medical Oncology, National Hospital Organization Kumamoto Medical Center, Kumamoto, Japan 14Department of Breast Medical Oncology, National Center for Global Health and Medicine, Tokyo, Japan
+
+## Abstract
+
+BACKGROUND: We conducted a phase I/II clinical trial to evaluate the efficacy of eribulin and olaparib in a tablet form (EO study) for triple-negative breast cancer (TNBC) patients. We hypothesized that somatic BRCA mutations and homologous recombination repair (HRR)-related gene alterations might affect efficacy. METHODS: Our analyses identifiedmutations in HRR-related genes and BRCA1/2, and we subsequently evaluated their association to response by the EO study participants. Tissue specimens were obtained from primary or metastatic lesion. Tissue specimens were examined for gene mutations or protein expression using a Foundation Medicine gene panel and immunohistochem-
+Address all correspondence to: Akihiko Shimomura, MD, PhD, Department of Breast and Medical Oncology, National Cancer Center Hospital, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. E-mail: ashimomu@ncc.go.jp Received 29 April 2019; Revised 14 July 2019; Accepted 15 July 2019 istry. RESULTS: In the 32 tissue specimens collected, we detected 33 gene mutations, with the most frequent nonsynonymousmutations found in TP53. The objective response rates (ORRs) in patientswith andwithout HRR-related gene mutation were 33.3% and 40%, respectively (P ¼ .732), and the ORRs in patients with and without somatic BRCA
+mutations were 60% and 33.3%, respectively (P ¼ .264), with the ORR numerically higher in the somatic BRCA-mutation group but not statistically significant. There was no correlation between immunohistochemistry status and response or between BRCA status or HRR-related gene mutation and survival. Immunohistochemical analysis indicated that EGFRnegative patients had a tendency for better progression-free survival (log-rank P ¼ .059) and significantly better overall survival (log-rank P ¼ .046); however, there was no correlation between the status of other immunohistochemistry markers and survival. CONCLUSION: These findings suggested somatic BRCA mutation and EGFR-negativity as a potential biomarker for predicting the efficacy of eribulin/olaparib combination therapy. (UMIN000018721).
+
+Translational Oncology (2019) 12, 1386–1394
+
+## Introduction
+
+Breast cancer is the fourth most common cancer in Japanese women and accounted for an estimated 9806 deaths in 2003 [1]. Although there have been major improvements in oncologic treatments, breast cancers frequently recur after primary treatment, and following recurrence, these cancers are incurable. Treatment options for inoperable advanced, metastatic, or recurrent breast cancer includes hormone therapy, anti-human epidermal receptor type 2 (HER2)
+therapy, and chemotherapy, depending on results from pathological examination of the tumor specimens.
+
+Anthracycline and taxane are the major cytotoxic agents representing highly efficacious chemotherapeutics. A chemotherapy regimen including an anthracycline or taxane agent can also be administered as part of first- or second-line chemotherapy for inoperable advanced, metastatic, or recurrent breast cancer; however, these agents are frequently not selected for first-or second-line chemotherapy, because these drugs might have been administered as neoadjuvant or adjuvant chemotherapy during primary treatment. Other cytotoxic agents, such as eribulin, capecitabine, vinorelbine, and gemcitabine, can be selected for systemic chemotherapy in patients previously treated with anthracycline and taxane agents [2]. Eribulin is a non-taxane microtubule inhibitor reported to improve overall survival (OS) of patients with metastatic breast cancer previously treated with anthracycline and taxane and relative to treatment with physicians' choice according to findings from the EMBRACE trial [3]. Since report of the EMBRACE results, eribulin has been considered a standard of care for this population of breast cancer patients and was approved for use by the Food and Drug Administration (FDA) in the United States and the Pharmaceuticals and Medical Devices Agency in Japan in 2010.
+
+In breast cancer, hormone-receptor-negative, HER2-negative breast cancer is referred to as triple-negative breast cancer (TNBC) and estimated to account for 10% to 20% of all breast cancers. TNBC occurs at a younger age and with a higher tumor grade, highly proliferative tumor characteristics, and poor survival as compared with other types of breast cancers. The treatment options for this type of breast cancer are limited to chemotherapy; therefore, there is a large unmet medical need for new treatment strategies for patients with TNBC [4,5].
+
+Olaparib (AZD2281) is a potent polyadenosine 50-diphosphoribose (poly-ADP ribose) polymerase (PARP)-1, -2, and- 3 inhibitor currently being developed as an oral therapy both as monotherapy, including tumor maintenance, and in combination with other anticancer agents. PARP inhibition is a novel approach for targeting tumors with deficiencies in DNA-repair mechanisms. PARP enzymes are essential for repairing DNA single-strand breaks (SSBs), and inhibiting PARPs leads to the persistence of SSBs, which are then converted to more serious DNA double-strand breaks (DSBs) during DNA replication. During the process of cell division, DSBs can be efficiently repaired in normal cells through homologous recombination repair (HRR); however, tumors with HRR deficiencies (HRD),
+such as serous ovarian cancers and breast cancer, cannot accurately repair the DNA damage, resulting in accumulating damaged DNA becoming potentially lethal to tumor cells. In such tumor types, olaparib might potentially serve as an efficacious and less toxic cancer treatment as compared with currently available chemotherapy regimens.
+
+Olaparib inhibits selected tumor-cell lines in vitro and in xenograft and primary explant models, as well as in genetic breast cancer susceptibility gene (BRCA) knockout models, either as standalone treatment or in combination with established chemotherapeutics.
+
+Cells deficient in HRR factors, notably BRCA1/2, are particularly sensitive to olaparib treatment. PARP inhibitors, such as olaparib, might also enhance the DNA-damaging effects of other chemotherapy agents [6e9]. BRCA1-related breast cancer accounts for 5% of all breast cancers [10]. Although >50% of BRCA1-mutation carriers have TNBC according to pathological analyses [11,12], patients with TNBC do not necessarily harbor BRCA1 mutations. However, previous studies report that TNBC patients harboring wild-type BRCA1 frequently exhibit downregulated BRCA1 expression or alterations in BRCA1 function, which might occur through methylation of the BRCA1 promoter or overexpression of the protein that normally regulates BRCA1 expression [13e16].
+
+Mutations in numerous HRR genes lead to phenotypes similar to those associated with mutated BRCA1/2, a situation called BRCAness. Previous studies report an elevated risk for cancer development associated with HRR deficiencies, such those associated with as RAD51, BRIP1, PALB2, and FANCA mutation. Actually, 50% of high-grade serous ovarian cancers involve at least one HRR-modulating gene alteration. In a clinical trial for high-grade serous ovarian carcinoma (HGS-OvCa), olaparib maintenance therapy doubled the progression-free survival (PFS) of patients with BRCA1/2 germline--
+mutation, platinum-sensitive HGS-OvCa [17]. Additionally, in a phase II study, olaparib showed efficacy as maintenance therapy for platinum-sensitive relapsed HGS-OvCa patients, regardless of germline BRCA1/2 status [18]. Moreover, maintenance therapy involving niraparib and rucaparib showed a significant response and prolonged PFS for patients with HGS-OvCa, regardless of BRCA1/2 status but especially in those with non- BRCA1/2 -related HRR deficiency
+[19,20]. However, In TNBC, the clinical significance of HRD to PARP inhibition has been not evaluated.
+
+Here, we conducted a phase I/II clinical trial (EO study) to evaluate the efficacy of eribulin combined with the tablet form of olaparib for TNBC patients. We enrolled 24 patients in phase I, and one patient in the first cohort experienced dose-limiting toxicity
+(DLT). The recommended phase II dose (RP2D) was established as the approved dose for both drugs (olaparib: 300 mg twice daily with eribulin 1.4 mg/m2). All 24 patients received RP2D in phase II,
+with the median number of courses administered in phase II at 5.5 (range: 1e28). For the 22 evaluable patients, the response rate was 18.2% [complete response (CR): 0; partial response (PR): 4; 95% confidence interval (CI): 6.5e36.9)]. Median PFS and OS
+were 4.2 (95% CI: 3.0e7.4) and 14.5 (95% CI: 4.8e22.0),
+respectively [21].
+
+We hypothesized that BRCA mutations and HRR-related gene alterations might contribute to the efficacy results obtained from the EO study. Therefore, we identified the existence of mutations in HRR-related genes and BRCA1/2 and evaluated their association with patient response to eribulin/olaparib combination therapy among the participants in the EO study.
+
+## Materials And Methods Patients
+
+The study was approved by the institutional review board of the National Cancer Center, Tokyo (2012-236), and complied with the ethical guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained from each patient. All patients had participated in the phase I/II EO study. The EO study is an open-label, non-randomized, multi-center, dose-escalation study of olaparib in combination with eribulin mesylate aimed to assess safety, tolerability, and efficacy in patients with recurrent or metastatic TNBC. The EO study is a multi-institutional study that includes seven sites in Japan.
+
+Tissue specimens from metastatic/primary sites were obtained at the time of surgery or biopsy from each site. Germline BRCA
+mutations were analyzed in patients who had consented to provide two blood samples for confirmatory germline BRCA1/2-mutation testing using the Myriad Genetics BRCA test (Myriad BRACAnalysis; Myriad Genetics, Salt Lake City, UT, USA). Patients with a known BRCA1/2 mutation were analyzed on the basis of this information.
+
+## Gene Analysis
+
+Archival tissue samples were examined for gene alterations using a Foundation Medicine, Inc. (FMI) gene panel. Pathogenic or likely pathogenic gene alterations were extracted from all detected gene alterations using the FMI data dictionary. This dictionary uses the COSMIC database, relevant literature, and internal evidence to determine the reportable status of an alteration. We chose previously defined HRR-related genes, including BRCA1, BRCA2, PALB2, FANCA, FANCI, FANCL, FANCC, RAD50, RAD51, RAD51C,
+RAD54L, ATM, ATR, CHEK1, and CHEK2 [22]. Correlation between the presence of HRR-related gene mutation and patient cancer response to the combination therapy was determined.
+
+## Immunohistochemistry
+
+We examined epidermal growth factor receptor (EGFR), cytokeratin 5/6 (CK5/6), (BRCA1/ 2, vimentin, zinc finger E-box binding homeobox 1 (ZEB1), and E-cadherin. Primary antibodies are presented in Table S1. For all antibodies, except that for ZEB1, antigens were retrieved with citrate buffer treatment at 121 C for 10 min. The Envision method was used for the secondary antibody reaction, and diaminobenzidine tetrahydrochloride was used for the peroxidase reaction [23]. For the ZEB1 antibody, antigen was retrieved with Target Retrieval solution PH9 (TRS9; DAKO; Agilent Technologies, Santa Clara, CA, USA) at 98 C for 40 min, and Histofine Simple Stain MAX-PO(G) (Nichirei, Tokyo, Japan) was used for the secondary antibody reaction.
+
+For BRCA1 and BRCA2, the proportion of positive cells (score:
+0e5) and staining intensity (score: 0e3) were considered [24], and the expression was regarded as positive when the sum of these scores was >2. In cases of a score of 2 with weak-internal positive staining, expression was regarded as equivocal. Cytoplasmic and/or membranous immunoreactive staining was regarded positive if 10% of the cells were stained, regardless of intensity. Moderate-to-strong membranous/cytoplasmic staining (2þ or 3þ, respectively) in 10% of tumor cells was regarded as positive for EGFR only.
+
+Immunohistochemistry (IHC) results were independently evaluated by two researchers (A.S. and M.Y.) without knowledge of the clinical characteristics (Figure S1).
+
+## Results Gene Expression, Amplification, And Homologous Deletion
+
+A total of 32 tissue specimens were collected, with 19 samples collected from the phase I trial and 13 samples from the phase II trial. Seventeen patients were treated at the recommended dose. Figure 1 shows the landscape of the gene mutations.
+
+Thirty-three gene mutations were detected in the TNBC specimens. The most frequent nonsynonymous mutations were in TP53
+(n ¼ 27; 84.4%), PIK3CA (n ¼ 7; 21.9%), BRCA1 (n ¼ 5; 15.6%),
+MLL3 (n ¼ 5; 15.6%), AKT1 (n ¼ 4; 12.5%), NF1 (n ¼ 3; 9.4%),
+NFKKBIA (n ¼ 3; 9.4%), CHD1 (n ¼ 2; 6.3%), RB1 (n ¼ 2; 6.3%),
+and PTEN (n ¼ 2; 6.3%) (Figure 2A). Detection of gene amplifications revealed the most frequent for MYC (n ¼ 6; 18.8%), EGFR
+(n ¼ 2; 6.3%), KDM5A (n ¼ 2; 6.3%), LYN (n ¼ 2; 6.3%), and CDK6 (n ¼ 2; 6.3%) (Figure 2B). Eight homozygous deletions were detected, with the most frequent being the loss of PTEN (n ¼ 4; 12.5%). As shown in Figure 2C, other frequently observed homozygous deletions included loss of STK11 (n ¼ 2; 6.3%), TP53
+(n ¼ 2; 6.3%), and CDKN2A (n ¼ 2; 6.3%). Additionally, we detected 10 gene rearrangements, and HRD, including BRCA1/2 mutations, were observed in nine patients (Figure 1).
+
+Response was evaluable in 29 patients. CR was observed in one patient (3.4%), and PR was observed in seven (24.1%). The ORR for CR and PR and the clinical benefit rate (CBR) for CR, PR, and stable disease were 34.5% and 75.9%, respectively. The ORRs in patients with HRD and without HRD were 33.3% and 40%, respectively n=32 1. In 1944, 554, The B 9 (10) 11. 12 13 14 15 14 17 18 19 20 21 22 23 24 25 26 27 28 29 20 31 12
+
+![3_image_0.png](3_image_0.png)
+
+|                  |            |
+|------------------|------------|
+| 2                |            |
+| Xchange          |            |
+| PIKION           |            |
+| INCAL            |            |
+| MLL              |            |
+| AKTI             |            |
+| ee               |            |
+| 81               |            |
+| PTER             |            |
+| ATEX             |            |
+| PK38             |            |
+| CON2
+MLL2                  |            |
+| ESBE             |            |
+| NF2              |            |
+| ACYR1B
+CREEBP                  |            |
+| SMOTCHI
+SMARCH                  |            |
+| HER
+ SAGA                  |            |
+| CO
+SOM                  |            |
+| TET2             |            |
+| PAK              |            |
+| SPERV            |            |
+| PADS1            |            |
+| DNMT             |            |
+| GAINRY           |            |
+| FOXP             |            |
+| COR              |            |
+| KONS             |            |
+| LYN              |            |
+| CK               |            |
+| MO               |            |
+| 1111             |            |
+| CD274            |            |
+| PCD1LG           |            |
+| FORT             |            |
+| FOF              |            |
+| FGFR             |            |
+| FOR              |            |
+| ner
+ Miss                  |            |
+| M M I S          |            |
+| PIK3C            |            |
+| PIKAGE           |            |
+| PKKZ             |            |
+| MONTH,           |            |
+| KBK              |            |
+| AKTS             |            |
+| EMS              |            |
+| MAP26            |            |
+| klus             |            |
+| sees.
+One                  |            |
+| CNDI             |            |
+| IGFIR            |            |
+| C1 Torf          |            |
+| PTEN_Ja          |            |
+| STK11_loss
+TP53_loss                  |            |
+| CKNZA, Ice       |            |
+| TSC1_log         |            |
+| FAS              |            |
+| CDKN2B_loa       |            |
+| 13_8904_fasic    |            |
+| __               | m          |
+| CAFFIL           | N/A_funcat |
+| KAEP1_KAEP1_dup1 |            |
+
+arcitical
+
+![4_image_0.png](4_image_0.png)
+
+(P = .732), and those for patients with and without somatic BRCA
+mutation were 60% and 33.3%, respectively ( P = .264) ( Table 1 ).
+
+ORR was numerically higher in the somatic BRCA -mutation group, although the difference was not statistically significant.
+
+The response rate in patients with germline BRCA1/2 mutation
+(n = 5) was 40% (CR in 0 patients and PR in 2 patients). No PD was observed. This rate was higher than that in the overall population.
+
+## Immunohistochemistry
+
+IHC analysis of the evaluable specimens revealed that BRCA1 was positive in 58.6% of the specimens, BRCA2 was positive in 53.6%, EGFR was positive in 42.9%, CK5/6 was positive in 13.8%, E-cadherin was positive in 10.7%, ZEB1 was positive in 10.7%, and vimentin was positive in 67.9%. There was no correlation between the IHC status of any of the markers Table 1. Response to eribulin/olaparib combination treatment by patients with somatic BRCA
+mutation or homologous recombination repair deficiency (HRD)
+
+| Objective response (%)                         | P         |             |           |      |
+|------------------------------------------------|-----------|-------------|-----------|------|
+| HRD                                            | n ¼ 29    | pos (n ¼ 9) | 3 (33.3%) | .732 |
+| neg (n ¼ 20)                                   | 8 (40%)   |             |           |      |
+| Somatic BRCA Mutation                          | n ¼ 29    | pos (n ¼ 5) | 3 (60%)   | .264 |
+| neg (n ¼ 24)                                   | 8 (33.3%) |             |           |      |
+| pos: positive, neg: negative. chi-Square test. |           |             |           |      |
+
+evaluated and response to eribulin/olaparib combination therapy
+(Table 2).
+
+## Survival Analysis According To Gene Alteration And Ihc Status
+
+For the patients included in this study, the median PFS was 5.0 months (95% CI: 3.641e6.359) and median OS was 14.0 months
+(95% CI: 12.429e15.571) (Figure 3). There was no correlation between BRCA or HRD status and survival (Figure 4).
+
+EGFR-negative patients according to IHC tended to have a better PFS (log-rank P ¼ .059) and a significantly better OS (log-rank P ¼ .046) (Figure S2). There was no correlation between the IHC
+status of other markers evaluated and survival.
+
+## Discussion
+
+In this study, we investigated correlations between response and HRD or IHC status in patients treated with the combination therapy of eribulin and olaparib in a phase the I/II trial. The combination therapy of eribulin and olaparib was well-tolerated and showed antitumor activity in patients with advanced or metastatic TNBC
+(response rate: 18.2%), with median PFS and median OS at 4.2 months (95% CI: 3.0e7.4) and 14.5 months (95% CI, 4.8e22.0),
+respectively. These findings support combination therapy with eribulin and olaparib as a promising new treatment for patients with advanced or metastatic TNBC.
+
+Recent advances in research regarding HRD, including the use of olaparib for treating BRCA1/2-mutation carriers, has made HRD a potential biomarker for the treatment of TNBC. Assays that evaluate HRR capacity might help guide treatments using agents that induce or target DNA-damage repair. Niraparib is an FDA-approved PARP inhibitor used to treat ovarian cancer. In a phase-III study of Table 2. Response to eribulin/olaparib combination treatment by patients relative to immunohistochemistry status
+
+| Responders                                     | Non-responders   | P value    |    |    |      |
+|------------------------------------------------|------------------|------------|----|----|------|
+| BRCA1                                          | pos              | 17 (58.6%) | 5  | 12 | .260 |
+| neg                                            | 12 (41.4%)       | 6          | 6  |    |      |
+| n ¼ 29                                         |                  |            |    |    |      |
+| BRCA2                                          | pos              | 15 (53.6%) | 6  | 9  | .934 |
+| neg                                            | 13 (46.4%)       | 5          | 8  |    |      |
+| n ¼ 28                                         |                  |            |    |    |      |
+| EGFR                                           | pos              | 12 (42.9%) | 4  | 8  | .576 |
+| neg                                            | 16 (57.1%)       | 7          | 9  |    |      |
+| n ¼ 28                                         |                  |            |    |    |      |
+| CK5/6                                          | pos              | 4 (13.8%)  | 2  | 2  | .592 |
+| neg                                            | 25 (86.2%)       | 9          | 16 |    |      |
+| n ¼ 29                                         |                  |            |    |    |      |
+| E-cadherin                                     | pos              | 3 (10.7%)  | 0  | 3  | .140 |
+| neg                                            | 25 (89.3%)       | 11         | 14 |    |      |
+| n ¼ 28                                         |                  |            |    |    |      |
+| ZEB1                                           | pos              | 3 (10.7%)  | 2  | 1  | .304 |
+| neg                                            | 25 (89.3%)       | 9          | 16 |    |      |
+| n ¼ 28                                         |                  |            |    |    |      |
+| Vimentin                                       | pos              | 19 (67.9%) | 8  | 11 | .657 |
+| neg                                            | 9 (32.1%)        | 3          | 6  |    |      |
+| n ¼ 28                                         |                  |            |    |    |      |
+| pos: positive, neg: negative. chi-square test. |                  |            |    |    |      |
+
+niraparib, exploratory analyses were conducted in an HRD-positive subgroup, finding that the median PFS in patients with HRD-positive tumors harboring wild-type BRCA was longer in the niraparib group as compared with that in the placebo group [19]. Moreover, patients with HRD-positive tumors harboring a somatic BRCA
+mutation displayed similar reductions in the risk of disease progression as those observed in the cohort harboring a germline BRCA mutation. Additionally, niraparib treatment improved PFS in the HRD-negative subgroup.
+
+Rucaparib is another FDA-approved PARP inhibitor that shows antitumor activity in patients with ovarian cancer. Rucaparib is approved for use in the treatment of patients with platinum-sensitive ovarian cancer and induces prolonged PSF in patients with recurrent platinum-sensitive ovarian carcinoma and who responded to platinum-based therapy associated with HRD [25]. In the present study, the ORR and CBR for the 32-patient cohort were 34.5% and 75.9%, respectively, and the frequency of nonsynonymous mutations was 100%, with the most common nonsynonymous mutation in TP53. These observed rates were higher than those previously reported [26]. Additionally, the frequency of gene amplification in the patients was 48.3%, with the most frequent being MYC.
+
+Moreover, we investigated correlations between HRD-related gene mutations and response rate to eribulin/olaparib combination therapy, finding that the ORR was numerically higher in the HRD
+group, although the difference was not statistically significant, and the CBR was similar between the HRD and non-HRD groups. This suggested that HRD might represent a potential biomarker for predicting the efficacy of combination therapy using olaparib with eribulin in TNBC patients.
+
+In a previous study of combination therapy with carboplatin and eribulin in a neo-adjuvant setting, the pathological complete response
+(pCR) rate in BRCA1/2-mutation-positive patients was 66% (2/3 patients), and patients with HRD achieved higher pCR rates than non-HRD patients [27]. These data agreed with our results. Although phase-III studies of eribulin monotherapy [3,28] and phase-I/II studies of monotherapy or combined therapy with eribulin have been reported
+[29e32], there were data describing associated gene alterations.
+
+The only report focusing on predictive factors in the efficacy of eribulin involved a pooled analysis of two phase-III trials [33]. One of the suspected mechanisms of eribulin action involves activity in the tumor microenvironment. Abnormal tumor vasculature leads to tumor aggressiveness, and eribulin induces the remodeling of abnormal tumor vasculature and eliminates inner tumor hypoxia
+[34e36]. Another possible mechanism involves suppression of the epithelialemesenchymal transition (EMT). An in vivo xenograft model showed that eribulin treatment reverses EMT and induces mesenchymaleepithelial transition, and surviving TNBC cells pretreated in vitro with eribulin for 7 days displayed decreased lung metastasis when assessed in an in vivo experimental metastasis model
+[35,36]. Tumor-infiltrating lymphocytes are also expected to be useful as predictive markers for eribulin efficacy during the treatment of patients with TNBC [37], and another study suggests that immune system status might correlate with eribulin efficacy [38].
+
+In our study, EGFR-positive patients according to IHC tended to have a worse PFS and a significantly worse OS. EGFR-positivity has been reported as a prognostic factor of breast cancer [39]. But in in context of predicting efficacy of combination therapy of eribulin and olaparib, the importance of EGFR-positivity is unclear.
+
+![6_image_0.png](6_image_0.png)
+
+![6_image_1.png](6_image_1.png)
+
+In conclusion, we investigated the status of epithelial and mesenchymal markers by IHC in response to combined eribulin/
+olaparib combination therapy. Our results showed that EGFR status was associated with better PFS and OS. The suspected mechanism associated with these results involved suppression of EMT by eribulin. Our findings also suggest that somatic BRCA mutations might serve as potential biomarkers for predicting the efficacy of combination therapy with eribulin and olaparib in patients with TNBC. Further analysis using a larger cohort is needed.
+
+Supplementary data to this article can be found online at https://
+doi.org/10.1016/j.tranon.2019.07.013.
+
+## Acknowledgments
+
+We would like to acknowledge the study staff, Tamie Sukigara, Ritsuko Nagasaka, Tomomi Yoshino, Noriko Tanabe.
+
+## Funding
+
+This research was conducted with support from an Externally Sponsored Research Program of AstraZeneca (ESR-15-11262).
+
+## Ethical Standards
+
+The present study was approved by the NCCH Institutional Review Board and was conducted in accordance with the ethical standards of the institutional and national research committee.
+
+## Data Availability
+
+The datasets generated during and/or analyzed during the current study are not publicly available due to individual privacy, but are available from the corresponding author on reasonable request.
+
+## Author Contributions
+
+A.S. and K.T. conceived and designed the study. A.S. and M.S.
+
+reviewed the IHC specimen. A.S. interpreted data and wrote the manuscript. All the authors have read and approved the submission of the manuscript.
+
+## Compliance With Ethical Standards
+
+Conflicts of interest: A. Shimomura reports personal fees from Chugai Pharmatheutical, personal fees from Novartis Pharmatheutical, personal fees from Pfizer, personal fees from Nippon Kayaku, personal fees and other from AstraZeneca, personal fees from Eisai, other from Eli Lilly, outside the submitted work; K. Yonemori reports personal fees from Eisai, personal fees from Taiho, outside the submitted work; M. Yoshida has nothing to disclose; T. Yoshida has nothing to disclose; H. Yasojima has nothing to disclose; N. Masuda reports grants and personal fees from Chugai, grants and personal fees from AstraZeneca, grants and personal fees from Pfizer, grants and personal fees from Eisai, grants and personal fees from Eli-Lilly, personal fees from Takeda, grants from Kyowa Hakko Kirin, grants from MSD, grants from Novartis, grants from Daiichi Sankyo, outside the submitted work; and Board of directors: Japan Breast Cancer Research Group Association; K. Aogi reports grants and personal fees from Eisai, personal fees from AstraZeneca, outside the submitted work; M. Takahashi reports personal fees from AstraZeneca, personal fees from Eisai, personal fees from Pfizer, personal fees from Kyowa Hakko Kirin, personal fees from Eli Lilly, outside the submitted work; Y. Naito reports speakers' bureau from Pfizer, speakers' bureau from Taiho, speakers' bureau from Nippon Kayaku, speakers' bureau from Eli Lilly, speakers' bureau from AstraZeneca, speakers' bureau from Merck Serono, speakers' bureau from Bayer, speakers' bureau from Meiji Seika, research funding and speakers' bureau from Roche Diagnostics, speakers' bureau from Pfizer, speakers' bureau from Novartis, speakers' bureau from Chugai, speakers' bureau from Pfizer, speakers' bureau from Eisai, outside the submitted work; S. Shimizu has nothing to disclose; R. Nakamura has nothing to disclose; A. Hamada has nothing to disclose; H. Michimae has nothing to disclose; J. Hashimoto has nothing to disclose; H.
+
+Yamamoto has nothing to disclose; A. Kawachi has nothing to disclose; C. Shimizu reports personal fees from Astra Zeneca, personal fees from Eizai, during the conduct of the study; grants from Eli Lilly, grants and personal fees from Chugai, grants and personal fees from Pfizer, grants from MSD, outside the submitted work; Y. Fujiwara reports grants and other from Japan Agency for Medical Research and Development, grants and other from The Ministry of Health Labor and Welfare, Japan, during the conduct of the study; other from Astra Zeneca KK, other from Daiichi Sankyo Co., Ltd., other from Taiho Pharmaceutical Co., Ltd., other from Chugai Pharmaceutical Co.,
+Ltd., other from Novartis Pharma KK, other from SRL Inc., outside the submitted work; K. Tamura reports grants from AstraZeneca, during the conduct of the study.
+
+Ethical approval: All procedures performed in studies involving human participants were in accordance the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
+
+## Informed Consent
+
+Written informed consent was obtained from all of the patients. A
+copy of the written consent is available for review upon requests.
+
+## References
+
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+Cancer Genet 216-217, 61e66.

medical/md/PMC6790566.md

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+DOI: 10.1111/ajt.15339 ORIGINAL ARTICLE
+Noninvasive detection of graft injury after heart transplant using donor‐derived cell‐free DNA: A prospective multicenter study Kiran K. Khush1 | Jignesh Patel2 | Sean Pinney3 | Andrew Kao4 | Rami **Alharethi5** | Eugene DePasquale6 | Gregory Ewald7 | Peter Berman8 | Manreet **Kanwar9** | David Hiller10 | James P. Yee11 | Robert N. Woodward10 | Shelley **Hall12** | Jon **Kobashigawa2**
+1Division of Cardiovascular Medicine, Stanford University, Stanford, California 2Cedars‐Sinai Smidt Heart Institute, Los Angeles, California 3Mount Sinai Medical Center, New York, New York 4St. Luke's Hospital Mid America Heart Institute, Kansas City, Missouri 5Intermountain Healthcare, Salt Lake City, Utah 6University of California, Los Angeles, California 7Washington University School of Medicine, Saint Louis, Missouri 8Tampa General Hospital, Tampa, Florida 9Allegheny General Hospital, Pittsburgh, Pennsylvania 10Research and Development, CareDx, Brisbane, California 11Clinical Research, CareDx, Brisbane, California 12Baylor University Medical Center, Dallas, Texas Correspondence Kiran K. Khush Email: kiran@stanford.edu Funding information CareDx Standardized donor‐derived cell‐free DNA (dd‐cfDNA) testing has been introduced into clinical use to monitor kidney transplant recipients for rejection. This report de‐ scribes the performance of this dd‐cfDNA assay to detect allograft rejection in sam‐ ples from heart transplant (HT) recipients undergoing surveillance monitoring across the United States. Venous blood was longitudinally sampled from 740 HT recipients from 26 centers and in a single‐center cohort of 33 patients at high risk for antibody‐ mediated rejection (AMR). Plasma dd‐cfDNA was quantified by using targeted ampli‐ fication and sequencing of a single nucleotide polymorphism panel. The dd‐cfDNA levels were correlated to paired events of biopsy‐based diagnosis of rejection. The median dd‐cfDNA was 0.07% in reference HT recipients (2164 samples) and 0.17% in samples classified as acute rejection (35 samples; P = .005). At a 0.2% threshold, Abbreviations: ACR, acute cellular rejection; AMR, antibody‐mediated rejection; AR, acute rejection; AUC, area under the curve; cfDNA, cell‐free DNA; CLIA, Clinical Laboratories Improvements Act; dd‐cfDNA, donor‐derived cell‐free DNA; D‐OAR, Donor‐Derived Cell‐Free DNA‐Outcomes AlloMap Registry; LVEF, left ventricular ejection fraction; NPV, negative predictive value; NR, no rejection; OPTN, Organ Procurement and Transplantation Network; PPV, positive predictive value.
+
+This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2019 The Authors. American Journal of Transplantation published by Wiley Periodicals, Inc. on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons Am J Transplant. 2019;19:2889–2899. amjtransplant.com | **2889**
+dd‐cfDNA had a 44% sensitivity to detect rejection and a 97% negative predictive value. In the cohort at risk for AMR (11 samples), dd‐cfDNA levels were elevated 3‐ fold in AMR compared with patients without AMR (99 samples, P = .004). The stand‐ ardized dd‐cfDNA test identified acute rejection in samples from a broad population of HT recipients. The reported test performance characteristics will guide the next stage of clinical utility studies of the dd‐cfDNA assay.
+
+KEYWORDS
+biomarker, clinical research/practice, heart (allograft) function/dysfunction, heart transplantation/cardiology
+
+## 1 | **Introduction**
+
+Acute rejection (AR), including acute cellular rejection (ACR) and antibody‐mediated rejection (AMR), continues to be a complica‐
+tion after heart transplant (HT).1 AR is a major cause of hospi‐
+talization and graft dysfunction during the early years post HT. However, symptoms may develop late in the process of graft damage, which underscores the need for routine rejection sur‐ veillance. Endomyocardial biopsy remains the primary means for rejection monitoring, but it is associated with patient discomfort, expense, and potentially serious procedural complications.2-4 Noninvasive peripheral gene expression testing via the AlloMap assay (CareDx, Inc., Brisbane, CA) has been widely adopted for AR surveillance by HT centers in the United States. This gene expression assay can provide a high (>99%) negative predictive value5 and has proved to be useful for ruling out ACR. However, the gene expression profiling test has a limited positive predic‐ tive value (PPV) for cellular rejection, and the assay was not de‐ signed to detect AMR. For these reasons, the development of a more comprehensive, noninvasive assay for the surveillance of AR (both ACR and AMR) remains of great interest and clinical importance.6 Donor‐derived cell‐free DNA (dd���cfDNA), detected in the blood of transplant recipients, has been proposed as a noninvasive marker of graft injury, which can be caused by ACR as well as acute AMR. Early dd‐cfDNA studies were based on the hypothesis that AR causes cell death in the allograft, which leads to increased levels of dd‐cfDNA in the recipient's bloodstream.
+
+Data from single‐center studies have shown that elevated dd‐
+cfDNA levels can detect AR after HT, and an increase in levels can occur before rejection is detected on endomyocardial biopsy.7-9 Shotgun whole‐genome sequencing has been used to detect and quantify dd‐cfDNA, but the complexity and cost of the analyses limit its application as a clinically relevant surveillance tool. Targeted quantification of dd‐cfDNA provides a more rapid and cost‐effective surveillance strategy, but also requires genotyping of the transplant donor, which can be impractical.
+
+More recently, a targeted amplification, next‐generation se‐
+quencing assay (AlloSure®; CareDx, Inc.) has been analytically and clinically validated to quantify the percentage of dd‐cfDNA in trans‐ plant recipients' blood without the need for donor or recipient geno‐
+typing.10 This assay was shown to detect ACR in HT in a multicenter retrospective case‐control study10 and to detect active rejection in kidney transplant in a prospective, multicenter trial.11 This study is the first to report a large, prospective, multicenter clinical validation of the ability of a standardized dd‐cfDNA assay to detect AR in HT recipients.
+
+We conducted the Donor‐Derived Cell‐Free DNA‐Outcomes AlloMap Registry (D‐OAR) study to examine the characteristics of dd‐cfDNA in a routine, clinical surveillance setting with HT recipi‐ ents at 26 centers across the United States. The objective of D‐OAR was to determine the test performance of dd‐cfDNA for the detec‐ tion of ACR, AMR, and graft dysfunction.
+
+## 2 | **Methods** 2.1 | **D‐Oar Study Design**
+
+The D‐OAR (NCT02178943) is an observational, prospective, multi‐ center registry that aimed to evaluate clinical outcomes in HT recipi‐
+ents who were receiving regular allograft rejection surveillance.12 The primary objective was to determine whether the dd‐cfDNA level in an HT recipient's blood can differentiate rejection from the absence of rejection, as determined by endomyocardial biopsy in‐ terpretation. Secondary objectives were to determine whether graft dysfunction in the absence of rejection is associated with increased dd‐cfDNA levels and to characterize dd‐cfDNA levels in stable pa‐ tients who have no evidence of AR.
+
+Eligible study subjects were HT recipients ≥ 15 years and >
+55 days posttransplant who were undergoing AlloMap gene expres‐ sion profiling for rejection surveillance. Multiorgan transplant recip‐ ients were excluded.
+
+Pretransplant and serial data after transplant, including clinical status, hospitalizations, diagnostic tests (including en‐ domyocardial biopsies, AlloMap scores, and echocardiograms), immunosuppressive maintenance therapy and drug levels, and posttransplant adverse events, were collected. The D‐OAR study included 26 HT centers in the United States.12 The primary study outcomes were AR (ACR and AMR) and graft dysfunction, as de‐
+fined here later.
+
+Between July 2014 and September 2016, blood specimens were collected for quantification of dd‐cfDNA levels at each surveillance visit that occurred for AlloMap testing. The regular surveillance schedule for testing was determined by each participating center's standard of care. Following a protocol amendment in September 2016 (through 2018), the dd‐cfDNA specimen was drawn only when a patient had a clinical suspicion of rejection and a planned "for‐cause" biopsy. Surveillance biopsies and biopsies performed for cause were analyzed separately in addition to the combined anal‐ ysis, to determine whether the 2 clinical settings render differing relationships with regard to cell‐free DNA. Two follow‐up dd‐cfDNA specimens were collected within 8 weeks after "for‐cause" biopsies in patients who were treated for rejection and/or graft dysfunction.
+
+## 2.2 | **Cedars‐Sinai Study**
+
+A parallel single‐center study was conducted at Cedars‐Sinai Medical Center from March 2016 to May 2017. HT recipients identified as high risk for the development of AMR were enrolled and followed longitudinally. The inclusion criteria were pretransplant PRA ≥ 10%, presence of donor‐specific antibodies at the time of transplant, any posttransplant detection of DSA or biopsy‐proved AMR, or "for‐cause" biopsy due to reduced left ventricular ejection fraction (LVEF). Samples were collected beginning 14 days posttransplant from patients 18 years or older. This cohort included 110 samples from 33 patients and information on dd‐cfDNA and biopsy grades were collected for ACR and AMR. The objective of this study was to correlate dd‐cfDNA levels in patients with pAMR ≥ 1 in this inde‐ pendent sample set.
+
+## 2.3 | **Blood Samples And Dd‐Cfdna Measurements**
+
+Venous blood was collected in Streck Cell‐Free DNA BCT tubes be‐ fore the performance of endomyocardial biopsies and was shipped to the central Clinical Laboratories Improvements Act–certified laboratory at CareDx, Inc. Details of the standardized specimen processing and analytical methods to determine the percentage of dd‐cfDNA (AlloSure®) have been published.10 The targeted next‐
+generation sequencing assay uses highly polymorphic single nucleo‐ tide polymorphisms to quantify dd‐cfDNA without the need for sep‐
+arate genotyping of the recipient or the donor.10 All measurements were performed by laboratory technicians unaware of the clinical identity of the samples.
+
+## 2.4 | **Diagnosis Of Graft Dysfunction And Of Biopsy‐** Defined Rejection
+
+Information was collected on the number of, and clinical indica‐ tion for, endomyocardial biopsies for each patient. Biopsies were graded according to the International Society for Heart and Lung Transplantation revised classification scheme for ACR13 and patho‐
+logic diagnosis of AMR.14 Biopsy interpretation was performed by the pathologist at the participating transplant center.
+
+The AR group was defined as transplant recipients whose blood samples indicated either ACR (grade 2R or 3R), AMR (pAMR grade 1, 2, or 3), or mixed rejection (satisfying the requirements of both ACR and AMR). For analysis by type of rejection, mixed rejections were pooled with AMR. The no rejection (NR) group was defined as recipients whose samples had no biopsy evidence of AR (ACR grade 0R or 1R and AMR grade pAMR0). The graft dysfunction group was defined as recipients whose blood samples had no biopsy evidence of AR and who had ≥ 1 of the following: LVEF < 40% or a decrease of ≥ 25% from the prior visit.
+
+## 2.5 | **Reference Population**
+
+The reference population was defined as all D‐OAR patients with samples that did not indicate ACR (grade 2R or 3R), AMR (pAMR grade 1, 2, or 3), or mixed rejection during the course of the study. For sensitivity, we also define a restricted population in which pa‐ tients with samples with any ACR grade 1R as well as dd‐cfDNA samples not paired with biopsy were excluded (146 patients [214 samples]).
+
+## 2.6 | **Statistical Analyses**
+
+The primary objective of the statistical analysis was to determine whether the dd‐cfDNA level in an HT recipient's blood can differ‐ entiate AR from NR, as determined by local pathologists' endomyo‐ cardial biopsy diagnostic classification. Surveillance biopsies and biopsies performed for cause were analyzed separately, to determine whether the 2 clinical settings render differing relationships with re‐ gard to dd‐cfDNA testing. The blood was collected for dd‐cfDNA assays within 3 days before endomyocardial biopsy. Wilcoxon rank sum testing was used to compare dd‐cfDNA values associated with AR with dd‐cfDNA values associated with NR. Further comparisons were performed of ACR vs no ACR in patients without AMR, and of AMR vs no AMR. Performance characteristics of dd‐cfDNA to diag‐ nose AR were computed, including sensitivity, specificity, PPV, and NPV.
+
+Additional analyses assessed the correlation of dd‐cfDNA to graft dysfunction in the absence of AR. The graft dysfunction group and the no–graft dysfunction group were compared by using the Wilcoxon rank sum test. An additional objective was to character‐ ize the reported values of dd‐cfDNA in the reference population, including median levels and normal ranges.
+
+Power calculations were performed as follows. An effect size of 0.83 was estimated from a prior HT study of dd‐cfDNA in ref‐ erence cases of biopsy‐based AR compared with control cases with NR.10 We assumed that the [mean log(dd‐cfDNA) AR - [mean log(dd‐cfDNA) NR]/(standard deviation of log (dd‐cfDNA)] = 0.83. Accordingly, there is a 90% power to demonstrate a significant
+
+![3_image_0.png](3_image_0.png)
+
+## 3.2 | **Reference Population** 3 | **Results** 3.1 | **D‐Oar Patients**
+
+FIGURE 1 CONSORT diagrams for Donor‐Derived Cell‐Free DNA‐Outcomes AlloMap Registry (D‐OAR). A, Reference population (Box A) includes all 2164 D‐OAR samples from patients with no clinical signs or symptoms of rejection and no biopsy‐based evidence of rejection (no rejection [NR] = no acute cellular rejection [ACR] grade ≥ 2R or antibody‐mediated rejection [AMR] grade ≥ pAMR1). Eight hundred forty‐one samples (Box B) had biopsy paired with donor‐derived cell‐free DNA (dd‐cfDNA) results, of which 18 had AMR, including 2 mixed rejections (Box C), 17 had ACR (Box D), and 806 had NR (ACR grade 0R or 1R and AMR grade pAMR0, Box E). B, The 2405 D‐OAR samples that did not have a rejection diagnosis (Box F) include 31 graft dysfunction samples (left ventricular ejection fraction [LVEF] < 40 or drop in LVEF of ≥ 25% from previous visits, Box G) and 2374 no graft dysfunction samples (Box H). C, The 110 samples from Cedars‐Sinai patients (Box I) include 11 associated with biopsy evidence of AMR (Box J) and 99 with NR (Box K)
+difference in dd‐cfDNA in 17 cases of AR compared with dd‐cfDNA 
+levels in 323 NR cases, from a total of 340 visits, assuming ACR
+and/or AMR would be discovered at 5% of biopsy surveillance visits.
+
+The reference population (Table 1, Figure 1A, Box A) was com‐ posed of 676 patients who contributed 2164 samples and who had no clinical signs or symptoms of AR during the course of the study. The mean age at enrollment was 54 years, 73% were > 50 years old, 75% were male, 70% were white, and the most common indications for transplant were dilated cardiomyopathy (49%) and ischemic car‐ diomyopathy (32%). Patients were enrolled at a median of 170 days posttransplant (IQR 116‐249 days), and 3 samples were drawn, on average, per patient. This reference population is demographically similar to, but slightly older, than HT patients included in the Organ Procurement and Transplantation Network data,15 who are 74% male and 73% white, and 57% were > 50 years old at time of transplant.
+
+The median dd‐cfDNA level in this reference population was 0.07% (IQR 0.03%‐0.14%), as shown in Figure 2A. The 97.5th percentile was 1.29%. Sensitivity analysis on the restricted pop‐ ulation (all samples from patients with any ACR grade 1R, as well as dd‐cfDNA samples not paired with biopsy, were excluded)
+From September 2014 to October 2017, 740 HT recipients were enrolled at 26 clinical sites (Figure 1A; Table S1, participating sites) from which 2447 plasma dd‐cfDNA level samples were drawn. Blood samples were drawn for dd‐cfDNA quantification from 55 days to >5 years posttransplant. Most (81%) samples were drawn within the first year posttransplant, and 13% were drawn during the second year (Figure S1). Eight hundred forty‐one dd‐ cfDNA results were paired with a biopsy, of which 587 biopsies were performed for routine rejection surveillance and 254 biopsies were "for cause" based on clinical suspicion (Table 1, Figure 1A).
+
+| TABLE 1 Demographic and clinical characteristics of the reference population Variable Reference All biopsies   | NR          | AR          | P (AR vs NR)   |             |         |
+|----------------------------------------------------------------------------------------------------------------|-------------|-------------|----------------|-------------|---------|
+| No. of samples                                                                                                 | 2164        | 841         | 806            | 35          |         |
+| No. of patients                                                                                                | 676         | 443         | 409            | 34          |         |
+| Samples per patient                                                                                            | 3.2         | 1.9         | 2.0            | 1.0         |         |
+| Pretransplant diagnosis                                                                                        | .201        |             |                |             |         |
+| Congenital                                                                                                     | 18 (3%)     | 13 (3%)     | 11 (3%)        | 2 (6%)      |         |
+| Ischemic cardiomyopathy                                                                                        | 216 (32%)   | 139 (31%)   | 125 (31%)      | 14 (41%)    |         |
+| Multiple                                                                                                       | 14 (2%)     | 10 (2%)     | 10 (2%)        | 0 (0%)      |         |
+| Nonischemic cardiomyopathy                                                                                     | 329 (49%)   | 221 (50%)   | 209 (51%)      | 12 (35%)    |         |
+| Other                                                                                                          | 92 (14%)    | 55 (12%)    | 50 (12%)       | 5 (15%)     |         |
+| Retransplant                                                                                                   | 7 (1%)      | 5 (1%)      | 4 (1%)         | 1 (3%)      |         |
+| Race                                                                                                           | .605        |             |                |             |         |
+| Asian                                                                                                          | 20 (3%)     | 9 (2%)      | 8 (2%)         | 1 (3%)      |         |
+| Black                                                                                                          | 112 (17%)   | 59 (13%)    | 54 (13%)       | 5 (15%)     |         |
+| White                                                                                                          | 474 (70%)   | 326 (74%)   | 303 (74%)      | 23 (68%)    |         |
+| Hispanic                                                                                                       | 50 (7%)     | 34 (8%)     | 31 (8%)        | 3 (9%)      |         |
+| Other                                                                                                          | 20 (3%)     | 15 (3%)     | 13 (3%)        | 2 (6%)      |         |
+| Male sex                                                                                                       | 505 (75%)   | 330 (74%)   | 305 (75%)      | 25 (74%)    | .841    |
+| Cytomegalovirus serologic status                                                                               | .523        |             |                |             |         |
+| D− :R−                                                                                                         | 113 (17%)   | 70 (16%)    | 66 (16%)       | 4 (12%)     |         |
+| D− :R+                                                                                                         | 127 (19%)   | 76 (17%)    | 70 (17%)       | 6 (18%)     |         |
+| D+ :R−                                                                                                         | 171 (25%)   | 122 (28%)   | 115 (28%)      | 7 (21%)     |         |
+| D+ :R+                                                                                                         | 235 (35%)   | 147 (33%)   | 131 (32%)      | 16 (47%)    |         |
+| Unknown                                                                                                        | 30 (4%)     | 28 (6%)     | 27 (7%)        | 1 (3%)      |         |
+| Mechanical support                                                                                             | .381        |             |                |             |         |
+| None                                                                                                           | 326 (48%)   | 204 (46%)   | 184 (45%)      | 20 (59%)    |         |
+| Left ventricular assist device                                                                                 | 297 (44%)   | 201 (45%)   | 189 (46%)      | 12 (35%)    |         |
+| Temporary circulatory support                                                                                  | 42 (6%)     | 37 (8%)     | 35 (9%)        | 2 (6%)      |         |
+| Total artificial heart                                                                                         | 11 (2%)     | 1 (0%)      | 1 (0%)         | 0 (0%)      |         |
+| Age at enrollment, y                                                                                           | 54 ± 13     | 54 ± 12     | 54 ± 12        | 55 ± 13     | .721    |
+| LVEF at enrollment, %                                                                                          | 59 ± 9      | 59 ± 9      | 59 ± 9         | 61 ± 7      | .393    |
+| Days posttransplant at enrollment                                                                              | 292 ± 537   | 299 ± 391   | 280 ± 311      | 523 ± 892   | 0<0.001 |
+| Height, cm                                                                                                     | 174.6 ± 9.9 | 175 ± 9.6   | 174.9 ± 9.7    | 176.2 ± 9.2 | .495    |
+| Weight, kg                                                                                                     | 84.9 ± 18.3 | 86.7 ± 18.8 | 86.4 ± 18.4    | 90.7 ± 23.5 | .221    |
+| AR, acute rejection; NR, no rejection; LVEF, left ventricular ejection fraction.                               |             |             |                |             |         |
+
+showed similar characteristics: the median dd‐cfDNA level was 0.07% (IQR 0.03%‐0.12%). There was no statistical difference between the assessment of the reference population and the restricted population (P = .925, Kolmogorov–Smirnov test). The levels of dd‐cfDNA in HT recipients' blood remain very low and stable during the first 2 years posttransplant, in the absence of AR (Figure 2B, P = .182). The median dd‐cfDNA intrapatient vari‐
+ability (CVI
+) is 70%, and the interpatient coefficient of variation 
+(CVG) of patient median values is 86%, based on 350 patients from the reference population who had at least 3 test results per patient.
+
+## 3.3 | **Clinical Events** 3.3.1 | **Rejection**
+
+Of the total of 841 endomyocardial biopsies performed in study subjects and paired with dd‐cfDNA, there were 17 biopsy‐ proved ACRs (grade 2R or 3R, no AMR), 18 AMRs (grade pAMR1 or pAMR2 including 2 mixed rejections), and 806 NR samples (Figure 1A, Boxes B‐E). The greatest number of biopsies (384) and the most AR cases (14) occurred within the first 6 months post‐ transplant. Ten ARs were diagnosed in 279 biopsies performed
+
+2894  |      al.
+
+![5_image_0.png](5_image_0.png)
+
+during months 6 through 12, 4 ARs occurred in 122 biopsies dur‐ ing year 2, and 4 additional ARs occurred in 51 biopsies during years 3 to 5 posttransplant. Three additional AR cases were diag‐ nosed in 5 biopsies after 5 years posttransplant.
+
+The dd‐cfDNA levels differed significantly between patients with and without AR (Figure 3A). The median level of dd‐cfDNA in patients with AR was significantly higher (0.17%) than in the group of patient specimens without rejection (0.07%, P < .001). Median dd‐cfDNA levels were 0.17% for both ACR and AMR.
+
+ACR grade 1R had a similar median level (0.08%) to grade 0 biopsies (0.07%), whereas ACR 2R (moderate) had a median dd‐cfDNA level of 0.15%, and ACR 3R (severe) had a median dd‐cfDNA level of 0.30% (Figure 3B). dd‐cfDNA levels differ‐ entiated ACR grade ≥ 2R (P = .004) from NR. Biopsies graded as pAMR0 had median dd‐cfDNA levels of 0.07%, whereas the median was 0.12% in pAMR1 and 0.25% in pAMR2 (Figure 3C).
+
+The fractions of true‐ and false‐positive results for dd‐cfDNA to detect AR are shown in Figure S2. The area under the curve (AUC) was 0.64 (95% confidence interval 0.52 to 0.75). With a cutoff of 0.2%, the dd‐cfDNA assay had 80% specificity and 44% sensitivity to differentiate AR from NR. The PPV was 8.9%, and the NPV was 97.1%. When examined by type of rejection, the PPV for ACR detec‐ tion was 4.8% with an NPV of 98.6%, whereas for AMR, the PPV was 4.2% and the NPV was 98.6%.
+
+## 3.4 | **Surveillance Biopsy Analysis**
+
+Of the 587 surveillance group samples, there were 21 ARs, including 9 ACRs and 12 AMRs. Each of these cases was from a unique pa‐ tient. The median level of dd‐cfDNA in surveillance samples paired with rejection was 0.15% (IQR 0.04%‐0.23%) and 0.07% in samples paired with NR (IQR 0.02%‐0.14%). The sample size was too small to detect a statistically significant difference in median dd‐cfDNA levels (P = .140, Figure 3D). Sensitivity was 38.1% (21.4%‐54.5%), specificity was 84.0% (81.2‐86.7%), PPV was 8.1% (4.7%‐11.9%), NPV was 97.3% (96.7%‐98.0%), and AUC was 60.5% (46.0%‐74.2%) for identifying rejection.
+
+## 3.5 | **For‐Cause Biopsy Analysis**
+
+Of the 254 "for‐cause" samples, there were 14 ARs, including 8 bi‐ opsy‐provedACRs from unique patients (ACR 2R or 3R) and 6AMRs
+
+![6_image_0.png](6_image_0.png)
+
+(pAMR1 or pAMR2). The median level of dd‐cfDNA in for‐cause samples paired with rejection was 0.25% (IQR 0.10%‐0.31%) and 0.09% in samples paired with NR (IQR 0.04%‐0.19%). Statistically different median dd‐cfDNA levels were observed in patients with and without rejection (Figure 3E, P = .02). Sensitivity was 53.8% (33.3‐75.1%), specificity was 76.1% (71.6%‐80.2%), PPV was 11.6% (7.6%‐16.6%), NPV was 96.6% (95.2%‐98.2%), and AUC was 68.5% (48.0%‐86.4%) for identifying rejection.
+
+## 3.6 | **Graft Dysfunction**
+
+Thirty‐one graft dysfunction events paired with dd‐cfDNA occurred in 31 unique study subjects (Figure 1B, Box F‐H). The dd‐cfDNA lev‐ els (median 0.07%) were not significantly different from the median levels in the reference population. However, when the %dd‐cfDNA is plotted against either LVEF or LVEF change, those with lowest LVEF generally had the highest dd‐cfDNA levels (Figure 4). Of those with both low LVEF and a ≥ 25% reduction of LVEF from the prior visit (red circles in Figure 4), the median dd‐cfDNA value was 0.53% (n = 8, P = .007 compared with no AR or graft dysfunction; clinical details are given in Table S2).
+
+## 3.7 | **Cedars‐Sinai Study**
+
+Among 110 samples from 33 patients in the Cedars‐Sinai study, there were 99 samples from 33 patients with pAMR0, 3 sam‐ ples from 3 patients with pAMR1, and 8 samples from 3 patients with pAMR2 (Figure 1C, Box I‐K). Sixty‐seven percent of the patients were nonwhite, and the average age was 56 years. The patients with AMR were younger than those without AMR (47 vs 58 years). Moderate or severe ACR was not diagnosed in any of
+
+![7_image_0.png](7_image_0.png)
+
+the patients: 25 patients had 53 grade 0R results and 22 patients had 57 grade 1R results. Patients with pAMR1 or pAMR2 had higher median dd‐cfDNA levels (0.50%) than those with pAMR0 (0.16%) (P = .004) (Figure 5). The dd‐cfDNA level of pAMR0 was more than double that observed for pAMR0 in D‐OAR; never‐ theless, dd‐cfDNA could still differentiate patients with pAMR1 or pAMR2 from those without AMR with sensitivity of 88.0%, specificity of 61.5%, PPV of 20.2%, and NPV of 97.9% at a thresh‐ old of 0.2%.
+
+## 4 | **Discussion**
+
+This report presents the results of a large, multicenter, prospective study that was designed to establish the performance characteris‐ tics of a well‐validated, fully standardized dd‐cfDNA assay in a broad population of HT recipients in the United States. The size and design of the study estimated that the number of AR events was sufficient to demonstrate statistically significant performance characteristics of the assay. Critically, the dd‐cfDNA measurement is an analytically validated assay in a College of American Pathologists‐accredited, CLIA‐certified reference laboratory.10 In the study population of patients who had received an HT at least 55 days before enrollment, dd‐cfDNA testing detected AR with an AUC of 0.64 and provided an estimated NPV of 97.1% and PPV of 8.9%. These results, from a contemporary HT patient population that includes patients with both ACR and AMR, validate prior reports of the performance characteristics of this assay in a population in which only ACR was characterized.10 We demonstrated that dd‐cfDNA levels are significantly higher in patients with AR compared with patients with no biopsy evidence of rejection. The current report is strengthened
+
+     |  al.  **2897**
+
+![8_image_0.png](8_image_0.png)
+
+by inclusion of an independent patient set (Cedars‐Sinai cohort) that confirms the ability of dd‐cfDNA to detect AMR. dd‐cfDNA levels are also correlated with the presence of graft dysfunction, especially in patients with a large (≥25%) drop in LVEF. These results confirm the hypothesis that cell‐free DNA is released from cells within the donor organ during episodes of significant graft injury. Reassuringly, episodes of grade 1R (mild) ACR, which is usually considered clinically irrelevant, were not correlated with elevated dd‐cfDNA levels. These results seem to confirm the clinical suspicion that grade 1R (mild) ACR does not result in significant graft injury. The natural progression of ACR
+grade 1R is not well defined, but we observed that the majority of pa‐ tients did not progress to clinically overt rejection. In another recent study,16 the composite outcome of death, retransplant, rejection with hemodynamic compromise (defined as LVEF ≤ 40% or a drop ≥ 25% compared with baseline or use of inotropic drugs or mechanical sup‐ port), and nonspecific graft dysfunction (hemodynamic compromise without evidence of rejection) occurred in 103 patients during follow‐ up, and the occurrence of this composite endpoint at 1, 5, and 10 years was 4%, 15%, and 23%, respectively, whereas grade 1R ACR was found in 40.7% (456/1118) of biopsies performed between 2 and 6 months posttransplant.
+
+In the reference population of stable HT recipients free of rejec‐
+tion, dd‐cfDNA is present at very low levels (median 0.07%). This is in contrast to stable kidney transplant recipients, in whom dd‐cfDNA
+is detected at a median of 0.21% by using the AlloSure® assay.11 Differences in baseline dd‐cfDNA levels may reflect differences in the rate of cell turnover within the allograft. This is also in contrast to the median dd‐cfDNA level of 0.16% in pAMR0 patients in the Cedars‐Sinai study. This difference is likely due to the difference in patient populations between the 2 cohorts; the stable patients in D‐ OAR were thought to be at low risk for AR and tended to have less allograft injury than the patients in the Cedars‐Sinai study, who were all allosensitized patients.
+
+Prior studies have also shown that dd‐cfDNA levels may begin to rise weeks to months before AR is diagnosed on endomyocardial bi‐
+opsy.8,17 These elevated levels represent the early graft injury that occurs before myocyte damage is apparent on histology. Surveillance with dd‐cfDNA may therefore detect early rejection and thereby trigger augmentation of immunosuppression to prevent a more se‐ vere rejection event that may result in irreversible graft damage. This early detection of graft injury in the setting of a negative biopsy may account for some of the false‐positive results seen in this study. Similarly, the relatively low PPV of 8.9% for the detection of AR re‐ flects the low prevalence of rejection in this clinically stable patient population. Patients were enrolled in the D‐OAR study while under‐ going routine AlloMap peripheral gene expression testing for rejec‐ tion surveillance. In general, patients who undergo AlloMap testing tend to be "low risk" clinically—they have not had recent rejection events and are not highly allosensitized. Only 2.2% of the biopsies performed in D‐OAR patients were positive for ACR and 2.1% were positive for AMR. The incidence of treated rejection in the first year posttransplant, as reported by the International Society for Heart and Lung Transplantation thoracic transplant registry, is currently 13%.1 Early rejection events may have occurred in the first 2 months post‐ transplant, before patients were eligible to enroll in D‐OAR.
+
+Additionally, many patients were enrolled later than 2 months posttransplant and were tested over several years, during which time the prevalence of AR is very low. The distribution of tests was 81% in the first year and 14% in the second year (Figure S1).
+
+Another consideration when evaluating dd‐cfDNA test perfor‐
+mance is that endomyocardial biopsy is not a true "gold standard" for the diagnosis of AR. There are many limitations of the biopsy, including sampling error and interobserver variability in biopsy interpretation. A prior study that compared expert panel (core) biopsy interpretation with locally assigned grades showed that 52% of local ≥ 2R ACRs were assigned lower grades (no significant rejection) by the panel and that overall agreement for the diagnosis of ACR between panel and local reads was only 28.4%.18 dd‐cfDNA, which directly assesses damage to the transplanted organ, may therefore be a more objective and accu‐ rate assay for graft injury than the traditional biopsy.
+
+The dd‐cfDNA noninvasive monitoring test can reduce biopsy utilization and save health care costs, as has been modeled for the AlloMap test.19 It has been estimated that the cost‐effectiveness of a blood‐based biomarker compared with endomyocardial biopsy for 2898  |      al.
+
+the diagnosis of acute allograft rejection may result in a cost saving of $27,244 and quality adjusted life year gain of 0.046 on average during the first 5 years posttransplant.20 The limitations of this study include (1) the change in protocol designed to increase the number of rejection events. To adjust for this change in study methods, we have analyzed the "surveillance" and "for‐cause" results separately. (2) Concurrent dd‐cfDNA re‐
+sults were available for only 58% of biopsy specimens. (3) The only analyte quantified by the assay used in this study is the fraction of dd‐cfDNA in the total cfDNA in the recipient's plasma. It is possi‐ ble that conditions unrelated to AR, such as increased turnover or death of recipient cells (as seen in trauma21 and sepsis22), can result in elevated total cfDNA levels and thereby reduce the donor frac‐
+tion. Nevertheless, dd‐cfDNA levels have previously been shown by multiple independent groups to be elevated in the setting of AR
+after heart,8-10 kidney,7,11 liver,7,23,24 and lung25 transplant. (4) The Cedars‐Sinai cohort was small and was from a single center. Also, only AMR events were observed in this cohort, likely due to the patient selection criteria. However, the results are consistent with the D‐OAR study and confirm the ability of dd‐cfDNA to detect graft damage. (5) With the recommended cutoff of 0.2%, there will be some false‐positive results; however, the potential negative impact of these may be mitigated if the clinician considers other clinical information about the patient (including symptoms, signs, and imaging results) before deciding whether a biopsy should be performed.
+
+dd‐cfDNA is measured in plasma from a blood draw and there‐
+fore can be performed frequently after HT. This, combined with the potential of the assay to detect early signs of graft damage,8,17 opens the door to more personalized titration of immunosuppres‐ sive therapies. Immunosuppressive medications such as cortico‐ steroids and calcineurin inhibitors could potentially be weaned faster in patients with no evidence of graft injury and augmented in patients who demonstrate a rise in dd‐cfDNA levels. Thus, more effective immunosuppression could be administered to recipients at higher risk of AR, whereas the side effects and toxicities of these medications could be avoided in stable patients. Additionally, this test may be performed if there is clinical suspicion for rejection or graft injury, before deciding on the need for endomyocardial biopsy. HT recipients may present with nonspecific symptoms, such as dyspnea, that could be due to graft dysfunction or op‐ portunistic infection. The dd‐cfDNA result may thereby help to focus subsequent diagnostic testing. This is especially useful in patients who are receiving anticoagulation therapy, with anatomic challenges, or with other contraindications to biopsy procedures.
+
+As with all laboratory tests, clinical evaluation of the patient must be factored into the interpretation of test results. The dd‐cfDNA test results may not eliminate the need for biopsy, but a high level may increase the probability of a positive biopsy result and would provide further justification for initiating clinical treatment of AR. The high NPV of the assay, on the other hand, would reduce the need for biopsies in patients with low suspicion for AR.
+
+In summary, this study establishes the performance of the dd‐
+cfDNA assay to detect acute rejection and graft dysfunction after HT in a large and diverse patient cohort in the United States. These results set the stage for subsequent clinical utility studies of the dd‐ cfDNA assay in HT patient management.
+
+## Acknowledgments
+
+The authors would like to thank Preethi Prasad and Theresa Wolf at CareDx, Inc., the research coordinators at the participating trans‐ plant centers for supporting the study conduct as well as sample and data collection, and the many heart transplant recipients who self‐ lessly participated in this study.
+
+## Disclosure
+
+The authors of this manuscript have conflicts of interest to disclose as described by the *American Journal of Transplantation*. Dr Khush is an advisor to CareDx and has received research support from CareDx. Drs Yee, Woodward, and Hiller are employees of CareDx. Drs Hall and Kobashigawa serve as co‐PIs and as advisors (consult‐ ants) to CareDx. Drs Pinney, Kao, Alharethi, DePasquale, Ewald, Berman, and Kanwar were co‐PIs in the DOAR study. Dr Patel has no conflicts of interest to disclose.
+
+## Data Availability Statement
+
+The data that support the findings of this study are available from the corresponding author upon reasonable request.
+
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+
+## Supporting Information
+
+Additional supporting information may be found online in the Supporting Information section at the end of the article. 
+
+How to cite this article: Khush KK, Patel J, Pinney S, et al. Noninvasive detection of graft injury after heart transplant using donor‐derived cell‐free DNA: A prospective multicenter study. *Am J Transplant*. 2019;19:2889–2899. https://doi. org/10.1111/ajt.15339

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+# Finding The Reasons Of Decrease In The Rate Of Population Growth In Iran Using Causal Layered Analysis (Cla) Method
+
+Mohammad Hossein Mehrolhassani1, Saeid Mirzaei2, Samira Sadat Poorhoseini3, Nadia Oroomiei*3 Received: 5 Mar 2018 Published: 4 Sep 2019
+
+## Abstract
+
+ **Background:** According to census 2011, general fertility rate in Iran was 1.6 children. The United Nations published a low population growth scenario for Iran in 2010, and if Iran continues to experience population replacement and does not have a plan to balance it, it will experience a population of 31 million, with a high percentage of elderly people in the next 80 years. This study was conducted to identify the causes of a decrease in population growth rate. **Methods:** This was a secondary study conducted by reviewing the scientific texts, papers, and upstream documents. The upstream documents contain all national documents related to population decline in Iran. Causal layered analysis (CLA) was used for data analysis. **Results:** The 9 most important identified causes for a decrease in population were litany (child mortality, maternal mortality, diseases burden, fertility rate, marriage squeeze, abortion, marriage age, high-risk behaviors, and badly supervised and neglected children. Also, 5 causes in structural layer were urbanization, education rate, economic participation rate and unemployment rate, new structures, a change in family structure, and intergenerational gap. Moreover, three causes in discourse layer included welfare, materialism, individualism, and 2 causes in metaphor layer were changing the perception of life and family formation, and women as workforce. **Conclusion:** It seems that the decrease in population growth in Iranian society is less the result of social planning and population control and more the result of the value and structural changes that have been occurred due to modernization in the society. It is recommended that policymakers primarily address the discourse and metaphor layers to solve the problems. Keywords: Causal layered analysis (CLA), Population decreasing, Iran Conflicts of Interest: *None declared* Funding: *None* 
+*This work has been published under *CC BY-NC-SA 1.0 license.* Copyright© Iran University of Medical Sciences Cite this article as: Mehrolhassani MH, Mirzaei S, Poorhoseini SS, Oroomiei N. Finding the reasons of decrease in the rate of population growth in Iran using causal layered analysis (CLA) method. *Med J Islam Repub Iran. 2019* (4 Sep);33:92. https://doi.org/10.34171/mjiri.33.92 
+
+## Introduction
+
+The population control policies and quality of population have long since been considered because population is the source of power and is the pillar of the development and improvement of any society (1). Negative consequences of population decrease are lack of economic growth, aging, generation discontinuity, disruption of social communication among children of low-population families, and mental and psychological problems due to 
+
+## The Lack Of Familial Ties (2).
+
+Many developing countries face a declining population, a trend different from the declining population in developed countries. The population decline in developed countries has occurred after experiencing economic growth and improving the living conditions. The population decline in developing countries has occurred prior to the improvement of living conditions, and it is faster than that in de-
+↑*What is "already known" in this topic:* Iran has experienced an unprecedented decline in fertility rates over the past 30 years. Among the negative consequences of population decreasing, the lack of economic growth and the aging of the society can be mentioned. 
+
+→*What this article adds:*
+This article identified the causes of population decline in Iran by documentary review and CLA method to be used in population policy and planning. 
+
+veloped countries. Iran, as a developing country, is not an exception (3). 
+
+Throughout history, Iran has experienced an unprecedented decline in the population. In 1970, there was an average of 6.5 children per Iranian woman, which dropped to 1.6 child per woman in 2011, which is below the fertility replacement rate (4, 5). The fertility rate in Iran has had an ascending descending trend over the past few decades. In the early 1970s, a moderate decline occurred in fertility from 6.5 in 1970 to about 6 in 1976. Following a rebound between 1976 and 1980 (increase of up to 7 in 1980), which coincided with the Islamic Revolution in 1979, and Iran-Iraq war (1980), the decline has resumed since the mid-80s. The family planning plan was also accelerated in 1979 (decrease from 6.8 in 1984 to 6.3 in 1986, and to about 5.5 in 1988) (4, 6). The extreme decline rate from 5.5 in 1988 to about 2.8 in 1996 reached 2.2 in 2000 (7). 
+
+These changes can be clearly linked to the population policies at each stage. For example, an antinatalist policy, at the end of the imperial regime, was the family planning program that was not accepted by the public due to the lack of support from religious leaders, which led to a slight decline in fertility at that time. Subsequently, by criticizing the family planning program and encouraging early marriage in the post-Islamic revolution of Iran and at the end of Iran-Iraq war, it returned to its antinatalist policy (8), which is strongly opposed by the Supreme Leader, Ayatollah Ali Khamenei. He has been demanding the suspension of the antinatalist policy and finding a pronatalist policy due to issues related to the aging population. Although Iran has adopted pronaturalist policies, statistics shows that it has not yet been able to reach the population replacement rate (9). This can be due to the lack of determination in the reasons for the problem of population decline in the root and in the underlying layers of the problem (10). Although many studies have been conducted on the factors influencing the increase and decrease in Iranian population, few studies have been conducted on the causes of population decline by determining the root causes. Given the importance of population and its favorable growth in Iran, the present study aimed to identify the various causes of decrease in population growth rate in Iran using CLA technique. 
+
+Soheil Enayatolah proposed a method for a deep investigation in the layers producing a problem, called causal layered analysis (CLA) (11), which has been employed by a few researchers (12–14). In this method, the reason for the appearance of a phenomenon is examined in 4 layers, each more rooted and general than the previous one (15). 
+
+## Methods
+
+This was a secondary study conducted by reviewing scientific texts and papers and upstream documents which contain all national documents related to population decline in Iran, developed and published by government organizations. Documents were provided by private organizations, but the newspapers were excluded from the study. The research team identified key upstream documents through examining the websites of the ministries and referring to several government organizations including Iranian Statistics Organization, Ministry of Health and Medical Education, State Welfare Organization of Iran, and National Organization for Civil Registration. The research team selected relevant documents based on 4 key factors: authenticity (being original and genuine), credibility (accuracy), representativeness (being representative of the totality of the documents in their class), and meaning 
+(what they say) (16). 
+
+The documents included Iranian Demographic Policy 
+(considerations and perspectives) (17), Youth Health Document (18), issued missives by Supreme Leader Ayatollah Khamenei (19), Iran's Multiple Indicator Demographic and Health Survey (20), Health Indicators in the Islamic Republic of Iran (21), analysis of the general population policies of the Supreme Leader Ayatollah Khamenei (22), Population and Housing Census (9), time series of the population (23), time series for marriage and divorce (24), Abortion Report (25), the first to sixth program of economic, social and cultural development of the Islamic Republic of Iran (26–31), child mortality in Iranian indices and trends from 1956 to 2021 (32), IslamicIranian Model of Progress (33), indices of employment and unemployment over the period of 1997-2012 (34), 
+Health Indicators (35), and Adolescent and Youth Reproductive Health Documents of Islamic Republic of Iran 
+(36).
+
+The documents were repeatedly studied by 3 members of the research team. Some parts of the texts were cited for reasons and consequences of population decline were selected as units of analysis. Disputes were reviewed and agreed upon through a meeting with the research team. CLA framework was employed for data analysis. 
+
+Based on this method, in the first layer, called litany, the most superficial data are analyzed, representing the official and accepted view of reality. The second layer is social causes, indicating the organized views. The third layer concerns the analysis of worldview and discourse. At this layer, argumentation assumptions, which are unconscious, and based on the context of ideologies and world view, are analyzed. Finally, the fourth layer indicated myths and metaphors, representing the unconscious motivational dimensions of the subject embodied in the form of myth and metaphor (10). 
+
+In this study, the cause of population decrease was investigated according to CLA technique. In the first place, the most tangible causes appeared in 2 layers of litany and social causes. However, a deeper analysis of the subject led to the emergence of the 2 following layers (the discourse layer and the metaphor and myth layer), each with its cause, and more general and rooted in the problem than the previous layer. 
+
+## Results
+
+ The findings were identified through reviewing these documents. The identified causes were classified into 4 layers: litany, social causes, discourse, and myth/metaphor (Fig. 1). 
+
+As Figure 1 demonstrates, most causes belong to Litany, which is the first and most tangible layer with 9 causes: 
+
+![2_image_0.png](2_image_0.png)
+
+child mortality (32, 36), maternal mortality (21, 35), disease burden (20), fertility rate (9, 37), marriage constraints 
+(9, 23), abortion (25, 36), marriage age (18, 24), high-risk behaviors (18, 36), and badly-supervised and neglected children (38). The second layer (social causes) includes 5 causes: urbanization and education rate (9), economic participation rate and unemployment rate (34), new structures (especially supportive structures) (33, 36), changing family structures, and intergenerational gap (33). The discourse layer consists of 3 causes: welfare, materialism, and individualism (17, 19, 22, 26–31, 33). The metaphor layer includes 2 causes: changing the perception of life and family formation, and women as workforce (17, 19, 22, 26–31, 33). 
+
+## Discussion
+
+In the following section, the reasons for population decrease have been analyzed in 4 layers. 
+
+## Litany Layer
+
+ In this layer, the clear causes of population decrease led to the identification of 9 causes. 
+
+The mortality index of pregnant mothers and children is considered as one of the most important demographic indexes and is considered as a symbol of development (39). 
+
+The death rate of mothers in Iran over the past 40 years has had a dramatic reduction, showing the effectiveness of implementing health system programs and other development sectors in Iran. However, this reduction is not enough to reach the Millennium Development Goal in 2015 (40). High mortality rate in children has adverse economic and social consequences (41). The mortality of mothers and children reduces the current population and the possibility of increasing the population in the future. 
+
+The second cause of population decrease was the burden of diseases. Non transmissible diseases are responsible for the deaths of over 35 million people each year, nearly two-thirds of the total deaths in the world (42). Studies related to the burden of diseases show that cardiovascular diseases, mental disorders, cancers, skeletal and muscular disorders, and AIDS are among the main causes of death in Iran which cause population decrease (43). 
+
+Demographic statistics show that in recent years, the rate of childbearing in Iranian families has been lower than the "fertility replacement rate" (44). Some scholars believe that increasing the fertility rate in Iran in the future is unlikely; moreover, by listing causes such as the increase in the level of urbanization and increase in the age of marriage, they have predicted a further decrease in the fertility rate (45). The increase in marriage squeeze in Iran is yet another cause affecting population decrease. In a simple definition, marriage squeeze can be defined as the imbalance in the number of men or women in marriage age. The results of marriage squeeze measurement in Iran have shown that it occurred for girls in 2006 and for boys in 2011 and 2016, and its severity would be more than girls' marriage squeeze in 2006 (46). Another barrier to population growth at this level is abortion. High statistics of abortion in Iran is unimaginable, as population growth is moving towards zero (47). The increase in the age of marriage and pregnancy, unplanned pregnancies, and the lack of desire for childbearing can be noted among causes of abortion (48). 
+
+The age of marriage is important in controlling population, as the increase in the age of marriage reduces fertility. The average age of marriage has increased over the recent years, which can be attributed to the change in people's attitudes toward economic issues, such as the increase in living costs, housing, unemployment, and marriage (46). 
+
+The high-risk behaviors known among Iranian youths include violence, suicide, careless driving, tobacco use, alcohol and drugs, high-risk sexual behaviors, unhealthy nutrition, and lack of exercise and physical activities. 
+
+Risky behavior is considered as the most major risk for society health and the cause of many deaths (49, 50). 
+
+Therefore, paying attention to this issue conduces to increase in the population. 
+
+According to statistics provided by the State Welfare Organization of Iran, there are 23 000 orphans and badly supervised children in Iran (51) who experience parental separation and divorce and have irresponsible parents. The increase in badly supervised children without any support disturbs the future of the society in terms of creating a healthy family and childbearing (52). 
+
+Social causes layer In this layer, 5 causes of population decrease have been discussed. 
+
+When there is a prospect of having a metropolis in the community and wealth is only available in city expansion, immigration and urbanization will increase. Population density, increase in living costs, reduction in the size of residential houses as barriers for more childbearing, and having a larger family are the consequences of this perspective (53). 
+
+New perspectives are related to large families as barriers to progress. Studies show that higher level of education increases the age of marriage and reduces fertility (54). Women's participation in economy has had consequences such as increased social welfare, and production level, and rising per capita income; however, along with the positive consequences, it has reduced the fertility rate (55). 
+
+Further affecting population growth are new structures such as single lives, and cohabitation, which is called white marriage in Iran, and has become widespread in big cities (46). 
+
+Another undeniable fact in urban life is the transformation in the family, which has taken place in various dimensions in Iran, including selecting the spouse, marriage, relationship between family members, changing attitudes, changing members' function and relationships between men and women, and changing lifestyles. Changes in the value system is also another transformation. 
+
+These changes include infidelity and easy approach to divorce and disturbance in family members relationship (56). 
+
+## Discourse Layer
+
+If we look more closely at the causes of population decrease and look for more fundamental reasons, we can find worldviews and discourses. The causes that have emerged in this layer have been rooted in the beliefs and values of individuals. When welfare, comfort, pleasure, materialism, and individualism that are the outcomes of Western culture and lifestyle become common among the values of a society, then, success, progress, and welfare are seen as having fewer children; such lifestyle is finally considered as the right way of life and standard of desirable life. Other studies have shown modernity to be effective in population decrease due to its welfare and pleasurecentered nature (57). Many scholars have identified individualism as an important factor that changes the attitudes towards matrimony (58, 59). The results of Aghajanian and Thompson's (2013) study showed an increase in the tendency towards individualism in Iran (60). Rastegar Khaled and Mohammadi (2014) have identified 4 causes for a decreasing population in Iran. Their results showed that secularism, individualism, and less attention to family values have a positive and consistent relationship with low fertility rates (61). 
+
+Myth/metaphor layer When we analyze the underlying layer of the iceberg 
+(CLA is the iceberg and litany and social causes are the tip, and a huge part of the iceberg (Discourse and Myth Layers) is hidden and not seen), the concept of "changing the perception of a family" emerges. Cultural changes that have taken place over time among people have changed the concept of family and childbearing. According to Abbasi-Shavazi and Askari-Nodoushan (2012), Iranian families are transitioning from traditional families to modern ones, and the signs of the crisis in this transition are obvious. Because of this transition, the family value system has changed (62). Marriage or family formation is in a transition situation from an unknown and destroyed tradition to a vague and poorly understood modernity. Cultural change has occurred not only in the role of the spouse and mother, but also in the philosophy of marriage and individuals' understanding of marriage. As women's beliefs have changed their role from women as spouses and mothers to women as economic powers, a shift has been created in attitudes toward marriage and spousal and maternal roles. The change in the role of women, increase in divorce statistics, marriage decrease, view change on the role of wife, and infidelity in marital relationships are among the damages of cultural change (63). 
+
+One reason for such damages is that the change from tradition to modernity has occurred before the new and industrial values are internalized in the society. Modern families without value internalization have endangered the stability of societies and the institution of the family. The new generation in Iran prefers to enjoy new social pleasures than to have responsibilities and family commitment 
+(64–66). 
+
+## Conclusion
+
+The results of this study showed that various cultural, social, political, and economic causes affect population growth. However, it seems that the decrease in population growth in Iranian society is less the result of social planning and population control policies (although its role should not be ignored), and more the result of the value and structural changes that have occurred due to modernization in the society. It does not mean that the causes in the first layers have lower effects or importance. It is recommended that policymakers primarily address the discourse and metaphor layers to solve the problems. Welfare, materialism, and individualism, and the paradigm shift that have occurred in the concept and role of woman in the metaphor and discourse layers have changed the perception of life and family formation to single life or having fewer children. Therefore, phenomena such as urbanization and the increase in the age of marriage appear in the social causes and litany layers. The problems identified in the social causes and litany layers will be solved by changing the discourse and metaphor layers. For example, changing the lifestyle and family identity and increasing the fertility rate can be achieved through the struggle with the metaphor that childbearing prevents progress. Based on CLA, it is necessary to pay attention to all known causes at different layers in population planning for Iran. 
+
+Strengths and limitations CLA method was employed to identify the underlying reasons in the discourse and metaphor for the problem of population decline and the failure of policies in this area in Iran. The limitation of this study was that only documentary analysis was done, while interviews with policymakers and decision-makers in this area can enhance the findings of the study. 
+
+Conflict of Interests The authors declare that they have no competing interests. 
+
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medical/md/PMC6972599.md

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+DOI: 10.1002/ppul.24508 ORIGINAL ARTICLE: ASTHMA
+
+# Subcutaneous Mepolizumab In Children Aged 6 To 11 Years With Severe Eosinophilic Asthma
+
+Atul Gupta MD(Res)1 | Isabelle Pouliquen PharmD2 | Daren Austin PhD2 | Robert G. Price MSc3 | Rodger Kempsford PhD4 | Jonathan Steinfeld MD5 | Eric S. Bradford MD6 | Steven W. Yancey MSc6 1King's College Hospital NHS Foundation Trust, King's College London, London, UK
+2Clinical Pharmacology Modelling and Simulation, GSK, Uxbridge, Middlesex, UK
+3Clinical Statistics, GSK, Stevenage, Hertfordshire, UK
+4Clinical Pharmacology Modelling and Simulation, R&D Medicines Research Centre, GSK, Stevenage, Hertfordshire, UK
+5Respiratory TAU & Flexible Discovery Unit, GSK, Philadelphia, Pennsylvania 6Respiratory Therapeutic Area, GSK, Research Triangle Park, North Carolina Correspondence Jonathan Steinfeld, MD, Respiratory TAU & Flexible Discovery Unit, GSK, Upper Providence UP1210, 1250 South Collegeville Road, Collegeville, PA 19426‐0989.
+
+Email: jonathan.x.steinfeld@gsk.com Funding information GSK, Grant/Award Number: 200363
+
+## Abstract
+
+Objectives: There are no published reports for anti‐interleukin‐5 therapy in children
+<12 years with asthma. The primary objective of this study was to characterize the pharmacokinetics and pharmacodynamics of mepolizumab following subcutaneous
+(SC) administration in children 6 to 11 years‐of‐age with severe eosinophilic asthma.
+
+Hypothesis: Mepolizumab SC pharmacokinetics and pharmacodynamics in children with severe eosinophilic asthma are comparable with adults.
+
+Study Design: Multinational, nonrandomised, open‐label (NCT02377427).
+
+Patient Selection: Children 6 to 11 years‐of‐age with severe eosinophilic asthma
+(blood eosinophil count ≥150 cells/µL at screening or ≥300 cells/µL <12 months of screening) and ≥2 exacerbations in the prior year.
+
+Methodology: Children received mepolizumab SC 40 mg (bodyweight <40 kg) or 100 mg (≥40 kg) every 4 weeks for 12 weeks.
+
+Results: Thirty‐six children received mepolizumab (40 mg, n = 26; 100 mg, n = 10).
+
+Mepolizumab exposures were higher and apparent clearance lower than predicted based on prior existing data. Derived mepolizumab exposures normalized to mean bodyweight for the 40 mg and 100 mg dose groups were 454 μg * day/mL and 675 μg * day/mL, respectively. At week 12, blood eosinophils were reduced by 89%
+and 83% from baseline to 42 and 55 cells/µL, respectively. Mepolizumab was well tolerated; no new safety signals were observed compared with previous adult/ adolescent studies.
+
+Conclusion: In children 6 to 11 years‐of‐age with severe eosinophilic asthma, mepolizumab SC 40 or 100 mg provided bodyweight‐adjusted drug exposure within twofold of target adult exposure as well as marked reductions to blood eosinophil counts similar to adults, and although not designed to evaluate efficacy outcomes, demonstrated a positive clinical profile.
+
+Presented at the 37th Annual Congress of the European Academy of Allergy and Clinical Immunology, 26 to 30 May 2018, Munich, Germany.
+
+---------------------------------------------------------------------------------------------------------------------------
+This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
+
+© 2019 The Authors. Pediatric Pulmonology Published by Wiley Periodicals, Inc.
+
+## 1 | Introduction 2 | Materials And Methods 2.1 | Study Participants 2.2 | Study Design 2.3 | Treatments
+
+Asthma is the most common chronic childhood disease worldwide.1 Approximately 2% to 5% of children with asthma have persistent symptoms, repeated hospital admissions for asthma exacerbations, school absences and poor quality of life (QoL)
+despite treatment with optimized therapy.2-5 Compared with children with mild or moderate asthma, those with severe asthma are at increased risk of severe exacerbations and medication side effects, have impaired lung function, have significantly poorer QoL, and are greater risk of developing chronic obstructive pulmonary disease as adults.3,6,7 Thus, there is an unmet need for Participants were children aged 6 to 11 years with a diagnosis of severe asthma, as defined by regional guidelines, and eosinophilic airway inflammation demonstrated by peripheral blood eosinophil counts
+≥300 cells/μL within 12 months of screening or ≥150 cells/μL at screening. Eligible children had also experienced ≥2 exacerbations requiring treatment with systemic corticosteroids (SCS) ≤ 12 months before screening (an exacerbation in children receiving maintenance OCS
+must have necessitated a ≥twofold increase in their OCS
+dose). In the 12 months before screening, participants were receiving regular medium‐ or high‐dose ICS (>200 µg/day fluticasone propionate or equivalent) with or without maintenance OCS. They were also receiving
+≥1 additional controller medication (eg, long‐acting β‐2‐agonist, leukotriene receptor antagonist, or theophylline) for ≥3 months, or had a documented failure of the additional controller medication for ≥3 successive months, in the 12 months before screening. Participants who had received omalizumab within 130 days of screening or any other biologic to treat inflammatory disease within five half‐lives of screening were not included in the study. Enrolled children had a prebronchodilator forced expiratory volume in 1 second (FEV1) < 110% predicted; or, FEV1/
+forced vital capacity ratio <0.8.
+
+This was a non‐randomised, open‐label, repeat‐dose, phase 2 study conducted at 13 centers in Japan, Poland, UK, and United States (ClinicalTrials.gov number NCT02377427; GSK ID 200363). The study consisted of two parts. Part A, presented here, assessed the PK and PD of mepolizumab SC over a 12‐week treatment period and an 8‐week follow‐up (Figure 1A). Part B will assess long‐term safety and PD over a 52‐week treatment period and will be reported separately.
+
+Written informed consent was obtained from the parent/guardian of each child. Study protocol, amendments, and informed consent were reviewed and approved by national, regional, or investigational center ethics committees or institutional review boards, and the study was conducted according to the ethical principles outlined in the current Declaration of Helsinki (2013). Anonymised individual participant data and study documents can be requested for further research from www.
+
+clinicalstudydatarequest.com.
+
+additional, effective, and well‐tolerated treatment options for severe asthma in children.8 The anti‐interleukin [IL]‐5 monoclonal antibody mepolizumab is approved for the treatment of adults and children ≥6 years of age with severe eosinophilic asthma in Europe, and for adults and adolescents ≥12 years in a number of countries, including the United States and Japan.9,10 In the pivotal phase 3 studies, addition of mepolizumab subcutaneously (SC) to standard of care was associated with significant reductions in exacerbation rates11 and dependency on oral corticosteroid (OCS) use,12 and a significant improvement in symptom control and health‐related QoL.11,13 In the adolescents aged 12 to 17 years included in the mepolizumab clinical development program, the exacerbation rate trended in favor of mepolizumab and the adverse event (AE) profile was generally similar to the overall population.9 Eosinophilic airway inflammation is a feature of severe asthma in some children,14,15 and anti‐IL‐5 therapy may be an attractive treatment option. Mepolizumab, administered intravenously (IV; 0.55, 2.5, or 10 mg/kg), has been shown to markedly reduce blood eosinophil counts in children aged 2 to 17 years with eosinophilic esophagitis.16 The pharmacokinetics (PK) of mepolizumab IV in these children was similar to that observed in adults when dosed on a weight basis.16 However, mepolizumab PK, pharmacodynamics (PD)
+and effectiveness following SC administration have not been evaluated in children <12 years in any indication.
+
+The primary objective of the first part of this two‐part study was to characterize the PK and PD of repeat‐dose mepolizumab following SC administration in children 6 to 11 years with severe eosinophilic asthma after 12 weeks of treatment. Secondary objectives included comparison of bodyweight‐adjusted mepo‐lizumab clearance between children aged 6 to 11 years and historic adult values, effectiveness, and tolerability. The hypothesis of the study was that subcutaneous mepolizumab pharmacokinetics and pharmacodynamics in children with severe eosinophilic asthma are comparable with adults.
+
+Mepolizumab was administered SC once every 4 weeks for a total of three doses (weeks 0, 4, and 8), with the study active
+
+  | 1959
+
+![2_image_0.png](2_image_0.png)
+
+treatment period defined as weeks 0 to 12. Children were assigned to one of two dosing groups based on their bodyweight at baseline: mepo‐lizumab 40 mg for children <40 kg, and mepolizumab 100 mg for ≥40 kg. This dosing scheme was designed to provide similar exposure to that achieved in adults and adolescents with mepolizumab 100 mg SC.11,12 Assigned doses remained the same irrespective of bodyweight changes during part A. Mepolizumab was added to existing stable asthma treatment, with additional controller or rescue medication permitted.
+
+## 2.4 | Endpoints And Assessments
+
+The primary PK endpoints were the population PK model derived estimates of mepolizumab plasma clearance, area under the plasma concentration‐time curve to infinity (AUC[0‐inf]), maximum plasma concentration (Cmax), and terminal phase elimination half‐life (t1/2).
+
+The primary PD endpoint was the ratio of absolute blood eosinophil count at week 12 to baseline. Secondary endpoints included bodyweight‐adjusted plasma clearance estimates, change from baseline in Asthma Control Questionnaire 7‐item (ACQ‐7) score
+
+## 1960 |
+
+and Childhood Asthma Control Test (C‐ACT) score, both at weeks 4, 8, 12, 16, and 20. Mepolizumab safety and tolerability were assessed through AE reporting, immunogenicity, laboratory parameters, and vital signs.
+
+Exploratory endpoints included asthma exacerbation frequency during the treatment period (weeks 0–12) and throughout part A
+(weeks 0–20), change from baseline to week 12 in FEV1 and serum total IL‐5 levels. An exacerbation was defined as worsening of asthma that required SCS treatment and/or hospitalization and/or an emergency room (ER) visit.
+
+Blood samples for PK assessment were taken at every visit from weeks 4 to 20 (weeks 4, 8, 9, 12, 16, and 20); samples at weeks 4 and 8 were drawn before mepolizumab dosing. The ACQ‐7 and C‐ACT
+questionnaires were administered at baseline and every 4 weeks to week 20. Hematology, including eosinophils, and assessment of exacerbations, AEs, and vital signs were evaluated at every visit to week 20. Serum total IL‐5 levels were measured at baseline and week 12.
+
+## 2.5 | Sample Size And Statistical Analysis
+
+The sample size was determined by population PK trial simulation. A sample size of 16 to 32 children, combined with sparse PK sampling, was deemed sufficient to maintain precision of exposure estimates below 20% (compared with US Food and Drug Administration guideline recommendations of below 40% 17), provided that five PK
+samples (including one close to the time of Cmax) were collected and four model parameters were fixed to adult values in the population PK model. For blood eosinophils, assuming a similar residual variance to that observed in adults and adolescents,11 a sample size of 20 children provided sufficient precision for the 95% confidence interval (CI) of the ratio to baseline to fall within 50% of the observed geometric mean.
+
+Mepolizumab plasma PK concentrations were analyzed by nonlinear mixed‐effect modeling methods (SAS Institute Inc, Cary, NC). A two‐compartment model with first‐order absorption and elimination was used with PK distribution parameters and bioavailability fixed to previously estimated adult values. Bodyweight was incorporated into the model using physiological allometry with allometric exponents for clearance and volumes estimated. Final model appropriateness was assessed using goodness of fit plots, simulations, and statistical tests. The ratio of blood eosinophil count at week 12 to baseline was summarized descriptively, and the ratio at each visit to baseline presented graphically. Bodyweight‐adjusted apparent clearance point estimates with 90% CIs in children 6 to 11 years were presented alongside the historical estimated adult value of 0.29 L/day
+(unpublished data) with a proposed 80% to 125% interval around this estimate of 0.23 to 0.36 L/day. An exploratory population PK analysis using the most recent mepolizumab population PK model with minimal estimation (absolute bioavailability, allometric exponents, and residual error) was conducted using NONMEM
+software (version 7.2; ICON Development Solutions, Ellicott City, MD).
+
+## 3 | Results 3.1 | Patient Population
+
+The study was conducted between 25 August 2015 and 7 December 2016. A total of 44 children were assessed for eligibility of whom seven were excluded during screening (Figure 1B).
+
+One additional patient did not meet the study continuation criteria due to an exacerbation during the run‐in phase. The remaining 36 children were assigned to study treatment; 26 children to the 40 mg dose group (weight <40 kg) and 10 to the 100 mg dose group (weight ≥40 kg). All 36 children were included in the analysis population. Part A was completed by 32 (89%)
+children with four children, all in the 40 mg dose group, withdrawing prematurely.
+
+Enrolled children were predominantly male (69%), with a mean body mass index (BMI) of 16.1 kg/m2 in the 40 mg dose group
+(weight <40 kg) and 23.1 kg/m2 in the 100 mg dose group
+(weight ≥40 kg) (Table 1). In the 12 months before study entry, the median number of exacerbations requiring SCS was 3.0
+(range 2‐15), and 44% (16/36) of children had required hospitalization for an exacerbation. Eight (22%) children were receiving OCS at baseline.
+
+## 3.2 | Primary Endpoints: Population Pk Parameters
+
+Mepolizumab population PK parameter estimates and predicted and observed mepolizumab concentration‐time plots are presented in Table 2 and Figure 2. Apparent clearance, AUC(0‐inf), Cmax and t½
+population PK parameter estimates are presented normalized to 27 and 50 kg (mean bodyweight for the 40 and 100 mg dose groups, respectively), and to a typical adult bodyweight (70 kg; which was not observed in the study). Derived mepolizumab exposure (AUC(0‐inf])
+normalized to 27 and 50 kg were 454.4 and 675.2 μg * day/mL,
+respectively (Table 2). Mepolizumab estimated t½ normalized to 27 and 50 kg was 23.6 and 21.8 days, respectively.
+
+## 3.3 | Primary Endpoints: Blood Eosinophil Counts
+
+At baseline, respective geometric mean blood eosinophil counts were 386, 331, and 370 cells/µL in the 40 mg dose group (<40 kg), 100 mg dose group (≥40 kg), and overall (Table 1). Blood eosinophil counts showed a marked reduction by the first on‐treatment assessment at week 4 (Figure 3). Blood eosinophil count reductions were of similar magnitude irrespective of dose group and were sustained throughout the treatment period. By week 12, blood eosinophil counts were reduced from baseline by 88.5% in the 40 mg dose group (to 42 cells/µL; 95% CI: 26, 67) and by 83.4% in the 100 mg dose group (to 55 cells/µL; 95% CI: 31, 97) (Figure 3 and Table 4).
+
+Once mepolizumab treatment was stopped, geometric mean blood
+
+  | 1961
+
+| TABLE 1                                                                                                                                                                                                       | Summary of patient demographics and baseline characteristics (safety population) Mepolizumab 40 mg   | Mepolizumab 100 mg   |             |
+|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------|----------------------|-------------|
+| (weight <40 kg)                                                                                                                                                                                               | (weight ≥40 kg)                                                                                      | Total                |             |
+| (N = 26)                                                                                                                                                                                                      | (N = 10)                                                                                             | (N = 36)             |             |
+| Derived agea , years                                                                                                                                                                                          | 8.0 (1.8)                                                                                            | 10.0 (1.3)           | 8.6 (1.9)   |
+| Sex, n (%) Female                                                                                                                                                                                             | 6 (23)                                                                                               | 5 (50)               | 11 (31)     |
+| Male                                                                                                                                                                                                          | 20 (77)                                                                                              | 5 (50)               | 25 (69)     |
+| Weight, kg                                                                                                                                                                                                    | 27.4 (4.7)                                                                                           | 49.5 (6.3)           | 33.5 (11.2) |
+| Body mass index, kg/m2                                                                                                                                                                                        | 16.1 (1.7)                                                                                           | 23.1 (2.8)           | 18.0 (3.7)  |
+| Pre‐BD lung function FEV1 (mL)                                                                                                                                                                                | 1407 (365)                                                                                           | 1940 (310)           | 1555 (422)  |
+| Predicted normal FEV1 (%)                                                                                                                                                                                     | 89 (16.9)                                                                                            | 92 (6.9)             | 90 (14.8)   |
+| FVC (mL)                                                                                                                                                                                                      | 1805 (372)                                                                                           | 2436 (448)           | 1985 (484)  |
+| FEV1/FVC                                                                                                                                                                                                      | 0.78 (0.11)                                                                                          | 0.80 (0.09)          | 0.79 (0.11) |
+| Number of exacerbations requiring corticosteroids in prior 12 mo, median (range)                                                                                                                              | 3 (2–15)                                                                                             | 2 (2–10)             | 3 (2–15)    |
+| Patients with an exacerbation requiring hospitalization in prior 12 mo, n (%)                                                                                                                                 | 14 (54)                                                                                              | 2 (20)               | 16 (44)     |
+| Blood eosinophils, cells/uL, geometric mean (SD logs)                                                                                                                                                         | 386 (0.75)                                                                                           | 331 (0.91)           | 370 (0.78)  |
+| Blood eosinophil count, n (%) ≥150 cells/μL at screening                                                                                                                                                      | 20 (77)                                                                                              | 10 (100)             | 30 (83)     |
+| ≥300 cells/μL in 12 mo before screening                                                                                                                                                                       | 21 (81)                                                                                              | 9 (90)               | 30 (83)     |
+| ≥150 cells/μL at screening and ≥300 cells/μL in 12 mo before screening                                                                                                                                        | 15 (58)                                                                                              | 9 (90)               | 24 (67)     |
+| IgE U/mL, geometric mean (SD logs)                                                                                                                                                                            | 336 (1.48)                                                                                           | 379 (1.09)           | 348 (1.36)  |
+| OCS daily dose, n (%) Any use                                                                                                                                                                                 | 6 (23)                                                                                               | 2 (20)               | 8 (22)      |
+| <7.5 mg/day                                                                                                                                                                                                   | 3 (12)                                                                                               | 0                    | 3 (8)       |
+| ≥7.5 to <15 mg/day                                                                                                                                                                                            | 1 (4)                                                                                                | 0                    | 1 (3)       |
+| ≥15 to <30 mg/day                                                                                                                                                                                             | 0                                                                                                    | 1 (10)               | 1 (3)       |
+| ≥30 mg/day                                                                                                                                                                                                    | 2 (8)                                                                                                | 1 (10)               | 3 (8)       |
+| ICS daily dose (fluticasone propionate [DPI] equivalentb ), n (%) Any use                                                                                                                                     | 26 (100)                                                                                             | 10 (100)             | 36 (100)    |
+| >200 to ≤400 µg/day                                                                                                                                                                                           | 6 (23)                                                                                               | 1 (10)               | 7 (19)      |
+| >400 µg/day                                                                                                                                                                                                   | 20 (77)                                                                                              | 9 (90)               | 29 (81)     |
+| ACQ‐7 score                                                                                                                                                                                                   | 1.99 (1.21)                                                                                          | 1.39 (0.96)          | 1.82 (1.17) |
+| C‐ACT score                                                                                                                                                                                                   | 15.6 (5.7)                                                                                           | 20.4 (3.2)           | 16.9 (5.6)  |
+| Data are mean (standard deviation) unless stated otherwise. Abbreviations: ACQ, Asthma Control Questionnaire; C‐ACT, Childhood Asthma Control Test; DPI, dry powder inhaler; BD, bronchodilator; FEV1, forced |                                                                                                      |                      |             |
+
+eosinophil counts increased toward baseline values during the follow‐up period.
+
+## 3.4 | Secondary Endpoints: Bodyweight‐Adjusted Apparent Plasma Clearance
+
+The population bodyweight‐adjusted apparent clearance (ie, CL/F at 70 kg) was 0.20 L/day (90% CI: 0.17, 0.22) for children in this study, which fell outside the prespecified 80% to 125% range around the historical adult value of 0.29 L/day (80%, 125% interval: 0.23, 0.36).
+
+This value implies a lower CL/F in children aged 6 to 11 years compared with adults (Table 3).
+
+## 3.5 | Exploratory Population Pk Analysis Results
+
+An exploratory population PK analysis was performed to investigate potential explanations for the lower bodyweight‐
+adjusted apparent clearance in children. The analysis used the most recent mepolizumab population PK model from a comprehensive meta‐analysis of previous mepolizumab studies. All parameters were fixed except absolute bioavailability and allometric exponents for scaling of clearance and volumes by bodyweight. The estimated absolute bioavailability was 105% (95% CI: 55, 155%), and estimates for allometric exponents for clearance and volumes were 0.86 (95% CI: 0.29, 1.43) and 0.66 (95% CI: 0.11, 1.21), respectively.
+
+| TABLE 2                                                                                                                                  | Mepolizumab population pharmacokinetic parameter estimates from the final model (primary population pharmacokinetic analysis) Mepolizumab population pharmacokinetic parameter estimate (95% CIs) Normalized to Normalized to Normalized to 70 kg 27 kg 50 kg (for comparison with adults)   |                      |                      |
+|------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|----------------------|
+| AUC(0‐inf) (μg ⁎ day/mL)                                                                                                                 | 454.4 (422.1, 486.7)                                                                                                                                                                                                                                                                         | 675.2 (602.2, 748.2) | 508.2 (423.3, 593.2) |
+| Cmax (μg/mL)                                                                                                                             | 10.2 (9.5, 10.9)                                                                                                                                                                                                                                                                             | 16.3 (15.0, 17.6)    | 12.8 (11.2, 14.4)    |
+| Cmax SS (μg/mL)                                                                                                                          | 17.8 (15.3, 20.2)                                                                                                                                                                                                                                                                            | 28.5 (25.0, 31.9)    | 22.3 (19.2, 25.5)    |
+| CL/F (L/day)                                                                                                                             | 0.09 (0.08, 0.09)                                                                                                                                                                                                                                                                            | 0.15 (0.13, 0.16)    | 0.20 (0.16, 0.23)    |
+| Cav (μg/mL)                                                                                                                              | 16.2 (15.1, 17.4)                                                                                                                                                                                                                                                                            | 24.1 (21.5, 26.7)    | 18.2 (15.1, 21.2)    |
+| t½ (days)                                                                                                                                | 23.6 (21.9, 25.3)                                                                                                                                                                                                                                                                            | 21.8 (19.6, 24.1)    | 21.0 (17.6, 24.3)    |
+| KA (per day)                                                                                                                             | 0.17 (0.13, 0.20)                                                                                                                                                                                                                                                                            |                      |                      |
+| Between‐subject variability for CL/F (%)                                                                                                 | 15.5 (10.6, 19.3)                                                                                                                                                                                                                                                                            |                      |                      |
+| CL allometric exponent                                                                                                                   | 0.84 (0.65, 1.04)                                                                                                                                                                                                                                                                            |                      |                      |
+| V allometric exponent                                                                                                                    | 0.72 (0.56, 0.88)                                                                                                                                                                                                                                                                            |                      |                      |
+| Residual error                                                                                                                           | 0.029 (0.023, 0.036)                                                                                                                                                                                                                                                                         |                      |                      |
+| Data are normalized to 27 kg (mean in the <40 kg group receiving 40 mg mepolizumab SC), 50 kg (mean in the ≥40 kg group receiving 100 mg |                                                                                                                                                                                                                                                                                              |                      |                      |
+
+![5_image_0.png](5_image_0.png)
+
+FIGURE 2 Observed and population PK model predicted mepolizumab plasma concentrations in children aged 6 to 11 years with severe eosinophilic asthma. PK,
+pharmacokinetic; SC, subcutaneous [Color figure can beviewed at wileyonlinelibrary.com]
+FIGURE 3 Ratio of blood eosinophil counts to baseline (geometric mean).
+
+Vertical bars represent 95% confidence intervals. SC, subcutaneous
+
+  | 1963
+
+![6_image_0.png](6_image_0.png)
+
+## 3.6 | Effectiveness 3.6.1 | Secondary Endpoints: Acq‐7 And C‐Act
+
+Mepolizumab treatment was associated with a trend toward improved asthma control as indicated by numerical improvements in mean ACQ‐7 and C‐ACT total scores compared with baseline
+(Table 4). At week 12, a minimally clinically important improvement (≥0.5‐point reduction) in ACQ‐7 total score from baseline was reported for 48% of children, with similar response rates in the two mepolizumab dose groups (Table 4).
+
+## 3.6.2 | Exploratory Analyses: Exacerbation Rate, Fev1, And Serum Total Il‐5 Levels
+
+Ten (28%) children reported ≥1 on‐treatment exacerbation with a total of 13 events (Table 4). Four children (all in the 40 mg dose group [<40 kg]) required an on‐treatment hospitalization or ER
+visit. Thirteen (36%) children reported ≥1 exacerbation over the 20‐week study period. Mean baseline prebronchodilator FEV1 values were 1407 and 1940 mL in the mepolizumab 40 (<40 kg)
+and 100 mg (≥40 kg) dose groups, respectively. There was no clear pattern of change in FEV1 from baseline to week 12 (Table 4).
+
+Total serum IL‐5 levels at baseline were below the assay lower limit of quantification (<7.81 ng/L) for 78% of the children (28/36).
+
+Total serum IL‐5 levels (free and mepolizumab‐bound IL‐5) had increased in all subjects at week 12 from baseline except one, to a median of 137.1 ng/L in the 40 mg dose group (<40 kg) and 96.4 ng/L in the 100 mg dose group (≥40 kg).
+
+## 3.7 | Safety
+
+Overall, 72% (26/36) of children experienced an AE, with 67% (24/
+36) experiencing an on‐treatment AE (Table 5). Headache and injection‐site reactions were the most frequent on‐treatment AEs (both 14% [5/36] of the children). On‐treatment serious AEs (SAE)
+were reported for 17% (6/36) of children including two children with events considered related to the study drug by investigators. One 9‐
+year‐old male child in the mepolizumab 40 mg dose group (<40 kg) permanently discontinued due to a drug‐related SAE of severe asthma exacerbation 11 days after administration of the second dose. The child was hospitalized and treated with prednisolone and salbutamol; the event resolved after 10 days. A second 10‐year‐old male child experienced drug‐related SAEs of severe back pain, chest pain, dizziness, headache, nausea, and pain. There were no deaths.
+
+On‐treatment infections occurred in 50% (18/36) of children; infections reported in more than one child included nasopharyngitis
+
+| TABLE 3                                                                                                                                                                                                                          | Mean bodyweight‐adjusted apparent plasma clearance   |                          |                           |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------|--------------------------|---------------------------|
+| Historic adult data                                                                                                                                                                                                              | Apparent clearance (L/day)                           | 80% lower‐bound interval | 125% upper‐bound interval |
+| Adult target (70 kg)a                                                                                                                                                                                                            | 0.29                                                 | 0.23                     | 0.36                      |
+| PK/PD study of children aged 6–11 y                                                                                                                                                                                              | Apparent clearance (L/day)                           | Lower 90% CI             | Upper 90% CI              |
+| Children (70 kg)b                                                                                                                                                                                                                | 0.20                                                 | 0.17                     | 0.22                      |
+| Children (50 kg)c                                                                                                                                                                                                                | 0.15                                                 | 0.13                     | 0.16                      |
+| Children (27 kg)d                                                                                                                                                                                                                | 0.09                                                 | 0.08                     | 0.09                      |
+| Abbreviations: CI, confidence interval; PD, pharmacodynamic; PK, pharmacokinetic; SC, subcutaneous. a Historic adult data from the MENSA trial for a 70 kg individual; data in children aged 6–11 y. b Typical adult bodyweight. |                                                      |                          |                           |
+
+Abbreviations: CI, confidence interval; PD, pharmacodynamic; PK, pharmacokinetic; SC, subcutaneous.
+
+aHistoric adult data from the MENSA trial for a 70 kg individual; data in children aged 6–11 y. bTypical adult bodyweight.
+
+cMean bodyweight in the ≥40 kg group receiving 100 mg mepolizumab SC. dMean bodyweight in the <40 kg group receiving 40 mg mepolizumab SC.
+
+## 1964 |
+
+| TABLE 4                                                                                                                                                                                                                                                                                                                      | Summary of secondary and other endpoints: ACQ‐7 and C‐ACT total scores and exacerbations Mean (95% CI) Mepolizumab 40 mg Mepolizumab 100 mg SC (weight <40 kg) SC (weight ≥40 kg)   | Total               |                      |
+|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------|----------------------|
+| (N = 26)                                                                                                                                                                                                                                                                                                                     | (N = 10)                                                                                                                                                                            | (N = 36)            |                      |
+| Blood eosinophil counts, cells/µL Ratio to baseline at week 4                                                                                                                                                                                                                                                                | 0.19 (0.13, 0.28)                                                                                                                                                                   | 0.22 (0.08, 0.61)   | 0.20 (0.14, 0.29)    |
+| Ratio to baseline at week 8                                                                                                                                                                                                                                                                                                  | 0.11 (0.08, 0.16)                                                                                                                                                                   | 0.14 (0.07, 0.29)   | 0.12 (0.09, 0.16)    |
+| Ratio to baseline at week 12                                                                                                                                                                                                                                                                                                 | 0.12 (0.07, 0.20)                                                                                                                                                                   | 0.17 (0.09, 0.32)   | 0.13 (0.09, 0.19)    |
+| % reduction from baseline at week 12                                                                                                                                                                                                                                                                                         | 88.5                                                                                                                                                                                | 83.4                | 87.1                 |
+| ACQ‐7 scorea Change from baseline at week 4                                                                                                                                                                                                                                                                                  | –0.55 (–1.01, –0.09)                                                                                                                                                                | –0.47 (–1.16, 0.21) | –0.53 (–0.89, –0.16) |
+| Change from baseline at week 8                                                                                                                                                                                                                                                                                               | –0.65 (–1.15, –1.16)                                                                                                                                                                | –0.30 (–1.19, 0.59) | –0.55 (–0.97, –0.14) |
+| Change from baseline at week 12                                                                                                                                                                                                                                                                                              | –0.41 (–0.91, 0.08)                                                                                                                                                                 | 0.08 (–0.88, 1.04)  | –0.26 (–0.69, 0.16)  |
+| ≥0.5 point reduction from baseline, n/N (%)                                                                                                                                                                                                                                                                                  | 11/23 (48)                                                                                                                                                                          | 5/10 (50)           | 16/33 (48)           |
+| C‐ACT scoreb Change from baseline at week 4                                                                                                                                                                                                                                                                                  | 1.8 (0.2, 3.5)                                                                                                                                                                      | 2.4 (–0.9, 5.7)     | 2.0 (0.6, 3.4)       |
+| Change from baseline at week 8                                                                                                                                                                                                                                                                                               | 3.0 (0.7, 5.4)                                                                                                                                                                      | 1.5 (–1.6, 4.6)     | 2.6 (0.8, 4.4)       |
+| Change from baseline at week 12                                                                                                                                                                                                                                                                                              | 2.1 (0.2, 4.1)                                                                                                                                                                      | –0.3 (–4.0, 3.4)    | 1.4 (–0.3, 3.1)      |
+| Prebronchodilator FEV1 (mL) Change from baseline at week 4                                                                                                                                                                                                                                                                   | 93 (–19, 206)                                                                                                                                                                       | 55 (–52, 162)       | 83 (–1, 167)         |
+| Change from baseline at week 8                                                                                                                                                                                                                                                                                               | 90 (–17, 198)                                                                                                                                                                       | –63 (–314, 188)     | 48 (–52, 148)        |
+| Change from baseline at week 12                                                                                                                                                                                                                                                                                              | 72 (–37, 181)                                                                                                                                                                       | 2 (–175, 179)       | 51 (–37, 139)        |
+| Patients with on‐treatment exacerbations (weeks 0–12), n (%) Any                                                                                                                                                                                                                                                             | 8 (31)                                                                                                                                                                              | 2 (20)              | 10 (28)              |
+| 1 exacerbation                                                                                                                                                                                                                                                                                                               | 6 (23)                                                                                                                                                                              | 1 (10)              | 7 (19)               |
+| 2 exacerbations                                                                                                                                                                                                                                                                                                              | 2 (8)                                                                                                                                                                               | 1 (10)              | 3 (8)                |
+| Data are mean (95% CI) unless stated otherwise; a decreased scores, or b increased scores, from baseline indicate improvement in asthma control. Abbreviations: ACQ‐7, Asthma Control Questionnaire; C‐ACT, Childhood Asthma Control Test; CI, confidence interval; FEV1, forced expiratory volume in 1 s; SC, subcutaneous. |                                                                                                                                                                                     |                     |                      |
+
+(n = 4), upper respiratory tract infections (n = 3), lower respiratory tract infection (n = 2), sinusitis (n = 2), and viral upper respiratory tract infection
+(n = 2). Three on‐treatment infections were considered serious though not related to mepolizumab (cellulitis, n = 1; lower respiratory tract infection, n = 2); all resolved without modification to mepolizumab dosing.
+
+One 11‐year‐old child in the mepolizumab 40 mg dose group (<40 kg) had a systemic hypersensitivity reaction with symptoms of pruritus of mild intensity. The event occurred less than 24 hours after the first dose, lasted for 57 days, was considered related to treatment and resolved without mepolizumab dose modification or interruption. Injection‐site reactions were all mild in intensity and resolved without mepolizumab dose modification or discontinuation. There were no cases of anaphylaxis.
+
+No children tested positive for anti‐drug antibody (ADA) at baseline; two children had transient positive ADA results post baseline with low titers that were negative at subsequent visits. No children tested positive for neutralizing antibodies and there were no apparent drug‐related changes in clinical laboratory parameters or vital signs.
+
+Post‐treatment, one child reported an SAE of campylobacter infection 44 days after the last mepolizumab 40 mg dose and a second child had a nonserious valvulopathy (mild mitral valve incompetence not requiring hospitalization or treatment) diagnosed 78 days after the last mepolizumab 40 mg dose. Both events were considered unrelated to study drug and were ongoing at the end of the follow‐up period.
+
+## 4 | Discussion
+
+This study of mepolizumab SC at bodyweight‐defined doses of 40 or 100 mg is the first reported clinical evaluation of an anti‐IL‐5 therapy in children aged 6 to 11 years with severe eosinophilic asthma. In summary, results showed that derived exposures (AUC0‐inf) normalized to the average bodyweight of each dose group were within twofold (1.32‐fold at 40 mg SC and 1.97‐fold at 100 mg SC) of the historical target adult exposure of 343 μg ⁎ day/mL (unpublished data). The mepolizumab SC half‐life of 22 to 24 days in children is, however, aligned with historical adult values of 16 to 22 days.9,18 Mepolizumab was well tolerated with no specific AE patterns.
+
+Reported injection‐site reactions were mild in intensity and managed without mepolizumab dose modification or discontinuation. No new safety concerns were observed beyond those seen previously with mepolizumab in adults and adolescents.11-13,19 Mepolizumab exposures were higher than predicted, and plasma apparent clearance normalized to adult bodyweight was lower than reported previously for adults (0.20 vs 0.29 L/day). An additional exploratory population PK analysis conducted to assess these discrepancies showed mepolizumab SC bioavailability to be 105% (95% CI: 55%, 155%) for children compared with the historic adult value of 76% (95% CI: 71%, 79%). This disparity may reflect differences in BMI (18 kg/m2 in this study vs ~28 kg/m2 for adults in
+
+| TABLE 5                                                                                                                                                                                         | Summary of adverse events   | Mepolizumab 40 mg   | Mepolizumab 100 mg   |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------|---------------------|----------------------|
+| SC (weight <40 kg)                                                                                                                                                                              | SC (weight ≥40 kg)          | Total               |                      |
+| Patients, n (%)                                                                                                                                                                                 | (N = 26)                    | (N = 10)            | (N = 36)             |
+| Any AE                                                                                                                                                                                          | 20 (77)                     | 6 (60)              | 26 (72)              |
+| On‐treatmenta                                                                                                                                                                                   | 18 (69)                     | 6 (60)              | 24 (67)              |
+| Posttreatmentb                                                                                                                                                                                  | 12 (46)                     | 2 (20)              | 14 (39)              |
+| Related to treatmentc                                                                                                                                                                           | 8 (31)                      | 3 (30)              | 11 (31)              |
+| Leading to withdrawald                                                                                                                                                                          | 1 (4)                       | 0                   | 1 (3)                |
+| SAEs                                                                                                                                                                                            | 6 (23)                      | 1 (10)              | 7 (19)               |
+| On‐treatmenta                                                                                                                                                                                   | 5 (19)                      | 1 (10)              | 6 (17)               |
+| Posttreatmentb                                                                                                                                                                                  | 4 (15)                      | 0                   | 4 (11)               |
+| Related to treatmentc                                                                                                                                                                           | 2 (8)                       | 0                   | 2 (6)                |
+| Fatal                                                                                                                                                                                           | 0                           | 0                   | 0                    |
+| Most frequent on treatment AEs (≥3 patients) Headache                                                                                                                                           | 3 (12)                      | 2 (20)              | 5 (14)               |
+| Injection‐site reaction                                                                                                                                                                         | 5 (19)                      | 0                   | 5 (14)               |
+| Asthma                                                                                                                                                                                          | 4 (15)                      | 0                   | 4 (11)               |
+| Nasopharyngitis                                                                                                                                                                                 | 3 (12)                      | 1 (10)              | 4 (11)               |
+| Nausea                                                                                                                                                                                          | 3 (12)                      | 0                   | 3 (8)                |
+| Upper respiratory tract infection                                                                                                                                                               | 2 (8)                       | 1 (10)              | 3 (8)                |
+| Wheezing                                                                                                                                                                                        | 2 (8)                       | 1 (10)              | 3 (8)                |
+| On‐treatment AEs of special interest Anaphylaxis                                                                                                                                                | 0                           | 0                   | 0                    |
+| Systemic reactions                                                                                                                                                                              | 1 (4)                       | 0                   | 1 (3)                |
+| Local injection‐site reactions                                                                                                                                                                  | 5 (19)                      | 0                   | 5 (14)               |
+| All infections                                                                                                                                                                                  | 14 (54)                     | 4 (40)              | 18 (50)              |
+| Neoplasms                                                                                                                                                                                       | 0                           | 0                   | 0                    |
+| Malignancies                                                                                                                                                                                    | 0                           | 0                   | 0                    |
+| Cardiac disorders                                                                                                                                                                               | 0                           | 0                   | 0                    |
+| Serious CVT events                                                                                                                                                                              | 0                           | 0                   | 0                    |
+| Abbreviations: AE, adverse event; CVT, cardiac, vascular, thromboembolic; SAE, serious adverse event; SC, subcutaneous. a From the first mepolizumab dose to 4 wk of the last mepolizumab dose. |                             |                     |                      |
+
+the phase 3 trials11-13,19). Population PK parameter estimates for the allometric exponents for clearance and volumes in children were 0.86 (95% CI: 0.29, 1.43) and 0.66 (95% CI: 0.11, 1.21), respectively, and consistent with historic adult values.
+
+Blood eosinophil reductions were accompanied by indicators of improvement in asthma control, as reflected by ACQ‐7 and C‐ACT
+scores. Serum total IL‐5 levels were increased at week 12 from baseline, as anticipated since the assay measures both free and mepolizumab‐bound IL‐5. Serum IL‐5 levels confirmed antibody‐
+target engagement in children and were consistent with observations in adults at which saturation was observed.18 The on‐treatment incidence of asthma exacerbations (28% of children) was also similar to exacerbation rates observed in adults and adolescents over the first 12 weeks of mepolizumab treatment (approximately 12‐30%).20 However, there was no clear pattern of lung function changes in response to mepolizumab.
+
+There continues to be scientific debate on the role of homeostatic levels of eosinophils in human health and disease based on observations in exaggerated rodent models (eg, IL‐5 or eosinophil deficient mice).21-23 There is growing evidence, suggested by murine studies, that drugs targeting the number of eosinophils in patients with elevated eosinophil levels have therapeutic benefit without adverse events.23 In line with findings in adolescents and adults,11-13,19,20,24 we did not associate mepolizumab treatment with an increased incidence of opportunistic infections or neoplasms. It must however be acknowledged that the patients enrolled in the first part of this two‐part study were followed for a limited duration (20 weeks) and did not reside in countries where parasitic diseases are endemic. Limitations of the efficacy and safety data from this study include the lack of a control group as this was not a placebo‐controlled, double‐blind randomized study, the selection of the sample size to assess PK/PD rather than effectiveness, and the relatively short treatment period in this study. The use and adherence of background asthma therapies was also not systematically checked during the study; instead, it was at the investigator's discretion whether to reduce or change background therapies. This may have influenced the results to some extent, and in particular, any changes in bronchodilator therapy may have affected the results on lung function. Therefore, effectiveness and tolerability results in part A should be interpreted with caution while the long‐term safety phase (part B) will provide further insight.
+
+## 5 | Conclusion
+
+In conclusion, mepolizumab SC, administered at bodyweight‐
+defined doses of 40 or 100 mg, provides higher than predicted drug exposure in children aged 6 to 11 years, but that remains within twofold of adult levels. This difference is likely due to the increased bioavailability of mepolizumab SC in children versus adults. Mepolizumab SC was associated with a similar therapeutic effect in children to adults, with marked reductions in blood eosinophil counts, a trend toward improved asthma control compared with baseline, and a favorable safety profile. The 40 and 100 mg SC dosing regimens were deemed acceptable for children aged 6 to 11 years with severe eosinophilic asthma based on mepolizumab's wide therapeutic index. Results will be used as a basis for further dosing refinement.
+
+## Acknowledgments
+
+This study was funded by GlaxoSmithKline (GSK ID 200363 ClinicalTrials.gov number NCT02377427). The sponsor did not place any restrictions on access to the data or on the statements made in the manuscript. The decision to submit for publication was that of the authors alone. The authors would like to thank the patients and caregivers who took part in this study, and Marie Duggan and Dennis Kelleher (both employees of GSK) for their roles in leading management of the study. Editorial support (in the form of writing assistance, including development of the initial draft from the study report, assembling tables and figures, collating and incorporating authors comments, grammatical editing, and referencing) was provided by Elizabeth Hutchinson PhD, CMPP, at Fishawack Indicia Ltd, UK, and was funded by GSK.
+
+## Conflict Of Interests
+
+IP, DA, RGP, RK, JS, ESB, and SWY are all GSK employees and stockholders. AG was the principal investigator for the current study at the King's College Hospital, which received grants from GSK to conduct the study. AG has also received personal fees for attendance at Advisory Boards from GSK, Boehringer Ingelheim and Novartis.
+
+## Author Contributions
+
+All authors reviewed and revised the manuscript, approved the final version, and made the decision to submit the manuscript for publication. AG contributed to the acquisition of data and analysis and interpretation of the study data. IP, DA, RGP, RK, JS, ESB, and SWY were all involved in the conception or design of the study, and in analysis and interpretation of the study data.
+
+## Orcid References
+
+1. World Health Organization. Chronic respiratory diseases: Asthma. 2018; www.who.int/respiratory/asthma/en/ Last accessed February 2018.
+
+2. Coverstone A, Bacharier LB, Fitzpatrick AM. Severe asthma in school‐
+age children: evaluation and phenotypic advances. Curr Allergy Asthma Rep. 2015;15(5):20.
+
+3. Lang A, Carlsen KH, Haaland G, et al. Severe asthma in childhood:
+assessed in 10 year olds in a birth cohort study. Allergy.
+
+2008;63(8):1054‐1060.
+
+4. Nordlund B, Melén E, Schultz ES, Grönlund H, Hedlin G, Kull I.
+
+Prevalence of severe childhood asthma according to the WHO. Respir Med. 2014;108(8):1234‐1237.
+
+5. Chung KF, Wenzel SE, Brozek JL, et al. International ERS/ATS
+guidelines on definition, evaluation and treatment of severe asthma.
+
+Eur Respir J. 2014;43(2):343‐373.
+
+6. Fleming L, Murray C, Bansal AT, et al. The burden of severe asthma in childhood and adolescence: results from the paediatric U‐BIOPRED
+cohorts. Eur Respir J. 2015;46(5):1322‐1333.
+
+7. Tai A, Tran H, Roberts M, Clarke N, Wilson J, Robertson CF. The association between childhood asthma and adult chronic obstructive pulmonary disease. Thorax. 2014;69(9):805‐810.
+
+8. Martin Alonso A, Saglani S. Mechanisms mediating pediatric severe asthma and potential novel therapies. Front Pediatr. 2017; 5:154.
+
+9. GlaxoSmithKline. NUCALA (mepolizumab) prescribing information.
+
+2017; www.accessdata.fda.gov/drugsatfda_docs/label/…/125526Orig1s000Lbl.pdf Last accessed February 2018.
+
+10. GlaxoSmithKline. NUCALA (mepolizumab) summary of product characteristics. 2018; http://www.ema.europa.eu/docs/en_GB/document_
+library/EPAR_‐_Product_Information/human/003860/WC500198037.
+
+pdf Last accessed February 2018.
+
+11. Ortega HG, Liu MC, Pavord ID, et al. Mepolizumab treatment in patients with severe eosinophilic asthma. N Engl J Med. 2014;371(13):
+1198‐1207.
+
+12. Bel EH, Wenzel SE, Thompson PJ, et al. Oral glucocorticoid‐sparing effect of mepolizumab in eosinophilic asthma. N Engl J Med. 2014; 371(13):1189‐1197.
+
+13. Chupp GL, Bradford ES, Albers FC, et al. Efficacy of mepolizumab add‐on therapy on health‐related quality of life and markers of asthma control in severe eosinophilic asthma (MUSCA): a randomised, double‐blind, placebo‐controlled, parallel‐group, multicentre, phase 3b trial. Lancet Respir Med. 2017;5(5):390‐400.
+
+14. Guiddir T, Saint‐Pierre P, Purenne‐Denis E, et al. Neutrophilic steroid‐refractory recurrent wheeze and eosinophilic steroid‐
+refractory asthma in children. J Allergy Clin Immunol Pract. 2017;5(5):
+1351‐1361. e1352.
+
+15. Lezmi G, Deschildre A, Abou Taam R, et al. Remodelling and inflammation in preschoolers with severe recurrent wheeze and asthma outcome at school age. Clin Exp Allergy. 2018;48:
+806‐813.
+
+16. Assa'ad AH, Gupta SK, Collins MH, et al. An antibody against IL‐5 reduces numbers of esophageal intraepithelial eosinophils in children with eosinophilic esophagitis. Gastroenterology. 2011;141(5):1593‐1604.
+
+17. FDA. General clinical pharmacology considerations for pediatric studies for drugs and biological products. 2014; https://www.fda.gov/ downloads/drugs/guidances/ucm425885.pdf Last accessed February 2018.
+
+18. Pouliquen IJ, Kornmann O, Barton SV, Price JA, Ortega HG.
+
+Characterization of the relationship between dose and blood eosinophil response following subcutaneous administration of mepolizumab. Int J Clin Pharmacol Ther. 2015;53(12):1015‐1027.
+
+19. Pavord ID, Korn S, Howarth P, et al. Mepolizumab for severe eosinophilic asthma (DREAM): a multicentre, double‐blind, placebo‐
+controlled trial. Lancet. 2012;380(9842):651‐659.
+
+20. Gupta A, Steinfeld J, Price R, Azmi J, Bradford E, Yancey S. Mepolizumab for severe eosinophilic asthma: a comparison of efficacy in children, adolescents, and adults. Eur Respir J. 2018;51(Suppl):PA5447.
+
+21. Gleich GJ, Klion AD, Lee JJ, Weller PF. The consequences of not having eosinophils. Allergy. 2013;68(7):829‐835.
+
+22. Klion A. Recent advances in understanding eosinophil biology.
+
+F1000Research. 2017;6:1084.
+
+23. Ramirez GA, Yacoub M‐R, Ripa M, et al. Eosinophils from physiology to disease: a comprehensive review. BioMed Res Int. 2018;2018:1‐28.
+
+24. Khatri S, Moore W, Gibson PG, et al. Assessment of the long‐term safety of mepolizumab and durability of clinical response in patients with severe eosinophilic asthma. J Allergy Clin Immunol. 2019;143:
+1742‐1751.
+
+How to cite this article: Gupta A, Pouliquen I, Austin D, et al. Subcutaneous mepolizumab in children aged 6 to 11 years with severe eosinophilic asthma. Pediatric Pulmonology. 2019; 54:1957–1967. https://doi.org/10.1002/ppul.24508

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+| Chinese Pharmaceutical Association                                                                                                              |
+|-------------------------------------------------------------------------------------------------------------------------------------------------|
+| Institute of Materia Medica, Chinese Academy of Medical Sciences Acta Pharmaceutica Sinica B www.elsevier.com/locate/apsb www.sciencedirect.com |
+
+![0_image_0.png](0_image_0.png)
+
+ORIGINAL ARTICLE
+Potential therapeutic effects of dipyridamole in the severely ill patients with COVID-19 Xiaoyan Liua,y, Zhe Lib,y, Shuai Liua,c,y, Jing Sund,y, Zhanghua Chene,f,y, Min Jiangg,y, Qingling Zhangd,y, Yinghua Weig, Xin Wangh, Yi-You Huangb, Yinyi Shic, Yanhui Xue, Huifang Xiane, Fan Baif, Changxing Oud, Bei Xionga, Andrew M. Lewi, Jun Cuij, Rongli Fange, Hui Huangk, Jincun Zhaod,*, Xuechuan Hongl,m,*, Yuxia Zhange,*, Fuling Zhoua,*, Hai-Bin Luob,*
+aDepartment of Hematology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China bGuangdong Provincial Key Laboratory of New Drug Design and Evaluation, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China cDawu County People's Hospital, Xiaogan 432826, China dState Key Laboratory of Respiratory Diseases, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China eGuangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, State Key Laboratory of Respiratory Diseases, Guangzhou Medical University, Guangzhou 510623, China fBiomedical Pioneering Innovation Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871, China gDepartment of Infectious Disease and Department of Pediatrics, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China hCenter for Innovative Marine Drug Screening & Evaluation (QNLM), School of Medicine and Pharmacy, Ocean University of China, Qingdao 266100, China iWalter and Eliza Hall Institute of Medical Research and Department of Microbiology & Immunology, University of Melbourne, Parkville, Vic 3052, Australia jSchool of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China kCardiovascular Department, The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen 518000, China lState Key Laboratory of Virology, College of Science, Innovation Center for Traditional Tibetan Medicine Modernization and Quality Control, Medical College, Tibet University, Lhasa 850000, China https://doi.org/10.1016/j.apsb.2020.04.008 2211-3835 ª 2020 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+mKey Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE), Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China Received 26 March 2020; received in revised form 3 April 2020; accepted 6 April 2020
+
+![1_image_0.png](1_image_0.png)
+
+Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause acute respiratory distress syndrome, hypercoagulability, hypertension, and multiorgan dysfunction. Effective antivirals with safe clinical profile are urgently needed to improve the overall prognosis. In an analysis of a randomly collected cohort of 124 patients with COVID-19, we found that hypercoagulability as indicated by elevated concentrations of D-dimers was associated with disease severity. By virtual screening of a U.S. FDA approved drug library, we identified an anticoagulation agent dipyridamole (DIP) in silico, which suppressed SARS-CoV-2 replication in vitro. In a proof-of-concept trial involving 31 patients with COVID-19, DIP supplementation was associated with significantly decreased concentrations of D-dimers
+(P < 0.05), increased lymphocyte and platelet recovery in the circulation, and markedly improved clinical outcomes in comparison to the control patients. In particular, all 8 of the DIP-treated severely ill patients showed remarkable improvement: 7 patients (87.5%) achieved clinical cure and were discharged from the hospitals while the remaining 1 patient (12.5%) was in clinical remission.
+
+ª 2020 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
+(http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+## 1. Introduction
+
+As of April 3, 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, formerly known as 2019-nCoV)1,2 had infected over 1,000,000 patients in 200 countries, such as USA, Spain, Italy, Germany, France, and UK; this rapid spread has been declared a global pandemic. To date, no agents have been reported to be specific to treat severely ill patents. Identification of readily available drugs for repositioning in COVID-19 therapy avails a relatively rapid way to clinical treatment3.
+
+SARS-CoV-2, together with SARS-CoV and MERS-CoV, belongs to the Beta-coronavirus genus, which is an enveloped, positive-stranded RNA virus with approximately 30,000 nucleotides4,5. Angiotensin I converting enzyme 2 (ACE2) is the receptor that engages the Spike surface glycoprotein of SARS-CoV and SARS-CoV-26,7. ACE2 is highly expressed in many organs, including the lung, heart, kidney, and intestine. Notably, in experimental models of SARS-CoV infection, Spike protein engagement decreases ACE2 expression and activates the reninangiotensin system (RAS)6. RAS activation promotes platelet adhesion and aggregation, and increases the risk for pulmonary embolism, hypertension and fibrosis8e11. It also accelerates cardiac and kidney injury by increasing local angiotensin II concentrations12e14. Apart from affecting the classic RAS pathway, ACE2 deficiency in the intestine is associated with malnutrition and colonic inflammation15.
+
+Infection from SARS-CoV can result in severe lymphopenia, prolonged coagulation profiles, lethal acute respiratory distress syndrome (ARDS), watery diarrhea, cardiac disease, and sudden death9,16e18. Many features have also been reported for COVID-19, such as prolonged coagulation profiles, elevated concentrations of D-dimers, severe lymphopenia, ARDS, hypertension, and acute heart injury in ICU-admitted patients2,19.
+
+Given that angiotensin II concentrations were highly elevated in the SARS-CoV-2 infected patients20, RAS was likely a major pathogenic contributor of disease progression. Indeed, in a recent study describing 1099 patients with COVID-19, the concentrations of D-dimers were elevated in 40% and 60% of the non-severe and severe cases at hospital admission21, respectively. Furthermore, Zhou et al.22 showed that a concentration of D-dimer greater than 1 mg/L on admission was associated with significantly increased risk of mortality for patients with COVID-19. Thus, prophylactic anti-coagulation therapy should be considered for alleviating the multi-organ damage for patients with COVID-19.
+
+After viral entry to the host cells, the coronavirus messenger RNA is first translated to yield the polyproteins, which are subsequently cleaved by two viral proteinases, 3C-like protease
+(3CLP, aka nsp5 or Mpro) and papain-like protease (PLP, or nsp3), to yield non-structural proteins essential for viral replication23.
+
+Inhibitors that suppress the activity of these proteases may inhibit viral replication and offer an avenue for the SARS-CoV-2 therapy.
+
+Dipyridamole (DIP) is an antiplatelet agent and acts as a phosphodiesterase (PDE) inhibitor that increases intracellular cAMP/cGMP24. Apart from the well-known antiplatelet function, DIP may provide potential therapeutic benefits to patients with COVID-19. First, published studies25e30, including clinical trials conducted in China31e33, have demonstrated that DIP has a broad spectrum antiviral activity, particularly efficacious against the positive-stranded RNA viruses26. Second, it suppresses inflammation and promotes mucosal healing34.
+
+Third, as a pan-PDE inhibitor, DIP may prevent acute injury and progressive fibrosis of the lung, heart, liver, and kidney35.
+
+Here we provide evidence advocating DIP as an adjunctive therapy.
+
+## 2. Results
+
+2.1. DIP suppresses SARS-CoV-2 replication in Vero E6 cells We virtually screened a U.S. FDA approved drug library and found that DIP bound to the SARS-CoV-2 protease Mpro
+(Fig. 1A and Supporting Information Fig. S1). Hydrophobic and hydrogen bond (H-bond) interactions are the main driving forces for the binding between DIP and Mpro. By free energy perturbation calculations, the binding free energy of DGpred was 8.60 kcal/mol with a predicted IC50, pred value of 490 nmol/L
+by the equation DGpred Z -RT ln (IC50, pred). The inhibitory potency of DIP against Mpro was then subjected to an enzymatic assay using a previously published method36. As a result, DIP
+exhibited an IC50, exp value of 530  10 nmol/L (Fig. 1B), which was consistent with the theoretical prediction of the IC50, pred values.
+
+To directly demonstrate that DIP suppresses SARS-CoV-2 replication in vitro, we measured viral titers using a susceptible cell line, the Vero E6 cells. Chloroquine was used as a positive control37,38. Remarkably, at concentration 100 nmol/L, DIP suppressed more than 50% of SARS-CoV-2 replication (Fig. 1C).
+
+This is four times less than the predicted and experimentally confirmed IC50 to suppress Mpro activity, which is consistent with previous findings showing that DIP possesses additional antiviral effects25e30. DIP (50 mg oral TID) has been used in patients to prevent hypercoagulability39, and the serum drug concentration was reported to be around 3 mmol/L40. Collectively, these data suggest that the therapeutic dosages of DIP used to treat hypercoagulability could potentially suppress SARS-CoV-2 replication in the infected patients.
+
+2.2. Demographics and baseline characteristics of the study participants We first retrospectively analyzed a randomly collected cohort of 124 patients with COVID-19. This has revealed that decreased lymphocyte counts, increased concentrations of D-dimers, CRP,
+and IL-6 were significantly associated with disease severity (Table 1).
+
+To evaluate the therapeutic potential of DIP as an adjunctive therapy to promote virus clearance and reduce the risk of hypercoagulability, an open label clinical study involving 31 patients was conducted in Dawu County People's Hospital (1st hospital, Xiaogan) and Huangpi Chinese Medicine Hospital (2nd hospital, Wuhan), Hubei province, China from February 3 to March 8, 2020. 12 patients and 10 controls were recruited from the 1st hospital, and 2 patients and 7 controls were recruited from the 2nd hospital. Patients were treated in different isolation wards by different attending physicians. Standard treatment procedures were applied for all patients according to the guidelines formulated by the General Office of National Health Committee. DIP
+was used in all patients of the selected wards by two specialists each from the two hospitals. Patients from other wards without DIP adjunctive therapy were used as controls.
+
+Baseline characteristics of the two groups were shown in Table 2. The average ages of the patients were 56 years. All patients manifested a cough, >75% had shortness of breath, and 35%e 57% had nausea and vomiting. Chest CT scan revealed bilateral pneumonia in the 14 DIP-treated patients and 17 patients in the control group. In addition, RT-PCR test of SARS-CoV-2 RNA was positive for all patients. D-dimer concentrations were elevated in 50% (4/8) and 42% (5/12) of the severely ill patients in the DIPtreated group and the control group, respectively. Comorbidities,
+
+![2_image_0.png](2_image_0.png)
+
+We also examined two critically ill patients who received DIP
+adjunctive therapy. A 70-year-old man who had suffered from hypoxia and multiorgan dysfunction at hospital admission unfortunately died 5 days after initiation of DIP treatment. He had an extremely high concentration of D-dimer (16.2 mg/L, Fig. 3A)
+
+![3_image_0.png](3_image_0.png)
+
+![3_image_1.png](3_image_1.png)
+
+including diabetes mellitus, cardiovascular, and cerebrovascular diseases, were found in 6 patients in each of the DIP and control groups. Patients with diabetes (Table 2) were treated with insulin injections, and those with cardiovascular diseases were treated with nifedipine. 2.3. DIP adjunctive therapy increases the clinical cure and remission rates in the severely ill patients with COVID-19 DIP adjunctive therapy was provided in 14 patients. The treatment protocol comprised of 50 mg oral tablets administered thrice daily (a total of 150 mg) for 14 consecutive days. All patients received ribavirin, glucocorticoids, and oxygen therapy, but none received antifungal treatment. Mechanical ventilation was required for all the critically ill patients from the DIPtreated (n Z 2), and 1 each from the severely and critically ill patients in the control group (nZ 2). Other treatment included antibiotics (42.9% vs. 58.8%) and intravenous immunoglobulin (14.3% vs. 23.5%).
+
+DIP adjunctive therapy was associated with markedly improved clinical cure and remission rates in both the non-severe and severely ill patients (odds ratio 23.75,PZ 0.06, Tables 3 and 4). In particular, for the 8 severely ill patients in the DIPtreated group, 7 patients (87.5%) achieved clinical cure and were discharged from the hospitals, and the remaining 1 patient
+(12.5%) was in clinical remission. In contrast, for the 12 severely ill patients in the control group, 4 patients (33.3%) were discharged, 2 patients (16.7%) were in remission, and 2 patients
+(16.7%) died, respectively.
+
+It should be mentioned that due to the urgent situation and the lack of resources to perform viral RNA detection by the participating hospitals, we were unable to accurately determine the effects of DIP to viral clearance. However, according to the qualitative RT-PCR result of SARS-CoV-2 RNA provided by local Centers for Disease Control and Prevention, the average time for virus clearance was shortened by 1.6 days for the severe cases in the DIP-treated group in comparison to the control group. 2.4. DIP adjunctive therapy improves the coagulation profiles and promotes immune cell recovery in the severely ill patients In analysis of the laboratory indices, we observed continuously increased, albeit not statistically significant, counts of lymphocyte and platelet in patients receiving DIP treatment in comparison to the control patients (Fig. 2). Given that lymphocytopenia and thrombocytopenia are markers of disease severity for patients with COVID-1920, immune recovery may contribute to infection resolution in DIP-treated patients. It should be noted that 50% and 42% of the severely ill patients from the DIP-treated and control group had increased baseline concentrations of D-dimer, respectively (Table 2). We calculated the dynamic changes for each patient in reference to their own baseline value, and found that D-dimer rose continuously in the control group, whereas they were decreased in the DIP-treated group (Fig. 2). 2.5. DIP adjunctive therapy in the two critically ill patients and a very low lymphocyte count (0.37  109/L) at the time of receiving DIP adjunctive therapy. Additionally, his oxygen saturation remained low throughout. In contrast, the other severely ill patient who also had very low oxygen saturation and high D-dimer concentration (8.83 mg/L) at administration had been in clinically remission by the time of manuscript submission. His D-dimer concentration initially increased as high as 15.72 mg/L
+two days after DIP treatment, but has gradually declined to 2.79 mg/L 4e5 days after DIP adjunctive therapy. This reinforces that high concentrations of D-dimer and low lymphocyte counts are associated with poor prognosis and suggest that DIP treatment should be initiated before the progression to a critical state
+(Fig. 3B).
+
+## 2.6. Chest Ct Findings With Dip Adjunctive Therapy
+
+All patients received chest CT scans and showed typical multiple patchy ground-glass shadows in the lungs before the treatment. For the DIP-treated patients, the lesions from all patients had a varied degree of absorption after treatment. In the control group, CT images in 1 of the 12 severely ill patients showed progression (Fig. 4 and Supporting Information Table S1).
+
+## 3. Discussion
+
+Despite the enormous threat of SARS-CoV-2, no drugs have been claimed to be specific including the existing drugs used to treat other viruses. In reference to SARS-CoV-2 infection, we hypothesized that the SARS-CoV-2 Spike protein engagement may activate RAS in the lung6,41. This hypothesis was supported by published clinical characteristics and biochemical data of the severe and critically ill patients with COVID-19, who showed ARDS, hypertension, acute heart, kidney injury, and positive D-dimer results2,19,20. In searching for available anticoagulants, we focused on DIP because of its broad-spectrum antiviral, anti-inflammatory, and anti-fibrotic effects. Very importantly, we found that an EC50 value of 100 nmol/L to suppress SARS-CoV-2 replication in vitro, indicating that the therapeutic dosage of DIP may potentiate effective antiviral responses in infected patients. These findings are in concordance with our clinical findings of the overall remarkable outcomes in the severely ill patients receiving two weeks of DIP adjunctive therapy. All the 8 DIP-treated severely ill patients showed remarkable improvement after DIP treatment, with 87.5% discharged from the hospitals and a further 12.5% showing clinical remission. In contrast, for the 12 severely ill patients in the control group, only 33.3% were discharged and death occurred in 16.7%.
+
+In a recent publication describing 1099 patients with COVID-19, D-dimer concentrations were elevated in 40% and 60% of the non-severe and severe cases at hospital admission21.
+
+It has been reported that a D-dimer concentration greater than 1 mg/L on admission was associated with significantly increased risk of mortality for patients with COVID-1922.
+
+We found that DIP adjunctive treatment blunted the increase in D-dimer concentrations, and increased the counts of circulating platelets and leucocytes. High concentrations of D-dimers are closely correlated with pulmonary embolism42, vascular
+
+| Table 2                                                      | Baseline characteristics of the 31 enrolled patients.   |                        |
+|--------------------------------------------------------------|---------------------------------------------------------|------------------------|
+| Variable                                                     | Dipyridamole group (n Z 14)                             | Control group (n Z 17) |
+| General characteristic Age (yr)dmeanSD (range)                                                              | 56  12 (32e74)                                                         | 56  15 (23e74)                        |
+| Genderdmale no./female no.                                   | 8/6                                                     | 13/4                   |
+| Group Non-severe no./Severe no./Critical no.                 | 4/8/2                                                   | 3/12/2                 |
+| Clinical variables Coughdno. (%)                             | 14 (100.0%)                                             | 17 (100.0%)            |
+| Shortness of breathdno. (%)                                  | 11 (78.6%)                                              | 13 (76.5%)             |
+| Nausea and vomitingdno. (%)                                  | 8 (57.1%)                                               | 6 (35.3%)              |
+| Systolic blood pressure (mmHg)dmeanSD (range)                                                              | 127  12 (120e155)/81  11 (57e124) 128  13 (107e153)/78  10 (56e96)                                                         |                        |
+| Partial pressure of oxygen (mmHg)dmeanSD (range) 86  12 (58e93)                                                              | 92  9 (76e96)                                                         |                        |
+| Laboratory values Lymphocyte (109/L)dmeanSD (range)                                                              | 1.07  0.57 (0.29e2.28)                                                         | 0.82  0.45 (0.17e1.85)                        |
+| Decrease in concentrations of lymphocytedno. (%)             | 9 (64.3%)                                               | 12 (70.6%)             |
+| dno. of non-severe cases (%)                                 | 1/4 (25%)                                               | 2/3 (66.7%)            |
+| dno. of severe cases (%)                                     | 7/8 (87.5%)                                             | 9/12 (75%)             |
+| dno. of critical cases (%)                                   | 1/2 (50%)                                               | 1/2 (50%)              |
+| D-dimer (mg/L)dmeanSD (range)                                                              | 2.00  2.54 (0.19e6.84)                                                         | 1.50  2.73 (0.01e8.43)                        |
+| Increase in concentrations of D-dimerdno. (%)                | 6/14 (42.9%)                                            | 5/17 (29.4%)           |
+| dno. of non-severe cases (%)                                 | 1/4 (25%)                                               | 0                      |
+| dno. of severe cases (%)                                     | 4/8 (50%)                                               | 5/12 (41.7%)           |
+| dno. of critical cases (%)                                   | 1/2 (50%)                                               | 0                      |
+| Respiratory pathogens The nucleic acid of SARS-CoV-2dno. (%) | 14 (100.0%)                                             | 17 (100.0%)            |
+| Unilateral pneumoniadno. (%)                                 | 0                                                       | 1 (5.9%)               |
+| Bilateral pneumoniadno. (%)                                  | 14 (100.0%)                                             | 16 (94.1%)             |
+| Comorbidities Diabetes mellitusdno. (%)                      | 3 (21.4%)                                               | 1 (5.9%)               |
+| Cardiovascular diseasedno. (%)                               | 2 (14.3%)                                               | 3 (17.6%)              |
+| Cerebrovascular diseasedno. (%)                              | 1 (7.1%)                                                | 2 (11.8%)              |
+
+| Table 3                                       | Treatment and clinical outcomes of 31 enrolled patients.   |                        |
+|-----------------------------------------------|------------------------------------------------------------|------------------------|
+| Variable                                      | Dipyridamole group (n Z 14)                                | Control group (n Z 17) |
+| Group dNon-severe no./Severe no./Critical no. | 4/8/2                                                      | 3/12/2                 |
+| Treatment Oxygen therapydno. (%)              | 14 (100.0%)                                                | 17 (100.0%)            |
+| Mechanical ventilationdno. (%)                | 2 (14.3%)                                                  | 2 (11.8%)              |
+| dno. of non-severe cases (%)                  | 0                                                          | 0                      |
+| dno. of severe cases (%)                      | 0                                                          | 1/12 (8.3%)            |
+| dno. of critical cases (%)                    | 2/2 (100%)                                                 | 1/2 (50%)              |
+| Antibiotic treatmentdno. (%)                  | 6 (42.9%)                                                  | 10 (58.8%)             |
+| Antifungal treatmentdno. (%)                  | 0                                                          | 0                      |
+| Antiviral treatmentdno. (%)                   | 14 (100.0%)                                                | 17 (100.0%)            |
+| Glucocorticoidsdno. (%)                       | 14 (100.0%)                                                | 17 (100.0%)            |
+| Outcome Discharge ratedno. (%)                | 11/14 (78.6%)                                              | 7/17 (41.2%)           |
+| dno. of non-severe cases (%)                  | 4/4 (100%)                                                 | 3/3 (100%)             |
+| dno. of severe cases (%)                      | 7/8 (87.5%)                                                | 4/12 (33.3%)           |
+| dno. of critical cases (%)                    | 0                                                          | 0                      |
+| Average time for viral clearance (days)       | e                                                          | e                      |
+| dsevere cases (%)                             | 15.4                                                       | 17.0                   |
+| Remission ratedno. (%)                        | 2/14 (14.3%)                                               | 2/17 (11.8%)           |
+| dno. of severe cases (%)                      | 1/8 (12.5%)                                                | 2/12 (16.7%)           |
+| dno. of critical cases (%)                    | 1/2 (50%)                                                  | 0                      |
+| Progression ratedno. (%)                      | 0                                                          | 1/17 (5.9%)            |
+| dno. of non-severe cases (%)                  | 0                                                          | 0                      |
+| dno. of severe cases (%)                      | 0                                                          | 1/12 (8.3%)            |
+| Death ratedno. (%)                            | 1/14 (7.1%)                                                | 4/17 (23.5%)           |
+| dno. of severe cases (%)                      | 0                                                          | 2/12 (8.3%)            |
+| dno. of critical cases (%)                    | 1/2 (50%)                                                  | 2/2 (100%)             |
+
+thrombosis, and renal dysfunction43. It is a crucial prognostic factor and is important to determine whether ICU-patients recover from severe infections44,45. Thus, prophylactic anticoagulation therapy with DIP should be considered in patients with COVID-19 to reduce the risk of hypercoagulability and multi-organ damage.
+
+It should be mentioned that several factors have limited our ability to fully investigate the therapeutic effects of DIP adjunctive therapy, these include the small number of enrolled patients, the lack of resources to quantify viral replication, and the requirement to follow the treatment guidelines under the circumstances of SARS-CoV-2 outbreak. However, we advocate further trials for DIP adjunctive therapy for patients with COVID-19, particularly for those with early signs of elevated concentrations of D-dimer. DIP has been used world-wide to treat coagulopathy. Additionally, it also exerts anti-inflammatory and antiviral effects in experimental settings and clinical trials. The wide availability, safety, and affordability of DIP argue for further investigation into its therapeutic use in COVID-19, particularly as SARS-CoV-2 infection has been declared a global pandemic.
+
+## 4. Methods 4.1. Ethics Statement
+
+The Ethics Committees from Zhongnan Hospital of Wuhan University (Wuhan, China), Dawu County People's Hospital (Xiaogan, China), and the First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China) approved the study and all patients signed informed consents. Clinical trial (ChiCTR2000030055) was registered.
+
+## 4.2. Study Design
+
+A multicenter parallel randomized controlled clinical trial involving 31 patients was conducted in Dawu County People's Hospital (1st hospital) and Huangpi Chinese Medicine Hospital (2nd hospital) from February 3 to March 8, 2020. We recruited 12 patients and 10 controls from the 1st hospital, and 2 patients and 7 controls from the 2nd hospital. Patients were treated in different isolation wards by different attending physicians. Standard treatment procedures were applied for all patients according to the
+
+| Table 4      | Multivariate analyses of clinical outcome associated factors.   |              |              |              |            |            |
+|--------------|-----------------------------------------------------------------|--------------|--------------|--------------|------------|------------|
+| Variable     | Age                                                             | Gender       | Dipyridamole | Ventilation  | Antibiotic | IVIG       |
+| (M vs. F)    | (Yes vs. No)                                                    | (Yes vs. No) | (Yes vs. No) | (Yes vs. No) |            |            |
+| Coefficients | 0.01                                                            | 0.81         | 3.17         | 20.45              | 3.59            | 0.86       |
+| Odd ratio    | 1.01                                                            | 2.24         | 23.75        | 0            | 0.03       | 2.37       |
+| 95% CI       | 0.92e1.11                                                       | 0.09e55.44   | 0.87e648     | 0-inf        | 0e0.59     | 0.09e65.08 |
+| P value      | 0.918                                                           | 0.623        | 0.06         | 0.995        | 0.022      | 0.609      |
+
+![6_image_0.png](6_image_0.png)
+
+guidelines formulated by the Chinese General Office of National Health Committee (Beijing, China). DIP was used in all patients of the selected wards by two specialists each from the two hospitals. Patients from other wards without DIP adjunctive therapy were recruited as controls. Informed written consents were obtained from all patients. The condition of the patients was monitored daily by the attending physicians. Routine laboratory test of the coagulation variables and blood indexes were carried out before, during, and after the treatment. Clinical symptoms and laboratory data were independently validated by two independent investigators for assurance of data accuracy.
+
+4.3.
+
+## Sars-Cov-2 Rna Test By Rt-Pcr
+
+![6_image_1.png](6_image_1.png)
+
+SARS-CoV-2 RNA from nasopharyngeal swabs were detected upon request of the charging physicians by the local Centers for
+
+![7_image_0.png](7_image_0.png)
+
+4.4. Disease severity assessment All patients had positive RT-PCR test of SARS-CoV-2 RNA from the nasopharyngeal swab specimens, performed by the local Chinese Center for Disease Prevention and Control. The diagnosis of severe case was made if patients met any of the following criteria: (1) respiratory rate  30 breaths/min; (2)
+SpO2  93% while breathing room air; (3) PaO2/ FiO2  300 mmHg. A critically ill case was diagnosed if any of the following criteria was met: (1) respiratory failure which requiring mechanical ventilation; (2) shock; (3) combined with other organ failure and need to be admitted to ICU.
+
+4.5. Retrospective analysis of the coagulation indices in 124 patients As of February 8, 2020, 124 confirmed COVID-19 cases had been identified from Zhongnan Hospital of Wuhan University (Table 1).
+
+All patients met the diagnostic criteria of "Diagnosis and Treatment Scheme of Novel Coronavirus-Infected Pneumonia (trial 6th)" formulated by the General Office of National Health Committee46. A retrospective review of the medical records of these patients was conducted to retrieve coagulation indexes and platelet parameters, including prothrombin time (PT), activated partial thromboplastin time (APTT), plasma fibrinogen (FIB), D-dimer, platelet (PLT) count, and mean platelet volume (MPV). Systemic inflammation was assessed according to the C-reactive protein
+(CRP), procalcitonin (PCT), and interleukin 6 (IL-6)
+concentrations.
+
+## 4.6. Treatment Procedures
+
+Anticoagulant therapy was provided via oral DIP tablets. The daily treatment protocol comprised of 150 mg in three separate doses for 14 consecutive days. All patients were monitored daily for possible adverse events. All patients received antiviral (ribavirin, 0.5 g, Q12h), corticoid (methylprednisolone sodium succinate, 40 mg, QID), oxygen therapy, and nutritional support as necessary. Patients with diabetes were treated with insulin injections (Table 2), and those with cardiovascular diseases were treated with nifedipine.
+
+## 4.7. Free Energy Perturbation Prediction
+
+We virtually screened an U.S. FDA-approved drug database using the SARS-CoV-2 protease Mpro as a drug target. DIP
+(PubChem CID: 3108, Fig. 1A) was identified as a lead drug. In order to obtain the binding pattern and calculate the binding free energy between DIP and Mpro, DIP was firstly docked onto Mpro by using Glide-SP method with the default parameters47, and the optimal binding pose (Supporting Information Fig. S1) was further assessed by absolute binding free energy calculation with free energy perturbation48. The calculations were carried out in Gromacs 201949, and the thermodynamic cycle and procedure was similar to that used by Matteo et al50. In the calculation, the ligand electrostatic and van der Waals interactions were decoupled using a linear alchemical pathway with Dl Z 0.10 for the van der Waals and Dl Z 0.20 for electrostatic interactions. Restraints were added for keeping the relative position between receptor and ligand, which consist of one distance, two angles, and three dihedrals harmonic potentials with a force constant of 10 kcal/mol/A˚ 2 [rad2]. The distance and angles for the restraints were determined by the values of the last 2 ns of the 4 ns preliminary MD simulations. In the FEP calculations, 4 ns simulations were performed for each window. The sampled DU in the simulations were fitted by Gaussian algorithms and the free energy estimates were obtained by using the Bennet acceptance ratio (BAR) method51.
+
+## 4.8. Enzymatic Assays Of Mpro
+
+The detailed methods of enzymatic assays of Mpro are shown in Supporting Information S1.
+
+## 4.9. Foci Forming Assay
+
+Vero E6 cells were seeded in 96-well plates. The cells were pretreated with different dosages of DIP or chloroquine for 1 h before infected with SARS-CoV2 200 foci forming units (FFU) per well, and overlaid with 1.6% carboxymethylcellulose with different dosages of DIP or chloroquine. After 24 h incubation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2%
+Triton X-100. And then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Sino Biological, Inc., Beijing, China), followed by an HRP-labelled goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc.,
+West Grove, PA, USA). The foci were visualized by TrueBlue™
+Peroxidase Substrate (KPL, Gaithersburg, MD, USA), and counted with an ELISPOT reader (CTL, Shaker Heights, OH, USA).
+
+Viral titers were calculated as FFU per mL.
+
+## 4.10. Statistical Analysis
+
+Statistical analyses and graphics production were performed using R v3.5.3 (Foundation for Statistical Computing)52 and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Categorical variables were described as frequencies or percentages, and continuous variables were shown as mean with standard deviation/
+error. Comparison for two independent groups was conducted using Student's t test (for normally distributed data) or ManneWhitney test (for non-normally distributed data). Comparison for laboratory indices between the DIP-treatment and control groups during the treatment course was conducted using generalized mixed linear model. Logistic regression was performed to identify factors associated with the clinical outcomes.
+
+P < 0.05 was considered statistically significant. Detailed descriptions of data comparison and statistical tests were specified in the figure legends.
+
+## Acknowledgments
+
+We cordially acknowledge Tencent Cloud and National Supercomputing centers in Guangzhou, Shenzhen, and Tianjin, China for providing HPC resources for virtual screening and free energy perturbation calculations, and Prof. H. Ke at the University of North Carolina, Chapel Hill, NC, USA for comments. We cordially acknowledge National Key R&D Program of China
+(2017YFB0202600 and 2020YFC0841400), National Natural Science Foundation of China (91742109, 8152204, 31770978, 81773674, and 21877134), National Health & Medical Research of Australia (1080321, 1143976 and 1150425), Science Foundation of Guangzhou City (201904020023, China), Guangdong Province Higher Vocational Colleges and Schools Pearl River Scholar Funded Scheme (2016 and 2019, China), Guangdong Provincial Key Laboratory of Construction Foundation (2017B030314030, China), Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program
+(2017BT01Y093, China), Zhejiang University special scientific research fund for COVID-19 prevention and control (China), National Health & Medical Research of Australia (1080321, 1143976, and 1150425), Taikang Insurance Group Co., Ltd. and Beijing Taikang Yicai Foundation (Beijing, China), and philanthropy donation from individuals. The funders had no roles in the design and execution of the study.
+
+## Author Contributions
+
+Jincun Zhao, Xuechuan Hong, Yuxia Zhang, Fuling Zhou, and HaiBin Luo co-designed the study and co-led overall data interpretation. Shuai Liu, Xiaoyan Liu, Yinghua Wei, Qingling Zhang, Yinyi Shi, Bei Xiong, and Min Jiang provided patient care and collected clinical data. Zhe Li, Xin Wang, Jun Cui, Hui Huang, and Yi-You Huang performed the virtual screening and enzymatic assay. Jing Sun and Jincun Zhao performed viral suppression assay. Zhanghua Chen, Yuxia Zhang, and Hai-Bin Luo analyzed data and generated the tables and figures. Yuxia Zhang drafted the manuscript with significant input from Andrew M. Lew. All authors interpreted the results and critically revised the manuscript for scientific content. All authors approved the final version of the article.
+
+## Conflicts Of Interest
+
+The authors have no conflicts of interest to declare.
+
+## Appendix A. Supporting Information
+
+Supporting data to this article can be found online at https://doi. org/10.1016/j.apsb.2020.04.008.
+
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+39. Bjornsson TD, Mahony C. Clinical pharmacokinetics of dipyridamole.
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+Thromb Res Suppl 1983;4:93e104.
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+et al. Distribution of dipyridamole in blood components among poststroke patients treated with extended release formulation. Thromb Haemost 2009;102:538e43.
+
+41. Imai Y, Kuba K, Penninger JM. The discovery of angiotensinconverting enzyme 2 and its role in acute lung injury in mice. Exp Physiol 2008;93:543e8.
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+42. Kline JA, Garrett JS, Sarmiento EJ, Strachan CC, Courtney DM. Overtesting for suspected pulmonary embolism in American emergency departments: the continuing epidemic. Circ Cardiovasc Qual Outcomes 2020;13:e005753.
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+43. Schefold JC, Gerber JL, AngehrnMC,MullerM,Messmer AS, Leichtle AB,
+et al. Renal function-adjusted D-dimer levels in critically ill patients with suspected thromboembolism. Crit Care Med 2020;48:e270e6.
+
+44. Zhou J, Mao W, Shen L, Huang H. Plasma D-dimer as a novel biomarker for predicting poor outcomes in HBV-related decompensated cirrhosis. Medicine (Baltim) 2019;98:e18527.
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+45. Adekanmbi O, Lakoh S. A favorable outcome of dengue hemorrhagic fever despite poor prognostic indices: a case report with a mix of classic and unusual clinical and laboratory features. Pan Afr Med J 2019;34:74.
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+46. General Office of National Health Committee. Diagnosis and treatment scheme of novel coronavirus-infected pneumonia (trial version 6). 2020. Available from: http://www.nhc.gov.cn.
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+47. Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL,
+Pollard WT, et al. Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med Chem 2004;47:1750e9.
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+Berendsen HJ. GROMACS: fast, flexible, and free. J Comput Chem 2005;26:1701e18.
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medical/md/PMC7524418.md

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+# Editorial
+
+![0_Image_0.Png](0_Image_0.Png) Polygenic Risk Scores And Their Potential Clinical Use In Psychiatry: Are We There Yet?
+
+## Gabriel R. Fries1,2,30000-0000-0000-0000
+
+1Translational Psychiatry Program, Faillace Department of Psychiatry and Behavioral Sciences, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, USA. 2Center for Precision Health, School of Biomedical Informatics, The University of Texas Health Science Center at Houston, Houston, TX, USA. 3Neuroscience Graduate Program, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA.
+
+The genetics of psychiatric disorders has been known to be extremely complex and heterogeneous for many years. Multiple genome-wide association studies (GWAS)
+focusing on highly heritable traits (as seen in family and twin studies) have confirmed these disorders to be polygenic and multifactorial - i.e., the result of interactions between environmental stimuli and multiple genetic variants, each of which showing very small effects in determining the risk for the disorder alone. Such variants are believed to include common single nucleotide polymorphisms (SNPs - showing the smallest effects and a typical prevalence of at least 1% in the population), copy number variants, and rare variants (which have the largest effects), with their specific effects in conferring risk being additive or of higher-order interactions (epistatic). This complex scenario has led to the current understanding that candidate gene studies or single gene markers are not ideal for the field; in fact, the current standard in psychiatric genetics involves the comparison of thousands of genome-wide variants in massive samples of patients and controls collected through consortia
+(such as the Psychiatric Genomics Consortium [PGC]). Successful examples include the notorious PGC GWAS
+of schizophrenia, which compared 36,989 cases and 113,075 controls and found 108 loci to be independently associated with the disorder,1 and the latest PGC GWAS
+of bipolar disorder, which compared 20,352 cases and 31,358 controls and identified 30 loci at a genome-wide significance.2 As GWAS sample sizes continue to increase, more genome-wide significant variants are expected to be identified in the near future. Nevertheless, strategies to estimate the polygenicity of complex diseases using results from existing GWAS already exist and may provide extremely valuable biomarkers. For instance, a socalled ''polygenic risk score'' (PRS) can be calculated in subjects from a ''target GWAS'' by multiplying the number of risk alleles a person has by the effect size of each variant (as detected in an independent ''base GWAS''), and then summing each of these products across all risk loci.
+
+3 Overall, PRS captures the cumulative effects of Correspondence: Gabriel R. Fries, 1941 East Rd, Suite 3142, 77054, Houston, TX, USA. E-mail: Gabriel.R.Fries@uth.tmc.edu Submitted Jan 21 2020, accepted Jan 23 2020, Epub Mar 16 2020.
+
+multiple genetic risk variants (or ''genetic burden'') for a particular phenotype, and often includes thousands of independent SNPs for its calculation (in other words, it includes several SNPs that were only nominally significant in the base GWAS, in addition to the few genomewide significant ones). Typically, PRSs are calculated for many different p-value thresholds from the base GWAS,3 and the ones that most strongly associate with the phenotype in the target GWAS are chosen for follow-up analyses. This strategy has been used by many research groups, and many recent studies have reported on interesting associations between PRSs calculated for psychiatric disorders and important phenotypes in both patients and controls.
+
+Not surprisingly, the possibilities and implications of such measures are immense. From a theoretical standpoint, PRSs may be used not only to predict a person's risk for a disorder, but also assess its association with specific behavioral, cognitive, and prognostic outcomes.
+
+Moreover, PRSs may be used to estimate the genetic correlation between allegedly independent phenotypes, in addition to identifying more homogenous subgroups of patients that may differ in prognostic and treatment aspects based on their genetic burden for specific diseases. Finally, a particularly appealing use of such measures involves the possibility of providing early targeted interventions to disease-free subjects who are found to be at a higher risk of developing a disorder.
+
+Although exciting and while being currently explored in research settings, the clinical applicability of PRS measures is still limited. The main reason for this is the fact that most GWAS for complex diseases, including psychiatric disorders, are still underpowered (of note, the power and accuracy of a particular PRS can only be as high as the power of the original base GWAS that is used for its calculation). Moreover, current GWAS used for PRS calculations rely exclusively on common variants
+(SNPs), thus not including copy number variations and rare variants that may be extremely important for the heritability of these diseases. In addition, an important limitation that hinders the broader use of PRSs in the How to cite this article: Fries GR. Polygenic risk scores and their potential clinical use in psychiatry: are we there yet? Braz J Psychiatry. 2020;42:459-460. http://dx.doi.org/10.1590/1516-44462020-0865
+
+## 460 Gr Fries
+
+clinical setting is the lack of ethnic diversity of the GWAS performed for psychiatric disorders to date (the largest PGC studies are overwhelmingly dominated by Caucasian subjects of European descent); however, PRSs are known to be highly sensitive to ethnic background.4 Finally, PRSs may not have high specificities due to the known pleiotropic relationships between psychiatric disorders, in addition to the fact that they do not take environmental effects into account. All in all, the consensus is that PRSs are still not very informative at the individual level, and therefore are premature for any clinical use at this moment.4 The ability to calculate the genetic burden for psychiatric disorders using PRSs offers many important and clinically significant possibilities. However, it is clear that, at this point, PRSs alone are insufficient to fully capture and explain the genetic risk for these complex and multifactorial conditions.3 The discriminatory abilities and clinical applicability of PRSs are expected to significantly improve with more powerful base GWAS,4 a focus on studies of diverse ethnic populations, and the development of more sophisticated methods for their calculations. Importantly, given their potential groundbreaking clinical applications, it is imperative that researchers responsibly discuss and explain the limitations of PRSs to the lay public and be aware of the ethical implications these scores may have to society,5 including stigmatization and a potentially harmful reductive view of psychiatric disorders.
+
+## Acknowledgements
+
+GRF is supported by a career development grant from the Center for Clinical and Translational Sciences (CCTS), University of Texas Health Science Center at Houston
+(UTHealth).
+
+## Disclosure
+
+The author reports no conflicts of interest.
+
+## References
+
+1 Schizophrenia Working Group of the Psychiatric Genomics Consortium. Biological insights from 108 schizophrenia-associated genetic loci. Nature. 2014;511:421-7.
+
+2 Stahl EA, Breen G, Forstner AJ, McQuillin A, Ripke S, Trubetskoy V,
+et al. Genome-wide association study identifies 30 loci associated with bipolar disorder. Nat Genet. 2019;51:793-803.
+
+3 Martin AR, Daly MJ, Robinson EB, Hyman SE, Neale BM. Predicting polygenic risk of psychiatric disorders. Biol Psychiatry. 2019;86:97-109.
+
+4 Fullerton JM, Nurnberger JI. Polygenic risk scores in psychiatry: will they be useful for clinicians? F1000Res. 2019;8.
+
+5 Palk AC, Dalvie S, de Vries J, Martin AR, Stein DJ. Potential use of clinical polygenic risk scores in psychiatry - ethical implications and communicating high polygenic risk. Philos Ethics Humanit Med. 2019;14:4.

medical/md/PMC7544250.md

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+
+
+![0_image_0.png](0_image_0.png)
+
+# The Umbilicus Free Flap
+
+![0_Image_1.Png](0_Image_1.Png)
+
+Geoffrey G. Hallock, MD **Summary:** The morbidly obese patient has few reliable options if a single free flap is required for large surface area coverage. Usually, a latissimus dorsi muscle would be the primary option. If unavailable, a transverse-oriented abdominal flap based on deep inferior epigastric perforators as either a perforator flap or a muscle sparing type 2 transverse rectus abdominis musculocutaneous flap would be an alternative. A central panniculectomy type approach allows primary donor site closure by the cephalad advancement of the intentionally retained ptotic portion of the panniculus. Inclusion of the umbilicus with the free flap, which in this patient subgroup often is at risk for complications if excluded, mitigates against the need for undermining of the upper abdomen. The umbilicus free flap, as part of a panniculectomy, not only minimizes intrinsic flap risks, but also those of the abdominal donor site. (Plast Reconstr Surg Glob Open 2020;8:e3101; doi: 10.1097/ GOX.0000000000003101; Published online 21 September 2020.)
+The morbidly obese patient who requires a free flap that will provide significant surface area coverage always presents a dilemma, which becomes compounded when bilateral latissimus dorsi muscle free flaps have already been used. A transversely oriented abdominal flap should then be considered, often very acceptable for all because removal of the ptotic lower abdominal pannus will be an unexpected bonus!1 Almost always in this subset, any flap design will easily include the periumbilical deep inferior epigastric perforators (DIEP). Lee et al2 have shown that DIEP perforators are rarely found below the arcuate line; so that region is relatively underperfused when a DIEP flap is based on a periumbilical perforator. Therefore, for a wide and/or lengthy horizontal flap, it would be best to leave the lower pannus in situ; and instead retain also the supraumbilical region, as Boyd et al3 have shown this to be better perfused. Such removal of the central abdomen could only be executed like a panniculectomy, although leaving a higher donor site scar.
+
+Cho et al4 have shown that umbilical complications with DIEP flaps, including complete necrosis, occur more frequently the higher the body mass index, the wider the diastasis recti, and the greater the umbilical stalk height, so that they often just discard the umbilicus to forego such a sequela. Instead, if the umbilicus were kept intact with the flap, there would be no flap wound healing issues, nor any From the Division of Plastic Surgery, St. Luke's Hospital, Sacred Heart Division, Allentown, Pa. Received for publication May 18, 2020; accepted July 20, 2020. Copyright © 2020 The Author. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
+
+DOI: 10.1097/GOX.0000000000003101 in the upper abdomen because undermining would not be required as done in a conventional abdominoplasty. This case report demonstrates the advantages of transfer of the intact umbilicus itself as a free flap, and more importantly as a composite portion of a panniculectomy.
+
+## Case Report
+
+Ten years previously, this now 75-year-old woman still with a body mass index of 32 was in a motorcycle accident, sustaining circumferential skin degloving of the left lower extremity from the tibial tubercle to the ankle with multiple extremity fractures, left brachial plexus traction injury, pulmonary contusion, and splenic laceration. 
+
+Serial debridement exposed a left fibular fixation plate involving the ankle. A right latissimus dorsi muscle free flap was used for plate coverage with skin grafting of the rest of the leg. Following an hypotensive episode 48 hours later, irreversible pedicle vein and artery thrombosis was found at the time of reexploration. Because the initial posterior tibial recipient site was again widely exposed, an immediate left latissimus dorsi flap was transferred, but slowly afterward it underwent irreversible necrosis. Two weeks later, a third attempt was made using the left gracilis muscle, with the anterior tibial vessels as the recipient site. This was completely successful.
+
+Over the next 3 years, she had intermittent skin graft breakdown of the left pretibial area. It was suggested that her large, extremely ptotic abdominal pannus could cover this unstable area, allowing a concomitant abdominoplasty; but at that time, she wanted no part of another free flap as she preferred riding her motorcycle, which was Disclosure: The author has no financial interest to declare in relation to the content of this article. Related Digital Media are available in the full-text version of the article on www.PRSGlobalOpen.com.
+
+![1_image_1.png](1_image_1.png)
+
+2 her passion. She was not seen for 6 years until presenting with a large granulating and cellulitic 6 × 10cm wound of the distal left leg as a result of falling off a treadmill (Fig. 1). These events were happening repeatedly; so now she allowed us to use her abdomen as previously advised.
+
+Intraoperatively, the proximal posterior tibial vessels had an excellent flow. All sores and residual skin grafts overlying the left tibia over an area about 15 × 36cm were removed. 
+
+A preexisting right subcostal scar put at risk any undermining of the right side of the upper abdomen. Anticipating complications, a panniculectomy approach was designed to include the horizontal central 3/5ths of the abdominal skin of size slightly larger than the debrided recipient site 
+(Fig. 2), where Boyd et al3 have indicated would be found the majority of DIEP perforators. The retained inferior portion of her pannus would allow primary donor site closure.
+
+Using a lateral to medial suprafascial approach on the virgin left hemiabdomen, two 1.5mm lateral row periumbilical perforators were identified. The same approach from the medial side first revealed an umbilical hernia that would be repaired, additional justification for keeping the umbilicus within the flap. Further dissection demonstrated 2 similar caliber medial row perforators. 
+
+![1_image_0.png](1_image_0.png)
+
+![1_image_2.png](1_image_2.png)
+
+Considering the huge size (17 × 37cm) of the flap and patient fragility, this was kept as a muscle sparing type 2 transverse rectus abdominis musculocutaneous flap5 by harvesting a portion of the rectus abdominis muscle encompassing all these perforators to limit the risk of iatrogenic injury if they were individually dissected, which was more important than any minor amount of time saved. Their common deep inferior epigastric pedicle was then dissected into the groin to reach vessels of a reasonable caliber (Fig. 3).
+
+Insetting placed the left hemiabdomen oriented toward the ankle before appropriate microanastomoses. The entire flap at first exhibited excellent capillary refill. Then started a series of complications. Premature extubation required reintubation in the operating room, prolonged ventilator support, and hypotension that necessitated vasopressors for several weeks. A small portion of the proximal flap (zone IV), though originally well perfused, necrosed, but enough survived to accomplish the original goal of resilient leg padding. The patient has returned to motorcycle riding and is considering liposuction soon so 
+
+![2_image_0.png](2_image_0.png)
+
+that the flap will not appear to be so bulky (Fig. 4). She is also quite satisfied with her leaner abdomen (**See figure,** 
+Supplemental Digital Content 1, which demonstrates final appearance of abdomen donor site closure, http://links. lww.com/PRSGO/B469).
+
+## Discussion
+
+Morbid obesity has an adverse effect on all muscleconserving abdominal flaps.6 A muscle sparing type 2 transverse rectus abdominis musculocutaneous flap is not unreasonable7 to maximize perfusion with the least risk of perforator injury in this high-risk subset, while also minimizing the time of their incorporation with the flap dissection. Retention of lateral and medial row perforators in this location best ensured capture of adjacent perforasomes.8,9 Although Douglas et al10 state that a single perforator provides better zone IV perfusion in the standard DIEP flap, in this case the perfusion problem was instead secondary to prolonged postoperative hypotension and use of vasopressors. In spite of untoward events, this umbilicus free flap (a.k.a. panniculectomy free flap) achieved the primary goal of adequate pretibial padding, now permanently resistant to ongoing constant shear stresses.
+
+Geoffrey G. Hallock, MD
+Division of Plastic Surgery St. Luke's Hospital, Sacred Heart Division 1230 South Cedar Crest Boulevard, Suite 306 Allentown, PA 18103 E-mail: gghallock@hotmail.com
+
+## Acknowledgment
+
+David C. Rice, BS, PE, St. Luke's Hospital, Sacred Heart Division, Allentown, Pennsylvania, assisted with all surgical aspects.
+
+REFERENCES
+1. Hallock GG. Abdominoplasty as the patient impetus for selection of the deep inferior epigastric perforator free flap for knee coverage. *Microsurgery*. 2014;34:102–105. 
+
+2. Lee KT, Eom Y, Jeon BJ, et al. Vertical spacing of perforators in deep inferior epigastric perforator flap breast reconstruction can affect the outcomes. *Plast Reconstr Surg*. 2018;142:319–329. 
+
+3. Boyd JB, Taylor GI, Corlett R. The vascular territories of the superior epigastric and the deep inferior epigastric systems. *Plast* Reconstr Surg. 1984;73:1–16. 
+
+4. Cho MJ, Teotia SS, Haddock NT. Predictors, classification, and management of umbilical complications in DIEP flap breast reconstruction. *Plast Reconstr Surg*. 2017;140:11–18. 
+
+5. Nahabedian MY, Momen B, Galdino G, et al. Breast reconstruction with the free TRAM or DIEP flap: patient selection, choice of flap, and outcome. *Plast Reconstr Surg*. 2002;110:466–475; discussion 476. 
+
+6. Lee KT, Mun GH. Effects of obesity on postoperative complications after breast reconstruction using free muscle-sparing transverse rectus abdominis myocutaneous, deep inferior epigastric perforator, and superficial inferior epigastric artery flap, a systematic review and meta-analysis. *Ann Plast Surg*. 2016;76:576–584. 
+
+7. Lindsey JT. Integrating the DIEP and muscle-sparing (MS-2) free TRAM techniques optimizes surgical outcomes: presentation of an algorithm for microsurgical breast reconstruction based on perforator anatomy. *Plast Reconstr Surg*. 2007;119:18–27. 
+
+8. Saint-Cyr M. Perforasomes, venosomes, and perfusion zones of the DIEAP flap [Reply]. *Plast Reconstr Surg*. 2010;126:2284–2286.
+
+9. Hembd A, Teotia SS, Zhu H, et al. Optimizing perforator selection: a multivariable analysis of predictors for fat necrosis and abdominal morbidity in DIEP flap breast reconstruction. Plast Reconstr Surg. 2018;142:583–592. 
+
+10. Douglas HE, Wilkinson MJA, Mackay IR. Effects of perforator number and location on the total pedicle flow and perfusion of zone IV skin and fat of DIEP flaps. *J Plast Recons Aesthet Surg*. 2014;67:212–218. 

medical/md/PMC7671332.md

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+# Nanopore Sequencing Of Native Adeno-Associated Virus (Aav) Single-Stranded Dna Using A Transposase-Based Rapid Protocol
+
+Marco T. Radukic1,†, David Brandt2,†, Markus Haak2, Kristian M. Muller ¨ 1,* and Jorn Kalinowski ¨ 2,*
+1Faculty of Technology, Bielefeld University, D-33501 Bielefeld, Germany and 2Center for Biotechnology (CeBiTec),
+Bielefeld University, D-33501 Bielefeld, Germany Received April 22, 2020; Revised August 6, 2020; Editorial Decision August 26, 2020; Accepted September 21, 2020
+
+## Abstract
+
+Next-generation sequencing of single-stranded DNA
+(ssDNA) enables transgene characterization of gene therapy vectors such as adeno-associated virus
+(AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without second-strand synthesis using a transposase-based protocol. We show for bacteriophage M13 ssDNA that the MuA transposase has unexpected residual activity on ssDNA, explained in part by transposase action on transient double-stranded hairpins. In case of AAV, library creation is additionally aided by genome hybridization. We demonstrate the power of direct sequencing combined with nanopore long reads by characterizing AAV vector transgenes. Sequencing yielded reads up to full genome length, including GC-rich inverted terminal repeats. Unlike short-read techniques, single reads covered genome-genome and genome-contaminant fusions and other recombination events, whilst additionally providing information on epigenetic methylation. Single-nucleotide variants across the transgene cassette were revealed and secondary genome packaging signals were readily identified. Moreover, comparison of sequence abundance with quantitative polymerase chain reaction results demonstrated the technique's future potential for quantification of DNA impurities in AAV
+vector stocks. The findings promote direct nanopore sequencing as a fast and versatile platform for ssDNA characterization, such as AAV ssDNA in research and clinical settings.
+
+## Introduction
+
+Next-generation sequencing (NGS) techniques for singlestranded DNA (ssDNA) gained attention through the rise of the ssDNA adeno-associated virus (AAV) as a gene therapy vector. Recombinant AAV (rAAV) are preferred gene therapy vectors with currently two approved drugs in the United States (Luxturna and Zolgensma). Their clinical success is driven by a low immunogenic profile and extrachromosomal stability of its genomes. AAV vectors are produced in eukaryotic cell culture predominantly by plasmid transfection. For production in mammalian HEK-293 cells, the wild-type AAV genome is separated onto two plasmids such that one plasmid carries the AAV genes rep and cap (pRepCap) and the other carries the gene of interest (GOI) to be packaged into viral capsids (pITR). The GOI
+is flanked by the AAV inverted terminal repeat (ITR) sequences, which mediate genome replication and packaging. A third plasmid provides adenoviral helper functions
+(pHelper) (1,2) and can be combined with pRepCap to one AAV helper plasmid (3). Based on AAV biology, AAV vectors harbour an ssDNA, but this DNA can be designed to be self-complementary (4). For AAV applications with modified capsids such as tumor therapy (5), four-plasmid systems have been designed (6).
+
+For quality control of AAV vectors in a clinical context, the state of the encapsulated AAV genome must be tightly monitored. Vector genome quality issues arise from falsely packaged contaminating DNA, which was initially identified by Southern hybridization and quantitative polymerase chain reaction (qPCR) methods to be rep and cap sequences
+(7) or sequences from the bacterial plasmid backbone (8).
+
+These can make up 0.5 to 6.1% of the cargo DNAs of AAV
+vectors, dependent on the plasmids used for AAV production (9). The same study found that the amount of contaminating DNA is even higher in self-complementary vectors.
+
+These contaminants should be avoided, as the transfer of bacterial sequences is linked to inflammatory response and gene silencing (10,11) and cap-positive vectors have been shown to express AAV capsid proteins, potentially leading to an increased immune response to the vector and thereby impeding its efficacy (12).
+
+Whilst DNA probe-based methods enable investigation of known contaminants, they only allow for a partial view of the sample. In search for unbiased methods to assess contaminations, NGS protocols have been developed. A first approach to AAV single-molecule sequencing relied on the Helicos HeliScope sequencer and identified low levels of contaminating plasmid DNA, but was deemed too expensive for routine quality control (13). An advancement in this field was an Illumina platform-based method for singlestranded AAV vectors (SSV-seq), which identified––next to the known contaminations––randomly packaged host cell sequences and AAV purification-specific DNA impurities, as well as helper plasmid-derived impurities (14). For rAAV
+stocks produced in insect cells, the technique identified baculoviral DNA as the main source of impurities (15). AAV
+self-complementary vectors on the other hand are particularly amenable to NGS by the single-molecule real time sequencing (SMRT) approach, which revealed human DNAvector chimeras, but requires double-stranded substrates
+(16). Illumina and SMRT, in general, require extensive sample preparation, including second-strand synthesis which can induce sequencing bias and increases hands-on time.
+
+The rapid transposase-based protocol provided by Oxford Nanopore Technologies offers the advantage of amplification-free direct sequencing, thereby simplifying the sample preparation and potentially eliminating additional sources of bias. In regular rapid library generation, double-stranded samples are fragmented by a transposase and adapters are ligated to the sample fragments as part of the transposase reaction. The sample can then be directly used for nanopore sequencing. We report here the application of this convenient protocol for direct ssDNA sequencing of bacteriophage M13 ssDNA (circular) and of AAV
+ssDNA (linear) with results obtainable within one working day. In addition, we demonstrate possibilities of large-scale virus genome analysis in the context of basic research on AAV biology and manufacturing aided by the long reads of nanopore sequencing technology. In one use case, we evaluate how sequencing reads can be utilized to qualitatively and to some extend quantitatively determine undesired DNA
+packaged in a common type of vector preparation used for research. We establish the feasibility of the analysis, which thus, in the future, can be used for other AAV samples such as vector preparations for therapeutics.
+
+## Materials And Methods Aav Production And Ssdna Extraction
+
+AAV vectors were produced by the calcium phosphate triple transfection method in adherent HEK-293 cells grown in TC150 cell culture dishes (Sarstedt) with DMEM (Merck/Sigma), 10% FCS (Merck/Sigma), 1%
+penicillin/streptomycin (Merck/Sigma), 37◦C, 5% CO2.
+
+Standard reagents were purchased in analysis grade from Merck/Sigma or Carl Roth. Cells were co-transfected by plasmids pRepCap (lab code pZMB0504, encoding the replicases of AAV serotype 2 and the capsid of AAV
+serotype 9, Supplementary Figure S12), pHelper (Agilent Technologies, lab code pZMB0088, encoding AAV adenoviral helper functions, Supplementary Figure S12) and pITR (lab code pZMB0347, encoding fluorescence reporter mKate2 under control of a CMV promoter and human growth hormone polyadenylation signal, Supplementary Figure S14) with in total 37.5 -g DNA per dish in a molar plasmid ratio of 1:1:1. The pITR, the design of which has previously been published (6), provides the GOI to be packaged into viral capsids. Three days after transfection, cells were harvested by scraping, pelleted (5 min, 3000 rcf) and lysed by three freeze-thaw cycles (in 2.5 ml buffer per pellet from two dishes, 20 mM Tris, 150 mM NaCl, 10 mM MgCl2, pH 7.5, range −80◦C to 37◦C). The supernatant after centrifugation (5 min, 3000 rcf) was processed further. Free nucleic acids were then digested with 60 units/ml Benzonase nuclease (Merck) for at least 30 min at 37◦C. Then 3-[(3-Cholamidopropyl)dimethylammonio]-
+1-propanesulfonate hydrate (CHAPS) (Carl Roth) was added to 0.5% w/v and incubated at 37◦C for 30 min. Additionally, AAV from the culture media were precipitated with ammonium sulphate (3.13 g per 10 ml, incubation 30 min on ice; pelleting 8300 rcf, 10 min). The pellet was resuspended with the cleared lysate. Further contaminants from this solution were precipitated with a second ammonium sulphate precipitation (25% saturation; pelleting 7700 rcf, 10 min, 4◦C). AAV were then pelleted with a third round of ammonium sulphate precipitation of the supernatant from the second round (55% saturation; pelleting 17 000 rcf, 20 min, 4◦C). The obtained pellet was dissolved in phosphate-buffered saline (PBS) (137 mM NaCl, 2.6 mM
+KCl, 10 mM Na2HPO4, 1,8 mM KH2PO4, pH 7.4) and the solution was run through a bed of Poros CaptureSelect AAVX (Thermo Scientific) affinity resin at 1 ml/min. The affinity material was washed with PBS containing 0.05%
+Tween 20 using at least ten times the bed volume. AAV vectors were eluted with 100 mM citric acid at pH 2.0 (adjusted with HCl) and the eluate was immediately brought to a neutral pH with 1M Tris, pH 8.8. AAV vectors were finally rebuffered (100 kDa cut-off Amicon ultrafiltration spin tubes
+(Merck)) to PBS containing a total of 180 mM NaCl and 0.005% Pluronic-F68 and stored at −80◦C. A representative yield was about 5 × 1012 DNaseI-resistant from five TC150 cell culture dishes (1 × 1013 viral genomes per ml in a 490 -l preparation).
+
+Vector DNA was extracted by capsid disruption and subsequent silica-affinity based DNA purification. For this, the vector stock was first brought to 100 mM guanidine and 50 mM ethylenediaminetetraacetic acid (EDTA) from a sixfold stock solution (Tris-buffered pH 8.0). Proteinase K
+(New England Biolabs) was added to a final concentration of 4 units/ml and incubated 37◦C, 1 h and 95◦C, 20 min.
+
+Vector DNA was extracted from this solution by the NucleoSpin Gel Extraction and PCR Cleanup Kit (Macherey Nagel) as per the manufacturer's instruction.
+
+## Bacteriophage M13 Production And Ssdna Extraction
+
+M13mp18-phagemid dsDNA and the corresponding ssDNA were purchased from New England Biolabs.
+
+M13KO7 helper phage was produced as described in the Supplementary Methods.
+
+## Qpcr Analysis
+
+Quantitative PCR analysis was performed on a Roche LightCycler 480 II using the Promega GoTaq qPCR Master Mix. Primers, annealing temperatures and qPCR qualification data are given in the Supplementary Methods.
+
+## Nanopore Sequencing Sample Preparation
+
+A total of 9 -l or up to 400 ng equivalent of the vector DNA and 1 -l fragmentation mix (Oxford Nanopore RBK-004 or RAD-004) were used for preparation of barcoded libraries using the Oxford Nanopore Rapid Barcoding Kit (RBK-004) and sequenced with R9.4.1 MinION
+flow cells on the Oxford Nanopore GridION sequencing machine. Non-barcoded libraries were prepared using the RAD-004 kit. For the sequencing of the commercially obtained M13mp18 DNA, 400 ng (according to the manufacturer's concentration measurements) of each dsDNA and ssDNA were used.
+
+## Data Evaluation
+
+Basecalling was carried out using ont-guppy-forgridion (v3.0.6) with the high accuracy model
+(dna r9.4.1 450bps hac.cfg). Porechop (v0.2.4) was used for adapter-trimming and demultiplexing. Initially reads with read length ≥1000 bases and average Phred quality score >5 and later read length ≥500 bases and average Phred quality score >10 were kept for downstream analysis. Reads were mapped to the reference sequences using minimap2 (17) (v2.10-r761) with the map-ont preset.
+
+Per-base read coverage was calculated using BEDTools genomecov (18) (v2.27.1) separately for both strands.
+
+Assignment of reads to the respective subject sequence was done using BLASTn (19) (max hsps 3). BLASTn results were analyzed using a custom python script, counting high-scoring segment pairs (HSPs) to each subject and in the case of multiple HSPs of a single query making a subject assignment based on the highest bitscore. Detection of CpG methylation was carried out by realignment of the Nanopore raw data against the respective reference sequence using the re-squiggle algorithm of Tombo (v1.5)
+(20) and subsequent analysis using DeepSignal (21) with standard parameter settings and the supplied CpG model
+(model.CpG.R9.4 1D.human hx1.bn17.sn360). Singlenucleotide variants (SNVs) were called using Longshot
+(v0.3.5) (22), with a strand bias P-value cutoff of 0.01 and a maximum coverage of 500 000.
+
+Determination of transposase insertion sites was carried out using a custom python script. Reads not subjected to adapter trimming with more than 500 nt length were mapped to the reference genome using minimap2 (17)
+(v2.10-r761) and the map-ont preset, excluding secondary alignments and alignments shorter than 100 nt. For each read, the alignment closest to the read start was selected and the start coordinate on the reference sequence (the end coordinate for mappings against the negative strand) was taken as an estimate for the insertion site. Each estimate was refined by realignment of the read to a set of 31 reference sequences. These references were constructed by taking the transposase adapter sequence (Supplementary Data p.
+
+8) and adding 75 nt of the reference genome sequence with start positions within a 31 nt window around the original transposase insertion site estimate, which gives a total of 31 reference sequences for each insertion site estimate (Supplementary Figure S15). If the highest scoring realignment was longer than 100 nt and comprised at most three gaps and/or insertions in a sequence window of 10 nt around the start of the genomic sequence, it was considered as a transposase insertion site.
+
+Mappings of sequencing reads to reference sequences performed with minimap2 were analyzed and visualized using a custom python script (see 'Data Availability' section).
+
+In case of a circular genomic reference, a single sequencing read may produce two non-overlapping alignments against the reference, discontinuous at the end of the linear subject sequence. Two such alignments were joint if the endspanning distance between them on the subject sequence was ≤100 nt. In these cases, the subject start of the joint alignment becomes greater than its subject end. In case of visualizing reference mappings of reads with trimmed adapter sequences, the lengths of the non-mapping termini were added to the subject start and subject end as if they were matching. The reads were subsequently binned and plotted with regards to their subject start and end positions. For visualization of untrimmed read mappings, the lengths of these overhangs were displayed in individual subplots.
+
+The propensity of nucleotide regions in single stranded genomes to be double-stranded during transposase-based library preparation was estimated by calculating ss-counts, where a ss-count is the number of times a base is single stranded in a group of predicted foldings. Calculations were based on 100 predicted ssDNA folding structures using Mfold (v3.6) (23) with parameter settings 'W = 10', 'T = 25' and 'LC = circular' in case of circular genomes.
+
+## Results
+
+The initial intention of this study was to find a general and fast protocol for AAV ssDNA genome sequencing for characterization of virus-vector batches. We chose nanopore sequencing and reasoned that a convenient way of library creation would be a transposase-based protocol, in which a transposase randomly cleaves the DNA and ligates the fragments to sequencing adapters. If desired, DNA barcodes for sample assignment in multiplexed sequencing could be added. Tagmentation with Tn5 transposase has been used for Illumina dye sequencing of randomly primed AAV ssDNA before (24). However, the previous approach relied on a multi-step sample preparation to obtain a doublestranded tagmentation substrate. Direct adapter ligation is therefore desirable. Transposases used for rapid library creation in NGS are, to the best of our knowledge, not known to use ssDNA as transposition substrate. We considered methods to obtain double-stranded substrates such as priming the genome at the inverted terminal repeats (ITRs) and subsequently generating the complementary strand with a polymerase. On the other hand, we assume that the AAV
+ITR sequences located at both AAV genome termini are probably already present as dsDNA and could suffice for transposase fragmentation and adapter ligation. Furthermore, AAVs package either DNA strands of their genome with equal probability, with the minor exception of some ITR-modified variants that package only a single-polarity genome (25). DNA extracted from AAV vector stocks might therefore already be in a partly double-stranded state, which should enable direct library creation without prior secondstrand synthesis. We followed the two routes of either ITR
+priming and second-strand synthesis or direct library creation with 1011 vector genomes (measured before DNA extraction) in both cases. Indeed, sequencing reads of comparable quality were obtained from both samples (data not shown). The overall read count in this initial test was low, which we attributed to the low DNA input. Hence, we saw the potential for direct sequencing of AAV ssDNA as a convenient characterization tool. Direct single-molecule sequencing of the ssDNA genome is a preferred method, because hands-on time and thereby additional sources of bias are reduced. We did not observe insertion bias towards the ITRs in this initial experiment and therefore wondered, if ssDNA in general might be a valid substrate for the transposase reaction. At this point of course, we could not rule out strand hybridization as the cause of successful library generation.
+
+## Bacteriophage M13 Ssdna Is Amenable To Direct Nanopore Sequencing
+
+We tested our hypothesis of generalized transposase-based sequencing of ssDNA by sequencing of M13 phage DNA,
+which is a commonly used ssDNA reference. Unlike AAV,
+the bacteriophage M13 packages only one circular strand referred to as the (+) strand during propagation. DNA
+prepared from this phage therefore is uniform and double strand formation is unlikely. We obtained commercial M13mp18 ssDNA and corresponding dsDNA phagemids.
+
+The direct preparation of the transposase-based library from M13 ssDNA and nanopore sequencing was then carried out as before without prior second-strand synthesis.
+
+Conforming with our hypothesis, the M13 ssDNA sample was readily sequenced. Sequencing yielded 5841 reads with an N50 of 6887 bp for the single-stranded M13 DNA. A total of 5704 of 5841 reads mapped to the reference sequence of M13mp18. Thereof, 5591 reads mapped to the (+) strand and 113 reads mapped to the (−) strand, which corresponds to a ssDNA purity of 98%. Furthermore, 3165 (+) reads and 42 (−) reads passed the filtering criteria to estimate transposase insertion sites (Figure 1A).
+
+From the M13mp18 dsDNA sample, 384 091 reads with an N50 of 7224 bp were generated and in contrast to the ssDNA sample, reads of the phagemid sample mapped to both strands with near-equal distribution. Of 382 079 total mapped reads, 191 446 reads mapped to the (+) strand and 190 633 reads mapped to the (−) strand, respectively.
+
+For the estimation of transposase insertion sites, 110 994
+(+) reads and 105 783 (−) reads passed the filtering process
+(Figure 1B).
+
+We observed reduced sequence quality with the ssDNA
+samples. Average sequence similarities to the M13mp18 reference sequence were at 93.38% for M13 dsDNA and 89.88% for M13 ssDNA on single-read level. Similarities were calculated as weighted average considering varying alignment lengths from over 10 000 BLASTn hits against the reference sequence. It is possible that the helicases, which are located at each pore and are employed for doublestrand separation and the reduction of sequencing speed do not function properly with ssDNA substrates.
+
+Regarding reactivity, we found that the ssDNA samples gave a strongly reduced output compared to that of the dsDNA phagemid sample. On first sight, the mapped reads were evenly distributed over the reference sequence for all datasets, however when we plotted the corresponding transposase insertion sites, hot spots were much more apparent for the ssDNA sample (Figure 1A and B) and 18% of total reads started at these positions. In a first analysis there was no clear correlation of these hot spots to the substrates ss-count in Mfold (23), which would indicate transposase preference toward dsDNA stretches (Supplementary Figure S1).
+
+## The Transposase Acts On Hairpin Loops
+
+For a more detailed analysis of transposition insertion sites, we binned subject start positions and end positions for all mapped reads as detailed in the 'Materials and Methods' section and plotted subject start bins against subject end bins (Figure 1C shows an excerpt and Supplementary Figure S2 shows the full plot). Mapped reads of M13 dsDNA
+showed a nearly even distribution of start and end positions across the plot, indicating that transposase insertion is indeed random on dsDNA. Reads from M13 ssDNA on the other hand accumulated at distinct start-end positions, which fits the transposase insertion site preference seen in Figure 1 A and further shows that, as expected, reads generated from the circular M13 genome end at defined positions upstream of the observed read start. Figure 1C shows the range 4100–4700 of the M13 ssDNA reads start and end positions. Interestingly, for many bins below the diagonal line with subject start > subject end there is a corresponding bin mirrored above the diagonal line comprising very short reads with subject start < subject end. This points to the conclusion that transposition on ssDNA, at least to some extent, depends on the formation of hairpin loops. This is because transposase insertion in a hairpin yields one long fragment and one short fragment that contains the hairpin.
+
+Both resulting fragments can be sequenced provided that two individual transposase insertion events occur in which sequencing adapters are ligated to either of the two resulting 5-ends. Since the transposase will always produce a doublestrand break within the hairpin loop, the start position of one fragment should always match the end position of the other and *vice versa*, which results in the mirrored pattern. An illustration of this reasoning is provided in Figure 1D.
+
+Figure 1C shows three prominent long fragments (I, II and III) below the diagonal line. Fragments (II) and (III) have a corresponding short fragment in the upper part of the plot, whereas (I) does not have a corresponding fragment. To exemplarily confirm the presence of hairpins in M13 ssDNA,
+Mfold (23) was used to predict the secondary structure of bases 4,100 to 4,700 (Supplementary Figure S3). For each
+
+![4_image_0.png](4_image_0.png)
+
+putative hairpin the results show a predicted intramolecular interaction between two regions that fits the expectation regarding loop size, which can be derived from Figure 1C. Bins very closely below the diagonal line do not have a corresponding bin above the diagonal line, which means that, either the DNA loop did not yield a long enough fragment to be sequenced or the transposase also truly acts on ssDNA. We repeated the experiment with M13KO7 helper phage propagated in our lab and obtained a similar result
+(Supplementary Figure S4). The patent literature suggests that the transposase in the Oxford Nanopore protocol is the MuA transposase (26). The mechanism of this transposase is complex, and ssDNA has been shown to be a cleaved substrate to generate what was termed 'Mu-ends' (27), but not a target for a transposition event. Although our findings point to the transposase acting on hairpin loops resulting from self-annealing on short stretches of a few bases, it is unclear, whether there is additional activity on true ssDNA. For AAV vector sequencing by direct library generation and nanopore sequencing of ssDNA in general, these results mean that the presence of both strands in the sample is not a necessity and that also true ssDNA is accessible by this method.
+
+## Nanopore Ssdna Sequencing Allows For Direct, Amplificationfree Sequencing Of Aav Vectors
+
+As we had observed a relatively low AAV read count in our initial test, we next improved ssDNA extraction from AAV to gain more reads. In the end, we settled with an AAV purification protocol based on Benzonase nuclease digest of the producer cell lysate, ammonium sulphate precipitation and subsequent Poros Capture Select AAVX affinity chromatography. Inactivation of residual Benzonase and capsid disruption for ssDNA release was then performed with 50 mM EDTA, 100 mM guanidine and proteinase K at slightly basic pH. Afterwards, ssDNA purification from this solution was achieved by silica-adsorption chromatography with a commercial kit and the eluate was used for the transposase reaction. Using this protocol, we performed two independent sample preparations with a time delay in between of three months starting from individual cryo-cultures of producer cells, with five TC150 cellculture dishes each. These samples will be referred to hereafter as sample 1 and sample 2 with their sequencing runs being run 1 and run 2. We obtained 0.5 × 1013 DNaseIresistant viral genomes after affinity chromatography from both cultures. From these two AAV preparations we were able to obtain 50 -l DNA solutions with optical densities of OD260, 10 mm = 0.40 and OD260, 10 mm = 0.85, corresponding to an equivalent total of about 1.0 and 2.1 -g dsDNA
+(or 0.66 and 1.4 -g ssDNA, respectively). This means a viral genome recovery rate after DNA extraction between 25% (assuming only ssDNA is present) and 37% (dsDNA
+only). Since DNA in these samples might be partially singlestranded and double-stranded, and since these forms have different absorption coefficients at 260 nm, we prefer to use volumes and optical densities for indications of quantities. Agarose gel electrophoresis of these two samples, directly after extraction and after sample freeze-thaw, showed distinct bands attributable to the rAAV genome in singlestranded, hybridized and aggregated states (Supplementary Figure S5).
+
+We used 9 -l of each of the two AAV samples for the transposase reaction and sequenced sample 1 on a pre-used flow cell for 22 h with sample assignment by barcodes (run 1). Calculated from the concentration measurements, the theoretical input in this run was between 1.1 × 1011 and 1.6 × 1011 total viral genomes. A fresh flow cell was used for sample 2 without barcoding (run 2) (sequencing time:
+17.5 h). Here the theoretical input was between 2.2 × 1011 and 3.4 × 1011 total viral genomes. We titrated sample 2 by qPCR and found that 9 -l contained 1 × 1011 viral genomes according to this method (±1 × 1010 with 95%
+confidence interval). Notably, if we had used a full-length 4.7 kb genome roughly half the amount of viral genomes give the stated mass of input DNA.
+
+Again, as expected, both samples were readily sequenced, but gave vastly different read counts that passed our initial length quality threshold of ≥1000 bases read length (22 174 reads for run 1 versus 291 036 reads for run 2). We performed a first mapping analysis of these reads and found that the vast majority of raw reads mapped to the reference genome (Table 1A). Coverage steadily increased until it reached a stable plateau at about half the genome length.
+
+At the ITRs however, a sudden decrease in coverage was observed (Figure 1E displays run 2). However, we found that many mappings that end in the ITRs are stemming from reads that have an unmapped 3 extension of lowerquality bases, which can be attributed to the remaining ITR
+(Supplementary Figure S2). Nonetheless, ITR coverage is still 222 712-fold in run 2 and ITR sequences are thus accessible by nanopore sequencing despite their known tendency to form secondary structures. In the transposase insertion site analysis of reads longer than 500 nt of AAV
+sample 2 220 075 (+) and 189 481 (−) reads passed filtering. Strand-specific hot spots were again apparent, although overall, most reads started throughout the genome (Figure 1E). These hot spots again did not correlate with the DNA
+fold and correlations to the GC content were minimal (Supplementary Figure S6). Interestingly, the read start pattern seems to be a combination of the patterns observed for M13 ssDNA and dsDNA.
+
+## Direct Nanopore Aav Ssdna Sequencing Reveals Single-Base Heterogeneity And Methylation Status
+
+Comparison of the assembled genome to the reference sequence revealed SNVs, as seen before for rAAV (14). Despite the base accuracy of 93%, which is an intrinsic property of the current nanopore sequencing technology, variant calling is enabled by the high coverage we obtained.
+
+SNVs were located within ITRs in the short hairpins (BC internal palindromes according to common nomenclature) and were transversions as well as transitions with an individual abundance of about 20%. We were able to link ITR SNVs to the two possible ITR configurations in FLIP
+and FLOP orientation, so that the found ITR SNVs are in the end expected to arise. On the other hand, prominent SNVs across the transgene cassette were mostly transitions with a hot spot located in the polyadenylation signal and throughout the CMV promoter with an abundance up
+
+| Table 1. BLASTn read assignments and qPCR results for two independently produced and sequenced rAAV samples (sample 1 and 2) A: nanopore BLAST bins as percent of total hits Run 1 (sample 1) Run 2 (sample 2) Group\threshold >500 nt >1000 nt >500 nt >1000 nt rAAV genome 97.00% 97.34% 97.91% 97.96% pITR 1.11% 1.29% 0.97% 1.25% pRepCap 0.47% 0.49% 0.23% 0.27% pHelper 0.25% 0.24% 0.17% 0.17% hg38 1.18% 0.65% 0.72% 0.35% B: qPCR (and in silico fragmentation) results as percent of total measurable with 95% confidence interval Primer Sample 1 Sample 2 (in silico) bla 2.0 ± 0.3% 2.9 ± 0.4% (1.79%) Rep 0.22 ± 0.04% 0.24 ± 0.04% (0.13%) E4 0.062 ± 0.009% 0.08 ± 0.01% (0.10%) A: Total contamination levels in both samples are independent of the read-quality thresholds tested here, however the individual share of contaminations   |
+|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+
+to 30% (Figure 1F). Only two and zero variants could be called for ssDNA M13 and dsDNA M13 samples, respectively. Raw reads were also analyzed for methylated CpG
+sites separately for both strands using a custom software workflow with Tombo and DeepSignal (21). The studied rAAV genome as encoded on the pITR plasmid contains 129 CG dinucleotides, 123 of which have been mapped in run 2. When we compared reads from run 2 to reads of in vitro amplified rAAV genomes, no substantial methylation was identified. However, these results are based on the current algorithms used by the applied software and need to be verified by additional experiments.
+
+## Qualitative Statements On The Abundancy Of Impurities Are Possible
+
+Our nucleotide reference database so far contained only the rAAV genome. We next extended this database to include the human genome build hg38 (GCF 000001405.39)
+as well as the utilized producer plasmids. Now, of all reads that passed the length quality threshold of ≥1000 bases, 96.73% (run 1) and 99.92% (run 2) gave a BLASTn-hit with our database. Of the reads not assigned to our database, 17% (101 reads, run 1) and 13% (33 reads, run 2) gave a hit against the National Center for Biotechnology Information (NCBI) Nucleotide database. We performed further analysis only with reads assigned to our database.
+
+The assignments calculated as percent-contaminants are summarized in Table 1A. Of the contaminants, pITR sequences showed the highest prevalence, representing 1.29%
+(run 1) and 1.25% (run 2) of total reads attributable to our database. They were followed by hg38 sequences, representing 0.65 and 0.35% of all reads. The proportion of reads that map to the pRepCap was 0.49 and 0.27% each. About 0.24 and 0.17% of attributable reads in both runs were assigned to the adenoviral helper genes. Notably, these results also depended on where the origin of sequence numbering was set on the reference plasmid, which is a limitation of the mapper. We therefore set the numbering origins to 2000 bp downstream of the f1 ori for all three plasmids.
+
+We performed additional qPCR analysis with primer sets that allow for the amplification of the CMV promoter within the rAAV genome, rep gene, E1 gene of the adenoviral helper plasmid as well as the ampicillin resistance gene bla, which is present on all three producer plasmids. Results are given as percentage of the combined absolute copy number (equals 100%) of all four measurements (Table 1B). As expected for the contaminants, the bla gene was present with the highest proportion of 2.0 ± 0.3% (95% confidence interval) in sample 1 and 2.9 ± 0.4% in sample 2. This result can be compared to the nanopore reads that map to one of the three bla containing producer plasmids, which had a combined share of 2.0% (run 1) and 1.7% (run 2) of all referenced reads. The qPCR result for bla thereby matches very well for run 1 but is slightly higher than expected from the nanopore analysis for run 2. On the other hand, the proportion of rep genes found by qPCR and sequencing matched well for run 2 but were off by a factor of two for run 1. Furthermore, adenoviral helper genes were underrepresented by qPCR compared to the sequencing results in both samples.
+
+We next wondered, whether certain contaminations are present in the capsid as small fragments below 1000 nt and if our initial length quality threshold of ≥1000 nt would cause the deviations between qPCR and nanopore sequencing.
+
+We therefore re-analyzed our datasets and included all reads above 500 nt and by doing this, the accepted read count for run 2 increased from 291 036 reads to 647 810 reads.
+
+The results of this analysis showed that, whilst the overall share of contaminants within the sample stays roughly the same regardless of the thresholds tested, the share of individual contaminants shifts. We found that the proportion of plasmid-derived contaminants remained mostly constant for both analyses, whereas the proportion of human genomic contaminants doubles for the lower threshold.
+
+Since the BLASTn binning could be distorted compared to qPCR analysis, because long reads might span fusions that contain two or more qPCR target sites, we performed in silico read fragmentation to better compare nanopore sequencing and qPCR data. We subsequently split all reads from run 2 (>500 nt) into fragments of 500 bp and removed resulting fragments below 300 bp. Subjecting a subset of 100k read fragments to the same BLASTn analysis as conducted above gives slightly different results, as the fraction of reads attributed to the rAAV genome drops to 96.42%.
+
+Accordingly, pITR and hg38 contaminants rise to 1.35 and 1.55%, respectively. pHelper and pRepCap contaminants account for 0.47 and 0.21%, respectively. This leads to a total of 2.03% of reads attributed to one of the bla containing producer plasmids in sample 2 after *in silico* read fragmentation. Another possibility is to bin the fragmented reads to a reference library that only contains the qPCR target sequences for direct comparison (CMV promoter, rep, bla, E4 gene). Here, the share of E4 sequences is comparable between the methods, whilst bla and rep sequences are slightly underrepresented (Table 1B). Clearly, at this point, a more descriptive data evaluation tool is needed to find the source of this disparity.
+
+## Direct Sequencing Reveals The Molecular State Of The Genome And Its Contaminants
+
+As we use a direct sequencing approach, each read represents a single 3-end ssDNA fragment of a natively packaged nucleic acid, presuming that it was fragmented only once by a transposase. This makes the fragments' GC content a calculatable (from the known sequence) as well as measurable (from sequencing) quantity for a given fragment length, at least for the recombinant AAV genome.
+
+Conclusions on the molecular state of the genome and its contaminants can then be drawn from a %GC versus read length plot showing reads selected based on the BLAST assignment, as shown in Figure 2. In these plots, when assuming no sequence preference of the transposase, reads of originally circular molecules ideally appear as single points, as these have a constant %GC content and the same read length, independent of the cut site (for single cut genomes).
+
+Accordingly, reads of linear fragments will produce a vertical line (|), if the GC content is constant along the DNA
+and a slanting line (/ or \), if the GC content increases towards one end. The latter fragment will result in an uppercase lambda (-) structure when both strands are present, because both directions are sequenced. In such a plot and according to expectations, the M13 reads group around the theoretical GC content and length with conical tailing towards shorter reads (Supplementary Figure S7). We suspect the latter to arise from double transposase fragmentations and premature sequencing breakoffs. In the AAV sequencing runs on the other hand, reads that gave a BLAST hit with the rAAV genome showed a more complex mirrored lower-case lambda-like pattern. The pattern becomes easier to spot in the large data volume when reads are displayed in a heatmap, as shown in Figure 2A for run 2 (refer to Supplementary Figure S8 for run 1). The read-length histogram underlaid in Figure 2B for the same data set further shows that most read lengths are at or below the theoretical genome length, which is 2.2 kb. The shape of the data distribution in the plot is a function of the fragment's nucleic acid composition. We therefore simulated the transposase reaction for the rAAV genome and found that in the plot the measured data are shifted slightly towards lower GC content compared to that of the simulation (Figure 2A,
+green line and supplementary information for the simulation script). Single-read analysis showed that the GC-rich 3 ITR is often sequenced incompletely, which explains the shift.
+
+Hot spots of transposition sites are also apparent in this plot in accordance with Figure 1E. Notably, a larger proportion of AAV-assigned reads are shorter than 1000 nt, which hints on double fragmentations. Plotting all 632 687 mapped reads directly reveals additional reads of a distinct distribution, which are longer than the theoretical rAAV genome (Figure 2B), although of all reads in the rAAV
+genome bin, only 0.9% are longer than 2300 nt. We reasoned that these could be genome head-to-tail (or tail-tohead) fusions, where a head-to-tail fusion is a concatemer of two genomes fused over a junction ITR so that the terminator of one genome is adjacent to the promoter of the next genome. Indeed, a simulation of such a fusion (green line, Figure 2B) matches well with the oversized reads, except for a protruding point cloud at about 4 kb read length (Figure 2B, black arrow), which are genome-backbone fusions as determined by later single-read investigation. Similar plots for the other BLAST bins reveal differing molecular states of the individual contaminants. Reads assigned to hg38 appear to be of completely random human origin, with an exponentially decaying size distribution (Figure 2C). Reads assigned to pRepCap at first sight also appear to be randomly fragmented and packaged as indicated by the overall triangle shape of the point cloud (Figure 2D). However, the underlying size distribution does not show an exponential decay of read abundancy with read length and this could hint on a different mechanism of packaging compared to packaging of human genomic sequences. The same was observed for pHelper-derived sequences (Figure 2E). In contrast, the distribution of reads assigned to the pITR backbone appears to follow clear rules (Figure 2F) and three features of this bin come immediately to attention as labelled in Figure 2F: (a) an accumulation of reads is located at around 4.25 kb read length, (b) another accumulation at around 2.25 kb read length, as also evident from the underlying histogram plot and (c) a slanting cloud of data points spanning from the shorter length accumulation all the way to the lower limit of read length. Accumulations in this plot can indicate transposase insertion bias on linear DNA resulting in fragments of similar length, although the prevalence of this 2.2 kb point cloud is more prominent than other hot spots for the rAAV genome. Another explanation might be a circular fragment. Conveniently, individual read subsets can also be investigated on single-read level as further elaborated below.
+
+## Single-Read Investigation Confirms The Global Analysis And Highlights The System'S Packaging Flexibility And Susceptibility For Recombination
+
+We took the opportunity long-read sequencing data hold and investigated reads of interesting appearance individually by annotating producer plasmid features (PlasMapper tool of Geneious R9 software) on each sequencing read. We started analysis with a subset of 200 randomly chosen reads from the rAAV bin of run 2 and of these, 198 reads (99%) were full-size transgenes or fragments of it, present in both
+
+![8_image_0.png](8_image_0.png)
+
+![8_image_1.png](8_image_1.png)
+
+c
+polarities as exemplarily depicted in Figure 3 A. Of these, one (0.5%) was a head-to-tail fusion as depicted in Figure 3B. Two reads (1%) were transgene-backbone fusions with one junction ITR (Figure 3 C). Furthermore, we were able to annotate 3 ′ -terminal ITR sequences (similarity threshold: 65%) for 149 reads of the subset (74.5%). This is especially interesting, because AAV packages its genome 3 ′ to 5 ′ , whereas the sequencing direction is 5 ′ to 3 ′ . This means that we can directly sequence the packaging signal and it also provides a measure on double fragmentations. We repeated the analysis for all 647 810 reads longer than 500 nt using BLAST ( E -value-cutoff of 1e-5) and came to a very similar result of 493 916 reads (76%) with 3′-terminal ITR
+sequences. We next expanded the subset of the rAAV bin to 10 000 randomly selected reads, and of these, 98 reads
+(1%) were longer than 2500 nt. A total of 81 reads (83%)
+of these overlong genomes terminated in an ITR sequence.
+
+A total of 75 reads (76.5%) were rAAV genome-genome fusions. The remaining reads were genome-backbone fusions with a 3 ′ transgene (except for one read) and these findings were in good agreement with our %GC versus read length plots and simulations whilst also providing indications on the abundances of fusions. We can extrapolate from these numbers the total content of genome monomers in our sample to be 96.95% (abundancy of monomers multiplied with rAAV bin size), although notably, this extrapolation is only valid under the assumption of quantitative sample preparation, no transposase insertion bias and equally likely packaging of both polarity genomes.
+
+Next, we were interested in the junction ITRs between two fused genomes and genome-backbone fusions, to eventually find hints on their origins. We previously described that in our pITR plasmid, just as in many other available ITR plasmids, the ITR adjacent to the transgene's
+
+![9_image_0.png](9_image_0.png)
+
+polyadenylation signal harbours an 11 bp deletion within the B-C hairpins (6). In addition, the ITR complementary A-region is shorter in both ITRs compared to the ITR reference sequence. Mapping of genome-backbone fusions (n =
+12) to our plasmid reference showed identity (including the deletion) to the plasmid reference within the limitations of current nanopore sequencing technology (Supplementary Figure S9). Junction ITRs between two fused genomes on the other hand were much more diverse. We constructed a reference sequence with two genomes fused by a complete ITR including two D-sequences, either in FLIP or in FLOP
+orientation, and mapped fusions to this reference with respect to their orientation. It became immediately apparent that, independent of genome orientation, the mapping quality dropped downstream of the B-C hairpins, which either hints on difficulties in sequencing, error-prone recombination at this position or both (Supplementary Figure S10).
+
+Interestingly, of all manually investigated junctions (n =
+41), only two (5%) clearly harbored the 11 bp deletion, although the evaluation on single-read level was complicated by the overall elevated number of mismatches in this region compared to our reference. The 3-terminal ITRs of the same reads (n = 32) showed a similar deletion only in four cases. Regarding ITR orientation (the ITR internal palindrome B-C can be inverted compared to rest of the ITR
+due to the genome replication scheme), we found that both FLIP (not inverted) and FLOP (inverted) orientations were present at the junctions and at the 3 terminal ITRs and that genome orientation was biased towards FLOP orientation when the polyadenylation signal was 5 of the junction (n =
+17 in FLOP and n = 4 in FLIP orientation versus n = 6 in FLOP and n = 16 in FLIP when the CMV promoter was 5 of the junction). Junction ITRs furthermore had two Dsequences.
+
+Further single read investigation of the three regions of interest within the pITR bin (Figure 2F) showed that reads from the 4.25 kb accumulation (arrow a) were mostly backbone-genome fusions with conserved read starts within the bacterial origin of replication and a full 3 transgene
+(n = 13 of an 18 read subset with length threshold 4200–
+4300 nt). Reads from the 2.25 kb accumulation (arrow b)
+were mostly reads starting at the same conserved position within the plasmid's bacterial origin of replication, but ending in the adjacent 3 junction ITR between backbone and transgene (example in Figure 3D; n = 53 of a 75 read subset with length threshold 2200–2300 nt). Reads from the slanting cloud of data points (arrow c), on the other hand started throughout the bacterial backbone and terminated in an ITR as well. However, these were of the inverse orientation compared to the reads of the described accumulation (n =
+62 of a 200 read subset with length threshold 500–2000 nt).
+
+To a smaller extent, shorter reads of this orientation also ended in an ITR (n = 37 of the same 200 read subset with length threshold 500–2000 nt).
+
+## The P5 Promoter Is A Secondary Packaging Signal And Is Prone To Recombination With Itr Sequences
+
+Reads from the pRepCap bin often terminated 3 in a p5 promoter sequence, which lead us to further investigate on the phenomenon (example Figure 3E, top). We searched for 3-located p5 sequences using BLAST (E-value-cutoff of 1e5) and found 527 reads (0.08%), and these had the cap coding sequence 5. Of these, 303 reads had the p5 promoter strictly 3-terminal and the coverage sharply dropped at position +12 downstream of the promoters TATA-box (coverage at position +11: 71%, +12: 42%, +13: 13%, Supplementary Figure S11). On the other hand, 158 reads had the p5 promoter followed 3 by a terminal ITR. In 59 of these cases the ITR D-sequence was located directly adjacent to the p5 promoter and in the other cases a maximum of 154 nt lay between p5 and the D-sequence. Long reads in the pRepCap bin also had an ITR without p5 sequences, when the rep gene was 3 (Figure 3E, bottom, n = 3 in a subset of 47 of reads longer than 3 kb). Significant recombination was also evident in the pHelper bin. About 41% of reads in the investigated subset of this bin had 3 ITR sequences (n = 82 in a random 200 read subset), often in combination with CMV promoter sequences (n = 19). ITR sequences were furthermore especially prevalent in reads where binning had failed (560 reads total in run 2). A random subset of 260 reads of these had a mean length of 715 nt and, despite their short length compared to the other bins, 47 reads of the subset (18%) had more than one ITR sequence (read examples shown in Figure 3F).
+
+## Discussion
+
+Nucleic acid contaminants in AAV vector stocks for gene therapy are gaining attention alongside the increase of therapeutic doses from 1012 viral genomes per kg in the first authority approved product Glybera to recently approved 1014 viral genomes per kg for Zolgensma, both single-dose systemic applications (28,29). Potentially, even higher doses in multi-administration therapies, like cancer gene therapy, are conceivable. The United States Food and Drug Administration recommends for a vaccine dose that residual cell-substrate DNA should be ≤10 ng and the median DNA size should be of 200 bp or lower
+(30). Vector manufacturers take extensive precautionary measures to ensure a homogenous product and preempt tighter AAV-specific regulations. These measures include the use of bacterial backbone-depleted circular supercoiled plasmid-derivatives, which were shown to be effective in significantly reducing false encapsidation of prokaryotic sequences in rAAV (9). Other strategies involve the use of closed-linear derivatives (31) or plasmid insertions of uncritical stuffer DNA beyond the AAVs packaging limit, to avoid packaging of the bacterial backbone and antibiotic-resistance gene (32). Monitoring of contaminants is a routine task in vector manufacturing and new time-saving techniques with reduced hands-on time are appreciated.
+
+We report here the direct transposase-based library generation and nanopore sequencing of ssDNA as a convenient and versatile tool for characterization of AAV packaged DNA and ssDNA in general. We present proof for direct ssDNA sequencing by use of bacteriophage M13mp18 ssDNA as control (Figure 1A). Use of transposases for library generation was originally designed for dsDNA tagmentation (Nextera library preparation using Tn5 transposase) and sequencing on the Illumina platform (33) and it was adapted for direct dsDNA sequencing on nanopores by Oxford Nanopore Technologies with MuA transposase (26,34). MuA forms a homotetrameric synaptic complex around paired phage Mu genome ends and then catalyzes strand transfer, leaving behind nicks that act as replication primers in the wild-type (35). A transposome consisting of MuA and end substrates (mini-Mu) is sufficient for in vitro transposition (36) and it shows only a slight target DNA bias towards a 5-CYSRG pentamer (37,38). Hyperactive MuA variants with also low target bias have been reported (39,40). However, we were unable to find previous reports of MuA (or other DDE transposase superfamily members) activity on ssDNA targets. Our data confirm the relatively low insertion bias of MuA on dsDNA (Figure 1B), but not on M13 ssDNA, where hot spots of insertions are seen and activity is reduced. We observed three especially prominent hot spots of transposition sites for M13 ssDNA. Reads from M13 ssDNA exhibited lower sequence similarity compared to the reference than M13 dsDNA
+reads.
+
+## Transposase Activity And Substrate
+
+Our analysis showed that hot spots of transposase insertion sites can be in part explained by MuA action on (transient) hairpin loops within the ssDNA target. To fit into the MuA target binding pocket, hairpins require a stem of at least 23–25 nt (35,38), however we saw also smaller hairpins as possible transposase targets. Moreover, MuA
+exhibits increased activity on mismatched targets (41) and the extent to which mismatches are tolerated has not yet been investigated, impeding our efforts to predict the target structures. Nonetheless, as we found the corresponding pairs of long reads and short reads for a given transposase insertion point on the circular substrate, we conclude that hairpin loops are one mode of transposase action on ssDNA. Compared, the M13 ssDNA sample transposase insertion sites have the most heterogeneous distribution (sites without insertion: 83%; average of insertions per site (excluding zero insertion sites): 2.7; median 1; max insertions at one site: 232; ratio average/median: 2.7), the dsM13 DNA integration distribution is the most homogeneous (sites without insertions 0.6%; average (excluding 0):
+30.1, median: 19.0; max: 433, ratio average/median: 1.58)
+and the AAV sample is in the middle with a noteworthy symmetry and the distributions leaning towards the dsM13 DNA (sites without insertions 0.1%; average (excluding 0):
+187.8, median: 91.0; max: 10 964, ratio average/median:
+2.06). Sites without insertions could be the result of the method parameters and/or of the transposase biology and are here omitted from average/median to allow for comparison across experiments with varying read numbers. The distribution of transposase insertion into the rAAV DNA is probably due to hybridization of two ssDNA genomes of AAV to one dsDNA, which we also observed in agarose gel electrophoresis. In the period between publication of our preprint and preparation of the manuscript for journal submission, another group confirmed the feasibility of our technique by tagmentation of hybridized AAV genomes and Illumina dye sequencing (42). Overlaid, we further find hot spots of transposition sites on one strand only. This is probably a different effect of transposase action on ssDNA, as seen for the M13 ssDNA. Given that hot spots lay on one strand only, we deduce that the effect could be in part sequence-specific in addition to conformation-specific, because of the inherent symmetry of hairpins when both strands are present. Further work will be required to completely elucidate the MuA transposase action on ssDNA
+targets.
+
+## Itr Sequencing And Snvs
+
+Albeit the given insertion bias of the transposon on ssDNA,
+nanopore long reads compensated for this and still enabled full coverage of the recombinant AAV genome. We achieved a 356 009-fold coverage (run 2), which also enabled investigation of SNVs despite the lower base accuracy of nanopore sequencing compared to other NGS methods. The sudden halving of coverage within the ITRs can be explained by a strong reduction of base-quality in the ITR regions, either stemming from difficulties of the helicase with the strong ITR secondary structure, or as a result of back-folding of ITRs after passing through the transmembrane pore. Furthermore, ITR internal hairpins can be present in two possible states, FLIP and FLOP (43), which adds to the reduction in coverage. A previous AAV NGS study found SNVs within the rAAV genome, mostly located within one region in the coding sequence and both ITRs (14). Regarding the ITRs, we found variants that locate in the ITR B-C hairpins
+(according to the ITR naming convention) as well. However, these are again attributable to the two possible states of ITRs which arise from AAV genome replication: FLIP
+and FLOP, where FLIP ITRs harbor the inverse complement C-B hairpins compared to FLOP ITRs whilst the rest of the ITR sequence stays the same. Our producer plasmid pITR encodes FLOP ITRs on both sides and FLIP-specific SNVs appeared with a 20% frequency. We note that only those ITR conformation-specific (FLIP) SNVs were called, that lie in the outer arms of the respective hairpins, and we are so far unable to explain this finding. Also, SNVs within the coding sequence with frequencies up to 30% were observed. The same workflow resulted in only two called variants for the bacteriophage M13 sample as a control. Given that variant calling from nanopore data is a relatively recent technique, we suggest re-cloning of AAV DNA and Sanger sequencing of individual clones to confirm these high SNV abundancies. Nonetheless, our finding is overall in agreement with the previous study (14) which found SNVs with an abundance up to 15%. The locally high SNV
+abundance raises the question where these variants come from and what their implication for vector quality is. We find it unlikely that these SNVs are already present on the producer plasmid level, as this would render cloning in *Escherichia coli* in general impractical to impossible and is also not in accordance with our frequent Sanger sequencing of pITR plasmids after cloning steps. Since there is no amplification step with our method, a somewhat error-prone AAV genome replication during virus production may be responsible and it would be very interesting to compare different transgenes, producer cell lines (different mammalian, insect and yeast) and wild-type AAV under this aspect.
+
+## Methylation Of The Packaged Aav Genome
+
+Nanopore sequencing also offered us the convenient opportunity to investigate CpG methylations from raw reads.
+
+In a previous study, laborious bisulfite PCR sequencing for packaged AAV2 wild-type genomes showed little to no methylation with a maximum share of 1.7% methylated CpG dinucleotides, but revealed hypermethylation of integrated genomes (44). We used recently published deep learning tools to investigate CpG methylations from nanopore raw data, but we did not observe significant methylation above an unmethylated reference. The finding supports the previous study, which used AAV wild-type and highlights the similarity of wild-type and recombinant genome replication.
+
+## Sample Preparation And Library Preparation
+
+As we present a direct sequencing method, the sample input is higher compared to other NGS methods. We find that, in case of AAV, extracted DNA from 0.5 × 1013 DNase Iresistant viral genomes is enough for about five sequencing reactions. We also saw that a critical step in sample preparation is the Benzonase digest of the producer cell lysate. When performed for one hour with the given concentration, no fragments beyond the AAV packaging limit of about 5 kb are seen (Figure 2). Digestion for only 30 min on the other hand led to emergence of longer reads in small proportions (Supplementary Figure S8) and since we performed virus precipitation and antibody-based affinity chromatography for sample preparation, we attribute these to overlong fragments protruding the capsid and otherwise capsid-associated DNA. In the future, this incomplete (or omitted) digest could be used as a method to investigate rAAV genome replication and packaging intermediates directly. Also, there does not seem to be a linear correlation between sample DNA input and total read output, and we recommend using samples of OD260, 10 mm = 0.8 or higher for library preparation. Furthermore, the incubation time of the transposase can be optimized to yield longer fragments. In multiplexing we observed overspill and a lower read count, which may also be attributed to the differences in library preparation for multiplexing. We therefore recommend non-multiplexed sequencing, possibly on single-use flow cells (Flongle), for quality control settings.
+
+## Sequencing Of The Packaging Signal Sequences
+
+In our opinion, the relatively high amount of sample input is rewarded by the increased information content in long reads from direct sequencing. We were intrigued to see that 76% of AAV reads had a 3-ITR sequence which is the predominant packaging initiation signal. Since the packaging direction is 3 to 5 (45,46) and the sequencing direction is 5 to 3, reasons for reads terminating 3 without an ITR could be 2-fold: either the corresponding fragment was double-fragmented by transposases prior to sequencing, or packaging initiated without an ITR. Given that all investigated human genomic sequences had no 3-ITR, we find that both hypotheses remain valid, and no definite conclusion can be made from the given data. We can, however, safely say that the extent to which reads represent doublefragmented sequences was below 24% in our experimental setup. Another interesting analysis would be to investigate genomes that are incompletely packaged after packaging is initiated by the ITRs (truncated genomes). Since the sequencing direction opposes the packaging direction, this analysis is not possible with the provided method and, for example, 3 adapter ligation, complementary strand synthesis and nanopore sequencing of the newly synthesized strand would be required.
+
+As the AAV genome 3-packaging signal is so conveniently accessible, we took the opportunity to search for 3 p5 promoter sequences. The p5 promoter has been described to be a secondary packaging and genome replication signal (7,47) and the corresponding activity was mapped to nucleotide positions 250–304 of the AAV serotype 2 reference genome with the terminal resolution site between positions 287 and 288 (48). Our data directly show that reads which terminated within the p5 promoter of the pRepCap plasmid, did so preferably at position +12 upstream of the TATA-box (equivalent to position 273 of the AAV serotype 2 reference genome). We reason that this 14–15 nt discrepancy between read-end position and the proposed terminal resolution site is probably an artefact of nanopore sequencing, in which the bases closest to the nucleic acid fragments 3-end cannot be sequenced. We already observed this in our read-start and read-end analysis on M13 ssDNA, where the read-ends were shifted slightly by about 11 nt. Since the effect might also be sequence dependent and no systematic studies on this topic are available to us, we conclude that our data on the p5 promoter is in good agreement with previous studies. However, we also found previously undescribed recombination between p5 and ITR
+sequences. In these, the complete p5 promoter sequence was directly followed by the ITR, starting with the D-sequence.
+
+This means that the terminal resolution sites in the ITR and the p5 promoter are likely not involved in this recombination and it is unclear from our data how these sequences emerge. Although the overall proportion of these reads was small in our study, since p5 and ITR sequences were associated with rep and cap genes, we find that these sequences might contribute significantly to already observed packaging, persistence and expression of associated cap and also rep sequences from recombinant vectors (9,12,32,49).
+
+As one reviewer pointed out to us, permutations of the 12 nt Rep binding site of the ITRs that exist within the human genome were shown to direct AAV genome integration
+(50). Once nanopores with increased base accuracy become available, our method could also be used to study whether these sites can act as packaging signals for contaminating human genomic DNA in vector preps.
+
+## Contaminant Sequence Abundancies And Comparison To Qpcr
+
+We then wondered if our method also allows for quantification of contaminants in AAV vector stocks and we assigned reads to single entries of our reference database by BLASTn bit score as a first test and compared the results to our qPCR measurements. In general, the obtained share of contaminants was in the same range between the two methods.
+
+However, we found that the result depended also on the chosen read length threshold and in part also on the sequence numbering of the plasmid references. One source of deviation are long reads, that span more than one qPCR target. A
+distorted result is expected here, because of the unique read assignments which assigns one read to only the most prominent hit in the genome database and cannot assign possible fusions to both parental sequence sources. We tested a more advanced data evaluation technique with *in silico* read fragmentation and subsequent binning to a database reduced to the qPCR targets, but again, binning results were offset for bla and rep sequences. This hints on another source of error which might arise from the apparent transposase insertion bias we observed for ssDNA targets but might on the other hand also come from the qPCR where a primer set dependent bias was observed for AAV samples
+(51). Moreover, qPCR imposes a systematic error because human genomic sequences cannot be assessed. Normalization of reads by their respective read starts could level the transposase-induced error and might help to overcome the observed discrepancy to yield a quantitative method, which should then be qualified with different therapeutic transgenes to be applicable in a clinical setting. Nonetheless, we find that qualitative statements on contaminations are already feasible. Since the overall share of contaminant sequences likely also depends on the transgene size and sequence, these findings should not be generalized to other AAV vector preparations with different transgenes. Concerning the length thresholding of reads for the BLAST
+analysis, we find that a threshold of >1000 nt represents a good trade-off between depth of analysis, computing time and the emergence of double-fragmented sequences.
+
+## Molecular State Of Packaged Aav Genomes And Itr Correction
+
+To further characterize AAV packaged DNA, a %GC versus read length plot proved to be a very convenient visualization for our nanopore data, as both parameters are computable quantities for uniquely fragmented sequences.
+
+When we plotted individual BLAST bins, a diverse picture emerged. First, read length histogram analyses allowed us to estimate that more than 99% of the rAAV genome sequences are of the expected size. Even though each read represents a fragmented genome, we find this conclusion is feasible, because both strands are equally likely packaged and independently sequenced, so that the non-strand-specific coverage in total is roughly constant along the genome length (evident in Figure 1E). A simulation confirmed that we obtained mostly unique fragmentations, albeit with an observable data shift, probably due to incomplete sequencing of the 3 ITR.
+
+Second, further analysis showed that genome head-totail fusions are packaged in the capsid to a larger extent
+(Figure 2B). The postulated primary mechanism for AAV
+genome replication suggests resolution of head-to-head and tail-to-tail fusions as rolling hairpin replication intermediates (46,52), but we never observed such fusions. Head-totail fusions on the other hand could be the result of rolling circle replication, which requires a circular template. Circularized AAV genomes have long been described to occur in vivo as monomers or as head-to-tail fusions (53,54). Further studies showed that these circular forms have a single junction ITR with a 5 and a 3 D-sequence (55) and that ssDNA genomes, which are subsequently packaged, can be replicated from this form by rolling circle replication (56). Compliant with those findings, fusions observed by us also had two D-sequences at the junction ITR. A further implication of the rolling circle replication model as a reservoir for ssDNA genomes is that the 3 ITR sequence has to be repaired from the 5 ITR as a template prior to genome packaging (56). This process can correct defective ITR sequences and ITR correction was observed even before circular intermediates were discovered (57). In our recombinant system, the plasmid carrying the GOI (pITR) encodes one ITR with a 11 bp deletion seen also by other groups (6), however we find now that this deletion is mostly absent in the ITRs of packaged genomes (except in the junction ITRs of genome-backbone fusions), which again hints on packaging from rolling circle amplified genomes. Whilst the ITR
+'correction' mechanism in theory has equal probability to yield two ITRs with the deletion on the one hand and two correct ITRs on the other hand, it is not clear where the preference for correct ITRs in coming from. Given that fusions have correct ITRs hints on ITR correction as a preliminary requirement prior to genome amplification.
+
+Notably, the mapping quality in the junction ITRs is low and it is not clear, whether this is a result of sequencing artifacts due to the strong secondary structures or due to error-prone recombination at this position. Given that the mapping quality always drops 3 of the B-C hairpins, independent of the genome orientation, the former explanation seems likely, although error prone circularization has also been observed (53). Possible explanations for the combined findings of dominantly monomer genomes on the one hand and the observation of head-to-tail fusions on the other hand are 3-fold: either (i) rolling circle replication rather than rolling hairpin replication might be the predominant replication scheme in our setting, possibly enforced by the erroneous ITR, or (ii) after gene correction through rolling circle replication, replication could also proceed mostly by rolling hairpin replication, provided that genome monomer excision is more efficient in this replication scheme. Equally possible is that (iii) observed fusions occur because their respective rolling circle templates harbour erroneous ITRs due to erroneous recombination and the resulting concatemers cannot be resolved to monomers following genome amplification, whereas the majority of genomes stem from templates with correct ITRs, where resolution is efficient. Without further investigation, for example on cellular rAAV unpackaged DNA, no definite conclusion is possible.
+
+## Packaging Of Pitr Backbone And Other Contaminant Sequences
+
+We also observed AAV genome-plasmid backbone fusions with identity to the pITR plasmid. In these, the junction ITR still harbored the 11 nt deletion and we observed a higher mapping quality throughout the junction. It is possible that nanopore sequencing is facilitated by ITRs with the deletion, which does not form as-strong secondary structures as wild type ITRs. These fusions furthermore terminated mostly in an ITR and we observed them in both polarities (GOI located 3 and backbone located 3), so that we suggest that they might also be actively replicated and not packaged directly from the producer plasmid. Observations of solely bacterial backbone sequences with 3 terminal ITRs within the pITR bin add to this suggestion.
+
+ITRs were found also 3 terminal of rep and cap as well as adenoviral helper sequences providing direct confirmation of previously observed non-homologous recombination between ITRs and helper sequences (49,58) and warrants efforts for their elimination (59), although the overall quantity of these reads was low in our setting.
+
+In contrast, human genomic sequences did not harbour 3 ITR sequences. Instead, they were found to be randomly packaged and of exponentially decaying size distribution, which hints at the involvement of enzymatic digestion. This raises the question, where these fragments originate from.
+
+We find it unlikely that the host cell genome is highly fragmented during virus production, however, host cell DNA
+is treated with Benzonase nuclease during virus purification. We wonder whether residual helicase activity of the AAV replicases during Benzonase treatment of producer cell lysate is responsible for randomly packaged host cell DNA. Presumably, host cell DNA could be randomly packaged from free ends during purification and is then cut at random timepoints at the capsid surface, yielding the decaying size distribution. Work towards a specific replicase inhibitor that can be added during virus purification might be a chance to further improve vector quality.
+
+In conclusion, we present here unprecedented deep nanopore sequencing of packaged ssDNA in recombinant AAV with the possibility to expand the application range to other single-stranded viruses or bacteriophages, as demonstrated for bacteriophage M13 circular ssDNA. The technique dramatically simplifies sample preparation and reduces turnover times compared to other NGS characterization methods for ssDNA. In addition, the information content of the result increases due to long reads and direct sequencing. This allowed us to find direct conformation for several fundamental research discoveries on AAV biology including AAV genome primary and secondary packaging signals, genome orientation, the genome's replication scheme, SNVs and recombination events. Furthermore, direct sequencing allowed for qualitative statements on DNA
+contaminations in AAV vector stocks. The present study highlights the necessity to further understand the AAV basic biology to gain high-transducing vectors with homogeneous payloads for gene therapy applications. Analytical procedures must keep pace with the development of new therapeutics and we foresee that quantitative PCR will lose its status as the gold standard, as unbiased NGS protocols become cheaper, easier and readily available.
+
+## Data Availability
+
+Custom Python scripts are available in the GitHub repository (https://github.com/MarkusHaak/
+Radukic Brandt 2020).
+
+Sequencing data have been deposited with the Sequence Read Archive (SRA) under accession number PRJNA610225.
+
+## Supplementary Data
+
+Supplementary Data are available at NARGAB Online.
+
+## Funding
+
+European Commission [685778 to D.B.]; Funding for open access charge: Deutsche Forschungsgemeinschaft and the Open Access Publication Fund of Bielefeld University.
+
+Conflict of interest statement. None declared.
+
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medical/md/PMC8160180.md

@@ -0,0 +1,132 @@
+ **Viewpoint**
+
+![0_image_0.png](0_image_0.png)
+
+# Rethinking Innovation And The Role Of Stakeholder Engagement In Sport And Exercise **Medicine**
+
+To cite: Hendricks S. 
+
+Rethinking innovation and the role of stakeholder engagement in sport and exercise medicine. *BMJ Open* Sport & Exercise Medicine 2021;7:e001009. doi:10.1136/
+bmjsem-2020-001009 Sharief Hendricks 1,2,3
+
+## Abstract
+
+In sport and exercise medicine, increasing pressure to improve athlete health outcomes and performance with limited resources has prompted an emphasis on innovation. A key component to innovation is stakeholder participation and engagement, that is, the involvement of those affected by the outcomes, such as end users and actors (the person(s) 
+performing the required actions/behaviour change), of the research process. Several research frameworks in sport and exercise medicine highly recommend stakeholder engagement as part of the research process. There are, however, different levels to how engaged a stakeholder can be in a research project, and this level of engagement may be dependent on the researchers' goals. Stakeholder engagement can be organised on a continuum based on the stakeholder's relationship to the research and how involved they are in the project's decision-making process. This continuum can be used as a rating scale to evaluate and monitor the degree of perceived stakeholder participation in a research project. There are different paths to innovation in research, which are interlinked, and ideas and knowledge flow between them. Considering the continuum of stakeholder engagement and paths to innovation, this article highlights how different research types require different degrees of stakeholder engagement.
+
+## What Is Already Known
+
+► Innovation is a broad concept that can be defined as the successful implementation of a novel idea that creates value for some or all its stakeholders.
+
+► In sport and exercise medicine, increasing pressure to improve athlete health outcomes and performance with limited resources has prompted an emphasis on innovation.
+
+► A key component of innovation is stakeholder participation and engagement.
+
+## What Are The New Findings
+
+► Stakeholder engagement can be organised on a continuum based on the stakeholder's relationship to the research and how involved they are in the project's decision-making process.
+
+► This continuum can be used as a rating scale to evaluate and monitor the degree of perceived stakeholder participation in research projects.
+
+► There are different paths to innovation in research, which are interlinked, and ideas and knowledge flow between them.
+
+► Different types of research for innovation require different degrees of stakeholder engagement.
+
+© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
+
+1Division of Exercise Science and Sports Medicine, Department of Human Biology, University of Cape Town, Rondebosch, South Africa 2Carnegie Applied Rugby Research (CARR) centre, Institute for Sport, Physical Actvity and Leisure, Leeds Beckett University, Leeds, UK
+3Health, Physical Activity, Lifestyle and Sport Research Centre, University of Cape Town, Cape Town, South Africa Correspondence to Dr Sharief Hendricks; sharief.hendricks01@gmail.com
+
+## Introduction
+
+Innovation is a broad concept that can be defined as the successful implementation of a novel idea that creates value for some or all its stakeholders.1 In sport and exercise medicine, increasing pressure to improve athlete health outcomes and performance with limited resources has prompted an emphasis on innovation.2 3 A key component of innovation is stakeholder participation and engagement, that is, the involvement of those affected by the outcomes, such as end users and actors (the person(s) performing the required actions/
+behaviour change), of the research process.4 Arguably, by involving stakeholders in the research process, research objectives will be more aligned to the stakeholders' needs and context, thereby increasing the likelihood of successful implementation.5 6 For this paper, the term *stakeholders* may include patients, athletes, target populations, practitioners, clinicians, policy makers or administrators, and a *research outcome*(s) is any finding(s), programme, product, practice or technology that benefits stakeholders and, ultimately, the health, welfare and performance of the athlete(s) or target population. In sport and exercise medicine, several research frameworks highly recommend stakeholder engagement as part of the research process.
+
+5–8 For example, in 2014, Verhagen et al. described the knowledge transfer scheme to help bridge the gap between science and practice.5 A key step in applying this framework is establishing a group of stakeholders 
+(knowledge transfer group) to identify the problem, discuss the available evidence and develop the product/programme.5 There are, however, different levels to how engaged a stakeholder can be in a research project, and this level of engagement may be dependent on the researchers' goals. The aims of this viewpoint are to (1) 
+describe the different levels of stakeholder engagement and offer potential ways to improve it; (2) describe the different paths to innovation; then, (3) considering the different levels of stakeholder engagement and paths to innovation, argue why the degree of stakeholder engagement depends on the type of research and its purpose.
+
+## Levels Of Stakeholder Engagement
+
+Stakeholder engagement can be organised on a continuum based on the stakeholder's relationship to the research and how involved they are in the project's decision-making process.9–15 For the lowest level of engagement on the continuum, stakeholders' relationship to the research is 'on' or 'for', and their involvement is doing what the research project requires 
+(ie, researchers make all the decisions). For the highest level, stakeholders' relationship to the research is 'by', and they are involved in all the decision-making on the project. This level of engagement is modelled on community development and empowerment, and described as the 'bottom-up' or the people-centred/patient-centred approach.12 14 16 Midway between the two extreme levels of engagement is a moderate level of engagement where stakeholders' relationship to research is 'with' and collaborative in nature. At the moderate level of engagement, stakeholders contribute to the decision-making, although researchers still make the final call.
+
+## Assessing Stakeholder Engagement
+
+![1_Image_0.Png](1_Image_0.Png)
+
+Hendricks13 and others12 15 have applied this continuum of stakeholder engagement to a 5-point rating scale which can be used to evaluate and monitor the degree of perceived stakeholder participation of research projects (figure 1). The rating scale offers a simple and practical measure to characterise the nature of stakeholder engagement within a project and has been used considerably in community health research.12 13 15 Using the rating scale, stakeholders can provide their perceived level of engagement at different stages of the research process. For example, after a knowledge transfer group meeting to identify the problem,5 each stakeholder can rate their level of engagement in the problem identification process. If a particular stakeholder rated their engagement as low, the group could address it in the next meeting. In the ideal scenario, the person monitoring the engagement should be external to the project and should collect the perceived ratings of both the researcher(s) and stakeholders. The ratings could be from their perspective and that of others in the working group, which can be used to cross-check the consistency of the ratings. In cases where the researcher is also the practitioner,17 that is, where the researcher can also be considered a stakeholder that may potentially benefit from the research outcome(s), the researcher–practitioner will rate their engagement based on their primary role on the research project. At the end of the project, ratings can also be related to the research outcomes and for reporting purposes. For example, British Medical Journal (BMJ) journals, like BMJ Open *Sport and Exercise* Science, require authors to include a 'patient and public involvement statement' within their manuscript Method section. Instead of a statement, BMJ can use the aforementioned rating scale to ask authors to either rate their patients and public (stakeholders) engagement themselves or to submit stakeholder engagement ratings that were collected during the research project. This will help BMJ standardise the information and allow them to monitor the level of stakeholder engagement for all their submissions.
+
+## Paths To Innovation
+
+There are different paths to innovation in research, and all these paths are interlinked. Fundamentally, research is inspired and driven to achieve two outcomes—improved 
+
+![2_image_0.png](2_image_0.png)
+
+understanding of 'how it works' (basic research) and usefulness ('Can we use it in practice?') (applied research).18 The outcomes are not mutually exclusive, and a study can aim to improve our understanding of a subject and consider its usability in practice. Studies of this nature, that is, with both aims in mind, are called use-inspired basic research. To illustrate the relationship between these types of research, Stokes placed them in a 2×2 table—what is now famously known as Pasteur's quadrant (figure 2).18 The quadrant was for heuristic purposes only; however, to connect the cells, the literature has erroneously indicated the direction in which knowledge flows between them.19 Stokes reveals the directional and dynamic flow of ideas and knowledge between research types and outcomes 
+(figure 3).18 The model shows how basic research may improve our current understanding of a particular topic or subject without considering its application. In sport and exercise medicine, for example, basic research may be studying skeletal muscle adaptation. Likewise, applied research may improve practice without improving our understanding. In sport and exercise medicine, this may be, for example, studying the effectiveness and implementation of a training programme to reduce the risk of tackle injuries in rugby. However, basic research and applied research can strongly influence each other in either direction, with use-inspired basic research playing the linking role.18 With that said, use-inspired research may also independently increase our fundamental understanding or produce real-world applications. Staying with our example, if we understand the physiology behind how skeletal muscle adapts to different stimuli, we can design and develop better training programmes. Use-inspired 
+
+![2_image_1.png](2_image_1.png)
+
+research may include exploratory studies, efficacy studies and experiential studies. As such, it is more likely to be systematic in its approach, apply traditional study designs and have study-specific outcome variables. Because of this systematic approach and design, the value of use-inspired research, especially to practice, is not immediately appreciated. From an innovation perspective, though, unlike applied research that may be constrained to context and time, use-inspired research has the freedom to explore and experiment. Research that aims for both understanding and usability may lead to a paradigm shift in our thinking of a particular topic, discover a new direction of research or increase the potential for a breakthrough innovation.
+
+## Why Stakeholder Engagement Does Not Have To Be 'High' For A Project To Be Innovative
+
+Considering the continuum of engagement levels in figure 1 and the dynamical model for knowledge and innovation in figure 3, this articles highlights that the different types of research require different degrees of stakeholder engagement to achieve the desired research outcomes (figure 4). In other words, the level of engagement on a project depends on the type of research and its purpose. For applied research to be successful, stakeholder engagement needs to be high; context needs to be understood, along with implementation barriers/facilitators. For use-inspired research, researchers work with the stakeholders, and the project is collaborative. While stakeholders still provide valuable input, the researcher makes the final decisions on the study design and outcome measurements. For basic research, stakeholder engagement may still occur, but this will be at a very low level, for example, obtaining participant samples during testing. It is also worth noting that the innovation produced from each research type may also benefit the practice of the research itself across the different research types. For example, the outcome of use-inspired research may be a tool that accurately measures tackling technique—this outcome will improve practice, but at the same time, it can also be used to test players for research studies.
+
+## Conclusion
+
+There is increasing pressure to improve athlete health outcomes and performance with limited resources. This has prompted an emphasis on innovation. A key component to innovation in health research is stakeholder participation and engagement, that is, the involvement of end users and actors in the research process who may be affected by the research outcomes. In sport and exercise medicine, several research frameworks highly recommend stakeholder engagement as part of the research process. Stakeholder engagement can be organised on a continuum based on the stakeholder's relationship to the research and how involved they are in the project's decision-making process. This continuum can be used as a rating scale to evaluate and monitor the degree of perceived stakeholder participation in research projects. 
+
+![3_image_0.png](3_image_0.png)
+
+Figure 4 Dynamic model for innovation and stakeholder engagement.
+
+There are different paths to innovation in research, which are interlinked, and ideas and knowledge flow between them. Considering the continuum of stakeholder engagement and paths to innovation, this article highlights how different research types require different degrees of stakeholder engagement.
+
+Twitter Sharief Hendricks @Sharief_H Contributors SH is the sole author of the article. Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. Competing interests None declared. Patient consent for publication Not required. Provenance and peer review Not commissioned; externally peer reviewed. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iD Sharief Hendricks http://orcid.org/0000-0002-3416-6266
+
+## References
+
+1 Varkey P, Horne A, Bennet KE. Innovation in health care: a primer. 
+
+Am J Med Qual 2008;23:382–8.
+
+2 Ramírez-López C, Till K, Boyd A, *et al*. Coopetition: cooperation among competitors to enhance applied research and drive innovation in elite sport. *Br J Sports Med* 2020;55:522–3.
+
+3 Speed CA, Roberts WO. Innovation in high-performance sports medicine. *Br J Sports Med* 2011;45:949–51.
+
+4 Bowen DJ, Hyams T, Goodman M, *et al*. Systematic review of quantitative measures of Stakeholder engagement. *Clin Transl Sci* 2017;10:314–36.
+
+5 Verhagen E, Voogt N, Bruinsma A, *et al*. A knowledge transfer scheme to bridge the gap between science and practice: an integration of existing research frameworks into a tool for practice. Br J Sports Med 2014;48:698–701.
+
+6 Finch CF, Talpey S, Bradshaw A, *et al*. Research priorities of international sporting federations and the IOC research centres. *BMJ* 
+Open Sport Exerc Med 2016;2:e000168.
+
+7 Owoeye OBA, Rauvola RS, Brownson RC. Dissemination and implementation research in sports and exercise medicine and sports physical therapy: translating evidence to practice and policy. *BMJ* Open Sport Exerc Med 2020;6:e000974.
+
+8 Ahmed OH, Defoe J, West LR, *et al*. Creating the dream team: 
+introducing participatory sports and exercise medicine via 'Patient Voices'. *Br J Sports Med* 2018;52:1547–8.
+
+9 Arnstein S. A ladder of participation in the USA. *J R Town Plan Inst* 1971;16:176–82.
+
+10 Cornwall A, De Koning K. Towards participatory practice: 
+participatory rural appraisal (PRA) and the participatory process. In: De Koning K, Marion M, eds. *Participatory research in health*. London: Zed Books Ltd, 1996.
+
+11 Rifkin SB, Lewando-Hundt G, Draper A. Participatory approaches in health promotion and health planning: a literature review. London: Health Development Agency, 2000.
+
+12 Draper AK, Hewitt G, Rifkin S. Chasing the dragon: developing indicators for the assessment of community participation in health programmes. *Soc Sci Med* 2010;71:1102–9.
+
+13 Hendricks S, Conrad N, Douglas TS, *et al*. A modified stakeholder participation assessment framework for design thinking in health innovation. *Healthc* 2018;6:191–6.
+
+14 Rifkin SB. Paradigms lost: toward a new understanding of community participation in health programmes. *Acta Trop* 1996;61:79–92.
+
+15 Rifkin SB, Muller F, Bichmann W. Primary health care: on measuring participation. *Soc Sci Med* 1988;26:931–40.
+
+16 Morgan LM. Community participation in health: perpetual allure, persistent challenge. *Health Policy Plan* 2001;16:221–30.
+
+17 Jones B, Till K, Emmonds S, *et al*. Accessing off-field brains in sport; an applied research model to develop practice. *Br J Sports Med* 2019;53:791–3.
+
+18 Stokes DE. Pasteur's quadrant: Basic science and technological innovation. Washington DC: Brookings Institution Press, 1997.
+
+19 Klahr D. Learning sciences research and Pasteur's quadrant. J Learn Sci 2019;28:153–9.

medical/md/PMC8183208.md

@@ -0,0 +1,226 @@
+# Review
+
+![0_Image_0.Png](0_Image_0.Png)
+
+To cite: Ohrndorf S, Glimm A-M, Ammitzbøll-Danielsen M, 
+et al. Fluorescence optical imaging: ready for prime time?. *RMD Open* 2021;7:e001497. doi:10.1136/ rmdopen-2020-001497
+► Additional online supplemental material is published online only. To view, please visit the journal online (http://dx.doi.org/10.1136/ rmdopen-2020-001497). Received 26 February 2021 Accepted 22 May 2021 © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
+
+1Department of Rheumatology and Clinical Immunology, Charité - Universitätsmedizin Berlin, Campus Mitte, Humboldt - Universität zu Berlin, Freie Universität Berlin, Berlin, Germany 2Copenhagen Center for Arthritis Research, Center for Rheumatology and Spine Diseases, Rigshospitalet, Copenhagen, Denmark 3Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark Correspondence to Dr Gerd R Burmester; gerd.burmester@charite.de
+
+# Fluorescence Optical Imaging: Ready For Prime **Time?**
+
+Sarah Ohrndorf ,1 Anne-Marie Glimm,1 Mads Ammitzbøll-Danielsen ,2,3 Mikkel Ostergaard,2,3 Gerd R Burmester 1
+
+## Abstract
+
+The novel technique of fluorescence optical imaging (FOI, Xiralite), which is approved in the European Union and the USA for clinical use, has been the object of studies since 2009. Indocyanine green-based FOI can demonstrate an impaired microcirculation caused by inflammation in both hands in one examination. Several studies have investigated FOI for detection of joint inflammation by comparing FOI to magnetic resonance imaging (MRI) and/ or musculoskeletal ultrasound (MSUS). The results have shown a generally good agreement (>80%) between FOI and clinical examination, MRI and MSUS by power Doppler in inflammatory joint diseases. Moreover, characteristic enhancements in skin and nails are seen in PsA, which potentially can be useful in the diagnostic process of early undifferentiated arthritis. Furthermore, FOI has been investigated for the visualisation of a disturbed microcirculation in the hands and fingers of patients with systemic sclerosis (SSc), highlighting the potential of monitoring vascular changes in SSc and other vasculopathies. The available data indicate that it is time to consider FOI as a useful part of the imaging repertoire in rheumatology clinical practice, particularly where MSUS and MRI are not easily available.
+
+## Introduction
+
+With new therapeutic options for treating rheumatic inflammatory joint diseases to target, early diagnostic procedures have become increasingly important. In modern rheumatology, the process of establishing an early diagnosis may be greatly helped by imaging procedures. Thus, magnetic resonance imaging (MRI) and musculoskeletal ultrasound (MSUS) with their advantages such as valid detection of inflammation with high sensitivity and good interreader reliability and disadvantages such as costs, contraindications and dependence on the examiner are already established procedures. In recent years, novel imaging techniques have also been developed including the new technique of fluorescence optical imaging (FOI, Xiralite) that has been the object of studies since 2009, and is approved in the European Union and the USA for clinical use.
+
+## Key Messages
+
+► The novel indocyanine green-based fluorescence optical imaging (FOI) is a fast, easy, and welltolerated method to visualize a disturbed microcirculation in both hands.
+
+► FOI demonstrates an enhanced microcirculation in the joints of both hands due to inflammation caused by various rheumatic joint diseases.
+
+► Characteristic enhancements in skin and nails of both hands are visualized by FOI, especially in psoriatic arthritis.
+
+► FOI presents a reduced microcirculation in systemic sclerosis, which is associated with digital ulcer development.
+
+This comprehensive review will focus on FOI and address the question if it is ready for clinical use. We believe that FOI can complement our current imaging repertoire.
+
+## How Does Foi Work?
+
+The basis of FOI in rheumatology is the visualisation of an impaired microcirculation in the joints of both hands, which is caused by an inflammatory process due to vasodilatation, hypervascularisation, increased capillary permeability and neoangiogenesis. Following intravenous application of indocyanine green (ICG) as fluorescence-optical dye, disturbed microcirculation in the hands is visualised via a connected software. The principle of ICG 
+usage was extensively investigated by Cherrick *et al*.
+
+1 Today, it is used in ophthalmology, cardiology and gastroenterology and was there demonstrated to be a well-known and well-tolerated dye. A disadvantage of FOI is the invasiveness of the examination because of the need of an intravenous access. In addition, kidney and liver function should usually be within normal limits and hyperthyroidism must be excluded.
+
+The intensity of the dye concentration is displayed via a false-colour grading: white as a very high concentration and intensity 
+
+## Rmd Open
+
+followed by red, yellow and green in descending order. 
+
+The examination lasts 6min recording one image per second and adding up to a total cluster of 360 images.
+
+Advantages of this method are the high acceptance of the patients and the possibility to examine all joints of both hands and wrists (30 joints in total) within the described short period of time of 6min. FOI is a delegable task that can be performed by a medical assistant or nurse with a physician in the background. Accordingly, the images can be evaluated at remote sides. The acquisition costs of an FOI device are comparable with a high-end ultrasound machine and less expensive than an MRI machine.
+
+Efforts have been made by different research groups to standardise the FOI image analysis. The comparison of three scoring methods from Berlin, Stockholm and Copenhagen showed moderate to good inter-reader reliability for all three FOI scoring methods (Pabak-OS: 
+0.50–0.78, ICC: 0.43–0.85) used in patients with erosive hand osteoarthritis (OA) and rheumatoid arthritis (RA) with similar sensitivities (63%–65%) and specificities 
+(76%–91%) compared with MRI.2 Mostly, the 'Berlin scoring method' is used including three phases dependent on the blood flow of the fingertips and the automatically generated sum image (PrimaVistaMode, PVM). The evaluation of the inflammatory intensity in the phases 1–3 and PVM is performed by a semiquantitative (0–3) 
+score called FOIAS (FOI Activity Score).2–13 Recently, the 'Copenhagen scoring method' FOIE-GRAS (FOI Enhancement-Generated RA Score) has been published, which presents a feasible, reliable and responsive single score for synovitis assessment in RA.14
+
+## Preclinical And Animal Studies On Foi
+
+Fischer *et al* evaluated in vivo fluorescence imaging of experimental inflammatory joint disease in a model of Borrelia-induced Lyme arthritis presenting that the fluorescence signal differed significantly between controls and arthritic animals (p<0.05).15 Meier *et al* evaluated a combined X-ray/optical imaging system for ICGenhanced detection of arthritic joints in a rat model of antigen induced arthritis of the knee and ankle, which were compared for significant differences with controls. 
+
+The fluorescence signal of arthritic joints was significantly higher compared with the non-arthritic control joints (p<0.05).16 Moreover, Vollmer *et al* described an in vivo near-infrared (NIR) fluorescence imaging technique for therapy monitoring of ankle joints affected by collagen-induced arthritis before and after treatment demonstrating a statistically significant decrease in fluorescence intensity in ankle joints (p<0.05).17
+
+## What Is The Current Evidence Of Foi In Rheumatology?
+
+Different research groups all over the world have examined FOI in numerous studies in the last 10 years for different rheumatologic diseases and indications; please see online supplemental table 1 in which full papers 
+
+![1_image_0.png](1_image_0.png)
+
+(congress abstracts excluded) are listed according to their order in the text.
+
+## Inflammatory Joint Diseases
+
+Most notably, the validity studies in patients with inflammatory rheumatoid joint diseases have shown a mostly good agreement between FOI, clinical examination (CE), MSUS and MRI.
+
+Fischer *et al* presented first clinical data on five patients with RA and a corresponding number of volunteers that were examined using fluorescence imaging in the NIR 
+spectral range following the intravenous administration of an unspecific contrast agent with ICG. 0.2 Tesla 
+(T) MRI served as the reference method. In this study, inflammatory joints of RA patients were enriched by ICG and showed a different kinetic behaviour compared with normal joints presenting the capability of contrastenhanced fluorescence imaging to detect early inflammatory changes with good correlation to MRI (r=0.84).18 In a subsequent study including 252 patients with different forms of arthritis, Werner *et al* demonstrated a sensitivity of 76% and a specificity of 54% for FOI in detecting joint inflammation (synovitis/tenosynovitis)—compared with MRI. Here, FOI in phase 1 had a high specificity of 94%. Furthermore, FOI showed agreement rates up to 88% vs CE, 64% vs MSUS in grey scale, 88% vs MSUS in power Doppler and 83% vs MRI, depending on the compared phase and parameter.3 In another study by Werner *et al* including 32 patients with early and very early arthritis as well as 46 controls, the sensitivity of FOI was 86% and the specificity was 63%, while the sum image (PVM), phase 1 and phase 3 reached high specificities of 87%, 90% and 88%, respectively, compared with MRI.4 In contrast, Meier et al reported that FOI had a sensitivity of only 39.6% and a specificity of 85.2%, compared with 3.0T MRI. In this study, 45 patients with clinically suspected inflammatory 
+
+![2_image_0.png](2_image_0.png)
+
+arthropathy were included. Referring to the reduced sensitivity, the authors stated that FOI showed limitations for the detection of inflamed joints of the hand in comparison with MRI.19 In contrast, Krohn *et al* examined 31 early RA patients via FOI—compared with MSUS in grey scale and power Doppler as well as 0.31T MRI. The working group presented an overall sensitivity/specificity for FOI of 81%/0%, 49%/84% and 86%/38% for wrist, metacarpophalangeal (MCP) and proximal interphalangeal (PIP) joints, respectively, in comparison to MRI, and an overall sensitivity/specificity for FOI of 88%/15%, 81%/76% and 100%/27% for wrist, MCP and PIP joints, respectively—compared with power Doppler US. FOI in phase 2 was the most sensitive phase, while phase 1 showed the highest specificity,5 comparable to the results by Werner *et al.*3 4 An FOI image example of an active, early RA patient is given by figure 1. In addition, Schäfer et al analysed 90 individual joints from 18 patients with 
+
+![2_image_1.png](2_image_1.png)
+
+active RA via FOI and found a sensitivity of 26/39 (67%) and a specificity of 31/40 (77%) for fluorescence ratio of phase 3 using a cut-off value of more than 1.2 to detect 1.5T MRI-confirmed synovitis with FOI. They concluded that FOI has a potential for visualising synovitis in subjects with RA.20 Subclinical synovitis as clinically non-apparent but visible pathology in MSUS could also be detected by FOI with a sensitivity of 80% and specificity of 96%. Thus, Kisten *et al* suggested the potential use of FOI for the screening of synovitis and clinically inactive inflammatory joint disease (see figure 2 as an FOI example of a clinically inactive (DAS28 <2.6) RA patient), especially when MSUS is not available.21 Thuermel *et al* examined 20 patients with highly active RA and 13 healthy volunteers by CE, FOI and contrast-enhanced 3.0T-MRI and 
+
+![2_image_3.png](2_image_3.png)
+
+![2_image_2.png](2_image_2.png)
+
+![3_image_0.png](3_image_0.png)
+
+reported an overall sensitivity of 57.3% and specificity of 92.1% of FOI versus MRI for the detection of synovitis. In addition, they demonstrated that the sensitivity of FOI increased with the degree of synovitis to 65.0% for moderate and severe synovitis (specificity 88.1%) and 76.3% for severe synovitis (specificity 80.5%), while FOI's performance decreased with the degree of synovitis with false negative results predominantly for mild (45.5%) and moderate (46.6%) synovitis and false positive results predominantly based on mild (grade 1) signals (81.6%). Accordingly, the authors concluded that FOI has a lower sensitivity than 3.0T-MRI and its diagnostic performance decreases with the degree of synovitis correspondent to the strength of FOI signals.22 Hirano *et al* compared performance profiles of CE, MSUS and FOI using 1.5T MRI as a reference in six active RA patients. FOI showed sensitivities and specificities of 85% and of 94% (phase 
+
+![3_image_1.png](3_image_1.png)
+
+1), and 69% and 94% (phase 2) in the detection of synovitis, which were comparable to those of MSUS and more specific than CE. They concluded that FOI has a potential as an assessment modality of RA.12 An FOI image example of a longstanding, active RA is given by figure 3. Recently, Kawashiri *et al* explored the significance of the FOI findings based on the association between the FOI and MSUS 
+findings and serum biomarkers (including vascular endothelial growth factor) in 50 consecutive patients with active RA. They presented FOI to be highly sensitive in the detection of synovitis, however, the frequency of positive findings and the diagnostic performance with MSUS as the reference standard for FOI differed considerably among the phases of FOI as well as among the affected joint regions. Furthermore, the FOI scores were positively correlated with clinical disease activity, MSUS scores and serum biomarkers. Moreover, the severity of FOI-proven synovitis was associated with the presence of MSUS-proven bone erosion.13 Recently, AmmitzbøllDanielsen *et al* presented a moderate agreement of FOI 
+(using the FOIE-GRAS method; figure 4) with ultrasound (ICC 0.30–0.54) for total score and moderate correlation with clinical joint assessment and Disease activity score 28 
+(DAS28)-CRP in RA patients .14 In a study on patients with suspected or confirmed psoriatic arthritis (PsA) published by Erdmann-Keding et al, FOI was more sensitive than MSUS for the detection of inflammation in PIP/distal interphalangeal (DIP) 
+joints (p=0.035) and different findings of pathologic enhancement for confirmed (pathologic signal enhancement mainly in FOI phase 2 + phase 3) and suspected PsA 
+(pathologic signal enhancement mainly in FOI phase 1) were found, which may be relevant for determining the inflammatory status.9 An FOI image in a patient with active PsA is demonstrated in figure 5.
+
+Apart from the inflammatory joint changes, Schmidt et al investigated inflammatory enhancements in the skin area of both hands that were observed in patients with psoriasis vulgaris (PsO) and PsA compared with RA patients and healthy controls in a retrospective analysis. Subclinical skin enhancement on the back of the hands was more common in PsO/PsA (72.5%) than in RA patients (20.5%) and healthy individuals (28.0%) (p<0.001). Based on the FOI pattern, most patients with PsO/PsA (72.5%), RA (76.9%), and healthy controls (68.0%) were classified correctly, which indicate FOI's potential to study microcirculation in rheumatic diseases with skin involvement.23 An FOI image example of a patient with active PsA (dactylitis) and skin enhancement is given by figure 6. Moreover, the 'green nail' phenomenon has been described by Wiemann *et al* as a possible microcirculation disturbance in the area of the nail with a specificity of 87% for PsA in relation to an RA control cohort.24 Werner *et al* had described a triangular, slightly arcuate enhancement from the nail bed into the DIP that was observed in 60 out of 64 (94%) subjects with PsA 
+compared with only 8 out of 38 (21%) patients with definite RA.3
+
+## Osteoarthritis
+
+In a subsequent study by Glimm *et al*, the amount and distribution of inflammatory signs in wrist and finger joints via FOI and MSUS was compared in RA vs OA patients. FOI and MSUS detected inflammation in both RA and OA highlighting the inflammatory component in the course of OA. The authors assumed that the different inflammatory patterns and various shapes of fluorescence enhancement may offer opportunities to distinguish both diseases via FOI.6 An FOI image example in a patient with OA is given by figure 7. In contrast, a study by Maugesten *et al* of 221 hand OA patients showed limited correlations between FOI, MRI and MSUS, 
+questioning the value of FOI for the detection of synovitis in hand OA.7 In another publication by Maugesten et al it was investigated whether FOI enhancement and MRI-defined synovitis were associated with pain and physical function in hand OA. The authors found that FOI enhancement and MRI-defined synovitis were associated with pain in the same finger joint (with numerical stronger associations between MRI-defined synovitis and finger joint pain/tenderness). Nevertheless, none of the imaging modalities demonstrated consistent associations with pain, stiffness and physical function on subject level concluding that FOI enhancement was probably associated with more noise than established imaging modalities (MRI and MSUS), furthermore FOI did not show enhancement in the thumb base joints, which are commonly affected by OA.8
+
+## Juvenile Idiopathic Arthritis
+
+FOI can also be used in children, as the dosage of the dye ICG can be adapted to the patients' weight. Apart from the particularly good tolerability, interestingly, the children investigated accepted the procedure and showed interest in this method due to the colourful presentation.
+
+In paediatric patients with inflammatory (juvenile idiopathic arthritis, JIA) and non-inflammatory joint diseases, FOI was more sensitive for detecting clinically active joints than MSUS in grey scale and power Doppler (GSUS/
+PDUS) (75.2% vs 57.3%/32.5%), which was presented by Beck *et al.* The predictive value in this study for discrimination between inflammatory and non-inflammatory joint diseases was 0.79 for FOI and 0.80/0.85 for GSUS/PDUS. 
+
+The authors concluded that FOI may provide an additional diagnostic method in paediatric rheumatology.11 Klein *et al* presented a 24-week observational study in polyarticular JIA in which 37 patients were evaluated clinically, by MSUS (GS/PD) and FOI at baseline, week 12 and week 24. Twenty-four patients started therapy with methotrexate and 13 patients with a biological agent for the first time (etanercept n=11, adalimumab and tocilizumab n=1each). Improvement on treatment with either methotrexate or a biologic therapy could be visualised by FOI and MSUS. Both methods could detect clinical but also subclinical inflammation, which was to a higher extent visualised by FOI than MSUS.25 An FOI image example in a patient with JIA is given by figure 8.
+
+## Therapeutic Monitoring
+
+In addition to the diagnostic studies on joint diseases mentioned above, studies on therapeutic monitoring were conducted. Among these, Glimm *et al* included 35 patients with early RA that were followed-up within 1year after baseline by FOI both in responders and nonresponders according to EULAR response criteria by DAS28. A significant signal reduction in FOI phase 1 was observed, so that an objective evaluation of the therapeutic success appears possible via FOI.10 Moreover, Meier *et al* demonstrated a significant difference in the rates of early enhancement in FOI—as quantitative measurement—after 24 weeks of therapy start or escalation in different inflammatory arthritis patients. They stated a signal reduction in responders (−21.5%, p<0.001) and increase in non-responders (+10.8%, p=0.075) underlining the possibility of a therapy monitoring per FOI.26 In a recent study by Ammitzbøll-Danielsen *et al* a good sensitivity to change (standardised response mean>0.80) 
+was found.14
+
+## Systemic Sclerosis
+
+The FOI procedure was also applied to patients with systemic sclerosis (SSc) to detect soft tissue inflammation by ICG enhancement in 19 segments per hand, which was shown to be significantly reduced 7days after either iloprost or alprostadil therapy from 40.9% to 24.7% presented by Pfeil *et al*.
+
+27 Furthermore, FOI was investigated for the visualisation of a disturbed microcirculation in the hands and fingers of SSc patients and to link FOI findings to clinical signs of ischaemia such as digital ulcers (DU) and pitting scars, reflecting progressive vasculopathy in SSc. In the results presented by Friedrich et al, 93.6% of healthy subjects showed initial ICG signals in their fingertips compared with only 78.5% in limited SSc and 43.2% in diffuse SSc. Moreover, FOI findings of missing or reduced microcirculation were significantly 
+
+## Rmd Open
+
+associated with a late capillaroscopic pattern, disseminated SSc features, a diffuse SSc subtype and the presence of digital ulcers or pitting scars in SSc patients.28 In the 12 months follow-up study investigating 76 SSc patients, Friedrich *et al* could show that fingers with pathological staining by FOI at baseline had a higher risk for new ulcer development in the same finger (p=0.0153). Especially, a missing signal of FOI in the right third digit at baseline was associated with the subsequent development of DU.29 Recently, this group has introduced a composite score 
+(CIP-DUS-Clinical features, Imaging, Patient historyDigital Ulcer Score) to predict DU in SSc patients also including FOI, which was able to identify all patients at risk of digital ulcers throughout 12 months.30
+
+## Summary And Conclusion
+
+Several studies have been published for the use of FOI 
+in the detection of joint inflammation comparing the performance of FOI to MRI and/or MSUS. The results presented high sensitivities and specificities for FOI in phase 1 and phase 3 by all studies using FOIAS in inflammatory joint diseases.3–5 9 12 13 Thus, phase-specific evaluation by the FOIAS method with particular attention to phase 1 (highest specificity compared with both power Doppler US and MRI) is strongly recommended. 
+
+This was confirmed by Glimm *et al* who found an acceptable responsiveness in phase 1 for RA patients (<2years disease duration) initiating or escalating antirheumatic treatment.10 Recently, this was confirmed in a new study using FOIE-GRAS (a peak enhancement score) demonstrating a good responsiveness, which was in line with MSUS scores, joint evaluation and DAS28-CRP.14 These data support that FOI is an alternative for monitoring of RA patients in clinical trials and practice.
+
+The value of FOI in patients with OA might be limited since nearly no FOI enhancement in the thumb base joints could be detected. Furthermore, FOI did not demonstrate consistent associations with pain, stiffness and physical function on subject level.8 However, FOI 
+seems to offer opportunities to distinguish OA vs RA, therefore, it might become a useful tool in differential diagnosis.6 Moreover, inflammatory enhancements in the skin area of both hands were detected by FOI indicating its potential to study microcirculation in rheumatic diseases with skin involvement.22 This and other microcirculation disturbances ('green nail sign' etc) need to be confirmed by prospective studies on larger scales, but they give first hints that FOI has the potential to present both skin and joint involvement at the same time underlining its potential in patients with PsA for monitoring, for example, objective ('real') psoriatic skin and joint disease stages.
+
+In patients with SSc, a reduced microcirculation in the hands and fingers could be presented by FOI. Furthermore, it could be shown that fingers with pathologic staining detected by FOI at baseline had a higher risk for new ulcer development in the same finger during 12 months. In a composite score called CIP-DUS also including FOI-examination, all SSc patients at risk of digital ulcers throughout 12 months could be predicted. 
+
+Therefore, FOI also has the potential to be used in the detection of reduced microcirculation in soft tissue diseases.
+
+An advantage of FOI is that a scan of all finger and hand joints is performed within a short time period of 6min. Furthermore, the procedure can be delegated to a trained nurse—in the presence of a doctor in the background. Due to the weight-dependent application of the dye it is possible to perform FOI also in children.11 24 The procedure can be repeated within few hours due to a short half-life of ICG. A disadvantage is the necessary of an intravenous access making FOI an invasive method, but apart from this is a painless method. Also, the use of ICG rarely causes allergic reactions. Thousands of examinations have shown that it is well-tolerated and well-accepted procedure by the patients. We have experienced that patients follow the examination on the screen attentively and that the images enable them to visually understand the disease and to discuss response and non-response to therapy. Limitations of FOI is that the palmar aspects of the hands (eg, flexor tenosynovitis) can only be partly examined due to the limited penetration of infrared light, and that the method provides a twodimensional visualisation of the hand in contrast to MRI 
+and ultrasound. Furthermore, important pitfalls of this method need to be known (eg, wounds located over the joint), which should be limited by a distinct documentation (eg, photographs of both hands).
+
+To summarise, FOI is a fast, easy, and well-tolerated method to visualise a disturbed microcirculation in various rheumatic diseases, and thereby allows for the detection of joint inflammation, vasculopathic changes and skin involvement of the hands.
+
+Therefore, we believe that FOI is a feasible and reliable method which is ready for clinical practice to complement our current imaging repertoire in rheumatology, particularly at sites where US and MRI are not readily available.
+
+## Twitter Mads Ammitzbøll-Danielsen @Madsammitzboll
+
+Acknowledgements We would like to acknowledge the study nurse Gabriela Schmittat for logistical and technical assistance (eg, image acquisition). Contributors SO, A-MG and GRB contributed substantially to the conception of the review. SO, A-MG and GRB drafted the first version of the review. SO, A-MG, MA-D, MO and GRB revised the article for important intellectual content. All of the named authors drafted the review and revised it for important intellectual content. All authors gave final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Funding One of the authors (SO) was funded by the 'Advanced Clinician Scientist program' of the German Society of Internal Medicine (Deutsche Gesellschaft für Innere Medizin). Competing interests None declared. Patient consent for publication Not required. Provenance and peer review Commissioned; externally peer reviewed.
+
+Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iDs Sarah Ohrndorf http://orcid.org/0000-0001-5943-4688 Mads Ammitzbøll-Danielsen http://orcid.org/0000-0003-4878-0432 Gerd R Burmester http://orcid.org/0000-0001-7518-1131
+
+## References
+
+1 Cherrick GR, Stein SW, Leevy CM, *et al*. Indocyanine green: 
+observations on its physical properties, plasma decay, and hepatic extraction. *J Clin Invest* 1960;39:592–600.
+
+2 Maugesten Ø., Ohrndorf S, Glinatsi D, *et al*. Evaluation of three scoring methods for fluorescence optical imaging in erosive hand osteoarthritis and rheumatoid arthritis. *Osteoarthr Cartil Open* 2020;1:100017.
+
+3 Werner SG, Langer H-E, Ohrndorf S, *et al*. Inflammation assessment in patients with arthritis using a novel in vivo fluorescence optical imaging technology. *Ann Rheum Dis* 2012;71:504–10.
+
+4 Werner SG, Langer H-E, Schott P, *et al*. Indocyanine greenenhanced fluorescence optical imaging in patients with early and very early arthritis: a comparative study with magnetic resonance imaging. *Arthritis Rheum* 2013;65:3036–44.
+
+5 Krohn M, Ohrndorf S, Werner SG, *et al*. Near-Infrared fluorescence optical imaging in early rheumatoid arthritis: a comparison to magnetic resonance imaging and ultrasonography. *J Rheumatol* 2015;42:1112–8.
+
+6 Glimm A-M, Werner SG, Burmester GR, *et al*. Analysis of distribution and severity of inflammation in patients with osteoarthitis compared to rheumatoid arthritis by ICG-enhanced fluorescence optical imaging and musculoskeletal ultrasound: a pilot study. Ann Rheum Dis 2016;75:566–70.
+
+7 Maugesten Øystein, Mathiessen A, Hammer HB, *et al*. Validity and diagnostic performance of fluorescence optical imaging measuring synovitis in hand osteoarthritis: baseline results from the Nor-Hand cohort. *Arthritis Res Ther* 2020;22:98.
+
+8 Maugesten Øystein, Ohrndorf S, Slatkowsky-Christensen B, 
+et al. Associations between fluorescence optical imaging and magnetic resonance imaging and symptoms in hand osteoarthritis. Rheumatology 2021:keab085.
+
+9 Erdmann-Keding M, Ohrndorf S, Werner SG, *et al*. Fluorescence optical imaging for the detection of potential psoriatic arthritis in comparison to musculoskeletal ultrasound. *J Dtsch Dermatol Ges* 2019;17:913–21.
+
+10 Glimm A-M, Sprenger LI, Haugen IK, *et al*. Fluorescence optical imaging for treatment monitoring in patients with early and active rheumatoid arthritis in a 1-year follow-up period. *Arthritis Res Ther* 2019;21:209.
+
+11 Beck MC, Glimm A-M, Ohrndorf S, *et al*. Fluorescence optical imaging in pediatric patients with inflammatory and non-inflammatory joint diseases: a comparative study with ultrasonography. *Arthritis Res Ther* 2017;19:233.
+
+12 Hirano F, Yokoyama-Kokuryo W, Yamazaki H, *et al*. Comparison of fluorescence optical imaging, ultrasonography and clinical examination with magnetic resonance imaging as a reference in active rheumatoid arthritis patients. *Immunol Med* 2018;41:75–81.
+
+13 Kawashiri SY, Nishino A, Shimizu T, *et al*. Fluorescence optical imaging in patients with active rheumatoid arthritis: a comparison with ultrasound and an association with biomarkers. Scand J Rheumatol 2020;21:1–9.
+
+14 Ammitzbøll-Danielsen M, Glinatsi D, Terslev L, *et al*. A Novel Fluorescence Optical Imaging Scoring System for Hand Synovitis in Rheumatoid Arthritis - validity and agreement with ultrasound. Rheumatology 2021:keab377.
+
+15 Fischer T, Gemeinhardt I, Wagner S, *et al*. Assessment of unspecific near-infrared dyes in laser-induced fluorescence imaging of experimental arthritis. *Acad Radiol* 2006;13:4–13.
+
+16 Meier R, Krug C, Golovko D. ICG-enhanced imaging of arthritis with an integrated optical Imaging/X-ray system. *Arthritis Rheum* 2010;62:2322–7.
+
+17 Vollmer S, Gemeinhardt I, Vater A, *et al*. In vivo therapy monitoring of experimental rheumatoid arthritis in rats using near-infrared fluorescence imaging. *J Biomed Opt* 2014;19:036011–7.
+
+18 Fischer T, Ebert B, Voigt J, *et al*. Detection of rheumatoid arthritis using non-specific contrast enhanced fluorescence imaging. Acad Radiol 2010;17:375–81.
+
+19 Meier R, Thürmel K, Moog P, *et al*. Detection of synovitis in the hands of patients with rheumatologic disorders: diagnostic performance of optical imaging in comparison with magnetic resonance imaging. *Arthritis Rheum* 2012;64:2489–98.
+
+20 Schäfer VS, Hartung W, Hoffstetter P, *et al*. Quantitative assessment of synovitis in patients with rheumatoid arthritis using fluorescence optical imaging. *Arthritis Res Ther* 2013;15:R124.
+
+21 Kisten Y, Györi N, Af Klint E, *et al*. Detection of clinically manifest and silent synovitis in the hands and wrists by fluorescence optical imaging. *RMD Open* 2015;1:e000106.
+
+22 Thuermel K, Neumann J, Jungmann PM, *et al*. Fluorescence optical imaging and 3T-MRI for detection of synovitis in patients with rheumatoid arthritis in comparison to a composite standard of reference. *Eur J Radiol* 2017;90:6–13.
+
+23 Schmidt A, Glimm AM, Haugen IK, *et al*. Detection of subclinical skin manifestation in patients with psoriasis and psoriatic arthritis by fluorescence optical imaging. *Arthritis Res Ther* 2020;22:192.
+
+24 Wiemann O, Werner SG, Langer H-E, *et al*. The "green nail" phenomenon in ICG-enhanced fluorescence optical imaging - a potential tool for the differential diagnosis of psoriatic arthritis. J Dtsch Dermatol Ges 2019;17:138–47.
+
+25 Klein A, Just GW, Werner SG, *et al*. Fluorescence optical imaging and musculoskeletal ultrasonography in juvenile idiopathic polyarticular disease before and during antirheumatic treatment - a multicenter non-interventional diagnostic evaluation. *Arthritis Res* Ther 2017;19:147.
+
+26 Meier R, Thuermel K, Noël PB, *et al*. Synovitis in patients with early inflammatory arthritis monitored with quantitative analysis of dynamic contrast-enhanced optical imaging and MR imaging. Radiology 2014;270:176–85.
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+27 Pfeil A, Drummer KF, Böttcher J, *et al*. The application of fluorescence optical imaging in systemic sclerosis. *Biomed Res Int* 2015;2015:1–6.
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+28 Friedrich S, Lüders S, Werner SG, *et al*. Disturbed microcirculation in the hands of patients with systemic sclerosis detected by fluorescence optical imaging: a pilot study. *Arthritis Res Ther* 2017;19:87.
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+29 Friedrich S, Lüders S, Glimm AM, *et al*. Association between baseline clinical and imaging findings and the development of digital ulcers in patients with systemic sclerosis. *Arthritis Res Ther* 2019;21:96.
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medical/md/PMC8556492.md

@@ -0,0 +1,83 @@
+# Endovascular Treatment Of A Giant Infected Ascending Aortic Pseudoaneurysm With Occlusion Device And Coil Embolization
+
+Matthias De Boulle, MD,a Bert Vandeloo, MD,a Eric Eeckhout, MD, PhD,b and Stijn Lochy, MD,a Brussels, Belgium; and Lausanne, Switzerland
+
+## Abstract
+
+A patient with recurrent sepsis caused by an infected ascending aortic pseudoaneurysm was deemed unsuitable for surgery after the heart team evaluation. He successfully underwent percutaneous treatment with a combination of a septal occlusion device and coil embolization and remained free of sepsis 24 months after implantation. (J Vasc Surg Cases Innov Tech 2021;7:706-9.)
+Keywords: Ascending aorta; High surgical risk; Sepsis; Structural cardiac interventions Ascending aortic pseudoaneurysm is a rare pathology that can occur as a consequence of aortic injury caused by cardiac surgery, inflammatory processes, including infection, or blunt trauma.1 The clinical presentation can vary from asymptomatic to a potentially fatal rupture.
+
+The treatment consists of open surgery or an endovascular approach.2 In the present report, we have described the case of a patient with recurrent sepsis caused by an infected ascending aortic pseudoaneurysm, who successfully underwent percutaneous treatment with a combination of a septal occlusion device and coil embolization.
+
+The patient provided written informed consent for the report of his case details and imaging studies.
+
+## Case Report
+
+A 68-year-old man was admitted to our hospital with recurrent diabetic wound infections of his left lower limb, resulting in methicillin-resistant Staphylococcus aureus sepsis. He had an extensive cardiac history, with coronary artery bypass grafting in 1990 and multiple percutaneous coronary interventions afterward. He had type 2 diabetes mellitus, with severe micro- and macrovascular complications, including multiple vascular interventions on both lower limbs.
+
+He required several treatments with antibiotics. However, after surgical attempts for source control, all vascular prosthesis material was finally resected from his left groin, and his left upper leg was amputated. 18F-fluorodeoxyglucose positron emission tomography/computed tomography (CT) scans had also shown tracer uptake at the level of the aortic root and anterior mediastinum but without signs of abscess formation (Fig 1, A and B).
+
+Transesophageal echocardiography (TEE) repeatedly showed no signs of endocarditis.
+
+Eleven months after the previous scan, a repeat 18F-fluorodeoxyglucose positron emission tomography/CT scan showed increasing tracer uptake in the anterior ascending aortic wall, with a new cavity anterior of the ascending aorta (Fig 1, C and D). TEE confirmed the presence of a large cavity (Fig 2; Supplementary Video, online only). The cavity (ie, sac) was in direct communication with the aortic lumen through an irregularly shaped defect (ie, the neck) in the aortic wall. Based on these findings, the diagnosis of an infected ascending aortic pseudoaneurysm was confirmed. Coronary angiography revealed the close proximity of the pseudoaneurysm to the proximal anastomosis of the venous coronary bypass but without signs of fistulation from the bypass to the pseudoaneurysm.
+
+After successful antibiotic treatment with 6 weeks of intravenous vancomycin, followed by 12 weeks of oral linezolid, surgical resection of the pseudoaneurysm was discussed by the heart team. However, after the multidisciplinary discussion, the patient's perioperative risk for open aortic surgery was deemed too high. A percutaneous approach was considered.
+
+The procedure was performed with the patient under general anesthesia. A 6 F and 12F sheath was inserted in the left radial and right femoral artery, consecutively. First, the aneurysm was accessed from the radial artery using an Amplatz right 1 diagnostic catheter (Boston Scientific, Marlborough, Mass) and a Terumo 0.035-in. straight guidewire (Terumo, Tokyo, Japan), which was then exchanged for an internal mammary artery guide catheter over an Amplatz Extra-Stiff Straight guidewire
+(Cook Medical, Bloomington, Ind; Fig 3). From the femoral artery, using the same technique, an AMPLATZER TorqVue
+
+![1_image_0.png](1_image_0.png)
+
+180 delivery system (AGA Medical Corp, Golden Valley, Minn)
+was advanced into the pseudoaneurysm cavity. After failure to implant a 20-mm device, a 16-mm AMPLATZER septal occluder device (Abbott Laboratories, Minneapolis, Minn) was successfully deployed (Fig 4; Supplementary Video, online only). Parallel to the occlusion device, coil embolization of the pseudoaneurysm sac was performed through the trapped internal mammary artery catheter. A total of six detachable HydroCoils (AZUR; Terumo) were delivered into the pseudoaneurysm. All were 20 mm in diameter and 20 cm long
+(Supplementary Video, online only). During advancement of the seventh coil, the guide catheter prolapsed out of the sac into the aorta, making the advancement of additional coils into the pseudoaneurysm impossible. Releasing the closure device concluded the procedure. The final aortogram and TEE showed a good position of the closure device, with near complete cessation of flow into the pseudoaneurysm (Fig 5; Supplementary Video, online only).
+
+The patient was discharged from the hospital 10 days after the procedure. No adverse events were observed during the remainder of his hospitalization. A follow-up CT scan after 1 month showed only a minimal amount of contrast visible in the remaining pseudoaneurysm sac. The remainder of the pseudoaneurysm sac had thrombosed.
+
+After the procedure, the patient was prescribed lifelong treatment with doxycycline. During a follow-up period of 24 months, he remained free of any systemic infections, in particular methicillin-resistant S. aureus sepsis.
+
+## Discussion
+
+Aortic pseudoaneurysm is defined as a dilation of the aorta due to disruption of all wall layers that is only contained by the periaortic connective tissue. In patients with ascending aortic pseudoaneurysms, surgical intervention is indicated, independent of aneurysm size. The choice of treatment is usually determined by the anatomic features, clinical presentation, and comorbidities.2 Certain factors increase the complication risk for open surgery, including active infection, prior cardiac surgery, and proximity of the pseudoaneurysm with the posterior sternal wall.3 Our patient had an elevated surgical risk owing to active infection, prior cardiac surgery, and multiple comorbidities. In addition, the pseudoaneurysm was in close proximity to the sternum.
+
+![2_image_0.png](2_image_0.png)
+
+![2_image_1.png](2_image_1.png)
+
+![2_image_2.png](2_image_2.png)
+
+In high-risk settings, a minimally invasive approach using endovascular grafts or atrial septal occlusion devices can potentially be an alternative. Several percutaneous approaches have been reported. However, the combination of coil embolization with an occlusion device has only been described a few times.3,4 This combination seems to provide an additive prothrombotic effect, helping to achieve stasis within the pseudoaneurysm sac.
+
+The use of percutaneous occlusion devices in the setting of pseudoaneurysms is not without limitations. An important prerequisite for successful device placement is the De  et al 709
+
+![3_image_0.png](3_image_0.png)
+
+Volume 7, Number 4 presence of a narrow neck between the pseudoaneurysm and the aorta. This will ensure adequate sealing of the pseudoaneurysm neck on deployment of the device.3,4 Interference of the device disc with the aortic valve should be prevented during implantation. The feasibility of additional coil placement is also dependent on the pseudoaneurysm neck diameter, which should be large enough to allow placement of a catheter alongside the occlusion device. Furthermore, coil deployment can also be limited by the sac size, which should be large enough to accommodate the coils.4 The favorable anatomy of the pseudoaneurysm in the present patient made placement of both an occlusion device and coils possible.
+
+Deploying too many coils should be avoided, because they can push the occlusion device out of the pseudoaneurysm sac.3 Coil advancement can also cause the catheter to prolapse out of the sac into the aorta. This has been described in other case series and also occurred in our patient.4 Ideally, the source of infection (ie, the pseudoaneurysm)
+would have been completely resected surgically to minimize the risk of future infections. Because complete removal was not possible in our patient, we combined endovascular exclusion of the pseudoaneurysm with the use of long-term suppressive antibiotic therapy. With this approach, which has been described for other cases,5 the patient remained free of sepsis during the follow-up period.
+
+## Conclusions
+
+In high-risk patients with large, infected aortic pseudoaneurysms, who are unsuitable for open aortic surgery, percutaneous endovascular treatment with occlusion devices and coil embolization is feasible, considering certain technical aspects and limitations. In terms of safety and efficiency, long-term favorable outcomes are potentially achievable.
+
+## References
+
+1. Iacovelli F, Spione F, Contegiacomo G, Pepe M, Bortone AS,
+Pestrichella V. Percutaneous exclusion of ascending aorta pseudoaneurysms: still an interventional challenge? J Cardiol Cases 2020;21:
+130-3.
+
+2. Erbel R, Aboyans V, Boileau C, Bossone E, Bartolomeo RD,
+Eggebrecht H, et al. 2014 ESC guidelines on the diagnosis and treatment of aortic diseases: document covering acute and chronic aortic diseases of the thoracic and abdominal aorta of the adult. The Task Force for the Diagnosis and Treatment of Aortic Diseases of the European Society of Cardiology (ESC). Eur Heart J 2014;35:2873-926.
+
+3. Stamou SC, Conway BD, Nores MA. Management of aortic pseudoaneurysms: evolving concepts and controversies. Aorta (Stamford)
+2020;8:1-5.
+
+4. Lyen SM, Rodrigues JCL, Manghat NE, Hamilton MCK, Turner M.
+
+Endovascular closure of thoracic aortic pseudoaneurysms: a combined device occlusion and coil embolization technique in patients unsuitable for surgery or stenting. Catheter Cardiovasc Interv 2016;88: 1155-69.
+
+5. Sedivy P, Spacek M, El Samman K, Belohlavek O, Mach T, Jindrak V,
+et al. Endovascular treatment of infected aortic aneurysms. Eur J Vasc Endovasc Surg 2012;44:385-94.
+
+Submitted Jun 21, 2021; accepted Sep 22, 2021.

medical/md/PMC9118438.md

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+A Randomized Preference Trial Comparing Cognitive-Behavioral Therapy and Yoga for the Treatment of Late-Life Worry:
+Examination of Impact on Depression, Generalized Anxiety, Fatigue, Pain, Social Participation, and Physical Function Global Advances in Health and Medicine Volume 11: 1–10
+© The Author(s) 2022 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/2164957X221100405 journals.sagepub.com/home/gam Suzanne C. Danhauer, PhD1, Michael E. Miller, PhD2, Jasmin Divers, PhD3, Andrea Anderson, MS4, Gena Hargis, MPH5, and Gretchen A. Brenes, PhD5
+
+## Abstract
+
+Background: Depression, generalized anxiety, fatigue, diminished physical function, reduced social participation, and pain are common for many older adults and negatively impact quality of life. The purpose of the overall trial was to compare the effects of cognitive-behavioral therapy (CBT) and yoga on late-life worry, anxiety, and sleep; and examine preference and selection effects on these outcomes.
+
+Objective: The present analyses compared effects of the 2 interventions on additional outcomes (depressive symptoms, generalized anxiety symptoms, fatigue, pain interference/intensity, physical function, social participation); and examined whether there are preference and selection effects for these treatments.
+
+Methods: A randomized preference trial of CBT and yoga was conducted in adults ≥60 years who scored ≥26 on the Penn State Worry Questionnaire-Abbreviated (PSWQ-A), recruited from outpatient medical clinics, mailings, and advertisements.
+
+Cognitive-behavioral therapy consisted of 10 weekly telephone sessions. Yoga consisted of 20 bi-weekly group yoga classes.
+
+Participants were randomized to(1): a randomized controlled trial (RCT) of CBT or yoga (n = 250); or (2) a preference trial in which they selected their treatment (CBT or yoga; n = 250). Outcomes were measured at baseline and post-intervention.
+
+Results: Within the RCT, there were significant between-group differences for both pain interference and intensity. The pain interference score improved more for the CBT group compared with the yoga group [intervention effect of (mean (95% CI) =
+2.5 (.5, 4.6), P = .02]. For the pain intensity score, the intervention effect also favored CBT over yoga [.7 (.2, 1.3), P < .01]. Depressive symptoms, generalized anxiety, and fatigue showed clinically meaningful within-group changes in both groups. There were no changes in or difference between physical function or social participation for either group. No preference or selection effects were found.
+
+Conclusion: Both CBT and yoga may be useful for older adults for improving psychological symptoms and fatigue. Cognitivebehavioral therapy may offer even greater benefit than yoga for decreasing pain.
+
+1Department of Social Sciences and Health Policy, Wake Forest School of Medicine, Winston-Salem, NC, USA
+2Division of Public Health Sciences, Wake Forest School of Medicine, Winston-Salem, NC, USA
+3Division of Health Services Research, NYU Long Island School of Medicine, Mineola, NY, USA 4Department of Biostatistics and Data Science, Wake Forest School of Medicine, Winston-Salem, NC, USA 5Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA
+Corresponding Author: Suzanne C. Danhauer, PhD, Department of Social Sciences & Health Policy, Wake Forest School of Medicine, Division of Public Health Sciences Medical Center Blvd. Winston-Salem, NC 27157, USA. Email: danhauer@wakehealth.edu
+
+## Keywords
+
+yoga, cognitive behavioral therapy, anxiety, depression, fatigue, older adults Received November 19, 2021; Revised March 14, 2022. Accepted for publication April 26, 2022
+
+## Introduction
+
+Worry, a cognitive component of anxiety, is common in older adults.1,2 While cognitive-behavioral therapy (CBT) is the most studied non-pharmacological treatment for late-life anxiety/worry and has a strong evidence base,3-5 there is growing interest in and evidence for the use of yoga to reduce anxiety in older adults.6,7 To our knowledge, this study was the first to compare yoga and CBT for the treatment of latelife worry and a variety of related symptoms in a large sample of older adults. Findings from previously published analyses of primary and secondary outcomes showed that CBT and yoga were both effective at reducing worry and anxiety, and a greater impact was seen for CBT compared with yoga for improving sleep.8 The focus of this investigation is on a variety of prevalent and interrelated symptoms commonly reported by older adults with substantial worry: depressive symptoms, generalized anxiety symptoms, fatigue, and pain.
+
+Comorbid depressive symptoms are common in older adults who report anxiety/worry.9-13 High levels of worry are also associated with physical health consequences, including fatigue and pain.17,18 Fatigue becomes more prevalent with increased age.19 Fatigue is a complex and multi-dimensional symptom and has negative implications for quality of life and physical performance (including frailty).20-23 Similarly, pain is a significant issue for older adults and has been associated with worry/anxiety symptoms in multiple studies. In older adults, chronic pain is common and is associated with substantial suffering, isolation, disability, risk of falls, and greater health care costs.24-30 Recent work highlights the relationship between greater pain and worry in older adults.31,32 In addition to treating psychological symptoms such as worry/
+anxiety and depression, CBT has demonstrated efficacy for managing pain symptoms in older adults.33,34 We conducted a two-stage randomized preference trial comparing CBT and yoga for treating worry in older adults.
+
+This design was chosen because it allows for a test of traditional randomized effects as well as a test of preference effects.
+
+Preference has been found to have an effect on clinical outcomes, adherence, attrition, and stratification.35-37 Preference for treatment is also a key component of shared decisionmaking and best practice standards. Half of participants were randomized to CBT or yoga, while the remainder were randomized to choose their treatment. As reported above, findings for the primary outcome (worry) and secondary outcomes
+(anxiety, sleep) have been published elsewhere.8 In this secondary data analysis using data from the randomized arm (N =
+250), we compared the effects of CBT and yoga on several exploratory outcomes: depressive symptoms, generalized anxiety symptoms, fatigue, pain interference, pain intensity, physical function, and social participation. Using data from both the randomized and preference trial arms (N = 500), we examined the effects of preference (i.e., an effect on outcome from having a choice in treatments compared with being randomized) and selection (i.e., an effect on outcomes by making a specific treatment choice) on depressive symptoms, generalized anxiety symptoms, fatigue, pain interference, pain intensity, physical function, and social participation.
+
+## Methods
+
+This study received approval from the Wake Forest School of Medicine Institutional Review Board. Participants were adults aged ≥ 60 years with elevated self-reported worry scores (≥26)
+on a standardized measure [Penn State Worry QuestionnaireAbbreviated (PSWQ-A)].38,39 Exclusion criteria included the following: current psychotherapy, current yoga practice, current alcohol or substance abuse, dementia diagnosis, cognitive impairment (based on the Telephone Interview of Cognitive StatusModified),40 current psychotic symptoms, active suicidal ideation with plan and intent, change in psychotropic medications in the last 4 weeks, and hearing loss that would prevent participation in study interventions. In a two-step screening process, participants provided verbal informed consent for a brief initial screen and written informed consent for the full screen.
+
+## Procedure
+
+Study procedures have been described in detail previously.41 Participants were recruited through outpatient clinics, flyers, mailings, and newspaper advertisements. Eligible participants were randomized to one of the following 3 conditions:
+randomized controlled trial (RCT) CBT, RCT yoga, or preference trial. Randomization was electronically linked to eligibility based on entry of the baseline forms, and the sequence of random assignments was only available to the statisticians who generated the randomization list and the programmers who implemented the randomization process.
+
+Individuals assigned to the randomized trial were then further randomized into CBT or yoga, stratified by whether they used psychotropic medication. Randomization was completed using a permuted block algorithm with random block lengths over a secure web-based data management system.
+
+## Assessment
+
+All self-reported study assessments (described in detail elsewhere (41)) were completed by telephone, in person, or by mail. The current study presents analyses of exploratory outcomes collected at baseline (Week 0) and following intervention completion (Week 11). As exploratory analyses, nominal P-values are presented and significance for each outcome set at .05.
+
+## Outcomes
+
+PROMIS-29. Constructs from the PROMIS-29 measure included in these analyses were: depressive symptoms (8 items), fatigue (4 items), pain interference (4 items), physical function (11 items), ability to participate in social roles and activities (4 items), and pain intensity (1 item).
+
+Generalized anxiety disorder screener (GAD-7). The GAD-7 is a self-report measure of DSM-IV generalized anxiety symptoms that has been validated for use in the general population.42,43
+
+## Interventions
+
+Cognitive-behavioral therapy participants completed up to 10 weekly 50-minute telephone-based psychotherapy sessions using a standardized workbook (with homework/daily practice). Each session focused on an assigned workbook chapter with standard CBT topics (e.g., relaxation techniques, cognitive restructuring) to manage worry and anxiety.41 Cognitive-behavioral therapy sessions were conducted by 2 Licensed Clinical Social Workers who received weekly supervision from the senior author (GAB).
+
+Yoga participants completed up to 20 biweekly 75-minute yoga classes based on the Relax into Yoga for Seniors gentle Hatha yoga program.44 Classes included centering and breathing, a gentle physical posture practice, and meditation/
+relaxation. Yoga was provided by 7 yoga instructors with at least RYT 200 certification.
+
+## Fidelity
+
+All sessions were audio- or video-recorded; 10% were selected randomly for rating by CBT or yoga experts for protocol adherence and intervention delivery competence. On a 9-point scale, mean therapist ratings were >6 on adherence and competence items.3,45,46 Mean yoga instructor ratings were >6 on adherence and competence items for all but 1 instructor (who stopped teaching study classes prior to retraining).
+
+## Statistical Analyses
+
+Our general approach to analysis of this study has been previously described.8 Baseline descriptive statistics by intervention group and within both the randomized and preference trials were calculated, and t-tests and chi-square tests were used to compare the CBT and yoga preference groups on baseline characteristics.
+
+Within RCT analyses. Constrained, repeated measures analysis of covariance, with an unstructured covariance matrix to account for multiple measurements (Weeks 0 and 11) not being independent, was used to estimate the intervention effect on the exploratory outcomes.47,48 The intervention effect was estimated by comparing mean scores between CBT
+and yoga groups in the RCT.47,48 Pre-specified effects for baseline psychotropic medication use, sex, and race (both related to depression; race dichotomized) and intervention effects specific to each measurement time were contained in this model. A contrast was used to test for intervention effects at Week 11 using a two-sided .05 significance level.
+
+Analyses were performed consistent with an "intent to treat" philosophy (i.e., all randomized participants were included in their allocated groups). Participants who achieved a clinically meaningful change in outcomes from baseline to Week 11 were identified based on thresholds from published literature. Marginal standardization [46] of results from logistic regression (adjusted for baseline psychotropic medication use, sex, and race) was used to estimate the proportion of participants achieving minimally important differences (MIDs) in each intervention group (and risk differences between groups); 95% confidence intervals were calculated using a bootstrap confidence intervals with 1000 replications.
+
+Estimation of preference and selection effects using both the RCT
+and preference study data. Preference and selection effects were estimated for each outcome using a mixed-model repeated measures framework and data collected in both randomized and preference trials.49 Dummy variables were used to represent membership in the randomized and preference groups, intervention assignment in the randomized group, and intervention preference in the preference group. The fitted model was used to estimate the adjusted means and variance-covariance matrix needed to compute these effects and their standard errors. The standard errors associated with the preference and selection effects were derived using formulas provided by Walter and colleagues.50 Statistical significance was assessed at the two-sided, .05 level in these secondary analyses.
+
+## Results
+
+Recruitment of 500 participants took place between May 2017 and November 2018. Intervention completion rates were comparable between the 2 trials [60% RCT, 65%
+preference trial (P = .27)]. Table 1 presents descriptive statistics on baseline demographic characteristics.8 Participants self-reported the following health issues: anxiety (43.8%),
+depression (45.8%), hypertension (46.9%), myocardial infarction (2.8%), congestive heart failure (3%), stroke (5.2%),
+and diabetes (12%). Regarding psychotropic medication use, they reported taking the following: anti-depressants (29.2%), anxiolytics (20%), anti-psychotics (2%), stimulants (2%), and
+
+| Table 1. Baseline Characteristics of Study Participants. Randomized Trial   | Preference Trial   |             |             |             |             |             |             |             |
+|-----------------------------------------------------------------------------|--------------------|-------------|-------------|-------------|-------------|-------------|-------------|-------------|
+| Baseline Characteristicsa                                                   | Overall            | CBT         | Yoga        | Total       | CBT         | Yoga        | P-Value     | Total       |
+| Number of participants                                                      | 500                | 125 (25%)   | 125 (25%)   | 250 (50%)   | 120 (24%)   | 130 (26%)   | 250 (50%)   |             |
+| Age [mean (SD)]                                                             | 66.5 (5.2)         | 66.7 (5.7)  | 66.3 (4.9)  | 66.5 (5.3)  | 67.4 (5.7)  | 65.6 (4.3)  | .004        | 66.5 (5.1)  |
+| Sex Male                                                                    | 67 (13.4%)         | 13 (10.4%)  | 22 (17.6%)  | 35 (14%)    | 15 (12.5%)  | 17 (13.1%)  | .89         | 32 (12.8%)  |
+| Female                                                                      | 433 (86.6%)        | 112 (89.6%) | 103 (82.4%) | 215 (86%)   | 105 (87.5%) | 113 (86.9%) | 218 (87.2%) |             |
+| Race Black or African American                                              | 74 (14.8%)         | 18 (14.4%)  | 20 (16%)    | 38 (15.2%)  | 21 (17.5%)  | 15 (11.5%)  | .39         | 36 (14.4%)  |
+| Caucasian or White                                                          | 394 (78.8%)        | 93 (74.4%)  | 99 (79.2%)  | 192 (76.8%) | 94 (78.3%)  | 108 (83.1%) | 202 (80.8%) |             |
+| Other                                                                       | 32 (6.4%)          | 14 (11.2%)  | 6 (4.8%)    | 20 (8%)     | 5 (4.2%)    | 7 (5.4%)    | 12 (4.8%)   |             |
+| Enough money to meet needs                                                  | 459 (91.8%)        | 119 (95.2%) | 116 (92.8%) | 235 (94%)   | 106 (88.3%) | 118 (90.8%) | .53         | 224 (89.6%) |
+| a Values are N (%) unless otherwise specified.                              |                    |             |             |             |             |             |             |             |
+
+Table 2. Baseline Scores on Outcome Measures.
+
+| Table 2. Baseline Scores on Outcome Measures. Overall   | Randomized Trial   | Preference Trial   |      |     |       |      |     |       |      |
+|---------------------------------------------------------|--------------------|--------------------|------|-----|-------|------|-----|-------|------|
+| Variable                                                | N                  | Mean               | SD   | N   | Mean  | SD   | N   | Mean  | SD   |
+| PROMIS-29 T-scores Depression                           | 500                | 57.09              | 8.36 | 250 | 57.52 | 8.57 | 250 | 56.65 | 8.14 |
+| Physical unction                                        | 498                | 49.17              | 7.76 | 249 | 49.28 | 7.63 | 249 | 49.06 | 7.90 |
+| Fatigue                                                 | 500                | 58.16              | 8.84 | 250 | 58.14 | 8.86 | 250 | 58.18 | 8.83 |
+| Pain interference                                       | 500                | 53.86              | 9.46 | 250 | 53.47 | 9.47 | 250 | 54.25 | 9.45 |
+| Social participation                                    | 500                | 47.45              | 7.91 | 250 | 47.57 | 7.70 | 250 | 47.33 | 8.12 |
+| PROMIS pain intensity                                   | 500                | 3.33               | 2.47 | 250 | 3.14  | 2.51 | 250 | 3.52  | 2.42 |
+| GAD-7                                                   | 500                | 10.69              | 5.00 | 250 | 10.67 | 5.05 | 250 | 10.72 | 4.95 |
+
+sleep medications (14%). As reported in our prior publication, there were no significant differences between participants in the randomized trial and the preference trial for health issues or psychotropic medication use.52 Table 2 presents descriptive statistics on outcome measures at baseline.
+
+## Exploratory Outcomes For The Rct
+
+Primary and secondary study outcomes (worry, anxiety, insomnia) have been reported previously.8 Exploratory outcomes (depression, fatigue, pain interference and intensity, physical function, social participation, and generalized anxiety symptoms) were assessed immediately upon completing the intervention (Week 11; Table 3). Participants randomized to CBT experienced a greater reduction in pain interference and pain intensity at Week 11 compared with those randomized to yoga. For pain interference, Week 11 changes in means from baseline were 2.6 (95% confidence interval, 4.1, 1.0) for CBT participants, compared with
+.1 (95% confidence interval, 1.6, 1.5) for yoga participants, for a Week 11 adjusted overall treatment effect of 2.5
+(95% confidence interval, .5, 4.6), P-value = .02. Similar results were observed with pain intensity, where the adjusted intervention effect was .7 (95% confidence interval, .2, 1.3),
+P-value<.01 at Week 11.
+
+Participants in both the CBT and yoga groups experienced improvements in depressive symptoms, fatigue, generalized anxiety symptoms, and social participation upon completing the intervention. However, there were no significant differences in the amount of improvement between the CBT and yoga groups. Minimal changes were seen for physical function for either intervention group.
+
+## Meaningful Change
+
+To examine whether the magnitude of change seen between baseline and Week 11 scores was meaningful, we examined how changes in outcomes compared with published data on MIDs (Table 4). A meaningful change in depressive symptoms and fatigue from the PROMIS-29 has been defined as ≥3 points.53-55 A large proportion of participants in both the CBT
+and yoga arms of the RCT demonstrated meaningful
+
+| Table 3. Week 11 Results from the Randomized Trial (Exploratory Outcomes)a . CBT   | Yoga              |                   |                     |                |                   |                |              |      |
+|------------------------------------------------------------------------------------|-------------------|-------------------|---------------------|----------------|-------------------|----------------|--------------|------|
+| Adjusted Mean Score                                                                | Change from       |                   |                     |                |                   |                |              |      |
+| N                                                                                  | Effect (95% CI)   | P-Value           |                     |                |                   |                |              |      |
+| (95% CI)                                                                           | Baseline (95% CI) | N                 | Adjusted Mean Score | Change from    |                   |                |              |      |
+| (95% CI)                                                                           | Baseline (95% CI) | Intervention      |                     |                |                   |                |              |      |
+| Outcome PROMIS-29 T-scores Depression                                              | 101               | 52.3 (50.3, 54.4) 6.2 (7.8, 4.6) 106                   | 52.8 (50.8, 54.8)   | 5.8 (7.3, 4.2)                | .5 (1.6, 2.6)                   | .65            |              |      |
+| Physical function                                                                  | 100               | 48.8 (47.2, 50.5) | 1.0 (.1, 2.1)                     | 105            | 47.6 (46.0, 49.2) | .2 (1.2, .9)                | 1.2 (2.6, .3)              | .11  |
+| Fatigue                                                                            | 101               | 53.6 (51.5, 55.7) 5.2 (6.8, 3.5) 106                   | 54.6 (52.6, 56.7)   | 4.1 (5.7, 2.5)                | 1.0 (1.2, 3.3)                   | .35            |              |      |
+| Pain interference                                                                  | 101               | 52.5 (50.4, 54.6) 2.6 (4.1, 1.0) 106                   | 55.0 (53.0, 57.0)   | .1 (1.6, 1.5)                | 2.5 (.5, 4.6)     | .016           |              |      |
+| Social participation                                                               | 100               | 50.6 (48.7, 52.5) | 4.2 (2.6, 5.7)      | 105            | 49.3 (47.4, 51.1) | 2.8 (1.3, 4.3) | 1.3 (3.4, .7)              | .20  |
+| PROMIS pain                                                                        | 101               | 3.3 (2.7, 3.8)    | .4 (.8, .0)                     | 106            | 4.0 (3.5, 4.5)    | .3 (.0, .7)                | .7 (.2, 1.3) | .005 |
+| intensity                                                                          |                   |                   |                     |                |                   |                |              |      |
+| GAD-7                                                                              | 101               | 6.0 (5.0, 7.0)    | 5.2 (6.1, 4.4) 106                     | 6.7 (5.7, 7.6) | 4.5 (5.4, 3.7)                   | .7 (.4, 1.7)                | .20          |      |
+| a All models are adjusted for sex, race, and baseline psychotropic medication use. |                   |                   |                     |                |                   |                |              |      |
+
+Table 4. Minimally Important Differences (MID) for Exploratory Outcomes: Logistic Regression Results Modelling the Probability of Reaching the MID1.
+
+| Predicted Probability Expressed as Percentage (95% CI)              |                   |                   |                   |            |
+|---------------------------------------------------------------------|-------------------|-------------------|-------------------|------------|
+| Outcome                                                             | Mid               | CBT               | Yoga              | Difference |
+| PROMIS-29 T-scores Depression                                       | ≥3 point decrease | 65.4 (55.7, 75.0) | 59.4 (49.6, 68.9) | 6.0 (7.1, 18.6)            |
+| Physical function                                                   | ≥4 point increase | 19.7 (12.5, 28.4) | 23.1 (15.1, 32.3) | 3.4 (15, 7.8)            |
+| Fatigue                                                             | ≥3 point decrease | 55.4 (44.9, 64.8) | 55.7 (46.3, 65.6) | .3 (15.0, 12.4)            |
+| Pain interference                                                   | ≥3 point decrease | 43.6 (33.4, 54.5) | 32.0 (23.4, 41.3) | 11.6 (2.9, 25.3)            |
+| Social participation                                                | n/a               | n/a               | n/a               | n/a        |
+| PROMIS pain intensity                                               | ≥2 point decrease | 23.0 (14.7, 32.2) | 16.8 (10.2, 24.3) | 6.1 (5.2, 17.3)            |
+| GAD-7                                                               | ≥3 point decrease | 74.1 (65.3, 82.0) | 65.2 (55.5, 74.2) | 8.9 (3.7, 21.8)            |
+| 1 Adjusted for baseline psychotropic medication use, sex, and race. |                   |                   |                   |            |
+
+reduction in depressive symptoms (65.4% and 59.4%, respectively; treatment effect and 95% CI: 6.0 (7.1, 18.6)).
+
+Just over half of participants in both the CBT and yoga arms of the RCT demonstrated meaningful decrease in fatigue (55.4% and 55.7%, respectively; treatment effect and 95%
+CI: .3 (15.0, 12.4)). Similarly, a meaningful change in generalized anxiety symptoms from the GAD-7 has been defined as ≥3 points.56 Most participants in both the CBT and yoga arms of the RCT demonstrated meaningful change in decreased generalized anxiety symptoms (74.1% and 65.2%,
+respectively; treatment effect and 95% CI: 8.9 (3.7, 21.8)).
+
+A meaningful change has been defined as a 4-point change in physical function (PROMIS-29),55 a 3-point decrease (and more conservatively, a 4-point decrease) in pain interference t-scores (PROMIS-29),53,55,57 and a 2-point decrease in pain intensity rating (PROMIS-29)58 from baseline to postintervention. While the average changes from baseline for physical function, pain interference, and pain intensity (as shown in Table 3) were smaller than their MIDs, some participants did achieve meaningful changes in these areas. No information on meaningful change in social participation was available in the literature. Our sample appears to be within normal limits for social participation (t-score from the PROMIS-29 of 45-80 is within normal limits9 as the adjusted mean scores for social participation in this sample were 50.6 for CBT and 49.3 for yoga.
+
+## Preference And Selection Effects
+
+Preference and selection effects were estimated by combining randomized and preference trial data (Table 5). There were no statistically significant preference and selection effects for any of the exploratory outcomes.
+
+## Discussion
+
+We examined the effects of CBT and yoga on depressive symptoms, generalized anxiety symptoms, fatigue, pain
+(interference and intensity), physical function, and social participation in a sample of community-dwelling older adults who reported significant levels of worry. Findings from these analyses showed that depressive symptoms, generalized
+
+| Table 5. Preference and Selection Effects for Exploratory Outcomes. Preference   | Selection       |         |                 |         |
+|----------------------------------------------------------------------------------|-----------------|---------|-----------------|---------|
+| Outcome                                                                          | Effect (95% CI) | P-Value | Effect (95% CI) | P-Value |
+| PROMIS-29 T-scores Depression                                                    | .2 (5.5, 5.0)                 | .93     | 1.8 (3.5, 7.0)                 | .51     |
+| Physical function                                                                | 1.1 (5.2, 2.9)                 | .59     | .6 (3.4, 4.7)                 | .77     |
+| Fatigue                                                                          | 2.2 (7.6, 3.3)                 | .44     | 1.2 (4.3, 6.7)                 | .67     |
+| Pain interference                                                                | 1.0 (4.4, 6.4)                 | .71     | .0 (5.3, 5.4)                 | .99     |
+| Social participation                                                             | 2.4 (7.5, 2.6)                 | .34     | 2.2 (7.2, 2.8)                 | .40     |
+| PROMIS pain intensity                                                            | .3 (1.7, 1.1)                 | .71     | .0 (1.4, 1.4)                 | .98     |
+| GAD-7                                                                            | .8 (3.8, 2.3)                 | .63     | .6 (2.5, 3.6)                 | .71     |
+
+anxiety symptoms, fatigue, and social participation were improved in both intervention groups upon completion of the intervention with no statistically significant between-group differences. Results showed greater reductions in pain (both interference and intensity) favoring participants randomized to CBT vs yoga. Little change was noted in physical function for either intervention group. Our findings support the use of both CBT and yoga for improving depressive symptoms, generalized anxiety symptoms, fatigue, and social participation in older adults. For those reporting issues with pain, significantly greater declines in both pain interference and pain intensity were seen for those in the CBT intervention group (compared with yoga).
+
+There are strong data to show that CBT benefits the outcomes of interest examined here, and our results are consistent with prior findings.3,59,60 Yoga has demonstrated efficacy for anxiety reduction; however, little yoga research has focused specifically on older adults, especially those with high levels of worry.61-69 To our knowledge, there have been no other comparative effectiveness studies of CBT and yoga for improving worry and related outcomes in older adults.
+
+Our findings demonstrate that yoga is effective in reducing some of the additional symptoms associated with worry. This finding is important because older adults prefer nonpharmacological treatments (primarily psychotherapy) over medication for anxiety and depression.70,71 Prior to this study, no one examined relative preference for yoga for the treatment of anxiety. Analyses of data from the current study suggest that older adults do not differ in their preference for psychotherapy or yoga.72 This finding in conjunction with those reported in the current study suggest that yoga is an efficacious and well-liked intervention for late-life worry and associated symptoms.
+
+Interestingly, when the randomized and preference trial data were combined, no preference or selection effects were seen for any of the outcomes in these analyses. These findings were surprising as one would expect that being given a choice of interventions rather than being randomized (preference)
+would have a beneficial effect on study outcomes. It had no effect. One might also expect that choosing one's preferred intervention (selection) would impact study outcomes. It did not. We can conclude from these findings that the opportunity to select CBT or yoga and which of those behavioral interventions is selected will not impact the effect of the intervention on depressive symptoms, generalized anxiety symptoms, fatigue, physical function, social participation, and pain. Older adults and their health care providers can confidently recommend either CBT or yoga, as they have shown similar efficacy whether chosen or prescribed.
+
+Perhaps the most relevant question is whether the change observed in these outcomes is at a level that is meaningful to participants. Beyond statistical significance, were the changes we observed in the various exploratory outcomes in the randomized trial enough to make a difference in their daily lives? As we compared average improvements in exploratory outcomes with published data on MIDs, we found meaningful levels of change for the majority of participants in both intervention groups for depressive symptoms, fatigue, and generalized anxiety symptoms. While the average changes from baseline were less than the MID for physical function, pain interference, and pain intensity for either intervention group, some participants did achieve meaningful change in these areas as well. It is notable that while participants were not recruited into this study based on level of pain at baseline, we saw meaningful levels of change for pain interference for a substantial minority of RCT participants, and to a somewhat lesser extent for pain intensity.
+
+While no prior work has compared CBT and yoga to look specifically at worry, prior studies have compared CBT and mindfulness-based intervention approaches (of which yoga is one) for pain. In the present study, we saw a significant between-groups difference for both pain interference and pain intensity favoring the CBT group. This finding is in contrast to a relatively recent RCT conducted with adults (aged 20-70 years) with chronic low back pain comparing treatment with mindfulness-based stress reduction (MBSR), CBT, and usual care.74 They found that both MBSR and CBT participants demonstrated a greater improvement in pain and functional limitations related to pain than the usual care group, and no significant differences were seen between MBSR and CBT.
+
+(To our knowledge, no comparable study has been conducted with a sample of older adults.) There are data that suggest potential neural mechanisms for mindfulness-based interventions to impact pain. Specifically, recent experimental findings suggest that people reporting higher levels of mindfulness report less pain intensity and unpleasantness
+(i.e., feel less pain) and show greater deactivation of the posterior cingulate cortex (brain region involved in sensory, cognitive, and affective appraisals of pain).75 These data suggest that both CBT and yoga remain viable options that warrant investigation in older adults, particularly for a study focused on pain-related outcomes.
+
+There are several limitations of this study. First, despite substantial efforts to recruit a diverse sample in terms of gender and race/ethnicity, the ultimate sample consisted of White women which may somewhat limit the generalizability of our findings. Second, as a randomized preference trial, all potential participants had to be willing to be randomized to either an RCT or a preference trial, meaning that they needed to agree in advance to either be randomized or select their intervention. Potential participants not willing to agree to this type of randomization may have dropped out earlier in the recruitment process, thereby decreasing generalizability of study findings. Third, given that multiple comparisons were conducted, it is possible that findings are due to Type 1 error.
+
+Finally, there was no objective performance measure of physical function as we relied on a brief self-report measure for this key variable.
+
+These points notwithstanding, this study has a number of substantial strengths. Clearly, the two-stage randomized design allowed us to examine preference and selection effects in addition to the more traditional RCT findings, and inclusion of multiple exploratory measures allowed us to examine impact of both CBT and yoga interventions on depression, generalized anxiety symptoms, fatigue, pain, and social participation - all important constructs that contribute to quality of life in older adults.
+
+## Conclusions
+
+These findings suggest that both CBT and yoga may be useful approaches for depressive symptoms, generalized anxiety symptoms, fatigue, and social participation in communitydwelling older adults who report high levels of worry. CBT
+may show greater benefit for improving pain-related outcomes. Finally, recommending an intervention or allowing a person to choose an intervention had no effect on outcomes.
+
+## Authors Contributions
+
+Data Access, Responsibility, and Analysis: Suzanne C. Danhauer, PhD and Gretchen A. Brenes, PhD had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Andrea Anderson, M.S., Jasmin Divers, PhD, and Michael E. Miller, PhD conducted the analyses.
+
+Study conception and design: Brenes, Danhauer, Divers, Miller. Acquisition, Analysis, or Interpretation of Data: Anderson, Brenes, Danhauer, Divers, Hargis, Miller. Drafting of the Manuscript:
+Anderson, Brenes, Danhauer, Divers, Hargis, Miller. Critical Revision of the Manuscript for Important Intellectual Content: Anderson, Brenes, Danhauer, Divers, Hargis, Miller. Obtaining Funding: Brenes, Danhauer, Divers, Miller. Administrative, Technical, or Material Support: Anderson, Brenes, Danhauer, Divers, Hargis, Miller. Supervision: Brenes, Danhauer, Divers, Miller
+
+## Declaration Of Conflicting Interests
+
+The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
+
+## Funding
+
+The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported through a Patient-Centered Outcomes Research Institute (PCORI) Program Award (CER-1511-33007; MPIs Brenes and Danhauer) and by the Office of the Dean at Wake Forest School of Medicine. PCORI was not involved in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
+
+## Disclaimer
+
+All statements in this report, including its findings and conclusions, are solely those of the authors and do not necessarily represent the views of the Patient-Centered Outcomes Research Institute
+(PCORI), its Board of Governors or Methodology Committee.
+
+## Trial Registration
+
+www.clinicaltrials.gov Identifier NCT 02968238
+
+## Orcid Id
+
+Suzanne C. Danhauer  https://orcid.org/0000-0002-2003-9805
+
+## References
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medical/md/PMC9305358.md

@@ -0,0 +1,315 @@
+# Antibacterial Peptide Cyclomarina Creates Toxicity By Deregulating The Mycobacterium Tuberculosis Clpc1–Clpp1P2
+
+## Protease
+
+Received for publication, March 22, 2022, and in revised form, June 16, 2022 Published, Papers in Press, June 26, 2022, https://doi.org/10.1016/j.jbc.2022.102202 Gabrielle Taylor1 , Yannick Frommherz2,3, Panagiotis Katikaridis2,3, Dominik Layer4 , Irmgard Sinning4, Marta Carroni5 , Eilika Weber-Ban1,*, and Axel Mogk2,3,*
+From the 1ETH Zurich, Institute of Molecular Biology and Biophysics, Zurich, Switzerland; 2Center for Molecular Biology of the University of Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany; 3Division of Chaperones and Proteases, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany; 4Heidelberg University Biochemistry Center (BZH), Heidelberg, Germany; 5Swedish Cryo-EM Facility, Science for Life Laboratory Stockholm University, Solna, Sweden Edited by Ursula Jakob The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1–ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains
+(MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity.
+
+ATPase Associated with diverse cellular Activities (AAA+)
+proteases are central components of proteostasis networks in bacteria and play crucial roles for bacterial physiology and stress resistance by degrading not only aberrant proteins but also functional proteins harboring specific degrons for protease targeting (1, 2). AAA+ proteases are composed of a hexameric ring–forming ATPase (AAA+) component (e.g.,
+ClpA, ClpC), which associates with a barrel-forming peptidase (e.g., ClpP). The proteolytic active sites are hidden in the interior of the peptidase barrel, protecting cellular proteins from unwanted degradation (3). Substrates access the degradation chamber via a continuous channel, which starts at the entry pore of the AAA+ hexamer. ATP hydrolysis by the AAA+ protein fuels the unfolding and threading of bound substrates into the peptidase barrel (4).
+
+Protein degradation is irreversible and represents a potentially deleterious activity that has to be tightly controlled.
+
+Substrate selection and overall activity of AAA+ proteases are therefore precisely regulated. AAA+ proteins share the AAA domain, which is responsible for not only ATP binding and hydrolysis but also hexamerization. Substrate selectivity and activity control typically involve additional domains, which are either N-terminally fused to or inserted into the AAA module (N-terminal domain [NTD] and middle domain [MD],
+respectively). NTDs can directly recognize substrates, for instance, Bacillus subtilis (Bs) ClpC NTD recognizes phosphorylated arginine residues of substrates as degron (5). Alternatively, NTDs function as binding platforms for cooperating adaptor proteins that transfer their bound cargo to the AAA+ protein. The adaptor protein ClpS binds to N-end rule substrates, harboring destabilizing residues at their N termini and delivers them to the AAA+ partners ClpA or ClpC (6, 7). Adaptor proteins can in addition regulate the global activity of an AAA+ partner. Bs ClpC and Staphylococcus aureus (Sa)
+ClpC do not exhibit significant ATPase and proteolytic activities on their own but depend entirely on the presence of adaptors like MecA (8, 9). MecA triggers the formation of ClpC hexamers, a process that is linked to ATPase activation and the transfer of bound substrates (9, 10). MecA binds to both ClpC NTD and its coiled-coil MD (11). In the absence of MecA, MDs self-interact to form an alternative assembly state (resting state) that has very low ATPase activity and does not associate with ClpP (9). The resting state is formed by two * For correspondence: Axel Mogk, a.mogk@zmbh.uni-heidelberg.de; Eilika Weber-Ban, eilika@mol.biol.ethz.ch.
+
+pentameric half-spirals that are held together by head-to-head MD interactions. ClpC MD mutants that are deficient in MD
+self-interaction constitutively form hexamers and are toxic in vivo (9). This underlines the crucial role of MDs as negative regulators for ClpC activity control. The activation of ClpC by MecA is only temporary as the adaptor targets itself for degradation by ClpC–ClpP, ensuring ClpC inactivation once specifically targeted substrates have been degraded (8).
+
+AAA+ proteases can play crucial roles in bacterial virulence
+(12). The AAA+ component ClpC1 of the important pathogen Mycobacterium tuberculosis (Mtb) is of particular interest as it forms an essential protease with the ClpP1P2 peptidase
+(13, 14). The crucial role of ClpC1–ClpP1P2 for Mtb viability qualifies the protease as an attractive drug target. Indeed, antibacterial compounds that target either ClpC1 or ClpP1P2 have been identified (15, 16). Acyldepsipeptide (ADEP) antibiotics target ClpP in multiple bacterial species triggering toxic ATPase-independent peptidase activation (17). In addition, ADEP competes with AAA+ components for binding to ClpP,
+thus in addition abrogating cooperation of ATPase and peptidase (18). ADEP antibiotics thereby exert dual effects on AAA+ proteases: the activation of ClpP enabling it for degradation of unstructured polypeptides and inhibition of ClpP cooperation with the AAA+ partner causing stabilization of folded substrates harboring specific degrons for protease targeting. ADEP toxicity in Mtb cells is based on the latter consequence (14). Diverse cyclic peptides, including lassomycin, ecumicin, rufomycin, and CyclomarinA (CymA), which are highly toxic to Mtb cells, target ClpC1 (19–22). All these compounds bind to a hydrophobic groove of ClpC1 NTD
+(23–25); however, their effects on ClpC1 ATPase and proteolytic activities seem diverse. Lassomycin and ecumicin increase ClpC1 ATPase activity (20, 21), whereas rufomycin shows no impact (22). All three peptides inhibit the degradation of the disordered model substrate casein by ClpC1–
+ClpP1P2, indicating peptide-induced uncoupling of ATPase and proteolytic activities (20–22). CymA activity has been characterized using a hybrid protein (NMtb–ClpC), composed of the Mtb ClpC1 NTD and the AAA and MD domains of Sa ClpC, which is insensitive to CymA in its WT form carrying the native NTD. For the hybrid fusion construct NMtb–ClpC,
+CymA increases both ATPase and proteolytic activities of the fusion construct (26). Whether CymA exerts the same enhancing effects on authentic Mtb ClpC1 or acts as an inhibitor like other cyclic peptides remained unknown. The understanding of drug mechanisms is hampered by a lack of knowledge about ClpC1 activity control. ClpC1 consists of two AAA domains (AAA1 and AAA2), an NTD, and a coiled-coil MD. While sharing the same domain organization with other bacterial ClpC proteins, it is unclear whether Mtb ClpC1 is regulated by a similar mechanism involving adaptor-controlled hexamer formation. Mtb ClpC1 exhibits autonomous proteolytic activity in contrast to Sa ClpC and Bs ClpC (20, 27, 28), indicating adaptor-independent hexamerization and differences in regulation. The stand-alone activity of ClpC1 is, however, low in comparison to MecA-activated Sa ClpC or Bs ClpC. This opens up the possibility that activation pathways also exist for ClpC1, suggesting in turn regulatory mechanisms are in place that downregulate its activity.
+
+Here, we dissected ClpC1 activity control and its modulation by the drug CymA. We show that ClpC1 exists in different activity states caused by changes in assembly states. ClpC1 predominantly assembles into an inactive resting state, indicating that this regulatory mechanism is conserved among ClpC homologs. Abrogating the resting state by MD mutation leads to formation of ClpC1 hexamers that constitute the highest activity state of ClpC1. The drug CymA converts the resting state into a ClpC1 supercomplex built of multiple hexamers that interact with each other through their MDs. This assembly state shows increased proteolytic activities toward a diverse range of substrates tested, indicating that CymA toxicity toward Mtb cells is due to overactivation of the ClpC1–ClpP1P2 protease caused by resting state conversion to the hexamer supercomplex.
+
+## Results Mtb Clpc1 Forms An Md-Dependent Resting State
+
+Sa ClpC activity is controlled by its coiled-coil MD that mediates the formation of an inactive resting state (9). Binding of the adaptor MecA to Sa ClpC NTD and MD prevents resting state formation and triggers formation of active ClpC hexamers. Mtb ClpC1 differs in various aspects from Sa ClpC. In a phylogenetic tree, Mtb ClpC1 localizes to a distinct branch, separating it from Sa ClpC (Fig. S1A). Notably, bacteria belonging to the Mtb ClpC1 clade do not encode for the canonical adaptor MecA, whereas MecA is present in all bacteria belonging to the Sa clade (Fig. S1A). This might suggest differences in the regulatory principles of both Hsp100 proteins. In agreement with this notion, Mtb ClpC1, in contrast to Sa ClpC, exhibits autonomous ATPase and proteolytic activities (20, 27, 28). However, ClpC1 ATPase and degradation activities are low as compared with MecAactivated Sa ClpC. Furthermore, ClpC1 also possesses the conserved phenylalanine (F444) at the tip of its MD, which was shown to be essential for resting state formation of Sa ClpC (Fig. 1A). These observations support the possibility that Mtb ClpC1 activity is also controlled by its MD. We therefore determined the oligomeric states of ClpC1 WT and the F444S mutant, probing for a potential role of the MD in controlling the assembly state (Fig. 1B). In size-exclusion chromatography (SEC) runs, ClpC1-WT eluted prior to the ClpC1-F444S
+mutant (Fig. 1B). The elution profiles of WT and F444S in the presence of nucleotide were very similar to those of Sa ClpC
+and Escherichia coli (Ec) ClpB serving as references for decameric resting and hexameric states, respectively. Mass determination of ClpC1-WT and ClpC1-F444S mutant complexes by multiangle light scattering measurements coupled to SEC
+runs (SEC–MALS) revealed that the diverse elution profiles are based on differences in molecular weight rather than shape
+(Fig. S1B). ClpC1-WT complexes had masses of 600 to 1160 kDa. Peak fractions had a molecular weight of 863 ± 81 kDa, corresponding to a nonameric complex, which is similar to the decameric resting state determined for Sa
+
+![2_image_0.png](2_image_0.png)
+
+ClpC (9). ClpC1-F444S complexes had lower masses from 330 to 670 kDa and a molecular weight of 448 ± 58 kDa at the peak fraction corresponding to a pentameric complex. Such complex composition might result from partial dissociation of a ClpC1-F444S hexamer during the SEC–MALS run. To provide direct evidence for ClpC1-F444S hexamer formation, we performed glutaraldehyde crosslinking and analyzed the crosslink products by SDS-PAGE (Fig. S1C). In the presence of nucleotide, ClpC1-WT was mostly crosslinked to high molecular weight complexes that failed to enter the gel, whereas ClpC1-
+F444S predominantly formed crosslinked products that located at positions identical to crosslinked ClpB hexamers. Notably, ClpC1-WT crosslinking also yielded some hexameric products, which was not observed for Sa ClpC (Fig. S1C). This difference in crosslinking pattern correlates with the low autonomous activity described for Mtb ClpC1. We in addition employed mass photometry to determine molar masses of ClpC1 complexes in bulk solution avoiding complex disintegration during SEC runs. This technique allows to identify distinct protein complexes existing in a single preparation (Fig. 1C). At the low concentration of ClpC1 (60 nM) required for this methodology, both samples featured a mass peak of 86 to 91 kDa corresponding to a monomer was observed in the presence of slowly hydrolyzable ATPγS. ClpC1-WT in addition formed large complexes (mass range: 875–1260 kDa) with a mass of 968 ± 8 kDa at the most populated assembly state corresponding to a decameric (10.3-mer) resting state complex. Some smaller complexes (mass range: 450–700 kDa) with a mass of 563 ± 3 kDa at the peak were in addition present. This mass corresponds to a hexamer, which forms at much lower frequencies than the resting state (Fig. 1C). These data are consistent with crosslinking and former SEC results documenting the formation of high molecular weight complexes and the presence of some hexameric species. The formation of the larger complexes must involve the MD as the molar masses observed for ClpC1-F444S in the presence of nucleotide were much lower (480–620 kDa). The mass of the most populated ClpC1-F444S state was 563 ± 13 kDa, corresponding to a hexamer (Fig. 1C). We also noticed formation of larger complexes with ClpC1-F444S (1050–1250 kDa). Those had a different mass profile than ClpC1-WT complexes and were also much less populated. To directly visualize ClpC1-WT and ClpC1-F444S complexes, we performed negative-staining EM
+(Fig. 1D). EM analysis revealed the coexistence of diverse ClpC1-WT assemblies including hexamers and resting states, which constituted the most populated species. A lowresolution three-dimensional reconstruction (25 Å) of the resting state allowed rigid-body docking of up to 12 subunits, which were organized in two spiral subcomplexes that interacted via their MDs (Fig. S1D). ClpC1-F444S exclusively formed hexameric species (Fig. 1D). Together, our analysis of Mtb ClpC1 assembly states documents the formation of a resting state that is mediated by MD interactions.
+
+## Cyma Induces The Formation Of A Clpc1 Supercomplex
+
+CymA causes severe cellular toxicity in Mtb cells by binding to the ClpC1 NTD (19). We analyzed whether and how CymA binding modulates the ClpC1 assembly state. In crosslinking experiments, CymA hardly changed the crosslink patterns of ClpC1-WT or ClpC1-F444S (Fig. S1C). The intensity of the crosslinked band at the position of the hexamer remained the same for ClpC1-F444S, suggesting that it largely stayed hexameric. For ClpC1-WT, a slight decrease in the levels of ClpC1-WT hexamers was observed, but the main portion of ClpC1-WT was found in the band corresponding to higher molecular weight crosslinks (Fig. S1C). To better understand what constitutes the higher molecular weight fraction, SEC
+analysis was carried out. For this, we used the ATPasedeficient double Walker B variant ClpC1-E288A-E626A
+(DWB), which harbors mutations in the Walker B motifs of both AAA domains, to ensure highest complex stabilities
+(Fig. 2A). CymA binding caused a shift of ClpC1-DWB toward earlier elution fractions, implying the formation of larger complexes, which we confirmed by SEC–MALS analysis
+(Fig. S2A). CymA-induced ClpC1 complexes had a mass of 2500 to 3100 kDa; however, mass determination was not highly accurate as the concentration of eluting CymA-bound ClpC1-DWB remained low in multiple runs. CymA did not alter the elution profile of ClpC1-DWB-F444S in SEC runs
+(Fig. 2A), suggesting that CymA-induced changes observed for ClpC1-WT involve the MD. To overcome the limitations of the SEC–MALS runs, we determined the masses of CymAinduced ClpC1 assemblies by mass photometry in bulk solution (Fig. 2B). We observed large changes in mass distributions of ClpC1-WT complexes in the presence of CymA and ATPγS.
+
+The presence of CymA strongly reduced the fraction of ClpC1 masses representing the resting state while inducing the formation of larger complexes (2000–2500 kDa). The main peak of the mass distribution had a molecular weight of 2249 ± 111 kDa, corresponding to 24 ClpC1 subunits. No major changes in mass profiles were observed for ClpC1-F444S in the presence of CymA and ATPγS (Fig. 2B). This observation is consistent with SEC analysis (Fig. 2A) and confirms that CymA-induced changes in the ClpC1-WT assembly state involve the MD. To determine the nature of the 24-mer ClpC1–CymA complex, we applied EM; however, analysis suffered from poor image acquisition that could not be improved despite multiple attempts.
+
+The formation of a CymA-induced ClpC1 complex consisting of 24 subunits is highly reminiscent of the
+
+![4_image_0.png](4_image_0.png)
+
+CymA-induced oligomerization changes observed for the NMtb–ClpC fusion construct (26). Here, EM analysis revealed a complex consisting of four hexamers that formed a triangular pyramid (26). As the effects of CymA on Mtb ClpC1 oligomerization involve the MD residue F444, we tested whether the same MD residue (F436) is also crucial for formation of 24-meric NMtb–ClpC complexes (Fig. S2B). Indeed, CymA
+presence did not alter the NMtb–ClpC-F436A elution profile in SEC runs, whereas it induced the formation of larger NMtb–ClpC complexes. We infer that CymA is causing similar changes in the assembly states of ClpC1 and NMtb–ClpC,
+converting the resting state into hexamers that can form higher assembly states in an MD-dependent manner.
+
+## Cyma Enhances Mtb Clpc1 Atpase Activity And Substrate Binding
+
+We determined the consequences of altered assembly states induced by either CymA or the F444S mutation on ClpC1 ATPase activity. CymA enhanced the basal ATPase activity of ClpC1 by 2.5-fold (Fig. 3A). This observation is similar to the stimulatory effect of CymA on NMtb–ClpC ATPase activity
+(26). Notably, we determined a strong synergistic effect of cooperating ClpP1P2 peptidase and the disordered model substrate casein on ClpC1 ATPase activity, resulting in a fourfold increase in the presence of both factors. CymA further enhanced ATP hydrolysis in the presence of casein and ClpP1P2 and led to the highest ATPase activity (Fig. 3A).
+
+ATPase activities of ClpC1-F444S, either alone or in the presence of ClpP1P2, casein, or CymA, were always higher as compared with ClpC1-WT (Fig. 3A). This links constitutive hexamer formation triggered by the F444S mutation to higher ATPase activities. Similarly, the stimulation of ClpC1 ATPase activity by CymA correlates with the conversion of the resting state into an assembly composed of hexamers. Titration of ATPase activity with increasing concentrations of CymA
+yielded a sigmoidal dose–response curve with a Hill coefficient of 3.2 ± 0.5. This suggests that binding of multiple CymA
+molecules to a ClpC1 hexamer is required for maximal ATPase stimulation (Fig. 3B). In order to analyze how CymA
+modulates substrate interaction, we determined binding affinities to fluorescently labeled casein (FITC-casein) using fluorescence anisotropy (Fig. 3C). Mtb ClpC1 bound only weakly to FITC-casein. The binding was strongly enhanced in the presence of CymA yielding a Kd of 1 μM. A similar binding affinity was determined for ClpC1-F444S in the absence and presence of CymA (Fig. 3C). These findings are consistent with a CymA-triggered conversion of the resting state into hexameric assemblies, which bind casein more efficiently. ClpC1-
+F444S adopts a hexameric state without CymA, explaining why its binding affinity to FITC-casein in the absence of CymA
+corresponded to the enhanced value observed with ClpC1-WT only in the presence of CymA and remained unaltered upon addition of CymA.
+
+## Cyma Enhances Degradation Of Various Clpc1 Substrates
+
+We analyzed how CymA binding and the MD F444S mutation affects the proteolytic activity of ClpC1-WT–ClpP1P2.
+
+We first monitored the degradation of FITC-casein, which leads to an increase in fluorescence intensity because of fluorophore dequenching upon cleavage to short peptides
+(Fig. 4A). CymA increased the basal degradation activity of ClpC1-WT–ClpP1P2 by fourfold (Fig. 4B). ClpC1-F444S–
+ClpP1P2 exhibited threefold increased proteolytic activity as compared with ClpC1-WT–ClpP1P2, and FITC-casein
+
+![5_image_0.png](5_image_0.png) 
+
+degradation was further enhanced in the presence of CymA reaching degradation rates similar to CymA-bound ClpC1-
+WT–ClpP1P2 (Fig. 4, A and B). These findings largely correlate with the structural states and ATPase activities we determined before and indicate that CymA binding and the F444S mutation increase overall ClpC1 activity. The finding that ClpC1-WT–ClpP1P2 exhibits autonomous degradation activity is in agreement with former findings (20, 27, 28) and is different from Sa ClpC–ClpP (9). We observed that Mtb ClpC1-WT can cooperate with Sa ClpP in substrate degradation, allowing us to directly compare both Hsp100 proteins and confirm the fundamental difference between them
+(Fig. S3, A and B). Mtb ClpC1-WT–Sa ClpP but not Sa ClpC–
+Sa ClpP complexes exhibited proteolytic activity toward FITCcasein. The impact of CymA and the F444S mutation on the proteolytic activity of ClpC1–Sa ClpP was comparable to those determined for ClpC1–ClpP1P2 complexes. Similarly, we observed synergistic stimulation of ClpC1-WT ATPase activity by casein and Sa ClpP, confirming that Sa ClpP can functionally replace Mtb ClpP1P2 (Fig. S3C).
+
+We next studied whether CymA enhances the ATPase activity of a particular AAA domain. ClpC1 harbors two AAA
+domains (AAA1 and AAA2). We generated Walker B mutants that specifically inhibit ATP hydrolysis at either AAA1
+(E288A, WB1) or AAA2 (E626A, WB2). CymA stimulated ATP hydrolysis of ClpC1-E288A but not ClpC1-E626A, indicating that ATP hydrolysis at AAA2 is primarily enhanced upon drug binding (Fig. S3, D and E). The AAA2 domain constitutes the main threading motor of Hsp100 proteins (29, 30), rationalizing the impact of CymA on ClpC1 activity.
+
+Notably, CymA did not enhance FITC-casein degradation by either of the ClpC1 Walker B mutants, indicating that ATPase stimulation of the AAA2 domain is not sufficient for increased proteolytic activity, which requires two functional ATPase rings (Fig. S3, E and F).
+
+How general is the impact of CymA on ClpC1 proteolytic activity? To answer this question, we determined the effects of CymA and F444S on the degradation of a folded model substrate by monitoring degradation of GFP-SsrA, harboring the SsrA degron for Hsp100 targeting. CymA enhanced GFP-SsrA
+degradation of ClpC1-WT–ClpP1P2 by 1.8-fold. ClpC1-
+F444S–ClpP1P2 exhibited a 3.7-fold higher GFP-SsrA degradation rate compared with ClpC1–ClpP1P2, confirming its enhanced activity state. This activity was slightly reduced in
+
+![6_image_0.png](6_image_0.png)
+
+the presence of CymA (Fig. 4, C and D), in contrast to our findings for FITC-casein (Fig. 4, A and B), indicating substratespecific effects of the drug. Notably, for ClpC1-F444S–
+ClpP1P2, we also observed an inhibitory effect of CymA on the degradation of the antitoxin VapB15, which represents a natural ClpC1 substrate (Fig. S4A) (7). CymA did not increase VapB15 degradation by ClpC1-WT–P1P2, and we rather observed a minor inhibition, again documenting substratespecific effects (Fig. S4A).
+
+We infer that CymA and the F444S mutation cause increased ClpC1 proteolytic activities toward multiple substrates, irrespective of their folding states or targeting signals.
+
+Although CymA does not affect degradation of all substrates equally, we did not observe strong inhibition of ClpC1-WT–
+ClpP1P2 activity as reported for other antibacterial peptides
+(20–22).
+
+## Cyma And Md F444S Mutation Enhance Clps-Dependent Substrate Degradation
+
+ClpS currently represents the only known adaptor protein of ClpC1. ClpS targets N-end rule substrates that harbor destabilizing residues (e.g., Phe) at their N termini for degradation
+(6, 7, 31). We therefore studied how CymA and the F444S mutation affect degradation of the N-end rule model substrate FR-L-GFP (Fig. 5, A and B). CymA and F444S enhanced FR-LGFP degradation rates by 1.6-fold and 8-fold, resembling their effects on GFP-SsrA. ClpS did not alter the basal ATP hydrolysis rates of ClpC1-WT and ClpC1-F444S in the absence or the presence of CymA (Fig. S4B). We also did not observe an impact of ClpS on ClpC1 crosslinking patterns (Fig. S4C),
+suggesting that ClpS solely acts as specificity factor without enhancing overall ClpC1 activity. Notably, CymA presence reduced the ClpS-dependent degradation activity of ClpC1- F444S by 2.3-fold (Fig. 5, A and B). Both CymA and ClpS
+
+![6_image_1.png](6_image_1.png)
+
+bind to ClpC1 NTD; however, their interaction sites do not overlap (Fig. S4D). This implies that CymA might alter the relative orientation of NTDs and bound ClpS, thereby hampering transfer of FR-L-GFP to the processing of ClpC1 pore site.
+
+## Loss Of Clpc1 Activity Control By Md Mutation Increases Cellular Toxicity
+
+We finally tested whether the modulations of ClpC1 activity states by the F444S mutation causes cellular toxicity. We made use of our finding that Mtb ClpC1 cooperates with Sa ClpP
+and coexpressed both genes from IPTG-controlled expression vectors in Ec host cells. This heterologous expression enabled us to directly determine the potentially toxic consequences of ClpC1 deregulation in the absence of so far unknown regulatory proteins that might control ClpC1 activity in M. tuberculosis cells. Co-overexpression of Mtb ClpC1 and Sa ClpP was toxic to Ec cells in a temperature-dependent manner and was observed at 37 C (Figs. 6 and S5A). Coexpression of Sa clpP increased toxicity at least 100-fold, indicating that cells are killed by Mtb ClpC1–mediated protein degradation. This finding is consistent with the autonomous degradation activity of Mtb ClpC1–Sa ClpP determined in vitro. The presence of authentic Ec ClpP does likely not interfere with toxicity, as it does not cooperate with ClpC1-WT or ClpC1-F444S in vitro
+(Fig. S5C). Toxicity was increased approximately 10-fold upon expression of ClpC1-F444S (Fig. 6). Increased toxicity of ClpC1-F444S was also observed in the absence of Sa ClpP expression but not to the same extent as in its presence.
+
+Expression levels of ClpC1-WT and ClpC1-F444S were identical (Fig. S5B), indicating that the increased activity of ClpC1-
+F444S causes enhanced killing of Ec cells.
+
+## Discussion
+
+In the present work, we show that regulation of Mtb ClpC1 activity crucially involves the MD-mediated formation of an inactive resting state. We validated the formation of this structural state by multiple techniques and by showing that mutating a conserved aromatic residue, which is obligatory for resting state formation by Sa ClpC, also triggers constitutive hexamer formation and activation of ClpC1. A recent study also documents resting state formation for Bs ClpC (32),
+indicating that ClpC activity control via MD-mediated resting state formation is widespread and evolutionarily conserved.
+
+While sharing the basic regulatory mechanism with other bacterial ClpC homologs, Mtb ClpC1 also exhibits differences as it shows low autonomous proteolytic activity toward the disordered model substrate casein, which is not observed for Sa ClpC and Bs ClpC (9, 10). Similarly, coexpression of Mtb ClpC1 and cooperating Sa ClpP creates toxicity in Ec cells, which is not observed upon coexpression of Sa ClpC and Sa ClpP (9). We show here that this stand-alone ClpC1 activity is linked to adaptor-independent hexamer formation. We suggest that ClpC1 hexamers and the resting state exist in a dynamic equilibrium. This also implies that MD-mediated ClpC1 activity control is less stringent compared with other ClpC homologs and opens additional pathways for ClpC
+activation. We find that the simultaneous presence of substrate casein and a cooperating peptidase enhances ClpC1 ATPase activity, implying increased hexamer formation. ClpP1P2 forms a rigid double heptamer, which might serve as an assembly platform that facilitates ClpC1 hexamer formation or stabilizes ClpC hexamers upon interaction. The autonomous ClpC1-WT activity, however, remains low as evident from decreased proteolytic activities in comparison to ClpC1-F444S.
+
+This difference in proteolytic activity is particularly pronounced for tightly folded model substrates like GFP-SsrA and FR-L-GFP. The difference in ClpC1-WT and ClpC1-F444S activities remains in the presence of ClpS indicating that ClpS
+solely acts as specificity factor but does not increase overall ClpC activity as other adaptors like MecA. It is tempting to speculate that Mtb encodes for a ClpC1 adaptor that triggers an activity state comparable to ClpC1-F444S. This could be achieved by adaptor binding to the MDs or by covalently modifying ClpC1, ultimately abrogating resting state formation. However, such a putative activator would not be expected to be expressed constitutively, as persistent activation by MD mutation is toxic in case of Sa ClpC and Bs ClpC (9) and, similarly, we show increased toxicity of ClpC1-F444S as compared with ClpC1-WT upon heterologous expression in Ec. This predicts that an activation of Mtb ClpC1 will be tightly controlled and temporally limited.
+
+ClpC1 represents the target of various cyclic peptides with strong antibacterial activities. Most of these compounds inhibit ClpC1–ClpP1P2 proteolytic activity toward FITC-casein
+
+![7_image_0.png](7_image_0.png)
+
+in vitro, rationalizing toxicity toward Mtb cells. We show here that CymA acts differently, as it enhances ClpC1–ClpP1P2 degradation activity toward most substrates tested, including FITC-casein. A significant inhibition of proteolytic activity is not observed for ClpC1-WT. These findings are consistent with former results using the hybrid NMtb–ClpC fusion construct
+(26) indicating that CymA kills Mtb cells by inducing deregulated proteolysis. The effects of CymA on ClpC1–ClpP1P2 activity are in part substrate specific as an enhancement of VapB15 degradation was not observed. We envision a scenario in which CymA can have dual effects, negative and positive ones, on ClpC1 function. While CymA overrides MD-mediated ClpC1 activity control and enhances its ATPase activity, it might also hamper binding to substrates like VapB15. Our data on the constitutively hexameric population of ClpC1-F444S
+might serve as a model for a putative adaptor–activated state.
+
+For this variant, we observe a reduction in degradation activity toward the N-end rule model substrate. A scenario can thus be envisioned where under specific adaptor-activated conditions, CymA may inhibit degradation of specific substrates, like for example, N-end rule substrates. In the sum, these opposing effects level out, explaining the largely unchanged degradation of VapB15 by ClpC1-WT–ClpP1P2 in the presence of CymA.
+
+It remains unknown why CymA can increase ClpC1 activity in contrast to other cyclic peptides. All peptides bind to the same groove of the NTD, yet the binding modes can be different as for instance, two ecumicin peptides bind to the NTD (25)
+(Fig. S6A). It is conceivable that diverse binding modes of CymA
+and ecumicin have different consequences on ClpC1 activity. However, rufomycin and CymA bind in an almost identical manner to ClpC1 NTD (Fig. S6B), leaving the question why both peptides have opposing effects on ClpC1 activity unanswered.
+
+The stimulatory effect of CymA is based on its effect on the ClpC1 resting state, which is converted to an alternative assembly state built from hexamers. Mass determination of CymA-bound ClpC1 in an all-ATP state suggests a supercomplex composed of four hexamers, which is identical to the NMtb–ClpC assembly state triggered upon CymA binding (26).
+
+Notably, an analogous arrangement of ClpC hexamers has been recently reported for Bs ClpC bound to a substrate that harbors a phosphoarginine (pArg) group as targeting degron
+(32). Here, four ClpC hexamers form a tetrahedron, which is highly similar to the architecture of CymA-bound NMtb–ClpC
+(26). Remarkably, the four pArg-bound ClpC hexamers are held together via head-to-head MD interactions involving the MD tips (32). We show here that mutating F444 (or F436)
+located at the MD tip of Mtb ClpC1 (or NMtb–ClpC) prevents the formation of CymA-induced supercomplexes, providing further evidence that the organizations of CymA and pArginduced ClpC assemblies are largely similar (Fig. 7). In this ClpC1 supercomplex, the AAA2 ring is fully accessible for ClpP1P2 interaction, explaining why the increase in ClpC1 ATPase activity in the presence of CymA is linked to enhanced proteolytic activity. How the binding of CymA to ClpC1 NTDs, which are not directly involved in resting state formation, converts the resting state into the tetrahedral supercomplex remains to be explored. As CymA does not bind to
+
+![8_image_0.png](8_image_0.png)
+
+regulatory MDs, those are still free for self-interaction, now mediating the interaction of individual hexamers. The finding that CymA acts on the ClpC1 resting state implies that drugbased overactivation can also be applied as antibacterial strategy for other major pathogens (e.g., S. aureus) harboring ClpC homologs that are regulated via the same mechanism.
+
+The activity of CymA-activated ClpC1 is typically lower as compared with ClpC1-F444S, which only forms single hexamers as the mutated MDs can no longer interact. This observation indicates that ClpC can adopt diverse activity states depending on the assembly formed (Fig. 7). The factors triggering the individual structural and activity states are diverse and include not only specific adaptors (e.g., MecA) but also degrons (e.g., pArg) leading to full or partial ClpC activation, respectively. This way ClpC activities can be fine-tuned and adjusted to the particular cellular target. CymA interference can occur at multiple levels, including redistribution of the relative population of the various ClpC1 assembly states.
+
+The induction of active ClpC assemblies is typically transient as both substrates and activating adaptors are subjected to ClpC-dependent degradation, automatically limiting ClpC
+activation. In contrast, ClpC1 activation by CymA is constitutive. CymA thereby utilizes existing ClpC activation pathways while circumventing their built-in deactivation routines.
+
+## Experimental Procedures Strains, Plasmids, And Proteins
+
+Ec strains used were derivatives of XL1-blue or BL21. Mtb ClpC1 was amplified by PCR, inserted into pET24a or pUHE21
+
+expression vectors, and verified by sequencing. ClpC1-F444S
+was generated by PCR-based mutagenesis and verified by sequencing.
+
+Mtb ClpC1 was purified after overproduction from Ec BL21 cells using nickel–iminodiacetic acid (Macherey–Nagel)
+following standard protocols. Eluted samples containing ClpC1 were pooled and further purified by SEC (Superdex S200; GE Healthcare) in 50 mM Hepes (pH 7.5), 150 mM KCl, 20 mM MgCl2, 2 mM DTT, and 5% (v/v) glycerol. Mtb ClpP1P2, ClpS, and Sa ClpP were purified as described before
+(7, 9). Protein concentrations refer to monomers and were determined with the Bio-Rad Bradford assay using bovine serum albumin as standard.
+
+## Sec And Sec–Mals
+
+Oligomeric states of Mtb ClpC1, Sa ClpC, or Ec ClpB (each 6 μM) were monitored by SEC (Superose 6 HR10-30; GE
+Healthcare) in buffer A (50 mM Tris [pH 7.5], 25 mM KCl, 20 mM MgCl2, 2 mM DTT, and 10% [v/v] glycerol) supplemented with 2 mM ATP at 4 C. Samples were preincubated with 2 mM ATPγS or 2 mM ATP (ClpC1-DWB) for 5 min prior injection. CycA (12 μM) was added as indicated. Fractions were collected in 96-well plates, aliquots taken, and subjected to SDS-PAGE. Gels were stained using SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific) following the manufacturer's instructions.
+
+SEC–MALS experiments were performed using 6 μM
+ClpC1 and 12 μM CymA at room temperature using a Superose 6 HR10-30 column. The column was connected to a MALS system (Dawn Heleos II 8+ and Optilab T-rEX; Wyatt Technology). Data were analyzed using the Astra 6 software (Wyatt Technology).
+
+## Mass Photometry
+
+Mass photometry was used to compare the oligomeric state of ClpC1-WT and ClpC1-F444S in either the presence or the absence of CymA. About 0.5 μM ClpC1 was prepared in buffer B (50 mM Hepes [pH 7.5], 25 mM KCl, 5 mM ATPγS, 20 mM
+MgCl2, 1 mM DTT, and 5% [v/v] glycerol) with 10 μM CymA
+added as indicated. To find focus, 15 μl buffer B was loaded within CultureWell gaskets (Merck on high-precision coverslips (ThorLabs). After finding focus, 2 μl ClpC1 was added to the buffer, and 60 s movies of landing events were recorded on the Refeyn OneMP mass photometer instrument using a frame rate of 100 Hz. The interferometric scattering contrast values of landing events were used to determine molecular mass values in the DiscoverMP software following calibration with NativeMark unstained protein standard (Thermo Fisher Scientific). More than 2000 particles were detected in each movie
+(typically 3000–5000 landing events). Kernal density estimates were computed in the Python KDEpy 1.1.0 package (https://
+github.com/tommyod/KDEpy) using the FFTKDE function with a Gaussian kernel and a bandwidth of 20. Mass values were determined in the DiscoverMP software from the fitted Gaussian peaks of three independent measurements and reported as the mode of distribution ± standard deviation.
+
+## Glutaraldehyde Crosslinking
+
+Glutaraldehyde crosslinking was performed by incubating 1 μM ClpC1, Sa ClpC, or Ec ClpB in buffer C (50 mM Hepes, 150 mM KCl, 20 mM MgCl2, 5% [v/v] glycerol, and 2 mM
+DTT) in the absence or the presence of 2 mM ATPγS at 25 C
+for 5 min. About 5 μM CymA or 0.5 μM ClpS were added as indicated. Crosslinking was started by adding glutaraldehyde
+(Sigma) to a final concentration of 0.1%. Aliquots were taken at indicated time points, and crosslinking was quenched by adding Tris (pH 7.5) to a final concentration of 50 mM.
+
+Samples were subjected to SDS-PAGE, and gels were stained with SYPRO Ruby Protein Gel Stain.
+
+## Atpase Assay
+
+The ATPase activity of ClpC1 was determined using coupled reactions of pyruvate kinase (PK) and lactate dehydrogenase (LDH) in the presence of phosphoenolpyruvate
+(PEP) and NADH. Briefly, PK regenerates ADP into ATP
+under conversion of PEP to pyruvate. Pyruvate is then reduced to lactate by LDH with NADH as electron donor.
+
+The consumption of NADH can be followed by measuring the absorbance decrease at 340 nm. The reactions were started with the addition of ATP followed by a 10-min incubation to allow for the assembly of ClpC1. Measurements were carried out at 30 C in buffer D (50 mM Hepes [pH
+7.5], 150 mM KCl, 20 mM MgCl2, 1 mM DTT, and 10% [v/
+v] glycerol) in the presence of 5 mM ATP, 2 mM PEP (Sigma), 1 mM NADH (PanReak AppliChem), 6 U/ml PK (Roche), and 18 U/ml LDH (Sigma) using a BioTek Synergy 2 plate reader. Protein concentrations were as follows: 1 μM ClpC1, 2.3 μM Mtb ClpP1P2, 10 μM casein, and 5 μM
+CymA as indicated. Assays including Sa ClpP were done in buffer D at 30 C in the presence of 2 mM ATP. Proteins were used at the following concentration: 1 μM ClpC1, 1.5 μM Sa ClpP, 10 μM casein, 1.5 μM ClpS. 2 μM FR-LGFP, and 5 μM CymA as indicated. ATPase rates were calculated from the linear decrease of absorbance at 340 nm in at least three independent experiments, and standard deviations were calculated. All reactions included 1% (v/v) dimethyl sulfoxide (DMSO) to equal buffer conditions in the presence of CycA. The presence of DMSO did not affect ClpC1 activities.
+
+## Anisotropy Measurements
+
+Binding of ClpC1 to FITC-casein (100 nM) was monitored by fluorescence anisotropy measurements using a BMG
+Biotech CLARIOstar platereader. Samples were incubated in 50 mM Tris (pH 7.5), 25 mM KCl, 20 mM MgCl2, and 5% (v/v)
+glycerol for 10 min at 30 C in the presence of 2 mM ATP.
+
+About 10 μM CymA was added as indicated. Polarization of FITC-casein was determined in black 384-well plates (excitation: 482 nm; emission: 530 nm; and target mP: 35). A sample containing FITC-casein only served as reference. Kd values were determined using nonlinear regression curve fitting
+(Prism software; GraphPad Prism, Inc).
+
+## Degradation Assays
+
+All degradation assays were performed in buffer D (50 mM
+Hepes [pH 7.5], 150 mM KCl, 20 mM MgCl2, 2 mM DTT, and 10% [v/v] glycerol) using 3 μM ClpC1, 4.5 μM ClpP1P2, 0.4 mM
+activator peptide (benzyloxycarbonyl-L-leucyl-L-leucinal; PeptaNova) in the presence of an ATP-regenerating system (6 U/ml PK and 5 mM PEP). Reactions included 10 μM CymA as indicated. Sa ClpP was used at 1.5 μM concentration. Substrates were used at the following concentrations: 0.5 μM GFP-SsrA,
+0.5 μM FITC-casein (Sigma), 1 μM FR-L-GFP, and 5 μM
+VapB15. CymA was added to 10 μM final concentration. All reactions included 1% (v/v) DMSO to equal buffer conditions in the presence of CymA. The presence of DMSO did not affect ClpC activities. FITC-casein degradation was analyzed using a CLARIOstar plate reader, in black 96-well plates (Corning; nonbinding surface coated; flat bottom). The increase of FITCcasein fluorescence upon its degradation was monitored by using 483 and 520/530 nm as excitation and emission wavelengths, respectively. For data processing, the initial fluorescence intensities were set to 100. FITC-casein degradation rates were determined from the initial slopes of the fluorescence signal increase in at least three independent experiments, and standard deviations were calculated. For degradation of GFP-SsrA and FR-L-GFP, fluorescence was monitored in a 96-well plate
+(Corning; nonbinding surface coated, flat bottom) with a BioTek Synergy 2 Plate Reader using a 360/40 nm band-pass filter and 528/20 nm band-pass filter as excitation and emission wavelengths, respectively. Initial GFP fluorescence was set to 100.
+
+Degradation rates were determined from the initial slopes of fluorescence signal decrease in at least three independent experiments, and standard deviations were calculated.
+
+Degradation of VapB15 was followed by SDS-PAGE. Reactions were incubated at 37 C, and samples were taken at the indicated time points.
+
+## Negative-Staining Em
+
+ClpC1-WT and ClpC-F444S were in 20 mM Tris–HCl, pH
+7.5, 20 mM KCl, 15 mM MgCl2, and 1 mM DTT supplemented with 2 mM ATPγS. Proteins were applied to glow-discharged continuous carbon-coated grids (EM Sciences) and stained with 2% uranyl acetate. Micrograph movies were recorded on a Falcon 3 (Thermo Fisher Scientific) direct electron detector operated in linear mode at a magnification of 92,000× (pixel size = 1.54 Å/px; underfocus range = 0.5–1.2 μm) using a Talos Arctica microscope operated at 200 kV under low-dose conditions with a total dose of 20e− divided into 20 frames. Movies were aligned using MotionCor2 (33), contrast transfer function was estimated with Gctf (34), particles were automatically picked with X-mipp (35), extracted on boxes of 400 pixels, and 2D classified with Relion 3.0 (MRC Laboratory of Molecular Biology) (36), all within the Scipion Box frame (37). Around 12,000 particles from 2D classes corresponding to the ClpC1-
+WT resting state were selected and used for 3D ab initio generation and refinement using Relion 3.0 (36). The final structure envelope at 25 Å resolution shows a spiral assembly, reminiscent of the Sa ClpC decamer. The Sa ClpC model was rigid body fitted, and density was assigned to the head-to-head locked MDs recognized (Fig. S1D). When displayed at high threshold, the skeleton of two ClpC1 hexamers or pentamers is seen (Fig. S1D).
+
+## In Vivo Toxicity Assay
+
+Ec XL1 cells harboring pUHE21-encoded clpC1 alleles were grown in the absence of IPTG overnight at 30 C. Cells in addition harbored the plasmids pDMI.1 or pDMI.1-Sa clpP. Absorbance values at 600 nm were adjusted to 1. Serial dilutions (10−1–10−6) were prepared, spotted on LB plates containing different IPTG concentrations, and incubated for 24 h at indicated temperatures. Spot tests were performed in two or more independent experiments each, and representative results are provided.
+
+## Data Availability
+
+All data are contained within the article.
+
+Supporting information—This article contains supporting information (23–25, 38).
+
+Author contributions—E. W.-B. and A. M. conceptualization; G. T.,
+Y. F., P. K., D. L., M. C., and A.M. methodology; G. T., Y. F., P. K.,
+D. L., M. C., and A. M. investigation; G. T., Y. F., P. K., D. L., M. C.,
+E. W.-B., and A. M. formal analysis; A. M. writing–original draft; G. T., M. C., I.S., E. W.-B., and A.M. writing–review and editing; I. S., E. W.-B., and A. M. supervision; G. T., M. C., and A. M.
+
+visualization; E. W.-B. and A. M. funding acquisition.
+
+Funding and additional information—This work was supported by a grant of the Deutsche Forschungsgemeinschaft (grant no.:
+MO970/4-3) to A.M. and by an ETH Zurich research grant (grant no.: ETH-40 16-1) to E.W.-B. P.K. was supported by the Heidelberg Biosciences International Graduate School.
+
+Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.
+
+Abbreviations—The abbreviations used are: AAA, ATPase Associated with diverse cellular Activities; ADEP, acyldepsipeptide; Bs, Bacillus subtilis; CymA, CyclomarinA; DMSO, dimethyl sulfoxide; DWB, double Walker B variant; Ec, Escherichia coli; LDH, lactate dehydrogenase; MD, middle domain; Mtb, Mycobacterium tuberculosis; NTD, N-terminal domain; pArg, phosphoarginine; PEP,
+phosphoenolpyruvate; PK, pyruvate kinase; Sa, Staphylococcus aureus; SEC, size-exclusion chromatography; SEC–MALS, multiangle light scattering measurements coupled to SEC run.
+
+5. Trentini, D. B., Suskiewicz, M. J., Heuck, A., Kurzbauer, R., Deszcz, L.,
+Mechtler, K., et al. (2016) Arginine phosphorylation marks proteins for degradation by a Clp protease. Nature 539, 48–53 6. Erbse, A., Schmidt, R., Bornemann, T., Schneider-Mergener, J., Mogk, A.,
+Zahn, R., et al. (2006) ClpS is an essential component of the N-end rule pathway in Escherichia coli. Nature 439, 753–756 7. Ziemski, M., Leodolter, J., Taylor, G., Kerschenmeyer, A., and WeberBan, E. (2021) Genome-wide interaction screen for Mycobacterium tuberculosis ClpCP protease reveals toxin-antitoxin systems as a major substrate class. FEBS J. 288, 111–126 8. Schlothauer, T., Mogk, A., Dougan, D. A., Bukau, B., and Turgay, K.
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+(2003) MecA, an adaptor protein necessary for ClpC chaperone activity.
+
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+Mogk, A., et al. (2006) Adaptor protein controlled oligomerization activates the AAA+ protein ClpC. EMBO J. 25, 1481–1491 11. Wang, F., Mei, Z., Qi, Y., Yan, C., Hu, Q., Wang, J., et al. (2011) Structure and mechanism of the hexameric MecA-ClpC molecular machine. Nature 471, 331–335 12. Bhandari, V., Wong, K. S., Zhou, J. L., Mabanglo, M. F., Batey, R. A., and Houry, W. A. (2018) The role of ClpP protease in bacterial pathogenesis and human diseases. ACS Chem. Biol. 13, 1413–1425 13. Akopian, T., Kandror, O., Raju, R. M., Unnikrishnan, M., Rubin, E. J., and Goldberg, A. L. (2012) The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring. EMBO J. 31, 1529–1541 14. Famulla, K., Sass, P., Malik, I., Akopian, T., Kandror, O., Alber, M., et al.
+
+(2016) Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease. Mol. Microbiol. 101, 194–209 15. Brotz-Oesterhelt, H., and Vorbach, A. (2021) Reprogramming of the caseinolytic protease by ADEP antibiotics: molecular mechanism, cellular consequences, therapeutic potential. Front. Mol. Biosci. 8, 690902 16. Lee, H., and Suh, J. W. (2016) Anti-tuberculosis lead molecules from natural products targeting Mycobacterium tuberculosis ClpC1. J. Ind.
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+Schroeder, W., et al. (2005) Dysregulation of bacterial proteolytic machinery by a new class of antibiotics. Nat. Med. 11, 1082–1087 18. Kirstein, J., Hoffmann, A., Lilie, H., Schmidt, R., Rübsamen-Waigmann, H., Brötz-Oesterhelt, H., et al. (2009) The antibiotic ADEP reprogrammes ClpP, switiching it from a regulated to an uncontrolled protease. EMBO
+Mol. Med. 1, 37–49 19. Schmitt, E. K., Riwanto, M., Sambandamurthy, V., Roggo, S., Miault, C.,
+Zwingelstein, C., et al. (2011) The natural product cyclomarin kills Mycobacterium tuberculosis by targeting the ClpC1 subunit of the caseinolytic protease. Angew. Chem. Int. Ed. Engl. 50, 5889–5891 20. Gavrish, E., Sit, C. S., Cao, S., Kandror, O., Spoering, A., Peoples, A., et al.
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+(2014) Lassomycin, a ribosomally synthesized cyclic peptide, kills mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2. Chem. Biol. 21, 509–518 21. Gao, W., Kim, J. Y., Anderson, J. R., Akopian, T., Hong, S., Jin, Y. Y., et al.
+
+(2015) The cyclic peptide ecumicin targeting ClpC1 is active against Mycobacterium tuberculosis in vivo. Antimicrob. Agents Chemother. 59, 880–889 22. Choules, M. P., Wolf, N. M., Lee, H., Anderson, J. R., Grzelak, E. M.,
+Wang, Y., et al. (2019) Rufomycin targets ClpC1 proteolysis in Mycobacterium tuberculosis and M. Abscessus. Antimicrob. Agents Chemother.
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+63, e02204-18 23. Vasudevan, D., Rao, S. P., and Noble, C. G. (2013) Structural basis of mycobacterial inhibition by cyclomarin A. J. Biol. Chem. 288, 30883–30891 24. Wolf, N. M., Lee, H., Choules, M. P., Pauli, G. F., Phansalkar, R.,
+Anderson, J. R., et al. (2019) High-resolution structure of ClpC1rufomycin and ligand binding studies provide a framework to design and optimize anti-tuberculosis leads. ACS Infect. Dis. 5, 829–840 25. Wolf, N. M., Lee, H., Zagal, D., Nam, J. W., Oh, D. C., Lee, H., et al.
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+(2020) Structure of the N-terminal domain of ClpC1 in complex with the antituberculosis natural product ecumicin reveals unique binding interactions. Acta Crystallogr. D Struct. Biol. 76, 458–471 26. Maurer, M., Linder, D., Franke, K. B., Jager, J., Taylor, G., Gloge, F., et al.
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+Two-step activation mechanism of the ClpB disaggregase for sequential substrate threading by the main ATPase motor. Cell Rep. 27, 3433–3446.e4 30. Kotamarthi, H. C., Sauer, R. T., and Baker, T. A. (2020) The nondominant AAA+ ring in the ClpAP protease functions as an antistalling motor to accelerate protein unfolding and translocation. Cell Rep. 30, 2644–2654.e3 31. Marsee, J. D., Ridings, A., Yu, T., and Miller, J. M. (2018) Mycobacterium tuberculosis ClpC1 N-terminal domain is dispensable for adaptor protein-dependent allosteric regulation. Int. J. Mol. Sci. 19 32. Morreale, F. E., Kleine, S., Leodolter, J., Ovchinnikov, S., Kley, J., Kurzbauer, R., et al. (2021) BacPROTACs mediate targeted protein degradation in bacteria. Cell 185, 2338–2353.e18 33. Zheng, S. Q., Palovcak, E., Armache, J. P., Verba, K. A., Cheng, Y., and Agard, D. A. (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 34. Zhang, K. (2016) Gctf: Real-time CTF determination and correction. J.
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medical/md/PMC9431352.md

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+# P1735 Improvements In Patient-Reported Outcomes In Mitapivat-Treated Patients With
+
+![0_Image_0.Png](0_Image_0.Png)
+
+![0_Image_1.Png](0_Image_1.Png)
+
+![0_Image_2.Png](0_Image_2.Png) Pyruvate Kinase Deficiency: A Descriptive Analysis From The Phase 3 Activate Trial
+
+Topic: 35. Quality of life, palliative care, ethics and health economics Kevin H.M. Kuo1, Rachael F. Grace2, Eduard J. van Beers3, Wilma Barcellini 4, Andreas Glenthøj 5, Susanne Holzhauer 6, Parija Patel 7, Vanessa Beynon7, Junlong Li 7, Erin Zagadailov7, Hanny Al-Samkari 8 1 Division of Hematology, University of Toronto, Toronto, *Canada;*
+2 Dana-Farber/Boston Children's Cancer and Blood Disorder Center, Harvard *Medical* School, Boston, United *States;*
+3 Van Creveldkliniek, Department of Internal Medicine, University Medical Center Utrecht, *Netherlands;*
+4 Fondazione *IRCCS*
+Ca' Granda Ospedale MaggiorePoliclinico, Milan, *Italy;*
+5 Department of Hematology, Rigshospitalet, Copenhagen, *Denmark;*
+6 Department of *Pediatric* Hematology and Oncology, Charité University Medicine, Berlin, *Germany;*
+7 AgiosPharmaceuticals, Inc., Cambridge, United *States;*
+8 *Division* of Hematology, Massachusetts General Hospital, Harvard Medical School, Boston, United *States* Background: Pyruvate kinase (PK) deficiency is a rare, inherited, non-spherocytic hemolytic anemia associated with acute and long-term complications, as well as a spectrum of signs and symptoms including jaundice, fatigue, and dyspnea. PK deficiency has a profound wide-ranging impact on health-related quality of life (HRQoL). Mitapivat
+(AG-348), a first-in-class, oral, allosteric activator of PK, was shown to improve hemoglobin (Hb), hemolysis, and hematopoiesis in a global, phase 3, randomized, placebo (PBO)-controlled trial evaluating mitapivat efficacy and safety in adults with PK deficiency who were not regularly transfused (ACTIVATE; NCT03548220). Furthermore, significant improvements in patient-reported outcomes (PROs) (measured by disease-specific PRO instruments: the PK deficiency diary [PKDD] and the PK deficiency impact assessment [PKDIA]) were demonstrated in patients (pts)
+receiving mitapivat compared with PBO. The PKDD is a self-administered, 7-item daily diary to assess the signs and symptoms of PK deficiency (including jaundice, tiredness, and shortness of breath), while the PKDIA is a 12-item weekly measure assessing the impacts of PK deficiency (pts rated the frequency of occurrence or difficulty of various activities).
+
+Aims: To describe PKDD and PKDIA outcomes for the subset of pts in the ACTIVATE trial who achieved the primary endpoint of Hb response, defined as a ≥1.5 g/dL increase in Hb from baseline (BL), sustained at ≥2 scheduled assessments at weeks (wks) 16, 20, and 24.
+
+Methods: Eighty pts were randomized 1:1 to receive mitapivat (5/20/50 mg twice daily) or PBO for 24 wks in the ACTIVATE trial. Change from BL to wk 24 in PKDD and PKDIA scores were prespecified secondary endpoints. For this post hoc analysis, changes from BL in PKDD weekly mean scores and PKDIA scores measured at scheduled visits
+(wks 4, 8, 12, 16, 20, and 24) were summarized for Hb responders, the overall mitapivat treatment arm, and PBO, using mean and 95% confidence intervals (CIs). For both the PKDD and the PKDIA, a lower score represents a lower disease burden.
+
+Results: In the ACTIVATE trial, 16/40 (40%) pts receiving mitapivat met the primary endpoint of Hb response compared with none for PBO (0/40; 0%). At wk 24, mean (95% CI) change from BL in PKDD weekly mean score was ‒ 7.12 (‒ 10.98, ‒ 3.27) for pts who achieved Hb response, ‒ 5.43 (‒ 7.47, ‒ 3.40) for the overall mitapivat treatment arm, and ‒ 1.86 (–4.04, 0.32) for the PBO arm (Figure 1a). At wk 24, mean (95% CI) change from BL in PKDIA score was ‒ 8.07 (‒ 11.05, ‒ 5.08) for pts who achieved Hb response, ‒ 4.82 (‒ 7.18, ‒ 2.46) for the overall mitapivat treatment arm, and ‒ 1.06 (‒ 3.70, 1.59) for the PBO arm (Figure 1b). Across both PRO instruments, improvements among mitapivat pts who achieved Hb response were sustained over time.
+
+## Image:
+
+Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc.
+
+Summary/Conclusion: In the ACTIVATE trial, mitapivat-treated pts demonstrated significant improvements in signs,
+
+![1_image_0.png](1_image_0.png)
+
+![1_image_1.png](1_image_1.png)
+
+symptoms, and impacts based on PK deficiency-specific PRO instruments, compared with PBO. This analysis further reveals that improvements were greater in the subset of mitapivat-treated pts who achieved the primary endpoint.
+
+Together, these data indicate that mitapivat has the potential to improve HRQoL in pts with PK deficiency who are not regularly transfused.
+
+Copyright Information: (Online) ISSN: 2572-9241
+© 2022 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open access Abstract Book distributed under the Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) which allows third parties to download the articles and share them with others as long as they credit the author and the Abstract Book, but they cannot change the content in any way or use them commercially.
+
+Abstract Book Citations: Authors, Title, HemaSphere, 2022;6:(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx.
+
+Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc.

medical/md/PMC9434327.md

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+https://doi.org/10.1093/plphys/kiac271  2022: 190: 882–897
+
+# Histone Deacetylase 15 And Mos4-Associated Complex Subunits 3A/3B Coregulate Intron Retention Of Aba-Responsive Genes
+
+Yi-Tsung Tu ,
+1 Chia-Yang Chen,1 Yi-Sui Huang ,
+1 Chung-Han Chang ,
+1 Ming-Ren Yen ,
+2 Jo-Wei Allison Hsieh ,
+2,3 Pao-Yang Chen 2,3,*
+,† and Keqiang Wu 1,*
+,†
+1 Institute of Plant Biology, National Taiwan University, Taipei 10617, Taiwan 2 Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan 3 Genome and Systems Biology Degree Program, Academia Sinica and National Taiwan University, Taipei 10617, Taiwan
+*Authors for correspondence: kewu@ntu.edu.tw (K.W.), paoyang@gate.sinica.edu.tw (P.-Y.C.)
+†Senior authors These authors contributed equally (Y.-T.T. and C.-Y.C.) Y.-T.T., P.-Y.C., and K.W. designed the study, and wrote and edited the manuscript. Y.-T.T., C.-Y.C., Y.-S.H., C.-H.C., M.-R.Y., and J.-W.A.H. performed the experiments and analyzed the data. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Keqiang Wu (kewu@ntu.edu.tw).
+
+## Abstract
+
+Histone deacetylases (HDAs) play an important role in transcriptional regulation of multiple biological processes. In this study, we investigated the function of HDA15 in abscisic acid (ABA) responses. We used immunopurification coupled with mass spectrometry-based proteomics to identify proteins interacting with HDA15 in Arabidopsis (Arabidopsis thaliana).
+
+HDA15 interacted with the core subunits of the MOS4-associated complex (MAC), MAC3A and MAC3B, with interaction between HDA15 and MAC3B enhanced by ABA. hda15 and mac3a/mac3b mutants were ABA-insensitive during seed germination and hyposensitive to salinity. RNA sequencing analysis demonstrated that HDA15 and MAC3A/MAC3B coregulate ABA-responsive intron retention (IR). Furthermore, HDA15 reduced the histone acetylation level of genomic regions near ABA-responsive IR sites and the association of MAC3B with ABA-responsive pre-mRNA was dependent on HDA15. Our results indicate that HDA15 is involved in ABA responses by interacting with MAC3A/MAC3B to mediate splicing of introns.
+
+## Introduction
+
+Posttranslational modifications of histones including methylation, acetylation, phosphorylation, ubiquitination, and SUMOylation play important roles in modulating gene expression involved in many essential biological processes
+(Chen and Tian, 2007; Kim et al., 2015; Ueda and Seki, 2020). Histone acetylation catalyzed by histone acetyltransferases (HATs) relaxes chromatin structure, which is essential for gene activation (Fisher and Franklin, 2011). Histone deacetylases (HDAs or HDACs) remove the acetyl groups from histones, resulting in chromatin compaction and gene repression. Most studies have focused on histone H3 and H4 acetylation since H3 and H4 are more preferable substrates for HATs and HDAs. At least four lysine residues (K9, K14, K18, and K23) on H3 and 5 lysine residues (K5, K8, K12, K16, and K20) on H4 can be acetylated by HATs and Research Article deacetylated by HDAs (Chen and Tian, 2007). In Arabidopsis
+(Arabidopsis thaliana), HDAs are grouped into three families:
+the Reduced Potassium Dependency 3 (RPD3)/HDA1 superfamily, Silent Information Regulator 2 family, and HD2 family
+(Pandey et al., 2002; Liu et al., 2014).
+
+HDA15, a RPD3/HDA1-type HDA, has been reported to mediate different biological processes including photomorphogenesis, abscisic acid (ABA) response, and stress tolerance. HDA15 associates with PIF3 and is involved in the regulation of chlorophyll biosynthesis and photosynthesis by reducing histone H4 acetylation levels in the dark (Liu et al.,
+2013). HDA15 also interacts with PIF1 to regulate the transcription of the light-responsive genes involved in multiple hormonal signaling pathways and cellular processes in germinating seeds (Gu et al., 2017). Furthermore, HDA15 acts cooperatively with the NC-YC transcriptional factors to regulate light control of hypocotyl elongation by co-regulating the transcription of the light-responsive genes (Tang et al., 2017). In addition, HDA15 also interacts with ELONGATED
+HYPOCOTYL 5 involved in repressing hypocotyl cell elongation during photomorphogenesis (Zhao et al., 2019). More recent studies indicate that HDA15 is also involved in abiotic stress responses. HDA15 and LONG HYPOCOTYL IN
+FAR-RED 1 cooperatively regulate several warm temperature marker genes such as HEAT SHOCK PROTEIN 20 (HSP20),
+INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19), and IAA29 in plant response to elevated ambient temperature (Shen et al., 2019). Furthermore, MYB96 forms a complex with HDA15 to repress the expression of RHO GTPASE OF PLANTS in ABA and drought stress responses (Lee and Seo, 2019). In addition, HDA15 can be phosphorylated in vivo and HDA15 phosphorylation results in the loss of enzymatic activity and functions (Chen et al., 2020).
+
+MOS4-associated complex (MAC), a highly conserved complex in eukaryotes, is the Arabidopsis orthologous complex of the NINETEEN COMPLEX (NTC) or Prp19 complex
+(Prp19C) in yeast and humans (Palma et al., 2007). The NTC/Prp19C function is essential for the pre-mRNA splicing reaction and is involved in the progression of spliceosome rearrangement (Chan et al., 2003; Hogg et al., 2010). The Arabidopsis MAC complex consists of over 20 proteins, and many of them are involved in alternative splicing (AS) of mRNA (Monaghan et al., 2009, 2010; Zhang et al., 2014).
+
+MAC also controls miRNA levels through modulating primiRNA transcription, processing, and stability (Jia et al., 2017; Li et al., 2018a). The core subunits of the MAC complex, MOS4, CELL DIVISION CYCLE5 (CDC5), PLEIOTROPIC
+REGULATORY LOCUS 1 (PRL1), MAC3A/3B, and MAC5A/
+5B/5C are required for plant immunity, since their loss-offunction mutations result in plants exhibiting enhanced susceptibility to pathogen infection (Palma et al., 2007; Monaghan et al., 2009, 2010). More recently, it has been found that MAC3A/MAC3B interacts with three other splicing factors, SKI-INTERACTING PROTEIN, PRL1, and SPLICEOSOMAL TIMEKEEPER LOCUS1, to mediate the splicing of pre-mRNAs encoded by circadian clock- and abiotic stress response-related genes (Li et al., 2019).
+
+The involvement of histone modifications in splicing regulation has been reported in yeasts and animals (Pajoro et al., 2017). Two models have been proposed to explain how histone modifications regulate AS (Rahhal and Seto, 2019). In the kinetic coupling model, condensed chromatin caused by histone deacetylation slows down the transcription elongation rate. Therefore, the splicing factors are recruited to the weak splicing sites, causing exon inclusion. In contrast, acetylated histones lead to looser chromatin and a faster elongation rate. As a result, the splicing factors are recruited to the strong splicing sites, leading to exon skipping (ES) (Rahhal and Seto, 2019). In the chromatin-adaptor model, chromatin-binding proteins read the histone marks and recruit different splicing regulators. For example, the heterochromatin 1 protein HP1r recognizes the H3K9me3 mark to facilitate inclusion of the alternative exons via decreasing transcriptional elongation rates in human (Saint-Andre´ et al., 2011). In addition, deletion of the yeast HDACs Hos3 and Hos2 reduces the recruitment of Prp19 and small nuclear ribonucleoproteins (snRNP) to pre-mRNA, suggesting that histone acetylation is coupled with spliceosome dynamics (Gunderson et al., 2011). However, the role of HDAs in splicing regulation in plants remains elusive.
+
+In this study, we found that HDA15 interacts with the core subunits of the MAC complex, MAC3A/MAC3B. The loss of function mutants of both HDA15 and MAC3A/MAC3B are hyposensitive to ABA and salinity. Transcriptome analyses demonstrate that HDA15 and MAC3A/MAC3B are involved in ABA-mediated splicing regulation, especially IR. Furthermore, the association of MAC3B with ABA-responsive pre-mRNA is dependent on HDA15. Our studies indicate that HDA15 is involved in ABA responses by interacting with MAC3A/MAC3B to mediate splicing of introns.
+
+## Results
+
+HDA15 interacts with MAC3A and MAC3B in vivo and their interaction is enhanced by ABA
+A previous study has shown that HDA15 interacts with the transcription factor MYB96 to regulate the gene expression involved in ABA responses (Lee and Seo, 2019). To further investigate the function of HDA15 in ABA responses, we performed immunopurification coupled with mass spectrometry-based proteomics to identify the HDA15 interacting proteins. Proteins extracted from 10-day-old Col-0 wild-type (WT) and hda15-1 plants treated with or without
+(mock) ABA were used for immunopurification using an anti-HDA15 antibody. Proteins that were only identified in WT immunoprecipitation (IP) samples but not in the control (hda15-1) were considered to be the potential interaction proteins of HDA15. SIN3-LIKE 2 (SNL2), a core subunit of the SIN3-HDAC complex (Gonzalez et al., 2007), was found to be co-purified with HDA15 in both mock and ABA treatment. Interestingly, the core subunits of MAC, MAC3B, MAC5A, and CDC5 (Palma et al., 2007; Monaghan 884 |  2022: 190; 882–897 Tu et al.
+
+| Table 1 List of the proteins copurified with HDA15 by LC–MS/MS Mock   | ABA         |                               |                               |                          |                               |                          |       |                               |    |    |       |    |    |
+|-----------------------------------------------------------------------|-------------|-------------------------------|-------------------------------|--------------------------|-------------------------------|--------------------------|-------|-------------------------------|----|----|-------|----|----|
+| Replicate 1                                                           | Replicate 2 | Replicate 1                   | Replicate 2                   |                          |                               |                          |       |                               |    |    |       |    |    |
+| Accession                                                             | Symbol      | Score                         | Num. of significant sequences | Num. of unique sequences |                               |                          |       |                               |    |    |       |    |    |
+| AT3G18520                                                             | HDA15       | 1163                          | 21                            | 28                       | 1,433                         | 23                       | 29    | 1201                          | 20 | 28 | 1,121 | 22 | 29 |
+| AT5G15020                                                             | SNL2        | 32                            | 1                             | 13                       | 34                            | 1                        | 22    | 50                            | 1  | 14 | 32    | 1  | 25 |
+| AT5G40490                                                             | RBGD5       | 89                            | 2                             | 5                        | -                             | -                        | -     | 133                           | 2  | 5  | 106   | 2  | 6  |
+| AT2G33340                                                             | MAC3B       | -                             | -                             | -                        | -                             | -                        | -     | 10                            | 1  | 1  | 30    | 1  | 5  |
+| AT1G07360                                                             | MAC5A       | -                             | -                             | -                        | -                             | -                        | -     | 11                            | 1  | 1  | 34    | 1  | 9  |
+| AT1G09770                                                             | CDC5        | -                             | -                             | -                        | -                             | -                        | -     | 10                            | 1  | 1  | -     | -  | -  |
+| AT2G13540                                                             | ABH1        | -                             | -                             | -                        | -                             | -                        | -     | 9                             | 1  | 1  | -     | -  | -  |
+| AT3G19670                                                             | PRP40B      | -                             | -                             | -                        | -                             | -                        | -     | 12                            | 1  | 1  | -     | -  | -  |
+| AT1G20580                                                             | SMD3B       | -                             | -                             | -                        | -                             | -                        | -     | -                             | -  | -  | 39    | 1  | 6  |
+| Num. of unique sequences                                              | Score       | Num. of significant sequences | Num. of unique sequences      | Score                    | Num. of significant sequences | Num. of unique sequences | Score | Num. of significant sequences |    |    |       |    |    |
+
+et al., 2009, 2010), were identified only in ABA treatment conditions. Several splicing regulators such as PRP40B, ABH1, and SmD3b (Kanno et al., 2017; Hugouvieux et al., 2001, Deng et al., 2016) were also identified (Table 1).
+
+Previous studies indicate that MAC3A and MAC3B function redundantly in plant innate immunity (Monaghan et al., 2009) and salt tolerance (Li et al., 2019). The interaction between MAC3A/MAC3B and HDA15 was further confirmed by bimolecular fluorescence complementation (BiFC)
+assays and co-IP assays. As shown in Figure 1A, both MAC3A and MAC3B interacted with HDA15 in the nucleus in Arabidopsis protoplasts in BiFC assays.
+
+For Co-IP assays, MAC3A or MAC3B fused with GFP
+(Pro35S:GFP-MAC3A or Pro35S:GFP-MAC3B) was transformed into Arabidopsis protoplasts. An anti-HDA15 antibody was used for IP and an anti-GFP antibody was then used for immunoblot analysis. GFP-MAC3A and GFP-MAC3B proteins could be precipitated by the anti-HDA15 antibody in Arabidopsis protoplasts (Figure 1B), supporting that MAC3A and MAC3B interact with HDA15 in vivo.
+
+Moreover, we also examined the interaction of HDA15 and MAC3B under ABA treatment conditions by Co-IP assays and split-luciferase complementation (SLC) assays. In the Co-IP assay, the Pro35S:GFP-MAC3B/mac3a mac3b (mac3a3b) transgenic plants expressing GFP-MAC3B driven by the 35S
+promoter in the mac3a/mac3b background were treated with or without ABA. Although GFP-MAC3B was coimmunoprecipitated by endogenous HDA15 in both mock and ABAtreated conditions, the interaction between HDA15 and MAC3B was increased 2.6- to 4.1-fold in two independent biological replicates after ABA treatment (Figure 1C and Supplemental Figure S1). In the SLC assay, HDA15 also interacted with MAC3B in Nicotiana benthamiana leaves and ABA treatment promoted this interaction (Figure 1, D and E).
+
+Taken together, these experiments indicated that ABA promotes the interaction between HDA15 and MAC3B.
+
+mac3a/mac3b plants are hyposensitive to salt stress and ABA
+Since HDA15 is involved in abiotic stress and ABA responses (Lee and Seo, 2019), we also investigated whether MAC3A
+and MAC3B are also involved in abiotic stress and ABA
+responses by comparing the phenotypes of the hda15, mac3a, and mac3b single mutants as well as the mac3a/ hda15, mac3b/hda15, and mac3a/mac3b double mutants.
+
+After salt stress treatment, the survival rates of hda15-1 and mac3a/mac3b plants ranged from 50% to 60%, whereas the survival rate of WT plants was -30% (Figure 2, A and B).
+
+The water loss rates of detached leaves in hda15-1 and mac3a/mac3b plants were also higher compared with WT
+(Figure 2C). Furthermore, hda15 and mac3a/mac3b seeds had higher germination rates on the medium containing ABA (Figure 2D). We also generated Pro35S:GFP-MAC3A/
+mac3a3b and Pro35S:GFP-MAC3B/mac3a3b transgenic plants, in which GFP-MAC3A and GFP-MAC3B driven by the 35S promoter were transformed in to the mac3a mac3b double mutant, respectively. Both Pro35S:GFP-MAC3A/ mac3a3b and Pro35S:GFP-MAC3B/mac3a3b could rescue the ABA hyposensitive phenotype of mac3a/mac3b (Figure 2E).
+
+These results indicate that similar to HDA15, MAC3A and MAC3B are also involve in abiotic stress and ABA responses. However, no obvious phenotype difference was observed in the mac3a and mac3b single mutants compared with WT. Furthermore, the phenotypes of mac3a/hda15 and mac3b/
+hda15 double mutants were similar to that of the hda15 single mutant in salt and ABA responses as well as leaf transpiration rates (Figure 2). These observations support the notion that MAC3A and MAC3B function redundantly in abiotic stress and ABA responses.
+
+Transcriptome analysis of hda15-1 and mac3a/mac3b mutants To further characterize the role of HDA15, MAC3A, and MAC3B in ABA responses, we analyzed HDA15 and MAC3A/MAC3B regulated transcriptome changes. Ten-dayold Col-0, hda15-1, and mac3a/mac3b plants treated with or without (mock) ABA were used for RNA-sequencing (RNAseq) analysis. Two independent biological replicates for each condition were performed. After low-quality read trimming, more than 94% of the reads for every replicate were mapped to Araport11 (Supplemental Table S1). Compared
+
+![3_image_0.png](3_image_0.png)
+
+![4_image_0.png](4_image_0.png)
+
+with WT in mock conditions, 78 and 705 genes displayed higher transcript levels (fold change 51.5 with P50.05) in hda15-1 and mac3a/mac3b plants, respectively
+(Supplemental Figure S2 and Supplemental Data Set S1).
+
+Among these upregulated genes, only nine of them (Fisher's exact test, P56.9e–06) were overlapping genes that were upregulated in both hda15-1 and mac3a/mac3b
+(Supplemental Figure S2 and Supplemental Data Set S2). We also identified 115 and 965 downregulated genes in hda15-1 and mac3a/mac3b plants, respectively (Supplemental Figure S2 and Supplemental Data Set S1). Among these downregulated genes, 18 of them (Fisher's exact test, P51.8e–10)
+were overlapping genes (Supplemental Figure S2 and Supplemental Data Set S2). The stress-responsive genes RESPONSIVE TO DESICCATION 29B, GLUTATHIONE STRANSFERASE 7, and LATE EMBRYOGENESIS ABUNDANT 7 upregulated by salt and drought stress (Hundertmark and Hincha, 2008; Nakashima et al., 2006; Seok et al., 2020) were affected in both hda15 and mac3a/mac3b plants
+(Supplemental Data Set S2).
+
+Under ABA treatment, 108 and 1,058 genes were upregulated (fold change 51.5 with P50.05) in hda15-1 and mac3a/mac3b, respectively (Supplemental Figure S2 and Supplemental Data Set S1). Among these upregulated genes, 30 of them (Fisher's exact test, P55.8e–23) were overlapping genes that were upregulated in both hda15-1 and mac3a/mac3b (Supplemental Figure S2 and Supplemental Data Set S2). There were 137 and 1,334 downregulated genes in hda15-1 and mac3a/mac3b, respectively
+(Supplemental Figure S2 and Supplemental Data Set S1),
+and 34 of them (Fisher's exact test, P56.5e–21) were overlapping genes (Supplemental Figure S2 and Supplemental Data Set S2). The salt stress-responsive genes SODIUM HYDROGEN EXCHANGER 3 and HSP22 (Li et al., 2018a, 2018b; Ma et al., 2018) were affected in both hda15 and mac3a/mac3b plants under ABA treatment (Supplemental Data Set S2).
+
+Together, our genome-wide expression analyses suggest that HDA15 and MAC3A/MAC3B coregulate a subset of genes involved in ABA responses. In addition, a large number of differentially expressed genes that are not overlapped in hda15-1 and mac3a/mac3b plants indicate that HDA15 and MAC3A/MAC3B also function independently in gene regulation.
+
+HDA15 and MAC3A/MAC3B are involved in AS in ABA responses MAC3A and MAC3B are the core subunits of MAC, which plays a critical role in pre-mRNA splicing (Palma et al., 2007; Monaghan et al., 2009, 2010). We further explored whether HDA15 and MAC3A/MAC3B are involved in the coregulation of pre-mRNA splicing by analyzing genome-wide AS. AS events can be classified into three types: alternative donor and/or acceptor (AltD/A), ES, and intron retention (IR). Compared with Col-0 in mock treatment, 1,050 and 15,260 additional IR events were identified in hda15-1 and mac3a/mac3b, respectively (Figure 3A and Supplemental Data Set S3). In ABA treatment, IR events were increased to 2,177 and 26,555 in hda15-1 and mac3a/mac3b, respectively
+(Figure 3A and Supplemental Data Set S4). Since IR is the major mode of AS events, we further analyzed the patterns of the IR events by monitoring relative IR levels. The IR level for a given intron was defined as the read coverage depth of the intron divided by that of the two neighboring exons
+(Shih et al., 2019). hda15-1 and mac3a/mac3b had higher IR
+levels than Col-0 both in mock and ABA treatment (Figure 3B).
+
+We further analyzed the coregulated IR events in hda15-1 and mac3a/mac3b. The Venn diagram showed that among the 591 coregulated IR events in mock treatment, 441 of them (74.6%) were co-enhanced as shown in the quadrant plot (Figure 4A). In ABA treatment, among the 1,450 coregulated events, 1,027 of them (70.8%) showed a similar enhancement pattern in hda15-1 and mac3a/mac3b
+(Figure 4B). In the co-enhanced IR events, 418 and 1,014 events were specifically occurred in mock and in ABA treatment, respectively (Figure 4C). The Gene Ontology (GO)
+analysis showed that "response to salt stress" and "response to wounding" were enriched in both mock and ABA treatment (Figure 4D). Interestingly, ABA- and stress-related GO
+terms such as "response to salt stress, cold, and ABA" were highly increased in ABA treatment (Figure 4D). Taken together, these results indicate that HDA15 and MAC3A/
+MAC3B coregulate IR of ABA- and stress-related genes in ABA response.
+
+## Hda15 And Mac3A/Mac3B Affect Aba-Responsive
+
+IR
+To further uncover the involvement of HDA15 and MAC3A/MAC3B in ABA-responsive IR, we analyzed the
+"ABA-responsive IR events" in Col-0, hda15-1, and mac3a/
+mac3b. The IR events with more than two-fold enrichment
+(P50.005) after ABA treatment in Col-0 were defined as ABA-responsive IR events, and 2,252 ABA-responsive IR events were identified (Figure 5A and Supplemental Data Set S5). We further examined whether HDA15 and MAC3A/ MAC3B regulate these ABA-responsive IR events. After ABA treatment, 266 (12%) and 236 (11%) of these IR events were not altered in hda15 (HDA15-specific) and mac3a/mac3b (MAC3A/3B-specific), respectively. 144 (6%) of these IR
+events were also affected in hda15 and mac3a/mac3b
+(HDA15 or MAC3A/3B-unrelated) similar to Col-0.
+
+Interestingly, 1,606 (71%) of these IR events were not changed by ABA in both hda15 and mac3a/mac3b mutants
+(HDA15 and MAC3A/3B-codependent), indicating that HDA15 and MAC3A/MAC3B co-regulate these ABAresponsive IR events under ABA treatment (Figure 5A).
+
+Moreover, we compared the IR levels of the ABAresponsive IR events in Col-0 with those in hda15-1 and mac3a/mac3b. The ABA-responsive IR levels were slightly increased in Col-0, hda15-1, and mac3a/mac3b when treated with ABA (Figure 5B). Intriguingly, the IR level of mac3a/
+mac3b in mock or ABA was higher than that of Col-0, suggesting that MAC3A/MAC3B are important for ABAresponsive IR. We further divided the ABA-responsive IR
+events into the ABA-enhanced or ABA-reduced IR events. In the ABA-enhanced IR events, the IR level was increased 3.7fold in Col-0 after ABA treatment, compared with 1.5-fold increases in hda15-1 and mac3a/mac3b (Figure 5C). In the ABA-reduced IR events, the IR level was decreased to 4.8-
+
+![6_image_0.png](6_image_0.png)
+
+![6_image_1.png](6_image_1.png)
+
+fold in Col-0 after ABA treatment, whereas the IR level was not affected in hda15-1 and mac3a/mac3b (Figure 5D).
+
+Taken together, these data suggest that HDA15 and MAC3A/MAC3B play important roles in modulating ABAresponsive IR, especially in the ABA-reduced IR.
+
+The effect of ABA on IR of five selected ABA-responsive genes was further analyzed. Two genes with the ABA-enhanced IR events including SUCROSE
+NONFERMENTING 1-RELATED PROTEIN KINASE 2.3
+(SNRK2.3), and SDIR1-INTERACTING PROTEIN1 (SDIRIP1),
+and three genes with ABA-reduced IR events including PYRIMIDINE 1 (PYD1), ABA INSENSITIVE RING PROTEIN 2
+(AIRP2), and ENHANCED DOWNY MILDEW 1 (EDM1), were validated by reverse transcription quantitative PCR (RTqPCR). The SnRK2.3 protein kinase is an important positive regulator of ABA signaling (Fujita et al., 2009). AIRP2, a RING-type E3 ubiquitin ligase, modulates ABA signaling by ubiquitination of the substrate SDIRIP1 (Oh et al., 2017). EDM1, which may act as a scaffold protein to connect HSC70/HSP90 functions (Catlett and Kaplan, 2006), plays a role in ABA-mediated stomatal closure and seed germination (Cle´ment et al., 2011). PYD1, which promotes the degradation of pyrimidine nucleobases, is a dihydropyrimidine dehydrogenase as well as an ABA-responsive gene
+(Cornelius et al., 2011). The relative IR levels were shown in Supplemental Figure S3. As shown in Figure 6, the IR forms of SNRK2.3 and SDIRIP1 were significantly increased in ABA
+treatment in Col-0 compared with hda15 and mac3a/
+mac3b. The IR forms of PYD1, AIRP2, and EDM1 were significantly decreased in ABA treatment in Col-0 compared with hda15 and mac3a/mac3b. Together, these results indicate that HDA15 and MAC3A/MAC3B coregulate IR of ABAresponsive genes under ABA condition.
+
+HDA15 affects the histone acetylation levels near the ABA-responsive IRs Since HDAs remove acetyl groups from e-N-acetyl lysine residues of histones, we further investigated whether HDA15 and MAC3A/MAC3B affect the histone acetylation levels at the nearby introns of the ABA-responsive IR events by chromatin immunoprecipitation (ChIP) assays. We examined the H3K9ac levels of -250 bp upstream and downstream regions of the corresponding introns (P1 and P2), and 750 bp downstream regions as the control (P3). The H3K9ac
+
+![7_image_0.png](7_image_0.png)
+
+levels near the third intron of SNRK2.3, the first intron of SDIRIP1, the second intron of EDM1, the third intron of AIRP2, and the fourth intron of PYD1 were analyzed. We found that the P1 and P2 regions of the corresponding introns were increased in hda15 compared with Col-0
+(Supplemental Figure S4). Furthermore, ABA also increased the H3K9ac levels of these regions compared with mock conditions. Although the H3K9ac level of P3 region was also increased in hda15-1 compared with Col0, the signal was lower than that of P1 and/or P2 regions in both mock and ABA treatment. However, there was no significant difference in the H3K9ac levels in mac3a/
+mac3b compared with Col-0 (Supplemental Figure S4).
+
+These results show that HDA15 affects the histone acetylation level of the regions near ABA-responsive IRs, and ABA treatment enhances this effect. However, MAC3A/
+MAC3B do not affect the histone acetylation level of the regions near ABA-responsive IRs.
+
+The ABA-enhanced association of MAC3B with pre-mRNA depends on HDA15 Since deletion of the yeast HDACs Hos3 and Hos2 reduces the recruitment of Prp19 to pre-mRNA (Gunderson et al., 2011), we investigated whether HDA15 and MAC3A/ MAC3B associate with the pre-mRNA of the genes with ABA-responsive IR events. ProHDA15:HDA15-GFP/hda15-1 plants expressing HDA15 fused with GFP (HDA15-GFP)
+driven by the HDA15 native promoter in the hda15 mutant background were generated. ProHDA15:HDA15-GFP/hda15-1, Pro35S:GFP-MAC3B/mac3b, and Pro35S:GFP-MAC3B/mac3b hda15-1 transgenic plants were used for RNAimmunoprecipitation (RIP) assays and determined the RNA
+
+890 |  2022: 190; 882–897 Tu et al.
+
+![8_image_0.png](8_image_0.png)
+
+abundance by RT-qPCR. We used an anti-GFP antibody to immunoprecipitate GFP-fusion proteins extracted from the transgenic plants treated with or without ABA. ACTIN7 premRNA was used as a negative control (Supplemental Figure S5). SNRK2.3 and SDIRIP1 pre-mRNAs were abundantly detected in the immunoprecipitants from ProHDA15:HDA15-GFP/hda15-1, Pro35S:GFP-MAC3B/mac3b, and Pro35S:GFP-MAC3B/mac3b hda15-1, but not from Col-0 (Figure 7, B and C). Moreover, the levels of SNRK2.3 and SDIRIP1 pre-mRNAs in Pro35S:GFP-MAC3B/mac3b were substantially elevated by ABA treatment (Figure 7C). In addition, PYD1 pre-mRNAs could be detected in ProHDA15:HDA15-GFP/hda15-1, Pro35S:GFP-MAC3B/mac3b, and Pro35S:GFP-MAC3B/mac3b hda15-1 under ABA treatment (Figure 7, B and C). Interestingly, the increased enrichment of SNRK2.3, SDIRIP1, and PYD1 pre-mRNAs with GFPMAC3B by ABA treatment was disrupted in the hda15 mutant (Figure 7C), suggesting that the binding of MAC3B to these ABA-responsive pre-mRNAs depends on HDA15.
+
+HDA activity is not necessary for HDA15 in ABA-responsive IR
+We propose that HDA15 removes H3K9ac of the regions near ABA-responsive IRs and facilitates MAC3B binding to pre-mRNA. Our previous study has revealed that the amino acid residues H277 and D313 are important for the catalytic activity of HDA15 (Zhao et al., 2019). To investigate whether the deacetylase activity of HDA15 is essential for its function in pre-mRNA splicing, the transgenic plants expressing GFP-HDA15 bearing mutations in these active sites (H277A
+and D313A) in the hda15 mutant (Zhao et al., 2019) were used to analyze the effect of ABA on IR of five selected ABA-responsive genes. Both WT and mutated versions of GFP-HDA15 could rescue the IR level of the hda15 mutant
+(Supplemental Figure S6), indicating that the HDA activity is not necessary for HDA15 in regulation IR of ABA-responsive genes under ABA treatment.
+
+## Discussion
+
+In yeast and mammals, HDACs exist as components of multiprotein complexes (De Ruijter et al., 2003; Sengupta and Seto, 2004). These HDAC multiprotein complexes interact with a wide variety of transcription repressors and corepressors, providing flexibility and specificity in modulating chromatin structure and transcription (Sengupta and Seto, 2004; Grzenda et al., 2009; McDonel et al., 2009). Mammalian class I HDACs, HDAC1, HDAC2, and HDAC3, are found in four different multiprotein complexes including Sin3, NuRD, CoREST, and NCoR/SMRT complexes (Hayakawa and
+
+HDA15 and MAC3A/MAC3B coregulate IR  2022: 190; 882–897 | 891
+
+![9_image_0.png](9_image_0.png)
+
+Nakayama, 2010). The Sin3 and NuRD complexes are highly conserved from yeast to humans.
+
+Six Sin3 homologs, SNL1 (Sin3-Like1), SNL2, SNL3 (AtSin3),
+SNL4, SNL5, and SNL6, are found in Arabidopsis thaliana
+(Bowen et al., 2010). SNL1 and SNL2 are involved in seed dormancy through regulating the ABA-ethylene antagonism
+(Wang et al., 2013). Furthermore, SNL1 can also interact with HDA19 in vitro (Wang et al., 2013). Using immunopurification coupled with mass spectrometry-based proteomics, we found that HDA15 interacts with SNL2, indicating that HDA15 is also a component of the Sin3-HDAC complex. HDA15 also interacts with the core subunits of the MAC complex, MAC3A and MAC3B. Interestingly, the interaction between HDA15 and MAC3B is enhanced by ABA. Moreover, the increased association of MAC3B to the ABA-responsive premRNAs of SNRK2.3, SDIRIP1, and PYD1 depends on HDA15.
+
+Taken together, these results indicate that HDA15 and MAC3A/MAC3B interaction is involved in ABA responses.
+
+![10_image_0.png](10_image_0.png)
+
+The spliceosome is a large dynamic macromolecular complex, which removes the intron in the processes of AS (Lee and Rio, 2015). Two U1 snRNPs, AtU1A and LUC7, are found to be required for abiotic stress tolerance. The atu1a mutant showed a hypersensitive phenotype in salt stress responses. RNA-seq analysis indicated that AtU1A regulates AS in many genes by modulating recognition of 50 splice sites (Gu et al., 2018). Similar with the atu1a mutant, the luc7 triple mutants also exhibited hypersensitivity in salt stress responses in root length. Interestingly, LUC7 preferentially promotes in the removal of a subset of terminal introns (De Francisco Amorim et al., 2018). Our study indicated that hda15 and mac3a/mac3b mutants are ABAinsensitive in seed germination and hyposensitive to salinity.
+
+RNA-seq and RT-qPCR analysis indicated that the IR form of SDIRIP1 was reduced in hda15-1 and mac3a/mac3b mutants, which may result in more functional SDIRIP1. By contrast, the IR form of AIRP2 was increased in the mutants, which may generate more nonfunctional AIRP2. In ABA signaling, SDIRIP1, a substrate of AIRP2, selectively regulates the expression of the bZIP transcription factor ABI5 (Oh et al., 2017). Increased SDIRIP1 functions might attenuate ABI5 activity to affect ABA responses in hda15-1 and mac3a/mac3b mutants.
+
+Spliceosome assembly is strongly coupled with histone modifications including acetylation and deacetylation (Gunderson and Johnson, 2009; Hnilicova´ et al., 2011). It has been shown that the activity of HDACs modulates the AS in about 700 human genes (Hnilicova´ et al., 2011).
+
+Moreover, inhibition or depletion of HDAC1 increased histone H4 acetylation surrounding the alternative exon
+(Hnilicova´ et al., 2011). The yeast histone HAT Gcn5 is required for the association of U2 snRNP to the splicing branchpoint (Gunderson and Johnson, 2009). Mutation or deletion of Gcn5-targeted histone H3 residues leads to the intron accumulation of the Gcn5 target genes (Gunderson et al., 2011). In yeast, Prp19 is a core component of the Prp19C, also known as NTC, which functions in splicing and stabilizes U5/U6 snRNP in the spliceosomal complex (Chan et al., 2003). The deletion of the yeast HDACs Hos3 and Hos2 reduces the recruitment of Prp19 to pre-mRNA (Gunderson et al., 2011). MAC3A and MAC3B are the plant orthologs of Prp19 (Monaghan et al., 2009). The interaction of MAC3A and MAC3B with HDA15 indicates that they may be functionally associated. However, we found that the H3K9 acetylation levels of SNRK2.3, SDIRIP1, EDM1, AIRP2, and PYD1 are increased in the hda15 mutant but not in the mac3a/mac3b mutant, suggesting that the splicing mediated by MAC3A/MAC3B may not be dependent on histone deacetylation. However, the association of MAC3B with ABA-responsive pre-mRNA is dependent on HDA15, suggesting that HDA15 facilitates the function of MAC3B in splicing. Our genome-wide expression analyses suggest that HDA15 and MAC3A/MAC3B coregulate a subset of genes involved in ABA responses. It is possible that HDA15 and MAC3A/MAC3B also affect ABA-signaling, which may induce both increased and reduced IR events.
+
+## Materials And Methods
+
+Plant materials and growth conditions The T-DNA insertion mutants, hda15-1 (SALK_004027),
+mac3a (SALK_089300), and mac3b (SALK_050811), were described previously (Monaghan et al., 2009; Liu et al., 2013)
+and all of them are in the Col-0 background. Arabidopsis
+(Arabidopsis thaliana) plants were grown under long-day conditions (16-h light/8-h dark cycle) at 23C after seeds were subjected to a 3-day stratification period. To generate Pro35S:GFP-MAC3B transgenic lines, MAC3B complementary DNA (cDNA) was cloned into the pK7WGF2 binary vector. The transgenic plants were generated using the floral dip method (Clough and Bent, 1998).
+
+## Lc-Ms/Ms
+
+Ten-day-old Col-0 WT and hda15-1 were treated with or without 50 mM ABA for 3 h. Total proteins were extracted in an extraction buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 10% [v/v] glycerol, 1% [v/v] Igepal CA-630, and 1 mM
+phenylmethylsulfonyl fluoride) containing a protease inhibitor cocktail (Roche). The HDA15-associated proteins were immunoprecipitated by rabbit polyclonal anti-HDA15 (Liu et al., 2013). After trypsin digestion and desalting, peptides were used to perform LC–MS/MS by Thermo Orbitrap Elite Mass Spectrometer and for Mascot analysis. hda15-1 was used as a negative control for excluding the proteins that bind non-specifically to the HDA15 antibody in immunopurification. Two biological replicates for each sample were performed independently.
+
+## Bifc Assays
+
+HDA15, MAC3A, and MAC3B were cloned into the pCR8/
+GW/TOPO vector and then recombined into the YFPN
+(pEarleyGate201-YFPN) and YFPC (pEarleyGate202-YFPC)
+vectors (Lu et al., 2010). Constructed vectors were transiently transformed into Arabidopsis protoplasts by polyethylene-glycol (PEG)-mediated transfection (Yoo et al., 2007). Nuclear localization was detected by NLS-mCherry (Liu et al., 2013). The florescence was observed using Leica TCS SP5 confocal microscope. YFP florescence was excited by 514 nm of an argon laser and observed at 525 to 565 nm.
+
+mCherry florescence was excited by 561 nm of an argon laser and observed at 575–620 nm. Chloroplast autofluorescence was detected at 650–700 nm.
+
+## Slc Assays
+
+HDA15 and MAC3B were cloned into nLuc and cLuc vectors, respectively (Supplemental Table S2). SLC assay was performed as described previously (Chen et al., 2008; Liang et al., 2021). Constructed vectors were transformed into Agrobacterium tumefaciens GV3101 which was injected into leaves of Nicotiana benthamiana. Six hours before observation, 20 mM ABA or H2O were injected into corresponding
+
+## Accession Numbers
+
+leaves. After 48-h infection of GV3101, Luc activity was measured after 1 mM luciferin was sprayed onto the leaves. Luc images were captured by cooled CCD imaging apparatus. Relative Luc activities were defined as the ratio of relative light units (RLUs) to infiltrated leaf area and normalized by ImageJ software (https://imagej.nih.gov/ij/).
+
+The major types of AS events, IR, AltD/A, and ES, were analyzed by RackJ as described previously (Kanno et al., 2017).
+
+In brief, significant IR changes were defined as P-value50.005 (t test) with fold-change 42. The significant ES
+and AltD/A events were determined using a method similar to that for IR events following the criteria: P-value50.005
+(t test) with fold-change 42; the sum of the read counts in two samples supporting the ES or AltD/A event 520, the sum of the splice read counts aligned to the skipped exon 520 (for ES); the sum of the splice read count supporting all other junctions of the same intron 520 (for AltD/A).
+
+## Co-Ip Assays
+
+MAC3A or MAC3B cDNA was cloned into the pK7WGF2 binary vector for transient expressing GFP-MAC3A or GFPMAC3B by PEG-mediated transfection in Col-0 Arabidopsis protoplasts. Ten-day-old Col-0, hda15-1, and Pro35S:GFPMAC3B transgenic plants were treated with or without 50 mM ABA for 3 h. Total proteins were extracted in the extraction buffer containing protease inhibitor cocktail
+(Roche) as described above. Protein extracts were incubated with a rabbit polyclonal anti-HDA15 antibody and protein G
+Mag Sepharose beads (GE Healthcare) overnight at 4C. The beads were washed with a wash buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 10% [v/v] glycerol, and 1% [v/v] Igepal CA-630). Immunoblotting was carried out by using the rabbit polyclonal anti-HDA15 antibody, anti-GFP antibody (Abcam, ab290), and the secondary antibody CleanBlot IP Detection Reagent (Thermo Fisher Scientific, 21230).
+
+## Rt-Qpcr
+
+Total RNA was extracted with Trizol reagent (Invitrogen)
+according to the manufacturer's protocol and used to synthesize cDNA. RT-qPCR was performed using iQ SYBR
+Green Supermix (Bio-Rad) and the CFX96 real-time PCR system (Bio-Rad). The gene-specific primers used for quantitative PCR are listed in Supplemental Table S2. Each sample was quantified at least in triplicate and normalized using Ubiquitin10 (UBQ10) as an internal control.
+
+## Chip-Qpcr
+
+ChIP assays were performed as described (Gendrel et al., 2005; Liu et al., 2013). Chromatin was extracted from 10day-old seedlings. After fixation with 1% (v/v) formaldehyde, the chromatin was sheared to an average length of 500 bp by sonication and then immunoprecipitated with the H3K9ac antibody (Diagenode, C15410004). The cross-linking was then reversed, and the amount of each precipitated DNA fragment was determined by qPCR using specific primers in Supplemental Table S2. Three biological replicates were performed, and three technical repeats were carried out for each biological replicate. Representative results from one biological replicate were shown.
+
+## Abiotic Stress Tolerance Test And Seed Germination
+
+treated with ABA
+For the salt tolerance test, 3-week-old plants were watered with 300 mM NaCl for 2 weeks, and the survival rates were measured. For transpiration (water loss) measurements, detached leaves from 5-week-old plants were exposed to white light in a growth chamber at 23C. Leaves were weighed at various time intervals, and the loss of fresh weight (percentage) was used to indicate water loss. For seed germination test, imbibed seeds were cold treated at 4C in the dark for 3 days, and then sown on a half-strength Murashige and Skoog (MS) medium (pH 5.7) with 1% (w/v) sucrose, 0.8%
+(w/v) agar, and 2 mM ABA for 3 days.
+
+## Rip Assays
+
+RIP assays were performed as described (Xing et al., 2015).
+
+The nuclear extracts were extracted from 10-day-old seedlings with Ribonuclease Inhibitor (Promega, N2515). After fixation with 1% (v/v) formaldehyde, the nuclear extracts were sheared to an average length of 500 bp by sonication and then immunoprecipitated with a GFP antibody (Abcam, ab290). The cross-linking was then reversed, and the RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer's protocol and used to synthesize cDNA.
+
+The amounts of cDNA fragments were determined by RTqPCR using specific primers in Supplemental Table S2. Three biological replicates were performed, and three technical repeats were carried out for each biological replicate. Representative results from one biological replicate were shown.
+
+## Rna-Seq
+
+For RNA sequencing, 10-day-old seedlings of Col-0 WT,
+hda15-1, and mac3a/mac3b were treated with or without 50 mM ABA for 3 h. Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer's protocol, and then used for Illumina NovaSeq 6000 platform to generate paired-end reads of 150 bp. Two biological replicates for each sample were performed independently.
+
+After low-quality read trimming, over 73 million pairedend reads were obtained per sample. More than 94% of the reads for every replicate were mapped to Araport11 using Bowtie version 2 (Langmead and Salzberg, 2012) and BLAT (Kent, 2002). Reads Per Kilobase Million (RPKM) values were calculated using the RackJ software package (http://rackj. sourceforge.net/), and normalized by the TMM method (Robinson and Oshlack, 2010). In this study, differentially expressed genes were defined as P-value50.05 (t test) with fold-change 51.5.
+
+HDA15, AT3G18520; MAC3A, AT1G04510; MAC3B, AT2G33340; SNRK2.3, AT5G66880; SDIRIP1, AT5G51110; PYD1, AT3G17810; EDM1, AT4G11260; AIRP2, AT5G01520;
+
+## Supplemental Data References Acknowledgments Funding
+
+UBQ10, AT4G05320. All RNA-seq data have been uploaded to the Sequence Read Archive database (accession no. PRJNA678483) at the National Center for Biotechnology Information.
+
+(NTUCC-111L893001 to K.W.), and NTU-Academia Sinica joint grant (NTU-AS-108L104310 to K.W. and P.-Y.C.).
+
+Conflict of interest statement. None declared.
+
+The following materials are available in the online version of this article.
+
+Supplemental Figure S1. Co-IP analysis of interaction between HDA15 and MAC3B in Pro35S:GFP-MAC3B transgenic plants under mock and ABA treatments.
+
+Supplemental Figure S2. Genome-wide expression analysis in Col-0, hda15-1, and mac3a/mac3b by RNA-seq.
+
+Supplemental Figure S3. ABA-responsive IR event defects in hda15-1 and mac3a/mac3b.
+
+Supplemental Figure S4. H3K9ac levels of the retained introns of SNRK2.3, SDIRIP1, EDM1, AIRP2, and PYD1 in hda15 and mac3a/mac3b.
+
+Supplemental Figure S5. RIP assays followed by RT-qPCR
+showing the enrichment of HDA15 and MAC3B on ACTIN7 pre-mRNA in response to ABA.
+
+Supplemental Figure S6. ABA-responsive IR events of WT and mutated GFP-HDA15 transgenic plants treated with ABA.
+
+Supplemental Table S1. Read counts and mapping rates in RNA-seq analysis.
+
+Supplemental Table S2. List of primers used in this study.
+
+Supplemental Data Set S1. Genes differentially expressed in hda15-1 and mac3a/mac3b compared with Col-0 under mock and ABA treatments.
+
+Supplemental Data Set S2. Co-regulated genes in hda151 and mac3a/mac3b under mock and ABA treatments.
+
+Supplemental Data Set S3. List of AS events in hda15-1 and mac3a/mac3b compared with Col-0 under mocktreated conditions.
+
+Supplemental Data Set S4. List of AS events in hda15-1 and mac3a/mac3b compared with Col-0 under ABA-treated conditions.
+
+Supplemental Data Set S5. List of ABA-responsive IR
+events in Col-0, hda15-1 and mac3a/mac3b.
+
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medical/md/PMC9467385.md

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+Interventional Medicine
+
+![0_image_0.png](0_image_0.png)
+
+and Applied Science 11 (2019) 4, 216–220 DOI:
+10.1556/1646.2020.00004
+© 2019 The Author(s)
+
+RESEARCH ARTICLE
+
+![0_image_1.png](0_image_1.png)
+
+![0_image_2.png](0_image_2.png)
+
+# Efficacy, Safety And Tolerability Of Bosentan As An Adjuvant To Sildenafil And Sildenafil Alone In Persistant Pulmonary Hypertension Of Newborn (Pphn)
+
+J.R. VIJAY KUMAR, H.S. NATRAJ SETTYp ,
+M. JAYARANGANATH and C.N. MANJUNATH
+Sri Jayadeva Institute of Cardiovascular Sciences and Research, Bangalore, Karnataka, India Received: June 22, 2017 - Accepted: September 6, 2019 Published online: July 16, 2021
+
+## Abstract
+
+Background: Pulmonary Arterial Hypertension (PAH) carries a poor prognosis in both adult and pediatric patients. It is a life-threatening condition in newborns. Current recommendations advocate the use of targeted monotherapy as a first-line approach for the treatment of Persistent Pulmonary Hypertension of the Newborn (PPHN). In case of an inadequate clinical response to treatment, an addition of a second or third agent is considered. PAH is usually managed with a phosphodiesterase 5 inhibitor or an endothelin receptor blocker. There are limited pediatric studies that address questions like which class of therapy should be initiated first or if a combination should be initiated together. With this background, the present study was initiated to compare the efficacy, safety, and tolerability of bosentan as an adjuvant to sildenafil and sildenafil alone in PPHN. Results: A total of 40 patients were enrolled in the study. Out of them, 26 were males (65%) and 14 were females (35%). PPHN was most commonly seen in the 29 (72.5%) of participants with a history of first order birth. Mean duration of symptoms was 14.05 ± 2.06 days. The participants were randomized to two groups. Group A consisted of total 25 participants that received both bosentan and sildenafil and group B had 15 participants that received sildenafil alone. Both groups were comparable in terms of birth weight and present weight, consanguinity, and mode of delivery. Efficacy was determined by the reduction in mean baseline Pulmonary Artery Systolic Pressure (PASP). PASP in group A was 75.56 ± 10.62 mm Hg and in group B was 64.86 ± 12.25 mm Hg which was not statistically significant (P > 0.05). PASP on the third and seventh day in group A were 43.72 ± 8.63 and 24.47 ± 3.52 mm Hg compared to 42.28 ± 9.43 and 27.276 ± 8.38 respectively in group B which was statistically significant (P < 0.05).There were two deaths each in both groups. Two participants in Group A developed liver function abnormalities. None of the participants in Group B had adverse effects. Conclusion: Most common clinical manifestations were nonspecific. Cardiovocal syndrome was common in PPHN. We conclude that oral sildenafil treatment is a safe, simple and effective treatment for persistent pulmonary hypertension in newborn.
+
+Combination of bosentan with sildenafil is more effective and safe in reducing pulmonary artery (PA) pressures in high-risk patients with PPHN.
+
+## Keywords
+
+pulmonary hypertension, sildenafil, bosentan, cardiovocal syndrome pCorresponding author. Tel.:þ91 080 26580051; fax: þ91 080 22977261 (Mobile: þ91 9845612322).
+
+E-mail: drnatrajsetty75@gmail.com
+
+## Introduction
+
+Persistent Pulmonary Hypertension of the Newborn (PPHN) is a syndrome of acute respiratory failure. It is characterized by severe hypoxemia after birth, marked pulmonary hypertension, vasoreactivity with extrapulmonary right-to-left shunting of blood across the ductus arteriosus and/or foramen ovale with an absence of cyanotic congenital heart disease
+[1]. It is a common problem in the neonate and is associated with significant morbidity and mortality [1, 2]. PPHN occurs in approximately 1.9 per 1,000 live births but a wide variation in the incidence of about 0.43–6.82 per 1,000 live births has been reported between centers. The disease is believed to be because of an abnormal pulmonary vasoreactivity due to decreased production or responsiveness to vasodilator stimuli and/or increased production or responsiveness to vasoconstrictor stimuli. The principal goal of PPHN treatment is selective pulmonary vasodilatation. Various modalities of PPHN treatment include Ventilation strategies and pulmonary vasodilators like nitric oxide, prostacyclin analogues, phosphodiesterase inhibitors, and endothelin antagonists [3]. Although the above treatments approved for PAH in adults have shown favorable effects in children, pediatric treatment decisions largely depend on results from evidence-based adult studies and the experience of clinicians. Also, there are reports that mention Inhaled Nitric Oxide (iNO) or invasive treatment with Extra Corporeal Membrane Oxygenation (ECMO) as the gold standards of the PPHN therapy. But they are expensive therapeutic modalities associated with technical difficulties in developing countries. Though mortality varies from 10 to 20% of affected newborns in the developing countries, it is much higher when the therapies discussed above are not available. So alternative less expensive treatments are being sought [4].
+
+Bosentan is a dual endothelin receptor antagonist that has been established in double-blind, placebo-controlled studies in adults with PAH [5, 6] and in children over 3 years of age [7]. Recent studies have emphasized that oral bosentan may be a safe and effective treatment to improve oxygenation in neonates with PPHN. Sildenafil is a phosphodiesterase inhibitor type 5 (PDE5) that has been shown to selectively reduce pulmonary vascular resistance. Several case reports and controlled studies document improved oxygenation and echo cardio-graphic evidence of reduced pulmonary arterial pressures following the administration of Sildenafil therapy in newborns that had PPHN. This study was designed to assess the efficacy, safety, and tolerability of bosentan as an adjuvant to sildenafil and sildenafil alone in PPHN.
+
+This was a prospective, randomized, interventional, open-label and comparative study. It was conducted at Sri Jayadeva Institute of cardiovascular sciences and Research, Karnataka, Bengaluru, India. It was conducted from August 2013 to January 2015 after taking approval from the Institutional Ethical Committee (IEC) of the hospital. A
+consecutive series of 40 patients were enrolled in this study.
+
+The inclusion criteria were as follows: i) Newborns to 3 months of age diagnosed with a known diagnosis of PAH before the beginning of the study or those diagnosed during the recruitment period ii) Patients with PAH Congenital Heart Disease PAH-CHD were included if increased Pulmonary Vascular Resistance (PVR) was considered unrelated to CHD iii) Informed consent by parents or Legally Authorized Representative (LAR). The exclusion criteria included children with cyanotic congenital heart disease, bleeding disorders and liver dysfunction. Patients who met the inclusion and exclusion criteria were included in the study. The diagnosis of PAH was established using Doppler echocardiography based European Society of Cardiology guidelines [8]. PAH history included the date of the first presentation, date of diagnosis, demographic profile, clinical manifestations, and functional signs. These were documented in a case report form. The participants were randomized based on computer-generated numbers [9] into Group A or Group B. Group A participants received both bosentan and sildenafil; and Group B only sildenafil. Sildenafil was administered via a nasogastric tube or orally at a starting dose of 0.5 mg/kg. The dose of sildenafil was increased stepwise by 0.5 mg/kg every 4–6 h up to a maximum dose of 2 mg/kg with careful hemodynamic monitoring. Bosentan solution was prepared from a 125-mg tablet, which was crushed to one-fourth and then dissolved in sterile water of 10 mL (3 mg/mL). Bosentan (1 mg/kg twice a day) was administered via orogastric tube or oral.
+
+Opaque covers were used to cover the drug's container and orogastric tubes used for the administration of drugs. Hemodynamic parameters were documented at inclusion, at 3rd day, and 7th day after completion of treatment.
+
+The evaluation of efficacy was based on the reduction of Pulmonary Artery Systolic Pressure (PASP) from baseline to the 3rd and 7th day of post-drug therapy. The safety and tolerability of the drugs were assessed by review of all study Adverse Drug Reactions (ADR). Patients were monitored for ADR like hypotension, gastric intolerance, bleeding or pulmonary hemorrhage daily during the administration of study therapy by active surveillance. Serum bilirubin, liver enzymes, alkaline phosphatase, serum creatinine, serum electrolytes and complete blood count were performed twice 3rd and 7th day of the drug therapy.
+
+## Statistical Methods
+
+The data were analyzed by using SAS-16.50 version.
+
+Descriptive statistics such as mean and standard deviation (SD) for continuous variables and percentage for categorical variables was determined. The reduction in PASP from baseline was evaluated using one way ANOVA followed by posthoc test. Univariate and multivariate analysis were employed to draw significant inference. A p-value of 0.05 or less was considered for statistical significance.
+
+## Results Demographic Profile
+
+A total of 40 participants were enrolled in the study. Out of them, 26 were males (65%) and 14 were females (35%) (P >
+0.05). There were 13 (32.50%) males and 12 (30.0%) females in Group A, and 13 (35.20%) males and 02 (5.0%) females in Group B respectively. The mean age at presentation was 2.45 ± 1.25 years in Group A, and 2.52 ± 1.57 in Group B
+respectively. The mean birth weight of participants in Group A was 2.77 ± 0.24 kg, and in group B 2.65 ± 3.02 kg respectively. The weight at presentation was 4.10 ± 0.97 kg in Group A, and 3.85 ± 0.84 in Group B respectively. The gender distribution is shown in Fig. 1.
+
+![2_image_0.png](2_image_0.png)
+
+## Clinical Profile
+
+PPHN was most commonly seen in the participants that had first order of birth and the number was 29 (72.5%).
+
+![2_image_1.png](2_image_1.png)
+
+Mean duration of symptom was 14.05 ± 2.06 days.
+
+Regarding the mode of delivery, 25 (62.5%) were born by Full Term Normal Delivery (FTND), 13 (32.5%) by term Lower Segment Caesarian Section (LSCS) and 2 (5%) were born by preterm LSCS. Consanguinity was noted in 10
+(25%) participants (P > 0.05).The most common clinical symptom was vomiting. It was seen in 95% participants, followed by feeding difficulty in 87.5%, decrease in voice and cyanosis in 72.5%, dyspnea in 60%, cough in 55% and fever in 27.5% respectively. Regarding concomitant disease, hypothyroidism was noted in 2 (5%) of the participants. .
+
+Tricuspid Regurgitation (TR) was trivial in 4 (10%), mild in 20 (50%), moderate in 10 (25%) and severe in 6 (15%) of the participants. Patent Foramen Ovale (PFO) with bidirectional shunt was most commonly associated lesion seen in 22 (55%) of the participants. Other associated lesions seen were tiny mid muscular Ventricular Septal Defect (VSD) in 8 (20%), 1 (2.5%) apical VSD, 9 (22.5%) Atrial Septal Defect (ASD), 1 (2.5%) coronary Atrio Ventricular
+(AV) fistula and double aortic arch in 1 (2.5%) patient.
+
+Right Ventricular (RV) dysfunction was seen in 15 (37.5%)
+of the participants. The results are shown in Figs 2–4 and Table 1.
+
+## Efficacy
+
+Mean baseline PASP in group A was 75.56 ± 10.62 and Group B; 64.86 ± 12.25 mmHg respectively (P > 0.05). PASP on the third and seventh day in group A was 43.72 ± 8.63 and 24.47 ± 3.52 mmHg compared to 42.28 ± 9.43 and 27.26 ± 8.38 in group B respectively which was statistically significant (P < 0.05). Results are shown in Fig. 5.
+
+## Safety
+
+There were two deaths each in both groups. Two participants in group A developed liver function abnormalities.
+
+None of the patients on sildenafil alone had ADR.
+
+![2_image_2.png](2_image_2.png) 
+
+![3_image_0.png](3_image_0.png) 
+
+| Group A       | Group B   |      |    |      |
+|---------------|-----------|------|----|------|
+| Variable      | No        | %    | No | %    |
+| TR-1 TRIVIAL  | 01        | 2.50 | 03 | 7.5  |
+| TR 2 MILD     | 17        | 42.5 | 03 | 7.5  |
+| TR3- MODERATE | 03        | 7.50 | 07 | 17.5 |
+| TR4 SEVERE    | 04        | 10.0 | 02 | 5.0  |
+
+![3_image_1.png](3_image_1.png)
+
+## Discussion
+
+PPHN is a life-threatening condition, being among the most rapidly progressive and potentially fatal form of vasculopathy. Despite the recent advances, the clinical approach to PPHN still represents an important challenge for neonatologists. The care of newborns with PPHN requires meticulous therapeutic and ventilation strategies including, besides the stabilization of the newborn, the use of highfrequency ventilation, inhaled nitric oxide and ECMO. This prospective, randomized study was designed to assess the efficacy, safety and tolerability of bosentan in newborn infants with PPHN although not the first report use of bosentan in newborn infants with PPHN. Nakwan N et al.
+
+[4] reported an effective use of bosentan alone as an alternative treatment of PPHN in a full-term neonate. Goissen et al. [8] reported a successful use of bosentan as an adjunct therapy to inhaled Nitric Oxide (iNO) and sildenafil in two newborns with PPHN complicating transposition of the great arteries.
+
+To our knowledge, this is the first study on clinical manifestations of PPHN. The most common clinical manifestations are nonspecific like vomiting, feeding difficulties, decreased voice, cyanosis, cough and fever. The peculiar symptom noted in our study was decreased voice complained by mother. We attribute it to probably dilated pulmonary artery causing compression on recurrent laryngeal nerve and trachea (cardio-vocal syndrome). Patients had improvement in voice after PA pressures were reduced. The outcome in both the groups A and B were comparable with a reduction in PA pressures and resolution of symptoms. Most of the participants on sildenafil had mild-moderate pulmonary hypertension and less of RV dysfunction compared to patients on both sildenafil and bosentan. Four deaths were seen, two in each group especially without any PFO or septal defects. This is the first prospective randomized study compared the efficacy of sildenafil and bosentan with sildenafil alone for lowering elevated PA pressure and also the clinical manifestations of PPHN.
+
+## Limitations
+
+Our sample size is small to conclude and follow up was done only for seven days. Also, randomization was not proper as more participants in the sildenafil group had mild to moderate pulmonary hypertension compared with the combination of bosentan and sildenafil group.
+
+## Conclusion
+
+Pulmonary vasodilators are the mainstay of pulmonary arterial hypertension (PAH) treatment. Despite all the guideline recommendations, there is always a major issue with the cost of medications, resulting in poor adherence.
+
+Our study demonstrated that treatment with bosentan along with sildenafil is safe and well well-tolerated. And that the combination is more effective and safe in reducing PAH in high-risk patients with PPHN.
+
+Study type - Prospective Observational Study. Study population - 40 patients with PPHN. Study period - August 2013–January 2015. Ethical committee - Approved.
+
+Funding: None. Conflict of interest: None.
+
+## Type Of Contribution
+
+Conception Dr. Vijay Kumar Design Dr. Natraj Setty H.S
+Supervision Dr. C.N Manjunath Materials Dr. Vijay Kumar Data collection and Processing Dr. Natraj Setty H.S
+Analysis and Interpretation Dr. Jayaranganath M Writer Dr. Vijay Kumar
+
+## Acknowledgment N/A. Abbreviations
+
+PPHN Persistent Pulmonary Hypertension of Newborn PAH Pulmonary Arterial Hypertension PASP Pulmonary Artery Systolic Pressure PA Pulmonary artery iNO Inhaled Nitric Oxide ECMO Extra Corporeal Membrane Oxygenation IEC Institutional Ethical Committee PVR Pulmonary Vascular Resistance CHD Congenital Heart Disease
+
+LAR Legally Authorized Representative
+
+ADR Adverse Drug Reactions
+
+SD Standard deviation
+
+FTND Full Term Normal Delivery
+
+LSCS Lower Segment Caesarian Section
+
+TR Tricuspid Regurgitation
+
+PFO Patent Foramen Ovale VSD Ventricular Septal Defect ASD Atrial Septal Defect
+
+AV Atrio Ventricular
+
+RV Right Ventricular
+
+## References
+
+[1] Morin, III FC, Stenmark KR. Persistent pulmonary hypertension of the newborn: state of the art. Am J Respircrit Care Med 1995;151:
+2010–32.
+
+[2] Steinhorn RH, Millard SL, Moran, III FC. Persistent pulmonary hypertension of the newborn: role of nitric oxide and endothelin in pathophysiology and treatment. ClinPerinatol 1995;22:
+405–28.
+
+[3] Konduri GG. New approaches for persistent pulmonary hypertension of newborn. ClinPerinatol 2004;31:591–611.
+
+[4] Nakwan N, Choksuchat D, Saksawad R, Thammachote P, Nakwan N. Successful treatment of persistent pulmonary hypertension of the newborn with bosentan. ActaPaediatr 2009;98:1683–5.
+
+[5] Galie N, Hinderliter A, Torbicki A, Fourme T, Simonneau G,
+Pulido T, et al. Effects of the oral endothelin-receptor antagonist bosentan on echocardiographic and Doppler measures in patients with pulmonary arterial hypertension. J Am Collcardiol 2003;41:
+1380–6.
+
+[6] Rubin LJ, Badesch DB, Barst RJ, Galie N, Black CM, Keogh A, et al.
+
+Bosentan therapy for pulmonary arterial hypertension. N Engl J
+Med 2002;346:896–903.
+
+[7] Barst R, Ivy D, Dingemanse J, Widlitz A, Schmitt K, Doran A, et al.
+
+Pharmacokinetics, safety, and efficacity of bosentan in pediatric patients with pulmonary arterialhypertension. ClinPharmacolTher 2003;73:372–82.
+
+[8] Goissen C, Ghyselen L, Tourneux P, Krim G, Storme L, Bou P, et al.
+
+Persistent pulmonary hypertension of the newborn with transposition of the great arteries: successful treatment with bosentan.
+
+Eur J Pediatr 2008;167:437–40.
+
+[9] R Geggel, J Murphy, D Langleben, R Crone, J Vacanti, L Reid.
+
+Congenital diaphragmatic hernia: arterial structural changes and persistent pulmonary hypertension after surgical repair. J Pediatr 1985;107(3):457–64. https://doi.org/10.1016/s0022-3476(85)80534-5.
+
+PMID: 4032138.
+
+Open Access. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (https://
+creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium for non-commercial purposes, provided the original author and source are credited, a link to the CC License is provided, and changes - if any - are indicated.

medical/md/PMC9577825.md

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+Case Report A case report: Lateral medullary syndrome with facial nerve palsy and hemiparesis Ramesh Shrestha a,*, Ghanshyam Kharel b, Shraddha Acharya c, Rohit Pandit d, Nitu Limbu e a Department of Neurology/Neurointervention, Upendra Devkota Memorial National Institute of Neurological and Allied Sciences, Kathmandu, Nepal b Department of Neurology, Upendra Devkota Memorial National Institute of Neurological and Allied Sciences, Kathmandu, Nepal c Department of Medicine, Montefiore New Rochelle Hospital, USA d Department of Emergency Medicine, Om Saibaba Memorial Hospital Pvt. Ltd, Kathmandu, Nepal e *Department of Emergency Medicine, Helping Hands Community Hospital, Nepal* 
+
+| ARTICLE INFO  Keywords:  Lateral medullary syndrome  Hemiparesis  Facial palsy  Upper motor neuron  Stroke   |
+|--------------------------------------------------------------------------------------------------------------|
+
+Lateral medullary syndrome (LMS) is the most common and severe neurological syndrome associated with atherothrombotic occlusion of the intracranial vertebral artery, followed by posterior inferior cerebellar artery and medullary artery occlusion. It presents as a typical triad of oculosympathetic palsy (Horner's syndrome), 
+ipsilateral gait ataxia, and hypoalgesia with ipsilateral thermoanesthesia of the face. 
+
+In LMS, the upper motor neuron facial palsy is caused by the involvement of aberrant supranuclear fibers of the facial nerve. The caudal extension of the infarction to the pyramidal tracts may explain contralateral hemiparesis. Here, we have discussed a 42-year-old non-diabetic, hypertensive male with LMS, hemiparesis, and left UMN-type facial palsy. We reported this case because developing nations, have few tertiary level health facilities for neurological examination, and non-neurologists often miss the diagnosis; therefore, the characteristics must be known and understood. 
+
+## 1. **Introduction**
+
+| ABSTRACT   |
+|------------|
+
+Lateral medullary syndrome (LMS), also known as Wallenberg syndrome, was initially recognized and documented in 1961 [1]. This neurological syndrome is caused by involvement of the lateral region of the medulla and is most commonly caused by occlusion of the atherothrombotic vertebral artery, the posterior inferior cerebellar artery (PICA), and followed by the medullary artery. Magnetic resonance imaging is used to make the diagnosis [2]. 
+
+In addition to these classic clinical findings, Wallenberg syndrome can have various unusual manifestations. Here, the contralateral hemiparesis could be explained by the caudal extension of the infarct involving pyramidal tracts before crossing at the medulla [3]. The involvement of Dejerine's aberrant pyramidal tract might explain facial palsy caused by the infarction of the left facial colliculus and the left lateral medulla [4]. According to SCARE guidelines by Agha RA et.al., 
+we are presenting the case of a patient who experienced upper motor neuron (UMN) facial nerve palsy and hemiparesis after a left lateral medullary infarction [5]. 
+
+## 2. **Case Presentation**
+
+* Corresponding author. 
+
+![0_image_2.png](0_image_2.png)
+
+![0_image_3.png](0_image_3.png)
+
+E-mail addresses: the1ramesh.stha@gmail.com (R. Shrestha), gskharel@gmail.com (G. Kharel), acharya.shraddha54@gmail.com (S. Acharya), rpandit419@ 
+gmail.com (R. Pandit), nitulimbu76@gmail.com (N. Limbu). 
+
+![0_image_4.png](0_image_4.png)
+
+A 42-year-old gentleman visited the emergency department with right-side weakness, severe headache, and dizziness that had persisted for four days and was accompanied by significant dizziness and facial deviation. He experienced tingling on the left side of his face and later found that he could not feel pain or temperature changes on that side of his face. There had been several episodes of vomiting, as well as difficulty swallowing, hiccups, and coughing. 
+
+He had uncontrolled hypertension for five years and was taking antihypertensive medication, which he discontinued last year. He had a blood pressure of 170/95 mmHg and a pulse rate of 98 beats per minute. 
+
+He does not smoke cigarettes or drink alcohol. 
+
+On neurological evaluation and physical examination, the patient had right-sided hemiparesis with motor power of 3/5, loss of pain and temperature on the right side of the body, increased deep tendon reflex 
+(DTR), Babinski reflex positivity, and ataxia. A left-sided horizontal gaze-evoked nystagmus without ophthalmoplegia, Horner's syndrome 
+(Fig. 1), and cerebellar dysarthria were also discovered. The finger-tohttps://doi.org/10.1016/j.amsu.2022.104722 Received 28 July 2022; Received in revised form 10 September 2022; Accepted 11 September 2022 Available online 17 September 2022 2049-0801/© 2022 The Authors. Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY-NC-ND license
+(http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+![1_image_1.png](1_image_1.png)
+
+nose test demonstrated left-sided cerebellar symptoms. The diagnosis was further complicated by presence of UMN facial nerve palsy with partial upper eyelid ptosis on the left side of his face (Fig. 2). 
+
+The patient had an abnormal lipid profile and increased erythrocyte sedimentation rate (34mm/hr), but all other lab findings reported normal results, including the coagulation profile. A right dorsolateral medullary infarction was discovered during a MRI scan of the brain. In the magnetic resonance angiography (MRA) of the brain and neck arteries, PICA and the left vertebral artery were not visible (Fig. 3). 
+
+Following the clinicoradiological evaluation, the patient was diagnosed with LMS. 
+
+MRA: Magnetic Resonance Angiogram; PICA: Posterior Inferior Cerebellar Artery; FLAIR: Fluid Attenuated Inversion Recovery. 
+
+Carotid Doppler showed the course and caliber of the bilateral common carotid artery, carotid bulb, and internal carotid artery were narrowed and thickened, but no significant stenosis. The echocardiography demonstrated mild left ventricular wall thickness, Grade I diastolic dysfunction, normal ventricular wall motion, a 65% left 
+
+![1_image_2.png](1_image_2.png)
+
+ventricular ejection fraction, and no intracardiac clots or masses. 
+
+Thrombolysis wasn't done due to the late presentation(>4.5hr). He was managed conservatively with careful blood pressure control. A nasogastric tube was inserted to prevent aspiration due to dysphagia, and the patient received aspirin, clopidogrel, atorvastatin, amlodipine, and losartan. He had paroxysms of pain on the left side of his face at the ophthalmic, maxillary trigeminal nerve distributions, for which he was given pregabalin and amitriptyline; an antihistamine twice daily to control hiccups, as well as a fresh tear over the left eye. He reported less pain frequency and severity over a week. His symptoms improved after four weeks of medication, and he was discharged with a nasogastric tube and a strict physiotherapy training regime. There were no further pain episodes or facial deviation during the three-month period. 
+
+## 3. **Discussion**
+
+Twenty percent of ischemic events in the brain involve posterior circulation (vertebrobasilar). LMS is the most common syndrome caused by occlusion of the intracranial vertebral artery, the PICA, followed by the medullary artery [6]. 
+
+As we noted in our report, common vestibulocerebellar symptoms of LMS include nystagmus (horizontal and rotational), vertigo with falling to one side of the lesion, gait ataxia, and dysmetria [1,2]. Pain and temperature loss in the contralateral trunk/limbs (spinothalamic tract) and ipsilateral face (spinal trigeminal nucleus and tract) are among the sensory findings. Other signs of involvement of the nucleus ambiguous include ipsilateral bulbar muscular weakness (e.g., dysphagia, dysarthria, hoarseness) and autonomic dysfunction (e.g., ipsilateral Horner's syndrome, hiccups, and lack of autonomic respiration during sleep) [7]. 
+
+Sensory symptoms can be explained by the involvement of the dorsal sensory root, formed by central axons that extend into the brainstem and divide into short ascending and long descending fibers. The short ascending fibers are afferent for touch, light pressure, and proprioception. Long ascending fibers carry pain and temperature sensations and extend to the upper cervical segment [8]. 
+
+High blood pressure, diabetes, and smoking are the most common risk factors. LMS can also be caused by various conditions, such as EhlerDanlos syndrome, Marfan syndrome, fibromuscular dysplasia, and vertebral artery dissection [9]. 
+
+In this case, the contralateral hemiparesis can be explained by a caudal extension of the infarction to the pyramidal tract before the decussation at the medulla. Some studies have linked this weakness to the spinocerebellar hypotonic syndrome, while a positive Babinski reaction indicated pyramidal tract involvement in our case [3]. 
+
+Our patient's UMN facial palsy could be caused by the involvement of Dejerine's aberrant pyramidal tract [10]. At the medulla, the hypothetical loop of supranuclear corticobulbar fibers descends ventromedially, decussate at the superior medulla, and then ascends dorsolaterally to reach the facial nerve nucleus [11]. 
+
+![1_image_0.png](1_image_0.png)
+
+## 4. **Conclusions**
+
+Physicians should be aware of the most common manifestations of LMS, their appropriate management, and evaluation, including various atypical presentations. Regarding our case, UMN-type facial palsy with contralateral hemiparesis raised questions about a simple lateral medullary syndrome diagnosis. Furthermore, because there are few tertiary centers for neurological science in developing countries like ours, such presentations are frequently missed. 
+
+## Ethical Approval
+
+The IRC of Upendra Devkota Memorial National Institute of neurological and Allied Sciences granted ethical approval for this study. 
+
+## Please State Any Sources Of Funding For Your Research
+
+No fund was obtained to support this case report. 
+
+## Author Contribution
+
+RS, GK contributed to the study concept, data collection, manuscript outlining, writing, and revision. RP, NL and SA contributed to the study concept, and critical revision of the manuscript for content. GK contributed with supervision, and content revision. All named authors accept overall responsibility integrity of the work and have given final approval for its publication. 
+
+## Please State Any Conflicts Of Interest
+
+All authors must disclose any financial and personal relationships with other people or organisations that could inappropriately influence 
+(bias) their work. Examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. No conflicts of interest No fund was obtained to support this case report. 
+
+## Registration Of Research Studies
+
+1. Name of the registry: NA 2. Unique Identifying number or registration ID: NA 3. Hyperlink to your specific registration (must be publicly accessible and will be checked): NA 
+
+## Guarantor
+
+Ramesh Shrestha. 
+
+## Consent
+
+Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. 
+
+## Acknowledgments
+
+We would like to thank all attending physicians from the Department of Neurology and Radiology Department at Upendra Devkota Memorial Neurological Institute and Allied Sciences. 
+
+## References
+
+[1] R. Saha, S. Alam, M.A. Hossain, Lateral medullary syndrome (Wallenberg's Syndrome) - a Case Report, Faridpur Med. Coll. J. 5 (1) (2010) 35–36, https://doi. 
+
+org/10.3329/fmcj.v5i1.6813. Available from: https://www.banglajol.info/index. 
+
+php/FMCJ/article/view/6813. 
+
+[2] J.S. Kim, Pure lateral medullary infarction: clinical–radiological correlation of 130 acute, consecutive patients, Brain 126 (8) (2003 Aug 1) 1864–1872, https://doi. 
+
+org/10.1093/brain/awg169. Available from: https://academic.oup.com/brain/ar ticle/126/8/1864/307992. 
+
+[3] U. Chakraborty, B. Banik, A. Chandra, J. Pal, An atypical manifestation of lateral medullary syndrome, Oxf. Med. Case Rep. 2019 (12) (2019 Dec 31) 527–529, https://doi.org/10.1093/omcr/omz139. Available from: https://academic.oup.co m/omcr/article/2019/12/527/5691272. 
+
+[4] C. Terrence, R. Costa, G. Fromm, An unusual case of paroxysmal facial pain, J. Neurol. 221 (2) (1979 Aug 1) 73–76, https://doi.org/10.1007/BF00313104. 
+
+Available from: https://link.springer.com/article/10.1007/BF00313104. 
+
+[5] R.A. Agha, T. Franchi, C. Sohrabi, G. Mathew, A. Kerwan, SCARE Group, The SCARE 2020 Guideline: Updating Consensus Surgical CAse REport (SCARE) 
+Guidelines, Int. J. Surg. Lond. Engl. 84 (2020 Dec) 226–230. Available from: 
+https://pubmed.ncbi.nlm.nih.gov/33181358. 
+
+[6] T.R. Huynh, B. Decker, T.J. Fries, et al., Lateral medullary infarction with cardiovascular autonomic dysfunction: an unusual presentation with review of the literature, Clin. Auton. Res. 28 (2018) 569–576, https://doi.org/10.1007/s10286018-0502-6. Available from: https://link.springer.com/article/10.1007/s10286-0 18-0502-6. 
+
+[7] P. Kk, R. K, P C, S.K. Aiyappan, N D, A Rare Variant of Wallenberg's Syndrome: 
+Opalski syndrome, J. Clin. Diagn. Res.: J. Clin. Diagn. Res. 8 (7) (2014 Jul) 
+MD05–6, https://doi.org/10.7860/jcdr/2014/9547.4626. Available from: https:// 
+europepmc.org/article/PMC/4149101, PMID: 25177595; PMCID: PMC4149101. 
+
+[8] Notices of recent publications, Brain 62 (Issue 1) (1939) 125, https://doi.org/ 
+10.1093/brain/62.1.125. 
+
+[9] R.L. Sacco, L. Freddo, J.A. Bello, J.G. Odel, S.T. Onesti, J.P. Mohr, Wallenberg's Lateral Medullary Syndrome: Clinical-Magnetic Resonance Imaging Correlations, Arch. Neurol. 50 (6) (1993 Jun 1) 609–614, https://doi.org/10.1001/ 
+archneur.1993.00540060049016. Available from: https://jamanetwork.com/jour nals/jamaneurology/article-abstract/592384. 
+
+[10] W.W. Campbell, R.J. Barohn, DeJong's the Neurologic Examination, 2020. 
+
+[11] M. Srinivasan, B. Bindu, S. Gobinathan, S. Balasubramanian, A. Nithyanandam, K. 
+
+R. Shanbhogue, An Unusual presentation Of Lateral Medullary Syndrome With Ipsilateral UMN Facial Palsy - An Anatomical Postulate, Ann. Indian Acad. Neurol. 8 (1) (2005 Jan 1) 37. Available from: https://www.annalsofian.org/article.asp?iss n=0972-2327;year=2005;volume=8;issue=1;spage=37;epage=40;aulast=srini vasan;type=0. 
+
+3

medical/md/PMC9584785.md

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+# Erectile Dysfunction: Causes, Assessment And Management Options
+
+## Summary
+
+Erectile dysfunction is one of the most common male sexual dysfunctions. The diagnosis can usually be made by a detailed history and examination. Men with erectile dysfunction benefit from multimodal management strategies. These include lifestyle modification, medical treatment and psychosexual counselling and therapy.
+
+An oral phosphodiesterase-5 inhibitor is often prescribed for erectile dysfunction. Providing simple and clear instructions is critical to realise the full benefits of these drugs. Those with severe vascular disease or a history of pelvic surgery may not respond to phosphodiesterase-5 inhibitors. Anxiety or unrealistic expectations can also result in a poor response.
+
+## Introduction
+
+Erectile dysfunction is a prevalent sexual dysfunction in men.1 Male sexual dysfunction can occur at any age, but erectile dysfunction and diminished libido increase with age. There may be underlying causes. Multimodal management is needed, but when drugs are indicated, oral phosphodiesterase-5 inhibitors or self-injectables such as alprostadil are options for erectile dysfunction. It is important to initially discuss treatment objectives and outcomes, and set realistic expectations to avoid dissatisfaction. While there is information available about drugs to use in erectile dysfunction, the information is rarely accompanied with specific advice for the patient on timing and other details about how to use the drugs.
+
+## Erectile Dysfunction
+
+Men with erectile dysfunction are unable to achieve an erection firm enough for sexual intercourse.
+
+## Causes
+
+There are many causes and risk factors for erectile dysfunction (Box 1).2 These were traditionally classified as organic, psychogenic or mixed. However, with advancements in the fields of psychological science and sexual medicine, the current view is that the aetiological factors are multimodal3 - biological, psychological, sociocultural, relational and sexual.
+
+## Assessment
+
+Men presenting with erectile dysfunction are initially assessed with a comprehensive history (Box 1). This helps the clinician to understand and differentiate the causes as predisposing (why this person?), precipitating (why now?) and perpetuating (what is keeping the problem?) factors. The history includes lifestyle (quality and quantity of sleep, snoring and sleep apnoea, weight, exercise, alcohol, smoking history), general health (physical and mental, medicines) and a relationship and psychosexual history.4,5 Box 2 shows some key questions to ask. Eliciting details about the quality of morning erections and erectile capacity during other sexual activities (e.g. masturbation) are critical to understand the underlying aetiology.4 The history of past and current treatment for erectile dysfunction, and the response achieved, helps in tailoring further management. A distinction must be made whether the man has erectile dysfunction or premature ejaculation because some men are not good at describing their problem. The man with premature ejaculation may say he has erectile dysfunction because he loses his erection early after ejaculation. Conversely, the man with erectile dysfunction may complain of premature ejaculation as he rushes to ejaculate quickly before he loses his erection. Erectile dysfunction and premature ejaculation are often confused but can occur together. The history should include a review of medicines (as listed in Box 1). This could provide valuable insight about the sexual adverse effects of certain drugs and, more importantly, a timeline between starting a specific drug and the onset of erectile complaints. The physical examination should include, at a minimum, general parameters (weight, waist circumference, body mass index and blood pressure) and the genitals. If investigations are indicated, the Michael Lowy Sexual health physician, Double Bay, Sydney Vijayasarathi Ramanathan Lecturer in Sexual Health, University of Sydney Keywords erectile dysfunction, impotence, phosphodiesterase-5 inhibitors Aust Prescr 2022;45:159–61 https://doi.org/10.18773/ austprescr.2022.051
+
+## Box 1 **Risk Factors For Erectile Dysfunction**
+
+- Advanced age - Atherosclerosis-related risk factors (e.g. cardiovascular disease, cigarette smoking, hypertension, dyslipidaemia, diabetes mellitus)
+- Pelvic surgery (e.g. radical prostatectomy), radiation, trauma - Endocrinological conditions (e.g. hypogonadism, hyperprolactinaemia, thyroid disorder) - Obesity and metabolic syndrome - Substance abuse - alcohol, illicit drugs (e.g. cannabis, barbiturates, cocaine, heroin, methamphetamine)
+- Psychological (partner-related, stress, guilt, situational anxiety, self-image problems, low self-esteem, history of sexual abuse, highly restricted sexual upbringing, generalised anxiety disorder, depression, psychosis)
+- Erectile dysfunction associated with other sexual dysfunction(s) (e.g. premature ejaculation, sexual aversion disorder, anorgasmia)
+- Medicines:
+- antihypertensives (e.g. diuretics, alpha and beta blockers) - psychotropics (e.g. selective serotonin reuptake inhibitors and other antidepressants, antipsychotics, anxiolytics)
+- anticonvulsants, anti-Parkinson's drugs - hormone-affecting drugs - antiandrogens, corticosteroids, chronic opioid use
+- Neurological conditions (Alzheimer's disease, multiple sclerosis, Parkinson's disease, stroke), spinal cord and peripheral nerve disorders (diabetic neuropathy)
+- Penile abnormalities (e.g. Peyronie's disease, venous leak)
+
+## Box 2 **Key Questions In The Assessment Of Erectile Dysfunction**
+
+- Is the problem intermittent, global or situational? - Is the problem recent or long term? - Is there an unusual curvature of the erection or an episode of sexual trauma to the erect penis?
+
+- Has the patient ever suffered from mental health problems?
+
+minimum is serum lipids, fasting glucose or ideally glycated haemoglobin.4,5 Should hypogonadism be suspected, measure serum testosterone on a blood sample taken before 11 am.4 A validated questionnaire, for example the International Index of Sexual Function (IIEF-5),6 can be an adjunct to history and examination. However, such questionnaires should not be used alone for diagnosing erectile dysfunction.5
+
+## Management Options
+
+The initial treatment of erectile dysfunction addresses lifestyle changes and psychological or relationship problems. Sex therapy is indicated particularly when there is a significant psychological contribution to erectile dysfunction and when there is no response to medical management.7 Ideally, sex therapists should be healthcare professionals with specific qualifications in the field of human sexuality along with skills in counselling and psychosexual therapy. General practitioners, psychologists and sexual health physicians can offer certain aspects of sex therapy, whereas a well-qualified and trained sex therapist can offer comprehensive psychosexual education, counselling and therapy.
+
+## Phosphodiesterase-5 Inhibitors
+
+The first step of drug treatment is an oral phosphodiesterase-5 inhibitor:
+- sildenafil 25, 50 and 100 mg
+- vardenafil 5 and 20 mg - avanafil 50, 100 and 200 mg
+- tadalafil 5, 10 and 20 mg.
+
+Phosphodiesterase-5 inhibitors work best if taken 1–2 hours before sexual intercourse. Tadalafil has a two-hour lead-in time, when taken as required, so is often used as a daily low-dose (5 mg) treatment. Daily dosing may also benefit men with erectile dysfunction who have benign prostatic hyperplasia as it can improve lower urinary tract symptoms. Large meals and alcohol should be avoided before a dose, but when phosphodiesterase-5 inhibitors are taken daily, food and alcohol have less impact on the response. It is critical to educate patients that phosphodiesterase-5 inhibitors do not create sexual stimuli. They only help with getting and maintaining an erection when there is adequate external sexual stimulation. Depending on the severity of erectile dysfunction, the clinician decides on the appropriate starting dose. Importantly, patients should be made aware that they need to take the drug as prescribed and, on five to six occasions, to assess the treatment effect. Failure to provide this information could lead to a suboptimal or no response, which in turn could lead to an inappropriate use of higher doses or the addition of other treatment options. The response to phosphodiesterase-5 inhibitors can be affected by anxiety, alcohol, excessive expectations of how these drugs should work, and not waiting long enough for them to work. The American Urological Association Guideline states that sildenafil, tadalafil, vardenafil and avanafil have similar efficacy in men with erectile dysfunction and that dose-response effects across phosphodiesterase-5 inhibitors are small and nonlinear.8 While there is no firm evidence that switching from one phosphodiesterase-5 inhibitor to another will have a beneficial effect, it is worth a clinical attempt provided the expectations are discussed with the patient. The classic adverse effects of phosphodiesterase-5 inhibitors are flushed face, headaches, blocked nose, altered colour vision (mainly with sildenafil) and gastric reflux. Most of these adverse effects have a dose-response pattern. The average rates are similar across the phosphodiesterase-5 inhibitors except for dyspepsia (lowest rates reported with avanafil), 
+flushing (lowest rates reported with tadalafil), and myalgia (lowest rates reported with vardenafil and avanafil).8 Tadalafil is associated with low back and leg pain which often go away when the drug is stopped. Phosphodiesterase-5 inhibitors should not be prescribed if the patient is taking nitrates or uses 'recreational' amyl nitrite. There is a risk of a precipitous blood pressure drop.
+
+## Injectable Drugs
+
+Penile injections tend to be used when oral phosphodiesterase-5 inhibitors are not effective. The drugs used for intracavernosal penile injection are vasoactive. They include alprostadil, which may be combined with papaverine and phentolamine. Penile injections work rapidly so sexual activity may begin within 10–15 minutes of injecting. Care must be taken to use the lowest effective dose to avoid priapism which can be a medical emergency. The patient may also experience delayed post-injection pain. Patient education (by means of explaining or referring to product information, or video demonstrations) is very important. The drug needs to be injected into the shaft at 10 o'clock or 2 o'clock positions, altered between different attempts, avoiding obvious veins and fibrosis.
+
+## Devices
+
+High rates of patient satisfaction have been reported for vacuum erection devices. They can be an effective and low-cost treatment option for any men with erectile dysfunction but more so for those with diabetes, spinal cord injury or after prostatectomy.8 Older men may tend to use vacuum mechanical devices as they are drug free. However, vacuum erection devices can be cumbersome and require some training in correct use. Shockwave therapy applies acoustic shock waves to the penis. This aims to improve vascularisation. Shockwave therapy appears to work best for the older patient with vasculogenic erectile dysfunction, but lacks robust evidence of efficacy.9 A penile implant is a restorative treatment option. It is a very effective treatment no matter the aetiology or severity of the erectile dysfunction and even if all other treatments have failed or are not suitable. However, it is irreversible.
+
+## Evaluation Of Treatment Outcomes
+
+Evaluating treatment outcomes for erectile dysfunction depends on the management goals that were established before treatment. Erectile capacity across different sexual activities (intercourse, masturbation), quality of morning erections, reduction in distress and overall sexual satisfaction are some of the measures used to assess progress.
+
+## Conclusion
+
+Erectile dysfunction is a common male sexual dysfunction. It requires a comprehensive clinical assessment and multimodal management. This may involve GPs, specialists and allied health professionals trained in the field of sexology.
+
+Conflicts of interest: none declared
+
+## References

medical/md/PMC9595062.md

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+# Open Access Full Text Article O R I G I N A L R E S E A R C H Effectiveness Of Moringa Oleifera Leaves On Tnf-Α Expression, Insulin Levels, Glucose Levels And Follicle Count In Rattus Norvegicus Pcos Model
+
+Salmon Charles PT Siahaan1, Budi Santoso2, Widjiati 3 1Doctoral Program of Medical Science, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia; 2Department of Obstetrics and Gynecology, Faculty of Medicine, Dr. Soetomo Teaching Hospital, Universitas Airlangga, Surabaya, Indonesia; 3Department of Embryology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia Correspondence: Budi Santoso, Department of Obstetrics and Gynecology, Faculty of Medicine, Dr. Soetomo Teaching Hospital, Universitas Airlangga, Jl. Mayjen Prof. Dr. Moestopo No. 47, Surabaya, East Java, 60132, Indonesia, Tel +62 812-3581-706, Email budi.santoso@fk.unair.ac.id Background: Polycystic ovary syndrome (PCOS) is a syndrome characterized by ovulation disorders accompanied by hyperandrogens. Women with PCOS are prone to develop insulin resistance which has metabolic characteristics similar to type 2 diabetes and leads to disturbance of follicular formation. PCOS is also known to increase the concentration of proinflammatory cytokines, namely TNF-α. *Moringa oleifera* leaves have been shown to have compounds that can reduce insulin levels and glucose levels in diabetes mellitus and should be able to reduce TNF-α and follicle count.
+
+Purpose: This study aims to prove the effectiveness of *Moringa oleifera* leaf in reducing insulin, glucose levels, TNF-α and follicle count in PCOS.
+
+Methods: The three-month-old white rats Wistar (*Rattus norvegicus*) 150–170 grams were divided into four groups (n = 10), namely normal rats, PCOS model rats, PCOS model rats given metformin, and PCOS rats given 500mg of *Moringa oleifera*. The method of this study is taking PCOS model rats by injecting the 100mg/kg BW hormone testosterone propionate for 21 days. After 21 days of therapy, we analyzed insulin, glucose levels, TNF-α and follicle count.
+
+Results: The PCOS control group showed an increase in insulin level, glucose levels, TNF-α expression, and a decrease in the follicle count compared to the normal control group. The insulin level, glucose level, TNF-α and follicle count in the *Moringa oleifera* 500 mg/kg BW treatment group were significantly lower than in the PCOS control group.
+
+Conclusion: *Moringa oleifera* leaves have the potential in reducing insulin levels, blood glucose levels, TNF-α and follicle count in PCOS patients.
+
+Keywords: follicle, glucose, insulin, *Moringa oleifera*, polycystic ovary syndrome, TNF-α
+
+## Introduction
+
+Polycystic ovary syndrome (PCOS) is a syndrome characterized by ovulation disorders accompanied by hyperandrogen conditions. Women with PCOS are prone to develop insulin resistance which has metabolic characteristics similar to type 2 diabetes and leads to disturbance of follicular formation.1,2 In women with PCOS with insulin resistance, a state of hyperinsulinemia is found which will result in a decreased effect of ovulation induction.3 This happens because, in women with insulin resistance, LH/FSH ratio are found to be fairly high and will affect oocyte quality.4 Ovulation induction agents cannot suppress insulin levels, high levels of luteinizing hormone (LH), and androgens in the blood. Therefore, the treatment of hyperinsulinemia in patients with PCOS will lead to an improvement in folliculogenesis, resulting in ovulation and high pregnancy rates.5 Insulin resistance plays an important role in the pathogenesis of PCOS.6 Endometrial cell differentiation depends on adequate glucose metabolism. Glucose transport (insulin-mediated) is carried out by Glucose Transporter 4 (GLUT4) as a carrier. With a decrease in GLUT4, insulin resistance occurs and leads to an increase in glucose and insulin levels which can cause damage to endometrial cell metabolism in patients with PCOS.7 This process may affect downstream insulin signal transduction and insulin receptors, and may therefore be associated with insulin resistance and PCOS.8 Insulin resistance causes the frequency of GnRH and secretion of LH pulses to increase, causing compensatory hyperinsulinemia and increased production of androgens in the ovaries.2 Ovarian theca androgen production increases and the production of SHGB by the liver will decrease due to hyperinsulinemia.9 Insulin resistance also causes oxidative stress due to hyperglycemia and increased levels of free fatty acids will produce reactive oxygen species (ROS). ROS will induce oxidative stress (OS). Oxidative stress is increased due to an imbalance between ROS and antioxidant defences.
+
+Increased ROS will also increase NFkB which will then form TNF-α, AP-1 activation, HIF-1 as an inflammatory mediator. An increase in TNF-α, IL-6, IL-1β will then activate NF-κB. This increase will cause activation of the serine phosphorylation pathway in the IRS and will inhibit the formation of PI3K which will compensate for the occurrence of hyperandrogens.10 Several studies have reported that PCOS-IR mouse models can be made by subcutaneous injection of testosterone propionate in combination with a high-fat diet.11,12 These mice have features similar to the pathology of PCOS and endocrine disorders but externally added androgens can affect the endocrine level of PCOS.13 Metformin has been used as an insulin sensitizer for the treatment of polycystic ovary syndrome. Studies have shown that metformin can not only improve endocrine disorders in patients with PCOS but also regulate ovarian function and reduce body weight in overweight women with PCOS.14 Metformin acts by inhibiting hepatic glucose uptake, increasing peripheral glucose uptake, reducing peripheral insulin levels, and increasing GLUT-4. However, clinically, the results of long-term metformin treatment result in digestive disorders, such as diarrhea and other effects.15 The search for herbal plants that have the potential for prevention and treatment is certainly very much needed.
+
+Research conducted by Arti Verma et al in 2009 aimed to determine the antioxidant effect of Moringa oleifera lies in its content which consists of various nutritional sources such as phytochemicals, viz, carotenoids, vitamins, minerals, amino acids, sterols, glycosides alkaloids, flavonoids, and phenolics.16 *Moringa oleifera* extract can work as an antioxidant that binds to ROS and inhibits the oxidative stress process that occurs in PCOS patients. The ethanol extract of *Moringa oleifera* will inhibit the formation of ROS so that the production of SOD will increase. And the antiinflammatory effect of the ethanolic extract of *Moringa oleifera* will reduce inflammatory factors so that the activation of the JNK and NF-B pathways decreases.
+
+Research by Lakshmipriya et al in 2016 found that extracts from this plant have potential as antioxidants, antidiabetics, and antimicrobials.17 As an anti-diabetic, especially in diabetic cases with insulin resistance, research in mice found that *Moringa oleifera* can reduce fasting sugar levels. *Moringa oleifera* extract also plays a role in the treatment of insulin resistance, this is following the research of Chinedu et al, who showed that administration of the extract could increase insulin receptor sensitivity (p<0.0001).18 Based on the above background, we held a research to study the effect of *Moringa oleifera* on reducing insulin, glucose levels, TNF-α and follicle count in rats PCOS model through insulin resistance and inflammation mechanisms.
+
+## Materials And Methods Materials
+
+Moringa oleifera is a tree belonging to the Moringaceae family originating from South Asia. The leaves of this tree are rich in minerals, vitamins, and other important phytochemicals.19 *Moringa oleifera* extract process (Kelorina, Moringa Indonesia, Blora, Indonesia) was carried out using the ultrasonic method with 96% ethanol as solvent, the extraction results were then evaporated using a rotary evaporator and oven to form a dry extract. The extraction steps were: 1) Moringa oleifera simplicia was weighed as much as 50 grams, 2) The simplicia was put into a beaker glass (50 grams each) and 500 mL of 96% ethanol was added to the beaker, 3) The time for the extraction process is set using the UAE, which is 3×2 minutes while stirring at each time interval, 4) The extraction results are filtered, 5) The collected filtrate is put into a rotary evaporator flask, 6) The temperature of the tool is set at 40°C with a rotational speed of 70 rpm, 7) The extract from the rotary evaporator was re-evaporated (dried) in an oven at 40°C to obtain a solvent-free dry extract.
+
+Rattus norvegicus female rats with Wistar strain were 3 months old and weighed 150–170 grams. *Rattus norvegicus* was chosen because they have stable genetics, shorter reproductive life, short estrogen cycle and easy to be handled. 
+
+Previous research in 2020 also use *Rattus norvegicus* as a PCOS model with insulin resistance.20 Before the study began, an adaptation period was given for 1 week, with a health condition, normal behavior, and normal vaginal swab results. Exclusion criteria for rats are anatomic abnormalities (ie, ears are injured or not intact, tail is short or stump, one or all four legs are deformed, cannot stand, have sores on body parts, eyes are not clear) and pregnancy during the adaptation period. All of these procedures have been approved by the ethics committee of the Faculty of Veterinary Medicine, Airlangga University, Indonesia.
+
+All experiments were performed following Airlangga University ethic principal (5F: Freedom from hunger and thirst; Freedom from discomfort; Freedom for pain, injury or disease; Freedom to express normal behaviour; freedom from fear and distress), Indonesian National Law: UU no.23 1992 article 69 par 1; UU no.36 2009 article 44 par 4; and UU no.18 2009 article 66 par 2 point C), and these following guidelines: Guide for Care and Use of Laboratory Animals, 8th edition; Report of the AVMA Panel on Euthanasia: 2013 edition (updated on 2020); ARRIVE 2010 (Animal Research: 
+Reporting of In Vivo Experiments) point 8 (updated on 2020).
+
+This research was conducted in a laboratory experimental type with a post-test-only control group design. The rat experiment was conducted with the PCOS model. Rats were divided into 4 major groups, namely the normal control group (K-), PCOS control rats' model (K-), PCOS rats' model given metformin (P1), and PCOS rats model given Moringa oleifera (P2). This research was conducted in the laboratory of the Faculty of Veterinary Medicine, Airlangga.
+
+## Therapeutic Protocols And Blood Collection Time Points
+
+Forty *Rattus norvegicus* strain Wistar female rats were randomly divided into equal 4 large groups, consisting of a control group (K-) that was only given aquades for 42 days; a PCOS control group (K+) that was given the hormone testosterone propionate 100mg/kg body weight (BW) for 21 days and aquades for the next 21 days; the first treatment group (P1) that was given testosterone propionate for 21 days and Metformin 2mg/100gram BW for the next 21 days; and the second treatment group (P2) that was given testosterone propionate for 21 days and *Moringa oleifera* 500mg/kg BW for the next 21 days. Testosterone propionate injections were performed intraperitoneally at the proestrus stage on K+, P1 and P2 groups. On day 22, a vaginal swab was examined to determine the status of the rat's lust cycle. PCOS rats will show the stage of diestrus on vaginal examination which shows ovulation disturbance. After administering testosterone propionate for 21 days, PCOS model rats will become IR (insulin resistance), characterized by an increase in HOMA IR 
+(P<0.005).12 And in this study, it was seen from the increase in the ratio of glucose and insulin which increased in PCOS model rats compared to normal rats. Metformin was administered to the rats by sonde. Moringa oleifera leaf ethanol extract was administered to the rats by sonde. Testosterone propionate was used to simulate PCOS conditions in Rattus norvegicus because another research in 2009 has proved that prolonged exposure to androgens (testosterone propionate) affects insulin resistance index and free fatty acid levels in PCOS model rat serum.21 A significant increase in insulin resistance index in the group that got testosterone propionate for 14, 21, and 28 days was higher than the controls. Before and after treatment, a vaginal swab was performed to see changes in the cycle due to treatment. On the 43rd day, the rats were sacrificed by ester anesthesia and neck dislocation, then the ovaries and blood samples were taken.
+
+## Estimation Of Serum Insulin, Glucose Parameters, Expression Of Tnf-Α And Follicle Count
+
+Before the experimental animals were sacrificed, they fasted for 12 hours for blood collection. Glucose levels were measured from the blood taken through rats' blood vessels which are examined using glucometers. Blood glucose was collected at the end of the study. The units obtained are mg/dL. The data scale is a ratio.
+
+Insulin levels in the serum of PCOS model rats were measured from the blood taken through rats' blood vessels which were examined using Enzyme-linked immunosorbent assay (ELISA). ELISA is a labelled immunoassay that is considered the gold standard of immunoassays. This immunological test is very sensitive and is used to detect and quantify substances, including antibodies, antigens, proteins, glycoproteins, and hormones.22 Blood insulin was taken at the end of the study. The units obtained are mmol/L. The data scale is a ratio.
+
+TNF- expression is a picture of an increased cell signaling protein (cytokine) and plays an important role in the inflammatory process.23 TNF-α count in the ovarium of PCOS model rats, taken directly from the ovarium organ. The units obtained are Metabolite Insulin Receptor Substrate −1 or IRS-1 which is the insulin signal at the receptor which is measured using a microscope (Cx41 microscope (Olympus, Japan)) with a magnification of 400 times in 10 fields of view measurements using immunohistochemical method, the measurement results are in IRS and is a ratio/interval scale.
+
+Follicle count is the number of follicles in the ovarium tissue of PCOS model rats, taken directly from the ovarium organ which is examined microscopically (histopathology) to observe the follicular phase (primordial, primary, secondary, tertiary, DeGraff, and corpus luteum). Folliculogenesis is measuring the number of follicles that grow in the ovaries, through wedge cutting and microscopic observation. Anatomical pathology laboratory of the Faculty of Veterinary Medicine, Airlangga University conducts the examination. The result is an overview of folliculogenesis for each phase. In 5 visual fields with 400x magnification by counting the number of developments. The data scale was a ratio.
+
+## Statistical Analysis
+
+This research data will be recorded in a data collection form that is specifically designed for this study, to observe insulin, glucose levels, TNF-α and follicle count in the PCOS rats' model. First, the normality test (Shapiro-Wilk test) is carried out. When the distribution is normal, the ANOVA test or analysis of variance was used. However, if the distribution is not normal, the Kruskal Wallis non-parametric test or Mann–Whitney test will be carried out. Duncan's test will be carried out when there was a significant difference in variable between groups. Statistical calculations will use SPSS version 22 software tools.
+
+## Results Effect Of Therapy On Parameters Of Tnf-Α Expression
+
+This histopathological examination aims to determine the expression of TNF-α in the ovaries. The data for each sample was assessed semi-quantitatively according to the modified Remmele method, where the Remmele scale index (Immuno Reactive Score/IRS) is the result of multiplying the percentage score of immunoreactive cells with the color intensity score on immunoreactive cells.24 The data for each sample is the average IRS value observed in ten different fields of view at 100x and 400x magnification.
+
+The results of the examination carried out a normality test before data analysis was carried out. The results of the Shapiro–Wilk test showed that the TNF-α data in the K+ group was not normally distributed (p < 0.05), so it was analyzed using the Kruskal Wallis test. The results of the Kruskal Wallis test showed that there was a significant difference in TNF-α between groups (p < 0.05). The results of the Mann–Whitney test showed that the K- group was significantly different from K+ and P2 and the K+ group was significantly different from P1 and P2 (see the Supplemental File for raw data).
+
+## Effect Of Therapy On Parameters Of Serum Insulin
+
+The results of the blood insulin examination were carried out for normality tests first. The results of the Shapiro–Wilk test showed that the insulin data of all groups were normally distributed (p > 0.05) so the differences in insulin between groups were analyzed using the analysis of variance test.
+
+The results of the analysis of variance showed that there was a significant difference in insulin between groups (p < 
+0.05), so further tests were needed to determine which groups were different. Duncan's test results showed that the insulin in the negative control group was significantly different from the positive control group and all treatment groups, the positive control group was significantly different from the negative control group and all treatment groups, but there was no significant difference between the treatment groups.
+
+Table 1 Results of Differences in Insulin Levels Between Groups (p < 0.05)
+
+| Groups   | n   | Mean ± SD     | p-value   |
+|----------|-----|---------------|-----------|
+| K-       | 10  | 19.68 ± 1.080 | < 0.001   |
+
+K- 10 19.68 ± 1.080 < 0.001 K+ 10 25.60 ± 1.556 P1 10 22.15 ± 0.969 P2 10 22.23 ± 0.831 Notes: Description: different superscripts show significant differences. K-: Normal control group. K+: PCOS control group. P1: Treatment Group 1 (PCOS given 2mg/100gram BW of metformin). P2: Treatment Group 2 (PCOS treated with *Moringa oleifera* 500mg/kg BW).
+
+Table 2 Results of Differences in Blood Glucose Levels Between Groups (p < 0.05)
+
+K- 10 95 ± 2.98 < 0.001 K+ 10 126.2 ± 9.76 P1 10 105.8 ± 7.20 P2 10 112.7 ± 7.68
+
+Notes: Description: different superscripts show significant differences. K-: Normal control group. K+: PCOS control group. P1: Treatment Group 1 (PCOS given 2mg/100gram BW of metformin). P2: Treatment Group 2 (PCOS treated with *Moringa oleifera* 500mg/kg BW).
+
+| Groups                                                      | n   | Mean ± SD    | p-value   |
+|-------------------------------------------------------------|-----|--------------|-----------|
+| K-                                                          | 10  | 95 ± 2.98    | < 0.001   |
+| K+                                                          | 10  | 126.2 ± 9.76 |           |
+| P1                                                          | 10  | 105.8 ± 7.20 |           |
+| P2                                                          | 10  | 112.7 ± 7.68 |           |
+| Notes: Description: different superscripts show significant |     |              |           |
+
+![4_image_0.png](4_image_0.png)
+
+## Effect Of Therapy On Parameters Of Serum Glucose
+
+![4_Image_1.Png](4_Image_1.Png)
+
+The results of the blood glucose examination were carried out for normality tests first. The results of the Shapiro–Wilk test showed that the glucose data of the P1 group was not normally distributed (p < 0.05) so the differences in glucose between groups were analyzed using the Kruskal Wallis test.
+
+Figure 1 Graph of average insulin levels.
+
+The results of the Kruskal Wallis test showed that there was a significant difference in glucose between groups (p < 0.05),
+so it was necessary to carry out further tests with the Mann–Whitney test to find out which groups were different. The results of the Mann–Whitney test showed that the negative control group was significantly different from the positive control group and all treatment groups, as well as the positive control group which was significantly different from the negative control group and all treatment groups. However, there was no difference between the treatment groups.
+
+## Effect Of Therapy On Parameters Of Follicle Counts
+
+The number of follicles observed was primordial follicles, primary follicles, secondary follicles, tertiary follicles, and de Graff follicles and the corpus luteum. The results of the examination of the number of follicles and the corpus luteum were tested for normality first.
+
+![5_image_0.png](5_image_0.png)
+
+![5_image_1.png](5_image_1.png)
+
+The results of the Shapiro–Wilk test showed that the data on the number of primordial follicles in all groups were normally distributed (p > 0.05) so the differences between groups were tested using analysis of variance.
+
+The results of the Shapiro–Wilk test showed that data on the number of primary follicles in all groups were normally distributed (p > 0.05) so the differences between groups were tested using analysis of variance. The results of the analysis of variance with Brown-Forsythe showed that there was a significant difference between the primary follicles between groups (p < 0.05), so further tests were needed to determine which groups were different. The results of the further test using the Games Howell test showed that the K- group was significantly different from the K+ and P2 groups.
+
+The results of the Shapiro–Wilk test showed that data on the number of secondary follicles were not normally distributed in all groups (p > 0.05), so the differences between groups were tested using the Kruskal Wallis test. The results of the Kruskal Wallis test showed that there were significant differences in secondary follicles between groups (p < 0.05). The results of the Mann– Whitney test showed that the K- group was significantly different from the other three groups. The K- group was significantly different from the K+ group and the treatment group, but the K+ group was not significantly different from all treatment groups.
+
+The results of the Shapiro–Wilk test showed that the data on the number of tertiary follicles were not normally distributed in all groups (p > 0.05), so the differences between groups were tested using Kruskal Wallis analysis. The results of the Kruskal Wallis test showed that there was a significant difference in tertiary follicles between groups (p < 0.05). The results of the Mann–Whitney test showed that the K- group was significantly different from the K+ group and the P2 group, but not significantly different from the P1 group. The K+ group was significantly different from all groups, both the K- group and the treatment group. There were significant differences between treatment groups.
+
+The results of the Shapiro–Wilk test showed that the data on the number of de Graff follicles were not normally distributed in all groups (p > 0.05), so the differences between groups were tested using Kruskal Wallis analysis. The results of the Kruskal Wallis test showed that there were significant differences in DeGraff follicles between groups (p < 0.05). The results of the Mann–Whitney test showed that the K- group was significantly different from all groups, both K+ and the treatment group. The K+ group was significantly different from the K- group, but not significantly different from all treatment groups. There were significant differences between the treatment groups.
+
+The results of the Shapiro–Wilk test showed that the data on the number of corpus luteum in the K- and K+ groups were not normally distributed (p > 0.05), so the differences between groups were tested using Kruskal Wallis analysis. The results of the 
+
+![6_image_0.png](6_image_0.png)
+
+Table 3 Kruskal Wallis Test Results TNF-α 
+
+Expression Between Groups (p < 0.05)
+
+| Expression Between Groups (p < 0.05) Groups n Median (Min-Max)   | p-value   |                |         |
+|------------------------------------------------------------------|-----------|----------------|---------|
+| K-                                                               | 10        | 4.6 (3.6–5.7)  | < 0.001 |
+| K+                                                               | 10        | 5.7 (4.8–8.4)  |         |
+| P1                                                               | 10        | 3.9 (2.6–5.8)  |         |
+| P2                                                               | 10        | 3.45 (2.4–4.5) |         |
+| Notes: Description: different superscripts show significant differences. K-: Normal control group. K+: PCOS control group. P1:                                                                  |           |                |         |
+
+Kruskal Wallis test showed that there was a significant difference in the Corpus Luteum between groups (p < 0.05). The results of the Mann–Whitney test showed that the K- group was significantly different from K+ and P1.
+
+The K- group was significantly different from all groups, both K+ and the treatment group. The K+ group was significantly different from the K- group, but not significantly different from all treatment groups. There were significant differences between the treatment groups.
+
+## Discussion
+
+![7_Image_0.Png](7_Image_0.Png)
+
+Comparison between the normal control group and the PCOS control group with insulin resistance showed significant differences, both in insulin levels (Table 1) and glucose levels (Table 2). The PCOS group with insulin resistance showed a corresponding increase in insulin levels as evidenced by a significant value of p < 0.05 in the PCOS control group with insulin resistance compared to the normal group.
+
+The injected testosterone propionate hormone is an androgen that causes insulin resistance by decreasing the amount and effectiveness of glucose transport protein, especially GLUT-4 which plays a role in glucose transport in muscle and fat. Androgens directly inhibit insulin action in the periphery, and the liver, and indirectly affect insulin sensitivity by changing body composition through fat metabolism.25
+
+Table 4 The Results Showed That There Was No Significant Difference Between Primordial Follicles Between Groups (p > 0.05)
+
+| Follicles Between Groups (p > 0.05) Groups n Mean ± SD                                                               | p-value   |                |       |
+|----------------------------------------------------------------------------------------------------------------------|-----------|----------------|-------|
+| K-                                                                                                                   | 10        | 44.30 ± 8.111  | 0.375 |
+| K+                                                                                                                   | 10        | 35.60 ± 11.796 |       |
+| P1                                                                                                                   | 10        | 38.30 ± 11.006 |       |
+| P2                                                                                                                   | 10        | 36.80 ± 15.245 |       |
+| Notes: Description: different superscripts show significant  differences. K-: Normal control group. K+: PCOS control |           |                |       |
+
+Testosterone facilitates lipolysis and fat breakdown leading to an increase in free fatty acids. The increase in androgens and free fatty acids inhibits insulin excretion in the liver and glucose transport in muscle, which ultimately leads to hyperinsulinemia and insulin resistance.26 Glucose transporter type 4 (GLUT-4) is a transport protein for glucose that aims to carry glucose into cells. The process of translocation of GLUT-4 to the target cell surface begins with the binding of insulin and the insulin receptor subunit where this binding causes the subunit of the insulin receptor and other subunits of IRS-1 to be autophosphorylated. IRS-1 further activates PI3K which mediates the translocation of GLUT-4 to the target cell surface.27 The increase in GLUT-4 translocation causes an increase in glucose uptake from extra cells into cells. Increased glucose absorption increases the condition of insulin resistance.
+
+We determined the condition of insulin resistance in a PCOS model. Then, the comparison of insulin resistance between normal and PCOS control groups showed a significant difference. PCOS model insulin resistance in female rats injected with testosterone propionate increased insulin and glucose levels as evidenced by a significant increase in insulin levels (Figure 1) and glucose levels (Figure 2) in the PCOS control group with insulin resistance compared to the normal group.
+
+Metformin is the first therapy for PCOS by inhibiting hepatic glucose absorption, increasing peripheral glucose uptake, decreasing peripheral insulin levels, and increasing GLUT-4.15 Metformin also has a role in the endothelium and free adipose tissue in its role in insulin and glucose levels.28
+
+![8_image_0.png](8_image_0.png)
+
+![9_image_0.png](9_image_0.png)
+
+Furthermore, Moringa leaf extract can reduce insulin and glucose levels in PCOS models with insulin resistance. 
+
+Moringa leaf therapy treatment group and metformin treatment group for 21 days in female rats with PCOS insulin resistance model did not show significant differences. This shows that *Moringa oleifera* and metformin are equally good at lowering insulin levels and glucose levels. The Moringa leaf extract group and the metformin group showed significantly lower insulin (Figure 1) and glucose levels (Figure 2) than the insulin-resistant PCOS control group. This 
+
+| Between Groups (p < 0.05) Groups n Mean ± SD                                                                         | p-value   |               |       |
+|----------------------------------------------------------------------------------------------------------------------|-----------|---------------|-------|
+| K-                                                                                                                   | 10        | 32.0 ± 8.615  | 0.001 |
+| K+                                                                                                                   | 10        | 17.1 ± 3.510  |       |
+| P1                                                                                                                   | 10        | 19.8 ± 11.679 |       |
+| P2                                                                                                                   | 10        | 16.2 ± 10.497 |       |
+| Notes: Description: different superscripts show significant  differences. K-: Normal control group. K+: PCOS control |           |               |       |
+
+Table 6 Results of Secondary Follicle Differences Between Groups (p < 0.05)
+
+| Groups   | n   | Median (Min-Max)   | p-value   |
+|----------|-----|--------------------|-----------|
+| K-       | 10  | 12 (6–22)          | < 0.001   |
+| K+       | 10  | 3.5 (2–10)         |           |
+| P1       | 10  | 4 (2–7)            |           |
+| P2       | 10  | 2.5 (1–11)         |           |
+| Notes: Description: different superscripts show significant differences. K-: Normal control group. K+: PCOS control group. P1:          |     |                    |           |
+
+shows that the administration of Moringa leaf extract and metformin reduces insulin levels and glucose levels in female rats with PCOS insulin resistance.
+
+Moringa leaves contain high concentrations of flavonol quercetin.17 Quercetin is a flavonoid that has strong bioactive elements with free radical, anti-inflammatory, anticancer, antihyperlipidemic, and antiplatelet effects.29 Decreased insulin levels in peripheral tissues will cause a decrease in androgens in the ovaries. Decreased insulin and IGF-1 levels can also indirectly reduce androgen levels by increasing the production of SHGB in the liver and increasing IGFBP-1 synthesis directly, quickly, and completely in both the liver and ovaries, resulting in decreased levels of IGF-I, 
+IGF-I. II, and free testosterone.2 Moringa oleifera is a versatile plant that is consumed both as food and known in medicine. This plant is rich in nutrients with a high content of polyphenols in the form of phenolic acids, flavonoids, and glucosinolates. Moringa leaves have also been shown to have hypoglycemic activity, both aqueous extracts and organic solvents from leaves and seeds. This hypoglycemic effect is both acute and long-term administration, preventing other metabolic changes.
+
+Moringa oleifera extract has strong antioxidant activity, so it can prevent and protect pancreatic cells from oxidative stress associated with hyperglycemic conditions.30 This plant also has isothiocyanate compounds that are important in glycemic control related to its ability to reduce resistance to insulin action and hepatic gluconeogenesis.31 Moringa oleifera leaves which have a hypoglycemic effect are attributed to their fiber content and the presence of flavonoids and phenolic acids. Several components of Moringa leaves such as kaempferol, quercetin, chlorogenic acid, gallic acid, and ellagic acid, among others, have been studied as protective agents against ROS and free radical oxidation of DNA, proteins, and lipids.32 This plant is referred to as an antidiabetic agent because it has been shown to induce strong changes in glycemic levels.33 A study in 2018 stated that *Moringa oleifera* leaf extract could decrease the expression of IGF-1 and expression of androgen receptors so that it could also decrease the thickness of endometrium in PCOS-Insulin Resistance model.34 Another study suggested Moringa oleifera could also be of benefit for the treatment of various metabolic and neurological conditions, including diabetes.35,36 TNF-α is one of the aspirant molecules responsible for causing insulin resistance.37 TNF-α expression is important in stimulating, proliferating, and steroidogenesis in ovarian follicular theca cells. In the PCOS model rat group, TNF-α expression increased compared to the normal group. This proves that the administration of the hormone testosterone propionate for 21 days causes insulin resistance.
+
+![10_image_0.png](10_image_0.png)
+
+Metformin therapy and *Moringa oleifera* ethanol extract showed a decrease in TNF-α expression (Figure 3), this proves that metformin therapy can effectively attenuate the production of pro-inflammatory cytokines, especially TNF-α in macrophages.38 TNF-α which is an important cytokine not only affects inflammation, but also metabolic diseases.39 Increased TNF-α causes insulin resistance by inhibiting insulin signaling pathways, interfering with glucose transporter 4 expression and altering adipokine levels, downregulating adiponectin while increasing leptin.40 TNF-α also interferes with lipid metabolism by increasing triglyceride and free fatty acid concentrations in the blood and altering the composition of lipoproteins such as LDL and VLDL.40 Therapeutic administration of *Moringa oleifera* showed a significant reduction in pro-inflammatory mediators 
+(TNF-α) which is indicated by the presence of a chromogen brown color (Figure 4 and Table 3).41 This may play a role in increasing insulin sensitivity and reducing inflammation associated with metabolic syndrome.42 Moringa oleifera therapy could lower cholesterol, triglycerides, VLDL, LDL, and increase HDL in hypercholesterolemic rabbits, rats fed a high-fat diet and also STZ-induced diabetic rats.43 The growth and development of follicles are controlled by the complex system of the hypothalamic-pituitary axis of the ovary. Gonadotropin hormone plays an important role in controlling this system, the gonadotropin hormones involved in folliculogenesis are FSH and LH.44 The FSH hormone plays a role in initiating follicular growth, while LH functions in stimulating the growth and rupture of the follicle.
+
+Primordial follicles are small follicles, and usually found close to the outer edge of the cortex, which surrounded by a single layer of flattened ovarian follicular epithelial cells. Primordial follicles are small, and usually found close to the outer edge of the cortex. When the primordial follicle is stimulated, it becomes a primary follicle. The oocyte enlarges, and the follicular cells divide. A follicle that has two layers of follicular cells is called a primary follicle. The primary follicle develops into a secondary follicle. The secondary follicles look very similar to primary follicles, except that they 
+
+| Between Groups (p < 0.05) Groups n Median (Min-Max)   | p-value   |             |       |
+|-------------------------------------------------------|-----------|-------------|-------|
+| K-                                                    | 10        | 6 (4–8)     | 0.003 |
+| K+                                                    | 10        | 11.5 (3–26) |       |
+| P1                                                    | 10        | 6 (3–11)    |       |
+| P2                                                    | 10        | 4 (3–7)     |       |
+
+K- 10 6 (4–8) 0.003 K+ 10 11.5 (3–26) P1 10 6 (3–11) P2 10 4 (3–7)
+
+Notes: Description: different superscripts show significant differences. K-: Normal control group. K+: PCOS control group. P1: Treatment Group 1 (PCOS given 2mg/100gram BW of metformin). P2: Treatment Group 2 (PCOS treated with *Moringa oleifera* 
+
+500mg/kg BW).
+
+| Groups   | n   | Median (Min-Max)   | p-value   |
+|----------|-----|--------------------|-----------|
+| K-       | 10  | 1 (0–1)            | 0.001     |
+| K+       | 10  | 2 (0–2)            |           |
+| P1       | 10  | 2 (1–3)            |           |
+| P2       | 10  | 1 (0–4)            |           |
+| Notes: Description: different superscripts show significant differences. K-: Normal control group. K+: PCOS control group. P1:          |     |                    |           |
+
+K- 10 1 (0–1) 0.001 K+ 10 2 (0–2) P1 10 2 (1–3) P2 10 1 (0–4)
+
+Notes: Description: different superscripts show significant differences. K-: Normal control group. K+: PCOS control group. P1: 
+
+Treatment Group 1 (PCOS given 2mg/100gram BW of metformin). P2: Treatment Group 2 (PCOS treated with *Moringa oleifera* 
+
+500mg/kg BW).
+
+Dovepress Siahaan et al
+
+![12_image_0.png](12_image_0.png)
+
+are larger, there are more follicular cells, and there are small accumulations of fluid in the intracellular spaces called follicular fluid. The secondary follicle develops into a de Graff follicle and starts its second meiotic division. After ovulation, the ruptured follicle collapses and fills with a blood clot (corpus haemorrhagicum) which then forms the corpus luteum.45 Based on the results of the number of primordial follicles (Figure 5), all groups did not show a significant difference 
+(Table 4). However, there was a decrease in the number of primary (Figure 6) and secondary follicles (Figure 7) in the PCOS model rat group when compared to the normal group. While the treatment group, neither metformin therapy nor Moringa oleifera ethanol extract showed significant differences from the PCOS model rat group (Tables 5 and 6). The results of the number of tertiary follicles in the PCOS model rat group were higher than the normal group and the treatment group (Figure 8). The group given metformin showed no significant difference from the normal group but was significantly different from the group given *Moringa oleifera* ethanol extract therapy (Table 7.). This also happened to DeGraff follicles, the number of DeGraff follicles in the normal group was significantly different when compared to other groups, and the group treated with metformin was different from the group treated with *Moringa oleifera* ethanol extract 
+(Table 8 and Figure 9). Metformin therapy and ethanol extract of *Moringa oleifera* did not show a significant increase in the number of follicles compared to the PCOS model rat group.
+
+![12_image_1.png](12_image_1.png)
+
+| Groups   | n   | Median (Min-Max)   | p-value   |
+|----------|-----|--------------------|-----------|
+| K-       | 10  | 3 (1–3)            | 0.005     |
+| K+       | 10  | 4.5 (2–5)          |           |
+| P1       | 10  | 5.5 (2–8)          |           |
+| P2       | 10  | 4.5 (0–6)          |           |
+| Notes: Description: different superscripts show significant differences. K-: Normal control group. K+: PCOS control group. P1:          |     |                    |           |
+
+K- 10 3 (1–3) 0.005 K+ 10 4.5 (2–5) P1 10 5.5 (2–8) P2 10 4.5 (0–6)
+
+Notes: Description: different superscripts show significant differences. K-: Normal control group. K+: PCOS control group. P1: 
+
+Treatment Group 1 (PCOS given 2mg/100gram BW of metformin). P2: Treatment Group 2 (PCOS treated with *Moringa oleifera* 
+
+500mg/kg BW).
+
+![13_image_0.png](13_image_0.png)
+
+Corpus Luteum groups showed a significant difference between groups (Table 9). The normal control group was significantly different from PCOS model rat group and metformin treated group. The Normal control group was significantly different from all groups, both PCOS model rat group and the treatment group. The PCOS model rat group was significantly different from the K- group, but not significantly different from all treatment groups. There were differences between the treatment groups (Figure 10).
+
+Ethanol extract of *Moringa oleifera* proved effective in reducing TNF- expression, glucose level, insulin level, and the number of follicles in PCOS model rats. Based on the results of the study, the researchers suggest further research to isolate the active compounds contained in the *Moringa oleifera* extract and its effectiveness against other benefits of this extract.
+
+## Ethics Approval
+
+All of these procedures have been approved by the ethics committee of the Faculty of Veterinary Medicine, Airlangga University, Indonesia.
+
+## Acknowledgment
+
+We wish to thank our staff in the Faculty of Veterinary Medicine and Dr. Soetomo General Hospital, the doctors, veterinarians, nurses, and the administrator for granting us the permission and necessary support to conduct our research.
+
+## Disclosure
+
+This study was funded by the authors' personal fund. The authors declare that there is no conflict of interest in this work.
+
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+doi:10.3389/fphar.2012.00024 44. Orisaka M, Miyazaki Y, Shirafuji A, et al. The role of pituitary gonadotropins and intraovarian regulators in follicle development: a mini-review. 
+
+Reprod Med Biol. 2022;20(2):169. doi:10.1002/rmb2.12371 45. Paxton S, Peckham M, Knibbs A, Paxton S, Knibbs A, Peckham M. The Leeds histology guide; 2003.
+
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medical/md/PMC9639638.md

@@ -0,0 +1,417 @@
+RESEARCH ARTICLE
+
+![0_image_0.png](0_image_0.png)
+
+# Predicting The Disease Severity In Male Individuals With Ornithine Transcarbamylase Deficiency
+
+Svenja Scharre1,* , Roland Posset1,* , Sven F. Garbade1, Florian Gleich1, Marie J. Seidl1, Ann-Catrin Druck1, Jurgen G. Okun € 1, Andrea L. Gropman2, Sandesh C. S. Nagamani3, Georg F. Hoffmann1, Stefan Kolker € 1, Matthias Zielonka1,4 ,
+for the Urea Cycle Disorders Consortium (UCDC) and the European registry and network for Intoxication type Metabolic Diseases (E-IMD) Consortia Study Group, 1Division of Pediatric Neurology and Metabolic Medicine, Center for Child and Adolescent Medicine, University Hospital Heidelberg, Heidelberg, Germany 2Division of Neurodevelopmental Pediatrics and Neurogenetics, Children's National Health System and The George Washington School of Medicine, Washington, District of Columbia, USA 3Department of Molecular and Human Genetics, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas, USA
+4Heidelberg Research Center for Molecular Medicine (HRCMM), Heidelberg, Germany Correspondence Matthias Zielonka, Division of Pediatric Neurology and Metabolic Medicine, Center for Child and Adolescent Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany. E-mail:
+matthias.zielonka@med.uni-heidelberg.de Funding Information The Urea Cycle Disorders Consortium (UCDC; U54HD061221) is part of the National Institutes of Health (NIH) Rare Disease Clinical Research Network (RDCRN), supported through a collaboration between the Office of Rare Diseases Research (ORDR), the National Center for Advancing Translational Science (NCATS) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). The Urea Cycle Disorders Consortium is also supported by the O'Malley Foundation, the Rotenberg Family Fund, the Dietmar Hopp Foundation, the Kettering Fund and the National Urea Cycle Disorders Foundation. In addition, support for neuropsychological testing is provided by an NIH grant for Intellectual and Developmental Disability Research Centers (U54HD090257). This work was also supported in part by the Clinical Translational Core at Baylor College of Medicine which is supported by the IDDRC (grant number U54HD083092) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The E-IMD patient registry has received funding from the European Union (E-IMD; EAHC no 2010 12 01; coordinator: SK), in the framework of the Health Program. After the end of the EU funding
+
+## Abstract
+
+![0_Image_1.Png](0_Image_1.Png)
+
+![0_Image_2.Png](0_Image_2.Png)
+
+Objective: Ornithine transcarbamylase deficiency (OTC-D) is an X-linked metabolic disease and the most common urea cycle disorder. Due to high phenotypic heterogeneity, ranging from lethal neonatal hyperammonemic events to moderate symptoms and even asymptomatic individuals, the prediction of the disease course at an early disease stage is very important to individually adjust therapies such as medical treatment or liver transplantation. In this translational study, we developed a severity-adjusted classification system based on in vitro residual enzymatic OTC activity. Methods: Applying a cell-based expression system, residual enzymatic OTC activities of 71 pathogenic OTC variants were spectrophotometrically determined and subsequently correlated with clinical and biochemical outcome parameters of 119 male individuals with OTC-D (mOTC-D) as reported in the UCDC and E-IMD registries. Results: Integration of multiple data sources enabled the establishment of a robust disease prediction model for mOTC-D. Residual enzymatic OTC activity not only correlates with age at first symptoms, initial peak plasma ammonium concentration and frequency of metabolic decompensations but also predicts mortality. The critical threshold of 4.3% residual enzymatic activity distinguishes a severe from an attenuated phenotype. Interpretation: Residual enzymatic OTC activity reliably predicts the disease severity in mOTC-D and could thus serve as a tool for severity-adjusted evaluation of therapeutic strategies and counselling patients and parents.
+
+period, the E-IMD patient registry has been sustained by funding from the Kindness-forKids Foundation (Munich, Germany), the Kettering Fund, and the Dietmar Hopp Foundation. No external funding was secured for this study. MZ (Heidelberg, Germany) was supported by the Physician Scientist Program at the University of Heidelberg and by a Career Development Fellowship provided by the Heidelberg Research Center for Molecular Medicine (HRCMM) in the framework of Excellence Initiative II of the German Research Foundation.
+
+Received: 4 June 2022; Revised: 1 September 2022; Accepted: 6 September 2022
+
+## Annals Of Clinical And Translational Neurology 2022; 9(11): 1715–1726
+
+doi: 10.1002/acn3.51668
+*Both authors contributed equally to this work.
+
+## Introduction
+
+Ornithine transcarbamylase deficiency (OTC-D) is an Xlinked urea cycle disorder (UCD) caused by deficiency of the mitochondrial enzyme ornithine transcarbamylase
+(OTC; OMIM\# 311250) due to pathogenic variants in the ornithine transcarbamylase gene (OTC) located on chromosome Xp21.1. OTC-D is the most common UCD with an estimated incidence of 1: 56,500–63,000 newborns.1–3 Since the urea cycle is the only metabolic pathway of irreversible elimination of excess nitrogen through ureagenesis in humans, disruption of this pathway leads to accumulation of ammonia and glutamine in plasma and thus, affected individuals often present with severe lifethreatening hyperglutaminemia and hyperammonemic events (HAE) during the neonatal period (early onset, EO, defined as first 28 days of life) facing mortality rates as high as 25%–50%. Moreover, survivors often suffer from significant cognitive deficits, and the (cognitive) outcome has not improved within the last decades in neonatal-onset disease.4–8 Some individuals with OTC-D
+and other UCDs, however, may be less severely affected since the clinical phenotype is highly variable, ranging from severe HAE to mild-to-moderate symptoms with disease onset any time after the neonatal period (late onset, LO). Symptoms of LO disease characteristics comprise headache, recurrent vomiting, liver dysfunction, psychiatric symptoms or cognitive impairment, even in the absence of recurrent HAE.9–11 Clinical approaches to predict the disease course and cognitive outcome in individuals with OTC-D identified disease onset as well as the severity of initial HAE [peak plasma ammonium concentration during initial HAE
+(NH4
++
+max)] as determinants. However, a considerable variability persists throughout these clinical correlations.2,4,5,9,12–15 Importantly, on the genomic level, OTC
+variants leading to complete loss of enzymatic OTC function are thought to cause higher disease burden as reflected by EO of symptoms, NH4
++
+max, frequency of HAE, cognitive impairment and mortality as opposed to partly preserved enzymatic activity.4,12,16 Nevertheless, methods to systematically investigate enzymatic OTC
+function are still lacking since in silico modelling of pathogenic variants to predict protein function and stability is still not reliable and kinetic characterization based on liver biopsies or purified recombinant human OTC
+expressed in bacteria has not yet been adapted for systematic measurements.16 Due to the heterogeneity in various clinical prediction models and existing methodological shortcomings regarding the impact of the genotypic background on the clinical outcome, we developed a novel in vitro expression system for male individuals with OTC-D
+(mOTC-D) that enables to predict major clinical outcome parameters and fosters a precision-based medical approach for future evaluation of diagnostic and therapeutic interventions which are supposed to alter the clinical disease course.
+
+## Materials And Methods Research Strategy
+
+Recently, we demonstrated that the clinical disease course and neurocognitive outcome of individuals with citrullinemia type 1 (CTLN1) and argininosuccinic aciduria (ASA), the most prevalent cytosolic UCDs, can be reliably predicted by the enzymatic activity of argininosuccinate synthetase 1 (ASS1) and argininosuccinate lyase (ASL), respectively, as determined by a newly established biallelic mammalian expression system.17,18 As we wanted to know if enzymatic OTC activity can also predict clinical outcome in individuals with OTC-D, the most prevalent mitochondrial UCD and the only X-linked UCD, we systematically investigated all unequivocal pathogenic exonic OTC variants and correlated the spectrophotometrically determined enzymatic OTC activity with individuals' clinical, biochemical and neurological follow-up data. Data were retrieved from the North American Urea Cycle Disorders Consortium (UCDC;
+https://www.rarediseasesnetwork.org/cms/ucdc) and the European registry and network for Intoxication-type Metabolic Diseases (E-IMD; https://www.eimd-registry.
+
+org/), the two largest observational natural history studies on UCDs worldwide.
+
+## Eligibility Criteria
+
+Only hemizygous male individuals carrying known pathogenic exonic variations in the OTC gene, who were confirmed by molecular genetic testing and were enrolled in the observational longitudinal studies of UCDC or E-IMD, were included in this study. Due to individual variation of X-inactivation, the methodological approach used in this study cannot reliably determine enzymatic OTC activity in female individuals with heterozygous variations in the OTC gene. The data model of both registries, information on written informed consent as well as the follow-up protocols used have been previously described in detail.14,19 All procedures were in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 2013.
+
+Written informed consent was given by patients or their legal guardians before enrollment in this study. Data were retrieved from the UCDC and E-IMD electronic databases with the cut-off date for data retrieval being 10 October 2018. The UCDC database is registered at the US National Library of Medicine (https:// clinicaltrials.gov, NCT00237315), whereas the E-IMD
+registry is recorded on the German Clinical Trials Register (https://www.drks.de, DRKS00013085).
+
+## Plasmids
+
+To generate tagged wild-type OTC expression vectors, the OTC coding sequence has been modified with an MYC-tag introduced in between the N-terminal signalling peptide and the core protein, which was cloned into HindIII- and NotI-restriction sites in the openreading frame of the eukaryotic expression vector pcDNA5/FRT/TO (Thermo Fisher Scientific). The inserted OTC coding sequence corresponds to NCBI reference sequence NM_000531.6 (https://www.ncbi.nlm.
+
+nih.gov/nucleotide/NM_000531.6). Using the QuickChange II site-directed mutagenesis kit (Agilent), pathogenic OTC gene variants reported in mOTC-D
+individuals from the E-IMD and UCDC registries were introduced into the tagged OTC expression vector according to the manufacturer's protocol. The correct nucleotide sequence of every inserted variant was confirmed by Sanger-sequencing. All OTC variants reported in this study and their provided nomenclature have been checked by applying the Mutalyzer 2.0.34 software
+(https://mutalyzer.nl/). pSV-b-Galactosidase control vector was kindly provided by N. Himmelreich (Heidelberg University, Germany).
+
+## Cell Culture And Transfections
+
+COS-7 cells were maintained as adherent cell culture in 100 mm culture dishes using a DMEM medium
+(Thermo Fisher Scientific) supplemented with 10% heatinactivated fetal calf serum in a humified incubator at 37 °C and 5% CO2. Transfections were performed using Lipofectamine 2000 reagent (Thermo Fisher Scientific)
+with 5 lg of the wild-type or mutated MYC-tagged OTC expression vector, 2.5 lg of each ASS1 and ASL expression vector and 1 lg of b-galactosidase control vector. Forty-eight hours after transfection, cells were lysed and lysates were used to perform Western blot analysis or spectrophotometric determination of enzymatic OTC activity.
+
+## Western Blot
+
+Forty-eight hours after transfection, COS-7-cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (Sigma-Aldrich) followed by sonification. After centrifugation at 13,000gmax and 4°C
+for 10 min, supernatants were used for Western blotting according to standard laboratory protocols. For protein visualization, PVDF membranes were incubated with the following primary antibodies: anti-MYC
+(1:2000; Cell Signaling) or anti-b-actin (1:2000; SigmaAldrich).
+
+## Spectrophotometric Analysis Of Otc Enzyme Activity
+
+Purified citrate synthase, malate dehydrogenase and fumarase from the porcine heart were purchased from SigmaAldrich. Transfected COS-7 cells were washed in ice-cold PBS and lysed in a buffer containing 10 mmol/L potassium phosphate, 10 mmol/L TRIS–HCl at pH 7.4 and by additional sonification. OTC enzymatic activity was determined as NAD+ reduction at k = 340–400 nm measured in 40 ll of lysates (triplicates) diluted in 100 ll of the abovementioned buffer supplemented with 2 mmol/L ornithine, 2 mmol/L carbamyl phosphate, 2 mmol/L aspartic acid, 330 lmol/L NAD+, 100 lmol/L acetyl-CoA and 2 mmol/L
+ATP, 660 mU/ml fumarase, 1800 mU/ml malate dehydrogenase and 560 mU/ml citrate synthase, adjusted to pH
+7.4. Lysates of COS-7 transfected cells with only bgalactosidase control plasmid served as a negative control.
+
+Negative control values were subtracted from OTC activities to adjust for unspecific background activity. OTC
+activity values were normalized to b-galactosidase activity in each sample as determined by the b-galactosidase enzyme assay system (Promega, Germany) following the manufacturer's protocol to control for transfection efficacy.
+
+Adjusted OTC activities were then normalized to the protein content of each sample. Enzymatic activities are depicted as a percentage of the total (%) by dividing the normalized OTC activity of the respective variant by the normalized wild-type OTC activity.
+
+## Clinical Variables Used For Data Analyses
+
+Data on the following numerical clinical variables were collected: HAE (defined as plasma ammonium concentration (NH4
++) > 100 lmol/L), age at first symptoms, age at death, initial NH4
++
+max (defined as peak plasma ammonium concentration during first HAE), number of HAE
+per year of observation (defined as the time between the date of birth and the last regular visit), initial L-citrulline and L-glutamine concentrations in plasma and initial orotic acid concentration in urine. Moreover, the cognitive standard deviation score (SDS) at the most recent visit was calculated using the normative data from the standardization sample of each cognitive test. The most recent visit was chosen to include the latest developmental data in the analysis. For each Wechsler Adult Intelligence Scale
+(n = 3), Wechsler Abbreviated Scale of Intelligence
+(n = 11), Wechsler Intelligence Scale for Children (n = 6)
+and Wechsler test, unknown version (n = 1) full scale IQ
+were selected. For Bayley Scales of Infant Development
+(n = 15), mental developmental index and cognitive scale were used. For the Adaptive Behavior Assessment System
+(n = 4), the general adaptive composite was used.
+
+As for categorical clinical variables, data of the following items were collected: mortality, disease onset (EO,
+LO, asymptomatic), presence or absence of movement disorders (dystonia and/or chorea and/or ataxia), tone change (muscular hypotonia and/or muscular hypertonia and/or spasticity), hepatocellular injury (alanine aminotransferase ≥250 U/L or aspartate aminotransferase
+≥250 U/L), liver transplantation (LTx) and kidney dysfunction (full age spectrum glomerular filtration rate
+(FAS-GFR) < 90 mL/min/1.73 m2). For symptomatic individuals with reported HAE during the initial presentation, the biochemical data of initial NH4
++
+max, initial plasma L-glutamine and urinary orotic acid concentrations represent the highest values, respectively, and the lowest value for initial plasma L-citrulline concentration prior to initiation of treatment. For symptomatic individuals without reported HAE during initial presentation, initial NH4
++
+max was defined as the upper limit of the normal range (50 lmol/L). For untreated asymptomatic individuals, NH4
++ max values represent the highest reported follow-up values during the observation period; plasma L-citrulline, plasma L-glutamine and urinary orotic acid concentrations represent the arithmetic mean of all reported follow-up values. Asymptomatic individuals with mOTC-D receiving therapeutic agents (scavenger, for Lcitrulline concentration analysis additionally L-citrulline and/or L-arginine substitution) were not considered for biochemical sub-analysis in order to prevent a confounding bias with iatrogenic interventions.
+
+We investigated the impact of the residual enzyme activity of OTC in vitro as determined by the established expression system on clinical outcome parameters outlined above.
+
+## Data Availability Statement
+
+The data sets generated and analysed during the current study are not publicly available due to existing data protection laws. Furthermore, data ownership is retained by the members of the UCDC and E-IMD consortia making data only available for specific research purposes. Data availability is subject to the consent of both consortia upon request.
+
+## Statistical Analyses
+
+All analyses were performed using R (http://www.rproject.org). To evaluate the association between a continuous dependent variable and the residual enzyme in vitro activity of OTC as predictor variable, a linear regression or generalized additive regression model
+(GAM) with automated smoothing selections was used.
+
+In GAM, the linear relationship between the response variable and predictors is replaced by non-linear smooth functions to evaluate an apparently non-linear relationship between dependent and predictor variables. The R package 'mgcv' was used to fit GAM regressions. To compare a numeric dependent variable between two groups, a t-test with Welch correction was applied. We used unbiased recursive partitioning to determine cut-off values for the impact of the enzymatic OTC activity on outcome variables.20 Cox-proportional hazard regression and Kaplan–Meier estimates were used to analyse mortality. p values reported were two-sided. p ≤ 0.05 was considered statistically significant.
+
+## Results Description Of The Study Population And Otc Protein Functions
+
+In 119 male individuals with OTC-D and a total of 71 different OTC gene variants, we systematically determined OTC protein expression levels by Western Blot and enzymatic activities using the newly established OTC enzymatic assay. Results of Western blot analysis are shown in Figure S1. Residual enzymatic OTC activities per variant are illustrated in Figure S2. Detailed descriptive characteristics of the study population for each subsequent analysis are specified in Table S1.
+
+## Enzymatic Activity Is Inversely Correlated To The Severity Of The Initial Presentation
+
+First, we studied whether age at first symptoms and biochemical parameters such as initial NH4
++
+max, Lglutamine, L-citrulline in plasma and orotic acid level in urine correlate with in vitro enzymatic activity. Age at first symptoms correlated with residual enzymatic OTC
+activity (n = 100, p < 0.0001, R2 = 0.17, linear regression), demonstrating that individuals with high residual enzymatic OTC activity present with first symptoms later in life than those with low residual enzymatic OTC activity (Fig. 1). This association is particularly strong for variants leading to residual enzymatic activities below 10%.
+
+In analogy, the disease onset of mOTC-D is strongly associated with residual enzymatic OTC activity as determined by the enzymatic assay. Individuals with early onset (n = 50) showed significantly lower enzymatic activities than those with LO (n = 56, p < 0.001, Tukey multiple comparison of means) and asymptomatic individuals (n = 10, p < 0.01, Tukey multiple comparison of means) (Fig. S3).
+
+Initial NH4
++
+max is negatively correlated with residual OTC activity (n = 98, p < 0.0001, R2 = 0.10, GAM analysis). Moreover, recursive partitioning identified a
+
+![4_image_0.png](4_image_0.png)
+
+threshold separating mOTC-D individuals with a severe initial decompensation from those with an attenuated form. Residual enzymatic OTC activity of below or equal to 4.6% was associated with a more severe decompensation (n = 98, p < 0.001), showing mean initial NH4
++
+max of 1,703 lmol/L in comparison to 652 lmol/L (mean)
+for residual activities >4.6% (Fig. 2). Unlike age at first symptoms and initial NH4
++
+max, there was no significant correlation between residual enzymatic OTC activity and initial plasma L-glutamine (n = 32, p = 0.06, Pearson's product–moment correlation), L-citrulline (n = 25, p = 0.20, r = 0.26, Pearson's product–moment correlation) or urinary orotic acid concentrations (n = 34, p = 0.91, r = 0.02, Pearson's product–moment correlation).
+
+## Residual Otc Activity Correlates With The Frequency Of Hyperammonemic Events
+
+In the next step, we assessed whether residual enzymatic OTC activity could also predict the severity of disease course and found a correlation with the number of HAE
+per year of observation (n = 52, p < 0.05, R2 = 0.06, GAM analysis), showing a higher number of HAE below the threshold of 16.0% (n = 52, p < 0.05, recursive partitioning) (Fig. 3). In mean, individuals with residual
+
+![5_image_0.png](5_image_0.png)
+
+enzymatic activities below or equal to 16.0% (n = 35)
+present with 3.2 HAE per year of observation, whereas individuals with residual OTC activities above 16.0%
+(n = 17) present with 0.3 HAE per year of observation.
+
+## Low Residual Otc Activity Increases The Risk Of Mortality
+
+Mortality negatively correlates with residual enzymatic OTC activity (n = 16/119, p = 0.005, odds ratio = 0.87, Cox Proportional Hazard). Male individuals with residual OTC activity below or equal to 4.3% had a higher risk of mortality than those with higher residual activities
+(n≤4.3% = 9/28, n>4.3% = 7/91, p = 0.003, recursive partitioning) (Fig. 4). Corresponding mortality rates are 32.1% for residual OTC activities below or equal to 4.3%
+and 7.7% for residual activities above 4.3%.
+
+A correlation between residual OTC activity and cognitive SDS at last follow-up test (n = 40, p = 0.12, R2 = 0.04, linear regression) was not found. However, sub-analyses showed that cognitive SDS was associated with initial NH4
++
+max (n = 29, p = 0.002, R2 = 0.40, multiple linear regression) but not with age at first symptoms
+(n = 29, p = 0.56, R2 = 0.40, multiple linear regression).
+
+Residual enzymatic activity was not associated with tone change (N = 93, p = 0.06, Welch two-sample t test), movement disorder (n = 90, p < 0.93, Welch two-sample t-test), liver transplantation (n = 119, p = 0.09, Welch two-sample t-test), kidney dysfunction (n = 105, p = 0.44, Welch two-sample t-test) or hepatocellular injury (n = 83, p = 0.44, Welch two-sample t-test).
+
+## Discussion
+
+The major aim of this study was to evaluate whether residual enzymatic activity of mutated OTC protein could precisely predict disease severity. To this end, we compared in vitro enzymatic activity of unequivocal exonic OTC variants with clinical outcome data reported in the UCDC and E-IMD databases. Residual enzymatic OTC
+activity reliably predicts phenotypic severity as reflected by the initial presentation, metabolic disease course and mortality for male individuals. We found for male individuals with OTC-D that (I) residual enzymatic OTC activity correlates with age at first symptoms, (II) enzymatic OTC activity below or equal to 4.3% is associated with higher initial plasma NH4
++
+max and higher mortality rates and (III) residual enzymatic OTC activity below or
+
+![6_image_0.png](6_image_0.png)
+
+A
+B
+
+![6_image_1.png](6_image_1.png)
+
+equal to 16.0% is associated with a higher number of HAE per year of observation.
+
+## Residual Enzymatic Otc Activity Qualifies As A Predictive Biomarker For Disease Severity
+
+As in vitro residual enzymatic OTC activity reflects disease severity and reliably predicts major clinical and biochemical outcome parameters, it qualifies as a prognostic and predictive biomarker as defined by Katz21 and the US Food and Drug Administration.22 As there are currently no approved pharmacotherapies aiming at increasing enzymatic OTC activity, the required reflection of net treatment effects on the outcome to truly serve as a validated surrogate marker cannot be addressed at present.23 However, the demonstrated correlations could further encourage the development of new therapies targeting the increase of enzymatic OTC
+activity. In particular, the established disease prediction model enables a precision-based medical approach as it indicates to which level residual enzymatic OTC activity needs to be increased to convert the phenotype from severe to attenuated.
+
+Using the identified robust cut-off values of residual enzymatic OTC activities enables risk stratification of mOTC-D phenotypes and their underlying OTC variants.
+
+According to the above-mentioned thresholds, we suggest a new classification comprising two clinical phenotypes: Severe phenotype for OTC variants leading to residual enzymatic activity below or equal to 4.3% (high initial NH4
++
+max and mortality rate) and attenuated phenotype for OTC variants leading to residual enzymatic activity above 4.3% (lower initial NH4
++
+max and mortality rate).
+
+Showing that the onset type is associated with residual enzymatic OTC activity allowed us to validate the current classification system based on EO, LO and asymptomatic individuals. A major shortcoming of the existing clinical classification system is the lack of predictive options since patients will be attributed retrospectively to one class after the onset of first symptoms. The here presented complementary stratification system is able to further specify and predict the evolving phenotype before first symptoms might have manifested and is based on the genotypic background of affected individuals.
+
+This new system of risk stratification allows counselling of patients and their families with regard to relevant clinical and biochemical long-term outcome parameters as early as genetic testing is completed. However, with regard to counselling patients and families, it is important to emphasize that this disease prediction model cannot predict the exact individual disease course but is of importance to anticipate the expected outcome in an evidence-based manner. Moreover, it serves as a useful tool for the severity-adjusted evaluation of (future) therapies and care concepts and allows the evaluation of existing diagnostic concepts (e.g. newborn screening) in a standardized and severity-adjusted manner.
+
+## Modelling Evidence-Based Thresholds For Clinical Health Outcomes In Male Otc-D
+
+It has recently been shown that residual enzymatic activity of the cytosolic urea cycle enzymes ASS1 and ASL reliably predict disease severity of individuals with CTLN1 and ASA, respectively.17,18 As we wanted to know if these associations also hold true for OTC-D, the most prevalent urea cycle disorder, we investigated residual activities of the mitochondrial enzyme OTC in vitro. Comparable to CTLN1 and ASA, residual OTC activity correlates with initial NH4
++
+max and the number of annual HAE in mOTC-D. Intriguingly, it predicts further relevant clinical endpoints, which are mortality and age at first symptoms. Although individuals with mOTC-D present a higher metabolic disease burden with more severe and more frequent HAE and higher mortality rates, the neurocognitive outcome in survivors seems to be better than in individuals with distal UCDs, suggesting additional underlying pathomechanisms other than NH4
++-dependent neurotoxic alterations alone.14,18 In the context of determining the accuracy of our expression system, the cut-off value for mortality of 4.3% can be seen as similar to the one for initial peak plasma NH4
++
+max (4.6%), reinforcing the clinical relevance of this threshold. This goes in line with the clinical observation that the most severely affected individuals with mOTC-D
+still frequently die during their first metabolic decompensation in the neonatal period despite improved intensive care as well as efficacious intravenous and extracorporeal detoxification strategies. Thus, it is tempting to speculate that therapeutic interventions that are able to increase the residual enzymatic activity at an asymptomatic state early in life might be a very promising option to reduce mortality.
+
+This is reinforced by the interesting finding that the threshold for a lethal disease course in mOTC-D is similar for different mammalian species. Knocking out the OTC gene in mice results in high and early mortality, while different currently used mouse models (spf, spf-j and spfash) with residual OTC activities of 5%–20% present milder phenotypes with low mortality rates and onslight biochemical alterations.24–27 Our results support the notion of higher mortality rates among individuals carrying an OTC variant leading to complete loss of OTC function.2,4 The study cohort shows one-third of mortality rates for individuals with residual enzymatic OTC activities below or equal to 4.3% as opposed to mortality rates of less than 10% for individuals with higher residual enzymatic OTC activities. Moreover, only two of the observed deaths occurred after the neonatal period. This fact strengthens the need for early diagnosis and stratification of fatal mutations in order to reduce mortality.
+
+Low plasma L-citrulline, high plasma L-glutamine and the detection of orotic acid in urine are highly indicative of OTC-D in clinical practice.9,10 However, these biochemical parameters do not seem to qualify as reliable predictive biomarkers for the severity of the initial decompensation or the subsequent disease course. Similar limitations account for neurological alterations, such as tone change and movement disorders.
+
+Intriguingly, residual enzymatic OTC activity showed no correlation with the cognitive outcome as measured by IQ testing. However, in line with recent works, a subanalysis of our cohort revealed an association between cognitive SDS and initial NH4
++
+max.12–14 These findings indicate that the height of the initial NH4
++
+max appears not only to be determined by the underlying residual enzymatic activity but also by other non-standardized factors, such as but not limited to (1) timepoint and intensity of initial medical management and (2) effect size of the cause(s) of the initial decompensation (e.g. catabolic state, protein load, etc.), which are very heterogeneous between various individuals having similar or the same residual enzymatic activities.
+
+Although the UCDC and E-IMD registries have been systematically collecting follow-up data of UCD individuals since 2003 and 2011, respectively, we are still missing intra-individual long-term data on cognitive functioning and cerebral brain imaging to improve our understanding of the natural cognitive disease course and to reveal morphological changes as well as their underlying pathomechanisms of neurological impairment.
+
+## Directions For Future Research
+
+The exploratory data on alterations of protein expression of OTC variants captured in this study can be used as a basis for further characterization of underlying molecular pathomechanisms of the respective pathogenic variants.
+
+For more than two decades, researchers have aimed at developing gene therapies for OTC-D.28-33 Recently, a phase 1/2 study of adeno-associated virus 8 gene therapy in adults has been conducted (https://clinicaltrials.gov/,
+Identifier: NCT02991144). Using the newly established expression system could be of help to assess the treatment effects of new therapeutic approaches (e.g. by application of gene therapies, chaperones or antisense nucleotides).
+
+Chaperones improving the stability of translated protein and antisense nucleotides targeting altered splicing of the precursor mRNA to increase the concentration of correctly translated protein have shown beneficial effects in other inborn diseases.34,35 Achieving an increase of residual enzymatic OTC activity above the threshold of 5% by therapeutic means might not only reflect a reduction of mortality but also the phenotypic conversion from a severe to an attenuated disease course.
+
+## Limitations
+
+The here presented stratification system is not able to predict exact maximal heights of NH4
++, number of HAE or age at onset. It appears likely that other factors like diagnostic and therapeutic delay or severity of catabolic state influenced by protein intake, infections, drugs, awareness of preceding symptoms or compliance to life-long therapy modify the clinical severity of an individual affected by OTC-D. It has been reported that even the same mutations can cause clinically variable appearances in different individuals.1,5,16,36 The study results need to be evaluated prospectively in the future.
+
+Moreover, our study has some inherent limitations.
+
+First, due to the applied cloning technique utilizing the OTC coding sequence, we were not able to design intronic mutations or those variants associated with defective splicing. Second, variants that affect substrate binding affinity (Km-variants) could not be investigated as our assay uses supraphysiological substrate concentrations leading to artificially increased enzymatic activities in those variants. Third, we could not include female individuals with OTC-D because of variable X-inactivation.
+
+Our study does not claim to systematically analyse the underlying molecular events in every OTC variant including impaired protein folding or decreased protein stability. Rather, the results of protein expression levels should be considered somewhat exploratory and encourage further investigation in future studies.
+
+Furthermore, the structure of the UCDC and E-IMD
+registries did not enable the inclusion of data from both registries for every subanalysis. Despite systematic requirements for data collection, the given data density and quality reduced test power for some analyses. Further intraindividual long-term data are crucial to substantiate our findings, and longer observation periods and the inclusion of more individuals might help to reveal further and strengthen given genotype–phenotype correlations, in particular, with regard to classify cognitive alterations or to identify asymptomatic individuals.
+
+It seems likely that the group of male individuals with an attenuated phenotype comprises different subphenotypes, such as life-long asymptomatic individuals, which might be revealed by evaluating long-term data in the future.
+
+## Conclusion
+
+Given the genotype–phenotype correlations revealed in this study, we suggest a new severity-adjusted classification system and prediction model for male individuals with OTCD dividing affected individuals into two phenotypic classes:
+severe (residual enzymatic OTC activity ≤4.3%) and attenuated phenotypes (residual enzymatic OTC activity >4.3%).
+
+This classification system can be considered complementary to the current clinical classification system for OTC-D based on the disease onset (EO, LO and asymptomatic).
+
+Given the impact of enzymatic OTC, ASS1 and ASL activities as predictive and prognostic biomarkers for the clinical disease course and outcome of individuals with OTC-D, CTLN1 and ASA, respectively, we are confident that this approach can be adapted and applied to other monogenetic inborn errors of metabolism. This would not only enable early identification of severely affected individuals preferably before irreversible organ dysfunctions might have manifested but could also facilitate the development and implementation of individual therapeutic care concepts adjusted to the anticipated disease severity.
+
+## Acknowledgements
+
+All UCDC and E-IMD sites contributed to the data sets of the longitudinal studies used in this publication. Principal investigators and personnel with key contributions are listed as UCDC and E-IMD consortia study group members. Furthermore, we gratefully acknowledge subsequent study coordinators—Jennifer Seminara, Saima Ali, Sondra Bloxam, Kia Bryan, Sara Elsbecker, Joan Hart, Melanie Horn, Elijah Kravets, Audrey Lynn, Mary Mullins, Maya Muldowney, Kendall Parks, Ulrike Mutze, Thu €
+Quan, Kara Simpson, Julia Smith, Suzanne Hollander and Hayden Vreugdenhil—and study neuropsychologists— Fabienne Dietrich Alber, Talin Babikian, Heidi Bender, Christopher Boys, David Breiger, Mina Nguyen-Driver, Benjamin Goodlett, Elizabeth Kerr, Magdalena E. Kowoll, Casey Krueger, Eva Mamak, Jacqueline H. Sanz, David Schwartz, Arianna K. Stefanatos, Rachel Tangen and Greta N. Wilkening. We would also like to acknowledge the contributions of (former) longitudinal study principal investigators: Mark L. Batshaw, Stephen Cederbaum, Annette Feigenbaum, Douglas S. Kerr, Brendan Lee, Uta Lichter-Konecki, Margretta R. Seashore, Marshall L. Summar, Peter Burgard, Curtis R. Coughlin II, Gregory Enns, Renata C. Gallagher, Cynthia Le Mons, Shawn E.
+
+McCandless, Tamar Stricker, Mendel Tuchman, Susan Waisbren and James D. Weisfeld-Adams. In particular, we are indebted to all our UCD individuals and their families for their trust, patience and participation in both longitudinal registry studies for many years.
+
+The Urea Cycle Disorders Consortium (UCDC;
+U54HD061221) is part of the National Institutes of Health (NIH) Rare Disease Clinical Research Network
+(RDCRN), supported through a collaboration between the Office of Rare Diseases Research (ORDR), the National Center for Advancing Translational Science
+(NCATS) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD).
+
+The Urea Cycle Disorders Consortium is also supported by the O'Malley Foundation, the Rotenberg Family Fund, the Dietmar Hopp Foundation, the Kettering Fund and the National Urea Cycle Disorders Foundation. In addition, support for neuropsychological testing is provided by an NIH grant for Intellectual and Developmental Disability Research Centers (U54HD090257). This work was also supported in part by the Clinical Translational Core at Baylor College of Medicine which is supported by the IDDRC (grant number U54HD083092) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The E-IMD patient registry has received funding from the European Union (E-IMD;
+EAHC no 2010 12 01; coordinator: SK), in the framework of the Health Program. After the end of the EU funding period, the E-IMD patient registry has been sustained by funding from the Kindness-for-Kids Foundation (Munich, Germany), the Kettering Fund, and the Dietmar Hopp Foundation. No external funding was secured for this study. MZ (Heidelberg, Germany) was supported by the Physician Scientist Program at the University of Heidelberg and by a Career Development Fellowship provided by the Heidelberg Research Center for Molecular Medicine (HRCMM) in the framework of Excellence Initiative II of the German Research Foundation. Open Access funding enabled and organized by Projekt DEAL. WOA
+Institution: UNIVERSITATSKLINIKUM HEIDELBERG
+Consortia Name : Projekt DEAL
+
+## Author Contributions
+
+The STROBE statement was used when preparing this manuscript. SS, RP and MZ contributed to the conception and design of the study. SS, RP, SFG, FG, MJS,
+ACD, JGO, ALG, SCSN, GFH, SK, MZ and all individual contributors from the UCDC and E-IMD consortia study group (Table S2) contributed to the acquisition and analysis of data. SS, RP, SFG, SK and MZ contributed to drafting the text and preparing the figures.
+
+## Conflict Of Interest
+
+SK received EU funding for the European registry and network for Intoxication type Metabolic Diseases (E-IMD;
+CHAFEA agreement no. 2010 12 01). SK receives funding from Immedica Pharma AB for the European PostAuthorization Registry for Ravicti- (glycerol phenylbutyrate) oral liquid in partnership with the E-IMD (RRPE)
+(EU PAS Register no. EUPAS17267; http://www.encepp.
+
+eu/). SK and GFH receive funding from the Dietmar Hopp Foundation (St. Leon-Rot, Germany) for a pilot study on extended newborn screening evaluating the technical feasibility, diagnostic process quality and health benefits for 28 inherited metabolic diseases including UCDs
+(NBS 2025, project no. 1DH1911376, 1DH2011117). GFH received lecture fees from Swedish Orphan Biovitrum GmbH. RP receives consultancy fees from Immedica Pharma AB. The sponsors have in no way influenced the design, conductance, analysis and report of the present study. All other authors declare that they have no conflict of interest.
+
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+Sci Adv. 2020;6(7):eaax5701.
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+
+## Supporting Information
+
+Additional supporting information may be found online in the Supporting Information section at the end of the article.
+
+Figure S1. Overview of protein expression levels per variant.
+
+Figure S2. Overview of residual enzymatic OTC activity per variant. Figure S3. Correlation between disease onset and residual enzymatic OTC activity.
+
+Table S1. Descriptive characteristics of study cohort and subanalyses.
+
+Table S2. Additional members and affiliations of the UCDC and E-IMD consortia study group.

medical/md/PMC9649934.md

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+Original article
+
+![0_image_1.png](0_image_1.png)
+
+High prevalence of vitamin D deficiency in HIV-infected individuals in comparison with the general population across Punjab province, Pakistan Wajiha Kanwal a,b, Abdul Rehman a,⇑
+a Institute of Microbiology and Molecular Genetics, University of the Punjab, New Campus, Lahore 54590, Pakistan b Punjab AIDS Control Program, Lahore, Pakistan article info
+
+## Abstract
+
+Article history:
+
+Received 17 August 2022
+
+Revised 20 September 2022
+
+Accepted 25 October 2022
+
+Available online 31 October 2022
+
+![0_image_4.png](0_image_4.png)
+
+![0_image_5.png](0_image_5.png)
+
+| Keywords: Hypovitaminosis D HIV/AIDS HBV/HCV ELISA CD4 count   |
+|----------------------------------------------------------------|
+
+journal homepage: www.sciencedirect.com The status of vitamin D in individuals infected with human immunodeficiency virus (HIV), particularly in naïve as well as treated patients, has never been reported in the Pakistani population. A cross-sectional study was performed to measure vitamin D in individuals infected with HIV living in various districts of the Punjab, Pakistan. 1000 persons attending various treatment centers of the Punjab were screened for HIV, hepatitis B virus (HBV), hepatitis C virus (HCV), and Syphilis. Total 398 patients met inclusion criteria and vitamin D level was measured in respective cases by using enzyme-linked immunosorbent assay
+(ELISA) technique. 232 samples from the healthy population were also included in present research.
+
+Demographic history and clinical parameters regarding HIV disease were evaluated. Comparison of variables was done to find out the link between vitamin D levels and characteristics of HIV infected persons and comparison to that of healthy individuals was performed. Among 398 HIV patients vitamin D deficiency and insufficiency was found among 15 % and 39 % while majority of the control participants had sufficient levels of vitamin D (78 %). Most of the HIV infected individuals were males (68.6 %) and had age between 24 and 47 years (67.8 %). A significant relationship was found for vitamin D level, lifestyle and CD4 count among HIV + ve non acquired immunodeficiency syndrome (AIDS) subjects (95 % CI;
+p < 0.001, p = 0.09). For HIV + ve AIDS patients vitamin D had a significant relationship with lifestyle along with HIV viral load and CD4 count. Hypovitaminosis D prevails among the HIV infected population of Punjab, Pakistan. 2022 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+## 1. Introduction
+
+Vitamin D, a steroid hormone, can be synthesized endogenously or exogenously via food or dietary supplements (Ali, 2020) and is a standard hormone characterized for its pivotal role in skeletal and mineral homeostasis. After exposure to UV rays from the sun, 7dihydrocholesterol in the skin is converted to preVitD3. PreVitD3 is then transformed into cholecalciferol via spontaneous isomerization (VitD3) and is carried to the kidney and gets hydroxylated
+⇑ Corresponding author.
+
+![0_image_7.png](0_image_7.png)
+
+E-mail addresses: wajiha.phd.mmg@pu.edu.pk (W. Kanwal), rehman.mmg@pu.
+
+edu.pk (A. Rehman).
+
+Peer review under responsibility of King Saud University.
+
+to form 1, 25 dihydroxycholecalciferol (1,25(OH)2D) or calcitriol and locally activated by CYP27B1 (Kim et al., 2020) in numerous tissues including immune cells, brain, breast, prostate and smooth muscles (Dusso et al., 2005; Jiménez-Sousa et al., 2018). The major hormonal activity of vitamin D is linked with intestinal calcium transport, renal calcium absorption, insulin secretion, osteogenesis, blood pressure regulation, and apoptosis (Skrobo et al., 2018).
+
+In order to initiate all biological processes and to be transported to all tissues 1,25(OH)2D form a complex with vitamin D-binding protein (DBP), an essential substrate to maintain the level of vitamin D in the body (Callejo et al., 2020). Vitamin D action process is interceded by interaction with high-affinity transcription factor VDR. The D-VDR complex modulates expression of gene at the transcriptional stage (Prietl et al., 2013). In addition to classical function, vitamin D also involves the following non-classical functions; immune responses, hormonal secretion, cell proliferation, and maturation (Xu et al., 2020; Dimitrov et al., 2021).
+
+| Production and hosting by Elsevier   |
+|--------------------------------------|
+
+![0_image_8.png](0_image_8.png)
+
+https://doi.org/10.1016/j.sjbs.2022.103484 1319-562X/ 2022 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
+
+This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+Contents lists available at ScienceDirect
+
+![0_image_0.png](0_image_0.png)
+
+![0_image_2.png](0_image_2.png)
+
+![0_image_3.png](0_image_3.png)
+
+![0_image_6.png](0_image_6.png)
+
+ 
+Vitamin D acts as a potent immunomodulator expressing intracellular VDR on macrophages, monocytes, and lymphocytes (T and B cells) (Holick, 2007; Bishop et al., 2020). The first evidence of vitamin D's effect on the immune cells in both innate and adaptive immune responses emerged approximately-three years ago (Penna et al., 2005; Bikle, 2009). It regulates the innate immune system by increasing the synthesis of b2 defensins and cationic antimicrobial peptide (CAMP), cathelicidin by macrophages and monocytes, hence enhancinptig their antibacterial activity (Dai et al., 2010).
+
+The immunomodulatory effect of vitamin D in adave immune response suggests that it inhibits T-helper1 cell (Th1) activation and synchronizes T-helper2 cell (Th2), T regulatory cell (Treg) and T-helper17 cell activity (Th17) (Boonstra et al., 2001). Vitamin D effect on cell differentiation is affirmed through antigenpresenting (APC) cells and dendritic cells, involved in T cells differentiation (Bishop et al., 2020). Dendritic cell differentiation within a vitamin-D microenvironment regulates a ''tolerogenic state" characterized by decreased inflammatory cytokine levels (i.e., IL12 AND TNF-) and enhanced anti-inflammatory cytokine levels (IL-10), which predominantly upregulate the development of regulatory T cells and promote cell death of autoreactive T-cells (Unger et al., 2009; Bishop et al., 2020).
+
+Vitamin D importance in regulating immune responses revealed that its insufficiency is linked with an increased risk of a large number of comorbidities, including metabolic disorders, cardiovascular disorders, osteoporosis, diabetes, autoimmune diseases, cognitive disorders, and certain malignancies (Colotta et al., 2017; Charoenngam and Holick, 2020). Vitamin D deficiency is a worldwide condition with a high occurrence in the general population of both developing (Middle East and subcontinent) and developed nations (Raza et al., 2019).
+
+Clinically, hypovitaminosis is described as a serum D (25[OH]D)
+concentration of 20 ng/ml (Holick, 2007) in both the general population and HIV-infected individuals (Lappe et al., 2007). Sunlight deprivation, black race, malnutrition, low nutritional intake, obesity, high altitude living, and usage of medicines that stimulate catabolism of vitamin D, like glucocorticoids and anticonvulsants, are all well known vitamin D deficiency risk factors (Bischoff-Ferrari et al., 2009). The deficiency of vitamin D is also linked with the prevalence of opportunistic infections and HIV exacerbation, leading to death in untreated patients (Bischoff-Ferrari et al., 2009).
+
+Vitamin D helps to increase the lifespan of individuals infected with HIV through a therapy i.e., highly active antiretroviral therapy
+(HAART) but the risk of comorbidities remains elevated in comparison to the general population, most likely due to immunesuppression (Collins et al., 2020). Another study described that vitamin D and CD4 + T cells made an undeviating association and decreased absolute CD4 + T cells were found among HIV-positive people who were vitamin D deficient after starting HAART
+(Rosenvinge et al., 2010).
+
+A metabolite of vitamin D, (25[OH]D), is served to determine the serum vitamin D concentration in humans (DeLuca, 2004). Although the 1,25-dihydroxy vitamin D (1,25[OH]2D) is the active vitamin D metabolite, routinely it can not be estimated due to its stringent regulation and short half life (Wasserman and Rubin, 2010). Quantifying 1,25[OH]2D alongside 25[OH]D may help to identify other diseases of 1- hydroxylation, 25[OH]D renal conversion to its active form, and irregular extra-renal synthesis of 1,25
+[OH]2D (systemic infections, sarcoidosis, and hematological disorders) (Wasserman and Rubin, 2010). There is a pressing need to investigate the vitamin D levels of the HIV-infected population.
+
+Immunocompromised population has an increased risk of suffering hypovitaminosis-related consequences. Consequently, the present study intends to investigate vitamin D levels in immunocompromised populations, i.e. HIV-infected individuals from different areas of Punjab, Pakistan.
+
+## 2. Materials And Methods 2.1. Study Design And Sample Collection
+
+In order to conduct this study, n = 1000 subjects were recruited between 2018 and 2019 from Voluntary Confidential Counseling and Testing (VCCT) Centers in Punjab, including Allied hospital Faisalabad, DHQ hospital Chiniot, Aziz Bhatti Shaheed Hospital Gujrat, Benazir Bhutto Shaheed hospital Rawalpindi, DHQ hospital Bahawalnagar, and DHQ hospital DG Khan (Fig. S1). Control blood samples (n = 232) were drawn from healthy populations that have not any known infectious or metabolic disorder. An informed consent about the study was obtained from patients infected with HIV
+and controls prior to sample collection. A questionnaire was created per the sample collecting inclusion and exclusion criteria.
+
+HIV positive status was a prerequisite for inclusion criteria. The co-infected patients with HBV, HCV and syphilis were excluded. The research questionnaire assessed demographic history (age, gender, district, and history of HIV treatment) and lifestyle (urban and rural).
+
+The 5CC Ethylenediaminetetraacetic acid (EDTA) whole blood was taken using the World Health Organization (WHO)-
+recommended venipuncture method. The whole blood was used to obtain a CD4 count, after which plasma was isolated for HIVPCR by centrifugation. These specimens were then processed in the Advanced diagnostic laboratory. Following the rapid screening test, samples infected with hepatitis B, C, and/or syphilis were excluded from this investigation. Also, subjects who were drug addicts or had infections following surgery were excluded. The present research work was recommended by the research ethics and biosafety committee of the Institute.
+
+## 2.2. Rapid Testing For Hiv
+
+Initially, samples were tested for HIV Ag/Ab, HBV, and HCV
+using ''AlereTM HIV Ag/Ab," ''AlereTM HBs Ag," and ''SD Bioline anti-HCV Ab" quick diagnostic machines, respectively.
+
+## 2.3. Cd4 Cell Count
+
+The CD4 count was performed on ''PIMATM analyzer" using 25 ll whole blood in PIMATM CD4 cartridge. It is an image-based, automated immunological assay intended for rapid in vitro measurement of CD4 cells (T helper cells).
+
+## 2.4. Rna Extraction
+
+Selected samples were treated for RNA extraction using a kit
+(QIAamp Viral RNA Mini Kit), and a buffer containing guanidinethiocyanate was used to lyse and homogenize the samples in order to purify and collect the intact RNA. To ensure the appropriate binding conditions, ethanol was added and then any contaminants present were washed away using the RNeasy Mini spin column. Then, high-quality RNA was eluted in 70 ul of AVE buffer.
+
+## 2.5. Rna Amplification
+
+HIV RNA was amplified using the Artus HI Virus-1 RG RT-PCR
+Kit (the limit of detection for HIV viral load was 71 IU/ml). For PCR amplification, a 50 ll reaction mix was prepared in the ''Rotor Gene" cup by adding 30 ll MM (master mix) and 20 ll of extracted RNA. ''Rotorgene" was used to amplify the reaction mixture. After performing RT-PCR for HIV viral load, 398 HIV-positive samples were chosen for additional vitamin D analysis.
+
+## 2.6. 25(Oh)D Estimation
+
+The vitamin D assay was performed by EUROIMMUN 25-OH
+vitamin D test kit (Noori et al., 2018). The vitamin D status was categorized as sufficient (30 ng/ml), insufficient (11–29 ng/ml), or deficient (10 ng/ml) (Ginde et al., 2009).
+
+## 2.7. Statistical Analysis
+
+Using the laboratory management information system (LMIS)
+software of PACP-ADL, the patient's age, gender, and area of residence were recorded. SPSS 28.0.0.0 was used to tabulate and analyze the data for all conducted tests and demographic variables. Chi square test was done to see the association of serum 25(OH) D and p-value<0.05 was considered as significant.
+
+## 3. Results 3.1. Regional Distribution
+
+Blood samples received from various HIV treatment centers of the Punjab i.e., Aziz Bhatti Shaheed hospital (ABS) Gujrat, Allied hospital Faisalabad, Benazir Bhutto Shaheed hospital (BBSH) Rawalpindi, DHQ hospital Bahawalnagar, DHQ hospital Chiniot, DHQ
+hospital DG Khan and various places from Lahore (Fig. S1). Most of the HIV samples in Lahore (n = 196) region were collected at Jinnah Hospital Lahore 56 (28.6 %) followed by Services Hospital Lahore 51 (26 %) (Fig. 1).
+
+## 3.2. Clinical Characteristics Of Patients And Controls
+
+A total number of 789 HIV positive samples were obtained after
+
+![2_image_0.png](2_image_0.png)
+
+HIV ICT screening out of total 1000 samples and 181 samples were rejected for HCV, HBV and Syphilis co-infection as an exclusion criteria. A subject was considered hepatitis C or B positive if one had anti-HCV antibody or HBsAg positive on ICT device. The remaining 607 samples were further processed for PCR amplification to rule out for the detection of viral load. After following all the screening steps, 398 samples with HIV viral load were preferred for CD4+
+count assessment and Vitamin D analysis (Fig. 2). Demographic characteristics of the HIV positive patients such as age, gender, lifestyle and use of cART were investigated. Most of the studied patients were males 275 (69.09 %). TGs were also reported in present study but in low numbers i.e., 6 (1.5 %). Most of the studied population 270 (68 %) was aged between ''24–47 years" followed by ''1–23 years" 92 (23 %) and was mostly from urban areas i.e., 256 (64.30 %). HIV risk groups were also identified in the studied population. Among them 138 (34.6 %) were intravenous drug users (IDUs) and the general population also had high frequency 90 (22.6 %) after IDUs. In the present study, 279 (70.1 %) samples were of newly diagnosed cases. Controls were also categorized for age and gender (Table 1). Most of the controls were males 155 (67 %) and 139 (60 %) aged between ''24–47 years". In the present study, most of HIV cases have viral load  10,000 i.e., 233 (56.03 %). CD4 count was also categorized and most of the patients had ''200–499 count/ll" i.e., 154 (38.7 %).
+
+3.3. Deficiency of serum 25(OH)D in population infected with HIV and healthy individuals In the current investigation, deficiency and insufficiency of vitamin D was found in 62 (15.6 %) and 156 (39.2 %) patients, respectively. Most of the control participants had sufficient levels of vitamin D 182 (78 %). Results of statistical analysis indicate a noteworthy link of vitamin D deficiency with HIV cases (p < 0.001). The concentration of 25(OH) D in the population infected with HIV and non infected population (controls) has been shown in Fig. 3.
+
+3.4. Serum 25(OH)D association with HIV + ve and AIDS -ve subjects Among HIV positive, non AIDS cases, all vitamin D categories including vitamin D sufficiency, insufficiency, and deficiency were found in 130, 104, and 33 subjects, respectively. The male population had insufficient 25(OH)D levels (65.6 %). A comparable vitamin D association was found in relation to lifestyle and CD4 count of the participants (p < 0.001 and p = 0.009) as shown in Table 2.
+
+3.5. Serum 25(OH)D association with HIV + ve & AIDS + ve subjects Various parameters of AIDS patients (CD4 count h2 00) in different categories with respect to vitamin D were analyzed by using SPSS version 28.0. The categorization was done on the basis of amount of vitamin D such as  10 ng/ml was considered as deficient patient (n = 27), 11 to 29 ng/ml was insufficient patients
+(n = 51) and  30 ng/ml was considered as sufficient patients
+(n = 53). In the category of gender, male and female were included.
+
+Deficiency and insufficiency was prevalent among males (70.37 %
+& 76.4 %) as compared to female patients (29.6 % & 23.5 %). Lifestyles of AIDS patients were classified in terms of rural and urban.
+
+Vitamin D deficiency was more prevalent among rural area participants (70.4 %) while insufficiency was seen among urban cases in high frequency (70.5 %). Lifestyle of AIDS patients was significantly linked with concentration of vitamin D (p < 0.001). Among HIV viral load and CD4 count categories, a significant relationship was determined in relation to 25(OH)D with lower CD4 count and viral load  10,0000 copies/ml (p = 0.01) (Table 3).
+
+## 3.6. Relationship Among Different Variables
+
+Pearson correlation depicted the weak negative correlation of vitamin D with viral load and CD4 count (-0.02, 0.025). However, the correlation was non-significant at CI = 95, p > 0.05 (Table S1).
+
+## 4. Discussion
+
+A condition in which serum vitamin D levels are low known as hypovitaminosis D which correlates a number of medical conditions significantly transitioning bone health. In clinical practice, hypovitaminosis D is generally assessed by insufficient 25-
+Fig. 1. Samples collected from various places of Lahore district (Punjab) from period of March 2018 to July 2019.
+
+ 
+
+![3_image_0.png](3_image_0.png)
+
+hydroxy vitamin D status in serum. The worldwide cumulative agreement suggests that below the optimal range (<30 ng/ml) is considered as vitamin D deficiency (Holick, 2007; Vieth et al., 2007). However, some studies propose the 25–80 ng/ml as optimal range and consider < 20 ng/ml as more apt to explicate deficiency
+(Ross et al., 2011). The current work reports that deficiency (15 %)
+and insufficiency (39 %) of vitamin D was higher among HIV infected individuals than local Pakistani population. Oyedele and Adeyemi reported the variance in prevalence rate of deficiency of vitamin D from 10 to 73 % in the population infected with HIV
+(Oyedele and Adeyemi, 2012).
+
+Above and beyond the certain discrepancies in heterogeneous study population, the researchers also ruled out the geographic and demographic differences distressing the concentrations of vitamin D in the individuals under trials. They also appraise the schematic approaches and the controversial cut-off points for low vitamin D levels as significant limitations in cross-sectional research involving HIV patients. Other trials stated that HIVpositive carriers frequently encounter vitamin D deficiency
+(Rodriguez et al., 2009; Mueller et al., 2010) even on successful combined antiretroviral therapy (cART). Well-known risk factors impairing bone maintenance and attributing to low levels of 25 (OH)D in HIV infection include minimized exposure to sunlight and nutritive intake, insufficient absorption, fatty liver, and renal damage encouraged anomalous vitamin D activation, altered bioavailability of non-hydroxylated vitamin D in adipose tissue, and the antiretroviral treatment intervening vitamin D metabolism
+(Cozzolino et al., 2003). Though progressive age is a typical indicator for hypovitaminosis D (Vescini et al., 2011), this relationship was not significant in the present work (p = 0.29; p = 0.09), and is consistent with prior findings in HIV carriers (Wasserman and Rubin, 2010; Dao et al., 2011; Allavena et al., 2012). Allavena et al. did not observe substantial correlation between age and VDD, one of the justifications may comprise the samples from
+
+| Table 1 Demographic and clinical characteristics of cases and control subjects. Characteristics No. (%) of participants Cases n = 398 Sex Male 273 (68.6) Female 119 (29.9) Transgender 6 (1.5) Age, years 1–23 92 (23.1) 24–47 270 (67.8) 48–71 36 (9.1) Lifestyle Rural 141 (35.4) Urban 257 (64.6) Risk Groups Female sex workers 50 (12.5) General population 90 (22.6) Intravenous drug users 138 (34.6) Male sex workers 70 (17.5) Men who have sex with men 50 (12.5) Treatment Status On Therapy 119 (30) Newly Diagnosed 279 (70) CD4 count (cells/ul) <200 131 (33) 200–500 154(39) >500 113(28) Viral Load (copies/ml) 10,000 179 (45) >10,000 219 (55) Vitamin D Deficient 60 (15) Insufficient 155 (39) Sufficient 183 (46) Controls n = 232 Sex Male 136 (58.6) Female 96 (41.4) Age 1–23 74 (31.8) 24–47 139 (59.9) 48–71 19 (8.1) Vitamin D Deficient 2 (0.86) Insufficient 48 (20.62) Sufficient 182 (78.42)   |
+|---|
+
+![4_image_0.png](4_image_0.png)
+
+younger HIV cohorts with few patients over 60 years of age, identical to the current research, which consisted majority of patients ranging within 23–47 years of age (Allavena et al., 2012). The current work revealed that insignificant results of gender and hypovitaminosis D (p = 0.471) consistent with other studies conducted on HIV infected individuals (Wasserman and Rubin, 2010; Allavena et al., 2012; Kwan et al., 2012). The majority of the population in study belonged to urban areas 257 (64.6 %). A notable link was observed between lifestyle and vitamin D status of HIV/AIDS & HIV/non AIDS patients (p < 0.001). Vitamin DD was mostly observed among rural populations. Although there are certain confounding factors for VDD in urban populations such as the sedentary lifestyle, growing urbanization and industrial development may also reduce contact with sunlight but the people usually take supplements in order to meet vitamin deficiencies.
+
+The association among 25(OH)D concentrations along with viral load and CD4+ T-cell count is contradictory. Few clinical trials approved a positive correlation (Aziz et al., 2013) while some failed to reveal a significant association (Gedela et al., 2014)). Another study described that lower CD4+ T cell count is linked with deficiency of vitamin D in individuals infected with HIV (Zhang et al.,
+2017). Present study findings are in good agreement with the results of Zhang et al. (2017) i.e., a significant relationship is established in CD4 count and concentration of vitamin D among HIV/
+AIDS (p = 0.01) in the current study. Several other investigations have also associated lower 25(OH)D concentration to lower CD4+
+T cell counts (Welz et al., 2010; Sudfeld et al., 2012). Because of complexity in their nature, several mechanisms involved in elucidating the link between chronic HIV disease and resultant 25
+(OH)D decrease are unclear. The individuals with immunosuppression are usually susceptible to a number of clinical complications; one may hypothesize a cause of HIV infection related to VDD. Secondly, as the viral infection progresses to chronic inflammation, elevated certain pro-inflammatory cytokines like TNF-a may interfere with the parathyroid hormone secretion (PTH). The PTH is the stimulatory hormone for the active form of vitamin D i.e. 1,25dihydroxyvitamin D and its reduced release may result in renal 1a-hydroxylase impairment. Third, patients with low immunity and decreased CD4+ immune cells are more prone to infectious complications and they may have a reduced sun exposure as a contributing factor for hypovitaminosis D. Hospitalization may also be considered an option for food intake limitations, insufficient absorption and eventually malnutrition (Mansueto et al., 2015).
+
+In the current work, viral load showed no significant relationship with vitamin D status in HIV/Non-AIDS (p = 0.53) however, HIV/AIDS subjects had a link among these parameters (p = 0.01). Bearden et al. (2013) reported a delicate association, statistically insignificant (p = 0.36), between HIV viral load and vitamin D levels. Among HIV/Non-AIDS subjects, most of the patients had viral load  10000 while the majority in HIV/AIDS group had > 10000. So increased prevalence VDD in the HIV/AIDS subjects could be explained by the likelihood of interactions between lipopolysaccharide (LPS), toll-like receptor (TLR) signaling pathways, HIV viremia, and proinflammatory cytokines with activation of 25-hydroxyvitamin D-1a-hydroxylase (CYP27B1) in macrophages. Uncontrolled HIV viral loads elvate LPS and proinflammatory cytokines through impaired gut-linked tissues (Anselmi et al.,
+2007; Brenchley and Douek, 2008). The up-regulation of 1,25
+(OH)2D receptor-specific Cyp24 mRNA and cathelicidin mRNA
+was not determined in the absence of vitamin D [25(OH)D] indicating the crucial role of vitamin D in the regulation of such molecules
+(Liu et al., 2006).
+
+In the current investigation, a remarkable borderline relationship was determined in 25(OH)D levels between naïve and patients under antiretroviral therapy among HIV/AIDS subjects
+(p = 0.05). Aziz et al. (2013) findings showed recovered 25(OH)D3 levels in HAART-treated HIV carriers as compared to the newly infected patients. This recovery is ascribed to the sufficient vitamin D supplementation in patients infected with HIV. There are certain confounders such as the kind of ART given to the patient. Moreover, the present study didn't include certain individual antiretroviral drugs. Efavirenz interacts with the enzymes responsible for vitamin D metabolism (cytochrome P450 monooxygenases) and
+
+| Table2 Characteristics of HIV positive, Non-AIDS subjects (CD4 > 200; n = 267). D: <10   | IS: 11–29   | S: >30    | Chi2      | p Value   |      |        |
+|------------------------------------------------------------------------------------------|-------------|-----------|-----------|-----------|------|--------|
+| (n = 33)                                                                                 | (n = 104)   | (n = 130) |           |           |      |        |
+| Parameter                                                                                | Categories  | No. (%)   | No. (%)   | No. (%)   |      |        |
+| Gender                                                                                   | Male        | 16 (48.4) | 68 (65.6) | 90 (69.7) | *    | *      |
+| Female                                                                                   | 17 (51.6)   | 31 (30.3) | 39 (30.3) |           |      |        |
+| Transgender                                                                              | 0           | 5 (4.1)   | 1 (1)     |           |      |        |
+| Age                                                                                      | 1–23        | 7 (21)    | 21 (20)   | 37 (28)   | 4.93 | 0.29   |
+| 24–47                                                                                    | 25 (76)     | 78 (75)   | 82 (64)   |           |      |        |
+| 48–71                                                                                    | 1 (3)       | 5 (5)     | 11 (8)    |           |      |        |
+| Lifestyle                                                                                | Rural       | 26 (79)   | 28 (27)   | 32 (25)   | 76.2 | <0.001 |
+| Urban                                                                                    | 7 (21)      | 76 (73)   | 98 (75)   |           |      |        |
+| cART status                                                                              | On therapy  | 11 (33)   | 35 (34)   | 42 (32)   | 0.09 | 0.95   |
+| Treatment naive                                                                          | 22 (67)     | 69 (66)   | 88 (68)   |           |      |        |
+| CD4 count                                                                                | 200–500     | 24 (73)   | 62 (60)   | 68 (52)   | 9.5  | 0.09   |
+| >500                                                                                     | 9(27)       | 42 (40)   | 62 (48)   |           |      |        |
+| HIV Viral load                                                                           | 10,000             | 15 (45)   | 64 (62)   | 72 (55)   | 5.87 | 0.53   |
+| >10,000                                                                                  | 18 (55)     | 40 (38)   | 58 (45)   |           |      |        |
+| *Data was not distributed among all categories so chi2 was not applicable.               |             |           |           |           |      |        |
+
+| Table 3 HIV positive, AIDS patients (CD4 count < 200; n = 131).   | D: 10            | IS: 11–29   | S: 30           | Chi2      | p VALUE   |        |
+|-------------------------------------------------------------------|------------|-------------|-----------|-----------|-----------|--------|
+| (n = 27)                                                          | (n = 51)   | (n = 53)    |           |           |           |        |
+| Parameter                                                         | Categories | No.(%)      | No.(%)    | No.(%)    |           |        |
+| Gender                                                            | Male       | 19(70.4)    | 39(76.5)  | 41(77.4)  | 1.5       | 0.471  |
+| Female                                                            | 8(29.6)    | 12(23.5)    | 12(22.6)  |           |           |        |
+| Age                                                               | 1–23       | 6(22)       | 10 (19.6) | 11(20.7)  | 7.84      | 0.09   |
+| 24–47                                                             | 19(70)     | 35(68.6)    | 31(58.4)  |           |           |        |
+| 48–71                                                             | 2(7.4)     | 6(11.7)     | 11(20.7)  |           |           |        |
+| Lifestyle                                                         | Rural      | 19 (70.4)   | 15 (29.5) | 21 (39.6) | 36.22     | <0.001 |
+| Urban                                                             | 8 (29.6)   | 36 (70.5)   | 32 (60.4) |           |           |        |
+| cART status                                                       | On therapy | 6(22.3)     | 16(31.4)  | 9(16.9)   | 5.62      | 0.05   |
+| Treatment Naïve                                                   | 21(77.7)   | 35(68.6)    | 44(83.1)  |           |           |        |
+| CD4 count                                                         | 100            | 7(26)       | 24(47.1)  | 23(43.4)  | 10.48     | 0.01   |
+| 100–199                                                           | 20(74)     | 27(52.9)    | 30 (56.6) |           |           |        |
+| Viral load                                                        | 10,000            | 3(11.1)     | 15(29.4)  | 10(18.8)  | 10.29     | 0.01   |
+| >10,000                                                           | 24(88.9)   | 36 (70.5)   | 43 (81.2) |           |           |        |
+
+potentially induces the enzymes involved in hydroxylation of vitamin D3 to 25(OH)D3 (CYP3A4) and catabolism of 1,25(OH)2D to inactive forms (CYP24) along with reduced CYP2R1 transcription in HIV populations (Kim et al., 2012).
+
+Hypovitaminosis D is a risk factor for developing comorbidities including infectious diseases as well as immune disorders in patients infected with HIV. The status of vitamin D of a large cohort of HIV infected patients in comparison to that of the general population particularly in naïve as well as treated patients, is under researched in Pakistani population.
+
+## 5. Conclusion
+
+In conclusion, the present work documents the pervasiveness of vitamin D insufficiency or deficiency among a large (n = 398) group of HIV-infected subjects in comparison with that in the general population. HIV subjects have a greater pervasiveness of VDD deficiency in comparison to the local population. Vitamin D deficiency occurrence rate was greater in male population infected with HIV
+and those living in urban areas of Punjab, Pakistan. A strong relationship was determined between the lifestyle and vitamin D level.
+
+The significant associations of low concentrations of 25(OH)D, HIV
+viral load, and CD4 count among AIDS individuals culminate the requirement to assess insufficiency or deficiency of vitamin D as routine care of HIV infected population.
+
+## Funding No Funding Was Obtained. Declaration Of Competing Interest
+
+The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
+
+## Appendix A. Supplementary Material
+
+Supplementary data to this article can be found online at https://doi.org/10.1016/j.sjbs.2022.103484.
+
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+Vullo, V., Carosi, G., Antinori, A., Tozzi, V., Monforte, A.d., 2011. Prevalence of hypovitaminosis D and factors associated with vitamin D deficiency and morbidity among HIV-infected patients enrolled in a large Italian cohort. J. Acquir. Immune Defic. Syndr. 58 (2), 163–172.
+
+Vieth, R., Bischoff-Ferrari, H., Boucher, B.J., Dawson-Hughes, B., Garland, C.F.,
+Heaney, R.P., Holick, M.F., Hollis, B.W., Lamberg-Allardt, C., McGrath, J.J., Norman, A.W., Scragg, R., Whiting, S.J., Willett, W.C., Zittermann, A., 2007. The urgent need to recommend an intake of vitamin D that is effective. Am. J. Clin.
+
+Nutr. 5, 649–650.
+
+Wasserman, P., Rubin, D.S., 2010. (2010) Highly prevalent vitamin D deficiency and insufficiency in an urban cohort of HIV-infected men under care. AIDS patient care and STDs 24, 223–227.
+
+Welz, T., Childs, K., Ibrahim, F., Poulton, M., Taylor, C.B., Moniz, C.F., Post, F.A., 2010.
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+Efavirenz is associated with severe vitamin D deficiency and increased alkaline phosphatase. AIDS 24, 1923–1928.
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+# Cohort Profile:The Danish National Cohort Study Of Effectiveness And Safety Of Sars-Cov-2 Vaccines **(Enforce)**
+
+Nina Breinholt Stærke ,1,2 Joanne Reekie,3 Isik S Johansen,4,5 Henrik Nielsen,6,7 Thomas Benfield,8,9 Lothar Wiese,10 Ole S Søgaard,1,2 Martin Tolstrup,1,2 Kasper Karmark Iversen,9,11 Britta Tarp,12 Fredrikke Dam Larsen,1,2 Lykke Larsen ,4,5 Susan Olaf Lindvig,4 Inge Kristine Holden,4 Mette Brouw Iversen,10 Lene Surland Knudsen,10 Kamille Fogh,11 Marie Louise Jakobsen,3 Anna Katrin Traytel,3 Lars Ostergaard,1,2 Jens Lundgren,3 For the ENFORCE Study Group To cite: Stærke NB, Reekie J, 
+Johansen IS, *et al*. Cohort Profile:The Danish National Cohort Study of Effectiveness and Safety of SARS-CoV-2 vaccines (ENFORCE). *BMJ Open* 2022;12:e069065. doi:10.1136/ bmjopen-2022-069065
+► Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2022-069065). Received 09 October 2022 Accepted 16 December 2022 © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. For numbered affiliations see end of article. Correspondence to Dr Nina Breinholt Stærke; ninase@rm.dk
+
+## Abstract
+
+Purpose The ENFORCE cohort is a national Danish prospective cohort of adults who received a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine as part of the Danish National SARS-CoV-2 vaccination programme. It was designed to investigate the longterm effectiveness, safety and durability of SARS-CoV-2 vaccines used in Denmark.
+
+Participants A total of 6943 adults scheduled to receive a SARS-CoV-2 vaccine in the Danish COVID-19 vaccination programme were enrolled in the study prior to their first vaccination. Participants will be followed for a total of 2years with five predetermined follow-up visits and additional visits in relation to any booster vaccination. Serology measurements are performed after each study visit. T-cell immunity is evaluated at each study visit for a subgroup of 699 participants. Safety information is collected from participants at visits following each vaccination. Data on hospital admissions, diagnoses, deaths and SARS-CoV-2 PCR 
+results are collected from national registries throughout the study period. The median age of participants was 64 years (IQR 53–75), 56.6% were women and 23% were individuals with an increased risk of a serious course of COVID-19. A total of 340 (4.9%) participants tested positive for SARS-CoV-2 spike IgG at baseline. Findings to date Results have been published on risk factors for humoral hyporesponsiveness and nondurable response to SARS-CoV-2 vaccination, the risk of breakthrough infections at different levels of SARS-CoV-2 spike IgG by viral variant and on the antibody neutralising capacity against different SARS-CoV-2 variants following primary and booster vaccinations. Future plans The ENFORCE cohort will continuously generate studies investigating immunological response, effectiveness, safety and durability of the SARS-CoV-2 vaccines. Trial registration number NCT04760132.
+
+## Strengths And Limitations Of This Study
+
+⇒ The ENFORCE study combines repeated detailed SARS-CoV-2 specific immunological measurements prior to, and throughout the course of SARS-CoV-2 vaccination, with register-based follow-up of safety data and microbiological test results.
+
+⇒ The ENFORCE cohort includes a large proportion of elderly participants and participants with concomitant diseases.
+
+⇒ The three vaccine groups display a high degree of variation in demographic factors and distribution across risk groups, due to the prioritisation of specific vaccines to risk groups during the primary roll out of the SARS-CoV-2 vaccination programme.
+
+## Introduction
+
+On the emergence of SARS-CoV-2, the pathogen causing COVID-19, the development of effective vaccines quickly became of highest priority for decision-makers, researchers and pharmaceutical companies throughout the world. Subsequently, within 1year of the emergence of the virus, the first reports from clinical phase 1–2 trials of SARSCoV-2 vaccines in humans were published.1–5 The first SARS-CoV-2 vaccine approved for emergency use in Denmark was the Pfizer-BioNTech mRNA vaccine BNT162n2 (Pfizer, New York, USA; BioNTech SE, 
+Mainz, Germany).6 It was used from the initiation of the Danish COVID-19 vaccination programme on 27 December 2020. In January 2021 both the Moderna mRNA-1273 vaccine (Moderna, Massachusetts, USA)7 and the AstraZeneca adenovirus-vectored ChAd0×1 nCoV-19 vaccine (AstraZeneca, Cambridge, UK)8 were approved for emergency use. The adenoviral vectored vaccine Ad26.COV2.S (Johnson & 
+Johnson, New Jersey, USA)9 was used in a supplementary programme alongside the Danish National COVID-19 vaccination programme and only a small group of individuals received this vaccine in Denmark.
+
+As of September 2022, more than 600million cases and 6million deaths due to COVID-19 have been reported to the WHO. Globally, over 12billion vaccine doses against SARS-CoV-2 have been administered, however especially on the African continent, large parts of the population are still lacking their first SARS-CoV-2 vaccination.10 Conversely, in Denmark and similar countries, the fourth vaccination dose is being planned for selected parts of the population.11 Additionally, new Omicron specific and bivalent vaccines are being developed and introduced in vaccination programmes replacing the initial vaccines.12 13 The efficacy and safety of the SARS-CoV-2 vaccines were described in defined study populations and smaller cohorts as the vaccines were introduced,6–8 14–20 however, there is an ongoing need for knowledge on the long-term effectiveness and safety of the vaccines, the durability and magnitude of vaccine-induced immune response, protection against new emerging variants and the response to booster doses particularly in risk groups.
+
+## Cohort Description
+
+ENFORCE was designed as an open-labelled, nonrandomised, parallel group, phase IV study enrolling adults residing in Denmark before their first SARS-CoV-2 vaccination offered through the Danish COVID-19 vaccination programme. The inclusion period ran from 13 February 2021 to 5 August 2021.
+
+The study was planned by the ENFORCE Consortium and is being carried out as a collaborative effort with seven study sites in the cities Aalborg, Silkeborg, Aarhus, Odense, Roskilde, Hvidovre and Herlev covering all five Danish regions. A full list of members of the ENFORCE study group is provided as online supplemental material. The study was approved by the Danish Medicines Agency.
+
+## Study Population
+
+Participants had to be eligible for SARS-CoV-2 vaccination through the Danish COVID-19 vaccination programme, willing and able to provide informed consent and willing and able to comply with trial procedures. Potential participants would be excluded if they were below 18 years of age, belonged to a subgroup for which the SARS-CoV-2 vaccines were contraindicated or had received a previous SARS-CoV-2 vaccination.
+
+Potential study participants were invited to join the ENFORCE study through a number of channels selected to ensure a high proportion of persons at increased risk of a serious course of SARS-CoV-2 infection, and healthcare workers (HCW) at risk of SARS-CoV-2 exposure. A letter of invitation was sent to the selected risk groups such as patients with cancer in active therapy, organ transplant recipients, patients with immunodeficiencies, patients in haemodialysis treatment and patients with certain rheumatological and pulmonary diseases. HCW were invited to participate via hospital information channels. The general population was invited to participate through a letter of invitation sent via the vaccination centres to adults with a scheduled appointment for vaccination in any of the study site cities.
+
+Enrolment visits were performed by ENFORCE study personnel, either at vaccination centres located near study sites, or at hospital clinics. Participants could be enrolled from 14 days up to 30min before their first dose of a SARS-CoV-2 vaccine. All participants gave written informed consent prior to any study procedures.
+
+## Data Collection
+
+At enrolment, baseline information was collected on age, sex, focused medical history, concomitant medication, vaccination priority group (defined by the Danish COVID-19 vaccination programme21), scheduled vaccination date and vaccine manufacturer (Pfizer-BioNTech, Moderna, AstraZeneca or Johnson & Johnson). Participants also provided blood samples for baseline SARSCoV-2 serology. Persons at increased risk of a serious course of SARS-CoV-2 infection were classified as such, on the basis that they were referred to vaccination by a medical specialist tending to their disease. The group included patients with cancer in active treatment, patients with immunodeficiencies (acquired or inherent), organ transplant recipients, haemodialysis patients and some patients with haematological, pulmonary or rheumatological diseases. Individuals that were vaccinated because of an occupation as HCW were registered as such. Vaccine type was confirmed by cross referencing each individual's unique civil registration (CPR) number with data from the Danish Vaccination Register (Statens Serum Institut, Copenhagen, Denmark).
+
+Safety data is collected using a questionnaire (either online or physical) on solicited local and systemic reactions filled out by the participants at 1 and 2weeks following each vaccination. Additionally, information regarding adverse events is collected until the visits following each vaccination. Data on specific concomitant diseases, newly diagnosed medical conditions, hospitalisations and death are collected from the Danish National Patient Register (Danish Health Data Authority, Copenhagen, Denmark).
+
+Data on any SARS-CoV-2 PCR-tests or SARS-CoV-2 antibody measurements were extracted from the surveillance system Key Infectious Diseases System (Statens Serum Institut, Copenhagen, Denmark), whereas viral variant information is obtained through the Danish National Microbiology Database (Statens Serum Institut, Copenhagen, Denmark).
+
+## Follow-Up
+
+Study participants are followed with five predetermined clinical visits in addition to the baseline visit. The second study visit is scheduled at 0–5days prior to the 
+
+![2_image_0.png](2_image_0.png)
+
+participants second SARS-CoV-2 vaccination, while the following visits are at 3, 6, 12 and 24 months after the first vaccination. Furthermore, study visits are scheduled 0–14days before and 1 month after any SARSCoV-2 booster vaccination. An outline of the study visit schedule is shown in figure 1.
+
+## Serology Measurements
+
+At each study visit serum and plasma samples are collected from all participants for measuring specific SARS-CoV-2 serology. SARS-CoV-2 spike IgG is measured on serum samples using a Wantai ELISA based assay (Beijing Wantai Biological Pharmacy Enterprise, Beijing, China) performed at Statens Serum Institut, Copenhagen, Denmark. Additionally, a Multiantigen Serology Assay (Meso Scale Diagnostics, Maryland, USA) quantifying spike receptor binding domain, full spike and nucleocapsid directed IgG and ACE-2 competition is performed on plasma samples at the Research Laboratory at the Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark. Additional plasma and serum samples for future research analyses are collected and stored at the Danish National Biobank at Statens Serum Institut.
+
+## Cellular Immune Response
+
+A group consisting of 699 participants evenly distributed across vaccine and age groups were enrolled in a substudy evaluating SARS-CoV-2 spike specific T-cell immunity using peripheral blood mononuclear cells. For this subgroup additional blood samples, including RNA stabilising PAXgene tubes, are collected at each visit. Analyses of T-cell immunity are performed at the Research Laboratory at the Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark.
+
+## Vaccine Groups
+
+The first participant included in this study received the AstraZeneca adenovirus vectored vaccine on 13 February 2021. However, the use of the AstraZeneca vaccine in the Danish COVID-19 vaccination programme was stopped on 11 March 2021, and therefore only 474 participants received this vaccine. A small group of 25 participants received the adenovirus vectored vaccine manufactured by Johnson & Johnson. In total 499 participants received an adenovirus vectored vaccine at their first vaccination and are included in the study as the adenovirus vector/ messenger RNA (mRNA) group.
+
+Inclusion in the Pfizer-BioNTech group started on 16 February 2021. This group was temporarily paused from mid-April to mid-May 2021 when the initial target of 2500 participants was reached, but was subsequently resumed in order to enrol more participants in younger age groups and concluded at 3824 participants.
+
+Inclusion in the Moderna group started on 24 February 2021, but was slow-paced in the beginning due to limited vaccine deliveries, but finalised at 2620 participants. Enrolment progression during the inclusion period for each vaccine group is shown in figure 2.
+
+The timing of additional doses of any of the COVID-19 vaccines was determined by the schedule outlined in the Danish National COVID-19 vaccination programme at the time of vaccination. The duration between first and second dose varied between 3 and 12 weeks, depending on the vaccine and the calendar time.
+
+## Demographics
+
+A total of 6972 participants were enrolled in the study and 6943 participants (99.6%) are included in the analysis. The most common reason for exclusion was withdrawal of consent (n=23) and receiving a non-standard 
+
+![3_image_0.png](3_image_0.png)
+
+vaccine regimen (n=4). Reasons for exclusion are shown in figure 3.
+
+Overall, the median age was 64 years (IQR 53–75 years), 3929 (56.6%) participants were women, 590 (8.5%) were HCW and 1599 (23%) were classified as having an increased risk of a serious course of COVID-19 due to concomitant diseases. Overall, 3.4% (n=239) of the cohort had a positive SARS-CoV-2 PCR test prior to enrolment in the study. Participants in the adenoviral vector/mRNA group (N=499) were generally younger with a median age of 45 years (IQR 31–55 years) and 411 (82.4%) were women, while 453 (90.8%) were HCW. A higher proportion in the adenoviral vector/mRNA group (n=47, 9.4%) had a positive SARS-CoV-2 PCR prior to baseline compared with the other vaccine groups. The Pfizer-BioNTech group (N=3824) had a larger proportion of participants with an increased risk of a serious course of infection with SARS-CoV-2 (n=1437, 37.6%) and the participants were generally older, with a median age of 71 years (IQR 55–78 years), relative to the other vaccine groups. The demographic distribution overall and within the vaccine groups is summarised in table 1.
+
+## Baseline Wantai Serology Results
+
+A total of 6889 participants (99.2% of the analysed population) had a conclusive result of the baseline Wantai analysis measuring SARS-CoV-2 spike IgG. There were five participants (0.07%) with an inconclusive result of 
+
+![3_image_1.png](3_image_1.png)
+
+the Wantai analysis, and 49 participants (0.71%) with missing data, these participants were excluded from the Wantai results presented in figure 4. Of the participants with a conclusive result 340 participants (4.9%) 
+had a positive result, indicating previous infection with SARS-CoV-2. The proportion of positive participants was 17/157 (10.8%) among the youngest participants aged 18–25 years, while it was 26/893 (2.9%) among those over the age of 80 years. Serology results are summarised in figure 4.
+
+## Participant And Public Involvement
+
+Public reports on the preliminary study results are shared on the ENFORCE website and a public seminar was held, where study results were presented. Participants in ENFORCE receive regular updates on the study progress and results, including results of their own antibody measurements.
+
+## Findings To Date
+
+We assessed levels of SARS-CoV-2 spike IgG and SpikeACE2-receptor-blocking antibodies at visits up to 6months after the participants' first SARS-CoV-2 vaccination and identified risk factors for humoral hyporesponsiveness and lack of durability of the vaccine response. We found that male sex, level of comorbidity and vaccine type were all associated with hyporesponsiveness and non-durability of both antibodies assessed.22 After the emergence of the Omicron variant, we conducted a study comparing the risk of breakthrough infection at different levels of SARSCoV-2 spike IgG stratified by viral variant. We found an inverse association between increasing level of spike IgG and risk of breakthrough infection for the Delta variant, while there was no association between antibody levels and risk of breakthrough infection with the Omicron variant.23 Recently, a study was published comparing the vaccine-induced antibody response and neutralising capacity towards a range of SARS-CoV-2 variants after primary vaccination and first booster dose. Here it was found that the neutralising capacity was reduced for each of the emergent variants tested as compared with wild type, and most pronounced for the Omicron variant. However, it was also seen that neutralising capacity increased significantly after the admission of a booster dose.24
+
+## Strengths And Limitations
+
+The primary strength of the ENFORCE study is that it combines repeated measurements of SARS-CoV-2 specific humoral and cellular immunological response throughout the course of the SARS-CoV-2 vaccination, with register-based follow-up of safety data and microbiological test results. This design allows the ENFORCE 
+cohort to contribute with significant knowledge of the interplay between humoral and cellular immune response to SARS-CoV-2 vaccination and new emerging 
+
+Table 1 Participant demographics at study enrolment by vaccine type. Participant characteristics at baseline are presented 
+
+overall and compared across vaccine groups, using either the χ2 test for categorical variables or Kruskal-Wallis test for 
+
+continuous variables.
+
+Vaccine type
+
+| continuous variables.          | Vaccine type                  |             |                        |             |            |    |
+|--------------------------------|-------------------------------|-------------|------------------------|-------------|------------|----|
+| Total                          | Pfizer-BioNTech               | Moderna     | Adenoviral Vector/mRNA |             |            |    |
+| (N=6943)                       | (N=3824)                      | (N=2620)    | (N=499)                | P value     |            |    |
+| Age at enrolment (median, IQR) | 64 (53–75)                    | 71 (55–78)  | 61 (54–69)             | 45 (31–55)  | .          |    |
+| Age group (N, %)               | <0.0001                       |             |                        |             |            |    |
+|                                | 18–25                         | 159 (2.3)   | 59 (1.5)               | 64 (2.4)    | 36 (7.2)   | .  |
+|                                | 25–39                         | 384 (5.5)   | 152 (4.0)              | 62 (2.4)    | 170 (34.1) | .  |
+|                                | 40–64                         | 3191 (46.0) | 1326 (34.7)            | 1577 (60.2) | 288 (57.7) | .  |
+|                                | 65–79                         | 2313 (33.3) | 1557 (40.7)            | 752 (28.7)  | 4 (0.8)    | .  |
+|                                | ≥80                           | 896 (12.9)  | 730 (19.1)             | 165 (6.3)   | 1 (0.2)    | .  |
+| Sex (N, %)                     | <0.0001                       |             |                        |             |            |    |
+|                                | Male                          | 3014 (43.4) | 1841 (48.1)            | 1085 (41.4) | 88 (17.6)  | .  |
+|                                | Female                        | 3929 (56.6) | 1983 (51.9)            | 1535 (58.6) | 411 (82.4) | .  |
+| Risk group (N,%)               | <0.0001                       |             |                        |             |            |    |
+|                                | Individuals at increased risk | 1599 (23.0) | 1437 (37.6)            | 153 (5.8)   | 9 (1.8)    | .  |
+|                                | Healthcare workers            | 590 (8.5)   | 109 (2.9)              | 28 (1.1)    | 453 (90.8) | .  |
+|                                | General population            | 4754 (68.5) | 2278 (59.6)            | 2439 (93.1) | 37 (7.4)   | .  |
+| Ever SARS-CoV-2 PCR positive   | <0.0001                       |             |                        |             |            |    |
+| Never                          | 6704 (96.6)                   | 3718 (97.2) | 2534 (96.7)            | 452 (90.6)  | .          |    |
+| Positive ≤6months              | 180 (2.6)                     | 82 (2.1)    | 56 (2.1)               | 42 (8.4)    | .          |    |
+| Positive >6months              | 59 (0.8)                      | 24 (0.6)    | 30 (1.1)               | 5 (1.0)     | .          |    |
+
+SARS-CoV-2 variants, hybrid immunity and long-term 
+
+![4_image_0.png](4_image_0.png) safety of SARS-CoV-2 vaccination.
+
+The ENFORCE cohort has high proportions of participants over the age of 65 years and of participants with significant comorbidities. These groups are of particular interest in the context of vaccine effectiveness and durability as they have an increased risk of developing a serious course of COVID-19, and vaccine programmes are often designed to protect these individuals.
+
+The three vaccine groups display a large degree of variation in demographic factors and distribution across risk groups, which may be explained by the availability and prioritising of specific vaccines to specific groups at different time points. The AstraZeneca vaccine was specifically prioritised for HCW before it was removed from the vaccination programme, and therefore the adenovirus vectored/mRNA group includes higher proportions of HCW, women and younger age groups. 
+
+## 
+
+The Pfizer-BioNTech vaccine was used to a large extent for the elderly population and individuals at increased risk of a serious course of disease, as it was the vaccine mainly available in the first months of the vaccination programme. The between-group differences complicates the comparison of vaccine groups as age, sex and health status may be confounding factors. A randomised design would have eliminated this limitation, but due to vaccine shortage in the early stage of the vaccination programme and vaccine prioritisation for risk groups it was unfeasible to randomise participants to vaccine groups. 
+
+When conducting comparative analyses of the vaccine groups this limitation will be mitigated by adjusting for confounding factors.
+
+The use of registry data comprises a risk of misclassification due to incorrect registration. However, the large size of the ENFORCE cohort reduces the risk of misclassification affecting results significantly.
+
+At enrolment the number of individuals with missing data was low (<1%). As the study progresses this number will vary depending on the question being addressed. At this stage we do not plan to perform imputation on missing data, instead the characteristics of those excluded due to missing values will be compared with those included in analysis and any differences described.
+
+## Future Plans
+
+Studies are planned or ongoing investigating the T-cell immune response of the cohort at different time points relative to primary vaccination and boosters, the significance of hybrid immunity, morbidity and mortality compared with those of a historic control group and the correlation of local and systemic reactions to vaccination with antibody response. Data collection will proceed until July 2023.
+
+Collaboration The ENFORCE study encourages scientific collaboration. While the study is ongoing data may be made available to scientists on approval of an application sent to the ENFORCE Scientific Steering Committee and further approval by relevant authorities. Applications for data must be sent to enforce.rigshospitalet@regionh.dk. Detailed information about data access may be found here: https://chip.dk/Research/Studies/ENFORCE/ Study-Governance.
+
+Author affiliations 1Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark 2Department of Clinical Medicine, Aarhus University, Aarhus, Denmark 3Center of Excellence for Health, Immunity and Infections, Rigshospitalet, University of Copenhagen, Kobenhavn, Denmark 4Department of Infectious Diseases, Odense Universitetshospital, Odense, Denmark 5Department of Clinical Research, University of Southern Denmark, Odense, Denmark 6Department of Infectious Diseases, Aalborg University Hospital, Aalborg, Denmark 7Department of Clinical Medicine, Aalborg University, Aalborg, Denmark 8Department of Infectious Diseases, Copenhagen University Hospital - Amager and Hvidovre, Hvidovre, Denmark 9Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark 10Department of Medicine, Zealand University Hospital, Roskilde, Denmark 11Department of Cardiology and Department of Emergency Medicine, Herlev Hospital, Herlev, Denmark 12Diagnostic Centre, Silkeborg Regional Hospital, Silkeborg, Denmark Acknowledgements The ENFORCE study group members all contributed substantially to the study.
+
+Contributors JL, LØ, MT, OSS, NBS and JR contributed to the conception and design of the study. ISJ, HN, TB, LW, LSK, MBI, KF, IKH, KKI, BT, FDL, LL, SOL and NBS contributed to the acquisition of the data. AKT, MLJ and JR performed data curation. JR performed the statistical analyses. NBS wrote the first draft. All authors revised the manuscript critically and accepted the final version. LØ and JL act as guarantors. Funding Funding for this work was granted by the Danish Ministry of Health (legal deeds 150 28/1 2021 and 263 3/6 2021). Competing interests NBS declares to have served as investigator on studies sponsored by Pfizer, Gilead, Bavarian and Sanofi Pasteur. HN declares receiving a grant from Novo Nordisk Foundation and to have been on advisory boards for MSD and GSK. TB declares receipt of unrestricted research or travel grants from GSK, Pfizer, Gilead Sciences, MSD; and being principal investigator on trials conducted by Boehringer Ingelheim, Roche, Novartis, Kancera, Pfizer, MSD and Gilead; Board member on Pentabase, and advisory board member for MSD, Gilead, Pfizer, GSK, Janssen and AstraZeneca; consulting fees from GSK and Pfizer; receiving donation of study drug from Eli Lilly; and receiving honorarium for lectures from GSK, Pfizer, Gilead Sciences, Boehringer Ingelheim, AbbVie and AstraZeneca. Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details. Patient consent for publication Not applicable. Ethics approval This study involves human participants and was approved by the Ethical Committees of The Central Denmark Region. Participants gave informed consent to participate in the study before taking part. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data may be made available to researchers upon approval of an application to the scientific steering committee of the ENFORCE study. More information about data access may be found at https://chip.dk/ Research/Studies/ENFORCE/Study-Governance. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iDs Nina Breinholt Stærke http://orcid.org/0000-0002-5834-2033 Lykke Larsen http://orcid.org/0000-0002-4113-4182
+
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+Available: https://covid19.who.int/ [Accessed 13 Sep 2022].
+
+11 Danish Health Authority. Vaccination fall/winter 2022-2023. Available: 
+https://www.sst.dk/en/English/Corona-eng/Vaccination-againstcovid-19/Vaccination-fall-and-winter-2022-2023 12 Chalkias SH, Vrbicky C;, Walsh K;. R. a bivalent Omicron-containing booster vaccine against Covid-19. *MedRxiv* 2022.
+
+13 FDA. COVID-19 bivalent vaccine boosters. Available: https://www.
+
+fda.gov/emergency-preparedness-and-response/coronavirusdisease-2019-covid-19/covid-19-bivalent-vaccine-boosters 
+[Accessed 13 Sep 2022].
+
+14 Borobia AM, Carcas AJ, Pérez-Olmeda M, *et al*. Immunogenicity and reactogenicity of BNT162b2 booster in ChAdOx1-S-primed participants (CombiVacS): a multicentre, open-label, randomised, controlled, phase 2 trial. *Lancet* 2021;398:121–30.
+
+15 Parry H, McIlroy G, Bruton R, *et al*. Antibody responses after first and second Covid-19 vaccination in patients with chronic lymphocytic leukaemia. *Blood Cancer J* 2021;11:136.
+
+16 Frater J, Ewer KJ, Ogbe A, *et al*. Safety and immunogenicity of the ChAdOx1 nCoV-19 (AZD1222) vaccine against SARS-CoV-2 in HIV infection: a single-arm substudy of a phase 2/3 clinical trial. Lancet HIV 2021;8:e474–85.
+
+17 Pimpinelli F, Marchesi F, Piaggio G, *et al*. Fifth-week immunogenicity and safety of anti-SARS-CoV-2 BNT162b2 vaccine in patients with multiple myeloma and myeloproliferative malignancies on active treatment: preliminary data from a single institution. *J Hematol Oncol* 2021;14:81.
+
+18 Voysey M, Clemens SAC, Madhi SA, *et al*. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. *Lancet* 2021;397:99–111.
+
+19 Ou MT, Boyarsky BJ, Chiang TPY, *et al*. Immunogenicity and Reactogenicity after SARS-CoV-2 mRNA vaccination in kidney transplant recipients taking belatacept. *Transplantation* 2021;105:2119–23.
+
+20 Strauss AT, Hallett AM, Boyarsky BJ, *et al*. Antibody response to severe acute respiratory Syndrome-Coronavirus-2 messenger RNA vaccines in liver transplant recipients. *Liver Transpl* 2021;27:1852–6.
+
+21 Danish Health Authority. Vaccination calendar. Available: https://
+www.sst.dk/en/English/Corona-eng/Vaccination-against-COVID-19/
+When-will-you-get-vaccinated 22 Søgaard OS, Reekie J, Johansen IS, *et al*. Characteristics associated with serological COVID-19 vaccine response and durability in an older population with significant comorbidity: the Danish nationwide enforce study. *Clin Microbiol Infect* 2022;28:1126–33.
+
+23 Stærke NB, Reekie J, Nielsen H, *et al*. Levels of SARS-CoV-2 antibodies among fully vaccinated individuals with delta or omicron variant breakthrough infections. *Nat Commun* 2022;13:4466.
+
+24 Hvidt AK, Baerends EAM, Søgaard OS, *et al*. Comparison of vaccineinduced antibody neutralization against SARS-CoV-2 variants of concern following primary and booster doses of COVID-19 vaccines. 
+
+Front Med 2022;9:994160.

medical/md/PMC9911851.md

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+
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+![0_image_0.png](0_image_0.png)
+
+Review
+
+# Endovascular Versus Medical Management Of Acute 
+
+![0_Image_1.Png](0_Image_1.Png) Basilar Artery Occlusion: A Systematic Review And Meta-Analysis Of The Randomized Controlled Trials
+
+Mohamad Abdalkader,a Stephanos Finitsis,b Chuanhui Li,c Wei Hu,d Xinfeng Liu,d Xunming Ji,e Xiaochuan Huo,f Fana Alemseged,g Zhongming Qiu,h Daniel Strbian,i Volker Puetz,j,k James E. Siegler,l Shadi Yaghi,m Kaiz Asif,n Piers Klein,a Yuyou Zhu,d Bruce C.V. Campbell,g Hui-Sheng Chen,o Simon Nagel,p,q Georgios Tsivgoulis,r Zhongrong Miao,f Raul G. Nogueira,s Tudor G. Jovin,l Wouter J. Schonewille,t Thanh N. Nguyen;a on behalf of the BasilaR Artery Ischemia Network STudy of Endovascular versus Medical Management (BRAINSTEM) Group aDepartments of Radiology and Neurology, Boston Medical Center, Boston University Chobanian & Avedisian School of Medicine, Boston, MA, USA bDepartment of Radiology, Aristotle University of Thessaloniki, Thessaloniki, Greece cStroke Center and Department of Neurology, Xuanwu Hospital of Capital Medical University, Beijing, China dDepartment of Neurology and Stroke Center, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China eDepartment of Neurosurgery, Xuanwu Hospital of Capital Medical University, Beijing, China fInterventional Neuroradiology, Beijing Tiantan Hospital, Beijing, China gMedicine and Neurology, Melbourne Brain Centre at the Royal Melbourne Hospital, University of Melbourne, Parkville, VC, Australia hDepartment of Neurology, The 903rd Hospital of The Chinese People's Liberation Army, Hangzhou, China iNeurology, Helsinki University Hospital, University of Helsinki, Helsinki, Finland jNeurology, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany kDresden Neurovascular Center, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany lCooper Neurological Institute, Cooper University Hospital, Camden, NJ, USA 
+mRhode Island Hospital, Brown University, Providence, RI, USA nAmita Health and University of Illinois-Chicago, Chicago, IL, USA oDepartment of Neurology, General Hospital of Northern Theatre Command, Shenyang, China pNeurology, Heidelberg University Hospital, Heidelberg, Germany qNeurology, Klinikum Ludwigshafen, Ludwigshafen, Germany rDepartment of Neurology, Attikon University Hospital, National and Kapodistrian University of Athens, Athens, Greece sDepartment of Neurology, Neurosurgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA 
+tDepartment of Neurology, St. Antonius Hospital, Nieuwegein, the Netherlands Background and Purpose The optimal management of patients with acute basilar artery occlusion 
+(BAO) is uncertain. We aimed to evaluate the safety and efficacy of endovascular thrombectomy (EVT) compared to medical management (MM) for acute BAO through a meta-analysis of randomized controlled trials (RCTs). 
+
+Methods We performed a systematic review and meta-analysis of RCTs of patients with acute BAO. We analyzed the pooled effect of EVT compared to MM on the primary outcome (modified Rankin Scale [mRS] of 0–3 at 3 months), secondary outcome (mRS 0–2 at 3 months), symptomatic intracranial hemorrhage (sICH), and 3-month mortality rates. For each study, effect sizes were computed as odds ratios (ORs) with random effects and Mantel-Haenszel weighting. 
+
+Correspondence: Wouter J. Schonewille Department of Neurology, St. Antonius Hospital, Koekoekslaan 1, 3435 CM, Nieuwegein, the Netherlands Tel: +31-88-320-3000 E-mail: w.schonewille @antoniusziekenhuis.nl https://orcid.org/0000-0002-4070-9730 Results Four RCTs met inclusion criteria including 988 patients. There were higher odds of mRS of 
+
+![1_image_0.png](1_image_0.png)
+
+0-3 at 90 days in the EVT versus MM group (45.1% vs. 29.1%, OR 1.99, 95% confidence interval 
+[CI] 1.04–3.80; P=0.04). Patients receiving EVT had a higher sICH compared to MM (5.4% vs. 0.8%, OR 7.89, 95% CI 4.10–15.19; P<0.01). Mortality was lower in the EVT group (35.5% vs. 45.1%, OR 0.64, 95% CI 0.42–0.99; P=0.05). In an analysis of two trials with BAO patients and National Institutes of Health Stroke Scale (NIHSS) <10, there was no difference in 90-day outcomes between EVT versus MM. 
+
+Conclusion In this systematic review and meta-analysis, EVT was associated with favorable outcome and decreased mortality in patients with BAO up to 24 hours from stroke symptoms compared to MM. The treatment effect in BAO patients with NIHSS <10 was less certain. Further studies are of interest to evaluate the efficacy of EVT in basilar occlusion patients with milder symptoms. 
+
+Keywords Acute stroke; Acute basilar artery occlusion; Posterior circulation; Meta-analysis Co-correspondence: Thanh N. Nguyen Department of Neurology, Radiology, Boston Medical Center, Boston University Chobanian and Avedisian School of Medicine, Boston, MA 02118, USA Tel: +1-617-638-8000 E-mail: thanh.nguyen@bmc.org https://orcid.org/0000-0002-2810-1685 Received: November 29, 2022 Revised: December 29, 2022 Accepted: January 6, 2023
+
+## Introduction
+
+Endovascular thrombectomy (EVT) has become the standard of care for acute ischemic stroke due to large vessel occlusion (LVO) in the anterior circulation up to 24 hours from symptom onset after the publication of several randomized trials between 2015 and 2018.1-7 Patients with posterior circulation stroke were underrepresented in these trials.8,9 The efficacy and safety of EVT in posterior circulation stroke has been debated.10,11 Posterior circulation strokes, particularly those due to acute basilar artery occlusion (BAO), are devastating with high morbidity and mortality rates reaching up to 83%–96% in the absence of reperfusion.12,13 EVT was reported with good clinical outcomes in acute BAO with rates of favorable functional outcome (modified Rankin Scale [mRS] score 0–3) at 90 days ranging between 32% to 40% 
+in two prospective studies.14,15 However, these studies were limited by selection bias, unblinded 90-day assessments, and heterogeneous treatment approaches.14-16 Over the last three years, four randomized controlled studies (RCTs) reached different conclusions regarding the efficacy of EVT in acute BAO stroke.17-20 In 2020 and 2021, the BEST (Basilar Artery Occlusion Endovascular Intervention versus Standard Medical Treatment) and BASICS (Basilar Artery International Cooperation Study) trials demonstrated that in patients with BAO, there was equivocal benefit of EVT as compared to medical management (MM).17,18 However, these trials were underpowered and had limitations that may have hindered the validity of their results. Recently, two RCTs (ATTENTION [Endovascular Treatment For Acute Basilar Artery Occlusion: A Multicentre Randomised Clinical Trial] and BAOCHE [Basilar Artery Occlusion Chinese Endovascular Trial]) showed benefit of EVT compared to MM for acute BAO up to 24 hours from stroke onset.19,20 As these trials were of moderate size, pooling of their data may improve precision in the estimated treatment effect and reflect the diversity of patients across multiple countries. In this systematic review and study-level meta-analysis of these four RCTs, we aimed to analyze the clinical and safety outcomes in acute BAO patients treated with EVT compared to MM.
+
+## Methods
+
+This systematic review and meta-analysis is presented according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.21 The study protocol was registered on the International Prospective Register of Systemic Reviews on July 18, 2022 (PROSPERO CRD42022344565). Data are available on reasonable request to the corresponding authors.
+
+## Search Strategy
+
+The search was conducted with MEDLINE, EMBASE, Cochrane, and Web of Science using the following terms: ("Vertebrobasilar" OR "Vertebral" OR "Basilar") AND ("Stroke" OR "Ischemia") AND ("Thrombectomy" OR "Thrombolysis") AND ("RCT" OR "randomized controlled trial"). In addition, the RCTs of LVO patients including the anterior circulation were searched for inclusion of patients with BAO. Abstracts and articles were identified and reviewed by two authors independently (MA and TNN). Disagreement was then discussed with a third author (SF).
+
+## Eligibility Criteria
+
+We included studies that reported EVT versus MM of patients with acute BAO stroke from January 1, 2000 to October 28, 2022. The inclusion criteria were as follows: (1) RCT, (2) interventional arm receiving EVT and MM, (3) control arm receiving MM, and (4) report of mRS score of 0–3 at 3 months, 90-day mortality, and symptomatic intracranial hemorrhage (sICH). Patients were treated with intravenous thrombolysis (IVT) if eligible before randomization. We excluded RCTs in which less than 50 patients with BAO were enrolled.
+
+## Data Extraction
+
+We collected patient characteristics including: age, sex, baseline National Institutes of Health Stroke Scale (NIHSS), IVT, baseline posterior circulation Alberta Stroke Program Early Computed Tomography Score (pc-ASPECTS), cross-over rate, endovascular technique, and recanalization rate (Table 1). Using data from the trials, we compared outcomes between the EVT and MM groups for four RCTs. Our pre-specified primary outcome was the proportion of patients with a favorable functional outcome defined as mRS of 0 to 3 at 90 days. Secondary outcomes included the proportion of patients who achieved excellent outcome (mRS of 0–2 at 90 days) after treatment. Safety outcomes included sICH 
+as defined by each trial and 90-day mortality rate (Table 2). Supplementary data of the primary, secondary, and safety outcomes were obtained and analyzed from the BASICS and BAOCHE trials for patients with NIHSS <10.
+
+## Statistical Analysis
+
+Data were reported as intention-to-treat analysis. For each study, effect sizes were computed as logit transformed odds ratios (ORs) 
+with random effects and Mantel-Haenszel weighting. The between-study variance component of random-effect models was estimated using restricted effects maximum likelihood with 95% 
+confidence intervals (CIs). Because of the small number of patients included in the meta-analysis, 95% CIs around pooled effect sizes were calculated using Hartung-Knapp adjustment to provide a more conservative estimate of the true intervention and reduce the risk of false positives.22 For each pooled result, Higgin I2 statistics were used to measure the percentage to the total variability in effect estimates attributed to heterogeneity rather than sampling error. The absolute value of the true variance in effect sizes is indicated by τ 2 values in forest plots, estimated using restricted effects maximum likelihood. A funnel plot was used to evaluate potential publication or selection bias.
+
+## Results Summary Of Included Studies
+
+The initial search strategy yielded eight RCTs (Figure 1). One RCT was excluded because of use of an older intra-arterial treatment (urokinase), which was terminated in 2005 after enrollment of 16 patients.23 Three RCTs that included patients with both anterior and posterior circulation strokes were excluded because of the low number of BAO patients enrolled (10 patients in EASI [Endovascular Acute Stroke Intervention], 4 patients in THRACE [Mechanical Thrombectomy After Intravenous Alteplase versus Alteplase Alone After Stroke], and 4 patients in IMS III [Interventional Management of Stroke III]).8,9,24 After exclusion of these four trials, this yielded four published BAO RCTs meeting inclusion criteria with a total of 988 patients. A description of each trial's inclusion, exclusion, and selection criteria is presented (Table 1).
+
+## Primary Outcome
+
+Across the four RCTs encompassing 988 patients, the primary endpoint of a favorable functional outcome (mRS of 0–3) at 90 days was achieved in 251 of 556 patients (45.1%, 95% CI 41%– 49.3%) in the EVT group compared to 128 of 432 patients (29.6%, 95% CI 21.7%–36.4%) for the MM group (Figure 2A). The odds of a favorable functional outcome of mRS 0–3 were higher in the EVT compared to the MM group (OR 1.99; 95% CI 1.04–3.80, P=0.04). The between-study variability in effects estimates unrelated to sampling error was moderate (I2=54.65%, P=0.08).
+
+## Secondary Outcome
+
+Excellent clinical outcome (mRS of 0–2) occurred in 194 of 556 patients (34.8%, 95% CI 30.9%–38.8%) for the EVT group, and 89 of 432 patients (20.6%, 95% CI 10.4%–29.9%) for the MM group (Figure 2B). No statistical difference was observed in the odds of mRS 0–2 in the EVT compared to the MM group (OR 
+2.26; 95% CI 0.78–6.57, P=0.09). The between-study variability in effect estimates unrelated to sampling error was significantly high (I2=77.26%, P<0.01).
+
+## Safety Outcome
+
+The analysis of the pooled data showed an overall sICH rate of 5.4% (95% CI 3.5%–7.2%) for the EVT group, and of 0.8% (95% CI 0.0%–2%) for the MM group (Figure 2C). The EVT group had higher odds of sICH than the MM group (OR 7.89; 95% CI 4.10–
+15.19, P<0.01). The estimated between-study variability in effect estimates unrelated to sampling error was low (I2=0%, P=0.95).
+
+The mortality rate in the EVT group was 198 of 556 patients 
+(35.6%, 95% CI 31.5%–39.5%), and for the MM group 196 of 432 patients (45.4%, 95% CI 38.1%–52.1%) (Figure 2D). The EVT group had lower odds of mortality compared to the MM group 
+(OR 0.64; 95% CI 0.42–0.99, P=0.05). The estimated betweenstudy variability in effect estimates unrelated to sampling error was low (I2=18.61%, P=0.36).
+
+## Risk Of Bias
+
+No evidence suggestive of publication bias was found by examining the funnel plots.
+
+| Table 1. Characteristics of basilar artery occlusion randomized trials BEST18    | BASICS17                                                                                                                                                      | ATTENTION19                                                                                                                                                  | BAOCHE20                                               |                                             |
+|----------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------|---------------------------------------------|
+| Trial summary Publication year                                                   | 2020                                                                                                                                                          | 2021                                                                                                                                                         | 2022                                                   | 2022                                        |
+| Years trial conducted                                                            | 2015–2017                                                                                                                                                     | 2011–2019                                                                                                                                                    | 2021–2022                                              | 2016–2021                                   |
+| Screened patients                                                                | 288                                                                                                                                                           | 424                                                                                                                                                          | 507                                                    | 537                                         |
+| No. of patients enrolled/                                                        | 131/344 (66 EVT, 65 MM)                                                                                                                                       | 300/300 (154 EVT, 146 MM)                                                                                                                                    | 342/342 (228 EVT, 114 MM)                              | 217/318 (110 EVT, 107 MM)                   |
+| target sample size Countries                                                     | China                                                                                                                                                         | 7 Countries                                                                                                                                                  | China                                                  | China                                       |
+| Estimated symptom onset  0–8                                                     | 0–6                                                                                                                                                           | 0–12                                                                                                                                                         | 6–24*                                                  |                                             |
+| to randomization (hr)  Age inclusion criteria,                                   | ≥18                                                                                                                                                           | Initially18–85 (extended                                                                                                                                     | ≥18                                                    | 18–80                                       |
+| years (yr)                                                                       | later to ≥18)                                                                                                                                                 |                                                                                                                                                              |                                                        |                                             |
+| NIHSS inclusion criteria                                                         | Not specified                                                                                                                                                 | Initially ≥10 (extended later                                                                                                                                | ≥10                                                    | Initially ≥10 (extended to                  |
+| to include NIHSS <10)                                                            | include NIHSS ≥6                                                                                                                                              |                                                                                                                                                              |                                                        |                                             |
+| Pre-stroke mRS inclusion                                                         | 0–2                                                                                                                                                           | 0–2                                                                                                                                                          | ≤80 years: 0–2                                         | ≤1                                          |
+| criteria                                                                         | >80 years: 0                                                                                                                                                  |                                                                                                                                                              |                                                        |                                             |
+| Imaging selection criteria                                                       | No evidence of intracranial  hemorrhage, significant  cerebellar mass effect,  acute hydrocephalus,  or extensive bilateral  brainstem ischemia on  CT or MRI | No evidence of intracranial  hemorrhage, extensive  bilateral brainstem  infarction on CT, cerebellar  mass effect, or acute  hydrocephalus on  neuroimaging | NCCT/CTA-SI/DWI pc-ASPECTS ≤80 years: ≥6 >80 years: ≥8 | pc-ASPECTS ≥6 or  Pons-midbrain-index of ≤2 |
+| Endovascular treatment                                                           | At the discretion of treating                                                                                                                                 | At the discretion of treating                                                                                                                                | At the discretion of treating                          | Solitaire stent retriever                   |
+| modality                                                                         | physician                                                                                                                                                     | physician                                                                                                                                                    | physician                                              |                                             |
+| Crossover EVT to MM                                                              | 3/66, 5%                                                                                                                                                      | 3/154, 1.9%                                                                                                                                                  | 3/226, 1.3%                                            | 1/110, 0.9%                                 |
+| MM to EVT                                                                        | 14/65, 22%                                                                                                                                                    | 7/146, 4.8%                                                                                                                                                  | 3/114, 2.6%                                            | 4/107, 3.7%                                 |
+| Trial demographics Age (yr) EVT                                                  | 62 (50–74)                                                                                                                                                    | 66.8±13.1                                                                                                                                                    | 66.0±11.1                                              | 64.2±9.6                                    |
+| MM                                                                               | 68 (57–74)                                                                                                                                                    | 67.2±11.9                                                                                                                                                    | 67.3±10.2                                              | 63.7±9.8                                    |
+| Male sex  EVT (%)                                                                | 73                                                                                                                                                            | 64.9                                                                                                                                                         | 66                                                     | 73                                          |
+| MM (%)                                                                           | 80                                                                                                                                                            | 65.8                                                                                                                                                         | 72                                                     | 74                                          |
+| Baseline NIHSS  EVT                                                              | 32 (18–38)                                                                                                                                                    | 21                                                                                                                                                           | 24 (15–35)                                             | 20 (15–29)                                  |
+| MM                                                                               | 26 (13–37)                                                                                                                                                    | 22                                                                                                                                                           | 24 (14–35)                                             | 19 (12–30)                                  |
+| Baseline pc-ASPECTS EVT                                                          | 8 (7–9)                                                                                                                                                       | 10 (8–10)                                                                                                                                                    | 9 (8–10)                                               | 8 (7–10)                                    |
+| MM                                                                               | 8 (7–9)                                                                                                                                                       | 10 (8–10)                                                                                                                                                    | 10 (8–10)                                              | 8 (7–10)                                    |
+| IVT EVT                                                                          | 18 (27)                                                                                                                                                       | 121 (78.6)                                                                                                                                                   | 69 (31)                                                | 15 (13.6)                                   |
+| MM                                                                               | 21 (32)                                                                                                                                                       | 116 (79.5)                                                                                                                                                   | 39 (34)                                                | 23 (21.5)                                   |
+| ICAD† EVT                                                                        | 37 (56)                                                                                                                                                       | 53/146 (36.3)                                                                                                                                                | 90/226 (40)                                            | N/A                                         |
+| MM                                                                               | 32 (49)                                                                                                                                                       | 43/132 (32.6)                                                                                                                                                | 33/114 (29)                                            | N/A                                         |
+| Demographic data are median (IQR), n (%), or mean±SD unless otherwise indicated. |                                                                                                                                                               |                                                                                                                                                              |                                                        |                                             |
+
+![3_image_0.png](3_image_0.png)
+
+| Table 2. Primary, secondary, and safety outcomes of the basilar artery occlusion randomized controlled trials BEST18 BASICS17 ATTENTION19   | BAOCHE20             |                       |                       |                |
+|---------------------------------------------------------------------------------------------------------------------------------------------|----------------------|-----------------------|-----------------------|----------------|
+| Primary outcome: mRS 0–3 at 90 days EVT 28/66 (42%)                                                                                         | 68/154 (44%)         | 104/226 (46%)         | 51/110 (46.4%)        |                |
+| MM                                                                                                                                          | 21/65 (32%)          | 55/146 (38%)          | 26/114 (23%)          | 26/107 (24.3%) |
+| aOR: 1.74 (0.81–3.74)                                                                                                                       | RR: 1.18 (0.92–1.50) | RR: 2.06 (1.46–2.91)  | aRR: 1.81 (1.26–2.60) |                |
+| P=0.23                                                                                                                                      | P=0.19               | P<0.001               | P<0.001               |                |
+| mRS 0–2 at 90 days  EVT                                                                                                                     | 22 (33%)             | 54 (35.1%)            | 75 (33%)              | 43 (39.1%)     |
+| MM                                                                                                                                          | 18 (28%)             | 44 (30.1%)            | 12 (10.5%)            | 15 (14%)       |
+| aOR: 1.40 (0.64–3.10)                                                                                                                       | RR: 1.17 (0.87–1.57) | RR: 3.17 (1.84–5.46)  | RR: 2.75 (1.65–4.56)  |                |
+| P=0.48                                                                                                                                      |                      |                       |                       |                |
+| Artery patency at 24 to 72 hours EVT 45/63 (71.4%)                                                                                          | 93/110 (84.5%)       | 147/161 (91.3%)       | 89/101 (88.1%)        |                |
+| (TICI ≥2b)                                                                                                                                  | (CTA patency 24 h)   | (artery patency)      | (TICI ≥2b)            |                |
+| MM                                                                                                                                          | 9/14 (64.2%)         | 54/96 (56.3%)         | 26/69 (37.7%)         | N/A            |
+| (TICI ≥2b)                                                                                                                                  | (CTA patency 24 h)   | (artery patency)      |                       |                |
+| sICH* EVT                                                                                                                                   | 5/66 (7.6%)          | 7/154 (4.5%)          | 12 (5.3%)             | 6/102 (5.9%)   |
+| MM                                                                                                                                          | 0/65 (0%)            | 1/146 (0.7%)          | 0%                    | 1/88 (1.1%)    |
+| OR: N/A                                                                                                                                     | RR: 6.9 (0.9–53.0)   | RR: 5.18 (0.64–42.18) |                       |                |
+| P=0.06                                                                                                                                      | P=0.06               | P=0.125               |                       |                |
+| Mortality  EVT                                                                                                                              | 22/66 (33%)          | 59/154 (38.3%)        | 83/226 (36.7%)        | 34/110 (30.9%) |
+| MM                                                                                                                                          | 25/65 (38%)          | 63/146 (43.2%)        | 63/114 (55.3%)        | 45/107 (42.1%) |
+| OR: 0.80 (0.37–1.64)                                                                                                                        | RR: 0.87 (0.68–1.12) | RR: 0.66 (0.52–0.82)  | RR: 0.75 (0.54–1.04)  |                |
+| P=0.54                                                                                                                                      | P=0.29               |                       |                       |                |
+
+BEST, Basilar Artery Occlusion Endovascular Intervention versus Standard Medical Treatment; BASICS, Basilar Artery International Cooperation Study; ATTENTION, Endovascular Treatment for Acute Basilar Artery Occlusion: A Multicentre Randomised Clinical Trial; BAOCHE, Basilar Artery Occlusion Chinese Endovascular Trial; mRS, modified Rankin Scale; EVT, endovascular treatment; MM, medical management; aOR, adjusted odds ratio; RR, risk ratio; TICI, thrombolysis in cerebral infarction; CTA, computed tomography angiography; N/A, not available; sICH, symptomatic intracranial hemorrhage; OR, odds ratio; NIHSS, National Institutes of Health Stroke Scale; SITS-MOST, The Safe Implementation of Thrombolysis in Stroke-Monitoring Study.
+
+*sICH definitions: BEST, defined as evidence of intracranial hemorrhage on imaging and an increase of 4 or more points on the NIHSS within 24 h after randomization; BASICS, Heidelberg bleeding classification; ATTENTION, SITS-MOST criteria; BAOCHE, SITS-MOST criteria (deterioration in NIHSS score of >4 points within 24 h from treatment and evidence of intraparenchymal hemorrhage type 2 in the 22–36 h follow-up imaging scans). BAOCHE also included sICH according to ECASS II criteria.
+
+## Nihss <10 Subgroup
+
+Supplementary analysis of 78 patients with BAO and NIHSS <10 was obtained from the BASICS (n=61) and BAOCHE (n=17) trials. In this subgroup analysis, frequencies of favorable (mRS 0–3) or excellent (mRS 0–2) clinical outcome between the EVT and the MM groups were comparable. Favorable functional outcome (mRS of 0–3) at 90 days was achieved in 26 of 37 patients (70.3%) in the EVT group and in 30 of 41 patients (73.2%) for the MM group. Excellent clinical outcome (mRS of 0–2) occurred in 22 of 37 patients (59.5%) for the EVT group, and 24 of 41 patients (58.5%) for the MM group (Figure 3). The rate of sICH in patients with NIHSS <10 was 8.1% for the EVT group. There was no sICH for the MM group. The mortality rate in the EVT group was 18.9% (7 of 37 patients) which was comparable to that of the MM group 
+
+## (17.1%, 7 Of 41 Patients). Discussion
+
+In this intention-to-treat meta-analysis of the BEST, BASICS, ATTENTION, and BAOCHE randomized trials, we confirm that in patients with acute BAO stroke, there was a twofold increase in the odds of favorable outcome (mRS 0–3) at 3 months with EVT 
+compared to MM. No difference was observed in the odds of excellent outcome (mRS of 0–2) in the EVT compared to the MM 
+group, although the direction and magnitude of effect similarly favored EVT. An mRS 0–3 (rather than mRS 0–2) was likely chosen as the primary endpoint across the four BAO RCTs because it may be a more sensitive measure that accounts for the high-
+
+![5_image_0.png](5_image_0.png)
+
+er severity of BAO disease when patients are recovered. Despite an increase in sICH in the EVT group, patients treated with EVT 
+had lower odds of mortality compared to the MM group. In subgroup analysis of 78 BAO patients with NIHSS <10, the frequency of favorable or excellent outcomes at 90 days as well as mortality was similar in the EVT as compared to the MM group while the frequency of sICH was 8.1% in EVT group compared to none in the MM group.
+
+The overall results of this meta-analysis are in alignment with the two recent BAO RCTs (ATTENTION and BAOCHE),19,20 which contrast with the initial neutral BAO RCTs (BEST and BASICS).12,18 BEST and BASICS encountered formidable challenges with enrollment, selection bias, and crossovers. Additionally, the original BASICS inclusion criterion of NIHSS ≥10 was modified during the trial to include patients with milder stroke deficits.25 As was observed with the prospective BASICS and Helsinki registry studies,12,26 the BASICS RCT, with a high proportion of patients receiving IVT, revealed how well patients with mild stroke deficits can do with MM of BAO, thereby attenuating the potential treatment effect of EVT in the subgroup of BAO patients with low NIHSS. In the BEST trial, the progressive drop in recruitment over time, and the crossovers led to the premature termination of the trial by the data and safety monitoring board,18 hence reducing statistical power and lowering the treatment effect size for the intention-to-treat analysis. Moreover, the overwhelmingly positive anterior circulation trials favoring EVT published in 2015 may have led to a loss of equipoise in randomizing consecutive BAO 
+patients into the trials, and thereby contributing to potential selection bias and the neutral BEST and BASICS trial results.17,18 Perhaps as a result of the neutral trial results, BEST and BASICS 
+provided the foundations to reinforce the concept of equipoise regarding EVT versus MM management of BAO patients.10,11 BAOCHE and ATTENTION reinforced consecutive enrolment following the neutral results of BEST and BASICS, resulting in a high inclusion of eligible patients with BAO. In ATTENTION, 342 out of 507 screened patients were enrolled in 1 year compared to 300 patients in 8 years in BASICS. EVT in ATTENTION was discouraged outside of the trial. Extending the time windows up to 12 hours from estimated onset in ATTENTION and up to 24 hours in BAOCHE expanded the number of eligible patients for enrollment. The crossover rates were low in ATTENTION compared to the BEST trial (1.3% [3/226] crossed-over from EVT to MM in ATTENTION vs. 5% [3/66] in BEST; 2.6% crossed over from MM to EVT in ATTENTION compared to 22% in BEST). To decrease the risk of futile recanalization, both BAOCHE and ATTENTION 
+trials used an imaging scale (pc-ASPECTS)27 and included patients with small infarct at baseline (pc-ASPECTS ≥8 in ATTENTION and ≥6 in BAOCHE).
+
+In ATTENTION, a twofold favorable outcome was achieved in the EVT compared to MM group, with a number needed to treat of 4.19 The disability reduction benefit of EVT in BAO patients in ATTENTION was comparable to the anterior circulation trials (90day mRS ordinal shift analysis: OR 2.8 [95% CI: 1.8–4.4] in ATTENTION vs. 2.49 [1.76–3.53] in HERMES [Highly Effective Reperfusion Evaluated in Multiple Endovascular Stroke Trials]28 and 2.54 [1.83-3.54] in AURORA [Analysis of Pooled Data from Randomized Studies of Thrombectomy More Than 6 Hours after Last Known Well]29). ATTENTION also demonstrated a potential mortality benefit to EVT treatment, in contrast to most anterior circulation EVT trials.19 BAOCHE assessed the safety and efficacy of EVT versus MM 
+for acute BAO in patients presenting in a 6 to 24 hour time window after symptom onset.20 In BAOCHE, the proportion of patients achieving favorable outcomes defined as mRS 0–3 at 90 days was higher in the EVT versus MM group with a number needed to treat of 4.5. BAOCHE showed the benefit of BAO EVT 
+in the extended window without increased risk of sICH.20 In BAOCHE subgroup analysis, the point estimates for good functional outcome favored EVT in each of the 6–12 hour (adjusted rate ratio, aRR 1.89 [95% CI 1.15–3.09]) and 12–24 hour (aRR 1.71 
+[95% CI 1.01–2.90]) windows. In contrast to the anterior circulation late-window trials whereby advanced imaging was utilized to select patients, pc-ASPECTS was adequate for selection 
+
+![6_image_0.png](6_image_0.png)
+
+![6_image_1.png](6_image_1.png)
+
+![6_image_2.png](6_image_2.png)
+
+| EVT                                                 | MM                    | Ods ratio   | Weight   |                            |             |
+|-----------------------------------------------------|-----------------------|-------------|----------|----------------------------|-------------|
+| mRS 0-3                                             |                       |             |          |                            |             |
+| Study                                               | Yes No Yes No         | with 95% Cl | (%)      |                            |             |
+| BAOCHE trial, 2022                                  | 66                    | 6 5         | n        | 1.00 [ 0.50, 242.34] 39.46 |             |
+| 10                                                  |                       |             |          |                            |             |
+| BASICS trial, 2021 17                               | 20                    | 11 24       | 6        | 0.45 [ 0.14,               | 1.45] 60.54 |
+| - Overall I                                         | 1.60 [10.00, 6.3e+08] |             |          |                            |             |
+| Heterogeneity: τ 2 = 3.66, l 2 = 72.04%, H 2 = 3.58 |                       |             |          |                            |             |
+| Test of θ j = θ j : Q(1) = 3.58, p = 0.06           | favors MM  favors EVT |             |          |                            |             |
+| Test of 0 = 0; t(1) = 0.30, p = 0.81                | 1/2                   | 2           | 8        | 32                         | 128         |
+
+![7_image_0.png](7_image_0.png)
+
+Figure 3. Forest plots for the (A) primary outcome data (mRS 0–3), (B) secondary endpoints of excellent clinical outcome (mRS 0–2), (C) symptomatic intracranial hemorrhage, and (D) mortality for patients with acute basilar occlusion and NIHSS <10 treated with EVT or MM. mRS, modified Rankin Scale; EVT, endovascular thrombectomy; MM, medical management; CI, confidence interval; BAOCHE, Basilar Artery Occlusion Chinese Endovascular Trial; BASICS, Basilar Artery International Cooperation Study; REML, random effect restricted maximum likelihood.
+
+of patients into the late window BAOCHE trial, with the majority of patients (approximately two-thirds) selected by non-contrast CT scan and CT angiogram alone. These results support the concept that EVT for late presenting patients with acute BAO has similar efficacy compared to EVT for early presenting patients with BAO or for patients with anterior circulation proximal LVO. 30 As the enrollment window from estimated time of symptom onset was longer for the BAO trials conducted in China, there was less usage of IVT in these trials compared to the BASICS trial.
+
+Observational data showed that up to 50% of patients with BAO can achieve mRS 0–3 with mostly IVT treatment if presenting in extended time windows with at least pc-ASPECTS of 8.³¹ This outcome contrasts with that of the MM group in ATTENTION and BAOCHE. Further studies are needed to explore a potential superior recanalization response after IVT in the posterior circulation compared to the anterior circulation. An ongoing trial is testing the efficacy of IVT in patients with BAO in the late time window (NCT05105633). Moreover, whether intra-arterial thrombolysis with urokinase, alteplase, or tenecteplase confers benefit to patients with BAO also remains to be explored, either as primary or adjunctive treatment to mechanical thrombectomy.32-34 BASICS and BAOCHE included patients with acute BAO and mild symptoms (NIHSS <10) whereas BEST and ATTENTION included only patients with NIHSS ≥10. Including mild strokes in these trials may have diluted the treatment effect of EVT.25 In our subgroup analysis of patients with low NIHSS, the treatment effect of EVT was not significant. While our analysis was underpowered due to the low sample size, these data, along with the prospective BASICS registry findings, highlights this subgroup of patients as a population that would merit further study to evaluate the benefit of EVT in addition to MM. Management of patients with mild symptoms is challenging as some may improve without EVT while others may deteriorate. The NIHSS also has reduced sensitivity to detect potentially disabling deficits in patients with posterior circulation stroke.35 Prior studies showed divergent results of EVT in these patients as it carries increased procedural risks, especially in patients with underlying intracranial atherosclerotic disease (ICAD).12,36,37 Randomized trials comparing EVT 
+versus MM in BAO patients with mild strokes are warranted. 
+
+ICAD, which is considered a potential cause of EVT failure in acute BAO stroke,16,38 was present in approximately 52% of patients in BEST (56% in the EVT group and 49% in the control group),18 in approximately half of the patients in the ATTENTION 
+trial leading to high rates of intracranial angioplasty +/- stenting 
+(39%),19 and over half of patients in the BAOCHE trial (intracranial stenting or angioplasty in 54%).20 Despite a high frequency of ICAD, a subgroup analysis showed no treatment effect modification based on the presence of ICAD with a significant benefit in ICAD-related LVOs.19 This is similar to prior studies that showed that, compared to patients without ICAD, patients with ICAD who received rescue treatment achieved similar rates of successful recanalization, favorable outcome, sICH, and mortality at 90 days.16,39 The rates of sICH were significantly higher in the EVT compared to the control group in all the included BAO trials with rates ranging between 4.5%–8.5% and a pooled rate of 5.4% in the EVT group compared to 0%–1.1% in the control group. These higher rates of sICH could be related to the technical challenges of EVT especially in patients with ICAD who usually require angioplasty and stenting. However, despite the increased rates of sICH, patients treated with EVT had higher odds of favorable outcomes and lower odds of mortality compared to the control group in both the ATTENTION and BAOCHE trials. 
+
+The four RCTs included in this meta-analysis were of high quality and low risk of bias. The significant between-studies heterogeneity noted for the primary and secondary outcomes reflects 
+
+![8_image_0.png](8_image_0.png)
+
+the variation between the four trials especially in terms of inclusion criteria, number of patients, NIHSS thresholds, duration of the study, and different treatment windows. Despite this heterogeneity, the benefit of EVT in acute BAO was demonstrated in this meta-analysis. 
+
+There are several limitations in our study. The generalizability of these findings need to be considered as three of the trials were completed in China and Asians are known to have high rates of ICAD.40 However, as mentioned, prior studies showed that patients with ICAD who received rescue treatment achieved similar rates of successful recanalization and favorable outcome, compared to patients without ICAD.16,39,41 Data on the percentage of ICAD were not available in the BAOCHE trial and we were unable to compare outcomes of patients with ICAD and without ICAD in this meta-analysis. Further subgroup data of treatment effect by sex, ethnicity, IVT, time window of treatment, pc-ASPECTS, and ICAD would be of interest in a patient-level meta-analysis. Our supplementary meta-analysis for patients with mild BAO (i.e., 
+NIHSS <10) is limited by the fact that BAOCHE only included patients with an NIHSS ≥6. Moreover, the time window in which patients were enrolled between BASICS (6–24 h) and BAOCHE (0–6 h) did not overlap.
+
+## Conclusion
+
+This intention-to-treat meta-analysis of the BEST, BASICS, ATTENTION, and BAOCHE trials supports the overall benefit of endovascular treatment in acute BAO up to 24 hours. A treatment effect was not observed in the subgroup of patients with NIHSS <10, although our analysis was underpowered. Further studies are of interest to evaluate the efficacy and safety of EVT in acute basilar occlusion patients presenting with milder symptoms (i.e., NIHSS <10) and the comparison of patients treated with EVT versus IVT. 
+
+## Disclosure
+
+The authors have no financial conflicts of interest.
+
+## References
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medical/md/PMC9943831.md

@@ -0,0 +1,113 @@
+# Comparison Of Medium-Term Results Of Minimally Invasive Plating Osteosynthesis And Open Reduction And Internal Fixation For Mid-Distal Humeral Shaft Fractures Bo Yin1, Lianhua Li2, Zheng Lu3, Jie Gao2, Huayong Zeng2, Yanhong Cai2, Yuanming He1, Zhi Liu2
+
+1Department of Orthopedic Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China; 2Senior Department of Orthopedics, the Fourth Medical Center of Chinese People's Liberation Army (PLA) General Hospital, Beijing 100048, China; 3Medical Supplies Center of PLA General Hospital, Beijing 100700, China.
+
+To the Editor: There is controversy in the literature on the benefits of open reduction and internal fixation (ORIF)
+and minimally invasive plating osteosynthesis (MIPO)
+for mid-distal humeral shaft fractures.[1] ORIF has the advantages of anatomical reduction, strong fixation, and little impact on the function of the elbow and shoulder joints. Nevertheless, it has some disadvantages, including the need for a large incision and more dissection of soft tissues, interrupting blood supply to the fracture site, resulting in difficulties in healing and an increased risk of bone non-union, and a high risk of iatrogenic radial nerve injury. In contrast, treatment of mid-distal humeral shaft fractures by MIPO has the advantages of less dissection of soft tissues, avoidance of exposure of the radial nerve, and a low risk of iatrogenic radial nerve palsy. This study aimed to compare the medium-term safety and effectiveness of MIPO and conventional ORIF in the treatment of mid-distal humeral shaft fractures. This nested case-control study was performed at the Seventh Medical Center of Chinese People's Liberation Army (PLA) General Hospital and Beijing Chaoyang Hospital. The nesting method has been described in detail elsewhere.[2] All the mid-distal humeral shaft fractures treated between January 2012 and December 2016 were eligible for the study and were enrolled. The inclusion criteria were patients aged 18 to 60 years with an acute displaced mid-distal humeral shaft fracture who experienced at least 3 years of post-operative follow-up. Those suffering from intra-articular fractures of the elbow, vascular insufficiency, pathological fracture, and multiple or open fractures were excluded. The included patients were divided according to whether they underwent MIPO or ORIF with plating (the MIPO and ORIF operation procedures were described in Supplementary Figures 1 Access this article online
+
+| www.cmj.org DOI: 10.1097/CM9.0000000000002424   |
+|-------------------------------------------------|
+
+![0_image_0.png](0_image_0.png)
+
+and 2, http://links.lww.com/CM9/B239). Those who underwent MIPO were then matched for gender and age (±3 years) with those who underwent ORIF at a ratio of 1:2. All cases were evaluated at the first, third, sixth, and 12th months post-operatively and 3rd to 5th years thereafter. Their medical records and radiographs obtained during hospitalization and follow-up after discharge were reviewed. This study protocol was approved by the Research Ethics Committees of the Seventh Medical Center of Chinese PLA General Hospital (No. 2018-21) and Beijing Chaoyang Hospital (No. 2019-310) and all enrolled patients have signed the informed consent.
+
+The primary outcome compared was the overall major complications, including iatrogenic radial nerve palsy, infection, myositis ossificans, and bone non-union. The secondary outcomes were the recovery of shoulder and elbow joint function evaluated by the University of California at Los Angeles (UCLA) scoring system and Mayo Elbow Performance Score (MEPS).
+
+All statistical analyses were performed using SPSS version 23.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 8.3.1 (GraphPad Software, San Diego, CA, USA). Continuous variables were described as mean ± standard deviation and compared using the paired t-test when the normal distribution was met, otherwise the paired nonparametric test was used; while categorical variables were described as number (percentage) and compared using the McNemar's test. Survival analysis was performed using the Kaplan–Meier method, in which the total major complication rate was the outcome variable and the time interval between the day of surgery and the onset of the complication was the "time to event".
+
+The Mantel–Haenszel test was used to estimate hazard Correspondence to: Lianhua Li, Department of Orthopedics, The Fourth Medical Center of Chinese People's Liberation Army (PLA) General Hospital, Beijing 100048, China E-Mail: 15901170726@163.com Copyright © 2022 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
+
+Chinese Medical Journal 2022;135(22)
+Received: 29-08-2022; Online: 27-12-2022 Edited by: Rongman Jia and Xiuyuan Hao 2764
+
+| Characteristics                                                                                                                                                                                                | MIPO (n = 28)   | ORIF (n = 56)   | x2   | P values   |
+|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------|-----------------|------|------------|
+| Male                                                                                                                                                                                                           | 18 (64.3)       | 36 (64.3)       | -    | -          |
+| Age (years)                                                                                                                                                                                                    | 36.0 ± 12.3     | 36.8 ± 11.8     | -    | -          |
+| Injury causes                                                                                                                                                                                                  | 1.08            | 0.783           |      |            |
+| Wrestling                                                                                                                                                                                                      | 10 (35.7)       | 23 (41.1)       |      |            |
+| Throwing                                                                                                                                                                                                       | 9 (32.1)        | 17 (30.3)       |      |            |
+| Fall                                                                                                                                                                                                           | 6 (21.4)        | 9 (16.1)        |      |            |
+| Car accident                                                                                                                                                                                                   | 3 (10.7)        | 7 (12.5)        |      |            |
+| OTA classification                                                                                                                                                                                             | 6.66            | 0.353           |      |            |
+| A1                                                                                                                                                                                                             | 13 (46.4)       | 29 (51.8)       |      |            |
+| A2                                                                                                                                                                                                             | 1 (3.6)         | 3 (5.4)         |      |            |
+| A3                                                                                                                                                                                                             | 0 (0)           | 2 (3.6)         |      |            |
+| B1                                                                                                                                                                                                             | 12 (42.9)       | 18 (32.1)       |      |            |
+| B2                                                                                                                                                                                                             | 0 (0)           | 1 (1.8)         |      |            |
+| B3                                                                                                                                                                                                             | 1 (3.6)         | 0 (0)           |      |            |
+| C1                                                                                                                                                                                                             | 1 (3.6)         | 3 (5.4)         |      |            |
+| Preoperative radial nerve injury                                                                                                                                                                               | 4 (14.3)        | 7 (12.5)        | 0.06 | 0.801      |
+| Data are expressed as n (%) or mean ± standard deviation. MIPO: Minimally invasive plating osteosynthesis; ORIF: Open reduction and internal fixation; OTA: Orthopaedic Trauma Association; –: Not applicable. |                 |                 |      |            |
+
+ratio (HR) of total major complications. A P-value of
+
+<0.05 was considered statistically significant.
+
+Between January 2012 and December 2016, 216 patients with mid-distal humeral shaft fracture underwent orthopedic surgery at either of the participating institutions.
+
+According to the matching method, 28 patients were divided into the MIPO group and 56 into the ORIF group. The baseline characteristics were listed in Table 1. During medium-term follow-up, the overall major complication rate was 0% (0/28) in the MIPO group and 28.6% (16/56) in the ORIF group. The difference was significant (x 2 = 10.37, P < 0.001). The analysis on the cumulative incidence curves for complications showed that the total major complication rate was significantly lower in the MIPO group (HR 0.20, 95%
+confidence interval [CI] 0.07–0.56; P < 0.001, Mantel–
+Haenszel test) [Supplementary Figure 3, http://links.lww.
+
+com/CM9/B239].
+
+There were no cases of iatrogenic nerve injury in the MIPO
+group and four cases (7.1%) in the ORIF group
+(x 2 = 0.12, P = 0.357). Among the four patients, two recovered within 4 weeks after surgery without intervention; another two did not recover within six months, and the radial nerve exploration on them revealed that the nerve of one patient was severed and that of the other was pressed under the plate. There were no deep infections in the MIPO group; however, there were three cases (5.4%)
+in the ORIF group, all of which were treated with intravenous antibiotic therapy. The between-group difference was not statistically significant (x 2 = 0.16, P = 0.600).
+
+There was significant between-group difference in time to bone union (6.2 ± 1.6 [ranged 4.0–9.0] months in the MIPO group vs. 6.0 ± 3.3 [ranged 3.0–20.0] months in the ORIF group; z = 2.46, P = 0.014). Three patients (5.4%)
+in the ORIF group had bone non-union, which was managed by removing the soft tissue at the fracture end and internal fixation was performed again with bone graft.
+
+The between-group difference was not statistically significant (x 2 = 1.57, P = 0.600). Myositis ossificans was detected in four patients (7.1%) in the ORIF group and none in the MIPO group (x 2 = 2.12, P = 0.357). One case required release at the elbow joint to improve elbow function.
+
+The plate was removed without the occurrence of complications in 20 patients in the MIPO group and 14 in the ORIF group. Two cases in the ORIF group suffered from complications (intra-operative radial nerve injury in one case and post-operative fracture for the second time in the other case). The complication rate was not significantly different between the two groups
+(x 2 = 1.03, P = 0.584).
+
+The mean UCLA score was significantly higher in the MIPO group than that in the ORIF group (34.4 ± 1.7 vs.
+
+31.2 ± 3.9, P < 0.001) at the last follow-up. Twenty-seven patients in MIPO group had an excellent outcome and one
+(3.6%) had a poor result. Thirty-five patients (62.5%) in the ORIF group had an excellent outcome, 14 (25.0%) had a good outcome, and 7 (12.5%) had a poor outcome.
+
+The post-operative UCLA values were significantly higher in the MIPO group when compared with that of the ORIF
+group (P = 0.003). Similar results were observed for MEPS
+at the last follow-up. This study compared the medium-term results of MIPO with those of ORIF when treating mid-distal humeral shaft fractures. Our main finding showed that there was a statistically significant between-group difference in the overall major complication rate, with a hazard ratio of 0.20. Several comparative studies have investigated the short-term advantages of MIPO in terms of avoiding complications in patients with humeral shaft fractures.[3]
+which are consistent with our present findings.
+
+2765 In this study, the recovery of post-operative shoulder and elbow joint functions was better in patients who underwent MIPO than those who underwent ORIF.
+
+Our findings in this regard are similar to those of Mahajan et al[4] who evaluated the efficacy of MIPO in middle humeral fracture patients regularly performed overhead shoulder movements, such as athletes and manual workers, and found that most patients had good functional results. We attribute the good joint function achieved by MIPO to the ability of this procedure to obtain strong fixation without damaging the surrounding soft tissues.
+
+In the mid-distal humerus, the bone is irregularly bent and there is a 20° to 30° intorsion in the junction between the middle and distal humeral shaft. The MIPO plate is usually pre-contoured to allow better attachment. However, up until now, most studies have used straight plates, which can result in poor fracture reduction and malunion.[5] None of the patients in our MIPO group showed malunion because the locking compression plate was contoured to conform to the anterolateral surface of the mid-distal humerus.
+
+The strength of this investigation is the nested case-control study design, whereby patients who underwent MIPO
+were strictly matched with those who underwent ORIF.
+
+However, the study has some limitations. First, the population size was relatively small. However, in clinical practice, it is difficult to enroll large numbers of patients for this type of studies. Second, the included patients were recruited from two centers, and the possibility of a confounding effect in terms of surgical technique and functional assessment cannot be excluded. In the future study, a multi-center randomized controlled trial is needed for further verification.
+
+To conclude, MIPO has significant clinical advantages in comparison with ORIF, including few major complications and better shoulder and elbow joint function recovery during at least three years of post-operative follow-up. MIPO is a safe and effective technique in treating mid-distal humeral shaft fractures.
+
+Conflicts of interest
+
+## None.
+
+References 1. Yang J, Liu DP, Zhang LN, Lu ZX, Liu T, Tao C. Treatment of humeral shaft fractures: a new minimally-invasive plate osteosynthesis versus open reduction and internal fixation: a case control study. BMC Surg 2021;21:349. doi: 10.1186/s12893-021-01347-4.
+
+2. Li LH, Ren JX, Liu J,Wang H, Sang QH, Liu Z, et al.What are the risk factors for dislocation of hip bipolar hemiarthroplasty through the anterolateral approach? A nested case-control study. Clin Orthop Relat Res 2016;474:2622–2629. doi: 10.1007/s11999-016-5053-3.
+
+3. Keshav K, Baghel A, Kumar V, Neradi D, Kaustubh K, Mishra P. Is minimally invasive plating osteosynthesis better than conventional open plating for humeral shaft fractures? A systematic review and meta-analysis of comparative studies. Indian J Orthop 2021;55:
+283–303. doi: 10.1007/s43465-021-00413-6.
+
+4. Mahajan AS, Kim YG, Kim JH, D'sa P, Lakhani A, Ok HS. Is anterior bridge plating for mid-shaft humeral fractures a suitable option for patients predominantly involved in overhead activities?
+
+A functional outcome study in athletes and manual laborers. Clin Orthop Surg 2016;8:358–366. doi: 10.4055/cios.2016.8.4.358.
+
+5. Wang C, Li J, Li Y, Dai G, Wang M. Is minimally invasive plating osteosynthesis for humeral shaft fracture advantageous compared with the conventional open technique? J Shoulder Elbow Surg 2015;24:1741–1748. doi: 10.1016/j.jse.2015.07.032.
+
+ 
+2766
+How to cite this article: Yin B, Li L, Lu Z, Gao J, Zeng H, Cai Y, He Y, Liu Z. Comparison of medium-term results of minimally invasive plating osteosynthesis and open reduction and internal fixation for mid-distal humeral shaft fractures. Chin Med J 2022;135:2764–2766. doi:
+10.1097/CM9.0000000000002424

medical/md/PMC9979960.md

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+# Unusual Presentation Of Colon Cancer As Rectal Prolapse In Middle-Aged Male
+
+Ashley A. Penton1 | Sarah B. Jochum1 | **Joshua M. Eberhardt1,2**
+1Department of Surgery, Loyola University Medical Center, Maywood, Illinois, USA
+2Division of Colon and Rectal Surgery, Department of Surgery, Loyola University Medical Center, Maywood, Illinois, USA Correspondence Ashley A. Penton, Department of Surgery, Loyola University Medical Center, 2160 S. 1st Ave, Maywood, IL 60153, USA. Email: ashley.penton@luhs.org
+
+## Abstract
+
+Rectal prolapse is typically a benign idiopathic condition. Rarely, rectal prolapse can be due to or associated with colorectal carcinoma. Here we present a middleaged gentleman with no previous medical or surgical history, who presented with rectal prolapse secondary to sigmoid adenocarcinoma.
+
+KEYWORDS
+colorectal cancer, general surgery, intussusception, oncology, rectal prolapse, sigmoid 
+
+![0_image_0.png](0_image_0.png) adenocarcinoma
+
+## 1 | **Introduction**
+
+Rectal prolapse is typically an idiopathic benign condition characterized by an intussusception of the rectal wall resulting in a protrusion through the anus. Epidemiologic studies have shown that the usual demographics are elderly females. These patients are at higher risk of various anatomic abnormalities associated with rectal prolapse, such as: diastasis of the levator ani, deep cul-de-sac, redundant sigmoid, patulous anal sphincter and loss of rectal sacral attachments.1 Rarely, rectal prolapse secondary to colorectal carcinoma has been reported.2–11
+
+## 2 | **Case Presentation**
+
+A middle-aged man with no past medical or surgical history presented with a painful, irreducible anal bulge (Figure 1). He denied a prior history of constipation or frequent straining and he had never had a colonoscopy. On arrival, his white blood cell count was 20.4 K/
+μL, hemoglobin was 15.9 g/dL, liver function tests were within normal limits, and carcinoembryonic antigen FIGURE 1 Photo of rectal prolapse.
+
+(CEA) was 2.8 ng/mL. On physical examination, he appeared to have a characteristic rectal prolapse; however, one area of exposed mucosa was irregularly appearing This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
+
+© 2023 The Authors. *Clinical Case Reports* published by John Wiley & Sons Ltd.
+
+2 of 4 | 
+
+![1_image_0.png](1_image_0.png)
+
+FIGURE 2 CT scan demonstrating intussusception (A) Coronal View, (B) Sagittal view, (C) Axial View.
+
+and firm on palpation. Attempts at bedside reduction were unsuccessful.
+
+Given the mucosal abnormality and the fact that he fell outside of the typical demographic that presents with rectal prolapse, a bedside mucosal biopsy was performed and was normal on frozen section. Formal colonoscopy could not be obtained due to patient discomfort and refusal to drink the bowel prep. CT of the chest, abdomen, and pelvis demonstrated intussusception of colon into rectum with prolapse out of the anus 
+(Figure 2). There was no evidence of locally advanced or metastatic disease. The patient was operated on urgently due to increasing pain requiring narcotics and the concern for ischemia of the incarcerated prolapse. 
+
+A perineal approach was avoided due to concern for malignancy despite the negative biopsy. After laparotomy, intussusception due to a sigmoid mass as the lead point was found and an oncologic resection was done. Final pathology revealed moderately differentiated sigmoid adenocarcinoma 5.6 cm pT2N0 (0/17 lymph nodes) with no lymphovascular invasion.
+
+## 3 | **Discussion**
+
+Rectal prolapse is a rare disorder that occurs in approximately 0.5% of the population. The usual presentation is in elderly females.1,12–15 When rectal prolapse does present in male patients, they are usually young and often report a history of longstanding constipation due to pelvic floor disorders or medications, such as anticholinergic medications.14 There are a variety of operations for the management of rectal prolapse which are categorized as either intraabdominal or perineal approaches.12,13,15 None of the operations necessitate an oncologic resection, though abdominal approaches can achieve an oncologic resection while perineal approaches cannot. Thus, if malignancy is suspected, it should impact the operative approach chosen.
+
+Approximately nine cases describing rectal prolapse secondary to a colorectal carcinoma have been reported in the literature (Table 1).2–11 In these reports, similar to our case, an unusual mass was either visualized on the surface of the prolapsed rectum or found on subsequent imaging. Also, it is interesting to note that in all of the previous reports, including this case, the cancers associated with rectal prolapse on presentation were node negative.
+
+| TABLE 1                                                                         | Case reports to date.                                                                                                                                |             |                                                                                                                                                                            |                                                                                                                                                                                                            |
+|---------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------|-------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
+| Author (year)                                                                   | N                                                                                                                                                    | Demographic | Characteristic/Staging                                                                                                                                                     | Course                                                                                                                                                                                                     |
+| Rashid (1996)2                                                                  | 4/70 (5.7%)                                                                                                                                          | 74% F       | History of rectal bleeding, and/or                                                                                                                                         |                                                                                                                                                                                                            |
+| Retrospective study                                                             | mass                                                                                                                                                 |             |                                                                                                                                                                            |                                                                                                                                                                                                            |
+| 3/4 rectal carcinoma, 1/4 sigmoid  carcinoma 3/4 Duke stage A, 1/4 Duke stage B | Screening included rigid proctoscopy,  flexible sigmoidoscopy,  colonoscopy and/or barium  enema Surgical approach for malignancy  was not discussed |             |                                                                                                                                                                            |                                                                                                                                                                                                            |
+| Yamazaki (1999)10                                                               | 1                                                                                                                                                    | 76 F        | Sigmoid adenocarcinoma                                                                                                                                                     | Barium enema showing sigmoid                                                                                                                                                                               |
+| T3N0M0                                                                          | carcinoma. Sigmoidectomy and  proctopexy                                                                                                             |             |                                                                                                                                                                            |                                                                                                                                                                                                            |
+| Bounovas (2007)8                                                                | 1                                                                                                                                                    | 85 F        | Mass noted on prolapsed colon,  3×4 cm Rectal adenocarcinoma. T3N0M0                                                                                                       | Biopsy of mass on prolapsed colon Sigmoidectomy with proctopexy                                                                                                                                            |
+| Chen (2008)6                                                                    | 1                                                                                                                                                    | 75 F        | History of rectal prolapse Sigmoid adenocarcinoma Unable to identify staging                                                                                               | Double contrast barium enema  showing intussusception at  rectosigmoid junction Flexible sigmoidoscopy, with biopsy.  Rectosigmoidectomy with  rectopexy                                                   |
+| Nabi (2015)7                                                                    | 1                                                                                                                                                    | 77 F        | History of rectal prolapse for  5 years Full thickness rectal stump  prolapse, 15 cm of prolapse.  Large mass of 3.5 cm emanating  from stump Rectal adenocarcinoma T2N0M0 | Biopsy of suspicious large mass.  Staging CT performed (CT chest,  abdomen/pelvis) Intersphincteric perineal  proctectomy, with high ligation of  mesorectum                                               |
+| Yamamoto (2018)4                                                                | 1                                                                                                                                                    | 63 F        | 7 cm soft mass, 5 cm from anal  verge Rectal adenocarcinoma cT1N0M0                                                                                                        | Colonoscopy with biopsy Low anterior resection performed                                                                                                                                                   |
+| Akyuz (2019)9                                                                   | 1                                                                                                                                                    | 77 M        | Complete rectal prolapse 15 cm w/ mass palpated on DRE Sigmoid mucinous  adenocarcinoma T3N0M0                                                                             | Rectal digital examination Low anterior resection                                                                                                                                                          |
+| Nocera (2020)5                                                                  | 1                                                                                                                                                    | 92 F        | 4 cm malignant polyp, 3–4 cm  above anal verge cT3-T4, cN1, cM0 adenocarcinoma  –> ypT1 pN0, M0                                                                            | Pain with suppository for  hemorrhoids. MRI, colonoscopy  with biopsy. Electing palliative  approach with radiotherapy 2 months later presented with rectal  prolapse. MRI confirmed Altemeier's technique |
+| Mazumdar (2021)11                                                               | 1                                                                                                                                                    | 52 M        | Sigmoid moderately differentiated  adenocarcinoma, infiltrating the  submucosa and muscularis propria                                                                      | Non - contrast CT showing sigmoidrectal intussusception telescoping  through anal canal Hartmann's procedure and  sigmoidectomy                                                                                                                                                                                                            |
+
+This case illustrates the fact that when patients present with rectal prolapse and they fall outside of the usual demographic, malignancy should be considered. The prolapse may not be due to the expected anatomic abnormalities, such as levator diastasis or loss of rectal sacral attachments; it may, in fact, be due to a tumor causing an intussusception that looks identical to ordinary idiopathic rectal prolapse. In these abnormal presentations, a preoperative workup should include colonoscopy, if possible. 
+
+If malignancy cannot be excluded within reason prior to operation, then operations that are not oncologic should be avoided.
+
+## Author Contributions
+
+Ashley A. Penton: Writing - original draft; writing - review and editing. **Sarah B. Jochum:** Writing - original draft; writing - review and editing. **Joshua M. Eberhardt:**
+Supervision; writing - review and editing.
+
+## Acknowlegements None.
+
+CONFLICT OF INTEREST STATEMENT
+The authors declare there is no conflict of interest regarding the publication of this article.
+
+## Data Availability Statement
+
+The data that support the findings of this study are not openly available due to patient privacy and are available from the corresponding author upon reasonable request. Consent was obtained from the patient. CONSENT Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.
+
+## References
+
+1. Kairaluoma MV, Kellokumpu IH. Epidemiologic aspects of complete rectal prolapse. *Scand J Surg*. 2005;94(3):207-210. 
+
+doi:10.1177/145749690509400306 2. Rashid Z, Basson MD. Association of rectal prolapsed with colorectal cancer. *Surgery*. 1996;119:51-55.
+
+3. Bordeianou L, Paquette I, Johnson E, et al. Clinical practice guidelines for the treatment of Rectal prolapse. *Dis Colon* Rectum. 2017;60(11):1121-1131.
+
+4. Yamamoto R, Mokuno Y, Matsubara H, Kaneko H, Iyomasa S. Laparoscopic low anterior resection for Rectal cancer with Rectal prolapse: a case report. *J Med Case Reports*. 2018;12(1):28. 
+
+doi:10.1186/s13256-017-1555-1 5. Nocera F, von Flüe M, Steinemann DC. Rectal prolapse following short-course radiotherapy for Rectal cancer: report of a case. *J Surg Case Rep*. 2020;2020:rjaa529.
+
+6. Chen CW, Hsiao CW, Wu CC, Jao SW. Rectal prolapse as initial clinical manifestation of colon cancer. *Z Gastroenterol*. 
+
+2008;46:348-350.
+
+7. Nabi H. Rectal adenocarcinoma in prolapsed rectal stump following Hartmann's procedure. *Int J Colorectal Dis*. 
+
+2015;30:987-988.
+
+8. Bounovas A, Polychronidis A, Laftsidis P, Simopoulos C. 
+
+Sigmoid colon cancer presenting as complete rectal prolapse. 
+
+Colorectal Dis. 2007;9:665-666.
+
+9. Akyuz M, Topal U, Gok M, Arikan TB, Seizure E, Akyildiz HY. 
+
+Sigmoid colon cancer presenting as complete prolapse. Annali Italiani di Chirurgia. 2019;8:345-348.
+
+10. Yamazaki T, Sakai Y, Sekine Y, Nihei K, Hatakeyama K. Sigmoid colon cancer presenting as complete rectal prolapse: report of a case. *Surg Today*. 1999;29:266-267.
+
+11. Mazumdar P, Kumar P, Katiyar G, Mulla M, Sardessai S. Sigmoid carcinoma with sigmoid-rectal intussusception presenting as rectal prolapse and large bowel obstruction in the ED. *Egypt J* Radiol Nucl Med. 2021;52:34. doi:10.1186/s43055-021-00414-3 12. Wells K, Fleshman J. Management of rectal prolapse. In: 
+Cameron JL, Cameron AM, eds. *Current Surgical Therapy*. 13th ed. Elsevier; 2020:220-223.
+
+13. Gourgiotis S, Baratsis S. Rectal prolapse. *Int J Colorectal Dis*. 
+
+2007;22(3):231-243. doi:10.1007/s00384-006-0198-2 14. Varma M, Rafferty J, Buie WD, Standards Practice Task Force of American Society of C, Rectal S. Practice parameters for the management of rectal prolapse. *Dis Colon Rectum*. 
+
+2011;54(11):1339-1346. doi:10.1097/DCR.0b013e3182310f75 15. Bordeianou L, Hicks CW, Kaiser AM, Alavi K, Sudan R, Wise PE. Rectal prolapse: an overview of clinical features, diagnosis, and patient-specific management strategies. *J Gastrointest Surg*. 
+
+2014;18(5):1059-1069. doi:10.1007/s11605-013-2427-7 How to cite this article: Penton AA, Jochum SB, 
+Eberhardt JM. Unusual presentation of colon cancer as rectal prolapse in middle-aged male. Clin Case Rep. 2023;11:e6908. doi:10.1002/ccr3.6908