Drug repurposing, the practice of utilizing existing drugs for novel clinical indications, has tremendous potential for improving human health outcomes and increasing therapeutic development efficiency. The goal of multi-disease multitarget drug repurposing, also known as shotgun drug repurposing, is to develop platforms that assess the therapeutic potential of each existing drug for every clinical indication. Our Computational Analysis of Novel Drug Opportunities (CANDO) platform for shotgun multitarget repurposing implements several pipelines for the large-scale modeling and simulation of interactions between comprehensive libraries of drugs/compounds and protein structures. In these pipelines, each drug is described by an interaction signature that is compared to all other signatures that are subsequently sorted and ranked based on similarity. Pipelines within the platform are benchmarked based on their ability to recover known drugs for all indications in our library, and predictions are generated based on the hypothesis that (novel) drugs with similar signatures may be repurposed for the same indication(s). The drug-protein interactions used to create the drug-proteome signatures may be determined by any screening or docking method, but the primary approach used thus far has been BANDOCK, our in-house bioanalytical or similarity docking protocol. In this study, we calculated drug-proteome interaction signatures using the publicly available molecular docking method Autodock Vina and created hybrid decision tree pipelines that combined our original bio- and chem-informatic approach with the goal of assessing and benchmarking their drug repurposing capabilities and performance. The hybrid decision tree pipeline outperformed the two docking-based pipelines from which it was synthesized, yielding an average indication accuracy of 13.3% at the top10 cutoff (the most stringent), relative to 10.9% and 7.1% for its constituent pipelines, and a random control accuracy of 2.2%. We demonstrate that docking-based virtual screening pipelines have unique performance characteristics and that the CANDO shotgun repurposing paradigm is not dependent on a specific docking method. Our results also provide further evidence that multiple CANDO pipelines can be synthesized to enhance drug repurposing predictive capability relative to their constituent pipelines. Overall, this study indicates that pipelines consisting of varied docking-based signature generation methods can capture unique and useful signals for accurate comparison of drug-proteome interaction signatures, leading to improvements in the benchmarking and predictive performance of the CANDO shotgun drug repurposing platform. Pharmacological innovation reduces human mortality rates and provides substantial improvements to the quality of life . TherapeDrug discovery traditionally relies on screening a compound or set of compounds against a biological target, typically a protein for a specific indication. Generally, these approaches incorporate high-throughput in vitro compound screens  and/or cDrug repurposing is the practice of finding new uses for existing drugs, taking advantage of prior safety, efficacy, and pharmacological knowledge and data . Drug reComputational models that improve drug discovery and repurposing leverage rapidly increasing computer processing power and vast collections of preclinical  and clinical data . AlthougMolecular docking models the three-dimensional (3D) interaction between small molecule compounds and macromolecular protein structures ,30,31,32The Computational Analysis of Novel Drug Opportunities (CANDO) platform was developed to mitigate endemic problems in drug discovery and enable multitarget approaches to drug repurposing ,43,44,45In this study, we set out to extend the CANDO platform with an additional molecular docking pipeline using the popular software AutoDock Vina  to deterThe performance of two new primary pipelines and a hybrid one in the CANDO platform was investigated and compared to those previously created, including a random control (see The second pipeline used was the default CANDO v1.5 pipeline restricted to the same 134 protein subset (v1.5-134). We generated a hybrid decision tree pipeline drawn from a combination of the Vina-134 and the v1.5-134 pipelines. For comparison, we examined the performance of these pipelines with respect to a random control and the v1.5 pipeline implemented with the full CANDO proteome library consisting of 46,784 protein structures (v1.5-full).The pipelines differed at average indication accuracy thresholds and on a per indication basis. In some cases, a pipeline that performed worse overall may do better for a specific indication. In this investigation, we hypothesized that distinct docking methods would yield distinct drug-proteome interaction signatures due to differing simulation implementation, and correspondingly differing performance for shotgun drug repurposing: BANDOCK, the default bioanalytical or similarity docking in CANDO is a knowledge-based template/comparative modeling protocol ,41,44,45Our results demonstrated that the CANDO platform was not dependent on a single pipeline implementation, and that combining different virtual screening pipelines can yield better performance relative to using the individual ones. On a platform level, the drug-proteome signature ranking and indication recovery paradigm was viable using more than one means of signature generation. On a pipeline level, the pipelines  each demonstrated varying degrees of performance and instances of unique signal capture.The Vina-134 pipeline implemented in this study was viable in that it performed substantially better than the random control and performed at a significant fraction of the performance of the original default pipeline that utilized a bigger protein set. However, small protein libraries have been shown to perform relatively well, and some subsets of protein libraries performed better than others . As prevAlthough some pipelines yielded superior signal over others in specific circumstances, precisely identifying why this occurred warrants further investigation. On a per-indication basis, it is possible to identify the superior performance of one pipeline over another , but theThe default CANDO pipelines were implemented using bio-/chem-informatic docking protocols, where interactions were predicted from curated drug and protein libraries. The specific implementations and evolution of the libraries were reported extensively in several publications ,41,44,45The pipelines used in this study relied on curated sublibraries of the structures of 3733 drugs/compounds and 134 proteins and 13,746 drug-indication mappings, obtained from the same sources as above. We used the sublibraries to rapidly evaluate the utility of multiple molecular docking pipelines.A CANDO virtual screening pipeline simulates the interactions between all of its proteins and drugs/compounds, usually 3D structures, and is not dependent on any particular approach to accomplish this. These simulations generate proteomic similarity signatures (the vector of drug-protein interaction scores). The default CANDO platform pipelines generate drug-proteome interaction signatures using bioinformatic and cheminformatic docking protocols also described elsewhere extensively ,41,44,45Broadly, the CANDO platform works by sorting every drug/compound relative to every other one based on their similarity and then uses known drug-indication associations to assess performance . VariousCombined drug-proteome interaction signatures form an interaction matrix, with drugs along one axis and proteins on the other. These signatures are compared with one another and then ranked on a per-drug basis, and the quality of the resulting ranking is evaluated using the leave-one-out benchmarking protocol described below.Our benchmarking protocol calculates the performance for every indication with at least two approved drugs  at various cutoffs, considering only the the top10 (abbreviated as “top10”), top25, top37 or 1%, top50, top100, top5%, top10%, and top50% of similarly ranked drugs. For each indication, the accuracy was derived from calculating how many known drugs mapped to that indication were “recovered” and highly ranked at various cutoffs.c is a count of the number of times at least one approved drug for the indication was recovered within a particular cutoff and d is the total number of drugs approved for that indication. The other two benchmarking metrics were pairwise accuracy  and coverage (number of indications that have an accuracy greater than zero).We utilized three metrics to benchmark pipeline performance: average indication accuracy, pairwise accuracy, and coverage ,41,44,45The pipelines examined in this study were derived from similarity ranking and benchmarking drug-proteome interaction signatures generated by large-scale bioinformatic and molecular docking. The CANDO platform is not limited to using docking-based virtual screening pipelines and has the potential to incorporate many different approaches to pipeline implementation and data sets .We used a small sublibrary (134 proteins) of the full CANDO proteome library to create the new molecular docking virtual screening pipeline due to computational constraints and also because we previously have shown that appropriately selected sublibraries of a similar size from the full library yield similar or better benchmarking performance . We usedPrior CANDO platform investigations have demonstrated that multiple pipelines can be combined into a hybrid decision tree to maximize indication accuracy by drawing from pipelines that produce the best performance on a per-indication basis . We usedWe also compared the benchmarking performance of the pipelines to values obtained using a hypergeometric distribution that estimates the numerical probability of making a correct prediction by chance. This is one of the random control reference benchmarks used in the CANDO platform, the implementation of which was covered in detail in prior publications ,45. BencOur results indicated that the utilization of multiple diverse docking-based virtual screening approaches in drug repurposing contexts such as the CANDO platform improves benchmarking performance. The Vina-134 pipeline performance indicated that the CANDO platform hypothesis of drug behavior similarity is not limited to the original bionalytical or similarity docking protocol BANDOCK for interaction signature generation. The hybrid decision tree pipeline performance provided further evidence that multiple signature generation pipelines may be combined to yield improved performance. Ongoing and future platform enhancement will incorporate multiple signature generation protocols and pipeline synthesis using AI/machine learning approaches to optimize performance. These improvements in turn will lead to greater predictive power and higher confidence in novel drug candidates generated for specific indications, which will be verified via prospective preclinical and clinical studies.


HIV assays designed to detect recent infection, also known as “recency assays,” are often used to estimate HIV incidence in a specific country, region, or subpopulation, alone or as part of recent infection testing algorithms (RITAs). Recently, many countries and organizations have become interested in using recency assays within case surveillance systems and routine HIV testing services to measure other indicators beyond incidence, generally referred to as “non-incidence surveillance use cases.”This review aims to identify published evidence that can be used to validate methodological approaches to recency-based incidence estimation and non-incidence use cases. The evidence identified through this review will be used in the forthcoming technical guidance by the World Health Organization (WHO) and United Nations Programme on HIV/AIDS (UNAIDS) on the use of HIV recency assays for identification of epidemic trends, whether for HIV incidence estimation or non-incidence indicators of recency.To identify the best methodological and field implementation practices for the use of recency assays to estimate HIV incidence and trends in recent infections for specific populations or geographic areas, we conducted a systematic review of the literature to (1) understand the use of recency testing for surveillance in programmatic and laboratory settings, (2) review methodologies for implementing recency testing for both incidence estimation and non-incidence use cases, and (3) assess the field performance characteristics of commercially available recency assays.Among the 167 documents included in the final review, 91 (54.5%) focused on assay or algorithm performance or methodological descriptions, with high-quality evidence of accurate age- and sex-disaggregated HIV incidence estimation at national or regional levels in general population settings, but not at finer geographic levels for prevention prioritization. The remaining 76 (45.5%) described the field use of incidence assays including field-derived incidence (n=45), non-incidence (n=25), and both incidence and non-incidence use cases (n=6). The field use of incidence assays included integrating RITAs into routine surveillance and assisting with molecular genetic analyses, but evidence was generally weaker or only reported on what was done, without validation data or findings related to effectiveness of using non-incidence indicators calculated through the use of recency assays as a proxy for HIV incidence.HIV recency assays have been widely validated for estimating HIV incidence in age- and sex-specific populations at national and subnational regional levels; however, there is a lack of evidence validating the accuracy and effectiveness of using recency assays to identify epidemic trends in non-incidence surveillance use cases. More research is needed to validate the use of recency assays within HIV testing services, to ensure findings can be accurately interpreted to guide prioritization of public health programming. There are many reasons to identify recently acquired HIV infections on a population level, including to 1) better understand current transmission of HIV in a country, region, or population subgroup; (2) evaluate whether specific prevention interventions are having the desired impact; and (3) focus limited resources for prevention or treatment services on groups of people or geographic locations with the greatest potential benefit  [ better uP = I × D  [Recency assays discriminate recent from longstanding infection in an individual using 1 or more biomarkers, typically using an understanding of the typical patterns of immune response maturation following initial infection . Individfection) . Howeverfection) .To date, no recency assay has fully met the target product profile for HIV incidence estimation as set out by the Foundation for Innovative Diagnostics (FIND) and the World Health Organization (WHO) in 2016 . NumerouSince the release in 2011 of technical guidance on the use of recency assays to estimate population-level HIV incidence from the WHO and Joint United Nations Programme on HIV/AIDS (UNAIDS) ,15-18. NTo our knowledge, no previous systematic review has been completed of literature related to the use of HIV recency assays for surveillance purposes. We endeavored to identify published evidence that could be used to validate methodological approaches to HIV incidence estimation and other measures of recency of HIV infection using recency assays. Findings from this systematic review were designed to inform a revised technical guidance on the use of HIV recency assays for identification of epidemic trends, whether for HIV incidence estimation or for other non-incidence indicators of recency, to be released by the WHO and UNAIDS in 2022. This guidance is intended to help raise global awareness of benefits and pitfalls of the use of these assays for surveillance purposes, and set clear standards for their appropriate use.Our systematic review had 3 primary objectives:Understand the use of recency testing in surveillance and programmatic and laboratory settings (to provide incidence estimates or for non-incidence surveillance use cases);Review methodologies for implementing recency testing in population surveys, case surveillance systems, and routine monitoring and evaluation activities; andHighlight use cases that have employed a recency assay or recent infection testing algorithm (RITA) within specific populations, with special attention to variations in assays, settings, and methods of analysis for calculating HIV incidence estimates or employing recency assays for non-incidence surveillance use cases. Within this category, one of our specific goals was to identify evidence that not only presents results of “proportion testing recent” or similar, but also reviews the methodological choice to use a simple proportion of recency or assess “factors associated with testing recent” as a proxy for HIV incidence or other indicators of ongoing HIV transmission within case surveillance systems.and needed to address validation of method. As we were not looking to perform a meta-analysis of assay performance  but rather review the evidence regarding validity of various methodologies for the use of these assays for surveillance purposes, simply reporting the use of a recency assay with a specific MDRI and FRR was insufficient for inclusion. Rather, to be included articles needed to compare findings with those of another standard assay, or describe in detail the methodological choices made and rationale for doing so. They also needed to use commercially available assays/methodologies used to determine recency of infection  nfection , as the Strategy 2 involved looking for articles about surveillance and programmatic utilization of recency testing. To be eligible for inclusion, articles needed to describe some aspect of population-level utility , using commercially available recency assays/methodologies  to determine recency of HIV infection. Studies could present either qualitative or quantitative data and could be descriptive studies lacking a comparator, as long as studies clearly presented outcomes specific to HIV recency testing.The literature search for the systematic review was conducted in PubMed and Web of Science, and included literature published in any language and in any indexed journal including preprint servers without peer review, from January 1, 2010, to November 11, 2021, by searching title, abstract, and MeSH terms/author keywords.For the Strategy 1 search, search terms included HIV, recency assay, incidence assay, test for recent infection (TRI), recent infection testing algorithm (RITA), multiassay algorithm, performance, false recent rate/ratio (FRR), proportion false recent, and mean duration of recent infection (MDRI). For the Strategy 2 search, search terms included recent infection/acute infection, recent infection testing algorithm, multiassay algorithm, incidence estimates, case surveillance, hotspot identification, hotspot mapping, cluster detection, procedures and protocols, and HIV. See Given that much of the research output in the field of HIV recency assay utilization is published in formal reports or presented in conference abstracts, we extended the search beyond traditional literature databases to include “gray literature,” that is, literature that is not formally published in peer-reviewed journals or books. We conducted a search of the gray literature through internet search engines and through websites of major international funders, subject matter conferences, and organizations involved with HIV surveillance  employinWe used a step-wise approach during the screening and reviewing process. After search and duplicate removal, SF screened titles and abstracts to identify papers potentially related to the focus areas and eligibility criteria. After screening was complete, full text of remaining articles was then independently reviewed by DF and SS to determine if the study met eligibility criteria; SF served as a tiebreaker for any articles for which the 2 preliminary screeners were not in agreement about inclusion. Once the full-text review was complete, SF hand-searched the references of all included articles for additional, potentially eligible articles. DF and SS then reviewed these articles and determined eligibility according to the process outlined above.Prior to conducting our search, we developed a formal protocol and circulated it among stakeholders at the WHO and UNAIDS for approval; we have made the protocol available in unmodified form as The literature included in the systematic review was rated by strength of published evidence using a 23-point rubric that we designed custom for this purpose . For eacThe search was conducted on November 11, 2021, and resulted in 180 records identified via MEDLINE (PubMed) and 193 records identified via Web of Science. Of these, 104 were duplicates, which were removed. An additional 27 records were identified through an internet search of gray literature and 15 records were identified through a hand search of the references in previously identified records.After deduplication, the remaining 311 documents from the search were initially scanned by SF for eligibility. This initial “quick screen” excluded 94 articles that very clearly did not meet inclusion criteria for the review, or did not contain sufficient detail on methods to have utility in the review. The remaining 217 documents were then subjected to a full-text review, which was conducted independently by both DF and SS. After excluding 50 full-text articles that did not meet our predefined inclusion criteria, a total of 167 studies, reports, or presentations were retained across both focus areas  and wereAmong the 167 pieces of evidence that were identified through the systematic review and that met the inclusion criteria, 91 (54.5%) ,30-100 fnational surveillance in the form of population-based surveys (including 10 from the US-supported Population-based HIV Impact Assessment (PHIA) surveys). Another 12 (24%) [impact assessment use cases, because they are intended for repeat implementation to assess changes in incidence over time as a result of HIV prevention or care interventions. There were 3 more studies that also used recency assays to estimate incidence for intervention impact assessment, but in the more narrow context of blood donor policy implementation [key or sentinel populations, including those accessing routine HIV testing or blood donation programs. Key or sentinel population surveillance involves testing within populations that are either of specific interest because they are at higher risk for infection (key) or considered to be representative of a larger population (sentinel). Sentinel and key population surveillance may be facility based or community based. For example, needle and syringe distribution programs are a good point of contact with people who inject drugs, sexual health clinics may provide access to men who have sex with men and sex workers, and antenatal clinics are used to sample pregnant women. All evidence in this category was of “very strong” (5/20) or “strong” (15/20) quality  -116 desc12 (24%) -128 wereentation  or behaventation ,131. Theentation -151 desc quality .targeted prevention planning. A total of 6 used recency testing as part of cluster identification or analysis  [Among the 31 articles describing non-incidence use cases, 24 used recency testing to assess risk factors predicting recent infection ,152-170 recency) ,167,171,recency) ,173, andrecency)  and evalrecency) ,174-176.recency) . The quaThere were 51 documents included in this review that provided methods and findings related to the field use of recency assays for HIV incidence estimation. As detailed above, 32 studies in this review used recency assays to estimate incidence for surveillance of subnational regions or key or sentinel populations; however, these strategies have also been used extensively at a national level. In 2015 the UNAIDS and WHO released guidelines on monitoring the impact of the HIV epidemic using population-based surveys, including using recency assays for estimation of incidence . Since tIn addition to the 10 PHIA studies, another 8 studies ,145,147 One of our objectives in the review was to identify evidence that not only presents results of “proportion testing recent” or similar, but also reviews the methodological choice to use a simple proportion of recency or assess “factors associated with testing recent” as a proxy for HIV incidence or another indicator of ongoing HIV transmission within case surveillance systems. Although there were 31 documents identified across the 11-year review period that were reporting on the use of recency assays for non-incidence use cases, all of those papers reported their estimates of non-incidence recency indicators (such as “proportion recent”) without attention to whether these indicators were valid proxies of ongoing HIV transmission. As many as 19 of these studies used a recency assay as part of a RITA  to help reduce misclassification rates ,172-176.Of the 91 studies devoted to assays, algorithms, or methods of incidence estimation, 59 evaluated the performance of 1 or more assays. Among these, 46 (78%) evaluated avidity assays , 23 (39%) evaluated BED assays , 4 (7%) evaluated rapid assays , and 3 (5%) evaluated comparative antigen reactivity assays . These studies reported various aspects of assay performance, including FRR (38/59), MDRI (31/59), sensitivity and specificity (10/59), and correlation of results between different assays (12/59). In addition, 20 of the studies explored a range of assay cut-off thresholds, to identify a cut-off that would achieve optimal FRR and MDRI results. Details of which studies are in which categories can be found in While 16 of the 59 studies utilized standard sample panels from the Consortium for the Evaluation and Performance of HIV Incidence Assays (CEPHIA) or other sources, the majority evaluated assays against patients from 1 or more geographic regions, including Africa (20/59), North America , western Europe (11/59), east Asia (8/59), the Caribbean , eastern Europe , and south Asia  and changes in testing coverage and frequency of diagnostic testing in the population. An unexpectedly high or rising proportion of new diagnoses being classified as recent infections may indicate either (1) ongoing transmission or (2) that the testing program is capturing more recent infections because most older infections have already been diagnosed. More evidence about the appropriate interpretation and use of these types of indicators is necessary.There were 24 analyses included in this review that assessed predictors or correlates of recent infection. Implied in these analyses is an assumption that subgroups with significantly greater odds of recent infection are currently experiencing More reports of countries or studies using HIV recency assays for identification and mapping of geographic hotspots will likely emerge as a result of the US President’s Emergency Plan for AIDS Relief (PEPFAR) “TRACE” initiative (Tracking with Recency Assays to Control the Epidemic) in the near future. Beginning in fiscal year 2019, PEPFAR funded 16 countries  who are nearing the 90-90-90 targets to introduce the TRACE initiative . ThroughThere are several limitations to our systematic review. First, given our search strategy many of the articles included in this review involved findings relevant to the performance of specific commercially available recency assays. However, some of those assays  are technically available but no longer in wide use, due to inferior performance for HIV incidence estimation compared with other available assays. Further, some assays included in this review are not available in all countries globally. Second, as with all systematic reviews, our review was time limited. Therefore, it is possible that some relevant literature that has been recently published or that was missed by our choice of search terms in the prespecified protocol is not included in this review.Surveillance strategies to accurately estimate HIV incidence or detect patterns of recent transmission are critical to global efforts to end the HIV epidemic. However, these calculations are only useful if they are timely and accurate, with potential biases clearly defined. Calculations that are considerably higher or lower than reality may result in incorrect interpretations of the data, and misalignment of resources as a result. This review found ample evidence to guide the use of recency assays in population-based surveys to accurately estimate HIV incidence. However, more research is needed to validate their use within HIV testing services and to explore best practices for calculating HIV recency indicators other than incidence to ensure that findings from recency testing can be accurately interpreted to guide prioritization of public health programming.


Endothelial cells (ECs) lining the cardiovascular system are subjected to a highly dynamic microenvironment resulting from pulsatile pressure and circulating blood flow. Endothelial cells are remarkably sensitive to these forces, which are transduced to activate signaling pathways to maintain endothelial homeostasis and respond to changes in the environment. Aberrations in these biomechanical stresses, however, can trigger changes in endothelial cell phenotype and function. One process involved in this cellular plasticity is endothelial-to-mesenchymal transition (EndMT). As a result of EndMT, ECs lose cell-cell adhesion, alter their cytoskeletal organization, and gain increased migratory and invasive capabilities. EndMT has long been known to occur during cardiovascular development, but there is now a growing body of evidence also implicating it in many cardiovascular diseases (CVD), often associated with alterations in the cellular mechanical environment. In this review, we highlight the emerging role of shear stress, cyclic strain, matrix stiffness, and composition associated with EndMT in CVD. We first provide an overview of EndMT and context for how ECs sense, transduce, and respond to certain mechanical stimuli. We then describe the biomechanical features of EndMT and the role of mechanically driven EndMT in CVD. Finally, we indicate areas of open investigation to further elucidate the complexity of EndMT in the cardiovascular system. Understanding the mechanistic underpinnings of the mechanobiology of EndMT in CVD can provide insight into new opportunities for identification of novel diagnostic markers and therapeutic interventions. In terms of cyclic strain, the physiologic range is 5 to 10% and strain above 20% is pathological and often observed in hypertension  comprise a highly heterogeneous population of cells that line the vasculature and the endocardium in the cardiovascular system . The micrtension . In addirtension . Perturbrtension . A notabrtension . Howeverrtension .EndMT was first described in the context of heart development as important for valve formation and heart septation, which is described in detail in other reviews . Many siEndMT pathways can be activated through a variety of biochemical and biomechanical stimuli. Because the TGFβ signaling pathways are the most extensively studied in EndMT, many studies utilize TGFβ isoforms as the main stimulus for EndMT induction . TGFβ isin vitro experiments due to the heterogeneity in EC responses to these stimuli that depend on multiple factors including EC subtype and dose of stimuli. A deeper characterization of the differential properties of these EC populations is needed.EC responses to EndMT-promoting stimuli are highly heterogeneous and are dependent on multiple factors . For exaLike variations in responses to treatment with inflammatory cytokines and growth factors, ECs are also influenced differentially by biomechanical stimuli, such as flow. There are multiple mechanical stresses in the cardiovascular system that are highly variable under physiological and pathological conditions. It has been shown the pattern of expression of adhesion molecules and immune cell adhesion varied not only on which vascular bed the ECs were derived from but also on whether the ECs were exposed to arterial or venous flow profiles . The hetAs discussed in the previous section, EndMT can be induced by various biochemical and biomechanical triggers in the EC microenvironment. Understanding how ECs sense and respond to mechanical forces has been a key focus of vascular biology and is covered extensively in other reviews . Here, wEC mechanosensors for detecting cyclic strain include stretch-sensitive ion channels and G-protein coupled receptors-like Gαq/11 . Of noteEC responses to cyclic stretch, matrix stiffness, and shear stress are also mediated by integrins and vascular endothelial cadherin , and endothelial nitric oxide synthase 3 also indicates the loss of the endothelial cellular phenotype, and the increase in expression of markers, such as α-smooth muscle actin (α-SMA), calponin, transgelin (SM22α), vimentin, and versican, denotes the transition to a mesenchymal state .2), a potent inducer of EndMT, revealed that endothelial marker genes NOS3, VWF, and CD34 were downregulated as early as 6h after exposure to OSS, whereas mesenchymal marker genes CDH2, TPM1, and FBLN5 began to be upregulated around 12h (2) for 24h, there was no significant effect on the expression of endothelial markers vWF, CD31, Tie2, vascular endothelial growth factor receptor 2, and VE-Cadherin, whereas exposure to pulsatile shear stress, which is protective against EndMT, upregulated the expression of these specific markers  a mesenchymal pattern facilitated by matrix metalloproteinases (MMPs), which promotes degradation of the ECM and (2) an amoeboid pattern mediated by Rho/ROCK cytoskeletal contraction . When TGin vivo corresponded with decreased collagen deposition and cardiac fibrosis after MI , MMP14 expressed in macrophages induced their release of TGFβ1, which subsequently resulted in activation of SMAD2-mediated EndMT pathways in nearby ECs through paracrine signaling. MMP14 silencing after MI . Furtherin vitro. It is hypothesized that this transient EndMT allows for ECs to migrate and participate in vascularization of ischemic areas. This partial activation has been termed as endothelial-to-mesenchymal activation and opens new avenues of inquiry into how EndMT may not only be a mechanism of maladaptive remodeling following ischemic injury but could also positively influence the healing response through revascularization of ischemic tissue.The phenotypic alterations associated with EndMT appear to exist on a continuum . In an iThe sequence and progression of EndMT are still not yet well understood. In a pathological context of pulmonary arterial hypertension (PAH), cell lineage tracing allowed for the identification of cells in both partial and complete EndMT states with distinct patterns of marker expression and functional properties . The celin vitro studies demonstrated that SOX7 mutations can lead to impaired EndMT via regulation of VE-Cadherin, indicating that the inhibition of EndMT during development may contribute to CTD . Abnormalities in OFT development can result in congenital heart diseases (CHD), such as conotruncal defects (CTD). In patients with CTD, SOX7 mutations have been identified and additional e to CTD .There are important mechanical forces that regulate EndMT in normal OFT development . AberratIn addition to contributing to abnormalities of valve development associated with CHD, EndMT is also involved in other pathological settings of the valves. The following sections describe the role of EndMT in CAVD and ischemic mitral regurgitation (IMR) and how various types of biomechanical alterations can regulate EndMT of valvular ECs.Calcific aortic valve disease is the third most prevalent cause of CVD . The patvia EndMT and osteogenic differentiation when compared to hydrogels seeded with VICs only  and valvular interstitial cells (VICs). Interactions between these cells contribute to the formation of calcific lesions . IndirecICs only . FurtherICs only .While these studies highlight how different cell types interact in the context of EndMT in CAVD, the lineages of the cell types present in calcific lesions in CAVD was not explored. However, a recent single-cell RNA sequencing study detailing a transcriptomic atlas of human aortic valves from both healthy and CAVD patient samples not only demonstrated remarkable cellular heterogeneity in the aortic valve leaflets but also provided more direct evidence of EndMT as the process that leads to the differentiation of resident VICs and VECs into newly identified valve-derived stromal cells that are prominent in only the CAVD samples . The pseWhile single-cell RNA sequencing allowed for the identification of distinct subpopulations of cells and suggested that EndMT contributes to this cellular heterogeneity in human CAVD, it did not provide mechanistic insight into what initiates and promotes EndMT of the resident VECs and differentiation of VICs. This is not yet well understood, but there are studies that have characterized the effect of varying cyclic strain, shear stress, matrix stiffness, cell-cell, and cell-matrix interactions on VECs, VICs, and EndMT . These svia increased Wnt/β-catenin signaling  and OSS demonstrated increased expression of EndMT-related markers Snail and α-SMA, TGFβ1 along with inflammation-related markers ICAM1 and NF-κB1, when compared to cells exposed to high SSS (10 and 20dyne/cm2) or static conditions  is a prevalent cause of valvular disease and is a consequence of leaflet tethering following papillary displacement due to left ventricle dilation after MI . Tetheriin vitro when addition of TGFβ to mitral VECs induced increased expression of CD45 and α-SMA , HL81397 (KB), HL151391 (YT), HL137647 (YT), and HL139675 (YY)], NIH/NIA [grant number AG061586 (YT)], NIH/NINDS [grant number NS79353 (YY)], the Presidential Early Career Award for Scientists and Engineers [grant number N00014-16-1-2997 (DD)], and the American Heart Association [Student Scholarship in Cardiovascular Disease (SI) and Howard S. Silverman Scholarship (SI)].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The plant circadian clock has a pervasive influence on many aspects of plant biology and is proposed to function as a developmental manager. To do so, the circadian oscillator needs to be able to integrate a multiplicity of environmental signals and coordinate an extensive and diverse repertoire of endogenous rhythms accordingly. Recent studies on tissue-specific characteristics and spatial structure of the plant circadian clock suggest that such plasticity may be achieved through the function of distinct oscillators, which sense the environment locally and are then coordinated across the plant through both intercellular coupling and long-distance communication. This review summarizes the current knowledge on tissue-specific features of the clock in plants and their spatial organization and synchronization at the organismal level. A circadian clock is an endogenous molecular mechanism that generates 24 h rhythms in a wide array of biological processes. As a consequence of the Earth’s rotation, organisms have evolved these timing mechanisms to align their physiology and development with the periodic changes in environmental conditions that occur over the day-night cycle. In the natural environment, the ability to track time enables organisms to anticipate these conditions and adequately coordinate different processes to occur at the most appropriate times. The anticipatory behavior conferred by these biological oscillators thus allows for an efficient distribution and use of metabolic resources and is thought to provide an adaptive advantage . In factCCA1 and LHY, as well as each other [GIGANTEA (GI), LUX ARRYTHMO (LUX) and EARLY FLOWERING 4 (ELF4) [GI and represses PRR9, PRR7 and LUX and likely indirectly induces CCA1 and LHY expression [Almost every aspect of plant physiology and development is subject to some extent of circadian regulation and many efforts have been devoted towards the identification of the genes and proteins that constitute the core molecular mechanism driving this pervasive rhythmicity. As a result, multiple clock components and intricate reciprocal regulatory connections have been identified ,11,12 F. Similarch other ,19,20. Ipression .It is assumed that essentially every plant cell contains an autonomous circadian oscillator. However, different parts of the plant are in very different environments, perceive different environmental cues and are relevant to different biological processes. This heterogeneity translates in a variability in the individual cellular circadian rhythms, which need to be adequately integrated and coordinated both locally and across the plant to orchestrate organismal responses  levels also free-run with a different period than the expression of a light-harvesting complex (Lhc)b gene family member [2+ oscillations, in different tissues [2+ levels) driven by different promoters with distinct spatial patterns of expression were generated. Under free-running conditions, circadian oscillations in Ca2+ exhibited distinct phases in each line. While these findings do not necessarily imply the function of separate oscillators, they evidence the existence of distinct cellular control mechanisms contributing to circadian rhythms in Ca2+ levels [The existence of multiple oscillators in plants has long been proposed. Early studies in bean plants evidenced that the free-running period of stomatal opening and photosynthesis was different to that of leaflet movement  and study member . A singl tissues . For thi+ levels .2+ levels, as well as in CHALCONE SYNTHASE (CHS), CHLOROPHYLL A/B BINDING PROTEIN (CAB) and PHYTOCHROME B (PHYB) promoter activity, which owing to their distinct spatial distribution patterns and free-running periods were suggested to be regulated by separate oscillators located in different cell types [TOC1 and the light signaling component DE-ETIOLATED 1, DET1) similarly affected the period of CHS and CAB promoter activity [PHYB and the core clock genes CCA1, LHY and ELF3 also affected the period of both CAB and PHYB promoter activity in a similar fashion, hence suggesting that the separate oscillators share common components [2+ levels and CAB promoter activity were both dependent on CCA1, LHY and TOC1 function [TOC1 (toc1-1), which contains an amino acid change in the conserved CCT  domain, uncoupled both rhythms and only affected CAB oscillations [To inspect whether the different oscillators were composed of similar components or not, the effect of mutations in the central oscillator and light input pathway genes on presumably independent rhythms was analyzed. These rhythms comprised oscillations in cytosolic Call types ,28,29. Gactivity . LikewisLocal differences in circadian rhythmicity from a similar oscillator can be achieved through various mechanisms including diverging levels in core clock gene expression, functional modulation of these clock genes by tissue-specific regulators and/or through differential perception of input signals.CCA1 promoter activity under free-running conditions in the center of the leaf with that in the center of the rosette of Arabidopsis thaliana plants revealed differences in period length depending on the organ [CCA1 was also reported to display lower expression levels and a longer free-running period in guard cells compared to whole leaves [LHY, TOC1 and CCA1 HIKING EXPEDITION (CHE) [GI, also had a later phase and run with a longer period in guard cells, but displayed similar expression levels in this cell type compared to whole leaves [GI seems to be more highly expressed in the vasculature [PRR3, which is suggested to regulate TOC1 protein stability in this tissue [CCA1, LHY, PRR9 and PRR7 are more highly expressed in mesophyll, while expression of evening phased genes such as TOC1, ELF4 and LUX is higher in the vasculature [The majority of clock genes are rhythmically expressed across the entire plant ,31,32,33he organ . CCA1 wae leaves . A similON (CHE) . Interese leaves . Hence, culature , similars tissue . It was culature . Differeculature . Interesculature ,40,41,42Differential processing of environmental signals is another factor that may contribute to tissue-specific circadian regulation. Because different parts of the plant are exposed to different microenvironments, it is anticipated that the impact of specific environmental cues, such as light quality and quantity, temperature or nutrient levels, will differ. Local differences in the clock’s sensitivity to a wide array of signals would enhance plasticity and could allow the clock to better adapt to ambient conditions locally ,43.PHY and CRY genes varies among tissues [Perhaps the most important entraining signal in plants is light. Plants use different classes of photoreceptors to sense the light environment and set the clock to the actual pace of day-night cycles . These p tissues ,48,49. T tissues ,50, in a tissues . Recent  tissues . Additio tissues ,52 and C tissues , as well tissues , SPA1 [5 tissues  and PIFs tissues ,57,58, hCAB2 and CATALASE 3 (CAT3) expression had different sensitivity to light and temperature. The pacemaker regulating CAB2 gene expression seemed to preferentially respond to light–dark cycles, while the one controlling CAT3 expression, was more sensitive to temperature signals [TOC1 promoter activity oscillations in the vasculature compared to whole leaves under light-dark and temperature cycles, it was seen that the vascular clock has lower sensitivity to temperature and higher sensitivity to photoperiodic signals [PRR9 and PRR7 function [In addition to light, temperature is another signal that conveys important information about the surrounding environment. Early studies showed that two separate oscillators involved in the regulation of  signals . More re signals . In fact signals ,61. Convfunction ,40.Altogether, several pieces of evidence suggest that local differences in circadian function may arise from a combination of factors, including heterogeneity in the expression levels of core clock components, tissue-specific connections within the circadian network and differential sensitivity and processing of environmental input signals.As outlined above, the overall circadian system in plants seems to be spatially organized with the clocks in different tissues displaying distinct circadian properties (such as period and phase) and responding differently to environmental cues. These observations necessarily raise the question of how these clocks communicate with each other and how they are coordinated to achieve optimal regulation of biological rhythms at the organismal level. Although initially proposed to be functionally independent , severalIn terms of organization, analysis of the regulatory relationships between the clocks in two different tissues, mesophyll and vasculature, revealed the existence of a certain hierarchy among tissue-specific clocks. Perturbation of circadian rhythms in the vasculature (through overexpression of CCA1 in this tissue) affected the rhythmicity of both the vasculature and mesophyll clocks, while disruption of the mesophyll clock only affected rhythms in this cell-type . These fMathematical modelling together with experimental evidence suggest that intrinsic differences in period length together with cell-to-cell coupling may be sufficient to generate these waves in gene expression and coordinate the circadian system across the plant ,67. HencIt is becoming evident that the circadian system in plants is spatially structured and is composed by a complex web of distinct local clocks that communicate with each other and are coordinated across tissues and organs . As oppoAt least two points of coordination have been proposed, one in the shoot apex and one in the root tip ,67. Whilcis elements in the promoters of clock regulated genes have been identified [Distinct circadian properties of local oscillators may additionally arise from differences in the wiring and function of clock components, for example through the existence of tissue-specific regulators. In fact, tissue-specific functions of the clock in the regulation of output pathways such as cell elongation and photoperiodic flowering have been reported ,61 and tentified . Currententified , these sConsidering the complexity of natural environments, it is likely that an array of signals and mechanisms converge for the regulation of rhythms across the plant. In addition to the signals mentioned in the previous section, several other signaling molecules may be involved in clock synchronization, including ions , metabol


Transformation of astrocytes into reactive states is considered one of the major pathological hallmarks of prion and other neurodegenerative diseases. Recent years witnessed a growing appreciation of the view that reactive astrocytes are intimately involved in chronic neurodegeneration; however, little is known about their role in disease pathogenesis. The current article reviews the progress of the last few years and critically discusses controversial questions of whether reactive astrocytes associated with prion diseases are neurotoxic or neuroprotective and whether bidirectional A1–A2 model is applicable for describing polarization of astrocytes. Moreover, other important topics, including reversibility of a transition to a reactive state, along with the role of microglia and other stimuli in triggering astrocyte activation are reviewed. Defining the role of reactive astrocytes in pathogenesis of neurodegenerative diseases will open unrealized opportunities for developing new therapeutic approaches against prion and other neurodegenerative diseases. Astrocytes are responsible for a number of homoeostatic functions required for proper functioning of CNS . Under cAccording to the hypothesis introduced by Barres and co-authors, astrocytes can polarize into well-defined neurotoxic (A1) or neuroprotective (A2) reactive states, which exhibit distinct transcriptional profile and opposing effects on neuronal survival ,12. The Sc strains that target astrocytes often have shorter incubation times to the disease in comparison with the strains that colocalize with neurons [Do reactive astrocytes contribute to diseases pathogenesis? Considering that a mixture of A1- and A2-reactive markers were upregulated in prion diseases, while neither markers being liked to homoeostatic or toxic functions of astrocytes, the bidirectional A1-A2 concept does not provide a cue whether reactive astrocytes are expected to produce neuroprotective or neurotoxic effects. PrP neurons ,27. More neurons . These rSc and cell debris or, in opposite, spread of PrPSc, elimination of synapses along with viable neurons, loss of homoeostatic functions, gain of toxic functions, inflammatory signalling that stimulates microglia-mediated synapse elimination, recruitment of immune cells and others mechanisms, should be examined to determine the net impact of reactive astrocytes  along with the secretion of IL-6, suggesting that the reactive phenotype is relatively stable and can be maintained in vitro in the absence of pro-inflammatory CNS environment [Because microglia and astrocytes often become reactive in parallel and both contribute to neuroinflammation, it is difficult to pinpoint the contribution of each cell type. According to the Barres hypothesis that gained popularity in neurodegeneration field, reactive microglia drives the polarization of astrocytes into neurotoxic A1 state via the upregulation of three secreted factors TNF-α, IL-1α and C1qa . In conf animals . Moreove animals . These s animals . Astrocy animals . Furtherigration . Reactivironment . IL-33 sironment , while eironment . In mousironment . To summIs presence of a stimulus that triggers reactive states important for maintaining the reactive phenotype? Studies of optic nerve subjected to mild injury introduced by a brief ocular pressure showed that astrocyte reactivity could be fully resolved if the insult is removed . In moreIs it possible to reverse reactive states in the presence of persistent stimulus? Activation of STAT3 transcription factor was identified as universal feature of astrocyte reactivity in neurodegenerative diseases shared between different species, brain regions and different types of illnesses , which in neurons was found to be involved in endocytosis of PrPSc [Sc phagocytosis, including toll-like receptors (TLRs), which are involved in the recognition of pathogens and extracellular protein aggregates; Axl receptor activates JAK/STAT pathway; a heterogeneous family of scavenger receptors is capable of recognition of danger- and pathogen-associated molecular patterns along with extracellular protein aggregates [Sc by upregulating proinflammatory signalling [Sc [Sc with desialylated glycans [C, PrPSc is sialylated [Sc particles are variable among prion strains and dictated by a strain identity [Sc sialylation levels that accompanied cross-species adaptation of a strain to a new host produced a new strain characterized by a very profound neuroinflammation and the shortest incubation time to the diseases [Sc, the degree of glia activation and the rate of disease progression exists [In prion diseases, astrocytes respond to prion infection prior to clinical symptoms or neuronal damage . In micemulation . Phagocytrocytes ,32 along animals ,27,53 sune genes . It is nof PrPSc . Astrocygregates ; and MEGgregates . Culturegnalling . Notablyling [Sc , with a  glycans . Like noalylated ; howeveridentity . It is ndiseases . These rn exists .With the development of new tools and gaining more knowledge regarding the role of reactive astrocyte in neurodegenerative disease, new questions have to be answered. To what extent the heterogeneity in homoeostatic state dictates reactive state of astrocytes? Do reactive phenotypes drive diseases pathogenesis? Is it possible to fully and selectively reverse reactive states of astrocytes in the presence of persistent proinflammatory stimulus? Does reversing of the reactive states represent an effective therapeutic approach? Answering these question brings a new opportunity for developing unexplored therapeutic approaches.


Recently, we read the valuable article entitled “Low dose Bitter leaf improves sperm quality disrupted in immunosuppressed Wistar rats: An experimental study” with interest that has been published online in International Journal of Reproductive Biomedicine (2020) (1) that evaluated the positive fertility effects of Bitter leaf through biochemical, hematological, and histopathological evidences. The work is incredible, however, among the most important findings here are a few comments that the authors should have considered in their article:1) One of the important factors in analyzing the results of sperm and fertility parameters of the male sex is to measure the sex hormones such as testosterone, luteinizing hormone, and follicle-stimulating hormone of the intervention groups. Changes in the levels of these hormones can be an important indicator in determining the involvement of the treatment agent in the hypothalamic–pituitary–gonadal axis and changes in the level of activity of enzymes involved in the synthesis of testosterone (or dihydrotestosterone). Measuring the levels of these hormones for this study could help with the results .2) First, it was better to use hydroalcoholic extracts because water or alcohol alone can separate single-phase compounds. Many of the hydrophobic compounds do not come out of the plant powder during the preparation of aqueous extract and do not enter the final extract, and vice versa for alcoholic extracts. Second, it would be better to use the liquid chromatography electrospray ionization tandem mass spectrometric technique using M/Z (M-H3) In animal models and humans reactive oxygen species (ROS) by mediating cellular signal transduction pathways play a major role in testicular tissue and spermatogenesis process via lipid peroxidation (loss of membrane integrity), decrease in sperm motility, increase in abnormalities in spermatogenesis, increase in deoxyribonucleic acid damage, induced apoptosis, and finally initiating an ROS-related inflammatory reaction. Extracts with different polyphenolic compounds have the ability to change these active species. Various tissue and serum techniques, including malondialdehyde and nitric oxide levels, total antioxidant capacity, as well as glutathione peroxidase superoxide dismutase and catalase activity, can help with these findings and identify the pathways of ROS-mediated apoptosis pathways damages in testis paranchymal tissue (5).4) Serum and tissue measurements of pro-inflammatory  and anti-inflammatory  cytokines can also be used to evaluate the inflammatory pathways involved in the toxic or protective effect of bitter leaf extract (6).


We describe the rationale and broad study design of the Indian Diabetes and Metabolic Health (InDiMeT) study, a new prospective, observational study incorporating extensive epigenetic (DNA methylation) and lipidomic signatures to examine their association with the dysregulation of adipose de novo lipogenesis (DNL) in South Asian Indians. The InDiMeT study aims to use a case–control design to identify genetic and modifiable-environmental-lifestyle associated determinants of (1) epigenomic (DNA methylome) dysregulation of adipose DNL in type 2 diabetes mellitus (T2DM) adipose tissue, (2) identify correlates of epigenomic (DNA methylome) dysregulation of adipose DNL in peripheral blood mononuclear cells (PBMCs) from T2DM subjects and (3) elucidate plasma lipidomic correlates of adipose DNL in T2DM that can be used as biomarkers of adipose tissue dysfunction.The InDiMeT study will involve recruitment of 176 normoglycaemic and T2DM individuals who will be undergoing laparoscopic surgery for clinical conditions. Extensive phenotyping of the subjects will be conducted and DNA methylome and lipidomic measurements will be made. The adipose DNL pathway genes are likely to be hypermethylated in patients with T2DM with corresponding reduction of gene expression. Correlates of epigenomic (DNA methylome) dysregulation of adipose DNL pathway in PBMCs and their adipose and plasma lipidomic signatures in T2DM subjects could act as early markers of development of T2DM.For the InDiMeT study, ethical approval for addressing the specific aims has been obtained from the Institutional Ethics Committee, St John’s Medical College and Hospital, St John’s National Academy of Health Sciences, Bangalore. Findings from this study will be disseminated through scientific publications in peer-reviewed journals, research conferences and via presentations to stakeholders, patients, clinicians, public and policymakers through appropriate channels. The InDiMeT  study will assess the epigenome (DNA methylome)-lipidome connection in relation to their contribution to adipose de novo lipogenesis (DNL) and metabolic homoeostasis.The prospective case–control study design will permit us to assess the blood epigenetic (DNA methylation) and lipidomic markers that exhibit correlations with adipose DNL dysregulation and can also potentially serve as minimally invasive early biomarkers of dysglycaemia.Understanding the epigenetic (DNA methylation) mechanisms and their associated lipidomic profiles related to dysregulated adipose DNL in South Asian Indians will provide the rationale for effective interventional strategies targeted to this population and discriminative diagnostic/prognostic biomarkers for prevention of type 2 diabetes mellitus (T2DM) in this high-risk population.Though the sample size of 176 individuals will be sufficient to detect differences in adipose DNA methylation between T2DM and normoglycaemic control subjects, the primary specific aim of this study, it is unlikely to provide required sensitivity to detect dysregulation of adipose DNA methylation in specific subgroups of T2DM, such as smokers or those with chronic kidney disease.The subjects being recruited are the individuals who come for treatment of their pre-existing clinical conditions and could not be a random representation of the total population.th leading cause of death overall and 4th amongst the non-communicable diseases in 2019.3–5India is experiencing a type 2 diabetes mellitus(T2DM) epidemic, attributed to rapid sociodemographic and nutritional changes, making T2DM the 7et al reported negative correlations between adipose DNL and plasma glucose in humans.Unlike hepatic DNL, adipose DNL is understood to be associated with preservation of insulin sensitivity and an overall favourable metabolic phenotype (‘metabotype’).Understanding the epigenetic (DNA methylation) mechanisms and their associated lipidome profiles related to exacerbated adipose DNL in South Asian Indians will further provide the rationale for effective interventional strategies targeted to this population and discriminative diagnostic/prognostic biomarkers for prevention of T2DM in this high-risk population.The overarching goal of the Indian Diabetes and Metabolic Health (InDiMeT) study is to identify aetiologically valuable determinants that affect susceptibility to type 2 diabetes at individual, subgroup and population level by testing the hypothesis that the epigenetic (DNA methylation) dysregulation of adipose DNL induced by high carbohydrate diet and environmental factors is an important step in the development adipose tissue dysfunction leading to type 2 diabetes  in IndiaThe overall outline of the InDiMeT study is depicted in To identify epigenomic (DNA methylome) dysregulation of adipose DNL in T2DM adipose tissue.To identify correlates of epigenomic (DNA methylome) dysregulation of adipose DNL in PBMCs from T2DM subjects.To elucidate plasma lipidomic correlates of adipose DNL in T2DM that can be used as biomarkers of adipose tissue dysfunction.The InDiMeT study is being conducted at St John’s Medical College and Hospital, a tertiary care hospital in Bangalore district, India. It is surrounded by the state of Tamil Nadu in the southeast, the Bangalore Rural district in the northeast and northwest and the Ramanagara district in the southwest.The InDiMeT study has been designed as a prospective tertiary care hospital-based cohort study. One hundred and seventy-six normoglycaemic and T2DM individuals will be recruited into the study after explanation of details of the study and obtaining informed consent at the department of General Surgery, St John’s Medical College and Hospital, St John’s National Academy of Health Sciences, Bangalore. Dissemination of information about the broader study goals and likely benefits to the community as well as engagement of potential subjects from the hospital was initiated in August 2020. Recruitment of subjects commenced from 19 November 2020, after ethical approval for the study had been obtained from the Institutional Ethics Committee. Recruitment of subjects will continue until the recruitment target  is achieved. Strategies elaborated below are being employed for recruitment:Chart reviews—From the existing group of patients coming in to the Department of General Surgery, St John’s Medical College and Hospital, subjects are screened and then contacted to request for their participation.Databases—From the database of patients coming to the St John’s Medical College and Hospital, those who are eligible to participate in the study are contacted for possible recruitment.Inclusion criteria are 35–60 years age; normoglycaemic and T2DM; normal and high body mass index (BMI); men and women who are undergoing laparoscopic surgeries for their clinical conditions such as cholecystectomies, hernia or splenectomy are invited to participate in the study.Exclusion criteria are age outside the range of 35–60 years, individuals who are unwilling to participate in the study, individuals participating in any other study and those who tested positive for hepatitis (HBsAg: Hepatitis B surface antigen), HIV or syphilis  infections. Those who have serious pre-existing medical condition, those undergoing laparoscopic surgery for clinical conditions other than cholecystectomies, hernia or splenectomy will be excluded, and these will be defined as conditions that require chronic or daily medical therapy, including medications, for T2DM, connective tissue diseases, inflammatory bowel disease, active tuberculosis and symptomatic heart disease.et al the sample size required to observe a mean difference of 7% in DNA methylation in AT with an SD of 10% between T2DM and normoglycaemic controls with 90% power and 5% level of significance is 44 per group. As AT DNA methylation differences could be associated with BMI of the subjects, 44 individuals will be recruited in the normal (BMI <25 kg/m2) and high BMI (BMI ≥25 kg/m2) groups of normoglycaemic controls and T2DM .10Based on the study by Nilsson Findings of this study could be influenced by selection bias in recruitment of the study subjects. Known sources of selection bias, such as geographical location of the study, which influences wide ranging subject parameters such as dietary diversity, levels of sunlight and air pollutant exposures, as well as recruitment of subjects from a hospital, and not from the community, are being addressed by ensuring recruitment of case and control subjects in tandem from a single study site.Detailed socio-demographic data will be collected by trained personnel using well-structured questionnaires as elaborated in Box 1Socio-demographicAge, ethnicity and religion, family composition, education, employment status and type, household income, socioeconomic status.Medical historyMedical history related to birth weight, chronic and infectious diseases, last 1 month’s morbidity conditions, current and past usage of tobacco  and alcohol, family history of diabetes and cardiovascular disease, details on most recent routine clinical investigations .Medication and supplement useType, brand, dose and duration of medication, vitamin and mineral supplements (clinician prescribed or over the counter).Dietary habits3 months food-frequency questionnaire.Physical activityDaily, weekly and monthly frequency of sports activities, manual labour related hobbies and household chores, sedentary activities; occupational classification in terms of physical activity; sleep habits.Vital sign measurementsBlood pressure, heart rate and pulse.Body composition and anthropometry measurementsDXA scanning, measurement of height and weight.Laboratory assessmentsBlood investigations: Blood glucose level, HbA1C, serum insulin, C-peptide, C reactive protein, glucagon-like peptide and glucose-dependent insulinotropic polypeptide.Collection of blood samplesSeparation and storage of plasma for lipidomic analyses.Collection of abdominal visceral adipose tissueDone during laparoscopic surgeries; collection and storage of adipose tissue for lipidomic, epigenomic, genomic and gene expression analyses.Non-targeted lipidomic analysesOn adipose tissue and plasma samples.Epigenomic analysisOn adipose tissue samples and PBMCs.Gene expression analysisOn adipose tissue samples.Genomic analysisOn PBMCs.DXA, dual-energy X-ray absorptiometry; GGT, gamma-glutamyl transferase; HbA1C, glycosylated haemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; OGTT, oral glucose tolerance test; PBMC, peripheral blood mononuclear cells; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase; TSH, thyroid-stimulating hormone; VLDL, very low-density lipoprotein.Socioeconomic status will be calculated based on scoring relevant for the Indian scenario.16Participants will be asked regarding their birth weight, recent morbidity conditions and the medications and supplements they are currently taking. Details of the type and usage of these medications and supplements will be recorded by trained staff . ParticiParticipants will be requested to relax and then blood pressure, heart rate and pulse will be measured and recorded by trained staff.Participants will be weighed in minimal clothing using a digital scale, to a precision of 0.1 kg. The height of the subjects will be recorded to the nearest 0.1 cm. Whole body and regional body composition will also be estimated using dual-energy X-ray absorptiometry (DXA) . The total body fat (BF) will be measured and both fat and muscle (ALST mass: Appendicular Lean Soft Tissue mass) will be expressed as a percentage of body weight (% BF and % ALST).Blood samples before and after their surgery will be collected from the participants. The blood sample collected before surgery will be a fasting sample as the participants abstain from eating anything 10 hours before surgery, as a preoperative requirement for surgery. These samples will be used for assessments as elaborated in 17 18The dietary intake will be assessed using a validated food frequency questionnaire (FFQ). The FFQ will be administered by trained personnel to obtain information about the habitual dietary intake for the preceding 3 months.The habitual physical activity and sleep habits of the subjects will be assessed by detailed and validated physical activity questionnaire.20Blood samples will be collected before and after the surgery by phlebotomy in EDTA/serum tubes, kept on ice and immediately processed for aliquoting and storage at −80°C.Intra-abdominal omental adipose tissue will be collected during laparoscopic surgery with clinical indications of cholecystectomy, meshplasty (hernia) or splenectomy. The collected tissue will be appropriately stored for phenome measurements  as described below.Nucleic acids will be extracted from PBMCs and adipose tissue. Epigenome-wide assessment of DNA methylation levels in CpG islands will be done by reduced representation bisulfite sequencing (RRBS) after bisulfite conversion.10 21 22MLXIPL, SREBP1, ACLY, ACC1, FASN, CPT1A, CPT1B, SCD and ELOVL6. While lysis of citrate to acetyl CoA, the initial substrate of lipogenesis, is catalysed by ATP-citrate lyase (ACLY) in the cytosol, cytosolic acetyl-CoA carboxylase (ACC1) condenses acetyl CoA into malonyl CoA.24Gene expression by quantitative real-time PCR will be performed with SYBR Green chemistry for the top 10 differentially methylated loci and for the adipose DNL pathway related genes: Genotyping of the subjects will be done using Metabochip (a custom genotyping array for metabolic and cardiovascular traits) or equivalent single nucleotide polymorphism array, such as the Infinium Global Screening Array .25 26Plasma and omental AT samples will be used for non-targeted lipidomic analysis. Samples will be spiked with SPLASH Lipidomix Mass Spec Standard  before lipid extraction and reconstitution. Non-targeted lipidomic analysis will be performed on a high-resolution analytical platform consisting a Vanquish Flex Binary UHPLC coupled to a Q Exactive Orbitrap, high-resolution accurate-mass spectrometer . High-resolution accurate mass data generated by Thermo Scientific Q Exactive Orbitrap will be identified and quantified by Thermo Scientific Lipid Search software. The following lipid species are of special interest: palmitic acid, stearic acid, oleic acid, myristic acid, palmitoleic acid, acyl carnitines, palmitic acid esters of hydroxyl stearic acids, furan fatty acid metabolite 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid.27–30Assessment of adipose DNL will be done by determining AT triglyceride (TG) content of the fatty acids myristic acid (14:0), palmitic acid (16:0) and stearic acid (18:0) that have been reported to be correlated with plasma non-esterified fatty acids (NEFA) 14:0 and 18:0 but not with plasma NEFA 15:0, that is exclusively derived from dietary dairy sources.31Plasma NEFA correlates of adipose DNL will be identified in the T2DM and normoglycaemic subjects by correlation analysis of plasma lipidomic data with adipose DNL measures (AT TG content of the fatty acids myristic acid (14:0), palmitic acid (16:0) and stearic acid (18:0)). This is a novel strategy for estimating adipose DNL and identifying plasma correlates of adipose DNL as such indirect methods do not exist at present and the direct stable isotope based method of measuring DNL primarily measures postprandial hepatic DNL rates.322 test for categorical variables.Continuous data will be presented as either mean±SD or median (quartile 1 and quartile 3). The normality of distribution will be examined using the Shapiro-Wilk test and Q-Q plots. Demographic and clinical characteristics will be compared between cases of T2DM and controls by Student’s t-test, if normally distributed or Mann-Whitney U test, if non-normally distributed, for continuous variables and by ΧFor analysis of RRBS data, the read sequences from the RRBS libraries will be adapted/quality trimmed using Trim GaloreAssociation studies between the CpG methylated regions and clinical traits will be performed using linear mixed-model package pyLMM and significant associations will be determined using Bonferroni corrected p value cut-offs.Gene expression values will be estimated by normalising target gene expression against expression of housekeeping genes followed by comparison of target gene expression between the groups.Methylation quantitative trait loci will be identified by testing for association between methylation levels and genotypes of the samples using FDR correction.Correlation-based clustering analysis of the lipid profiles will be performed and visualised as heatmaps to evaluate the inter-relationships between the lipid species and for identifying signatures that differentiate between T2DM and control samples using supervised and unsupervised approaches. For heatmap analysis, only significantly altered features  will be analysed and data will be organised using a Euclidean distance map and complete clustering algorithm. Random forest will be used to assess the classification performance of the lipidomics signatures. The significantly perturbed pathways will be identified based on the differentially regulated lipids. For random forest and pathway impact analyses, data will be filtered using the IQR function and scaled using auto-scaling, in which values are mean-centred and divided by the SD of each variable. For the random forest analysis, the number of predictors to try for each node will be set to the square root of the total number of variables.To identify the adipose lipid species that correlate with transcript abundances of genes in the adipose DNL pathway, first correlation analysis  will be conducted. This will be followed by regression analysis, with levels of lipid species exhibiting significant correlations as dependent variable and transcript abundance of individual genes in the adipose DNL pathway as independent variables, adjusting for probable confounders such as BMI and sex, if found to be significantly correlated with the dependent variables.All statistical analysis will be processed and analysed using a combination of SPSS V.25 (IBM), Stata V.14 (StataCorp) and R .Patients and general public were not invited to comment on the study design and were not consulted to formulate study outcomes. Results for the InDiMeT study will be communicated to the patients and public in two complementary ways. Data related to individual participants of the study will be shared with them, if deemed beneficial for improving their health status, in consultation with clinician collaborators on this study and specific participant or their primary care providers. This is mandated by our Institutional Ethics Committee as well. The overall study results will be disseminated to participating individuals and public via appropriate forums for public science outreach.Institutional ethical approval for the study has been obtained from the Institutional Ethics Committee (IEC), St John’s Medical College and Hospital, St John’s National Academy of Health Sciences, Bangalore . Subject recruitment has been initiated from 19 November 2020.The objectives and aims of the study are being explained in subject’s preferred local language to interested volunteers who fulfil the inclusion criteria. The patient information sheet contains relevant and specific information about the study, collection and storage of samples, genetic analysis and safe storage of subject data. A written, informed consent is being obtained from the subjects before the start of the protocol.Data and resources generated as part of this study will be managed, stored and shared in line with institutional, state and national policies. In brief, specifics are as follows:Conventions for naming and storage organisation of data files as well as samples from human subjects (adipose and blood) will be set up, documented and circulated among study team members at the beginning of the study.Data will be stored in a secure manner in three copies in two different media. Back-up copies of data will be accompanied with metadata that describe the stored data in a standard, understandable format. Data storage and protection support will be obtained from the Institutional Information Technology team.Samples from the study subjects will be processed and stored as per the experimental protocols. Samples in excess of the requirements of the study objectives will be stored appropriately as per the institutional policies as well as policies of the peer-reviewed journals where findings from the study will be submitted for possible publications.Sequence data generated from this study will be deposited in GenBank and epigenetic data in Gene Expression Omnibus, while raw and processed lipidomic data will be deposited in appropriate repositories such as Metabolomics Workbench or MetaboLights during the time of publication. Findings from the InDiMeT study will be appropriately communicated in refereed journals and forums for scientific and community science outreach.The InDiMeT study will provide the base and act as a robust tool for mechanistic interpretations of public health and epidemiological aspects of developing T2DM and its complications in the Indian setting. Though a few large, prospective cohort studies to identify the determinants of T2DM are being conducted in India, to the best of our knowledge, none of them involve stated deep phenotyping or extensive phenome measurements of the participants.The InDiMeT study will not be without limitations. Notable ones will include the low probability of participation by individuals of specific subsets of the population due to the voluntary nature of participation. The subjects being recruited are the individuals who come for treatment of their pre-existing clinical conditions could not be a random representation of the total population. Further, the study setting being a tertiary care hospital in a large, urban area in India, a country known for its large regional variations in cuisine and culture-associated dietary habits, composition and amounts of habitual dietary intakes of the study subjects are likely to be heterogenous. These factors could act as confounders and limit our ability to detect differences in DNA methylation and lipid profiles that are of low effect sizes. Detailed recording of these factors as proposed, will however help us in understanding if these factors do act as confounders in our study and allow us and others to improve design of similar studies in future.


Turdus philomelos) sera sampled in autumn, and lineage 1 in kidneys of six trans-Saharan birds sampled in spring. Neutralizing antibodies against WNV and USUV were found in 5.80%  and 0.32%  of the tested sera, respectively. Our results do not exclude the role of migratory birds as potential spreaders of WNV and USUV from Africa and Central Europe to Mediterranean areas and highlight the importance of a more extensive active surveillance of zoonotic viruses.The actual contribution of migratory birds in spreading West Nile (WNV) and Usutu virus (USUV) across Europe and from Africa to old countries is still controversial. In this study, we reported the results of molecular and serological surveys on migrating birds sampled during peaks of spring and autumn migration at 11 Italian sites located along important flyways, from 2012 to 2014. A total of 1335 specimens made of individual or pooled sera, and organs from 275 dead birds were tested for WNV and USUV RNA by real time PCR (RT-PCR). Furthermore, sera were tested by serum neutralization assay for detecting WNV and USUV neutralizing antibodies. Molecular tests detected WNV lineage 2 RNA in a pool made of three Song Thrush ( Flavivirus genus, Flaviviridae family, within the Japanese encephalitis virus serocomplex. They use birds as amplifying hosts and mosquitoes as vectors, particularly those belonging to the Culex genus. In Europe, their circulation is mainly associated to a sylvatic cycle involving wild birds and ornithophilic mosquitoes, and to an urban cycle based on synanthropic or domestic birds and species of mosquitoes capable of feeding on both birds and mammals [Among terrestrial vertebrates, birds have the physical ability to move through continents for thousands of kilometers in a few days, crossing ecological barriers, such as mountains, deserts and seas. Migratory avian species of the Western Palearctic  have developed countless seasonal North-South routes represented either by movements within the region itself  or by reaching sub-Saharan Africa (long-distance or trans-Saharan migrants). These birds leave the breeding sites and move southward in the late boreal summer/autumn for then flying back northwards in spring for the following breeding season. Migratory behavior can be different among species. It can always be present and genetically determined (obligate migrants) or even be completely absent (resident species). Even within the same species there could be resident and migratory individuals . In some species, finally, the migratory behavior is context dependent and it is just triggered by specific habitat conditions at the breeding grounds (irregular migrants). During their journey, migratory birds can vehicle and spread zoonotic pathogens, including viruses, and they are thought to be responsible for the wide geographic distribution of some important arboviruses. West Nile and Usutu viruses are African mosquito-borne viruses, belonging to the  mammals . Humans, mammals  and bats mammals , are inc mammals . Thus, m mammals .West Nile virus (WNV) is nowadays the most widespread virus among the flaviviruses reported in almost all continents. Detected for the first time in Europe in 1958, during the last two decades its presence has been confirmed in several European and Mediterranean countries ,11 becomUsutu virus (USUV) was originally isolated in South Africa and detected for the first time in Europe in 2001 where it caused severe mortality in the Austrian Blackbird populations ,27. In tWe report the results of three-year sampling on birds captured at stop-over sites in Italy while flying from Africa in spring and from Central/East Europe during fall migrations. The aim of this large-scale investigation on migratory birds was to provide evidences by using molecular and serologic methods of their role in introducing West Nile and Usutu viruses in Italy during the three-year  study period.The study was carried out during a three-year period, from fall 2012 to fall 2014. Migratory birds were caught and sampled during ringing activities at some stopover sites located along the migration routes followed across the Italian territory. Stations with the highest capture rates and number of species per season were chosen to provide the most abundant and heterogeneous samples. All personnel manipulating wild birds was authorized by local administrators and by the Italian Institute for Environmental Protection and Research (ISPRA). The investigation was focused on the avian species belonging to the orders considered highly competent for flaviviruses infection, such as Passeriformes and Charadriiformes , but alsg for 10 min and the serum harvested and stored at −20 °C until analyzed.Once captured, birds were identified by species, marked with metal rings, measured, sexed and aged where possible, weighed and then bled before being released. Blood was collected from the brachial vein, in non-heparinized microhematocrit capillary glass tubes and dispensed in 1.5 mL tubes using a pipette bulb aid. The volume of blood taken from a single individual varied depending on the size of the bird, but never exceeded 1% of the total body mass (bm) . Thus, bFurthermore, brain and kidneys were collected from net casualties (due to predation or starvation) during the spring campaigns in 2013 and 2014 in the islands of Ventotene and Zannone. The presence of viral RNAs was assessed by molecular analysis. Organs were excised and collected in cryotubes, then kept in a liquid nitrogen portable tank and stored at −80 °C once in the laboratory.50 (tissue culture infectious dose) of reference WNV and USUV field strains were added to each dilution. In both, WNV and USUV SN assays, positive control sera of either viruses were included to assess possible cross-reactions. After 1 h at 37 °C and 5% CO2 in a humidified incubator, 100 µL of 105 Vero cells were added to each well. The plates were incubated at 37 °C for 5 d. Starting from the third day after incubation, the plates were checked for cytopathic effect (CPE). The positive threshold was set at the 1:10 dilution. The sample was considered positive when it showed more than 90% CPE neutralization at the lowest dilution (1:10). WNV and the USUV may cross react but the detection of neutralizing antibody titers at least 4-fold higher for a given flavivirus over the other(s) is considered a proof of specificity [Total RNA was extracted from all the sera and the supernatant of organs previously homogenized  using thcificity . The posPossible differences between the WNV and USUV prevalence found in birds according to orders or migratory season (spring vs. autumn) have been analyzed by using a Chi-square test at a significance level of 0.01.n = 2126) but because of their light body mass, the samples were pooled and thus the number of samples tested was 521 (39.02%). Detailed information about serum collection is reported in During the 3-year period, 3188 birds belonging to 52 species and 14 different orders were ringed and bled, leading to the collection of 1335 serum specimens. Of the 52 bird species, 39 were long-distance, 3 short-distance, 9 partial and 1 resident/irregular migratory species. When distributed according to the migratory season, 823 specimens from 25 species were sampled during the spring activities while 512 samples from 35 species were collected during the autumn fieldwork sessions. Passeriformes were the most abundant among the collected individuals , while the serological survey was limited to the serum specimens collected between 2013 and 2014 (n = 1241 from 44 species).During necropsies, 529 organs (269 kidneys and 260 brains) were collected from 275 dead birds belonging to 30 different species of Passeriformes , 22 of wTurdus philomelos) sera collected during the 2014 fall migration in Bocca Caset, northern Italy  (n = 1), Common Whitethroat (Sylvia communis) (n = 2), Wood Warbler (Phylloscopus sibilatrix) (n = 1), Pied Flycatcher (Ficedula hypoleuca) (n = 2). Except for the Icterine Warbler collected in 2013, the positive carcasses were collected in 2014 on the island of Ventotene (The presence of WNV RNA lineage 1 was detected in the kidneys of 6 birds belonging to 4 different trans-Saharan Passeriformes species: Icterine Warbler (entotene .Usutu virus genome was never detected in any of the samples collected in the three-year period.Accipiter nisus), spring specimens showing neutralizing antibodies against WNV were all trans-Saharan migrants. European Turtle Dove (Streptopelia turtur)  and Eurasian Golden Oriole (Oriolus oriolus)  were the species in which WNV antibodies were more frequently detected. Together with the Common Quail (Coturnix coturnix), they were the species most commonly tested, representing almost 50% of the serum samples collected in spring. The three species are long-distance migrants, their samples were mostly tested individually due to the large body size (>50 g). During the autumn migration, 418 serum samples belonging to 27 species were tested. WNV antibodies were detected in 10 samples . These belonged to 5 species (11%) represented by 4 long-distance and 1 short-distance migrant, respectively.Of the 1241 sera tested by WNV SN, 72 (5.80%) resulted positive . In partUSUV neutralizing antibodies were detected in bird samples collected in 2013 only . In spriSylvia borin), Common Quail and European Turtle Dove. Being impossible to exclude any cross reaction between the viruses, the samples were considered inconclusive. In two pool samples, one from Common Whitethroats and the other from Garden Warblers, different neutralizing antibodies against WNV and USUV were also found. In these cases, the lowest titer was regarded as a result of a cross-reaction and the samples were counted as positives only against the virus with the highest antibody titer  were observed in three samples from Garden Warbler (dy titer .p < 0.01) than that found in birds captured during the autumn migratory seasons. Other statistical analyses could not be performed due to the exiguous number of positive samples.The chi-squared test revealed that the WNV prevalence found in birds migrating during the spring migratory seasons was significantly higher  and from Central-Northern Europe (autumn).In this study, WNV lineage 2 (L2) RNA was detected in a pooled sample derived from three Song Thrushes captured in the Trentino Region while crossing the Alps following the NE-SW direction in October 2014. The question might arise as to whether these birds were infected in or outside Italy. The phenology of the Song Thrush, supported by rings’ recovery data, clearly indicates that these birds entered Italy crossing the Alpine arch following the NE/SW route ,55. AccoHistorically, African countries have been claimed as the source of WNV introduction in the European and Mediterranean regions . MigratoAlthough it does not necessarily imply the presence of infectious virus, the detection of the WNV genome in the sample of the Song Thrush appears to reinforce what was previously observed. Data from WNV experimental infection have shown 5–7 days of high level of viremia in Passerines . This isThe presence of WNV RNA in kidneys of six birds belonging to trans-Saharan species found dead in Ventotene island during spring, suggests a possible remnant of a recent infection. In fact, kidney is, among others, the organ in which WNV has been found most abundant and persistent (up to 6 weeks) ,65,66,67A broad spectrum of different bird species, mainly belonging to Passeriformes and Columbiformes, showed WNV neutralizing antibodies. Indeed, the size of most Passeriformes is smaller than that of birds belonging to the other orders. As a consequence, the number of tested specimens were much lower than the number of sampled animals. With regard to this order, pooling can have influenced the results: in fact, if it is true that a pooled sample represents more individuals and thus increases the probability to include at least a seropositive bird, it is also true that the higher the number of birds per pool, the greater is the dilution effect of the antibody titer if present in few birds only. This however was a necessary compromise to maximize the number of sera collected and to provide a descriptive assessment of WNV and USUV in migratory birds reaching Italian sites.Accipiter gentilis), is regarded as particularly susceptible to WNV infection as noticed in 2004 and 2005 in Hungary and in 2017 in Czech Republic, where many birds died due to severe encephalitis [WNV neutralizing antibodies in birds sampled during spring were detected in 12 out of 22 species. Eleven of them were long-distance migrants reaching Europe after leaving non-breeding grounds in sub-Saharan Africa. Overall, titers varied from 1:10 to 1:320, but titers ≥ 1:80 were detected only in six samples from four species: European Turtle Dove, Eurasian Golden Oriole, Garden Warbler and Sparrowhawk. The latter species, as well as other closely related species such as Goshawks  which is known to be a long-distance migrant susceptible to WNV  and ableSerological results confirmed that migratory birds, at least some of them, have been exposed to WNV and USUV infection during their lifetimes. In this survey, it appears that the WNV exposure was higher in migratory birds coming from sub-Saharan Africa compared to those from Mediterranean countries, confirming what has been already reported by previous similar surveys ,78. HoweIn conclusion, we provided a descriptive assessment of potential flavivirus dispersal through migratory birds crossing Italy. The results on WNV obtained by our study do not exclude the “Migrant Bird as Introductory Host” hypothesis  and whatConversely, the results of this survey seem to indicate that the probability of new introductions of USUV to Italy through migratory birds is very low. This conclusion supports the findings of a recent study on the evolutionary processes and spatial spread of USUV strains circulating in the European context and in Italy in which the European origin of the four different lineages circulating in Italy was confirmed .Through our survey it was possible to identify several wild species as potential amplifier hosts which are hardly detectable by passive surveillance either in urban or rural areas. Most of them, in fact, are strictly associated to different wild habitats which are usually not investigated, and the mortality rate is often very low and clinical signs are rarely reported. Furthermore, results of this work show how ringing activities can contribute to providing relevant information on status of bird-related diseases from a wide range of species. In Italy, as in Europe, a widespread network of hundreds of ringing sites operating through standardized protocols for monitoring projects could represent a new connection between the study of bird movements and pathogen circulation, within the perspective of a more extensive and active surveillance of zoonotic viruses.