However, since not all eplets are equally capable of inducing an immune response, antibody verification is needed to confirm their ability to be bound by antibodies, such that only clinically relevant eplets are considered. The HLA Epitope Registry has documented all theoretically defined HLA eplets along with their antibody verification status and has been the foundation for many clinical studies investigating eplet mismatch in transplantation. The verification methods for eplets in the Registry range from polyclonal sera from multi- and uni-parous women to murine and human monoclonal antibodies (mAbs), and antibodies purified by adsorption and elution from sera of HLA immunized individuals. The classification of antibody verification based on different methods for validation is problematic, since not all approaches represent the same level of evidence. In this study, we introduce a classification system to evaluate the level of evidence for the antibody-verified status of all eplets in the HLA Epitope Registry. We demonstrate that for a considerable number of eplets, the antibody-verified status is solely based on polyclonal serum reactivity of multiparous women or on reactivity of murine mAbs. Furthermore, we noted that a substantial proportion of patient sera analyses and human mAb data presented in the HLA Epitope Registry Database has never been published in a peer-reviewed journal. Therefore, we tested several unpublished human HLA-specific mAbs by luminex single antigen beads assay to analyze their HLA reactivity for eplet antibody verification. Although the majority of analyzed mAbs indeed verified their assigned eplets, this was not the case for a number of eplets. This comprehensive overview of evidence for antibody verification of eplets in the HLA Epitope Registry is instrumental for future investigations towards eplet immunogenicity and clinical studies considering antibody-verified eplet mismatch in transplantation and warrants further standardization of antibody verification using high quality data.Matching strategies based on HLA eplets instead of HLA antigens in solid organ transplantation may not only increase the donor pool for highly sensitized patients, but also decrease the incidence of  Donor-specific antibodies (DSA) are formed against mismatched polymorphic amino acid residues on donor human leukocyte antigens (HLA) and are a major complication in renal transplantation, leading to chronic rejection and graft loss , 2. HLA th International HLA and Immunogenetics Workshop in 2012, which has documented all theoretically defined eplets, as well as their antibody verification status  assays in twelve laboratories worldwide . Only cehttp://www.allelefrequencies.net/pop6001c.asp?pop_id=0003257). If the immunizer was unknown, the specificity of the bead with the highest MFI in SAB assay was used to determine amino acid mismatches. Next, we determined whether these solvent accessible amino acid mismatches were uniquely shared by the reactive HLA alleles and absent on the non-reactive HLA alleles. In order to visualize amino acid positions and to establish whether amino acids were within 3-3.5 Å to form an eplet, the following HLA crystal structures were visualized in Swissviewer . When HLA crystal structures were not available, modelled PBD structures were used; 3PL6, 3WEX, 4I5B, 4NT6 and 4Z7U . For HLAMatchmaker analysis, ABC Antibody Analysis Program V3.1 and DRDQDP Antibody Analysis Program v3.1 were used (http://www.epitopes.net/).HLA Epitope Mismatch Algorithm (HLA-EMMA) version 1.05  was usedssviewer : ProteinFor every eplet with the antibody-verified status in the HLA Epitope Registry that consisted of more than one polymorphic residue, it was determined whether the involved amino acids were indeed within 3-3.5 Å using Swissviewer . If not,Box 1Functional epitope: The functional epitope determines the specificity of the antibody through its interaction with the complementarity-determining region 3 (CDR3) of the heavy chain of the antibody.Eplet: The definition of an eplet resembles the functional epitope and comprises the minimal amino acid configuration on the HLA-molecule that is needed to induce an antibody response. Involved residues must be within 3-3.5 Å.Structural epitope: The structural epitope comprises all amino acids of the HLA-molecule that are involved in the binding to the antibody paratope and spans a radius of approximately 15 Å.Reactivity pattern: In some cases, the SAB analysis of a human mAb yields multiple uniquely shared residues or multiple uniquely shared combinations of residues that are not within 3-3.5 Å, indicating that there are multiple possible eplets that could have induced the formation of the antibody. Often, these amino acids are simultaneously present on HLA alleles, which limits the possibilities of determining the actual eplet using SAB or cellular assays. However, the fact that the residues involved always occur together on these HLA alleles, also means that these residues can be regarded as a ‘’reactivity pattern’’ and can be used as a single entity in matching strategies and immunological risk assessment for the vast majority of transplant patients.For 13 HLA class I eplets in the HLA Epitope Registry, antibody-verified status was based on data of 15 mAbs that had not been published in a peer-reviewed journal. Therefore, these mAbs were re-tested in luminex SAB assay to determine whether they would indeed provide evidence for antibody verification of these eplets. SAB analysis of the mAbs was performed by comparison of amino acid sequences of the reactive alleles in SAB assay with non-reactive alleles to identify uniquely shared amino acids that could have induced the antibody response. These uniquely shared residues were then mapped to corresponding eplets . TherefoAnalysis of SAB data of three HLA class I mAbs did not verify the eplets which they were attributed to by the HLA Epitope Registry. Firstly, according to the Registry, mAb VIE6C10 verifies eplet 65GK. However, SAB and CDC results demonstrated that mAb VIE6C10 is negative for allele A*24:02, which bears eplet 65GK . Howeveror 142T + 149A are uniquely shared by the reactive alleles , it was negative in SAB analysis. Considering A*02:03 non-reactive, the combinations of 145H + 149A Lastly, SAB analysis of mAb VDK1D12 yielded three uniquely shared residues; 44K, 150V and 158V , including the mAb data presented in this paper, and adsorption and elution studies (n=22). A number of 22 eplets were considered provisionally antibody-verified based on reactivity analysis of patient sera (n=15), CDC with serologically typed cells only (n=1) and murine mAbs (n=6). The HLA Epitope Registry included four eplets that were listed as ‘eplet pairs’. Eplet pairs were considered not antibody-verified since they consist of two separate eplets that are located too far from each other to form a single eplet (> 3.5 Å), and thus are not jointly capable of inducing an antibody response. For two eplets, 44KM and 193PL, the residues that comprise these eplets as defined by the HLA Epitope Registry exceed the 3.5 Å range  and based on reactivity analysis of adsorbed and eluted antibodies (n=1). Three previously not-verified eplets could be verified based on recent literature that was not yet included in the Registry (eplets 31FY and 70QA)  and baseThe residues defining eplet 98ES exceed the 3.5 Å distance, which is therefore considered as an antibody-verified reactivity pattern .For HLA-DQ, 10 of the antibody-verified eplets exceed the 3.5 Å radius and are therefore considered as antibody-verified reactivity patterns  and a murine mAb (n=1). An exception regarding antibody verification classification was made for eplets 56A and 85GPM, of which the highest level of evidence is patient sera. These eplets were considered antibody-verified because of the extensive analysis on multiple sera performed by Cano et al. , and theOverall, we consider 44 HLA class I eplets and 19 HLA class II eplets as being truly antibody-verified and a total of two HLA class I and 11 HLA class II reactivity patterns as being antibody-verified.The HLA Epitope Registry and HLAMatchmaker have formed the foundation for the vast majority of clinical studies investigating the role of HLA eplets in transplantation. In this study, we have critically reviewed the evidence for the antibody verification status of eplets included in the HLA Epitope Registry. The different methodologies that are currently used for antibody-verification do not represent the same level of evidence for the antibody-verified status of eplets. However, while previously a category of ‘provisionally verified’ was present, the current dichotomous yes or no antibody-verified status in the HLA Epitope Registry does not take the heterogeneity in the level of evidence into account. To provide insight on what basis an eplet is considered antibody-verified by the Registry, we have introduced a classification system to score the level of evidence. Our results show that for many eplets, especially for HLA class II, the antibody-verified status is based on sera from multi- or uni-parous women or transplant patients, experiments with only serologically typed cells, or murine mAbs. However, we argue that these methods are not suitable for definitive antibody verification of eplets. Although SAB analysis of sera from immunized individuals can be informative, the reactivity of sera tested in SAB is in most, if not all cases the result of a polyclonal antibody response. These patterns are often broad and do not permit the identification of a single HLA eplet, since the pattern of reactive HLA alleles is caused by multiple antibodies recognizing several HLA epitopes. Even seemingly narrow SAB reactivity may be caused by more than one eplet mismatch. For several other eplets, antibody verification status was based on experiments using serologically typed cells only. These cells are not suitable for state-of-the-art reactivity analysis due to the low resolution of HLA typing, which makes definitive assignment of the inducing eplet very difficult. Furthermore, for 11 eplets only reactivity analysis of murine mAbs was available. Murine mAbs are generated by immunization with HLA but do not necessarily recognize the same epitopes as human mAbs, since immunogenicity of HLA antigens is affected by the recipients’ HLA type . TherefoAside from eplets, the HLA Epitope Registry also includes ‘‘eplet pairs’’, of which a number have been assigned the antibody-verified status. HLA eplets are based on the concept that one or multiple mismatched amino acid residues induce the humoral immune response through interaction with the CDR3 region of the B cell receptor heavy chain. Accordingly, the residues that constitute an eplet should be in a 3.5 Å radius . HoweverOur review of the HLA Epitope Registry does not only provide insight in the heterogeneity of the level of evidence of eplet antibody verification, but also demonstrates that a substantial portion of the presented mAb data and patient sera analyses had not been published in peer-reviewed journals. Aiming to substantiate the antibody-verified status of eplets based on human mAbs which reactivity analyses have not been published previously, we tested these mAbs in SAB assays and performed reactivity analysis. For the majority of mAbs tested, the identified uniquely shared amino acids indeed corresponded with the eplet. However, SAB analysis of three mAbs did not confirm the antibody verification of the eplet as assigned by the Registry. The analyses of mAbs SN66E3 and RTLK1E2 supported the verification of two different eplets, while no inducing eplet could be determined for mAb VIE6C10. Furthermore, the reactivity analyses of SN607D8 and SN66E3 identified multiple uniquely shared residues or uniquely shared combinations of two residues, while the corresponding eplets, 144TKH and 145KHA respectively, are defined by three residues. Based on our analyses, it is possible that not all three, but only one or two residues are crucial for the induction of anti-HLA antibodies. For these eplets, this difference in possible eplet definitions is clinically relevant, since there are less common, but intermediately and well-documented alleles that bear only one of the uniquely shared residues . ConsequDetailed analysis of the localization of antibody-verified eplet configurations on crystalized HLA structures demonstrated that not all antibody-verified eplets in the HLA Epitope Registry comply with the eplet definition. Especially for HLA-DQ, the polymorphic residues that comprise the eplet configuration are often too distant (> 3.5 Å) from each other to form an eplet. Re-analysis of previously published SAB data of human mAbs and eluted antibodies from patient sera demonstrated that for 10 HLA-DQ, one HLA-DR and two HLA class I eplets multiple uniquely shared amino acids could be identified that were not within 3.5 Å. Because these residues are simultaneously present on the Common HLA alleles in the CIWD 3.0.0  with onlThe rationale for this explicit and precise definition of eplets and reactivity patterns also follows from the need to define the most immunogenic eplets in transplantation . MultiplThe SAB assays in this study were performed with the Lifecodes SAB assay from Immucor. It has been demonstrated that the beads of the other manufacturer of these assays  are bound with an admixture of intact and denatured HLA , 62, whiThis critical review of the antibody-verified status of eplets in the HLA Epitope Registry has demonstrated that the level of evidence of antibody-verified eplets is heterogeneous and that not all data have been published in peer-reviewed journals. Analysis of luminex SAB data of human mAbs showed that not all mAbs verified the eplets they were assigned to. Since an increasing number of clinical studies investigate eplet mismatch load as a risk factor for inferior transplant outcomes and seek to identify the most immunogenic eplet mismatches, it is vital to define a set of well-defined antibody-verified eplets in a transparent manner. Our list of antibody-verified eplets and reactivity patterns is the first step towards a uniform and transparent method of eplet definition and antibody verification. However, eplets that are considered provisionally verified, or not antibody-verified could still play a clinically relevant role in transplantation, since antibody verification is limited by the available reagents and patient material. In this respect it is important for our field to collaborate in the yet uncompleted endeavor of eplet antibody verification. For future publications, we propose to set a standard of required data regarding antibody verification that should be published. Preferably, this includes reactivity analysis of human mAb data tested in SAB assay or antibodies eluted from immunized patient sera. The used SAB panel and HLA typing of the antibody producer and immunizer should also be included to make re-analysis and thorough interpretation of the data possible. Finally, we propose to establish an international committee that oversees nomenclature and antibody verification of eplets to facilitate the establishment of a well-documented, transparent list of eplets with antibody verification status classified by the level of evidence, striving for better comparable results in clinical and immunogenicity studies on the road to eplet matching in transplantation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SB reviewed the literature and analyzed the data. KB performed laboratory experiments. SB and SH wrote the manuscript. CK, JF, MR, AM, and FC critically reviewed the manuscript. All authors contributed to the article and approved the final version of the manuscript.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.


Oat1 and Oat3 knockout mice revealed changes in tryptophan derivatives involved in metabolism and signaling. Several of these metabolites are derived from the gut microbiome and are implicated as uremic toxins in chronic kidney disease. Direct interaction with the transporters was supported with cell-based transport assays. To assess the impact of the loss of OAT1 or OAT3 function on the kidney, an organ where these uptake transporters are highly expressed, knockout transcriptomic data were mapped onto a “metabolic task”-based computational model that evaluates over 150 cellular functions. Despite the changes of tryptophan metabolites in both knockouts, only in the Oat1 knockout were multiple tryptophan-related cellular functions increased. Thus, deprived of the ability to take up kynurenine, kynurenate, anthranilate, and N-formylanthranilate through OAT1, the kidney responds by activating its own tryptophan-related biosynthetic pathways. The results support the Remote Sensing and Signaling Theory, which describes how “drug” transporters help optimize levels of metabolites and signaling molecules by facilitating organ cross talk. Since OAT1 and OAT3 are inhibited by many drugs, the data implies potential for drug–metabolite interactions. Indeed, treatment of humans with probenecid, an OAT-inhibitor used to treat gout, elevated circulating tryptophan metabolites. Furthermore, given that regulatory agencies have recommended drugs be tested for OAT1 and OAT3 binding or transport, it follows that these metabolites can be used as endogenous biomarkers to determine if drug candidates interact with OAT1 and/or OAT3.How organs sense circulating metabolites is a key question. Here, we show that the multispecific organic anion transporters of drugs, OAT1 (SLC22A6 or NKT) and OAT3 (SLC22A8), play a role in organ sensing. Metabolomics analyses of the serum of  This group of drug transporters includes other members of the SLC (solute carrier) and the ABC (ATP-binding cassette) superfamilies , 8. TherBecause of the vast array of small organic anion compounds transported by OAT1 and OAT3, a wide range of xenobiotics and endogenous molecules can conceivably compete for access to and renal clearance by these OAT transporters , 15. ThiOat1 and Oat3 knockout (KO) mice to analyze the major metabolic functions that are influenced by OAT1 and OAT3. The serum metabolomic data from the Oat1 and Oat3 KO mice represented the impact these transporters have on endogenous systemic metabolism. We used the transcriptomic data from the kidney, which is the site of expression of the encoding transporter genes, to evaluate the activity of hundreds of metabolic functions using metabolic task analysis. In doing so, we were able to complement an understanding of extracellular metabolite changes due to the loss of the OAT1 or OAT3 transporters with an analysis of affected intracellular metabolic pathways.We applied a systems biology approach that employed metabolomic and transcriptomic data from Together, the multi-omics approach and systems biology analysis used here provide a portrait of the local and systemic metabolic and signaling pathways modulated separately and jointly by OAT1 and OAT3. We show that OAT1 not only regulates tryptophan metabolism systemically, but it also plays a key role in the tryptophan metabolic pathways inside the tissue where it is found. Thus, our results indicate that studies of new drug entities should not only consider drug–metabolite interaction (DMI) effects in the serum but also evaluate potential shifts in cellular metabolism due to loss of metabolite influx through competition at the level of the transporter. In support of the human relevance of our work, we show that many of the alterations in serum levels of tryptophan metabolites seen in the knockout mice are also observed in humans after administration of probenecid, a drug used to treat gout that inhibits OAT1 and OAT3.Oat1 or Oat3 and their wild-type controls. These data sets have previously been examined from a chemoinformatic perspective  encodes for transporters that participate in the uptake of many unique compounds across several tissues, though much of the research has focused on a handful of members  that handle many common drugs , 21. Howspective  and to pspective , but herOat1 or Oat3 are similar to those of wild-type mice  or trending toward significantly increased , suggesting that they are either OAT1 substrates or that their serum concentrations depend on OAT1. For Oat3 KO mice, we found that ten metabolites in the Amino Acid Superpathway were significantly increased, while 11 were trending toward significantly increased  for tryptophan metabolites. Consistent with other pharmacological and physiological data obtained from the OAT1 and OAT3 knockout mice or their tissue indicating important functional differences (Oat1 KO (63 metabolites) and Oat3 KO (21 metabolites) mice revealed only ten overlapping metabolites in these subsets  that was not measured in the serum of the Oat3 KO  and one of 90 Subpathways. Of the 17 metabolites that significantly accumulated in the serum of both the tabolism . Indepentabolism . Sixteen Oat3 KO . Analysisyndrome , 40. TheOat1 KO mouse and humans treated with probenecid. All eight metabolites that were significantly elevated in the Oat1 KO were also significantly elevated in the drug-treated humans. Only serotonin, which was elevated in the Oat3 KO mouse, was not altered in humans.Probenecid, a drug used to treat gout, was administered to humans to determine the short-term impact of a high-affinity OAT-binding drug on the levels of circulating metabolites. Probenecid has three well-established targets: SLC22A12 , OAT1, and OAT3, all of which are closely related genes in the organic anion transporter group of the SLC22 family of solute carriers. URAT1 is a uric acid transporter located on the apical membrane (urine-facing side) of the proximal tubule, so OAT1 and OAT3, which are multispecific and expressed on the blood-facing side of the proximal tubule, are expected to exert a more profound effect on the serum metabolome. Comparisons of metabolic measurements on the blood before and 5 h after dosage show major changes to the Tryptophan Metabolism Subpathway . Due to As an intermediate between our knockout mouse models and humans, we treated wild-type mice with probenecid, a well-established OAT-inhibiting drug. This drug treatment led to the elevation of six tryptophan metabolites, including kynurenine, kynurenate, and indolelactate . These mThus, the overall results in probenecid-treated humans and mice, as well as the knockout mice, support the role of OAT1 and OAT3 in regulating circulating levels of tryptophan metabolites.in vitro transport assays using cells overexpressing human OAT1 and OAT3. Many of the metabolites in this Subpathway have been tested against these transporters  (data not shown). The IC50 values were then integrated with known Km values to calculate inhibitory constants. The inhibitory constant (Ki) for ILA with OAT1 was 119.3 μM.To confirm our findings, we performed sporters , but othsporters . FurtherOat1 KO, Oat3 KO, and their wild-type controls to identify how the transporter alterations influence the kidney metabolic functions. To this end, we used a systems biology method .We used microarray data from the kidneys of analysis  from theanalysis , coverinin vitro transport assays and other analyses have shown that OAT1 and OAT3 interact with metabolites in each of the general systems . As regulators of systemic metabolism, as well as local metabolism within their tissue of expression, the type of studies and analyses we have described here, if performed for all “drug” transporters, may radically alter our physiological views of these evolutionarily conserved proteins.These multispecific drug transporters are highly expressed in nearly all tissues and play a particularly critical role at epithelial and endothelial barriers between blood and other body fluids/compartments  to limit the possibility of drug–drug interactions (DDI)—cases where two or more drugs compete for access to the same transporter. Transporter-mediated DDIs can alter the concentration of the drugs in the blood, potentially leading to adverse clinical effects . ConsideOat1 and Oat3 knockout mice, there were marked alterations in tryptophan-related metabolites. The increases in systemic levels of metabolites, due to the absence of these transporters at the blood interface with the kidney, raised the possibility of diminished intracellular concentration in kidney proximal tubule cells. How might these cells in turn respond? We used transcriptomics-based metabolic task analysis to address this question. We identified multiple local (kidney) tryptophan-related metabolic tasks affected by the absence of the transporters. These changes in metabolic task scores are caused by compensatory increases in the expression of the metabolic task-associated genes in the context of OAT1 deficiency. While many metabolic pathways were surveyed by both metabolomics and transcriptomics-based metabolic task analysis, the use of both approaches provided us with unique insights into how certain drug transporters participate in the communication between the tissue and the extracellular environment. Thus, whereas tryptophan metabolites were regulated by both transporters at the systemic level, tryptophan-related metabolic tasks within the kidney were primarily dependent upon OAT1.In our metabolomics analyses of the serum of The results indicate that the proximal tubule of the kidney, where OATs are found, is not simply a conduit for renal elimination of tryptophan metabolites; it senses tryptophan metabolites and responds to changes in their intracellular abundance. Collectively, the data supported the view that OAT1 plays a key role not only in clearing tryptophan-related metabolites from the circulation by promoting their uptake by the kidney, but also that this transporter regulates intracellular metabolism, notably tryptophan metabolism.This supports the view that OAT1-mediated transport of kynurenine, kynurenate, anthranilate, and N-formylanthranilate are important for kidney function . These fNearly all ingested tryptophan is metabolized into three main subpathways: kynurenine, serotonin, and indole , 48. In Considering the myriad of signaling roles the elevated metabolites have, there is potential for OAT1 and OAT3 to influence many aspects of physiology. For example, kynurenate activates GPR35, a drug target and a GPCR involved in inflammatory responses and cardiovascular disease . Many ofThe metabolomics studies of the two knockout mice showed that a key endogenous function of OAT1 and OAT3 is to regulate systemic levels of tryptophan metabolites. Thus, a drug targeting OAT1 and OAT3 would be expected to have a similar impact on the metabolome. As predicted, the translational potential of our knockout mice as models of DMIs was strongly supported by the results from humans treated with probenecid, an OAT-inhibiting drug that is used worldwide to treat gout. Several of the tryptophan metabolites were elevated in both the serum of humans and the knockout mice.via SLC and ABC drug transporters  and the probenecid-treated humans were healthy, some of the elevated metabolites are known uremic toxins that are increased in the serum of humans suffering from chronic renal failure and associated with negative outcomes , 55. Thesporters , 58.Oat1 KO and Oat3 KO mice—together with our demonstration that some are ligands in vitro, provide additional support for the importance of these transporters in regulating communication between the host and commensal organisms. Altogether, our results align with the Remote Sensing and Signaling Theory, which proposes that drug transporters and drug metabolizing enzymes participate in interorgan and interorganismal communication through the transport and modification of small molecules to maintain homeostasis . All animals were handled in accordance with the Institutional Guidelines on the Use of Live Animals for Research. Adult WT, ications , 27. Proications , 24. Theerformed . The OatAll experimental protocols were reviewed and approved by the Institutional Review Board and abide by the Declaration of Helsinki Ethical Principles. Whole-blood samples were collected from 20 individuals . The average age was 30.85 ± 10.98, and the average BMI was 24.18 ± 3.52. Participants were not taking any medications and were on vegetarian diets for the duration of the study. An oral dose of 1 gram of probenecid was administered, and after 5 h, whole blood was collected again. Each sample was kept frozen at –80 °C until metabolomic analysis.Human serum samples were immediately stored at –80 °C and shipped on dry ice to Metabolon Inc. Mouse serum samples were collected and analyzed, as previously described , 24. To t-test with metabolites that achieved statistical significance (p ≤ 0.05), as well as those approaching significance (0.05 ≤ p ≤ 0.10) included in subsequent analyses. Enrichment was determined using Equation k is the number of significantly altered metabolites in a Subpathway, m is the number of metabolites in a Subpathway, n is the number of significantly altered metabolites in the total data set, and N is the number of measured metabolites in the total data set.For both human and mouse samples, raw values were normalized to volume, log transformed, and missing values were replaced with the lowest observed value for each compound. In the human serum samples, statistical significance was determined using ANCOVA contrasts that incorporate BMI and age. For mouse serum samples, significance was determined using Welch's two-sample i.e., microarray data from the kidneys of Oat1 KO, Oat3 KO, and their wild-type controls). This analysis predicts the activity of a curated collection of hundreds of tasks covering seven major metabolic activities of a cell  directly from transcriptomic data by using genome-scale models of human metabolism. More specifically, the computation of the relative activity of a metabolic task  relies first on the preprocessing of the available transcriptomic data and the attribution of a gene activity score for each gene . Therefore, the metabolic task score is computed as the average activity score of all the genes contributing to a metabolic function. Doing so, transcriptomic data can be directly used to quantify the relative activity of each metabolic function in a specific condition.We used the metabolic task analysis, as implemented in the CellFie module in GenePattern, to quantify the kidney's metabolic functions and the influence of OAT transporter alterations from gene expression data  -293 cells that stably overexpress human OAT1 and OAT3 (Solvo Biotechnology) were grown to confluence in Dulbecco's Modified Eagle's Medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and maintained in 5% COFluorescent intensity values were normalized so that the lowest value was set to 0% and the highest value was set to 100%. Following normalization, the data was fit to a nonlinear model, and the IC50 was determined using Equation Ki was then calculated for each metabolite using the Cheng–Prusoff equation Equation , with thsnigam@health.ucsd.edu.All relevant metabolomics data are contained within the article and supplementary material. Transcriptomic data are available upon request from This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Individuals with disabilities and type 2 diabetes require self-management programs that are accessible, sustainable, inclusive, and adaptable. Health coaching has been shown to be an effective approach for improving behavioral changes in self-management. Health coaching combined with telehealth technology has the potential to improve the overall quality of and access to health services.This protocol outlines the study design for implementing the Artificial Intelligence for Diabetes Management (AI4DM) intervention. The protocol will assess the feasibility, acceptability, and preliminary efficacy of the AI4DM telehealth platform for people with disabilities.The AI4DM study is a 2-arm randomized controlled trial for evaluating the delivery of a 12-month intervention, which will involve telecoaching, diabetes educational content, and technology access, to 90 individuals with diabetes and physical disabilities. The hypothesis is that this pilot project is feasible and acceptable for adults with permanently impaired mobility and type 2 diabetes. We also hypothesize that adults in the AI4DM intervention groups will have significantly better glycemic control (glycated hemoglobin) and psychosocial and psychological measures than the attention control group at the 3-, 6-, and 12-month follow-ups.The AI4DM study was approved by the university’s institutional review board, and recruitment and enrollment will begin in October 2021.The AI4DM study will improve our understanding of the feasibility and efficacy of a web-based diabetes self-management program for people with disabilities. The AI4DM intervention has the potential to become a scalable and novel method for successfully managing type 2 diabetes in people with disabilities.ClinicalTrials.gov NCT04927377; https://clinicaltrials.gov/ct2/show/NCT04927377PRR1-10.2196/31689 Diabetes mellitus is one of the most common metabolic disorders; it affects approximately 12.2% of the overall adult population in the United States and 25.25% of the population aged above 65 years [Although physical disabilities increase the risk of developing diabetes, an inverse may also occur. Diabetes is associated with a significant increase in the risk of mobility disability . It has 1c) in both the general population and people with intellectual and developmental disabilities [In diabetes management, PA, medication adherence, and glucose tracking have been shown to be effective in reducing glycated hemoglobin  is another major problem among people with disabilities . SimilarPrevent T2 for ALL, an inclusive adaptation of the CDC’s Prevent T2 program. However, the Prevent T2 program is a diabetes prevention program (not a diabetes management program), and the primary focus is on BMI control, not glycemic control.However, people with disabilities face inordinate barriers to achieving a healthy diet and recommended PA levels -31. PeopAs our next step toward diabetes management for people with disabilities, we propose the development of an inclusive telecoaching self-management program for diabetes management in people with disabilities. To make the telecoaching approach sustainable and scalable, artificial intelligence techniques are employed to reduce the time health coaches spend on each participant.The primary objective of this project is to evaluate the preliminary efficacy and feasibility of an accessible and inclusive artificial intelligence–assisted, individualized, family-focused lifestyle modification intervention  for glycemic control in people with disabilities.1c) associated with type 2 diabetes mellitus (T2DM) self-management and self-efficacy outcomes would have a greater effect in the AI4DM intervention arm than in the attention control arm at the 3-, 6-, and 12-month follow-ups. We also hypothesize that the AI4DM intervention is feasible and acceptable for adults with permanent impaired mobility and T2DM, their caregivers, and health coaches and that the fidelity of the program will be maintained. A fidelity monitoring protocol was developed by the research team for the AI4DM study based on the five domains recommended by the National Institutes of Health Behavior Change Consortium [We hypothesize that the clinical outcome  the AI4DM intervention group with telecoaching support and (2) the attention control group. The active intervention will include 6 months of telecoaching followed by 6 months of follow-up and access to the technology but with no telecoaching calls . For theThe AI4DM intervention is designed to evaluate the feasibility and preliminary efficacy of a web-based diabetes self-management program for people with disabilities. Participants will either be assigned to the intervention group, which includes telehealth coaching calls, a technology package, and access to diabetes educational content, or an attention control group, which will receive the same number of telecoaching calls at the same frequency as the intervention group. These calls generally serve as courtesy calls or general wellness calls.1c level of ≥8%; (3) an age of 18-65 years; (4) individuals living with a permanent physical disability such as spinal cord injury, spina bifida, multiple sclerosis, or stroke and (5) the ability to speak and read English. To screen for permanently impaired mobility, the NHANES Physical Functioning Survey [Eligible participants must meet the following inclusion criteria: (1) a diagnosis of T2DM; (2) an HbAg Survey  will be Exclusion criteria include (1) the use of insulin medication for diabetes treatment; (2) current enrollment in any diabetes-related intervention; (3) severe cognitive impairment; (4) severe untreated depression in the past 6 months; (5) a major cardiac event in the past 12 months; (6) uncontrolled blood pressure; (7) resting tachycardia; (8) renal failure; (9) severe peripheral neuropathy; and (10) the unavailability of a smartphone.1c kits for glucose data collection.The total study duration for both groups is 12 months. The protocol includes both groups receiving weekly health coaching calls weekly for 12 weeks, and one biweekly coaching call (every other week) for 12 weeks, for a total of 18 coaching calls. The intervention group, AI4DM, will receive access to home and web-based technology and diabetes-specific educational content through a telehealth app. The attention control group will serve as an untreated comparison group for the AI4DM intervention group. Both groups will also receive 4 HbAAfter enrolling in the study, participants will automatically be emailed a Health Insurance Portability and Accountability Act (HIPAA)–compliant Research Electronic Data Capture (REDCap) link to a survey packet. REDCap is a secure program developed by a collaboration between Vanderbilt University and the National Institutes of Health National Center for Research Resources that manages and stores clinical trial data. This survey packet included a demographic survey and the secondary outcome measures listed in 1c KitsHbA).After the first survey packet is emailed to the participants, a study team member will call the participants. The purpose of this call is to welcome them into the study and explain the study elements. During the call, the staff will inform the participant that they will receive a package within the next few days , 3 months, 6 months, and postintervention (12 months). The HbA1c kit will include a testing kit and return packaging materials. Participants will be asked to complete and return the kit at their earliest convenience.All participants, regardless of study arm allocation, will receive 4 HbA1c kit are completed.A randomization sequence will be generated and stored within REDCap, which automatically assigns a participant to the AI4DM intervention group or the attention control group. Randomization will only occur after the baseline survey packet and baseline HbAAfter randomization, a study health coach will be notified from REDCap that a participant has been randomized to one of the study arms and will conduct an orientation call with the participant. The purpose of this call is to introduce themselves to the participants as their primary health coach and to schedule the first weekly coaching call. The first weekly call, regardless of the study arm assignment, will be scheduled for the next calendar week.If a participant is randomized into the AI4DM intervention group, the research staff will coordinate the shipment of a technology package that will be delivered to the participant’s residence within 1 to 2 days after randomization. The contents of this intervention package will include (1) an Amazon Echo, (2) a Fitbit Flex device, and (3) a wireless glucometer. Participants will also be given supplemental instructions for signing in to the mobile health  app and device setup instructions. This package is used for intervention delivery and not for outcome measurements.Coaching calls for the intervention group will be guided by a prepared outline of diabetes-related content that will be delivered to the participant through the mHealth app. This content will be delivered at the beginning of each week before the coaching call. The educational content is derived from using the Partnership to Improve Diabetes Education program and delivered to the participant as multimedia content . Other hBiweekly coaching calls during the following 12 weeks will cover nutrition and eating habits, exercise and PA, glucose monitoring, medication adherence, general well-being, and Partnership to Improve Diabetes Education content, as needed.The coaching content for the attention control group will refrain from any diabetes-specific content; rather, the health coach will only focus on general well-being. This will apply to all phone calls during weekly and biweekly coaching calls.After the study duration, up to 30 participants from the AI4DM intervention group will be contacted for a follow-up interview to obtain their experiences and feedback with the AI4DM telehealth platform. These interviews will be conducted virtually.1c with the secondary outcome measures described in Primary and secondary outcome measures will be assessed at baseline, 3 months, 6 months, and postintervention (12 months). The primary clinical outcome measure is HbA1c kits will be distributed and received by a clinical at-home laboratory testing service. Study personnel will use the company’s web-based resources to ship at-home test kits to all participants. The test kit will contain all necessary items to collect, prepare, and mail dried blood specimens to the laboratory for testing. Upon receiving the kit, the testing company will report the values through their network, and study personnel will obtain the values and record them directly into REDCap.HbAAll other measures, including demographic data and secondary outcome measures, will be retrieved and automatically stored through electronic surveys delivered by REDCap. All self-reported items within the questionnaires will be required to prevent missing data. The questionnaire packet will be delivered electronically to the participants at 4 time points during the study .To measure psychological distress, participants will complete the Diabetes Distress Scale (DDS). This 17-item questionnaire assesses diabetes-related emotional distress using Likert-style questions for the previous month. The DDS has been shown to be a valid and reliable tool for measuring diabetes-related emotional distress ,38.Participants will complete the Diabetes Quality of Life Measure, which is a 15-item questionnaire asking about perceptions of one’s ability to manage their diabetes in conjunction with other life areas .To measure psychosocial self-efficacy in diabetes management, participants will complete the Diabetes Empowerment Scale Short Form (DES-SF). The DES-SF is an 8-item questionnaire asking about attitudes toward diabetes and the ability to successfully manage diabetes. The DES-SF has been shown to be a valid and reliable measure of psychosocial self-efficacy .Participants will complete the Diabetes Social Support Questionnaire, which is a 52-item questionnaire that asks questions related to familial support for individuals with diabetes . This quTo measure PA, participants will complete the Godin Leisure-Time Exercise Questionnaire. This short questionnaire asks three questions related to strenuous, moderate, and light exercises performed during the past seven days .Participants will complete the UK Diabetes and Diabetes Questionnaire to assess nutrition and dietary behaviors within the past month . This 24To measure medication adherence, participants completed the Medication Adherence Rating Scale. The Medication Adherence Rating Scale is a general, 10-item binary questionnaire that asks questions related to regularly taking medication .Participants will complete the eHealth Literacy Scale, which is an 8-item survey that measures perceived knowledge, comfort, and skill when using technology to address overall health .To measure the usability of the telehealth dashboard, participants will complete the System Usability Scale and the Health Information Usability Evaluation Scale. These Likert scales include items asking about the dashboard’s effectiveness (ability to complete tasks), efficiency (level of dashboard use), and satisfaction (subjective reactions to the dashboard) ,47.Participants will be asked to provide current medications and dosages throughout the study duration to control medication data during the statistical analysis.This study will obtain measures related to the feasibility and acceptability of the AI4DM intervention on diabetes self-management. Measures will include process, resource, scientific, and management feasibility outcomes. All elements of intervention delivery will be collected, including but not limited to adherence, retention, attrition, coach and participant communication needs, staff preparation, and adverse events. Obtaining these measures will inform future considerations in the delivery of a web-based diabetes self-management program for people with disabilities.The study duration for those enrolled in the study will be 12 months, regardless of study arm allocation.A total of 90 participants will be enrolled. The sample size for this protocol is based on a primary analytic strategy of analysis of covariance, a two-sided test, type 1 error rate of 0.05, and intention-to-treat analysis with multiple imputations. Assuming a correlation between baseline and follow-up outcomes of at least 0.7, we will have 80% power to detect an effect size of 0.65.The entire recruitment will be conducted on the web nationally through the National Center on Health, Physical Activity and Disability website and its associated social media. Through these mechanisms, interested individuals will be directed to a landing website page that will have promotional media content and information about the study. From there, participants will be directed to a separate link through HIPAA-compliant REDCap and complete a screening eligibility form. If deemed eligible, the participants will be sent an electronic consent form to be completed using the HIPAA-compliant REDCap. On the basis of our recruitment methods, we expect that approximately 180 individuals will be screened for eligibility.1c kit has been completed by the participant. The computer-generated randomization process will be implemented in REDCap after the data from the baseline surveys and HbA1c kit are entered into the REDCap database. This will trigger the allocation procedure to randomly assign participants to one of the two study arms. The health coach will be notified about the randomization through the telecoaching dashboard and will inform the participants about their randomization into the AI4DM intervention or the attention control arm during the orientation call.Participants will be randomized to the two study arms in a 1:1 allocation ratio using a computer-generated randomization procedure. The allocation sequence will be implemented after the baseline surveys, and the baseline HbAThe principal investigator and primary statistician will be blinded to the randomization of the participants into the study arms. All other study staff will be unblinded for purposes of recruitment, consent, orientation calls, welcome calls, coaching calls, and intervention package shipping. There are no circumstances in which the principal investigator or statistician will become unblinded during this protocol.1c values are completed.All data collected will be entered directly into REDCap. Participants will be assigned a subject number upon enrollment in the study. Only approved study personnel will have access to the REDCap program, where data will be stored. Data checking will involve confirming that surveys, questionnaires, and HbA1c) will control for the impact of medication and dosage during the intervention period.All data will be examined for normality violations, outliers, errors, and patterns of missing values. For primary and secondary outcome measures, the sample and variables will be described using frequency distributions and appropriate summary statistics. Randomization across the study arms will be checked. If any baseline variable differs by group, it will be included as a covariate in subsequent analyses. Our primary analysis for the hypothesis that adults in the AI4DM intervention group will result in better glycemic control than the attention control group will be tested using analysis of covariance with baseline measurement as a covariate. Multiple imputations will be used to impute missing data. The study data will also explore the pattern and missing mechanism, and additional sensitivity analyses will be performed to test parametric assumptions as well as assumptions of missingness. Outliers and normality of quantitative variables will be evaluated. This study is powered to enable the estimation of effect sizes. Analysis of the clinical outcome  to ensure proper and consistent adherence to the study design and implementation. Elements of auditing will include checklists, coaching call logs, audio recordings or coaching calls, content resource banks, telehealth platform reviews and event logs, review of participant food, PA, medication, glucose entries, time spent on the platform, and team meetings to discuss participant progress and protocol adherence.All proposed elements of this protocol will be approved by the university’s institutional review board before beginning the study.Any changes deemed necessary by the principal investigator will be submitted as amendments to the university’s institutional review board and will only make changes after approval.All consent processes will be conducted on the web through predetermined screening questionnaires provided through the landing website and REDCap link. If a participant is deemed eligible through the web-based process, an electronic consent form will be sent to the participant to complete. Only approved study staff will have access to complete records of the consent form.Personal information will be collected in this study. All personal information shared, such as demographic details, will be collected and stored through HIPAA-compliant REDCap. Only approved study staff will have access to securely stored personal information. All information will be subject to the university’s institutional review board and the policies of affiliated entities surrounding confidentiality.There are no declarations of interests for all study staff for this protocol.This is not applicable. No provisions for posttrial care are included in the study.The results of this study will be disseminated publicly through publications in peer-reviewed journals and presented at regional and national conferences.1c analysis at 4 different time points . The laboratory testing service will be responsible for mailing, receiving, and processing completed HbA1c kits. The study personnel will not interact or have access to blood samples at the testing laboratory.Blood samples will be collected for HbAThe AI4DM protocol has been approved by the university’s institutional review board and has been registered at ClinicalTrials.gov . The mHealth app  and landing website are currently under development by the study technical support team, and research personnel are developing the database (REDCap) that will store data and communication mechanisms . We estimate that enrollment for the study will begin in October 2021.Although diabetes mellitus is one of the most common metabolic disorders affecting millions in the US population, this disorder has become more prevalent in people with disabilities . Compare1c) for people with disabilities using mobile and web-based apps, telehealth coaching sessions, and diabetes-related educational multimedia content. The AI4DM study will also focus on quality-of-life measures during the intervention and will include qualitative interviews following the intervention to provide a holistic evaluation of the intervention program as a viable mode of diabetes management. Next, to reduce barriers experienced by people with disabilities, the AI4DM program will be conducted completely on the web with all coaching sessions taking place over the telephone, thereby allowing all participants to manage their diabetes in a home environment. Finally, the results of this feasibility study will inform how web-based chronic disease management programs can be improved for future studies.As multiple barriers exist that would hinder a person’s ability to successfully manage diabetes, AI4DM seeks to create a sustainable, scalable, accessible, and inclusive diabetes management program for people with disabilities, which currently does not exist. This study will examine the feasibility and preliminary efficacy of AI4DM for glycemic control (HbA


BSX and NRGN genes were not included, though 30 of the 96 HPO identifiers associated with this OMIM were identified in the patient. The deletion of the FLI-1, ETS1, JAM3 and THYN1 genes was considered to be directly associated with the immunodeficiency exhibited by the patient. Although immunodeficiency is widely accepted as a major sign of JBS, only constipation, bone marrow hypocellularity and recurrent respiratory infections have been included in the HPO as terms used to refer to the immunological defects in JBS. Exhaustive functional analysis and individual monitoring are required and should be mandatory for these patients.Jacobsen syndrome or JBS (OMIM #147791) is a contiguous gene syndrome caused by a deletion affecting the terminal q region of chromosome 11. The phenotype of patients with JBS is a specific syndromic phenotype predominately associated with hematological alterations. Complete and partial JBS are differentiated depending on which functional and causal genes are haploinsufficient in the patient. We describe the case of a 6-year-old Bulgarian boy in which it was possible to identify all of the major signs and symptoms listed by the Online Mendelian Inheritance in Man (OMIM) catalog using the Human Phenotype Ontology (HPO). Extensive blood and marrow tests revealed the existence of thrombocytopenia and leucopenia, specifically due to low levels of T and B cells and low levels of IgM. Genetic analysis using whole-genome single nucleotide polymorphisms (SNPs)/copy number variations (CNVs) microarray hybridization confirmed that the patient had the deletion arr[hg19]11q24.3q25x1 in heterozygosis. This alteration was considered causal of partial JBS because the essential  BSX, NRGN, ETS-1, FLI-1 and RICS (ARHGAP32) genes are deleted 11q24.3q25x1 . PartialIM genes . This waThe rest of the detected CNVs were considered normal due to their existence in the reference population database, which suggests that they do not have any relation to the phenotype or deleterious capability. Loss of heterozygosity regions did not reveal consanguinity or uniparental disomy. Germline mosaicism was also ruled out as a cause.BSX, NRGN, ETS-1, FLI-1 and RICS (ARHGAP32) are included in the deletion [http://human-phenotype-ontology.github.io, accessed on 12 February 2021). The relationship between combinations of deleted genes and distinct sets of characteristics is being investigated in patients with a molecular diagnosis of partial JBS to determine correlations between specific genetic alterations and phenotypes of JBS. The existence of 30 out of 96 HPO terms or identifiers in our case was more concordant with the clinical phenotype described for complete JBS, although the underlying causal genetic alteration corresponded to partial JBS.Since the description of JBS in 1973, over 200 cases have been reported; all of them have been characterized by deletions affecting the telomeric q region of chromosome 11. This terminal haploinsufficiency in chromosome 11 could affect more than 100 different genes. The diagnosis of complete JBS is established when the genes Face2Gene is an increasingly used tool for phenotyping and is considered to be a standard of professional excellence in genetics services due to its proven reliability. However, in our case, Face2Gene did not correlate the patient’s appearance with facial schemes associated with Jacobsen syndrome. The software instead suggested a differential diagnosis of NBS, which we considered to be a plausible suggestion, and had we not had a molecular diagnosis of JBS, we would have investigated the susceptibility gene for NBS (NBS1).Reports of JBS cases in the literature have found quite distinct cytogenetic alterations, making the diagnosis of JBS more difficult. Gadzickit and colleagues described a case of JBS caused by an inversion: inv(11)p15q24) . Other cq24 . OthThe phenotype of JBS patients specifically includes defects in immune system function. Some authors have suggested that JBS should be considered primarily as an immunodeficiency syndrome . Early rFLI-1) [FL1-1 has been proposed to cause functional impairment and deficiency of T helper cells, dysmegakaryopoiesis and low serum level of IgM in patients [FL1-1 is considered to be the most relevant gene for inducing thrombocytopenia, and deletion of FL1-1 has also been associated with high levels of megakaryocytes and T and B leukocytes and low serum levels of IgM. However, FLI-1 is not the only candidate gene for causing immunological defects in JBS and PTS patients. ETS1, NFRKB, JAM3 and THYN1 genes could also play a role in the presence of immunodeficiency. ETS1 encodes a transcription factor from the ETS family, which has been proposed to be involved in hematopoiesis, thrombocytopenia, pancytopenia and even in the formation of micromegakaryocytes. NFRKB encodes a DNA-binding protein (R-κB) that regulates IL2R α-chain gene expression, which is crucial for the activation of T cells. JAM3 is a member of the junction adhesion molecule family located in the cell surface, with a high expression in CD8+ T cells, as well as activated T lymphocytes. Additionally, JAM3 is highly expressed on megakaryocytes and platelets and has been proposed to participate in the inflammatory process mediated by monocytes. Finally, the THYN1 gene encodes a thymocyte nuclear protein that has been suggested to be involved in apoptosis and is present in CD34+ hematopoietic stem and progenitor cells. The expression of the THYN1 gene in these immune cells leads us to propose its implication in the immunodeficiency of JBS patients, along with FLI-1, ETS1, NFRKB and JAM3.Occasionally, authors have related the observed immunodeficiency seen in JBS to Paris-Trousseau syndrome . PTS is characterized by thrombocytopenia with abnormal platelets with alpha-granules and dysmegakaryopoiesis and is caused by deletions in the 11q23 region, which is located centromeric to the susceptibility region for JBS ,21. Due FLI-1) . This gepatients ,19,22, aThe first case of JBS with immunologic deficiency was reported in 1998 . The patBARX2 gene because of its role in the formation of the ectodermal lining of the mandibular and maxillonasal tissues and the tissues surrounding the eye during craniofacial development. The dysmorphic facies seen in patients with JBS have been correlated with the deletion of the ST14 was also included in the deletion in our patient. Per OMIM, ST14 is related to ichthyosis with hypotrichosis syndrome in a recessive inheritance manner. Thinning hair and hyperkeratosis were evident in our patient, suggesting an alteration associated with the structure of the stratum corneum.The HPO database only includes eczema (HPO: 964) among the features of JBS that relate to skin and hair. The gene The congenital heart defects described in some of the reported cases in the literature were also observed in our patient. The 7 Mb terminal region of the 11q chromosome has been proposed as a critical region for a putative causative gene or genes for human congenital heart disease, such as hypoplastic left heart associated with severe thrombocytopenia ,12,27. TThe mild hyponatremia detected recurrently in our patient will be controlled, but the disequilibrium of ions was not considered until the last medical follow-up.Following an exhaustive review of the signs and symptoms of partial and complete JBS phenotypes, we suggest a revision of the described phenotype and the inclusion of additional identifiers. Primary immune deficiency should be included as a major criterion of diagnosis, with a specific description of the functional effect on the immune system. Immunological analysis should be performed in each patient diagnosed with JBS, since early recognition of immunodeficiency enables earlier therapeutic intervention and prevention of recurrent infections.For genotype–phenotype correlation, phenotype description with the HPO database and molecular characterization of the genetic alteration in each case are essential. A detailed clinical description and the differentiation between Jacobsen and Paris-Trousseau syndromes should determine if they are two variants of the same syndrome or different syndromes. The failure of the Face2Gene software in predicting the Jacobsen syndrome phenotype based on photographs of the patient suggests high variability in facial features of patients with JBS.


Physical activity alleviates chronic stress. The latest research suggests a relationship between resilience and physical fitness. Beneficial adaptations of the hypothalamic-pituitary-adrenal axis, sympathetic nervous system, endocannabinoid system, and tryptophan pathway, which are induced by an active lifestyle, are considered to be conducive to resilience. However, detailed knowledge on the molecular link between the effects of acute and chronic physical exercise and improved resilience to stress in humans is missing. Moreover, the relationship between innate and acquired aerobic capacity and resilience is poorly understood.The aim of this study is to implement a human exercise intervention trial addressing the following main hypotheses: a high innate aerobic capacity is associated with high resilience to stress, and web-based physical exercise training improves aerobic capacity of physically inactive adults, which is accompanied by improved resilience. In this setting, we will analyze the relationship between resilience parameters and innate and acquired aerobic capacity as well as circulating signaling molecules.A total of 70 healthy, physically inactive  adults (aged 18-45 years) will be randomly assigned to an intervention or control group. Participants in the intervention group will receive weekly training using progressive endurance and interval running adapted individually to their remotely supervised home training performance via web-based coach support. A standardized incremental treadmill exercise test will be performed before and after the intervention period of 8 weeks to determine the innate and acquired aerobic capacity (peak oxygen uptake). Before and after the intervention, psychological tests and questionnaires that characterize parameters implicated in resilience will be applied. Blood and saliva will be sampled for the analysis of cortisol, lactate, endocannabinoids, catecholamines, kynurenic acid, and further circulating signal transducers. Statistical analysis will provide comprehensive knowledge on the relationship between aerobic capacity and resilience, as well as the capacity of peripheral factors to mediate the promoting effects of exercise on resilience.The study was registered in October 2019, and enrollment began in September 2019. Of the 161 participants who were initially screened via a telephone survey, 43 (26.7%) fulfilled the inclusion criteria and were included in the study. Among the 55% (17/31) of participants in the intervention group and 45% (14/31) of participants in the control group who completed the study, no serious adverse incidents were reported. Of 43 participants, 4 (9%) withdrew during the program  and 8 (19%) have not yet participated in the program; moreover, further study recruitment was paused for an indeterminate amount of time because of the COVID-19 pandemic.Our study aims to further define the physiological characteristics of human resilience, and it may offer novel approaches for the prevention and therapy of mental disorders via an exercise prescription.DERR1-10.2196/29712 Physical inactivity is one of the major risk factors for global mortality; therefore, an active lifestyle is highly important to promote health and longevity. The risk for cardiovascular diseases, type 2 diabetes, osteoporosis, metabolic syndrome, specific cancers, and several other diseases is reduced by being physically active , and phyTo show the influence of exercise type, frequency, duration, and intensity on mental health, a cross-sectional study was conducted with 1.2 million individuals in the United States between 2011 and 2015 . It was 2peak), it has been shown that individuals who perform interval training have a higher increase in VO2peak ) is the most common German version of the Center for Epidemiologic Studies Depression Scale ) ) . The MDB [MDBF]) -92. As i [MDBF]) ,93, we wThe Habitual Physical Activity Questionnaire is divided into 3 indices evaluating (1) occupational physical activity, (2) sports, and (3) leisure time, and it originally included 16 items . The GerThe long version of the International Physical Activity Questionnaire contains 27 items and collects physical activity values in different domains  and different intensities (moderate and vigorous) within the past 7 days . The GerThe 36-item Short-Form Health Survey is a cross-disease assessment instrument for measuring the health-related quality of life of the general population and patients . It consno. This questionnaire is used as an exclusion criterion in this study and is used in the telephone survey. If a question is answered with yes, the person is not included in the study.The Physical Activity Readiness Questionnaire is an internationally renowned preparticipation screening tool that was based on expert opinion  . It inteAs the study design aims to investigate acute stress effects at several time points (T0 and T2) by focusing on the change in saliva cortisol levels as a primary end point, 2 alternative paradigms for stress induction are required, which are described below. Thus, habituation and expectation effects can be reduced based on the first stress induction paradigm. These psychological stress tests will be conducted between 1:30 PM and 4 PM to control the circadian cortisol level. To eliminate possible contaminations of saliva, participants will be instructed to refrain from eating or drinking for 1 hour before the stress tests. In addition, participants will watch a relaxing movie for approximately 20 minutes to minimize baseline differences in cortisol concentrations.Stress induction at T0 will be performed by means of the SECPT . The parStress induction at T2 will be performed using the compact version of the psychosocial stress task for scanner environments (ScanSTRESS) paradigm ,52. FirsTo validate the effectiveness of the stress tests performed, 6 saliva samples will be collected throughout the experiment to detect the stress hormone cortisol. Commercially available Salivettes (Sarstedt) will be used for this purpose. All saliva samples will be stored at –20 °C and sent to the Institute of Biopsychology at the Technical University Dresden, Germany, for analysis. Salivary concentrations will be measured using commercially available chemiluminescence immunoassays with high sensitivity .To detect differences in the ability to regulate emotions, 2 different tests will be used in parallel to the stress tests.view condition). In the second task condition, the participants are asked to cognitively modify the content of the presented image by assigning it a positive content  and thereby regulating their emotions. In the third task condition, participants are asked to indicate as fast as possible if the given mathematical equation is correct or incorrect via a button press (distraction). For this purpose, the participants are presented with both negative (first image category) and neutral images (second image category). The images are taken from the internationally standardized and evaluated databases International Affective Picture System) ) of the calves (CTsr). The infrared camera will be placed on a tripod behind the participant at the height of 85 cm. The distance between the camera and participant will be 2.30 m. The focus will be set perpendicular to the backside of the calves to capture the area from the popliteal space to the ankle. To stabilize the running position on the treadmill, a horizontal barrier made of elastic-plastic will be fixed behind the participant, and the foot position will be marked on the treadmill with adhesive tape. The infrared camera temperature scale will be set from 25 °C to 35 °C. An acclimatization period of 10 minutes will be implemented before the exercise test. The participants will only be allowed to wear ankle socks and shorts; should sleep and fast for 8 hours; should drink not more than 1.5 L of water; should be physically inactive; and should avoid cosmetics, showering, and sunbathing for at least 8 hours before the test.Infrared thermography is a noninvasive, contact-free, nonradiating tool used to measure surface radiation temperature and patterns . In thisAnalysis of thermal images–TDM V2.0 (Optoprecision GmbH). The report of the entire infrared thermography procedure is in accordance with that of Moreira et al . I. I121]. All data will be recorded and stored in the Department of Sports Medicine, Prevention, and Rehabilitation , and access will be restricted to authorized personnel. Confidential information will be stored safely in the trial files and will not be shared with any other third institution or entity except in response to a legal requirement. Electronic data will be stored on password-protected computers in a restricted-access building. All data will be pseudonymized and submitted to the evaluation team for analysis. A data manager from each department is nominated, who will be responsible for data management and processing. The data and safety management team will be formed by the members of the project. Only the data manager will have access to all personal data. Trial documentation and data will be archived for 10 years after completion of the trial. All dropouts and the reason for dropping out of the study will be reported. Any harm or unintended events during the examinations will be recorded and reported to the Landesärztekammer  Rhineland-Palatinate. The results will be publicly available in open-access journals and presented via contributions at conferences and congresses according to good scientific practice.2peak) and experimental stress reactivity . On the basis of the results of Bacon et al [f(V)=0.35. An alpha error set to .05 and statistical power of 0.80 were assumed for a multivariate analysis of variance (MANOVA) with 2 repeated measurements, 2 groups, and interaction effects. Descriptive statistics will be used for all outcome measures at each time point to give an overview of all results. First, MANOVA will be performed with the main primary outcomes as dependent variables, that is, aerobic capacity (VO2peak) and experimental stress reactivity  to maximize the power of the analyses and to be able to investigate the main and interaction effects of time  and group (IG vs CG). Groups should be equal at preintervention (T0 and T1). A time effect should be observed for the IG. Furthermore, we expect a significant time×group interaction as the IG should react differently over time, with a higher increase in aerobic capacity (VO2peak). Significant effects of the MANOVA will be followed up by posthoc analyses of variance. As a secondary aim of our study, we aim to identify variables  that mediate the relationship between physical exercise and resilience outcomes. To that end, we plan to calculate multiple regression analyses that include potential mediators. Principal component analyses regarding potential mediating variables will precede the mediator analyses to avoid redundancy in our regression models. This will be statistically detailed with posthoc tests and corrections for multiple comparisons . Missin2peak and cortisol levels) because of the lockdown of the laboratory facilities. Further study recruitment will resume when COVID-19 restrictions are completely lifted. On the basis of the experiences from the first recruitment period, the study is expected to continue for another 6 months to complete the intended sample size.The study was registered in October 2019 (DRKS00018078). Enrollment began in September 2019 and was paused from April 2020 until submission of this study protocol because of the COVID-19 pandemic. Of the 161 interested people who have contacted us so far, 26.7% (43/161) fulfilled the inclusion criteria. Among the 55% (17/31) participants in the IG and 45% (14/31) participants in the CG, who completed the study (N=31), no serious adverse incidents were reported. Within the program, 9% (4/43) of participants  withdrew because of individual reasons (dropout rate 11.4%). Approximately 19% (8/43) have not yet participated in the program because of the COVID-19 pandemic. Although the COVID-19 pandemic does not affect the web-based exercise training approach, the study had to be paused in April 2020, as it was not possible to determine the primary and secondary end points  participants with a high innate aerobic capacity will show a higher resilience to stress compared with participants with low aerobic capacity. Furthermore, we hypothesize that (2) 8 weeks of individually structured interval-type and continuous endurance exercise training remotely supervised by a web-based approach will improve the aerobic capacity of healthy inactive adults and that (3) the change from sedentary to an active lifestyle will improve resilience. In this study setting, we will be able to exploratively examine the molecular link between the effects of acute and chronic physical exercise and improved resilience to stress.Evidence that exercise can lead to various benefits in mental health and resilience is increasing ,124-129.2peak in healthy, physically inactive adults are as follows: (1) participants are not location dependent for the training sessions; (2) individualized, gradual, and timely adjustment of training content based on the individual’s feedback reduces the risk of injury, avoids overtraining, and avoids ineffective training; and (3) only 1 sports therapist or trainer is needed for training management of several training groups or trainees, which obviously has economic advantages and reduces bias from different sports therapists. However, web-based training support also has disadvantages compared with presence (face-to-face) interventions. Notably, it requires access to the internet, which limits the study population. Furthermore, we are dependent on the correct and complete documentation of the training sessions by the participants. For an objective way to control reports on the training and to remotely supervise the participants in the IG, we included a smartwatch (Polar M430) that records the heart rate and distance of the individual training sessions. These are given to both the IG and CG participants to exclude an effect on the study outcome only by wearing the smartwatch. Furthermore, group or partner training can bring motivational and social benefits [The strengths of our web-based, supervised physical exercise training approach with a focus on increasing VObenefits -132, whibenefits -45.2peak more strongly in comparison with continuous exercise training [2peak of 3%-19% [Here, we selected an exercise intervention period of 8 weeks using a combination of interval-type training and continuous endurance exercise to efficiently enhance aerobic capacity in a short period. Interval training improves VOtraining -135. Howf 3%-19% . Althougf 3%-19% , it has f 3%-19% . TherefoRepeated stress induction and the estimation of resilience factors in a test-retest setting, as chosen in our study, are obstacles to resilience research. It was shown that for psychological stress tests, a test-retest design could lead to a learning effect, resulting in lower stress response ,137. Her2peak and its effect on resilience only. However, the identified relationships between molecular markers and exercise-induced resilience parameters in our setting may be further elucidated in mixed model interventions in follow-up studies.Several psychological interventions have been similarly used to increase resilience , and it We cannot exclude a selection bias so that persons who are generally interested in (1) psychological questions, (2) physical exercise, or (3) web-based services are rather addressed by the study announcement. Furthermore, because of a high time commitment for presence tests (2 measurement time points before and after the intervention), (4) people who live close to the survey site are more likely to participate and complete the study. On the basis of the World Health Organization guidelines on physical activity and sedentary behavior, we included individuals who were physically active for <150 minutes/week . These iResilience is a dynamic and variable state that can be represented in a multifactorial manner. So far, individual instruments have failed to fully capture the variance of resilience ,141,142.This study will allow us to investigate whether an increase in physical activity with a simultaneous improvement in aerobic capacity is associated with an increase in resilience and whether this effect is reflected in circulating molecular marker levels. These findings will help to reveal novel characteristics of human resilience and may offer novel approaches for the prevention and therapy of mental disorders by exercise prescription.


Background: The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has placed a significant demand on healthcare providers (HCPs) to provide respiratory support for patients with moderate to severe symptoms. Continuous Positive Airway Pressure (CPAP) non-invasive ventilation can help patients with moderate symptoms to avoid the need for invasive ventilation in intensive care. However, existing CPAP systems can be complex (and thus expensive) or require high levels of oxygen, limiting their use in resource-stretched environments.Technical Development + Testing: The LeVe (“Light”) CPAP system was developed using principles of frugal innovation to produce a solution of low complexity and high resource efficiency. The LeVe system exploits the air flow dynamics of electric fan blowers which are inherently suited to delivery of positive pressure at appropriate flow rates for CPAP. Laboratory evaluation demonstrated that performance of the LeVe system was equivalent to other commercially available systems used to deliver CPAP, achieving a 10 cm H2O target pressure within 2.4% RMS error and 50–70% FiO2 dependent with 10 L/min oxygen from a commercial concentrator.Pilot Evaluation: The LeVe CPAP system was tested to evaluate safety and acceptability in a group of ten healthy volunteers at Mengo Hospital in Kampala, Uganda. The study demonstrated that the system can be used safely without inducing hypoxia or hypercapnia and that its use was well-tolerated by users, with no adverse events reported.Conclusions: To provide respiratory support for the high patient numbers associated with the COVID-19 pandemic, healthcare providers require resource efficient solutions. We have shown that this can be achieved through frugal engineering of a CPAP ventilation system, in a system which is safe for use and well-tolerated in healthy volunteers. This approach may also benefit other respiratory conditions which often go unaddressed in Low and Middle Income Countries (LMICs) for want of context-appropriate technology designed for the limited oxygen resources available. The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has placed a significant demand on healthcare providers (HCPs) to provide respiratory support for patients with moderate to severe symptoms , a chang2 (Fraction of inspired oxygen) which allows clinicians to specify an appropriate valve from a measurement of the patient's oxygen saturation levels, with pressure in the circuit controlled using a Positive End-Expiratory Pressure (PEEP) valve design  , where limitations in healthcare infrastructure often precludes oxygen delivery from a centralised, pressurised source. Instead, portable oxygen concentrators provide a sustainable, reliable and cost-effective solution  in contrast to supply from compressed oxygen cylinders which require a reliable supply infrastructure and continual monitoring , 20. NGO2) , 22. Thi2) . The ove2)  and in t2) .LeVe (“light”)—CPAP systems with low complexity and high resource efficiency—both from a design and oxygen supply (flow rate and pressure) perspective. “Frugal innovation” provides a development approach to target this region. Weyrauch and Herstatt , which was adopted successfully within the Leeds Teaching Hospitals NHS Trust to support COVID-19 patients. Such systems have also been demonstrated in an open-source framework with a low raw component cost although the expertise to assemble and guarantee the quality of such a system is still relatively high . The flowrate required to maintain positive pressure is not set a priori, but determined to ensure that the pressure remains positive for all parts of the breathing cycle, nevertheless a typical guide value of 60 L/min provided an initial starting point for a suitable flow  supplied at the modest delivery pressures that can be achieved by oxygen concentrators (2O)  . A generThe heterogenous nature of healthcare settings means that adoption of a specific technology requires local assessment against availability of parts, manufacturing facilities and healthcare services. To support this, a wider discussion of overall system requirements is included within this work.The LeVe CPAP Blower system is based on the premise that a category of Brushless DC Current (BLDC) “fan blowers” typically used for thermal management in electrical equipment inherently have the flow dynamics required for provision of CPAP, in general providing relatively high flow rates at low but stable pressures. We propose a design compatible with an oxygen concentrator which intrinsically drives an oxygen efficient solution. Based on frugal engineering principles, the design centers around an appropriately specified, single electric fan-blower without the need for control features of more complex fan-based CPAP systems. The resultant breathing circuit also minimises the number of parts required for effective CPAP since careful choice of the fan and the use of a simple expiration port  means that no PEEP valve (which brings greater complexity and cost) is required.2O  at flow rates of ~100 L/min, thus ensuring the flow is greater than the peak inspiratory flow rate to maintain continuous positive pressure through the breathing cycle. By limiting the peak delivery pressure in this selection ensures there is a degree of safety for the patient should any speed regulation fail.We examined a range of BLDC fans capable of generating pressures in the range of 12–20 cm HCUI Devices CBM-979533B-168 , Sanyo Denki San Ace B97 9BMB  and B97 9BMC ].Two multinational manufacturers (selected to help ensure supply-chain availability) supply models which fall within these criteria [Sanyo Denki San Ace 9BMB model because it allows  control of the fan speed either by varying the supply voltage or by fixing this and using a Pulse Width Modulation (PWM) control input (the robust 555 timing chip can thus be used). The latter can reduce system complexity and potential failure modes (in comparison to directly varying the fan supply voltage). This model is available in 12 or 24 V models.Of these possibilities, we selected the 2O, selected to match typical requirements in COVID-19 treatment and with an upper limit advised by our clinical team  mains source to achieve a working pressure of 10 cm H2O , 29. The bar main2 of inspired air for a given oxygen flow rate. These aspects were investigated in controlled laboratory conditions to ensure appropriate performance prior to human use.The fundamental performance characteristics of a CPAP system can be measured through: (i) the pressure at the patient mask during a breathing cycle; and (ii) the FiOTo measure the flow dynamics of the system in a controlled and repeatable manner, a breathing simulator was developed. The simulator, summarised in Key parameters measured within the system were mask pressure , outlet flowrate of the blower , and oxygen concentration in the mask . Pressure and flowrate data were logged using a data acquisition system  at 200 Hz whilst the oxygen concentration was recorded once a steady state was reached. In each configuration, measurements were averaged over 10 cycles. Oxygen was supplied using a concentrator . Each system was connected to the breathing simulator using standard medical-grade components.2O. In general, a drop in pressure is observed during inspiration as air is drawn from the breathing limb, and a rise observed during expiration. For the baseline respiratory regime 1 (see 2O (2.4% error), in comparison the Nippy 3+ achieves 9.32 cm H2O (6.8% error). Across the breathing cycle the pressure range of the LeVe is 7.04 cm H2O and for the Nippy 3+ is 7.29 cm H2O. These characteristics are similar to those recorded for other commercial and open source CPAP systems (me 1 see  the RMS  systems .2O (Regime 1) and 4.32 cm H2O (Regime 4). It should be noted that pressures remained positive in all cases, which is important to ensure the lungs remain open.2 achieved for a given oxygen supply flowrate) than the LeVe system. More significantly, the respiratory regime has an effect on oxygen performance, in particular lower peak inspiration rates  resulting in increased efficiency and a reduction in the difference between systems. In both systems, the maximum FiO2 that can be achieved at 10 L/min O2  ranges from ca. 50 to 55%  to 70% .A pilot study was conducted to evaluate the safety and acceptability of the LeVe CPAP Flow Generator in a group of healthy volunteers at Mengo Hospital in Kampala, Uganda. Two research questions were set:Primary: Can the LeVe CPAP Flow Generator be used safely without inducing hypoxia or hypercapnia? Secondary: Is the LeVe CPAP Flow Generator well-tolerated by users?The healthy participants in the study are likely to present with lungs with normal lung compliance. While this does not necessarily replicate the lung condition of those with COVID-19 (the motivation for this work) it is a necessary first stage in demonstrating that the system can provide a safe and tolerable intervention.This study took place in the Intensive Care Unit at Mengo Hospital, Kampala, Uganda. A sample of 10 participants was recruited, all of whom were members of staff at the hospital. All participants were provided with a written information sheet, gave informed written consent and were not offered any financial incentive to participate in the study. Approval was obtained from the Mengo Hospital Research and Ethics Committee (M 02/01-2021) and the Uganda National Council for Science and Technology (HS1250ES). The protocol was registered and approved by the Pan African Clinical Trials Registry (PACTR202105734146484), a summary of key aspects is as follows. The inclusion criteria were for participants to be staff members at Mengo Hospital aged 20–50 years with no involvement in the research study. The key exclusion criteria were:Current or ex-smokerUnderlying respiratory conditionsBMI > 30Any contraindications from previous use of CPAP or oxygen therapy.2O). A commercially available oxygen concentrator was connected to the breathing circuit (as shown in 2) and end tidal CO2 (ETCO2) level were monitored continuously throughout the study. Oxygen saturation was measured by a pulse oximeter (Mindray VS900) with finger attachment, ETCO2 using a capnograph with the sample line attached to a port on the face mask. Hypoxia was defined as oxygen saturations <94% and hypercapnia was defined as ETCO2 > 5.7 kPa  and 50% were female. Measured data for end-tidal CO00% SpO2  across aercapnia .P = 0.0002) across the five groups with significant pairwise comparisons noted for both groups as denoted by CPAP values of 10 and 12 cm H2O when compared with the base readings in the absence of CPAP. This indicates a small but detectable improvement in oxygen saturation in participants with healthy lungs at the two highest values of CPAP pressure, potentially because this acts to reduce the physiological shunt. However, this outcome requires further study in a non-healthy population before any clinical significance can be attributed.In terms of tolerability, overall comfort was measured on a scale of 1–5 where 1 indicated “not at all comfortable” and 5 indicated “very comfortable.” 2O target pressure within 2.4% RMS error) and 50–70% FiO2 over representative respiratory regimes, despite their lack of closed-loop pressure regulation. This has a small impact on oxygen performance . This places an emphasis on the need to accompany such systems with appropriate training and clinical use protocols to ensure quality of care.The results of the study demonstrate that in a healthy cohort, the LeVe system is safe for use and well-tolerated by the participants. Measures of oxygen saturation demonstrate that the system does not induce hypoxia or reduce oxygen saturation, and in fact it may actually increase saturation levels. In a fit and well-population, the significance of any increase in SpOConsidering comfort, it is important to note that the provision of positive pressure through a face mask will inherently impact on “natural” inhalation and expiration, thus may cause anxiety and discomfort, regardless of the air source . Within Research has shown how the clinical efficacy of CPAP systems has evolved rapidly during the COVID-19 pandemic . ParticuCPAP ventilation systems provide an important treatment option for COVID-19 patients, particularly in the early stages before invasive ventilation strategies are required, to deliver oxygen-enriched air to stabilise patients until they can be escalated or de-escalated. To deliver this for the high patient numbers associated with the COVID-19 pandemic, healthcare providers require resource efficient solutions. We have shown that this can be achieved through frugal engineering of a CPAP ventilation system.The data from the pilot study indicate that the LeVe CPAP Flow Generator is safe to use in healthy volunteers and was well-tolerated by the cohort. This solution has different merits in clinical performance and efficiency to existing CPAP systems but provides resource-limited healthcare providers with a more resource-efficient solution to support flexible treatment pathways that can be rapidly deployed to reduce the burden on ICU during the COVID-19 pandemic. Beyond this immediate need, there is also evidence that CPAP can help provide much needed therapeutic benefit to address other respiratory conditions  which often go unaddressed in LMICs for want of context-appropriate technology.Our ambition is that this work will support the treatment of patients suffering from COVID-19 and (beyond the current pandemic) expand treatment options available to healthcare professionals targeting respiratory distress syndromes. Our ongoing work will build on these foundations; firstly by evaluating the clinical efficacy of using the LeVe system to provide respiratory support in patients with COVID-19, secondly by exploring opportunities to make the technology commercially available, coupled with the requisite regulatory approval, for use in LMIC settings.https://licensing.leeds.ac.uk/.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Mengo Hospital  IRB and the Uganda National Council for Science and Technology approved this study. The patients/participants provided their written informed consent to participate in this study.PC and NK supervised the project and wrote the manuscript. WD and IW manufactured, tested, and analysed results from the systems. DJ, RH, AK, and CO provided technical assistance in the system development. GB, SA, ML, TB, and TR provided technical information to inform development of the system. TL, JP, RMi, and SM provided clinical expertise to guide development and evaluation. DB coordinated independent evaluation of the system. RMu, GN, AL coordinated and conducted the safety study at Mengo Hospital. EN was principal clinical investigator, supervising the safety study at Mengo Hospital. All authors contributed to the article and approved the submitted version.TR and RMi were employed by company Medical Aid International Ltd. The remaining 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.