The European Bioinformatics Institute (EMBL-EBI) maintains a comprehensive range of freely available and up-to-date molecular data resources, which includes over 40 resources covering every major data type in the life sciences. This year's service update for EMBL-EBI includes new resources, PGS Catalog and AlphaFold DB, and updates on existing resources, including the COVID-19 Data Platform, trRosetta and RoseTTAfold models introduced in Pfam and InterPro, and the launch of Genome Integrations with Function and Sequence by UniProt and Ensembl. Furthermore, we highlight projects through which EMBL-EBI has contributed to the development of community-driven data standards and guidelines, including the Recommended Metadata for Biological Images (REMBI), and the BioModels Reproducibility Scorecard. Training is one of EMBL-EBI’s core missions and a key component of the provision of bioinformatics services to users: this year's update includes many of the improvements that have been developed to EMBL-EBI’s online training offering. Based in Hinxton , EMBL-EBI maintains the world's most comprehensive range of freely available and up-to-date molecular data resources. EMBL-EBI’s activities take place within the broader context of service provision at EMBL, which enables scientists from around the world to take advantage of a broad portfolio of world-class infrastructures and resources through a single Europe-wide partner.EMBL-EBI has a 5-fold mission, including providing scientific services and training, carrying out bioinformatics research, disseminating cutting-edge technologies to industry, and supporting the coordination of biomolecular data provision in Europe. The synergy between these areas is one of the Institute's unique strengths. EMBL-EBI hosts a suite of over 40 open data resources, which encompass every data type in molecular biology. Data resources include deposition databases  and added-value databases , as well as open-source software tools. EMBL-EBI services are provided collaboratively, and many of its data resources are run in partnership with other organisations all over the world.www.embl.org/documents/document/embl-ebi-impact-report-2021). The same study highlighted research impacts worth £1.3 billion annually that could not have been realised in the absence of EMBL-EBI managed resources. These figures show the importance of EMBL-EBI managed resources for the global life sciences community, and more generally they demonstrate how open data resources are central to scientific progress.EMBL-EBI services are crucial to the life science community. On an average day in 2020, EMBL-EBI resources received over 81 million web and API requests. Throughout 2020, EMBL-EBI services were visited via almost 41 million IP addresses , and over 545,000 IP addresses visited its Train online platform. Moreover, by the end of 2020 EMBL-EBI reached over 390 petabytes of raw data storage. An economic Value and Impact study commissioned by EMBL-EBI in 2020 on its open data resources estimated a use value of £5.5 billion per annum for EMBL-EBI resources  , in collaboration with the artificial intelligence company DeepMind. AlphaFold DB provides open access to the predicted protein structures for the entire human proteome, alongside that of 20 other organisms important to biomedical research, including mice, rats, Caenorhabditis elegans and Drosophila melanogaster. The initial release of AlphaFold DB contained over 350,000 structures. The predictions are generated using AlphaFold 2, an artificial intelligence (AI) system developed by DeepMind . An extensive description of AlphaFold DB and its features is available in this NAR Database Issue at .EMBL-EBI launched the AlphaFold Protein Structure Database, AlphaFold DB  is an open resource for polygenic scores. A polygenic score (PGS) is a measurement for genetic predisposition for a trait or phenotype that aggregates the effects of many genetic variants into a single number.PGS Catalog . PGS are increasingly used in the study of various diseases, including heart disease, cancer and psychiatric illnesses. The PGS Catalog was instantiated to respond to a community need to systematically evaluate PGS and provide access to the information necessary to calculate the scores themselves ,7. The CAll EMBL-EBI data resources are continuously updated to ensure that they are relevant for the research community. EMBL-EBI databases that have had major updates are detailed in articles elsewhere in this issue of Nucleic Acids Research and include Ensembl , EnsemblThe European COVID-19 Data Platformhttps://www.covid19dataportal.org/), which was launched in April 2020 .During its first year, the European COVID-19 Data Portal received over 4.5 million requests by over 180,000 users. During the same period of time, users accessed over 1.5 million records, including viral genomic sequences, SARS-CoV-2 protein entries, and gene and protein expression data of human genes implicated in the virus infection of the host cells. Moreover, since March 2020 users of the Portal have submitted over 2.2 million datasets, spanning across the various data types present in the COVID-19 Data Portal, in addition to > 450,000 linked publications. This includes >1.1 million assembled SARS-CoV-2 nucleotide sequences  to produce its reports on mutations and variation in SARS-CoV-2 to a large number of public health and other stakeholders. The most recent VEO report at the time of writing, its seventh, is available at We are keen to acknowledge the strong partnerships that have enabled EMBL-EBI to respond rapidly and at scale with the European COVID-19 Data Platform, in particular EMBL-EBI’s partnership with ELIXIR. Through its network of bioinformatics experts and capacity around Europe, ELIXIR has been particularly important in the mobilisation of European data activities and National Portals.Europe PMC is now offering access to preprintshttps://europepmc.org/). Since 2018 Europe PMC has been aggregating and indexing preprint abstracts alongside peer-reviewed full text articles  makes them significantly more discoverable. The COVID-19 full text preprint collection can also be accessed programmatically, with over 20,000 open access preprints available via bulk download as of September 2021. This enables large-scale analyses of the COVID-19 preprint corpus and provides a basis to explore new approaches to open and rapid publication systems.EBI Search has grown and further developed its Search-as-a-Service featureshttps://www.ebi.ac.uk/ebisearch/s4). Through EBI Search, users can navigate across different data resources via a network of cross-references. EBI Search is maintained in sync with all established EMBL-EBI Data Resources and is currently indexing ∼5 billion entries.EBI Search  is a seaEBI Search can be accessed in a web browser or programmatically using the RESTful Web Services interface. The EBI Search API  is being used behind the scenes by a growing number of EMBL-EBI data resources and tools, including the COVID-19 Data Portal, Ensembl, RNAcentral, OmicsDI, ENA, MGnify, InterPro, and the HMMER web server. Programmatic use of EBI Search allows it to be used in analytical pipelines and workflows, enriching the results from analysis tools , such astrRosetta and RoseTTAfold models were introduced in Pfam and InterProhttp://pfam.xfam.org/; https://www.ebi.ac.uk/interpro/) databases made available a set of 6370 structural models created by researchers at the University of Washington using the trRosetta software . To enable users to view and interpret the structural models, the team developed a new interface within the InterPro website, under the trRosetta tab of Pfam families  is the world's largest database of non-coding RNA sequences  or run R2DT locally. In addition, R2DT is integrated into the sequence similarity search deployed on the RNAcentral and Rfam websites https://www.ebi.ac.uk/gifts/) connects Ensembl genes and transcripts to UniProt protein sequences: through its user-friendly platform, researchers can access human and mouse data and connect genomic and protein information. At the time of writing, GIFTS mapped 99% of UniProtKB human canonical sequences. Through GIFTS, biocurators at EMBL-EBI have the tools to annotate and exchange transcript and protein sequence information to map content between Ensembl and UniProt, improving the consistency of annotation available to users of these services.In 2021, the Uniprot and Ensembl teams launched the Genome Integrations with Function and Sequence (GIFTS) service, which aims to provide a common framework for Ensembl and UniProt entity mappings. GIFTS  as possible . The prohttps://bit.ly/rembi_v1) have been divided into eight categories, including study information, study components (e.g. imaging method), biosample information , specimen data (e.g. preparation method), image acquisition (e.g. acquisition parameters), image data (e.g. format and compression), image correlation (e.g. related images and their relationship), and analysed data . The BioImage Archive is adopting REMBI for its submission pipeline: submitters will be guided through the process of supplying metadata for their imaging datasets, supported by clear examples and explanations at each stage. This process will provide integration with other existing data resources, for example by sharing sample metadata with BioSamples  is the repository of systems biology models managed by EMBL-EBI  with eiwww.ebi.ac.uk/training). External training activities across all parts of EMBL-EBI are coordinated with the goal of creating a coherent, high-quality programme with global reach. An ongoing project involving the EMBL-EBI web development team to make training content FAIR . This provides open access to competency frameworks of relevance to the molecular life sciences, enabling learning professionals to determine the needs of their audiences and analyse training gaps. The Competency Hub provides a stable, version-controlled home for the ISCB’s Competency Framework .Several ‘behind the scenes’ improvements contribute to the new site's adherence to FAIR principles. Bioschemas tags for live courses and webinars enable EMBL-EBI Training content to be pulled by other aggregators, such as ELIXIR’s Training e-Support System. Course information can also seamlessly be transferred from the training content database to the new embl.org events page ; this has enabled the Training team to achieve a clear visual identity for all of EMBL’s external training.The authoring tool for self-paced tutorials has been re-developed. The new WordPress-based authoring tool streamlines the writing, publishing and updating process and brings it in line with the content authoring mechanism for EMBL team websites. Finally, the EMBL-EBI training website was an early adopter of EMBL’s open Visual Framework (As the world and the scientific community recover from the ongoing COVID-19 pandemic, there is ample opportunity to reflect on the importance of open science and open data. Open science and FAIR data depend on continuously evolving data resources and community-driven data standards and guidelines. The work carried out at EMBL-EBI during the past year underscores these principles, offering new data resources for emerging data types, new features and updates to the current portfolio of data resources, and work on establishing new data standards and guidelines to drive the quality of the data contained within databases.


Slackiaisoflavoniconvertens, were encoded only to a minor extent in Slackia MAGs, but were more abundant in Adlercreutzia equolifaciens and an uncharacterized Eggerthellaceae species. In addition, enzymes with a sequence identity of about 35% were encoded in highly abundant MAGs of uncultivated Collinsella species, which suggests a hitherto uncharacterized daidzein-to-equol potential in these bacteria. Of all potential flavonoid modification steps, O-deglycosylation (including derhamnosylation) was by far the most abundant in this analysis. In contrast, enzymes putatively involved in C-deglycosylation were detected less often in human gut bacteria and mainly found in Agathobacter faecis (formerly Roseburia faecis). Homologs to phloretin hydrolase, flavanonol/flavanone-cleaving reductase and flavone reductase were of intermediate abundance  and mainly prevalent in Flavonifractor plautii. This first comprehensive insight into the black box of flavonoid modification in the human gut highlights many hitherto overlooked and uncultured bacterial genera and species as potential key organisms in flavonoid modification. This could lead to a significant contribution to future biochemical-microbiological investigations on gut bacterial flavonoid transformation. In addition, our results are important for individual nutritional recommendations and for biotechnological applications that rely on novel enzymes catalyzing potentially useful flavonoid modification reactions.Flavonoids are a major group of dietary plant polyphenols and have a positive health impact, but their modification and degradation in the human gut is still widely unknown. Due to the rise of metagenome data of the human gut microbiome and the assembly of hundreds of thousands of bacterial metagenome-assembled genomes (MAGs), large-scale screening for potential flavonoid-modifying enzymes of human gut bacteria is now feasible. With sequences of characterized flavonoid-transforming enzymes as queries, the Unified Human Gastrointestinal Protein catalog was analyzed and genes encoding putative flavonoid-modifying enzymes were quantified. The results revealed that flavonoid-modifying enzymes are often encoded in gut bacteria hitherto not considered to modify flavonoids. The enzymes for the physiologically important daidzein-to-equol conversion, well studied in  O-deglycosylation by epithelial enzymes. Flavonoid aglycons then undergo phase I/II transformation [Bifidobacterium and Lactobacillus were observed to O-deglycosylate, e.g., flavanone and isoflavone glycosides [O-deglycosylation was the most extensively described flavonoid deglycosylation, with many glycosides from flavonols , flavanones (often found in citrus fruits) and isoflavones (present mainly in soy products) as substrates, several bacteria also mediate C-deglycosylation from compounds such as isovitexin, e.g., from buckwheat. Additional flavonoid transformations involve demethylation, dehydroxylation, reduction, and C-ring cleavage [Flavonoids, which are exclusively plant secondary metabolites, are considered to bear many beneficial effects on health ,2,3,4,5 ormation ,9,10. Thormation ,12,13,14ycosides . While Ocleavage .O-deglycosylating enzymes, six beta-galactosidases [C-deglycosylating enzyme systems were studied in fewer gut bacteria [C-deglycosylation system described in strain PUE is dependent on at least three enzymes, DgpABC, which are part of a large gene cluster including transporters and other accessory genes whose function in C-deglycosylation is unclear [C-deglycosylating enzyme system of Eubacterium cellulosolvens includes the five enzymes DfgABCDE [O-demethylation of flavonoids are not yet described. However, an O-demethylase from Eubacterium limosum ZL-II demethylates polyphenolic lignans, [O-demethylases [O-demethylation of flavonoid carried out by E. limosum and E. ramulus [Many described gut bacterial flavonoid-converting activities lack biochemical or genetic analyses and, therefore, sequences of the involved enzymes. Of the osidases ,16,17 anosidases  that cleosidases ,22,23,24bacteria ,26,27  hydrolytically cleaves the C-C bond adjacent to the aromatic A-ring of phloretin and thereby produces phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The first Phy was discovered in  ramulus ,44. The  ramulus .E. ramulus was described to catalyze the conversion of, e.g., naringenin to phloretin also without CHI. Still, as CHIs [The flavanone- and flavanol-cleaving reductase (Fcr), cleaving the heterocyclic C-ring of flavanones and flavanonols, was recently characterized in detail . Earlier as CHIs ,47,48,49 as CHIs .All of the above studies were performed with few flavonoid-modifying bacteria and the corresponding enzymes originate from a limited number of bacterial species. Isolation of these strains from fecal samples could be biased because of selective enrichment of culturable bacteria. Therefore, the research on gut-bacterial flavonoid modification published up to now does not display a complete picture of flavonoid transformation in the human gut: phenotypic studies on human fecal samples rely mainly on  16S rRNA gene sequencing and do not resolve bacterial species. Thus, flavonoid-converting species might be missed. In addition, quantitative studies of flavonoid-converting bacteria are scarce. A recent study on the quantification of daidzein-to-equol-converting bacteria relied on primers targeting genes of involved reductases . With thHere, we aim to identify and quantify flavonoid-modifying bacteria in the human gut microbiota. For this, we screened the most recent combined human gut MAGs collection, the Unified Human Gastrointestinal Protein or Genome (UHGP/UHGG) catalog of more than 280,000 assembled genomes, with a BLAST search using protein sequences of characterized flavonoid-modifying enzymes  as queriO-deglycosylation was the most abundant flavonoid modification performed by human gut bacteria (31 strains). C-deglyocsylation, C-ring cleavage, reduction, dehydroxylation or O-demethylation of flavonoids were far less abundant (between three and seven strains each).First, we screened the accompanying metadata of the MAGs database for the abundance of bacterial species described to transform flavonoids. For this, a literature-based overview of currently known flavonoid-converting bacterial strains present in the human gut was compiled, resulting in 45 distinct bacterial strains of 43 different species . Of theshttps://gtdb.ecogenomic.org/, accessed on 1 July 2020) in the UHGG metadata. Thus, species without a sequenced genome  or species lacking taxonomic assignment in the GTDB to a given genome (S. isoflavoniconvertens) could not be found. Besides the overall abundance of MAGs, we also calculated the prevalence of MAGs across individuals as percentages of the overall number of metagenome samples in the metadata.We then performed a text-based filtering and counting of species in the lineage column of the UHGG metadata with the aim of specifying the MAG abundances of described flavonoid-transforming species as percentages of the overall number of MAGs in the human gut. Important to note here is that the taxonomic assignments were based on the genome taxonomy database , Parabacteroides distasonis (14.5%), Bifidobacterium infantis (8.4%), and Bi. adolescentis  of 35 to each other, resulted in a high number of hits in MAGs classified to six different Bifidobacterium sp. , Bi. infantis and Bi. pseudocatelunatum  as the most numerous  and CAG-217 sp000436335 of the same family with about 1000 occurrences. All of these show a PID of about 50 to Bi. pseudocatenulatum GluD. The high abundance and similarity to BpGluD indicates a potentially prominent role of these Acutalibacteriaceae members in deglycosylation of flavonoids. Furthermore, Blautia wexlerae and two taxonomically undescribed Eubacterium spp. with a high PID to BpGluD were abundant as well. More distantly related glucosidases (PID between 40 and 50 to the queries) include those of Faecalicatena gnavus, several Clostridium and Ruminococcus species and Gemmiger sequences more closely related to the Bifidobacterium breve enzyme and a second Blautia enzyme  a potena enzyme . Since oium spp. .Bl. wexlerae sequences form a large distinct clade that also includes sequences from Facealicatena gnavus, while the Gemmiger glucosidases forms another clade, also distinct from all clades harboring characterized flavonoid O-glycosidases  and the Bacteroides sequence form larger clades, with the Bl. wexlerae, the CAG-180 and the F. prausnitzii clades being much larger, suggesting that the O-deglycosylating potential of the human gut microbiota is largely uncharacterized and awaits biochemical investigation.To unravel the phylogenetic relationships between the observed enzymes and to see whether the novel sequences can be classified into distinct phylogenetic classes with potential new catalytic properties, a phylogenetic tree was created. The largest part of the osidases . The latO-glycosidases was observed in Catenibacillus scindens, where a cluster of two genes, dfgCD, was characterized to encode an enzyme that 7-O-deglycosylated isoflavones and flavones [Hungatella hathewayi, Faecalicatena species, and Eisenbergiella sp900066775, all with approximately 100 occurrences  was expected, given the low abundance of Lactobacillus species in the gut MAGs metadata . With a lower abundance (about 50 appearances), metagenome-derived rhamnosidase 2 orthologs were detected in MAGs of the genus CAG-170 (family Oscillospiraceae). Orthologs (PID of about 98) of the metagenome-derived rhamnosidase 1 were detected in only five MAGs from the family Monoglobaceae, which could not yet be assigned to a genus and differ from the only described member of this genus, the pectin-degrading intestinal Monoglobus pectinilyticus [Parabacteroides and Bacteroides and the species Alistipes onderdonkii (approximately 45 to 50 PID to MGR2) were found to be highly abundant  as queries, since only these genes were shown to catalyze reactions in the C-deglycosylation pathway [The current gene clusters reported to play a role in rain PUE B, we use pathway ,26.dgpABC or at least three of dfgABCDE. The latter resulted in only a few similar hits with a PID higher than 40  plus several dozen in Blautia, Dorea, Enterococcus and Faecalicatena MAGs , so the occurrence of A. faecis cannot necessarily be linked to the C-deglycosylation of flavonoids. The occurrence of C-deglycosylating genes in Faecalicatena gnavus seems even less specific, with only 84 MAGs of 1197 encoding DgpABC  as queries. Up- and downstream genes were not considered in this study, since they were suggested to play accessory roles [Slackia, Lactococcus or Eggerthella were scarce, which contradicts reports in the literature on a widely distributed daidzein-to-equol conversion by these species in the human gut. Four hits of Slackia MAGs containing the daidzein-to-equol gene cluster were assigned to the same species represented by GUT_GENOME145587. A comparison of this representative genome to the available S. isoflavoniconvertens DSM22006 genome (GenBank assembly number GCA_003725955.1) using ANI calculator [S. isoflavoniconvertens is justified. Since the other 261 MAGs representing this species do not encode the daidzein-to-equol pathway enzymes, S. isoflavoniconvertens appears not to be a typical equol producer in the human gut. Therefore, conclusions of equol production in an individual should not be based on 16S rRNA analyses, which obviously cannot resolve the daidzein-to-equol transforming potential of Slackia spp. Another bacterial species described to transform daidzein to equol, Adlercreutzia equolifaciens, was more abundant, with 33 occurrences of the ddr gene, but also here, the percentage of MAGs carrying the daidzein-to-equol gene cluster was relatively low with less than ten percent . This observation is strengthened by a report of a human gut A. equolifaciens isolate that lacks the cluster and is not able to transform daidzein to equol . Another species whose MAGs frequently carried daidzein-to-equol cluster genes was CAG-1427 sp000435475  and several, mostly uncharacterized, Collinsella species  plus the optional dihydrodaidzein racemase encoded by  garviae  are encory roles . Hits inlculator  resultedcreutzia , which t species . The occzr genes .Since these four reactions, the reduction of flavons and flavonols to flavanones and flavanonols, respectively , the C-ring cleavage of these products (via Fcr), isomerization of chalcones and auronols (via CHI), and the hydrolysis of the dihydrochalcone phloretin (via Phy) can be viewed as steps in the same pathway of flavone/flavonol degradation , and, with a lower PID, in MAGs of the Olsenella, Eubacterium and Fusobacterium species  harbor two copies of the flr gene, with one of them more closely related (PID of ~60) to that of Clostridium ljungdahlii than to the “main” Flr of F. plautii. This second putative Flr of F. plautii did not show any apigenin-reducing activity [The very recently characterized Flrs from ngdahlii  resulted species . Most ofactivity , but migEubacterium ramulus resulted in hits identified in F. plautii MAGs  encoded an Fcr-like enzyme with approximately 85% identity to Fcr from E. ramulus. Besides these three main species, a number of MAGs from Clostridium boltae encoded a putative Fcr with a PID of approximately 42 to the Fcr from F. plautii. Most hits to Fcr  showed around 35 PID  from d 35 PID  and freqFlavonifractor plautii  and Clostridoides difficile (375) and, to a lesser extent, were present in Eubacterium ramulus (137), in which it was initially described. Below a PID of 40, only MAGs from Fusobacterium mortiferum were detected in a higher number (ca. 100).Genes similar to that encoding CHI  were higE. ramulus resulted in several hits with a PID of more than 90, assigned to 159 E. ramulus MAGs  and Anaerostipes hadrus (119 occurrences), were abundant with a PID of approximately 60 and 75 to the Phy query. Opposed to the Phy of E. ramulus, the BLAST search with the Phy of M. abscessis resulted in only few hits below 40 PID in a Dakarella species.The BLAST search using the phloretin hydrolase (Phy) sequence of lus MAGs . The moslus MAGs  were fouhloretin . Genes oF. plautii and E. ramulus MAGs, emphasizing the co-functionality of these enzymes in a single pathway of flavone degradation. F. plautii was by far the most abundant species in this analysis, pointing toward its major role in flavone degradation in the human gut. Of the 610 MAGs assigned to F. plautii, most (509 to 610) contained at least one of the genes. In contrast, A. hadrus, containing only Fcr and Phy, showed only a minor amount of MAGs carrying flavonoid-degrading enzymes .The co-occurrence of hits to all enzyme sequences  was observed in most of the C-deglycosylation, O-demethylation requires several proteins  acting together in a complex reaction pathway. Since no specific flavonoid O-demethylation system has been characterized, the four enzyme sequences of the O-demethylase system from Eubacterium limosum, which acts on the polyphenolic lignan secoisolariciresinol [Eubacterium genus, notably from the species E. callenderi and to a lesser extent E. limosum (characterized for flavonoid O-demethylation), were observed . The high PID of MT1, which defines the substrate specificity of O-demethylation [I. bartlettii (never tested for O-demethylation of flavonoids) has a similar substrate spectrum for O-demethylation as E. limosum. The MTI gene is part of a complete O-demethylase gene cluster in I. bartlettii , but their ability to convert flavonoids may have been overlooked. An example is Anaerostipes hadrus, so far mainly known for butyrate production [Flavonifractor plautii. Most of its MAGs contain the Flr, Fcr, Phy and CHI genes, and it is relatively abundant. Thus, F. plautii might serve as a good indicator of the flavonoid degradation potential in 16S rRNA gene-based gut microbiota analyses. On the other hand, our quantitative results on flavonoid-modifying bacteria imply that several key flavonoid modification processes in the human gut, which might have been so far assigned to bacteria, are neither representative nor the main flavonoid-transforming bacteria in this ecosystem. For example, equol formation from daidzein appears to mainly be catalyzed in the human intestine by Eggerthella, and it may be catalyzed by Collinsella species more frequently than by the thoroughly studied Slackia isoflavoniconvertens and Adlercreutzia equolifaciens.The in silico analysis of potential flavonoid-modifying enzymes of human gut bacteria presented here is a first step to uncover the complete picture of flavonoid transformation in the human intestine. The findings constitute a basis for targeted testing of the detected potential novel enzymes and their bacterial sources. oduction . An  and subsequently extracted. With BLAST+  from 286,997 MAGs assembled from gut metagenomes  was downon 2.9.0 ) installO-glycosidases, rhamnosidases) and 1e-20 for the C-deglycosylation enzymes, which contained two sequences below 150 amino acids. The maximal number of target hits was set to 100,000 via the max_target_seqs argument. Blast table output format 6 was chosen with the specified columns  or 1e-60 for longer sequences  server . The sequence comparison tool was used to compare up to ten genomes.Phylogenetic trees of the rhamnosidase and O-glycosidase sequences were constructed after clustering of the sequences with CD-hit (v 4.8.1 ) using s


Background: Physiologically, the aorta is less stiff than peripheral conductive arteries, creating an arterial stiffness gradient, protecting microcirculation from high pulsatile pressure. However, the pharmacological manipulation of arterial stiffness gradient has not been thoroughly investigated. We hypothesized that acute administration of nitroglycerin (NTG) may alter the arterial stiffness gradient through a more significant effect on the regional stiffness of medium-sized muscular arteries, as measured by pulse wave velocity (PWV). The aim of this study was to examine the differential impact of NTG on regional stiffness, and arterial stiffness gradient as measured by the aortic-brachial PWV ratio (AB-PWV ratio) and aortic-femoral PWV ratio (AF-PWV ratio).Methods: In 93 subjects , aortic, brachial, and femoral stiffnesses were determined by cf-PWV, carotid-radial (cr-PWV), and femoral-dorsalis pedis artery (fp-PWV) PWVs, respectively. The measurements were repeated 5 min after the sublingual administration of NTG (0.4 mg). The AB-PWV and AF-PWV ratios were obtained by dividing cf-PWV by cr-PWV or fp-PWV, respectively. The central pulse wave profile was determined by radial artery tonometry through the generalized transfer function.Results: At baseline, cf-PWV, cr-PWV, and fp-PWV were 12.12 ± 3.36, 9.51 ± 1.81, and 9.71 ± 1.89 m/s, respectively. After the administration of NTG, there was a significant reduction in cr-PWV of 0.86 ± 1.27 m/s (p < 0.001) and fp-PWV of 1.12 ± 1.74 m/s (p < 0.001), without any significant changes in cf-PWV (p = 0.928), leading to a significant increase in the AB-PWV ratio  and AF-PWV ratio . There was a significant correlation between changes in the AF-PWV ratio and changes in the timing of wave reflection  and the amplitude of the heart rate-adjusted augmented pressure .Conclusion: This study shows that acute administration of NTG reduces PWV of muscular arteries  without modifying aortic PWV. This results in an unfavorable profile of AB-PWV and AF-PWV ratios, which could lead to higher pulse pressure transmission into the microcirculation. Aortic stiffness, determined by aortic pulse wave velocity (PWV), is a hallmark of vascular aging and an independent cardiovascular risk factor in various clinical conditions ranging from hypertension to end-stage kidney disease . The conIn the context of chronic kidney disease (CKD), we have previously used the PWV ratio between cf-PWV and cr-PWV as a surrogate measure of arterial stiffness gradient and have shown that it was a better prognostic indicator than cf-PWV alone . In CKD Therefore, we aimed to examine the effect of acute administration of NTG on arterial stiffness gradient. Second, we aimed to study the effect of NTG on local stiffness parameters of the brachial artery and its relation to changes in cr-PWV. Finally, we investigated whether the vascular response to NTG is different in patients with CKD than subjects without a significant decline in renal function.n = 55) was composed of apparently healthy subjects or hypertensive subjects with an estimated glomerular filtration rate (eGFR) ≥ 45 ml/min/1.73 m2 [CKD-EPI equation (n = 38) was composed of patients with an eGFR < 45 ml/min/1.73 m2   medication and without any acute episode of illness (infection or recent cardiovascular events). Patients were excluded if they had a history of adverse reactions to NTG or were on renal replacement therapy. The control group (equation ]. The CKd above) . This cu disease . The parbPP). Immediately after BP measurements, radial pulse wave profile was recorded using applanation tonometry after calibration with brachial SBP and DBP , followed by the determination of PWVs in peripheral  and central (aortic) arterial segments using Complior ® Analyse . Brachial BP, PWVs, and radial pulse wave profile measurements were repeated 5 min after the sublingual administration of 0.4 mg of NTG with the same described procedures except that pulse wave profile has been taken once instead of thrice. Radial pulse wave profile post-NTG was introduced later in the measurement protocol and performed in the last 56 patients. Wall properties of the brachial artery were assessed in 57 subjects by a radiofrequency-based wall-tracking system  before and continuously during 10 min after NTG administration.All measurements were performed after 15 min of rest in a supine position. Brachial artery BP was recorded using an automatic oscillometric sphygmomanometer BPM-100  by an experienced operator who was present in the room. BP was recorded six times, with a 2-min interval between each measurement, and the average of the last five measurements was used to determine the brachial systolic BP (SBP), diastolic BP (DBP), and brachial artery pulse pressure , and carotid-femoral PWV /(cp transit time − cf transit time). Stiffness gradients from the aorta to the brachial artery (upper limb) and from the aorta to the femoral artery (lower limb) were obtained using the aortic-brachial PWV ratio (AB-PWV ratio = cf-PWV/cr-PWV) and aortic-femoral PWV ratio (AF-PWV ratio = cf-PWV/fp-PWV), respectively.d), intima-media thickness (IMT), and distension of the brachial artery (∼3 cm above the elbow crease). The ART-LAB probe (10 MHz linear probe) was fastened by a locking articulating arm mounted on a platform that allowed two-dimensional microadjustments and good stability for a 10 min-period of real-time measurements after sublingual NTG administration. Arterial wall properties were calculated as follows:With the right arm stabilized in an inflated cushion, pre-NTG end-diastolic diameter , heart rate-adjusted central augmented pressure (AP@75), and augmentation index (AIx@75), as well as end systolic pressure, were measured. Period, ejection duration, diastolic duration, the first peak of pressure height (P1) with the respective time of each peak , time to the reflection of the reflected pressure wave (TR), and subendocardial viability ratio (SEVR) were also obtained .n (%), or median (25th–75th percentiles). Differences between groups were assessed using t-test, Mann-Whitney U-test, or chi-square accordingly. The effects of NTG on vascular parameters were examined using paired t-test. Linear regression was used to examine the relationship between renal function, PWVs, and PWV ratios. We used generalized estimating equations (GEE) to adjust changes in vascular parameters for changes in DBP. Linear regressions were performed to determine the relationship between cr-PWV and brachial artery diameter. GEEs were also used to compare if the vascular response to NTG was different between CKD and the control group. Forward conditional linear regression was used to identify the determinants of changes in cr-PWV after NTG administration. We used the Spearman rank correlation coefficient to assess the association between TR and AP@75 with both AB-PWV and AF-PWV ratios. Finally, as part of sensitivity analysis, we examined if there were any significant interactions between age, sex, and established cardiovascular disease, and the vascular response to NTG (GEE). All statistical analyses were performed using SPSS Statistics for Windows, Version 27.0 . A two-tailed p-value <0.05 was considered to be statistically significant.The results are expressed as mean ± SD, r = − 0.37, p < 0.001), AB-PWV ratio , AF-PWV ratio , but not with cr-PWV , and fp-PWV  in this study population.p < 0.001) and AIx@75 . The end-systolic pressure decreased despite a reduction in ejection duration, but no changes were observed in the period. Finally, there was an increase in SEVR .As shown in p = 0.928), but there were significant reductions in both cr-PWV  and fp-PWV . The results remained consistent even after adjustment for the small changes in DBP (As shown in s in DBP . Consequp < 0.001) and the luminal cross-sectional area  increased significantly. However, systolic-diastolic distention  and distensibility  decreased, incremental elastic modulus increased , without any significant changes in brachial artery compliance . These results are summarized in R2 = 0.42; To examine if the change in cr-PWV was related to brachial artery diameter, we performed a linear regression analysis and found that higher baseline cr-PWV and smaller baseline diameter were significant determinants in the decline of cr-PWV after NTG administration , sex, and established cardiovascular disease on the extent of studied vascular responses to NTG.This study shows that acute administration of NTG reduces brachial and femoral PWVs without affecting aortic PWV, causing an increase in the AB-PWV and AF-PWV ratios. The delay in the timing of the reflected wave and the lowering of the amplitude of the reflection wave after NTG were best correlated with the changes in the AF-PWV ratio.These observations are in line with previous studies showing that NTG reduces PWV in muscular peripheral arteries but has no effect in attenuating aortic PWV . These cThe clinical and biological determinants of a lower cr-PWV remain largely unknown and understudied. However, data are emerging that in certain conditions such as heart failure, kidney disease, or obesity in adolescents, the cr-PWV may be lower, hence contributing to the attenuation of the arterial stiffness gradient beyond the sole increase in aortic PWV . Moreovep = 0.045) but not cf-PWV (p = 0.501), and only the AB-PWV ratio has been associated with eGFR and albuminuria in a cohort of middle-aged women with increased cardiovascular risk   .In the ARIC study population, a cross-sectional analysis showed that a low arterial stiffness gradient  was associated with diabetes, coronary heart disease, heart failure, and stroke, whereas a high cf-PWV was only associated with hypertension . It shouh is the wall thickness and r is the radius of the artery, and ρ is the blood density. However, we found no statistical relationship between measured changes in cr-PWV and changes in either diameter or wall-to-lumen ratio. This discrepancy could be explained by several reasons. First, it could be that our spatial resolution was too limited for a reliable assessment of in vivo changes in wall-to-lumen ratio. Second, it could be related to the violation of basic assumptions underlying the equation, i.e., that the arterial wall is isotropic and behaves in an isovolumetric manner in response to pulse pressure , but this increase in diameter was accompanied by a lower degree of distention, showing no significant changes in the brachial artery compliance and even an increase in incremental elastic modulus. This increase in incremental elastic modulus could be explained by the transfer of pressure load from the vascular smooth muscle cells to the stiffer collagens of the extracellular matrix . Therefopressure . In addi of Einc , therefoper se. This observation is in line with the findings of Besides arterial stiffness gradient, this study underlines the importance of proper assessment of regional vascular stiffness and its response to therapy. Given the nature of translational medicine, we have been eager to take the arterial stiffness assessments out of specialized laboratories and into the large-scale epidemiological studies and clinical practice through simplification of arterial stiffness assessment. For example, our study clearly shows that PWV from the carotid to the dorsal pedis artery declined significantly after NTG, but upon closer look, we saw that this decline was related to the reduction in PWV from the femoral to the dorsal pedis segment and not related to aortic PWV via transfer function from the radial pressure waveform and the validity of central AIx as a measure of wave reflection (This study has several strengths and limitations. First, it involves a large number of subjects with a detailed evaluation of various regional pulse wave velocities, central pulse wave profile, and brachial stiffness parameters. Second, given the heterogeneity of the subjects, we were able to perform various sensitivity analyses showing the robustness of our findings. Indeed, older subjects may have a more hypotensive response to NTG . Howeverflection . Indeed,flection . Furtherflection . FinallyWhile there are statistical associations between markers of arterial stiffness gradient and certain clinical conditions in both cross-sectional and longitudinal studies, there has not been a systematic focus on the impact of pharmacological interventions on the arterial stiffness gradient. In light of recent evidence supporting the potential usefulness of arterial stiffness gradient assessment, and our present data showing an alteration in arterial stiffness gradient in response to NTG, we would like to underline the importance of assessing regional PWV in order to develop an integrated approach into the arterial tree and its response to therapy. As such, these results are hypothesis generating and their clinical and pharmacological usefulness should be assessed in properly designed studies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Comité d’Éthique de la Recherche du CHU de Québec. The patients/participants provided their written informed consent to participate in this study.MA and CF provided the conception and design of the study. CF, C-AG, MP, HO, NC, KD, and MA recruited participants and collected data. CF, C-AG, and MA performed the statistical analysis and wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.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.


Saccharomyces cerevisiae. However, a huge mass of recorded images makes the data processing labor-intensive and time-consuming to determine yeast replicative lifespan (RLS), a primary criterion in yeast aging. To address this limitation and pursue label-free RLS assays, electrical impedance spectroscopy (EIS) that can be easily functionalized through microelectrodes in microfluidic devices, was introduced to monitor cell growth and division of budding yeast. Herein, a microfluidic device integrated with EIS biosensor was proposed to perform in-situ impedance measurement of yeast proliferation in single-cell resolution so as to identify the momentary events of daughter dissection from its mother. Single yeast cells were reliably immobilized at the bottleneck-like traps for continuous culturing, during which daughter cells were effectively detached from their mother cells by hydraulic shear forces. Time-lapse impedance measurement was performed every 2 min to monitor the cellular process including budding, division and dissection. By using the K-means clustering algorithm to analyze a self-defined parameter “Dissection Indicator,” to our knowledge for the first time, the momentary event of a daughter removing from its mother cell was accurately extracted from EIS signals. Thus, the identification of daughter dissection events based on impedance sensing technology has been validated. With further development, this microfluidic device integrated with electrical impedance biosensor holds promising applications in high-throughput, real-time and label-free analysis of budding yeast aging and RLS.Microfluidic devices in combination with fluorescent microscopy offer high-resolution and high-content platforms to study single-cell morphology, behavior and dynamic process in replicative aging of budding yeast,  Saccharomyces cerevisiae (S. cerevisiae), benefiting from its fast proliferation, short lifespan, easy maintenance, and fully-sequenced genome, has been extensively used as a model organism in aging studies , as an alternative to optical imaging, is a non-invasive, label-free and multiparametric tool to probe biophysical properties of biological specimens, such as cells and tissues . By inted motion , mitosisd motion , as welld motion . Althougin-situ electrical impedance monitoring of single budding yeast cells. The microfluidic device features: i) bottleneck-like traps for reliable capture, long-term retention and continuous culturing of yeast cells, as well as automatic and effective removal of daughter cells through hydraulic shear forces; and ii) localized coplanar microelectrode pairs for real-time and label-free detection of daughter-cell dissections using time-lapse in-situ electrical impedance biosensor. In addition, the K-means clustering algorithm was used to compensate any baseline drift in the recorded EIS signals and accurately extract momentary information of daughter-cell dissection events.In this work, we report on an EIS-biosensor-integrated microfluidic device, which is capable of hydrodynamic trapping, continuous culturing and in-situ impedance monitoring of single budding yeast cells. The microdevice consists of 5 layers, i.e., a glass substrate, Pt electrodes, a SiNx passivation layer, SU-8 microfluidic channels and a polydimethylsiloxane (PDMS) cover. The SU-8 fluidic network comprises a main channel (width: 150 μm) and a parallel side channel (width: 300 μm), with 10 bottleneck-like traps (narrow neck width: 4 μm) connecting those two channels. Budding yeast cell suspension and culturing medium were infused into the main channel via their corresponding inlets , both at a flow rate of 1.5 μL/min. By precisely controlling the underpressure  applied to the pressure port, single yeast cells were reliably captured at the trap orifices. After cell immobilization, the pressure was raised to a constant value  to prevent redundant cells from being captured at the same traps. To integrate electrical impedance biosensor, Pt microelectrode pairs were patterned underneath the SU-8 structures, with the SiNx passivation layer in between. To implement in-situ impedance measurement, long common electrode in the main channel serves as the stimulus electrode to supply an excitation alternating current (AC) voltage, and tip individual electrodes around trap orifices serve as recording electrodes to receive the weak response current, which is then amplified and converted into a detectable voltage signal. The SiNx layer was reopened in the sensing region to reduce potential electric crosstalk between adjacent electrodes, and in the contact pads around the device border to connect electrodes to external circuits. Thus, the sensing unit, which features a trap for cell capture and retention and a pair of microelectrodes for in-situ electrical impedance measurement, is the core composition of the EIS-integrated microdevice.in-situ electrical impedance biosensor, single cells are firstly immobilized at the traps and continuously cultured under laminar-perfused medium in the microfluidic device. During cell culturing for an extended time period, electrical impedance measurement is carried out every 2 min to monitor the dynamic cell process, such as budding, cytokinesis and daughter dissection. As soon as the cytokinesis-completed daughter cell is detached from its mother by hydraulic shear forces, it is immediately dragged away by the continuous medium flow, thereby suddenly cutting down the cell impedance between the stimulus and recording electrodes. This drastic decrease in impedance, corresponding to the momentary event of daughter dissection, can be detected by the time-lapse impedance measurement, showing a steep signal jump on the amplitude curve of EIS signal , followed by reactive ion etching (RIE) to define the opening in sensing region and contact pads. Subsequently, 30-μm-thick SU-8 3,025 photoresist  was spin-coated on the wafer with patterned microelectrodes, and structured into microfluidic channels by using standard photolithography. After wafer dicing, an unstructured PDMS  layer, punched with holes as fluidic inlets and outlets was used to seal the microfluidic channel network. For an irreversible bond between PDMS and SU-8, both SU-8 surface of each die and oxygen-plasma-activated PDMS cover were modified with 3-aminopropyltriethoxysilane  in aqueous solution  from the impedance spectroscope was applied to the stimulus electrode at an interval of 2 min, and the response current For time-lapse EIS measurement, a swept-frequency AC voltage S. cerevisiae) cell strain, derived from BY4743, was used in the experimental verification of the EIS-biosensor-integrated microfluidic device. Yeast cells were cultured using standard methods as described in literature  flows mostly around cells and very few penetrates cell membrane. Thus, EIS signal at 1 MHz can be directly used to characterize cell size or morphology, which has been validated previously ”  curve . During in-situ impedance monitoring of daughter dissection events of budding yeast cells. In the microfluidic device, narrow bottleneck-like traps enable reliable retention of yeast mothers in single-cell resolution and provide high hydraulic shear forces for the automatic removal of daughters after the completion of cell division. The EIS sensing unit allows for label-free and sensitive detection of daughter dissections during continuous replication of budding yeast. “Dissection Index (DI)” minimizing signal fluctuations has been successfully used to compensate the EIS signal drifts derived from bud rotation, cell movement, or dirt attachment in the sensing unit. Associated with K-means cluster analysis, momentary events of daughters separating from their mother can be clearly identified from EIS signals.In this work, a microfluidic device integrated with EIS biosensor has been developed to perform real-time and With regard to present design of the microfluidic device, we have noticed a few problems or limitations on real-time monitoring of budding yeast replication and identification of daughter-dissection events by using the integrated electrical impedance biosensor. i) Although higher traps provide adequate space in vertical direction for cell rotation and reorientation, they increase the risk of stacking more cells in vertical space. Lower microfluidic channels and traps might reduce such risks, but would increase the hydraulic resistance in channels for cell delivery and cause channel clogging by big clusters of yeast cells. ii) Since the EIS sensing unit, especially the long common stimulus electrode, has much free space exposed in the fluidic channel, passage of cell suspension or capture of dirt may affect the measured impedance and give rise to noise or drift on the recorded EIS signal. iii) Current microfluidic device is limited to a throughput of 10 independent cell traps. Also, these traps feature non-uniform distribution of hydraulic pressure drops so that they cannot be simultaneously occupied by single cells under the same setting of medium flow rate and underpressure. As such, the present device is feasible to verify the concept of yeast-daughter-dissection monitoring and investigate the sensitivity of the integrated electrical impedance biosensor, but poses challenges to integrate more traps for high-throughput culturing and monitoring of single budding yeast cells. iv) Due to accidental cell loss or trapping of additional cell during the EIS recording, current version of the microfluidic device allows for only several-hour monitoring of cell growth and division in a single experiment, which cannot cover the whole replicative lifespan of budding yeast. The cell-culturing system, especially the microfluidic structure for single-cell trapping and long-term retention, needs to be optimized in terms of stability and robustness so that it can monitor single-cell replication over 48 h without interruption. Accordingly, the design of addressable electrodes to implement EIS measurement requires optimization to adapt for the new cell-culturing system.To summarize, this proof-of-concept study has demonstrated that the EIS-integrated microfluidic device is capable of automatic removal of daughter cells by hydraulic shear forces and sensitive monitoring of daughter-dissection events by real-time EIS measurement. With further optimization and development in terms of long-term stability and throughput, the microdevice potentially provides an automatic and non-optical platform for yeast replicative aging studies and RLS determination.


Dental caries is a multifactorial disease with high prevalence in both children and adults. Recent genome-wide association studies (GWASs) have revealed that genetic factors play an important role in caries incidence. However, existing methods are not sufficient to identify caries-associated genes, due to the complex correlation structure of caries GWAS data, and lack of appropriate summarization at the gene level. This paper attempts to address that by analyzing data from the Gene, Environment Association Studies (GENEVA) consortium.We investigated gene-based genetic associations for dental caries based on genome-wide data derived from the GENEVA database, with adjustment to covariates, linkage disequilibrium among single-nucleotide polymorphisms, and family relations, in sampled individuals.Several suggestive genes were identified, in which some of them have been previously found to have potential biological functions on cariogenesis.By comparing the gene sets identified from gene-based and SNP-based association testing methods, we found a non-negligible overlap, which indicates that our gene-based analysis can provide substantial supplement to the traditional GWAS analysis. Dental caries is a chronic, transmissible disease caused by activities of bacteria . Among tAnalyzing genome-wide data generated from epidemiological studies of dental health opens up cost-effective opportunities to uncover how genetic factors affect inherited risks for dental caries. Previous genome-wide association studies (GWASs) for dental caries have identified several genes that may be associated with caries heritability , includiIn this paper, we propose to use a novel gene-based association mapping strategy to identify caries-associated genes based on genome-wide data from the GENE enVironment Association studies (GENEVA) consortium. Several suggestive genes were identified, in which some (such as PTPRD) have been previously found in other single-SNP-based GWASs to play plausible biological roles for dental caries. An interesting finding was obtained by comparing the gene sets identified separately from gene-based and SNP-based tests. The non-negligible overlap between these two sets suggests that the two types of analyses are not independent of each other and therefore results from gene-based association analyses may contain important signals relevant to cariogenesis, complementing those from the traditional single-SNP-based GWASs.S. mutans present . The genotype data were collected for a total of 589,735 SNPs on an Illumina BeadChip, among which 289,629 (49.11%) are located in 26,449 entrez genes (according to the human gene Ensembl dataset GRCh37). Based on the cleaned genotype data, a total of 20,197 (76.36%) entrez genes were found to contain more than one SNP in the genotype dataInitiated in 2006, the GENEVA program aims to enhance the benefit of collaborative work to further maximize knowledge obtainable through GWAS [As part of the quality-control procedure, the genotyped individuals were further filtered by excluding those who met either of the following two criteria:Empirical self-kinship coefficient Completeness ≤ 96%, where completeness is defined as the proportion of SNPs for which a given individual had genotypes called. Under these criteria, a subset of 652 samples from the GENEVA dental caries data, which have complete data in both phenotypes and genotypes, were selected for the analysis.Among these 652 participants, 571 are from 201 families (nuclear families or parent-offspring trios), and the rest are unrelated individuals. Quality control on SNPs was conducted based on the following conditions:Call rate ≥ 96%, andMinor allele frequency (MAF) ≥ 1%.To perform the gene-based test, the entire genotype matrix  was split according to the starting and ending positions of each gene.Each partition of the genotype matrix was further cleaned in order to avoid the possible non-singular problem regarding the following aspects: (1) columns corresponding non-polymorphic SNPs were excluded, and (2) given limited sample size, only columns with unique SNP data were retained whereas duplicated ones were eliminated. These quality assurance steps resulted in 26,831 genes in consideration.The adaptive-weight burden test (ABT) is a retrospective, mixed model test for genetic association of quantitative traits on genotype data with complex correlations. In the GENEVA dental caries study, a group of individuals with known pedigree information were sampled for phenotype (the DMFT index), covariates , and genotypes (multiple SNPs in pre-defined gene regions). Under the gene-based testing setup, we denote the phenotype vector by Y, covariate matrix by Z, and genotype matrix by G , i.e., by treating genotypes as random response in regression. The main advantage of such a retrospective model lies in its ability to directly model genotypic correlations across both SNPs (caused by LD) and samples  , as wellABT can be explicitly derived as a mixture of TPGW, whereIt has been shown that the ABT statisticividuals . When usThe GENEVA dental caries data (dbGaP accession: phs000095. v3.p1) include 5,291 phenotyped and 4,020 genotyped individuals. In this study, we focus on a subset of 652 participants who have complete data in the following phenotypic characteristics: gender, age, education group, water source, presence/absence of S. mutans, home tap water fluoride level, saliva flow, brush frequency, and the Decay-Missing-Filled (DMF) index. These characteristics are summarized in We note that, the 652 participants include both children and adults. The age ranges from 7 to 61 years, and 406 out of the total 652 are older than 18 at the time of examination. The majority of these participants are white , therefore race was not included as a covariate in our study.We use the adaptive-weight burden test  to perfoS. mutans are significantly associated with the DMFT trait with positive coefficient estimates, indicating that patients with male gender, younger age, and absence of S. mutans are more likely to have lower DMFT measurements. The brush frequency, while expected to be positively associated with dental caries, seems to have a “boundary” effect, which turns out to be significant at nominal level 0.1 but not at 0.05.In the step of null phenotype model identification, two variance-component parameters and 10 fixed-effect regression coefficients for the non-genetic covariates (two indicators for water source categories) are estimated by the maximum likelihood method. These estimates are shown in es GWASs –25. In cIn this gene-based association analysis, the Manhattan plot and Quantile-Quantile (Q-Q) plot for a total number of 26,831 entrez genes on the human genome are shown in Our study also identified five other genes that were reported in the GWAS catalogue  to be re−16), indicating that the results from these two types of tests are not independent.The comparison of gene-based test and SNP-based test shows that, out of the total 26,831 genes, 1,053 exhibit significance by MASTOR, slightly less than the 1,468 genes found by ABT, and the intersection set, i.e., identified by both MASTOR and ABT, contains 688 genes. The contingency table is shown in Traditional GWASs rely on screening the genome on the basis of SNPs. Though such a SNP-based association testing strategy has been shown successful in identifying susceptibility loci for several complex genetic diseases ,32, chalS. mutans were found significantly associated with the DMFT trait, and the narrow-sense heritability was found comparable with traditional heritability estimates in previous family-based dental caries GWASs. Due to the differences between study designs and testing methods, it is not reasonable to compare the gene set identified by this study with that by other SNP-based GWASs. However, we attempted to compare the results from both gene- and SNP-based association testing on the basis of the GENEVA dental caries data. The comparison revealed that the gene-based test captured 65.34% genes that are significant in the SNP-based test. A further test for independence illustrated that, though gene-based association testing has a totally different mechanism, the identified genes are significantly overlapped with those by SNP-based association testing, given that two tests are performed on the same GENEVA data. Therefore, findings from this gene-based association testing may contain important signals relevant to cariogenesis and could complement those from the traditional SNP-based GWASs.In this study, we performed gene-based association testing for dental caries data from the GENEVA consortium, with adjustment of phenotype covariates and accounting for LD in SNPs and familial relation in samples. We observed suggestive associations between the DMFT trait and several genes, some of which have been found to have plausible biological functions relevant to cariogenesis. Three non-genetic covariates sex, age, and


In childhood tuberculosis (TB), with an estimated 69% of missed cases in children under 5 years of age, the case detection gap is larger than in other age groups, mainly due to its paucibacillary nature and children’s difficulties in delivering sputum specimens. Accurate and accessible point-of-care tests (POCTs) are needed to detect TB disease in children and, in turn, reduce TB-related morbidity and mortality in this vulnerable population. In recent years, several POCTs for TB have been developed. These include new tools to improve the detection of TB in respiratory and gastric samples, such as molecular detection of Mycobacterium tuberculosis using loop-mediated isothermal amplification (LAMP) and portable polymerase chain reaction (PCR)-based GeneXpert. In addition, the urine-based detection of lipoarabinomannan (LAM), as well as imaging modalities through point-of-care ultrasonography (POCUS), are currently the POCTs in use. Further to this, artificial intelligence-based interpretation of ultrasound imaging and radiography is now integrated into computer-aided detection products. In the future, portable radiography may become more widely available, and robotics-supported ultrasound imaging is currently being trialed. Finally, novel blood-based tests evaluating the immune response using “omic-“techniques are underway. This approach, including transcriptomics, metabolomic, proteomics, lipidomics and genomics, is still distant from being translated into POCT formats, but the digital development may rapidly enhance innovation in this field. Despite these significant advances, TB-POCT development and implementation remains challenged by the lack of standard ways to access non-sputum-based samples, the need to differentiate TB infection from disease and to gain acceptance for novel testing strategies specific to the conditions and settings of use. In recent years, it was estimated that approximately 10 million individuals had tuberculosis (TB) each year, of which almost one-third were not diagnosed or reported. This detection gap has further increased in 2020 as reported cases fell from 7.1 million to 5.8 million, with an estimated 4.1 million cases thought to be unreported . Thus, aGenerally, a POCT aims to facilitate the clinical decision making of the health care provider close to the patient by providing fast and accurate information independent of an access to laboratory facilities. Examples of implemented POCTs for the diagnosis of infectious diseases include rapid diagnostic tests for malaria, human immunodeficiency virus (HIV), Ebola, dengue and most recently severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) ,5. POCTsMycobacterium tuberculosis (Mtb) that is released from metabolically active or degenerating mycobacterial cells into urine. The Alere Determine™ TB LAM Ag (AlereLAM) was the first commercially available assay using lateral flow for LAM detection in TB patients. As the sensitivity of the AlereLAM is limited, the WHO, however, recommends its use mostly in inpatient settings [Tests based on detection of mycobacterial lipoarabinomannan (LAM) antigen in urine have been in use since 2015. LAM is a constituent glycolipid from settings . In addisettings . Additiosettings .M. tuberculosis-specific LAM epitopes and a silver amplification step to increase the visibility of test and control lines. Studies show better sensitivity in both HIV-infected and uninfected patients than its predecessor [In recent years, a new LAM detection assay, Fujifilm SILVAMP TB LAM  has been introduced. The improvements include a combination of high-affinity monoclonal antibodies against decessor ,11. The decessor .A Nigerian study found similar sensitivities of FujiLAM in HIV-infected and -uninfected adults , whereas a study in Zambia found a sensitivity of 75% in both HIV-infected and -uninfected groups ,14.Mtb to the kidneys in those patients [Mtb bacterial load and TB severity may influence the detection of LAM in urine [The biological mechanisms for the possible better performance of LAM assays in HIV-infected adults are not fully understood. It has been speculated that the higher concentration of LAM in the urine of HIV-infected individuals may reflect an increased hematogenous spread of patients . In thatpatients ,16. Intepatients . The reain urine ,18. In cin urine . This suin urine ,20. ThesMtb complex deoxyribonucleic acid (DNA) in sputum samples. The test was officially endorsed by the WHO in 2016 [The TB loop-mediated isothermal amplification (LAMP) assay, developed by Eiken Chemical Company Ltd. , is a molecular diagnostic test that detects  in 2016 . The LAM in 2016 . Further in 2016 . AdditioA systematic review and meta-analysis performed in 2016 found that in adults with culture-confirmed pulmonary TB the performance characteristics of the TB-LAMP assay were very similar to that of the Xpert MTB/RIF assay, with pooled sensitivity and specificity estimates of 78% and 99% versus 81% and 98%, respectively . HoweverA large number of studies have evaluated the performance of the “classical” Xpert MTB/RIF assay across various locations and using a broad range of clinical specimens . A recent, detailed meta-analysis estimated the sensitivity of the MTB/RIF assay performed on sputum in children with pulmonary TB to be 65% with a specificity estimate of 99% . The samMtb detection are more frequent, which also results in loss of rifampicin-resistance results. Despite its limitations, Xpert testing allows for a more rapid diagnosis awaiting culture confirmation, or it can even substitute culture testing in settings where the latter is not available, which is already a considerable progress. Importantly, Xpert using stool specimen has become of interest, because its collection is easier compared to sputum collection in children. Two systematic reviews and meta-analyses showed a pooled sensitivity of 50% (95% CI 0.44–0.55) and 67%  compared with the reference standard of culture and/or respiratory samples using Xpert [Additionally, as children have distinctively lower bacterial load compared to adults, “trace” results for ng Xpert ,33. Despng Xpert .The GeneXpert Omni instrument developed by Cepheid  is a portable system designed specifically for POCT use . The OmnIn 2020, a further PCR-based assay was endorsed by the WHO. The Truenat assays, developed by MolBio Diagnostics Pvt. Ltd. , are based on real-time PCR chips that are analyzed on the company’s Truelab instruments. The instruments are compact, portable and can be operated with a battery-pack, making them ideal for use as a POCT. So far, all studies evaluating these assays were performed in India, with some including head-to-head comparisons between Xpert MTB/RIF and Truenat MTB indicating both assays had similar performance characteristics . A largeMtb. However, several of those manufacturers are currently working on diagnostic TB assays for their POCT platforms. There are several other compact POCT molecular test platforms commercially available that enable the detection of a variety of pathogens, including the Alere q system , ID Now , Revogene , Biofire Filmarray , Q-POC  and Cobas Liat , but at present, none of those systems have commercially available assays for the detection of Traditionally, ultrasound imaging has been provided within radiology departments. In contrast, point-of-care ultrasonography (POCUS), is performed by the healthcare provider responsible for frontline clinical decision making. Continuing emergence of POCUS applications availability have been fueled substantially by technological advances. Portable devices, handheld smartphone- or tablet-based, as well as wireless systems, have become available at comparatively low costs. Point of care acquisition and interpretation of ultrasound imaging facilitates accurate and timely diagnosis and improves patient outcome. The advantages of POCUS have also been embraced in pediatric care . ChildreThe first TB-focused POCUS application named ‘focused assessment with sonography’ (FASH) for HIV-associated TB aimed at improving diagnosis of HIV-associated extrapulmonary TB in adults in South Africa . The FASExperience using POCUS to detect extrapulmonary TB in children is limited. It is expected, however, that the benefit in this patient group is high as the proportion of extrapulmonary TB is relatively high in children ,50. A fiChest radiographs play a key role in screening for and diagnosis of childhood TB ,53,54. FLung ultrasound imaging is a rapidly emerging imaging modality, and for childhood pneumonia, lung ultrasound imaging shows good diagnostic accuracy and diagnostic performance . Lung ulData on chest-POCUS in children with pulmonary TB are limited to one study from South Africa ,67. ClinIn summary, current results of POCUS for pulmonary and extrapulmonary TB suggest the potential for rapid radiological detection, and a beneficial role in monitoring treatment response, although sensitivity and specificity remain to be determined. Currently, experiences with TB-focused POCUS in children appear limited to research cohorts ,66,67 orIn a next step, integration of extrapulmonary and pulmonary focused POCUS is important as shown in a multi-center study in sub-Saharan Africa . The impThe C-reactive protein (CRP), a well-established host immune biomarker of infection, received renewed attention after the WHO recently endorsed CRP as a screening marker in HIV-infected individuals . AdvantaAmong newer host immune biomarkers currently evaluated for TB diagnosis, antibodies, cytokines, ribonucleic acid (RNA) signatures and host cellular markers are the most commonly investigated. A recently published systematic review  showed tAntibodies were the most studied host immune biomarkers. Despite this, older studies evaluating the diagnostic performance of TB-specific antibodies were rather discouraging . HoweverThe potential advantages of antibody biomarkers include high stability of the marker of interest, exciting robust low-cost assays for other pathogens , and limited operator training. Those features highlight the potential for antibody-based tests to be translated into POCT format.Urine is a readily available and non-invasive specimen, and therefore particularly useful for the diagnosis of TB in patients unable to expectorate sputum, such as children, the elderly and adults without a productive cough ,84. In aA recent development is PCR-based detection of TB-specific cell-free DNA fragments excreted in the urine . Using aArtificial intelligence (AI)-based interpretation of medical imaging has recently been extensively researched and developed further, and multiple commercial products have become available for clinical use ,90. CompDigitalization and development of multi-layer detectors have enabled increased availability of radiography and enhanced the development of portable systems for POCT use. The newest portable systems, called ultra-portable or handheld x-ray systems, come with compacted technology that allows safe TB screening in remote locations or home-based environments . At leasResearch and development into AI for ultrasound imaging is less advanced compared to radiography. Obstacles comprise operator-dependency and lack of large data sets with standardized views. So far, AI endeavors for ultrasound imaging focused on algorithms for respiratory conditions because of the comparably consistent acquisition of images and homogenous sonographic pattern of healthy lungs ,96,97. PM. tuberculosis, and therefore lipidomics may be a further promising way to screen for specific mycobacterial components and diagnose TB [Advancing TB diagnostics by investigating signatures from “omic” analyses is a promising multi-dimensional and unbiased approach . Transcrgnose TB ,103. Furgnose TB . In addignose TB . Sweat, gnose TB . The “omics” approach and detection of biomarkers is inevitably connected to digital development. The internet of  things—which means medical devices and applications are connected to healthcare systems through online computer networks—provides a continuously growing field in which patients and healthcare workers are connected and machine-to-machine communication allows for the rapid exchange of results ,109,110.Despite significant advances in TB point-of-care diagnostics over the past decades , many baNone of the POCTs currently in use have focused on TB infection, as the implication of detecting TB infection has until recently been underestimated . NeverthApart from the challenges due to the complexities of the host and the pathogen, sociopolitical hurdles need to be overcome. POCTs require delivery to remote places with limited infrastructure, and inequities in access to diagnostics also must be addressed. Novel biomarker identification should focus on children, rather than extrapolation from ongoing studies from adults, as the pediatric population presents with distinct clinical picture and has other requirements for adequate testing. Whilst much development is still required, the ongoing COVID-19 pandemic shows strikingly that tools to prevent, diagnose and treat a disease can be developed with unprecedented speed, resulting from joint collaborations and prioritization of scientific funding. Notably, in 2020, research and development investment in diagnostics have been nearly ten times higher for COVID-19 compared to TB diagnostics in 2019 ,121. Ult


Integration of clinical skills, ethical decision-making, and reflection skills have emerged as cornerstones of clinical teaching in medical schools. This study aimed to detect whether a multimodal learning environment approach consisting of lectures, a drill, post-drill video debriefing, and written reflection in an emergency medicine rotation boot camp improves interns’ patient management skills, ethical decision-making, and reflection skills.A multimodal learning environment was created by the collaboration of emergency medicine, ethics, and medical education specialists. Multiple educational techniques involving lectures, case discussions, and role-playing a crisis scenario were applied. Pre-test and post-test, debriefing on performances on video records, video-recorded performance assessment, and reflective essays about their own and group’s performances were used to assess various aspects of the student performances. Additionally, a meeting was organized with the presence of the authors to create qualitative data obtained through the program evaluation meeting conducted on three themes: influences of teaching methods, students’ performances, and common achievements and mistakes of students.133 students participated. Post-test multiple-choice question (MCQ) test scores were slightly higher than pre-test. A low and medium correlation was detected among pre-test and post-test patient management problem (PMP) and reflection scores, which was more prominent for female students. Multiple linear regression showed that pre-test and post-test PMP scores significantly contributed to reflection scores. These results might support that better patient management predicts more robust reflective practice. Teachers observed that students appreciated being inspired by well-performing peers, particularly noting the empathic needs of patients, companions, and other health professionals. However, students overlooked summoning forensic or social services and were inhibited by the pressure of the contextual traits of the drill.The multimodal learning environment created by multidisciplinary collaboration contributed to the improvement of components of situational awareness of the interns: patient management skills, ethical decision-making, and reflective practice. During this research, we created a toolbox better to capture the richness and diversity of student interactions. Considering the scarcity of context-specific assessment methods and widespread use of MCQs or generic scales for higher-order thinking skills in medicine, this study might be regarded as a step forward in that context.The online version contains supplementary material available at 10.1186/s12909-021-02970-8. Emergency medicine operates against time pressures in the care of patients in life-threatening situations and entails numerous ethical dilemmas. Although fundamental ethical obligations such as informed consent and confidentiality might sometimes not be fully met for patients in emergency care, respect for patient autonomy, beneficence, nonmaleficence, privacy and confidentiality, and fairness must be balanced with optimal care , 2 in liReflection is an important step in ethical reasoning and decision-making and involves the following steps: analyzing the underlying dilemma, realizing duties and responsibilities in terms of conflicting values, presenting a solution in the light of ethical principles, and decision testing , 4. StraTsai and Harasym noted the importance of considering medical indications, quality of life, the patient’s preferences, and the current context in ethical reasoning . This reDebriefing is a purposeful, structured reflection technique that involves reflecting on the experienced event, discussing experiences with others, and learning and modifying behaviors . EvaluatIn the last decades, integration of clinical skills, ethical decision-making, and reflection skills have emerged as cornerstones of clinical teaching in medical schools, but all are seldom linked in curricula . The innIt was noticed that in the literature, assessment of ethics skills was solely based on pre- post-tests, multiple-choice questions (MCQs), or generic test batteries such as Defining Issues Test or Moral Judgment Tests –24. AddiAt the Acibadem Mehmet Ali Aydinlar University School of Medicine (SoM), medical ethics, bioethics, and professionalism are integrated into the curriculum longitudinally in phase one (years 1–3). In phase 2, (years 4–5) “Ethics Rounds” are practiced within the clinical clerkships . The aimDuring the emergency medicine internship in phase 3, a novel module has been implemented to blend clinical reasoning, ethical decision-making, and reflection in an emergency care drill during the rotation before emergency department attachment. The novel module was included in the abdominal pain day of a 5-day long boot camp that focused on diagnosing and managing different life-threatening situations. Each day, situations such as altered mental status, chest pain and dyspnea, multiple traumas, and a busy emergency department were taught. In addition, high-fidelity simulators were utilized, the faculty and intern doctors were included in role-playing scenarios, and the interns were given a chance for reflection throughout . The leaThis study aimed to examine the relationships between patient management skills, ethical decision-making, and reflective practice to introduce the multimodal learning environment designed for interns at the Acibadem Mehmet Ali Aydinlar University SoM to improve clinical management, ethical reasoning, and reflection skills. The environment was introduced in the five-day-long boot camp of the emergency medicine rotation. Lastly, it was aimed to present the study results obtained through the quantitative assessment methods and the qualitative data obtained through a program evaluation meeting.This study was approved by the Acibadem Mehmet Ali Aydinlar University Research Ethics Committee (ATADEK) with the decision number: 2018–2/68. The written informed consent was obtained from the participants before the practice. All methods were performed in accordance with the relevant guidelines and regulations.In an emergency medicine rotation intern boot camp, does a multimodal learning environment approach consisting of lectures, a drill, post-drill video debriefing, and written reflection improve the patient management skills, ethical decision-making, and reflective practice of interns?A multimodal learning environment was created by the multidisciplinary collaboration of emergency medicine, ethics, and medical education specialists. This study was created as a multi-method design consisting of quantitative student assessment methods, and also presents the qualitative data obtained through the program evaluation meeting conducted with the presence of the authors SY, YIU, GS, KV.Multiple educational techniques involving lectures, case discussions, and role-playing a crisis scenario were applied. Pre-test and post-test, debriefing on performances on video records, video-recorded performance assessment (VPA), and reflective essays about their own and group’s performances were used for the assessment of various aspects of the student performances. Additionally, a meeting was organized with the presence of the authors SY, YIU, KV, and GS to evaluate the program. During the meeting, observations, and opinions of authors as teachers were obtained on three themes: (1) influences of teaching methods, (2) students’ performances, and (3) common achievements and mistakes of students.The study group consisted of interns from the Acibadem Mehmet Ali Aydinlar University SoM in their emergency medicine rotations. The research was conducted over two years 2018–2019) with eight consecutive groups undergoing “Abdominal Pain Day” of the five-day-long boot camps before the beginning of the emergency medicine rotation  for assessing the cognitive skills of applying and analyzing were used  evaluating the influences of teaching content and methods, (2) students’ performances, and (3) common achievements, mistakes, and stereotypes of the interns. Meeting notes were discussed in the presence of all and decided to be reported in this manuscript as qualitative data.The drill scenario consisted of five roles: physician, nurse, patient, the patient’s mother, and the patient’s brother. These were designed according to a hidden curriculum aspect consisting of one adult and one minor patient, both of whom were hemodynamically unstable due to a rupture of ectopic pregnancy as a result of an extramarital relationship. All roles were played by the interns. The video recording was then watched with the interns, and a debriefing was given. The drill was concluded with the reflective essays from the interns.The footage recorded during the drill was assessed by two teachers (one ethicist and one emergency medicine physician) and was scored in line with the VPA rubric form. If the scores of two teachers were inconsistent, they negotiated and settled on an agreed score. The group performance results were adopted as the individual interns’ VPA scores. The VPA topics on the rubric form are presented in Tables Descriptive statistics were presented using mean, standard deviation, median, minimum, maximum. Paired Samples t-test was used to compare two normally distributed dependent groups.Spearman’s rho correlation analysis was used to analyze relationships between two non-normally distributed variables. Multiple linear regression analysis was used to predict interns’ reflective essay scores. The data categories from 133 interns were as follows: reflective essay scores, gender, VPA scores, pre-test and post-test MCQs and PMPs. The backward variable selection method was used.p values were lower than 0.05.Statistical significance was accepted when two-sided Statistical analysis was performed using MedCalc Statistical Software version 12.7.7.± 1.26 years .The research sample consisted of all 133 interns of the Acibadem Mehmet Ali Aydinlar University SoM attending emergency medicine internship in eight groups consisting of 12–21 students (Mean: 16.63). Seventy-seven (57.9%) students were women and 56 (42.1%) were men. Students’ mean age was 24.1 Table The total post-test MCQ score was slightly higher than the pre-test MCQs (p: 0.010). The difference in the post-test PMPs was not found to have a statistically significant difference with the pre-test PMPs. The mean VPA scores were 28.86 out of 40, the mean reflective writing score was 18.76 out of 30, and the mean total post-test score was 63.40 out of 86.Correlation coefficients of total and all reflection questions’ scores, and VPA scores with pre-test and post-test MCQ and PMP are presented in Table VPA Video-recorded Performance Assessment, MCQ Multiple-Choice Questions, PMP Patient Management Problems) Spearman’s rho,p * < 0.05, ** < 0.001.Total scores of pre-test and post-test MCQ were found to be moderately correlated (0.46). Pre-test and post-test PMP scores were also found to have a moderate correlation (0.404). A weak correlation between pre-test MCQ and VPA was detected (0.272). For female students, post-test MCQ scores showed a moderate correlation with VPA scores (0.399). Reflection essay scores had no correlation with the pre-test and post-test MCQs, except low correlation of reflection question 6 for male students 0.345). There was a weak correlation of all reflection essay questions and total scores with pre-test PMP and post-test PMP scores, changing from 0.182 to 0.279 . Pre-test and post-test PMP scores significantly contributed to the prediction of reflection scores (p < 0.05) with one unit change in the pre-test PMP increasing the reflection score 0.538 points and one unit of change in the post-test PMP increasing the reflection score 0.404 points. The equation for predicting reflection scores from pre-test and post-test PMPs was =12.039 + 0.538 (pre-test PMP score) + 0.559 (post-test PMP score).The backward variable selection method was used. No autocorrelation  or multicollinearity (VIF = 1.082 < 10) were present. The model was found to be significant (F = 7039, Opinions and observations of teachers obtained in the program evaluation meeting revealed that the boot camp had increased both the performance and enthusiasm of the students in the following seven-week rotation. Also, the scenario had worked well, and students made genuine efforts to find solutions. In the video debriefing sessions, students appreciated the opportunity of peer learning through inspiration from good performances of role model peers. Students’ approaches were those of novices, and the contextual features of the case had affected the decision-making process and the intervention. Likewise, interns established less contact with forensic or social services than expected. While role-playing, students mostly portrayed the conservative brother as a man of low socioeconomic status. This highlights an important clue to a student prejudice, that conservative men are of low socioeconomic status. Another noteworthy observation was that female students exhibited a more patient and genuine approach toward an interfering companion. It was remarkable that students frequently emphasized that they had learned the empathy needs of the patient, companions, and other team members.This study describes a one-day drill program using multiple methods of teaching and assessment and aiming to teach clinical decision-making, ethical decision-making, and reflection skills in medical emergency cases, in an integrated approach.There are several studies in medical education and health professionals’ education literature that integrate either clinical reasoning/decision-making with ethical reasoning/decision-making or reflection skills –19. ThusDespite the fact that integration of clinical skills, ethical decision-making, and reflection skills have emerged as cornerstones of clinical teaching in medical schools recently , assessmIn this study, a slight but statistically significant difference was detected between pre-test and post-test MCQ scores. In the literature, there are studies on ethics education in medical schools reporting no statistically significant difference  or signiThe correlation of pre-test and post-test PMP scores with reflection essay scores implied that when students acquire higher levels of cognitive skills, i.e., applying and analyzing, they might perform better in terms of higher-order thinking skills such as reflection. Moreover, the multiple linear regression results support this finding and show that the pre-test and post-test PMP scores contribute significantly to the prediction of reflection scores. These results might support that, better patient management predicts more robust reflective practice.The spiral model of ethics education in the Acibadem Mehmet Ali Aydinlar University SoM which starts from the first year and introduces more advanced applications or new skills related to those previously covered in the previous years might have contributed to this result. The effect of the contextual features of the case on students’ decision-making and intervention pointed out the need for greater opportunity to work independently and to encounter more complex cases in the previous clinical education. Observing students’ prejudice towards the patient’s brother evoked the need for structured skills training regarding difficult situation management. Besides, fewer students contacted with forensic or social services than expected implied the need to emphasize more when and how to involve forensic and social services in difficult situations.During this research, we created a toolbox to better capture the richness and diversity of student interactions. A transformative loop, capable of triggering a transformation in self-confidence, supporting the questioning of assumptions, and experiences giving the opportunity to reflect on the actions in both the teachers and the students was formed in the integrated, effective, and supportive learning environment. Students’ situational awareness was improved, and both parties had a view of the traits in the hidden curriculum, such as pre-acceptance of conservative men belonging to low socioeconomic status.The scarcity of context-specific assessment methods and widespread use of MCQs or generic scales for ethical reasoning in the literature revealed the need for implementing innovative and multi-method assessing ethical reasoning, clinical judgment, and reflection skills in difficult situations . The preFuture studies in which students reflect on their performances and the use of these writings for following up on their progress might be conducted to assess their reflection skills and self-directed learning.Students’ reflection essays were not used for following up on their improvement throughout the rotation or the year. No written or structured oral feedback was obtained from the students. Structured qualitative data was not collected.This study describes a novel and innovative program utilizing multiple teaching and assessment methods to teach and assess the interrelation between clinical decision-making, ethical decision-making, and reflection with an integrated approach.Lectures helped students to retrieve previous learning and provided a focus for ethical dilemmas in medical emergency cases. Role-playing the opposite gender and acting as a patient and interfering companion helped the interns to see the gaps in their empathic behaviors and provided a framework for improvement in handling a disorienting dilemma. Debriefing sessions and reflection essays gave them the opportunity for premise reflection, by asking them to reflect on their feelings, thoughts, performances, and their own and peers’ weaknesses or strengths. Reflection essays supplied them with the clues for self-directing their learning and improving their clinical and ethical decision-making skills by formulating action plans to learn further.Additional file 1. Drill scenarios, multiple-choice questions, and patient management problems.Additional file 2.  Research data.