Small extracellular vesicles (small EVs) are nanovesicles found in tissues and body fluids that contain regulatory molecules including microRNAs, termed exomiRs. Research in murine models has demonstrated that exercise can trigger the release of small EVs into the circulation. The aim of this study was to study exomiR release in humans pre and post exercise and to characterise the function of these microRNAs especially in relation to thermogenic fat. We found that exercise increased the release of exomiR-196a-5p in endurance athletes, a microRNA that induces UCP1 expression and browning of white adipocytes. We observed that myotubes specifically release miR-196a-5p within small EVs after in vitro exercise-mimicking conditions such as electrical pulse stimulation and cAMP treatment. Likewise, the expression at basal levels of the exercise-induced exomiR-342-3p negatively correlated with BMI and age. EV proteomics revealed a positive correlation between FABP4+ and miR-342-3p, suggesting an adipocyte cell origin. Overexpression of miR-342-3p increased Myogenin levels during skeletal muscle cell differentiation, indicating a positive role in muscle differentiation. Our results suggest that oxidative extreme metabolic capacities in endurance athletes contribute to the enhanced release of circulatory exomiRs after exercise mediating bi-directional crosstalk between skeletal muscle and thermogenic adipose tissue. Graphical abstract ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, 450149205 Deutsche Forschungsgemeinschaft, 388171357 [1]: pending:yes

Familial adenomatous polyposis (FAP) is an inherited gastrointestinal syndrome associated with duodenal adenoma formation. Even among carriers of the same genetic variant, duodenal phenotypes vary, indicating that additional factors, such as the local immune system, play a role. We observe an increase in duodenal IL-17A(+)NKp44(−) innate lymphoid type 3 cell (ILC3) in FAP, localized near the epithelium and enriched in adenomas and carcinomas. Elevated IL1B , IL23A , and DLL4 transcript levels correlate with IL-17A(+)NKp44(−)ILC3 accumulation, and in vitro studies with duodenal organoids confirmed this relationship. Bulk RNA sequencing reveals upregulated Reactive oxygen species (ROS)-inducing enzymes DUOX2 and DUOXA2 in FAP adenomas. IL-17A-stimulated FAP organoids show increased DUOX2/DUOXA2 expression, Duox2 protein, and ROS production, leading to DNA damage, suggesting a mechanism by which these immune cells promote tumorigenesis. These findings suggest IL-17A(+)NKp44(–)ILC3s may contribute to a local environment that makes the epithelium more submissive for oncogenic transformation in FAP.

Immune response genes are highly polymorphic in humans and mice, with heterogeneity amongst loci driving strain-specific host defence responses. The inadvertent retention of polymorphic loci can introduce confounding phenotypes, leading to erroneous conclusions, and impeding scientific advancement. In this study, we employ a combination of RNAseq and variant calling analyses to identify a substantial region of 129S genome, including the highly polymorphic Nlrp1 locus, proximal to Nlrp3, in one of the most commonly used mouse models of NLRP3 deficiency (Nlrp3tm1Flv). We show that the presence of the Nlrp1129S locus leads to an increase in NLRP1B protein expression, and a sensitising of Nlrp3tm1Flv macrophages to NLRP1 inflammasome activation, independent of NLRP3 deficiency. Retention of 129S genome further leads to protein sequence differences and altered gene regulation across multiple cell types, including of the key tissue-resident macrophage marker, TIM4. Using alternative models of NLRP3 deficiency, including a previously undescribed conditional Nlrp3 allele enabling precise temporal and cell-type specific control over Nlrp3 deletion, we further show that NLRP3 contributes to Talabostat-driven IL-1β release. Our study also establishes a generic framework to identify functionally relevant SNPs and assess genomic contamination in transgenic mice using RNAseq data. This allows for unambiguous attribution of phenotypes to the target gene and advances the precision and reliability of research in the field of host defence responses.

We present the results of the human genomic small variant calling benchmarking initiative of the German Research Foundation (DFG) funded Next Generation Sequencing Competence Network (NGS-CN) and the German Human Genome-Phenome Archive (GHGA). In this effort, we developed NCBench, a continuous benchmarking platform for the evaluation of small genomic variant callsets in terms of recall, precision, and false positive/negative error patterns. NCBench is implemented as a continuously re-evaluated open-source repository. We show that it is possible to entirely rely on public free infrastructure (Github, Github Actions, Zenodo) in combination with established open-source tools. NCBench is agnostic of the used dataset and can evaluate an arbitrary number of given callsets, while reporting the results in a visual and interactive way. We used NCBench to evaluate over 40 callsets generated by various variant calling pipelines available in the participating groups that were run on three exome datasets from different enrichment kits and at different coverages. While all pipelines achieve high overall quality, subtle systematic differences between callers and datasets exist and are made apparent by NCBench.These insights are useful to improve existing pipelines and develop new workflows. NCBench is meant to be open for the contribution of any given callset. Most importantly, for authors, it will enable the omission of repeated re-implementation of paper-specific variant calling benchmarks for the publication of new tools or pipelines, while readers will benefit from being able to (continuously) observe the performance of tools and pipelines at the time of reading instead of at the time of writing.

Immune response genes are highly polymorphic in humans and mice, with heterogeneity amongst loci driving strain-specific host defense responses. The inadvertent retention of polymorphic loci can introduce confounding phenotypes, leading to erroneous conclusions, and impeding scientific advancement. In this study, we employ a combination of RNAseq and variant calling analyses and identify a substantial region of 129S genome, including the highly polymorphic Nlrp1 locus proximal to Nlrp3 , in one of the most commonly used mouse models of NLRP3 deficiency. We show that increased expression of 129S NLRP1b sensitizes Nlrp3 −/− macrophages to NLRP1 inflammasome activation. Furthermore, the presence of 129S genome leads to altered gene and protein regulation across multiple cell-types, including of the key tissue-resident macrophage marker, TIM4. To address the challenge of resolving NLRP3-dependent phenotypes, we introduce and validate a conditional Nlrp3 allele, enabling precise temporal and cell-type-specific control over Nlrp3 deletion. Our study establishes a generic framework to identify functionally relevant SNPs and assess genomic contamination in transgenic mice. This allows for unambiguous attribution of phenotypes to the target gene and advances the precision and reliability of research in the field of host defense responses.

Bone marrow fibrosis in myeloproliferative neoplasm (MPN), myelodysplastic syndromes (MDS), MPN/MDS overlap syndromes and acute myeloid leukemia (AML) is associated with poor prognosis and early treatment failure. Myelofibrosis (MF) is accompanied by reprogramming of multipotent bone marrow mesenchymal stromal cells (MSC) into osteoid and fiber-producing stromal cells. We demonstrate NRP2 and osteolineage marker NCAM1 (neural cell adhesion molecule 1) expression within the endosteal niche in normal bone marrow and aberrantly in MPN, MDS MPN/MDS overlap syndromes and AML (n = 99), as assessed by immunohistochemistry. Increased and diffuse expression in mesenchymal stromal cells and osteoblasts correlates with high MF grade in MPN (p < 0.05 for NRP2 and NCAM1). Single cell RNA sequencing (scRNAseq) re-analysis demonstrated NRP2 expression in endothelial cells and partial co-expression of NRP2 and NCAM1 in normal MSC and osteoblasts. Potential ligands included transforming growth factor β1 (TGFB1) from osteoblasts and megakaryocytes. Murine ThPO and JAK2V617F myelofibrosis models showed co-expression of Nrp2 and Ncam1 in osteolineage cells, while fibrosis-promoting MSC only express Nrp2. In vitro experiments with MC3T3-E1 pre-osteoblasts and analysis of Nrp2−/− mouse femurs suggest that Nrp2 is functionally involved in osteogenesis. In summary, NRP2 represents a potential novel druggable target in patients with myelofibrosis.

Abstract Background Acute abdominal pain is a common presenting symptom in the emergency department and represents heterogeneous causes and diagnoses. There is often a decision to be made regarding emergency surgical care. Machine learning (ML) could be used here as a decision-support and relieve the time and personnel resource shortage. Methods Patients with acute abdominal pain presenting to the Department of Surgery at Bonn University Hospital in 2020 and 2021 were retrospectively analyzed. Clinical parameters as well as laboratory values were used as predictors. After randomly splitting into a training and test data set (ratio 80 to 20), three ML algorithms were comparatively trained and validated. The entire procedure was repeated 20 times. Results A total of 1357 patients were identified and included in the analysis, with one in five (n = 276, 20.3%) requiring emergency abdominal surgery within 24 hours. Patients operated on were more likely to be male (p = 0.026), older (p = 0.006), had more gastrointestinal symptoms (nausea: p < 0.001, vomiting p < 0.001) as well as a more recent onset of pain (p < 0.001). Tenderness (p < 0.001) and guarding (p < 0.001) were more common in surgically treated patients and blood analyses showed increased inflammation levels (white blood cell count: p < 0.001, CRP: p < 0.001) and onset of organ dysfunction (creatinine: p < 0.014, quick p < 0.001). Of the three trained algorithms, the tree-based methods (h2o random forest and cforest) showed the best performance. The algorithms classified patients, i.e., predicted surgery, with a median AUC ROC of 0.81 and 0.79 and AUC PRC of 0.56 in test sets. Conclusion A proof-of-concept was achieved with the development of an ML model for predicting timely surgical therapy for acute abdomen. The ML algorithm can be a valuable tool in decision-making. Especially in the context of heavily used medical resources, the algorithm can help to use these scarce resources more effectively. Technological progress, especially regarding artificial intelligence, increasingly enables evidence-based approaches in surgery but requires a strictly interdisciplinary approach. In the future, the use and handling of ML should be integrated into surgical training.

Subclinical inflammation induced by obesity, unhealthy diet and sedentary behavior is closely linked to the risks of cardiovascular disease and type 2 diabetes. Weight loss is recommended to resolve inflammation which involves the inflammasome in the inborn immune system.

Aims Type 2 diabetes (T2D) is related to chronic low-grade inflammation and innate immune system dysfunction. The underlying mechanisms of immune-metabolic programming in diabetic remission by weight loss are still unclear. We therefore performed fluorescence-activated cell sorting (FACS) of peripheral blood mononuclear cells (PBMC) and determined inflammatory bio-molecules in T2D patients with diabetes remission after weight loss by hypocaloric diet.

Sex-specific differences in schizophrenia (SCZ) are pervasive and have been reported for various aspects of the disease. Previous authors have therefore hypothesized that genetic risk factors on the X chromosome play a role in the complex interplay that leads to the development of this multifactorial and highly polygenic disorder. Nevertheless, few studies specifically targeted the investigation of these sex-specific genetic risk factors. In recent years, large-scale systematic genetic studies in SCZ have broadened the spectrum of genetic variation contributing to the disease and identified, among others, several X-chromosomal risk-associated loci. The world´s largest copy number variant (CNV) analysis for SCZ reported the first potential risk-associated CNV on the X chromosome. Duplications in Xq28,distal confer risk to SCZ in both sexes and span eight different protein-coding genes. Due to the shared genomic breakpoints of the duplications described to date, the issue of which gene/s at this locus is/are relevant in terms of SCZ pathogenesis remains unclear. In order to detect an enrichment of rare and potentially damaging variants in possible disease-relevant genes, the objective was to provide a detailed analysis of the genetic burden within the Xq28,distal locus using high-throughput, multiplex targeted sequencing. All coding regions of the implicated genes were sequenced in a large, European SCZ case-control cohort (n= 1935 patients with SCZ and n=1905 population-based controls) using the single molecule molecular inversion probe method on an Illumina HiSeq2500. To identify rare and potentially damaging variants, the analyses focused on non-synonymous single-nucleotide variants and Indels with a minor allele frequency ≤ 0.1% and a Combined Annotation Dependent Depletion score ≥ 20. The burden analysis was performed in a gene-wise manner and with sex as a covariate using the X-chromosomal model of the Optimal Unified Sequence Kernel Association Test adjusting for possible sex-specific effects. A total of n=73 independent variants passed the applied quality control criteria. After filtering for rare and potentially damaging variants, a total of n= 13 independent variants, as identified in four cases and eleven controls, were subjected to the consecutive burden analysis. For the genes within the Xq28,distal locus, no significant single-gene association was identified (smallest value in BRCC3 with p=0.103, p-adjusted=0.515), on the level of rare and potentially damaging variants. Overall, the present study confirms the recent gene-based results for the implicated genes of the Xq28, distal locus in the latest meta-analysis of exome sequencing data in SCZ conducted by the SCHEMA Consortium. The small number of identified variants is partly explained by the strict filter criteria and limited sample size. These findings might indicate a possible contribution of ultra-rare variants locus and a high degree of conservation within this region. Moreover, this may point towards alternative pathomechanisms not investigated in the present analyses; such as the involvement of sex-dependent risk factors, a genuine overexpression of a disease-relevant gene or the disruption of topologically associated domains in the context of duplications at Xq28,distal. Although inconclusive, the present findings highlight the need for more research into the role of X-chromosomal genetic risk factors in SCZ.

BACKGROUND:A relevant part of the genetic architecture of complex traits is still unknown; despite the discovery of many disease-associated common variants. Polygenic risk score (PRS) models are based on the evaluation of the additive effects attributable to common variants and have been successfully implemented to assess the genetic susceptibility for many phenotypes. In contrast, burden tests are often used to identify an enrichment of rare deleterious variants in specific genes. Both kinds of genetic contributions are typically analyzed independently. Many studies suggest that complex phenotypes are influenced by both low effect common variants and high effect rare deleterious variants. The aim of this paper is to integrate the effect of both common and rare functional variants for a more comprehensive genetic risk modeling.METHODS:We developed a framework combining gene-based scores based on the enrichment of rare functionally relevant variants with genome-wide PRS based on common variants for association analysis and prediction models. We applied our framework on UK Biobank dataset with genotyping and exome data and considered 28 blood biomarkers levels as target phenotypes. For each biomarker, an association analysis was performed on full cohort using gene-based scores (GBS). The cohort was then split into 3 subsets for PRS construction and feature selection, predictive model training, and independent evaluation, respectively. Prediction models were generated including either PRS, GBS or both (combined).RESULTS:Association analyses of the cohort were able to detect significant genes that were previously known to be associated with different biomarkers. Interestingly, the analyses also revealed heterogeneous effect sizes and directionality highlighting the complexity of the blood biomarkers regulation. However, the combined models for many biomarkers show little or no improvement in prediction accuracy compared to the PRS models.CONCLUSION:This study shows that rare variants play an important role in the genetic architecture of complex multifactorial traits such as blood biomarkers. However, while rare deleterious variants play a strong role at an individual level, our results indicate that classical common variant based PRS might be more informative to predict the genetic susceptibility at the population level.

Dowling-Degos disease (DDD) is a rare autosomal-dominant hyperpigmentation disorder caused by mutations in KRT5, POFUT1, POGLUT1 and PSENEN. Our results suggest that dysfunctional Notch signalling in melanocytes plays a key role in DDD pathogenesis, and that altered biogenesis and intracellular trafficking of melanosomes, receptor tyrosine kinase signalling and oestrogen signalling receptor-mediated signalling may represent downstream molecular mechanisms through which decreased Notch signalling leads to hyperpigmentation in DDD. Furthermore, a common downstream pathomechanism for both POGLUT1 and PSENEN mutation carriers can be assumed.

Male-pattern hair loss (MPHL) is a highly heritable and prevalent form of hair loss. While genome-wide association studies (GWAS) have provided insights into the contribution of common genetic variants to MPHL etiology, the relevance of rare variants is unclear. In this study, we tested for a contribution of rare coding variants to MPHL etiology based on exome data of 72,469 men from the UK Biobank. We performed SKAT-O and GenRisk gene-based association analyses as well as single-variant tests, followed by interpretation, risk prediction modelling and enrichment testing. Men were classified as cases or controls based on self-reported hair loss pattern (1-unaffected, 2-frontotemporal balding, 3-frontotemporal and vertex balding, 4-baldness of the top of the scalp). We used three classification schemes: (i) controls (pattern 1) were compared to cases (patterns 2-4), (ii) supercontrols (pattern 1, age ≥ 60) were compared to severe cases (pattern 4, age < 60) and (iii) controls (patterns 1-2) were compared to cases (patterns 3-4), addressing a plausible self-report misclassification between patterns 1 and 2. The overall contribution of rare variants to MPHL based on risk prediction was minimal. Nevertheless, our analyses identified significant associations of MPHL and rare variants in 125 genes and 2 single variants. Associated genes include previously implicated candidate genes (e.g. WNT10A) and novel candidate genes at and beyond GWAS risk loci (e.g. CDH1, SPINK5). We further observe an enrichment of genes causative for monogenic trichoses. In summary, while our data point to a low contribution of rare coding variants to MPHL, they provide evidence for candidate genes and variants at and beyond known risk loci and suggest an association between monogenic trichoses and the common MPHL phenotype.

Anorectal malformations (ARM) represent a spectrum of rare malformations originating from a perturbated development of the embryonic hindgut. Approximately 60% occur as a part of a defined genetic syndrome or within the spectrum of additional congenital anomalies. Rare copy number variations (CNVs) have been associated with both syndromic and non-syndromic forms. The present study represents the largest study to date to explore the contribution of CNVs to the expression of ARMs. SNP-array-based molecular karyotyping was applied in 450 individuals with ARM and 4392 healthy controls. CNVs were identified from raw intensity data using PennCNV. Overlapping CNVs between cases and controls were discarded. Remaining CNVs were filtered using a stringent filter algorithm of nine filter steps. Prioritized CNVs were confirmed using qPCR. Filtering prioritized and qPCR confirmed four microscopic chromosomal anomalies and nine submicroscopic CNVs comprising seven microdeletions (del2p13.2, del4p16.2, del7q31.33, del9p24.1, del16q12.1, del18q32, del22q11.21) and two microduplications (dup2p13.2, dup17q12) in 14 individuals (12 singletons and one affected sib-pair). Within these CNVs, based on their embryonic expression data and function, we suggest FOXK2 , LPP , and SALL3 as putative candidate genes. Overall, our CNV analysis identified putative microscopic and submicroscopic chromosomal rearrangements in 3% of cases. Functional characterization and re-sequencing of suggested candidate genes is warranted.

Milk is a nutrient-rich biofluid that contains several biocomponents with distinctive functions, including extracellular vesicles (EV). Milk EV have been associated with the regulation of the newborn's immune system and to influence essential cellular development. The EV proteome comprises the protein constituents and cargo; changes in these compartments could impact their role mediating communication. The ratio of dietary ω-6 to ω-3 polyunsaturated fatty acids (PUFA) is known to affect health and inflammation, and to induce changes in milk fatty acid composition, but no reports have included the milk EV fraction so far. We isolated EV from milk samples obtained on days 0, 7, and 14 after parturition from sows receiving either a standard diet or a test diet enriched in ω-3 (ω6:ω3 = 4:1). Small milk-derived EV were isolated using ultracentrifugation coupled with size exclusion chromatography, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Using a TMT-based high-resolution quantitative approach, the proteomics analysis revealed variations in the milk EV proteome within the diet groups with differences in the abundance of spondin-2 and 78 kDa glucose-regulated protein. Future studies are encouraged to explore further dietary effects on milk EV composition and their relation to the offspring's development. SIGNIFICANCE: Milk EV are known as key players mediating the regulation of the infant's immune system and growth. The EV proteome comprises the protein constituents and protein cargo, and any changes in this system could impact their role in intercellular communication. This study aimed at evaluating how different ω-6:ω-3 ratios in the maternal diet could translate to the milk EV proteome. This is relevant for basic research, but also has applied aspects in animal nutrition and health and may provide new perspectives for feeding additives.

The first major COVID-19 outbreak in Germany occurred in Heinsberg in February 2020 with 388 officially reported cases. Unexpectedly, the first outbreak happened in a small town with little to no travelers. We used phylogenetic analyses to investigate the origin and spread of the virus in this outbreak. We sequenced 90 (23%) SARS-CoV-2 genomes from the 388 reported cases including the samples from the first documented cases. Phylogenetic analyses of these sequences revealed mainly two circulating strains with 74 samples assigned to lineage B.3 and 6 samples assigned to lineage B.1. Lineage B.3 was introduced first and probably caused the initial spread. Using phylogenetic analysis tools, we were able to identify closely related strains in France and hypothesized the possible introduction from France.

Defects in nucleic acid metabolizing enzymes can lead to spontaneous but selective activation of either cGAS/STING or RIG-like receptor (RLR) signaling, causing type I interferon–driven inflammatory diseases. In these pathophysiological conditions, activation of the DNA sensor cGAS and IFN production are linked to spontaneous DNA damage. Physiological, or tonic, IFN signaling on the other hand is essential to functionally prime nucleic acid sensing pathways. Here, we show that low-level chronic DNA damage in mice lacking the Aicardi-Goutières syndrome gene SAMHD1 reduced tumor-free survival when crossed to a p53-deficient, but not to a DNA mismatch repair-deficient background. Increased DNA damage did not result in higher levels of type I interferon. Instead, we found that the chronic interferon response in SAMHD1-deficient mice was driven by the MDA5/MAVS pathway but required functional priming through the cGAS/STING pathway. Our work positions cGAS/STING upstream of tonic IFN signaling in Samhd1-deficient mice and highlights an important role of the pathway in physiological and pathophysiological innate immune priming.