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Abstract There is currently no medical therapy to prevent calcific aortic valve stenosis (CAVS). Multi-omics approaches could lead to the identification of novel molecular targets. Here, we perform a genome-wide association study (GWAS) meta-analysis including 14,819 cases among 941,863 participants of European ancestry. We report 32 genomic loci, among which 20 are novel. RNA sequencing of 500 human aortic valves highlights an enrichment in expression regulation at these loci and prioritizes candidate causal genes. Homozygous genotype for a risk variant near TWIST1, a gene involved in endothelial-mesenchymal transition, has a profound impact on aortic valve transcriptomics. We identify five genes outside of GWAS loci by combining a transcriptome-wide association study, colocalization, and Mendelian randomization analyses. Using cross-phenotype and phenome-wide approaches, we highlight the role of circulating lipoproteins, blood pressure and inflammation in the disease process. Our findings pave the way for the development of novel therapies for CAVS.

Abstract Development of high yielding cowpea varieties coupled with good taste and rich in essential minerals can promote consumption and thus nutrition and profitability. The sweet taste of cowpea grain is determined by its sugar content, which comprises mainly sucrose and galacto-oligosaccharides (GOS) including raffinose and stachyose. However, GOS are indigestible and their fermentation in the colon can produce excess intestinal gas, causing undesirable bloating and flatulence. In this study, we aimed to examine variation in grain sugar and mineral concentrations, then map quantitative trait loci (QTLs) and estimate genomic-prediction (GP) accuracies for possible application in breeding. Grain samples were collected from a multi-parent advanced generation intercross (MAGIC) population grown in California during 2016–2017. Grain sugars were assayed using high-performance liquid chromatography. Grain minerals were determined by inductively coupled plasma–optical emission spectrometry and combustion. Considerable variation was observed for sucrose (0.6–6.9%) and stachyose (2.3–8.4%). Major QTLs for sucrose (QSuc.vu-1.1), stachyose (QSta.vu-7.1), copper (QCu.vu-1.1) and manganese (QMn.vu-5.1) were identified. Allelic effects of major sugar QTLs were validated using the MAGIC grain samples grown in West Africa in 2017. GP accuracies for minerals were moderate (0.4–0.58). These findings help guide future breeding efforts to develop mineral-rich cowpea varieties with desirable sugar content.

AimsElevated lipoprotein(a) [Lp(a)] levels are associated with the risk of coronary artery disease (CAD) and calcific aortic valve stenosis (CAVS). Observational studies revealed that Lp(a) and C-reactive protein (CRP) levels, a biomarker of systemic inflammation, may jointly predict CAD risk. Whether Lp(a) and CRP levels also jointly predict CAVS incidence and progression is unknown.Methods and resultsWe investigated the association of Lp(a) with CAVS according to CRP levels in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk study (n = 18 226, 406 incident cases) and the UK Biobank (n = 438 260, 4582 incident cases), as well as in the ASTRONOMER study (n = 220), which assessed the haemodynamic progression rate of pre-existing mild-to-moderate aortic stenosis. In EPIC-Norfolk, in comparison to individuals with low Lp(a) levels (50 mg/dL) and low CRP levels (50 mg/dL) and elevated CRP levels (>2.0 mg/L) had a higher CAVS risk [hazard ratio (HR) = 1.86 (95% confidence intervals, 1.30-2.67) and 2.08 (1.44-2.99), respectively]. A comparable predictive value of Lp(a) in patients with vs. without elevated CRP levels was also noted in the UK Biobank. In ASTRONOMER, CAVS progression was comparable in patients with elevated Lp(a) levels with or without elevated CRP levels.ConclusionLp(a) predicts the incidence and possibly progression of CAVS regardless of plasma CRP levels. Lowering Lp(a) levels may warrant further investigation in the prevention and treatment of CAVS, regardless of systemic inflammation.

Summary: Background: The most near-term clinical application of genome-wide association studies in lung cancer is a polygenic risk score (PRS). Methods: A case-control dataset was generated consisting of 4002 lung cancer cases from the LORD project and 20,010 ethnically matched controls from CARTaGENE. A genome-wide PRS including >1.1 million genetic variants was derived and validated in UK Biobank (n = 5419 lung cancer cases). The predictive ability and diagnostic discrimination performance of the PRS was tested in LORD/CARTaGENE and benchmarked against previous PRSs from the literature. Stratified analyses were performed by smoking status and genetic risk groups defined as low (80th percentile) PRS. Findings: The phenotypic variance explained and the effect size of the genome-wide PRS numerically outperformed previous PRSs. Individuals with high genetic risk had a 2-fold odds of lung cancer compared to low genetic risk. The PRS was an independent predictor of lung cancer beyond conventional clinical risk factors, but its diagnostic discrimination performance was incremental in an integrated risk model. Smoking increased the odds of lung cancer by 7.7-fold in low genetic risk and by 11.3-fold in high genetic risk. Smoking with high genetic risk was associated with a 17-fold increase in the odds of lung cancer compared to individuals who never smoked and with low genetic risk. Interpretation: Individuals at low genetic risk are not protected against the smoking-related risk of lung cancer. The joint multiplicative effect of PRS and smoking increases the odds of lung cancer by nearly 20-fold. Funding: This work was supported by the CQDM and the IUCPQ Foundation owing to a generous donation from Mr. Normand Lord.

This study examined student experiences participating in a peer-led three-day wilderness canoe excursion in Northern Ontario, Canada. This program is a mandatory outdoor experience program (MOEP) for third-year students and is the final course in a progressive series of outdoor experiences offered at the beginning of the first, second, and third year. Guided by principles of realist evaluation, the written reflections from consenting students in the 2015 MOEP cohort were analyzed to identify the contexts and mechanisms associated with enjoyment or lack of enjoyment of their trip. These findings will help improve the MOEP program and have implications for similar intentionally led outdoor programs offered at other higher education institutions.

Microbiome research has grown substantially over the past decade in terms of the range of biomes sampled, identified taxa, and the volume of data derived from the samples. In particular, experimental approaches such as metagenomics, metabarcoding, metatranscriptomics and metaproteomics have provided profound insights into the vast, hitherto unknown, microbial biodiversity. The ELIXIR Marine Metagenomics Community, initiated amongst researchers focusing on marine microbiomes, has concentrated on promoting standards around microbiome-derived sequence analysis, as well as understanding the gaps in methods and reference databases, and solutions to computational overheads of performing such analyses. Nevertheless, the methods used and the challenges faced are not confined to marine studies, but are broadly applicable to all other biomes. Thus, expanding this Community to a more inclusive ELIXIR Microbiome Community will enable it to encompass a broad range of biomes and link expertise across ‘omics technologies. Furthermore, engaging with a large number of researchers will improve the efficiency and sustainability of bioinformatics infrastructure and resources for microbiome research (standards, data, tools, workflows, training), which will enable a deeper understanding of the function and taxonomic composition of the different microbial communities.

Small GTPases are highly regulated proteins that control essential signaling pathways through the activity of their effector proteins. Among the RHOA subfamily, RHOB regulates peculiar functions that could be associated with the control of the endocytic trafficking of signaling proteins. Here, we used an optimized assay based on tripartite split-GFP complementation to localize GTPase-effector complexes with high-resolution. The detection of RHOB interaction with the Rhotekin Rho binding domain (RBD) that specifically recognizes the active GTP-bound GTPase, is performed in vitro by the concomitant addition of recombinant GFP1–9 and a GFP nanobody. Analysis of RHOB-RBD complexes localization profiles combined with immunostaining and live cell imaging indicated a serum-dependent reorganization of the endosomal and membrane pool of active RHOB. We further applied this technology to the detection of RHO-effector complexes that highlighted their subcellular localization with high resolution among the different cellular compartments.

Abstract Features of the gut microbiota have been associated with several chronic diseases and longevity in preclinical models as well as in observational studies. Whether these relations underlie causal effects in humans remains to be established. We aimed to determine whether the gut microbiota influences cardiometabolic traits as well as the risk of chronic diseases and human longevity using a comprehensive 2-Sample Mendelian randomization approach. We included as exposures 10 gut-associated metabolites and pathways and 57 microbial taxa abundance. We included as outcomes nine cardiometabolic traits (fasting glucose, fasting insulin, systolic blood pressure, diastolic blood pressure, HDL cholesterol, LDL cholesterol, triglycerides, estimated glomerular filtration rate, body mass index [BMI]), eight chronic diseases previously linked with the gut microbiota in observational studies (Alzheimer’s disease, depression, type 2 diabetes, non-alcoholic fatty liver disease, coronary artery disease (CAD), stroke, osteoporosis and chronic kidney disease), as well as parental lifespan and longevity. We found 7 associations with evidence of causality before and after sensitivity analyses, but not after multiple testing correction (1198 tests). Most effect sizes (4/7) were small. The two largest exposure-outcome effects were markedly attenuated towards the null upon inclusion of BMI or alcohol intake frequency in multivariable MR analyses. While finding robust genetic instruments for microbiota features is challenging hence potentially inflating type 2 errors, these results do not support a large causal impact of human gut microbita features on cardiometabolic traits, chronic diseases or longevity. These results also suggest that the previously documented associations between gut microbiota and human health outcomes may not always underly causal relations.

Background The impact of an elevated body mass index (BMI) on atherosclerotic cardiovascular disease (ASCVD) risk in individuals who are metabolically healthy is debated. We investigated the respective contributions of BMI as well as lifestyle and cardiometabolic risk factors combined to ASCVD incidence in 319 866 UK Biobank participants. Methods and Results We developed a cardiovascular health score (CVHS) based on 4 lifestyle and 6 cardiometabolic parameters. The impact of the CVHS on incident ASCVD (15 699 events) alone and in BMI and waist‐to‐hip ratio categories was assessed using Cox proportional hazards in women and men separately. In participants with a high CVHS (8–10), those with a BMI ≥35.0 kg/m2 had a nonsignificantly higher ASCVD risk (hazard ratio [HR], 1.20 [95% CI, 0.84–1.70]; P=0.32) compared with those with a BMI of 18.5 to 24.9 kg/m2. In participants with a BMI of 18.5 to 24.9 kg/m2, those with a lower CVHS (0–2) had a higher ASCVD risk (HR, 4.06 [95% CI, 3.23–5.10]; P

Summary: Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent disease with no specific drug therapy. High-throughput metabolomics present an unprecedented opportunity to identify biomarkers and potentially causal risk factors for NAFLD. Here, we determined the impact of 21 circulating metabolites, 17 lipids, and 132 lipoprotein particle characteristics on NAFLD combining prospective observational and two-sample Mendelian randomization (MR) analyses in 121,032 UK Biobank participants. We identified several metabolic factors associated with NAFLD risk in observational and MR analyses including triglyceride-rich and high-density lipoprotein particles composition, as well as the ratio of polyunsaturated fatty acids to total fatty acids. This study, is one of the largest to investigate incident NAFLD, provides concordant observational and genetic evidence that therapies aimed at reducing circulating triglycerides and increasing large HDL particles, as well as interventions aimed at increasing polyunsaturated fatty acid content may warrant further investigation into NAFLD prevention and treatment.

Background Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan-dioxygenase (TDO) are enzymes catabolizing the essential amino acid tryptophan into kynurenine. Expression of these enzymes is frequently observed in advanced-stage cancers and is associated with poor disease prognosis and immune suppression. Mechanistically, the respective roles of tryptophan shortage and kynurenine production in suppressing immunity remain unclear. Kynurenine was proposed as an endogenous ligand for the aryl hydrocarbon receptor (AHR), which can regulate inflammation and immunity. However, controversy remains regarding the role of AHR in IDO1/TDO-mediated immune suppression, as well as the involvement of kynurenine. In this study, we aimed to clarify the link between IDO1/TDO expression, AHR pathway activation and immune suppression.Methods AHR expression and activation was analyzed by RT-qPCR and western blot analysis in cells engineered to express IDO1/TDO, or cultured in medium mimicking tryptophan catabolism by IDO1/TDO. In vitro differentiation of naïve CD4+ T cells into regulatory T cells (Tregs) was compared in T cells isolated from mice bearing different Ahr alleles or a knockout of Ahr, and cultured in medium with or without tryptophan and kynurenine.Results We confirmed that IDO1/TDO expression activated AHR in HEK-293-E cells, as measured by the induction of AHR target genes. Unexpectedly, AHR was also overexpressed on IDO1/TDO expression. AHR overexpression did not depend on kynurenine but was triggered by tryptophan deprivation. Multiple human tumor cell lines overexpressed AHR on tryptophan deprivation. AHR overexpression was not dependent on general control non-derepressible 2 (GCN2), and strongly sensitized the AHR pathway. As a result, kynurenine and other tryptophan catabolites, which are weak AHR agonists in normal conditions, strongly induced AHR target genes in tryptophan-depleted conditions. Tryptophan depletion also increased kynurenine uptake by increasing SLC7A5 (LAT1) expression in a GCN2-dependent manner. Tryptophan deprivation potentiated Treg differentiation from naïve CD4+ T cells isolated from mice bearing an AHR allele of weak affinity similar to the human AHR.Conclusions Tryptophan deprivation sensitizes the AHR pathway by inducing AHR overexpression and increasing cellular kynurenine uptake. As a result, tryptophan catabolites such as kynurenine more potently activate AHR, and Treg differentiation is promoted. Our results propose a molecular explanation for the combined roles of tryptophan deprivation and kynurenine production in mediating IDO1/TDO-induced immune suppression.

Summary: Therapeutic neoantigen cancer vaccines have limited clinical efficacy to date. Here, we identify a heterologous prime-boost vaccination strategy using a self-assembling peptide nanoparticle TLR-7/8 agonist (SNP) vaccine prime and a chimp adenovirus (ChAdOx1) vaccine boost that elicits potent CD8 T cells and tumor regression. ChAdOx1 administered intravenously (i.v.) had 4-fold higher antigen-specific CD8 T cell responses than mice boosted by the intramuscular (i.m.) route. In the therapeutic MC38 tumor model, i.v. heterologous prime-boost vaccination enhances regression compared with ChAdOx1 alone. Remarkably, i.v. boosting with a ChAdOx1 vector encoding an irrelevant antigen also mediates tumor regression, which is dependent on type I IFN signaling. Single-cell RNA sequencing of the tumor myeloid compartment shows that i.v. ChAdOx1 reduces the frequency of immunosuppressive Chil3 monocytes and activates cross-presenting type 1 conventional dendritic cells (cDC1s). The dual effect of i.v. ChAdOx1 vaccination enhancing CD8 T cells and modulating the TME represents a translatable paradigm for enhancing anti-tumor immunity in humans.

Background Despite their revolutionary success in cancer treatment over the last decades, immunotherapies encounter limitations in certain tumor types and patients. The efficacy of immunotherapies depends on tumor antigen-specific CD8 T-cell viability and functionality within the immunosuppressive tumor microenvironment, where oxygen levels are often low. Hypoxia can reduce CD8 T-cell fitness in several ways and CD8 T cells are mostly excluded from hypoxic tumor regions. Given the challenges to achieve durable reduction of hypoxia in the clinic, ameliorating CD8 T-cell survival and effector function in hypoxic condition could improve tumor response to immunotherapies.Methods Activated CD8 T cells were exposed to hypoxia and metformin and analyzed by fluorescence-activated cell sorting for cell proliferation, apoptosis and phenotype. In vivo, metformin was administered to mice bearing hypoxic tumors and receiving either adoptive cell therapy with tumor-specific CD8 T cells, or immune checkpoint inhibitors; tumor growth was followed over time and CD8 T-cell infiltration, survival and localization in normoxic or hypoxic tumor regions were assessed by flow cytometry and immunofluorescence. Tumor oxygenation and hypoxia were measured by electron paramagnetic resonance and pimonidazole staining, respectively.Results We found that the antidiabetic drug metformin directly improved CD8 T-cell fitness in hypoxia, both in vitro and in vivo. Metformin rescued murine and human CD8 T cells from hypoxia-induced apoptosis and increased their proliferation and cytokine production, while blunting the upregulation of programmed cell death protein 1 and lymphocyte-activation gene 3. This appeared to result from a reduced production of reactive oxygen species, due to the inhibition of mitochondrial complex I. Differently from what others reported, metformin did not reduce tumor hypoxia, but rather increased CD8 T-cell infiltration and survival in hypoxic tumor areas, and synergized with cyclophosphamide to enhance tumor response to adoptive cell therapy or immune checkpoint blockade in different tumor models.Conclusions This study describes a novel mechanism of action of metformin and presents a promising strategy to achieve immune rejection in hypoxic and immunosuppressive tumors, which would otherwise be resistant to immunotherapy.

It remains unclear whether the increased risk of new-onset type 2 diabetes (T2D) seen in statin users is due to low LDL-C concentrations, or due to the statin-induced proportional change in LDL-C. In addition, genetic instruments have not been proposed before to examine whether liability to T2D might cause greater proportional statin-induced LDL-C lowering. Using summary-level statistics from the Genomic Investigation of Statin Therapy (GIST, nmax = 40,914) and DIAGRAM (nmax = 159,208) consortia, we found a positive genetic correlation between LDL-C statin response and T2D using LD score regression (rgenetic = 0.36, s.e. = 0.13). However, mendelian randomization analyses did not provide support for statin response having a causal effect on T2D risk (OR 1.00 (95% CI: 0.97, 1.03) per 10% increase in statin response), nor that liability to T2D has a causal effect on statin-induced LDL-C response (0.20% increase in response (95% CI: -0.40, 0.80) per doubling of odds of liability to T2D). Although we found no evidence to suggest that proportional statin response influences T2D risk, a definitive assessment should be made in populations comprised exclusively of statin users, as the presence of nonstatin users in the DIAGRAM dataset may have substantially diluted our effect estimate.

Gagnon et al. perform a Mendelian randomization study to investigate whether excess liver fat underpins adiposity’s effect on cardiometabolic disease risk. They show that abdominal adiposity strongly increases disease risk independently of liver fat, suggesting that targeting liver fat alone may not be sufficient to prevent cardiometabolic disease.

Abstract Background Heart failure (HF) is a prevalent cause of mortality and morbidity. The molecular drivers of HF are still largely unknown. Results We aimed to identify circulating proteins causally associated with HF by leveraging genome-wide genetic association data for HF including 47,309 cases and 930,014 controls. We performed two-sample Mendelian randomization (MR) with multiple cis instruments as well as network and enrichment analysis using data from blood protein quantitative trait loci (pQTL) (2,965 blood proteins) measured in 3,301 individuals. Nineteen blood proteins were causally associated with HF, were not subject to reverse causality and were enriched in ligand-receptor and glycosylation molecules. Network pathway analysis of the blood proteins showed enrichment in NF-kappa B, TGF beta, lipid in atherosclerosis and fluid shear stress. Cross-phenotype analysis of HF identified genetic overlap with cardiovascular drugs, myocardial infarction, parental longevity and low-density cholesterol. Multi-trait MR identified causal associations between HF-associated blood proteins and cardiovascular outcomes. Multivariable MR showed that association of BAG3, MIF and APOA5 with HF were mediated by the blood pressure and coronary artery disease. According to the directional effect and biological action, 7 blood proteins are targets of existing drugs or are tractable for the development of novel therapeutics. Among the pathways, sialyl Lewis x and the activin type II receptor are potential druggable candidates. Conclusions Integrative MR analyses of the blood proteins identified causally-associated proteins with HF and revealed pleiotropy of the blood proteome with cardiovascular risk factors. Some of the proteins or pathway related mechanisms could be targeted as novel treatment approach in HF.

Summary: Genome-wide association studies (GWAS) have identified hundreds of genetic variants associated with body weight but the biological relevance of most remains unexplored. Given the critical role of the brain in body weight regulation, we set out to determine whether genetic variants linked with body mass index (BMI) could be mapped to brain proteins. Using genetic colocalization, we mapped 25 loci from the largest BMI GWAS (n = 806,834) to brain protein concentrations obtained from publicly available datasets. We also performed a proteome-wide Mendelian randomization on 696 brain proteins followed by genetic colocalization and identified 35 additional brain proteins. Only a minority of these proteins (