Clostridioides difficile infections are associated with gut microbiome dysbiosis and are the leading cause of hospital-acquired diarrhoea. The infectious process is strongly influenced by the microbiota and successful infection relies on the absence of specific microbiota-produced metabolites. Deoxycholate and short-chain fatty acids are microbiota-produced metabolites that limit the growth of C. difficile and protect the host against this infection. In a previous study, we showed that deoxycholate causes C. difficile to form strongly adherent biofilms after 48 h. Here, our objectives were to identify and characterize key molecules and events required for biofilm formation in the presence of deoxycholate. We applied time-course transcriptomics and genetics to identify sigma factors, metabolic processes and type IV pili that drive biofilm formation. These analyses revealed that extracellular pyruvate induces biofilm formation in the presence of deoxycholate. In the absence of deoxycholate, pyruvate supplementation was sufficient to induce biofilm formation in a process that was dependent on pyruvate uptake by the membrane protein CstA. In the context of the human gut, microbiota-generated pyruvate is a metabolite that limits pathogen colonization. Taken together our results suggest that pyruvate-induced biofilm formation might act as a key process driving C. difficile persistence in the gut.

PURPOSE: DYRK1A syndrome is among the most frequent monogenic forms of intellectual disability (ID). We refined the molecular and clinical description of this disorder and developed tools to improve interpretation of missense variants, which remains a major challenge in human genetics. METHODS: We reported clinical and molecular data for fifty individuals with ID harboring DYRK1A variants and developed i) a specific DYRK1A clinical score, ii) amino acid conservation data generated from one hundred of DYRK1A sequences across different taxa, iii) in vitro overexpression assays to study level, cellular localization, and kinase activity of DYRK1A mutant proteins, and iv) a specific blood DNA methylation signature. RESULTS: This integrative approach was successful to reclassify several variants as pathogenic. However, we questioned the involvement of some others, such as p.Thr588Asn, still reported as likely pathogenic, and showed it does not cause an obvious phenotype in mice. CONCLUSION: Our study demonstrated the need for caution when interpreting variants in DYRK1A, even those occurring de novo. The tools developed will be useful to interpret accurately the variants identified in the future in this gene. ACCEPTED MANUSCRIPT -CLEAN COPYand analyzed as previously described 20 , a total of n=774,590 probes were analyzed for differential methylation. Standard quality control metrics showed good data quality for all samples except Ind#20. Briefly, limma regression with covariates age, sex, and five predicted blood cell types identified a DNAm signature with a Benjamini-Hochberg adjusted p-value<0.05 and 10% methylation difference. Next, we developed a support vector machine (SVM) model with linear kernel trained on including non-redundant CpG sites 20 using the methylation values for the discovery cases vs. controls. The model generated scores ranging between 0 and 1 for tested samples, classifying samples as "positive" (score>0.5) or "negative" (score<0.5). Additional neurotypical controls (n=94) and DYRK1A LoF validation samples (n=6) were scored to test model specificity and sensitivity respectively, and samples with pathogenic KMT2A (n=8) and ARID1B (n=4) variants and DYRK1A missense and distal frameshift (n=11) variants were tested. RESULTS Identification of genetic variants in DYRK1A in individuals with IDWe collected molecular and clinical information from 50 individuals with ID (44 never reported and six previously reported 6,7 ) carrying a variant in DYRK1A identified in clinical and diagnostic laboratories: structural variants deleting or interrupting DYRK1A and recurrent or novel nonsense, frameshift, splice and missense variants (Table 1, Figure S1). When blood or fibroblast samples were available, we characterized the consequences of these variants on DYRK1A mRNA by RNA-sequencing and RT-qPCR (Figure S2, Supplementary Text). For one variant, c.1978del, occurring in the last exon of the gene (Ind #18), the mutant transcripts escape to nonsense mRNA mediated decay (NMD) and result in a truncated protein p.Se...

Clostridioides difficile infections are associated with gut microbiome dysbiosis and are the leading cause of hospital acquired diarrhoea. The infectious process is strongly influenced by the microbiota and successful infection relies on the absence of specific microbiota-produced metabolites. Deoxycholic acid (DOC) and short chain fatty acids are microbiota-produced metabolites that limit the growth of C. difficile and protect the host against this infection. In a previous study, we showed that DOC causes C. difficile to form strongly adherent biofilms after 48 h. Here, our objectives were to identify and characterize key molecules and events required for biofilm formation in the presence of DOC. We applied time-course transcriptomics and genetics to identify sigma factors, metabolic processes and type IV pili that drive biofilm formation. These analyses revealed that extracellular pyruvate induces biofilm formation in the presence of DOC. In the absence of DOC, pyruvate supplementation was sufficient to induce biofilm formation in a process that was dependent on pyruvate uptake by the membrane protein CstA. In the context of the human gut, microbiota-generated pyruvate is a metabolite that limits pathogen colonization. Taken together our results suggest that pyruvate-induced biofilm formation might act as a key process driving C. difficile persistence in the gut.

BackgroundHigh-impact pathogenic variants in more than a thousand genes are involved in Mendelian forms of neurodevelopmental disorders (NDD).MethodsThis study describes the molecular and clinical characterisation of 28 probands with NDD harbouring heterozygous AGO1 coding variants, occurring de novo for all those whose transmission could have been verified (26/28).ResultsA total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Recurrently identified variants were present in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linker domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behaviour and additional behavioural manifestations.ConclusionOur study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to the recently reported AGO2-related NDD.

ABBSTRACTDYRK1A-related intellectual disability (ID) is among the most frequent monogenic form of ID. We refined the description of this disorder by reporting clinical and molecular data of forty individuals with ID harboring DYRK1A variants. We developed a combination of tools to interpret missense variants, which remains a major challenge in human genetics: i) a specific DYRK1A clinical score, ii) amino acid conservation data generated from one hundred of DYRK1A sequences across different taxa, iii) in vitro overexpression assays to study level, cellular localization, and kinase activity of DYRK1A mutant proteins, and iv) a specific blood DNA methylation signature. This integrative approach was successful to reclassify several variants as pathogenic. However, we questioned the involvement of some others, such as p.Thr588Asn, yet reported as pathogenic, and showed it does not cause obvious phenotype in mice, emphasizing the need to take care when interpreting variants, even those occurring de novo.

Chronic wounds, defined by their resistance to care after four weeks, are a major concern, affecting millions of patients every year. They can be divided into three types of lesions: diabetic foot ulcers (DFU), pressure ulcers (PU), and venous/arterial ulcers. Once established, the classical treatment for chronic wounds includes tissue debridement at regular intervals to decrease biofilm mass constituted by microorganisms physiologically colonizing the wound. This particular niche hosts a dynamic bacterial population constituting the bed of interaction between the various microorganisms. The temporal reshuffle of biofilm relies on an organized architecture. Microbial community turnover is mainly associated with debridement (allowing transitioning from one major representant to another), but also with microbial competition and/or collaboration within wounds. This complex network of species and interactions has the potential, through diversity in antagonist and/or synergistic crosstalk, to accelerate, delay, or worsen wound healing. Understanding these interactions between microorganisms encountered in this clinical situation is essential to improve the management of chronic wounds.

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