Génétique Moléculaire Génomique Microbiologie

Metformin, a widely prescribed antidiabetic drug, is frequently detected in aquatic environments due to its limited removal during wastewater treatment. Yet its environmental behaviour and ecological effect on microbial communities at the biogeochemically active sediment-water interface remains poorly understood. We examined metformin degradation and its effect on prokaryotic communities under diverse controlled oxygenation conditions in laboratory microcosms mimicking the sediment-water interface. In autoclaved microcosms, metformin showed slow dissipation without significant formation of transformation products, irrespective of oxygenation conditions. In contrast, biotic microcosms showed metformin biodegradation within 13 days following a lag phase of up to 28 days. Degradation was faster under anoxic conditions. Guanylurea was the sole transformation product detected, suggesting enzymatic hydrolysis and potential use of dimethylamine and guanylurea as carbon and nitrogen sources for microbial growth. Prokaryotic community composition was significantly affected by oxygenation conditions and repeated metformin contamination. Metformin exposure and changes in oxygenation regime had predominantly additive effects. Nevertheless, non-additive effects on procaryotic community composition emerged over time, particularly after repeated metformin exposure. Twenty-one bacterial taxonomic biomarkers of metformin exposure were tentatively identified, including methylotrophic taxa potentially associated with the utilisation of metformin and its metabolites. Microbial activity was essential for metformin dissipation at the sediment-water interface, and oxygenation regime modulated the effect of metformin and its transformation on prokaryotic communities. Our study shows the importance of oxygenation conditions and microbial community responses in assessing the behaviour and associated risks of pharmaceuticals in dynamic aquatic environments such as the sediment-water interface.

Huntington’s disease (HD) is a rare genetic neurodegenerative disorder which generally manifests in mid-adulthood and primary affects the striatum, leading to the development of cognitive, motor and neuropsychiatric symptoms. It is due to a CAG expansion in exon 1 of the HTT gene, and leads to various dysfunctions. We recently showed that HD striatal neurons undergo accelerated epigenetic aging characterized by a loss of epigenetic information, i.e. a decrease in the expression of striatal identity genes (e.g. Pde10a) and a re-expression of developmental genes (e.g. Onecut1). Although there is currently no cure available, various studies have demonstrated the beneficial effect of environmental enrichment (EE) in patients and animal models of HD. Using enrichment cages, we demonstrate a beneficial effect on cognition and other behavioral components, such as anxiety or apathy-like behaviour, in HD R6/1 transgenic mice. In order to characterize the underlying molecular mechanism, using OMICS techniques, we focus on the impact of EE housing on epigenetic and transcriptional regulations. Interestingly, our striatal RNAseq data generated from R6/1 mice show that EE housing induces an upregulation of genes linked to the adiponectin pathway (e.g. AdipoR2, adiponectin receptor 2), and partially normalizes the expression of developmental genes. This analysis is paralleled by CUT&Tag data, where we focus on neuronal H3K9 and H3K27 acetylation levels known to be deregulated in the disease, in order to specify the role of epigenetic regulations in this effect. Further analysis will enable us to identify which pathways respond positively to EE housing, potentially uncovering new therapeutic targets.

The photocatalyzed dearomative reaction between various electron-deficient aromatic compounds and a non-stabilized azomethine ylide is successfully performed in a flow system. Whereas the use of supported eosin as organic photocatalyst exhibits limited efficiency, turning to the soluble Rose Bengal allows to transform a broad range of substrates from hetarenes (indole, benzofuran, quinoline, pyridine) to naphthalenes and benzenes. This photocatalyzed (3+2) dearomative cycloaddition under green light irradiation provides a simple and efficient access to tridimensional pyrrolidino scaffolds with a tetrasubstituted carbon center at ring junction and can be performed in the friendly ethyl acetate. Computational studies support the mechanism involving azomethine ylide as reactive species toward the electron-poor arene.

BackgroundPrimary Sjögren’s syndrome (pSS) is a B-cell driven systemic auto-immune disease associated with the highest risk of lymphoma among systemic auto-immune diseases. In the prospective ASSESS cohort (395 patients enrolled, with a 10-year follow-up), transcriptomic analysis recently showed the association between baseline peripheral blood overexpression of Bruton’s tyrosine kinase (BTK) and lymphoma.ObjectivesThis study aimed to confirm the transcriptomic findings at the mRNA and protein level and to evaluate the relevance of BTK as a lymphoma predictor with regards to other predictors.MethodsBTK mRNA expression was assessed using qRTPCR in whole blood samples from 63 patients (12 patients with a history of lymphoma, 9 patients with incident lymphoma occurring during the 10-year follow-up, 21 patients without lymphoma and no risk factors for lymphoma and 21 patients without lymphoma and ESSDAI ≥ 5). BTK protein expression in peripheral blood was analyzed by flow cytometry in 7 healthy controls, 5 pSS patients without risk factors for lymphoma, 8 pSS patients with ESSDAI ≥5, and 6 pSS patients with lymphoma. In 346 patients of the ASSESS cohort for whom all data were available, the association between pSS-related lymphoma and BTK expression at enrolment, with 9 validated lymphoma predictors (parotid enlargement, purpura, ESSDAI ≥ 5, lymphocytopenia, CD4/CD8 ratio ≤ 0.8, RF positivity, cryoglobulinemia, monoclonal component, and low C4) and other potential biomarkers (serum FLT3L, BAFF, digital interferon alpha, gammaglobulins, IgG, beta2microglobulin, kappa and lambda free light chains, quantitative levels of anti-SSA/SSB) was analyzed. Multivariate analyses took into account in a first model, BTK and the 9 validated predictors of pSS-related lymphoma, and in a second model variables associated with lymphoma in univariate analysis selected by a stepwise procedure.ResultsIn qRTPCR, BTK was significantly upregulated between pSS patients with and without lymphoma. In flow cytometry, BTK protein expression in CD19+ B cells (p=0.03), and in most B-cell subpopulations, was significantly increased in patients with lymphoma compared to healthy controls. A gradual increase was observed in BTK expression of CD19+ B cells, between patients without risk factors for lymphoma, those with ESSDAI ≥5, and those with lymphoma. In multivariate analysis, taking into account 9 validated predictors of lymphoma in pSS, BTK expression at enrolment was significantly associated with lymphoma (adjusted OR [aOR] 1.45; 95% CI [1.19-1.82]; p<0.001), along with CD4/CD8 ratio ≤ 0.8 (aOR 6.03; 95% CI [1.23-26.13]; p=0.02). In a second model including 4 biomarkers selected by a stepwise procedure, BTK expression remained significantly associated with lymphoma (aOR 1.36; 95% CI [1.14-1.64]; p<0. 001), as well as serum BAFF levels (mean [SD]:1028.5 pg/ml [710.3]; per 1000 BAFF units, aOR 2.04; 95% CI [1.3-3.19]; p=0.001), low C4 (aOR 4.16; 95% CI [1.43-13.88]; p=0.01) and CD4/CD8 ratio ≤0.8 (aOR 10.62; 95% CI [2.49-44.15]; p=0.001).ConclusionThese results confirm that increased expression of BTK in peripheral blood is associated with pSS-related lymphoma. These results also show the potential interest of combining B-cell (BTK and BAFF) and T-cell (CD4/CD8 ratio)-related biomarkers to evaluate the risk of lymphoma in pSS.AcknowledgementsTo the patients for their participation to the ASSESS cohort; To the investigators of the ASSESS cohort; plateforme de transcriptomique de l’Institut Cochin; centre de ressources biologiques de l’Hôpital Bichat; The French Society of Rheumatology.Disclosure of InterestsPierre-Marie DURET: None declared, Cédric Schleiss: None declared, Nathanaël Sedmak: None declared, Nicolas Meyer: None declared, Tao Ye: None declared, Lou Kawka: None declared, Alain Saraux: None declared, Valerie Devauchelle-Pensec: None declared, Divi Cornec: None declared, Claire Larroche: None declared, Aleth Perdriger: None declared, Raphaèle Seror: None declared, Renaud FELTEN: None declared, Jean Sibilia: None declared, Gaetane Nocturne: None declared, Xavier Mariette Consultant of: Astra Zeneca, BMS, Galapagos, GSK, Novartis, Pfizer, Jacques-Eric Gottenberg Consultant of: Astra Zeneca, BMS, Galapagos, GSK, Novartis, Pfizer.

Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host-pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles. Key features • Tested on human mitochondria; potentially applicable to cell cultures, non-model organisms, extracellular vesicles, enveloped viruses, tissues; does not require genetic manipulations or highly pure organelles. • In the case of human cells, the required amount of starting material is ~2,500 cm2 of 80% confluent cells (or ~3 × 108 HEK293 cells). • CoLoC-seq implements a special RNA-seq strategy to selectively capture intact transcripts, which requires RNases generating 5'-hydroxyl and 2'/3'-phosphate termini (e.g., RNase A, RNase I). • Relies on nonlinear regression software with customisable exponential functions.

Metformin is one of the most prescribed antidiabetic agents worldwide and is also considered for other therapeutic applications including cancer and endocrine disorders. It is largely unmetabolized by human enzymes and its presence in the environment has raised concern, with reported toxic effects on aquatic life and potentially also on humans. We report on the isolation and characterisation of strain MD1, an aerobic methylotrophic bacterium growing with metformin as its sole carbon, nitrogen and energy source. Strain MD1 degrades metformin into dimethylamine used for growth, and guanylurea as a side-product. Sequence analysis of its fully assembled genome showed its affiliation to Aminobacter niigataensis. Differential proteomics and transcriptomics, as well as mini-transposon mutagenesis of the strain, point to genes and proteins essential for growth with metformin and potentially associated with hydrolytic C-N cleavage of metformin or with cellular transport of metformin and guanylurea. The obtained results suggest the recent evolution of the growth-supporting capacity of strain MD1 to degrade metformin. Our results identify candidate proteins of the enzymatic system for metformin transformation in strain MD1 and will inform future research on the fate of metformin and its degradation products in the environment and in humans.