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Dosage of short chain fatty acids (SCFAs) according to food diet showed that the content of acetate was high in newborn feaces. Infants receiving food supplements have a complex and diverse gut microbiota. Moreover, the results show that infants from poor districts have an abundant concentration of SCFAs in their faeces compared to those living in places with a relatively high standard of living. Among infants receiving milk, the highest proportion of SCFA is acetate in breastfed infants (BF) at a rate of 15.025 ± 2.23 μmol/g, followed by propionate in infants receiving mixed feeding (BF+FF), at a rate of 13.58 ± 1.03 μmol/g and butyrate in infants taking mixed feeding at a rate of 0.32 ± 0.72 μmol/g. However, among infants starting early diet diversification, acetate is higher in infants receiving milk formula and diet diversification (FF+FD) with a concentration of 25.4 ± 0 μmol/g, followed by propionate (2.36 ± 0 μmol/g) in infants receiving mixed feeding (BF+FF) and butyrate in those fed with (BF+FD). Partial breastfeeding is associated with a higher proportion of acetate, butyrate and propionate. The study of the correlation between the different SCFAs produced and the ASV (Variants of Microbial Amplicon Sequences) of the intestinal community of the child, shows that acetate is positively correlated with Bifidobacterium and negatively with Streptococcus and Escherichia-Shigella. Propionate is positively correlated with Bifido bacterium and negatively with Escherichia-Shigella. Similarly, butyrate is positively correlated with Bifidobacterium and negatively with Escherichia-Shigella.

Dissemination of antibiotic resistance, a current societal challenge, is often driven by horizontal gene transfer through bacterial conjugation. During conjugative plasmid transfer, single-stranded (ss) DNA is transferred from the donor to the recipient cell. Subsequently, a complete double-stranded (ds) plasmid molecule is generated and plasmid-encoded genes are expressed, allowing successful establishment of the transconjugant cell. Such dynamics of transmission can be modulated by host- or plasmid-encoded factors, either in the donor or in the recipient cell. We applied transposon insertion sequencing to identify host-encoded factors that affect conjugative transfer frequency in Escherichia coli. Disruption of the recipient uvrD gene decreased the acquisition frequency of conjugative plasmids belonging to different incompatibility groups. Results from various UvrD mutants suggested that dsDNA binding activity and interaction with RNA polymerase are dispensable, but ATPase activity is required for successful plasmid establishment of transconjugant cells. Live-cell microscopic imaging showed that the newly transferred ssDNA within a uvrD− recipient often failed to be converted to dsDNA. Our work suggested that in addition to its role in maintaining genome integrity, UvrD is also key for the establishment of horizontally acquired plasmid DNA that drives genome diversity and evolution.

International audience; In this study, we report on the fabrication of photo/thermo dual stimulus-responsive liquid marbles (LMs) that can be disrupted by light irradiation and/or heating. To stabilize the LMs, we synthesized micrometer-sized stearic acid (SA) particles coated with overlayers of polypyrrole (PPy) by aqueous chemical oxidative seeded dispersion polymerization. The SA/PPy core–shell particles could adsorb at the air–water interface to stabilize LMs by rolling water droplets on the particle powder bed. The presence of SA, known as a phase-change material, which undergoes a transition from solid to liquid by heating, and PPy, which can transduce light to heat, gives rise to the photo and thermo dual stimulus-responsive characters of the LMs. The disruption of the LMs could be induced in a cascade manner: light irradiation on the LM induced a temperature increase, followed by melting of the SA component on the LM surface, leading to its disruption and release of the inner water. The disruption time is linked to the PPy loading and light irradiation power, and it can be tuned from quasi-instantaneous to a few tens of seconds. The melting of SA due to a light-induced phase change from the solid to liquid state is a new mechanism to trigger the disruption of LMs. We finally demonstrated two applications of the LMs as a light-responsive microreactor and a sensor.

International audience; This oral presentation sums up different approaches explored during the past years to manage dairy fouling over stainless steel substrates.

Background Bacteria and their viruses, bacteriophages, are the most abundant entities of the gut microbiota, a complex community of microorganisms associated with human health and disease. In this ecosystem, the interactions between these two key components are still largely unknown. In particular, the impact of the gut environment on bacteria and their associated prophages is yet to be deciphered. Results To gain insight into the activity of lysogenic bacteriophages within the context of their host genomes, we performed proximity ligation-based sequencing (Hi-C) in both in vitro and in vivo conditions on the 12 bacterial strains of the OMM12 synthetic bacterial community stably associated within mice gut (gnotobiotic mouse line OMM12). High-resolution contact maps of the chromosome 3D organization of the bacterial genomes revealed a wide diversity of architectures, differences between environments, and an overall stability over time in the gut of mice. The DNA contacts pointed at 3D signatures of prophages leading to 16 of them being predicted as functional. We also identified circularization signals and observed different 3D patterns between in vitro and in vivo conditions. Concurrent virome analysis showed that 11 of these prophages produced viral particles and that OMM12 mice do not carry other intestinal viruses. Conclusions The precise identification by Hi-C of functional and active prophages within bacterial communities will unlock the study of interactions between bacteriophages and bacteria across conditions (healthy vs disease).

Integrative and conjugative elements (ICEs) are important drivers of horizontal gene transfer in prokaryotes. They are responsible for antimicrobial resistance spread, a major current health concern. ICEs are initially processed by relaxases that recognize the binding site of oriT sequence and nick at a conserved nic site. The ICESt3/Tn916/ICEBs1 superfamily, which is widespread among Firmicutes, encodes uncanonical relaxases belonging to a recently identified family called MOBT. This family is related to the rolling circle replication initiators of the Rep_trans family. The nic site of these MOBT relaxases is conserved but their DNA binding site is still unknown. Here, we identified the bind site of RelSt3, the MOBT relaxase from ICESt3. Unexpectedly, we found this bind site distantly located from the nic site. We revealed that the binding of the RelSt3 N-terminal HTH domain is required for efficient nicking activity. We also deciphered the role of RelSt3 in the initial and final stages of DNA processing during conjugation. Especially, we demonstrated a strand transfer activity, and the formation of covalent DNA-relaxase intermediate for a MOBT relaxase.

Posté le 17 septembre 2021.; International audience; ObjectivesA nationwide cross-sectional epidemiological survey was conducted to capture the true extent of coronavirus disease 2019 (COVID-19) exposure in Senegal.MethodsMulti-stage random cluster sampling of households was performed between October and November 2020, at the end of the first wave of COVID-19 transmission. Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies were screened using three distinct ELISA assays. Adjusted prevalence rates for the survey design were calculated for each test separately, and thereafter combined. Crude and adjusted prevalence rates based on test performance were estimated to assess the seroprevalence. As some samples were collected in high malaria endemic areas, the relationship between SARS-CoV-2 seroreactivity and antimalarial humoral immunity was also investigated.ResultsOf the 1463 participants included in this study, 58.8% were female and 41.2% were male; their mean age was 29.2 years (range 0.20–84.8.0 years). The national seroprevalence was estimated at 28.4% (95% confidence interval 26.1–30.8%). There was substantial regional variability. All age groups were impacted, and the prevalence of SARS-CoV-2 was comparable in the symptomatic and asymptomatic groups. An estimated 4 744 392 (95% confidence interval 4 360 164–5 145 327) were potentially infected with SARS-CoV-2 in Senegal, while 16 089 COVID-19 RT-PCR laboratory-confirmed cases were reported by the national surveillance. No correlation was found between SARS-CoV-2 and Plasmodium seroreactivity.ConclusionsThese results provide a better estimate of SARS-CoV-2 dissemination in the Senegalese population. Preventive and control measures need to be reinforced in the country and especially in the south border regions.

International audience; Clustered organization of biosynthetic non-homologous genes is emerging as a characteristic feature of plant genomes. The co-regulation of clustered genes seems to largely depend on epigenetic reprogramming and three-dimensional chromatin conformation. In this study, we identified the long non-coding RNA (lncRNA) MARneral Silencing (MARS), localized inside the Arabidopsis marneral cluster, which controls the local epigenetic activation of its surrounding region in response to abscisic acid (ABA). MARS modulates the POLYCOMB REPRESSIVE COMPLEX 1 (PRC1) component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) binding throughout the cluster in a dose-dependent manner, determining H3K27me3 deposition and chromatin condensation. In response to ABA, MARS decoys LHP1 away from the cluster and promotes the formation of a chromatin loop bringing together the MARNERAL SYNTHASE 1 (MRN1) locus and a distal ABA-responsive enhancer. The enrichment of co-regulated lncRNAs in clustered metabolic genes in Arabidopsis suggests that the acquisition of novel non-coding transcriptional units may constitute an additional regulatory layer driving the evolution of biosynthetic pathways

Chromosome condensation and segregation are two essential processes for the transmission of genetic material in all living organisms. Indeed, chromosomes are composed of a DNA molecule and several families of proteins that perform various functions related to DNA. Ubiquitous SMC complexes in the living world are among these proteins that constitute the chromosome, and their study is of great importance in understanding the mechanisms of condensation, segregation, and their possible links.The MukBEF SMC complex is the condensin of the bacterium Escherichia coli. Although MukBEF is essential under fast growth conditions, its deletion is tolerated under slow growth, and is characterized by the production of anucleate cells reflecting a defect in chromosome segregation. The chromosome conformation capture technique (3C-seq/HiC) has revealed that MukBEF promotes long-distance contacts throughout the chromosome, except in the Ter domain where it is inhibited by the Matp/matS system.During my thesis, I focused on characterizing the activity of MukBEF on the chromosome. I was able to demonstrate that the loading of MukBEF, unlike other bacterial condensins that have been studied, occurs without site specificity.Additionally, I identified that this loading is more efficient on newly replicated DNA. I also investigated the characterization of the inhibition mechanism of MukBEF by the MatP/matS system in the Ter domain. MatP is a protein that specifically binds to 28 matS sites distributed in the Ter domain. My results suggest a mechanism of inhibition that involves unloading MukBEF from the chromosome near the borders of the Ter domain. Furthermore, I demonstrated that 3 matS sites are sufficient to inhibit MukBEF, but the addition of additional sites strengthens the effectiveness of this inhibition. Moreover, the analysis of the distribution of matS sites in several bacteria suggests that increasing the number of matS sites and their density near the dif site aims to prevent MukBEF loading near the dif site.; La condensation des chromosomes et leur ségrégation sont deux processus essentiels à la transmission du matériel génétique chez tous les êtres vivants. En effet, les chromosomes sont constitués d'une molécule d'ADN et de plusieurs familles de protéines qui assurent plusieurs fonctions liées à l'ADN. Les complexes SMC ubiquitaires dans le monde vivant font partie de ces protéines qui constituent le chromosome, et leur étude est d'une grande importance dans la compréhension des mécanismes de condensation, de ségrégation et des liens possibles entre eux.Le complexe SMC MukBEF est la condensine de la bactérie Escherichia coli. Bien que MukBEF soit essentiel dans des conditions de croissance rapide, sa délétion est tolérée en croissance lente, et se caractérise par la production de cellules anucléées reflétant un défaut de ségrégation des chromosomes. La technique de capture de conformation du chromosome (3C-seq/HiC) a révélé que MukBEF favorise des contacts de longue distance sur tout le chromosome, sauf dans le domaine Ter où elle est inhibée par le système Matp/matS.Durant ma thèse, je me suis intéressé à la caractérisation de l'activité de MukBEF sur le chromosome. J'ai pu mettre en évidence que le chargement de MukBEF, contrairement aux autres condensines bactériennes étudiées, se fait de manière non spécifique de site. De plus, j'ai identifié que ce chargement est plus efficace sur de l'ADN néo-répliqué. Je me suis également intéressé à la caractérisation du mécanisme d'inhibition de MukBEF par le système MatP/matS dans le domaine Ter. MatP est une protéine qui se fixe spécifiquement à 28 sites matS répartis dans le domaine Ter. Mes résultats permettent de proposer un mécanisme d'inhibition qui consiste en un déchargement de MukBEF du chromosome à proximité des bordures du Ter. J'ai également pu montrer que 3 sites matS suffisent pour inhiber MukBEF, mais que l'ajout de sites supplémentaires renforce l'efficacité de cette inhibition. De plus,l'analyse de la répartition des sites matS dans plusieurs bactéries suggère que l'augmentation du nombre de sites matS et de leur densité à proximité du site dif vise à empêcher le chargement de MukBEF à proximité du site dif.

As part of the fight against the Covid-19 pandemic, messenger RNA vaccines (mRNA vaccines) have been widely distributed among the world's population – at least the Western population – and it is clear that they have saved many lives. The official discourse of scientists and public health officials ensures that they are generally without harmful consequences for human beings and that, despite certain incidents and some contraindications, they can be used without danger. In particular, it seems excluded that they can pass into the genome (or all of the genetic information of an organism, genes and chromosomes included) and modify the host DNA. Some biologists however point out that, by reverse transcriptase, and under certain very specific conditions, an mRNA can trigger the synthesis of a DNA, a phenomenon observed on several occasions, in particular in vitro, which could then authorize, contrary to what has always been claimed, its entry into cell nuclei. What to think of this controversy which opposes on the one hand, an overwhelming majority of "mainstream" biologists and, on the other, a few mavericks who, for the moment, have not convinced the scientific community? Should we be wary of this kind of vaccines and avoid, if possible, their use? We will attempt to inform the incriminating and exculpatory debate, within the limits, of course, of the current state of knowledge.; Dans le cadre de la lutte contre la pandémie Covid-19, les vaccins à ARN messager (mRNA vaccines) ont été largement diffusés dans la population mondiale – au moins la population occidentale – et il est clair qu'ils ont sauvé de nombreuses vies. Le discours officiel des scientifiques et des responsables de la santé publique assure qu'ils sont généralement sans conséquences néfastes pour l'être humain et que, malgré certains incidents et quelques contre-indications, on peut les utiliser sans danger. En particulier, il semble exclu qu'ils puissent passer dans le génome (ou ensemble de l'information génétique d'un organisme, gènes et chromosomes compris) et modifier l'ADN-hôte. Certains biologistes font cependant remarquer que, par transcriptase inverse, et sous certaines conditions bien particulières, un ARNm peut déclencher la synthèse d'un ADN, phénomène observé à plusieurs reprises, en particulier in vitro, ce qui pourrait ensuite autoriser, contrairement à ce qu'on a toujours prétendu, son entrée dans des noyaux cellulaires. Que penser de cette controverse qui oppose d'un côté, une écrasante majorité de biologistes "mainstream" et, de l'autre, quelques francs-tireurs qui, pour l'instant, n'ont pas convaincu la communauté scientifique? Faut-il se méfier de ce genre de vaccins et éviter, si possible, leur utilisation? Nous tenterons d'instruire le débat à charge et à décharge, dans la limite, bien sûr, de l'état actuel des connaissances.

Extensive intestinal resection leads to Short Bowel Syndrome (SBS), the main cause of chronic intestinal failure. Colon preservation is crucial for spontaneous adaptation, to improve absorption and reduce parenteral nutrition dependence. Fecal microbiota transplantation (FMT), a promising approach in pathologies with dysbiosis as the one observed in SBS patients, was assessed in SBS rats with jejuno‐colonic anastomosis. The evolution of weight and food intake, the lenght of intestinal villi and crypts and the composition of fecal microbiota of Sham and SBS rats, transplanted or not with high fat diet rat microbiota, were analyzed. All SBS rats lost weight, increased their food intake and exhibited jejunal and colonic hyperplasia. Microbiota composition of SBS rats, transplanted or not, was largely enriched with Lactobacillaceae, and α‐ and β‐diversity were significantly different from Sham. The FMT altered microbiota composition and α‐ and β‐diversity in Sham but not SBS rats. FMT from high fat diet rats was successfully engrafted in Sham, but failed to take hold in SBS rats, probably because of the specific luminal environment in colon of SBS subjects favoring aero‐tolerant over anaerobic bacteria. Finally, the level of food intake in SBS rats was positively correlated with their Lactobacillaceae abundance. Microbiota transfer must be optimized and adapted to this specific SBS environment.

Contrary to the vast majority of bacteria that possesses a unique chromosome, Vibrio cholerae, the pathogenic agent responsible for cholera disease, contains a genome divided into two replicons: a main chromosome (Chr1) and a secondary chromosome (Chr2). Secondary chromosomes appeared independently in many bacterial phylum and are often associated with pathogenic or symbiotic traits (i.e. Leptospira, Agrobacterium, Burkholderia). Secondary chromosomes result from the acquisition and domestication of large plasmids1. Indeed, some “megaplasmids” have cohabited with their host over a long evolutionary period and became an obligatory component of their host’s genome, essential for their normal metabolism. During their domestication, secondary chromosomes acquired different features allowing their maintenance through cell divisions ensuring their vertical transfer to subsequent generations. The study of multipartite genome bacteria allows questioning the selective benefit of an organism to possess several chromosomes. In addition, how can a horizontally acquired genetic element connect to a pre-established genetic network? One of the major steps in megaplasmid domestication is their integration into the host cell cycle. In V.cholerae, Chr2 replication is coordinated with that of Chr1. Chr1, which is 3 times larger than Chr2, is initiated first and when 2/3 has replicated, an initiation signal triggers the replication of Chr2 so that both chromosomes terminate replicating concomitantly. This initiation signal is emitted when the replication fork passes through a non-coding Chr1 locus named crtS (Chr2 Replication Triggering Site)2,3. However, the molecular mechanism behind this initiation signal is still very elusive. Our work aims to elucidate the mechanism of this novel bacterial replicative checkpoint. In order to tackle this problem we developed several cutting-edge approaches to study the mechanism at different levels: -Genomic scale: high resolution ChIPseq -Single cell: fluorescent microscopy -Molecular: CryoEM.; Contrairement à la grande majorité des bactéries qui possèdent un chromosome unique, Vibrio cholerae, l'agent pathogène responsable de la maladie du choléra, contient un génome divisé en deux réplicons : un chromosome principal (Chr1) et un chromosome secondaire (Chr2). Les chromosomes secondaires sont apparus indépendamment dans de nombreux phylum bactériens et sont souvent associés à des caractères pathogènes ou symbiotiques (par exemple Leptospira, Agrobacterium, Burkholderia). Les chromosomes secondaires résultent de l'acquisition et de la domestication de grands plasmides. En effet, certains "mégaplasmides" ont cohabité avec leur hôte au cours d'une longue période évolutive et sont devenus un composant essentiel du génome de leur hôte. Au cours de leur domestication, les chromosomes secondaires ont acquis différentes caractéristiques permettant leur maintien à travers les divisions cellulaires assurant leur transfert vertical aux générations suivantes. L'étude des bactéries à génome multipartite permet de s'interroger sur le bénéfice sélectif d'un organisme à posséder plusieurs chromosomes. Ou encore, comment un élément génétique acquis horizontalement peut-il se connecter à un réseau génétique préétabli ? L'une des principales étapes de la domestication des mégaplasmides est leur intégration dans le cycle cellulaire de l'hôte. Chez V.cholerae, la réplication de Chr2 est coordonnée avec celle de Chr1. Chr1, qui est 3 fois plus grand que Chr2, est initié en premier et lorsque les 2/3 se sont répliqués, un signal d'initiation déclenche la réplication de Chr2 de sorte que les deux chromosomes terminent leur réplication de façon concomitante. Ce signal d'initiation est émis lorsque la fourche de réplication passe par un locus non codant de Chr1 nommé crtS (Chr2 Replication Triggering Site)2,3. Cependant, le mécanisme moléculaire à l'origine de ce signal d'initiation est encore très difficile à élucider. Notre travail vise à élucider le mécanisme de ce nouveau point de contrôle bactérien. Afin de s'attaquer à ce problème, nous avons développé plusieurs approches de pointe pour étudier le mécanisme à différents niveaux : -Echelle génomique : ChIPseq à haute résolution -Single-cell : microscopie à fluorescence -Moléculaire : CryoEM.

FUS is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, translation, miRNA processing, and replication-dependent histone gene expression. In this work, we show that FUS depletion results in the differential expression of numerous small nucleolar RNAs (snoRNAs) that guide 2’-O methylation (2’-O-Me) and pseudouridylation of specific positions in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Using RiboMeth-seq and HydraPsiSeq for the profiling of 2’-O-Me and pseudouridylation status of rRNA species, we demonstrated considerable hypermodification at several sites in HEK293T and SH-SY5Y cells with FUS knockout (FUS KO) compared to wild-type cells. We observed a similar direction of changes in rRNA modification in differentiated SH-SY5Y cells with the FUS mutation (R495X) related to the severe disease phenotype of amyotrophic lateral sclerosis (ALS). Furthermore, the pattern of modification of some rRNA positions was correlated with the abundance of corresponding guide snoRNAs in FUS KO and FUS R495X cells. Our findings reveal a new role for FUS in modulating the modification pattern of rRNA molecules, that in turn might generate ribosome heterogeneity and constitute a fine-tuning mechanism for translation efficiency/fidelity. Therefore, we suggest that increased levels of 2’-O-Me and pseudouridylation at particular positions in rRNAs from cells with the ALS-linked FUS mutation may represent a possible new translation-related mechanism that underlies disease development and progression.

Monogenic disorder are inherited mutation(s) in a single gene that result in abnormal protein synthesis impairing normal cellular homeostasis and organ function that reduces patient life-pan. Recently autologous transplantation of genetically corrected patient's hematopoietic stem cells (HSCs) defined as ex-vivo gene therapy (GT) has emerged as a promising curative approach for such genetic diseases. In ex-vivo GT, targeted integration of a therapeutic transgene into a tolerant and safe locus appears to be a powerful approach to produce the therapeutic protein above physiological level in a broad type of hematopoietic cells using endogenous promoter. Such genome editing strategy relies on DNA double strand break and delivery of a functional copy of the mutated gene in the locus of interest. Here, we exploited the α-globin locus (HBA) as a safe harbor locus for transgene integration to develop a universal platform for expression and secretion of therapeutic proteins into erythroid cells. In HSCs, we induced double strand breaks (DSBs) in HBA locus with CRISPR-Cas9 and delivered a therapeutic transgene using AAV-6 to target integration by homology directed repair. Due to the abundance of erythroid cells in human (5X10^6 RBCs/microliter of blood) and the high HBA expression in erythroid population (around 1,5g/day), the HBA platform would require minimal transgene integration events in HBA locus to provide sufficient amount of therapeutic proteins reducing as consequence the potential off-target and toxicity due to editing and DNA donor delivery. First, we applied the HBA platform for β-thalassemia : a genetic condition impairing the hemoglobin formation and triggering toxic α-globin chain precipitation. The genetic correction of β-thalassemia using our HBA platform results in 1 copy of A-globin gene deletion decreasing the α-globin chain precipitation while α-globin gene integration into HBA locus restore the normal hemoglobin formation. Then, we evaluated the HBA platform for erythroid-specific secretion of therapeutic proteins into bloodstream and, in particular,we focused on a genetic metabolic disorder: lysosomal acid lipase (LAL) deficiency caused by LIPA gene mutation(s) that lead to toxic lipid accumulation especially in liver. We achieved efficient expression and secretion of LAL enzyme that retained enzymatic activity and cross-corrected the lipid accumulation in patients' fibroblasts. We then focused on the optimization of LIPA cDNA transgene using lentiviral vectors (LVVs) to maximize the LAL expression and secretion. The optimized LIPA transgene will serve for LVV-based ex-vivo GT and for our HBA targeted platform in the purpose of enhancing the LAL expression while minimizing the targeting integration events required to fully correct the disorder. Finally, to better characterize LAL-D phenotype and to evaluate the therapeutic potency of our GT strategy, we set-up a multimodal cytometry approach using flow cytometry and image flow cytometry. We evaluated this pipeline on mainly patient's fibroblasts and on peripheral mononuclear blood mononuclear cells (PBMCs) for LAL-D mouse model.; Les maladies monogéniques sont des mutations héréditaires d'un seul gène qui entraînent une synthèse anormale de protéines, altérant l'homéostasie cellulaire normale et la fonction des organes et réduisant la durée de vie des patients. Récemment, la greffe autologue de cellules souches hématopoïétiques (HSCs) de patients génétiquement corrigés appelée thérapie génique (TG) ex-vivo, est apparue comme une approche curative prometteuse pour ces maladies. Dans la TG ex-vivo, l'intégration ciblée d'un transgène thérapeutique dans un locus tolérant apparaît comme une approche puissante pour produire les protéines thérapeutiques à un niveau supérieur au niveau physiologique dans un large type de cellules hématopoïétiques en utilisant un promoteur endogène. Une telle stratégie d'édition du génome repose sur la cassure double brin d'ADN et la livraison d'une copie fonctionnelle du gène muté dans le locus d'intérêt. Ici, nous avons exploité le locus de l'α-globine (HBA) comme un locus tolérant pour l'intégration de transgènes afin de développer une plateforme universelle pour l'expression et la sécrétion de protéines thérapeutiques par les globules rouges (GR). Dans les HSCs, nous avons induit des cassures double brin dans le locus HBA avec CRISPR-Cas9 et délivré un transgène thérapeutique en utilisant un AAV-6 pour cibler l'intégration par réparation dirigée par l'homologie. En raison de l'abondance des globules rouges chez l'homme (5X10^6 globules rouges/microlitre de sang) et de la forte expression de HBA dans cette population (environ 1,5g/jour), la plateforme HBA nécessiterait un minimum d'événement d'intégration du transgène pour fournir une quantité suffisante de protéines thérapeutiques, réduisant ainsi les risques de toxicité et d'édition non spécifique induit par CRISPR-Cas9 ou l'AAV6. Dans un premier temps, nous avons appliqué la plateforme HBA à la β-thalassémie : une condition génétique altérant la formation de l'hémoglobine (composé de 2 α-globine et 2 β-globine) et déclenchant la précipitation toxique de la chaîne α-globine. La correction génétique de la β-thalassémie à l'aide de notre plateforme HBA entraîne la délétion d'une copie du gène de l'α-globine, ce qui diminue la précipitation de cette chaîne, tandis que l'intégration du gène de la β-globine dans le locus HBA rétablit la formation normale d'hémoglobine. Ensuite, nous avons évalué la plateforme HBA pour la sécrétion spécifique de protéines thérapeutiques par les GRs dans la circulation sanguine et, en particulier, nous nous sommes concentrés sur un trouble métabolique génétique : la déficience en lipase acide lysosomale (LAL) causée par une ou plusieurs mutations du gène LIPA qui entraînent une accumulation toxique de lipides, notamment dans le foie. Nous avons obtenu une expression et une sécrétion efficaces de l'enzyme LAL qui a conservé son activité enzymatique et corrigé l'accumulation de lipides dans les fibroblastes des patients. Nous nous sommes ensuite concentrés sur l'optimisation du transgène LIPA en utilisant des vecteurs lentiviraux (LVVs) pour maximiser l'expression et la sécrétion de la LAL. Le transgène LIPA optimisé servira à développer une TG ex-vivo basée sur les LVVs et à notre plateforme ciblée HBA dans le but d'améliorer l'expression de LAL tout en minimisant les événements d'intégration nécessaires pour corriger complètement la maladie. Enfin, pour mieux caractériser le phénotype la déficience en LAL et évaluer le pouvoir thérapeutique de notre stratégie de GT, nous avons mis en place une approche de cytométrie multimodale utilisant la cytométrie en flux et la cytométrie en flux d'images. Nous avons évalué ces techniques principalement sur les fibroblastes du patient et sur les cellules mononucléaires du sang périphérique (PBMCs) provenant de souris déficiente en LAL.

Monogenic disorder are inherited mutation(s) in a single gene that result in abnormal protein synthesis impairing normal cellular homeostasis and organ function that reduces patient life-pan. Recently autologous transplantation of genetically corrected patient's hematopoietic stem cells (HSCs) defined as ex-vivo gene therapy (GT) has emerged as a promising curative approach for such genetic diseases. In ex-vivo GT, targeted integration of a therapeutic transgene into a tolerant and safe locus appears to be a powerful approach to produce the therapeutic protein above physiological level in a broad type of hematopoietic cells using endogenous promoter. Such genome editing strategy relies on DNA double strand break and delivery of a functional copy of the mutated gene in the locus of interest. Here, we exploited the α-globin locus (HBA) as a safe harbor locus for transgene integration to develop a universal platform for expression and secretion of therapeutic proteins into erythroid cells. In HSCs, we induced double strand breaks (DSBs) in HBA locus with CRISPR-Cas9 and delivered a therapeutic transgene using AAV-6 to target integration by homology directed repair. Due to the abundance of erythroid cells in human (5X10^6 RBCs/microliter of blood) and the high HBA expression in erythroid population (around 1,5g/day), the HBA platform would require minimal transgene integration events in HBA locus to provide sufficient amount of therapeutic proteins reducing as consequence the potential off-target and toxicity due to editing and DNA donor delivery. First, we applied the HBA platform for β-thalassemia : a genetic condition impairing the hemoglobin formation and triggering toxic α-globin chain precipitation. The genetic correction of β-thalassemia using our HBA platform results in 1 copy of A-globin gene deletion decreasing the α-globin chain precipitation while α-globin gene integration into HBA locus restore the normal hemoglobin formation. Then, we evaluated the HBA platform for erythroid-specific secretion of therapeutic proteins into bloodstream and, in particular,we focused on a genetic metabolic disorder: lysosomal acid lipase (LAL) deficiency caused by LIPA gene mutation(s) that lead to toxic lipid accumulation especially in liver. We achieved efficient expression and secretion of LAL enzyme that retained enzymatic activity and cross-corrected the lipid accumulation in patients' fibroblasts. We then focused on the optimization of LIPA cDNA transgene using lentiviral vectors (LVVs) to maximize the LAL expression and secretion. The optimized LIPA transgene will serve for LVV-based ex-vivo GT and for our HBA targeted platform in the purpose of enhancing the LAL expression while minimizing the targeting integration events required to fully correct the disorder. Finally, to better characterize LAL-D phenotype and to evaluate the therapeutic potency of our GT strategy, we set-up a multimodal cytometry approach using flow cytometry and image flow cytometry. We evaluated this pipeline on mainly patient's fibroblasts and on peripheral mononuclear blood mononuclear cells (PBMCs) for LAL-D mouse model., Les maladies monogéniques sont des mutations héréditaires d'un seul gène qui entraînent une synthèse anormale de protéines, altérant l'homéostasie cellulaire normale et la fonction des organes et réduisant la durée de vie des patients. Récemment, la greffe autologue de cellules souches hématopoïétiques (HSCs) de patients génétiquement corrigés appelée thérapie génique (TG) ex-vivo, est apparue comme une approche curative prometteuse pour ces maladies. Dans la TG ex-vivo, l'intégration ciblée d'un transgène thérapeutique dans un locus tolérant apparaît comme une approche puissante pour produire les protéines thérapeutiques à un niveau supérieur au niveau physiologique dans un large type de cellules hématopoïétiques en utilisant un promoteur endogène. Une telle stratégie d'édition du génome repose sur la cassure double brin d'ADN et la livraison d'une copie fonctionnelle du gène muté dans le locus d'intérêt. Ici, nous avons exploité le locus de l'α-globine (HBA) comme un locus tolérant pour l'intégration de transgènes afin de développer une plateforme universelle pour l'expression et la sécrétion de protéines thérapeutiques par les globules rouges (GR). Dans les HSCs, nous avons induit des cassures double brin dans le locus HBA avec CRISPR-Cas9 et délivré un transgène thérapeutique en utilisant un AAV-6 pour cibler l'intégration par réparation dirigée par l'homologie. En raison de l'abondance des globules rouges chez l'homme (5X10^6 globules rouges/microlitre de sang) et de la forte expression de HBA dans cette population (environ 1,5g/jour), la plateforme HBA nécessiterait un minimum d'événement d'intégration du transgène pour fournir une quantité suffisante de protéines thérapeutiques, réduisant ainsi les risques de toxicité et d'édition non spécifique induit par CRISPR-Cas9 ou l'AAV6. Dans un premier temps, nous avons appliqué la plateforme HBA à la β-thalassémie : une condition génétique altérant la formation de l'hémoglobine (composé de 2 α-globine et 2 β-globine) et déclenchant la précipitation toxique de la chaîne α-globine. La correction génétique de la β-thalassémie à l'aide de notre plateforme HBA entraîne la délétion d'une copie du gène de l'α-globine, ce qui diminue la précipitation de cette chaîne, tandis que l'intégration du gène de la β-globine dans le locus HBA rétablit la formation normale d'hémoglobine. Ensuite, nous avons évalué la plateforme HBA pour la sécrétion spécifique de protéines thérapeutiques par les GRs dans la circulation sanguine et, en particulier, nous nous sommes concentrés sur un trouble métabolique génétique : la déficience en lipase acide lysosomale (LAL) causée par une ou plusieurs mutations du gène LIPA qui entraînent une accumulation toxique de lipides, notamment dans le foie. Nous avons obtenu une expression et une sécrétion efficaces de l'enzyme LAL qui a conservé son activité enzymatique et corrigé l'accumulation de lipides dans les fibroblastes des patients. Nous nous sommes ensuite concentrés sur l'optimisation du transgène LIPA en utilisant des vecteurs lentiviraux (LVVs) pour maximiser l'expression et la sécrétion de la LAL. Le transgène LIPA optimisé servira à développer une TG ex-vivo basée sur les LVVs et à notre plateforme ciblée HBA dans le but d'améliorer l'expression de LAL tout en minimisant les événements d'intégration nécessaires pour corriger complètement la maladie. Enfin, pour mieux caractériser le phénotype la déficience en LAL et évaluer le pouvoir thérapeutique de notre stratégie de GT, nous avons mis en place une approche de cytométrie multimodale utilisant la cytométrie en flux et la cytométrie en flux d'images. Nous avons évalué ces techniques principalement sur les fibroblastes du patient et sur les cellules mononucléaires du sang périphérique (PBMCs) provenant de souris déficiente en LAL.

International audience; Purpose Protein synthesis and proteolysis are known to be controlled through mammalian target of rapamycin, AMP-activated kinase (AMPK) and general control non-derepressible 2 (GCN2) pathways, depending on the nutritional condition. This study aimed at investigating the contribution of liver AMPK and GCN2 on the adaptation to high variations in protein intake. Methods To evaluate the answer of protein pathways to high- or low-protein diet, male wild-type mice and genetically modified mice from C57BL/6 background with liver-specific AMPK- or GCN2-knockout were fed from day 25 diets differing in their protein level as energy: LP (5%), NP (14%) and HP (54%). Two hours after a 1 g test meal, protein synthesis rate was measured after a C-13 valine flooding dose. The gene expression of key enzymes involved in proteolysis and GNC2 signaling pathway were quantified. Results The HP diet but not the LP diet was associated with a decrease in fractional synthesis rate by 29% in the liver compared to NP diet. The expression of mRNA encoding ubiquitin and Cathepsin D was not sensitive to the protein content. The deletion of AMPK or GCN2 in the liver did not affect nor protein synthesis rates and neither proteolysis markers in the liver or in the muscle, whatever the protein intake. In the postprandial state, protein level alters protein synthesis in the liver but not in the muscle. Conclusions Taken together, these results suggest that liver AMPK and GCN2 are not involved in this adaptation to high- and low-protein diet observed in the postprandial period.

International audience; By selecting two electroactive species immobilized in a layered double hydroxide backbone (LDH) host, one able to act as a positive electrode material and the other as a negative one, it was possible to match their capacity to design an innovative energy storage device. Each electrode material is based on electroactive species, riboflavin phosphate (RF) on one side and ferrocene carboxylate (FCm) on the other, both interleaved into a layered double hydroxide (LDH) host structure to avoid any possible molecule migration and instability. The intercalation of the electroactive guest molecules is demonstrated by X-ray diffraction with the observation of an interlayer LDH spacing of about 2 nm in each case. When successfully hosted into LDH interlayer space, the electrochemical behavior of each hybrid assembly was scrutinized separately in aqueous electrolyte to characterize the redox reaction occurring upon cycling and found to be a rapid faradic type. Both electrode materials were placed face to face to achieve a new aqueous battery (16C rate) that provides a first cycle-capacity of about 7 mAh per gram of working electrode material LDH/FCm at 10 mV/s over a voltage window of 2.2 V in 1M sodium acetate, thus validating the hybrid LDH host approach on both electrode materials even if the cyclability of the assembly has not yet been met.

International audience; On one hand, studies on Salmo biological variations during the last centuries have led to the morphological description of several Salmo species (>50). On the other hand, Salmo trutta is seen as a polymorphic species, i.e. including populations with different morphotypes and ecotypes, subdivided into nine genetically divergent evolutionary lineages. For 30 years, phylogeographic and phylogenetic investigations tried to solve the Salmo systematic problem using the mitochondrial control region, sometimes combined with other mitochondrial (e.g. protein coding region or rRNA genes) or nuclear (e.g. allozymes or microsatellites) markers. With the advent of high throughput next-generation sequencing, complete mitogenomes were made available for Salmo phylogenetic studies. Even better, complete genome or chromosomes with annotations as well as genes of interest can now be loaded from public database. However, in the genomic era, some challenges still need to be addressed such as an appropriate taxon sampling or the identification of orthologous genes before having an accurate phylogeny. In the present review, I examine how traditional molecular markers contributed to our knowledge of trout systematics, and what we can expect from the genomics revolution.

The tumour suppressor SLX4 plays multiple roles in the maintenance of genome stability, acting as a scaffold for structure-specific endonucleases and other DNA repair proteins. It directly interacts with the mismatch repair (MMR) protein MSH2 but the significance of this interaction remained unknown until recent findings showing that MutSβ (MSH2-MSH3) stimulates in vitro the SLX4-dependent Holliday junction resolvase activity. Here, we characterize the mode of interaction between SLX4 and MSH2, which relies on an MSH2-interacting peptide (SHIP box) that drives interaction of SLX4 with both MutSβ and MutSα (MSH2-MSH6). While we show that this MSH2 binding domain is dispensable for the well-established role of SLX4 in interstrand crosslink repair, we find that it mediates inhibition of MutSα-dependent MMR by SLX4, unravelling an unanticipated function of SLX4.

International audience; V enezuelan equine encephalomyelitis (VEE) complex viruses consist of antigenically related arboviruses widely distributed throughout the Americas (1). Only subtype I varieties AB and C cause severe equine epizootics and human outbreaks marked by the occurrence of encephalitis and fetal damage (2). The other subtypes are endemic in small areas of South America (3). In 1973, subtype III-B, the Tonate virus (TONV), was isolated in birds from French Guiana (4). It has since been found in neighboring countries and in South Dakota and Colorado in the United States (5,6). The wild cycle of TONV is still poorly understood. Transmission by Culicidae insects has been observed during the rainy season (4). Birds and bats are the only identifi ed vertebrate hosts (7). In humans in French Guiana, TONV seroprevalence suggests endemic transmission, particularly along the coast of the Bas Maroni region (8). However, clinical descriptions remain scarce, and no adverse pregnancy outcomes or vertical transmission have been reported (9,10). We report a case of vertical transmission of TONV from a pregnant woman to her fetus and describe ultrasonographic and fetopathological fi ndings. The Study During the 2019 rainy season, a 33-year-old woman living in the Bas Maroni region of French Guiana was referred to the prenatal diagnosis unit at West French Guiana Hospital Center (Saint-Laurent-du-Maroni, French Guiana) for fetal anomalies. This healthy G8P7 woman had no history of genetic disorders or birth defects from previous pregnancies. She was asymptomatic during the fi rst trimester of pregnancy and tested negative for syphilis, toxoplasmosis, rubella, cytomegalovirus, chikungunya, and Zika. An ultrasound screening performed at 20 weeks of gestation showed a hydropic fetus with microcephaly. The atrophic cerebral mantle exhibited calcifi cations and moderate ventriculomegaly. The corpus callosum, the cerebellum, and the brain stem were dysplastic. The fetus manifested limb malformations and an absence of swallowing at the time of the serially performed sonograms (Appendix Figure, https:// wwwnc.cdc.gov/EID/article/28/2/21-0884-App1. pdf). Therefore, we performed amniocentesis for etiological investigation. Because of the poor prognosis, the mother elected to terminate the pregnancy. After approval by the multidisciplinary center for prenatal diagnosis, the pregnancy was terminated without complication. The patient gave written informed consent for the publication of her case. Karyotype and array comparative genomic hybridization were normal. Results of screening for metabolic diseases were negative. All PCR and reverse transcription PCR (RT-PCR) for toxoplasmosis, rubella, cytomegalovirus, herpes simplex virus, and common arboviruses from the Amazon were negative. However, we reproducibly detected the presence of a VEE complex virus in the amniotic fl uid with a real-time RT-PCR test yielding cycle threshold values of 30. Furthermore, although maternal serum samples collected 2 months before pregnancy were negative for TONV IgM, the test was positive at the time of pregnancy termination.

The Mendelian inheritance pattern of acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria is autosomal dominant, but the clinical phenotype is heterogeneous. Within the general population, penetrance is low, but among first-degree relatives of a symptomatic proband, penetrance is higher. These observations suggest that genetic factors, in addition to mutation of the specific enzyme of the biosynthetic pathway of heme, contribute to the clinical phenotype. Recent studies by others suggested that the genotype of the transporter protein ABCB6 contribute to the porphyria phenotype. Identifying the molecule(s) that are transported by ABCB6 has been problematic and has led to uncertainty with respect to how or if variants/mutants contribute to phenotypic heterogeneity. Knockout mouse models of Abcb6 have not provided a direction for investigation as homozygous knockout animals do not have a discrete phenotype. To address the proposed link between ABC6 genotype and porphyria phenotype, a large cohort of patients with acute hepatic porphyria and erythropoietic protoporphyria was analyzed. Our studies showed that ABCB6 genotype did not correlate with disease severity. Therefore, genotyping of ABCB6 in patients with acute hepatic porphyria and erythropoietic protoporphyria is not warranted.

Purpose: Mutations in STK11 (LKB1) occur in 17% of lung adenocarcinoma (LUAD) and drive a suppressive (cold) tumor immune microenvironment (TIME) and resistance to immunotherapy. The mechanisms underpinning the establishment and maintenance of a cold TIME in LKB1-mutant LUAD remain poorly understood. In this study, we investigated the role of the LKB1 substrate AMPK in immune evasion in human non—small cell lung cancer (NSCLC) and mouse models and explored the mechanisms involved. Experimental Design: We addressed the role of AMPK in immune evasion in NSCLC by correlating AMPK phosphorylation and immune-suppressive signatures and by deleting AMPKα1 (Prkaa1) and AMPKα2 (Prkaa2) in a KrasG12D-driven LUAD. Furthermore, we dissected the molecular mechanisms involved in immune evasion by comparing gene-expression signatures, AMPK activity, and immune infiltration in mouse and human LUAD and gain or loss-of-function experiments with LKB1- or AMPK-deficient cell lines. Results: Inactivation of both AMPKα1 and AMPKα2 together with Kras activation accelerated tumorigenesis and led to tumors with reduced infiltration of CD8+/CD4+ T cells and gene signatures associated with a suppressive TIME. These signatures recapitulate those in Lkb1-deleted murine LUAD and in LKB1-deficient human NSCLC. Interestingly, a similar signature is noted in human NSCLC with low AMPK activity. In mechanistic studies, we find that compromised LKB1 and AMPK activity leads to attenuated antigen presentation in both LUAD mouse models and human NSCLC. Conclusions: The results provide evidence that the immune evasion noted in LKB1-inactivated lung cancer is due to subsequent inactivation of AMPK and attenuation of antigen presentation.

Prior to their analysis, crude DNA samples have to be processed through consecutive steps of purification, concentration, and size selection. These steps generally involve a number of time-consuming and low-yield tools, which are most often based on affinity columns and/or fractioning by gel electrophoresis. The fraction of DNA circulating in the blood is considered to be a promising biomarker, but its preparation is suffering from many of the aforementioned pitfalls because it is highly diluted, distributed in size (between 100 and 2000 base pairs (bp)), and solubilized in complex biofluids with salts and proteins.The µLAS technology, which consists in conveying DNA molecules in electrohydrodynamic viscoelastic flows, has shown a great potential for DNA analysis with a sensitivity of 1 fg/µL. It is however limited in throughput because its analytical flow rate is 1 µL/min. In this PhD, we aimed at intensifying the potential of µLAS for purification by parallelizing this technology. For this, we used a format based on an isoporous membrane integrated within a 3D printed macrometric system, which offers a platform to operate hundred of thousand of channels simultaneously. We report a high throughput associated to a flow rate of 300 µL/min, and demonstrate efficient processing of a few mL of solution in the range of 200 bp to 50 kbp, with a concentration of up to 10 times, a size accordable DNA size selection threshold, and the purification of salts in a process of less than ten minutes. It also has the advantage to be functional at low amplitudes of actuation : a pressure lower than 2 bar and a voltage lower than 50 V making it easily accessible. Finally, we discuss its application potential for the preparation of sequencing samples.; La préparation d’un échantillon d’ADN est une étape obligatoire de sa chaîne analytique. Réalisée concomitamment ou postérieurement à l’extraction d’ADN de l’organisme ciblé, la préparation inclut une étape de concentration voire de fractionnement en fonction de la taille des chaînes d’ADN. Les outils de préparation d’ADN sont le plus souvent fondés sur des colonnes de silice et quelques fois aussi l’électrophorèse sur gel. Ils sont laborieux en temps de main d’œuvre et leur rendement pas toujours au rendez-vous. Ces considérations sont particulièrement critiques pour la préparation de l’ADN circulant dans le sang, un biomarqueur prometteur, car il est très dilué, présentant des tailles variées de petits fragments de 100 pb (paires de bases) à plus de 2 kpb et dissous dans des fluides biologiques complexes avec des sels et des protéines.Nous avons investigué le potentiel de la technologie µLAS pour la préparation d’ADN. Fondée sur la migration d’ADN dans un fluide viscoélastique et sous écoulement électro-hydrodynamique, elle permet d’analyser des solutions complexes avec des sensibilités au fg/µL. Cela dit, son débit actuel de quelque µL/min n’est pas adapté à préparation d’ADN. Aussi, nous avons parallélisé µLAS avec une membrane isopore intégrée à un système macrométrique imprimé en 3D pour atteindre des performances adaptées à la purification à travers des centaines de milliers de canaux microfluidiques en simultané. Nous avons réalisé le traitement haut débit (300 µL/min) de quelques mL d’ADN dans une gamme de taille comprise entre 200 paires de bases (pb) et 50 kpb, avec une concentration jusqu’à 10 fois et la purification des sels. Le procédé, d’une durée inférieure à dix minutes, présente également l’avantage de fonctionner à faibles amplitudes d’actionnement : une pression inférieure à 2 bar et une tension inférieure à 50 V rendant cette technologie facilement accessible. Pour terminer, nous discutons le potentiel de notre technologie pour la préparation d’échantillons pour leur caractérisation par séquençage.

International audience; Peptidoglycan, the major structural polymer forming the cell wall of bacteria, is an important mediator of physiological and behavioral effects in mammalian hosts. These effects are frequently linked to its translocation from the intestinal lumen to host tissues. However, the modality and regulation of this translocation across the gut barrier has not been precisely addressed. In this study, we characterized the absorption of peptidoglycan across the intestine and its systemic dissemination. We report that peptidoglycan has a distinct tropism for host organs when absorbed via the gut, most notably by favoring access to the brain. We demonstrate that intestinal translocation of peptidoglycan occurs through a microbiota-induced active process. This process is regulated by the parasympathetic pathway via the muscarinic acetylcholine receptors. Together, this study reveals fundamental parameters concerning the uptake of a major microbiota molecular signal from the steady-state gut.

Natural transformation plays a major role in establishing the amazing genomic plasticity of Helicobacter pylori, a Gram-negative bacterium that infects the stomach of half of the human population and is at the origin of multiple pathologies including gastritis, ulcers and cancer. Horizontal gene transfer through natural transformation is thought to be a major contributor for the increase in antibiotic resistant strains observed in H. pylori infections. In this project, we will address some of the unique characteristics of the natural transformation mechanisms used by this human pathogen. Whilst many of the players in natural transformation are conserved among naturally competent bacteria, other components are absent in H. pylori. Furthermore, some seem to be unique for this pathogen. We focused our attention on two of those proteins that we have shown to be essential for transformation. The main part of this thesis aims at characterizing the role of HP0792, which is a homologue of ComM, a protein conserved in Gram-negative bacteria. ComM is a helicase potentially involved in the homologous recombination during natural transformation. Our project combined genetics and biochemical approaches to identify two functions for ComM, in natural transformation and in intragenomic recombination. A comM deletion leads to a 200-fold reduction of the natural transformation rate when inserting a point mutation (StrepR). A similar reduction of the transformation rate is observed when integrating an 842 bp heterologous region. Moreover, we show that ComM is not involved in defining the length of tDNA inserted in the genome. Interestingly, these results are different from what was reported for Vibrio cholera and Acinetobacter baylyi in the literature. By bacterial two hybrid, we have also unveiled an interaction between ComM and DprA and have explored the surface interaction between the two proteins. In parallel, we discovered a new role for ComM in the recombination of genomic repeats. The second part addresses the role of HP1421, an ATPase involved in the first steps of the natural transformation in H. pylori and potentially associated with the ComB Type IV secretion system.; La transformation naturelle joue un rôle clé dans l'importante plasticité génomique de la bactérie à Gram-négatif Helicobacter pylori. Ce pathogène humain, qui infecte l'estomac de la moitié de la population, est à l'origine de nombreuses pathologies, telles que des gastrites, les ulcères et des cancers. Le transfert horizontal de gènes à travers la transformation naturelle est considéré comme un contributeur majeur à la propagation de résistances aux antibiotiques observée actuellement dans les infections par H. pylori. Chez H. pylori la capture et l'internalisation de l'ADN présentent des caractéristiques uniques. Certains des acteurs conservés dans la plupart des bactéries compétentes sont absents chez H. pylori alors que de nouvelles protéines impliquées dans la transformation y ont été identifiés. Parmi elles, nous nous sommes particulièrement intéressés à deux candidates que nous avons découvertes comme essentielles pour la transformation. La majeure partie du travail de thèse se concentre sur HP0792, un homologue de ComM, une protéine présente chez les Gram négatifs. ComM est une hélicase jouant un rôle dans la recombinaison homologue lors de la transformation naturelle. Par des approches génétiques et biochimiques, nous avons identifié deux fonctions de cette protéine chez H. pylori, dans la transformation naturelle et dans la recombinaison intra-génomique. Une délétion de comM entraine une diminution de la transformation par un ADN portant une mutation ponctuelle (StrepR) d'un facteur 200 et d'un facteur similaire lors de l'intégration d'un ADN possédant 842 bp hétérologues. De plus, nous avons mis en évidence que ComM n'a pas d'impact sur la taille d'ADNt inséré dans le génome. Ces résultats diffèrent des études publiées sur ComM chez Vibrio cholerae et Acinetobacter baylyi. Nous avons également mis en évidence par double hybride bactérien une interaction avec DprA et nous avons exploré les interactions de surface entre ces deux protéines. En parallèle, nous avons découvert un second rôle à ComM qui intervient dans la recombinaison de séquences répétées dans le génome. La seconde partie, mineure, explore le rôle de HP1421, une ATPase impliquée dans les premières étapes de la transformation naturelle chez H. pylori et potentiellement liée au système de sécrétion de type IV ComB.

The COVID-19 pandemic has more than 600 million reported cases worldwide and has resulted in more than 6 million deaths (as of September 2022). Men with COVID-19 have been found to be more likely than women to be hospitalized, admitted to intensive care, or die from the disease. Physiological effects of androgens are among the factors that may contribute to this difference. In addition, it is already known that the expression of the TMPRSS2 gene, which encodes a protease that is crucial for virus entry, is regulated by androgens in the prostate. Thus, the possible regulation of TMPRSS2 by androgens in the lung could explain, at least in part, the heightened severity observed in men with SARS-CoV-2.Our objective is to study the effect of gene expression modulation by androgens on SARS-CoV-2 infection and COVID-19 severity.We established a hormone-dependent prostate cell line stably overexpressing ACE2, the protein essential for SARS-CoV-2 infection, to be able to measure the effect of hormonal modulation of the TMPRSS2 gene on different stages of SARS-CoV-2 infection in vitro. The results demonstrate a hormone-dependent modulation of the cellular infection rate. More precisely, using pseudoviruses, we showed that this effect takes place during virus entry.In parallel, in vivo, in order to study the effect of androgens on SARS-CoV-2 infection and disease severity over time, we used a hamster model, for which we performed androgen suppression by castration. Our results show an earlier pulmonary inflammatory response in castrated hamsters, assessed by anatomo-pathological diagnosis, inflammatory cytokine monitoring and transcriptomic analysis. This kinetic shift in response to SARS-CoV-2 infection reveals an effect of androgens in the lung via an altered transcriptional program. In men, these results were supported by an analysis of the CACOVID cohort, which identifies patients with cancer, including prostate cancer treated or not with hormone therapy, who had been diagnosed with COVID-19.Together, these results indicate a role for androgens in the SARS-CoV-2 infection process and in particular in the kinetics of disease progression.; La pandémie de COVID-19 compte plus de 600 millions de cas recensés dans le monde et a fait plus de 6 millions de morts (chiffres en septembre 2022). Il a été constaté que les hommes atteints par la COVID-19 ont plus de risques que les femmes d'être hospitalisés, admis en soins intensifs ou de décéder de la maladie. Les effets physiologiques des androgènes figurent parmi les facteurs qui pourraient contribuer à cette différence. En outre, on sait déjà que l'expression du gène TMPRSS2, qui code une protéase cruciale dans l'entrée du virus, est régulée par les androgènes dans la prostate. Ainsi, la possible régulation de TMPRSS2 par les androgènes dans le poumon pourrait expliquer, au moins en partie, la sévérité accrue observée chez les hommes face au SARS-CoV-2.Notre objectif est d'étudier l'effet de la modulation d'expression des gènes par les androgènes, en particulier TMPRSS2, sur l'infection au SARS-CoV-2 et la sévérité de la COVID-19.Nous avons établi une lignée de cellules prostatiques hormono-dépendantes surexprimant stablement ACE2, la protéine indispensable à l'infection au SARS CoV 2, pour pouvoir mesurer l'effet de la modulation hormonale du gène TMPRSS2 sur différentes étapes de l'infection au SARS-CoV-2 in vitro. Les résultats démontrent une modulation du taux d'infection cellulaire de manière hormono-dépendante. Plus précisément, grâce à l'utilisation de pseudovirus, nous avons montré que cet effet a lieu lors de l'entrée du virus.En parallèle, in vivo, pour étudier l'effet des androgènes sur l'infection au SARS CoV-2 et la sévérité de la maladie au cours du temps, nous avons utilisé un modèle de hamster, pour lequel nous avons réalisé la suppression androgénique par castration. Nos résultats montrent une réponse inflammatoire pulmonaire plus précoce chez les hamsters castrés, évaluée à la fois par un diagnostic anatomo-pathologique, par le suivi de cytokines inflammatoires et par analyse transcriptomique. Ce décalage de cinétique en réponse à l'infection au SARS-CoV-2 révèle un effet des androgènes dans le poumon via un programme transcriptionnel modifié. Ces résultats ont été complétés par une analyse de la cohorte CACOVID.L'ensemble de ces résultats témoigne d'un rôle des androgènes dans le processus d'infection au SARS-CoV-2 et notamment dans la cinétique d'évolution de la maladie.

International audience; The ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity.

International audience; The molecular factors and genetic adaptations that contributed to the emergence of Mycobacterium tuberculosis (MTB) from an environmental Mycobacterium canettii -like ancestor, remain poorly investigated. In MTB , the PhoPR two-component regulatory system controls production and secretion of proteins and lipid virulence effectors. Here, we describe that several mutations, present in phoR of M . canettii relative to MTB , impact the expression of the PhoP regulon and the pathogenicity of the strains. First, we establish a molecular model of PhoR and show that some substitutions found in PhoR of M . canettii are likely to impact the structure and activity of this protein. Second, we show that STB-K, the most attenuated available M . canettii strain, displays lower expression of PhoP-induced genes than MTB . Third, we demonstrate that genetic swapping of the phoPR allele from STB-K with the ortholog from MTB H37Rv enhances expression of PhoP-controlled functions and the capacities of the recombinant strain to colonize human macrophages, the MTB target cells, as well as to cause disease in several mouse infection models. Fourth, we extended these observations to other M . canettii strains and confirm that PhoP-controlled functions are expressed at lower levels in most M . canettii strains than in M . tuberculosis . Our findings suggest that distinct PhoR variants have been selected during the evolution of tuberculosis bacilli, contributing to higher pathogenicity and persistence of MTB in the mammalian host.

International audience; Extracellular glutamate levels are maintained low by efficient transporters, whose dysfunction can cause neuronal hyperexcitability, excitotoxicity, and neurological disease. While many methods estimate glutamate uptake in vitro/ex vivo, a limited number of techniques address glutamate transport in vivo. Here, we used in vivo microdialysis in a two-in-one approach combining reverse dialysis of isotopic glutamate to measure uptake ability and zero-flow (ZF) methods to quantify extracellular glutamate levels. The complementarity of both techniques is discussed on methodological and anatomical basis. We used a transgenic mouse model of human disease, expressing low levels of the EAAT-2/GLT1 glutamate transporter, to validate our approach in a relevant animal model. As expected, isotopic analysis revealed an overall decrease in glutamate uptake, while the ZF method unveiled higher extracellular glutamate levels in these mice. We propose a sensitive and expedite two-in-one microdialysis approach that is sufficiently robust to reveal significant differences in neurotransmitter uptake and extracellular levels through the analysis of a relatively low number of animals.

Environmental conditions in food and medical fields enable the bacteria to attach and grow on surfaces leading to resistant bacterial biofilm formation. Indeed, the first step in biofilm formation is the bacterial irreversible adhesion. Controlling and inhibiting this adhesion is a passive approach to fight against biofilm development. This strategy is an interesting path in the inhibition of biofilm formation since it targets the first step of biofilm development. Those pathogenic structures are responsible for several foodborne diseases and nosocomial infections. Therefore, to face this public health threat, researchers employed cold plasma technologies in coating development. In this review, the different factors influencing the bacterial adhesion to a substrate are outlined. The goal is to present the passive coating strategies aiming to prevent biofilm formation via cold plasma treatments, highlighting antiadhesive elaborated surfaces. General aspects of surface treatment, including physico-chemical modification and application of cold plasma technologies, were also presented. Key points • Factors surrounding pathogenic bacteria influence biofilm development. • Controlling bacterial adhesion prevents biofilm formation. • Materials can be coated via cold plasma to inhibit bacterial adhesion.

The bacterial DNA sliding clamp (SC), or replication processivity factor, is a promising target for the development of novel antibiotics. We report a structure-activity relationship study of a new series of peptides interacting within the Escherichia coli SC (EcSC) binding pocket. Various modifications were explored including N-alkylation of the peptide bonds, extension of the N-terminal moiety, and introduction of hydrophobic and constrained residues at the C-terminus. In each category, single modifications were identified that increased affinity to EcSC. A combination of such modifications yielded in several cases to a substantially increased affinity compared to the parent peptides with Kd in the range of 30-80 nM. X-ray structure analysis of 11 peptide/EcSC co-crystals revealed new interactions at the peptide-protein interface (i.e., stacking interactions, hydrogen bonds, and hydrophobic contacts) that can account for the improved binding. Several compounds among the best binders were also found to be more effective in inhibiting SC-dependent DNA synthesis.

Lactic acid bacteria, including the microorganisms formerly designated as Lactobacillus, are the major representatives of Live Biotherapeutic Microorganisms (LBM) when used for therapeutic purposes. However, in most cases, the mechanisms of action remain unknown. The antifungal potential of LBM has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding their mechanisms of action is strategic for the development of new therapeutics for humans. Here, Caenorhabditis elegans was used as an in vivo model to analyze pro-longevity, anti-aging and anti-candidiasis effects of the LBM Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) Lcr35®. A high-throughput transcriptomic analysis revealed a specific response of C. elegans depending on whether it is in the presence of the LBM L. rhamnosus Lcr35® (structural response), the yeast Candida albicans (metabolic response) or both (structural and metabolic responses) in a preventive and a curative conditions. Studies on C. elegans mutants demonstrated that the p38 MAPK (sek-1, skn-1) and the insulin-like (daf-2, daf-16) signaling pathways were involved in the extended lifespan provided by L. rhamnosus Lcr35® strain whereas the JNK pathway was not involved (jnk-1). In addition, the anti C. albicans effect of the bacterium requires the daf-16 and sek-1 genes while it is independent of daf-2 and skn-1. Moreover, the anti-aging effect of Lcr35®, linked to the extension of longevity, is not due to protection against oxidative stress (H2O2). Taken together, these results formally show the involvement of the p38 MAP kinase and insulin-like signaling pathways for the longevity extension and anti-Candida albicans properties of Lcr35® with, however, differences in the genes involved. Overall, these findings provide new insight for understanding the mechanisms of action of a probiotic strain with antimicrobial potential.

Apicomplexa is a phylum of obligate intracellular protozoan parasites responsible for serious diseases in both humans and animals. T. gondii and P. falciparum are notable members of this phylum and possess, like their counterparts, an assortment of exclusive apical secretory organelles (micronemes and rhoptries) that orchestrate their entry into the host cell. Micronemes are involved in motility, attachment, and finally escape from the host cell after parasite replication. Rhoptry proteins act in synergy with microneme proteins to enable invasion and are involved in the hijacking of host functions and the establishment of the parasitophorous vacuole. In T. gondii, our model of study, these organelles are dependent on TgSORT, a transmembrane cargo receptor residing in the post-Golgi and endosome-like compartment (ELC) that sorts proteins from these organelles. Indeed, the N-terminal domain (TgN-SORT) binds microneme and rhoptry proteins to transport them in the ELC in order to allow the biogenesis of these organelles. The receptor is thus essential for the virulence and survival of the parasite. TgSORT is homologous to human sortilin, a multifunctional receptor required for protein transport to lysosome using a mannose-6-phosphate independent pathway. A homolog is also present in P. falciparum where it plays an equally crucial role for parasite survival. The main objective of this thesis is to study mechanisms of interaction between TgSORT and its ligands. Using the yeast Pichia pastoris, we successfully expressed and purified TgN-SORT, which was previously insoluble when expressed in E. coli. We also produced its partners MIC5, ROP18 and ROP16. In addition, we performed cryoEM experiments of TgN-SORT, which showed that TgN-SORT has a toroidal structure, which is consistent with predictions. This observation was confirmed by the powerful Alphafold 2 algorithm, which allowed us to obtain with a satisfying confidence score the 3D structure of TgSORT. This structure is similar to that of human sortilin and is in the form of 10 β-propeller domains arranged as a helix followed by two 10CC motifs with a central tunnel. In the parasite, three loops are found outside the tunnel. In addition, Alphafold 2 revealed direct binding between TgN-SORT and rhoptries proteins through the same motifs that human sortilin uses to bind neurotensin. Subsequently, we tested on T. gondii, 12 positive compounds identified from an automated screening of a library of 1121 compounds on a transgenic strain of T. gondii SORT-GFP/ROP1-mCherry that we generated. The objective is to identify small molecules that could profoundly alter the subcellular distribution of the TgSORT receptor and the rhoptries that depend on its biological function. The activities of 4 of the 12 compounds were validated on the reference strain P. falciparum 3D7.; Les Apicomplexa sont un embranchement de parasites protozoaires intracellulaires obligatoires responsables de graves pathologies aussi bien chez l'homme que chez les animaux. T. gondii et P. falciparum sont des membres notables de ce phylum et possèdent, à l'instar de leurs homologues d'un assortiment d'organites sécrétoires apicaux exclusifs (micronèmes et rhoptries) qui orchestrent leur entrée dans la cellule hôte. Les micronèmes interviennent dans la motilité, l'attachement, et enfin l'échappement de la cellule hôte après la réplication du parasite. Les rhoptries agissent en synergie avec les protéines des micronèmes pour permettre l'invasion et sont également impliqués dans le détournement des fonctions de l'hôte et l'établissement de la vacuole parasitophore. Cependant, chez T. gondii, notre modèle d'étude, ces organites sont sous la dépendance de TgSORT, un récepteur cargo transmembranaire résidant dans le post-Golgi et l'ELC et qui assure le tri des protéines de ces organites. En effet, le domaine N-terminal (N-SORT) fixe les protéines des micronèmes et des rhoptries qu'il conduit à l'ELC afin de permettre la biogenèse des protéines des micronèmes et des rhoptries. Il est de ce fait indispensable à la virulence et à la survie du parasite. TgSORT est homologue à la sortiline humaine, un récepteur multifonctionnel requis pour le transport des protéines vers le lysosome par une voie indépendante du mannose-6-phosphate. Un homologue est également présent chez P. falciparum où il joue un rôle tout aussi crucial pour la survie du parasite. Cette thèse a pour objectif principal d'étudier les mécanismes d'interaction entre TgSORT et ses ligands. Nous avons établi un protocole et exprimé dans Pichia pastoris N-SORT, qui était précédemment insoluble lorsqu'elle était exprimée dans E. coli. Nous avons également exprimé ses partenaires MIC5, ROP18 et ROP16. Par ailleurs, nous avons réalisé des expériences de cryoEM de N-SORT qui ont montré que N-SORT présente une structure torique, ce qui est conforme aux prédictions. Cette observation a été confirmée par le puissant algorithme Alphafold 2 qui nous a permis d'obtenir avec un score de confiance satisfaisant la structure 3D de TgSORT. Cette structure est semblable à celle de la sortiline humaine et se présente sous la forme de 10 domaines β-propellers arrangés sous forme d'hélice suivis de deux motifs 10CC avec un tunnel central. Chez le parasite, trois boucles sont retrouvées à l'extérieur du tunnel. En outre, Alphafold 2 a révélé une liaison directe entre N-SORT et les protéines de rhoptries grâce aux mêmes motifs qu'utilise la sortiline humaine pour lier la neurotensine. Par la suite, nous avons testé sur T. gondii, 12 composés positifs identifiés à partir d'un screening automatisé d'une librairie de 1121 composés sur une souche transgénique de T. gondii SORT-GFP/ROP1-mCherry que nous avons générée. L'objectif est d'identifier des petites moléculaires qui pourraient modifier profondément la distribution subcellulaire du récepteur TgSORT et les rhoptries qui dépendent de sa fonction biologique. Les activités de 4 composés parmi les douze ont été validées sur la souche de référence P. falciparum 3D7.

Enhancers play a central role in the spatiotemporal control of gene expression and tend to work in a cell-type-specific manner. In addition, they are suggested to be major contributors to phenotypic variation, evolution and disease. There is growing evidence that enhancer dysfunction due to genetic, structural or epigenetic mechanisms contributes to a broad range of human diseases referred to as enhanceropathies. Such mechanisms often underlie the susceptibility to common diseases, but can also play a direct causal role in cancer or Mendelian diseases. Despite the recent gain of insights into enhancer biology and function, we still have a limited ability to predict how enhancer dysfunction impacts gene expression. Here we discuss the major challenges that need to be overcome when studying the role of enhancers in disease etiology and highlight opportunities and directions for future studies, aiming to disentangle the molecular basis of enhanceropathies., All authors are members of the EU-funded Innovative Training Network ‘Molecular Basis of Human Enhanceropathies’ (Enhpathy, www.enhpathy.eu) and received funding from the Horizon 2020 research and innovation program under Marie Sklodowska-Curie grant agreement no. 860002.

International audience

Introduction: The aim of this study was to investigate endogenous retrovirus (ERV) expression and type I interferon (IFN) activation in human pleural mesothelioma (PM) and their association with clinical outcome. Methods: The expression of ERV was determined from PM cohorts and mesothelial precursor RNA sequencing data. The expression of ERV was confirmed by quantitative polymerase chain reaction (qPCR). Methylation of genomic DNA was assessed by quantitative methylation-specific PCR. DNA demethylation was induced in cells by demethylating agent 5-Aza-2’-deoxycytidine (5-Aza-CdR) treatment. To block type I IFN signaling, the cells were treated with ruxolitinib or MAVS silencing. The expression of IFN-stimulated genes (ISGs) was determined by qPCR and Western blot. Circulating ERVs were detected by qPCR. Results: Long terminal repeats (LTRs) represent the most abundant transposable elements up-regulated in PM. Within the LTR, ERVmap_1248 and LTR7Y, which are specifically enriched in PM, were further analyzed. The 5-Aza-CdR treatment increased the levels of ERVmap_1248 expression and induced ERVmap_1248 promoter demethylation in mesothelial cells. In addition, ERVmap_1248 promoter was more demethylated in the mesothelioma tissue compared with nontumor tissue. The 5-Aza-CdR treatment of the mesothelial cells also increased the levels of ISGs. Basal ISG expression was higher in the mesothelioma cells compared with the mesothelial cells, and it was significantly decreased by ruxolitinib treatment or MAVS silencing. Furthermore, ISG expression was higher in the tumor tissue with high expression levels of ERVmap_1248. High expression of ERVmap_1248 was associated with longer overall survival and BAP1 mutations. ERVmap_1248 and LTR7Y can be detected in the PM plasma. Conclusions: We provide clues for patient stratification especially for immunotherapy where best clinical responses are associated with an activated basal immune response., JTO Clinical and Research Reports, 3 (12), ISSN:2666-3643

France went through three deadly epidemic waves due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing major public health and socioeconomic issues. We proposed to study the course of the pandemic along 2020 from the outlook of two major Parisian hospitals earliest involved in the fight against COVID-19. Genome sequencing and phylogenetic analysis were performed on samples from patients and health care workers (HCWs) from Bichat (BCB) and Pitié-Salpêtrière (PSL) hospitals. A tree-based phylogenetic clustering method and epidemiological data were used to investigate suspected nosocomial transmission clusters. Clades 20A, 20B and 20C were prevalent during the spring wave and, following summer, clades 20A.EU2 and 20E.EU1 emerged and took over. Phylogenetic clustering identified 57 potential transmission clusters. Epidemiological connections between participants were found for 17 of these, with a higher proportion of HCWs. The joint presence of HCWs and patients suggest viral contaminations between these two groups. We provide an enhanced overview of SARS-CoV-2 phylogenetic changes over 2020 in the Paris area, one of the regions with highest incidence in France. Despite the low genetic diversity displayed by the SARS-CoV-2, we showed that phylogenetic analysis, along with comprehensive epidemiological data, helps to identify and investigate healthcare associated clusters.

International audience; L’obésité est une maladie complexe dont la prévalence continue à augmenter chez les enfants et les adolescents dans de nombreuses régions du monde. La SFN et l’ECOG (European Childhood Obesity Group), avec le soutien financier d’Aprifel, ont organisé un webinaire sur les dernières avancées autour des stratégies de prise en charge de l’obésité infantile. Cette session s’est tenue en digital le 19 mai 2022. L’intégralité des conférences est disponible gratuitement sur le site de la SFN à l’adresse suivante : https://sf-nutrition.fr/workshop-de-la-sfn/. Nous en présentons ici une synthèse.

Background In all living organisms, DNA replication is exquisitely regulated in a wide range of growth conditions to achieve timely and accurate genome duplication prior to cell division. Failures in this regulation cause DNA damage with potentially disastrous consequences for cell viability and human health, including cancer. To cope with these threats, cells tightly control replication initiation using well-known mechanisms. They also couple DNA synthesis to nutrient richness and growth rate through a poorly understood process thought to involve central carbon metabolism. One such process may involve the cross-species conserved pyruvate kinase (PykA) which catalyzes the last reaction of glycolysis. Here we have investigated the role of PykA in regulating DNA replication in the model system Bacillus subtilis. Results On analysing mutants of the catalytic (Cat) and C-terminal (PEPut) domains of B. subtilis PykA we found replication phenotypes in conditions where PykA is dispensable for growth. These phenotypes are independent from the effect of mutations on PykA catalytic activity and are not associated with significant changes in the metabolome. PEPut operates as a nutrient-dependent inhibitor of initiation while Cat acts as a stimulator of replication fork speed. Disruption of either PEPut or Cat replication function dramatically impacted the cell cycle and replication timing even in cells fully proficient in known replication control functions. In vitro, PykA modulates activities of enzymes essential for replication initiation and elongation via functional interactions. Additional experiments showed that PEPut regulates PykA activity and that Cat and PEPut determinants important for PykA catalytic activity regulation are also important for PykA-driven replication functions. Conclusions We infer from our findings that PykA typifies a new family of cross-species replication control regulators that drive the metabolic control of replication through a mechanism involving regulatory determinants of PykA catalytic activity. As disruption of PykA replication functions causes dramatic replication defects, we suggest that dysfunctions in this new family of universal replication regulators may pave the path to genetic instability and carcinogenesis.

The d,d-transpeptidase activity of penicillin-binding proteins (PBPs) is the well-known primary target of β-lactam antibiotics that block peptidoglycan polymerization. β-lactam-induced bacterial killing involves complex downstream responses whose causes and consequences are difficult to resolve. Here, we use the functional replacement of PBPs by a β-lactam-insensitive l,d-transpeptidase to identify genes essential to mitigate the effects of PBP inactivation by β-lactams in actively dividing bacteria. The functions of the 179 conditionally essential genes identified by this approach extend far beyond l,d-transpeptidase partners for peptidoglycan polymerization to include proteins involved in stress response and in the assembly of outer membrane polymers. The unsuspected effects of β-lactams include loss of the lipoprotein-mediated covalent bond that links the outer membrane to the peptidoglycan, destabilization of the cell envelope in spite of effective peptidoglycan cross-linking, and increased permeability of the outer membrane. The latter effect indicates that the mode of action of β-lactams involves self-promoted penetration through the outer membrane.

AMPK has been reported to facilitate hypoxic pulmonary vasoconstriction but, paradoxically, its deficiency precipitates pulmonary hypertension. Here we show that AMPK-α1/α2 deficiency in smooth muscles promotes persistent pulmonary hypertension of the new-born. Accordingly, dual AMPK-α1/α2 deletion in smooth muscles causes premature death of mice after birth, associated with increased muscularisation and remodeling throughout the pulmonary arterial tree, reduced alveolar numbers and alveolar membrane thickening, but with no oedema. Spectral Doppler ultrasound indicates pulmonary hypertension and attenuated hypoxic pulmonary vasoconstriction. Age-dependent right ventricular pressure elevation, dilation and reduced cardiac output was also evident. KV1.5 potassium currents of pulmonary arterial myocytes were markedly smaller under normoxia, which is known to facilitate pulmonary hypertension. Mitochondrial fragmentation and reactive oxygen species accumulation was also evident. Importantly, there was no evidence of systemic vasculopathy or hypertension in these mice. Moreover, hypoxic pulmonary vasoconstriction was attenuated by AMPK-α1 or AMPK-α2 deletion without triggering pulmonary hypertension.

Nitrogen (N2) fixation, or diazotrophy, supports a large part of primary production in oceans. Culture-independent approaches highlighted the presence in abundance of marine non-cyanobacterial diazotrophs (NCD) but their ecophysiology remains elusive, mostly because of the low number of isolated NCD and because of the lack of available genetic tools for these isolates. Here, a dual genetic and functional approach allowed unveiling the ecophysiology of a marine NCD affiliated to the species Vibrio diazotrophicus. Physiological characterization of the first marine NCD mutant obtained so far was performed using a soft-gellan assay, demonstrating that a ΔnifH mutant in not able to grow in nitrogen-deprived media. Furthermore, we demonstrated that V. diazotrophicus produces a thick biofilm under diazotrophic conditions, suggesting biofilm production as an adaptive response of this NCD to cope with the inhibition of nitrogen-fixation by molecular oxygen. Finally, the genomic signature of V. diazotrophicus is essentially absent from metagenomic data of Tara Ocean expeditions, despite having been isolated from various marine environments. We think that the genetically tractable V. diazotrophicus strain used in this study may serve as an ideal model to study the ecophysiology of these overlooked procaryotic group.

Background Myotonic dystrophy type 1 (DM1) is an incurable multisystem disease caused by a CTG-repeat expansion in the DM1 protein kinase (DMPK) gene. The OPTIMISTIC clinical trial demonstrated positive and heterogenous effects of cognitive behavioral therapy (CBT) on the capacity for activity and social participations in DM1 patients. Through a process of reverse engineering, this study aims to identify druggable molecular biomarkers associated with the clinical improvement in the OPTIMISTIC cohort. Methods Based on full blood samples collected during OPTIMISTIC, we performed paired mRNA sequencing for 27 patients before and after the CBT intervention. Linear mixed effect models were used to identify biomarkers associated with the disease-causing CTG expansion and the mean clinical improvement across all clinical outcome measures. Results We identified 608 genes for which their expression was significantly associated with the CTG-repeat expansion, as well as 1176 genes significantly associated with the average clinical response towards the intervention. Remarkably, all 97 genes associated with both returned to more normal levels in patients who benefited the most from CBT. This main finding has been replicated based on an external dataset of mRNA data of DM1 patients and controls, singling these genes out as candidate biomarkers for therapy response. Among these candidate genes were DNAJB12, HDAC5, and TRIM8, each belonging to a protein family that is being studied in the context of neurological disorders or muscular dystrophies. Across the different gene sets, gene pathway enrichment analysis revealed disease-relevant impaired signaling in, among others, insulin-, metabolism-, and immune-related pathways. Furthermore, evidence for shared dysregulations with another neuromuscular disease, Duchenne muscular dystrophy, was found, suggesting a partial overlap in blood-based gene dysregulation. Conclusions DM1-relevant disease signatures can be identified on a molecular level in peripheral blood, opening new avenues for drug discovery and therapy efficacy assessments.

International audience; The development of ultrafast kinetic methods is one of the factors that allowed the research on protein folding to flourish over the last twenty years. The introduction of new optical triggering techniques enabled to experimentally investigate the protein dynamics at the nanosecond to millisecond timescale, allowing researchers to test theoretical predictions and providing experimental benchmarks for computer simulations. In this work the details of how to perform kinetic experiments by the laserinduced temperature jump technique, using the two most commonly used probing techniques (namely infrared absorption and fluorescence spectroscopy) are given, with a strong emphasis on the practical details.

IntroductionDespite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful.MethodsTo this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established.Results and discussionMutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing.

In eukaryotic cells, DNA is compacted in chromatin and all DNA-related processes including transcription occur in this context. Mediator of transcription regulation is a conserved mutliprotein complex which integrates the transcription factors' regulatory signals and transmits them to the basal transcription machinery. Given its essential role in gene transcription, mutations in its subunits are invovled in a broad range of diseases including cancers. Despite intensive studies, it remains largely unknown how Mediator's functions are coordinated with the other transcription coregulators. We found that several Mediator subunits contact RSC, which is an abundant and essential chromatin remodeling complex in cells. Importance of this complex is highlighted by the involvment of its human homolog named PBAF in cancer : about 20% of cancers have mutations in at least one subunit of this chromatin remodeler family. In this project, we studied Mediator's mechanisms in relation with RSC in transcription. We characterized a physical and functional interaction between the two complexes, mainly by using yeast as a biological model. This project represents an important issue in gene transcription field et allows us to better understand this fundamental cellular process, involved in initiation and development of several serious human diseases.; Dans les cellules eucaryotes, l'ADN est compacté sous forme de chromatine et tous les processus qui y sont liés y compris la transcription se déroulent dans ce contexte chromatinien. Le Médiateur de la régulation transcriptionnelle est un complexe multiprotéique essentiel et conservé qui intègre les signaux de régulation des facteurs de transcription et les transmet à la machinerie transcriptionnelle. Etant donné son rôle essentiel dans la transcription des gènes, des mutations qui l'affectent sont impliquées dans un large spectre de maladies comme le cancer. Bien que le Médiateur ait fait l'objet de nombreuses études, on ne sait pas comment il coordonne ses fonctions avec les autres corégulateurs transcriptionnels. Nous avons observé que plusieurs de ses sous-unités sont en contact avec le remodeleur de la chromatine RSC, très abondant dans la cellule et essentiel pour sa viabilité. L'importance de ce complexe est soulignée par la grande implication de son homologue humain PBAF dans les pathologies cancéreuses : 20 % des cancers portent une mutation dans au moins une sous-unité de cette famille de remodeleurs. Dans ce projet, nous avons étudié les mécanismes d'action du Médiateur en relation avec RSC dans la transcription. Nous avons caractérisé une interaction physique et fonctionnelle entre les deux complexes, en utilisant principalement la levure comme modèle d'étude. Ce projet représente un enjeu majeur dans le domaine de la transcription génique et nous permet de mieux comprendre ce processus cellulaire fondamental, impliqué dans l'établissement et le maintien de nombreuses maladies grave.

The brown trout form the Salmo trutta complex, a diversified assemblage of salmonids. Its native area mainly covers Europe. It can develop three ecological forms or ecotypes, depending on its migratory behaviours: resident, anadromous (going to sea) and lacustrine (going to lakes). The sea trout is the anadromous ecotype, born up river, living at sea where it reaches salmon size, and returning to the river of its birth for spawning. Like other anadromous fish species, this natural ecotype is protected in France. While its distribution along the Atlantic coasts is known, the sea trout is considered absent in the Mediterranean basin. However, some isolated individuals have been observed in the Rhône River and some other rivers from southern France. In order to understand the genetic position of these large specimens swimming upstream in Mediterranean rivers, and despite the degraded DNA due to bad tissue preservation, eight samples of these trout, mainly caught by anglers, were successfully genotyped at seven microsatellite loci and three sequenced at the mitochondrial Control Region. All specimens tested belong to the Atlantic lineage and are probably stocked domestic trout. This study provides preliminary elements for the conservation status of this ecotype in the Mediterranean basin.

Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types, we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading and migration. RNA-dependent toxicity and phenotypes was also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses revealed extensive expression and accumulation of toxic RNA in astrocytes, which resulted in RNA spliceopathy that was remarkably more severe than in neurons. Astrocyte missplicing affected primarily transcripts that regulate cell adhesion, cytoskeleton and morphogenesis, and it was confirmed in human brain tissue. We demonstrate for the first time that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function.

SLX4 disabled in Fanconi anemia group P is a multifunctional scaffold protein that coordinates the action of structure-specific endonucleases and other DNA repair proteins to ensure genome stability. We show here that SLX4 drives the regulated assembly of an extensive protein network cross-linked by SLX4 dimerization and SUMO-SIM interactions that yield liquid-like nuclear condensates. SLX4 condensates compartmentalize the SUMO system and the STUbL RNF4 to enhance selectively the modification of substrate proteins by SUMO and ubiquitin. Specifically, we find that the assembly of SLX4 condensates induces the processing of topoisomerase 1 - DNA protein crosslinks (TOP1cc), as well as the resection of newly synthesized DNA. This unanticipated function of SLX4 emerges from the collective behavior of proteins that compose SLX4 condensates in live cells. We conclude that SLX4 foci are functional compartments maintained by site-specific protein-protein interactions that control key biochemical reactions in DNA repair.

International audience; Soil, surface water and groundwater in Martinique and Guadeloupe (French West Indies) are contaminated with chlordecone (CLD; C10Cl10O), a very persistent organochlorine pesticide. In soils, its persistence is estimated as several decades in nitisols, centuries in ferralsols and half a millennium in andosols. CLD accumulates in food chains. Long-term chronic exposure to CLD through food and drinking water can have consequences for human health.