A new type of IRES within gag coding region recruits three initiation complexes on HIV-2 genomic RNA

Genomic RNA of primate lentiviruses serves both as an mRNA that encodes Gag and Gag-Pol polyproteins and as a propagated genome. Translation of this RNA is initiated by standard cap dependant mechanism or by internal entry of the ribosome. Two regions of the genomic RNA are able to attract initiation complexes, the 5′ untranslated region and the gag coding region itself. Relying on probing data and a phylogenetic study, we have modelled the secondary structure of HIV-1, HIV-2 and SIVMac coding region. This approach brings to light conserved secondary-structure elements that were shown by mutations to be required for internal entry of the ribosome. No structural homologies with other described viral or cellular IRES can be identified and lentiviral IRESes show many peculiar properties. Most notably, the IRES present in HIV-2 gag coding region is endowed with the unique ability to recruit up to three initiation complexes on a single RNA molecule. The structural and functional properties of gag coding sequence define a new type of IRES. Although its precise role is unknown, the conservation of the IRES among fast evolving lentiviruses suggests an important physiological role

CRISPR Typing and Subtyping for Improved Laboratory Surveillance of Salmonella Infections

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool

Genomic history of the seventh pandemic of cholera in Africa.

The seventh cholera pandemic has heavily affected Africa, although the origin and continental spread of the disease remain undefined. We used genomic data from 1070 Vibrio cholerae O1 isolates, across 45 African countries and over a 49-year period, to show that past epidemics were attributable to a single expanded lineage. This lineage was introduced at least 11 times since 1970, into two main regions, West Africa and East/Southern Africa, causing epidemics that lasted up to 28 years. The last five introductions into Africa, all from Asia, involved multidrug-resistant sublineages that replaced antibiotic-susceptible sublineages after 2000. This phylogenetic framework describes the periodicity of lineage introduction and the stable routes of cholera spread, which should inform the rational design of control measures for cholera in Africa

NADPH oxidase 4 inhibition is a complementary therapeutic strategy for spinal muscular atrophy

IntroductionSpinal muscular atrophy (SMA) is a fatal neurodegenerative disorder, characterized by motor neuron (MN) degeneration and severe muscular atrophy and caused by Survival of Motor Neuron (SMN) depletion. Therapies aimed at increasing SMN in patients have proven their efficiency in alleviating SMA symptoms but not for all patients. Thus, combinational therapies are warranted. Here, we investigated the involvement of NADPH oxidase 4 (NOX4) in SMA-induced spinal MN death and if the modulation of Nox4 activity could be beneficial for SMA patients.MethodsWe analysed in the spinal cord of severe type SMA-like mice before and at the disease onset, the level of oxidative stress and Nox4 expression. Then, we tested the effect of Nox4 inhibition by GKT137831/Setanaxib, a drug presently in clinical development, by intrathecal injection on MN survival and motor behaviour. Finally, we tested if GKT137831/Setanaxib could act synergistically with FDA-validated SMN-upregulating treatment (nusinersen).ResultsWe show that NOX4 is overexpressed in SMA and its inhibition by GKT137831/Setanaxib protected spinal MN from SMA-induced degeneration. These improvements were associated with a significant increase in lifespan and motor behaviour of the mice. At the molecular level, GKT137831 activated the pro-survival AKT/CREB signaling pathway, leading to an increase in SMN expression in SMA MNs. Most importantly, we found that the per os administration of GKT137831 acted synergistically with a FDA-validated SMN-upregulating treatment.ConclusionThe pharmacological inhibition of NOX4 by GKT137831/Setanaxib is neuroprotector and could represent a complementary therapeutic strategy to fight against SMA

Genomic insights into the 2016-2017 cholera epidemic in Yemen.

Yemen is currently experiencing, to our knowledge, the largest cholera epidemic in recent history. The first cases were declared in September 2016, and over 1.1 million cases and 2,300 deaths have since been reported1. Here we investigate the phylogenetic relationships, pathogenesis and determinants of antimicrobial resistance by sequencing the genomes of Vibrio cholerae isolates from the epidemic in Yemen and recent isolates from neighbouring regions. These 116 genomic sequences were placed within the phylogenetic context of a global collection of 1,087 isolates of the seventh pandemic V. cholerae serogroups O1 and O139 biotype El Tor2-4. We show that the isolates from Yemen that were collected during the two epidemiological waves of the epidemic1-the first between 28 September 2016 and 23 April 2017 (25,839 suspected cases) and the second beginning on 24 April 2017 (more than 1 million suspected cases)-are V. cholerae serotype Ogawa isolates from a single sublineage of the seventh pandemic V. cholerae O1 El Tor (7PET) lineage. Using genomic approaches, we link the epidemic in Yemen to global radiations of pandemic V. cholerae and show that this sublineage originated from South Asia and that it caused outbreaks in East Africa before appearing in Yemen. Furthermore, we show that the isolates from Yemen are susceptible to several antibiotics that are commonly used to treat cholera and to polymyxin B, resistance to which is used as a marker of the El Tor biotype

Etude de la fonction d'ARN structurés (recherche d'un ribozyme de fonction inédite et initiation de la traduction de l'ARN génomique d'HIV-2)

Dans le but d'étudier le rôle et l'évolution des enzymes ARN, nous cherchons à substituer une enzyme protéique, la S-Adénosyl-Méthionine synthétase par un ARN catalytique. L'originalité de cette démarche impose que l'enzyme agisse en trans par rapport à ces deux substrats, or les techniques de sélection in vitro classiques permettent d'isoler des catalyseurs agissant exclusivement en cis. Les premières étapes de ce projet ont été réalisées : la sélection et la caractérisation de motifs ARN liant l'ATP, l'introduction de ces motifs dans une banque combinatoire pour la sélection du ribozyme et la fonctionalisation de l'extrémité d'un ARN pour donner une indépendance structurale à l'ATP en le liant à la librairie au moyen d'un linker flexible.La traduction d'un messager eucaryote est initiée par la liaison d'un complexe ribosomal 43S sur la coiffe située à l'extrémité 5' de l'ARN. Ce complexe migre ensuite jusqu'au codon initiateur. Alternativement, la 5'UTR de certains ARN attire la sous unité 40S du ribosome grâce à un site d'entrée interne des ribosomes (IRES). L'ARN génomique d'HIV2 code la polyprotéine Gag. En collaboration avec l'équipe de T.Ohlmann (U412 INSERM), nous avons découvert que trois isoformes de Gag sont produites par entrée interne des ribosomes. De manière étonnante, L'IRES est situé dans la phase codante, c'est à dire en aval du premier codon d'initiation. Plus surprenant encore, il est possible de traduire un gène gag débutant directement par l'AUG initiateur alors que la traduction d'un gène classique nécessite au moins 8 nucléotides en amont du codon d'initiation. L'ensemble de ces résultats peuvent être étendu à deux autres lentivirus, SIV et HIV1. Ces propriétés reposent sur la structure adoptée par les 450 premiers nucléotides de la phase codante. Nous avons étudié le mécanisme moléculaire de l'entrée interne des ribosomes sur ces ARN par une combinaison d'approches; mutagenèse dirigée, essais in vivo, et analyse des complexes ribosomaux.The purpose of my first project was to evaluate the role of active RNA during evolution and in modern genomes and the plausibility to have implication for the use of RNA as therapeutic agents. In order to substitute in vivo metabolic enzymes by a catalytic RNA, we first need to isolate a ribozyme by in vitro selection. I have resolved several technical difficulties and provided the laboratory with all the tools necessary to start the real process. The second project was to study the translation initiation of HIV2 genomic RNA. We showed that the HIV2 genomic RNA encodes to the full length Gag p57 and two, yet undescribed, shorter isoforms. The synthesis of these proteins is driven by internal ribosome entry site entirely located in the coding region. Gag p57 is produced by a novel mechanism of the ribosome recruitment requires structural element located downstream the start codon (Herbreteau et al. 2005).ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Selection and evolution of NTP-specific aptamers

ATP occupies a central position in biology, for it is both an elementary building block of RNA and the most widely used cofactor in all living organisms. For this reason, it has been a recurrent target for in vitro molecular evolution techniques. The exploration of ATP-binding motifs constitutes both an important step in investigating the plausibility of the ‘RNA world’ hypothesis and a central starting point for the development of new enzymes. To date, only two RNA motifs that bind ATP have been characterized. The first one is targeted to the adenosine moiety, while the second one recognizes the ‘Hoogsteen’ face of the base. To isolate aptamers that bind ATP in different orientations, we selected RNAs on an affinity resin that presents ATP in three different orientations. We obtained five new motifs that were characterized and subsequently submitted to a secondary selection protocol designed to isolate aptamers specific for cordycepin. Interestingly, all the ATP-binding motifs selected specifically recognize the sugar-phosphate backbone region of the nucleotides. Three of the aptamers show some selectivity for adenine derivatives, while the remainder recognize any of the four nucleotides with similar efficiency. The characteristics of these aptamers are discussed along with implications for in vitro molecular evolution

Apport de la génomique microbienne à l’épidémiologie du choléra

International audienceIn 2022, the burden of cholera—an acute watery diarrheal disease caused by Vibrio cholerae serogroup O1 (or more rarely O139) bacteria, which produce cholera toxin—remains high in many African and Asian countries. In the last few years, microbial genomics has made it possible to define the bacterial populations responsible for cholera more precisely. It has been shown that the current, seventh pandemic is due to a single lineage with a reservoir in the countries of the Bay of Bengal (India and Bangladesh). There have been several transmissions of the causal agent of cholera from this region to Africa, Asia and Latin America, suggesting a human-to-human transmission of the disease. Microbial genetics can help to fight this scourge by providing insight into cholera epidemiology and through its use in disease monitoring, thereby contributing to the achievement of the World Health Organization’s goal of reducing cholera deaths by 90% by 2030.En 2022, de nombreux pays d’Afrique et d’Asie restent des foyers épidémiques de choléra, maladie diarrhéique causée par la bactérie Vibrio cholerae de sérogroupe O1 (ou plus rarement O139) produisant la toxine cholérique. La génomique microbienne a permis ces dernières années de mieux définir les populations bactériennes responsables du choléra. Il a ainsi été montré qu’il n’existait qu’une seule lignée génétique de Vibrio cholerae O1 responsable de la septième pandémie dont le réservoir se situe dans la région du golfe du Bengale (Inde et Bangladesh). Plusieurs évènements de transmission de l’agent du choléra vers l’Afrique, l’Europe ou l’Amérique latine ont été identifiés et suggèrent une transmission interhumaine de la maladie. Les données issues des travaux de génomique microbienne ainsi que son utilisation pour la surveillance globale du choléra vont permettre de mieux lutter contre ce fléau et participer à l’objectif de l’Organisation mondiale de la Santé de réduire de 90 % les décès dus à cette maladie en 2030

Analyse de sensibilité globale (Sobol) d'un modèle couplé Surface/Subsurface de flux d'eau et de transport de solutés réactifs sur un versant réel

International audienceThe migration and fate of pesticides in natural environments is highly complex. At the hillslope scale, the quantification of contaminant fluxes and concentrations requires a physically based model. This class of model has recently been extended to include coupling between the surface and the subsurface domains for both the water flow and solute transport regimes. Due to their novelty, the relative importance of and interactions between the main model parameters has not yet been fully investigated. In this study, a global Sobol sensitivity analysis is performed on a vineyard hillslope for a one hour intensive rain event with the CATHY (CATchment HYdrology) integrated surface/subsurface model. The event-based simulation involves runoff generation, infiltration, surface and subsurface solute transfers, and shallow groundwater flow. The results highlight the importance of the saturated hydraulic conductivity Ks and the retention curve shape parameter n and they reveal a strong role for parameter interactions associated with the exchange processes represented in the model. The mass conservation errors generated by the model are lower than 1% in 99.7% of the simulations. Boostrapping analysis of sampling methods and errors associated with the Sobol indices highlights the relevance of choosing a large sampling size (at least N = 1000) and raises issues associated with rare but extreme output results