Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans.

Epub ahead of printInternational audienceRNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene (1). While the creation of libraries of RNAi clones covering most of the C. elegans genome (2,3) opened the way for true functional genomic studies (see for example (4-7)), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens (8). The approach relies on microscopic imaging and image analysis. Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture. We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing(9). When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months

Contrôle de l'anoïkis par les petites GTPases de la famille Rho

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

C. elegans: model host and tool for antimicrobial drug discovery

For almost four decades, the nematode Caenorhabditis elegans has been of great value in many fields of biological research. It is now used extensively in studies of microbial pathogenesis and innate immunity. The worm lacks an adaptive immune system and relies solely on its innate immune defences to cope with pathogen attack. Infectious microbes, many of which are of clinical interest, trigger specific mechanisms of innate immunity, and provoke the expression of antifungal or antibacterial polypeptides. In this review, we highlight some of these families of antimicrobial peptides (AMPs) and proteins that are candidates for the development of novel antibiotics. In addition, we describe how systems of C. elegans infection provide an increasing number of possibilities for large-scale in vivo screens for the discovery of new antimicrobial drugs. These systems open promising perspectives for innovative human therapies

Le senior connecté

La présente proposition souhaite éclairer les pratiques et les usages des seniors vis-à-vis du numérique, afin de démontrer que, derrière l'appellation « seniors », se cache un ensemble de profils très hétérogènes et que leur relation au numérique mérite un examen plus attentif. This article aims to highlight digital practices and uses, in order to demonstrate that the term " senior " refers to numerous and heterogeneous profiles. We also aim to prouve the eldery's relationship with digital technologies deserve a minutest examination

Gut bacteria-derived peptidoglycan induces a metabolic syndrome-like phenotype via NF-κB-dependent insulin/PI3K signaling reduction in Drosophila renal system

International audienceAlthough microbiome-host interactions are usual at steady state, gut microbiota dysbiosis can unbalance the physiological and behavioral parameters of the host, mostly via yet not understood mechanisms. Using the Drosophila model, we investigated the consequences of a gut chronic dysbiosis on the host physiology. Our results show that adult flies chronically infected with the non-pathogenic Erwinia carotorova caotovora bacteria displayed organ degeneration resembling wasting-like phenotypes reminiscent of Metabolic Syndrome associated pathologies. Genetic manipulations demonstrate that a local reduction of insulin signaling consecutive to a peptidoglycan-dependent NF-κB activation in the excretory system of the flies is responsible for several of the observed phenotypes. this work establishes a functional crosstalk between bacteria-derived peptidoglycan and the immune NF-κB cascade that contributes to the onset of metabolic disorders by reducing insulin signal transduction. Giving the high degree of evolutionary conservation of the mechanisms and pathways involved, this study is likely to provide a helpful model to elucidate the contribution of altered intestinal microbiota in triggering human chronic kidney diseases

Le senior connecté

La présente proposition souhaite éclairer les pratiques et les usages des seniors vis-à-vis du numérique, afin de démontrer que, derrière l'appellation « seniors », se cache un ensemble de profils très hétérogènes et que leur relation au numérique mérite un examen plus attentif. This article aims to highlight digital practices and uses, in order to demonstrate that the term " senior " refers to numerous and heterogeneous profiles. We also aim to prouve the eldery's relationship with digital technologies deserve a minutest examination

The function and expansion of the Patched- and Hedgehog-related homologs in C. elegans

The Hedgehog (Hh) signaling pathway promotes pattern formation and cell proliferation in Drosophila and vertebrates. Hh is a ligand that binds and represses the Patched (Ptc) receptor and thereby releases the latent activity of the multipass membrane protein Smoothened (Smo), which is essential for transducing the Hh signal. In Caenorhabditis elegans, the Hh signaling pathway has undergone considerable divergence. Surprisingly, obvious Smo and Hh homologs are absent whereas PTC, PTC-related (PTR), and a large family of nematode Hh-related (Hh-r) proteins are present. We find that the number of PTC-related and Hh-r proteins has expanded in C. elegans, and that this expansion occurred early in Nematoda. Moreover, the function of these proteins appears to be conserved in Caenorhabditis briggsae. Given our present understanding of the Hh signaling pathway, the absence of Hh and Smo raises many questions about the evolution and the function of the PTC, PTR, and Hh-r proteins in C. elegans. To gain insights into their roles, we performed a global survey of the phenotypes produced by RNA-mediated interference (RNAi). Our study reveals that these genes do not require Smo for activity and that they function in multiple aspects of C. elegans development, including molting, cytokinesis, growth, and pattern formation. Moreover, a subset of the PTC, PTR, and Hh-r proteins have the same RNAi phenotypes, indicating that they have the potential to participate in the same processes

Health Literacy in Complex Digital Information Environments

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