Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis

Aromatic amino and nitro compounds are potent carcinogens found in the environment that exert their toxic effects by reacting with DNA following metabolic activation. One important adduct is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF), which has been extensively used in studies of the mechanisms of DNA repair and mutagenesis. Despite the importance of dG-AAF adducts in DNA, an efficient method for its incorporation into DNA using solid-phase synthesis is still missing. We report the development of a modified 'ultra-mild' DNA synthesis protocol that allows the incorporation of dG-AAF into oligonucleotides of any length accessible by solid-phase DNA synthesis with high efficiency and independent of sequence context. Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF. We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction. Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DN

Ultrastructural and quantitative analysis of the lipid droplet clustering induced by hepatitis C virus core protein.: HCV-induced lipid droplet clustering

International audienceHepatitis C virus (HCV) release is linked to the formation of lipid droplet (LD) clusters in the perinuclear area of infected cells, induced by the core protein. We used electron microscopy (EM) to monitor and compare the number and size of LD in cells producing the mature and immature forms of the HCV core protein, and 3D EM to reconstruct whole cells producing the mature core protein. Only the mature protein coated the LD and induced their clustering and emergence from endoplasmic reticulum membranes enriched in this protein. We found no particular association between LD clusters and the centrosome in reconstructed cells. The LD clustering induced by the mature core protein was associated with an increase in LD synthesis potentially due, at least in part, to the ability of this protein to coat the LD. These observations provide useful information for further studies of the mechanisms involved in HCV-induced steatosis

Templeuve – Rue Grande Campagne

Suite à un projet immobilier déposé par la société Bouygues, une intervention d’archéologie préventive a été prescrite par le service régional de l’archéologie du Nord-Pas-de-Calais à Templeuve-en-Pévèle, rue Grande Campagne. Le site avait fait l’objet d’un diagnostic préalable en 2014, lequel révéla la présence d’un axe routier gallo-romain associé à une tombe à caractère privilégié (Neaud 2014). Durant le mois de mai 2015, une campagne de fouille a été menée sur une emprise de 6 123 m2. À l..

Contribution d’une démarche quantitative à l’analyse des flux médiatiques d’information

Comment appréhender les flux médiatiques d’information qui fabriquent l’actualité ? Cette contribution propose de restituer à l’information une partie de son sens, en la replaçant dans le flux dont elle procède et qu’elle produit. L’attribution standardisée de descripteurs rédactionnels permet de multiplier les « prises » sur des corpus volumineux de sujets : le matériau, devenu descriptible dans le temps, gagne en intelligibilité par le recours à l’inférence statistique. Une saisie macroscopique et diachronique de l’information est rendue possible, qui invite à découvrir les structures, les dynamiques et les événements saillants à partir desquels se fabrique l’actualité médiatique et, au-delà, l’espace public. Cette méthode est présentée par les résultats qu’elle a fournis pour analyser dix ans d’information sportive dans les journaux télévisés hertziens français. Pour autant, l’outil n’explique pas par lui-même, et suppose de multiplier les questions, de recourir à d’autres savoirs pour rendre intelligible ce qu’il donne à lire autrement. Cette forme de travail ouvre ainsi moins des questions que de nouvelles voies pour produire des indicateurs permettant d’y répondre.How to grasp the media streams of information which make the current events ? This contribution suggests restoring to the information a part of its meaning, by replacing it in the stream of which it proceeds and which it produces. The standardized attribution of editorial descriptors allows to multiply the "grips" on voluminous corpuses of subjects : the material, become descriptible in time, wins in comprehensibility by the appeal to statistical inference. A macroscopic and diachronic seizure of the information is made possible, which invites to discover the structures, the dynamics and the striking events from which are made the media current events and, beyond, the public space. This method is presented by the results that it supplied to analyze ten years of sports information in the French Hertzian television news. For all that, the tool does not explain by itself, and supposes to multiply the questions, to resort to other knowledges to make understandable what it gives to read otherwise. This working shape so opens fewer questions than new ways to produce indicators allowing to answer it

Author Correction: Combining CRISPRi and metabolomics for functional annotation of compound libraries

No abstract available

High-throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification

Formalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here, we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh-frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between 1 and 15 years in a biobank and show a high degree of the proteome pattern similarity between two types of histological regions in small FFPE samples, that is, punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of a certain degree of biological variations. Applying the method to two independent DLBCL cohorts, we identified myeloperoxidase, a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered

Genetic effects on molecular network states explain complex traits

The complexity of many cellular and organismal traits results from the integration of genetic and environmental factors via molecular networks. Network structure and effect propagation are best understood at the level of functional modules, but so far, no concept has been established to include the global network state. Here, we show when and how genetic perturbations lead to molecular changes that are confined to small parts of a network versus when they lead to modulation of network states. Integrating multi-omics profiling of genetically heterogeneous budding and fission yeast strains with an array of cellular traits identified a central state transition of the yeast molecular network that is related to PKA and TOR (PT) signaling. Genetic variants affecting this PT state globally shifted the molecular network along a single-dimensional axis, thereby modulating processes including energy and amino acid metabolism, transcription, translation, cell cycle control, and cellular stress response. We propose that genetic effects can propagate through large parts of molecular networks because of the functional requirement to centrally coordinate the activity of fundamental cellular processes

Combining CRISPRi and metabolomics for functional annotation of compound libraries

Molecular profiling of small molecules offers invaluable insights into the function of compounds and allows for hypothesis generation about small-molecule direct targets and secondary effects. However, current profiling methods are limited in either the number of measurable parameters or throughput. Here we developed a multiplexed, unbiased framework that, by linking genetic to drug-induced changes in nearly a thousand metabolites, allows for high-throughput functional annotation of compound libraries in Escherichia coli. First, we generated a reference map of metabolic changes from CRISPR interference (CRISPRi) with 352 genes in all major essential biological processes. Next, on the basis of the comparison of genetic changes with 1,342 drug-induced metabolic changes, we made de novo predictions of compound functionality and revealed antibacterials with unconventional modes of action (MoAs). We show that our framework, combining dynamic gene silencing with metabolomics, can be adapted as a general strategy for comprehensive high-throughput analysis of compound functionality from bacteria to human cell lines

Puf6 primes 60S pre-ribosome nuclear export at low temperature

Productive ribosomal RNA (rRNA) compaction during ribosome assembly necessitates establishing correct tertiary contacts between distant secondary structure elements. Here, we quantify the response of the yeast proteome to low temperature (LT), a condition where aberrant mis-paired RNA folding intermediates accumulate. We show that, at LT, yeast cells globally boost production of their ribosome assembly machinery. We find that the LT-induced assembly factor, Puf6, binds to the nascent catalytic RNA-rich subunit interface within the 60S pre-ribosome, at a site that eventually loads the nuclear export apparatus. Ensemble Förster resonance energy transfer studies show that Puf6 mimics the role of Mg2+ to usher a unique long-range tertiary contact to compact rRNA. At LT, puf6 mutants accumulate 60S pre-ribosomes in the nucleus, thus unveiling Puf6-mediated rRNA compaction as a critical temperature-regulated rescue mechanism that counters rRNA misfolding to prime export competence.ISSN:2041-172