In silico identification, synthesis and biological evaluation of novel tetrazole inhibitors of MurB

In the context of antibacterial drug discovery resurgence, novel therapeutic targets and new compounds with alternative mechanisms of action are of paramount importance. We focused on UDP-N- acetylenolpyruvylglucosamine reductase (i.e. MurB), an underexploited target enzyme that is involved in early steps of bacterial peptidoglycan biosynthesis. On the basis of the recently reported crystal structure of MurB in complex with NADP+ , a pharmacopohore model was generated and used in a virtual screening campaign with combined structure-based and ligand-based approaches. In order to explore chemical space around hit compounds, further similarity search and organic synthesis was employed to obtain several compounds with micromolar IC50 values on MurB. The best inhibitors in the reported series of 5-substituted tetrazol-2-yl acetamides were compounds 13, 26 and 30 with IC50 values of 34, 28 and 25 µM, respectively. None of the reported compounds possessed in vitro antimicrobial activity against S. aureus and E. coli

EP-1502: High resolution portal image prediction for radiotherapy treatment verification & in vivo dosimetry

International audiencePurpose/Objective: Historically designed as a control system for patient positioning for radiotherapy treatment, Electronic Portal Imaging Devices (EPIDs) are nowadays widely used for quality assurance and dosimetric verifications in new irradiation techniques. One of the main advantages of the EPID is its high resolution which can detect small details. The objective of this study is to compare the EPID image acquired during the treatment with a predicted high resolution portal image computed by Monte Carlo (MC) simulation. A new method for prediction of high resolution EPID images is tested for in vivo treatment verification. Materials and Methods: Experiments were carried out on a Siemens ARTISTETM, equipped with a 160-MLCTM, and its Siemens OptivueTM 1000 EPID. This EPID has an active detection area of 41 x 41 cm2 and a matrix of 1024 x 1024 pixels. A model of this linac and the EPID was developed with the MC code Penelope, and commissioned. We focus on a breast treatment conformational beam (6 MV) on the CIRS adult female phantom. The CT-scan of the phantom was used as input, and Hounsfield numbers were converted in density and atomic composition, so as to obtain a voxelized geometry used in the Penelope code. Particles exiting the phantom and impinging on the EPID are simulated up to the EPID in order to compute the predicted portal image by scoring the energy deposited in the phosphor layer on a 1024 x 1024 virtual grid. The simulated image was then smoothed using a denoising algorithm in order to keep the high resolution advantage. Several denoising algorithms were tested, among them IRON, LASG and a recently developed one called DPGLM. For now, we use the gamma-index technique to evaluate the accuracy of the simulated image against the experimental one. Results: Figure 1 shows the acquired image and the simulated one. The gamma-index is satisfied for 94.4 % of the pixels for 3.5 % and 3.5 mm criterion. The DPGLM gives the best result toward accuracy and computed time. Indeed, the denoising of 1024 x 1024 images takes about 1h30 mn, 2h and 5 mn using DPGLM, IRON, and LASG, respectively. The LASG algorithm is really fast but the result is too smoothed for the high resolution purpose. Conclusions: This work is the first step in the aim of in vivo dosimetry by comparing experimental portal images with high resolution predicted images obtained using MC simulations in a voxelized geometry. First results obtained on a breast treatment are encouraging, and we can expect to detect treatment errors

The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis

Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE-MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE-MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation9FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP11/52067-6; 2017/12436-9; 2013/02451-0FRISBI [ANR-10-INSB-05-02]; GRAL within the Grenoble Partnership for Structural Biology (PSB) [ANR-10-LABX-49-01]; Rhone-Alpes RegionRegion Auvergne-Rhone-Alpes; Fondation pour la Recherche Medicale (FRM)Fondation pour la Recherche Medicale; fonds FEDER; Centre National de la Recherche Scientifique (CNRS)Centre National de la Recherche Scientifique (CNRS); Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA)French Atomic Energy Commission; University of Grenoble Alpes; EMBL; GIS-Infrastructures en Biologie Sante et Agronomie (IBISA); Laboratoire International Associe BACWALL (CNRS); FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [11/52067-6, 2017/12436-9]; Agence Nationale de la RechercheFrench National Research Agency (ANR) [ANR-13-BSV8-0015-01]; ANRFrench National Research Agency (ANR); Fondation pour la Recherche Medicale (FRM)Fondation pour la Recherche Medicale [FDT20160435484]; FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2013/02451-0

Le travail de groupe en physique-chimie en classe de 5e

In physical chemistry, for making work students, group work is a very used teaching tool. But all teachers ask themselves the question: how do we ensure that group work be efficient and skill’s builder and no an entertainment time? Thanks to a session analysis in the form of investigation approach, this reflexive writing, showed that it existed essential rules to respect in work group. Firstly, in order to be most involved, each student should have a specific job in the group. Next, during this work group, two things should be assess, the final production as well as the involvement in the work group. We also be able to see that the final pooling should be made by students in order to better build and retain knowledge. So, the essential teacher task is to manage the operating of different groups.En physique chimie, le travail de groupe est un outil pédagogique très utilisé pour faire travailler les élèves. Mais la question qui se pose à tous les professeurs est : comment faire pour que le travail de groupe soit efficace et constructeur de compétences et non un moment de distraction ? Grâce à l’analyse d’une séance sous forme de démarche d’investigation, cet écrit réflexif, a montré qu’il existait des règles indispensables à respecter dans le travail de groupe. Tout d’abord, pour être plus impliqué, chaque élève doit avoir un rôle particulier dans le groupe. Ensuite, lors de ce travail de groupe, deux choses doivent être évaluées, la production finale ainsi que l’implication dans le travail de groupe. Nous avons également pu voir que la mise en commun finale devait être faite par les élèves afin de mieux construire et retenir les savoirs. La tâche essentielle du professeur est donc de gérer le fonctionnement des différents groupes

The phonology / morphology Interface

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The Biology of Colicin M and Its Orthologs

The misuse of antibiotics during the last decades led to the emergence of multidrug resistant pathogenic bacteria. This phenomenon constitutes a major public health issue. Consequently, the discovery of new antibacterials in the short term is crucial. Colicins, due to their antibacterial properties, thus constitute good candidates. These toxin proteins, produced by E. coli to kill enteric relative competitors, exhibit cytotoxicity through ionophoric activity or essential macromolecule degradation. Among the 25 colicin types known to date, colicin M (ColM) is the only one colicin interfering with peptidoglycan biosynthesis. Accordingly, ColM develops its lethal activity in E. coli periplasm by hydrolyzing the last peptidoglycan precursor, lipid II, into two dead-end products, thereby leading to cell lysis. Since the discovery of its unusual mode of action, several ColM orthologs have also been identified based on sequence alignments; all of the characterized ColM-like proteins display the same enzymatic activity of lipid II degradation and narrow antibacterial spectra. This publication aims at being an exhaustive review of the current knowledge on this new family of antibacterial enzymes as well as on their potential use as food preservatives or therapeutic agents

Introduction

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Introduction

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Purification and biochemical characterisation of GlmU from Yersinia pestis.

International audienceAntibiotic resistance has emerged as a real threat to mankind, rendering many compounds ineffective in the fight against bacterial infection, including for significant diseases such as plague caused by Yersinia pestis. Essential genes have been identified as promising targets for inhibiting with new classes of compounds. Previously, the gene encoding the bifunctional UDP-N-acetylglucosamine pyrophosphorylase/glucosamine-1-phosphate N-acetyltransferase enzyme GlmU was confirmed as an essential gene in Yersinia. As a step towards exploiting this target for antimicrobial screening, we undertook a biochemical characterisation of the Yersinia GlmU. Effects of pH and magnesium concentration on the acetyltransferase and uridyltransferase activities were analysed, and kinetic parameters were determined. The acetyltransferase activity, which is strongly increased in the presence of reducing agent, was shown to be susceptible to oxidation and thiol-specific reagents

A WASp–VASP complex regulates actin polymerization at the plasma membrane

Proteins of the Wiskott–Aldrich syndrome and Ena/VASP families both play essential functions in the regulation of actin dynamics at the cell leading edge. However, possibilities of functional interplay between members of these two families have not been addressed. Here we show that, in hemopoietic cells, recruitment of the C-terminal VCA (Verprolin homology, Cofilin homology, Acidic) domain of WASp at the plasma membrane by a ligand technique using rapamycin as an intermediate is not sufficient to elicit efficient Arp2/3 complex-mediated actin polymerization. Other domains of WASp, in particular the proline-rich domain, are required for the formation of actin-rich structures. An in vitro analysis demonstrates that the proline-rich domain of WASp binds VASP with an affinity of ∼10(6) M(–1). In addition, WASp and VASP both accumulate in actin-rich phagocytic cups. Finally, in a reconstituted motility medium, VASP enhances actin-based propulsion of WASp-coated beads in a fashion reminiscent of its effect on Listeria movement. We propose that VASP and WASp cooperation is essential in stimulating actin assembly and membrane protrusion at the leading edge