In vitro prediction of the evolution of the GES-1 β-lactamase hydrolytic activity

Resistance to ß-lactams is constantly increasing, due to the emergence of totally new enzymes, but also to the evolution of pre-existing ß-lactamases. GES-1 is a clinically-relevant extended-spectrum β-lactamase (ESBL) hydrolyzing penicillins and broad-spectrum cephalosporins, but sparing monobactams and carbapenems. However, several GES-1 variants (i.e. GES-2 and GES-5) previously identified among clinical isolates display an extended spectrum of activity toward carbapenems. To study the evolution potential of the GES-1 β-lactamase, this enzyme was submitted to in-vitro directed evolution, with selection on increasing concentrations of the cephalosporin cefotaxime, the monobactam aztreonam, or the carbapenem imipenem. The highest resistance levels were conferred by the combination of up to four substitutions. The A6T, E104K, G243A variant selected on cefotaxime, and the A6T, E104K, T237A, G243A variant selected on aztreonam, conferred high resistance to cefotaxime, ceftazidime, and aztreonam. Conversely, the A6T, G170S variant selected on imipenem conferred high resistance to imipenem and cefoxitin. Noteworthy, the A6T substitution involved in higher MICs for all ß-lactams is located in the leader peptide of the GES enzyme, therefore not present in the mature protein. Acquired cross resistance was not observed since selection with CTX or ATM did not select for resistance to IPM and vice versa. Here we demonstrated that β-lactamase GES-1 exhibits peculiar properties with a significant potential to gain activity toward broad-spectrum cephalosporins, monobactams, and carbapenems

Real-time PCR for detection of plasmid-mediated polymyxin resistance (mcr-1) from cultured bacteria and stools

The aim of the study was to develop a simple assay for rapid detection of the mcr-1 gene, recently identified as a source of plasmid-mediated acquired resistance to polymyxins in Enterobacteriaceae.Methods: A SYBR Green-based real-time PCR assay was designed for detection of the mcr-1 gene. This assay was applied to cultured bacteria and to spiked human and cattle stools.Results: The mcr-1 gene could be detected with a lower limit of 102 cultured bacteria. This test was highly sensitive and specific, and generated no false-positive results. The assay was also conclusive when applied to stools spiked with mcr-1-positive Escherichia coli.Conclusions: This simple, rapid, sensitive and specific assay will be useful for rapid screening of this resistance trait in both human medicine and veterinary medicine

Quantification of mRNA stability of stress-responsive yeast genes following conditional excision of open reading frames

Eukaryotic cells rapidly adjust the levels of mRNAs in response to environmental stress primarily by controlling transcription and mRNA turnover. How different stress conditions influence the fate of stress-responsive mRNAs, however, is relatively poorly understood. This is largely due to the fact that mRNA half-life assays are traditionally based on interventions (e.g., temperature-shifts using temperature-sensitive RNA polymerase II alleles or treatment with general transcription inhibitory drugs), which, rather than blocking, specifically induce transcription of stress-responsive genes. To study the half-lives of the latter suite of mRNAs, we developed and describe here a minimally perturbing alternative method, coined CEO, which is based on discontinuance of transcription following the conditional excision of open reading frames. Using CEO, we confirm that the target of rapamycin complex I (TORC1), a nutrient-activated, central stimulator of eukaryotic cell growth, favors the decay of mRNAs that depend on the stress- and/or nutrient-regulated transcription factors Msn2/4 and Gis1 for their transcription. We further demonstrate that TORC1 controls the stability of these mRNAs via the Rim15-Igo1/2-PP2ACdc55 effector branch, which reportedly also controls Gis1 promoter recruitment. These data pinpoint PP2ACdc55 as a central node in homo-directional coordination of transcription and post-transcriptional mRNA stabilization of a specific array of nutrient-regulated genes

Sumoylation of Drosophila SU(VAR)3-7 is required for its heterochromatic function

In Drosophila, SU(VAR)3-7 is an essential heterochromatin component. It is required for proper chromatin condensation, and changing its dose modifies position-effect variegation. Sumoylation is a post-translational modification shown to play a role in diverse biological processes. Here, we demonstrate that sumoylation is essential for proper heterochromatin function in Drosophila through modification of SU(VAR)3-7. Indeed, SU(VAR)3-7 is sumoylated at lysine K839; this modification is required for localization of SU(VAR)3-7 at pericentric heterochromatin, chromosome 4, and telomeres. In addition, sumoylation of SU(VAR)3-7 is a prerequisite for its ability to enhance position-effect variegation. Thus, these results show that the heterochromatic function of SU(VAR)3-7 depends on its own sumoylation, and unveil a role for sumoylation in Drosophila heterochromati

Les fonctions des principes fondamentaux de la commande publique

The principles of the Public Commission suffer a legal indistinctness, inasmuch as, aspiring to common purposes by means of identical legal instruments, none of the general objectives that they pursue together cannot be specifically connected to one of them. To mitigate this insufficiency of the substantive law, a new key to the interpretation of principles must be discovered. A detailed analysis of the substantive law reveals that the time constitutes the pivot of a certain legibility of the fonctions of the principles. Structuring Public Commissions Law, this element allows to identify different time pursuing specific objectives, of which the concordance with the principles allows to delimit the characteristic purposes of each.From this temporal application of the principles, it is possible to define their own fonctions, in this sense that each of the rules specific of Public Commissions Law can be connected to one of them. A confrontation of this temporal conceptualisation reveals that of this definition of appropriate functions, ensue positives implications of the Public Commission Law. On one hand, some latent grey areas of this branch of the law are clarified. On the other hand, the office of the judge of pre-contractual and contractual disputes is specified. A singular reading of the fonctions of the principles allows thereby to mitigate partially to the obvious complexity of the Public Commission Law.Les principes de la commande publique souffrent d’une imprécision juridique dans la mesure où, aspirant à des finalités communes au moyen d’instruments juridiques identiques, aucun des objectifs généraux qu’ils poursuivent ensemble ne peut être spécifiquement rattaché à l’un d’eux. Afin de pallier cette insuffisance du droit positif, une nouvelle clé de lecture des principes doit être découverte. Une analyse détaillée du droit positif dévoile que le temps constitue le pivot d’une lisibilité certaine des fonctions des principes. Structurant le droit de la commande publique, cet élément permet d’identifier différents temps poursuivant des objectifs spécifiques, dont la concordance avec les principes permet de délimiter les finalités caractéristiques à chacun. À partir de cette application temporelle des principes, il est possible de définir leurs fonctions propres, en ce sens que chacune des règles spécifiques du droit de la commande publique peut être rattachée àl’un d’entre eux. Une confrontation de cette conceptualisation temporelle au droit positif dévoile que de cette définition des fonctions propres, découlent des implications positives sur le droit de la commande publique. D’une part, certaines zones d’ombres latentes de cette branche du droit sont clarifiées. D’autre part, l’office du juge des contentieux précontractuel et contractuel est précisé. Une lecture singulière des fonctions des principes permet ainsi de pallier en partie la complexité patente du droit de la commande publique

Le monde rural : un concept en évolution

Les tendances générales d’évolution dans les pays de l’OCDE bouleversent les représentations traditionnelles qui associaient, du point de vue économique, social et démographique, le milieu rural au secteur agricole. Dans des situations nationales certes fort diversifiées, ces transformations récentes doivent être prises en compte dans la problématique de la politique scolaire

Precipitation forecasting through an analog sorting technique: a comparative study

This study aims at comparing two quantitative precipitation forecasting techniques based on the meteorological analogy concept. Method A considers first a selection of analogous situations at synoptic scale. Second a subset of the most similar situations in terms of hygrometry is extracted. Method B extends method A by two innovative ways, which are restricting the search for analogues with temperature information instead of the common season criterion, and exploiting the information about vertical motion considering vertical velocity. Forecasts are evaluated in a perfect prognosis context and in operational conditions as well, by mean of verification measures (Continuous Ranked Probability Skill Score and scores computed from contingency tables). Results of the case study in France show that: (1) there is an increase in forecast skill when temperature and vertical velocity are included in the procedure, (2) it is possible to anticipate rainfall events up to one week ahead and (3) the introduction of new variables such as vertical velocity may be useless beyond few days ahead if the forecast of the weather model is not reliable

CIITA-induced occupation of MHC class II promoters is independent of the cooperative stabilization of the promoter-bound multi-protein complexes

Precise regulation of MHC class II expression plays a crucial role in the control of the immune response. The transactivator CIITA behaves as a master controller of constitutive and inducible MHC class II gene activation, but its exact mechanism of action is not known. Activation of MHC class II promoters requires binding of at least three distinct multi-protein complexes (RFX, X2BP and NF-Y). It is known that the stability of this binding results from cooperative interactions between these proteins. We show here that expression of CIITA in MHC class II- cells triggers occupation of the promoters by these complexes. This observation raised the possibility that the effect of CIITA on promoter occupation is mediated by an effect on the cooperative stabilization of the DNA-bound multi-protein complexes. We show, however, that the presence of CIITA does not affect the stability of the higher-order protein complex formed on DNA by RFX, X2BP and NF-Y. This suggests other mechanisms for CIITA-induced promoter occupancy, such as an effect on chromatin structure leading to increased accessibility of MHC class II promoters. This ability of CIITA to facilitate promoter occupation is undissociable from its transactivation potential. Finally, we conclude that this effect of CIITA is cell-type specific, since expression of CIITA is not required for normal occupation of MHC class II promoters in B lymphocyte

La prévision des précipitations par recherche d'analogues : état de l'art et perspectives

La prévision des précipitations par analogie, adaptée des sorties de modèles numériques de prévision, s'est améliorée ces dix dernières années et est actuellement implémentée opérationnellement dans diverses infrastructures françaises. Le premier objectif de cette étude est de dresser l'état de l'art de cette approche. L'application de cette méthode nécessite une base de données contenant les champs des variables qui permettent de caractériser les situations météorologiques passées. Etant donné qu'il existe deux archives de génération différente (réanalyses ERA-40 et NCEP/NCAR), la sensibilité de cette méthode de prévision au choix de l'archive a été étudiée. Les résultats révèlent une faible sensibilité, même si de manière générale les performances sont légèrement supérieures avec l'archive ERA-40, notamment pour les événements pluvieux extrêmes. Enfin, des perspectives d'amélioration de la méthode susceptibles d'être exploitées à court terme sont évoquées. / Precipitation forecasting based on an adaptation of model outputs through an analog sorting technique has been improved for around ten years. The method runs operationally in several French institutions. First, this short paper presents the state of the art of this approach and the more recent developments. Second, a sensitivity analysis to the choice of the database from which the variables that characterise the past meteorological situations are extracted is performed. Two available archives are tested (ERA-40 and NCEP/NCAR re-analyses). The results show that despite the performances obtained with the ERA-40 database are slightly better, especially for heavy rainfall events, the sensitivity is weak. Finally, further ways for improvement that could be investigated are suggested