Erythromycin degradation by an esterase in enzymatic membrane reactors

1 Introduction Pharmaceuticals products (PPs) and endocrine disrupting chemicals (EDCs) as well as their transformation products have been detected in almost all effluents from sewage facilities, in surface water, in groundwater, adsorbed on sediments and even in drinking water [1,2]. Ecotoxicity studies have demonstrated that pharmaceutical pollutants could affect the growth, reproduction and behavior of birds, fishes, invertebrates, plants and bacteria [3,4]. Some recently published studies report that the presence of low concentrations of antibiotics in the wastewaters may develop antibiotic resistance in the whole environment [5, 6]. As previously reported by Demarche et al. [7], the use of enzymes might be beneficial to enhance or complement conventional wastewater treatments. As far as enzymes are relatively expensive the reuse of the biocatalyst appears to be essential to ensure the economic and industrial viability of the process. Enzymatic membrane reactors appear to be an interesting alternative since they enable to couple reaction and separation [8]. In fact, in such enzymatic reactors, the substrate is continuously brought in contact with the biocatalyst, which is retained by the membrane, either freely circulating with the retentate or fixed on or within the membrane and the reaction products are recovered in the permeate. This work describes the study of erythromycin degradation by an EreB esterase in free and immobilized forms. It focuses on the comparison between 3 different enzymatic membrane reactors for erythromycin degradation by esterase EreB. In the first configuration the free biocatalyst was confined in the reaction media by a ceramic membrane. In the two other cases, the enzyme was immobilized in the membrane either covalently grafted or adsorbed. Please click Additional Files below to see the full abstract

Microbiota-based markers predictive of development of Clostridioides difficile infection

Antibiotic-induced modulation of the intestinal microbiota can lead to Clostridioides difficile infection (CDI), which is associated with considerable morbidity, mortality, and healthcare-costs globally. Therefore, identification of markers predictive of CDI could substantially contribute to guiding therapy and decreasing the infection burden. Here, we analyze the intestinal microbiota of hospitalized patients at increased CDI risk in a prospective, 90-day cohort-study before and after antibiotic treatment and at diarrhea onset. We show that patients developing CDI already exhibit significantly lower diversity before antibiotic treatment and a distinct microbiota enriched in Enterococcus and depleted of Ruminococcus, Blautia, Prevotella and Bifidobacterium compared to non-CDI patients. We find that antibiotic treatment-induced dysbiosis is class-specific with beta-lactams further increasing enterococcal abundance. Our findings, validated in an independent prospective patient cohort developing CDI, can be exploited to enrich for high-risk patients in prospective clinical trials, and to develop predictive microbiota-based diagnostics for management of patients at risk for CDI

Incidence and predictive biomarkers of Clostridioides difficile infection in hospitalized patients receiving broad-spectrum antibiotics

Trial enrichment using gut microbiota derived biomarkers by high-risk individuals can improve the feasibility of randomized controlled trials for prevention of Clostridioides difficile infection (CDI). Here, we report in a prospective observational cohort study the incidence of CDI and assess potential clinical characteristics and biomarkers to predict CDI in 1,007 patients ≥ 50 years receiving newly initiated antibiotic treatment with penicillins plus a beta- lactamase inhibitor, 3rd/4th generation cephalosporins, carbapenems, fluoroquinolones or clindamycin from 34 European hospitals. The estimated 90-day cumulative incidences of a first CDI episode is 1.9% (95% CI 1.1-3.0). Carbapenem treatment (Hazard Ratio (95% CI): 5.3 (1.7-16.6)), toxigenic C. difficile rectal carriage (10.3 (3.2-33.1)), high intestinal abundance of Enterococcus spp. relative to Ruminococcus spp. (5.4 (2.1-18.7)), and low Shannon alpha diversity index as determined by 16 S rRNA gene profiling (9.7 (3.2-29.7)), but not nor- malized urinary 3-indoxyl sulfate levels, predicts an increased CDI risk

EFFECTEURS ET REGULATEURS DES GTPASES RAS ET RAP2 (ETUDE DES RALGEFS ET DE LA PDE)

LES PROTEINES RAP2 PRESENTENT 50% D'IDENTITE AVEC LES PROTEINES RAS ET COMPORTENT UN DOMAINE EFFECTEUR CONSERVE. A CE JOUR, LA FONCTION CELLULAIRE DE CES PROTEINES DEMEURE INCONNUE. AFIN D'IDENTIFIER CETTE FONCTION, UN CRIBLE DOUBLE HYBRIDE A ETE REALISE A PARTIR D'UNE BANQUE D'ADNC DE CELLULES DE LA LIGNEE DE LYMPHOME T JURKAT, AVEC LA PROTEINE RAP2B SAUVAGE ET ENTIERE COMME APPAT. DEUX PARTENAIRES CELLULAIRES POTENTIELS ONT FAIT L'OBJET D'UNE ETUDE PLUS APPROFONDIE. TOUT D'ABORD, LES RALGEFS QUI SONT DES FACTEURS D'ECHANGE DE LA GTPASE RAL ET DES EFFECTEURS DE LA GTPASE RAS. NOUS AVONS MONTRE QUE LES RALGEFS SONT DES EFFECTEURS POTENTIELS DE LA GTPASE RAP2. LA FORMATION DES COMPLEXES RAP2/RALGEFS AU NIVEAU DU RETICULUM ENDOPLASMIQUE N'ABOUTIT PAS A L'ACTIVATION DE LA GTPASE RAL, NI N'INTERFERE AVEC LA CASCADE ACTIVATRICE RAS/RAL. PAR CONSEQUENT, CES DONNEES SUGGERENT L'EXISTENCE D'UNE NOUVELLE VOIE DE SIGNALISATION AU NIVEAU DU RETICULUM ENDOPLASMIQUE, IMPLIQUANT LES PROTEINES RAP2 ET RALGEFS. UNE PROTEINE DE 220 KDA INTERGISSANT IN VITRO AVEC LA REGION N-TERMINALE DE LA PROTEINE RALGDS POURRAIT INTERVENIR DANS CETTE VOIE. ENSUITE, LA SOUS-UNITE DE LA PHOSPHODIESTERASE A GMPC DES CELLULES EN BATONNETS DE LA RETINE (PDE), UNE ENZYME IMPLIQUEE DANS LA PHOTOTRANSDUCTION. NOUS AVONS MONTRE QUE LA PDE INTERAGIT DANS LE SYSTEME DOUBLE HYBRIDE AVEC DIVERSES GTPASES DOTEES D'UN MOTIF CAAX, SIEGE DE LEURS MODIFICATIONS POST-TRADUCTIONNELLES. ALORS QU'IL A ETE DEMONTRE QUE LA PDE EXERCE UNE ACTIVITE GDI SUR LES SOUS-UNITES ET DE LA PHOSPHODIESTERASE ET SUR LA GTPASE RABL3, NOUS AVONS DEMONTRE QUE CE N'EST PAS LE CAS POUR LES GTPASES RAS ET RAP2. NOS DONNEES CONVERGENT VERS L'IMPLICATION DE LA PDE DANS LE TRAFIC DES GTPASES AU COURS DE LEUR MATURATION, LORSQUE LA FORME ISOPRENYLEE DES GTPASES VA S'ANCRER AU RETICULUM ENDOPLASMIQUE. ENFIN, D'AUTRES DONNES INDIQUENT QUE LA PDE POURRAIT ETRE IMPLIQUEE DANS LE TRANSPORT VESICULAIRE INTRACELLULAIRE.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Etude de la protéine Gem, un membre de la superfamille Ras

Gem est une nouvelle protéine de la superfamille Ras (famille RGK) dont l'expression est induite dans différents types cellulaires après stimulation par des agents mitogènes. Gem possède des extensions N-terminale et C-terminale par rapport à Ras de fonctions inconnues, ainsi que des substitutions d'acides aminés dans des positions clés pour la liaison et hydrolyse du GTP. Afin de rechercher les partenaires cellulaires de Gem, est plus particulièrement ceux de cette région N-terminale, nous avons mis au point un système double hybride modifié où la protéine appât est fusionnée en position N-terminale par rapport au domaine de liaison à l'ADN de la protéine bactérienne LexA, c'est-à-dire, de polarité inversée à celle des systèmes double-hybride classiques. Nous l'avons validé en montrant qu'il conservait la spécificité d'interaction entre les GTPases Ras et Ral et ses régulateurs et effecteurs, et possédait même dans certains cas une sensibilité de détection accrue. Nous avons construit une banque d'ADNc à partir de cellules de la lignée de lymphome T humain Jurkat, que nous avons criblée en utilisant comme appât : (i) les 82 acides aminés N-terminaux de Gem fusionnés à l'extrémité N-terminale du domaine de liaison à l'ADN (DBD) de LexA ; (ii) la protéine Gem entière fusionnée à l'extrémité C-terminale du DBD de GAL4. Parmi les 20 clones positifs obtenus contenant des ADNc distincts, nous avons décidé d'en étudier deux. Ces ADNc codent pour une protéine homologue à la protéine 4.1N, et une nouvelle protéine avec un domaine RhoGAP. Nous avons mis en évidence que la nouvelle protéine à domaine RhoGAP est bien capable d'augmenter l'activité GTPase intrinsèque de GTPases de la sous-famille Rho/Rac/Cdc42, aussi bien in vitro que in vivo, et nous avons démontré par coimmunoprécipitation que Gem est capable d'interagir avec cette protéine dans des cellules eucaryotes. Ces données suggèrent que Gem pourrait jouer un rôle dans la régulation de la fonction des protéines Rho.Gem is a recently identified protein belonging to the branch RGK of the Ras superfamily of GTPases. Gem is induced in several cell types upon mitogen stimulation, and it presents N- and C-terminal extensions of unknown function when compared to Ras as well as several amino acid substitutions in key positions for GTP binding and hydrolysis. With the aim of identifying Gem partners, and in particular those interacting with its N-terminal extension, we have developed a new vector for two-hybrid studies where the bait is fused through its C-terminus with the N-terminus of the DNA-binding domain (DBD) of LexA, therefore, possessing an inverted polarity as compared with a classical two-hybrid vector. We have validated this system by showing that it allows the same specific interactions between Ras and Ra1 GTPases with their effectors and regulators as a classical two-hybrid vector, and that it even shows an increased sensibility. We have built a two-hybrid cDNA library from Jurkat cells that we have screened for Gem partners with two different baits: (i) the first 82 amino acids of Gem fused to the N-terminus of LexA DBD; (ii) full-length Gem fused to the C-terminus of GAL4 DBD. Amongst the 20 different clones obtained, two have been studied further. Their cDNAs coded for an isoform of the 4.1N protein, and for a novel protein containing a RhoGAP domain. We have shown that the new RhoGAP protein is capable of increasing the intrinsic GTPase activity of GTPases belonging to the Rho/Rac/Cdc42 branch, in vitro as well as in vivo, and we have demonstrated by co-immunoprecipitation that Gem is able to interact with this protein in eukaryotic cells. These results suggest that Gem could be implicated in the regulation of Rho GTPases function.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Génération de nouveaux mutants dominants négatifs de la GTPases Rap1 application à la mise en évidence du rôle de Rap1 dans la dynamique cellulaire

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

A novel Rho GTPase-activating-protein interacts with Gem, a member of the Ras superfamily of GTPases.

Gem is a Ras-related protein whose expression is induced in several cell types upon activation by extracellular stimuli. With the aim of isolating the cellular partners of Gem that mediate its biological activity we performed a yeast two-hybrid screen and identified a novel protein of 970 amino acids, Gmip, that interacts with Gem through its N-terminal half, and presents a cysteine-rich domain followed by a Rho GTPase-activating protein (RhoGAP) domain in its C-terminal half. The RhoGAP domain of Gmip stimulates in vitro the GTPase activity of RhoA, but is inactive towards other Rho family proteins such as Rac1 and Cdc42; it is also specific for RhoA in vivo. The same is true for the full-length protein, which is furthermore able to down-regulate RhoA-dependent stress fibres in Ref-52 rat fibroblasts. These findings suggest that the signalling pathways controlled by two proteins of the Ras superfamily, RhoA and Gem, are linked via the action of the RhoGAP protein Gmip (Gem-interacting protein)

Armand Tavitian (1931-2020)

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