The putative Escherichia coli dehydrogenase YjhC metabolises two dehydrated forms of N-acetylneuraminate produced by some sialidases

Homologues of the putative dehydrogenase YjhC are found in operons involved in the metabolism of N-acetylneuraminate (Neu5Ac) or related compounds. We observed that purified recombinant YjhC forms Neu5Ac from two dehydrated forms of this compound, 2,7-anhydro-N-acetylneuraminate (2,7-AN) and 2-deoxy-2,3-didehydro-N-acetylneuraminate (2,3-EN) that are produced during the degradation of sialoconjugates by some sialidases. The conversion of 2,7-AN into Neu5Ac is reversible and reaches its equilibrium when the ratio of 2,7-AN to Neu5Ac is ≈1/6. The conversion of 2,3-EN is irreversible, leading to a mixture of Neu5Ac and 2,7-AN. NMR analysis of the reaction catalysed by YjhC on 2,3-EN indicated that Neu5Ac was produced as the α-anomer. All conversions require NAD+ as a cofactor, which is regenerated in the reaction. They appear to involve the formation of keto (presumably 4-keto) intermediates of 2,7-AN, 2,3-EN and Neu5Ac, which were detected by liquid chromatography-mass spectrometry (LC-MS). The proposed reaction mechanism is reminiscent of the one catalysed by family 4 β-glycosidases, which also use NAD+ as a cofactor. Both 2,7-AN and 2,3-EN support the growth of Escherichia coli provided the repressor NanR, which negatively controls the expression of the yjhBC operons, has been inactivated. Inactivation of either YjhC or YjhB in NanR-deficient cells prevents the growth on 2,7-AN and 2,3-EN. This confirms the role of YjhC in 2,7-AN and 2,3-EN metabolism and indicates that transport of 2,7-AN and 2,3-EN is carried out by YjhB, which is homologous to the Neu5Ac transporter NanT

The metalloprotein YhcH is an anomerase providing N-acetylneuraminate aldolase with the open form of its substrate

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alphaanomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate

Mice prenatally exposed to valproic acid do not show autism-related disorders when fed with polyunsaturated fatty acid-enriched diets

AbstractDietary supplementations with n-3 polyunsaturated fatty acid (PUFA) have been explored in autism spectrum disorder (ASD) but their efficiency and potential in ameliorating cardinal symptoms of the disease remain elusive. Here, we compared a n-3 long-chain (LC) PUFA dietary supplementation (n-3 supp) obtained from fatty fish with a n-3 PUFA precursor diet (n-3 bal) obtained from plant oils in the valproic acid (VPA, 450 mg/kg at E12.5) ASD mouse model starting from embryonic life, throughout lactation and until adulthood. Maternal and offspring behaviors were investigated as well as several VPA-induced ASD biological features: cerebellar Purkinje cell (PC) number, inflammatory markers, gut microbiota, and peripheral and brain PUFA composition. Developmental milestones were delayed in the n-3 supp group compared to the n-3 bal group in both sexes. Whatever the diet, VPA-exposed offspring did not show ASD characteristic alterations in social behavior, stereotypies, PC number, or gut microbiota dysbiosis while global activity, gait, peripheral and brain PUFA levels as well as cerebellar TNF-alpha levels were differentially altered by diet and treatment according to sex. The current study provides evidence of beneficial effects of n-3 PUFA based diets, including one without LCPUFAs, on preventing several behavioral and cellular symptoms related to ASD

Interrogating the lactate dehydrogenase tetrameric interface using (stapled) peptides and short proteins : an original approach to protein inhibition

Lactate Dehydrogenases (LDHs) are tetrameric enzymes of major significance in cancer metabolism and promising cancer therapy targets. However, their wide and polar catalytic sites make them a challenging target for orthosteric inhibition. In this work, we conceived to target LDH tetrameric interface with the ambition of disrupting their oligomeric state. To do so, we designed a protein model of a dimeric LDH. We exploited this model using orthogonal biophysical techniques to identify and characterize two families of α-helical peptides and stapled derivatives that targeted the LDH tetrameric interface and destabilized the tetrameric protein. These (stapled) peptides, along with the dimeric model of LDH, constitute promising pharmacological tools for the de novo design and identification of LDH tetramerization disruptors. Overall, this work demonstrates that targeting the LDH interface is achievable and paves the way toward LDH inhibition through this novel molecular mechanism.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 202

Altérations comportementales et striatales au cours du vieillissement dans deux modèles murins de troubles du spectre autistique

Autism spectrum disorders are neurodevelopmental disorders affecting one person in 160 around the world. They are characterized by communication and social impairments, associated with a restricted pattern of interests and behaviors. Alterations in GABA levels have been highlighted in patients with autism spectrum disorders (ASD), but mechanisms underlying these alterations remain unclear. One of the key structure in ASD physiopathology is the striatum, as it is involved in motor, associative and cognitive functions, all three altered in ASD. In this thesis work, two transgenic mouse models of ASD have been used (Shank3∆C/∆C and Cntnap2-/-) as they display a strong face validity. Indeed, both mutations are found in syndromic and non-syndromic ASD patients. This PhD thesis are about the evolution of ASD motor symptoms with aging as well as the evolution of related striatal alterations, in three experimental groups: 10weeks, 20weeks, 40weeks, with both males and females. Results exposed here show the presence of motor stereotypies at different stages, worsening with aging. Patch-clamp recordings of DLS MSNs show dysfunctions in GABAergic transmission, evolving with aging. To go further, we examined the neuronal morphology as well as the functionality of PV interneurons, which are the main source of GABA onto the MSNs, with an optogenetic approach. Finally, a chemogenetic approach allowed us to study the phenotypic restoration in ASD mice models. The work presented here allow to characterize striatal dysfunctions in our models, and to establish a timeline for their evolution.Les troubles du spectre autistique sont un ensemble de troubles neurodéveloppementaux affectant 1 personne sur 160 dans le monde et se traduisant par des troubles sociaux et de communication, ainsi qu’un ensemble restreint d’intérêts et de comportements. Des altérations dans les niveaux de GABA ont été mises en évidences chez les patients atteints de troubles du spectre autistique (TSA) depuis plusieurs décennies déjà, mais les mécanismes qui sous-tendent ces altérations ne sont pas clairement définis à ce jour. L’une des structures impliquée dans la physiopathologie des TSA est le striatum, qui est impliqué dans des fonctions motrices, associatives et cognitives, toutes trois altérées dans les TSA. Dans les travaux présentés ici, deux modèles murins transgéniques de TSA ont été utilisés (Shank3∆C/∆C et Cntnap2-/-) en raison de leur forte validité conceptuelle, ces mutations étant identifiées dans des formes syndromiques et non-syndromiques de TSA chez l’Homme.Les travaux présentés ici portent sur l’évolution des symptômes autistiques moteurs avec l’âge et des altérations striatales dans des groupes expérimentaux composés de souris âgées de 10 semaines, 20 semaines et 40 semaines, mâles et femelles, dans ces deux modèles murins transgéniques de TSA. Les résultats présentés ici montrent la présence de stéréotypies motrices aux différents âges, s’aggravant avec l’âge. L’enregistrement électrophysiologique des neurones de projection (MSN) du striatum dorsolatéral montre des dysfonctions dans la transmission GABAergique évoluant avec l’âge. Afin d’étudier les possibles causes de ces altérations fonctionnelles, la morphologie de ces neurones a été évaluée d’une part afin de déterminer l’impact du vieillissement sur celle-ci dans les deux modèles. D’autre part, l’étude des interneurones parvalbumine (PV), principale source de GABA sur les neurones de projection a été réalisée par électrophysiologie ex-vivo, incluant une approche optogénétique pour mesurer la communication PV-MSN. Enfin, une approche chimiogénétique a permis d’étudier la restauration d’un phénotype typique chez les souris TSA. Les travaux présentés ici permettent donc de caractériser les dysfonctions striatales dans les deux modèles utilisés, ainsi que d’établir une chronologie de leur apparition et de leur mise en place

La lecture courante expressive à l’École primaire

Thabault Roger. La lecture courante expressive à l’École primaire. In: La revue pédagogique, tome 84, Janvier-Juin 1924. pp. 171-197

La culture intellectuelle des instituteurs (2e article)

Thabault Roger. La culture intellectuelle des instituteurs (2e article). In: La revue pédagogique, tome 77, Juillet-Décembre 1920. pp. 270-282

La culture intellectuelle des instituteurs

Thabault Roger. La culture intellectuelle des instituteurs. In: La revue pédagogique, tome 77, Juillet-Décembre 1920. pp. 185-198