Histoire du LSD. De l’ergot de seigle à l’utilisation thérapeutique

National audienceLe LSD, de l’allemand Lysergsäurediethylamid, est une substance hallucinogène utilisée à but récréatif. Également connue sous le nom d’« acide », cette molécule à propriétés psychotropes est classée en France comme un stupéfiant illicite selon l’arrêté du 22 février 1990..

Novel genes upregulated when NOTCH signalling is disrupted during hypothalamic development.

International audienceBACKGROUND: The generation of diverse neuronal types and subtypes from multipotent progenitors during development is crucial for assembling functional neural circuits in the adult central nervous system. It is well known that the Notch signalling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. However, the role of Notch during hypothalamus formation along with its downstream effectors remains poorly defined. RESULTS: Here, we have transiently blocked Notch activity in chick embryos and used global gene expression analysis to provide evidence that Notch signalling modulates the generation of neurons in the early developing hypothalamus by lateral inhibition. Most importantly, we have taken advantage of this model to identify novel targets of Notch signalling, such as Tagln3 and Chga, which were expressed in hypothalamic neuronal nuclei. CONCLUSIONS: These data give essential advances into the early generation of neurons in the hypothalamus. We demonstrate that inhibition of Notch signalling during early development of the hypothalamus enhances expression of several new markers. These genes must be considered as important new targets of the Notch/proneural network

Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNA(ala )T-loops in slow- and fast-growing mycobacteria

BACKGROUND: Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNA(ala )genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNA(ala )loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNA(ala )genes. RESULTS: The three tRNA(ala )genes are highly conserved in M. smegmatis and BCG. However, in the T-loop of tRNA(alaU )and tRNA(alaV )containing the attB site, a single base difference was observed between the two species. We observed that the tRNA(alaU )gene was the only site into which Ms6-derived integration-proficient vectors integrated in M. smegmatis, whereas in BCG, the tRNA(alaV )gene was used as the target. No integration occurred in the BCG tRNA(alaU )T-loop, despite a difference of only one base from the 26-base Ms6 attP core. We mutated the attP core to give a perfect match with the other tRNA(ala )T-loops from M. smegmatis and BCG. Modification of the seven-base T-loop decreased integration efficiency, identifying this site as a possible site of strand exchange. Finally, two Ms6 vectors were constructed to integrate two reporter genes into the tRNA(alaU )and tRNA(alaV )T-loops of the same BCG chromosome. CONCLUSION: Small changes in the 7 bp T-loop attP site of Ms6 made it possible to use another attB site, albeit with a lower integration efficiency. These molecular studies on BCG tRNA(ala )genes made it possible to create valuable tools for the site-directed insertion of several genes in the same BCG strain. These tools will be useful for the development of novel multivalent vaccines and genetic studies

A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: application to acetaminophen

International audienceObesity and nonalcoholic fatty liver disease (NAFLD) can increase susceptibility to hepatotoxicity induced by some xenobiotics including drugs, but the involved mechanisms are poorly understood. For acetaminophen (APAP), a role of hepatic cytochrome P450 2E1 (CYP2E1) is suspected since the activity of this enzyme is consistently enhanced during NAFLD. The first aim of our study was to set up a cellular model of NAFLD characterized not only by triglyceride accumulation but also by higher CYP2E1 activity. To this end, human HepaRG cells were incubated for one week with stearic acid or oleic acid, in the presence of different concentrations of insulin. Although cellular triglycerides and the expression of lipid-responsive genes were similar with both fatty acids, CYP2E1 activity was significantly increased only by stearic acid. CYP2E1 activity was reduced by insulin and this effect was reproduced in cultured primary human hepatocytes. Next, APAP cytotoxicity was assessed in HepaRG cells with or without lipid accretion and CYP2E1 induction. Experiments with a large range of APAP concentrations showed that the loss of ATP and glutathione was almost always greater in the presence of stearic acid. In cells pretreated with the CYP2E1 inhibitor chlormethiazole, recovery of ATP was significantly higher in the presence of stearate with low (2.5 mM) or high (20 mM) concentrations of APAP. Levels of APAP-glucuronide were significantly enhanced by insulin. Hence, HepaRG cells can be used as a valuable model of NAFLD to unveil important metabolic and hormonal factors which can increase susceptibility to drug-induced hepatotoxicit

T-cell and serological responses to Erp, an exported Mycobacterium tuberculosis protein, in tuberculosis patients and healthy individuals

<p>Abstract</p> <p>Background</p> <p>The identification of antigens able to differentiate tuberculosis (TB) disease from TB infection would be valuable. Cellular and humoral immune responses to Erp (Exported repetitive protein) – a recently identified <it>M. tuberculosis </it>protein – have not yet been investigated in humans and may contribute to this aim.</p> <p>Methods</p> <p>We analyzed the cellular and humoral immune responses to Erp, ESAT-6, Ag85B and PPD in TB patients, in BCG⁺individuals without infection, BCG⁺individuals with latent TB infection (LTBI) and BCG^-controls. We used lymphoproliferation, ELISpot IFN-γ, cytokine production assays and detection of specific human antibodies against recombinant <it>M. tuberculosis </it>proteins.</p> <p>Results</p> <p>We included 22 TB patients, 9 BCG⁺individuals without TB infection, 7 LTBI and 7 BCG^-controls. Erp-specific T cell counts were higher in LTBI than in the other groups. Erp-specific T cell counts were higher in LTBI subjects than TB patients (median positive frequency of 211 SFC/10⁶PBMC (range 118–2000) for LTBI subjects compared to 80 SFC/10⁶PBMC (range 50–191), p = 0.019); responses to PPD and ESAT-6 antigens did not differ between these groups. IFN-γ secretion after Erp stimulation differed between TB patients and LTBI subjects (p = 0.02). Moreover, LTBI subjects but not TB patients or healthy subjects produced IgG3 against Erp.</p> <p>Conclusion</p> <p>The frequencies of IFN-γ-producing specific T cells, the IFN-γ secretion and the production of IgG3 after Erp stimulation are higher in LTBI subjects than in TB patients, whereas PPD and ESAT-6 are not.</p

High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials

Diagnosis and management of Silver–Russell syndrome: first international consensus statement

This Consensus Statement summarizes recommendations for clinical diagnosis, investigation and management of patients with Silver–Russell syndrome (SRS), an imprinting disorder that causes prenatal and postnatal growth retardation. Considerable overlap exists between the care of individuals born small for gestational age and those with SRS. However, many specific management issues exist and evidence from controlled trials remains limited. SRS is primarily a clinical diagnosis; however, molecular testing enables confirmation of the clinical diagnosis and defines the subtype. A 'normal' result from a molecular test does not exclude the diagnosis of SRS. The management of children with SRS requires an experienced, multidisciplinary approach. Specific issues include growth failure, severe feeding difficulties, gastrointestinal problems, hypoglycaemia, body asymmetry, scoliosis, motor and speech delay and psychosocial challenges. An early emphasis on adequate nutritional status is important, with awareness that rapid postnatal weight gain might lead to subsequent increased risk of metabolic disorders. The benefits of treating patients with SRS with growth hormone include improved body composition, motor development and appetite, reduced risk of hypoglycaemia and increased height. Clinicians should be aware of possible premature adrenarche, fairly early and rapid central puberty and insulin resistance. Treatment with gonadotropin-releasing hormone analogues can delay progression of central puberty and preserve adult height potential. Long-term follow up is essential to determine the natural history and optimal management in adulthood

Mise au point du dosage du LSD et ses métabolites dans les matrices biologiques (application en toxicologie médico-judiciaire)

Le diéthylamide de l'acide lysergique (LSD) est un puissant hallucinogène dérive de l'ergot de seigle. L'analyse du LSD dans les fluides biologiques est complexe car le LSD est consommé à de faibles doses, est rapidement métabolisé avec une demi-vie courte et est un composé instable. Ainsi, une méthode analytique rapide et sensible a été développée et validée pour doser simultanément le LSD et ses métabolites (NOR-LSD, ISO-LSD, O-H-LSD) dans le sang et l'urine. Cette méthode inclut une étape d'extraction en phase solide (SPE) et une séparation par un système de chromatographie en phase liquide couplée à la spectrométrie de masse en tandem(LC-MS/MS) équipé d'un spectromètre de masse avec triple quadripôle couplée à une source d'ionisation par électrospray (ESI). Cette méthode a été validée puis utilisée avec succès dans un cadre médico-judiciaire de dosage postmortem de LSD dans les fluides biologiques d'un consommateur.Lysergic acid diethylamide (LSD) is a highly potent psychedelic drug derived from ergot alkaloids. The analysis of lysergic acid diethylamide (LSD) in biological fluids is a challenge since LSD is taken in low doses, is rapidly metabolized with a short half-life and is an unstable compound. Therefore, a fast and sensitive method has been was developed and validated for simultaneous determination of LSD and its metabolites (NOR-LSD, ISO-LSD, O-H-LSD) in complex matrixes, such as urine and blood. This method included a step of solid phase extraction (SPE) and a chromatographic separation on a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system equipped with an electrospray ionization source and a tandem triple quadrupole mass spectrometer in multiple reaction monitoring mode (MRM). The LC-MS/MS method was validated and successfully applied to the forensic determination of postmortem LSD levels in the biological fluids of a drug consumer.RENNES1-BU Santé (352382103) / SudocLYON1-BU Santé (693882101) / SudocSudocFranceF

Immunogénicité de souches de Mycobacterium bovis BCG recombinant exprimant des gènes du Virus de l'Immunodéficience Simienne VISmac251 chez le macaque Cynomolgus

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Ethylglucuronide et éthylsulfate, marqueurs biologiques de la consommation d’alcool

National audienceEthanol is the most frequently psychoactive substance used in France, leading to major public health pro-blems. Alcohol intake detection might be important in a variety of clinical and forensic settings. Interestin-gly, ethylglucuronide (EtG) and ethylsulfate (EtS) are potentially direct markers of alcohol consumption, being detected in various biological samples such as blood, urine or hair. Moreover, since their detection timeframe corresponds to few hours until several days, these two biomarkers can prove ethanol intake even after the complete elimination of alcohol from the body. We describe herein the metabolic pathway of ethanol leading to EtG and EtS production, the currently used methods for their determination, the precautions in data interpretation and clinical applications of the measurement of these two biomarkers of alcohol misuse.L’éthanol est la substance psychoactive la plus consommée en France et pose de réel problème de santé publique. La mise en évidence de sa consommation par des méthodes biologiques peut s’avérer importante aussi bien dans des contextes cliniques que dans des affaires médico-judiciaires. L’éthylglucuronide (EtG) et l’éthylsulfate (EtS) sont des marqueurs directs d’une consommation d’éthanol pouvant être identifiables dans différentes matrices biologiques comme le sang, l’urine ou les cheveux, ce qui représente un intérêt majeur à leur utilisation. De plus, leur détection peut se faire plusieurs heures voire même plusieurs jours après ingestion, ce qui permet de mettre en évidence une consommation d’éthanol après que celui-ci ait disparu de l’organisme. Cet article présente de manière synthétique les voies de production de l’EtG et l’EtS lors du métabolisme de l’éthanol, les méthodes actuellement employées pour leur mise en évidence, ainsi que les précautions d’interprétation des résultats et les applications du dosage de ces marqueurs dans différents contextes