Identification des dermatophytes par spectrométrie de masse MALDI-TOF

Introduction L’identification des dermatophytes par les méthodes microbiologiques conventionnelles est souvent longue et fastidieuse. La technique de spectrométrie de masse et sa variante MALDI-TOF (Matrix Assisted Laser Desorption Ionisation-Time of Flight) est un nouvel outil utilisé pour l’identification des bactéries et des levures dans les laboratoires d’analyses médicales. Nous avons récemment développé une méthode standardisée pour l’identification en routine des champignons filamenteux à partir de culture en milieu solide. L’objectif de cette étude est d’étendre cette méthode standardisée à l’identification des dermatophytes dans l’activité de routine du laboratoire. Matériel et méthode Une banque de référence contenant les spectres de masse de 44 souches parfaitement caractérisées correspondants à 13 espèces de dermatophytes a été générée sur un UltraFlex (BruckerDaltonics, Allemagne) couplé au logiciel MaldiBiotyper v2.1. Par la suite, 133 souches isolées de prélèvements cliniques ont été identifiées en comparant leur spectre à ceux inclus dans la banque de référence : l’identification d’espèce a été retenue si le Log Score (LS) obtenu était supérieur ou égal à 1,7. Enfin, l’identification par MALDI-TOF a été considérée comme correcte en cas de concordance avec l’identification morphologique ou moléculaire des isolats cliniques. Résultats L’identification par spectrométrie de masse(SM) a été correcte pour 130 (97,8 %) des isolats. Pour 2 isolats identifiés conventionnellement comme Microsporum canis, l’identification par SM n’a pas pu générer de spectre avec un LS valide. Pour un isolat correspondant à Microsporum audouinii, la SM a généré une mauvaise identification. Tous les isolats ont pu être identifiés après seulement 3 à 6 jours de culture avant l’apparition des caractères morphologiques conventionnels d’identification. Conclusion Le protocole de SM utilisé pour l’identification des champignons filamenteux au laboratoire est applicable aux dermatophytes. Une identification d’espèce peut être obtenue en 3 à 6 jours alors qu’une identification conventionnelle qui nécessite notamment des milieux de cultures complémentaires demande 2 à 3 semaines

Anopheles gambiae TEP1 forms a complex with the coiled-coil domain of LRIM1/APL1C following a conformational change in the thioester domain

The complement-like protein thioester-containing protein 1 (TEP1) is a key factor in the immune response of the malaria vector Anopheles gambiae to pathogens. Multiple allelic variants of TEP1 have been identified in laboratory strains and in the field, and are correlated with distinct immunophenotypes. TEP1 is tightly regulated by conformational changes induced by cleavage in a protease-sensitive region. Cleaved TEP1 forms exhibit significant variation in stability from hours to days at room temperature. In particular, the refractory allele TEP1*R1 is significantly more stable than the susceptible allele TEP1*S1. This raises the question of whether the stability of cleaved TEP1 is linked to allelic variation and varying immunophenotypes. We have analyzed the stability of the cleaved form of additional TEP1 alleles and constructs. We show that stability is correlated with allelic variation within two specific loops in direct proximity to the thioester bond. The variable loops are part of an interface between the TED and MG8 domains of TEP1 that protect the thioester from hydrolysis. Engineering specific disulfide bonds to prevent separation of the TED-MG8 interface stabilizes the cleaved form of TEP1 for months at room temperature. Cleaved TEP1 forms a soluble complex with a heterodimer of two leucine-rich repeat proteins, LRIM1 and APL1C, and precipitates in the absence of this complex. The molecular structure and oligomeric state of the TEP1/LRIM1/APL1C complex is unclear. The C-terminal coiled-coil domain of the LRIM1/APL1C complex is sufficient to stabilize the cleaved form of TEP1 in solution but cleaved forms of disulfide-stabilized TEP1 do not interact with LRIM1/APL1C. This implies that formation of the TEP1cut/LRIM1/APL1C complex is related to the conformational change that induces the precipitation of cleaved TEP1

Virus-mediated shRNA knockdown of prodynorphin in the rat nucleus accumbens attenuates depression-like behavior and cocaine locomotor sensitization

Dynorphins, endogenous opioid peptides that arise from the precursor protein prodynorphin (Pdyn), are hypothesized to be involved in the regulation of mood states and the neuroplasticity associated with addiction. The current study tested the hypothesis that dynorphin in the nucleus accumbens (NAcc) mediates such effects. More specifically, we examined whether knockdown of Pdyn within the NAcc in rats would alter the expression of depressive-like and anxiety-like behavior, as well as cocaine locomotor sensitization. Wistar rats were injected with adeno-associated viral (AAV) vectors encoding either a Pdyn-specific short hairpin RNA (AAV-shPdyn) or a scrambled shRNA (AAV-shScr) as control. Four weeks later, rats were tested for anxiety-like behavior in the elevated plus maze test and depressive-like behavior in the forced swim test (FST). Finally, rats received one daily injection of saline or cocaine (20 mg/kg, i.p.), followed by assessment of locomotion for 4 consecutive days. Following 3 days of abstinence, the rats completed 2 additional daily cocaine/saline locomotor trials. Pdyn knockdown in the NAcc led to a significant reduction in depressive-like behavior in the FST, but had no effect on anxiety-like behavior in the elevated plus maze. Pdyn knockdown did not alter baseline locomotor behavior, the locomotor response to acute cocaine, or the initial sensitization of the locomotor response to cocaine over the first 4 cocaine treatment days. However, following 3 days abstinence the locomotor response to the cocaine challenge returned to their original levels in the AAV-shPdyn rats while remaining heightened in the AAV-shScr rats. These results suggest that dynorphin in a very specific area of the nucleus accumbens contributes to depressive-like states and may be involved in neuroadaptations in the NAcc that contribute to the development of cocaine addiction as a persistent and lasting condition

Amygdala 14-3-3ζ as a Novel Modulator of Escalating Alcohol Intake in Mice

Alcoholism is a devastating brain disorder that affects millions of people worldwide. The development of alcoholism is caused by alcohol-induced maladaptive changes in neural circuits involved in emotions, motivation, and decision-making. Because of its involvement in these processes, the amygdala is thought to be a key neural structure involved in alcohol addiction. However, the molecular mechanisms that govern the development of alcoholism are incompletely understood. We have previously shown that in a limited access choice paradigm, C57BL/6J mice progressively escalate their alcohol intake and display important behavioral characteristic of alcohol addiction, in that they become insensitive to quinine-induced adulteration of alcohol. This study used the limited access choice paradigm to study gene expression changes in the amygdala during the escalation to high alcohol consumption in C57BL/6J mice. Microarray analysis revealed that changes in gene expression occurred predominantly after one week, i.e. during the initial escalation of alcohol intake. One gene that stood out from our analysis was the adapter protein 14-3-3ζ, which was up-regulated during the transition from low to high alcohol intake. Independent qPCR analysis confirmed the up-regulation of amygdala 14-3-3ζ during the escalation of alcohol intake. Subsequently, we found that local knockdown of 14-3-3ζ in the amygdala, using RNA interference, dramatically augmented alcohol intake. In addition, knockdown of amygdala 14-3-3ζ promoted the development of inflexible alcohol drinking, as apparent from insensitivity to quinine adulteration of alcohol. This study identifies amygdala 14-3-3ζ as a novel key modulator that is engaged during escalation of alcohol use

Comparative genetic analysis: the utility of mouse genetic systems for studying human monogenic disease

One of the long-term goals of mutagenesis programs in the mouse has been to generate mutant lines to facilitate the functional study of every mammalian gene. With a combination of complementary genetic approaches and advances in technology, this aim is slowly becoming a reality. One of the most important features of this strategy is the ability to identify and compare a number of mutations in the same gene, an allelic series. With the advent of gene-driven screening of mutant archives, the search for a specific series of interest is now a practical option. This review focuses on the analysis of multiple mutations from chemical mutagenesis projects in a wide variety of genes and the valuable functional information that has been obtained from these studies. Although gene knockouts and transgenics will continue to be an important resource to ascertain gene function, with a significant proportion of human diseases caused by point mutations, identifying an allelic series is becoming an equally efficient route to generating clinically relevant and functionally important mouse models

MtNramp1 mediates iron import in rhizobia-infected Medicago truncatula cells.

Symbiotic nitrogen fixation is a process that requires relatively high quantities of iron provided by the host legume. Using synchrotron-based X-ray fluorescence, we have determined that this iron is released from the vasculature into the apoplast of zone II of M. truncatula nodules. This overlaps with the distribution of MtNramp1, a plasma membrane iron importer. The importance of MtNramp1 in iron transport for nitrogen fixation is indicated by the 60% reduction of nitrogenase activity observed in knock-down lines, most likely due to deficient incorporation of this essential metal cofactor at the necessary levels

Deciphering the interaction of the corticotropin-releasing factor and serotonin brain systems in anxiety-related disorders.

Contains fulltext : 79988.pdf (publisher's version ) (Open Access

A comparison of 129S2/SvHsd and C57BL/6JOlaHsd mice on a test battery assessing sensorimotor, affective and cognitive behaviours: implications for the study of genetically modified mice.

Mice from the 129S2/SvHsd (129) and C57BL/6JOlaHsd (C57) strains were assessed for performance on a test battery including sensorimotor, affective and cognitive measures, using established as well as modified paradigms. Marked strain differences were observed in measures of locomotion, motor coordination and anxiety: the 129 mice were typically less active and more anxious. In contrast, the strains did not markedly differ in muscular strength, habituation to an open field or spatial working memory. The data provide baseline parameters of behaviour against which genetically modified lines derived from these particular parental strains can be assessed