Exhaustive Screening of the Acid β-Glucosidase Gene, by Fluorescence-Assisted Mismatch Analysis Using Universal Primers: Mutation Profile and Genotype/Phenotype Correlations in Gaucher Disease

SummaryGaucher disease (GD) is one of the most prevalent lysosomal storage disorders and one of the rare genetic diseases now accessible to therapy. Outside the Ashkenazi Jewish community, a high molecular diversity is observed, leaving ∼30% of alleles undetected. Nevertheless, very few exhaustive methods have been developed for extensive gene screening of a large series of patients. Our approach for a complete search of mutations was the association of fluorescent chemical cleavage of mismatches with a universal strand-specific labeling system. The glucocerebrosidase (GBA) gene was scanned by use of a set of six amplicons, comprising 11 exons, all exon/intron boundaries, and the promoter region. By use of this screening strategy, the difficulties due to the existence of a highly homologous pseudogene were easily overcome, and both GD mutant alleles were identified in all 25 patients studied, thus attesting to a sensitivity that approaches 100%. A total of 18 different mutations and a new glucocerebrosidase haplotype were detected. The mutational spectrum included eight novel acid β-glucosidase mutations: IVS2 G(+1)→T, I119T, R170P, N188K, S237P, K303I, L324P, and A446P. These data further indicate the genetic heterogeneity of the lesions causing GD. Established genotype/phenotype correlations generally were confirmed, but notable disparities were disclosed in several cases, thus underlining the limitation in the prognostic value of genotyping. The observed influence of multifactorial control on this monogenic disease is discussed

Adjustment and Characterization of an Original Model of Chronic Ischemic Heart Failure in Pig

We present and characterize an original experimental model to create a chronic ischemic heart failure in pig. Two ameroid constrictors were placed around the LAD and the circumflex artery. Two months after surgery, pigs presented a poor LV function associated with a severe mitral valve insufficiency. Echocardiography analysis showed substantial anomalies in radial and circumferential deformations, both on the anterior and lateral surface of the heart. These anomalies in function were coupled with anomalies of perfusion observed in echocardiography after injection of contrast medium. No demonstration of myocardial infarction was observed with histological analysis. Our findings suggest that we were able to create and to stabilize a chronic ischemic heart failure model in the pig. This model represents a useful tool for the development of new medical or surgical treatment in this field

Evidence of a relation between hippocampal volume, white matter hyperintensities, and cognition in subjective cognitive decline and mild cognitive impairment

Objective: The concepts of mild cognitive impairment (MCI) and subjective cognitive decline (SCD) have been proposed to identify individuals in the early stages of Alzheimer’s disease (AD), or other neurodegenerative diseases. One approach to validate these concepts is to investigate the relationship between pathological brain markers and cognition in those individuals. Method: We included 126 participants from the Consortium for the Early Identification of Alzheimer’s disease-Quebec (CIMA-Q) cohort (67 SCD, 29 MCI, and 30 cognitively healthy controls [CH]). All participants underwent a complete cognitive assessment and structural magnetic resonance imaging. Group comparisons were done using cognitive data, and then correlated with hippocampal volumes and white matter hyperintensities (WMHs). Results: Significant differences were found between participants with MCI and CH on episodic and executive tasks, but no differences were found when comparing SCD and CH. Scores on episodic memory tests correlated with hippocampal volumes in both MCI and SCD, whereas performance on executive tests correlated with WMH in all of our groups. Discussion: As expected, the SCD group was shown to be cognitively healthy on tasks where MCI participants showed impairment. However, SCD’s hippocampal volume related to episodic memory performances, and WMH to executive functions. Thus, SCD represents a valid research concept and should be used, alongside MCI, to better understand the preclinical/prodromal phase of AD

Qualifications et parcours - Qualification des parcours. XXVèmes journées du longitudinal, Nantes, 20 et 21 juin 2019

International audienceLes XXVèmes journées du longitudinal (JDL) se proposaient de questionner deux notions : la qualification et les parcours ainsi que leur articulation. Elles furent l'occasion d'évoquer les parcours biographiques et de saisir la qualification des personnes comme un construit dans le temps, dans une perspective de « formation tout au long de la vie ». Ces journées ont permis de discuter de la polarisation des parcours tels les parcours d'exclusion et parcours d'accès aux qualifications les plus socialement valorisées mais aussi de la multiplication des parcours atypiques remettant en cause les relations directes entre réussite ou échec scolaire d'une part, insertion professionnelle durable ou précaire d'autre part. À cet égard, les JDL ont permis de discuter des méthodes statistiques permettant d'identifier des parcours-types, notamment à travers les techniques de classification

Imbalanced Lignin Biosynthesis Promotes the Sexual Reproduction of Homothallic Oomycete Pathogens

Lignin is incorporated into plant cell walls to maintain plant architecture and to ensure long-distance water transport. Lignin composition affects the industrial value of plant material for forage, wood and paper production, and biofuel technologies. Industrial demands have resulted in an increase in the use of genetic engineering to modify lignified plant cell wall composition. However, the interaction of the resulting plants with the environment must be analyzed carefully to ensure that there are no undesirable side effects of lignin modification. We show here that Arabidopsis thaliana mutants with impaired 5-hydroxyguaiacyl O-methyltransferase (known as caffeate O-methyltransferase; COMT) function were more susceptible to various bacterial and fungal pathogens. Unexpectedly, asexual sporulation of the downy mildew pathogen, Hyaloperonospora arabidopsidis, was impaired on these mutants. Enhanced resistance to downy mildew was not correlated with increased plant defense responses in comt1 mutants but coincided with a higher frequency of oomycete sexual reproduction within mutant tissues. Comt1 mutants but not wild-type Arabidopsis accumulated soluble 2-O-5-hydroxyferuloyl-l-malate. The compound weakened mycelium vigor and promoted sexual oomycete reproduction when applied to a homothallic oomycete in vitro. These findings suggested that the accumulation of 2-O-5-hydroxyferuloyl-l-malate accounted for the observed comt1 mutant phenotypes during the interaction with H. arabidopsidis. Taken together, our study shows that an artificial downregulation of COMT can drastically alter the interaction of a plant with the biotic environment

Exploration clinique et biologique du NO

En 1980, Furchgott et al. démontrent que la relaxation des cellules musculaires lisses artérielles en réponse à l’acétylcholine est dépendante de l’intégrité anatomique de l’endothélium. Ils baptisent le principe à l’origine de cette relation intercellulaire EDRF (Endothelium Derived Relaxing Factor), qui fut identifié 7 ans plus tard comme étant le monoxyde d’azote (NO), un gaz radicalaire. Au niveau des vaisseaux, le NO produit localement est généré par la NO synthase endothéliale et a une action essentiellement paracrine (relaxation des cellules musculaires lisses sous- jacentes, mais aussi l’inhibition de l’agrégation plaquettaire). La demi-vie du NO est brève in vivo, et son étude est donc difficile. On dispose actuellement de techniques invasives et non invasives qui permettent d’explorer les variations de calibre ou de débit artériels. L’observation princeps de Furchgott préfigure de façon magistrale toute la physiopathologie de la fonction relaxante de l’endothélium. En effet, de nombreuses affections, et en particulier l’athérosclérose, s’accompagnent d'anomalies de la vasodilatation endothélium-dépendante (« dysfonction endothéliale »). Tandis que l’acétylcholine (ou la sérotonine) injectée dans une artère saine entraîne une vasodilatation, elle provoque en revanche une vasoconstriction dans une artère athéroscléreuse, du fait d’une diminution de la biodisponibité du NO. Ce déficit de NO pourrait précipiter la maladie coronaire en favorisant, outre le spasme artériel, l'interaction entre les plaquettes et la paroi artérielle et donc la thrombose. En clinique, le NO ne peut pas être mesuré directement, à l’exception du NO détecté dans l’air exhalé. In vivo, le NO est rapidement oxydé en nitrite ([math]) et en nitrate ([math]), l’ensemble étant appelé NOx. Nous détaillons ici les limites de ce dosage comme index biochimique de la production de NO d’origine « endothéliale »

Intérêt des légumineuses fourragères pâturées et/ou récoltées dans l'alimentation des vaches laitières en France

Forage legumes play a central role in bovine dairy systems in France because of their high nutritional value. In particular, they are protein rich, which means that they can partially or entirely replace protein supplements. Legumes may be used in diverse, complementary ways: they can serve as forage in grasslands or generate different types of fodder. Milk production by dairy cows fed forage legumes is often equal to, if not greater than, that by cows fed diets that do not contain legumes. In this work, we discuss the dietary benefits and diverse uses (across different dietary regimes) of lucerne, white clover, and red clover in bovine dairy systems

Evaluation of seafood toxicity in the Australes archipelago (French Polynesia) using the neuroblastoma cell-based assay

Ciguatera fish poisoning (CFP), a disease caused by consuming fish that have accumulated ciguatoxins (CTXs) in their tissue, is regarded as the most prevalent form of intoxication in French Polynesia. Recently, the Australes, one of the least affected archipelago until the early 1980s, has shown a dramatic increase in its incidence rates in 2009 with unusual CFP cases. In the present work, potential health hazards associated with the proliferation of various marine phytoplankton species and the consumption of fish and marine invertebrates highly popular among local population were assessed in three Australes islands: Raivavae, Rurutu and Rapa. Extracts from the marine dinoflagellates Gambierdiscus, Ostreospis and mat-forming cyanobacteria as well as fish, giant clams and sea urchin samples were examined for the presence of CTXs and palytoxin (PLTX) by using the neuroblastoma cell-based assay (CBA-N2a). Cytotoxic responses observed with both standards (Pacific CTX-3C and PLTX) and targeted marine products indicate that CBA-N2a is a robust screening tool, with high sensitivity and good repeatability and reproducibility. In Rurutu and Raivavae islands, our main findings concern the presence of CTX-like compounds in giant clams and sea urchins, suggesting a second bio-accumulation route for CFP toxins in the ciguatera food chain. In Rapa, the potential CFP risk from Gambierdiscus bloom and fish was confirmed for the first time, with levels of CTXs found above the consumer advisory level of 0.01ng Pacific CTX-1Bg(-1) of flesh in three fish samples. However, despite the presence of trace level of PLTX in Ostreopsis natural assemblages of Rapa, no sign of PLTX accumulation is yet observed in tested fish samples. Because this multi-toxinic context is likely to emerge in most French Polynesian islands, CBA-N2a shows great potential for future applications in the algal- and toxin-based field monitoring programmes currently on hand locally

Alteration of plasmalemmal caveolae mimics endothelial dysfunction observed in atheromatous rabbit aorta.

OBJECTIVE: In endothelial cells, nitric oxide (NO) is produced by the endothelial isoform of nitric oxide synthase (eNOS), which is localized in the cholesterol-rich plasmalemmal microdomains involved in signal transduction, known as caveolae. The present study was undertaken to evaluate the effect of hypercholesterolemia and fatty streak formation on the endothelial caveolae and on endothelial function, and attempted to determine to what extent the caveolae were involved in endothelium-derived NO production. METHODS AND RESULTS: We first studied the effect of atheroma on endothelial NO production. Fatty streak infiltrated aorta of cholesterol-fed New Zealand White rabbits demonstrated an impairment of acetylcholine-induced relaxation and nearly normal calcium ionophore A23187-induced maximal relaxation. The abundance of caveolae in the endothelium covering the fatty streak, as well as their 'grape-like' clustering, appeared to be decreased. We therefore investigated the effect, on endothelial NO production, of the cholesterol-binding agents 2-hydroxypropyl-beta-cyclodextrin (hp-beta-CD) and filipin, known to alter caveolae structure and/or function. Treatment with either hp-beta-CD (2%) or filipin (4 microg/ml) did not affect contraction to phenylephrine or relaxant responses to A23187 or to the NO donor sodium nitroprusside. In contrast, both treatments impaired acetylcholine-induced relaxation. Cultured bovine aortic endothelial cells (BAEC) similarly treated with hp-beta-CD demonstrated a 50% decrease of total cellular cholesterol and a decreased abundance of caveolae as well as their 'grape-like' clustering. Cholesterol depletion decreased the bradykinin-induced transient peak of free intracellular calcium and subsequent receptor-stimulated NO production (assessed using reporter cells rich in soluble guanylyl cyclase), whereas that elicited by A23187 remained unaltered. CONCLUSION: Fatty streak deposit is associated with a decrease in caveolae 'transductosomes' abundance which appears to represent a novel mechanism of endothelial dysfunction