The dystroglycan: Nestled in an adhesome during embryonic development

AbstractInvertebrate and vertebrate development relies on complex processes that require many coordinated cell functions including cell adhesion, migration, proliferation and polarization. These processes depend on tissues and are spatio-temporally regulated by specific interactions between cells and between cells and the extracellular matrices. The dystroglycan, a transmembrane receptor that binds multiple extracellular matrix proteins, is expressed from oogenesis to organogenesis. There are increasing data suggesting that the axis, consisting of extracellular component–dystroglycan–cytoplasmic proteins, controls both the adhesion of cells to matrices as well as the transduction of signals coming from or directed to matrices. In this article, we review current advances leading to consider that the dystroglycan is a key protein nestled in an adhesome involved in mechanisms of cell adhesion during embryonic development

Heterologous expression of a plant uracil transporter in yeast: improvement of plasma membrane targeting in mutants of the Rsp5p ubiquitin protein ligase.

Plasma membrane proteins involved in transport processes play a crucial role in cell physiology. On account of these properties, these molecules are ideal targets for development of new therapeutic and agronomic agents. However, these proteins are of low abundance, which limits their study. Although yeast seems ideal for expressing heterologous transporters, plasma membrane proteins are often retained in intracellular compartments. We tried to find yeast mutants potentially able to improve functional expression of a whole set of heterologous transporters. We focused on Arabidopsis thaliana ureide transporter 1 (AtUPS1), previously cloned by functional complementation in yeast. Tagged versions of AtUPS1 remain mostly trapped in the endoplasmic reticulum and were able to reach slowly the plasma membrane. In contrast, untagged AtUPS1 is rapidly delivered to plasma membrane, where it remains in stable form. Tagged and untagged versions of AtUPS1 were expressed in cells deficient in the ubiquitin ligase Rsp5p, involved in various stages of the intracellular trafficking of membrane-bound proteins. rsp5 mutants displayed further plasma membrane stabilization of untagged AtUPS1, and improved steady state amounts of tagged versions of AtUPS1. rsp5 cells are thus powerful tools to solve the many problems inherent in heterologous expression of membrane proteins in yeast, including ER retention

De novo TUBB2B mutation causes fetal akinesia deformation sequence with microlissencephaly: an unusual presentation of tubulinopathy

International audienceTubulinopathies are increasingly emerging major causes underlying complex cerebral malformations, particularly in case of microlissencephaly often associated with hypoplastic or absent corticospinal tracts. Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. We report on an early foetal case with FADS and microlissencephaly due to TUBB2B mutation. Neuropathological examination disclosed virtually absent cortical lamination, foci of neuronal overmigration into the leptomeningeal spaces, corpus callosum agenesis, cerebellar and brainstem hypoplasia and extremely severe hypoplasia of the spinal cord with no anterior and posterior horns and almost no motoneurons. At the cellular level, the p.Cys239Phe TUBB2B mutant leads to tubulin heterodimerization impairment, decreased ability to incorporate into the cytoskeleton, microtubule dynamics alteration, with an accelerated rate of depolymerization. To our knowledge, this is the first case of microlissencephaly to be reported presenting with a so severe and early form of FADS, highlighting the importance of tubulin mutation screening in the context of FADS with microlissencephaly

Comment former des enseignants pour une éducation à l'environnement et au développement durabl

Nous présentons, dans cette contribution, un atelier de formation proposé à des stagiaires IUFM et l’évaluation qui en a été faite quelques mois plus tard. Cet atelier a été élaboré en référence à une identifi cation des compétences professionnelles nécessaires à la mise en oeuvre d’une Éducation à l’environnement et au développement durable (ÉEDD). Une enquête par questionnaire permet d’évaluer l’impact de cet atelier ainsi que les freins et les motivations des enseignants par rapport aux spécifi cités de l’intégration de l’ÉEDD dans leurs pratiques d’enseignement. L’ensemble des informations recueillies permet d’envisager des propositions d’évolution du dispositif proposé

Exon skipping as a therapeutic strategy applied to an RYR1 mutation with pseudo-exon inclusion causing a severe core myopathy.

International audienceCentral core disease is a myopathy often arising from mutations in the type 1 ryanodine receptor (RYR1) gene, encoding the sarcoplasmic reticulum calcium release channel RyR1. No treatment is currently available for this disease. We studied the pathological situation of a severely affected child with two recessive mutations, which resulted in a massive reduction in the amount of RyR1. The paternal mutation induced the inclusion of a new in-frame pseudo-exon in RyR1 mRNA that resulted in the insertion of additional amino acids leading to the instability of the protein. We hypothesized that skipping this additional exon would be sufficient to restore RyR1 expression and to normalize calcium releases. We therefore developed U7-AON lentiviral vectors to force exon skipping on affected primary muscle cells. The efficiency of the exon skipping was evaluated at the mRNA level, at the protein level, and at the functional level using calcium imaging. In these affected cells, we observed a decreased inclusion of the pseudo-exon, an increased RyR1 protein expression, and a restoration of calcium releases of normal amplitude either upon direct RyR1 stimulation or in response to membrane depolarization. This study is the first demonstration of the potential of exon-skipping strategy for the therapy of central core disease, from the molecular to the functional level

Absence of triadin, a protein of the calcium release complex, is responsible for cardiac arrhythmia with sudden death in human

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease so far related to mutations in the cardiac ryanodine receptor (RYR2) or the cardiac calsequestrin (CASQ2) genes. Because mutations in RYR2 or in CASQ2 are not retrieved in all CPVT cases, we searched for mutations in the physiological protein partners of RyR2 and CSQ2 in a large cohort of CPVT patients with no detected mutation in these two genes. Based on a candidate gene approach, we focused our investigations on triadin and junctin, two proteins that link RyR2 and CSQ2. Mutations in the triadin (TRDN) and in the junctin (ASPH) genes were searched in a cohort of 97 CPVT patients. We identified three mutations in triadin which cosegregated with the disease on a recessive mode of transmission in two families, but no mutation was found in junctin. Two TRDN mutations, a 4 bp deletion and a nonsense mutation, resulted in premature stop codons; the third mutation, a p.T59R missense mutation, was further studied. Expression of the p.T59R mutant in COS-7 cells resulted in intracellular retention and degradation of the mutant protein. This was confirmed after in vivo expression of the mutant triadin in triadin knock-out mice by viral transduction. In this work, we identified TRDN as a new gene responsible for an autosomal recessive form of CPVT. The mutations identified in the two families lead to the absence of the protein, thereby demonstrating the importance of triadin for the normal function of the cardiac calcium release complex in humans

Epilepsy with migrating focal seizures

To report new sporadic cases and 1 family with epilepsy of infancy with migrating focal seizures (EIMFSs) due to KCNT1 gain-of-function and to assess therapies' efficacy including quinidine. We reviewed the clinical, EEG, and molecular data of 17 new patients with EIMFS and KCNT1 mutations, in collaboration with the network of the French reference center for rare epilepsies. The mean seizure onset age was 1 month (range: 1 hour to 4 months), and all children had focal motor seizures with autonomic signs and migrating ictal pattern on EEG. Three children also had infantile spasms and hypsarrhythmia. The identified KCNT1 variants clustered as "hot spots" on the C-terminal domain, and all mutations occurred de novo except the p.R398Q mutation inherited from the father with nocturnal frontal lobe epilepsy, present in 2 paternal uncles, one being asymptomatic and the other with single tonic-clonic seizure. In 1 patient with EIMFS, we identified the p.R1106Q mutation associated with Brugada syndrome and saw no abnormality in cardiac rhythm. Quinidine was well tolerated when administered to 2 and 4-year-old patients but did not reduce seizure frequency. The majority of the KCNT1 mutations appear to cluster in hot spots essential for the channel activity. A same mutation can be linked to a spectrum of conditions ranging from EMFSI to asymptomatic carrier, even in the same family. None of the antiepileptic therapies displayed clinical efficacy, including quinidine in 2 patients

Colonization with Enterobacteriaceae producing ESBLs in children attending pre-school childcare facilities in the Lao People's Democratic Republic.

OBJECTIVES: Intestinal carriage constitutes an important reservoir of antimicrobial-resistant bacteria, with some of the highest rates reported from Asia. Antibiotic resistance has been little studied in Laos, where some antibiotics are available without restriction, but others such as carbapenems are not available. PATIENTS AND METHODS: We collected stools from 397 healthy children in 12 randomly selected pre-school childcare facilities in and around Vientiane. Colonization with ESBL-producing Enterobacteriaceae (ESBLE) and carbapenemase-producing Enterobacteriaceae (CPE) was detected using a disc diffusion screening test and ESBLE were characterized using WGS. Risk factor data were collected by questionnaire. RESULTS: Ninety-two children (23%) were colonized with ESBLE, mainly Escherichia coli carrying blaCTX-M and Klebsiella pneumoniae carrying blaSHV or blaCTX-M, which were frequently resistant to multiple antibiotic classes. Although residence in Vientiane Capital, foreign travel, higher maternal level of education, antibiotic use in the preceding 3 months and attending a childcare facility with a 'good' level of hygiene were all associated with ESBLE colonization on univariable analysis, a significant association remained only for antibiotic use when a stepwise approach was used with a multivariate random-effects model. WGS analysis suggested transmission in both childcare facilities and community settings. CONCLUSIONS: The high prevalence of paediatric colonization with ESBLE in Laos, one of the highest reported in Asia, is probably the result of inappropriate antibiotic use. Paediatric colonization with CPE was not identified in this study, but it is important to continue to monitor the spread of antibiotic-resistant Enterobacteriaceae in Laos

Functional diversity measures revealed impacts of non-native species and habitat degradation on species-poor freshwater fish assemblages

International audienceTrail-based ecology has been developed for decades lo infer ecosystem responses to stressors based on the functional structure of communities, yet its value in species-poor systems is largely unknown. Here, we used an extensive clataset in a Spanish region highly prone to non-native fish invasions (15 catchments, N 389 sites) to assess for the first time how species-poor communities respond to large-scale environmental gradients using a taxonomic and functional trait-based approach in riverine fish. We examined total species richness and three functional trait-based indices available when many sites have \textless= 3 species (specialization, FSpe; onginaliy, FOri and entropy, FEnt). We assessed the responses of these taxonomic and functional indices along gradients of altitude, water pollution, physical habitat degradation and non-native fish biomass. Whilst species richness was relatively sensitive to spatial effects, functional diversity indices were responsive across natural and anthropogenic gradients. All four diversity measures declined with altitude but this decline was modulated by physical habitat degradation (richness, FSpe and FEnt) and the non-native total fish biomass ratio (FSpe and FOri) in ways that varied between indices. Furthermore, FSpe and FOri were significantly correlated with Total Nitrogen. Non-native fish were a major component of the taxonomic and functional structure of fish communities, raising concerns about potential misdiagnosis between invaded and environmentally-degraded river reaches. Such misdiagnosis was evident in a regional fish index widely used in official monitoring programs. We recommend the application of FSpe and FOri to extensive clatasets from monitoring programs in order to generate valuable cross-system information about the impacts of non-native species and habitat degradation, even in species-poor systems. Scoring non-native species apart from habitat degradation in the indices used to determine ecosystem health is essential to develop better management strategies. (C) 2013 Elsevier B.V. All rights reserved

Trafficking of Siderophore Transporters in Saccharomyces cerevisiae and Intracellular Fate of Ferrioxamine B Conjugates

We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1-mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process