19 research outputs found
New correctors of the protein F508del-CFTR in the context of cystic fibrosis
La mucoviscidose est la maladie gĂ©nĂ©tique rĂ©cessive la plus frĂ©quente dans les populations caucasiennes. Elle est dĂ»e Ă des mutations dans le gĂšne CFTR qui code pour une protĂ©ine qui a une fonction canal chlorure et qui est exprimĂ©e au pĂŽle apical des Ă©pithĂ©liums sĂ©crĂ©toires. La mutation la plus frĂ©quente F508del altĂšre le repliement de la protĂ©ine, induisant sa dĂ©gradation prĂ©coce par le protĂ©asome et son absence Ă la membrane plasmique. La recherche fondamentale se focalise sur la protĂ©ine mutĂ©e et cherche Ă dĂ©couvrir des molĂ©cules capables dâadresser celle-ci au pĂŽle apical des cellules Ă©pithĂ©liales oĂč elle pourra remplir sa fonction de canal chlorure. Au sein du laboratoire, nous nous intĂ©ressons aux interactions de la protĂ©ine CFTR/ F508del-CFTR avec dâautres protĂ©ines. Nous avons dĂ©jĂ montrĂ© lâexistence dâune interaction non voulue entre F508del-CFTR et un filament intermĂ©diaire, la cytokĂ©ratine 8. En combinant une approche in-silico et du criblage haut dĂ©bit, nous avons identifiĂ© des molĂ©cules capables dâinterrompre ces interactions. Des tests fonctionnels sur des lignĂ©es cellulaires ainsi que sur des modĂšles murins ont montrĂ© une restauration de la fonction canal chlorure aprĂšs traitements avec ces molĂ©cules. Au cours de ma thĂšse, je me suis intĂ©ressĂ©e Ă lâune dâentre elles qui est la molĂ©cule « c407 ». Lâobjectif de ma thĂšse Ă©tait de comprendre les mĂ©canismes dâaction de cette molĂ©cule. Jâai Ă©galement Ă©valuĂ© lâefficacitĂ© des traitements actuels dans le contexte des allĂšles complexes de F508del-CFTR. Dans une seconde partie de ma thĂšse, jâai Ă©tudiĂ© lâeffet dâune cytokine (TNFα) sur la protĂ©ine mutĂ©e F508del-CFTR. De façon inattendue, jâai observĂ© que le TNFα, Ă des concentrations physiologiques, corrige le dĂ©faut de routage de la protĂ©ine F508del-CFTR. Cette observation pourrait expliquer une fonction rĂ©siduelle de la F508del-CFTR chez certains patients atteints de mucoviscidose. En conclusion, mes travaux ont permis de prĂ©ciser les mĂ©canismes dâaction dâun correcteur et de dĂ©couvrir un effet inattendu dâune cytokine pro-inflammatoire. Ces travaux permettent de relier la correction dâun dĂ©faut de routage au processus inflammatoire ouvrant ainsi un nouveau champ dâinvestigation.Cystic fibrosis is due to the loss of epithelial chloride transport caused by mutations in the CFTR gene, the most frequent mutation being F508del. One of the strategies developed to find new treatment for Cystic fibrosis (CF) is to discover compounds that correct the trafficking of F508del-CFTR to the plasma membrane. Using hypothesis-driven approach and combining modeling of NBD1, molecular docking and functional assays, we identified 4 compounds that correct F508del-CFTR function in cells (including human primary bronchial cells in culture) and F508del mice. New correctors probably act by interrupting the interaction between F508del-CFTR with keratin 8 (Odolczyk et al EMBO Mol Med 2013). During my PhD, I focused on one of those molecules, the "c407" molecule. The aim of my thesis was to investigate the mechanisms of action of this molecule. I have also evaluated the effectiveness of current treatments in the context of complex alleles F508del-CFTR. In the second part of my thesis, I studied the effect of a cytokine (TNFα) on the protein F508del-CFTR. Unexpectedly, I observed that the TNFα at physiological concentrations, corrects the trafficking of F508del-CFTR protein. This observation could explain a residual function of F508del-CFTR in some CF patients. In conclusion, my thesis helped to clarify the mechanisms of action of new correctors of F508del-CFTR
Molecular evidence of vector-borne pathogens in dogs and cats and their ectoparasites in Algiers, Algeria
International audienceIn Algeria, only limited information is currently available on the prevalence of emergent canine and feline vector-borne diseases. The aim of the present work was to detect by qPCR vector-associated bacteria in stray dogs and cats and their ectoparasites from Algiers. 18/117 (15.38%) dogs and 2/107 (1.87%) cats were positive for at least one vector-borne agent. Coxiella burnetii and Bartonella henselae were identified in 1/117 (0.85%) dog individually. Ehrlichia canis DNA was detected in 17/117 (14.52%) dogs. 1/107 (0.93%) cat was positive to C. burnetii and another 1/107 (0.93%) to B. henselae. DNA of Rickettsia massiliae, Rickettsia conorii and E. canis was detected in Rhipicephalus sanguineus. Cat fleas were infected with Rickettsia felis, B. henselae and Bartonella clarridgeiae. B. vinsonii subsp. berkhoffii was identified in Xenopsylla cheopis collected from dogs. The findings of this study indicate that dogs and cats from Algeria are exposed to multiple tick and flea-borne pathogens. (C) 2016 Elsevier Ltd. All rights reserved
Urinary shedding of pathogenic Leptospira in stray dogs and cats, Algiers: A prospective study
International audienceBackground Leptospirosis is an important worldwide zoonosis. This disease is caused by pathogenic species of the genus Leptospira which are maintained in the environment via chronic renal infection of carrier animals which can be asymptomatic excretors of the organisms in their urines and become a source of infection for humans and other hosts. The prevalence of animal leptospirosis in Algiers, Algeria, is unknown.Methodology/principal findings Real-time PCR and standard PCR and sequencing were used to detect pathogenic Leptospira organisms in the urines of stray dogs and cats in Algiers. In the presence of appropriate controls, none of the 107 cat urine samples were positive while 5/104 (4.8%) canine urine samples (asymptomatic mixed-breed dogs, three females and two males) were positive in two real-time PCR assays targeting the rrs and hsp genes. The positivity of these samples was confirmed by partial PCR-sequencing of the rpoB gene which yielded 100% sequence similarity with Leptospira interrogans reference sequence. In this study, L. interrogans prevalence was significantly higher in dogs aged < one year (16.46% - 29.41%) than in adults (0%) (P value = 0.0001) and then in the overall dog population (2.68% -4.8%) (P = 0.0007).Conclusions/significance These results suggest that dogs are maintenance hosts for zoonotic leptospirosis in Algiers, Algeria. To face this situation, effective canine vaccination strategies and raising public health awareness are mandatory. Further investigations incorporating a larger sample in more localities will be undertaken to document the epidemiology of urban animal leptospirosis in Algeria at large
Analysis of CLCNKB mutations at dimerâinterface, calciumâbinding site, and pore reveals a variety of functional alterations in ClCâKb channel leading to Bartter syndrome
International audiencePathological missense mutations in CLCNKB gene give a wide spectrum of clinical phenotypes in Bartter syndrome type III patients. Molecular analysis of the mutated ClC-Kb channels can be helpful to classify the mutations according to their functional alteration. We investigated the functional consequences of nine mutations in the CLCNKB gene causing Bartter syndrome. We first established that all tested mutations lead to decreased ClC-Kb currents. Combining electrophysiological and biochemical methods in Xenopus laevis oocytes and in MDCKII cells, we identified three classes of mutations. One class is characterized by altered channel trafficking. p.A210V, p.P216L, p.G424R, and p.G437R are totally or partially retained in the endoplasmic reticulum. p.S218N is characterized by reduced channel insertion at the plasma membrane and altered pH-sensitivity; thus, it falls in the second class of mutations. Finally, we found a novel class of functionally inactivated mutants normally present at the plasma membrane. Indeed, we found that p.A204T alters the pH-sensitivity, p.A254V abolishes the calcium-sensitivity. p.G219C and p.G465R are probably partially inactive at the plasma membrane. In conclusion, most pathogenic mutants accumulate partly or totally in intracellular compartments, but some mutants are normally present at the membrane surface and simultaneously show a large range of altered channel gating properties
An unexpected effect of TNF-α on F508del-CFTR maturation and function
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An unexpected effect of TNF-α on F508del-CFTR maturation and function [v1; ref status: indexed, http://f1000r.es/5jf]
Cystic fibrosis (CF) is a multifactorial disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which encodes a cAMP-dependent Cl- channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, with major consequences in the airways of patients with CF, characterized by both fluid transport abnormalities and persistent inflammatory responses. The relationship between the acute phase of inflammation and the expression of wild type (WT) CFTR or F508del-CFTR is poorly understood. The aim of the present study was to investigate this effect. The results show that 10 min exposure to TNF-alpha (0.5-50ng/ml) of F508del-CFTR-transfected HeLa cells and human bronchial cells expressing F508del-CFTR in primary culture (HBE) leads to the maturation of F508del-CFTR and induces CFTR chloride currents. The enhanced CFTR expression and function upon TNFα is sustained, in HBE cells, for at least 24 h. The underlying mechanism of action involves a protein kinase C (PKC) signaling pathway, and occurs through insertion of vesicles containing F508del-CFTR to the plasma membrane, with TNFα behaving as a corrector molecule. In conclusion, a novel and unexpected action of TNFα has been discovered and points to the importance of systematic studies on the roles of inflammatory mediators in the maturation of abnormally folded proteins in general and in the context of CF in particular
In silico model of the human ClC-Kb chloride channel: pore mapping, biostructural pathology and drug screening
International audienceThe human ClC-Kb channel plays a key role in exporting chloride ions from the cytosol and is known to be involved in Bartter syndrome type 3 when its permeation capacity is decreased. The ClC-Kb channel has been recently proposed as a potential therapeutic target to treat hypertension. In order to gain new insights into the sequence-structure-function relationships of this channel, to investigate possible impacts of amino-acid substitutions, and to design novel inhibitors, we first built a structural model of the human ClC-Kb channel using comparative modeling strategies. We combined in silico and in vitro techniques to analyze amino acids involved in the chloride ion pathway as well as to rationalize the possible role of several clinically observed mutations leading to the Bartter syndrome type 3. Virtual screening and drug repositioning computations were then carried out. We identified six novel molecules, including 2 approved drugs, diflusinal and loperamide, with Kd values in the low micromolar range, that block the human ClC-Kb channel and that could be used as starting point to design novel chemical probes for this potential therapeutic target
Concentration of components in the final reaction mixtures of the standard polymerase chain reaction assay used in this study.
<p>Concentration of components in the final reaction mixtures of the standard polymerase chain reaction assay used in this study.</p
Concentration of components in the final reaction mixtures of the real-time polymerase chain reaction (qPCR) assays used in this study.
<p>Concentration of components in the final reaction mixtures of the real-time polymerase chain reaction (qPCR) assays used in this study.</p