Heterogeneity in the processing of ClC-5 mutants related to Dent disease

International audienceMutations in the electrogenic Cl-/H+ exchanger ClC-5 gene CLCN5 are frequently associated with Dent disease, an X-linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in X. laevis oocytes and in HEK293 cells of 9 previously reported, pathogenic, missense mutations of ClC-5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild-type ClC 5, but exhibited reduced currents. Three mutants displayed improper N-glycosylation, and were non-functional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC-5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC-5 loss-of-function in Dent disease

Modelling the response to vaccine in non-human primates to define SARS-CoV-2 mechanistic correlates of protection

The definition of correlates of protection is critical for the development of next-generation SARS-CoV-2 vaccine platforms. Here, we propose a model-based approach for identifying mechanistic correlates of protection based on mathematical modelling of viral dynamics and data mining of immunological markers. The application to three different studies in non-human primates evaluating SARS-CoV-2 vaccines based on CD40-targeting, two-component spike nanoparticle and mRNA 1273 identifies and quantifies two main mechanisms that are a decrease of rate of cell infection and an increase in clearance of infected cells. Inhibition of RBD binding to ACE2 appears to be a robust mechanistic correlate of protection across the three vaccine platforms although not capturing the whole biological vaccine effect. The model shows that RBD/ACE2 binding inhibition represents a strong mechanism of protection which required significant reduction in blocking potency to effectively compromise the control of viral replication.Initiative for the creation of a Vaccine Research InstituteInfrastructure nationale pour la modélisation des maladies infectieuses humaine

Recherche et pratique musicales en bibliothèque : une expérience partagée entre bibliothécaires, élèves et professeurs

La cité de la musique à Paris Comme toutes les bibliothèques d’écoles spécialisées, celle du Conservatoire national supérieur de musique et de danse de Paris (CNSMDP) a toujours entretenu un lien très fort avec l’institution et ses pratiques pédagogiques. Ouverte à tous, son principal public est aujourd’hui celui des musiciens et dans une moindre mesure des danseurs élèves du Conservatoire. La manière dont ils fréquentent ou « contournent » le lieu est une source d’interrogation : malgré des..

Launcher Sequential Analysis

International audienceThis paper addresses the use of a set of techniques (domain specific language, constraint’s solving, model checking and simulation) to capture, implement and verify the sequential analysis of a launcher

NMR-Based Method for Intramolecular 13 C Distribution at Natural Abundance Adapted to Small Amounts of Glucose

International audienceQuantitative nuclear magnetic resonance (NMR) for isotopicmeasurements,known as irm-NMR (isotope ratio measured by NMR), is well suited forthe quantitation of C-13-isotopomers in position-specificisotope analysis and thus for measuring the carbon isotope composition(& delta;C-13, mUr) in C-atom positions. Irm-NMR has alreadybeen used with glucose after derivatization to study sugar metabolismin plants. However, up to now, irm-NMR has exploited a "single-pulse"sequence and requires a relatively large amount of material and longexperimental time, precluding many applications with biological tissuesor extracts. To reduce the required amount of sample, we investigatedthe use of 2D-NMR analysis. We adapted and optimized the NMR sequenceso as to be able to analyze a small amount (10 mg) of a glucose derivative(diacetonide glucofuranose, DAGF) with a precision better than 1 mUrat each C-atom position. We also set up a method to correct raw dataand express C-13 abundance on the usual & delta;C-13 scale (& delta;-scale). In fact, due to the distortion associatedwith polarization transfer and spin manipulation during 2D-NMR analyses,raw C-13 abundance is found to be on an unusual scale. Thiswas compensated for by a correction factor obtained via comparativeanalysis of a reference material (commercial DAGF) using both previous(single-pulse) and new (2D) sequences. Glucose from different biologicalorigins (CO2 assimilation metabolisms of plants, namely,C-3, C-4, and CAM) was analyzed with the two sequencesand compared. Validation criteria such as selectivity, limit of quantification,precision, trueness, and robustness are discussed, including in theframework of green analytical chemistry

Combination of C-13 and H-2 SNIF-NMR isotopic fingerprints of vanillin to control its precursors

Synthetic vanillin is the flavouring most used in agro-food industries, but more and more frequently consumers are now turning to natural origin. Vanilla planifolia (Andrews) is the most commercialized species of pods in the world and is mainly produced in Madagascar. However, periodically the production of V. planifolia suffers from poor climatic conditions, with the resulting degradation of both the quality and the quantity of the pods. Consequently, the price of vanilla pods rose to record in 2017 and the risk of fraud has increased in response. Analytical methods based on isotopic compositions have already proven their ability to ensure the authenticity of natural vanillin. In 2006, H-2 SNIF-NMR (site-specific natural isotopic fractionation by nuclear magnetic resonance) was approved as official method by the Association of Official Analytical Chemists (AOAC 2006.05). However, this method is time consuming and the extraction of 1 g is cumbersome for the finished products. The information brought by combined H-2 and C-13 SNIF-NMR profiles was compared using chemometric tools to determine the best routine tool to improve both the global analysis time and the potential of detection. As a result of this work the ability of the SNIF-NMR method to verify the authenticity of vanillin has been improved, in particular by providing the first means to discern the geographical origin of vanilla pods. Furthermore, C-13 NMR using pulse sequences such as INEPT (insensitive nuclei enhanced by polarization transfer) offers the possibility to improve the sensitivity of the analysis, with a reduced quantity of product (less than 50 mg) and a shorter analysis time, which will facilitate the study of the finished matrices as well as clearly discriminating the origins of the vanilla flavourings

Difficulties in Differentiating Natural from Synthetic Alkaloids by Isotope Ratio Monitoring using (13) C Nuclear Magnetic Resonance Spectrometry

International audienceWithin the food and pharmaceutical industries, there is an increasing legislative requirement for the accurate labeling of the products origin. A key feature of this is to indicate whether the product is of natural or synthetic origin. With reference to this context, we have investigated three alkaloids commonly exploited for human use: nicotine, atropine, and caffeine. We have measured by (13) C nuclear magnetic resonance spectrometry the position-specific distribution of (13) C at natural abundance within several samples of each of these target molecules. This technique is well suited to distinguishing between origins, as the distribution of the (13) C isotope reflects the primary source of the carbon atoms and the process by which the molecule was (bio)synthesized. Our findings indicate that labeling can be misleading, especially in relation to a supplied compound being labeled as synthetic even though its (13) C profile indicates a natural origin

FullSpectrumIsotopic(13)CNMRUsingPolarizationTransferforPosition-Specifi cIsotopeAnalysis

International audienceFor the last ten years, quantitative isotope ratio monitoring C-13 NMR (irm-C-13 NMR) has been successfully tested and proven as an efficient tool for the determination of position-specific C-13/C-12 ratios. Several applications in different domains have shown the interest in this technique. In the context of origin assignment, the possibility to track the distribution network of illicit drugs or cutting agents is of prime importance. However irm-C-13 NMR still suffers from a relative lack of sensitivity limiting its dissemination among control laboratories. Improvements were proposed to reduce experiment time by using the INEPT sequence (''Insensitive Nuclei Enhanced by Polarization Transfer'') based on polarization transfer from highly sensitive H-1 to less sensitive( 13)C. Several applications based on the use of the one bond scalar coupling between H-1 and C-13 ((1)J(CH)) have shown the potential of this methodology in terms of short experimental duration. However, the isotopic information given by quaternary carbons was lost. The aim of this study is to extend this approach by using short- and long-range coupling ((1)J(CH), (2)J(CH), and (3)J(CH)) in order to have access to all C-13/C-12 position-specific ratios, i.e., acquisition of the full spectrum (FS-INEPT). It is shown that this innovative tool provides both sensitivity gain-thanks to the long-range polarization transfer-and appropriate repeatability. The relative isotopic profiles allowed the classification of two cutting agents, caffeine and paracetamol (acetaminophen), according to their origin, as it was previously observed with ``classical'' irm-C-13 NMR but consuming much less sample and/or reducing the experimental time

Comparative study of (13)C composition in ethanol and bulk dry wine using isotope ratio monitoring by mass spectrometry and by nuclear magnetic resonance as an indicator of vine water status

The potential of wine (13)C isotope composition (δ(13)C) is presented to assess vine water status during grape ripening. Measurements of δ(13)C have been performed on a set of 32 authentic wines and their ethanol recovered after distillation. The data, obtained by isotope ratio monitoring by mass spectrometry coupled to an elemental analyser (irm-EA/MS), show a high correlation between δ(13)C of the bulk wine and its ethanol, indicating that the distillation step is not necessary when the wine has not been submitted to any oenological treatment. Therefore, the ethanol/wine δ(13)C correlation can be used as an indicator of possible enrichment of the grape must or the wine with exogenous organic compounds. Wine ethanol δ(13)C is correlated to predawn leaf water potential (R (2) = 0.69), indicating that this parameter can be used as an indicator of vine water status. Position-specific (13)C analysis (PSIA) of ethanol extracted from wine, performed by isotope ratio monitoring by nuclear magnetic resonance (irm-(13)C NMR), confirmed the non-homogenous repartition of (13)C on ethanol skeleton. It is the δ(13)C of the methylene group of ethanol, compared to the methyl moiety, which is the most correlated to predawn leaf water potential, indicating that a phase of photorespiration of the vine during water stress period is most probably occurring due to stomata closure. However, position-specific (13)C analysis by irm-(13)C NMR does not offer a greater precision in the assessment of vine water status compared to direct measurement of δ(13)C on bulk wine by irm-EA/MS