Caractérisation des complexes contenant l'hélicase à motif DEAD DDX6 dans les cellules humaines

P-bodies are cytoplasmic granules. Their function is unknown, but they are conserved from the yeast to humans, suggesting an important role through eukaryotes. The expression inhibition of several proteins localized in P-bodies leads to their disassembly. In most cases, a cellular stress induced by arsenite treatment causes P-body assembly, except in cells depleted for DDX6. Since this observation showed that DDX6 is necessary for P-body assembly, to study this protein is a good starting point to further understand the role of P-bodies. This 54 kDa DEAD-box helicase interacts with proteins of the CPEB repression complex in xenopus oocytes, but also with protein of the déadénylation and dacapping complex in yeasts, drosophila and mammals, and with proteins of the miRISC complex. Our project was to determine the DDX6 main protein partners in human cells. To do so, DDX6 containing complexes were purified using HEK293 cells transfected with a plasmid encoding for the FLAG-DDX6-HA. Over 300 partners were identified by mass spectrometry. Three main DDX6 containing complexes were highlighted in human cells: A “CPEB-like” repression complex, the decapping complex, and a complex containing ATXN2 and ATXN2L proteins. Exon junction complex core proteins were also identified as DDX6 partners, raising the possibility that DDX6 interacts with mRNA not yet translated. A large number of ribosomal proteins were also identified with high scores, suggesting that DDX6 interacts with actively translated mRNA. Analyze of cytoplasmic lysates on sucrose gradients showed that DDX6 is partially associated with polysomes. To conclude, these observations showed the multiple roles of DDX6 in human cell, between mRNA repression and degradation. The helicase could allow the recruitment of the repression complex on actively translated mRNA. In a nutshell, the multiple binding of DDX6 on one mRNA could be a way to enable the fixation of repression and degradation complexes at the same time, on the same mRNA.Les P-bodies sont des granules cytoplasmiques de fonction inconnue. Ils sont néanmoins conservés de la levure à l’homme, suggérant un rôle important chez les eucaryotes. L’analyse de l’influence de l’expression de certaines protéines pouvant se localiser dans ces granules a permis de mettre en évidence le rôle crucial de DDX6 dans l’assemblage des P-bodies. En effet, l’inhibition de l’expression de DDX6 dans les cellules humaines empêche l’assemblage des P-bodies. L’étude de la protéine DDX6 semblait donc être un bon point de départ pour comprendre d’avantage le rôle des P-bodies. Cette hélicase à motif DEAD de 54 kDa interagit avec des protéines du complexe répression de la traduction CPEB chez le Xénope, mais également avec des protéines des complexes de dégradation 5’-3’ et 3’-5 ‘ des ARNm chez les mammifères, les levures et les drosophiles, ou encore avec les protéines Argonautes du complexe miRISC chez les mammifères. Nos travaux se sont donc intéressés à déterminer les principales fonctions de DDX6 dans les cellules humaines. Les complexes protéiques contenant DDX6 ont été purifiés à partir de lysats cytoplasmiques de cellules HEK293 transfectées avec un plasmide codant pour la protéine FLAG-DDX6-HA. Plus de 300 partenaires protéiques ont été identifiés en spectrométrie de masse. Trois complexes majeurs contenant DDX6 ont été mis en évidence : un complexe de répression « CPEB-like », le complexe de décoiffage des ARNm, et un complexe contenant les ataxin-2 et ataxin-2-like. Les protéines du cœur du complexe de jonction d’exons sont également en interaction avec DDX6, suggérant que DDX6 interagit avec des ARNm tout juste sortis du noyau. Enfin, le grand nombre et les hauts scores avec lesquels ont été identifiés les protéines ribosomales nous ont conduit à analyser la présence de DDX6 au niveau des polysomes. L’analyse de lysats cytoplasmiques sur gradient de sucrose nous a permis de mettre en évidence l’association d’une fraction de DDX6 aux polysomes. Toutes ces observations mettent en évidence le rôle multiple de DDX6 entre répression et dégradation des ARNm, dans les cellules humaines. L’hélicase pourrait permettre le recrutement du complexe de répression par des ARNm activement traduits. La fixation multiple de DDX6 à l’ARNm pourrait être un moyen de recruter simultanément les complexes de répression et de dégradation des ARNm sur un même ARNm

How can we combat multicenter variability in MR radiomics? Validation of a correction procedure

International audienc

DMTs and Covid-19 severity in MS: a pooled analysis from Italy and France

We evaluated the effect of DMTs on Covid-19 severity in patients with MS, with a pooled-analysis of two large cohorts from Italy and France. The association of baseline characteristics and DMTs with Covid-19 severity was assessed by multivariate ordinal-logistic models and pooled by a fixed-effect meta-analysis. 1066 patients with MS from Italy and 721 from France were included. In the multivariate model, anti-CD20 therapies were significantly associated (OR = 2.05, 95%CI = 1.39–3.02, p < 0.001) with Covid-19 severity, whereas interferon indicated a decreased risk (OR = 0.42, 95%CI = 0.18–0.99, p = 0.047). This pooled-analysis confirms an increased risk of severe Covid-19 in patients on anti-CD20 therapies and supports the protective role of interferon

Characterization of the DEAD-box DDX6 containing complexes in human cells

Les P-bodies sont des granules cytoplasmiques de fonction inconnue. Ils sont néanmoins conservés de la levure à l’homme, suggérant un rôle important chez les eucaryotes. L’analyse de l’influence de l’expression de certaines protéines pouvant se localiser dans ces granules a permis de mettre en évidence le rôle crucial de DDX6 dans l’assemblage des P-bodies. En effet, l’inhibition de l’expression de DDX6 dans les cellules humaines empêche l’assemblage des P-bodies. L’étude de la protéine DDX6 semblait donc être un bon point de départ pour comprendre d’avantage le rôle des P-bodies. Cette hélicase à motif DEAD de 54 kDa interagit avec des protéines du complexe répression de la traduction CPEB chez le Xénope, mais également avec des protéines des complexes de dégradation 5’-3’ et 3’-5 ‘ des ARNm chez les mammifères, les levures et les drosophiles, ou encore avec les protéines Argonautes du complexe miRISC chez les mammifères. Nos travaux se sont donc intéressés à déterminer les principales fonctions de DDX6 dans les cellules humaines. Les complexes protéiques contenant DDX6 ont été purifiés à partir de lysats cytoplasmiques de cellules HEK293 transfectées avec un plasmide codant pour la protéine FLAG-DDX6-HA. Plus de 300 partenaires protéiques ont été identifiés en spectrométrie de masse. Trois complexes majeurs contenant DDX6 ont été mis en évidence : un complexe de répression « CPEB-like », le complexe de décoiffage des ARNm, et un complexe contenant les ataxin-2 et ataxin-2-like. Les protéines du cœur du complexe de jonction d’exons sont également en interaction avec DDX6, suggérant que DDX6 interagit avec des ARNm tout juste sortis du noyau. Enfin, le grand nombre et les hauts scores avec lesquels ont été identifiés les protéines ribosomales nous ont conduit à analyser la présence de DDX6 au niveau des polysomes. L’analyse de lysats cytoplasmiques sur gradient de sucrose nous a permis de mettre en évidence l’association d’une fraction de DDX6 aux polysomes. Toutes ces observations mettent en évidence le rôle multiple de DDX6 entre répression et dégradation des ARNm, dans les cellules humaines. L’hélicase pourrait permettre le recrutement du complexe de répression par des ARNm activement traduits. La fixation multiple de DDX6 à l’ARNm pourrait être un moyen de recruter simultanément les complexes de répression et de dégradation des ARNm sur un même ARNm.P-bodies are cytoplasmic granules. Their function is unknown, but they are conserved from the yeast to humans, suggesting an important role through eukaryotes. The expression inhibition of several proteins localized in P-bodies leads to their disassembly. In most cases, a cellular stress induced by arsenite treatment causes P-body assembly, except in cells depleted for DDX6. Since this observation showed that DDX6 is necessary for P-body assembly, to study this protein is a good starting point to further understand the role of P-bodies. This 54 kDa DEAD-box helicase interacts with proteins of the CPEB repression complex in xenopus oocytes, but also with protein of the déadénylation and dacapping complex in yeasts, drosophila and mammals, and with proteins of the miRISC complex. Our project was to determine the DDX6 main protein partners in human cells. To do so, DDX6 containing complexes were purified using HEK293 cells transfected with a plasmid encoding for the FLAG-DDX6-HA. Over 300 partners were identified by mass spectrometry. Three main DDX6 containing complexes were highlighted in human cells: A “CPEB-like” repression complex, the decapping complex, and a complex containing ATXN2 and ATXN2L proteins. Exon junction complex core proteins were also identified as DDX6 partners, raising the possibility that DDX6 interacts with mRNA not yet translated. A large number of ribosomal proteins were also identified with high scores, suggesting that DDX6 interacts with actively translated mRNA. Analyze of cytoplasmic lysates on sucrose gradients showed that DDX6 is partially associated with polysomes. To conclude, these observations showed the multiple roles of DDX6 in human cell, between mRNA repression and degradation. The helicase could allow the recruitment of the repression complex on actively translated mRNA. In a nutshell, the multiple binding of DDX6 on one mRNA could be a way to enable the fixation of repression and degradation complexes at the same time, on the same mRNA

Estimation of purkinje activation from ECG:An intermittent left bundle branch block study

In pressInternational audienceModelling the cardiac electrophysiology (EP) can help understand pathologies and predict the response to therapies such as cardiac resynchronization. To this end, estimating patient-specific model parameters is crucial. In the case of patients with bundle branch blocks (BBB), part of the Purkinje system is often affected. The aim of this work is to estimate the activation of the right and left Purkinje systems from standard non-invasive techniques: magnetic resonance imaging (MRI) and 12-lead electrocardiogram (ECG). As it is difficult to differentiate the contribution of the Purkinje system, this work relies on a particular intermittent left BBB (LBBB) case where both LBBB and absence of LBBB (ALBBB) were recorded on different 12-lead ECGs. First, an efficient forward EP model is proposed by coupling a Mitchell-Schaeffer cardiac model with a current dipole formulation that simulates the ECG. We used the Covariance Matrix Adaptation Evolution Strategy (CMA-ES) algorithm to optimize the 3 parameters by minimizing the error with the real ECG. The estimation of conduction velocity (CV) parameters for LBBB and ALBBB shows a good agreement on the myocardial CV (0.39m/s for ABBB, 0.40m/s for LBBB), while the estimation of the left Purkinje CV seems to identify the pathology (1.32m/s for ALBBB, 0.49m/s for LBBB). Finally, the plots of the simulated 12-lead ECGs together with the ground truth ECGs indicate similar shapes

Multivesicular Vesicles: Preparation and Applications

Multivesicular vesicles, i. e. vesicles containing internal, non-concentrically arranged smaller vesicles, are artificial, polymolecular compartment systems, which can be prepared from naturally occurring or fully synthetic bilayer-forming amphiphiles in aqueous solution through various guided assembly procedures. The general concepts for the preparation of such “vesicles-inside-vesicles” systems (also called “vesosomes”) are summarized, and the different methods used are compared. Selected applications of multivesicular vesicles in the field of drug delivery, cell-mimicking model systems, and as versatile compartments for the investigation of confined reactions are discussed

Transfer Learning from Simulations on a Reference Anatomy for ECGI in Personalised Cardiac Resynchronization Therapy

International audienceGoal: Non-invasive cardiac electrophysiology (EP) model personalisation has raised interest for instance in the scope of predicting EP cardiac resynchronization therapy (CRT) response. However, the restricted clinical applicability of current methods is due in particular to the limitation to simple situations and the important computational cost. Methods: We propose in this manuscript an approach to tackle these two issues. First, we analyse more complex propagation patterns (multiple onsets and scar tissue) using relevance vector regression and shape dimensionality reduction on a large simulated database. Second, this learning is performed offline on a reference anatomy and transferred onto patient-specific anatomies in order to achieve fast personalised predictions online. Results: We evaluated our method on a dataset composed of 20 dyssynchrony patients with a total of 120 different cardiac cycles. The comparison with a commercially available electrocardiographic imaging (ECGI) method shows a good identification of the cardiac activation pattern. From the cardiac parameters estimated in sinus rhythm, we predicted 5 different paced patterns for each patient. The comparison with the body surface potential mappings (BSPM) measured during pacing and the ECGI method indicates a good predictive power. Conclusion: We showed that learning offline from a large simulated database on a reference anatomy was able to capture the main cardiac EP characteristics from non-invasive measurements for fast patient-specific predictions. Significance: The fast CRT pacing predictions are a step forward to a non-invasive CRT patient selection and therapy optimisation, to help clinicians in these difficult tasks

Nuclear expression of mitochondrial ND4 leads to the protein assembling in complex I and prevents optic atrophy and visual loss

Leber hereditary optic neuropathy is due to mitochondrial DNA mutations; in â¼70% of all cases, a point mutation in the mitochondrial NADH dehydrogenase subunit 4, ND4, gene leads to central vision loss. We optimized allotopic expression (nuclear transcription of a gene that is normally transcribed inside the mitochondria) aimed at designing a gene therapy for ND4; its coding sequence was associated with the cis-acting elements of the human COX10 mRNA to allow the efficient mitochondrial delivery of the protein. After ocular administration to adult rats of a recombinant adeno-associated viral vector containing the human ND4 gene, we demonstrated that: (i) the sustained expression of human ND4 did not lead to harmful effects, instead the human protein is efficiently imported inside the mitochondria and assembled in respiratory chain complex I; (ii) the presence of the human protein in the experimental model of Leber hereditary optic neuropathy significantly prevents retinal ganglion cell degeneration and preserves both complex I function in optic nerves and visual function. Hence, the use of optimized allotopic expression is relevant for treating mitochondrial disorders due to mutations in the organelle genome

Smurfness‐based two‐phase model of ageing helps deconvolve the ageing transcriptional signature

International audienceAgeing is characterised at the molecular level by six transcriptional ‘hallmarks of ageing’, that are commonly described as progressively affected as time passes. By contrast, the ‘Smurf’ assay separates high‐and‐constant‐mortality risk individuals from healthy, zero‐mortality risk individuals, based on increased intestinal permeability. Performing whole body total RNA sequencing, we found that Smurfness distinguishes transcriptional changes associated with chronological age from those associated with biological age. We show that transcriptional heterogeneity increases with chronological age in non‐Smurf individuals preceding the other five hallmarks of ageing that are specifically associated with the Smurf state. Using this approach, we also devise targeted pro‐longevity genetic interventions delaying entry in the Smurf state. We anticipate that increased attention to the evolutionary conserved Smurf phenotype will bring about significant advances in our understanding of the mechanisms of ageing