Transient Alteration of Cellular Redox Buffering before Irradiation Triggers Apoptosis in Head and Neck Carcinoma Stem and Non-Stem Cells

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive and recurrent malignancy owing to intrinsic radioresistance and lack of induction of apoptosis. The major focus of this work was to design a transient glutathione depleting strategy during the course of irradiation of HNSCC in order to overcome their radioresistance associated with redox adaptation. Methodology/Principal Findings: Treatment of SQ20B cells with dimethylfumarate (DMF), a GSH-depleting agent, and L-Buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis 4 h before a 10 Gy irradiation led to the lowering of the endogenous GSH content to less than 10 % of that in control cells and to the triggering of radiation-induced apoptotic cell death. The sequence of biochemical events after GSH depletion and irradiation included ASK-1 followed by JNK activation which resulted in the triggering of the intrinsic apoptotic pathway through Bax translocation to mitochondria. Conclusions: This transient GSH depletion also triggered radiation-induced cell death in SQ20B stem cells, a key event to overcome locoregional recurrence of HNSCC. Finally, our in vivo data highlight the relevance for further clinical trials o

Freins et facilitateurs de la relation entre le médecin généraliste et le patient autiste sans déficience intellectuelle : point de vue des patients et pistes de réflexion pour une amélioration de la relation de soins

Thèse d'exercice soutenue par Tiphaine Robert (spécialité : psychiatrie) sous la direction de Julien Dubreucq, et par Raphaëlle Rousson (spécialité : médecine générale) sous la direction de Virginie Ardito.INTRODUCTION : Les personnes autistes présentent un risque accru de comorbidités médicales comme les troubles intestinaux, l’hypertension, les troubles du sommeil et les troubles anxieux, et la dépression. Cependant elles rapportent des difficultés et appréhensions communicationnelles, relationnelles et organisationnelles qui freinent l’accès aux soins. Le MG est un acteur de soin de première ligne et le coordonnateur du parcours de santé des patients. Il existe peu d’études sur la relation MG / patients chez les patients TSA-SDI. L’objectif de cette étude est de recueillir leur ressenti à propos de cette relation, et de dégager des pistes pour en optimiser la qualité. METHODOLOGIE : Une méthodologie qualitative a été utilisée. Les participants ont été recrutés au Centre Expert des Troubles du Spectre Autistique Sans Déficience Intellectuelle de Grenoble. Les données ont été recueillies au moyen d’entretiens individuels semi-dirigés présentiels ou téléphoniques, enregistrés puis retranscrits intégralement par écrit et anonymisées. Les données ont été codées par les deux chercheuses en aveugle, afin d’en dégager des codes, sous-thèmes et thèmes, puis a eu lieu une triangulation de l’analyse des données. RESULTATS : La qualité des relations entretenues par les participants avec leur MG était hétérogène. Elle était notamment influencée par les caractéristiques liées au TSA (particularités sensorielles, spécificités communicationnelles et cognitives) et les antécédents médicaux (souvent marqués par des errances diagnostiques, des prises en charges multiples et des ruptures de soin). Il a été retrouvé une influence positive des qualités humaines telles que l’empathie, la bienveillance et l’écoute, en opposition au sentiment d’être jugé négativement ou remis en cause. Une attitude calme est souhaitée, ainsi qu’une attention portée aux explications et au consentement, et un positionnement d’égal à égal. Ont été évoquées des adaptations possibles dans la prise en charge médicale, comme l’attention aux particularités sensorielles, aux comorbidités somatiques et psychiatriques courantes chez les autistes, et aux difficultés quotidiennes (ex : le travail). Des efforts de formation sont souhaités pour mieux répondre aux besoins des autistes TSA-SDI. CONCLUSION : La qualité de la relation avec son MG, évaluée par les autistes SDI, est influencée par les facteurs suivants : expériences préalables négatives, perception que son médecin n’est pas formé sur les TSA et/ou présente des attitudes stigmatisantes, difficulté voire refus du professionnel de mettre en place des adaptations répondant aux besoins des personnes autistes. A l’inverse, la perception que son interlocuteur présente des qualités humaines et d’écoute, et l’installation d’un véritable rapport collaboratif, facilite l’alliance thérapeutique. Nous avons pu dégager des adaptations concrètes possibles, qu’il conviendrait de personnaliser en interrogeant les patients sur leurs besoins, difficultés et particularités propres : par exemple veiller à être calme et à expliquer clairement, demander le consentement pour tout geste, passer par l’écrit, réduire nos attentes en matière de « politesse sociale », diminuer la charge sensorielle du cabinet (ex : luminosité). Ce travail pourrait donner lieu à l’élaboration d’outils pratiques à l’usage des MG pour l’évaluation des besoins et les adaptations possibles. Des études interrogeant le ressenti des MG envers les patients TSA pourraient compléter ce travail

Combined Use of In Silico and In Vitro Splicing Assays for Interpretation of Genomic Variants of Unknown Significance in Cardiomyopathies and Channelopathies

The identification of molecular anomalies in patients with inherited arrhythmias or cardiomyopathies is a multi challenge due to: i) the number of genes involved; ii) the number of polymorphisms and the fact that most mutations are private; and iii) the variable degree of penetrance which complicates family segregation study. Consequently, a number of unclassified variants (UV) are found in patients’ DNA and some (outside the canonical GT/AG) may affect splicing. Mutational screening on the most prevalent genes involved in arrythmias syndromes or in cardiomyopathies was performed on a cohort made up of 740 unrelated French index probands. To identify splice variants among the identified UVs, a combination of available in silico and in vitro tools were used since transcript is not available. Using this approach, 10 previously identified UVs were reclassified as disease-causing mutations and, more precisely, as haploinsufficiency mutations rather than dominant-negative mutations. Most of them (7 of 10) were observed in MYBPC3. Our study highlighted the importance of the combined use of in silico and in vitro splicing assays to improve the prediction of the functional impact of identified genetic variants. The primary challenge now, with new sequencing technologies, is to distinguish between background polymorphisms and pathogenic mutations. Since splice site mutations can account for almost 50% of disease-causing mutations, recognizing them from among other variations is essential

Diversity and complexity of ceramide generation after exposure of jurkat leukemia cells to irradiation.

International audiencePURPOSE: To define which intracellular pools of sphingomyelin and ceramide are involved in the triggering of apoptosis of Jurkat leukemia cells in response to gamma-ray exposure. METHODS AND MATERIALS: We examined the kinetics of ceramide generation at the whole-cell level and in different subcellular compartments (plasma membrane rafts, mitochondria, and endoplasmic reticulum) after irradiation with photons. Ceramide was measured by high-performance liquid chromatography or after pulse labeling experiments, and the presence of sphingomyelinase within mitochondria was assessed by electron microscopy. RESULTS: Irradiation of Jurkat leukemia cells resulted in the sequential triggering of sphingomyelin hydrolysis, followed by de novo synthesis that led to a late ceramide response (from 24 h) correlated with the triggering of apoptosis. At the subcellular level, pulse-label experiments, using [(3)H]-palmitate as a precursor, strengthened the involvement of the radiation-induced sphingomyelin breakdown and revealed a very early peak (15 min) of ceramide in plasma membrane rafts. A second peak in mitochondria was measured 4 h after irradiation, resulting from an increase of the sphingomyelin content relating to the targeting of acid sphingomyelinase toward this organelle. CONCLUSION: These data confirm that ceramide is a major determinant in the triggering of radiation-induced apoptosis and highlight the complexity of the sequential compartment-specific ceramide-mediated response of Jurkat leukemia cells to gamma-rays

A new C-terminal hERG mutation A915fs+47X associated with symptomatic LQT2 and auditory-trigger syncope.: New LQT2-related hERG mutation A915fs+47X

Expected publication in November issue of Heart Rhythm. Now after proof correction.International audienceBACKGROUND: A novel mutation of hERG (A915fs+47X) was discovered in a 32-year-old woman with torsades de pointes, long QTc interval (515 ms), and syncope upon auditory trigger. OBJECTIVE: We explored whether the properties of this mutation could explain the pathology. METHODS: Whole-cell A915fs+47X (del) and wild-type (WT) currents were recorded in transiently transfected COS7 cells or Xenopus oocytes. Western blots and sedimentation analysis of del/WT hERG were used to analyze protein expression, assembly, and trafficking. RESULTS: The tail current density at -40 mV after a 2-s depolarization to +40 mV in COS7 cells expressing del was 36% of that for WT. Inactivation was 1.9-fold to 2.8-fold faster in del versus WT between -60 and +60 mV. In the range -60 to -10 mV, we found that a nondeactivating fraction of current was increased in del at the expense of a rapidly deactivating fraction, with a slowly deactivating fraction being unchanged. In Xenopus oocytes, expression of del alone produced 38% of WT currents, whereas coexpression of 1/2 WT + 1/2 del produced 49.8%. Furthermore, the expression of del protein at the cell surface was reduced by about 50%. This suggests that a partial trafficking defect of del contributes to the reduction in del current densities and to the dominant negative effect when coexpressed with WT. In model simulations, the mutation causes a 10% prolongation of action potential duration. CONCLUSION: Decreased current levels caused by a trafficking defect may explain the long QT syndrome observed in our patient

Chromosomal translocations t(4;14), t(11;14) and proliferation rate stratify patients with mature plasma cell myelomas into groups with different survival probabilities: a molecular epidemiologic study on tissue microarrays

Plasma cell myelomas (PMs) exhibit clinical and molecular heterogeneity. To date, morphology and immunohistochemistry on bone marrow trephines are of limited value to stratify patients into different prognostic categories. However, some chromosomal translocations are of prognostic and/or of predictive importance in PMs. In this study, the prognostic significance of morphology, CyclinD1 expression, proliferation index (Mib1) and presence of the translocations FGFR3/IgH [t(4;14)] and CCND1/IgH [t(11;14)] are compared in 119 patients with PM. Immunohistochemistry and fluorescence in situ hybridization analysis were carried out on a tissue microarray containing bone marrow trephines. Hundred and one PMs showed a mature morphology whereas 10 were immature. All but one PM carrying a translocation showed a mature morphology. Patients with a t(4;14) (12%) had a statistically significant shorter 1-year survival (P=0.004), whereas those with a t(11;14) (21%) had a trend towards a better clinical outcome. CyclinD1 protein expression was not significantly associated with survival. Besides the t(4;14), an immature morphology (P<0.001) and a proliferation index (Mib1) of more than 10% (P=0.002) were associated with a significantly worse outcome. A high occurrence of strong CyclinD1 protein expression in the tumor cells was predictive of either a t(11;14) or of a low level amplification of the CCND1 gene, suggesting that different molecular mechanisms may have lead to an over-expression of the CyclinD1 protein in PMs. These findings demonstrate that a high proliferation rate and translocations involving the IgH locus can stratify mature PMs into groups with distinct survival probabilities