New Molecular Diagnosis Approaches — From the Identification of Mutations to their Characterization

Molecular diagnosis of cystic fibrosis is based on the detection of mutation in the CFTR gene, identified in 1989. During the past 20 years, thanks to evolutions of diagnostic techniques, our knowledge of mutation spectrum and pathophysiological mechanisms involved in the disease has significantly improved. Sanger sequencing and quantitative methods greatly contributed to the identification of the 2,000 sequence variations reported worldwide in CFTR. We are now entering the new technological age with the generalisation of Next Generation Sequencing (NGS) technologies in diagnostics laboratories. These high throughput approaches allow scanning for the entire CFTR locus, including deep intronic regions, and in parallel other candidate genes that possibly influence the clinical evolution of patients. However, this powerful technology poses new challenge in test interpretation. In this chapter, we review the current and new technologies used in molecular diagnostics of cystic fibrosis, particularly NGS approaches. We also present current and new bioinformatics tools available for the interpretation of variants and in vitro/ex vivo and in vivo techniques that can be used to improve the characterization of the functional impact of CFTR variations

Pregnane × Receptor (PXR) expression in colorectal cancer cells restricts irinotecan chemosensitivity through enhanced SN-38 glucuronidation

<p>Abstract</p> <p>Background</p> <p>Clinical efficacy of chemotherapy in colorectal cancer is subjected to broad inter-individual variations leading to the inability to predict outcome and toxicity. The topoisomerase I inhibitor irinotecan (CPT-11) is worldwide approved for the treatment of metastatic colorectal cancer and undergoes extensive peripheral and tumoral metabolism. PXR is a xenoreceptor activated by many drugs and environmental compounds regulating the expression of drug metabolism and transport genes in detoxification organs such as liver and gastrointestinal tract. Considering the metabolic pathway of irinotecan and the tissue distribution of Pregnane × Receptor (PXR), we hypothesized that PXR could play a key role in colon cancer cell response to irinotecan.</p> <p>Results</p> <p>PXR mRNA expression was quantified by RT-quantitative PCR in a panel of 14 colon tumor samples and their matched normal tissues. PXR expression was modulated in human colorectal cancer cells LS174T, SW480 and SW620 by transfection and siRNA strategies. Cellular response to irinotecan and its active metabolic SN38 was assessed by cell viability assays, HPLC metabolic profiles and mRNA quantification of PXR target genes. We showed that PXR was strongly expressed in colon tumor samples and displayed a great variability of expression. Expression of hPXR in human colorectal cancer cells led to a marked chemoresistance to the active metabolite SN38 correlated with PXR expression level. Metabolic profiles of SN38 showed a strong enhancement of SN38 glucuronidation to the inactive SN38G metabolite in PXR-expressing cells, correlated with an increase of UDPglucuronosyl transferases UGT1A1, UGT1A9 and UGT1A10 mRNAs. Inhibition of PXR expression by lentivirus-mediated shRNA, led to SN38 chemoresistance reversion concomitantly to a decrease of UGT1A1 expression and SN38 glucuronidation. Similarly, PXR mRNA expression levels correlated to UGT1A subfamily expression in human colon tumor biopsies.</p> <p>Conclusion</p> <p>Our results demonstrate that tumoral metabolism of SN38 is affected by PXR and point to potential therapeutic significance of PXR quantification in the prediction of irinotecan response. Furthermore, our observations are pharmacologically relevant since many patients suffering from cancer diseases are often exposed to co-medications, food additives or herbal supplements able to activate PXR. A substantial part of the variability observed among patients might be caused by such interactions</p

Ascospore release and survival in Sclerotinia sclerotiorum

The release and survival of ascospores of a UK Sclerotinia sclerotiorum isolate were studied. Apothecia placed in a spore clock apparatus with different lighting regimes at 15 °C released ascospores continuously with an increasing rate for the duration of experiments (72–84 h). Spore release was not confined to light or dark periods in alternating regimes and occurred in continuous dark or light. Ascospores were released in both saturated air (90–95% rh) and at 65–75% rh. High temperature and rh were detrimental to ascospore survival but spore viability was maintained for longer periods than previously reported. The significance of these results in relation to disease control is discussed

Reference gene selection for head and neck squamous cell carcinoma gene expression studies

<p>Abstract</p> <p>Background</p> <p>It is no longer adequate to choose reference genes blindly. We present the first study that defines the suitability of 12 reference genes commonly used in cancer studies (<it>ACT, ALAS, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPL27, RPS29, SHAD </it>and <it>TBP</it>) for the normalization of quantitative expression data in the field of head and neck squamous cell carcinoma (HNSCC).</p> <p>Results</p> <p>Raw expression levels were measured by RT-qPCR in HNSCC and normal matched mucosa of 46 patients. We analyzed the expression stability using geNorm and NormFinder and compared the expression levels between subgroups. In HNSCC and/or normal mucosa, the four best normalization genes were <it>ALAS, GAPDH, RPS18 </it>and <it>SHAD </it>and the most stable combination of two genes was <it>GAPDH-SHAD</it>. We recommend using <it>KALPHA-TBP </it>for the study of T1-T2 tumors, <it>RPL27-SHAD </it>for T3-T4 tumors, <it>KALPHA-SHAD </it>for N0 tumors, and <it>ALAS-TBP </it>for N+ tumors. <it>ACT, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPS29, SHAD </it>and <it>TBP </it>were slightly misregulated (<1.7-fold) between tumor and normal mucosa but can be used for normalization, depending on the resolution required for the assay.</p> <p>Conclusion</p> <p>In the field of HNSCC, this study will guide researchers in selecting the most appropriate reference genes from among 12 potentially suitable reference genes, depending on the specific setting of their experiments.</p

Multiscale structures of lipids in foods as parameters affecting fatty acid bioavailability and lipid metabolism.

This review is respectfully dedicated to the memory of Michel Ollivon, Research Director at CNRS (Châtenay-Malabry, France), outstanding physico-chemist specialist of lipid organization, recipient of the Hilditch Memorial Lecture award, who was the initiator of the network RMT LISTRAL. We are also sadly paying tribute to Jean-Luc Vendeuvre, Food Engineer at the French Pork and Pig Institute (IFIP, Maisons-Alfort, France), outstanding expert in meat products who participated actively in RMT LISTRAL and provided unpublished data for figures in the present review, who passed away during review submission. RMT LISTRAL: Mixed Technological Network combining academic and industrial partners, devoted to the enhancement and divulgation of knowledge regarding structured dietary lipids.International audienceOn a nutritional standpoint, lipids are now being studied beyond their energy content and fatty acid (FA) profiles. Dietary FA are building blocks of a huge diversity of more complex molecules such as triacylglycerols (TAG) and phospholipids (PL), themselves organised in supramolecular structures presenting different thermal behaviours. They are generally embedded in complex food matrixes. Recent reports have revealed that molecular and supramolecular structures of lipids and their liquid or solid state at the body temperature influence both the digestibility and metabolism of dietary FA. The aim of the present review is to highlight recent knowledge on the impact on FA digestion, absorption and metabolism of: (i) the intramolecular structure of TAG; (ii) the nature of the lipid molecules carrying FA; (iii) the supramolecular organization and physical state of lipids in native and formulated food products and (iv) the food matrix. Further work should be accomplished now to obtain a more reliable body of evidence and integrate these data in future dietary recommendations. Additionally, innovative lipid formulations in which the health beneficial effects of either native or recomposed structures of lipids will be taken into account can be foreseen

Single-cell transcriptomics reveals shared immunosuppressive landscapes of mouse and human neuroblastoma

BACKGROUND High-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies. METHODS We combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays. RESULTS We show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes. CONCLUSIONS Our study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment

Reversible transitions between noradrenergic and mesenchymal tumor identities define cell plasticity in neuroblastoma

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity

Clinical relevance of nine transcriptional molecular markers for the diagnosis of head and neck squamous cell carcinoma in tissue and saliva rinse

<p>Abstract</p> <p>Background</p> <p>Analysis of 23 published transcriptome studies allowed us to identify nine genes displaying frequent alterations in HNSCC (<it>FN1, MMP1, PLAU, SPARC</it>, <it>IL1RN, KRT4, KRT13, MAL</it>, and <it>TGM3</it>). We aimed to independently confirm these dysregulations and to identify potential relationships with clinical data for diagnostic, staging and prognostic purposes either at the tissue level or in saliva rinse.</p> <p>Methods</p> <p>For a period of two years, we systematically collected tumor tissue, normal matched mucosa and saliva of patients diagnosed with primary untreated HNSCC. Expression levels of the nine genes of interest were measured by RT-qPCR in tumor and healthy matched mucosa from 46 patients. <it>MMP1 </it>expression level was measured by RT-qPCR in the salivary rinse of 51 HNSCC patients and 18 control cases.</p> <p>Results</p> <p>Dysregulation of the nine genes was confirmed by the Wilcoxon test. <it>IL1RN, MAL </it>and <it>MMP1 </it>were the most efficient diagnostic markers of HNSCC, with ROC AUC > 0.95 and both sensitivity and specificity above 91%. No clinically relevant correlation was found between gene expression level in tumor and T stage, N stage, tumor grade, global survival or disease-free survival. Our preliminary results suggests that with 100% specificity, <it>MMP1 </it>detection in saliva rinse is potentially useful for non invasive diagnosis of HNSCC of the oral cavity or oropharynx, but technical improvement is needed since sensitivity was only 20%.</p> <p>Conclusion</p> <p><it>IL1RN, MAL </it>and <it>MMP1 </it>are prospective tumor diagnostic markers for HNSCC. <it>MMP1 </it>overexpression is the most promising marker, and its detection could help identify tumor cells in tissue or saliva.</p

GASCON : Gestion agro-écologique de la santé des cultures et des organismes nuisibles

Le croisement des sciences agronomiques, de l’écologie appliquée à la gestion des agroécosystèmes,et des sciences humaines et sociales, qu’implique la transition agroécologique, pose de nouveaux défis pour répondre aux enjeux agricoles: intégrer des connaissances de différentes disciplines et produites à différentes échelles d’organisation pour agir en situation; développer des cadres d’analyse et démarches intégrant la diversité de situations à gérer par les acteurs et permettant de construire des réponses adaptées à chaque situation; et concevoir et mettre en œuvre des pratiques d’enseignement et d’apprentissage, qui dotent les apprenants de capacités à penser leur action en contexte, en mobilisant des savoirs et savoir-faire multiples en termes de contenus disciplinaires et des savoir-être pour construire des solutions avec une diversité d’acteurs. Dans le champ de la formation, ces défis nécessitent dès lors de revisiter les contenus des enseignements dispensés, les modalités pédagogiques et les dispositifs de formation existants, de manière à appréhender au mieux la complexité des processus à l’œuvre. Pour autant, peu de travaux s’attardent sur les modalités pratiques de ce changement et de ses implications, alors même que de nombreuses initiatives en matière de pédagogie et d’agroécologie se développent ces dernières années. L’objectif de ce séminaire est de promouvoir une information partagée et l’échange d’expériences pour répondre aux enjeux posés par l’agroécologie dans la formation (transversalité, pluridisciplinarité, approche systémique, pédagogies actives). Ces enjeux peuvent se décliner suivant plusieurs entrées : les thématiques enseignées (agriculture, élevage, territoire, alimentation, ...); les pratiques et les dispositifs pédagogiques mis en œuvre pour aborder ces questions (enseignement numérique, dispositifs expérimentaux, projets professionnels, référentiels, ...);les publics d’apprenants: élèves, étudiants, professionnels, ..

The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis : toward an international consensus

Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented