Development of new genetic resources for faba bean (Vicia faba L.) breeding through the discovery of gene-based SNP markers and the construction of a high-density consensus map.

Faba bean (Vicia faba L.) is a pulse crop of high nutritional value and high importance for sustainable agriculture and soil protection. With the objective of identifying gene-based SNPs, transcriptome sequencing was performed in order to reduce faba bean genome complexity. A set of 1,819 gene-based SNP markers polymorphic in three recombinant line populations was selected to enable the construction of a high-density consensus genetic map encompassing 1,728 markers well distributed in six linkage groups and spanning 1,547.71 cM with an average inter-marker distance of 0.89 cM. Orthology-based comparison of the faba bean consensus map with legume genome assemblies highlighted synteny patterns that partly reflected the phylogenetic relationships among species. Solid blocks of macrosynteny were observed between faba bean and the most closely-related sequenced legume species such as pea, barrel medic or chickpea. Numerous blocks could also be identified in more divergent species such as common bean or cowpea. The genetic tools developed in this work can be used in association mapping, genetic diversity, linkage disequilibrium or comparative genomics and provide a backbone for map-based cloning. This will make the identification of candidate genes of interest more efficient and will accelerate marker-assisted selection (MAS) and genomic-assisted breeding (GAB) in faba bean.

[Sodium restriction prevents cardiovascular remodeling associated with insulin-resistance in the rat]. / La restriction sodée prévient le remodelage cardiovasculaire dans l'insulinorésistance chez le rat.

AIM OF THE STUDY: In the present work, the objective was to evaluate the influence of a dietary sodium restriction on cardiovascular morphology changes associated with insulin-resistance. ANIMALS AND PROTOCOL: At 8 weeks of age, rats were fed for 12 weeks a 60%-fructose diet containing a regular sodium content (0.64%) or totally lacking in sodium chloride (<0.01%). A group of rats fed a wheat starch-based diet with regular sodium content served as control group. RESULTS: Elevated HOMA index and plasma insulin confirm the presence of insulin-resistance in fructose-fed rats. Concomitantly, an increase in cardiac mass and in cardiac collagen (Sirius red staining) was detected without obvious change in arterial pressure or cardiac aldosterone synthase mRNA expression. In addition, cross-sectional area of the carotid artery was higher in fructose-fed rats. Production of superoxide anion, equated with dihydroethidium (DHE) staining, was enhanced in cardiac tissue of rats with insulin-resistance. Withdrawal of sodium from the fructose diet prevented all the cardiovascular effects of fructose consumption, including DHE staining. CONCLUSION: These results are in favor of the participation of oxidative stress normalization in the beneficial influence of dietary sodium deprivation on cardiovascular remodeling in this model of insulin-resistance in rats.

Perinatal xenohormone exposure impacts sweet preference and submandibular development in male rats.

OBJECTIVE: To determine the effect of perinatal exposure to low doses of genistein and/or vinclozolin on submandibular salivary gland (SSG) development in juvenile and adult male rats and to establish a link with sweet preference. MATERIAL AND METHODS: Female rats received orally (1 mg kg(-1) body weight/day) genistein and vinclozolin, alone or in combination, from the first gestational day up to weaning. Sweet preference was assessed at weaning and in adulthood in male offspring; submandibular glands were then collected to study the morphogenesis and mRNA expression of steroid receptors, growth factors and taste related proteins. RESULTS: Exposure to genistein and/or vinclozolin resulted in a higher saccharin intake on postnatal day 25 (P < 0.05) linked to a higher number of pro-acinar cells (P < 0.01) and mRNA expression of progesterone receptor, growth factors and gustine (P < 0.01). These increases disappeared in adulthood, but mRNA expressions of sex hormone receptors and growth factors were strongly repressed in all treated groups (P < 0.01). CONCLUSION: Our findings confirm that the SSG are target for xenohormones and provide evidence that perinatal exposure to low doses of genistein and/or vinclozolin could simultaneously disrupt not only the salivary gland prepubertal development and sweet intake but also endocrine gene mRNA expression later in life.

Autoantibody signatures: progress and perspectives for early cancer detection.

Becoming invasive is a crucial step in cancer development, and the early spread of tumour cells is usually undetected by current imaging technologies. In patients with cancer and no signs of overt metastases, sensitive methods have been developed to identify circulating autoantibodies and their antigen counterparts in several cancers. These technologies are often based on proteomic approaches, and recent advances in protein and antibody microarrays have greatly facilitated the discovery of new antibody biomarkers in sera from cancer patients. Interestingly, in a clinical application setting, combinations of multiple autoantibody reactivities into panel assays have recently been proposed as relevant screening tests and validated in several independent trials. In addition, autoantibody signatures seem to be particularly relevant for early detection of cancer in high-risk cancer patients. In this review, we highlight the concept that immunogenic epitopes associated with the humoural response and key pathogenic pathways elicit serum autoantibodies that can be considered as relevant cancer biomarkers. We outline the proteomic strategies employed to identify and validate their use in clinical practice for cancer screening and diagnosis. We particularly emphasize the clinical utility of autoantibody signatures in several cancers. Finally, we discuss the challenges remaining for clinical validation.

Humoral response to cancer as a tool for biomarker discovery.

There is an important need to find relevant biomarkers that show high sensitivity and specificity for early diagnosis and prognosis of cancer. An immune response to cancer is elicited in humans, as demonstrated in part by the identification of autoantibodies against a number of tumor-associated antigens in sera from patients with different types of cancer. Identification of tumor-associated antigens and their cognate autoantibodies is a promising strategy for the discovery of relevant biomarkers. During the past few years, proteomic approaches, including SEREX, SERPA and, more recently, protein microarrays, have been the dominant strategies used to identify tumor-associated antigens and their cognate autoantibodies. In this review, we aim to describe advantages, drawbacks, and recent improvements of these approaches for the study of humoral responses.

Proteomic analysis of RCL2 paraffin-embedded tissues.

Histopathological diagnosis in most of the world's hospitals is based upon formalin-fixed and paraffin-embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non-toxic fixative, for sparing proteins preserved in paraffin-embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi-dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western-blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser-captured microdissection were found to be identical for frozen and RCL2-fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin-embedded tissue sample, and can be a new method for investigating protein biomarkers.

Proteomics-based identification of HSP60 as a tumor-associated antigen in early stage breast cancer and ductal carcinoma in situ.

The detection of autoantibodies in cancer patients has been shown to constitute an excellent tool for early diagnosis. Because breast cancer still lacks early diagnostic markers, we investigated novel tumor-associated antigens and related autoantibodies in sera from patients with early stage breast cancer compared to autoimmune disease, other cancers, and healthy volunteers, using a proteomics-based approach. Among the 26 protein antigens specifically recognized by early stage breast cancer sera, we focused on Heat Shock Protein 60 (HSP60). Using ELISA, we investigated the frequency of autoantibodies directed against this protein in the sera of 240 individuals, comprising patients with either ductal carcinoma in situ (DCIS) ( n = 49) or early stage breast cancer ( n = 58), other cancers ( n = 20), autoimmune disease ( n = 20), and healthy subjects ( n = 93). Autoantibodies directed against HSP60 were present in 16/49 (31%) early stage breast cancer and 18/58 (32.6%) DCIS patients, compared to 4/93 (4.3%) healthy subjects. In particular, autoantibodies were present in 11/23 patients (47.8%) with high-grade DCIS, compared to 5/26 (19.2%) with low-grade DCIS. HSP60 mRNA levels were significantly higher in primary breast cancer compared to healthy breast tissues. Using immunohistochemistry, we found that HSP60 expression gradually increases from normal through DCIS to invasive tissues. Our results indicate that HSP60 autoantibodies may be of interest in terms of clinical utility for the early diagnosis of breast cancer and more particularly in DCIS. Moreover, HSP60 overexpression during the first steps of breast carcinogenesis may be functionally correlated to tumor growth and/or progression.

Molecular changes associated with the agonist activity of hydroxy-tamoxifen and the hyper-response to estradiol in hydroxy-tamoxifen-resistant breast cancer cell lines.

The aim of this study was to explore the pharmacological response to 4-hydroxy-tamoxifen (OH-Tam) and to estradiol (E2) in three cell lines: MVLN, a human breast carcinoma cell line derived from MCF-7, and two MVLN-derived OH-Tam-resistant (OTR) cell lines, called CL6.8 and CL6.32. The OH-Tam response in the OTR cells was associated with the development of both an agonist activity of the drug on cell proliferation and the resistance of the cells to OH-Tam-induced apoptosis. The OTR cells also developed an increased sensitivity to the E2 growth-stimulating activity. To delineate the genes that determine such responses, we combined a mini-array-based gene-selection approach and an extensive real-time quantitative PCR exploration in the MVLN and OTR cell lines exposed to three pharmacological conditions: a 4-day treatment with E2, OH-Tam or both E2 and OH-Tam. Compiled data revealed a hyper-response to E2 and a modification of the OH-Tam pharmacological response (loss of antagonist action and agonist activity) at the gene-expression level. The proteins encoded by the genes selected in this study have been reported to be involved in the regulation of cell proliferation, cell transformation, DNA repair and apoptosis, or belong to the ErbB/epidermal growth factor receptor-driven pathway. Our data also provide evidence of changes in transcriptional co-regulator expression, elevated mitogen-activated protein kinase activity and increase in the phosphorylation status of estrogen receptor alpha on serine residue 118 in the OTR cell lines, suggesting the possible involvement of such mechanisms in the agonist activity of OH-Tam and/or the hyper-response of cells to E2. Taken together, our study should enhance our knowledge of the multifactorial events associated with the development of Tam resistance in two independent cell lines issued from the same selection process and should help in the identification of potential molecular targets for diagnosis or therapy.