Cross-Sample Validation Provides Enhanced Proteome Coverage in Rat Vocal Fold Mucosa

The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high Mr ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high Mr glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories

Evaluation of nanostructured vectors for the treatment of osteoarticular pathologies

World Congress of the Osteoarthritis-Research-Society-International (OARSI), Paris, FRANCE, APR 24-27, 2014International audiencePurpose: One of the major problems in treatment of osteoarticular diseases is to reach cells inside the matrix to provide drug. Indeed, cartilage is an avascular tissue with a few cells feed by diffusion through a dense protein network (collagens, glycosaminoglycans). In this work we have designed polymeric nanoparticles (NPs) of poly (D, L-lactic/glycolic acid)(PLGA) synthesized by a double emulsion method, which are biocompatible, biodegradable and can encapsulate water-soluble agents. Our NPs are labelled with BSA coupled to a fluorescent dye (Cyanine-3) to follow them by epifluorescent microscopy. As articular cells expressed CD44, one receptor of hyaluronic acid (HA)(a main component of synovial fluid), the nanoparticles are recovered with HA in order to enhance targeting of cells. Here, we have studied the internalization kinetics of “empty” nanoparticles, and we have evaluated their neutrality on chondrocytes matrix synthesis, mesenchymal stem cell (MSC) differentiation and inflammatory response. Innocuity has been also evaluated in healthy animals, after direct intraarticular injection of labelled nanoparticles (inflammatory response, Extracellular matrix integrity).Methods: Articular cells (chondrocytes, synoviocytes) and MSC are isolated from human donors, and cultured as primary culture. First, cells are exposed with 100 μg/mL of NPs from 2 to 12 hours. At the end of the kinetic immunofluorescence pictures with DAPI (nuclear staining) are realized to assess of the internalization of NPs, expression of inflammatory markers (IL1ß, TNFα and Cox2) are monitored by RT-qPCR analysis, and confirmed by PGE2 and nitrites measurement in supernatant.In other hand we evaluate, with pellets culture system, the effect of NPs exposition on extracellular matrix synthesis by chondrocytes, with RT-qPCR analysis of specific markers (Col2, Aggrecan and COMP), and by histological study of pellets (Alcian blue staining of proteoglycans, Sirius Red staining of collagen). Finally by growing MSC into 3 different differentiation media, we investigate if NPs pre-treatment can interfere with differentiation ability of MSC onto chondrogenic, adipogenic or osteogenic pathway. RT-qPCR assays for differentiation markers according to culture conditions and specific staining of lipid vesicles or calcium deposits, allow us to confirm the differentiation of cells.Intraarticular injections were realized in healthy rat’s Knees. Structure of joint (synovium, cartilage, subchondral bone) was assessed by histological studies, performed at 7 and 10 days after injection (single and repeated).Results: For the different cell types, NPs are found into cytoplasm after 6 hours of exposition

Genetic control of oil content in oilseed rape (Brassica napus L.)

International audienceIn oilseed rape (Brassica napus L.) like in most oleaginous crops, seed oil content is the main qualitative determinant that confers its economic value to the harvest. Increasing seed oil content is then still an important objective in oilseed rape breeding. In the objective to get better knowledge on the genetic determinism of seed oil content, a genetic study was undertaken in two genetic backgrounds. Two populations of 445 and a 242 doubled haploids (DH) derived from the crosses "Darmor-bzh" x "Yudal" (DY) and "Rapid"" x "NSL96/25" (RNSL), respectively, were genotyped and evaluated for oil content in different trials. QTL mapping in the two populations indicate that additive effects are the main factors contributing to variation in oil content. A total of 14 and 10 genomic regions were involved in seed oil content in DY and RNSL populations, respectively, of which five and two were consistently revealed across the three trials performed for each population. Most of the QTL detected were not colocalised to QTL involved in flowering time. Few epistatic QTL involved regions that carry additive QTL in one or the other population. Only one QTL located on linkage group N3 was potentially common to the two populations. The comparisons of the QTL location in this study and in the literature showed that: (i) some of the QTL were more consistently revealed across different genetic backgrounds. The QTL on N3 was revealed in all the studies and the QTL on N1, N8 and N13 were revealed in three studies out of five, (ii) some of the QTL were specific to one genetic background with potentially some original alleles, (iii) some QTL were located in homeologous regions, and (iv) some of the regions carrying QTL for oil content in oilseed rape and in Arabidopsis could be collinear. These results show the possibility to combine favourable alleles at different QTL to increase seed oil content and to use Arabidopsis genomic data to derive markers for oilseed rape QTL and identify candidate genes, as well as the interest to combine information from different segregating populations in order to build a consolidated map of QTL involved in a specific trait

Progress Towards a Reference Genome for Sunflower

The Compositae is one of the largest and most economically important families of flowering plants and includes a diverse array of food crops, horticultural crops, medicinals, and noxious weeds. Despite its size and economic importance, there is no reference genome sequence for the Compositae, which impedes research and improvement efforts. We report on progress toward sequencing the 3.5 Gb genome of cultivated sunflower (Helianthus annuus), the most important crop in the family. Our sequencing strategy combines whole-genome shotgun sequencing using the Solexa and 454 platforms with the generation of high-density genetic and physical maps that serve as scaffolds for the linear assembly of whole-genome shotgun sequences. The performance of this approach is enhanced by the construction of a sequence-based physical map, which provides unique sequence-based tags every 5–6 kb across the genome. Thus far, our physical map covers ∼85% of the sunflower genome, and we have generated ∼80× genome coverage with Solexa reads and 15.5× with 454 reads. Preliminary analyses indicated that ∼78% of the sunflower genome consists of repetitive sequences. Nonetheless, ∼76% of contigs \u3e5 kb in size can be assigned to either the physical or genetic map or to both, suggesting that our approach is likely to deliver a highly accurate and contiguous reference genome for sunflower