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- Effect of variable levels of dietary cholesterol and plant sterols on the growth performance and bone metabolism in gilthead seabream (Sparus aurata) juvenilesPublication . Dias, J.; Colen, Rita; Rodrigues, V.; Aragão, C.; Engrola, S.; Viegas, Michael; Laizé, Vincent; Gavaia, Paulo J.; Cancela, LeonorCholesterol is found in all animal tissues and is an important component of biological cell membranes with functions such as precursor to bile acids, hormones and vitamins. Fish meal and fish oil are cholesterol-rich ingredients. Replacement of these marine-derived ingredients by plant proteins and vegetable oils tends to reduce dietary cholesterol levels.
- Identification of a new cartilage-specific S100-like protein up-regulated during endo/perichondral mineralization in gilthead seabreamPublication . Fonseca, V. G.; Rosa, Joana; Laizé, Vincent; Gavaia, Paulo J.; Cancela, LeonorCalcium ions and calcium-binding proteins play a major role in many cellular processes, in particular skeletogenesis and bone formation. We report here the discovery of a novel S100 protein in fish and the analysis of its gene expression patterns. A 648-bp full-length cDNA encoding an 86-amino acid S100-like calcium-binding protein was identified through the subtractive hybridization of a gilthead seabream (Sparus aurata) cDNA library constructed to identify genes associated with in vitro mineralization. Deduced protein lacks an identifiable signal peptide and exhibits two EF-hand motifs characteristic of S100 proteins. Phylogenetic and bioinformatic analyses of S100 sequences suggested that gilthead seabream protein represents a novel and fish-specific member of the S100 protein family. Expression of S100-like gene was up-regulated during the in vitro mineralization of bone-derived cell lines and during seabream development, from larvae throughout adulthood, reflecting skeletogenesis. Restriction of S100-like gene expression to chondrocytes of cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish further confirmed the mineralogenic role of the protein in fish and emphasized the potential of S100-like as a marker of mineralizing cartilage in developing fish.
- Matrix Gla protein expression: a complex process involving the use of alternative promoters, multiple splicing events and microRNAsPublication . Cancela, Leonor; Laizé, Vincent; Conceição, N.; Tiago, Daniel; Maia, Ana-Teresa; Bensimon-Brito, A.; Gavaia, Paulo J.Matrix Gla protein (MGP) is a secreted vitamin K-dependent protein (VKD) located in the extracellular matrix and capable of binding calcium through its -carboxyglutamate residues. Although identified in 1983, transcriptional and post-transcriptional mechanisms regulating its expression remain unclear.
- Quantitative assessment of the regenerative and mineralogenic performances of the zebrafish caudal finPublication . Cardeira Da Silva, João; Gavaia, Paulo J.; Fernandez, Ignacio; Cengiz, Ibrahim Fatih; Moreira-Silva, Joana; Oliveira, Joaquim Miguel; Reis, Rui L.; Cancela, Leonor; Laizé, VincentThe ability of zebrafish to fully regenerate its caudal fin has been explored to better understand the mechanisms underlying de novo bone formation and to develop screening methods towards the discovery of compounds with therapeutic potential. Quantifying caudal fin regeneration largely depends on successfully measuring new tissue formation through methods that require optimization and standardization. Here, we present an improved methodology to characterize and analyse overall caudal fin and bone regeneration in adult zebrafish. First, regenerated and mineralized areas are evaluated through broad, rapid and specific chronological and morphometric analysis in alizarin red stained fins. Then, following a more refined strategy, the intensity of the staining within a 2D longitudinal plane is determined through pixel intensity analysis, as an indicator of density or thickness/volume. The applicability of this methodology on live specimens, to reduce animal experimentation and provide a tool for in vivo tracking of the regenerative process, was successfully demonstrated. Finally, the methodology was validated on retinoic acid-and warfarin-treated specimens, and further confirmed by micro-computed tomography. Because it is easily implementable, accurate and does not require sophisticated equipment, the present methodology will certainly provide valuable technical standardization for research in tissue engineering, regenerative medicine and skeletal biology.
- ESSA1 embryonic stem like cells from gilthead seabream: a new tool to study mesenchymal cell lineage differentiation in fishPublication . Parameswaran, V.; Laizé, Vincent; Gavaia, Paulo J.; Cancela, LeonorEmbryonic stem (ES) cells are a promising tool for generation of transgenic animals and an ideal experimental model for in vitro studies of embryonic cell development, differentiation and gene manipulation. Here we report the development and initial characterization of a pluripotent embryonic stem like cell line, designated as ESSA1, derived from blastula stage embryos of the gilthead seabream (Sparus aurata, L). ESSA1 cells are cultured in Leibovitz’s L-15 medium supplemented with 5% fetal bovine serum and, unlike other ES cells, without a feeder layer. They have a round or polygonal morphology, grow exponentially in culture and form dense colonies. ESSA1 cells also exhibit intense alkaline phosphatase activity, normal karyotype and are positive for stage-specific embryonic antigen-1 (SSEA1) and octamer-binding transcription factor 4 (Oct4) markers for up to 30 passages. Upon treatment with all-trans retinoic acid, ESSA1 cells differentiate into neuron-like, oligodendritic, myocyte and melanocyte cells; they can also form embryoid bodies when seeded in bacteriological plates, a characteristic usually associated with pluripotency. The capacity of ESSA1 cells to differentiate into osteoblastic, chondroblastic or osteoclastic cell lineages and to produce a mineralized extracellular matrix in vitro was demonstrated through histochemical techniques and further confirmed by immunocytochemistry using lineage-specific markers. Furthermore, ESSA1 cells can be used to produce chimera, where they contribute to the development of a variety of tissues including the trunk and gut of zebrafish embryos and fry. Thus, ESSA1 cells represent a promising model for investigating bonelineage cell differentiation in fish and also highlight the potential of piscine stem cell research.
- Dietary lipid quality regulates bone composition and metabolism in gilthead seabream (Sparus aurata) juvenilesPublication . Dias, J.; Rodrigues, V.; Colen, Rita; Rosa, Joana; Viegas, Michael; Cardeira Da Silva, João; Cancela, Leonor; Gavaia, Paulo J.; Laizé, VincentReplacement of significant amounts of marine fish oils by vegetable oils is a major trend in the aquaculture feed industry. However, knowledge on the mechanisms underlying the nutritional regulation of bone metabolism is extremely scarce in fish. We speculate that changes in the dietary ratio of fatty acids may modulate tissue eicosanoids production and affect bone formation in fastgrowing gilthead seabream, an important fish species for aquaculture in the Mediterranean region.
- Red algal extracts from Plocamium lyngbyanum and Ceramium secundatum stimulate osteogenic activities in vitro and bone growth in zebrafish larvaePublication . Carson, Matthew A.; Nelson, John; Cancela, M. Leonor; Laizé, Vincent; Gavaia, Paulo J.; Rae, Margaret; Heesch, Svenja; Verzin, Eugene; Maggs, Christine; Gilmore, Brendan F.; Clarke, Susan A.Through the current trend for bioprospecting, marine organisms-particularly algae-are becoming increasingly known for their osteogenic potential. Such organisms may provide novel treatment options for osteoporosis and other musculoskeletal conditions, helping to address their large healthcare burden and the limitations of current therapies. In this study, extracts from two red algae-Plocamium lyngbyanum and Ceramium secundatum-were tested in vitro and in vivo for their osteogenic potential. In vitro, the growth of human bone marrow stromal cells (hBMSCs) was significantly greater in the presence of the extracts, particularly with P. lyngbyanum treatment. Osteogenic differentiation was promoted more by C. secundatum (70 mu g/ml), though P. lyngbyanum had greater in vitro mineralisation potential. Both species caused a marked and dose-dependent increase in the opercular bone area of zebrafish larvae. Our findings therefore indicate the presence of bioactive components in P. lyngbyanum and C. secundatum extracts, which can promote both in vitro and in vivo osteogenic activity.