Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell

Background: The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised.Results: Recent expressed sequence tag (EST) investigations of the mantle tissue from the tropical abalone (Haliotis asinina) provide an opportunity to further characterise the proteins in the shell by a proteomic approach. In this study, we have identified a total of 14 proteins from distinct calcified layers of the shell. Only two of these proteins have been previously characterised from abalone shells. Among the novel proteins are several glutamine- and methionine-rich motifs and hydrophobic glycine-, alanine- and acidic aspartate-rich domains. In addition, two of the new proteins contained Kunitz-like and WAP (whey acidic protein) protease inhibitor domains.Conclusion: This is one of the first comprehensive proteomic study of a molluscan shell, and should provide a platform for further characterization of matrix protein functions and interactions

Near-infrared fluorescence imaging-guided surgery improves recurrence-free survival rate in novel orthotopic animal model of head and neck squamous cell carcinoma

International audienceBackground. Appropriate animal models are required to test novel therapeutics for head and neck squamous cell carcinoma (HNSCC) such as near-infrared (NIR) imaging-guided surgery. Methods. We developed an optimized animal model of orthotopic HNSCC (in female athymic NMRI (Naval Medical Research Institute) nude mice) with a prolonged survival time. Resection of the orthotopic tumors was performed 30 days after implantation with or without the aid of a minia-turized clinical grade NIR optical imaging device, after systemic administration of a fluorescent RGD-based probe that targets a v b 3 integrin. Results. NIR optical imaging-guided surgery increased the recurrence-free survival rate by 50% through the detection of fluorescent cancer residues as small as 185 mm; these fragments could remain unidentified if resection was performed exclusively under unaided visual guidance. Conclusion. NIR optical imaging-guided surgery showed an improved HNSCC tumor resection quality in our optimized orthotopic animal model

Identification et caractérisation des molécules de la matrice organique de la nacre de l'huître Pinctada margaritifera, actives sur les cellules de la lignée ostéoclastique

La nacre comme l'os est une structure squelettique biominéralisée, composée d'une matrice organique et de minéral. Il a été démontré que la nacre de la coquille de l'huître Pinctada margaritifera contient dans sa fraction organique des molécules hydrosolubles qui stimulent l'activité du tissu osseux en agissant sur la différenciation des ostéoblastes (cellules formatrices d'os) et la minéralisation. Nous avons démontré dans cette étude que la matrice organique de la nacre contient également des molécules signal actives sur les cellules de la lignée ostéoclastique ce qui explique l'activité de la nacre sur le remodelage osseux. Les résultats font apparaître trois groupes de molécules d'intérêt: des molécules de faibles masses moléculaires apparentes qui stimulent la différenciation des ostéoclastes; des molécules associées à une fraction de haute masse moléculaire apparente qui inhibent la différenciation des ostéoclastes et des molécules inhibitrices de protéases à cystéine et particulièrement de la cathepsine K humaine, protéase essentielle de la résorption ostéoclastique. Au total, 15 protéines spécifiquement exprimées par les cellules responsables de la biominéralisation de la nacre sont caractérisées : 13 protéines de structure de la matrice organique de la nacre et 2!protéines intracellulaires impliquées dans la régulation de la biominéralisation, l'une au niveau de la mobilisation du calcium et l'autre par une activité inhibitrice de protéases. Les séquences des protéines de structure caractérisées suggèrent qu'elles possèderaient d'intéressantes propriétés d'agrégation et participeraient de ce fait à la réticulation de la trame organique de la nacre et à l'adsorption de molécules de faible masse moléculaire, mobilisables et porteuses de l'activité biologique. Le potentiel d'application de ces recherches dans les domaines industriels liés à la santé a conduit à la protection des molécules valorisables par le dépôt de 3 brevetsMother-of-pearl, from the shellfish Pinctada margaritifera, like bone in vertebrates is made-up of an organic matrix and mineral. Nacre water-soluble organic matrix was previously shown, by in vitro and in vivo experiments, to contain signal molecules that stimulate bone tissue, especially osteoblastic differentiation and mineralization. These studies demonstrate the presence of signal molecules, in the nacre organic matrix, active on osteoclasts, the bone resorbing cells, explaining the outcome of bone remodeling. Nacre water-soluble matrix contains 3 groups of significant molecules: low-molecular weight molecules stimulating osteoclastic differentiation, molecules within the high-molecular weight fraction of nacre organic matrix inhibiting osteoclastic differentiation and molecules inhibiting cysteine proteases and particularly cathepsin K, the main hydrolytic protease in bone resorbing cells. Fifteen proteins specifically expressed by the nacre-forming cells have been characterized : 13!proteins scaffolding the nacre organic matrix and 2 proteins involved in the control of biomineralization, calcium transport and protease inhibition. The matrix proteins characterized in these studies can form aggregates and thus contribute in threading the cross-linked network of the nacre organic matrix and in maintaining inside, the diffusible low-molecular weight molecules bearing the biological activity. The high rate of valuation of this research for health industry leads to the deposit of 3 patentsPARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Analyse sensorielle. Une méthode pour les rouges d'Anjou

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Ynol Ethers from Dichloroenol Ethers: Mechanistic Elucidation Through 35 Cl Labeling

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Role of near-infrared fluorescence imaging in head and neck cancer surgery: from animal models to humans

International audienceComplete resection of head and neck cancers with negative surgical margins improves the prognosis of the disease and decreases the recurrence rate. Near-infrared fluorescence-guided surgery of head and neck cancer is a rapidly evolving field that represents an invaluable tool for tumor detection and resection. Here, we present a literature review of the principles of near-infrared fluorescence imaging and its use in head and neck cancer surgery. We discuss important studies in both animal models and humans that have been carried out up to this point. We also outline the important fluorescent molecules and devices used in head and neck fluorescence imaging-guided surgery. Although near-infrared fluorescence-guided surgery for head and neck cancers showed efficacy in animal models, its use in humans is limited by the small number of fluorescent probes that are approved for clinical use. However, it is considered as a novel surgical aid that helps delineate tumor margins preoperatively and could spare patients from the added morbidity that is associated with additional surgery or chemoradiation. In addition, it is a useful tool to detect sentinel lymph nodes as well as met-astatic lymph nodes

Nacre calcification in the freshwater mussel Unio pictorum: carbonic anhydrase activity and purification of a 95 kDa calcium-binding glycoprotein.

9 pagesInternational audienceThe formation of the molluscan shell is finely tuned by macromolecules of the shell organic matrix. Previous results have shown that the acid-soluble fraction of the nacre matrix of the freshwater paleoheterodont bivalve Unio pictorum shell displays a number of remarkable properties, such as calcium-binding activity, the presence of extensive glycosylations and the capacity to interfere at low concentration with in vitro calcium carbonate precipitation. Here we have found that the nacre-soluble matrix exhibits a carbonic anhydrase activity, an important function in calcification processes. This matrix is composed of three main proteinaceous discrete fractions. The one with the highest apparent molecular weight is a 95 kDa glycoprotein that is specific to the nacreous layer. P95, as it is provisionally named, is enriched in Gly, Glx and Asx and exhibits an apparent pI value of approximately 4, or approximately 7 when chemically deglycosylated. Furthermore, its glycosyl moiety, consisting of sulfated polysaccharides, is involved in calcium binding. Purified fractions of the three main proteins were digested with trypsin, and the resulting peptides were analysed by mass spectrometry. Our results suggest that identical peptides are constitutive domains of the different proteins. Partial primary structures were obtained by de novo sequencing and compared with known sequences from other mollusc shell proteins. Our results are discussed from an evolutionary viewpoint

Proteomic strategy for identifying mollusc shell proteins using mild chemical degradation and trypsin digestion of insoluble organic shell matrix: a pilot study on Haliotis tuberculata.

13 pagesInternational audienceA successful strategy for the identification of shell proteins is based on proteomic analyses where soluble and insoluble fractions isolated from organic shell matrix are digested with trypsin with the aim of generating peptides, which are used to identify novel shell proteins contained in databases. However, using trypsin as a sole degradative agent is limited by the enzyme's cleavage specificity and is dependent upon the occurrence of lysine and arginine in the shell protein sequence. To bypass this limitation, we investigated the ability of trifluoroacetic acid (TFA), a low-specificity chemical degradative agent, to generate clusters of analyzable peptides from organic shell matrix, suitable for database annotation. Acetic acid-insoluble fractions from Haliotis tuberculata shell were processed by trypsin followed by TFA digestion. The hydrolysates were used to annotate an expressed sequence tag library constructed from the mantle tissue of Haliotis asinina, a tropical abalone species. The characterization of sequences with repeat motifs featured in some of the shell matrix proteins benefited from TFA-induced serial cutting, which can result in peptide ladder series. Using the degradative specificities of TFA and trypsin, we were able to identify five novel shell proteins. This pilot study indicates that a mild chemical digestion of organic shell matrix combined with trypsin generates peptides suitable for proteomic analysis for better characterization of mollusc shell matrix proteins

Ultrastructure, chemistry and mineralogy of the growing shell of the European abalone Haliotis tuberculata

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