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Terrat Y.,CEA Saclay Nuclear Research Center | Biass D.,Atheris Laboratories | Dutertre S.,Atheris Laboratories | Favreau P.,Atheris Laboratories | And 4 more authors.
Toxicon | Year: 2012

Although cone snail venoms have been intensively investigated in the past few decades, little is known about the whole conopeptide and protein content in venom ducts, especially at the transcriptomic level. If most of the previous studies focusing on a limited number of sequences have contributed to a better understanding of conopeptide superfamilies, they did not give access to a complete panorama of a whole venom duct. Additionally, rare transcripts were usually not identified due to sampling effect. This work presents the data and analysis of a large number of sequences obtained from high throughput 454 sequencing technology using venom ducts of Conus consors, an Indo-Pacific living piscivorous cone snail. A total of 213,561 Expressed Sequence Tags (ESTs) with an average read length of 218 base pairs (bp) have been obtained. These reads were assembled into 65,536 contiguous DNA sequences (contigs) then into 5039 clusters. The data revealed 11 conopeptide superfamilies representing a total of 53 new isoforms (full length or nearly full-length sequences). Considerable isoform diversity and major differences in transcription level could be noted between superfamilies. A, O and M superfamilies are the most diverse. The A family isoforms account for more than 70% of the conopeptide cocktail (considering all ESTs before clustering step). In addition to traditional superfamilies and families, minor transcripts including both cysteine free and cysteine-rich peptides could be detected, some of them figuring new clades of conopeptides. Finally, several sets of transcripts corresponding to proteins commonly recruited in venom function could be identified for the first time in cone snail venom duct. This work provides one of the first large-scale EST project for a cone snail venom duct using next-generation sequencing, allowing a detailed overview of the venom duct transcripts. This leads to an expanded definition of the overall cone snail venom duct transcriptomic activity, which goes beyond the cysteine-rich conopeptides. For instance, this study enabled to detect proteins involved in common post-translational maturation and folding, and to reveal compounds classically involved in hemolysis and mechanical penetration of the venom into the prey. Further comparison with proteomic and genomic data will lead to a better understanding of conopeptides diversity and the underlying mechanisms involved in conopeptide evolution. © 2011 Elsevier Ltd.


Joubert C.,Center Oceanologique du Pacifique | Piquemal D.,Skuldtech | Marie B.,University of Burgundy | Manchon L.,Skuldtech | And 5 more authors.
BMC Genomics | Year: 2010

Background: The shell of the pearl-producing bivalve Pinctada margaritifera is composed of an organic cell-free matrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increase genomic resources and identify shell matrix proteins implicated in biomineralization in P. margaritifera, high-throughput Expressed Sequence Tag (EST) pyrosequencing was undertaken on the calcifying mantle, combined with a proteomic analysis of the shell.Results: We report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedented catalog of 82 P. margaritifera biomineralization-related mantle protein sequences. Components of the current "chitin-silk fibroin gel-acidic macromolecule" model of biomineralization processes were found, in particular a homolog of a biomineralization protein (Pif-177) recently discovered in P. fucata. Among these sequences, we could show the localization of two other biomineralization protein transcripts, pmarg-aspein and pmarg-pearlin, in two distinct areas of the outer mantle epithelium, suggesting their implication in calcite and aragonite formation. Finally, by combining the EST approach with a proteomic mass spectrometry analysis of proteins isolated from the P. margaritifera shell organic matrix, we demonstrated the presence of 30 sequences containing almost all of the shell proteins that have been previously described from shell matrix protein analyses of the Pinctada genus. The integration of these two methods allowed the global composition of biomineralizing tissue and calcified structures to be examined in tandem for the first time.Conclusions: This EST study made on the calcifying tissue of P. margaritifera is the first description of pyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data set covers most of the diversity of the matrix protein of P. margaritifera shell, but also that the mantle transcripts encode proteins present in P. margaritifera shell, hence demonstrating their implication in shell formation. Combining transcriptomic and proteomic approaches is therefore a powerful way to identify proteins involved in biomineralization. Data generated in this study supply the most comprehensive list of biomineralization-related sequences presently available among protostomian species, and represent a major breakthrough in the field of molluskan biomineralization. © 2010 Joubert et al; licensee BioMed Central Ltd.


Jackson D.J.,University of Queensland | Jackson D.J.,University of Gottingen | McDougall C.,University of Queensland | Woodcroft B.,University of Queensland | And 9 more authors.
Molecular Biology and Evolution | Year: 2010

The capacity to biomineralize is closely linked to the rapid expansion of animal life during the early Cambrian, with many skeletonized phyla first appearing in the fossil record at this time. The appearance of disparate molluscan forms during this period leaves open the possibility that shells evolved independently and in parallel in at least some groups. To test this proposition and gain insight into the evolution of structural genes that contribute to shell fabrication, we compared genes expressed in nacre (mother-of-pearl) forming cells in the mantle of the bivalve Pinctada maxima and the gastropod Haliotis asinina. Despite both species having highly lustrous nacre, we find extensive differences in these expressed gene sets. Following the removal of housekeeping genes, less than 10% of all gene clusters are shared between these molluscs, with some being conserved biomineralization genes that are also found in deuterostomes. These differences extend to secreted proteins that may localize to the organic shell matrix, with less than 15% of this secretome being shared. Despite these differences, H. asinina and P. maxima both secrete proteins with repetitive low-complexity domains (RLCDs). Pinctada maxima RLCD proteins-for example, the shematrins-are predominated by silk/fibroin-like domains, which are absent from the H. asinina data set. Comparisons of shematrin genes across three species of Pinctada indicate that this gene family has undergone extensive divergent evolution within pearl oysters. We also detect fundamental bivalve-gastropod differences in extracellular matrix proteins involved in mollusc-shell formation. Pinctada maxima expresses a chitin synthase at high levels and several chitin deacetylation genes, whereas only one protein involved in chitin interactions is present in the H. asinina data set, suggesting that the organic matrix on which calcification proceeds differs fundamentally between these species. Large-scale differences in genes expressed in nacre-forming cells of Pinctada and Haliotis are compatible with the hypothesis that gastropod and bivalve nacre is the result of convergent evolution. The expression of novel biomineralizing RLCD proteins in each of these two molluscs and, interestingly, sea urchins suggests that the evolution of such structural proteins has occurred independently multiple times in the Metazoa.


Rabhi I.,CNRS PASTEUR Laboratory | Rabhi S.,CNRS PASTEUR Laboratory | Ben-Othman R.,CNRS PASTEUR Laboratory | Rasche A.,Max Planck Institute for Molecular Genetics | And 6 more authors.
PLoS Neglected Tropical Diseases | Year: 2012

We analyzed the transcriptional signatures of mouse bone marrow-derived macrophages at different times after infection with promastigotes of the protozoan parasite Leishmania major. Ingenuity Pathway Analysis revealed that the macrophage metabolic pathways including carbohydrate and lipid metabolisms were among the most altered pathways at later time points of infection. Indeed, L. major promastiogtes induced increased mRNA levels of the glucose transporter and almost all of the genes associated with glycolysis and lactate dehydrogenase, suggesting a shift to anaerobic glycolysis. On the other hand, L. major promastigotes enhanced the expression of scavenger receptors involved in the uptake of Low-Density Lipoprotein (LDL), inhibited the expression of genes coding for proteins regulating cholesterol efflux, and induced the synthesis of triacylglycerides. These data suggested that Leishmania infection disturbs cholesterol and triglycerides homeostasis and may lead to cholesterol accumulation and foam cell formation. Using Filipin and Bodipy staining, we showed cholesterol and triglycerides accumulation in infected macrophages. Moreover, Bodipy-positive lipid droplets accumulated in close proximity to parasitophorous vacuoles, suggesting that intracellular L. major may take advantage of these organelles as high-energy substrate sources. While the effect of infection on cholesterol accumulation and lipid droplet formation was independent on parasite development, our data indicate that anaerobic glycolysis is actively induced by L. major during the establishment of infection. © 2012 Rabhi et al.


Violette A.,Atheris Laboratories | Biass D.,Atheris Laboratories | Dutertre S.,Atheris Laboratories | Dutertre S.,University of Queensland | And 5 more authors.
Journal of Proteomics | Year: 2012

Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~ 400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~ 7 kDa) and two proteins in the 25 and 50. kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery. © 2012 Elsevier B.V.


Patent
Skuldtech and University of Liège | Date: 2013-10-09

The invention provides an in vitro method or assay for the diagnosis of osteochondrosis or prediction of the likelihood of its onset in a terrestrian mammal, comprising measuring the expression level of a marker in a sample obtained from said terrestrian mammal and comparing said expression level to the expression level of said marker measured in a sample obtained from one or more terrestrian mammals of the same species not affected by osteochondrosis, wherein the marker is ApoB-3G, and wherein an increase in the expression level of ApoB-3G is indicative of osteochondrosis. The invention also provides a diagnostic kit for use in said method, which comprises at least one agent, which binds specifically to the product of the gene of the respective marker and which can be used to determine the expression level of said marker, wherein the marker is ApoB-3G.


Rosa R.D.,Montpellier University | de Lorgeril J.,Montpellier University | Tailliez P.,CNRS Microbial Ecology | Bruno R.,Skuldtech | And 2 more authors.
BMC Genomics | Year: 2012

Background: The complex balance between environmental and host factors is an important determinant of susceptibility to infection. Disturbances of this equilibrium may result in multifactorial diseases as illustrated by the summer mortality syndrome, a worldwide and complex phenomenon that affects the oysters, Crassostrea gigas. The summer mortality syndrome reveals a physiological intolerance making this oyster species susceptible to diseases. Exploration of genetic basis governing the oyster resistance or susceptibility to infections is thus a major goal for understanding field mortality events. In this context, we used high-throughput genomic approaches to identify genetic traits that may characterize inherent survival capacities in C. gigas.Results: Using digital gene expression (DGE), we analyzed the transcriptomes of hemocytes (immunocompetent cells) of oysters able or not able to survive infections by Vibrio species shown to be involved in summer mortalities. Hemocytes were nonlethally collected from oysters before Vibrio experimental infection, and two DGE libraries were generated from individuals that survived or did not survive. Exploration of DGE data and microfluidic qPCR analyses at individual level showed an extraordinary polymorphism in gene expressions, but also a set of hemocyte-expressed genes whose basal mRNA levels discriminate oyster capacity to survive infections by the pathogenic V. splendidus LGP32. Finally, we identified a signature of 14 genes that predicted oyster survival capacity. Their expressions are likely driven by distinct transcriptional regulation processes associated or not associated to gene copy number variation (CNV).Conclusions: We provide here for the first time in oyster a gene expression survival signature that represents a useful tool for understanding mortality events and for assessing genetic traits of interest for disease resistance selection programs. © 2012 Rosa et al.; licensee BioMed Central Ltd.


Patent
Skuldtech and Ab Science | Date: 2014-04-09

The present invention relates to an in vitro method for determining the prognosis of pancreatic cancer in a patient. More particularly, this method comprises the following steps:c) measuring the expression level of at least two genes chosen in the group consisting in: ING5, RUNDC1, ABCC1, FGFR3, S100A6, ANKRD55, and CD8A or homologous genes, in a blood sample of said patient,d) predicting the outcome of the pancreatic cancer in said patient. The invention also relates to a kit specifically designed to carry out such a method.


Patent
Skuldtech | Date: 2014-04-16

The invention provides an in vitro method or assay for the diagnosis of osteochondrosis or prediction of the likelihood of its onset in a terrestrian mammal, comprising measuring the expression level of a marker in a sample obtained from said terrestrian mammal with an agent that can be used to determine the expression level of said marker, and comparing the expression level measured in step a) to the expression level of said marker measured in a sample obtained from one or more terrestrian mammals of the same species not affected by osteochondrosis, wherein the marker is ApoB-3G, and wherein an increase in the expression level of ApoB-3G is indicative of osteochondrosis. Methods are provided for the diagnosis and/or prediction whether the fetlock joint, the hock joint or the stifle joint is or will be affected by osteochondrosis. The invention also provides a diagnostic kit for use in said method, which comprises at least one agent, which binds specifically to the product of the gene of the respective marker and which can be used to determine the expression level of said marker, wherein the marker is ApoB-3G.


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