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- Transcriptional profiling of populations in the clam Ruditapes decussatus suggests genetically determined differentiation in gene expression along parallel temperature gradients and between races of the Atlantic ocean and west Mediterranean seaPublication . Saavedra, Carlos; Milan, Massimo; M. Leite, Ricardo; Cordero, David; Patarnello, Tomaso; Cancela, M. Leonor; Bargelloni, LucaOngoing ocean warming due to climate change poses new challenges for marine life and its exploitation. We have used transcriptomics to find genetically based responses to increased temperature in natural populations of the marine clam Ruditapes decussatus, which lives along parallel thermal gradients in southern Europe. Clams of the Atlantic and West Mediterranean races were collected in northern (cool) and a southern (warm) localities. The animals were kept in running seawater in the warm, southern Atlantic locality for a 15-week period. During this period, water temperature was raised to typical southern European summer values. After this period, an expression profile was obtained for a total of 34 clams and 11,025 probes by means of an oligonucleotide microarray. We found distinct transcriptional patterns for each population based on a total of 552 differentially expressed genes (DEGs), indicating innate differences which probably have a genetic basis. Race and latitude contributed significantly to gene expression differences, with very different sets of DEGs. A gene ontology analysis showed that races differed mainly in the genes involved in ribosomal function and protein biosynthesis, while genes related to glutathione metabolism and ATP synthesis in the mitochondria were the most outstanding with respect to north/south transcriptional differences.
- Shikimate and folate pathways in the protozoan parasite, Perkinsus olseniPublication . Elandalloussi, Laurence M.; Rodrigues, Pedro; Afonso, Ricardo; Leite, Ricardo; Nunes, Patrícia A.; Cancela, LeonorWe have exploited the experimental accessibility of the protozoan parasite Perkinsus olseni and its similarities to apicomplexan parasites to investigate the influence of specific drugs on its proliferation. For this purpose, shikimate and folate pathways present an attractive target for parasitic therapy given their major differences with mammalian pathways. Glyphosate, a potent inhibitor of the shikimate pathway enzyme EPSP synthase inhibited the in vitro proliferation of P. olseni in a dose-dependent manner and this effectwas reversed by addition of chorismate, indicating the presence of a shikimate pathway. However, this effect was not antagonised by p-aminobenzoate or folic acid. Furthermore, antagonism was observed, via pyrimethamine to glyphosate inhibitory effect, suggesting that the shikimate pathway is not essential for the biosynthesis of folate precursors and is therefore crucial for another pathway downstream from chorismate. In addition, sulfadiazine, a well known inhibitor of dihydropteorate synthase, an enzyme of the folate biosynthetic pathway,had no inhibitory effect on P. olseni proliferation. In view of these results, the parasite does not appear to require the folate biosynthesis pathway for its survival and is most likely able to use exogenous folate. Even though pyrimethamine was found to inhibit P. atlanticus growth, this inhibitory effect could not be reversed by co-addition of folic acid. Therefore, we propose that the effect of pyrimethamine observed in this study results from the inhibition of a target other than dihydrofolate reductase. Similarly, proguanil target is likely to be separate from DHFR since only its metabolite cycloguanil has been shown to have inhibitory properties on DHFR.
- Increasing genomic information in bivalves through new EST collections in four species: Development of new genetic markers for environmental studies and genome evolutionPublication . Tanguy, A.; Bierne, N.; Saavedra, Carlos; Pina, B.; Bachere, E.; Kube, M.; Bazin, E.; Bonhomme, F.; Boudry, P.; Boulo, V.; Boutet, I.; Cancela, Leonor; Dossat, C.; Favrel, P.; Huvet, A.; Jarque, S.; Jollivet, D.; Klages, Sven; Lapegue, S.; Leite, Ricardo; Moal, J.; Moraga, D.; Reinhardt, Richard; Samain, J. F.; Zouros, E.; Canario, Adelino V. M.The generation of EST information is an essential step in the genomic characterisation of species. In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs in commercial marine mollusk species and more specifically in the less studied but ecologically and commercially important groups, such as mussel and clam genera. Normalized cDNA libraries were constructed for four different relevant bivalves species (Crassostrea gigas, Mytilus edulis, Ruditapes decussatus and Bathymodiolus azoricus), using numerous tissues and physiological conditions. In this paper, we present the analysis of the 13,013 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1300–3000 unique sequences were identified in each species. For the different species, functional categories could be assigned to only about 16 to 27% of ESTs using the GO annotation tool. All sequences have been incorporated into a publicly available database and form the basis for subsequent microarray design, SNP detection and polymorphism analysis, and the placement of novel markers on genetic linkage maps.
- A microelectrode impedance method to measure interaction of cellsPublication . Gomes, Henrique L.; Leite, Ricardo; Afonso, R.; Stallinga, Peter; Cancela, M. LeonorAn impedance method was developed to determine how immune system cells (hemocyte) interact with intruder cells (parasites). When the hemocyte cells interact with the parasites, they cause a defensive reaction and the parasites start to aggregate in clusters. The level of aggregation is a measure of the host-parasite interaction, and provides information about the efficiency of the immune system response. The cell aggregation is monitored using a set of microelectrodes. The impedance spectrum is measured between each individual microelectrode and a large reference electrode. As the cells starts to aggregate and settle down towards the microelectrode array the impedance of the system is changed. It is shown that the system impedance is very sensitive to the level of cell aggregation and can be used to monitor in real time the interaction between hemocyte cells and parasites.
- Peroxides with antiplasmodial activity inhibit proliferation of Perkinsus olseni, the causative agent of Perkinsosis in bivalvesPublication . Araujo, Nuna C. P.; Afonso, Ricardo; Bringela, A.; Cancela, Leonor; Cristiano, Maria Lurdes Santos; Leite, RicardoPerkinsus olseni, the causative agent of Perkinsosis, can drastically affect the survival of target marine mollusks, with dramatic economic consequences for aquaculture. P. olseni is a member of the Alveolata group, which also comprises parasites that are highly relevant for medical and veterinary sciences such as Plasmodium falciparum and Toxoplasma. P. olseni shares several unique metabolic pathways with those pathological parasites but is not toxic to humans. In this work, six antimalarially active peroxides, derived from the natural product artemisinin or synthetic trioxolanes, were synthesized and tested on P. olseni proliferation and survival. All peroxides tested revealed an inhibitory effect on P. olseni proliferation atmicromolar concentrations. The relevance of the peroxide functionality on toxicity and the effect of Fe(II)-intracellular concentration on activity were also evaluated. Results demonstrated that the peroxide functionality is the toxofore and intracellular iron concentration also proved to be a crucial co-factor on the activation of peroxides in P. olseni. These data points to a mechanismof bioactivation in P. olseni sharing similaritieswith the one proposed in P. falciparumparasites. Preliminary studies on bioaccumulation were conducted using fluorescent-labeled peroxides. Results show that synthetic trioxolanes tend to accumulate on a vacuolewhile the labeled artemisinin accumulates in the cytoplasm. Preliminary experiments on differential genes expression associated to Fe(II) transport protein (Nramp) and calcium transport protein (ATP6/SERCA) were also conducted by qPCR. Results point to a fourfold increase in expression of both genes upon exposure to trioxolanes and approximately twofold upon exposure to artemisinin derivatives. Data obtained in this investigation is relevant for better understanding of the biology of Perkinsus andmay also be important in the development of new strategies for Perkinsosis prevention and control.
- Two isoforms of vasa maternal factor in Senegalese sole: Biotechnological applicationsPublication . Pacchiarini, T.; Cabrita, Elsa; Cross, I.; Ortiz-Delgado, J. B.; Leite, Ricardo; Gavaia, Paulo J.; Herráez, M. P.; Rebordinos, L.; Sarasquete, C.Primordial Germ Cells (PGCs) identification and manipulation present considerable potential for hatchery practice and surrogate broodstocks. To carry out the PGCs characterization a specific molecular marker is required. The vasa gene is a good candidate to identify PGCs and others germinal cells (Nagasawa et al., 2009). The aim of this study was the cloning of the Solea senegalensis vasa cDNA and its expression pattern during early development and adulthood.
- mRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interactionPublication . Leite, Ricardo; Milan, Massimo; Coppe, Alessandro; Bortoluzzi, Stefania; Dos Anjos, António; Reinhardt, Richard; Saavedra, Carlos; Patarnello, T.; Cancela, Leonor; Bargelloni, LucaBackground: The Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis. Results: A database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed.Conclusions: This study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.
- Evolutionary conservation of TFIIH subunits: Implications for the use of zebrafish as a model to study TFIIH function and regulationPublication . Silva, IAL; Cox, C. J.; Leite, Ricardo; Cancela, Leonor; Conceição, N.Transcriptional factor IIH (TFIIH) is involved in cell cycle regulation, nucleotide excision repair, and gene transcription. Mutations in three of its subunits, XPB, XPD, and TTDA, lead to human recessive genetic disorders such as trichothiodystrophy and xeroderma pigmentosum, the latter of which is sometimes associated with Cockayne's syndrome. In the present study, we investigate the sequence conservation of TFIIH subunits among several teleost fish species and compare their characteristics and putative regulation by transcription factors to those of human and zebrafish. We report the following findings: (i) comparisons among protein sequences revealed a high sequence identity for each TFIIH subunit analysed; (ii) among transcription factors identified as putative regulators, OCT1 and AP1 have the highest binding-site frequencies in the promoters of TFIIH genes, and (iii) TFIIH genes have alternatively spliced isoforms. Finally, we compared the protein primary structure in human and zebrafish of XPD and XPB – twoimportant ATP-dependent helicases that catalyse the unwinding of the DNA duplex at promoters during transcription – highlighting the conservation of domain regions such as the helicase domains. Our study suggests that zebrafish, a widely used model for many human diseases, could also act as an important model to study the function of TFIIH complex in repair and transcription regulation in humans.
- Exploring an uncommon host-parasite interaction to unveil the uniqueness of Perkinsus sp as a model organismPublication . Leite, Ricardo; Cancela, Maria LeonorWe report the exploration of some unique metabolic pathways in Perkinsus olseni a marine protist parasite, responsible to significant mortalities in mollusks, especially in bivalves all around the world. In Algarve, south of Portugal carpet shell clam Ruditapes decussatus mortalities can reach up to 70%, causing social and economic losses. The objective of studying those unique pathways, is finding new therapeutic strategies capable of controlling/eliminating P. olseni proliferation in clams. In that sense metabolic pathways, were explored, and drugs affecting these cycles were tested for activity. The first step involved the identification of the genes behind those pathways, the reconstitution of the main steps, and molecular characterization of those genes and later on, the identification of possible targets within the genes studied. Metabolic cycles were screened due to the fact of not being present in host or differ in a critical way, such as the following pathways: shikimate, MEP-‐ isoprenoids, Leloir cycle for chitin production, purine biosynthesis (unique among protists), the de novo synthesis of folates (absent in metazoa) and some unique genes like, the alternative oxidase (a branch of respiratory chain) and the hypoxia sensor HPH. All those pathways were covered and possible chemical inhibition using therapeutic drugs was tested with positive results. The relation between the common host Ruditapes decussatus and P. olseni was also explored in a dimension not possible some years ago. With the accessibility to second generation sequencers and microarray analysis platforms, genes involved in host defense or parasite virulence and resistance to the host were deciphered, allowing aiming to new targets (mechanisms and pathways), offering new possibilities for the control of Perkinsus in close environments. The thousands of genes, generated by this work, sequenced and analyzed from this commercial valuable clam and for Perkinsus olseni will be an important and value tool for the scientific community, allowing a better understanding of host-‐parasite interactions, promoting the usage of P. olseni as an emerging model for alveolata parasites.
- Localization of sites of expression of the BAT-1 gene enconding a RNA HelicasePublication . Leite, Ricardo; Gavaia, Paulo J.; Cancela, LeonorBAT-1 is a member of the DEAD-box family of ATP-dependent helicases involved in a number of cellular functions including initiation of translation, RNA splicing, and ribosome assembly.
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