Browsing by Author "Vieira, Florbela A."
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- Calcitonin receptor family evolution and fishing for function using in silico promoter analysisPublication . Martins, R.; Vieira, Florbela A.; Power, DeborahIn the present study the calcitonin receptor (CTR) sub-family of family B G-protein coupled receptors (GPCRs) in teleosts is evaluated and put in the context of the families overall evolution from echinodermates to vertebrates. Echinodermates, hemichordates, cephalochordates and tunicates have a single gene that encodes a receptor that bears similarity to the vertebrate calcitonin receptor (CTR) and calcitoninlike receptor (CTR/CLR). In tetrapods one gene encodes the calcitonin receptor (CALCR) and another gene the calcitonin receptor-like receptor (CALCRL). The evolution of CALCR has been under strong conservative pressure and a single copy is also found in fishes and high conservation of gene organisation and synteny exits from teleosts to human. A teleost specific CTR innovation that occurred after their divergence from holostei is the presence of several HBDs in the N-terminus. CALCRL had a different evolutionary trajectory from CALCR and although a single gene copy is present in tetrapods the sarcopterygii fish, the coelacanth, has 1 copy of CALCRL but also a fish specific form CALCRL3. The ray-finned fish, the spotted gar, has 1 copy of CALCRL and 1 of CALCRL3 but the teleost specific whole genome duplication has resulted in a CALCRL1 and CALCRL2 in addition to the fish specific CALCRL3. Strong conservation of CALCRL gene structure exists from human to fish. Promoter analysis in silico reveals that the duplicated CALCRL genes in the teleosts, zebrafish, takifugu, tetraodon and medaka, have divergent promoters and different putative co-regulated gene partners suggesting their function is different.
- Comparative analysis of a teleost skeleton transcriptome provides insight into its regulationPublication . Vieira, Florbela A.; Thorne, Michael A. S.; Stueber, K.; Darias, M.; Reinhardt, Richard; Clark, M. S.; Gisbert, Enric; Power, DeborahAn articulated endoskeleton that is calcified is a unifying innovation of the vertebrates, however the molecular basis of the structural divergence between terrestrial and aquatic vertebrates, such as teleost fish, has not been determined. In the present study long-read next generation sequencing (NGS, Roche 454 platform) was used to characterize acellular perichondral bone (vertebrae) and chondroid bone (gill arch) in the gilthead sea bream (Sparus auratus). A total of 15.97 and 14.53 Mb were produced, respectively from vertebrae and gill arch cDNA libraries and yielded 32,374 and 28,371 contigs (consensus sequences) respectively. 10,455 contigs from vertebrae and 10,625 contigs from gill arches were annotated with gene ontology terms. Comparative analysis of the global transcriptome revealed 4249 unique transcripts in vertebrae, 4201 unique transcripts in the gill arches and 3700 common transcripts. Several core gene networks were conserved between the gilthead sea bream and mammalian skeleton. Transcripts for putative endocrine factors were identified in acellular gilthead sea bream bone suggesting that in common with mammalian bone it can act as an endocrine tissue. The acellular bone of the vertebra, in contrast to current opinion based on histological analysis, was responsive to a short fast and significant (p < 0.05) down-regulation of several transcripts identified by NGS, osteonectin, osteocalcin, cathepsin K and IGFI occurred. In gill arches fasting caused a significant (p < 0.05) down-regulation of osteocalcin and up-regulation of MMP9.
- Divergent responsiveness of the dentary and vertebral bone to a selective estrogen-receptor modulator (SERM) in the teleost Sparus auratusPublication . Vieira, Florbela A.; Pinto, Patricia IS; Guerreiro, P. M.; Power, DeborahIn teleosts the regulation of skeletal homeostasis and turnover by estrogen is poorly understood. For this reason raloxifene, a selective estrogen-receptor modulator (SERM), was administered to sea bream (Sparus auratus) and its effect on plasma calcium balance and transcript expression in dentary (dermal bone) and vertebra (perichondral bone) was studied. The concentration of total calcium or phosphorus in plasma was unchanged by raloxifene treatment for 6 days. The activity of alkaline phosphatase (ALP) in dentary bone of raloxifene treated fish was significantly (p < 0.05) higher than control fish but it was not changed in vertebral bone. Transcripts for estrogen receptor (ER) a were in very low abundance in the sea bream dentary and vertebra and were unchanged by raloxifene treatment. In contrast, raloxifene caused significant (p < 0.05) up-regulation of the duplicate ERb transcripts in the dentary but did not affect specific transcripts for osteoclast (TRAP), osteoblast (ALP, Runx2, osteonectin) or cartilage (IGF1, CILP2, FN1a). In the vertebra ERbb was not changed by raloxifene but ERba was significantly (p < 0.05) down-regulated as was the skeletal specific transcripts, TRAP, ALP, CILP2, FN1a. In summary, ERbs regulate estrogen sensitivity of the skeleton in sea bream, which responds in a non uniform manner. In common with mammals raloxifene appears to have an anti-resorptive role (in sea bream vertebra), but also an osteoblast stimulatory role, inducing ALP activity in the dentary of sea bream. Overall, the results indicate bone specific responsiveness to raloxifene in the sea bream. Further work will be required to understand the basis of bone responsiveness and the role of E2 and ERs in teleost bone homeostasis.
- Evolution of secretin family GPCR members in the metazoaPublication . Cardoso, João CR; Pinto, Vanda C.; Vieira, Florbela A.; Clark, M. S.; Power, DeborahBackground: Comparative approaches using protostome and deuterostome data have greatly contributed to understanding gene function and organismal complexity. The family 2 G-protein coupled receptors (GPCRs) are one of the largest and best studied hormone and neuropeptide receptor families. They are suggested to have arisen from a single ancestral gene via duplication events. Despite the recent identification of receptor members in protostome and early deuterostome genomes, relatively little is known about their function or origin during metazoan divergence. In this study a comprehensive description of family 2 GPCR evolution is given based on in silico and expression analyses of the invertebrate receptor genes. Results: Family 2 GPCR members were identified in the invertebrate genomes of the nematodes C. elegans and C. briggsae, the arthropods D. melanogaster and A. gambiae (mosquito) and in the tunicate C. intestinalis. This suggests that they are of ancient origin and have evolved through gene/ genome duplication events. Sequence comparisons and phylogenetic analyses have demonstrated that the immediate gene environment, with regard to gene content, is conserved between the protostome and deuterostome receptor genomic regions. Also that the protostome genes are more like the deuterostome Corticotrophin Releasing Factor (CRF) and Calcitonin/Calcitonin Gene-Related Peptide (CAL/CGRP) receptors members than the other family 2 GPCR members. The evolution of family 2 GPCRs in deuterostomes is characterised by acquisition of new family members, with SCT (Secretin) receptors only present in tetrapods. Gene structure is characterised by an increase in intron number with organismal complexity with the exception of the vertebrate CAL/CGRP receptors. Conclusion: The family 2 GPCR members provide a good example of gene duplication events occurring in tandem with increasing organismal complexity during metazoan evolution. The putative ancestral receptors are proposed to be more like the deuterostome CAL/CGRP and CRF receptors and this may be associated with their fundamental role in calcium regulation and the stress response, both of which are essential for survival.
- Identification of molecular and physiological responses to chronic environmental challenge in an invasive species The Pacific oyster, Crassostrea gigasPublication . Clark, M. S.; Thorne, Michael A. S.; Araújo Amaral, Ana Margarida; Vieira, Florbela A.; Batista, Frederico; Reis, João; Power, DeborahUnderstanding the environmental responses of an invasive species is critical in predicting how ecosystem composition may be transformed in the future, especially under climate change. In this study, Crassostrea gigas, a species well adapted to the highly variable intertidal environment, was exposed to the chronic environmental challenges of temperature (19 and 24°C) and pH (ambient seawater and a reduction of 0.4 pH units) in an extended 3-month laboratory-based study. Physiological parameters were measured (condition index, shell growth, respiration, excretion rates, O:N ratios, and ability to repair shell damage) alongside molecular analyses. Temperature was by far the most important stressor, as demonstrated by reduced condition indexes and shell growth at 24°C, with relatively little effect detected for pH. Transcriptional profiling using candidate genes and SOLiD sequencing of mantle tissue revealed that classical “stress” genes, previously reported to be upregulated under acute temperature challenges, were not significantly expressed in any of the treatments, emphasizing the different response between acute and longer term chronic stress. The transcriptional profiling also elaborated on the cellular responses underpinning the physiological results, including the identification of the PI3K/AKT/mTOR pathway as a potentially novel marker for chronic environmental challenge. This study represents a first attempt to understand the energetic consequences of cumulative thermal stress on the intertidal C. gigas which could significantly impact on coastal ecosystem biodiversity and function in the future.
- Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyroseqeuencingPublication . Clark, M. S.; Thorne, Michael A. S.; Vieira, Florbela A.; Cardoso, João CR; Power, Deborah; Peck, Lloyd S.Abstract Background The Antarctic clam, Laternula elliptica, is an infaunal stenothermal bivalve mollusc with a circumpolar distribution. It plays a significant role in bentho-pelagic coupling and hence has been proposed as a sentinel species for climate change monitoring. Previous studies have shown that this mollusc displays a high level of plasticity with regard to shell deposition and damage repair against a background of genetic homogeneity. The Southern Ocean has amongst the lowest present-day CaCO3 saturation rate of any ocean region, and is predicted to be among the first to become undersaturated under current ocean acidification scenarios. Hence, this species presents as an ideal candidate for studies into the processes of calcium regulation and shell deposition in our changing ocean environments. Results 454 sequencing of L. elliptica mantle tissue generated 18,290 contigs with an average size of 535 bp (ranging between 142 bp-5.591 kb). BLAST sequence similarity searching assigned putative function to 17% of the data set, with a significant proportion of these transcripts being involved in binding and potentially of a secretory nature, as defined by GO molecular function and biological process classifications. These results indicated that the mantle is a transcriptionally active tissue which is actively proliferating. All transcripts were screened against an in-house database of genes shown to be involved in extracellular matrix formation and calcium homeostasis in metazoans. Putative identifications were made for a number of classical shell deposition genes, such as tyrosinase, carbonic anhydrase and metalloprotease 1, along with novel members of the family 2 G-Protein Coupled Receptors (GPCRs). A membrane transport protein (SEC61) was also characterised and this demonstrated the utility of the clam sequence data as a resource for examining cold adapted amino acid substitutions. The sequence data contained 46,235 microsatellites and 13,084 Single Nucleotide Polymorphisms(SNPs/INDELS), providing a resource for population and also gene function studies. Conclusions This is the first 454 data from an Antarctic marine invertebrate. Sequencing of mantle tissue from this non-model species has considerably increased resources for the investigation of the processes of shell deposition and repair in molluscs in a changing environment. A number of promising candidate genes were identified for functional analyses, which will be the subject of further investigation in this species and also used in model-hopping experiments in more tractable and economically important model aquaculture species, such as Crassostrea gigas and Mytilus edulis.
- PACAP, VIP and their receptors in the metazoa Insights about the origin and evolution of the ligand-receptor pairPublication . Cardoso, J. C. R.; Vieira, Florbela A.; Power, DeborahThe evolution, function and interaction of ligand–receptor pairs are of major pharmaceutical interest. Comparative sequence analysis approaches using data from phylogenetically distant organisms can provide insights into their origin and possible physiological roles. The present review focuses on the pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP) and their receptors in the metazoa. A PACAP-like peptide is present in tunicates and chordates while VIP- and PACAP/VIP-specific receptors have only been isolated in the latter phyla. The apparently disparate evolution of the ligands and their specific receptors raises questions about their evolution during the metazoan radiation and also about how the ligands may have acquired new functions.
- Putative ancestral receptor genes of vertebrate family 2 GPCR in the invertebrate genome of the nematode Caenorhabditis elegansPublication . Cardoso, João CR; Vieira, Florbela A.; Clark, M. S.; Power, DeborahThree putative ancestral genes for the family 2 of GPCRs were identified in the invertebrate genome of the nematode Caenorhabditis elegans (C. elegans). The nematode clones namely CB13B9.4, ZK643.3 and C18B12.2 are localised on chromosomes III (CB13B9.4, ZK643.3) and X (C18B12.2) and were identified by searching the nematode genome database with the amino acid sequences of the vertebrate family 2 receptor genes. In human and in the teleost Takifugu rubripes genomes, 15 and 21 members of the family 2 GPCRs have been identified, respectively. Linkage mapping analysis revealed that the gene environment of family 2 GPCRs is highly conserved between human and Takifugu genomes and some conservation also occurs with C. elegans chromosome III and X regions containing the putative family 2 GPCRs. Sequence comparative analysis revealed that the putative C. elegans receptors contained seven transmembrane (TM) domains and long N-terminal regions with conserved cysteine residues typical of the family 2 GPCR members. Phylogenetic analysis carried out with the TM domains of all vertebrate, chordate (Ciona) and insect (fruit fly) family 2 receptor members has shown that the nematode receptors identified tend to group with the clade containing the CRF and Calcitonin/CGRP family 2 GPCRs.
- Skin healing and scale regeneration in fed and unfed sea bream, Sparus auratusPublication . Vieira, Florbela A.; Gregorio, Silvia; Ferraresso, Serena; Thorne, Michael A. S.; Costa, Rita; Milan, Massimo; Bargelloni, Luca; Clark, M. S.; Canario, Adelino V. M.; Power, DeborahAbstract Background Fish scales are an important reservoir of calcium and phosphorus and together with the skin function as an integrated barrier against environmental changes and external aggressors. Histological studies have revealed that the skin and scales regenerate rapidly in fish when they are lost or damaged. In the present manuscript the histological and molecular changes underlying skin and scale regeneration in fed and fasted sea bream (Sparus auratus) were studied using a microarray 3 and 7 days after scale removal to provide a comprehensive molecular understanding of the early stages of these processes. Results Histological analysis of skin/scales revealed 3 days after scale removal re-epithelisation and formation of the scale pocket had occurred and 53 and 109 genes showed significant up or down-regulation, respectively. Genes significantly up-regulated were involved in cell cycle regulation, cell proliferation and adhesion, immune response and antioxidant activities. 7 days after scale removal a thin regenerated scale was visible and only minor changes in gene expression occurred. In animals that were fasted to deplete mineral availability the expression profiles centred on maintaining energy homeostasis. The utilisation of fasting as a treatment emphasised the competing whole animal physiological requirements with regard to barrier repair, infection control and energy homeostasis. Conclusions The identification of numerous genes involved in the mitotic checkpoint and cell proliferation indicate that the experimental procedure may be useful for understanding cell proliferation and control in vertebrates within the context of the whole animal physiology. In response to skin damage genes of immune surveillance were up-regulated along with others involved in tissue regeneration required to rapidly re-establish barrier function. Additionally, candidate fish genes were identified that may be involved in cytoskeletal re-modelling, mineralization and stem cells, which are of potential use in aquaculture and fish husbandry, as they may impact on the ability of the fish to produce structural proteins, such as muscle, efficiently.
- The secretin G-protein-coupled receptor family: teleost receptorsPublication . Cardoso, João CR; Clark, M. S.; Vieira, Florbela A.; Bridge, P. D.; Gilles, A.; Power, DeborahTwenty-one members of the secretin family (family 2) of G-protein-coupled receptors (GPCRs) were identified via directed cloning and data-mining of the Fugu Genome Consortium database, representing the most comprehensive description of secretin GPCRs in a teleost fish to date. Duplicated genes were identified for many of the family members, namely the receptors for pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP), calcitonin, calcitonin gene-related peptide (CGRP), growth hormone releasing hormone (GHRH), glucagon receptor/glucagon-like peptide (GLP) and parathyroid hormone-related peptide (PTHrP)/PTH. Mining of other teleost genomes (zebrafish and Tetraodon) revealed that the duplicated genes identified in the Takifugu genome were also present in these fish. Additional database searching of the Escherichia coli, yeast, Drosophila, Caenorhabditis elegans and Ciona genomes revealed that the family 2 of GPCRs were only present in the multicellular organisms. Orthologues of all the human secretin receptors were identified with the exception of secretin itself. Additional database searches in the Fugu Genome Consortium database also failed to reveal a secretin ligand and so it is hypothesised that both the receptor and the ligand evolved after the divergence of teleost/tetrapod lineages. Phylogenetic analysis at both the protein and the DNA level provided strong support for each of the individual receptor family groupings, but weak support between groups, making evolutionary inferences difficult. A more critical analysis of the PACAP/VIP receptor family confirmed previous hypotheses that the vasoactive intestinal peptide receptor (VPAC1R) gene is the ancestral form of the receptor.