Browsing by Author "Alves, Ricardo N."
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- Bioaccessibility of lipophilic and hydrophilic marine biotoxins in seafood: an in vitro digestion approachPublication . Alves, Ricardo N.; Rambla-Alegre, Maria; Braga, Ana Catarina; Maulvault, Ana L.; Barbosa, Vera; Campàs, Mònica; Reverté, Laia; Flores, Cintia; Caixach, Josep; Kilcoyne, Jane; Costa, Pedro Reis; Diogène, Jorge; Marques, AntónioThis study aimed to assess the bioaccessibility of different marine biotoxins in naturally contaminated shellfish and fish gonads using an in vitro digestion methodology. In general, hydrophilic toxins (domoic acid, paralytic shellfish poisoning toxins and tetrodotoxins) showed higher bioaccessibility than lipophilic ones (okadaic acid and azaspiracids). The bioaccessibility of toxins from the okadaic acid group ranged from 69% (raw European razor clams) to 74% (raw donax clams). Regarding azaspiracids, 47% of the initial content was bioaccessible in steamed blue mussel. As for hydrophilic toxins, 100% of the initial content was bioaccessible after digestion in raw shellfish and puffer fish gonads. The total tetrodotoxin bioaccessibility in puffer fish gonads decreased significantly after steaming. The profile of tetrodotoxins changed during the digestion process: TTX and 11-norTTX-6S-ol analogues decreased significantly after digestion, but the 5,6,11-trideoxy TTX analogue increased in both raw and steamed puffer fish gonads. These preliminary findings confirm the need to consider bioaccessibility data in future seafood risk assessment, as such information enables a more accurate and realistic estimation of potential seafood hazards, particularly in what concerns lipophilic toxins, therefore, constituting a crucial tool in the refinement of regulatory limits for the presence of biotoxins in seafood.
- Changes in liver proteome expression of Senegalese sole (Solea senegalensis) in response to repeated handling stressPublication . Cordeiro, O.; Silva, Tomé S.; Alves, Ricardo N.; Costas, B.; Wulff, Tune; Richard, Nadège; Vareilles, Mahaut; Conceição, L. E. C.; Rodrigues, PedroThe Senegalese sole, a high-value flatfish, is a good candidate for aquaculture production. Nevertheless, there are still issues regarding this species’ sensitivity to stress in captivity. We aimed to characterize the hepatic proteome expression for this species in response to repeated handling and identify potential molecular markers that indicate a physiological response to chronic stress. Two groups of fish were reared in duplicate for 28 days, one of them weekly exposed to handling stress (including hypoxia) for 3 min, and the other left undisturbed. Two-dimensional electrophoresis enabled the detection of 287 spots significantly affected by repeated handling stress (Wilcoxon–Mann–Whitney U test, p<0.05), 33 of which could be reliably identified by peptide mass spectrometry. Chronic exposure to stress seems to have affected protein synthesis, folding and turnover (40S ribosomal protein S12, cathepsin B, disulfide-isomerase A3 precursor, cell-division cycle 48, and five distinct heat shock proteins), amino acid metabolism, urea cycle and methylation/folate pathways (methionine adenosyltransferase I α, phenylalanine hydroxylase, mitochondrial agmatinase, serine hydroxymethyltransferase, 3-hydroxyanthranilate 3,4-dioxygenase, and betaine homocysteine methyltransferase), cytoskeletal (40S ribosomal protein SA, α-actin, β-actin, α-tubulin, and cytokeratin K18), aldehyde detoxification (aldehyde dehydrogenase 4A1 family and aldehyde dehydrogenase 7A1 family), carbohydrate metabolism and energy homeostasis (fatty acid-binding protein, enolase 3, enolase 1, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, aconitase 1, mitochondrial ATP synthase α-subunit, and electron-transfer flavoprotein α polypeptide), iron and selenium homeostasis (transferrin and selenium binding protein 1), steroid hormone metabolism (3-oxo-5-β-steroid 4-dehydrogenase), and purine salvage (hypoxanthine phosphoribosyltransferase). Further characterization is required to fully assess the potential of these markers for the monitoring of fish stress response to chronic stressors of aquaculture environment.
- Identificação de indicadores moleculares de bem-estar durante o cultivo de dourada (Sparus aurata, Linnaeus 1758), utilizando técnicas de proteómicaPublication . Alves, Ricardo N.; Rodrigues, Pedro; Conceição, LuísO bem-estar dos organismos produzidos em aquacultura tornou-se, na actualidade, um dos aspectos fundamentais desta indústria. O principal objectivo deste trabalho foi tentar identificar indicadores moleculares de bem-estar durante o cultivo de Sparus aurata, através da análise da expressão das proteínas do fígado. O fígado será o órgão alvo deste estudo, pois é onde estão activos a maioria dos processos chave do metabolismo dos peixes, e como tal, será o órgão mais rapidamente afectado em condições de stress crónico. Foi comparado o proteoma entre peixes sujeitos a condições óptimizadas de cultivo (CTRL) e condições indutoras de stress crónico (manuseamento regular – MAN e densidades de cultivo elevadas – DEN), utilizando a técnica de separação de proteínas, a electroforese bidimensional. Esta técnica demonstrou ser eficaz na identificação de biomarcadores relacionados com o stress. Foram detectados 560 spots (PDQuest 2-D Analysis Software – BioRad) e a análise estatística (teste não paramétrico Mann-Whitney, p < 0,05) revelou a existência de proteínas com expressão diferenciada entre as diferentes condições: 235 proteínas diferem entre CTRL e MAN e 223 proteínas diferem entre CTRL e DEN. Apenas 70 proteínas diferem entre as duas condições de stress. As proteínas com um nível de expressão ≥ 2x entre as várias condições, foram consideradas relevantes e sequenciadas por LC- MS/MS, de modo a serem identificadas por peptide fragmentation fingerprinting. Os resultados obtidos neste estudo revelaram a expressão diferencial de proteínas, muitas delas já previamente relacionadas com stress, estando envolvidas em vários processos e vias metabólicas. Os dois principais indicadores moleculares identificados foram: a heat shock cognate protein 70 (Hsc70) e a fatty acid binding protein (FABP). Outras proteínas, tais como, as proteínas envolvidas no metabolismo dos carbohidratos (isomerase da triose fosfato, desidrogenase do gliceraldeído 3-fosfato, alfa enolase, desidrogenase beta do piruvato) e a sintetase da glutamina poderão possivelmente ser utilizados no futuro, após a realização de mais estudos acerca destas enzimas e do seu papel em situações de stress crónico. Futuramente, com o auxílio destes indicadores moleculares, poderão ser desenvolvidos testes práticos e ferramentas genómicas para estudar o bem-estar de peixes em aquacultura. Estes biomarcadores poderão ainda ser utilizados em processos de certificação de produtos de elevada qualidade produzidos pelos sistemas semiintensivos que caracterizam a aquacultura portuguesa.
- In vitro bioaccessibility of the marine biotoxin okadaic acid in shellfishPublication . Braga, Ana C.; Alves, Ricardo N.; Maulvault, Ana L.; Barbosa, Vera; Marques, António; Costa, Pedro R.Okadaic acid (OA) and their derivatives are marine toxins responsible for the human diarrhetic shellfish poisoning (DSP). To date the amount of toxins ingested in food has been considered equal to the amount of toxins available for uptake by the human body. In this study, the OA fraction released from the food matrix into the digestive fluids (bioaccessibility) was assessed using a static in vitro digestion model. Naturally contaminated mussels (Mytilus galloprovincialis) and donax clams (Donax sp.), collected from the Portuguese coast, containing OA and dinophysistoxin-3 (DTX3) were used in this study. Bioaccessibility of OA total content was 88% and 75% in mussels and donax clams, respectively. Conversion of DTX3 into its parent compound was verified during the simulated digestive process and no degradation of these toxins was found during the process. This is the first study assessing the bioaccessibility of OA group toxins in naturally contaminated seafood. This study provides relevant new data that can improve and lead to more accurate food safety risk assessment studies concerning these toxins. (C) 2016 Elsevier Ltd. All rights reserved.
- In vitro bioaccessibility of the marine biotoxins okadaic acid, dinophysistoxin-2 and their 7-O-acyl fatty acid ester derivatives in raw and steamed shellfishPublication . Manita, Diana; Alves, Ricardo N.; Braga, Ana Catarina; Fogaca, Fabiola H. S.; Marques, Antonio; Reis Costa, PedroOkadaic acid (OA), Dinophysistoxins (DTX1 and DTX2) and their acyl-derivatives (DTX3) are marine toxins responsible for the human diarrhetic shellfish poisoning. To date the amount of toxins ingested from consumption of shellfish has been considered equal to the amount of toxins available for uptake by the human body. The aim of this study is to assess the OA, DTX2 and DTX3 fractions released from raw and steamed mussels and cockles into the digestive fluids (bioaccessibility) using a static in vitro digestion model. Higher bioaccessibility was found in mussels (86 +/- 4%) than in cockles (59 +/- 9%). A significant reduction of ester derivatives of OA and an increase of OA were observed in the bioaccessible fraction of mussel samples, suggesting that DTX3 undergo conversion into their more toxic parent compounds during human digestion. However, similar increase of DTX2 and reduction of the respective acyl derivatives was not observed. Steaming lead to significant reduction of OA and analogues bioaccessibility in both species even though increased concentrations of toxins are obtained after this treatment. Risk assessment based solely on DSP toxins occurrence in seafood can conduct to an overestimation of the exposure and lead to more conservative regulatory measures. (C) 2017 Elsevier Ltd. All rights reserved.
- Metabolic molecular indicators of chronic stress in gilthead seabream (Sparus aurata) using comparative proteomicsPublication . Alves, Ricardo N.; Cordeiro, O.; Silva, Tomé S.; Richard, Nadège; De Vareilles, M.; Marino, G.; Di Marco, Patrizia; Rodrigues, Pedro; Conceição, L. E. C.The aim of this study was to identify possible metabolic molecular indicators of chronic stress in gilthead sea bream Sparus aurata. Two potential stressful conditions were tested: repeated handling and crowding at high stocking density. Gilthead seabream kept under optimized rearing conditions were used as control fish. Cortisol was measured as primary stress indicator and the liver proteome of stressed fish was compared to that of control fish using comparative proteomics. Plasma cortisol levels in sea bream repeatedly handled and crowded at high stocking density were significantly higher than in undisturbed control fish. A total of 560 spots were detected and the statistical analysis revealed a differential expression in about 50% of all detected proteins. Spots with greater than 2-fold or lower than 0.5-fold changes were identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Proteins like fatty acid binding protein (lipid transport and antioxidant role), heat shock cognate protein (chaperoning), calmodulin (Ca2+ signaling), mitochondrial porine — voltage-dependent anion channel (lipid oxidation), glutamine synthetase (ammonia metabolism), cofilin and beta-tubulin (cytoskeleton), hemoglobin and several other proteins involved in carbohydrate metabolism (triose-phosphate isomerase, pyruvate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, alfa-enolase) were differentially expressed in fish under chronic stress. Some of these proteins may be used in the future as chronic stress and/or part of a panel of welfare biomarkers, after validation studies using RT-PCR and ELISA assays.
- Molecular and cellular changes in skin and muscle during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus) are accompanied by changes in deiodinases expressionPublication . Campinho, Marco António; Galay-Burgos, M.; Silva, Nádia; Costa, R. A.; Alves, Ricardo N.; Sweeney, Glen E.; Power, DeborahFlatfish metamorphosis is the most dramatic postnatal developmental event in teleosts. Thyroid hormones (TH), thyroxine (T4) and 3,3′-5′-triiodothyronine (T3) are the necessary and sufficient factors that induce and regulate flatfish metamorphosis. Most of the cellular and molecular action of TH is directed through the binding of T3 to thyroid nuclear receptors bound to promoters with consequent changes in the expression of target genes. The conversion of T4 to T3 and nuclear availability of T3 depends on the expression and activity of a family of 3 selenocysteine deiodinases that activate T4 into T3 or degrade T4 and T3.
- The transcriptome of metamorphosing flatfishPublication . Alves, Ricardo N.; Stueber, Kurt; Tine, Mbaye; Thorne, M. A. S.; Smáradóttir, H.; Reinhard, Richard; Clark, M. S.; Rønnestad, Ivar; Power, DeborahBackground Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. Metamorphosis in vertebrates is driven by thyroid hormones (THs), but how they orchestrate the cellular, morphological and functional modifications associated with maturation to juvenile/adult states in flatfish is an enigma. Since THs act via thyroid receptors that are ligand activated transcription factors, we hypothesized that the maturation of tissues during metamorphosis should be preceded by significant modifications in the transcriptome. Targeting the unique metamorphosis of flatfish and taking advantage of the large size of Atlantic halibut (Hippoglossus hippoglossus) larvae, we determined the molecular basis of TH action using RNA sequencing. Results De novo assembly of sequences for larval head, skin and gastrointestinal tract (GI-tract) yielded 90,676, 65,530 and 38,426 contigs, respectively. More than 57 % of the assembled sequences were successfully annotated using a multi-step Blast approach. A unique set of biological processes and candidate genes were identified specifically associated with changes in morphology and function of the head, skin and GI-tract. Transcriptome dynamics during metamorphosis were mapped with SOLiD sequencing of whole larvae and revealed greater than 8,000 differentially expressed (DE) genes significantly (p < 0.05) up- or down-regulated in comparison with the juvenile stage. Candidate transcripts quantified by SOLiD and qPCR analysis were significantly (r = 0.843; p < 0.05) correlated. The majority (98 %) of DE genes during metamorphosis were not TH-responsive. TH-responsive transcripts clustered into 6 groups based on their expression pattern during metamorphosis and the majority of the 145 DE TH-responsive genes were down-regulated. Conclusions A transcriptome resource has been generated for metamorphosing Atlantic halibut and over 8,000 DE transcripts per stage were identified. Unique sets of biological processes and candidate genes were associated with changes in the head, skin and GI-tract during metamorphosis. A small proportion of DE transcripts were TH-responsive, suggesting that they trigger gene networks, signalling cascades and transcription factors, leading to the overt changes in tissue occurring during metamorphosis.