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- Fish allergenicity modulation using tailored enriched diets—Where are we?Publication . Schrama, Denise; Czolk, Rebecca; Raposo de Magalhães, Cláudia; Kuehn, Annette; Rodrigues, PedroFood allergy is an abnormal immune response to specific proteins in a certain food. The chronicity, prevalence, and the potential fatality of food allergy, make it a serious socio-economic problem. Fish is considered the third most allergenic food in the world, affecting part of the world population with a higher incidence in children and adolescents. The main allergen in fish, responsible for the large majority of fish-allergic reactions in sensitized patients, is a small and stable calcium-binding muscle protein named beta-parvalbumin. Targeting the expression or/and the 3D conformation of this protein by adding specific molecules to fish diets has been the innovative strategy of some researchers in the fields of fish allergies and nutrition. This has shown promising results, namely when the apo-form of beta-parvalbumin is induced, leading in the case of gilthead seabream to a 50% reduction of IgE-reactivity in fish allergic patients.
- Domestic animal proteomics in the 21st century: a global retrospective and viewpoint analysisPublication . Almeida, André M.; Ali, Syed Azmal; Ceciliani, Fabrizio; Eckersall, P. David; Hernández-Castellano, Lorenzo E.; Han, Rongwei; Hodnik, Jaka J.; Jaswal, Shalini; Lippolis, John D.; McLaughlin, Mark; Miller, Ingrid; Mohanty, Ashok Kumar; Mrljak, Vladimir; Nally, Jarlath E.; Nanni, Paolo; Plowman, Jeffrey E.; Poleti, Mirele D.; Ribeiro, David M.; Rodrigues, Pedro; Roschitzki, Bernd; Schlapbach, Ralph; Starič, Jože; Yang, Yongxin; Zachut, MayaAnimal production and health are of significant economic importance, particularly regarding the world food supply. Animal and veterinary sciences have evolved immensely in the past six decades, particularly in genetics, nutrition, housing, management and health. To address major challenges such as those posed by climate change or metabolic disorders, it is of utmost importance to use state-of-the-art research tools. Proteomics and the other post-genomic tools (transcriptomics or metabolomics) are among them. Proteomics has experienced a considerable development over the last decades. This brought developments to different scientific fields. The use and adoption of proteomics tools in animal and veterinary sciences has some limitations (database availability or access to proteomics platforms and funding). As a result, proteomics' use by animal science researchers varies across the globe. In this viewpoint article, we focus on the developments of domestic animal proteomics over the last decade in different regions of the globe and how the researchers have coped with such challenges. In the second part of the article, we provide examples of funding, educational and laboratory establishment initiatives designed to foster the development of (animal-based) proteomics. International scientific collaboration is a definitive and key feature in the development and advancement of domestic animal proteomics. SIGNIFICANCE: Animal production and health are very important for food supply worldwide particularly as a source of proteinaceous foods. Animal and veterinary sciences have evolved immensely in the last decades. In order to address the major contemporary challenges facing animal and veterinary sciences, it is of utmost importance to use state-of-the-art research tools such as Proteomics and other Omics. Herein, we focus on the major developments in domestic animal proteomics worldwide during the last decade and how different regions of the world have used the technology in this specific research field. We address also major international efforts aiming to increase the research output in this area and highlight the importance of international cooperation to address specific problems inherent to domestic animal proteomics.
- Changes in the soluble bone proteome of reared white seabream (Diplodus sargus) with skeletal deformitiesPublication . Silva, Tomé S.; Cordeiro, O.; Richard, Nadège; Conceição, L. E. C.; Rodrigues, PedroOne of the main constrains for commercial aquaculture production of white seabream (Diplodus sargus) is the high incidence of skeletal malformations in reared fish. The purpose of this study was to obtain a better understanding of the mechanisms involved in the development of these types of skeletal malformations by comparative proteomic analysis of the vertebral column of normal and deformed fish using 2DE for protein separation and MS for protein identification. We observed a 3.2 and 3.4-fold increase in the expression of two tropomyosin isoforms, one of which (tropomyosin-4) is essential for the motility and polarization cycles of osteoclasts. Furthermore, a 1.6, 1.7 and 1.8-fold increase in three parvalbumin spots was detected, suggesting a cellular response to increased intracellular Ca2+ levels. These results can be interpreted as signs of increased cellular activity in the bone of white seabream with skeletal deformities coupled to a higher degree of calcium mobilization, which elicits further studies into the use of these proteins as indicators of skeletal metabolic state.
- Impact of dietary protein hydrolysates on skeleton quality and proteome in Diplodus sargus larvaePublication . Vareilles, Mahaut; Richard, Nadège; Gavaia, Paulo J.; Silva, Tomé S.; Cordeiro, O.; Guerreiro, Inês; Yúfera, M.; Batista, I.; Pires, C.; Pousão-Ferreira, P.; Rodrigues, Pedro; Rønnestad, I.; Fladmark, K. E.; Conceição, L. E. C.In order to investigate the effects of dietary protein hydrolysates (PH) on larval growth performance, skeleton quality and proteome expression, triplicate groups of white seabream (Diplodus sargus) larvae were co-fed from first-feeding with live feed and three microencapsulated diets differing in the molecular weight of their PH fraction (Control – inclusion of CPSP-90; H – inclusion of a high amount in 0.5–30 kDa hydrolysates; L – inclusion of a high amount in <0.5 kDa hydrolysates). At 15 days after hatching (DAH), proteome expression changes were assessed in entire larvae by twodimensional gel electrophoresis and the quality of larval skeleton was analysed at 28 DAH through double staining of cartilage and bone. Dietary PH fractions tested affected growth, the larvae fed diet L being significantly larger than those fed diet H, but it did not affect the incidence of deformed larvae, nor the number of deformities per fish. Two-dimensional analysis of larvae proteome allowed the detection and the comparative quantification of a total of 709 protein spots having a pI between 4 and 7, around half of which had an expression significantly affected by dietary treatment, the main difference being between proteome of Control larvae with those of both groups L and H. From these spots, 52 proteins involved in diverse processes such as cytoskeletal dynamics, energetic, lipoprotein, amino acid (AA), and nucleotide metabolisms, protein chaperoning and degradation, and signal transduction, were identified. This study revealed that the molecular weight of the dietary protein hydrolysate fraction had a minor impact on skeletal deformities in white seabream larvae, but affected growth performance and had a strong impact on larvae whole body proteome.
- How tryptophan levels in plant-based aquafeeds affect fish physiology, metabolism and proteomePublication . Cerqueira, Marco; Schrama, Denise; Silva, Tome S.; Colen, R.; Engrola, Sofia; Conceicao, Luis E. C.; Rodrigues, Pedro; Farinha, Ana PaulaFish meal replacement by plant-protein sources is a priority in aquaculture feeds. Within this framework, dietary supplementation with essential amino acids (EAA), as tryptophan (TRP), is strategic to ensure that the individual nutritional needs are met, besides promoting enhanced immunological status. The purpose of this study was to examine the beneficial effects of TRP incorporation in plant-protein source diets on fish growth performance and nutritional status. We tested diets with 20% lower (LTRP) and 27% higher (HTRP) of the putative requirements of TRP for seabream (Sparus aurata) and assessed its impact on fish physiology and liver metabolism and proteome. After 12 weeks, growth performance, body proximate, hepatic composition and liver metabolic profiling were similar between diets. Nevertheless, liver proteome analysis indicated a higher accumulation of proteins involved in acute-phase responses, typically triggered by infection, inflammation or trauma, in fish fed with HTRP diet as compared with those fed with LTRP. The overall results obtained suggest a potential beneficial effect of TRP supplementation in terms of immune stimulation, without compromising growth or feed intake. Moreover, proteomics and metabolic profiling demonstrate to be valuable tools in this endeavour. Significance: Nutritional needs are hard to assess in aquaculture fisheries, and many times controversial depending on the methodology employed. The estimated amino acid requirements depend on both fish species and stage development, making it extremely hard to standardise. On the other hand, the substitution of fish-based to plant-based protein sources diets towards a sustainable aquaculture, may imbalance these requirements, being necessary further studies to assess the impact on fish growth and development. Finally, the incorporation of crystalized amino acids such as TRP into diets aims global better performance both at fish health/immune condition and growth development. This work focused on the potential beneficial effects of TRP supplementation into diets with a plant-based protein source, addressing the effects on the liver metabolism and proteome, and on growth performance of Gilthead seabream juveniles, a species with special relevance and economical importance in the Mediterranean region. The present study by employing proteomics together with metabolic profiling shows that TRP supplementation at the tested doses, does not compromise growth performance, and seems to stimulate the immune system. Our findings can contribute to the development of new feed formulations for Gilthead seabream species, therefore, reinforcing the resilience and competitiveness of the on-growing aquaculture industry and impact directly the sustainability of living resources with the decrease of the fisheries needs to fulfil the human search for quality proteins consume.
- Protein changes as robust signatures of fish chronic stress: a proteomics approach to fish welfare researchPublication . Raposo de Magalhães, Cláudia; Schrama, Denise; Farinha, Ana Paula; Revets, Dominique; Kuehn, Annette; Planchon, Sébastien; Rodrigues, Pedro; Cerqueira, MarcoBackground Aquaculture is a fast-growing industry and therefore welfare and environmental impact have become of utmost importance. Preventing stress associated to common aquaculture practices and optimizing the fish stress response by quantification of the stress level, are important steps towards the improvement of welfare standards. Stress is characterized by a cascade of physiological responses that, in-turn, induce further changes at the whole-animal level. These can either increase fitness or impair welfare. Nevertheless, monitorization of this dynamic process has, up until now, relied on indicators that are only a snapshot of the stress level experienced. Promising technological tools, such as proteomics, allow an unbiased approach for the discovery of potential biomarkers for stress monitoring. Within this scope, using Gilthead seabream (Sparus aurata) as a model, three chronic stress conditions, namely overcrowding, handling and hypoxia, were employed to evaluate the potential of the fish protein-based adaptations as reliable signatures of chronic stress, in contrast with the commonly used hormonal and metabolic indicators. Results A broad spectrum of biological variation regarding cortisol and glucose levels was observed, the values of which rose higher in net-handled fish. In this sense, a potential pattern of stressor-specificity was clear, as the level of response varied markedly between a persistent (crowding) and a repetitive stressor (handling). Gel-based proteomics analysis of the plasma proteome also revealed that net-handled fish had the highest number of differential proteins, compared to the other trials. Mass spectrometric analysis, followed by gene ontology enrichment and protein-protein interaction analyses, characterized those as humoral components of the innate immune system and key elements of the response to stimulus. Conclusions Overall, this study represents the first screening of more reliable signatures of physiological adaptation to chronic stress in fish, allowing the future development of novel biomarker models to monitor fish welfare.
- Metabolic fingerprinting of gilthead seabream (Sparus aurata) liver to track interactions between dietary factors and seasonal temperature variationsPublication . Silva, Tomé S.; Costa, Ana M. Rosa da; Conceição, L. E. C.; Dias, Jorge P.; Rodrigues, Pedro; Richard, NadègeFarmed gilthead seabream is sometimes affected by a metabolic syndrome, known as the "winter disease", which has a significant economic impact in the Mediterranean region. It is caused, among other factors, by the thermal variations that occur during colder months and there are signs that an improved nutritional status can mitigate the effects of this thermal stress. For this reason, a trial was undertaken where we assessed the effect of two different diets on gilthead seabream physiology and nutritional state, through metabolic fingerprinting of hepatic tissue. For this trial, four groups of 25 adult gilthead seabream were reared for 8 months, being fed either with a control diet (CTRL, low-cost commercial formulation) or with a diet called "Winter Feed" (WF, high-cost improved formulation). Fish were sampled at two time-points (at the end of winter and at the end of spring), with liver tissue being taken for FT-IR spectroscopy. Results have shown that seasonal temperature variations constitute a metabolic challenge for gilthead seabream, with hepatic carbohydrate stores being consumed over the course of the inter-sampling period. Regarding the WF diet, results point towards a positive effect in terms of performance and improved nutritional status. This diet seems to have a mitigating effect on the deleterious impact of thermal shifts, confirming the hypothesis that nutritional factors can affect the capacity of gilthead seabream to cope with seasonal thermal variations and possibly contribute to prevent the onset of "winter disease".
- Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe-4S] clusterPublication . Rodrigues, Pedro; Macedo, Anjos L.; Goodfellow, B. J.; Moura, Isabel; Moura, José J. G.Abstract Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe3S4 cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdIIint) with four 1H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdIIox and FdIIint were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same 1H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe–4S] core in the oxidized state (DgFdIIox) was used here for the assignment of the same three ligands in the intermediate state (DgFdIIint). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm 1 and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdIIint. This study, together with the spin delocalization mechanism proposed here for DgFdIIint, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdIIox and DgFdIIint.
- Animal board invited review: advances in proteomics for animal and food sciencesPublication . Almeida, A. M.; Bassols, A.; Bendixen, E.; Bhide, M.; Ceciliani, F.; Cristobal, S.; Eckersall, P. D.; Hollung, K.; Lisacek, F.; Mazzucchelli, G.; McLaughlin, M.; Miller, I.; Nally, J. E.; Plowman, J.; Renaut, J.; Rodrigues, P.; Roncada, P.; Staric, J.; Turk, R.Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid - i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002 - Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East-West and North-South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.
- The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from desulfovibrio gigasPublication . Goodfellow, B. J.; Macedo, Anjos L.; Rodrigues, Pedro; Moura, Isabel; Wray, Victor; Moura, José J. G.The use of standard 2D NMR experiments in combination with 1D NOE experiments allowed the assignment of 51 of the 58 spin systems of oxidised [3Fe-4S] ferredoxin isolated from Desulfovibrio gigas. The NMR solution structure was determined using data from 1D NOE and 2D NOESY spectra, as distance constraints, and information from the X-ray structure for the spin systems not detected by NMR in torsion angle dynamics calculations to produce a family of 15 low target function structures. The quality of the NMR family, as judged by the backbone r.m.s.d. values, was good (0.80 Å), with the majority of f/c angles falling within the allowed region of the Ramachandran plot. A comparison with the X-ray structure indicated that the overall global fold is very similar in solution and in the solid state. The determination of the solution structure of ferredoxin II (FdII) in the oxidised state (FdIIox) opens the way for the determination of the solution structure of the redox intermediate state of FdII (FdIIint), for which no X-ray structure is available.