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- Listeria monocytogenes em condições de simulação do sistema gastrointestinal e a infecção no modelo de Galleria mellonellaPublication . Schrama, Denise; Faleiro, Maria LeonorA bactéria Listeria monocytogenes é o agente etiológico da listeriose, uma doença de origem alimentar. No grupo de risco estão incluídos os idosos, grávidas e doentes imunocomprometidos. Embora rara, esta doença tem uma taxa de mortalidade associada muito elevada (cerca de 20 a 30%). A maioria dos surtos são provocados pelos serovares 1/2a, 1/2b e 4b. Entre os alimentos implicados destacam-se os produtos lácteos, entre os quais o queijo tem especial relevância. No presente estudo pretendeu-se avaliar o impacto de uma exposição prévia a condições sub-letais de pH e sal (pH 5,5 e 3,5% (p/v) NaCl) na protecção das células a subsequentes condições de stress designadamente ao passar pelo sistema gastrointestinal. Para atingir este objectivo as células da estirpe L. monocytogenes C882 foram submetidas a uma exposição de condições de adaptação num meio de simulação de queijo durante 2h a 20ºC e em seguida foram transferidas para um modelo de simulação do sistema gastrointestinal. Como controlo foram utilizadas células que não sofreram uma adaptação prévia (pH 7,0 sem adição de sal). As respostas fisiológicas foram avaliadas através da análise do proteoma intracelular. Foi ainda avaliado o impacto das condições de adaptação na patogenicidade desta estirpe através do modelo Galleria mellonella L.(Lepidoptera: Pyralidae). Os resultados obtidos evidenciam uma capacidade de sobrevivência no modelo de simulação do sistema gastrointestinal semelhante entre as células adaptadas e não adaptadas. No entanto, a análise do proteoma intracelular evidencia a montagem de diferentes estratégias moleculares pelas células adaptadas em comparação com as células não adaptadas. Com o modelo de G. mellonella não foram encontradas diferenças significativas (P>0,05) no potencial de virulência entre as células adaptadas e não adaptadas das estirpes C882 e Scott A. Contudo foi possível verificar que a estirpe C882 é mais virulenta que a estirpe clínica Scott A.
- 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.
- 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.
- Effects of dietary lipid sources on hepatic nutritive contents, fatty acid composition and proteome of Nile tilapia (Oreochromis niloticus)Publication . Boonanuntanasarn, Surintorn; Nakharuthai, Chatsirin; Schrama, Denise; Duangkaew, Rungsun; Rodrigues, Pedro M.Dietary oils of varying fatty acid composition have been used in tilapia feeds; nevertheless, investigation of their effects on metabolism and physiological processes has been limited. Therefore, in this study, using proteomic technology, the effects of different dietary lipids (DLs) on hepatic lipid metabolism and physiological processes were investigaed in Nile tilapia (Oreochromis niloticus). Fish were fed with different DL, which included palm oil (PO), linseed oil (LO) and soybean oil (SBO) for 90 days. Growth performance appeared to be similar among the dietary group. Hepatic FA contents were reflected by DL. Dietary PO (source of saturated fatty acids; SFA) led to an increase in the hepatosomatic index as well as lipid and protein contents in liver. Dietary SBO (source of n-6 polyunsaturated fatty acids; n-6 PUFAs) increased hepatic carbohydrate contents, but decreased the hepatic protein. The proteomic analysis showed that these nutritive changes in the liver were mediated by several proteins involved in lipid, carbohydrate, and amino acid metabolism. Dietary SBO showed an increased accumulation in proteins related to oxidative stress, immune and inflammatory processes. Dietary LO (source of n-3 PUFAs) increased abundance of cytoskeleton-related proteins. qRT-PCR was performed to provide supportive information for the result of proteomic study. Similar effects of DLs on mRNA levels were observed for atp5b, krt18, and selenbp1. Combined together, dietary SFA could supply as energy reservoir for regular activites. Dietary n-3 and n-6 PUFAs led to induce vital metabolic and physiologic processes which would contribute to maintaining normal health and/or providing health-related benefits. Significance: Dietary SBO increased hepatic carbohydrate contents, but decreased the hepatic protein. Different dietary lipid led to alter the abundance of several proteins (i.e., DHRS1, ATP5B, PLA2G12B, APO, AMY2A, GRP78, PRSS1, FAH, and PRSS36) involved in lipid, carbohydrate, and amino acid metabolism. Dietary SBO showed an increased accumulation in proteins (i.e., QDPR, CABC1, and PRDX6) that respond to oxidative stress, suggesting that n-6 PUFAs induce oxidative conditions. Dietary SBO led to increase the accumulation of proteins (i.e., NITR26, NCCRP1, and LCN) involved in immune and inflammatory processes. Dietary LO increased the abundant levels of cytoskeleton-related proteins (i.e., ACTB, AHNAK, ERC2, KRT18, and RILP1). Other proteins (i.e., SELENBP1, FAM46C, and ANC1) involved in other physiological processes were also modulated by DL. qRT-PCR was performed to provide supportive information of proteomic study. Similar effects of DLs on mRNA levels were observed for atp5b, krt18, and selenbp1.
- Fish pathology research and diagnosis in aquaculture of farmed fish; a proteomics perspectivePublication . Moreira, Márcio; Schrama, Denise; Farinha, Ana Paula; Cerqueira, Marco; Raposo de Magalhães, Cláudia; Carrilho, Raquel; Rodrigues, PedroOne of the main constraints in aquaculture production is farmed fish vulnerability to diseases due to husbandry practices or external factors like pollution, climate changes, or even the alterations in the dynamic of product transactions in this industry. It is though important to better understand and characterize the intervenients in the process of a disease outbreak as these lead to huge economical losses in aquaculture industries. High-throughput technologies like proteomics can be an important characterization tool especially in pathogen identification and the virulence mechanisms related to host-pathogen interactions on disease research and diagnostics that will help to control, prevent, and treat diseases in farmed fish. Proteomics important role is also maximized by its holistic approach to understanding pathogenesis processes and fish responses to external factors like stress or temperature making it one of the most promising tools for fish pathology research.
- Amyloodiniosis in aquaculture: A reviewPublication . Moreira, Márcio; Costas, Benjamín; Rodrigues, Pedro; Lourenço‐Marques, Cátia; Sousa, Rui; Schrama, Denise; Raposo de Magalhães, Cláudia; Farinha, Ana Paula; Soares, FlorbelaFish ectoparasites are one of the pathogens groups that pose great concern to the aquaculture industry. The dinoflagellate Amyloodinium ocellatum is responsible for amyloodiniosis, a parasitological disease with a strong economic impact in temperate and warm water aquaculture, mainly in earthen pond semi-intensive systems. Amyloodiniosis represents one of the most important bottlenecks for aquaculture and, with the predictable expansion of the area of influence of this parasite to higher latitudes due to global warming it might also be a threat to other aquaculture species that are not yet parasitized by A. ocellatum. This review made a compilation of the existing knowledge about this parasite and the disease associated with it. It was noticed that, except from the life cycle characterisation, detection methods, histopathological analysis, and treatments, there are still a lot of areas that need a further investment in research. Areas like parasite-host interactions, epidemiological models, taxonomy, host physiological responses to parasitism, and genome sequencing, amongst others, can contribute to a better understanding of this disease. These proposed approaches and routes of investigation will enhance and contribute to a more standardised knowledge, creating the opportunity for a better understanding of amyloodiniosis impacts on fish and contributing for the development of new tools against A. ocellatum, that may reduce fish mortality in aquaculture production due to amyloodiniosis outbreaks.
- Effect of creatine and EDTA supplemented diets on European seabass (Dicentrarchus labrax) allergenicity, fish muscle quality and omics fingerprintPublication . Schrama, Denise; Raposo de Magalhães, Cláudia; Cerqueira, Marco; Carrilho, Raquel; Farinha, Ana Paula; Costa, Ana Rosa da; Gonçalves, Amparo; Kuehn, Annette; Revets, Dominique; Planchon, Sébastien; Engrola, Sofia; Rodrigues, PedroThe relatively easy access to fish worldwide, alongside the increase of aquaculture production contributes to increased fish consumption which result in higher prevalence of respective allergies. Allergies to fish constitute a significant concern worldwide. beta-parvalbumin is the main elicitor for IgE-mediated reactions. Creatine, involved in the muscle energy metabolism, and ethylenediamine tetraacetic acid (EDTA), a calcium chelator, are potential molecules to modulate parvalbumin. The purpose of this study was to test creatine (2, 5 and 8%) and EDTA (1.5, 3 and 4.5%) supplementation in fish diets to modulate beta-parvalbumin expression and structure and its allergenicity in farmed European seabass (Dicentrarchus labrax) while assessing its effects on the end-product quality. Fish welfare and muscle quality parameters were evaluated by plasma metabolites, rigor mortis, muscle pH and sensory and texture analysis. Proteomics was used to assess alterations in muscle proteome profile and metabolic fingerprinting by Fourier transform infrared spectroscopy was used to assess the liver metabolic profile. In addition, IgE-reactivity to parvalbumin was analysed using fish allergic patient sera. Metabolic fingerprinting of liver tissue revealed no major alterations in infrared spectra with creatine supplementation, while with EDTA, only absorption bands characteristic of lipids were altered. Comparative proteomics showed up regulation of (tropo) myosin and phosphoglycerate mutase 2 with Creatine supplementation. In the case of EDTA proteomics showed up regulation of proteins involved in cellular and ion homeostasis. Allergenicity seems not to be modulated with creatine or EDTA supplementation as no decreased expression levels were found and IgE-binding reactivity showed no quantitative differences.
- Effect of EDTA enriched diets on farmed fish allergenicity and muscle quality; a proteomics approachPublication . Raposo de Magalhães, Cláudia; Schrama, Denise; Fonseca, Flavio; Kuehn, Annette; Morisset, Martine; Ferreira, Sara; Goncalves, Amparo; Rodrigues, PedroFish is one of the most common elicitors of food-allergic reactions worldwide. These reactions are triggered by the calcium-binding muscle protein beta-parvalbumin, which was shown to have reduced immunoglobulin E (IgE)-binding capacity upon calcium depletion. This work aimed to reduce gilthead seabream allergenicity using diets supplemented with a calcium chelator. Three experimental feeds were tested, differing in ethylenediaminetetraacetic acid (EDTA) supplementation, and its effects on muscle and parvalbumin's IgE-reactivity were analyzed. Chromatographic determination of EDTA showed no accumulation in the muscle and sensory results demonstrated that the lowest concentration did not affect fish quality as edible fish. Proteomics revealed one protein related to muscle contraction with significantly different relative abundance. Immunoblot assays performed with fish-allergic patients sera indicated a 50% reduction in IgE-reactivity upon EDTA presence. These preliminary results provide the basis for the further development of a non-GMO approach to modulate fish allergenicity and improve safety of aquaculture fish.
- EAACI molecular allergology user's guide 2.0Publication . Dramburg, Stephanie; Hilger, Christiane; Santos, Alexandra F.; de las Vecillas, Leticia; Aalberse, Rob C.; Acevedo, Nathalie; Aglas, Lorenz; Altmann, Friedrich; Arruda, Karla L.; Asero, Riccardo; Ballmer‐Weber, Barbara; Barber, Domingo; Beyer, Kirsten; Biedermann, Tilo; Bilo, Maria Beatrice; Blank, Simon; Bosshard, Philipp P.; Breiteneder, Heimo; Brough, Helen A.; Bublin, Merima; Campbell, Dianne; Caraballo, Luis; Caubet, Jean Christoph; Celi, Giorgio; Chapman, Martin D.; Chruszcz, Maksymilian; Custovic, Adnan; Czolk, Rebecca; Davies, Janet; Douladiris, Nikolaos; Eberlein, Bernadette; Ebisawa, Motohiro; Ehlers, Anna; Eigenmann, Philippe; Gadermaier, Gabriele; Giovannini, Mattia; Gomez, Francisca; Grohman, Rebecca; Guillet, Carole; Hafner, Christine; Hamilton, Robert G.; Hauser, Michael; Hawranek, Thomas; Hoffmann, Hans Jürgen; Holzhauser, Thomas; Iizuka, Tomona; Jacquet, Alain; Jakob, Thilo; Janssen‐Weets, Bente; Jappe, Uta; Jutel, Marek; Kalic, Tanja; Kamath, Sandip; Kespohl, Sabine; Kleine‐Tebbe, Jörg; Knol, Edward; Knulst, André; Konradsen, Jon R.; Korošec, Peter; Kuehn, Annette; Lack, Gideon; Le, Thuy‐My; Lopata, Andreas; Luengo, Olga; Mäkelä, Mika; Marra, Alessandro Maria; Mills, Clare; Morisset, Martine; Muraro, Antonella; Nowak‐Wegrzyn, Anna; Nugraha, Roni; Ollert, Markus; Palosuo, Kati; Pastorello, Elide Anna; Patil, Sarita Ulhas; Platts‐Mills, Thomas; Pomés, Anna; Poncet, Pascal; Potapova, Ekaterina; Poulsen, Lars K.; Radauer, Christian; Radulovic, Suzana; Raulf, Monika; Rougé, Pierre; Sastre, Joaquin; Sato, Sakura; Scala, Enrico; Schmid, Johannes M.; Schmid‐Grendelmeier, Peter; Schrama, Denise; Sénéchal, Hélène; Traidl‐Hoffmann, Claudia; Valverde‐Monge, Marcela; van Hage, Marianne; van Ree, Ronald; Verhoeckx, Kitty; Vieths, Stefan; Wickman, Magnus; Zakzuk, Josefina; Matricardi, Paolo M.; Hoffmann‐Sommergruber, KarinSince the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.
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