Pedro M. Rodrigues

Animal board invited review: advances in proteomics for animal and food sciences

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.

PROTEOMICS in aquaculture: applications and trends.

Over the last forty years global aquaculture presented a growth rate of 6.9% per annum with an amazing production of 52.5 million tonnes in 2008, and a contribution of 43% of aquatic animal food for human consumption. In order to meet the worlds health requirements of fish protein, a continuous growth in production is still expected for decades to come. Aquaculture is, though, a very competitive market, and a global awareness regarding the use of scientific knowledge and emerging technologies to obtain a better farmed organism through a sustainable production has enhanced the importance of proteomics in seafood biology research. Proteomics, as a powerful comparative tool, has therefore been increasingly used over the last decade to address different questions in aquaculture, regarding welfare, nutrition, health, quality, and safety. In this paper we will give an overview of these biological questions and the role of proteomics in their investigation, outlining the advantages, disadvantages and future challenges. A brief description of the proteomics technical approaches will be presented. Special focus will be on the latest trends related to the aquaculture production of fish with defined nutritional, health or quality properties for functional foods and the integration of proteomics techniques in addressing this challenging issue.

Proteomics in food: Quality, safety, microbes, and allergens.

Food safety and quality and their associated risks pose a major concern worldwide regarding not only the relative economical losses but also the potential danger to consumers health. Customers confidence in the integrity of the food supply could be hampered by inappropriate food safety measures. A lack of measures and reliable assays to evaluate and maintain a good control of food characteristics may affect the food industry economy and shatter consumer confidence. It is imperative to create and to establish fast and reliable analytical methods that allow a good and rapid analysis of food products during the whole food chain. Proteomics can represent a powerful tool to address this issue, due to its proven excellent quantitative and qualitative drawbacks in protein analysis. This review illustrates the applications of proteomics in the past few years in food science focusing on food of animal origin with some brief hints on other types. Aim of this review is to highlight the importance of this science as a valuable tool to assess food quality and safety. Emphasis is also posed in food processing, allergies, and possible contaminants like bacteria, fungi, and other pathogens.

The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from Desulfovibrio gigas.

Abstract 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 φ/ψ 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.

Metabolic fingerprinting of gilthead seabream (Sparus aurata) liver to track interactions between dietary factors and seasonal temperature variations

Farmed 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”.

Changes in the soluble bone proteome of reared white seabream (Diplodus sargus) with skeletal deformities.

One 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.

Dietary tools to modulate glycogen storage in gilthead seabream muscle: glycerol supplementation.

The quality and shelf life of fish meat products depend on the skeletal muscles energetic state at slaughter, as meat decomposition processes can be exacerbated by energy depletion. In this study, we tested dietary glycerol as a way of replenishing muscle glycogen reserves of farmed gilthead seabream. Two diets were tested in duplicate (n = 42/tank). Results show 5% inclusion of crude glycerol in gilthead seabream diets induces increased muscle glycogen, ATP levels and firmness, with no deleterious effects in terms of growth, proximate composition, fatty acid profile, oxidative state, and organoleptic properties (aroma and color). Proteomic analysis showed a low impact of glycerol-supplementation on muscle metabolism, with most changes probably reflecting increased stress coping capacity in glycerol-fed fish. This suggests inclusion of crude glycerol in gilthead seabream diets (particularly in the finishing phase) seems like a viable strategy to increase glycogen deposition in muscle without negatively impacting fish welfare and quality.

Nutritional mitigation of winter thermal stress in gilthead seabream associated metabolic pathways and potential indicators of nutritional state

A trial was carried out with gilthead seabream juveniles, aiming to investigate the ability of an enhanced dietary formulation (diet Winter Feed, WF, containing a higher proportion of marine-derived protein sources and supplemented in phospholipids, vitamin C, vitamin E and taurine) to assist fish in coping with winter thermal stress, compared to a low-cost commercial diet (diet CTRL). In order to identify the metabolic pathways affected by WF diet, a comparative two dimensional differential in-gel electrophoresis (2D-DIGE) analysis of fish liver proteome (pH 4–7) was undertaken at the end of winter. A total of 404 protein spots, out of 1637 detected, were differentially expressed between the two groups of fish. Mass spectrometry analysis of selected spots suggested that WF diet improved oxidative stress defense, reduced endoplasmic reticulum stress, enhanced metabolic flux through methionine cycle and phenylalanine/tyrosine catabolism, and induced higher aerobic metabolism and gluconeogenesis. Results support the notion that WF diet had a positive effect on fish nutritional state by partially counteracting the effect of thermal stress and underlined the sensitivity of proteome data for nutritional and metabolic profiling purposes. Intragroup variability and co-measured information were also used to pinpoint which proteins displayed a stronger relation with fish nutritional state. SIGNIFICANCE Winter low water temperature is a critical factor for gilthead seabream farming in the Mediterranean region, leading to a reduction of feed intake, which often results in metabolic and immunological disorders and stagnation of growth performances. In a recent trial, we investigated the ability of an enhanced dietary formulation (diet WF) to assist gilthead seabream in coping with winter thermal stress, compared to a standard commercial diet (diet CTRL). Within this context, in the present work, we identified metabolic processes that are involved in the stress-mitigating effect observed with diet WF, by undertaking a comparative analysis of fish liver proteome at the end of winter. This study brings information relative to biological processes that are involved in gilthead seabream winter thermal stress and shows that these can be mitigated through a nutritional strategy, assisting gilthead seabream to deal better with winter thermal conditions. Furthermore, the results show that proteomic information not only clearly distinguishes the two dietary groups from each other, but also captures heterogeneities that reflect intra-group differences in nutritional state. This was exploited in this work to refine the variable selection strategy so that protein spots displaying a stronger correlation with “nutritional state” could be identified as possible indicators of gilthead seabream metabolic and nutritional state. Finally, this study shows that gel-based proteomics seems to provide more reliable information than transmissive FT-IR spectroscopy, for the purposes of nutritional and metabolic profiling.

Data Visualization and Feature Selection Methods in Gel-based Proteomics

Despite the increasing popularity of gel-free proteomic strategies, two-dimensional gel electrophoresis (2DE) is still the most widely used approach in top-down proteomic studies, for all sorts of biological models. In order to achieve meaningful biological insight using 2DE approaches, importance must be given not only to ensure proper experimental design, experimental practice and 2DE technical performance, but also a valid approach for data acquisition, processing and analysis. This paper reviews and illustrates several different aspects of data analysis within the context of gel-based proteomics, summarizing the current state of research within this field. Particular focus is given on discussing the usefulness of available multivariate analysis tools both for data visualization and feature selection purposes. Visual examples are given using a real gel-based proteomic dataset as basis.

Open Source Technologies Involved in Constructing a Web-Based Football Information System

The current information systems and match analysis software associated to professional football output a huge amount of statistics. Many football professionals are particularly interested in real time information about the tactical plan occurring during the match, and the relations between that information and what was prepared in the training sessions. It is fundamental to have on the bench, and on-the-fly, the most relevant information each time they have to take a decision. In this paper, we present a set of open source technologies involved in building a multi-platform web based integrated football information system, supported in three main modules: user interfaces, databases, and the tactical plan detection and classification. We show that the selected technologies are suitable for those modules, allowing field occurrences to trigger meaningful information.