NOTCH1 Gene as a Novel Cause of Thoracic Aortic Aneurysm in Patients with Tricuspid Aortic Valve: Two Cases Reported.

Thoracic aortic aneurysms (TAA) consist of abnormal dilation or the widening of a portion of the ascending aorta, due to weakness or destructuring of the walls of the vessel and are potentially lethal. The congenital bicuspid aortic valve (BAV) is considered a risk factor for the development of TAA because asymmetric blood flow through the bicuspid aortic valve detrimentally influences the wall of the ascending aorta. NOTCH1 mutations have been associated with non-syndromic TAAs as a consequence of BAV, but little is known regarding its haploinsufficiency and its relationship with connective tissue abnormalities. We report two cases in which there is clear evidence that alterations in the NOTCH1 gene are the cause of TAA in the absence of BAV. On the one hand, we describe a 117 Kb deletion that includes a large part of the NOTCH1 gene and no other coding genes, suggesting that haploinsufficiency can be considered a pathogenic mechanism for this gene associated with TAA. In addition, we describe two brothers who carry two variants, one in the NOTCH1 gene and another in the MIB1 gene, corroborating the involvement of different genes of the Notch pathway in aortic pathology.

Entropy and Fractal Techniques for Monitoring Fish Behaviour and Welfare in Aquacultural Precision Fish Farming-A Review.

In a non-linear system, such as a biological system, the change of the output (e.g., behaviour) is not proportional to the change of the input (e.g., exposure to stressors). In addition, biological systems also change over time, i.e., they are dynamic. Non-linear dynamical analyses of biological systems have revealed hidden structures and patterns of behaviour that are not discernible by classical methods. Entropy analyses can quantify their degree of predictability and the directionality of individual interactions, while fractal dimension (FD) analyses can expose patterns of behaviour within apparently random ones. The incorporation of these techniques into the architecture of precision fish farming (PFF) and intelligent aquaculture (IA) is becoming increasingly necessary to understand and predict the evolution of the status of farmed fish. This review summarizes recent works on the application of entropy and FD techniques to selected individual and collective fish behaviours influenced by the number of fish, tagging, pain, preying/feed search, fear/anxiety (and its modulation) and positive emotional contagion (the social contagion of positive emotions). Furthermore, it presents an investigation of collective and individual interactions in shoals, an exposure of the dynamics of inter-individual relationships and hierarchies, and the identification of individuals in groups. While most of the works have been carried out using model species, we believe that they have clear applications in PFF. The review ends by describing some of the major challenges in the field, two of which are, unsurprisingly, the acquisition of high-quality, reliable raw data and the construction of large, reliable databases of non-linear behavioural data for different species and farming conditions.

Addressing the Joint Impact of Temperature and pH on Vibrio harveyi Adaptation in the Time of Climate Change.

Global warming and acidification of the global ocean are two important manifestations of the ongoing climate change. To characterize their joint impact on Vibrio adaptation and fitness, we analyzed the temperature-dependent adaptation of Vibrio harveyi at different pHs (7.0, 7.5, 8.0, 8.3 and 8.5) that mimic the pH of the world ocean in the past, present and future. Comparison of V. harveyi growth at 20, 25 and 30 °C show that higher temperature per se facilitates the logarithmic growth of V. harveyi in nutrient-rich environments in a pH-dependent manner. Further survival tests carried out in artificial seawater for 35 days revealed that cell culturability declined significantly upon incubation at 25 °C and 30 °C but not at 20 °C. Moreover, although acidification displayed a negative impact on cell culturability at 25 °C, it appeared to play a minor role at 30 °C, suggesting that elevated temperature, rather than pH, was the key player in the observed reduction of cell culturability. In addition, analyses of the stressed cell morphology and size distribution by epifluorescent microscopy indicates that V. harveyi likely exploits different adaptation strategies (e.g., acquisition of coccoid-like morphology) whose roles might differ depending on the temperature-pH combination.

Antioxidant Activities and Selenogene Transcription in the European Sea Bass (Dicentrarchus labrax) Liver Depend, in a Non-linear Manner, on the Se/Hg Molar Ratio of the Feeds.

Feeding 3.9 and 6.7 mg Hg/kg (Se/Hg molar ratios of 0.8 and 0.4, respectively) for 14 days negatively affected Dicentrarchus labrax growth and total DNTB- and thioredoxin-reductase (TrxR) activities and the transcription of four redox genes (txn1, gpx1, txnrd3, and txnrd2) in the liver, but a diet with 0.5 mg Hg/kg (Se/Hg molar ratio 6.6) slightly increased both reductase activities and the transcription of txn1, gpx1, and txnrd2. Feeding 6.7 mg Hg/kg for 53 days downregulated the genes of the thioredoxin system (txn1, txnrd3, and txnrd2) but upregulated gpx1, confirming the previously proposed complementarity among the antioxidant systems. Substitution of 20% of the feed by thawed white fish (hake) slightly counteracted the negative effects of Hg. The effects were not statistically significant and were dependent, in a non-linear manner, on the Se/Hg molar ratio of the feed but not on its Hg concentration. These results stress the need to consider the Se/Hg molar ratio of the feed/food when evaluating the toxicity of Hg.

Protein Signatures to Trace Seafood Contamination and Processing.

This review presents some applications of proteomics and selected spectroscopic methods to validate certain aspects of seafood traceability. After a general introduction to traceability and the initial applications of proteomics to authenticate traceability information, it addresses the application of proteomics to trace seafood exposure to some increasingly abundant emergent health hazards with the potential to indicate the geographic/environmental origin, such as microplastics, triclosan and human medicinal and recreational drugs. Thereafter, it shows the application of vibrational spectroscopy (Fourier-Transform Infrared Spectroscopy (FTIR) and Fourier-Transform Raman Spectroscopy (FT Raman)) and Low Field Nuclear Magnetic Resonance (LF-NMR) relaxometry to discriminate frozen fish from thawed fish and to estimate the time and temperature history of frozen fillets by monitoring protein modifications induced by processing and storage. The review concludes indicating near future trends in the application of these techniques to ensure seafood safety and traceability.

Proteomic Strategies to Evaluate the Impact of Farming Conditions on Food Quality and Safety in Aquaculture Products.

This review presents the primary applications of various proteomic strategies to evaluate the impact of farming conditions on food quality and safety in aquaculture products. Aquaculture is a quickly growing sector that represents 47% of total fish production. Food quality, dietary management, fish welfare, the stress response, food safety, and antibiotic resistance, which are covered by this review, are among the primary topics in which proteomic techniques and strategies are being successfully applied. The review concludes by outlining future directions and potential perspectives.

Reducing the Number of Individuals to Monitor Shoaling Fish Systems - Application of the Shannon Entropy to Construct a Biological Warning System Model.

The present study aims at identifying the lowest number of fish (European seabass) that could be used for monitoring and/or experimental purposes in small-scale fish facilities by quantifying the effect that the number of individuals has on the Shannon entropy (SE) of the trajectory followed by the shoal's centroid. Two different experiments were performed: (i) one starting with 50 fish and decreasing to 25, 13, and 1 fish, and (ii) a second experiment starting with one fish, adding one new fish per day during 5 days, ending up with five fish in the tank. The fish were recorded for 1h daily, during which time a stochastic event (a hit in the tank) was introduced. The SE values were calculated from the images corresponding to three arbitrary basal (shoaling) periods of 3.5 min prior to the event, and to the 3.5 min period immediately after the event (schooling response). Taking both experiments together, the coefficient of variation (CV) of the SE among measurements was largest for one fish systems (CV 37.12 and 17.94% for the daily average basal and response SE, respectively) and decreased concomitantly with the number of fish (CV 8.6-10% for the basal SE of 2 to 5 fish systems and 5.86, 2.69, and 2.31% for the basal SE of 13, 25, and 50 fish, respectively). The SE of the systems kept a power relationship with the number of fish (basal: R2= 0.93 and response: R2= 0.92). Thus, 5-13 individuals should be the lowest number for a compromise between acceptable variability (<10%) in the data and reduction in the number of fish. We believe this to be the first scientific work made to estimate the minimum number of individuals to be used in subsequent experimental (including behavioral) studies using shoaling fish species that reaches a compromise between the reduction in number demanded by animal welfare guidelines and a low variability in the fish system's response.

The Shannon Entropy Trend of a Fish System Estimated by a Machine Vision Approach Seems to Reflect the Molar Se:Hg Ratio of Its Feed.

The present study investigates the suitability of a machine vision-based method to detect deviations in the Shannon entropy (SE) of a European seabass (Dicentrarchus labrax) biological system fed with different selenium:mercury (Se:Hg) molar ratios. Four groups of fish were fed during 14 days with commercial feed (control) and with the same feed spiked with 0.5, 5 and 10 mg of MeHg per kg, giving Se:Hg molar ratios of 29.5 (control-C1); 6.6, 0.8 and 0.4 (C2, C3 and C4). The basal SE of C1 and C2 (Se:Hg > 1) tended to increase during the experimental period, while that of C3 and C4 (Se:Hg < 1) tended to decrease. In addition, the differences in the SE of the four systems in response to a stochastic event minus that of the respective basal states were less pronounced in the systems fed with Se:Hg molar ratios lower than one (C3 and C4). These results indicate that the SE may be a suitable indicator for the prediction of seafood safety and fish health (i.e., the Se:Hg molar ratio and not the Hg concentration alone) prior to the displaying of pathological symptoms. We hope that this work can serve as a first step for further investigations to confirm and validate the present results prior to their potential implementation in practical settings.

Comparative study of muscle proteins in relation to the development of yake in three tropical tuna species yellowfin (Thunnus albacares), big eye (Thunnus obesus) and skipjack (Katsuwonus pelamis).

Burnt tuna (BT), or yake-niku, is a quality flaw of the muscle characterised by a pale colour and grainy and exudative texture. Cathepsin-L, water soluble and total protein components from normal and BT muscles, from three tropical tuna species - yellowfin (YFT, Thunnus albacares), bigeye (BET, Thunnus obesus) and skipjack (SKJ, Katsuwonus pelamis) - were compared by electrophoretic and western blot analyses to identify biomarkers for BT. As expected, SDS-PAGE patterns were species-specific but differences, due to BT, were observed only between some low ionic strength extracts of BET and YFT. Protein oxidation and cell proliferation analysed by immunoblotting did not show differences between BT and normal muscles. Gelatine zymography revealed different gelatinase activity patterns that, although not linked to BT, may affect the final texture of the muscle. A 43 kDa band, identified as creatine kinase by proteomic analysis, showed the potential to be a good indicator for BT in BET and YFT.

The role of environmental biotechnology in exploring, exploiting, monitoring, preserving, protecting and decontaminating the marine environment.

In light of the Marine Strategy Framework Directive (MSFD) and the EU Thematic Strategy on the Sustainable Use of Natural Resources, environmental biotechnology could make significant contributions in the exploitation of marine resources and addressing key marine environmental problems. In this paper 14 propositions are presented focusing on (i) the contamination of the marine environment, and more particularly how to optimize the use of biotechnology-related tools and strategies for predicting and monitoring contamination and developing mitigation measures; (ii) the exploitation of the marine biological and genetic resources to progress with the sustainable, eco-compatible use of the maritime space (issues are very diversified and include, for example, waste treatment and recycling, anti-biofouling agents; bio-plastics); (iii) environmental/marine biotechnology as a driver for a sustainable economic growth.

Development of a real-time PCR method for the identification of Atlantic mackerel (Scomber scombrus).

A Real Time-PCR method based on TaqMan technology for the identification of Scomber scombrus has been developed. A system of specific primers and a Minor Groove Binding (MGB) TaqMan probe based on sequences of the mitochondrial cytochrome b region was designed. The method was successfully tested in 81 specimens of S. scombrus and related species and validated in 26 different commercial samples. An average Threshold Cycle (Ct) value of 15.3 was obtained with S. scombrus DNA. With the other species tested fluorescence signal was not detected or Ct was significantly higher (P<0.001). The efficiency of the assay was estimated to be 92.41%, with 100% specificity, and no cross reactivity was detected with any other species. These results reveal that the developed method is a rapid and efficient tool to unequivocally identify S. scombrus and may aid in the prevention of fraud or mislabelling in mackerel products.

Estimation of freezing storage time and quality changes in hake (Merluccius merluccius, L.) by low field NMR.

The potential of low field NMR (LF NMR) as a fast monitoring technique to estimate the quality of hake (Merluccius merluccius) frozen stored at -10°C for up to 6months was evaluated. LF NMR clearly detected three populations of water: water strongly bound to macromolecules (T(2b)), trapped water (T(21)) and free water (T(22)). As storage time increased, and concomitant with an increase in the T(22) and a decrease in the T(21) water populations, the water holding capacity (WHC) and apparent viscosity values decreased and the shear strength increased, reflecting the characteristic loss of juiciness and tougher texture developed by hake during frozen storage. Two mathematical models were constructed: a simple regression using the biexponential analysis of the relaxation times (T(21), T(22)) and amplitudes (A(21), A(22)) and a partial least square regression (PLS) of CONTIN analysis. Both models seemed suitable to estimate the quality of the product.

Classification of wild and farmed salmon using bayesian belief networks and gas chromatography-derived fatty acid distributions.

In this study, we present the use of Bayesian Belief Networks (BBN) for the classification of wild versus farmed Atlantic salmon (Salmo salar L.). Using a data set of 131 salmon samples from several geographical origins and the gas chromatography-derived distributions of 12 fatty acids (FAs), a Bayesian Belief Network was constructed, ultimately using only the three most important FAs (16:1n-7, 18:2n-6, and 22:5n-3). The training data set yielded a prediction error of 0% (68/68 farmed; 20/20 wild correct) while the validation data set prediction error was 4.65% (32/32 farmed; 9/11 wild correct). Different randomly chosen validation sets yielded similar prediction accuracies. This model was then applied to 30 market (store-bought) samples where predictions were compared with the product labels.

Bioactive compounds in cod (Gadus morhua) products and suitability of 1H NMR metabolite profiling for classification of the products using multivariate data analyses.

This work investigates the suitability of (1)H NMR spectroscopy to identify the fate of some bioactive compounds in seafood submitted to several processing conditions and examines the possibility of using (1)H NMR spectroscopy profiling to classify such products. Perchloric acid extracts of cod white muscle from newly killed and (i) unprocessed, (ii) boiled, and (iii) fried fillets and from (iv) frozen fillets, (v) the frozen fillets after thawing, and (vi) their drip loss and from (vii) rehydrated cod klippfish (n = 5) were analyzed by 500 MHz (1)H NMR spectroscopy. It was possible to identify taurine, betaine, anserine, creatine, and trimethylamine oxide (TMAO) in all extracts examined, and frozen fish was recognizable by the presence of dimethylamine (DMA). None of the heating procedures seemed to induce the loss of bioactive compounds from the fillet, but freezing and thawing did: the compounds were lost in what is known as drip loss. About 80% of the samples were correctly classified using a probabilistic neural network procedure having as inputs the scores of the first 20 principal components of the principal component analysis of a selected region of the NMR spectra.

Application of proteome analysis to seafood authentication.

Part of our work aims at studying the modifications that proteins suffer in foods and use them as markers to estimate the origin and history of the product. Proteomics is a powerful approach to do this: comparison of the two-dimensional (2-D) maps of the intact and treated samples would permit to identify marker spots so that in the future it may be possible to estimate the treatment a foodstuff has suffered by examining its 2-D protein map or just the selected markers. This work summarizes some of our previous studies showing the application of proteomics to the (i) identification of species and muscle tissues, (ii) characterization of post-mortem changes in arctic and tropical species, and (iii) study of the effect of some additives during the processing of fish muscle.

Genetic variability among isolates of Listeria monocytogenes from food products, clinical samples and processing environments, estimated by RAPD typing.

RAPD analysis with four primers was used to examine the genetic relationship among 432 strains of Listeria monocytogenes isolated from clinical and veterinarian cases of listeriosis, dairy, vegetable, meat- and fish-based food items, environmental samples and samples collected from one transport terminal, one poultry-processing company and four Atlantic salmon-processing plants. The purpose of the study was to determine whether clinical isolates belonged to a specific genetic group, whether links could be made between food groups and clinical cases and whether specific genetic groups were associated with specific food products or processing units. There was great genetic variability among the isolates, which produced a total of 141 RAPD composites based on the RAPD analysis with four primers. The RAPD composites divided in two major clusters and clinical isolates were evenly distributed in both of them. None of the isolates from food products had the same RAPD composite as isolates from human patients, thus, no particular food commodity could be linked to clinical cases. Each food-processing environment was contaminated with more than one RAPD composite and the genetic variability found within each company was, in most cases, of approximately the same magnitude as the variability found when considering all the samples. In each plant, one or a few types persisted over time, indicating the presence of an established in-house flora. Our results indicate that most of the analysed cases of listeriosis were sporadic and, further, that these cases cannot be traced to a few specific food sources. We also found that no particular RAPD composite was better suited for survival in specific food types or food-processing environments, indicating that although differences may be found in virulence properties of individual strains, all L. monocytogenes must be treated as potentially harmful.