Exploring the Anti-Inflammatory Effect of Inulin by Integrating Transcriptomic and Proteomic Analyses in a Murine Macrophage Cell Model.

Inulin is a natural polysaccharide classified as a soluble fiber with demonstrated prebiotic activity. Prebiotics can reduce intestinal and systemic inflammation through modulation of the gut microflora and their metabolites. Additionally, extensive research is illuminating the role of macrophages in the interaction between gut microbiota and many systemic inflammatory diseases. In this study, the anti-inflammatory properties of inulin were evaluated using a murine macrophage cell model (RAW 264.7) of inflammation, and the immunomodulatory mechanism was investigated using omics technologies. The cells underwent comprehensive transcriptomic and proteomic analyses to identify the mechanisms responsible for the observed anti-inflammatory phenotype. Functional analyses of these omics results revealed two potential mechanisms that may lead to an overall reduction in cytokine and chemokine transcription: the inhibition of the NF-κB signaling pathway, leading to the downregulation of proinflammatory factors such as COX2, and the promotion of the phase II defense protein Hmox1 via the Nrf2 pathway. This study provides promising targets for research on immune modulation by dietary fibers and offers new strategies for the design of functional ingredients, foods, and nutraceutical products, which could ultimately lead to personalized nutrition and improved consumer health.

Fast Real-Time PCR for the Detection of Crustacean Allergens in Foods.

Crustaceans are one of the most common allergens causing severe food reaction. Hypersensitivity reactions associated with seafood intake are one of the most common food allergies in adults. Crustaceans including shrimps, prawns, crabs, lobster, and crayfish are a common cause of anaphylaxis or hypersensitivity, with shrimps and crabs being the most common causes of allergy. Symptoms occur most often when food or cooking water are ingested.These food allergens are a health problem, and they have become very important; as evidenced by the existence of several regulations that establish that labeling must be present regarding these allergens to warn consumers.The methodology herein exposed allows the detection of crustaceans in any type of product, including those where very aggressive treatments of temperature and pressure are used during the manufacturing process.The main features of this method are its high sensitivity and specificity, and reduced analysis time of real-time PCR (40 min). This assay is a potential tool in issues related to the labeling of products and food security to protect the allergic consumer.

Fast Real-Time PCR Method for Detection of Soy in Foods.

Soy is used as an additive in the manufacturing of diverse products, because of their ability of emulsification, water and fat absorption, contributing to the consistency of food products. Moreover, soy is recognized as a potential allergen, so its presence should be indicated in all the food products.These issues highlight the need for techniques that allow the detection of soy in foods. This work describes a real-time PCR method for the detection of soy in a wide range of foodstuffs. The main features of this technique are its reliability and sensitivity, allowing the detection of trace amounts of soybean in processed products. TaqMan real-time PCR is one of the simplest and fastest molecular biology techniques, with a high potential for automation. Therefore, it is one of the techniques most used for screening a variety of substances.The methodology herein described is of great value in issues regarding the presence of soy protein in processed products, especially in verifying labeling and security regulations to protect consumer's rights.

FAST Real Time PCR for control of intra-species recycling in aquaculture feed, focused to the most relevant fish species farmed in Europe.

Recent regulations in animal feed composition prohibit intra-species recycling, the recycling of one given animal species to the same species, in order to avoid potential safety risks to human and animal health. These regulations have generated the need of their control in aquaculture by effective and specific analytical techniques. To date, most studies of species identification and detection in feedstuffs are focused on land species, but few studies are focused on species composition in fish feed. The present work describes five methodologies based in Real Time PCR for detection of the most relevant fish species farmed in Europe: gilthead sea bream (Sparus aurata); sea bass (Dicentrarchus labrax); turbot (Scophthalmus maximus); rainbow trout (Onchorynchus mykiss); and salmon (Salmo salar), in order to guarantee the intra-species recycling regulation in aquaculture feedstuffs.

Detection of the causal agents of Keriorrhea (Lepidocybium flavobrunneum and Ruvettus pretiosus) by means of Real Time PCR.

Consumption of Lepidocybium flavobrunneum and Ruvettus pretiosus is related with the gastrointestinal disease called Keriorrhea. Sometimes, intentionally or not, these species are mislabelled as other harmless species, causing severe disruptions to consumers. The correct identification of these species helps to avoid food fraud and health problems. For this reason, a multiplex Real Time-PCR method based on TaqMan technology for the correct authentication of L. flavobrunneum and R. pretiosus has been developed. The method is based on a species-specific set of primers and TaqMan probe which amplifies a 276 bp fragment of the cytochrome oxidase I (COI) mitochondrial DNA region. This methodology allows your application to any type of product, regardless of the degree of processing it has undergone with high specificity, sensitivity and rapidity. Also, it might be a useful tool in monitoring and verifying labelling regulation and protect consumer rights.

Development of an in-house fast real-time PCR method for detection of fish allergen in foods and comparison with a commercial kit.

Food allergy is recognised as an important human health problem. Fish represent one of the most important causes of food hypersensitivity reaction. Small amounts of the allergen can cause severe reactions in sensitive individuals, so correct labelling is essential to ensure the protection of consumers. The objective of the present work was to develop a reliable, sensitive and specific real-time PCR method for the detection of fish and traces of fish in all kind of products included those that have undergone aggressive treatments such as high temperature or pressure. This methodology was validated simulating products likely to contain this allergen and spiking them with fish cooking water. In addition, a comparison between the performance of in-house methodology and a commercial kit, both of them based on real-time PCR, was carried out. This work is relevant because it is the first, rapid real-time PCR method developed to date for the detection of fish in processed food products. The results obtained confirm the present assay is a useful tool in detecting fish and, therefore, minimising exposure and reducing incidences of allergic reaction to fish in contaminated products.

Outlier SNP markers reveal fine-scale genetic structuring across European hake populations (Merluccius merluccius).

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome-wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large- and fine-scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F(CT) = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F(CT) range 0.275-0.705) and fine-scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.

Authentication of male beef by multiplex fast real-time PCR.

Meat is a significant source of high-quality nutrients, which are very important in the diet. Among meat products, one of the most prized is bovine meat, of which male beef has been designated to be of a higher quality. However, because of its similarity with female beef, deliberate or unintentional substitutions can occur. To avoid this, methodology based on the fast real-time polymerase chain reaction has been developed to authenticate the species and gender origin of beef. This technique consists of two polymerase chain reactions: one bovine-specific reaction and another Y-chromosome-specific multiplex reaction. This methodology has been validated for all kinds of beef products, including those subjected to intensive processing treatments, and it has subsequently been applied to 10 commercial samples labelled as ox to determine whether they are properly labelled. This assay has been shown to have high specificity, sensitivity and rapidity, with the potential to be a powerful tool in enforcing food labelling regulations.

Fast Real-Time PCR assay for detection of Tetramicra brevifilum in cultured turbot.

Global aquaculture production of turbot has rapidly increased worldwide in the last decade and it is expected to have even bigger growth in the next years due to new farms operating. The losses caused by pathogen infections have grown at the same time as the production of this species. Parasitological infections are among the main relevant pathologies associated with its culture and produce serious losses in aquaculture, reduce the growth rate in fish and may lead to unmarketable fish due to skeletal muscle abnormalities in cases with high intensity of infection. The microsporidian parasite Tetramicra brevifilum causes severe infections and generates major losses in farmed turbot. Infections are difficult to control due to spore longevity and its direct transmission. To facilitate the infection management, an effective tool for fast detection and identification of T. brevifilum is needed. This study provides a molecular methodology of fast Real-Time PCR for T. brevifilum detection to the aquaculture industry, useful for routine control of T. brevifilum at turbot farms. The method is characterized by its high specificity and sensitivity, and it can be applied to cultured turbot for parasite detection regardless of the life-cycle stage of the pathogen or the infection intensity.

Rapid method for controlling the correct labeling of products containing common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas) by fast real-time PCR.

The TaqMan real-time PCR has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of species. This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas). This technique is notable for the combination of simplicity, speed, sensitivity and specificity in an homogeneous assay. The method can be applied to all kinds of products; fresh, frozen and processed, including those undergoing intensive processes of transformation. This methodology was validated to check how the degree of food processing affects the method and the detection of each species. Moreover, it was applied to 34 commercial samples to evaluate the labeling of products made from them. The methodology herein developed is useful to check the fulfillment of labeling regulations for seafood products and to verify traceability in commercial trade and for fisheries control.

Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

Fast real-time PCR for the detection of crustacean allergen in foods.

Crustaceans are one of the most common allergens causing severe food reaction. These food allergens are a health problem, and they have become very important; there are various regulations that establish that labeling must be present regarding these allergens to warn consumers. In the present work a fast real-time PCR, by a LNA probe, was developed. This allows the detection of crustaceans in all kinds of products, including processed products in which very aggressive treatments of temperature and pressure during the manufacturing process are used. This methodology provides greater sensitivity and specificity and reduces the analysis time of real-time PCR to 40 min. This methodology was further validated by means of simulating products likely to contain this allergen. For this, products present on the market were spiked with crustacean cooking water. The assay is a potential tool in issues related to the labeling of products and food security to protect the allergic consumer.

Molecular cloning, sequence analysis and expression of Fein-Penaeidin from the haemocytes of Indian white shrimp Fenneropenaeus indicus.

Penaeidins are members of a special family of antimicrobial peptide existing in penaeid shrimp and play an important role in the immunological defense of shrimp. Here, we report a penaeidin sequence cloned from the Indian white shrimp Fenneropenaus indicus (Fein-Penaeidin). The Fein-Penaeidin open reading frame encodes a 77 amino acid peptide including a 19 amino acid signal peptide. The deduced amino acid sequences of Fein-Penaeidin include a proline rich N-terminal domain and a carboxyl-domain that contains six cysteine residues. Structural analysis revealed an alpha-helix in its secondary structure and the predicted 3D structure indicated two-disulphide bridges in the alpha-helix. Phylogenetic analysis and sequence comparison with other known peaneidin suggest the gene shows high similarity to that of penaeidin from Peneaus monodon (95%), F. indicus (80%) and Fenneropenaeus chinensis (74%). Fein-Penaeidin was examined in normal and microbial challenged shrimp and was found to be constitutively expressed in haemocytes, Heart, gills, muscles, intestine, hepatopancreas and eyestalk. Bacterial challenge resulted in mRNA up-regulation, inducing expression at 6 h post injection indicating the penaeidin involved in the innate immunity.

Real-time PCR method applied to seafood products for authentication of European sole (Solea solea) and differentiation of common substitute species.

Judged by quality and taste, the European sole (Solea solea) is considered one of the finest flatfish and is, thus, of considerable commercial value. In the present work, a specific fast real-time PCR was developed for the authentication of S. solea, i.e. to distinguish it from other related species and avoid substitution of this species, either deliberately or unintentionally. The method is based on a species-specific set of primers and MGB Taqman probe which amplifies a 116-bp fragment of the internal transcribed spacer 1 (ITS 1) ribosomal DNA region. This assay combines the high specificity and sensitivity of real-time PCR with the rapidity of the fast mode, allowing the detection of S. solea in a short period of time. The present methodology was validated for application to all types of manufactured products for the presence of S. solea, with successful results. Subsequently, the method was applied to 40 commercial samples to determine whether correct labeling had been employed in the market. It was demonstrated that the assay is a useful tool in monitoring and verifying food labeling regulations.

Novel tools for conservation genomics: comparing two high-throughput approaches for SNP discovery in the transcriptome of the European hake.

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).

Genetic identification of horse mackerel and related species in seafood products by means of forensically informative nucleotide sequencing methodology.

In the present study, a methodology based on the amplification of a fragment of mitochondrial cytochrome b and subsequent phylogenetic analysis (FINS: forensically informative nucleotide sequencing) to genetically identify horse mackerels have been developed. This methodology makes possible the identification of more than 20 species belonging to the families Carangidae, Mullidae, and Scombridae. The main novelty of this work lies in the longest number of different horse mackerel species included and in the applicability of the developed methods to all kinds of processed products that can be found by consumers in markets around the world, including those that have undergone intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 15 commercial samples, all of them canned products. Therefore, these methods are useful for checking the fulfillment of labeling regulations for horse mackerels and horse mackerel products, verifying the correct traceability in commercial trade, and fisheries control.

Authentication of Atlantic salmon (Salmo salar) using real-time PCR.

A real-time PCR assay based on LNA TaqMan probe technology was developed for the detection and identification of Atlantic salmon (Salmo salar). Among the advantages it is worth highlighting simplicity, rapidity, highest potential for automation and minor risk of contamination of this technique. The TaqMan real-time PCR is the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabelling of this species. The method can be applied to all kind of products, fresh, frozen and processed products, including those undergoing intensive processes of transformation. The developed methodology using specific primers-probe set was validated and further applied to 20 commercial samples labelled as salmon or S. salar in order to determinate if the species used for their manufacturing corresponded to this species. The methodology herein developed is useful to check the fulfilment of labelling regulations for seafood products, verify the correct traceability in commercial trade and for fisheries control.

Population proteomics of the European Hake (Merluccius merluccius).

We report the novel use of proteomics to investigate protein variation among populations of the European hake (Merluccius merluccius). The liver and brain extracts of 18 hake (N = 36) captured in the Mediterranean Sea, Cantabrian Sea, and Atlantic Ocean were examined by 2D/DIGE and mass spectrometry. Significant differences in protein expression among populations were revealed by 84 spots obtained in the gels for the liver and 145 spots for the brain. Population groups of samples were defined by multivariate analysis (PCA and hierarchical clustering). According to protein expression levels and the functions of the 55 candidate protein spots identified, which showed significant expression differences, highest population discrimination was rendered by brain proteins involved in cell signaling and metabolism/energy and by liver proteins involved in protein fate. Finally, we present a statistically robust framework to accurately classify individuals according to their population of origin. Thus, purposely identified protein isoforms were found to be competent at discriminating populations. These results suggest the possibility of identifying protein biomarkers related to environmental changes in a nonmodel species such as the hake and pave the way for more extensive research on protein variation among populations of marine fishes.

Rapid identification of seaweeds in food products by PCR combined with ALF-RFLP and FINS methodologies.

In the present study, two methods for the genetic identification of the most important seaweed species used for human consumption were developed. Both are carried out through PCR amplification of an 18S rRNA gene fragment. The first one is based on the phylogenetic analysis of DNA sequences (FINS), while the second is based on length polymorphism and RFLP visualized by means of an ALF system. The main novelty of this work lies in the fact that it allows genetic identification of the main commercial species of seaweed. Moreover, the developed systems can be applied to all kinds of processed products, including those that have undergone intensive transformation, as for instance canned foods. These methodologies also permit the detection of species in complex matrixes where more than one algal species is present. The methods were validated using products manufactured in a pilot plant showing correct functioning. Finally, the methods were applied to 23 commercial samples including some that had been subjected to intensive thermal treatment, allowing the detection of those that were incorrectly labeled (30%). Therefore, these molecular tools can be used for clarifying questions related to the correct labeling and traceability of commercial products that include some seaweeds in their composition.

Authentication of Atlantic cod (Gadus morhua) using real time PCR.

This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of Atlantic cod (Gadus morhua). Among the advantages of this technique, it is worth highlighting that this is reliable in terms of specificity and sensitivity. The TaqMan real-time PCR is the simplest, fastest testing process and has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of this species. The method can be applied to all kinds of products, fresh, frozen, and processed products, including those undergoing intensive processes of transformation. The developed methodology using specific primer-probe set was validated and further applied to 40 commercial samples labeled as cod in order to determinate if the species used for their manufacturing corresponded to G. morhua, detecting 20% that were incorrectly labeled. A C(t) value of about 19 was obtained when G. morhua was present. In samples with a species mixture, all samples that had a fluorescence signal were positive (C(t) < 30) for the presence of G. morhua by conventional end-point RT-PCR, and the estimated limit of detection for these type of samples was of 20 pg of DNA. The methodology herein developed is useful to check the fulfilment of labeling regulations for seafood products and verify the correct traceability in commercial trade and for fisheries control.