The ability of polyphenols to ameliorate potential oxidative damage of ω-3 PUFAs when they are consumed together and then, to enhance their potentially individual effects on metabolic health is discussed through the modulation of fatty acids profiling and the production of lipid mediators. For that, the effects of the combined consumption of fish oils and grape seed procyanidins on the inflammatory response and redox unbalance triggered by high-fat high-sucrose (HFHS) diets were studied in an animal model of Wistar rats. A standard diet was used as control. Results suggested that fish oils produced a replacement of ω-6 by ω-3 PUFAs in membranes and tissues, and consequently they improved inflammatory and oxidative stress parameters: favored the activity of 12/15-lipoxygenases on ω-3 PUFAs, enhanced glutathione peroxidases activity, modulated proinflammatory lipid mediators synthesis through the cyclooxygenase (COX) pathways and down-regulated the synthesis de novo of ARA leaded by Δ5 desaturase. Although polyphenols exerted an antioxidative and antiinflammatory effect in the standard diet, they were less effective to reduce inflammation in the HFHS dietary model. Contrary to the effect observed in the standard diet, polyphenols up-regulated COX pathways toward ω-6 proinflammatory eicosanoids as PGE2 and 11-HETE and decreased the detoxification of ω-3 hydroperoxides in the HFHS diet. As a result, additive effects between fish oils and polyphenols were found in the standard diet in terms of reducing inflammation and oxidative stress. However, in the HFHS diets, fish oils seem to be the one responsible for the positive effects found in the combined group.

Food contains bioactive compounds that may prevent changes in gut microbiota associated with Westernized diets. The aim of this study is to explore the possible additive effects of D-fagomine and ω-3 PUFAs (EPA/DHA 1:1) on gut microbiota and related risk factors during early stages in the development of fat-induced pre-diabetes. Male Sprague Dawley (SD) rats were fed a standard diet, or a high-fat (HF) diet supplemented with D-fagomine, EPA/DHA 1:1, a combination of both, or neither, for 24 weeks. The variables measured were fasting glucose and glucose tolerance, plasma insulin, liver inflammation, fecal/cecal gut bacterial subgroups and short-chain fatty acids (SCFAs). The animals supplemented with D-fagomine alone and in combination with ω-3 PUFAs accumulated less fat than those in the non-supplemented HF group and those given only ω-3 PUFAs. The combined supplements attenuated the high-fat-induced incipient insulin resistance (IR), and liver inflammation, while increasing the cecal content, the Bacteroidetes:Firmicutes ratio and the populations of Bifidobacteriales. The functional effects of the combination of D-fagomine and EPA/DHA 1:1 against gut dysbiosis and the very early metabolic alterations induced by a high-fat diet are mainly those of D-fagomine complemented by the anti-inflammatory action of ω-3 PUFAs.

Parvalbumins beta (β-PRVBs) are the main fish allergens. The only proven and effective treatment for this type of hypersensitivity is to consume a diet free of fish. We present the molecular characterization of B-cell epitopes by shotgun proteomics of different β-PRVBs combined with protein-based bioinformatics and IgE-reactive approaches. The final goal of this work is to identify potential peptide vaccine candidates for fish allergy. Purified β-PRVBs from the main fifteen different fish species that cause allergy were analyzed by shotgun proteomics. Identified β-PRVBs peptide sequences and ninety-eight β-PRVB protein sequences from UniProtKB were combined, aligned and analyzed to determine B-cell epitopes using the Kolaskar and Tongaonkar algorithm. The highest rated predicted B-cell peptide epitopes were evaluated by ELISA using the corresponding synthetic peptides and sera from healthy and fish allergic patients. A total of 35 peptides were identified as B-cell epitopes. The top B-cell peptide epitopes (LKLFLQV, ACAHLCK, FAVLVKQ and LFLQNFV) that may induce protective immune responses were selected as potential peptide vaccine candidates. The 3D model of these peptides were located in the surface of the protein. This study provides the global characterization of B-cell epitopes for all β-PRVBs sequences that will facilitate the design of new potential immunotherapies. SIGNIFICANCE: This work provides the global characterization of B-cell epitopes for all β-PRVBs sequences by Shotgun Proteomics combined with Protein-based Bioinformatics and IgE-reactive approaches. This study will increase our understanding of the molecular mechanisms whereby fish allergens elicit allergic reactions and will facilitate the design of new potential peptide vaccine candidates.

Obesity has been recognized as a major risk factor for chronic kidney disease, insulin resistance being an early common metabolic feature in patients suffering from this syndrome. This study aims to investigate the mechanism underlying the induction of kidney dysfunction and the concomitant onset of insulin resistance by long-term high-fat and sucrose diet feeding in Sprague Dawley rats. To achieve this goal, our study analyzed renal carbonylated protein patterns, ectopic lipid accumulation and fatty acid profiles and correlated them with biometrical and biochemical measurements and other body redox status parameters. Rats fed the obesogenic diet developed a prediabetic state and incipient kidney dysfunction manifested in increased plasma urea concentration and superior levels of renal fat deposition and protein carbonylation. An obesogenic diet increased renal fat by preferentially promoting the accumulation of saturated fat, arachidonic, and docosahexaenoic fatty acids while decreasing oleic acid. Renal lipotoxicity was accompanied by selectively higher carbonylation of proteins involved in the blood pH regulation, i.e., bicarbonate reclamation and synthesis, amino acid, and glucose metabolisms, directly related to the onset of insulin resistance. This study also tested the combination of antioxidant properties of fish oil with the anti-diabetic properties of buckwheat D-Fagomine to counteract diet-induced renal alterations. Results demonstrated that bioactive compounds combined attenuated lipotoxicity, induced more favorable lipid profiles and counteracted the excessive carbonylation of proteins associated with pH regulation in the kidneys, resulting in an inhibition of the progression of the prediabetes state and kidney disease.

The present study addressed the ability of long-chain ω-3 polyunsaturated fatty acids (ω-3 PUFA), i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), to ameliorate liver protein damage derived from oxidative stress and induced by consumption of high-caloric diets, typical of Westernized countries. The experimental design included an animal model of Sprague-Dawley rats fed high-fat high-sucrose (HFHS) diet supplemented with ω-3 EPA and DHA for a complete hepatic proteome analysis to map carbonylated proteins involved in specific metabolic pathways. Results showed that the intake of marine ω-3 PUFA through diet significantly decreased liver protein carbonylation caused by long-term HFHS consumption and increased antioxidant system. Fish oil modulated the carbonylation level of more than twenty liver proteins involved in critical metabolic pathways, including lipid metabolism (e.g., albumin), carbohydrate metabolism (e.g., pyruvate carboxylase), detoxification process (e.g., aldehyde dehydrogenase 2), urea cycle (e.g., carbamoyl-phosphate synthase), cytoskeleton dynamics (e.g., actin), or response to oxidative stress (e.g., catalase) among others, which might be under the control of diet marine ω-3 PUFA. In parallel, fish oil significantly changed the liver fatty acid profile given by the HFHS diet, resulting in a more anti-inflammatory phenotype. In conclusion, the present study highlights the significance of marine ω-3 PUFA intake for the health of rats fed a Westernized diet by describing several key metabolic pathways which are protected in liver.

Our goal was to establish panels of protein biomarkers that are characteristic of patients with microbiologically confirmed pulmonary tuberculosis (TB) and their contacts, including latent TB-infected (LTBI) and uninfected patients. Since the first pathogen-host contact occurs in the oral and nasal passages the saliva and sputum were chosen as the biological fluids to be studied. Quantitative shotgun proteomics was performed using a LTQ-Orbitrap-Elite platform. For active TB patients, both fluids exhibited a specific accumulation of proteins that were related to complement activation, inflammation and modulation of immune response. In the saliva of TB patients, a decrease of in proteins related to glucose and lipid metabolism was detected. In contrast, the sputum of uninfected contacts presented a specific proteomic signature that was composed of proteins involved in the perception of bitter taste, defense against pathogens and innate immune response, suggesting that those are key events during the initial entry of the pathogen in the host. SIGNIFICANCE: This is the first study to compare the saliva and sputum from active TB patients and their contacts. Our findings strongly suggest that TB patients show not only an activation of processes that are related to complement activation and modulation of inflammation but also an imbalance in carbohydrate and lipid metabolism. In addition, those individuals who do not get infected after direct exposure to the pathogen display a typical proteomic signature in the sputum, which is a reflection of the secretion from the nasal and oral mucosa, the first immunological barriers that M. tuberculosis encounters in the host. Thus, this result indicates the importance of the processes related to the innate immune response in fighting the initial events of the infection.

Some functional food components may help maintain homeostasis by promoting balanced gut microbiota. Here, we explore the possible complementary effects of d-fagomine and ω-3 polyunsaturated fatty acids (ω-3 PUFAs) eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA 1:1) on putatively beneficial gut bacterial strains. Male Sprague-Dawley rats were supplemented with d-fagomine, ω-3 PUFAs, or both, for 23 weeks. Bacterial subgroups were evaluated in fecal DNA by quantitative real-time polymerase chain reaction (qRT-PCR) and short-chain fatty acids were determined by gas chromatography. We found that the populations of the genus Prevotella remained stable over time in animals supplemented with d-fagomine, independently of ω-3 PUFA supplementation. Animals in these groups gained less weight than controls and rats given only ω-3 PUFAs. d-Fagomine supplementation together with ω-3 PUFAs maintained the relative populations of Bacteroides. ω-3 PUFAs alone or combined with d-fagomine reduced the amount of acetic acid and total short-chain fatty acids in feces. The plasma levels of pro-inflammatory arachidonic acid derived metabolites, triglycerides and cholesterol were lower in both groups supplemented with ω-3 PUFAs. The d-fagomine and ω-3 PUFAs combination provided the functional benefits of each supplement. Notably, it helped stabilize populations of Prevotella in the rat intestinal tract while reducing weight gain and providing the anti-inflammatory and cardiovascular benefits of ω-3 PUFAs.

Tuberculosis (TB) is the most lethal infection among infectious diseases. The specific aim of this study was to establish panels of serum protein biomarkers representative of active TB patients and their household contacts who were either infected (LTBI) or uninfected (EMI-TB Discovery Cohort, Pontevedra Region, Spain). A TMT (Tamdem mass tags) 10plex-based quantitative proteomics study was performed in quintuplicate containing a total of 15 individual serum samples per group. Peptides were analyzed in an LC-Orbitrap Elite platform, and raw data were processed using Proteome Discoverer 2.1. A total of 418 proteins were quantified. The specific protein signature of active TB patients was characterized by an accumulation of proteins related to complement activation, inflammation and modulation of immune response and also by a decrease of a small subset of proteins, including apolipoprotein A and serotransferrin, indicating the importance of lipid transport and iron assimilation in the progression of the disease. This signature was verified by the targeted measurement of selected candidates in a second cohort (EMI-TB Verification Cohort, Maputo Region, Mozambique) by ELISA and nephelometry techniques. These findings will aid our understanding of the complex metabolic processes associated with TB progression from LTBI to active disease.

The regular intake of diets high in saturated fat and sugars increases oxidative stress and has been linked to cognitive decline and premature brain aging. The cerebellum is highly vulnerable to oxidative stress and thus, obesogenic diets might be particularly detrimental to this tissue. However, the precise molecular mechanisms behind obesity-related brain damage are still not clear. Since protein carbonylation, a biomarker of oxidative stress, influences protein functions and is involved in metabolic control, the current investigation addressed the effect of long-term high-fat and high-sucrose diet intake on the cerebellum of Sprague-Dawley rats by deciphering the changes caused in the carbonylated proteome. The antioxidant effects of fish oil supplementation on cerebellar carbonylated proteins were also investigated. Lipid peroxidation products and carbonylated proteins were identified and quantified using immunoassays and 2D-LC-MS/MS in the cerebellum. After 21 weeks of nutritional intervention, the obesogenic diet selectively increased carbonylation of the proteins that participate in ATP homeostasis and glutamate metabolism in the cerebellum. Moreover, the data demonstrated that fish oil supplementation restrained carbonylation of the main protein targets oxidatively damaged by the obesogenic diet, and additionally protected against carbonylation of several other proteins involved in amino acid biosynthesis and neurotransmission. Therefore, dietary interventions with fish oils could help the cerebellum to be more resilient to oxidative damage. The results could shed some light on the effect of high-fat and high-sucrose diets on redox homeostasis in the cerebellum and boost the development of antioxidant-based nutritional interventions to improve cerebellum health.

High consumption of fish carries a lower risk of cardiovascular disease as a consequence of dietary omega-3 long chain polyunsaturated fatty acid (n-3 PUFA; especially EPA and DHA) content. A controversy exists about the component/s responsible of these beneficial effects and, in consequence, which is the best proportion between both fatty acids. We sought to determine, in healthy Wistar rats, the proportions of EPA and DHA that would induce beneficial effects on biomarkers of oxidative stress, and cardiovascular disease risk.

Marine polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been associated with improvement in the Metabolic Syndrome (MS). The aim of this study is to evaluate how three fish-oil diets with different eicosapentaenoic acid/docosahexaenoic acid ratios (EPA/DHA ratio) affect the histology of liver, kidney, adipose tissue and aorta in a preliminary morphological study. This work uses an animal model of metabolic syndrome in comparison with healthy animals in order to provide information about the best EPA:DHA ratio to prevent or to improve metabolic syndrome symptoms.

A novel approach of bioactive lipid extraction by different green solvents was carried out on squid (Doryteuthis gahi) by-products. By-products (viscera, heads, skin, tails, etc.), considered as a single product, were subjected to the following solvent systems: ethanol, acetone, ethyl acetate, 1/1 ethanol/acetone, 1/1 ethanol/ethyl acetate, and 1/1 acetone/ethyl acetate. Analyses carried out included lipid yield, lipid class content, and fatty acid (FA) composition. Results were compared to the lipid extract obtained by the traditional procedure (1/1 chloroform/methanol). Lipid yields obtained by green solvents led to a 33.4−73.2% recovery compared to traditional extraction; the highest values (p < 0.05) were obtained by ethanol-containing systems. Compared to the traditional procedure, ethanol systems showed an 85.8−90.3% recovery of phospholipid compounds and no differences (p > 0.05) in the ω3/ω6 ratio. Green-extracting systems led to higher average values for eicosapentaenoic acid content (15.66−18.56 g·100 g−1 total FAs) and polyene index (1.93−3.29) than chloroform/methanol extraction; differences were significant (p < 0.05) for systems including acetone and ethyl acetate. No differences (p > 0.05) were detected for docosahexaenoic acid content between the traditional procedure and green systems, with all values being included in the 31.12−32.61 g·100 g−1 total FA range. The suitability of EtOH-containing green systems for extraction of bioactive lipid compounds from squid by-products was concluded.

This study focuses on the extraction of nutritional and healthy constituents of whole by-products resulting from the commercialisation of Patagonian squid (Doriteuthis gahi). By-products corresponding to squid individuals captured at different seasons were comparatively analysed for proximate composition, lipid classes content, fatty acid (FA) profile, and macroelement and trace element composition. As a result, moisture, lipid, protein, and ash values were included in the ranges 829.0-842.8, 17.5-21.8, 106.0-123.7, and 9.3-13.3 g·kg-1 by-products, respectively. Phospholipids showed to be the most abundant lipid class (359.2-463.5 g·kg-1 lipids), while triacylglycerols were only present in a 9.5-13.1 g·kg-1 lipids range. Valuable levels were detected for α-tocopherol (539.6-973.3 mg·kg-1 lipids), polyunsaturated fatty acids (PUFA; 50.5-52.6 g·100 g-1 FA), ω3 PUFA (47.0-48.6 g·100 g-1 FA), PUFA/saturated FA ratio (1.4-1.6), and ω3/ω6 ratio (12.1-13.4). Among macroelements, S, P, and Na showed to be more abundant than K, Mg, and Ca. Profitable levels of Co, Cu, Fe, Mn, Se, and Zn were detected in all kinds of individuals. In spite of content variations found as a result of the capture season of Patagonian squid individuals, whole by-products of this cephalopod species can be considered as a profitable source to provide the food and pharmaceutical industries with useful value-added constituents.

The goal of this work is to explore if the changes induced by d-fagomine in the gut microbiota are compatible with its effect on body weight and inflammation markers in rats. Methods: Sprague Dawley rats were fed a standard diet supplemented with d-fagomine (or not, for comparison) for 6 months. The variables measured were body weight, plasma mediators of inflammation (hydroxyeicosatetraenoic acids, leukotriene B4, and IL-6), and the concentration of acetic acid in feces and plasma. The composition and diversities of microbiota in cecal content and feces were estimated using 16S rRNA metabarcoding and high-throughput sequencing. We found that after just 6 weeks of intake d-fagomine significantly reduced body weight gain, increased the plasma acetate concentration, and reduced the plasma concentration of the pro-inflammatory biomarkers' leukotriene B4, interleukin 6 and 12 hydroxyeicosatetraenoic acids. These changes were associated with a significantly increased prevalence of Bacteroides and Prevotella feces and increased Bacteroides, Prevotella, Clostridium, and Dysgonomonas while reducing Anaerofilum, Blautia, and Oribacterium in cecal content. In conclusion, d-fagomine induced changes in the composition and diversity of gut microbiota similar to those elicited by dietary fiber and compatible with its anti-inflammatory and body-weight-reducing effects.

Adipose tissue is now recognized as an active organ with an important homeostatic function in glucose and lipid metabolism and the development of insulin resistance. The present research investigates the role of lipid mediators and lipid profiling for controlling inflammation and the metabolic normal function of white adipose tissue from rats suffering from diet-induced prediabetes. Additionally, the contribution to the adipose lipidome induced by the consumption of marine ω-3 PUFAs as potential regulators of inflammation is addressed. For that, the effects on the inflammatory response triggered by high-fat high-sucrose (HFHS) diets were studied in male Sprague-Dawley rats. Using SPE-LC-MS/MS-based metabolo-lipidomics, a range of eicosanoids, docosanoids and specialized pro-resolving mediators (SPMs) were measured in white adipose tissue. The inflammatory response occurring in prediabetic adipose tissue was associated with the decomposition of ARA epoxides to ARA-dihydroxides, the reduction of oxo-derivatives and the formation of prostaglandins (PGs). In an attempt to control the inflammatory response initiated, LOX and non-enzymatic oxidation shifted toward the production of the less pro-inflammatory EPA and DHA metabolites rather than the high pro-inflammatory ARA hydroxides. Additionally, the change in LOX activity induced the production of intermediate hydroxides precursors of SPMs as protectins (PDs), resolvins (Rvs) and maresins (MaRs). This compensatory mechanism to achieve the restoration of tissue homeostasis was significantly strengthened through supplementation with fish oils. Increasing proportions of ω-3 PUFAs in adipose tissue significantly stimulated the formation of DHA-epoxides by cytochrome P450, the production of non-enzymatic EPA-metabolites and prompted the activity of 12LOX. Finally, protectin PDX was significantly reduced in the adipose tissue of prediabetic rats and highly enhanced through ω-3 PUFAs supplementation. Taken together, these actively coordinated modifications constitute key mechanisms to restore adipose tissue homeostasis with an important role of lipid mediators. This compensatory mechanism is reinforced through the supplementation of the diet with fish oils with high and balanced contents of EPA and DHA. The study highlights new insides on the targets for effective treatment of incipient diet-induced diabetes and the mechanism underlying the potential anti-inflammatory action of marine lipids.

A non-targeted strategy to simultaneously screen for over 100 lipid mediators from ω-6 ARA and ω-3 EPA and DHA fatty acids is presented. The method based on an extensive study of fragmentation patterns obtained by SPE-LC-MS/MS analysis-provided fingerprints to comprehensively elucidate and identify lipid mediators in biological samples. Many of these metabolites are associated to metabolic disorders, inflammatory, immune and oxidative stress. The methodology consisted of a three-step procedure. (1) SPE extraction of compounds from plasma and adipose tissue was followed by LC-MS/MS analysis operating in full scan mode. The methodology was validated for a group of 65 metabolites using standards. SPE recoveries ranged from 29-134% and matrix effect from 10-580%. LOD and LOQ ranged from 0.01 to 1765 ng/mL and 0.03 to 5884 ng/mL respectively, similarly than current analytical strategies based on MRM mode. (2) An extensive study of the mass spectra of a wide range of compounds was done to stablish a specific fragmentation pattern. Interestingly, illustrative fragmentations and new specific transitions to identify EPA and DHA lipid mediators have been innovatively established. (3) After analysis, 30 lipid mediators were tentatively identified in plasma and 35 in adipose tissue of rats according to the pre stablished fragmentation patterns. The hypothetical identification of compounds was validated by using reference standards. Around 85-90% of proposed identifications were correctly assigned and only 4 and 3 identifications failed in adipose tissue and plasma, respectively. The method allowed the identification of these metabolites without losing information by the use of predefined ions list. Therefore, the use of full scan mode together with the study of fragmentation patterns provided a novel and stronger analytical tool to study the complete profile of lipid mediators in biological samples than the analysis through MRM based methods. Importantly, no analytical standards were required at this qualitative screening stage and the performance and sensitivity of the assay were very similar to that of a MRM method.

Anisakids are fish-borne parasites that are responsible for a large number of human infections and allergic reactions around the world. World health organizations and food safety authorities aim to control and prevent this emerging health problem. In the present work, a new method for the fast monitoring of these parasites is described. The strategy is divided in three steps: (i) purification of thermostable proteins from fish-borne parasites (Anisakids), (ii) in-solution HIFU trypsin digestion and (iii) monitoring of several peptide markers by parallel reaction monitoring (PRM) mass spectrometry. This methodology allows the fast detection of Anisakids in <2h. An affordable assay utilizing this methodology will facilitate testing for regulatory and safety applications.

The potential effects of various dietary eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6) ratios (1:1, 2:1, and 1:2, respectively) on protein redox states from plasma, kidney, skeletal muscle, and liver were investigated in Wistar rats. Dietary fish oil groups were compared with animals fed soybean and linseed oils, vegetable oils enriched in ω6 linoleic acid (LA; 18:2) and ω3 α-linolenic acid (ALA; 18:3), respectively. Fish oil treatments were effective at reducing the level of total fatty acids in plasma and enriching the plasmatic free fatty acid fraction and erythrocyte membranes in EPA and DHA. A proteomic approach consisting of fluorescein 5-thiosemicarbazide (FTSC) labeling of protein carbonyls, FTSC intensity visualization on 1-DE or 2-DE gels, and protein identification by MS/MS was used for the protein oxidation assessment. Albumin was found to be the most carbonylated protein in plasma for all dietary groups, and its oxidation level was significantly modulated by dietary interventions. Supplementation with an equal EPA:DHA ratio (1:1) showed the lowest oxidation score for plasma albumin, followed in increasing order of carbonylation by 1:2 <2:1 ≈ linseed < soybean. Oxidation patterns of myofibrillar skeletal muscle proteins and cytosolic proteins from kidney and liver also indicated a protective effect on proteins for the fish oil treatments, the 1:1 ratio exhibiting the lowest protein oxidation scores. The effect of fish oil treatments at reducing carbonylation on specific proteins from plasma (albumin), skeletal muscle (actin), and liver (albumin, argininosuccinate synthetase, 3-α-hydroxysteroid dehydrogenase) was remarkable. This investigation highlights the efficiency of dietary fish oil at reducing in vivo oxidative damage of proteins compared to oils enriched in the 18-carbon polyunsaturated fatty acids ω3 ALA and ω6 LA, and such antioxidant activity may differ among different fish oil sources because of variations in EPA/DHA content.

Anisakis simplex is a parasitic nematode that can cause anisakiosis and/or allergic reactions in humans. The presence of invasive third-stage larvae (L3) in many different consumed fish species and the fourth-stage larvae (L4) in marine mammals, where L3 can accidentally affect to humans and develop as far as stage L4. World Health Organization and food safety authorities aim to control and prevent this emerging health problem. In the present work, using Tandem Mass Tag (TMT)-based quantitative proteomics we analyzed for the first time the global proteome of two A. simplex development stages, L3 and L4. The strategy was divided into four steps: (a) protein extraction of L3 and L4 development stages, (b) high intensity focused ultrasound (HIFU)-assisted trypsin digestion, (c) TMT-isobaric mass tag labeling following by high-pH reversed-phase fractionation, and (d) LC-MS/MS analysis in a LTQ-Orbitrap Elite mass spectrometer. A total of 2443 different proteins of A. simplex were identified. Analysis of the modulated proteins provided the specific proteomic signature of L3 (i.e. pseudocoelomic globin, endochitinase 1, paramyosin) and L4 (i.e. neprilysin-2, glutamate dehydrogenase, aminopeptidase N). To our knowledge, this is the most comprehensive dataset of proteins of A. simplex for two development stages (L3 and L4) identified to date. SIGNIFICANCE: A. simplex is a fish-borne parasite responsible for the human anisakiosis and allergic reactions around the world. The work describes for the first-time the comparison of the proteome of two A. simplex stages (L3 and L4). The strategy is based on four steps: (i) protein extraction, (ii) ultra-fast trypsin digestion under High-Intensity Focused Ultrasound (HIFU), (iii) TMT-isobaric mass tag labeling followed by high-pH reversed-phase fractionation and (iv) peptide analysis using a LTQ-Orbitrap Elite mass spectrometer. The workflow allows to select the most modulated proteins as proteomic signature of those specific development stages (L3 and L4) of A. simplex. Obtained stage-specific proteins, could be used as targets to control/eliminate this parasite and in future eradicate the anisakiosis disease.

The combined supplementation of buckwheat D-fagomine (FG) and fish omega-3 polyunsaturated fatty acids (ω-3 PUFA) attenuates the development of insulin resistance in rats fed a high-fat (HF) diet. This study aimed to examine the effects of combined supplementation with FG and ω-3 PUFA on dyslipidemia, transaminases, interleukin-6, and oxidative stress. Forty-five male Sprague-Dawley rats were fed a standard diet, an HF diet, an HF diet supplemented with FG, an HF diet supplemented with ω-3 PUFA, or an HF diet supplemented with FG and ω-3 PUFA for 21 weeks. Triacylglycerol, cholesterol, aspartate aminotransferase, alanine aminotransferase, and interleukin-6 were measured. The assessment of oxidative stress included plasma antioxidant capacity, antioxidant enzyme activities, glutathione content, lipid peroxidation, and protein carbonylation. The combined supplementation with FG and ω-3 PUFA did not attenuate the slight accumulation of liver cholesterol induced by the HF diet but normalized the plasma alanine aminotransferase activity. Rats fed the HF diet supplemented with the combination showed a lower amount of plasma interleukin-6 than those fed a standard diet. The combination attenuated oxidative damage induced by the HF diet, decreased antioxidant enzyme activities, and enhanced glutathione status. The beneficial effects of the combination of FG and ω-3 PUFA on oxidative stress and related risk factors in pre-obese rats were mainly modulated by ω-3 PUFA.