A sandwich ELISA for the detection of mollusks and mollusk products.

Mollusks are a major allergenic food under the food allergen regulations of many countries and must be declared on food products to reduce the risk of allergic reactions. However, a reliable immunoassay for detecting edible mollusks (cephalopods, gastropods, and bivalves) has not been reported. In this study, the developed sandwich enzyme-linked immunosorbent assay (sELISA) detected 32 edible mollusk species in raw and heated without cross-reaction with non-mollusk species. The detection limits of the assay were 0.1 ppm for heated mollusks and 0.1-0.5 ppm for raw mollusks, depending on the mollusk species tested. The inter-assay and intra-assay coefficients of variation (CVs) were ≤14.83 and ≤8.11, respectively. The assay detected steamed, boiled, baked, fried, and autoclaved mollusk samples and all commercial mollusk products tested. In this study, we developed a mollusk-specific sELISA to protect people allergic to mollusks.

Effect of species, muscle location, food processing and refrigerated storage on the fish allergens, tropomyosin and parvalbumin.

Fish tropomyosin is a latest identified fish allergen without full understanding of its biochemical characteristics from the perspective of food allergen. Accordingly, the objective of this study was to investigate the effects of species, muscle location, food processing, and refrigerated storage on fish tropomyosin and compare with main fish allergen, parvalbumin. The result of mass spectrometry analysis revealed tropomyosin as the most abundant thermally stable protein in fish muscle. Fish tropomyosin was ubiquitous among all 28 edible fish species tested, abundant in fish skeletal muscle, resistant to common food processing, and resistant to refrigerated storage up to six days. By contrast, parvalbumin content varied between fish species and was not as thermally stable as tropomyosin under autoclaving. This study demonstrates the intrinsic and processing factors affecting fish allergens and provides valuable information for the presence of major fish allergens and practical consideration of fish allergen detection.

Combined Low Plant and High Animal Dietary Pattern Is Associated with a Lower Risk of Anemia among Dyslipidemic Adult Women in Taiwan: A Retrospective Study from 2001 to 2015.

Anemia and dyslipidemia often occurs in premenopausal women. This study investigated the association between dietary patterns and anemia among dyslipidemic women in Taiwan. This study recruited 22,631 dyslipidemic women aged 20-45 years between 2001 and 2015. The dietary assessment was collected by a validated food frequency questionnaire. The biochemical data including blood lipids, red blood cells, hemoglobin, hematocrit, and C-reactive protein (CRP) were retrieved from the database. Women with a combined high plant diet (HP) and low animal diet (LA) were associated with a lower prevalence of obesity (11.7%), central obesity (16.0%), high total cholesterol (16.4%), and high low-density lipoprotein cholesterol (11.9%), and had lower hemoglobin (12.9 ± 1.4 g/dL), hematocrit (38.8 ± 3.6%), and CRP levels (20.6 ± 31.4 nmol/L). The low plant diet (LP) + high animal diet (HA) pattern was negatively associated with moderate to severe anemia (OR: 0.76, 95% CI: 0.64-0.92, p = 0.004) compared to the low plant diet (LP) + low animal diet (LA) pattern. However, the HP + LA pattern was positively correlated with moderate to severe anemia (OR: 1.22, 95% CI: 1.04-1.43, p = 0.015). In conclusion, a low plant and high animal diet plays a role in preventing anemia development among dyslipidemic women.

Characterization of a 36 kDa antigenic protein of fish-specific monoclonal-antibody 8F5.

The 36 kDa antigenic protein of a fish-specific monoclonal antibody (mAb), 8F5, previously developed to detect fish in foodstuffs to protect fish allergic individuals, was characterized to establish its identity and to identify the fish-specific epitope. We hypothesized that this antigenic protein is tropomyosin based on its thermal stability and molecular weight. Western blot showed that both the 36 kDa protein and fish tropomyosin were recognized by mAb 8F5, and their molecular weight migration in urea gel electrophoresis was identical. In addition to matching the amino acid composition profile, this 36 kDa protein's sequences precisely correspond to those in fish tropomyosin fragments. Further analysis revealed the sequence of the fish-specific epitope bound by mAb 8F5 to be EDDLVALQKK. These results confirm that the 36 kDa protein is indeed tropomyosin and will be a suitable biomarker for the immunodetection of fish in cooked food.

Development and Characterization of Monoclonal Antibodies for the Detection of Fish Protein.

This study developed and characterized anti-fish monoclonal antibodies (mAbs) capable of detecting fish, a major allergenic food, in processed food products to protect fish sensitized individuals. Of the three mAbs raised against crude protein extract of cooked fish muscle, mAb 8F5 exhibited a positive reaction to all 50 common food fish species tested with no cross-reactions to shellfish, land animals, or food additives. Although the ELISA results were negative against swordfish and yellowfin tuna, western blot clearly detected both after cooking. The ~36 kDa antigenic protein of mAb 8F5, which was found in all fish species, was detectable by mAb 8F5 in all of the fish samples even after prolonged heat treatment (100 °C, up to 60 min). These findings suggest that mAb 8F5 has great potential utility as a probe for the immunochemical detection of fish tissue in cooked food.

Dietary exposure to chlorpyrifos affects systemic and hepatic immune-cell phenotypes in diabetic mice.

Hyperglycemia induces low-grade systemic inflammation and immune dysregulation, leading to overstated reactions to immune stimuli and diabetes-related organ damage. Tissue inflammation is characterized by leukocyte infiltration, and T cells play crucial roles in directing leukocyte-mediated inflammatory responses. The aim of the study was to investigate the effects of dietary exposure to chlorpyrifos (CPF) on systemic and hepatic immune-cell phenotypes in C57BL/6 mice with streptozotocin (STZ)-induced diabetes. Mice received an intraperitoneal injection of STZ for 5 consecutive days to induce diabetes, and diabetic mice were given either an AIN-93-based control diet or a CPF-containing diet at doses of 0.5, 1, or 2 mg/kg body weight/day for 28 days. Results showed that dietary exposure to CPF had no influence on the body weight or the erythrocyte hemoglobin A1c level in diabetic mice. Both blood and hepatic neutrophil populations were enhanced by CPF exposure. CPF-exposed groups had lower percentages of blood T cells without altering the proportions of CD4+ and CD8+ T-cell subsets, and lower expression levels of the Bcl-2 antiapoptotic gene in the spleen. CPF exposure reduced the percentage of blood regulatory T cells (Tregs); however, the Treg population was upregulated in the liver even when hepatic T cells were not affected by CPF in diabetic mice. Hepatic expressions of Treg-related genes were suppressed in all CPF-exposed groups. Higher plasma levels of aspartate aminotransferase and expression levels of the hepatic interleukin-1ß gene were observed in diabetic mice exposed to medium and high doses of CPF. These findings suggest that dietary exposure to CPF affects the distribution of both myeloid and lymphoid immune cells in the blood and liver under hyperglycemic conditions, which may lead to hyperinflammation when encountering immune stimuli.

Self-Healing Concrete by Biological Substrate.

At present, the commonly used repair materials for concrete cracks mainly include epoxy systems and acrylic resins, which are all environmentally unfriendly materials, and the difference in drying shrinkage and thermal expansion often causes delamination or cracking between the original concrete matrix and the repair material. This study aimed to explore the feasibility of using microbial techniques to repair concrete cracks. The bacteria used were environmentally friendly Bacillus pasteurii. In particular, the use of lightweight aggregates as bacterial carriers in concrete can increase the chance of bacterial survival. Once the external environment meets the growth conditions of the bacteria, the vitality of the strain can be restored. Such a system can greatly improve the feasibility and success rate of bacterial mineralization in concrete. The test project included the microscopic testing of concrete crack repair, mainly to understand the crack repair effect of lightweight aggregate concrete with implanted bacterial strains, and an XRD test to confirm that the repair material was produced by the bacteria. The results show that the implanted bacterial strains can undergo Microbiologically Induced Calcium Carbonate Precipitation (MICP) and can effectively fill the cracks caused by external concrete forces by calcium carbonate deposition. According to the results on the crack profile and crack thickness, the calcium carbonate precipitate produced by the action of Bacillus pasteurii is formed by the interface between the aggregate and the cement paste, and it spreads over the entire fracture surface and then accumulates to a certain thickness to form a crack repairing effect. The analysis results of the XRD test also clearly confirm that the white crystal formed in the concrete crack is calcium carbonate. From the above test results, it is indeed feasible to use Bacillus pasteurii in the self-healing of concrete cracks.

Oligonol Alleviates Sarcopenia by Regulation of Signaling Pathways Involved in Protein Turnover and Mitochondrial Quality.

SCOPE: Oligonol has been shown to moderate mitochondrial biogenesis, protein synthesis, and protein degradation in diabetic mice in a previous study. It is therefore hypothesized that oligonol alleviated sarcopenia by regulating pathways involved in protein turnover and mitochondrial quality. METHODS AND RESULTS: The 32-week-old senescence-accelerated mouse prone 8 (SAMP8) mice are fed with chow diet containing 200 mg kg-1 oligonol for 8 weeks. Oligonol supplementation increased skeletal muscle mass, cross-sectional areas, and grip strength in SAMP8 mice. Oligonol increased phosphorylation of AKT/mTOR/p70sk6, inhibited nuclear localization of FoxO3a and NFκB, and decreased transcription of MuRF-1 and MAFbx in skeletal muscle of SAMP8 mice. Downregulation of mitochondrial biogenesis genes (PGC-1α and Tfam) and mitochondrial fusion genes (Mfn2 and Opa1), loss of PINK1, overexpression of Atg13, LC3-II, and p62, and abundant accumulation of autophagosomes and lysosomes in skeletal muscle of SAMP8 mice are limited by oligonol. Furthermore, oligonol reduced expression of released cytochrome c and cleaved caspase-9 in skeletal muscle of SAMP8 mice. CONCLUSION: Regulating pathways involved in protein synthesis and degradation, mitochondrial biogenesis, mitochondrial fusion/fission, autophagy, and mitochondria-dependent apoptosis by oligonol contribute to positive protein turnover and mitochondrial quality, thus increasing muscle mass and strength in SAMP8 mice.

Resveratrol protects muscle cells against palmitate-induced cellular senescence and insulin resistance through ameliorating autophagic flux.

Skeletal muscle, a highly metabolic tissue, is particularly vulnerable to increased levels of saturated free fatty acids (FFAs). The role of autophagy in saturated FFAs-induced cellular senescence and insulin resistance in skeletal muscle remains unclear. Therefore, the present study was aimed to explore autophagic flux in cellular senescence and insulin resistance induced by palmitate in muscle cells, and whether resveratrol limited these responses. Our results showed that palmitate induced cellular senescence in both myoblasts and myotubes. In addition, palmitate delayed differentiation in myoblasts and inhibited expression of insulin-stimulated p-AKTSer473 in myotubes. The accumulations of autophagosome assessed by tandem fluorescent-tagged LC3 demonstrated that autophagic flux was impaired in both palmitate-treated myoblasts and myotubes. Resveratrol protected muscle cells from palmitate-induced cellular senescence, apoptosis during differentiation, and insulin resistance via ameliorating autophagic flux. The direct influence of autophagic flux on development of cellular senescence and insulin resistance was confirmed by blockage of autophagic flux with chloroquine. In conclusion, impairment of autophagic flux is crucial for palmitate-induced cellular senescence and insulin resistance in muscle cells. Restoring autophagic flux by resveratrol could be a promising approach to prevent cellular senescence and ameliorate insulin resistance in muscle.

Viral infection upregulates myostatin promoter activity in orange-spotted grouper (Epinephelus coioides).

Myostatin is a negative regulator of myogenesis and has been suggested to be an important factor in the development of muscle wasting during viral infection. The objective of this study was to characterize the main regulatory element of the grouper myostatin promoter and to study changes in promoter activity due to viral stimulation. In vitro and in vivo experiments indicated that the E-box E6 is a positive cis-and trans-regulation motif, and an essential binding site for MyoD. In contrast, the E-box E5 is a dominant negative cis-regulatory. The characteristics of grouper myostatin promoter are similar in regulation of muscle growth to that of other species, but mainly through specific regulatory elements. According to these results, we conducted a study to investigate the effect of viral infection on myostatin promoter activity and its regulation. The nervous necrosis virus (NNV) treatment significantly induced myostatin promoter activity. The present study is the first report describing that specific myostatin motifs regulate promoter activity and response to viral infection.

Monoclonal antibody-based sandwich ELISA for reliable identification of imported Pangasius catfish.

Pangasius catfish, primarily tra (Pangasius hypophthalmus) and basa (Pangasius bocourti), are farm-raised catfish imported from Asia and have become a common substitute for domestic catfish, grouper, and other high-valued fish in restaurant-served dishes in the United States. This article reports on the development of a monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) for the identification of cooked Pangasius fish, basa, and tra. The assay uses a pair of monoclonal antibodies (MAbs F7B8 and T7E10) specific to a heat-stable 36-kDa protein present in a saline extraction of the fish muscle; MAb F7B8, which cross-reacts to all fish species, is the capture antibody and the biotin-conjugated MAb T7E10, which is specific to Pangasius fish, is the detection antibody. This sandwich ELISA reliably identified fully cooked basa and tra from more than 70 common finfish, shellfish, land animal species, and other protein materials tested. It can also sensitively detect 0.5% of adulterated basa or 0.1% tra in a mixed crabmeat products with low intra-assay (%CV: 2.59 to 4.14) and inter-assay (%CV: 3.36 to 3.71) variabilities. The new assay provides a rapid and reliable means of distinguishing fish in the Pangasiidae family from other common food fish and nonfish species and will greatly assist efforts to discourage the illegal practice of substituting high-value popular fish species by the cheaper farm-raised imported Pangasius fish at the retail and restaurant levels.

Receptor for advanced glycation endproducts mediates neutrophil migration across intestinal epithelium.

Receptor for advanced glycation endproducts (RAGE) is an Ig superfamily cell surface receptor that interacts with a diverse array of ligands associated with inflammatory responses. In this study, we provide evidence demonstrating that RAGE is involved in inflammatory responses in the intestines. We showed that RAGE is expressed in intestinal epithelial cells, primarily concentrated at the lateral membranes close to the apical cell junction complexes. Although RAGE expression was low in epithelium under normal conditions, this protein was up-regulated after treatment with the inflammatory cytokines IFN-gamma and/or TNF-alpha. RAGE expression was also elevated in colon tissue samples from patients with inflammatory bowel diseases. Using in vitro transmigration assays, we found that RAGE mediates neutrophil (polymorphonuclear leukocytes (PMN)) adhesion to, and subsequent migration across, intestinal epithelial monolayers. This activity appears to be mediated by the binding of RAGE to the PMN-specific beta(2) integrin CD11b/CD18. Thus, these results provide a novel mechanism for the regulation of PMN transepithelial migration and may suggest a new therapeutic target for intestinal inflammation.

Functional elements on SIRPalpha IgV domain mediate cell surface binding to CD47.

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.