The macrophage polarization in allergic responses induced by tropomyosin of Macrobrachium nipponense in cell and murine models.

Macrophages are one of the important immune cells, which play important roles in innate and adaptive immune. However, the roles of macrophages in food allergy are not thoroughly understood. To investigate the roles of macrophages during food allergy, we focused on the relationship between macrophage polarization and allergic responses induced by tropomyosin (TM) in the present study. Arg 1 and CD206 expressions in the TM group were significantly higher than those of the PBS group, while iNOS and TNF-α expressions were no obvious difference, moreover, the morphology of macrophages stimulated by TM was similar to that of M2 macrophages. These results indicated macrophages were mainly polarized toward M2 phenotypes in vitro. The antibodies, mMCP-1, histamine and cytokines, revealed that macrophages could participate in food allergy, and macrophage polarization was associated with changes in allergic-related factors. The cytokine levels of M2 phenotypes were significantly higher than those of M1 phenotypes in peripheral blood. The mRNA expressions and protein levels of Arg1 and iNOS in the jejunum and peritoneal cells indicated that M2 phenotypes were the major macrophage in these tissues compared with M1 phenotypes. Hence, macrophage polarization plays an important role in food allergy.

The pathogenesis of food allergy and protection offered by dietary compounds from the perspective of epigenetics.

Food allergy is a global food safety concern, with an increasing prevalence in recent decades. However, the immunological and cellular mechanisms involved in allergic reactions remain incompletely understood, which impedes the development of effective prevention and treatment strategies. Current evidence supports those epigenetic modifications regulate the activation of immune cells, and their dysregulation can contribute to the development of food allergies. Patients with food allergy show epigenetic alterations that lead to the onset, duration and recovery of allergic disease. Moreover, many preclinical studies have shown that certain dietary components exert nutriepigenetic effects in changing the course of food allergies. In this review, we provide an up-to-date overview of DNA methylation, noncoding RNA and histone modification, with a focus on their connections to food allergies. Following this, we discuss the epigenetic mechanisms that regulate the activation and differentiation of innate and adapted immune cell in the context of food allergies. Subsequently, this study specifically focuses on the multidimensional epigenetic effects of dietary components in modulating the immune response, which holds promise for preventing food allergies in the future.

Large-flake graphene-modified biochar for the removal of bisphenol S from water: rapid oxygen escape mechanism for synthesis and improved adsorption performance.

The combined effects of graphene and biochar for enhanced adsorption of organic pollutants have not been demonstrated yet. Therefore, the mechanisms of graphene-modified biochar synthesis and its application to adsorption of contaminants remain unclear. In this study, the effect of flake-size graphene on biochar modification and its bisphenol S (BPS) adsorption performance was explored for the first time. Three sizes of graphene oxide were used as the precursor to prepare graphene/biochar composites using pyrolysis. It was found that the graphene with a small flake size was interspersed in the macropores of biochar, while the biochar was completely or mostly wrapped by the large-sized graphene sheet, which effectively prevented the agglomeration and pore blockage of biochar. Large-flake graphene oxide modified biochar (LGB) showed the highest adsorption capacity towards BPS, exhibiting 2.8 times higher adsorption than pristine biochar. Density functional theory (DFT) calculation suggested that the maximum diffusion barrier of O atoms in graphene coated cellulose (most frequently used biochar representative) could be reduced significantly (∼46%) at pyrolysis temperature of 873 K. Taking the advantage of small amount of graphene and enhanced adsorption performance, LGB could be a promising adsorbent for the removal of certain organic pollutants from wastewater and is conducive for the development of high-valued biochar modification.

Dietary Linolenic Acid Increases Sensitizing and Eliciting Capacities of Cow's Milk Whey Proteins in BALB/c Mice.

α-Lactalbumin (BLA) and ß-lactoglobulin (BLG) are the major whey proteins causing allergic reactions. Polyunsaturated fatty acids (PUFAs) stand among the extrinsic factors of the food matrix that can bind BLA and BLG and change their bioactivities, but their contribution to change the allergenic properties of these proteins has not been investigated. Here, we aimed to determine how PUFAs influence BLA and BLG to sensitize and trigger allergic responses in BALB/c mice. First, tricine-SDS-PAGE and spectroscopic assays identified that α-linolenic acid (ALA, as a proof-of-concept model) can induce BLA and BLG to form cross-linked complexes and substantially modify their conformation. Then, BALB/c mice (n = 10/group) were orally sensitized and challenged with BLA and BLG or ALA-interacted BLA and BLG, respectively. Allergic reactions upon oral challenge were determined by measuring clinical allergic signs, specific antibodies, levels of type-1/2 cytokines, the status of mast cell activation, and percentage of cell populations (B and T cells) in different tissues (PP, MLN, and spleen). Overall, systemic allergic reaction was promoted in mice gavage with ALA-interacted BLA and BLG by disrupting the Th1/Th2 balance toward a Th2 immune response with the decreased number of Tregs. Enhanced induction of Th2-related cytokines, as well as serum-specific antibodies and mast cell activation, was also observed. In this study, we validated that ALA in the food matrix promoted both the sensitization and elicitation of allergic reactions in BALB/c mice.

Characterization of systemic allergenicity of tropomyosin from shrimp (Macrobrachium nipponense) and anaphylactic reactions in digestive tract.

BACKGROUND: Tropomyosin (TM) is the major allergen of crustaceans. The allergenicity of TM from Macrobrachium nipponense (MnTM) and the anaphylactic reaction in the digestive tract are still unclear. The aim of this study was to evaluate the allergenicity of MnTM and the anaphylactic reaction in the digestive tract. RESULTS: Serum IgE and IgG1 binding ability in the TM group were significantly higher than those in the PBS and CT groups (P < 0.01) and CP group (P < 0.05), while serum IgG and IgG2a binding ability showed no obvious difference between the four groups (P > 0.05). The levels of cytokines IL-4, IL-5 and IL-13 in TM and CP groups were significantly higher than those in PBS and CT groups. Histamine and ß-hexosaminidase in the TM and CP groups from basophil cell models were significantly higher than those in the PBS group. The highest mRNA expression of IL-4 and IL-13 was in the jejunum from TM-sensitized mice. Histopathological analysis showed that more immune cells infiltrated into the jejunum than the duodenum and ileum from the TM-sensitized mice. CONCLUSIONS: MnTM could promote an allergic response in mice and lead to degranulation in basophil cells. The jejunum was more easily affected by MnTM than duodenum and ileum, and the jejunum may be the major site of allergic response in the digestive tract. © 2020 Society of Chemical Industry.

Molecular characterization of cu/Zn SOD gene in Asian clam Corbicula fluminea and mRNA expression and enzymatic activity modulation induced by metals.

The copper/zinc superoxide dismutase (Cu/Zn SOD) could effectively eliminate reactive oxygen species (ROS). In this study, the cDNA sequence of Cu/Zn SOD from Corbicula fluminea (designated as CfCu/Zn SOD) was cloned by the rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfCu/Zn SOD was of 1288 bp, including a 465 bp ORF encoding a protein of 154 amino acids. Two SOD family signatures were identified in CfCu/Zn SOD amino acids sequence. Multiple sequence alignments indicated that CfCu/Zn SOD amino acid sequences exhibited high similarities to those of other species. The tissue distribution of the CfCu/Zn SOD of C. fluminea was detected by fluorescent real-time quantitative PCR. The mRNA expression levels in digestive gland and gill were significantly higher than those of other four tissues. In response to metal ions (Cd2+, Cu2+, and Pb2+) challenge, the expression and enzymatic activities of CfCu/Zn SOD in the gills were measured after exposure for 0, 6, 12, 24, 48, and 72 h. The results showed that the mRNA expression and enzymatic activities of CfCu/Zn SOD could be induced by three metal ions. After Cd2+ and Cu2+ exposure, the mRNA expression levels increased gradually and reach the peak at 24 h, afterwards it slowed down. The change trends of enzymatic activities were similar to the mRNA expression. After Pb2+ exposure, the expression and activities CfCu/Zn SOD were raised up gradually and reach the highest value at 72 h. All these results indicated that the mRNA expression and enzymatic activity of CfCu/Zn SOD are sensitive to Cd2+, Cu2+ and Pb2+ ions and can be used as molecular biomarkers of metal pollution in water.

Molecular cloning and functional characterization of a novel i-type lysozyme in the freshwater mussel Cristaria plicata.

The freshwater bivalve Cristaria plicata, which is widely distributed in Eastern Asia, is a key species in the pearl culture industry. In this study, a novel invertebrate-type lysozyme, designated as CpLYZ2, was cloned from hemocytes of C. plicata. This lysozyme shares high sequence identity and is homologous to a previously identified lysozyme CpLYZ1 isolated from C. plicata and with HcLyso3 isolated from Hyriopsis cumingii. The full-length cDNA of CpLYZ2 is 913 bp long, which includes an open reading frame (ORF) of 486 bp, a 3' untranslated region (UTR) of 389 bp and a 5' UTR of 38 bp. The ORF encodes a putative polypeptide of 161 amino acids with a predicted molecular mass of 18.2 kDa and a theoretical isoelectric point of 6.56. CpLYZ2 mRNA transcripts can be detected in hemocytes, hepatopancreas, muscle, gills and mantle tissues, the greatest expression being observed in the gills. CpLYZ2 expression in hemocytes, hepatopancreas and gills increased significantly after the mussel was challenged with Aeromonas hydrophila. Furthermore, the optimal pH and temperature for enzyme activity of the recombinant CpLYZ2 were 5.5 and 50°C, respectively. The recombinant lysozyme protein exhibited bacteriolytic activity against Escherichia coli, A. hydrophila, Staphylococcus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis. The findings of this study help to elucidate immune responses in molluscs and will thus expedite disease management of these key freshwater species, in turn boosting pearl culture in eastern Asia.

Molecular cloning and characterization of cathepsin L from freshwater mussel, Cristaria plicata.

Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. The Cathepsin L cDNA and genome of Cristaria plicata（CpCL） was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends (RACE) PCR. The genomic DNA was 9353 bp long and had a total of six introns and seven exons. The full-length cDNA of CpCL was 1144 bp, the cDNA contained a 5' untranslated region (UTR) of 34 nucleotides, the 3' UTR of 108 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1002 bp, encoding 333 amino acid residues with 37.65 kDa predicted molecular weight. The theoretical isoelectric point was 8.61. The prepro-cathepsin L was consisted of a typical signal peptide (Met1-Gly20), a pro-region peptide (Leu21-Glu116) and a mature peptide (Tyr117-Val333). Many members of the papain family possessed of a proline residue at position 2 in the mature enzymem, this was also observed in CpCL. The preproprotein included an oxyanion hole (Gln 135), the active center formed by Cys141, His280 and Asn 300, the potential N-glycosylation site (Asn38, Asn 113 and Asn 272) and the conserved GCXGG motifs, which was characteristic of cathepsin, the conserved ERWNIN and GNFD motifs, which were characteristic for cathepsin L. Homology analysis revealed that the CpCL shared 49-87% identity to other known cathepsin L sequences. The phylogenetic tree showed that the CpCL clustered with the invertebrate cathepsin L cysteine proteases, and was closely related to the cathepsin L of Hyriopsis cumingii. The expression of CpCL mRNA was detected in hepatopancreas, hemocytes, mantle, gills and adductor muscle, and the higher expression level was in hepatopancreas. After A. hydrophila stimulation, the expression of the CpCL mRNA was up-regulated in hemocytes and hepatopancreas, and the expression level was significantly lower in gill than one after PBS challenge group.

Gene identification and recombinant protein of a lysozyme from freshwater mussel Cristaria plicata.

Lysozymes are important proteins to bivalve in the innate immune responses against bacterial infections, and provide nutrition as digestion enzymes. A new LYZ1 from the freshwater mussel Cristaria plicata was cloned by rapid amplification of cDNA ends (RACE) and nested PCR method. The full-length cDNA sequence of CpLYZ1 was 763 bp. The cDNA contained a 5'-terminal untranslated region (UTR) of 21 bp, a 3'- terminal UTR of 259 bp with a 29 bp poly(A) tail, a tailing signal (AATAAA) and the open reading frame of 483 bp. The CpLYZ1 cDNA encoded a polypeptide of 160 amino acids with a predicted molecular mass of 17.8 kDa, and a theoretical isoelectric point of 6.07. The comparison of the deduced amino acid sequences with LYZs from other species showed that the enzyme belonged to i-type lysozyme. The mRNA transcript of CpLYZ1 could be detected in all the examined tissues with the highest expression level in hepatopancreas. The expression levels of CpLYZ1 in hemocytes, hepatopancreas and gill significantly increased after Aeromonas hydrophila challenge. The expression level of CpLYZ1 in hemocytes sharply decreased from 6 h to 24 h and significantly increased at 48 h, and was the highest level in hepatopancreas at 24 h, and was the maximum level in gill at 48 h. Furthermore, the recombinant CpLYZ1 was induced to be expressed as an inclusion body form by IPTG at 37 °C for 4 h, and then was purified by using the Ni(2+) affinity chromatography. The relative enzyme activity of the recombinant CpLYZ1 was influenced on pH and temperature. The optimal pH and temperature was 5.5 and 50 °C, respectively. Against Escherichia coli, A. hydrophila, Staphyloccocus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis, the recombinant CpLYZ1 had bacteriolytic activity.

Cloning, identification and functional characterization of a pi-class glutathione-S-transferase from the freshwater mussel Cristaria plicata.

Glutathione-S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione and play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). The piGST cDNA was cloned and sequenced after rapid amplification of cDNA ends (RACE) from the freshwater mussel Cristaria plicata. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzymes belonged to the pi-class and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. The Cp-piGST cDNA is 816 nucleotides (nt) in length and contained a 615 nt open reading frame (ORF) encoding 205 amino acid residues, and has 19 nt of 5' untranslated region (UTR) and a 3' UTR of 182 nt including a tailing signal (AATAAA) and a poly (A) tail. The molecular weight of the predicted piGST is 23.4 kDa, with the calculated PI being 5.2. The mRNA transcript of Cp-piGST could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of Cp-piGST in hepatopancreas and gill showed similar trend that were significantly increased after bacterial challenge compared to the control group at 12 h. Furthermore, the recombinant Cp-piGST with high enzyme activity was induced to be expressed as a soluble form by IPTG at 20°C for 8 h, and then was purified by using the native Ni(2+) affinity chromatography. The specific activity of the purified soluble Cp-piGST enzyme into pET30 was 2.396 µmol/min/mg, and which into pET32 was 1.706 µmol/min/mg. The recombinant Cp-piGST had a maximum activity at approximately pH 8.0, and its optimum temperature was 37°C. The recombinant Cp-piGST enzyme activity became lower gradually with the denaturant concentration increasing.

A catalase from the freshwater mussel Cristaria plicata with cloning, identification and protein characterization.

Catalase is an important antioxidant protein which can protect organisms against various oxidative stresses by eliminating hydrogen peroxide. The catalase cDNA of Cristaria plicata (cpCAT) was cloned from the haemocytes using degenerate primers by the method of 3' and 5' rapid amplification of cDNA ends PCR. The gene is 4863 bp long and has a total of two introns and three exons. The precise size and location of the introns and exons have been determined. In addition the full-length cDNA of cpCAT contained 2618 bp, The cDNA contained a 5' untranslated region (UTR) of 136 nucleotides, the 3' UTR of 979 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1503 bp, encoding 501 amino acid residues with 56.86 kDa predicted molecular weight. The theoretical isoelectric point was 6.77. BLAST analysis showed that the deduced amino acid sequence of cpCAT had significant homology to catalases from animals, plants and bacteria. The deduced amino acid sequence of cpCAT had characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77), heme-ligand signature motif (351RLYSYSDTH359), two glycosylation sites (N145, N436), NADPH binding site and the three catalytic amino acid residues (His72, Asn145 and Tyr355). It had no signal peptide. The phylogenetic tree indicated that cpCAT gene was very close to the gene of scallops, Chlamys farreri. The enzymatic activity of purified recombinant cpCAT was 11194.4 ± 40.4 U/mg, it might resist against H(2)O(2). The recombinant enzyme held higher thermal stability, the optimum temperature was 25 °C, it retained more than 82% activity between 25 and 60 °C. The stability of the recombinant enzyme were higher between pH 5 and 10, and the optimal pH value was 7.0. When cpCAT was treated with 2-4 moL/L urea and 1%-3% SDS, the activity was also stable, it kept more than 80% activity.