Antioxidant and Immunomodulatory Activities of Polysaccharides from Fermented Wheat Products of Grifola frondosa: In Vitro Methods.

Despite the well-known health benefits of Grifola frondosa, there is a lack of understanding regarding the potential antioxidant and immunomodulatory properties of different varieties when fermented with wheat grains. We aimed to explore the potential of G. frondosa-fermented wheat flour as a functional food. Three varieties of G. frondosa (GFA, GFB, and GFC) were fermented with wheat grains for solid-state fermentation. Polysaccharides were extracted and analyzed for total sugar content, monosaccharide composition, Mw profile, antioxidant activity, cytotoxicity, and immunomodulatory properties. Results were evaluated using HPLC, DPPH assay, MTS assay, Griess reagent, and ELISA method. Our study found variations in three different varieties of G. frondosa-fermented wheat polysaccharides. Glucose was the predominant monosaccharide, followed by galactose and mannose. Each variety had a different molecular weight distribution, with GFA-wheat mainly present in fraction II, GFB-wheat in fraction I, and GFC-wheat in fraction III. At a concentration of 1.25 mg/mL, GFA-wheat and GFB-wheat polysaccharides increased DPPH scavenging ability by 76.8% and 58.7%, respectively. The polysaccharides showed no apparent toxic effect and enhanced the production of NO, IL-6, and TNF-α in RAW 246.7 macrophages. GFB-wheat polysaccharides demonstrated remarkable immunomodulatory properties at a concentration of 5 µg/mL. Our study provides a theoretical basis for using G. frondosa in wheat staple agricultural products to improve human health.

Heterologous expression of pediocin/papA in Bacillus subtilis.

Listeria monocytogenes is a food-borne pathogen. Pediocin is a group IIα bacteriocin with anti-listeria activity that is naturally produced by Pediococcus acidilactic and Lactobacillus plantarum. The pedA/papA gene encodes pediocin/plantaricin. In native hosts, the expression and secretion of active PedA/PapA protein rely on the accessory protein PedC/PapC and ABC transporter PedD/PapD on the same operon. The excretion machines were also necessary for pediocin protein expression in heterologous hosts of E. coli, Lactobacillus lactis, and Corynebacterium glutamicum. In this study, two vectors carrying the codon sequence of the mature PapA peptide were constructed, one with and one without a His tag. Both fragments were inserted into the plasmid pHT43 and transformed into Bacillus subtilis WB800N. The strains were induced with IPTG to secrete the fused proteins PA1 and PA2. Supernatants from both recombinant strains can inhibit Listeria monocytogenes ATCC54003 directly. The fused protein possesses inhibition activity as a whole dispense with removal of the leading peptide. This is the first report of active pediocin/PapA expression without the assistance of PedCD/PapCD in heterogeneous hosts. In addition, the PA1 protein can be purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography.

[IL-7/IL-15/IL-21/IL-23 effectively promote the generation of human CD8+ central memory T cells in vitro].

Objective To compare the effect of different cytokine combinations combined with anti-CD3/CD28 beads in vitro inducing the generation of central memory T cell (Tcm). Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors. Naive CD8+ T cells were purified using immunomagnetic beads and stimulated with CD3/CD28 antibody for 48 hours. Cells were treated with different cytokine combinations as follows: Interleukin-2(IL-2), IL-7/IL-15, IL-7/IL-15/IL-21, and IL-7/IL-15/IL-21/IL-23. The automatic blood cell counting instrument was used for cell counting. 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE)was employed to test the cell proliferation and flow cytometry was adopted to measure the immune memory phenotype, apoptosis and intracellular factor expression of CD8+ T cells induced by different cytokine combinations. The expression of Bcl-2 was determined by Western blot analysis. Results Unlike other cytokine combinations, IL-7/IL-15/IL-21/IL-23 promoted the proliferation of CD8+ T cells and significantly increased the expression of CD8+CD62L+CD45RA- central memory T cell subsets. At the same time, IL-7/IL-15/IL-21/IL-23 treatment significantly reduced the secretion levels of IFN-γ, perforin, and granzyme B. The level of cell apoptosis was also significantly decreased. Conclusion The cytokines combination of IL-7/IL-15/IL-21/IL-23 can effectively induce the differentiation of naive CD8+T cells to CD8+ Tcm cells in vitro, which provides a new strategy for the generation of human CD8+ Tcm in immunotherapy.

An altered HLA-A0201-restricted MUC1 epitope that could induce more efficient anti-tumor effects against gastric cancer.

MUC1 is a tumor-associated antigen (TAA) overexpressed in many tumor types, which makes it an attractive target for cancer immunotherapy. However, this marker is a non-mutated antigen without high immunogenicity. In this study, we designed several new altered peptides by replacing amino acids in their sequences, which were derived from a low-affinity MUC1 peptide, thus bypassing immune tolerance. Compared to the wild-type (WT) peptide, the altered MUC1 peptides (MUC11081-1089L2, MUC11156-1164L2, MUC11068-1076Y1) showed higher affinity to the HLA-A0201 molecule and stronger immunogenicity. Furthermore, these altered peptides resulted in the generation of more cytotoxic T lymphocytes (CTLs) that could cross-recognize gastric cancer cells expressing WT MUC1 peptides, in an HLA-A0201-restricted manner. In addition, M1.1 (MUC1950-958), a promising antitumor peptide that has been tested in multiple tumors, was not able to induce stronger antitumor responses. Collectively, our results demonstrated that altered peptides from MUC1, as potential HLA-A0201-restricted CTL epitopes, could serve as peptide vaccines or constitute components of peptide-loaded dendritic cell vaccines for gastric cancer treatment.

Crystal structure of bacterial cyclopropane-fatty-acyl-phospholipid synthase with phospholipid.

The lipids containing cyclopropane-fatty-acid (CFA) protect bacteria from adverse conditions such as acidity, freeze-drying desiccation and exposure to pollutants. CFA is synthesized when cyclopropane-fatty-acyl-phospholipid synthase (CFA synthase, CFAS) transfers a methylene group from S-adenosylmethionine (SAM) across the cis double bonds of unsaturated fatty acyl chains. Here, we reported a 2.7-Å crystal structure of CFAS from Lactobacillus acidophilus. The enzyme is composed of N- and C-terminal domain, which belong to the sterol carrier protein and methyltransferase superfamily, respectively. A phospholipid in the substrate binding site and a bicarbonate ion (BCI) acting as a general base in the active site were discovered. To elucidate the mechanism, a docking experiment using CFAS from L. acidophilus and SAM was carried out. The analysis of this structure demonstrated that three groups, the carbons from the substrate, the BCI and the methyl of S(CHn)3 group, were close enough to form a cyclopropane ring with the help of amino acids in the active site. Therefore, the structure supports the hypothesis that CFAS from L. acidophilus catalyzes methyl transfer via a carbocation mechanism. These findings provide a structural basis to more deeply understand enzymatic cyclopropanation.