Intake of compound probiotics accelerates the construction of immune function and gut microbiome in Holstein calves.

Acquired immunity is an important way to construct the intestinal immune barrier in mammals, which is almost dependent on suckling. To develop a new strategy for accelerating the construction of gut microbiome, newborn Holstein calves were continuously fed with 40 mL of compound probiotics (containing Lactobacillus plantarum T-14, Enterococcus faecium T-11, Saccharomyces cerevisiae T-209, and Bacillus licheniformis T-231) per day for 60 days. Through diarrhea rate monitoring, immune index testing, antioxidant capacity detection, and metagenome sequencing, the changes in diarrhea incidence, average daily gain, immune index, and gut microbiome of newborn calves within 60 days were investigated. Results indicated that feeding the compound probiotics reduced the average diarrhea rate of calves by 42.90%, increased the average daily gain by 43.40%, raised the antioxidant indexes of catalase, superoxide dismutase, total antioxidant capacity, and Glutathione peroxidase by 22.81%, 6.49%, 8.33%, and 13.67%, respectively, and increased the immune indexes of IgA, IgG, and IgM by 10.44%, 4.85%, and 6.12%, respectively. Moreover, metagenome sequencing data showed that feeding the compound probiotics increased the abundance of beneficial strains (e.g., Lactococcus lactis and Bacillus massionigeriensis) and decreased the abundance of some harmful strains (e.g., Escherichia sp. MOD1-EC5189 and Mycobacterium brisbane) in the gut microbiome of calves, thus contributing to accelerating the construction of healthy gut microbiome in newborn Holstein calves. IMPORTANCE: The unstable gut microbiome and incomplete intestinal function of newborn calves are important factors for the high incidence of early diarrhea. This study presents an effective strategy to improve the overall immunity and gut microbiome in calves and provides new insights into the application of compound probiotics in mammals.

Association of serum CTRP4 levels with vascular endothelial function in patients with type 2 diabetes mellitus: CTRP4 ameliorating inflammation, proliferation and migration in human umbilical vein endothelial cells.

OBJECTIVE: We investigated the correlation between serum C1q/TNF-related protein 4 (CTRP4) level and flow-mediated dilation (FMD) in patients with type 2 diabetes mellitus (T2DM), and evaluated the biological effects of CTRP4 on human umbilical vein endothelial cells (HUVECs). METHODS: A group of 165 patients diagnosed with T2DM were included in this study. Endothelial function was measured with the examination of brachial artery FMD. ELISA kit was used to measure the levels of CTRP4 in serum. HUVECs were stimulated with recombinant CTRP4 protein to assess its biological functions. RESULTS: The levels of CTRP4 showed a significant variation among three groups based on FMD tertiles (p = 0.001). What's more, FMD had a significant difference among three CTRP4 tertile groups (p < 0.05) and was negatively related to serum CTRP4 levels (r = -0.270, p < 0.001). In T2DM patients, logistic regression analysis demonstrated that CTRP4 was the primary influence factor of low FMD (p < 0.01). In receiver operating characteristic curve analysis, the area under the curve of CTRP4 for predicting low FMD was 0.66 (95%CI 0.58-0.75). When stimulated HUVECs with recombinant CTRP4 protein, we found that CTRP4 could concentration-dependently ameliorate proliferation and migration of HUVECs in wounding healing and transwell assay. This protein could also decrease the expression of IL-6 and TNF-α and promote the release of NO in HUVEC supernatants, with suppression of NF-κB and STAT3 phosphorylation. CONCLUSIONS: Serum CTRP4 concentrations were negatively associated with FMD. CTRP4 alleviated proliferation, migration and inflammation in HUVECs through the suppression of NF-κB and STAT3 signaling pathways.

Reduced Branched-Chain Amino Acid Intake Improved High-Fat Diet-Induced Nonalcoholic Fatty Pancreas Disease in Mice.

OBJECTIVE: To explore the effects of branched-chain amino acids (BCAAs) on nonalcoholic fatty pancreas disease (NAFPD) and its possible mechanism in high-fat diet (HFD) induced mice. MATERIALS AND METHODS: Pancreatic morphology and lipid infiltration was assessed by hematoxylin-eosin staining and immunohistochemistry, and lipid levels in the pancreas were determined using colorimetric enzymatic method. Relevant mechanism was investigated using western blotting and biochemical test. RESULTS: In HFD-fed mice, dietary BCAAs restriction could attenuate body weight increase, improve glucose metabolism, and reduce excessive lipid accumulation in the pancreas. Furthermore, expression of AMPKα and downstream uncoupling protein 1 were upregulated, while genes related to mammalian target of rapamycin complex 1 (mTORC1) signal pathway and lipid de novo synthesis were suppressed in HFD-BCAA restriction group compared with HFD and HFD-high BCAAs fed mice. In addition, BCAA restriction upregulated expression of BCAAs related metabolic enzymes including PPM1K and BCKDHA, and decreased the levels of BCAAs and branched chain keto acid in the pancreas. However, there was no difference in levels of lipid content in the pancreas and gene expression of AMPKα and mTORC1 between HFD and HFD-high BCAAs groups. CONCLUSIONS: Branched-chain amino acid restriction ameliorated HFD-induced NAFPD in mice by activation of AMPKα pathway and suppression of mTORC1 pathway.

Salt stress perception and metabolic regulation network analysis of a marine probiotic Meyerozyma guilliermondii GXDK6.

Introduction: Extremely salt-tolerant microorganisms play an important role in the development of functional metabolites or drug molecules. Methods: In this work, the salt stress perception and metabolic regulation network of a marine probiotic Meyerozyma guilliermondii GXDK6 were investigated using integrative omics technology. Results: Results indicated that GXDK6 could accept the salt stress signals from signal transduction proteins (e.g., phosphorelay intermediate protein YPD1), thereby contributing to regulating the differential expression of its relevant genes (e.g., CTT1, SOD) and proteins (e.g., catalase, superoxide dismutase) in response to salt stress, and increasing the salt-tolerant viability of GXDK6. Omics data also suggested that the transcription (e.g., SMD2), translation (e.g., MRPL1), and protein synthesis and processing (e.g., inner membrane protein OXA1) of upregulated RNAs may contribute to increasing the salt-tolerant survivability of GXDK6 by improving protein transport activity (e.g., Small nuclear ribonucleoprotein Sm D2), anti-apoptotic ability (e.g., 54S ribosomal protein L1), and antioxidant activity (e.g., superoxide dismutase). Moreover, up to 65.9% of the differentially expressed genes/proteins could stimulate GXDK6 to biosynthesize many salt tolerant-related metabolites (e.g., ß-alanine, D-mannose) and drug molecules (e.g., deoxyspergualin, calcitriol), and were involved in the metabolic regulation of GXDK6 under high NaCl stress. Discussion: This study provided new insights into the exploration of novel functional products and/or drugs from extremely salt-tolerant microorganisms.Graphical Abstract.

The average 30-minute post-prandial C-peptide predicted diabetic retinopathy progress: a retro-prospective study.

BACKGROUND: The conclusion between Connecting peptide (C-peptide) and diabetic chronic complication was controversial. The purpose of this study is to explore the possible association between average C-peptide with diabetic retinopathy (DR) progression in Chinese patients with type 2 diabetes. METHODS: This is a retro-prospective study. 622 patients with type 2 diabetes were included. DR was evaluated using non-mydriatic fundus photography and DR progression was defined as any deterioration of either eye. Fasting and postprandial c-peptide levels were assayed at baseline and follow-up period. Differences between continuous variables were compared using the Mann-Whitney U test; and categorical variables were analyzed by the chi-square test. Correlation between parameters and 30-minute postprandial C-peptide were determined by Spearman correlation test. The relationship between C-peptide and DR progression was evaluated by multivariable binary logistic regression. Two-tailed P-values < 0.05 were regarded as statistically significant. RESULTS: DR was present in 162 (26.0%) patients at baseline, and 26.4% of patients were found progression of DR at follow-up. Patients with progression of DR had lower average levels of 30-minute postprandial C-peptide (2.01 ng/ml vs. 2.6 ng/ml, p = 0.015) and 120-minute postprandial C-peptide (3.17 ng/ml vs. 3.92 ng/ml, p < 0.029), as well as average increment of 30-minute (0.41 ng/ml vs. 0.64 ng/ml, p = 0.015) and 120-minute postprandial C-peptide (1.48 ng/ml vs. 1.93 ng/ml, p < 0.017), than those without DR aggravation. Multivariate logistic regression analysis determined that 30-minute postprandial C-peptide and its increment were related to reduced odds ratios for DR progression (odds ratios [OR] = 0.83 and 0.74, respectively). CONCLUSION: Our results suggest that the Average 30-minute post-prandial C-peptide and increment were negatively correlated with DR progression, which further demonstrates the importance to preserve ß-cell residual function in the prevention for DR progression. TRIAL REGISTRATION: Not applicable.

Copper Tolerance Mechanism of the Novel Marine Multi-Stress Tolerant Yeast Meyerozyma guilliermondii GXDK6 as Revealed by Integrated Omics Analysis.

Various agricultural products used in food fermentation are polluted by heavy metals, especially copper, which seriously endangers human health. Methods to remove copper with microbial strategies have gained interests. A novel Meyerozyma guilliermondii GXDK6 could survive independently under high stress of copper (1400 ppm). The copper tolerance mechanism of GXDK6 was revealed by integrated omics in this work. Whole-genome analysis showed that nine genes (i.e., CCC2, CTR3, FRE2, GGT, GST, CAT, SOD2, PXMP4, and HSP82) were related to GXDK6 copper tolerance. Copper stress elevated glutathione metabolism-related gene expression, glutathione content, and glutathione sulfur transferase activity, suggesting enhanced copper conjugation and detoxification in cells. The inhibited copper uptake by Ctr3 and enhanced copper efflux by Ccc2 contributed to the decrease in intracellular copper concentration. The improved expression of antioxidant enzyme genes (PXMP4, SOD2, and CAT), accompanied by the enhanced activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase), decreased copper-induced reactive oxygen species production, protein carbonylation, lipid peroxidation, and cell death. The metabolite D-mannose against harsh stress conditions was beneficial to improving copper tolerance. This study contributed to understanding the copper tolerance mechanism of M. guilliermondii and its application in removing copper during fermentation.

Whole genome sequencing and metabolomics analyses reveal the biosynthesis of nerol in a multi-stress-tolerant Meyerozyma guilliermondii GXDK6.

BACKGROUND: Nerol (C10H18O), an acyclic monoterpene, naturally presents in plant essential oils, and is used widely in food, cosmetics and pharmaceuticals as the valuable fragrance. Meanwhile, chemical synthesis is the only strategy for large-scale production of nerol, and the disadvantages of chemical synthesis greatly limit the production and its application. These defects drive the interests of researchers shift to the production of nerol by eco-friendly methods known as biosynthesis methods. However, the main technical bottleneck restricting the biosynthesis of nerol is the lacking of corresponding natural aroma-producing microorganisms. RESULTS: In this study, a novel multi-stress-tolerant probiotics Meyerozyma guilliermondii GXDK6 with aroma-producing properties was identified by whole genome sequencing and metabolomics technology. GXDK6 showed a broad pH tolerance in the range of 2.5-10.0. The species also showed salt tolerance with up to 12% NaCl and up to 18% of KCl or MgCl2. GXDK6 exhibited heavy-metal Mn2+ tolerance of up to 5494 ppm. GXDK6 could also ferment with a total of 21 kinds of single organic matter as the carbon source, and produce abundant aromatic metabolites. Results from the gas chromatography-mass spectrometry indicated the production of 8-14 types of aromatic metabolites (isopentanol, nerol, geraniol, phenylethanol, isobutanol, etc.) when GXDK6 was fermented up to 72 h with glucose, sucrose, fructose, or xylose as the single carbon source. Among them, nerol was found to be a novel aromatic metabolite from GXDK6 fermentation, and its biosynthesis mechanism had also been further revealed. CONCLUSION: A novel aroma-producing M. guilliermondii GXDK6 was identified successfully by whole genome sequencing and metabolomics technology. GXDK6 showed high multi-stress-tolerant properties with acid-base, salty, and heavy-metal environments. The aroma-producing mechanism of nerol in GXDK6 had also been revealed. These findings indicated the aroma-producing M. guilliermondii GXDK6 with multi-stress-tolerant properties has great potential value in the fermentation industry.

Multi-Omics Analysis of Lipid Metabolism for a Marine Probiotic Meyerozyma guilliermondii GXDK6 Under High NaCl Stress.

Investigating microbial lipid regulation contributes to understanding the lipid-dependent signal transduction process of cells and helps to improve the sensitivity of microorganisms to environmental factors by interfering with lipid metabolism, thus beneficial for constructing advanced cell factories of novel molecular drugs. Integrated omics technology was used to systematically reveal the lipid metabolism mechanism of a marine Meyerozyma guilliermondii GXDK6 under high NaCl stress and test the sensitivity of GXDK6 to antibiotics when its lipid metabolism transformed. The omics data showed that when GXDK6 perceived 10% NaCl stress, the expression of AYR1 and NADPH-dependent 1-acyldihydroxyacetone phosphate reductase was inhibited, which weaken the budding and proliferation of cell membranes. This finding was further validated by decreased 64.39% of OD600 under 10% NaCl stress when compared with salt-free stress. In addition, salt stress promoted a large intracellular accumulation of glycerol, which was also verified by exogenous addition of glycerol. Moreover, NaCl stress remarkably inhibited the expression of drug target proteins (such as lanosterol 14-alpha demethylase), thereby increasing sensitivity to fluconazole. This study provided new insights into the molecular mechanism involved in the regulation of lipid metabolism in Meyerozyma guilliermondii strain and contributed to developing new methods to improve the effectiveness of killing fungi with lower antibiotics.

Optimization of monomethoxy polyethyleneglycol-modified oxalate decarboxylase by response surface methodology.

In order to improve the stability of oxalate decarboxylase (Oxdc), response surface methodology (RSM), based on a four-factor three-level Box-Behnken central composite design was used to optimize the reaction conditions of oxalate decarboxylase (Oxdc) modified with monomethoxy polyethyleneglycol (mPEG5000). Four independent variables such as the ratio of mPEG-aldehyde to Oxdc, reaction time, temperature, and reaction pH were investigated in this work. The structure of modified Oxdc was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy, the stability of the modified Oxdc was also investigated. The optimal conditions were as follows: the mole ratio of mPEG-aldehyde to Oxdc of 1:47.6, time of 13.1 h, temperature at 29.9 °C, and the reaction pH of 5.3. Under optimal conditions, experimental modified rate (MR = 73.69%) and recovery rate (RR = 67.58%) were matched well with the predicted value (MR = 75.11%) and (RR = 69.17%). SDS-PAGE and FTIR analysis showed that mPEG was covalently bound to the Oxdc. Compared with native Oxdc, the modified Oxdc (mPEG-Oxdc) showed higher thermal stability and better tolerance to trypsin or different pH treatment. This work will provide a further theoretical reference for enzyme modification and conditional optimization.

Chemical modification of oxalate decarboxylase to improve adsorption capacity.

In order to enhance the adsorption capacity of oxalate decarboxylase (Oxdc) on calcium oxalate monohydrate crystals and improve the application performance of Oxdc, chemical modification of Oxdc with ethylenediaminetetraacetic dianhydride (EDTAD) was investigated in this work. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography tandem mass spectrometry (LC/MS) analysis results demonstrated that Oxdc and EDTAD have been covalently bound, and suggested that the chemical modification occurred at the free amino of the side chain and the α-amine of the N-terminus of Oxdc. Fluorescene and circular dichroic measurement showed that the structure and conformation of Oxdc were tinily altered after modification by EDTAD. The optimum pH of EDTAD-modified Oxdc was shifted to the alkaline side about 1.5 unit and it has a higher thermostability. The analysis of kinetic parameters indicated that the EDTAD-modified Oxdc showed a higher affinity towards the substrate. Through modification the adsorption capacity of Oxdc onto CaOx monohydrate crystals was increased by 42.42%.