Characteristics of Gut Microbiota in Female Patients with Diabetic Microvascular Complications.

Objective: To explore the characteristics and analyze the gut microbiota in female patients with diabetic microvascular complications (DMC). Methods: Thirty-seven female patients with type 2 diabetes mellitus (T2DM) were included in the study. These patients were divided into DM group with microvascular complications (T2DM-MC, n = 17) and no microvascular complications group (T2DM-0, n = 20). Patients in the microvascular group presented with the involvement of at least one of the following: kidney, retinal, or peripheral nerves. Using real-time fluorescence quantitative polymerase chain reaction, fecal samples from the two groups were tested for Bacteroides, Prevotella, Bifidobacterium spp, Lactobacillus, Faecalibacterium prausnitzii, Enterococcus spp, Eubacterium rectale, Veillonellaceae, Clostridium leptum, and Roseburia inulinivorans. Levels of fasting and 2 h postprandial blood glucose, glycosylated hemoglobin (HbA1c), lipids, and creatinine were determined to explore the correlation between gut microbiota and blood sugar. Mann-Whitney U test was used to analyze the differences between the two groups. Spearman correlation analysis was used to determine the correlation between gut microbiota and blood glucose. Multifactor logistic regression was used to analyze the risk factors for DMC. Results: The HbA1c levels in the T2DM-MC group were higher than those in the T2DM-0 group. The abundances of Bacteroides and Enterococcus spp in the T2DM-MC group were higher than that in the T2DM-0 group. The abundances of Bacteroides and Enterococcus spp in the T2DM-MC group were lower than that in the T2DM-0 group. Spearman's correlation analysis showed that Bacteroides, Prevotella, Lactobacillus, C. leptum, and R. inulinivorans were related to the levels of HbA1c or blood glucose (p < 0.05). Logistic regression analysis showed that after adjusting for confounding factors such as age, body mass index, family history, HbA1c, hypertension, dyslipidemia, and creatinine, Bacteroides remained an independent risk factor in female patients with DMC. Conclusion: Gut microbiota is related to blood glucose levels. Female patients with DMC experience gut microbiota disorders. The abundances of Bacteroidesare related to DMC, and the abundances of intestinal flora may affect the blood sugar levels of the body.

[Construction of the mutants of rice nonspecific lipid transfer protein and expression comparison in two kinds of thioredoxin fusion expression vectors].

Five structural important residues of rice nonspecific lipid transfer protein LTP110 were mutated by site-directed mutagenesis. Sequence results showed that they were all mutated successfully. After trying various E. coli expression systems, thioredoxin fusion expression system was found to be a proper system to express wild type and mutant LTP110. cDNA sequences encoding wild type LTP110 and the mutants Y17A, P72L, R46A, D43A, C50A were cloned into two kinds of thioredoxin fusion expression vectors. The expression results were compared. In pTrxFus/GI724 expression system, wild type LTP110 and the mutants Y17A, P72L, R46A could be expressed at low level while D43A and C50A could not be expressed normally; in pET32a(+)/BL21 (DE3) trxB- expression system, wild type LTP110 and all mutant proteins could be expressed very well and the levels were higher than that in pTrxFus/GI724 system. LTP110 fusion protein expressed in pET32a(+) vector was purified and its activity was checked by fluorescence labeled fatty acid. Results indicated that the recombinant LTP110 fusion protein has lipid binding activity. This work provides good basis for the further study.

[Expression, purification and function of rice nonspecific lipid transfer protein].

Plant nonspecific lipid transfer protein(nsLTP) is a class of protein which has in vitro lipid transferring activity between biomembranes. In order to study the antimicrobial function of rice nonspecific lipid transfer protein, a gene LTP110 encoding rice nsLTP was cloned into ThioFusion expression vector pET32a (+) and expressed in host strain Bl21(DE3)trxB-. After induction by IPTG at 30 degrees C for 5 h, the fusion protein thio-LTP110 was in large amount produced. The expressed protein was purified by Ni2(+)-chelating Sepharose fast flow column, then digested by enterokinase. By passing through nickel affinity column again, the cleavage product, LTP110, was obtained. CD spectrum scanning from 185 nm to 250 nm showed that the recombinant protein LTP110 had similar secondary structure with the nsLTP purified from rice etiolated seedlings. Activity determination by fluorescent lipid P-96 showed that it had lipid binding activity. Microbial inhibition test results revealed that LTP110 deterred germination of the spores of rice pathogen P. oryzae, showing it might be involved in plant microbial resistance function. Therefore, it has the potential to be used in plant transgene engineering to improve plant resistance.