Effects of Stool Sample Preservation Methods on Gut Microbiota Biodiversity: New Original Data and Systematic Review with Meta-Analysis.

Here, we aimed to compare the effects of different preservation methods on outcomes of fecal microbiota. We evaluated the effects of different preservation methods using stool sample preservation experiments for up to 1 year. The stool samples from feces of healthy volunteers were grouped based on whether absolute ethanol was added and whether they were hypothermically preserved. Besides, we performed a systematic review to combine current fecal microbiota preservation evidence. We found that Proteobacteria changed significantly and Veillonellaceae decreased significantly in the 12th month in the room temperature + absolute ethanol group. The four cryopreservation groups have more similarities with fresh sample in the 12 months; however, different cryopreservation methods have different effects on several phyla, families, and genera. A systematic review showed that the Shannon diversity and Simpson index of samples stored in RNAlater for 1 month were not statistically significant compared with those stored immediately at -80°C (P = 0.220 and P = 0.123, respectively). The -80°C refrigerator and liquid nitrogen cryopreservation with 10% glycerine can both maintain stable microbiota of stool samples for long-term preservation. The addition of absolute ethanol to cryopreserved samples had no significant difference in the effect of preserving fecal microbial characteristics. Our study provides empirical insights into preservation details for future studies of the long-term preservation of fecal microbiota. Systematic review and meta-analysis found that the gut microbiota structure, composition, and diversity of samples preserved by storage methods, such as preservation solution, are relatively stable, which were suitable for short-term storage at room temperature. IMPORTANCE The study of gut bacteria has become increasingly popular, and fecal sample preservation methods and times need to be standardized. Here, we detail a 12-month study of fecal sample preservation, and our study provides an empirical reference about experimental details for long-term high-quality storage of fecal samples in the field of gut microbiology research. The results showed that the combination of -80°C/liquid nitrogen deep cryopreservation and 10% glycerol was the most effective method for the preservation of stool samples, which is suitable for long-term storage for at least 12 months. The addition of anhydrous ethanol to the deep cryopreserved samples did not make a significant difference in the preservation of fecal microbiological characteristics. Combined with the results of systematic reviews and meta-analyses, we believe that, when researchers preserve fecal specimens, it is essential to select the proper preservation method and time period in accordance with the goal of the study.

Dexamethasone impairs the differentiation and maturation of murine dendritic cells by Toll-like receptor 4-nuclear factor-kappaB pathway.

BACKGROUND: Recent studies have demonstrated that dexamethasone (DEX) interferes with immune responses by targeting key functions of dendritic cells (DCs) at the earliest stage. However, the cellular and molecular mechanisms are still incompletely understood. This study aimed to explore the possible mechanisms by investigating the roles of DEX on differentiation, maturation & function of murine DCs and the effects of DEX on DCs via Toll-like receptor 4 (TLR4)-nuclear factor (NF)-kappaB mediated signal pathway. METHODS: Immature DCs (imDCs) were cultured from murine bone marrow (BM) cells. We added DEX into culture medium at different time. The expression of CD11c, CD86 and I-A(b) (mouse MHC class II molecule) was determined by flow cytometry. We determined the expression of NF-kappaB and its inhibitory protein I-kappaBalpha by electrophoretic mobility shift assay (EMSA) and Western blotting, respectively. The productions of interleukin (IL)-12p70 and IL-10 in cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: DEX impaired differentiation of DCs from murine bone marrow progenitors, and inhibited lipopolysaccharide (LPS) induced maturation of DCs. DEX significantly inhibited NF-kappaB expression of normal DCs, the higher the DEX concentration or the longer the DEX treatment time, the more obvious the effect. However, DEX had little effect on LPS-induced NF-kappaB activation, and partially impaired LPS-induced I-kappaBalpha degradation. DEX significantly decreased LPS induced IL-12p70 production by DCs. Interestingly, our results showed a synergistic effect between DEX and LPS on the production of IL-10 by DCs. CONCLUSIONS: DEX inhibits the differentiation and maturation of murine DCs involved in TLR4-I-kappaB-NF-kappaB pathway, and also indirectly impairs Th1 development and interferes with the Th1-Th2 balance through IL-12 and/or IL-10 secretion by DCs.

[Prokaryotic expression of S2 extracellular domain of SARS coronavirus spike protein and its fusion with Hela cell membrane].

OBJECTIVE: To construct the expression plasmid of S2 extracellular domain (S2ED) of SARS-coronavirus (SARS- Cov) spike protein (S protein) and enhanced green fluorescent protein (EGFP) to obtain the fusion protein expressed in prokaryotic cells. METHODS: S2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fractions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion of S2ED with Hela cell membranes was observed with fluorescent microscope. RESULTS: The pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 (DE3). When incubated with Hela cells, the purified protein could not internalize through membrane fusion. CONCLUSIONS: The expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.

[The analysis in the expression of Toll-like receptor 4 mRNA and the effect of dexamethasone on mice dendritic cells by real-time PCR].

AIM: To establish a SYBR Green I quantitative real-time PCR method for detecting the expression of the TLR4 mRNA of mice and to monitor the dynamics for TLR4 mRNA level of mice bone marrow-derived dendritic cells (DC) in the process of the maturation and the effect of dexamethasone (DEX) on TLR4 expression. METHODS: (1) DC from mice bone marrow were induced by cytokines and separated by magnetic beads. (2) The combined plasmid pUCm-T/TLR4 and pUCm-T/beta-actin for the standard materials in the real-time PCR was reconstructed. (3) The dynamics for the express TLR4 mRNA of DC cultivated in vitro for 4, 6, 8, 10, 12 days was detected respectively. (4) The TLR4 mRNA expression between DC treated with or without DEX was detected and compared. RESULTS: (1) The DC with the purity over 90% were fully separated in success. (2) A SYBR Green I quantitative real-time PCR method for analyzing the TLR4 mRNA expression level of the mice was successful established. (3) TLR4 mRNA level was stable early during the culture of DC and then rise obviously at last. CONCLUSION: There exists a close relationship between the level of TLR4 mRNA and the period of maturation of DC and the expression of TLR4 could be increased by DEX.