Molecular characterization of gut microbial shift in SD rats after death for 30 days.

To observe the temporal shifts of the intestinal microbial community structure and diversity in rats for 30 days after death. Rectal swabs were collected from rats before death (BD) and on day 1, 5, 10, 15, 20, 25, and 30 after death (AD). Bacteria genomic DNA was extracted and V3 + V4 regions of 16S rRNA gene were amplified by PCR. The amplicons were sequenced at Illumina MiSeq sequencing platform. The bacterial diversity and richness showed similar results from day 1 to 5 and day 10 to 25 all presenting downtrend, while from day 5 to 10 showed slightly increased. The relative abundance of Firmicutes and Proteobacteria displayed inverse variation in day 1, 5, 10 and that was the former decreased, the latter increased. Bacteroidetes, Spirochaete and TM7 in day 15, 20, 25, 30 was significantly decline comparing with BD. Enterococcus and Proteus displayed reduced trend over day 1, 5, 10 and day 10, 15, 20, 25, respectively, while Sporosarcina showed obvious elevation during day 15, 20, 25. Accordingly, there was a certain correlation between intestinal flora succession and the time of death. The results suggested that intestinal flora may be potential indicator to aid estimation of post-mortem interval (PMI).

Microbiota Analysis of Eggshells in Different Areas and During Different Storage Time by Non-cultural Methods.

This study is to investigate and characterize the microbiota composition on eggshells from 3 different areas of Shaanxi province (Yulin, Hanzhong and Xi'an). The eggs were stored at 25 °C for 56 days and bacterial samples were collected from eggshells on day 0, 14, 28, 42 and 56. Denaturing gradient gel electrophoresis and high-throughput sequencing of 16S rRNA hypervariable region V3-V4 were performed. Alpha diversity was applied for analyzing the diversity of samples through 6 indices, including Observed-species, Chao1, Shannon, Simpson, ACE and Good's-coverage. Beta diversity was used to study the similarities or differences in the community composition of the samples. Totally, 36 phyla and 595 genera were classified by 16S rRNA gene sequencing. The composition of the microbial communities of different regions was quite different. Firmicutes (33-38% of total phyla) and Actinobacteria (36-61% of total phyla) were the most abundant phyla in all three regions. Proteobacteria were relatively more abundant (about 18% of total phyla) on eggs from Hanzhong. During storage time, the microbial communities mainly changed from Firmicutes to Actinobacteria on eggs from Yulin and Xi'an. Lactobacillus, Kocuria and Streptomyces were much higher at the genus level. Spoilage bacteria Staphylococcus, Streptococcus, Pseudomonas and Enterococcus were detected at the genus level. Campylobacter jejuni (< 1% of total bacteria), which might be related to human illness, was also detected. In conclusion, the structure, abundance, and composition of microbiota on eggshells differ among areas. The microbiota changed regularly during storage time. The current study may offer a new insight into bacterial species on eggshells.

Down-regulated in OA cartilage, SFMBT2 contributes to NF-κB-mediated ECM degradation.

The interplay between anabolic and catabolic factors regulates cartilage matrix homoeostasis. In OA, this balance is disrupted which results in cartilage degradation involving a plethora of inflammatory factors. Here, we identify a novel gene "Scm-like with four MBT domains protein 2" (SFMBT2) negatively regulated in OA cartilage. Articular cartilage from human OA patients undergoing knee arthroplasty surgery exhibited significantly decreased levels of SFMBT2 compared to the normal controls. Down-regulation of SFMBT2 by specific siRNA disturbed the metabolic homoeostasis and led to decreased expression of anabolic genes (SOX9, COL2A1) while increasing the expression of catabolic genes (MMP13 and ADAMTS4), in human chondrocytes. Finally, we revealed that SFMBT2 intervention by siRNA contributed to the catabolic phenotype of human chondrocytes mediated by NF-kB pathway.

Molecular characterization of gut microbiota in high­lipid diet­induced hyperlipidemic rats treated with simvastatin.

Hyperlipidemia is a major risk factor for cardiovascular diseases. Simvastatin (SV), a cholesterol­lowering agent, has been widely used in the treatment of hyperlipidemia. Gut microbiota is known to influence drug response, including that to statins. However, the effect of SV on the gut microbiota of hyperlipidemic rats is not fully understood. To investigate the influence of SV on gut microbiota in hyperlipidemic rats, the molecular characterization of gut microbiota and the potential functions of genes involved in the downstream metabolic pathways were analyzed using high­throughput sequencing technology and the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States approach. The results revealed that SV treatment could reduce the gut microbial diversity and drive marked remodeling of the fecal bacterial community composition. At the phylum level, the relative abundance of Firmicutes and Actinobacteria was decreased following SV therapy, whereas that of Bacteroidetes was elevated. At the genus level, the percentage of the genera Bacteroides, Sutterella and Phascolarctobacterium was significantly increased, but that of Bifidobacterium, Ruminococcaceae_NK4A214, Ruminococcaceae_UCG­009, Intestinimonas and Tyzzerella was significantly decreased. Additionally, functional prediction analysis indicated that in the SV­associated microbiota, genes involved in energy, carbohydrate, amino acid and nucleotide metabolism likely exhibited enrichment. Briefly, to the best of our knowledge, the present study was the first to establish a profound and comprehensive association between the SV­induced alterations of the gut flora and the consequent influences of downstream metabolic pathways by gut microbiota. These findings suggested that the gut microbiota may contribute to the SV hypolipidemic efficacy in the progression of hyperlipidemia, which could provide insights for the prevention and treatment of hyperlipidemia.

Molecular characterization of alterations in the intestinal microbiota of patients with grade 3 hypertension.

Hypertension has become a major risk factor for many diseases, including cardiovascular, cerebrovascular and kidney disorders. It has been reported that the composition of human gut microbiota is changed during the progression of cardiovascular and kidney diseases. The current study aimed to qualitatively and quantitatively compare the composition of gut microbiota between patients with hypertension and healthy controls. Fecal samples were collected from 50 patients diagnosed with grade 3 hypertension and 30 healthy controls. Touchdown PCR­denaturing gradient gel electrophoresis with primers specifically targeting the V3 region of 16S ribosomal RNA, and quantitative PCR, were performed to characterize all the samples. High­throughput sequencing of the V3­V4 regions was performed on 30 randomly selected samples. By comparing diversity and richness indices, the gut microbiome of the hypertensive individuals was found to be more diverse than that of the healthy controls. Among the main bacterial phlya that reside in the gut, Bacteroidetes, Firmicutes and Proteobacteria were dominant in all the samples; however the Firmicutes to Bacteroidetes ratio was variable, with a significant increase in the patients with hypertension compared with the healthy control group. In addition, at the genus level, there was an increased abundance of Prevotella_9, Megasphaera, Parasutterella and Escherichia­Shigella in patients with hypertension, while Bacteroides and Faecalibacterium were decreased. These results suggested that the human gut microbiota is altered in hypertension, and understanding the mechanism of these changes in microbial composition may open up new insights, and help to treat hypertension and other related diseases.

SFMBT2 positively regulates SOX9 and chondrocyte proliferation.

SRY­box 9 (SOX9) is the master regulator of the chondrocyte phenotype, which is essential for differentiating chondrogenic mesenchymal condensations into chondrocytes, and is involved in regulating every stage of chondrocyte differentiation. SOX9 deletion in chondrocytes at the late stages of cartilage development results in decreased chondrocyte proliferation; inhibited expression of cartilage matrix genes, including Indian hedgehog and the downstream parathyroid hormone­related protein; and premature conversion of proliferating chondrocytes into hypertrophic chondrocytes, which mineralize their matrix prematurely. Therefore, SOX9 is considered vital for the majority of phases of chondrocyte lineage, from early condensations to the differentiation of proliferating chondrocytes, leading to chondrocyte hypertrophy. It has been reported that SOX9 expression is decreased in osteoarthritis (OA) cartilage. Regeneration or repair of cartilage degradation in OA remains a challenge. Previous studies have indicated that overexpression of SOX9 can promote cartilage repair and can be used as a potential therapeutic agent at the early stages of human OA. The present study identified Scm­like with four malignant brain tumor domains 2 (SFMBT2) as a novel regulator of SOX9 expression in human chondrocytes. Our previous study revealed that SFMBT2 is negatively regulated in OA cartilage, and decreased levels of SFMBT2 contribute to the catabolic phenotype of chondrocytes. The present study detected increased expression levels of SFMBT2 in early cartilage development and during the early phases of chondrogenesis. Overexpression of SFMBT2 in C28/I2 cells upregulated SOX9 expression in a dose­dependent manner. Furthermore, SFMBT2 positively regulated C28/I2 cell proliferation and restored the decreased levels of SOX9 in chondrocytes following tumor necrosis factor­α treatment. Additional studies may reveal novel insights into the molecular mechanism involved and the potential role of SFMBT2 in cartilage repair and OA management.