Comparison of microbial diversity determined with the same variable tag sequence extracted from two different PCR amplicons.

BACKGROUND: Deep sequencing of the variable region of 16S rRNA genes has become the predominant tool for studying microbial ecology. As sequencing datasets have accumulated, meta-analysis of sequences obtained with different variable 16S rRNA gene targets and by different sequencing methods has become an intriguing prospect that remains to be evaluated experimentally. RESULTS: We amplified a group of fecal samples using both V4F-V6R and V6F-V6R primer sets, excised the same V6 fragment from the two sets of Illumina sequencing data, and compared the resulting data in terms of the α-diversity, ß-diversity, and community structure. Principal component analysis (PCA) comparing the microbial community structures of different datasets, including those with simulated sequencing errors, was very reliable. Procrustes analysis showed a high degree of concordance between the different datasets for both abundance-weighted and binary Jaccard distances (P < 0.05), and a meta-analysis of individual datasets resulted in similar conclusions. The Shannon's diversity index was consistent as well, with comparable values obtained for the different datasets and for the meta-analysis of different datasets. In contrast, richness estimators (OTU and Chao) varied significantly, and the meta-analysis of richness estimators was also biased. The community structures of the two datasets were obviously different and led to significant changes in the biomarkers identified by the LEfSe statistical tool. CONCLUSIONS: Our results suggest that beta-diversity analysis and Shannon's diversity are relatively reliable for meta-analysis, while community structures and biomarkers are less consistent. These results should be useful for future meta-analyses of microbiomes from different data sources.

Illumina sequencing of 16S rRNA tag revealed spatial variations of bacterial communities in a mangrove wetland.

The microbial community plays an essential role in the high productivity in mangrove wetlands. A proper understanding of the spatial variations of microbial communities will provide clues about the underline mechanisms that structure microbial groups and the isolation of bacterial strains of interest. In the present study, the diversity and composition of the bacterial community in sediments collected from four locations, namely mudflat, edge, bulk, and rhizosphere, within the Mai Po Ramsar Wetland in Hong Kong, SAR, China were compared using the barcoded Illumina paired-end sequencing technique. Rarefaction results showed that the bulk sediment inside the mature mangrove forest had the highest bacterial α-diversity, while the mudflat sediment without vegetation had the lowest. The comparison of ß-diversity using principal component analysis and principal coordinate analysis with UniFrac metrics both showed that the spatial effects on bacterial communities were significant. All sediment samples could be clustered into two major groups, inner (bulk and rhizosphere sediments collected inside the mangrove forest) and outer mangrove sediments (the sediments collected at the mudflat and the edge of the mangrove forest). With the linear discriminate analysis scores larger than 3, four phyla, namely Actinobacteria, Acidobacteria, Nitrospirae, and Verrucomicrobia, were enriched in the nutrient-rich inner mangrove sediments, while abundances of Proteobacteria and Deferribacterias were higher in outer mangrove sediments. The rhizosphere effect of mangrove plants was also significant, which had a lower α-diversity, a higher amount of Nitrospirae, and a lower abundance of Proteobacteria than the bulk sediment nearby.

[Study on mitochondrial function of ND1 gene with 3316 G-->A mutation in human diabetes].

OBJECTIVE: To explore the effects of ND1 gene with 3316 G-->A mutation upon mitochondrial function and elucidate its role in the development of human diabetes. METHODS: The eukaryotic expression vector pcDNA3.1B and E. coli DH5alpha were used to construct the recombinant plasmid (pcDNA3.1B-ND1) of wild-type and 3316 G-->A mutant type ND1 gene. And the recombinant plasmids were analyzed by restriction enzyme digestion and DNA sequencing. Two siRNAs (mtND11 and mtND12) specific for human mtNDA ND1 gene were designed, synthesized and then transfected into Hela cells for silencing endogenous mtDNA ND1 gene. The gene-silencing effects were analyzed by RT-PCR, SDS-PAGE and MitoCapture mitochondrial apoptosis detection kit. Later the two types recombinant plasmids were transfected into Hela cells in which endogenous mtDNA ND1 gene was silenced. After 48 h culture, the Hela cells were collected for determination of mitochondrial proteins by SDS-PAGE. RESULTS: Both mtND11 and mtND12 could decrease mtDNA ND1 expression and mtND11 caused a smaller decrease. The expression of mitochondrial protein in 3316 G-->A mutant type recombinant decreased. CONCLUSION: The normal expression of mitochondrial ND1 gene maintain the function of mitochondrial respiratory chain and cell proliferation. The 3316 G-->A mutation in mitochondrial ND1 gene might be related to the down-regulated expression of mitochondrial protein and the diabetes mellitus pathogenesis.

[Analysis of features for suicide attempters at emergency departments in general hospitals].

OBJECTIVE: To investigate the prevalence of various mental disorders of suicide attempters and analyze the clinical features of suicide attempters with mental disorders. METHODS: The investigators randomly selected four Class III general hospitals from different parts of Shenyang and collected 239 suicide attempters over 15 years old at emergency room. When the conditions of suicide attempters improved after rescue, the investigators studied by suicide general table, self-injury questionnaire, Hamilton Depression Rating Scale for Depression-24 and Structure Clinical Interview for DSM-IV Axis I Disorders-Patient Edition. RESULTS: The overall prevalence of mental disorders in attempted suicides was 69.46% while self-injury, one of the highest prevalent mood disorders, accounted for 48.12%. During the year before suicide, only 7.23% of the suicide attempters with mental disorders consulted a psychologist or psychiatrist, and took anti-psychosis drugs, anti-depression or anti-anxiety drugs. Suicide attempters with mental disorders often committed suicide less impulsively. And their purpose was to relieve themselves and reduce the burden of others so that the rate of self-help was lower after their injuries occurred; suicide attempters with mental disorders had more obvious symptoms. And their degrees of depression were greater (P < 0.05) than ones without mental disorders. CONCLUSION: The prevalence of mental disorders at emergency rooms in general hospitals is high in suicide attempts, but the pre-suicide consulting rate remains so low. The depressive components of mental disorders are most directly related to suicidal behaviors of suicide attempters.

Comparison of direct boiling method with commercial kits for extracting fecal microbiome DNA by Illumina sequencing of 16S rRNA tags.

Low cost and high throughput capacity are major advantages of using next generation sequencing (NGS) techniques to determine metagenomic 16S rRNA tag sequences. These methods have significantly changed our view of microorganisms in the fields of human health and environmental science. However, DNA extraction using commercial kits has shortcomings of high cost and time constraint. In the present study, we evaluated the determination of fecal microbiomes using a direct boiling method compared with 5 different commercial extraction methods, e.g., Qiagen and MO BIO kits. Principal coordinate analysis (PCoA) using UniFrac distances and clustering showed that direct boiling of a wide range of feces concentrations gave a similar pattern of bacterial communities as those obtained from most of the commercial kits, with the exception of the MO BIO method. Fecal concentration by boiling method affected the estimation of α-diversity indices, otherwise results were generally comparable between boiling and commercial methods. The operational taxonomic units (OTUs) determined through direct boiling showed highly consistent frequencies with those determined through most of the commercial methods. Even those for the MO BIO kit were also obtained by the direct boiling method with high confidence. The present study suggested that direct boiling could be used to determine the fecal microbiome and using this method would significantly reduce the cost and improve the efficiency of the sample preparation for studying gut microbiome diversity.

Diverse vaginal microbiomes in reproductive-age women with vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC) is one of the most prevalent vaginal infectious diseases, and there are controversial reports regarding the diversity of the associated vaginal microbiota. We determined the vaginal microbial community in patients with VVC, bacterial vaginosis (BV), and mixed infection of VVC and BV using Illumina sequencing of 16S rRNA tags. Our results revealed for the first time the highly variable patterns of the vaginal microbiome from VVC patients. In general, the alpha-diversity results of species richness and evenness showed the following order: normal control < VVC only < mixed BV and VVC infection < BV only. The beta-diversity comparison of community structures also showed an intermediate composition of VVC between the control and BV samples. A detailed comparison showed that, although the control and BV communities had typical patterns, the vaginal microbiota of VVC is complex. The mixed BV and VVC infection group showed a unique pattern, with a relatively higher abundance of Lactobacillus than the BV group and higher abundance of Prevotella, Gardnerella, and Atopobium than the normal control. In contrast, the VVC-only group could not be described by any single profile, ranging from a community structure similar to the normal control (predominated with Lactobacillus) to BV-like community structures (abundant with Gardnerella and Atopobium). Treatment of VVC resulted in inconsistent changes of the vaginal microbiota, with four BV/VVC samples recovering to a higher Lactobacillus level, whereas many VVC-only patients did not. These results will be useful for future studies on the role of vaginal microbiota in VVC and related infectious diseases.