In this study, we successfully demonstrated that 454 pyrosequencing was a powerful approach for investigating the bacterial communities in the activated sludge, digestion sludge, influent, and effluent samples of a full scale wastewater treatment plant treating saline sewage. For each sample, 18,808 effective sequences were selected and utilized to do the bacterial diversity and abundance analysis. In total, 2,455, 794, 1,667, and 1,932 operational taxonomic units were obtained at 3 % distance cutoff in the activated sludge, digestion sludge, influent, and effluent samples, respectively. The corresponding most dominant classes in the four samples are Alphaproteobacteria, Thermotogae, Deltaproteobacteria, and Gammaproteobacteria. About 67 % sequences in the digestion sludge sample were found to be affiliated with the Thermotogales order. Also, these sequences were assigned into a recently proposed genus Kosmotoga by the Ribosomal Database Project classifier. In the effluent sample, we found high abundance of Mycobacterium and Vibrio, which are genera containing pathogenic bacteria. Moreover, in this study, we proposed a method to differentiate the "gene percentage" and "cell percentage" by using Ribosomal RNA Operon Copy Number Database.
High throughput sequencing of 16S rRNA gene leads us into a deeper understanding on bacterial diversity for complex environmental samples, but introduces blurring due to the relatively low taxonomic capability of short read. For wastewater treatment plant, only those functional bacterial genera categorized as nutrient remediators, bulk/foaming species, and potential pathogens are significant to biological wastewater treatment and environmental impacts. Precise taxonomic assignment of these bacteria at least at genus level is important for microbial ecological research and routine wastewater treatment monitoring. Therefore, the focus of this study was to evaluate the taxonomic precisions of different ribosomal RNA (rRNA) gene hypervariable regions generated from a mix activated sludge sample. In addition, three commonly used classification methods including RDP Classifier, BLAST-based best-hit annotation, and the lowest common ancestor annotation by MEGAN were evaluated by comparing their consistency. Under an unsupervised way, analysis of consistency among different classification methods suggests there are no hypervariable regions with good taxonomic coverage for all genera. Taxonomic assignment based on certain regions of the 16S rRNA genes, e.g. the V1&V2 regions -provide fairly consistent taxonomic assignment for a relatively wide range of genera. Hence, it is recommended to use these regions for studying functional groups in activated sludge. Moreover, the inconsistency among methods also demonstrated that a specific method might not be suitable for identification of some bacterial genera using certain 16S rRNA gene regions. As a general rule, drawing conclusions based only on one sequencing region and one classification method should be avoided due to the potential false negative results.
Schisandrin B (Sch B) is a monomer with anti-cancer and anti-inflammatory effects, which are isolated from the plant Schisandra chinensis (Turcz) Baillon. We investigated the anti-gastric cancer (GC) effects of Sch B and its underlying molecular mechanisms. The Cell Counting Kit-8 assay was used to determine the effects of Sch B on the viability of GC and normal cell lines. Hoechst/propidium iodide staining and flow cytometry were used to assess the apoptosis induction of Sch B. Western blotting was used to evaluate the effects of Sch B on downstream apoptotic proteins. The DCFH-DA fluorescent probe was used to assess the regulatory effects of Sch B on reactive oxygen species (ROS) levels and related signaling pathways in GC cells. The results showed that Sch B could regulate the phosphorylation level of mitogen-activated protein kinase (MAPK) by upregulating ROS accumulation in gastric cancer cells, and then reduce the expression of nuclear factor kappa B (NF-κB) and phosphorylated transcription 3 (p-STAT3). In addition, Sch B downregulated the cell cycle proteins cyclin-dependent kinase 2/4/6 and cyclin D1/E, and arrested cells in the G0/G1 phase. Moreover, it also inhibited cell migration, which was reversed with Nacetylcysteine pretreatment. In summary, Sch B has killing effects on GC cells by upregulating the production of intracellular ROS and regulating the MAPK/STAT3/NF-κB signaling pathway, leading to the migration arrest and apoptosis of GC cells.
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