Nasopharyngeal carcinoma derived exosomes regulate the proliferation and migration of nasopharyngeal carcinoma cells by mediating the miR-99a-5p BAZ2A axis

Abstract Objectives Nasopharyngeal Carcinoma (NPC) is a common malignant tumor of nasopharyngeal mucosal epithelium in clinical practice. Radiotherapy and chemotherapy are the main treatment methods at present, but the therapeutic effect is still unsatisfactory. Studies have shown that exosomes and microRNAs (miRNAs) play an important role in the development of cancer. Therefore, this study aimed to investigate the effects of NPC derived exosomes on NPC and their molecular mechanisms. Methods Serum was collected from healthy subjects, Epstein-Barr Virus (EBV) infected patients and NPC patients (n = 9 group) and exosomes were extracted separately. High-throughput sequencing of exosomes was performed to screen differentially expressed miRNAs. The function of the screened miRNA was identified by treating NPC cells with exosomes. The target gene of miRNA was identified using the dual-luciferase assay. Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) was used to determine the levels of miR-99a-5p and Bromodomain Adjacent Tozinc finger domain protein 2A (BAZ2A). Cell Counting Kit-8 assay, flow cytometry, and wound healing assay were utilized to detect cell viability, cell cycle and apoptosis, and migration ability. The protein levels were evaluated by Western blot. Results MiR-99a-5p was identified as the most significant differentially expressed miRNA in exosomes (p< 0.05). The proliferation and migration of NPC cells were extremely facilitated by exosomes, accompanied by the suppressed apoptosis, upregulated BAZ2A, Monocyte Chemotactic Protein-1 (MCP1), and Vascular Endothelial Growth Factor A (VEGFA), and downregulation of Interleukin (IL)-1β and Nuclear Transcription Factor-κB (NF-κB) (p< 0.05). BAZ2A was a target gene of miR-99a-5p. Furthermore, the regulatory effect of exosomes on the proliferation, migration, and apoptosis was significantly abolished by overexpression of miR-99a-5p or downregulation of BAZ2A (p< 0.05). Conclusion NPC derived exosomes facilitated the proliferation and migration of NPC through regulating the miR-99a-5p/BAZ2A axis.

Association analysis of various obesity-related indices and vitamin D deficiency in middle-aged and elderly population in Lanzhou / 中华临床营养杂志

Objective:To analyze and compare the association between different obesity-related indices and vitamin D deficiency in middle-aged and elderly population dwelled in Lanzhou city.Methods:From May, 2011 to September, 2012, middle-aged and elderly individuals with complete baseline data were included via randomly cluster sampling from 3 communities in Lanzhou. The subjects were divided into 4 subgroups by vitamin D levels and various obesity-related indices were compared across subgroups with the same gender. The relationship between the obesity-related indices and the severity of vitamin D deficiency was analyzed using Spearman correlation analysis, and the effects of different obesity-related indices on the severity of vitamin D deficiency was analyzed using multivariate logistic regression analysis.Results:A total of 9 437 residents were included. The overall prevalence of vitamin D deficiency was 97.7%. Compared with the group with lower vitamin D level, participants in the group with higher vitamin D level showed evidently lower body mass index (BMI), waist circumference (WC), lipid accumulation product (LAP), visceral adiposity index (VAI) and triglyceride/ high density lipoprotein cholesterol (TG/HDL-C) ratio in the total population and females, while only WC, LAP, VAI and TG/HDL-C in the males (all P<0.05). Spearman correlation analysis showed that BMI, WC, LAP, VAI and TG/HDL-C were positively correlated with the severity of vitamin D deficiency in the total population and the females, while only LAP, VAI and TG/HDL-C in the males (all P<0.05) . Multivariate logistic regression analysis showed that higher levels of these obesity related indices were correlated with more severe vitamin D deficiency in the total population and the females, while only higher LAP, VAI and TG/HDL-C in the males (all P<0.05). The effects of higher LAP was the most prominant in the total population ,the females and the males. Conclusion:Various obesity phenotypes are closely related to vitamin D deficiency in middle-aged and elderly women, while only visceral obesity and abnormal lipid metabolism are related to vitamin D deficiency in middle-aged and elderly men, with LAP being the most important influencing factor.

Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp1 protein is 520 amino acids long and is comparable to the Ytp1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP (green fluorescent protein) fusion expression results indicate that Mtp1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Deltamtp1 mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1x10(6) ml(-1), the percentages of conidium germination and appressorium formation were (97.98+/-0.67)% and (97.88+/-0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 microg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.

Promoter trapping in Magnaporthe grisea / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Application of promoter trapping based on transformation in Magnaporthe grisea is reported in this paper. Two promoter-trapping vectors, designated as pCBGFP and pEGFPHPH, were constructed and transformed into protoplasts of M. grisea. A library of 1,077 transformants resistant to hygromycin B was generated. Of which, 448 transformants were found to express eGFP gene in different structures of M. grisea. Three transformants grew slowly, 5 transformants decreased in conidiation and 7 transformants reduced in pathogenicity greatly among these 448 transformants. Eleven transformants were checked by genomic southern blot randomly, and 9 of which were single-copy insertions. The promoter trapping technique has been applied successfully in M. grisea and can be used as a tool for functional genomic analysis.

Response of superoxide dismutase, catalase, and ATPase activity in bacteria exposed to acetamiprid / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate how acetamiprid, a new insecticide, affects the activity of superoxide dismutase (SOD), catalase (CAT), and ATPase and the SOD isozyme patterns in two G bacteria, E. coli K12 and Pse.FH2, and one G+ bacterum, B. subtilis.</p><p><b>METHODS</b>The SOD, CAT, and ATPase specific activities of cell lysates were determined spectrophotometrically at 550 nm, 240 nm, and 660 nm, respectively, with kits A001, A016, and A007. SOD isozyme patterns were detected by native PAGE analysis.</p><p><b>RESULTS</b>SOD and CAT activities in the tested bacteria increased significantly in a concentration-dependent manner after different concentrations of acetamiprid were applied. The activity of SOD in B. subtilis and Pse.FH2 was stimulated and reached the highest level after treatment with 100 mg/L acetamiprid for 0.5 h. For Pse.FH2, there was another stimulation of SOD activity after acetamiprid application for about 8.0 h and the second stimulation was stronger than the first. The stimulation by acetamiprid showed a relative lag for E. coli K12. Acetamiprid seemed to exhibit a similar effect on CAT activity of the two G bacteria and had an evident influence on ATPase activity in the three bacteria within a relatively short period. Only one SOD isozyme was detectable in Pse.FH2 and B. subtilis, while different isozyme compositions in E. coli could be detected by native PAGE analysis.</p><p><b>CONCLUSION</b>Acetamiprid causes a certain oxidative stress on the three bacteria which may not only elevate SOD and CAT activities but also generate new SOD isozymes to antagonize oxidative stress. However, this oxidative stress lasts for a relatively short time and does not cause a long-term damage.</p>

Influences of quinclorac on culturable microorganisms and soil respiration in flooded paddy soil / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the potential effects of herbicide quinclorac (3,7-dichloro-8-quinoline-carboxylic) on the culturable microorganisms in flooded paddy soil.</p><p><b>METHODS</b>Total soil aerobic bacteria, actinomycetes and fungi were counted by a 10-fold serial dilution plate technique. Numbers of anaerobic fermentative bacteria (AFB), denitrifying bacteria (DNB) and hydrogen-producing acetogenic bacteria (HPAB) were numerated by three-tube anaerobic most-probable-number (MPN) methods with anaerobic liquid enrichment media. The number of methanogenic bacteria (MB) and nitrogen-fixing bacteria (NFB) was determined by the rolling tube method in triplicate. Soil respiration was monitored by a 102G-type gas chromatography with a stainless steel column filled with GDX-104 and a thermal conductivity detector.</p><p><b>RESULTS</b>Quinclorac concentration was an important factor affecting the populations of various culturable microorganisms. There were some significant differences in the aerobic heterotrophic bacteria. AFB and DNB between soils were supplemented with quinclorac and non-quinclorac at the early stage of incubation, but none of them was persistent. The number of fungi and DNB was increased in soil samples treated by lower than 1.33 micro x g(-1) dried soil, while the CFU of fungi and HPAB was inhibited in soil samples treated by higher than 1.33 microg x g(-1) dried soil. The population of actinomycete declined in negative proportion to the concentrations of quinclorac applied after 4 days. However, application of quinclorac greatly stimulated the growth of AFB and NFB. MB was more sensitive to quinclorac than the others, and the three soil samples with concentrations higher than 1 microg x g(-1) dried soil declined significantly to less than 40% of that in the control, but the number of samples with lower concentrations of quinclorac was nearly equal to that in the control at the end of experiments.</p><p><b>CONCLUSION</b>Quinclorac is safe to the soil microorganisms when applied at normal concentrations (0.67 microg x g(-1)).</p>

A METHOD FOR ISOLATION AND PURIFICATION OF THERMOPHILIC CELLULOLYTIC ANAEROBES / 微生物学通报

A new method based on adherence of cellulolytic bacteria to insoluble cellulose for isolation and purification of thermophilic cellulolytic anaerobes was reported, in which Hungate anaerobic operating techniques were used to roll tubes with insoluble cellulose powder as substrate.