Structural and Functional Microbial Ecology of Denitrifying Bacteria Using Different Organic Carbon Sources

This dissertation research represents one of the first attempts to investigate the structural and functional microbial ecology of methanol, ethanol and glycerol fostered denitrification. The overarching goal of this research was to elucidate the link between the structure and function of denitrifying microbial populations grown on different carbon sources. Specific objectives were to: 1) diagnose bacteria specifically assimilating methanol and ethanol and determine denitrification kinetics induced by the two carbon sources; 2) investigate factors leading to nitrous oxide (N2O) and nitric oxide (NO) emissions from methanol and ethanol feeding denitrification reactors; 3) characterize glycerol assimilating populations that perform suspended- and biofilm-growth denitrification; 4) examine the potential of using alcohol dehydrogenase gene as a biomarker for methanol and glycerol induced denitrification activity; 5) evaluate the impact of different carbon sources (methanol and ethanol) on the transcript and proteome of a model facultative methylotroph, Methyloversatilis universalis FAM5. First, the technique of DNA stable isotope probing and quantitative polymerase chain reaction were adapted to diagnose and track methylotrophic denitrifying bacteria in activated sludge. Methanol assimilating populations in the methanol fed denitrifying sequencing batch reactor (SBR) were Methyloversatilis spp. and Hyphomicrobium spp. related species. Upon switching to ethanol, only Methyloversatilis spp. was sustained pointing to their metabolic versatility at least with respect to carbon assimilation. This study represents one of the first investigations of the existence and utilization of facultative methylotrophy in activated sludge. Second, the potential of N2O and NO emitted from methanol and ethanol fed denitrifying SBRs was studied during different transient shocks, including organic carbon limitation, nitrite inhibition and oxygen inhibition. Organic carbon limitation and exposure to nitrite did not result in statistically significant emissions over the control. However, statistically higher N2O emissions were observed during exposure to oxygen on the ethanol fed biomass and coincided with sustained denitrification activity in the presence of oxygen. Therefore, the results suggest that the dosage of ethanol to anoxic zones needs to be strictly controlled to minimize N2O emissions in the downstream aerobic zones. Third, the structure-function analysis of denitrification was extended to glycerol (the main component of biodiesel waste and a potential replacement for methanol) in both suspended and biofilm phases of a hybrid integrated fixed-film bioreactor. During long-term operation on glycerol, the biofilm community had a higher phylogenetic diversity (dominated by Comamonas spp., Bradyrhizobium spp., and Tessaracoccus spp.), and lower denitrification kinetics than the suspended community (dominated by Comamonas spp. and Diaphorobacter spp.). Distinct identities of glycerol assimilating populations due to the different substrate availability in the suspended and biofilm phases were shown for the first time. Fourth, carbon source-specific biomarkers of denitrification activity based on gene expression were developed. Based on short-term batch denitrification activity assays as well as long-term bioreactor operation, the applicability of alcohol dehydrogenase gene expression as quantitative descriptors of denitrification activity on methanol and glycerol in mixed cultures was demonstrated. Finally, Methyloversatilis universalis was selected as model organism to study the effects of varying electron donors (from methanol to ethanol) on its gene and protein expression profiles. Genes encoding essential enzymes that involve carbon oxidation, C1 assimilation and central metabolism were found to be differentially expressed during growth on methanol and ethanol. Several physiological and metabolic responses by M. universalis pointed to a well-defined strategy to overcome carbon limitation for surviving in engineered or natural denitrifying environments. In sum, the structural and functional ecology and the metabolism of heterotrophic denitrification on methanol, ethanol and glycerol as applicable to engineered denitrifying bioreactors was investigated in detail. From an engineering perspective, the knowledge gained can help to guide the selection and application of potential organic carbon sources for denitrification in biological nitrogen removal systems. It is expected that such judicious selection can also eventually result in better design, operation and control of engineered nitrogen removal processes and thus help attain ever more stringent nitrogen standards

Molecular Mechanisms of Metformin for Diabetes and Cancer Treatment

Metformin has been the first-line drug treatment for hyperglycemia and insulin resistance for over 50 years. However, the molecular basis of its therapeutic role remained incompletely understood. Recent advances demonstrate that metformin could exert its glucose-lowering effect by multiple mechanisms, including activation of 5′-AMP-activated protein kinase, decreasing production of cyclic AMP, suppressing mitochondrial complex I of the electron transport chain, targeting glycerophosphate dehydrogenase, and altering the gut microbiome. In addition, epidemiological and clinical observation studies suggest that metformin reduced cancer risk in patients with type 2 diabetes and improved survival outcome of human cancers. Experimental studies have shown that this drug can inhibit cancer cell viability, growth, and proliferation through inhibiting mTORC1 signaling and mitochondrial complex I, suggesting that it may be a promising drug candidate for malignancy. Here, we summarize recent progress in studies of metformin in type 2 diabetes and tumorigenesis, which provides novel insight on the therapeutic development of human diseases

Novel genetic reassortants in H9N2 influenza A viruses and their diverse pathogenicity to mice

<p>Abstract</p> <p>Background</p> <p>H9N2 influenza A viruses have undergone extensive reassortments in different host species, and could lead to the epidemics or pandemics with the potential emergence of novel viruses.</p> <p>Methods</p> <p>To understand the genetic and pathogenic features of early and current circulating H9N2 viruses, 15 representative H9N2 viruses isolated from diseased chickens in northern China between 1998 and 2010 were characterized and compared with all Chinese H9N2 viruses available in the NCBI database. Then, the representative viruses of different genotypes were selected to study the pathogenicity in mice with the aim to investigate the adaptation and the potential pathogenicity of the novel H9N2 reassortants to mammals.</p> <p>Results</p> <p>Our results demonstrated that most of the 15 isolates were reassortants and generated four novel genotypes (B62-B65), which incorporated the gene segments from Eurasian H9N2 lineage, North American H9N2 branch, and H5N1 viruses. It was noteworthy that the newly identified genotype B65 has been prevalent in China since 2007, and more importantly, different H9N2 influenza viruses displayed a diverse pathogenicity to mice. The isolates of the 2008-2010 epidemic (genotypes B55 and B65) were lowly infectious, while two representative viruses of genotypes B0 and G2 isolated from the late 1990s were highly pathogenic to mice. In addition, Ck/SD/LY-1/08 (genotype 63, containing H5N1-like NP and PA genes) was able to replicate well in mouse lungs with high virus titers but caused mild clinical signs.</p> <p>Conclusion</p> <p>Several lines of evidence indicated that the H9N2 influenza viruses constantly change their genetics and pathogenicity. Thus, the genetic evolution of H9N2 viruses and their pathogenicity to mammals should be closely monitored to prevent the emergence of novel pandemic viruses.</p

The role of brevican in glioma: promoting tumor cell motility in vitro and in vivo

Background: Malignant glioma is a common primary tumor of the central nervous system. Brevican, an abundant extracellular matrix component in the adult brain, plays a critical role in the process of glioma. The mechanisms for the highly invasive behavior of gliomas are still poorly understood. The aim of this study was to examine whether brevican is a predictor of glioma and its roles in glioma cell motility. Methods: In this study, immunohistochemistry staining for brevican expression was performed in malignant gliomas and benign controls. We also explored the effects of brevican on cell adhesion and migration in brevican-overexpressed cells. Knockdown of brevican expression was achieved by stable transfection of U251 cells transduced with a construct encoding a short hairpin DNA directed against the brevican gene, which correspondingly, down-regulated the proliferation, invasion and spread of brevican-expressing cells. Moreover, the role of brevican in the growth and progression of glioma was demonstrated by in vivo studies. Results: Our results provide evidence for the molecular and cellular mechanisms that may underlie the motility-promoting role of brevican in the progression of glioma. The role of brevican as a target for immunotherapy might be taken into consideration in future studies. Conclusions: This study suggests that expression of brevican is associated with glioma cell adhesion, motility and tumor growth, and also is related to glioma cell differentiation, therefore it may be a marker for malignance degree of gliom

Evaluating two concepts for the modelling of intermediates accumulation during biological denitrification in wastewater treatment

The accumulation of the denitrification intermediates in wastewater treatment systems is highly undesirable, since both nitrite and nitric oxide (NO) are known to be toxic to bacteria, and nitrous oxide (N2O) is a potent greenhouse gas and an ozone depleting substance. To date, two distinct concepts for the modelling of denitrification have been proposed, which are represented by the Activated Sludge Model for Nitrogen (ASMN) and the Activated Sludge Model with Indirect Coupling of Electrons (ASM-ICE), respectively. The two models are fundamentally different in describing the electron allocation among different steps of denitrification. In this study, the two models were examined and compared in their ability to predict the accumulation of denitrification intermediates reported in four different experimental datasets in literature. The N-oxide accumulation predicted by the ASM-ICE model was in good agreement with values measured in all four cases, while the ASMN model was only able to reproduce one of the four cases. The better performance of the ASM-ICE model is due to that it adopts an “indirect coupling” modelling concept through electron carriers to link the carbon oxidation and the nitrogen reduction processes, which describes the electron competition well. The ASMN model, on the other hand, is inherently limited by its structural deficiency in assuming that carbon oxidation is always able to meet the electron demand by all denitrification steps, therefore discounting electron competition among these steps. ASM-ICE therefore offers a better tool for predicting and understanding intermediates accumulation in biological denitrification

PA-X is a virulence factor in avian H9N2 influenza virus

H9N2 influenza viruses have been circulating worldwide in multiple avian species, and regularly infect pigs and humans. Recently, a novel protein, PA-X, produced from the PA gene by ribosomal frameshifting, was demonstrated to be an antivirulence factor in pandemic 2009 H1N1, highly pathogenic avian H5N1 and 1918 H1N1 viruses. However, a similar role of PA-X in the prevalent H9N2 avian influenza viruses has not been established. In this study, we compared the virulence and cytopathogenicity of H9N2 WT virus and H9N2 PA-X-deficient virus. Loss of PA-X in H9N2 virus reduced apoptosis and had a marginal effect on progeny virus output in human pulmonary adenocarcinoma (A549) cells. Without PA-X, PA was less able to suppress co-expressed GFP in human embryonic kidney 293T cells. Furthermore, absence of PA-X in H9N2 virus attenuated viral pathogenicity in mice, which showed no mortality, reduced progeny virus production, mild-to-normal lung histopathology, and dampened proinflammatory cytokine and chemokine response. Therefore, unlike previously reported H1N1 and H5N1 viruses, we show that PA-X protein in H9N2 virus is a pro-virulence factor in facilitating viral pathogenicity and that the pro- or antivirulence role of PA-X in influenza viruses is virus strain-dependent

Reliability and validity of the Chinese version of St Andrew′s Nutrition Screening Instrument

Objective·To translate St Andrew′s Nutrition Screening Instrument (SANSI), and test its reliability and validity.Methods·With the consent of the authors, the SANSI was translated into Chinese in accordance with the "Brislin translation-back translation method" and the Chinese version underwent cross-cultural debugging. Five clinical medical staff were selected to a pre-survey on 10 patients, and the expression and structure of the scale items were fine-tuned according to the feedback. A total of 221 inpatients with mental disorders were selected by convenient sampling method in Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine as research objects from January to February 2022, and the general clinical data of these patients were collected. The correlation between the items in Part Ⅲ and the identified risks in that part was evaluated by Spearman correlation analysis, and some items were deleted according to the results. Eight experts in mental disorders or nutrition were selected to evaluate the items of Chinese version of SANSI, and then the content validation indexes (CVIs) were calculated. With the standard of Nutritional Risk Screening 2002 (NRS2002), Spearman correlation analysis was used to evaluate the calibration validity of Chinese version of SANSI. Cronbach′s α coefficient, split half reliability and inter-rater reliability (κ consistency coefficient) were used to evaluate the reliability of the Part Ⅲ of the scale.Results·Through literal translation, back translation, cross-cultural debugging and pre-investigation, Chinese version of SANSI was formed. Among the 221 patients with mental disorders, 174 (78.73%) were males, the mean age was (64.12±13.87) years, and the duration of diseases was (26.06±17.65) years. There were 128 cases of schizophrenia (57.92%), 75 cases of organic mental disorders (33.94%), 10 cases of mood disorders (4.52%) and 8 cases of other mental disorders (3.62%). According to the results of correlation analysis in Part Ⅲ, the original items 1, 3, and 10 were deleted. The correlation coefficients between the remaining 7 items and the risk degree in this part were 0.391－0.734 (all P0.05).Conclusion·Chinese version of SANSI has good reliability and validity for hospitalized patients with mental disorders

PPAR-α Agonist Fenofibrate Upregulates Tetrahydrobiopterin Level through Increasing the Expression of Guanosine 5′-Triphosphate Cyclohydrolase-I in Human Umbilical Vein Endothelial Cells

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide (NO) synthase. Guanosine 5′-triphosphate cyclohydrolase-I (GTPCH-I) is a key limiting enzyme for BH4 synthesis. In the present in vitro study, we investigated whether peroxisome proliferator-activated receptor α (PPAR-α) agonist fenofibrate could recouple eNOS by reversing low-expression of intracellular BH4 in endothelial cells and discussed the potential mechanisms. After human umbilical vein endothelial cells (HUVECs) were treated with lipopolysaccharide (LPS) for 24 hours, the levels of cellular eNOS, BH4 and cell supernatant NO were significantly reduced compared to control group. And the fluorescence intensity of intracellular ROS was significantly increased. But pretreated with fenofibrate (10 umol/L) for 2 hours before cells were induced by LPS, the levels of eNOS, NO, and BH4 were significantly raised compared to LPS treatment alone. ROS production was markedly reduced in fenofibrate group than LPS group. In addition, our results showed that the level of intracellular GTPCH-I detected by western blot was increased in a concentration-dependent manner after being treated with fenofibrate. These results suggested that fenofibrate might help protect endothelial function and against atherosclerosis by increasing level of BH4 and decreasing production of ROS through upregulating the level of intracellular GTPCH-I