Isolation and Characterization of a Novel Lytic Halotolerant Phage from Yuncheng Saline Lake.

Halophilic phage are a type of virus that exist in salty environments within halophilic archaeal or bacterial hosts. However, relatively few reports on halophilic bacteriophages exist, and our overall understanding of halophilic bacteriophages is quite limited. We used SYBR Green I fluorescent staining to detect the abundance of viruses in Yuncheng Saline Lake, China. Using the double-layer plate method, a lytic phage that could infect halophilic bacterium Salinivibrio sp. YM-43 was isolated and named YXM43. We studied host range, optimal host, morphological characteristics, nucleic acid type, protein composition, and other biological characteristics of the virus. Results reveal a high abundance of this halophilic virus in Yuncheng Saline Lake. The newly isolated bacteriophage YXM43 has a narrow host range, with the most suitable host being Virgibacillus sp. SK39. After purification and enrichment, YXM43 is observed as a spherical particle with a diameter of approximately 30 nm, with no tail. No lipid envelope can be seen in YXM43. The capsid protein of the virus can be separated into seven proteins with molecular weights ranging from 62.0 to 13.0 kDa. YXM43 is a DNA virus with a genome approximately 23 kb. The virus is tolerant of low salinity, and its activity is highest at a temperature of 60 °C and a pH of 10. YXM43 is temperature and pH tolerant, and can adapt to environmental change, even withstanding chloroform treatment. The results indicate that bacteriophage YXM43 is a novel halophilic bacteriophage with broad tolerance to environmental change.

Leptin receptor overlapping transcript (LepROT) gene participates in insulin pathway through FoxO.

Leptin receptor overlapping transcript (LepROT) is co-transcribed with the leptin receptor (LepR). However, the function and mechanism of LepROT in insulin pathway is unclear. In this study, we report the function of LepROT in maintaining consistent FoxO transcription. LepROT is constitutively expressed during larval development. 20-Hydroxyecdysone, methoprene, and insulin have no effect on the transcription of LepROT. However, the knockdown of LepROT by dsRNA injection in larvae causes delay of the development of Helicoverpa armigera. Knockdown of LepROT results in the upregulation of FoxO and downregulation of PI3K. The knockdown of LepROT also results in the subcellular translocation of FoxO from cytoplasm to nuclei. By contrast, overexpression of LepROT in the HaEpi cell line inhibits FoxO expression. Results suggest that LepROT participates in insulin signaling.

MicroRNA-125b attenuates epithelial-mesenchymal transitions and targets stem-like liver cancer cells through small mothers against decapentaplegic 2 and 4.

UNLABELLED: Emerging evidence suggests that epithelial-mesenchymal transitions (EMTs) play important roles in tumor metastasis and recurrence. Understanding molecular mechanisms that regulate the EMT process is crucial for improving treatment of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play important roles in HCC; however, the mechanisms by which miRNAs target the EMT and their therapeutic potential remains largely unknown. To better explore the roles of miRNAs in the EMT process, we established an EMT model in HCC cells by transforming growth factor beta 1 treatment and found that several tumor-related miRNAs were significantly decreased. Among these miRNAs, miR-125b expression was most strongly suppressed. We also found down-regulation of miR-125b in most HCC cells and clinical specimens, which correlated with cellular differentiation in HCC patients. We then demonstrated that miR-125b overexpression attenuated EMT phenotype in HCC cancer cells, whereas knockdown of miR-125b promoted the EMT phenotype in vitro and in vivo. Moreover, we found that miR-125b attenuated EMT-associated traits, including chemoresistance, migration, and stemness in HCC cells, and negatively correlated with EMT and cancer stem cell (CSC) marker expressions in HCC specimens. miR-125b overexpression could inhibit CSC generation and decrease tumor incidence in the mouse xenograft model. Mechanistically, our data revealed that miR-125b suppressed EMT and EMT-associated traits of HCC cells by targeting small mothers against decapentaplegic (SMAD)2 and 4. Most important, the therapeutic delivery of synthetic miR-125b mimics decreased the target molecule of CSC and inhibited metastasis in the mice model. These findings suggest a potential therapeutic treatment of miR-125b for liver cancer. CONCLUSION: miR-125b exerts inhibitory effects on EMT and EMT-associated traits in HCC by SMAD2 and 4. Ectopic expression of miR-125b provides a promising strategy to treat HCC.

A distinct glucose metabolism signature of acute myeloid leukemia with prognostic value.

Acute myeloid leukemia (AML) is a group of hematological malignancies with high heterogeneity. There is an increasing need to improve the risk stratification of AML patients, including those with normal cytogenetics, using molecular biomarkers. Here, we report a metabolomics study that identified a distinct glucose metabolism signature with 400 AML patients and 446 healthy controls. The glucose metabolism signature comprises a panel of 6 serum metabolite markers, which demonstrated prognostic value in cytogenetically normal AML patients. We generated a prognosis risk score (PRS) with 6 metabolite markers for each patient using principal component analysis. A low PRS was able to predict patients with poor survival independently of well-established markers. We further compared the gene expression patterns of AML blast cells between low and high PRS groups, which correlated well to the metabolic pathways involving the 6 metabolite markers, with enhanced glycolysis and tricarboxylic [corrected] acid cycle at gene expression level in low PRS group. In vitro results demonstrated enhanced glycolysis contributed to decreased sensitivity to antileukemic agent arabinofuranosyl cytidine (Ara-C), whereas inhibition of glycolysis suppressed AML cell proliferation and potentiated cytotoxicity of Ara-C. Our study provides strong evidence for the use of serum metabolites and metabolic pathways as novel prognostic markers and potential therapeutic targets for AML.

[Odor emission rate of municipal solid waste from landfill working area].

Emission rates of volatile organic compounds (VOCs), H2S and odor unit from the surface of a municipal solid waste (MSW) landfill working area were measured with a wind tunnel sampler. The results show that the emission rate of odor from the non-point source of landfill is the function of environmental temperature and surface sweeping velocity. The emission rate measured in the high temperature season can be 6 times higher of that in the low temperature season. Within the experimental range of 0.6-4 m x s(-1) wind sweeping velocity, the emission rate shows a linear relationship with wind sweeping velocity. In summer, the emission rates of VOCs (measured by PID as isobutylene), H2S and odor unit are 385-680 microg x (m(2) x s)(-1), 4-7 microg x (m(2) x s)(-1), and 46.5-136 OU x (m(2) x s)(-1) respectively. The continuous sweeping experiment shows that the emission rate measured with clean air sweeping is the maximum possible emission rate, which needs to be adjusted when it is used to estimate the odor concentration of more than 10 min sample time or an area emission load.

Plasma metabolite profiles of Alzheimer's disease and mild cognitive impairment.

Previous studies have demonstrated altered metabolites in samples of Alzheimer's disease (AD) patients. However, the sample size from many of them is relatively small and the metabolites are relatively limited. Here we applied a comprehensive platform using ultraperformance liquid chromatography-time-of-flight mass spectrometry and gas chromatography-time-of-flight mass spectrometry to analyze plasma samples from AD patients, amnestic mild cognitive impairment (aMCI) patients, and normal controls. A biomarker panel consisting of six plasma metabolites (arachidonic acid, N,N-dimethylglycine, thymine, glutamine, glutamic acid, and cytidine) was identified to discriminate AD patients from normal control. Another panel of five plasma metabolites (thymine, arachidonic acid, 2-aminoadipic acid, N,N-dimethylglycine, and 5,8-tetradecadienoic acid) was able to differentiate aMCI patients from control subjects. Both biomarker panels had good agreements with clinical diagnosis. The 2 panels of metabolite markers were all involved in fatty acid metabolism, one-carbon metabolism, amino acid metabolism, and nucleic acid metabolism. Additionally, no altered metabolites were found among the patients at different stages, as well as among those on anticholinesterase medication and those without anticholinesterase medication. These findings provide a comprehensive global plasma metabolite profiling and may contribute to making early diagnosis as well as understanding the pathogenic mechanism of AD and aMCI.

Prognostic significance of 2-hydroxyglutarate levels in acute myeloid leukemia in China.

The 2-hydroxyglutarate (2-HG) has been reported to result from mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes and to function as an "oncometabolite." To evaluate the clinical significance of serum 2-HG levels in hematologic malignancies, acute myeloid leukemia (AML) in particular, we analyzed this metabolite in distinct types of human leukemia and lymphoma and established the range of serum 2-HG in appropriate normal control individuals by using gas chromatograph-time-of-flight mass spectrometry. Aberrant serum 2-HG pattern was detected in the multicenter group of AML, with 62 of 367 (17%) patients having 2-HG levels above the cutoff value (2.01, log2-transformed from 4.03 µg/mL). IDH1/2 mutations occurred in 27 of 31 (87%) AML cases with very high 2-HG, but were observed only in 9 of 31 (29%) patients with moderately high 2-HG, suggesting other genetic or biochemical events may exist in causing 2-HG elevation. Indeed, glutamine-related metabolites exhibited a pattern in favor of 2-HG synthesis in the high 2-HG group. In AML patients with cytogenetically normal AML (n = 234), high 2-HG represented a negative prognostic factor in both overall survival and event-free survival. Univariate and multivariate analyses confirmed high serum 2-HG as a strong prognostic predictor independent of other clinical and molecular features. We also demonstrated distinct gene-expression/DNA methylation profiles in AML blasts with high 2-HG compared with those with normal ones, supporting a role that 2-HG plays in leukemogenesis.

Inhibitory effect of Ginkgo biloba extract on fatty liver: regulation of carnitine palmitoyltransferase 1a and fatty acid metabolism.

OBJECTIVE: To investigate the potential effect of Ginkgo biloba extract (GBE) on the prevention and treatment of nonalcoholic fatty liver disease (NAFLD). METHODS: Male Wistar rats were divided into 4 groups (the control group, GBE group, high-fat diet [HFD] group and HFD + GBE group). The human hepatocellular carcinoma cell line (HepG2) was treated with GBE and its flavonoid ingredients. The fatty acid composition of the rat liver was analyzed with gas chromatography/time-of-flight mass spectrometry (GC/TOFMS). Triglyceride contents of both the rat liver and HepG2 cells were measured by enzymatic colorimetric method. The expressions of fatty acid metabolism-related genes were analyzed with real-time reverse transcription-polymerase chain reaction (RT-PCR). The protein expression and enzymatic activity were subsequently measured. RESULTS: In rat livers, GBE reduced the elevations of hepatic triglyceride contents caused by HFD and the increased hepatic fatty acids were differentially affected by GBE. Notably, the expression and total activity of the fatty acid ß-oxidation rate-limiting enzyme, carnitine palmitoyltransferase 1a (CPT1A), were also promoted with GBE ingestion. In HepG2 cells, GBE and its ingredients, quercetin, kaempferol and isorhamnetin, could decrease the cellular triglyceride content and upregulate the expression and total activity of CPT1A, respectively. CONCLUSIONS: The triglyceride-lowering effect of GBE on the HFD rat liver is closely associated with the increased expression and activity of CPT1A, and the flavonoid ingredients are the major contributors of GBE.

Effect of carbonate on anaerobic acidogenesis and fermentative hydrogen production from glucose using leachate as supplementary culture under alkaline conditions.

Carbonate was added into a co-culture of glucose and fresh leachate under alkaline condition to enhance batched acidogenesis and fermentative hydrogen production simultaneously. Results indicated carbonate has positive effect on both H(2) production and acetic acid generation. The highest hydrogen yield (about 1.40 mol/mol glucose) was obtained at [CO(3)(2-)] = 280 mg/L with pH 8.0 and [CO(3)(2-)] = 560 mg/L with pH 9. The dominant liquid metabolites were ethanol, acetic and butyric acid. The highest total volatile fatty acid yield (0.38 g/g glucose) was achieved at [CO(3)(2-)] = 560 mg/L with pH 9. In this case, the acetic acid yield reached 0.13 g/g glucose. Verification tests using simulated wastewater as substrate were also carried out at pH 9. Results demonstrated calcium ions inhibit hydrogenogens activity while carbonate addition can alleviate the suppression effect caused by Ca(2+).

Use of the metabolomics approach to characterize Chinese medicinal material Huangqi.

Integration of the genetic and metabolic fingerprinting can provide a new approach to differentiate similar Traditional Chinese Medical (TCM) materials. Two leguminous plants, Mojia Huangqi and Menggu Huangqi, are important medical herbs and share great similarities in morphology, chemical constituent, and genomic DNA sequence. The taxonomy of Mojia Huangqi and Menggu Huangqi has been debated for more than 50 years and discrimination of TCM materials directly affects the pharmacological and clinical effects. AFLP based genetic fingerprinting and GC-TOF/MS-based metabolic fingerprinting were used to successfully discriminate the two species. The results of AFLP supported the opinion that Menggu Huangqi was a variant of Mojia Huangqi. The metabolic fingerprinting showed growth locations have greater impacts on the metabolite composition and quantity than the genotypes (cultivated versus wild) in Menggu Huangqi. The difference of some soluble sugars, fatty acids, proline, and polyamine reflected plant adaptation to different growth environments. Using multivariate and univariate statistical analysis, three AFLP markers and eight metabolites were identified as candidate DNA and metabolic markers to distinguish the two herb materials. The correlation network between AFLP markers and metabolites revealed a complex correlation network, which indicated the special metabolic pathways and the regulation networks of Huangqi.

The development and preliminary evaluation of a new Mycobacterium tuberculosis vaccine comprising Ag85b, HspX and CFP-10:ESAT-6 fusion protein with CpG DNA and aluminum hydroxide adjuvants.

Ag85b and HspX of Mycobacterium tuberculosis (Mtb) (H37Rv) were expressed and purified in this study. These two proteins were combined with another fusion protein CFP-10:ESAT-6 (C/E) (Ag), then mixed with the adjuvants CpG DNA and aluminum hydroxide and used to vaccinate mice and guinea pigs challenged with Mtb (H37Rv). The number of spleen lymphocytes secreting Ag85b, HspX and C/E-specific interferon-gamma were significantly higher in the Ag+Al+CpG group than in the Ag and CpG groups. The combination of Ag, Al and CpG induced the highest concentrations of anti-Ag85b, anti-HspX and anti-C/E immunoglobulin G in mouse serum. Mouse peritoneal macrophages from the Ag+Al+CpG group secreted significantly higher levels of interleukin-12 compared with macrophages from the other groups. The total mean liver, lung and spleen lesion scores and bacterial loads in the spleen in guinea pigs vaccinated with Ag+Al+CpG were lower than those of the other groups, but no significant difference was found. These results show that the mixture of Ag85b, HspX and C/E with a combination of CpG and aluminum adjuvants can induce both humoral and cellular immune responses in mice, whereas it plays only a small role in the control of disease progression in guinea pigs challenged with Mtb.

[Study on effects of baicalin, berberine and Astragalus polysaccharides and their combinative effects on aldose reductase in vitro].

OBJECTIVE: To observe the activities of baicalin, berberine and Astragalus polysaccharides and their combinative effects on aldose reductase (AR) by a screening model of aldose reductase inhibitor (ARI) in vitro. METHODS: The inhibition of AR by baicalin, berberine and Astragalus polysaccharides and positive drug (Epalrestat) in different concentrations were evaluated, and their combinative effects were studied according to orthogonal t design. RESULTS: Baicalin and berberine had remarkable inhibitory effects on AR, the inhibitory rates were (88.4 +/- 7.4)% and (69.0 +/- 9.4)% at the concentration of 300 microg/mL. However, the combinative effect of the inhibition on AR by the two compounds was antagonistic action. Astragalus polysaccharides had no activity of inhibition on AR. CONCLUSION: Baicalin and berberine are the potential AR inhibitors as they can inhibit the activity of AR in vitro.

[Study on extraction and purification of polysaccharides from Astragalus membranaceus].

OBJECTIVE: To optimize the extraction and purification procedure for polysaccharides from Astragalus membranaceus. METHODS: The orthogonal test was used to determine the optimal extraction technology in terms of the extraction rate of crude polysaccharides and the concentration of polysaccharides. The efficiency of deproteinization and decoloration with different methods were evaluated with the ration of protein and colorant removing as the indices. Their influence on the content of Astragalus polysaccharides was calculated as well. RESULTS: The optimum extraction procedure was as follows: 8 times of water extracting three times at 100 degrees C, 90 minutes per time. Deproteinization using pepsase combined with Sevage solvent and depigmenting with DEAE fibrin achieved a high purification rate. CONCLUSION: The optimized extraction and purification procedure is simple, quick and efficient. The result establishes the foundation for the further study of the polysaccharides from Astragalus membranaceus.

[Analysis of matrine and oxymatrine in Kushensu injection by UPLC/Q-TOF-MS].

OBJECTIVE: To develop a method for the determination of matrine and oxymatrine in Kushensu injection by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). METHOD: The analysis was performed on a Acquity UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 microm) with the gradient elution of 10 mmol x L(-1) amine acetate (pH 8, adjusted by aqueous ammonia) and methanol. The flow rate was set as 0.30 mL x min(-1). The column temperature was maintained at 30 degrees C. The peaks were detected at 210 nm and the injection volume was 2 microL. RESULT: The calibration curves showed good linearity within the test ranges of 0.084-3.36 microg for matrine and 0.086-3.44 microg for oxymatrine, respectively. The mean recoveries were 103.2%, 98.7%, and the RSD were 1.5%, 1.2%, respectively. CONCLUSION: The established method is simple, rapid and sensitive, can be used for the quality control of matrine and oxymatrine in Kushensu injection through the manufacturing process and clinical implement.

[Quality control of non-alkaloids from Radix Sophorae tonkinensis].

OBJECTIVE: To establish a method for quality control of non-alkaloids from Radix Sophorae tonkinensis. METHODS: Sugar, organic-acid, polyphenolic and saponin were identified by TLC. The contents of Sugar, polyphenolic and saponin were determined by spectrophotometry and the content of organic-acids was determined by acid-based titration. RESULTS: The contents of sugar, polyphenolic and saponin were 54.37%, 4.74%, 4.40%, respectively. The average content of organic-acid was 9.40%. CONCLUSIONS: The established method is simple, accurate, reproducible and suitable for the quality control of non-alkaloids from Radix Sophorae tonkinensis.

[Anti-inflammatory and anti-allergic effects of acidic fraction of Pheratima extract in asthma mice induced by ovalbumin].

OBJECTIVE: To explore the effects of acidic fraction of Pheratima extract in an ovalbumin (OVA) induced asthma mouse model, and to provide the experimental evidences for the anti-asthmatic application of Pheratima extract with further purification and development. METHOD: Mice model of allergic asthma was established through the OVA challenge. To investigate the inflammatory cell level and Th1/Th2 levels as well as the therapeutic effects of acidic fraction from Pheratima extract, cell count of bronchoalveolar lavage fluid (BALF) was performed to evaluate the secretion of eosinophils (EOS) cells, and IgE, IL-4, IL-5, IL-13, IFN-y levels were detected by ELISA. RESULT: Compared with control group, the EOS count of BALF in the model group was remarkably increased (P<0.01), and IgE, IL-4, IL-5, IL-13 levels were also increased (P<0.01), while IFN-gamma decreased (P<0.01). Acidic fraction from Pheratima (S) extract and its 30% ethanol washed fraction (S30) significantly inhibited the increase of EOS count (P<0.01), decreased the IgE, IL-4, IL-5, IL-13 levels (P<0.05), and inhibited the decrease of IFN-gamma level (P<0.01). CONCLUSION: The results indicate that inhibition of the EOS secretion and balancing of the altered Th1/Th2 levels may be important mechanisms of Pheratima's therapeutic effect in asthmatic mice model, and S30 is pharmacologically effective as evaluated with the above mentioned parameters, as a representative fraction of the Pheratima extract.

[Effect of jinfu kang to experimental precancerous colon lesions and urinary metabolites in rat].

OBJECTIVE: : To profile urinary metabolite variations from 1, 2-dimethylhydrazine (DMH)-induced precancerous colon rats, Jinfu Kang treated rats and healthy controls. METHOD: We used ethyl chloroformate derivatization and gas chromatography-mass spectrometry (GC-MS) based metabonomic method to analyze rat urines. RESULT: The time-dependent variations of metabolite profile showed a progressive deviation of the metabolism in the model group from the initial pattern over time and a systemic recovery of the metabolism in the treatment group, which is consistent with the histological results. The in-depth analysis indicated that the disorder of tricarboxylic acid cycle (TCA), tryptophan metabolism, polyamine metabolism and gut flora structure were associated with DMH intervention. CONCLUSION: Metabolic study revealed that Jinfu Kang can effectively reverse metabolic departures in DMH-induced precancerous colon rat, which is consistent with pathological results.

Antitumor constituents from Alternanthera philoxeroides.

Two new compounds, alternanthin B (1) and N-trans-feruloyl-3,5-dimethoxytyramine (2), along with four known compounds (3-6) were isolated from the aerial parts of Alternanthera philoxeroides. Their structures were elucidated on the basis of spectroscopic methods. The antitumor activity of the isolated compounds was also evaluated.

[Application of metabolomics in research of plant metabolites].

Metabolomics, a branch of systems biology, has gained extensive attention and profound achievements in the plant. Although plant metabolomics is to be explored, it has been one of the most effective methods to study the physiological and biochemical process and gene modification in pattern plants. We herein summarized the concept, development, and application of metabolomics and prospected the potentials in the metabolite profiling for plant. Metabolomics provides an omics' methodology to elucidate the whole biological process, identify and quantify the complex components in the plant. A number of metabolites present in the plant are active components of traditional Chinese medicine, and these bioactive components are influenced by the multi-factors such as environment, species, and processing methods etc. Therefore, it is of great importance to analyze a wide spectrum of compositions with diverse chemical characteristics and varied concentration, which is the foundation to quality control, allowing the elucidation of the pharmacological effectiveness, and further exploiting of traditional Chinese medicine.

Three new compounds from Isodon melissoides.

Reinvestigation of the aerial parts of Isodon melissoides has afforded two new ent-diterpenoids, melissoidesins V (1) and W (2), together with five known ones, glabcensin W (3), melissoidesin C (4), melissoidesin A (5), melissoidesin B (6) and melissoidesin D (7), one new ionone derivative 3alpha,4alpha-isopropyliden-beta-ionol (8), as well as three analogues, 3-hydroxy-4-oxo-beta-ionol (9), megastigma-7-en-3,5,6,9-tetraol (10) and blumenol A (11), and three phenolic compounds salicylic acid (12), syringic acid (13) and cirsiliol (14). The new structures were elucidated on the basis of spectroscopic techniques, especially the 2D-NMR spectral analysis.