Discovery and identification of quality markers of Sparganii Rhizoma based on zebrafish thrombosis model / 中草药·英文版

Objective: The aim of the present study was to determine the quality marker (Q-Markers) of Sparganii Rhizoma against thrombus through an integration of investigations on its antithrombotic effect, content determination and spectrum-effect correlation analysis. Methods: Based on the concept of Q-Marker, Sparganii Rhizoma was investigated for the identification of chemical component. The pharmacological effects on arachidonic acid-induced thrombosis in zebrafish were also investigated. The material basis in ethanol extract was determined by HPLC-UV. Furthermore, the potential Q-Markers were analyzed and predicted according to the effect-chemical correlation analysis. Finally, the anti-thrombotic Q-Markers were verified through the anti-thrombotic test of monomer components. Results: The model of thrombosis zebrafish was established with larvae exposed to 100 µmol/L arachidonic acid for 1 h. Nine ingredients in Sparganii Rhizoma were identified as 5-hydroxymethylfurfural, vanillic acid, ferulic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, vanillin, protocatechuic acid, p-coumaric acid and isoferulic acid. According to the determination effect of zebrafish thrombosis model and HPLC content analysis results, all the other contents present positive correlation except 5-hydroxymethylfurfural, and the P values of three representative potential Q-Markers (ferulic acid, protocatechuic acid and p-coumaric acid) were 0.002, 0.001 and 0.026, respectively. Conclusion: Sparganii Rhizoma showed a dose-dependent effect on the recovery of reducing cardiac red blood cell on zebrafish model. Three phenolic acids (ferulic acid, protocatechuic acid and p-coumaric acid) were proved to possess the anti-thrombotic effects which could be regarded as the potential Q-Markers for quality assessment of Sparganii Rhizoma.

Molecular Mechanism of Anti-thrombosis and Anti-platelet Aggregation of Sparganii Rhizoma Based on Integrated Pharmacological Platform / 中国实验方剂学杂志

Objective: To study on the molecular mechanism of anti-platelet aggregation and anti-thrombosis of Sparganii Rhizoma. Method: Based on the data information and analysis functions of traditional Chinese medicine(TCM) database,TCM composition database and target database in TCM platform for integration of pharmacology,information of chemical compositions in Sparganii Rhizoma was retrieved,interaction network between potential targets of Sparganii Rhizoma and disease targets was built,and the key targets were enriched and calculated,the gene functions and pathways were analyzed,multidimensional relationship network of " herbal medicines-chemical components-key targets-key pathways" was built. Result: Active ingredients of Sparganii Rhizoma mainly included flavonoids and phenolic acids,such as mountain kaempferol,sanleng acid,linoleic acid,etc.Anti-thrombosis involved 202 key targets,including protein kinase Cδ(PRKCD),glucose kinase(GCK),(PRKAA2),adenosine ribonucleotide activated protein kinase α-2 catalytic subunit,etc;and it was related to the endocrine system,circulatory system,neurodegenerative diseases and other related biological processes and signal pathways.Anti-platelet aggregation involved 136 key targets,including PRKCD,cytochrome C oxidase protein 7C(COX7C),GCK,etc;and it was involved in Parkinson's disease,circulatory system,neurodegenerative diseases and other related biological processes and signal pathways. Conclusion: Sparganii Rhizoma regulates vascular endothelial cell adhesion molecule expression and platelet mitochondrial function mediated apoptosis of platelets mainly through regulating neurotransmitter activity in the central nervous system,in order to exert antithrombotic effect and anti-platelet aggregation effect.

Molecular Mechanism of Banxia Baizhu Tianma Tang in Treating Hypertension Based on Integrated Pharmacological Platform / 中国实验方剂学杂志

Objective:To explore the molecular mechanism of Banxia Baizhu Tianma Tang in treating hypertension. Method:Based on an internet-based computation platform for integrated pharmacology of traditional Chinese medicine(TCM),TCM prescription database,TCM database,TCM component database and disease/symptoms target database,information on the chemical compositions contained in TCM was retrieved,an interaction network between potential targets and disease targets of Banxia Baizhu Tianma Tang was built,then core target was enriched and calculated,gene function and pathway analysis was carried out,the multi-dimensional relationship network of "Chinese herbs-ingredients-critical targets-key pathways" of Banxia Baizhu Tianma Tang was further constructed. Result:A total of 90 active ingredients in Banxia Baizhu Tianma Tang were obtained,including alkaloids,nucleosides,flavonoids,saponins,organic acids and other ingredients;they involved 287 core targets,including 13 direct targets,such as adenylate cyclase,G protein coupled with β1 receptor,glucose kinase,etc;they also involved nervous system,endocrine system,circulatory system,estrogen signaling pathway,chemokine signaling pathway and other related biological processes and signaling pathways. Conclusion:Banxia Baizhu Tianma Tang can regulate neurotransmitter concentration and activity abnormalities,improve the protective effect of vascular endothelial cells by improving insulin resistance,regulating the production of inflammatory factors,and inhibiting inflammatory reactions,thereby exerting pharmacological effects on the treatment of hypertension.This study can provide a scientific basis for comprehensive interpretation of mechanism of Banxia Baizhu Tianma Tang.

Immunochromatographic Assay for Detection of Imidaclothiz Based on Upconversion Fluorescence Labeling / 分析化学

A simple,rapid and sensitive upconversion immunochromatographic assay(UICA) was developed to detect imidaclothiz using NaYF4:Yb,Er upconversion nanoparticles(UCNPs) labeled with anti-imidaclothiz monoclonal antibody. The amino-modified UCNPs were conjugated with anti-imidaclothiz monoclonal antibody to prepare the UICA strip,which could realize the quantitative detection of imidaclothiz using a fluorescence photometer with an external 980 nm laser source. The working conditions of the UICA were systematically optimized, and the sensitivity, specificity, precision and accuracy were assessed by the studies of cross-reactivity (CR), spiked recovery and validation with HPLC. Under the optimal conditions (pH 8. 0, 0.3 mol/L NaCl,2.5% methanol and 0.2% PEG2000), the UICA could be completed in 25 min for the detection of imidaclothiz. The half-maximal inhibition concentration (IC50), limit of detection (IC10) and linear range (IC10-IC90) were 97.37 ng/mL,26.30 ng/mL and 26.30-363.08 ng/mL, respectively. The UICA had no CR with the analogues of imidaclothiz except for imidacloprid. The average spiked recoveries were 71.8%-97.2% with the relative standard deviations of 0.7%-10.7% in the matrices of paddy water, soil,pear,peach,wheat,cucumber,tomato and rice. The detection results of UICA for the authentic paddy water and pear samples were consistent with that of high performance liquid chromatography (HPLC).

Long-term effects of heme oxygenase 1 overexpression on post-infarction heart function in diabetic rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To clarify the impact of increased heme oxygenase-1 (HO-1) expression on cardiac function of diabetic rats with myocardial infarction and its mechanism.</p><p><b>METHODS</b>Sixty adult male Wistar rats were randomly divided into five groups (n = 12): sham operation group (sham), diabetes + sham operation group (DM + sham), diabetes + MI group (DM + MI) , diabetes + myocardial infarction + cobalt original porphyrin (CoPP) group (DM + MI + CoPP), diabetes + myocardial infarction + CoPP+ tin porphyrin (SnMP) group (DM + MI + CoPP + SnMP). CoPP 4.5 mg/kg or SnMP 15 mg/kg were administered at the day next to MI operation, for six weeks, once a week. At the 28th week post operation, the echocardiography, left heart via the carotid artery indoor intubation were used to observe the long-term influence of HO-1 inducer (cobalt protoporphyrin, CoPP) and activity of HO inhibitor (tin porphyrin, SnMP) on the indices of left ventricular remodeling and cardiac function after the intervention. Blood glucose (GLU), total cholesterol (TC), C-reactive protein (CRP), serum creatinine (Cr), aminotransferase (ALT) and other indicators were measured. ELISA was used to test interleukin-6 (IL-6), tumor necrosis factor (TNF), nitric oxide (NO), prostacyclin (PGI2), adiponectin, and ultra sensitive CRP (HsCRP) level.</p><p><b>RESULTS</b>HO-1 inducer, CoPP, could ameliorate ± dp/dtmax, left ventricular ejection fraction and left ventricular shortening fraction in diabetic myocardial infarction rats. It could also decrease left ventricular end-diastolic diameter. The serum bilirubin, NO and PGI2 levels, myocardial phosphorylated endothelial nitric oxide synthasee(peNOS), phosphorylated activated protein kinase (pAkt), phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) expression were also significantly elevated, and the serum hs-CRP and TNF levels were significantly inhibited. Compared to inducer group, cardiac function were worse in the inhibitor group.</p><p><b>CONCLUSION</b>Upregulated HO-1 level can improve the endothelial function, inhibite of the inflammatory response and enhance the antioxidant substances in serum bilirubin via peNOS-pAMPK pathway, which effectively inhibit ventricular remodeling and improve the long-term cardiac function after infarction in diabetic rats.</p>

Expression and purification of four single-stranded DNA-binding proteins and their binding on HCV RNA / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Express and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.</p><p><b>METHODS</b>The expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel.</p><p><b>RESULTS</b>Suitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA.</p><p><b>CONCLUSIONS</b>We have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.</p>

Comparison of ELISA diagnostic kits used in China for hepatitis C virus IgG antibody detection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To compare the commercial diagnostic reagent available in China for hepatitis C virus ( HCV) IgG antibody detection in order to provide some useful information for HCV prevention.</p><p><b>METHODS</b>The HCV-IgG detection reagents produced by six different Enterprises named A, B, C, D, E and F were chosen and applied to detect 160 HCV specious sera samples. HCV-IgG reagent from ABBOTT was adopted as gold-standard and the samples in gray zone were determined by RIBA method finally.</p><p><b>RESULTS</b>160 sera samples comprised 88 positive samples and 72 negative samples. The total conformity ranged from 88.13% to 98.13% and the Youden indexes ranged from 0.74 to 0.96 when the reagents from six different Enterprises were compared with gold-standard. The highest conformity was 98.13%, observed in D reagent with the highest Youden index of 0.96.</p><p><b>CONCLUSION</b>The total conformity rates were more than 88% when the HCV-IgG antibody detection reagents from six different Enterprises were compared with ABBOTT reagent. It was highly conformable. However, some reagent proved to be less conformable in negative samples detection.</p>

Investigation on the difference of intolerance to food between southern and northern middle-aged Chinese and its association with eating habits / 中国应用生理学杂志

<p><b>OBJECTIVE</b>The aim of the present study was to investigate the difference of intolerance to food between southern and northern middle-aged Chinese, and furthermore analyze its association with eating habits in both study population.</p><p><b>METHODS</b>ELISA was applied to determine the serum concentrations of specific IgG of 14 food anaphylactogen in 1568 healthy subjects from totally 9 districts in both southern and northern China. Life style questionnaire was also applied to investigate the daily intake of six categorizes of food associated with food intolerance.</p><p><b>RESULTS</b>45.8% of all subjects were found to be intolerant to certain food. 62.3% of subjects from southern China and 40.4% of subjects from northern China were found to be intolerant to certain food, the difference between southern and northern Chinese was statistically significant. Top three foods intolerant by southern Chinese were crab, egg, and cold fish, while top three food intolerant by northern Chinese were egg, crab, and milk. The differences of intolerance to crab, cold fish, soy bean, rice, and tomato between southern and northern Chinese were statistically significant. Investigation on eating habits revealed that cereals and fish were the major food consumed by subjects in our study. There was no certain association between food intolerance and eating habits.</p><p><b>CONCLUSION</b>Considering that there are differences between southern and northern Chinese, southern and northern Chinese should pay attention to their daily food in order to avoid food allergy.</p>

Establishing a high-titer infectious avian influenza A (H5N1) pseudotyped viral particle / 病毒学报

A transient four-plasmid cotransfection system was used to construct avian influenza A (H5N1) pseudotyped viral particle (H5N1Pp) by incorporating hemagglutinin (HA) protein and neuraminidase (NA) protein from H5N1 avian influenza virus onto Murine leukemia virus pseudotyped viral particles, the transmission electron microscopy, infectivity titer assay, hemagglutination assay, neutralization assay of H5N1Pp were studied. We established a pseudotyped H5N1 viral particle at a high titer of 10(8) Pp/mL, the morphology,the hemagglutination activity and neutralization specificity of H5N1Pp is simililar to wild H5N1 virus. The research result sets a platform for studying this virus, including its receptors, the functional analysis of HA and NA, neutralizing antibodies and anti-H5N1 drug development.

Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription.</p><p><b>METHODS</b>We express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction.</p><p><b>RESULTS</b>The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15.</p><p><b>CONCLUSIONS</b>kod-ssb may in future be used to enhance DNA and cDNA amplification.</p>