Objective To compare the relative efficacy and quality of extraction of human fecal DNA using four methods.Methods Real-time PCR were utilized for analysis both quantification and quality of the fecal targeted bacteria(including gut all eubaeterium,Bacteriodes-PrevoteUa group,Bifidobacterium spp Enterobacteriaceae and Enterococcus spp)by using 16s rRNA gene-targeted genus or group-specific primer sets.Results The negative rat of PCR product from method 3(phenol-chloroform plus bead-beating) was about 40%(4/10)by using universal primers,the PCR inhibition disappeared after fecal DNA purified with column.The total fecal 16s rRNA gene copy numbers(per gram of wet weight of feces)as well as the numbers of Bacteriodes-Prevotella group from method 1(QIAamp~DNA stool mini kit)and 4(QIAamp~ DNA stool mini kit combined with bead-beating)was higher significantly than that from method 2(FastDNA ~Kit,Biol01)and 3(P

Based on the gene sequence of AIV matrix protein 2(M2) and two cytotoxic T-lymphocyte epitopes derived from nucleoprotein,the prokaryotic expression vector pET-3M2e-NP1-NP2 was constructed.The target gene was expressed in the solvable form in BL21(DE3) when induced with 1.0 mmol/L IPTG and Western blot analysis showed that the expression product had good immunogencity.Purified fusion protein was mixed with various amount of adjuvant,including Freund's,Vash oil and chitosan,and then immunized 20-day-old chicken by intramuscular injection and boosted 3weeks later.Blood samples were collected weekly following the primary vaccination.The anti-M2e antibody was detected with ELISA method with the synthesized peptide as coated antigen;the neutralizing ability of anti-serum was evaluated on MDCK cell line and chick embryo,the CD4+ and CD8+ T lymphocyte amounts in peripheral blood of immunized chicken was measured by flow cytometry.Results showed that the fusion protein can induce immunological reaction,and the antibody can bind with the viral M2 protein that expressed on the surface of MDCK cells.Flow cytometry result showed that CD4+ and CD8+ T lymphocyte in peripheral blood increased obviously following immunization(P

Humans ; Nasal Obstruction ; surgery ; Treatment Outcome ; Turbinates ; surgery

Red in vivo recombination is a new kind of genetic engineering technique based on homologous recombination. In this work, plasmid pKD46 which expresses Red recombination proteins is transferred into Escherichia coli strain DH5?.The kanamycine resistant gene is generated by PCR by using primers with homology to hisDCB gene of E.coli chromosome. Thus, the hisDCB gene was replaced with kanamycine resistant gene by the plasmid recombination system, then the resistant gene was eliminated by a helper plasmid encoding the FLP recombinase. At last, a E.coli histidine auxotroph which is sensitive to kanamycine was got. The results indicate that Red in vivo recombination is a convenient method to construct auxotrophs.

The known members of inhibitor of growth (ING) gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to regulate brain tumour angiogenesis through transcriptional repression of NF-?B-responsive genes, induce G2/M arrest by the increased p21 expression in a p53-dependent manner, suppress the loss of contact inhibition and represses activation of the hypoxia inducible factor, which plays an important role in the progression of tumorigenesis. However, seldom studies about ING4 inducing tumor cells apoptosis were reported.The C6 cells (mouse glioma cells) were infected respectively with the blank adenovirus carrying GFP (Ad) and the recombinated Ad-hING4-His, then RT-PCR assay was used to detect the transcriptions of hING4, as well Western-blotting assay was ued to detect the expressions of hING4. The effects of hING4 expression upon C6 cells were observed, and the growth curve was drawed and tumor control rates were calculated. The C6 cells, which were affected by blank Ad and Ad-hING4-His, were respectively observed by LSCM (laser scan confocal microscope) and transmission electron microscope (TEM), detected by flow cytometry; and the genomic DNA of both groups were extracted and electrophoresised in agarose gel to examinate the DNA fragments. The results showed hING4 can significantly inhibit the growth of C6 cells by promoting the cell’s apoptosis, which probably is the first one to prove this property of ING4.The experimental and theoretical foundation for gene therapy for gliomas with ING4 in the future was established.

Aim The effect of tetrandrine on TGF-?1 mRNA expression in glomerulosclerosis rat was observed. Methods The rats were randomly divided into four groups, such as the normal control group (sham operative rat), glomerulosclerosis model group,tetrandrine group and amlodipine group. The expression of TGF-?1 mRNA was analyzed by Northern blot hybridization. Results The expressions of TGF-?1 mRNA in two treating groups were much lower than untreated model group. There were no difference between these two treating groups. Conclusion Tetrandrine can decrease the expression of TGF-?1 mRNA in glomerulosclerosis rat induced by unilateral renctomy plus adriamycin.

Objective To evaluate the possibility of short interfering RNA (siRNA) inhibiting Coxsackievirns B3 (CVB3) infection in vitro, and discover the mechanism initially. Methods We obtained proper effective dosage of siRNA by observing cytopathic effect (CPE). Estimate its antiviral activities and its pathway of siRNA by Western Blot assay and RT-PCR. Results Results showed that siRNA-3753 can be effectively transfected into HeLa cells, we can achieve a high transfection efficiency up to 98.77 % and its effect can last for 48 h stably in cells. 0.6 μmol/L siRNA-3753 got a high inhibiting effect of virus and didn't show any toxicity to cells. So we consider this concentration as the experimental concentration, siRNA-3753 can debase virus reproduction. The antiviral effect is sequence-specific and is not attributable to either interferon or the interferon response effectors protein kinase R (PKR). Conclusion The data confirmed that siRNA can effectively inhibit CVB3 infection in vitro, its antivirus effect was gained from specific debase of virus genome.

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.
Animals ; Antibodies, Viral ; blood ; Bacteriophage T7 ; genetics ; immunology ; metabolism ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Viral ; Immunization ; Influenza A virus ; genetics ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; immunology ; metabolism ; prevention & control ; Peptides ; genetics ; immunology ; metabolism ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Specific Pathogen-Free Organisms ; Viral Matrix Proteins ; genetics ; immunology ; metabolism

In order to explore the scientific connotation of "Fangzhengduiying (formula corresponding to pattern types)", "Qiyinliangxuzheng (Qi and Yin deficiency pattern)" of myocardial ischemia rat model and GC-TOF/MS based metabonomic method were used for comparing the effects of Sheng-mai injection, Salvia injection and propranolol in the present study. After data processing and pattern recognition, Sheng-mai injection showed better efficacy than the other two drugs in accordance with not only visual observation from PLS-DA scores plots but also the number of abnormal endogenous compounds restored to the normal level. Further studies showed that Sheng-mai injection could normalize the level of plasma endothelin-1, the index related to cardiovascular diseases and sleep disorders, which verified the results of metabonomics. Finally, the regulated metabolites and related metabolic pathways were analyzed, and it was supposed that the effects of Sheng-mai injection involved in the alternation of energy metabolism, lipid metabolism, amino acids metabolism, and so on. These findings provided scientific evidence to Shengmai "Fang" used for "Qi and Yin deficiency pattern" correspondingly, indicating that metabonomics has great potential in traditional Chinese medical research, which provides a novel approach and way to modernization of traditional Chinese medicine.
Animals ; Anti-Arrhythmia Agents ; pharmacology ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Endothelin-1 ; blood ; Gas Chromatography-Mass Spectrometry ; methods ; Injections ; Male ; Medicine, Chinese Traditional ; Metabolomics ; methods ; Myocardial Ischemia ; blood ; metabolism ; pathology ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Propranolol ; pharmacology ; Qi ; Rats ; Rats, Sprague-Dawley ; Salvia ; chemistry ; Schisandraceae ; chemistry ; Yin Deficiency ; metabolism

Isoproterenol (ISO)-induced myocardial ischemia animal model has been widely applied to the study of myocardial ischemia and evaluation of drug efficacy. Metabolic profiling of endogenous compounds can make a deep insight into biochemical process of the ISO-induced myocardial ischemia rats. Herein, rats were treated with ISO (2 mg x kg(-1)) for 10 days. After the model was established by measuring myocardial histopathology and plasma creatine kinase, GC/TOF-MS was used to determine endogenous metabolites in plasma and cardiac muscle of rats, and pattern recognition was used to process the data. Results showed that the plasma metabolic profiling of ISO-induced myocardial ischemia rats was significantly different from that of the control, and it had the tendency to the normal state after the discontinue of ISO injection. Besides, the cardiac muscle of rats treated with ISO for 10 days and the normal cardiac muscle could also be separated clearly. The potential biomarkers in plasma and cardiac muscle of model rats had homogeneity and their own specialty. Biochemical metabolic pathway analysis indicated that this myocardial ischemia model was involved in the alternation of energy metabolism, saccharometabolism, lipid metabolism, nucleoside metabolism and amino acid metabolism, and in relationship with oxidative stress. These findings revealed that metabonomics may be a promising tool to evaluate myocardial ischemia rat model induced by ISO and could further extend the study of pharmacodynamic action of drugs at the molecular level.
Animals ; Creatine Kinase ; blood ; Energy Metabolism ; Isoproterenol ; Lipid Metabolism ; Male ; Metabolome ; Metabolomics ; methods ; Myocardial Ischemia ; blood ; chemically induced ; metabolism ; Myocardium ; metabolism ; Oxidative Stress ; Random Allocation ; Rats ; Rats, Sprague-Dawley