OBJECTIVETo establish real-time PCR and loop-mediated isothermal amplification (LAMP) systems for detecting Cryptococcus neoformans CAP10 gene.

METHODSSpecific primers were designed targeting CAP10 gene of Cryptococcus neoformans, and the plasmid was constructed. After optimization of the reaction condition, the plasmid was quantitatively detected using real-time PCR and LAMP, and the detection sensitivity and specificity were evaluated. Clinical samples were also detected using the two methods.

RESULTSThe detection sensitivity of real-time PCR and LAMP was 6.8×10(1) and 6.8×10(3) copies, respectively. Real-time PCR yielded a higher positivity rate than LAMP. Both of the two methods showed a high detection specificity and produced negative results in the detection of Neisseria meningitidis, Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus niger and Escherichia coli.

CONCLUSIONReal-time PCR is highly sensitive and specific for detecting Cryptococcus neoformans CAP10 gene but requires sophisticated equipment. LAMP, though with a relatively lower sensitivity, is simple to operate without the need of special equipment, and the result can be conveniently observed. Both of the two methods are suitable for detecting Cryptococcus neoformans and evaluating the treatment outcomes.

Cryptococcus neoformans ; genetics ; Fungal Proteins ; genetics ; Nucleic Acid Amplification Techniques ; methods ; Plasmids ; RNA, Fungal ; genetics ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Objective The primary culture of synovial fibroblasts is a convenient tool to study the pathology and physiology of synovial tissues .An improved method was constructed in this study by C57BL /6 mice to study the mechanism of rheumatoid ar-thritis(RA) .Methods The synovium around the hip joints were collected .Attention should be paid to eliminate the egg-yolk like yellow oval substance in the middle of the synovium .The synovium was transferred into a 1 .5 mL Eppendorf tube containing 0 .5%type Ⅳ collagenase and cut into 1 mm3 blocks or so .The Eppendorf tube was placed in 37 ℃ Constant temperature orbital shaker incubator for 60 min .After digestion ,the tube was placed on the Vortex for a high-speed oscillation for 1 .5 minutes to guarantee the separation of cells .Results Within about 1 week ,the first passage was performed by the trypsin digestion method .On day 10 , the number of synovial macrophages reached the maximum and then decreased gradually .After the third generation (day 15 to 20) , the synovial macrophages generally disappeared .Vimentin was suitable for the immunofluorescence cytochemical staining for the synovial fibroblasts .The cell purity was indicated as > 95% .The cytometric analysis indicated that purity of Vimentin and CD90 .2-labelled cells was over 95% ;the purity of CD54-labelled cells was 80% approximately .Conclusion It is a simple and effective method for primary culture of synovial fibroblasts in mice .

OBJECTIVETo explore the effect of quercetin on the invasion, migration, proliferation and cell cycle of glioma U87 cells.

METHODSGlioma U87 cells were treated with 50, 100, or 150 µmol/L quercetin (Q(50), Q(100) and Q(150) groups, respectively) or with DMSO (Q(0) group). Transwell in vitro invasion and migration assays, Click-iT Edu test and flow cytometry were performed to evaluate the effect of quercetin on the invasion, migration, proliferation and cell cycle of U87 cells.

RESULTSAfter 36 h of quercetin treatment, the cells in Q(50), Q(100) and Q(150) groups showed invasive cell percentages (relative to Q(0) group) of 52.08%, 24.63%, and 13.13%, respectively (P<0.05). After quercetin treatment for 12 h, the migrating cell percentages (relative to Q(0) group) in Q(50), Q(100) and Q(150) groups were 49.46%, 26.78%, and 14.56%, respectively (P<0.05). After 24 h of quercetin treatment, the cell proliferation ratios in Q(0), Q(50), Q(100) and Q(150) groups were 25.21%, 18.38%, 16.74% and 15.24%; the cell percentages in phase G0/Gl were 71.14%, 72.71%, 69.29%, and 66.47%, phase S were 25.32%, 22.48%, 21.96%, and 23.32%, and phase G(2)/M were 3.53%, 4.80%, 8.75%, and 10.25% in the 4 groups, respectively, showing a significant difference between groups Q(100), Q(150) and group Q(0) in phase G(2)/M cell percentages (P<0.05).

CONCLUSIONSQuercetin can significantly inhibit the invasion, migration and proliferation of glioma U87 cells by blocking the cell cycle progression.

Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Glioma ; pathology ; Humans ; Quercetin ; pharmacology

OBJECTIVETo apply pyrosequencing technique in the detection of the common pathogens in sepsis.

METHODSThe primers for amplification and sequencing in pyrosequencing were designed according to alignment of the bacterial 16S rRNA sequence. Bacterial genomic DNA was extracted for pyrosequencing, and the pathogen species were determined according to the sequencing data obtained.

RESULTSPyrosequencing effectively yielded the sequencing data of the 28 bp sequences of the pathogens and clearly distinguished the pathogen species of Streptococcus pyogenes, Streptococcus pneumonia, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Neisseria meningitides, and Salmonella, but failed to distinguish Staphylococcus epidermidis from Staphylococcus aureus.

CONCLUSIONPyrosequencing technique can effectively distinguish the common pathogens in sepsis at the species level.

Bacteria ; classification ; isolation & purification ; DNA Primers ; DNA, Bacterial ; genetics ; Microbiological Techniques ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Sepsis ; microbiology

OBJECTIVETo investigate the effect of quercetin on apoptosis and feedback regulation of MDM2-p53 in multiform glioblastoma U87 cells in vitro.

METHODSU87 cells exposed to different concentrations of quercetin (50, 100, and 150 µmol/L) were examined with flow cytometry, RT-PCR and Western blotting for detecting the cell apoptosis, MDM2 mRNA expression, and p53 and caspase-3 expressions.

RESULTSQuercetin induced obvious apoptosis in U87 cells in a concentration-dependent manner, with apoptosis rates of (12.40∓0.70)% at Q0, (22.53∓0.72)% at Q50, (29.06∓0.81)% at Q100, and (31.5∓0.45)% at Q150. Quercetin significantly increased the expressions of MDM2 mRNA and active caspase-3 protein but decreased the expression of p53 in the cells.

CONCLUSIONQuercetin promotes the apoptosis of multiform glioblastoma U87 cells mediated by caspase-3 and influences the feedback balance of MDM2-p53.

Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; drug effects ; Glioma ; metabolism ; pathology ; Humans ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Quercetin ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene.

METHODSRtM204I (ATT) mutant and rtM204 (ATG) nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing (Sanger) for comparison of the detection sensitivity and specificity.

RESULTSThe fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing (R(2)>0.99, P<0.05). Nested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing (92% vs 100%, P<0.01). The detection sensitivity of Sanger sequencing varied with the viral loads, especially in samples with low viral copies (HBV DNA ≤3log10 copies/ml), where the sensitivity was 78%, significantly lower than that of pyrosequencing (100%, P<0.01). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity.

CONCLUSIONCompared with nested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.

Adolescent ; Adult ; Aged ; DNA, Viral ; genetics ; Drug Resistance, Viral ; genetics ; Female ; Hepatitis B virus ; drug effects ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Phosphoric Acids ; Plasmids ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Young Adult

Objective To investigate the effect of quercetin on apoptosis and feedback regulation of MDM2-p53 in multiform glioblastoma U87 cells in vitro. Methods U87 cells exposed to different concentrations of quercetin (50, 100, and 150μmol/L) were examined with flow cytometry, RT-PCR and Western blotting for detecting the cell apoptosis, MDM2 mRNA expression, and p53 and caspase-3 expressions. Results Quercetin induced obvious apoptosis in U87 cells in a concentration-dependent manner, with apoptosis rates of (12.40 ± 0.70)% at Q0, (22.53 ± 0.72)% at Q50, (29.06 ± 0.81)% at Q10 , and (31.5 ± 0.45)% at Q150. Quercetin significantly increased the expressions of MDM2 mRNA and active caspase-3 protein but decreased the expression of p53 in the cells. Conclusion Quercetin promotes the apoptosis of multiform glioblastoma U87 cells mediated by caspase-3 and influences the feedback balance of MDM2-p53.

Objective To investigate the effect of quercetin on apoptosis and feedback regulation of MDM2-p53 in multiform glioblastoma U87 cells in vitro. Methods U87 cells exposed to different concentrations of quercetin (50, 100, and 150μmol/L) were examined with flow cytometry, RT-PCR and Western blotting for detecting the cell apoptosis, MDM2 mRNA expression, and p53 and caspase-3 expressions. Results Quercetin induced obvious apoptosis in U87 cells in a concentration-dependent manner, with apoptosis rates of (12.40 ± 0.70)% at Q0, (22.53 ± 0.72)% at Q50, (29.06 ± 0.81)% at Q10 , and (31.5 ± 0.45)% at Q150. Quercetin significantly increased the expressions of MDM2 mRNA and active caspase-3 protein but decreased the expression of p53 in the cells. Conclusion Quercetin promotes the apoptosis of multiform glioblastoma U87 cells mediated by caspase-3 and influences the feedback balance of MDM2-p53.

Objective To investigate the intervention mechanism of Shenqi Yiliu Prescription in a rat model of precancerous lesions of gastric carcinoma(PLGC)based on transcriptomics.Methods A PLGC rat model was constructed using composite factor modeling method.Rats were randomly divided into blank group,model group,folic acid group(0.002 g/kg),and Shenqi Yiliu Prescription high-,medium-and low-dosage groups(39.6,19.8 and 9.9 g/kg),with 10 rats in each group.They were given corresponding solutions for gavage for 90 consecutive days.The general condition of rats was observed,HE staining was used to observe the morphology gastric mucosa,immunofluorescence staining was used to detect the expression of PCNA protein in gastric tissue,transcriptomics obtains differentially expressed mRNA in gastric tissue and enriches differentially expressed pathways,ELISA was used to detect the contents of Bcl-xL,C-myc and Cyclin D1 in gastric tissue,RT-qPCR was used to detect the expression of Bcl-xL,C-myc and Cyclin D1 mRNA in gastric tissue,Western blot was used to detect the expressions of IL-6,JAK2,STAT3,p-JAK2 and p-STAT3 protein in gastric tissue.Results Compared with the blank group,rats in the model group showed decreased body mass(P<0.05),with structural disorders of the gastric mucosa,the expression of PCNA protein in gastric tissue increased(P<0.05),the contents and mRNA expression Bcl-xL,C-myc and Cyclin D1 in gastric tissue significantly increased(P<0.05),the expression of IL-6,JAK2,STAT3,p-JAK2,p-STAT3 protein significantly increased(P<0.05).Compared with the model group,the body mass of rats in Shenqi Yiliu Prescription high-and medium-dosage groups increased to varying degrees(P<0.05),the abnormal morphology of the gastric mucosa were improved to different degrees,and the expression of PCNA protein in Shenqi Yiliu Prescription high-,medium-and low-dosage groups significantly decreased(P<0.05).The results of transcriptomics experiments confirmed that the JAK-STAT signalling pathway showed significant differences between the blank group and model group,as well as the model group and Shenqi Yiliu Prescription high-dosage group.The content and mRNA expression of Bcl-xL,C-myc and Cyclin D1 in gastric tissue of Shenqi Yiliu Prescription high-and medium-dosage groups significantly decreased(P<0.05),and the protein expression of IL-6,JAK2,STAT3,p-JAK2 and p-STAT3 significantly decreased(P<0.05).Conclusion Shenqi Yiliu Prescription can improve the abnormal morphology of gastric mucosa in PLGC model rats,and its mechanism is related to regulating the IL-6/JAK2/STAT3 signaling pathway,thereby inhibiting cell proliferation.