Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs.

Objective To construct the recombinant plasmid of protein CFP10-MPT48-TB8.4 of Mycobacterium tuberculosis and to investigate the diagnosis potential of this fusion protein in tuberculosis serodiagnosis.Methods The recombinant fusion protein CFP10-MPT48-TB8.4 was expressed, and identified by Western blot.The ELSIA based on the purified fusion protein was done,and used for screening in 230 cases of clinical serum samples including pulmonary tuberculosis patients ( n =150 ),pulmonary disease patients other than tuberculosis (n =70) and health controls (n =103 ).The test result was analyzed by Medcale11.5 software.Results The fusion protein CFP10-MPT48-TB8.4 was successfully expressed with a purity over 95％.Specific immunogenicity of the recombinant protein was confirmed by Western blot.The overall sensitivity and specificity obtained of ELISA were 56.7％ (85/150) and 90.8％ ( 157/173 ),respectively.The specificity was 85.7 ％ (60/70) in non-tuberculosis group and 94.2％ (97/103 ) in healthy group,respectively.Conclusion The recombinant protein of CFP10-MPT48-TB8.4 has a high sensitivity and specificity and may be a potential candidate antigen in tuberculosis serodiagnosis.

Objective To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX(systematic evolution of ligands by exponential enrichment)technology. Methods A random ssDNA library with in vitro synthesized 78 nucleotides in length was subiected to 12 rounds of selection by SELEX method against MPT64 protein. The binding ability of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. Results The selective system used was as follows:in PCR amplification,annealing temperature was 65℃ and the concentration of Mg2+ was 1.5 mmol/L in optimizing library, and when preparation of ssDNA with asymmetrical PCR amplification, 0.75 mmol/L of Mg2+ was used. When using the plate for ELISA as the substrate for the selection, the pattern of electrophoretic band of PCR product after the tenth round selection became unitary and denser than that of the first round. The binding assay demonstrated that A value at 450 nm of the tenth round increased by 9.18 times as compared with that of the first round. The results showed that the affinities of the aptamers were different. The highest A value at 450 nm was 1.606, and the lowest 0.572. Conclusion A set of aptamers with considerable binding affinity to MPT64 protein are successfully picked out from the initial random DNA library.

Objective To clone and express of Rv0091 encoding protein in Mycobacterium tuberculosis,identify and characterize of the enzyme activities.Methods Construct the Rv0091 prokaryotic expression plasmid,the vector was transformed into E.coli strain BL21trxB.After induced by IPTG,recombinant protein was purified by Ni2+-NTA chromatography and analyzed for purity by SDS-PAGE gels stained with Coomassie Blue.Immunological activity was identified by Western blot.The recombinant protein molecular weight was identified by Mass spectrometry.The enzyme-coupled assay detectes enzyme activity.Results The expression plasmid pET32a-Rv0091 was constructed and expressed in E.coli.BL21trxB,and the optimum expression system was conformed.The purity of the recombinant protein was more than 95％.Western blot analysis confirmed that recombinant protein was one of Mycobacterium tuberculosis proteins.Mass spectrometry identified the relative molecular weight and theoretical molecular weight was basically the same.Enzyme assay showed the recombinant protein able to catalyze the substrate MTA.Enzymatic properties showed that the optimal buffer for the phosphate and Hepes buffer,the poor thermal stability of the enzyme,the optimal temperature of 37℃,optimal pH10-12,when the pH ≤7,the protein denaturation and loss of some vitality.Conclusion The recombinant protein methylthioadenosine nucleosidase(MTAN) was obtained and enzyme activity was detected and plays a key role in the metabolism of Mycobacterium tuberculosis.

OBJECTIVETo find an inhibitor to reduce the volatilization of formalin.

METHODThe saturated solution of sodium hydrosulphite (SHS) was sprayed on the surface of the anatomy specimens, then the concentration of formaldehyde in the air was tested.

RESULTSThe concentration of formaldehyde in the air of SHS sprayed group [(3.10 +/- 1.22) mg/m3] was significantly lower than that of the control group [(8.36 +/- 4.11) mg/m3, P < 0.01].

CONCLUSIONSHS may be a volatilization inhibitor for formalin, which could reduce the concentration of formaldehyde in the air.

Air Pollution, Indoor ; prevention & control ; Anatomy ; Formaldehyde ; analysis ; chemistry ; Sulfites ; chemistry ; Volatilization

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.
Erythrocytes ; immunology ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

Objective To study the relationship of two variants( -871A/G and -336A/G) polymorphisms of the DC-SIGN gene with the susceptibility to pulmonary tuberculosis in Chinese population.Methods Two hundred and thirty-seven tuberculosis cases and 244 controls were genotyped by pyrosequencing in this case-control study. The analysis of the relationship of the -871A/G and -336A/G polymorphisms with their susceptibility of pulmonary tuberculosis(PTB) and the relationship of the two variants with their clinical correlation of tuberculosis was performed by chi-square test. Results The genotypic frequencies of A/G + G/G and A/A of - 871, 37.6%, 62.4% respectively in cases, and 43.4%, 56. 6%respectively in controls, had no significant difference in statistics. And the genotypic frequencies of A/G + G/G and A/A of -336, 12. 2% ,87.8% respectively in cases, and 14.3% ,85.7% respectively in controls, had also no statistical difference between two groups. Interestingly, a significant association is disclosed between the promoter variant - 336G allele and fever in patients ( P = 0. 037, OR = 0. 191, 95 % CI:0. 040-0. 907 ). Conclusion The single nucleotide polymorphism of -871A/G and -336A/G in DCSIGN gene promoter might not be associated with the susceptibility to tuberculosis in Chinese. Tuberculosis patients with -336G allele are significantly protected fever.

OBJECTIVE: To explore the role of Long noncoding RNA UFC1 (lincRNA-UFC1) in modulating the metastasis and invasion of hepatocellular carcinoma (HCC) cells and the underlying mechanism. METHODS: Human HCC cell line Huh7 was infected with the lentiviral vector carrying lincRNA-UFC1 to obtain a cell line with lincRNA-UFC1 overexpression. A short hairpin RNA (shRNA) targeting lincRNA-UFC1 was delivered in human HCC BEL-7402 cells via a lentiviral vector to obtain a cell line with lincRNA-UFC1 knockdown. Expression levels of lincRNA-UFC1 in the two HCC cell lines were detected using real-time PCR, and the changes in the cell invasion and migration in response to lincRNA-UFC1 overexpression or knockdown were analyzed using Transwell and wound-healing assays. The expressions of GSK-3β/β-catenin-related proteins in the cells were detected with Western blotting. XAV-939, a GSK-3β/β-catenin inhibitor, was used for assessing the impact of lincRNAUFC1 overexpression on the invasion and migration of the HCC cells through Transwell and wound-healing assays. RESULTS: Overexpression of lincRNA-UFC1 significantly promoted the invasion and migration of Huh7 cells as compared with the control cells ( < 0.001), while lincRNA-UFC1 knockdown obviously suppressed the invasion and migration of BEL-7402 cells ( < 0.001). The results of Western blotting showed that the expressions of proteins associated with the cell invasion and migration, namely β-catenin and P-GSK-3β, were significantly upregulated in response to lincRNA-UFC1 overexpression, and were obviously lowered after lincRNA-UFC1 knockdown. Treatment of the cells with XAV-939 significantly reversed the effect of lincRNA-UFC1 overexpression on the cell invasion and migration ( < 0.001). CONCLUSIONS lincRNA-UFC1 overexpresison promotes cell invasion and migration through the GSK-3β/β-catenin axis in HCC cells .
Carcinoma, Hepatocellular ; genetics ; Cell Line, Tumor ; Glycogen Synthase Kinase 3 beta ; Humans ; Liver Neoplasms ; genetics ; Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Long Noncoding ; beta Catenin

Objective To evaluate the therapeutic efficacies of neoadjuvant immunochemotherapy (NAIC )and neoadjuvant chemotherapy (NAC )for local advanced gastric adenocarcinoma and analyze the clinicopathological characteristics.Methods A retrospective cohort study was conducted on 243 patients with locally advanced gastric cancer admitted in Army Medical Center of PLA (Daping Hospital)and the First Affiliated Hospital of China Medical University from January 2017 to July 2023. After balancing the confounding factors by inclusion and exclusion criteria and propensity score matching (PSM),the tumor pathological regression (TRG)rate and safety of the 2 neoadjuvant treatment regimens were analyzed,and the clinical pathological characteristics were analyzed to find clinical pathological characteristics related to efficacy.Results After using PSM to balance the baseline characteristics of the 2 groups of patients,53 subjects in each group were included in the analysis.In terms of TRG,the pathological complete response (pCR)rate in the NAIC group (13 patients,25％)was significantly higher than that in the NAC group (2 patients,3.8％,P<0.05 ).Similar results were observed in terms of major pathological response (MPR),with 23 patients (43％)in the NAIC group achieving MPR,while 9 patients (17％)in the NAC group achieved MPR (P<0.05 ).In terms of safety,the incidence of treatment-related adverse events (TRAEs) of any grade in the NAIC group and the NAC group was comparable (96.2％ and 96.2％,respectively).In an exploratory subgroup analysis of tumor pathological regression,the patients with clinicopathological features such as age<65 years,male,stage Ⅲ～Ⅳ A of American Joint Committee on Cancer (AJCC)staging,histological type of adenocarcinoma,high-moderate differentiated,intestinal-type gastric cancer,stage T3～4 of clinical T-staging,and stage N2～3 of clinical N-staging were more likely to benefit from NAIC.Conclusion NAIC results in a higher rate of pathological regression and a comparable incidence of adverse events when compared with chemotherapy alone for patients with local advanced gastric adenocarcinoma.