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BACKGROUND:Numerous studies have demonstrated that mild hypothermia has a better protective effect on neurons after cerebral infarction. OBJECTIVE:To investigate theeffect of mild hypothermia on nerve regeneration microenvironment of infarcted area in rat models of cerebral infarction and analyze its possible effects on neural functional recovery after cerebral infarction. METHODS:Twenty out of 65 adult femaleSprague-Dawleyrats were randomly selected as the sham group. The remaining 45 rats were subjected to carotid artery ligation to establish rat models of cerebral infarction. Five rats were rejected because of modeling failureor death, the remaining 40 rats were randomly and evenly divided into cerebralinfarction and mild hypothermia groups. The head temperature of rats in the cerebral infarction group was downregulated to (37±1)℃ using a semiconductor refrigeration instrument. The rats were transferred to the room with the temperature of 25℃ after the operation. Brain hypothermia was also induced in rats from the mild hypothermia group. At 13.0-14.0 minutes after establishing rat models of cerebral ischemia, the head on the side of cerebral ischemia was tightly connected with the probe of the semiconductor refrigeration instrument. The refrigerator temperature was adjusted to 6-8℃, so as to make the temperature of brain tissue on the lesion side at 32.0-33.0℃ for 4 hours. RESULTS AND CONCLUSION:Compared with the cerebral infarction group, the BBB scores of rats inthe mild hypothermia group were distinctly increased, and the volume of infarcted area decreased. At 1 day after modeling, the expression level of growth associated protein 43 mRNA in brain tissue of rats in the mild hypothermia group was close to that in the cerebral infarction group. At 2 weeks after modeling, the expression level of growthassociated protein 43 mRNAin brain tissue of rats in the mild hypothermia group was significantly increased compared with that in the cerebral infarction group. These results suggest that mild hypothermia therapy can protect nerve cels against injury caused by cerebral infarction and promote the recovery of neurological function. Its underlying mechanism may be related to the up-regulation of the expression of growth associated protein 43 in ischemic penumbra .
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Objective To evaluate the accuracy of pyrosequencing for the mutation detection of katG gene in isoniazid resistance in Mycobacterium tuberculosis using Meta-analysis.Methods Searching PubMed,Web of Science,Elsevier,and China National Knowledge Infrastructure (CNKI),Weipu and WANFANG DATA to obtain the relevant English and Chinese-language articles,respectively.A written protocol and explicit study selection criteria was followed.Quality of included trials was assessed by QUADAS (quality assessment of diagnostic accuracy studies).Subsequently,the characteristics of the included articles were appraised and extracted.Heterogeneity of the included articles was tested by using STATA 10.0,which was used to select proper effect model.The fixed effects model was adopted using Meta-Disc software.Finally,sensitivity analysis was performed.Results Totally 114 research papers were collected and 9 articles were selected.The accordance between pyrosequencing and conventional sequencing result was 100％.Eight studies were involved including 945 specimens when katG gene was detected.The overall sensitivity and specificity were 0.77 (0.73,0.80) and 1.00 (0.99,1.00),respectively.The area under the SROC was 0.9882.As inhA gene was detected,the overall sensitivity and specificity were 0.19 (0.15,0.24) and 1.00 (0.98,1.00).The test was stable.Conclusions Our meta-analysis reveals that pyrosequencing is a highly specific tool for detection mutation of katG gene of isoniazid resistance.This result suggests that it is useful for screening of isoniazid resistance in diagnostic test.(Chin J Lab Med,2013,36:329-332)
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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.
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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.
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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.
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Objective To test the drug susceptibilities in Mycobacterium tuberculosis by microscopic observation drug susceptibility(MODS)and evaluate the method for the detection susceptibility in second-line drugs.Methods To set up the MODS method.the drug susceptibilities of 4 second-line drugs in Mycobacterium tuberculosis were tested in 24 well plates.The test conditions were discussed.The resistance to protionamide(PTH),amikacin(AMK),capreomycin(CPM)and levofloxacin(LVF)of 60 MTB isolated from 2007 to 2008 in Shanghai Pulmonary Hospital were detected and compared with that of L-J method.The isolates were tested by minimum inhibitory concentration(MIC)method when the drug susceptibilities were not consistent.Results Among the 60 Mycobacterium tuberculosis clinical isolates.the results of drug susceptibility were confirmed by MODS,absolute concentration method and the accordance rate of PTH,AMK,CPM and LVF were 96.7%(58/60),98.3%(59/60),91.7%(55/60)and 96.7%(58/60),respectively.If the result of absolute concentration method was as the standard.the sensitivity.specificity.positive and negative predictive value as well as accuracy were 100%,87.5%,87.5%,100%and 96.7%in PTH;100%,90.0%,90.0%,100%and 98.3% in AMK;76.9%,95.7%,83.3%,93.8%,91.7% in CPM;100%,96.0%,83.3%,100% and 96.7% in LVF respectively detected by MODS assay.Conclusion MODS is a rapid and simple method for susceptibility testing of second-line drug in Mycobacterium tuberculosis.
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OBJECTIVE To set up and evaluate the microscopic observation drug susceptibility assay(MODS)technology and use it to detect rapidly Mycobacterium tuberculosis(MTB).METHODS The 24-hole cell culture plate method of liquid culture were used to set up MODS technology.The MODS technology was used to detect MTB and non-M.tuberculosis(NMT)isolates comparing with Lowenstein-Jensen(L-J) method.RESULTS When bacterial concentration was 3?103 CFU/ml,the time of reading test-result was the seventh day by MODS;four kinds of NMT(M.phlei,M.kansasii,M.chelonaeand M.marinum) in the liquid medium in the observation was similar to the growth of cording,it was difficult to distinct from the cording of MTB in the liquid medium;When using the 4-Nitro-benzoic acid 800 ?g/ml,thiophene-2-carboxylic acid hydrazine 2.5 ?g/ml for testing conditions,the correct diagnosis can be improved;the test results of clinical isolates by MODS were highly concordance rate with the results of L-J.If the results of L-J was the golden standard,the sensitivity,specificity,positive and negative predictive value as well as accuracy by MODS was 95.7%,100%,100%,99.9% and 97% respectively.CONCLUSIONS The results of MODS in detection of MTB are highly concordance wih the results of L-J method;MODS assay can be used for rapid detection of tuberculosis,with a rapid,simple,inexpensive,and other advantages.
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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.