The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol （EMB） resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization （RDBH...The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol （EMB） resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization （RDBH） in addition to evaluating the clinical value with application of PCR-RDBH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates （65.9%） showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation （38/91, 41.8% ） and the ATA mutation （16/91, 17.6% ） were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing fcr one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.展开更多
Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a...Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a result, after mycobacterium and non-mycobacterium standard specimens were duplicated by PCR, mycobacterium standard specimens reproduced 360bp DNA fragments; on the other hand, non-mycobacterium specimens did not reproduce any fragments except for hemolytic streptococcus and corynebacterium pseudodiphtheriticum which had the same results as mycobacterium standard specimens. Sensitive test is able to detect lpg tuberculosis mycobacterium DNA. The probe test showed that, among 21 oligonucleotide probes, probe-M. fortuitum and M. marinum were cross-hybrid; the other probes were specific. We used the new method to identify 126 mycobacterium clinical isolated specimens. The test results of this new method matched with conventional method. In conclusion, compared to the traditional method, the use of rpob gene chip technology to identify mycobacterium species will be faster, more accurate and higher value in application.展开更多
The coding regions of Ag85B MPT-64, and ESAT-6 secreted proteins were cloned initially into the eu-karyotic expression vector pJW4303, then transformed to E. coli Top 10 strain for plasmid DNA extraction and further a...The coding regions of Ag85B MPT-64, and ESAT-6 secreted proteins were cloned initially into the eu-karyotic expression vector pJW4303, then transformed to E. coli Top 10 strain for plasmid DNA extraction and further analysis. Plasmids containing the right insertion were se-quenced to confirm their identity. COS7 cells were trans-fected with a mixture containing serially diluted plasmid DNA encoding three secreted proteins and Lipofectin (Gibco). The supernatants and pellets prepared from various cell lines were run on SDS-PAGE gel and the expression of these proteins in COS7 cells were demonstrated by immunoblot using polyclonal or monoclonal antiserum of M.TBH37Rv. 21 days after first vaccination of C57BL-6 mice by all three recombinant eukaryotic expressing vectors, antibody titer for Ag85B reached 1:3200. 21 days after second vaccination, the antibody titer reached 1:102400. The highest antibody levels induced by multivalent vaccines after the second injection were equal to or even greater than the展开更多
The situation of tuberculosis and M. tuberculosis （MTB） drug resistance is severe in China.Among the MTB isolates, 27.8% were resistant to one primary antituberculosis drug at least. Isoniazid （INH） is one of the ...The situation of tuberculosis and M. tuberculosis （MTB） drug resistance is severe in China.Among the MTB isolates, 27.8% were resistant to one primary antituberculosis drug at least. Isoniazid （INH） is one of the first-line anti-TB drugs. The rates of primary resistance and acquired resistance to INH were 11.0% and 31.0%, respectively. MTB strains resistance to INH is mainly caused by the alterations in several genes encoding the molecular targets as follows： catalase peroxidase （katG）,展开更多
Tuberculosis(TB),caused by Mycobacterium tuberculosis,continues to pose a significant threat to global health.The resilience of TB is amplified by a myriad of physical,biological,and biopharmaceutical barriers that ch...Tuberculosis(TB),caused by Mycobacterium tuberculosis,continues to pose a significant threat to global health.The resilience of TB is amplified by a myriad of physical,biological,and biopharmaceutical barriers that challenge conventional therapeutic approaches.This review navigates the intricate landscape of TB treatment,from the stealth of latent infections and the strength of granuloma formations to the daunting specters of drug resistance and altered gene expression.Amidst these challenges,traditional therapies often fail,contending with inconsistent bioavailability,prolonged treatment regimens,and socioeconomic burdens.Nanoscale Drug Delivery Systems(NDDSs)emerge as a promising beacon,ready to overcome these barriers,offering better drug targeting and improved patient adherence.Through a critical approach,we evaluate a spectrum of nanosystems and their efficacy against MTB both in vitro and in vivo.This review advocates for the intensification of research in NDDSs,heralding their potential to reshape the contours of global TB treatment strategies.展开更多
The situation of tuberculosis （TB） and Mycobacterium tuberculosis （ M. tuberculosis） drug resistance is still very serious in China. Improper treatment for TB may lead to a prolonged course of antimicrobial therap...The situation of tuberculosis （TB） and Mycobacterium tuberculosis （ M. tuberculosis） drug resistance is still very serious in China. Improper treatment for TB may lead to a prolonged course of antimicrobial therapy and spreading of drug resistant strains. Thus, rapid detection of drug resistant strains will allow prompt initiation and adjustment of effective chemotherapeutic treatment of TB. Ethambutol （EMB） is a first line anti-TB drug. embB Met306 is located in a cytoplasmic loop that forms an EMB resistance determining region,展开更多
文摘The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol （EMB） resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization （RDBH） in addition to evaluating the clinical value with application of PCR-RDBH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates （65.9%） showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation （38/91, 41.8% ） and the ATA mutation （16/91, 17.6% ） were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing fcr one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.
文摘Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a result, after mycobacterium and non-mycobacterium standard specimens were duplicated by PCR, mycobacterium standard specimens reproduced 360bp DNA fragments; on the other hand, non-mycobacterium specimens did not reproduce any fragments except for hemolytic streptococcus and corynebacterium pseudodiphtheriticum which had the same results as mycobacterium standard specimens. Sensitive test is able to detect lpg tuberculosis mycobacterium DNA. The probe test showed that, among 21 oligonucleotide probes, probe-M. fortuitum and M. marinum were cross-hybrid; the other probes were specific. We used the new method to identify 126 mycobacterium clinical isolated specimens. The test results of this new method matched with conventional method. In conclusion, compared to the traditional method, the use of rpob gene chip technology to identify mycobacterium species will be faster, more accurate and higher value in application.
基金This work was supported by the Chinese National High-Tech "863" Project (Grant No. 2001AA213141).
文摘The coding regions of Ag85B MPT-64, and ESAT-6 secreted proteins were cloned initially into the eu-karyotic expression vector pJW4303, then transformed to E. coli Top 10 strain for plasmid DNA extraction and further analysis. Plasmids containing the right insertion were se-quenced to confirm their identity. COS7 cells were trans-fected with a mixture containing serially diluted plasmid DNA encoding three secreted proteins and Lipofectin (Gibco). The supernatants and pellets prepared from various cell lines were run on SDS-PAGE gel and the expression of these proteins in COS7 cells were demonstrated by immunoblot using polyclonal or monoclonal antiserum of M.TBH37Rv. 21 days after first vaccination of C57BL-6 mice by all three recombinant eukaryotic expressing vectors, antibody titer for Ag85B reached 1:3200. 21 days after second vaccination, the antibody titer reached 1:102400. The highest antibody levels induced by multivalent vaccines after the second injection were equal to or even greater than the
基金This study was supported by the a grant from Army Research Foundation for Outstanding Person (No. 01J020).
文摘The situation of tuberculosis and M. tuberculosis （MTB） drug resistance is severe in China.Among the MTB isolates, 27.8% were resistant to one primary antituberculosis drug at least. Isoniazid （INH） is one of the first-line anti-TB drugs. The rates of primary resistance and acquired resistance to INH were 11.0% and 31.0%, respectively. MTB strains resistance to INH is mainly caused by the alterations in several genes encoding the molecular targets as follows： catalase peroxidase （katG）,
基金support from the S˜ao Paulo Research Foundation(FAPESP,Brazil),Grant numbers#01664-1,#2022/02661-3 and#2020/16573-3,respectivelyNational Council for Scientific and Technological Development(CNPq):Productivity Research Fellows(PQ CNPq):305408/2022-4This study is part of the National Institute of Science and Technology in Pharmaceutical Nanotechnology:a transdisciplinary approach,INCT-NANOFARMA,which is supported by S˜ao Paulo Research Foundation(FAPESP,Brazil)Grant#2014/50928-2,and by“Conselho Nacional de Desenvolvimento Científico e Tecnol′ogico”(CNPq,Brazil)Grant#465687/2014-8.Prof.H.A.Santos acknowledges financial support from the Research Council of Finland(Grant No.331151)and the UMCG Research Funds.
文摘Tuberculosis(TB),caused by Mycobacterium tuberculosis,continues to pose a significant threat to global health.The resilience of TB is amplified by a myriad of physical,biological,and biopharmaceutical barriers that challenge conventional therapeutic approaches.This review navigates the intricate landscape of TB treatment,from the stealth of latent infections and the strength of granuloma formations to the daunting specters of drug resistance and altered gene expression.Amidst these challenges,traditional therapies often fail,contending with inconsistent bioavailability,prolonged treatment regimens,and socioeconomic burdens.Nanoscale Drug Delivery Systems(NDDSs)emerge as a promising beacon,ready to overcome these barriers,offering better drug targeting and improved patient adherence.Through a critical approach,we evaluate a spectrum of nanosystems and their efficacy against MTB both in vitro and in vivo.This review advocates for the intensification of research in NDDSs,heralding their potential to reshape the contours of global TB treatment strategies.
基金This research was supported by the Army Research Foundation forthe Outstanding Person with Ability (No.01J020).
文摘The situation of tuberculosis （TB） and Mycobacterium tuberculosis （ M. tuberculosis） drug resistance is still very serious in China. Improper treatment for TB may lead to a prolonged course of antimicrobial therapy and spreading of drug resistant strains. Thus, rapid detection of drug resistant strains will allow prompt initiation and adjustment of effective chemotherapeutic treatment of TB. Ethambutol （EMB） is a first line anti-TB drug. embB Met306 is located in a cytoplasmic loop that forms an EMB resistance determining region,
