Role of acetylation modification of host proteins in tuberculosis / 中华微生物学和免疫学杂志

Post-translational modification of host proteins induced by pathogenic microorganism plays a critical role in the development, treatment and prevention of diseases. Mycobacterium tuberculosis ( Mtb) is an intracellular pathogen that causes tuberculosis. The post-translational modification induced by Mtb infection is essential in the development and progression of tuberculosis. In recent years, it has been found that Mtb-induced host protein acetylation plays an important role in the regulation of host immunity against tuberculosis, which significantly affects the development of tuberculosis. This review focused on the role and mechanism of Mtb in regulating host protein acetylation, aiming to provide reference for future investigation on potential immunotherapy for tuberculosis.

Observation on the functional characteristics of multidrug-resistant Mycobacterium tuberculosis-infected macrophage model / 中华微生物学和免疫学杂志

Objective:To observe the characteristics of the phagocytosis and bactericidal function of multidrug-resistant Mycobacterium tuberculosis(MDR- Mtb)-infected macrophage model, and the changes of the immune response and metabolic function in the process of phagocytosis and bactericidal function, aiming to provide reference for studying the role and mechanism of macrophages in the occurrence and development of multidrug-resistant tuberculosis(MDR-TB). Methods:We established MDR- Mtb and H37Rv-infected macrophage models, and used the colony-forming unit (CFU), Magnetic Luminex ? Assay and Cholesterol Assay kit to observe the effects on phagocytosis and bactericidal function, the secretion of Th1(IL-12/23 p40, IL-27 and TNF-α) and Th2 cytokines (IL-6 and IL-10) and cholesterol metabolism. The data were analyzed by SPSS25.0 software. The data were expressed as Mean± SD and analyzed by t test or F test. P<0.05 was considered statistically significant. Results:(1) After MDR- Mtb-infected macrophages, the intracellular CFU gradually increased and reached the highest at 24 h, while the extracellular CFU gradually decreased and reached the lowest at 24 h. The intracellular CFU at 48 h was lower than that at 24 h, while the extracellular CFU was higher than that at 24 h ( P<0.05). Both intracellular and extracellular CFU at 48 h were close to those at 4 h ( P>0.05). The intracellular CFU was lower than the H37Rv group at 8-48 h, while the extracellular CFU was higher than the H37Rv group ( P<0.05). (2) The level of IL-12/23 p40, IL-27, TNF-α, IL-6 and IL-10 of MDR-TB group were higher than those of blank group ( P<0.05), but the level of TNF-α and IL-6 at 24 h and 48 h were higher than that at 4 h ( P<0.05). IL-12/23 p40 and TNF-α at 48 h and IL-6 at 24 h were lower than those of the H37Rv group, while IL-27 at 48 h was higher than that of the H37Rv group ( P<0.05). (3) The levels of cholesterol of MDR-TB group at 24 h and 48 h were lower than those of 4 h and blank group ( P<0.05), but the level of cholesterol was similar to the H37Rv group at any time ( P>0.05). (4) TNF-α reached the highest when the intracellular CFU reached the highest at 24 h, and IL-6 reached the highest when the intracellular CFU decreased at 48 h. With the decreasing of cholesterol expression, the intracellular CFU increased and then decreased. Conclusions:MDR- Mtb could induce the phagocytosis and bactericidal function of macrophages, increase the expression of Th1 and Th2 cytokines and promote the utilization and consumption of cholesterol, but this function was weaker than that of H37Rv strain.

Research on the construction of scientific research performance evaluation system for specialized hospitals based on disciplinary development / 中华医学科研管理杂志

Objective:To construct a scientific research performance evaluation index system for specialized hospitals based on disciplinary development.Methods:Building a preliminary evaluation index framework of scientific research performance through literature review and expert consultation meetings, combined with the strategic goals of strengthening specialization and weakening generalization; Delphi method was adopted to conduct two rounds questionnaires survey to collect the importance scores for each index, which confirmed the content of the index system; finally, applied the analytic hierarchy process to calculate the weight coefficient of each index.Results:The responding rates of the two rounds questionnaire survey reached 95%, and the authoritative coefficients were 0.75 and 0.76, respectively. The research results were reliable. The coordination coefficients of all levels of the two surveys were greater than 0.1, according to the significance testing, the P values were less than 0.05, and the expert opinions were well coordinated. Finally, we developed a three-level scientific research performance evaluation system under the guidance of disciplinary development, which including 3 first-level indicators, 20 second-level indicators, as well as 56 third-level indicators. Conclusions:A scientific research performance evaluation system was constructed based on scientific theories. This system combined relevant indicators, such as the development of special disciplinary of specialized hospitals with scientific validity and operability. Besides, it could serve as a tool to evaluate the scientific research performance of specialized hospitals taking into account of the practical application value for guiding scientific research development and disciplinary improvement.

The optimal reference genes for miRNA quantitative analysis in cerebrospinal fluid of patients with tuberculous meningitis / 中华检验医学杂志

Objective:To explore the feasibility of U6 and Cel-miR-39 as reference genes for quantitative detection of microRNA (miRNA) in cerebrospinal fluid (CSF) of tuberculous meningitis (TBM), and validate the difference of miRNAs between tuberculous and viral meningitis (VM).Methods:The remaining CSF specimens after routine examination were collected in Beijing Chest Hospital of Capital Medical University. A total of 36 TBM and 34 VM patients were enrolled based on the information in the medical records. Total RNA were extracted from the CSF samples, and Taqman based real-time quantitative PCR (RT-CR) analysis were performed to determine the concentration of the miRNAs in CSF. GeNorm, NormFinder and Bestkeeper software were used for stability analysis of the two reference genes. 2 -ΔCt method was used to determine the relative gene expression. Accordance of repeated tests was analyzed by Pearson correlation test. Continuous variables were compared by the t-test. Results:Among the 70 samples, the average cycle threshold (Ct) value of U6 was 30.40±3.30, while the average Ct value of Cel-miR-39 was 21.49±0.70. The expression level of Cel-miR-39 was higher than that of U6. Correlation analysis showed good accordance of the repeated tests among the reference genes and target genes analysis in the randomly selected 10 samples ( r>0.931, P<0.001). Based on the analyses results of the three software, including GeNorm, NormFinder and Bestkeeper, Cel-miR-39 presented better stability in RT-PCR analysis and was more suitable as a reference gene for miRNA quantitative determination in CSF sample of TBM patients. The relative expression levels of the three target miRNAs were calculated using Cel-miR-39 as the reference gene, and miR-126-3p (1.13±0.41 vs 3.34±0.82, t=2.452, P=0.016), miR-130a-3p (0.56±0.10 vs 2.59±0.70, t=2.960, P=0.004) and miR-151a-3p (0.64±0.25 vs 2.11±0.33, t=3.536, P<0.001) were showed significant lower expression levels in CSF in TBM group than that in VM group. Conclusions:Cel-miR-39 can be used as a reference gene for quantitative detection of miRNAs in CSF of TBM patients. Significant differences were detected in expression level of miR-126-3p, miR-130a-3p and miR-151a-3p between TBM and VM group.

Clinical Application of Plasma miR-34b-3p and miR-302a-5p in the Diagnosis of Non-small Cell Lung Cancer / 中国肺癌杂志

BACKGROUND@#MicroRNA is a kind of single-stranded non-coding RNA whose length is about 22 nucleotides and its abnormal expression is related to disease closely. This study is aiming to explore the relative expression of miR-34b-3p and miR-302a-5p in the plasma of non-small cell lung cancer (NSCLC) patients and its clinical value.@*METHODS@#The levels of miR-34b-3p and miR-302a-5p in plasma were detected by real-time polymerase chain reaction (RT-PCR) in 86 patients with NSCLC, 64 patients with pulmonary tuberculosis (PTB) and 39 healthy subjects. Analyze their value in diagnosing NSCLC by contrasting and combining carcino-embryonic antigen (CEA), neuron-specific enolase (NSE), and cytokeratin 19 fragments 21-1 (CYFRA21-1).@*RESULTS@#The levels of plasma miR-34b-3p and miR-302a-5p in NSCLC group were significantly higher than those in the PTB group and the healthy group (P<0.05). In patients with NSCLC, the levels of plasma miR-34b-3p was correlated with the diameter of tumor (P<0.01). When using one plasma marker to diagnose NSCLC, miR-302a-5p had the highest sensitivity (82.6%) and CEA had the highest specificity (81.6%). While combined two plasma markers, miR-34b-3p+miR-302a-5p had the highest sensitivity (80.2%) and miR-34b-3p+CEA had the highest specificity (81.4%). As detected multiple markers, miR-302a-5p+NSE+CYFRA21-1 had the highest sensitivity (81.4%) and miR-34b-3p+CEA+NSE had the highest specificity (90.3%). The combination of miR-34b-3p, miR-302a-5p and CEA obtained the highest area under the curve (AUC), which was 0.832. Logistic regression model indicated that miR-34b-3p was independent risk factor for NSCLC compared to control groups.@*CONCLUSIONS@#Plasma miR-34b-3p and miR-302a-5p could be used as biological markers for the diagnosis of NSCLC.

Construction of recombinant mycobacteriophage TM4-RpfE / 上海交通大学学报（医学版）

Objective·To construct recombinant mycobacteriophage TM4-RpfE to lay a foundation for experimental research about how to eradicate Mycobacterium tuberculosis in combination with anti-tuberculosis drugs,and how to shorten treatment for tuberculosis ultimately.Methods·Electrotransformation was used to introduce pJV53 plasmid into Mycobacterium smegmatis to prepare recombinant engineering bacteria.After amplification of hsp60-RpfE fusion gene by overlap PCR,a long gene fragment (homologous +hsp60-RpfE+homologous,HHRH) was amplified by multi-step overlap PCR.The DNA of mycobaeteriophage TM4 and HHRH fragment were cotransfected into the recombinant engineering bacteria by electrotransformation,then the recombinant phage from the single primary plaques were confirmed by PCR and sequencing.SDS-PAGE was used to analyze the protein expression in recombinant phage.Results·The hsp60-RpfE fusion gene at the length of 901 bp and HHRH fragment at the length of 1 873 bp were identified by overlap PCR.The PCR product produced 955 bp and 301 bp DNA bands in the first generation plaques colony.SDS-PAGE analysis showed a specific protein band at 21 000 in the recombinant phages.Conclusion·The recombinant mycobacterium phage TM4-RpfE was successfully constructed and the expression of target gene RpfE was initially verified.

Study on relation between regulation of MBL level and pulmonary tuberculosis susceptibility / 国际检验医学杂志

Objective To study the relation between the regulation of mannose‐binding lectin (MBL) level and pulmonary tuber‐culosis(TB) susceptibility .Methods A total of 142 inpatients with pulmonary TB and 120 healthy controls were recruited to par‐ticipate in this case‐control study .Serum MBL level was detected ,meanwhile the restriction fragment length polymorphism (RFLP) was adopted to detect MBL2 gene polymorphism .Results The one‐way analysis of variance was adopted to analyze the MBL level in different genotype groups ,including the group YA/YA ,XA/YA ,XA/XA ,YA/YB ,XA/YB and YB/YB ,it was found that the MBL level had statistical differences among 3 groups and between any two groups(P1 000 ng/mL ,while which in 100% individuals(26/26) carrying geno‐type XA/XA or allele B was≤1 000 ng/mL .Conclusion The MBL level may be associated with the susceptibility to pulmonary TB .The YA/YA gene for determining high MBL level may be a protected gene .

The auxiliary diagnosis value of enzyme-linked immunosorbent spot assay and flow cytometry in detection of IFN-γ in tuberculous pleurisy / 中华检验医学杂志

Objective To explore the value of IFN-γ produced or secreted by CD+4 T Lymphocytes from pleural effusion mononuclear cells for the diagnosis of tuberculous pleurisy(plTB).Methods The PEMCs of 40 patients with tuberculous pleural effusion and 30 patients with malignancy pleural effusion were selected as the tuberculosis and disease control groups, then co-cultured with the early secretory antigenic target 6 (ESAT-6) and culture filtered protein 10 (CFP-10) fusion protein (E/C).The numbers of spot forming cells(SFC) secreting IFN-γ were enumerated by ELISpot and the ratios of cells producing IFN-γ were detected by flow cytometry and intracellular cytokine staining.Moreover, the two indicators were compared between tuberculosis and disease control groups to evaluate the 2 methods detecting IFN-γ in the diagnosis of plTB.Results After E/C stimulation, the numbers of SFC were 205(125-450)SFC/5×104 PEMC in tuberculosis group and 5(2-18)SFC/5×104 PEMC in disease control group by ELISpot.The difference between two groups was statistically significant (U= 20.00, P<0.01).The proportion of IFN-γ-secreting CD+4 T lymphocytes was 3.27% (1.81%-7.34%) in tuberculosis group and 0.12% (0.06%-0.46%) in control group detected by FCM. The difference between the two groups was statistically significant (U=45.00, P<0.01).The indicators of ELISpot in detection of IFN-γ which was secreted by PEMC after co-cultured with E/C were as follows: sensitivity 92.5% (37/40), specificity 80.0% (24/30), positive predictive value 0.86, negative predictive value 0.89, positive likelihood ratio 4.63, negative likelihood ratio 0.09 and accuracy 87.1%;and for FCM, they were 87.5% (35/40), 90.0% (27/30), 0.92, 0.84, 8.75 and 0.14, respectively and accuracy 88.6%.Conclusion After E/C stimulation, the assay for IFN-γ-secreting CD+4 T lymphocytes by FCM and ELISpot is highly sensitive and specific for diagnosis of plTB as an auxiliary method.

Prognostic value of methylation status of RASSF1A gene as an independent factor of non-small cell lung cancer / 中国肺癌杂志

<p><b>BACKGROUND AND OBJECTIVE</b>Promoter hypermethylation of the RASSF1A gene is among the most abundant epigenetic deregulations in human cancer. The aim of this study is to investigate the relationship between the methylation status of RASSF1A promoter and the prognoses of non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>The methylation status of RASSF1A promoter in 150 NSCLC and 25 non-malignant tissues was determined using a methylation-specific polymerase chain reaction (MSP).</p><p><b>RESULTS</b>RASSF1A promoter hypermethylation was detected in 38.7% (58/150) of NSCLC tissues, but in none of the non-malignant tissues. The patients with hypermethylation of RASSF1A had a poor survival rate, and the relationship between the survival rate and hypermethylation of RASSF1A was statistically significant (P = 0.004). Then by using stepwise Cox proportional hazard regression testing, methylation status of RASSF1A was an independent factor affecting the NSCLC patients' survival (RR = 1.584, 95% CI: 1.040-2.411, P = 0.032).</p><p><b>CONCLUSION</b>The hypermethylation of the RASSF1A promoter may be an independent prognostic factor of NSCLC after operation.</p>

The value of the enzyme-linked immunospot assay detecting spot forming cells in the diagnosis of latent tuberculosis infection / 中华检验医学杂志

stinguish active tuberculosis and healthy cases with tuberculosis exposure history according SFC count.

Study of MTT and resazurin assays for susceptibility test to first-line anti-tuberculosis drugs / 中华检验医学杂志

Objecflve To evaluate the performance of two rapid and low-cost metheds(MTT test,and rosazurin mierotitre assay)for the detection of resistance to first-line drugs in Mycobacterium tuberculosis.Methods sixty-four Myeobaeterium tuberculosis clinical isohtes were tested by the MTT test and the rosazuxin microtitre assay(REMA)respectively,and the results were compared with those obtained with the absolute concentration method on L(o)wenstein Jensen medium.Results The MTT test and the resazurin microtitre assay showed a good agreement compared with the absolute concentration method for all first-line drugs tested.The sensitibity,specificity and accuracy of the MTT test were 94.8%,96.0%,95.3%,for RFP;93.8%,93.8%,93.8% for INH;92.9%,96.O%,95.3% for EMB,90.6%,87.5%,89.1% for SM,respectively.The sensitivity,specificity and accuracy of the resazurin microtitre assay were 92.3%,96.0%,93.8%,for RFP;90.6%,90.6%,90.6% for INH;92.9%,94.0%,93.8% for EMB,87.5%,87.5%,87.5% for SM,respectively.The Kappa value of the MTT test and the absolute concentration method for the detection of resistance to RFP,INH,EMB,SM were 0.857,0.831,0.714,0.792.respeedvely;The Kappa value of the regazurin mierotitre assay and the absolute concentration method for the detection of resistance to RFP,INH,EMB,SM were 0.871,0.826,0.826,0.750,respectively.The Kappa value of the MTT test and the resazurin microtitre assay for the detection of resistance to RFP,INH,EMB,SM wefe 0.889,0.875.0.787,0.844,respectively.Conclusions Both MTT test and the resazurin microtitre assays are simple,rapid,low-cost and sensitive for rapid detection of resistance to first-line drugs.They could be promising methods for susceptibility assay of the first-line antituberculosis drugs in low-resource countries.