Aim To establish and optimize a method for screening HIV-1 integrase 3′-processing inhibitor. Methods Fluorescence resonance energy transfer ( FRET) was used to create an assay for screening in-tegrase 3′-processing inhibitors; wavelength was de-fined by DNaseⅠ; factors affecting IN activity were optimized, including buffer composition, substrate con-centration, enzyme concentration, metal ion concentra-tion. Results Integrase 3′-processing optimizing reac-tion conditions were buffer 1 , 500 nmol · L-1 sub-strate, 1 μmol·L-1 integrase, 20mmol·L-1 magne-sium ion. Positive drug raltegravir and myricetin could effectively inhibit integrase 3′-processing activity using this assay. Two integrase 3′-processing inhibitors were screened by this method. Conclusion The method for screening HIV-1 integrase 3′-processing inhibitor is successfully established and optimized.

Objective To comparatively evaluate the effects of a recombinant Mtb protein ESAT 6-CFP10 ( rESAT6-CFP10 ) and a purified protein derivative ( PPD ) as skin test reagents in guinea pigs . Methods Guinea pigs were sensitized with different Mycobacteria species .After sensitization , all guinea pigs were intradermally injected with rESAT6-CFP10 and PPD.At 48 h after the injection, the size of ery-thema at injection sites was measured by using a double-blind method .For guinea pigs sensitized with viable Mtb, the size of erythema at injection sites were measured at 24 h after the injection .The positive conversion rates of skin test with rESAT 6-CFP10 and PPD were calculated .Results The results of PPD skin test were positive in all guinea pigs sensitized with viable Mtb , killed Mtb and BCG with erythema diameters of (11.4 ±0.9) mm, (11.8±1.1) mm and (13.2±0.8) mm, respectively.Positive skin test with rESAT6-CFP10 was only observed in guinea pigs infected by viable Mtb-showing erythema diameters of (13.7±5.7) mm. The skin test with rESAT6-CFP10 was negative in guinea pigs sensitized by killed Mtb-and vaccinated by BCG.The skin tests by using rESAT6-CFP10 and PPD were performed on randomly selected guinea pigs at ninth day after infection by Mycobacterium tuberculosis H37Rv.At the 2nd week, totally 24 selected guinea pigs showed positive skin test results with rESAT6-CFP10 (24/24) with erythema diameters of (19.9± 3.0) mm, while only 15 out of 24 had positive PPD skin test with erythema diameters of (6.1±5.5) mm. At the 4th week, all guinea pigs showed positive PPD skin test (3/3) with erythema diameters of (12.7± 2.5) mm.Conclusion The skin test by using recombinant ESAT 6-CFP10 protein can effectively distin-guish viable Mtb infection from BCG vaccination and killed Mtb sensitization , which is a more suitable anti-gen than PPD for the early diagnosis of Mtb infection .

Objective To establish a suitable guinea pig model for evaluating the protective effects of new therapeutic TB vaccines in preclinical study .Methods The guinea pigs were subcutaneously injected with single-cell suspension of multi-drug resistant M.tuberculosis at a dose of 1000 CFU, 0.2 ml per animal.The study was divided into experimentⅠand experimentⅡ.In experimentⅠ, the guinea pigs were given immuno-therapy and/or chemical treatment on day 3 after infection .Four guinea pigs in each group were dissected at weeks 5, 7 and 9 after infection for evaluating lesion scores and histopathology changes of liver , spleen and lung, as well as bacterial load in spleen .In experimentⅡ, the guinea pigs were given immunotherapy and/or chemical treatment with different doses on day 14 after infection .All guinea pigs were dissected at week 5 after infection for evaluating lesion scores of liver , spleen and lung , as well as bacterial load in spleen .Results In experimentⅠ, all of the three treatments including immunotherapy combined with chemotherapy , immunotherapy alone and chemotherapy alone could effectively prevent organ lesion and reduce bacterial load in spleen , which were significantly different from negative control group .The immunotherapy combined with chemotherapy showed better treatment effects than other two treatments .Along with a prolonged period after drug withdrawal , the de-gree of organ lesion in immunotherapy group and chemotherapy group rebounded sharply , but only slightly in im-munotherapy combined with chemotherapy group .In experimentⅡ, animals in all treatment groups showed alle -viated organ lesion and reduced bacterial load in spleen .A relatively better treatment effect was observed in im-munotherapy combined with chemotherapy group .Conclusion The established guinea pig model of M.tubercu-losis infection showed an advantage of good repeatability .It might be used to evaluate the protective effects of new therapeutic vaccines against tuberculosis in preclinical study .

Objective To identify the cross-reactive antigens shared by Mycobacteria smegmatis(MS) and Mycobacteria tuberculosis(MTB) and to analyze their antigenicity.Methods Bacterial antigens were extracted from strains of MS and MTB by ultrasonication.Western blot assay was performed to analyze common antigens that reacted with both of the antiserum samples against MS and MTB.The extracted bacterial antigens were mixed with incomplete Freund′s adjuvant and then were injected into muscles of mice.Cytokines secreted by murine spleen lymphocytes following stimulation with various antigens of MS and MTB were determined by ELISPOT and flow cytometry on the 7th day.IgG levels in serum samples were detected by ELISA 7 days after injection.Results There were cross-reactive antigens shared by MS and MTB.Potent humoral immune responses and cellular immunity against both MS and MTB could be induced by those cross-reactive antigens after sensitization the mice by either MS or MTB antigens.Cytokines of IL-2 and IFN-γ in CD4+ and CD8+T cells of mice stimulated with MS or MTB antigens were significantly increased as compared with those of non-sensitization group and those of Brucella antigens stimulation group.ConclusionCross-reactive antigens shared by MS and MTS can effectively promote specific immune reactions to the infection of MTB, which provides a scientific basis for the development of tuberculosis vaccines.

Objective To study the expressions of osteopontin (OPN),caspase-3 and mt-P53 proteins, and their relationship in gliomas. Methods Seventy gliomas specimens of patients (glioma group) were selected, and 10 samples of non-glioma brain tissue were used as control group. The SP method was used to detect the positive rates of protein expressions of OPN, caspase-3 and mt-P53 between two groups. The relationship between protein expressions of OPN, caspase-3 and mt-P53 in gliomas and grade of gliomas were detected by Western blot assay. Spearman rank correlation was compared between the positive expression of OPN, caspase-3 and rate mt-P53. Results The positive expression rates of OPN and mt-P53 were significantly higher in glioma group (64.29%and 60%) than those of control group (no positive expression), but the positive expression rate of caspase-3 was significantly lower than that of control group (47.14%vs. 90%, P<0.05). There were no significant differences in OPN, caspase-3 and mt-P53 expressions between different gender, age, tumor size and tumor position (P>0.05). The higher the WHO classification, the higher the positive expression rates of OPN and mt-P53 (P<0.001), and the lower the positive expression rate of caspase-3 (P<0.001). With the increased level of glioma grade, OPN and mt-P53 protein levels were increased, but caspase-3 protein expression level was decreased. There was a negatively correlation between OPN and the positive expression of caspase-3, but there was a positive correlation between OPN and the expression of mt-P53 (rs=-0.720 and 0.722, P<0.05). There was a negative correlation between caspase-3 and mt-P53 expressions (rs=-0.556, P<0.05). Conclusion The higher the WHO classification, the higher the positive expression rates of OPN and mt-P53, while the lower the positive expression rate of caspase-3. The study reveals that OPN, caspase-3 and mt-P53 expressions are associated with the occurrence and the progress of gliomas. The combined detection of them can contribute to the judgment of biological behavior of gliomas.

Objective To establish a guinea pig model of latent Mycobacterium tuberculosis infec-tion for evaluating the effects of therapeutic vaccines .Methods Guinea pigs were subcutaneously inocula-ted with 5.0×103 CFU Mtb.The skin test was performed with 0.5μg recombinant ESAT6-CFP10 protein to detect positive conversion rates at different time points .Two weeks after Mtb inoculation , guinea pigs in model group received 5 mg isoniazid treatment ( three times a week for four weeks ) by oral gavage , while those in control group received normal saline .At the sixth week after Mtb infection , guinea pigs with and without isoniazid treatment were dissected for pathology examination .The pathological scores of liver , spleen and lung, as well as bacteria loads in spleen were compared between two groups .The established guinea pig model of latent infection was then validated by testing two reference vaccines ( AEC/BC02 and AEC/BC03 ) . Results Two weeks after Mtb inoculation , all guinea pigs showed positive EC skin test with induration area of (19.9±3.0) mm.Upon four weeks of isoniazid treatment , the guinea pigs in model group showed no pathological changes with zero scores in the examined organs .No bacterium was detected in spleen of ani-mals from model group.However, the total pathological score was 38.8±16.5 and bacteria load in spleen was (5.1±0.3) Log10 CFU with the guinea pigs from control group .Natural recurrence of tuberculosis in model group was observed after drug withdrawal .The total pathological scores were 48.5±23.9 and 51.3± 23.41.The bacterial loads in spleen were (4.5±1.3) and (4.2±1.1) Log10 CFU and bacterial loads in lung were (4.1±1.2) and (3.4±1.3) Log10 CFU respectively as verified with reference vaccines of AEC /BC02 and AEC/BC03.Conclusion Isoniazid treatment inhibited the proliferation of inoculated Mtb in guinea pigs.A guinea pig model of latent Mycobacterium tuberculosis infection is successfully established with an advantage of good repeatability .Therefore, it can be used to evaluate the effects of therapeutic vaccines on latent Mycobacterium tuberculosis infection.

Objective To establish a suitable guinea pig model for evaluating the protective effects of new TB vaccines in BCG prime-boost regimen .Methods Two different immunization strategies by using the recombinant TB vaccine were employed to boost BCG primed guinea pigs in this study .One was for short-term evaluation with 14 weeks interval between prime and boost immunization and another was for long -term evaluation with 54 weeks interval .In the short-term evaluation group , guinea pigs were boosted twice with the recombinant TB vaccine ( AEC/BC02 ) in every two weeks , while guinea pigs in the long-term evaluation group were boosted for three times with two weeks interval between each injection .A negative con-trol group ( NS→NS) and a BCG control group ( BCG→NS) were both set up in two evaluation groups .One week after the last immunization , all guinea pigs were challenged with M.tuberculosis.Six to seven weeks after bacteria challenge , all animals were euthanized and dissected to evaluate lesion scores of liver , spleen and lung, as well as the viable bacterial load in spleen .Results In the short-term evaluation group , the le-sion scores in those boosted with vaccine (3.33±5.00) was lower than that of BCG control group (5.56± 7.27) (P>0.05) and negative control group (47.00±28.11) (P=0.0001).The difference between BCG control group and negative control group in lesion score was also significant .The animals in vaccine boosted group had lower bacterial loads (0.78±1.55 log10 ) in spleen than that in BCG control group (1.06±1.87) (P>0.05) and negative control group (5.47±0.61) (P=0.0003).In the long-term evaluation group, the lesion score in those boosted with vaccine was lower (5.0±7.6) than that in BCG control group (14.4± 13.5) (P=0.0394) and negative control group (56.9±14.1) (P<0.0001).The animals in vaccine boos-ted group (1.00±1.86 log10) had lower bacterial loads in spleen than that in BCG control group (1.46± 1.94) (P>0.05) and negative control group (5.43±0.56) (P=0.01).There was a significant difference in bacterial load between BCG control group and negative group (P=0.0089).Conclusion The results suggest that the interval time between BCG-prime and boost immunization should be properly prolonged in the guinea pig model used for evaluating the protective effects of new TB vaccines in BCG prime -boost regimen .

More than 60% of active tuberculosis(TB) patients are smear- and culture-negative, constituting a prime group in the prevention and control of TB in China. In the existing laboratory testing technologies, immunological diagnosis is more advantageous than etiological diagnosis in the detection of smear-and culture-negative TB. Serum antibody detection reagents are cheap,easy to operate and time-sav-ing,and have been widely used in China. However,these agents are not stable in sensitivity and specificity, and because of that their accuracy in the diagnosis of smear-and culture-negative TB is doubtful. In this re-view,we summarize some problems in the use of serum antibody detection among smear- and culture-nega-tive pulmonary TB patients and discuss possible methods to solve these problems expecting to provide some ideas for promoting its development,application and policy formulation.

Objective To assess the efficacy of injection of sodium hyaluronate in the treatment of patients with disc perforation by using cone-beam computed tomography (CBCT) and temporomandibular joint (TMJ) function.Methods Twenty-eight patients (38 joints) with disc perforation observed through arthrography who received injection of sodium hyaluronate and other 20 patients (29 joints) who received disc repair were included in this study.Pain (visual analog scale,scores 0 to 100),maximal mouth opening,modified Helkimo's clinical dysfunction index and TMJ examinations with CBCT were obtained before and 3,9 months after treatment.Results A statistically significant increase (P＜0.05) in TMJ clinical outcome was observed at each follow-up.The maximal mouth opening (MMO) increased 5.89 mm and 7.64 mm at 3 and 9 months follow-up in the hyaluronic acid (HA) group,and the MMO increased 2.75 mm and 6.10 mm at 3 and 9 months follow-up in the operation group.Osseous remodeling occurred in 66％ (25 of 38 joints) of patients after HA injection and 55％(16 of 29 joints) of patients appeared bone remodeling after disc repair.Conclusions Both injection of hyaluronic acid and disc repair are effective for treatment of temporomandibular joint disc perforation.

RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.
Aptamers, Nucleotide ; Gene Library ; Heterogeneous Nuclear Ribonucleoprotein A1 ; genetics ; High-Throughput Nucleotide Sequencing ; Humans ; RNA ; SELEX Aptamer Technique