The crystal structure and biochemical properties of DHBPS from Streptococcus pneumoniae, a potential anti-infective target for Gram-positive bacteria.

The enzymes involved in riboflavin biosynthesis are considered to be potential anti-bacterial drug targets because these proteins are essential in bacterial pathogens but are absent in humans. 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) is one of the key enzymes in the biosynthesis of riboflavin. DHBPS catalyzes the conversion of ribulose-5-phosphate (Ru5P) to 3,4-Dihydroxy-2-butanone-4-phosphate (DHBP) and formate. The purified SpDHBPS enzyme, in the presence of Mg(2+) ion, catalyzed the conversion of Ru5P to DHBP at a rate of 109nmolmin(-1)mg(-1) with an apparent Km value of 181µM at 37°C. Surprisingly, our experiments first revealed that DHBPS showed activity in the presence of the trivalent metal ion, Fe(3+). Furthermore, we determined the crystal structure of DHBPS from Gram-positive bacteria, Streptococcus pneumoniae, with 2.0Å resolution. The overall architecture of SpDHBPS was similar to its homologs, which comprise one ß-sheet (five-stranded) and eight α-helices, adopting a three-layered α-ß-α sandwich fold. Similar to the homologs, gel-filtration experiments verified that the enzyme was arranged as a dimer. Although the overall fold of DHBPS was similar, the significant structural differences between the species at the active site region may be utilized to develop antibacterial agents that are species-specific.

Chronic infection of hepatitis B virus and cellular immunity / 中华微生物学和免疫学杂志

The persistent infection of hepatitis B virus (HBV) is the result of lacking specific immunity against the virus. This state is also called immune tolerance to HBV. In most cases, acute HBV infection in adults can induce specific immune response which can clear the virus. Perinatal HBV infection, however, usually progresses to chronic infection, indicating a defect in HBV-specific immune response. A typical specific immune response includes four processes, which were antigen presentation, specific CD4 + T cell activation, specific CD8 + T cell activation and B cell activation. There must be some dysfunctions in some or all of the four processes during chronic HBV infection. This article discussed the relationship between chronic HBV infection and cellular immunity, hoping to provide a reference for further study on the reconstitution of specific immunity against HBV.

Functional cure of hepatitis B from the perspective of hepatitis B virus covalently closed circular DNA / 临床肝胆病杂志

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is the template for HBV transcription and replication and exists stably in hepatocytes in the form of minichromosome. Based on the mechanism of cccDNA inactivation or clearance and the development of related drugs, this article introduces the current knowledge on the formation, transcription, and degradation of cccDNA and reviews the research advances in cccDNA-targeted drugs and biological techniques.

Comparative study and enlightenment on the evaluation system of domestic and foreign universities under the background of "Double First-Class" / 中华医学教育探索杂志

The construction of "Double First-class" has started a new journey of higher education in China. Scientific, reasonable and objective evaluation on universities is the foundation and important guarantee for the construction of first-class universities. On the basis of introducing the main university evaluation system at home and abroad, the selection of representative Chinese and world university evaluation system for comparative study has strong guiding significance for the establishment of first-class university evaluation system. Under the background of "Double First-Class" construction, based on Chinese characteristics, the author puts forward some thoughts and suggestions on perfecting the university evaluation system in China.

Ultrasound-mediated microbubble destruction enhances VEGF gene delivery to the infarcted myocardium in rats.

OBJECTIVE: To investigate the possibility of improving the delivery of vascular endothelial growth factor (VEGF) gene to the myocardium in rats by using ultrasound-mediated microbubble destruction (UMMD). METHODS: Fifteen male Wistar rats underwent left anterior descending coronary artery ligation in this study. The rats were divided into three groups 3 days after ligation. Ultrasound microbubble vectors (UMVs) attaching to pcD2VEGF121 gene were injected into the tail vein of rats with or without simultaneous echocardiographic microbubble destruction in two groups. The third group was used as control group. VEGF protein expression and formation of new blood vessels were evaluated by immunohistochemical technique during autopsy on 15 rats at 2 weeks after gene transformation. Microvascular density (MVD) in the area with myocardial infarction was counted under a microscope. RESULTS: VEGF protein expression and MVD in the ischemic myocardium were higher in the rats receiving UMMD than in the group that did not receive UMMD. CONCLUSION: UMMD is a noninvasive method to effectively improve the delivery of targeted genes to the heart.

Epigenetic regulation of hepatitis B virus cccDNA and related prospects for antiviral therapy / 临床肝胆病杂志

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is stably maintained in hepatocytes in the form of minichromosome and is considered the most important cause of chronicity of HBV infection, presence of HBV after antiviral therapy, and recurrence of hepatitis after drug withdrawal. However, due to a lack of antiviral regimens targeting cccDNA itself or the formation and transcription of cccDNA, there is an urgent need for treatment strategies targeting cccDNA. With the gradual understanding of epigenetic modification of histones of the cccDNA minichromosome, epigenetic therapy is expected to become a potential therapy for HBV. This article reviews the current status and future directions of HBV DNA methylation and cccDNA-bound histone modification, in order to provide new thoughts for epigenetic therapy for HBV.

Seroepidemiology of Epstein Barr virus in men who have sex with men in Chongqing area / 中国感染与化疗杂志

Objective To investigate the seroepidemiology of Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV) in adult men who have sex with men (MSM) in Chongqing area. Methods Nonprobability sampling method was used to test EB-CA-IgG, EB-NA-IgG and EB-VCA-IgM in the sera of 1082 MSMs from the clinical trials of HIV/AIDS treatments in Chongqing area from 2012 to 2015, and 1059 healthy individuals by means of enzyme-linked immunosorbent assay. The results were analyzed by Chi-square test. The difference was considered statistically significant when P<0.05. Results The 1082 MSM included 130 HIV positive and 952 HIV negative subjects. The prevalence of prior EBV infection was 92.6% in total MSM population, 88.5% in HIV-positive MSM, and 93.2% in HIV-negative MSM. The prevalence in total MSM and HIV negative MSM was significantly higher than that in control group (89.9%). Prior EBV infection was not?found?in?0.5%?of?the?total?MSM,?0.8%?of?HIV?positive?MSM?and?0.4%?of?HIV?negative?MSM,?all?significantly?lower?than?that?of control group (5.0%) (P<0.05).?Finally,?the?rate?of?EBV?reactivation?in?HIV?positive?MSM?(10.0%)?was?significantly?higher?than?that in control group (3.8%) and in HIV negative MSM group(4.1%) (P<0.005). Conclusions EBV infection is highly prevalent in MSM, higher than that in the general population. The rate of EBV reactivation in HIV negative MSM is similar to that in general population. The rate of seroepidemiology-based EBV reactivation is significantly higher in HIV positive MSM, which may be associated with the immunocompromised status post HIV infection.

Study on HIV prevention related knowledge-motivation-psychological model in men who have sex with men, based on a structural equation model / 中华流行病学杂志

Objective Knowledge-motivation-psychological model was set up and tested through structural equation model to provide evidence on HIV prevention related strategy in Men who have Sex with Men (MSM).Methods Snowball sampling method was used to recruit a total of 550 MSM volunteers from two MSM Non-Governmental Organizations in Urumqi,Xinjiang province.HIV prevention related information on MSM was collected through a questionnaire survey.A total of 477 volunteers showed with complete information.HIV prevention related Knowledge-motivationpsychological model was built under related experience and literature.Relations between knowledge,motivation and psychological was studied,using a ‘structural equation model’ with data from the fitting questionnaires and modification of the model.Results Structural equation model presented good fitting results.After revising the fitting index:RMSEA was 0.035,NFI was 0.965 and RFI was 0.920.Thereafter the exogenous latent variables would include knowledge,motivation and psychological effects.The endogenous latent variable appeared as prevention related behaviors.The standardized total effects of motivation,knowledge,psychological on prevention behavior were 0.44,0.41 and 0.17 respectively.Correlation coefficient of motivation and psychological effects was 0.16.Correlation coefficient on knowledge and psychological effects was-0.17 (P＜0.05).Correlation coefficient of knowledge and motivation did not show statistical significance.Conclusion Knowledge of HIV and motivation of HIV prevention did not show any accordance in MSM population.It was necessary to increase the awareness and to improve the motivation of HIV prevention in MSM population.

Study on HIV prevention related knowledge-motivation-psychological model in men who have sex with men, based on a structural equation model / 中华流行病学杂志

Objective Knowledge-motivation-psychological model was set up and tested through structural equation model to provide evidence on HIV prevention related strategy in Men who have Sex with Men (MSM).Methods Snowball sampling method was used to recruit a total of 550 MSM volunteers from two MSM Non-Governmental Organizations in Urumqi,Xinjiang province.HIV prevention related information on MSM was collected through a questionnaire survey.A total of 477 volunteers showed with complete information.HIV prevention related Knowledge-motivationpsychological model was built under related experience and literature.Relations between knowledge,motivation and psychological was studied,using a ‘structural equation model’ with data from the fitting questionnaires and modification of the model.Results Structural equation model presented good fitting results.After revising the fitting index:RMSEA was 0.035,NFI was 0.965 and RFI was 0.920.Thereafter the exogenous latent variables would include knowledge,motivation and psychological effects.The endogenous latent variable appeared as prevention related behaviors.The standardized total effects of motivation,knowledge,psychological on prevention behavior were 0.44,0.41 and 0.17 respectively.Correlation coefficient of motivation and psychological effects was 0.16.Correlation coefficient on knowledge and psychological effects was-0.17 (P＜0.05).Correlation coefficient of knowledge and motivation did not show statistical significance.Conclusion Knowledge of HIV and motivation of HIV prevention did not show any accordance in MSM population.It was necessary to increase the awareness and to improve the motivation of HIV prevention in MSM population.

Establishment and optimization for TALE-TFs construction / 重庆医学

Objective To optimize the method of transcription activator‐like effector transcription factors (TALE‐TFs) con‐struction ,some improvement and adaption were made based on the traditional methods .Methods We first constructed the basic tandem fragments with different length ,including trimer ,tetramer ,pentamer and hexamer by Golden Gate cloning technique and PCR ,then the procedure with the highest efficacy was chosen to construct our TALE‐TFs .To determine the function of the TALE‐TFs ,the plasmid pminCMV with the specific binding sequence of TALE‐TFs was constructed by fragment substitution reaction (FSR) .The transcription activating function of TALE‐TFs was confirmed by the intensity of red fluorescence ,after TALE‐TFs , pEGFP‐N1 and pminCMV plasmid were co‐transfected into 293HEK cells .Results An optimized method for TALE‐TFs construc‐tion and functional assay was established .Conclusion This method can potentially be wildly used in fields that the expression of some constitutively expressed genes needs to be modified .

Establishment of an HBV chronic hepatitis B infection mouse model by vivo transduction of HBV cccDNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To generate a mouse model of chronic hepatitis B (CHB) infection by performing in vivo transduction of hepatitis B virus (HBV) covalently closed circular (ccc)DNA.</p><p><b>METHODS</b>Nude mice were injected with HBV cccDNA at doses of 1.5, 1.0 or 0.5 mug/ml. A control group was generated by giving equal injection volumes of physiological saline. The serum levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) on post-injection days 1 and 3, weeks 1-6, 8 and 10 were assayed by reflection immunoassay. At post-injection week 10, all animals were sacrificed and liver tissues were collected. Copies of HBV DNA in serum and liver tissue were detected by real-time PCR. HBV antigens in liver tissue were detected of by immunohistochemistry. Pathological analysis of liver tissue carried out with hematoxylin-eosin staining. Linear correlation of data was determined by statistical analysis.</p><p><b>RESULTS</b>HBsAg and HBeAg were detected in sera from all three groups of cccDNA-injected mice staring at post-injection day 1 and lasting through week 10. The levels of HBsAg over the 10-week period showed two patterns of increase-decrease;the lowest level was detected at week 4 and the highest level was detected at week 8. In contrast, the levels of HBeAg over the 10-week period showed three patterns of increase-decrease; the lower levels were detected at weeks 2 and 4 and the higher levels at weeks 3 and 6. HBV DNA copies in liver tissues showed a cccDNA dose-dependent descending trend over the 10-week study period (1.5 mug/ml:1.14E+07 ± 6.51E+06 copies/g, 1.0 mug/ml:9.81E+06 ± 9.32E+06 copies/g, and 0.5 mug/ml:3.72E+06 ± 2.35E+06 copies/g; Pearson's r =0.979). HBV DNA copies in sera showed the pattern of 1.0 mug/ml cccDNA more than 1.5 mug/ml cccDNA more than 0.5 mug/ml cccDNA, and in general were higher than those detected in the liver tissues. Liver tissues from all cccDNA-injected mice showed positive immunohistochemistry staining for both HBsAg and HBeAg. HE staining showed that the liver tissues of all cccDNA-injected mice had severe fatty and vacuolar degeneration and less obvious structure of liver lobules (compared to the liver tissues from control mice).</p><p><b>CONCLUSION</b>The CHB mouse model successfully established in this study by in vivo transduction of HBV cccDNA may represent a useful tool to study the pathogenic mechanisms and potential antiviral treatments of human CHB.</p>

Impacts on hepatitis B virus replication by gene engineering at apical loop region of capsid protein / 生物工程学报

Hepatitis B virus (HBV) DNA replication takes place in the viral capsid that consists of 180 or 240 copies of HBV capsid (HBc or core) protein. The monomeric core protein contains an apical loop region that forms the spikes on the surface of viral capsid upon core dimerization and capsid assembly. To investigate the impact on HBV DNA replication through gene engineering at the spike of HBV capsid. plasmids expressing engineered HBc with linker-fused enhanced green fluorescent protein (EGFP) or shortened EGFP insertion at the spike region were constructed by Restriction Digestion and Ligation-independent Cloning (RLIC). The wildtype or mutant HBc construct was cotransfected with HBV1.1c(-), a plasmid containing 1.1 unit-length HBV genome with deficiency in HBc expression, into HEK293 cells, respectively. GFP signal was observed through a fluorescence microscope and HBV DNA replicative intermediates were assayed by Southern blotting to determine the expression and functions of different recombinants. Our results demonstrated that the RLIC method was effective to generate deletion or insertion in the apical loop region of HBc. Both HBc-EGFP recombinants with different linkers produced green fluorescence but with different subcellular distribution pattern. However, HBV DNA replication was not detected with the trans-complementation of these two HBc recombinants. In addition, other recombinants including the one only with the deletion of aa79-80 failed to support HBV replication. Taken together, our results suggest that RLIC is a robust method which can be broadly applied in gene engineering; different peptide linkers may have different influences on the functions of an engineered fusion protein; and HBc aa79-80 play a critical role for HBc to support HBV DNA replication.

Regulation of microRNA-122 on HBV replication by targeting HBx sequence / 生物医学工程学杂志

In order to find microRNA associated with HBV infection and to explore the mechanism of the infection, first of all, we found in our preliminary study that in HepG2 cells transfected with HBV expression plasmid, miR-122 expression was up-regulated, suggesting that miR-122 was related to the HBV infection. On this basis, in the present study, miR-122 and pCH9-HBV1.1 plasmid were cotransfected into HepG2 cells. Southern blot detection result showed that miR-122 can inhibit HBV replication. Using MiRanda computer software, HBx was predicted to be the target sequence of miR-122; Luciferase reporter gene system and Western blot detection of HBx protein expression changes were further used to verify the HBx expression regulated by miR-122. And finally, it can be speculated that miR-122 may affected HBV replication by regulating the expression of HBx.

Identification of recombinant intermediates of hepatitis B virus between genotype B and C in vitro / 中南大学学报(医学版)

Objective To detect the recombinant intermediates of hepatitis B virus (HBV) between genotype B and C in vitro. Methods Vector Plenti6/V5-D-topo-X was genetically modified by genotype B and C to transfect HepG2 cells. Then the HepG2 cells were amplified and sequence of the nucleic acid after the transinfection was tested and compared with RDP3Beta40 software package and bootscanning procedure in SimPlot program package. Results Three recombinant intermediates of HBV between genotype B and C were identified in vitro. Genotype C in the precore region plus the core gene spanning nucleotide positions from 1740-1838 to 2443-2485 contributed to the recombination with genotype B. Isolate R1 recombinant intermediate had 2 break points at nt2170-2172 and nt2188-2189. Nucleic acid changed from CAC to TGT and from GA to AC, respectively. Isolate R2 recombinant intermediate had a break point at nt1740-1 838, and 3 bases changed in different nucleic acid sites: from A to T at nt1740, from C to T at nt1753, and from G to A at nt1838, respectively. Isolate R3 recombinant intermediate had a break point at nt2443-2483, and 4 bases changed in different nucleic acid sites: from C to T at nt2443, from A to G at nt2452, from T to C at nt2480, and from C to T at nt2483, respectively. Conclusion The recombinant intermediates of HBV between genotype B and C have been detected in vitro and the changes have been identified in the precore region plus the core gene in genotype B and C.

Experience of scientific research file management at university / 中华医学科研管理杂志

The management of the scientific research files is an important basic work at the college,which is of great significance to the research work in both school and the scientific research organizations.However,some researchers and managers are lack of file consciousness and the management system remains unadapted to the new condition.These reasons result in the use of the dossiers inadequate or even absent.Therefore,it seems necessary and important for the perfection of the scientific research file work to take essential measures:establishing a scientific and reasonable file system,improving the file management regulation,and increasing the utilization ratio of the dossiers.

Comparison of real-time genotyping and quantitative PCR,multiplex-PCR and sequence analysis for hepatitis B virus genotypes B and C / 中华微生物学和免疫学杂志

Objective To evaluate the real-time genotyping and quantitative PCR(RT-GQ-PCR)method by comparing it with direct sequence analysis and the multiplex-PCR method.Methods RT-GQ-PCR,direct sequence analysis and the multiplex-PCR method were used to detect HBV genotypes of 113 patient samples with HBV-DNA positive.ResultsThe detection rate of RT-GQ-PCR and direct sequence analysis was 100%,and the multiplex-PCR is 94.69%.The concordance between RT-GQ-PCR and the multiplex-PCR is perfect(Kappa value =0.915),and the consistency of RT-GQ-PCR and direct sequence analysis is pretty good(Kappa value = 0.742),specially at detecting single genotype.Twenty-eight samples with genotypes B and C dual infections were detected by RT-GQ-PCR,but only 19 samples by the multiplexPCR and 13 samples by direct sequence analysis.Conclusion The RT-GQ-PCR is convenient,rapid and accurate in HBV genotyping,especially more sensitive than direct sequence analysis and the multiplex-PCR for detecting dual genotypes.The method is applicable for large-scale epidemiological study.

Construction of pGL3-SM22-SCAP (D443N) eukaryotic expression vector and its expression in CHO cells / 生物工程学报

The experiment was designed to investigate the function of SREBP cleavage-activating protein (SCAP) mutant (D443N) by constructing an eukaryotic expressive vector using a smooth muscle specific promoter SM22 (pGL3-SM22-SCAP(D443N)). SM22 promoter (pSM22) was amplified from genome DNA of mice by nested PCR, and then cloned into pMD-T vector. The SM22 promoter fragment released from the vector by Kpn I and Hind III digestion was sub-cloned into pGL3-control-Luc vector, to form pGL3-SM22-Luc. The activity of pSM22 in human vascular smooth muscle cells (VSMCs) was tested using Dual-Luciferase Reporter System. SCAP(D443) mutant amplified from plasmid pTK-HSV-SCAP(D443N) and pSM22 from mice liver were cloned into pGL3-control vector to construct pGL3-SM22-SCAP(D443N) which was transfected into Chinese hamster ovary cells (CHO) to test SCAP(D443) expression by real-time PCR and Western blot. The sequence and construction of pGL3-SM22-SCAP(D443N) were correct. SM22 promoter activity initiated the expression of luciferase in VSMCs and also drove SCAP(D443) expression in transfected CHO cells. The pGL3-SM22-SCAP(D443N) eukaryotic expression vector was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of SCAP and also in producing vascular smooth muscle specific SCAP transgenic mice.

Study on the characteristics and hepatotropism of the negative regulatory element of the HBVnt453-250 / 中华微生物学和免疫学杂志

Objective To analyze the characteristics and hepatotropism of this negative element HBVnt453-250 sequence.Methods pHBv453-250,pHBV250-453.plucHBV453-250 and plucHBV250-453 were constructed,with luciferase and enhanced green fluorecence protein(EGFP)gene as the reporter gene,respectively.After transfection of HepG2 cells with these plasmids,luciferase assays,real-time PCR and western blot assays were used to detect the gene transcription and expression level.The SV40 promoter of pGL3 control and pHBV453-250 were replaced by the cytomegalovirus early promoter,resulting in plasmids pCMVcontrol(luc)and pCMV453-250(luc).Results Compared with pHBV453-250,the mutant plasmids.with the inhibitory element inserted in difierent site or inverted orientation.exerted similar downregulation of Juciferase gene transcription and expression.Western blot analysis demonstrated the similar repression when EGFP was used as the reporter gene.By transfeeted to HepG2 cell line,the plasmid pCMV453-250(1UC)could reduce lneiferase activity(36.56%)compared with pCMLcontrol(luc).When the plasmids plueHBV453-250 and plucHBV250-453 were transfected to non-liver cell lines(A549,HeLa),luciferase gene was expressed weakly,compared with that of pGL3control(P＜0.05).Conclusion The inhibitory effect of HBVnt453-250 sequence acted in both orientation-and position-independent manners,and had no promoter selectivity and funotioned in hepatocyte-independent manner.

Mechanisms of clearance of duck hepatitis B virus from infected adult ducks / 中国免疫学杂志

Objective:To gain insight into the mechanism responsible for clearance of natural hepa DNA virus infections.Methods:A group of seven 2～3 month old ducks were infected intravenously with 10～20 ml DHBV positive serum containing 5?10 7 genomes/ml.Following inoculation,ducks were bled at weekly intervals to obtain serum samples for analysis of DHBV DNA and DHBsAg and anti DHBV antibodies.Peripheral blood mononuclear cells were collected at 10,35 days postinoculation(p.i) and used to conduct antigen specific blastogenesis assay.Liver samples were obtained at 5,30,60 days p.i for analysis of DHBV DNA and surface antigens and liver histology.Results:Infection of all 7 animals with approximately 5?10 8～1?10 9 DHBV genomes led to a transient viremia after an incubation period of 1 to 2 weeks.Liver samples contained multiple copies of all of the expected species of DHBV DNA replicative intermediates,including DHBV cccDNA during the peak of viremic phase.Further analysis showed that the absence of a prolonged viremia could be explained by immediate antigen specific blastogenesis and high titer of antibody response.Meanwhile,there was no obvious evidence of liver cell injury during transient DHBV infection.Conclusion:These results demonstrate that noncytopathic antiviral mechanisms make a role in hepa DNA virus clearance.

Screening of proteins interacting with hepatitis C virus core protein from T7-phage display library / 第三军医大学学报

Objective To screen and identify proteins that interact with the hepatitis C virus core protein by means of T7-phage display system. Methods The hepatitis C virus core protein was expressed by prokaryotic expression and used as selected molecule to biopan the T7 human liver cDNA library. The selected positive clones were identified by DNA sequence and analyzed with BLAST program in GenBank. Results After BLAST in all positive clones, one protein--Smad interacting protein 1 (SIP1) was found to interact with the hepatitis C virus core protein. Conclusion T7-phage display system is a convenient, rapid and effective method for screening interacting proteins.