Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein,infected-cell protein 22 (ICP22), in the nucleus by observing the localization of ICP22-EGFP fusion protein.Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus,persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracyclinedependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of internal ribosome entry sites (IRES) on transcriptional regulation.

The three immediate-early proteins of HSV-1, ICPO, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.

HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off. Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.

Objective To detect and analyze thyroid tumor by two-dimensional sonogram and Doppler parameter, and evaluate the value of high-frequency ultrasound in diagnosing thyroid benign and malignant tumors. Methods The ultrasonic images of 104 thyroid tumor from 80 patients with typical features were collected. Thyroid tumor was classified into benign and malignant nodules, based on the shape, border, or the rear wall echo, echo attenuation loss, internal echo, and microcalcifications in two-dimensional sonogram and systolic blood peak velocity (Vmax) and resistant index (RI) in Doppler examination. The expected results of high frequency ultrasound were compared with pathological results on consistency and error rate. Results Prediction of benign tumor by high frequency ultrasound was 66, and pathology 61, consistency rate of the two was 92.4%. Prediction of malignant lesions was 38, and pathological examination 32, consistency rate of the two was 84.2%. The total coincidence rate was 89.4%(93/104) and the error rate was 11.6%( 11/104). Conclusions The typical features of thyroid tumor on high-frequency ultrasound are helpful in diagnosis of benign or malignant nodules, which is valuable in guiding clinical treatment.

Objective To study the effects of semaphorin 5A (SEMA5A) gene silencing by lentivirus?mediated short hairpin RNA(shRNA)on biological activity of malignant melanoma cell line A375. Methods Two pairs of interference sequences for SEMA5A gene(shRNA1 and A375?shRNA2)and a pair of control interference sequences were designed to build lentiviral vectors, which were then transfected into HEK293T cells to gain lentivirus. A375 cells were divided into three groups:experimental group(A375?shRNA1 and A375?shRNA2 cells)transfected with the lentivirus containing shRNA1 or shRNA2, negative control group (A375?con cells) transfected with that containing the control shRNA, and blank control group(A375 cells)receiving no transfection. The A375 cells with stable knockdown of SEMA5A gene expression were screened by puromycin. Subsequently, reverse transcription?PCR and Western?blot analysis were performed to detect mRNA and protein expressions of Semaphorin 5A in these cells, and methyl thiazolyl tetrazolium(MTT)assay was applied to evaluate the growth of cells. The scratch assay and invasion assay were conducted to estimate migration and invasion ability of cells. Results The lentivirus containing the SEMA5A?targeting shRNAs or control shRNA was successfully transfected into A375 cells, and stably transfected cells were gained after puromycin selection. The expressions of semaphorin 5A mRNA and protein in the A375?shRNA2 cells were significantly reduced compared with those in the A375?con and A375 cells(all P < 0.05). MTT assay showed that the growth of A375?shRNA2 cells was significantly slower than that of A375?con and A375 cells(both P<0.05), while there was no significant difference in the growth rate between A375?con and A375 cells(P>0.05). The scratch assay showed that there was no obvious cell migration into the scratch in the experiment group, whereas the scratch was almost covered by cells in the negative control group and blank control group. The invasion assay showed that the number of A375?shRNA2 cells passing through the Transwell chamber was significantly smaller than that of A375 and A375?con cells(both P<0.05), while there was no significant difference between that of A375 and A375?con cells(P > 0.05). Conclusion The silencing of SEMA5A gene by lentivirus?mediated shRNA could effectively down?regulate the expression of semaphorin 5A, and inhibit the growth, invasion and migration of A375 cells.

Highly Active Anti-Retroviral Therapy (HAART) has effectively inhibited the prevalence of HIV-1 and reduced the death rate caused by AIDS. In recent years,the emergence of resistance-conferring RT gene mutations in HIV-1 strains has become the major reason for HAART failure. The detection of drug resistance is important for the HAART regimen choice and novel drug development. A novel assay for the detection of HIV-1 RT drug resistance mutations was developed. HIV-1 drug resistance and wild strains in B subtypes were investigated using Two-Step Mutagenically-Separated PCR (MS-PCR),and point mutations including M41L,K70R,K103N,Y181C,T215F were detected. A longer mutant type primer was designed,using microplates hybridization and ELISA technique to detect several point mutations within a mixed mutant-wild type population. The results indicate that the Two-Step MS-PCR is as sensitive and specific as that in the traditional MS-PCR and MS-PCR combined with ELISA can give a good P/N quotient with better sensitivity,low cost,relatively less time consumption and high-throughput screening. It will be used in clinic usage for the detection of HIV-1 drug resistance mutations as well as other point mutations.

Herpes simplex virusⅠ(HSVⅠ) regulating the pathway of transcription and translation modify in host cell is a very systematic and complicate system. A clear understanding of the concrete mechanisms of infection will greatly help to comprehend the virus replication and the interaction with the host cell. By the analysis of 2-DE, the heterogeneous nuclear ribonucleoprotein H2 in human fetal liver cell represent distinction after the HSVⅠinfection.Utilization of Northern blot and Western blot technologies verified the expression of hnRNP H2 in different stage of virus infection is varied.

HIV-1 envelope glycoprotein gp41,which is a hopeful target for HIV-1 fusion inhibitors,plays a critical role in the fusion of viral and cellular membranes.In order to build up the screening assay of HIV-1 fusion inhibitors targeting gp41,HIV-1 gp41 5-helix and 6-helix were expressed in prokaryotic cells.Gp41 5-helix and 6-helix recombined plasmids were constructed by using PCR,enzyme digestion and ligation taking the clade B HIV-1 genome as a template.The plasmid was transferred into E.coli BL21(DE3)and then induced by IPTG.The expressed protein was purified by affinity chromatography after denaturation and renaturation.The SDS-PAGE analysis was used during expression and purification.Native-PAGE was used to identify the interaction between gp41 5-helix and T-20.The result will be helpful to build up the screening assay of HIV-1 fusion inhibitors targeting gp41.

The pol gene of HIV-1 encodes mainly three enzymes: reverse transcriptase (RT), protease (PR) and integrase (IN). Currently, FDA approved drugs targeting RT and PR are available and administered in various combinations, while no anti-IN drug was approved. HIV-1 integrase is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multi-drug therapy of AIDS. The 288 amino acids of IN (32kDa) recognizes specific sequences in the long terminal repeats (LTRs) of the retroviral DNA. The IN protein catalyzes the 3′-processing step and the 5′-strand transfer step reaction in vivo, which was called integration and this reaction could be analysed by ELISA Assay in vitro. It has been reported that F185K and C280S mutations of HIV-1 integrase would improve the enzyme solubility, and the catalytical activity of the enzyme was the same as that of the wild-type enzyme in vitro. In order to build the platform of screening inhibitor against integrase of HIV-1 virus, the IN enzyme was expressed and the function of integrase protein was assayed. The cDNA of clade B HIV-1 genome was used as a template, overlapped PCR was used to construct site mutagenesis of F185K/C280S and NdeI/Xho I restriction sites were brought in. The PCR product was cloned into the prokaryotic vector pET-28a(+) to form a recombined plasmid, transferred into the host cell E.coli(BL21 DE3). The recombined clones were identified by PCR and Nde I/Xho I digestion .The positive plasmid was sequenced, and the successfully recombined plasmid in the host cell was induced by IPTG. The expressed IN protein was puriied sy the Co+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombined IN protein. The recombinant protein was soluble, and expressed highly and stably in E.coli. The molecular weight of the expression product was identical to the expectation. The IN protein was proved to be functional in 3′ processing and 5′strand transfer by ELISA. It will be helpful to build the platform of screening inhibitors against HIV-1 integrase.

Objective: To explore the relationship between plasma Jagged1 protein level and coronary collateral circulation (CCC) formation in patients with coronary artery disease(CAD). Methods: According to coronary angiography (CAG) examination, our research was categorized in 2 groups: CAD group, n=89 patients with at least one of left anterior descending (LAD), left circumflex(LCX) or right coronary artery(RCA) stenosis ≥ 95％ and Control group, n=30 subjects without abnormal findings by CAG. Based on Rentrop grading system, CAD group was further divided into 2 subgroups: Good CCC subgroup, n=42 patients with Rentrop grade ≥ 2 and Poor CCC subgroup, n=47 patients with Rentrop grade≤1. Plasma levels of Jagged1 protein,vascular endothelial growth factor (VEGF) were measured by ELISA and the relevant correlation study was conducted by multivariate regression analysis. Results: Compared with Control group, CAD group had increased plasma levels of Jagged1 protein (38.74±10.60)ng/L vs (23.04±8.97)ng/L and elevated VEGF (113.98±30.80)pg/L vs (72.73±14.55)pg/L. Compared with Poor CCC subgroup, Good CCC subgroup presented increased Jagged1 protein (46.77±8.49)ng/L vs (31.56±6.26)ng/L and elevated VEGF (128.10±20.24) pg/L vs (92.43±21.09)pg/L. Correlation study showed that Jagged1 protein was positively related to VEGF in CAD patients (r=0.730, P<0.01); multivariate regression analysis indicated that Jagged1 protein (OR=1.318, P=0.000) and VEGF (OR=1.043, P=0.043) were the independent predictors for CCC processing.Conclusion: CAD patients with good CCC had the higher plasma Jagged1 protein level than the patients with poor CCC which implied that Jagged1 protein played important role in CCC processing, such finding may provide a new direction for treating CAD patients in clinical practice.