Objective To construct and identify the screening vector pUC18-EGFP,using EGFP as an indicator. Methods The EGFP gene was prepared by PCR and cloned into pUC18 resulting the vector pUC18-EGFP. Then DNA fragment was inserted into the MCS of pUC18-EGFP to test its practicability based on green fluorescence. Results The pUC18-EGFP was confirmed correctly by restriction enzyme analyses. The pUC18-EGFP was used to select recombinants. The green strains on the plate were confirmed by restriction enzyme and DNA analyses,which were E.coli harboring recombinants. Conclusion The screening vector PUCl8-EGFP was constructed successfully. Thus,we can select the positive clones on plates based on the green fluorescence of EGFP.

To establish a cell-based rapid luciferase suppression assay for high-throughput screening (HTS) anti-alphaviruses compounds screening, which could cause viral encephalitis, raise the social issues associated directly with public health and huge economic burden to the society. The Gaussia luciferase assay system was used for HTS model for identifying inhibitors of labeled virus XJ160-GLUC. The decreased 50% GLUC activity inhibition ratio was deemed to be the screening positive index. The reaction system in this model was optimized, and the reliability of the model was evaluated. For HTS model's optimization, cells were infected with XJ160-GLUC at an MOI of 0.025 PFU/cell. The supernatant treated with compounds 48h were collected for GLUC expression detection. In the model, Z' factor was up to 0.71, demonstrating that HTS assay for identifying inhibitors that target all aspects of the viral life cycle of XJ160-GLUC was stable and reliable. After screening 8080 compounds (five-in-one), 341 positive samples were selected, and the positive rate was 4.2% with a cutoff at 50% inhibition. Then 1705 compounds were screened subsequently and the positive rate was 1.1% with obtaining 19 positive compounds. These results will lay the foundation for finding the anti-alphaviruses' drug targets.
Alphavirus ; drug effects ; genetics ; metabolism ; Animals ; Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Genes, Reporter ; High-Throughput Screening Assays ; methods ; Luciferases ; genetics ; metabolism

Objective To analyze the differentiation status of CTL and to evaluate its clinical val-ue in patients with HIV/HCV coinfection .Methods Twenty-eight patients with HIV/HCV coinfection and twelve patients with single HCV infection were enrolled in this study .The technique of Fibro-Scan was used to evaluate liver fibrosis .The viral load of HCV was detected by real-time quantitative PCR .Flow cytometry analysis was performed to measure the differentiation status of CTL .Results Both of the levels of alanine transaminase ( ALT) and alkaline phosphatase ( ALP) in patients with HIV/HCV coinfection were signifi-cantly higher than those in patients with single HCV infection [(53.7464±48.1180) U/L vs (27.4750± 13.9850) U/L, P=0.012;(24.5071±8.1940) g/L vs (16.9667±7.1890) g/L, P=0.009].The liver stiffness of patients with HIV/HCV coinfection was higher than that of patients with single HIV infection [(5.9500, 5.8250) Kpa vs (5.1500, 1.0500) Kpa, P=0.117].Compared with the patients with single HCV infection, the patients with HIV/HCV coinfection showed higher viral loads of HCV [( 6.4768, 5.3434) lg copy/ml vs (2.6815, 1.6990) lg copy/ml, P=0.012], but lower clearance rate of HCV [32.14%vs 75%, P=0.032].Compared with the patients with single HCV infection , the patients with HIV/HCV coinfection showed lower percentages of CD 27+CD28+CTL [(28.265±15.095)%vs (18.068±10.263)%, P=0.017), but higher percentages of CD27+/-CD28+CTL [(62.449 ±14.561)% vs (71.111±12.681)%, P=0.066].A trend toward negative correlation was observed between the percent -age of CD27+CD28+CTL and the degree of liver stiffness (r=-0.310, P=0.058).Conclusion HIV in-fection could accelerate the progression of liver disease in patients with HIV /HCV coinfection by affecting the differentiation of CTL .

Objective To understand the influence of HIV infection on hepatitis C progress in patients co-infected with HIV and hepatitis C virus (HCV) and related immune mechanism. Methods Twenty eight patients co-infected with HIV/HCV and 12 patients with simplex HCV infection were enrolled. The liver function and hepatic fibrosis progress were evaluated by detecting peripheral blood and with Fibro-Scan. The viral load of HCV was detected by using real time quantitative PCR. And the percentage of Treg/CD4 +T lymphocyte cell was tested by using flow cytometry. Results The levels of ALT and ALP in HIV/HCV co-infection group were (76.16 ± 81.248)U/L,(24.507 1 ± 8.194)g/L respectively,higher than those of simplex HCV infection group [(27.475 0±13.985)U/L,(16.966 7±7.189)g/L],the differences were statistical significant. P value was 0.012 and 0.009 respectively. The liver fibrosis index in HIV/HCV co-infection group was 5.950 0-5.825 0 Kpa,higher than that in simplex HIV infection group(5.150 0-1.050 0 Kpa),and the difference was nearly statistical significant(P=0.077). The HCV viral load in HIV/HCV co-infection group was(6.476 8-5.343 4)lg copy/ml,higher than that in simplex HCV infection group[(1.699 0-2.681 5)lg copy/ml],and the rate of HCV clearance in HIV/HCV co-infection group was 32.14%, lower than that in simplex HCV infection group(75.00%). P value was 0.012 and 0.032 respectively. The percentage of Treg/CD4+T lymphocyte cell in HIV/HCV co-infection group was (7.460 0%-2.287 5%),higher than that in simplex HCV infection group (5.965 0%-2.105 0%),and the difference was significant (P=0.032). The percentage of Treg/CD4 + T lymphocyte cell was significantly related with HCV viral load(ρ=0.350,P=0.027),and HCV viral load was significantly related with the liver fibrosis index(ρ=0.487,P=0.001). Conclusion HIV infection could accelerate the progress of hepatitis C,and Treg cells were involved in this progress.

Objective To investigate the effect of time interval between diagnosis and surgical treatment on the prognosis of breast cancer.Methods A retrospective study that include a total of 252 female patients who underwent breast cancer diagnosis and treatment in the Second Affiliated Hospital of Xi'an Jiaotong University from April 2012 to August 2014 were included in the present study,the average age was (58.2 ± 10.8) years old,range from 31 to 67 years old.General demographic information and data of tumor were collected.Information on postoperative recurrence,metastasis,death,and disease-free survival status of breast cancer patients were followed up 5 years by outpatient follow-up or telephone follow-up.All participants were divided into four groups (＜2 weeks,2-4 weeks,4-8 weeks,≥8 weeks) by the time interval between diagnosis and surgical treatment,including 26,118,78 and 30 cases,respectively.In addition,according to the diameter of breast cancer tumors,all participants were divided into three groups (＜20 mm,20-40 mm,and ≥40 mm),including 99,124,and 29 cases,respectively.According to the results of pathological examination of the lymph nodes obtained during intraoperative dissection,the all participants were divided into three groups (lymph nodes without metastasis,1 to 3 metastasis,and ≥3 metastasis),including 66,124,and 62 cases,respectively.The Cox proportional regression risk models were used to assess the hazard ratio (HR) and its 95％ confidence interval (CI) of time interval between diagnosis and surgical treatment with the prognosis of breast cancer,with adjustment for age,education levels and body mass index.Further,stratified analysis by tumor characteristics,including pathological type,histological grade,tumor diameter,lymph node metastasis,and receptor expression were also conducted to evaluated the above association.Kaplan-Meier survival curve was used to evaluate the effects of time interval between diagnosis and surgical treatment on the prognosis of breast cancer.Results The Kaplan-Meier survival curves for the five-year follow-up of total survival time between 4 different time intervals groups showed significantly different (P ＜0.001),and patients with a pre-treatment interval of ＜2 weeks had the longest survival time,while those with ≥8 weeks had the lowest survival time.With a one-week interval before treatment,the overall risk of death in breast cancer patients increased by 6％ (HR =1.06,95％ CI:1.01-1.1 l),and the risk of breast cancer death increased by 8％ (HR =1.08,95％ CI:1.02-1.14),the risk of distant metastasis of breast cancer cells increased by 10％ (HR =1.10,95％ CI:1.08-1.13).With the increase in breast cancer tumor diameter (＜20 mm,20-40 mm,≥40 mm),the overall risk of death due to prolonged treatment interval increased gradually,with HR (95％CI) were 1.06 (1.03-1.09),1.08 (1.02-1.12) and 1.11 (1.05-1.17),respectively.With the increase of lymph node metastasis in breast cancer (no metastasis,metastasis at 1-3,≥ 3 metastasis),the total mortality risk caused by prolonged treatment time interval also showed an increasing trend,with HR (95％CI) were 1.04 (1.02-1.08),1.06 (1.04-1.08) and 1.08 (0.99-1.11),respectively.The same results were also shown in the effect of tumor diameter or distant lymph node metastasis on the association between treatment time interval and breast cancer survival and distant metastasis of breast cancer cells.Conclusion With the prolongation of the time interval between the diagnosis of the breast cancer and the surgical treatment of breast cancer patients,the risk of postoperative death is significantly increased,and the association is more pronounced in breast cancer patients with larger tumor volume or higher distant lymph node metastasis.

The treatment of bi （痹） disease in The Inner Canon of Yellow Emperor （《黄帝内经》） is mainly based on acupuncture therapy. There are differences in needling depth， angle， needling techniques， number of needles and the selection of needle instruments. By reviewing literature， it is found that in The Inner Canon of Yellow Emperor， five body constituents （skin， vessel， flesh， sinew， bone） disease location differentiation is taken as the principle for the diagnosis and treatment of bi disease， guiding the needle to the lesion level where the disease is located， and according to the severity of the disease， the characteristics of pathological qi and other factors， the choice of specific acupuncture method and needles are made. This paper summarized and grouped the 17 kinds of acupuncture methods for the treatment of bi disease according to the different five body consitutuents disease location. For needle instruments， filiform needles which can softly unblock and regulate qi are often used in the treatment of bi disease. Lance needles are good at treating vessel bi with the function of clearing blood and moving qi. Round-sharp needles and fire needles are applicable for sinew bi， among which fire needles are especially good at that induced by cold. Long needles have advantages for deep-seated bi disease due to their long needle body. The puncturing method should be in accordance with the needle instruments. The five body instituents disease location differentiation and treatment is a unique system of acupuncture in the treatment of bi disease， which is worthy of more inheritance and application.

Objective To establish a quantitative assay of serum Golgi protein 73 ( GP73) using xMAP technology and evaluate its performance. Methods Monoclonal antibodies against GP73 were prepared and purified, and antibody pair screening was performed by double?antibody sandwich enzyme?linked immunosorbent assay. The screened antibodies were used to construct a Luminex liquid chip detection system, and the analysis performance of the detection system was evaluated. The serum levels of GP73 were detected in 90 clinical samples from healthy controls and patients with chronic hepatitis B infection ( CHB) and hepatocellular carcinoma ( HCC). Results Five anti?GP73 monoclonal antibodies were prepared and purified, and 5 antibody pairs were successfully screened.The Luminex liquid chip detection system of GP73 was successfully constructed using 8F10D1 and 10B9F11 antibody pairs. The analytical performance evaluation showed that the sensitivity of this system was 0.25 ng／ml and the dynamic range was 0.25?100 ng／ml. No cross reactivity was observed. The intra? and inter?assay variation for GP73 was ＜8% and ＜11%, respectively. The recovery was 83%?92%. The linear regression equation was y＝1.141x+6.436 ( r2 ＝0.998 4, P＜0.001). The GP73 concentrations in the serum samples of healthy control, CHB group, and HCC group were 42.8 (38.68, 55.90) ng／ml, 61.49 (43.59, 81) ng／ml, and 122.78 (49.36 liter, 264.55) ng／ml, respectively.The levels of GP73 in HCC group were significantly higher than those in CHB group and healthy controls (P＜0.05). Moreover, the levels of GP73 in CHB group were significantly higher than those in healthy controls ( P＜0.05). Conclusions A liquid chip detection system of GP73 was successfully constructed. It provides a powerful tool for the clinical application of GP73 in the future.

OBJECTIVETo understand the influence of HIV infection on hepatitis C progress in patients co-infected with HIV and hepatitis C virus (HCV) and related immune mechanism.

METHODSTwenty eight patients co-infected with HIV/HCV and 12 patients with simplex HCV infection were enrolled. The liver function and hepatic fibrosis progress were evaluated by detecting peripheral blood and with Fibro-Scan. The viral load of HCV was detected by using real time quantitative PCR. And the percentage of Treg/CD4⁺ T lymphocyte cell was tested by using flow cytometry.

RESULTSThe levels of ALT and ALP in HIV/HCV co-infection group were (76.16 ± 81.248) U/L, (24.507 1 ± 8.194) g/L respectively, higher than those of simplex HCV infection group [(27.475 0 ± 13.985) U/L, (16.966 7 ± 7.189) g/L], the differences were statistical significant. P value was 0.012 and 0.009 respectively. The liver fibrosis index in HIV/HCV co-infection group was 5.950 0-5.825 0 Kpa, higher than that in simplex HIV infection group (5.150 0-1.050 0 Kpa), and the difference was nearly statistical significant (P = 0.077). The HCV viral load in HIV/HCV co-infection group was (6.476 8-5.343 4) lg copy/ml, higher than that in simplex HCV infection group [(1.699 0-2.681 5) lg copy/ml], and the rate of HCV clearance in HIV/HCV co-infection group was 32.14%, lower than that in simplex HCV infection group (75.00%). P value was 0.012 and 0.032 respectively. The percentage of Treg/CD4⁺ T lymphocyte cell in HIV/HCV co-infection group was (7.460 0%-2.287 5%), higher than that in simplex HCV infection group (5.965 0%-2.105 0%), and the difference was significant (P = 0.032). The percentage of Treg/CD4⁺ T lymphocyte cell was significantly related with HCV viral load (ρ = 0.350, P = 0.027), and HCV viral load was significantly related with the liver fibrosis index (ρ = 0.487, P = 0.001).

CONCLUSIONHIV infection could accelerate the progress of hepatitis C, and Treg cells were involved in this progress.

CD4-Positive T-Lymphocytes ; Coinfection ; Disease Progression ; HIV Infections ; complications ; Hepacivirus ; Hepatitis C ; complications ; virology ; Humans ; Liver Cirrhosis ; virology ; Viral Load

Objective: To establish a quantitative assay of serum Golgi protein 73 (GP73) using xMAP technology and evaluate its performance. Methods: Monoclonal antibodies against GP73 were prepared and purified, and antibody pair screening was performed by double-antibody sandwich enzyme-linked immunosorbent assay. The screened antibodies were used to construct a Luminex liquid chip detection system, and the analysis performance of the detection system was evaluated. The serum levels of GP73 were detected in 90 clinical samples from healthy controls and patients with chronic hepatitis B infection (CHB) and hepatocellular carcinoma (HCC). Results: Five anti-GP73 monoclonal antibodies were prepared and purified, and 5 antibody pairs were successfully screened. The Luminex liquid chip detection system of GP73 was successfully constructed using 8F10D1 and 10B9F11 antibody pairs. The analytical performance evaluation showed that the sensitivity of this system was 0.25 ng/ml and the dynamic range was 0.25-100 ng/ml. No cross reactivity was observed. The intra- and inter-assay variation for GP73 was <8% and <11%, respectively. The recovery was 83%-92%. The linear regression equation was y=1.141x+ 6.436 (r²=0.998 4, P<0.001). The GP73 concentrations in the serum samples of healthy control, CHB group, and HCC group were 42.8 (38.68, 55.90) ng/ml, 61.49 (43.59, 81) ng/ml, and 122.78 (49.36 liter, 264.55) ng/ml, respectively. The levels of GP73 in HCC group were significantly higher than those in CHB group and healthy controls (P<0.05). Moreover, the levels of GP73 in CHB group were significantly higher than those in healthy controls (P<0.05). Conclusions A liquid chip detection system of GP73 was successfully constructed. It provides a powerful tool for the clinical application of GP73 in the future.