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Inhibition of HBV replication and gene expression in vitro and in vivo with a single AAV vector delivering two shRNA molecules

  • Li, Zhi;He, Ming-Liang;Yao, Hong;Dong, Qing-Ming;Chen, Yang-Chao;Chan, Chu-Yan;Zheng, Bo-Jian;Yuen, Kwok-Yung;Peng, Ying;Sun, Qiang;Yang, Xiao;Lin, Marie C.;Sung, Joseph J.Y.;Kung, Hsiang-Fu
    • BMB Reports
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    • v.42 no.1
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    • pp.59-64
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    • 2009
  • Hepatitis B virus (HBV) infection is highly prevalent worldwide. The major challenge for current antiviral treatment is the elevated drug resistance that occurs via rapid viral mutagenesis. In this study, we developed AAV vectors to simultaneously deliver two or three shRNAs targeting different HBV-related genes. These vectors showed markedly better antiviral effects than ones that delivered a single shRNA in vitro. A dual shRNA expression vector (AAV-157i/1694i), which simultaneously expressed two shRNAs targeted the S and X genes of HBV, reduced HBsAg, HBeAg and HBV DNA levels by $87{\pm}4$, $80.3{\pm}2.6$ and $86.2{\pm}7%$ respectively, eight days post-transduction. In a mouse model of prophylactic treatment, HBsAg and HBeAg were reduced to undetectable levels and the serum HBV DNA level was reduced by at least 100 fold. These results indicate that AAV-157i/1694i generates potent anti-HBV effects and that the strategy of constructing multi-shRNA expression vectors may lead to enhanced anti-HBV efficacy and overcome the evading mechanism of the virus and thus the development of drug resistance.

BRI3 associates with SCG10 and attenuates NGF-induced neurite outgrowth in PC12 cells

  • Gong, Yanhua;Wu, Jing;Qiang, Hua;Liu, Ben;Chi, Zhikai;Chen, Tao;Yin, Bin;Peng, Xiaozhong;Yuan, Jiangang
    • BMB Reports
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    • v.41 no.4
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    • pp.287-293
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    • 2008
  • In a yeast two-hybrid screen, we identified the microtubule-destabilizing protein SCG10 as a potential effector protein of $BRI_3$. The association was verified using GST pull-down, Co-IP, and their perinuclear co-localization. The analysis of in vitro microtubule polymerization/depolymerization showed that the binding of $BRI_3$ to SCG10 effectively blocked the ability of SCG10 to induce microtubule disassembly, as determined by turbidimetric assays. In intact PC12 cells, $BRI_3$ exhibited the ability to stabilize the microtubule network and attenuate the microtubule-destabilizing activity of SCG10. Furthermore, co-expression of $BRI_3$ with SCG10 attenuated SCG10-mediated PC12 cell neurite outgrowth induced by NGF. These results identify a novel connection between a neuron-specific BRI protein and the cytoskeletal network, suggesting possible roles of BRI3 in the process of neuronal differentiation.

A LysM Domain-Containing Protein LtLysM1 Is Important for Vegetative Growth and Pathogenesis in Woody Plant Pathogen Lasiodiplodia theobromae

  • Harishchandra, Dulanjalee Lakmali;Zhang, Wei;Li, Xinghong;Chethana, Kandawatte Wedaralalage Thilini;Hyde, Kevin David;Brooks, Siraprapa;Yan, Jiye;Peng, Junbo
    • The Plant Pathology Journal
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    • v.36 no.4
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    • pp.323-334
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    • 2020
  • Lysin motif (LysM) proteins are reported to be necessary for the virulence and immune response suppression in many herbaceous plant pathogens, while far less is documented in woody plant pathogens. In this study, we preliminarily characterized the molecular function of a LysM protein LtLysM1 in woody plant pathogen Lasiodiplodia theobromae. Transcriptional profiles revealed that LtLysM1 is highly expressed at infectious stages, especially at 36 and 48 hours post inoculation. Amino acid sequence analyses revealed that LtLysM1 was a putative glycoprotein with 10 predicted N-glycosylation sites and one LysM domain. Pathogenicity tests showed that overexpressed transformants of LtLysM1 displayed increased virulence on grapevine shoots in comparison with that of wild type CSS-01s, and RNAi transformants of LtLysM1 exhibited significantly decreased lesion length when compared with that of wild type CSS-01s. Moreover, LtLysM1 was confirmed to be a secreted protein by a yeast signal peptide trap assay. Transient expression in Nicotiana benthamiana together with protein immunoblotting confirmed that LtLysM1 was an N-glycosylated protein. In contrast to previously reported LysM protein Slp1 and OsCEBiP, LtLysM1 molecule did not interact with itself based on yeast two hybrid and co-immunoprecipitation assays. These results indicate that LtLysM1 is a secreted protein and functions as a critical virulence factor during the disease symptom development in woody plants.

Wind loads on a moving vehicle-bridge deck system by wind-tunnel model test

  • Li, Yongle;Hu, Peng;Xu, You-Lin;Zhang, Mingjin;Liao, Haili
    • Wind and Structures
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    • v.19 no.2
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    • pp.145-167
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    • 2014
  • Wind-vehicle-bridge (WVB) interaction can be regarded as a coupled vibration system. Aerodynamic forces and moment on vehicles and bridge decks play an important role in the vibration analysis of the coupled WVB system. High-speed vehicle motion has certain effects on the aerodynamic characteristics of a vehicle-bridge system under crosswinds, but it is not taken into account in most previous studies. In this study, a new testing system with a moving vehicle model was developed to directly measure the aerodynamic forces and moment on the vehicle and bridge deck when the vehicle model moved on the bridge deck under crosswinds in a large wind tunnel. The testing system, with a total length of 18.0 m, consisted of three main parts: vehicle-bridge model system, motion system and signal measuring system. The wind speed, vehicle speed, test objects and relative position of the vehicle to the bridge deck could be easily altered for different test cases. The aerodynamic forces and moment on the moving vehicle and bridge deck were measured utilizing the new testing system. The effects of the vehicle speed, wind yaw angle, rail track position and vehicle type on the aerodynamic characteristics of the vehicle and bridge deck were investigated. In addition, a data processing method was proposed according to the characteristics of the dynamic testing signals to determine the variations of aerodynamic forces and moment on the moving vehicle and bridge deck. Three-car and single-car models were employed as the moving rail vehicle model and road vehicle model, respectively. The results indicate that the drag and lift coefficients of the vehicle tend to increase with the increase of the vehicle speed and the decrease of the resultant wind yaw angle and that the vehicle speed has more significant effect on the aerodynamic coefficients of the single-car model than on those of the three-car model. This study also reveals that the aerodynamic coefficients of the vehicle and bridge deck are strongly influenced by the rail track positions, while the aerodynamic coefficients of the bridge deck are insensitive to the vehicle speed or resultant wind yaw angle.

Utrecht Interstitial Applicator Shifts and DVH Parameter Changes in 3D CT-based HDR Brachytherapy of Cervical Cancer

  • Shi, Dan;He, Ming-Yuan;Zhao, Zhi-Peng;Wu, Ning;Zhao, Hong-Fu;Xu, Zhi-Jian;Cheng, Guang-Hui
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.9
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    • pp.3945-3949
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    • 2015
  • Background: For brachytherapy of cervical cancer, applicator shifts can not be avoided. The present investigation concerned Utrecht interstitial applicator shifts and their effects on organ movement and DVH parameters during 3D CT-based HDR brachytherapy of cervical cancer. Materials and Methods: After the applicator being implanted, CT imaging was achieved for oncologist contouring CTVhr, CTVir, and OAR, including bladder, rectum, sigmoid colon and small intestines. After the treatment, CT imaging was repeated to determine applicator shifts and OARs movements. Two CT images were matched by pelvic structures. In both imaging results, we defined the tandem by the tip and the base as the marker point, and evaluated applicator shift, including X, Y and Z. Based on the repeated CT imaging, oncologist contoured the target volume and OARs again. We combined the treatment plan with the repeated CT imaging and evaluated the change range for the doses of CTVhr D90, D2cc of OARs. Results: The average applicator shift was -0.16 mm to 0.10 mm for X, 1.49 mm to 2.14 mm for Y, and 1.9 mm to 2.3 mm for Z. The change of average physical doses and EQD2 values in Gy${\alpha}/{\beta}$ range for CTVhr D90 decreased by 2.55 % and 3.5 %, bladder D2cc decreased by 5.94 % and 8.77 %, rectum D2cc decreased by 2.94 % and 4 %, sigmoid colon D2cc decreased by 3.38 % and 3.72 %, and small intestines D2cc increased by 3.72 % and 10.94 %. Conclusions: Applicator shifts and DVH parameter changes induced the total dose inaccurately and could not be ignored. The doses of target volume and OARs varied inevitably.

Impact of IL-2 and IL-2R SNPs on Proliferation and Tumor-killing Activity of Lymphokine-Activated Killer Cells from Healthy Chinese Blood Donors

  • Li, Yan;Meng, Fan-Dong;Tian, Xin;Sui, Cheng-Guang;Liu, Yun-Peng;Jiang, You-Hong
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.18
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    • pp.7965-7970
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    • 2014
  • One of the goals of tumor immunotherapy is to generate immune cells with potent anti-tumor activity through in vitro techniques using peripheral blood collected from patients. However, cancer patients generally have poor immunological function. Thus using patient T cells, which have reduced in vitro proliferative capabilities and less tumor cell killing activity to generate lymphokine-activated killer (LAK) cells, fails to achieve optimal clinical efficacy. Interleukin-2 (IL-2) is a potent activating cytokine for both T cells and natural killer cells. Thus, this study aimed to identify optimal donors for allogeneic LAK cell immunotherapy based on single nucleotide polymorphisms (SNP) in the IL-2 and IL-2R genes. IL-2 and IL-2R SNPs were analyzed using HRM-PCR. LAK cells were derived from peripheral blood mononuclear cells by culturing with IL-2. The frequency and tumor-killing activity of LAK cells in each group were analyzed by flow cytometry and tumor cell killing assays, respectively. Regarding polymorphisms at IL-2-330 (rs2069762) T/G, LAK cells from GG donors had significantly greater proliferation, tumor-killing activity, and IFN-${\gamma}$ production than LAK cells from TT donors (P<0.05). Regarding polymorphisms at IL-2R rs2104286 A/G, LAK cell proliferation and tumor cell killing were significantly greater in LAK cells from AA donors than GG donors (P<0.05). These data suggest that either IL-2-330(rs2069762)T/G GG donors or IL-2R rs2104286 A/G AA donors are excellent candidates for allogeneic LAK cell immunotherapy.

Association of XRCC3 Thr241Met Polymorphisms and Gliomas Risk: Evidence from a Meta-analysis

  • Liang, Hong-Jie;Yan, Yu-Lan;Liu, Zhi-Ming;Chen, Xu;Peng, Qi-Liu;Wang, Jian;Mo, Cui-Ju;Sui, Jing-Zhe;Wu, Jun-Rong;Zhai, Li-Min;Yang, Shi;Li, Tai-Jie;Li, Ruo-Lin;Li, Shan;Qin, Xue
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.7
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    • pp.4243-4247
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    • 2013
  • The relationship between the X-ray repair cross-complementing group 3 (XRCC3) Thr241Met polymorphism and gliomas remains inclusive or controversial. For better understanding of the effect of XRCC3 Thr241Met polymorphism on glioma risk, a meta-analysis was performed. All eligible studies were identified through a search of PubMed, Elsevier Science Direct, Excerpta Medica Database (Embase) and Chinese Biomedical Literature Database (CBM) before May 2013. The association between the XRCC3 Thr241Met polymorphism and gliomas risk was conducted by odds ratios (ORs) and 95% confidence intervals (95% CIs). A total of nine case-control studies including 3,533 cases and 4,696 controls were eventually collected. Overall, we found that XRCC3 Thr241Met polymorphism was significantly associated with the risk of gliomas (T vs. C: OR=1.10, 95%CI=1.01-1.20, P=0.034; TT vs. CC: OR=1.30, 95%CI=1.03-1.65, P=0.027; TT vs. TC/CC: OR=1.29, 95%CI=1.01-1.64, P=0.039). In the subgroup analysis based on ethnicity, the significant association was found in Asian under four models (T vs. C: OR=1.17, 95%CI=1.07-1.28, P=0.00; TT vs. CC: OR=1.79, 95%CI=1.36-2.36, P=0.00; TT vs. TC/CC: OR=1.75, 95%CI=1.32-2.32, P=0.00; TT/TC vs. CC: OR=1.11,95% CI=1.02-1.20). This meta-analysis suggested that the XRCC3 Thr241Met polymorphism is a risk factor for gliomas, especially for Asians. Considering the limited sample size and ethnicities included in the meta-analysis, further large scale and well-designed studies are needed to confirm our results.

Colorectal Cancer Concealment Predicts a Poor Survival: A Retrospective Study

  • Li, Xiao-Pan;Xie, Zhen-Yu;Fu, Yi-Fei;Yang, Chen;Hao, Li-Peng;Yang, Li-Ming;Zhang, Mei-Yu;Li, Xiao-Li;Feng, Li-Li;Yan, Bei;Sun, Qiao
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.7
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    • pp.4157-4160
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    • 2013
  • Objectives: Understanding the situation of cancer awareness which doctors give to patients might lead to prognostic prediction in cases of of colorectal cancer (CRC). Methods: Subsets of 10,779 CRC patients were used to screen the risk factors from the Cancer Registry in Pudong New Area in cancer awareness, age, TNM stage, and gender. Survival of the patients was calculated by the Kaplan-Meier method and assessed by Cox regression analysis. The views of cancer awareness in doctors and patients were surveyed by telephone or household. Results: After a median observation time of 1,616 days (ranging from 0 to 4,083 days) of 10,779 available patients, 2,596 of the 4,561 patients with cancer awareness survived, whereas 2,258 of the 5,469 patients without cancer awareness and 406 of the 749 patients without information on cancer awareness died of the disease. All-cause and cancer-specific survival were poorer for the patients without cancer awareness than those with (P < 0.001 for each, log-rank test). Cox multivariate regression analysis showed that cancer concealment cases had significantly lower cancer-specific survival (hazard ratio (HR) = 1.299; 95 % confidence interval (CI): 1.200-1.407)and all-cause survival (HR = 1.324; 95 % CI: 1.227-1.428). Furthermore, attitudes of cancer awareness between doctors and patients were significantly different (P < 0.001). Conclusion: Cancer concealment, not only late-stage tumor and age, is associated with a poor survival of CRC patients.

Prediction of Phase Behavior of ε-caprolactam Derivatives and Carbon Dioxide using a Group Contribution Method (그룹 기여 방법을 이용한 ε-caprolactam 유도체와 이산화탄소의 상거동 예측에 관한 연구)

  • Kwon, Soyoung;Bae, Won;Lee, Kyoungwon;Kim, Hwayong
    • Clean Technology
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    • v.11 no.3
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    • pp.117-122
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    • 2005
  • N-vinyl caprolactam (NVCL), a kind of N-vinyl amide monomer, must be dissolved in continuous phase ($scCO_2$) for dispersion polymerization in supercritical carbon dioxide. Phase behavior of $CO_2$+NVCL is very important and necessary for determining initial polymerization condition and for monomer extraction from final polymer. There is the limitation of experimental method for obtaining pure properties of the monomer because of the possibility of polymerization. And N-methyl caprolactam (NMCL) is the useful solvent for the gas treating process. In the viewpoint of molecular thermodynamics, NVCL and NMCL have same functional group i.e. ${\varepsilon}$-caprolactam. In the case of NVCL, hydrogen of amide group is substituted with vinyl group and for NMCL, hydrogen of amide group is substituted with methyl group. We suggested modified group contribution method for this ${\varepsilon}$-caprolactam derivatives. This new group contribution parameter was applied to correlate $CO_2$ + N-vinyl caprolactam or N-methyl caprolactam system.

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Regulatory effects of saponins from Panax japonicus on colonic epithelial tight junctions in aging rats

  • Dun, Yaoyan;Liu, Min;Chen, Jing;Peng, Danli;Zhao, Haixia;Zhou, Zhiyong;Wang, Ting;Liu, Chaoqi;Guo, Yuhui;Zhang, Changcheng;Yuan, Ding
    • Journal of Ginseng Research
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    • v.42 no.1
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    • pp.50-56
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    • 2018
  • Background: Saponins from Panax japonicus (SPJ) are the most abundant and main active components of P. japonicus, which replaces ginseng roots in treatment for many kinds of diseases in the minority ethnic group in China. Our previous studies have demonstrated that SPJ has the effects of anti-inflammation through the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-${\kappa}B$) signaling pathways. The present study was designed to investigate whether SPJ can modulate intestinal tight junction barrier in aging rats and further to explore the potential mechanism. Methods: Aging rats had been treated with different doses (10 mg/kg, 30 mg/kg, and 60 mg/kg) of SPJ for 6 mo since they were 18 mo old. After the rats were euthanized, the colonic samples were harvested. Levels of tight junctions (claudin-1 and occludin) were determined by immunohistochemical staining. Levels of proinflammatory cytokines (interleukin-$1{\beta}$ and tumor necrosis factor-${\alpha}$) were examined by Western blot. NF-${\kappa}B$ and phosphorylation of MAPK signaling pathways were also determined by Western blot. Results: We found that SPJ increased the expression of the tight junction proteins claudin-1 and occludin in the colon of aging rats. Treatment with SPJ decreased the levels of interleukin-$1{\beta}$ and tumor necrosis factor-${\alpha}$, reduced the phosphorylation of three MAPK isoforms, and inhibited the expression of NF-${\kappa}B$ in the colon of aging rats. Conclusion: The studies demonstrated that SPJ modulates the damage of intestinal epithelial tight junction in aging rats, inhibits inflammation, and downregulates the phosphorylation of the MAPK and $NF-{\kappa}B$ signaling pathways.