Identification of Halohydrin Dehalogenase Mutants that Resist COBE Inhibition

Shao-Yun Chen,Xiu-Juan He,Jian-ping Wu,Gang Xu,Li-Rong Yang 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.1

The biocatalytic cascade conversion of ethyl4-chloroacetoacetate (COBE) to ethyl (R)-4-cyano-3-hydroxybutyrate ((R)-HN) for the preparation of atorvastatinrepresents significant economic and environmental benefits,and is catalyzed by alcohol dehydrogenase and halohydrindehalogenase (HHDH). However, as the activity of HHDHis inhibited by COBE, the cascade reaction is an inefficientone-pot reaction. In this study, substrate inhibition kineticsanalysis was performed and the inhibition by COBE wasfound to be competitive reversible inhibition. Molecularsimulation analysis was used to determine the inhibitionmechanism by COBE. The results showed that COBEbound to the active center of HHDH via the formation ofhydrogen bonds with the OH groups of S132 and Y145. Site saturation mutagenesis of residues around the activesite and at the entrance of the access tunnel was performed,and two target mutant residues were identified, F136 andW249. Small focused mutagenesis on these two residueswas performed and the F136V/W249F mutant wassuccessfully found to relieve the activity inhibition ofHHDH by COBE. The half inhibiting concentration ofmutant F136V/W249F was found to be 20-fold higher thanwild-type HHDH. The efficiency of the multi-enzymaticone-pot system for the synthesis of (R)-HN from COBEusing mutant F136V/W249F was improved significantly.

Triaxial Mechanical Characteristics and Microscopic Mechanism of Graphene-Modified Cement Stabilized Expansive Soil

Chen Zhang,Wei Wang,Zhi-Duo Zhu,Na Li,Shao-Yun Pu,Yu Wan,Wang-Wen Huo 대한토목학회 2022 KSCE Journal of Civil Engineering Vol.26 No.1

To investigate the effect of graphene (G) on the triaxial mechanical characteristics of cement stabilized soil, a triaxial unconsolidated and undrained (UU) test was conducted on cement stabilized expansive soil (CES) and graphene-modified cement stabilized expansive soil (GCES) with 0.05%, 0.1%, 0.15%, and 0.2%G content, respectively. The experimental results showedthat compared with CES, the deviatoric stress-strain curve of GCES showed a more obvious softening characteristic. The addition of G had a good modification effect on the triaxial mechanical characteristics of CES, and with the increase of the content of G, the compressive strength and shear strength of CES increased first and then decreased, and reached the best value when the G content was 0.1%. The failure angle predicted by the Mohr-Coulomb failure angle formula had a good correlation with the measured failure angle of soil specimens, so it could provide help for the prediction of soil failure surface in practical engineering. Besides, the scanning electron microscope (SEM) analysis indicated that the internal structure of CES was improved due to G addition.

Chinese herbal injections in combination with radiotherapy for advanced pancreatic cancer: A systematic review and network meta-analysis

Yun-Ru Chen,Ruo-Tong Zhao,Yi-Fang Xu,Yin-Jie Ma,Shao-Bo Hu,Xue-Hui Wang,Bing-Bing Fan,Yan-Ji Zhou,Yu-Bei Huang,Nicola Robinson,Jian-ping Liu,Zhao-Lan Liu 한국한의학연구원 2023 Integrative Medicine Research Vol.12 No.4

Background: Advanced pancreatic cancer (APC) is a fatal disease with limited treatment options. This study aims to evaluate the effectiveness and safety of different Chinese herbal injections (CHIs) as adjuvants for radiotherapy (RT) in APC and compare their treatment potentials using network meta-analysis. Methods: We systematically searched three English and four Chinese databases for randomized controlled trials (RCTs) from inception to July 25, 2023. The primary outcome was the objective response rate (ORR). Secondary outcomes included Karnofsky performance status (KPS) score, overall survival (OS), and adverse events (AEs). The treatment potentials of different CHIs were ranked using the surface under the cumulative ranking curve (SUCRA). The Cochrane RoB 2 tool and CINeMA were used for quality assessment and evidence grading. Results: Eighteen RCTs involving 1199 patients were included. Five CHIs were evaluated. Compound Kushen injection (CKI) combined with RT significantly improved ORR compared to RT alone (RR 1.49, 95 % CrI 1.21–1.86). Kanglaite (KLT) plus RT (RR 1.58, 95 % CrI 1.20–2.16) and CKI plus RT (RR 1.49, 95 % CrI 1.16–1.95) were associated with improved KPS score compared to radiation monotherapy, with KLT+RT being the highest rank (SUCRA 72.28 %). Regarding AEs, CKI plus RT was the most favorable in reducing the incidence of leukopenia (SUCRA 90.37 %) and nausea/vomiting (SUCRA 85.79 %). Conclusions: CKI may be the optimal choice of CHIs to combine with RT for APC as it may improve clinical response, quality of life, and reduce AEs. High-quality trials are necessary to establish a robust body of evidence. Protocol registration: PROSPERO, CRD42023396828.

Intensity-Modulated Radiotherapy versus Three-Dimensional Conformal Radiotherapy in Definitive Chemoradiotherapy for Cervical Esophageal Squamous Cell Carcinoma: Comparison of Survival Outcomes and Toxicities

Nai-Bin Chen,Bo Qiu,Jun Zhang,Meng-Yun Qiang,Yu-Jia Zhu,Bin Wang,Jin-Yu Guo,Ling-Zhi Cai,Shao-Min Huang,Meng-Zhong Liu,Qun Li,Yong-Hong Hu,Qi-Wen Li,Hui Liu 대한암학회 2020 Cancer Research and Treatment Vol.52 No.1

Purpose The purpose of this study was to compare the survival and toxicities in cervical esophageal squamous cell carcinoma (CESCC) treated by concurrent chemoradiothrapy with either three-dimensional conformal radiotherapy (3D-CRT) or intensity-modulated radiotherapy (IMRT) techniques. Materials and Methods A total of 112 consecutive CESCC patients were retrospectively reviewed. 3D-CRT and IMRT groups had been analyzed by propensity score matching method, with sex, age, Karnofsky performance status, induction chemotherapy, and tumor stage well matched. The Kaplan- Meier method and Cox proportional hazards model were used for overall survival (OS) and progression-free survival (PFS). Toxicities were compared between two groups by Fisher exact test. Results With a median follow-up time of 34.9 months, the 3-year OS (p=0.927) and PFS (p=0.859) rate was 49.6% and 45.8% in 3D-CRT group, compared with 54.4% and 42.8% in IMRT group. The rates of grade ! 3 esophagitis, grade ! 2 pneumonitis, esophageal stricture, and hemorrhage were comparable between two groups, while the rate of tracheostomy dependence was much higher in IMRT group than 3D-CRT group (14.3% vs.1.8%, p=0.032). Radiotherapy technique (hazard ratio [HR], 0.09; 95% confidence interval [CI], 0.01 to 0.79) and pretreatment hoarseness (HR, 0.12; 95% CI 0.02 to 0.70) were independently prognostic of tracheostomy dependence. Conclusion No survival benefits had been observed while comparing IMRT versus 3D-CRT in CESCC patients. IMRT with fraction dose escalation and pretreatment hoarseness were considered to be associated with a higher risk for tracheostomy dependence. Radiation dose escalation beyond 60 Gy should be taken into account carefully when using IMRT with hypofractionated regimen.

Involvement of Cdc25c in Cell Cycle Alteration of a Radioresistant Lung Cancer Cell Line Established with Fractionated Ionizing Radiation

Li, Jie,Yang, Chun-Xu,Mei, Zi-Jie,Chen, Jing,Zhang, Shi-Min,Sun, Shao-Xing,Zhou, Fu-Xiang,Zhou, Yun-Feng,Xie, Cong-Hua Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.10

Cancer patients often suffer from local tumor recurrence after radiation therapy. Cell cycling, an intricate sequence of events which guarantees high genomic fidelity, has been suggested to affect DNA damage responses and eventual radioresistant characteristics of cancer cells. Here, we established a radioresistant lung cancer cell line, A549R, by exposing the parental A549 cells to repeated ${\gamma}$-ray irradiation with a total dose of 60 Gy. The radiosensitivity of A549 and A549R was confirmed using colony formation assays. We then focused on examination of the cell cycle distribution between A549 and A549R and found that the proportion of cells in the radioresistant S phase increased, whereas that in the radiosensitive G1 phase decreased. When A549 and A549R cells were exposed to 4 Gy irradiation the total differences in cell cycle redistribution suggested that G2-M cell cycle arrest plays a predominant role in mediating radioresistance. In order to further explore the possible mechanisms behind the cell cycle related radioresistance, we examined the expression of Cdc25 proteins which orchestrate cell cycle transitions. The results showed that expression of Cdc25c increased accompanied by the decrease of Cdc25a and we proposed that the quantity of Cdc25c, rather than activated Cdc25c or Cdc25a, determines the radioresistance of cells.

VSV-G Viral Envelope Glycoprotein Prepared from Pichia pastoris Enhances Transfection of DNA into Animal Cells

( Xin Liu ),( Ying Dong ),( Jingquan Wang ),( Long Li ),( Zhenmin-zhong ),( Yun-pan Li ),( Shao-jun Chen ),( Yu-cai Fu ),( Wen-can Xu ),( Chi-ju Wei ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.6

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones (Mut^s) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

HiF-1α siRNA and Cisplatin in Combination SuppressTumor Growth in a Nude Mice Model of Esophageal Squamous Cell Carcinoma

Liao, Hong-Ying,Wang, Gui-Ping,Gu, Li-Jia,Huang, Shao-Hong,Chen, Xiu-Ling,Li, Yun,Cai, Song-Wang Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.2

Introduction: The esophagus squamous cell carcinoma (ESCC) is one of the most deadly malignances, and a current challenge is the development of effective therapeutic agents. Our present work addressed the effect of HIF-$1{\alpha}$ siRNA alone or in combination with cisplatin on the growth of ESCC in nude mice. Materials and Methods: Xenografts were established by inoculating ESCC TE-1 cells in nude mice, and transplanted tumors were treated with HIF-$1{\alpha}$ siRNA, cisplatin alone or together. Growth was assessed by measuring tumor volume. HIF-$1{\alpha}$ mRNA and protein expression were detected using RT-PCR and immunohistochemistry, respectively. Apoptosis of ESCC TE-1 cells was analyzed by flow cytometry. Results: In our nude mice model, HIF-$1{\alpha}$ siRNA effectively inhibited the growth of transplanted ESCC, downregulating HIF-$1{\alpha}$ mRNA and protein expression, and inducing ESCC TE-1 cell apoptosis. Notably when combinated with cisplatin, HIF-$1{\alpha}$ siRNA showed synergistic interaction in suppressing tumor growth. Furthermore, the proportion of apoptotic cells in HIF-$1{\alpha}$ siRNA plus cisplatin group was significantly higher than that in cisplatin or HIF-$1{\alpha}$ siRNA-treated groups (P<0.05). Conclusions: Down-regulated HIF-$1{\alpha}$ expression induced by siRNA could effectively suppress the growth of transplanted ESCC $in$ $vivo$. HIF-$1{\alpha}$ siRNA could enhance the cytotoxicity of cisplatin, which suggests that a combination of these two agents may have potential for therapy of advanced ESCC.