Production of a Recombinant Laccase from Pichia pastoris and Biodegradation of Chlorpyrifos in a Laccase/Vanillin System

( Hui fang Xie ),( Qi Li ),( Min Min Wang ),( Lin Guo Zhao ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.6

The recombinant strain P. pastoris GS115-lccC was used to produce laccase with high activity. Factors influencing laccase expression, such as pH, methanol concentration, copper concentration, peptone concentration, shaker rotate speed, and medium volume were investigated. Under the optimal conditions, laccase activity reached 12,344 U/L on day 15. The recombinant enzyme was purified by precipitating and dialyzing to electrophoretic homogeneity, and was estimated to have a molecular mass of about 58 kDa. When guaiacol was the substrate, the laccase showed the highest activity at pH 5.0 and was stable when the pH was 4.5~6.0. The optimal temperature for the laccase to oxidize guaiacol was 60℃, but it was not stable at high temperature. The enzyme could remain stable at 30℃ for 5 days. The recombinant laccase was used to degrade chlorpyrifos in several laccase/mediator systems. Among three synthetic mediators (ABTS, HBT, VA) and three natural mediators (vanillin, 2,6-DMP, and guaiacol), vanillin showed the most enhancement on degradation of chlorpyrifos. Both laccase and vanillin were responsible for the degradation of chlorpyrifos. A higher dosage of vanillin may promote a higher level of degradation of chlorpyrifos, and the 2-step addition of vanillin led to 98% chlorpyrifos degradation. The degradation of chlorpyrifos was faster in the L/V system (kobs = 0.151) than that in the buffer solution (kobs = 0.028).

Liquid Chromatography Quadrupole Time-Of-Flight Tandem Mass Spectrometry for Selective Determination of Usnic Acid and Application in Pharmacokinetic Study

Fang, Minfeng,Wang, Hui,Wu, Yang,Wang, Qilin,Zhao, Xinfeng,Zheng, Xiaohui,Wang, Shixiang,Zhao, Guifang Korean Chemical Society 2013 Bulletin of the Korean Chemical Society Vol.34 No.6

A rapid and sensitive method for determining usnic acid of Lethariella cladonioides in rat was established using high performance liquid chromatography (HPLC) quadrupole time-of-flight (QTOF) tandem mass (MS/MS). Rat plasma was pretreated by mixture of acetonitrile and chloroform to precipitate plasma proteins. Chromatographic separation was achieved on a column ($50{\times}2.1$ mm, $5{\mu}m$) with a mobile phase consisting of water (containing $5{\times}10^{-3}$ M ammonium formate, pH was adjusted to 3.0 with formic acid) and acetonitrile (20:80, v/v) at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via collision induced dissociation (CID) under negative ionization mode. The MS/MS transitions monitored were m/z 343.0448 ${\rightarrow}$ m/z 313.2017 for usnic acid and m/z 153.1024 ${\rightarrow}$ m/z 136.2136 for protocatechuic acid (internal standard). The linear range was calculated to be 2.0-160.0 ng/mL with a detection limit of 3.0 pg/mL. The inter- and intra-day accuracy and precision were within ${\pm}7.0%$. Pharmacokinetic study showed that the apartment of usnic acid in vivo confirmed to be a two compartment open model. The method was fully valid and will probably be an alternative for pharmacokinetic study of usnic acid.

Serum Uric Acid Relation for Hearing Threshold Shiftt

Hui-Fang Yang,Tung-Wei Kao,Tao-Chun Peng,Yu-Shan Sun,Fang-Yih Liaw,Chung-Ching Wang,Ju-Ting Hsueh,Wei-Liang Chen 대한이비인후과학회 2017 Clinical and Experimental Otorhinolaryngology Vol.10 No.2

Objectives. The effects of serum uric acid (UA) level on a variety of diseases were found from experimental and observational studies via oxidative stress and anti-oxidants. However, research on the association of UA and hearing thresholds is relatively sparse. We investigated this issue in the U.S. general population to evaluate the relationship of serum UA levels and pure tone threshold of hearing. Methods. Forty four thousand eighty four eligible participants aged 20 to 69 years who have serum UA data and received Audiometry Examination Component were enrolled from the National Health and Nutrition Examination Survey 1999–2004. Hearing thresholds (dB) as a pure tone average at low frequencies (0.5, 1, 2 kHz) and at high frequencies (3, 4, 6, and 8 kHz) were computed. Multivariate linear regression models and tertile-based analysis with an extended-model approach for covariates adjustment were used to assess the correlation between serum UA level and hearing thresholds. Results. In the adjusted mode of tertile-based analysis, the regression coefficients elucidated as the change of log-transformed mean hearing thresholds upon comparing participants in the highest tertile of serum UA to those in the lowest tertile were –0.067 (P=0.023) in high frequency and –0.058 (P=0.054) in low frequency. After adjusting for multiple pertinent covariates, inverse association between tertiles of serum UA and hearing thresholds remained essentially unchanged. The negative trends between serum UA and hearing thresholds were statistically significant (P for trends <0.05) in tertile-based multiple linear regressions. Conclusion. Individuals with elevated UA levels independently were found to be inversely associated with hearing thresholds for pure tone audiometry in a nationally representative sample of U.S. adults.

Human Papillomavirus Vaccine Awareness, Acceptability, and Decision-Making Factors among Chinese College Students

Wang, Shao-Ming,Zhang, Shao-Kai,Pan, Xiong-Fei,Ren, Ze-Fang,Yang, Chun-Xia,Wang, Zeng-Zhen,Gao, Xiao-Hong,Li, Man,Zheng, Quan-Qing,Ma, Wei,Zhao, Fang-Hui,Qiao, You-Lin,Sivasubramaniam, Priya Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.7

Background: College students are recommended as the target groups for catch-up human papillomavirus (HPV) vaccination. Systematical exploration of awareness, acceptability, and decision-making factors of HPV vaccination among Chinese college students has been limited. Materials and Methods: A multi-center survey was conducted in mainland China between November 2011 and May 2012. College students aged 18-22 years were stratified by their grade, gender, and major for sampling. Socio-demographic and HPV-related information such as knowledge, perceptions, acceptability, and attitudes were collected through a questionnaire. Results: A total of 3,497 undergraduates completed the questionnaire, among which 1,686 were males. The acceptability of the HPV vaccine was high (70.8%). Undergraduates from high-level universities, at lower grade, or with greater prior knowledge of HPV vaccines showed higher acceptability of HPV vaccination ($p_{trend}$ <0.001). Additionally, undergraduates with vaccination experience outside the National Expanded Program on Immunization (OR=1.29; 95%CI: 1.10-1.51) or fear of HPV-related diseases (OR=2.79; 95%CI: 2.28-3.41) were more willing to accept HPV vaccination. General knowledge of HPV vaccine was low among undergraduates, and safety was a major concern (71.05%). The majority of students wished to pay less than 300RMB for HPV vaccine and chose the Chinese Center for Disease Control and Prevention as the most appropriate venue for vaccination. Conclusions: Although most undergraduates demonstrate positive attitudes towards HPV vaccination, challenges pertaining to introduction exist in China. Corresponding proactive education and governmental subsidy to do so are urgently needed by this age-group population. Suggestions and potential strategies indicated may help shape the future HPV vaccination program in China.

Comprehensive Analysis of Magnetization, Magnetostriction, Hysteresis and Kinematical Characteristics for Precision Magnetostrictive Actuator

Hui-Fang Liu,Jie Wang,Han-Yu Wang,Qing-Wei Shao 한국정밀공학회 2016 International Journal of Precision Engineering and Vol.17 No.12

In order to systematically know the operating characteristics and further improve accuracy of precision magnetostrictive actuator used in precision and ultra precision machining fields, a model considering magnetization hysteresis mechanism and kinematics is established based on the domain wall magnetization essential theory. Magnetization, magnetostriction and kinematical characteristics, and the origin of hysteresis are discussed. According to input energy per unit volume of magnetostrictive materials is the sum of static magnetic energy and hysteresis loss energy, magnetization is divided into reversible and irreversible processes. Moreover, hysteresis loop is represented as an offset of irreversible magnetization relative to anhysteretic magnetization. Actuator’s model is deduced through dividing working process into four stages. Experiments show that the predicted values show good agreement with experimental data and average relative error of displacement is less than 5%. Magnetic hysteresis is derived from irreversible process in which magnetic domain cannot return back to initial distribution state completely. And hysteresis is more serious with the increasement of frequency. Research results reveal magnetization and kinematic characteristics from intrinsic physical mechanism, thus it provides a theoretical guidance for further improve the accuracy of magnetostrictive actuator and promotes the practical application of actuator in precision and ultra precision machining fields.

Ultrasound Targeted Microbubble Destruction for Novel Dual Targeting of HSP72 and HSC70 in Prostate Cancer

Wang, Hang-Hui,Song, Yi-Xin,Bai, Min,Jin, Li-Fang,Gu, Ji-Ying,Su, Yi-Jin,Liu, Long,Jia, Chao,Du, Lian-Fang Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.3

The aim was to determine whether ultrasound targeted microbubble destruction (UTMD) promotes dual targeting of HSP72 and HSC70 for therapy of castration-resistant prostate cancer (CRPC), to improve the specific and efficient delivery of siRNA, to induce tumor cell specific apoptosis, and to find new therapeutic targets specific of CRPC.VCaP cells were transfected with siRNA oligonucleotides. HSP70, HSP90 and cleaved caspase-3 expression were determined by real-time quantitative polymerase chain reaction and Western blotting. Apoptosis and transfection efficiency were assessed by flow cytometry. Cell viability assays were used to evaluate safety. We found HSP72, HSC70 and HSP90 expression to be absent or weak in normal prostate epithelial cells (RWPE-1), but uniformly strong in prostate cancerous cells (VCaP). UTMD combined with dual targeting of HSP72 and HSC70 siRNA improve the efficiency of transfection, cell uptake of siRNA, downregulation of HSP70 and HSP90 expression in VCaP cells at the mRNA and protein level, and induction of extensive tumor-specific apoptosis. Cell counting kit-8 assays showed decreased cellular viability in the HSP72/HSC70-siRNA silenced group. These results suggest that the combination of UTMD with dual targeting HSP70 therapy for PCa may be most efficacious, providng a novel, reliable, non-invasive, safe targeted approach to improve the specific and efficient delivery of siRNA, and achieve maximal effects.

miR-638 is a new biomarker for outcome prediction of non-small cell lung cancer patients receiving chemotherapy

Fang Wang,Jian-fang Lou,Yan Cao,Xin-hui Shi,Peng Wang,Jian Xu,Er-fu Xie,Ting Xu,Rui-hong Sun,Jianyu Rao,Pu-wen Huang,Shi-yang Pan,Hong Wang 생화학분자생물학회 2015 Experimental and molecular medicine Vol.47 No.-

MicroRNAs (miRNAs), a class of small non-coding RNAs, mediate gene expression by either cleaving target mRNAs or inhibiting their translation. They have key roles in the tumorigenesis of several cancers, including non-small cell lung cancer (NSCLC). The aim of this study was to investigate the clinical significance of miR-638 in the evaluation of NSCLC patient prognosis in response to chemotherapy. First, we detected miR-638 expression levels in vitro in the culture supernatants of the NSCLC cell line SPC-A1 treated with cisplatin, as well as the apoptosis rates of SPC-A1. Second, serum miR-638 expression levels were detected in vivo by using nude mice xenograft models bearing SPC-A1 with and without cisplatin treatment. In the clinic, the serum miR-638 levels of 200 cases of NSCLC patients before and after chemotherapy were determined by quantitative real-time PCR, and the associations of clinicopathological features with miR-638 expression patterns after chemotherapy were analyzed. Our data helped in demonstrating that cisplatin induced apoptosis of the SPC-A1 cells in a dose- and time-dependent manner accompanied by increased miR-638 expression levels in the culture supernatants. In vivo data further revealed that cisplatin induced miR-638 upregulation in the serum derived from mice xenograft models, and in NSCLC patient sera, miR-638 expression patterns after chemotherapy significantly correlated with lymph node metastasis. Moreover, survival analyses revealed that patients who had increased miR-638 levels after chemotherapy showed significantly longer survival time than those who had decreased miR-638 levels. Our findings suggest that serum miR-638 levels are associated with the survival of NSCLC patients and may be considered a potential independent predictor for NSCLC prognosis.

Liquid Chromatography Quadrupole Time-Of-Flight Tandem Mass Spectrometry for Selective Determination of Usnic Acid and Application in Pharmacokinetic Study

Minfeng Fang,Hui Wang,Yang Wu,Qilin Wang,Xinfeng Zhao,Xiao-Hui Zheng,Shixiang Wang,Guifang Zhao 대한화학회 2013 Bulletin of the Korean Chemical Society Vol.34 No.6

A rapid and sensitive method for determining usnic acid of Lethariella cladonioides in rat was established using high performance liquid chromatography (HPLC) quadrupole time-of-flight (QTOF) tandem mass (MS/MS). Rat plasma was pretreated by mixture of acetonitrile and chloroform to precipitate plasma proteins. Chromatographic separation was achieved on a column (50 × 2.1 mm, 5 μm) with a mobile phase consisting of water (containing 5 × 10−3 M ammonium formate, pH was adjusted to 3.0 with formic acid) and acetonitrile (20:80, v/v) at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via collision induced dissociation (CID) under negative ionization mode. The MS/MS transitions monitored were m/z 343.0448 → m/z 313.2017 for usnic acid and m/z 153.1024 → m/z 136.2136 for protocatechuic acid (internal standard). The linear range was calculated to be 2.0-160.0 ng/mL with a detection limit of 3.0 pg/mL. The inter- and intra-day accuracy and precision were within ± 7.0%. Pharmacokinetic study showed that the apartment of usnic acid in vivo confirmed to be a two compartment open model. The method was fully valid and will probably be an alternative for pharmacokinetic study of usnic acid.

Evolutionary and functional implications of 3′ untranslated region length of mRNAs by comprehensive investigation among four taxonomically diverse metazoan species

Wei Wang,Dong‑hui Fang,Jia Gan,Yi Shi,Hui Tang,Huai Wang,Mao‑zhong Fu,Jun Yi 한국유전학회 2019 Genes & Genomics Vol.41 No.7

Background In eukaryotic organisms, it has been well acknowledged that 3′ untranslated regions (3′ UTRs) of mRNA are actively involved in post-transcriptional regulations of gene expression. Although both shortening and lengthening of 3′ UTRs of specific candidate genes were explicitly documented to have functional consequences, landscape of 3′ UTR lengths in relation to evolutionary dynamics and biological meanings remains to be elucidated when large-scale data become available. Objectives The primary objective of this study was to revealed different inter- and intra-species patterns on length distribution of 3′ UTRs in comparison with 5′ UTRs and coding regions. Methods In the present study, we investigated 3′ UTR lengths in a highly curated set of 57,135 mRNA sequences among four well-studied and taxonomically diverse metazoan species (fruit fly, zebrafish, mouse and human). Results The average length ratio of 3′–5′ UTRs considerably increased from fruit fly (twofold) to human (fivefold). Moreover, genes can be characterized by the obviously different patterns of evolutionary change on 3′ UTR lengths. By utilizing the Gene Ontology annotations, genes with differential lengths of 3′ UTRs were suggested to have the divergent functional implications. In human, we further revealed that ubiquitously transcribed genes had higher median lengths of 3′ UTRs than the genes of tissue-restricted expressions. Conclusion We conducted a comprehensive analysis and provided an overview regarding 3′ UTRs biology of mRNAs in animals, whereas the mechanistic explanations of 3′ UTRs length variation in correlation to regulation of gene expression still remain to be further studied.

Combined Screening of Cervical Cancer, Breast Cancer and Reproductive Tract Infections in Rural China

Li, Zhi-Fang,Wang, Shao-Ming,Shi, Ju-Fang,Zhao, Fang-Hui,Ma, Jun-Fei,Qiao, You-Lin,Feng, Xiang-Xian Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.7

Objectives: To investigate the current prevalence and knowledge of cervical cancer, breast cancer and reproductive tract infections (RTIs) in rural Chinese women, and to explore the acceptance and feasibility of implementing a combined screening program in rural China. Methods: A population-based, cross-sectional study was conducted among women aged 30 to 59 years old in Xiangyuan County, Shanxi Province from 2009 to 2010. Socio-demographic characteristics, knowledge of cervical cancer, breast cancer and RTIs, and the attitude toward single or combined screening were collected by an interview questionnaire. Each participant received a clinical examination of the cervix, breast and reproductive tract. Examinations included visual inspection, mammography, laboratory tests and pathological diagnosis. Results: A total of 1,530 women were enrolled in this study. The prevalence of cervical precancerous lesions, suspicious breast cancer, suspicious benign breast disease and RTIs was 1.4%, 0.2%, 14.0% and 54.3%, respectively. Cervicitis, trichomonas vaginitis, and bacterial vaginitis were the three most common RTIs among our participants. Television, radio broadcast, and public education during screening were the major source of healthcare knowledge in rural China. Moreover 99.7% of women expressed great interest in participating in a combined screening project. The affordable limit for combined screening project was only 50 RMB for more than half of the rural women. Conclusion: A combined screening program would be more effective and popular than single disease screening projects, while appropriate accompanied education and a co-pay model for its successful implementation need to be explored, especially in low-resource settings.