Biocatalysis and Bioprocess Engineering : Heterologous Expression and Characterization of a Thermostable Exo-β-D-Glucosaminidase from Aspergillus oryzae

( Ding Xin Wu ),( Lin Chun Wang ),( Yuwei Li ),( Shu Miao Zhao ),( Nan Peng ),( Yun Xiang Liang ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.2

An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50°C; however, the enzyme expressed Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50°C than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed comparison of V_{max} and k_{cat} values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.

Relationship among porcine lncRNA TCONS_00010987, miR-323, and leptin receptor based on dual luciferase reporter gene assays and expression patterns

Ding, Yueyun,Qian, Li,Wang, Li,Wu, Chaodong,Li, DengTao,Zhang, Xiaodong,Yin, Zongjun,Wang, Yuanlang,Zhang, Wei,Wu, Xudong,Ding, Jian,Yang, Min,Zhang, Liang,Shang, Jinnan,Wang, Chonglong,Gao, Yafei Asian Australasian Association of Animal Productio 2020 Animal Bioscience Vol.33 No.2

Objective: Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987. Methods: Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3'-UTR fragments were generated and cloned into pmiRRB-REPORT^TM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting. Results: Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3'-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3'-UTR (p>0.05 for both). Backfat expression levels of TCONS_00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01). Conclusion: LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.

High Mobility Group Box 1 Protein Is Methylated and Transported to Cytoplasm in Clear Cell Renal Cell Carcinoma

Wu, Fei,Zhao, Zuo-Hui,Ding, Sen-Tai,Wu, Hai-Hu,Lu, Jia-Ju Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.10

Background: The high mobility group box 1 (HMGB1) protein is a widespread nuclear protein present in most cell types. It typically locates in the nucleus and functions as a nuclear cofactor in transcription regulation. However, HMGB1 can also localize in the cytoplasm and be released into extracellular matrix, where it plays critical roles in carcinogenesis and inflammation. However, it remains elusive whether HMGB1 is relocated to cytoplasm in clear cell renal cell carcinoma (ccRCC). Methods: Nuclear and cytoplasmic proteins were extracted by different protocols from 20 ccRCC samples and corresponding adjacent renal tissues. Western blotting and immunohistochemistry were used to identify the expression of HMGB1 in ccRCC. To elucidate the potential mechanism of HMGB1 cytoplasmic translocation, HMGB1 proteins were enriched by immunoprecipitation and analyzed by mass spectrometry (MS). Results: The HMGB1 protein was overexpressed and partially localized in cytoplasm in ccRCC samples (12/20, 60%, p<0.05). Immunohistochemistry results indicated that ccRCC of high nuclear grade possess more HMGB1 relocation than those with low grade (p<0.05). Methylation of HMGB1 at lysine 112 in ccRCC was detected by MS. Bioinformatics analysis showed that post-translational modification might affect the binding ability to DNA and mediate its translocation. Conclusion: Relocation of HMGB1 to cytoplasm was confirmed in ccRCC. Methylation of HMGB1 at lysine 112 might the redistribution of this cofactor protein.

Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays

Yueyun Ding,Shujiao Zhu,Chaodong Wu,Li Qian,DengTao Li,Li Wang,Yuanlang Wang,Wei Zhang,Min Yang,Jian Ding,Xudong Wu,Xiao-Dong Zhang,Yafei Gao,Zongjun Yin 아세아·태평양축산학회 2019 Animal Bioscience Vol.32 No.7

Objective: Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. Methods: Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3′-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3′-UTR and pGL3 Control LDLR mutant-3′-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3′-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. Results: Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3′-UTR-driven (p<0.01), but not mutant LDLR-3′-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = –0.656, p<0.05). Conclusion: LDLR is a potential target of miR-20a, which might directly bind the LDLR 3′-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

Knowledge roadmap of sustainable development in the textile and apparel industry: a scientometric analysis

Zhaoshan Wu,Liya Zhou,Xuemei Ding,Xiongying Wu,Laili Wang 한국의류학회 2022 Fashion and Textiles Vol.9 No.1

Practices in the textile and apparel industry (TAI) have led to numerous environmental and social problems, which have prompted extensive research on the sustainable development of the textile and apparel industry (SDTAI). This paper presents a comprehensive and quantitative analysis of the status quo in the SDTAI domain using scientometrics. From 1987 to 2019, the Web of Science core collection databases (SCI and SSCI) included 863 journal articles related to SDTAI, and our analysis results were as follows: (1) 60 critical research keywords occur in the knowledge base; (2) four research hotspots were identifed; (3) fve themes constituted the main knowledge area; and (4) based on the knowledge base, research hotspot, and knowledge domain, the knowledge structure consisted of nine subjects and fve systems. This paper proposes a knowledge roadmap that can be helpful for practitioners and academicians to better understand the current sustainable development status and trends in the TAI.

Short-circuiting in fullerene devices studied by in situ electrical measurement in high vacuum and infrared imaging analysis?

H.R. Wu,M.L. Wang,Q.L. Song,Y. Wu,Z.T. Xie,X.D. Gao,X.M. Ding,X.Y. Hou 한국물리학회 2007 Current Applied Physics Vol.7 No.3

In the present work, currentvoltage (IV) characteristics of fullerene devices (ITOnC60nAl) are reexamined byin situelectrical mea-surement in high vacuum and by infrared imaging analysis. Two kinds ofIV curves are detected: ‘ohmic’ and nonohmic. Degradationprocesses of the two dierent devices are measured, and ‘ohmic’ degradation processes are ascribed to short-circuiting. ITOnC60nAldevices in high vacuum are conrmed to be intrinsically nonohmic. Surface temperature distribution of the two dierent devices is mea-buers are inserted between fullerene layer and cathode and this is found to be eective.

Development of Enzymatic Recombinase Amplification Assays for the Rapid Visual Detection of HPV16/18

Ding Ning,Qi Wanwan,Wu Zihan,Zhang Yaqin,Xu Ruowei,Lin Qiannan,Zhu Jin,Zhang Huilin 한국미생물·생명공학회 2023 Journal of microbiology and biotechnology Vol.33 No.8

Human papillomavirus (HPV) types 16 and 18 are the major causes of cervical lesions and are associated with 71% of cervical cancer cases globally. However, public health infrastructures to support cervical cancer screening may be unavailable to women in low-resource areas. Therefore, sensitive, convenient, and cost-efficient diagnostic methods are required for the detection of HPV16/18. Here, we designed two novel methods, real-time ERA and ERA-LFD, based on enzymatic recombinase amplification (ERA) for quick point-of-care identification of the HPV E6/E7 genes. The entire detection process could be completed within 25 min at a constant low temperature (35–43°C), and the results of the combined methods could be present as the amplification curves or the bands presented on dipsticks and directly interpreted with the naked eye. The ERA assays evaluated using standard plasmids carrying the E6/E7 genes and clinical samples exhibited excellent specificity, as no cross-reaction with other common HPV types was observed. The detection limits of our ERA assays were 100 and 101 copies/μl for HPV16 and 18 respectively, which were comparable to those of the real-time PCR assay. Assessment of the clinical performance of the ERA assays using 114 cervical tissue samples demonstrated that they are highly consistent with real-time PCR, the gold standard for HPV detection. This study demonstrated that ERA-based assays possess excellent sensitivity, specificity, and repeatability for HPV16 and HPV18 detection with great potential to become robust diagnostic tools in local hospitals and field studies.

The Effectiveness of OSCE Staged Training Program on Nurse Practitioner Learning

Wu Shu-Chen,Chen Yen-Chin,Ding Jian-Siou,Lin Hui-Shan,Lin Chia-Ying,Change Wan- Ling 한국간호과학회 2023 한국간호과학회 학술대회 Vol.2023 No.11

Hierarchical porous ECM scaffolds incorporating GDF-5 fabricated by cryogenic 3D printing to promote articular cartilage regeneration

Wu Jiang,Fu Liwei,Yan Zineng,Yang Yu,Yin Han,Li Pinxue,Yuan Xun,Ding Zhengang,Kang Teng,Tian Zhuang,Liao Zhiyao,Tian Guangzhao,Ning Chao,Li Yuguo,Sui Xiang,Chen Mingxue,Liu Shuyun,Guo Quanyi 한국생체재료학회 2023 생체재료학회지 Vol.27 No.00

In recent years, there has been significant research progress on in situ articular cartilage (AC) tissue engineering with endogenous stem cells, which uses biological materials or bioactive factors to improve the regeneration microenvironment and recruit more endogenous stem cells from the joint cavity to the defect area to promote cartilage regeneration.In this study, we used ECM alone as a bioink in low-temperature deposition manufacturing (LDM) 3D printing and then successfully fabricated a hierarchical porous ECM scaffold incorporating GDF-5.Comparative in vitro experiments showed that the 7% ECM scaffolds had the best biocompatibility. After the addition of GDF-5 protein, the ECM scaffolds significantly improved bone marrow mesenchymal stem cell (BMSC) migration and chondrogenic differentiation. Most importantly, the in vivo results showed that the ECM/GDF-5 scaffold significantly enhanced in situ cartilage repair.In conclusion, this study reports the construction of a new scaffold based on the concept of in situ regeneration, and we believe that our findings will provide a new treatment strategy for AC defect repair.

Reliability Evaluation of a Distribution System with wind Turbine Generators Based on the Switch-section Partitioning Method

Wu, Hongbin,Guo, Jinjin,Ding, Ming The Korean Institute of Electrical Engineers 2016 Journal of Electrical Engineering & Technology Vol.11 No.3

Considering the randomness and uncertainty of wind power, a reliability model of WTGs is established based on the combination of the Weibull distribution and the Markov chain. To analyze the failure mode quickly, we use the switch-section partitioning method. After defining the first-level load zone node, we can obtain the supply power sets of the first-level load zone nodes with each WTG. Based on the supply sets, we propose the dynamic division strategy of island operation. By adopting the fault analysis method with the attributes defined in the switch-section, we evaluate the reliability of the distribution network with WTGs using a sequential Monte Carlo simulation method. Finally, using the IEEE RBTS Bus6 test system, we demonstrate the efficacy of the proposed model and method by comparing different schemes to access the WTGs.