Structural-based Screening of L-phenylglycine Aminotransferase using L-phenylalanine as the Optimal Amino Donor: Recycling of L-phenylalanine to Produce L-phenylglycine

Shuang Ping Liu,Rui Xia Liu,Jian Mao,Liang Zhang,Zhongyang Ding,Zhenghua Gu,Gui-Yang Shi 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.1

The aproteinogenic amino acid, L-phenylglycine, is an important side chain building block for some drugs. It would be of great commercial and environmental value to biocatalyse L-phenylalanine to L-phenylglycine, and thus replace the organic synthesis method. To produce Lphenylglycine from L-phenylalanine, an L-phenylglycine aminotransferase was screened and characterized. HpgTAO showed high homology to α-aminoadipate aminotransferase. The L-phenylalanine binding site was near the residues S26, R401, N201, and G46 in HpgTAO, and L-phenylalanine formed a hydrogen bond with Asn20, which was similar to the substrate binding mechanism of α-aminoadipate aminotransferase. HpgTAO showed increased activity in alkalescent environment below 40°C. The kinetic analysis showed that L-phenylalanine had the highest affinity to HpgTAO, which ensured the recycle biosynthesis of Lphenylglycine from L-phenylalanine. To date, it was the only aminotransferase using L-phenylalanine as an optimal amino donor. The L-phenylglycine biocatalysis operon was also constructed by co-expressing the hmaS, hmo and hpgT by a single plasmid. The first in vitro conversion of Lphenylalanine to L-phenylglycine was achieved by directly using the L-phenylalanine fermentation broth as the raw material.

The effect of dietary asparagine supplementation on energy metabolism in liver of weaning pigs when challenged with lipopolysaccharide

Kang, Ping,Liu, Yulan,Zhu, Huiling,Zhang, Jing,Shi, Haifeng,Li, Shuang,Pi, Dinan,Leng, Weibo,Wang, Xiuying,Wu, Huanting,Hou, Yongqing Asian Australasian Association of Animal Productio 2018 Animal Bioscience Vol.31 No.4

Objective: This experiment was conducted to investigate whether asparagine (Asn) could improve liver energy status in weaning pigs when challenged with lipopolysaccharide. Methods: Forty-eight weaned pigs ($Duroc{\times}Large\;White{\times}Landrace$, $8.12{\pm}0.56kg$) were assigned to four treatments: i) CTRL, piglets received a control diet and injected with sterile 0.9% NaCl solution; ii) lipopolysaccharide challenged control (LPSCC), piglets received the same control diet and injected with Escherichia coli LPS; iii) lipopolysaccharide (LPS)+0.5% Asn, piglets received a 0.5% Asn diet and injected with LPS; and iv) LPS+1.0% Asn, piglets received a 1.0% Asn diet and injected with LPS. All piglets were fed the experimental diets for 19 d. On d 20, the pigs were injected intraperitoneally with Escherichia coli LPS at $100{\mu}g/kg$ body weights or the same volume of 0.9% NaCl solution based on the assigned treatments. Then the pigs were slaughtered at 4 h and 24 h after LPS or saline injection, and the liver samples were collected. Results: At 24 h after LPS challenge, dietary supplementation with 0.5% Asn increased ATP concentration (quadratic, p<0.05), and had a tendency to increase adenylate energy charges and reduce AMP/ATP ratio (quadratic, p<0.1) in liver. In addition, Asn increased the liver mRNA expression of pyruvate kinase, pyruvate dehydrogenase, citrate synthase, and isocitrate dehydrogenase ${\beta}$ (linear, p<0.05; quadratic, p<0.05), and had a tendency to increase the mRNA expression of hexokinase 2 (linear, p<0.1). Moreover, Asn increased liver phosphorylated AMP-activated protein kinase (pAMPK)/total AMP-activated protein kinase (tAMPK) ratio (linear, p<0.05; quadratic, p<0.05). However, at 4 h after LPS challenge, Asn supplementation had no effect on these parameters. Conclusion: The present study indicated that Asn could improve the energy metabolism in injured liver at the late stage of LPS challenge.

Research on the Strategy of Role Management Based on Grid Environment

Yi He LIU,Yu Ping QIN,Shuang ZHANG 보안공학연구지원센터 2016 International Journal of Security and Its Applicat Vol.10 No.2

The grid security affects directly the development of the grid and the practical application of grid system software. The access control is one of important contents of grid security research. The traditional access control models have ignored the subject security, and can not be solved with the dynamic grid, and the characteristics of the coexistence of multiple security strategy etc problems. Regarding expanded the concepts under the common network environment subject, the object, safe service, granularity control, the concept of subject/ object decomposition and the organization classifications are defined. Using the RBAC model and BLP the model basic principle, some of the new access control security strategy based on the grid environment has been defined in the paper. After discussion shows that the new rules emphasis on the subject security, and adapt to the dynamic nature of grid environment and characteristics of the coexistence of multiple security strategy. The new rules are compatibility with existing network access control model, they are secure and are also an expansion under the common network environment access control strategy, and this has certain positive significance to the grid security research.

Molecular characterization and functional analysis of two trehalose transporter genes in the cabbage beetle, Colaphellus bowringi

Li Jia-Xu,Cao Zhen,Guo Shuang,Tian Zhong,Liu Wen,Zhu Fen,Wang Xiao-Ping 한국응용곤충학회 2020 Journal of Asia-Pacific Entomology Vol.23 No.3

The trehalose, major blood sugar in insects, enhances stress resistance of diapausing individuals in adverse environment and provides an energy source for reproduction. Trehalose transporters (TRETs) play an important role in transport of trehalose from trehalose-producing tissues, e.g. fat body, to trehalose-consuming tissues. Although studies have shown that trehalose contributes to diapause and reproduction, the function of TRETs in these processes remains unclear. In this work, we cloned two TRET genes, TRET1a and TRET1b, from the cabbage beetle Colaphellus bowringi, which is capable of entering reproductive diapause under long-day conditions. We also analyzed the expression profiles of these two genes and investigated their potential roles in diapause and reproduction. The results suggested that both TRET1a and TRET1b belong to sugar-transporter and major facilitator superfamilies. Interestingly, TRET1a was highly expressed in the fat bodies of diapause-destined (DD) females but TRET1b was predominantly expressed in the ovaries of non-diapause-destined (NDD) females. Hormonal induction indicated that juvenile hormone induced TRET1b but repressed TRET1a at transcriptional levels. Methoprene-tolerant and Krüppel homolog 1 mediated the JH-suppressed TRET1a expression but were not involved in the regulation of TRET1b expression by JH. RNAi of TRET1a in DD females elevated the trehalose content in the fat bodies and suppressed the expression of a couple of genes related to stress resistance, which is a critical diapause trait. Knockdown of TRET1b in NDD females reduced the trehalose content in the ovaries but had no apparent effect on the ovary development and yolk deposition. These data suggest that TRET1a and TRET1b could regulate the trehalose content in specific tissues and may play potential roles in reproductive diapause in the females of C. bowringi. Introduction Diapause is a survival strategy for many insects in adapting to adverse environmental conditions (Tauber and Tauber, 1976; Tougeron, 2019). Diapause is a dynamic successive process that consists of diapause induction, preparation, initiation, maintenance, termination, and post-diapause quiescence (Kostal, 2006). Insects accumulate enormous nutrients such as sugars and lipids to enter diapause during diapause preparation phase (Hahn and Denlinger, 2007, 2011). The trehalose is the major hemolymph sugar in insects. The sugar not only provides source of energy but also protects proteins and cellular membranes from dehydration, desiccation, heat, cold, and oxidation (Elbein et al., 2003; Li et al., 2002; Richards et al., 2002; Wyatt and Kalf, 1957; Yancey, 2005; Zhu et al., 2008). Therefore, trehalose can play roles in reproduction and diapause by mediating energy supply and stress tolerance, respectively (Kamei et al., 2011; Lu et al., 2019; Xu et al., 2009).

Add-on Tiotropium in Chinese Patients With Moderate Asthma: A Pooled Subgroup Analysis of MezzoTinA-Asthma 1 and 2

Jiangtao Lin,Huanying Wan,Jian Kang,Qianli Ma,Ping Chen,Meiling Jin,Haoyan Wang,Shuang Liu,Qinglin Hao,Yong Lin,Lin Su,Na Hu 대한천식알레르기학회 2019 Allergy, Asthma & Immunology Research Vol.11 No.4

Purpose: Asthma affects approximately 30 million patients in China; however, tiotropium data for Chinese patients is limited. This study aimed to assess the efficacy and safety of tiotropium in Chinese patients with moderate symptomatic asthma. Methods: A post hoc subgroup analysis was conducted on 430 Chinese patients pooled from two 24-week, replicate phase 3 trials (NCT01172808 and NCT01172821), in which they received once-daily tiotropium 2.5 μg (Tio R2.5) or 5 μg (Tio R5) (n = 106 or 109, respectively), twice-daily salmeterol 50 μg (Sal 50) (n = 110), or placebo (n = 105), while maintaining inhaled corticosteroids (ICS). The co-primary endpoints assessed in week 24 were forced expiratory volume in 1 second (FEV1) peak0–3h response, trough FEV1 response, and responder rate as assessed using the Asthma Control Questionnaire (ACQ). Results: For both FEV1 peak0–3h responses and trough FEV1 responses, the mean treatment differences were greater for Tio R2.5, Tio R5, and Sal 50 compared with placebo at 0.249 L, 0.234 L, and 0.284 L, and 0.172 L, 0.180 L, and 0.164 L, respectively (P< 0.001). The ACQ responder rate in placebo, Tio R2.5, Tio R5, and Sal 50 was 58.7%, 62.3%, 59.3%, and 69.1%, respectively. Furthermore, 11 (2.6%) of 430 patients had serious adverse events (Tio R5, n = 4; Tio R2.5, n = 1; Sal 50, n = 1; and placebo, n = 5). Conclusions: Once-daily tiotropium, as add-on to medium-dose ICS, was effective and well tolerated for Chinese patients with moderate symptomatic asthma, consistent with the main analysis.

The Relationship between HIF-2α and VEGF with Radiographic Severity in the Primary Osteoarthritic Knee

Zhou Jian-lin,Fang Hong-song,Peng Hao,Deng Shuang,Chen Shen,Li Jian-ping,Qiu Bo,Weng Jin-qing,Liu Feng 연세대학교의과대학 2016 Yonsei medical journal Vol.57 No.3

Purpose: The aim of this study was to determine the relationship of hypoxia-inducible factor-2 (HIF-2α) and vascular endothelial growth factor (VEGF) with radiographic severity in primary osteoarthritis (OA) of the knee. Expression of these two factors in cartilagesamples from OA knee joints was examined at mRNA and protein levels. Materials and Methods: Knee joints were examined using plain radiographs, and OA severity was assessed using the Kellgren and Lawrence (KL) grading system. Specimens were collected from 29 patients (31 knees) who underwent total knee replacementbecause of severe medial OA of the knee (KL grades 3 and 4), 16 patients who underwent knee arthroscopy (KL grade 2), and 5 patients with traumatic knees (KL grade 0). HIF-2α and VEGF expression was quantified by real-time polymerase chain reactionand western blotting. Results: Cartilage degeneration correlated with the radiographic severity grade. OA severity, determined using the Mankin scale, correlated positively with the KL grade (r=0.8790, p<0.01), and HIF-2α and VEGF levels with the radiographic severity of knee OA (r=0.7001, p<0.05; r=0.6647, p<0.05). Conclusion: In OA cartilage, HIF-2α and VEGF mRNA and protein levels were significantly and positively correlated. The expressionof both factors correlated positively with the KL grade. HIF-2α and VEGF, therefore, may serve as biochemical markers as well as potential therapeutic targets in knee OA.

Determination of Protein, Fat, Starch, and Amino Acids in Foxtail Millet [Setaria italica (L.) Beauv.] by Fourier Transform Near-Infrared Reflectance Spectroscopy

Xiu-Shi Yang,Li-Li Wang,Xian-Rong Zhou,Shaomin Shuang,Zhi-Hua Zhu,Nan Li,Yan Li,Fang Liu,San-Cai Liu,Ping Lu,Guixing Ren,Chuan Dong 한국식품과학회 2013 Food Science and Biotechnology Vol.22 No.6

Quantitative detection of protein, fat, starch,and amino acids in foxtail millet using Fourier transformnear-infrared spectroscopy (NIRS) was investigated. Foxtail millet samples (n=259) were analyzed using NIRS. Spectral data were linearized with data from chemicalanalyses. Calibration models were established using apartial least-squares (PLS) algorithm with cross-validation. Optimized models were tested using external validation setsamples with coefficients of determination in the externalvalidation (R2val) of >0.90. Residual predictive deviation(RPD) values were nearly equal to or >2.5 for crudeprotein, alanine, aspartic acid, glutamic acid, isoleucine,leucine, and serine. However, for glycine, histidine,phenylalanine, proline, threonine, tyrosine, and valine, theR2val values were >0.83 and RPD values were nearly equalto or >2.0. For crude fat, total starch, arginine, and lysine,the R2val values were >0.70 and RPD values were >1.5. NIRS is a rapid determination tool for foxtail milletbreeding, and for quality control.

TSHR Variant Screening and Phenotype Analysis in 367 Chinese Patients With Congenital Hypothyroidism

Zhang Hai-Yang,Wu Feng-Yao,Li Xue-Song,Tu Ping-Hui,Zhang Cao-Xu,Yang Rui-Meng,Cui Ren-Jie,Wu Chen-Yang,Fang Ya,Yang Liu,Song Huai-Dong,Zhao Shuang-Xia 대한진단검사의학회 2024 Annals of Laboratory Medicine Vol.44 No.4

Background: Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype–phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. Methods: In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results: Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. Conclusions: We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.