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Quantifying the effects of short-term heat stress at booting stage on nonstructural carbohydrates remobilization in rice

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The Crop Journal 2020年第2期8卷 194-212页

作者： Fengxian Zhen Junjie Zhou Aqib Mahmood Wei Wang Xini Chang Bing Liu Leilei Liu Weixing Cao Yan Zhu Liang Tang National Engineering and Technology Center for Information Agriculture Key Laboratory for Crop System Analysis and Decision MakingMinistry of Agriculture and Rural Affairs of the People’s Republic of ChinaJiangsu Key Laboratory for Information AgricultureJiangsu Collaborative Innovation Center for Modern Crop ProductionNanjing Agricultural UniversityNanjing 210095JiangsuChina

Extreme heat stress events are becoming more frequent under anticipated climate change,which can have devastating impacts on rice growth and *** quantify the effects of short-term heat stress at booting stage on nonstructural carbohydrates(NSC)remobilization in rice,two varieties(Nanjing 41 and Wuyunjing 24)were subjected to 32/22/27°C(maximum/minimum/mean),36/26/31°C,40/30/35°C,and 44/34/39°C for 2,4 and 6 days in phytotrons at booting stage during 2014 and *** and yield components,dry matter partitioning index(DMPI),NSC accumulation and translocation were measured and *** results showed that the increase of high-temperature level and duration significantly reduced grain yield by suppressing spikelet number per panicle,seed-setting rate,and grain *** stress at booting decreased DMPI in panicles,increased DMPI in stems,but had no significant effect on photosynthetic *** NSC concentration increased whereas panicles NSC concentration,stem NSC translocation efficiency,and contribution of stem NSC to grain yield *** heat stress even transformed the stem into a carbohydrate sink during grain *** heat-tolerant Wuyunjing 24 showed a higher NSC transport capacity under heat stress than the heat-sensitive Nanjing *** degree-days(HDD),which combines the effects of the intensity and duration of heat stress,used for quantifying the impacts of heat stress indicates the threshold HDD for the termination of NSC translocation is 9.82°C *** yield was negatively correlated with stem NSC concentration and accumulation at maturity,and yield reduction was tightly related to NSC translocation *** results suggest that heat stress at booting inhibits NSC translocation due to sink size ***,genotypes with higher NSC transport capacity under heat stress could be beneficial for rice yield formation.

关键词： Rice(Oryza sativa L.) Heat stress Yield components nonstructural carbohydrates Translocation

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Construction of infectious cDNA clones of PRRSV:Separation of coding regions for nonstructural and structural proteins

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Science China(Life Sciences) 2008年第3期51卷 271-279页

作者： YUAN ShiShan & WEI ZuZhang Department of Animal Infectious Diseases, Key Laboratory of Animal Parasitology Chinese Ministry of Agriculture, Shanghai Vet- erinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200232, China Department of Animal Infectious Diseases Key Laboratory of Animal Parasitology Chinese Ministry of Agriculture Shanghai Veterinary Research Institute Chinese Academy of Agricultural Sciences Shanghai 200232 China

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the causative agent of the ongoing "porcine high fever syndrome" in China, is capable of genetic and antigenic mutations at high fre- quency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great interest. We developed an infectious cDNA clone of an attenuated strain of Type II PRRSV, and further manipulated the infectious cDNA clone by inserting polylinker between ORF1 and ORF2, en- coding for nonstructural- or structural-protein, respectively. The cDNA was generated from the cell-attenuated virus strain, APRRS, via RT-PCR, and followed by nucleotide sequencing and molecular cloning. The full-length of the APRRS genomic RNA was determined as 15521 nucleotides in length excluding poly(A) tail, which has a 99.7% nucleotide identity with that of PRRSV Nsp strain, also a vac- cine strain. Based on the nucleotide sequencing results, the full-length cDNA clone was assembled in pBlueScript vector, under the control of T7 promoter at the immediate 5′ terminus of genome. To dis- cern the rescued viruses from that of parental virus, a Mlu I restriction site was engineered into ORF5 coding region. Upon transfection of the in vitro transcripts of both the original and Mlu I-tagged cDNAs into MA-104 cells, typical PRRSV cytopathic effects were observed. The rescued viruses from the full-length cDNA clones displayed the same virological and molecular properties. Subsequently, PCR-based mutagenesis was conducted to separate the coding regions between PRRSV nonstructural genes, ORF1, and structural proteins, ORF2-ORF7. The synthetic RNA of such mutant clone, pCSA, was infectious and the rescued virus shared similar properties with that of the parental virus. This study provided a valuable tool for development of chimeric PRRSV as vaccine candidate offering cross-protection to various genetically diversified PRRSV strains, and a platform for further develop- ment of PRRSV a

关键词： PRRSV reverse genetic system coding region nonstructural protein

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Challenge to overcome: nonstructural protein 5A-P32 deletion in direct-acting antiviral-based therapy for hepatitis C virus

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World Journal of Gastroenterology 2018年第38期24卷 4304-4310页

作者： Ken Sato Toshio Uraoka Department of Gastroenterology and Hepatology Gunma University Graduate School of Medicine

Interferon(IFN)-based therapy for hepatitis C virus(HCV) infection has recently been replaced by IFNfree direct-acting antiviral(DAA)-based therapy, which has been established as a 1^(st) line therapy with high efficacy and tolerability due to its reasonable safety profile. Resistance-associated substitutions(RASs) have been a weakness of DAA-based therapy. For example, combination therapy with daclatasvir and asunaprevir(DCV/ASV) is less effective for HCV genotype 1-infected patients with Y93H as a nonstructural protein 5A RAS. However, the problem regarding RASs has been gradually overcome with the advent of recently developed DAAs, such as sofosbuvir-based regimens or combination therapy with glecaprevir and pibrentasvir. Despite the high efficiency of DAA-based therapy, some cases fail to achieve viral eradication. P32 deletion, an NS5A RAS, has been gradually noticed in patients with DCV/ASV failure. P32 deletion has been sporadically reported and the prevalence of this RAS has been considered to be low in patients with DCV/ASV failure. Thus, the picture of P32 deletion has not been fully evaluated. Importantly, currently-commercialized DAA-based combination therapy was not likely to be effective for patients with P32 deletion. Exploring and overcoming this RAS is essential for antiviral therapy for chronic hepatitis C.

关键词： Chronic hepatitis C Direct-acting antivirals Resistant-associated substitution P32 deletion nonstructural protein 5A

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Primary structure and variability of partial nonstructural gene 5 region of hepatitis G virus

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Chinese Medical Journal 1997年第10期 29-33页

作者：常锦红魏来陶其敏

Objective To sequence partial nonstructural 5 (NS5) gene of hepatitis G virus (HGV) in the sera of Chinese patients with chronic non A E hepatitis or hepatitis B virus(HBV) infection. Methods Total nucleic acid was extracted from the sera of 35 patients with chronic non A E hepatitis and 20 patients with HBV infection, Then it was subjected to reverse transcriptase nested polymerase chain reaction (RT nested PCR) with primers derived from the putative nonstructural gene 5 (NS5) region of HGV. The positive PCR products(994 bp in length) were directly sequenced using the Sang's method after purification. Results HGV RNA was detected in 1 of the 35 patients (2.9%) with chronic non A E hepatitis and 5 of the 20 patients (25%) with HBV infection. All the 6 positive samples were sequenced. These 6 Chinese isolates exhibited 87.21% to 93.92% nucleotide sequence identities with 3 reported isolates (GBV C, PNF2161, R10292) over the 994 nucleotides examined,while the nucleotide homologies among the 6 isolates were 90.09% to 93.82%. And 93.61% to 98.72% homologies were found at the deduced amino acid level in comparing the 6 isolates with 3 reported ones, while 93.61% to 98.40% amino acid identities were found among the 6 isolates. In the region sequenced, there existed 16 conserved proline residues and 8 conserved cysteine residues. Conclusions Six isolates of HGV were obtained from different Chinese patients in this study. The partial nucleotide sequences, as well as amino acid sequences, of the putative NS5 region of HGV in Chinese patients are relatively conserved. The overall degree of nucleotide conservation (90.09% to 93.82%)among Chinese isolates may make this region of the genome suitable for the development of diagnosis reagents.

关键词：陶其敏 structure nonstructural partial gene and variability 锦红 Primary

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登革病毒2型非结构蛋白NS2B的原核表达、纯化及其抗体的制备

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免疫学杂志 2008年第2期24卷 135-138页

作者：刘丽梅陈宗涛田衍平陈炜江雯徐小峰高娜安静第三军医大学基础医学部微生物学教研室重庆400038 第三军医大学基础医学部组织胚胎学教研室重庆400038 首都医科大学基础医学院微生物学教研室北京100069

目的原核表达登革病毒(Dengue Virus,DV)NS2B蛋白并纯化,制备NS2B的小鼠多克隆抗体。方法RT-PCR扩增DV2全长的NS2B基因序列,构建表达带有6×His标签的NS2B的原核表达载体,进行表达与纯化,免疫Balb/c小鼠,制备NS2B多克隆抗体。采用ELISA... 详细信息

目的原核表达登革病毒(Dengue Virus,DV)NS2B蛋白并纯化,制备NS2B的小鼠多克隆抗体。方法RT-PCR扩增DV2全长的NS2B基因序列,构建表达带有6×His标签的NS2B的原核表达载体,进行表达与纯化,免疫Balb/c小鼠,制备NS2B多克隆抗体。采用ELISA检测抗体效价,Western-blot及免疫荧光染色检测其特异性。结果成功构建原核表达载体pQE-31/NS2B,进一步获得相对分子质量为16000的纯化蛋白及多克隆抗体,ELISA显示抗体效价达1∶25600。Western-blot和免疫荧光染色显示多克隆抗体与NS2B蛋白特异性结合。结论本研究获得了纯化的DV2NS2B蛋白,成功地制备了NS2B多克隆抗体,为进一步研究NS2B的作用机制奠定了基础。

关键词：登革病毒 nonstructural Protein 2B(NS2B) 原核表达载体纯化蛋白多克隆抗体

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Recent Progress on Functional Genomics Research of Enterovirus 71

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Virologica Sinica 2019年第1期34卷 9-21页

作者： Huiqiang Wang Yuhuan Li Beijing Key Laboratory of Antimicrobial Agents Institute of Medicinal BiotechnologyChinese Academy of Medical Sciences and Peking Union Medical College NHC Key Laboratory of Biotechnology of Antibiotics Institute of Medicinal BiotechnologyChinese Academy of Medical Sciences and Peking Union Medical College

Enterovirus 71(EV71) is one of the main pathogens that causes hand-foot-and-mouth disease(HFMD). HFMD caused by EV71 infection is mostly self-limited; however, some infections can cause severe neurological diseases, such as aseptic meningitis, brain stem encephalitis, and even death. There are still no effective clinical drugs used for the prevention and treatment of HFMD. Studying EV71 protein function is essential for elucidating the EV71 replication process and developing anti-EV71 drugs and vaccines. In this review, we summarized the recent progress in the studies of EV71 noncoding regions(50 UTR and 30 UTR) and all structural and nonstructural proteins, especially the key motifs involving in viral infection, replication, and immune regulation. This review will promote our understanding of EV71 virus replication and pathogenesis, and will facilitate the development of novel drugs or vaccines to treat EV71.

关键词： Enterovirus 71 (EV71) Functional genomics Structural protein nonstructural protein Viral protein function

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Hepatitis C virus NS5A promotes insulin resistance through IRS-1 serine phosphorylation and increased gluconeogenesis

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World Journal of Gastroenterology 2015年第43期21卷 12361-12369页

作者： Fahed Parvaiz Sobia Manzoor Jawed Iqbal Mehuli Sarkar-Dutta Muhammad Imran Gulam Waris Atta-urRahman School of Applied Biosciences National University of Sciences and Technology Department of Microbiology and Immunology Rosalind Franklin University of Medicine and Science

AIM: To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus(HCV) nonstructural protein 5A(NS5A).METHODS: The human hepatoma cell lines,Huh7 and Huh7.5,were infected with HCV or transientlytransfected with a vector expressing HCV NS5 A. The effect of HCV NS5 A on the status of the critical players involved in insulin signaling was analyzed using realtime quantitative polymerase chain reaction and Western blot assays. Data were analyzed using Graph Pad Prism version ***: To investigate the effect of insulin treatment on the players involved in insulin signaling pathway,we analyzed the status of insulin receptor substrate-1(IRS-1) phosphorylation in HCV infected cells or Huh7.5 cells transfected with an HCV NS5 A expression vector. Our results indicated that there was an increased phosphorylation of IRS-1(Ser307) in HCV infected or NS5 A transfected Huh7.5 cells compared to their respective controls. Furthermore,an increased phosphorylation of Akt(Ser473) was observed in HCV infected and NS5 A transfected cells compared to their mock infected cells. In contrast,we observed decreased phosphorylation of Akt Thr308 phosphorylation in HCV NS5 A transfected cells. These results suggest that Huh7.5 cells either infected with HCV or ectopically expressing HCV NS5 A alone have the potential to induce insulin resistance by the phosphorylation of IRS-1 at serine residue(Ser307) followed by decreased phosphorylation of Akt Thr308,Fox01 Ser256 and GSK3β Ser9,the downstream players of the insulin signalingpathway. Furthermore,increased expression of PECK and glucose-6-phosphatase,the molecules involved in gluconeogenesis,in HCV NS5 A transfected cells was ***: Taken together,our results suggest the role of HCV NS5 A in the induction of insulin resistance by modulating various cellular targets involved in the insulin signaling pathway.

关键词： Hepatitis C virus nonstructural protein 5A Insulin

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Expression of Major B-cell Epitopes within the 2C Non-structural Protein of FMDV and Their Immunoreactivity

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畜牧兽医学报 2009年第S1期40卷 39-42页

作者： FU Yuan-fang LU Zeng-jun TIAN Mei-na ZHANG Xiao-li LIU Zai-xin CAI Xue-peng State Key Laboratory of Veterinary Etiological Biology National Foot-and-Mouth Disease Reference LaboratoryLanzhou Veterinary Research InstituteChinese Academy of Agricultural Sciences

A pair of specific primers was designed from the 2C gene sequence of foot-and-mouth disease virus(FMDV)for amplification of a fragment including 174bp of the 5'-end and 279bp of the 3'-end of the2C gene,which encoded an abundance of known B-cell epitopes of the *** amplified fragment was inserted into pET-30a plasmid(Novagen)via two unique endonuclease restriction sites,Nco I and Sal *** confirmed that the open reading frame of interest was correctly inserted into the positive recombinant *** positive plasmid was transformed into the host bacteria BL21(DE3)pLys for protein *** induction by IPTG at 37℃for 5 hours,the expressed product was analyzed by SDSPAGE and Western blotting,confirming successful *** product is a 23kDa fusion protein and was shown to react with sera derived from FMDV-infected *** approach provides an useful antigen for establishing an enzyme-linked immunoelectro-transfer blot assay(EITB)diagnostic method,useful for differentiating FMDV-infected animals from those that been vaccinated.

关键词： FMDV nonstructural protein 2C gene EITB

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Molecular Characteristics,Functions,and Related Pathogenicity of MERS-CoV Proteins

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Engineering 2019年第5期5卷 940-947页

作者： Yan-Hua Li Chen-Yu Hu Nan-Ping Wu Hang-Ping Yao Lan-Juan Li State Key Laboratory for Diagnosis and Treatment of Infectious Diseases Hangzhou 310003China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases The First Affiliated HospitalSchool of MedicineZhejiang UniversityHangzhou 31003China

Middle East respiratory syndrome(MERS)is a viral respiratory disease caused by a de novo coronavirus—MERS-CoV—that is associated with high ***,the mechanism by which MERS-CoV infects humans remains *** date,there is no effective vaccine or antibody for human immunity and treatment,other than the safety and tolerability of the fully human polyclonal Immunoglobulin G(IgG)antibody(SAB-301)as a putative therapeutic agent specific for *** rapid diagnostic and public health measures are currently being implemented,new cases of MERS-CoV infection are still being ***,various effective measures should be taken to prevent the serious impact of similar epidemics in the *** investigation of the epidemiology and pathogenesis of the virus,as well as the development of effective therapeutic and prophylactic anti-MERS-CoV infections,is *** this purpose,detailed information on MERS-CoV proteins is *** this review,we describe the major structural and nonstructural proteins of MERS-CoV and summarize different potential strategies for limiting the outbreak of *** combination of computational biology and virology can accelerate the advanced design and development of effective peptide therapeutics against *** summary,this review provides important information about the progress of the elimination of MERS,from prevention to treatment.

关键词： MERS-CoV Spike protein Structural protein Accessory protein nonstructural protein

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Metabolic alterations and hepatitis C:From bench tobedside

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World Journal of Gastroenterology 2016年第4期22卷 1461-1476页

作者： Ming-Ling Chang Liver Research Center Department of Gastroenterology and HepatologyChang Gung Memorial HospitalTaoyuan 33305Taiwan Graduate Institute of Clinical Medical Sciences Chang Gung UniversityTaoyuan 33302Taiwan

In addition to causing cirrhosis and hepatocellular carcinoma, hepatitis C virus(HCV) is thought to cause hypolipidemia, hepatic steatosis, insulin resistance, metabolic syndrome, and diabetes. The viral life cycle of HCV depends on cholesterol metabolism in host cells. HCV core protein and nonstructural protein 5A perturb crucial lipid and glucose pathways, such as the sterol regulatory element-binding protein pathway and t he prot e in kinase B /mammal ian t arget of rapamycin/S6 kinase 1 pathway. Although several lines of transgenic mice expressing core or full HCV proteins exhibit hepatic steatosis and/or dyslipidemia, whether they completely reflect the metabolic alterations in humans with HCV infection remains unknown. Many cross-sectional studies have demonstrated increased prevalences of metabolic alterations and cardiovascular events in patients with chronic hepatitis C(CHC); however, conflicting results exist, primarily due to unavoidable individual variations. Utilizing anti-HCV therapy, most longitudinal cohort studies of CHC patients have demonstrated the favorable effects of viral clearance in attenuating metabolic alterations and cardiovascular risks. To determine the risks of HCV-associated metabolic alterations and associated complications in patients with CHC, it is necessary to adjust for crucial confounders, such as HCV genotype and host baseline glucose metabolism, for a long follow-up period after anti-HCV treatment. Adipose tissue is an important endocrine organ due to its release of adipocytokines, which regulate lipid and glucose metabolism. However, most data on HCV infection and adipocytokine alteration are inconclusive. A comprehensive overview of HCV-associated metabolic and adipocytokine alterations, from bench to bedside, is presented in this topic highlight.

关键词： Lipid Glucose Cardiovascular Hepatitis Cvirus Core nonstructural protein 5 A Transgenic mice

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