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Science China(Life Sciences) 2008年第7期51卷 640-647页

作者： SUN DanFeng,TIAN XiaoQing,ZHANG YanJie,CHEN YingXuan,LU Rong,WANG Xia &FANG JingYuan Shanghai Jiao-Tong University Sc0hool of Medicine Renji Hospital,Shanghai Institute of Digestive Disease,Shanghai 200001, China Shanghai Jiao-Tong University Sc0hool of Medicine Renji Hospital Shanghai Institute of Digestive Disease Shanghai China

The present study aimed to evaluate the relationship between mTOR signaling pathway and DNA methylation in cell survival,cell cycle,gene expression and protein level on human gastric cancer cells. Human gastric cancer cell lines,MKN45 and SGC7901 were treated with 5-aza-dC,rapamycin and/or LY294002.Cell viability was analyzed by MTT.Cell cycle distribution was evaluated by flow cytometry (FCM).The transcription level of PTEN and p27 Kip1 genes was detected by using real-time PCR.Protein expressions were detected by Western blotting.We found that cell viability was moderately reduced when treated with 5-aza-dC alone,but remarkably reduced when mTOR pathway was inhibited together (P<0.01).mTOR inhibition enhances the effects of 5-aza-dC on arresting cell cycle at G2 phase in human gastric cancer cell lines.The expression of PTEN and p27 Kip1 mRNA was remarkably increased in the gastric cancer cells treated with combind drugs(P<0.01).Phosphorylation of Akt,p70S6K and 4E-BP1 were significantly reduced in the cells treated with LY294002 or RAPA(P<0.01),but we failed to find that 5-aza-dC enhance these effects.We suggested that mTOR inhibition could enhance the effects of 5-aza-dC on suppressing cell proliferation and arresting cell cycle in human gastric cancer cell lines, which might be a potential target for tumor therapy.

关键词： mammalian target of rapamycin rapamycin DNA methylation 5-aza-dC gastric cancer

Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing

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The Innovation 2022年第4期3卷 74-82页

作者： Yangcan Chen Yanping Hu Xinge Wang Shengqiu Luo Ning Yang Yi Chen Zhikun Li Qi Zhou Wei Li State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China Institute for Stem Cell and Regenerative Medicine Chinese Academy of SciencesBeijing 100101China Bejing Institute for Stem Cell and Regenerative Medicine Beijing 100101China

The naturally occurring prokaryotic CRISPR-Cas systems provide valuable resources for the development of new genome-editing tools.However,the majority of prokaryotic Cas nucleases exhibit poor editing efficiency in mammalian cells,which significantly limits their utility.Here,we have developed a method termed Improving Editing Activity by Synergistic Engineering(MIDAS).This method exerts a synergistic effect to improve mammalian genome-editing efficiency of a wide range of CRISPR-Cas systems by enhancing the interactions of Cas nuclease with the protospacer adjacent motif(PAM)and the single-stranded DNA(ssDNA)substrate in the catalytic pocket simultaneously.MIDAS robustly and significantly increased the gene-editing efficiency of Cas12i,Cas12b,and CasX in human cells.Notably,a Cas12i variant,Cas12iMax,exhibited robust activity with a very broad PAM range(NTNN,NNTN,NAAN,and NCAN)and higher efficiency than the current widely used Cas nucleases.A high-fidelity version of Cas12iMax(Cas12iHiFi)has been further engineered to minimize off-target effects.Our work provides an expandable and efficacious method for engineering Cas nucleases for robust mammalian genome editing.

关键词： editing engineering mammalian

mammalian target of rapamycin inhibitors after post-transplant hepatocellular carcinoma recurrence:Is it too late?

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World Journal of Gastrointestinal Surgery 2020年第4期12卷 149-158页

作者： Kin Pan Au Kenneth Siu Ho Chok Department of Surgery Queen Mary HospitalHong Kong 999077China Department of Surgery and State Key Laboratory for Liver Research The University of Hong KongHong Kong 999077China

BACKGROUND mammalian target of rapamycin(mTOR)inhibitors have been shown to reduce the risk of tumour recurrence after liver transplantation for hepatocellular carcinoma(HCC).However,their role in established post-transplant HCC recurrence is uncertain.AIM To investigate whether mTOR inhibitor offers a survival benefit in posttransplant HCC recurrence.METHODS A retrospective study of 143 patients who developed HCC recurrence after liver transplantation was performed.They were divided into 2 groups based on whether they had received mTOR inhibitor-based immunosuppression.The primary endpoint was post-recurrence survival.RESULTS Seventy-nine(55%)patients received an mTOR inhibitor-based immunosuppressive regime,while 64(45%)patients did not.The mTOR inhibitor group had a lower number of recurrent tumours(2 vs 5,P=0.02)and received more active treatments including radiotherapy(39 vs 22%,P=0.03)and targeted therapy(59 vs 23%,P<0.001).The median post-recurrence survival was 21.0±4.1 mo in the mTOR inhibitor group and 11.2±2.5 mo in the control group.Multivariate Cox regression analysis confirmed that mTOR inhibitor therapy was independently associated with improved post-recurrence survival(P=0.04,OR=0.482,95%CI:0.241-0.966).The number of recurrent tumours and use of other treatment modalities did not affect survival.No survival difference was observed between mTOR inhibitor monotherapy and combination therapy with calcineurin inhibitor.CONCLUSION mTOR inhibitors prolonged survival after post-transplant HCC recurrence.

关键词： mammalian target of rapamycin inhibitor Hepatocellular carcinoma Recurrence Liver transplant Survival Outcomes

Improving Baculovirus Transduction of mammalian Cells by Incorporation of Thogotovirus Glycoproteins

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Virologica Sinica 2019年第4期34卷 454-466页

作者： Liangbo Hu Yimeng Li Fei Deng Zhihong Hu Hualin Wang Manli Wang State Key Laboratory of Virology Wuhan Institute of VirologyChinese Academy of SciencesWuhan 430071China University of the Chinese Academy of Sciences Beijing 100049China

Baculovirus can transduce a wide range of mammalian cells and is considered a promising gene therapy vector. However,the low transduction efficiency of baculovirus into many mammalian cells limits its practical application. Co-expressing heterologous viral glycoproteins(GPs), such as vesicular stomatitis virus G protein(VSV G), with baculovirus native envelope protein GP64 is one of the feasible strategies for improving virus transduction. Tick-borne thogotoviruses infect mammals and their GPs share sequence/structure homology and common evolutionary origins with baculovirus GP64.Herein, we tested whether thogotovirus GPs could facilitate the entry of the prototype baculovirus Autographa californica multiple multiple nucleopolyhedrovirus(AcMNPV) into mammalian cells. The gp genes of two thogotoviruses, Thogoto virus and Dhori virus, were inserted into the AcMNPV genome. Both GPs were properly expressed and incorporated into the envelope of the recombinant AcMNPVs. The transduction rates of recombinant AcMNPVs expressing the two thogotovirus GPs increased for approximately 4–12 fold compared to the wild type AcMNPV in six of the 12 tested mammalian cell lines. It seemed that thogotovirus GPs provide the recombinant AcMNPVs with different cell tropisms and showed better performance in several mammalian cells compared to VSV G incorporated AcMNPV. Further studies showed that the improved transduction was a result of augmented virus-endosome fusion and endosome escaping, rather than increased cell binding or internalization. We found the AcMNPV envelope protein GP64-mediated fusion was enhanced by the thogotovirus GPs at relatively higher p H conditions. Therefore, the thogotovirus GPs represent novel candidates to improve baculovirus-based gene delivery vectors.

关键词： Autographa californica multiple nucleopolyhedrovirus(AcMNPV) Baculovirus Thogotovirus Glycoprotein Transduction mammalian cells

The D253N Mutation in the Polymerase Basic 2 Gene in Avian Influenza(H9N2) Virus Contributes to the Pathogenesis of the Virus in mammalian Hosts

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Virologica Sinica 2018年第6期33卷 531-537页

作者： Jinfeng Zhang Rong Su Xiaoyun Jian Hongliang An Ronbing Jiang Chris Ka Pun Mok Laboratory Medicine Center Foshan Hospital of Traditional Chinese Medicine Guangzhou University of Chinese Medicine Department of Respiratory Medicine Foshan Hospital of Traditional Chinese Medicine Guangzhou University of Chinese Medicine State Key Laboratory of Respiratory Disease National Clinical Research Center for Respiratory Disease Guangzhou Institute of Respiratory Health The First Affiliated Hospital of Guangzhou Medical University HKU-Pasteur Research Pole School of Public Health HKU Li Ka Shing Faculty of Medicine The University of Hong Kong

Mutations in the polymerase basic 2(PB2) gene of avian influenza viruses are important signatures for their adaptation to mammalian hosts. Various adaptive mutations have been identified around the 627 and nuclear localization sequence(NLS) domains of PB2 protein, and these mutations contribute to the replicative ability of avian influenza viruses.However, few studies have focused on adaptive mutations in other regions of PB2. In this study, we investigated the functional roles of the D253N mutation in PB2 in an H9N2 virus. This mutation was found to affect an amino acid residue in the middle domain of the PB2 protein. The virus with the D253N mutation showed higher polymerase activity and transiently increased viral replication in human cells. However, the mutant did not show significant differences in viral replication in the respiratory tract of mice upon infection. Our results supported that the D253N mutation in the middle domain of PB2, similar to mutations at the 627 and NLS domains, specifically contributed to the replication of avian influenza viruses in human cells.

关键词： Avian influenza virus mammalian adaptation D253N Polymerase basic 2(PB2) H9N2

Conditional Genome Editing in the mammalian Brain Using CRISPR-Cas9

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Neuroscience Bulletin 2021年第3期37卷 423-426页

作者： Haojie Sun Jie Zheng Ming Yi You Wan Neuroscience Research Institute Department of NeurobiologySchool of Basic Medical SciencesPeking UniversityBeijing 100083China Key Laboratory for Neuroscience Ministry of Education/National Health Commission of ChinaPeking UniversityBeijing 100083China Department of Pathophysiology Huazhong University of Science and TechnologyWuhan 430030China

Neuronal ensembles with distinct morphological,biochemical,and functional identities are organized into complex circuits in the mammalian brain,and malfunction of specific neuronal types in different networks contributes to diverse pathological symptoms.Taking memory as an example,specific memories are held in a subset of neurons,referred to as engram cells.

关键词： mammalian networks organized

A “GC-rich” method for mammalian gene expression:A dominant role of non-coding DNA GC content in the regulation of mammalian gene expression

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Science China(Life Sciences) 2010年第1期53卷 94-100页

作者： Gilbert Rishton Matthew (Mizhou) HUI AmProtein Inc NCPC R&D Center Harbin Pharmaceutical Group R&D Center AmProtein China

High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein.The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment(including intron) of extremely GC-rich at the 5’ or/and 3’ flanking regions of these protein genes or their gene promoters.This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super "chromatin opening element" and plays an important role in mammalian gene expression.This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure,namely DNA GC-content.

关键词： GC-rich GC content DNA structure stable expression mammalian cell expression mechanism

Amino acids regulate energy utilization through mammalian target of rapamycin complex 1 and adenosine monophosphate activated protein kinase pathway in porcine enterocytes

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Animal Nutrition 2020年第1期6卷 98-106页

作者： Hao Xiao Cuifang Zha Fangyuan Shao Li Wang Bi’e Tan State Key Laboratory of Livestock and Poultry Breeding Ministry of Agriculture Key Laboratory of Animal Nutrition and Feed Science in South ChinaGuangdong Public Laboratory of Animal Breeding and NutritionGuangdong Key Laboratory of Animal Breeding and NutritionInstitute of Animal ScienceGuangdong Academy of Agricultural SciencesGuangzhou510640China Hunan Provincial Key Laboratory of Animal Nutritional Physiology and Metabolic Process Key Laboratory of Agro-ecological Processes in Subtropical RegionNational Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry ProductionInstitute of Subtropical AgricultureChinese Academy of SciencesChangsha410125China Faculty of Health Sciences University of MacaoMacaoChina College of Animal Science and Technology Hunan Agricultural UniversityChangsha410128China

As major fuels for the small intestinal mucosa,dietary amino acids(AA)are catabolized in the mitochondria and serve as sources of energy production.The present study was conducted to investigate AA metabolism that supply cell energy and the underlying signaling pathways in porcine enterocytes.Intestinal porcine epithelial cells(IPEC-J2)were treated with different concentrations of AA,inhibitor,or agonist of mammalian target of rapamycin complex 1(mTORCl)and adenosine monophosphate activated protein kinase(AMPK),and mitochondrial respiration was monitored.The results showed that AA treatments resulted in enhanced mitochondrial respiration,increased intracellular content of pyruvic acid and lactic acid,and increased hormone-sensitive lipase mRNA expression.Meanwhile,decreased citrate synthase,isocitrate dehydrogenase alpha,and carnitine palmitoyltransferase 1 mRNA expression were also observed.We found that AA treatments increased the protein levels of phosphorylated mammalian target of rapamycin(p-mTOR),phosphorylated-p70 ribosomal protein S6 kinase,and phosphorylated-4 E-binding protein 1.What is more,the protein levels of phosphorylated AMPKα(pAMPKa)and nicotinamide adenine dinucleotide(NAD)-dependent protein deacetylase sirtuin-1(SIRT1)were decreased by AA treatments in a time depending manner.Mitochondrial bioenergetics and the production of tricarboxylic acid cycle intermediates were decreased upon inhibition of mTORCl or AMPK.Moreover,AMPK activation could up-regulate the mRNA expressions of inhibitor of nuclear factor kappa-B kinase subunit beta(Ikbk(3),integrin-linked protein kinase(ILK),unconventional myosin-Ic(Myolc),ribosomal protein S6 kinase beta-2(RPS6 Kβ2),and vascular endothelial growth factor(VEGF)-β,which are downstream effectors of mammalian target of rapamycin(mTOR).The mRNA expressions of phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform(PIK3 CD)and5’-AMP-activated protein kinase subunit gamma-1(PRKAG1),which are upstream regulators of mTOR,were also up-regulated b

关键词： Amino acids mammalian target of rapamycin Adenosine monophosphate activated protein kinase Mitochondrial respiration Energy utilization

Paleoenvironments indicated by the fossil mammalian assemblages from red clay-loess sequence in the Chinese Loess Plateau since 8.0 Ma B.P.

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Science China Earth Sciences 2006年第5期49卷 518-530页

作者： XUE Xiangxu1,2, ZHANG Yunxiang1,2 & YUE Leping1,2 1. State Key Laboratory of Continent Dynamics (Northwest University), Institute of Cenozoic Geology and Environment, De- partment of Geology, Northwest University, Xi’an 710069, China 2. Institute of Earth Environment, State Key Laboratory of Loess and Quaternary Geology, Chinese Academy of Sciences, Xi’an 710075, China State Key Laboratory of Continent Dynamics (Northwest University) Institute of Cenozoic Geology and Environment Department of Geology Northwest University Xi’an China Institute of Earth Environment State Key Laboratory of Loess and Quaternary Geology Chinese Academy of Sciences Xi’an China

Information from eleven profiles of eolian earthy red silty clay and loess of the middle of the Late Miocene to Holocene age have been studied and correlated. A complete summary profile with projected isotopic ages and fossil-bearing beds has been assembled. The profile is subdivided into 12 stratigraphic units proceeding from the lower (older) to the upper (younger) one. The character of mammalian assemblages contained in each unit was analyzed and the corresponding pa- leo-climatic environments were deduced. The environmental character of each period and the change from the middle of the Late Miocene at about 8.0 Ma B.P. up to the Holocene progressed from the hot semiarid and semi-moist, warmer-moist, warm-moister of the Late Miocene. To slightly less warm moist, cool-dry, slight-warmer-moister of the Pliocene; later the cool to cold dry periods alternated frequently with the mild semiarid and semi-moist periods of the Quaternary. Vegetation progressed from the grasslands with sparse woods, wooded shrub-grasslands and subtropical forest-grasslands of the Late Miocene age to sparse grasslands and dry grasslands of the Pliocene; and to sparse grasslands, dry grasslands and tundras of the Quaternary. The climate changes in the Neogene were of low-amplitute in a generally warm-humid background. Those of the Quaternary were of higher am- plitude with a longer phase. Cool or cold dry and slightly warm semiarid and semi-moist climates al- ternated on a generally cool-dry background. The tendency since the start of the Quaternary was the change to more northern and western shift in the Loess Plateau, with progressively cooler and dryer conditions. The dramatic uplift of the Qinghai-Xizang (Tibet) Plateau and the forming and expansion of the Arctic ice sheet might be the main reasons of the more pronounced changes in the Quaternary.

关键词： paleoenvironment, mammalian fossils, eolian red clay-loess sequence, Loess Plateau.

Secretory expression of the fusion protein composed of human IL-2 and PreS antigen of hepatitis B virus in mammalian cells

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Chinese Science Bulletin 1998年第9期43卷 781-784页

作者： Zhangguo Chen Dalong Ma Jing Wang Yingmei Zhang Yong Yuan Wenling Han Hongtao Liu Beijing Med Univ Dept Immunol Beijing 100083 Peoples R China

In order to make entire HBV preSAg secrete from mammalian cells, we constructed an eukaryotic expression vector by using leader sequence of human interleukin-2 (IL-2) as secretory signal peptide, and using high hydrophilic amino acids as the linker between IL-2 C end and preSAg N end. As a result, the IL-2preS fusing protein could be secreted from mammalian cells transfected with the reconstructed vector and the expression efficiency was identical to that of natural IL-2. It was considered that the retentive effect of preS1Ag could be successfully bypassed. The results not only laid a theoretical and practical foundation for constructing specific gene vaccine against HBV persistent infection, but also supplied experimental evidence for studying modulation of protein secretory expression.

关键词： hepatitis B gene vaccine gene immunization preS antigen mammalian cells secretion.