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replication-defective

By Jorge Campbell,2014-07-08 06:24
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replication-defective

    Task4 of BioBio Group

    sample 1: (contributed by )

    Robust hepatitis C virus infection in vitro

Jin Zhong*, Pablo Gastaminza*, Guofeng Cheng*, Sharookh Kapadia*, Takanobu Kato, Dennis R. Burton,

    Stefan F. Wieland*, Susan L. Uprichard*, Takaji Wakita, and Francis V. Chisari*

    Departments of *Molecular and Experimental Medicine and ?Immunology, The Scripps Research Institute, La

    Jolla, CA 92037; and Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo

    183-8526, Japan

    Contributed by Francis V. Chisari, May 1, 2005

Materials and Methods

HCV Constructs and Transcription.

    The HCV consensus clone used was derived from a Japanese patient with fulminant hepatitis and

    has been designated JFH-1 (23). Wakita et al. (22) cloned this HCVcDNA behind a T7 promoter

    to create the plasmid pJFH-1, as well as a replication-defective NS5B negative control construct

    pJFH-1-GND (22). To generate genomic JFH-1 and JFH-1-GND RNA, the pJFH-1 and

    pJFH-1-GND plasmids were linearized at the 3 end of the HCV cDNA by XbaI digestion. The

    linearized DNA was then purified and used as a template for in vitro transcription (MEGAscript; Ambion, Austin, TX).--

Cell Culture.

    The hepatic (Huh-7 and Huh-7.5.1) -- and nonhepatic HEK293 (27) and

    HeLa (28) cells were maintained in complete DMEM supplemented with 10% FCS with10 mM Hepes; 100 units/ml penicillin 100 mg/ml streptomycin; 2 mM L-glutamine (Invitrogen) --

     at 5% CO2. The human promyeloblastic HL-60 and monoblastoid U-937 cell lines were obtained from American Type Culture Collection and cultivated as recommended. The human hepatocarcinoma cell line HepG2 (American Type Culture Collection) -- (29) and Epstein-Barr virus-transformed B cells were maintained in RPMI medium 1640 with the same supplements described above (Invitrogen) --. Huh-7.5.1 cells were derived

    from the Huh-7.5 GFP-HCV replicon cell line NS5A-GFP-6 (30), kindly provided by Charles Rice (Rockefeller University, New York) --. The NS5A-GFP-6 replicon cells were cultured 3 weeks in the presence of 100 units/ml human IFN-γ to eradicate the NS5A-GFP-6 replicon. Clearance of the HCV replicon bearing the neomycin resistance gene was confirmed by G418 sensitivity and HCV-specific reverse transcription real-time quantitative PCR

    (RT-QPCR) -- analysis.

HCV RNA Transfection.

    In vitro transcribed genomic JFH-1 RNA was delivered to cells by electroporation or

    liposome-mediated transfection. Electroporation was performed as described by Krieger et al.

    7(31). Briefly, trypsinized cells were washed twice with PBS and then resuspended in serum-free cells per ml. Ten micrograms of JFH-1 RNA was

    Opti-MEM (Invitrogen) --at 1 * 10mixed with 0.4 ml of the cells in a 4-mm cuvette, and a Bio-Rad Gene Pulser system was used to

    deliver a single pulse at 0.27 kV, 100 ohms, and 960 µF and the cells were plated in a T162 Costar

    flask (Corning) --. Liposome-mediated transfection was performed with Lipofectamin 2000 (Invitrogen) --at an RNA:lipofectamin ratio of 1:2 by using 5 µg

    4of JFH-1 RNA in cell suspensions containing 10 cells. Cells were then plated in DMEM with

    20% FCS for overnight incubation. In both cases, transfected cells were transferred to complete

    DMEM and cultured for the indicated period. Cells were passaged every 3-5 days; the presence

    of HCV in these cells and corresponding supernatants were determined at the indicated time

    points.

RNA Analysis.

    Total cellular RNA was isolated by the guanidine thiocyanate method by using standard protocols (32). RT-QPCR analysis (for primer sequences, see Fig. 7, which is published as

    supporting information on the PNAS web site) --was performed as described (19, 33), and HCV and GAPDH transcript levels were determined relative to a standard curve comprised of serial dilutions of plasmid containing the HCV JFH-1

    cDNA or human GAPDH gene.

Indirect Immunofluorescence.

    Intracellular staining was performed as described (33). Polyclonal anti-NS5A rabbit antibody MS5

    [a gift from Michael Houghton (Chiron)] -- was used at a dilution of 1:1,000 followed by incubation with a 1:1,000 dilution of Alexa555- conjugated goat anti-rabbit IgG

    (Molecular Probes) --for 1 h at room temperature. Cell nuclei were stained by

    Hoechst dye.

Titration of Infectious HCV.

    Cell supernatants were serially diluted 10-fold in complete DMEM and used to infect 104 naıve Huh-7.5.1 cells per well in 96-well plates (Corning) --. The inoculum was incubated

    with cells for 1 h at 37?C and then supplemented with fresh complete DMEM. The level of HCV

    infection was determined 3 days postinfection by immunofluorescence staining for HCV NS5A.

    The viral titer is expressed as focusforming units per milliliter of supernatant (ffu/ml) --, determined by the average number of NS5A-positive foci detected at the highest dilutions.

Amplification of HCV Viral Stocks.

    To generate viral stocks, infectious supernatants were diluted in complete DMEM and used to

    inoculate naıve 10-15% confluent Huh-7.5.1 cells at an moi of 0.01 in a T75 flask (Corning) --

    . Infected cells were trypsinized and replated before confluence at day 4-5 postinfection

    (p.i.) --. Supernatant from infected cells was then harvested 8-9 days p.i. and aliquoted for

    storage at 80?C. The titer of viral stock was determined as described above.

Concentration and Purification of HCV.

Sucrose density-gradient ultracentrifugation analysis was performed as described (34). Pooled

    supernatant from two mock or two HCV-infected T162 cm2 Costar flasks (Corning) --were centrifuged at 4,000 rpm for 5 min to remove cellular debris and then pelleted through a

    20% sucrose cushion at 28,000 rpm for 4 h by using a SW28 rotor in an L880M ultracentrifuge (Beckman) --. The pellet was resuspended in 1 ml of TNE buffer (50 mM Tris-HCl,

    pH 8;100 mM NaCl; 1 mM EDTA) -- containing protease inhibitors

    (Roche Applied Science, Indianapolis) --, loaded onto a 2060% sucrose gradient (12.5-ml total volume) --, and centrifuged at 120,000 g for 16 h at 4?C in a SW41Ti rotor (Beckman) --. Fractions of 1.3 ml were collected from the top of the gradient. The fractions were analyzed by RT-QPCR to detect HCV RNA. To determine the infectivity titer of each fraction, fractions were titrated on

    Huh7.5.1 cells as described above.

Blocking Infection with CD81- and E2-Specific Antibodies.

    Recombinant human monoclonal (IgG1) --anti-E2 antibody was derived from a cDNA expression library (prepared from mononuclear cells of a HCV patient) --HCVcDNA that was screened against recombinant HCV genotype 1a E2 protein

    (GenBank accession no. M62321) -- by phage display. The antibody

    was serially diluted and preincubated with 15,000 ffu of JFH-1 virus in a volume of 250:l for 1 h

    at 37?C. The virus antibody mixture was then used to infect 45,000 Huh-7.5.1 cells in a 24-well

    plate (Corning) --for 3 h at 37?C. Mouse monoclonalanti-human CD81 antibody 5A6

    (35) at 1 mg/ml (a gift from Shoshana Levy, Stanford University, Stanford, CA) --

    was serially diluted (1:2,000, 1:200, and 1:20) --and preincubated in a volume of 50:l with 104 Huh-7.5.1 cells seeded in a 96-well plate for 1 h at 37?C.

    Cells were subsequently inoculated with infectious JFH-1 supernatant at an moi of 0.3 for 3 h at

    37?C. The efficiency of the infection in the presence of antibodies was monitored 3 days p.i. by RT-QPCR and immunofluorescence.

1.

2.

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    3. 4.

    Contributed by

    sample 2: contributed by IkappaBalpha gene promoter polymorphisms are associated with

    hepato- carcinogenesis in patients infected with hepatitis B virus

    genotype C

Yongchao He, Hongwei Zhang, Jianhua Yin, Jiaxin Xie, Xiaojie Tan, Shijian Liu,

    1123Qian Zhang, Chengzhong Li,Jun Zhao, Hongyang Wang and Guangwen Cao*

    1Department of Epidemiology, Department of Infectious Diseases, the 1st Affiliated

    23Hospital, Department of Hepatobiliary Surgery and Laboratory for Signal Transduction, the 3rd Affiliated Hospital, Second Military Medical University,

    Shanghai 200433, People’s Republic of China

Patients and methods

    Clinical data

    The study involved 404 HBV-infected patients treated at the first and third affiliated

    hospitals of this university from October 2006 to October 2008, 404 healthy controls

    without HBV infection from the physical examination center of the First affiliated

    hospital from January 2007 to October 2008 and 280 asymptomatic hepatitis B

    surface antigen carriers (ASCs)-前面名称的简写recruited in an epidemiological survey from September to December 2006. All participants were ethic Chinese.

    Diagnostic criteria of ASCs, the patients with CHB and the patients with HCC, and

    exclusion criteria of the subjects were as described previously (6). Only newly

    diagnosed HCC patients were included; patients with a prior history of HCC or other

    cancers were excluded. LC was diagnosed by using a Philips iU22 scanner (Philips Medical Systems, Best, The Netherlands)-对仪器的介绍equipped with a 24 MHz variable convex probe. The ultrasonography scoring system consisting of liver surface, parenchyma, vascular structure and splenic size was used to describe the

    existence and severity of cirrhosis. The scores ranged from four for a normal liver to

    11 for advanced cirrhosis (26). A score of eight or more was used as the cutoff point for HBV- related ultrasonographic cirrhosis. In addition to ultrasonographic findings, esophageal of gastric varices, low platelet count and cirrhotic complications such as

    ascites or encephalopathy were used for the diagnosis of clinical cirrhosis. The study

    protocol conformed to the 1975 Declaration of Helsinki and was approved by the

    ethics committees of the institutions, and an informed consent was obtained from each participant.

Examination of HBV serological parameters

    Before any treatment, 5 ml fasting blood was split into two blood collection tubes

    ? and with or without anticoagulant. The serum was separated by centrifugation at 4

    stored in a sterile tube at -80? within 4 h of sample collection. Serological testing for

    hepatitis B surface antigen, HBeAg, alpha-fetoprotein and liver function examination

    including alanine aminotransferase (ALT)-前面单词的简写 was performed as described previously (6). Upper limit of normal ALT was 45 U/l. HBV DNA

    concentration was measured in the Light Cycler TM480 (Roche, Basel,

    Switzerland)-介绍仪器产地 using quantitative HBV polymerase chain reaction

    (PCR) 简称 fluorescence diagnostic kits (Fuxun Diagnostics, Shanghai, China)-使用试剂的介绍. The kit has a certified lower limit of detection of 500 copies/ml.

    HBV genotype and subgenotype were determined by a multiplex PCR assay (6,27).

SNP analysis

    Genomic DNA was extracted from a 200 ul peripheral blood sample using a QIAamp

    blood kit (QIAGEN, Hilden, Germany)-使用试剂的介绍 according to the manufacturer’s instructions. Genotyping for the polymorphisms of NFKB1-94

ins/del ATTG (rs 28720239), NFKBIA 3’-UTR A/G (rs 57898959), NFKBIA -519

    C/T (rs 2233408), NFKBIA -826 C/T (rs 2233406) and NFKBIA-881 A/G (rs

    3138053) was conducted using PCRrestriction fragment length polymorphism

    method. Primers for NFKB1-94 were 5’-TGGGCACAAGT CGTTTATGA-3’ and 5’-CTGGAGCCGGTAGGGAAG-3’, amplifying a fragment of 281 bps or 285 bps

    (23). Primers for NFKBIA 3’-UTR were 5’-GGCTGAAAGAACATGGAC TTG-3’ and 5’-GTACACCATTTACAGGAGGG-3’ that amplify a fragment of 424 bps (23).

    Primers for NFKBIA-519C/T were 5’-GCTTTCACAACTTCTACCTG-3’ and 5’-AGAGT GGAAATGATGGCTG-3’, which amplify a fragment of 188 bps (24). Primers for NFKBIA -826 C/T and -881 A/G were

    5’-GGTCCTTAAGGTCCAATCG-3’ and 5’-GTTGTGGATA CCTTGCACTA-3’ (underlined, mismatched nucleotide)-对引物序列的补充说明,which amplify a fragment of 200 bps (28). PCR was performed in 25ul mixture containing 50100 ng DNA, 1×PCR buffer, 1 U Taq polymerase (TaKaRa Biotechnology, Dalian,

    China)-对使用的聚合酶生产厂家说明, 0.5uM each primer (synthesized by

    Sangon Biotechnology, Shanghai, China)- 对使用的引物生产厂家说明 and 0.25 mM deoxynucleoside triphosphate (TaKaRa Biotechnology)- 对使用的dNTP生产厂家说明. The amplification was performed with annealing temperature of 60? (NFKB1 -94 or NFKBIA 3’-UTR) or 55?(NFKBIA -519 or NFKBIA -826 and -881)

    for 30 s for 35 cycles by using an Autorisierter thermocycler (Eppendorf AG,

    Humburg, Germany)-对使用PCR扩增仪器厂家说明. The PCR product of NFKB1

    -94 was digested with Van91 I (New England Biolabs, Ipswitch, MA) at 37? for 18 h. The PCR product of NFKBIA 3’-UTR was digested with HaeIII (TaKaRa) at 37? for 6 h. The PCR product of NFKBIA -519 was digested with MnlI (New England

    Biolabs) at 37? for 6 h. The PCR product of NFKBIA -826 was digested with XspI

    (TaKaRa) at 37? for 8h, whereas that of NFKBIA -881 was digested with TspRI

    (New England Biolabs) at 65? for 5 h. Both the PCR and restriction products were

    visualized in 2% agarose gel with 0.2 lg/ml ethidium bromide, together with the DNA

    ladders, as shown in supplementary Figure 1 (available at Carcinogenesis Online).

    Genotypes of NFKB1 polymorphisms were distinguished as wild type (WT) (281 bps,

ATTG1/ATTG1), heterozy gote (ATTG1/ATTG2) and polymorphic homozygote (45

    and 240 bps, ATTG2/ ATTG2). Genotypes of NFKBIA 3’-UTR polymorphisms were determined as WT (424 bps, AA),heterozygote (AG) and polymorphic homozygote

    (108 and 316 bps, GG).Genotypes of NFKBIA -519 polymorphisms were determined

    as WT (188 bps, CC), heterozygote (CT) and polymorphic homozygote (121 and 67

    bps,TT). Genotypes of NFKBIA -826 polymorphisms were determined as WT (200

    bps, CC), heterozygote (CT) and polymorphic homozygote (180 and 20bps, TT).

    Genotypes of NFKBIA -881 polymorphisms were determined as WT (200 bps, AA),

    heterozygote (200, 129 and 71 bps, AG).

    Statistical analysis

    The frequencies of all SNPs in health controls were tested for conformation to Hardy

    Weinberg equilibrium online (http://ihg.gsf.de/ihg/snps.htm1)-对使用软件的介绍.

    Statistical analyses were performed using the Statistical Program for Social Sciences

    (SPSS15.0 for Windows, SPSS, Chicago, IL) -对使用软件的介绍. Categorical

    2 test. variables, like the frequencies of SNPs and HBV genotypes, were tested by X

    Continuous variables, like serum viral load and ALT level with skewed distribution,

    were adjusted to normal distribution by transformation into logarithmic function and

    then tested by Student’s t-test. To determine the factors contributing independently to HCC, forward stepwise multivariate regression analysis (Pentry 5 0.05, Premoval 5

    0.10) was performed to obtain the adjusted odds ratios (AOR) of polymorphisms for

    HCC risk and their 95% confidence intervals (CI). The estimated haplotype

    frequencies were determined by the estimating haplotype frequencies program

    (http://linkage.rockefeller.edu/soft/). A P value of<0.05 was considered as

    statistically significant. All statistical tests were two sided..

    1.这篇论文研究的是宿主IkappaBalpha基因启动子区域多态性与C型乙肝病毒感染所导致的肝癌的关系;

    2.方法部分对研究对象的选择(包括入选标准,排除标准以及临床诊断指标)、

    实验方法(包括乙型肝炎病毒血清标志物的检测,SNP检测与分析等)和统计方

法都作了详细介绍;

    3.方法部分中有很多括号对本研究所使用的仪器、试剂以及统计分析使用的软件

    进行补充说明;

    4.方法部分中用下划线着重标示的单词或短语是该部分写作常见的词汇或比较

    特殊的用法。

    contributed by 谢佳新)

sample 3: contributed by 唐晓露

    Quantitative associations between osteocyte density and

    biomechanics, microcrack and microstructure in OVX rats vertebral

    trabeculae

    Yu-Lin Ma, Ru-Chun Dai, Zhi-Feng Sheng, Yan Jin, Yu-Hai Zhang, Ling-Na Fang, Hui-Jie

    Fan, Er-Yuan Liao_

    Institute of Metabolism and Endocrinology, The Second Xiang-Ya Hospital, Central South

    University, Changsha, 410011, Hunan, PR China

    Accepted 10 January 2008

    Journal of Biomechanics 41 (2008) 13241332

Materials and methods

Animal preparation

    All animal procedures were approved by the Institutional Animal Care and

    Use Committee of People’s Republic of China. Forty female Sprague–Dawley

    (7-month-old) rats were obtained from the Animal Center of the Second Xiang-Ya Hospital (Central South University, China). The rats were single-fed, housed at 2325 ?C with a 12 h light/dark cycle, and allowed free access to water.

    The rats underwent either ovariectomy (n = 30, OVX) or a sham-operation (n =

    10, SHAM). The OVX rats were randomly divided into three groups: OVX and

treated with 17b-estradiol (Sigma, Chemical, St. Louis, MO, USA, 10 mg/kg/day,

    EST) ——(Dai et al., 2004) and genistein (5mg/kg/day,

    GEN) (Fanti et al., 1998). At 15 weeks post-operation, all the rats were

    anesthetized with Phenobarbital 1 mg/kg, injected intraperitoneally) and

    sacrificed by bloodletting from the ventral aorta. The vertebrae were dissected

    and stored at-70 ?C until micro-computed tomography (m-CT) scanning was

    performed.——

Mechanical tests

    The L-5 vertebral body specimen was prepared as previously described (Dai

    et al., 2004). The vertebral cylinder samples were attached to the material-testing

    machine and analysis systems (CSS44100, Changchun Research Institute of Test

    Machines, Changchun, China). The vertebral height and diameters were

    measured. A compression force was applied in the craniocaudal direction using a

    steel disk (1.8 cm diameter) at a nominal deformation rate of 2 mm/min.

    Loaddeformation curves were recorded continually in the computerized

    monitor linked to the tester. The ultimate compressive load was obtained directly from the loaddeformation curves at the level of maximum load (ML) ——.

    The elastic modulus (EM) ——was estimated using the following equation:

    EM = stiffness × (height/CSA), where stiffness is the slope of the fitting linear

    portion of the load/ displacement curve and CSA is the cross-sectional area

    calculated asrectangle area.——

Micro-computed tomography (m-CT) (Sheng et al., 2007)

    The L-6 vertebral body specimens were scanned by a micro-CT specimen scanner (GE eXplore LocusSP Specimen Scanner; GE Healthcare Company,

    London, Canada),—— which is a cone-beam scanning system. The

    scanning protocol was 80 kV and 80 mA, with an isotopic resolution of

    6.5×6.5×6.5 mm voxel size and an exposure time of 3000 ms per frame. ——

    Trabecular bone volume fraction (BV/TV)——, bone surface density (BS/BV) ——, trabecular number (Tb.N) ——, trabecular separation (Tb.Sp) ——, trabecular thickness (Tb.Th) ——, geometric degree of anisotropy (DA) ——, connectivitydensity (Conn.D) ——, structure model index (SMI) ——and trabecular volumetric BMD——at both organ and tissue levels were determined.——

Fatigue damage testing (Dai et al., 2004)

    After micro-CT scanning, the L-6 vertebra were treated in the same way as the L-5 vertebra. Fatigue damage tests were performed on the PLD 5010 bone

    fatigue damage testing machine (Changchun Research Institute of Test Machines,

    Changchun, China),—— with a loading frequency of 2.5 Hz, a

    loading number of 10,000 times for each vertebra, and a loading strength of 60 N.

    After the fatigue damage tests, the vertebral bodies were fixed in 70% ethanol, followed by en-bloc basic fuchsin staining, plastic embedding and sectioning. 2.5.

    En-bloc basic fuchsin staining the en-bloc basic fuchsin staining method as described above (Dai et al., 2004) ——was used to detect microcracks. After staining, basic fuchsinstained specimens were immersed in

    xylene for 1 day, followed by routine plastic embedding for bone

    histomorphometry analysis.——

Observation of microcracks and quantification of osteocyte density

    (Dai et al., 2004; Da Costa Go0mez et al., 2005)

    Two serial coronal sections of 5080 mm were prepared for morphometric analysis using polarizing microscopy at 400×magnification and Leica DMLA

    microscope-image analysis system (Leica Corporation, Wetzla, Germany).——

     The following histomorphometric variables were determined:

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