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test3

2_test

{"project":"2_test","denotations":[{"id":"22396652-20530205-98461890","span":{"begin":612,"end":614},"obj":"20530205"},{"id":"22396652-10621974-98461891","span":{"begin":664,"end":666},"obj":"10621974"},{"id":"22396652-21841782-98461892","span":{"begin":827,"end":829},"obj":"21841782"},{"id":"22396652-21187318-98461893","span":{"begin":3492,"end":3494},"obj":"21187318"}],"text":"Materials and Methods\n\nEthics statement\nAll experiments on mice were conducted according to the national (Belgian Law 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European (EU Directives 2010/63/EU, 86/609/EEG) animal regulations. Animal protocols were approved by the ethics committee of Ghent University (permit number LA1400091, approval ID 2010/001). All efforts were made to ameliorate suffering of animals. Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of ketamine (12 mg/kg) and xylazine (60 mg/kg).\n\nMice\nA20fl/fl mice were generated as previously described [56]. A20fl/fl mice were crossed with LysM-Cre mice [84] (provided by I. Förster, Institute of Genetics, University of Cologne, Germany) to generate A20fl/fl LysMCre transgenes and are described in detail elsewhere [48]. Mice were housed in individually ventilated cages at the VIB Department of Molecular Biomedical Research in specific pathogen-free animal facilities. Influenza infections were performed on age- (between 7 and 9 weeks old) and sex-matched littermates. A20fl/fl LysM-Cre animals were backcrossed three times to the C57Bl/6 background. A20fl/fl mice expressing or lacking the LysM-Cre transgene were termed A20myel-KO and wild type (A20myel-WT) respectively.\n\nViral infection and determination of viral titers\nMouse adapted IAV X-47 (H3N2; PR8×A/Victoria/3/75) was propagated in MDCK cells. For viral inoculation, mice were anesthetized by i.p. injection with ketamine (12 mg/kg) and xylazine (60 mg/kg) and 50 µl X-47 diluted in PBS was administered intranasally. For lethal and sublethal infection, mice received respectively 2-LD50 or 0.05-LD50 X-47. To determine pulmonary viral titers, median tissue culture infectious dose (TCID50) was measured as follows: lungs were homogenized with a Polytron homogenizer (Kinematica) in PBS. Eight-fold serial dilutions of lung homogenates were incubated on MDCK cells for 5 days in DMEM supplemented with trypsin (1 µg/ml), 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics. For read-out, 0.5% chicken red blood cells (RBC) were added and end-point dilution of hemagglutination was monitored. TCID50 titers were then calculated according to the method of Reed and Muench [85].\n\nDetermination of HAI (hemagglutination inhibition) titers\nTo determine the HAI titers in infected mice, sera of these were treated with receptor-destroying enzyme (RDE/Cholera filtrate; Sigma) to remove sialic acids from serum proteins capable of aspecific inhibition of agglutination. After incubation overnight at 37°C, the RDE was inactivated by addition of 0.75% sodium citrate in PBS and heating to 56°C for 30 min. To remove sialic acid binding proteins, sera were cleared with 1/10 volume 50% chicken RBC. Titration was done by incubating a two-fold dilution series of sera with 4 HA units of X-47 virus for 1 hour at room temperature in 96-well U-bottom plates. Finally, an equal volume of 0.5% chicken RBC was added and titers were read 30 min later. Negative controls included PBS instead of immune serum (agglutination control) or PR8 instead of X-47 virus (control for agglutination effect of sera); as positive control, serum from a mouse infected twice with a sublethal dose of X-47 was used.\n\nIn vivo intracellular GrB and IFNγ staining of activated CD8+ T cells\nGranzyme B (GrB) and IFNγ expressing CD8+ T cells were determined by treating the mice intranasally with 50 µg Brefeldin A (Sigma) as previously described [86]. 6 h later, BAL and lungs were isolated and single cell suspensions were prepared from the lung in the presence of 3 µg/ml Brefeldin A. Cells were stained, fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences) according to the manufacturer's instructions. Activated CD8+ T cells were analyzed by flow cytometry based on CD62lo CD3+ and CD8+ expression. Live/Dead fixable aqua dead cell stain kit (Molecular Probes) was used to discriminate live from dead cells.\n\nCells and transfection\nHEK293T and MDCK cells were grown in DMEM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics. HEK293T cells were transfected using the calcium phosphate precipitate transfection method with specific expression vectors (pCAGGS-E-hA20 (LMBP 3778), pCAGGS-E-RIG-I-CARD (LMBP 6517), pEF-HA-IRF-7 (kindly provided by T. Taniguchi, Graduate School of Medicine and Faculty of Medicine, University of Tokyo)), NF-κB, IRF3, IRF7 reporter plasmids (respectively pConLuc (LMBP3248), pISRE-luc (LMBP4011), pGL3-IFNα4-luc (kindly provided by J. Hiscott, McGill University, Montreal, Quebec, Canada), and pACTbetagal (LMBP4341) for transfection efficiency normalization. Details of plasmids are presented along with detailed sequence maps at the BCCM-LMBP plasmid databank http://bccm.belspo.be/index.php.\nFor the generation of BMDM, bone marrow cells were cultured 7 days in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate, antibiotics and 40 ng/ml recombinant M-CSF. BMDM were of ≥95% purity as measured by flow cytometry using F4/80 and CD11b specific antibodies. For the isolation of alveolar macrophages, the trachea was canulated and the lung was flushed 4 times with HBSS containing 1 mM EDTA. Alveolar macrophages were cultured in RPMI 1640 (Gibco) supplemented with 1% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.\n\nWestern blotting\nFor total lysates, cells were lysed at 4°C for 15 min in lysis buffer (200 mM NaCl, 1% NP-40, 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 2 mM DTT) supplemented with protease and phosphatase inhibitors. Nuclear and cytoplasmic lysates were prepared by resuspending cells in B1 (10 mM Hepes pH 7.5, 10 mM KCl, 1 mM MgCl2, 5% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. Next, NP-40 detergent was added to a final concentration of 0.65% and cells were centrifuged at 500 g for 5 min. The nuclear fraction containing pellet was lyzed in B2 (20 mM Hepes pH 7.5, 1% NP-40, 400 mM NaCl, 10 mM KCl, 1 mM MgCl2, 20% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. The lysates were subsequently separated by SDS-PAGE and analyzed by western blotting and ECL detection (Perkin Elmer Life Sciences). Immunoblots were revealed with anti-A20, anti-IκBα, anti-p65, and anti-histon H1 (Santa Cruz), anti-IRF3 (Invitrogen), anti-phospho-IRF3 and anti-phospho-IκBα (Cell Signaling) and anti-actin (MP Biomedicals). The density of the bands was quantified (fold induction) with the ImageJ (http://rsbweb.nih.gov/ij) Gel analyzer tool. All intensities were calculated relative to the first lane ( = time 0).\n\nFlow cytometry\nLungs were dissected and incubated with collagenase type IV (1 mg/ml; Sigma) and DNAse (100 U/ml; Roche) at 37°C for 30 min. Subsequently, samples were filtered through a 70 µm and 40 µm nylon mesh. For the preparation of BAL, trachea were canulated and airway lumen was flushed 4 times with HBSS with 1 mM EDTA. Cells were stained with monoclonal antibodies directed against MHC-II (I-A/I-E) FITC (M5/114.15.2), CD11c PerCP-Cy5.5 (N418), F4/80 APC (BM8), CD62L PE (MEL-14), Granzyme B FITC (NGZB) from eBiosciences and CD3 Molecular Complex Horizon v450 (17A2), Ly6C Horizon v450 (AL-21), Ly6G PE (1A8), CD11b APC-Cy7 (M1/70), CD8α PerCP (53-6.7), IFNγ Alexa 647 (XMG1.2) and CD16/32 (2.4G2) from BD Pharmingen. Samples were acquired on a LSRII Cytometer and analyzed using FACSDiva software (BD Biosciences). Propidium iodide was used to discriminate between live and dead cells.\n\nCytokine quantification\nFor TNF ELISA, 96-well plates were coated with TNF coating (TN3-19, eBioscience) and detection (R4-6A2, eBioscience) antibodies. IFNα and IFNβ protein levels were determined with an ELISA kit (PBL Biomedical Laboratories). For IFNγ ELISA, 96-well plates were coated with IFNγ coating (XMG1.2) and detection (R4-6A2) antibodies (eBiosciences). Detection of MCP-1, KC, TNF, IL-1β and IL-6 in BAL fluid was performed using Bioplex (BioRad) technology according to the manufacturer's instructions. Milliplex technology (Millipore) was used for the detection of MIP-2 in BAL fluid.\n\nRNA isolation, cDNA synthesis and qPCR\nTotal RNA was extracted using Aurum Total RNA mini kit (BioRad) and reverse transcribed into cDNA with iScript cDNA synthesis kit (BioRad) according to the manufacturer's instructions. qPCR was performed by using SYBR Green I master mix I (Roche) in the Lightcycler 480 detection system (Roche) with the following primers: HPRT: 5′-AGTGTTGGATACAGGCCAGAC-3′ and 5′CGTGATTCAAATCCCTGAAGT-3′; IL-6: 5′-GAGGATACCACTCCCAACAGACC-3′ and 5′-AAGTGCATCATCGTTGTTCATACA-3′; IFNβ: 5′-TCAGAATGAGTGGTGGTTGC-3′ and 5′-GACCTTTCAAATGCAGTAGATTCA-3′; A20: 5′-AAACCAATGGTGATGGAAACTG-3′ and 5′-GTTGTCCCATTCGTCATTCC-3′; CCL2: 5′-TTAAAAACCTGGATCGGAACCAA-3′ and 5′-GCATTAGCTTCAGATTTACGGGT-3′; CXCL1: 5′-GAGCCTCTAACCAGTTCCAG-3′ and 5′-TGAGTGTGGCTATGACTTCG-3′ and IFNα4: 5′-TGATGAGCTACTACTGGTCAGC-3′ and 5′-GATCTCTTAGCACAAGGATGGC-3′. Primers were designed with PerlPrimer (http://perlprimer.sourceforge.net). Quantification was performed using the comparative CT method (ΔΔCT). Results are expressed relative to HPRT values.\n\nStatistics\nResults are expressed as the mean ± SEM. Statistical significance between groups was assessed using two-way ANOVA. The differences for in vivo experiments (at least 5 mice per group) were calculated using the Mann-Whitney U-test for unpaired data. Statistical significance of differences between survival rates was analyzed by comparing Kaplan-Meier curves using the log-rank test (GraphPad Prism version 5, GraphPad, San Diego, CA)."}

pmc-enju-pas

bionlp-st-ge-2016-test-proteins

bionlp-st-ge-2016-uniprot

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T17825","span":{"begin":6964,"end":6971},"obj":"http://purl.obolibrary.org/obo/UBERON_0003126"},{"id":"T17824","span":{"begin":6737,"end":6742},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T16542","span":{"begin":5196,"end":5200},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T16541","span":{"begin":5166,"end":5173},"obj":"http://purl.obolibrary.org/obo/UBERON_0003126"},{"id":"T16540","span":{"begin":4848,"end":4859},"obj":"http://purl.obolibrary.org/obo/UBERON_0002371"},{"id":"T16108","span":{"begin":3587,"end":3591},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T16107","span":{"begin":3516,"end":3521},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T15281","span":{"begin":2086,"end":2091},"obj":"http://purl.obolibrary.org/obo/UBERON_0000178"},{"id":"T15280","span":{"begin":1895,"end":1899},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T15279","span":{"begin":1792,"end":1797},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T15278","span":{"begin":1727,"end":1733},"obj":"http://purl.obolibrary.org/obo/UBERON_0000479"},{"id":"T15659","span":{"begin":3191,"end":3196},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T15658","span":{"begin":3067,"end":3072},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T15657","span":{"begin":2479,"end":2484},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"}],"text":"Materials and Methods\n\nEthics statement\nAll experiments on mice were conducted according to the national (Belgian Law 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European (EU Directives 2010/63/EU, 86/609/EEG) animal regulations. Animal protocols were approved by the ethics committee of Ghent University (permit number LA1400091, approval ID 2010/001). All efforts were made to ameliorate suffering of animals. Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of ketamine (12 mg/kg) and xylazine (60 mg/kg).\n\nMice\nA20fl/fl mice were generated as previously described [56]. A20fl/fl mice were crossed with LysM-Cre mice [84] (provided by I. Förster, Institute of Genetics, University of Cologne, Germany) to generate A20fl/fl LysMCre transgenes and are described in detail elsewhere [48]. Mice were housed in individually ventilated cages at the VIB Department of Molecular Biomedical Research in specific pathogen-free animal facilities. Influenza infections were performed on age- (between 7 and 9 weeks old) and sex-matched littermates. A20fl/fl LysM-Cre animals were backcrossed three times to the C57Bl/6 background. A20fl/fl mice expressing or lacking the LysM-Cre transgene were termed A20myel-KO and wild type (A20myel-WT) respectively.\n\nViral infection and determination of viral titers\nMouse adapted IAV X-47 (H3N2; PR8×A/Victoria/3/75) was propagated in MDCK cells. For viral inoculation, mice were anesthetized by i.p. injection with ketamine (12 mg/kg) and xylazine (60 mg/kg) and 50 µl X-47 diluted in PBS was administered intranasally. For lethal and sublethal infection, mice received respectively 2-LD50 or 0.05-LD50 X-47. To determine pulmonary viral titers, median tissue culture infectious dose (TCID50) was measured as follows: lungs were homogenized with a Polytron homogenizer (Kinematica) in PBS. Eight-fold serial dilutions of lung homogenates were incubated on MDCK cells for 5 days in DMEM supplemented with trypsin (1 µg/ml), 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics. For read-out, 0.5% chicken red blood cells (RBC) were added and end-point dilution of hemagglutination was monitored. TCID50 titers were then calculated according to the method of Reed and Muench [85].\n\nDetermination of HAI (hemagglutination inhibition) titers\nTo determine the HAI titers in infected mice, sera of these were treated with receptor-destroying enzyme (RDE/Cholera filtrate; Sigma) to remove sialic acids from serum proteins capable of aspecific inhibition of agglutination. After incubation overnight at 37°C, the RDE was inactivated by addition of 0.75% sodium citrate in PBS and heating to 56°C for 30 min. To remove sialic acid binding proteins, sera were cleared with 1/10 volume 50% chicken RBC. Titration was done by incubating a two-fold dilution series of sera with 4 HA units of X-47 virus for 1 hour at room temperature in 96-well U-bottom plates. Finally, an equal volume of 0.5% chicken RBC was added and titers were read 30 min later. Negative controls included PBS instead of immune serum (agglutination control) or PR8 instead of X-47 virus (control for agglutination effect of sera); as positive control, serum from a mouse infected twice with a sublethal dose of X-47 was used.\n\nIn vivo intracellular GrB and IFNγ staining of activated CD8+ T cells\nGranzyme B (GrB) and IFNγ expressing CD8+ T cells were determined by treating the mice intranasally with 50 µg Brefeldin A (Sigma) as previously described [86]. 6 h later, BAL and lungs were isolated and single cell suspensions were prepared from the lung in the presence of 3 µg/ml Brefeldin A. Cells were stained, fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences) according to the manufacturer's instructions. Activated CD8+ T cells were analyzed by flow cytometry based on CD62lo CD3+ and CD8+ expression. Live/Dead fixable aqua dead cell stain kit (Molecular Probes) was used to discriminate live from dead cells.\n\nCells and transfection\nHEK293T and MDCK cells were grown in DMEM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics. HEK293T cells were transfected using the calcium phosphate precipitate transfection method with specific expression vectors (pCAGGS-E-hA20 (LMBP 3778), pCAGGS-E-RIG-I-CARD (LMBP 6517), pEF-HA-IRF-7 (kindly provided by T. Taniguchi, Graduate School of Medicine and Faculty of Medicine, University of Tokyo)), NF-κB, IRF3, IRF7 reporter plasmids (respectively pConLuc (LMBP3248), pISRE-luc (LMBP4011), pGL3-IFNα4-luc (kindly provided by J. Hiscott, McGill University, Montreal, Quebec, Canada), and pACTbetagal (LMBP4341) for transfection efficiency normalization. Details of plasmids are presented along with detailed sequence maps at the BCCM-LMBP plasmid databank http://bccm.belspo.be/index.php.\nFor the generation of BMDM, bone marrow cells were cultured 7 days in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate, antibiotics and 40 ng/ml recombinant M-CSF. BMDM were of ≥95% purity as measured by flow cytometry using F4/80 and CD11b specific antibodies. For the isolation of alveolar macrophages, the trachea was canulated and the lung was flushed 4 times with HBSS containing 1 mM EDTA. Alveolar macrophages were cultured in RPMI 1640 (Gibco) supplemented with 1% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.\n\nWestern blotting\nFor total lysates, cells were lysed at 4°C for 15 min in lysis buffer (200 mM NaCl, 1% NP-40, 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 2 mM DTT) supplemented with protease and phosphatase inhibitors. Nuclear and cytoplasmic lysates were prepared by resuspending cells in B1 (10 mM Hepes pH 7.5, 10 mM KCl, 1 mM MgCl2, 5% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. Next, NP-40 detergent was added to a final concentration of 0.65% and cells were centrifuged at 500 g for 5 min. The nuclear fraction containing pellet was lyzed in B2 (20 mM Hepes pH 7.5, 1% NP-40, 400 mM NaCl, 10 mM KCl, 1 mM MgCl2, 20% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. The lysates were subsequently separated by SDS-PAGE and analyzed by western blotting and ECL detection (Perkin Elmer Life Sciences). Immunoblots were revealed with anti-A20, anti-IκBα, anti-p65, and anti-histon H1 (Santa Cruz), anti-IRF3 (Invitrogen), anti-phospho-IRF3 and anti-phospho-IκBα (Cell Signaling) and anti-actin (MP Biomedicals). The density of the bands was quantified (fold induction) with the ImageJ (http://rsbweb.nih.gov/ij) Gel analyzer tool. All intensities were calculated relative to the first lane ( = time 0).\n\nFlow cytometry\nLungs were dissected and incubated with collagenase type IV (1 mg/ml; Sigma) and DNAse (100 U/ml; Roche) at 37°C for 30 min. Subsequently, samples were filtered through a 70 µm and 40 µm nylon mesh. For the preparation of BAL, trachea were canulated and airway lumen was flushed 4 times with HBSS with 1 mM EDTA. Cells were stained with monoclonal antibodies directed against MHC-II (I-A/I-E) FITC (M5/114.15.2), CD11c PerCP-Cy5.5 (N418), F4/80 APC (BM8), CD62L PE (MEL-14), Granzyme B FITC (NGZB) from eBiosciences and CD3 Molecular Complex Horizon v450 (17A2), Ly6C Horizon v450 (AL-21), Ly6G PE (1A8), CD11b APC-Cy7 (M1/70), CD8α PerCP (53-6.7), IFNγ Alexa 647 (XMG1.2) and CD16/32 (2.4G2) from BD Pharmingen. Samples were acquired on a LSRII Cytometer and analyzed using FACSDiva software (BD Biosciences). Propidium iodide was used to discriminate between live and dead cells.\n\nCytokine quantification\nFor TNF ELISA, 96-well plates were coated with TNF coating (TN3-19, eBioscience) and detection (R4-6A2, eBioscience) antibodies. IFNα and IFNβ protein levels were determined with an ELISA kit (PBL Biomedical Laboratories). For IFNγ ELISA, 96-well plates were coated with IFNγ coating (XMG1.2) and detection (R4-6A2) antibodies (eBiosciences). Detection of MCP-1, KC, TNF, IL-1β and IL-6 in BAL fluid was performed using Bioplex (BioRad) technology according to the manufacturer's instructions. Milliplex technology (Millipore) was used for the detection of MIP-2 in BAL fluid.\n\nRNA isolation, cDNA synthesis and qPCR\nTotal RNA was extracted using Aurum Total RNA mini kit (BioRad) and reverse transcribed into cDNA with iScript cDNA synthesis kit (BioRad) according to the manufacturer's instructions. qPCR was performed by using SYBR Green I master mix I (Roche) in the Lightcycler 480 detection system (Roche) with the following primers: HPRT: 5′-AGTGTTGGATACAGGCCAGAC-3′ and 5′CGTGATTCAAATCCCTGAAGT-3′; IL-6: 5′-GAGGATACCACTCCCAACAGACC-3′ and 5′-AAGTGCATCATCGTTGTTCATACA-3′; IFNβ: 5′-TCAGAATGAGTGGTGGTTGC-3′ and 5′-GACCTTTCAAATGCAGTAGATTCA-3′; A20: 5′-AAACCAATGGTGATGGAAACTG-3′ and 5′-GTTGTCCCATTCGTCATTCC-3′; CCL2: 5′-TTAAAAACCTGGATCGGAACCAA-3′ and 5′-GCATTAGCTTCAGATTTACGGGT-3′; CXCL1: 5′-GAGCCTCTAACCAGTTCCAG-3′ and 5′-TGAGTGTGGCTATGACTTCG-3′ and IFNα4: 5′-TGATGAGCTACTACTGGTCAGC-3′ and 5′-GATCTCTTAGCACAAGGATGGC-3′. Primers were designed with PerlPrimer (http://perlprimer.sourceforge.net). Quantification was performed using the comparative CT method (ΔΔCT). Results are expressed relative to HPRT values.\n\nStatistics\nResults are expressed as the mean ± SEM. Statistical significance between groups was assessed using two-way ANOVA. The differences for in vivo experiments (at least 5 mice per group) were calculated using the Mann-Whitney U-test for unpaired data. Statistical significance of differences between survival rates was analyzed by comparing Kaplan-Meier curves using the log-rank test (GraphPad Prism version 5, GraphPad, San Diego, CA)."}

GO-BP

GO-MF

GO-CC

sentences

ICD10

{"project":"ICD10","denotations":[{"id":"T15291","span":{"begin":1289,"end":1304},"obj":"http://purl.bioontology.org/ontology/ICD10/B34.9"}],"text":"Materials and Methods\n\nEthics statement\nAll experiments on mice were conducted according to the national (Belgian Law 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European (EU Directives 2010/63/EU, 86/609/EEG) animal regulations. Animal protocols were approved by the ethics committee of Ghent University (permit number LA1400091, approval ID 2010/001). All efforts were made to ameliorate suffering of animals. Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of ketamine (12 mg/kg) and xylazine (60 mg/kg).\n\nMice\nA20fl/fl mice were generated as previously described [56]. A20fl/fl mice were crossed with LysM-Cre mice [84] (provided by I. Förster, Institute of Genetics, University of Cologne, Germany) to generate A20fl/fl LysMCre transgenes and are described in detail elsewhere [48]. Mice were housed in individually ventilated cages at the VIB Department of Molecular Biomedical Research in specific pathogen-free animal facilities. Influenza infections were performed on age- (between 7 and 9 weeks old) and sex-matched littermates. A20fl/fl LysM-Cre animals were backcrossed three times to the C57Bl/6 background. A20fl/fl mice expressing or lacking the LysM-Cre transgene were termed A20myel-KO and wild type (A20myel-WT) respectively.\n\nViral infection and determination of viral titers\nMouse adapted IAV X-47 (H3N2; PR8×A/Victoria/3/75) was propagated in MDCK cells. For viral inoculation, mice were anesthetized by i.p. injection with ketamine (12 mg/kg) and xylazine (60 mg/kg) and 50 µl X-47 diluted in PBS was administered intranasally. For lethal and sublethal infection, mice received respectively 2-LD50 or 0.05-LD50 X-47. To determine pulmonary viral titers, median tissue culture infectious dose (TCID50) was measured as follows: lungs were homogenized with a Polytron homogenizer (Kinematica) in PBS. Eight-fold serial dilutions of lung homogenates were incubated on MDCK cells for 5 days in DMEM supplemented with trypsin (1 µg/ml), 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics. For read-out, 0.5% chicken red blood cells (RBC) were added and end-point dilution of hemagglutination was monitored. TCID50 titers were then calculated according to the method of Reed and Muench [85].\n\nDetermination of HAI (hemagglutination inhibition) titers\nTo determine the HAI titers in infected mice, sera of these were treated with receptor-destroying enzyme (RDE/Cholera filtrate; Sigma) to remove sialic acids from serum proteins capable of aspecific inhibition of agglutination. After incubation overnight at 37°C, the RDE was inactivated by addition of 0.75% sodium citrate in PBS and heating to 56°C for 30 min. To remove sialic acid binding proteins, sera were cleared with 1/10 volume 50% chicken RBC. Titration was done by incubating a two-fold dilution series of sera with 4 HA units of X-47 virus for 1 hour at room temperature in 96-well U-bottom plates. Finally, an equal volume of 0.5% chicken RBC was added and titers were read 30 min later. Negative controls included PBS instead of immune serum (agglutination control) or PR8 instead of X-47 virus (control for agglutination effect of sera); as positive control, serum from a mouse infected twice with a sublethal dose of X-47 was used.\n\nIn vivo intracellular GrB and IFNγ staining of activated CD8+ T cells\nGranzyme B (GrB) and IFNγ expressing CD8+ T cells were determined by treating the mice intranasally with 50 µg Brefeldin A (Sigma) as previously described [86]. 6 h later, BAL and lungs were isolated and single cell suspensions were prepared from the lung in the presence of 3 µg/ml Brefeldin A. Cells were stained, fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences) according to the manufacturer's instructions. Activated CD8+ T cells were analyzed by flow cytometry based on CD62lo CD3+ and CD8+ expression. Live/Dead fixable aqua dead cell stain kit (Molecular Probes) was used to discriminate live from dead cells.\n\nCells and transfection\nHEK293T and MDCK cells were grown in DMEM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics. HEK293T cells were transfected using the calcium phosphate precipitate transfection method with specific expression vectors (pCAGGS-E-hA20 (LMBP 3778), pCAGGS-E-RIG-I-CARD (LMBP 6517), pEF-HA-IRF-7 (kindly provided by T. Taniguchi, Graduate School of Medicine and Faculty of Medicine, University of Tokyo)), NF-κB, IRF3, IRF7 reporter plasmids (respectively pConLuc (LMBP3248), pISRE-luc (LMBP4011), pGL3-IFNα4-luc (kindly provided by J. Hiscott, McGill University, Montreal, Quebec, Canada), and pACTbetagal (LMBP4341) for transfection efficiency normalization. Details of plasmids are presented along with detailed sequence maps at the BCCM-LMBP plasmid databank http://bccm.belspo.be/index.php.\nFor the generation of BMDM, bone marrow cells were cultured 7 days in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate, antibiotics and 40 ng/ml recombinant M-CSF. BMDM were of ≥95% purity as measured by flow cytometry using F4/80 and CD11b specific antibodies. For the isolation of alveolar macrophages, the trachea was canulated and the lung was flushed 4 times with HBSS containing 1 mM EDTA. Alveolar macrophages were cultured in RPMI 1640 (Gibco) supplemented with 1% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.\n\nWestern blotting\nFor total lysates, cells were lysed at 4°C for 15 min in lysis buffer (200 mM NaCl, 1% NP-40, 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 2 mM DTT) supplemented with protease and phosphatase inhibitors. Nuclear and cytoplasmic lysates were prepared by resuspending cells in B1 (10 mM Hepes pH 7.5, 10 mM KCl, 1 mM MgCl2, 5% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. Next, NP-40 detergent was added to a final concentration of 0.65% and cells were centrifuged at 500 g for 5 min. The nuclear fraction containing pellet was lyzed in B2 (20 mM Hepes pH 7.5, 1% NP-40, 400 mM NaCl, 10 mM KCl, 1 mM MgCl2, 20% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. The lysates were subsequently separated by SDS-PAGE and analyzed by western blotting and ECL detection (Perkin Elmer Life Sciences). Immunoblots were revealed with anti-A20, anti-IκBα, anti-p65, and anti-histon H1 (Santa Cruz), anti-IRF3 (Invitrogen), anti-phospho-IRF3 and anti-phospho-IκBα (Cell Signaling) and anti-actin (MP Biomedicals). The density of the bands was quantified (fold induction) with the ImageJ (http://rsbweb.nih.gov/ij) Gel analyzer tool. All intensities were calculated relative to the first lane ( = time 0).\n\nFlow cytometry\nLungs were dissected and incubated with collagenase type IV (1 mg/ml; Sigma) and DNAse (100 U/ml; Roche) at 37°C for 30 min. Subsequently, samples were filtered through a 70 µm and 40 µm nylon mesh. For the preparation of BAL, trachea were canulated and airway lumen was flushed 4 times with HBSS with 1 mM EDTA. Cells were stained with monoclonal antibodies directed against MHC-II (I-A/I-E) FITC (M5/114.15.2), CD11c PerCP-Cy5.5 (N418), F4/80 APC (BM8), CD62L PE (MEL-14), Granzyme B FITC (NGZB) from eBiosciences and CD3 Molecular Complex Horizon v450 (17A2), Ly6C Horizon v450 (AL-21), Ly6G PE (1A8), CD11b APC-Cy7 (M1/70), CD8α PerCP (53-6.7), IFNγ Alexa 647 (XMG1.2) and CD16/32 (2.4G2) from BD Pharmingen. Samples were acquired on a LSRII Cytometer and analyzed using FACSDiva software (BD Biosciences). Propidium iodide was used to discriminate between live and dead cells.\n\nCytokine quantification\nFor TNF ELISA, 96-well plates were coated with TNF coating (TN3-19, eBioscience) and detection (R4-6A2, eBioscience) antibodies. IFNα and IFNβ protein levels were determined with an ELISA kit (PBL Biomedical Laboratories). For IFNγ ELISA, 96-well plates were coated with IFNγ coating (XMG1.2) and detection (R4-6A2) antibodies (eBiosciences). Detection of MCP-1, KC, TNF, IL-1β and IL-6 in BAL fluid was performed using Bioplex (BioRad) technology according to the manufacturer's instructions. Milliplex technology (Millipore) was used for the detection of MIP-2 in BAL fluid.\n\nRNA isolation, cDNA synthesis and qPCR\nTotal RNA was extracted using Aurum Total RNA mini kit (BioRad) and reverse transcribed into cDNA with iScript cDNA synthesis kit (BioRad) according to the manufacturer's instructions. qPCR was performed by using SYBR Green I master mix I (Roche) in the Lightcycler 480 detection system (Roche) with the following primers: HPRT: 5′-AGTGTTGGATACAGGCCAGAC-3′ and 5′CGTGATTCAAATCCCTGAAGT-3′; IL-6: 5′-GAGGATACCACTCCCAACAGACC-3′ and 5′-AAGTGCATCATCGTTGTTCATACA-3′; IFNβ: 5′-TCAGAATGAGTGGTGGTTGC-3′ and 5′-GACCTTTCAAATGCAGTAGATTCA-3′; A20: 5′-AAACCAATGGTGATGGAAACTG-3′ and 5′-GTTGTCCCATTCGTCATTCC-3′; CCL2: 5′-TTAAAAACCTGGATCGGAACCAA-3′ and 5′-GCATTAGCTTCAGATTTACGGGT-3′; CXCL1: 5′-GAGCCTCTAACCAGTTCCAG-3′ and 5′-TGAGTGTGGCTATGACTTCG-3′ and IFNα4: 5′-TGATGAGCTACTACTGGTCAGC-3′ and 5′-GATCTCTTAGCACAAGGATGGC-3′. Primers were designed with PerlPrimer (http://perlprimer.sourceforge.net). Quantification was performed using the comparative CT method (ΔΔCT). Results are expressed relative to HPRT values.\n\nStatistics\nResults are expressed as the mean ± SEM. Statistical significance between groups was assessed using two-way ANOVA. The differences for in vivo experiments (at least 5 mice per group) were calculated using the Mann-Whitney U-test for unpaired data. Statistical significance of differences between survival rates was analyzed by comparing Kaplan-Meier curves using the log-rank test (GraphPad Prism version 5, GraphPad, San Diego, CA)."}

simple1

BioNLP16_DUT

BioNLP16_Messiy

DLUT931

bionlp-st-ge-2016-test-ihmc

bionlp-st-ge-2016-spacy-parsed

bionlp-st-ge-2016-test-tees

testone

PMC:3291650 / 32109-41812 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:3291650 / 32109-41812 JSON TXT