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Find patient medical information for Hydrocil Instant Oral on WebMD including its uses, side effects and safety, interactions, pictures, warnings and user ratings. Skip to main content.
Hydroxychloroquine (HCQ) is an antimalarial drug also used in treating autoimmune diseases. Its antiviral activity was demonstrated in restricting HIV infection in vitro; however, the clinical implications remain controversial.
Infection with dengue virus (DENV) is a global public health problem, and we lack an antiviral drug for DENV. Here, we evaluated the anti-DENV potential of treatment with HCQ. Immunofluorescence assays demonstrated that HCQ could inhibit DENV serotype 1–4 infection in vitro. RT-qPCR analysis of HCQ-treated cells showed induced expression of interferon (IFN)-related antiviral proteins and certain inflammatory cytokines. Mechanistic study suggested that HCQ activated the innate immune signaling pathways of IFN-β, AP-1, and NFκB.
Knocking down mitochondrial antiviral signaling protein (MAVS), inhibiting TANK binding kinase 1 (TBK1)/inhibitor-κB kinase ɛ (IKKɛ), and blocking type I IFN receptor reduced the efficiency of HCQ against DENV-2 infection. Furthermore, HCQ significantly induced cellular production of reactive oxygen species (ROS), which was involved in the host defense system. Suppression of ROS production attenuated the innate immune activation and anti-DENV-2 effect of HCQ. In summary, HCQ triggers the host defense machinery by inducing ROS- and MAVS-mediated innate immune activation against DENV infection and may be a candidate drug for DENV infection. Virus, cell lines, and chemicals We used local Taiwanese strains of DENV-1 766733A and DENV-2 PL046 (Genbank accession no. ) isolated from patients with dengue fever and DENV-4 466088A isolated from a patient with DHF.
DENV-3 H87 strain was kindly provided by D. Gubler (Lin and others ). These viruses were propagated in mosquito cell line C6/36 (ATCC: CRL-1660) grown in RPMI 1640 medium containing 5% fetal bovine serum (FBS).
A549 human lung epithelial carcinoma cells (ATCC: CCL-185), Hepa1-6 hepatoma cells (BCRC: 60051), WS1 human fetal skin normal fibroblasts (BCRC: 60300), J774A.1 mouse macrophages (BCRC: 60140), and HEK-293T cells (ATCC: CRL-3216) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Invitrogen). The reagents HCQ (Sigma-Aldrich; H0915), Baf A1 (Calbiochem; #196000), and recombinant human IFN-α 2a (Prospec; CYT-204) were used. TBK1/IKKɛ inhibitors BX795 (InvivoGen; tlrl-bx7) and Amlexanox (Sigma-Aldrich; SML0517) (Reilly and others ) and reactive oxygen species (ROS) inhibitor N-tert-Butyl-(-phenylnitrone (PBN) (Sigma-Aldrich; B7263) (Fidanboylu and others ) were used. Real-time quantitative PCR TRIzol reagent (Invitrogen) was used for total RNA extraction, and cDNA was synthesized from 0.5 μg total RNA by use of Superscript III reverse transcriptase (Invitrogen).
QPCR amplification involved 3 ng cDNA in 10 μL SYBR Green PCR master mix (Applied Biosystems) with 3 μM primers in ABI StepONE Plus Real-Time PCR system (Applied Biosystems). Transcript levels were normalized to that of hypoxanthine phosphoribosyltransferase. The primer sequences for gene detection are in and (Supplementary Data are available online at ). Luciferase reporter assay TurboFect transfection reagent (Thermo Scientific) was used for transient transfection following the manufacturer's protocol. Cells cultured in 12-well plates were transfected with NFκB-, AP-1-, ISRE-, or IFN-β-Luc reporter plasmids (Chang and others ). Mayavi mari chan tamil serial. PRL-TK (Promega), encoding Renilla luciferase under an herpes simplex virus thymidine kinase promoter, was an internal control.
In some cases, pcDNA3.1 V5-tagged-MAVS was used (Yu and others ). Cell lysates were collected for dual-luciferase assay (Promega). Firefly luciferase activity was normalized relative to that of Renilla luciferase. Immunofluorescence assay Cells were fixed with 4% paraformaldehyde for 30 min, then permeabilized with 0.5% Triton X-100 for 10 min. After 2 washes with phosphate-buffered saline (PBS), cells were blocked with 10% skim milk in PBS.
DENV-2 NS3 was detected by incubation with a monoclonal antibody against NS3 (#YH3304, 1:500 dilution; Yao-Hong Biotechnology) (Chang and others ), plus Alexa Fluor-488-conjugated goat anti-mouse IgG antibody (Invitrogen). Fluorescence signals were observed by fluorescence microscopy (ZEISS Observer. The 50% inhibition concentration (IC 50) of HCQ against DENV-2 in cells was estimated by immunofluorescence intensity measured with use of a microplate reader (Fluroskan Ascent FL; Thermo Scientific). Anti-E antibody (#YH3304, 1:100 dilution; Yao-Hong Biotechnology) was used for antibody-dependent enhancement (ADE) of DENV-2 infection in J774A.1 cells. IRF3 and NFκB p65 nuclear translocation assay was described previously (Chang and others ). IRF3 and NFκB p65 location was detected on incubation with the primary antibody anti-IRF3 (#sc-9082) and anti-NFκB p65 (#sc-372; both Santa Cruz Biotechnology), respectively, then Alex 568-conjugated anti-rabbit IgG antibody (Invitrogen). Nuclei were stained with DAPI.
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Find patient medical information for Hydrocil Instant Oral on WebMD including its uses, side effects and safety, interactions, pictures, warnings and user ratings. Skip to main content.
Hydroxychloroquine (HCQ) is an antimalarial drug also used in treating autoimmune diseases. Its antiviral activity was demonstrated in restricting HIV infection in vitro; however, the clinical implications remain controversial.
Infection with dengue virus (DENV) is a global public health problem, and we lack an antiviral drug for DENV. Here, we evaluated the anti-DENV potential of treatment with HCQ. Immunofluorescence assays demonstrated that HCQ could inhibit DENV serotype 1–4 infection in vitro. RT-qPCR analysis of HCQ-treated cells showed induced expression of interferon (IFN)-related antiviral proteins and certain inflammatory cytokines. Mechanistic study suggested that HCQ activated the innate immune signaling pathways of IFN-β, AP-1, and NFκB.
Knocking down mitochondrial antiviral signaling protein (MAVS), inhibiting TANK binding kinase 1 (TBK1)/inhibitor-κB kinase ɛ (IKKɛ), and blocking type I IFN receptor reduced the efficiency of HCQ against DENV-2 infection. Furthermore, HCQ significantly induced cellular production of reactive oxygen species (ROS), which was involved in the host defense system. Suppression of ROS production attenuated the innate immune activation and anti-DENV-2 effect of HCQ. In summary, HCQ triggers the host defense machinery by inducing ROS- and MAVS-mediated innate immune activation against DENV infection and may be a candidate drug for DENV infection. Virus, cell lines, and chemicals We used local Taiwanese strains of DENV-1 766733A and DENV-2 PL046 (Genbank accession no. ) isolated from patients with dengue fever and DENV-4 466088A isolated from a patient with DHF.
DENV-3 H87 strain was kindly provided by D. Gubler (Lin and others ). These viruses were propagated in mosquito cell line C6/36 (ATCC: CRL-1660) grown in RPMI 1640 medium containing 5% fetal bovine serum (FBS).
A549 human lung epithelial carcinoma cells (ATCC: CCL-185), Hepa1-6 hepatoma cells (BCRC: 60051), WS1 human fetal skin normal fibroblasts (BCRC: 60300), J774A.1 mouse macrophages (BCRC: 60140), and HEK-293T cells (ATCC: CRL-3216) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Invitrogen). The reagents HCQ (Sigma-Aldrich; H0915), Baf A1 (Calbiochem; #196000), and recombinant human IFN-α 2a (Prospec; CYT-204) were used. TBK1/IKKɛ inhibitors BX795 (InvivoGen; tlrl-bx7) and Amlexanox (Sigma-Aldrich; SML0517) (Reilly and others ) and reactive oxygen species (ROS) inhibitor N-tert-Butyl-(-phenylnitrone (PBN) (Sigma-Aldrich; B7263) (Fidanboylu and others ) were used. Real-time quantitative PCR TRIzol reagent (Invitrogen) was used for total RNA extraction, and cDNA was synthesized from 0.5 μg total RNA by use of Superscript III reverse transcriptase (Invitrogen).
QPCR amplification involved 3 ng cDNA in 10 μL SYBR Green PCR master mix (Applied Biosystems) with 3 μM primers in ABI StepONE Plus Real-Time PCR system (Applied Biosystems). Transcript levels were normalized to that of hypoxanthine phosphoribosyltransferase. The primer sequences for gene detection are in and (Supplementary Data are available online at ). Luciferase reporter assay TurboFect transfection reagent (Thermo Scientific) was used for transient transfection following the manufacturer's protocol. Cells cultured in 12-well plates were transfected with NFκB-, AP-1-, ISRE-, or IFN-β-Luc reporter plasmids (Chang and others ). Mayavi mari chan tamil serial. PRL-TK (Promega), encoding Renilla luciferase under an herpes simplex virus thymidine kinase promoter, was an internal control.
In some cases, pcDNA3.1 V5-tagged-MAVS was used (Yu and others ). Cell lysates were collected for dual-luciferase assay (Promega). Firefly luciferase activity was normalized relative to that of Renilla luciferase. Immunofluorescence assay Cells were fixed with 4% paraformaldehyde for 30 min, then permeabilized with 0.5% Triton X-100 for 10 min. After 2 washes with phosphate-buffered saline (PBS), cells were blocked with 10% skim milk in PBS.
DENV-2 NS3 was detected by incubation with a monoclonal antibody against NS3 (#YH3304, 1:500 dilution; Yao-Hong Biotechnology) (Chang and others ), plus Alexa Fluor-488-conjugated goat anti-mouse IgG antibody (Invitrogen). Fluorescence signals were observed by fluorescence microscopy (ZEISS Observer. The 50% inhibition concentration (IC 50) of HCQ against DENV-2 in cells was estimated by immunofluorescence intensity measured with use of a microplate reader (Fluroskan Ascent FL; Thermo Scientific). Anti-E antibody (#YH3304, 1:100 dilution; Yao-Hong Biotechnology) was used for antibody-dependent enhancement (ADE) of DENV-2 infection in J774A.1 cells. IRF3 and NFκB p65 nuclear translocation assay was described previously (Chang and others ). IRF3 and NFκB p65 location was detected on incubation with the primary antibody anti-IRF3 (#sc-9082) and anti-NFκB p65 (#sc-372; both Santa Cruz Biotechnology), respectively, then Alex 568-conjugated anti-rabbit IgG antibody (Invitrogen). Nuclei were stained with DAPI.