Journal: Journal of immunoassay & immunochemistry

Article Title: A Comparison of Nanoparticle-Antibody Conjugation Strategies in Sandwich Immunoassays

doi: 10.1080/15321819.2016.1269338

Figure Lengend Snippet: Evaluation of the effect of conjugation strategy on assay performance for fixed NP concentration. a) LOD measurement for active (left), passive (middle), and commercial (right) conjugation for 0.06 nM conjugate concentration, immobilized antibody at 0.4 μg/mm, and serial dilution of purified Dengue NS1 in human serum. Scale bars = 5 mm. (b) Left, image analysis for active (red circles), passive (blue squares), and commercial conjugation (green triangles) with sigmoidal fits to calculate LOD values (dotted lines). Inset: zoom in for concentration 0–80 ng/ml. Right, Bar graph comparing LOD values from each conjugation. p<0.001.

Article Snippet: Supernatants from Vero cells infected with dengue virus serotype 2 and recombinant dengue NS1 serotype 2 produced in 293 human cells (The Native Antigen Company, United Kingdom) were used as a source of NS1 protein for dipstick chromatography experiments.

Techniques: Conjugation Assay, Concentration Assay, Serial Dilution, Purification

Journal: Tropical Medicine and Infectious Disease

Article Title: Resurgence of Dengue Virus Serotype 4 in Malaysia: A Comprehensive Clinicodemographic and Genomic Analysis

doi: 10.3390/tropicalmed8080409

Figure Lengend Snippet: NS1 mutation mapping for the recently sequenced DENV-4 genotype II in comparison with reference sequences.

Article Snippet: The X-ray crystal structure of DENV-2 NS1 was retrieved from the Protein Databank (RCSB-PDB) (PBD ID: 4O6B) and used as a template to build the wild type (WT) and mutant DENV4 NS1 model. Once the WT and mutant DENV-4 protein models were generated, energy minimisations were performed using the Swiss-PDB viewer [ ] program to remove unwanted contacts.

Techniques: Mutagenesis, Comparison

Journal: Tropical Medicine and Infectious Disease

Article Title: Resurgence of Dengue Virus Serotype 4 in Malaysia: A Comprehensive Clinicodemographic and Genomic Analysis

doi: 10.3390/tropicalmed8080409

Figure Lengend Snippet: The homo-dimer X-ray crystal structure of DENV-2 NS1 (PDB ID: 4O6B) and wild type of the DENV-4 NS1 structure model: ( A ). X-ray crystal structure of DENV-2 NS1 with missing residue (red circle); ( B ). Complete wild type DENV-4 NS1 structure model developed based on the X-ray crystal structure of DENV-2 NS1. Blue colour represents the β-roll domain, yellow indicates the wing domain, green shows the connector sub-domain and orange signifies the central β-ladder domain. The red dotted circle represents unresolved regions (amino acids 7 to 11 and amino acids 108 to 128).

Article Snippet: The X-ray crystal structure of DENV-2 NS1 was retrieved from the Protein Databank (RCSB-PDB) (PBD ID: 4O6B) and used as a template to build the wild type (WT) and mutant DENV4 NS1 model. Once the WT and mutant DENV-4 protein models were generated, energy minimisations were performed using the Swiss-PDB viewer [ ] program to remove unwanted contacts.

Techniques: Residue

Journal: Tropical Medicine and Infectious Disease

Article Title: Resurgence of Dengue Virus Serotype 4 in Malaysia: A Comprehensive Clinicodemographic and Genomic Analysis

doi: 10.3390/tropicalmed8080409

Figure Lengend Snippet: Quality assessment of the predicted WT and mutant DENV-4 NS1 structures using SWISS-MODEL and AlphaFold2.

Article Snippet: The X-ray crystal structure of DENV-2 NS1 was retrieved from the Protein Databank (RCSB-PDB) (PBD ID: 4O6B) and used as a template to build the wild type (WT) and mutant DENV4 NS1 model. Once the WT and mutant DENV-4 protein models were generated, energy minimisations were performed using the Swiss-PDB viewer [ ] program to remove unwanted contacts.

Techniques: Mutagenesis

Journal: Tropical Medicine and Infectious Disease

Article Title: Resurgence of Dengue Virus Serotype 4 in Malaysia: A Comprehensive Clinicodemographic and Genomic Analysis

doi: 10.3390/tropicalmed8080409

Figure Lengend Snippet: Mutations mapped onto subunits A and B of the homo-dimer wild type DENV-4 NS1.

Article Snippet: The X-ray crystal structure of DENV-2 NS1 was retrieved from the Protein Databank (RCSB-PDB) (PBD ID: 4O6B) and used as a template to build the wild type (WT) and mutant DENV4 NS1 model. Once the WT and mutant DENV-4 protein models were generated, energy minimisations were performed using the Swiss-PDB viewer [ ] program to remove unwanted contacts.

Techniques:

Journal: Tropical Medicine and Infectious Disease

Article Title: Resurgence of Dengue Virus Serotype 4 in Malaysia: A Comprehensive Clinicodemographic and Genomic Analysis

doi: 10.3390/tropicalmed8080409

Figure Lengend Snippet: Thermodynamic stability of the predicted WT and mutant DENV-4 NS1 structures using FoldX.

Article Snippet: The X-ray crystal structure of DENV-2 NS1 was retrieved from the Protein Databank (RCSB-PDB) (PBD ID: 4O6B) and used as a template to build the wild type (WT) and mutant DENV4 NS1 model. Once the WT and mutant DENV-4 protein models were generated, energy minimisations were performed using the Swiss-PDB viewer [ ] program to remove unwanted contacts.

Techniques: Mutagenesis

Journal: Tropical Medicine and Infectious Disease

Article Title: Resurgence of Dengue Virus Serotype 4 in Malaysia: A Comprehensive Clinicodemographic and Genomic Analysis

doi: 10.3390/tropicalmed8080409

Figure Lengend Snippet: Hydrogen bond interactions between the wild type (His50) and the mutant (Tyr50) with their neighbouring residues. The wild type residue is presented as the yellow stick and sphere ( A ), while the mutant residue is presented as the green stick and sphere ( B ). Hydrogen bonds are indicated by black dotted lines. The DENV-4 NS1 structures are presented in cyan and salmon cartoons, respectively.

Article Snippet: The X-ray crystal structure of DENV-2 NS1 was retrieved from the Protein Databank (RCSB-PDB) (PBD ID: 4O6B) and used as a template to build the wild type (WT) and mutant DENV4 NS1 model. Once the WT and mutant DENV-4 protein models were generated, energy minimisations were performed using the Swiss-PDB viewer [ ] program to remove unwanted contacts.

Techniques: Mutagenesis, Residue

Journal: Tropical Medicine and Infectious Disease

Article Title: Resurgence of Dengue Virus Serotype 4 in Malaysia: A Comprehensive Clinicodemographic and Genomic Analysis

doi: 10.3390/tropicalmed8080409

Figure Lengend Snippet: Hydrogen bond interactions between the wild type (Pro144) and the mutant (Ser144) with their neighbouring residues. The wild type residue is denoted as a yellow stick and sphere ( A ), while the mutant residue is presented as the green stick and sphere ( B ). Hydrogen bonds are indicated by black dotted lines. The DENV-4 NS1 structures are presented in cyan and salmon cartoons, respectively.

Article Snippet: The X-ray crystal structure of DENV-2 NS1 was retrieved from the Protein Databank (RCSB-PDB) (PBD ID: 4O6B) and used as a template to build the wild type (WT) and mutant DENV4 NS1 model. Once the WT and mutant DENV-4 protein models were generated, energy minimisations were performed using the Swiss-PDB viewer [ ] program to remove unwanted contacts.

Techniques: Mutagenesis, Residue

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) HEK293T cells were co-transfected with HA-MMP-9 and Flag-Cap, Flag-M, Flag-Prm, Flag-E, Flag-NS1, Flag-NS2A, Flag-NS2B, Flag-NS3, Flag-NS4A, or Flag-NS4B. Cell lysates were immunoprecipitataed using anti-Flag antibody, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input. ( B ) HEK293T cells were co-transfected with HA-MMP9 and Flag-NS1, Cell lysates were immunoprecipitataed using anti-HA antibody, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input. ( C, D ) HEK293T cells were co-transfected with empty vector or VC-155-MMP-9 and VN173-NS1/E/NS4A. At 24 h post-transfection, living cells were observed by confocal microscopy. The quantification of YFP-positive cells was determined by ImageJ software (D). ND means not detection. ( E ) Purified His-SARS-CoV-2-N (5 μg) or His-DENV2-NS1 (5 μg) was incubated with purified no-tagged MMP-9 protein (3 μg) for 24 h, Mixtures were incubated with Ni-NTA Agarose beads. Mixtures were analyzed by immunoblotting using anti-MMP9, anti-NS1, anti-His antibody. Untreated protein including His-SARS-CoV-2-N (1 μg), His-DENV2-NS1 (1 μg), or no-tagged MMP-9 protein (1 μg) were analyzed by immunoblotting using anti-MMP9, anti-NS1, and anti-His antibody (as input). ( F ) Yeast strain AH109 were co-transformed with combination of binding domain (BD-p53, BD-MMP-9, and BD-Lam) and activation domain (AD-T, AD-NS1) plasmid. Transfected yeast cells were grown on SD-minus Trp/Leu double dropout plates, and colonies were replicated on to SD-minus Trp/Leu/Ade/His fourth dropout plates to check for the expression of reporter genes. ( G, H ) Schematic diagram of wild-type MMP-9 protein and truncated mutants MMP-9 protein (D1 to D9) (G). HEK293T cells were co-transfected with HA-NS1 and Flag-MMP-9 truncated mutants (D1 to D9). Cell lysates were immunoprecipitated using anti-Flag antibody, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input (H). Dates were representative of three independent experiments.

Article Snippet: The concentration of culture supernatants and MMP-9 or NS1 were measured by Human MMP-9 ELISA Kit (Invitrogen) or DENV2-NS1 ELISA Kit (Arigo biolaboratories) according to manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Software, Purification, Incubation, Western Blot, Transformation Assay, Binding Assay, Activation Assay, Expressing, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) PMA-differentiated THP-1 macrophages (top) or HUVEC cells (bottom) were treated with infectious or UV- inactivated DENV2 at MOI = 5 for 48h. Intracellular DENV2 E RNA (bottom) was determined by qRT-PCR analysis. Mock: untreated cells. Control: supernatant of C6/36 cells without DENV2 infection. ( B and C ) PMA-differentiated THP-1 macrophages were infected with DENV2 for different times at MOI = 5 (B) or at different MOI for 24 h (C). Intracellular MMP-9 RNA (top) and DENV2 E RNA (bottom) was determined by qRT-PCR analysis, MMP-9 proteinase activity in the supernatants was determined by gelatin zymography assays and proteins in cell extract (middle) were analyzed by Western blotting. ( D and E ) HUVEC cells were infected with DENV2 for different times at MOI = 5 (D C ) and at different MOI for 24 h (E). Intracellular MMP-9 RNA (top) and DENV2 E RNA (bottom) was determined by qRT-PCR analysis, MMP-9 proteinase activity in the supernatants was determined by gelatin zymography assays and proteins in cell extract (middle) were analyzed by Western blotting. ( F ) HUVEC cells or PMA-differentiated THP-1 macrophages were infected with DENV2 at MOI = 5 for 24 h. Viral copies were quantified by RT-PCR. ( G ) HUVEC cells or PMA-differentiated THP-1 macrophages were equally distributed to four 12-hole plates and infected with DENV2 at MOI = 5 for 24 h. MMP-9 protein in cell supernatants were measured by ELISA (top) and indicated proteins in cell extract were analyzed by WB (bottom). Dates were representative of three independent experiments. ns means not significant. Values are mean ± SEM, P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***).

Article Snippet: The concentration of culture supernatants and MMP-9 or NS1 were measured by Human MMP-9 ELISA Kit (Invitrogen) or DENV2-NS1 ELISA Kit (Arigo biolaboratories) according to manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Infection, Activity Assay, Zymography, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) PMA-differentiated THP-1 macrophages were infected with DENV2 for different times at MOI = 5, NS1 protein in Supernatants were analyzed by ELISA (top). Cell lysates were analyzed by immunoblotting (bottom). ( B ) Confluent monolayers of HUVEC cells were grown on polycarbonate membrane system and treated with the supernatants came from DENV2 infected HUVEC cells or THP-1 cells for 24 h or pre-incubated with 600nM SB-3CT (a specific inhibitor of MMP-9 protein) or 600 nM SC75741 for 1h. Endothelial permeability was evaluated by measuring trans-endothelial electrical resistance (TEER) (ohm) using EVOM2 epithelial voltohmmeter. ( C – I ) IFNAR -/- C57BL/6 mice were intravenously injected with 300 μl DENV2 at a dose of 1×10 6 PFU/mouse (n = 6), pre-treated with 300 μl PBS containing MMP-9 specific inhibitor SB-3CT (5 mg/kg per mice) by intraperitoneal injection for 90 min and then treated with DENV2 (1×10 6 PFU/mouse), repeat treated with SB-3CT (5 mg/kg per mice) on the fourth day after DENV2 (NGC) infection (n = 6), or 300 μl PBS containing the same volume DMSO as a control group (n = 4). 7 days after infection, mice were euthanasia, and the tissues were collected. MMP-9 RNA in the blood was determined by qRT-PCR (upper) and MMP-9 protein in the serum was measured by ELISA (lower). Points represent the value of each serum samples (C). Evans blue dye was intravenously injected into mice 7 days after DENV infected groups (n = 5), control groups (n = 4) and DENV+SB-3CT (n = 5) (C–E). The dye was allowed to circulate for 2 hours before mice were euthanasia, tissues include liver (D), spleen (E) and lung (F) were collected, and the value of Evans blue was measured at OD 610 . Histopathology analysis of tissues includes Liver (G), Spleen (H) and Lung (I) after DENV infection. ( J ) Monolayers of HUVEC cells grown on Transwell inserts were incubated for 48 h with MMP-9 protein (100 ng/ml) or NS1 protein (5 μg/ml) or NS1 (5 μg/ml) plus different concentration of MMP-9 (50 ng/ml to 100ng/ml) or pre-treated with 600 nM SB-3CT or 600 nM SC7574 for 1 h, then incubated with NS1 plus MMP-9. The TEER (ohm) was measured at indicated time points. Dates were representative of two to three independent experiments. ns means not significant. Values are mean ± SEM, P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***).

Article Snippet: The concentration of culture supernatants and MMP-9 or NS1 were measured by Human MMP-9 ELISA Kit (Invitrogen) or DENV2-NS1 ELISA Kit (Arigo biolaboratories) according to manufacturer’s instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Permeability, Injection, Quantitative RT-PCR, Histopathology, Concentration Assay

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A – F ) IFNAR -/- C57BL/6 mice were intravenously injected with 300 μl DENV2 at a dose of 1×10 6 PFU/mouse (n = 6), pre-treated with 300 μl PBS containing MMP-9 specific inhibitor SB-3CT (5 mg/kg per mice) by intraperitoneal injection for 90 min and then treated with DENV2 (1×10 6 PFU/mouse), repeat treated with SB-3CT (5 mg/kg per mice) on the fourth day after DENV2 (NGC) infection (n = 6), or 300 μl PBS containing the same volume DMSO as a control group (n = 4). 7 days after infection, mice were euthanasia, and the tissues were collected. The indicated proteins in Lung (A), spleen (B) and Liver (C) were measured by Western-blot. The expression of β-catenin in Liver (D), spleen (E), and Lung (F) by Immunohistochemistry analysis. ( G ) HUVEC cells were respectively transfected with plasmid encoding MMP-9 (2 μg) or NS1 (2 μg) or NS1 (1 ug) plus MMP-9 (1 μg) for 24 h or firstly co-transfected with plasmid encoding NS1 (1ug) plus MMP-9 (1 μg) for 12 h, then treated with 600nM SB-3CT for 12 h. The indicated proteins in cell extract were analyzed by WB. ( H–K ) HUVEC cells were treated with NS1 protein (5 μg/ml) or MMP-9 protein (100 ng/ml) or NS1 (5 μg/ml) plus MMP-9 (100 ng/ml) or pre-incubated with 600 nM SB-3CT for 1 h, then treated with NS1 (5 μg/ml) plus MMP-9 (100 ng/ml) for 6 h, The distribution of endogenous β-catenin (H) or ZO-1 (J) protein were visualized under confocal microscope. The quantifications of relative β-catenin (I) or ZO-1 (K) intensities were determined by ImageJ software. The quantification of protein was used by ImageJ software (A–G). (D–F) +++ means Percentage contribution of high positive cells; ++ means Percentage contribution of positive cells; + means Percentage contribution of low positive cells;—means Percentage contribution of negative cells. ns means not significant. All dates were representative of two to three independent experiments.

Article Snippet: The concentration of culture supernatants and MMP-9 or NS1 were measured by Human MMP-9 ELISA Kit (Invitrogen) or DENV2-NS1 ELISA Kit (Arigo biolaboratories) according to manufacturer’s instructions.

Techniques: Injection, Infection, Western Blot, Expressing, Immunohistochemistry, Transfection, Plasmid Preparation, Incubation, Microscopy, Software

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with plasmid encoding HA-NS1 plus Flag-β-catenin. Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-Flag, anti-HA, or anti-β-catenin antibody. Cell lysates (40 μg) were used as Inputs. ( B ) Hela cells were transfected with plasmid encoding HA-NS1, Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-HA or anti-β-catenin antibody. Cell lysates (40 μg) was used as Input. ( C , D ) HEK293T cells (C) or Hela cells (D) were co-transfected with plasmid encoding HA-NS1 plus Flag-ZO-1, Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-Flag or anti-HA antibody. Cell lysates (40 μg) were used as Inputs. ( E , F ) HEK293T cells were transfected with Flag-β-catenin (E) or Flag-ZO-1 (F) and incubated with purified His-SARS-CoV-2-N (5 μg) or His-DENV2-NS1 (5 μg) for 24 h, cell extracts were incubated with Ni-NTA Agarose beads. Mixtures were analyzed by immunoblotting using anti-β-catenin, anti-NS1, anti-His, anti-ZO-1 antibody. Untreated proteins including His-SARS-CoV-2-N (1 μg) and His-DENV2-NS1 (1 μg) and HEK293T cell lysates were analyzed by immunoblotting using anti-β-catenin, anti-NS1, anti-His, and anti-ZO-1 antibody (as input). ( G , H ) Hela cells were co-transfected with plasmid encoding HA-NS1 plus Flag-MMP-9, Cell lysates were immunoprecipitated using anti-Flag (G) or anti-β-catenin antibody (H), and analyzed using anti-Flag, anti-HA or anti-β-catenin antibody. Cell lysates (40 μg) was used as Input. ( I, J ) Hela cells were co-transfected with plasmid encoding HA-NS1, HA-MMP-9, and Flag-ZO-1 (I) or Flag-NS1, HA-MMP-9, and Flag-ZO-1 (J). Cell lysates were immunoprecipitated using anti-Flag (top) or anti-HA antibody (bottom), and analyzed using anti-Flag, anti-HA or anti-MMP-9 antibody. Cell lysates (40 μg) was used as Input. ( K , L ) HUVEC cells were treated with NS1 protein (5 μg/ml), MMP-9 protein (100 ng/ml), and NS1 protein (5 μg/ml) plus MMP-9 protein (100 ng/ml), respectively, for 6 h. The distributions of the membrane marker (Dil) (yellow), the endogenous β-catenin (yellow) and ZO-1 (yellow) proteins and the extracellular NS1 (red) and MMP-9 (green) proteins were visualized under confocal microscope. The quantifications of co-localization fluorescence were determined by ImageJ software (L). ND means not detected. All dates were representative of three independent experiments.

Article Snippet: The concentration of culture supernatants and MMP-9 or NS1 were measured by Human MMP-9 ELISA Kit (Invitrogen) or DENV2-NS1 ELISA Kit (Arigo biolaboratories) according to manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Incubation, Purification, Western Blot, Marker, Microscopy, Fluorescence, Software

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: C57BL/6 mice and MMP-9 -/- mice were injected intravenously DENV2 NS1 protein [10 mg/kg (n = 5)], the same volume of PBS was also tail vein injected to C57BL/6 mice and MMP-9 -/ - mice (n = 5) as control group. Another group of MMP-9 -/- mice (n = 5) were injected intravenously DENV2 NS1 protein (10 mg/kg) plus recombinant mouse MMP-9 protein (70 μg /kg). ( A – C ) After 24 h post-injection, mice were intravenously injected with Evans blue dye. The dye was allowed to circulate for 2h before mice were euthanized, and tissue include Lung (A), spleen (B), and Liver (C) were collected. The value of Evans blue was measured at OD 610 . ( D – I ) After 24 h post-injection, mice were euthanized and tissue were collected. The indicated proteins in Lung (D), spleen (E), and Liver (F) were measured by Western-blot. The expression of β-catenin in Lung (G), spleen (H), and Liver (I) were analyzed by Immunohistochemistry. All dates were representative of two to three independent experiments. The quantification of protein was used by ImageJ software (D–I). (G–H) +++ means Percentage contribution of high positive cells; ++ means Percentage contribution of positive cells; + means Percentage contribution of low positive cells;—means Percentage contribution of negative cells. ns means not significant. Values are mean ± SEM, P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***).

Article Snippet: The concentration of culture supernatants and MMP-9 or NS1 were measured by Human MMP-9 ELISA Kit (Invitrogen) or DENV2-NS1 ELISA Kit (Arigo biolaboratories) according to manufacturer’s instructions.

Techniques: Injection, Recombinant, Western Blot, Expressing, Immunohistochemistry, Software

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 interacts with MMP-9 and facilitates the enzyme to alter the adhesion and tight junctions and vascular leakage in human endothelial cells and mice tissues. Moreover, NS1 recruits MMP-9 to interact with β-catenin and Zona occludens protein-1/2 to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mice tissues.

Article Snippet: The concentration of culture supernatants and MMP-9 or NS1 were measured by Human MMP-9 ELISA Kit (Invitrogen) or DENV2-NS1 ELISA Kit (Arigo biolaboratories) according to manufacturer’s instructions.

Techniques: Expressing

Journal: The Journal of Infectious Diseases

Article Title: Zika Virus Seroprevalence in Urban and Rural Areas of Suriname, 2017

doi: 10.1093/infdis/jiz063

Figure Lengend Snippet: ZIKV IgG ELISA and VNT results in 2017 cohort and pre-ZIKV cohort

Article Snippet: The 44 samples of the pre-ZIKV cohort were tested with a DENV-2 virus particle-based commercial DENV ELISA kit (Euroimmun) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell Biochemistry and Biophysics

Article Title: ROCK is Involved in Vimentin Phosphorylation and Rearrangement Induced by Dengue Virus

doi: 10.1007/s12013-013-9665-x

Figure Lengend Snippet: Vimentin is recruited to the perinuclear region after DENV2 infection. At different time points p.i., the cells were fixed and stained with an anti-vimentin antibody. Immunostaining images show that DENV2 infection induces rearrangement of vimentin as early as 30 min p.i. A marked redistribution of the organization of vimentin was observed at 1, 8, and 24 h p.i. Scale bars 10 μm

Article Snippet: Then, the cells were incubated with anti-DENV2 NS1 protein antibody at 4 °C overnight, and Cy5-conjugated antibody (Beyotime, China) was added for 1 h at 37 °C.

Techniques: Infection, Staining, Immunostaining

Journal: Cell Biochemistry and Biophysics

Article Title: ROCK is Involved in Vimentin Phosphorylation and Rearrangement Induced by Dengue Virus

doi: 10.1007/s12013-013-9665-x

Figure Lengend Snippet: DENV2 infection induced aggregation of vimentin and Ser71-phosphorylated vimentin into dense structures at the perinuclear area. ECV304 cells were infected with DENV2 for 24 h. Immunostaining shows that the characteristic distribution of vimentin in control cells ( a ) completely changes in infected cells where vimentin ( b ) and phosphorylated vimentin ( c ) move to the perinuclear region and co-localize with DENV2 NS1 (arrows). Scale bars 10 μm

Article Snippet: Then, the cells were incubated with anti-DENV2 NS1 protein antibody at 4 °C overnight, and Cy5-conjugated antibody (Beyotime, China) was added for 1 h at 37 °C.

Techniques: Infection, Immunostaining

Journal: Cell Biochemistry and Biophysics

Article Title: ROCK is Involved in Vimentin Phosphorylation and Rearrangement Induced by Dengue Virus

doi: 10.1007/s12013-013-9665-x

Figure Lengend Snippet: DENV2 infection induced ROCK activation and phosphorylation of vimentin at Ser71. At various time points after DENV2 infection, cell lysates were analyzed by ELISA and Western blotting. a ROCK levels were measured by ELISA. A histogram depicts the change in ROCK activation. Each reaction was performed in duplicate, and each bar represents the mean + the standard deviation for three experiments. * P < 0.05. b Western blot analysis was performed to detect Ser71-phosphorylated vimentin and vimentin expression, respectively. The results show that DENV2 induced an increase in the phosphorylation of vimentin at Ser71 from 15 min to 24 h, with a maximum increase of 7.8-fold above control cells at 8 h p.i. Each blot represents data from a minimum of six separate experiments

Article Snippet: Then, the cells were incubated with anti-DENV2 NS1 protein antibody at 4 °C overnight, and Cy5-conjugated antibody (Beyotime, China) was added for 1 h at 37 °C.

Techniques: Infection, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation, Expressing

Journal: Cell Biochemistry and Biophysics

Article Title: ROCK is Involved in Vimentin Phosphorylation and Rearrangement Induced by Dengue Virus

doi: 10.1007/s12013-013-9665-x

Figure Lengend Snippet: Inactivation of ROCK with Y-27632 inhibited DENV2-induced vimentin rearrangement and phosphorylation of vimentin at Ser71. ECV304 cells were pretreated with 10 μM Y-27632 or mock treated for 1 h at 37 °C prior to infection with DENV2. The cells were collected at different time points p.i. and then stained with the anti-vimentin antibody or analyzed by Western blotting with antibodies specific for Ser71-phosphorylated vimentin and vimentin, respectively. a Effect of ROCK on vimentin distribution. Immunostaining images show that Y-27632 prevented the rearrangement of vimentin aggregates into dense structures at the perinuclear area in DENV2-infected cells at 30 min, 1 h, 8 h, and 24 h p.i. Scale bars 10 μm. b Effect of ROCK on the phosphorylation of vimentin at Ser71. The results show that in Y-27632-treated cells, phosphorylation of vimentin at Ser71 was decreased at 30 min, 1 h, 8 h, and 24 h p.i. Each blot represents data from a minimum of six separate experiments

Article Snippet: Then, the cells were incubated with anti-DENV2 NS1 protein antibody at 4 °C overnight, and Cy5-conjugated antibody (Beyotime, China) was added for 1 h at 37 °C.

Techniques: Infection, Staining, Western Blot, Immunostaining

Journal: Cell Biochemistry and Biophysics

Article Title: ROCK is Involved in Vimentin Phosphorylation and Rearrangement Induced by Dengue Virus

doi: 10.1007/s12013-013-9665-x

Figure Lengend Snippet: Inactivation of ROCK with Y-27632 partially inhibited DENV2-induced ER rearrangement. Cells were infected for 24 h with DENV2. Viral replication sites were located using antibodies specific for the NS1 protein ( red ), and the ER was visualized using antibodies specific for calnexin ( green ). The Golgi complex was labeled with an anti-giantin antibody ( green ), and mitochondria were labeled with MitoTracker Green. Normal distribution of the ER ( a ), Golgi complex ( c ), and mitochondria ( e ) in uninfected cells is shown. In DENV2-infected cells, the ER staining redistributed to the perinuclear region and co-localized with DENV2 NS1 ( b , arrows ). No rearrangement of the Golgi apparatus or mitochondria was obvious in DENV2-infected cells ( d , f ). Y-27632 treatment partially blocked ER aggregation into dense structures ( g ). Scale bars 10 μm (Color figure online)

Article Snippet: Then, the cells were incubated with anti-DENV2 NS1 protein antibody at 4 °C overnight, and Cy5-conjugated antibody (Beyotime, China) was added for 1 h at 37 °C.

Techniques: Infection, Labeling, Staining