Journal: Journal of Virology

Article Title: ApoA1 Neutralizes Proinflammatory Effects of Dengue Virus NS1 Protein and Modulates Viral Immune Evasion

doi: 10.1128/JVI.01974-20

Figure Lengend Snippet: ApoA1 inhibits NS1-induced cell activation and the NS1 facilitation effect on DENV2 attachment to the cell membrane. (A) RAW 264.7 cells were incubated with NS1 for 24 h in the presence of different concentrations of ApoA1. Secreted NO was quantified in culture supernatants by the Griess assay. (B) Cells were fixed and labeled with CTB-FITC, and lipid rafts were analyzed by flow cytometry. The MFI of CTB was quantified relative to control cells incubated with PBS (pH 7.4). (C) Cells were preincubated for 24 h with 50 μg/ml NS1, in the presence or absence of 100 μg/ml ApoA1, and then challenged with DENV2 for 1 h at 4°C for virus attachment. Unbound virus was washed out with PBS, and total RNA was extracted. vRNA was quantified by qRT-PCR. Error bars represent the SDs of at least three biological replicates. Asterisks represent significant differences, compared to control. ***, P < 0.001; ns, not significant.

Article Snippet: ApoA1 mimetic peptide 4F (acetyl- DWFKAFYDKVAEKFKEAF -NH 2 ) was synthetized by GenOne (Rio de Janeiro, Brazil).

Techniques: Activation Assay, Membrane, Incubation, Griess Assay, Labeling, Flow Cytometry, Control, Virus, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: ApoA1 Neutralizes Proinflammatory Effects of Dengue Virus NS1 Protein and Modulates Viral Immune Evasion

doi: 10.1128/JVI.01974-20

Figure Lengend Snippet: NS1 interacts with ApoA1 via nonpolar interfaces. (A) Purified NS1 was incubated with 10% human serum (HS) and immunoprecipitated with purified anti-NS1 antibody. The input (IN) and elution (E) fractions were analyzed by Western blotting with anti-NS1 and anti-ApoA1 monoclonal antibodies. (B) Microplates were coated with purified ApoA1 and incubated with increasing amounts of recombinant NS1 protein expressed in bacteria (NS1bac) or in Sf9 cells (NS1Sf9). Bound NS1 was detected using anti-NS1 polyclonal antibody and HRP-conjugated secondary antibody. OD490 values were normalized to negative-control (BSA) values. (C) ApoA1-coated microplates were incubated with 0.6 μM NS1bac in the presence of Triton X-100 or PEG. Bound NS1 was detected as described previously. (D) A monolayer of RAW 264.7 cells was fixed with 4% paraformaldehyde, blocked, and coincubated with purified ApoA1 and NS1Sf9. Bound NS1 was detected using anti-NS1 polyclonal antibody and HRP-conjugated secondary antibody. OD490 values were normalized to negative-control (BSA) values. Error bars indicate SDs of two independent experiments. *, P < 0.5; **, P < 0.05.

Article Snippet: ApoA1 mimetic peptide 4F (acetyl- DWFKAFYDKVAEKFKEAF -NH 2 ) was synthetized by GenOne (Rio de Janeiro, Brazil).

Techniques: Purification, Incubation, Immunoprecipitation, Western Blot, Bioprocessing, Recombinant, Bacteria, Negative Control

Journal: Journal of Virology

Article Title: ApoA1 Neutralizes Proinflammatory Effects of Dengue Virus NS1 Protein and Modulates Viral Immune Evasion

doi: 10.1128/JVI.01974-20

Figure Lengend Snippet: ApoA1-mediated lipid raft depletion from ABCA1-expressing cells inhibits DENV2 attachment to the cell membrane. (A) RAW 264.7 cells were treated with 0.3 mM 8-Br-cAMP for 16 h. Total cell lysate was analyzed by Western blotting with anti-ABCA1. (B) Lipid rafts were quantified by flow cytometry of cells labeled with CTB-FITC. Control cells were treated with PBS (pH 7.4) or depleted with 3 mM MβCD for 2 h. ABCA1-expressing cells were treated with 100 μg/ml ApoA1 for 2 h. MFI was calculated relative to control cells. (C) After induced lipid raft depletion, RAW 264.7 cells were challenged with DENV2 for 1 h at 4°C for viral attachment. Unbound virus was washed out with PBS, and total RNA was extracted. vRNA was quantified by qRT-PCR. (D) ABCA1-expressing cells were incubated with ApoA1 for 2 h in the presence of different concentrations of NS1. Lipid rafts were quantified by flow cytometry of CTB-FITC. Each bar represents the SD of at least three biological replicates. Asterisks represent significant differences, compared to control. *, P < 0.01; **, P < 0.01; ***, P < 0.001.

Article Snippet: ApoA1 mimetic peptide 4F (acetyl- DWFKAFYDKVAEKFKEAF -NH 2 ) was synthetized by GenOne (Rio de Janeiro, Brazil).

Techniques: Expressing, Membrane, Western Blot, Flow Cytometry, Labeling, Control, Virus, Quantitative RT-PCR, Incubation

Journal: Journal of Virology

Article Title: ApoA1 Neutralizes Proinflammatory Effects of Dengue Virus NS1 Protein and Modulates Viral Immune Evasion

doi: 10.1128/JVI.01974-20

Figure Lengend Snippet: Serum ApoA1 concentrations are decreased in DENV-infected patients. (A) Total ApoA1 was quantified in human serum using the human ApoA1 ELISA kit (Thermo Fisher Scientific). Of samples from 33 patients, 23 were DENV positive and 10 were DENV negative. Samples from 7 healthy donors were also quantified as control samples. (B) Among DENV-positive patients, 16 were diagnosed with dengue fever and 7 were diagnosed with severe dengue. Bars represent mean and SDs. (C) HepG2 cells were mock infected or infected with DENV2 (multiplicity of infection of 1) for 48 h, and total cell lysates were analyzed by Western blotting. The ApoA1 band was quantified and expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars represent means and SDs from three experiments. Asterisks represent significant differences, compared to mock-infected cells. ****, P < 0.005; ns, not significant.

Article Snippet: ApoA1 mimetic peptide 4F (acetyl- DWFKAFYDKVAEKFKEAF -NH 2 ) was synthetized by GenOne (Rio de Janeiro, Brazil).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: Critical Care

Article Title: ApoA1/HDL and sepsis-associated vascular endothelial injury: a narrative review

doi: 10.1186/s13054-025-05668-1

Figure Lengend Snippet: Mechanism by which ApoA1 Inhibits Vascular Endothelial Inflammation. Lipid-free ApoA1 protein or its mimetic peptides interact with the ABCA1 receptor (a 12-transmembrane protein located in non-raft membrane domains, which has poor cholesterol) on macrophages. Concurrently, ApoA1 within (r)HDL interacts with the scavenger receptor SR-B1 (located in cholesterol-rich lipid raft domains). Both interactions promote the efflux of free cholesterol from macrophages to ApoA1 (indicated by a yellow straight arrow). Notably, the cholesterol efflux regulatory protein AIBP (ApoA1-Binding Protein) enhances the binding of ApoA1 to the ABCA1 receptor. Pathway ①: Inhibition of TLR4 consequently suppresses its downstream pro-inflammatory signaling pathways. Pathways ② and ③: Signaling pathways that inhibit inflammation, initiated respectively by the binding of ApoA1 to the ABCA1 receptor and HDL to the SR-B1 receptor. Pathway ④: Reconstituted HDL (rHDL) inhibits LPS-stimulated activation of the NLRP3 inflammasome. Created with BioGDP.com

Article Snippet: APOA1 mimetic peptide 4 F , In vivo , Sprague - Dawley rats , 10 mg/kg; i.p. , CLP , , IL-6↓;HDL↑; survival rate↑ , [ ] .

Techniques: Membrane, Binding Assay, Inhibition, Protein-Protein interactions, Activation Assay

Journal: Critical Care

Article Title: ApoA1/HDL and sepsis-associated vascular endothelial injury: a narrative review

doi: 10.1186/s13054-025-05668-1

Figure Lengend Snippet: ApoA1 maintains endothelial barrier integrity. Pathway ①: ApoA1 and ApoA1 mimetic peptides (such as D-4 F) inhibit EndMT in endothelial cells. Pathway ②: (r)HDL binds to the SR-B1 receptor, initiating signaling pathways that upregulate ANGPTL4 levels, thereby protecting the endothelial glycocalyx. Created with BioGDP.com

Article Snippet: APOA1 mimetic peptide 4 F , In vivo , Sprague - Dawley rats , 10 mg/kg; i.p. , CLP , , IL-6↓;HDL↑; survival rate↑ , [ ] .

Techniques: Protein-Protein interactions

Journal: Critical Care

Article Title: ApoA1/HDL and sepsis-associated vascular endothelial injury: a narrative review

doi: 10.1186/s13054-025-05668-1

Figure Lengend Snippet: ApoA1 prevents vascular occlusion, thrombosis, and maintains vascular tone. Pathway ①: (r)HDL binds to the PDZK1 domain of SR-B1, leading to eNOS phosphorylation, producing NO to prevent microvascular occlusion and reducing vWF. Pathway ②: Lipid-free ApoA1 or (r)HDL binds to Ecto-F1-ATPase, activating the PI3Kβ/AKT signaling pathway. Pathway ③ and ④ upregulate COX-2 and PGI-2. Created with BioGDP.com

Article Snippet: APOA1 mimetic peptide 4 F , In vivo , Sprague - Dawley rats , 10 mg/kg; i.p. , CLP , , IL-6↓;HDL↑; survival rate↑ , [ ] .

Techniques: Phospho-proteomics