Journal: iScience

Article Title: A micro-electroporation/electrophoresis-based vaccine screening system reveals the impact of vaccination orders on cross-protective immunity

doi: 10.1016/j.isci.2023.108086

Figure Lengend Snippet:

Article Snippet: WT SARS-CoV-2 spike , Sino Biological , Cat #40589-V08B1.

Techniques: Virus, Recombinant, Cream, Enzyme-linked Immunosorbent Assay, Luciferase, Lysis, Staining, Membrane, Western Blot, Software

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae prevents the invasion of SARS-CoV-2 particles into host cells. (A and B), immunofluorescence staining (A) or enzyme-linked immunosorbent assay (ELISA) analysis (B) of SARS-CoV-2 virus-like particles (VLPs) invasion into human induced pluripotent stem cells (hiPSC)-derived alveolar epithelial cells type II (AECII). Control: dimethyl sulfoxide (DMSO)-treated cells; spike (S) protein neutralizing antibody (S NAb): positive control; and Kae: 10 µM in DMSO.(C and D), the entry of SARS-CoV-2 VLPs into lungs of hACE2 transgenic mice evaluated by immunofluorescence staining (C) or luciferase activity (D).Control: 0.5% carboxymethylcellulose sodium-treated mice; ursodeoxycholic acid (UDCA): positive control; and Kae: indicated dosages in 0.5% carboxymethylcellulose sodium. (A and C), VLPs, ACE2, F-actin and nucleus are shown as indicated colors. Scale bar = 25 µm. (B), ***, p < 0.005 vs Blank. #, p < 0.05 and ##, p < 0.01 vs Control. (D), n = 4, ***, p < 0.005 vs Blank. ##, p < 0.01; and ###, p < 0.005 vs Control. ns: no statistical differences vs Control.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Virus, Derivative Assay, Control, Positive Control, Transgenic Assay, Luciferase, Activity Assay

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae targets coronaviral fusion. (A), flow cytometry analyses of the binding affinity between spike (S) protein receptor-binding domain (S-RBD) and HEK-293F cells expressing angiotensin-converting enzyme 2 (ACE2) in the presence or absence of Kae with the S protein neutralizing antibody (S NAb) as a positive control. ***, p < 0.005 vs Blank. ###, p < 0.005, and ns, no statistical differences vs Control. (B), impacts of Kae on SARS-CoV-2 endocytosis into Vero E6 cells with S NAb as a positive control. ***, p < 0.005, and ns, no statistical differences vs Control. (C, D and E), effects of Kae on membrane fusion mediated by SARS-CoV-2 variants (C), or SARS-CoV (D), or MERS-CoV (E) with transmembrane serine protease 2 (TMPRSS2) inhibitor, camostat mesylate (CM), as a positive control. Scale bar = 300 µm. **, p < 0.01; and ***, p < 0.005 vs Control. Control: dimethyl sulfoxide (DMSO)-treated cells; DPP4: dipeptidyl peptidase 4; ns: no statistical differences vs Control.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Flow Cytometry, Binding Assay, Expressing, Positive Control, Control, Membrane

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae interferes with the S2 subunit of SARS-CoV-2 spike protein. (A), immunoblots representing the cleavage and abundance of SARS-CoV-2 spike (S) protein induced by Kae, with camostat mesylate (CM) as a positive control. (B), thermal stability determinations for full-length S protein, S1 and S2 subunits of SARS-CoV-2 in the presence or absence of Kae. Control: dimethyl sulfoxide (DMSO)-treated cells.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Western Blot, Positive Control, Control

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae impairs HR1 and HR2 of SARS-CoV-2 S2 subunit. (A and B), circular dichroism (CD) spectra analyses of synthesized heptad repeat 1 (HR1) and heptad repeat 2 (HR2) peptide, the formation of six-helix bundle (6-HB, HR1+HR2), and impacts of Kae on 6-HB, HR1 and HR2 using isopropanol as solvent controls. (C, D, E and F), native polyacrylamide gel electrophoresis (N-PAGE) assessment of effects of Kae on HR1, HR2 and 6-HB. *, p < 0.05; **, p < 0.01; and ***, p < 0.005 vs Control. ns: no statistical differences vs Blank. (E and F), the stability of wildtype (WT) and mutant (MT) HR2 in the absence (E) or presence (F) of Kae compared to corresponding wildtype peptide. (G), atomic force microscopy (AFM) topographic images before and after adding Kae to HR1, HR2 and 6-HB. Scale bar = 4 µm. (C, D, F and G), Control: dimethyl sulfoxide (DMSO)-treated peptides.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Circular Dichroism, Synthesized, Solvent, Polyacrylamide Gel Electrophoresis, Control, Mutagenesis, Microscopy

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Schematic diagram of the inhibitory mechanism of Kae against SARS-CoV-2 invasion. Instead of blocking the interaction between angiotensin-converting enzyme 2 (ACE2) and spike (S) protein receptor-binding domain (S-RBD), Kae disrupts heptad repeat 1 (HR1) and heptad repeat 2 (HR2) segments in the S2 subunit of SARS-CoV-2 S protein, leading to inhibitions of viral fusion-mediated invasion of SARS-CoV-2.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Blocking Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Spike-Dependent Opsonization Indicates Both Dose-Dependent Inhibition of Phagocytosis and That Non-Neutralizing Antibodies Can Confer Protection to SARS-CoV-2

doi: 10.3389/fimmu.2021.808932

Figure Lengend Snippet: Neutralization assays identify one monoclonal as blocking the ACE2-Spike protein interaction. (A) SPR analysis of the binding of monoclonal antibodies to the RBD domain of the Spike protein. PBS served as a negative control, and the intact RBD was our positive control for ACE2 binding. (B) The 96 antibodies which we produced were assayed for neutralization potential in a Spike-bead-based neutralization assay. Spike-beads [such as the ones used in (B) ] were opsonized with the antibodies in 96 well plates. The beads were then centrifuged, reconstituted in fresh media, and added to HEK293-ACE2 cells at a ratio of 20 beads per cell and imaged with automated microscopy. The data is from 4 pooled experiments and is presented as bead association normalized percentage. Error bars indicate the SEM for the replicate experiments. (C) The 10 Spike-ELISA reactive antibodies were assayed for pseudovirus neutralization. A firefly luciferase encoding pseudotype lentivirus was used to infect HEK239-ACE2 cells. Antibody serial dilutions were used to block the viral entry into the HEK293-ACE2 cells. Nonlinear regression lines were fitted for the three antibodies that showed a higher than 50% reduction of infectivity. Those antibodies were highlighted in green (Ab57), blue (Ab36), and red (Ab59). Created with BioRender.com .

Article Snippet: The antigens, produced in HEK293 cells, were obtained from SinoBiological (Beijing, China; product numbers: SARS-CoV-2 Spike RBD: 40592-V08H; SARS-CoV-2 Spike RBD-N501Y: 40592-V08H82; SARS-CoV-2 Spike RBD-E484K: 40592-V08H84; SARS-CoV-2 Spike RBD-K417N, E484K, N501Y: 40592-V08H85; SARS-CoV-2 Spike S1 HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H: 40591-V08H12).

Techniques: Neutralization, Blocking Assay, Binding Assay, Negative Control, Positive Control, Produced, Microscopy, Enzyme-linked Immunosorbent Assay, Luciferase, Infection