Journal: Immunology and Cell Biology

Article Title: The vaccinia‐based Sementis Copenhagen Vector coronavirus disease 2019 vaccine induces broad and durable cellular and humoral immune responses

doi: 10.1111/imcb.12539

Figure Lengend Snippet: Spike‐specific antibody and SARS‐CoV‐2 neutralization responses following SCV‐S vaccination. (a) S1 and S2 subunit endpoint IgM and IgG ELISA titers determined from serum of female C57BL/6J mice ( n = 3) at the indicated times after a single vaccination with 10 7 PFU of SCV‐S, with (b) ratio of S1‐ and S2‐specific IgG2c to IgG1 endpoint ELISA titers determined 28 days after vaccination. (c) S1 IgG ELISA binding titers (left panel) and cPass neutralization titers (right panel) in outbred ARC(s) and inbred C57BL/6J female mice ( n = 5) 21 days after a single vaccination with 10 7 PFU of SCV‐S or vector control, with (d) durability of response in the outbred cohort shown by S1‐specific endpoint IgG ELISA titers (left panel) and neutralization titers (right panel) at the indicated times. (e) S1‐specific IgG ELISA (left panel) and neutralization titers (right panel) 50 days after a single‐dose (day 0) or prime‐boost (day 0 and 28) vaccination of female C57BL/6J mice ( n = 5) with 10 7 PFU SCV‐S or control vector, with (f) neutralizing activity in ACE2 and RBD blocking ELISA, and (g) neutralizing activity (IC 80 titers) against lenti‐SARS‐CoV‐2‐S pseudoviruses bearing spike protein from the Wuhan reference strain, the alpha or beta variant, shown for prime‐boost samples. (h) Neutralization titers in young (6–8 weeks old; n = 5) and middle‐aged (9–10 months old; n = 10) C57BL/6J mice at the indicated times after prime‐boost vaccination with 10 7 PFU SCV‐S. Results shown are representative of four independent experiments (indicated above) with binding and neutralizing antibody levels comparable at similar doses and time points across all experiments. Symbols represent individual mice and bars show the mean ± s.e.m. from independent experiments. Data were log transformed and statistical significance determined using Brown–Forsythe and Welch ANOVA with Dunnett T3 multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ACE2, angiotensin‐converting enzyme‐2; IC 80 , 80% inhibitory concentration; Ig, immunoglobulin; ns, not significant; PFU, plaque‐forming units; RBD, receptor‐binding domain; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; SCV‐S, Sementis Copenhagen Vector spike protein.

Article Snippet: ACE2‐HEK293 recombinant cell line (catalog number 79951; BPS Bioscience, San Diego, CA, USA) was maintained in Dulbecco’s Modified Eagle Medium supplemented (Sigma Aldrich, MO, USA) with 10% fetal bovine serum, 2 mM l ‐glutamine and penicillin–streptomycin.

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Binding Assay, Plasmid Preparation, Activity Assay, Blocking Assay, Variant Assay, Transformation Assay, Concentration Assay

Journal: eLife

Article Title: A mosaic-type trimeric RBD-based COVID-19 vaccine candidate induces potent neutralization against Omicron and other SARS-CoV-2 variants

doi: 10.7554/eLife.78633

Figure Lengend Snippet: ( A ) Schematic illustration of the designed mos-tri-RBD. In mos-tri-RBD, three heterologous RBDs were connected end to end into a single chain and co-assembled into a trimeric structure. For the three RBDs, one was derived from the Omicron (BA.1) variant (green color), and the other two were artificially designed harboring the key immune-evasion-related mutations that emerged in SARS-CoV-2 variants, in which one contained the mutations of K417N, L452R, T478K, F490S, and N501Y (cyan color), and the other one contained K417T, S477N, and E484K (blue color). These mutations are highlighted in the red ball-and-stick model in the figure. Each RBD subunit in mos-tri-RBD was composed of the residues 319–537 from the spike protein. The dotted curves in the figure represent the direct connection between the C-terminus of the former RBD and the N-terminus of the latter RBD. The schematic structure of mos-tri-RBD was drawn by Chimera software based on the PDB file with accession number 6zgi. ( B ) SDS-PAGE analysis of the recombinant mos-tri-RBD. ( C ) Concentration-dependent binding ability of mos-tri-RBD with an RBD-specific monoclonal neutralizing antibody MM117 tested using ELISA. ( D ) Binding avidity of mos-tri-RBD with the receptor hACE2 measured using SPR assay. In this figure, different curves represent different concentrations of analyte (top to bottom: 263.70 ng/ml, 131.85 ng/ml, 65.93 ng/ml, 32.96 ng/ml, and 16.48 ng/ml). Both the original (color curves) and fitted (black curves) data are displayed. Figure 1—source data 1. The raw files of SDS-PAGE results. Figure 1—source data 2. Concentration-dependent binding ability of mos-tri-RBD with the antibody MM117.

Article Snippet: Peptide, recombinant protein , Recombinant hACE2 protein (mammalian cell-expressed) , Sino Biological Inc, China , Cat#10108-H08H , .

Techniques: Derivative Assay, Variant Assay, Software, SDS Page, Recombinant, Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay

Journal: eLife

Article Title: A mosaic-type trimeric RBD-based COVID-19 vaccine candidate induces potent neutralization against Omicron and other SARS-CoV-2 variants

doi: 10.7554/eLife.78633

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Recombinant hACE2 protein (mammalian cell-expressed) , Sino Biological Inc, China , Cat#10108-H08H , .

Techniques: Virus, Control, Isolation, Enzyme-linked Immunosorbent Assay, Recombinant, Plasmid Preparation, Expressing, Purification, Adjuvant, Produced, Software

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Reporter gene fusion assay. Donor cells (293T) were co-transfected with plasmids encoding the T7 polymerase together with either the empty vector (negative control) or a viral envelope protein i.e., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or vesicular stomatitis virus glycoprotein (VSV-G). The viral envelope proteins contain authentic signal peptides and transmembrane domains to target to the plasma membrane. Although the SARS-CoV-2 spike protein was localized to the endoplasmic reticulum-Golgi intermediate compartment, leaky transport would still target a proportion of the spike protein to the plasma membrane. Target cells (either 293T-ACE2 or 293T-ACE2-TMPRSS2) were co-transfected with two reporter genes expressing the luciferase protein under the T7 promoter and the internal control, β-galactosidase under the CMV promoter. Binding of the spike protein with the angiotensin-converting enzyme 2 (ACE2) protein will trigger fusion of the donor and target cells. Mixing of cytoplasmic contents enabled the T7 polymerase from the donor cells to bind to the T7 promoter in the target cells to drive transcription of the luciferase gene. The transcript was then translated from the encephalomyocarditis (EMCV) internal ribosome entry site (IRES) element to produce a luciferase read-out. Fusion was measured as a ratio of the luciferase activity to the β-galactosidase activity.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Single Vesicle Fusion Assay, Transfection, Plasmid Preparation, Negative Control, Expressing, Luciferase, Binding Assay, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Vesicular stomatitis virus glycoprotein triggers universal acidic pH fusion. (A,B) Donor 293T cells were transfected with empty vector (negative control) or the vesicular stomatitis glycoprotein (VSV-G) together with a plasmid encoding the T7 polymerase. 19.000 donor cells were co-cultured with 19,000 target cells (A) (293T, 293T-ACE2, 293T-ACE2-TMPRSS2) or (B) Vero cells transfected with the luciferase and β-galactosidase reporter genes. The co-cultures were treated as indicated. Fusion activity was measured as luciferase activity normalized against β-galactosidase activity and expressed as a ratio to their respective vector control in the respective cell type and fusion condition. Data are presented as mean +/− SD of 3 repeats. * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) Donor 293T cells transfected with 0.15 μg of VSV-G (left and right) or 0.074 μg of VSV-G (middle) were co-cultured with target 293T cells and fusion triggered by incubating with pH7.4 (left) or pH5 (middle and right) fusion buffers. Cells were fixed and stained with methylene blue. A syncytium was outlined. Fusion index was calculated as (S–N)/T where S = number of nuclei in syncytia; N = number of syncytia; T = total number of nuclei. All photomicrographs are of the same magnification and scale.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Transfection, Plasmid Preparation, Negative Control, Cell Culture, Luciferase, Activity Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Severe acute respiratory syndrome coronavirus 2 spike protein triggers fusion in different cell types and fusion conditions. (A,B) Donor 293T cells were transfected with empty vector (negative control) or the severe acute respiratory syndrome coronavirus 1 and 2 spike proteins (SARS-1-S, SARS-2-S), respectively together with a plasmid encoding the T7 polymerase. 19,000 donor cells were co-cultured with 19,000 target cells (A) (293T, 293T-ACE2, 293T-ACE2-TMPRSS2) or (B) Vero cells transfected with the luciferase and β-galactosidase reporter genes. The co-cultures were treated as indicated. Fusion activity was measured as luciferase activity normalized against β-galactosidase activity and expressed as a ratio to their respective vector control in the respective cell type and fusion condition. Data on the vesicular stomatitis virus glycoprotein (VSV-G) from are included for comparison purpose. Data are presented as mean +/− SD of 3 repeats. (C) Donor 293T cells transfected with an empty vector or serial doses of the plasmid expressing the SARS-2-S, as indicated, were co-cultured with target 293T-ACE2 cells under various fusion condition ( n = 1). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Transfection, Plasmid Preparation, Negative Control, Cell Culture, Luciferase, Activity Assay, Expressing

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Syncytia formation is enhanced by TMPRSS2 or trypsin. 19,000 target cells (293T-ACE2, 293T-ACE2-TMPRSS2) transfected with the luciferase and β-galactosidase reporter genes were co-cultured with 19,000 donor 293T cells transfected with an empty vector or the severe acute respiratory syndrome coronavirus 2 spike protein (SARS-2-S) together with a plasmid encoding the T7 polymerase. The co-cultures were treated as indicated. Photomicrographs illustrate intact single cells and syncytia (arrowheads). Bright-field images are of the same magnification x100 and scale and from the same repeat.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Transfection, Luciferase, Cell Culture, Plasmid Preparation

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Specificity of different modes of fusion. (A) 19,000 target cells (293T-ACE2, 293T-ACE2-TMPRSS2) transfected with the luciferase and β-galactosidase reporter genes were pre-incubated with 10 μg/ml of anti-spike monoclonal antibody (ACROB SPD-M128), 75 μg/ml soybean trypsin inhibitor (SBTi), 10 μg/ml leupeptin or 10 μM of individual drugs before co-cultured with 19,000 donor 293T cells transfected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins (SARS-2-S) together with a plasmid encoding the T7 polymerase. The co-cultures were treated as indicated. Fusion activity was measured as luciferase activity normalized against β-galactosidase activity and expressed as a ratio to the solvent control in the respective cell type and fusion condition which is set as 1. Data are presented as mean+/-SD of >3 repeats. (B) Photomicrographs of fusion cell morphology. 50,000 target cells (293T-ACE2, 293T-ACE2-TMPRSS2) pre-incubated with 28 μg/ml anti-spike monoclonal antibody (Sino Biol, 40592-R001) or 10 μM of individual drugs were co-cultured with 50,000 donor 293T cells transfected with an empty vector or the SARS-CoV-2 spike protein. The co-cultures were treated as indicated. Cells were fixed and stained with methylene blue. Bright-field images are of the same magnification x100 and scale. Fields of entire or mostly syncytia formation are framed with red squares. Fields of entire or mostly intact cells are framed with orange squares. Scattered syncytia are circled or outlined red and labelled S. Scattered intact cells are circled or outlined orange and labelled C. Sporadic intact cells and syncytia are marked by orange and red arrowheads, respectively. (C) Viability was measured by XTT assays in fusion cells (293T + 293T-ACE2 or 293T + 293T-ACE2-TMPRSS2) under physiological pH and treated with compounds as in (A) . Data are expressed as % viability to solvent control which is set as 100%. Data are presented as mean+/-SD of three repeats. (D) Negative screen. One of the three cell types: 293T, 293T-ACE2 and 293T-ACE2-TMPRSS2 (30,000 cells) was co-transfected with plasmids encoding the T7 polymerase, luciferase and β-galactosidase reporter genes and then treated with compounds as in (A) . Luciferase, β-galactosidase and the luciferase/β-galactosidase ratio are presented as ratios to the solvent controls which are set as 1. Data are presented as mean+/-SD of three repeats * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Transfection, Luciferase, Incubation, Cell Culture, Plasmid Preparation, Activity Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Heatmap of drug inhibition profiles of different modes of fusion. 19,000 target cells (293T-ACE2, 293T-ACE2-TMPRSS2) transfected with the luciferase and β-galactosidase reporter genes were pre-incubated with 10 μM of individual drugs before co-cultured with 19,000 donor 293T cells transfected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins together with a plasmid encoding the T7 polymerase. The co-cultures were treated as indicated. Fusion activity was measured as luciferase activity normalized against β-galactosidase activity and expressed as a ratio to the solvent control in the respective cell type and fusion condition which is set as 1. Data are presented as mean of 3 repeats. Viability was measured by XTT assays in fusion cells (293T + 293T-ACE2 or 293T + 293T-ACE2-TMPRSS2) under physiological pH and expressed as % viability to solvent control which is set as 100%. Data are presented as mean of three repeats. Data outside the range are depicted as dark purple.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Inhibition, Transfection, Luciferase, Incubation, Cell Culture, Plasmid Preparation, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Negative screen. One of the three cell types: 293T, 293T-ACE2 and 293T-ACE2-TMPRSS2 (30,000 cells) was co-transfected with plasmids encoding the T7 polymerase, luciferase and β-galactosidase reporter genes and then treated with 10 μM of individual drugs. Luciferase, β-galactosidase and the luciferase/β-galactosidase ratio are presented as ratios to the solvent controls which are set as 1. Data are presented as mean +/− SD of three repeats. * p < 0.05

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Transfection, Luciferase

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Fusion cell morphology. 50,000 293T-ACE2-TMPRSS2 pre-incubated with 10 μM of individual drugs were co-cultured with 50,000 donor 293T cells transfected with an empty vector or the SARS-CoV-2 spike protein (S2). 293T-ACE2 cells treated with tenofovir disoproxil fumarate under physiological pH was displayed at the bottom right corner; otherwise, all images represent fusion of 293T-ACE2-TMPRSS2 cells. Cells were fixed and stained with methylene blue. Bright-field images are of the same magnification x100 and scale. Fields of entire or mostly syncytia formation are framed with red squares. Fields of entire or moslty intact cells are framed with orange squares. Scattered syncytia are circled or outlined red and labelled S. Scattered intact cells are circled or outlined orange and labelled C. Sporadic intact cells and syncytia are marked by orange and red arrowheads, respectively. Drug images are from PubChem.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Incubation, Cell Culture, Transfection, Plasmid Preparation, Staining

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Rimonabant and anidulafungin inhibit ACE2-spike binding. Enzyme-linked immunosorbent assay of spike neutralizing antibody (S NAb) and drug inhibition of ACE2-spike binding. Data are presented as % binding of their respective solvent control and represent mean +/− SD of 2 repeats.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Rimonabant, rucaparib, carvedilol and anidulafungin inhibit SARS-CoV-2 infection of TMPRSS2 cells. Mouse leukaemia virus pseudotyped with the spike protein (S) from severe acute respiratory syndrome coronavirus (SARS-1-S) and SARS-CoV-2 (SARS-2-S) was used to infect 293T-ACE2-TMPRSS2 cells, in a 96-well plate for 48 h in the presence of the drug, as indicated, with 1 h pre-treatment. Infectivity was measured as luciferase activity and expressed as % infectivity versus infected, solvent control. Viability was measured by XTT assays in un-infected samples and expressed as % viability versus solvent control. Data are presented as a heat map of the mean of three repeats.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Infection, Luciferase, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Pan-coronaviral and pan-viral fusion inhibitors. Mouse leukaemia virus pseudotyped with glycoprotein from vesicular stomatitis virus (VSV-G) and spike protein (S) from severe acute respiratory syndrome coronavirus (SARS-1-S), SARS-CoV-2 (SARS-2-S) and Middle East respiratory syndrome coronavirus (MERS-S), was used to infect 293T-ACE2 (for SARS-1-S, SARS-2-S, VSV-G) or Huh-7 (for MERS-S) cells, in a 96-well plate for 48 h in the presence of the drug, as indicated, with 1 h pre-treatment. Infectivity was measured as luciferase activity and expressed as % infectivity versus infected, solvent control. Viability was measured by XTT assays in un-infected samples and expressed as % viability versus solvent control. Data are presented as a heat map of the mean of three repeats.

Article Snippet: Proteins from equal number of cells together with recombinant ACE2 protein (BIOSS) or spike protein (BEI Resources NR-53937) standards were separated on TGX Stain-Free SDS-PAGE gel (Bio-Rad), transferred to polyvinylidene difluoride membrnaes (Millipore), blocked in 5% semi-skimmed milk (Marvel) in 0.1% Tween 20 (Sigma)/TBS (50 mM Tris pH 7.4, 150 mM NaCl) before being probed against primary and HRP-conjugated secondary antibodies in blocking buffer.

Techniques: Infection, Luciferase, Activity Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Biological Evaluation of Honokiol Derivatives Bearing 3-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)oxazol-2(3H)-ones as Potential Viral Entry Inhibitors against SARS-CoV-2

doi: 10.3390/ph14090885

Figure Lengend Snippet: Antiviral properties of honokiol derivatives 6a-p against the entry of SARS-CoV-2 spike pseudovirus into HEK-293T-ACE2 h cells.

Article Snippet: The binding kinetics of compounds with the protein of recombinant histidine-tagged human ACE2 (Sino Biological, Beijing, China) were analyzed by bio-layer interferometry on an Octet system (Octet RED96, ForteBio, Fremont, CA, USA) [ , ].

Techniques:

Journal: Pharmaceuticals

Article Title: Synthesis and Biological Evaluation of Honokiol Derivatives Bearing 3-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)oxazol-2(3H)-ones as Potential Viral Entry Inhibitors against SARS-CoV-2

doi: 10.3390/ph14090885

Figure Lengend Snippet: Effect of honokiol, representative 6a and 6p on the entrance of SARS-CoV-2 pseudovirus into HEK-293T-ACE2 h cells. The luciferase luminescence value of the pseudovirus control was defined as 100%. The values of luminescence of either antibody inhibitor-treated or compound-treated groups were normalized accordingly. Results were expressed as mean ± standard error (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with pseudovirus control group. The experiments were repeated at least three times.

Article Snippet: The binding kinetics of compounds with the protein of recombinant histidine-tagged human ACE2 (Sino Biological, Beijing, China) were analyzed by bio-layer interferometry on an Octet system (Octet RED96, ForteBio, Fremont, CA, USA) [ , ].

Techniques: Luciferase

Journal: Pharmaceuticals

Article Title: Synthesis and Biological Evaluation of Honokiol Derivatives Bearing 3-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)oxazol-2(3H)-ones as Potential Viral Entry Inhibitors against SARS-CoV-2

doi: 10.3390/ph14090885

Figure Lengend Snippet: Binding curves of 6p and 6a with human ACE2 and SARS-CoV-2 spike RBD proteins. ( A ) The interaction of SARS-CoV-2 spike RBD (0.03125, 0.0625, 0.125, 0.25, 0.5 and 1 nM) with human ACE2 protein. ( B ) Compound 6p (0.3125, 0.625, 1.25, 2.5, 5 and 10 µM) binding to human ACE2 protein. ( C ) Compound 6a (6.25, 12.5, 25, 50, 100 and 200 µM) binding to human ACE2 protein. ( D ) Honokiol (3.125, 6.25, 12.5, 25 and 50 µM) binding to human ACE2 protein. ( E ) The interaction of 6p (3.125, 6.25, 12.5, 25, 50 and 100 µM) with spike RBD protein.

Article Snippet: The binding kinetics of compounds with the protein of recombinant histidine-tagged human ACE2 (Sino Biological, Beijing, China) were analyzed by bio-layer interferometry on an Octet system (Octet RED96, ForteBio, Fremont, CA, USA) [ , ].

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Biological Evaluation of Honokiol Derivatives Bearing 3-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)oxazol-2(3H)-ones as Potential Viral Entry Inhibitors against SARS-CoV-2

doi: 10.3390/ph14090885

Figure Lengend Snippet: Equilibrium dissociation constant (K D ) values of 6a , 6p, and honokiol with human ACE2 protein by BLI binding assay and their binding energy by a molecular docking study.

Article Snippet: The binding kinetics of compounds with the protein of recombinant histidine-tagged human ACE2 (Sino Biological, Beijing, China) were analyzed by bio-layer interferometry on an Octet system (Octet RED96, ForteBio, Fremont, CA, USA) [ , ].

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Biological Evaluation of Honokiol Derivatives Bearing 3-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)oxazol-2(3H)-ones as Potential Viral Entry Inhibitors against SARS-CoV-2

doi: 10.3390/ph14090885

Figure Lengend Snippet: Docking simulation of 6p and honokiol in the active sites of human ACE2 receptor (PDB code: 1R4L). ( A ) 3D binding models of 6p (gray) and honokiol (green) with human ACE2. ( B ) 2D binding model of 6p with human ACE2. ( C ) 2D binding model of honokiol with human ACE2.

Article Snippet: The binding kinetics of compounds with the protein of recombinant histidine-tagged human ACE2 (Sino Biological, Beijing, China) were analyzed by bio-layer interferometry on an Octet system (Octet RED96, ForteBio, Fremont, CA, USA) [ , ].

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Biological Evaluation of Honokiol Derivatives Bearing 3-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)oxazol-2(3H)-ones as Potential Viral Entry Inhibitors against SARS-CoV-2

doi: 10.3390/ph14090885

Figure Lengend Snippet: Binding activity of SARS-CoV-2 spike RBD with ACE2 protein in the absence (control) and presence of honokiol and 6p (12.5, 25 and 50 µM) by a competitive ELISA assay. A SARS-CoV-2 antibody inhibitor (1 nM) was utilized as a positive control. The OD value of the control group was defined as 100%. The OD values of either antibody inhibitor-treated or compound-treated groups were normalized accordingly. Results were expressed as mean ± standard error (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with the control group. The experiments were repeated three times.

Article Snippet: The binding kinetics of compounds with the protein of recombinant histidine-tagged human ACE2 (Sino Biological, Beijing, China) were analyzed by bio-layer interferometry on an Octet system (Octet RED96, ForteBio, Fremont, CA, USA) [ , ].

Techniques: Binding Assay, Activity Assay, Competitive ELISA, Positive Control

Journal: Biomarker Insights

Article Title: Serum ACE2 Level is Associated With Severe SARS-CoV-2 Infection: A Cross-Sectional Observational Study

doi: 10.1177/11772719221125123

Figure Lengend Snippet: Baseline ACE2 level, demographical, clinical characteristics of the Control group and the COVID-19 group.

Article Snippet: ACE2 was measured in 100 µL of serum samples in duplicates using the human ACE2 DuoSet enzyme-linked immunosorbent assay plates (R&D Systems, Minneapolis, USA; DY933-05, lot: P266918) in duplicates according to the manufacturer’s instructions.

Techniques: Control

Journal: Biomarker Insights

Article Title: Serum ACE2 Level is Associated With Severe SARS-CoV-2 Infection: A Cross-Sectional Observational Study

doi: 10.1177/11772719221125123

Figure Lengend Snippet: Association of ACE2 level with Age and Days of hospital admission the Control group and the COVID-19 group.

Article Snippet: ACE2 was measured in 100 µL of serum samples in duplicates using the human ACE2 DuoSet enzyme-linked immunosorbent assay plates (R&D Systems, Minneapolis, USA; DY933-05, lot: P266918) in duplicates according to the manufacturer’s instructions.

Techniques: Control

Journal: Biomarker Insights

Article Title: Serum ACE2 Level is Associated With Severe SARS-CoV-2 Infection: A Cross-Sectional Observational Study

doi: 10.1177/11772719221125123

Figure Lengend Snippet: Comparison of ACE2 level between Control and COVID-19 group in association with different clinical parameters.

Article Snippet: ACE2 was measured in 100 µL of serum samples in duplicates using the human ACE2 DuoSet enzyme-linked immunosorbent assay plates (R&D Systems, Minneapolis, USA; DY933-05, lot: P266918) in duplicates according to the manufacturer’s instructions.

Techniques: Comparison, Control

Journal: Biomarker Insights

Article Title: Serum ACE2 Level is Associated With Severe SARS-CoV-2 Infection: A Cross-Sectional Observational Study

doi: 10.1177/11772719221125123

Figure Lengend Snippet: Association of ACE2 level with variable clinical parameters in COVID-19 group.

Article Snippet: ACE2 was measured in 100 µL of serum samples in duplicates using the human ACE2 DuoSet enzyme-linked immunosorbent assay plates (R&D Systems, Minneapolis, USA; DY933-05, lot: P266918) in duplicates according to the manufacturer’s instructions.

Techniques: Significance Assay

Journal: iScience

Article Title: Activation of limbal epithelial proliferation is partly controlled by the ACE2-LCN2 pathway

doi: 10.1016/j.isci.2024.110534

Figure Lengend Snippet:

Article Snippet: Rabbit anti-Ace2 , Proteintech , Cat:# 21115-1-AP; RRID: AB_10732845.

Techniques: Virus, Recombinant, Bicinchoninic Acid Protein Assay, RNA Sequencing, Software