Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: (A) Average force profiles of WT (red), Alpha (blue), Beta (orange), Gamma (sky blue), Epsilon (green), Kappa (pink), and Delta (gray) variants as a function of the distance between the centers of mass of RBD and ACE2. (B) Initial snapshot of WT. Residues subjected to each mutation are shown as solid sticks (N501, K417, E484, L452, and T478). RBD and ACE2 are, respectively, colored in light gray and yellow. All N-glycans, water, and ions are hidden for clarity. (C) Initial snapshot of WT with clockwise 90° rotation along the normal from (B). All N-glycans are depicted in different colors. Any other residues, water, and ions are not shown for clarity.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Mutagenesis

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: Two-dimensional contact maps at D = 53 Å. (A) Interacting residue pairs between RBD WT and ACE2. RBD residues subjected to mutation are shown in colored boxes at the bottom: (B) blue for Alpha, (C) orange for Beta, and (D) green for Epsilon. The contact frequency is numbered with colors from light blue to dark blue. Dark red and yellow colors on the map, respectively, represent increased and decreased interactions between RBD and ACE2 upon mutations.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Mutagenesis

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: (A) The average number of contacts between RBD residue 501 and ACE2. (B, C) Representative snapshots at D = 53 Å of (B) Alpha variant and (C) WT. (D) Average number of contacts between RBD residue 417 and ACE2 and (E, F) their interacting residue pairs at D = 53 Å of (E) Beta and (F) Alpha variants. (G) Average number of contacts between RBD residue 478 and ACE2 and (H, I) key interaction pairs at D = 78 Å of (H) Delta and (I) Epsilon variants. The overall color scheme is the same as in Figure , and each mutated residue in each variant is shown using the same colors (i.e., red for WT, blue for Alpha, orange for Beta, green for Epsilon, and gray for Delta). Interacting residues are depicted as solid sticks, and residues losing their interactions are shown as transparent sticks. RBD and ACE2 are presented in light gray and yellow, respectively.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Variant Assay

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: Binding affinities between RBD variants and ACE2 and its comparison with the simulation results. K d is obtained from microscale thermophoresis experiments. F WT / F is a ratio, where F WT and F are the respective maximum pulling force of WT and of each variant obtained from the SMD simulations.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Binding Assay, Microscale Thermophoresis, Variant Assay

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Comparison between RBDCoV-ACE2 and a virus neutralization test (VNT). Serum samples (n=16) of pre-pandemic (n=4) and COVID-19 convalescent (n=12) individuals were measured using both assays and analyzed by linear regression. The equation of the dashed regression line is shown next to the graph. VNT results are depicted as half-maximal inhibiting serum dilutions (VNT 50 ), RBDCoV-ACE2 results are shown in percentage inhibition of ACE2 binding. Correlation analysis was performed after Spearman and the correlation coefficient r is shown.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Neutralization, Inhibition, Binding Assay

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation between SARS-CoV-2 NeutraLISA and VNT and comparison to RBDCoV-ACE2. (a) Correlation and linear regression between NeutraLISA and VNT results for pre-pandemic (n=4) and COVID-19 infected (n=12) samples. Correlation analyses were performed after Spearman and correlation coefficients r are shown. (b) Descriptive statistics of the (c) correlation between NeutraLISA and RBDCoV-ACE2. One sample from each individual (n=168) was measured using both assays correlation was calculated after Spearman. Samples were classified as being negative (non-neutralizing) if they had a value below 20% (red lines).

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Infection

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: ACE2 binding inhibition varies between RBD mutants. Violin plots showing ACE2 binding inhibition (%) of individual serum samples from 7 to 49 days post PCR (n=50, depicted as dots) against RBD mutants. Black horizontal lines represent medians. Fold-decrease of ACE2 binding inhibition in comparison to wild-type corresponds to the ratio between the medians of wild-type and the respective RBD mutant. VOC-RBDs are shown in blue. Mutations of each RBD mutant are shown in the box above the violin plot.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Mutagenesis

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation between anti-RBD IgG MFI signals and ACE2 binding inhibition (%) of serum samples from COVID-19 patients for wild-type and 11 RBD mutants. Regression analysis comparing ACE2 binding inhibition (%) and IgG responses (MFI) for wild-type and all RBD mutants included in the study. Each circle represents one sample (n=168). For longitudinal donors with more than one sample available, the sample closest to 20 days post positive PCR diagnosis was selected. The percentage next to the bracket indicates the proportion of samples with ACE2 binding inhibition ≤ 20% (in orange). Spearman’s correlation coefficient (r) is specified for every correlation.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Longitudinal analysis of ACE2 binding inhibition and anti-RBD IgG levels in Covid-19 patients. Mean ACE2 binding inhibition (%) and IgG responses (MFI) for wild-type RBD against time post positive PCR test for samples (n=149) taken from 1 to 92 days post PCR are shown (a, b). Black dots indicate mean responses with standard deviation indicated by the error bars. The same analysis is then shown for longitudinal samples of selected donors (n=6) for wild-type (c, d) and RBD delta (e, f). For all RBD mutants, mean ACE2 binding inhibition (%) and mean IgG responses (MFI) 1 to 92 days post PCR included in the study is shown (g, h). Each variant is illustrated by a different color according to the figure key.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Standard Deviation, Variant Assay

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation of anti-RBD IgG levels and ACE2 binding inhibition with SARS-CoV-2 disease severity. Bar charts showing mean ACE2 binding inhibitions (%) against wild-type and RBD delta are correlated with WHO grades for disease severity for samples 7-49 days post PCR (a, b) and ≥ 50 days post PCR (c, d). Mean anti-RBD WT IgG and anti-RBD delta IgG levels are shown for samples 7-49 days post PCR (e, f) and ≥ 50 days post PCR (g, h). Individual samples are displayed as colored dots, bars indicate the mean of the dataset with error bars representing standard deviation. Number of samples is given below the columns (n). If no samples for a group were available, the column is labeled with “n/a”. WHO grade 1 - ambulatory / no limitations of activities, 2 - ambulatory / limitation of activities, 3 - hospitalized, mild disease / no oxygen therapy, 4 - hospitalized, mild disease / mask or nasal prongs, 6 - hospitalized, severe disease / intubation + mechanical ventilation, 7 - hospitalized, severe disease / ventilation + additional organ support (pressors, RRT, ECMO), 8 – Death. The study did not contain samples of WHO grade 5.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Standard Deviation, Labeling

Journal: bioRxiv

Article Title: SARS-CoV-2 susceptibility of cell lines and substrates commonly used in diagnosis and isolation of influenza and other viruses

doi: 10.1101/2021.01.04.425336

Figure Lengend Snippet: Whole cell lysate from uninoculated Vero E6, CV-1, A549, Mv1Lu, CRFK, MDCK-NBL-2 and MDCK-SIAT1 cell lines were immunoblotted for endogenous ACE2 expression. Recombinant hACE2 (Sino Biological) was used as a positive control for detection of hACE2. 20 μg of cell lysates or 0.2 ng of recombinant hACE2 protein were loaded. β-actin was also immunoblotted from samples as a loading control.

Article Snippet: Cell lysates and recombinant ACE2 protein control (Sino Biological) were immunoblotted for ACE2 and β-actin using primary antibodies (1:500 polyclonal goat anti-human ACE2, R&D Systems, AF933; 1:1000 monoclonal mouse anti-β-Actin, Abcam, AB8226) followed by secondary antibodies (1:4000 donkey anti-goat, Abcam; 1:4000 goat anti-mouse, Biorad).

Techniques: Expressing, Recombinant, Positive Control

Journal: bioRxiv

Article Title: SARS-CoV-2 susceptibility of cell lines and substrates commonly used in diagnosis and isolation of influenza and other viruses

doi: 10.1101/2021.01.04.425336

Figure Lengend Snippet: ACE2 protein sequences from human, rhesus macaque, African green monkey, cat, dog, American mink, mouse, and chicken were aligned using MUSCLE. Residues involved in interaction with SARS-CoV-2 spike protein (based on ref ( – )) are shown using hACE2 numbering, and residues varying from hACE2 are highlighted in yellow. A gap in alignment is indicated with a dash. Percent identity to hACE2 across the entire protein is shown.

Article Snippet: Cell lysates and recombinant ACE2 protein control (Sino Biological) were immunoblotted for ACE2 and β-actin using primary antibodies (1:500 polyclonal goat anti-human ACE2, R&D Systems, AF933; 1:1000 monoclonal mouse anti-β-Actin, Abcam, AB8226) followed by secondary antibodies (1:4000 donkey anti-goat, Abcam; 1:4000 goat anti-mouse, Biorad).

Techniques:

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: a SARS-CoV-2 replication kinetics in HAE from different donors, HCoV-NL63 was used as a control ( n = 3). b Transepithelial electrical resistance (TEER in Ω cm 2 ) between the apical and basal poles was measured at each time point ( n = 3). c SARS-CoV-2 infected both ciliated cells (72 h pi) and secretory cells (72 h pi). arrows: virus particles, arrowhead: cilium, asterisk: secretory vesicle, insets dashed-line squares indicate magnification of arrowed areas. d Costaining of SARS-CoV-2 N protein (green) with ciliated cell marker β-tubulin-IV (red), goblet cell marker Muc5AC (red), club cell marker CCSP (red), and ACE2 (red) positive cells. HCoV-NL63 N protein (green) staining was used as a control (72 h pi). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Data a , b are the means ± s.d. of three independent biological replicates. Source data a – d are provided as a Source Data file.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Infection, Marker, Staining

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: Source of antibodies and dyes with work concentration for immunofluorescence.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Concentration Assay, Immunofluorescence

Journal: Nature Communications

Article Title: Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 infection

doi: 10.1038/s41467-021-22781-1

Figure Lengend Snippet: a Representative expression of angiotensin-converting enzyme-II (ACE2) in kidney tissues from COVID-19 post-mortem (case #2) was detected by immunohistochemistry. Scale bars = 100 μm. b The co-expression of ACE2 and SARS-CoV-2 NP (nucleocapsid protein) or S (spike) antigens in kidney sections from COVID-19 post-mortem (case #2), hepatitis B virus-associated membranous nephropathy (HBV-MN) and trauma victims. Arrow indicates positive tubules. Scale bars = 100 μm. Data represent one of at least three technical replication each.

Article Snippet: For immunofluorescence double-staining, the sections were incubated with primary mouse originated antibodies including anti-SARS NP antibodies (ab273434, 1:500, mouse IgG), anti-SARS S antibodies (ab273433, 1:500, mouse IgG), or anti-DP2 (sc-271898, 1:200, mouse monoclonal C-5; Santa Cruz Biotechnology) and rabbit originated antibodies including anti-SARS NP antibodies (clone ID: 019, 1:200, rabbit IgG; Sino Biological, Beijing), anti-ACE2 (clone ID: 10108-RP01, 1:100, rabbit IgG; Sino Biological) or anti-PDS (ab182141, 1:100, rabbit IgG1; Abcam) at 4 °C overnight, respectively.

Techniques: Expressing, Immunohistochemistry