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: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Identification of neutralizing antibodies with a PtY display platform. We first used our preconstructed naïve phage displayed human scFv library to screen binders with biotinylated SARS-CoV-2 RBD protein in the solution phase. After enrichment of phage binders, the scFv DNA from enriched binders was cloned into the yeast display plasmid, resulting in display of scFv on the yeast cell surface. We then performed FACS to isolate potential blocking antibodies that could prevent binding of the SARS-CoV-2 RBD to hACE2. The 0.013% gate contained blocking antibodies with high affinity toward RBD. That is, higher Y axis signal represented higher affinity to labeled RBD, whereas lower X signal represented higher potency in blocking the binding of differently labeled hACE2 to RBD. The potential blocking antibodies were sent for sequencing and transient expression. The purified antibodies were evaluated for affinity, blocking activity, biophysical properties, and virus-neutralizing activity

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Clone Assay, Plasmid Preparation, Blocking Assay, Binding Assay, Labeling, Sequencing, Expressing, Purification, Activity Assay

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characteristics of potential blocking antibodies

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Blocking Assay, Expressing, Binding Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of potential blocking antibodies. (a) Blocking assay was performed by immobilizing 1 µg/ml hACE2 on a plate. Serially diluted antibodies and biotinylated SARS-CoV-2 RBD protein were added for competitive binding to hACE2. IC 50 values were calculated with Prism V8.0 software using a four-parameter logistic curve fitting approach. (b) Epitope binning was carried out by BLI. Biotinylated SARS-CoV-2 RBD was immobilized onto the SA sensor, and a high concentration of the primary antibody was used to saturate its own binding site. Subsequently, a second antibody was applied to compete for the binding site on the SARS-CoV-2 RBD protein. Data were analyzed with Octet Data Analysis HT 11.0 software. (c) Neutralization activities of Ab2001.08 and Ab2001.10 were assessed by live virus assay. Live SARS-CoV-2 and serially diluted (3-fold) antibodies were added to VERO E6 cells. The PRNT 50 values were determined by plotting the plaque number (neutralization percentage) against the log antibody concentration in Prism V8.0 software

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Blocking Assay, Binding Assay, Software, Concentration Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of JMB2002. Binding affinity of JMB2002 for the SARS-CoV-2 RBD (a)/S1 (b) prototype and its variants was determined by BLI. JMB2002 was loaded onto the AHC sensor, and serially diluted antigens were bound to JMB2002 on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. Blocking activity was assessed using ELISA with hACE2-coated plates. A mixture of biotinylated SARS-CoV-2 RBD (c)/S1 (d) proteins and JMB2002 was added for competitive binding to hACE2. IC 50 values were calculated by Prism V8.0 software using a four-parameter logistic curve fitting approach. Values are displayed as the mean ± standard deviations from three independent experiments. (e) The pseudovirus neutralization activity of JMB2002 was evaluated using a pseudotyped SARS-CoV-2 system, which contained a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to hACE2-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. IC 50 values were calculated by plotting the inhibition rate against the log antibody concentration in Prism V8.0 software

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Binding Assay, Software, Blocking Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Luciferase, Expressing, Incubation, Infection, Inhibition, Concentration Assay

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 1 Schematic Illustration. a Preparation of rhACE2-loaded electrospun nanofiber patch by hyaluronan (HA) micro-sol electrospun and the mechanism of formation of core-shell structure. b Myocardial infarction mice model established by precise ligation of the left anterior descending (LAD) coronary artery along with the illustration of rhACE2 patch implantation. c In situ rhACE2 patch niche degrading angiotensin II (AngII) into a cardioprotective heptapeptide, Ang1–7, which counter the AngII/AT1R mediated effects by inhibiting cardiac fibrosis and cardiomyocyte apoptosis. PLLA poly(L-lactic acid), rhACE2 recombinant human angiotensin-converting enzyme 2, AT1R angiotensin II receptor type 1, PKC protein kinase C, MAPK mitogen-activated protein kinase, ECM extracellular matrix.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: Ligation, In Situ, Recombinant

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 2 Characterization of the rhACE2 patch. The general view, SEM and TEM micrographs demonstrating the core-shell structure (a), dynamic light scattering (b), water contact angle (c) and stress-strain curve (d) from PLLA-HA/ACE2 electrospun nanofibers. Scale bars represent 1 cm in general view, 20 μm in SEM and 1 μm in TEM.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques:

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 3 Biocompatibility tests in vitro. a Representative live/dead fluorescence stained by Calcein AM (green) and Propidium Iodide (red) at day 3. Scale bars represented 50 and 20 μm for zoomed pictures. b Live and dead cell count per HPF of control, PLLA, PLLA-HA/ACE2 groups. n = 4/group. c CCK-8 cell viability quantification results in different groups after 1, 2, or 3 days. n = 4/group. Data were represented as the mean ± SEM and analyzed for statistical significance using One-way ANOVA followed by Tukey’s multiple comparison test; NS no significant difference.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: In Vitro, Staining, Cell Counting, Control, CCK-8 Assay, Comparison

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 5 rhACE2 patch sustained-release activity test. a In vitro releasing curve of PLLA-HA/ACE2 fibrous membranes. b Releasing buffer collected at specified time points during release study was added into culture media of NRCMs undergone 6 h hypoxia. Representative immunofluorescence image of TUNEL (red) and nuclear visualized by DAPI (blue). Scale bars represented 50 μm. c Statistical analysis of TUNEL- positive cell counts. n = 3/group. Data were represented as the mean ± SEM and analyzed for statistical significance using One-way ANOVA followed by Tukey’s multiple comparison test; NS no significant difference; **P < 0.01 compared to the control group.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: Activity Assay, In Vitro, TUNEL Assay, Comparison, Control

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 6 rhACE2 patch preserve left ventricular function after acute myocardial infarction in vivo. a The animal research timeline design with images represented the acute MI model and successful implantation of rhACE2 patch. Echo, echocardiography. b Representative M-mode parasternal long axis view of left ventricle echocardiographic images of different groups at day 28 after LAD coronary artery ligation. Statistical analysis of left ventricular ejection fraction (c), shortening fraction (d), heart weight/body weight ratio (e), LV end-diastolic diameter (f), LV end- systolic diameter (g) and heart weight/tibial length (h) determined by echocardiography obtained from PLLA, intramyocardial injection (IM) and PLLA-HA/ACE2 treatment group at day 7, day 14 and day 28 after operation. n = 5/group. Data were represented as the mean ± SEM and analyzed for statistical significance using two-way ANOVA followed by Tukey’s multiple comparison test; NS no significant difference, *P < 0.05, **P < 0.01.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: In Vivo, Ligation, Injection, Comparison

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Antibody Table

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Sequencing, Affinity Purification

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Shedding of ACE2 in insulinoma cells. The 832/13 cells were cotransfected with 0.5 μg ACE2 expression plasmids per well and the indicated amounts of the ADAM17 expression plasmid pAd17 or pcDNA3.1(−) in two experiments, with each treatment given to duplicate wells. A–C, Western blots of cellular ACE2, ACE2 released into the cell culture medium, and cellular ADAM17, respectively. D–F, Densitometric analyses of band intensities observed in the Western blots for cellular ACE2, shed ACE2, and cellular ADAM17, respectively. Bands were normalized relative to the average intensity of bands of interest on each blot, which was set at a value of 100. *,***, P < .05, P < .001 vs 0 μg pAd17; ###, P < .001 vs 0.5 μg pAd17 for post hoc contrasts of means after an ANOVA.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Expressing, Plasmid Preparation, Western Blot, Cell Culture

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Enzymatic activity of shed ACE2. A, Maintenance medium for 832/13 cells and select culture broths from the experiments outlined in were tested for hydrolytic activity against the ACE2 substrate Mca-APK(Dnp) in the absence and presence of the ACE2-specific inhibitor DX600. B, The ACE2 activities of 832/13 maintenance medium stored at 4°C, after an overnight incubation at 37°C, and after an overnight incubation with untransfected 832/13 cells at 37°C, were compared by measuring six aliquots of each. C, The ACE2 activities of the cell culture media from the experiments outlined in were determined. **,***, P < .01, P < .001 vs 0 μg pAd17; #, ###, P < .05, P < .001 vs 0.5 μg pAd17 for post hoc contrasts of means after an ANOVA.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Activity Assay, Incubation, Cell Culture

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Overexpressed ADAM17 affects ACE2 levels. A, ACE2 and ADAM17 mRNA concentrations were determined from four experiments in which 832/13 cells were transfected with 0.5 μg/well mACE2/pcDNA3.1 and cotransfected with 2 μg/well of pAd17, pAd17E406A (an expression plasmid for inactive ADAM17), or pcDNA3.1(−). B–F, In six experiments, 832/13 cells were transfected with 4, 20, 100, or 500 ng mACE2/pcDNA3.1 per well and cotransfected with 2 μg/well of pAd17, pAd17E406A, or pcDNA3.1(−). We measured the activity (SensoLyte activity) with a SensoLyte ADAM17 activity kit (B), the activity by a more specific ADAM17 assay (C), the shed ACE2 activity (D), and the cellular ACE2 activity (E) compared with the average ACE2 activity level from five wild-type mice. We calculated the ratio of total shed ACE2 activity to total cellular ACE2 (F). G, The total cellular ACE2 and ADAM17 activities were determined at the beginning and end of a 24-hour incubation period for 832/13 cells that were cotransfected four times with 25 ng mACE2/pcDNA3.1 and 2 μg pAd17 per well. H, 832/13 cells were individually transfected with 25 ng/well mACE2/pcDNA3.1, 2 μg/well pAd17, and 2 μg/well pAd17E406A. The next day, cells were trypsinized and mixed for coculture of ACE2-expressing cells and cells overexpressing either active or inactive ADAM17. We measured ADAM17 activity, cellular ACE2 activity, and shed ACE2 activity. I, The control strengths of the amount of the ACE2 expression plasmid mACE2/pcDNA3.1 on cellular and shed ACE2 are determined as the slopes of the indicated regression lines for the natural logarithms of ACE2 activities plotted against the natural logarithms of the amount of mACE2/pcDNA3.1. Data were analyzed by an ANOVA (A–F) and t tests (H). Due to the large range in SDs for ACE2 activity measurements occurring with the different levels of the ACE2 expression plasmids, separate ANOVAs for the ACE2 parameters were conducted for each level of mACE2/pcDNA3.1(−) (D–F). *, **, ***, P < .05, P < .01, P < .001 vs. pAd17E406A.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Transfection, Expressing, Plasmid Preparation, Activity Assay, Incubation

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Effect of ADAM17-mediated ACE2 shedding on ACE2 protein stability. The 832/13 cells that were cotransfected in four experiments with 100 μg/well mACE2/pcDNA3.1 and 2 μg/well of either pAd17 or pAd17E406A were treated with cycloheximide. The total ACE2 activity (A) and ADAM17 activity (B) were measured for up to 21 hours. Exponential decay curves were fitted to the data between the 0- and 8-hour time points. The ratio of total shed ACE2 to total cellular ACE2 activity was also determined (C). *, ***, P < .05, P < .001 vs pAd17E406A for post hoc contrasts after an ANOVA.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Activity Assay

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Determination of the control strength of ADAM17 on ACE2 activity. 832/13 cells were cotransfected with 25 ng/well mACE2/pcDNA3.1 and 2 μg/well of a mix of pAd17 and pAd17E406A. The pAd17 content in the mix were 0, 0.4, 0.8, 1.2, 1.6, and 2 μg/well in four experiments and 0, 0.05, 0.1, 0.2, 0,4, and 2 μg/well in eight experiments. ADAM17 activities (A) and ACE2 activities (B) were measured. C, For estimation of transfection efficiencies, green fluorescent 832/13 cells transfected with 2 μg pEGFP-C3 and 25 ng mACE/pcDNA3.1 were determined as in the upper right panel compared with control cells in the upper left panel. To estimate cotransfection efficiency, cells cotransfected with a plasmid encoding the red fluorescent tdTomato and pAd17 (lower left panel) were compared to cells cotransfected with plasmids for eGFP and tdTomato expression (lower right panel). For the different amounts of pAd17 used in the transfection experiments, shed ACE2 (D) and cellular ACE2 (E) were plotted against the estimated ADAM17 activity of the transfected cells, ie, corrected for the transfection efficiency. Curves describing saturation kinetics were fitted to the data. F, Based on the model fits, the control strengths were calculated and plotted as a function of the ADAM17 activity of transfected cells.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Expressing

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: ACE2 and ADAM17 in mouse pancreatic islet cells. A, Dispersed islet cells were assessed for red tdTomato fluorescence. The lower panel shows a cell subpopulation with high red fluorescence that is sorted from the non-red cells. B, Quantitative RT-PCR was performed to measure ACE2, ADAM17, insulin 2, glucagon, and somatostatin mRNA for sorted red and non-red islet cells from four female Tom +/− , Ins2-Cre +/− mice. C, ACE2 activity was determined for sorted red and non-red islet cells from two female and one male Tom +/− , Ins2-Cre +/− mice. *, **, P < .05, P < .01 vs non-red cells as determined by paired t tests.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Fluorescence, Quantitative RT-PCR, Activity Assay

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Expression of ACE2 and ADAM17 in diabetes. RNA from pancreatic islets was isolated from db/db and db/m mice. There were four mice in each group, except for only three db/db mice at 12 weeks of age. The concentrations of ACE2 mRNA (A), ADAM17 mRNA (B), and insulin 2 mRNA (C) were determined. Protein extracts from pancreatic islets were isolated from db/db and db/m mice with four mice in each group. ACE2 activities (D) and ADAM17 activities (E) were measured. F, The hydrolytic activity against the ADAM17 substrate in the absence of the ADAM17 inhibitor, DPC-333, was increased in db/db mice. *, ***, P < .05, P < .001 vs db/m mice in the same age group for post hoc contrasts of means after a two-way ANOVA.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Expressing, Isolation, Activity Assay

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Effects of inhibiting ADAM17 activity on ACE2 expression. 832/13 cells were transfected in four experiments with 100 ng/well mACE2/pcDNA3.1 and cotransfected with 2 μg/well of pAd17 or pAd17E406A. After transfection, cells were incubated in the absence or presence of 1 μM DPC-333. We determined the cellular ACE2 activity (A) and calculated the ratio of total shed ACE2 activity to total cellular ACE2 (B). *, **, ***, P < .05, P < .01, P < .001 vs no DPC-333; ###, P < .001 vs pAd17 in the absence of DPC-333 for post hoc contrasts of means after an ANOVA. Pancreatic islets were isolated from C57BL/6 mice aged 4–12 months. Pools of islets from four mice each were generated for a total of three pools from male mice and two pools from female mice. Aliquots of the islets were incubated for 24 hours in RPMI 1640 medium without serum. We determined the cellular ACE2 activity (C) and calculated the ratio of total shed ACE2 activity to total cellular ACE2 (D). There were no significant differences for post hoc contrasts of means in the absence and presence of DPC-333 after an ANOVA, but the main effect for sex was significant at P < .001 for cellular ACE2 activity.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Activity Assay, Expressing, Transfection, Incubation, Isolation, Generated