Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Serum ACE2 activity is significantly correlated with SBP in stroke-alert patients and healthy young adults, but not AIS patients. Correlation graphs of ACE2 activity and SBP among stroke-alert patients (a) and healthy young adults (b) as compared to stroke patients (c). Young adult blood plasma samples in panel (b) were from a biorepository established by Wegman et al., which were obtained from research participants undergoing baseline measurements. (d) Correlation graph of ACE activity and mRS at discharge from hospital among AIS patients. ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; mRS: modified Rankin score; RFU: relative fluorescence unit; SBP: systolic blood pressure.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Clinical Proteomics, Modification, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Activity of ACE2 and ACE in serum is altered following stroke. For human serum, bar graphs are means ± SEM and represent enzyme activity levels of ACE2 (a) and ACE (c) from control, stroke-alert, or AIS patients at an average of 3.6 hours and again at 3 days after stroke. Individual differences and means ± SEM in ACE2 (b) and ACE (d) are shown. * P <0.05 versus control and † P <0.05 versus stroke-alert. ‡ P <0.05 versus AIS <6 hours. ACE: angiotensin converting enzyme; ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; RFU: relative fluorescence unit.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Control, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Predictors of acute ischemic stroke by multiple linear regression analysis.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay

Journal: medRxiv

Article Title: A multiplexed high-throughput neutralization assay reveals a lack of activity against multiple variants after SARS-CoV-2 infection

doi: 10.1101/2021.04.08.21255150

Figure Lengend Snippet: ( a ) Schematic outline of the S 3 /ACE2 neutralization assay. Anti-SARS-CoV-2 serum antibodies are monitored for their capacity in blocking the S 3 /ACE2 interaction. ACE2 binding to S protein was detected though use of a fluorescently labeled secondary antibody and the signal intensities are inversely proportional to the neutralizing potential of the anti-S protein antibodies. ( b ) Serum dilution IC 50 values were calculated for 104 healthy adult donor samples collected prior to November 2019 (pre-COVID-19 pandemic). The mean IC50 values and SD were used to establish a lower limit cutoff of 50 indicated by the dashed line (mean IC 50 of 12.5 + 4 × 9.0 SD = 50 serum dilution). ( c ) Representative concentration response curves for ten healthy donor serum samples and ( d ) ten SARS-CoV-2 seropositive donors with varying levels of anti-S protein IgG antibody. Mean ± s.d. shown in graph b . S protein / ACE2 structure was generated with PDB 7a98.

Article Snippet: Plates were agitated on a plate shaker for 60 minutes then the ACE2 mouse Fc fusion protein (Creative Biomart or produced by EPFL Protein Production and Structure Core Facility) was then added to each well at a final concentration of 1 μg/ml and agitated for a further 60 minutes.

Techniques: Neutralization, Blocking Assay, Binding Assay, Labeling, Concentration Assay, Generated

Journal: medRxiv

Article Title: A multiplexed high-throughput neutralization assay reveals a lack of activity against multiple variants after SARS-CoV-2 infection

doi: 10.1101/2021.04.08.21255150

Figure Lengend Snippet: ( a ) Cross-validation studies between S 3 /ACE2 surrogate neutralization assay and the “gold standard” SARS-CoV-2 cytopathic effect (CPE) neutralization assay. Seropositive donors (n=206) with varying levels of anti-S protein antibodies were selected for comparison of the assays. Donors consisted of COVID-19 hospitalized patients (n=31), symptomatic infected donors with RT-PCR-documentation (n=64) and other seropositive donors identified through random sampling, volunteers or contact with a confirmed SARS-CoV-2 infected individual in a Swiss population serological survey (n=111). ( b ) Correlation between the live SARS-CoV-2 virus cytopathic effect and S protein pseudotyped neutralization assays. A group of 74 samples from S protein seropositive donors were compared in the live virus CPE and S protein pseudotyped virus cell based neutralization assays. Correlation between the two neutralization assays is represented by the black dashed line and the 20 serum dilution IC 50 cutoff for positivity in the CPE assay is shown with the blue dashed line.

Article Snippet: Plates were agitated on a plate shaker for 60 minutes then the ACE2 mouse Fc fusion protein (Creative Biomart or produced by EPFL Protein Production and Structure Core Facility) was then added to each well at a final concentration of 1 μg/ml and agitated for a further 60 minutes.

Techniques: Biomarker Discovery, Neutralization, Comparison, Infection, Reverse Transcription Polymerase Chain Reaction, Sampling, Virus

Journal: medRxiv

Article Title: A multiplexed high-throughput neutralization assay reveals a lack of activity against multiple variants after SARS-CoV-2 infection

doi: 10.1101/2021.04.08.21255150

Figure Lengend Snippet: The surrogate neutralization assay was performed with two panels of S 3 coupled beads consisting of the 2019-nCov S protein and S mutations produced with one or more amino acid substitutions or deletions. ( a ) Concentration response curves of the REGN10933 therapeutic antibody evaluated against S protein mutations from three viral variants in the S 3 /ACE2 assay. ( b-c ) Serum dilutions from RT-PCR positive donors 3506 and 9504 ( b ) or 1034 and 5537 ( c ) in the S 3 -ACE2 assay with two separate panels of S 3 coupled beads. ( c ) Spider plot and heatmap showing IC 50 serum dilutions for donors 1034 and 5537 serums samples against the indicated S protein mutations found in variants of concern including B.1.1.7 and P1. In these graphs, green background corresponds to an IC 50 dilution >100 for strong blocking of the S 3 /ACE2 interaction, yellow background for moderate blocking with IC 50 dilutions from 50 to 100 and red background for low to no blocking activity against the indicated S protein mutant.

Article Snippet: Plates were agitated on a plate shaker for 60 minutes then the ACE2 mouse Fc fusion protein (Creative Biomart or produced by EPFL Protein Production and Structure Core Facility) was then added to each well at a final concentration of 1 μg/ml and agitated for a further 60 minutes.

Techniques: Neutralization, Produced, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Activity Assay, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Enhanced Cardiorenal Protective Effects of Combining SGLT2 Inhibition, Endothelin Receptor Antagonism and RAS Blockade in Type 2 Diabetic Mice

doi: 10.3390/ijms232112823

Figure Lengend Snippet: Kidney ACE2/ACE mRNA expression and enzymatic activity ratio. ( A ) ACE2/ACE mRNA expression ratio. ( B ) ACE2/ACE kidney activity ratio. ( C ) ACE2/ACE serum activity ratio. db/m : non-diabetic mice treated with vehicle. db/db : diabetic mice treated with vehicle. db/db EMP : diabetic mice treated with empagliflozin. db/db EMP + RAM : diabetic mice treated with empagliflozin and ramipril. db/db EMP + RAM + ATR : diabetic mice treated with empagliflozin, ramipril, and atrasentan. db/db ATR : diabetic mice treated with atrasentan. db/db ATR + RAM : diabetic mice treated with atrasentan and ramipril. db/db RAM : diabetic mice treated with ramipril. Factorial ANOVA main effect results are displayed next to the graph. P Diabetes : diabetes’ main effect. P EMP : empagliflozin’s main effect. P RAM : ramipril’s main effect. P ATR : atrasentan’s main effect. $ p < 0.05 vehicle-treated db/db mice vs. vehicle-treated db/m mice. * p < 0.05 any treated db/db mice compared with vehicle-treated db/db mice.

Article Snippet: In addition, two wells with 0.008 ng/μL of recombinant mouse ACE2 (3437-ZN-010, R&D Systems, Minneapolis, MN, USA) and two wells with lysis buffer alone were included as positive and negative control, respectively.

Techniques: Expressing, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Enhanced Cardiorenal Protective Effects of Combining SGLT2 Inhibition, Endothelin Receptor Antagonism and RAS Blockade in Type 2 Diabetic Mice

doi: 10.3390/ijms232112823

Figure Lengend Snippet: Proposed modulation of intrarenal renin angiotensin system (RAS) in db/db mice treated with empagliflozin, atrasentan, ramipril, or their combination. Treatment with empagliflozin ( B ) or atrasentan ( D ) alone did not significantly change intrarenal RAS but ramipril alone increases kidney renin levels ( C ) when compared to vehicle-treated db/db mice ( A ). However, either empagliflozin or atrasentan combined with ramipril (( E , F ), respectively) increased renin activity and directed the intrarenal RAS towards the Ang (1–7) protective axis. These effects were highest in the combination therapy of empagliflozin, atrasentan and ramipril ( G ). Ang I : angiotensin I. Ang II : angiotensin II. Ang (1-7) : angiotensin (1-7). ACE : angiotensin converting enzyme. ACE2 : angiotensin-converting enzyme 2. db/db : diabetic mice treated with vehicle. db/db EMP : diabetic mice treated with empagliflozin. db/db EMP + RAM : diabetic mice treated with empagliflozin and ramipril. db/db EMP + RAM + ATR : diabetic mice treated with empagliflozin, ramipril, and atrasentan. db/db ATR : diabetic mice treated with atrasentan. db/db ATR + RAM : diabetic mice treated with atrasentan and ramipril. db/db RAM : diabetic mice treated with ramipril.

Article Snippet: In addition, two wells with 0.008 ng/μL of recombinant mouse ACE2 (3437-ZN-010, R&D Systems, Minneapolis, MN, USA) and two wells with lysis buffer alone were included as positive and negative control, respectively.

Techniques: Activity 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

Journal: Allergy

Article Title: Development and preclinical evaluation of virus‐like particle vaccine against COVID‐19 infection

doi: 10.1111/all.15091

Figure Lengend Snippet: Immunoprotective activity of the VLP vaccine in K18‐hACE2 transgenic mice. K18‐hACE2 transgenic mice ( n = 10/group) were subcutaneously immunized with 2 µg (low dose; LD) or 8 µg (high dose; HD) of the VLP vaccine on days 0 and 14. Two weeks after booster injection, (A) RBD‐specific IgG, IgG1, IgG2c antibody titers were determined by ELISA and (B) neutralizing antibody titers against the authentic Wuhan strain and the B.1.1.7. Alpha variant were determined. Groups were compared by one‐way ANOVA with Dunnett's multiple comparisons test. * P < .05, ** P < .01, *** P < .001, **** P < .0001. On day 21 after booster, mice were challenged intranasally on 3 consecutive days with 50 µl of 1 × 10 5 pfu/mouse of SARS‐CoV‐2 (Wuhan strain). Lungs were collected 7 days after last virus instillation. (C) Infectious virus loads in lung homogenates were assessed by qRT‐PCR against the nucleocapsid (NC1 and NC2). Bars represent the mean virus load ( n = 10/group) as 1/ct values. Comparisons were performed by unpaired Student's t test; * P < .05, ** P < .01, *** P < .001, **** P < .0001. (D) Histological micrographs showing healthy (first column), placebo (second column), low‐dose vaccine (third column), and high‐dose vaccine (fourth column) groups. (A–D) Hematoxylin‐eosin (H&E), areas marked green shows inflamed parts of the lungs; (E–L) H&E, 20×; M‐P, Gomori Trichrome (GT), 40×. a, alveoli; b, bronchiole; v, blood vessel; blue arrow, protein debris; red arrow, hyaline membrane. (E) Histomorphometric measurements. The descriptive statistics were presented as median and interquartile range in all graphs except for inflamed area percent (mean ± SD). Statistical significance ( P < .05): a, compared to healthy group; b, compared to placebo group; c, compared to low‐dose vaccine group, d, compared to high‐dose vaccine group. Nonparametric variables were compared between groups using Kruskal‐Wallis test. Pairwise comparisons were made with Dunn's test. Parametric variables were compared in multiple groups using one‐way analysis of variance. Pairwise comparisons were performed with the Tukey test

Article Snippet: Carboxyl modified latex beads (2 mg of 4% (w/v), Thermo Fisher Scientific) were coated with 5 μg recombinant hACE2 (ProSci) or anti–IL‐1β in PBS and blocked in 5% BSA in PBS.

Techniques: Activity Assay, Transgenic Assay, Injection, Enzyme-linked Immunosorbent Assay, Variant Assay, Virus, Quantitative RT-PCR, Membrane