Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a – c , Based on simulations of RBD-bound ACE2, new polar interactions were identified between the ACE2.v2.4 (orange) and RBD (yellow) loops (red) ( a ). ACE2 mutations are presented as sticks colored cyan with nearby RBD residues as sticks colored yellow. New polar interactions between ACE2.v2.4 and RBD loops 1 ( b ) and 2 ( c ) are indicated by the dotted blue lines. d , MSM-weighted distance distributions of newly formed hydrogen bonds. For each interacting pair, the ACE2.v2.4 residue is listed first and the RBD residue is listed second. e , Probabilities for stable hydrogen bond interactions using a 4-Å distance criterion between accepter and donor. f , Root mean square fluctuation (RMSF) of RBD residues when bound to WT (cyan) or v2.4 (orange) ACE2 receptors. The RBD regions that interface with ACE2 are shaded gray. The absolute difference in RMSF between WT and v2.4 proteins is shown in black. Distance distributions, hydrogen bond probabilities and RMSF calculations were based on 40,000 frames from the simulations. Frames were selected based on the MSM stationary probability to represent the entire conformational ensemble. The error bars represent the 95% confidence intervals calculated from 20 bootstrapped samples.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Residue

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a , mRNA expression of ACE2 (NM_001371415.1) and TMPRSS2 (NM_001135099) in human lung epithelial A549 cells. A549 cells stably expressing hACE2 and hLMVECs were analyzed by one-step RT–PCR (top) and real-time PCR (bottom). Relative expression was normalized to glyceraldehyde 3-phosphate dehydrogenase expression. b , Cultured hACE2-A549 cells, A549 cells and hLMVECs were preincubated with sACE2 2 -IgG1 or sACE2 2 .v2.4-IgG1 at 5 µg ml −1 or 25 µg ml −1 for 1 h. SARS-CoV-2 pseudovirus (MOI = 0.1) was added to the cells, which were collected at 24 h. Virus entry was evaluated by luciferase activity. n = 4 replicates. c , A dose of 10 mg kg −1 sACE2 2 -IgG1, sACE2 2 .v2.4-IgG1 or buffer (PBS + 0.2% BSA) was administrated intravenously into K18-hACE2 transgenic mice for 30 min before SARS-CoV-2 pseudovirus (10 6 PFU) intraperitoneal injection. Tissue lysates were prepared at 24 h and virus entry in the selected organs was evaluated by luciferase activity. Buffer was applied as the control group, n = 4. d – g , K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 isolate WA-1/2020 at 1 × 10 4 PFU. Mice received control PBS or sACE2 2 .v2.4-IgG1 10 mg kg −1 via intravenous injection 12 h before inoculation. Mice were observed for survival ( d ) and body weight ( e ), n = 5. EBA as a marker of pulmonary endothelial permeability ( f ) and lung wet/dry ratio ( g ) as a measure of lung edema were quantified. Data are presented as the mean ± s.e.m., n = 4. b , c , f , g , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Expressing, Stable Transfection, One Step RT-PCR, Real-time Polymerase Chain Reaction, Cell Culture, Virus, Luciferase, Activity Assay, Transgenic Assay, Injection, Control, Marker, Permeability

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a – d , sACE2 carrying the v2.4 mutations has increased S binding compared with WT sACE2. Human Expi293F cells expressing myc-tagged S from 4 SARS-CoV-2 variants (Wuhan ( a ), B.1.1.7/Alpha ( b ), B1.351/Beta ( c ) and P.1/Gamma ( d )) were incubated with monomeric sACE2-8 h (black) or dimeric sACE2 2 -IgG1 (gray); bound protein was detected by flow cytometry. WT ACE2 proteins are shown as broken lines, v2.4 proteins are shown as solid lines. n = 3 independent replicates; data are shown as the mean ± s.e.m. e – i , Binding of sACE2 2 .v2.4-IgG1 is comparable to clinically effective mAbs. Binding of mAbs versus sACE2 2 .v2.4-IgG1 to the S proteins of SARS-CoV-2 VOCs (B.1.1.7/Alpha ( e ), B1.351/Beta ( f ), B.1.617.2/Delta ( g ) and P.1/Gamma ( h )) and S protein of SARS-CoV-1 ( i ), as measured by flow cytometry. n = 3 independent replicates; data are shown as the mean ± s.e.m.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: Phylogenetic tree of bat ACE2 protein sequences with mammalian or avian ACE2 sequences. Phylogenetic trees were constructed by aligning CVB and PB ACE2 protein sequence with previously studied mammalian or bat ACE2 sequences. Alignments were performed using the Jukes–Cantor genetic distance model, tree was built using neighbor-joining algorithm using Chicken-ACE2 as an outgroup since it does not bind to SC2 S protein. Consensus tree was generated by resampling with 500 bootstraps. Branch labels indicate substitutions per site. Scale bar is shown.

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Construct, Sequencing, Generated

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: CVB-ACE2 and PB-ACE2 cells support infection of SC2 variants. Line graphs show log 10 TCID 50 titers/mL of WA1, Delta, Lambda, and Omicron lineage SC2 viruses in CVB-ACE2/hTMPRSS2 ( A ), PB-ACE2/hTMPRSS2 expressing DF-1 cells ( B ) and DF-1 cells expressing human ACE2 and TMPRSS2 ( C ). Data represent mean ± SD from three independent experiments for each time point. Statistical comparisons were conducted with 2-way ANOVA using repeated measures with Geisser–Greenhouse correction and Tukey multiple comparisons test with individual variances computed for each comparison. Lines with different lowercase letters indicate statistically significant differences ( p < 0.05). Titers are indicated on Y-axis and time points of infection are indicated on the X-axis. CVB = Common vampire bat (Desmodus rotundus ), PB = Pallid bat ( Antrozous pallidus ), Hs = Homo sapiens .

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Infection, Expressing, Comparison

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: Immunofluorescence microscopy of SC2 infected CVB-ACE2 cells. Confluent (75%) monolayers of CVB-ACE2 expressing DF-1 cells on iBID chamber slides were infected with WA1 ( A ), Delta ( B ), Lambda ( C ) or Omicron ( D ) variant of SC2 for 48 h and then stained for SC2 S protein and counterstained for nuclei using DAPI as stated in materials and methods. Scale bars at the bottom right represent 10× magnification.

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Immunofluorescence, Microscopy, Infection, Expressing, Variant Assay, Staining

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: Immunofluorescence microscopy of SC2 infected PB-ACE2 cells. Confluent (75%) monolayers of PB-ACE2 expressing DF-1 cells on iBID chamber slides were infected with WA1 ( A ), Delta ( B ), Lambda ( C ) or Omicron ( D ) variant of SC2 for 48 h and then stained for SC2 S protein and counterstained for nuclei using DAPI as stated in materials and methods. Scale bars at the bottom right represent 10× magnification.

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Immunofluorescence, Microscopy, Infection, Expressing, Variant Assay, Staining

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a – c , Based on simulations of RBD-bound ACE2, new polar interactions were identified between the ACE2.v2.4 (orange) and RBD (yellow) loops (red) ( a ). ACE2 mutations are presented as sticks colored cyan with nearby RBD residues as sticks colored yellow. New polar interactions between ACE2.v2.4 and RBD loops 1 ( b ) and 2 ( c ) are indicated by the dotted blue lines. d , MSM-weighted distance distributions of newly formed hydrogen bonds. For each interacting pair, the ACE2.v2.4 residue is listed first and the RBD residue is listed second. e , Probabilities for stable hydrogen bond interactions using a 4-Å distance criterion between accepter and donor. f , Root mean square fluctuation (RMSF) of RBD residues when bound to WT (cyan) or v2.4 (orange) ACE2 receptors. The RBD regions that interface with ACE2 are shaded gray. The absolute difference in RMSF between WT and v2.4 proteins is shown in black. Distance distributions, hydrogen bond probabilities and RMSF calculations were based on 40,000 frames from the simulations. Frames were selected based on the MSM stationary probability to represent the entire conformational ensemble. The error bars represent the 95% confidence intervals calculated from 20 bootstrapped samples.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Residue

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a , mRNA expression of ACE2 (NM_001371415.1) and TMPRSS2 (NM_001135099) in human lung epithelial A549 cells. A549 cells stably expressing hACE2 and hLMVECs were analyzed by one-step RT–PCR (top) and real-time PCR (bottom). Relative expression was normalized to glyceraldehyde 3-phosphate dehydrogenase expression. b , Cultured hACE2-A549 cells, A549 cells and hLMVECs were preincubated with sACE2 2 -IgG1 or sACE2 2 .v2.4-IgG1 at 5 µg ml −1 or 25 µg ml −1 for 1 h. SARS-CoV-2 pseudovirus (MOI = 0.1) was added to the cells, which were collected at 24 h. Virus entry was evaluated by luciferase activity. n = 4 replicates. c , A dose of 10 mg kg −1 sACE2 2 -IgG1, sACE2 2 .v2.4-IgG1 or buffer (PBS + 0.2% BSA) was administrated intravenously into K18-hACE2 transgenic mice for 30 min before SARS-CoV-2 pseudovirus (10 6 PFU) intraperitoneal injection. Tissue lysates were prepared at 24 h and virus entry in the selected organs was evaluated by luciferase activity. Buffer was applied as the control group, n = 4. d – g , K18-hACE2 transgenic mice were inoculated with the SARS-CoV-2 isolate WA-1/2020 at 1 × 10 4 PFU. Mice received control PBS or sACE2 2 .v2.4-IgG1 10 mg kg −1 via intravenous injection 12 h before inoculation. Mice were observed for survival ( d ) and body weight ( e ), n = 5. EBA as a marker of pulmonary endothelial permeability ( f ) and lung wet/dry ratio ( g ) as a measure of lung edema were quantified. Data are presented as the mean ± s.e.m., n = 4. b , c , f , g , P values were calculated by one-way ANOVA with Tukey post hoc test.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Expressing, Stable Transfection, One Step RT-PCR, Real-time Polymerase Chain Reaction, Cell Culture, Virus, Luciferase, Activity Assay, Transgenic Assay, Injection, Control, Marker, Permeability

Journal: Nature Chemical Biology

Article Title: Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants

doi: 10.1038/s41589-021-00965-6

Figure Lengend Snippet: a – d , sACE2 carrying the v2.4 mutations has increased S binding compared with WT sACE2. Human Expi293F cells expressing myc-tagged S from 4 SARS-CoV-2 variants (Wuhan ( a ), B.1.1.7/Alpha ( b ), B1.351/Beta ( c ) and P.1/Gamma ( d )) were incubated with monomeric sACE2-8 h (black) or dimeric sACE2 2 -IgG1 (gray); bound protein was detected by flow cytometry. WT ACE2 proteins are shown as broken lines, v2.4 proteins are shown as solid lines. n = 3 independent replicates; data are shown as the mean ± s.e.m. e – i , Binding of sACE2 2 .v2.4-IgG1 is comparable to clinically effective mAbs. Binding of mAbs versus sACE2 2 .v2.4-IgG1 to the S proteins of SARS-CoV-2 VOCs (B.1.1.7/Alpha ( e ), B1.351/Beta ( f ), B.1.617.2/Delta ( g ) and P.1/Gamma ( h )) and S protein of SARS-CoV-1 ( i ), as measured by flow cytometry. n = 3 independent replicates; data are shown as the mean ± s.e.m.

Article Snippet: For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry

Journal: EBioMedicine

Article Title: Alveolar macrophage dysfunction and cytokine storm in the pathogenesis of two severe COVID-19 patients

doi: 10.1016/j.ebiom.2020.102833

Figure Lengend Snippet: COVID-19-infected cells in lung and pulmonary hilum lymph nodes. Proliferation of the type II pneumocytes by H&E stain (a, 400 ×) and expression of Rp3-NP by immunohistochemistry in type II pneumocytes (b, 400 ×, arrow); Membrane positive of ACE2 expression in type II pneumocytes and macrophages respectively (c, 400 ×, arrow and f, 400 ×, arrow); Aggregation of macrophages in alveolar by H&E stain (d, 400 ×) and expression of Rp3-NP of SARS-CoV-2 (e, 400 ×, arrow); Macrophages in the cortical sinuses of pulmonary hilum lymph nodes (g, 200 ×), expression of Rp3-NP of SARS-CoV-2 (h, 200 ×, arrow) and ACE2 (i, 200 ×, arrow).

Article Snippet: In addition, ACE2 protein was revealed using a mouse monoclonal anti-human ACE2 antibody (clone 1G4; 1:160; ORIGENE) while the detection of SARS-CoV-2 was performed using the antibody against SARS-CoV-2 Rp3 N-protein (Rp3-NP) (1:100; the Rp3-NP antibody was provided by Prof. Zheng-Li Shi, Wuhan Institute of Virology, Chinese Academy of Sciences).

Techniques: Infection, Staining, Expressing, Immunohistochemistry, Membrane

Journal: EBioMedicine

Article Title: Alveolar macrophage dysfunction and cytokine storm in the pathogenesis of two severe COVID-19 patients

doi: 10.1016/j.ebiom.2020.102833

Figure Lengend Snippet: SARS-CoV-2 Spike protein interaction with CD68 macrophage. (a-b) Flow cytometry analysis showing the distribution of S protein bound immune cells in lung tissues (a) and peripheral blood. (b) CD14 markers were used for labeling monocytes/ macrophages while CD3 for T cells. (c-d ) Flow cytometry analysis showing the expression of ACE2 on the surface of monocytes/macrophages, but not of T cells in lung tissues (c) and peripheral blood (d).

Article Snippet: In addition, ACE2 protein was revealed using a mouse monoclonal anti-human ACE2 antibody (clone 1G4; 1:160; ORIGENE) while the detection of SARS-CoV-2 was performed using the antibody against SARS-CoV-2 Rp3 N-protein (Rp3-NP) (1:100; the Rp3-NP antibody was provided by Prof. Zheng-Li Shi, Wuhan Institute of Virology, Chinese Academy of Sciences).

Techniques: Flow Cytometry, Labeling, Expressing

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: Development of Mice Sensitized to SARS-CoV-2 Infection (A and B) To assess hACE2 expression and surface localization, 17CL-1 cells were transduced with Ad5-hACE2 or Ad5-Empty at MOI of 100 at 37°C for 4 h. hACE2 expression was monitored by western blot assay (A) or flow cytometry (B). (C) Ad5-hACE2 transduced 17CL-1 cells were infected with SARS-CoV-2 at MOI of 0.5 at 48 h post transduction, and virus titers were determined by foci forming assay (FFA) at 24, 48, and 72 hours post infection (h.p.i.). (D) Five days after transduction with 2.5 × 10 8 FFU of Ad5-hACE2 or Ad5-Empty in 75 μL of DMEM intranasally, lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with an anti-hACE2 antibody (brown color). hACE2 protein (brown color) was detected only in Ad-hACE2-treated mice and was predominantly localized to alveolar epithelial cells. Scale bars, 467 and 94 μm, top and bottom panels, respectively. (E and F) Ad5-hACE2- or Ad5-Empty-transduced BALB/c or C57BL/6 mice were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2 in 50 μL of DMEM. Weight changes in 6-to-8-week old BALB/c (E) and C57BL/6 (F) mice were monitored daily (n = 5 mice per group). To obtain virus kinetics in BALB/c (E) and C57BL/6 (F) mice, lungs were harvested and homogenized at the indicated time points, and virus was titered by plaque assay. Titers are expressed as PFU/g lung tissue (n = 3 mice per group per time point). Data are representative of two independent experiments. (G) 2 d.p.i., lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with anti-SARS-CoV-2 N protein. Scale bar, 476 μm. (H) Representative Hematoxylin-eosin (HE) staining of lungs from BALB/c and C57BL/6 mice harvested at the indicated time points p.i. Scale bars, 443 and 88 μm, top and bottom panels, respectively. Asterisk, edema. (I) Summary histology scores determined at the indicated time points (n = 4 to 5 mice per group). PMN, neutrophils. (J) Photographs of lung specimens isolated from infected mice at indicated time points are shown. Arrowheads indicate regions with vascular congestion and hemorrhage.

Article Snippet: Membranes were stained with a mouse anti-human hACE2 antibody (clone OTI2G7, Origene, Rockville, MD), a mouse anti-C9 antibody (clone Rho 1D4, EMD Millipore, Temecula, CA), or a mouse anti–β-actin (Cat: A01546, Genscript, Nanjing).

Techniques: Infection, Expressing, Transduction, Western Blot, Flow Cytometry, Virus, Staining, Plaque Assay, Isolation

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: The Role for IFN and STAT1 Signaling in SARS-CoV-2 Infection (A) 5 days after transduction with 2.5 × 10 8 FFU of Ad5-hACE2, C57BL/6 mice were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2. Weight changes were monitored daily (n = 5 mice per group), and virus titers in the lungs were measured at the indicated time points using FFA (n = 3–4 mice per group per time point). Titers are expressed as FFU/g tissue. (B) Sections of paraffin embedded lungs from SARS-CoV-2-infected Ad5-hACE2-transduced, wild-type and genetically modified C57BL/6 mice at 4 d.p.i. were stained with hematoxylin/eosin. Scale bar, 100 μm. (C) Photographs of gross pathological lung specimens isolated from infected C57BL/6 mice at 4 d.p.i. Arrowheads indicate regions with vascular congestion and hemorrhage. (D) Ad5-hACE2-transduced C57BL/6 mice were treated with 80 μg of poly I:C in 50 μL of PBS 6 h before intranasal infection with SARS-CoV-2. Weight changes were monitored daily, and viral titers in lungs were measured at the indicated time points. ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values ≤ 0.0001.

Article Snippet: Membranes were stained with a mouse anti-human hACE2 antibody (clone OTI2G7, Origene, Rockville, MD), a mouse anti-C9 antibody (clone Rho 1D4, EMD Millipore, Temecula, CA), or a mouse anti–β-actin (Cat: A01546, Genscript, Nanjing).

Techniques: Infection, Transduction, Virus, Genetically Modified, Staining, Isolation

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: Requirements for T Cells and Antibodies for SARS-CoV-2 Clearance and Protection from Subsequent Challenge Ad5-hACE2-transduced mice were infected with 1 × 10 5 PFU of SARS-CoV-2. (A) For systemic depletion of CD4 + or CD8 + T cells, mice were injected intraperitoneally (i.p.) with 0.5 mg anti-CD4 antibody (clone GK1.5) and/or 0.5 mg anti-CD8 antibody (clone 2.43) or 0.5 mg rat IgG at days −2 and 0 p.i. Virus titers in the lungs were measured at the indicated time points. Titers are expressed as FFU/g tissue (n = 4 mice per group per time point) (B–D) To identify SARS-CoV-2 T cell responses, single-cell suspensions were prepared from the BALF of transduced/infected BALB/c mice and stimulated with 2 μM structural protein peptide pools for 5–6 h in the presence of brefeldin A. Flow plots (B, 7 d.p.i), and summary of frequencies and cell numbers of SARS-CoV-2-N pool specific CD4 + T cells (C) and S1 pool specific CD8 + T cells (D) (determined by IFN-γ intracellular staining) are shown (n = 3 to 4 mice per time point). (E) PRNT 50 titers in the sera of transduced/infected C57BL/6 mice at indicated time points p.i. are shown. (F) BALB/c and C57BL/6 mice were immunized with 1 × 10 5 infectious units (IU) of VRP-S intranasally in 50 μL of PBS. Mice were transduced and infected with 1 × 10 5 PFU of SARS-CoV-2 3 weeks after vaccination. Virus titers in the lungs were measured at the indicated time points (n = 4 mice per group per time point). (G) For adoptive transfer of serum, BALB/c mice were immunized with 1 × 10 5 IU of VRP in the footpad in 50 μL of PBS and boosted with the same dose 3 weeks later. Sera were obtained 1–2 weeks after VRP booster. Then, 150 μL of serum was transferred into transduced mice intravenously (i.v.) 1 day before SARS-CoV-2 infection (n = 3 mice per group per time point). ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values ≤ 0.0001.

Article Snippet: Membranes were stained with a mouse anti-human hACE2 antibody (clone OTI2G7, Origene, Rockville, MD), a mouse anti-C9 antibody (clone Rho 1D4, EMD Millipore, Temecula, CA), or a mouse anti–β-actin (Cat: A01546, Genscript, Nanjing).

Techniques: Infection, Injection, Virus, Staining, Adoptive Transfer Assay

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: Convalescent Plasma from COVID-19 Patients and Remdesivir Protect Mice from SARS-CoV-2 Infection (A and B) For plasma adoptive transfer, Ad5-hACE2-transduced mice were injected with 150 μL of plasma i.v. from a healthy donor or COVID-19, MERS, or SARS convalescent patients, at −1 d.p.i. Weight and virus titers in lung tissues were monitored (A) and expressed as FFU/g tissue (n = 4 mice per group per time point). Sections of paraffin embedded lungs from plasma adoptive transferred and infected mice were stained with HE at day 4 p.i. (B). Scale bar, 100 μm. (C and D) For remdesivir treatment, Ad5-hACE2-transduced mice were treated with remdesivir (25 mg/kg, bid s.c.) or vehicle at −1 d.p.i. Weight loss of infected mice and virus titers in the lungs were monitored (C), and hematoxylin/eosin staining of sections of paraffin-embedded lungs is shown at 4 d.p.i. (D) (n = 4 mice per group per time point). Data are representative of two independent experiments. Scale bar, 100 μm. ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values of ≤ 0.0001.

Article Snippet: Membranes were stained with a mouse anti-human hACE2 antibody (clone OTI2G7, Origene, Rockville, MD), a mouse anti-C9 antibody (clone Rho 1D4, EMD Millipore, Temecula, CA), or a mouse anti–β-actin (Cat: A01546, Genscript, Nanjing).

Techniques: Clinical Proteomics, Infection, Adoptive Transfer Assay, Injection, Virus, Staining

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet:

Article Snippet: Membranes were stained with a mouse anti-human hACE2 antibody (clone OTI2G7, Origene, Rockville, MD), a mouse anti-C9 antibody (clone Rho 1D4, EMD Millipore, Temecula, CA), or a mouse anti–β-actin (Cat: A01546, Genscript, Nanjing).

Techniques: Virus, Blocking Assay, Plasmid Preparation, Software

Journal: Clinical Science

Article Title: Antiepileptic effects of long-term intracerebroventricular infusion of angiotensin-(1-7) in an animal model of temporal lobe epilepsy

doi: 10.1042/cs20200514

Figure Lengend Snippet: Figure 7. Effects of TLE and Ang-(1-7) treatment on hippocampal protein levels Representative Western blot bands (A) and protein levels of ACE2 (B), NEP (C), AT2R (D), AT1R (*P=0.0406 CT vs EP) (E), Mas [*P=0.0406 CT vs EP + Ang-(1-7)] (F), IL-6 (G), SOD (H), CAT (*P=0.0389 CT vs EP) (I), Bcl-2 (*P=0.0468 CT vs EP) (J), and

Article Snippet: Published by Portland Press Limited on behalf of the Biochemical Society 2265 D ow nloaded from http://portlandpress.com /clinsci/article-pdf/134/17/2263/892258/cs-2020-0514.pdf by U niversidade Federal de Sao Paulo (U N IFESP) user on 14 M ay 2024 Table 1 Host, specificity, dilution, catalog number and source of the antibodies used in the present study Antibody Host Specificity Dilution Catalog number Source ACE2 Rabbit Monoclonal 1:1000 GTX01160 GeneTex, CA, U.S.A. NEP Mouse Monoclonal 1:1000 sc-46656 Santa Cruz Biotechnology, CA, U.S.A. AT1 Mouse Monoclonal 1:500 sc-515884 Santa Cruz Biotechnology, CA, U.S.A. AT2 Rabbit Monoclonal 1:1000 M00432 Boster Biological Technology, CA, U.S.A. Mas Rabbit Polyclonal 1:500 ab66030 Abcam, MA, U.S.A. IL-6 Mouse Monoclonal 1:500 IM-0407 Imuny Biotechnology, SP, Brazil SOD Mouse Monoclonal 1:1000 sc-17767 Santa Cruz Biotechnology, CA, U.S.A. CAT Mouse Monoclonal 1:1000 LS-B2554 Lifespan Biosciences, WA, U.S.A. Bcl-2 Mouse Monoclonal 1:1000 sc-7382 Santa Cruz Biotechnology, CA, U.S.A. mTOR Mouse Monoclonal 1:1000 #4517 Cell Signaling Technology, MA, U.S.A. Phospho-mTOR Rabbit Polyclonal 1:1000 #2971 Cell Signaling Technology, MA, U.S.A. GAPDH Mouse Monoclonal 1:2000 sc-365062 Santa Cruz Biotechnology, CA, U.S.A. 2266 © 2020 The Author(s).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Role of Circulating Angiotensin Converting Enzyme 2 in Left Ventricular Remodeling following Myocardial Infarction: A Prospective Controlled Study

doi: 10.1371/journal.pone.0061695

Figure Lengend Snippet: Panel A: Box-plot showing the differences in the median (horizontal lines), interquartile range (boxes) and 95% of the observed values (whiskers) for sACE2 activity between controls and STEMI patients. Panel B: Scatter-plot detailing individual changes in sACE2 activity among patients. *: p<0.001 for comparison between baseline measurements in patients and controls. †: p<0.01 for comparison between baseline and 7 days measurements by paired analysis. ACE2: Angiotensin Converting Enzyme 2; RFU: relative fluorometric units.

Article Snippet: The ACE2 fluorescent enzymatic assay protocol was performed as previously described with modifications, using an ACE2 quenched fluorogenic substrate (Mca-Ala-Pro-Lys(Dnp)-OH, Enzo Life Sciences) , .

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Role of Circulating Angiotensin Converting Enzyme 2 in Left Ventricular Remodeling following Myocardial Infarction: A Prospective Controlled Study

doi: 10.1371/journal.pone.0061695

Figure Lengend Snippet: A weak but significant correlation was observed between infarct size and sACE2 activity at 7 days (panel A) and at 6 months follow-up (panel B). FU: Follow-up. sACE2: Serum Angiotensin Converting Enzyme 2; LV: left ventricular; RFU: relative fluorometric units.

Article Snippet: The ACE2 fluorescent enzymatic assay protocol was performed as previously described with modifications, using an ACE2 quenched fluorogenic substrate (Mca-Ala-Pro-Lys(Dnp)-OH, Enzo Life Sciences) , .

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Role of Circulating Angiotensin Converting Enzyme 2 in Left Ventricular Remodeling following Myocardial Infarction: A Prospective Controlled Study

doi: 10.1371/journal.pone.0061695

Figure Lengend Snippet: Scatter-plots showing a statistically significant correlation between ACE2 activity at 7 days and EF during admission (panel A) and at 6 months follow-up (panel B). sACE2: Serum Angiotensin Converting Enzyme 2; EF: ejection fraction; RFU: relative fluorometric units.

Article Snippet: The ACE2 fluorescent enzymatic assay protocol was performed as previously described with modifications, using an ACE2 quenched fluorogenic substrate (Mca-Ala-Pro-Lys(Dnp)-OH, Enzo Life Sciences) , .

Techniques: Activity Assay

Journal: Nano-Micro Letters

Article Title: Human ACE2-Functionalized Gold “Virus-Trap” Nanostructures for Accurate Capture of SARS-CoV-2 and Single-Virus SERS Detection

doi: 10.1007/s40820-021-00620-8

Figure Lengend Snippet: Schematic diagram of COVID-19 SERS sensor design and single-virus detection mechanism. a Schematic diagram of COVID-19 SERS sensor design and operation procedure. SARS-CoV-2 can be localized by “virus-traps” nanoforest composed of oblique gold-nanoneedles array (GNAs), and be captured by ACE2 anchored on amide-modified GNAs from virus-containing urines even with complex multi-proteins circumstance. Through machine-learning and identification techniques, the identification standard of virus signals are established, and utilized for virus diagnoses. b Schematic diagrams of single-virus detection by selectively capturing and trapping virus, and the multi-SERS enhancement mechanism

Article Snippet: Human ACE2 (2.62 mg mL −1 ), SARS-CoV spike glycoprotein (0.58 mg mL −1 ), SARS-CoV-2 spike glycoprotein (2.38 mg mL −1 ), SARS-CoV-2 nucleocapsid phosphoprotein (1.3 mg mL −1 ) were obtained from Sanyou Biopharmaceuticals Co. Ltd, obtained from 293 T protein expression system.

Techniques: Virus, Modification

Journal: Nano-Micro Letters

Article Title: Human ACE2-Functionalized Gold “Virus-Trap” Nanostructures for Accurate Capture of SARS-CoV-2 and Single-Virus SERS Detection

doi: 10.1007/s40820-021-00620-8

Figure Lengend Snippet: Morphology analysis of gold-nanoneedles array and SERS spectra of viral protein. a SEM images of Au nanoneedles array fabricated by Ar + ions irradiation at a tilted angle of 45° on an Au film of 500 nm in thickness. b Schematics of “virus-traps” nanoforest composed of tilted gold-nanoneedles array. c Calculated intensity distribution (|E| 2 ) at 785 nm for a tilted Au nanoneedle array with the polarization of the incident laser along the x-axis. d Structure schematics of SARS-CoV-2 (left), and SERS spectra (right) of SARS-CoV-2 S protein and nucleocapsid protein, SARS-CoV S protein, and Human ACE2 protein at 100 nM level. e Calculated static Raman spectra of 4 types of main individual amino acids Tyr, Trp, His, Phe encoded in SARS-CoV-2 S on Au cluster

Article Snippet: Human ACE2 (2.62 mg mL −1 ), SARS-CoV spike glycoprotein (0.58 mg mL −1 ), SARS-CoV-2 spike glycoprotein (2.38 mg mL −1 ), SARS-CoV-2 nucleocapsid phosphoprotein (1.3 mg mL −1 ) were obtained from Sanyou Biopharmaceuticals Co. Ltd, obtained from 293 T protein expression system.

Techniques: Irradiation, Virus

Journal: Nano-Micro Letters

Article Title: Human ACE2-Functionalized Gold “Virus-Trap” Nanostructures for Accurate Capture of SARS-CoV-2 and Single-Virus SERS Detection

doi: 10.1007/s40820-021-00620-8

Figure Lengend Snippet: Affinity analysis of SARS-CoV-2 S, SARS-CoV S with ACE2. a Schematic illustration of S proteins’ binding on ACE2-functionalized GNAs of SARS-CoV-2 and SARS-CoV. SERS spectra of b SARS-CoV and c SARS-CoV-2 S proteins at the same concentration of 177 nM after bound to ACE2-functionalized GNAS for different binding time durations. d Intensity of detected SERS signals of SARS-CoV-2 S and SARS-CoV S after bound to ACE2-functionalized GNAs with different binding times. e Intensity of Raman bands (1027 cm −1 ) of SARS-CoV-2 S with different concentration detected with ACE2-functionalized GNAs by immersing in the diluted protein solution, and without ACE2-functionalized GNAs by dropping the corresponding concentration of diluted protein solution. It is worth noting that the application of dropping method is because GNAs without ACE2 modification have little affinity with SARS-CoV-2 S protein. The value marked on the line represents the number of S proteins in one Raman-focused window. η represents enrichment multiple by ACE2

Article Snippet: Human ACE2 (2.62 mg mL −1 ), SARS-CoV spike glycoprotein (0.58 mg mL −1 ), SARS-CoV-2 spike glycoprotein (2.38 mg mL −1 ), SARS-CoV-2 nucleocapsid phosphoprotein (1.3 mg mL −1 ) were obtained from Sanyou Biopharmaceuticals Co. Ltd, obtained from 293 T protein expression system.

Techniques: Binding Assay, Concentration Assay, Modification