Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: Molecular models for HDP-2P (gold) complexes with ACE2 (red), and RBD (turquoise) obtained by protein docking. A Structures of HDP-2P complexed with ACE2 (left panel) and S-protein RBD (right panel) determined using Frodock. The central (control) panel shows re-docking of ACE2 to RBD, which results in a structure that resembles published X-ray structures; B A Rosetta-optimized binding interface between HDP-2P and RBD intersects the ACE2-RBD binding surface, which could prevent RBD from interacting with ACE2; C A different Rosetta-optimized binding interface between HDP-2P and ACE2 where HDP-2P docks away from the ACE2-RBD binding surface, implying no direct interplay between viral protein and PLA 2

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Control, Binding Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: PLA 2 s exhibit antiviral activity against viruses containing mutations and interfere with different stages of the SARS-CoV-2 replication cycle. a CPE inhibition assay. Vero E6 cells were infected with SARS-CoV-2 strain PMVL-19 or PMVL-20 at 100 TCID 50 in the presence of different concentrations of HDP-2 for 72 h. CPE inhibition was then measured by colorimetric assay with MTT. b Time-of-drug-addition assay. Vero E6 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01, and virus yield in the infected cell supernatants was quantified by qRT-PCR 18 h after infection. c 293T/ACE2 were infected with different pseudo-SARS-CoV-2-GFP either in the presence of vehicle (PBS, Control) or HDP-2 (10 µg/ml). Representative fluorescent microscopy images of 293T/ACE2 cells infected with the pseudo-SARS-CoV-2 and treated with HDP-2. HDP-2 treatment led to a decrease in the entry of pseudoviruses, which was manifested in a decrease in the number of GFP-positive cells compared to the control. Scale bars, 100 µm. d Infectivity of pseudo-SARS-CoV-2 particles on 293T/ACE2 cells was quantified by measuring GFP fluorescence. Wuhan, B.1.1.7 and B.1.351 are the Wuhan reference strain, the lineage B.1.1.7 (United Kingdom) and the lineage B.1.351 (South Africa), respectively. Significant difference was determined using a Student’s t test: *p ˂0.05; **p ˂0.01; ***p ˂0.001. All results are shown as mean ± SD of n = 3 or 5 biologically independent samples. RFU relative fluorescence units

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Activity Assay, Inhibition, Infection, Colorimetric Assay, Virus, Quantitative RT-PCR, Control, Microscopy, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: HDP-2P reduces the binding of an anti-ACE2 antibody and RBD of glycoprotein S to ACE2 receptor at 293T/ACE2 cells. a , b Inhibition of anti-ACE2 antibody binding to 293T/ACE2 cells by HDP-2P. 293T/ACE2 cells were incubated with (100 µg/ml) or without (control) HDP-2P for 30 min and stained using human phycoerythrin (PE) conjugated anti-mouse ACE2 antibody. MFI mean fluorescence intensity. c , d Cells were incubated with PBS (control) or HDP-2P. The RBD protein fused with human Fc was then added for 1 h. After washing, the binding of RBD was detected using a DyLight 650-conjugated secondary anti-human Fc antibody. e Representative RBD binding profile in control and HDP-2P-treated cells. The cell populations binding high (RBD hi ) and low (RBD lo ) amount of RBD are shown. The percentage of positive cells was determined using flow cytometry analysis. Significant difference was determined using a Student’s t test: *p ˂0.05, ** p ˂0.01. Results are mean ± SD and are representative of at least three independent determinations

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Binding Assay, Inhibition, Incubation, Control, Staining, Fluorescence, Flow Cytometry

Journal: Hypertension

Article Title: Origin of the Y Chromosome Influences Intrarenal Vascular Responsiveness to Angiotensin I and Angiotensin (1-7) in Stroke-Prone Spontaneously Hypertensive Rats

doi: 10.1161/hypertensionaha.114.03756

Figure Lengend Snippet: Figure 2. Renal and renal artery mRNA gene expression of angiotensin-converting enzyme (ACE), ACE2, neprilysin, angiotensin II receptor type 1a (AT1aR), angiotensin II receptor type 2 (AT2R), and Mas receptor (MasR) in Wistar Kyoto (WKY), WKY.SPGlaY, spontaneously hypertensive stroke-prone rats (SHRSP), and SP.WKYGlaY (n>5 per group). All relative gene expression data are expressed relative to the WKY group and are presented as mean±SEM. *P<0.05, **P<0.01, ***P<0.005 compared with WKY; #P<0.05, ##P<0.01 compared with SHRSP.

Article Snippet: Taqman predesigned assays (Applied Biosystems) for ACE (Rn00561094_m1), ACE2 (Rn01416293_m1), angiotensin II receptor type 1a (Rn02758772_s1), angiotensin II receptor type 2 (AT2R, Rn00560677_s1), and Mas receptor (MasR, Rn00562673_s1) were multiplexed with Actb (4352340E-labeled VIC).

Techniques: Gene Expression

Journal: Hypertension

Article Title: Origin of the Y Chromosome Influences Intrarenal Vascular Responsiveness to Angiotensin I and Angiotensin (1-7) in Stroke-Prone Spontaneously Hypertensive Rats

doi: 10.1161/hypertensionaha.114.03756

Figure Lengend Snippet: Figure 3. Plasma angiotensin II (Ang II) and Ang (1-7) concentration, angiotensin-converting enzyme (ACE), and ACE2 activity in Wistar Kyoto (WKY), WKY.SPGlaY, spontaneously hypertensive stroke-prone rats (SHRSP), and SP.WKYGlaY (n≥5/ strain). Data are presented as mean±SEM with *P<0.05, **P<0.01 compared with WKY; #P<0.05, ##P<0.01 compared with SHRSP.

Article Snippet: Taqman predesigned assays (Applied Biosystems) for ACE (Rn00561094_m1), ACE2 (Rn01416293_m1), angiotensin II receptor type 1a (Rn02758772_s1), angiotensin II receptor type 2 (AT2R, Rn00560677_s1), and Mas receptor (MasR, Rn00562673_s1) were multiplexed with Actb (4352340E-labeled VIC).

Techniques: Clinical Proteomics, Concentration Assay, Activity Assay

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet: Characterization of an AAV6.2FF-hACE2 transduction model for SARS-CoV-2 infection of wild-type mice (A) Diagram of AAV genome expressing hACE2 from the CASI promoter. (B) Western blot of HEK293 cells transduced with AAV6.2FF-hACE and probed with an anti-hACE2 antibody. (C) BALB/c mice were administered 1 x 10 11 vg of AAV-Luc intranasally and imaged 10 days later using an IVIS imager. (D–F) (D) IFA images of lungs harvested from BALB/c mice infected intranasally with 1 x 10 11 vg of AAV-hACE2 or AAV-Luc and euthanized 10 days later. Lungs were stained with a rabbit anit-hACE2 antibody and imaged at 20 X (scale bar, 50mM). Viral RNA (E), and virus TCID50 titers (F) were determined in respiratory tissues on days 2 and 4 post-infection. n = 6 (3M, 3F). Statistical significance determined by Mann-Whitney test. ∗ = p < 0.05, ∗∗ = p < 0.01.

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: Transduction, Infection, Expressing, Western Blot, Staining, MANN-WHITNEY

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet: SARS-CoV-2 spike DNA antigens protect from viral replication in vivo (A) Mice were immunized once or twice separated by four weeks with 10ug of pS via electroporation. Serum was collected at day 18 post-final immunization. At 35 days post-final immunization mice were infected intranasally with adeno-associated virus expressing human ACE2 (white). 17 days following AAV6-ACE2 transduction, animals were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2 VIDO-01 P2. Four days post infection, animals were sacrificed to quantify viral replication. SARS-CoV-2 specific serum IgG endpoint titers (B) and pseudoviral neutralization titers (C) at day 18 post-final immunization. Replication competent virus (D), and viral RNA (E) in the lungs four-days post-infection. Pearson correlations between virus titer and serum IgG endpoints (F) and neutralization titers (G). Pearson correlations between viral RNA copies and serum IgG endpoints (H) and neutralization titers (I). Each point represents the average of duplicate samples from an individual animal, bars represent the mean, lines represent the median, and error bars represent the SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by student's t-test (A and B), or Kruskall-Wallis ANOVA (D and E). Spearman correlations were used to determine relationships (F-I). Data are representative of one experiment with n = 5 males (squares) and 5 females (circles) per group.

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: In Vivo, Electroporation, Infection, Expressing, Transduction, Neutralization

Journal: iScience

Article Title: A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens

doi: 10.1016/j.isci.2021.102699

Figure Lengend Snippet:

Article Snippet: The cDNA for human ACE2 (SinoBiological; HG10108-M) was cloned into an AAV genome plasmid (pACASI-MSC-WPRE) containing the composite CASI promoter ( ) consisting of the human cytomegalovirus immediate-early gene enhancer region, the chicken beta actin promoter, and the human ubiquitin C promoter, as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and simian virus 40 polyadenylation sequence downstream of hACE2 with flanking AAV2 inverted terminal repeats (ITRs).

Techniques: Recombinant, Plasmid Preparation, Enzyme-linked Immunospot, Software

Journal: Nature Communications

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

doi: 10.1038/s41467-020-17796-z

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

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

Techniques: Infection, Marker, Staining

Journal: Nature Communications

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

doi: 10.1038/s41467-020-17796-z

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

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

Techniques: Concentration Assay, Immunofluorescence