Journal: Biochemical and Biophysical Research Communications

Article Title: Highly polymerized proanthocyanidins (PAC) components from blueberry leaf and stem significantly inhibit SARS-CoV-2 infection via inhibition of ACE2 and viral 3CLpro enzymes

doi: 10.1016/j.bbrc.2022.04.072

Figure Lengend Snippet: An experiment to examine the ability of BB-Fr7 to inhibit virus replication at different times of treatment. A. As in Fig. S2 , the effect of BB-Fr7 treatment on VeroE6/TMPRSS2 cells was investigated using three different treatment methods: before (Pre), during (On), and after (Post) the virus infection period as indicated in Materials and Methods. As a control, we included those not treated with BB-Fr7 (No) and those treated with BB-Fr7 (All) at all experimental times. The same experiment (N = 3) was repeated three times. ∗; p < 0.05, ∗∗; p < 0.005, ∗∗∗; p < 0.001. B. To examine the inhibition of infection by BB-PAC, infection of HeLa-ACE2-TMPRSS2 cells with SARS pseudovirus was measured by luciferase activity. The left figure shows the results for Fr4 (dotted line) and Fr7 (solid line) in blueberry leaves, and the right figure shows the results for the fractions of Fr4 and Fr7 from blueberry stems. Luciferase activity is shown relative to the condition without BB-PAC fraction as 100%.

Article Snippet: A. ACE2 enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the ACE2 activity assay kit (BPS Bioscience).

Techniques: Infection, Inhibition, Luciferase, Activity Assay

Journal: Biochemical and Biophysical Research Communications

Article Title: Highly polymerized proanthocyanidins (PAC) components from blueberry leaf and stem significantly inhibit SARS-CoV-2 infection via inhibition of ACE2 and viral 3CLpro enzymes

doi: 10.1016/j.bbrc.2022.04.072

Figure Lengend Snippet: Inhibitory activity of ACE2, BSS, and virus-derived 3CL protease enzymes by BB-PAC. A. ACE2 enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the ACE2 activity assay kit (BPS Bioscience). The right figure shows the results using the blueberry leaf fraction and the left figure shows the results using the blueberry stem fraction. B. TMPRSS2 enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the TMPRSS2 activity assay kit (BPS Bioscience). C. 3CLpro enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the 3CLpro activity assay kit (BPS Bioscience).

Article Snippet: A. ACE2 enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the ACE2 activity assay kit (BPS Bioscience).

Techniques: Activity Assay, Derivative Assay

Journal: Kidney international

Article Title: Chronic AT2 receptor activation attenuates renal AT1 receptor function and blood pressure in obese Zucker rats

doi: 10.1038/ki.2013.193

Figure Lengend Snippet: (A) ACE2 expression (B) ACE2 activity and (C) Mas receptor expression in the kidney cortex of control and CGP-treated lean and obese Zucker rats. Upper panels: Representative Western blots for respective proteins with loading control β-actin. For Western blot only, bar graphs represent the ratios of densities of respective protein bands and β-Actin i.e. ACE2/β-Actin, MasR/β-Actin $ significantly different compared with lean control rats, # significantly different compared with obese control rats. Values are represented as mean±SEM; One-way ANOVA followed by Neuman-Keuls test, p<0.05; N=5-7 in each group). (LCT- lean control, LCGP-lean treated with CGP42112A, OCT-obese control, OCGP-obese treated with CGP42112A).

Article Snippet: The expression of AT 1 R, AT 2 R, and renin, ACE, ACE2 and MasR in the idney cortex was determined by Western blotting and ACE2 activity was measured by Sensolyte 390 ACE2 activity kit (Anaspec Inc, CA) using spectrofluorometer (Cytofluor Series 4000, Applied Biosystem).For western blotting, the kidney cortices were homogenized in the buffer containing (in mM) Tris 50, EDTA 10, PMSF 1, cocktail of protease inhibitors (aprotinin, calpain inhibitors, leupeptin, pepstatin and trypsin inhibitor).

Techniques: Expressing, Activity Assay, Control, Western Blot

Journal: Kidney international

Article Title: Chronic AT2 receptor activation attenuates renal AT1 receptor function and blood pressure in obese Zucker rats

doi: 10.1038/ki.2013.193

Figure Lengend Snippet: Effect of AT 2 R agonist and antagonist on (A) ACE2 activity (B) MasR expression (C) Renin activity and (D) AT 1 R expression. Upper panels: Representative Western blots for respective proteins with loading control β-actin. Bar graphs: represent the ratios of densities of respective protein bands and β-Actin i.e AT 1 /β-Actin, Mas/β-Actin. *significantly different compared with control. Values are represented as mean ± SEM; One-way ANOVA followed by Neuman-Keuls test, p<0.05, N=6-7 in each group). (CT-control, CGP-CGP42112A, PD-PD123319).

Article Snippet: The expression of AT 1 R, AT 2 R, and renin, ACE, ACE2 and MasR in the idney cortex was determined by Western blotting and ACE2 activity was measured by Sensolyte 390 ACE2 activity kit (Anaspec Inc, CA) using spectrofluorometer (Cytofluor Series 4000, Applied Biosystem).For western blotting, the kidney cortices were homogenized in the buffer containing (in mM) Tris 50, EDTA 10, PMSF 1, cocktail of protease inhibitors (aprotinin, calpain inhibitors, leupeptin, pepstatin and trypsin inhibitor).

Techniques: Activity Assay, Expressing, Western Blot, Control

Journal: Scientific Reports

Article Title: HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells

doi: 10.1038/srep15085

Figure Lengend Snippet: ( A ) DU145 prostate tumor cells, incubated with recombinant human HMGB1 (rhHMGB1) for 24–48 h, were lysed and analyzed by western blot for presence of clusterin, and for β-actin for equal loading. ( B ) DU145 tumor cells were treated with docetaxel (DTX) for 1–4 days and their supernatants were evaluated for HMGB1 by ELISA. ( C ) DU145 tumor cells were treated with the indicated chemotherapeutic agents for 24 h and the supernatants analyzed for HMGB1.

Article Snippet: DU145 cells or PC3 cells were treated with docetaxel (DTX) for 1–4 days or were treated with the indicated chemotherapeutic agents for 24 h. Supernatants were collected and analyzed for the presence of human HMGB1 by an ELISA kit (LS-F4038) from LifeSpan Biosciences.

Techniques: Incubation, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: SARS-CoV-2 infection in male and female k18-hace2 mice over 7 days post-infection (dpi) (A) Body weight loss in response to intranasal inoculation of SARS-CoV-2 (4 × 10 5 TCID50/50μL/mouse). ∗p < 0.05 vs. male. Data represented in median ± IQR. (B) Lung injury scoring assessed on a scale of 0–4 for each of the following criteria: 1) neutrophil numbers in the alveolar space, 2) alveolar septal thickening, 3) number of hyaline membranes, 4) alveolar hemorrhage, and 5) cellular hyperplasia. ∗p < 0.05 vs. Ctrl and male. Data represented in mean ± SEM. (C) Viral RNA levels in oropharyngeal swabs at 2, 4, and 6 dpi. ∗p < 0.05 vs. male. Data represented in median ± IQR. (D–G) Copy numbers of E-gene and RdRp-gene in multiple organ tissues. ∗p < 0.05 vs. Ctrl. Data represented in mean ± SEM. (H) Detection of SARS-CoV-2 nucleocapsid protein (red) in lung tissues. Scale bars, 50 μm (main images) and 20 μm (magnified). (I) Protein expression in lung tissue detected by western blots in vehicle control (C) and at 7 dpi. (J) Concentration of soluble RAGE protein in plasma in vehicle control and at 7 dpi. ∗p < 0.05 vs. other groups. Data represented in mean ± SEM. (K) Correlation of protein levels of ACE2 and ERα in lung tissue of male mice before and after infection at 7 dpi. (L) Estrogen receptor alpha (ERα)/androgen receptor (AR) ratio assessed from western blot analysis in relation to β-actin loading control. ∗p < 0.05 vs. male. Data represented in mean ± SEM. (M) Concentration of 17β Estradiol in lung tissue homogenates in vehicle control and at 7 dpi. ∗p < 0.05 vs. male. n = 5–7 biologically independent mice for all groups. Data represented in mean ± SEM. (see also Figure S1 ).

Article Snippet: The hACE2 concentration in both rabbit and mouse plasma and lung tissue of mice was measured by hACE2 ELISA (#ab235649: Abcam, UK) following the manufacturer’s instructions.

Techniques: Infection, Expressing, Western Blot, Concentration Assay

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: Inhalation of rACE2 attenuates SARS-CoV-2 infection, replication, and lung injury and recovers expression of endogenous ACE2 and ERα in K18-hACE2 male mice at 7 dpi (A) Study scheme. Mice were intranasally inoculated with SARS-CoV-2 (4 × 10 5 TCID50/50μL/mouse) and 48 h later received daily nebulization of rACE2 (12 mg/kg) or vehicle control solution for 5 days. (B and C) Copy numbers of viral envelope (E) gene by RT-qPCR and titers (TCID50) in lung tissue in the SARS-CoV-2 infection alone and rACE2-treated groups. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (D) Gene expression of IL-6 in lung tissue in the infection alone and rACE2-treated groups. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (E) Concentration of soluble RAGE protein in plasma in infection alone and rACE2-treated mice. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (F) Detection of SARS-CoV-2 nucleocapsid protein (red) in lung tissues. Scale bars, 50 μm (main images) and 20 μm (magnified). (G) Representative lung histology by H&E staining. Arrows indicate neutrophil infiltration in the alveolar space. Lung injury scores from 6 animals per group are also shown. ∗p < 0.05 vs. naive control (C), ♰p < 0.05 vs. SARS-CoV-2 alone & (C). Data represented in mean ± SEM. (H and I) Protein expression in lung tissue detected by western blots in naive control (C), SARS-CoV-2 (CoV2) alone, and rACE2-treated groups. ∗p < 0.05 vs. C & rACE2-treated groups. Data represented in mean ± SEM. (J and K). Fold changes in the concentrations of 17β Estradiol and ERα/AR ratio assessed from western blot analysis in relation to β-actin loading control in lung tissue in naive control (C), SARS-CoV-2 (CoV2) alone, and rACE2-treated groups. ∗p < 0.05 vs. C and SARS-CoV-2, respectively. n = 5–7 biologically independent mice for all groups. Data represented in mean ± SEM. (see also Figure S3 ).

Article Snippet: The hACE2 concentration in both rabbit and mouse plasma and lung tissue of mice was measured by hACE2 ELISA (#ab235649: Abcam, UK) following the manufacturer’s instructions.

Techniques: Infection, Expressing, Quantitative RT-PCR, Concentration Assay, Staining, Western Blot

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet:

Article Snippet: The hACE2 concentration in both rabbit and mouse plasma and lung tissue of mice was measured by hACE2 ELISA (#ab235649: Abcam, UK) following the manufacturer’s instructions.

Techniques: Isolation, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software