Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The inhibition of the interaction between the spike protein S1 receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor by kuwanon C (KC). Spike protein coated on a 96-well plate interacted with a preincubated mixture of the ACE2 receptor and ( A ) anti-SARS-CoV-2 spike S1 antibody as the positive control and ( B ) 0, 3.125, 6.25, 12.5, 25, 50, or 100 μM KC. The inhibition of the spike S1 RBD:ACE2 receptor interaction by KC was determined based on chemiluminescence measurements.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Inhibition, Binding Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The global kinetic analysis of KC binding to biotinylated ( A ) spike S1 RBD- and ( B ) ACE2 receptor-immobilized BLI sensors. The kinetics for the binding of KC to the spike S1 RBD or the ACE2 receptor were measured by the association of 0, 50, 200, and 400 μM of KC in PBS containing 1% DMSO with immobilized spike S1 or ACE2 receptor and the subsequent dissociation in PBS containing 1% DMSO.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The binding kinetics of KC to spike S1 RBD and ACE2 receptor.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: In silico docking simulation between KC and the spike protein/ACE2 receptor. KC was docked onto the SARS-CoV-2 spike protein and ACE2 receptor (PDB code: 6M0J) using AutoDock Vina. The pharmacophore of KC with each target proteins was analyzed using BIOVIA Discovery Studio Visualizer.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: In Silico

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: KC inhibits SARS-CoV-2 lentiviral pseudovirus infection in HEK293T cells stably expressing human ACE2 and TMPRSS2. ( A ) The cytotoxic effect of KC in HEK293T cells stably expressing human ACE2 and TMPRSS2 was determined using the MTT assay. HEK293T cells were cultured in 96-well plates (5 × 10 4 cells/well) for 18 h. ( B ) The ACE2 expression level in HEK293T cells was monitored during KC treatment using real-time quantitative PCR analysis. Then, WT or mutant (D614G) SARS-CoV-2 spike pseudovirus (at a final concentration of 1 × 10 4 TU/mL to each well) were mixed with different concentrations of KC (2 and 20 μM) or anti-SARS-CoV-2 antibody, and the mixtures were incubated at 37 °C for 1 h. Then, these mixtures were added to HEK293T cells. ( C , D ) Green fluorescent protein (GFP) expression levels using flow cytometry were assessed at 72 h after viral infection, scale bar = 100 μm. ( E , F ) The inhibitory effect of SARS-CoV-2 spike pseudovirus infection was determined by measuring GFP expression using flow cytometry and measured under a fluorescence microscope. Bar graph (mean ± SEM) statistics were determined from three experimental data sets using one-way ANOVA with Tukey’s post hoc test, *** p < 0.001, compared with the CON (KC-untreated) samples. ### p < 0.001, compared with the cell-only sample.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Infection, Stable Transfection, Expressing, MTT Assay, Cell Culture, Real-time Polymerase Chain Reaction, Mutagenesis, Concentration Assay, Incubation, Flow Cytometry, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: KC suppresses the infection of a clinical isolate of SARS-CoV-2 alpha strain (βCoV/Korea/KCDC03/2020) in Vero cells. Vero cells were cultured on 384-well plates (1.2 × 10 4 cells/well) for 24 h. Then, Vero cells were infected with SARS-CoV-2 (MOI 0.0125) immediately after being treated with serially diluted KC and incubated at 37 °C for 24 h. The cells were then stained using anti-SARS-CoV-2 nucleocapsid (N) primary antibody, Alexa Fluor 488-conjugated goat antirabbit IgG secondary antibody, and Hoechst 33342.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Infection, Cell Culture, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: Schematic of the blockade of the SARS-CoV-2 spike S1 RBD:ACE2 receptor interaction by KC.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques:

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: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) GDF15 levels; B) ACE2 levels ; C) ratio GDf15/ACE2 ; The box plots represent the maximum and minimum levels (whiskers), the upper and lower quartiles, and the median. The length of each box represents the interquartile range. Dots represent outliers. Statistical significance between groups was determined using the ANOVA test. ** p <0.001, # p <0.001 UCI vs Control, n/s (non significant); GDF15 (Growth differentiation factor 15); ACE2 (angiotensin-converting enzyme 2).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Control

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 is positive associated with age (A), while ACE2 is not correlated with age (B). Each dot represents an individual value. The solid blue line represents the regression line. The grey shade represents the confidence interval.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: The box plots represent the maximum and minimum levels (whiskers), the upper and lower quartiles, and the median. The length of each box represents the interquartile range. Dots represent outliers. Statistical significance between groups was determined using the ANOVA test. ** p <0.001, # p <0.001 UCI vs Control, n/s (non significant); ACE2 (angiotensin-converting enzyme 2).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Control

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) SARS-CoV-2 cell entry assay strategy: Lentiviral-based replication-defective pseudovirus were generated in HEK293T cells from lentiviral parental genes, SARS-CoV-2 Spike and encapsulating a mCherry reporter. Since the entry steps of the SARS-CoV-2 pseudovirions are governed by the coronavirus Spike protein at their surface, they enter cells in a similar fashion to native counterparts. A549 airway cells were transfected with exogenous GFP-hACE2 enabling SARS-CoV-2 pseudovirus to effectively infect the cells with mCherry reporter. Double-positive GFP/mCherry cells were quantified by flow cytometry to assess viral infection capacity. B,C) Following the strategy described in A, A549 cells expressing GFP-hACE2 were assayed for cell entry by SARS-CoV-2 pseudovirus expressing either empty vector (Δ Spike) or Spike protein corresponding to origin variant (Wuhan-1) or variants-of-concern Alpha, Beta, Delta or Zeta. A representative flow cytometry experiment is shown. Bars demonstrate mean and Standard Error of Mean while each data point represents a unique experiment; ****P < 0.0001; **P < 0.01; *P < 0.1 by t test.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Generated, Transfection, Flow Cytometry, Infection, Expressing, Plasmid Preparation, Variant Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A549 cells were transfected with either GFP-ACE2 WT, GFP-ACE2 polymorphisms or GFP alone. Then, ACE2 protein expression was analyzed by A) Western Blot with MA5-32307 antibody B) Immunocytochemistry (red) with either MA5-32307 antibody (left panel) or MAB933 antibody (right panel). Nuclei was stained with DAPI (blue). Transfected cells contain GFP (green). CT: secondary antibody control to detect unspecific binding. Images were acquired with Cell Observer-Zeiss. Scale bar: 50 μm

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Transfection, Expressing, Western Blot, Immunocytochemistry, Staining, Control, Binding Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) Studying the effect of ACE2 SNPs. Non-synonymous ACE2 single nucleotide polymorphism were selected among those fulfilling the triple criteria of high allelic frequency (Allele freq > 1.00e-4; Allele count > 20); involved in ACE2-claw S-protein RBD-binding interface and previously associated to clinical outcome. B, C) Following the strategy described in , A549 cells expressing either GFP-ACE2 either WT or polymorphisms were assayed for cell entry by SARS-CoV-2 pseudovirus expressing either empty vector (Δ Spike) or Spike protein corresponding to origin variant (Wuhan-1) or variants-of-concern Alpha, Beta, Delta or Zeta. A representative flow cytometry experiment is shown. Bars demonstrate mean and Standard Error of Mean while each data point represents a unique experiment; ***P < 0.001, **P < 0.01, *P < 0.1, ns P >0.1 to WT by t test.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Plasmid Preparation, Variant Assay, Flow Cytometry

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: A) Heatmap showing the distribution of ACE2 variants in the hospitalization severity groups. Coloured squares indicate de presence of the ACE2 variants. Red: Promoting; Green: Protective; Yellow: No-effect. B) Frequencies of ACE2 SNPs among hospitalization severity groups. Bars represent frequencies of the SNP in each group C) Frequencies of ACE2 SNPS among susceptibility groups.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 and ACE2 levels across ACE2 variants.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: GDF15 and ACE2 levels among ICU and non-ICU COVID-19 patients and ACE2 genotypes.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: Genotype 0 corresponding to subjects that did not carry any variant; Genotype 1, subjects that carry at least one promoting variant; Genotype 2, subjects that carry at least one protective variant; Genotype 3, subjects that carry at least one promoting and one protective variant ( ; ). We only found differences in the levels of the ACE2 mRNA ( P <0.05).

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Variant Assay

Journal: bioRxiv

Article Title: GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

doi: 10.1101/2022.05.06.490907

Figure Lengend Snippet: Then, PCR1, PCR2 and PCR3 were performed in order to amplify the regions comprising the studied SNPs. PCR products were sequenced and aligned against reference ACE2 (NM_021804). PCR1: residues from Ser3 to Met249 (743 bp), PCR2: residues Phe308 to Arg621 (944 bp), PCR3: residues from Val670 to Val752 (250 bp) A) Agarose gel electrophoresis with the PCR products of several patients was performed to confirm specificity. B) Representative image of the alignment of the sequenced (forward and reverse) PCR products against reference ACE2 using SnapGene® Software. In particular, the image corresponds to the PCR2 of the non-ICU patient 10 that presents the L351V variant.

Article Snippet: Circulating levels of ACE2 from plasma samples were quantified using the Human ACE2 SimpleStep ELISA® Kit (#ab235649, Abcam) following the manufacturer’s instructions.

Techniques: Agarose Gel Electrophoresis, Software, Variant Assay

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