Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Sequences of small-interfering RNAs

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques:

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Sequences of the primers used for RT-qPCR

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Sequencing

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Correlation analysis between LPS, inflammatory factors, and RAS pathway members. A Heat map showing the correlation between LPS, inflammatory cytokines and RAS members. Blue dots represent positive correlation and red dots represent negative correlation. Larger dots and darker colour correspond to greater correlation. Lower correlation is represented by smaller dots and lighter colour. B Ratio of ACE/ACE2 after treatment of BMEC with different concentrations of LPS. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques:

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Distribution of ACE2 in BMEC. Cells were stained with antibodies against ACE2 and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Representative images were visualized by confocal laser microscopy. Red, ACE2; blue, DAPI. Scale bar: 10 μm.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Staining, Microscopy

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: DAD3 induces ACE2 expression in LPS-induced BMEC. BMEC were treated with LPS (0.5 μg/mL), DA (20 μg/mL), DAD3 (20 μg/mL), LPS+DA (0.5 μg/mL+20 μg/mL) and LPS+DAD3 (0.5 μg/mL+20 μg/mL) for 24 h. A Relative ACE2 mRNA levels were determined by RT-qPCR. B ACE2 protein expression levels were determined by Western blotting. β-actin was used as a control. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Effects of ACE2 silencing and DAD3 treatments on the transcriptional or expression levels of RAS members. A-E BMEC were treated with either control siRNA (scrambled) or ACE2-siRNA for 24 h, followed by treatment with 0.5 μg/mL LPS for 24 h. After washing with PBS, cells were treated with 20 μg/mL DA or 20 μg/mL DAD3 for 24 h. The ACE2, AT1R and AT2R relative mRNA levels (transcriptional levels) of RAS members were determined by RT-qPCR. The Ang II and Ang-(1–7) protein expression levels of RAS members were determined by ELISA. A Relative ACE2 mRNA levels. B Expression levels of Ang II. C Expression levels of Ang-(1–7). D Relative AT1R mRNA levels. E Relative AT2R mRNA levels. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Effects of ACE2 silencing and DAD3 treatments on the transcriptional levels of pro-inflammatory factors. A–D BMEC were treated with either control siRNA (scrambled) or ACE2-siRNA for 24 h, followed by treatment with 0.5 μg/mL LPS for 24 h. After washing with PBS, cells were treated with 20 μg/mL DA or 20 μg/mL DAD3 for 24 h. Relative mRNA levels of pro-inflammatory factors were determined by RT-qPCR. A Relative IL-1β mRNA levels. B Relative IL-6 mRNA levels. C Relative IL-8 mRNA levels. D Relative TNF-α mRNA levels. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Quantitative RT-PCR

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Effects of ACE2 silencing and DA and DAD3 treatment on the expression levels of components of the MAPK and NF-κB pathways. A, B BMEC were treated with either control siRNA (scrambled) or ACE2-siRNA for 24 h, followed by treatment with 0.5 μg/mL LPS for 24 h. After washing with PBS, cells were treated with 20 μg/mL DA or 20 μg/mL DAD3 for 24 h. The phosphorylation levels of p38, JNK1/2/3, ERK1/2 and IκB-α (p-p38, p-JNK, p-ERK1/2 and p-IκB-α) were determined by Western blotting. Activation of NF-κB pathway was indicated by p-IκB-α, an indicator for the activation of NF-κB. The band intensity of all detected proteins was normalized to β-actin and the expressions of p-p38, p-JNK, and p-ERK1/2 were normalized to p38, JNK, and ERK1/2, respectively. A DA treatments on the expression levels of components of the MAPK and NF-κB pathways; B DAD3 treatments on the expression levels of components of the MAPK and NF-κB pathways. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Expressing, Western Blot, Activation Assay

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Immunodetection of ACE2. ( a ) Vero or A549 cells were incubated with the indicated anti-ACE2 antibodies prior to fixation. Right panels show the background of the secondary anti-rabbit IgG antibody. Insets show the cell contours. ( b ) A549 cells cultured for 5 days or 7 days after plating were stained with the indicated anti-ACE2 antibodies after fixation with 4% (w/v) PFA and permeabilization with 0.1% (v/v) Triton X-100 for 5 min. Nuclei were counterstained with DAPI. (p, rabbit polyclonal; ab, rabbit polyclonal antibody from Abcam). Bars, 20 μm. ( c ) Detection of ACE2 by western blot. Total cell lysates from the indicated cell types containing 30 μg of protein, or 100 ng of purified ACE2 protein (ACE2-His) were analyzed by SDS-PAGE followed by immunoblot with the indicated anti-ACE2 antibodies. Left panels show a long and a short exposure of the blot of cell lysates and recombinant protein, respectively. Middle panels show a short exposure in both cases. In the right panel, the total protein on blots was visualized by staining with Simply Blue. ( d ) Scheme depicting the sequence of ACE2, the location of the epitopes of the antibodies used and the region spanned by the recombinant protein. SP, signal peptide; TM, transmembrane domain.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Immunodetection, Incubation, Cell Culture, Staining, Western Blot, Purification, SDS Page, Recombinant, Sequencing

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Detection of Spike constructs and ACE2 in several cell types employing different sequences for immunodetection. ( a ) Scheme of the incubation and washing steps performed for immunodetection. Incubations were carried out for 1 h in the cold. After each incubation coverslips were washed three times with 200 μl of cold PBS. At the end of the procedure, an additional washing step with water was performed before coverslips were allowed to dry and and mounted. ( b ) Representative images from the detection of Spike constructs and ACE2 in the indicated cell lines employing the different immunodetection sequences. The graph shows the colocalization between Spike S1-Fc and ACE2 fluorescent signals for every cell type and immunodetection sequence assayed. Colocalization is expressed as the proportion of Spike S1 colocalizing with ACE2 signal (Manders’ coefficient), measured applying the automatic Costes’ threshold (n ≥ 8 per condition). Results are shown as mean values ± SEM; ns, non-significant, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 by ANOVA with Tukey’s post-test. Bars, 20 μm.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Construct, Immunodetection, Incubation, Sequencing

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Detection of vimentin and ACE2 in A549 cells. ( a ) Live A549 cells were incubated simultaneously with anti-vimentin and anti-ACE2 antibodies, after which they were incubated with a combination of the corresponding secondary antibodies prior to fixation. An illustrative image is depicted showing points of colocalization at intercellular contacts at mid-cell height, highlighted in the colocalization mask. In addition, an overlay of the region of interest delimited by the dotted square with the bright field image is shown to illustrate the juxtanuclear position of one of the colocalization points (white arrow); the contour of the nucleus is highlighted in magenta. The far right panel shows the top section of the region of interest along with the orthogonal projections centered in the colocalization point marked with the arrow. Bars, 20 μm. ( b – d ) Cells were fixed and permeabilized as specified below before immunodetection. ( b ) A549 cells were fixed with 4% (w/v) PFA and permeabilized with 0.1% (v/v) Triton for 5 min for staining with monoclonal antibodies against acetylated tubulin (acTubulin, left panels) or ARL13B (right panels). Nuclei were stained with DAPI. ( c ) A549 cells fixed and permeabilized as in ( b ), were stained with anti-ACE2 and anti-acetylated tubulin antibodies. ( d ) A549 cells were fixed with 2% (w/v) PFA, permeabilized with 0.1% (v/v) Triton X-100 and stained with anti-vimentin antibodies (SP20 or C-end) and either anti-acetylated tubulin or anti-ARL13B antibodies, as indicated. Images in ( b – d ) are single sections. In each case, the regions of interest (dotted square) are enlarged at the right. Bars, 10 μm.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Incubation, Immunodetection, Staining

Journal: Scientific Reports

Article Title: Cell surface detection of vimentin, ACE2 and SARS-CoV-2 Spike proteins reveals selective colocalization at primary cilia

doi: 10.1038/s41598-022-11248-y

Figure Lengend Snippet: Detection of Spike S1, ACE2 and vimentin at primary cilia. A549 cells were incubated with Spike S1-Fc at 37 °C for 90 min in complete medium. Non-permeabilized cells were stained with ( a ) anti-ACE2 and anti-vimentin 84-1 antibodies, ( b ) anti-ACE2 and anti-acetylated tubulin or ( c ) anti-vimentin SP20 and anti-ARL13B antibodies, followed by the corresponding secondary antibodies, and finally with anti-human IgG antibody to detect the Spike S1 Fc tag, before fixation. In ( a , b ) cells were fixed with 4% (w/v) PFA and in ( c ) with 2% (w/v) PFA. Images shown are single sections for each channel obtained by confocal microscopy, at middle height of the cells, and the corresponding overlays. Regions of interest (dotted squares) are enlarged in the lower panels for each condition. Colocalization masks for the signals of ( a ) vimentin and Spike, ( b ) ACE2 and Spike, and ( c ) vimentin and Spike, are shown at the right as white signals on black background; numbers in insets correspond to the Pearson’s coefficient and the percentage of colocalization for the regions shown. Colocalization analysis was performed with Leica software. Arrows in c point to structures showing colocalization. Bars, 10 μm.

Article Snippet: SARS-CoV-2 Spike protein constructs, Spike S1-Fc-Avi and Spike S1-His-Avi (Spike protein residues Gln14 to Arg683), Spike S-Fc-Avi (S1 + S2; amino acids Gln14 to Trp1212), and human ACE2-His-Avi (residues Gln18 to Ser740), all expressed in HEK293 cells, were from Bioss Antibodies.

Techniques: Incubation, Staining, Confocal Microscopy, Software

Journal: World Journal of Gastroenterology

Article Title: Hepatic angiotensin-converting enzyme 2 expression in metabolic dysfunction-associated steatotic liver disease and in patients with fatal COVID-19

doi: 10.3748/wjg.v30.i31.3705

Figure Lengend Snippet: Angiotensin-converting enzyme 2 protein expression pattern in normal and metabolic dysfunction-associated steatotic liver disease livers. A: Representative images of angiotensin-converting enzyme 2 immunostaining across the metabolic dysfunction-associated steatotic liver disease histological spectrum. Scale bars: 200 µm; B: Representative views of boxed areas in panel A (positive and specific staining is indicated with arrowheads). Scale bars: 50 µm. MASH: Metabolic dysfunction-associated steatohepatitis; MASLD: Metabolic dysfunction-associated steatotic liver disease.

Article Snippet: Following this, slides were incubated in hydrogen peroxide for 5 min and then with either the primary anti-ACE2 antibody (Abcam, ab15348; 1:1200 dilution), a primary isotype control antibody as negative control (Thermofisher, AB_2532938), or a second primary anti-ACE2 antibody (Bioss, bs-1004R-A488, 1:200) for 1 hour, followed by incubation with the Refine Detection Kit Polymer for 15 minutes, DAB refine reagent for 10 minutes, and finally haematoxylin counterstain for 5 minutes.

Techniques: Expressing, Immunostaining, Staining

Journal: World Journal of Gastroenterology

Article Title: Hepatic angiotensin-converting enzyme 2 expression in metabolic dysfunction-associated steatotic liver disease and in patients with fatal COVID-19

doi: 10.3748/wjg.v30.i31.3705

Figure Lengend Snippet: Hepatic angiotensin-converting enzyme 2 immunolocalization in cholangiocytes and hepatocytes in normal and metabolic dysfunction-associated steatotic liver disease. Representative images of liver sections showing strong and precise immunostaining for angiotensin-converting enzyme 2 (ACE2) on the apical membrane of cholangiocytes. A: Normal liver; B: Metabolic dysfunction-associated steatotic liver disease (MASLD)-related cirrhosis (arrowheads); C: Normal liver showing punctuate granular intracellular ACE2 immunostaining in hepatocytes (arrowhead); D: Metabolic dysfunction-associated steatohepatitis showing ACE2 immunostaining localised close to the plasma membrane in some steatotic hepatocytes (arrowhead). Scale bars: 20 µm.

Article Snippet: Following this, slides were incubated in hydrogen peroxide for 5 min and then with either the primary anti-ACE2 antibody (Abcam, ab15348; 1:1200 dilution), a primary isotype control antibody as negative control (Thermofisher, AB_2532938), or a second primary anti-ACE2 antibody (Bioss, bs-1004R-A488, 1:200) for 1 hour, followed by incubation with the Refine Detection Kit Polymer for 15 minutes, DAB refine reagent for 10 minutes, and finally haematoxylin counterstain for 5 minutes.

Techniques: Immunostaining, Membrane

Journal: World Journal of Gastroenterology

Article Title: Hepatic angiotensin-converting enzyme 2 expression in metabolic dysfunction-associated steatotic liver disease and in patients with fatal COVID-19

doi: 10.3748/wjg.v30.i31.3705

Figure Lengend Snippet: Hepatic angiotensin-converting enzyme 2 protein expression is elevated in metabolic dysfunction-associated steatohepatitis without fibrosis and correlates with hepatocyte lipid droplet content. A: Computational quantification of hepatic angiotensin-converting enzyme 2 (ACE2) immunostaining in tissue sections across the histopathological metabolic dysfunction-associated steatotic liver disease (MASLD) spectrum. Metabolic dysfunction-associated steatohepatitis (MASH) - fib = MASH without fibrosis, MASH + fib = MASH with fibrosis. Data was analysed using the Kruskal-Wallis test and Dunn’s post-hoc multiple comparisons (Normal vs MASH - fib: P < 0.05; other comparisons not statistically significant); B: Correlation between histological fibrosis (picrosirius red-positive area) and hepatic ACE2 protein levels (ACE2-positive area) across the MASLD spectrum assessed by Spearman’s rank correlation coefficient: r s = 0.3, I = 0.09, not statistically significant); C: Correlation between lipid droplet content (lipid droplet-positive area) and hepatic ACE2 protein levels (ACE2-positive area) across the MASLD spectrum assessed by Spearman’s rank correlation coefficient: r s = 0.5, P = 0.01). ACE2: Angiotensin-converting enzyme 2.

Article Snippet: Following this, slides were incubated in hydrogen peroxide for 5 min and then with either the primary anti-ACE2 antibody (Abcam, ab15348; 1:1200 dilution), a primary isotype control antibody as negative control (Thermofisher, AB_2532938), or a second primary anti-ACE2 antibody (Bioss, bs-1004R-A488, 1:200) for 1 hour, followed by incubation with the Refine Detection Kit Polymer for 15 minutes, DAB refine reagent for 10 minutes, and finally haematoxylin counterstain for 5 minutes.

Techniques: Expressing, Immunostaining

Journal: World Journal of Gastroenterology

Article Title: Hepatic angiotensin-converting enzyme 2 expression in metabolic dysfunction-associated steatotic liver disease and in patients with fatal COVID-19

doi: 10.3748/wjg.v30.i31.3705

Figure Lengend Snippet: Angiotensin-converting enzyme 2 protein levels are increased in post-mortem livers of patients who died with severe coronavirus disease 2019. A: Computational quantification of hepatic angiotensin-converting enzyme 2 (ACE2) protein immunostaining in post-mortem tissue sections from the coronavirus disease-19 ‘Exploration of Critical Aspects of Pathogenesis’ (ICECAP) patient cohort with and without histopathological evidence of liver injury (LI) (ICECAP + LI and ICECAP no LI), including steatosis and/or fibrosis. Groups were compared by one-way ANOVA with Bonferroni’s post-hoc test (Normal vs ICECAP + LI: P < 0.05, comparison of Normal vs ICECAP no LI not statistically significant); B: Correlation between histological fibrosis (picrosirius red-positive area) and hepatic ACE2 protein levels (ACE2-positive area) assessed by Spearman’s correlation coefficient: r s = 0.29, P = 0.39, not statistically significant; C: Correlation between lipid droplet content (lipid droplet positive-area) and hepatic ACE2 protein levels (ACE2-positive area) assessed by Spearman’s correlation coefficient: r s = 0.04, P = 0.89, not statistically significant; D: Representative image showing granular ACE2 immunostaining pattern (arrowhead) in the cytoplasm of hepatocytes in pericentral areas of the liver lobule (central vein) in a steatotic post-mortem liver. Scale bar: 50 µm; E: Representative image showing a bile duct is shown with strong and specific staining localised to the apical membrane of cholangiocytes (arrowhead). Scale bar: 20 µm. ACE2: Angiotensin-converting enzyme 2; LI: Liver injury. CV: Central vein; BD: Bile duct.

Article Snippet: Following this, slides were incubated in hydrogen peroxide for 5 min and then with either the primary anti-ACE2 antibody (Abcam, ab15348; 1:1200 dilution), a primary isotype control antibody as negative control (Thermofisher, AB_2532938), or a second primary anti-ACE2 antibody (Bioss, bs-1004R-A488, 1:200) for 1 hour, followed by incubation with the Refine Detection Kit Polymer for 15 minutes, DAB refine reagent for 10 minutes, and finally haematoxylin counterstain for 5 minutes.

Techniques: Immunostaining, Comparison, Staining, Membrane

Journal: World Journal of Gastroenterology

Article Title: Hepatic angiotensin-converting enzyme 2 expression in metabolic dysfunction-associated steatotic liver disease and in patients with fatal COVID-19

doi: 10.3748/wjg.v30.i31.3705

Figure Lengend Snippet: Hepatic lipid droplet content correlates with detectable severe acute respiratory syndrome coronavirus 2 RNA. A: Representative image showing extensive hepatocellular steatosis (arrowhead) in a post-mortem liver section from an ICECAP study patient who died with severe coronavirus disease-19. Scale bar: 250 µm; B: Hepatic lipid droplet content (lipid droplet-positive area); C: Hepatic angiotensin-converting enzyme 2 (ACE2) protein levels (ACE2-positive area); D: Histological fibrosis (picrosirius red-positive area) in ICECAP patient post-mortem liver sections were compared with the presence or absence of detectable liver severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (liver SARS-Cov-2 PCR result), in the ICECAP study patient cohort, using the Mann-Whitney test for unpaired group comparisons (hepatic lipid droplet content vs detectable liver SARS-CoV-2 viral RNA P < 0.05; other comparisons not statistically significant). PSR: Picrosirius red; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2.

Article Snippet: Following this, slides were incubated in hydrogen peroxide for 5 min and then with either the primary anti-ACE2 antibody (Abcam, ab15348; 1:1200 dilution), a primary isotype control antibody as negative control (Thermofisher, AB_2532938), or a second primary anti-ACE2 antibody (Bioss, bs-1004R-A488, 1:200) for 1 hour, followed by incubation with the Refine Detection Kit Polymer for 15 minutes, DAB refine reagent for 10 minutes, and finally haematoxylin counterstain for 5 minutes.

Techniques: MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

doi: 10.1371/journal.ppat.1010242

Figure Lengend Snippet: (A) Phylogenetic trees depicting N genes isolated from nose-throat swabs by SGS. IDs of individuals from whom swabs were isolated are indicated in the figure. Black rectangles depict the dominant N gene isolated from each swab sample. Red rectangles depict N genes with missense mutations, grey rectangles depict N genes with silent mutations and blue rectangles depict N genes with nonsense mutations. Mutations that are not documented in the GSAID are marked with an asterisk. For each N-gene variant, the nucleotide substitution is indicated (based on Wuhan-Hu-1 numbering). The segment with the number below each phylogenetic tree shows the length of branch that represents an amount genetic change. The amount of genetic change is the number of nucleotide substitutions divided by the length of the N-gene sequence. (B) Pie charts showing the proportion of all N-gene sequences isolated from each swab sample. The White pie slice depicts unmutated sequences. The Colored pie slices depict mutated N genes. The different mutations are indicated in the figure. The number in the middle of the pie chart depicts the total number of N-gene variants that were sequenced. (C) Relative p24 value as measured by adding 20 μl of supernatant containing the pseudovirus to Lenti-X GoStix Plus. The x-axis depicts the SARS-CoV-2 pseudoviruses that were produced, and the y-axis depicts the relative p24 protein (GoStix values) in the supernatant of each pseudovirus. (D) 293T-ACE2 infection with SARS-CoV-2 pseudovirses expressing mutated N proteins and unmutated (Wuhan-Hu-1) N protein (WT). The x-axis depicts the mutation in the N proteins of the pseudovirus that was used for infection. The y-axis depicts the luminescence levels that were measured 48 hours post infection. Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. Statistically significant differences in comparison to the WT SARS-CoV-2 pseudovirus are indicated (student’s t test, p < 0.005). Figure was generated using biorender.com .

Article Snippet: 293T-ACE2 (5x10 4 ) cells were seeded in a 96-well plate, washed, and were incubated with polyclonal anti-ACE2 antibody (Bioss, BS-23028R) or RBD-Ig [ ] for 1 hour at 4°C at 1:20 dilution, followed by incubation with PE-conjugated goat anti-human IgG (Jackson, Cat #109-116-088, 1:200) for 45 minutes.

Techniques: Isolation, Variant Assay, Sequencing, Produced, Infection, Expressing, Mutagenesis, Generated

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

doi: 10.1371/journal.ppat.1010242

Figure Lengend Snippet: (A) Schematic representation of the ten spike variants that were tested for infectivity. Amino acids substitution in each spike mutants is shown in the figure. NTD = N-terminal domain, RBD = receptor binding domain, FP = fusion peptide, HR1 = heptad repeat 1, HR2 = heptad repeat 2, TM = transmembrane domain, CT = cytoplasmic domain. (B) Relative p24 value as measured by adding 20 μl of supernatant containing the pseudovirus to Lenti-X GoStix Plus. The y-axis depicts the SARS-CoV-2 pseudoviruses that were produced, and the x-axis depicts the relative p24 protein (GoStix values) in the supernatant of each pseudovirus. WT = pseudovirus that expresses an unmutated (Wuhan-Hu-1) spike. (C) FACS staining of 293T-ACE2 cells. The gray histogram shows the staining of the 293T-ACE2 cells with secondary antibody only. The empty black histograms depict the staining anti-ACE2 antibody or RBD-Ig. Shown is one representative experiment out of three preformed. (D) 293T-ACE2 infection with SARS-CoV-2 pseudovirses expressing mutated spikes, unmutated (Wuhan-Hu-1) spike protein and bald pseudovirus. The x-axis depicts the mutation in the spike of the pseudovirus that was used for infection. The y-axis depicts the luminescence levels that were measured 48 hours post infection. Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. WT = pseudovirus that expresses an unmutated (Wuhan-Hu-1) spike. Statistically significant differences in comparison to the WT SARS-CoV-2 pseudovirus are indicated (student’s t test, *p < 0.05, **p < 0.005). Figure was generated using biorender.com .

Article Snippet: 293T-ACE2 (5x10 4 ) cells were seeded in a 96-well plate, washed, and were incubated with polyclonal anti-ACE2 antibody (Bioss, BS-23028R) or RBD-Ig [ ] for 1 hour at 4°C at 1:20 dilution, followed by incubation with PE-conjugated goat anti-human IgG (Jackson, Cat #109-116-088, 1:200) for 45 minutes.

Techniques: Infection, Binding Assay, Produced, Staining, Expressing, Mutagenesis, Generated

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

doi: 10.1371/journal.ppat.1010242

Figure Lengend Snippet: (A, C) OD values of ELISA against spike S1 subunit of plasma from vaccinated individuals (A) and of convalescent plasma (C). Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. OD values of control plasma (unvaccinated healthy individual) are shown in the red dashed graph. (B) The luminescence values derived from 293T-ACE2 cells 48 hours after infection with nanoluc-expressing SARS-CoV-2 pseudovirus and following incubation with increasing concentrations of plasma from vaccinated individuals. The plasma dilution is shown in the x-axis. Luminescence values after incubation with control (unvaccinated healthy individual) is shown in the red-dashed graph. Experiments were done in triplicates and repeated two times. Mean values and standard errors are shown; representative of two independent experiments is shown. (D) The luminescence values derived from 293T-ACE2 cells 48 hours post infection with nanoluc-expressing SARS-CoV-2 pseudovirus and following incubation with convalescent plasma at 10 −3 dilution. The study ID of the plasma samples is shown in the x-axis. Experiments were done in triplicates and repeated two times. Mean values and standard errors are shown; representative of two independent experiments is shown.

Article Snippet: 293T-ACE2 (5x10 4 ) cells were seeded in a 96-well plate, washed, and were incubated with polyclonal anti-ACE2 antibody (Bioss, BS-23028R) or RBD-Ig [ ] for 1 hour at 4°C at 1:20 dilution, followed by incubation with PE-conjugated goat anti-human IgG (Jackson, Cat #109-116-088, 1:200) for 45 minutes.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Infection, Expressing, Incubation

Journal: Physical chemistry chemical physics : PCCP

Article Title: Corilagin and 1,3,6-Tri- O -galloy-β-D-glucose: potential inhibitors of SARS-CoV-2 variants.

doi: 10.1039/d1cp01790j

Figure Lengend Snippet: Fig. 5 Characterization of molecular interactions by surface plasmon resonance. (A and B) Binding kinetics of corilagin and TGG on immobilized (A) RBD and (B) ACE2. The recombinant proteins RBD and ACE2 are respectively immobilized on a CM5 sensor chip and increasing concentra- tions of polyphenols are injected to evaluate binding kinetics. (C) Kinetics of ACE2 binding to immobilized RBD (left panel) and kinetics of RBD binding to immobilized ACE2 (right panel). (D) Pre-incubation of RBD (50 nM) for 30 minutes with increasing concentrations of corilagin or TGG inhibit the binding of RBD to immobilized ACE2.

Article Snippet: Recombinant Human ACE2 Protein was purchased from Bioss Inc. (Cat number: BS-46110P).

Techniques: SPR Assay, Binding Assay, Recombinant, Injection, Incubation

Journal: Physical chemistry chemical physics : PCCP

Article Title: Corilagin and 1,3,6-Tri- O -galloy-β-D-glucose: potential inhibitors of SARS-CoV-2 variants.

doi: 10.1039/d1cp01790j

Figure Lengend Snippet: Fig. 6 Inhibitory effects of TGG, corilagin and their mixture on the interaction between SARS CoV-2 Spike protein and human ACE2. TGG (A) and corilagin (B) are tested at different concentrations (0.1, 1, 5 and 10 mM) and their mixture (C) (0,1, 1, 5 mM) to evaluate their ability to inhibit the binding of immobilized Spike protein (0.5 mg ml1) to human biotin labeled ACE2 (0.5 mg ml1), by using the ELISA assay. The absorbance values at 450 nm of human ACE2 (0.5 mg ml1) are set to 100%. Results are expressed as mean standard error of the mean (SEM) of two (combined effect) or three independent assays. Statistical analysis is performed using the One-way ANOVA followed by the Dunnetts post hoc test with *p o 0.05, **p o 0.01, ***p o 0.001 compared to human ACE2 (0.5 mg ml1).

Article Snippet: Recombinant Human ACE2 Protein was purchased from Bioss Inc. (Cat number: BS-46110P).

Techniques: Binding Assay, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Physical chemistry chemical physics : PCCP

Article Title: Corilagin and 1,3,6-Tri- O -galloy-β-D-glucose: potential inhibitors of SARS-CoV-2 variants.

doi: 10.1039/d1cp01790j

Figure Lengend Snippet: Fig. 7 Inhibitory effects of TGG, corilagin and their mixture on the interaction between human ACE2 and ACE2 antibody (18–740 AA). TGG (A) and corilagin (B) are tested at different concentrations (0.1, 1, 5 and 10 mM) and their mixture (C) (0,1, 1, 5 mM) to study their ability to inhibit the binding of immobilized ACE2 antibody (0.5 mg ml1) to human biotin labeled ACE2 (0.5 mg ml1), by using the ELISA assay. The absorbance values at 450 nm of human ACE2 (0.5 mg ml1) were set to 100%. Results are expressed as mean standard error of the mean (SEM) of two (combined effect) or three independent assays. Statistical analysis was performed using the One-way ANOVA followed by the Dunnetts post hoc test with *p o 0.05, **p o 0.01, ***p o 0.001 compared to human ACE2 (0.5 mg ml1).

Article Snippet: Recombinant Human ACE2 Protein was purchased from Bioss Inc. (Cat number: BS-46110P).

Techniques: Binding Assay, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Physical chemistry chemical physics : PCCP

Article Title: Corilagin and 1,3,6-Tri- O -galloy-β-D-glucose: potential inhibitors of SARS-CoV-2 variants.

doi: 10.1039/d1cp01790j

Figure Lengend Snippet: Fig. 8 Mutations effect on the solvent accessibility of RBD alone. RBD’s per residue solvent accessible surface area (SASA) difference between the MD and the experimental structure. Only the residues of RBD interacting with ACE2 (contact probability greater than 60% during the MD simulation) in the complex structure are shown. The red residue number indicates the position of a mutation. The SASA of wildtype (blue), E484K (teal), N501Y (yellow) and E484K/N501Y (red) are compared. The error bars correspond to the standard deviation over the 250–500 ns interval.

Article Snippet: Recombinant Human ACE2 Protein was purchased from Bioss Inc. (Cat number: BS-46110P).

Techniques: Solvent, Residue, Mutagenesis, Standard Deviation