Journal: International journal of molecular sciences

Article Title: Long Chain N3-PUFA Decreases ACE2 Protein Levels and Prevents SARS-CoV-2 Cell Entry.

doi: 10.3390/ijms232213825

Figure Lengend Snippet: Figure 1. DHA reduces both ACE1 and ACE2 levels in key rat tissues. Western blotting was used to measure ACE1 and ACE2 levels relative to total protein load, as

Article Snippet: Rabbit anti-ACE2 polyclonal primary antibody (Catalog #: 3217, ProSci, San Diego, CA, USA) and StarBright Blue 700 Goat Anti-Rabbit secondary antibody (Catalog#: 12004162, Bio-Rad, Hercules, CA, USA) were used to detect ACE2.

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: Long Chain N3-PUFA Decreases ACE2 Protein Levels and Prevents SARS-CoV-2 Cell Entry.

doi: 10.3390/ijms232213825

Figure Lengend Snippet: Figure 2. DHA differentially modulates ACE1 and ACE2 levels in growing and quiescent EA.hy926 en- dothelial cells. Western blotting was used to compare ACE1 and ACE2 relative to total protein load, as measured by Ponceau S in growing cells treated with ALA, EPA, or DHA at the indicated concentrations (µM) for (a) 8 and (b) 24 h, as well as in quiescent cells treated with n3-PUFA for (c) 8 and (d) 24 h. The band intensities of ACE2 and ACE1 were quantified and are graphically presented in panels (e,f), respectively; the 100 kDa band (grey bars) represents non-glycosylated ACE2, whereas the 130 kDa band (black bars) corresponds to N-glycosylated ACE2. Data are presented as mean ± SEM, n = 3; bars not sharing a common letter in the graphs are significantly different (p < 0.05) based on Duncan’s multiple range or LSD post-hoc tests.

Article Snippet: Rabbit anti-ACE2 polyclonal primary antibody (Catalog #: 3217, ProSci, San Diego, CA, USA) and StarBright Blue 700 Goat Anti-Rabbit secondary antibody (Catalog#: 12004162, Bio-Rad, Hercules, CA, USA) were used to detect ACE2.

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: Long Chain N3-PUFA Decreases ACE2 Protein Levels and Prevents SARS-CoV-2 Cell Entry.

doi: 10.3390/ijms232213825

Figure Lengend Snippet: Figure 5. Schematic of proposed mechanism of action. LCn3-PUFAs, EPA and DHA, reduce ACE2 protein levels. These n3-PUFAs may also decrease ACE2 glycosylation. These mechanisms may explain how DHA blocks SARS-CoV-2 pseudovirus entry into HEK293 cells. LCn3-PUFAs also downregulate ACE1 protein levels, thus maintaining the balance between ACE1 and ACE2, and diminishing the risk of adverse CVD outcomes. This figure was prepared by S. Huang using Microsoft PowerPoint software version 16.16.27.

Article Snippet: Rabbit anti-ACE2 polyclonal primary antibody (Catalog #: 3217, ProSci, San Diego, CA, USA) and StarBright Blue 700 Goat Anti-Rabbit secondary antibody (Catalog#: 12004162, Bio-Rad, Hercules, CA, USA) were used to detect ACE2.

Techniques: Glycoproteomics, Software

Journal: Cell Metabolism

Article Title: Expression of SARS-CoV-2 Entry Factors in the Pancreas of Normal Organ Donors and Individuals with COVID-19

doi: 10.1016/j.cmet.2020.11.005

Figure Lengend Snippet:

Article Snippet: Thereafter, the membranes were incubated at 4°C overnight with one of four primary antibodies (rabbit monoclonal anti-ACE2 (1:1,000 dilution, Abcam, Cambridge, MA), rabbit polyclonal anti-ACE2 (1:500 dilution, Abcam), mouse monoclonal anti-ACE2 (1:1,000 dilution, R&D Systems), goat polyclonal anti-ACE2 (1:500 dilution, R&D Systems, Minneapolis, MN)) and mouse monoclonal anti-b-actin (1:10,000 dilution; Sigma-Aldrich, St. Louis, MO) in Intercept Antibody Diluent (LI-COR Biosciences).

Techniques: Plasmid Preparation, Control, Recombinant, Protease Inhibitor, Blocking Assay, Avidin-Biotin Assay, Staining, RNAscope, Multiplex Assay, Fluorescence, BIA-KA, Generated, Gene Expression, Software

Journal: Hypertension

Article Title: Overexpression of Angiotensin-Converting Enzyme 2 in the Rostral Ventrolateral Medulla Causes Long-Term Decrease in Blood Pressure in the Spontaneously Hypertensive Rats

doi: 10.1161/01.hyp.0000259942.38108.20

Figure Lengend Snippet: Figure 1. ACE2 immunoreactivity in the rat medulla oblongata. A, Coronal sections of the medulla oblongata were immunostained with polyclonal anti-ACE2 antibody (sc-20998, Santa Cruz Biotechnology) as described in the Methods section. Sections incubated without primary antibody were used as negative control (a and e). The low-magnification photographs of sections (top) show the distribution of ACE2 immunoreactivity in the caudal part of medulla oblongata including area postrema (AP) and the dorsal motor nucleus of the vagus (b), the NTS (c), and the ventrolateral medulla, including nucleus ambiguus and RVLM (d). The high-magnification photographs of sections (bottom) show ACE2 immunoreactivity of neurons in the AP (f), neurons (arrowhead) and neuropils (arrow) of the NTS (g) and neurons of the RVLM (h). The immunoreactivity in the RVLM neurons was observed in cytoplasm of neurons (arrowhead) and its proxi- mal dendrite (arrow). The scale bar denotes 100 m in the top and 20 m in the bottom. B, Coronal sections of the medulla oblongata were immunostained with polyclonal anti-ACE2 antibody (GTX15348, GeneTex) as described in the Methods section. The low- magnification photographs of sections show negative immunostaining controls consisted of exclusion and preadsorption of the primary antibodies for ACE2 (a and b, respectively). The corresponding low-magnification photograph (c) shows the distribution of ACE2 immunoreactivity in the ventrolateral medulla. The distribution of ACE2 immunoreactivity using this antibody was similar to that of immunoreactivity using the Santa Cruz antibody. The high-magnification photograph shows ACE2 immunoreactivity in the RVLM neurons (d). The scale bar denotes 100 m in a, b, and c, and 20 m in d.

Article Snippet: They were incubated with rabbit polyclonal antibody to ACE2 (1:50, sc-20998, Santa Cruz Biotechnology) or another rabbit polyclonal antibody to ACE2 (1:500, GTX15348, GeneTex) containing 0.3% BSA in PBS containing 0.3% Triton X100 overnight at 4°C followed by incubation with biotinylated goat anti-rabbit IgG for 120 minutes and avidin–biotin–peroxidase complex reagents for 60 minutes and stained with diaminobenzidine solution for 8 minutes according to the manufacturer’s instructions (Vector Laboratories).

Techniques: Incubation, Negative Control, Immunostaining

Journal: Hypertension

Article Title: Overexpression of Angiotensin-Converting Enzyme 2 in the Rostral Ventrolateral Medulla Causes Long-Term Decrease in Blood Pressure in the Spontaneously Hypertensive Rats

doi: 10.1161/01.hyp.0000259942.38108.20

Figure Lengend Snippet: Figure 2. ACE2 protein levels in the RVLM of WKY rats and SHRs. A, Representative autoradiogram of ACE2 protein levels in the RVLM: Western blot analysis was used to measure ACE2 levels from 20 g of total cell lysate isolated from the RVLM punches as described in the Methods section. Data were nor- malized using -tubulin. B, Quantitation of the ACE2 protein band. *P0.05 vs WKY rats. Data were meanSEM (n4 in each strain).

Article Snippet: They were incubated with rabbit polyclonal antibody to ACE2 (1:50, sc-20998, Santa Cruz Biotechnology) or another rabbit polyclonal antibody to ACE2 (1:500, GTX15348, GeneTex) containing 0.3% BSA in PBS containing 0.3% Triton X100 overnight at 4°C followed by incubation with biotinylated goat anti-rabbit IgG for 120 minutes and avidin–biotin–peroxidase complex reagents for 60 minutes and stained with diaminobenzidine solution for 8 minutes according to the manufacturer’s instructions (Vector Laboratories).

Techniques: Western Blot, Isolation, Quantitation Assay

Journal: Hypertension

Article Title: Overexpression of Angiotensin-Converting Enzyme 2 in the Rostral Ventrolateral Medulla Causes Long-Term Decrease in Blood Pressure in the Spontaneously Hypertensive Rats

doi: 10.1161/01.hyp.0000259942.38108.20

Figure Lengend Snippet: Figure 3. Transduction of the SHR RVLM with lenti-ACE2. After termination of the experiment, lentiviral-injected rats were used to eval- uate transgene expression by GFP fluorescence and ACE2 immunostaining as described in the Methods section. A, Representative photograph of lenti-ACE2–injected medulla oblongata: the GFP expression was restricted to the bilateral RVLM. The scale bar denotes 1 mm at the top and 50 m at the bottom. B, Overexpression of ACE2: low-magnification photographs show the lenti-ACE2–injected RVLM. ACE2 immunoreactivity was stronger in the lenti-ACE2–injected site (right) compared with its endogenous expression in the neu- rons (left). The scale bar denotes 200 m at the top and 50 m at the bottom. C, Colocalization of GFP and ACE2 in the lenti-ACE2– injected RVLM: GFP was colocalized with ACE2 in most of the cells. Arrows identify colocalization in individual cells, whereas areas of cells clusters representing both GFP and ACE2 are marked with asterisks. The scale bars denote 200 m.

Article Snippet: They were incubated with rabbit polyclonal antibody to ACE2 (1:50, sc-20998, Santa Cruz Biotechnology) or another rabbit polyclonal antibody to ACE2 (1:500, GTX15348, GeneTex) containing 0.3% BSA in PBS containing 0.3% Triton X100 overnight at 4°C followed by incubation with biotinylated goat anti-rabbit IgG for 120 minutes and avidin–biotin–peroxidase complex reagents for 60 minutes and stained with diaminobenzidine solution for 8 minutes according to the manufacturer’s instructions (Vector Laboratories).

Techniques: Transduction, Injection, Expressing, Immunostaining, Over Expression

Journal: Hypertension

Article Title: Overexpression of Angiotensin-Converting Enzyme 2 in the Rostral Ventrolateral Medulla Causes Long-Term Decrease in Blood Pressure in the Spontaneously Hypertensive Rats

doi: 10.1161/01.hyp.0000259942.38108.20

Figure Lengend Snippet: Figure 4. ACE2 protein levels in the RVLM after ACE2 gene transfer. A, Representative autoradiogram of ACE2 protein levels in the RVLM: Western blot analysis was used to measure ACE2 protein levels from the RVLM punches of noninjected WKY rats and SHRs and SHRs 6 weeks after lenti-ACE2 injection. Data were normalized using -tubulin. B, Quantitation of the ACE2 protein band. Data were meanSEM (n4 in each group).

Article Snippet: They were incubated with rabbit polyclonal antibody to ACE2 (1:50, sc-20998, Santa Cruz Biotechnology) or another rabbit polyclonal antibody to ACE2 (1:500, GTX15348, GeneTex) containing 0.3% BSA in PBS containing 0.3% Triton X100 overnight at 4°C followed by incubation with biotinylated goat anti-rabbit IgG for 120 minutes and avidin–biotin–peroxidase complex reagents for 60 minutes and stained with diaminobenzidine solution for 8 minutes according to the manufacturer’s instructions (Vector Laboratories).

Techniques: Western Blot, Injection, Quantitation Assay

Journal: Hypertension

Article Title: Overexpression of Angiotensin-Converting Enzyme 2 in the Rostral Ventrolateral Medulla Causes Long-Term Decrease in Blood Pressure in the Spontaneously Hypertensive Rats

doi: 10.1161/01.hyp.0000259942.38108.20

Figure Lengend Snippet: Figure 5. Effect of lenti-ACE2 on MAP and HR in WKY rats and SHRs. Lenti-GFP or lenti-ACE2 was injected into the bilateral RVLM of WKY rats (A) and SHRs (B), and MAP (left) and HR (right) were recorded in a conscious state using radioteremetry as described in the Methods section. Lenti- ACE2–injected SHRs showed a time- dependent decrease in MAP and HR. Data are meanSEM (n4 per group in WKY rats and n6 per group in SHRs) *P0.05 vs GFP control groups.

Article Snippet: They were incubated with rabbit polyclonal antibody to ACE2 (1:50, sc-20998, Santa Cruz Biotechnology) or another rabbit polyclonal antibody to ACE2 (1:500, GTX15348, GeneTex) containing 0.3% BSA in PBS containing 0.3% Triton X100 overnight at 4°C followed by incubation with biotinylated goat anti-rabbit IgG for 120 minutes and avidin–biotin–peroxidase complex reagents for 60 minutes and stained with diaminobenzidine solution for 8 minutes according to the manufacturer’s instructions (Vector Laboratories).

Techniques: Injection, Control

Journal: medRxiv

Article Title: SARS-CoV-2 mRNA vaccination exposes progressive adaptive immune dysfunction in patients with chronic lymphocytic leukemia

doi: 10.1101/2022.12.19.22283645

Figure Lengend Snippet: (A and B) Comparison of SARS-CoV-2 vaccine-elicited spike (A) and RBD (B) ELISA IgG antibody titers in HC (n=30) and CLL (n=95) patients expressed as half-maximal effective concentrations (EC 50 ). (C and D) SARS-CoV-2 vaccine-elicited spike (C) and RBD (D) ELISA IgG antibody titers stratified by CLL disease status: treatment-naïve (Naïve) (n=45), on-therapy (On Tx) (n=34), off-therapy in clinical remission (CR) (n=9), and off therapy and relapsed or refractory (R/R) (n=7). (E-G) Comparison of SARS-CoV-2 vaccine-elicited neutralizing antibody titers for HC (n=30) and CLL patients against (E) D614G (CLL; n=95) and (F) delta (CLL; n=93) spike variants expressed as the reciprocal half-maximal inhibitory dilution (ID 50 ) as determined in an HIV-based pseudovirus neutralization assay or by (G) ACE2/RBD (Wuhan) binding inhibition (CLL; n=95) at a 1:25 dilution. (H-J) SARS-CoV-2 vaccine elicited NAb titers for D614G (H) and delta (I) S variants and ACE2/RBD binding frequencies (J) stratified by CLL disease status. Bars indicate the median with 95% CI. Dotted black lines indicate assay sensitivity cutoffs (EC 50 values of <100 by ELISA, ID 50 values of <20 in the neutralization assay, and >90% ACE2 binding in the RBD-inhibition assay). P values were determined by the Mann-Whitney test (A-B, E-G) or Dunn’s test of multiple comparisons following a Kruskal-Wallis test (C-D, H-J).

Article Snippet: Plates were incubated at room temperature for 1 h, washed 4 times with PBST, and 50 µl of biotinylated goat anti-human ACE2 (R&D) diluted at 0.1 µg/ml in PBST was added to the wells.

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Binding Assay, Inhibition, MANN-WHITNEY

Journal: Nature Communications

Article Title: Trimethylamine N-oxide impairs β-cell function and glucose tolerance

doi: 10.1038/s41467-024-46829-0

Figure Lengend Snippet: a Schematic diagram of tests in male mice during chow diet and choline diet feeding. b Plasma TMAO concentration of male chow- and choline diet-fed (4 weeks) mice. n = 6 mice. c IPGTT of chow- and choline diet-fed (5 weeks) mice. n = 15 mice. The glucose dose was 2 g/kg of body weight. This step was followed by determining the AUC of the GTT. d – g Plasma insulin ( d ), increase in plasma insulin (%, e ), C-peptide ( f ) and increase in C-peptide (%, g ) in male chow- and choline diet-fed (9 weeks) mice after 0, 2, and 5 min of intraperitoneal injection of glucose. n = 15 mice ( d , e ); n = 7 mice ( f , g ). h – j Plasma insulin and AUC of first-phase (0–5 min) and second-phase (5–120 min) insulin levels during hyperglycemic clamp of male chow- ( n = 7 mice) and choline diet-fed (13 weeks) mice ( n = 9 mice). k GSIS of primary islets from male chow- and choline diet-fed (10 weeks) mice. n = 4 biologically independent islet samples. l Pancreatic HE staining in male chow- and choline diet-fed (13 weeks) mice. The arrowhead indicates inflammatory cells. Scale bar, 100 μm. m – o Immunofluorescence of insulin (green), glucagon (red), and DAPI (blue) in paraffin-embedded pancreas sections from male chow- and choline diet-fed mice ( m ). This analysis was followed by measurements of % β-cell area ( n ) and % α cell area ( o ). n = 6 mice. Scale bar, 20 μm. p Plasma glucagon levels of male chow- ( n = 10 mice) and choline diet-fed (6 weeks) mice ( n = 9 mice). Statistical significance was calculated ( b – e , g – k , n – p ) by two-sided Student’s t -test. P values in ( h ) denoted by asterisks (from left to right): P = 0.049, P = 0.046, P = 0.026, P = 0.006. Mice: C57BL/6 J, choline diet feeding from 8 weeks old ( a – p ). Ins, insulin; Gcg, glucagon; AUC, area under the curve. Source data are provided as a Source Data file.

Article Snippet: Normal β cells were obtained through negative selection with magnetic activated cell sorting (MACS) using the α cell marker ACE2 (anti-ACE2-Biotin (Novus, cat. #NBP1-76614B, 1:10)), and the δ-cell marker CD24 (anti-CD24-Biotin (Miltenyi Biotec, clone M1/69, cat. #130-101-982, 1:10)) and identified by insulin staining.

Techniques: Clinical Proteomics, Concentration Assay, Injection, Staining, Immunofluorescence

Journal: Nature Communications

Article Title: Trimethylamine N-oxide impairs β-cell function and glucose tolerance

doi: 10.1038/s41467-024-46829-0

Figure Lengend Snippet: a Schematic diagram of tests in male Fmo3 +/+ and Fmo3 −/− mice fed a choline diet. b Hepatic Fmo3 mRNA levels in male Fmo3 +/+ and Fmo3 −/− mice. n = 3 mice. c Plasma TMAO levels in male Fmo3 +/+ and Fmo3 −/− mice after 6 weeks of a choline diet. n = 6 mice. d IPGTTs of male choline diet-fed (9 weeks) Fmo3 +/+ ( n = 7 mice) and Fmo3 −/− mice ( n = 10 mice). Then, the AUC of the GTT was determined. e Plasma insulin levels and increases in plasma insulin (%) after intraperitoneal injection of glucose for 0, 2, and 5 min in male choline diet-fed (14 weeks) Fmo3 +/+ and Fmo3 −/− mice. n = 7 mice. f Plasma C-peptide levels and increases in plasma C-peptide (%) after intraperitoneal injection of glucose for 0, 2, and 5 min in male choline diet-fed (15 weeks) Fmo3 +/+ ( n = 6 mice) and Fmo3 −/− mice ( n = 10 mice). g , h Plasma insulin levels and AUC of first-phase (0–5 min) and second-phase (5–120 min) insulin levels during hyperglycemic clamp in male choline diet-fed (18 weeks) Fmo3 +/+ and Fmo3 −/− mice. n = 5 mice. i – k Immunofluorescence of insulin (green), glucagon (red) and DAPI (blue) in male Fmo3 +/+ and Fmo3 −/− choline diet-fed (18 weeks) mice ( i ). This step was followed by measurements of % β-cell area ( j ) and % α cell area ( k ). n = 8 mice. Scale bar, 10 μm. Statistical significance was calculated ( b – h , j ) by two-sided Student’s t -test. The data are presented as mean ± SEM. Mice: C57BL/6 J, choline diet feeding from 8 weeks old ( a – k ). Source data are provided as a Source Data file.

Article Snippet: Normal β cells were obtained through negative selection with magnetic activated cell sorting (MACS) using the α cell marker ACE2 (anti-ACE2-Biotin (Novus, cat. #NBP1-76614B, 1:10)), and the δ-cell marker CD24 (anti-CD24-Biotin (Miltenyi Biotec, clone M1/69, cat. #130-101-982, 1:10)) and identified by insulin staining.

Techniques: Clinical Proteomics, Injection, Immunofluorescence

Journal: Nature Communications

Article Title: Trimethylamine N-oxide impairs β-cell function and glucose tolerance

doi: 10.1038/s41467-024-46829-0

Figure Lengend Snippet: a Schematic diagram of tests during ASO injection. b , c Hepatic Fmo3 mRNA and protein levels after 10 weeks of ASO treatment in male db/db mice. n = 5 ( b ), or 4 ( c ) mice. d Plasma TMAO concentration after 4 weeks of ASO treatment in male db/db mice. n = 10 mice. e Serca2 protein levels in primary islets from ASO-treated (6 weeks) male db/db mice. n = 3 mice. f IVGTTs after 6 weeks of ASO treatment in male db/db mice. n = 9 mice. g , h Plasma insulin ( g ) and increase in plasma insulin (%, h ) during IVGTT. n = 7 (control ASO), or n = 6 ( Fmo3 ASO) mice. i , j C-peptide ( i ) and increase in C-peptide (%, j ) during IVGTT. n = 7 mice. k GSIS in primary islets after 10 weeks of ASO treatment in male db/db mice. n = 4 (control ASO at 2.8 mM Glu), 5 ( Fmo3 ASO at 2.8 mM Glu), 6 (control ASO at 16.8 mM Glu), or 8 ( Fmo3 ASO at 16.8 mM Glu) biologically independent islet samples. l – n Immunofluorescence of insulin (green), glucagon (red) and DAPI (blue) in male control and Fmo3 ASO-treated db/db mice ( l ). This step was followed by measurements of % β-cell area ( m ) and % α cell area ( n ). n = 10 mice. Scale bar, 50 μm. o – t Immunofluorescence for insulin (green; o , q , s ), Sox9 (red; o ), Pdx1 (red; q ), CC3 (red; s ) and DAPI (blue; o , q , s ) in male control and Fmo3 ASO-treated db/db mice. This step was followed by measurements of the % Sox9 + /Ins + area ( p ), % Pdx1 + /Ins + area ( r ), % CC3 + /Ins + area ( t ). n = 13 (control ASO), or 8 ( Fmo3 ASO) ( p ); n = 9 (control ASO), or 12 ( Fmo3 ASO) ( r ); n = 10 ( t ). Scale bar, 10 μm ( o ), or 20 μm ( q , s ). Statistical significance was calculated ( b–i , k , m , n , p , r , t ) by two-sided Student’s t -test. The data are presented as mean ± SEM. Mice: db/db , ASO treatment (50 mg/kg body weight) from 6 weeks old ( a – t ). ASO, antisense oligonucleotides. Source data are provided as a Source Data file.

Article Snippet: Normal β cells were obtained through negative selection with magnetic activated cell sorting (MACS) using the α cell marker ACE2 (anti-ACE2-Biotin (Novus, cat. #NBP1-76614B, 1:10)), and the δ-cell marker CD24 (anti-CD24-Biotin (Miltenyi Biotec, clone M1/69, cat. #130-101-982, 1:10)) and identified by insulin staining.

Techniques: Injection, Clinical Proteomics, Concentration Assay, Control, Immunofluorescence

Journal: iScience

Article Title: Predicted risk of heart failure pandemic due to persistent SARS-CoV-2 infection using a three-dimensional cardiac model

doi: 10.1016/j.isci.2023.108641

Figure Lengend Snippet: Deterioration of the tissue function of persistent infection model of SARS-CoV-2 triggered by hypoxic stress (A and B) Assessment of cardiac function. “SARS-CoV-2” indicates persistent infection model of SARS-CoV-2. (A) Beats per minute (BPM) at pre-treatment and after 48h of reperfusion condition (n = 6 each). Error bars show S.D. B, PIPM over time (n = 6 each). (C and D) IFA for persistent infection model of SARS-CoV-2 before treatment (Pre-treatment), 18h of hypoxia treatment (Hypoxia) or Normoxia followed by 48h reperfusion treatment. (C) cTnT (Green) and ACE2 (Red). (D) cTnT (Green) and S protein (Red). (E) CD31 (Green). Scale bars: C,D,100 μm. (E) 500 μm. Nuclei were stained with DAPI (Blue).

Article Snippet: For IFA, CMTs were stained with cTnT antibody (Thermo Fisher) (1:250), CD31 (monoclonal mouse IgG1, clone 9G11) (R&D) (1:250), ACE2 (polyclonal antibody) (Bioss, Woburn, MA, USA) (1:250), S protein (ab272504), Abcam (1:250) or monoclonal mouse antibody provided by Dr. Hisashi Arase (Osaka University) (1:50), with DAPI (4‘,6-diamidino-2-phenylindole) (Thermo Fisher) (1:1000).

Techniques: Infection, Staining

Journal: iScience

Article Title: Predicted risk of heart failure pandemic due to persistent SARS-CoV-2 infection using a three-dimensional cardiac model

doi: 10.1016/j.isci.2023.108641

Figure Lengend Snippet:

Article Snippet: For IFA, CMTs were stained with cTnT antibody (Thermo Fisher) (1:250), CD31 (monoclonal mouse IgG1, clone 9G11) (R&D) (1:250), ACE2 (polyclonal antibody) (Bioss, Woburn, MA, USA) (1:250), S protein (ab272504), Abcam (1:250) or monoclonal mouse antibody provided by Dr. Hisashi Arase (Osaka University) (1:50), with DAPI (4‘,6-diamidino-2-phenylindole) (Thermo Fisher) (1:1000).

Techniques: Virus, Recombinant, Modification, Staining, Labeling, In Situ, Avidin-Biotin Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Software

Journal: BioMed Research International

Article Title: Mas -Mediated Antioxidant Effects Restore the Functionality of Angiotensin Converting Enzyme 2-Angiotensin-(1–7)- Mas Axis in Diabetic Rat Carotid

doi: 10.1155/2014/640329

Figure Lengend Snippet: ACE2, angiotensin-(1–7) and Mas receptors staining in carotid arteries from nontreated control or diabetic rats. ACE2 expression in nontreated control (a) or diabetic (b) rat carotid, angiotensin-(1–7) levels in nontreated control (c) or diabetic (d) rat carotid, and Mas receptors expression in nontreated control (e) or diabetic (f) rat carotid. E: endothelium; M: media; Adv: adventitia. The immunostaining is denoted in red (magnification: 100x).

Article Snippet: They are STZ, angiotensin II, angiotensin-(1–7), losartan, PD123,319, hydroxocobalamin, L-NNA, 1400 W, apocynin, tiron, PEG-catalase, CDCF-DA, and DCPIB (Sigma, St. Louis, Mo., USA); DX600 (Anaspec Inc., Fremont, CA, USA); A779 (Bachem California Inc., Torrance, CA, USA); L-NPA (Tocris, Avonmouth, UK); DHE (Invitrogen, Carlsbad, CA, USA); ketamine (União Química, Jabaquara, SP, Brazil); xylazine (Calier Laboratory, Jubatuba, MG, Brazil); rabbit polyclonal anti-ACE2 antibody (Abcam, Cambridge, MA, USA); rabbit anti-angiotensin-(1–7) antibody (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA); rabbit polyclonal anti-angiotensin-(1–7)- Mas receptor antibody (Alomone Labs, Jerusalem, Israel); biotinylated universal secondary antibody, avidin-biotin peroxidase complex (Vectastain Elite ABC kit, Universal, Vector Laboratories Inc. U.S. Headquarters, Burlingame, CA, USA).

Techniques: Staining, Control, Expressing, Immunostaining

Journal: BioMed Research International

Article Title: Mas -Mediated Antioxidant Effects Restore the Functionality of Angiotensin Converting Enzyme 2-Angiotensin-(1–7)- Mas Axis in Diabetic Rat Carotid

doi: 10.1155/2014/640329

Figure Lengend Snippet: Conclusive graphical abstract. Endothelial AT 1 -activated NAD(P)H oxidase-driven generation of O 2 − and H 2 O 2 in carotid arteries from type I-diabetic rats impairs the functionality of the local vasoprotective ACE2-angiotensin-(1–7)- Mas axis, which in turn impairs carotid blood flow. In this mechanism, H 2 O 2 derived from O 2 − dismutation inhibits ACE2 activity in generating angiotensin-(1–7) by activating I Cl,SWELL , while O 2 − inhibits the nitrergic vasorelaxant effect evoked by angiotensin-(1–7) upon Mas receptors activation. The chronic treatment of diabetic rats with angiotensin-(1–7) restores the functionality of carotid ACE2-angiotensin-(1–7)- Mas axis by triggering a positive feedback on this axis, played by a residual population of endothelial Mas -receptors that blunts the endothelial AT 1 -activated NAD(P)H oxidase-driven generation of reactive oxygen species in rat carotid. Mas -mediated antioxidant effects evoked by the chronic treatment with angiotensin-(1–7) also restores carotid resistance and blood flow in diabetic rats, pointing the important contribution of the ACE2-angiotensin-(1–7)- Mas axis in maintaining carotid function.

Article Snippet: They are STZ, angiotensin II, angiotensin-(1–7), losartan, PD123,319, hydroxocobalamin, L-NNA, 1400 W, apocynin, tiron, PEG-catalase, CDCF-DA, and DCPIB (Sigma, St. Louis, Mo., USA); DX600 (Anaspec Inc., Fremont, CA, USA); A779 (Bachem California Inc., Torrance, CA, USA); L-NPA (Tocris, Avonmouth, UK); DHE (Invitrogen, Carlsbad, CA, USA); ketamine (União Química, Jabaquara, SP, Brazil); xylazine (Calier Laboratory, Jubatuba, MG, Brazil); rabbit polyclonal anti-ACE2 antibody (Abcam, Cambridge, MA, USA); rabbit anti-angiotensin-(1–7) antibody (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA); rabbit polyclonal anti-angiotensin-(1–7)- Mas receptor antibody (Alomone Labs, Jerusalem, Israel); biotinylated universal secondary antibody, avidin-biotin peroxidase complex (Vectastain Elite ABC kit, Universal, Vector Laboratories Inc. U.S. Headquarters, Burlingame, CA, USA).

Techniques: Derivative Assay, Activity Assay, Activation Assay

Journal: Vaccines

Article Title: Development of a Vaccine against SARS-CoV-2 Based on the Receptor-Binding Domain Displayed on Virus-Like Particles

doi: 10.3390/vaccines9040395

Figure Lengend Snippet: Coupling of spike–RBD with CuMV TT viruslike particle (VLP). ( A ) Outline of the strategy to display RBD on CuMV TT surface. ( B ) Analysis of the RBD and coupling reactions of the RBD to CuMV TT by SDS-PAGE. Coupling band is indicated by an arrow. Lane 1: CuMV TT linked to SMPH; lane 2: RBD; lane 3: coupled CuMV TT –RBD with free RBD; lane 4: coupled CuMV TT –RBD without free RBD. An amount of 5 μg of each sample was loaded. ( C ) Binding of the CuMV TT –RBD vaccine to the anti-RBD antibody and human ACE2 by ELISA. An amount of 5 μg of the anti-CuMV TT antibody was coated to capture different concentrations of the CuMV TT –RBD vaccine. ( D ) Transmission electron microscope (TEM) image of the coupled CuMV TT –RBD vaccine. Yellow stars indicate the CuMV TT –RBD VLP.

Article Snippet: Then human anti-RBD antibody (Sanyou Biopharmaceuticals, Shanghai, China) or biotinylated ACE2 (Sino Biological, Beijing, China) was added and incubated on plates for 1 h at room temperature.

Techniques: SDS Page, Binding Assay, Enzyme-linked Immunosorbent Assay, Transmission Assay, Microscopy

Journal: Vaccines

Article Title: Development of a Vaccine against SARS-CoV-2 Based on the Receptor-Binding Domain Displayed on Virus-Like Particles

doi: 10.3390/vaccines9040395

Figure Lengend Snippet: Sera of mice immunized with the CuMV TT –RBD vaccine inhibit the interaction of the RBD to ACE2. ( A ) Competition ELISA results using immobilized ACE2 (1 μg/mL). Five mouse sera per group (1:40 dilution, d31 after first immunization) were incubated with RBD–His (0.15 μg/mL) before adding to ACE2. ( B ) Competition BLI results using the RBD immobilized on a biosensor. Sera of immunized mice at d31 after first immunization (1:20 dilution) were used to compete for the binding of ACE2 (50 nM) to the RBD. Shown are a scatter plot of individual mice ( n ≥ 3/group), and area under the curve (binding of ACE2 to the RBD with time) was assessed. Statistical analysis was performed with unpaired t -test, p ≤ 0.005 (***), p ≤ 0.001 (****).

Article Snippet: Then human anti-RBD antibody (Sanyou Biopharmaceuticals, Shanghai, China) or biotinylated ACE2 (Sino Biological, Beijing, China) was added and incubated on plates for 1 h at room temperature.

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