Journal: Biomaterials and Biosystems

Article Title: Disruptive 3D in vitro models for respiratory disease investigation: A state-of-the-art approach focused on SARS-CoV-2 infection

doi: 10.1016/j.bbiosy.2023.100082

Figure Lengend Snippet: Morphological analysis and development of BEAS-CTL and BEAS-ACE2 spheroids and 2D cells . (A) Scaffold-free spheroid-producing technique using agarose micro-molds. (B) Bright-field images of the spheroids on days 1, 7, and 14. (C) Spheroids historesin assay. Comparison of spheroids’ (D) diameter and (E) volume. (F) Bright-field images of 2D cultures of BEAS-CTL and BEAS-ACE2 cells. (G) Area measurement of 2D cultures of BEAS-CTL and BEAS-ACE2 cells. Scale bar: 50-100 µm. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001. n = 25-50.

Article Snippet: Then, they were resuspended in TBSTX (TBS [pure Tris base (Trimethamine, Bio-Rad, Brazil) + NaCl] + Triton [Triton X-100 detergent, Bio-Rad, Brazil]), and later, incubated with primary antibodies (Polyclonal Anti-Spike rabbit IgG [Anti-SARS-CoV-2 spike glycoprotein antibody – Coronavirus (ab272504), Abcam, United States] and Monoclonal Anti-ACE2 mouse IgG [ACE2 antibody (E-11) - sc-390851, Santa Cruz Animal Health, Brazil]) overnight at 4°C.

Techniques:

Journal: Biomaterials and Biosystems

Article Title: Disruptive 3D in vitro models for respiratory disease investigation: A state-of-the-art approach focused on SARS-CoV-2 infection

doi: 10.1016/j.bbiosy.2023.100082

Figure Lengend Snippet: MatriWells representative scheme and SARS-CoV-2 kinetics in BEAS-ACE2 MatriWells and HUVEC cells . (A) Pulmonary ALI creation using the MatriWells, BEAS-ACE2 and HUVEC cells. (B) Intracellular viral load in BEAS-ACE2 MatriWells. (C) Number of viral particles per mL(PFU[plaque-forming units]/mL) in HUVEC's supernatant. (D) Intracellular viral load in HUVEC cells. *p ≤ 0.05; ***p ≤ 0.001. n = 12.

Article Snippet: Then, they were resuspended in TBSTX (TBS [pure Tris base (Trimethamine, Bio-Rad, Brazil) + NaCl] + Triton [Triton X-100 detergent, Bio-Rad, Brazil]), and later, incubated with primary antibodies (Polyclonal Anti-Spike rabbit IgG [Anti-SARS-CoV-2 spike glycoprotein antibody – Coronavirus (ab272504), Abcam, United States] and Monoclonal Anti-ACE2 mouse IgG [ACE2 antibody (E-11) - sc-390851, Santa Cruz Animal Health, Brazil]) overnight at 4°C.

Techniques:

Journal: Biomaterials and Biosystems

Article Title: Disruptive 3D in vitro models for respiratory disease investigation: A state-of-the-art approach focused on SARS-CoV-2 infection

doi: 10.1016/j.bbiosy.2023.100082

Figure Lengend Snippet: BEAS-CTL and BEAS-ACE2 spheroids viability over time . The Viability over time was quantified using the trypan blue staining. *p ≤ 0.05, n = 3.

Article Snippet: Then, they were resuspended in TBSTX (TBS [pure Tris base (Trimethamine, Bio-Rad, Brazil) + NaCl] + Triton [Triton X-100 detergent, Bio-Rad, Brazil]), and later, incubated with primary antibodies (Polyclonal Anti-Spike rabbit IgG [Anti-SARS-CoV-2 spike glycoprotein antibody – Coronavirus (ab272504), Abcam, United States] and Monoclonal Anti-ACE2 mouse IgG [ACE2 antibody (E-11) - sc-390851, Santa Cruz Animal Health, Brazil]) overnight at 4°C.

Techniques: Staining

Journal: Biomaterials and Biosystems

Article Title: Disruptive 3D in vitro models for respiratory disease investigation: A state-of-the-art approach focused on SARS-CoV-2 infection

doi: 10.1016/j.bbiosy.2023.100082

Figure Lengend Snippet: Viral load quantification and immunofluorescence assay of BEAS-CTL and BEAS-ACE2 spheroids . (A) Spheroid intracellular viral concentration. (B) Immunofluorescence microscopy images of the spheroids with nucleus (Hoechst), ACE2, and Spike protein staining. Fluorescence quantification of (C) Spike protein and (D) ACE2 expression. (E) Pearson correlation comparing ACE2 and Spike protein expression. Scale bar: 50 µm. *p ≤ 0.05; **p ≤ 0.01. n = 3-5

Article Snippet: Then, they were resuspended in TBSTX (TBS [pure Tris base (Trimethamine, Bio-Rad, Brazil) + NaCl] + Triton [Triton X-100 detergent, Bio-Rad, Brazil]), and later, incubated with primary antibodies (Polyclonal Anti-Spike rabbit IgG [Anti-SARS-CoV-2 spike glycoprotein antibody – Coronavirus (ab272504), Abcam, United States] and Monoclonal Anti-ACE2 mouse IgG [ACE2 antibody (E-11) - sc-390851, Santa Cruz Animal Health, Brazil]) overnight at 4°C.

Techniques: Immunofluorescence, Concentration Assay, Microscopy, Staining, Fluorescence, Expressing

Journal: Biomaterials and Biosystems

Article Title: Disruptive 3D in vitro models for respiratory disease investigation: A state-of-the-art approach focused on SARS-CoV-2 infection

doi: 10.1016/j.bbiosy.2023.100082

Figure Lengend Snippet: SARS-CoV-2 infection of the 3D models . (A) SARS-CoV-2 infection of spheroids and MatriWells. (B) Comparison of intracellular viral load in BEAS-CTL spheroids and MatriWells. (C) Comparison of intracellular viral load in BEAS-ACE2 spheroids and MatriWells. ****p ≤ 0.0001, *p≤0.05. n = 5-15

Article Snippet: Then, they were resuspended in TBSTX (TBS [pure Tris base (Trimethamine, Bio-Rad, Brazil) + NaCl] + Triton [Triton X-100 detergent, Bio-Rad, Brazil]), and later, incubated with primary antibodies (Polyclonal Anti-Spike rabbit IgG [Anti-SARS-CoV-2 spike glycoprotein antibody – Coronavirus (ab272504), Abcam, United States] and Monoclonal Anti-ACE2 mouse IgG [ACE2 antibody (E-11) - sc-390851, Santa Cruz Animal Health, Brazil]) overnight at 4°C.

Techniques: Infection

Journal: Viruses

Article Title: SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells

doi: 10.3390/v15030736

Figure Lengend Snippet: Distribution of ACE2 receptor and attachment of SARS-CoV-2 and human coronavirus (HCoV-NL63) to tissue sections of human trachea. These images show formalin-fixed paraffin-embedded cross-sections of human tracheal sections stained with ImmPRESS VR anti-mouse/rabbit IgG horseradish peroxidase (HRP) polymer detection kit. Dark brown represents the presence of a protein interacting with a specific antibody and is considered a positive expression. Pale brown background, and the nucleus counterstained with hematoxylin is blue. ( a ) Expression of ACE2 in human trachea revealed by immunostaining with mouse anti-ACE2 monoclonal antibody (4 μg/mL); and ( d ) corresponding negative control tissue sections stained with secondary anti-mouse HRP antibody only. ( b , c , e , f ) The tissues sections presented in this panel show virus binding in formalin-fixed paraffin-embedded cross-sections after overnight incubation with 250 μL ( b ) heat-inactivated SARS-Related Coronavirus 2, Isolate USA-WA1/2020 or ( c ) HCoV-NL63. ( e , f ) Virus-incubated sections that were stained with secondary anti-rabbit HRP antibody only. Scale bar—100 μm.

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal anti-ACE2 (4 μg/mL; E-11 Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-pan-cytokeratin (0.5 μg/mL; AE1/AE3; Bio-Rad Laboratories, Hercules, CA, USA); anti-HCoV-NL63 nucleocapsid (N) protein monoclonal antibody (2D4; Ingenasa-Eurofins, Madrid, Spain) (0.25 µg/mL) and a recombinant anti-SARS-CoV-2 nucleocapsid (N) protein rabbit monoclonal antibody (0.75 μg/mL) (BEI Resources) [The following reagent was obtained through BEI Resources, NIAID, NIH: Monoclonal Anti-SARS Coronavirus/SARS-Related Coronavirus 2 Nucleocapsid Protein (produced in vitro), NR-53791; SinoBio Cat: 40143-R001].

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Expressing, Immunostaining, Negative Control, Binding Assay, Incubation

Journal: Viruses

Article Title: SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells

doi: 10.3390/v15030736

Figure Lengend Snippet: Characterization of human respiratory epithelial cells (HRECs) for pan-cytokeratin and angiotensin-converting enzyme-2 (ACE2) using target-specific markers. Primary HRECs stained for mouse monoclonal to anti-pan-cytokeratin (epithelial cell marker; 0.5 μg/mL) showed strong expression both in ( a ) IHC and ( c ) flow cytometry analysis. ( b ) HRECs incubated with a secondary antibody only displayed minimum background. ( c ) HRECs were also quantified for the ACE2 expression using flow cytometry. Flow cytometry data was collected using an Attune NxT flow cytometer. A representative of 10,000 events were acquired and analyzed for each sample. Cells were gated for singlet population using forward (FSA) and side-scatter (SSA) properties, and the mean of percent live cell population was used to quantify the levels of ( c-i ) pan-cytokeratin and ( c-ii ) ACE2 ( n = 4). The bar graph represents the mean with the standard error of the mean (SEM).

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal anti-ACE2 (4 μg/mL; E-11 Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-pan-cytokeratin (0.5 μg/mL; AE1/AE3; Bio-Rad Laboratories, Hercules, CA, USA); anti-HCoV-NL63 nucleocapsid (N) protein monoclonal antibody (2D4; Ingenasa-Eurofins, Madrid, Spain) (0.25 µg/mL) and a recombinant anti-SARS-CoV-2 nucleocapsid (N) protein rabbit monoclonal antibody (0.75 μg/mL) (BEI Resources) [The following reagent was obtained through BEI Resources, NIAID, NIH: Monoclonal Anti-SARS Coronavirus/SARS-Related Coronavirus 2 Nucleocapsid Protein (produced in vitro), NR-53791; SinoBio Cat: 40143-R001].

Techniques: Staining, Marker, Expressing, Flow Cytometry, Incubation

Journal: Scientific Reports

Article Title: A pseudovirus-based platform to measure neutralizing antibodies in Mexico using SARS-CoV-2 as proof-of-concept

doi: 10.1038/s41598-022-22921-7

Figure Lengend Snippet: Expression of ACE2 on the surface of HEK-293 T, Caco-2, Vero, and Vero E6 cells. ( A ) Proportion of cells expressing ACE2 ( B ) Relative expression of ACE2 on cell surface as expressed by median fluorescence intensity (MFI) of ACE2-AF647. Average of 3 separate experiments done in duplicate with standard deviations shown. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Article Snippet: Briefly, 3 × 10 5 cells were stained with a mouse anti-human ACE2 monoclonal antibody conjugated to AF-647 (Santa Cruz Biotechnology, USA, cat. SC-390851) following manufacturer’s instructions.

Techniques: Expressing, Fluorescence

Journal: Antioxidants

Article Title: An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System

doi: 10.3390/antiox11040764

Figure Lengend Snippet: Features of primer sequences for real-team PCR expression analysis.

Article Snippet: Blots were blocked in TBS-T containing 5% non-fat dry milk and incubated overnight at 4 °C with either mouse monoclonal antibody against ACE2 (cat# Sc-73668; dilution 1:500), or rabbit polyclonal antibodies directed against Akt1/2/3 (Santa Cruz Biotechnology, cat# Sc-8312), pAkt1/2/3-Ser473 (cat# Sc-7985-R), AMPKα (Cell Signaling Technology, cat# 5831; dil 1:500), pAMPKα (Thr172) (Cell Signaling Technology, cat# 2535; dil 1:500), eNOS (cat# N3893), or goat polyclonal antibody directed against pNOS3-Ser1177 (cat# Sc-12972; dil. 1:500).

Techniques: Expressing

Journal: Antioxidants

Article Title: An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System

doi: 10.3390/antiox11040764

Figure Lengend Snippet: ( A ) ace2 mRNA expression levels in goldfish C. auratus tissues. The amounts of target mRNA are calculated as 2-ΔCt mean values obtained from the output Ct values of two rounds of real-time PCR assays for each of three independent biological replicates. Statistics were assessed by one-way ANOVA followed by a Sidak’s multiple comparison test (**** p < 0.0001); ( B ) Representative immunoblotting of ACE2 expression in the goldfish heart. M: marker; H: heart; ( C ) Representative HPLC chromatogram showing Alamandine (Ala) elution from C. auratus plasma compared to Alamandine standard (red dotted line).

Article Snippet: Blots were blocked in TBS-T containing 5% non-fat dry milk and incubated overnight at 4 °C with either mouse monoclonal antibody against ACE2 (cat# Sc-73668; dilution 1:500), or rabbit polyclonal antibodies directed against Akt1/2/3 (Santa Cruz Biotechnology, cat# Sc-8312), pAkt1/2/3-Ser473 (cat# Sc-7985-R), AMPKα (Cell Signaling Technology, cat# 5831; dil 1:500), pAMPKα (Thr172) (Cell Signaling Technology, cat# 2535; dil 1:500), eNOS (cat# N3893), or goat polyclonal antibody directed against pNOS3-Ser1177 (cat# Sc-12972; dil. 1:500).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Marker

Journal: Antioxidants

Article Title: An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System

doi: 10.3390/antiox11040764

Figure Lengend Snippet: ( A ) Quantitative real-time PCR analysis of ace2 mRNA expression in cardiac extracts of goldfish C. auratus exposed to normoxia and hypoxia. Comparison of 2-ΔCt mean values of normoxic and hypoxic hearts, reported as percent fold change ( y -axis). Statistic was assessed by one-way ANOVA followed by a Sidak’s multiple comparison test ( n = 3); ( B ) Representative immunoblot and densitometric analysis of ACE2 expression in cardiac extracts of goldfish C. auratus exposed to normoxia and hypoxia. Data were expressed as means ± s.e.m. of absolute values from individual experiments ( n = 3). Statistical analysis was performed by two-tailed unpaired t -test (* p < 0.05). ( C ) Representative HPLC chromatogram showing Alamandine (Ala) elution from plasma samples of goldfish C. auratus exposed to hypoxia compared with standard (red dotted line).

Article Snippet: Blots were blocked in TBS-T containing 5% non-fat dry milk and incubated overnight at 4 °C with either mouse monoclonal antibody against ACE2 (cat# Sc-73668; dilution 1:500), or rabbit polyclonal antibodies directed against Akt1/2/3 (Santa Cruz Biotechnology, cat# Sc-8312), pAkt1/2/3-Ser473 (cat# Sc-7985-R), AMPKα (Cell Signaling Technology, cat# 5831; dil 1:500), pAMPKα (Thr172) (Cell Signaling Technology, cat# 2535; dil 1:500), eNOS (cat# N3893), or goat polyclonal antibody directed against pNOS3-Ser1177 (cat# Sc-12972; dil. 1:500).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Two Tailed Test

Journal: Antioxidants

Article Title: An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System

doi: 10.3390/antiox11040764

Figure Lengend Snippet: Analysis of the protein–protein interaction network (PPI) by the STRING online suite (version 11.5). The PPI network includes 14 proteins (number of nodes: 14; number of edges: 38; average node degree: 5.43; avg. local clustering coefficient: 0.623; expected number of edges: 2; PPI enrichment p -value: <1.0 × 10 −16 ). ace2-related proteins: ace2, Angiotensin I converting enzyme 2; agtr2, Angiotensin II receptor, type 2; ace, Angiotensin I converting enzyme 1; agtr1a, Angiotensin II receptor, type 1a; agtr1b, Angiotensin II receptor, type 1b; ren, Renin; nos1, nitric oxide synthase; agt, Angiotensinogen; hif1-related proteins: egln1a, Egl-9 family hypoxia-inducible factor 1; hif1an, Hypoxia-inducible factor 1-alpha inhibitor; tceb1b, Transcription elongation factor B (SIII), polypeptide 1b; hif1al, Hypoxia-inducible factor 1, alpha subunit, -like; hif1ab, Hypoxia-inducible factor 1, alpha subunit b; hif1aa, Hypoxia-inducible factor 1, alpha subunit a.

Article Snippet: Blots were blocked in TBS-T containing 5% non-fat dry milk and incubated overnight at 4 °C with either mouse monoclonal antibody against ACE2 (cat# Sc-73668; dilution 1:500), or rabbit polyclonal antibodies directed against Akt1/2/3 (Santa Cruz Biotechnology, cat# Sc-8312), pAkt1/2/3-Ser473 (cat# Sc-7985-R), AMPKα (Cell Signaling Technology, cat# 5831; dil 1:500), pAMPKα (Thr172) (Cell Signaling Technology, cat# 2535; dil 1:500), eNOS (cat# N3893), or goat polyclonal antibody directed against pNOS3-Ser1177 (cat# Sc-12972; dil. 1:500).

Techniques: