Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: ACE2 is a key molecule potentially linking COVID-19 to associated metabolic defects (A) Volcano plot of differentially expressed genes after in vitro infection of SARS-CoV-2 (MOI = 0.005) in HUVECs for 24 h. (B) DisGeNET pathway enrichment of differentially expressed genes after infection (combined 685 upregulated and 774 downregulated genes from A). (C–E) Bioinformatics analysis of the mutual target disease-associated genes of diabetes mellitus (DM), hypertension (HTN), diabetic nephropathy (DN), and atherosclerosis (AS) that were queried from DisGeNET and Open Target database. The overlapping 48 shared genes from DisGeNET (C), the 72 shared genes from Open Target (D), and 20 genes identified on both DisGeNET (left) and Open Target database (right) as mutual target disease-associated genes of four diseases (E) were shown. (F) The heatmap to represent the alterations of the above 20 genes upon SARS-CoV-2 infection. Data were shown as log 2 fold change. The cross mark represents the undetectable gene from the transcriptomic study. (G) ELISA quantification of plasma angiotensin II (Ang II) and angiotensin-(1–7) (Ang-(1–7)) in COVID-19 patients, shown in Ang II against Ang-(1–7) ratio. HC, healthy control; I, infection phase; R, recovery phase. Health control group, n = 10, mean age ± SD = 42.30 ± 7.35, male/female = 5/5; non-severe group, n = 18, mean age ± SD = 51.61 ± 10.13, male/female = 9/9; severe group, n = 12, mean age ± SD = 57.75 ± 14.55, male/female = 6/6. (H and I) HUVECs were infected with SARS-CoV-2 (MOI = 0.005) for 24 h and were subjected to real-time PCR of ACE2 (H) and immunoblotting (I, blot shown on the left, quantification on the right, n = 4). (J) HUVECs were infected with SARS-CoV-2 (MOI = 0.005) for 48 h, and then the medium was removed and 100 nM Ang II in 1 mL HEPES solution was applied for the cells. After 1 h treatment in the incubator at 37°C, the supernatant containing Ang II and cleaved Ang-(1–7) was examined (n = 6). (K and L) Sixteen-week-old human ACE2 transgenic mice were intranasally challenged with 4 × 10 4 FFU SARS-CoV-2 after 10-week high-fat-diet treatment. After 7 days post-infection, all mice were fasted for 6 h and sacrificed. Livers (K) and kidneys (L) were subjected to real-time PCR (n = 3–4). (M–O) HUVECs were treated with a “cocktail” of different inflammatory factors (IFs), namely the combination of 10 or 50 ng/mL of TNF-α, IL-4, IL-6, and IFN-γ or 1 μM DX600 for 48 h and were subjected to real-time PCR of ACE2 (M) (n = 6), immunoblotting (N, blot shown on the left, quantification on the right, n = 4), and enzymatic activity assay (O, data were shown as the area under the kinetic activity curves, n = 6). DX600 was used as a negative control. Error bars represent SEM; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. HUVECs, human umbilical vein endothelial cells; AU, arbitrary unit; CoV-2, SARS-CoV-2. See also Figure S1 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: In Vitro, Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Transgenic Assay, Enzyme Activity Assay, Activity Assay, Negative Control

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: ACE2 plays an important role in maintaining metabolic homeostasis (A) HUVECs were infected with 10 nM control siRNA (Con) or ACE2 siRNA (siACE2) for 24 h and subjected to real-time PCR (n = 6). (B and C) Eight-week-old male ob/ob mice were treated with intravenous injection of AAV9-CAG-hACE2-EGFP (ob/ob-ACE2) and corresponding control virus (ob/ob-Con) and their wild-type littermates with control virus (WT-Con). Glucose tolerance testing (GTT) was performed at 10 weeks (B), and insulin tolerance testing (ITT) was performed at 11 weeks (C) (n = 6). The significance of ob/ob-Con versus WT-Con was shown as ∗ ; ob/ob-ACE2 versus ob/ob-Con as #. (D–F) At 12 weeks, all mice were fasted for 6 h and sacrificed. Quantification of fasting blood glucose (D), plasma insulin (E), and homeostatic model assessment of insulin resistance (HOMA-IR) (F) were shown (n = 5). (G and H) Plasma TNF-α (G) and IL-6 (H) were measured (n = 4). (I and J) Livers were subjected to oil red O (ORO) staining (I, images shown on the left, quantification on the right; n = 4) and real-time PCR (n = 6) (J). (K) Plasma KIM-1 (n = 4). (L and M) Kidneys were subjected to periodic acid-Schiff (PAS) staining (L, images shown on the left, quantification on the right; n = 8) and real-time PCR (M) (n = 5). (N) Aortas were subjected to real-time PCR (n = 5). (O) Hearts were subjected to echocardiography (representative M-mode echocardiography images shown on the top, quantification of LVEF (%) and FS (%) on the bottom; n = 8). Error bars represent SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; # p < 0.05, ## p < 0.01, and ### p < 0.001. AUC, area under the curve; KIM-1, kidney injury molecule-1; LVEF, left ventricular ejection fraction; FS, fractional shortening. See also Figure S2 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: Infection, Real-time Polymerase Chain Reaction, Injection, Staining

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: Imatinib, harpagoside, and methazolamide are identified as ACE2 activators (A) Flow chart depicting the process of in silico identification of potential ACE2 activators with Tianhe-2 supercomputer virtual screening and CMAP bioinformatic analysis (top) and detailed information of the overlapped compounds (bottom, # and ∗ indicated different overlapping strategy). (B) The docking site (gray) of ACE2 protein (PDB: 1R42 ). (C) Schematic of the in vitro experimental workflow to identify the final three compounds. (D–F) Real-time PCR of gene expression in HUVECs. Log 10 fold change was calculated based on treated samples against control samples. HUVECs were treated with 15 compounds for 16 h (D), inflammatory factors for 32 h (50 ng/mL TNF-α, IL-4, IL-6, and IFN-γ) following eight compounds for 16 h (E), or 10 nM control siRNA (siCon) and ACE2 siRNA (siACE2) for 8 h following six compounds for 16 h (F) (n = 3). The selected compounds based on the scoring system were highlighted in red and applied for the following screening. (G) The low (L), medium (M), and high (H) concentrations of 15 compounds applied in the above three experiments. See also Figure S3 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: In Silico, In Vitro, Real-time Polymerase Chain Reaction, Expressing

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: Imatinib, harpagoside, and methazolamide directly bind to and activate ACE2 (A–E) The direct binding was illustrated by surface plasmon resonance (SPR) assay of purified ACE2 protein with imatinib (Ima) (A), harpagoside (Har) (B), methazolamide (Met) (C), diminazene aceturate (DIZE) (D), or xanthone (Xan) (E). Xanthone was used as a negative control. The Kd values (equilibrium dissociation constant) of compounds binding to ACE2 protein were calculated based on the fitted curves. (F and G) The detailed binding between ACE2 protein (gray) and imatinib (F, blue) and methazolamide (G, blue) was simulated with molecular dynamics simulation by GROMACS. Residues involved in binding were marked in green. (H) The HUVECs were treated with 25 μM imatinib, 100 μM harpagoside, 100 μM methazolamide, or 1 μM DX600 for 16 h, then subjected to ACE2 enzymatic activity assay. Data shown as the area under the kinetic activity curves. The ACE2 enzymatic inhibitor DX600 was used as a negative control (n = 3). (I–L) The HUVEC lysates were prepared and treated with 1.5 × 10 −6 M imatinib, 1.5 × 10 −6 M harpagoside, 1.5 × 10 −6 M methazolamide, or 1.0 × 10 −6 M DX600 for 30 min, then subjected to ACE2 enzymatic activity assay in a kinetics model. The kinetic activity curves were shown (I) (n = 3). The effects of imatinib (J) at concentrations of 1.5 × 10 −7 M (Ima-1) and 1.5 × 10 −6 M (Ima-2), harpagoside (K) at concentrations of 0.5 × 10 −7 M (Har-1) and 1.5 × 10 −6 M (Har-2), or methazolamide (L) at concentrations of 1.5 × 10 −7 M (Met-1) and 1.5 × 10 −6 M (Met-2) on the enzymatic activity of ACE2 in HUVEC lysates were shown as the area under the kinetic activity curves (n = 3). Error bars represent SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figure S4 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: Binding Assay, SPR Assay, Purification, Negative Control, Enzyme Activity Assay, Activity Assay

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: Imatinib and methazolamide ameliorate metabolic defects in insulin-resistant mice via ACE2 (A–I) Twenty-eight-week-old male mice with 23-week high-fat-diet treatment (DIO) and controlled lean mice (Lean) were treated with vehicle, 250 mg/kg imatinib (DIO + Ima), or 100 mg/kg methazolamide (DIO + Met) through gavage once each day for 4 weeks. (A and B) Glucose tolerance testing (GTT) was performed at 30 weeks (A); insulin tolerance testing (ITT) was performed at 31 weeks (B) (n = 6). The significance of lean versus DIO was shown as ∗ , DIO versus DIO + Ima as #, and DIO versus DIO + Met as $. ∗,#,$ p < 0.05, ∗∗,##,$$ p < 0.01, and ∗∗∗,###,$$$ p < 0.001. At 32 weeks, all mice were fasted for 6 h and sacrificed. (C–F) Fasting blood glucose (C), plasma insulin (D), homeostatic model assessment of insulin resistance (HOMA-IR) (E), and plasma TNF-α (F) (n = 6). (G and H) Livers were subjected to oil red O (ORO) staining (G, images shown on the left, quantification on the right) and real-time PCR (H) (n = 6). (I) Aortas were subjected to real-time PCR (n = 6). (J–P) For kidney conditional knockdown of ACE2 (ACE2 C-kd), 26-week-old male mice with 21-week high-fat-diet treatment (DIO) and controlled lean mice (Lean) were treated with transparenchymal renal pelvis injection of AAV9-CAG-mACE2shRNA-EGFP or control virus. After 2-week recovery, mice were given vehicle, 250 mg/kg imatinib (DIO + Ima and DIO + Ima + ACE2 C-kd), or 100 mg/kg methazolamide (DIO + Met and DIO + Met + ACE2 C-kd) through gavage once each day for 4 weeks. At 32 weeks, all mice were fasted for 6 h and sacrificed. (J and K) Plasma KIM-1 (J) and CREA (K) (n = 6). (L and M) Kidneys were subjected to periodic acid-Schiff (PAS) staining (L, images and quantification were shown; n = 5) and real-time PCR (M) (n = 6). (N) Quantification of plasma ratio of Ang II against Ang-(1–7) (n = 5). (O and P) The lysates of kidneys (O) and aortas (P) were subjected to ACE2 enzymatic activity assay, and data were shown as the area under the kinetic activity curves (n = 3). (Q–U) For A779 (MasR inhibitor) intervention, 28-week-old male mice with 23-week high-fat-diet treatment were given vehicle (DIO and DIO + A779), 250 mg/kg imatinib (DIO + Ima and DIO + Ima + A779), or 100 mg/kg methazolamide (DIO + Met and DIO + Met + A779) with or without 3 mg/kg A779 once each day for 4 weeks. (Q and R) Glucose tolerance testing (GTT) was performed at 30 weeks (Q), and insulin tolerance testing (ITT) was performed at 31 weeks (R) (n = 6). The significance of DIO versus DIO + Ima was shown as ∗ , DIO versus DIO + Met as #, DIO + Ima versus DIO + Ima + A779 as $ and DIO + Met versus DIO + Met + A779 as &. ∗,#,$,& p < 0.05, ∗∗,##,$$,&& p < 0.01, and ∗∗∗,###,$$$,&&& p < 0.001. At 32 weeks, all mice were fasted for 6 h and sacrificed. (S–U) Livers (S), kidneys (T), and aortas (U) were subjected to real-time PCR. Error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CREA, creatinine. See also Figure S5 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: Staining, Real-time Polymerase Chain Reaction, Injection, Enzyme Activity Assay, Activity Assay

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: ACE2 enzymatic activators improve metabolic defects and inhibit virus entry upon SARS-CoV-2 infection (A–Q) Twelve-week-old human ACE2 transgenic mice were given vehicle (Mock, CoV-2), 250 mg/kg imatinib (CoV-2 + Ima), and 100 mg/kg methazolamide (CoV-2 + Met) through gavage once each day for 4 weeks after 6-week high-fat-diet treatment and were intranasally challenged with 4 × 10 4 FFU SARS-CoV-2. After 7 days of infection, all mice were fasted for 6 h and sacrificed. (A–H) Quantification of the ratio of Ang II against Ang-(1–7) in plasma (A), fasting blood glucose (B), plasma insulin (C), homeostatic model assessment of insulin resistance (HOMA-IR) (D), plasma triglyceride (E), plasma total cholesterol (F), plasma TNF-α (G), and plasma IL-6 (H) (n = 4). (I–K) Livers were subjected to lipid assay (I and J) and real-time PCR (K) (n = 3–4). (L) Plasma KIM-1 (n = 4–5). (M and N) Kidneys were subjected to periodic acid-Schiff (PAS) staining (M, images and quantification were shown; n = 4) and real-time PCR (N) (n = 4). (O–Q) Lungs were subjected to H&E staining (O, images and quantification were shown; n = 4), real-time PCR (P) (n = 4), and immunoblotting of viral nucleocapsid protein (NP) (Q, blot shown on the left, quantification on the right; n = 4). (R) Vero E6 cells were pretreated with 25 μM imatinib, 100 μM harpagoside, or 100 μM methazolamide for 6 h, followed by SARS-CoV-2 (MOI = 0.005) infection for 42 h; the supernatant was subjected to real-time PCR (n = 4). (S) HEK293T cells expressing hACE2 were pretreated with 25 μM imatinib, 100 μM harpagoside, or 100 μM methazolamide for 6 h, followed by pseudovirion treatment for 66 h, and were observed with fluorescence microscopy (images shown on the left, quantification on the right; n = 5). (T) The HEK293T cells overexpressing spike-FLAG and ACE2-HA were treated with 25 μM imatinib, 100 μM harpagoside, or 100 μM methazolamide for 48 h. Precipitated proteins were subjected to immunoblotting (blot shown on the left, quantification on the right; n = 4). Error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figure S6 .

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: Infection, Transgenic Assay, Real-time Polymerase Chain Reaction, Staining, Western Blot, Expressing, Fluorescence, Microscopy

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet:

Article Snippet: Human angiotensin-I converting enzyme 2 (ACE2) , Sino Biological Company , Cat. 10108-H05H.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, RNA Extraction, SYBR Green Assay, Activity Assay, Cholesterol Assay, Software

Journal: Parasitology

Article Title: Identification of Leishmania infantum in blood donors from endemic regions for visceral leishmaniasis

doi: 10.1017/S0031182020001936

Figure Lengend Snippet: Results of the analysis of κ agreement between ELISA and qPCR and conventional PCR and qPCR for the diagnosis of asymptomatic visceral leishmaniasis

Article Snippet: ELISA rK39 and conventional polymerase chain reaction (PCR) ELISA rK39 (Kalazar Detect ELISA InBios ® Seattle, Washington, USA) was performed following the manufacturer's instructions, and conventional PCR was conducted as described by Ferreira-Silva et al . ( 2018 ).

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Parasitology

Article Title: Identification of Leishmania infantum in blood donors from endemic regions for visceral leishmaniasis

doi: 10.1017/S0031182020001936

Figure Lengend Snippet: Comparative results between the techniques used in the 10 samples sequenced to identify infection by Leishmania infantum

Article Snippet: ELISA rK39 and conventional polymerase chain reaction (PCR) ELISA rK39 (Kalazar Detect ELISA InBios ® Seattle, Washington, USA) was performed following the manufacturer's instructions, and conventional PCR was conducted as described by Ferreira-Silva et al . ( 2018 ).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: Frontiers in Veterinary Science

Article Title: Use of ATP-Binding Cassette Subfamily A Member 13 (ABCA13) for Sensitive Detection of Focal Pathological Forms of Subclinical Bovine Paratuberculosis

doi: 10.3389/fvets.2022.816135

Figure Lengend Snippet: ABCA13 and MMP8 expression levels in the 704 animals included in the study. (A) ABCA13 expression levels in sera of Holstein Friesian cattle showing different types of histological lesions consistent with paratuberculosis (PTB) in their intestinal tissues [focal ( n = 447), multifocal ( n = 59), and diffuse ( n = 60)] and control animals from PTB-free farms ( n = 138). (B) MMP8 expression levels in serum of Holstein Friesian cattle showing different types of histological lesions consistent with PTB in their intestinal tissues [focal ( n = 442), multifocal ( n = 58), and diffuse ( n = 60)] and control animals from PTB-free farms ( n = 138). Biomarkers were quantified by specific ELISAs supplied by MyBioSource, San Diego, CA, USA; ABCA13, bovine ATP binding cassette subfamily A member 13; MMP8, bovine matrix metallopeptidase 8. The data are represented as scatter plots with each dot representing a single animal. The mean of each histopathological group is represented by a gross black point and the standard deviation by a vertical line. The asterisks indicate whether differences between each histopathological group and the control are or not significant (*** p < 0.001).

Article Snippet: Given the good discriminatory power of the ABCA13-based ELISA its diagnostic performance was compared with that of other conventional PTB diagnostic methods (IDEXX ELISA for serum detection of Map-specific antibodies, PCR and bacteriological culture of feces and tissues) and the Ziehl-Neelsen staining (see ).

Techniques: Expressing, Control, Binding Assay, Standard Deviation

Journal: Frontiers in Veterinary Science

Article Title: Use of ATP-Binding Cassette Subfamily A Member 13 (ABCA13) for Sensitive Detection of Focal Pathological Forms of Subclinical Bovine Paratuberculosis

doi: 10.3389/fvets.2022.816135

Figure Lengend Snippet: ABCA13 and MMP8 mean concentration values in the different histopathological groups.

Article Snippet: Given the good discriminatory power of the ABCA13-based ELISA its diagnostic performance was compared with that of other conventional PTB diagnostic methods (IDEXX ELISA for serum detection of Map-specific antibodies, PCR and bacteriological culture of feces and tissues) and the Ziehl-Neelsen staining (see ).

Techniques: Concentration Assay, Control

Journal: Frontiers in Veterinary Science

Article Title: Use of ATP-Binding Cassette Subfamily A Member 13 (ABCA13) for Sensitive Detection of Focal Pathological Forms of Subclinical Bovine Paratuberculosis

doi: 10.3389/fvets.2022.816135

Figure Lengend Snippet: Diagnostic performance of the ABCA13 and MMP8-based ELISAs for the detection of animals with different types of PTB histological lesions.

Article Snippet: Given the good discriminatory power of the ABCA13-based ELISA its diagnostic performance was compared with that of other conventional PTB diagnostic methods (IDEXX ELISA for serum detection of Map-specific antibodies, PCR and bacteriological culture of feces and tissues) and the Ziehl-Neelsen staining (see ).

Techniques: Diagnostic Assay, Control

Journal: Frontiers in Veterinary Science

Article Title: Use of ATP-Binding Cassette Subfamily A Member 13 (ABCA13) for Sensitive Detection of Focal Pathological Forms of Subclinical Bovine Paratuberculosis

doi: 10.3389/fvets.2022.816135

Figure Lengend Snippet: Receiver Operator Characteristic Curves (ROC curves) of the ABCA13 biomarker-based ELISA in Holstein Friesian cows with focal ( n = 447), multifocal ( n = 59), diffuse histological lesions ( n = 60) and any type of paratuberculosis (PTB) lesions ( n = 566) vs . control animals from PTB-free farms ( n = 138); All, includes all animals with focal, multifocal and diffuse lesions; ABCA13, bovine ATP binding cassette subfamily A member 13.

Article Snippet: Given the good discriminatory power of the ABCA13-based ELISA its diagnostic performance was compared with that of other conventional PTB diagnostic methods (IDEXX ELISA for serum detection of Map-specific antibodies, PCR and bacteriological culture of feces and tissues) and the Ziehl-Neelsen staining (see ).

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Control, Binding Assay

Journal: Frontiers in Veterinary Science

Article Title: Use of ATP-Binding Cassette Subfamily A Member 13 (ABCA13) for Sensitive Detection of Focal Pathological Forms of Subclinical Bovine Paratuberculosis

doi: 10.3389/fvets.2022.816135

Figure Lengend Snippet: Receiver Operator Characteristic Curves (ROC curves) of the MMP8 biomarker-based ELISA in Holstein Friesian cows with focal ( n = 442), multifocal ( n = 58), diffuse lesions ( n = 60) and any type of lesions ( n = 560) vs . control animals from PTB-free farms ( n = 138); All, includes all animals with focal, multifocal and diffuse lesions; MMP8, bovine matrix metallopeptidase 8.

Article Snippet: Given the good discriminatory power of the ABCA13-based ELISA its diagnostic performance was compared with that of other conventional PTB diagnostic methods (IDEXX ELISA for serum detection of Map-specific antibodies, PCR and bacteriological culture of feces and tissues) and the Ziehl-Neelsen staining (see ).

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Veterinary Science

Article Title: Use of ATP-Binding Cassette Subfamily A Member 13 (ABCA13) for Sensitive Detection of Focal Pathological Forms of Subclinical Bovine Paratuberculosis

doi: 10.3389/fvets.2022.816135

Figure Lengend Snippet: Comparison of the diagnostic performance of the ABCA13-based ELISA with that of other conventional paratuberculosis (PTB) diagnostic methods.

Article Snippet: Given the good discriminatory power of the ABCA13-based ELISA its diagnostic performance was compared with that of other conventional PTB diagnostic methods (IDEXX ELISA for serum detection of Map-specific antibodies, PCR and bacteriological culture of feces and tissues) and the Ziehl-Neelsen staining (see ).

Techniques: Comparison, Diagnostic Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Epigenetic Alterations in the Brain Associated with HIV-1 Infection and Methamphetamine Dependence

doi: 10.1371/journal.pone.0102555

Figure Lengend Snippet: The content of HIV DNA and cellular HIV RNA was profiled in the frontal cortex area of HIV seropositive individuals with and without history of METH use, by real-time PCR and normalized to Actin or GAPDH levels respectively. A. and B. HIV-1 DNA and cellular HIV RNA did not show significant changes among groups. C. HIV-1 RNA/DNA ratio (as a marker of average HIV transcription) was significantly higher in METH user group, suggesting increases on viral transcription in the brain. *p<0.05 by Mann Whitney pairs test.

Article Snippet: Bisulfite-converted DNA templates (40 ng) from the studied cases underwent PCR alongside with standards ranging from 0% to 100% methylation using MeltDoctor HRM Dye (Life Technologies).

Techniques: Real-time Polymerase Chain Reaction, Marker, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Epigenetic Alterations in the Brain Associated with HIV-1 Infection and Methamphetamine Dependence

doi: 10.1371/journal.pone.0102555

Figure Lengend Snippet: A. Global methylation was determined by ELISA quantification of 5-mC on genomic DNA from frontal cortex samples. Significant increase in DNA methylation was observed in HIV seropositive METH users. *p<0.05 by Wilcoxon matched pairs test. B. Cellular HIV-1 RNA levels correlate with DNA methylation levels as determined by Spearman correlation between global DNA methylation and log HIV-1 RNA in the brain R = 0.527 with p = 0.01. C. Quantitative real-time PCR showed significant increase in DNMT1 transcript levels on HIV seropositive individuals who used METH. D. METH exposure did not alter mRNA levels of DNMT3B, a closely related family member reported to have redundant functions to DNMT1 in the brain. *p<0.05 by Wilcoxon matched pairs test. E. Western blot analysis of DNMT1 protein content in the nucleus showing representative HIV+METH− and HIV+METH+ cases. F. Image analysis showing integrated pixel intensity of Dnmt1 immunoreactivity. G. Immunofluorescence detection of DNMT1 on frontal cortex sections. Green fluorescent signal correspond to DNMT1 immunoreactivity and blue signal corresponds to DAPI nuclear staining. H. Image analysis showing average DNMT1 positive nuclear counts. Bar represents 10 µm. One way ANOVA was used to determine statistical significance, *p<0.05.

Article Snippet: Bisulfite-converted DNA templates (40 ng) from the studied cases underwent PCR alongside with standards ranging from 0% to 100% methylation using MeltDoctor HRM Dye (Life Technologies).

Techniques: Methylation, Enzyme-linked Immunosorbent Assay, DNA Methylation Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Epigenetic Alterations in the Brain Associated with HIV-1 Infection and Methamphetamine Dependence

doi: 10.1371/journal.pone.0102555

Figure Lengend Snippet: Quantitative analysis of transcript abundance by real-time PCR showed significant increases of TGFBR3 ( A ) and NET1 ( C ) on the HIV+METH+ group in comparison to HIV+METH− subjects. *p<0.05 by Student’s t Test. Linear regression analysis of gene expression and DNA methylation showed positive correlations for TGFBR3 on HIV+METH− and on HIV+METH+ groups ( B ) and a significant correlation between gene expression and DNA methylation for NET1 ( D ) . **p<0.01 and ***p<0.001 by Linear regression analysis with a 95% confidence interval.

Article Snippet: Bisulfite-converted DNA templates (40 ng) from the studied cases underwent PCR alongside with standards ranging from 0% to 100% methylation using MeltDoctor HRM Dye (Life Technologies).

Techniques: Real-time Polymerase Chain Reaction, Expressing, DNA Methylation Assay

Journal: bioRxiv

Article Title: Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy

doi: 10.1101/2021.03.22.436337

Figure Lengend Snippet: (A) Experimental strategy utilizing SARS-CoV-2 carrying nLuc reporter in ORF7a for non-invasive BLI of virus spread following intranasal (i.n.) challenge of B6 or K18-hACE2 mice. (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions at the indicated dpi and after necropsy (C) Temporal quantification of nLuc signal as flux (photons/sec) acquired non-invasively in the indicated tissues of each animal. The color bar above the x-axis (yellow to orange) represents computed signal intensities in K18-hACE2 mice that are significantly above those in B6 mice. (D) Temporal changes in mouse body weight with initial body weight set to 100%. (E) Kaplan-Meier survival curves of mice for experiment as in A statistically compared by log-rank (Mantel-Cox) test. (F) Ex vivo imaging of indicated organs and quantification of nLuc signal as flux(photons/sec) at 6 dpi after necropsy. (G, H) Viral loads (FFUs/mg or nLuc activity/mg) in indicated tissue measured on Vero E6 cells as targets. Non-detectable virus amounts were set to 1. (I) Ratio of C t values for SARS-CoV-2 nucleocapsid (N) and nLuc estimated by RT-PCR using RNA extracted from input virions (inoculum) and virions from sera of mice at 6 dpi. (J, K) Fold changes in cytokine mRNA levels in lung and brain tissues at 6 dpi after normalization to GAPDH mRNA in the same sample and that in uninfected mice. Each curve in (C) and (D) and each data point in (F), (I), (J), and (K) represents an individual mouse. Scale bars in (B and (F) denote radiance (photons/sec/cm 2 /steradian). p values obtained by non-parametric Mann-Whitney test for pairwise comparison. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant; Mean values ± SD are depicted.

Article Snippet: Briefly, 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (pLenti-C-mGFP-P2A-Puro-ACE2) (OriGene).

Techniques: Infection, Ex Vivo, Imaging, Activity Assay, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: bioRxiv

Article Title: Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy

doi: 10.1101/2021.03.22.436337

Figure Lengend Snippet: (A) NAb binding to SARS-CoV-2 Spike ectodomain (S-6P) or RBD estimated by ELISA. Relative light units (RLU) were normalized to the cross-reactive SARS-CoV-1 mAb CR3022. NAb binding to SARS-CoV-2 S2 N-His tag protein on cell-surface of transfected 293T cells analyzed by flow cytometry. Median fluorescence intensities (MFIs) for anti-Spike NAbs were normalized to the signal obtained with an anti-His tag mAb. (B) Flow cytometric detection of 293T cells expressing S from the indicated human CoVs. MFI from 293T cells transfected with empty vector was used for normalization. (C) Pseudoviruses bearing SARS-CoV-2 or SARS-CoV-1 S were tested for capture by anti-Spike NAbs. The cross-reactive CR3022 mAb was used for normalization. (D-E) NAb binding affinity and kinetics to SARS-CoV-2 S using Surface Plasmon Resonance (SPR). SARS-CoV-2 S-6P or S2 ectodomain was immobilized as the ligand on the chip and CV3-1 or CV3-25 Fab was used as analyte at concentrations ranging from 1.56 to 100 nM for both Fabs to S-6P and 3.125nM to 200nM for CV3-25 to S2 (2-fold serial dilution, see Methods for details). Alternatively, CV3-1 IgG was immobilized on the chip and SARS-CoV-2 RBD used as analyte from 1.56 to 50 nM (2-fold serial dilution). Kinetic constants were determined using a 1:1 Langmuir model in BIA evaluation software (experimental readings depicted in blue and fitted curves in black). (F-H) FRET histograms of ligand-free S on S-MEN coronavirus-like particles (VLPs) or in presence of 50 µg/mL of CV3-1 (G) or CV3-25 (H). VLPs were incubated for 1 h at 37°C before smFRET imaging. N m is the number of individual FRET traces compiled into a conformation-population FRET histogram (gray lines) and fitted into a 4-state Gaussian distribution (solid black) centered at 0.1-FRET (dashed cyan), 0.3-FRET (dashed red), 0.5-FRET (dashed green), and 0.8- FRET (dashed magenta). (I) Neutralizing activity of CV3-1 and CV3-25 alone or in combination (1:1 ratio) on SARS-CoV-2 S bearing pseudoviruses using 293T-ACE2 cells. (J) Microneutralization activity of anti-Spike NAbs on live SARS-CoV-2 virus using Vero E6 cells. (K) Inhibition of cell-to-cell fusion between 293T cells expressing HIV-1 Tat and SARS-CoV-2 S and TZM-bl-ACE2 cells by NAbs. Half maximal inhibitory antibody concentration (IC 50 ) values in I-K were determined by normalized non-linear regression analyses. (L) MFI of CEM.NKr cells expressing SARS-CoV-2 Spike (CEM.NKr-Spike) stained with indicated amounts of NAbs and normalized to parental CEM.NKr. (M) % ADCC in the presence of titrated amounts of NAbs using 1:1 ratio of parental CEM.NKr cells and CEM.NKr-Spike cells as targets when PBMCs from non-infected donors were used as effector cells (N) % ADCP in the presence of titrated amounts of NAbs using CEM.NKr-Spike cells as targets and THP-1 cells as phagocytic cells.

Article Snippet: Briefly, 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (pLenti-C-mGFP-P2A-Puro-ACE2) (OriGene).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection, Flow Cytometry, Fluorescence, Expressing, Plasmid Preparation, SPR Assay, Serial Dilution, Software, Incubation, Imaging, Activity Assay, Inhibition, Concentration Assay, Staining, Infection

Journal: bioRxiv

Article Title: Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy

doi: 10.1101/2021.03.22.436337

Figure Lengend Snippet: (A) Experimental design to test in vivo efficacy of NAbs CV3-1 and CV3-25 administered alone (12.5 mg/kg body weight) or as a 1:1 cocktail (6.25 mg/kg body weight each) 1 day prior to challenging K18-hACE2 mice (i.n.) with SARS-CoV-2-nLuc followed by non-invasive BLI every 2 days. Human IgG1-treated (12.5 mg/kg body weight) mice were the control cohort (Iso). (B) Representative BLI images of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions. (C-D) Temporal quantification of nLuc signal as flux (photons/sec) computed non-invasively. (E) Temporal changes in mouse body weight with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test. (G, H) Ex-vivo images of organs and nLuc signal quantified as flux (photons/sec) after necropsy. (I) Viral loads (nLuc activity/mg tissue) measured in Vero E6 cells as targets. Non-detectable virus amounts were set to 1. (J, K) Fold changes in cytokine mRNA levels in lung and brain tissues normalized to GAPDH mRNA in the same sample and that in non-infected mice after necropsy. Viral loads (I) and inflammatory cytokine profile (J, K) were determined after necropsy for mice that succumbed to infection at 6dpi and in mice surviving at 22 dpi. Scale bars in (B) and (G) denote radiance (photons/sec/cm 2 /steradian). Each curve in (C)-(E) and each data point in (H)-(K) represents an individual mouse. Grouped data in (C)-(K) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and for those to CV3-25 are shown in red. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

Article Snippet: Briefly, 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (pLenti-C-mGFP-P2A-Puro-ACE2) (OriGene).

Techniques: In Vivo, Infection, Ex Vivo, Activity Assay

Journal: bioRxiv

Article Title: Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy

doi: 10.1101/2021.03.22.436337

Figure Lengend Snippet: (A) Experimental design to test in vivo efficacy of CV3-1 administered i.p. (12.5 mg/kg body weight) at indicated times after i.n. challenge of K18-hACE2 mice with SARS-CoV-2 nLuc followed by non-invasive BLI every 2 days. Human IgG1 treated (12.5 mg/kg body weight) mice were the control cohort (Iso). (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions. Scale bars denote radiance (photons/sec/cm 2 /steradian). (C-D) Temporal quantification of nLuc signal as flux (photons/sec) computed non-invasively. (E) Temporal changes in mouse body weight with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test. (G, H) Ex vivo imaging of organs and quantification of nLuc signal as flux(photons/sec) after necropsy. (I) Viral loads (nLuc activity/mg tissue) measured in Vero E6 cells as targets. Non-detectable virus amounts were set to 1. (J, K) Fold changes in cytokine mRNA levels in lung and brain tissues normalized to GAPDH mRNA in the same sample and that in non-infected mice after necropsy. Viral loads (I) and inflammatory cytokine profile (J, K) were determined after necropsy at 6dpi. Each curve in (C)-(E) and each data point in (H)-(K) represents an individual mouse. CV3-1 treatment times are indicated in (C)-(E). Grouped data in (C)-(K) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and for groups under CV3-1 therapies to 4 dpi-treatment shown in red. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

Article Snippet: Briefly, 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (pLenti-C-mGFP-P2A-Puro-ACE2) (OriGene).

Techniques: In Vivo, Infection, Ex Vivo, Imaging, Activity Assay

Journal: bioRxiv

Article Title: Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy

doi: 10.1101/2021.03.22.436337

Figure Lengend Snippet: (A) Experimental design to test therapeutic efficacy of NAb CV3-1 and its corresponding Leucine to Alanine (LALA) mutant administered ip (12.5 mg/kg body weight) in K18-hACE2 mice 3 dpi with SARS-CoV-2 nLuc followed by non-invasive BLI every 2 days. Human IgG1-treated (12.5 mg/kg body weight) mice were used as the control cohort (Iso). (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions. Scale bars denote radiance (photons/sec/cm 2 /steradian). (C-D) Temporal quantification of nLuc signal as flux (photons/sec) computed non-invasively. (E) Temporal changes in mouse body weight with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test. (G) Viral loads (nLuc activity/mg tissue) measured in Vero E6 cells as targets. Non-detectable virus amounts were set to 1. (H, I) Fold changes in cytokine mRNA levels in lung and brain tissues normalized to Gapdh mRNA in the same sample and that in non-infected mice after necropsy. Viral loads (G) and inflammatory cytokine profile (H, I) were determined after necropsy at 6dpi. Each curve in (C)-(E) and each data point in (G)-(I) represents an individual mouse. CV3-1 treatment times are indicated in (C)-(E). Grouped data in (C)-(I) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance for group comparisons to isotype control are shown in black and between CV3-1 and CV3-1 LALA treated cohorts are shown in red. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

Article Snippet: Briefly, 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (pLenti-C-mGFP-P2A-Puro-ACE2) (OriGene).

Techniques: Mutagenesis, Infection, Activity Assay

Journal: bioRxiv

Article Title: Live Imaging of SARS-CoV-2 Infection in Mice Reveals Neutralizing Antibodies Require Fc Function for Optimal Efficacy

doi: 10.1101/2021.03.22.436337

Figure Lengend Snippet: (A) Experimental design to test the contribution of NK cells, neutrophils (CD11b + Ly6G + ) and monocytes (CCR2 + Ly6 hi CD11b + ) in K18-hACE2 mice therapeutically treated with CV3-1 NAb (i.p.,12.5 mg/kg body weight) at 3 dpi after challenge with SARS-CoV-2-nLuc. αNK1.1, αLy6G and αCCR2 mAbs (i.p., 20, 20 and 2.5 mg/kg body weight respectively) were used to deplete NK cells, neutrophils and monocytes respectively every 48h starting at 1 dpi. Human and/or rat isotype mAb treated cohorts served as controls (Iso). The mice were followed by non-invasive BLI every 2 days from the start of infection. (B) Representative images from temporal BLI of SARS-CoV-2-nLuc-infected mice in ventral (v) and dorsal (d) positions. Scale bars denote radiance (photons/sec/cm 2 /steradian). (C-D) Temporal quantification of nLuc signal as flux (photons/sec) computed non-invasively. (E) Temporal changes in mouse body weight with initial body weight set to 100%. (F) Kaplan-Meier survival curves of mice statistically compared by log-rank (Mantel-Cox) test. (H) Viral loads (nLuc activity/mg tissue) measured in Vero E6 cells as targets. Non-detectable virus amounts were set to 1. (I, J) Fold change in cytokine mRNA levels in lung and brain tissues normalized to GAPDH mRNA in the same sample and that in non-infected mice after necropsy. Viral loads (H) and inflammatory cytokine profile (I, J) were determined after necropsy at 6dpi. Each curve in C-E and each data point in H-J represents an individual mouse. Grouped data in (C)-(I) were analyzed by 2-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison tests. Statistical significance: group comparisons to isotype control are shown in black; group comparisons to Iso+CV3-1 within the NK and neutrophil depleted cohorts are shown in purple; group comparisons to Iso+CV3-1 within the monocyte-depleted cohorts are shown in red. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Mean values ± SD are depicted.

Article Snippet: Briefly, 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (pLenti-C-mGFP-P2A-Puro-ACE2) (OriGene).

Techniques: Infection, Activity Assay

Journal: Scientific Reports

Article Title: Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

doi: 10.1038/s41598-021-90348-7

Figure Lengend Snippet: Schematic representation of selection and screening strategies for identification of SARS-CoV-2 neutralizing mAbs. ( a ) Cartoon representing SARS-CoV-2 host cell attachment mediated via ACE2-Spike interaction. The ACE2-RBD interaction in the box is adapted from PDB 6M0J . ( b ) Phage display of scFv library was carried out on recombinant SARS-CoV-2 RBD-Fc protein; acidic or competitive elution were implemented. ( c ) Variable heavy chains of scFvs were extracted from sub-libraries and sequenced by MiSeq Illumina. ( d ) The trend of enrichment of given clones was evaluated within and between the selection cycles. ( e ) Potential binders (scFvs) were converted into fully human IgG4 mAbs in a high yield expressing cell line (HEK293ES_1). ( f ) Binding to rRBD and competition with rACE2 were evaluated in vitro with recombinant protein assay. Neutralization capacity of RBD-specific mAbs was further evaluated for blocking SARS-CoV-2 replication.

Article Snippet: The following human recombinant proteins were used: SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein, SARS-CoV-2 (2019-nCoV) Spike RBD-His protein and ACE2 His Protein (all from Sino Biological, 10108-H08H Düsseldorfer Eschborn, Germany); Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (from Abcam, ab273068, Cambridge UK); Recombinant ACE-2 Fc chimeric protein (Z03484 GenScript, Piscataway NJ); recombinant IgG1 Fc protein (R & D Systems, 110-HG Minneapolis MN).

Techniques: Selection, Cell Attachment Assay, Recombinant, Clone Assay, Expressing, Binding Assay, In Vitro, Neutralization, Blocking Assay

Journal: Scientific Reports

Article Title: Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

doi: 10.1038/s41598-021-90348-7

Figure Lengend Snippet: Screening by ELISA and expression of anti-Spike scFvs for analysis of their binding to Spike RBD and their competition with ACE2. ( a ) Representative image of scFv-phages screening by ELISA to test their binding to Spike-RBD recombinant protein. ( b ) Screening of positive phage clones by ELISA assay on human Spike RBD-Fc recombinant protein (black bars) or human recombinant IgG Fc used as a negative control in parallel assays (grey bars). ( c ) Western blotting analysis with the anti-c-myc antibody of the periplasmic extracts of the cells transformed with the two selected positive clones, D3 and F12, expressed in the absence or in the presence of IPTG, used for induction (The blot was obtained by grouping two different parts of the same blot and the black line has been inserted to indicate the two distinct parts. The corresponding full-length blot has been inserted in Supplementary Data set as full-length blot of Figure . The samples were processed in parallel in the same experiment). ( d ) Representative image of soluble scFvs binding to Spike-RBD recombinant protein by ELISA ( e ) at two different concentrations: 45 nM (grey bars) or 90 nM (black bars). ( f ) Representative image of soluble scFvs interference in Spike-ACE2 interaction tested by ELISA ( g ). The binding of ACE2-His to immobilized Spike RBD protein in the absence (dark grey bars) or in the presence (light grey bars) of the indicated soluble scFvs (90 nM). ( h ) Competitive ELISA assay was performed by measuring the binding of Spike RBD-Fc protein on ACE2-positive VERO E6 cells in the absence or in the presence of D3 and F12 scFvs.

Article Snippet: The following human recombinant proteins were used: SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein, SARS-CoV-2 (2019-nCoV) Spike RBD-His protein and ACE2 His Protein (all from Sino Biological, 10108-H08H Düsseldorfer Eschborn, Germany); Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (from Abcam, ab273068, Cambridge UK); Recombinant ACE-2 Fc chimeric protein (Z03484 GenScript, Piscataway NJ); recombinant IgG1 Fc protein (R & D Systems, 110-HG Minneapolis MN).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Binding Assay, Recombinant, Clone Assay, Negative Control, Western Blot, Transformation Assay, Competitive ELISA

Journal: Scientific Reports

Article Title: Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

doi: 10.1038/s41598-021-90348-7

Figure Lengend Snippet: Binding affinity of D3 and F12 mAbs for Spike RBD and their competition with ACE2. ( a ) The binding affinity of ACE2 receptor to immobilized SARS-CoV-2 RBD recombinant protein was tested by ELISA as a positive control. ( b , c ) ELISA assays were performed by testing D3 ( b ) and F12 ( c ) mAbs at increasing concentrations (0.5–100 nM) on human Spike RBD-Fc chimeric protein (black curves). In parallel the Fc domain (grey curves) was used as a negative control. ( d , e ) Competitive ELISA assays were performed by measuring the binding of ACE2-His protein to Spike RBD in the absence or in the presence of D3 ( d ) and F12 ( e ) mAb used at a concentration of 100 nM. ( f ) Competitive ELISA assays were performed by measuring the binding of ACE2-His to RBD in the absence (striped bar) or in the presence of the indicated mAbs used alone (gray bars) or in combination (black bars) at a concentration of 100 nM. The binding values were reported as the mean of at least three determinations obtained in three independent experiments. Error bars depicted means ± SD .

Article Snippet: The following human recombinant proteins were used: SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein, SARS-CoV-2 (2019-nCoV) Spike RBD-His protein and ACE2 His Protein (all from Sino Biological, 10108-H08H Düsseldorfer Eschborn, Germany); Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (from Abcam, ab273068, Cambridge UK); Recombinant ACE-2 Fc chimeric protein (Z03484 GenScript, Piscataway NJ); recombinant IgG1 Fc protein (R & D Systems, 110-HG Minneapolis MN).

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Competitive ELISA, Concentration Assay

Journal: Scientific Reports

Article Title: Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

doi: 10.1038/s41598-021-90348-7

Figure Lengend Snippet: ELISA assays on Spike RBD to test the binding affinity of S96 and AC2 mAbs and their competition with ACE-2. ( a ) The binding affinity and specificity for Spike RBD of S96 and AC2 mAbs was evaluated by testing each mAb at increasing concentrations (0.5–100 nM) on Spike RBD-Fc chimeric protein or Fc, used as a negative control. ( b ) Competitive ELISA assays were performed by measuring the binding of ACE2-His to RBD in the absence (white bars) or in the presence of the indicated mAbs (gray and black bars) at a concentration of 100 nM. ( c ) Competitive ELISA assays to determine the epitope binning were performed by measuring the binding of Biotinylated F12 (F12-B) mAb to RBD, pre-incubated in the absence (white bars) or in the presence of the indicated mAbs (grey and black bars). The binding values were reported as the mean of at least three determinations obtained in three independent experiments. Error bars depicted means ± SD .

Article Snippet: The following human recombinant proteins were used: SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein, SARS-CoV-2 (2019-nCoV) Spike RBD-His protein and ACE2 His Protein (all from Sino Biological, 10108-H08H Düsseldorfer Eschborn, Germany); Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (from Abcam, ab273068, Cambridge UK); Recombinant ACE-2 Fc chimeric protein (Z03484 GenScript, Piscataway NJ); recombinant IgG1 Fc protein (R & D Systems, 110-HG Minneapolis MN).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control, Competitive ELISA, Concentration Assay, Incubation

Journal: Scientific Reports

Article Title: Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

doi: 10.1038/s41598-021-90348-7

Figure Lengend Snippet: Analysis of human primary cells, infected with the VOC 202012/01 lineage B.1.1.7 variant in the absence or presence of D3, by immunofluorescence assays and RT-PCR. ( a ) The cells untreated or infected with the VOC 202012/01 variant of SARS-CoV-2, in the absence or presence of D3 for 72 h at 37 °C, were fixed, washed and permeabilised, as described in the Methods. After blocking, the slides were incubated with the relevant primary antibodies overnight at 4 °C: anti-ACE2 (1:100; ab15348; Abcam) or anti-SARS-CoV-2 Nucleoprotein (N) Antibody (1:100; No. 35-579, ProSci), followed by the relevant secondary anti-mouse Alexa Fluor 488 (1:200; ab150113; Abcam) and anti-Rabbit Alexa Fluor 546 (1:200; A-11035; ThermoFisher), respectively. DNA was stained with DAPI (1:5000; #62254; Thermo Fisher). Microscopy images were obtained with the Elyra 7 platform (Zeiss) with the optical Lattice SIM technology, using the 63 × oil immersion objective. ( b ) Quantification of the mean fluorescence intensity was performed by using the ZEN software (Zeiss, black edition). ( c ) Cell extracts were analyzed by RT-PCR for measuring the expression of N1 viral gene and the levels of the indicated cytokines. In parallel, the extract of uninfected cells was used as negative control.

Article Snippet: The following human recombinant proteins were used: SARS-CoV-2 (2019-nCoV) chimeric Spike RBD-Fc protein, SARS-CoV-2 (2019-nCoV) Spike RBD-His protein and ACE2 His Protein (all from Sino Biological, 10108-H08H Düsseldorfer Eschborn, Germany); Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (from Abcam, ab273068, Cambridge UK); Recombinant ACE-2 Fc chimeric protein (Z03484 GenScript, Piscataway NJ); recombinant IgG1 Fc protein (R & D Systems, 110-HG Minneapolis MN).

Techniques: Infection, Variant Assay, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Incubation, Staining, Microscopy, Fluorescence, Software, Expressing, Negative Control

Journal: Journal of Virology

Article Title: Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

doi: 10.1128/JVI.01207-18

Figure Lengend Snippet: Polarization of HepG2/C3A subclone F2 cells on semipermeable collagen inserts. (A to D) Immunostaining of F2 cells on semipermeable collagen inserts. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Bar =10 μm. (A and B) Maximum-intensity projections of x-y stacks (A) and x-z sections (B) for the tight-junction protein ZO-1. (C and D) x-y (C) and x-z (D) sections for DPP4. (E) Percentages of albumin exported from F2 cells. The albumin in the apical (black bars) and basolateral (gray bars) supernatants was quantified by ELISA. The data shown are means and standard deviations (SD) (n = 12) of the results of four experiments performed in triplicate. *, P < 0.05; ***, P < 0.001. (F) Total bile acids from 21-day cultures of F2 cells quantified by LC-MS. The results are expressed as percentages of exported bile acids in the apical (black bars) and basolateral (gray bars) supernatants. The data shown are means and SD (n = 4). *, P < 0.05. (G) Bile acids were quantified by LC-MS on day 21 postseeding. The amounts of each detected species in the apical (hatched bars) and basolateral (gray bar) supernatants are shown (n = 4). The bile acids quantified were allolithocholic acid (alloLCA), alpha-muricholic acid (aMCA), beta-muricholic acid (bMCA), cholic acid (CA), 3-sulfo-cholic acid (CA-3S), chenodeoxycholic acid (CDCA), 3-sulfo-cheno deoxycholic acid (CDCA-3S), deoxycholic acid (DCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), gamma-muricholic acid (gMCA), glycoursodeoxycholic acid (GUDCA), hyodeoxycholic acid (HDCA), isodeoxycholic acid (isoDCA), isolithocholic acid (isoLCA), lithocholic acid (LCA), tauroalfamuricholic acid (TaMCA), taurobetamuricholic acid (TbMCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA), 3-sulfotaurolithocholic acid (TLCA-3S), ursodeoxycholic acid (UDCA), and omega muricholic acid (wMCA). For clarity, only detected bile acids are shown. (H) HepG2/C3A (circles) and F2 (squares) cells were infected with nHEV genotype 3 (1.35 × 106 HEV RNA copies/106 cells; white symbols) or eHEV genotype 3 (3.3 × 106 HEV RNA copies/106 cells; black symbols). Supernatants were collected every 2 days, and HEV RNA was quantified by RT-PCR. (I) HepG2/C3A (circles) and F2 (squares) cells were infected with eHEV genotype 3 (2.5 × 108 HEV RNA copies/106 cells). Supernatants were collected on day 15 postinfection, and HEV RNA was quantified by RT-PCR. The data shown are from three independent experiments performed in triplicate. The horizontal bars represent medians. *, P < 0.05. (J) Immunofluorescence of ORF2 protein in HepG2/C3A (white bars) and F2 (black bars) cells 21 to 30 days postinfection. Nuclei were stained with DAPI. The results are expressed as percentages of cells containing ORF2. The data shown are means and SD of the results of three independent experiments. ***, P < 0.001.

Article Snippet: Rabbit anti-CD26/DPP4 (Ab28340) was from Abcam (Paris, France).

Techniques: Immunostaining, Staining, Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy, Infection, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: Journal of Virology

Article Title: Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

doi: 10.1128/JVI.01207-18

Figure Lengend Snippet: Colocalization of ORF2 with Rab27A and DPP4 at the apical sides of hepatocytes. (A to E) Immunostaining of ORF2 protein (red) and Rab27a (green) in F2 cells grown on inserts for 14 days, infected with HEV genotype 3, and sampled 14 days postinfection. The arrows across the cells on the merged image indicate the paths of the 2-pixel-wide scans shown in panel E. (C and D) Stacks of 22 (x-y) sections were acquired, slices were separated, and Pearson’s (C) or Manders (D) coefficients were calculated on each section. The mean proportion of ORF2 intensities in Rab27a (red) and of Rab27a intensities in ORF2 (green) are shown. The error bars represent SD; n = 10. (F to J) Immunostaining of ORF2 protein (red) and DPP4 (green) in F2 cells grown on inserts for 14 days, infected with HEV genotype 3, and sampled 14 days postinfection. The arrows across the cells in the merged image indicate the paths of the 2-pixel-wide line scans shown in panel J. (H and I) Stacks of 22 (x-y) sections were acquired, slices were separated, and Pearson’s (H) or Manders (I) coefficients were calculated on each section. The mean proportions of ORF2 intensities in DPP4 (red) and of DPP4 intensities in ORF2 (green) are shown. The error bars represent SD; n = 10. Scale bars = 10 μm.

Article Snippet: Rabbit anti-CD26/DPP4 (Ab28340) was from Abcam (Paris, France).

Techniques: Immunostaining, Infection

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet: Discovery of C-type lectins and TTYH2 as interacting partners of the SARS-CoV-2 S protein (A) Schematic of the myeloid cell receptor discovery approach. Individual plasmids of genes encoding myeloid cell receptors were transfected into HEK293T cells, and a human Fc-tagged SARS-CoV-2 S protein mixture (S-Fc, S1-Fc, and RBD-Fc) and anti-human immunoglobulin G (IgG) Fc detection antibody were added to the cell culture to assess binding (see for more details). S protein subunits and subdomains relative to ACE2 binding (RBD) are also shown. (B) Other than ACE2 (purple), DC-SIGN, L-SIGN, LSECtin, ASGR1, CLEC10A, and TTYH2 (all in red) were identified as binding partners for the SARS-CoV-2 S protein (n = 2). Fc receptors (blue) served as positive controls. (C) Representative images of the binding of the Fc-tagged S protein, its subunits, or Fc control (Fc Ctr) to the indicated receptors, captured by the cellular detection system (CDS) (n = 3). (D) Quantification of the interaction between S protein subdomains/subunits and different receptors, indicated on the x axis. Normalized binding capacity is shown on the y axis (the sum of the total fluorescence intensity to the indicated receptor was set to 100). (E) Binding between HEK293T cells expressing the indicated receptors and HIV-GFP virus pseudotyped with the SARS-CoV-2 S protein was detected by an anti-S polyclonal antibody and analyzed by flow cytometry (n = 4). Data are presented as the mean ± SEM of five pooled independent experiments (D) or a representative experiment (E); ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA (E). n refers to the number of independent experiments. See also Figure S1 .

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Transfection, Cell Culture, Binding Assay, Fluorescence, Expressing, Flow Cytometry

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet: Glycan-dependent SARS-CoV-2 S interaction with C-type lectins via residues distinct from ACE2 (A) Quantification of S-Fc or RBD-Fc binding to HEK293T cells expressing the indicated receptors in the presence or absence of a 100× excess (in the mass ratio) of His-tagged ACE2 ectodomain recombinant protein (ACE2-His) (n = 3). (B) The hydrogen bond formation between the Lys352-Asp353, Tyr41 of human ACE2 (green) and the Thr500-Asn501-Gly502 loop segment of the wild-type (WT) SARS-CoV-2 receptor binding motif (RBM) (PDB: 6M0J ; left , cyan ) or the Ala500-Ala501-Glu502 SARS-CoV-2 RBM mutant (right, cyan). (C) Quantitative comparison of binding to the indicated receptors of Fc-tagged WT S1 and the T500A/N501A/G502A mutant S1 recombinant protein (n = 3). (D) Representative images (left) and quantification (right) of binding of S-Fc or RBD-Fc (for TTYH2) to the indicated receptors in the presence or absence of 20 μg/mL mannan (n = 3). (E) Glycosylated SARS-CoV-2 S protein model highlighting Asn(N)165, N282, N343, and N603 glycans. The SARS-CoV-2 RBD is colored cyan, and the ACE2 N-terminal peptidase domain is shown in green. (F) Quantification of representative S1 Asn mutants that enhanced interaction with ACE2 (N282Q, left) or some of the C-type lectins (N165Q, right) (n = 3). (G) Quantification of representative S1 Asn mutants that reduced interaction with ACE2 (N343Q, left) or C-type lectins (N603Q, right) (n = 3). (H) Schematic of the distribution of N- or O-glycosylation sites in the S1 subunit (top panel) and existence of natural mutations related to some of these sites among ∼5,000 SARS-CoV-2 viral genomes as well as quantitative analysis of the effect of individual mutations of these sites on S protein binding to the indicated receptors (bottom panel). All fluorescence images were captured by CDS and analyzed by CellProfiler software. Data are presented as the mean ± SEM of a representative experiment (A, C, D, F, and G) or three pooled independent experiments (H). ns, not significant. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by two-tailed unpaired Student’s t test (A, C, D, F, and G) or one-way ANOVA (H). n refers to the number of independent experiments. See also Figure S2 .

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Binding Assay, Expressing, Recombinant, Mutagenesis, Protein Binding, Fluorescence, Software, Two Tailed Test

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet: Myeloid cell receptors mediate ACE2-independent SARS-CoV-2 virus-immune interactions (A) HEK293T cells transfected with the indicated receptors or vector control were co-cultured with HIV-GFP virus pseudotyped with SARS-CoV-2 WT or mutant (T500A/N501A/G502E) S protein (SARS-CoV-2 pseudovirus) in the presence or absence of mannan (100 μg/mL) for 48 h, followed by flow cytometry analysis of GFP expression (n = 3). (B) Human PBMC-derived myeloid cells were co-cultured with SARS-CoV-2 pseudovirus with WT S protein in the presence or absence of mannan (100 μg/mL) for 48 h, followed by flow cytometry analysis (n = 3). (C) HEK293T cells with or without ACE2 overexpression (left) and human PBMC-derived myeloid cells (right) were co-cultured with SARS-CoV-2 pseudovirus in the presence of Fc Ctr or anti-ACE2 antibody (20 μg/mL). GFP-positive cells were quantified by flow cytometry after 48-h incubation (n = 3). (D and E) Human PBMC-derived myeloid cells were co-cultured with SARS-CoV-2 pseudovirus with WT S protein (D) or a clinical isolate of SARS-CoV-2 (MOI = 1) (E), with or without Fc-tagged S-interacting decoy receptor(s) (25 μg/mL of each). Cells were analyzed by flow cytometry after 48-h incubation (D) or lysed for RNA extraction and RT-PCR analysis after 24-h incubation (E). The viral mRNA level was normalized to the host GAPDH level, and the average value of the Fc Ctr group was set to 100 (E) (n = 3). Representative flow cytometry plots (left) and statistical analysis (right) are shown in (A), (B), and (D). Data are presented as mean ± SEM of a representative experiment. ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by two-way ANOVA (A), two-tailed unpaired Student’s t test (B and right panel in C), or one-way ANOVA (left panel in C, D, and E). n refers to the number of independent experiments. See also Figure S4 .

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Mutagenesis, Flow Cytometry, Expressing, Derivative Assay, Over Expression, Incubation, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet: Identification of nanobodies capable of blocking SARS-CoV-2-induced hyperinflammatory responses (A) Schematic of the nanobody screening program to develop bispecific nanobodies that block S protein interaction with ACE2 and myeloid cell receptors (see for details). (B) Clustered heatmap of the relative blocking score for each VHH nanobody. Clones A8 and G11 are highlighted in the rectangular areas, and non-RBD interacting nanobodies were labeled with an asterisk (n = 3). (C) Probelife kinetics analysis of binding of A8-Fc, G11-Fc, and A8-G11-Fc to the S protein. K D , K on , and K off values of individual nanobodies are shown in the table. A representative experiment is shown. (D) Human PBMC-derived myeloid cells were incubated with HIV-GFP virus pseudotyped with SARS-CoV-2 S protein in the presence of Fc Ctr, A8-Fc, G11-Fc, or A8-G11-Fc (50 μg/mL) for 48 h, followed by flow cytometry (n = 3). (E) Neutralization of mNeonGreen SARS-CoV-2 reporter virus (MOI = 1) infection of HEK293T cells expressing ACE2 by an S-interacting decoy receptor cocktail (ACE2-Fc/L-SIGN-Fc, 25 μg/mL of each) or a bi-specific nanobody (A8-G11-Fc, 50 μg/mL). (F and G) Human PBMC-derived myeloid cells from two healthy cohorts were incubated with a clinical isolate of SARS-CoV-2 (MOI = 0.5) in the presence of Fc Ctr protein (50 μg/mL), an ACE2-Fc/L-SIGN-Fc cocktail (25 μg/mL of each), or an A8-G11-Fc bi-specific nanobody (50 μg/mL). Mock control was performed using conditioned medium. Cells were lysed for RNA extraction, and the supernatant was harvested 24 h after incubation. mRNA levels of the indicated cytokines and chemokines were measured by RT-PCR and normalized to that of GAPDH (F), and cytokines in the supernatant were quantified by a multiplex magnetic bead assay (G). Data are presented as mean ± SEM of a representative experiment (D and E) or four independent pooled experiments (F and G). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA (D, F, and G). n refers to the number of independent experiments. See also Figure S6 .

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Blocking Assay, Clone Assay, Labeling, Binding Assay, Derivative Assay, Incubation, Flow Cytometry, Neutralization, Infection, Expressing, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Multiplex Assay

Journal: Immunity

Article Title: SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2

doi: 10.1016/j.immuni.2021.05.006

Figure Lengend Snippet:

Article Snippet: The wells were incubated with recombinant ACE2 (Sino Biological), DC-SIGN (Sino Biological), L-SIGN (Sino Biological), LSECtin (Acro Biosystems), ASGR1 (R&D Systems), and CLEC10A (Sino Biological) with human Fc tag at 37°C for 6 hr.

Techniques: Staining, Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot, SYBR Green Assay, Multiplex Assay, Infection, shRNA, cDNA Library Assay, Software, Luminex

Journal: Cell Reports

Article Title: Evolutionary loss of inflammasomes in the Carnivora and implications for the carriage of zoonotic infections

doi: 10.1016/j.celrep.2021.109614

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-mouse, rat caspase-1 p10 (M-20) , Santa Cruz Biotechnology , Cat# sc-514, RRID: AB_2068895.

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Cytotoxicity Assay, Sequencing, Real-time Polymerase Chain Reaction, Software, CRISPR