Journal: Frontiers in Medicine

Article Title: Urinary Levels of SARS-CoV-2 Nucleocapsid Protein Associate With Risk of AKI and COVID-19 Severity: A Single-Center Observational Study

doi: 10.3389/fmed.2021.644715

Figure Lengend Snippet: Association between AKI in COVID-19 and urinary SARS-CoV-2 N, ACE2, and TMPRSS2.

Article Snippet: According to the manufacturer's protocols, the urinary levels of SARS-CoV-2 N (KIT40588, Sino Biological), human ACE2 (NBP2-78734, Novus Biologicals), and human TMPRSS2 (NBP2-89170, Novus Biologicals) were analyzed.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin

doi: 10.3389/fimmu.2022.958581

Figure Lengend Snippet: Effect of LF and the LF-derived peptides pLF1-pLF3 on the activity of serine proteases. Proteolytic activities of purified TMPRSS2 (A) , plasmin (B) , elastase (C) , and trypsin (D) were measured with or without peptides pLF1, pLF2, pLF3, LF or the control peptide pCTR at 37°C in Tris-HCl buffer using the fluorogenic substrate Boc-Gln-Ala-Arg-AMC. The reaction was monitored using an ELISA reader for the indicated time intervals. The insets in (A) and (B) show concentration-dependent inhibition of TMPRSS2 and plasmin activities by pLF1 but not by LF after 30 min and 4 h, Values of p*<0.05, p**<0.005, p***<0.0005, p****<0.0001 (as indicated) were considered to be significant or highly significant, respectively.

Article Snippet: The proteolytic activities of plasmin (10 nM; from human plasma; #10602361001, Roche Diagnostics GmbH, Mannheim, Germany), trypsin (10 nM; from bovine pancreas; #T1005, Sigma-Aldrich, St. Louis, MO), elastase (20 nM; from porcine pancreas; #E1250, Sigma-Aldrich) and TMPRSS2 (25 nM; recombinant protein produced in mammalian cells; #MBS1193731, MyBioSource, San Diego, CA) were measured in a Nunc black fluorescence-based cell assay plate (Sigma-Aldrich) in Tris-HCl buffer (20 mM, pH 8.0, 150 mM NaCl).

Techniques: Derivative Assay, Activity Assay, Purification, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin

doi: 10.3389/fimmu.2022.958581

Figure Lengend Snippet: Effect of LF and the LF-derived peptides on SARS-CoV-2 S protein proteolytic processing. (A) The purified recombinant 6x-His-tagged S protein was exposed to TMPRSS2 with or without pLF1 or pCTR at 37°C in Tris-HCl buffer for 2 h. The digestion of the S protein was analyzed by immunoblotting of the reaction mixtures followed by an incubation with the specific anti-6x-His mAb. Upon cleavage by TMPRSS2, a digestion product of the S protein of about 75 kD became visible (marked by *). (B) Densitometric quantifications of the proteolytic processing of the S protein by TMPRSS2. Peptide pCTR served as a control. Four independent immunoblotting experiments were evaluated. The bands corresponding to the full-length S protein in the absence of TMPRSS2 were set as a relative maximum of 100%. The immunoblots were quantified by the AzureSpot software. (C) Purified recombinant 6x-His-tagged S protein was exposed to plasmin instead of TMPRSS2 and treated afterwards as in (A) . (D) Densitometric quantifications of the proteolytic processing of the S protein by plasmin. Peptide pCTR served as a control. Densitometric evaluation of three independent immunoblotting experiments was performed as in (B) . (E-G) The purified recombinant 8x-His-tagged mutated S protein was treated in in vitro proteolysis assay with TMPRSS2 (E) and plasmin (F) as in (A, C) , and the cleavage (G) was evaluated as in (B, D) Values of p*<0.05, p**<0.005 (as indicated) were considered to be significant..

Article Snippet: The proteolytic activities of plasmin (10 nM; from human plasma; #10602361001, Roche Diagnostics GmbH, Mannheim, Germany), trypsin (10 nM; from bovine pancreas; #T1005, Sigma-Aldrich, St. Louis, MO), elastase (20 nM; from porcine pancreas; #E1250, Sigma-Aldrich) and TMPRSS2 (25 nM; recombinant protein produced in mammalian cells; #MBS1193731, MyBioSource, San Diego, CA) were measured in a Nunc black fluorescence-based cell assay plate (Sigma-Aldrich) in Tris-HCl buffer (20 mM, pH 8.0, 150 mM NaCl).

Techniques: Derivative Assay, Purification, Recombinant, Western Blot, Incubation, Control, Software, In Vitro, Proteolysis Assay

Journal: Frontiers in Immunology

Article Title: Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin

doi: 10.3389/fimmu.2022.958581

Figure Lengend Snippet: Generation and testing of human LFC. (A) Optimizations of HCl and pepsin concentrations (mM and % (w/w), respectively) on LF cleavage. LF (10.5 mg/mL) was exposed to pepsin (3% or 10% per amount of LF) and HCl (10-90 mM, as indicated) for 30 min (left panel) or various time points (15, 30, 45 or 60 min, right panel) at 37°C. Digestion mixtures were analyzed by 10% tricine-SDS-PAGE and subjected to Coomassie blue staining. (B) Separation of LF cleavage products by cation exchange chromatography. Individual fractions were analyzed by 10% tricine-SDS-PAGE and subjected to Coomassie blue staining: L – digestion mixture, X1 - material not captured on the column, A2, A4, A5, A6 - eluted fractions. (C) Effect of LFC (fraction A6) on the activity of TMPRSS2. Proteolytic activity of TMPRSS2 was measured with or without LFC or pCTR at 37°C in Tris-HCl buffer using the fluorogenic substrate Boc-Gln-Ala-Arg-AMC. The reaction was monitored using an ELISA reader for the indicated time intervals. (D) Effect of LFC on the infection capability of SARS-CoV-2. Vero cells were incubated with or without LFC or pCTR (both 80 µg/mL) for approx. 1 h, and then infected with the authentic SARS-CoV-2 (300 TCID50/well; MOI ≈0.02). After 48 h, the cells were fixed and the infection rate was assessed by In-Cell ELISA. Values show mean inhibitory capacity of LFC and pCTR ± SD from three independent experiments. p*<0.05.

Article Snippet: The proteolytic activities of plasmin (10 nM; from human plasma; #10602361001, Roche Diagnostics GmbH, Mannheim, Germany), trypsin (10 nM; from bovine pancreas; #T1005, Sigma-Aldrich, St. Louis, MO), elastase (20 nM; from porcine pancreas; #E1250, Sigma-Aldrich) and TMPRSS2 (25 nM; recombinant protein produced in mammalian cells; #MBS1193731, MyBioSource, San Diego, CA) were measured in a Nunc black fluorescence-based cell assay plate (Sigma-Aldrich) in Tris-HCl buffer (20 mM, pH 8.0, 150 mM NaCl).

Techniques: SDS Page, Staining, Chromatography, Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Incubation, In-Cell ELISA