Journal: International Immunopharmacology

Article Title: Dual mechanism: Epigenetic inhibitor apabetalone reduces SARS-CoV-2 Delta and Omicron variant spike binding and attenuates SARS-CoV-2 RNA induced inflammation

doi: 10.1016/j.intimp.2023.109929

Figure Lengend Snippet: BETi reduces Delta- and Omicron-spike protein binding to Calu-3 cells. (A) Soluble ACE2 blocks wild-type SARS-CoV-2 spike-RBD binding to ACE2 expressing Calu-3 cells. Competitive binding of wild-type spike-RBD Fc to membrane-bound ACE2 on Calu-3 cells with soluble recombinant human ACE2 (rhACE2, R&D Systems) was examined for ACE2-dependent binding. (B) Quantification of BETi impact on the binding of spike-RBD-Fc (wild-type versus Delta) to Calu-3 cells following 48 h or 72 h of BETi treatments. (C-D) Assessment of binding of Delta (C) or Omicron (D) spike-ectodomain (coupled with Alexa Fluor TM 647) to Calu-3 cells following 48 h or 72 h of BETi treatments. WT = wild-type, apa = apabetalone, ctr = control. Data are mean ± SD from four independent experiments. For A-B, binding results are shown as mean fluorescent intensity (MFI) ratio to human IgG1 Fc ctr. For C-D, binding results are shown as MFI ratio to no binding control (cells alone). Statistical analysis was done by 1-way ANOVA followed by Dunnett's multiple comparison test relative to Fc ctr (A) or DMSO (B-D). Also, 2-way ANOVA was performed followed by Sidak’s multiple comparison test when comparing wild-type and Delta in B. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In this assay, we used two types of recombinant SARS-CoV-2 spike RBD regions fused to the human IgG1 Fc fragment (spike-RBD-Fc, R&D Systems) including wild-type (wild-type spike-RBD-Fc) and its Delta variant form (Delta spike-RBD-Fc).

Techniques: Protein Binding, Binding Assay, Expressing, Membrane, Recombinant, Control, Comparison

Journal: Frontiers in Immunology

Article Title: Adrenomedullin Expression Is Associated With the Severity and Poor Prognosis of Interstitial Lung Disease in Dermatomyositis Patients

doi: 10.3389/fimmu.2022.885142

Figure Lengend Snippet: Adrenomedullin mRNA levels from PBMCs were elevated in DM patients with RP-ILD. (A) Adrenomedullin mRNA levels in DM patients were significantly higher than those in IMNM patients and HCs. (B) Adrenomedullin mRNA levels in DM patients with ILD and those without ILD. (C) The adrenomedullin mRNA levels in DM patients with RP-ILD, chronic ILD, and those without ILD. DM, dermatomyositis; IMNM, immune-mediated necrotizing myopathy; HC, healthy control; ILD, interstitial lung disease; RP-ILD, rapidly progressive interstitial lung disease. NS indicates no significant difference. Differences between the levels were expressed as relative expression via the 2 -ΔCt method. Data were expressed as the mean ± standard deviation (SD).

Article Snippet: The tissue sections were incubated with rabbit anti-adrenomedullin polyclonal antibody (1:500 dilution; Proteintech, Wuhan, China) overnight at 4°C, and then incubated with a goat anti-rabbit IgG secondary antibody (Gene Tech Shanghai Company Limited, Shanghai, China) for 30 min at room temperature.

Techniques: Control, Expressing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Adrenomedullin Expression Is Associated With the Severity and Poor Prognosis of Interstitial Lung Disease in Dermatomyositis Patients

doi: 10.3389/fimmu.2022.885142

Figure Lengend Snippet: Elevated adrenomedullin mRNA levels in PBMCs were correlated with the severity of lung involvement in DM patients with ILD. (A) Adrenomedullin mRNA Levels were positively correlated with ferritin levels in DM-ILD. (B) Adrenomedullin mRNA Levels were positively correlated with LDH levels in DM-ILD. (C) Correlation between adrenomedullin mRNA levels and lung VAS in DM-ILD. (D) Adrenomedullin mRNA levels were negatively correlated with FVC% in DM-ILD. (E) Adrenomedullin mRNA levels were negatively correlated with FEV1% in DM-ILD. (F) Adrenomedullin mRNA levels were negatively correlated with DLco% in DM-ILD. (G) Adrenomedullin mRNA expression levels in patients with severe ILD (n = 3) and mild-moderate ILD (n = 15). DM, dermatomyositis; ILD, interstitial lung disease; LDH, lactate dehydrogenase; VAS, visual analogue scale; FVC, forced vital capacity; FEV1, forced expiratory volume in 1s; DLco, carbon monoxide diffusion capacity.

Article Snippet: The tissue sections were incubated with rabbit anti-adrenomedullin polyclonal antibody (1:500 dilution; Proteintech, Wuhan, China) overnight at 4°C, and then incubated with a goat anti-rabbit IgG secondary antibody (Gene Tech Shanghai Company Limited, Shanghai, China) for 30 min at room temperature.

Techniques: Expressing, Diffusion-based Assay

Journal: Frontiers in Immunology

Article Title: Adrenomedullin Expression Is Associated With the Severity and Poor Prognosis of Interstitial Lung Disease in Dermatomyositis Patients

doi: 10.3389/fimmu.2022.885142

Figure Lengend Snippet: Enhanced adrenomedullin expression in the lung tissue of DM patients with RP-ILD. (A, B) The lung of a DM patient with chronic ILD. Positive adrenomedullin staining was detected in macrophages (black arrowhead) and alveolar epithelial cells (white arrowhead). (C, D) The lung of a DM patient with RP-ILD. Positive adrenomedullin staining was detected in macrophages (black arrowhead) and alveolar epithelial cells (white arrowhead). Scale bar = 50 μm. DM, dermatomyositis; ILD, interstitial lung disease; RP-ILD, rapidly progressive ILD.

Article Snippet: The tissue sections were incubated with rabbit anti-adrenomedullin polyclonal antibody (1:500 dilution; Proteintech, Wuhan, China) overnight at 4°C, and then incubated with a goat anti-rabbit IgG secondary antibody (Gene Tech Shanghai Company Limited, Shanghai, China) for 30 min at room temperature.

Techniques: Expressing, Staining

Journal: Frontiers in Immunology

Article Title: Adrenomedullin Expression Is Associated With the Severity and Poor Prognosis of Interstitial Lung Disease in Dermatomyositis Patients

doi: 10.3389/fimmu.2022.885142

Figure Lengend Snippet: Prognostic value of adrenomedullin in DM patients with ILD and survival analysis. (A) The adrenomedullin mRNA levels in PBMCs were higher in decedents than survivors among DM patients with ILD. (B) Receiver operating characteristic curve analyses to predict the mortality of DM patients with ILD. (C) The cumulative survival rate was significantly lower in the group with adrenomedullin mRNA levels >0.053 than in those with adrenomedullin mRNA levels <0.053 (62.5% vs 100%, log-rank test, P = 0.005). (D) Kaplan–Meier curves showed that the cumulative survival rate was significantly lower in DM patients with RP-ILD than in those without RP-ILD (53.8% vs 100%, log-rank test, P < 0.001). DM, dermatomyositis; ILD, interstitial lung disease; RP‐ILD, rapidly progressive ILD.

Article Snippet: The tissue sections were incubated with rabbit anti-adrenomedullin polyclonal antibody (1:500 dilution; Proteintech, Wuhan, China) overnight at 4°C, and then incubated with a goat anti-rabbit IgG secondary antibody (Gene Tech Shanghai Company Limited, Shanghai, China) for 30 min at room temperature.

Techniques:

Journal: Frontiers in Immunology

Article Title: Adrenomedullin Expression Is Associated With the Severity and Poor Prognosis of Interstitial Lung Disease in Dermatomyositis Patients

doi: 10.3389/fimmu.2022.885142

Figure Lengend Snippet: A proposed role of adrenomedullin in the develop and progress of ILD in DM. Adrenomedullin stimulates macrophage activations and activated macrophage could also secrete adrenomedullin and other inflammatory cytokines, type I interferon, promote the production of TGF-β, and collagen-I which could contribute to the develop and progress of ILD in DM.

Article Snippet: The tissue sections were incubated with rabbit anti-adrenomedullin polyclonal antibody (1:500 dilution; Proteintech, Wuhan, China) overnight at 4°C, and then incubated with a goat anti-rabbit IgG secondary antibody (Gene Tech Shanghai Company Limited, Shanghai, China) for 30 min at room temperature.

Techniques:

Journal: bioRxiv

Article Title: Investigation of ACE2 N-terminal fragments binding to SARS-CoV-2 Spike RBD

doi: 10.1101/2020.03.19.999318

Figure Lengend Snippet: (A) RMSD for SBP1 docked to SARS-CoV-2-RBD during 200 ns MD simulation. (B) Binding interface between SARS-CoV-2-RBD and SBP1 after 200 ns simulation. Individual RMSD (C) and average RMSD (D) values for SBP1 residues over the course of the 200 ns simulation. Arrows indicate residues contributing key hydrogen bonding interactions (determined using UCSF Chimera, Version 1.12). Individual residues with RMSD below 5 Å arbitrarily colored green. (E) Binding affinity of SBP1 and a scrambled SBP1 sequence to various sources of glycosylated SARS-CoV-2-RBD proteins determined by bio-layer interferometry (BLI). (F) BLI association response of SBP1 to negative control human protein menin and (G) BLI association response of 12-mer SBP2 peptide to Sino Biological insect-derived SARS-CoV-2-RBD.

Article Snippet: However, competition was not observed when using non-biotinylated SBP1 pre141 mixed in solution with Sino Biological insect-derived SARS-CoV-2-RBD, even with a 1000-fold excess of the peptide ( ).

Techniques: Binding Assay, Sequencing, Negative Control, Derivative Assay

Journal: bioRxiv

Article Title: Investigation of ACE2 N-terminal fragments binding to SARS-CoV-2 Spike RBD

doi: 10.1101/2020.03.19.999318

Figure Lengend Snippet: (A) Association responses of biotinylated ACE2 to Sino Biological insect derived SARS-CoV-2-RBD at different concentrations. The kinetic dissociation constant determined under these conditions was K D , ACE2 = 15 nM. (B and D) Association responses of biotinylated ACE2 with Sino Biological insect-derived SARS-CoV-2-RBD (kept constant at 100 nM) after mixing with soluble human ACE2 at different concentrations. (B) shows the BLI traces and (D) shows re-plots of the endpoint association response (nm) as a function of ACE2 concentration. (C and E) Association responses of biotinylated ACE2 with Sino Biological insect-derived SARS-CoV-2-RBD (kept constant at 100 nM) after mixing with SBP1 at different concentrations. (C) shows the BLI traces and (E) shows re-plots of the endpoint association response (nm) as a function of SBP1 concentration.

Article Snippet: However, competition was not observed when using non-biotinylated SBP1 pre141 mixed in solution with Sino Biological insect-derived SARS-CoV-2-RBD, even with a 1000-fold excess of the peptide ( ).

Techniques: Derivative Assay, Concentration Assay

Journal: bioRxiv

Article Title: Investigation of ACE2 N-terminal fragments binding to SARS-CoV-2 Spike RBD

doi: 10.1101/2020.03.19.999318

Figure Lengend Snippet: Total ion current chromatograms (TIC) obtained by LC-MS analysis of commercial samples of (A) glycosylated Sino Biological HEK-expressed SARS-CoV-2-RBD (solution in phosphate-buffered saline) and (B) glycosylated Sino Biological insect-derived SARS-CoV-2-RBD with associated deconvoluted mass spectra obtained by integration over the protein peak at ~8 min. The broad bands in the TIC chromatograms of (B) are due to additives present in the vendor-formulated solid powder (10% glycerol, 5% trehalose, 5% mannitol and 0.01% tween-80).

Article Snippet: However, competition was not observed when using non-biotinylated SBP1 pre141 mixed in solution with Sino Biological insect-derived SARS-CoV-2-RBD, even with a 1000-fold excess of the peptide ( ).

Techniques: Liquid Chromatography with Mass Spectroscopy, Derivative Assay