Journal: Science Advances

Article Title: Leukotriene A4 hydrolase inhibition improves age-related cognitive decline via modulation of synaptic function

doi: 10.1126/sciadv.adf8764

Figure Lengend Snippet: ( A ) Brain homogenate concentration of AKST1220 (nanomolar) following a single oral dose of 10, 1, or 0.1 mg/kg. ( B ) Brain homogenate concentration of SC57461A (nanomolar) following a single oral dose of 10 or 5 mg/kg. ( C ) Comparison of the brain homogenate concentrations of AKST1220 (A) and SC57461A (B) at 10 mg/kg. All data contain n = 3 mice per treatment group per time point at 20 months of age. All data are shown as means ± SEM. ( D ) Representative image of mouse midbrain after a single oral dose of 14 C-AKST1220 (small black particles) and subsequent immunohistochemical staining of biotinylated lectin (brown). Inset zoom in of the dentate gyrus (DG) with black arrows identifying blood vessels and arrowheads identifying AKST1220 in the brain parenchyma. H&E, with hematoxylin and eosin. ( E ) Dark-field image of (D) to visualize 14 C-AKST1220 silver grains as bright white particles on a dark background.

Article Snippet: Five minutes before administration of 14 C-AKST1220, a single intravenous dose of biotinylated lectin (Vector Laboratories) was administered at 1.5 mg/kg.

Techniques: Concentration Assay, Comparison, Immunohistochemistry, Staining

Journal: Frontiers in Immunology

Article Title: Interactions between Siglec-8 and endogenous sialylated cis ligands restrain cell death induction in human eosinophils and mast cells

doi: 10.3389/fimmu.2023.1283370

Figure Lengend Snippet: Treatment of human eosinophils with sialidase from V. cholerae selectively cleaves α2,3-linked sialic acids from the cell surface. (A) Human eosinophils isolated from peripheral blood were incubated with biotinylated 1-MDa polyacrylamide decorated with the Siglec-8 glycan ligand 6′-sulfo-3′-sialyl-LacNAc for 20 min at 4°C prior to detection of surface-bound ligand with fluorophore-conjugated streptavidin. Sialidase (Sia) from V. cholerae was used at 50 mU/mL for 1 hr at 37°C to cleave sialic acid from the ligand or from the eosinophils prior to ligand binding. Data are representative (top) or show the quantified normalized Siglec-8 binding to eosinophils relative to control untreated cells as well as overall mean and standard deviations of seven independent experiments (bottom). **, p<0.01; ***, p<0.001; ****, p<0.0001 by one-way ANOVA and Tukey multiple comparisons test. (B, C) Eosinophils were incubated with or without 30 ng/mL rhIL-5 for 18–24 hr. Unprimed cells were then treated with sialidase at the indicated activities for 1 hr prior to detection of cell-surface sialic acids by lectin binding. Surface-bound biotinylated MAL-II (B) or SNA (C) lectin was detected using fluorophore-conjugated streptavidin by flow cytometry. Similarly treated eosinophils incubated without lectin (No Lectin) were analyzed for background streptavidin binding. Data are representative (top) or show quantified normalized lectin binding from three independent experiments (bottom). **, p<0.01; ****, p<0.0001 vs. untreated control sample by two-way ANOVA and Dunnett multiple comparisons test.

Article Snippet: To detect cell-surface sialic acid, eosinophils (2×10 5 cells per condition) were incubated with biotinylated Maackia amurensis lectin (MAL)-II or Sambucus nigra agglutinin (SNA) (both from Vector Laboratories, Newark, CA) at 10 μg/mL for 30 min. After washing, cell surface-bound lectin was stained with DyLight488-conjugated streptavidin (Vector Laboratories) at 10 μg/mL for 30 min and detected by flow cytometry.

Techniques: Isolation, Incubation, Ligand Binding Assay, Binding Assay, Flow Cytometry