Journal: Theranostics

Article Title: Inactivation of the NLRP3 inflammasome mediates exosome-based prevention of atrial fibrillation

doi: 10.7150/thno.89520

Figure Lengend Snippet: Intramyocardial injection of human extracellular vesicles (EVs) reduce activation of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome in a model of rat sterile pericarditis. (A) Schematic outline of the inflammatory effect of post operative pericarditis on NLRP3 activation, atrial remodeling and EV effects (red boxes). (B) Effect of pericarditis and EV treatment on the atrial content of NLRP3 and downstream mediators (interleukin-1 beta (IL-1β) and IL-18). NLRP3 data was analyzed using a one-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons test. *P<0.05 vs. talc + vehicle treated animals. Relationship between NLRP3 and IL-1β or IL-18 abundance within atrial tissue was analyzed using simple linear regression. (C) Rank order plot highlighting the abundance of miRNAs (left panel) and proteins (right panel) found in EVs suggested to directly inhibit NLRP3 activation. (D) In vitro effect of explant-derived cell (EDC) EVs on caspase-1 activity, a measure of NLRP3 activation, in THP-1 cells (macrophages) and primary cultured atrial fibroblasts after activation by lipopolysaccharide (LPS) and nigericin. One-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons test. *P<0.05 vs. untreated cells; #P<0.05 vs. LPS+nigericin treated cells. (E) In vitro effect of EDC EVs on downstream mediators of NLRP3 activation (IL-1β and IL-18) in THP-1 cells (macrophages) after activation by LPS and nigericin. One-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons test. *P<0.05 vs. untreated cells; #P<0.05 vs. LPS+nigericin treated cells. (F) Comparative effect of EVs from bone marrow mesenchymal stromal cells (BM-MSCs), umbilical cord mesenchymal stromal cells (UC-MSCs) or EDCs on caspase-1 activity, the principal effector of NLRP3 activation. One-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons test. *P<0.05 vs. untreated cells; #P<0.05 vs. BM-MSC or UC-MSC treated cells. NEDD4, neural precursor cell expressed developmentally down-regulated protein 4; PRDX1, Peroxiredoxin-1; SQSTM1, Sequestosome 1.

Article Snippet: The specificity of caspase-1 activation was validated by including the caspase-1 inhibitor (YVAD-CHO, Promega).

Techniques: Injection, Activation Assay, Binding Assay, Sterility, In Vitro, Derivative Assay, Activity Assay, Cell Culture

Journal: Biochemistry

Article Title: C2 Domain of Protein Kinase C?: Elucidation of the Membrane Docking Surface by Site-Directed Fluorescence and Spin Labeling †

doi: 10.1021/bi026596f

Figure Lengend Snippet: Deduced orientation of the membrane-bound PKCα C2 domain relative to the membrane surface. Shown is a model membrane of pure lipids (44, 45) to which the crystal structure of the isolated PKCα C2 domain has been docked (13). The headgroup layers of the membrane exhibit high heavy atom densities due to the presence crystallographic waters, some of which are highlighted (small space filled molecules), while the central hydrocarbon layer exhibits a lower atom density. For each labeling site on the protein surface, a nitroxide spin label (ball-and-stick, with the nitrogen indicated by sphere) was modeled onto the crystal structure using the program INSIGHT II (Biosym Technologies). Most of the spin labels have been given the g+, g+ dihedral angles believed to be most common (46) except for those where the dihedral angles are constrained to fit the EPR data (M186S1, N189S1, N206S1, R216S1, K230S1, S234S1, R249S1, and R252S1). Also shown are two bound Ca2+ ions (large yellow spheres), the Ca2+ binding loops 1 and 3 that directly contact the membrane, and the phosphate ion (red) bound in the anion binding site on strands 3–4.

Article Snippet: For each labeling site on the protein surface, a nitroxide spin label (ball-and-stick, with the nitrogen indicated by sphere) was modeled onto the crystal structure using the program INSIGHT II (Biosym Technologies).

Techniques: Membrane, Isolation, Labeling, Binding Assay