The strategy of developing NAAA inhibitors.

<p>A–C, the chemical structures of classic NAAA inhibitors including PEA (A), CCP (B), and (S)-OOPP (C); (D) SAR study of 1-Pentadecanyl-carbonyl pyrrolidine; (E) Dose-dependent inhibition of 1-Pentadecanyl-carbonyl pyrrolidine on NAAA activity.</p

Compound 16 reduced LPS-induced inflammation.

<p>(A) Effect of compound 16 (concentrations in µM) or Vehicle on PEA levels (A), mRNA expression levels of iNOS (B) and IL-6 (C) in RAW264.7 treated with vehicle (open bars) or LPS (closed bars). vehicle, 0.1% DMSO; LPS, 0.5 µg/mL. **, P<0.01; ***, P<0.001 vs. vehicle; ##, P<0.01; ###, P<0.001 vs. LPS control, n = 5.</p

Characterization of compound 16 as a reversible and competitive NAAA inhibitor.

<p>(A) Effect of compound 16 (10 µM) on NAAA activity in HEK293 cells heterogeneously overexpressing NAAA. ***, P<0.001 vs. vehicle, n = 4. (B) Concentration-dependent inhibition of NAAA by compound 16 using NAAA recombinant protein derived from HEK293 cell heterogeneously expressing NAAA. (C) Rapid dilution NAAA assay in the presence of vehicle (1% DMSO, open circles) or compound 16 (closed circles). (D) Effect of NAAA activity in the presence of vehicle (open bars) or compound 16 (closed bars) before dialysis (0) and 8 hr after dialysis (8). ***, P<0.001 vs vehicle, n = 4; (E) Michaelis-Menten analysis of the NAAA reaction in the presence of vehicle (open circles) or compound 16 (closed circles). Insert is shown in a Lineweaver-Burk plot.</p

Compound 16 interacted with NAAA protein.

<p>(A) Computational model illustrated docking of compound 16 at the active site of rat NAAA. (B) Effect of mutant Ala²⁰⁹-NAAA on NAAA activity. Mock, HEK293 cell heterogeneously overexpressing vector control; NAAA, HEK293 cell heterogeneously overexpressing NAAA; Ala209, HEK293 cell heterogeneously overexpressing mutant Ala²⁰⁹-NAAA. ***, P<0.001 vs. NAAA, n = 5.</p

Atheroprotective Effect of Oleoylethanolamide (OEA) Targeting Oxidized LDL

<div><p>Dietary fat-derived lipid oleoylethanolamide (OEA) has shown to modulate lipid metabolism through a peroxisome proliferator-activated receptor-alpha (PPAR-α)-mediated mechanism. In our study, we further demonstrated that OEA, as an atheroprotective agent, modulated the atherosclerotic plaques development. <i>In vitro</i> studies showed that OEA antagonized oxidized LDL (ox-LDL)-induced vascular endothelial cell proliferation and vascular smooth muscle cell migration, and suppressed lipopolysaccharide (LPS)-induced LDL modification and inflammation. <i>In vivo</i> studies, atherosclerosis animals were established using balloon-aortic denudation (BAD) rats and ApoE^-/- mice fed with high-caloric diet (HCD) for 17 or 14 weeks respectively, and atherosclerotic plaques were evaluated by oil red staining. The administration of OEA (5 mg/kg/day, intraperitoneal injection, i.p.) prevented or attenuated the formation of atherosclerotic plaques in HCD-BAD rats or HCD-ApoE^−/− mice. Gene expression analysis of vessel tissues from these animals showed that OEA induced the mRNA expressions of PPAR-α and downregulated the expression of M-CFS, an atherosclerotic marker, and genes involved in oxidation and inflammation, including iNOS, COX-2, TNF-α and IL-6. Collectively, our results suggested that OEA exerted a pharmacological effect on modulating atherosclerotic plaque formation through the inhibition of LDL modification in vascular system and therefore be a potential candidate for anti-atherosclerosis drug.</p></div

DataSheet1_Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing.docx

Moraxella catarrhalis (M. catarrhalis) was an important pathogen closely associated with respiratory tract infections. We employed the loop-mediated isothermal amplification (LAMP) coupled with nanoparticle-based lateral flow biosensor (LFB) and fluorescence testing technique for formulating two diagnostic methods for M. catarrhalis detection, termed M. catarrhalis-LAMP-LFB assay and M. catarrhalis-LAMP-FRT, respectively. The M. catarrhalis-LAMP-LFB system incorporated the use of biotin-14-dCTP and a forward loop primer (LF) with a hapten at the 5′ end. This design in LAMP reaction enabled the production of double-labeled products that could be effectively analyzed using the lateral flow biosensor (LFB). For the M. catarrhalis-LAMP-FRT assay, the LF was modified with a sequence at 5′ end, and a fluorophore, as well as a black hole quencher, were strategically labeled at the 5′ end and within the middle of the new LF. The restriction endonuclease Nb.BsrDI could accurately recognize and cleave the newly synthesized double-strand terminal sequences, resulting in the separation of the fluorophore from the black hole quencher and releasing fluorescence signals. Both assays have been proven to be highly sensitive and specific, capable of detecting genomic DNA of M. catarrhalis at concentrations as low as 70 fg, with no cross-reactivity observed with non-M. catarrhalis pathogens. Furthermore, both methods successfully identified M. catarrhalis in all clinical samples within 1 h that were confirmed positive by real-time PCR, exhibiting superior sensitivity than conventional culture methods. Herein, the newly developed two LAMP-based assays were rapid and reliable for M. catarrhalis detection and hold significant promise for deployment in point-of-care (POC) settings.</p

OEA reduced atherosclerotic plaque formation in ApoE^−/− mice.

<p>Oil red O staining (A) and H & E staining (B–E) showed aortic atherosclerotic formation in wild-type C57mice fed with HCD (A₁, B), ApoE<b>⁻</b>^/<b>−</b> mice fed with normal diet (A₂, C), ApoE<b>⁻</b>^/<b>−</b> mice fed with HCD (A₃, D), and ApoE<b>⁻</b>^/<b>−</b>-HCD mice with OEA administration (5 mg/kg, i.p.) (A₄, E). Scale bar, 2 mm or 0.5 mm. (F–K), The effect of OEA on mRNA expression levels of PPAR-α (F), M-CSF (G), COX-2 (H), CRP (I), TNF-α (J) and IL-6 (K) in aorta tissues of wt-HCD mice, ApoE<b>⁻</b>^/<b>−</b>-ND mice and ApoE<b>⁻</b>^/<b>−</b>-HCD mice. Vehicle, 5% PEG/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p.; * p<0.05, ** p<0.01, *** p<0.001, one-way ANOVA, n = 6–8 mice/group.</p

Luteolin down-regulated interacting proteins of Hsp90 and induced degradation of prospho-STAT3 and Akt.

<p>A, HeLa cells were treated with 0, 20, 30, 40, 50 µM luteolin for 24 h and then were lysed. Protein levels of Akt, IKKα, IKKβ and Hsp90 in cell lysates were detected by using indicated antibody respectively. B, As a control, indicated dose of flavone was used to treat HeLa cells for 24 h. The levels of phosphor-STAT3 (tyr705), phosphor-STAT3 (ser727), STAT3, Akt and Hsp90 were detected by Western blotting. C, HeLa cells were treated with luteolin (50 µM) for 24 h in the presence of proteasome inhibitor MG-132 (30 µM), protein levels of phosphor-STAT3 (tyr705), phosphor-STAT3 (ser727), STAT3 and Akt were measured by Western blot analysis.</p

OEA prevented atherosclerotic plaque formation in BAD rats.

<p>(A–D), The effect of vehicle (A–C) or OEA (D) on atherosclerotic plaque formation in BAD rats fed with normal diet (BAD-ND, A), sham-operated rats fed with high-caloric diet (sham-HCD, B), and BAD rats fed with HCD (BAD-HACD, C-D). Scale bar, 50 μm. (E–J), The effect of vehicle (−) or OEA (+) on mRNA expression levels of PPAR-α (E), M-CSF (F), iNOS (G), CRP (H), TNF-α (I) and IL-6 (J) in aorta tissues of BAD-ND rats, sham-HCD rats and BAD-HCD rats. Vehicle, 5% PEG/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p.; * p<0.05, ** p<0.01, *** p<0.001, one-way ANOVA, n = 7–9 rats/group.</p

Luteolin induced carcinoma cells apoptosis.

<p>A, Carcinoma cells (HeLa, HepG2), and normal cells (WRL-68, HEK293, XJH B) were treated with 20, 30, 40, 50, 60 µM luteolin for 24 h and followed by CytoTox-Glo™ cytotoxicity assays. The data above are plotted as “dead cell” rate versus the concentration of Luteolin. B. HeLa cells were treated with indicated concentration of luteolin for 24 h and subjected to immunoblot analysis for pro-caspase3 and PARP. C. HeLa cells were treated with indicated concentration of luteolin for 24 h and were harvested, and then stained with propidium iodidle and AnnexinV-FITC for detecting the apoptosis by flow cytometry. D. HeLa cells were transfected with indicated concentration of HA-Hsp90 and 24 h after transfection cells were treated with 50 µM luteolin for 24 h and were harvested, and then stained with propidium iodidle and AnnexinV-FITC for detecting the apoptosis by flow cytometry. E. HeLa cells were treated with 50 µM luteolin and 10 µM GA, for 24 h, and then cells were incubated with primary antibodies against PARP. HeLa cells were immunostained with anti-mouse alexa fluor 488 secondary antibody and then stained with DAPI and Alexa Fluor 555 phalloidin derivatives for labeling F-actin. The specimens were visualized by confocal laser scanning microscopy. Blue depicts the nucleus, red depicts localization of F-actin and green depicts localization of cleaved-PARP. F. HeLa cells were pretreated with 25 µM z-VAD-fmk for 1 h and then treated with 50 µM luteolin for 24 h followed by immunoblotting with pro-caspase3 and cleaved PARP antibodies.</p