The role of retinoid-related orphan receptor-α in cigarette smoke-induced autophagic response.

BACKGROUND: Retinoid-related orphan receptor-α (RORα) and autophagy dysregulation are involved in the pathophysiology of chronic obstructive pulmonary disease (COPD), but little is known regarding their association. We investigated the role of RORα in COPD-related autophagy. METHODS: The lung tissues and cells from a mouse model were analyzed for autophagy markers by using western blot analysis and transmission electron microscopy. RESULTS: Cigarette smoke increased the LC3-II level and decreased the p62 level in whole lung homogenates of a chronic cigarette smoking mouse model. Although cigarette smoke did not affect the levels of p62 in Staggerer mutant mice (RORαsg/sg), the baseline expression levels of p62 were significantly higher than those in wild type (WT) mice. Autophagy was induced by cigarette smoke extract (CSE) in Beas-2B cells and in primary fibroblasts from WT mice. In contrast, fibroblasts from RORαsg/sg mice failed to show CSE-induced autophagy and exhibited fewer autophagosomes, lower LC3-II levels, and higher p62 levels than fibroblasts from WT mice. Damage-regulated autophagy modulator (DRAM), a p53-induced modulator of autophagy, was expressed at significantly lower levels in the fibroblasts from RORαsg/sg mice than in those from WT mice. DRAM knockdown using siRNA in Beas-2B cells inhibited CSE-induced autophagy and cell death. Furthermore, RORα co-immunoprecipitated with p53 and the interaction increased p53 reporter gene activity. CONCLUSIONS: Our findings suggest that RORα promotes autophagy and contributes to COPD pathogenesis via regulation of the RORα-p53-DRAM pathway.

Highly Sensitive Ratiometric Fluorescent Detection of Indium(III) Using Fluorescent Probe Based on Phosphoserine as a Receptor.

Indium is one of the most widely used scarce metals for manufacturing various electronic devices including notebooks, mobile phones, and PC monitors. Recent studies revealed that indium and its compound could cause several toxicities to human beings and animals. However, there is no report about ratiometric fluorescent detection of In(III) in aqueous solutions. We synthesized a fluorescent probe (1) for In(III) based on a phosphoserine as a receptor with a pyrene fluorophore using solid phase synthesis. 1 showed highly sensitive ratiometric response to In(III) in purely aqueous solutions by increasing excimer emission intensity at 476 nm with a concomitant decrease in monomer emission intensity at 395 nm. 1 showed sensitive ratiometric responses to In(III) over a wide range of pH (2 < pH < 8) and exhibited a highly selective ratiometric response to In(III) among 18 tested metal including Al(III) and Ga(III). Job's plot analysis indicated that 1 preferred to form a 2:1 complex with In(III) and the binding affinity for In(III) was measured to be 2.3 × 1012 M-2 ( R2 = 0.989). 1 showed linear ratiometric responses to nanomolar concentrations (0-750 nM) of In(III) and the detection limit was calculated to be 64 nM ( R2 = 0.992) in aqueous solution. The binding mode study using NMR, IR, and CD spectroscopies revealed that the phosphate and the amide groups of the receptor of 1 played an important role for the binding with In(III). Moreover, 1 was suitable for the ratiometric detection of In(III) in tap water and groundwater. 1 showed much better detection properties than those of the colorimetric methods using EDTA with Eriochrome black T (EBT) and 4-(2-pyridylazo) resorcinol (PAR) for the detection of In(III) in tap water and groundwater.

Highly sensitive and selective detection of Al(III) ions in aqueous buffered solution with fluorescent peptide-based sensor.

A fluorescent sensor based on a tripeptide (SerGluGlu) with a dansyl fluorophore detected selectively Al(III) among 16 metal ions in aqueous buffered solutions without any organic cosolvent. The peptide-based sensor showed a highly sensitive turn on response to aluminium ion with high binding affinity (1.84×10(4)M(-1)) in aqueous buffered solutions. The detection limit (230nM, 5.98ppb) of the peptide-based sensor was much lower than the maximum allowable level (7.41µM) of aluminium ions in drinking water demanded by EPA. The binding mode of the peptide sensor with aluminium ions was characterized using ESI mass spectrometry, NMR titration, and pH titration experiments.

The arachidonic acid metabolite 11,12-epoxyeicosatrienoic acid alleviates pulmonary fibrosis.

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid that are rapidly metabolized into diols by soluble epoxide hydrolase (sEH). sEH inhibition has been shown to increase the biological activity of EETs, which are known to have anti-inflammatory properties. However, the role of EETs in pulmonary fibrosis remains unexplored. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to analyze EETs in the lung tissues of patients with idiopathic pulmonary fibrosis (IPF, n = 29) and controls (n = 15), and the function of 11,12-EET was evaluated in in vitro and in vivo in pulmonary fibrosis models. EET levels in IPF lung tissues, including those of 8,9-EET, 11,12-EET, and 14,15-EET, were significantly lower than those in control tissues. The 11,12-EET/11,12-DHET ratio in human lung tissues also differentiated IPF from control tissues. 11,12-EET significantly decreased transforming growth factor (TGF)-ß1-induced expression of α-smooth muscle actin (SMA) and collagen type-I in MRC-5 cells and primary fibroblasts from IPF patients. sEH-specific siRNA and 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU; sEH inhibitor) also decreased TGF-ß1-induced expression of α-SMA and collagen type-I in fibroblasts. Moreover, 11,12-EET and TPPU decreased TGF-ß1-induced p-Smad2/3 and extracellular-signal-regulated kinase (ERK) expression in primary fibroblasts from patients with IPF and fibronectin expression in Beas-2B cells. TPPU decreased the levels of hydroxyproline in the lungs of bleomycin-induced mice. 11,12-EET or sEH inhibitors could inhibit pulmonary fibrosis by regulating TGF-ß1-induced profibrotic signaling, suggesting that 11,12-EET and the regulation of EETs could serve as potential therapeutic targets for IPF treatment.

Tuning of the selectivity of fluorescent peptidyl bioprobe using aggregation induced emission for heavy metal ions by buffering agents in 100% aqueous solutions.

Smart fluorescent probes of which the detection of specific target molecules can be controlled are attracting remarkable interest. A fluorescent peptidyl bioprobe (1) was rationally synthesized by conjugating tetraphenylethylene, an aggregation-induced emission (AIE) fluorophore with a peptide receptor (AspHis) that acted as hard and intermediate bases. The selective detection of 1 for specific metal ion in 100% aqueous solutions was controlled by the buffering agents with the chelate effect without the change of pH. In distilled water and phosphate buffered aqueous solution at neutral pH, 1 exhibited a selective Off-On response to a soft metal ion, Hg2+ among test metal ions by 100-fold enhancement of the emission at 470nm. 1 showed a selective Off-On response (180-fold enhancement) to a hard metal ion, Al3+ ions among test metal ions in Tris buffered aqueous solution at neutral pH and Hexamine (hexamethylenetetramine) buffered aqueous solution at acidic pH. The detection limit of 0.46 ppb for Hg2+ and 2.26 ppb for Al3+ in each condition was lower than the maximum allowable level of the metal ions in drinking water by EPA. This research helps to understand how buffering agents participate in the complex formation and aggregation of fluorescent probes using an AIE process for the selective detection of specific metal ions in aqueous solutions.