Access to cyclohexadiene and benzofuran derivatives via catalytic arene cyclopropanation of α-cyanodiazocarbonyl compounds.

The arene cyclopropanation between diazo compounds and benzene is well known to produce a tautomeric mixture of norcaradiene and cycloheptatriene in favour of the latter species. Nevertheless, previous studies have suggested that the initially formed norcaradiene can be stabilized by a C-7 cyano group with prevention of its 6π-electrocyclic ring opening. According to this feature, a synthetic route to functionalized cyclohexadienes has been designed using α-cyanodiazoacetates and α-diazo-ß-ketonitriles as the starting materials, respectively. The Rh2(esp)2-catalyzed arene cyclopropanation of α-cyanodiazoacetates in benzene afforded the expected 7-alkoxycarbonyl-7-cyanonorcaradienes as isolable compounds, which then served as templates for the second cyclopropanation with ethyl diazoacetate or α-cyanodiazocarbonyls to enable the formation of bis(cyclopropanated) adducts. Their subsequent treatment with SmI2 triggered a double ring-opening process, allowing for the generation of 1,4- and/or 1,3-cyclohexadienes as either regio- or diastereomeric mixtures. On the other hand, the norcaradienes generated from phenyl- or methyl-substituted α-diazo-ß-ketonitriles were found to undergo an in situ rearrangement to yield dihydrobenzofurans that could be converted to benzofuran derivatives by DDQ oxidation.

Angiopoietin-2 inhibition attenuates kidney fibrosis by hindering chemokine C-C motif ligand 2 expression and apoptosis of endothelial cells.

Plasma levels of angiopoietin-2 are increased in patients with chronic kidney disease (CKD). Moreover, mouse models of progressive kidney disease also demonstrate increased angiopoietin-2 in both plasmas and kidneys. The role of dysregulated angiopoietins in the progression of kidney disease has not been thoroughly investigated. Here, we found in a cohort of 319 patients with CKD that plasma angiopoietin-2 and angiopoietin-2/angiopoietin-1 ratios were positively associated with the development of kidney failure. In mice with progressive kidney disease induced by either ureteral obstruction or ischemia-reperfusion injury, overexpression of human angiopoietin-1 in the kidney tubules not only reduced macrophage infiltration in the initial stage post-injury but also attenuated endothelial cell apoptosis, microvascular rarefaction, and fibrosis in the advanced disease stage. Notably, angiopoietin-1 attenuated chemokine C-C motif ligand 2 (CCL2) expression in the endothelial cells of the fibrosing kidneys, and these protective effects led to attenuation of functional impairment. Mechanistically, angiopoietin-1 reduced CCL2-activated macrophage migration and protected endothelial cells against cell apoptosis induced by angiopoietin-2 and Wnt ligands. Based on this, we applied L1-10, an angiopoietin-2 inhibitor, to the mouse models of progressive kidney disease and found inhibitory effects on macrophage infiltration, microvascular rarefaction, and fibrosis. Thus, we defined the detrimental impact of increased angiopoietin-2 on kidney survival of patients with CKD which appears highlighted by angiopoietin-2 induced endothelial CCL2-activated macrophage infiltration and endothelial cell apoptosis in their kidneys undergoing fibrosis.

Effective disinfection of airborne microbial contamination in hospital wards using a zero-valent nano-silver/TiO2 -chitosan composite.

A novel antimicrobial composite of zero-valent silver nanoparticles (AgNPs), titania (TiO2 ), and chitosan (CS) was prepared via photochemical deposition of AgNPs on a CS-TiO2 matrix (AgNPs@CS-TiO2 ). Electron microscopy showed that the AgNPs were well dispersed on the CS-TiO2 , with diameters of 6.69-8.84 nm. X-ray photoelectron spectra indicated that most of the AgNPs were reduced to metallic Ag. Fourier-transform infrared spectroscopy indicated that some AgNPs formed a chelate with CS through coordination of Ag+ with the CS amide II groups. The zones of inhibition of AgNPs@CS-TiO2 for bacteria (Escherichia coli and Staphylococcus epidermidis) and fungi (Aspergillus niger and Penicillium spinulosum) were 6.72-11.08 and 5.45-5.77 mm, respectively, and the minimum (critical) concentrations of AgNPs required to inhibit the growth of bacteria and fungi were 7.57 and 16.51 µg-Ag/mm2 , respectively. The removal efficiency of a AgNPs@TiO2 -CS bed filter for bioaerosols (η) increased with the packing depth, and the optimal filter quality (qF) occurred for packing depths of 2-4 cm (qF = 0.0285-0.103 Pa-1 ; η = 57.6%-98.2%). When AgNPs@TiO2 -CS bed filters were installed in the ventilation systems of hospital wards, up to 88% of bacteria and 97% of fungi were removed within 30 minutes. Consequently, AgNPs@TiO2 -CS has promising potentials in bioaerosol purification.

Evaluation of PM1, PM2.5, and PM10 exposure and the resultant health risk of preschool children and their caregivers.

Preschool children have a higher respiratory rate per unit body weight than adults, and their respiratory systems are not mature. Hence, children may have more health risks associated with particulate matter (PM) exposure. In this study, we assessed the exposure of preschool children and their caregivers to PM and the resulting health risks. The PM concentrations at heights of 60-80 cm (preschool children) and 150 cm (adults) were measured at ten indoor and eight outdoor sites in the Taipei metropolitan area from March 2015 to February 2017. Four PM2.5 and seven PM10 indoor measurements exceeded the indoor air quality standard of Taiwan, whereas only two PM2.5 outdoor measurements exceeded the ambient air quality standard. The outdoor PM concentrations were related to traffic emissions, whereas the indoor PM concentrations were associated with ventilation rate and occupant density. The chronic daily PM1, PM2.5, and PM10 intakes of preschool children were notably higher than those of adults. In addition, the hazard quotient resulting from PM2.5 exposure indicated a significant health risk for preschool children (93.74% greater than 1). Consequently, reducing the exposure of preschool children to PM2.5 is an emerging issue in the Taipei metropolitan area.

IP3 and calcium signaling involved in the reorganization of the actin cytoskeleton and cell rounding induced by cigarette smoke extract in human endothelial cells.

Smoking increases the risk of cardiovascular disorders and leads to damage caused by inflammation and oxidative stress. The actin cytoskeleton is a key player in the response to inflammatory stimuli and is an early target of cellular oxidative stress. The purpose of this study was to investigate the changes in actin cytoskeleton dynamics in human endothelial EA.hy926 cells exposed to cigarette smoke extract (CSE). Immunostaining revealed that CSE exposure resulted in modification of the actin cytoskeleton and led to cell rounding in a dose- and time-dependent manner. In addition, the intracellular calcium concentration was increased by treatment with CSE. Pretreatment with antioxidants (lipoic acid, glutathione, N-acetyl cysteine, aminoguanidine, α-tocopherol, and vitamin C) significantly attenuated the CSE-induced actin cytoskeleton reorganization and cell rounding. Calcium ion chelators (EGTA, BAPTA-AM AM) and a potent store-operated calcium channel inhibitor (MRS 1845) also reduced CSE-induced intracellular calcium changes and attenuated actin cytoskeleton reorganization and cell morphology change. Moreover, the CSE-induced intracellular calcium increase was suppressed by pretreatment with the inositol trisphosphate receptor (IP3R) inhibitor xestospongin C, the phospholipase C (PLC) inhibitor U-73122, and the protein kinase C (PKC) inhibitor GF109203X. These results suggest that reactive oxygen species production and intracellular calcium increase play an essential role in CSE-induced actin disorganization and cell rounding through a PLC-IP3-PKC signaling pathway. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1293-1306, 2016.

Angiopoietin-2-induced arterial stiffness in CKD.

The mechanism of vascular calcification in CKD is not understood fully, but may involve collagen deposition in the arterial wall upon osteo/chondrocytic transformation of vascular smooth muscle cells (VSMCs). Increased levels of circulating angiopoietin-2 correlate with markers of CKD progression and angiopoietin-2 regulate inflammatory responses, including intercellular and vascular adhesion and recruitment of VSMCs. Here, we investigate the potential role of angiopoietin-2 in the pathogenesis of arterial stiffness associated with CKD. In a cohort of 416 patients with CKD, the plasma level of angiopoietin-2 correlated independently with the severity of arterial stiffness assessed by pulse wave velocity. In mice subjected to 5/6 subtotal nephrectomy or unilateral ureteral obstruction, plasma levels of angiopoietin-2 also increased. Angiopoietin-2 expression markedly increased in tubular epithelial cells of fibrotic kidneys but decreased in other tissues, including aorta and lung, after 5/6 subtotal nephrectomy. Expression of collagen and profibrotic genes in aortic VSMCs increased in mice after 5/6 subtotal nephrectomy and in mice producing human angiopoietin-2. Angiopoietin-2 stimulated endothelial expression of chemokines and adhesion molecules for monocytes, increased Ly6C(low) macrophages in aorta, and increased the expression of the profibrotic cytokine TGF-ß1 in aortic endothelial cells and Ly6C(low) macrophages. Angiopoietin-2 blockade attenuated expression of monocyte chemokines, profibrotic cytokines, and collagen in aorta of mice after 5/6 subtotal nephrectomy. This study identifies angiopoietin-2 as a link between kidney fibrosis and arterial stiffness. Targeting angiopoietin-2 to attenuate inflammation and collagen expression may provide a novel therapy for cardiovascular disease in CKD.

Role of pirenoxine in the effects of catalin on in vitro ultraviolet-induced lens protein turbidity and selenite-induced cataractogenesis in vivo.

PURPOSE: In this study, we investigated the biochemical pharmacology of pirenoxine (PRX) and catalin under in vitro selenite/calcium- and ultraviolet (UV)-induced lens protein turbidity challenges. The systemic effects of catalin were determined using a selenite-induced cataractogenesis rat model. METHODS: In vitro cataractogenesis assay systems (including UVB/C photo-oxidation of lens crystallins, calpain-induced proteolysis, and selenite/calcium-induced turbidity of lens crystallin solutions) were used to screen the activity of PRX and catalin eye drop solutions. Turbidity was identified as the optical density measured using spectroscopy at 405 nm. We also determined the in vivo effects of catalin on cataract severity in a selenite-induced cataract rat model. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied to analyze the integrity of crystallin samples. RESULTS: PRX at 1,000 µM significantly delayed UVC-induced turbidity formation compared to controls after 4 h of UVC exposure (p<0.05), but not in groups incubated with PRX concentrations of <1,000 µM. Results were further confirmed by SDS-PAGE. The absolute γ-crystallin turbidity induced by 4 h of UVC exposure was ameliorated in the presence of catalin equivalent to 1~100 µM PRX in a concentration-dependent manner. Samples with catalin-formulated vehicle only (CataV) and those containing PRX equivalent to 100 µM had a similar protective effect after 4 h of UVC exposure compared to the controls (p<0.05). PRX at 0.03, 0.1, and 0.3 µM significantly delayed 10 mM selenite- and calcium-induced turbidity formation compared to controls on days 0~4 (p<0.05). Catalin (equivalent to 32, 80, and 100 µM PRX) had an initial protective effect against selenite-induced lens protein turbidity on day 1 (p<0.05). Subcutaneous pretreatment with catalin (5 mg/kg) also statistically decreased the mean cataract scores in selenite-induced cataract rats on post-induction day 3 compared to the controls (1.3±0.2 versus 2.4±0.4; p<0.05). However, catalin (equivalent to up to 100 µM PRX) did not inhibit calpain-induced proteolysis activated by calcium, and neither did 100 µM PRX. CONCLUSIONS: PRX at micromolar levels ameliorated selenite- and calcium-induced lens protein turbidity but required millimolar levels to protect against UVC irradiation. The observed inhibition of UVC-induced turbidity of lens crystallins by catalin at micromolar concentrations may have been a result of the catalin-formulated vehicle. Transient protection by catalin against selenite-induced turbidity of crystallin solutions in vitro was supported by the ameliorated cataract scores in the early stage of cataractogenesis in vivo by subcutaneously administered catalin. PRX could not inhibit calpain-induced proteolysis activated by calcium or catalin itself, and may be detrimental to crystallins under UVB exposure. Further studies on formulation modifications of catalin and recommended doses of PRX to optimize clinical efficacy by cataract type are warranted.

Ditopic complexation of selenite anions or calcium cations by pirenoxine: an implication for anti-cataractogenesis.

This study investigated whether and how pirenoxine (PRX) interacts with selenite or calcium ions, as these two ions have been proven respectively a factor leading to the formation of lens cataract. UV, NMR, and isothermal titration calorimetry (ITC) analysis indicated that PRX could bind maximum up to six selenite anions and the binding site preference was concentration dependent with the peripheral binding first followed by the π-π interactions with the aromatic moiety; while for calcium cation interaction the 3-carboxylate and ß-ketoimine functional groups were responsible for chelating calcium ions. The results obtained by MP2/6-31+G(d) molecular orbital calculations provided theoretical evidence in support of the π-π interactions between selenite and the PRX aromatic framework, and further analysis of the binding energies with the aromatic moiety indicates that these interactions take place most likely at the benzoquinone (ring I) π-system. The calcium binding preferences with PRX were also determined based on the stabilization energy obtained by B3LYP/6-31+G(d) calculations, showing the binding preferences were site 2 > site 1 > site 3 > ring II, consistent with the experimental data. The in vitro study of the reduction of selenite or calcium ions-induced lens turbidity by PRX with ditopic recognition properties was thus demonstrated. These results may provide a rationale for using PRX as an anti-cataract agent and warrant further biological studies.

Effectiveness of the Nanosilver/TiO2-Chitosan Antiviral Filter on the Removal of Viral Aerosols.

Background: The precaution of airborne transmission of viruses, such as influenza, SARS, MERS, and COVID-19, is essential for reducing infection. In this study, we applied a zero-valent nanosilver/titania-chitosan (nano-Ag0/TiO2-CS) filter bed, whose broad-spectrum antimicrobial efficacy has been proven previously, for the removal of viral aerosols to minimize the risk of airborne transmission. Methods: The photochemical deposition method was used to synthesize the nano-Ag0/TiO2-CS antiviral material. The surface morphology, elemental composition, and microstructure of the nano-Ag0/TiO2-CS were analyzed by a scanning electron microscopy/energy dispersive X-ray spectroscopy and a transmission electron microscopy, respectively. The MS2 bacteriophages were used as surrogate viral aerosols. The antiviral efficacy of nano-Ag0/TiO2-CS was evaluated by the MS2 plaque reduction assay (PRA) and filtration experiments. In the filtration experiments, the MS2 aerosols passed through the nano-Ag0/TiO2-CS filter, and the MS2 aerosol removal efficiency was evaluated by an optical particle counter and culture method. Results and Conclusions: In the MS2 PRA, 3 g of nano-Ag0/TiO2-CS inactivated 97% of MS2 bacteriophages in 20 mL liquid culture (2 ± 0.5 × 1016 PFU/mL) within 2 hours. The removal efficiency of nano-Ag0/TiO2-CS filter (thickness: 6 cm) for MS2 aerosols reached up to 93%. Over 95% of MS2 bacteriophages on the surface of the nano-Ag0/TiO2-CS filter were inactivated within 20 minutes. The Wells-Riley model predicted that when the nano-Ag0/TiO2-CS filter was used in the ventilation system, airborne infection probability would reduce from 99% to 34.6%. The nano-Ag0/TiO2-CS filter could remain at 50% of its original antiviral efficiency after continuous operation for 1 week, indicating its feasibility for the control of the airborne transmission.

DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys.

Renal erythropoietin-producing cells (REPCs) remain in the kidneys of patients with chronic kidney disease, but these cells do not produce sufficient erythropoietin in response to hypoxic stimuli. Treatment with HIF stabilizers rescues erythropoietin production in these cells, but the mechanisms underlying the decreased response of REPCs in fibrotic kidneys to anemic stimulation remain elusive. Here, we show that fibroblast-like FOXD1+ progenitor-derived kidney pericytes, which are characterized by the expression of α1 type I collagen and PDGFRß, produce erythropoietin through HIF2α regulation but that production is repressed when these cells differentiate into myofibroblasts. DNA methyltransferases and erythropoietin hypermethylation are upregulated in myofibroblasts. Exposure of myofibroblasts to nanomolar concentrations of the demethylating agent 5-azacytidine increased basal expression and hypoxic induction of erythropoietin. Mechanistically, the profibrotic factor TGF-ß1 induced hypermethylation and repression of erythropoietin in pericytes; these effects were prevented by 5-azacytidine treatment. These findings shed light on the molecular mechanisms underlying erythropoietin repression in kidney myofibroblasts and demonstrate that clinically relevant, nontoxic doses of 5-azacytidine can restore erythropoietin production and ameliorate anemia in the setting of kidney fibrosis in mice.

Methylomics analysis identifies ZNF671 as an epigenetically repressed novel tumor suppressor and a potential non-invasive biomarker for the detection of urothelial carcinoma.

The molecular mechanism underlying the lethal phenomenon of urothelial carcinoma (UC) tumor recurrence remains unresolved. Here, by methylation microarray, we identified promoter methylation of the zinc-finger protein gene, ZNF671 in bladder UC tumor tissue samples, a finding that was independently validated by bisulphite pyrosequencing in cell lines and tissue samples. Subsequent assays including treatment with epigenetic depressive agents and in vitro methylation showed ZNF671 methylation to result in its transcriptional repression. ZNF671 re-expression in UC cell lines, via ectopic expression, inhibited tumor growth and invasion, in possible conjunction with downregulation of cancer stem cell markers (c-KIT, NANOG, OCT4). Clinically, high ZNF671 methylation in UC tumor tissues (n=96; 63 bladder, 33 upper urinary tract) associated with tumor grade and poor locoregional disease-free survival. Quantitative MSP analysis in a training (n=97) and test (n=61) sets of voided urine samples from bladder UC patients revealed a sensitivity and specificity of 42%-48% and 89%-92.8%, respectively, for UC cancer detection. Moreover, combining DNA methylation of ZNF671 and 2 other genes (IRF8 and sFRP1) further increased the sensitivity to 96.2%, suggesting a possible three-gene UC biomarker. In summary, ZNF671, an epigenetically silenced novel tumor suppressor, represents a potential predictor for UC relapse and non-invasive biomarker that could assist in UC clinical decision-making.

Distinct DNA methylation epigenotypes in bladder cancer from different Chinese sub-populations and its implication in cancer detection using voided urine.

BACKGROUND: Bladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. Previous studies have identified several tumor-related genes that are hypermethylated in bladder cancer; however the DNA methylation profile of bladder cancer in Taiwan is not fully understood. METHODS: In this study, we compared the DNA methylation profile of multiple tumor suppressor genes (APC, DAPK, E-cadherin, hMLH1, IRF8, p14, p15, RASSF1A, SFRP1 and SOCS-1) in bladder cancer patients from different Chinese sub-populations including Taiwan (104 cases), Hong Kong (82 cases) and China (24 cases) by MSP. Two normal human urothelium were also included as control. To investigate the diagnostic potential of using DNA methylation in non-invasive detection of bladder cancer, degree of methylation of DAPK, IRF8, p14, RASSF1A and SFRP1 was also accessed by quantitative MSP in urine samples from thirty bladder cancer patients and nineteen non-cancer controls. RESULTS: There were distinct DNA methylation epigenotypes among the different sub-populations. Further, samples from Taiwan and China demonstrated a bimodal distribution suggesting that CpG island methylator phentotype (CIMP) is presented in bladder cancer. Moreover, the number of methylated genes in samples from Taiwan and Hong Kong were significantly correlated with histological grade (P < 0.01) and pathological stage (P < 0.01). Regarding the samples from Taiwan, methylation of SFRP1, IRF8, APC and RASSF1A were significantly associated with increased tumor grade, stage. Methylation of RASSF1A was associated with tumor recurrence. Patients with methylation of APC or RASSF1A were also significantly associated with shorter recurrence-free survival. For methylation detection in voided urine samples of cancer patients, the sensitivity and specificity of using any of the methylated genes (IRF8, p14 or sFRP1) by qMSP was 86.7% and 94.7%. CONCLUSIONS: Our results indicate that there are distinct methylation epigenotypes among different Chinese sub-populations. These profiles demonstrate gradual increases with cancer progression. Finally, detection of gene methylation in voided urine with these distinct DNA methylation markers is more sensitive than urine cytology.