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Cellular Physiology of Cholesterol Efflux in Vascular Endothelial Cells
Background— Of the cells that compose the atherosclerotic plaque, vascular endothelial cells are the most resistant to cholesterol accumulation. Cholesterol efflux pathways may play an important role in endothelial cholesterol homeostasis.
Methods and Results— We examined the global genetic response of endothelial cells to cholesterol and in particular the contribution of the cholesterol efflux proteins ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor B-I (SR-BI) to endothelial cell cholesterol efflux. The ABCG1 gene is induced in endothelial cells by cholesterol, whereas ABCA1 is not. Using specific chemical inhibitors of ABC transporters and SR-BI, we have shown that neither ABC transporters nor SR-BI is required for apolipoprotein A-1–mediated endothelial cholesterol efflux.
Conclusions— Endothelial cells may use nontraditional pathways for cholesterol efflux.
Received August 19, 2002; de novo received June 3, 2004; accepted July 1, 2004.
Endothelial dysfunction is a key factor in the pathogenesis of atherosclerosis and is characterized by an increased expression of leukocyte adhesion molecules, increased permeability of the endothelium to LDLs, and decreased bioavailability of nitric oxide. Endothelial cells do not, however, undergo the foam cell phenotype change seen in macrophages and smooth muscle cells in the atherosclerotic plaque, a change embodied by the unrestricted accumulation of cellular cholesterol. This is surprising because endothelial cells express receptors for oxidized lipoproteins (eg, CD36, LOX-1), receptors that are not subject to negative feedback regulation by cholesterol and are thought to underlie foam cell formation.
Numerous processes, including de novo synthesis of cholesterol, lipoprotein uptake, cholesterol esterification, and reverse cholesterol transport, control cellular cholesterol metabolism. Genes that govern reverse cholesterol transport may be particularly important in determining the fate of cells within the atherosclerotic plaque. Mutations in the gene encoding the ATP-binding cassette transporter A1 (ABCA1) cause cellular phospholipid and cholesterol efflux defects that result in a severe loss of plasma HDL.1 Low plasma HDL cholesterol is a risk factor for the development of atherosclerosis,2 but loss of ABCA1 activity specifically in macrophages may predispose them to foam cell formation and contribute directly to the development of atherosclerotic lesions.3 Because HDL is thought to have important effects on endothelial health,4 it is appealing to hypothesize that ABCA1 is active in endothelium. However, the evidence supporting a role for ABCA1 in endothelial cells is equivocal.5,6
The ATP-binding cassette transporter G1 (ABCG1) is a poorly characterized half-transporter relative of ABCA1, originally described in macrophages.7,8 It is thought to participate in reverse cholesterol transport by promoting HDL subfraction 3 (HDL3)–mediated cellular cholesterol efflux.7 Like the full transporter ABCA1, transcriptional regulation of ABCG1 is controlled by the liver X receptor/retinoid X receptor (LXR/RXR) pathway of nuclear hormone receptors.9 This pathway is induced by specific hydroxylated derivatives of cholesterol, eg, 27-hydroxycholesterol, which can serve as a proxy indicator for the presence of cholesterol in cells. Overexpression of ABCG1 in mouse liver results in decreased plasma levels of cholesteryl esters and HDL cholesterol, although the mechanism for this effect is not known.10 A recent report shows that ABCG1 expression in the liver is highest in Kupffer cells (tissue macrophages) and endothelial cells and lowest in parenchymal cells.11
The scavenger receptor B-I (SR-BI, also known as CLA-1 in humans) is an HDL receptor that can mediate selective uptake of HDL cholesteryl esters by cells but can also promote cellular free cholesterol efflux to HDL and the reorganization of a cholesterol oxidase–sensitive pool of cellular cholesterol.12 In endothelial cells, SR-BI is required for endothelial nitric oxide synthase (eNOS) activation by HDL.13 SR-BI has been localized within caveolae, cholesterol-rich plasma membrane microdomains that are abundant in endothelial cells.14
In the present study we examined the expression of cholesterol-regulated genes in endothelial cells in general using functional genomics and the genes ABCA1, ABCG1, and SR-BI in particular. We also investigated the contribution of ABCA1, ABCG1, and SR-BI to endothelial cholesterol efflux.
Pooled human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were from Cambrex and were used before the fourth passage. Normal human skin fibroblasts were obtained by biopsy from a healthy human volunteer, and Tangier fibroblasts were from a patient with compound ABCA1 mutations (ABCA1 C1477R amino acid substitution and an ABCA1 IVS25+1 [G→C] mRNA splice site mutation). Fibroblasts were used before the 13th passage. COS cells were from Invitrogen. Cell medium for HUVECs and HAECs was EGM2 (Cambrex), and cell medium for human skin fibroblasts and COS cells was Dulbecco’s modified Eagle’s medium (Invitrogen), supplemented with 100 U/mL penicillin and 100 U/mL streptomycin (Invitrogen), nonessential amino acids (Invitrogen), and 10% FBS (Biomedia). Cell medium for HepG2 cells was the same as for fibroblasts, except that Dulbecco’s modified Eagle’s medium-F12 was used (Invitrogen). Bovine serum albumin (BSA), cholesterol, 22(R)-hydroxycholesterol, and 8-bromo-cAMP were from Sigma. For the SR-BI dose-response experiment, a variety of concentrations of cholesterol (0 to 50 μg/mL) and 22(R)-hydroxycholesterol (0 to 5 μg/mL) were used. No significant cell death was observed through a light microscope at the reported doses of reagents.
We seeded 250 000 HUVECs in 100-mm cell culture dishes. At confluence, the medium was changed to serum-free medium containing 2 mg/mL BSA and either 20 μg/mL cholesterol in an ethanol carrier or the ethanol carrier alone and incubated for 24 hours. Total RNA was isolated with the use of the RNeasy Mini Kit (Qiagen). RNA was tested for quality on an agarose gel. We used 1 μg of total RNA on a HU95A DNA microarray (Affymetrix) containing probe sets for >12 000 gene sequences; results were analyzed with GeneSpring software (Silicon Genetics). Criteria for differential regulation by cholesterol treatment were set as ≥2-fold upregulation or downregulation at a probability value of <0.05 with n=3.
We seeded 250 000 HUVECs, HAECs, and human skin fibroblasts on 100-mm cell culture plates. On reaching confluence, growth medium was changed to serum-free medium with 2 mg/mL BSA and either 20 μg/mL cholesterol in an ethanol carrier, 0.5 mmol/L 8-bromo-cAMP, or ethanol carrier alone and incubated for 24 hours. Total RNA was isolated with the RNeasy Mini Kit, and 15 μg of RNA was loaded on a 1% formaldehyde agarose gel. RNA was then transferred to a Hybond N+ membrane (Amersham) and hybridized with a 460-bp ABCG1 probe prepared by polymerase chain reaction amplification (forward=5′-GTA TGG GTT CGA AGG GGT CAT-3′, reverse=5′-CAC CAA TCT GCC TAC ATC TTC-3′). A Phosphorimager (Molecular Dynamics) was used to capture the 32P image. The membrane was stripped and rehybridized with a ABCA1 probe prepared as described previously.15 Signal from a glyceraldehyde-3-phosphate dehydrogenase DNA probe was used to control for consistent loading. Radiolabeled probes were prepared with the use of a nick translation kit and Redivue 32P-dCTP supplied by Amersham. ImageQuant 5.1 software (Molecular Dynamics) was used to quantify RNA expression.
We seeded 250 000 HepG2 cells and 50 000 each of HUVECs/HAECs/fibroblasts on either 12-well or 24-well plates. One day after seeding, cellular cholesterol pools were radiolabeled for 24 hours with medium containing 0.2 μCi/mL [3H]cholesterol (Amersham) for 12-well plates or 1 μCi/mL [3H]cholesterol for 24-well plates. Except where indicated, the cells were washed and incubated for 24 hours in serum-free medium containing 2 mg/mL BSA and 20 μg/mL cholesterol. The kinetic study used 2 mg/mL BSA alone, BSA with 20 μg/mL cholesterol, BSA with 2.5 μg/mL 22(R)-hydroxycholesterol, or BSA with 20 μg/mL cholesterol plus 2.5 μg/mL 22(R)-hydroxycholesterol. The cells were equilibrated in serum-free medium containing 1 mg/mL BSA for 6 hours. Cholesterol efflux was performed over 24 hours except in the case of the kinetic study. Efflux substrate was serum-free medium containing 1 mg/mL BSA, serum-free medium containing 1 mg/mL BSA and apolipoprotein A-1 (apoA-I) at 5 μg/mL (Biodesign), or serum-free medium containing 1 mg/mL BSA and 50 μg/mL HDL3 (Biodesign). The ABC inhibitor glyburide was purchased from Calbiochem, and a stock solution of 25 mg/mL was prepared in dimethyl sulfoxide (DMSO). A novel inhibitor of SR-BI function, BLT-1, was purchased as compound 5234221 from Chembridge, and a 100-mmol/L stock solution was prepared in DMSO. Glyburide was added to the efflux medium for the final 24 hours of the experiment. BLT-1 was added to the efflux medium, and efflux was for 6 hours to limit cell toxicity. BLT-1 medium contained 5 mg/mL BSA and 0.5% DMSO to ensure solubility of the compound. Radioactivity was counted in both the efflux medium and the cells. Efflux was expressed as the radioactivity counted in the medium divided by the sum total of the radioactivity in the medium and the cells (n=3). In some cases, HUVEC cellular lipids from 12-well plates were isolated with 2:3 hexane:isopropanol and dried, and total cholesterol was measured with the Infinity total cholesterol kit (ThermoDMA) according to the manufacturer’s instructions.
The ABCG1 expression vector was from the Invitrogen Genestorm library and contained a full-length ABCG1 cDNA tagged with a V5 epitope and a 6Xhis in a pcDNA 3.1 plasmid. The ABCA1 expression vector contained a full-length ABCA1 cDNA cloned in a pcDNA 3.0 plasmid. Empty pcDNA 3.0 plasmid containing no cDNA was used as a control vector. COS cells were seeded at 100 000 cells per well in 12-well plates. At half-confluence, cellular cholesterol pools were radiolabeled with 1 μCi/mL [3H]cholesterol. At confluence, medium was changed to OptiMEM (Invitrogen), and cells were transfected overnight with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. At this point, cells were either incubated for 24 hours with 50 μg/mL HDL3 to measure efflux or their cell lysate was isolated for use in an immunoblot.
Cell lysates were harvested in lysis buffer (20 mmol/L Tris, 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, 0.5% Triton X-100, Complete protease inhibitors [Roche]), homogenized, and centrifuged for 10 minutes at 3500 rpm in a tabletop centrifuge at 4°C to pellet nuclei. The protein concentration of postnuclear supernatants was measured with the BCA protein assay (Pierce). We loaded 45 μg of cell lysate onto an 8% polyacrylamide gel. After electrophoresis, proteins were transferred to an Immobilon P membrane (Millipore). Primary anti–SR-BI antibody (Novus Biologicals catalog No. NB400-104B1) was used at a concentration of 1:750, anti-V5 antibody (Invitrogen) was used at 1:2500, anti-ABCA1 antibody was used at 1:500 (Novus Biologicals), anti-ABCG1 antibody was used at 1:100 (Santa Cruz Biotechnologies), and anti-HSP70 antibody was used at 1:1000 (Transduction Laboratories). Anti-mouse and anti-rabbit secondary antibodies coupled to horseradish peroxidase (Amersham) were used at a concentration of 1:5000, and the donkey anti-goat secondary antibody (Santa Cruz Biotechnologies) was used at a concentration of 1:10 000. Signal was revealed with the use of an enhanced chemiluminescence kit (Pierce) and captured with X-Omat Blue XB-1 film (Kodak). ImageQuant software was used to quantify protein signals. For the dose-response experiments, semiquantitative exposures were selected.
We used a DNA microarray to search for changes in the expression of large numbers of HUVEC genes induced by cholesterol. Gene expression in HUVECs treated with 20 μg/mL cholesterol for 24 hours was compared with basal gene expression with the use of Affymetrix HU95A DNA microarrays. The Table shows those HUVEC mRNA transcripts found to be significantly upregulated or downregulated by cholesterol treatment. A large group of downregulated transcripts is required for de novo cholesterol biosynthesis. ABCG1, which has been proposed to enhance HDL3-mediated cellular cholesterol and phospholipid efflux, was upregulated by cholesterol treatment. Other transcripts affected by cholesterol treatment are implicated in proliferation, inflammation, cytoskeletal function, and protein chaperone function. A number of HUVEC expressed sequence tags or otherwise uncharacterized transcripts were also affected by cholesterol treatment. The increase in cellular cholesterol did not increase the mRNA levels of the cell adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and e-selectin (data not shown).
Expression and Regulation of the ABC Transporters ABCA1 and ABCG1
Because our microarray study showed ABCG1 to be upregulated by cholesterol and because ABCG1 is proposed to enhance cholesterol efflux, we further studied the regulation of this gene and its close relative ABCA1 in HAECs and HUVECs. We treated endothelial cells, normal fibroblasts, and Tangier fibroblasts with cholesterol or the LXR agonist 22(R)-hydroxycholesterol. Total RNA from cells was analyzed by Northern blot with the use of specific radiolabeled ABCA1 and ABCG1 DNA probes (Figure 1A). In HUVECs and HAECs, cholesterol and 22(R)-hydroxycholesterol were potent inducers of ABCG1 expression, whereas ABCA1 expression was low or absent throughout. This pattern was reversed in fibroblasts, in which ABCA1 could be highly induced by cholesterol and 22(R)-hydroxycholesterol but ABCG1 levels remained low. Similar results were obtained at the protein expression level (Figure 1B). 8-Bromo-cAMP, which increases ABCA1 transcription in mouse macrophages,16 had no effect on endothelial or fibroblast ABC transporter expression (data not shown).
ABCA1, ABCG1, and Endothelial Cholesterol Efflux
To examine possible pathways utilized by endothelial cells for reverse cholesterol transport, we measured efflux to a variety of potential substrates. Efflux to BSA alone, apoA-1, and HDL3 was measured in cholesterol-treated HUVECs, normal fibroblasts, and Tangier fibroblasts deficient in ABCA1 (Figure 2). ApoA-I stimulated cholesterol efflux in control fibroblasts but not in HUVECs or Tangier fibroblasts. This is consistent with the low ABCA1 expression seen in HUVECs and the absent ABCA1 activity in Tangier fibroblasts. HDL3 stimulated cholesterol efflux in all cell types but was especially effective in promoting cholesterol efflux in ABCA1-expressing normal fibroblasts. In a dose-response experiment, we compared cholesterol efflux in cholesterol-treated normal fibroblasts with efflux in cholesterol-treated Tangier fibroblasts using 10, 25, 50, and 100 μg/mL HDL3; efflux in normal fibroblasts was 2.45-, 2.69-, 2.15-, and 1.85-fold higher than in Tangier fibroblasts at these concentrations (data not shown). HAEC efflux was also tested and showed a pattern similar to that of HUVECs among efflux substrates (data not shown). Because HDL can mediate a bidirectional cholesterol flux and our standard efflux assay only measures the efflux component of total flux, we measured total cholesterol mass in cholesterol-treated HUVECs incubated with BSA alone or with HDL3. Total cholesterol mass was significantly less in cholesterol-treated HUVECs incubated with HDL3 than in cells incubated with BSA alone, demonstrating that the net cholesterol flux between HUVECs and HDL3 is efflux (data not shown).
We took advantage of the fact that ABCG1 transcription is induced by 22(R)-hydroxycholesterol to determine whether ABCG1 expression could promote cholesterol efflux to HDL3. We treated HUVECs with increasing doses of the LXR agonist 22(R)-hydroxycholesterol, from 0 to 2.5 μg/mL, and measured cholesterol efflux to HDL3. No significant change in efflux was associated with 22(R)-hydroxycholesterol treatment (data not shown). A kinetic study of cholesterol efflux to HDL3 showed that endothelial cells had the same rate of efflux to HDL3 regardless of sterol treatment (Figure 3A). In contrast, normal fibroblasts had increased efflux when treated with various sterols (Figure 3B). We also treated HUVECs, normal fibroblasts, and Tangier fibroblasts with the ABC inhibitor glyburide and measured cholesterol efflux to HDL3. Glyburide can inhibit a broad range of ABC transporters, including ABCA1 and the CFTR protein.17 At high doses, glyburide inhibited cholesterol efflux in normal fibroblasts but not in Tangier fibroblasts, indicating that glyburide was indeed inhibiting ABCA1 activity (Figure 4). HUVEC efflux was not affected by glyburide treatment, suggesting that cholesterol efflux in endothelial cells was not dependent on ABC-type transporters that are sensitive to glyburide.
ABCA1, ABCG1, and COS Cell Cholesterol Efflux
To extend our observation that ABCG1 expression in HUVEC was not associated with cholesterol efflux, we transfected COS cells with ABCG1 and ABCA1 expression vectors and measured their efflux to apoA-I and HDL3 (Figure 5A and 5B). Cells transfected with ABCA1 showed increased expression of ABCA1 in immunoblots with the use of anti-ABCA1 antibody; this increased expression was associated with large increases in cholesterol efflux to apoA-I and HDL3 compared with cells transfected with empty vector. Immunoblots of lysates from cells transfected with tagged ABCG1 with an anti-V5 epitope antibody showed expression of a protein with a molecular weight of ≈65 kDa. This is smaller than the predicted molecular weight of ABCG1 but is consistent with data from another group that used a recombinantly tagged ABCG1 construct.18 ABCG1-transfected COS cells had no increase in cholesterol efflux to HDL3 compared with cells transfected with empty vector.
SR-BI Expression and Activity
We considered the possibility that an alternate efflux protein mediates endothelial cholesterol efflux to HDL3. SR-BI is an HDL receptor that has been described in endothelial cells and is known to promote cellular cholesterol efflux. We compared the expression of SR-BI in HepG2 cells, normal fibroblasts, and HUVECs. HepG2 lysates show very high levels of SR-BI protein expression (Figure 6). SR-BI expression in HUVECs was present, although lower than in HepG2 cells. SR-BI expression in fibroblasts was undetectable. In HUVECs, cholesterol and 22(R)-hydroxycholesterol treatment decreased SR-BI expression. A dose-response experiment observing SR-BI expression in HUVECs treated with increasing concentrations of cholesterol and 22(R)-hydroxycholesterol was performed. In both cholesterol- and 22(R)-hydroxycholesterol–treated HUVECs, increasing sterol doses led to a decrease in SR-BI protein expression, to a maximum of ≈65% of basal expression (Figure 7). To determine whether the SR-BI expressed in HUVECs increases HDL3-mediated cholesterol efflux, we treated cells with the specific SR-BI inhibitor BLT-1 and measured cholesterol efflux to HDL3. Nieland et al19 characterized BLT-1, showing it to be a highly effective blocker of selective cholesteryl ester uptake from HDL by SR-BI, as well as the SR-BI component of HDL3-mediated cellular cholesterol efflux. High doses of BLT-1 significantly attenuated cholesterol efflux to HDL3 in control HepG2 cells but not HUVECs (Figure 8). These data suggest that cholesterol efflux to HDL3 in HUVEC is independent of SR-BI.
Cholesterol homeostasis in endothelial cells remains poorly understood. In contrast to other cells that form the atherosclerotic plaque (smooth muscle cells and macrophages), vascular endothelial cells do not accumulate cholesterol. We addressed this question by investigating the global genetic response of endothelial cells to cholesterol and by examining in particular the role of 3 prominent proteins thought to promote cellular cholesterol efflux: ABCA1, ABCG1, and SR-BI.
The DNA microarray study we performed on HUVEC RNA indicates that the endothelial cholesterol biosynthetic apparatus is profoundly affected by cholesterol treatment. Although many cholesterol biosynthesis genes are known to be regulated by sterol-responsive cis-acting DNA elements, the scale of the effect of cholesterol in HUVECs is notable, going beyond the rate-limiting step catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (Table). The expression of galectin-3, a macrophage recruitment protein, was also induced in HUVECs by cholesterol treatment. Endothelial activation and leukocyte recruitment into the arterial intima is of prime importance in the pathogenesis of atherosclerosis. Hypercholesterolemia is a major risk factor for the development of atherosclerosis, and therefore the induction of HUVEC galectin-3 by cholesterol may have in vivo significance. However, we believe that if cholesterol were truly a strong proinflammatory stimulus for endothelial cells, an increase in the expression of classic markers of endothelial activation, such as vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and e-selectin, would also have been seen. Probe sets for these genes were present on the DNA microarray used in our experiments, but no significant changes in gene expression were detected. Other lipoprotein constituents besides cholesterol, such as lysophosphatidylcholine,20 remain good candidates as mediators of endothelial activation.
Our DNA microarray also showed that cholesterol upregulated the expression of ABCG1 mRNA in HUVECs. ABCG1 has been implicated previously in HDL3-mediated cholesterol and phospholipid efflux in macrophages.7 We used Northern blots to study the expression of ABCG1 and the related transporter ABCA1 in endothelial cells and fibroblasts. ABCG1 mRNA and protein expression in HUVECs and HAECs was inducible by cholesterol and the LXR ligand 22(R)-hydroxycholesterol, but ABCA1 expression was low or absent (Figure 1). In contrast, ABCG1 expression in fibroblasts was low, whereas ABCA1 could be strongly upregulated by cholesterol and 22(R)-hydroxycholesterol. Both the ABCA1 and ABCG1 genes are known to contain LXR/RXR-binding DNA consensus sequences that confer responsiveness to specific oxysterols.9 Because endothelial ABCG1 was inducible by cholesterol and 22(R)-hydroxycholesterol alike, it is likely that endothelial cells possess the hydroxylating enzymes that convert cholesterol to oxysterol LXR ligands. Other studies on the expression of 27-hydroxylase in endothelium support this conclusion as well.21,22 It is unclear why ABCA1 and ABCG1 are differentially regulated in endothelial cells and fibroblasts, but this may be related to tissue-specific expression of transcription factors that regulate these genes.
A functional assay for cholesterol efflux demonstrated no ABCA1 activity in HUVEC, consistent with its low expression, because HUVECs, like Tangier fibroblasts, were unable to efflux cholesterol to apoA-I (Figure 2). HUVECs and Tangier fibroblasts were able to efflux cholesterol to HDL3, which is consistent with the 2-step model of cholesterol efflux proposed by Fielding et al.6 In this model, as lipid-poor apoA-I gains phospholipids from ABCA1, it gains the ability to promote cholesterol efflux through an ABCA1-independent pathway. Fibroblasts expressing normal ABCA1 still had higher efflux to HDL3 than either Tangier fibroblasts or endothelial cells (Figure 2). It has been shown that ABCA1 has a higher affinity for lipid-free apoA-I than HDL.23,24 Our data suggest that ABCA1 is able to enhance HDL3-mediated cholesterol efflux over a range of HDL concentrations. Although this does not show a direct physical interaction between HDL and ABCA1, our data demonstrate that regardless of the specific mechanism, ABCA1 and HDL3 can cooperate at some level to promote cholesterol efflux.
Using sterol induction and a chemical inhibitor of ABC transporters, we have shown that ABCG1 expression is not associated with cholesterol efflux to HDL3 in endothelial cells (Figures 3 and 4⇑). Furthermore, exogenous expression of ABCG1 in COS cells also failed to increase cholesterol efflux to HDL3 (Figure 5). However, it is possible that coexpression of an as yet unknown heterodimeric partner of ABCG1 may be required for full functionality of the protein. This is in striking contrast to the work of Klucken et al,7 which demonstrated an association between ABCG1 expression and cholesterol efflux to HDL3 in macrophages. In vivo the functional interaction between HDL and ABCG1 could be indirect; by promoting cholesterol and phospholipid excretion into the bile, ABCG1 may promote liver uptake of HDL and lowering of plasma HDL-cholesterol.10
We also investigated the role of SR-BI in endothelial cholesterol efflux. SR-BI is being scrutinized increasingly for its antiatherogenic properties, which appear to stem from its key position in the reverse cholesterol pathway and its ability to provoke nitric oxide synthesis in endothelial cells.13 SR-BI is an HDL receptor that mediates selective cholesteryl ester uptake from HDL but can also promote cellular free cholesterol efflux to HDL.12 Expression of SR-BI was high in liver-derived HepG2 cells, lower in HUVECs, and absent altogether in fibroblasts (Figure 6). SR-BI expression was decreased in a dose-dependent manner in HUVECs treated with cholesterol or 22(R)-hydroxycholesterol (Figure 7). SR-BI mediates the selective uptake of cholesteryl esters, and therefore downregulation of the SR-BI gene may protect HUVECs from excessive sterol uptake and accumulation. Keratinocytes behave in a fashion similar to endothelial cells, downregulating SR-BI in response to cholesterol and oxysterols.25 A specific chemical inhibitor of SR-BI, BLT-1, inhibited cholesterol efflux to HDL3 in HepG2 cells but not HUVECs (Figure 8). This is likely due to the higher expression of SR-BI in HepG2 cells, which itself implies that efflux may be more dependent on SR-BI in that cell type (Figure 6). Another study of SR-BI in porcine brain capillary endothelial cells showed a correlation between SR-BI expression and efflux to HDL, but this required high expression of SR-BI using cells transfected with SR-BI–bearing adenoviruses.26 Although SR-BI expression may not be high enough in endothelial cells to make SR-BI a major pathway for cholesterol efflux to HDL, this does not exclude SR-BI from playing a major role in HDL-endothelium eNOS signaling. Our finding that cholesterol and oxysterols can decrease expression of SR-BI may provide an additional mechanistic link between hypercholesterolemia and decreased endothelial function.
Our present study shows that endothelial cells have a strong ability to efflux cellular cholesterol to HDL3 that may be independent of ABCA1, ABCG1, and SR-BI expression or activity. Although some groups have already shown no ABCA1 activity in endothelial cell lines,6 the finding that modifying the expression or activity of ABCG1 and SR-BI has little influence on endothelial efflux is novel. There may be other mechanisms and genes that have been overlooked or have gone undiscovered that mediate endothelial cholesterol efflux. Efflux to HDL may occur simply through passive diffusion or microsolubilization. In endothelial cells, oxysterol formation may itself be a means for removing excess cholesterol independent of HDL. We saw evidence of endothelial conversion of cholesterol to oxysterols. It has been suggested elsewhere that oxysterol formation may be the predominant mechanism for removal of excess brain sterols because the blood-brain barrier prevents normal reverse cholesterol transport from taking place.26 Further investigation into the mechanisms of endothelial-HDL interactions will be required to shed light on HDL3-mediated cholesterol efflux in endothelium.
This study was supported by grants MOP 15042 and MOP 62834 from the Canadian Institute of Health Research.
↵*The first 2 authors contributed equally to this work.
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Venkateswaran A, Repa JJ, Lobaccaro JM, et al. Human white/murine ABC8 mRNA levels are highly induced in lipid-loaded macrophages: a transcriptional role for specific oxysterols. J Biol Chem. 2000; 275: 14700–14707.
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Hoekstra M, Kruijt JK, Van Eck M, et al. Specific gene expression of ATP-binding cassette transporters and nuclear hormone receptors in rat liver parenchymal, endothelial, and Kupffer cells. J Biol Chem. 2003; 278: 25448–25453.
Yancey PG, Bornick AE, Kellner-Weibel G, et al. Importance of different pathways of cellular cholesterol efflux. Arterioscler Thromb Vasc Biol. 2003; 23: 712–719.
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Hasko G, Deitch EA, Nemeth ZH, et al. Inhibitors of ATP-binding cassette transporters suppress interleukin-12 p40 production and major histocompatibility complex II up-regulation in macrophages. J Pharmacol Exp Ther. 2002; 301: 103–110.
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domain: medical


On a Class of Minimum Energy Controls Related to Spline Functions
Netravali, Arun N.; de Figueiredo, Rui J.P.
The problem of determining a minimum energy control for a dynamically interconnected set of p single-input single-output finite-dimensional linear time-varying dynamical systems, for which the outputs are constrained to assume prescribed values at different points in time is considered. It is shown that the solution (optimal controller) is obtained by performing a linear operation on an appropriate vector-valued generalized interpolating spline.
control; spline; control; spline


text:
Divorced and Deadly Paperback
A light-hearted and comic romp from the nation's favourite storyteller and author of The Loner.Footloose, fancy free and ready to get back in the game, newly divorced Ben is back home with mum which is just where she wants him.
But best mate Dickie has other ideas, and soon they are sharing a flat, up to all sorts and plotting Ben's future romantic adventures.But being single isn't all it's cracked up to be.Ben staggers from one disaster to another - all under the beady gaze of his ex, who may not want him but doesn't want anyone else to have him either! And then there's Ben's co-worker Poppy, who seems very interested in his escapades too - could she have plans of her own?Laugh-out-loud funny and based on true stories that you just couldn't make up, Divorced and Deadly is the perfect feel-good read.
- Format: Paperback
- Pages: 240 pages
- Publisher: HarperCollins Publishers
- Publication Date: 13/05/2010
- Category: Modern & contemporary fiction (post c 1945)
- ISBN: 9780007301430
- eAudiobook MP3 from £24.96
- EPUB from £5.49
Showing 1 - 2 of 2 reviews.
Review by callmejacx
Not as funny as I was expecting. Maybe it is just my sense of humour, but when things get far fetched I no longer find humour in it. This is suppose to be a No. 1 bestseller. Not sure it if is my type of funny.
Review by shelleyraec
This missed the mark for me - I was expecting something a lot wittier but Ben is just a jerk, the humour mostly falls flat. The book description doesn't quite fit either.

domain: entertainment


TEHRAN—Iran said Wednesday it foiled an attempt to sabotage a facility belonging to its atomic agency near the capital, the latest attack on its nuclear program and one that follows the election of a hard-line cleric as the country’s president.
Saboteurs targeted the building in Karaj, west of Tehran, earlier in the day but were thwarted, according to Iran’s semiofficial Tasnim news agency. Iranian authorities didn’t say what the building contained or give any other details about the attack.
State-run PressTV said the “hostile attempt” hadn’t caused damage or casualties due to precautions taken after previous attacks on the country’s nuclear facilities and the killing of one its top nuclear scientists. Investigators were trying to identify the perpetrators, it said.
The alleged sabotage attempt comes at a politically sensitive time for Iran. The country last week elected a new conservative president,
who has said he supports reviving the 2015 nuclear deal with the U.S. and other world powers. But on Monday he rebuffed a key goal of the Biden administration as it negotiates a revival of the multilateral accord, hinting that Tehran wouldn’t stop supporting Shiite militia groups fighting across the Middle East or rein in its missile program.
Mr. Raisi faces daunting challenges as he prepares to take office in August. A day after his election, one of Iran’s main nuclear facilities experienced an unexplained blackout. On Tuesday, U.S. agencies seized more than 30 Iranian web domains, including sites operated by government-run PressTV as well as social media channels affiliated with Iran-backed militias in Iraq.
Over the past two years, Iran has scaled up its nuclear activities to force the U.S. to roll back crushing sanctions—breaching multiple key limitations in the 2015 multilateral nuclear deal—and its atomic ambitions have come under greater international scrutiny.
Tehran is currently negotiating with U.S., European, Russian and Chinese diplomats in Vienna about reviving the 2015 agreement, which put curbs on its enrichment activities in return for relief from U.S. sanctions. Former President
withdrew the U.S. from that accord in 2018, saying the deal didn’t go far enough, and didn’t curtail Iran’s other military activities.
Iran’s nuclear program has been targeted in a number of previous attacks.
One in April on its main nuclear plant in Natanz potentially destroyed hundreds of uranium-enrichment centrifuges. In November, one of its top nuclear scientists,
was killed outside Tehran. A fire at Natanz in July badly damaged a centrifuge assembly hall.
Iran has accused Israel of orchestrating most of the recent attacks that have targeted its nuclear program. Israel, which is vehemently opposed to the nuclear deal, hasn’t commented on the allegations. But it has said it is willing to act on its own, if necessary, to prevent Iran from acquiring a nuclear weapon.
Iran has long insisted its nuclear enrichment program is meant for civilian energy purposes. With the nuclear agreement, the International Atomic Energy Agency gained unprecedented monitoring access to Iran’s facilities, but has since criticized Iran for insufficient cooperation, including its failure to explain the presence of uranium particles at three undeclared sites.
In recent months, Iran has threatened to further limit access to United Nations nuclear inspectors if the U.S. doesn’t lift sanctions.
The alleged sabotage attempt comes days after one of the country’s main nuclear power plants in Bushehr went partially offline in an unexplained blackout.
More on Iran’s Nuclear Program
A unit of the Bushehr nuclear power plant started to lose power around 8 p.m. local time on Saturday, which led to the unit being disconnected from the circuit early the next day, according to Iran’s atomic energy agency.
An energy ministry spokesman said Monday the disconnection of the unit was due to a technical error, and that the plant would probably be back in service within four days.
Banners on the websites seized by U.S. agencies said the domains had been taken by the U.S. government as part of enforcement action taken by the Federal Bureau of Investigation and the U.S. Department of Commerce’s Bureau of Industry and Security. The U.S. is able to seize the sites because they use internet infrastructure either in the U.S. or owned by U.S.-based firms.
Iranian Minister of Information and Communications Technology
Mohammad-Javad Azari Jahromi
told Iranian media Wednesday that the U.S. had seized the websites out of “desperation” and that the “U.S. regime is trampling its own claimed values.”
Write to Sune Engel Rasmussen at email@example.com
Copyright ©2020 Dow Jones & Company, Inc. All Rights Reserved. 87990cbe856818d5eddac44c7b1cdeb8


text:
RAF woman wins sex discrimination case
The RAF was condemned in a judgment that recognised a culture of sexism among senior levels of staff.
The sexual discrimination the woman received was "illegal" and there were too many instances of "senior officers with sexist views", it was found.
The flight lieutenant - who cannot be identified for legal reasons - claimed she was driven out of the RAF by sexism and harassment senior officers did little to stop.
The tribunal failed to find that the woman was constructively dismissed from her job, but commented that the RAF "should take little comfort from that".
"We heard too many instances of senior officers with sexist views, which have no place in an organisation committed to equal opportunities and are quite frankly illegal," the tribunal panel said in its judgment.
One of a small band of females to fly an RAF Nimrod, the woman became pregnant by a married high-ranking officer 20 years her senior in 2000.
After maternity leave, she was treated for anxiety and depression, but was not allowed to fly again and took premature voluntary retirement in 2001.
She told the tribunal she had been determined to maintain her career but that she had been removed from flying, had become ill and had not had her concerns noted by senior officers.
In hearings at Croydon Employment Tribunal that spread over six months this year, the woman claimed RAF personnel were "macho, chauvinist and almost tribal".
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domain: law


2.3 acre lot at the corner of East St. and Telegraph Hill Rd. Zoned Hillside. Will require soil test for permit applications. Road frontage on East St. and Telegraph Hill Rd. Madison water and sewer available per Water/Sewer Dept. Tap fees $700 as of 2/23/18
- Site Link
- Style Not Applicable
Land -Agricultural (Not Zoned)
Many possibilities with a River View!! Fenced 3 acre lot with a 30x60 barn that features 6 bay doors. Electricity, water & sewer is available on site. The property has 3 drive thru gates and a small pond. Acessible from SR56 on Bridge St. The front of the proerty is divided into lots and zoned...
MLS ID 20180197
Courtesy of: River Valley Real Estate, Inc.
Office Phone: +18122731234
Listing Agent: Deborah Hardy
Updated: 11th December, 2018 2:33 PM.
606-608 East St Madison, IN


PHPRunner 2019 9.0 Free Download Latest Version and Single Link for Windows. It is Also offline Setup and standalone installer and Compressed Version Of PHPRunner 2019 Free Download.
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text:
Last summer, Pima County Interfaith Council (PCIC) leaders learned that local residents would be required to use an out-of-state bank for loans under Tucson Electric Power's 2011 Energy Efficiency Proposal. Knowing this would create a stumbling block for individuals, and knowing Tucson's local economy would benefit from local financing, PCIC requested that the Arizona Corporation Commission (ACC) require a local bank. ACC staff agreed to make this recommendationâ€”IFâ€”PCIC could find a local lender. PCIC leaders met with many bank and credit union officers.
Result: an innovative agreement between Tucson Electric Power and Vantage West Credit Union for loans to local ratepayers.

domain: news


text: Bootleg Tower, Day Canyon, Moab. Day Canyon is one of dozens of pristine canyons, rarely visited, only a few miles from Moab. Though few have towers, Day Canyon, home of an early bootlegger, has several challenging ones. In 1974 Terry McKenna and I made the first ascent of sandstone Bootleg Tower. NCCS I, F8, A2.

domain: geography


text:
By Ed Frankl and Joshua Kirby
Henkel AG shares dived Friday after it said it expects a lower earnings margin this year, as raw-material prices surge on the war in Ukraine.
Shares were down 8.0% to EUR58.12 at 0730 GMT.
The German consumer-goods company now expects an adjusted earnings before interest and taxes margin of 9%-11%, from 11.5%-13.5% previously.
Full-year adjusted earnings per share is now expected to decline 15%-35%, from between a positive rise of 5% and a decline of 15% previously, Henkel said.
The new outlook is due to rising raw-material prices and higher logistics costs stemming from the Russia-Ukraine war, as well as the effects of exiting operations in Russia and Belarus following the invasion, the Dusseldorf-based company said.
"Due to these developments, we now expect significantly higher pressure on our earnings for the rest of the year than at the beginning of the year," Chief Executive Carsten Knobel said.
The company now sees additional raw-material costs for the full year at around 2 billion euros ($2.10 billion), twice as much as had been assumed at the end of January.
The company nevertheless said it expects higher sales growth for the year than previously targeted, after first-quarter sales rose on higher prices and with a slight decline in volumes, it said.
According to preliminary figures, the company's sales rose by 7.1% to around EUR5.3 billion in the quarter.
Henkel said it is now aiming for full-year organic growth between 3.5% and 5.5% in fiscal 2022, compared with a previous targeted range of 2% to 4%.
The company will set out full first-quarter results on May 5.
Write to Ed Frankl at email@example.com
(END) Dow Jones Newswires

domain: finance


", "FutureSound Keynotes: Pandora CEO Joe Kennedy, Superfly Co-Founder Rick Farman, S-Curve Founder Steve Greenberg", "NoCAPRICHO 2012: We the Kings, We Are the in Crowd e Before You Exit estão confirmados! 84 on the Pop 100 and No. I love them and @jennyrobinson1…. Each person who purchased a package had their face placed on the album cover along with the band members' faces in a yearbook format. Travis Clark is the lead vocalist for the famous rock band, We The Kings. While teaming with Bret Disend in Fall of 2005, We the Kings went on its first tour under that name as a five piece, with the then-unknown Boys Like Girls as the group supported the new release of the "Great Escape" music video. 350.3k Followers, 175 Following, 2,277 Posts - See Instagram photos and videos from Travis Clark (@traviswethekings)  The band also made a stop in Rhode Island to perform at Roger Williams University.  The single was later certified gold in Australia for shipments exceeding 35,000. We the Kings also played with New Found Glory at Fitchburg State College's annual spring concert. and drummer Danny Duncan. On October 4, Clark previewed a song off of the new album titled “All The Way” in his YouTube vlog. The Summer Set, The Downtown Fiction, Hot Chelle Rae and Action Item provided support. By Jag. 185 cm / 6 ft 1 in: Born: 24 April 1985 : How old is Travis Clark in 2021? 1.6K likes.  On November 26, Charles Trippy announced on Twitter that Vitam Regum would be released on December 16, 2013. The current members of We The Kings include Hunter Thomsen, Danny Duncan, Coley O’Toole, Charles Trippy, and Travis Clark who took the time to speak with us about life on the road, overcoming challenges, and becoming a working parent in the music business today. We the Kings also sports their own miniature series of webisodes, called 'The King's Carriage," detailing life on their tour bus. PureVolume, a place where people could upload their music, helped the band's popularity.  The band's line-up consisted of vocalist Travis Clark, guitarist Hunter Thomsen, bassist Drew Thomsen. It featured the single "Runaway". , In February 2009, the band headlined a tour called The Secret Valentine Tour with The Maine, The Cab, There for Tomorrow, and Versa. On August 25, 2015, Trippy started tracking bass for the album in his home studio in Tampa Bay, Florida. - News - Fuse", "We The Kings to release new acoustic album, 'Stripped, "We The Kings announce the 'From Here To Mars Tour, "We the Kings member O'Toole talks tour and newest single", "Vans Warped Tour, with New Found Glory, We The Kings and more, sets Jones Beach date", "We the Kings Announce WTK 10 Tour to Celebrate 1oth Anniversary of Their Debut Album", "We the Kings' Travis Clark on the Band's 10th Anniversary Tour: 'I Will Remember This For the Rest of My Life, "Travis Clark on Instagram: "Love doesn't even come close to explaining my heart right now.  Charles Trippy is married to Allie Wesenberg since March 11, 2017, and they have a daughter.  In summer, the group toured with Don't Die Cindy. , Friends since childhood, the four bandmates (singer/guitarist Travis Clark, guitarist Hunter Thomsen, his brother bassist Drew Thomsen, and drummer Danny Duncan) formed the group while attending high school. … ", Australian Recording Industry Association, Learn how and when to remove this template message, "We The Kings release title track from upcoming album, 'Strange Love, "We the Kings – Chart History: Pop Songs", "2013 Vans Warped Tour / Date And Band Details Coming This Fall", "We the Kings – Chart History: Billboard 200", "Billboard's FutureSound conference is coming up fast! We The Kings are a band who have seen and done an awful lot in their career. "I speak with her frequently, so I knew [many of the rumors out there] were lies," Travis told the magazine, "It's a classic, 'Don't believe everything you hear' story.  Coley O'Toole is married to Monika and together they have a son and daughter. The group's sophomore album, which was released in December 2009, reached 112 on the Billboard 200 and spent three weeks on the chart. Since the mid-2000s, We The Kings vocalist/lyricist Travis Clark has been rocking out onstage and engaging crowds around the world. , According to Travis Clark, the album was "really guitar driven, with those poppy catchy choruses". On September 20, Elena Coats entered the studio to record vocals for a track on the new album called “XO”. The band got its name from the middle school that all the members attended called Martha B.  The group supported Boys Like Girls on their US headlining tour, dubbed Tourzilla, from late September to late October. The band has also played in Warped Tour 2010 from June 26 to August 2. Avril Lavigne made a triumphant return to music this year with her sixth record, Head Above Water, but now she’s giving the title track an update with We The Kings ‘ … When Travis proposed to Jenny it was a huge surprise and you can watch a video of that proposal below: . : We The Kings Reveal New Album 'Vitam Regum' Release Date", "Alter The Press! The King's Carriage detail the band's life on the road and give the fans insight into the band members' lives. The band's line-up consisted of vocalist Travis Clark, guitarist Hunter Thomsen, bassist Drew Thomsen.  In July, the band supported All Time Low on their US east coast tour.. ", On December 5, 2016, We the Kings announced a tour that will commemorate the ten year anniversary of their debut album, We the Kings, that was released in 2007. , After Warped Tour 2014, Clark started working on new songs as well as re-recording songs from the previous album for an acoustic album. The first single off of the album, "Love Again", came out October 30, as well as the pre-order. The song, "Just Keep Breathing", is about how frontman Clark had been bullied as a kid. The band was on tour in Fall 2009, again supporting All Time Low on the Glamour Kills Tour, with Hey Monday and The Friday Night Boys. Biography. Alongside him in We the Kings are bassist Charles Trippy, lead guitarist Hunter Thomsen, keyboard player Coley O'Toole and drummer Danny Duncan. , Under the guidance of manager Bret Disend, the band placed a number of tracks on the social networking site Purevolume in 2007 to build online buzz. Travis was born and raised in Bradenton, Florida. Travis Clark is the lead vocalist and songwriter for We the Kings. On September 12, 2017, We the Kings released a tenth anniversary album called Self-Titled Nostalgia. We The Kings is a melodic emo-pop band from Bradenton, Florida. Sep 30, 2012 - We The Kings Milwaukee, WI Warped Tour 2012 .. <3 Abby <3 Victoria <3 Kennedy <3 Sarah “We could release ‘Check Yes Juliet’ over and over but it would lose the feeling. During February, the band headlined another tour throughout Europe, visiting numerous countries including the United Kingdom, The Netherlands, Germany, Italy and Switzerland. He formed the group in 2006 during high school along with his childhood friends Hunter Thomsen, Drew Thomsen, and Danny Duncan. No one expects We the Kings to break new ground; we just expect them to play familiar emo pop far better than their contemporaries. Travis Randall Clark (born: March 25, 1985 [age 35]) is a YouTube vlogger and the vocalist/guitarist for pop-punk/pop rock band We The Kings.. (7.2.15 - Day 2256)" 'CTFXC', July 2, 2015. In February 2016, We The Kings put out a single called “The Story of Tonight”. Biography. On a recent Janua… , On February 18, 2014, Clark announced via YouTube that the band would be traveling on Warped Tour 2014. In summer, the group toured with Don't Die Cindy. Travis Clark Height, Weight, Wiki, Biography, Age, Net Worth. On March 2, 2015 Duncan entered the studio in Los Angeles to track drums, and finished on March 4. However, partway through the tour Valencia had to drop off the tour due to their vocalist Shane Henderson having strep throat.  The second single, "Find You There", was released on May 3. Bands also playing the Bamboozle Road Show tour included Forever the Sickest Kids, The Cab, Never Shout Never, and Mercy Mercedes. , In January 2016, We The Kings released a video announcing a U.S. headlining tour that would go from March to April 2016 called the From Here To Mars Tour. On this episode of Inside Music, We The Kings frontman Travis Clark hops on the phone with host James Shotwell to discuss his unique career in entertainment. He surprised us all and came 3 weeks early... We think he just couldn't…, "Ryan Hunter Thomsen on Instagram: "Jack Kelly Thomsen - Welcome to our family So thankful for everyone involved. — TRAVIS CLARK of WE THE KINGS - Vans Warped Tour... 1.5M ratings 277k ratings See, that’s what the app is perfect for. Lipshutz, Jason. Clark spoke with the Bradenton Herald about the inspiration behind the new album, … Before Bradenton native Travis Clark became a rock star playing packed venues on both side of the Atlantic with 200,000-plus people awaiting his every Tweet, he got picked on for something as silly as his hair color. Dem's good friend Travis Clark, the frontman for We The Kings, tells J-14 magazine that the public shouldn't believe the rumors about Demi. , In January 2013, We the Kings began recording the group's fourth album. TRAVIS CLARK of WE THE KINGS - Vans Warped Tour 2018. Yay for more/new songs!  On October 24, 2015 We The Kings announced Strange Love as the album's title and revealed its cover art. The group toured in support of Cobra Starship in early 2008 along with Metro Station and The Cab. Travis Clark is the singer and the songwriter in We the Kings band. The tour will go from February to May 2017 with 32 dates in the US, 3 dates in the UK, and 1 date in Canada.. "Music: Happening Now-Pop: Kingdom Come. Travis Clark of We Are Kings married Jenny in Orlando, Florida at Bella Collina. The event raised thousands of dollars for ClearWater Initiative and was the organization's biggest fundraiser up to that date. : We The Kings Release New Song "Art Of War, "We the Kings - Chart history (Billboard 200)", "Warped Tour 2014 Lineup: See All the Bands Playing This Year! Writing Head Above Water with Travis Clark from We The Kings was such a beautiful experience. "It’s always been ride or die" - Travis Clark We The Kings are a band who have seen and done an awful lot in their career. On October 4, We The Kings announced that the album's release date would be November 20, 2015. Just weeks after We The Kings dropped their pre-quarantine-reminiscent single “These Nights,” Travis Clark has reimagined the song in a new acoustic video, …  In February and March 2009, the group went on a headlining US tour, dubbed The Secret Valentine Tour, with support from the Maine, the Cab, There for Tomorrow and VersaEmerge. ", "Ryan Hunter Thomsen on Instagram: "Mr. and Mrs. Thomsen #happilyeverThomsen, "Ryan Hunter Thomsen on Instagram: "We welcomed Oliver Regal Thomsen to the world on December 4, 2017. The album peaked at number 151 on the Billboard 200 and spent fifteen weeks on the chart. He discovered his passion for songwriting while at his high school when he wrote a song titled “Gonna Love You”. Get all the lyrics to songs by Travis Clark and join the Genius community of music scholars to learn the meaning behind the lyrics.  In April 2010 the band performed a benefit concert with fellow pop punk band Voted Most Random at a local venue in New Haven, Connecticut. With their 2007 debut, the group followed the same path that the Academy Is and Jimmy Eat World helped construct, mixing slickly produced pop songs and bleeding-heart lyrics in equal doses. (C) 2008 S-Curve Records#WeTheKings #CheckYesJuliet #Vevo Travis Randall Clark is listed as a President with We The Kings Inc. in North Carolina.  In April, the band performed on the Bamboozle Roadshow and appeared at The Bamboozle festival in early May. By March 2013, the band members announced that a new song would be released in the first week of April. and drummer Danny Duncan. The album spawned a second single, "Say You Like Me", which peaked at number 32 on the Pop Songs chart, and number 29 in Australia. The album was released July 6, 2018. The group's second album Smile Kid (2009) included Top 40 singles "Heaven Can Wait" and "We'll Be a Dream" (featuring Demi Lovato), as well as the single "She Takes Me High". 5. Like the previous two We The Kings records, this album was released without a label. The group performed "Secret Valentine" along with a few other songs and closed with "Check Yes Juliet". In February 2015, Clark, O'Toole, and Thomsen started tracking guitar and vocals for the fifth album in O'Toole's studio in Connecticut. We the Kings' Travis Clark on Bradenton, band's history . The album's second single "Check Yes Juliet", gained commercial success. Throughout much of 2011 and 2012, the band toured various countries on numerous tours including the End of the World Tour, performing with Anarbor, The Downtown Fiction and Mayday Parade. Travis Clark Fans. Singers. The album peaked at No. On Saturday (December 19), Clark responded to a fan's … He discovered his passion for songwriting while at his high school when he wrote a song titled “Gonna Love You”. Born 12-12-2015. "Coley O'Toole on Instagram: "Those teeth!!! We the Kings is the self-titled debut studio album by American rock band We the Kings.  It was released on October 2, 2007. ITS JUST FANS THAT LOVES HIM AND WE THE KINGS!! The group toured Australia in early March 2011, including performing at the Soundwave festival. In December 2017, We the Kings released a video announcing the start of production for their sixth studio album. We the Kings formed in 2005 while its member attended Manatee High School. Yay for the studio being in my house! Lead vocalist, guitar player, keyboardist, and songwriter for the rock group We The Kings. / Age: 35 years: Where was Travis Clark born? Kya Leigh Robinson Clark aka my baby girl was born 9.25.17 at 4:09am.…, "Travis Clark on Instagram: "Our beautiful baby girl. I like to Tweet and some times even Instagram. The band A Heartwell Ending (later renamed Call the Cops) supported for the final leg of the tour. The album was fully released on November 24, 2014. Demi Lovato Responds to Anti-Vaccine Post From We The Kings Frontman Travis Clark popcrush.com - PopCrush Staff. He previewed a song off the album titled “From Here to Mars” on his YouTube vlog series, "Internet Killed Television". We the Kings formed in 2005 while its member attended Manatee High School. "Just Keep Breathing", released on April 5, reached number 101 on the UK Singles Chart and number 13 in the Official Alternative chart after only three days of sales. Check Yes Juliet Charles Trippy We The Kings Child Smile The Way Back Rock Groups Sunshine State Debut Album Beautiful Men. In May and June, We the Kings supported Cute Is What We Aim For and Boys Like Girls on the group's UK tour. Web. 350.3k Followers, 175 Following, 2,277 Posts - See Instagram photos and videos from Travis Clark (@traviswethekings) Travis explains the critical moments on the band’s journey where one or two decisions shaped everything that followed, as well as the influence of his personal life on his creative output. The album was announced to include "Planes, Trains, and Cars", "Festival Music", and eight to ten more songs. Hi, I'm Travis! Travis Clark is best known as a Rock Singer. We the Kings is an American rock band from Bradenton, Florida. 3.  The album was also released in 2011 and was certified platinum by the ARIA for sales of 70,000 copies. Dem's good friend Travis Clark, the frontman for We The Kings, tells J-14 magazine that the public shouldn't believe the rumors about Demi. Coats also came on tour as a special guest to sing “XO” and “Sad Song” with We The Kings as well as perform her own music. The album was released on November 20. We the Kings completed a short, Florida-only tour for this album, and released a lyric video for "Stone Walls"..  After its first week of release as an iTunes exclusive the album debuted at 44 on the Billboard 200 and at 88 on the UK Albums Chart. We the Kings have become famous for creating lean, catchy pop-punk bursts but the band’s latest song is more richly textured with moody atmospherics driving home the deeper message of perseverance. , In October 2011, Charles Trippy and Coley O'Toole joined We the Kings with Trippy playing bass guitar (replacing Drew Thomsen who left the group earlier that year) and O'Toole playing keyboard and rhythm guitar. Blake Healy of Metrostation, who produced Somewhere Somehow, also produced this album. , For part of the fall of 2007, the band played a tour called Tourzilla with headliner Boys Like Girls, All Time Low, and The Audition. Lead vocalist, guitar player, keyboardist, and songwriter for the rock group We The Kings.He has a popular YouTube channel called TravisClarkTV where he posts video blogs of his life. The band members (Travis Clark - Lead Vocals/Rhythm Guitar/Piano, Hunter Thomsen - Lead Guitar ,Coley O'Toole - Backing Vocals/Piano, … read more. Trivia. The company is a North Carolina Business Corporation, which was filed on August 26, 2009. ", Lipshutz, Jason. The music video debuted on The American Top 40 with Ryan Seacrest on April 12, 2016. The band also headlined two sold-out sideshows in Sydney and Melbourne with The Maine and Never Shout Never. ", "We The Kings release new single, "Just Keep Breathing, "Need U 100% holds on to Official Singles Chart Update Number 1", "Alter The Press! , Almost two years after the release of the band's second album, a follow-up, titled Sunshine State of Mind, was released. Meanwhile, the band also pursued a deal with EMI's imprint S-Curve Records, which eventually signed the group. Registrati a Deezer gratuitamente e ascolta We The Kings: discografia, brani top e playlist. "We The Kings: Check Yes Juliet (Run Baby Run).". A conversation with We The Kings’ Travis Clark. We the Kings played on its first headlining tour Long Hair Don't Care with Valencia, The Cab, Sing It Loud, and Charlotte Sometimes during late March and throughout April.  The band released the fourth single for Somewhere Somehow, entitled "Art of War", on December 9, 2013.  In April, the band appeared at the Bamboozle Left festival.  We the Kings was certified platinum by the Australian Recording Industry Association (ARIA) for the shipment of 75,000 copies. We the Kings is an American rock band from Bradenton, Florida, currently signed to record label S-Curve Records. He discovered his passion for songwriting while at his high school when he wrote a song titled “Gonna Love You”. WE THE KINGS are on an emotional roller coaster. The re-recording's lyrics were altered to include a reference to Tampa Bay Rays. He's doing everything he wants to be doing, and music is the reason that any of it is possible.  On August 30, 2008 the band was a part of The Rays Summer Concert Series, playing on the field after a Tampa Bay Rays game. The band's self-titled full-length debut album, released in 2007, included the platinum single "Check Yes Juliet", and went on to sell over 250,000 copies in the US. The band also performed at Warped Tour 2009. At the start the band name was Broken Image, but then they changed it to We the Kings and they are still using that name now. We The Kings is scheduled to perform on the Vans Warped Tour 2016. Brothers James, a band consisting of We The Kings members Coley O'Toole and Hunter Thomsen, also performed on the tour.. The group's fourth album Somewhere Somehow was released in late 2013 and includes singles "Just Keep Breathing", "Find You There", "Any Other Way", and "Art of War". “We continue to try and grow with our band,” Clark said. King Middle School. He has a popular YouTube channel called TravisClarkTV where he posts video blogs of his life.  The band's first tour was with Don't Die Cindy in the summer of 2005 where the group went by Broken Image, then later went under the moniker De Soto. Coats was also featured on a track off of the previous record, Somewhere Somehow, called “Sad Song”. , Travis Clark is married to Jenny Robinson Clark; they have three daughters. After Warped Tour, the band plan on a world tour in 2011.  In October and November, the band supported the Academy Is... on their headlining tour of the US. I also have a clothing line called King Travis Clothing. Kinsley James Robinson Clark. Ahead of We The Kings hitting Aussie shores for their 10 Year Anniversary Tour, Don’t Bore Us had a chat with vocalist, Travis Clark.These are the 7 things we learnt outside the fact he has the voice of a … Sad Song Lyrics: You and I / We're like fireworks and symphonies exploding in the sky / With you, I'm alive / Like all the missing pieces of my heart, they finally collide / So stop time right here The singer is happy and healthy behind the DJ booth with We The King’s Travis Clark, belting out vocals that prove she’ll always be a forced to be reckoned with. 3 on Billboard's Top Heatseekers chart, and at No. We The Kings Milwaukee, WI Warped Tour 2012. On September 1, 2013, the band finished the Summerfest 2013 tour with Breathe Carolina, T. Mills, and The Ready Set. We the Kings played tour dates at the Vans Warped Tour 2012.  Hunter Thomsen is married to Caitlin, they have two sons. "It’s always been ride or die" - Travis Clark. Hola, it’s Alyssa! "I speak with her frequently, so I knew [many of the rumors out there] were lies," Travis told the magazine, "It's a classic, 'Don't believe everything you hear' story. 151 on the Billboard 200. The album, Stripped, featured acoustic versions of songs from Somewhere Somehow, as well as some new material, including "Stone Walls" and the bonus track, "Is This the End?". After the band returned home from their Australian tour in March 2016, a music video was filmed for “The Story Of Tonight”. and drummer Danny Duncan. "Music Happening Now-Pop: Kingdom Come. , On November 22, 2013, We the Kings announced the title of the group's upcoming fourth album would be Vitam Regum.  From mid-January to early March 2008, the band supported Cobra Starship on their North America tour.  In June, the band supported Cobra Starship on their US tour, dubbed Really Really Really Ridiculously Good Looking Tour Out to Warped Tour Tour. Travis Clark. The band's third single, "Any Other Way" was released on June 14. Full name : Travis Clark How old is Travis Clark: 35 years Male Birthday: April 24, 1985 Sun sign: Taurus Nationality: Florida, United States Travis Clark Spouse: Jenny Robinson-Clark (m. 2014) Travis Clark siblings: Taylor Clark TRAVIS CLARK of WE THE KINGS - Vans Warped Tour 2018. With six full-length albums under their belt, countless miles out on the road and thousands of adoring fans dotted through every continent, there is … The group's second album Smile Kid (2009) included Top 40 singles "Heaven Can Wait" and "We'll Be a Dream" (featuring Demi Lovato), as well as the single "She Takes Me High". Everyone is happy and healthy! ❤Hannah and Elliot at the park with mama. 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Convenient and Healthy: The Rise of Organic Food Delivery Services
The demand for organic food has been steadily rising over the past few years, as people become increasingly aware of the benefits it offers for both their health and the environment. However, finding high-quality organic produce can sometimes be a challenge, especially for those with busy lifestyles. This is where organic food delivery services come in, providing a convenient solution that combines the benefits of organic food with the ease of home delivery. In this article, we will explore the rise of organic food delivery services and how they are revolutionizing the way people access healthy and sustainable food options.
The Growth of Organic Food Delivery Services
Organic food delivery services have seen a significant increase in popularity in recent years, with more and more people opting for the convenience and quality these services offer. These services work by partnering with local organic farmers and producers to source fresh and seasonal produce, which is then delivered directly to the customers’ doorsteps. By eliminating the need for multiple trips to the grocery store and connecting consumers with local farmers, organic food delivery services have simplified the process of obtaining organic produce.
One of the key reasons behind the growth of organic food delivery services is the increasing demand for organic products. Consumers are seeking healthier and more sustainable alternatives to conventionally grown food, which often contains harmful pesticides and chemicals. Organic food, on the other hand, is grown without the use of synthetic fertilizers, pesticides, and genetically modified organisms (GMOs), making it a safer and more nutritious option. By offering a wide range of organic fruits, vegetables, dairy products, and pantry staples, these delivery services cater to the growing health-conscious population.
The Benefits of Organic Food Delivery Services
Convenience: One of the primary advantages of organic food delivery services is the convenience they provide. With just a few clicks on a website or a mobile app, customers can easily browse through a variety of organic products and have them delivered to their doorstep within a specified time frame. This saves both time and effort, especially for individuals with busy schedules who may not have the time to visit multiple stores in search of organic produce.
Quality and Freshness: Organic food delivery services prioritize quality and freshness, ensuring that customers receive the best possible products. Most services source their produce directly from local organic farmers, guaranteeing that the food is picked at its peak ripeness and delivered within a short time frame. This ensures that the produce retains its nutritional value and flavors, making it both delicious and nutritious.
Environmental Sustainability: By supporting organic food delivery services, consumers contribute to a more sustainable food system. Organic farming practices promote biodiversity, conserve resources, and reduce pollution by avoiding the use of synthetic inputs. Additionally, these delivery services often use eco-friendly packaging and delivery methods, further reducing their environmental impact.
Supporting Local Communities: Organic food delivery services often collaborate with local farmers and producers, providing them with a platform to sell their products directly to consumers. This creates a direct connection between farmers and consumers, ensuring that farmers receive fair compensation for their hard work. By supporting local organic farmers, customers contribute to the sustainability of local food systems and the preservation of traditional agricultural practices.
Frequently Asked Questions (FAQs) about Organic Food Delivery
Q: How do organic food delivery services source their products?
A: Organic food delivery services typically partner with local organic farmers, cooperatives, and producers to source their products. This allows them to provide customers with fresh and locally grown organic produce.
Q: Are organic food delivery services more expensive than traditional grocery stores?
A: While organic food delivery services may sometimes have slightly higher prices due to their focus on high-quality organic produce, they often offer competitive pricing compared to traditional grocery stores. Additionally, customers may find that the convenience and time-saving aspects of these services outweigh the slightly higher costs.
Q: Can I customize my organic food delivery?
A: Yes, most organic food delivery services offer customization options, allowing customers to choose the specific fruits, vegetables, dairy products, and pantry staples they want. Some services also offer various dietary preferences, including vegan, gluten-free, and paleo options.
Q: Is organic food delivery available nationwide?
A: Organic food delivery services operate in various regions across the country. While some services are limited to specific cities or states, many are expanding their delivery areas to serve a larger customer base. It is advisable to check the availability of these services in your specific area.
Q: How are organic food delivery services ensuring food safety during the delivery process?
A: Organic food delivery services prioritize food safety and take measures to ensure that products remain fresh and safe during the delivery process. This includes using insulated packaging, refrigerated delivery vehicles, and adhering to strict food handling practices.
In conclusion, organic food delivery services have emerged as a convenient and healthy solution for those looking to incorporate organic food into their lifestyles. With their focus on quality, freshness, and sustainability, these services offer a wide range of organic products delivered directly to customers’ homes. By supporting local farmers and providing accessible organic options, organic food delivery services are empowering consumers to make healthier and more environmentally conscious food choices.
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- Organic Food Delivery


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Many people today respect the search for a spiritually grounded life. By studying the formation of early Friends’ experience of transformation, Doug Gwyn offers an understanding of the doctrine of atonement that offers depth and hope to those with a hunger for truth.
About the Author(s)
Doug Gwyn grew up in the pastoral stream of Friends in Indiana. After experiencing a call to ministry in 1968, he attended Union Theological Seminary in New York City, where he began to know unprogrammed Friends better. Over the years, he has followed his calling to ministry into work as a Friends pastor, as a writer for the American Friends Service Committee, and as a teacher at the Quaker study centers Pendle Hill and Woodbrooke. His training in biblical studies has informed his research and writing on early Friends and on current issues among Friends today. Doug has coined the term “bispiritual” to describe Friends like himself who are engaged and nurtured by both pastoral and unprogrammed Friends, in different ways. His wife, Caroline Jones, is a Friend and dharma teacher in the Insight Buddhist tradition.
His books include Apocalypse of the Word: The Life and Message of George Fox (1986), The Covenant Crucified: Quakers and the Rise of Capitalism (1995), A Sustainable Life: Quaker Faith and Practice in the Renewal of Creation (2014), and Conversation with Christ: Quaker Meditations on the Gospel of John (2011). Doug is currently completing a history of Pendle Hill.

domain: christianity


Romoss Simple 20 20000mAh Input: Type C|Lightning|Micro USB|Output: 2 x USB Power Bank – White
The Romoss Simple20 2000mAh Power Bank is a handheld hero with its high power 2.1A charging that is widely compatible with most mobile devices. This sleek and compact power bank boasts a total of three input ports and two output ports, which can be used to simultaneously charge two devices.
Out of stock
Charge your phone and tablet with the compact Romoss Simple20 20000mAh Power Bank. This power bank offers a wide range of compatibility with its three input ports, a Micro USB port, a Apple Lightning port and a Type-C port. It only gets better from there, with its dual USB outputs that allow for 1A or 2.1A power output, ensuring a fast charge for your devices.
- 20000mAh Capacity power bank
- Handheld design
- 5V 2.1A charging
- Simultaneous dual device charging
- Input ports – Lightning, Micro USB, Type-C
- Output ports – Dual USB, Type-C
- LED Charge indicators
- Battery Type: Lithium Polymer
- Battery Capacity: 20000mAh/(74Wh)
- Input: 5V 2.1A (Lightning/Micro USB/Type-C)
- Output: 5V 2.1A | 5V 1A | 2.1A
What’s In The Box:
- Romoss Simple 20 20000mAh Power Bank x1
- User Guide x1
*No other products depicted in media is included with this product.
|Dimensions||9 × 3.2 × 16.4 cm|


text:
Happy Pride 2021 and welcome to the first episode of GWK the Podcast! Join GaysWithKids.com's executive editor David Dodge, founder Brian Rosenberg, contributing writer Giovhan Dejoie and assistant editor Brit Smith to talk about the latest news and issues that matter to the LGBTQ+ parenting community.
This week; 21-year-old Weston Charles-Gallo, a gay former-foster youth from Missouri who found a loving family with two dads, testified to Congress in support of the Every Child Deserves A Family Act. The organizers of NYC's Pride celebrations have canceled all contracts with New York Police Department, and have banned officers from marching in uniform. And Twitter has been lighting up about a real-life "Won't Someone Think of the Children" moment when a parent requested all gays keep their leather at home this Pride so the kids can safely attend.
Check out GaysWithKids.com for more news that matters to gay parents, and lots of great gay/bi/trans dad family stories. Follow us on social media @gays_with_kids and remember to vote for the winner of the GWKHero Award from June 14-17.
Learn more about GaysWithKids at NYC Pride's Family Move Night at www.nycpride.org/event/family-movie-night


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WARRNAMBOOL City Council local laws officers are using a softly-softly approach to change a long-held culture where motorists park on nature strips and parks.
During last week’s influx for the Fun4Kids Festival 200 notes were placed on vehicles along Timor Street between Banyan and Japan streets warning that further parking on the grassed verge would incur an $89 fine.
It obviously worked because only one penalty notice was issued and a visitor sent an email thanking the council department for issuing a warning rather than an immediate fine.
“A ticket would have soured our experience of Warrnambool,” the email said.
Dozens more vehicles overflowed on to the Cannon Hill lawns, which technically is also illegal.
But local laws manager Ian Fitzgibbon said no action was taken other than monitoring the situation.
“We are reviewing the whole issue of overflow parking for major events and will determine the best consistent approach for future instances,” he said.
“The parking on grass beside Pertobe Road that happens in summer is also technically illegal and has been discussed with stakeholders.
“Nature-strip parking is against the law across Victoria and for Warrnambool the issue will involve a cultural change.
“The law is designed for safety, as well as protecting the amenity.
“Drivers should be aware of road rules.”
A specific overflow parking area at the showgrounds and shuttle bus transfer has been used for Fun4Kids in the past and is likely to be continued.
Mr Fitzgibbon said the council would be reluctant to implement a widespread crackdown on nature-strip parking in residential areas unless there were regular complaints.
Two zones which have been on the radar are Koroit Street between Banyan and Japan streets, plus the base hospital precinct where residents have been complaining about hospital staff and visitors hogging nature strips all day.
Koroit Street concerns were resolved earlier this year by lowering prices in all-day designated parking zones and more stringent enforcement patrols.
A comprehensive report on the hospital precinct is being compiled by council officers in consultation with local residents and hospital management.
“It’s a cultural thing and we are working with the community to raise awareness,” he said.
“Some other cities have chosen to put a blanket ban on nature-strip parking.”
Meanwhile, there will be no relaxing of parking patrols on street meters and designated off-street areas during festivals.
“It’s business as usual,” Mr Fitzgibbon said. “We need to keep the vehicle turnover happening and treat everyone equally — locals and visitors alike.”


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These luxurious soft 600 thread count, easy-care, blended cotton bed sheets are a great product that will make your bed feel super comfortable. The variety of colors will give your room a beauitufl look. The cotton and polyester blend creates a wrinkle-resistant effect on the fabric that is soft and easy to wash.


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Extension > Agriculture > Crops > Soybean > Soybean cyst nematode management guide > How often, when, and how should I take soil samples for soybean cyst nematode counts to help make management decisions?
How often, when, and how should I take soil samples for soybean cyst nematode counts to help make management decisions?
Fields infested with SCN should be managed to minimize yield loss. In order to manage SCN populations effectively, it is important to monitor SCN populations over time. Distributions of SCN are generally uneven in most fields, and nematode egg numbers can vary with sampling technique. So, to minimize the variability for a representative SCN egg count, it is very important to use recommended sampling procedures:
- Divide a field into 5- to 10-acre areas.
- Use a 1-inch-diameter soil probe to collect soil cores to a depth of 6 to 8 inches.
- Collect soil cores from about 20 different locations in a zigzag pattern for each area to be sampled (Fig. 9).
- Place the soil cores in a plastic bag.
- Label the sample with field identification using a water- resistant marker.
- Store the samples at a cool temperature if they cannot be sent within a few days to a professional laboratory for analysis.
- If a soil sample is used for both SCN and soil fertility analyses, mix the soil sample thoroughly before sending subsamples to different laboratories.
There are a number of factors that contribute to the variability of egg numbers from soil samples. SCN eggs are deposited in a cluster, and the spatial distribution of SCN in many fields is an aggregated pattern. In some fields, soil cores may contain high egg numbers from hot spots and low, even zero, egg numbers from non-infested or recently infested areas.
Increasing the number of soil cores collected in each 10-acre area or reducing the size of the area for each sample can increase the precision of the sample. If the hot spots in the field cannot be managed separately from the rest of the field, the best option is to manage the entire field according to the higher population density.
The efficiency of extracting SCN from the soil is dependent on soil characteristics such as texture and moisture content at the time of sampling. Some variability may be associated with the actual laboratory processing of the sample, leading to a rough estimate of the average SCN population rather than an exact measure. However, an effective management program can be implemented using the rough estimate of the average SCN population in a field.
There are other reasons why SCN population densities may vary in two soil samples taken from the same field. SCN egg counts will be highest if samples are collected in the soybean row at the end of the growing season. Due to the variability, it is difficult to compare SCN samples taken from a field at different areas and times of the year.
Long-term SCN management based on soil samples is best done with a sampling plan that is consistent in area(s) sampled, crop sampled, and time of year samples are taken. Since SCN egg population densities are reduced during a year when a nonhost crop is grown, SCN egg counts from samples taken after corn harvest, but before soybean planting, are the most useful in estimating potential soybean yield loss. Sampling in the fall rather than spring allows more time for the soybean producer to develop an appropriate SCN management plan.

domain: agronomy


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When preparing this photograph for use here it was noticed that there appeared to be something slightly odd about it. Not particularly the fact that this sporting vehicle had been snapped against a less than ideal background, what seemed unusual was that an Italian car of 1947 was still apparently of right-hand drive configuration.
Quite by chance, a close look at the front hub-cap showed a word thereon, which read something like: ITAЯЭՑAM. It took a moment or two for the penny to drop – the negative had been reversed when the photograph had been printed. This is not an unknown phenomenon by any means, but thanks to modern technology it takes only seconds to flip the image over and restore the car’s correct stance – which is how we now see it.
Had this transformation not been achievable the photograph would not have been used and an opportunity would have been lost to explore at least some of the story about this eye-catching Maserati A6-1500.
In 1937 the Maserati brothers sold their company to Adolfo Orsi and their activities were moved from Bologna to Modena, but they remained as consultants for 10 years before returning home to form their OSCA business. The Tipo A6 was the last Maserati model for which the brothers were responsible, it having a tubular ‘ladder’ type chassis with coil-spring suspension, into which was fitted a 1488cc straight-six single overhead camshaft engine. Later in 1947 a 2-litre version was produced and variations on this Tipo A6G-2000 theme continued to be made for the next 10 or so years, although output does not seem to have averaged out at much more than about one car a month.
The prototype A6 chassis passed to Zagato and they built their aerodynamic ‘Panoramica’ coupé on it, in due course they made related versions of this, and from 1954 a run of open A6G ‘Spyder’ versions were produced.
The first production A6 – the car pictured here – was bodied by Pinin Farina and exhibited in March 1947 at the Geneva Motor Show. Its most unusual feature was the retractable headlights, a very early use of such a system. The next example that the firm clothed had the headlights conventionally placed in the front wings and this presaged the more flowing body shape on the Cisitalia 202 that they built later in the year, which Pinin Farina consider ‘marked a decisive turning point in the development of automotive style’.
A point for debate perhaps, but all of the coachbuilt cars referred to here were attractive-looking vehicles.

domain: automotive


Feb 23, 2017 · Do you want a gas/oxygen setup or just a gas/air setup? Any of these common gases (Acetylene, Propane, Butane, Mapp, Natural Gas), when mixed with oxygen will be much hotter than with air alone. With the O2 setup, you need two regulators, 2 hoses, 2 tanks, etc.
May 12, 2020 · MAPP (AKA MAP-Pro) gas burns at a higher temperature than butane and propane. Propane is the next step up and can be used for tasks like loosening rusted bolts. MAP-Pro is used for the more robust jobs like soldering copper pipes and heat treating knives.