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Diesel exhaust particulate (DEP), a major component of urban air pollution, has been linked to atherogenesis and precipitation of myocardial infarction. We hypothesized that DEP exposure would increase and destabilise atherosclerotic lesions in apolipoprotein E deficient (ApoE−/−) mice.
ApoE−/− mice were fed a 'Western diet' (8 weeks) to induce 'complex' atherosclerotic plaques, with parallel experiments in normal chow fed wild-type mice. During the last 4 weeks of feeding, mice received twice weekly instillation (oropharyngeal aspiration) of 35 μL DEP (1 mg/mL, SRM-2975) or vehicle (saline). Atherosclerotic burden was assessed by en-face staining of the thoracic aorta and histological examination of the brachiocephalic artery.
Brachiocephalic atherosclerotic plaques were larger in ApoE−/− mice treated with DEP (59±10%) than in controls (32±7%; P = 0.017). In addition, DEP-treated mice had more plaques per section of artery (2.4±0.2 vs 1.8±0.2; P = 0.048) and buried fibrous layers (1.2±0.2 vs 0.4±0.1; P = 0.028). These changes were associated with lung inflammation and increased antioxidant gene expression in the liver, but not with changes in endothelial function, plasma lipids or systemic inflammation.
Increased atherosclerosis is caused by the particulate component of diesel exhaust producing advanced plaques with a potentially more vulnerable phenotype. These results are consistent with the suggestion that removal of the particulate component would reduce the adverse cardiovascular effects of diesel exhaust.
Keywords:Diesel; Air pollution; P; Atherosclerosis; ApoE; Oxidative stress
Particulate matter (PM) air pollution is associated with an increase in cardiovascular morbidity and mortality [1,2]. In epidemiologic studies, residents of areas with high air pollution have evidence of increased atherosclerosis [3-6]. This relationship is supported by pre-clinical studies showing that exposure to traffic-derived pollutants can increase atherosclerotic lesion size and alter lesion composition (comprehensively reviewed in [7]). The mechanisms responsible for these changes remain unclear, but there is evidence implicating altered endothelial cell function [8] and increased oxidative stress [9-11].
Diesel exhaust (DE) emissions are the major source of nanoparticles in the urban environment that, while contributing little to the airborne mass, produce very high numbers of ambient particles and a large reactive surface area, as well as the potential to deposit throughout the lungs [12,13]. We have previously shown that inhalation of environmentally-relevant concentrations of DE impairs endothelial and fibrinolytic function [14,15], promotes blood thrombogenicity [16] and exacerbates cardiac ischemia [17] in man. Inhaled DE also promotes atherosclerosis in mice [10,18-20]. Importantly, the procoagulant changes and impairment of vascular function induced by DE can be prevented by filtration [21,22], consistent with the hypothesis that the particulate component of DE drives the adverse cardiovascular effects (reviewed in [23]). To date, no investigation has directly determined the effect of diesel exhaust particulate (DEP) on the development of atherosclerotic lesions. We, therefore, addressed the hypothesis that exposure to DEP would increase lesion size and complexity in a murine model of atherosclerosis, and that this would be mediated by inflammation, endothelial dysfunction and oxidative stress.
Instillation and pulmonary inflammation
Pulmonary instillation of DEP was chosen as the exposure method; to administer only the particulate components of diesel exhaust, to ensure the complete delivery of the dose to the lungs, and establish this model as a basis for future experiments looking at the effects of fractionated DEP. Instillation was well tolerated, with no mortality and no effect on body weight (Additional file 1: Figure S1). Lungs of DEP-instilled mice showed fibrotic thickening of the alveolar septae and cuffing of alveolar blood vessels (Figure 1a,b). Particulate was visible deep in the airways, largely within alveolar macrophages. Instillation of DEP produced similar increases in the total cell count in bronchoalveolar lavage fluid (BALF) from atherosclerosis-prone apoliprotein E knockout (ApoE−/−) mice to that from wild-type (C57bl6) mice (P < 0.03, unpaired t-test; n = 7-9; Figure 1c). Macrophages were the predominant cell type in BALF (Figure 1d), although DEP also increased the number of neutrophils (P < 0.011) but not lymphocytes (P > 0.23) in both strains (n = 7-9 for all). DEP had no effect on interleukin-6 (IL-6; P > 0.13; n = 4-6 for all), tumour necrosis factor alpha (TNFα; P > 0.25; n = 4-5 for C57bl6, n = 6-7 for ApoE−/−) or monocyte chemotactic protein-1 (MCP-1; CCL2/JE; P > 0.23; n = 4-5 for all) levels in BALF (Additional file 1: Table S2).
Additional file 1. On-line supplementary information.
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Figure 1. Instillation of DEP induces pulmonary inflammation and alveolar remodelling. Representative lung sections from (a) saline-instilled and (b) DEP-instilled ApoE−/− mice showing airway fibrosis and cuffing of pulmonary blood vessels. Inset: High power image showing particle-laden macrophages deep within the airspaces. Scale bars = 100 μm. (c) Total cell counts in BALF. (d) Cell differentials in BALF. Mean ± S.E.M. (n = 7-9 for all) *P < 0.05, **P < 0.01, unpaired t- test comparing DEP with saline control in same animal type. ApoE−/− were fed Western diet for 8 weeks, whereas wild-type mice were fed normal chow.
Distribution and size of atherosclerotic plaques
Aortae from C57bl6 mice exhibited little Sudan IV staining in either the saline- or DEP-instilled groups. In saline-treated ApoE−/− mice, lipid-rich lesions were visible on the surface of the thoracic aorta largely confined to the aortic arch (Figure 2a). Lipid incorporation was greater in DEP- than saline-instilled ApoE−/− mice (P = 0.046, n = 6-7; Figure 2b).
Figure 2. Instillation of DEP increases atherosclerosis. (a) Representative images showing lipid-rich lesions in the aorta following staining with Sudan IV (red stain; orange is non-specific staining). Scale bars = 1 mm. (b) Percentage of the intimal surface staining positive for lipids. Mean ± S.E.M. (n = 3-4 for C57bl6, n = 6-7 for ApoE−/−), *P < 0.05, Mann–Whitney test, saline vs DEP in ApoE−/− mice. (c) Representative transverse sections of atherosclerotic plaques in the brachiocephalic artery stained with United States Trichrome (UST). Scale bars = 100 μm. (d) Plaque size, as a percentage of medial area was increased in DEP-treated ApoE−/− mice. Mean ± S.E.M. (n = 3-5 for C57bl6, n = 7-9 for ApoE−/−), *P < 0.05, unpaired t-test, saline vs DEP in ApoE−/− mice. Mean value obtained from analysis of serial sections at 100 μm intervals for each animal; Mean number of sections per group = 4.4 ± 0.6 for ApoE-saline (n = 9), 4.4 ± 0.8 for ApoE-DEP (n = 7). ApoE−/− were fed Western diet for 8 weeks, whereas wild-type mice were fed normal chow. (e) Plaque size in the brachiocephalic artery correlated with the extent of pulmonary inflammation (BALF total cell count) at time of necropsy (R2 = 0.45, P = 0.0043, n = 16).
The brachiocephalic artery, which develops complex atherosclerotic plaques, was chosen for histological analysis [24]. No lesions were evident in C57/bl6 mice (saline, n = 3; DEP, n = 5). In contrast, ApoE−/− mice developed large lesions throughout the length of the brachiocephalic artery, particularly in the 400 μm proximal to the bifurcation of the aortic arch (Figure 2c). Mean plaque size was markedly increased (P = 0.017) in DEP-treated ApoE−/− mice (59±10%, n = 7) compared with controls (32±7%, n = 9; Figure 2d). This increase was seen using several different measures of lesion size (Additional file 1: Table S3). The size of atherosclerotic lesions was associated with levels of pulmonary inflammation (BALF total cell count) on the day of necropsy (P = 0.004, n = 16; Figure 2e).
Plaque composition and structure
Plaque composition was studied in detail to provide insight into the potential ability of DEP to promote plaque instability. ApoE−/− mice developed complex atherosclerotic lesions with variable proportions of smooth muscle cells, foam cells, lipid, cholesterol crystals and fibrous tissue (Figure 3). Exposure to DEP had little effect on lesion composition (n = 5-6/group; Additional file 1: Figure S2). Quantification of foam cells (MAC-2-positive), lipid cavities, smooth muscle cells and collagen (Figure 3a-d) showed no differences between saline- and DEP-treated ApoE−/− mice (n = 7-9/group; Additional file 1: Figure S2). Similarly, no differences were detected in the levels of matrix metalloproteinases (MMP-2 and MMP-9; saline n = 9, DEP n = 4; Additional file 1: Figure S3) or fibrin/ fibrinogen (Additional file 1: Figure S4) in the plaques of DEP-treated mice (n = 5-6/group for all). However, DEP-treated animals had a greater number of distinct plaques per section of lesion (2.4±0.2 plaques) compared with saline-treated controls (1.8±0.2 plaques; P = 0.022, n = 7-8, Figure 3e-g). DEP also trebled the frequency of buried fibrous layers per section of lesion (1.2±0.2 vs 0.4±0.1; P = 0.013, n = 7-8; Figure 3e-g).
Figure 3. Assessment of plaque composition and structure. Representative brachiocephalic plaque sections showing (a) lipid cavities: plaque regions showing absence of all components of UST stain, (b) inflammatory cells: MAC2 immunohistochemistry (brown), (c) collagen: picrosirius red (red stain), (d) smooth muscle: SMA immunohistochemistry (brown). Scale bar = 100 μm. (e) Example of a large atherosclerotic plaque from a saline-treated ApoE−/− mouse, showing only a single plaque per section and no buried fibrous layers. (f) Example of three buried fibrous layers in a plaque (black, green and red arrows mark each fibrous layer) taken from a DEP-treated ApoE−/− mouse. UST staining, scale bar = 100 μm. (g) DEP instillation increased the average number of distinct plaques within each cross-section of lesion and the number of buried fibrous layers per section. Mean ± S.E.M. Values obtained from analysis of serial sections at 100 μm intervals for each animal; Mean number of sections per group = 4.4 ± 0.7 for ApoE-saline (n = 8), 4.4 ± 0.8 for ApoE-DEP (n = 7), *P < 0.05, Mann–Whitney test comparing saline with DEP. All images shown are from arteries from DEP-instilled ApoE−/− mice, except panel 3e, taken from saline-treated ApoE−/− mouse.
Blood lipids, C-reactive protein and fibrinogen
Serum cholesterol concentrations were increased in ApoE−/− mice fed a Western diet (20.1±0.7 mM) compared to C57bl6 mice fed standard chow (1.3±1.5 mM; n = 16, combined saline and DEP, for both genotypes; P < 0.001, Figure 4a) whereas serum triglyceride concentrations were similar in both ApoE−/− (0.34±0.03 mM) and C57bl6 mice (0.36±0.02 mM; n = 16, combined saline and DEP, for both genotypes; Figure 4b). Both variables were unaffected by DEP instillation. Similarly, serum C-reactive protein (CRP) and plasma fibrinogen concentrations were unaffected by DEP instillation (both P > 0.05; n = 8 for all; Figure 4c,d; Additional file 1: Table S2). Total and active tissue plasminogen activator (t-PA) levels were below the limit of detection (n = 7-8 for all; Additional file 1: Table S2).
Figure 4. DEP instillation did not alter lipid or markers of systemic inflammation and coagulation within the blood. (a) Serum cholesterol (n = 10), (b) serum triglyceride (n = 10), (c) serum CRP (n = 8) and (d) plasma fibrinogen (n = 8) levels. Mean ± S.E.M. ***P < 0.001, unpaired t-test of C57bl6 vs ApoE; nsP > 0.05, Bonferroni post-hoc tests, following one-way ANOVA. ApoE−/− were fed Western diet for 8 weeks, whereas wild-type mice were fed normal chow.
Vascular function
To determine whether DEP-instillation caused endothelial dysfunction, aortic rings were isolated for functional analysis. Phenylephrine caused an identical concentration-dependent contraction in C57bl6 and ApoE−/− mice and was unaltered by DEP instillation (P > 0.24, DEP vs saline; n = 6-7; Figure 5a). Relaxations to the endothelium-dependent vasodilator, acetylcholine, or the endothelium-independent nitric oxide (NO) donor, sodium nitroprusside (SNP), were unaffected by DEP-instillation in C57bl6 and ApoE−/− mice (P > 0.29; n = 4-5 for all; Figure 5b,c). Similar responses to relaxants were seen if the arteries were pre-contracted with noradrenaline rather than phenylephrine (n = 3-7; Additional file 1: Figure S5).
Figure 5. Vascular function was unchanged by DEP instillation. (a) Contraction of aortic rings to phenylephrine, (b) endothelium-dependent relaxation to acetylcholine and (c) endothelium-independent relaxation to sodium nitroprusside. Responses in C57bl6 mice (grey lines) or ApoE−/− mice (black lines) receiving either saline (circles, broken line) or DEP (triangles, solid line) instillation. All dose response curves were performed in the same vessel from a single animal. ApoE−/− were fed Western diet for 8 weeks, whereas wild-type mice were fed normal chow. Mean ± S.E.M. (n = 6-7 for phenylephrine, n = 4-5 for acetylcholine and SNP), n.s.P > 0.05, two-way ANOVA saline vs DEP in C57bl6 mice, N.S.P > 0.05, two-way ANOVA saline vs DEP in ApoE−/− mice.
Hepatic antioxidant expression
Expression of antioxidants in liver was used to as an indicator of a response to a systemic oxidative stress. Instillation of DEP increased NF-E2-related factor-2 (Nrf2) expression in C57bl6 mice (P < 0.01; n = 5 for all; Figure 6). Exposure to DEP induced an increase in gene expression of Nrf2 (P = 0.023; n = 5 for all), NAD(P)H-quinone oxidoreductase 1 (NQO1; P = 0.034; n = 7 for C57bl6 groups, n = 5 for ApoE−/− groups) and hemeoxygenase-1 (HO-1; P = 0.027; n = 8 for all) in ApoE−/− mice (Figure 6).
Figure 6. Instillation of DEP increased expression of antioxidant genes in the liver, particularly in ApoE−/− mice. (a) NF-E2-related factor-2 (Nrf2), (b) NAD(P)H-quinone oxidoreductase-1 (NQO1) and (c) hemeoxygenase-1 (HO-1). Effect of DEP shown as fold-increase in the expression in saline-treated mice. Mean ± S.E.M. (n = 5-8), n.s.P > 0.05, *P < 0.05, ***P < 0.001, unpaired t-test comparing saline vs DEP. ApoE−/− were fed Western diet for 8 weeks, whereas wild-type mice were fed normal chow.
We have here demonstrated that, in the murine apolipoprotein E deficiency model, instillation of DEP increased lesion size, produced more lesions per vessel and generated more buried fibrous caps. The proatherosclerotic effect was concomitant with pulmonary inflammation and systemic oxidative stress. We conclude that the particulate components of DE accelerate plaque development and are associated with a more complex plaque phenotype. The processes involved may contribute to the association between exposure to urban air pollution, atherosclerosis and acute myocardial infarction in man.
The influence of DEP was assessed using a recognised model of atherosclerosis at a period of extensive plaque expansion and remodelling [25,26]. Serial sections of artery were used to provide a surrogate marker of plaque volume and a sensitive approach to quantifying changes in plaque burden. We found that four weeks of repeated DEP exposure caused an almost doubling of plaque size from 32% to 59%. The 4-week exposure to DEP was consistent with pre-clinical investigations using concentrated ambient particles (CAPs) [8,9,27-30], whole DE [11,19] and, to a lesser extent, non-carbon nanoparticles [31]. These previous studies have been performed in several different animal models (ApoE−/− mice, low density lipoprotein receptor Apo E double knockout mice and Watanabe heritable hyperlipidaemic rabbits) using either inhalation [8,10,11,19,30] or instillation [27,29,31]. The present study is the first to assess whether direct exposure to DEP increases atherosclerosis.
The marked increases in plaque size with DEP instillation are striking given the short duration of the exposure period. In the present study, long-term exposure has been emulated by administration of higher doses of particles over a shorter length of time. The instillation technique has been well validated [32-36] and the dose employed is similar to that of other studies using repeated instillations [37,38], and below those commonly used for single instillation exposures. While we cannot rule out that a proportion of the particulate administered deposits in the upper airways, studies have shown that the aspiration instillation technique to provide excellent delivery and dispersity of particulate throughout the alveoli [39,40]. The MPPD particle deposition model [41] estimates that the average 24-h instilled dose used here (assuming all DEP reaches the alveoli) is approximately 40-fold higher than the comparable alveolar deposition from a 24-h inhalation of 100 μg/m3 in man (adjusted for mice). It is important to highlight that these calculations are based on inhalation of particles with a primary particle diameter of that of diesel exhaust (~60 nm), whereas, urban levels of air pollution will contain a wide range of particulate sizes of which only a lower proportion of the larger particulates will penetrate past the tracheobronchial regions. Nevertheless, it is estimated that ~20% (6-41%; [42,43]) of the mass of PM2.5 is in the ultrafine range, and concentrations frequently reach 100 μg/m3 (a high percentage of which will originate from vehicle exhaust) for sustained periods in heavily polluted cities in both developed and developing nations. Furthermore, the airborne mass concentration of 100 μg/m3 represents a moderate/high PM levels over a 24-h period and does not take into account peaks in PM levels that regularly occur in cities, or the increased PM deposition produced by exercise such as brisk walking or cycling in urban environments. In comparison to a long-term continuous inhalation, the instillation regimen will unavoidably induce intermittent very high doses and dose-rate interspersed with long periods of non-exposure [44]. Nevertheless, the similarity of the current results with that of DE inhalation in ApoE−/− mice [10,11,19,20,45] supports the contention that the biological pathways identified using the instillation protocol are both relevant and important. While a role for the gaseous component of DE cannot be excluded, our results suggest that the particulate component of DE alone is sufficient to enhance plaque formation. It is possible that the gaseous constituents could drive other responses leading to altered plaque composition [10] which we did not see in our study. The large magnitude of the pro-atherosclerotic effect of DEP will allow us to use this model in the future to explore which attributes and constituents of DEP (e.g. transition metals, polyaromatic hydrocarbons and quinones), and urban PM in general, are responsible for these effects.
Transient exposure to road-traffic has been linked to acute coronary events [46] and several pre-clinical investigations have suggested that exposure to atmospheric pollution increases markers of lesion vulnerability [8,10,11,27,29]. Urban PM [8,27,30,47,48], whole DE [11,19,20] or the gaseous components of DE [10], increase lipid content and inflammatory cells in plaques and the underlying vessel wall. Our results indicated that lipid and inflammatory cells increased in lesions from DEP-instilled mice, but only in proportion to lesion size.
While there was no change in the proportion of individual plaque constituents, or other typical markers associated with plaque vulnerability (e.g. MMPs), the complexity of the lesions in DEP-instilled mice was increased, with more lesions per section and more buried caps within lesions compared with controls. The results are similar to that previously observed in ApoE−/− mice exposed to whole DE by inhalation: an effect that was prevented by addition of a cerium additive to the diesel fuel that decreased the number of particles in the exhaust [20]. It has been suggested that the presence of buried fibrous layers in the brachiocephalic artery are signs of plaque erosion or a previous plaque rupture [49,50]. The question as to whether buried fibrous layers are evidence of plaque rupture, or is merely a feature of the on-going development of a single plaque, remains a subject of debate [25,51]. Atherosclerotic lesions in ApoE−/− mice do not rupture catastrophically, but similar changes in human lesions would be associated with increased plaque vulnerability [48,52]. Thus, these findings would suggest that the ability of DEP to increase plaque size and complexity are likely to be an important contributor to the cardiovascular manifestations of urban air pollution in man.
Previous investigations have suggested that increased atherosclerosis following exposure to DE or CAPs may be due to increased systemic inflammation [11,27-29], vascular dysfunction [8,19,30], or increased oxidative stress [9-11,30]. In the current investigation, DEP did not increase circulating concentrations of plasma lipids or plaque lipid content, therefore, the exacerbation of lesion formation cannot be attributed to altered lipid handling.
It has been hypothesised that inhaled particles exert their cardiovascular effects indirectly through the passage of inflammatory mediators from the lung to the systemic circulation [53]. DEP caused a clear lung inflammation characterised by infiltration of macrophages and neutrophils into the airways. Interestingly, there was an association with levels of lung inflammation and the size of atherosclerotic plaques. Other studies have failed to find an association between the ability of nanoparticles to induce pulmonary inflammation and their actions on a range of cardiovascular end-points (reviewed in [54]). Concentrations of acute phase proteins (CRP) in the blood were unchanged by DEP exposure. Although we cannot exclude transient surges in cytokines in response to the particulate, or the involvement of other cytokines (e.g. tumour necrosis factor alpha, interleukins, serum amyloid A3), we have not found consistent changes in any blood cytokine at either early (2 h) or late (24 h) time-points after DE exposure in man [14,15,55]. Future studies will address these possibilities in more detail. Additionally, in the present study DEP did not change the inflammatory cell content of atherosclerotic plaques, a finding similar to that after 40 days inhalation of CAPs [9]. Overall, while inflammatory pathways are likely to contribute to the cardiovascular effects of DEP, neither pulmonary nor systemic inflammation alone can account for the atherogenic actions of DEP.
Repeated instillation of DEP was not associated evidence of vascular dysfunction. Previous investigations have reported increased [8,19] or reduced [30] vascular contractility accompanying atherosclerosis following pollutant exposure. There have also been contradictory indications of normal [30] or impaired [8] acetylcholine-mediated relaxation. However, in the latter, the magnitude of the impairment was small and followed prolonged (>10 weeks) exposure and high-fat feeding. Alternatively, vasomotor impairment may be restricted to resistance arteries [56] and not the large conductance arteries where atherogenesis takes place. Our results suggest that vascular dysfunction is not required for DEP-induced acceleration of atherosclerosis.
The hepatic up-regulation of several protective antioxidant genes in response to DEP suggests a counter-regulatory response to the systemic pro-oxidative effects of DEP. We have previously shown that the Nfr2 pathway is also upregulated in systemic tissues in response to inhalation of ultrafine urban PM in ApoE−/− mice [9] and whole diesel exhausts [45]. Interestingly, up-regulation of Nrf2 could trigger vascular proatherogenic effects as we have recently reported that systemic Nrf2 deletion inhibits rather than promotes atherosclerotic lesion formation in the aorta of ApoE null mice [57]. DEP itself can generate free radicals in solution [58], and oxidative stress is one of the most consistently proposed links between the pulmonary and systemic effects of particulate exposure [59]. Vehicle exhaust promotes lipid peroxidation in plasma lipoproteins and systemic tissues [60], consistent with recent studies where DE led to increased plasma levels of 8-isoprostanes, 12-HETEs, 13-HODEs, the development of dysfunctional prooxidative and proinflammatory high density lipoprotein (HDL) [45], and increased isoprostanes in urine [11]. Although we did not evaluate the status of lipid peroxidation in our study, we suggest that the systemic pro-oxidative effects of the particulates in DE could drive a significant portion of the proatherosclerotic actions of urban PM. Interestingly, antioxidant upregulation was especially notable in ApoE−/− mice compared to wild-type mice. This observation adds to the growing evidence that animals/individuals with pre-existing vascular disease (or their risk factors), or diseases with associated cardiovascular complications (e.g. diabetes), may be particularly susceptible to the effects of air pollution [1,7,61,62].
Exposure of the particulate constituents of diesel exhaust increases both the size and complexity of atherosclerotic plaques in vivo, and may therefore increase the susceptibility of plaques to rupture. The increased atherogenesis was not directly related to raised levels of systemic inflammation, changes in lipid handling or endothelial dysfunction, but was associated with pulmonary inflammation and systemic changes in antioxidant expression. Similar changes in man would be predicted to increase plaque vulnerability and rupture, helping to explain the associations between urban air pollution and acute myocardial infarction. These findings provide support for the implementation of strategies to remove vehicle-derived particulate emissions as a means to reduce the detrimental health effects of air pollution.
Detailed Methods in Additional file 1.
Animals, instillations and necropsy
All experiments were performed according to the Animals (Scientific Procedures) Act 1986 (UK Home Office). Adult male ApoE−/− mice (N = 20) and the background strain (C57bl6 mice; N = 16) were purchased from Charles River (Margate, UK) aged 8–9 wks (20–25 g). C57bl6 mice were fed 8 weeks standard chow, whereas ApoE−/− mice received a high fat 'Western Diet' (21% fat; Research Diets, New Brunswick, USA). This approach was used to allow a direct comparison of the effect of DEP on animals with no atherosclerosis with that of animals exhibiting large 'complex' atherosclerotic plaques, respectively.
For the final four weeks of feeding, mice underwent twice weekly instillation (oropharnyngeal aspiration) of 35 μg DEP (35 μL of 1 mg/mL; National Institute of Standards and Technology (NIST); SRM-2975; Gaithersburg, U.S.A.), to represent an average daily dose of 10 μg/mouse (see Discussion). This reference material is a commonly used source of DEP, allowing comparability with other researchers, and we have previously shown it has the capacity to generate superoxide free radicals in vitro, stimulate cultured macrophages and can directly impair arterial function [58,63]. The mean particle size of DEP in saline buffer prior to administration was 257 ± 46 nm (n = 6; dynamic light scattering, Brookhaven PS90 Particle Size Analyser; data not shown). Animals were sacrificed 3–4 days after their last instillation and blood (0.6-1 mL) withdrawn from the heart. Lungs were lavaged with sterile saline (3x 0.8 mL) to collect bronchoalveolar lavage fluid (BALF), and the carotid arteries, brachiocephalic artery, aortic arch, thoracic aorta and liver biopsies collected.
Measures of pulmonary inflammation
BALF samples were centrifuged (180 g, 5 min, 4°C) and the cell pellets combined, resuspended, stained with Diff-Quik (Raymond A Lamb, London, UK) and prepared into cytocentrifuge smears for differential cell counts. The inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-α) and monocyte chemotactic protein-1 (MCP-1; CCL2/JE) were measured in the primary cell-free BALF by enzyme-linked immunosorbent assay (ELISA; R&D Duoset Systems; Patricell Ltd, Nottingham, UK).
Assessment of atherosclerosis
The aortic arch and descending thoracic aorta were randomly selected from half of the mice from each treatment group. The artery was cut longitudinally and lipid-rich atherosclerotic plaques were stained en-face using Sudan IV.
Brachiocephalic arteries were fixed in formalin and histological sections were taken in triplicate at 100 μm intervals, beginning at the first section of artery with a fully intact media. Sections were stained with United States Trichrome (UST). The cross-sectional area of the plaque was measured and standardised to the medial area. The medial wall was chosen for standardisation rather than luminal area, as vessels could not be perfusion-fixed in situ, as they were also required for assessment of vascular function by myography (see below). A single mean value of atherosclerotic burden for each animal was calculated from the plaque size from each complete serial section throughout the brachiocephalic artery.
Plaque composition was assessed by both semi-quantitative scoring of fibrous matrix, foam cell and lipid content, and thickness of plaque cap, in UST-stained sections. Quantitative measurement of plaque constituents was performed using histology (collagen, fibrin) and immunohistochemistry (lipids, macrophages, smooth muscle cells, fibrinogen, metalloproteinases).
Plaque structure was assessed by counting distinct adjoining, or overlying, plaques within each section [20]. Additionally, the number of buried fibrous layers (defined as 'a length of fibrous/cellular matter that completely bisects a lipid-rich regions of two overlying plaque sections [24]) was counted in each section throughout the artery and a mean value obtained. Samples were randomised before assessment and all scores independently verified by a second blinded assessor. See Additional file 1 for full details on methods used to assess atherosclerosis.
Measurement of blood lipids, C-reactive protein and fibrinogen
Non-citrated blood was allowed to clot on ice (>2 hours, 4°C), centrifuged (10,000 rpm, 10 min) and used for colormetric measurement of cholesterol and triglycerides (Microgenics, St Albans, UK), and C-reactive protein (CRP) by ELISA (Innovative Research kits; Patricell, Nottingham, UK). Citrated blood was centrifuged (3,000 g, 15 min) to collect platelet-poor plasma for measurement of fibrinogen (Innovative Research ELISA kits; Patricell, UK).
Vascular function
Segments (1–2 mm length) of the distal portion of the thoracic aorta were mounted in a multi-myograph system (610M; Danish Myo Technology, Aarhus, Denmark). Concentration-response curves were generated for phenylephrine (PE), the endothelium-dependent vasodilator, acetylcholine (ACh), and the endothelium-independent nitric oxide (NO) donor sodium nitroprusside (SNP). Only a single segment was used from each animal, and all dose response curves were performed in the same vessel. Drugs were washed out for >30 min, and prior dose response curves did not affect the responses to subsequent drugs.
Expression of hepatic antioxidants
The expression of tissue mRNA for hemeoxygenase-1 (HO-1), NAD(P)H-quinone oxidoreductase 1 (NQO1), catalase, and NF-E2-related factor-2 (Nrf2) were measured by quantitative real-time polymerase chain reaction [9].
Statistical analysis
Statistical comparisons were made using unpaired Student's t-test or analysis of variance (ANOVA) with Bonferroni post-hoc tests where appropriate. Kruskal-Wallis or Mann–Whitney tests were used for non-parametric or ordinal level data (1–5 scoring of plaque components). Two-sided P < 0.05 was taken as statistically significant.
ACh: Acetylcholine; ANOVA: Analysis of variance; ApoE−/−: Apolipoprotein E knockout; BALF: Bronchoalveolar lavage fluid; CAPs: Concentrated ambient particles; CRP: C-reactive protein; DE: Diesel exhaust; DEP: Diesel exhaust particulate; ELISA: Enzyme-linked immunosorbent assay; HO-1: Hemeoxygenase-1; IL-6: Interleukin-6; MCP-1: Monocyte chemotactic protein-1; MMP: Matrix metalloproteinase; NIST: National Institute of Standards and Technology; NQO1: NAD(P)H-quinone oxidoreductase 1; Nrf2: NF-E2-related factor-2; NO: Nitric oxide; PE: Phenylephrine; PM: Particulate matter; S.E.M.: Standard error of the mean; SMA: Smooth muscle actin; SNP: Sodium nitroprusside; TNF-α: Tumour necrosis factor alpha; UST: United States Trichrome.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MRM designed the study, carried out the experimental work, carried out the statistical analysis and drafted the manuscript. SGM carried out the experimental work, and assisted with the data and image analysis. RD assisted with the particle instillations and pulmonary assays, and contributed to the drafting of the manuscript. AOL carried out the antioxidant expression work and analysis. JAA contributed to the interpretation of the data and helped draft the manuscript. CAS contributed to the drafting of the manuscript. NLM contributed to the drafting of the manuscript. KD participated in the design and interpretation of the study, and helped to draft the manuscript. DEN participated in the design and interpretation of the study, and helped to draft the manuscript. PWFH participated in the design and interpretation of the study, and helped to draft the manuscript. All authors have read and approved the final version of the manuscript.
We acknowledge the support of the British Heart Foundation Centre of Research Excellence (CoRE) award.
Sources of funding
This work was funded by British Heart Foundation Programme (RG/10/009) and Project (PG/10/042/28388) Grants. AOL and JAA are funded by an Outstanding New Investigator in the Environmental Sciences Award from the National Institute of Environmental Health Sciences (RO1 ES016959). DEN is funded by the British Heart Foundation (CH/09/002).
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The Brass Verdictby Michael Connelly
Staff Pick
Thriller writer extraordinaire, Connelly combines his two most indelible series characters for one white-knuckled novel. The Brass Verdict finds Connelly's storytelling bravado in first-rate form.
Synopses & Reviews
Publisher Comments:
Things are finally looking up for defense attorney Mickey Haller. After two years of wrong turns, Haller is back in the courtroom. When Hollywood lawyer Jerry Vincent is murdered, Haller inherits his biggest case yet: the defense of Walter Elliott, a prominent studio executive accused of murdering his wife and her lover. But as Haller prepares for the case that could launch him into the big time, he learns that Vincent's killer may be coming for him next.
Enter Harry Bosch. Determined to find Vincent's killer, he is not opposed to using Haller as bait. But as danger mounts and the stakes rise, these two loners realize their only choice is to work together.
Bringing together Michael Connelly's two most popular characters, The Brass Verdict is sure to be his biggest book yet.
"Bestseller Connelly delivers one of his most intricate plots to date in his 20th book, a beautifully executed crime thriller. When L.A. lawyer Mickey Haller, last seen in The Lincoln Lawyer (2005), inherits the practice and caseload of a fellow defense attorney, Jerry Vincent, who's been murdered, the high-profile double-homicide case against famed Hollywood producer Walter Elliot, accused of shooting his wife and her alleged lover, takes top priority. As Haller scrambles to build a defense, he butts heads with LAPD Det. Harry Bosch, the stalwart hero of Connelly's long-running series (The Black Echo, etc.), who's working Vincent's murder. When Haller realizes that the Elliot affair is bigger than simply a jealous husband killing his cheating wife, he and Bosch grudgingly agree to work together to solve what could be the biggest case in both their careers. Bosch might have met his match in the wily Haller, and readers will delight in their sparring. 10-city author tour. (Oct.)" Publishers Weekly (Starred Review) (Copyright Reed Business Information, Inc.)
Graham Greene liked to distinguish between his serious novels and those he called his "entertainments," though given the complexity of the man and his work it wasn't always easy for readers to draw the distinction. Probably Michael Connelly would be the last to compare himself with Greene, but he, too, writes at differing levels of seriousness. If at first encounter he seems primarily an exceptionally... Washington Post Book Review (read the entire Washington Post review) accomplished writer of crime novels, at closer examination he is also a mordant and knowing chronicler of the world in which crime takes place, i.e., our world. Three years ago, within the space of only a few months, Connelly published two novels notable for the serious business underlying the entertainment. The first, "The Closers," published in May 2005, found his noted Los Angeles police detective Harry Bosch trying to solve a "cold case" and thus trying to bring justice to victims on whom the law has turned its back. Then, in October of the same year, he published "The Lincoln Lawyer," his first novel told from a lawyer's point of view, about an ambulance chaser named Mickey Haller, who, in the course of pursuing a lucrative case, finds himself seeking justice for a man he believes he failed to represent fairly when his case was being heard. Now, in "The Brass Verdict," Connelly brings Bosch and Haller together for the first time. Though the novel has some serious things to say about the workings, and occasional failures, of the jury system, it is primarily an entertainment, and more than welcome purely as such. It's narrated by Mickey, a criminal-defense lawyer who is just coming off a year's self-imposed sabbatical — he'd been shot in the gut and then had become addicted to painkillers in various forms — and plans to ease slowly back into his practice. He's no K Street lawyer, as he tells a young man he takes on as his driver: "I haven't had an office since I left the Public Defenders Office twelve years ago. My car is my office. I've got two other Lincolns just like this one. I keep them in rotation. Each one's got a printer, a fax and I've got a wireless card in my computer. Anything I have to do in an office I can do back here while I'm on the road to the next place. There are more than forty courthouses spread across L.A. County. Being mobile is the best way to do business." Mickey's hopes of easing back in are quickly deep-sixed when a lawyer he's known slightly, Jerry Vincent, is found murdered in his car. He and Vincent had worked the occasional case together but hadn't been close. Still, Mickey is called into the office of the chief judge of the Los Angeles Superior Court and informed that Vincent "filed a motion with the court ten years ago that allowed for the transfer of his practice to you should he become incapacitated or deceased." Most of the 31 active cases in Vincent's file are minor stuff, but one is huge: "Walter Elliot ... was the chairman/owner of Archway Pictures and a very powerful man in Hollywood. He had been charged with murdering his wife and her lover in a fit of rage after discovering them together in a Malibu beach house. The case had all sorts of connections to sex and celebrity and was drawing wide media attention. It had been a publicity machine for Vincent and now it would go up for grabs." Obviously, Mickey would love to have the case, but first he has to persuade Elliot — who most emphatically is not a nice man — to take him on. Once he does, Mickey is off and running. One of the people he runs into is Bosch, who is back on the active force and investigating Vincent's murder. Bosch wants access to Vincent's past and present case files because he believes the murderer was a client who'd crossed swords with him, but Mickey refuses on the grounds that to release the information would violate lawyer/client confidentiality. Bosch has 33 years on the force and is "a man on a mission" to seek justice wherever he can find it. He's a tough cop and an honest one, and there are angry sparks between him and Mickey from the moment they first meet. Mickey would just as soon have nothing to do with Harry — Connelly's faithful readers don't have to be told that his real name is Hieronymus, "like the painter" — but there's a problem: The deeper both men dig into Vincent's past, the more suspicions are raised. Vincent had received a lot of money, presumably from Elliot, and much of it — $100,000, to be precise — had disappeared. Mickey says Vincent claimed that "he needed the money to buy a boat and that if he made the deal in cash, he would get the best deal and save a lot of money," to which Harry replies: "There is no boat. The story was a lie." Vincent "bought something," Harry says, "and your client Walter Elliot probably knows what it was" — something, for starters, like a potential juror. "You should take it as a warning, Counselor," Harry continues. When Mickey scoffs, he says, "His lawyer got killed, not him. Think about it. And remember, that little trickle on the back of your neck and running down your spine? That's the feeling you get when you know you have to look over your shoulder. When you know you're in danger." Mickey doesn't want to be scared, but as things unfold it appears he doesn't have much choice. One of those things is, how much — if at all — can he trust his client? Walter Elliot loudly and frequently proclaims his innocence and insists he wants a speedy trial to clear his name as rapidly as possibly, but though Mickey wants to believe him, experience teaches him to be cautious: "Over the years I had represented and been in the company of a couple dozen killers. The one rule is that there are no rules. They come in all sizes and shapes, rich and poor, humble and arrogant, regretful and cold to the bone. The percentages told me that it was most likely Elliot was a killer. That he had calmly dispatched his wife and her lover and arrogantly thought he could and would get away with it. But there was nothing about him on first meeting that told me one way or the other for sure. And that's the way it always was." If you're beginning to get a whiff of the O.J. Simpson case, well, that's pretty obviously how Connelly planned it. Not merely is the accused murderer a Los Angeles celebrity and the victims his wife and her lover, but Connelly drops in the occasional teasing reference as well. When Elliot blusters in court that "the sooner Mr. Haller gets to prove my innocence to the world, the better," Mickey dismisses it as "O.J. 101," and when another lawyer offers to pitch in and help, Mickey tells him: "He wants only one lawyer at the table. ... He said no dream team." But all of that is just a little juice on the side; the main story is strictly Connelly's. The essence of it is this, as Mickey puts it: "I was defending a man I believed was innocent of the murders he was charged with but complicit in the reason they had occurred. I had a sleeper on the jury whose placement was directly related to the murder of my predecessor. And I had a detective watching over me whom I was holding back on and couldn't be sure was considering my safety ahead of his own desire to break open the case." Yet how does Mickey feel? "I felt like a guy flipping a three-hundred-pound sled in midair. It might not be a sport but it was dangerous as hell and it did what I hadn't been able to do in more than a year's time. It shook off the rust and put the charge back in my blood." Mickey is pumped, and, take my word for it, you will be too. Even though the way it ends is just a wee bit contrived, it's still a terrific ride. Jonathan Yardley's e-mail address is yardleyj(at symbol)washpost.com. Reviewed by Jonathan Yardley, Washington Post Book World (Copyright 2006 Washington Post Book World Service/Washington Post Writers Group)
(hide most of this review)
"Connelly is justly celebrated for his characters and his ability to create mood from the sights and sounds of L.A., but he's also a terrific plotter, and that skill is in high relief here. Essential for fans; a great read for anybody." Booklist (Starred Review)
"Connelly is firing on all cylinders in this epic page-turner. The intriguing story line, the chance to view Bosch from another perspective, and Haller's reappearance as a main character add up to a fantastic read. One of the best thrillers of the year..." Library Journal (Starred Review)
"The answer to every Connelly fan's dream....Even if the case is less than baffling, Connelly brings his two sleuths together in a way that honors them both." Kirkus Reviews
"The Brass Verdict has the sneaky metabolism of any Connelly book....Like Harry Bosch's mojo, Mickey Haller's is liable to work well for a long time." Janet Maslin, New York Times
"Haller is a mixture of light and dark, a flesh-and-blood guy who, as with Harry Bosch, we'll look forward to meeting again." Los Angeles Times
"Connelly's skills at melding plot, character and scenery into a cohesive unit shine in this book. The Brass Verdict is gold." South Florida Sun-Sentinel
"Connelly writes detective and legal thrillers that elevate the genre with authentic characters, realistic dialogue and knowing details." Oregonian
"Connelly builds to some breathtaking twists before all comes to a close. And a more perfect end to the maze he has drawn is difficult to imagine." Denver Post
At last, Mickey Haller, hero of The Lincoln Lawyer, Connelly's bestselling hardcover yet, meets the legendary LAPD Detective Harry Bosch. Mickey and Harry are flip-sides of the same coin, one for the prosecution and one for the defense. Each must work with the other before someone — namely Mickey — gets killed.
About the Author
Michael Connelly, a #1 New York Times bestselling novelist and a former journalist, has won numerous crime fiction prizes. He lives in Florida.
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Here is a source for 3M propore it is a king size materus cover made out of 3M propore: http://www.improvementsplanner.com/home-improvements-mattress-cover-king-10002814.html Is this the same material that is used in waterproof rain jackets like Rainshield O2 rainwear? Is there a better sources for a microporous polpropylene laminate? The Jackets made from this are very light does any one know the actual weight of a square yard of 3M propor? Has any one made anything out of this? I know that are not as durable as other water proof breathable fabrics but might this be durable enough for the top of a bivy? Thanks for the info.
I've used propore from one of the weathershield jacks r better things for a bivy top. it worked out well, deffinitly waterproof, i ended up using the wrong side out, but no difference. it is deffinitly a more fragile option and i wouldn't recomend a really tight stitch so you dont weaken it to much. i've slept with it for some 8 nights without problems, there were some condensation sometimes at the foot, but i think it's because i made the footend with silnylon, which was a mistake :xgood luck,mike!
I have 5 yards of the Yellow Propore and it weighs 2.37 ounces per sq yard. What I have came from 3M when they were still making it.
Edited by bfornshell on 02/01/2007 22:37:29 MST.
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|When flying machines were all the rage
A half-century and more ago, Delaware County was dotted with airfields, and planes might swoop along a street.
Feb. 9, 1992
Delaware County has had a long-standing romance with flying machines.
Even before Charles Lindbergh soloed the Atlantic in 1927, and long before Silvio "Babe" Dignazio buzzed the Media courthouse during World War II - in a B-24 bomber, no less - planes and airfields were part of the local scene.
The county seat, for example, had a licensed landing field as far back as 1924. It is said to have been the setting for one of the nation's earliest air shows and numerous visits by barnstorming pilots.
Other fields included Buckman Airport in Chester's West End and Benedict Airport in rural Booths Corner, Bethel Township, which boasted 20 hangars in a building near Foulk and Kirk Roads.
All three were out of business by the 1950s, sold for residential development or, in the case of Benedict, an oil tank farm. Smaller back-yard runways that dotted the countryside from the Linville farm in Middletown to Bishop Road in Springfield have also disappeared.
The oldest landing "field" in the county isn't on land, and is still in business. It's the Philadelphia Seaplane Base in Essington, started in 1915 by Col. Robert Glendinning, a wealthy Philadelphian who owned a Curtiss Flying Boat.
Today, the base is owned and operated by Bob Mills, 72, whose father, Frank, was Glendinning's chief pilot, instructor and mechanic before he purchased the facility, which includes 10 acres of land, in 1936. Frank Mills died in 1940.
The Media Flying Field was just outside the borough limits in Nether Providence Township. Leased and operated by the Media Business Men's Association, it was on 50 acres on the south side of Baltimore Pike between Beatty and Turner Roads.
Media lawyer Hugh Bonner, 86, recalled recently that the field consisted of no more than a 2,500-foot dirt landing strip, a small building and a windsock on a pole. But it was "home" to a number of flying enthusiasts, instructors and a glider club that he helped to organize.
The inspiration for the field, according to records in the Media Historic Archives, is credited to C. Frank Williamson, president of the businessmen's group in 1924, and T. Elwood Allison, a member who operated a local drugstore chain.
Williamson apparently saw the promotional value of having airplanes, which then attracted great public interest, take off and land in the vicinity of the retail district. Allison owned the field and lived in a castle-like mansion that stood nearby on Plush Mill Road.
Dick Wolff, 74, of Lima, who piloted bombers during World War II, remembers the day in 1947 that he flew Santa Claus to the Media field in his two-passenger Aeronca. Children who had gathered to greet Santa became so excited, he recalled, that they almost swamped the plane.
Even greater excitement gripped Media residents one summer afternoon in 1944 when, as local businessman I. Frank Lees recounted it, "the most god-awful roar I ever heard chased workers out of their stores and offices."
"Babe" Dignazio, owner of the Towne House Restaurant in Media and an Air Force captain during the war, flew a huge four-engine B-24 Liberator down State Street en route to an airfield in Washington.
Dignazio, now 73, said the escapade brought him before a court-martial, which ultimately cleared him. He said he was transporting the bomber from England to the United States when he "flew a little off course."
Buckman Airport was laid out in the early 1930s on the west side of Highland Avenue in Chester, overlapping what is now Interstate 95. It was built and operated by Norman Smith and was designated as an airport because, unlike Media, it had a bona fide hangar and a maintenance facility.
Alfred Graeber of Springfield, whose father owned a Piper Cub J-3, recalled visits to Buckman, where the county's earliest airmail pickups took place. Graeber said children would gather just to witness the novel system.
"Post office people strung the mall bag on a special line between two poles, and the airmail plane would swoop down, hook the line and zoom away with the bag bouncing in the air," he said.
Graeber said his father, Alfred Sr., maintained a private landing strip behind the family residence at 228 S. Bishop Rd,, Springfield, until 1941, when the outbreak of war forced its closing.
"The government required all licensed airstrips to have 24-hour security, and my father couldn't afford the cost, so he closed down and moved his plane to Buckman," Graeber explained.
Paul Nelson, 75, a retired pilot from West Chester, said he helped build Buckman's hangar using materials reclaimed from a trolley barn at Fifth and Penn Streets in Chester.
"I was pretty young then, and I did it for flying time rather than money," he said. He recalled that the airmail service was operated by All-American Aviation, a company founded by Wilmington flyer Richard Dupont.
Wolff, who operates Red Maple Nurseries in Lima, flew 28 bomber missions over Germany during World War II. He was the first president of the Delaware County Aviation Association in 1946, and for a time leased Benedict Airport with a partner.
"We made money on airplane rides, flying instruction, plane rentals, gas and maintenance, and did a booming business on weekends," he recalled. "The war created a tremendous interest in flying, and lots of veterans used their GI Bill to take lessons."
Wolff said rides costs $6 per person and lessons were $12 per hour. In those days, it took a minimum of 35 hours to obtain a flying license, he noted.
Nelson remembered that the Army captured a German helicopter during the war and shipped it to Benedict because the field was so remote. "It was a big attraction, watching them test it, because it was supposed to be secret," he said.
Part of the History of Delaware County Website


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Indoor Bonsai Plants
Indoor Bonsai Plants or simply "bonsai" basically made known by Japanese people. Bonsai plants were basically trained to by japanese gardeners to shape and form their leaves, trunks and branches.
There are many types of bonsai plants with benefits. Trees specially tropical trees are the trees that are usually trained to become mini-trees. Japanese people train these trees by cutting a few branches and leaves and placing garden wires around their branches and trunks to form a more attractive mini-tree suitable inside a room or house.
Taking care of these beautiful indoor bonsai plants also requires garden skills so that the bonsai will be able to sustain its life. Read the tips on how to take care of bonsai plants. Read some tips on how to grow indoor bonsai plants out bonsai tree seeds and where you can buy them. Read some bonsai tree seeds guidelines. Also find out more about automatic plant watering systems which is suitable for use at home. Automatic plant watering systems have won numerous awards because of its benefits. See how automatic plant watering systems work.
Here are sample indoor bonsai plants below. Both sample indoor bonsai plants have their own characteristics and special features.
This Brussel's Jade Bonsai is a plant suitable for gardeners who are newbies when it comes to taking care of bonsai plants. The adaptability nature of this indoor bonsai plant is astonishing. It is flexible enough to live in various types of climates.
This indoor bonsai plant is an aromatic and herbal type of bonsai plant. This bonsai plant is one of the most beautiful bonsai plant with countless special features which attracts most bonsai buyers. Its leaves can be use for herbal purposes and edible.
One of the other plants that can be placed indoors and might replace the expensive Indoor Bonsai Plants are Indoor Bamboo Plants. Bamboo symbolizes luck in Ancient Chinese and one of the few plants that are easy to manage at home. They also come in various shapes and designs with ribbons and can also be braided. Find out the numerous benefits of having an indoor bamboo plants at home.


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Adjudication Power of Attorney Form
Tuesday, April 24, 2012
Registered owner authorizing someone to act on his/her behalf for ticket issues.
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The "B" Stands For "Bae"
Written by Moksha August
Monday, July 7th, 2014 at 10:31am
You see that big black "B" embroidered onto the breast of this perfectly gray BWGH sweater? Yeah man, that stands for "Bae," as in, you should buy this shit before anything else because the Internet told you to. It's also


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About Us
Named by Playboy as one of the "coolest stores" in all of America, Big Fun Toy Store provides a shopping experience that is second to none. Learn more about our history.Read more »
Social Networking
We hear all this social networking stuff is kind of "hip" these days. So we've made it easy to follow us on Facebook or Twitter. You know if you'd like too.Read more »
Each and every second there is a lot of "neat" stuff on the intertubes you might miss. Well we post a lot of that stuff on our blog so you don't have to search for it.Read more »


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|Meet the Candidates
|Wednesday, 12 September 2007 07:01
A "round robin" formatted Meet the Candidates forum will be held tomorrow night at the Watauga County Courthouse.Candidates for Boone Mayor and Boone Town Council will be present to discuss the issues- and answer questions provided by community citizens. If Appalachian State University's Monday forum was any indication, it should be an interesting evening. At Monday's forum, so called "progressive candidates" like incumbent Mayor Loretta Clawson, were on the offensive- responding to radio and print ads submitted by new Political Action Committee, Citizens for Change. Monday's table turned into a battle between the Progressives and the PAC- setting the stage for what should be a heated forum tomorrow night.
Can't make it to the courthouse? We'll be broadcasting the program live from 6 to 7:30 p.m. on WATA and WXIT.
The forum is organized by the Boone Area Chamber of Commerce.The municipal election is October 9.
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Image tracing
In computer graphics, image tracing or vectorization is the conversion of raster graphics into vector graphics.
- In computer-aided design (CAD) drawings (blueprints etc.) are scanned, vectorized and written as CAD files in a process called paper-to-CAD conversion or drawing conversion.
- In geographic information systems (GIS) satellite or aerial images are vectorized to create maps.
- In graphic design and photography, graphics can be vectorized for easier usage and resizing.
- Vectorization is often the first step in OCR solutions for handwritten text or signatures.
Signature of Christopher Columbus as JPEG image (1,308 × 481 pixel), 63 kB
Vectorized two-color variant of the signature of Christopher Columbus, 19 kB
Unlike the opposite process, rasterization, vectorization is not well defined, meaning there is no single "correct" method. Many different algorithms exist, and each gives different results, as vector representations are more abstract than pixels. The following tracing examples of a hard to vectorize photo illustrate these limits.
A photograph in JPEG format, 25 KB
Same photograph vectorized with AutoTrace in the Delineate GUI, 677 KB
Same photograph vectorized with Scan2CAD, 340 KB
Vectorization algorithms[edit]
Classic edge detection methods are for example Roberts Cross, Canny edge detector and Sobel.
See also[edit]
- Image scanner
- Digitizing
- CAD data exchange
- Comparison of raster to vector conversion software
- Optical character recognition
- Feature detection (computer vision)
|This computer graphics–related article is a stub. You can help Wikipedia by expanding it.


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price fixing
Price fixing represents an attempt by suppliers within a market to control supply and fix price at a level close to the level we would expect from a monopoly.
In recent years we have seen the effective collapse of several high profile price-fixing agreements. Good examples to draw on would include the following:
The OPEC oil cartel (this is still in existence but there have been frequent tensions within the cartel - particularly when oil prices have been heading lower and leading oil producers have seen a slump in their oil export revenues and tax receipts)
The Net Book Agreement (this cartel between publishers and retailers ended in 1995)
The Coffee Export Cartel (this export-retention agreement between members of the Association of Coffee producers collapsed in April 2001 - leading in part to a further collapse in world coffee prices throughout most of 2001)
The cartel in 'Over the counter' pharmaceutical products (This ended May 2001 and was given much media coverage at the time as it was the last legal price-fixing agreement in the UK economy)
The End of Price Fixing in Over-The-Counter Pharmaceuticals
Britain's last bit of government-sanctioned price-fixing was ruled illegal on May 15th, 2001. The Restrictive Practices Court decided that makers of over-the-counter remedies could no longer set minimum retail prices for their products. The Office of Fair Trading (OFT) brought the suit because of big gaps between prices for branded and unbranded drugs. In countries without price-fixing, such as America, prices for equivalent products are lower. Supermarkets such as Asda, Sainsbury and Tesco announced responded immediately by cutting prices by as much as 50% for popular medicines. Small pharmacists argued that the end of the cartel was potentially a disaster for them. They cannot compete on price with the supermarkets that exploit economies of size to negotiate big discounts from the manufacturers. The pharmacists say that consumers will suffer too, because those who do not live within easy reach of a big supermarket will find it hard to buy medicines. Small pharmacies may have to change to survive. Even if small pharmacies do disappear, that matters less to consumers than it might have a few years ago.
Price Fixing
To fix prices, the producers in the market must be able to exert control over supply. In the diagram above a producer cartel is assumed to fix the cartel price at the joint profit maximising output Qm and price Pm. This price is agreed among the members of the cartel and market supply is allocated on the basis of a quota system. Although the cartel as a whole is maximising profits, the individual firm's output quota is unlikely to be at their profit maximising point. This is shown in the left hand side of the diagram above. For this firm, expanding output and selling at a price that undercuts the cartel price can achieve extra profits. Unfortunately if one firm does this, it is in each firm's interests to do the same.
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The 3nd Global Home DIY Summit 2015
Approximately 900 suppliers - manufacturers, wholesalers
and importers - are arranged according to twelve main product
categories. Also included are their postal addresses, telephone
and fax numbers, plus email and Internet addresses.
This reference work also contains the addresses of associations, institutions,
service companies and fair organisers, etc..
The product categories specified:
The information from the PET Einkaufsführer
/ PET Buyers' Guide can now also be downloaded
on the Internet at PET-global.com. The companies listed
can be searched for and selected according to different
criteria: by product category, product, company name or
Published once each year.


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Our husband's don't really have a clue. My husband came home one day and asked what all I had done because even though I had cleaned, there were toys every where. The next day, I did absolutely nothing, didn't lift a finger on anything! He figured out really quick what I did in a days time! Of course, it was a pain getting all of it done after letting it sit for the whole day but it was well worth him saying he would never question me again!
Also, there were 2 years in a row that I had to go home for a funeral. I knew that both were dieing so I had it all planned out for him. I made a list for the older 2 that he was staying home with as to who was putting them on the bus, him or my friend, who was feeding them when, a menu as to what he could cook, if I hadn't frozen something for him, etc. I took the baby with me (who is now 3)! He had it made! Now, he always likes to tell me that he can do because he did it two separate times. Next, there won't be a schedule and lets see if he gets is all figured out!
hi! can i join you? i am a sahm of 16 month old twin boys, alexander and jonathan. my mother comes over every day to help me (and to keep me sane) and 4 days a week, we have my 19 month old nephew. all three boys keep us very busy, since they are getting into everything, walking, running, dancing and unfortunately my nephew is bullying.
i am also going to photography school. it's nice because it's a distance learning school, so i actually don't have to go anywhere.
my husband is in a band, so once a week, he has band practice and once a week or so, he has a show. i am with the boys when he's away, which used to be hard when they were really little because one was extremely colicky. i love it now, we get to play together and have our quiet time before bed.
thanks for letting me join in.
I don't have my little one in any activities right now outside of the home, so I don't go out much except for errands to the grocery store or drug store. I don't know any other SAHMs in my neighborhood so I don't interact much either so it can get a little lonely at times.
I don't know if this would even interest you, but there's a Mom's group called MOPS (Mothers of Preschoolers) and it's international so there are groups all over. It is a Christian based group but not specific to any one religion. I go to meetings at a Lutheran school and I'm Catholic . . . it doesn't matter what religion you are. It's mostly a support group for mom's and it's fantastic. You meet twice a month for the most part and you bring your kids along - they've got on-site child care so while you have your 2 hour meeting, the kids are playing, eating snack, etc . . . with other kids their age. In the meetings, the moms eat breakfast together, chat, share ideas, do crafts sometimes . . . it really is a great network of other mom's so you can all commisserate together. It's not just for SAHM's, either, as I know the one I go to also has an evening meeting for working mom's. It's just a great way to meet other mom's, get some time just for you to unwind and relax. Meetings cover all sorts of topics . . . our group has had speakers on topics like digital photography and kids, fashion, organization of the home, and we even had a spa day where people were there doing paraffin wax hand dips, massages, make-up consultations, etc . . . AND, the best part is that it isn't expensive. You only pay $75 for the YEAR (from Sept. through May) and that helps cover things like child care costs and dues to the international group. And even if that's a bit much, I know that they have scholarships because they don't want any mom to feel like they don't have a place.
Just a thought . . . I've gotten so much out of my group and thought someone else might enjoy it, too. You're eligible up until your kids are kindergarteners so as long as you've got a preschooler at home, you can attend. If you want to look into it, you can go to www.MOPS.org and just click on "join a group" to find one in your area.
And no, I'm not on their "recruitment committee" I just really appreciate that there's a group out there that specifically caters to mom's and totally "gets" how hard our jobs are.
It's not as if we could just pick up and go on vacation with our girlfriends whenever we want, but just for fun, imagine what it would be like for our husbands if we were gone for a week. What if we didn't prepare things and make lists like navywifetrat did? What if they were on their own, like we are every day? What would the house look like? What would the children look like? How many pizza boxes would there be in the garbage can when we returned? I'm not complaining - I have a wonderful, supportive husband. I'm just saying if he could be in my shoes for at least a few days, he would know that we are not the ladies of leisure people think we are.
You can always tell when our church has a ladies weekend retreat by looking at the little ones that come to the nursery on that Sunday morning. The little girls don't have their hair as pretty as usual, some of the clothes don't match. Some kids are missing socks or shoes. It's pretty funny. Their daddies do their best, but it's not quite the same as when Mom's home, is it?
A friend of mine told me that when she has gone away for a weekend the house was great when she came home. The husband made it look like everything went well and she was impressed. Then the kids opened their little mouths and told the mom how things really went! I can see my husband doing the same thing! Of coure, he would probably bribe my kids because they are older not to tell! But it would still come out! My kids would love it because they would love to eat out more, order pizza, etc! They don't appreciate my home cooked meals! Maybe one day they will!
My husband is pretty good when I am not home. I had to leave to Puerto Rico for my brother's wedding and left my husband home with the youngest (3 yrs old).
When I came back home the house was immaculate! he is always like that. He is very supportive of me staying home and is actually insisting that I stay longer that planned.
My hubby is pretty good, too. He can't match clothes worth a darn but since we have two little boys, it's basically navy, gray, and denim so there's not much room for error. He can handle the kids every bit as well as I can. He doesn't do much cooking so they'd eat out more but other than that he'd do just fine. He does know what's involved with staying home all day, day in and day out and he's the one looking for me to get relief. I think that because he works so much and is gone a lot, he feels guilty and makes the most of every minute that he is home.
husbands always say they can handle the house even when they cant I went to visit my family for 16 days I took both of our children with me I handled all the bills online and gave him list for things I couldnt do online and when I would talk to him he would say everything is good no problems at all well the night i get home the house looks ok on the surface until I open the dishwasher and there sat all the dishes I had loaded into the dishwasher 16 days before talk about gross I mean how hard is it to turn on the dishwashwer I also found out from his coworkers that he ate nothing but frozen burrittos and fast food for 2 weeks I just laughed men do not know how much we do for them and for our houses and kids and everyone else.
I have a question for you SAHMs. Do you guys wear makeup every day?
Maybe I should have started a new thread?
I do not wear make-up unless I have somewhere to go and the grocery store does not count but if were going to a play group or a little mini field trip or a teacher confrence I will put on makeup but day makeup if that makes since not what I would wear if i were say going out to dinner with my husband. It took me awhile before I was confident going out withough a full overhall from head to toe but in the time it takes to do all of that my 3 year old would have the house turned upside down and id be to busy cleaning to go out.
so i guess i just jump right in then.
i don't wear makeup everyday unless i am going out somewhere (not like the store or my pt appointments or anything).
my dh is not too bad with the house. for the most part, he will clean or pick up if i go out, but i have never been gone over night. he does have some trouble with meals though. he will only feed the boys gerber pasta pickups or hotdogs. he tries, though and i appreciate it. he does a lot around here normally. i really can't complain.
does anyone else have twins in this group?
I used to be really fussy about my appearance while I was working. Once I quit my job, I quit putting my face on every day. Now I only put it on if I have to go somewhere. Even then sometimes I don't wear it. I feel so free! I love not worrying about it. I do kind of feel bad for my husband, though. He's so sweet though, he doesn't complain.
Not everyday. Since I am at the house during the day and then go pick up my daughter at school and the occasional trip to supermarket, I don't see the need to.
On Saturdays and Sundays I do.
No makeup except for more special trips out. Although I could use it, one of those people where makeup and hairspray go along way.
I have had to leave DH at home from time to time for 4 or 5 days while I went to conventions. Leave detailed lists. Come home. he never looked at the list, homework for 9 year old not done all week, his sister and mom show up and bail him out on the housework and meals. Was aggrevated the first time but have learned to let it go.
My youngests are almost twins, 5 months apart, attend 1/2 day preschool together.
I'm one of those make-up everyday kind of people. I wear light make up everyday, more make-up for date nights and social events. But I have really let go on the wardrobe (I'm a sweats almost every day kind of girl) so it makes me feel better about myself to be made up.
Thanks for posting that about the MOPS group... I searched for one in my area and I think I'm going to join! I just moved to a new area and don't have many contacts here so I really need to meet new people.
If I am just staying home all day I don't put on make up, but if I go out at all I wear light make up every day. My skin is too uneven not to
I am so glad you started this post! It's good to have association, you don't feel so alone. I only have one other sahm friend and she has 4 kids, so she's in a whole different league. I have a very busy 7 month old and am not planning on "going back" to work. but I am in the process of starting an event planning business - of course including cakes....
Things have been interesting around here lately, my husband and I just bought our first investment home and are completely renovating it. So as we both decided he is working on it about 4 days a week after his full time job. So that's pretty much my story....
Thanks for posting that about the MOPS group... I searched for one in my area and I think I'm going to join! I just moved to a new area and don't have many contacts here so I really need to meet new people.
If I am just staying home all day I don't put on make up, but if I go out at all I wear light make up every day. My skin is too uneven not to
Let me know what you think about it . . . I know we have a lot of groups in my immediate area and some have waiting lists to get in so if you call and that group is full, check others in the area. Also, if there aren't others close by definitely get on the waiting list. We have new people joining almost every meeting so there is turn over due to people moving, etc . . . I really like the group because it focuses specifically on moms and what we do all day, day in and day out and you're with a big group of other moms going through the same things you are. My group does lots of social things, too. Some are daytime events with the kids, some are evening or weekend events with just adults (moms or couples). We do an annual trivia night, have movie nights or dinners out, etc . . . so you can get as involved as you want to get. I've really gotten a lot out of it.
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Radio Noon Montreal | Apr 26, 2013 | 18:59
Tragedy underlines cellphone use risks for pedestrians
The coroner's office is investigating the death of a young woman in the Montreal metro last week. The victim was using her cell phone when she walked right between two metro cars. We speak to experts and hear from our listeners about cellphone use risks.


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Put the pedal to the metal in these studded stunners. Open toe wedge with stud detail. Genuine leather. BRUENS works wonders with jeans and tucked in tees. Pair her up with some knee high socks when the weather gets chillier.
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