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Ceramide accumulation mediates the pathogenesis of chronic obstructive lung diseases. Although an association between lack of cystic fibrosis transmembrane conductance regulator (CFTR) and ceramide accumulation has been described, it is unclear how membrane-CFTR may modulate ceramide signaling in lung injury and emphysema. Cftr+/+ and Cftr−/− mice and cells were used to evaluate the CFTR-dependent ceramide signaling in lung injury. Lung tissue from control and chronic obstructive pulmonary disease patients was used to verify the role of CFTR-dependent ceramide signaling in pathogenesis of chronic emphysema. Our data reveal that CFTR expression inversely correlates with severity of emphysema and ceramide accumulation in chronic obstructive pulmonary disease subjects compared with control subjects. We found that chemical inhibition of de novo ceramide synthesis controls Pseudomonas aeruginosa-LPS–induced lung injury in Cftr+/+ mice, whereas its efficacy was significantly lower in Cftr−/− mice, indicating that membrane-CFTR is required for controlling lipid-raft ceramide levels. Inhibition of membrane-ceramide release showed enhanced protective effect in controlling P. aeruginosa-LPS–induced lung injury in Cftr−/− mice compared with that in Cftr+/+ mice, confirming our observation that CFTR regulates lipid-raft ceramide levels and signaling. Our results indicate that inhibition of de novo ceramide synthesis may be effective in disease states with low CFTR expression like emphysema and chronic lung injury but not in complete absence of lipid-raft CFTR as in ΔF508-cystic fibrosis. In contrast, inhibiting membrane-ceramide release has the potential of a more effective drug candidate for ΔF508-cystic fibrosis but may not be effectual in treating lung injury and emphysema. Our data demonstrate the critical role of membrane-localized CFTR in regulating ceramide accumulation and inflammatory signaling in lung injury and emphysema.
Chronic obstructive pulmonary disease (COPD), emphysema, asthma, and cystic fibrosis (CF) subjects suffer from severe tissue-debilitating lung inflammation that is induced by exposure to environmental contaminants like cigarette smoke (CS) and bacterial infections (1–3). The Pseudomonas aeruginosa bacterial infection has been shown to have a critical role in pathogenesis of both CF and COPD (4–6), but it is not clear why these patients are highly sensitive to P. aeruginosa infections. The absence of cystic fibrosis transmembrane conductance regulator (CFTR) protein from the plasma membrane is known to result in an inherent hyperinflammatory lung phenotype causing chronic obstructive lung disease in both human (CF) and Cftr-deficient (Cftr−/−) mice (7–11). It is evident that CFTR has other critical signaling and/or transport functions (in addition to its well-documented chloride efflux functions) that control the chronic inflammatory response (12–14). In COPD, although inflammatory lung exacerbations cause most of the lung tissue damage, genetic risk factors can modify disease susceptibility. Moreover, emphysema is a disease of the alveoli, and functional CFTR is known to be expressed in alveolar epithelial cells (type I/II) (15, 16) and macrophages (17). The earlier studies indicate that genetic mutations in CFTR may be a risk factor for chronic lung diseases like COPD, emphysema, and asthma, which warrants further investigation (18, 19). In addition, CFTR is known to regulate membrane accumulation of the bioactive lipid ceramide, which is proposed as a mechanism for pathogenesis of emphysema, COPD (20), CF (7), and chronic lung inflammation (21, 22).
Recent data from CF cell lines and Cftr−/− mice demonstrate that CFTR also acts as a transporter for sphingolipids (13). Moreover, the studies of Hamai (23) show that expression of defective CFTR in lung epithelial cells results in increased mass and synthesis of sphingolipids, including various ceramide species. They demonstrate that expression of wild-type (WT)-CFTR controls ceramide accumulation (23). The authors of these studies propose that deficiency of functional CFTR (Cftr−/− mice) results in an alteration of the sphingolipid metabolism and an accumulation of cellular ceramide, but how CFTR regulates inflammatory signaling and ceramide accumulation is unclear. It has previously been demonstrated that the last three amino acids in the C terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane, essential for its chloride channel function (24, 25). We demonstrate here that expression of the mutant form of CFTR lacking the PDZ-interacting domain (ΔTRL), modulates its role as a pattern recognition molecule (26) and results in ceramide accumulation.
Our current work supports and expands these important findings and correlates the expression of membrane and lipid-raft (27, 28) localized CFTR with ceramide signaling and severity of lung disease. Our data show that CFTR regulates tight junction formation (29), ceramide accumulation, and inflammatory signaling in lung injury and emphysema.
Materials and Methods
Reagents and treatments
The cells were cultured at 37°C with 5% CO2P. aeruginosa-LPS (Pa Pa-LPS, 50 μM FB1, 50 μM AMT, 5 mM CD, 5 or 10 μg/ml Con A, 10 ng/ml TNF-α, and/or 0–160 μg/ml CSE as described. Mice were treated by intratracheal (i.t.) instillation with 20 μg Pa-LPS, 50 μg FB1, 50 μg AMT, and/or 50 μg CD as indicated in 100 μl total volume of PBS, and control mice received PBS alone.
Murine experiments and human subjects
All animal experiments were carried out in accordance with Johns Hopkins University (Baltimore, MD) Animal Care and Use Committee-approved protocols. We used age-, weight-, and sex-matched (24 wk old), B6- 129S6- Cftr−/− (Cftrtm1Kthc–TgN(FABPCFTR)) (30, 31) and Cftr+/+ inbred mice strains (procured from Case Western Reserve University Animal Resource Center, Cleveland, OH; n = 3–5 for all experiments). The changes in cytokine and inflammatory markers between Cftr+/+ and Cftr−/− mice were verified by multiple (2, 3) experiments, and representative data are shown. All mice were housed in a controlled environment and pathogen-free conditions. We induced lung injury in these mice by i.t. instillation of Pa-LPS (20 μg in 100 μl PBS) for 12 h, which resulted in ∼1–2 g loss in body weight. The de novo ceramide synthesis or membrane-ceramide release was partially inhibited by i.t. (50 μg in 100 μl PBS) FB1 or AMT administration 12 h after Pa-LPS treatment. Mice were sacrificed 24 h after drug treatment, and the bronchoalveolar lavage fluid (BALF) was collected for cytokine ELISAs. The lungs were fixed in 10% buffered formalin phosphate (Fisher Scientific, Pittsburgh, PA), paraffin embedded, and cut into longitudinal sections (5 μm thick) on glass slides for immunostaining. For depletion of membrane-CFTR in murine lungs, we used 72-h i.t. CD treatment, and lung tissues were collected as above. The mice (three to four mice per group, 8 to 10 wk old) were exposed to CS using the TE-2 cigarette smoking machine (Teague Enterprises, Davis, CA). The CS was generated by burning research-grade cigarettes (3R4F; 0.73 mg nicotine per cigarette) purchased from the Tobacco Research Institute (University of Kentucky, Lexington, KY) for 5 h/d for 5 d. An average total particulate matter of 150 mg/m3 was recorded in real time during the smoking protocols. The control group of mice was exposed to filtered room air, and all the mice were sacrificed 2 h after the last CS exposure. The human lung tissue samples from Gold stage I (mild, forced expiratory volume [FEV1]% predicted >80%), Gold stage II (moderate, FEV1% predicted = ∼50–80%), and Gold stage III–IV (severe/very severe, FEV1% predicted <50%) nontumor COPD (with FEV1/forced vital capacity [FVC] ratio of <70%) and Gold stage 0 (at risk) control subjects (procured from Lung Tissue Research Consortium [Bethesda, MD], National Heart, Lung and Blood Institute, National Institutes of Health) were used for quantification and localization of indicated proteins by immunostaining. All the subjects were stable, and Gold I–IV subjects had emphysema. Moreover, one patient in each group (Gold I–IV) had first-degree blood relatives with chronic bronchitis. A detailed description of the human subjects is shown in Table I.
In vitro and ex vivo experiments
The macrophages and neutrophils from Cftr+/+ and Cftr−/− mice were isolated by i.p. injection of 1 ml 4% thioglycolate broth (Fluka, St. Louis, MO). The peritoneal cavity was flushed as indicated after 6 h (32) (neutrophils) or 4 d (33) (macrophages) with 10 ml RPMI 1640 medium (Life Technologies, Carlsbad, CA) containing 10% FBS (Life Technologies) and 1% PSA (Life Technologies) (complete RPMI medium). The lavage was centrifuged at 1200 rpm for 8–10 min at 4°C followed by RBC lysis in LCK lysis buffer (Quality Biologicals, Gaithersburg, MD). The 3 × 105 cells per well were plated in a 6-well plate and cultured overnight in complete RPMI medium. The culture supernatants were collected for cytokine ELISAs and myeloperoxidase (MPO) measurements. The spleens were dissected from Cftr+/+ and Cftr−/− mice and macerated using the plunger of a 5-ml BD (San Diego, CA) syringe. The suspension was subjected to RBC lysis as described above, and 2 × 105 splenocytes per well were cultured in a 96-well plate. The cells were treated with 5 or 10 μg/ml Con A for 72 h. For splenocyte proliferation assay, 20 μl of the Cell Titer 96 AQueousOne Solution (Promega, Madison, WI) was added at the 60-h time point, and the plate was incubated at 37°C, 5% CO2 for another 12 h. The OD at 490 nm was recorded by a 96-well microplate reader (Molecular Devices, Sunnyvale, CA) using SOFT-MAX-Pro software (Molecular Devices) as a measure of cell proliferation. For immunoblotting, splenocytes (2 × 106 Pa-LPS, 5 mM CD, and/or 10 ng/ml TNF-α. The CFBE41o−WT-CFTR cells were treated with PBS or 5 mM CD for 24 h on a 24-well plate, and IL-8 secretion in the cell supernatants was quantified by sandwich ELISA (R&D Biosystems, Minneapolis, MN). The HEK-293 cells were transiently transfected with WT-CFTR and incubated with increasing doses (0, 40, 80, 120, and 160 μg/ml) CSE for 12 h. The total protein cell lysate from these samples was extracted as described above, and levels of mature (C form) and immature (B form) CFTR were quantified by Western blotting. The lipid-rafts were isolated from CFBE41o−WT-CFTR and CFBE41o− cells treated with PBS, Pa-LPS (10 ng/ml), or FB1 (50 μM) for 24 h. For the ΔTRL/WT-CFTR experiments, HEK-293 cells were transiently transfected with pEGFP-WT-CFTR or pEGFP-ΔTRL-CFTR (a gift from Dr. William B. Guggino, Johns Hopkins University) constructs (24Escherichia coli LPS (Molecular Probes, Carlsbad, CA) for the final 3 h and analyzed by flow cytometry without permeabilizing the cells. The same set of transfections was also performed with or without 50 ng/ml TNF-α treatment for 6 h, and lipid-raft proteins were isolated to detect CFTR expression by Western blotting.
Immunofluorescence microscopy and flow cytometry
The longitudinal tissue sections from murine or human lungs or CFBE41o− and CFBE41o−WT-CFTR cells were immunostained with the primary Abs (1:50 to 1:200 dilution) for CFTR (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), ceramide (mouse monoclonal; Alexis Biochemicals, Plymouth Meeting, PA), Foxp3 (rabbit polyclonal; Santa Cruz Biotechnology), NF-κB (rabbit polyclonal; Santa Cruz Biotechnology), zona occludens (ZO)-1 (rabbit polyclonal; Santa Cruz Biotechnology), ZO-2 (goat polyclonal; Santa Cruz Biotechnology), and neutrophil marker NIMP-R14 (rat monoclonal; Abcam, Cambridge, MA) followed by the secondary Abs (1:200 dilution), using our previously described protocol (34Cftr+/+ and Cftr−/−
ELISA, MPO activity, and reporter assay
The BALF and cell culture supernatants (n28). Data were normalized with internal renilla luciferase control for each sample, and the changes in reporter activities with CFTR overexpression were calculated.
Immunoblotting and lipid-raft isolation
Splenocytes from Cftr+/+ and Cftr−/− mice were isolated and stimulated with 5 μg/ml Con A for 12 h. Cells were washed in PBS, and total protein was isolated using the 1× M-PER Mammalian protein extraction reagent (Pierce) supplemented with protease inhibitor mixture (Sigma). The protein lysate was immunoblotted for Foxp3 primary (Santa Cruz Biotechnology) or β-actin (Sigma) loading control and anti-rabbit IgG HRP secondary Abs (Amersham, Piscataway, NJ) and developed using the Super Signal West Pico Chemiluminescent Substrate kit (Pierce). Similarly, the total cell lysates from HEK-293 cells transiently transfected with the WT-CFTR and treated with increasing doses of CSE were immunoblotted with CFTR (Cell Signaling Technologies, Danvers, MA) or β-actin (Sigma) loading control and anti-rabbit or anti–mouse-HRP Ab, respectively. The mouse lung tissue from air and CS exposed mice was homogenized in cold tissue lysis buffer (T-PER; Pierce) supplemented with protease inhibitor mixture. The lung lysate was immunoprecipitated with CFTR 169 Ab (rabbit polyclonal), followed by Western blot with CFTR (M3A7) Ab (Abcam). For lipid-raft isolation, CFBE41o− and CFBE41o-WT-CFTR cells were plated in a 25 cm2 tissue culture flask and treated with Pa-LPS (10 ng/ml) and/or FB1 (50 μM) for 24 h. The cells were washed with cold PBS, and raft proteins were isolated using the Signal Protein Isolation kit (G Biosciences, Maryland Heights, MO). The lung tissue from air and CS exposed mice was similarly harvested in signal protein extraction (SPE) buffer-I and subjected to raft isolation as described below. Briefly, cells or lung tissue were resuspended in SPE buffer-I and sonicated for 10 s to disrupt the cells or tissue. Total protein was quantified in each sample, and equal amount of protein (cells, 300 μg; and lung tissue, 500 μg) was used to purify the raft fraction. The SPE buffer-II was added followed by incubation on ice for 15 min with intermittent vortexing. The lysate was centrifuged at 20,000 × g for 15 min and the supernatant discarded. The pellet containing signal proteins was solubilized in adequate amount of focus protein solubilization buffer and used for immunoblotting of ZO-2 (Santa Cruz Biotechnology, goat primary and anti-goat IgG HRP) and α-actin (Sigma, rabbit primary and anti-rabbit IgG HRP). The raft protein from mouse lungs or HEK-293 cells was immunoblotted with CFTR 570 Ab (mouse polyclonal Ab; procured from University of North Carolina, Chapel Hill and Cystic Fibrosis Foundation Therapeutics under a material transfer agreement).
Statistical analysis
Data are represented as the mean ± SEM of at least three experiments, and Student t test and ANOVA were used to determine the statistical significance. The murine and human microscopy data were analyzed by densitometry (MATLAB R2009b; Mathworks, Natick, MA) followed by Spearman's correlation coefficient analysis to calculate the significance among the indicated groups.
CFTR regulates innate and adaptive immune response
To confirm and expand the hypothesis that functional CFTR is a critical regulator of inflammatory signaling (28), we compared the immune profile of the gut-corrected Cftr−/− mice with that of the Cftr+/+ mice. We quantified the constitutive levels of proinflammatory cytokine IL-6 ex vivo in peritoneal macrophages and neutrophils isolated from Cftr+/+ and Cftr−/− mice (n = 3) and found significantly (p < 0.001) higher basal IL-6 levels in Cftr−/− compared with that in the Cftr+/+ (Fig. 1A). We also found a significant increase (p < 0.01) in constitutive neutrophil-MPO levels (Fig. 1B) in Cftr−/− compared with those in the Cftr+/+, which is indicative of the activated state of neutrophils in the absence of CFTR. We confirmed this in vivo using the murine model and observed a significant increase (p < 0.05) in basal and Pa-LPS–induced MPO levels in BALF of Cftr−/− mice compared with those in the Cftr+/+ mice (Fig. 1C). To test the outcome of CFTR deficiency on the adaptive immune response, we quantified differences in cell proliferation and IL-6 secretion in splenocytes from Cftr+/+ and Cftr−/− mice. We did not find a significant difference in the nonactivated splenocytes, but Con A induced a significantly higher (**p < 0.01, ***p < 0.001) splenocyte proliferation and IL-6 secretion in Cftr−/− compared with that in Cftr+/+ (Fig. 1D, 1E). We confirmed that CFTR is expressed on murine splenocytes (Fig. 1Fi). The CFTR-deficient splenocytes demonstrate higher numbers of CD4+IFN-γ+ T cells (Fig. 1Fii) supporting the notion that the absence of CFTR results in a constitutive hyperinflammatory state by inducing the proinflammatory response. In addition, prevalence of regulatory T cells is reported in the hyperinflammatory COPD lungs (35). We compared the expression of Foxp3 in Cftr+/+ and Cftr−/− mice and found constitutively higher numbers of CD4+Foxp3+ splenocytes in the Cftr−/− (0.55%) compared with that in the Cftr+/+ (0.32%) (Fig. 1Fiii). We also confirmed this by Foxp3 immunostaining and Western blotting in lung sections and splenocytes, respectively (Fig. 1G, 1H). The data substantiate the previous observations (28, 36–40) and strongly suggest that CFTR is a critical regulator of both innate and adaptive immune responses.
CFTR expression in inflammatory cells inversely correlates with the levels of ceramide and lipid-raft marker (ZO-1)
Ceramide is a critical regulator of inflammatory and apoptotic signaling (20) and mediates these processes in lung injury (41), asthma (21), emphysema, COPD (20), and CF (7). Moreover, CFTR is present in the lipid-rafts (27, 42), and its role in regulating TNF-R1 and lipid-raft signaling has been examined previously (27). We tested the hypothesis that CFTR may be regulating inflammatory signaling via ceramide by inhibiting the formation of membrane and lipid-raft platforms, which would hamper proper clustering of signaling receptor complexes on the plasma membrane. Evidence from previous studies (7) and our data show that macrophages from Cftr−/− mice have significantly higher ceramide levels compared with those of the Cftr+/+ mice (Fig. 2A, left panel), which concurs with increased expression of lipid-raft marker ZO-1 (Fig. 2A, right panel). Although the Cftr−/− neutrophils show a similar increase in ZO-1 expression, ceramide levels remain unchanged (Fig. 2B). We speculate that other mechanisms may be involved in constitutive increase of neutrophil (MPO) activity in the absence of CFTR (14, 17). Our data indicate a mechanism by which CFTR regulates lipid-raft signaling and inflammatory cell function(s). The constitutive defect in the absence of CFTR compromises the ability of these inflammatory cells to respond to infection or injury resulting in pathogenesis of chronic lung disease.
CFTR regulates membrane-ceramide signaling and pathogenesis of chronic emphysema
Ceramide upregulation was recently correlated with emphysema (20), and it is known that CFTR deficiency leads to increased ceramide accumulation and lung injury (7). We verified this observation in lung sections from control (Gold 0, at risk) and COPD (Gold I, mild; II, moderate; and III–IV, severe and very severe emphysema) human subjects (Table I) and found that CFTR expression significantly decreases with disease severity while ceramide levels increase (Fig. 3A, 3B, p < 0.001). Although CFTR is not completely absent in severe COPD lungs, its expression is significantly downregulated. We anticipate this as an outcome of lung injury. These data imply that lipid-raft localization of CFTR (Fig. 3A, inset) controls ceramide accumulation and possibly severity of emphysema. We confirmed our findings in HEK-293 cells transfected with WT-CFTR and show that CSE treatment decreased cell surface expression of CFTR (mature, band C) in a dose-dependent manner (Fig. 3C, left panel). The non-transfected HEK-293 cells do not show the CFTR at this Ab concentration (Fig. 3C, right panel). Extending our findings in the murine model (C57BL/6 mice), we found that acute CS exposure (5 h/d for 5 d) diminished CFTR expression both in the mouse lung lysate (Fig. 3D, upper panel, and Fig. 3E, left panel, p < 0.01) and in the purified lipid-raft fraction (Fig. 3D, lower panel, and Fig. 3E, right panel, p < 0.001). Moreover, we also demonstrate that lungs of CS-exposed mice have significantly (p = 0.004, ρ = 0.9316) increased ceramide accumulation that is colocalized with ZO-1 (Fig. 3F), which implies that CS-mediated decrease in CFTR expression results in lipid-raft ceramide accumulation. Therefore, in accord with our previous observation (28), the current data verify that decreased cell surface and lipid-raft expression of CFTR correlates with the increased inflammation and emphysema (Fig. 3A, H&E staining, bottom panel).
CFTR expression regulates ceramide signaling in lung injury
To verify whether CFTR regulates ceramide signaling and outcome of lung injury, we used the Pa-LPS–induced mouse model of lung injury. We treated Cftr+/+ and Cftr−/− mice with 20 μg/mouse Pa-LPS i.t. for 12 h, followed by either FB1 or AMT (50 μg/mouse) for another 24 h. We inhibited either the de novo ceramide synthesis (FB1) or membrane-ceramide release (AMT), as they have been shown to mediate the pathogenesis of emphysema and CF lung disease, respectively (7, 20). We measured BALF cytokines IL-6 and IL-1β in all the groups as a marker of Pa-LPS–induced proinflammatory insult and the efficacy of the drugs. We found that inhibition of de novo ceramide synthesis by FB1 in Cftr+/+ mice shows a 2-fold reduction (p < 0.05) in the Pa-LPS–induced IL-6 levels (Fig. 4Ai) and a very significant decrease (p < 0.001) in IL-1β secretion (Fig. 4Aii). In the absence of Cftr (Cftr−/− mice), FB1 treatment decreased Pa-LPS–induced IL-6 (Fig. 4Aiii), but the magnitude of rescue was not as efficient as that in Cftr+/+ mice. In addition, IL-1β levels were unaltered by FB1 treatment in the Cftr−/− mice (Fig 4Aiv). This was also verified by immunostaining of lung sections from these mice for ceramide, NF-κB, and neutrophil marker NIMP-R14 (Supplemental Fig. 1A, 1B).
In contrast, inhibition of membrane-ceramide release by AMT was unable to rescue Pa-LPS–induced IL-6 or IL-1β secretion in Cftr+/+ mice (Fig. 4Bi, 4Bii), whereas inhibition of membrane-ceramide in the Cftr−/− mice showed a significant decrease (p < 0.05) in Pa-LPS–induced IL-6 and IL-1β levels (Fig. 4Biii, 4Biv). The ceramide, NF-κB, and NIMP-R14 immunostaining of murine lungs verified these findings (Supplemental Fig. 2A, 2B). Our data concur with findings of Teichgräber et al. (7) who showed that normalization of acid sphingomyelinase (Asm) levels by AMT treatment or partial genetic deficiency reduced pulmonary ceramide levels that protected Cftr-deficient mice from P. aeruginosa infection. Our results indicate that inhibition of de novo ceramide synthesis (not the release) by FB1 may be effective in disease states with low CFTR expression like emphysema and lung injury but not in total absence of apical or lipid-raft CFTR, for instance in ΔF508-cystic fibrosis (ΔF508-CF), where phenylalanine mutation impairs the folding and trafficking of CFTR to the cell surface. In contrast, inhibition of Asm activity or membrane-ceramide release by AMT has potential application as a more effective drug treatment for ΔF508-CF but may not be effectual in treating lung injury and emphysema.
CFTR expression negatively regulates membrane-ceramide and lipid-raft signaling
To elucidate the mechanism by which CFTR regulates lipid-raft signaling, we quantified the expression of tight junction protein ZO-2 in purified raft-protein extract from CFBE41o−WT-CFTR and CFBE41o− cells with or without Pa-LPS or FB1. We found that ZO-2 expression was downregulated by Pa-LPS or FB1, only in the presence of WT-CFTR (Fig. 5A). It is possible that Pa-LPS may induce more recruitment of WT-CFTR to the raft (41, 43), which in turn inhibits raft formation (low ZO-2). FB1 is also able to modulate ZO-2 expression by an unknown mechanism that needs further investigation. Moreover, in the absence of functional CFTR in CFBE41o− cells, we observed higher basal expression of ZO-2 compared with that in CFBE41o−WT-CFTR cells. We also observed that neither Pa-LPS nor FB1 is able to modulate ZO-2 expression in these cells (Fig. 5A). To confirm these data, we stained the lung sections from Cftr+/+ and Cftr−/− mice with ZO-2 and found a constitutively higher ZO-2 expression in the Cftr-deficient mouse lungs (Fig. 5B). We also tested another marker of tight junctions, ZO-1, and analyzed it by co-immunostaining with ceramide using the lung sections from Cftr+/+ and Cftr−/− mice that were treated with Pa-LPS or PBS. We found a constitutive increase in ceramide levels in the Cftr−/− mice lungs compared with that in the Cftr+/+ mice, which was significantly enhanced by Pa-LPS treatment. Moreover, ceramide was colocalized with ZO-1 indicating its presence in the membrane lipid-rafts. (Fig. 5C, 5D).
Lack of PDZ binding domain modulates CFTR-dependent ceramide accumulation
Our data demonstrate the importance of cell surface and lipid-raft CFTR in regulating ceramide-mediated inflammatory signaling. The C-terminal PDZ-interacting domain of CFTR protein is crucial for its apical membrane polarization and functional robustness (24, 25). To investigate the role of this domain in CFTR-dependent inflammatory responses, we overexpressed WT- or ΔTRL- (CFTR lacking the PDZ binding domain) CFTR-GFP in HEK-293 cells and quantified ceramide levels by flow cytometry. We found that expression of ΔTRL-CFTR triggers higher ceramide accumulation (Fig. 6A, upper panel), which is more prominent upon CSE treatment (Fig. 6A, lower panel). Expression of ΔTRL-CFTR also decreases the binding of E. coli LPS-Alexa Fluor 488 to the plasma membrane (Fig. 6B). Because CFTR has been described as a pattern recognition molecule for LPS binding (26), our data demonstrate that the PDZ binding domain of CFTR may be crucial for its function as a pattern recognition molecule. We also demonstrate that expression of ΔTRL-CFTR leads to less CFTR protein reaching the lipid-raft fraction (Fig. 6Ca, 6Cb [6Ca, 30-s exposure; 6Cb, 20-min exposure], 6D). Treatment with TNF-α induces the localization of CFTR to the lipid-rafts, but ΔTRL-CFTR mutation compromises its translocation to lipid-raft. Our data suggest that PDZ binding domain is required for CFTR membrane stability, and lipid-raft-localization and signaling (Fig. 7). We anticipate binding to PDZ domain-containing proteins (ZO-1/2) may be critical for this process.
We and others have recently shown that apical lipid-raft–localized functional WT-CFTR is critical for controlling the innate immune response (7, 28, 36, 39, 44). Although the link between CFTR dysfunction and inflammatory pathophysiology of CF lung disease has been a subject of debate (45), recent work clarifies and discusses these findings that we have recently reviewed in detail (11, 28). In this study, we verify that CFTR is not only critical for regulating the innate immune response in epithelial cells but also regulates the adaptive immune response as lack of functional CFTR confers a hyperinflammatory phenotype to the splenocytes. It has been reported that CD4+ T cells from CF patients have lower IFN-γ response (46). We report in this study that mouse CD4+ T cells lacking CFTR (Cftr−/−) secrete higher amounts of IFN-γ compared with that of the Cftr+/+. A recent study by Carrigan et al. (47) showed that although natural regulatory T cells (Tregs) were increased in P. aeruginosa-infected Cftr+/+ mice, depletion of Tregs did not alter the disease outcome. Our original finding shows that lack of functional CFTR was able to modulate Foxp3 expression in the lungs and the peripheral tissues indicative of increased number of Tregs. The lungs of COPD patients similarly harbor higher number of Tregs that are proposed to be involved in controlling pulmonary inflammation or autoimmunity (48). We anticipate that a similar mechanism may be triggered in the absence of functional CFTR, and strategies directed to modulate functional Tregs to revert acute or chronic lung disease warrant further investigation (49, 50).
The proinflammatory response in the Cftr-deficient mice is known to be mediated by neutrophils and macrophages, the primary cells of the innate immune response (40, 51–54). We evaluated whether the defect in lipid metabolism in the absence of CFTR (7) extends to these immune effector cells. For these studies, we used the common Pa-LPS–induced acute lung injury model (55) that is also a component of air pollutants that cause lung inflammation (56). Notably, we observed increased ceramide staining in macrophages (Fig. 2A, left panel) but not neutrophils (Fig. 2B, left panel) from uninfected Cftr−/− mice, which correlates with the higher constitutive and Pa-LPS–induced proinflammatory cytokine levels. We also observed an increase in ZO-1 staining in both macrophages and neutrophils in the absence of CFTR. Some recent studies support our finding and have shown the expression of tight junction proteins like ZO-2 in human macrophages (57, 58). Our data support the recent findings that CFTR inhibition by CFTR small interfering RNA in human alveolar macrophages renders them a proinflammatory phenotype along with an increase in caveolin-1 expression, as it is related to inflammation and apoptosis of macrophages (59). Although constitutive activation of neutrophils in CF is well documented (44, 60), CFTR expression in neutrophils is a subject of debate. Based on current literature, CFTR expression in neutrophils is either very low or absent. It may be possible that lack of CFTR regulates neutrophil function in a ceramide-independent manner. The lower expression of functional CFTR protein on murine and human neutrophils compared to that of epithelial or other inflammatory cells (52) may account for lack of ceramide accumulation in the Cftr−/− over Cftr+/+. Moreover, a recent study inversely correlates CFTR-mediated SCN(−) transport with the MPO activity (14). We anticipate this as a potential mechanism of neutrophil activation in the Cftr−/− mice that mediates the pathogenesis of chronic lung disease in the presence of Pseudomonas aeruginosa infection or lung injury.
It is proposed that changes in sphingosine and sphingosine-1–phosphate uptake in the absence of CFTR may result in membrane-ceramide accumulation (13) that triggers a proinflammatory and proapoptotic response in the respiratory tract. Ceramide forms membrane platforms and alters small lipid-rafts that consist of sphingomyelin and cholesterol. We anticipate that ceramide accumulation in the absence of CFTR might change the function of proteins in the membrane by altering the composition of sphingomyelin–cholesterol-rich lipid-rafts. In favor of this hypothesis, WT-CFTR expression in Cftr−/− cells controls ceramide accumulation and inflammatory signaling (13). The data also support the critical role of membrane or lipid-raft CFTR in ceramide biogenesis and pathogenesis of lung disease (21, 41). This raises the important question whether modulation of CFTR expression in airway diseases can contribute to pathogenesis of chronic lung disease. Notably, CSE has previously been shown to inhibit chloride secretion in human bronchial epithelial cells (61). A more direct correlation between CSE and CFTR expression was established by Cantin et al. (62), showing that CSE decreased expression of CFTR- gene, protein, and function in Calu-3 cells. Our in vitro studies in HEK-293 cells transfected with WT-CFTR confirm their observation. We document here the first report to our knowledge showing that decrease in CFTR expression correlates with severity (Gold 0 [at risk] versus Gold I [mild], II [moderate], and Gold III–IV [severe and very severe]) of lung emphysema and ceramide accumulation (Fig. 3A). We verified that acute CS exposure of Cftr+/+ (C57BL/6) mice decreases cell surface and lipid-raft expression of CFTR in murine lungs (Fig. 3D, 3E). We also found an increase in colocalization of ceramide and ZO-1 (Fig. 3F) in the murine lungs after CS exposure. In support of our findings, a recent study (63) demonstrates that CS induces ceramide accumulation in human bronchial epithelial cells. We anticipate based on these data that ceramide accumulation and chronic P. aeruginosa infections in severe COPD patients (5) and CF (5, 64) may be an outcome of decreased CFTR expression.
The previous clinical studies showing the association of CFTR mutations with asthma and COPD (18, 19, 65, 66) were not conclusive due to lack of sufficient controls. Moreover, only few reports have verified emphysema development in CF subjects (67). Our data suggest the critical modifier role of membrane CFTR and ceramide levels in pathogenesis of severe emphysema. An interesting question here is why CF subjects with ΔF508-mutation, resulting in very low membrane-CFTR levels, do not develop severe emphysema? The paucity of emphysema in ΔF508-CF patients may be due to the absence of other contributors like CS or lack of detection as the patients die before severe emphysema develops or is recognized. Nonetheless, our data suggest that pathogenetic changes in membrane and lipid-raft CFTR may have a modifier function in pathogenesis of COPD and emphysema. Based on our data, we propose that the association of apical and lipid-raft CFTR expression with COPD disease severity and ceramide accumulation and signaling has a clinical application as both prognostic marker and therapeutic. Further clinical studies are warranted to confirm the role of CFTR as a modifier or pathogenetic susceptibility factor for COPD, emphysema, and asthma.
Because ceramide is an important component of lipid-rafts (43), we hypothesized that disruption of raft CFTR by CD (28) may trigger ceramide accumulation and NF-κB activation. We selected CD treatment as a method to deplete selectively CFTR from the lipid-rafts over CFTR small interfering RNA or inhibitor as it would result in an overall decrease of CFTR expression and/or function. We found that depletion of raft-CFTR by CD abrogated its regulatory function, marked by a significant increase in NF-κB activity, ceramide levels, and IL-8 secretion (Supplemental Fig. 3A, 3B). In vivo depletion of lipid-raft CFTR also showed an increase in ceramide, NF-κB, and neutrophil (Supplemental Fig. 3C) levels and activity, confirming our hypothesis that lipid-raft–localized CFTR controls ceramide and NF-κB mediated proinflammatory signaling. We further verified these results by depleting (CD treatment) (28, 42) or inducing (TNF-α) (41, 43) lipid-raft CFTR in CFBE41o−WT-CFTR cells and observed that lipid-raft CFTR expression controls membrane-ceramide accumulation (Supplemental Fig. 4), although we understand that CD and TNF-α may modulate NF-κB signaling by CFTR-independent mechanisms, which warrants further investigation and identification of small molecules that can selectively modulate lipid-raft CFTR expression. Nonetheless, our preliminary studies indicate that membrane-localized WT-CFTR inhibits lipid-raft formation as the expression of lipid-raft marker ZO-1/2 was elevated in the absence of CFTR, which is known to induce immune receptor clustering and signaling (68). We anticipate this as a potential mechanism by which CFTR regulates ceramide-mediated NF-κB signaling.
We and others observed that ceramide-mediated lung injury and NF-κB signaling is prevented by inhibiting de novo ceramide synthesis (FB1) (20); therefore, we tested the efficacy of FB1 to suppress TNF-α–induced NF-κB reporter activity in the presence or absence of CFTR. FB1 was able to suppress NF-κB reporter activity only in the CFBE41o−WT-CFTR cells but not in the CFBE41o− cells (Supplemental Fig. 3D). We speculated that WT-CFTR might be regulating membrane-ceramide levels by its interaction with lipid-raft signaling complex (TNF-R1–sphingomyelin) while FB1 suppresses the de novo ceramide hydrolysis. We anticipated that in the CFTR-deficient scenario, membrane-ceramide accumulation is catalyzed by ASM; hence, inhibition of de novo ceramide synthesis is rendered ineffective. The importance of the ASM pathway in several disease models has been comprehensively reviewed (69). Recently, Teichgräber et al. (7) demonstrated that Cftr−/− mice induce lung ceramide accumulation via Asm, and its inhibition by AMT rescued the mice from P. aeruginosa infection. A clinical trial using AMT in CF patients also demonstrates its safety and efficacy as a potent drug candidate (70). In the current study, we demonstrate that inhibition of de novo (FB1) or membrane-ceramide (AMT) synthesis/release has differential outcomes in controlling the Pa-LPS–induced lung injury in the presence and absence of CFTR. We found that in the presence of WT-CFTR, inhibition of de novo ceramide synthesis by FB1 inhibits Pa-LPS–induced NF-κB activity and recruitment of neutrophils in the lungs of Cftr+/+ mice while its inhibitory effect was significantly lower in Cftr−/− mice indicating that WT-CFTR depletes NF-κB activity by controlling TNF-R1 or sphingomyelin (Fig. 7). Moreover, treatment with FB1 may not only prevent the ceramide synthesis but also deplete sphingomyelin levels. This may indirectly modulate the function of ASM that leads to lower ceramide generation and thereby decreased inflammation in Cftr+/+ mice. In contrast, inhibition of ASM by AMT showed an enhanced protective effect in controlling the Pa-LPS–induced lung injury in Cftr−/− mice compared with that in the Cftr+/+ indicating that inhibition of de novo ceramide synthesis by FB1 can be a more potent therapeutic strategy in lung injury, emphysema, and COPD where CFTR raft expression is depleted but not absent, whereas AMT may be more effective in absence of cell surface CFTR, as in the case of ΔF508-CF.
The previous observations that PDZ-interacting domain in CFTR is required for its apical polarization and Cl− channel function (24, 25) led us to investigate its role in CFTR-dependent ceramide and lipid-raft signaling. Our data demonstrate that the absence of CFTR PDZ binding domain (ΔTRL) leads to 1) reduction in membrane CFTR levels (Fig. 6C), 2) decrease in binding of E. coli LPS to the plasma membrane (Fig. 6B), and 3) increased ceramide accumulation in both constitutive and CSE-induced states (Fig. 6A). These findings elucidate a potential mechanism by which CFTR may be sequestered to the lipid-rafts, where it regulates ceramide-mediated inflammatory signaling. We anticipate binding to PDZ domain-containing proteins (ZO-1/2) is required for CFTR membrane stability and lipid-raft translocation. The current study not only describes the critical role of CFTR in pathogenesis of obstructive lung diseases but also demonstrates the scope of an intervention strategy targeting CFTR-dependent lipid-rafts and ceramide for treatment of lung injury and emphysema. In addition, we evaluate CFTR-dependent lipid-rafts as a biomarker for lung injury and emphysema and demonstrate its potential utility as a prognosticator of the aforementioned therapeutic strategy. It remains an open question whether the development of a potent CFTR corrector (CF-ΔF508) and potentiator (COPD and emphysema) drug (currently under phase II–III clinical trial for CF by Vertex Pharmaceuticals, Cambridge, MA) may serve as an effective therapeutic strategy to overcome the ceramide-induced pathological conditions emerging from decreased membrane or lipid-raft CFTR expression. Because the Vertex drugs were identified based on their ability to correct CFTR chloride transport function only (71), we anticipate that the development of selective strategies to modulate CFTR-dependent lipid-rafts and ceramide signaling as proposed in this study will have a more specific therapeutic outcome for treating the chronic stages of lung disease.
We thank Drs. Dieter Gruenert (University of California) and William B. Guggino (Johns Hopkins University) for providing CFBE41o− and CFBE41o−WT-CFTR cell lines and the CFTR-ΔTRL construct, respectively. The human lung tissue samples were provided by the Lung Tissue Research Consortium, National Heart, Lung and Blood Institute, National Institutes of Health.
Disclosures The authors have no financial conflicts of interest.
This work was supported by Cystic Fibrosis Foundation (R025-CR07 and VIJ07IO), Flight Attendant Medical Research Institute, National Aeronautics and Space Administration (NNJ06HI17G), and National Institutes of Health (CTSA UL RR 025005 and RHL096931) grants (to N.V.).
The online version of this article contains supplemental material.
Abbreviations used in this paper:
- Asm
- acid sphingomyelinase
- amitriptyline
- bronchoalveolar lavage fluid
- methyl-β-cyclodextrin
- cystic fibrosis
- cystic fibrosis transmembrane conductance regulator
- chronic obstructive pulmonary disease
- cigarette smoke
- cigarette smoke extract
- ΔF508-cystic fibrosis
- fumonisin-B1
- forced expiratory volume; FVC, forced vital capacity
- i.t.
- intratracheal
- myeloperoxidase
- Pa-LPS
- Pseudomonas aeruginosa-LPS
- penicillin, streptomycin, and amphotericin B
- signal protein extraction
- Treg
- regulatory T cell
- zona occludens.
- Received August 23, 2010.
- Accepted November 1, 2010.


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Sports Day October 6
Monday, October 5, 2009 - 20:42 By Editor
Competition !
The best picture from Sports Day uploaded to the IEGS Facebook Fan Page wins you a school T-shirt. All entries must be tagged "Sports Day 2009" and be submitted by 9AM Thursday October 8th.
If you're looking for instructions for Sports Day you can get them here:

subset: commoncrawl


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It's True- Your Headlines Are Terrible.
100% of your audience needs you to write a better headline.
100% of your audience needs you to write a better headline.
Connell Aviation Group experts say the best results come from an integrated strategy: a healthy blend of traditional marketing and PR with a strong mix of social media. But what's the best social media strategy for your business? The answer is in these three steps:
Your website is often the first impression the customer has of your business. It's important to have your branding apparent throughout the website and your logo on each site page. Simplicity is also key.


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Another great thing about fall is that the voice is back! It's a show on chanel 5. It has 4 judges. Celo green, Christina Agulara, Adam Leveine,and Blake Shelton! I'm obsessed with it. This season my favorite so far is James W. He sang Love Interruption.
It is on Mondays and Tuesdays.


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Men's Golf hosts Bethel, St. Thomas, UW-Eau Claire
May 4All day
TimeMay 4All day
LocationLe Sueur, Minn.
The Gustavus golf team hosts a quadrangular with Bethel, St. Thomas, and UW-Eau Claire on May 3-4 at Le Sueur Country Club.
Contactsportsinfo — 507-933-7647
SponsorAthletic Department
Additional Times
PostedApr 17, 2019
|Apr 05
|12:00 am
|Men's Golf at Camp Lejeune The Men's Golf Team will play at Camp Lejeune on April 5-7.
|Apr 14
|12:00 am
|Men's Golf at Bobby Krig Invite The Men's team competes in the Bobby Krig Invite at Ridges at Sand Creek…
|Apr 19
|12:00 am
|Men's Golf at Millikin Men's Golf competes at Millikin at the Red Rail Run and Southside CC on April 19-20.
|Apr 27
|12:00 am
|Men's Golf at Saint John's Invite The Men's team competes in the Saint John's Invite at Territory Golf…


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I am thinking about my final program when doing downloads and interactions with the internet. I'm careful not to use my personal stuff. That's a good point.
I am thinking about my final program when doing downloads and interactions with the internet. I'm careful not to use my personal stuff. That's a good point.
Last edited by goodman441; 10-04-2019 at 12:19 PM.
I've built my app and installed it on my pc. Very cool! I understand the Automation Server now.... One thing I noticed is the Run Scripts button in the ribbon is not active until a record is selected, I wanted the user to be able to load current data so there is no data there to start, but I think I can work thru that by setting up an Update process instead of a complete load of data. One question came to mind while testing my program. The Automation..Run Scripts button, can it be re-labeled to be something more descriptive to the app, Like I would like Functions...Run I looked around the model editor and could not find where I could change that.
No way to change the name of the button.
You will probably want to use Schedule scripts if all you are doing is going to the web and getting data. Action scripts are typically created to respond to a action taken on a record. Like if someone saved a new record and they wanted a script to run. Or if they set a particular field to something they can have set a script to run. It is based on scenarios. That is why you need to select a record in order to run a script.
Schedule actions do not care what is happening with the end user. It just runs on a hourly, daily, weekly, monthly basis.
Like I said earlier, you can preload data that will be installed. The only thing is any data you add after they install won't be carried over.
If this is the Clickbank program, I would think about having that data on my webserver. The new version will be able to access that data on your webserver. So the data will be there when they open it up and all you need to do is update your webserver (can be done through the automation) and they will get the updated data.


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A PLAY park has been saved from housing development after a dramatic U-turn by council bosses - but two other recreational sites remain on the chopping block.
Councillors decided not to proceed with the sale of land in Staines Lane, Chertsey, due to flooding concerns, contradicting months of previous decisions in favour of selling.
But green space in Marley Close, Addlestone, and Marshall Place, New Haw, will be sold off.
Village green bid to protect open spaces
During a lengthy debate at a meeting of Runnymede Borough Council's corporate management committee last Thursday (June 28), councillors apologised to residents for the manner in which the land sales had been handled.
Cllr Peter Waddell said that while the process followed by officers may have been adequate in the eyes of the law, "ticking all the boxes" simply "isn't enough".
He said: "Really the whole thing has escalated into a saga. We need to be very clear about what transparency means. I think we have let our residents down here and I think we have let our elected members down and I think we need to do far better."
Councillors were told there was a "serious risk" the Chertsey site would not be suitable for development due to the potential for flooding in the area.
The vote was unanimous in favour of saving the land, as were the decisions to sell the two remaining sites.
However, it was not all bad news for families around Marshall Place as the sale of the land in New Haw was made subject to an adjacent piece of green space being brought into public use.
All of the decisions will be taken as recommendations at a meeting of Runnymede's full council later this month.
For more on this story, see this week's Surrey Herald, out on Thursday, July 5.


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Introduction|Intro|Background} Hero Market Alam Megah
Within this Market Hero review, I will show some email marketing secrets which are so great, you are going to want to crawl buck-naked across broken glass simply to find them. Hero Market Alam Megah
But , I wish to say"thank you" into Alex Becker, the founder of Market Hero.
I started watching Alex Becker's YouTube movies back in 2016. He taught me the best way to run a Shopify shop, and a lot of stuff regarding entrepreneurship, online advertising.
It was right around the time that Alex published his book, The 10 Pillars of Household: Mind-Sets of The World's Richest People. I got that book once I saw it and I jumped to highlight it and mark it up. I could move re-read it as soon as I finish this.
Truly, Market Hero isn't even his sole multi-million dollar company. He founded the search engine optimization software development firm Source Wave and today he's running a supplement manufacturer new. He got a couple of courses out there from the marketplace teaching individuals market more about Shopify and to grow their email lists.
The dude is a vending machine. My hat's off to you, Alex.
Sign up for the free trial here: Market Hero
What Is Market Hero?
Anyhow, Market Hero is one of the best email Autoresponders available on the market nowadays and it is so much better than its rivals because it focuses on in-depth revenue metrics.
I have used a bunch of email autoresponder software over the years, such as MadMimi and Drip. Drip has been my favorite up before I started using Market Hero. Drip allows you like monitor your open rates to do a lot of stuff and tag your readers.
But if you're anything like me, while you care about open prices and click-through rates, the thing that matters most is SALES. Earnings are the lifeblood of your business and earnings is what places cold, hard cash in your pocket. Market Hero helps you monitor sales, plain and simple.
Market Hero Support
While I didn't have to use Market Hero service that They return to me Much because everything is, the couple times that I did reach out to them. Hero Market Alam Megah
The service isn't amazing to the point at which they provide you with detailed step-by-step videos and hold your hand through each step of the way, but it is not terrible. In any case, if you want that level of hand-holding and coddling, possibly Market Hero is not for you.
Market Hero has English-speaking support team available to respond to your queries through live chat at no cost. You do not need to be a part of any plan or upgraded package to possess priority service, like some companies do. You just log in and begin chatting.
Could Market Hero Truly 4X Your Enterprise?
On Market Hero's homepage You will see states,"4X Your Email Business With Unequaled Delivery and Sales Metrics". This immediately set my B.S. detector because I understand how hard it is to 4X an email arrangement, especially if you're any good at what you're doing.
But Market Hero offers a free trial so I figured I'd register and see whether it was any good. I figured I only require a little increase in earnings for Market Hero to pay for itself and if it did 4X my enterprise, I would be flying high.
Now, in the interest of full disclosure I really do want to tell you Market Hero has NOT improved my earnings 4X (yet), but I am getting more sales than before and Market Hero IS paying for itself. Easily, actually. I had my own game tight and was already doing well, so that I understood a 4X increase could be rough.
But if you are new to email marketing or if you have a smaller internet company, Market Hero could probably 4X your sales. Here's the way:
It monitors your customers' lifetime value.
I know I already mentioned this, but the key to Market Hero is how it retains your customers' lifetime value (LTV) in front of you at all times and it changes as you send out emails and tweak your own autoresponder. Once you know your client value, you can literally"buy" customers. What Market Hero does is actively track every sale and each conversion for every guide on your listing. It then averages everything together and gives you the stats you need to cultivate your organization.
If you were to do this manually, you would probably have to go through different softwares, different spreadsheets, and go back through your previous earnings. You may need to crunch a lot of amounts that you most likely don't have anymore. Market Hero solves this problem better than anybody else on the market.
You're able to segment using tags.
Subscribe to the free trial : Market Hero
I know that you're probably thinking,"Tags? Okay… big deal." But the manner that Market Hero allows you to use tags is better. Here's why…
Let us say that I'm running two different campaigns for the same audience. Anyone who opts-in into the first campaign will be labeled as"A" and anybody who opts-in to the second campaign is going to be labeled as"B".
I will go into Market Hero and filter out all of the"B" opt-ins and find out just how much they are worth compared to"A" opt-ins. When I find out that the"B" opt-ins are earning more money, I know that I should be pouring more ad dollars into advertising the"B" opt-in. Hero Market Alam Megah
Should you do this again and again, you can create a powerful business as you'll always be split-testing to see which types of individuals are worth more to your business.
And getting this up and running is super-easy too. All you have to do is choose the pixel which Market Hero provides you and put it in the areas that you want to track. From that point, Market Hero will keep tabs on all your clients, sales, conversions, etc. to give you the data that you need to cultivate your business.
You can integrate with a lot of other services.
Inside Market Hero, there is a button called"integrations" that – you guessed it helps you incorporate with things like ClickFunnels, SamCart, Shopify, Leadpages, Zapier, and more.
I think this is a smart move by Alex Becker because it indicates he understands his market. He understands that are using things and he would like to make sure his software integrates.
My only criticism is that a lot of the software I use, such as OptinMonster and Thrive Themes hasn't yet been put in the"integrations" tab. That does not mean that you CAN'T incorporate them, because you can (you utilize Zapier to make them speak to each other) but it's not as simple as something like ClickFunnels or Shopify.
Speaking of Shopify…
How Is This Different From Different Autoresponders?
The best method for me is of The other autoresponders were built for email marketers. Their thinking has its own limits and I send emails although I've nothing against email marketers. Market Hero was built by means of a company owner FOR company owners.
Market Hero differs since it provides you with a lot of different ways that will assist you track ROI and generate more profit from your emails.
By way of instance, among the coolest features is the fact that it shows you just how much each lead is worth. Imagine logging in to your email autoresponder and seeing that every guide is worth $6.87 to you. Meaning that you can go out and"buy" email addresses to create money. Everything you do is you purchase paid visitors from Google, Facebook, etc. and send that traffic to a opt-in page. You get a business, if it's possible to get a successful than it's worth. As you can simply wash and repeat as many times.
Additionally, it also lets you segment your list in far deeper ways than other autoresponder services out there. I utilized Drip plus they have some decent segmentation tools from the ability to apply tags to your prospects like I said, but it's small potatoes in comparison Market Hero. I don't need to give any of Market Hero's proprietary info, but let us just say that you will most likely be pleased with the segmentation ability. Hero Market Alam Megah
Is Market Hero a Scam?
From what I have seen, absolutely not. I think that At this point, by being a scam artist Alex Becker has too much to lose. Businesses's running and he is smart enough to realize that if his reputation was tarnished by him, he'd be screwed.
From what I have seen, people have complained about his refund policy. However, these individuals are the individuals who have utilized Market Hero for weeks and attempt to get all their money back. These are the scammers. You are scum, if you aren't willing to cover for a service that can improve your organization and your life. Plain and simple.
Market Hero's free trial offer is pretty straightforward. It says that in case you cancel within 14 days then anything won't be charged.
Rest assured, gentle reader. Market Hero is untrue.
If You Have Shopify (Or Any Online Business…)
When I began writing this Market Hero review, I Was worried it was going to lean a bit too much towards Shopify entrepreneurs. That is why I want to stress that Market Hero will assist you in ANY business, not Shopify.
On his YouTube station, Alex has published a lot of different videos about Shopify. When you get beyond the titles that are clickbait-style, you find that he provides a lot of info about the best way to earn money. Part of the reason that he does so is because he needs to get folks to check out Market Hero because he knows it might help them with their Shopify company.
By way of instance, Market Hero lets you create abandoned cart strings. When someone begins the checkout procedure in Shopify (by entering their email) but does not finish it, that gets tracked in Market Hero. From that point, you can follow up with that person and get them back to complete their purchase.
The capacity to create those abandoned cart strings is just one of the ways that you can earn Market Hero literally cover itself. Let's do some math…
Depending on the analysis that you read, the ordinary cart abandonment rate for an internet shop is generally somewhere in the neighborhood of 70-80 percent. Are there individuals with much lower abandonment rates? Sure, but this is the average.
Imagine you have 100 people go through your checkout process and 80 of these individuals abandon your cart. That means you have 20 clients and 80 non-customers. What if a cart recovery sequence only got a 10% of this non-customers to purchase?
That would get you an extra 8 customers. This means that you would literally boost your sales 40%! (Because 8 extra sales is 40 percent of the 20 earnings you had.)
In the event that you boosted your sales 40 percent, would that justify the investment in Market Hero? But that is not all…
Market Hero also allows you to make upsell sequences. Can you create such sequences? Yes, you can. I am totally transparent about that, but it is nowhere near as fluid and it doesn't give you that crucial lifetime customer value. Hero Market Alam Megah
The motive Market Hero is so powerful is because you're able to apply tags and track LTV. When you connect Market Hero into Shopify, it starts monitoring immediately. Buy tags are implemented that show.
Imagine you have a Shopify shop selling print-on-demand dog shirts. It'd be silly to attempt to sell Golden Retriever stuff to this person if someone purchased a pug shirt? But that is exactly what a lot of entrepreneurs do because they don't have the information in front of them. Because Market Hero enables you to determine what the individual bought, you can focus on selling only stuff for your pug customers.
You are able to totally geek out on this stuff. You can get as comprehensive as intricate as you like. In my own business, I use it to section the subjects that advisors are interested in. I don't want to bombard them with emails about GETTING clients, because they'd be immaterial Since I sell products that assist financial advisors GET clients and let them KEEP customers, if someone is interested in KEEPING clients. That person would finally unsubscribe.
Oh, and if you're interested, here is a movie of Alex Becker showing you ways to raise your own Shopify revenue 300% with Market Hero. Keep in mind the Market Hero may be used with ANY business, not Shopify.
Market Hero is an Wonderful email software because It's built for people who are attempting to earn money on the internet. If you haven't completed So already, go over there, import your list and make the most of the free trial.
You can't afford not to understand your numbers for another month. That's like Driving blind. If you don't understand your numbers, you are leaving a huge Amount of money on the table. Imagine how much more money you could make if you Knew precisely what every lead was worth. How would that change your decisions? How would that change your life? Hero Market Alam Megah
Subscribe to the free trial : Market Hero


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The Duffer brothers had so many good ideas for Stranger Things season 2, that they had to shelve some. The first season of Netflix's '80s themed sci-fi show saw Will Byers being abducted by a Demagorgan and spending time in the Upside Down, while Eleven, a young girl who was being held as part of a scientific experiment, made her way into our world and became friends with Will's classmates. With a strong ensemble cast, great plotlines and outstanding direction from Matt and Ross Duffer, Stranger Things was renewed for a second season, set to arrive on the streaming service this Fall.
We already know some of plotlines that Stranger Things will examine in its second season. This includes seeing the effect that the Upside Down has had on Will, who was seen vomiting slugs at the end of last season. Eleven is hiding someplace, Sheriff Hopper is leaving Eggos out in the woods for her, and there are new monsters lurking. The Hawkins National Laboratory is now under new management, though our guess is there's still a lot of sinister goings on up there.
In an interview with Mashable, executive producer Shawn Levy talked about Stranger Things season two, including the fact that the Duffer brothers had so many ideas for the season, that some have been saved for (hopefully) future seasons:
"As the episodes were written by (the Duffer Brothers), they realized that there was too much story for nine episodes. So it forced us to be judicious in which stories we tell this season. So one of the surprises was, not all of our grand ideas are going to be serviced in one season…If we had 40 ideas going into the season, 30 (we) are going to pick, and the rest go in the back of our head for, hopefully, future chapters."
Though the plot of Stranger Things is undoubtedly what got people watching in the first place, viewers stayed based on the strength of the cast; especially the young kids who are at the center of it all. Levy confirmed that Will (Noah Schnapp) is very much at the center of the second season, and added that the show will continue to root itself in character:
"As much density of story as our show has, we have to service the characters — and the moments and relationships between characters — above everything. If we stop being character-anchored, and we become pure genre, I think we take the beating heart out of Stranger Things, and we take the thing that people maybe don't write articles about the most, but I think it's what they love the most — these characters on screen."
Levy is correct; while most articles focus on what's to come in terms of plot, it really is a character-driven show. Season one provided many moments of light relief, as well as lots of emotionally heavy scenes between the whole cast, which is a big part of the show's charm. Going forward, it's good to see that the Duffer brothers are acutely aware of this, so much so that they know it's better to save some plotlines for a later date in order that the characters they've created, and the cast that play them, can shine.
NEXT: What to Watch For on Netflix in 2017
Stranger Things season 2 arrives on Netflix in time for Halloween 2017.
Source: Mashable


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Brian Horan - Agent
- NameBrian Horan
- Cell216-906-8082
- Office440-389-3062
- E-mail Me
- Visit My Website
About Brian
Hello, my name is Brian Horan. I have been in sales and finance for the last 10 years. I also have an extensive appraisal background. My #1 motivation is to build long term relationships with my clients. I look forward to the opportunity to work with you and would appreciate the opportunity to do so! Have a good day!


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CCTV Monitoring Services
Outsourcing CCTV Monitoring
Trusting that your property is secured 24/7 is a difficult task and we would rather you allow us to monitor the security for you. Outsource cctv monitoring service is one of our areas of specialization at SupportNinja. We will meticulously tailor a plan to protect your property and your employees so that you can feel safe and secure while attending to business. The service we provide includes ensuring that you receive periodic reports on the standards being maintained and any occurrence of a breach or non-compliance to safety regulations. Outsourcing cctv monitoring is a valuable investment when you use SupportNinja. Our recipe for effective CCTV monitoring outsourcing has three main ingredients prevention, protection and the cure.
1. Prevention
Our systems will allow us to use our intelligent and trained monitoring experts and effective tools to prevent any security breach.
2. Protection
Our safety mechanisms are built to lessen your exposure to any criminal or fraudulent activity, both internally and externally. Our methodology also ensures that employees on site comply with safety and security regulations.
3. Cure
In the highly unlikely event that there is an unexpected breach of security or safety protocols, you can expect a detailed report of the occurrence, the measures that were taken to prevent the activity and the reason why the incident was not preventable. In any event, you will be informed of irregularities and the authorities will be alerted to intervene when necessary.
Advantages of Our CCTV Monitoring Service:
Effectively resolving emergencies
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Preventing criminal or accidental loss
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Assessing regulation compliance
When you are serious about protecting property, securing lives and maintaining standards you will make every effort to mitigate against losses. When you're ready to make this step get in touch with SupportNinja.
If I had to choose 1 word to describe these guys, it'd be BADASSES. I make and review apps across the IOS and Android platform and found myself struggling to keep up with my users so I could effectively improve each apps performance. We discussed a few possible solutions and although I was hesitant at first, I'm glad I made the leap. Today I've got 3 full time guys working for me and they all sit right next to eachother. 1 guy does dev work and fixes user reported bugs, another helps me with app reviews and the ladder handles full-on support mon-fri. Every monday we have a team meeting and they go off and do their thing for most of the week. I typically don't write reviews but these guys are amazing with everything they've been able to offer and I look forward to growing my app business with them every step of the way!
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The demon again comes to Aksinya that evening. This time he comes with a gift. Isn't this demon clever?
In the middle of the night, a knock at Aksinya's door woke her. She sat up and answered without thinking, "Come."
The dark heavy shape of the demon entered her room. He was more massive in the darkness than she ever remembered, "Countess, as I promised, I brought your mother's jewelry box. She had more than one. You were not specific so I brought the largest."
Aksinya's voice trembled, "That will be sufficient for now. What about my sister's jewelry?"
"I have not been able to find it."
Aksinya wanted to cry out, "You liar." Instead she took a deep breath, "Bring it when you can find it."
"Yes, Countess." The demon still held the ornate box, "Where should I put it?"
Aksinya scooted toward the head of the bed, "Here on the bed."
"It's heavy."
"Do I need to repeat myself? Place it on the bed."
The demon's fangs were evident in the light from the streetlamps. He might have been smiling. He placed the box near the middle of Aksinya's bed. Then he stepped back, "You should not leave this in your room."
"Why not, demon."
He snarled, "Frau Drescher will find it."
"Will she steal it?"
"I wish she would. That would be a definite sin. In any case, I do not wish any other undue attention toward you at this point. In this place, they worship your aristocracy. This is a pleasant little lust for all of them here. A little more mystery might enhance that mystique at some point, but not now."
After Aksinya works on German with Natalya, she is sleeping. The Demon waits, this time, for her summons. Do you see how he is playing her. At one moment demanding, the next responding as her servant. All the demon's actions are intended to make Aksinya follow his purpose. The play here, is we think the demon doesn't fully understand Aksinya's actions. That is a reasonable thought. I will go further and reveal, the demon can't read her mind. I think I mentioned this before. There are secrets Aksinya can hide from the demon, therefore, the demon's actions will be to try to force the secrets from her. We will see some of these tricks later. Right now, the demon is accommodating Aksinya because he believes this will aid in his purpose. The demon doesn't realize that Aksinya intends to use the jewelry boxes to discover more of his bounds (weaknesses).
Aksinya trembles--it isn't in fear. It is in excitement. She will now know the answer to her question--can the demon touch or move evident crosses? This is an incredibly important question to her. The answer means that Aksinya might have another specific means to oppose the demon. That's why the question about her sister's jewelry. At that moment, Aksinya knows the truth. The demon can't move an evident cross--therefore her reaction.
Now there is a set up for a problem for Aksinya. You don't get to see it for a little. The demon asks, where he can put the box. Aksinya is so excited, she doesn't stop to think. The demon's smile should have warned Aksinya. Then Asmodeus tells her not to leave the jewelry box in her room. We'll see more about this tomorrow, but you might wonder about the demons statement. Aksinya does, she asks the obvious, but not the best question, and the demon's answer is Frau Drescher.
Then the wake up statement from Asmodeus that we will address from Aksinya tomorrow. He wishes Frau Drescher would steal the jewelry, but that would cause undue attention toward Aksinya. His point: they worship her aristocracy. This is very evident, but the rest is interesting. "This is a pleasant little lust for all of them here. A little more mystery might enhance that mystique at some point, but not now." This is interesting enough that Aksinya ponders it tomorrow.


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DIY Shirt by yours truly.
I really had fun cutting out this old Mango shirt from my college days and turning into a skull masterpiece. hehe! I saw similar shirts like these and they sell it for a whole lot of $$$! Why not make a DIY out of old shirts and a pair of scissors? =p
Gold Dot gold clutch/ WAGW gold necklace
Forever21 leather pants
Forever21 leopard pumps
You might all be wondering how harsh the title of this post. haha! Well, its a line from one of my favorite tracks from Lana del Rey. If you're a fan, you'll know which part i got it from. I love her, I never got tired of her songs 'till now.
Hope you like today's post guys!


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George Bush has pipped John Kerry to the post of US Prez. We don't want to get into the realpolitik of it all, but Indians at large aren't certainly happy with what has happened to the Candidate-with-his-heart-in-the-right-place, John Kerry!However, those in the tech industry are happy that outsourcing will continue under the Bush administration.
Says fashion designer Tarun Tahiliani, who has just opened a designer mall in the upmarket Defence Colony, "I am disappointed. I was all for Kerry's win. I think Bush was a very retarded President. Have you watched the film, Fahrenheit 9/11 ? It has a lot to say about Prez Bush", said Tahiliani.
The Indian IT industry Thursday expressed delight at US President George W Bush's re-election, hoping he would continue to be a proponent of free trade and outsourcing.I guess those of us who voted for Kerry should move to India.
"The US is a powerhouse of the global economy and the Indian IT industry is delighted in being a partner to US corporations, helping to make them more competitive," India's National Association for Software and Service Companies (NASSCOM) president Kiran Karnik said in a statement.


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Their social media profiles may resemble The Rich Kids of Instagram but our 3 young cover personalities are enterprising and inspiring millennials who will stop at nothing to achieve what they set out to do. Presenting for the first-time ever, 3 of Chennai's youngest successful businessmen on the cover of South India's largest lifestyle magazine, RITZ.
Photographs: Ganesh Toasty
Location: Feathers Hotel
Styling: Sunil Karthik (Team Rehane)
Special thanks to leading fashion designer Rehane for her inputs and ideas
Hair and Makeup: Page 3 Luxury Salon and Makeover Studio, Khader Nawaz Khan Road, Chennai
Desi Dan The Business Man – Thirukumaran Roopkumar
Thirukumaran Roopkumar hails from a family that is steeped in the business fabric of Tamilnadu. So when he says, "It's not about making it, the key is maintaining it," you know it harks back to the values he was raised with. Still, not one to hop on solely to the family business bandwagon, Thiru, as the handsome 25-year-old businessman is fondly called, has started his own venture TRK Global which has just forayed into the sports arena among other fields. Quizzing one of Chennai's most eligible bachelors on a range of topics from his expansion plans to workout routine and his charm offensive with the ladies, Diya Subramanian captures this spicy chat.
2017 has started off as a blockbuster year for Thiru with the launch of his venture 'Turf 137' football arena which guarantees a five-star experience for footballers. The idea struck him over a casual conversation with friends and today stands transformed into South India's finest of its kind, for which Thiru has spared no expense to ensure comfort and safety for players. The turf area is FIFA-approved and imported from Amsterdam; this is in stark contrast to the cement flooring underneath turfs at most arenas, which could lead to injuries in the long term. "People start playing football as early as 4 years of age. I don't want my customers to hit age 40 with knee problems and multiple injuries because the turf they started out on compromised on quality. The highest standards of quality adherence is an essential in any business I do." The space for Turf 137 comes from one of Thiru's sprawling homes where the family used to live before they moved to their current residence in Adyar.
As a business proposition, Turf 137 has already received a fair share of interest from potential franchisees. Thiru says, "Yes, we are planning to franchise; we already have three offers that we are considering."
As we chat further sitting in the plush and verdant environs of Chennai's poshest club, The Madras Club, I can't help but notice the number of older people who walk up to Thiru and exchange pleasantries with him with a lot of fondness. "Guys my age usually spend time with those in similar age groups but I am a total people person. I've always been a good listener and I take keen interest in the lives of people I interact with."
Thiru's friends call him Desi Dan, after the American Instagram superstar Dan Bilzerian.
The topic takes us back to his family and Thiru's eyes light up when he talks about the man who he adores and is inspired by – his grandfather. "People tell me I was born with a golden spoon. But my family never believed in spoiling me with privileges. My upbringing has been quite grounded and rooted in culture. For instance, my grandfather always taught me to be humble. He was in fact the man who was instrumental in making SPIC a formidable industrial giant in those days. Despite the 20,000-strong workforce, my granddad was the kind of leader who would know the security guard at the gate by name. I admire him for the respect with which he treated others which they reciprocated to him multifold." Considering his family legacy, Thiru learnt the trappings of business at the family's polyurethane foam manufacturing unit. "That was my first job. We manufacture mattresses, cushions and foam for the domestic and automobile sector. I also dabbled in our real estate business and in the textile sector, where we are one of the largest manufacturers for H&M in addition to supplying to Diesel, Hanes and other international brands."
So what prompted him to branch away on his own instead of staying put in familiar well-heeled territory? "I still run the fabric export division in the textile business. But I started TRK Global because I have always been a guy who is independent – I have always wanted to stand on my own feet. There is tremendous expectation from me, in terms of my success in business given my family's goodwill. Well, what can I say? I am all for challenges and meeting those expectations!" So what is the future for TRK Global? "Next up for us in the short term is a couple of bars in Chennai – high-end and upscale. The future for TRK Global would be in the fields of Energy, Construction and Real Estate."
But a little birdie told us that Thiru has already been offered acting roles and is considering them. So where does cinema fit into his game plan? Thiru laughs, "I have always had a fascination for Tamil movies since 8th grade. I have always loved acting, it's a passion I have never explored. Yes there have been offers but the timing has never been right. I have always been groomed for business so before I decide to sign on for a movie, I would definitely like to stabilise my new ventures. Acting is definitely on the cards though. Even at my workplace, I am constantly goaded by my team to get into movies. They even tell me they will be my fan club president, secretary etc.(laughs)!"
Tough indeed to resist so much temptation and pressure.
But taking it slow with regard to an acting career hasn't stopped Thiru from dating actresses, as we hear that he has been been romantically involved with at least 2 South Indian heroines. So how can one interview one of Chennai's most eligible and handsome bachelors without broaching the topic of his romantic life? We hear Thiru's friends fondly call him Desi Dan, after the American Instagram superstar, poker champ and trust fund beneficiary Dan Bilzerian. True to his nickname, we are told by reliable sources that Thiru once flew down Swedish and European models who are his friends and partied with them in the city with his gang of Indian pals! Apparently, his mom even ribs him quite a lot joking that his list of girlfriends spans the English alphabet twice over! So I coax him to spill the beans. In true movie star-Casanova style, Thiru replies with a big smile which breaks into a chuckle, "No comments, please." I refuse to relent. He offers, "See, I am a diehard romantic but I've also had my heart broken. You live and learn, as the saying goes."
Taking it slow with regard to an acting career hasn't stopped Thiru from dating actresses, as we hear that he has been been romantically involved with at least 2 South Indian heroines
With his hectic schedule, Thiru makes it a point to stay fit, spending 2 hours in the gym least three a week and is also a champion swimmer. "I like to look good. A big part of looking good, as you know, comes from feeling good. Workouts and keeping fit are essential to me. But I must say, my workout regime tends to shock people. I've been known to do pull ups with about 25 kilos suspended to my waist. Upside down crunches with weights are my thing. Naturally, I get a lot of eyeballs (laughs).
Chennai's very own Dan Bilzerian is sure making the city look good on the sports map and has exciting plans to make South India a lifestyle and entertainment hotspot. Well, we can't wait!
Quick Takes:
Favourite Holiday destination?
Maldives, because it's my scuba diving paradise. London would be one place I would like to settle down in for sure. I spend at least 3 months of the year in New York as well.
Favourite places in Chennai?
Radio Room and Q Bar are regular hangout spots. Prego at the Taj Coromandel is my place to eat.
Favourite TV show character?
I am a big fan of Harvey Specter from Suits. And my current favourite show is the Night Manager.
Favourite business quote?
"Do what you love and give it your very best"
Being Young is: "Exhilarating. You can't expect to be old and wise if you've never been young and crazy"


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Maintenance Load Bank Testing Load bank testing of standby gensets is a preventive maintenance practice for an emergency power supply system (EPSS). The testing is primarily meant for diesel-powered gensets. The main purpose of this testing is to help ensure the gensets reliable operation during utility power failure. If this testing is performed regularly, it […]
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In a mark of solidarity, at 7 o'clock this evening, church bells rang out across the nation. The scenes witnessed this week in France haveRead more
Category: International
Brexit Update: Flextension, European Elections and Legal Complications
On this political update, host Douglas M. Worth and Wesley Manta, review the latest Brexit news.Read more
Brexit: MP's reject May's 'deadline day proposal'
Today, 29 March 2019, was the original deadline for the government to deliver Brexit, but instead another series of votes in parliament on the UK's future relationship with the European Union took place without coming to an agreement.Read more
We Stand Together: Lincoln citizens unite in light of New Zealand terrorist attacks
A vigil took place in Lincoln outside the Lincoln Central Mosque & Cultural Centre on the 22nd March. Amber Marshall event organiser and University ofRead more
Women's History Month and a Reflection on the world today
We're midway through March which is Women's History Month celebrating the amazing achievements of women around the world.Read more
Help underprivileged children this International Book-Giving Day
International Book-Giving Day returns this Thursday, with the aim of giving books to under-privileged children from around the world. Founded in 2012, it asking people toRead more
Lincoln Japan Festival returns, encouraging the community to embrace a different lifestyle
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Welcome the year of the Pig
Celebrations have begun all over the UK to bring in the start of the Chinese New Year. Tower bar in Lincoln hosted an eventRead more


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Democratic congressional candidate Dan Helmer's latest TV ad starts out normal, which is to say boring. "I'm different: I'm not a politician. I'm a Rhodes scholar who's served in combat," he tells the camera. OK, cool. "I'm for a Medicare for all option, and against drug companies that rip off seniors. I'll fight the gun lobby to protect children, not guns," he adds. Alright, alright.
Then the ad takes a turn:
"After 9/11, the greatest threat to our democracy lived in a cave," Helmer, who is running in a primary for Virginia's tenth congressional district, says as footage of Osama bin Laden appears. "Today, he lives in the White House. No one, even the president, is above the law."
Hmmmmm. HMMMMMMM.
First of all, you might point out that the notion that bin Laden and al Qaeda were existential threats to the US is the kind of alarmism that led to the worst excesses of the war on terror. Bin Laden and his followers killed a lot of Americans, but there were never going to destroy America. Terrorism, then and now, is extremely unlikely to be the reason you die.
But mostly, it seems like a bad strategic move to compare Donald Trump to the most infamous terrorist in American history. The White House said the ad, which first aired Wednesday, was "reprehensible"—which it is—and called on House Minority Leader Nancy Pelosi to denounce it. Her spokesman said that "while the leader does not condone the end of this ad, if the president wants to join in raising the level of civility in politics, he should begin with himself," a fairly weak attempt to turn things around on Trump.
Helmer is no stranger to making terrible political ads. Before this, the Army veteran was maybe best known outside Virginia for a web-only commercial where he rolls up to a bar on a motorcycle, then sings an extremely lame parody song called "You've Lost That Centrist Feeling" at someone who is supposed to be Republican incumbent Barbara Comstock. It's under two minutes long but the poor production values make it feel much longer. There were also some Top Gun references in the spot, which was so bad Stephen Colbert took time to make fun of it.
This new ad at least looks like something a professional political campaign made—as it should, since according to OpenSecrets, Helmer has raised over $1 million for his campaign, more than all but one of the six Democrats competing seriously to challenge Comstock in the general election this November.
Dumbass campaign commercials crop up every election cycle, but this one highlights a broader problem facing Democrats in 2018 and 2020. While there's plenty of solid policy grounds for their anti-Trump fervor, it's easy for that to bleed into outright hysteria. Notably, in the wake of the controversy, the Helmer campaign doubled down on the message, telling Politico, "What is abhorrent is the way this President has failed to uphold his oath of office, to preserve, protect and defend the Constitution of the United States."
If your goal is to nudge American politics in a more positive direction, to push back against the extremism and demagoguery of Trump and many other Republicans, it seems like comparing Trump to Osama bin Laden is exactly the sort of thing you shouldn't do. It may serve to convince voters that both parties are consumed by partisan hatred and there's no meaningful difference between them. The rest of the ad, while not exactly the kind of thing that makes the national news, actually does represent the kind of positive message liberals should get behind: healthcare for all, pushback against drug companies, a real effort to change gun laws.
That these worthy causes have been overshadowed by a head-ass 9/11 comparison suggests the "Trump derangement syndrome" thing Republicans like to complain about is real, and in a worst-case scenario might even cost Democrats their shot at taking back Congress.
Follow Harry Cheadle on Twitter.


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Is New Construction in Naples New Communities causing a glut in homes?
Posted by Joseph Epifanio on Monday, February 20th, 2017 at 10:43am
If Real Estate is your passion and is also what makes your daily bread, then when my Real Estate Advisors tell me that there is a large number of homes available on the MLS inventory and that sales are slowing down, it makes sense to look more closely at the figures right?
But figures can be elusive, manipulated and pretty much made to say what you want them to say. So I decided to take another tack, yes it would appear that there is a large number of existing homes on the market for sale. So why are they not selling? I took a look at the latest figures from Collier County, particularly at new construction and new communities. As a town, Naples is growing, anyone driving around would be able to see the huge amount of new construction that is taking place. After the last downturn in the economy, everything stopped, construction almost came to a standstill and half-built communities had empty homes with very little activity going on.
Now compare that with the number of new communities recorded by Collier County in 2015 (43 communities) and 2016 (46 communities), already 2017 has seen 8 new communities and it is only early February. So lets be cautious and half that number to say 4 new communities for January 2017, if the trend continues, then it looks like 2017 is set to have a minimum 48 new communities, and if you took the 8 already proposed then it could be as high as 96 new communities to be approved this year. Remember, of course, figures can be manipulated, it may be that those 8 new communities are the last ones to be approved and the trend stops there, no more building and no more new homes, there are always extremes right? What we do know is that somewhere in the middle is the figure for the number of new communities that will be approved by Collier County this year.
So where is all this leading? My Advisors tell me there is a large amount of property for sale in Naples Florida on the market that is having a hard time selling, some of them interpret that as a slowing of the market. While that may be true to some extent for existing homes, the number of new homes being built definitely puts up a defense to that argument.
What we do know is that if you have to say $500,000 to spend on a home, when you compare an existing home to a new build, then there is a lot going for the new home. Newer building techniques, lower running costs, very low maintenance, no roofs to replace and of course all the model homes look like a million dollars so they heavily influence the buyers' decision on which home to put their money into.
As a Realtor I know that the things that appeal to buyers are often superficial, in a model home, for instance, designers make the most of what they have, they put in the top of the line appliances, tile choices, and options. When buyers go to actually buy their new home, what they have in mind is the beautiful model home, what they actually buy is a downgraded version of the model, which in some cases will never become the perfect home they first envisaged.
On the other hand, yes there is a glut in the market of older homes which are being passed over for the shiny new version. But, what I tell my agents is this, the location has always been and always will be the number one influencer for a buyer. With so many of the new communities in Naples being built out of the East side of town, the drive to the beach becomes a consideration, as does easy access to shopping and nightlife.
So if you are a Realtor representing a home for sale, the most sound advice you can give your customers is to upgrade, either spend some money to make the home more modern, or drop the price to compete with the newer homes, the only way to do that is to price the home so that the buyer has funds left to be able to modernize the home themselves. If the home is in a better location and has had the upgrades already made, then those sellers are going to be in a strong position to compete with the newer homes. If the Realtor and the seller get the price right to sell the property in Naples in the first place, giving buyers the opportunity to do a little work and have the home they want in the area they want, then the older homes have a better chance of selling.So when I am told that there is a downturn and a large number of homes on the market, the truth is that homes are selling are the newer homes. With land in Naples being a vanishing commodity, eventually the new Naples communities will be being built too far out to be attractive, then the balance of new and old will be addressed. But until then, getting the price right in the first place for both sellers and buyers is the only way to compete.


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Class 3 Snow Learning
Please complete the Multiplication and Division work I have uploaded. There are different files for Year 3 and Year 4 with a range of activities in each file. Also, please can you log into TT Rockstars or Doodle Maths and complete some practise.
I have uploaded a file for your English work, it is linked to The Hodgeheg and our instruction writing.
Please practise your spellings on Spelling Shed or in your spelling book. In case anyone is struggling to access Spelling Shed, I have attached the files with the spellings in on Class Dojo.
There is also an activity below for you to complete linked to our Science topic, Forces.
I have uploaded some general snow day sheets. One is a review of your snow day, to let me know what you have been getting up to.
The next has different challenges on in a bingo grid which you can complete.
Have a lovely day and I can't wait to see you all tomorrow!


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- Electro plating technology
- Multiple plating material options
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- Complete and consistent part coverage
- Get the right plating for your application at the most competitive price
- Highly repeatable quality
Zinc Phosphate Coating for Fasteners
Elgin Fastener Group has numerous capabilities to offer its customer base. From hot and cold forging fasteners to wire form to closed die cold forming, we have the answers to improve your overall business performance. One of these Elgin capabilities is metal finishing. Poor planning or missed deliveries from plating suppliers is not acceptable. Elgin …
What is NIST-Certified?
Elgin Fastener Group uses the latest in computer-aided design technology in combination with computer-aided machining to create the custom tooling needed to meet a company's precise wire product fabrication requirements. We have decades of experience on the shop floor to assure consistency and quality. The inspection process for wire forming is just as rigorous. Our …


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Key Features
Buy it now for
To reserve this car please call us on 0191 266 8686 and the reservation team will be happy to help.
Finance from
16in Alloy Wheels, Electric Heated Front Seats, Air Conditioning, Bluetooth Connectivity, Cruise Control, Multi function steering wheel, Heated steering wheel, Automatic headlight elevation, Electrically Operated Front Windows, Auto dimming rear view mirror, Heated Windscreen, Digital Radio, Hill Start Assist, IntelliLink Audio System, Rain-Sensitive Windscreen Wipers, Tyre Pressure Monitoring System, Anti lock brakes, Anti theft system, Electric door mirrors, Cloth upholstery, Folding rear seats, Leather steering wheel, Trip computer.
Henson Motor Group have been supplying cars to the UK since 1996. We are committed to supplying quality new and used cars at competitive prices. We offer the best in part exchange prices and can also settle your existing finance agreement. We have a range of finance packages to suit all budgets, we also help customers with poor credit history. Each car comes with a free RAC warranty, 12 months MOT, full vehicle history check and a pre-delivery inspection. We don't charge any admin fees. We have 100's of cars on display, with new vehicles arriving daily. Open 7 days a week till late. Please call for further information or to arrange a test drive. We look forward to speaking with you. (Showroom price £6,499)


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The first time I came across the name of Ignaz Moscheles was in the context of the "Sonntagsmusiken", organized by Fanny Mendelssohn at the Mendelssohns' mansion in Berlin. Moscheles, one of the greatest piano virtuosos of his time and a first-rate music teacher, was a regular guest at the Mendelssohns' and both Fanny and Felix visited him while he stayed on London. His friendship with Fanny's brother Felix led to his appointment as principal professor of piano at the newly founded Leipzig Conservatory in 1846. He had taught the 15 year old Felix in the 20s in Berlin, Edvard Grieg was one of his students many years later in Leipzig.
Moscheles composed six piano concertos, a symphony, an overture, a sextet, a septet and several sets of smaller piano works. His Piano Concerto No. 6 in B flat major (Op. 90), written in 1833 in London, also goes by the name "Fantastique". It was first performed earlier in 1834 at London's Philharmonic Society, where it was rather coolly received, in contrast to the enormous enthusiasm when Moscheles played it in Leipzig in October the following year.
Romantic dynamism
While the concerto's structure follows the traditional form in three movements, its many thematic links give it an overall structural unity highly original for its time. Robert Schumann was rather critical about this "unstable form", however he rated the concerto nevertheless well-written. Jerome and Henry Roche write in a piece for Oxford Music Online that "Moscheles brought a crisp and incisive touch to his own piano playing […] He admired the pianistic innovations of Chopin and Liszt." In his own compositions for orchestra he tempered thematic ideas is with an early Romantic dynamism, they say. "Pathos in general, and chromaticism in particular, are not overplayed, and his music is never sentimental."
The fanfare-like three-note opening of the piano concerto in B flat major shows the Romantic dynamism right at the beginning – a forceful start, immediately balanced by a lyrical theme. Ludwig van Beethoven may have served as an example, Moscheles was an eager student of the Vienna master. And this opening was one of the reasons I picked the piece for this post. Overcoming the last lethargic feelings that may linger on, out, out, into the fresh air, the sunshine, the lush green!
The Frankfurt Brandenburg State Orchestra has recorded the piano concerto and the first and last movement – allegro con spirito and allegro agitato – rip through any dark brooding like a laser beam, the pulse of the music compels you to move, to run, to spring, to dance, out, out, out! Now!
© Charles Thibo