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Empathetic Engineering: Watch my Keynote and a Shaper Spotlight from AltiumLive 2018
October 13, 2018 | 0 comments
I'm an Electrical Engineer developing open-source hacks, building robots, blogging, promoting sustainability, and heralding entrepreneurship. I believe that creative engineering is indistinguishable from fine artwork.
October 2015 - Present
Shaper Origin is the world'd first handheld CNC.

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The ubiquitous pannexin 1 (Panx1) ion- and metabolite-permeable channel mediates the release of ATP, a potent signalling molecule. In the present study, we provide striking evidence that ATP, in turn, stimulates internalization of Panx1 to intracellular membranes. These findings hold important implications for understanding the regulation of Panx1 when extracellular ATP is elevated. In the nervous system, this includes phenomena such as synaptic plasticity, pain, precursor cell development and stroke; outside of the nervous system, this includes things like skeletal and smooth muscle activity and inflammation. Within 15 min, ATP led to significant Panx1–EGFP internalization. In a series of experiments, we determined that hydrolysable ATP is the most potent stimulator of Panx1 internalization. We identified two possible mechanisms for Panx1 internalization, including activation of ionotropic purinergic (P2X) receptors and involvement of a putative ATP-sensitive residue in the first extracellular loop of Panx1 (Trp74). Internalization was cholesterol-dependent, but clathrin, caveolin and dynamin independent. Detailed analysis of Panx1 at specific endosome sub-compartments confirmed that Panx1 is expressed in endosome membranes of the classical degradation pathway under basal conditions and that elevation of ATP levels diverts a sub-population to recycling endosomes. This is the first report detailing endosome localization of Panx1 under basal conditions and the potential for ATP regulation of its surface expression. Given the ubiquitous expression profile of Panx1 and the importance of ATP signalling, these findings are of critical importance for understanding the role of Panx1 in health and disease.
- adenosine 5′-triphosphate (ATP)
- endocytosis
- endosomes
- pannexin 1
- pannexin1
- trafficking
Six individual pannexin 1 (Panx1) proteins come together to form channels that facilitate the regulated passage of Ca2+ and small molecules across the plasma membrane in cells throughout the body [1]. Panx1 channels are perhaps best known for their association with ATP release from cells and can be opened by several stimuli including mechanical stretch and depolarization of the plasma membrane. ATP, in turn is a key signalling molecule that acts through various purinergic receptors to modulate a number of important signalling events.
The ubiquitously expressed Panx1 is particularly enriched in the nervous system in developing [2,3] and mature neurons [4,5] and astrocytes [6,7]. There is a growing body of evidence that Panx1 plays key roles in regulating synaptic plasticity [8,9] and neural cell responses to oxygen and glucose deprivation [10,11]; recently reviewed in [12,13]. The factors regulating Panx1 surface expression in neural cells and therefore its influence on synaptic plasticity and neurobiology are poorly understood.
In the course of our studies on Panx1 in neural precursor cells and neurons, we observed a sizeable, stable sub-population of Panx1 on intracellular membranes, consistent with reports from several other groups studying both neural and non-neural cell types [14–17]. Whether this population resulted from diversion from the secretory pathway or internalization (retrograde trafficking) of mature Panx1 from the plasma membrane was unknown. We reasoned that stable intracellular expression resulting from retrograde Panx1 trafficking would require a stimulus that is episodically released from cells. ATP fits this criterion; it is constitutively released in episodic bursts from many types of neural cells [18,19]. In the present study, we tested the prediction that elevated extracellular ATP stimulates Panx1 internalization.
Cell culture
Mouse Neuro2a (N2a) neuroblastoma cells (procured from the A.T.C.C.) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin (all obtained from Gibco/Life Technologies). Human embryonic kidney (HEK)293T cells (procured from the A.T.C.C.) were cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. Where indicated, N2a and HEK293-T cells were transfected using jetPEI reagent (Polyplus transfection/VWR) according to the manufacturer's protocol.
A mutant Panx1–W74A–EGFP plasmid was created by site-directed mutagenesis of the Panx1–EGFP plasmid [20] with the following primers (forward: 5′-CGAGTTCTTTCTCCGCGCGACAGGCTGCCTTTG-3′; reverse: 5′-CAAAGGCAGCCTGTCGCGCGGAGAAAGAACTCG-3′) using the QuikChange II site-directed mutagenesis kit following manufacturer's protocols (Agilent Technologies) and confirmed by sequencing (Eurofins MWG Operon). Where indicated, N2a cells stably expressing equivalent levels of Panx1–EGFP or Panx1–W74A–EGFP were maintained in DMEM/F12 containing 10% FBS and 100 units/ml penicillin, 100 μg/ml streptomycin and 400 μg/ml geneticin 418 (all obtained from Gibco/Life Technologies).
Primary antibodies used were anti-GFP (1:500, Roche Applied Science), anti-β-actin (1:160000; Sigma–Aldrich) anti-early endosome antigen 1 (EEA1; 1:200, Cell Signaling Technology Inc.), anti-lysosomal-associated membrane protein 1 (Lamp1; 1:200, StressMarq Biosciences Inc.), anti-caveolin-1 (Cav-1; 1:100, Novus Biologicals LLC), anti-clathrin heavy chain (1:50; Novus Biologicals LLC), anti-Ras related protein Rab 14 (1:400, Abcam Inc.), anti-Rab4 (1:200, BD Biosciences), anti-mannose-6-phosphate receptor (M6PR; 1:100, Abcam Inc.), anti-Rab7 (1:200, Abcam Inc.), anti-Rab11 (1: 50, Santa Cruz Biotechnology) and anti-giantin (1: 500, Lifespan Biosciences Inc.). The Panx1 primary antibody used was anti-Panx1–extracellular loop 2 (EL2; 1:200) [21]. Secondary antibodies included horseradish peroxidase (HRP)-conjugated AffiniPure donkey anti-rabbit IgG and HRP-conjugated AffiniPure donkey anti-mouse IgG (both at 1:4000; Jackson ImmunoResearch), Alexa Fluor® 647-conjugated AffiniPure donkey anti-rabbit IgG (1:600; Jackson ImmunoResearch), Alexa Fluor® 568 donkey anti-mouse IgG and Alexa Fluor® 568 donkey anti-rabbit IgG (both at 1:600; Life Technologies).
Live and fixed cell imaging
Cells were treated in media containing 20 μg/ml cycloheximide (CHX; Sigma–Aldrich) for 8 h prior to experimental treatments. Confocal imaging was performed with a Leica TCS SP8 confocal microscope and analysis was performed with Leica Application Suite software (version 3.1.3). All imaging and quantification were performed double-blinded to treatment conditions. Comparisons were made under identical conditions. Confocal micrographs displayed as representative images were adjusted for contrast uniformly using Adobe Photoshop for display purposes only; no contrast adjustments were made prior to analysis. Stimulated emission depletion (STED) confocal microscopy [22] was performed on a Leica TCS SP8 STED confocal microscope and deconvolution was performed using the Huygens Professional Deconvolution software (Suite 15.05) [23].
Live-cell Panx1 tracking
N2a cells stably expressing Panx1–EGFP were plated on 100 μg/ml poly-D-lysine (PDL)-coated eight-well chambered coverglass (Nunc LabTek/ ThermoScientific) and maintained at a temperature of 37°C and 5% CO2. Tetramethylrhodamine (TRITC)-conjugated wheat germ agglutinin (WGA; 1 μg/ml; Life Technologies) was added 5 min prior to imaging. ATP (100 μM, 200 μM, 500 μM; Sigma–Aldrich) or vehicle control (water) was added to individual wells and images were collected at 1-min intervals for 30 min using a 20× (0.7 numerical aperture (NA)) objective. Quantification of Panx1–EGFP fluorescence intensity to describe 'intracellular Panx1' was performed at time zero and each 5-min interval thereafter, as follows: a polygonal trace was drawn 1 μm inside of the peak WGA intensity at the cell periphery and the encapsulated average Panx1–EGFP fluorescence intensity per pixel was computed (Figure 1). Data were normalized to values obtained at time zero. n ≥ 25 cells were analysed per experimental condition per biological replicate. Only cells that were stable in the z-axis for the entire imaging window were counted.
Fixed-cell Panx1 localization
N2a cells stably expressing Panx1–EGFP were plated on PDL-coated coverslips in 24-well plates. Cells were cultured and pre-treated briefly with CHX as described above in the live-cell confocal experiments. Where indicated, other stimuli or drugs were added to the culture media, in the presence of CHX, including 30 mM potassium chloride (K+), 10 μM Ca2+ ionophore A23187, chemical oxygen glucose deprivation (OGD; 100 μM potassium cyanide and 1 μg/ml oligomycin), 100 μM or 500 μM 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate triethylammonium salt (BzATP), 500 μM UTP Tris salt, 500 μM adenosine-5'-(β-thio)-diphosphate trilithium salt (ADPβS; all obtained from Sigma–Aldrich) or 500 μM adenosine-5'-(γ-thio)-triphosphate tetralithium salt (ATPγS; Tocris Bioscience). For the P2X7 or endocytosis inhibitor experiments, cells were pre-treated for 1 h prior to 500 μM ATP or control treatment with 100 μM A438079 (Tocris Bioscience) or 10 μg/ml chlorpromazine (CLPZ), 1 μg/ml filipin III (Fil III), 10 mM methyl-β-cyclodextrin (MβCD) or vehicle control (equal volume of DMSO; all endocytosis inhibitors obtained from Sigma–Aldrich) respectively. Following ATP/control treatment, cells were fixed with 4% paraformaldehyde in PBS for 10 min. Coverslips were then incubated with primary antibodies in antibody buffer (3% BSA, 0.3% Triton-X-100 in PBS) overnight at 4°C, washed and incubated with corresponding fluorophore-conjugated secondary antibody and Hoechst 33342 nuclear counterstain in antibody buffer for 1 h at room temperature, then washed and mounted in VectaShield (Vector Labs). n ≥ 50 cells were analysed per experimental condition per biological replicate. Confocal images were obtained at 4× optical zoom in 1024 × 1024 format with a pixel area of 71 nm2 at room temperature using a 40× (1.3 NA) oil immersion objective as confocal z-stacks and quantification was performed in the confocal plane displaying the largest central plane of the nuclei in the region of interest. Intracellular Panx1 was quantified as described above (Figure 1) and normalized to values obtained for vehicle-treated controls. STED imaging of selected regions of interest was performed to support localization to specific compartments obtained with conventional confocal microscopy with enhanced resolution. STED images were obtained with a pixel area of 39 nm2 using a 100× (1.4 NA) oil immersion STED white objective. This allowed for a theoretical maximum resolution of approximately 80 nm prior to deconvolution. Deconvolution removes aberrations and increases the contrast of the image allowing for an increase in resolution by a factor of 1.5–2 [23]. Analysis of background specular structures (reflecting single antibody molecules or aggregates of antibody molecules) revealed an apparent resolution of 42 nm.
Mechanism of endocytosis
The co-distribution of Panx1 and proteins involved in clathrin or caveolin-mediated endocytosis in either 500 μM ATP or vehicle control-treated N2a cells was evaluated using Pearson's correlation coefficient for Panx1–EGFP and antibodies for these proteins. Pearson's correlation coefficient is a pixel-by-pixel measure of the covariance in the intensities of two fluorophores. Pearson's values can range from 1 for linearly related distribution of two intensities to −1 for two intensities that have a perfectly inverse relationship, a value of 0 reflects an uncorrelated relationship [24]. Following an 8 h CHX treatment, cells were fixed at 30 min post-stimulus and co-distribution data were collected for each Panx1–EGFP-positive cell in a region of interest within 1 μm of any unopposed plasma membrane. The data for ≥50 cells was averaged for each biological replicate (n=4). To assay for dynamin-dependent endocytosis, stable Panx1–EGFP N2a cells grown on PDL-coated coverslips were first incubated for 6 h in complete DMEM/F12 containing CHX. The cells were then rinsed and incubated in DMEM/F12 containing CHX supplemented with 2 mg/ml BSA for 1 h (all at 37°C). Cells were then incubated for an additional hour in DMEM/F12/CHX/BSA supplemented with 80 μM Dynasore (Sigma–Aldrich) or DMSO control. Following this, cells were pre-incubated at 4°C for 10 min, then in the same media supplemented 80 μM Dynasore or DMSO control and 25 μg/ml transferrin-Alexa Fluor 647 (Life Technologies) for an additional 45 min at 4°C. Cells were then rinsed in ice-cold DMEM/F12/CHX/BSA supplemented with 80 μM Dynasore or DMSO control and fixed immediately or media were replaced with warm media containing 500 μM ATP or vehicle control. Following a 30-min incubation period at 37°C (internalization period), cells were fixed and intracellular Panx1–EGFP and transferrin were measured as described above.
Sub-cellular distribution
The co-distribution of internalized Panx1–EGFP with antibody markers for specific sub-cellular compartments was determined by analysis of confocal micrographs with the Leica Application Suite software (version 3.1.3) Pearson's correlation tool. Following 8 h of CHX treatment, cells were fixed at 0.5, 1 and/or 2 h after 500 μM ATP stimuli. Vehicle-treated cells (2 h) served as control. Panx1–EGFP-expressing N2a cells were then immunostained with antibodies for sub-cellular compartment-specific markers. Images were obtained at 4× optical zoom in 1024 × 1024 format with a pixel area of 71.02 nm2 at room temperature using a 40× (1.3 NA) oil immersion objective as z-stacks and quantification was performed in the confocal plane displaying the largest central plane of the nucleus (Hoechst 33342) in the region of interest. For this analysis we re-named the intracellular analysis region as 'central' and we added a 1-μm thick region of analysis, termed 'peripheral', immediately outside the 'central' region (and bounded by the plasma membrane) because early endosomes are primarily located in the cell periphery [25]. The Pearson's correlation coefficient of Panx1–EGFP and antibodies for sub-cellular compartment-specific markers was determined per cell in each region of interest. The data for ≥50 cells was averaged for each biological replicate (n=4).
Western blotting
N2a cells stably expressing Panx1–EGFP were treated with CHX, as described above in the live cell confocal experiments. Cells were either collected immediately (0 h) or stimulated with vehicle-control for 2 h or with ATP for 0.5, 1 or 2 h and collected at the end-point. Cells were homogenized in a Tris-based lysis buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0), supplemented with protease inhibitor cocktail at 1 μl/106 cells [stock: 0.104 mM 4-(2-aminoethyl) benzenesulfoyl fluoride hydrochloride, 0.08 mM aprotinin, 4 mM bestatin hydrochloride, 1.4 mM n-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide, 2 mM leupeptin hemisulfate salt, 1.5 mM pepstatin A; Sigma–Aldrich), PMSF at 2 μl/106 cells, sodium orthovanadate at 2 μl/106 cells and 1 mM EDTA and incubated on ice for 30 min. Homogenates were then passed through a 27-gauge needle three times and centrifuged at 17500 g for 15 min. Supernatant was collected as whole cell lysate. Samples were boiled (100°C) for 20 min in SDS/PAGE loading dye under reducing conditions (DTT and β-mercaptoethanol). Western analysis was performed as previously described [2,3,26].
Reverse transcriptase-PCR
Total RNA was isolated and first-strand synthesis (Superscript II; according to the manufacturer's protocol; Life Technologies) was performed followed by PCR using cycling parameters of 94°C for 5 min, 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min and a final step at 72°C for 7 min for the following primers: 5′-CTTGGCCTTCATTGCGGGTA-3′, 5′-GGAAGGCAAGACCATTAGGCA-3′ defining a 100-bp Cav-2 amplicon, accession number (GenBank: NM_016900) and 5′-TGGTGCTGAGTATGTCGTGGAGT-3′, 5′-AGTCTTCTGAGTGGCAGTGATGG-3′ defining a 292-bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplicon, accession number (GenBank: NM_008084.2), whereas the cycling parameters of 94°C for 5 min, 35 cycles of 94°C for 30 s, 53°C for 30 s and 72°C for 1 min and a final step at 72°C for 7 min were used for the primers 5′-CATGTCTGGGGGCAAATACG-3′, 5′-GTCGTTGAGATGCTTGGGGT-3′ defining a 184-bp Cav-1 amplicon, accession number (GenBank: NM_007616.4).
Statistical analysis
Western blot densitometry was quantified using ImageJ 1.45 (http://rsbweb.nih.gov/ij/index.html). Significance was determined using unpaired Student's t test. Results from live-cell imaging experiments were analysed using a two-way ANOVA for time and treatment. Fixed cell confocal microscopy results were analysed using a one-way ANOVA or a Student's t test, where applicable. Data analysed using ANOVA were subsequently subjected to a Dunnett's post-hoc test, a multiple comparison procedure that compares all treatments to a single control. All data are presented as mean ± S.E.M.
We tracked the movement of Panx1 in response to ATP in living N2a cells (Figure 1). These cells have a large cytoplasm to nucleus ratio, facilitating visualization of channel internalization. To create a relatively homogeneous, relatively low level of Panx1–EGFP, we established a Panx1–EGFP stable line that we pre-treated with the protein synthesis inhibitor CHX to eliminate a potential confounding fluorescence signal from de novo Panx1–EGFP in the secretory pathway (Figure 1A). We then used confocal microscopy to image Panx1–EGFP trafficking dynamics over a period of 25 min in the presence of ATP (0, 100, 200, 500 μM; Figure 1Bi). Supplementary Movie S1 clearly demonstrates the Panx1–EGFP signal overlapping with the red fluorescence signal of the labelled plasma membrane marker WGA, producing yellow vesicles internalizing from the plasma membrane in response to treatment with 500 μM ATP. To quantify intracellular Panx1, a trace was drawn 1 μm inside the WGA-defined plasma membrane to compute the average EGFP fluorescence intensity of the encapsulated area (Figure 1Bii). ATP triggered a significant time (≥15 min)- and dose (≥200 μM)-dependent increase in intracellular Panx1 after stimulation (Figure 1Biii). No significant increase in Panx1 internalization was detected with control treatment. A small, non-significant increase was observed with 100 μM ATP treatment. ATP treatment did not have an appreciable effect on cell size and shape or viability. To investigate if the effect was specific to the ATP stimulus directly or could be attributed to a generalized increase in excitability triggered by the ATP stimulus, we compared 30-min ATP treatment with exposure to more generalized stimuli associated with increased excitability and/or excito-toxicity (Figure 1C). Following the treatment, the cells were fixed and intracellular Panx1 levels were measured by confocal microscopy. As expected from the live-cell assay, treatment with 200 μM ATP elicited a significant increase in intracellular Panx1 (∼180% of control), whereas K+, OGD and A23187 Ca2+ ionophore treatment did not significantly increase the intracellular population of Panx1. Although our earlier observations [27] suggested that these general stimuli can weakly influence Panx1 motility or surface expression, they did not have a long lasting effect on the size of the internal pool of Panx1. These data are further supported by a biochemical cell surface luminometry assay in HEK293-T cells [28] (Supplementary Figure S1). This assay requires a primary antibody to an extracellular epitope of the protein of interest. Abundance of the target protein is then measured by ECL with a standard plate reader. The signal from unpermeabilized cells (channels at the surface membrane) is normalized to the signal from permeabilized cells (channels on all cell membranes) to obtain a relative measure of channel cell surface expression amongst different treatments/experimental groups. We treated HEK293-T cells overexpressing Panx1–EGFP with K+ (10 mM), ATP (200 μM) or vehicle control. Cells were then processed for detection of surface and whole cell Panx1 using an antibody that recognizes an epitope in its second extracellular loop [21]. We found that ATP treatment (at a concentration associated with Panx1 channel inhibition [29,30]) significantly decreased surface Panx1 compared with control, whereas elevated extracellular K+ had no effect.
Next we sought to determine the nucleotide (nt) dependence of the effect and relatedly the possible involvement of purinergic receptors. Numerous studies have described functional and physical interactions of Panx1 with purinergic P2 receptors in various systems [31–36]. Screening the nt dependency of this effect can simultaneously provide insight into potential ionotropic (P2X) and metabotropic (P2Y) purinergic receptor involvement by capitalizing on the differential potency of different nt analogues. Following a 30-min incubation with the indicated nts (Figure 2), we fixed the cells and measured intracellular Panx1 levels using confocal microscopy. In comparison with control treated cells, 500 μM ATP elicited a significant increase in intracellular Panx1 to ∼280% of the control (Figure 2A), whereas treatment with the slowly hydrolysable ATP analogue, ATPγS (500 μM; P2X receptors) elicited a significant but smaller increase in intracellular Panx1 levels (∼150% of control). The increase in intracellular Panx1 elicited by the slowly hydrolysable ADP analogue, ADPβS (500 μM; P2Y receptors) was not significantly different from control-treated cells. In comparison with control, BzATP (P2X receptors) had a small but non-significant effect on Panx1 internalization (Figure 2B), whereas UTP (P2Y receptors) had no effect. These results suggest a lack of involvement of P2Y receptors: ADPβS and UTP, the two analogues that act on P2Y receptors [37–39], had no effect on Panx1 internalization. Further supporting a lack of P2Y involvement, the previously described mode of Panx1 activation via P2Y receptors involved a P2Y-mediated increase in intracellular Ca2+, which can be mimicked by Ca2+ ionophore treatment. As demonstrated by the data presented in Figure 1, Ca2+ ionophore treatment had no effect on Panx1 internalization. The nt data were inconclusive with respect to P2X receptors. Because BzATP had a small albeit non-significant effect and since these cells express P2X4 and P2X7 receptors (with P2X7 being the primary functional isoform in these cells [40]), to rule out involvement of P2X receptors, we used the selective P2X7 blocker A438079 [41,42]. Somewhat surprisingly, given the non-significant effect of BzATP, A438079 completely blocked Panx1 internalization. Despite the enigmatic nt dependence, this result implicates P2X7 receptors in ATP-stimulated Panx1 internalization.
Putative ATP-sensitive residues have also been identified within the Panx1 extracellular loops [29,30,43]. Qiu and Dahl [30] identified these in the course of investigating inhibition of Panx1 currents by ATP (≥200 μM) using in vitro expression systems [29,44]. One of the residues at which ATP had a large effect was a tryptophan residue at amino acid 74 in the first extracellular loop of Panx1 [29]. To test the effect of this amino acid substitution on ATP-mediated Panx1 internalization, we generated the mutant EGFP-tagged construct, Panx1–W74A–EGFP and stably expressed it in N2a cells (Figure 3A). The ATP-insensitive Panx1 mutant (Panx1–W74A–EGFP) exhibited robustly reduced ATP-mediated internalization (Figure 3B). In the Qiu et al. [29] study first describing the ATP-sensitive residue W74A, the selective P2X7 blocker A438079 also inhibited Panx1 currents. Importantly the expression systems used in studies of ATP inhibition of Panx1 currents [29,30] endogenously express P2X receptors (Xenopus oocytes HEK293-T cells [44–46]). This suggests that the intrinsic ATP-sensitivity of Panx1 could interplay with P2X receptors. Note that there is no evidence, as of yet, that these are functionally expressed in Xenopus oocytes.
Next we sought to gain insight into the endocytosis mechanism (Figure 4). Endocytosis mechanisms are commonly classified into two major groups: clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE); recently reviewed in [47]. Although CIE had historically been linked with a protein called caveolin, numerous caveolin-independent CIE mechanisms have now been described [47]. Because of its effects on membrane curvature and stability, cholesterol is known to play an important role in the majority of endocytic mechanisms [48]. To investigate the potential cholesterol-dependence of ATP-stimulated Panx1 internalization, we first used Fil III, which binds to and aggregates cholesterol [49,50]. In the presence of Fil III, ATP (500 μM) was not able to stimulate an increase in intracellular Panx1, whereas ATP had the expected effect in the presence of vehicle (an equal volume of DMSO; Figures 4A and 4B). To confirm the cholesterol dependence of internalization, we next employed the cholesterol-disrupting agent MβCD (Figure 4C), which acts by removing cholesterol from membranes [51]. MβCD also blocked Panx1 internalization, confirming the importance of cholesterol. Therefore, we next tested the effects of CLPZ (Figure 4D), a phenothiazine-derived anti-psychotic drug (APD) and established CME inhibitor [52–54]. ATP-stimulated Panx1 internalization was preserved in the presence of CLPZ, suggesting that CME is not involved. To confirm these results, we drew on previous work demonstrating that transmembrane proteins employing caveolin-dependent CIE or CME mechanisms exhibit co-distribution with caveolin or clathrin [55] respectively. Consistent with previous reports [56,57], we found that N2a cells express low levels of Cav-1, but we did not detect Cav-2 (Figure 4; Supplementary Figure S2). Note that the expression of a third caveolin, caveolin-3 is muscle-specific [58]. We found that Panx1 did strongly not co-distribute with either caveolin-1 or clathrin heavy chain. We quantitatively examined co-distribution of Panx1 with Cav-1 (Figure 4E) and clathrin heavy chain (Figure 4F) following ATP stimulus. To confirm our data suggesting the mechanism of Panx1 internalization is independent of both CME and caveolin-dependent CIE pathways, we capitalized on the fact that CME and caveolin-dependent CIE converge mechanistically at the dynamin-dependent scission step. We therefore used the pharmacological inhibitor of dynamin I, dynamin II and mitochondrial dynamin called Dynasore [59,60]. We observed similar significant ATP-induced increases in intracellular Panx1 in the presence of Dynasore or vehicle control (Figure 5C). Together, these data suggest that ATP-stimulated Panx1 internalization is dependent on cholesterol, but is most probably independent of dynamin, pointing to a non-caveolin CIE mechanism. The study of non-caveolin CIE pathways involving ion channels and receptors is complex and rapidly evolving [47]. Given that Panx1 was recently identified as an ubiquitinated protein [61], which is a key CIE mechanism [62], the putative role of ubiquitination (and associated protein machinery) in ATP-dependent Panx1 internalization will be the focus of future study. Consistent with our results, an earlier report [63] demonstrated that basal (un-stimulated) Panx1 internalization did not occur by CME or caveolin-dependent CIE. However, in contrast with our findings on stimulated internalization, basal internalization was unaffected by the cholesterol disruptor MβCD. It is reasonable to speculate that Panx1 could be internalized through different pathways depending on the context (basal compared with stimulated), as has been observed with other proteins [47].
Since internalized surface proteins are often targeted for degradation, we examined whole-cell Panx1–EGFP (in the presence of CHX) in ATP treated (0.5, 1 and 2 h after treatment) and control vehicle treated (0 and 2 h after treatment) cells. Figure 6(A) demonstrates that overall Panx1 levels (normalized to β-actin levels) were unchanged throughout the analysis period in both control and ATP-treated cells suggesting that internalized Panx1 is not targeted for degradation over this 2-h period. We next sought to determine Panx1's intracellular destination over time (0.5, 1 and 2 h) after 500 μM ATP stimulation by measuring Pearson's correlation coefficient for Panx1 and the markers outlined in Figure 6(B). Upon internalization from the plasma membrane, transmembrane proteins first encounter the early endosome (EEA1). From there, they can follow the degradation pathway, through the late endosome (Rab7 [64]) and endolysosome (M6PR [65,66]) to end up in the lysosome (Lamp1 [67]) or they can be diverted from the early endosome to recycling endosomes (Rab14 [68,69], Rab11, Rab4 [70]) with potential to return to the plasma membrane. Transmembrane proteins also shuttle to and from the trans-Golgi network (Giantin [71]). Supplementary Figure S3 demonstrates that, in N2a cells, these markers can be spatially distinguished from one another using confocal microscopy. As expected, EEA1 and Golgi demonstrated limited co-distribution (Pearson's: 0.029±0.003). EEA1 and Lamp1 shared minor co-distribution (Pearson's: 0.16±0.01). Although these are at opposite ends of the degradation pathway, one would expect a degree of overlap reflecting the constant membrane trafficking between the various endosomal compartments and the lysosome during endosomal maturation [25]. As anticipated given their shared localization within recycling endosomes, Rab4 and Rab14 had a much higher degree of co-distribution (0.53±0.03).
Reflecting the peripheral to central distribution of endosomes with progression through the degradation pathway [25], we defined an additional 1-μm thick region of analysis, 'peripheral', immediately outside the intracellular region (defined in Figure 1) that we newly defined here as 'central'. We anticipated that 0.5 h after ATP treatment we would expect to see increased Panx1 at the most immature early endosomes and that we could potentially miss this observation if we did not add this peripheral region of analysis where the most immature early endosomes are most likely to be found. Given the limits of resolution of light microscopy, we would expect this 'peripheral' region to include populations of Panx1 (1) at the plasma membrane, (2) immediately beneath the plasma membrane and (3) in peripherally-located early endosomes. As expected, 0.5 h following ATP treatment peripheral Panx1/EEA1 co-distribution was significantly increased (∼150%) in comparison with control treated cells (Figure 6C). Moreover, higher resolution STED microscopy confirmed the localization of Panx1 to EEA1-positive early endosomes (Figure 6Ciii). At the same time point, there was a significant decrease (by ∼50%) in central Panx1/Rab7 and Panx1/Lamp1 co-distribution (Supplementary Figure S4), suggesting the sub-population of Panx1 in the degradation pathway is decreasing in response to increased extracellular ATP. At 1 h after ATP treatment, central Panx1/Rab14 co-distribution significantly increased (∼220%) relative to control and remained high (∼240%) at 2 h (Figure 5D). The localization of Panx1 at Rab14-positive recycling endosomes was confirmed with STED microscopy (Figure 6Diii). Additional recycling endosome markers Rab 11 and Rab4 exhibited a similar result to Rab14, albeit non-significant for Rab4 (Supplementary Figure S4). This sub-population of Panx1 in the recycling endosome could either be en-route to return to the plasma membrane or could be recruited to the recycling endosome to play a yet-to-be-determined functional role therein.
Our analysis also revealed that under control conditions intracellular Panx1, when present, co-distributed most strongly with markers for the early endosome, late endosome, endolysosome, and lysosome (Supplementary Figure S4). These results suggest a sub-population of intracellular Panx1 is expressed in endosomes under control conditions and primarily localizes to the degradation pathway. Here, Panx1 could either play a functional role, as suggested by a recent study demonstrating the Panx1 blocker probenecid blocks lysosome function [72] or simply be constitutively targeted for degradation. We did not see a reduction in Panx1 over the 2-h observation period in the present study; however, previous work indicated that disruption of the lysosome led to accumulation of Panx1 [63] (albeit in a different cell line). A longer analysis period in future studies will help to resolve this potential discrepancy. Note that Panx1 exhibited limited expression in the Golgi under control conditions, thus the physiological relevance of the significant relative decrease in co-distribution with Giantin at 0.5 h with ATP treatment is uncertain (Supplementary Figure S4). Together these results suggest that ATP stimulus increases the trafficking of a sub-population of Panx1 to recycling endosomes.
Our results, in the present study, identify a novel localization of intracellular Panx1 within endosomal systems and raise important considerations with regards to Panx1 surface stability in diverse physiological and pathophysiological scenarios in which ATP can be rapidly elevated in the extracellular space. In the presence of elevated extracellular ATP, intracellular Panx1 is increased and the co-distribution of Panx1 with markers for recycling endosomes is increased. ATP is episodically released from developing neurons in the ventricular zone of the lateral ventricles, acting as an important autocrine and paracrine regulator of their proliferation, differentiation and migration through activation of various purinergic receptors that are developmentally regulated [18,19]. Furthermore, ATP is co-released with other neurotransmitters as an important modulator of synaptic plasticity and is also released from immune cells and dead and dying cells, recently reviewed in [73]. Locally surrounding the ATP release sites (from vesicle/granule release or ATP-permeable channels), ATP levels would be predicted to be very high, analogous to Ca2+ micro and nano domains surrounding Ca2+-permeable channels [74,75]. Intracellular ATP concentrations range from 3 to 5 mM and secretory granules can contain ATP on the order of 100 mM [76,77]. Consequently, taking into account extracellular nucleotidase activity, ATP levels would be comparable to those tested in the present study (if not higher) in the microdomain surrounding vesicular release sites or ATP channels. It is reasonable to propose that in the context of Panx1 as an ATP-release channel, ATP-mediated internalization of Panx1 could represent an important homoeostatic means of regulating ATP release within a range of extracellular concentrations.
A putative functional role of Panx1 in the degradation and recycling endosome systems remains to be determined. A recent report demonstrated neuroprotection in the context of stroke was contingent on double knockout of both Panx1 and Panx2, suggesting functional overlap [78,79]. This suggests there could be a cross-talk between Panx1 and resident Panx2 [15,26,80,81] in endolysosomal membranes. Interestingly, Boassa et al. [80], observed no co-distribution between Panx1 and Panx2 (in MDCK, HeLa and HEK293-T cells), but did observe co-distribution between Panx2 and Rab4. Our data suggest that in N2a cells, Panx1 and Panx2 could co-distribute in Rab4-positive endosomes and potentially other endosomal compartments that were not explored in that study. Whether Panx1 and Panx2 physically or functionally co-operate in one or both of the endosomal systems, remains to be determined.
Overall, the present study enhances our understanding of the localization of Panx1 within cells and the factors that contribute to the regulation of Panx1 channel trafficking. These novel findings have significant implications for scenarios in which extracellular ATP levels are dynamic. Based on our pharmacological data with the selective P2X7 receptor blocker A438079, our data suggest that P2X7 receptors could be involved in ATP-stimulated Panx1 internalization, possibly in combination with putative ATP-sensitive residues in the Panx1 sequence. This suggests that a similar internalization phenomenon could be expected in cells expressing both Panx1 and P2X7 receptors, as was the case with our N2a cell and HEK293-T cell assays. The important next steps will be to understand the precise contribution and mechanisms of P2X7 receptor involvement as well as their interplay with ATP-sensitive residues in Panx1 and to extend these results into in vivo systems.
Leigh Anne Swayne directed the study, wrote and revised the manuscript, helped to prepare the figures and performed the cell surface luminometry experiments. Andrew Boyce performed the majority of the experiments, prepared the figures and contributed to the writing and revision of the manuscript. Michelle Kim created the W74A mutant Panx1EGFP construct. Leigh Wicki-Stordeur assisted with cell surface luminometry and cell culture.
This work was supported by operating grant support to L.A.S. from the Natural Sciences and Engineering Research Council (NSERC) [grant number 402270-2011]; infrastructure support from the Canadian Foundation for Innovation (CFI) [grant number 29462]; and the BC Knowledge Development Fund (BCKDF) [grant number 804754]. L.A.S. was also supported by a salary grant from the Michael Smith Foundation for Health Research (MSFHR) [grant number 5900]. Student scholarships were awarded to A.K.J.B. from NSERC [grant number PGSD 459931-2014] and from the University of Victoria (President's Research Scholarship), to M.S.K. from NSERC (USRA), and to L.E.W.-S. from NSERC [grant number Vanier Canada Graduate Scholarship CGV-NSERC-00184].
We thank Dr Silvia Penuela and Dr Dale Laird (Western University) for the Panx1–EGFP plasmid and the Panx1–EL2 antibody. We thank Reg Sidhu (Leica Microsystems) for providing access to the Leica TCS SP8 STED system.
Abbreviations: ADPβS, adenosine-5'-(β-thio)-diphosphate trilithium salt; ATPγS, adenosine-5'-(γ-thio)-triphosphate tetralithium salt; BzATP, 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate triethylammonium salt; Cav-1/2, caveolin-1/2; CHX, cycloheximide; CIE, clathrin-independent endocytosis; CLPZ, chlorpromazine; CME, clathrin mediated endocytosis; DMEM, Dulbecco's modified Eagle's medium; EEA1, early endosome antigen 1; EL2, extracellular loop 2; Fil III, filipin III; HEK293-T, human embryonic kidney 293T; HRP, horseradish peroxidase; Lamp1, lysosomal-associated membrane protein 1; M6PR, mannose 6-phosphate receptor; MβCD, methyl-β-cyclodextrin; NA, numerical aperture; N2a, Neuro2a; OGD, oxygen glucose deprivation; Panx1, pannexin 1; PDL, poly-D-lysine; P2X, ionotropic purinergic receptor; P2Y, metabotropic purinergic receptor; Rab, Ras related protein Rab; STED, stimulated emission depletion; WGA, wheat germ agglutinin
- © 2015 Authors; published by Portland Press Limited


text: Back to Results Listed Building The only legal part of the listing is the address/name of site. Addresses and building names may have changed since the date of listing – see 'About Listed Buildings' below for more information. KNOCKDOW HOUSELB11886 Status: Designated Jump To: Documents Summary Description Statement References About Images Documents There are no additional online documents for this record. Summary CategoryB Date Added20/07/1971 Local AuthorityArgyll And Bute Planning AuthorityArgyll And Bute ParishInverchaolain NGRNS 10464 70653 Coordinates210464, 670653 Description 18th-19th cents. ORIGINAL MANSION: Georgian. 2 storeys and garret. Harled. Slated roof. Gabled dormers. Corner stones. Pedimented upper windows; gabled lower ones.Additions: c.1815 and later. 2 long wings flanking the original mansion; terminating in front in bow windows and returning to meet at rear. E. wing has segmental pediment over central bay with rusticated porte-cochere flanked by bay windows. W. wing has high crenellated tower and bay window. Statement of Special Interest Lands at Kilmichael were purchased by Lamonts of Knockdow 1753 Kilmichael House was enlarged for James Lamont, 14th of Knockdow and re-named after old seat (fragmentary remains) near the Kirk. Seat of Lamonts of Knockdow until 1958. References Bibliography N.S.A.T.S.A.GroomeH. McKechnie, 'The Lamont Clan' pp.433-448 (ills.) About Listed Buildings Historic Environment Scotland is responsible for designating sites and places at the national level. These designations are Scheduled monuments, Listed buildings, Inventory of gardens and designed landscapes and Inventory of historic battlefields. We make recommendations to the Scottish Government about historic marine protected areas, and the Scottish Ministers decide whether to designate. Listing is the process that identifies, designates and provides statutory protection for buildings of special architectural or historic interest as set out in the Planning (Listed Buildings and Conservation Areas) (Scotland) Act 1997. We list buildings which are found to be of special architectural or historic interest using the selection guidance published in Designation Policy and Selection Guidance (2019) Listed building records provide an indication of the special architectural or historic interest of the listed building which has been identified by its statutory address. The description and additional information provided are supplementary and have no legal weight. These records are not definitive historical accounts or a complete description of the building(s). If part of a building is not described it does not mean it is not listed. The format of the listed building record has changed over time. Earlier records may be brief and some information will not have been recorded. The legal part of the listing is the address/name of site which is known as the statutory address. Addresses and building names may have changed since the date of listing. Even if a number or name is missing from a listing address it will still be listed. Listing covers both the exterior and the interior and any object or structure fixed to the building. Listing also applies to buildings or structures not physically attached but which are part of the curtilage (or land) of the listed building as long as they were erected before 1 July 1948. While Historic Environment Scotland is responsible for designating listed buildings, the planning authority is responsible for determining what is covered by the listing, including what is listed through curtilage. However, for listed buildings designated or for listings amended from 1 October 2015, legal exclusions to the listing may apply. If part of a building is not listed, it will say that it is excluded in the statutory address and in the statement of special interest in the listed building record. The statement will use the word 'excluding' and quote the relevant section of the 1997 Act. Some earlier listed building records may use the word 'excluding', but if the Act is not quoted, the record has not been revised to reflect subsequent legislation. Listed building consent is required for changes to a listed building which affect its character as a building of special architectural or historic interest. The relevant planning authority is the point of contact for applications for listed building consent. Find out more about listing and our other designations at www.historicenvironment.scot/advice-and-support. You can contact us on 0131 668 8914 or at firstname.lastname@example.com. Images There are no images available for this record, you may want to check Canmore for images relating to KNOCKDOW HOUSE There are no images available for this record. Search Canmore Printed: 15/06/2019 21:42


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Course summary
The comprehensive one-day in-house training course in Microsoft SharePoint is designed to cover the essential content management operations in Microsoft SharePoint. The training approach developed by SquareOne will enable your company staff to immediately apply the acquired skills and develop a successful company-wide content management strategy.
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When you have a small emergency fund, you won't have to reach for your credit cards when disaster strikes. You can use the saved money and not slide back into debt. Of course, when you use the money in your emergency fund, you may need to pause debt payment to rebuild it again. But at least you won't have gone deeper into debt when trying to climb out of the hole.
Reputable credit counseling organizations have staff counselors who are certified and trained. These counselors can discuss client financial situations to help them develop a personalized plan for their economic issues. An initial counseling session typically lasts an hour, with an offer of follow-up sessions. A reputable credit counseling agency should offer free information about itself and the services it provides without requiring potential clients to release any details about their situation.
Credit counselors at nonprofit credit counseling agencies operate under strict state and organizational guidelines designed to insure they act in their client's best interests. Non-profits are frequently audited by states to insure they comply with all of that state's regulations, and they must demonstrate that they are acting in the best interests of all of their clients. For example, InCharge offers clients monthly newsletters with money-saving tips and stories of people who have gotten out of debt to help motivate clients to do the same.
The great thing about Clearpoint is that their debt management program allowed me to consolidate the payments of 9 different credit cards into one single payment… They were the ones that contacted all the credit card companies and got the lowest APR possible. And they were very supportive too—there was never any judgment about what had happened or anything like that. They were just there to help, completely on board with me as a part of my team.
One thing to consider: If you're eligible for Chapter 7 bankruptcy, the process can be over fairly quickly and with reasonable certainty that your unsecured debts will be forgiven. Debt management, on the other hand, is more of a question mark. The process can take years, and many people who start debt management plans ultimately drop out and may have to consider bankruptcy anyway.
Starting your own business has never been easier! Do you have a knack for making things? Sell your products online! Are you an animal lover? Take up dog walking or pet sitting. Do you have a good eye and a nice camera? Start taking on clients for photo sessions. Christy Wright's Business Boutique is a great resource to walk you through the process!
We find that because our financial counseling is free, confidential, and carries no obligation, the best course of action if you may be interested in a Debt Management Plan is to call and speak directly to one of our certified coaches. In addition to the valuable budgeting assistance, we will help you assess whether a DMP is the right path for you.
Credit card modifications are becoming more common. For example, Bank of America expects to modify the credit card terms of over 1 million cardholders, Chase is rewriting the terms of thousands of card agreements, and almost every other lender as well as bank offers some form of modifications. It is more possible than ever today to get out of debt with help from credit card issuers. Continue learning about credit card modifications.
Watch out for common red flags. While good credit counseling agencies are transparent about their fees and services, unscrupulous ones can be evasive and pushy. Red flags include demands for payment before services start, failure to provide a contract, insisting on access to your bank account and promises of debt repair that sound too good to be true.
Before discussing anything else, we'd like you to know that you have the option to fix everything yourself. Unfortunately success is not guaranteed as you have proven that you haven't been able to manage your finances. But it could be worth a try. The good thing is that you will not be burdened with the additional costs of hiring someone to help you out. You will be able to concentrate all your funds on paying off your debts.
Avoid outrageous upfront fees. A small initial fee (up to $50 or, in rare cases, as much as $100 if you have a lot of debt or high income) is normal, but large upfront fees are out of line. If any agency asks for a fee (or donation) make sure that you know what it will cover, and get it in writing. Find out if you'll have to pay any additional fees to start the program. Don't get tricked into paying one "consultation fee," and then an "application fee" or "an enrollment fee." If you're truly unable to pay, look for an agency that is willing to waive the fee or spread it out (without charging additional fees for doing so).
Editorial Note: Compensation does not influence our rankings and recommendations. However, we may earn a commission on sales from the companies featured in this post. To view a list of partners, click here. Opinions expressed here are the author's alone, and have not been reviewed, approved or otherwise endorsed by our advertisers. Reasonable efforts are made to present accurate info, however all information is presented without warranty. Consult our advertiser's page for terms & conditions.
*Clients who are able to stay with the program and get all their debt settled realize approximate savings of 50% before fees, or 30% including our fees, over 24 to 48 months. All claims are based on enrolled debts. Not all debts are eligible for enrollment. Not all clients complete our program for various reasons, including their ability to save sufficient funds. Estimates based on prior results, which will vary based on specific circumstances. We do not guarantee that your debts will be lowered by a specific amount or percentage or that you will be debt-free within a specific period of time. We do not assume consumer debt, make monthly payments to creditors or provide tax, bankruptcy, accounting or legal advice or credit repair services. Not available in all states. Please contact a tax professional to discuss tax consequences of settlement. Please consult with a bankruptcy attorney for more information on bankruptcy. Depending on your state, we may be available to recommend a local tax professional and/or bankruptcy attorney. Read and understand all program materials prior to enrollment, including potential adverse impact on credit rating.
It will decimate your credit scores and stay on your credit report for up to 10 years even as you restore your credit history. That's no small thing, because poor credit history can affect your eligibility for certain jobs, your chances of getting an apartment lease, and how much you pay for car insurance. When your credit is already bad, a bankruptcy may allow you to rebuild your credit much sooner than continuing to try to repay. (Learn more about when bankruptcy is the best option.)
Many banks and credit card issuers, such as Bank of America, HSBC, Wells Fargo, and Capital One offer consumers their own debt management plans (DMP) as part of the Call to Action. This is a government supported debt assistance program that will reduce interest rates, eliminate fees, and help in other ways. It often involves some form of payment plan as well. Continue.
This is where it helps to talk to a professional. Consumer credit counselors understand all the options available to pay off credit card debt. They can impartially evaluate your debt, credit and budget to help you identify the best solution for your needs. You get an unbiased, expert opinion on your best course of action so you can move forward with confidence.
If you're interested in starting a debt management plan, you'll first need to find a credit counselor. The Federal Trade Commission recommends you never agree to any debt management plan until a reputable credit counselor has thoroughly reviewed your financial situation with you. The U.S. Department of Justice maintains a state-by-state list of approved credit counseling agencies, so you can search for someone near you.
Chapter 13: Most debts are discharged. Some debts that are not dischargeable in a Chapter 7 case have to be paid in full in a Chapter 13 plan. To keep your secured debts like a car loan or mortgage, you have to continue making monthly payments. There are circumstances in which you can add your car into your plan payment. You can also use the plan payment to catch up past due house payments and prevent a foreclosure.
"There are hundreds of companies that claim to offer consumers ways to erase bad credit, create a new credit identity, or even remove bankruptcies, judgments or liens from credit files. Many of these services are outright scams and should be avoided," says Mike Long, executive vice president and chief credit officer at UW Credit Union in Wisconsin.
Hi Angry – This is a great question and one that we may be covering in a future story with a more in-depth look at how sports teams manage season ticket holder accounts. Out of curiosity, which team were the season tickets for? We may be able to reach out to them for comment about how they handle collection proceedings with defaulted ticket holders.
People are creatures of habits and spending money is no exception. We shop at the same stores, eat in the same restaurants and drive the same car, because it's comfortable. It's also costing you more than you can handle financially. Remedy: If you won't change your spending habits, you won't ever get out of debt. Start with your morning habits (have your coffee and breakfast at home). Go to lunch with a brown bag, not a wallet. In the evening, watch games or movies on TV, while eating a home cooked meal. You will see an immediate impact on your daily spending habits. You don't have to do without. You just have to make better choices with what you do.
I've only been in the program a few weeks. I'm rather disappointed with their csr. At first they treat you like you're golden and give the impression that they actually care. Once I signed up I tried reaching out to the lady who originally helped me,she completely ignored me and has not replied to any of my emails. It just feels awful being tossed to the side like garbage after initially being treat with attention and support.
Find information on credit counseling agencies in Texas near you. Numerous non-profit organizations operate in the state and can provide mortgage, debt, and various other forms of assistance to consumers and homeowners. The agencies deal with issues ranging from credit repair to debt management plans, bankruptcy filing, and other financial matters in Texas, and most services are free of charge. More information is below, including how to get help by county.
The content on this page provides general consumer information. It is not legal advice or regulatory guidance. The CFPB updates this information periodically. This information may include links or references to third-party resources or content. We do not endorse the third-party or guarantee the accuracy of this third-party information. There may be other resources that also serve your needs.
If you're looking specifically for a nonprofit credit counseling agency to work with, explore NFCC member agencies, all of which are nonprofit. NFCC member agencies are required to meet eligibility criteria that ensure they are accredited by a third party, upfront about included fees and provide consumers with counseling and financial guidance that can help them improve their finances over time.
Global criticism of credit counseling comes primarily from predatory practices that take advantage of debtors that are already struggling.[4] These practices include failing to meet required standards, charging unlawful or unreasonable fees, failing to provide affordable solutions for consumers, and neglecting to make customers aware of free debt services available elsewhere.[5]
My first week of training was taught by the Chief Sales Officer. That set the tone for how leadership operates. They care and are involved. All my coworkers and leadership are willing to help regardless of what team you are on and who you report to. There is A LOT of recognition for all kinds of successes. There are plenty of spiffs throughout the week/month. The money potential is real. If you are a worker, willing...
There's also a substantial risk associated with taking out a loan on your home, because the house secures the loan. When you owe unsecured debt, such as credit card debt, personal loan debt, or medical debt, there's nothing guaranteeing the loan except your promise to repay it. While lenders could sue you for unpaid debt and perhaps get an order to garnish wages or put a lien on your house, it's very unlikely your home could ever be put at risk of a forced sale because of unpaid unsecured debt. But when you've borrowed against your home, the house is collateral, and if you don't pay, the lender will probably foreclose and take the house. Converting unsecured debt to debt secured by your home isn't typically advisable for that reason.
Consolidate debt using a low interest rate credit card. Discover the 10 best low interest rate credit cards as determined by CardRatings and Consumer Reports. They also tend to have very competitive fees. A low interest rate credit card can greatly reduce the amount you need to pay on your outstanding credit card bills and debt. Find a list of the 10 best credit cards for consolidation.
While maybe not as widely known, this may be the leading non-profit organization to call for debt management plans, help for paying loans, and other credit counseling in western Texas. They can help qualified individuals refinance their debts and pay off outstanding bills over a period of time using DMPs, consolidation, or so called hardship programs. Services are offered in Spanish too.
Start paying into your settlement fund. National Debt Relief asks you to make monthly payments into an escrow account that it can eventually use to pay your debt settlement costs. This monthly payment is typically lower than monthly payments on your debt. While you can stop making payments on your debt if it's unaffordable, you'll end up paying more in the end.
This isn't good news for the millions of American consumers who struggle with mounting debts and less-than-perfect credit scores. Since carrying long-term debts increases your chances of missing a payment, running up excessive balances or damaging your credit in either ways, debt consolidation lenders don't have a very big pool of potential applicants at their disposal. Unless you've been fortunate enough to maintain a stellar credit score during your debt struggles, you might have to look elsewhere for help.
When it comes to student loan debt, you might consolidate federal student loans into one loan through the Department of Education's Direct Consolidation Loans. Also, or alternatively, you might take out a private loan to consolidate debts -- that's generally referred to as refinancing student loan debt. The advantages of consolidating can include lower monthly payments (if you extend your payment period) and getting out of default, while drawbacks can include less flexibility and a longer payback period -- which can mean more interest paid, overall.
Elsewhere in the European Union, regulation and non-regulation of credit counseling agencies and their approaches, including DMPs, are widely varied. In Sweden, guidelines for credit counseling are loosely provided by the Swedish Confederation of Professional Employees (TCO) and creditors are encouraged to use them in lieu of the court system. In Ireland, the Irish Congress of Trade Unions (ICTU) provides debt resolution information directly to debtors. In Latvia, a debt advisory company called LAKRA works with employers to assist indebted employees.[6]
Consumer Credit Counseling Service of Greater Dallas, Inc. - Consult with a specialist for free/low cost, and most importantly, confidential advice. They will help you get out of debt, find resources to pay bills, and offer budgeting advice as well which can lead to long term self-sufficiency. They are also a federal government HUD certified housing agency.
This involves opening several bank accounts — your regular current account, one for your own wage, another for tax and finally one for a rainy day. You then apply the percentages to your income and as soon as you get paid, you transfer these percentages into the accounts. For example, you have 70 percent as your wages, 10 percent tax and 5 percent for rainy days. This leaves 15 percent in your current account for expenses. After this, you'll hopefully be in a position to reach your earning target with the sales you already have. However, you can also work backward, using these percentages, to price your services and products.
InCharge Debt Solutions clients have access to a Debt Management App that makes managing your accounts, checking your balances, and rescheduling payments easy and convenient. The Debt Management App also allows you to check your up-to-the-minute "debt free" percentage: "You Are 55 percent Debt Free." Research shows that tracking a goal makes you more likely to stay motivated and accomplish it. With the Debt Management App, InCharge strives to be the "Fitbit" of the personal finance world.


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The field is replete with terms that might confuse and perplex the average individual. What is a squeeze page? What's a sales funnel? What's a CPA? What's SEO? How do you setup a good blog to filter the right type of relevant traffic and get your offer in front of eligible users? What's a massive value post (MVP) really mean? Clearly, there are an endless array of terms, some of which you might already know or might not depending on how much you presently know about the field.
As Affiliate Manager you will work with international affiliate networks and merchants directly to build Loylogic's worldwide points/miles accrual network. You will be an important part of our expansions plans which include growing our offerings with the top affiliate networks, partnering with the top merchants and building revenue on an international scale. You will also be responsible for sourcing promotions and offers to drive the overall revenue performance for our solutions. You will assist in negotiating deals / contracts and work with our operations team to set up the new merchants, deals and offers on our websites. less more
All the girls are then seen performing dance routines inside the hall of the house, with Lauren in the middle and two girls at each side of the staircase. Ty Dolla Sign appears while singing with a sledgehammer over his shoulders. He is seen for the first time together with the group, leaning against a wall, while the girls dance off-cam and perform twerking moves. The girls are now outside of the house, where night has approached, as each girl performs synchronized dance moves.
Online advertising, also called online marketing or Internet advertising or web advertising, is a form of marketing and advertising which uses the Internet to deliver promotional marketing messages to consumers. Consumers view online advertising as an unwanted distraction with few benefits and have increasingly turned to ad blocking for a variety of reasons. When software is used to do the purchasing, it is known as programmatic advertising.
Websites and services based on Web 2.0 concepts—blogging and interactive online communities, for example—have impacted the affiliate marketing world as well. These platforms allow improved communication between merchants and affiliates. Web 2.0 platforms have also opened affiliate marketing channels to personal bloggers, writers, and independent website owners. Contextual ads allow publishers with lower levels of web traffic to place affiliate ads on websites.[citation needed]
The Super Affiliate Network could have been a great program. At least that's how i feel about it. Instead, it's a high-ticket membership program that will basically teach you how to promote SAN and not a training course that will teach how to start an affiliate marketing business. There are also many things the training doesn't cover. They are just mentioned.
Still, before we get there, there's a whole lot of information to grasp. As an online marketer myself, it's important that I convey the truth about the industry to you so that you don't get sucked up into the dream. While there are legitimate marketers like Sharpe out there ready and willing to help, there are loads of others that are simply looking to help part you from your hard-earned cash. Before you do anything, gather all of the information you can.
Understanding Mobiles: Understanding mobile devices is a significant aspect of digital marketing because smartphones and tablets are now responsible for 64% of the time US consumers are online (Whiteside, 2016).[42] Apps provide a big opportunity as well as challenge for the marketers because firstly the app needs to be downloaded and secondly the person needs to actually use it. This may be difficult as 'half the time spent on smartphone apps occurs on the individuals single most used app, and almost 85% of their time on the top four rated apps' (Whiteside, 2016).[42] Mobile advertising can assist in achieving a variety of commercial objectives and it is effective due to taking over the entire screen, and voice or status is likely to be considered highly; although the message must not be seen or thought of as intrusive (Whiteside, 2016).[42] Disadvantages of digital media used on mobile devices also include limited creative capabilities, and reach. Although there are many positive aspects including the users entitlement to select product information, digital media creating a flexible message platform and there is potential for direct selling (Belch & Belch, 2012).[44]
Provide your affiliate the necessary tools. Many marketers assume that once an affiliate is given the responsibility of promoting a product, their job is done. That, however, is not the case because nobody else can promote your product better than you. Therefore, you must take control and develop resources and tools for your affiliates to use. Providing some well-designed tools, such as attractive banners, will not only enhance the overall effectiveness of his promotions but will also encourage him to put you offer on top of his priority list.
Your Brand Persona and Target Audience. When you eventually start creating content, you have to know who you're talking to and tailor your brand voice to appeal to them uniquely. If you aren't targeting the right audience (those people who will lean in to hear what you're saying), you won't find success. And, if you can't find a way to stand out, you'll blend into the hordes of other brands competing for attention in your industry.
Affiliate marketing occurs when advertisers organize third parties to generate potential customers for them. Third-party affiliates receive payment based on sales generated through their promotion.[24]:22 Affiliate marketers generate traffic to offers from affiliate networks, and when the desired action is taken by the visitor, the affiliate earns a commission. These desired actions can be an email submission, a phone call, filling out an online form, or an online order being completed.
Affiliate marketing is also called "performance marketing", in reference to how sales employees are typically being compensated. Such employees are typically paid a commission for each sale they close, and sometimes are paid performance incentives for exceeding objectives.[25] Affiliates are not employed by the advertiser whose products or services they promote, but the compensation models applied to affiliate marketing are very similar to the ones used for people in the advertisers' internal sales department.
Companies often use email marketing to re-engage past customers, but a "Where'd You Go? Want To Buy This?" message can come across as aggressive, and you want to be careful with your wording to cultivate a long-term email subscriber. This is why JetBlue's one year re-engagement email works so well -- it uses humor to convey a sense of friendliness and fun, while simultaneously reminding an old email subscriber they might want to check out some of JetBlue's new flight deals.
The problem with affiliate marketing, like many other home business options, are the so-called gurus and get-rich-quick programs that suggest affiliate marketing can be done fast and with little effort. Odds are you've read claims of affiliate marketing programs that say you can make hundreds of thousands of dollars a month doing almost nothing ("Three clicks to rich!"). Or, they suggest you can set up your affiliate site, and then forget it, except to check your bank deposits.


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Frequently used programs are automatically displayed in the left column, newly installed programs are highlighted, and the user may opt to "pin" programs to the start menu so that they are always accessible without having to navigate through the Programs folders.The default internet browser and default email program are pinned to the Start menu.
There are significant changes made to Windows Explorer in Windows XP, both visually and functionally.
Microsoft focused especially on making Windows Explorer more discoverable and task-based, as well as adding a number of features to reflect the growing use of a computer as a "digital hub".
The GDI dynamic library can be shipped with an application and used under older versions of Windows.
The total number of GDI handles per session is also raised in Windows XP from 16384 to 65536 (configurable through the registry).
The user's name and user's account picture are also shown on the Start menu.
The taskbar buttons for running applications and Quick Launch have also been updated for Fitt's law.To help the user access a wider range of common destinations more easily from a single location, the Start menu was expanded to two columns; the left column focuses on the user's installed applications, while the right column provides access to the user's documents, and system links which were previously located on the desktop.Links to the My Documents, My Pictures and other special folders are brought to the fore.Direct X 9 was released in 2003, which also sees major revisions to Direct3D, Direct Sound, Direct Music and Direct Show.support for floating-point texture formats, Multiple Render Targets, and texture lookups in the vertex shader.The task pane is displayed on the left side of the window instead of the traditional folder tree view when the navigation pane is turned off.


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text: http://llvm.org/bugs/show_bug.cgi?id=1160 Summary: loop code badly optimized Product: libraries Version: trunk Platform: PC OS/Version: Linux Status: NEW Severity: normal Priority: P2 Component: Backend: X86 AssignedTo: unassignedbugs at nondot.org ReportedBy: nicholas at mxc.ca This small program produces bad x86 assembly code: unsigned int huge[2048]; unsigned int target[2048]; extern void foo(); int main(int argc, char *argv[]) { foo(); for (unsigned int i = 0; i < 2048; i++) { target[i] |= huge[i]; } unsigned int x = 0; for (unsigned int i = 0; i < 2048; i++) { x += target[i]; } return x; } There are two problems with the resulting x86 assembly. For the body of the first loop, LLVM produces: movl huge(,%eax,4), %ecx orl target(,%eax,4), %ecx movl %ecx, target(,%eax,4) when it should really be loading from huge, then or'ing into target. In the second loop, LLVM mis-predicts the exit branch, which is inexcusable seeing as the loop is countable: .LBB1_5: #bb23 cmpl $2048, %ecx jne .LBB1_3 #bb15 <--- should be reversed .LBB1_6: #bb27 addl $4, %esp ret .LBB1_3: #bb15 addl target(,%ecx,4), %eax incl %ecx jmp .LBB1_5 #bb23 ------- You are receiving this mail because: ------- You are on the CC list for the bug, or are watching someone who is.


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