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Cells respond to poliovirus infection by switching on the apoptotic program, implementation of which is usually suppressed by viral antiapoptotic functions. We show here that poliovirus infection of HeLa cells or derivatives of MCF-7 cells was accompanied by the efflux of cytochrome c from mitochondria. This efflux occurred during both abortive infection (e.g., interrupted by guanidine-HCl and ending with apoptosis) and productive infection (leading to cytopathic effect). The former type of infection, but not the latter, was accompanied by truncation of the proapoptotic protein Bid. The virus-triggered cytochrome c efflux was suppressed by overexpression of Bcl-2. Both abortive and productive infections also resulted in a decreased level of procaspase-9, as revealed by Western blotting. In the former case, this decrease was accompanied by the accumulation of a protein with the electrophoretic mobility of active caspase-9. In contrast, in the productively infected cells, the latter protein was absent but caspase-9-related polypeptides with altered mobility could be detected. Both caspase-9 and caspase-3 were shown to be essential for the development of such hallmarks of virus-induced apoptosis as chromatin condensation, DNA degradation, and nuclear fragmentation. These and some other results suggest the following scenario. Poliovirus infection activates the apoptotic pathway, involving mitochondrial damage, cytochrome c efflux, and consecutive activation of caspase-9 and caspase-3. The apoptotic signal appears to be amplified by a loop which includes secondary processing of Bid. The implementation of the apoptotic program in productively infected cells may be suppressed, however, by the viral antiapoptotic functions, which act at a step(s) downstream of the cytochrome c efflux. The suppression appears to be caused, at least in part, by aberrant processing and degradation of procaspase-9.
Poliovirus, a small unenveloped virus containing a 7.4-kb RNA genome (an enterovirus representative of the family Picornaviridae), is usually cytocidal. Two types of death of the poliovirus-infected cell have been described. The canonical cytopathic effect (CPE), known for several decades (35), consists of a dramatic change in the cytoplasmic infrastructure with accumulation of numerous membranous vesicles, increased plasma membrane permeability, distortion and displacement of the nuclei, partial condensation of chromatin (pyknosis), rounding up of the infected cells, and eventually their lysis. On the other hand, poliovirus-infected cells may develop an apoptotic response (64) with cytoplasmic blebbing, heavy condensation and fragmentation of chromatin, and degradation of chromosomal DNA. The process is completed with cell fragmentation into small membrane-coated entities, the so-called apoptotic bodies. In HeLa cells, productive infection results primarily in CPE, whereas abortive infection caused by some viral mutations or the presence of some inhibitors may trigger the apoptotic reaction (3, 64). However, in some other cell cultures, e.g., CaCo-2 colon carcinoma cells (5) and a line of promonocytic cells (42), signs of apoptosis could also be observed in a proportion of productively infected cells.
Apoptosis is a result of the implementation of a complex cellular death program evolved to eliminate various forms of damaged or unwanted cells both during normal development (44, 67) and under different pathological conditions, including infectious diseases (19, 23). Apoptosis can be triggered by a broad variety of external and internal stimuli. Schematically, three major primary targets for these stimuli are known: (i) receptors on the cell surface, such as FAS, tumor necrosis factor (TNF) receptors, and so on (37, 70); (ii) mitochondria (2, 17, 57); and (iii) the endoplasmic reticulum (ER) (46, 53). The pathways starting from these primary targets generally include, among other components, a cascade of cysteine proteinases or caspases (12, 14, 21, 62). The upstream components of the cascades, initiator caspases, are specific for each primary target. Thus, the upstream caspase in the receptor-mediated apoptotic pathway is caspase-8, the mitochondrion-related pathway exploits caspase-9 as the initiator caspase, and the pathway triggered by ER stress is based on caspase-12. The above-mentioned pathways converge on downstream, or effector, caspases, exemplified by caspase-3. Each pathway also includes a variety of activating and inhibiting proteins, and there is cross talk between different pathways (for recent reviews, see references 2, 7, 11, 30, and 68). In addition, forms of apoptosis or apoptosis-like death which are accomplished through caspase-independent mechanisms are known (9, 28, 38, 59, 60, 66).
Against the background of this wealth of information about the cellular apoptotic machinery, little is known about the mechanisms of virus-triggered apoptosis in general (48, 55) and of apoptosis induced by poliovirus in particular. Expression of poliovirus proteases 2A (26) and 3C (6) has been reported to result, under certain conditions, in the apoptotic response. The implementation of poliovirus-induced apoptosis involves activation of caspases and is prevented in the presence of a broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD.fmk) (3). However, the nature of the virus-activated apoptotic pathway and the identities of the components involved were not elucidated.
Although poliovirus infection switches on a defensive apoptotic reaction, other viral functions exhibit antiapoptotic activities (64). In fact, the development of CPE rather than apoptosis in certain cells, such as HeLa cells, is caused by the early interruption of implementation of the activated cellular apoptotic program due to the expression of viral antiapoptotic functions (4). At what step and how the apoptotic pathway is interrupted remain unknown.
The major goal of the present study was to identify the nature of the pathway resulting in the apoptotic death of poliovirus-infected cells and to map the step of this pathway that is suppressed by the viral antiapoptotic function. By making use of cells deficient in caspase-3 or caspase-9 activity and by studying some markers of specific apoptotic pathways, we came to the conclusion that the viral infection primarily activates the apoptotic pathway involving consecutive activation of caspase-9 and caspase-3 due to cytochrome c release from damaged mitochondria. The results also suggest that productive viral infection suppresses implementation of this apoptotic program downstream of the cytochrome c release and that the suppression is caused, at least in part, by aberrant processing and degradation of procaspase-9.
Cells and virus.A subline of HeLa-S3, HeLa-B (64), and Bcl-2-expressing HeLa-Bcl2 (4) were cultivated in Eagle medium supplemented with 10% bovine serum, while cultivation of derivatives of MCF-7 cells (see below) was carried out in Dulbecco's modified Eagle medium with 10% calf embryonal serum. A derivative of poliovirus type 1 Mahoney strain, Mgs (63), was used.
Derivation of cells with altered properties of caspases.Cells differentially devoid of functional caspase-3 or caspase-9, or both, were derived from MCF-7 human breast carcinoma cells, known to have an inactivating deletion in the gene encoding caspase-3 (34). The genes encoding intact procaspase-3 and procaspase-3 harboring a loss-of-function mutation (24) fused to the gene encoding green fluorescent protein (GFP; Clontech) were cloned into the MaRX-IV(puro) retroviral vector, while cDNA of caspase 9DN (a dominant-negative mutant of caspase 9 [25]) was cloned into the MaRX-IV(neo) vector (27). The caspase-3-competent MCF-Cas3-GFP and the caspase-3-negative MCF-Cas3cs-GFP cells were obtained from MCF-7 cells after transduction with MaRX-IV/Cas3-GFP and MaRX-IV/Cas3cs-GFP(puro) vectors, respectively. The transduction was performed essentially as described previously (24). The transduced cells were selected in the presence of 1 μg of puromycin/ml for 4 days and were subsequently maintained in the presence of 0.5 μg of puromycin/ml. The fluorescence of GFP in all these cultures was relatively weak and did not interfere with any of our specific assays. For brevity, the suffix GFP will hereafter be omitted from the names of the cultures.
The caspase-3+ caspase-9+ MCF-Cas3/MIV cells and caspase-3+ caspase-9− MCF-Cas3/Cas9DN cells were similarly obtained by retroviral transduction of MCF-Cas3 cells with MaRX-IV(neo) and Marx-IVCas9DN(neo), respectively. MCF-Cas3cs/Cas9DN cells devoid of both caspase-3 and caspase-9 activities were prepared by using the same procedure after transduction of MCF-Cas3cs cells with the MaRX-IVCas9DN(neo) vector. Cells were subjected to selection with 500 μg of G-418 (Sigma)/ml for approximately 2 weeks, and the resistant clones were pooled. Subsequent cultivation was carried out in the presence of 300 μg of G-418/ml.
The overexpression of relevant caspases in derivatives of MCF-7 was confirmed by Western blotting (Fig. 1A).
Poliovirus infection and single-cycle growth experiments.Aliquots of EDTA-treated cells were plated on 35- or 60-mm-diameter petri dishes (Corning-Costar) at a density of approximately 105/cm2 and cultivated overnight under 5% CO2 at 37°C in Eagle medium with 5% bovine serum (HeLa cells) or Dulbecco's modified Eagle medium with 5% calf embryonal serum (MCF-7 derivatives). The growth medium was discarded, and the virus was added in a volume of 2 ml to provide an effective multiplicity of infection of ∼100 (HeLa cells) or ∼40 (MCF-7 derivatives) PFU/cell. After a 30-min incubation at 18°C, the cells were washed off with Earle's solution, and 1 or 3 ml (for 35- or 60-mm-diameter dishes, respectively) of serum-free Eagle medium was added. After incubation at 37°C for the appropriate time intervals, the viral harvest was plaque titrated on RD cells.
Apoptosis induction.The protein synthesis inhibitor cycloheximide (CHI; 100 μg/ml) or the transcription inhibitor actinomycin D (ActD; 1 μg/ml) was used as a nonviral inducer of apoptosis. To produce apoptotic response to poliovirus infection, the infected cells were treated with guanidine-HCl (100 μg/ml) at an early step (1.5, 2.0, or 2.5 h postinfection [p.i.]) and fixed for microscopic analysis several hours thereafter. When necessary, the development of apoptosis was prevented by 100 μM zVAD.fmk (Enzyme Systems Products, Dublin, Calif.).
Fluorescence and immunofluorescence microscopy.For observation of the status of chromatin, cells were grown on coverslips placed in petri dishes (see above). The permeable fluorescent dye Hoechst 33342 (Sigma) was added at a final concentration of 5 μg/ml 30 min before cell fixation with Safe Fix (Curtin Matheson Scientific, Houston, Tex.). For the immunofluorescence cytochrome c assay, cells were grown on coverslips, fixed with 4% paraformaldehyde in Dulbecco's phosphate-buffered saline (PBS) for 10 min, and permeabilized with 0.2% Triton X-100 in 4% paraformaldehyde in PBS for 10 min. Anti-cytochrome c rabbit polyclonal antibodies were from Santa Cruz Biotechnology (catalog no. sc-7159), and anti-rabbit immunoglobulin G fluorescein isothiocyanate-conjugated antibodies were from Sigma (catalog no. F-0382). Three percent nonfat dry milk in PBS was used for blocking nonspecific binding sites and for dilution of primary and secondary antibodies. The DNA dye Hoechst 33342 was added to the blocking solution at a concentration of 5 μg/ml. Subsequent incubations in blocking solution with primary and secondary antibodies were done for 1 h at 37°C. The cells were washed twice for 5 min each time in PBS with agitation at room temperature after incubation with the primary and secondary antibodies. The stained coverslips were placed on a drop of a mixture of glycerol-100 mM Tris, pH 9, and examined with a Leica DMLS epifluorescent microscope equipped with a Leica DC 100 digital camera and with filter cube A or I3 used for the observation of blue (Hoechst) and green (immunostaining) fluorescence, respectively.
TUNEL assay.Cells were grown on coverslips and stained with Hoechst 33342 as described above but were fixed at room temperature with Safe Fix for 30 min. Then, the cells were treated with 96 and 70% ethanol and stored at −20°C. The assay was performed with the kit from Promega according to the manufacturer's instructions. The filter cube I3 was used for the registration of terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL)-positive cells.
DNA fragmentation electrophoretic assay.DNA fragmentation was assayed as described previously (64). EDTA-treated cells suspended in a buffer containing 20 mM EDTA and 10 mM Tris-HCl, pH 7.4, were lysed with 0.5% Triton X-100 for 20 min in an ice bath. The nuclei were pelleted in an Eppendorf 5415C minicentrifuge (12,000 rpm; 15 min; 4°C), and the resulting supernatants were treated with phenol-sodium dodecyl sulfate. After ethanol precipitation, the nucleic acids were dissolved in 10 μl of H2O and treated with RNase A (10 μg/ml; 37°C; 30 min). Samples were subjected to electrophoresis on 1.5% agarose gels.
Western blotting.Cells were treated with EDTA and collected, together with the detached floating material, by centrifugation in an Eppendorf minicentrifuge at minimal speed for 4 min. The supernatant was discarded, and the pellet was lysed with a buffer containing 2% sodium dodecyl sulfate-35 mM β-mercaptoethanol-50 mM Tris-HCl, pH 6.8, supplemented immediately before use with 1 mM phenylmethylsulfonyl fluoride and a cocktail of protease inhibitors containing chymostatin, pepstatin, leupeptin, and antipain (2 μg of each/ml). Following addition of the lysis buffer, the cells were incubated in a boiling-water bath for 10 min, and the lysates were sonicated to reduce viscosity. The protein concentration was measured using the Bradford protein concentration assay (Bio-Rad) according to the manufacturer's recommendations. The total amount of protein loaded into one well of the discontinuous denaturing polyacrylamide minigel was 30 μg. After electrophoresis, the proteins were transferred onto a 0.45-μm polyvinylidene difluoride membrane (Amersham) using the Bio-Rad minigel transfer system. The membrane was incubated overnight in blocking solution (135 mM NaCl, 17.3 mM Tris-HCl [pH 7.0], 0.1% Tween-20, 5% nonfat dry milk) at 4°C with agitation. This blocking solution was also used for the dilution of primary and secondary antibodies. The following preparations were used as primary antibodies: anti-caspase-9 rabbit polyclonal and anti-caspase-3 mouse monoclonal antibodies (25), anti-Bid rabbit polyclonal antibodies from Pharmingen (catalog no. 550365), anti-cytochrome c mouse monoclonal antibodies from Santa Cruz Biotechnology (catalog no. sc-13156), and anti-actin mouse monoclonal antibodies from Sigma (catalog no. A-4700). The secondary antibodies were represented by anti-mouse or anti-rabbit horseradish peroxidase conjugates from Amersham. Incubation with antibodies was performed for 1 h at room temperature, followed by triple washing with the blocking solution without milk. Protein bands were visualized with the ECL+ detection system (Amersham).
Cell fractionation.EDTA-treated cells from 6-cm-diameter petri dishes were pelleted for 5 min by centrifugation in an Eppendorf minicentrifuge at minimal speed. The pellet was resuspended in 200 μl of a hypotonic lysis buffer (20 mM HEPES [pH 7.4], 10 mM KCl, 2 mM EGTA, 1 mM dithiothreitol), supplemented immediately before use with the cocktail of protease inhibitors (see above). The cells were allowed to swell on ice for 15 min and were lysed by 15 strokes of an insulin syringe with a 27.5-gauge needle. The lysis was controlled under a phase-contrast microscope. Organelles, nuclei, and unbroken cells were collected at 15,000 × g for 20 min, and the supernatant was used as the soluble cytoplasmic fraction.
Role of caspase-3 in poliovirus-triggered apoptosis.The converging downstream parts of different caspase cascades include effector caspase-3, caspase-6, and caspase-7. Our preliminary experiments demonstrated that MCF-7 human breast carcinoma cells, known to lack functional caspase-3 due to a deletion in the appropriate gene (34), failed to elicit the apoptotic response to poliovirus infection either under permissive conditions or after interruption of productive infection by guanidine-HCl (not shown), a condition known to trigger the apoptotic response in HeLa cells (3, 64). These observations were consistent with a key role of caspase-3 in the implementation of the apoptotic program in poliovirus-infected cells. However, MCF-7 cells are deficient not only in caspase-3 but also in another component(s) of the standard apoptotic machinery (33).
To gain a deeper insight into the role of caspase-3 in poliovirus-triggered apoptosis, we made use of two derivatives of MCF-7 cells. One, MCF-Cas3, was stably transformed with a retroviral vector carrying the intact caspase-3 gene under a cytomegalovirus promoter, while the other, MCF-Cas3cs, was transformed with a similar vector having a loss-of-function mutation in the caspase-3 gene. When tested with a nonviral inducer of apoptosis (the protein synthesis inhibitor CHI), MCF-Cas3 cells, but not MCF-Cas3cs cells, developed a marked apoptotic response, as judged by nuclear staining with a permeable nuclear dye, Hoechst 33342, and electrophoretic assay for DNA degradation (not shown). The CHI-induced signs of apoptosis in MCF-Cas3 cells were essentially prevented in the presence of a 100 μM concentration of a broad caspase inhibitor, zVAD.fmk (not shown). Similar results were obtained with another apoptotic inducer, the transcription inhibitor ActD (not shown). These results demonstrated that the responses of MCF-Cas3 cells to CHI and ActD were similar to those of HeLa cells. On the other hand, MCF-Cas3cs cells proved to be refractory to these apoptotic inducers (not shown).
In single-cycle growth experiments, the efficiency and time course of poliovirus reproduction were essentially the same in the cells with and without caspase-3, with a yield of ∼104 PFU per cell by 6 h p.i. (Fig. 1B). The productive infection of MCF-Cas3 or MCF-Cas3cs produced a typical pattern of CPE, revealed by nuclear staining (nuclear deformation and partial condensation of chromatin) (Fig. 2A) and phase-contrast microscopy (not shown). The electrophoretic assay showed no appreciable DNA degradation in the productively infected MCF-Cas3cs cells (Fig. 3A, lane 7) and only a small accumulation of high-molecular-mass DNA in MCF-Cas3 cells (Fig. 3A, lane 1), which was prevented in the presence of zVAD.fmk (Fig. 3A, lane 2). The TUNEL assay for DNA degradation in productively infected cells revealed only a very small proportion of positive cells, ∼6 and 0% for MCF-Cas3 and MCF-Cas3cs cells, respectively (Fig. 2B). Even these small proportions of TUNEL-positive signals corresponded largely to dead cells, as judged by their staining with trypan blue (not shown). Also, a similar proportion of TUNEL-positive infected cells could be revealed in the cultures treated with zVAD.fmk (not shown), suggesting that the degradation of DNA in a few productively infected cells was caused largely by caspase-independent postmortem alterations.
When ongoing poliovirus infection was interrupted by the addition of millimolar concentrations of guanidine-HCl, a marked proportion of MCF-Cas3 cells developed apoptotic reactions, as evidenced by strong condensation of chromatin revealed by Hoechst staining (Fig. 2C), positive TUNEL reactions (Fig. 2B and C), and DNA fragmentation, predominantly into high-molecular-mass species (Fig. 3A, lanes 3 and 5). These effects were suppressed by zVAD.fmk (Fig. 3A, lane 6, and results not shown). Thus, the response to nonpermissive poliovirus infection of the MCF-7 derivative with active caspase-3 was very similar to that of HeLa cells. In contrast, no significant morphological signs of apoptosis or DNA degradation could be observed under these conditions in MCF-Ca3cs cells (Fig. 2B and C and 3A, lane 8).
The above-mentioned facts clearly demonstrate that caspase-3 is an essential component of the apoptotic pathway triggered by abortive poliovirus infection in MCF-Cas3 cells and that the failure of MCF-Cas3cs cells to undergo apoptosis under these conditions is due to the absence of active caspase-3.
Role of caspase-9 in poliovirus-triggered apoptosis.Caspase-3 is an effector caspase which can be activated through specific proteolytic cleavage by upstream components of different apoptotic pathways. One such upstream caspase is caspase-9, which is involved in the mitochondrion-dependent pathway.
To ascertain the role of caspase-9 in poliovirus-triggered apoptosis, we generated a derivative of MCF-Cas3 cells in which caspase-9 was inactivated by stable transfection with a retrovirus vector expressing a dominant-negative mutant of this enzyme with a Cys287-to-Ser substitution (25, 51). These cells, lacking active caspase-9 but possessing caspase-3, were named MCF-Cas3/Cas9DN. As controls, MCF-Cas3 cells transformed with empty vector (MCF-Cas3/MIV cells) were used. In contrast to control cells, the MCF-Cas3/Cas9DN cells failed to develop appreciable apoptosis when treated with nonviral inducers of apoptosis, such as CHI or ActD (not shown).
The absence of caspase-9 did not affect the yield (about 104 PFU/cell) or time course of poliovirus reproduction in MCF-Cas3/Cas9DN cells (Fig. 1B), nor could any significant differences be observed in the appearance of nuclear (Fig. 4A) and cytoplasmic (not shown) CPE upon productive infection of these two cultures. At a late stage of infection, only a small proportion (∼4%) of productively infected MCF-Cas3/MIV cell appeared to be TUNEL positive (Fig. 4B), and only limited DNA degradation to high-molecular-mass species could be detected in appropriate samples upon electrophoresis (Fig. 3B, lane 2). As indicated above, a limited DNA degradation in productively infected MCF-Cas3 cells was not necessarily apoptosis related.
A significant proportion (20 to 30%) of control MCF-Cas3/MIV cells, which contained functional caspase-9 and caspase-3, efficiently responded by apoptosis to abortive poliovirus infection (i.e., when reproduction was interrupted at an early stage by guanidine-HCl), as judged by strong chromatin condensation revealed by Hoechst staining (Fig. 4C), as well as DNA degradation seen with both the TUNEL assay (Fig. 4B and C) and gel electrophoresis (Fig. 3B, lane 1). On the other hand, MCF-Cas3/Cas9DN cells lacking caspase-9 failed to develop apoptosis upon nonpermissive poliovirus infection, judging by morphological and cytochemical criteria (Fig. 4B and C) and DNA electrophoresis (Fig. 3B, lane 4).
We concluded that caspase-9 activity is essential for efficient apoptotic response to poliovirus infection.
Cytochrome c release from mitochondria caused by poliovirus infection.A major mechanism of caspase-9 activation involves its autoproteolysis after recruitment into the apoptosome, a complex also containing Apaf-1 and cytochrome c (25, 40). Formation of this complex requires release of the latter from mitochondria damaged by more upstream apoptotic signals (2, 16). Therefore, the distribution of cytochrome c in cells undergoing permissive and abortive (apoptogenic) infection with poliovirus was studied by immunostaining. In intact or mock-infected cells, this technique revealed punctate entities, obviously corresponding to mitochondria, concentrated predominantly in the perinuclear region (Fig. 5A and B). The efflux of cytochrome c from mitochondria was seen as a diffuse staining of the entire cytoplasm in a significant proportion of HeLa cells, not only when they were undergoing abortive infection (resulting in apoptosis) but, surprisingly, also upon productive infection (resulting in CPE-type death) (Fig. 5A). The cytoplasmic extramitochondrial accumulation of cytochrome c in the abortively and productively infected cells could also be registered by Western blotting (Fig. 5C), being clearly observed by 4 h p.i. under both conditions and developing even faster in the case of productive infection (Fig. 5C, compare lanes 2 and 6 or lanes 3 and 7). Similarly, efflux of cytochrome c from mitochondria could be registered in MCF-Cas3 cells (i.e., cells containing both caspase-3 and caspase-9) destined for either CPE or apoptosis (not shown). The cytochrome c efflux from the mitochondria of infected HeLa cells was markedly suppressed by overexpression of the antiapoptotic Bcl-2 protein (Fig. 5B), a condition shown previously to postpone the development of apoptosis in abortively infected cells but exerting no appreciable effect on either the time course of virus reproduction or CPE development (4). The absence of both caspase-9 and caspase-3 in MCF-Cas3cs/Cas9DN cells did not significantly affect either the time course or the extent of cytochrome c release (Fig. 5B), in line with the notion that this release was an upstream event.
We concluded that poliovirus infection is accompanied by mitochondrial damage and exit of cytochrome c, which may or may not lead to development of apoptosis, depending on the conditions of infection.
Processing of Bid upon abortive poliovirus infection.Different factors may result in the mitochondrial damage that leads to cytochrome c efflux. Among them is Bid, a proapoptotic protein of the Bcl-2 family, the truncated form of which, together with another Bcl-2-related protein, Bax, induces permeabilization of the outer mitochondrial membrane (1, 10, 31). When the content of the unprocessed form of Bid was assayed in extracts from productively and abortively infected HeLa cells by Western blotting, a marked decrease was detected only in the latter, e.g., when the infection was interrupted by the early addition of guanidine at 1.5 h p.i. (Fig. 6, lane 6). Under these conditions, the processing of Bid was even more efficient than in the cells in which apoptosis was triggered by the addition of CHI (Fig. 6, lane 5). On the other hand, no appreciable processing of Bid was detected under productive infection (Fig. 6, lanes 1 to 3). If guanidine was added at 3 h p.i., that is, when the viral antiapoptotic function was expressed sufficiently to suppress the cellular apoptotic response to infection (4), processing of Bid was also markedly suppressed (Fig. 6, lane 7).
Since cytochrome c release from mitochondria was observed in both types of infection while truncation of Bid occurred only upon abortive infection, we concluded that processing of Bid was not the primary cause of the permeabilization of mitochondrial membranes in poliovirus-infected cells but rather was involved in the amplification of the apoptotic signal in the cells destined for apoptosis.
Aberrant caspase-9 processing upon productive infection.As was shown above, productive infection of HeLa or MCF-Cas3 cells resulted in the efflux of cytochrome c into the cytoplasm, but this event, generally associated with the induction of apoptosis, failed to trigger such a response under these conditions. This failure obviously reflected the interruption of the implementation of the activated apoptotic program by antiapoptotic viral functions (4). We decided to ascertain whether the block in the implementation of the apoptotic program took place upstream or downstream of caspase-9. This enzyme is normally stored in inactive proenzyme form (procaspase-9), and its activation is accomplished by autoproteolysis within the apoptosome, a structure composed of cytochrome c, Apaf-1, and procaspase-9. The amount of procaspase-9 diminished under conditions of both productive (destined for CPE) (Fig. 6, lanes 2 and 3) and abortive (destined for apoptosis) infection of HeLa cells (Fig. 6, lane 6). However, in abortive infection, the decrease in procaspase-9 was accompanied by the accumulation of a 35-kDa protein band identical to that generated upon CHI-triggered apoptosis (Fig. 6, compare the bands marked with the arrowhead in lanes 5 and 6) and apparently corresponding to the active enzyme, while this band was virtually absent upon productive infection (Fig. 6, lanes 2 and 3). Under these conditions, the appearance of caspase-9-related protein species with aberrant electrophoretic mobility could be detected instead. The processing of procaspase-9 and the appearance of the aberrant bands were not prevented by zVAD.fmk (Fig. 6, lane 4), suggesting that other caspases, at least those sensitive to zVAD.fmk, could hardly be involved.
A similar assay with antibodies against procaspase-3 demonstrated that processing of this enzyme occurred upon abortive infection (Fig. 6, lane 6), as expected (4, 42), but no appreciable loss of procaspase-3 could be detected in productively infected HeLa cells (Fig. 6, lanes 2 and 3) or when guanidine was added too late to suppress the viral antiapoptotic activity (Fig. 6, lane 7).
Viruses can trigger the defensive apoptotic response of the infected host cell by a variety of mechanisms, but many viruses possess different tools to overcome this defense (48, 55, 65). The results of this study allowed us to identify the major apoptotic pathway activated upon poliovirus infection and to ascertain which part of this pathway is targeted by the viral antiapoptotic activity.
The efflux of cytochrome c from mitochondria and the degradation of procaspase-9 in the poliovirus-infected cells were the most upstream of the identified apoptosis-related events studied here. These effects became prominent between 2 and 4 h p.i. (Fig. 5 and 6). Remarkably, switching on of the apoptotic program in a significant proportion of infected cells could be detected as early as 1.5 to 2 h p.i. (4). The upstream reactions occurring at the very early stages of infection prior to the mitochondrial damage have yet to be elucidated. Since several poliovirus proteins, i.e., 2A (26), 3C (6), and possibly some others, exhibit apoptosis-inducing activity, more than one mechanism of the poliovirus-triggered mitochondrial damage may conceivably be involved. Theoretically, the virus-activated pathway leading to cytochrome c detachment from cardiolipin and its subsequent efflux into the cytoplasm through an altered outer mitochondrial membrane (2, 17, 43, 49, 71) may include activation of some proapoptotic members of the Bcl-2 family of proteins, such as Bid (1, 10, 31); direct interaction of the viral protein and mitochondria (22); some other indirect effects; or a combination of these factors. Processing of Bid was indeed detected in abortively infected HeLa cells destined for apoptosis. However, since permeabilization of the outer mitochondrial membrane occurred upon productive infection as well, when no appreciable Bid processing was observed, we concluded that Bid was not the primary inducer of mitochondrial damage. More likely, processing of Bid was accomplished by an already activated effector caspase, e.g., caspase-3 (61), and represented a component of the loop involved in the amplification of the apoptotic signal. Caspase-2, which was recently demonstrated to be a key component in the mitochondrial-damage pathway activated by certain apoptotic inducers (26a, 37a), does not appear to play such a role in MCF-7 cells (37a) and therefore could hardly be responsible for the poliovirus-triggered apoptosis in the derivatives of these cells.
It is perhaps appropriate to note that alterations in the oxidative functions of mitochondria have been reported to occur early in poliovirus infection (36).
Although they were not studied here, mitochondrial alterations may also be accompanied by the release of other components of the apoptotic machinery, such as the mitochondrial stores of some procaspases (e.g., procaspase-9 and procaspase-3) (43, 71) and the Smac/Diablo protein acting as an inhibitor of the cellular antiapoptotic IAP proteins (18, 20, 69), as well as proteins capable of killing cells in a caspase-independent way, such as the apoptosis-inducing factor AIF (59), endonuclease G (39, 66), and Omi/HtrA2 serine protease (28). The fact that in the absence of caspase-9 and caspase-3 the virus-triggered apoptotic response was strongly suppressed indicated that mechanisms independent of these caspases (e.g., AIF mediated), if involved, hardly played a decisive role in this response.
It may be noted that apoptosis induced by proteins of some other viruses, e.g., human immunodeficiency virus (32) or chicken anemia virus (16), may also involve the mitochondrion-dependent pathway.
Once cytochrome c is released, the energy-dependent generation of high-molecular-mass complexes, the apoptosomes, also involving Apaf-1 and procaspase-9, is expected to occur, ensuring limited autoproteolysis of the latter and the appearance of active caspase-9 (40, 54). In poliovirus-infected cells destined for apoptosis, the proper procaspase-9 cleavage was documented here by Western blotting, and the key role of caspase-9 in the poliovirus-triggered apoptotic pathway was demonstrated by a dramatic suppression of the development of apoptosis-related nuclear damage in cells lacking appropriate activity (i.e., MCF-Cas3/C9DN).
The next downstream essential component of the poliovirus-induced apoptotic pathway is caspase-3. Previously, activation of Asp-Glu-Val-Asp (DEVD)-specific caspases (4) and cleavage of procaspase-3 (42) have been observed in poliovirus-infected cells undergoing apoptosis. As shown here, the lack of caspase-3 in MCF-Cas3cs cells resulted in the nearly complete absence of key signs of apoptosis. Thus, caspase-3 is an essential component of the poliovirus-triggered apoptotic pathway, while other known effector caspases, e.g., caspase-6 and caspase-7, appear to be unable to efficiently replace caspase-3 in this process.
One may speculate that the activation of caspase-3 in abortively infected cells might be accomplished not only by the mitochondrion-dependent pathway but also by some other mechanisms. For example, the involvement of caspase-12 known to be activated in response to ER stress (45, 46, 53) cannot be ruled out, since drastic ER alterations accompany poliovirus infection (56). As shown recently, this enzyme is able to activate caspase-9 through a mitochondrion-independent mechanism (44a, 52a). The ER-dependent pathway was reported to be activated, for example, upon infection with respiratory syncytial virus (8). Although the potential role of this and some other mechanisms in poliovirus-induced apoptosis has yet to be explored, the marked antiapoptotic activity of Bcl-2 argues for a mitochondrion-mediated mode. Thus, our results suggest that the major poliovirus-triggered apoptotic pathway in the cells studied here includes consecutive activation of caspase-9 and caspase-3 and appears to be caused by cytochrome c release (Fig. 7).
Concurrently with turning on the apoptotic program and despite the potential availability of all of the necessary components of the major apoptotic pathway, the overwhelming majority of productively infected HeLa and MCF-Cas3 cells develop CPE rather than typical apoptosis. This is due to the antiapoptotic function(s) of poliovirus capable of interrupting implementation of the ongoing apoptotic program (4, 64). Since cytochrome c release proceeds upon productive infection essentially as efficiently as upon abortive infection, we conclude that the major target of the putative viral antiapoptotic function(s) is downstream of the mitochondrial damage. In this respect, the antiapoptotic functions of poliovirus differ from those of several other viruses, e.g., some herpesviruses and adenoviruses, which encode proteins able to stabilize mitochondria and thus prevent the release of cytochrome c (22).
The molecular basis of poliovirus interference with the activation of caspase-9 is still unclear. The appearance of caspase-9-related proteins with unexpected electrophoretic mobility in productively infected cells may suggest that the viral antiapoptotic function involves aberrant processing, e.g., alternative proteolysis, of procaspase-9. The amino acid sequence of human procaspase-9 contains a YG dipeptide, a canonical cleavage site for the poliovirus 2A protease (50). The putative cleavage should generate peptides of 251 and 165 amino acid residues, respectively. Neither of them could be related to the observed aberrant polypeptide bands. Since the specificity of poliovirus 2A protease, especially its trans-acting activity, is highly dependent on the context of the cleavage site (29), it is not surprising that the YG bond in procaspase-9 does not appear to be the target for this protease. No canonical cleavage site (QG [50]) for the poliovirus protease 3C is present in procaspase-9. Thus, either a viral protease cleaves the proenzyme at suboptimal sites or an infection-stimulated cellular proteolytic activity is involved. Interestingly, a Ca-dependent protease, calpain, is known to degrade and inactivate procaspase-9, as well as some other caspases (15). In this regard, it may be noted that a misbalance of ions (Ca2+ included) caused by alterations in the permeability of cellular membranes in poliovirus-infected cells is a well-documented phenomenon (13).
If the aberrant cleavage of procaspase-9 occurs closer to its N terminus, it may be accompanied by the removal or inactivation of the caspase recruitment domain (CARD), preventing recruitment of the proenzyme into the apoptosome (72). It may be appropriate to note that the liberated CARD itself may perform an antiapoptotic function by activating NF-κB (58).
The identification of aberrant processing of procaspase-9 as a putative component of the poliovirus antiapoptotic activity does not necessarily rule out the existence of additional relevant mechanisms. A variety of factors are known to impair the recruitment of caspase-9 into apoptosomes or its subsequent activation (7, 41, 52, 68), and they may be regarded as potential targets for viral antiapoptotic functions.
Although the major apoptotic pathway operating in poliovirus-infected cells is mitochondrion mediated, other pathways may potentially be involved under some settings. As already mentioned, the ER stress-mediated pathway also potentially contributes to the cellular response to poliovirus infection. The infection may modulate the extrinsic, receptor-mediated apoptotic pathway as well. For example, expression of poliovirus protein 2A renders the cells more sensitive to the apoptosis-inducing effect of TNF (47). On the other hand, viral infection and, in particular, expression of the viral protein 3A results in depletion of TNF receptor (47) and receptors for some other cytokines (47a) from the plasma membrane, decreasing the sensitivity of the cells to these cytokines.
Thus, although we did not study it, the fate of the infected cell depends on a complex network of interacting cellular and viral proteins and is host specific as well as virus specific. The results reported here contribute to a deeper understanding of this network. The type of death of infected cells is a significant factor in the pathogenesis of viral diseases. CPE, but not apoptosis, is generally accompanied by an inflammatory reaction, and the presence or absence of this reaction should markedly affect the clinical patterns of viral diseases.
This study was supported by grants from INTAS, the Ludwig Institute for Cancer Research, the Russian Foundation for Basic Research, the Scientific School Support Program, and the Russian Federal Program "Integration."
- Received 17 April 2002.
- Accepted 23 September 2002.
- ↵*Corresponding author. Mailing address: Institute of Poliomyelitis, Moscow Region 142782, Russia. Phone: 7 (095) 439 9026. Fax: 7 (095) 439 9321. E-mail: agol{at}belozersky.msu.ru.
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- American Society for Microbiology


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investment property cash out refinancing Real Estate Matters | How to maximize rental property investment – Let's start with your questions about refinancing. While you might be able to do a cash. property. Another strategy is to buy a multi-unit building, live in one of the units and rent out the others.construction to permanent home loans 2 Types Of Construction Loans Explained | Bankrate.com – There are two main types of home construction loans: Construction-to-permanent: You borrow to pay for construction. When you move in, the lender converts the loan balance into a permanent mortgage.
How to Get Money for a Down Payment on a House – 16. – 2. Shrink Your Required Down Payment With a Special Loan. If you're looking to buy on an accelerated timetable, live in an expensive housing market, or doubt your ability to save for a 20% down payment on an acceptable house in your target neighborhood, look into special loan programs with lower down payment requirements.
What Are the Rules for Down Payment Gifts? – SmartAsset – In that scenario, you'd be responsible for paying at least 3.5% of the down payment yourself. Regardless of whether you're getting a conventional, FHA or VA loan, a down payment gift is only acceptable when the house you're purchasing will be your primary residence or second home.
While you can't use a loan for a down payment on a house, here are some other ways you can come up with your down payment. gift funds. Some mortgages, like FHA loans, allow for the down payment to be a gift from a friend or family member. 100 percent of the 3.5% down payment required for FHA loans may be gifted.
Your down payment plays an important role when you're buying a home. A down payment is a percentage of your home's purchase price that you pay up front when you close your home loan. lenders often look at the down payment amount as your investment in the home. Not only will it affect how much you'll need to borrow, it can also influence:
what is interest rate and apr fha restrictions on buying check eligibility for a FHA Home Loan – · To finance the program and obtain mortgage insurance from the FHA, buyers are required to pay an upfront mortgage insurance premium equal to 1.75 percent of the cost of the loan they are seeking. For example, if a borrower took out a loan for $100,000, they would be required to pay an upfront premium of $1,750. · Interest is essentially a rental or leasing charge to the borrower for the use of an asset. In the case of a large asset, such as a vehicle or building, the lease rate may serve as the interest rate.refinance to avoid foreclosure applying for a mortgage with bad credit Can We Get a Mortgage If One Spouse Has Bad Credit? – You could also have a family member with good credit replace the person with the bad credit when applying for a mortgage, but this may not be the best solution since doing business with family can lead to bad results.buying new house tax deductions hud homes for sale good neighbor next door current 30 year refinance mortgage rates current mortgage rates for April 7, 2019 are still near their historic lows. compare 30-year, 15-year fixed rates, and ARMs to find the best home loan offer all in one place at LendingTree.Chapter 8: Good Neighbor Next Door Program | RealtyStore – Available to teachers and first responders, the Good Neighbor Next door program offers some excellent benefits to qualifying people who want to purchase HUD homes. This special program allows qualifying individuals to enjoy a 50% discount from the HUD home's listing price.What Deductions Can You Claim When Buying a Home? | Finance. – Whether you're buying a new primary residence or buying a vacation home, your purchase makes you eligible for several tax deductions, especially if you finance your purchase with a mortgage.Loan modification could help you avoid foreclosure until the divorce is final and the house is sold or one of you qualifies to refinance the mortgage on your own..
Down payment – Wikipedia – Down payment (or downpayment, also called a deposit in British English), is a payment used in the context of the purchase of expensive items such as a car and a house, whereby the payment is the initial upfront portion of the total amount due and it is usually given in cash at the time of finalizing the transaction.
What Is the Standard Down Payment on a House? | realtor.com – "Down payment": It's amazing that these two little words have such a profound influence on your homeownership process-and your life! Ask most people what is an acceptable down payment on a house.
The Average. A down payment of 20% or more reducing the need for expensive Private Mortgage Insurance (PMI). PMI is there to insure that the lenders funds are protected should a buyer no longer make the mortgage payments. Thus a down payment in Silicon Valley where home prices.

subset: commoncrawl


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Watch The Texas Chainsaw Massacre 2 (1986) : Full Movie Online Free A radio host is victimized by the cannibal family as a former Texas Marshall hunts them.
|The Texas Chainsaw Massacre 2
|Release Date
|Aug 22, 1986
|Production Company
|Cannon Group, Golan-Globus Productions
|Production Countries
|United States of America
|Dennis Hopper, Caroline Williams, Jim Siedow, Bill Moseley, Bill Johnson, Ken Evert , Harlan Jordan, Kirk Sisco, James N. Harrell, Lou Perryman, Kinky Friedman
|Plot Keywords
|gore, b movie, slasher, chainsaw,
Lieutenant 'Lefty' EnrightDennis Hopper
Vanita 'Stretch' BrockCaroline Williams
Drayton Sawyer, the CookJim Siedow
'Chop-Top' SawyerBill Moseley
Leatherface 'Bubba' SawyerBill Johnson
Grandpa SawyerKen Evert
PatrolmanHarlan Jordan
DetectiveKirk Sisco
Cut-Rite ManagerJames N. Harrell
L.G. McPetersLou Perryman
Sports AnchormanKinky Friedman
Man in Hotel CorridorTobe Hooper


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- Wednesday 2 January 2013
- Embracing their passion for local produce, John and Deborah Marten from Fruity Fanatics, squeeze, crush, puree, blend and freeze their fruit and turn it into handcrafted treats. ...Monday 25 June 2012
- With the recent rainfall across the Southwest Land Division many farmers are breathing a big sigh of relief....Tuesday 19 June 2012
- With the recent rainfall across the Southwest Land Division many farmers are breathing a big sigh of relief....Tuesday 19 June 2012
- A citrus orchard at Moora, 200 kilometres north of Perth, hasn't looked back since planting their first trees in 2005. ...Friday 15 June 2012
Explore by Topic
Latest Fruits stories on ABC Esperance
Latest Fruits stories on ABC Online
- Across the country, there have been more than 100 reports of needles found inside strawberries picked and packed for public consumption. The contamination scandal brought an industry to its knees and police are no closer to the truth. It's a mystery ripe for investigation. Hagar Cohen reports. ...Sunday 21 October 2018
- Thursday 22 November 2018
- Wednesday 4 April 2018
- Wednesday 22 November 2017
- Friday 4 August 2017
Explore ABC Esperance by Topic
Latest videos
Latest audio
- Perth Mint chief executive Richard Hayes talks about plans to develop a crypto-gold product
- Remembering Ally; David and Dorothy Henderson talk about their daughter Alexandra
- WA Minister for Education Sue Ellery talks about changes at Muresk
- Curtin vice chancellor Deborah Terry talks about the university's return to Muresk
- Family pays tribute to teenage shark attack victim


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Getting to and from your Cotswold Way hiking tour is easy… when you know how!
In your walking pack, you will receive all the necessary public transport information you need to make your journey as convenient as possible.
In the meantime, if you'd like to do some advance planning yourself, here are our tips:
Most Convenient Airports:
3. London's Airports (Heathrow, Stansted, Gatwick, Luton, London City)
How to Get to the Start-point of your Hiking Tour:
Most starting locations can easily be reached by train or bus. See this link for further information.
Departing from the Cotswolds after your Hiking Tour:
Use train or bus services to Birmingham, Bristol or London
Travelling by car?
It is usually possible to park at your first B&B and simply pick your car up again after your hiking tour.
Please contact us in advance so that we can make these arrangements on your behalf.


text: constantly seeking beauty: an instagram round-up My creativity is fueled by beauty. Here's a round-up of some things that truly inspire that spark. Note to self: Make a balloon ceiling in the near future. 🎈🎈🎈 Regram from @elsielarson via our book #HappyHandmadeHome A photo posted by Elsie + Emma A Beautiful Mess (@abeautifulmessofficial) on Jun 28, 2015 at 4:30pm PDT Taking this beauty along my morning commute tomorrow. Please pray that I don't pop a tire or hit a car or die. E #thankgodforsidewalks A photo posted by Flourish Collaborative (@flourishcollaborative) on Jun 2, 2015 at 3:57pm PDT Endless coffee and planning #naturally #designloveplanner #cactus #almondmilklatte #dairyfree #blush A photo posted by Design Love Co. (@designloveco) on May 21, 2015 at 11:25am PDT Taking this box of goodies over to Dad's (card by @kardzkouture) Full blown family BBQ with all the fixing tonight! Hope you are enjoying your weekend💕 #fathersday #familyfirst #sunday A photo posted by Sydney Roberts (@workinggirlinc) on Jun 21, 2015 at 6:20pm PDT today is the perfect day for a little song and a masala chai #oldcrowcoffeeco A photo posted by Old Crow Coffee Co. (@oldcrowcoffeeco) on May 25, 2015 at 2:03pm PDT Mental HealthMegan te BoekhorstJune 29, 2015New Westminster Facebook0 Twitter LinkedIn0 StumbleUpon Tumblr Pinterest0 0 Likes


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Grand Champions Polo Club Spring Season Gets Under Way Friday With Santa Rita Abierto/
By Sharon Robb
WELLINGTON, FL—April 20, 2017----Grand Champions Polo Club opens its spring season Friday with the Santa Rita Abierto Tournament.
The first of six tournaments kicks off what is expected to be the best spring season in the club's eleven-year history.
Five teams are entered for the Santa Rita Abierto and Grand Champions Cup.
Two games are scheduled for Friday at 3 and 5 p.m. The winners advance into the final and runner-up teams play for the Grand Champions Cup.
In Game One, Dutta Corp (Timmy Dutta, Lucas Diaz Alberdi, Piki Diaz Alberdi, Alex Webb) plays Altair Polo (Ashley Busch, Brandon Phillips, Kris Kampsen, Moritz Gaedeke) and Deeridge (Will Jacobs, Grant Ganzi, Ezequiel Ferrario, Carlitos Gracida) at 3 p.m. on Field 3.
In Game Two, Sebucan (Josh Dubarry, Pablo Pulido, Alejandro Novillo Astrada, Julio Gracida) plays Hidden Creek (Sean Keys, Pablo Dorignac, Jesse Bray, Marc Ganzi) at 5 p.m. on Field One.
On Saturday, Grand Champions will host the Governor's Cup on Field 1 at 1:30 p.m. followed by the National President's Cup on Field 2 at 3 p.m. featuring Deeridge (Will Jacobs, Juan Olivera, Juancito Bollini, Tito Gaudenzi) against Aspen Valley Polo Club (Rich Desich, Melissa Ganzi, Grant Ganzi, Juan Bollini).
On Sunday, the Grand Champions Cup will get underway at 9:45 a.m. on Field One followed by the Santa Rita Abierto Tournament at 11 a.m. on Field 2.
Dutta Corp is two-time defending champion. Two years ago, as a 15-goal team, Dutta Corp won the 16-goal rated tournament with pro Matias Magrini, brothers Carlitos and Mariano Gracida and Timmy Dutta. Last year a talented 11-goal team of Tim Dutta, Timmy Dutta, Piki Diaz Alberdi and Carlitos Gracida won the 14-goal tournament with an impressive 12-5 victory over Whitehall Ranch.
Several top and up-and-coming players and patrons will compete during the fun and competitive GCPC Spring Invitational League that features six 12-16-goal tournaments in April and May.
During the Grand Champions' spring season, amateur polo players get the chance to play with and against some of the world's top professional polo players including former 10-goaler Piki Diaz Alberdi, Alejandro Novillo Astrada, Pablo Dorignac, Carlitos Gracida, Brandon Phillips, Kris Kampsen and Jesse Bray. It is geared towards improving polo skills and having fun.
The spring league is also a great opportunity for younger players to develop and hone their skills against their peers and more experienced players.
The GCPC spring league features:
*Medium Goal practice games at least once a week.
*Stick-and-ball sessions available during the week.
*Teams get two medium goal tournament games every weekend.
*Ambulance and umpires for all tournament games.
*Complete turn-key tournament polo with some of the top professionals in the world.
*Playing polo on some of Wellington's most prestigious and well-manicured fields.
*Club-sponsored social events every weekend for all players.
*Excellent trophies for all teams every weekend.
The spring season is being live-streamed on Wellington-based ChukkerTV, worldwide leaders in polo broadcasting.
Grand Champions Polo Club and Santa Rita Polo Farm is the largest and most unique private 100-acre polo facility in Wellington with 120 stalls in five self-contained barns, exercise track, five climate-controlled tack rooms, vet room, staff quarters, guest house and polo fields with state-of-the-art underground irrigation and stick-and-ball fields.
During fall, winter and spring seasons, Grand Champions Polo Club, the nation's largest club with 10 well-manicured fields, hosts polo tournaments ranging from six to 26-goal in addition to special events.
The club features monthly 6, 8 and 12-goal tournaments and women's weekly league play during the winter season in addition to two 16-20 tournaments, $50,000 National 12 Goal, $100,000 World Cup winner-take-all 0-40-goal tournament, Sunny Hale's Legacy WCT Final and USPA National President's Cup.
During the 2017 winter season Grand Champions attracted a record 72 teams and another 18 teams during the spring season.
Grand Champions caters to men, women and youth polo players at all levels. Its expert staff can customize a complete playing experience including horses, pros and certified umpires in addition to lessons and practice sessions as part of its' Polo On Demand program, the only polo club in the U.S. to offer the unique program.
The Club Hotline is 561-644-5050. It will be updated every day at 8 a.m. for both players and fans.
2018 Spring Schedule
(Subject to change because of weather and scheduling conflicts).
Friday, Santa Rita Abierto, 3 p.m. Dutta Corp vs. Altair Polo vs. Deeridge; 5 p.m. Sebucan vs. Hidden Creek; Sunday, 9:45 a.m., Grand Champions Cup, Field One; 11 a.m., Santa Rita Abierto Final, Field Two.
Saturday, 1:30 p.m., Governor's Cup, Field One; 3 p.m., The National President's Cup, Field Two, Grand Champions vs. Aspen Valley Polo Club.


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Shop our selection of , Decking in the Lumber & Composites Department at The Home Depot.
The cost of decking materials will vary depending on the type and quality of material . back; Building Permits · How To Hire A Contractor · Contracts . Home Depot, Lowes, and Menards usually only stock short lengths of . With over 100 brands to choose from there is a wide range of prices for vinyl and composite decking.
Have any of you used the newer composite decking materials for decks or porches? . Veranda Composite Decking — sold exclusively at Home Depot.
How do I know I can trust these reviews about Composite Decking? .. I have other decking materials on my home and none have ever 'spotted' as does. .. We purchase it on 9/12/08 at Home Depot after we did some research and.
11 Apr 2016 . Wood or synthetic decking is fastened to the deck frame over the . install waterproofing on an outdoor structure, you can visit the Home Depot.
UDECX is a modular, portable and easy to install DIY composite decking . The surface pads are made of composite material to ensure strength and durability . My wife and I walked in to Home Depot and they had a UDECX system set up. . We ordered the udecx starter kit because we are military and currently renting.
Recycled Plastic/Composite/PVC: we've got it. Take advantage of unbeatable inventory and prices from Quebec's expert in construction & renovation.
Find and save ideas about Composite decking on Pinterest. | See more about . Deck & Fence Inspiration | The Home Depot Canada. Timber DeckWood . pool wood plastic decking materials in the UK, Artificial wood plastic decking Square.
Composite deck installation can cost $25-$70 or more a square foot (8,000 -$22,400 for 16'x20"). Typically . Home Depot offers an index of decking materials .
14 May 2009 . Composite Deck Recall: Home Depot Veranda, ABTCo and LP WeatherBest . ABTCo and Home Depot Veranda composite decking were recalled after dozens of reports were received . The lumber yard I bought it from had a good sale on it. . I hope they take care of it all, so I don't need to hire a lawyer!


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Jess will come in and fill out the details, but in a nutshell, we decided to remodel the kitchen on a whim and completed the project in only 3 weeks.
The pictures cover the before, during and after shots and I think everyone will agree, it's a HUGE improvement.
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Copyright © 2002-2019 4homepages.de


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Offices for lease
We are seeking a single tenant for the 633 sq m (6,813 sq ft) ground floor offices at a quoting rent of £25.00 per sq ft or £170,000 per annum
This would suit a creative industry, such as fashion, film, design, performing arts, publishing, R&D, software, food, architecture, art & crafts, advertising or post-production. The accommodation would also be suitable for co-working or artists studios.
Space planning shows the potential for a floorplate which can accommodate 30-60 staff together with a meeting room for 10 people.
The accommodation is to be leased through Dutch and Dutch https://www.dutchanddutch.com
The office specification includes:-
Super fast fibre optic broadband (56 Mbps) with CAT6 repeaters
Entry via double height roller shutter door with 4.5m eaves and space for unloading vehicles
Triple height communal space & access to garden
Kitchenette with induction hob and double fridge freezer
Possibility to create an additional internal courtyard
Defra approved 16 KW multi fuel wood burner with log store
Electric car charging by negotiation + on-street business permits readily available
Polished concrete floor & exposed conduits over original brickwork
Male and female WCs with a single unisex shower
Aluminium / Crittal argon filled double glazed windows
Galvanished ceiling cable handling trays (wide as possible for voice and data)
Suspended slimline lighting and warm LED strip lights·
Ceiling mounted infrared heating panel (these radiate heat in a similar way to the sun, which is very effective at warming the thermal mass).
Emergency lighting with fire, smoke and integral carbon monoxide alarms
Ethernet CCTV and app based door entry systems
The images below are illustrative and for space planning exercises only to show the potential layouts that could be achieved. Once a preferred layout has been agreed with a tenant we would be happy to procure the "fit-out", which could then be "rentalised" over the duration of the lease.


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Express your love with our 9 Hearts Box. It is the perfect introduction to our chocolates. Our standard box has one of each of all our current flavors, but you are welcome to choose from the list below.
is backordered. We will ship it separately in 10 to 15 days.
© 2019 Chocolats Passion
Powered by Shopify


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The benefits of drinking water are well known, being well hydrated can boost metabolism, help you lose weight, keep skin looking healthy, tackle headaches, and so much more! But, drinking plain water can be boring and shop bought fruit flavoured waters are costly, not to mention the extra plastic waste you create each time you buy one. That's why an infuser water bottle is so great. You can create a wide variety of different flavours by mixing fruits, vegetables and even herbs and spices, which makes achieving your 2 litres of water per day a much tastier prospect.
This fruit infuser water bottle is made from strong and durable BPA free plastic and has a silicone seal to stop leaks and spills. There's also a flip up spout to make grabbing a sip of water on the go easy - no need to unscrew or remove lids. We've also included a carabiner clip so you can attach it to your bag or backpack whilst you're out walking, jogging or running and ensure you drink is easy to reach.
You can put this bottle through the dishwasher on a low heat cycle (under 60˚C), or wash by hand with warm soapy water and a soft cloth. Each bottle infuser water bottle comes with a handy cleaning brush so you can make sure your infuser core is free of any pips or pulp from your fruit.
The classic fruit infused water recipes include lemon and lime, cucumber and mint, strawberries and blueberries, but whether you go for something traditional, or more experimental recipes, there are some good tips to follow:
Check out the 'Extra Info' tab for some inspiring recipe ideas.
Your infusion water bottle core can be used for much more than just fruit. It's a great way to quickly add ice and chill down your water on a warm day. You could also put in herbal tea bags (or the new cold infusion sachets) for a quick and easy burst of flavour. There are many herbs and spices that are fantastic in an infusion, for example, try adding a sprig of rosemary, mint or basil, of try a cinnamon stick, cardamom pod or vanilla beans. Did you know you can also use edible flowers? So long as they are pesticide free, the petals from roses, lavender, pansies and violets are both colourful and tasty.
|Product Dimensions
|7.3cm L x 7.3cm W x 25.5cm H
|Dishwasher Safe
|BPA Free
|Primary Material
|BPA Free Tritan Plastic
Try some of these recipe ideas in your water infusion bottle:


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The practice of daily gratitude not only changes your life in a profoundly positive way, it also impacts the lives of those around you! Just think what kind of changes could take place if everyone began this practice!
Why not start today? Join the Finding Gratitude Group & see what being grateful for even just one thing per day for 30 days does!If you're already a member, invite a friend & let's expand this!
Love & Light,
Laura Lum Corby


text: Stratco Manhattan Patio Setting See In-Store Or Online Buy Now Find Store Add to List T & Cs Print Share Terms and Conditions Catalogue Terms and ConditionsPLEASE NOTE: Stratco have made every effort in this Catalogue to ensure all pictures and information are accurate at time of publication. Rainchecks may be taken in some stores. Pictures are used for illustrative purposes only and may show extras, fittings and features not included in price. Some products shown are not available at all stores but may be transferred from other stores upon request. Please check availability. For more advice on Stratco steel products, please refer to our 'Selection Use And Maintenance Of Stratco Steel Products' brochure. PATIO-9/18 Starts 01 Jul - Ends 31 Oct Category: Homewares , Outdoor Living , Outdoor Furniture , Stratco Painted steel construction 5 Piece setting. CO-4032


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Brisbane Bookfest
We are so pleased to say that funds raised from the Lifeline Bookfest Brisbane reached $1,130,023. That's over 28,000 Lifeline 13 11 14 suicide prevention calls.
To everyone who attended the Lifeline Bookfest Brisbane, a heart felt thank-you!
Watch this space for date announcements of the next Lifeline Bookfest Brisbane.
Event info
Bookfest Holiday Competition Winner
Blake from Lutwyche!
Where and when
Watch this space for date announcements of the next Lifeline Bookfest Brisbane.
Entry is free; books from just $1
- Hire a trolley for as long as you like for $2.
- BYO bags or purchase an enviro bag for $1.
- Food and drink available for purchase.
- EFTPOS available.
- ATMs on main exhibition concourse.
- View our Brisbane Bookfest map to plan your day.
Parking and transport
- Catch the train to South Brisbane Railway Station, bus to the Cultural Centre Station or ferry to South Bank Terminal
- Park at the venue or on the surrounding streets
- Taxi rank directly outside the venue.
Bookfest categories
The books are divided into categories for ease of browsing:
Art/Music, Australiana, Biography, CDs, Children's, Comics, Cooking, DVDs, Family, Foreign Language, Games/Puzzles, Hardback Fiction, Health, History, Humour/New Age, Lifestyle, Literature/Classics, Magazines, Paperback Fiction, Rare and Collectables, Records, Reference, Religion, Science Fiction, Science/Politics, Sports, Stationery, Textbooks, Travel, True Crime, War/Vehicles, Young Adult
Book donations
We would be delighted to accept your donation! Thanks so much for thinking of us.
Donating at the event
You can donate your books at the Brisbane Bookfest, so please see one of our volunteers at the event for assistance.
Donating anytime
We welcome donations all year round through your nearest:
- Lifeline shop
- Lifeline donation bin
- Lifeline Warehouse — to organise collection of bulk book donations please ring 07 3632 1010 or fill out the pick-up request form
Lifeline Bookfest Brisbane
Brisbane Convention & Exhibition Centre
Merivale St & Glenelg Street
South Brisbane, QLD, 4101
Get Directions


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Situated in close proximity to Florida Road, the business and recreational 'high street' ofMorningside, the hotel enjoys the best of the quiet private street and the convenience of theaccessible entertainment and public activities.
Facilities include 36 air-conditioned bedrooms offering all the comforts and amenities such as tea and coffee making facilities, DSTV with 12 channels, LCD TV, Secure parking. 24- hour security, PL8 restaurant, meeting room, secretarial services, Wi-Fi. Transport shuttles to and from the airport.
Designed by ProSysCom.


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Volkswagen Touran
Source : Volkswagen Touran
Third-gen MPV exudes characteristic Volkswagen refinements, so the idea's solid, upmarket, smart along with practical – albeit a bit bland This specific can be the all-brand new product of Volkswagen's ultra-grown-up, medium-sized MPV, the Touran. The last Touran carved out a niche as the MPV for the sensible family man, along with This specific brand new one develops the same theme. So the idea isn't 'quirky', 'funky' or 'sporty'; the idea's just an MPV. yet, as the idea happens a particularly smart, solid along with upmarket one.The headline alterations to the auto because of This specific third full product generation can be summarised pretty simply: the idea's slightly bigger, lighter along with pricier.The adoption of the Volkswagen Group's MQB platform makes the first two possible, adding the flexibility needed to stretch the wheelbase, shorten the front overhang along with still take 62kg out of the auto's kerb weight – despite simultaneously adding a not-inconsiderable 130mm to the overall length.The subtle cost repositioning can be nothing unusual at the introduction of a brand new-generation car along with justified by a more generous standard equipment across the four trim levels. Entry-level cars get a crash mitigation system, touchscreen multimedia system, DAB, Bluetooth media streaming, air conditioning, roof rails along with seven seats all as standard. With the flagship SEL trim equipping the Toruan using a panoramic sunroof, sat nav, adaptive cruise control along with three-zone climate control.The engine range for the UK market will consist of two turbocharged petrols along with three turbodiesels. The former can be made up of VW's 1.2 along with 1.4 TSI engines producing 108 along with 148bhp respectively, the 1.4 being available using a manual or a DSG dual-clutch automatic gearbox. The latter are the familiar 1.6 along with 2.0 TDIs, with peak power outputs ranging via 108 to 187bhp. Both of the lesser-powered diesels are offered in manual or DSG forms, while the 187bhp oil-burner can be DSG-only.The mid-spec 1.6-litre TDI SE should be the most favorite product of the Touran from the UK. the idea's a pragmatic, practical along with classy car which's comfy, quite refined along with very easy to drive – albeit no doubt a bit bland for some tastes.The Inner surface can be the VW's strongest selling point. Even on a mid-range SE-spec car, the fascia looks along with feels tactile along with plush. On perceived quality, the idea's a cut above which which you'll find in a Citroën Grand Picasso or a Vauxhall Zafira Tourer along with on a level footing using a BMW 2 Series Gran Tourer.Soft-touch plastics cover the whole of the dashboard along with the tops of the doors along with feel as expensive to the touch as VW's smooth leather facings for the primary controls. the idea's perhaps more important which every fitting can be substantial, robust along with ready for the rigours of everyday family life.Occupant space can be not bad by the strictest like-for-like standards, although there are still more accommodating seven-seaters.The Touran along with its ilk are narrower along with shorter than full-sized rivals such as the Seat Alhambra, So from the second row the Touran offers not bad leg along with headroom yet its seats are a touch smaller for grown adults. the auto's third-row seats are usable along with easier to access thanks to some bigger back door apertures – yet you still wouldn't call them adult-sized. yet all a few back seats have ISOFIX childseat anchorages, along with while the middle three slide along with fold independently, the rearmost two collapse into the boot floor easily enough.The Touran's boot in a few-seat mode, meanwhile, measures a class-leading 743 litres up to the window line, along with which's big enough to beat a brand new Ford S-Max.To drive, the Touran can be comfy, slick along with consistent. the idea's also somewhat middle of the road along using a bit forgettable, yet which's mostly the cost of its supreme ease-of-use nature. the auto rides with supple calm, steers using a directness along with weight perfectly matched to its moderate grip along with Centeng control, along with isolates both wind along with road noise well.VW's 1.6-litre diesel powertrain continues to feel humdrum along with undernourished; the idea's less refined than the 2.0 TDI along with falls short of the standards of the best low-emission diesels on responsiveness along with flexibility. yet its outright performance levels are more than acceptable along with its real-world economy easily surpassed 50mpg on our mostly urban test route. the idea's smart, assured, relaxing, robust, full of practical features along with has been turned with the kind of distinguishing attention to detail you expect of a VW. the idea's also lighter, more CO2-efficient along with more economical than much of its opposition, too, along with has many of the premium-brand lures which BMW's brand new 2 Series Gran Tourer offers yet for a lower list cost.The only question can be whether you're comfortable enough about your own need for an MPV to actually become a Touran owner, because Volkswagen evidently doesn't see the need to add much from the way of spice to an otherwise very complete recipe.Volkswagen Touran 1.6 TDI SEPrice £24,000 Engine 4 cyls, 1598cc, turbodiesel; Power 108bhp at 3200-4000rpm; Torque 184lb ft at 1500-3000rpm; 0-62mph 11.9sec; Top speed 116mph; Gearbox 6-spd dual-clutch automatic; Kerb weight 1539kg; Economy 64.2mpg (combined); CO2 116g/km
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Interested in booking a private workshop for a group of friends, corporate event, or holiday party?
We can accommodate groups of up to 30 people in our new facility!
Workshop attendees also receive 15% off any in-store purchases during the event!
Drop us a line at email@example.com for more information!
- This workshop has passed.
Wreath and Birdhouse Workshop
June 4 @ 7:00 pm - 9:00 pm
Tuesday, June 4, 2019 – 7:00-9:00pm
With warm weather finally here, it's starting to feel like we can open our doors and let that fresh air in!
So no better time to fancy up the outside of your door with a snazzy new hand made 15″ wreath~
The workshop is a blend of hand made goodness, and painting techniques. Painting the decorative birdhouse, you'll have your choice of Fusion Mineral Paint colours, and then learn an added ageing process. Dry brushing, waxing, or antiquing glaze.
You then get to whip and weave some burlap, and add hydrangeas until your heart is content!
Many colours will be available of course, as well as other spring/summer florals.
Only $45+tax!
Workshop attendees also receive 15% off any in-store purchases during the event!
Please note that payments for the workshop are non-refundable.Share this Event!


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Should Chapels Be Exempt from Paying Levy?
Chapels in the United States have been able to get tax exemption since the earlier nineteenth hundred years; even before it turned out written in law, several experienced such privileges currently anyway. While in the American Movement, nine of this original colonies were churning out some form of tax burden relief to their churches. In 1777, number exemption to churches was in fact formally printed into Va state laws.
Should churches be exempt from paying taxing?
New York put into practice their example right before 1780 and the Improve Congress lasted a federal rules to exempt churches with paying site taxes in 1894. All fifty towns exempt orlando organizations right from paying assets taxes and perhaps they are able to take any contributions from their taxation assessments. Providing chapels with taxing exemption has many supporters. They will feel that the exemption preserves religion and government split up and thus honors the United States Make-up. To tax bill churches, there is a saying, would give the government power to regulate religious corporations. In addition , these organizations more often than not provide a significant amount in public decent and have results on their groups.
However , there is opponents to such permission as well. Many people feel that to exempt orlando organizations with having to pay buildings taxes or perhaps report support does, in fact , violate the Constitution. To exempt chapels form forking out taxes is undoubtedly showing favoritism and extra treatment. This forces visitors to financially support religious loan companies that they may not agree with or perhaps believe in and is seen as an equivalent to the governing administration giving the churches profit the amount they will have to pay through taxes. In addition, they feel that the exemption simply does not make sense financially. Though church buildings have been besides from paying out taxes in the most common of American heritage, the question over no matter whether this should continue have become popular as of late.
Churches must be tax exempt
A very popular reason for the support of keeping church buildings tax exempt is that it will be protected through First Upbeat of the combinaison. They think that extending a tax-exempt position to areas of worship is included in the Primary Amendment's Office Clause , which prohibits the government from establishing any sort of national religion. In 1970, when a case the fact that challenged this brought to the Supreme Trial, it was determined that the taxing exemption can provide less of a relationship concerning church and state when compared to taxing them would, mainly because it restricts their very own financial supervision and helps to strengthen the desired separation. Furthermore, supporters of the bible exemption think that if the administration were to require places in worship to shell out taxes, may violate the First Amendment's Free Physical fitness Clause .
If virtually any places of worship defaulted on their repayments, the government would be able to shut these people down. As stated by the The best Court once more in 1819, if the federal government is given the strength to duty something, they may have the power to 'destroy this as well. Those who believe tiring places in worship might possibly be unconstitutional even feel that to tax these people would be adding government previously religion.
When it is able to regulate churches so, it would a number of circumstances government operate over foi, which is is really important by the composition. Opponents in tax permission often suggest that the permission is a security to religious beliefs, making it out of constitute. However , granting a religious efficiency exemption right from paying consider cannot be believed to be a sponsorship because the governing administration does not handover any of their income on the churches. Alternatively, the permission does not grant churches the obligation to on a financial basis support the federal government.
Admirers of number exemption to have religious establishments feel that the tax permission is validated because chapels contribute really to the society. They offer services such as f-r-e-e meals and shelter in the destitute, food assistance and after-school programs to low-income parents, etc . A large number of disadvantaged many people rely on all these programs of course, if churches are not afforded income tax exempt status, they may not be allowed to fund these kind of projects. Most of these duties may fall towards the government without churches. With additional programs to support, at this time there would need to stay cuts someplace and some of a programs might undoubtedly bear. The money the fact that government supposedly loses by simply granting taxation exemption is made up in the cash they rescue by without having to do the task the churches do.
Another reason that many think that churches must be exempt from forking over taxes is it would result in double taxation. If religious organizations been required to pay taxation, one result may be that they need to start recharging money intended for membership or perhaps events. Simply because individuals are taxed on their own as well and do not receive any money from their destinations of praise, to duty them another time for begin, you can in these establishments would be two times taxation. Additionally , it could get the better of people from donating additional time and money to their churches, which would probably lead to a decrease in good influence the churches can easily have individual communities.
Churches should certainly pay property taxes
Many who all feel that spots of praise should have to pay for taxes the actual everyone else believe that, rather than protecting the Initially Amendment, that violates it. In Walz v. Tax burden Commission of your City of Los angeles, Justice Bill O. Douglas defended his dissenting judgment of the reaches verdict when he stated,
'If believers are entitled to public personal support, so are non-believers. A good believer and nonbeliever in the present legislation are prepared differently because the articles with their faith…
For churches to be sum exempt as they are religious gives them valuable treatment, that is not in conformity with the Primary Amendment.
Set up government would have been to grant the exemption due to its positive support made by makes of praise to the large public, there is nothing dictating this inside the Constitution. Church buildings also get different cure from the government than other nonprofits do; even while regular benevolent organizations of a boring nature be required to report cash flow and record form 990 with the IRS, churches do not need to report their annual monetary information at all. This kind of favoritism granted to places of worship is normally agreed to come to be unconstitutional. Also considered out of constitute is the govt subsidizing religion, but sum exemption is a form of security. Tax deductibility and exemptions are a method of subsidy carried out by the government's tax program. Those who rebel ? go against sb/sth ? disobey tax exemption for church buildings feel that a tax exemption is basically your cash approve in the amount the church buildings would have to fork out in taxing if these were not exempt.
Rivals of tax-exemption, including Draw Twain, believe that tax exemption for orlando organizations enforced taxpayers to compliment religion. Twain said,
'No house of worship property is normally taxed restrictive infidel as well as atheist plus the man not having religion happen to be taxed to create up the shortage in the public court income this caused.
Churches gain benefit from the government getting involved in collecting taxes and yet do not have to contribute to the money in any respect. In addition , there is nothing to distinguish the best religion right from fraudulent lists seeking to work with tax exemption.
One of the most debatable religious corporations is the Religious organization of Scientology. In 1991, the press shown Scientology to become UFO-based conspiracy aimed at getting power and feeding avarice; still, they already have enjoyed their very own tax exempt status for pretty much a quarter 100 years, saving the church approximately millions of dollars in taxes. Besides the fact that most people profess that Scientology is more from a cult than the religion, they may be still specified exemption via having to pay taxations.
Another reason that some feel that churches won't receive ChiefEssays.Net number exemption is the fact to do so just simply does not produce economic awareness. Tax faveur to secular not-for-profit firms would make more sense mainly because such places, like the work place and soups kitchens, will give you results and take care of the things that would decline to the federal otherwise. Though churches may take part during charitable will work and projects, their key purpose lies in religious praise, which the government would not be capable of do properly anyway. You cannot find any fiscal help for government entities to exempt churches and also other places in worship from having to pay property taxes.
Another reason the fact that providing tax bill exempt situation for church buildings does not appear sensible economically is the fact is costs the government quantities of dollars for lost earnings. It is forecasted by the Brown House scheme analysts the fact that churches in the us own up to 500 billion us dollars in property that can not be legally taxed. The city of New York individual loses much more than six million dollars dollars through property tax revenue yearly.
A number of churches in the United States try to make millions of dollars in revenue each year, sometimes more, and zero of that income is taxed or regulated by the government at all. For instance , one megachurch in Texas earns seventy five million dollars every year while Church in Scientology may possibly make well over half a billion us dollars annually. They can collect pretty much everything money and not have to pay the federal government taxes for any of the idea, which will not make sense, monetarily.
In sum
This pattern essay with the writers available at Ultius exposes even before income tax exemption with churches and other places of worship is officially a law, these types or institutions have been getting tax breaks meant for as long as set up religion has become present in the. While the most of states offered these cracks without any legal motivation or maybe obligation, a lot of states up and running establishing establish rules to get exempting everything churches by normal place a burden on responsibilities. Other country implemented suit and currently, most fifty expresses do not call for churches to shell out property take into account or report their contributions and financial information. Nonetheless despite the fact that that exemptions has long been going on to receive so long, at the same time faced opposition from the very beginning.
During the nineteenth century very own, Presidents Garfield, Grant, and Madison mentioned disapproval in support of exempting destinations of praise from spending money on taxes. The moment this permission was referred to as written right into law, it absolutely was the first time that your United States federal had called any company to be forgiven from paying out taxes; alternatively, it was more usual at the time to list agencies that could be taxed by the authorities. To this day, controversy still proceeds on over if religious organisations should be taxed by the authorities.
Many feel that the exemptions are rationalized because the chapels contribute efficiently to the network, they function public good and assist that would normally fall for the government, plus they honor the constitution that dictates an important separation in church and state. Continue to, though, there are numerous who feel that churches needs to be taxed a non-threatening organization. Pertaining to the government to look at special treatment is out of constitute, it draws citizens to assist the exempt organizations, and does not make monetary sense in the present American current economic climate.


text: I saw in my 'device manager' that 'PCI simple communications controller' and 'USB IMAGING DEVICE' had yellow signs next to them, and when I checked it out, it said the drivers weren't installed. I tried to install them by the 'internet search' choice, and also 'searching' the hard drive, neither worked(It said the drivers couldn't be found.) Also, when I plug into the front USB port, a message comes up that says it isn't running at top speed, I should turn the port(s) into hi-speed ports. This is recent, previously, the device manager didn't show any issues, and the front ports were 3.0, hi-speed. What is the issue here, and where can I find the drivers to correct the problem, or is that the problem at all? Thanks!


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Top 10 Guide: Marketing your Start-Up and SME
Immerse yourself with international brand man and entrepreneur, Graham Dodridge who shares his insight and experience.Download Now
I was blankley staring at my smartphone in the lift this morning making a concerted effort not to make eye contact with anyone (as is standard lift protocol). As I was reading through the random emails a popular travel app caught my eye. Oooh, I thought: 'Dizzo, look what we found for you'……'hotels in Venice'. This would be very helpful if I was looking to travel to Venice but here's the thing; I stayed in Venice six-months ago and since then I have been to Cornwall, Spain, the Lake District and Ireland. I no longer give a monkeys about Venice and yet I get this same email at least every couple of weeks – only this time I thought I would open it to see what happens next. Nice try at being relevant 'popular travel app'.
Most of us accept that the old-style marketing campaigns don't work. It has to be different, relevant to me, and not the pray and spray one-size-fits-all baloney from when the dinosaurs walked the earth (and by the way a few still do).
If you send me a blanket email. I will ignore it.
If you send me something I actually care about and is relevant to me. I might open it.
I am in control. I set the pace. I will buy when I am ready to buy (unless you show me something really shiny!)
These days it is all about the 'segmentation of one'. You know my every move, there are people and machines crunching data… they think they know me better than I know myself. And yet, if this is the case, then how can some brands still get it so wrong?
The truth is, most large companies have multiple marketing teams, multiple products they want me to buy, multiple systems and a fragmented or piecemeal marketing strategy. There is limited insight into whether the messages really hit the mark, or in 'popular travel apps' case they're misunderstanding customer behaviours and not intervening with common sense to interpret what the data is really telling them. "He's been to Venice, you've seen the dates, he used your app when he was there and six months on he is probably thinking about the next trip to somewhere different".
A customer-centric marketing strategy is far more than a state of mind and some whizzy digital tools. Successful campaigns require real people thinking, questioning and interpreting what the whizzy platforms and insights tell them; drawing on experience and sometimes (dare I say it) using a dose of common sense.
It's a symptom of the complicated world we live in. But let's try to keep it simple and not rely on just data to really understand customers.
1. Connect with your buyers by understanding their personas. People are smart, so don't try to be too clever with what the data is telling you.
2. Use multiple channels to paint a rich picture of your customers behaviours and find new ways to reach them.
3. Analytics will help you to optimise campaign effectiveness but look further than just the numbers.
4. Content is still king. Make it relevant and don't create content for contents sake.
5. Manage your database. It's the foundation for all your campaigns and the key to successful campaigns.
Of course 'popular travel app' isn't getting it all wrong. The regular emails, no matter how wide of the mark, still keep them front of mind and make me go back for more. The question is whether this is because they are the market leaders or is there trying to be customer-centric marketing strategy is actually working. If a successful challenger brand enters the market, how long will 'popular travel app' stay on top? It's now more important to recognizse the differing customer requirements, no matter how subtle. Like the zebras in Madagascar II say. "I'm unique…. We're all unique".
Dan is Business Director at Silver. He is a new advanced breed of marketing expert, creating next generation customer engagement programmes for Silver brand customers.
Immerse yourself with international brand man and entrepreneur, Graham Dodridge who shares his insight and experience.Download Now


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Pianist-composer John Kameel Farah returns to Toronto for a special concert, celebrating the release of his new album "STRANDS", 9 improvisations for solo piano.
Farah will also perform new & older works for piano, synth and electronics, which combine elements of Early Music, Middle-Eastern, Experimental and Electronic Dance Music.
Doors open at 8:00pm, music begins 8:30pm
Entrance on a sliding scale – $5 minimum / $10 recommended


text: "esmith1" wrote : Portal 2.6.6 | AS 4.2.2 | JDK 1.5.0_12 | DB = sqlserver using sql_latin1_general_cp1_cs_as (case sens) collation | | I am getting an error during startup (below) because the JBP_REG_PROPERTY table has a (capital) "NAME" field but the hibernate definition of the map-key in JBP_REGISTRATION_INFO that references it is lowercase. | As a quick test I changed jboss-portal.sar\portal-wsrp.sar\conf\hibernate\consumer\domain.hbm.xml | JBP_REGISTRATION_INFO class map persistentRegistrationProperties property map-key from | | <map-key type="string" formula="name"/> to <map-key type="string" formula="NAME"/> and the error went away. Can a JIRA be entered for this? | Please do so. Don't forget to link to this forum topic in the appropriate field of the JIRA task. Thanks for the report! View the original post : http://www.jboss.com/index.html?module=bb&op=viewtopic&p=4169059#4169059 Reply to the post : http://www.jboss.com/index.html?module=bb&op=posting&mode=reply&p=4169059