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Numerous studies have demonstrated that the hepatitis B virus HBx protein stimulates signal transduction pathways and may bind to certain transcription factors, particularly the cyclic AMP response element binding protein, CREB. HBx has also been shown to promote early cell cycle progression, possibly by functionally replacing the TATA-binding protein-associated factor 250 (TAFII250), a transcriptional coactivator, and/or by stimulating cytoplasmic signal transduction pathways. To understand the basis for early cell cycle progression mediated by HBx, we characterized the molecular mechanism by which HBx promotes deregulation of the G0 and G1 cell cycle checkpoints in growth-arrested cells. We demonstrate that TAFII250 is absolutely required for HBx activation of the cyclin A promoter and for promotion of early cell cycle transit from G0 through G1. Thus, HBx does not functionally replace TAFII250 for transcriptional activity or for cell cycle progression, in contrast to a previous report. Instead, HBx is shown to activate the cyclin A promoter, induce cyclin A–cyclin-dependent kinase 2 complexes, and promote cycling of growth-arrested cells into G1 through a pathway involving activation of Src tyrosine kinases. HBx stimulation of Src kinases and cyclin gene expression was found to force growth-arrested cells to transit through G1 but to stall at the junction with S phase, which may be important for viral replication.
Chronic infection with hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma in humans. Consequently, there has been intense interest in HBV gene products that can alter cellular gene activity. The smallest open reading frame of the mammalian HBVs, including the human virus, encodes a 17-kDa regulatory protein known as HBx (or X protein) (reviewed in references 2 and 92). HBx is essential for productive infection by the mammalian HBVs (12, 95). Studies initially characterized HBx as a transcriptional transactivator of weak to moderate strength. Transcription factors activated by HBx include NF-κB, NF-AT, AP-1, and ATF/CREB (4, 6, 8, 45, 48, 51, 52, 55, 68, 73, 77, 81,82, 90). In addition to stimulation of RNA polymerase II-directed transcription, HBx also stimulates transcription by RNA polymerases I and III (3, 43, 86, 88). HBx is therefore a modest activator of many types of transcription elements and factors (28, 29, 46, 51, 54, 60, 61, 90). Many of the reported activities of HBx have been shown to result from its ability to activate cytoplasmic signal transduction pathways, particularly the Ras–Raf–mitogen-activated protein kinase (MAPK) pathway (6, 18, 55, 86), the cell stress-induced MEKK1–p38–c-Jun N-terminal kinase (JNK) pathway (8, 73), and the family of Src tyrosine kinases (41). HBx activation of Src may be important for viral replication (40). In vitro, HBx can interact with several components of the transcriptional apparatus, including factors TATA-binding protein (TBP), TFIIB, TFIIH, and the RPB5 subunit of RNA polymerases (14, 60, 61). HBx also possesses nuclear transcription-activating functions (23) that may involve interaction with CREB (51, 59, 77, 90) or possibly a reported coactivator activity (29, 30, 83).
HBx has been shown to stimulate deregulation of early cell cycle checkpoint controls (7, 42, 71). Expression of HBx in cells that have had their growth arrested by serum withdrawal results in their transit through the G1 phase of the cell cycle but without progression into S phase (71). Thus, in the absence of serum, HBx-expressing cells are stalled at the G1-S phase junction, whereas control cells without HBx remain in G0. If serum is provided to growth-arrested cells that express HBx, these cells advance more rapidly through G1 and may enter S phase, in contrast to cells without HBx, particularly if the cell is transformed (7, 42). Although HBx activation of Ras has been shown to be necessary for deregulation of the G0 checkpoint (7), there is little understanding of the mechanism by which HBx promotes early cell cycle progression. In this regard, HBx was also reported to functionally replace TBP-associated factor 250 (TAFII250) in transcriptional activation, induction of cell cycle progression, and inhibition of apoptosis ints13 cells. Thus, these data implicate both cytoplasmic signal transduction and nuclear transcription functions in the ability of HBx protein to promote cell cycle progression. ts13 cells are from a hamster cell line containing a temperature-sensitive defect in TAFII250 caused by a single amino acid change in the TAFII250 protein sequence (31, 76). The TAFII250ts13 mutation causes cell cycle arrest and apoptosis when these cells are shifted from the permissive temperature (33°C) to the nonpermissive temperature (39.5°C). TAFII250, the largest TAF identified, interacts with TBP and several other TAFs (reviewed in references 1,11, and 58), and it is essential for assembly of the TFIID complex (89). It was recently established that TAFII250 targets specific chromatin-bound promoters via multiply acetylated histone H4 proteins. By utilizing its intrinsic histone acetyltransferase (HAT) activity, TAFII250 activates transcription through chromatin remodeling and recruitment of the transcription complex (36). TAFII250 is essential for activation of A- and D-type cyclin genes, a necessary event for progression through the cell cycle (67). Importantly, extensive analyses of ts13 cells have demonstrated that it is the HAT activity of TAFII250, and not other activities, that is impaired at the restrictive temperature (24). Thus, at the restrictive temperature, failure of TAFII250 to induce cyclin A and D genes results in cell cycle arrest and ultimately apoptosis (67), which results from the loss of TAFII250 HAT activity. In addition, the effect of the TAFII250 mutation in ts13 cells on cellular transcription is not global, and only a small number of genes are affected (47, 67, 84). Transcription of the c-fos gene, for example, is not influenced by a shift ofts13 cells to 39.5°C, whereas the activity of the cyclin A promoter is decreased by 8- to 10-fold.
The simian virus 40 large T antigen (SV40 T-Ag) and the human cytomegalovirus (HCMV) major immediate-early proteins (MIEPs) can overcome the transcriptional defect of ts13 cells at the restrictive temperature for TAFII250 (19,49). Rescue of ts13 cell transcription is partially restored by the IEP86 protein, although near-complete rescue requires expression from the entire major immediate-early transcription unit, implying that at least several proteins are required to overcome the loss of TAFII250 (49, 50). Moreover, studies demonstrate that while SV40 T-Ag and HCMV MIEPs can compensate for some of the functions of TAFII250, it is unlikely that a single viral protein is able to replace the multiple functions of TAFII250. For instance, the HCMV MIEPs cannot rescue the cell cycle defect in ts13 cells grown at the nonpermissive temperature for TAFII250 function, although their expression does prevent apoptosis (50). Genetic evidence also indicates that different MIEP functions prevent apoptosis and stimulate transcription (50). This is consistent with the multiple activities of TAFII250, such as transcriptional activation, cell cycle progression, and effects on apoptosis, involving distinct and separable functions of TAFII250. Thus, the multifunctional nature of TAFII250 could explain why transcriptional activation of cyclin promoters by a rescuing protein does not necessarily result in cell cycle progression or inhibition of apoptosis when ts13 cells are shifted to the nonpermissive temperature. HBx protein was reported to promote cell cycling in ts13 cells incubated at the restrictive temperature for endogenous mutant TAFII250, as well as to overcome the transcriptional defect (27). As HBx has been shown to be an inducer of apoptosis in many cells (9, 16, 39, 66, 70, 74, 78, 79), it is unclear how expression of HBx in a TAFII250 mutant cell line can block the antiapoptotic effect that results from the loss of TAFII250 activity.
Studies have characterized the temperature-responsive component of the cyclin A promoter, demonstrating that this element contains an ATF/CREB transcription factor binding site which is involved in its activation (85). ATF and CREB factors and the ATF–CREB-responsive element (CRE) site in cyclin A and D promoters can be induced via a Src kinase signaling pathway (44). HBx can activate Src kinases (40, 41), implicating an established mechanism of action in HBx induction of cyclin gene activity. Moreover, HBx is widely reported to bind to and activate ATF/CREB (reviewed in reference 2). Thus, there are potentially multiple mechanisms by which HBx promotes early cell cycling. In this report, we characterized the mechanism by which HBx promotes cycling of growth-arrested cells. We examined whether HBx induction of cell cycling occurs through its nuclear functions by replacing TAFII250 or through its cytoplasmic functions by activating Src kinases. We show that the induction of the cyclin A promoter by HBx in ts13 cells, which is essential for cell transit through G1, is dependent on activation of the Src family of tyrosine kinases. HBx could not functionally replace the loss of TAFII250 activity for activation of the cyclin A promoter or for early cell cycle progression. Furthermore, HBx induction of the cyclin A promoter through cytoplasmic activation of Src kinases is shown to be involved in the release of cells from quiescence and their transit through G1 to the junction with S phase. HBx is shown to be located predominately in the cytoplasm of ts13 cells regardless of temperature, consistent with an absolute requirement for induction of cytoplasmic signaling cascades but inconsistent with an essential requirement for nuclear transcription functions. In fact, an HBx protein that was engineered to concentrate in the nucleus by fusion to a nuclear localization signal (NLS) failed to substitute for TAFII250 function or to promote cell cycle progression. Finally, HBx is shown to activate the endogenous cyclin A promoter and cyclin A–cyclin-dependent kinase 2 (cdk2) complexes in wild-type (TAFII250 containing) cells through cytoplasmic activation of Src signaling pathways.
Cell culture. ts13 cells were maintained at 33°C in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing penicillin (100 U/ml) and streptomycin (100 μg/ml), supplemented with 10% fetal bovine serum. Chang cells were maintained in DMEM at 37°C as described above.
Plasmids and viruses.The HBx-expressing plasmids, pAd-CMVX, pAd-HBxFlag, pAd-HBxo, pAd-HBxNLSFlag, and pAd-HBxSLNFlag, the TAFII250-expressing plasmid, the Csk-expressing plasmid (pCaCsk), and the cyclin A promoter-driven luciferase reporter plasmid (pCyA-Luc) have been described previously (23, 41,84). High-concentration stocks of plasmids were purified using the Concert High Purity Plasmid Maxiprep System (Life Technologies) according to the manufacturer's instructions. Replication-defective recombinant adenovirus (Ad) vectors have been described previously (23). Ad-HBx and Ad-HBxo express wild-type HBx and an HBx mutant mRNA that lacks all potential translation initiation codons and does not synthesize HBx protein, respectively. HBx was inserted in place of Ad region E1. Ad vectors were propagated and titers were determined on 293 cells, which complement the loss of the E1 transcription unit.
Transfection, luciferase assays, and survival curves.All transient transfections were performed by both standard calcium phosphate transfection methods and lipid methods using Lipofectamine-Plus (Life Technologies) according to the manufacturer's instructions. Both methods gave similar results, and only those from the Lipofectamine-Plus transfections are reported here. Luciferase reporter assays were performed using the Promega luciferase assay system according to the manufacturer's instructions. To analyze HBx-induced ts13 survival at the nonpermissive temperature, cells were cotransfected with 0.5 μg of a green fluorescent protein (GFP)-expressing plasmid (pGFP) and amounts of pAd-CMV control vector or pAd-CMVX (23) ranging from 1 to 10 μg/5 × 106 cells. After 24 h of recovery to ensure protein expression, cells were either maintained at 33°C (permissive for TAFII250) or shifted to 39.5°C (restrictive). At indicated times after the temperature shift, relative cell survival was calculated from the mean of surviving GFP-expressing cells, collected by viewing 10 fields at 40× power using a standard UV light microscope outfitted with GFP filters.
Flow cytometry.These studies were carried out as previously described (7), with minor changes as described below. Briefly, Chang cells were made quiescent by cultivation in DMEM without serum for 30 h, infected with replication-defective Ad vectors expressing HBx (Ad-CMVX) or a mutant devoid of all potential HBx AUG codons, known as HBxo (Ad-CMVXo), at 25 PFU per cell, and maintained in DMEM in the absence of serum for up to 24 h. Cells were lysed in a solution containing 0.1% Nonidet P-40, 50 μg of propidium iodide per ml, 100 μg of DNase-free RNase A per ml, 5 mM NaCl, and 10 mM trisodium citrate. Flow cytometry was performed without modification as described previously (34). Results shown are typical of three independent trials.
Kinase assays.For the cyclin A-cdk2 complex assay, serum-starved Chang cells were transfected with plasmid DNAs for 24 h and lysed, and extracts were prepared as described previously (7). Cyclin A was specifically immunoprecipitated with a commercial monoclonal antibody to cyclin A (Upstate Biotechnology, Lake Placid, N.Y.), and equal fractions were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% gel) and immunoblotted with the same antibody, followed by visualization with the enhanced chemiluminescence system (ECL; Amersham). The remaining immunoprecipitate was added to in vitro kinase reactions containing 5 μCi (1 Ci = 37 GBq) of [γ-32P]ATP and 50 mg of histone H1 per ml in 50-μl reaction volumes as described previously (7, 33). Proteins were resolved by SDS-PAGE (12% gel) and quantitated and detected by phosphorimage analysis. Data represent typical results from three independent experiments. The Src kinase assay was carried out as described previously (41), with minor changes. Briefly, cells were lysed in buffer containing 1% Nonidet P-40, 150 mM NaCl, 20 mM Tris-HCl (pH 8), 2.5 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 μg of aprotinin per ml, and 10 μg of leupeptin per ml. c-Src was immunoprecipitated from equal amounts of extract using a commercial monoclonal antibody (Upstate Biotechnology), and equal amounts were resolved by SDS-PAGE (12% gel) and immunoblotted with the same antibody. Equal fractions of the remaining immunoprecipitate were resuspended in kinase buffer (20 mM HEPES [pH 7.4], 10 mM MnCl2) with 0.2 μg of acid-denatured enolase (Sigma), 20 μCi of [γ-32P]ATP, and 10 μM ATP, as previously described (41). Samples were resolved by SDS-PAGE (12% gel) and subjected to phosphorimage analysis. Data shown are typical of three independent experiments.
Indirect immunofluorescence.Immunofluorescent antibody staining of ts13 cells transfected with HBx (pAd-HBxFlag) was performed as previously described (23). Briefly, cells were grown on collagen-coated coverslips, transfected with either pAd-HBxFlag, pAd (DNA vector control), pAd-HBxNLSFlag, or pAd HBx SLNFlag, and allowed to recover for 24 h. For fixation and permeabilization, the medium was removed and cells were washed twice with phosphate-buffered saline (PBS) and then treated in 95% ethanol plus 5% acetic acid overnight at −20°C or in 70% acetone plus 30% methanol for 10 min at −20°C. Fixed and permeabilized cells were washed with PBS and then blocked in PBS plus 1% nonfat dry milk for 30 min at 37.5°C. Cells were incubated with anti-Flag M1 antibody (Kodak) for 1 h at 37°C, washed four times with PBS, and then incubated with secondary donkey anti-mouse fluorescein isothiocyanate (FITC)-conjugated antibody. Cells were visualized and photographed using a Zeiss Axiophot fluorescence photomicroscope. Both fixation methods gave identical results.
HBx requires TAFII250 activity for cyclin A promoter transcription.Studies first examined the ability of HBx to stimulate transcription of a cyclin A promoter-luciferase reporter construct in ts13 cells at the permissive temperature for TAFII250 transcriptional activity (33°C). Cells were transiently transfected with a plasmid expressing wild-type HBx or control plasmid DNA and were maintained at the permissive temperature. Cotransfection with pGFP demonstrated equal transfection efficiencies in all samples (∼70%; data not shown). HBx was found to reproducibly induce the ectopic cyclin A promoter two- to threefold at the permissive temperature (Fig.1). These results are consistent in magnitude with the widely reported weak to moderate transactivation activity of HBx in a variety of cells and with different promoters (typically two- to sixfold). To evaluate whether HBx can rescue thets13 transcription defect resulting from inactivation of TAFII250 at the restrictive temperature, cells were transfected for 5 h at 33°C with expression plasmids for HBx and the cyclin A promoter-controlled luciferase reporter and were maintained at the nonrestrictive temperature (33°C) or shifted to 39.5°C. At the restrictive temperature for TAFII250 activity, HBx only marginally stimulated transcription of the transfected cyclin A promoter just slightly above that of vector alone (Fig. 1). Importantly, HBx did not even stimulate the cyclin A promoter to the basal levels of transcription that were observed at the permissive temperature. In fact, the experiment could not be continued past 30 h of transfection due to cell death, indicating the failure of HBx to rescue TAFII250 function. Transfection of cells with the HBx expression plasmid, ranging from 0.25 to 10 μg per 107 cells (a 40-fold concentration range), also failed to recover cyclin A promoter activity at the restrictive temperature (data not shown). Identical results were obtained when cells were transfected at 33°C and then shifted to 39.5°C 1 day later or when they were transfected at 39.5°C and maintained at the nonpermissive temperature. Consequently, there were no experimental conditions in which HBx replaced TAFII250 function for cyclin A promoter activity. In contrast, cotransfection of ts13 cells at the restrictive temperature (39.5°C) with the cyclin A promoter-reporter construct and a wild-type TAFII250 expression vector recovered almost full cyclin A promoter transcriptional activity compared to control cells expressing functional endogenous TAFII250 at the permissive temperature. Since about 70% of the cells were transfected (data not shown), these data demonstrate that ectopic expression of TAFII250 can rescue the transcription defect of ts13 cells within 24 h at 39.5°C. Ectopic expression of wild-type TAFII250 in ts13 cells at the permissive temperature actually reduced cyclin A promoter activity slightly, possibly as a result of squelching due to supraphysiological expression levels. HBx did not lose function at 39.5°C, since at 39.5 and 33°C it stimulated transcription equally well of a reporter controlled by a minimal promoter and four AP-1 transcription factor binding sites in Chang cells (Table1). In addition, HBx is stably synthesized at 39.5°C (shown later in Fig. 2). Collectively, these data demonstrate that HBx requires TAFII250 activity for cyclin A promoter activation, suggesting that nuclear HBx functions are either insufficient or unnecessary to promote early cell cycling, which was investigated next.
HBx protein engineered to concentrate in the nucleus requires TAFII250 function.Most studies have found HBx protein to be largely, but not exclusively, in the cytoplasm of a variety of cell types, whether expressed by transient transfection or in the context of viral infection (e.g., see references 20, 21, and 23). The cellular location of HBx in ts13 cells was investigated first using an HBx construct containing a C-terminal foreign Flag epitope. HBxFlag was shown previously to behave identically to unmodified wild-type HBx (23).ts13 cells were transfected with the HBxFlag expression plasmid or control vector DNA, and then indirect immunofluorescence analysis was carried out on cells fixed to coverslips and stained with anti-Flag antibodies followed by FITC-conjugated secondary antibodies. Although there was a low level of nuclear staining, the majority of HBx was found in the cytoplasm in all cells observed (Fig.2). Furthermore, there was no change in wild-type HBx intracellular distribution when ts13 cells were shifted to the nonpermissive temperature for TAFII250 activity 24 h prior to fixation (Fig. 2). An HBx variant engineered to contain an N-terminal NLS from SV40 T-Ag (HBxNLS) (23) was concentrated in the nucleus but retained some cytoplasmic distribution. An HBx control which contains a defective NLS sequence (HBxSLN) (23) remained cytoplasmic. HBxNLS and HBxSLN have been characterized extensively in a variety of cell types (e.g., see references 23 and 77). Shifting of the cells to 39.5°C did not alter the intracellular distribution of HBxNLS or HBxSLN proteins from that observed at 33°C (data not shown). It can be concluded, therefore, that HBx proteins are synthesized and retain their typical intracellular distribution at both 33 and 39.5°C.
Studies were then conducted to examine whether HBx failed to functionally replace TAFII250 at 39.5°C because too little HBx accumulates in the nucleus in ts13 cells. Cells at the permissive or restrictive temperature were transfected with wild-type HBx, HBxNLS, or wild-type TAFII250 expression plasmids, and the effect on cyclin A promoter activity was determined (Fig.3A). At the restrictive temperature, ectopic expression of wild-type TAFII250 recovered normal levels of cyclin A promoter transcriptional activity compared to the nonrestrictive temperature. There was no recovery of cyclin A promoter activity at the restrictive temperature by HBx or HBxNLS proteins. Thus, nuclear HBx protein does not functionally replace TAFII250 transcriptional activity at the restrictive temperature. Studies performed at the nonrestrictive temperature for TAFII250 activity demonstrate similar activation by HBxNLS and HBx. Since some HBxNLS remains cytoplasmic whereas the nuclear level of HBx is increased strongly, these results indicate that even with increased levels of HBx in the nucleus, like wild-type HBx, TAFII250 function is still required for activation of the cyclin A promoter. Because titration analysis of HBx showed that even 10-fold-lower levels could activate transcription, we suspect that the small amount of cytoplasmic HBxNLS in ts13 cells is sufficient for activation of the cyclin A promoter. These results also suggest that nuclear and cytoplasmic HBx functions are involved in early cell cycle progression, but the nuclear function does not include a previously reported TAFII250 activity which could not be reproduced here.
Studies were therefore performed to determine whether the nuclear HBx function involves activation of the transcription factor CREB, since HBx has been shown to bind and activate CREB in vitro and since CREB activation is required for stimulation of the cyclin A promoter. Cotransfection of ts13 cells at the permissive temperature with a CRE-luciferase reporter and wild-type HBx (Fig.3B) demonstrated two- to threefold activation of CREB-dependent transcription by wild-type HBx. Similar analysis using HBxNLS, which is largely but not entirely distributed in the nucleus, demonstrated slightly lower activation of the CREB-dependent reporter. This is consistent with a previous report which suggested that HBx activation of CREB might involve both cytoplasmic and nuclear HBx functions (90). At the restrictive temperature for TAFII250, neither wild-type HBx nor HBxNLS proteins stimulated CREB-dependent transcription (Fig. 3B). This is consistent with an essential requirement for TAFII250 function in transcriptional activation by HBx. Studies presented later demonstrate that HBx activation of Src kinase signaling is involved in CREB activation as well.
HBx requires TAFII250 activity and cytoplasmic functions to promote cell viability and cycling.We sought to determine whether HBx promotion of ts13 cell viability and cycling involves nuclear HBx functions that require or are independent of TAFII250 activity. Cells were cotransfected with a GFP expression vector and pAd-CMVX or control plasmid DNA and then maintained at the permissive temperature (33°C) or shifted to a restrictive temperature (39.5°C) for endogenous TAFII250 activity. Under these conditions, all GFP-expressing cells were cotransfected with either TAFII250, HBx, or vector DNA at ∼70% efficiency, regardless of temperature (data not shown). At different times following the temperature shift, cells were observed by light microscopy, and the number of viable GFP-expressing cells was quantified as described in Material and Methods. Approximately 90% of the transfected ts13 cells containing wild-type TAFII250 and GFP survived at the nonpermissive temperature, indicating inhibition of cell death (Fig.4). Cells expressing HBx were identical to control cells transfected with vector alone at the nonpermissive temperature, demonstrating widespread cell death. Thus, HBx did not delay the onset or rate of cell death and therefore did not promote cell cycling in the absence of TAFII250 activity. Identical results were obtained with HBxNLS, which concentrates in the nucleus (data not shown). We attempted to isolate surviving colonies of ts13 cells that were transformed by HBx at 39.5°C. Cells were transformed with vector alone or plasmids expressing HBx, HBxNLS, or wild-type TAFII250, at concentrations ranging from 1 to 10 μg of plasmid per 5 × 106 cells. The numbers of surviving transformants after 2 weeks of selection at 39.5°C are shown in Table2. HBx, HBxNLS, and vector DNA all gave rise to fewer than two colonies per 107 cells, a transformation frequency of 2 × 10−7. In comparison, cells transformed with TAFII250 averaged 7 × 106 transformants per 107 cells, a transformation efficiency almost equal to the efficiency of transfection, indicating that the majority of cells transfected with TAFII250 could be rescued. In addition, attempts to produce stably selected ts13 cells expressing HBx under a regulated promoter failed due to cell death, likely resulting from leaky synthesis of HBx protein (data not shown). Collectively, these results demonstrate that nuclear HBx functions are not sufficient to promote cell viability and cycling in the absence of TAFII250 activity.
HBx stimulates the cyclin A promoter, promotes formation of cyclin A-cdk2 complexes, and deregulates early cell cycle checkpoints in a Src kinase-dependent manner.It was shown previously that activation of transcription by HBx in part involves its cytoplasmic stimulation of Src kinase signaling and the Ras-Raf-MAPK/JNK pathway (6, 8, 40, 41, 73, 86). Moreover, HBx was shown previously to stimulate cyclin E and A synthesis by acting on cytoplasmic signal transduction pathways (7). The failure of HBx to stimulate cell cycling and the cyclin A promoter in the absence of TAFII250 activity at 39.5°C, even when relocated to the nucleus (Fig. 1 and 3), implicated HBx activation of signaling pathways in this action. We therefore characterized HBx activation of the endogenous cyclin A promoter and cyclin A-cdk2 complexes. Previous studies have shown that HBx can deregulate the G0 cell cycle control checkpoint in a variety of cell types with arrested growth and can facilitate transit into or through G1 to the early S phase transition (7, 42, 71). We first show that HBx promotes transit of G0-G1-arrested cells through G1to the G1-S junction in the absence of serum. It was not possible to conduct these studies in ts13 cells, which apoptose when growth is arrested by serum depletion (67), particularly with expression of HBx (data not shown). Consequently, Chang cells, a human liver cell line which can have its growth arrested by serum withdrawal without inducing apoptosis, were used.
Quiescent cells (cells in G0 and at the G0-G1 junction) were accumulated by serum withdrawal. Cells were then transduced by a replication-defective Ad vector lacking the Ad E1 region, which in its place expresses the HBx gene, or by a control HBxo gene which expresses an mRNA that cannot synthesize HBx protein (7, 23, 40, 73). Studies have shown that these vectors remain genetically silent for the time course of these experiments (73). Cells were lysed at various times after transduction with Ad vectors, and flow cytometry was performed on propidium iodide-treated nuclei (Fig.5). In the absence of serum stimulation, cells expressing the HBxo gene did not exit from the G0-G1fraction upon the termination of these studies (24 h posttransfection). Control cells expressing HBxo in the presence of serum proliferated more rapidly and entered S phase during this same time course. In comparison, HBx-expressing cells in the absence of serum showed a gradual increase in accumulation at the G1 junction with early S phase which was striking by 24 h of expression. Although cells expressing HBx in the absence of serum did not enter S phase and replicate, there was an evident increase in cell number at the rightward G1 transition with S phase. This likely represents the initiation of DNA decompaction in the nucleus which precedes entry into S phase and causes an increased fluorescent signal (62). The disappearance of the small fraction of cells in the G2-M phase in the HBx-expressing sample at time zero probably represents cells that cycled through to G1phase and then stopped (71).
Studies next determined whether HBx activation of Src kinases is important for deregulation of early cell cycle checkpoints. Chang cells were transfected with a plasmid expressing the kinase Csk, which phosphorylates and downregulates Src kinases. Csk was chosen for these studies because it very specifically blocks the family of Src kinases. At 8 h following transfection, cells were transduced with an Ad vector expressing HBx or HBxo. Approximately 70% of the cells were found to be transfected, whereas 95% were transduced by the Ad vector (data not shown). Previous studies demonstrated that Csk does not inhibit the expression of HBx controlled by the CMV promoter (40, 41). Expression of Csk in HBx-expressing cells reduced the fraction of cells that accumulated at the G1-S phase junction by about half at 24 h in the absence of serum. Various chemical inhibitors of Src kinases were found to provide equivalent results and will be published elsewhere. Cells expressing both Csk and HBxo were identical to the HBxo controls in the absence of serum (data not shown). Since Src kinases are stimulated by HBx acting in the cytoplasm (41), which is required for HBx promotion of cell cycling as shown here, we did not evaluate the effect of HBxNLS. These results demonstrate that HBx deregulates early cell cycle checkpoints in a Src kinase-dependent manner, an event that includes synthesis of cyclin A and activation of cyclin A-cdk2 complexes.
Studies therefore examined the role of cytoplasmic HBx activities in deregulation of early cell cycle control. Since many cytoplasmic functions of HBx can be accounted for by its activation of Src signaling, the role of Src activation in the stimulation of the endogenous cyclin A promoter was investigated. Serum-starved Chang cells were transfected with HBx or HBxo expression vectors, with or without cotransfection of a plasmid expressing the kinase Csk. Cell extracts were prepared 30 h later, c-Src was specifically immunoprecipitated, and HBx activation was shown by the ability of immunoprecipitated Src to phosphorylate the substrate enolase in vitro (Fig. 6A). HBx induced a fourfold activation of Src compared to cells transfected with the HBxo vector which was prevented by coexpression with Csk (Fig. 6A). Equal amounts of Src were assayed, as shown by the immunoblot analysis of the immunoprecipitates. Studies then determined whether HBx stimulates endogenous cyclin A gene expression through a Src kinase pathway in growth-arrested Chang cells. Cells were assayed for cyclin A protein levels and cyclin A-cdk2 activity 24 h after transfection, corresponding to the accumulation of cells in G1-early S phase (Fig. 5). Compared to the HBxo control, HBx induced about a fivefold increase in cyclin A protein levels in these cells, which was blocked by coexpression of Csk (Fig. 6B). The activity of cyclin A-cdk2 complexes was determined by immunoprecipitation of cyclin A followed by an in vitro assay of cdk2 phosphorylation of the substrate histone H1 (7) (Fig. 6B). Compared to the HBxo control, HBx induced a fivefold stimulation of cyclin A-cdk2 complexes in serum-starved cells by 24 h after transfection, which was prevented by coexpression with Csk. These data therefore demonstrate that HBx stimulation of Src kinases is an important cytoplasmic component of deregulation of early cell cycle control.
Studies next showed that HBx activation of Src signaling is also vital for stimulation of the ectopic cyclin A promoter-reporter ints13 cells. Cells were cotransfected with vectors expressing HBx or TAFII250, plus the cyclin A promoter-reporter, with or without cotransfection of a plasmid expressing Csk. Cells maintained at the permissive temperature for thets13 mutation in TAFII250 showed a consistent increase in cyclin A promoter activity of two- to threefold with HBx expression, which was blocked by overexpression of Csk (Fig. 7). At the restrictive temperature for TAFII250, HBx stimulated the cyclin A promoter only very slightly, and this stimulation was also blocked by Csk expression. As a control, cells maintained at the restrictive temperature and expressing wild-type TAFII250 were shown to be unaffected by overexpression of Csk. Furthermore, Csk did not downregulate transcription of a CMV promoter-controlled reporter construct (data not shown), excluding inhibition of HBx activity by a general decrease in transcription (Fig. 7). Thus, HBx stimulates the cyclin A promoter and deregulates early cell cycle control through a pathway that requires TAFII250 function and activation of Src kinases.
The HBV HBx protein possesses a wide variety of activities, such as activation of cytoplasmic signal transduction pathways, induction or sensitization of cells to apoptosis, loss of early cell cycle control checkpoints, possibly direct interaction with several components of the transcription apparatus, and a weak to moderate activation of transcription directed by RNA polymerases I, II, and III. Given the variety of activities attributed to HBx and the fact that its ability to activate transcription is at best moderate, there has been considerable difficulty in firmly establishing the mechanism(s) through which HBx activates transcription. The in vitro demonstration of interactions between HBx and components of the transcriptional machinery such as RBP5 or TFIIH or the transcription factor ATF/CREB has led to the suggestion that HBx functions directly in the nucleus (14, 51, 60, 61, 90). Transcriptional activation by HBx, according to this model, is a consequence of direct interaction with any of several transcription factors in the nucleus. While most evidence points to a largely cytoplasmic location for HBx, some of the protein is typically in the nucleus, providing support for this model (20, 21, 23, 63,72). Moreover, HBx is thought to activate the transcription factor CREB, at least in part by direct interaction in the nucleus (90). Another large body of evidence also supports a cytoplasmic function for HBx in transcriptional activation. Activation of NF-κB, NF-AT, and AP-1 by HBx as well as stimulation of transcription directed by RNA polymerases, I, II, and III involve HBx activation of cytoplasmic signaling (6-8,15, 17, 18, 23, 32, 38, 40, 41, 43, 48, 55, 73, 74, 86-88). Furthermore, most studies on HBx cellular location have demonstrated that most of the protein is located in the cytoplasm, regardless of whether it is synthesized during transient transfection or in the context of woodchuck hepatitis virus or human HBV infection of liver cells in vivo (20, 21, 23, 35, 63, 72). This is in accord with the observation that cytoplasmic HBx activation of Src signaling, in particular, strongly stimulates viral replication in cultured cells (40).
Ras and Src signal transduction pathways, both of which are activated by HBx, are critical effectors for progression of cells to the G1-early S phase transition of the cell cycle. Transit of quiescent cells through G1 involves stimulated synthesis of cyclin D followed by cyclin E, both of which associate with and activate cdk-4, -6, and -2 (reviewed in reference 65). Transit of cells to the S phase junction involves synthesis of cyclin A, which associates with and activates cdk2. Ras and Src signaling are involved in multiple events for progression to the G1-S transition (65), which include activation of Fos and Jun (AP-1) and ATF/CREB family members (5, 10, 37, 44, 53, 56, 64,91; reviewed in reference 80). Studies have demonstrated that a key target of these signaling events for G1 progression is activation of cyclin D and A promoters by ATF/CREB transcription factors (69, 75, 85) in conjunction with specific activation of TAFII250 (85). The critical function of TAFII250 for activation of cyclin D and A promoters was shown to be its intrinsic HAT activity, as the ts13 cell mutation renders TAFII250 HAT defective at the restrictive temperature, prevents D and A cyclin gene transcription, and blocks progression to the G1-early S transition (24).
In this study, we explored the functions of HBx in promoting early cell cycling, including its purported functional replacement of TAFII250 (27). Since TAFII250 couples transcriptional activation to cell proliferation, functional substitution by HBx would be unprecedented, given that it is only 17 kDa in size and possesses no known enzymatic activities. It would also be expected to reflect a novel and underscribed proto-oncogenic activity of HBx, because continuous expression of HBx is presumably required during virus replication (21). We showed that HBx does not functionally replace known activities of TAFII250. HBx did not stimulate the cyclin A promoter in the absence of TAFII250 activity (Fig. 1), regardless of whether the nuclear levels of HBx were elevated (Fig. 2 and 3), and it did not rescue cells from death induced by the absence of TAFII250 activity (Table 1; Fig. 4). However, it was found that HBx required TAFII250 function to stimulate the cyclin A promoter (Fig. 1, 6, and 7). HBx also required TAFII250 function to induce quiescent cells to transit from G1 to the S phase (Fig. 5 and 6), since switching cells to the restrictive temperature for TAFII250 blocked cell cycling. Since the only defect at the restrictive temperature is in TAFII250 function and since TAFII250 function is essential for cell cycle progression (24, 49, 50, 67, 75, 84, 85), these data demonstrate that HBx does not override the requirement for TAFII250 in stimulating cell cycling. These data are consistent with other previous reports demonstrating induction of early cell cycling by HBx (and presumably activation of the cyclin D promoter as well) (6, 7, 42, 71), possibly to the G1-early S phase junction (71). In addition, HBx activation of Src signaling, which is coupled to activation of Ras (41), was shown to be important for progression of cells through G1 and activation of the cyclin A promoter (Fig. 6 and 7).
The results reported here are consistent with critical cytoplasmic functions of HBx in the stimulation of early cell cycling. HBx stimulation of Src signaling was found to be essential and is consistent with the established critical importance of Src and Ras in promoting progression of cells through G1. The inability of HBx to functionally replace TAFII250 during 24 to 30 h of expression is also consistent with the established longevity of the TAFII250-TFIID complex. Most studies demonstrate that this complex is very stable. In ts13 cells at the restrictive temperature for TAFII250, almost 24 h was required for replacement of the mutant form of TAFII250 by ectopically expressed wild-type protein (19, 49). Thus, the reported rescue by HBx ofts13 TAFII250 within 5 h of the inactivating temperature shift, if correct, would be unprecedented (27). However, HBx has been shown to bind in vitro to the CREB transcription factor (4, 51, 77, 90) and to stimulate CREB-dependent transcription in vivo (59, 90). Furthermore, an ATF/CREB element can function in transcriptional activation of cyclin D and A genes. Taken together, these observations raised the possibility that perhaps HBx might circumvent TAFII250 function by activating CREB, leading to cell cycling and prevention of cell death. Nevertheless, neither wild-type HBx nor a variant engineered to concentrate in the nucleus blocked cell death or displayed any TAFII250-independent activity, including transcriptional activation of the cyclin A promoter and CREB-dependent transcription at the restrictive temperature for TAFII250 (Fig. 3). Although nuclear HBx was not sufficient to activate cell cycling, studies need to determine its involvement in promotion of early cell cycling, possibly by direct interaction between HBx and CREB.
HBx stimulation of the cyclin A promoter and G1 cell cycle progression may represent a significant activity in the HBV life cycle. HBx expression during infection by HBV may stimulate quiescent hepatocytes, not to divide but to transit the G1-to-S-phase transition. A similar function is well established for regulatory proteins of many oncogenic viruses. It is thought that release of cells from quiescence may aid in viral replication by expanding the pool of deoxynucleoside triphosphates (dNTPs), which is significantly restricted during G0 and is elevated during the G1 transition (22). Moreover, dNTP pools are much lower in the cytoplasm, where HBV replicates, than in the nucleus, which could present a considerable impediment to HBV replication in quiescent cells. Transit through G1 would therefore increase dNTP metabolism and elevate the available pool for HBV replication. In contrast, if HBx were to function as a TAFII250-like protein, promoting cells into cycling, this would be deleterious, as studies have shown greatly impaired HBV replication in cells during S phase (57). Importantly, for classic retroviruses, depletion of dNTP pools, particularly dCTP, has been shown to be responsible for arrest of viral reverse transcription in G0cells, which is relieved by transit into the G1-S transition or by supplementation of G0-arrested cells with high levels of dNTPs (13, 25, 26, 93, 94). HBx has been shown to be required for HBV replication (12, 95), which also utilizes reverse transcription of the viral RNA genome. In striking similarity to classic retroviruses, HBx promotes HBV reverse transcription and second-strand DNA synthesis in cultured cells through a pathway that involves HBx activation of Src kinases (40). Thus, our data support a role for HBx stimulation of early cell cycle transit, in part through Src kinase and TAFII250 stimulation of cyclin promoters and activation of cdks, so as to promote viral replication.
This work was supported by NIH grants CA56533 (R.J.S.), CA74476 (M.B.), and GM51314 (N.T.) and by American Cancer Society grant RPG-98-201-CCG (E.H.W.).
- Received 29 November 2000.
- Accepted 5 February 2001.
↵* Corresponding author. Mailing address: Department of Microbiology, NYU School of Medicine, 550 First Ave., New York, NY 10016. Phone: (212) 263-6006. Fax: (212) 263-8276. E-mail:schner01{at}popmail.med.nyu.edu.
- Copyright © 2001 American Society for Microbiology


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Some people are destined to be popular and wealthy in one's life because they are extremely lucky in their life.They always find themselves at the right place at the right time,getting help from right kind of people making them highly successful in one's life.
Let's see some of the lucky lines which are found on the person's hand.
1)Fate line from the wrist or bracelet
If the fate line starts from the wrist or bracelet and flawlessly goes to the mount of Saturn as shown is considered as very lucky.Such person frequently gets promoted in one's career and does not have to worry about money.
On the top of it,if the Rahu mount is elevated as shown will take the person to the top position in a very short period of time.Rahu is a very powerful planet.On a good hand,it confers the person with sudden wealth and power and even has the potential to convert your enemies into friends.
2)An Arc like formation
If the line starts between the Apollo and the Saturn finger and goes all the way to the Jupiter mount forming an arc like formation as shown indicates the bearer is going to inherit significant portion of property either through one's spouse or family members.
Also there are high chances of person winning hugh amount of money in lottery or gambling.
3)Two parallel fate lines
If two fate lines of equal depth and color running parallel to each other as shown is considered as very lucky because the person will be earning two incomes from two different professions or business at the same time making the person quite rich in one's life.
Remember guys, the two fate lines should be distinct and without any defects.
4)Single vertical line on the Apollo finger
If a single vertical line is found on the second and third section of the Apollo finger as shown indicates the person will be very lucky in getting the help from the famous and powerful people in his or her life.
With the constant support and favor from these people the person will become rich and famous in one's life.
5)Triangle formation between Headline and Sun line
If the line from the headline joins the Sun line making a triangle like formation as shown indicates the person is gifted with a vision and intelligence which will make him or her immensely popular and lucky in one's career or business.
6)Flawless Sun line and the Fateline
If the fate line and the Sun line are starting from the base of the palm without any defects is considered as one of the most fortunate and lucky signs that can be found on the person's hand. Such person is born to be famous and rich in one's life as the opportunities keeps running at his or her doorstep.
Sun line starting from the base of the palm makes the person famous at the beginning of one's career and the flawless fate line maintains the stability in the bearer's life.

subset: commoncrawl


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Construction Work Tips
Use a palette on the floor covering, and the use of 4 of them making walls which would roughly an hour. Pallets come in many sizes, 36×36 rectangle in the palette is common 48×40.I always thought of pallets as undesirable, but not anymore! Two pallets on top of each other are a sofa or a large futon frame. This is one of the home remodeling ideas we share inside this article.
Love the veritable army of ideas to use the palette humble. The ideas that I am about to share with you that can be made using any size pallet, but if possible, the 48×40 is the best and the strongest. Bill, with the price of wood these days, they are good ideas. Anyway, thank you for your comment and have added ideas.You gave us some great ideas to recycle and make great additions to the comforts of home.
Go to the construction site for more tips. The morning after a midweek stay. Construction is defined as "a process that involves the construction or assembling of infrastructure. When it comes to building materials, you should not only price but also to compare the composition element compare. Cities are increasingly in need of a project manager to work on their construction projects.
I once heard an artist teaching a rookie to the industry how to really make money: marking materials . to collect the materials used as appliances, doors, bricks, wood, and much more! When you are ready for the materials always ask in advance the contractor. Includes materials manufacturer in his citation for the overall task. Cost of labor and material costs.
Look at their work in progress and completed work. Finding building construction customers in both directions – the methods for screening qualified contractors. I'm glad you liked it and I hope it works for you. Even if you make a residential project, there are some safety tips for construction that you need to remember to keep your workplace safe. Last seen working for a construction company nationally renounced which had a branch in our region.


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Western Sydney hospitals are under an avalanche of pressure with emergency department waiting times blowing out.
The latest data from the Bureau of Health Information showed over the April to June quarter, patients at Westmead (47.3%), Blacktown (46.9%), Liverpool (40.9%) Campbelltown (40.7%) and Nepean (39.5%) waited longer than four hours in an emergency department.
"It would appear that in Western Sydney in the last quarter there were particular issues," Health Minister Brad Hazzard said.
Fairfield and Blacktown Hospitals both had an 11% increase in patients, while there was a 38.9% and 16.8% spike respectively for people arriving by ambulance.
Acting Chief Executive Dr Kim Sutherland from the Bureau of Health Information said many of these were sicker patients.
"There's been an increase in the number of more urgent cases that are presenting to the emergency department," Dr Sutherland said.
The Opposition said the spike is due to tight family budgets.
"Patients are flooding emergency department because they cannot get into a GP's practice or they are unable to pay extra for a GP – putting unnecessary pressure on the state's emergency departments," Shadow Health Minister Walt Secord said.
Ambulance response times have improved overall with "Priority 1A" cases (like cardiac arrest) waiting seven-and-a-half minutes on average.
Minister Hazzard is expecting much worse results next quarter when the BHI reports on Sydney's record breaking flu season.


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2015-04-29 | Peer Reviewed
Contingent valuation of community forestry programs in Ethiopia: Controlling for preference anomalies in double-bounded CVM
Koch, Steven. 2015. "Contingent valuation of community forestry programs in Ethiopia: Controlling for preference anomalies in double-bounded CVM." Ecological Economics 114: 79-89.
Download reference Doi:doi:10.1016/j.ecolecon.2015.03.014
This study examines the welfare effects of community plantations in Ethiopia via contingent valuation. Both single-bounded and double-bounded survey methods were considered, and, with respect to double-bounded methods, the potential for anomalous response behaviour was also taken into account. The results generally confirm that there are statistically significant welfare benefits to be derived from community forestry; however, the range of the estimated benefits is large. After controlling for anomalous response behaviour, the range of estimated benefits narrows, and our preferred estimates place the welfare gain between Ethiopian Birr (ETB) 20.14 and 30.41 per household, which is much lower than the estimated benefits without controlling for anomalous preference responses.
- Forest Collaborative
- South Africa
Center of origin
- Forestry


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Steve noticed some trouble with his vision which led him to the eye doctor and then to a retinal specialist. When his eyes checked out fine, he had an MRI done of his brain and the doctors discovered a pituitary adenoma. This tumor on the pituitary gland is normally non-cancerous, but it is about the size of a walnut, and it is putting pressure on his optical nerve. He expects to have neuro-surgery in late June or July and endocrine therapy depending on how the pituitary is affected. Please pray for healing and for wisdom for the doctors involved in his care. Pray for God's provision to cover these medical expenses too. Thank you for your prayers and concern!
Steve's Health Concerns


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Score: Eastern Nazarene 8, Dean College 4
Records: Eastern Nazarene (14-10), Dean College (7-16-1)
Location: Adams Field, Quincy, Mass.
Date: Tuesday, April 10, 2018
Time: 3:30 PM
Eastern Nazarene erased a 4-0 deficit and the host Lions scored eight unanswered runs to top Dean College, 8-4, in non-league baseball action on Tuesday afternoon at Adams Field.
Dylan Louviaux broke a 4-4 tie with a two-run double in the bottom of the seventh to highlight a three-run inning that catapulted the Lions to victory.
Dean jumped out to the aforementioned 4-0 lead as Landon Fish (New Port Richey, Fla.), Brian Hadden (Newburyport, Mass.) and Vincent Buonaiuto (Danbury, Conn.) all drove in runs in the top of the first.
Unfortunately for the Bulldogs, that would be all the offense they would be able to muster as Eastern Nazarene slowly erased the four-run deficit.
First, they pushed two runs across on a Steven Pesce single and a Connor Simpson groundout in the second. After single runs in the fourth and fifth, Louviaux delivered the go ahead runs in the seventh, which were followed by a run scoring groundout by Pesce and an unearned run in the eighth.
Xavier Faria came on in relief and tossed four innings, allowing just three hits to improve to 3-0 on the season.
Travis Dinsdale (Worcester, Mass.) went 3-for-5 with a run scored for the Bulldogs in his leadoff spot, while Will Kulcher (Crown Point, Ind.) and Hadden each had two hits.
The Bulldogs return to action on Wednesday when they visit Eastern Connecticut State at 4:00 p.m.


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Used corrugated plastic totes provide secure storage of papers and other small items, and are less expensive than molded plastic. Used corrugated plastic totes are nestable and lightweight for convenient storage.
Used corrugated plastic bins are produced from extruded plastic similar in appearance to corrugated paper. Both upper and lower surfaces are supported by fluted ribs.
The corrugated plastic most used is polypropylene because it is light weight, tough and abrasion resistant with excellent chemical resistance and is unaffected by water.
Used corrugated plastic bins are an amazing value when purchased "used" due to their low cost. Make the right choice when searching for low cost light weight storage containers. Speak with one of our storage technicians who will explain the many choices available for your storage needs.


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Should you have any questions about an order or our services then we hope you will find an answer in our most Frequently Asked Questions page FAQ's .
Our Customer Service Teams are available to answer your emails between 9:00am and 5:00 pm, Monday to Friday. Please contact us using the form below Or Please email to firstname.lastname@example.com .


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RamFanatic Bio or More Than You Ever Wanted To Know About RamFanatic
RamFanatic was born on Feb 19,1932 in Rocky Mount, NC also the birth place of Kay Kyser, Buck Williams and Phil Ford.
He tired of the scenery at the age of 6 months and took his family to Raleigh.
At the age of 4, he moved for a year to Woodville, NC in the Ahoskie-Aulander area of eastern North Carolina. He also took his family with him on this journey where he had found his father as job as foundryman at the Gay Harrington Manufacturing Co. Not completely happy in a village as small as Woodville, RamFanatic packed up and moved his family back to Raleigh where he remained until he entered East Carolina University in the fall of 1950. While at ECU, (3 months) he handled the publicity for the Department of Athletics and was paid with meal tickets which he quickly squandered away on his friends. In the fall of 1950, he entered the U.S. Air Force where he served in the Strategic Air Command (SAC) for four years with tours in Texas, Florida, England and French Morocco. Having learned in the Air Force how little he knew and faced with the responsibility of supporting a wife and child, RamFanatic decided to wipe the slate clean and begin anew at UNC. One of the best, if not the best decisions, he ever made in his entire life.
Upon graduation from UNC, RamFanatic was employed at Central Prison in Raleigh as Asst. Director of the Reception Center until he was employed on Jan. 1, 1960 as Asst. Director of Personnel at the University of Virginia. He remained there for 5 years and then moved on to Vanderbilt University in Nashville, TN., where he fell in love with the banjo. After two years, he accepted the position of Director of Personnel for the Duke University Medical Center where he remained for 4 years. In Feb. of 1971, he accepted the job of Director of Personnel for the Department of Alcoholic Beverage Control for the Commonwealth of Virginia where he remained until retirement in Sept. of 1991.
RamFanatic has played the tenor banjo in various musical groups since "discovering" the instrument during his stay in Nashville. While at Duke, he played with a banjo group named the Executives and played at halftime at a Duke-State, State-Wake Forest and an NCAA playoff game between Davidson and St. Johns. which was nationally televised.
Another hobby of RamFanatic is antique advertising and collection of county store items. His living room is set up like a turn of the century rural county store and does not have a piece of furniture in it. He and his wife Betty Lou have had their collection of items featured in 3 books, 2 magazines and a full page feature in the Richmond News Leader.
Another resident of the RamFanatic household in Richmond, Virginia is Leroy, a red dachshund who is also a "fierce" Tar Heel fan.
About this Site
I never aspired to having my own web site, but that didn't prevent me from getting one. A telephone call from a relative in Raleigh who works with computers, internet, web sites, etc. informing me that I now "had my own web site" came as a complete surprise since he and I had never discussed the possibility of establishing my own web site. The relative was aware of the fact that I had been posting on Tar Heel Talk for about a year, and correctly surmised that I was enjoying it. In addition, he also noticed that the response to my posts had been positive. In his mind, two plus two equaled four.
The initial response to the web site has exceeded my expectations. We have operated with certain handicaps by virtue of being a "static" web site, I refer to the site being static because we make no effort to follow the news of the day and we have no message board. Both of these features would insure a base following who would probably make frequent visits to the board for the latest developments. We have decided to try and carve out a much narrower niche among viewers by concentrating on sports events and personalities from the past and the examination of larger sports issue like officiating, the recruiting process, etc. Only time will tell if we are to succeed in this endeavor. We received close to 1000 visits in the first 18 days but the challenge will be to sustain this traffic over a period of time. We will be trying various methods to attract viewers back to the site. I have asked my webmaster if he can develop a method of spotlighting new entries on the site so viewers can tell immediately if additional material has been added since their last visit. We may go to a procedure which will have material added only at certain times of the week like Monday mornings. That way, viewers could expect additions since their last visit. Any ideas on how we can attract return viewers would be welcomed and can be communicated to me through the e-mail address on the front page.
We plan to continue the site as long as it is fun and there seems to be sufficient interest to justify the time and effort involved. The site is not a memorial to my ego. I've already had my 15 minutes at center stage and it was great. Hope you enjoy the site and will share your thoughts with me through the guest book.


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In keeping with its name, The Black Angel, a passive magnetic soundhole pickup, is matte black in color. Like The Angel, The Black Angel "hears" the ...Click To Read More About This Product
High-quality passive magnetic soundhole pickup for use with any dual or multi-source system.
In keeping with its name, The Black Angel, a passive magnetic soundhole pickup, is matte black in color. Like The Angel, The Black Angel "hears" the entire range of the acoustic guitar from top to bottom, with no gaps anywhere. Its "feel" is just as important. The Black Angel tracks right- and left-hand dynamics, and responds immediately to the sound coming off the string.
The Black Angel is quiet and, although it's magnetic, it doesn't sound electric - there's no hum and no battery. Both coils are on a parallel axis to the strings, featuring a humbucking magnetic circuit that is acoustically isolated to reduce unwanted finger and pick noises. Players who like to mix two or more pickups together will appreciate the built-in phase switch making The Black Angel ideal for use in any dual or multi-source system, and it can be easily combined with any piezo or microphone.
The Black Angel installs easily and quickly in the soundhole, with no modification to your guitar, and fits soundholes of 3-1/2" (89 mm) and larger. The 2-1/16 "E"-to-"E" string spacing on The Black Angel easily accommodates guitar nut dimensions ranging from 1-11/16" to 1-3/4". Both a 1/8-inch to 1/4-inch Switchcraft endpin jack (install version) and a quick mount 10-foot cable (standard phone jack) are included with The Black Angel.
Give your guitar an upgrade with this DiMarzio pickup. Order today!
Our product catalog varies by country due to manufacturer restrictions. If you change the Ship-To country, some or all of the items in your cart may not ship to the new destination.


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Tips For Removing Push4check.com from Windows 2000
More infection related to Push4check.com
|Browser Hijacker
|Aprotectservice.com, Cloud-connect.net, Search.easylifeapp.com, Speedtestbeta.com, Int.search-results.com, SmartSearch, CoolWebSearch.cpan, 7000n, needupdate.com, Search.iMesh.net, Asafetywarning.com, HeadlineAlley Toolbar, Questdns.com, VacationXplorer, Websearch.simplespeedy.info, HeretoFind
|Fuck_You Ransomware, Dot Ransomware, [email protected] Ransomware, BandarChor Ransomware, .aes256 File Extension Ransomware, BitStak Ransomware, [email protected] Ransomware
|Infoaxe, Spyware.PowerSpy, Surfcomp, FinFisher, VirusEffaceur, WebHancer, ANDROIDOS_DROISNAKE.A, SpamTool.Agent.bt, Toolbar888, Egodktf Toolbar, Modem Spy
|BitGrabber, Vanish, WinProtect, Golden Palace Casino, Adware:Win32/InfoAtoms, Adware.SearchExeHijacker, Win32.Adware.AdPromo, IAGold, Vapsup.ctc, OneStep.c
|Transponder.LocalNRD (threat-c), TrojanDownloader:Win32/Karagany.I, Trojan.Tobfy.J, Troj/DwnLdr-KJW, Trojan.Kimejkay.B, Suspect-AB!E923A5A32CE9, TSPY_ZBOT.ADD, Trojan.Agent-FPE, Google WebHP Virus, JS:ScriptSH-inf, Mosaic 2.0
Quick Steps To Get Rid Of Push4check.com In Simple Clicks From PC
What do you know about Push4check.com?
Generally speaking, Push4check.com is term refer to a type of dangerous computer virus which aims to gain complete access over infected machine. It easily exploit the installed program vulnerabilities and then take advantage of user's incompetence regarding safe web browsing. The threat displays flashing pop-ups, banners, auto-play video commercials and in-text links onto your system's screen. It is capable of hijacking installed Internet Explorer for collecting sensitive information about user's online browsing activities. The malware can know what websites you visit the most, what search terms you use and what data you enter. This type of infection blocks the entire machine and seeks to threaten the computer users into paying an invented fine. Additionally, Push4check.com can used to get an unauthorized access to the affected PC, infecting crucial files and damaging the computer. Therefore, it is very important to Get Rid Of the virus safely from your Windows 8.
Main Principles of Push4check.com Distribution
The threat may apply few major principles for the PC infiltration and it does need an additional program or special conditions in order to arrange and carry out a successful malicious attack. Typically, Push4check.com rely on some deceptive techniques, such as:
Misleading Official Web Portals: Most of the rogue programs have their official domains presenting them as an useful tools, for example, handy browser toolbars, helpful shopping assistants or amusing game plug-ins. If you fails to recognize that such softwares is capable of performing annoying activities on your PC, then you may end up with Push4check.com onto the system.
Fake Pop-up Adverts & Links: Clicking on misleading advertisements or redirect links can also be malicious and may lead you to the infiltration of such noxious virus. This type of notifications typically suggest downloading missing program updates, several system security softwares, free computer scans and attention-grabbing offers.
Bundling: There are plenty of cost-free applications that have been bundled with Push4check.com. Such deceptive program packages are usually promoted on unsafe software sharing domains or P2P networks. If you tend to visit these websites, you may end up downloading rogue programs associated with dangerous viruses.
Effect on the System After Push4check.com Invasion
Once the malware successfully enters the machine, it typically performs various changes onto installed web browsers. When infected with this parasite, you may notice an unknown homepage, search engine and a new tab page. Besides, it can also add and delete entries on your Registry Editor. It initiates these changes without user's permission. It is mostly used to redirect users to phishing web pages and infect their machine with other sorts of noxious viruses. Push4check.com can seriously decrease your system's security. Hence, Removing the malware is considered very important for you.
Click Here To Download Push4check.com Free Scanner
Process To Get Rid of Push4check.com From PC & Internet Explorer
Process 1: Quick Steps To Boot Windows System in Safe Mode To Get Rid Of Push4check.com
Process 2: Steps To Get Rid Of Push4check.com From Control Panel of PC
Process 3: Quick Steps To To Clean Push4check.com Related Malicious Registry Entries
Process 4: Push4check.com Removal Report For Affected Internet Explorer
Process 5: Get Rid Of Unwanted Processes of Push4check.com From Windows Task Manager
Process 6: How To Reset Internet Explorer Settings To Delete Push4check.com
Process 1: Instructions To Boot PC in Safe Mode To Get Rid Of Push4check.com
Removing Push4check.com From Windows 8 or Windows 10 Computers
Press "Win key + C" keys and click on Settings. Then after, tap on Power icon, hold down the Shift key and click "Restart" button.
Select "Troubleshoot option" and click on the "Advanced" options.
Select "Startup Settings" and then press Restart.
To enable "Safe Mode with Networking" option, press F5 key.
Now, your PC starts in Safe Mode with Networking and you can find and delete Push4check.com as quickly as possible.
Push4check.com Removal From Windows XP/ Win Vista/ 7 Systems
Press F8 key continuously when the PC is powered on or restarted.
Here, you will notice Advanced Boot Options on your computer screen.
To select Safe Mode or Safe Mode with Networking, use arrow keys and hit Enter button.
Now, identify and Get Rid Of Push4check.com from your system as early as possible.
Process 2: Quick Steps To To Eliminate Push4check.com From Control Panel of PC
Click on Start button and go to Control Panel option.
Now, under Programs category, click on Uninstall a Program option.
Then after, you can select Push4check.com and its associated annoying programs and click on Uninstall button.
Process 3: Method To Get Rid Of Push4check.com Related Malicious Registry Entries
To open Registry Editor, press Start button and choose Run option.
In the open field, type "regedit" and hit Enter key.
- Get Rid Of malicious registry entries of Push4check.com without any delay.
Process 4: Push4check.com Removal Report For Affected Internet Explorer
Push4check.com Removal From Chrome 49.0.2623
Open Chrome browser and tap on gear icon at the top right corner.
Click on More Tools option and then go to Extensions.
Select Push4check.com related unwanted extensions and click on Trash bin icon.
Removing Push4check.com From Mozilla Firefox:38.2.1
Launch Mozilla Firefox and click on gear icon to open browser menu.
Select Add-ons and choose Extensions panel.
Then after, Get Rid Of Push4check.com related add-ons by clicking on Remove button.
Get Rid Of Push4check.com From IE 10:10.0.8400.00000
Open Internet Explorer and click on Gear Icon from the right-top corner to open Tools menu.
Now, tap on Manage Add-ons option and select Toolbars and extension tab.
Find all the suspicious add-ons related to Push4check.com and tap on "Disable" button.
Process 5: Get Rid Of Unwanted Processes of Push4check.com From Windows Task Manager
To open Windows Task Manager, press "Ctrl + Alt + Del" keys altogether.
Click on Processes tab in order to identify malicious processes of malware.
Select Push4check.com and its associated processes and click on "End Process" button.
Process 6: How To Reset Internet Explorer Settings To Get Rid Of Push4check.com
Reset Mozilla Firefox:38.2.1
Start Mozilla FF, tap on Firefox menu, select Help option and then click on Troubleshooting Information option.
From top of the page, click on he "Refresh Firefox" button.
Tap on "Refresh Firefox" button in the appeared dialog box.
After resetting Firefox, Push4check.com will be removed In Simple Clicks from your browser.
Reset Chrome 49.0.2623
Launch Chrome browser, tap on Chrome menu >> Settings option.
In the search box, type Reset and click on Reset button.
Hence, Push4check.com will be deleted completely when you reset your Chrome 49.0.2623.
Reset IE 10:10.0.8400.00000
Open Internet Explorer, tap on >> Tools menu >> select Internet options.
Click on Advanced Tab button and hit Reset.
Check mark the "Delete Personal Settings" option and tap on Reset button.
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Denver office buildings draw yield-hungry Korean funds
A consortium of Hana Financial Investment Co. Ltd. and Hyundai Asset Management Co. Ltd. has recently closed a $238 million acquisition of an office tower in downtown Denver, Colorado, with Samsung SRA Asset Management in talks to buy another skyscraper US Bank Tower in the city, according to industry sources.
The purchase of 1670 Broadway from a UBS Realty Investors LLC came after a consortium of the Construction Workers' Mutual Aid Association (CWMA) and an unidentified South Korean insurer bought an office building used as Re/Max Plaza International's headquarters in a 132-billion-won ($115 million) deal in March.
The two property deals in Denver underlined South Korean institutional investors' effort to diversify into second-tier cities in developed countries or niche markets for higher returns, after valuations of landmark buildings in gateway cities have soared.
To finance the acquisition of 1670 Broadway, Hana Financial committed 120 billion won to a domestic fund launched for the transaction by Hyundai Asset Management.
The brokerage firm is selling down the equity interest in the property to domestic institutional investors.
Additionally, Hana Financial borrowed 100 billion won in a five-year loan at a fixed rate of 4.10% in the United States in September, secured on the 36-story tower, according to South Korea's Internet media Global Economic News.
Teachers Insurance and Annuity Association (TIAA) occupies 51% of the space on a long-term lease.
The building's cap rate, or net rental incomes divided by the purchasing price, is mid-5%, according to online news provider edaily in early October.
Meanwhile, Samsung SRA was recently chosen as the preferred buyer of US Bank Tower in central business district of Denver.
It is seeking to acquire the multi-tenant property via a blind-pool fund. No further details of the possible deal were available.
Driven by youth population growth and high average incomes relative to other US cities, property value in Denver is expected to rise further.
Separately, Jack Kim, founder and principal of US-based KORE Investments, recently told the Korean Investors that South Korean investment funds are diversifying away from landmark buildings in big US cities in search of higher returns.
In doing so, they prefer local management firms to global managers so that they can secure high-yielding real estate assets, he noted.
KORE, focusing on assets in Denver and Chicago, arranged the Re/Max Plaza building transaction for the CWMA and the insurance company.
<Edited by Yeonhee Kim>
Photo: Getty Images Bank


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Kala jadu specialist tantrik baba ji in Chennai
Kala jadu specialist tantrik baba ji in Chennai
Kala jadu in itself is one of the most Incomprehensible things because it can complete all those desires of the person who makes it in use. Similarly, also can ruin someone completely who for once come under its approach. Therefore, this is one of the most relevant reasons that why does it ha considered one of the most Incomprehensible things. In addition, our kala jadu specialist tantrik baba ji in Chennai believes positively in its use. However, people think that its use can only attempt to achieve some selfish perspective. Furthermore, it is not true.
Because the tantriks belongs to the world of tantra sadhna. Similarly, the means of kala jadu shall not be considered only for the negative purpose when it is done by tantrik. Alike of our black magic specialist in chennai, who has become the master of tantra vidya. With the assistance of he has been now doing successful kala jadu tricks. On the base of he has been helping people.
What is kala jadu?-why should one need to use it by specialist tantrik baba ji in Chennai?
When it comes to the definition of evil powers then the words that instantly comes in mind is kala jadu. Which is one of the most authoritative options for one who wants some special powers likewise control someone by black magic? Especially for some evil purpose to deliberately achieve some target. Because it is the inclusion of all the evil and dark energy that easily attempt any activity. Resulting easily grant one its desired results. This is the also a mandatory reason according to our kala jadu tantrik baba ji that why do people afraid of it.
While it is also a fact that the use of black magic can also put you in troubles. Especially when you are in its grip. In addition, only a few of the people know well about it that if it is bad then simultaneously it can also help one. Likewise, our kala jadu specialist tantrik baba ji in Chennai, also makes the use of black magic. Specifically, for the person that is relevant to the people's general life issues. Such as
- Love Troubles
- Enemy problems
- Husband-wife relationship problems
- Business issues
- Financial problems
And many other problems of a common individual's life can be easily resolved with the help of our specialist. Therefore, you can also contact him for consultation about your problem's solution.
How our kala jadu specialist tantrik baba ji in Chennai-known as a successful black magic specialist?
However, there are often a lot of people present on the name of kala jadu specialist baba ji. But only a few of them may be genuine still if they are genuine then there is a rare chance that their black magic is successful. Because the kala jadu tricks are really very intricate thing that never allows a common individual to make it in use. Even though, if any other fresher makes it use with some efforts. Then also they don't get to see all those results that other desires.
Therefore, if you are ever trying to make the use of black magic through any other tantrik. Then the primary thing that you should need to give importance. Is the same that always get the feedback of that person through his experience. His experience in tantra mantra can make you believe how much does he is genuine. In the second place, you often can say that our kala jadu specialis tantrik baba ji in Chennai. Is in actuality is a real specialist.
Because of his extreme efforts. He has attained a lot of experience in the field of black magic. Therefore you can easily and without doubt, call him a black magic specialist. Who truly uses black magic spells and those all come true.
Now get rid of problems- Only by black magic(kala jadu) specialist tantrik baba ji in Chennai
Do you think that it is enough that you are not getting any special moment in life? Or you think that you are of the most unlucky person in this world? Then my advice for you, to not to blame yourself. Because in this world people may not get that all that they desire. But now our kala jadu specialist tantrik baba ji in Chennai has come to change fortune. With his practices of tanra mantra. As well as with his efforts he has secure a lot of people's ruined life. He has saved much life from getting destroyed. Similarly, if you also think that you should quit your life. Then before doing anything just take the consultation of our black magic specialist in Chennai. Who knows if this comes true as the way out of your troubles.


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Dura Racking
Shelving Shop's Dura Racking System has been accurately engineered and calculated for Safe Working Load (SWL) Limits. It is recognised as one of the safest and most durable Racking Systems in the market.
Shelving Shop is at the forefront of warehouse racking system design, combining durability with precision engineering for a heavy duty, high-performance storage solution for all of your warehousing needs.
Our selective pallet racking allows you to utilise as much space as possible through innovation. Adjust beam levels and add to them with ease. Completely modular and versatile, it maximises your floor space for higher density storage.
We know the demands imposed by the unpredictable seismic conditions in New Zealand, and our Dura Pallet Racking is designed to meet the highest quality and safety specifications.


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At iQ Dental, patients are proud of the service and support they recieve. Based in West Byfleet, Surrey, our premises have been adapted to help our patients feel comfortable and relaxed. With our excellent customer service you can be sure to enjoy coming to see us, like the following patients who were happy to act as case studies.
Debbie(Office Manager), Addlestone, Surrey
Orthodontic tooth coloured fixed braces
Debbie had tooth coloured (aesthetic) fixed braces for 18 months. We were set a target completion date due to an important family holiday for her anniversary.
I thought about wearing braces for several years and it took that long to pluck up the courage as I am extremely nervous about going to the dentist. I was referred to iQ Orthodontics by my dentist as he informed me that they were very helpful, caring and professional – what an understatement!
All members of the team, from the reception staff, Bilal and in particular his nurse Donna, who would always do her best to reassure me when I was nervous (which I frequently was!), made me feel relaxed and at ease. Thank you for holding my hand!!!
Appointments were made to fit around me and I never had to wait to be seen, every stage of the process was explained in great detail and every care was taken during my appointments to put me at ease. Any questions were answered and they were always quick to reply to any phone calls/e-mails when I had any concerns, with Bilal even giving me his personal mobile number in case I had any problems out of hours.
After 18 months of treatment my braces were removed and I can honestly say I have never smiled so much. I feel more confident in myself and as an added bonus I have lost weight as well –'the brace diet'.
If you are thinking of having this treatment I cannot recommend iQ Orthodontics highly enough. It is well worth the cost and I won't lie the occasional discomfort to achieve the end result.
.Lastly I actually consider Bilal and Donna to be not only my Orthodontist but friends as well and even once my care treatment is over I hope to keep in touch with them as they are such lovely people
Chris(Sales Manager aged 32), Addlestone, Surrey
Orthodontic fixed braces
Chris lacked confidence when talking to clients and was very embarrased about his smile. We removed 3 of his adult teeth and provided upper and lower fixed braces (train tracks). The treatment lasted 18 months.
I can't stop smiling, my family and friends love the result. Soon after my brace was removed I felt more self assured in my job and this gave me the confidence to smile more with my clients. My career has improved as a result and even I managed to get a promotion at work!
If you would like to add a testimonial to this page, please call 019 3235 2486 or send an email. These testimonials are from NHS Choices. Another way you can review the service you received from us is by leaving feedback on the NHS Outpatients Survey – just click on the link to leave your feedback.
I have no hesitation in strongly recommending this orthodontist. My daughter has just had her top and bottom braces removed. At the beginning the dentist explained clearly what our options were and ensured at every step that we understood fully what was involved and were happy in all respects with the proposed treatment. They are an extremely polite and courteous person to whom my daughter responded well. The dental nurse and reception staff could not have been more helpful in all our dealings with them.
The Practice supported me throughout the process and I am thankful to everyone. The treatment and care I was given was outstanding, and all the information I needed was given and was relevant and helpful. This resulted in a shortened treatment of just under a year rather than the 18 months originally stated.
Excellent Orthodontist and practice staff – I highly recommend. My 12 year old son had braces fitted (top and bottom) by the dentist 15 months ago and during that time has been treated incredibly professionally by the dentist and thier team. The dentist has just had his braces removed and my son has gorgeous, straight teeth. The difference is quite staggering. My son had a very pronounced under-bite and now his bite is perfect! The dentist has a good rapour with the young patients and is kind but firm in what is expected of them – looking after their brace and teeth. Every stage was explained clearly. I would highly recommend the dentist and thier practice.I cannot fault the service we have received
Excellent service from start to finish. They explained everything very clearly to me and my daughter. We knew what to expect, how to care for the teeth at home and approximately how long it would take. Our dentist has a lovely 'chair side' manner, which immediately puts patient and mother at ease. They are very knowledgeable in their profession and my daughters' teeth look amazing. She has not stopped smiling since the brace has been removed. Thank you very much. I would highly recommend them. First class treatment from beginning to end. J.M.
Very pleased with the work done. Very impressed with the result.
The end result of my son's treatment is fantastic, and he now has teeth to be proud of. Thank you! I would thoroughly recommend this practice.
You are outstanding and simply the best. My daughter has had braces for the last year or so. She has just had her top and bottom braces removed. At the outset, the dentist explained clearly what our options were given the circumstances. They ensured at every step that we understood fully what was involved and were happy with the proposed treatment. In fact my daughter's teeth made it difficult and embarrassing for her to smile. She used to but, behind her hands. She was told that she would potentially lose at least a couple of her teeth. They were so bad, sorry. But, the incredible dentist managed to correct them without a single extraction. We thought the treatment would take two years, but it took less than a year. The entire staff are extremely polite and courteous. There is something very contagious about this dentist, they put their patients and parents at ease. The dental nurse and reception staff could not have been more helpful in all our dealings with them. My daughter has a big, big smile on her face and mine too. Outstanding, no words to describe. Thank you indeed.
Excellent orthodontist! Outstanding treatment and care, all of the staff are very friendly and helpful. I would highly recommend this practice.
Excellent orthodontist! So friendly and always willing to explain everything to both the children and the parents. Son's teeth are looking fantastic compared to where they started. Thanks.
We are extremely happy with the results and would recommend this practice. All of the staff are very friendly and helpful, each stage of the treatment was well explained, and great results have been achieved.
Highly professional, effective and extremely caring. My son's orthodontic treatment was a wonderfully resounding success. Right from his initial assessment through to his final retainer fitting we were kept extremely well informed by the doctor and their warm and caring team. P.J.
I would recommend this practice to friends and family. My son had a really good experience having his orthodontic work done. The treatment was completed in a shorter time than first expected. I never had any trouble getting appointments, all the team were friendly and helpful.
I would recommend this to all of my friends who are getting braces. Very impressed, kind and well mannered.
Excellent – I would be happy to recommend this practice. I was kept informed at all stages of my daughter's treatment, the outcome of which has been very successful in a much shorter time than we were originally advised. The dentist and staff were always helpful and polite. Best of all, my daughter is no longer embarrassed to smile properly!
My daughter had a very positive experience with the dentist, they were fantastic at all times as my daughter was very nervous at first, the treatment was much quicker than predicted lasting 12 months instead of 17, the results are fantastic and I would recommend anyone to this Orthodontist.
I have had a very good experience with my treatment and have always felt comfortable and relaxed throughout it. I would advise and recommend anyone to undergo treatment themselves. I am very happy with the outcome of my treatment, and have seen a huge improvement in my teeth. It was definitely worth my time and money. S.K.
The treatment and patient care was outstanding. My daughter's teeth look fantastic now, she has not stopped smiling. The treatment was carefully explained both to me and my daughter. Explicit instructions were given on care with braces and now with the retainers. The end result is wonderful. Nothing was too much trouble. Excellent. Thank you. J.D.
I am writing to express how pleased I have been with the service of IQ orthodontics. My treatment was started under G Spriggs & when he unfortunately retired my treatment was continued by Bilal. The transition was extremely smooth & I was made to feel as if my treatment was important to the practice as the new customers you had.
Time was taken not to rush my treatment, but to ensure that the best possible result was achieved. Questions have been answered to give me clarity of all the different options, as when, they have occurred. Reassurance & care as given when I expressed concern about the final removal of the braces.
Anyone considering having treatment should voice any concerns or questions, no matter how small, directly to Bilal, as he is very approachable & knowing what to expect is so important. I was able to choose retainers which were best for me, although I am sure another person would suit an alternative.
Although it is not cheap to have the treatment & it did take me three years to pluck up the courage & finance to start, I do not regret it for a minute. Having invested the time & money I do not want to lose all the benefits this treatment has given me. So I will be wearing my retainers as instructed by Bilal. I still have support & back up appointments from IQ Orthodontics to keep me on track.
Many thanks for your care, concern & expertise in my treatment. F.F.
Having my braces fitted was surprisingly quick and completely painless. Bilal Qureshi and staff were extremely friendly and supportive and are always there to answer any questions and give advice. I felt entirely comfortable throughout the whole process.
My advice for anyone having braces is to be patient and not to panic. As i said, having the braces fitted doesn't hurt but it can be painful to bite when the teeth start moving. However, the pain goes away within a couple of days and you get completely used to having braces -you hardly even notice they are even there! It is also very important to take extra care brushing your teeth – it all pays off in the end! There is definitely nothing to be nervous about.
I was extremely pleased with the results, not only do they look fantastic they feel great and make you feel much more confident! It's definitely worth investing the time and money as it pays off in the long run. S.B.
The orthodontist and the practice staff have been friendly and professional throughout and it always felt that they were working together to achieve the results I was looking for.
When initially discussing the proposed treatment, time was taken to talk me through the various options, costs and the advantages and disadvantages of each. In the end, I felt the course of action recommended was the most clinically appropriate to my situation even though it wasn't the most expensive.
Committing to having fixed braces as an adult working in a professional environment was not an easy decision to make but iQ were always sensitive to this, for example, allowing me quickly to arrange an appointment to come in and have my white elastics changed when I ate something that turned them bright yellow! I was continually surprised how many people said that they didn't notice my braces unless I mentioned them.
Since having the treatment I am free of the self-consciousness that I've always felt about my teeth. Previously, I would put my hand up to cover my mouth when laughing, and when looking at photographs my eye was always immediately drawn to the gap in my teeth. Now I don't have to feel conscious of this and, while it may seem like a little thing to other people, it makes a huge difference to me.
The treatment in itself was not cheap by its nature (that's not to say that iQ were more expensive than anywhere else – in fact the opposite is true). It would also be disingenuous to say that it wasn't a little uncomfortable at times (though the discomfort was limited to just the day or two after an appointment as the teeth adjusted). Looking back, however, the months have passed quickly and the end result is very much worth the investment of time and money. I would say to anyone considering treatment that, although the prospect may seem unappealing at the outset, it is really only for a relatively short period but the end result is worth it and the benefits should last a lifetime. G.E.
The clear, comfortable and removable way to straighten teeth To receive a Free Consultation, Please call our friendly reception team on
West Byfleet: 019 3235 2486 or Woking: 014 8390 1082
OrBook An Appointment


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The date of payment from buyers deliver within days
Long-term effective
2017-10-29 01:02
Company ProfileShenzhen Better Lighting Co.,Ltd
By certification [File Integrity]
Website:http://www.btledlightings.com/ http://biyite.sztolips.com/
2016 new fluorescent shop light are good office lighting fixtures . It is hot sale hanging lamp. Concise design popular used in modern office suspension pendant lamp fixtures.
Specification of suspension hanging light fixtures
Size:1220x125x70mm (length can be customized)
Color :Silver, Black
Material : Aluminum + Acrylic cover
Power : 2X14W LED (traditional tube T5 2*28W)
Color temperature : warm white 3000K, natural white 4500K, cold white 6500K
Voltage : 110v , 220v, AC85-265v
Lighting space : 8-10 square meters
fluorescent shop light hanging lamp fixtures application :
Widely used in Study, office buildings pendant ceiling lights, teaching buildings, libraries, airports, offices office lighting, commercial shops modern ceiling lights, stores, fine homes, hotels commercial lighting , museums, art centers
Feature of led suspension light fixture
1. LED pendant light with IP64 protection level.
2. Extrusion aluminum heat sink, perfect and reliable thermal system.
3. Patent and functional design, with imaginative appearance, visual texture and impressive uniformity.
4. Support for pendant, ceiling, wall mounting solutions. More flexible, and easy for installation.
Better Lighting is one of the leading China hanging office light fixtures 2 t5 led tube pendant light manufacturers. Welcome to wholesale products from us.


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This MFC's research paper considers the (mis)match between the financing needs of European MSMEs and the perception of availability of external financing. Publisher: Microfinance Centre Authors: Justyna Pytkowska, Piotr Koryński Year: 2018 Download "Between Needs and Availability: The External Financing Gap Indicator" Our question is: how do changes in the perceived need for external financing (and its availability) line up with the patterns ...Read More
Blog Archives
EIF Working Papers 2018/46-47: EIF SME Access to Finance Index and European Small Business Finance Outlook
EIF Working Paper 2018/47 – EIF SME Access to Finance Index This Working Paper outlines the methodology underlying the new EIF SME Access to Finance (ESAF) Index, which aims oat monitoring developments in SMEs' access to financial resources in the EU. The index, which is newly developed and will be periodically updated, fills a gap since such an approach did ...Read More
Financing Gap and SME Employment Growth: Beyond Access to Finance
The key aim of these MFC research paper "Financing Gap and SME Employment Growth: Beyond Access to Finance" was to investigate the relation between the perceived changes in financing gap and employment growth by SMEs in the EU. The paper builds on and extends the analysis of SAFE survey data, in order to focus on the question of employment creation which ...Read More


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Returning Customer
Hello, welcome back to your account.
New Customer
Pastry Chef's Boutique supplies a full line of professional artisanal bread making products and accessories from bread molds, stencils, rings, and displays to fermentation cloths, pate molds, bakers blades, bannetons and proofing baskets, and so much more
Bannetons and Proofing Baskets
Fermentation Clothes
Baker's Blade
Bread Ring
Silform Bread Molds
These SIlform® non-stick mats have been especially developed for bread and are ideal for a variety of small bread shapes including: round breads, small rounds, hot dog rolls, soft rolls, submarines, hamburger buns, sandwiches, rolls and more! The perforated texture of Silform® is designed to provide optimal heat transfer which results in the perfect crunch and color every time!
Pate Mold
Baguette and Tuiles Pan
Panettone Baking
Bread Display
At Vero Accusams Etiusto Odio.


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Brazil: City mayor arrested for corruption
The mayor of the Brazilian city of Presidente Kennedy (named after JFK) was arrested Thursday for corruption in a Federal Police operation ironically dubbed "Lee Harvey Oswald." The city was named after U.S. President John F. Kennedy, in a tribute to his persona. The city received donations from his Alliance for Progress plan. Reginaldo Quinta was detained along with 27 other people on charges they conspired to embezzle 50 million reais ($26.6 million) in public funds, the Federal Police said in a statement.
The original article can be found at ldcfitzgerald.com.


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Archive Productions Inc. Archive Productions Inc.
Here's a sample of our volunteer opportunities:VIEW AS
Please wait . . .
Mission Statement
1) Archive our history through videos of events, interviews, and documentaries; 2) Promote other nonprofits, the arts, and social issues by providing free video services; 3) Train film makers and videographers.
Archive Productions Inc. was created over ten years ago and incorporated as a nonrpfoti in 2009. Our 501(c)(3) status was updated 2/11/16. We are an all volunteer organization in the San Francisco Bay Area, composed of retired seniors and film students from the local colleges (SFSU, CCSF, AI, Academy of Art, UC Berkeley, etc).
We are the official videographers for over sixty Bay Area Nonprofits. We neither charge for nor sell our nonprofit work. We also video tape senior interviews and documentaries.
Would you recommend Archive Productions Inc.?


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A subsistence farm used RDP support in order to become commercially viable by investing in organic production.
Projects & Practice
Building a reservoir to catch runoff water and provide protection against floods in a vulnerable area.
An agricultural cooperative invested in setting up organic apple orchards with anti-hail nets. They also acquired agricultural machinery to support the production of organic beef.
The future-proof peat meadow polder 'Lange Weide', is unique bottom-up initiative and the largest submerged drainage project in the Netherlands.
Adjustments to water management and remedial measures help to preserve protected areas and conserve the landscape of the Western Langstraat.
Ensuring the preservation and restoration of species and habitats in Saxony, by offering free advice and relevant qualifications to land users.
An educational institution for farmers in Austria used RDP support to develop a training programme using digital, web-based technologies and applications.
Amstelland Natuurzuivel is a cooperative of farmers in the Amstelland region who set up a milk factory to produce regional dairy products for the Amsterdam area.
Expanding a water reservoir and installing a solar farm to reduce the energy costs of irrigation.
On-farm processing can add value to the product and can also make farming a more attractive choice for young farmers - as demonstrated by a berry farm in Finland.


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Some call then extras, sparkle, zest. We call them "garnishes" Just a fun little accent to add zip to your pieces. These are best used in wall hangings and pillows, small things, not your rugs on the floor. Sari Ribbon is made from fine, lightweight silk fabrics. Use it for fringing, embellishing, surface design, quilting, scrap-booking, mixed media art, etc.
Approx 10mm to 20mm wide. Material: silk, rayon, cotton, metal threads. Each bundle has approximately 12-15 pieces 30 inches long or about 12 yards total.