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TGF-β is a pluripotent cytokine that mediates its effects through a receptor composed of TGF-β receptor type II (TGFBR2) and type I (TGFBR1). The TGF-β receptor can regulate Smad and nonSmad signaling pathways, which then ultimately dictate TGF-β's biological effects. We postulated that control of the level of TGFBR2 is a mechanism for regulating the specificity of TGF-β signaling pathway activation and TGF-β's biological effects. We used a precisely regulatable TGFBR2 expression system to assess the effects of TGFBR2 expression levels on signaling and TGF-β mediated apoptosis. We found Smad signaling and MAPK-ERK signaling activation levels correlate directly with TGFBR2 expression levels. Furthermore, p21 levels and TGF-β induced apoptosis appear to depend on relatively high TGFBR2 expression and on the activation of the MAPK-ERK and SMAD pathways. Thus, control of TGFBR2 expression and the differential activation of TGF-β signaling pathways appears to be a mechanism for regulating the specificity of the biological effects of TGF-β.
Transforming growth factor β (TGF-β) is a secreted multifunctional cytokine that regulates a variety of cellular processes including growth inhibition secondary to G1/S phase arrest, differentiation, apoptosis, immunosuppression, stimulation of connective tissue deposition, secondary induction of angiogenesis, and probably maintenance of genomic stability. TGF-β induces these effects by activating the TGF-β receptor, a heterodimeric complex composed of type I (TGFBR1) and type II (TGFBR2) subunits, which are both serine-threonine kinases with single transmembrane domains [1]. Both TGFBRI and TGFBR2 appear to be essential for TGF-β induced effects on the cell [2, 3]. The TGF-β receptor is activated through a sequence of events that is initiated by the TGFBR2 subunit binding to the activated TGF-β ligand. The activated TGFBR2 associates with TGFBRI, and phosphorylates a glycine-serine rich sequence in TGFBRI. TGFBRI is then activated and can phosphorylate downstream targets [4]. The TGF-β receptor complex activates both the Smad signaling pathway, which includes Smad2, Smad3, and Smad4, and the nonSmad signaling cascades (e.g. PI3K/AKT, p38MAPK, MAPK-ERK, JNK, etc.) to produce the full spectrum of TGF-β responses [5-7].
With regard to the nonSmad signaling pathway, TGF-β can activate a variety of nonSmad signaling pathways, including the MAPK, JNK, p38MAPK, NFκB, RhoA, and PI3K pathways. TGF-β can regulate the nonSmad signaling pathways both directly and indirectly. For instance, direct physical interactions between the TGF-β receptor and TAB1 (TAK1-binding protein 1), which can regulate TAK1 (TGF-β Activated Kinase 1) and p38MAPK, and PI3K/AKT, have been shown [8]. Other nonSmad signaling pathways are also clearly TGF-β responsive but, in most cases, it has not been shown whether the effect is a direct or indirect effect of TGF-β. The nonSmad signaling pathways have been implicated in regulating TGF-β mediated apoptosis [8, 9], extracellular matrix production [10-12], and differentiation [11, 13].
The Smad signaling pathway is more completely understood than the nonSmad signaling pathways that are activated by the ligand bound TGF-β receptor. Ligand binding to the TGF-β receptor complex results in TGFBRI-mediated phosphorylation of Smad2 and Smad3 on two serine residues in a conserved Ser-Ser-X-Ser motif located at the C-terminus of the R-Smads [14, 15]. Phosphorylation of these serine residues activates the signaling pathway and is required for the function of Smads in transcription factor complexes [16, 17]. The phosphorylated Smad2 and Smad3 bind to Smad4, and the complex can then translocate into the nucleus to function as a transcription factor complex. The details of this pathway have been the subject of a recent review (see [18]).
Although initially perceived as a binary (i.e. "on" or "off") and linear mechanism, it is now clear that the TGF-β signaling pathways' effects on the biological responses of a cell depend on the intensity and duration of the activation of the pathways [19]. Indeed, a substantial number of studies have demonstrated that the TGF-β signaling pathways are regulated by a variety of mechanisms that control the cellular localization, phosphorylation state, and expression levels of the post-receptor signaling elements [19, 20]. Moreover, these studies have provided a plausible explanation for why the cell state and cell type, referred to as the "cellular context" can have a profound effect on the way in which TGF-β affects a cell [21-23]. Recent data has shown that the degree of activation of the TGF-β signaling pathways is subject to regulation by a large number of intracellular and extracellular agonists and antagonists, including decorin, Smad7, Smurf, SARA, etc [24, 25].
The discovery of these regulatory mechanisms demonstrates that not only is the absolute activation state of the pathway important for determining TGF-β's effects on cells but that the degree of pathway activation and the duration of pathway activation is also likely important. In fact, it is plausible that the paradoxical effects of TGF-β on tumor cells could be at least partly explained through differences in the intensity of activation of the pathways or by differential activation of Smad vs. nonSmad signaling pathways. Regulation of the expression levels of TGFBR1 and TGFBR2 may be a mechanism through which the activation state of the TGF-β signaling pathways can be regulated [8]. Consistent with this model is the recent demonstration that levels of TGFBR1 have been shown to correlate with an increased risk of colorectal cancer [26].
In order to assess whether the regulation of TGFBR2 expression could be a mechanism for determining the response of cells to TGF-β, we generated a system using the RheoSwitch® inducible gene expression system to regulate the expression of TGFBR2. We have found that the level of TGFBR2 affects the ability of TGF-β to induce p21 and apoptosis in the V-400 colorectal cancer cell line. In addition, TGF-β's ability to activate the MAPK pathway is directly dependent on the expression level of TGFBR2. These results demonstrate that the regulation of the receptor level for TGF-β is a potential mechanism for determining the specificity of the response of a cell to TGF-β.
V-400 is a microsatellite stable (MSS), TGF-β resistant colon cancer cell line, which was kindly provided by James K.V. Willson (UT Southwestern Medical School, Dallas, TX). It carries biallelic missense TGFBR2 mutations that inactivate the endogenous receptor, and it has an intact Smad signaling pathway [27]. The V-400 cell line was grown in Dulbecco's Modified Eagle Medium (Gibco, Grand Island, NY). The media was supplemented with 10% FBS (Cambrex, East Rutherford, NJ). The MEK1/2 inhibitor U0126 (cat# 662005), and the PIK3 inhibitor LY294002 (cat# 440204) were obtained from Calbiochem (San Diego, CA). The Smad7 plasmid was kindly provided by Neil Bhowmick (Vanderbilt University, Nashville, TN).
The inducible system used in this project was developed by RheoGene, Inc. (Norristown, PA), now Intrexon Corporation (Blacksburg, VA), and is a highly engineered system based on a modified version of the ecdysone receptor (EcR) [28, 29]. Important modifications of this system include mutations in the ecdysone receptor to increase potency to synthetic diacylhydrazine agonists; trans-positioning of the VP16 activation domain and the Gal4 DNA binding domain as fusions to the RXR and EcR components, respectively; and development of potent and bioavailable synthetic agonists. Two agonists, RG-102240 and RG-115819, were used in these studies. They are closely related diacylhydrazine analogs, with the latter being slightly more potent. These modifications significantly improve the precision of the transgene expression regulation by the synthetic agonist.
A single construct carrying both the RheoSwitch® receptor genes and an HA-tagged TGFBR2 transgene was made. The TGFBR2 transgene was inserted into the cloning site present in the lentiviral vector p5004, provided by RheoGene. (Figure 1) The TGFBR2 transgene was obtained from the vector pRC/CMV kindly provided by Dr. Sandford Markowitz (Case Western Reserve University, Cleveland, OH) [27]. The TGFBR2 gene was released from its original backbone using the enzymes NotI and EcoRI and then treated with Klenow. After cloning the TGFBR2 transgene into the p5004 vector, plasmids were screened for inserts with the correct orientation and were confirmed to carry a wild-type TGFBR2 transgene by sequencing
A lentivirus carrying the TGFBR2 transgene was produced in the Gene Therapy core lab at the Fred Hutchinson Cancer Research Center using a 293T-based packaging cell line. V-400 cells were infected with 0.5 ml of viral supernatant. The cells were incubated for 24 hours with the viral supernatant in presence of polybrene. The viral supernatant was then removed and Blasticidin (6 μg/ml) was added into the complete media (DMEM in presence of 10% FBS). The transduced cells were then subjected to cloning by limiting dilution. An evaluation to identify clones that displayed minimal expression in the baseline state but showed adequate induction of TGFBR2 after treatment with RG-115819 was performed in different clonal populations. The control cells were treated with DMSO vehicle alone.
In order to evaluate TGF-β-mediated transcription, the cell lines were transiently transfected with the p3TP-lux reporter (kindly provided by Joan Massagué, Memorial Sloan-Kettering Cancer Center, New York, NY) or with the CAGA reporter assay (kindly provided by Bert Vogelstein, Johns Hopkins University, Baltimore, MA) concomitantly with the pRL-TK reporter construct (Promega, Madison, WI). The cells were treated with TGF-β1 (10ng/ml), and luciferase activity was evaluated 48h after transfection using the Dual Luciferase Reporter Assay System (Promega, Madison, WI) with a Veritas luminometer (Turner Biosystems, Sunnyvale, CA). The p21 expression was evaluated using a luciferase reporter assay (kindly provided by Dr. Xiao-Fan Wang's Laboratory) [30]. The luciferase activity was evaluated 24 and 48 hr post-transfection as noted above. All the transfections were performed using FuGENE 6 (Roche, Basel, Switzerland) following the protocol provided by the manufacturer. A 3:1 FuGENE-DNA ratio was used.
V-400R2 and V-400 cells treated for 48 hours with RG-115819 and TGF-β1 were used for these studies. The cells were collected after being briefly treated with trypsin (0.125%) to detach them from the flasks, washed in media with 20% FBS (4°C) followed by 1X PBS (4°C), and then fixed in 3% formaldehyde for 15 minutes. The cells were then incubated with 10% normal horse serum for 30 minutes, washed with PBS 3 times, and incubated with an anti-HA antibody (1:100, Cat # 2367 Cell Signaling, Danvers, MA), followed by incubation with a goat-anti-mouse secondary antibody labeled with fluorescein (FITC) (1:100, Cat#:115-095-146, Jackson ImmunoResearch, West Grove, PA). The cells were then washed with PBS (3 times) and subjected to FACS analysis using FACScan (Becton Dickenson, Franklin Lakes, NJ). The results were analyzed using CellQuest software (Becton Dickenson, Franklin Lakes, NJ).
Cell lysates were prepared using RIPA buffer supplemented with a complete protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich, St. Louis, MO). The lysates were then used for SDS-PAGE (10% polyacrylamide gels and PVDF membranes, Pierce, Rockford, IL). Immunoblots were performed following the manufacturer's recommendations. The following antibodies were used: pERK1/2 (Cat # 9101 Cell Signaling, Danvers, MA 01923), ERK1/2 (Cat # 9102 Cell Signaling, Danvers, MA 01923), p21(Cat # OP68 EMD San Diego California), anti-TGFBR1 ((H-100 antibody; sc-904, 1;200), HRP-goat anti-mouse (Cat # SC 2031) and donkey anti-rabbit (Cat # SC 2317) (Santa Cruz Biotechnology). A commercial ECL kit (RPN 3004, GE Healthcare, Piscataway, NJ) was used to detect the HRP-labeled secondary antibodies and the signal was detected using autoradiography. Reagents used in conjunction with this kit included n-butyric acid sodium salt (Cat# B-5887), Luminol (Cat# A8511), and p-coumaric acid (Cat# C9008) (Sigma-Aldrich, St. Louis, MO).
Phosphorylated AKT was detected using a commercially available ELISA assay (SuperArray, SABiosciences, Frederick, MD). Ten thousand cells/well were seeded in a 96 well plate, and were treated with different concentrations of RG-115819 in order to differentially induce the expression of the TGFBR2 transgene. TGF-β (10 ng/ml) was then added to the cells and the cells were harvested after 48 hours. The ELISA was performed following the manufacturer's recommendations (cat # FE-001 SuperArray), and read in a plate reader at 450 nm (VERSAmax, Molecular Devices, Sunnyvale, CA).
One μg of RNA extracted from V-400 and V-400R2, was reverse transcribed using oligo d(T) priming and Superscript-II reverse transcriptase (Invitrogen) and 2 nM dNTPs following the manufacturer's protocol. TaqMan On-Demand primers and probes were used to determine the relative expression levels of CDKN1A/p21 (Assay number Hs99999142_m1, Applied Biosystems, Foster City, CA) and 18S in all samples (Assay number Hs99999901_s1, Applied Biosystems,). The reactions were run in triplicate in the ABI Prism 7700 detection system (Applied Biosystems) and results were analyzed with SDS 2.1 software.
Apoptosis was measured with two different assays: the Cell Death ELISA assay (Roche, Cat# 11 774 425 001), which detects histone-complexed DNA fragments, following the manufacturer's protocol, and the Caspase-Glo Luciferase-based reporter assay, which measures caspase 3 and 7 activity (Promega, Catalog number TB323) following the manufacturer's protocol.
In order to assess the effect of the expression level of TGFBR2 on the activation of TGF-β signaling pathways, we employed the RheoSwitch® gene regulation system that can precisely regulate the transgene of interest [28, 29, 31]. An HA-tagged TGFBR2 transgene was cloned into a lentiviral expression vector and used to generate lentivirus for transducing the target cell line, V-400. This cell line lacks a functional TGF-β receptor and becomes TGF-β responsive when it is reconstituted with TGFBR2 [27]. (See Materials and Methods section for information on the plasmids used.) The V-400 cells were transduced with TGFBR2 and clones expressing the RheoSwitch® receptor and TGFBR2 transgene were selected and then assessed for TGF-β responsiveness using the 3TP-lux or CAGA reporters. Two clones displayed a linear dose response of 3TP-Lux activity in relation to RG-102240 concentration. (Figure 2)
The expression levels of the TGFBR2 transgene were then assessed by FACS analysis in order to determine the amount of TGFBR2 expressed on the surface of the transduced cells. (Immunoblotting of total protein lysates from the cells for the HA-tagged TGFBR2 transgene confirmed the results of the FACS analysis but does not demonstrate the same linear response to the RG-115819 because of the lower precision of immunoblotting to detect small differences in protein expression. (Figure 3A-B). Clones 1 and 2 showed a linear increase in 3TP-Lux induction and TGFBR2 transgene expression after treatment with increasing concentrations of RG-115819. (Figure 3A) A linear dose response for TGFBR2 expression between 0.04 μM to 1 μM RG-115819 was demonstrated, and thus this range of RG-115819 was used for the subsequent experiments. We did not observe any change in TGFBR1 expression with increasing concentrations of RG-115819, which led us to interpret that any RG-115819 dependent phenomenon were strictly the result of changes in the expression level of TGFBR2. (Figure 3C)
TGF-β can regulate the expression of a variety of genes that control a myriad of cell responses including proliferation, apoptosis, differentiation, etc [30, 32, 33]. A significant question that arises is how the specificity of the response is determined. One of the TGF-β regulated genes that highlights the issues related to control of the specificity of the TGF-β response is the CDK inhibitor CDKN1A/p21, which plays an important role in several cellular processes including apoptosis, cell cycle arrest, induction of cell differentiation, and cellular senescence [32, 34]. Thus, we assessed the regulation of CDKN1A/p21 expression in the setting of different levels of TGFBR2 expression to determine if the regulation of the expression of the TGF-β receptor may be a mechanism through which TGF-β mediated responses are regulated. In order to determine how changes in the expression levels of TGFBR2 affect the expression of CDKN1A/p21, V-400R2 cells were treated for 24 and 48 hours with four different concentrations of RG-115819 (0.04, 0.08, 0.4 and 1 μM) and TGF-β (10ng/ml). The induction of CDKN1A/p21 expression was assessed using a CDKN1A/p21 luciferase reporter [35]. CDKN1A/p21 reporter activity was increased at 24 hours but no correlation between increasing TGFBR2 expression and reporter activity was observed at that time point. At 48 hours, a direct relation between TGFBR2 expression level and CDKN1A/p21 reporter activity was apparent (Figure 4A). Importantly, no basal CDKN1A/p21 reporter luciferase activity was detected in the V-400R2 without RG-115819 or in the parental cell line V-400. (Figure 4A) CDKN1A/p21 protein expression also correlated with the CDKN1A/p21 luciferase reporter activity. (Figure 4B) Localization of p21 was also assessed in light of the possibility that MAPK signaling could regulate p21 nuclear localization in these cells. We observed increased nuclear p21 in association with increased p21 expression induced by increased TGFBR2 expression. These results suggest that the increased p21 is being retained in the nucleus despite the presence of increased activated ERK and that p21 is acting to affect the behavior of the cell through actions on gene transcription (Supplemental Figure 1).
The expression of CDKN1A/p21 is regulated by several signaling pathways and transcription factors, including the Smad signaling pathway, ERK signaling pathway, myc, etc. [23, 36-38]. In light of the differing effect of low and high levels of TGFBR2 expression on CDKN1A/p21 expression, we assessed the effect of different levels of TGFBR2 expression on the activation status of Smad and nonSmad signaling pathways. V-400R2 cells were treated with RG-115819 (0.4-1.0μM) and assessed for MAPK-ERK activation at 24 and 48 hours. ERK activation was not present at 24 hours but was present at 48 hours after treatment with RG-115819 and TGF-β. (Figure 5A) The level of phosphorylated ERK directly correlated with the expression level of TGFBR2 as well as with CDKN1A/p21 expression (Figure 5A). Of interest, we also assessed the effect of TGFBR2 expression on PI3K pathway activation and found that phosphorylated AKT is present in the parental cell line and that TGF-β does not induce an increase in phosphorylated AKT in the TGFBR2 reconstituted cell line at any level of TGFBR2 expression (data not shown). After assessing the effect of TGFBR2 on the MAPK-ERK pathway, we next assessed the activation state of the Smad signaling pathway. Unlike with the MAPK-ERK pathway, Smad2 phosphorylation was increased with moderate to high levels of TGFBR2 but not with low expression levels of the receptor. There was also a modest increase in phosphorylated Smad2 with increasing levels of TGFBR2 expression, but the range of increased phosphorylated Smad2 was more limited than that for phosphorylated ERK1/2. (Figure 5B) The functional effect of the increase in phosphorylated Smad2 can be observed by a linear increased in CAGA luciferase reporter activity, which is specific for Smad mediated transcription. (Figure 8)
It has been previously shown in other cell line systems that the MAPK-ERK pathway can control the expression of CDKN1A/p21 and that TGF-β can regulate CDKN1A/p21 through the MEK pathway [30, 39, 40]. Consequently we assessed the effect of inhibition of ERK activation on the ability of TGF-β to induce CDKN1A/p21 expression in the V-400R2 cell line in order to confirm that this was also true in this colorectal cancer cell line. TGF-β mediated activation of ERK was inhibited with the selective MAPK inhibitor U0126 (5μM) in V-400R2 cells treated with RG-115819 (0.04-1.0 μM) and TGF-β (10 ng/ml). The induction of TGFBR2 expression induced the expression of CDKN1A/p21, but blockade of the MAPK-ERK pathway prevented the differential increase in CDKN1A/p21 expression observed in the prior studies. These results suggest that CDKN1A/p21 expression is regulated by both MAPK-ERK and nonMAPK-ERK signaling pathways. (Figure 6A) We next assessed the effect of TGF-β mediated Smad pathway activation on the expression of CDKN1A/p21 using the inhibitory Smad, Smad7. Transfection of the V-400R2 cells with SMAD7 almost completely blocked the induction of the CAGA luciferase reporter and modestly suppressed the induction of CDKN1A/p21 reporter activity, but did not prevent the linear increase in CDKN1A/p21 expression observed with increasing TGFBR2 expression indicating the TGF-β mediated regulation of the precise expression levels of CDKN1A/p21 in V-400R2 is regulated predominantly by nonSmad signaling. (Figure 6B-C)
In light of our results showing that TGFBR2 expression directly correlates with increased ERK activation and CDKN1A/p21 expression, we carried out a series of studies to determine whether the expression level of TGFBR2 can directly affect the ability of TGF-β to induce apoptosis. The V-400R2 cell line was treated with RG-115819 in order to induce low and high levels of TGFBR2 and then assessed for TGF-β mediated apoptosis. A direct relationship between TGFBR2 expression and apoptosis was demonstrated showing that high levels of TGFBR2 are permissive for TGF-β mediated apoptosis. (Figure 7) These results suggest that TGF-β can mediate apoptosis in the V-400R2 cell line through the induction of the MAPK-ERK pathway and subsequent increase in CDKN1A/p21 expression. Thus, TGFBR2 levels may be a mechanism for determining whether cells are sensitive or resistant to TGF-β mediated apoptosis.
In order to establish if the induction of increased CDKN1A/p21 resulting from the increased TGFBR2 expression and ERK activation is a central mechanism for TGF-β mediated apoptosis in the V-400R2 cells, CDKN1A/p21 expression was inhibited with p21-siRNA in the presence of low and high levels or TGFBR2 and TGF-β (10ng/ml). (Figure 8A) Interestingly we found that inhibition of CDKN1A/p21 prevented the increase in apoptosis observed with increased TGFBR2 levels providing support for CDKN1A/p21 regulation being a mechanism for TGF-β mediated apoptosis in colon cancer. (Figure 8B) However, we also observed that inhibition of Smad signaling with SMAD7 impaired TGF-β mediated apoptosis presumably through a p21 independent mechanism, given that we did not observe an effect of SMAD7 on p21 expression. These results demonstrate TGFBR2 can regulate apoptosis through both p21 dependent and independent mechanisms. (Figure 8C).
TGF-β can induce a variety of biological responses in cells, and these responses appear to be dependent on the cell type and context of the cell [41]. Some of the mechanisms that appear to play a role in determining the specificity of the response of a cell to TGF-β include signal pathway cross-talk, regulation of the expression levels of cytoplasmic and nuclear antagonists, and control of the composition of transcription factor complexes [20, 33]. In addition, over a decade ago, Derynck and Feng proposed the possibility of a receptor threshold model for determining the specificity of TGF-β's effects. This model proposes that there is a critical expression level of the TGF-β receptor that determines the specific TGF-β responses of a cell [42, 43]. Several lines of indirect evidence have supported this model, but to date there has been no direct assessment of this hypothesis [8, 43-47]. We now provide direct evidence that the regulation of the expression level of TGFBR2 can affect the specificity of the TGF-β response. We have shown that the activation of the Smad and nonSmad signaling pathways can be modulated by the expression level of TGFBR2 and that the activation state of the nonSmad signaling pathway principally determines whether TGF-β can induce CDKN1A/p21 mediated apoptosis in an epithelial cell line.
TGF-β and the TGF-β receptors are expressed in nearly all cell types and in developing and adult organisms. In embryonic development there is clear evidence that the concentration of active TGF-β and the extent of TGF-β signal pathway activation is tightly regulated and creates a gradient of responses that determines cell fate, among other things [41]. The majority of these studies have provided indirect and correlative evidence that the concentration of the ligands, receptors, and intracellular Smad proteins can determine the specific response of a cell to TGF-β. Further indirect evidence that the expression level can affect the specificity of the TGF-β response comes from studies of dominant negative TGFBR2 transgenes and chimeric receptors [22, 24, 48-50]. Our studies now provide direct evidence that the TGFBR2 expression level can regulate the intensity of activation of the Smad and nonSmad signaling pathways. Furthermore, the differences in activation of the Smad and MAPK-ERK pathways at different TGFBR2 expression levels suggests that not only can the level of TGFBR2 determine the intensity of pathway activation but also which pathways are activated [33]. The differential regulation of the post-TGF-β receptor pathways would be expected to lead to differences in transcription factor activation and transcription factor complex formation, which could then induce different patterns of gene expression.
Although the model above predicts that TGFBR2 expression levels will cause the differential expression of a variety of genes, we chose to specifically study CDKN1A/p21 because of its well-demonstrated regulation by TGF-β and because of its clear biological role in the inhibition of proliferation and induction of apoptosis. In addition, CDKN1A/p21 is regulated by TGF-β both by Smad and nonSmad signaling pathways and thus allows an assessment of the effect of Smad and nonSmad signaling on gene regulation [10, 51]. Our data suggest that CDKN1A/p21 is regulated by both the MAPK-ERK and Smad signaling pathways, which is true of other genes, such as FURIN [32]. These pathways may crosstalk at the level of Smad phosphorylation, nuclear localization of Smads, or at the level of transcription factor complex formation [33, 52, 53]. The differential activation of MAPK-ERK and Smad pathways suggests that at low levels of TGF-β receptor activation the nonSmad signaling pathways predominate over the Smad signaling pathways in determining the effect of TGF-β on the cell. The differences in activation of the MAPK and Smad pathways likely reflects the fact that the MAPK signaling pathway is a catalytic pathway whereas the Smad pathway is non-catalytic [8]. An interesting observation related to this explanation for the differential activation of the Smad and nonSmad pathways is the fact that in V-400R2 the MAPK-ERK pathway appears to be the predominant pathway regulating the level of CDKN1A/p21 expression whereas the Smad pathway has a more modest effect. These results are consistent with those of Hu et al who observed a similar phenomenon in the HaCaT keratinocyte cell line, but are in contrast to other studies that have shown that both Smad and SP1 mediated signaling are necessary for CDKN1A/p21 expression [20, 30, 54, 55]. These differences may reflect cell line specific differences in the expression of other regulators of CDKN1A/p21, such as c-Myc or MIZ-1 [56, 57].
There are several limitations related to our studies of the effect of TGFBR2 expression levels on TGF-β signaling. At this time, it is not clear whether the effects we have observed involve direct or indirect mechanisms for regulating the Smad and nonSmad signaling pathways. This limitation reflects our incomplete understanding of the mechanisms through which TGF-β affects the nonSmad signaling pathways [20]. In addition, we have not assessed the effect of TGFBR2 expression levels on the regulation of other signaling pathways and cannot exclude the possibility that these pathways are also playing a role in the regulation of CDKN1A/p21 and apoptosis that is affected by the expression level of the TGF-β receptor. Nonetheless, our results demonstrate that TGF-β receptor levels can regulate MAPK-ERK activation and that the control of this pathway has a central role in regulating CDKN1A/p21 expression and CDKN1A/p21 mediated apoptosis in the V-400R2 cell line.
Our results have several implications for the role of TGF-β in regulating normal cellular responses and for the role of TGF-β in cancer. The relevance of the effect of TGFBR2 expression levels on the regulation of TGF-β's effects on cells is appreciated in light of prior studies that have shown decreased TGFBR2 expression in a variety of cancers and in some inflammatory states [58]. In fact, many cancers have suppressed TGFBR2 expression without detectable mutations in any of the TGF-β signal pathway genes [59] [60-62]. In these cancers with an intact TGF-β receptor that is expressed at a low level, TGF-β appears to have the potential to act as an oncogene as well as a tumor suppressor gene [63]. This paradoxical role of TGF-β has been associated with its ability to activate Smad independent pathways (MAPK, PI3K and Rho) and with the modulation of TGF-β signaling pathway activation through interactions with other signaling pathways induced by mutated oncogenes and tumor suppressor genes [8, 18, 21, 22]. Our results suggest that an additional mechanism by which TGF-β may mediate paradoxical effects on cells is through the down-regulation of the expression of TGFBR2, which could differentially activate signaling pathways and alter the gene expression patterns of the cells. Thus, our results not only demonstrate that the expression level of TGFBR2 can influence the pathway activation status of TGF-β signaling pathways and the specific response of cells to TGF-β, but also that the regulation of the expression level is a plausible mechanism for the paradoxical effects of TGF-β on cancer cells. In summary, regulation of TGFBR2 expression levels is an additional mechanism for controlling the specificity of TGF-β's effects on cells and is a potential mechanism for the paradoxical effects of TGF-β observed in cancer.
Supplemental Figure 1: Immunostaining for p21 in the V-400R2 cell line. The V-400R2 cell line was grown with increasing concentrations of RG-115819 and then immunostained with an anti-p21 antibody (OP#68, EMD) and detected with a FITC-tagged secondary antibody (SC2012 in a 1:1000 dilution). Predominantly cytoplasmic p21 is observed in the cells treated with 0 or 0.5 μM RG-115819 whereas nuclear p21 can be seen in the cells treated with 1μM RG-115819.
These studies were conducted as follows. The V-400R2 cells were seeded in a chambered slide and grown for 48 hr with RG-115819 and TGF-β (10ng/ml). Then cells were fixed in −10aC methanol for 5 minutes and washed 3 times with 1X PBS. The slides were blocked with 10% normal blocking serum diluted in PBS (20 minute incubation at room temperature) and the washed with 1X PBS. The slides were then incubated with the primary antibody for 60 minutes at room temperature, washed in 1XPBS (three times), incubated in the secondary antibody (30 minutes at room temperature), and washed in 1X PBS (three times). The slides were then subjected to fluorescence microscopy.
This work was supported by RO1 CA115513, VA Dept. of R&D Presidential Early Career Award for Scientists and Engineers, and Mallinckrodt Scholar Award from the Edward Mallinckrodt Jr Foundation (to WMG).
We wish to acknowledge Patty Trobridge for her technical assistance with immunoblotting studies.
Dr's Dean Cress and Malla Padidam performed this work while employees of RheoGene, which was acquired by Intrexon Corporation. Dr. Cress is an employee of Intrexon. Dr. William Grady and Dr. Andres Rojas have no conflicts of interest to report.
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Last week, we posted about Jet Airways' new Disney-themed aircraft:
The aircraft, VT-JBC, is a 5 year old 737-800.
This promotional livery looks much better than the disastrous Nokia Lumia livery which Jet tried out a few months ago. Daisy, Goofy, Mickey Mouse, Minnie Mouse, Donald Duck, and Pluto grace the aircraft, which was used today to take 37 families to Hong Kong's Disneyland.
Here is a time-lapse video of the livery being painted on:

subset: commoncrawl


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CHICAGO, IL--(Marketwired - July 31, 2014) - As part of its commitment to help physicians navigate evolving issues in medicine, the American Medical Association (AMA) today released a new resource to help physicians negotiate business and legal issues common to group practice employment contracts.
The Annotated Model Physician-Group Practice Employment Agreement is intended to meet increased physician interest in employed positions. The handbook offers prospective employees and practice owners a valuable primer when preparing to negotiate an employment agreement, including thorough descriptions of common contract terms, in-depth explanations of business and legal consequences, negotiation tips and sample contract provisions.
"Physicians often need to navigate complex issues when negotiating agreements with their current or prospective employer and this new AMA resource will ensure physician interests can be fully taken into account in these discussions," said AMA President Robert M. Wah, M.D. "By helping physicians successfully navigate this choice, the AMA hopes to establish sustainable medical practices that result in optimal health outcomes for patients and greater professional satisfaction for physicians."
Physician interest in employed positions is expected to continue with more physicians entering into employment and other contractual relationships with group practices, hospitals, and health systems. According to a recent AMA study, nearly 42 percent of patient care physicians are in employed positions.
Additional AMA resources on employment and contractual relationships include:
- Physician Employment Resources Website - An online repository for a variety of resources to help meet the unique needs of a growing population of employed physicians.
- Annotated Model Physician-Hospital Employment Agreement - An educational primer addressing the specific needs of physicians who are preparing to negotiate employment contracts with hospitals.
- Principles for Physician Employment - A framework to help guide physicians and their employers as they collaborate on providing safe, high-quality and cost-effective patient care.
AMA members can obtain the new handbook on group practice employment contracts free of charge. Non-members may purchase the handbook through the AMA Store.
About the AMA
The American Medical Association is the premier national organization dedicated to empowering the nation's physicians to continually provide safer, higher quality, and more efficient care to patients and communities. For more than 165 years the AMA has been unwavering in its commitment to using its unique position and knowledge to shape a healthier future for America.


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Different interpretations
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Consider the following essay topic on the ethics of abortion. Then look at interpretations of the topic made by two students (A and B).
Whose interpretation, in your view, is more adequate?
Essay topic
Is the right to choose abortion just the right to choose what happens in and to one's own body - as Judith Jarvis Thomson suggests - or is it the right to choose the death of the foetus? Discuss with reference to the unconscious violinist.
Student A's interpretation:
This question is asking about whether abortion is justified or not. In my essay I would consider the arguments on both sides of the debate - the right-to-lifers and the pro-choice lobby - and then present my opinion. I believe that women should have the right to choose. I would also mention the unconscious violinist by Judith Jarvis Thomson.
Student B's interpretation:
This question is concerned with a specific issue in the abortion debate - whether the argument about an individual having rights over their own body is sufficient to justify abortion. This is Judith Jarvis Thomson's position as laid out in 'the unconscious violinist'. In my essay, I will carefully analyse Thomson's argument, along with some objections to it put forward by other philosophers. My tentative position is that whilst 'the unconscious violinist' is a valid enough argument, on its own it does not provide sufficient justification.
Whose is the more adequate interpretation? Why?
Check your answers
Student A's interpretation:
This is not an adequate interpretation of the topic. First note how this student plans to focus on the abortion issue in general, rather than on the specific issue raised in the question: whether the rights of the carrier of the foetus are the only consideration.
Further, the student doesn't seem to have thought about how 'the unconscious violinist' argument relates to the topic as a whole. One can imagine from this interpretation, that Thomson's argument will be tacked on at the end of the essay, instead of being integrated into the whole work.
The problem with Student A's interpretation can be seen in the following summary:
Student B's interpretation:
This is a well-considered interpretation of the topic; it can be summarised thus:
Note that there may be other valid ways of approaching this topic. These however, would need to be based on a careful reading of the topic.
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An alleged gay bashing at a popular Portland gay dance night in April 2009 finally got its day in court today.
Update 1/19: The jury found Blake McCune not guilty of intimidation, but guilty of interfering with a police report. /end update
The case became a major discussion point last spring, when it was brought up at a public forum as a key example of gay-bashing in Portland going unprosecuted. It's a bit of a he-said-she-said case and the conflicting stories make it difficult to pin down exactly what happened. But they both paint a tense portrait of group, and one person specifically, not willing to turn their cheek to the word "fag".
Both sides agree that on April 12th, 2009 a scuffle occurred outside Blow Pony, a queer dance night at Casey's bar on NW 6th and Couch, and the word "fag" was shouted. When Blow Pony organizer Airick Heater tried to call the police, a man named Blake McCune knocked the phone out of his hand.
The prosecution says the scuffle clearly amounted to gay bashing. The defense says it was some heated words that sparked an over-reaction from a gay man burning with anger about homophobia in Portland.
What's up for debate is whether McCune shouted at Heater and hit his phone specifically because he thought Heater was gay, or just because McCune was angry. McCune and two of his friends dismiss the prosecution's claim that McCune shouted terrible anti-gay slurs at the crowd and punched Heater in the face.
This is actually the second time around for the case. The DA admitted that it screwed up when Heater originally pressed charged in 2009, copping to a clerical error that accidentally mailed Heater's trial notices to Minnesota. McCune is currently facing charges of intimidation based on sexual orientation and interfering with a police report.
After hearing testimony from both sides all day, the jury is deliberating on the case now. I'll update when they make a decision tomorrow morning.
On the one side is Heater, a well-known and outspoken activist in Portland's queer community. As an organizer and DJ for Blowpony, Heater stands out with his bright red hair and knuckle tattoos. In his opening statement to the jury today, defense attorney Troy Pickard described Heater as a "man who felt besieged by anti-gay sentiment from all sides in Portland."
On the other is McCune, a 23-year-old from Vancouver who admits that he seems physically intimidating, at a burly 6'5 ("I look like a big football player. I look like I'm joining the army," he told me in an interview) and admits that, in the heat of the moment, he got angry and called Heater a fag, but says he never punched him.
The whole thing started outside Blow Pony, when after a night of bar-going, McCune and two friends were waiting on NW 6th and Couch to be buzzed into a friend's apartment above Casey's bar. Around them, the crowd from Blow Pony spilled out onto the street. McCune hadn't had a drink all night, but his friend Nick Woytuk was drunk and started asking people in the crowd for cigarettes. Somehow, the two groups started sniping at each other.
"It turned into a little pissing match," McCune told me. "'Fuck you guys!' 'No fuck you guys!' No one said anything about sexuality." Heater came out of the bar and got between the two groups. "He's right in my face, like right up against me," says McCune. "He grabs onto my shirt as I'm going in and says, 'Have you ever heard of the Hells Angels?'"
In court today, McCune's friend Torin Daniels, told the jury he also heard this line.
"And this is the moment that I'm not proud of and I wish I had not done," continued McCune, in our interview. "I said, 'I'm pretty sure the Hells Angels don't accept fags.'"
Heater says that's not how it happened at all. when he he heard the ruckus outside Blow Pony, he heard McCune shout, "You AIDS-infested faggots!" When he got between the two groups, Heater says McCune asked if he was "one of these faggots." Heater says he never said anything about the Hells Angels.
Finally, the apartment door opened, the friends rushed inside, but Heater and some of the Blow Pony crowd followed after them. Heater grabbed Woytuk and put him against the wall, pulling out his phone to call the police.
McCune says he tried to leave, but Heater blocked his path, so he angrily hit the phone out of his hand.
Heater says McCune approached him, called him a "fucking faggot bitch" and punched him in the face, causing him to drop the phone. A defense witness who was at the party, Daniel Perez, says he definitely saw McCune punch Heater in the face. McCune's friend Torin Daniels says he definitely say McCune just slap the phone with a hand motion "like I would high-five my little sister."
The call was recorded by the 911 dispatcher, but the audio just before the phone hits the ground is not clearly audible. What does come through clearly is Heater telling the police, "They're coming through the door." A mocking voice—McCune's—shouts over the line, "Yeah! We're coming through the door!" The phone hits the ground and someone else picks it up, saying that McCune knocked the phone out of Heater's hand and is "still yelling at everyone."
In closing, defense attorney Troy Pickard told the jury that Heater was right to stand up to bigotry—but in this case, Heater got the wrong guy, assuming he was being target as a gay man when he wasn't.
District Attorney Adam Gibbs said Heater was in the right. "If they wanted to beat up these guys who were making offensive comments, they outnumbered them. But what did he do? He called the police... At any point during this, he could have just backed off, let them go about their business, let them know it's okay to make these kinds of comments and just walk away."


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The village church, a testament to stunning construction techniques that have stood the test of time, is dedicated to St. Giles.
The village, and more specifically Netherwitton Hall, played a small but important role in the Civil War. Cromwell used the hall as lodging for his troops for a single night. Unfortunately, his troopers were quite rowdy and Cromwell ending up having to pay nearly £100 for the damage his men caused!
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ADELAIDE Football Club is building its interest in a city training base — but any move away from AAMI Stadium would likely hinge on coat-tailing a long-term Commonwealth Games bid.
Crows chairman Rob Chapman says the club would "love" a city training facility close to its new game-day home at Adelaide Oval, Sunday Mail columnist Dwayne Russell reveals.
Adelaide was perceived to have been dragged from West Lakes to North Adelaide under cricket and football's reunion at the renovated venue.
The club's $20-million investment in its Westpac Centre and staff offices adjoining AAMI Stadium has been seen as an added anchor.
But Chapman, speaking to Russell, said while the club was open to shifting its non-gameday operation from West Lakes, it would not independently fund such a move.
"We'd love to be in the city," Chapman said.
"It's on our agenda but it's a dream more than an aspiration.
"If the Commonwealth Games bid did get a positive vote from our government, and if you are going to build infrastructure, you would ask how can everyone get full advantage out of that, and that (key, long-term AFL involvement) would be a great way if you were going to build.
"And you would like to think those committees would engage both Port Adelaide and us."
A group led by SA businessman Rob Gerard is pushing for Adelaide to bid for the 2026 or 2030 Commonwealth Games. Bids for the 2026 Games must be lodged by 2018.
A successful Commonwealth Games bid would mean building new, or upgrading, facilities. Adelaide would hope to occupy a new or renovated venue near Adelaide Oval outside of Commonwealth Games commitments.
Russell, a former Magpies player, also suggests Port Adelaide is divided on the benefits of a city training base against the threat of losing its "historical mojo" by leaving its suburban Alberton roots.
Russell says both clubs have investigated city sites including the ovals at the intersection of West Terrace and Port Road, opposite the former rail yards.
Adelaide, at least, dismissed the site and Chapman said a base neighbouring Adelaide Oval, in the northern Parklands, was the only option for a move to the city.
"It would be highly unlikely it would happen unless you could build something on the doorstep of Adelaide Oval," Chapman said.
"You would want to make sure all or your admin and staff are together."
Originally published as Crows keen to move to a city base


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Thursday, March 31, 2016
Blood Rubies and Wicked Stepmother
3 comments:
Yes! Good stuff, Ryan, will definitely pick these up.ReplyDelete
Read BLOOD RUBIES this weekend... more suspense melodrama than horror, with a kinda Bret Easton Ellis vibe in one storyline!ReplyDeleteReplies
I kind of got that too. Out of the two I think I prefer Wicked Stepmother but I enjoyed both. I ordered a print of the photograph used on the original Wicked Stepmother paperback. Which reminds me I should post that on here.Delete
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Discussion in 'Android Devices' started by FreakyLocz14, Oct 2, 2010.
Does the G2 have these features?
Well thats disappointing. That would have been a deal sealer for me.
FFC is something I would never use. I hate calling people and talking, why would I wanna see their face also? Lame.
Don't get me wrong, the G2 is an awesome device. I even held one in my hand and it felt awesome! Even if you don't use video chat, you can use the FFC for taking self-portrait photos or recording yourself.
I read about an FFC being installed on the Samsung Vibrant. If this could be done for the G2 then I'm buying one! Sure the plan is a tad more pricey than Sprint, but T-Mobile gets better coverage where I live. And I can always take it back after 30 days if things don't work out.
Front cameras ca come in handy sometimes
My Touch HD will fill that roll... T-Mobile Announces the Newest myTouch with HSPA+, Front-Facing Camera, and Froyo | Android Phone Fans
No physical QWERTY?
I just got my g2 today and I think I see a front facing camera on the phone. Its on th top left hand corner. Or it could be a light senor I'm not sure. Maybe if it is it will be in a update like when the g1 first came out it didn't record video and look it what it can do now
i believe what your referring to is the proximity sensor or the light sensor, no front camera
Yeah I noticed that in mine also..looks just like a lil camera eye in the top left corner..
@Warbird87 please explain what that dose. I will be the first to admit that Im not the greatest when it comes to these things.
Clearly not a front facing camera. Things like this would be said already. T-Mobiles first phone with this feature will be the Mytouch aka Mytouch HD.
Personally I think they should of added that feature to the G2 as well. That would of been perfect. But no it does not have it.
i realize i am late to this party, but i just discovered this little speck of an eye on my G2 today while sitting in the sunlight on a break. whaaaa????!!! surprise surprise! so i was curious too. so it is absolutely determined this is a light sensor (perhaps for the "auto" brightness feature?) and NOT a camera?
Any insight into the actual use of this hardware? As I said, my best guess is to sense the auto-brightness feature (if enabled in display) to brighten or dim the screen in different light settings. Otherwise, how can I use or control this hardware?
Anyone know of any apps or ROMS that offer control over this hardware?
there is no camera there, the phone has been taken apart and looked at and there is no hardware to support a camera in the front.
Soooo, there's no software to support the hardware You have investigated the device and determined that it is really just a light sensor? So it's a little-known extra bit of hardware that is most likely used for...? My guess is the auto-display brigtness control. Am I on the right track? Do you know what that speck of an eye does?
one is for proximity, one is for auto lighting. no camera sorry.


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This pillow harness is exactly what you think it is. It allows you to attach your favorite wand massager, so you can grind and hump your way to amazing pleasure. Straddle your favorite pillow. Ride your favorite cushion. The straps of the harness are fully adjustable.
All prices are in USD.


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GraphicRiver Sack of Eggplants 7863697
Stock Vector \ Objects \ Food
Eggplants \ Storage \ Purple \ Vegetable \ Violet \ Background
Stock Vector - Sack of Eggplants 7863697 by interactimages @ GraphicRiver
Stock Vector \ Objects \ Food
Illustration of a sack of eggplants on a white background
Layered: Yes; Graphics Files Included: JPG Image, Vector EPS; Minimum Adobe CS Version: CS.
Toys; Sports Silhouettes; Letter D; Four Boats in Different Colors; Board Game Theme for Children; Pumpkins; Sweets.
Keywords: bag, brown, bulk, cartoon, container, delivery, edible, eggplants, farm, farmer, farming, food, green, group, harvest, harvesting, isolated, lavender, order, purple, sack, set, sets, storage, vegetable, violet, white.


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- Please select a date to see the proper prices!
Description of the accommodation:
Rooms, services When taking a trip to China, booking a room at Yiyi Fashion Hotel Linyi may be a great decision. The guests of the hotel may stay in 129 nicely furnished, comfortable rooms in Linyi. The exact scope of services offered by Yiyi Fashion Hotel Linyi (including those for an extra fee) is available on the information sheet below.
Payment Advance payment by credit card or providing credit card guarantee may be necessary for finalizing the reservation, depending on the chosen date, room and price.
Arrival, opening hours You will be able to speak with the personnel in Chinese - China and English languages.
Yiyi Fashion Hotel Linyi rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
Desk in the room, Tea/coffee-maker


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Over 2,000 costumes, accessories and masks to choose from!
Browse our huge range of Star Wars costumes.
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Sports Programs
Ditch the mouse and get with your active side
Well-structured sports programs can provide children and adults with opportunities to participate in physical activity that has immediate and long-term benefits. Higher fitness levels, better health and enjoyable social interaction are a few of the fringe benefits of participating in sports activities. Fun and sports go together, whether it's basketball, golf, gymnastics, volleyball or badminton that appeals to you, just get active.


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Interest rate cut delivered
Interest rates were reduced by 0.25 of a per cent today, it is the first rate cut since November, 2001. Market analysts argue that neither side of politics can take the credit.
KERRY O'BRIEN, PRESENTER: Welcome to the program, on the day the interest rate arrow took a U-turn and headed south for the first time since December 2001 - nearly seven years.
No surprise of course. A quarter of one per cent, as predicted by just about everyone. A drop in the bucket for lower and middle income homeowners after 12 straight increases, but the Government saw it as a brief ray of sunshine before Australian Bureau of Statistics quarterly national accounts are released tomorrow, which are expected to reveal a sharp slowdown in the nation's growth.
The Opposition wants the Government to wear responsibility for that round its neck like a bell, so of course the debate in Parliament today was fierce.
But according to market analysts neither side can shift blame or take the credit.
In a moment I'll be talking to shadow treasurer, Malcolm Turnbull, but first political editor Michael Brissenden.
MICHAEL BRISSENDEN, REPORTER: Politics is often reduced to a numbers game, but as they all know - some numbers have more significance than others.
Today's small and anticipated numeric shift was one of those. We knew what was coming, but it was the talking point of the day well before it was formally delivered.
DAVID BRADBURY, LABOR BACKBENCHER: I know that there'll be many people in my electorate that will be leaving their house this morning, heading off to work. They'll be anxiously awaiting the outcome of the Reserve Bank's deliberations today.
JASON CLARE, LABOR BACKBENCHER: We all wait with baited breath, and everyone in my electorate has their fingers crossed.
WAYNE SWAN, TREASURER: We can't necessarily predict the outcome of the meeting today.
MALCOLM TURNBULL, SHADOW TREASURER: They were going to bring down interest rates by putting downward pressure on inflation.
MICHAEL BRISSENDEN: But by Question Time both sides were reserving their rhetoric with the banks formal announcement not due until 2:30pm AEST the Government spent the first half hour filling us in on the plans for nation building - sure ports, roads, rail links and infrastructure are important. But there was only one ever going to be one dominant theme, and the politicians, like everyone else, had to wait for it to arrive.
By 2:32pm the Reserve Bank's verdict was delivered and the tactics shifted immediately from filler buster to afraid.
KEVIN RUDD, AUSTRALIAN PRIME MINISTER: And Mr Speaker, I can inform the Parliament that the Reserve Bank of Australia has just announced it will cut interest rates by 0.25 per cent.
Mr Speaker, this will take the official cash rate down to 7 per cent. The first interest rate cut in seven years, and will provide some relief, some modest relief, to mortgage-holders right across Australia.
I'm advised that for 740,000 first-time buyers, this will be the first time they have ever experienced an interest rate cut.
MICHAEL BRISSENDEN: Well, we knew it was coming having been goaded after the May Budget by the Shadow Treasurer Malcolm Turnbull to accept full responsibility for every future rate rise, there was no doubting the Government was fully intending to completely own this interest rate movement.
And the Opposition leader has already been manoeuvring to undermine whatever political mileage there might be in it, with a strategy that began yesterday with his unusual call for the RBA to cut by 0.5 per cent, twice as much as usual. Today, once the news was out, Dr Nelson was again insisting that a rate cut wasn't necessarily a sign of good economic management.
DR BRENDAN NELSON, OPPOSITION LEADER: It is good news that we are getting an interest rate cut, but the reasons that we are getting it with the slowing economy, rising unemployment and still high inflation is not good news.
The Reserve Bank has cut interest rates basically because business and consumer confidence has flat-lined the economy is slowing very quickly, and as we know, the Reserve Bank says that the labour market is loosening up, which in plain language means that Australians are loosing their jobs.
MICHAEL BRISSENDEN: An interest rate movement, whichever way it goes, is always a political opportunity, and the interpretation can be entirely different, depending on who is doing the talking.
Interest rate increases are not good, but they can be the sign of a strong economy. Rate drops are welcome, but is it good management or the sign of a weakening economy?
MALCOLM TURNBULL: My question is addressed to the Treasurer. I refer to the statement issued today by the Reserve Bank governor Glenn Stevens which stated, and I quote, "It is looking more likely that household demand will remain subdued and overall economic growth slow over the period ahead". Is this the slow down we had to have?
WAYNE SWAN: Well, Mr Speaker, I was talking before about the political opportunism and the stupidity of those opposite, and it is just so evident in the question.
Why is the economy slowing, Mr Speaker? It's slowing under the weight of 10 interest rate rises in a row under those opposite, Mr Speaker, something they want to forget about.
MICHAEL BRISSENDEN: As you can see the Government argues that the legacy of past economic management, 10 interest rate rises and the highest inflation in 16 years coupled with the global financial market turbulence is the reason for the slowing economy.
The RBA statement reads in part, "Inflation in Australia has been high over the past year in an environment of limited spare capacity and earlier strong growth in demand", the period that covers both governments.
WAYNE SWAN: I was talking about the political opportunism from plan A there. Plan B up there and plan C over here, and I tell you what it's based on, Mr Speaker.
It's based on the notion that there was no inflation in the Australian economy until the 24 November last year. That's what it's based on. It's based on the notion, Mr Speaker, that 10 interest rate rises in a row never had any impact on economic growth, Mr Speaker.
MICHAEL BRISSENDEN: So what do the experts think? Saul Eslake from the ANZ Bank has read the entrails of the RBA's deliberations for years and he says neither side of politics can claim credit or lay blame in particular.
SAUL ESLAKE, CHIEF ECONOMIST, ANZ BANK: The Reserve Bank has been responding over a long period of time to an accumulation of inflationary pressures as a result of strong demand and the income produced by the commodities boom.
MICHAEL BRISSENDEN: Saul Eslake says the global credit crunch and fuel prices have compounded the impact of the RBA's previous rate increases and he says we should expect quite a few more cuts over the next few years.
SAUL ESLAKE: If the Reserve Bank's forecasts as outlined in the statement are correct for household demand to remain subdued, for overall economic growth to slow and for inflation to fall back to three per cent or less by 2010 are all borne out, then we are likely to see a gradual series of interest rate reductions over the next 18 months to two years.
I would venture that we'll see one more this year, three during 2009, and possibly two in the first half of 2010, which would take us down to 5.75 per cent by the middle of 2010.
MICHAEL BRISSENDEN: So, a slowing economy is good news? Well, once again it depends on who is doing the talking.
KERRY O'BRIEN: Political editor Michael Brissenden.
Author Michael Brissenden


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Detalii loc de munca - .
Descrierea jobului - Finlanda
Desired Candidate Profile - Good knowledge of iPhone solutions as well as interest in contributing to their development; - Excellent software development and problem solving skills, in particular with iOS platform; - Several years of hands on experience in object oriented C and C# languages; - Customer orientation and consultative work approach; - Experience with Core Audio and Core Animation are considered as an advantage; - Familiarity with Xcode development environment is a plus; - Excellent interpersonal communication and presentation skills; - B.Sc. or M.Sc. degree in the relevant field; - Fluency in Finnish and English.
Permis de conducere : categoria B: minim 2 ani Limba straina: Engleză: Avansat ; Finlandeză: Incepator We offer: - Challenging and interesting projects; - Possibility to grow in a large company; - International working environment; - Competitive salary package.


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Kitchen and Bath Experts
- 7475 Clairemont Mesa Blvd #100
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Anyone who has ridden Disaster Transport, the indoor "dark" rollercoaster at Cedar Point can tell you that the coaster is showing it's age. There are numerous light leaks in the dark portions, the theme consists of poorly painted florescent paint, and it seems the coaster itself is screeching for mercy. SO, since we are Disney addicts, we're going to give this coaster a Disney sized makover! Tell me your ideas for this poor, rundown coaster!
Disaster Transport at Cedar Point
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Re: Disaster Transport at Cedar Point
Sounds like the "Runaway Mountain" at Six Flags over Texas. It has some rockwork on the outside, but it is literaly a cube of fake rocks. Its obvious that there is a steel building under it (and you can see it in some spots). It isn't much better on the inside (the cieling in the loading room is horible). And, there are lots of light spills.
Both of these rides could use a new structure around them, one that will keep all natural light out. Perhaps there could be a few animatronics to help tell some kind of story?
Re: Disaster Transport at Cedar Point
I think it needs more then just a new structure though. The coaster itself is having issues. It was screeching really loud when we rode it, and it didn't seem safe. I would definitely like to see some nice animatronics though. There is one in the loading area, but it's very cheap...
I almost wonder if it would be better to demolish it and start from scratch. I'll see if I can find some pictures.
EDIT: Found some pics.
Here is the show building.
This is inside the coaster. The robotic arm in the middle didn't move when I rode it. (above)
This is in the que. The animatronics are really jerky, but at least it's something.
Last edited by BANANAZ; 02-21-2010, 12:02 PM.
Re: Disaster Transport at Cedar Point
I haven't ridden Disaster Transport since it was brand new. But even back in 1990 I thought it was a bad Space Mountain ripoff. :lol:
The ride that was there before it was a bobsled ride called Avalance Run. It was weird, but different at the same time. You rode in these giant bobsleds down a slide track. Found some pics here:
Disaster Transport (Cedar Point)
Re: Disaster Transport at Cedar Point
Disaster Transport IS Avalanche Run, in a box. Intamin Bobsled ride.
Re: Disaster Transport at Cedar Point
I don't know why they felt like they needed to box it in though. And it's so HOT inside the show building. And that was on a fall day. Does anyone know if they have air conditioning?
EDIT: I found an article that stated it was closed in because blowing sand got in the troughs. Makes sense. Although it stated that the effects used to be a lot better, and they let it run down and took a lot of effects out. How sad, huh?Last edited by BANANAZ; 02-21-2010, 07:05 PM.
- May 2005
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Re: Disaster Transport at Cedar Point
I love Disaster Transport. I would hate to see it go. It sounds like it needs refurbishment. So just fix exerything up and leave it at that.
Re: Disaster Transport at Cedar Point
It's also got a leaky roof- so it closes on rainy days, too
Re: Disaster Transport at Cedar Point
'No need to waste money' as they would say.
It's at the end of its career, give it five or ten years and America's Roller Coast will have it long replaced with another record-breaking monster.Don't think of a gun as a weapon.
Think of it as a third arm that magically
shoots bullets!
Re: Disaster Transport at Cedar Point
I also wasn't aware that it was one of the last bobsled coasters left. I would really hate to see it replaced with some record breaking monster, because I wouldn't ride it if it was replaced. I don't like Dragster or Millennium Force. Too much is just too much.
Anyway, this thread is about ideas. Anyone got some rethemeing ideas for this coaster?
Re: Disaster Transport at Cedar Point
Make it a bobsled-themed coaster again, keep it indoors but like theme it to the Olympics and call it "Cool Runnings". Jamaican music can play in the queue and have an AA of Doug E Doug kissing his lucky egg. :lol:
Re: Disaster Transport at Cedar Point
Originally posted by aimster View PostMake it a bobsled-themed coaster again, keep it indoors but like theme it to the Olympics and call it "Cool Runnings". Jamaican music can play in the queue and have an AA of Doug E Doug kissing his lucky egg. :lol:
Re: Disaster Transport at Cedar Point
Are you kidding? That movie & the whole story of the Jamaican bobsled team in '88 got me into really loving to watch bobsled every winter Olympics. I was sooo disappointed to see the Jamaicans don't have a bobsled team this year though.
I even remember when Prince Albert was on Monaco's bobsled team. I thought that was pretty wild.
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November 28th, 2011, 08:37 PM
manderee _3
Mega Super Mommy
Join Date: Mar 2011
Location: tn
Posts: 301
My IL s would only get more annoying if I spout facts. They are a whole new level of bad. They would tell me I'm brain washed. I have no credibility in their eyes. Feeding my daughter the right foods are still a freaking fight. I've brought literature home from Dr and they still think I'm crazy and a little coke/chocolate/taco soup at 6 months old (!!!) Isn't gonna hurt her. I screamed no peanut butter for months but since Nana babysits I lost that fight. No, she isn't allergic but it's my call. I seriously despise their ignorance. Sadly they're highly educated backwoods ignorant which is a whole new level. Ask my may mommies. This is a daily struggle for me.
I have to ignore them.
Dd1 my water broke and I went through the natural stages of labor in 11 hours. I didn't have normal labors. I was having contrax 2 mins apart but they weren't awful painful. I went in, was at 3-4 they gave me epidural and stalled my labor. Broke my water. Started pitocin and the babies were here an hour later. I was only 37 weeks with them. (36 with dd1 when water broke)
I am not going with my contractions this time because I'm really thinking I was very early in labor and they sped it up when I might've had days left. Being this is #4 I'm worried he'll come quickly. Time lines and all that go put the window.
mommy to:
Stasia Kaleigh 12-27-02
Robert "Bob" Clay 2-19-05
Elise Gabrielle 5-3-10
Jackson Reed EDD 4-27-12
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Had a great time here this year. Kids loved it, a big part of this is due to the caravan, great welcome, nice & clean area.
Connor said: "This was the best summer holiday."
Lewis said: "It was ace. I would love to stay for all of the summer holidays."
To view the poster from Lewis, please click here
All the best from
Andy & Angela, Lewis, Connor & Ellis from Macduff
Thanks again for the use of your superb caravan. Hope to see you next year again.
Best wishes
Margaret-Anne Malcolm
To all the comments said before - just like to add a little more!
Comments from the Reid family can be read by clicking here
Hope you can accommodate us again another time! Laura & Arthur, you are simply the best! Many thanks,
Janette, Bob & Sandra Reid, Alloa, Clacks
Mum & I had a lovely time here in your lovely caravan. That wee special touch of tea, coffee & sugar was most appreciated. What a lovely spot. Hope to be back again, possibly next year,
Christine McIntyre & Mum, Annie


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Be Too Loft Hotel
Be Too Loft is a 3-star stylish hotel offering a spacious accommodation within easy reach of Hippodrome, Bosques de Palermo and Recoleta.
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The bar's menu includes barbeque and grills as well as complimentary drinks. The pizzeria Palermo Urbano is within easy reach of the property.
Guests can reach Be Too Loft Hotel by car from Airport. Jorge Newbery airport in 5 minutes.
The hotel offers airport transfer, tour assistance and dry cleaning together with a baggage storage, parking and an elevator for guests' comfort.


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Analysis Fighting for the Spoils: The Real Lesson of the Netanyahu Newspaper Scandal
It's not about right or left. It's about money and power.
Israeli politics is often portrayed, in Israel and especially abroad, as an ongoing struggle between right and left on security...
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Stratford's blight officer position will go full-time for the remainder of the 2016-17 fiscal year.
Town Council members voted unanimously at Monday's meeting to make the blight officer post a full-time position through the end of the fiscal year. Current blight officer Rich Fredette will remain in the post, according to Chief Administrative Officer Chris Tymniak. The town will spend $21,000 to make the position into a full-time job.
Council members compromised after initial discussion to make the job full-time going in future years, which would cost about $54,000 a year.
Fourth District Councilman David Harden was the biggest proponent for expanding the blight officer's hours, saying there were many areas in his district with blighted conditions in need of attention, including on Hollister Street, Boston Avenue and Jackson Avenue.
"A lot of buildings there … had a lot of waste and materials on the street that should not be on the street. So that was my concern that these things are considered," Harden said. "It's important that we take care of the safety issues and the blight issues in town."
Harden had a testy exchange with 10th District Councilman Tina Manus in pushing for the blight officer expansion. Second District Councilman Scott Farrington-Posner said he understood Harden advocating for his district, but noted that residents in his district are concerned about taxes.
Farrington-Posner said the No. 1 issue he heard about when walking his district was taxes and how the recent tax increase made it difficult for residents to stay in their homes.
Harden was one of five council members who voted for the current fiscal year's $215.3-million spending plan. The 5-5 tie was broken by Mayor John Harkins. The budget included a 2.01-mill increase.
Manus and Farrington-Posner also took issue with Harden for criticizing Stratford firefighters and Department of Public Works employees for their workmanship on preparing the new town ice rink at Longbrook Park. Manus said she was "offended" by Harden's critiques of the town employees.
"You don't go after our firemen and our public works people. Not with me around, you don't," Manus said after the meeting. "It's one thing to want things for your district. It's quite another to want things and throw Department of Public Works and fire under the bus at the same time."


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Domestic violence initiative launched in County Wexford
A group of statutory, voluntary and community organisations have launched a new domestic violence awareness initiative in Wexford which will see materials about domestic violence support services, as well as counselling and social work information, on display and available in the foyer areas of local courts throughout the county.
Speaking at the launch at Wexford District Court, Tusla's Chief Operations Officer John Gibson said it was important that awareness was raised about the issue of domestic violence and the supports. 'It is imperative that we raise awareness of this issue and that people affected have access to information about local services and support, to ensure they get the assistance they need. The launch of this initiative shows what can be achieved when statutory agencies come together with our colleagues in voluntary and community organisations to bring about local change,' he said. Chief Superintendent John Roche said the gardai were well aware of the special place victims have in the Garda Service and the traumatic effect domestic violence has on families. 'We are committed to fully supporting victims of domestic violence. 'We are delighted to launch this initiative today and would like to acknowledge the support of the courts service in making this possible,' he said. The initiative is the result of collaboration to raise awarness between organisations including Tusla, the gardai, Wexford Women's Refuge, Wexford Rape and Sexual Abuse Support Service, Barnardos and Youth New Ross.
Wexford People


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Hello ladies and gentlemen! So as of lately, I have been feeling a stuck. What do I mean? Well, I mean I still have a year left in college and I'm stuck living in my hometown until then. Also, I feel stuck financially because I don't have the means to do what i want to do. So I am already feeling "stuck" and now it seems like all of my peers are moving and getting these great jobs, basically moving up in their life! Of course, I am so very happy for all of my peers, but I just feel discontent where I am! Then, God convicted me! FYI, I have a love/hate relationship with conviction! Anyhow, God led me to a couple of scriptures.
Let us not become conceited, or provoke one another, or be jealous of one another.-Galatians 5:26
"I knew you before I formed you in your mother's womb. Before you were born I set you apart and appointed you as my prophet to the nations." Jeremiah 1:5,
This verse should give put your mind to ease. It is an amazing feeling knowing God thought us of us BEFORE we were even born! Most importantly, we should take account into the "set apart" in this verse. The definition of apart is used after a noun to indicate that someone or something has distinctive qualities that mark them out from other people or things. You have distinct qualities that no one else has! These qualities will help you fulfill God's plan for your life!
A heart at peace gives life to the body, but envy rots the bones. Proverbs 14:30
Because of our faith, Christ has brought us into this place of undeserved privilege where we now stand, and we confidently and joyfully look
forward to sharing God's glory.-Romans 2:5
Let me summarize these scriptures for you. Galatians 5:26 and Proverbs 14:30 are very similar because they talk about becoming content. They also talk about not becoming jealous about others accomplishments. Jeremiah 1:5 and Romans 2:5 are similar because they discuss how God has brought us to this specific place in our lives.
How Can I Get Over This Discontentment?
1) Spend time with God daily!
- Try to wake up every morning before you start you day and spend time with Him! This will change your outlook on the day! Trust me, I have done it before!
2) Don't compare yourself to others!
- Meditate on scripture that will help you remember God's has specific plans just for you!
3) Have a mind of of thankfulness!
- Be thankful for every circumstance!
4) Remove Distractions out of your life!
- It's easy to get down and out when you are scrolling on social media all day! Go on a fast from social media! Do something meaningful with your life! Don't just wallow in your discontentment!


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Expats Blog Interview
February 22, 2013 1:00 pm | by Stuart | Posted in The Blog
A few weeks ago I was asked by the people from Expatsblog.com to take part in one of their Expats Blog Interview. Naturally I jumped at the chance (while trying keep my calm British composure).
"Stuart's transition from Britain to The Netherlands didn't exactly go to plan. In fact, it wasn't even part of the plan. That's what happens when you are a self-declared accident prone Englishman. Originally from London Stuart has spent over the last decade living in the land of clogs and windmills otherwise known as The Netherlands and has even started a family of his own. Language misunderstandings, cultural confusion, bizarre adventures and a few cases of mistaken identity are just a few of the things that have happened to him during his time in the country."
In the full Expats Blog Interview I explain the bizarre events which led to my arrival in Holland, the virtues of having a friend who regularly calls you an idiot, and how a broken ankle can sometimes be a good cure for home sickness… Click here to read the full thing
3 responses to "Expats Blog Interview"
Well done, Stu! It's great that people are latching on to your blog and stories. The word is spreading. Holland's conquest is complete!
Great interview, and as always, very funny…
Every time I read your blog it makes me long to be back living in Holland so much. And now reading that interview has only amped it up ten fold!


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That's how one of the friends said it. And I'm not sure exactly what does that 'K' stands for. So, I'm assuming it is K for kurus. LOL.
Since the day I was back from Launnie, I've been receiving a lot of that K comment. Which I doubt. Hahaha. So, most probably, it's the camera angle, or the tudung I wore, or the clothes. Yes, I seriously lost weight during finals. Due to workloads and kesedihan melampau probably. During that time I no longer can fit all the pants. I need to wear either skirts or jeggings. And wearing belts didn't help much. Not that I like being fat. But I don't think losing weight that way is healthy. And yesterday, I re-insta my date with ziq n qil, and received a lot of text asking whether I am ok. (Sorry that I only realised the message once I woke up this morning).
I'm not sure how kurus I look like in the pics. But with all the text and all. I presume I look kurus than normally I do. And maybe look a bit 'ill'. Tapi sebenarnya sebab penat kot....~
So. To answer all the questions.
I'm good. Healthy Alhamdulillah. And. No. Saya tidak diet. Itu semua ilusi. Haha.
And thank you for all the love shown. Sayang korang! Terima lah daku yang tak berapa kurus ni seadanya...hahahahaha..


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text: President Bush today nominated Vice Admiral Thomas H. Collins, 55, to be the 22nd Commandant of the United States Coast Guard. Secretary of Transportation Norman Y. Mineta, the Coast Guard's Service Secretary, praised the President's nomination, saying, "Admiral Collins reflects the best leadership traits of the dedicated men and women of the United States Coast Guard. He is the right person to lead the modern Coast Guard through today's challenges to tomorrow's promise of a safer, more secure American coastline and maritime transportation system. "Tom Collins brings a combination of scholarly intelligence, practical experience at sea, and Washington policy expertise to the critical job of commandant at a time when the Coast Guard is facing a host of new missions," Secretary Mineta said. "I look forward to working with him." Once confirmed by the Senate, Vice Admiral Collins will relieve the current Commandant, Admiral James M. Loy, at a formal change of command ceremony in Washington, DC. The ceremony is being planned for May 30, 2002. Admiral Loy, who has held the Commandant position since May 1998, will retire at the May ceremony after serving with the Coast Guard for forty-two years. Vice Admiral Collins is currently serving as the Coast Guard's Vice Commandant, the service's second-in-command. Prior to that he served as Commander of the Coast Guard Pacific Area. Vice Admiral Collins began his Coast Guard service at sea aboard the Coast Guard Medium Endurance Cutter Vigilant. He later commanded the Patrol Boat Cape Morgan, a Coast Guard Group at Long Island Sound, NY, and the 14th Coast Guard District at Honolulu, HI. He also served in several Headquarters assignments including Deputy Chief of Staff. Vice Admiral Collins graduated from the Coast Guard Academy, New London, CT, in 1968 and returned as a faculty member within the Humanities Department from 1972 to 1976. He earned a Master of Arts degree from Wesleyan University in 1972 and a Master of Business Administration from the University of New Haven, in 1976.


text: Hi, I got my HTC Desire a few days ago, and as I needed a Google account for the Market I rememberd one I had created/palyed around with some months ago. The credentials were stored in my KeePass file, but the password was like 20 random letters and numbers. So I changed it to something typeable and entered it in my phone. Needles to say I forgot to change it in keepass and now I can't remember it. Is there any practicable way to show the password stored in my device? Thanks in advance Aaron


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Anthropology of the Spirit World. Anthropology of the spirit world: Characteristics "ad intra" . Two general clusters of features Characteristics of immateriality Characteristics of personality //. Invisible Intangible Immortal //.
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Two general clusters of features
Immortal //The immateriality of the spirits
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Discussion in 'Football' started by ChrisMWilliams, Mar 28, 2012.
Chris Williams' Blog on Iowa State Football: Whose stock is up? | Cyclone Fanatic
Good to hear about Miller. We're going to have a drop off in LB production next year. No two ways about it. Hearing that he's progressing nicely will hopefully soften the blow a little.
I always thought Miller was supossed to be like 6'4". I know they lie about heights, but isu is usually right on for football
First of all, thanks Chris! Good stuff.
Miller has been listed everywhere from 6'-2" to 6'-4", prior to this year. Last year, he was listed at 6'-4", and I thought he looked taller than Knott out there. Either way, he's tall enough, I guess. I wonder if there's any thought to him being our 3rd big linebacker when we go to those sets, or if it will be George.
Hey Chris, do you happen to have insight into the staff's "grading" system of players? What does it mean to us that TJ is at 50-60 percent?
Can't speak to ISU's system, but most defensive grading systems give/take away points based on pre-snap reads/checks/alignment, post-snap mental errors/technique, and overall making a play. Bonus points for forcing turnoves/TFL's big play.
In the programs I've been a part of, championship-level football is usually considered somewhere in the 80-ish percent range
I'm pretty sure that the specifics vary from program to program but I assume it's similar to what wart listed above.
On most of the videos I've seen, it seems like there are lots of guys standing around waiting a turn. I've heard of other programs whose practices have tons of stations so that everyone is doing something all the time (particularly so that the backup players get more "reps" just like the starters). Is ours not like that? Or am I just not seeing the whole picture?
Just curious--if Farniok has put on 18 pounds, why is his roster size the same as last season? Is he 298 now--or was he only really 262 last season?
a 50 for a true freshman isnt too bad. TJ will be a solid DB for us in the years to come
pretty sure the sizes on the site havent been officially updated yet
Well, 298 - 18 = 280. I think he was in that range last season.


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Here is an unique app for fitness professionals and their clients named FLIP. FLIP (Fitness Liability Insurance Protector) is a pre-questionnaire that you can take with you everywhere you take your mobile or tablet with you.This App has a series of questions related to the Personal trainers clients wellbeing.
Last updated 6 Aug 2015