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Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G1 checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that γ-irradiated murine myeloid 32D cells arrest in G1 with active cyclin D–cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21Cip1 and p27Kip1, yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G1 arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.
As with other cell types, hematopoietic cells initiate apoptotic and cell cycle arrest checkpoints in response to DNA-damaging agents, such as γ irradiation (15). This has biological significance since the failure of DNA-damaged cells to die or growth arrest allows the accumulation of new mutations and may contribute to tumorigenic development (36,45). However, both apoptotic and growth arrest responses to DNA damage in hematopoietic cells can be overridden by treatment with cytokine growth factors, such as erythropoietin (Epo) and interleukin (IL)-3. The ability of these growth factors to bypass apoptotic responses to DNA damage is likely associated with their ability to induce expression of the antiapoptotic proteins Bcl-2 and Bcl-xL (1, 40). By comparison, the mechanism by which cytokines override the growth arrest checkpoints remains undefined.
Normal progression through the mammalian cell cycle is controlled by the sequential activation of cyclin-dependent kinases (Cdks) (34). The transition from G1 to S phase is initiated by the expression of D-type cyclins and their assembly into kinase complexes with Cdk 4 (Cdk4) and Cdk6. Once activated, the cyclin D-dependent kinases phosphorylate and inactivate the tumor suppressor protein pRb (23). In early to mid-G1 phase, hypophosphorylated pRb associates with the transcription factor E2F and actively represses transcription through the recruitment of histone deacetylase. The initial phosphorylation of pRb by cyclin D-Cdk4 complexes in mid-G1 negates its ability to repress transcription and results in increased expression of cyclin E, the regulatory partner for Cdk2. Subsequently, cyclin E-Cdk2 complexes phosphorylate pRb on additional sites, causing the release of free E2F and the activated transcription of genes required for S-phase entry, including cyclin A.
Cdks are regulated by several mechanisms in addition to their association with cyclins. Activation of Cdk2 requires phosphorylation at threonine-160 by a Cdk-activating kinase (CAK), as well as the removal of inhibitory phosphorylations by Cdc25 phosphatases (34). Cdks may also be directly inhibited by association with Cdk inhibitors (CKIs), including p21Cip1 and p27Kip1 (46). Expression of p27 is induced by antiproliferative stimuli, including growth factor withdrawal, transforming growth factor beta (TGF-β), contact inhibition, and differentiation. By comparison, p21 is largely regulated by p53-dependent responses to cellular stresses, such as DNA damage.
The ability of most cells to arrest following DNA damage is dependent on the tumor suppressor p53 (29). When activated in response to a variety of cellular stresses, p53 functions as a transcription factor to induce expression of genes which elicit cell cycle arrest or programmed cell death. In so doing, p53 plays a crucial role in removing damaged cells from an organism. The importance of this response in preventing tumorigenesis is demonstrated by the frequent loss or mutation of p53 in human cancer (22). However, p53 is not commonly inactivated in leukemias (8), and DNA damage-induced cell cycle arrest checkpoints are retained in p53-deficient hematopoietic cells (40, 48). Thus, it seems likely that hematopoietic cell cycle checkpoints are mechanistically distinct.
It has previously been shown that the ability of Epo to override γ irradiation-induced growth arrest is dependent on the ability of the Epo receptor (EpoR) to activate a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway (24, 40). For example, truncated EpoRs lacking PI 3-kinase recruitment sites retain mitogenic activity under normal culture conditions but fail to promote proliferation in irradiated myeloid cell lines. In the present study, we have examined the molecular basis of the γ irradiation-induced G1 checkpoint in hematopoietic cell lines expressing truncated EpoRs. We show that this G1arrest correlates with induced expression of p27 and inhibits phosphorylation of Cdk2 at threonine-160. Moreover, the ability of cytokines to override this checkpoint specifically correlates with phosphorylation and activation of Cdk2 but not with altered expression of CKIs. These data define a novel DNA damage-induced checkpoint in hematopoietic cells targeting the activating phosphorylation of Cdk2.
Cell lines and culture conditions.32D murine myeloid cells (5) stably expressing either the wild-type (EpoR[wt]) (11)or the truncated (EpoR[H]) (33) cDNA were electroporated with 20 μg of thebcl-xL cDNA (20) in the pXM expression vector plus 2 μg of pSV2-NEO. Transfected cells were selected in 1 mg of G418/ml plus 70 pg of recombinant IL-3/ml. Clonal lines were obtained by dilution, and the constitutive expression of Bcl-xL was confirmed by Western blotting. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum plus either recombinant human Epo (5 U/ml) or recombinant murine IL-3 (70 pg/ml). In certain experiments, the PI 3-kinase inhibitor LY294002 (Calbiochem) was added to cell culture medium to a final concentration of 10 μM.
Flow cytometry.Cells (n = 106) were washed with phosphate-buffered saline and were resuspended in 1 ml of propidium iodide staining solution (0.05 mg/ml propidium iodide, 01.% [wt/vol] sodium citrate, and 0.1% [wt/vol] Triton X-100) containing 2 μg of DNase-free RNase A/ml. Samples were incubated for 30 min at room temperature, and the DNA content of cells was analyzed on a FACScan (Benton Dickinson). Cell cycle distributions were determined using ModFit LT software (Verity).
Cell synchronization and γ irradiation.Cells were synchronized in G0/G1 by culturing for 24 h in the absence of cytokine (RPMI 1640 medium supplemented with 10% fetal bovine serum). One hour prior to γ irradiation, Epo (5 U/ml) or IL-3 (1.4 ng/ml) was added to cultures as indicated. Cells were then exposed to 4 Gy of γ irradiation from a 137Cs source or were left untreated.
Immunoprecipitations.Cells were washed twice with phosphate-buffered saline and were lysed in Tween 20 buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 4-(2-ominoethyl)-benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin) for 30 min at 4°C. Insoluble material was removed by centrifugation at 10,000 × gfor 10 min. Specific proteins were precipitated with 2 μg of antiserum/ml specific for Cdk4 (Santa Cruz C-22), cyclin E (Santa Cruz M-20), or Cdk7 (Santa Cruz M-19) and were used in kinase reactions or for Western blotting.
Kinase assays.Immune complexes were washed twice with Tween 20 buffer and twice with Cdk buffer (50 mM HEPES, pH 8.0, and 10 mM MgCl2). Glutathione S-transferase–Rb was added as a substrate for Cdk4 kinase reactions as described earlier (32). For Cdk2 kinase reactions, 10 μg of histone H1 (Roche) was added. For Cdk7 kinase reactions, 1 μg of glutathioneS-transferase–Cdk2 (Santa Cruz) was used. All reactions were initiated by the addition of 0.1 pmol of ATP plus 10 μCi of [γ-32P]ATP (ICN) and proceeded at 30°C for 30 min. Reaction products were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were visualized by autoradiography or on a PhosphorImager (Molecular Dynamics).
Western blot analysis.Total cell lysates were prepared by direct lysis in SDS-PAGE sample buffer. Lysates or immunoprecipitated proteins were resolved on SDS–12.5% PAGE gels and were transferred to nitrocellulose membrane. Membranes were probed with specific antibodies. Antibodies to Cdk2 (D-12), Cdk4 (C-22), Cdk7 (M-19), cyclin D2 (M-20), cyclin D3 (H-292), cyclin E (M-20), Kip2 p57 (M-20), Kip1 p27 (F-8), and Mat1 (FL-309) were obtained from Santa Cruz Biotechnology. Antibodies to cyclin A (Ab-3) and p21WAF1 (Ab-4) were from Oncogene Research Products. Antibodies to Rb (G3–245) were from BD Pharmingen. Cdk2 phosphorylation at tyrosine-15 was detected using antibodies raised against the conserved, phosphorylated tyrosine-15 site of Cdc2 (New England BioLabs); Cdk2 phosphorylation at threonine-160 was detected using antiserum (provided by Philipp Kaldis, NCI Frederick) raised against the phosphorylated, conserved, activating site of Cdc28p (42). Blotted proteins were visualized with Lumi-Light Blotting Reagents (Roche), as directed by the manufacturer.
Synchronized 32D-EpoR[H] cells arrest at a G1checkpoint following irradiation.Previous studies have demonstrated that treatment of hematopoietic cells with cytokines, such as Epo or IL-3, overrides DNA damage-induced checkpoints in both G1 and G2/M phases of the cell cycle (24, 40). However, this activity is absent in cells expressing cytokine receptor mutants that are mitogenically competent but which lack a PI 3-kinase recruitment site, such as the truncated form of EpoR, EpoR[H] (Fig.1A). In order to specifically characterize the γ irradiation-induced G1checkpoint in hematopoietic cells, murine myeloid 32D cells expressing wild-type EpoR (32D-EpoR[wt]) or truncated EpoR (32D-EpoR[H]) were stably transfected with the antiapoptotic genebcl-xL . Bcl-xL was used in these studies to protect against cell death during growth factor withdrawal, thereby enabling synchronization of these cells. Notably, for all assays that could be performed in the absence of synchronization, identical results were obtained using cells lacking enforced expression of Bcl-xL (data not shown). Clonal cell lines were established that could be synchronized in G0/G1 following culture for 24 h in the absence of cytokine, as indicated by the predominance of 2N DNA content in the culture (Fig. 1B). All synchronized cells could subsequently be induced to reenter the cell cycle following 24 h of treatment with Epo or IL-3. Following treatment with 4 Gy of γ irradiation, synchronized 32D-EpoR[H] cells were unable to reenter the cell cycle when cultured in Epo. The G1 arrest of this population was confirmed by a complete absence of bromodeoxyuridine labeling (not shown). By contrast, Epo efficiently induced the proliferation of irradiated 32D-EpoR[wt] cells. As a control, IL-3 induced the proliferation of all cell lines exposed to γ irradiation. Thus, synchronized 32D-EpoR[H] cells treated with Epo following γ irradiation provided a source of cells predominantly arrested at the G1 checkpoint.
γ irradiation-induced G1 arrest of 32D cells correlates with inhibition of cyclin E-Cdk2 activity and with increased expression of p27Kip1.To begin characterizing the nature of the G1 cell cycle checkpoint in hematopoietic cells, the expression of common regulators of G1 cell cycle progression was evaluated. Synchronized 32D-EpoR[H] cells were restimulated with Epo or IL-3 in the presence or absence of γ irradiation, and lysates were subjected to Western blotting with specific antibodies. As shown in Fig.2, expression of cyclins D2, D3, and E was not significantly different in Epo-treated cultures with or without γ irradiation. Cyclin D1 is not expressed in 32D cells (not shown). By contrast, irradiated cells cultured in Epo specifically lacked any detectable expression of cyclin A. Since cyclin A is first expressed in early S phase, this result is consistent with the G1 arrest of this population. Expression levels of Cdk4 and Cdk6 remained the same regardless of treatment. By contrast, two forms of Cdk2 were detected in proliferating cells, whereas only one species was expressed in growth-arrested cells. The faster-migrating form of Cdk2 (pCdk2) has been shown to result from phosphorylation on threonine-160, and it represents the active form of this kinase (21, 34). This activated form of Cdk2 was absent in cells which were arrested following irradiation (Epo, + 4 Gy), as well as in control cells which exit the cell cycle due to growth factor withdrawal (no factor).
Cyclin A is an E2F-regulated gene whose induction depends on the sequential phosphorylation of pRb by cyclin D- and E-dependent kinases (23). To assess the status of this pathway, the phosphorylation status of pRb in each lysate was evaluated. pRb migrates as three distinct forms on denaturing gels with the fastest-migrating form representative of hypophosphorylated pRb. Two slower-migrating forms represent differentially hyperphosphorylated species (ppRb). The intermediate form of ppRb contains phosphorylations at Cdk4-specific sites, while the slowest-migrating form is phosphorylated at both Cdk4- and Cdk2-specific sites (7). As shown in Fig. 2, cells arrested by γ irradiation (Epo, + 4 Gy) contained a significant portion of hypophosphorylated pRb as well as Cdk4-phosphorylated ppRb but gave no evidence of Cdk2-phosphorylated ppRb. By comparison, irradiated cells cultured in IL-3 contained all three forms of pRb, albeit with lower levels of the Cdk2-phosphorylated form than did nonirradiated cells.
To directly assess the activity of pRb kinases in checkpoint arrested cells, Cdk4 or Cdk2 was immunoprecipitated from lysates of 32D-EpoR[H] cells and in vitro kinase reactions were performed (Fig.3). As expected, no kinase activity was present in lysates from cells synchronized by growth factor withdrawal (no factor). Cdk4-associated kinase activity was present in lysates from arrested cells cultured in Epo following irradiation and was comparable to the activity present in lysates from cells restimulated with Epo in the absence of irradiation. Similar results were obtained when cyclin D2 complexes were assayed for kinase activity (not shown). Notably, no detectable Cdk2-associated kinase activity was present in cells arrested following irradiation (Epo, + 4 Gy). By contrast, irradiation did not inhibit IL-3-induced Cdk2 activity compared to cells restimulated with IL-3 or Epo in the absence of irradiation.
To assess the influence of cyclin E complex formation on Cdk2 activity in irradiated cells, cyclin E-specific immunoprecipitations were Western blotted for the presence of Cdk2 (Fig. 3). Cdk2 coprecipitated with cyclin E from all cell lysates except those arrested by growth factor withdrawal. However, cyclin E immune complexes from irradiated, arrested cells (Epo, + 4 Gy) lacked any detectable kinase activity. High levels of cyclin E-associated kinase activity were obtained from all proliferating cells, including irradiated cells cultured in IL-3. Thus, γ irradiation-induced G1 arrest of 32D cells correlates with an inhibition of cyclin E-Cdk2 activity while retaining active cyclin D-Cdk4 complexes. These observations are consistent with the partially hyperphosphorylated state of pRb in these cells (Fig. 2).
Similar to the analysis of total cell lysates given for Fig. 2, anti-Cdk2 Western blotting of either Cdk2 or cyclin E immunoprecipitates appeared to detect only the slower-migrating, inactive form of Cdk2 from irradiated, arrested cells (Fig. 3; Epo, + 4 Gy), while the faster-migrating, active form (pCdk2) was present in immunoprecipitates from all proliferating cells. However, the two forms of Cdk2 were poorly resolved on SDS-PAGE gels when obtained from immunoprecipitated material. To confirm that the faster-migrating form of Cdk2 indeed corresponds to active Cdk2 phosphorylated at threonine-160, immunoprecipitated Cdk2 was Western blotted with antiserum specific for the phosphorylated, conserved activation site of Cdks. Threonine-160-phosphorylated Cdk2 was detected only in immunoprecipitates from proliferating cells, including irradiated cells cultured in IL-3. No threonine-160 phosphorylation was observed in cells arrested following irradiation (Epo, + 4 Gy). No significant differences were observed between the phosphorylated status of cyclin E-associated Cdk2 and that of the total Cdk2 population obtained from each culture.
Inhibition of Cdk2 activity may also be mediated by phosphorylation at tyrosine-15. However, no tyrosine-15 phosphorylation was detected in either Cdk2 or cyclin E immunoprecipitates from irradiated, arrested cells (Fig. 3; Epo, + 4 Gy) using phosphotyrosine-15-specific antibodies. Consistent with previous observations (21,44), tyrosine-15 phosphorylation of Cdk2 was present in all proliferating cultures having significant S and G2 phase populations. Irradiation did not alter the amount of tyrosine-15 phosphorylation of Cdk2 or alter its distribution between total cellular versus cyclin E-associated subpopulations.
Phosphorylation of the activating threonine-160 on Cdk2 is mediated by a CAK whose identity has not been firmly established. One candidate mediator of this activity is the cyclin H-Cdk7 complex (18,31). Cdk7-associated immune complex kinase reactions were performed from lysates of 32D-EpoR[H] cells (Fig. 3). Notably, Cdk7-associated kinase activity was present in all lysates tested, with no significant differences observed in cells arrested following irradiation compared to cycling populations. The Mat1 protein has been reported to alter the specificity of cyclin H-Cdk7 complex to favor substrates other than Cdk2 (52). As shown in Fig. 3, Mat1 was absent from active Cdk7 complexes in cells withdrawn from growth factor. However, Mat1 was associated with Cdk7 in all cytokine-treated cells, including cells arrested following irradiation and Epo treatment. Thus, there are no apparent alterations in Cdk7 activity or association with Mat1 that could account for the lack of Cdk2 phosphorylation in irradiated 32D-EpoR[H] cells cultured in Epo.
Inhibition of cyclin-Cdk activity by p21 is commonly associated with p53-dependent responses to DNA damage (29, 46). Expression of p21 and other Cip/Kip family members was evaluated by Western blot analysis of 32D-EpoR[H] cell lysates (Fig.4). Cells synchronized in G0/G1 by growth factor withdrawal contained high levels of p27 and low levels of p21. Restimulation of cells with Epo or IL-3 significantly reduced p27 expression. Earlier studies also showed p53-dependent expression of p21 in response to DNA damage in hematopoietic cells, including 32D cells (8, 41). Indeed, p21 expression was elevated in irradiated 32D cell cultures. However, p21 expression was equivalently elevated in arrested cells (Epo, + 4 Gy) compared with proliferating cells (IL-3, + 4 Gy). By contrast, p27 expression was high in checkpoint-arrested cells (Epo, + 4 Gy) but was absent in irradiated cells cultured in IL-3. p57 expression was not detectable in any 32D cell lysates (not shown).
To assess the potential contribution of p21 and p27 to the inhibition of cyclin E-Cdk2 activity in checkpoint-arrested cells, Cdk-immunoprecipitated proteins were Western blotted for the presence of p21 or p27 (Fig. 4). p21 was detected in catalytically active Cdk4 immune complexes present in irradiated cells, but this inhibitor was not observed in any Cdk2 complexes analyzed. No p27 protein coprecipitated with either Cdk4 or Cdk2, regardless of treatment conditions. In keeping with these observations, we have been unable to detect p27 or p21 in cyclin E immunoprecipitations; nor was cyclin E, Cdk2, or Cdk4 present in p27 immunoprecipitates from these 32D cell lysates (not shown). To confirm that the Cdk2 antibodies and conditions used supported the precipitation of complexes with CKIs, similar immunoprecipitations were performed from lysates of murine 3T3 cells (Fig. 4B). Both p21 and p27 were readily detectable in Cdk2 immunoprecipitates from contact-inhibited 3T3 cells. As anticipated, the degree of CKI coprecipitation was significantly enhanced in contact-inhibited versus asynchronously growing cells.
Ability of cytokines to override γ irradiation-induced p27 expression and Cdk2 inactivation is dependent on a PI 3-kinase signaling pathway.The data described above indicate that γ irradiation-induced arrest of 32D cells correlates with both inhibition of cyclin E-Cdk2 activation and elevated expression of p27 protein. In related studies, it has been recently shown that the ability of cytokines to override γ irradiation-induced cell cycle arrest requires activation of a PI 3-kinase signaling pathway (24). To determine if inhibition of this signaling pathway had similar effects on cell cycle regulators in irradiated cells, the effects of the PI 3-kinase inhibitor, LY294002, on 32D-EpoR[wt] cells were assessed. LY294002 treatment did significantly reduce the S-phase population in cytokine-treated cultures in the absence of irradiation (Fig. 5A). However, these cultures still retained greater than 40% of their population in S phase. By contrast, LY294002 completely blocked the ability of IL-3 or Epo to stimulate cell cycle progression following irradiation of synchronized cells, as indicated by the near absence of S phase. Interestingly, LY294002 treatment also abolished the ability of Epo and IL-3 to induce threonine-160 phosphorylation of Cdk2, coinciding with the inactivation of Cdk2 kinases in irradiated cells (Fig. 5B). Inhibition of PI 3-kinase in nonirradiated cells had little effect on Cdk2 activity. LY294002 also had no effect on cytokine-induced Cdk4 activity in either irradiated or nonirradiated cells. By comparison, LY294002 inhibited the Epo- and IL-3-dependent suppression of p27 expression following irradiation. Thus, the γ irradiation-induced arrest of LY294002-treated 32D-EpoR[wt] cells had a molecular phenotype identical to the arrest in 32D-EpoR[H] cells, which express receptors lacking the PI 3-kinase activation domain.
Cytokine-induced release from the G1 checkpoint correlates with phosphorylation and activation of Cdk2.Together, the data above indicate that through activation of a PI 3-kinase signaling pathway, cytokines override DNA damage-induced expression of p27 and inactivation of Cdk2. To determine which of these activities is most closely associated with the G1 arrest in 32D cells, we examined the stability of and the kinetics of release from this checkpoint. To first assess the permanence of the cell cycle block, 32D-EpoR[H] and 32D-EpoR[wt] cells were irradiated and cultured in Epo for up to 5 days. As shown in Fig.6A, irradiated 32D-EpoR[H] cells failed to progress out of G1 for the entire 5-day period. These cells remained viable during this time period, but by day 9 the viability of this culture rapidly declined (not shown). By contrast, irradiated 32D-EpoR[wt] cells continued to proliferate over the same time course. Cell lysates prepared from each culture at 24-h intervals were assayed for p21-, p27-, and Cdk2-associated kinase activity. Growth-arrested 32D-EpoR[H] cells showed elevated p27 expression and no Cdk2-associated kinase activity up to 5 days following irradiation (Fig. 6B), consistent with their inability to proliferate. Conversely, proliferating 32D-EpoR[wt] cells retained active Cdk2 and showed no increased p27 expression through the 5-day time course (not shown). Interestingly, irradiation-induced p21 expression displayed different kinetics in the proliferating versus arrested cell cultures (Fig. 6C). Arrested 32D-EpoR[H] cells had their highest level of p21 expression within the first 24 h following irradiation, after which p21 expression steadily diminished to background levels. In irradiated 32D-EpoR[wt] cells, p21 levels continued to increase up to 48 h following irradiation and remained high through the remainder of the time course. Irradiated 32D-EpoR[H] cells cultured in IL-3 remained asynchronous and displayed a pattern of p21 expression identical to that of 32D-EpoR[wt] cells cultured in Epo (not shown). Together, these data show that while p21 expression declined in arrested 32D-EpoR[H] cells, p27 expression remained high and Cdk2 remained inactive.
We next tested whether reversal of p27 expression or Cdk2 inactivation was more closely associated with a release from an established G1 checkpoint. Synchronized 32D-EpoR[H] cells remained uniformly arrested in G1 phase when cultured in Epo for 24 h following γ irradiation (Fig.7A; Checkpoint Arrested). Arrested cells were then induced to override the G1 checkpoint by addition of IL-3 and began exiting G1 phase after 16 h, as indicated by the increasing percentage of S-phase cells. Lysates from cells at various time points following restimulation with IL-3 were assayed for the activities of cell cycle regulators (Fig. 7B). Cdk2 activity was undetectable up to 14 h following IL-3 restimulation, as was cyclin A expression, a marker of S-phase entry. Concurrent with the appearance of increasing S-phase populations (Fig. 7A), cells restimulated with IL-3 for 16 h contained detectable levels of Cdk2 phosphorylated at threonine-160 (pCdk2), Cdk2-associated kinase activity, and cyclin A expression. Each of these activities increased through 24 h of IL-3 restimulations in parallel with the increase in S-phase content of this culture. By comparison, expression of p27 protein showed little if any change during the 24-h period following IL-3 addition and remained elevated for up to 36 h (latter time point not shown). Consistent with earlier results (Fig. 6C), p21 protein levels were upregulated by IL-3 treatment throughout the entire time course in a manner that did not correspond to cell cycle entry.
Normal cellular responses to DNA-damaging agents include cell cycle arrest in G1 and G2/M phases. Previously, we have shown that treatment of hematopoietic cell lines with growth-promoting cytokines can override these cell cycle checkpoints (40). Moreover, the ability of cytokines to override these checkpoints is dependent on their ability to activate a PI 3-kinase/Akt signaling pathway (24). In the present study, we report that the DNA damage-induced G1arrest in hematopoietic cells correlates with the absence of Cdk2 phosphorylation at threonine-160 and that this appears to be a target of the cytokine-activated PI 3-kinase signaling pathway.
In many cell types, DNA damage-induced G1 arrest is associated with p53-dependent expression of p21 and with subsequent inhibition of cyclin-Cdk complexes. In murine embryonic fibroblasts, p21 expression is required for G1 arrest in response to irradiation and results in specific inhibition of cyclin E-Cdk2 but not cyclin D-Cdk4 (7). This differential inhibition of Cdk2 versus Cdk4 is similar to the irradiation-induced arrest in 32D cells. However, hematopoietic cells lacking functional p53 still retain the ability to arrest in response to DNA damage (40, 48), suggesting that other arrest mediators are functional in these cells. In fact, the results presented here show that γ irradiation does induce expression of p21 in 32D cells, but p21 levels are equally high in cells arrested at the G1 checkpoint and in those in which the arrest is overridden by cytokine treatment. Thus, p21 expression does not appear to be sufficient to induce arrest of hematopoietic cells treated with growth-promoting cytokines. Moreover, p21 expression decreased to near-background levels in cells that remained arrested 5 days after irradiation, indicating that p21 is not likely to contribute to the long-term maintenance of the G1 checkpoint.
In a separate study, exposure of hematopoietic cells to low doses of γ irradiation caused a transient slowing in G1-to-S phase progression when cells were cultured in IL-3, and this slowing could be alleviated by overexpression of Cdk4 (41). Similar to our findings, p21 was found associated with cyclin D-Cdk4 but not cyclin E-Cdk2 in irradiated cells. Notably, Cdk4 is not as effectively inhibited by p21 as is Cdk2 (46). Thus, cytokine-treated hematopoietic cells appear to maintain a sufficient pool of Cdk4 to sequester p21 and prevent inhibition of Cdk2 following irradiation. However, this apparent ability to sequester p21 is not sufficient to prevent a γ irradiation-induced arrest in the absence of PI 3-kinase activity.
Unlike p21, increased p27 expression did correlate with the establishment of the G1 arrest following γ irradiation. However, we have not been able to demonstrate the presence of p27 in any cyclin-Cdk complexes derived from irradiated or nonirradiated 32D cell lysates. Moreover, there was no apparent decrease in p27 expression as cells progressed into S phase following cytokine-induced release from the G1 checkpoint (Fig. 7B). It is not surprising that a decrease in p27 expression was not detectable prior to activation of Cdk2 in this experiment, since only a small percentage of cells begins to enter S phase 16 h following release from the checkpoint. However, by 24 h, 42% of the culture had exited G1 phase and still there was no detectable reduction in p27 expression. Thus, it does not appear that significant reduction in p27 expression is required for cytokine treatment to override the G1 checkpoint. There is also a notable difference in the pattern of p27 expression in cells that initially arrest and are subsequently released from the G1 checkpoint, compared to cells that never arrest following irradiation. Specifically, p27 levels are low in cells that do not arrest, whereas p27 levels remain high in cells released from an established G1 arrest (compare Fig. 4 and7). The basis for this distinction remains unclear at present, but it raises the question of whether p27 plays any functional role in γ irradiation-induced arrest of hematopoietic cells. There is no prior association of p27 with established models of DNA damage response pathways, although deficiencies in p27 expression have been associated with an increased susceptibility to irradiation-induced tumor formation in mice (17). However, no significant increase in leukemias or tumors derived from hematopoietic compartments were found in p27-null mice.
The γ irradiation-induced G1 arrest of 32D cells was consistently associated with a complete lack of Cdk2 activity despite the continued presence of cyclin E-Cdk2 complexes. Recently, degradation of Cdc25A and persistent inhibitory phosphorylation of Cdk2 were shown to mediate a UV-induced, p53-independent G1 arrest of an osteosarcoma cell line (30). However, we have not observed any detectable phosphorylation of Cdk2 at inhibitory sites in irradiated, arrested 32D cells (Fig. 3). We have also not observed any detectable reduction in Cdc25A protein levels in these cells (not shown). Thus, this novel DNA damage checkpoint mechanism does not appear to be activated in γ-irradiated 32D cells.
The absence of an activating phosphorylation at threonine-160 of Cdk2 consistently correlated with establishment of the irradiation-induced G1 arrest. Moreover, Cdk2 phosphorylation and activation are the earliest events that we have observed following release from this checkpoint in response to cytokine treatment. Phosphorylation of Cdk2 is mediated by a CAK. Association of Cdks with either p21 or p27 has been reported to inhibit their phosphorylation by CAK (4, 25, 28, 47). Thus, it remains possible that Cip/Kip family members play a role in preventing access to threonine-160 of Cdk2 in irradiated 32D cells, despite our inability to detect p21 or p27 in cyclin E-Cdk2 complexes from these cells. Notably, in vitro CAK phosphorylation of Cdk4 has been found to be inhibited by stoichiometric association with p27, which could be overcome by increased concentrations of Cdk4 in the reaction (28). We have tested for a similar effect in 32D cells by enforced overexpression of Cdk2. However, 32D-EpoR[H] cells expressing up to sixfold-higher levels of Cdk2 still completely arrest in G1 phase with inactive Cdk2 following γ irradiation when cultured in Epo (A. K. Eapen, unpublished data). This implies that a more significant change in stoichiometry must be required, if indeed p27 is inhibiting Cdk2 activation in irradiated 32D cells. However, the lack of any detectable reduction in p27 expression as irradiated 32D cells reenter cycle (Fig. 7) does not support such a possibility, unless subcellular relocalization of p27 plays a significant role. Based on our observations, a more plausible explanation may be that cytokine-activated signaling pathways more directly regulate Cdk2 phosphorylation through presently undefined mechanisms.
Initially, a mammalian CAK was identified as the cyclin H-Cdk7 complex based on its ability to phosphorylate Cdk2 in vitro (18,31). However, the Cdk7-associated kinase activity was not found to vary in a cell cycle-dependent manner in vivo. Other work suggests that addition of Mat1 to this complex can shift its specificity from Cdk2 to other substrates, including RNA polymerase II and pRB (52). In the present study, we found no alteration in Cdk7-associated kinase activity or levels of Cdk7-associated Mat1 in irradiated versus nonirradiated cells. Thus, it seems unlikely that the lack of threonine-160 phosphorylated Cdk2 in checkpoint-arrested 32D cells results from altered cyclin H-Cdk7 activity or specificity.
In yeast, CAK activity is not a function of the Cdk7 homolog Kin28. Instead, it is mediated by a monomeric kinase, Cak1 (16, 27,51). Although a mammalian homolog of Cak1 has not been conclusively identified, a similar activity has been observed in lysates of human cells (26) and may be downregulated in a TGF-β-induced G1 arrest (35). Notably, the TGF-β-induced arrest was associated with active cyclin D-Cdk4 complexes and inactive cyclin E-Cdk2 complexes in which Cdk2 lacked phosphorylation at threonine-160. This is similar to the irradiation-induced arrest in 32D cells. Thus, it is tempting to speculate that these two growth inhibitory responses may be mediated through inactivation of the same or similar CAK activity.
Alternatively, the lack of Cdk2 phosphorylation in irradiated 32D cells could result from activation of a phosphatase. Phosphatase activity toward threonine-160 has been associated with the Cdk-associated phosphatase (37) and members of the protein phosphatase 2C family (9). However, activity for both the Cdk-associated phosphatase and protein phosphatase 2C is restricted to monomeric Cdk2 and would likely not be effective against the cyclin E-Cdk2 complexes present in G1-arrested 32D cells. Since there is no prior evidence that a CAK is inhibited or a phosphatase activated in response to DNA damage in other cell types, it is possible that this response to DNA damage is a unique property of hematopoietic cells.
Growth-promoting cytokines, such as Epo and IL-3, normally regulate progression through early G1 phase in factor-dependent hematopoietic cells. Previously, the normal growth response to cytokines has been most closely associated with induced expression of D-type cyclins and the inhibited expression of p27 (2, 3, 6). Indeed, these activities can be regulated through PI 3-kinase pathways in response to cytokines and other growth factors. PI 3-kinase activity can contribute to induced expression of D-type cyclins (19) and to increased cyclin D1 stability through Akt-dependent phosphorylation of glycogen synthase kinase 3β (13). Also, PI 3-kinase activation can effectively downregulate expression of p27, since various inhibitors of PI 3-kinase pathways have been shown to cause enhanced expression of p27 protein (6, 14, 49, 50).
Through these or other activities, PI 3-kinase activation clearly contributes to the efficiency of cytokine-induced cell cycle progression. Notably, cells expressing truncated EpoR or those treated with PI 3-kinase inhibitors (e.g., LY294002) reproducibly exhibit a reduced S-phase population compared to cells growing under control of wild-type cytokine receptors (approximately 40% S phase compared to 60% S phase, respectively). Yet, under normal culture conditions, cytokine receptors lacking the ability to directly activate PI 3-kinase do support long-term proliferation of hematopoietic cells (10,12, 33, 38, 39, 43). By contrast, the ability of cytokines to override DNA damage-induced cell cycle checkpoints in both G1 and G2 phases is absolutely dependent on their ability to activate PI 3-kinase (24). We now find that following DNA damage, the most demonstrable effect of cytokine-induced PI 3-kinase activation is the maintenance of Cdk2 in its phosphorylated, active state. Possible effects on cyclin D stability do not appear to be significant since cyclin D-Cdk4 complexes remain active in irradiated, arrested cells. Similarly, the lack of detectable reductions in p27 expression as irradiated, arrested 32D cells reenter the cell cycle suggests that inhibited p27 expression is not a major effect of PI 3-kinase activation in this context. Instead, the ability to regulate Cdk2 phosphorylation appears to represent a novel activity for the PI 3-kinase signaling pathway. However, there remains a significant 16-h time lag between cytokine treatment and the appearance of detectable Cdk2 activity in irradiated cells. This suggests that multiple steps connect PI 3-kinase activation to the phosphorylation of Cdk2 in irradiated, hematopoietic cells.
We thank Philipp Kaldis (NCI Frederick) for providing anti-threonine-160-Cdk2 antiserum. This work was performed with assistance from the Flow Cytometry Facility (University of Iowa), a137Cs source operated by James W. Osborne (Department of Radiation Biology, University of Iowa), and core facilities of the Diabetes and Endocrinology Research Center at The University of Iowa.
D. E. Quelle was supported by a grant from the American Cancer Society (RPG-98–254-01-MGO). This work was supported by a Howard Hughes Medical Institute Biomedical Research Support Program grant and by Public Health Service grant CA-79889 from the National Cancer Institute.
- Received 1 February 2001.
- Returned for modification 13 March 2001.
- Accepted 18 June 2001.
- Copyright © 2001 American Society for Microbiology


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To promote the new Gen 2 Electron Wheel, the rider will bike for 10 days and 2,800 miles from New York to Las Vegas
One of cycling's best examples of "If you build it, they will come"
The handbuilt frames have an ingenious coupling system so they break down in minutes and pack up small for travel
These quirky bike trips will have you begging for more vacation days
The best ways to find safe cycling routes in your city, region, or state
Don't have time for the epic bikepacking journey of your dreams? Here's how to get the most out of your days off.
The project, called Route Feminent, raises money for ambitous cyclists
Bestselling author Susan Orlean goes bike touring in France's Loire Valley
Exactly how good does a Hefeweizen taste after 136 miles of riding?

subset: commoncrawl


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The 2017 Ride the Rockies tour will stop in Durango June 12-13, bringing 2,000 cyclists to town.
We know you love "The Walking Dead." We do, too. Now you can get a taste of what your favorite TV characters go through while you try to "outrun and outsmart a field full of zombies" during the 7th annual Zombie Challenge at 2:30 p.m. Saturday, Oct. 26, in the Three Springs neighborhood. During the 1.5-mile course, participants will have to navigate zombie-infested obstacle courses and challenge zones, all while dodging zombies that are… Read More
If dressing up in your Halloween costume just isn't enough fun for you, how about running in your Halloween costume? If running in a tutu is more your speed, you might want to sign up for the Great Pumpkin Chase on Saturday, Oct. 26, in Bayfield, Colorado, just a 30-minute drive east of Durango. The Great Pumpkin Chase 5K and 1-mile run starts at 10 a.m. Oct. 26 at 480 Wolverine Drive in… Read More
If you're looking to get down and dirty, you still have time to register for one of three Durango Muck & Mire on the Mountain events Saturday, Aug. 10, at Durango Mountain Resort. Early registration for the 5k, 10k or "Mini Muck & Mire Mile" fun course (for the kiddos) ends Aug. 2, so act soon if you're looking to roll around in some mud in a socially sanctioned way. You can… Read More


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Parks and Trails in Muncie
Muncie offers some of the most extensive biking, hiking, and commuting trails in the state of Indiana. See below for some maps and resources and we'll see you around town!
Click here for a printable map of Muncie's Bike Lanes
Click here for an interactive map!
Visit Bike Muncie for more information on bike friendly routes!
Built in 1928 at a cost of over $400,000, the Muncie Fieldhouse was built to house Muncie Central Bearcat basketball games. The Fieldhouse contains 7,635 seats, making it one of the largest high school gymnasiums in the country--a reflection on the importance of the game of basketball to Indiana.


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|PEDODONTIA (4 credits)
|GENERAL AND SPECIALISED PEDIATRICS (1 credits)
|PEDODONTICS TRAINEESHIP (1 credits)
Recommended readings
1) R.E. Behrman, R.M.Kliegman, A.M. Arvin, W.E.Nelson. TRATTATO DI PEDIATRIA. Ed. Minerva Medica.
2) G. Bartolozzi, G. Maurizio. PEDIATRIA. PRINCIPI E PRATICA PEDIATRICA. Ed. Masson
Antonella Polimeni- Odontoiatria Pediatrica- Elsevier srl 2012
3) G.L.de'Angelis. L'ENDOSCOPIA DIGESTIVA IN ETA' PEDIATRICA E GIOVANILE. Ed. Mediche Scientifiche Internazionali - Roma
Teaching methods
Class lessons: the program themes will be discussed with the aid of slides. The proposed topics will be the subject of collective discussion and study on the basis of ideas and any questions raised during the lessons themselves.
Assessment methods and criteria
The achievement of the goals of the course is verified by an oral exam.
Answering questions about the contents of the course, the student will demonstrate whether he/she has achieved the goals of knowledge and understanding of the contents


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Superior Tecnico (IST) is the Engineering School of the Technical
University of Lisbon, which is committed to education and research
in various areas of engineering and technology. It has an academic
staff of approximately 800, about 8000 undergraduate students, 750
MSc. students and 250 PhD students. The Unit of Marine Technology
and Engineering is the Research Unit of Instituto Superior Técnico
that will be participating in this project. The Unit of Marine Technology
and Engineering has total research personnel around 40, organised
in five groups: Marine Environment, Dynamics of Marine Vehicles
and Platforms, Marine Structures, Ship Design and Maritime Transportation
and Safety, Reliability and Maintainability. This Unit has been
involved in or is currently involved in about 40 European projects.
The Group of Marine Environment has been involved in or is currently
involved in 11 European Projects and has experience in analysis
and modelling of wave data, including buoy data, hindcast models
and more recently remote sensed wave and current data. Also work
has been done in connection with modelling the fate of oil spills
and with oceanographic instrumentation.
For further information visit the IST website on: http://mar.ist.utl.pt


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What is it?
This is the revised Mitsubishi Colt. Mitsubishi has given the Colt range a mild refresh, with this CZ2 three-door being a representative mid-range example.
The biggest visual difference is the revised headlight treatment, a part-successful attempt to transfer the look of the Lancer to the Colt. Other changes are more minor, with new trim and revised spec levels.
What's it like?
The Mitsubishi Colt itself remains a thoroughly likeable car, but not one that's going to cause much anxiety for more modern rivals.
The mid-ranking 1.3-litre engine delivers impressively keen performance when worked hard, and the Colt seems to enjoy making reasonably rapid progress.
The ride still feels bouncy over anything other than the smoothest road surfaces, and the steering is short of the feedback necessary to allow the Colt to be considered a true warm hatch.
It's better on the motorway than most supermini rivals, though, with decent refinement and reasonably long gearing.
The Colt brings plenty of kit in CZ2 spec, including air-con, alloys and cruise control, but the Colt feels small compared with more modern supermini rivals, with poor space for adults in the rear seats and a dinky boot.


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text:
- Description
Tekna Nautic Montrose Large Pendant-C
Hanging light in Dark Bronze with pagoda structure, closed top, outside fitted clear glass, water-resistant cable entry and handle (eyelet).
Width - 436 x 436 mm
Height - 922 mm.
4-Arm lampholder with Caret Squirrel Cage lamp 230V E27 4 x 8W. - main power 230V 50Hz.
Use: Indoor and Outdoor (IP43)
For suspension: See accessories in options list.
Allow up to 2-4 weeks lead time as these are made to order.
- Reviews


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Econometrist by training, after 10 years of experience in quantitative studies, Arnaud joined Utopies in September 2013 as manager of the "Local Footprint" department, whose goal is to help firms in measuring the economic impact of their activities, in order to, in particular, maintain their operating licences, to improve communication with stakeholders and to improve the environment in which they are evolving.
Arnaud has developed the LOCAL FOOTPRINT ® for Utopies, making it possible to conduct impact studies in more than 193 countries, on a national scale as well as on a local scale. The completed studies make it possible to precisely assess the direct, indirect and induced impacts of a business, a site or a project in terms of economic flow, jobs and public revenues. In addition, Arnaud supports teams on problems of quantitative studies (inquiries by stakeholders, leaders of opinion and consumers).
His secret for committing so many double faults in his tennis game.
AccorHotels and Utopies share findings from the first study into worldwide socio-economic impacts of a hotel group
> Read more
A "productive city" is a city that aims to increase its self-production capacity - to manufacture the products it needs - by relocating the necessary manufacturing production, by mobilizing local material resources (renewable resources, circular economy...), while connecting to global collaborative networks (data, design, know-how, etc.).
> Read more
LOCAL SHIFT ® models the economic metabolism of a territory: it tracks trade flows between 382 sectors of activity, households and the non-market sector within and outside the territory thanks to an economic matrix modelling.
> Read more


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BUDAPEST (Reuters) - Twelve hours after Germany's soccer team had won the World Cup in Brazil, the country's water polo team jumped in the pool for the opening game of the European Championship in the Hungarian capital.
It is probably not the result - Germany suffered a sound 10-5 defeat at the hands of Croatia - that kept news of the game out of the limelight. Professional water polo has been shunned for so long that the sport's leaders now fear for its survival.
That is not the conclusion one might come to at the Margaret Island Aquatics Complex, which has been outfitted with 8,000 seats and fills to capacity on most nights, especially when hosts and world champions Hungary play.
Television is so saturated with other sports, however, that water polo sponsors get nowhere near the kind of media exposure they would through other events.
"Water polo has been around forever, and some games induce downright euphoria out here," said Tamas Gyarfas, the Hungarian vice president of the international water sports association FINA. "But their TV appeal is weaker for a number of reasons.
"We worry that it might hit a point where it can no longer be considered a top priority sport."
Gyarfas, who has also had a career as a media businessman, said it is hard to popularize, and therefore make money on a team sport where the athletes are mostly submerged in water and the action is interrupted every few seconds by referees.
"Viewers have little idea what's going on," he said. "It is not enjoyable the same way as athletics, where people dash about, or gymnastics, where they fly around. Still, we would like to save this game."
Water polo leaders have sensed this challenge for years.
FINA dedicated a conference to reforming water polo earlier this year, inviting marketing and advertising experts from other professional sports like the U.S. National Basketball Association to learn how to sell a game well.
One presentation compared the introductory show at an NBA game with the start of a water polo match.
The contrast was huge: fanfare and fireworks on one side, a solemn announcer reading out the players' names on the other.
With that in mind, Hungary has put on a big show at the aquatics center, with boisterous sound effects and a cheerleader who directs the fans.
For lasting change, however, more will be needed. Pushing back the playoffs into the summer months so spectators have more fun at open air games is one idea for reform.
Other ideas include smaller pools to allow faster games, fewer players to fit in the smaller pools, or a smaller ball to allow more powerful shots and more goals.
The long-term goal is to spread the sport, which is only really big in the Balkans and some southern European countries; but water polo officials are reluctant to change a sport that has taken a century to develop.
FINA water polo director Gianni Lonzi said any changes need to be tested before they become adopted, a process that could start next year but will take time to calcify.
"It is like when I have an old car which I want to change," he said. "Until I see a car that gives me the same satisfaction, I don't change."
Apart from a regular contract with the Hungarian team Szeged, three-times Olympic gold medallist Tamas Molnar has also played for the last five years in the summer league in Malta, where people adore the sport like nowhere else.
"To the 500,000 people who live in Malta, there are ten teams," Molnar said. "Prominent games often have 3,000 fans, not just spectators, but proper fans, with flags and drums and all. They create a fantastic atmosphere. It's exceptional."
He said part of the secret is that the summer league takes water polo from its regular-season professional roots and puts it where it belongs: in the sun, by the water, where fans get to enjoy the games while they spend a day at the beach.
Molnar's generation had exceptional opportunities which allowed them to focus on the game and earn a very decent living doing it. But he says those circumstances may be hard to emulate in the future.
"I don't know how long this type of a professional game can be sustained," he said. "It is impossible to plan for the long term. Clubs struggle to secure sponsors on a business basis."
The annual budget of a championship-level water polo club can be close to 1.5 million euros ($2.03 million), he said. That may sound like peanuts for some other professional sports, but even that money is often from sponsors who like the game and not investors.
"We hoped that the U.S. would show the way and start a well-financed pro league when the men played in the Beijing Olympic final and the American women won (the 2012) Olympics."
To this day there is no professional water polo league in the United States, and not because Molnar and the Hungarians beat them in that Beijing final. It is because the sport just won't lend itself to mass media.
When big companies finance water polo it is usually a case-by-case affair. Britain's Vodafone PLC has sponsored Champions League football and Formula One on a global basis. It only backs water polo in Hungary.
"The bigger Vodafone units get to choose one sport beyond football and Formula 1," Vodafone Hungary CEO Gyorgy Beck said. "The parent company was surprised when we picked water polo over rugby but when they saw the fan numbers they had no questions."
Vodafone's annual water polo sponsorship budget was more than 100 million forints ($436,200) until recently, Beck said. It has been cut back now, although it still sponsors youth games and special events.
The company constructed a temporary water polo arena on a downtown Budapest square for the European Championships at a cost Beck would not disclose. The arena will emulate the beach atmosphere and broadcast games live.
"It has been a very positive investment," Beck said. "Water polo helped us establish our brand in Hungary. But water polo would not have worked this well in other countries."
($1 = 229.2700 Hungarian Forints)($1 = 0.7400 Euros)
Editing by Rex Gowar


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"The true measure of a man is how he treats someone who can do him absolutely no good." — Samuel Johnson
- Genuine kindness doesn't have ulterior motives.
- Being kind without expectation is the truest kind of kindness.
- Doing Good
- The right thing to do
NEW BOOK BY ZERO DEAN: Lessons Learned from The Path Less Traveled™ Volume 1
Get the details at: Amazon.com


text:
Broadcast Media Spokespeople
Gareth Shaw
Gareth Shaw is one of Which?'s money experts, and our lead spokesperson on personal finance issues.
A journalist since 2007, Gareth worked for the Financial Times before joining Which? in 2010, going on to become editor of Which? Money magazine until 2015. He spent a year working for over 50s company Saga, before returning to Which? in 2017.
Follow Gareth on Twitter at @GarethRShaw
Harry Rose
Harry Rose is editor of Which? magazine. Prior to becoming editor, Harry edited Which? Money for three years, our monthly personal finance magazine. Harry also spent three years covering investments for the magazine, building on six years' experience working in the investment industry.
Follow Harry on Twitter at @Harry_Rose
Richard Headland
Richard Headland is editor-in-chief of Which? magazine. During his time at Which?, Richard has previously worked on technology and cars before being promoted to editor in 2013.
Sue Davies
Sue Davies is celebrating 20 years at Which? and is our Chief Policy Adviser on food issues. Because of her extensive work in this field at Which?, she was awarded an MBE for services to food safety.
Adam French
Adam is a consumer rights expert at Which?, and the editor of our online consumer rights advice website. Prior to becoming editor, Adam spent three years as a digital journalist at Which?. Adam has previously worked in commercial marketing roles as an editor and copywriter.
Follow Adam on Twitter @thatadamfrench


text:
Virtalis has proved that VR delivers vital time and cost financial savings throughout the whole asset administration lifecycle, together with the design, danger assessments, development, commissioning, handover, operation, sustaining, and decommissioning of bodily and infrastructure property. Believable: You really want to really feel like you're in your virtual world (on Mars, or wherever) and to keep believing that, or the illusion of virtual reality will disappear. This narrows down potential users to people who already own compatible Samsung phones, since buying one simply to make use of with the Gear VR pushes the worth to HTC Vive ranges.
eighty Philosophical implications of VR are mentioned in books, including Philip Zhai 's Get Actual: A Philosophical Journey in Virtual Reality (1998) and Digital Sensations: House, Identification and Embodiment in Virtual Reality (1999), written by Ken Hillis.
As the price of digital reality goes down and it becomes extra mainstream you may count on extra critical makes use of, comparable to schooling or productivity applications, to come back to the fore. Digital Reality by Howard Rheingold. Digital actuality has been promised for decades, but in my conversations with the highest builders within the subject, it quickly became clear that never earlier than have a lot money and expertise bet on its imminent arrival.
Virtual reality is the time period used to describe a three-dimensional, pc generated setting which will be explored and interacted with by a person. Architects used to construct fashions out of card and paper; now they're much more prone to build virtual reality computer models you may stroll by means of and explore.
Mychilo S. Cline argued in 2005 that through virtual reality techniques will probably be developed to influence human habits, interpersonal communication , and cognition seventy seven seventy eight 79 Within the guide The Metaphysics of Virtual Reality by Michael R. Heim , seven different ideas of digital reality are recognized: simulation, interaction, artificiality, immersion, telepresence , full-physique immersion , and community communication.


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Do you ever find it tiresome trying to get children to brush their teeth. The earlier you start instilling good oral health habits into your children the easier it will be.. it will just seem as normal to them as getting dressed on a daily basis. And remember, toddlers learn most of what they do through watching what you do, so include them in your daily tooth brushing routine. Learn more about how to create a healthy brushing routine with young children .
Power of Nature recommends using OraWellness HealThy Mouth Blend and OraWellness Shine Remineralizing Tooth Whitening Powder for better oral health.
Happi Earth Organic Laundry Liquid
Power of Nature Instagram
Nutritious vegetarian and vegan recipes
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Meridian Tooth Chart
Food Intolerance Testing Service
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text:
New Delhi: BCCI and Anushka Sharma's spokespersons quickly issued statements clarifying the news about Virat-Anushka marriage in Italy is just a rumor, there is no truth in it.
Worried media houses have written to both Anushka and Virat to get married soon or at least confirm we will marry within next six months otherwise lot of their effort will go for a toss.
"I am more worried how I will fill the TV show slots for my channel in the coming weekend. We have prepared a six hour show and I was planning to show it 2 times each on Saturday and Sunday," said Pratap Sharma, working as programming head of a leading news channel.
Mr. Sharma added, "Today's celebrities have no heart. Nahin to koi iss tarah se news ko kharij nahin karta. Itna kya jaldi tha. On Monday, they could have said this is just rumor, by then we would have shown our programs. This is not the first time Virat and Anushka are doing something like this, last few occasions whenever news came, they have done the same."
"Now I am compelled to show same Raju Srivastav youtube videos in repeat mode that we showed last weekend also. Acha kaam ka koi kadar nahin hai iss desh mein," said Mr. Sharma who was checking also how many speeches Rahul Gandhi and Modi will give in Gujrat and what will be their probable length.
"Mani Shankar Aiyar is our true friend for every season. He has given some sound bites, around which some debates we can arrange, but I am disappointed with Kohli and Anushka to say the least," said Mr. Sharma when one reporter of his channel came inside.
The reporter who did not know this marriage news is a rumor, had gone to Pallika Bazar to print a wedding card which they were supposed to claim during the show as Virat-Anushka's true wedding card, jo unke haath laga hai.


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Health care giant Anthem could be eligible for more than $50 million in incentives from the state if it meets certain benchmarks in establishing a technology center in Atlanta, according to an analysis by The Atlanta Journal-Constitution.
A state Department of Economic Development spokeswoman said the parent of Blue Cross Blue Shield of Georgia will file for what's known as Mega Project Tax Credits, which are reserved for high job-count projects.
Economic development officials and elected leaders say incentives often are a necessary part of winning corporate recruitment battles, but critics deride such perks as corporate welfare.
Anthem said Wednesday it plans to create more than 1,800 jobs at the new $20 million center in Bank of America Plaza.
"We have a long history in the state already and have made other significant investments including our Innovation Center in Tech Square and a new facility in Columbus," Gene Rodriguez, a spokeswoman for Anthem, said in an email. "So, we are committed to the state and its workforce. Our plan is to continue to grow our footprint in the state over the next six years and tap into its talent pipeline."
To receive the mega credits, companies must create at least 1,800 jobs and meet one of two other requirements: spending more than $450 million on a facility, or having an annual payroll of $150 million.
If Anthem meets the goals, the company could receive a $5,250 credit for each new jobs for five years. That could total $47.25 million over five years.
On Wednesday, the state also announced Anthem will receive a $3.6 million grant to help induce the move, boosting the potential total to $50.85 million, or more than $28,000 per job.
Local incentives also are possible. A spokesman for Invest Atlanta, the city's economic development arm, said the agency intends to ask the Atlanta City Council for an Economic Opportunity Fund grant, which comes from the city's general fund.
"While [an incentive package] was not the primary reason Atlanta was chosen as a core location, we are excited to partner with the state to build a relationship with the community while growing our footprint in the area," Anthem said in a statement.
The Anthem announcement was the 18th new or expanded technology or software development center announced in Georgia in the past year, state officials said. It's a boost for Atlanta both as a tech hub and for Bank of America Plaza, the city's tallest tower, which has been about half-empty for years.
The tower rises in what's known as an Opportunity Zone, an area designated for redevelopment. If Anthem does not meet the thresholds for mega credits, the company could still be eligible to receive Opportunity Zone credits totaling $3,500 per new job for five years.
Anthem's new tech hub will develop apps and other platforms to engage customers and help them shop for health plans and connect with care providers, company officials said.
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Get your glow on!
Bike the Night takes place this Saturday in the heart of Downtown Vancouver and offers thousands of cyclists the opportunity to ride in style after dark.
Presented by MEC, the fourth annual event is the province's largest of its kind, drawing more than 5,000 riders on an epic 10-kilometre journey through open city streets. The party starts and ends at Sunset Beach, and the Burrard Street Bridge will also be closed for the ride.
The event will offer music, food trucks, live entertainment and the Vancouver International Airport will be giving away thousands of MEC rechargeable lights and other decorations. Riders are also encouraged to rock glowing enhancements on their bodies and bikes such as lights, reflectors, and stickers, as the organizers hope to make the parade a luminous affair.
The pre-party begins at 6 p.m. at Sunset Beach where participants can enjoy tunes, games, food trucks, ping pong and giveaways. Stretch Vancouver will be offering a "Yoga for Cyclists" session. The ride starts at 7:45 p.m. for families, kids, slower riders, and other riders will start at 8 p.m.
Bike the Night presented by MEC
When: Saturday, Sept. 7, 6-9 p.m.
Where: Pre party at Sunset Beach Park, Beach Avenue and Bute Street
Cost: $20 tickets | $5 student special | Youth 16 and under ride free
All registered participants will also be entered to win a $500 gift card to MEC.
Find out more information and register for the ride here.
Click here for original story.


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5 Uses For
The Ultimate Marketing Tools for Your Book
It is always a joyful moment when a writer completes writing a book. To provide the successful marketing of a book, a writer should have an advertisement plan to make his book popular. But the joy might be short-lived if the writer finds out that his marketing tools are inadequate. In most cases, readers who have no established followers are likely to be disappointed because of lack of exposure. Nowadays there are very many authors, and this makes it necessary to use all the tools at your disposal to stand out. The major tools in marketing your book are indicated in this article.
The first important promotional tool is WordPress. To market your book successfully the author needs to have a website where he can build an online presence. Websites are vital when the author wants to interact with his readers and potential followers. WordPress comes in handy when an author is looking to develop a site. WordPress is an essential marketing tool for which the author can provide a sneak peek of the contents of the book to attract readers and where readers can comment on the book. Creating a website using WordPress does not require special skills like graphic design. There are templates for creating a website in WordPress, and all that is required is your information to fill them. For those who are not financially stable, there is also a platform for you in WordPress. The site also gives tutorials and information about their services.
The second tool you need is social media. Social press is an essential marketing tool for online marketing campaigns. There are several social media tools where authors can set up their presence including Facebook, Instagram and Twitter and more. Social media platforms are easily manageable promotional tools. Holders of social media account for promotion have to engage their followers regularly and remain active on the sites. The author can also create an online community where he interacts with his satisfied readers and urges them to promote the book if they find it interesting.
To conclude, you need to create an Amazon account. Through Amazon, writers are given a platform to sell their books which is essential. Amazon is suitable for both new and established writers. Amazon deals with selling the physical book from their stores and even e-books of the same books.
Amazon is very efficient because it can be linked with other online marketing tools like Facebook which direct potential buyers to the site. Hence the author should ensure that his Amazon profile, book description, and reviews are optimal to attract more readers. There are articles and blogs which give more tips on setting up the best amazon account.


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When an abuser attempts to get custody of the children, it can lead to the most agonizing situations. These links might be helpful for those facing this nightmare.
Please note that apart from Lundy's work, I don't have enough knowledge of any of these organizations to recommend or endorse their work. So please use this merely as a springboard to do your own research — test all things!
- Part Three of Domestic Violence in Popular Culture, presentation by Lundy Bancroft — Lundy speaks about abusers, courts and child custody.
- Child Custody Justice by Lundy Bancroft
- Battered Mothers Custody Conference
- Domestic Violence Legal Empowerment Custody and Abuse Project
Note: when researching the issue of child custody justice, you may get the impression that there is disharmony between the advocates. I have not had the time to drill down into it all, but here is a post by Lundy Bancroft that you…
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Before finalizing plans to travel to India it is advisable to check the travel advisory notifications of respective countries. However, while planning to travel to India visitors may also note the followings:
Foreign nationals wishing to travel to India are required to possess a valid passport of their country and a valid Indian visa. However, nationals of Nepal and Bhutan do not require visa to enter India and nationals of Maldives do not require visa for entry in India for a period up to 90 days (a separate visa regime exists for diplomatic/official passport holders). Visas are granted through various Indian missions abroad. However, the Embassy/High Commission/Consulate reserves the right on granting and deciding the type/duration of visa irrespective of the fees tendered at the time of making the application.
Specific visas are granted for different purposes:
Visa for NRIs and PIOs
Persons of Indian Origins and Non-Resident Indians who possess either OCI (Overseas Indian Citizenship) or PIO card don't need to apply separately for an Indian Visa. OCI and PIO cards give them the freedom to visit India without visa within rules. However, those NRIs and PIOs who don't have OCI or PIO card can apply for and get Indian visa through the procedure mentioned above.


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How can data tell a story that keeps a vulnerable person safe?
Read more about how the councils that are piloting a different approach are unlocking the potential of early intervention and transforming the way services address the needs of the most vulnerable people.
Please enter your details below to download EY's report.


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I organize private tours in Porto
Check this page for more information or send an email to email@example.com.
As Der Spiegel quite rightfully wrote, Porto is being flooded by tourists which is phenomenal for the local economy, but it also brings its downsides, such as an up rise of tourists traps.This is the main reason why I think it is my moral obligation to write about the few surviving traditional restaurants in Porto, hoping that you might like them as much as we do and contribute to its survival.
A few things before we dive into the amazing world of the traditional restaurants in Porto:
- Don't expect vegetarian / vegan / gluten free options in traditional restaurants in Porto. Out of courtesy they might come up with an omelet or with a fresh salad to accommodate your dietary restrictions – but then again you will not really be taking advantage of eating in such restaurants, would you?
Therefore if you are a vegetarian or a vegan, I strongly recommend you to stick to the restaurants mentioned of our Survival Guide for Vegetarians, in order to avoid having a disappoint meal.
- These are traditional restaurants in Porto, so don't expect to find proper English menus. Instead, make sure you are patient enough to use google translator and creative communication techniques with the waiters!
Before reading this article: have you already decided where to stay? If not, read my article about the Best neighborhoods to stay in Porto! There I give you plenty of recommendations of great hotels and apartments in Porto.
With this in mind, it's time to unveil the best traditional restaurants in Porto:
O Ernesto
Rua da Picaria, 85
I've been going to Ernesto since I am a little girl, to eat hake fillets with my family. In fact, Ernesto is one of the few Portuguese restaurants that has been part of the families from Porto from generation to generation.The restaurant's menu consists of the traditional Portuguese flavours couple with daily dishes that depend on the day of the week:
- Monday: bacalhau à Gomes Sá (traditional codfish dish from Porto) and coelho assado no forno (roasted rabbit)
- Tuesday: tripas à moda do porto (beef's stomach cooked in a stew with white beans, carrots and cumin)
- Wednesday: filetes de pescada com arroz de feijão, de grelos ou de legumes (hake fillets with vegetables' or beans' rice)
- Thursday: filetes de polvo com arroz do mesmo (octopus fillets) and cozido à portuguesa (amazing Portuguese dish with different types of meats and sausages served with rice, potatoes and boiled vegetables)
- Friday: arroz de pato (roasted duck rice)
- Saturdays: rojões (pork cubes cooked in pork lard) and cabrito assado no forno (baby goat cooked in the oven, it's to die for!)
There are those who recommend Ernesto's famous fruit salad to finish off the meal, but I prefer a burnt crème brûlée. It never fails to amaze me!
How to get there: Aliados metro station is a 5min walk away.
Cozinha do Manel
Rua do Heroísmo, 215
One of the most traditional restaurants in Porto is hidden in Bonfim, the neighborhood that divides the tourist area from the the oriental part of the city. I'm talking about Cozinha do Manel, a restaurant that has been cheering Porto's palates for the past 30 years.
Cozinha do Manel is one of the most welcoming traditional restaurants in Porto: you enter through a narrow hallway, from where you can take a sneak peek at the delicious desserts displayed on the fridge. The hallway gives access to a cozy dining room, covered in granite and tiles.
The starters are simply to die for: alheira assada (traditional Portuguese sausage) from Trás os Montes and queijo da Serra (soft sheep cheese) are a mandatory presence at my dining table.
As main dishes I recommend cabrito assado no forno (baby goat cooked in the oven – you need to order it with at least two days in advance!), arroz de pato (roasted duck rice) and vitela assada (roasted veal – even though, to be honest, it always feels a bit rubbery). If you prefer to go for fish, then I highly recommend to try bacalhau assado no forno (oven roasted codfish) or hake / octopus fillets.
You should not leave Cozinha do Manel without trying its fabulous rabanadas – the Portuguese twist on French toasts -, served throughout the entire year.
How to get there: Heroísmo metro station is a 5min walk away.
Adega do Carregal
Travessa do Carregal, 102
I ate my very first rojões in Adega do Carregal, a couple of years ago. Even though I am not a huge fan of this traditional Portuguese dish, that consists of pork's cubes cooked in pork lard, I couldn't resist to try one of the famous dishes at one of the best traditional restaurants in Porto.
At Adega do Carregal I recommend you to try the codfish dishes as well as posta à mirandesa (an amazing steak with a olive oil & vinegar sauce). There are always daily dishes like the infamously good tripas à moda do Porto (beef's stomach cooked in a stew with white beans, carrots and cumin) and anho assado no forno (roasted lamb).
The service is very attentive, without fake pretentiousness; we're at home.
How to get there: Aliados metro station is a 10min walk away.
Garfada do Ouro
Rua Senhor da Boa Morte, 84
This is one of the well-kept secrets of Porto: Helder, one of the former owners of the amazing restaurant O Carteiro, fell in love with an Italian woman and they both created a new project that rescues the concepts of sustainable food using local, seasonal and biological ingredients.
Helder opens the doors if his house – and what a house, located in Foz Velha – every Tuesday and Thursday night.
They are often wonderful events for meeting new people, but it is also a good place to have private dinners with friends or business colleagues.
You have to subscribe to attend these amazing dinners so there is no point in showing up unannounced. To book your place, send an email to email@example.com. The dinners cost € 60 / person, which include all dishes plus wines. Do not forget to use the code PORTOALITIES5 which gives you a discount of 5 € / person on your meal.
In order to have dinner at Helder's place just send an email to firstname.lastname@example.com to check his availability.
How to get there: take the tram Line 1 until Ouro. The house is a 2min walking distance.
Rua da Picaria, 26 | Rua Dr. Alberto Macedo, 437
Purists will have to forgive me, but the way I see it, one could not miss Badalhoca in this list of best traditional restaurants in Porto.It is true that Badalhoca is not quite a restaurant, it a down-to-earth tavern where we lay our appetite in smoked ham sandwiches and small plates of pork liver baits or octopus cooked with green sauce. Always watered with a tap Super Bock – the traditional beer from Porto-, as if we order a soft drink we are greeted with a bell ring that will embarrass you in front of everyone. It's really worth going there!
How to get there: For the downtown location, Aliados metro station is a 5min walk away. For the other location, take bus 502 (direction Matosinhos Mercado) until Gomes da Costa bus stop. The restaurant is 6min walking distance.
O Gaveto
Rua Roberto Ivens, 824 | Matosinhos
O Gaveto is undoubtedly my favorite restaurant in Porto. It helps the fact that fish and seafood always have primacy on my table and that Gaveto serve both in an irreproachable way.
In this beautiful (almost) seafront restaurant, I strongly recommend you to try the amêijoas à Bulhão Pato (steamed clams with a garlic and white wine sauce – who would have guessed that such a simple dish could be so delicious!), the monkfish rice and the pièce de resistance, the seafood açorda (bread stew with seafood and loads of spices).
In my opinion Gaveto is also one of the most interesting traditional restaurants in Porto as it has a superb wine list, making it a mandatory stop for wine lovers like myself.
How to get there: Matosinhos Sul metro station is a 5min walk away.
O Buraquinho
Praça dos Poveiros, 33
Another controversial option on this list of the best traditional restaurants in Porto is Buraquinho, an impeccable tavern in the Bonfim neighborhood.
The reason that makes me come back to this tiny room in a basement, practically without ventilation and with very few tables available, is the honesty of the flavors served: the combinados dishes (tiny plates filled with different types of Portuguese sausages and cured meats, alheira com ovo (Portuguese sausage served with a fried egg on top), mushrooms sautéed in white wine make out for a meal shared among friends.
At Buraquinho, I only regret the absence of a proper wine list and a couple desserts, which would prolong my stay in this secret corner of Porto.
How to get there: metro station 24 Agosto is a 5min walk away.
Manuel Alves
Avenida de Fernão de Magalhães, 782
Hidden from the tourist sights, in the Paranhos neighborhood, is one of the best traditional restaurants in Porto, Manuel Alves.The menu of this restaurant is very diverse, consisting of countless fish options (baked shrimp and cod are always safe bets) and meat (we particularly like the veal medallions and the roasted beef).
How to get there: Campanhã and Estádio do Dragão metro stations are both a 15-minute walk away.
Extra: Go on a private tour in Porto
The best way to get to know Porto is to go on a private tour with me!Check this page to learn more about my tours in Porto, or simply send an email to email@example.com. I will shortly get back to you with a tailor made tour designed specifically for you.It will be a pleasure to welcome you in Porto!
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I have listed a new property at 310 13727 74TH AVE in Surrey.
Bright and open 2 bedroom and 2 full bathroom unit in Kings Court. This bright and spacious home features large windows to let in lots of light, has been updated with fresh paint and is move in ready. Enjoy BBQs on your huge patio with access from both living area and master bedroom in the summer, or warm up beside your gas fireplace in the winter. This worry free, well managed building features thermal pane windows, concrete sub flooring, prewired alarm system and insuite laundry. Conveniently located within walking distance to all amenities, shopping and transportation. Gas and hotwater are included in the already low maintenance fees. Hurry, this one won't last!