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The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)→Akt→mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.
Author Summary
Growth factor pathways are required for normal development but are often inappropriately activated in many cancers. One growth-factor–sensitive pathway of increasing interest to cancer researchers relies on the mammalian target of rapamycin (mTOR), a kinase that (like all kinases) delivers phosphate groups from ATP to amino acid residues of downstream proteins. TOR proteins were first discovered in yeast as the cellular targets of rapamycin, a small, naturally occurring molecule derived from bacteria that is widely used as an immunosuppressant and more recently in some cancer therapies. The study of TOR proteins has relied heavily on the use of rapamycin, but rapamycin does not directly inhibit TOR kinase activity; rather, rapamycin influences TOR's enzymatic activities by binding to a domain far from the kinase's active site. Some mTOR functions are resistant to rapamycin, as a result of the kinase activity of one kind of multiprotein complex, the mTOR complex 2 (mTORC2), whereas rapamycin-sensitive functions of mTOR are due to the mTOR complex 1 (mTORC1). We have developed new inhibitors of mTOR that bind to the ATP-binding site of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2 without inhibiting other kinases. Unexpectedly, these inhibitors had profound effects on protein synthesis and cell proliferation due to their inhibition of mTORC1 rather than mTORC2. We found that the phosphorylation of a protein that controls protein synthesis, the mTORC1 substrate 4E binding protein (4EBP) is partially resistant to rapamycin but fully inhibited by our new inhibitors. The finding that 4EBP phosphorylation is resistant to rapamycin suggests that active-site inhibitors may be more effective than rapamycin in the treatment of cancer and may explain why rapamycin is so well tolerated when taken for immunosuppression.
Citation: Feldman ME, Apsel B, Uotila A, Loewith R, Knight ZA, Ruggero D, et al. (2009) Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2. PLoS Biol 7(2): e1000038. https://doi.org/10.1371/journal.pbio.1000038
Academic Editor: Tony Hunter, Salk Institute for Biological Studies, United States of America
Received: November 19, 2008; Accepted: January 12, 2009; Published: February 10, 2009
Copyright: © 2009 Feldman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: BA thanks the UCSF Cancer Research Coordinating Committee Fellowship for funding, and MF thanks the ARCS Foundation for funding. This work was supported by the Howard Hughes Medical Institute (HHMI). Neither HHMI nor the other funding agencies had a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: MEF, BA, ZAK, and KMS are inventors on patent applications owned by UCSF related to PP242 and licensed to Intellikine. ZAK and KMS are consultants to Intellikine.
Abbreviations: IRES, internal ribosome entry site; MEF, mouse embryonic fibroblast; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; RNAi, RNA interference; TORKinibs, TOR kinase domain inhibitors
The mammalian target of rapamycin (mTOR) is a serine-threonine kinase related to the lipid kinases of the phosphoinositide 3-kinase (PI3K) family. mTOR exists in two complexes, mTORC1 [1,2] and mTORC2 [3,4], which are differentially regulated, have distinct substrate specificities, and are differentially sensitive to rapamycin. mTORC1 integrates signals from growth factor receptors with cellular nutritional status and controls the level of cap-dependent mRNA translation by modulating the activity of key translational components such as the cap-binding protein and oncogene eIF4E [5].
mTORC2 is insensitive to rapamycin, and selective inhibitors of this complex have not been described. Partly because acute pharmacological inhibition of mTORC2 has not been possible, the functions of mTORC2 are less well understood than those of mTORC1. mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt [3,6] and SGK [7] although other kinases, including DNA-PK and Ilk, have also been implicated in Akt hydrophobic motif phosphorylation [8–11]. Growth factor stimulation of PI3K causes activation of Akt by phosphorylation at two key sites: the activation loop (T308) and the C-terminal hydrophobic motif (S473). Active Akt promotes cell survival in many ways, including suppressing apoptosis, promoting glucose uptake, and modifying cellular metabolism [12]; consequently, there is significant interest in identifying the kinase(s) responsible for each activating phosphorylation, the relationship between these phosphorylation sites, and the role of differential Akt phosphorylation on Akt substrate phosphorylation. Of the two phosphorylation sites on Akt, activation loop phosphorylation at T308, which is mediated by PDK1, is indispensable for kinase activity, whereas hydrophobic motif phosphorylation at S473 enhances Akt kinase activity by approximately 5-fold [13].
The disruption of mTORC2 by different genetic and pharmacological approaches has variable effects on Akt phosphorylation. Targeting mTORC2 by RNA interference (RNAi) [6,14], homologous recombination [15–17], or long-term rapamycin treatment [18] results in loss of Akt hydrophobic motif phosphorylation (S473), strongly implicating mTORC2 as the kinase responsible for phosphorylation of this site. RNAi targeting mTORC2 and long-term rapamycin result in loss of Akt phosphorylation on its activation loop (T308), but this phosphorylation remains intact in mouse embryonic fibroblasts (MEFs) lacking the critical mTORC2 component SIN1. It cannot be inferred from this genetic data whether acute pharmacological inhibition of mTORC2 would block the phosphorylation of Akt only at S473, resulting in partial Akt deactivation, or also disrupt phosphorylation at T308, resulting complete Akt inhibition.
Several small molecules have been identified that directly inhibit mTOR by targeting the ATP binding site; these include LY294002, PI-103, and NVP-BEZ235 [19–22]. These molecules were originally discovered as inhibitors of PI3Ks and later shown to also target mTOR. Because all of these molecules inhibit PI3Ks and mTOR with similar potency, they cannot be used to selectively inhibit mTOR or PI3Ks in cells. Indeed, because mTORC1 and mTORC2 function downstream of PI3Ks in most settings, it is unclear to what extent the ability of these molecules to block the activation of signaling proteins such as Akt reflects PI3K versus mTOR inhibition. It is possible that some of the functions attributed to PI3Ks using the classical inhibitor LY294002 are a consequence of mTOR inhibition [19,23], but it is has not been possible address this, because small molecules that inhibit mTOR without inhibiting PI3Ks have not been available.
We recently reported the synthesis of pyrazolopyrimidines that inhibit members of the PI3K family, including mTOR [24]. Two of these molecules, PP242 and PP30, are the first potent, selective, and ATP-competitive inhibitors of mTOR. Unlike rapamycin, these molecules inhibit both mTORC1 and mTORC2, and, unlike PI3K family inhibitors such as LY294002, these molecules inhibit mTOR with a high degree of selectivity relative to PI3Ks and protein kinases. To distinguish these molecules from the allosteric mTORC1 inhibitor rapamycin, we are calling them "TORKinibs" for TOR kinase domain inhibitors. The dual role of mTOR within the PI3K→Akt→mTOR pathway as both an upstream activator of Akt and the downstream effector of pathway activity on cell growth and proliferation has excited interest in active-site inhibitors of mTOR [25–30]. We describe here the biological activity of these molecules.
Another small-molecule ATP-competitive mTOR inhibitor called Torin1 was reported while our manuscript was in the process of publication [56].
Specific Active-Site Inhibition of mTOR by the TORKinibs PP242 and PP30
PP242 and PP30 inhibit mTOR in vitro with half-maximal inhibitory concentrations (IC50 values) of 8 nM and 80 nM, respectively. As expected for active-site inhibitors, PP242 and PP30 inhibit mTOR in both mTORC1 and mTORC2 (Table S1). Both compounds are selective within the PI3K family, inhibiting other PI3Ks only at substantially higher concentrations (Figure 1). Testing of PP242 against 219 purified protein kinases at a concentration 100-fold higher than its mTOR IC50 value revealed exceptional selectivity with respect to the protein kinome; most protein kinases were unaffected by this drug, and only four—PKC-alpha, PKC-beta, RET, and JAK2 (V617F)—were inhibited more than 80% [24]. We determined IC50 values for PP242 against these kinases in vitro using purified proteins. In these assays, PP242 was relatively inactive against PKC-beta, RET, or JAK2 but inhibited PKC-alpha with an in vitro IC50 of 50 nM (Figure 1). Importantly, PP30 showed no activity against PKC-alpha or PKC-beta in the same assay (Figure 1). These data indicate that PP242 is a highly selective inhibitor of mTOR and that PP30 can be used to confirm that the effects of PP242 are due to inhibition of mTOR and not PKC-alpha. The availability of a second structurally dissimilar mTOR inhibitor—PP30—provides additional control for unanticipated off-targets of PP242.
Inhibition of mTORC2 and Akt Phosphorylation by TORKinibs
We characterized the effect of PP242 on the PI3K→Akt→mTOR pathway. PP242 and PP30 both inhibited insulin-stimulated phosphorylation of Akt at S473, confirming that mTOR kinase activity is required for hydrophobic motif phosphorylation (Figure 2A). The inhibition of mTOR by PP242 and PP30 also resulted in loss of Akt phosphorylation at T308, but significantly higher doses of PP242 and PP30 were required to inhibit T308 as compared with S473 (Figure 2A and 2B). PP242 inhibited S473-P and T308-P at both early and late time points after insulin stimulation, indicating that the differential sensitivity of these sites to PP242 does not reflect differing kinetics of phosphorylation (Figure S1). By comparison, the PI3K inhibitor PIK-90, which does not inhibit mTOR, inhibited the phosphorylation of both Akt sites equipotently (Figure 2B), as observed previously [21].
(A) Serum-starved L6 myotubes were pre-treated with kinase inhibitors prior to stimulation with insulin for 3 min. Lysates were analyzed by Western blotting.
(B) PP242 inhibits pS473 (red) of Akt more potently than pT308 (gray). Serum-starved L6 myotubes were treated with kinase inhibitors prior to stimulation with insulin for 10 min. Akt phosphorylation was measured by in-cell Western and is shown relative to serum starvation and insulin stimulation (n = 3 for each inhibitor dose). EC50 values from the best fit curves are plotted. ***p < 0.001, F test. EC50 values for PIK-90 on pS473 and pT308 were not significantly different (p = 0.2, F test).
We sought to confirm that the loss of T308-P caused by PP242 and PP30 results from inhibition of mTOR-mediated phosphorylation of S473, rather than from inhibition of an off-target kinase, or from an effect of mTOR inhibition unrelated to S473-P. To do this, we examined the effect of PP242 on T308 phosphorylation in two situations in which Akt could not be phosphorylated on S473.
First, we overexpressed S473A mutant Akt and stimulated these cells with insulin (Figure 3A). S473A Akt was phosphorylated on T308 to a similar level as wild-type, yet in contrast to the wild-type, T308-P on S473A Akt was not inhibited by PP242. The lack of effect of PP242 on S473A Akt confirms that PP242 inhibition of pT308 requires S473 and also that PP242 does not inhibit PDK1 in cells, as was suggested by direct testing of PDK1 in vitro (Figure 1).
(A) pT308 is not inhibited by PP242 in cells overexpressing S473A Akt. HEK293 cells were transfected with wild-type Akt, S473A Akt, or not transfected (Mock) and were treated with 2.5 μM PP242 or 625 nM PIK-90 as indicated prior to insulin stimulation. Lysates were analyzed by Western blotting. Quantitation of pT308 relative to insulin treated cells overexpressing wild-type Akt (lane 2) is shown below that blot. Data are representative of two independent experiments.
(B) pT308 is not inhibited by PP242 in SIN1−/− MEFs, which lack pS473. Primary wild-type (WT) and SIN1−/− MEFs were pre-treated with 625 nM PIK-90, 10 μM BX-795, or 100 nM rapamycin for 24 h, 100 nM rapamycin for 30 min, or the indicated concentrations of PP242 prior to stimulation with insulin. Lysates were analyzed by Western blotting.
As a further test of the specificity of PP242 and the requirement for functional S473 phosphorylation in order for PP242 to inhibit T308-P, we examined the effect of PP242 on the phosphorylation of Akt in primary MEFs from embryos that lack SIN1 [16] (Figure 3B). SIN1 is a component of mTORC2, and knockout of SIN1 compromises the physical integrity of mTORC2 leading to a complete loss of Akt phosphorylation at S473 without affecting its phosphorylation at T308. Consistent with our results from L6 cells, PP242 inhibited the phosphorylation of Akt at both S473 and T308 in wild-type MEFs. By contrast, PP242 had no effect on the phosphorylation of T308 in SIN1−/− MEFs that lack mTORC2. Furthermore, PP242 had no effect on the constitutive phosphorylation of the turn motif of Akt at T450 [16,31]. As a further comparison, we examined the effect of long-term rapamycin, which is known to block the assembly of mTORC2 is some cell lines [18]. Similar to PP242, long-term rapamycin treatment of wild-type MEFs inhibited S473-P and reduced the phosphorylation of T308-P, as was seen previously [18]. Importantly, the PI3K inhibitor PIK-90 and the PDK1 inhibitor BX-795 [32] blocked phosphorylation of T308 in SIN1−/− MEFs, indicating that the failure of PP242 to block T308 in SIN1−/− MEFs does not reflect a general resistance of T308 to dephosphorylation in cells that lack mTORC2. From these data, we conclude that PP242′s effect on T308-P is dependent on its inhibition of Akt phosphorylation by mTOR at S473. It remains unclear why mTORC2 knockout cells, but not cells treated with RNAi or pharmacological inhibitors of mTORC2, are able to retain T308 phosphorylation in the absence of phosphorylation at S473. However, there are a growing number of examples in which genetic deletion of a kinase results in compensatory changes that mask relevant phenotypes observed with the corresponding small molecule inhibitor [33].
Akt Substrate Phosphorylation Is Only Modestly Inhibited by PP242
Akt requires phosphorylation at both S473 and T308 for full biochemical activity in vitro [13], but it is unclear whether all of the cellular functions of Akt require it to be dually phosphorylated. Singly phosphorylated (T308-P) Akt from SIN1−/− MEFs is competent to phosphorylate the cytoplasmic Akt substrates GSK3 and TSC2, but not the nuclear target FoxO [16]. Because low concentrations of PP242 inhibit the phosphorylation of S473 and higher concentrations partially inhibit T308-P in addition to S473-P, we used PP242 to examine whether some substrates of Akt are especially sensitive to loss of S473-P (Figure 4). We compared PP242 to the PI3K inhibitor PIK-90 and the allosteric Akt inhibitor Akti-1/2 [34], which inhibit the phosphorylation of Akt at both sites. In contrast to PIK-90 and Akti-1/2, which completely inhibited the phosphorylation of Akt and its direct substrates, PP242 only partially inhibited the phosphorylation of cytoplasmic and nuclear substrates of Akt. This suggests that phosphorylation of the Akt substrates we examined is only modestly sensitive to loss of S473-P. A caveat of comparing Akt substrates in Sin1−/− MEFs with PP242-treated cells is the different turn motif (T450-P) status in these two conditions (Figure 3B).
Lysates from L6 myotubes treated with kinase inhibitors and stimulated with insulin were analyzed by Western blotting. Quantitation of pAkt and pTSC2 relative to the insulin control (lane 2) is show below these blots.
In contrast to Akt, which maintains T308-P, SGK activity is completely inhibited by genetic disruption of mTORC2 [7]. Because SGK can phosphorylate FoxO and its activity is completely inhibited by disruption of mTORC2, it was suggested that the loss of FoxO phosphorylation in SIN1−/− MEFs indicates that FoxO is primarily phosphorylated by SGK rather than Akt [7]. Because Akti-1/2 does not inhibit SGK [34] but inhibits FoxO1/O3a phosphorylation at T24/T32 in L6 myotubes (Figure 4), our data suggests that the major kinase for T24/T32 of FoxO1/O3a in L6 myotubes is Akt and not SGK.
PP242 Does Not Have an Obvious Effect on Actin Stress Fibers
TORC2 is required for the generation of a polarized actin cytoskeleton in yeast [35]. Previous analysis of mTORC2 function using RNAi revealed a role for mTORC2 in the control of the actin cytoskeleton [3,4], yet these findings were not confirmed in primary MEFs lacking mTORC2 [15,17]. We examined actin stress fibers in NIH 3T3 cells (Figure 5) and in primary MEFs (unpublished data) treated with PP242. After 8 h of treatment with PP242, we found no obvious effect on the morphology or abundance of actin stress fibers (Figure 5), suggesting that mTORC2 activity is not required for the maintenance of actin stress fibers in these cells. That PP242 didn't obviously affect the morphology or abundance of actin stress fibers, does not rule out a role for mTOR in the control of the actin cytoskeleton, but it does show that pharmacological inhibition of mTORC2 does not affect the obvious changes in actin structure seen with RNAi.
(A) NIH 3T3 cells were stained for actin with Alexa 488-phalloidin (green) and for DNA with DAPI (blue). Images are representative of greater that 100 cells.
(B) Differential inhibition of cell proliferation by PP242 and rapamycin does not require mTORC2. Proliferation of primary MEFs cultured for 3 d in the presence of kinase inhibitors was assayed by resazurin fluorescence (RF) and is shown in arbitrary units.
PP242 Inhibits Proliferation More Completely Than Rapamycin
We next measured the effect of dual mTORC1/mTORC2 inhibition by PP242 on the proliferation of primary MEFs (Figure 5B). For this analysis, we compared PP242 to selective mTORC1 inhibition by rapamycin. Rapamycin was tested at concentrations above its mTOR IC50, and at all concentrations tested, it inhibited growth to the same extent. By contrast, PP242 had a dose-dependent effect on proliferation and at higher doses was much more effective than rapamycin at blocking cell proliferation. The ability of PP242 to block cell proliferation more efficiently than rapamycin could be a result of its ability to inhibit mTORC1 and mTORC2, because rapamycin can only inhibit mTORC1. To test this possibility, we measured the effects of both compounds on the proliferation of SIN1−/− MEFs, which lack mTORC2. In SIN1−/− MEFs, rapamycin was also less effective at blocking cell proliferation than PP242. That PP242 and rapamycin exhibit very different anti-proliferative effects in SIN1−/− MEFs suggests that the two compounds differentially affect mTORC1.
Rapamycin-Resistant mTORC1
mTORC1 regulates protein synthesis by phosphorylating the hydrophobic motif of p70S6-Kinase (S6K) at T389 and the eIF4E-binding-protein, 4EBP1, at multiple sites. Our proliferation experiments suggest that rapamycin and PP242 have distinct effects on mTORC1. We compared the effects of acute treatment with rapamycin and PP242 on S6K, ribosomal protein S6 (S6), and 4EBP1 phosphorylation (Figure 6A) to see if these inhibitors differentially affect the phosphorylation of these canonical substrates of mTORC1. Both rapamycin and PP242 inhibited the phosphorylation of S6K and its substrate S6, and neither rapamycin nor PP242 affected the phosphorylation of 4EBP1 on T70 (Figure S2A). In contrast, PP242 fully inhibited the phosphorylation of 4EBP1 at T36/45 and S65, whereas rapamycin only had a modest affect on these same phosphorylations. Treatment of cells with PP30 was also effective at reducing the phosphorylation of 4EBP1 at T36/45 (Figure S3), indicating that the block of T36/45 phosphorylation by PP242 is due to its inhibition of mTOR and not PKC-alpha. PIK-90 did not reduce the phosphorylation of 4EBP1 at T36/45, demonstrating that inhibition of PI3K and Akt activation alone is not sufficient to block the phosphorylation of 4EBP1 at T36/45 (Figure S3).
(A) Western blots of lysates from L6 myotubes treated with kinase inhibitors and stimulated with insulin for 10 min.
(B) Western blots of lysates from Figure 3B. Actin loading control is repeated here for clarity.
The enhanced dephosphorylation of 4EBP1 caused by PP242 as compared with rapamycin could be due to incomplete inhibition of mTORC1 by rapamycin or involvement of mTORC2 in the phosphorylation of 4EBP1. To examine these alternatives, we analyzed the effect of PP242 and rapamycin on the phosphorylation of 4EBP1 in SIN1−/− MEFs that lack mTORC2 (Figure 6B). SIN1−/− MEFS showed higher levels of p4EBP1, suggesting that due to the lack of mTORC2, these cells have more mTORC1 activity, although stronger S6K phosphorylation in wild-type cells contradicts this simple interpretation. Despite an increase in p4EBP1 in SIN1−/− compared with wild-type MEFs, shorter exposures of the p4EBP1 blots (Figure S2B) show that PP242 inhibits p4EBP1 with the same potency in both cells. The fuller inhibition of p4EBP1 by PP242 than by rapamycin in wild-type and SIN1−/− MEFs indicates that the presence of mTORC2 is not required for rapamycin and PP242 to have distinct effects on 4EBP1 phosphorylation, and suggests that PP242 is a more complete inhibitor of mTORC1 than rapamycin.
Inhibition of Translation by TORKinibs
While the precise role of S6K in translation control is still poorly understood, it is known that the hypophosphorylated 4EBP1 protein acts as negative regulator of the major cap-binding protein eIF4E. We directly assessed the effect of PP242 on cap-dependent translation downstream of mTOR activation. The phosphorylation of 4EBP1 by mTOR in response to growth factor and nutrient status causes it to dissociate from eIF4E allowing eIF4G and associated factors to bind to the 5' cap, recruit the 40S subunit of the ribosome, and scan the mRNA for the start codon to initiate translation. The phosphorylation of 4EBP1 by mTOR is complicated in that it occurs at multiple sites, and not all sites are equally effective at causing dissociation of 4EBP1 from eIF4E [36]. Furthermore, a hierarchy is thought to exist whereby the N-terminal threonine phosphorylations at 36/45 precede and are required for the C-terminal phosphorylations at S65 and T70 [37,38]. Phosphorylation at S65 causes the greatest decrease in affinity of 4EBP1 for eIF4E [39,40], and S65 is probably the most important site in cells for dissociation of 4EBP1 from eIF4E [41], but other sites are also important [36,42].
We examined the effect of PP242 on the active eIF4E initiation complex of translation by using a cap-binding assay. eIF4E binds tightly to beads coated with the cap analogue 7-methyl GTP (m7GTP), allowing proteins bound to eIF4E to be examined. Rapamycin caused partial inhibition of the insulin-stimulated release of 4EPB1 from eIF4E (Figure 7A), consistent with its partial inhibition of S65 phosphorylation (Figure 6A). The rapamycin-induced retention of 4EBP1 was accompanied by a loss of recovery of eIF4G, because the binding of 4EBP1 and eIF4G to eIF4E are mutually exclusive. In contrast, treatment with PP242 caused a much larger retention of 4EBP1, raising the retention of 4EBP1 above the level seen in unstimulated serum-starved cells, which are known to have low levels of protein translation [43].
(A) Cap-binding proteins in lysates from Figure 6A were purified by 7-methyl GTP (m7GTP) affinity and analyzed by Western blotting.
(B) Primary MEFs were transfected with a bicistronic reporter vector. The ratio of renilla (cap-dependent) to firefly (IRES-dependent) luciferase activity was measured after incubation overnight in either 10% serum (steady state) or with the indicated inhibitors in the presence of 10% serum (n = 3). *p < 0.05, **p < 0.01, ANOVA with Tukey's post test.
(C) Primary MEFs were incubated overnight as in (B) prior to labeling new protein synthesis with 35S. Newly synthesized proteins were separated by SDS-PAGE, transferred to nitrocellulose and visualized by autoradiography.
(D) Newly synthesized protein from three experiments as in (C) was quantified. *p < 0.05, ANOVA with Tukey's post test.
Translation initiation depending on eIF4E activity is the rate-limiting step in cap-dependent protein translation [44]. PP242 caused a higher level of binding between 4EBP1 and eIF4E than rapamycin (Figure 6A), suggesting that cap-dependent translation will be more highly suppressed by PP242 than by rapamycin. To quantify the efficiency of cap-dependent translation in the presence of PP242 and rapamycin, we used the well-established bicistronic reporter assay where translation initiation of the first cistron is dependent on the 5′ cap, whereas initiation of the second cistron depends on a viral internal ribosome entry site (IRES) that bypasses the need for cap-binding proteins such as eIF4E [45]. PP242 caused a significant decrease in cap-dependent, but not IRES-dependent (Figure S4), translation, whereas rapamycin did not have a statistically significant effect on cap-dependent translation (Figure 7B), consistent with the modest effect of rapamycin on 4EBP1 phosphorylation (Figure 6A). Based on this assay, inhibition of mTOR and p4EBP1 reduces cap-dependent translation by about 30%, suggesting that cap-dependent translation is only partially inhibited by hypophosphorylated 4EBP1. The majority of protein synthesis is thought to be cap-dependent [44], and consistent with this we find that PP242 also reduces total protein synthesis by about 30%, whereas rapamycin does not have a significant effect (Figure 7C and 7D).
Inhibition of mTORC1 and mTORC2 In Vivo
Mouse knock-outs of mTORC1 or mTORC2 result in embryonic lethality and thus it has been difficult to examine the effects of loss of mTOR in animals. To begin to explore the tissue specific roles of mTORC1 and mTORC2 and confirm the pathway analysis from cell culture experiments, we treated mice with PP242 and rapamycin and examined the acute effect of these drugs on insulin signaling in fat, skeletal muscle, and liver tissue (Figure 8). In fat and liver, PP242 was able to completely inhibit the phosphorylation of Akt at S473 and T308, consistent with its effect on these phosphorylation sites observed in cell culture. Surprisingly, PP242 was only partially able to inhibit the phosphorylation of Akt in skeletal muscle and was more effective at inhibiting the phosphorylation of T308 than S473, despite it's ability to fully inhibit the phosphorylation of 4EBP1 and S6. These results will be confirmed by in vivo dose-response experiments, but, consistent with the partial effect of PP242 on pAkt in skeletal muscle, a muscle-specific knockout of the integral mTORC2 component rictor resulted in only a partial loss of Akt phosphorylation at S473 [46]. These results suggest that a kinase other than mTOR, such as DNA-PK [8,9], may contribute to phosphorylation of Akt in muscle.
Rapamycin (5 mg/kg), PP242 (20 mg/kg), or vehicle were injected into the intraperitoneal (IP) cavity of mice, followed by IP injection of 250 mU insulin or saline. Lysates were prepared from perigenital fat, leg muscle, and liver and analyzed by Western blotting.
Rapamycin often stimulates the phosphorylation of Akt [47,48], probably by relieving feedback inhibition from S6K to the insulin receptor substrate 1 (IRS1) [49], a key signaling molecule that links activation of the insulin receptor to PI3K activation. In all tissues examined, and especially in fat and muscle, acute rapamycin treatment activated the phosphorylation of Akt at S473 and T308 (Figure 8). In contrast to rapamycin, by inhibiting both mTORC2 and mTORC1, PP242 suppresses rather than enhances Akt activation.
As was seen in cell culture, rapamycin and PP242 also differentially affect the mTORC1 substrates S6K and 4EBP1 in vivo. S6 phosphorylation was fully inhibited by rapamycin and PP242 in all tissues examined. While PP242 was effective at blocking the phosphorylation of 4EBP1 on both T36/45 and S65 in all tissues examined, rapamycin did not block 4EBP1 phosphorylation as completely as PP242. Further experiments will be required to identify the mechanism by which 4EBP1 phosphorylation is partially resistant to rapamycin.
Rapamycin has been a powerful pharmacological tool allowing the discovery of mTOR's role in the control of protein synthesis. Since the discovery of a rapamycin-insensitive mTOR complex, there has been a significant effort to develop pharmacological tools for studying this complex. We have used two structurally distinct compounds to pharmacologically dissect the effects of mTOR kinase inhibition toward mTORC1 and mTORC2 activity.
We have shown through the use of these inhibitors that the inhibition of mTOR kinase activity is sufficient to prevent the phosphorylation of Akt at S473, providing further evidence that mTORC2 is the kinase responsible for Akt hydrophobic motif phosphorylation upon insulin stimulation. We also find that phosphorylation at T308 is linked to phosphorylation at S473, as had been observed in experiments where mTORC2 was disabled by RNAi and long-term rapamycin, but not homologous recombination. Surprisingly however, inhibition of mTORC2 does not result in a complete block of Akt signaling, as T308P is partially maintained and Akt substrate phosphorylation is only modestly affected when S473 is not phosphorylated.
Despite its modest effect on Akt substrate phosphorylation, PP242 was a strikingly more effective anti-proliferative agent than rapamycin. These results were reproduced even in cells lacking mTORC2 (SIN1−/−), suggesting that downstream mTORC1 substrates might be responsible for PP242′s strong anti-proliferative effects. Interestingly, we observe that phosphorylation of the mTORC1 substrate 4EBP1 is partially resistant to rapamycin treatment at concentrations that fully inhibit S6K, whereas PP242 completely inhibits both S6K and 4EBP1. Because rapamycin can only partially inhibit the phosphorylation of 4EBP1, but it can fully in inhibit the phosphorylation of S6K, rapamycin appears to be a substrate-selective inhibitor of mTORC1. Consistent with this finding, experiments with purified proteins have shown that rapamycin/FKBP12 only partially inhibits the in vitro phosphorylation of 4EBP1 at Ser 65 by mTOR but can fully inhibit the in vitro phosphorylation of S6K [50]. By contrast, LY294002, a direct inhibitor of many PI3K family members including mTOR, was equally effective at inhibiting the phosphorylation of S6K and 4EBP1 by mTOR in vitro [50] and in cells [23], although this finding is complicated by LY294002′s inhibition of multiple lipid and protein kinases [51] including PIM, a kinase potentially upstream of 4EBP1 phosphorylation [52,53]. These results argue that PP242, in addition to being useful for investigating mTORC2, can reveal rapamycin-resistant components of mTORC1 function. Indeed, proliferation of SIN1−/− MEFs is more sensitive to PP242 than rapamycin (Figure 5B), suggesting that rapamycin-resistant functions of mTORC1, including the aspects of translation initiation highlighted in Figure 7, are key to the anti-proliferative effects of PP242. Furthermore, our findings suggest that the inhibition of translational control and the anti-proliferative effects of PP242 require inhibition of 4EBP1 phosphorylation and eIF4E activity.
Using TORKinibs to acutely inhibit mTOR has surprisingly led to the identification of outputs from mTORC1 that are rapamycin-resistant. These observations should motivate further studies aimed at understanding how rapamycin is able to selectively affect different outputs downstream of mTORC1. As active site inhibitors of mTOR join rapamycin and its analogs in the clinic [22,27,30], it will be important to understand the distinct effects of these pharmacological agents on cellular and organismal physiology and to evaluate their efficacy in the treatment of disease and cancer caused by hyperactivation of the PI3K→Akt→TOR pathway.
Materials and Methods
Ethics statement.
Mice were handled in accordance with protocols approved by the committee for animal research at the University of California San Francisco, United States of America.
Cell culture.
Cells were grown in DMEM supplemented with 10% FBS, glutamine, and penicillin/streptomycin. Confluent L6 myoblasts were differentiated into myotubes by culturing them for 5 d in medium containing 2% FBS. L6 myotubes were maintained in medium containing 2% FBS until use. Primary wild-type MEFs used in Figure 7 were isolated at embryonic day 13.5 as previously described [54]. Primary SIN1−/− MEFs and matching wild-type controls were provided by B. Su and isolated as previously described [16].
Cell lysis and Western blotting.
Except where indicated otherwise, cells were serum starved overnight and incubated with inhibitors or 0.1% DMSO for 30 min prior to stimulation with 100 nM insulin for 10 min. All inhibitors were either synthesized as previously described [21,24,55] or were from Calbiochem (rapamycin and Akti-1/2). Cells were lysed by scraping into ice cold lysis buffer followed by brief sonication. Lysates were cleared by centrifugation, resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies from Cell Signaling Technology. Unless otherwise indicated, cells were lysed in 300 mM NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.02% NaN3, 20 nM microcystin (Calbiochem), Sigma phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail, and 2 mM PMSF. For Figures 6A and 7A, and Figure S2A, cells were lysed in cap lysis buffer (140 mM KCl, 10 mM Tris pH 7.5, 1 mM EDTA, 4 mM MgCl2, 1 mM DTT, 1% NP-40, 20 nM microcystin, Sigma phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail without EDTA and 2 mM PMSF).
Cap pull-down assay.
L6 myotubes from one well of a six-well plate were lysed in 300 μl of cap lysis buffer as described above. 50 μl of detergent-free cap lysis buffer and 20 μl of pre-washed cap beads were added to 150 μl of cleared lysate and incubated at 4 °C overnight with tumbling. The beads were washed twice with 400 μl of cap wash buffer (cap lysis buffer with 0.5% NP-40 instead of 1% NP-40) and twice with 500 μl of PBS. The beads were boiled in SDS-PAGE sample buffer and the retained proteins analyzed by Western blot. All antibodies were from Cell Signaling Technologies except for the anti-eIF4E antibody, which was from BD Biosciences.
Kinase assays.
Phosphorylation of histone H1 (4 μM) by PKC was assayed in a buffer containing 200 ng/ml recombinant kinase, 25 mM HEPES pH 7.5, 10 mM MgCl2, 5 mM ß-glycerol phosphate, 0.05 mg/ml phosphatidylserine, 0.03% Triton X-100, 0.5 mg/ml BSA, 2.5 mM DTT, 100 μM CaCl2, 1 μM PMA, 10 μM ATP, and 15 μCi/ml of γ-32P-ATP. Inhibitors were tested in a four-fold dilution series from 10 μM to 600 pM, and four measurements were made at each concentration. The kinase reaction was terminated by spotting onto nitrocellulose, which was washed 5 times with 1 M NaCl/1% phosphoric acid. The radioactivity remaining on the nitrocellulose sheet was quantified by phosphorimaging, and IC50 values were determined by fitting the data to a sigmoidal dose-response curve using the Prism software package.
PDK1, mTORC1, and mTORC2 were assayed as previously described [21].
In-cell Western.
L6 myotubes were grown and differentiated in 96-well plates. The outside wells of the plate were not used for the experiment, but were kept filled with media. Following stimulation, cells were fixed for 15 min with 4% formaldehyde in PBS with Ca++ and Mg++. The cells were washed three times with PBS and the blocked and permeabilized with 5% goat serum in PBS with 0.3% Triton X-100 (PBS-GS-TX). Primary antibodies to S473 (Cell Signaling #4060) and T308 (Cell Signaling #2965) were added at 1:1000 and 1:500, respectively, in PBS-GS-TX, and the plates were incubated at 4 °C overnight. The plates were then washed three times with PBS, and goat anti-rabbit secondary antibody (Pierce Biotechnology) was added at 0.01 μg/ml in PBS-GS-TX. After 1 h at room temperature, plates were washed three times with PBS. ELISA chemiluminescent reagent (Femto, Pierce Biotechnology) was added to each well and after 1 min, the plate was read in a luminescence plate reader using a 100-ms integration time. The pAkt signal from pT308 and pS473 was normalized to control wells, so that 0 represents the level of pAkt in serum starved cells and 1 represents the level upon insulin stimulation. EC50 values were determined by fitting the data to a sigmoidal dose-response curve using the Prism software package. The significance of differences between EC50 values was evaluated using the F test.
Akt transfection.
Akt was transfected into HEK293 cells using Lipofectamine 2000 according the manufacturers protocol. Two days after transfection, cells were serum starved overnight and the next day they were treated with inhibitors and processed for western blotting as described above.
Actin cytoskeleton staining.
NIH 3T3 cells were plated on poly-lysine coated coverslips at 30% confluence the day before the experiment. Following treatment with PP242 or 0.1% DMSO for 8 h in 10% serum growth medium, the actin cytoskeleton was stained as previously described [24].
Bicistronic reporter assay.
Primary MEFs were transfected with a bicistronic reporter [54] containing a viral IRES using Lipofectamine 2000 according to the manufacturers protocol. At 2 d post transfection, cells were treated overnight with compounds as indicated or starved of serum. The next day, Renilla and Firefly luciferase activity were measured using the Dual-Luciferase kit (Promega). Differences in the ratio of Renilla to Firefly luciferase signals were analyzed for statistical significance by one-way ANOVA with Tukey's post test using the Prism software package.
35S labeling of new protein synthesis.
Primary MEFs grown to 70% confluence in six-well plates were incubated overnight in either 10% Serum (Steady State), kinase inhibitors in 10% serum, or 0.1% serum (starved). Cells were then washed once with DMEM lacking cysteine and methionine (DMEM-noS), and the medium was replaced with DMEM-noS including dialyzed serum and kinase inhibitors as indicated. After incubation for 1 h, 50 μCi of Expre35S35S (NEN) was added to each well and the cells were labeled for 4 h. Cells were washed once with ice-cold PBS, and lysed as described above for Western blotting. Following separation by SDS-PAGE, and transfer to nitrocellulose, 35S-labeled proteins were visualized by autoradiography with film. For quantitation, the membrane was exposed to a phosphorimager screen and the resulting image was quantified in ImageJ. Differences in 35S incorporation were analyzed for statistical significance by one-way ANOVA with Tukey's post test using the Prism software package.
In vivo drug treatment and Western blotting.
Drugs were prepared in 100 μl of vehicle containing 20% DMSO, 40% PEG-400, and 40% saline. Six-wk-old male C57BL/6 mice were fasted overnight prior to drug treatment. PP242 (0.4 mg), rapamycin (0.1 mg), or vehicle alone was injected IP. After 30 min for the rapamycin-treated mouse or 10 min for the PP242 and vehicle-treated mice, 250 mU of insulin in 100 μl of saline was injected IP. 15 min after the insulin injection, the mice were killed by CO2 asphyxiation followed by cervical dislocation. Tissues were harvested and frozen on liquid nitrogen in 200 μl of cap lysis buffer. The frozen tissue was thawed on ice, manually disrupted with a mortar and pestle, and then further processed with a micro tissue-homogenizer (Fisher PowerGen 125 with Omni-Tip probe). Protein concentration of the cleared lysate was measured by Bradford assay and 5–10 μg of protein was analyzed by Western blot as described above.
Cell proliferation assay.
Wild-type and SIN1−/− MEFs were plated in 96-well plates at approximately 30% confluence and left overnight to adhere. The following day cells were treated with PP242, rapamycin, or vehicle (0.1% DMSO). After 72 h of treatment, 10 μl of 440 μM resazurin sodium salt (Sigma) was added to each well, and after 18 h, the florescence intensity in each well was measured using a top-reading florescent plate reader with excitation at 530 nm and emission at 590 nm.
Supporting Information
Figure S1. PP242 Inhibits Akt Phosphorylation over the Course of 1 h
L6 myotubes were pre-treated with PP242 or DMSO for 30 min and stimulated with insulin for the indicated times prior to lysis and analysis by Western blotting.
Figure S2. Additional Analysis of 4EBP1 Phosphorylation
(A) 4EBP1 phosphorylation at T70 is not inhibited by either PP242 or rapamycin. L6 myotube lysates from Figure 6A were analyzed by Western blotting.
(B) 4EBP1 phosphorylation is inhibited by PP242 with similar potency in SIN1−/− and wild-type (WT) MEFs. Western blotting from Figure 6B is shown with shorter exposures of p4EBP1.
Figure S3. Rapamycin-Resistant Phosphorylation of 4EBP1 Is Sensitive to the TORKinibs PP30 and PP242, but Not the PI3K Inhibitor PIK-90
L6 myotube lysates from Figure 2A were analyzed by Western blotting.
Figure S4. PP242 Inhibits Cap, but Not IRES-Dependent, Translation
(A) Renilla (cap-dependent) luciferase activity from samples in Figure 7B.
(B) Firefly (IRES-dependent) luciferase activity from samples in Figure 7B. Firefly luciferase activity of the PP242 treated sample is not significantly different from control (p = 0.4, ANOVA).
Table S1. In Vitro IC50 Determinations Using Three Forms of mTOR
We thank J. Taunton for suggesting the SIN1−/− experiments and B. Su for providing the SIN1−/− MEFs. We thank A. Pusic for providing primary MEFs and for experimental advice. We thank Yi Liu and Pingda Ren at Intellikine for providing PP242 for animal studies. We thank A. Dar for performing the JAK kinase assay, and W. Weiss and M. Barna for advice on this manuscript. We thank C. Zhang and D. Fiedler for providing BX-795.
Author Contributions
MEF, AU, RL, ZAK, DR, and KMS conceived and designed the experiments. MEF, BA, and AU performed the experiments. MEF, BA, AU, and KMS analyzed the data. BA, RL, ZAK, DR, and KMS contributed reagents/materials/analysis tools. MEF, BA, ZAK, DR, and KMS wrote the paper.
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One of the most popular features of this magazine, besides this one, of course, is the Julia Street column where questions are asked and answers are provided about all matters New Orleans.
Those of us madly in love with this town, or at any given moment just mad at it, are happy to know about times past in this most fascinating place. We don't just like nostalgia; we revel in it.
And we keep reinventing it. The (re)birth of the Loyola streetcar line is an example of our desire to move forward while respecting the past. It even sloshes over into our drinking selections. New is good and pleasurable, but what is the beverage based on?
With the new streetcar line comes a new drink, "A Streetcar Named Loyola," from a rebirthed place, the Hyatt Hotel, in a bar named after an historic Canal Street movie house, Vitascope Hall.
A Streetcar Named Loyola
1 1/2 ounces Cathead Honeysuckle Vodka
1/2 ounce white ginger syrup
1/2 ounce lemon juice
1/4 ounce St. Germain
Ginger ale
Peychaud's Bitters
Rosemary sprig for garnish
Build in a rocks glass fill with ice. Top with ginger ale.
Garnish with 8 drops of Peychaud's Bitters and a sprig of rosemary.
Thank you to the bar staff at Vitascope Hall located in the Hyatt Regency Hotel located right on the Loyola Streetcar line.


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Consciousness is a term related to awareness. Awareness of what? Awareness of ourselves and others. Those things we can observe and those that we cannot. Those we can measure and those we intuit.
Individually, our consciousness is a product of our capacities and capabilities in relation to our life conditions, those elements that impact how we comprehend ourselves, others, and the contexts that shape our understanding. Collectively, we help to shape each individual consciousness and bring that consciousness into a set of understandings, values, aspirations, and intentions that we call culture.
As with many words in English, the term culture means different things to different people. For example, it is often used to refer to a set of shared worldviews and values held by a group of people in a family, a village, an organization, or a nation.
Alternatively, I like to think of culture as the constellation of values, beliefs, worldviews, aspirations, and intentions in a human system, whether that system is tribal, regional or national. If we take this approach, we can be aware of not only what is shared, but also what is not. This, in turn, provides us with a richer fabric to understand what we observe in human systems.
An approach such as this—whether we are trying to manage or lead a change in agricultural practices, the degradation of water systems by the poisons used in mining, shift to the use of renewable resources for energy production, political and business processes and institutions, or any of a myriad of other changes that potentiate generativity in our world—supports our comprehending the complexity of what we are working with, individually and collectively. And keep in mind that generativity is essential for the evolution and sustenance of the human race, as well as all of the other non-human phenomena in the universe. It resembles the Shiva process of destruction and creation.
The path to building our individual capabilities lies through life-long learning. It is commonplace today to see this notion urged upon us. It is a pathway of letting go of what no longer supports generativity. We experience along this pathway little deaths (à la Stanley Kellerman), as old mental models and beliefs are replaced by new. We experience stagnation, not only as we age, but as we make choices that reduce our use of aspects of our being. In the face of stagnation, we learn new skills and competencies to meet the changing requirements of the contexts in which we live and work, as well as developing and using practices that support such learning. It also involves learning about ourselves as sense- and meaning-making beings.
Psychologist Erik H. Erikson once said that "reconciling lifelong generativity and stagnation involves the elder in a review of his or her own years of active responsibility for nurturing the next generations, and also in an integration of earlier-life experiences of caring and of self-concern in relation to previous generations."
With the pace of change and challenges to our very existence, it is no longer just the elders who need to review, but each of us frequently, for this supports our learning. Used wisely, this is essential to generativity.
One caution: This post does not constitute actionable advice. Each of us must find the practices that support our learning and development. Because their effectiveness also depends on variable life conditions, there is not likely to be a best practice for all at all times.
Read other posts by Russ Volckmann
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Silicon Laboratories, Inc. EFM8BB31F16G-QFN24
The Silicon Laboratories, Inc. EFM8BB31F16G-QFN24 is a High-speed pipelined CIP-51 (8051) MCU, 16KBytes Flash, 256 Bytes Data RAM, 2048 Bytes XRAM. 20 Digital I/O lines, 1 12-bit ADC Unit with 12 Channels (up to 400 ksps), 2 12-bit DACs (up to 200 ksps), 4 Configurable Logic Units 2 Low-current analog comparators with adjustable reference [up to 6 (CMP0) or 6 (CMP1) external positive\negative inputs], 6-Channel Programmable Counter Array (PWM, Clock Generation, Capture/Compare) 2 UARTs (up to 3 Mbaud), 1 SPI (Master/Slave up to 12 Mbps), 1 SMBus / I2C (up to 400 kbps), and 1 High-Speed (3.4 Mbps) I2C-Slave bus. 1 16-bit CRC unit, 6 16-bit general-purpose timers, 1 Watchdog timer, On-Chip: Low-dropout (LDO) regulator, 3 x Oscillators (80kHz, 24,5MHz 49MHz), Power-On reset circuit, brownout detector, and the non-intrusive Silicon Labs 2-Wire (C2) debug interface.
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Google will let some users of its Apps suite test-drive in the fall its Wave collaboration and communication tool, the company said on Tuesday in an official blog.
Wave, announced in May, is currently available in a preview version to application developers because the product is still in an early stage.
Apps administrators interested in making Wave available to their end-users can sign up by filling out this online form. Google plans to make Wave available to all Apps users next year.
Wave generated a lot of buzz when it was first demoed in May at Google's I/O Conference for developers. It is a sort of Swiss army knife for communication and collaboration, consolidating in a single tool features from e-mail, instant messaging, blogging, wikis, multimedia management and document sharing.
As such, it will be interesting to see how it fits in Apps, since Wave could possibly overlap with individual components of that suite, like Gmail, Talk, Docs and Sites.
Google officials have acknowledged that, although Wave is very promising and potentially groundbreaking, its success will depend to a large extent on Google's ability to communicate clearly to people what it is, what it does and what are its use cases.
In its announcement on Tuesday, Google took another crack at describing Wave and suggesting ways in which it could be useful for users of Apps, a collaboration and communications suite designed for use in organizations of all sizes and educational institutions.
"A wave is equal parts conversation and document, where individuals communicate and work together in a multimedia environment – the wave itself," reads the blog posting. "Whether there's a report to write, an event to plan, research to do or communications to conduct, we're building Google Wave so people can be more productive and collaborate more effectively in a real time environment."
For example, Wave lets users collaborate on a document by adding elements like text, photos, lightweight "gadget" applications and maps, according to Google. "It's communication and collaboration, conversation and document, in one unified, cloud-based space," reads the posting.


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The price listed is the retail price, buy more and more of course the cheaper (Check Wholesale Prices). This zipper you can buy Roll-an, can also be in the form of a sack (1 Sack contents 20 Roll) Wholesale. If you want to buy Per-Bolt please select the purchase amount 90 and multiples (1 Roll: 90 Meter / 100 Yard).
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Yarn Knitting and Embroidery
Untuk keterangan lebih lanjut, silahkan download PDF ini :
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Here we go, after you enter here make sure you go over and check out Misadventures in Baby Raising and her amazing giveaways too!
Today I bring you one of my favorite things an amazing clothing store! LayersClothing is an wonderful online store for Tees Dresses Pants Swimwear. It was all so beautiful I had such a hard time picking something. I just loved their Swimwear and just had to get the Gidget! I know don't you just love the name. It so reminded me of her too, and since I'm the same bubbly person I just knew it was perfect for me. Plus check out those super cute ruffles and of course hello pink is in there! Sorry everyone no picture of me in the actual Swimwear, I want you all coming back here and not to scare you off. Soon when I've dropped all the baby weight I'll put it on for you, for now it's a backyard item. A girl still needs to tan :)
A little about the Swimwear I received believe me the spandex is wonderful, yup holds you in wonderful :):
Gidget: Pink gingham halter tankini. Mini accordian pleatdetail along v-neck line and bustline. Front linedtop with removable bra pads. Fully lined bottomswith fold over band detail. Nylon/Spandex. XS-XL.
Currently I see some wonderful deals going on their site you'll just have to check out! They also have one retail store in Sandy, Utah. 426 W Universal Circle Sandy, UT 84070. Come shop with us Monday-Friday 9-7 and Saturdays 10-5. For my Utah friends you'll have to check them out! For everyone else you must log on LayersClothing (yes its a link I make it easy) and check out the wide variety they have. I have my eye on this super cute Bianca that comes in Coral, now would that be an amazing color on me?
The amazing people at LayersClothing has offered one of my readers a $25 E-Gift Certificate. How they work:
E-gift certificates come in the form of email. You can purchase an e-gift
certificate online or in our store. The e-gift certificate will be emailed
directly to the recipient or to yourself, just enter in the valid email address
of the intended recipient along with a message upon checkout. We do not offer
paper certificates or gift cards at this time.
WIN IT: Mandatory got to LayersClothing website and tell me what you would spend you $25 E-Gift Certificate! Super easy plus you may just find a SALE you have to have!
EXTRA ENTRIES (Do one, Do them all or Don't do any it's up to you) ~ 1 extra per each unless otherwise stated:
- Follow Me (2 EXTRA Entries)
- Have "Running Away" Button up (2 EXTRA Entries)
- Have Fun in the Sun Button up (Remember for those who did it early get 5 extra entries)
- Subscribe via Email (up on the top left) (2 EXTRA Entries)
- Blog about the giveaway (3 EXTRA Entries)
- Become a Fan @ LAYERS Clothing Official Blog
- Sign up for LayersClothing Newsletter (bottom of their page)
- Enter My Fun In the Sun ~ Munchie Mug Giveaway (By 7/10/09)
- Enter My Fun In the Sun ~ Skin MD Natural Giveaway (By 7/8/09)
- Enter My Fun In the Sun ~ Ouchies 2 Winners (By 7/6/09)
- Enter My Protect a Bed Giveaway (By 7/4/09)
- Enter My My Birthday Gift to My Readers (by 7/4/09)
- Enter PopATot @ Misadventures in Baby Raising (Come back here and say "I entered PopATot")
- Enter Piggy Paint! @ Misadventures in Baby Raising (Come back here and say "I entered Piggy Paint")
Check that out that is A LOT of chances to win! Keep the entries coming and remember if you Grabbed the Button before today you get 5 EXTRA entries in both of our giveaways!!! Also if you blogged about it you get 10 EXTRA entries in both mine and Misadventures in Baby Raising "Fun in The Sun" Giveaways!
This giveaway ends on July 11, 2009 at 11:59 pm PST. 1 winner will be chosen by Random.org and posted in a separate post. Winner will have 48 hours to contact me after the drawing or forfeit their prize.


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Tan Lin
Tan Lin is the author of Lotion Bullwhip Giraffe, BlipSoak01, Ambience is a Novel with a Logo, Heath (Plagiarism/Outsource) and 7 Controlled Vocabularies and Obituary 2004. The Joy of Cooking (2010). His work has appeared in numerous journals including Conjunctions, Artforum, Cabinet, New York Times Book Review, Art in America, and Purple. His video, theatrical and LCD work have been shown at the Marianne Boesky Gallery, Yale Art Museum, Sophienholm Museum (Copenhagen), Ontological Hysterical Theatre, and as part of the Whitney Museum of American Art's Soundcheck Series. Lin is the recipient of a Getty Distinguished Scholar Grant for 2004-2005 and a Warhol Foundation/Creative Capital Arts Writing Grant to complete a book-length study of the writings of Andy Warhol. He has taught at the University of Virginia and Cal Arts, and currently teaches creative writing at New Jersey City University.
Seven Controlled Vocabularies and Obituary 2004. The Joy of Cooking (Wesleyan Poetry Series, 2010)
HEATH (PLAGIARISM/OUTSOURCE), NOTES TOWARDS THE DEFINITION OF CULTURE, UNTILTED HEATH LEDGER PROJECT, A HISTORY OF THE SEARCH ENGINE, DISCO OS (Counterparth, 2011)
(first edition: Zasterle, 2009)
ambience is a novel with a logo (Katalanche Press, 2007)
Blipsoak01 (Atelos, 2003)
Lotion Bullwhip Giraffe (Sun & Moon Press, 2000)
|Twelve POD publications expanding 7CV (Edit Publications, 2010)
the patio and the index, from Triple Canopy (2011)
RPT MC-60 00.27 8, from 7CV, Poets.org, August 2010
SOFT INDEX (OF repeating PLACES, PEOPLE, AND WORKS), boundary 2 36(3) 2009 (with an abstract)
"Diary/Blog", The Brooklyn Rail, May 2007
from "Ambient Stylistics", Boston Review, April/May 1999 (with an introduction by Charles Bernstein)
"The Imitation of the Imitation of History", Lingo 6, 1996
with David Foote on Inomnia and the Aunt (2015)
with Angela Genusa (on the book), 2012 (Rhizome)
with Danny Snelson, Gordon Tapper, Kristen Gallagher, Chris Alexander, 2011 (Jacket 2)
with Katherine Elaine Sanders, Bombsite, March 2010
with Asher Penn, "Tan Lin: Enhancing the Book," Art in America, June 2010
with Jeremy JF Thompson, Keiran Daly, and Danny Snelson, Talk:7CV Event Inventory and Documentation, Edit Wiki, June 2010
with Colin Marshall, "Experimental poet Tan Lin: ambiently breaking reading conventions," The Marketplace of Ideas, June 2010
with Kristen Gallagher, Chris Alexander, and Gordon Tapper, Galatea Resurrects #12, May 2009


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While technically the recession is over, again technically, the reality is that many jobs that are gone will not be revived.
In other words, these jobs are deader than a doornail.
The jobless recovery, with millions out of work for a very long time.
This is what November will be about.
Fewer construction workers will be needed. Don't expect as many interior designers or advertising copywriters, either. Retailers will get by with leaner staffs.
The economy is strengthening. But millions of jobs lost in the recession could be gone for good.
And unlike in past recessions, jobs in the beleaguered manufacturing sector aren't the only ones likely lost forever. What sets the Great Recession apart is the variety of jobs that may not return.
That helps explain why economists think it will take at least five years for the economy to regain the 8.2 million jobs wiped out by the recession — longer than in any other recovery since World War II.
It means that even as the economy strengthens, more Americans could face years out of work. Already, the percentage of the labor force unemployed for six months or longer is 4.3 percent. That's the highest rate on records dating to 1948.
Behind the trend are the cutbacks businesses made in the recession to make up for a loss of customers. To sustain earnings, they became more productive: They found ways to produce the same level of goods or services with fewer workers. Automation, global competition and technological efficiencies helped solidify the trend.
Diminished home equity and investment accounts have made shoppers more cautious, too. And their frugality could endure well into the recovery. That's why fewer retail workers, among others, will likely be needed. read more here...
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Even for Yahoo Inc., a company with a lengthy track record of failure, this was an epic fail.
For five days this week an untold number of its 100 million users couldn?t send or receive email, pro mpting many longtime, loyal users to savage the company on social media and threaten to leave for another email service.
Despite the misery the outage caused people and businesses ? who could have sent a letter faster than they could an email ? Yahoo was slow to respond and even slower to inform users about it.
And that has cast a shadow over Chief Executive Marissa Mayer?s yearlong effort to turn around the troubled Internet company.
Peter Sealey, adjunct professor of marketing at Claremont Graduate University, said the handling of the email outage was a major public relations stumble and a ?blemish? on Mayer?s tenure as CEO.
Colin Gillis, an analyst with BGC Partners, called the outage ?an embarrassment.?
For all of the Sunnyvale, Calif., company?s challenges over the years, some 800 million people log into Yahoo each month to check their email, get stock quotes and scan headlines.
Yahoo Mail is one of the main ways the company keeps a hold on users. Mayer identified Yahoo Mail ? one of the world?s largest email services ? as one of the company?s biggest missed opportunities that she planned to fix.
But so far, Yahoo email users have not been happy with the results. A redesign has triggered loud protests, and many users say the service is no longer reliable.
Even Yahoo employees don?t want to use it. In an internal memo leaked to All Things D tech blogger Kara Swisher, Yahoo management begged employees to switch their corporate email accounts from Microsoft Outlook to Yahoo Mail.
According to Swisher, the executive in charge of Yahoo Mail, Jeff Bonforte, joked at a staff meeting last month that while users might be dissatisfied with changes to the product, the company would have to give them a swift kick in a delicate body part to get them to stop using it.
Yahoo is now issuing frequent updates on its progress in fixing the outage. Still, many users may be missing a couple of weeks of email, and some users were still reporting problems Friday.
Yahoo did not respond to questions about the outage, including how many people were affected and how many may have dropped the service.
Dwana De La Cerna, a government worker who lives in Chicago, says she has been frustrated because her 17-year-old son is waiting to hear from his No. 1 college choice, Indiana University. He has only one email account, Yahoo, and that?s the one he gave the school, she said.
?We kept wondering why we hadn?t heard anything,? she said.
Kathy Badertscher, director of content with public relations agency Knock Twice, says she?s had a Yahoo Mail account since 2000. It was her first personal email address. She stuck with Yahoo after opening a Google Gmail account in 2007.
Even before the latest prolonged outage, Badertscher said email access had become ?more spotty.? And she was not a fan of the redesign. She says she has slowly begun to use Gmail more, and now she?s thinking about switching over permanently to Gmail.
?Frustrated? Yep. Disgusted? A little,? she said.
Alienating loyal users such as Badertscher won?t help Mayer in her difficult quest to breathe new life into Yahoo products, jump-start its advertising business and transform the company culture.
The former Google Inc. executive joined Yahoo in July 2012 to help revive a company that had not kept up with the rapid migration to mobile devices.
Yahoo shares, which rose 38 cents, or 1 percent, to $39.73 on Friday, have more than doubled this year. But investors are not valuing the stock on Yahoo?s business, but on that of Chinese e-commerce giant Alibaba, in which Yahoo has a 24 percent stake. Alibaba accounts for about half of Yahoo?s market valuation.
Mayer has warned investors that it will take years to turn Yahoo around. But Wall Street won?t necessarily be that patient. Analysts note that she has not managed to grow the company?s core advertising business even as digital advertising spending surges and competitors Google and Facebook capture big gains.
In October, Yahoo reported that its third-quarter profit fell 13 percent, excluding a gain from the sale of some of its stake in Alibaba, while revenue fell 1 percent. And Yahoo again lowered its revenue and profit outlook for 2013.
Yahoo this week was also forced to reveal that it is more dependent on its search deal with Microsoft Corp. than it had previously said. That search deal accounted for 25 percent of its $5 billion in revenue last year and 31 percent of its $1.1 billion in revenue last quarter. Yahoo had previously said the contract with Microsoft was responsible for more than 10 percent of its revenue in 2011 and 2012 without being more specific.
On the plus side, Mayer has been given credit for improving morale at Yahoo and pushing the company to develop products for mobile devices. She has purchased several startups to bring new blood and ideas into the company, including spending $1 billion on blogging platform Tumblr. She is also pushing redesigns of Yahoo websites and mobile apps to make them appeal to a younger crowd.
Yahoo has made some strides in growing its audience. The company said in October that its websites now draw more than 800 million users a month, up 15 percent since Mayer took over as CEO. Monthly mobile users rose 15 percent in the third quarter to 390 million.
Alibaba has given Mayer some breathing room on Wall Street, but that won?t last, said Gillis, the BGC Partners analyst.
?Most investors are eyeing the Alibaba piece, not the core business,? Gillis said. ?But one day the core business is going to be valued on its own merits.?
Source: MCT Information Services


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Stopend for plain half round gutter - primed
Stopend for plain half round gutter
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Free Arkansas CDL Tanker Vehicles Practice Test 2023
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The MLS Commissioner said Donovan won't be transferred for the 3-plus years remaining on his Galaxy contract. Some people aren't happy, but to that MLS says, who cares what you want?
He's become a real soccer hero," Don Garber said. "MLS needs soccer heroes, and we have a great American soccer hero playing for us in LA, holding the torch for the sport in our country, and that's very important. I don't believe that it's something we can do without."
While it's certainly a coup for a player of Donovan's quality to spend the majority of his career in our domestic league, he didn't really become a soccer hero until South Africa. Sure he was good, but is the face of American soccer we want to show the world the type of guy who couldn't cut it at Bayer Leverkusen or Bayern Munich?
No, the real ambassadors of American soccer over the past decade have been guys like Tim Howard, Brian McBride and Clint Dempsey, who have shown they can play in one of the best leagues in the world, and hold their own.
Donovan's always been a big fish in a small pond, and that's better suited his confidence and his image than his stints in the Bundesliga. And that was fine, until he was able to perform on a larger stage (Everton, then the MNT).
So why wouldn't US Soccer export their best product to somewhere he can gain more attention? Well, MLS isn't US Soccer.
MLS is a business, concerned with growing their brand. And now that Donovan is a brand name, they're not going to forgo the ratings, attendance and attention boosts he gives them. So even though more Americans watch Premier League matches than MLS games every week, Garber can't be concerned with raising America's international profile.
So despite what fans want, or what Donovan himself wants, in keeping him in Los Angeles through 2013, MLS is making the right decision — for MLS.


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I was testing BlogJet today as a possible desktop blogging tool. Strangely, what caught my eye first was not the tool, but the quote, attributed to Albert Einstein, that they used in the sample post… I found it a little hard to believe that Einstein who died in 1955 would have a quote about computers…especially about computers being fast. Here is the quote:
"Computers are incredibly fast, accurate and stupid; humans are incredibly slow, inaccurate and brilliant; together they are powerful beyond imagination." — Albert Einstein (or was it Leo Cherne?…read on)
I decided to see if I could find out starting, as expected, with a simple Google search. When you search the web using Google for confirmation, its easy to find:
"Computers are incredibly fast, accurate and stupid" – You get 21,800 results
"Computers are incredibly fast, accurate and stupid" + Einstein – 12,100 results
"The computer is incredibly fast, accurate, and stupid." Cherne – 509 results
We might stop there and say "21,000 web pages is enough evidence for meâ€, but WHEN did Einstein say it? WHERE? No one seems to know. A large number of these quotes seem respectable enough – many are on university web sites, computer science portals, and in textbooks. The quote was even used as the motto for Super-computing 2006 and used in the key note speech. It seems in fact to be everywhere except in any book, paper, speech, or a citable source by Albert Einstein. By this point I'm 99.9% sure he never said it – but lets keep looking.
Wikiquote (a wikipedia project) tries to track down the actual original source of all quotes lists the "powerful beyond imagination†quote as on of its unverified on the talk page. No verified quote mentions computers at all.
So in this case the web is no help, so let's try books.
So I switched from Google web search to Google Book search. Book authors normally spend a little more time researching before they publish…most of the time. I was able to find the quote in over 190 books that Google has scanned. Unfortunately the full text is rarely available. Interestingly, most of the newer books – after 2004 readily attribute the quote to Einstein.
But no book actually about Einstein mentions the quote…or anything else about computers.
According to the book – "The New Quotable Einstein†by Freeman Dyson (take a look at the index), Einstein never mentioned computers at all. Why would he, he died in 1955, the best computer of the time looked like this:
The ENIAC (Electronic Numerical Integrator And Calculator), began construction in 1943 and was completed 1946. It occupied about 1,800 square feet, used about 18,000 vacuum tubes, and weighed almost 50 tons. When it was turned off in 1955, (the year Einstein died) its estimated to have done more arithmetic than the entire human race had done prior to 1945. That IS a lot of math, maybe its possible Einstein said it after all, but we still need evidence.
1955 in Computer history did seem to be a big year:
|Steve Jobs is born February 24, 1955
|John McCarthy coins the term Artificial Intelligence (AI) in 1955 at Dartmouth University.
|Dartmouth Colleges John McCarthy coins the term "artificial intelligence."
|Tim Bernes-Lee is born June 8, 1955.
|William (Bill) H. Gates is born October 28, 1955.
|IBMÂ introduces the first IBMÂ 702.
|Bell Labs introduces its first transistor computer. Transistors are faster, smaller and create less heat than traditional vacuum tubs, making these computers more reliable and efficient.
|The ENIAC is turned off for the last time. Its estimated to have done more arithmetic than the entire human race had done prior to 1945.
So if not Einstein, then who?
"Speaking of Scienceâ€, a book of science quotations by Jon Fripp includes the quote – but doesn't attribute it to Einstein. The entry appears like this:
The Computer is incredibly fast, accurate and stupid. Man is unbelievably slow, inaccurate and brilliant. The marriage of the two is a challenge and an opportunity beyond imagination.
– Walesh, 1989 (Summarizing the reasons for using computer modeling for hydrologic and water quality analysis.)
Walesh huh…who's that? While this is a book of quotes… and the while the back of his book does state that "each quote is carefully referencedâ€, I know I have seen it earlier than 1989
Lets see if we can find any thing before 1989
Going back to Google book search I see the quote in a fiction book – Fort Momma (Page 37 by Al Gowan – Fiction – 2003) this time attributed to Leo Cherne. Cherne's version is slightly different:Â
Microcomputers and Children in the Primary School: Proceedings of … – Google Books Result
Leo Cherne (1977) wrote: The computer is incredibly fast, accurate and stupid. … Themarriage of the two is a force beyond calculation. …
Let's just find the oldest damn reference to it!
Here it is (that I can find) – 1969. In a journal called "Advances in Instrumentation†v.24 pt.4, 1969, page 691, published by Instrument Society of America. Google book search found it on a shelf at the University of Michigan and digitized it on Nov 28, 2007. Here is a bit of context I was able to tease out of Google by searching over and over…
"The net result of the overall program was a group of well trained men that had been training in the same way and, as a result, now operate the machine more uniformly. If you get nothing else out of computer installation other than a well trained crew, it alone makes it worth the effort. Even with a computer there is no substitute for a good operator. Computers are incredibly fast, accurate and stupid. On the other hand, a well trained operator as compared with a computer is incredibly slow, inaccurate and brilliant. We think of this feature as "intelligent override" in our control system. We feel you will always have to have this to make decisions about some phases of paper machine operation. Another fringe benefit is accurate production logs. Ours are set up on a 24-hour basis, but can be gotten on demand, to evaluate our progress on a given run. We also integrate stock, chemical, and steam consumption figures to give us a good reliable picture of grade manufacturing costs. In conclusion, we at Eastex feel that what we have done on our No. 4 Machine is merely the beginning. There is no question in our minds that in the future, DDC and the systems engineering method will become the industry standard for process design, installations and operation.â€
But who is the author? – Alas, I do not know. Again, I can't get to the article. If you have access to this book, please let me know. Something tells me there are earlier versions than this, and I doubt this is the reference that popularized it, but due to the copyright fear that grips the internet, I can not get to the top of the page…
Why does this bother me?
People use quotes as a way to strengthen their own position. If I can quote someone you respect, it adds credibility to whatever argument I'm making. Over time, the truth gets further and further away. The biggest names have always attracted people who are more than willing to put words into their mouths for their own gain – Confucious, Jesus, Aristotle, Shakespere, Einstein, and the biggest, most misquoted, of them all – God. All of them have probably been quoted more for the things they never said, than things they actually did say. I imagine this is the most disappointing part of time travel, waiting around to witness words never spoken and deeds never done.


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These beautiful Enamel Bead Personalised Name Bracelets are made to be treasured. Customize your word, your color, and your size Description enamel tiles with touches of colored alphabet beads carefully placed within a chain linked bracelet for an added touch.
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A full store credit of the purchase price (minus the cost of shipping) will be issued once the merchandise is received and in its original condition.
Due to the nature of our products, we do not offer refunds.
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Once you place your order, we start working right away. Custom made products require, three (3) to five (5) weeks production time from the date of order. If your order is not shipped at the 5th week please contact us at firstname.lastname@example.com. If it is within the 3-5 week timeframe please allow time for your product to be created.
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Please allow between 2-7 days for your order to arrive after it has been shipped. If more than 14 business days have passed after the order has shipped please email us at firstname.lastname@example.com to review the status of your order.
Once your order is shipped, tracking information will be sent via e-mail.
Please note: The Signature Collection is NOT, under any circumstances responsible for packages lost/damaged in transit, incorrectly supplied addresses, unclaimed/refused packages, or lost/stolen packages marked "Delivered" by USPS or UPS. If your package falls under any of these categories, please contact USPS directly to file a claim at 800-275-8777.