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Positive feedback between Dia1, LARG, and RhoA regulates cell morphology and invasion
- Gertrude Poole
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1 RESEARCH COMMUNICATION Positive feedback between Dia1, LARG, and RhoA regulates cell morphology and invasion Thomas M. Kitzing, 1,4 Arul S. Sahadevan, 1,4 Dominique T. Brandt, 1 Helga Knieling, 1 Sebastian Hannemann, 2 Oliver T. Fackler, 2 Jörg Großhans, 3 and Robert Grosse 1,5 1 Institute of Pharmacology, University of Heidelberg, Heidelberg, Germany; 2 Department of Virology, University of Heidelberg, Heidelberg, Germany; 3 Zentrum für Molekulare Biologie Heidelberg, University of Heidelberg, Heidelberg, Germany The RhoA-effector Dia1 controls actin-dependent processes such as cytokinesis, SRF transcriptional activity, and cell motility. Dia1 polymerizes actin through its formin homology (FH) 2 domain. Here we show that Dia1 acts upstream of RhoA independently of its effects on actin assembly. Dia1 binds to the leukemia-associated Rho-GEF (LARG) through RhoA-dependent release of Dia1 autoinhibition. The FH2 domain stimulates the guanine nucleotide exchange activity of LARG in vitro. Our results reveal that Dia1 is necessary for LPA-stimulated Rho/ROCK signaling and bleb-associated cancer cell invasion. Thus, Dia1-dependent RhoA activation constitutes a positive feedback mechanism to modulate cell behavior. Supplemental material is available at Received January 11, 2007; revised version accepted May 8, Diaphanous-related formins (DRFs) are Rho GTPasebinding proteins that possess conserved functions in actin cytoskeleton dynamics exerted through their formin homology (FH) 2 domains (Goode and Eck 2007). DRFs are involved in essential cellular processes such as cytokinesis, cell movement, and polarity (Faix and Grosse 2006; Gomez et al. 2007), which are frequently deregulated during pathological situations like tumor cell transformation and metastasis (Sahai 2005). The dormant conformation of the DRF Dia1 is maintained by intramolecular association of its regulatory N terminus to the diaphanous autoregulatory domain (DAD), which is relieved through binding of active RhoA (Lammers et al. 2005; Otomo et al. 2005a). The catalytic FH2 domain is believed to become exposed by conformational changes in the DRFs to promote barbed end actin polymerization by forming a tethered dimer (Xu et al. 2004; [Keywords: Diaphanous-related formins; RhoA; LARG; actin polymerization; LPA; Rho-kinase] 4 These authors contributed equally to this work. 5 Corresponding author. FAX Article is online at Otomo et al. 2005a). The FH2 domain of Dia1 promotes stress fiber formation and transcriptional activation of the MAL/SRF pathway through its actin-polymerizing activity (Copeland and Treisman 2002; Grosse et al. 2003; Miralles et al. 2003). A Dia1 mutant defective in FH2 dimerization interferes with lysophosphatidic acid (LPA)-induced stress fiber formation and SRF activity (Copeland and Treisman 2002), suggesting that Dia1 is part of LPA signal transduction known to play an important role in cell proliferation and metastasis of a variety of human cancers (Mills and Moolenaar 2003). LPA receptors belong to the group of G-protein-coupled receptors that activate the heterotrimeric G-proteins G 12 and G 13, which can bind to RGS-containing Rho-GEFs such as leukemia-associated Rho-GEF (LARG), initially isolated from a patient with acute myeloid leukemia (AML) (Kourlas et al. 2000; Vazquez-Prado et al. 2004). Rhodependent mechanisms have emerged as critical processes in tumor progression (Sahai and Marshall 2002; Lozano et al. 2003), and evidence exists that Rho/ROCK function is essential to promote a specific type of rounded bleb-associated mode of cell invasion (Sahai and Marshall 2003; Wyckoff et al. 2006). However, the role of DRFs in tumor cell behavior has not been investigated. In this study, we provide evidence for an essential role of the Rho-effector Dia1 in LPA-mediated Rho/ROCK activity for tumor cell morphology and invasion that involves LARG, thereby constituting a positive feedback loop toward RhoA. Results and Discussion To explore the role of Dia1 in the regulation of RhoA, we performed GTPase activity assays (Goulimari et al. 2005). HEK293 cells were used to achieve transfection efficiencies >70% to determine whether versions of Dia1 lacking the regulatory N terminus ( DiaN) (Fig. 1A) required for autoinhibition could induce endogenous RhoA activity. Interestingly, DiaN caused substantial activation of RhoA (Fig. 1B). Active DAD-deletion mutants ( DAD) of mouse Dia1, Dia2, and Dia3 also stimulated RhoA but not Cdc42 activity, indicating that this is a shared characteristic among DRFs of this family (Fig. 1C). Dia1 also activated Rac as previously suggested (Tsuji et al. 2002; data not shown). Further analysis revealed that the FH2 domain was sufficient to activate RhoA (Fig. 1D). Since the FH2 is also sufficient for actin assembly and activation of SRF, we tested their possible involvement, indicative of an indirect mechanism. For this we treated cells with Latrunculin B, which sequesters actin monomers and prevents SRF activation (Sotiropoulos et al. 1999). Although Latrunculin B effectively blocked FH2-induced SRF activation as expected (Fig. 1E), it failed to inhibit RhoA activation by FH2 and LARG, while transfection of C3 transferase as a control blocked RhoA activity (Fig. 1D). Interestingly and in agreement with previous findings (Copeland and Treisman 2002), we observed that FH2-induced SRF activity was significantly inhibited by C3 (Fig. 1E), suggesting that upstream RhoA contributes to FH2-exerted actin dynamics. The FH2 domain dimerizes involving its N-terminal lasso loop linker in order to nucleate actin filaments, whereas the core FH2 (FH2core) lacking the lasso behaves as a barbed end capper (Shimada et al. 2004; 1478 GENES & DEVELOPMENT 21: by Cold Spring Harbor Laboratory Press ISSN /07;
2 Activation of LARG and RhoA by Dia1 Figure 1. Role of Dia1 in activation of RhoA. (A) Schematic representation of mdia1. (B) Cells were transfected with control plasmid or increasing amounts of plasmids (0.2, 0.5, 1.0 µg) encoding DiaN and subjected to RhoA-GTP pull-down assays. RhoA and Flag- DiaN were analyzed by Western blotting. (C) Activation of RhoA but not Cdc42 by DAD expression plasmids for Dia1 3 is shown. Active RhoA and Cdc42 were precipitated simultaneously from cell extracts. (D) FH2-induced RhoA activity in the absence or presence of 0.5 µm Latrunculin B (LatB) or in the absence or presence of C3 plasmid (0.5 µg). (E) Cells expressing FH2 (0.1 and 0.3 µg) together with the SRF reporter were cotransfected with C3 (0.1 µg) or pretreated with LatB as indicated. Reporter activity is shown as the mean ± SEM of three independent experiments. (F) FH2 or FH2core was transfected. The amount of Rho-GTP bound, of total RhoA, and of expressed FH2 and FH2core proteins was analyzed by Western blotting. (G) Shown is FH2-induced actin polymerizing versus FH2core capping activity using pyreneactin assembly assays with indicated components. (H) Expression plasmids for FH2 or FH2core were transfected with an SRF reporter. Data represent the mean ± SEM of three independent experiments. (I) Dia1 is required for RhoA activation by LPA as shown by sirna. Cells were untreated or stimulated with LPA (10 µm, 5 min) as indicated, and activated RhoA was precipitated. Cell extracts were immunoblotted for the indicated proteins. (J) HEK293 cells were transfected with Flag- Dia1 DAD and increasing amounts of expression plasmids encoding dnlarg (1.0, 5.0 µg). Activated RhoA and cell extracts were immunoblotted for the indicated proteins. All Western blot quantifications shown are the mean ± SEM of at least two independent experiments.. Otomo et al. 2005b). Therefore, we compared FH2core and FH2 for RhoA activity and actin polymerization. We found that both domains activate RhoA (Fig. 1F), but only the FH2 was able to stimulate SRF and actin polymerization, whereas the FH2core inhibited SRF and basal actin polymerization due to its capping activity (Fig. 1G,H). These data demonstrate that Dia1 stimulates RhoA activity independently of its actin nucleation activity, thus representing a novel function of this conserved protein. Next we tested the possibility that Dia1 may be part of a signal transduction pathway toward RhoA. For this we stimulated cells with LPA under conditions in which Dia1 expression was suppressed by RNA interference. We observed that Dia1 is required for full RhoA activation by LPA (Fig. 1I), indicating that Dia1 is an essential component of LPA signal transduction toward RhoA. Since this function is separable from the actin nucleation activity, it is an independent and additional function of Dia1. Activation of RhoA requires GEF activity. We speculated about an involvement of LARG, based on the facts that (1) LARG, like Dia1, is implicated in LPA signaling by interaction with G 12/13 as well as by using a dominant-negative mutant of LARG (dnlarg) (Vogt et al. 2003; Tanabe et al. 2004; Vazquez-Prado et al. 2004); and (2) the Drosophila LARG homolog drhogef2 colocalizes with Diaphanous at the furrow canal during cellularization (Grosshans et al. 2005). To investigate whether LARG is involved in Dia1-induced stimulation of RhoA, we used the previously characterized dnlarg lacking the DH/PH domains (Fig. 2A; Vogt et al. 2003). Despite relatively low expression levels, dnlarg partially but significantly inhibited Dia1 DAD-induced RhoA activity (Fig. 1J), indicating that LARG is involved in RhoA-GTP formation through Dia1. How can Dia1 regulate RhoA activity? Since dnlarg interfered with Dia1-induced RhoA activity and both proteins function in LPA signaling toward RhoA, we tested whether LARG and Dia1 associate. Using Dia1 antibodies, we were able to coimmunoprecipitate Dia1 together with endogenous or transfected LARG (Fig. 2B; Supplementary Fig. 1). We also tested if dishevelled is GENES & DEVELOPMENT 1479
3 Kitzing et al. Figure 2. Dia1 interacts with LARG. (A) Schematic representation of LARG. (RGS) Regulator of G protein signaling; (DH) Dbl homology; (PH) pleckstrin homology. (B) Cells maintained in 10% serum were lysed before immunoprecipitation with -Dia1 or control antiserum (co). Cell lysates and precipitates (IP) were analyzed by immunoblot as indicated. (C) Lysates were prepared from cells expressing myc-larg derivatives or full-length LARG. Immunoprecipitates (IP) prepared using -Dia1 were analyzed for associating LARG derivatives (Co-IP) by immunoblotting as indicated. (D) Lysates from cells expressing FH2core and LARG C-terminal mutants were immunoprecipitated and analyzed by immunoblotting as indicated. present, as it was shown to bind the DRF Daam1 for Wnt/Frizzled activation of RhoA during Xenopus gastrulation (Habas et al. 2001), but we did not detect dishevelled in the Dia1/LARG complex (data not shown). LARG belongs to the RGS domain-containing RhoGEFs, consisting of two additional members, p115rhogef and PDZ-RhoGEF (PRG) (Vazquez-Prado et al. 2004). Hence, we assessed the specificity of the Dia1/ LARG association. We found that Dia1 interaction with LARG is specific among this RhoGEF family since PRG or p115rhogef did not coimmunoprecipitate with Dia1 (Supplementary Fig. 1). However, we cannot exclude the possibility that Dia1 influences additional RhoGEFs or RhoGTPase-regulating proteins. To determine the regions required for association with Dia1, a series of LARG truncation mutants was tested. Only LARG mutants containing the C terminus coimmunoprecipitated with Dia1 (Fig. 2C). Since the FH2core domain of Dia1 is sufficient to induce RhoA activity, we tested whether the LARG C terminus and the FH2core domain would associate. Indeed, we could coimmunoprecipitate the FH2core domain with the C terminus of LARG (Fig. 2D). To further define the region within the LARG C terminus, we coexpressed its N- or C-terminal regions (CT-N and CT-C, respectively) with FH2core. As shown in Figure 2D, only the C-terminal half of the LARG C terminus efficiently coimmunoprecipitated the FH2core domain, indicating that these regions are responsible for the association. These data were confirmed using protein overlay assays (Supplementary Fig. 2). The association of FH2core with the C terminus of LARG suggested that activated Dia1 regulates the LARG C terminus. The release of Dia1 autoinhibition through binding of active RhoA to Dia1-NT (Fig. 1A) can be reconstituted in vitro by measuring FH2-DAD (CT) domain-induced actin polymerization (Fig. 3A; Li and Higgs 2005; Brandt et al. 2007). To address whether autoinhibition of Dia1 regulates its interaction with LARG, we performed GST pull-down assays with LARG CT-C and the Dia1-NT and Dia1-CT fragments in the absence or presence of active RhoAV14. Interestingly, association between Dia1-CT and LARG-CT-C was strongly inhibited by the addition of Dia1-NT (Fig. 3B). Moreover, this inhibition was overcome by the addition of active RhoA to the complex (Fig. 3C). These data demonstrate that the interaction between Dia1 and LARG is controlled by RhoA-induced release of Dia1 autoinhibition. This suggests the existence of a positive feedback loop involving RhoA, Dia1, and LARG. Interestingly, conformational changes of the C terminus of LARG (CT) appear to be mediated by the interaction of N- and C-terminal regions, while the FH2core domain can interfere with CT-N/CT-C binding in a dose-dependent manner (Supplementary Fig. 2), indicating that Dia1 may modulate LARG C-terminal conformation, known to control LARG activity in vivo (Chikumi et al. 2004). To directly assess if Dia1 affects LARG activity, we performed in vitro GEF assays using a recombinant LARG consisting of the DH/PH domain and the C terminus. LARG efficiently accelerated the release of GDP from RhoA in a dose-dependent manner (Fig. 3D). Under these conditions, the lowest measurable concentration of LARG was used as a baseline activity to which recombinant FH2 was added. This resulted in a concentrationdependent acceleration of LARG activity (Fig. 3E). These Figure 3. Dia1 stimulates LARG GEF activity. (A) Actin assembly assays of Dia1-CT show release of NT-induced inhibition by RhoAV14. (B) His-Dia-NT and His-Dia-CT proteins were incubated with GST-LARG C-terminal regions, and complexes were analyzed by immunoblot using -His antibody. Coomassie staining for GST- LARG CT-N and CT-C is shown in the bottom panel. (C) GST pull-downs show association of LARG CT-C with Dia1-CT and the release of NT-induced binding inhibition by RhoAV14. Complexes were analyzed by immunoblot as indicated. Coomassie staining for GST-LARG CT-N and CT-C is shown in the bottom panel. One representative out of four independent experiments is shown. (D) Indicated concentrations of GST-LARG incubated with [8-3 H]GDPloaded RhoA. [8-3 H]GDP-bound RhoA (%) is plotted against incubation time. Shown is the mean ± SEM of three independent experiments. (E) One nanomolar GST-LARG was incubated with [8-3 H]GDP-loaded RhoA with 100-fold molar excess of GST or with 10- and 100-fold molar excess of GST-FH2. [8-3 H]GDP-bound RhoA (%) is plotted against incubation time. Shown is the mean ± SEM of three independent experiments GENES & DEVELOPMENT
4 Activation of LARG and RhoA by Dia1 results show that Dia1 can stimulate the GEF activity of LARG. We propose that the Dia1/LARG interaction results in conformational changes of regulatory regions within the LARG C terminus. LARG was identified as a gene fusion rearranged in AML (Kourlas et al. 2000). Up-regulated activities of RhoA have been reported for a variety of human cancers (Sahai and Marshall 2002; Lozano et al. 2003). Here we show that Dia1 promotes RhoA activity via interaction with LARG and that Dia1 is required for LPA-mediated RhoA activity. Since LPA is known to play an important role in tumor development and progression promoting cancer cell metastasis and invasion (Mills and Moolenaar 2003), we tested the role of Dia1 in tumor cell invasion. One mode of tumor cell invasion in three-dimensional (3D) matrices is represented by the rounded mode of motility, for which Rho/ROCK signaling is necessary and sufficient (Sahai and Marshall 2003; Wyckoff et al. 2006). After optimizing the conditions for small interfering RNA (sirna_ toward Dia1 (Fig. 4A,B), we used highly invasive MDA-MB-435 human cancer cells (Sellappan et al. 2004; Rae et al. 2006), which express Dia1, Dia2 (Fig. 4B), and Dia3 (data not shown), and tested their ability to migrate into LPA-containing 3D matrices when Dia1 or Dia2 expression was down-regulated. Interestingly, knockdown of Dia1 efficiently and significantly inhibited the total number of tumor cells that have invaded as well as the overall invasion distance (Fig. 4D,E; Supplementary Videos 1, 2), whereas random MDA-MB-435 cell migration under two-dimensional (2D) tissue culture conditions was slightly increased (Fig. 4C). Knockdown of Dia2 did not inhibit cell invasion (data not shown). Similar results were obtained in A375m2 melanoma cells (data not shown), which also use the rounded mode of motility (Sahai and Marshall 2003). We next determined the involvement of LARG in MDA-MB-435 cell invasion. For this, we transfected GFP-dnLARG, GFP alone, or cotransfected with LARG1 820 as a control (transfection efficiencies were 1%) and assessed the GFP-positive cells invaded into the Matrigel. This revealed that dnlarg inhibited cell invasion (Fig. 4F) but not random 2D migration (Fig. 4C). Inhibition of ROCK with 10 µm Y27632 blocked invasion of MDA-MB-435 cells as expected, while treatment with the Src-kinase inhibitor PP1 had no effect (Fig. 4G). Consistently, usage of sirnas against LARG significantly reduced cell invasion (Fig. 4H,I). Together, these data suggest that LARG is involved in cancer cell invasion. In order to understand better the mechanisms underlying the dependence on Dia1 for tumor cell invasion, we analyzed the MDA-MB-435 cell morphologies in Matrigel. We observed that these cells use the rounded blebassociated mode of motility (Supplementary Video 3), while in 2D culture these cells displayed a mesenchymal-like cell morphology (data not shown). Analysis of F-actin using phalloidin staining revealed that untreated or control sirna-treated MDA-MB-435 cells showed extensive formation of membrane blebs when contacting and invading Matrigel (Fig. 5B; Supplementary Video 4). However, cells that were silenced for Dia1 (Fig. 5A) displayed strikingly different morphologies and lost their ability to promote membrane blebbing (Fig. 5B; Supplementary Video 5). Bleb-associated cell invasion requires ROCK (Sahai and Marshall 2003) to control contractile force through Figure 4. Dia1 is required for MDA-MB-435 cancer cell invasion. (A) Cells treated with different amounts of sirna as indicated and analyzed by immunoblotting. (B) Cells treated with control sirna or sirna against Dia1 were immunoblotted for the indicated proteins. (C) Cells transfected with indicated sirnas or plasmids were analyzed for random motility. Data are expressed as mean ± SD of three independent experiments. (D) Representative 3D reconstructions of invaded cells transfected with sirna as indicated. Cells were stained for F-actin (red); dashed line indicates the approximate position of the Transwell membrane; the arrow (50 µm) indicates the direction of movement. (E) Cells were allowed to invade for 48 h into Matrigel before counting at indicated invasion distances. The average of cell number from three independent experiments ±SD is shown. (F) MDA-MB-435 cells transfected with indicated plasmids were analyzed in invasion assays. The percentage of GFP-positive cells invaded is shown from two independent experiments counting >300 cells. (G) The number of cells invaded into Matrigel containing LPA and indicated inhibitors is shown. (H,I) Cells treated with indicated sirnas were analyzed for invasion. myosin activity (Riento and Ridley 2003). When basal as well as LPA-stimulated phosphorylation of myosin light chain (MLC), a ROCK substrate (Riento and Ridley 2003), was determined in MDA-MB-435 cells in the presence or absence of sirna against LARG or Dia1, we found that MLC phosphorylation was strongly reduced (Fig. 5C E) in contrast to Dia2 sirna (Fig. 5F). To investigate whether Dia1 promotes ROCK activity through RhoA, we used HEK293 cells to efficiently express the FH2 domain with ROCK-I before determining in vitro kinase activities using either MBP or MLC as substrates. Interestingly, we found that active Dia1 increased ROCK activity in a Rho-dependent manner (Fig. 5G). These results clearly show that activation of RhoA by Dia1 leads to an increase in ROCK activity to promote the MLC phosphorylation required for bleb-associated cell invasion. Dia1 is a well-established downstream effector of ac- GENES & DEVELOPMENT 1481
5 Kitzing et al. Figure 5. Dia1 is required for ROCK signaling and cancer cell morphology. (A) MDA-MB-435 cells were treated with control sirna or sirna against Dia1, and cell extracts were immunoblotted for the indicated proteins. (B) Representative 3D reconstructions of MDA-MB-435 cells treated in A invading through the porous membrane into the Matrigel. Cells were stained using DAPI (blue) to visualize nuclei and rhodaminephalloidin (red) to visualize F-actin. Bar, 20 µm. (C F) MDA-MB-435 cells treated with indicated sirnas were stimulated with 10 µm LPA for 10 min. Cell extracts were immunoblotted for the indicated proteins. (G) myc-rock-i was immunoprecipitated from HEK293 cells cotransfected with Flag-FH2 or C3 or treated with 10 µm Y27632 as indicated, and subjected to kinase assays using MBP or MLC as substrates. Kinase reactions were separated by SDS-PAGE, and phosphorylated substrates were visualized by autoradiography ( 32 P-MBP, 32 P-MLC). Immobilized rabbit IgG was used as a control (co). One representative out of three independent experiments with similar results is shown. (H) Proposed hypothetical model for Dia1-mediated feedback activation of RhoA through LARG. tive RhoA. Our results presented here reveal an unexpected and essential role for Dia1 in the activation of the Rho/ROCK signal transduction pathway and subsequent bleb-associated cancer cell motility. We provide evidence that Dia1 is required and sufficient for full LPAinduced activation of RhoA and downstream ROCK signaling. This effect can be mediated through interaction of Dia1 with LARG. The data shown here imply a novel signaling module by which Dia1, in addition to its role as a downstream RhoA effector, can function upstream of RhoA. This constitutes a positive feedback mechanism (Fig. 5H) amplifying signal-regulated cellular effects such as tumor cell invasion by activating RhoA and its effector ROCK. Materials and methods Plasmids, sirna, transfections, and cell-based assays Plasmids were generated using standard procedures. All sirna oligos were from IBA. GTPase pull-down assays, immunoprecipitations, and Rho-kinase and SRF activity assays were performed in HEK293 or NIH3T3 cells. See the Supplemental Material for details. Protein purification, actin assembly, and guanyl-nucleotide exchange assays Proteins were produced and purified in Escherichia coli strain DE3 as GST or His fusions. Actin polymerization was monitored as described (Brandt et al. 2007). Guanyl-nucleotide exchange assays were performed using GST-RhoA loaded with [8-3 H]GDP. See the Supplemental Material for full details. 3D matrigel invasion and confocal analysis Human MDA-MB-435 cancer cells were used for invasion assays. Assays were analyzed using confocal microscopy (Leica TCS SP2). See the Supplemental Material for full details. Acknowledgments We thank R. Treisman and B. Di Ventura for critical reading of the manuscript. We are indebted to G. Stier for plasmids, reagents, and expertise in protein purification. We thank S. Offermanns for support. We thank S. Narumiya and S. Gutkind for plasmids and A. Rippberger and J. Stastná for technical assistance. O.T.F. is supported by the CHS Stiftung. This work was funded by the Emmy Noether Program of the DFG (GR2111/ 1-2) to R.G GENES & DEVELOPMENT
6 Activation of LARG and RhoA by Dia1 References Brandt, D.T., Marion, S., Griffiths, G., Watanabe, T., Kaibuchi, K., and Grosse, R Dia1 and IQGAP1 interact in cell migration and phagocytic cup formation. J. Cell Biol. (in press). Chikumi, H., Barac, A., Behbahani, B., Gao, Y., Teramoto, H., Zheng, Y., and Gutkind, J.S Homo- and hetero-oligomerization of PDZ- RhoGEF, LARG and p115rhogef by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential. Oncogene 23: Copeland, J.W. and Treisman, R The diaphanous-related formin mdia1 controls serum response factor activity through its effects on actin polymerization. Mol. Biol. Cell 13: Faix, J. and Grosse, R Staying in shape with formins. Dev. Cell 10: Gomez, T.S., Kumar, K., Medeiros, R.B., Shimizu, Y., Leibson, P.J., and Billadeau, D.D Formins regulate the actin-related protein 2/3 complex-independent polarization of the centrosome to the immunological synapse. Immunity 26: Goode, B.L. and Eck, M.J Mechanism and function of formins in control of actin assembly. Annu. Rev. Biochem. 76: Goulimari, P., Kitzing, T.M., Knieling, H., Brandt, D.T., Offermanns, S., and Grosse, R G 12/13 is essential for directed cell migration and localized Rho-Dia1 function. J. Biol. Chem. 280: Grosse, R., Copeland, J.W., Newsome, T.P., Way, M., and Treisman, R A role for VASP in RhoA-diaphanous signalling to actin dynamics and SRF activity. EMBO J. 22: Grosshans, J., Wenzl, C., Herz, H.M., Bartoszewski, S., Schnorrer, F., Vogt, N., Schwarz, H., and Muller, H.A RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation. Development 132: Habas, R., Kato, Y., and He, X Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell 107: Kourlas, P.J., Strout, M.P., Becknell, B., Veronese, M.L., Croce, C.M., Theil, K.S., Krahe, R., Ruutu, T., Knuutila, S., Bloomfield, C.D., et al Identification of a gene at 11q23 encoding a guanine nucleotide exchange factor: Evidence for its fusion with MLL in acute myeloid leukemia. Proc. Natl. Acad. Sci. 97: Lammers, M., Rose, R., Scrima, A., and Wittinghofer, A The regulation of mdia1 by autoinhibition and its release by Rho*GTP. EMBO J. 24: Li, F. and Higgs, H.N Dissecting requirements for auto-inhibition of actin nucleation by the formin, mdia1. J. Biol. Chem. 280: Lozano, E., Betson, M., and Braga, V.M Tumor progression: Small GTPases and loss of cell cell adhesion. Bioessays 25: Mills, G.B. and Moolenaar, W.H The emerging role of lysophosphatidic acid in cancer. Nat. Rev. Cancer 3: Miralles, F., Posern, G., Zaromytidou, A.I., and Treisman, R Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 113: Otomo, T., Otomo, C., Tomchick, D.R., Machius, M., and Rosen, M.K. 2005a. Structural basis of Rho GTPase-mediated activation of the formin mdia1. Mol. Cell 18: Otomo, T., Tomchick, D.R., Otomo, C., Panchal, S.C., Machius, M., and Rosen, M.K. 2005b. Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain. Nature 433: Rae, J.M., Creighton, C.J., Meck, J.M., Haddad, B.R., and Johnson, M.D MDA-MB-435 cells are derived from M14 melanoma cells A loss for breast cancer, but a boon for melanoma research. Breast Cancer Res. Treat. doi: /s Riento, K. and Ridley, A.J Rocks: Multifunctional kinases in cell behaviour. Nat. Rev. Mol. Cell Biol. 4: Sahai, E Mechanisms of cancer cell invasion. Curr. Opin. Genet. Dev. 15: Sahai, E. and Marshall, C.J RHO GTPases and cancer. Nat. Rev. Cancer 2: Sahai, E. and Marshall, C.J Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat. Cell Biol. 5: Sellappan, S., Grijalva, R., Zhou, X., Yang, W., Eli, M.B., Mills, G.B., and Yu, D Lineage infidelity of MDA-MB-435 cells: Expression of melanocyte proteins in a breast cancer cell line. Cancer Res. 64: Shimada, A., Nyitrai, M., Vetter, I.R., Kuhlmann, D., Bugyi, B., Narumiya, S., Geeves, M.A., and Wittinghofer, A The core FH2 domain of diaphanous-related formins is an elongated actin binding protein that inhibits polymerization. Mol. Cell 13: Sotiropoulos, A., Gineitis, D., Copeland, J., and Treisman, R Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell 98: Tanabe, S., Kreutz, B., Suzuki, N., and Kozasa, T Regulation of RGS-RhoGEFs by G 12 and G 13 proteins. Methods Enzymol. 390: Tsuji, T., Ishizaki, T., Okamoto, M., Higashida, C., Kimura, K., Furuyashiki, T., Arakawa, Y., Birge, R.B., Nakamoto, T., Hirai, H., et al ROCK and mdia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts. J. Cell Biol. 157: Vazquez-Prado, J., Basile, J., and Gutkind, J.S Modular architecture and novel protein protein interactions regulating the RGS-containing Rho guanine nucleotide exchange factors. Methods Enzymol. 390: Vogt, S., Grosse, R., Schultz, G., and Offermanns, S Receptordependent RhoA activation in G12/G13-deficient cells: Genetic evidence for an involvement of Gq/G11. J. Biol. Chem. 278: Wyckoff, J.B., Pinner, S.E., Gschmeissner, S., Condeelis, J.S., and Sahai, E ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo. Curr. Biol. 16: Xu, Y., Moseley, J.B., Sagot, I., Poy, F., Pellman, D., Goode, B.L., and Eck, M.J Crystal structures of a Formin Homology-2 domain reveal a tethered dimer architecture. Cell 116: GENES & DEVELOPMENT 1483
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text:
Chin Augmentation
If you're unhappy with the size or shape of your chin, you may want to consider chin augmentation surgery. Otherwise known as genioplasty, chin augmentation can increase or reduce the length and projection of the chin. This can give you a stronger, more proportional look that is balanced with the rest of the face.*
At Florida Oral & Facial Surgical Associates, we invite all new patients from Daytona Beach, Palm Coast, New Smyrna Beach and the surrounding areas to set up a complimentary consultation today to find out how they can benefit from this surgery. In the meantime, take a look at some chin augmentation before and after pictures to get a better idea of what this procedure can do for you. During your consultation, your surgeon will be happy to discuss chin augmentation prices, as well as any other concerns you may have.
How is Chin Augmentation Performed?
There are two basic procedures used in genioplasty; one involves moving the chin bone in any direction, then fixing it securely with tiny wires or screws, and the other uses a chin implant. During the implant procedure, your surgeon will make a small incision under the chin or inside the mouth. He will first place a sizer to determine what size implant best enhances the proportion of your face. He will then remove the sizer, place the implant, and close your incision with sutures.*
Am I a Good Candidate for Chin Augmentation?
The best candidate for chin augmentation surgery has a receding or protruding chin and a normal dental bite. For patients needing bite correction, jaw surgery in conjunction with genioplasty may be necessary.
Your medical background will also play a part in whether or not you are a good candidate for this procedure. If you have any immune system problems, if you're suffering from hypertension or other cardiac problems, or if you're a smoker, you may not be a suitable candidate. Discuss your medical history with your doctor prior to the surgery.
What Kind of Post-Operative Chin Augmentation Care Can I Expect?
The most common side effects of chin augmentation surgery are mild soreness and swelling, which may cause your chin to feel tight. To reduce the swelling, your surgeon will advise you to keep your head elevated as much as possible. You should be back to work and normal activities within one week.*
What are the Typical Results of Chin Augmentation?
Chin augmentation surgery can provide dramatic results for patients who are unhappy with their weak or receding chin. Their new, stronger profile provides a much more balanced appearance, thereby providing a newfound self-confidence. While the results are subtle, they can make a drastic improvement. See for yourself in these chin implant before and after pictures.*
*Individual results may vary.

subset: commoncrawl


text:
October is Scrapbusters Month over at http://www.sewmamasew.com/ and I decided to get into the spirit and use up some of my scraps. I ended up making lots of things, but I thought that this would be pretty useful for most as an alternative to the rolls of plastic produce bags in the grocery store. The measurements are very flexible, and I think that having bars of cotton fabric instead of total construction of mesh will help it hold up longer. Another bonus is that this is a project that it doesn't judge...if you stitch a seam crooked...it still functions!
I clarified reinforcing the mesh in this video. It is a little rough holding the camera and recording, but I hope it helps!
scraps of mesh (I found this as a remnant in the fabric store, but repurposed sheer curtains, and netting works also.)
Length of ribbon (mine was 24 inches)
Fabric scraps--I used pieces from my cut 2.5 inch strip bin (which is the same as a jelly roll width)-what a great way to use some of those leftover prints!
Getting Started:
1. Cut the mesh you have to a rectangle shape. This can vary--I made a bag large enough for a few pounds of apples...you may want just enough for a few onions. I will give my measurements throughout. Mesh = 12 inches x 22 inches
2. Cut Fabric to size.
Top fabric: 4" x 22"
Two pieces of side fabric: 1.5" x 12"
Bottom piece: 2.5" x 22"
3. Prepare Top Casing
Fold in half (wrong side together) along the 22" length and iron. Fold 1/2" in on each side and press. (see drawing)
4. Attach Top Casing
Open up and use a zig-zag stitch to attach mesh to casing. I made sure the stitching was about 1/2" into the mesh to help it not tear too easily.
Fold over casing and stitch in place.
5. Attach the bottom piece to the mesh with a zig-zag stitch (outside of mesh bag is touching the right side of fabric.


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The earth has begun to shed the heavy, gray death coat of winter; to stretch yearning green fingers toward the returning sun. I feel the same primal urge to sweep the clutter from my life; to shed the physical and spiritual costumes that distract me from myself. On my windowsill, the single gray eye of a moonshell regards me with pointed intent. It is a reminder of purpose, of the need for the soul to remain single-minded amidst the whirlwind of daily life.
Purpose and meaning. For the bumblebee hovering on the other side of my window, purpose is found in his delicate dance with the lavender blossoms in my herb garden. This simple recognition of meaning and place is why the natural world offers such tranquility to us mere mortals. The bumblebee doesn't fret about career options; the cedar tree doesn't bemoan its lack of advanced education. Each living creature accepts and cleaves to its individual purpose. I yearn for the simplicity and focus of the moonshell. As a woman, a mother, a writer, the daily chores of raising a family and making a living often fragment my soul's sense of purpose, so that my life resembles a treadmill on high speed.
In one of my favorite books, "Gift From the Sea," Anne Morrow Lindbergh wrote about the challenge women face in fulfilling their obligations to others while retaining a stable and defined sense of self. Almost 40 years later, with all of our technological advances, the challenge to remain centered is greater than ever. A jangling and ongoing litany of noise and words assault the senses and starve the spirit. How do we silence the circus so that simplicity of purpose may be discovered?
I believe it's all about choices; about shedding the unnecessary aspects of our lives that prevent us from being true to our purpose. It's about being selfish, but not stingy, with one's time – and about pausing to reflect on the interconnectedness of each life within this brilliant and vast web. So here's my role and purpose…to bring light through my writing to the experiences that define and illuminate the human condition; the acts, great and small, that bring us closer to our own meaning and lift us up to God.


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Joshua 2:1-21 New International Version - UK (NIVUK)
Rahab and the spies
2 Then Joshua son of Nun secretly sent two spies from Shittim. 'Go, look over the land,' he said, 'especially Jericho.' So they went and entered the house of a prostitute named Rahab and stayed there.
2 The king of Jericho was told, 'Look, some of the Israelites have come here tonight to spy out the land.' 3 So the king of Jericho sent this message to Rahab: 'Bring out the men who came to you and entered your house, because they have come to spy out the whole land.'
4 But the woman had taken the two men and hidden them. She said, 'Yes, the men came to me, but I did not know where they had come from. 5 At dusk, when it was time to close the city gate, they left. I don't know which way they went. Go after them quickly. You may catch up with them.' 6 (But she had taken them up to the roof and hidden them under the stalks of flax she had laid out on the roof.) 7 So the men set out in pursuit of the spies on the road that leads to the fords of the Jordan, and as soon as the pursuers had gone out, the gate was shut.
8 Before the spies lay down for the night, she went up on the roof 9 and said to them, 'I know that the Lord has given this land to you and that a great fear of you has fallen on us, so that all who live in this country are melting in fear because of you. 10 We have heard how the Lord dried up the water of the Red Sea[a] for you when you came out of Egypt, and what you did to Sihon and Og, the two kings of the Amorites east of the Jordan, whom you completely destroyed.[b] 11 When we heard of it, our hearts sank[c] and everyone's courage failed because of you, for the Lord your God is God in heaven above and on the earth below.
12 'Now then, please swear to me by the Lord that you will show kindness to my family, because I have shown kindness to you. Give me a sure sign 13 that you will spare the lives of my father and mother, my brothers and sisters, and all who belong to them – and that you will save us from death.'
14 'Our lives for your lives!' the men assured her. 'If you don't tell what we are doing, we will treat you kindly and faithfully when the Lord gives us the land.'
15 So she let them down by a rope through the window, for the house she lived in was part of the city wall. 16 She said to them, 'Go to the hills so that the pursuers will not find you. Hide yourselves there for three days until they return, and then go on your way.'
17 Now the men had said to her, 'This oath you made us swear will not be binding on us 18 unless, when we enter the land, you have tied this scarlet cord in the window through which you let us down, and unless you have brought your father and mother, your brothers and all your family into your house. 19 If any of them go outside your house into the street, their blood will be on their own heads; we will not be responsible. As for those who are in the house with you, their blood will be on our head if a hand is laid on them. 20 But if you tell what we are doing, we will be released from the oath you made us swear.'
21 'Agreed,' she replied. 'Let it be as you say.'
So she sent them away, and they departed. And she tied the scarlet cord in the window.
New International Version - UK (NIVUK)
Holy Bible, New International Version® Anglicized, NIV® Copyright © 1979, 1984, 2011 by Biblica, Inc.® Used by permission. All rights reserved worldwide.
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Increase Performance of Any Dust CollectorFebruary 24, 2009 4:34 pm
American Fabric Filter Co., Wesley Chapel, FL, reduces back-pressure and improves the vacuum of any dust collector by pairing a large felt filter bag with a non-breathing plastic collector bag. The increased surface area provided by the oversized felt bag allows filtration of dust particles down to 1 micron as compared to the manufacturer's standard 30 micron woven bag. The sealed collector bag offers total containment of wood dust along with convenient disposal.
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There are many reasons to consider refinancing a mortgage whether it be due to a lifestyle change, consolidating your debts or, you quite simply just want a better deal. At Illawarra Home Loans we have refinancing options available at great rates. Have questions? check out our refinancing guides.
Best Reasons to Refinance Your Mortgage
There are so many different reasons that people look to refinance a mortgage and chances are, it's related to either unlocking equity, getting a better interest rate or changing to either a fixed or variable rate to suit your changing needs.
Read on for more detail on the main reasons you should refinance your mortgage.
5 Wise Strategies To Refinance Your Mortgage
There are plenty of reasons why people choose to refinance their loan to benefit from a lower interest rate, or switch to a different loan type, or access equity to finance renovations or the deposit on an investment property.
While you can refinance to a better overall loan, what that means to each person is quite individual.
1000′s Locked Into Fixed Mortgages Now Looking To Refinance
Thousands of home owners who locked into fixed mortgage rates before the Reserve Bank of Australia (RBA) significantly reduced the official cash rate three years ago are now looking to refinance, says Illawarra Home Loans director Martin Anstee.
So what's our answer for people looking to switch from a fixed to a variable interest rate?
Refinance With The Bank Beater Home Loan Now
If you're paying too much on your home loan or you want to consolidate you debts, apply for an obligation free online pre-approval now. Then simply complete the 3 step online application form, select a time that suits you and we'll call you with details of your Bank Beating rate and pre-approval.
It's that simple to refinance with Illawarra Home Loans.


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It is a question that I would like to ask everybody. Naruto is, probably one of the oldest series I loved before I actually fell in love with Anime as I do today. I was a big fan of it since the Anime adaptation of the series. I loved the story and it's a typical Shounen manga (少年漫画) with action and fights, featuring attractive female protagonists with exaggerated or overemphasized features. And in a word, attractive.
But as time passed, the series grew into a black hole which I felt I can never see the end of the story. When a story becomes too long, it usually become difficult to catch up with, and it is hard to understand the relationships among all these characters. Especially when the Anime started to drive away from the path of the original Manga. So I dropped this series at around 60 episodes (TV Anime). It was not a black stormy night that I made this decision, but something really nature.
Asking the commenters, have you watched this series before? If you have, are you still following it, either Anime or Manga? If you have already dropped it, when did you do?


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On Valentine's Day, the company that owns Anthropologie, Free People, and Urban Outfitters is set to unveil a new bridal line, BHLDN. The line will be filled with vintage-inspired wedding gowns, jewels, lovely bridesmaid dresses, and shoes.
I can hardly wait to sneak a peek at the beautiful lovelies this line is sure to have. I absolutely adore modern-vintage style weddings! :)


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At Trinity, we intentionally provide three specific kinds of care: common, crisis, and chronic.
Common Care
Common care is the day-in and day-out maintenance of gospel "life together." Common care happens at both the congregational and parish/small group levels.
• Marriage and parenting seminars, conferences, mentors, and adult education/Bible studies offered by the Community & Care Team and Spiritual Formation Team
• Discipleship and mentoring of children—from birth to college
• Discipleship and mentoring of local college students by Reformed University Fellowship and other college ministries supported by Trinity
• Pastoral counseling and "on call" pastor available for care emergencies
Geographically focused care by elders and deacons takes place as our officers are assigned the task of caring for the members and attenders in specific parishes. Sometimes this parish care will take the form of elders and deacons contacting parishioners not currently in small groups, "checking in" with them and encouraging them toward small groups. For those in a small group, that group's assigned elder will provide care on a personal basis. More on Parish Care
Chronic Care
Chronic care encompasses all situations that require ongoing, long-term care. These include issues of marital infidelity, long-term joblessness, and long-term health/medical/mental health issues. At Trinity, chronic care happens in the following ways:
• Small Groups—In all circumstances, chronic or otherwise, community in the form of a parish small group is key.
• Elders/Deacons—Through regular contact with small group leaders and parish leadership teams, the elders and deacons are aware of and intervene in situations of chronic care.
• Supplemental Care ministry teams—Ministry teams in various areas (divorce, financial, addiction, etc.) supplement the care provided by small groups, parishes, and New City Counseling, providing specific expertise and experience, as well as resources and materials.
To let us know about a chronic care need, contact the church office: 434.977.3700.
Crisis Care
For crisis care, call the church office at 434.977.3700 between 10am and 4pm. After these hours, and on weekends and holidays, call our emergency number, 434.260.0001, to reach a pastor. Please use the emergency number only for situations that require immediate attention, such as a hospitalization or death.
Crisis care takes place in crucial moments as people experience pain surrounding loss and/or tragedy. This could include a death in the family, the loss of a job, and other trauma-inducing situations. Crisis care happens at Trinity in the following ways:
• Small Group—Times of crisis are one of the exact moments when we need a community of people who know us well and with whom we feel comfortable asking for help. A person's small group spearheads efforts to care for a person or family during crisis.
• Elders/Deacons/Pastor on Call—When a crisis need comes to the attention of a small group leader, the church receptionist, or the Director of Community and Care, the elders, and the pastor on call are contacted and work together to help in the situation.
• Care Teams—In some circumstances a specific Care Team made up of small group leaders, elders, deacons, staff, and counselors may be put together.
• Hospital Visitation Team—Along with small group leaders and members visiting parishioners who are in the hospital, a Trinity pastor or elder makes an initial hospital contact and, if possible and necessary, takes a member of the hospital visitation team with him.
• Funeral Service Team—This team helps coordinate efforts surrounding funeral services. They engage the family's parish in helping with funeral arrangements, namely those items surrounding the service and reception itself.


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i really did try and do a wordless post but i cant resist!!
here's a few new additions to the pretty vintage household!
a new light for the landing... well we've only lived here 3.5 yrs and have had a bare bulb on the landing all that time.. urgh i can hear you say..a bare bulb it must have looked like a squat!!
please ignore the extremely ugly artex ice cream ceiling.. its my bit of retro on the ceiling!.. well i have to tell myself that!.. we bought this from Homebase and its fab.. you don't even need to do any wiring or anything just pop onto the light fitting great stuff!
pea green coat not pea green boat!
we bought this last week it is Edwardian .. and its huge!!..its obviously missing the Pitcher to go with it. so I'll have to pair it up with something else for now
this is the top of the biggest tin.. so sweet isn't it


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|Pat Glass MP
Some basic text you might want to use or adapt is below. You might also want to have a look at Why Nursery Schools Matter from the National Campaign for Real Nursery Education.
If you have some time to personalise what you say and make it more relevant to your MP, then your email will have even more effect. If it helps, have a look at what I wrote to Stephen Timms MP.
Dear [insert the name of your MP]
On Tuesday there is an adjournment debate on the future of nursery schools. The continued closure of nursery schools is a matter of great concern to me, and I think it is urgent that parliament speaks up to support them.
Maintained nursery schools are DFE-registered schools, like primaries and secondaries. There is a substantial evidence-base for their effectiveness. The DFE-sponsored Effective Provision of Pre-School Education (EPPE) project found that nursery schools had the highest quality and the best outcomes for children. As recently as this year, Ofsted's first Annual Report on the Early Years found that "looking only at the overall judgements given, nursery schools perform considerably better than other types of early years provision". In fact, figures from the National Campaign for Nursery Education show that 55% of nursery schools inspected between 1st January and 31st March 2014 were judged outstanding in comparison to 8% of Primaries and 14% of Secondary schools.
Perhaps even more impressively, Ofsted commented in its annual Early Years Report that nursery schools are the only part of the school system which "perform as strongly in deprived areas as more affluent ones".
So, the evidence points strongly to the quality of nursery schools, and their particularly beneficial impact for disadvantaged children. Nursery Schools do not just benefit the children on their roll: they are often at the heart of Children's Centres, providing support and early intervention for thousands of children. Increasingly, they work with other early years settings and with childminders to support quality improvement for all. The national charity Early Education reports that more than 80% of nursery schools are involved in training and placements for training, with more than one in five leading or being part of a Teaching School Alliance.
Yet they are closing fast. In 1980, there were 599 nursery schools in England. According to the DFE, there are now just 418. And the rate of closure is getting faster all the time: figures from the national charity Early Education indicate that over 100 nursery schools in Britain have been closed since 1999.
Across the country, many nursery schools report that they are feeling vulnerable to closure. In a recent survey conducted by Early Education, 77% of nursery schools reported that they were concerned about their future viability or faced imminent loss of their independence. Only 12% felt positive about the future. Ignoring the evidence about quality and wider impact, the previous minister for Early Years and Childcare, Liz Truss, sat back and allowed the continued loss of nursery schools to accelerate, telling the Select Committee that "nursery schools should not get special treatment".


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Singapore has been known as a center for a wide range of products around the world. There is a quoted that says "There product is not regard as quality if it has not yet entered Singapore". This is what makes Singapore a very attractive place to shop.
|The Uniqueness of the History of Singapore (picture)
The uniqueness of the History of Singapore
Singapore gained independence from Malaysia after being together with Malaysia formed Union. Singapore's past history was recorded as one of the important areas for the royal Malay Kingdoms before the Kingdom move to Melaka.
It was said that at one time, Maharaja Prameswara met with a sleeping Lion while resting on the small island (Singapore). But when the Lion approached it even ran away that what make origins name of the word Singapore. Lion = Singa, Pretend = Pura.
There is also another version, that says at the time of Maharaja Prameswara was looking for a suitable place to serve the capital of the kingdom. The King encounter large building in the form of "Pura = Temple", on the temple also standing Lion statues. Then from a combination of two words that later became the name of Singapore. Namely Singa = Lion and Pura = Temple, or the Lion Temple.
I wonder which version is actually a reference to the name of Singapore's up to day. Clear during the reign of the Johor-Riau, Singapore was known as Temasek. In a further development, Singapore is located in the territory of Johor-Riau Kingdon until finally purchased by TS Raffles. In era of Raffles, Singapore began as the foundation initially as regional distribution centers across the continent.
Singapore uniqueness Language
Singapore community living in the present era, is a unique community and consist of various tribes. Singapore national language is English, but in fact they have a distinct British accent. Language of Singapore is finally known as Singlish abbreviation of Singaporean English. The uniqueness of this Singlish accent has become the main attraction for the tourists who visit the Singapore.
Singlish used by different ethnic communities. There are also some Chinese, Indian or Malay who use their Singlish to communicate. However, Singlish is using by people who come from different native language.
For those of you who are planning to shop in Singapore do not forget to follow the style of Singapore language society, because usually by mimicking the native accent an area we will get a slightly cheaper price.
"How's much for this?" Is change to "how much for this one ha?"
Then let them answer "20 Dollars" ex.
The Uniqueness of the History of Singapore
Replied again with intonation Singlish style, just acting like you are Chinese people who just learn speak English "Haiya .... Too expensive ar, make it less little bit lah" or "Haiya could you make it less ar? "
If it's true by following local dialect will make the prices little cheap, coupled with a bit of luck then you will definitely get a little discount that expected.
Singlish is usually synonymous with the word "Lah" behind the sentence as a flavoring language. For example, "This one is pretty expensive lah" or "that's very beautiful girl lah". Interestingly despite having Singlish as a unifying language, but Singapore still use Malay as their national anthem. Singapore national anthem is "Majulah Singapura". Here are the lyrics of the song "onward Singapore".
Singapore National Anthem Lyrics
Mari Kita Rakyat Singapura Sama-sama menuju bahagia
Cita-Cita kita yang mulia
Majulah Singapura
Marilah kita bersatu dengan semangat yang baru
Semua kita berseru Majulah Singapura
Singapore Foods
Singapore also has a unique food. Singapore food in general is a blend of India and Chinese food culture. Try a taste of some culinary Singapore among Nasi Goreng, Mee Goreng or Martabak along Teh Tarek as the drink. Of course you will feel the sensation of extraordinary blend of seasonings. And make you yearn to return to Singapore. (Zulfahri)


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Well the celica's taking a back seat for awhile..... kinda getting bored with her.....
so i bought this fun little thing.
1990 Nissan 240sx
5 speed with only 95k on a swapped ka24de motor....
I bought it from a good friend so i know the mileage is about right.
stripped interior but i have all the pieces...ill leave it this way for a bit.
only issue is a crack in the gas tank......so i got a new one in
anyways just popping on here to let everyone know what ive been doing.
moar pics


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culture vulture May 22 to 28 - events in Colombo
Culture Vulture Colombo© is a selective, non exhaustive, noncomprehensive
listing of Colombo's cultural offering, updated on an ad-hoc basis, and
mailed out absolutely free. Its views are un-biased but totally subjective. If you would like to list a non-political event here, please send us an e-mail. Please
confirm show-times etc. with event organisers, as Artistes and venue
owners alike tend to lead interesting and at times chaotic lives. To offset the environmental pollution caused by printing books, Perera Hussein grows trees in
Puttalam – Sri Lanka's semiarid zone. Protected content ph-books.com
WEEKLY ROUND UP 22 TO 28 MAY Protected content
On going
EXHIBITION, Loot from the Loft –
Assorted work for viewing &
for sale. To 30/5
V: Barefoot Gallery (FREE)
T: Protected content
EXHIBITION, Life Acrylic on
canvas paintings by Rohan
Amarasinghe. To 24/5
V: Saskia Fernando Gallery
T: Protected content
22 May (Tue)
15:00 & 18:30 MOVIE, La
Petite Jerusalem – a beautiful
young Jewish woman falls in
love with a Muslim co-worker.
(FREE) to 23/5
V: Allaince Fr. Colombo
24 May (Thu)
17:00 LECTURE, Out of Africa
& the Significance of the South
Asian Archaeological record. By
prof. Michael
Petraglia of the Uni. of Oxford
Mythil's Secret (Rs. Protected content
Prashani Rambukwella
"Go outside
and play Mythil," standard
mother's command.
"With who" ponders Mythil?
He knows this is all a shallow
prelude to his parent's regular
squabbling which is nothing
but "pus wedilli" that he sees
often. Starts at nothing and
ends up covering everything
from who ran up the phone
bill to why Archchi should be
gifted apple juice and then
they hold hands, Ammi and
Thaththi, cease fire negotiateed
and all quiet on the home
Such is the conversation of
the book, of day to day family
feuds and banter that we
know so well; easy to relate to
and all so familiar to a child
Author Rambukwella has got
into the imaginative mind of
a pre-teenage boy and has
given a wonderful story for
children with a lot of local
Winner of a previous year's
Gratiaen Prize. Available
everywhere books are sold.
19:00 MUSIC, Chamber Music
Plus- Mahler, Schubert etc. w/
Lakshman Joseph de Saram.
V: Goethe Hall (TKTS: Rs. Protected content
T: Protected content
ANNOUNCEMENT, Gratiaen
Prize Protected content from the shortlist
of: Mariam Riza, Charulatha
Thewarathanthri, Lucky de
Chickera, Malinda Seneviratne
& Madhubashini Ratnayake.
What are the odds? Two of
them have been shortlisted
before; two new writers and
one veteran with two books
under his belt.
V: (by invitation G. Trust)
28 May (Mon)
19:00 DANCE, Another
Tempest # 4, an adaptation of
Shakespeare & Aime Cesare's
une tempete, recited on a music
collage of live percussion juxtaposed
w/ western classical
music. Choreography Nalin
Maligaspe, Dramaturgy Asoka
de Zoysa. (TKTS Protected content Protected content
V: Warehouse Project
T: Protected content
V: 96 Nelum Pokuna Mw.
19:30 PLAY, Cast as Mother – a
Reading – based on the
experience & writings of 13
women on motherhood.
Ruwanthi de Chickera, Tracy
Holsinger etc.
V: Lionel Wendt
T: Protected content invitations)
25 May (Fri)
18:30 LECTURE, Geoffrey Bawa
Memorial Lecture – on Praxis,
delivered by Indian architect
Bijoy Jain of Studio Mumbai.
V: Ananda College (Kularatne)
T: Protected content
18:30 MOVIE, The Pianist
Award winning
Roman Polanski film showing
Szpilman as a holocaust
survivor. Starring Brody.
V: Russian Centre (Rs. Protected content
26 May (Sat)
EXHIBITION, Skzin & Bones,
art & design by Dr. Milinda
Salpitikorala (FREE) To 27/5
V: Lionel Wendt (HPG)


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we are getting a new kitchen, but the Work will leve us whit out a kitchen for 3 weeks. We have a microwave and are buying one burnerplate. Any ideas for what to make? I have some frezermeals (variasion on meatsause) but not enoff for 3 weeks.
Take out are expensiv in Denmark, so we cant live of that for so long.
One-pot pasta dinners are great!
Salads are easy too. Actually just google posh student meals and you will find a ton of recipes! And congrats with the new kitch! :)
Any chance you have a rice cooker around? Or are they are as cheap in Denmark as in the US? Making things in the rice cooker was something my parents relied on when they were renovating their kitchen because they also had to clean up in buckets or the bathroom sink and it is very easy to clean in a bucket compared to a full pot/pan.
You can hydrate lots of grains besides rice following the regular directions, to eat with a simple salad, seared meat (and if it were me a vinaigrette over all three). Simple food, but you can change up each component to have increased variety. In addition, there are a handful of weird things you can make in a rice cooker that you would normally bake in the oven, like frittatas and curries, this lifehaker list looks like it has all of the techniques I know: http://lifehacker.com/15...
Eat out and explore some new restaurants for inspiration you can use in your new kitchen.
My neighbor remodeled her kitchen after I had remodeled mine. She did 2 things I wish I'd done - she had her kitchen sink temporarily plumbed into her basement (I missed the sink more than anything) and she used a slow cooker.
I've been using the utility sink in the laundry with a dish pan for washing the dishes. I can't wait for this to be completed! Thankfully, we have the microwave and oven but still hard to prepare anything without counters. I feel for you, Jenny Marie!
I think a slow cooker would be very handy. Great for whole chickens cooked down in own juice, pot roast, soups, stews. If you have any interest in owning a toaster oven you might find that helpful also as long as you are cooking for only a few people. I have baked many a pasta or casserole type dish in toaster oven.
Electric tea kettles are also a huge help--great for boiling water for couscous and bulgur, and for giving a head start to that pasta water on that single burner!
slow cooker meals, they are so easy or your grill. I re-did my kitchen and made soup each weekend in the slow cooker and just has a microwave in my living room. But I also did a lot of grilling or foil packets on the grill with veggies, etc.
I'm in the same situation, kitchen remodel. I guess I didn't really think it was going to be that big of an inconvenience....I was so wrong! Like others have suggested, I pulled out the slow cooker and made exception soup and stews. We have the fridge available, which is a HUGE plus. Canned potatoes, green beans, tuna, all wonderful as well.


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SAN FRANCISCO (MarketWatch) — Netflix Inc.'s strong results and better-than-expected gains in subscriber growth came at the same time that the video-streaming company instigated a price hike.
But unlike in 2011, when Netflix lost about 1 million subscribers in the fallout from a price hike, this time around Netflix NFLX, -0.97% says it added 1.69 million video streaming subscribers in its June quarter, which was above its previous forecast of 1.46 million. Those additional customers joined in spite of the price hike to $8.99 per month for streaming for new customers in the U.S.
CEO Reed Hastings told analysts in an earnings interview that the price hike was "only a dollar difference, so I really think it's background noise, which is what we want it to be." Company executives also touted some of the company's exclusive offerings, including the show "Orange is the New Black," as attracting and keeping viewers.
"The content is so great and there is so much of it being produced, you could barely watch it all in your lifetime," said Theordore Sarandos, Netflix's Chief Content Officer.
Investors were pleased that the company has learned from its past missteps and drove up the shares in after-hours trading. Looking ahead, though, how to keep getting new subscribers is going to be an issue. Investors are now hoping that Netflix will eventually enter China.
Want this type of analysis sent to your inbox? Subscribe to MarketWatch's free MarketWatch First Takes newsletter. Sign up here.


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Let's start where we can all agree: The overuse of medically important antibiotics in food animals should stop. We can focus on the past and point fingers, or we can address it now.
Sponsored Links
Last week, the Food and Drug Administration (FDA) initiated an approach that we believe will reduce the risk of antibiotic resistance and protect the public health.
We'll do this by working with the animal drug industry, ranchers and farmers, and veterinarians. Our strategy will help ensure the health of America's families as well as the health of the animals on farms and ranches across the country. These ideas are not mutually exclusive and can be achieved by eliminating production uses of certain medically important drugs in livestock, while preserving therapeutic purposes upon the advice of a veterinarian.
Some critics argue that FDA's strategy is too soft and call for regulatory action. In reality, initiating an enforcement approach in this instance would mean going drug by drug and could take many years.
About Editorials/Debate
Opinions expressed in USA TODAY's editorials are decided by its Editorial Board, a demographically and ideologically diverse group that is separate from USA TODAY's news staff.
Most editorials are accompanied by an opposing view — a unique USA TODAY feature that allows readers to reach conclusions based on both sides of an argument rather than just the Editorial Board's point of view.
Our strategy is immediate and industrywide: All companies are being asked to remove production uses from their drugs. FDA is proposing a time frame for these changes, and we're confident it can be met. And we have been working with the U.S. Department of Agriculture to strike the right balance and help ensure our success.
To those who say we are simply "trusting" the industry, we would reply, "Trust but verify." It will be clear how industry responds, and we have not yielded any of our enforcement options.
It is important to recognize that the economic realities of the marketplace are already bringing change. More and more companies — such as McDonald's and KFC— require their suppliers to verify that they don't use antibiotics for production purposes, because that's what consumers want. We understand that phasing out these production uses of antibiotics as quickly as possible is a public health priority.
FDA's actions provide an orderly, timely path forward that will reduce antibiotic resistance to protect both human and animal health. We can focus on the past, or we can move ahead. We chose to move ahead.
Michael R. Taylor is deputy commissioner for foods at the Food and Drug Administration.
Looking beyond the spin of Big Pharma PR. But encouraging gossip. Come in and confide, you know you want to! "I'll publish right or wrong. Fools are my theme, let satire be my song." Email: jackfriday2011(at)hotmail.co.uk
Tuesday, April 17, 2012
FDA: 'Trust but verify' – USATODAY.com
via usatoday.com
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This blog was originally posted as part of i-ACT21 Day 11.
One of our outstanding Little Ripples teachers attended our assessment training today. It was such a pleasure to see her. She is always so sharp and engaged in whatever training and work we're doing. On one of our previous trips, I asked how many children Fatima wanted, at the time she had two, and she responded, "more, more!" For some reason her response has stayed with me. She exemplifies the woman who does it all. She's a wife, a mother, a teacher, a friend, a leader, and I'm sure the list goes on.
When we were walking to lunch together, she pulled out her phone to show me that the background image was a picture of her and me! I couldn't believe it. I was so touched and it struck a chord with me. I always wonder what it must be like for the teachers and others we work with to see us come so briefly and go. It's something I give a lot of thought to. So to see that Fatima carries an image of me with her was so surprising and so humbling, and a reminder that there are few things in life that more important than connecting with others and continuing to show up.
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