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GATA factors establish transcriptional networks that control fundamental developmental processes. Whereas the regulator of hematopoiesis GATA-1 is subject to multiple posttranslational modifications, how these modifications influence GATA-1 function at endogenous loci is unknown. We demonstrate that sumoylation of GATA-1 K137 promotes transcriptional activation only at target genes requiring the coregulator Friend of GATA-1 (FOG-1). A mutation of GATA-1 V205G that disrupts FOG-1 binding and K137 mutations yielded similar phenotypes, although sumoylation was FOG-1-independent, and FOG-1 binding did not require sumoylation. Both mutations dysregulated GATA-1 chromatin occupancy at select sites, FOG-1-dependent gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, FOG-1- and SUMO-1-independent genes persisted at the periphery. These results illustrate a mechanism that controls trans-acting factor function in a locus-specific manner, and differentially regulated members of the target gene ensemble reside in distinct subnuclear compartments.
Posttranslational modifications establish versatile molecular switches that control fundamental cellular processes. A classic example is cAMP-dependent phosphorylation of CREB, which enhances its affinity for the coregulator and histone acetyltransferase CBP/p300 (Goodman and Smolik, 2000; Mayr and Montminy, 2001). By contrast to phosphorylation and other simple chemical modifications, certain posttranslational modifications involve the conjugation of small proteins, including ubiquitin or the related SUMO proteins, to recipient proteins. Ubiquitination functions to control proteasome-mediated proteolysis and also via proteasome-independent mechanisms (Kerscher et al., 2006). By controlling protein-protein interactions, sumoylation regulates diverse processes (Geiss-Friedlander and Melchior, 2007). Whereas transcription factor sumoylation can increase transcriptional repression by facilitating corepressor recruitment (Rosendorff et al., 2006), considerably less is known about how sumoylation promotes transcriptional activation (Gill, 2005; Lyst and Stancheva, 2007).
The consequences of signal-dependent posttranslational modifications can differ in distinct experimental and biological contexts. Given that transcription factors can utilize distinct mechanisms to regulate different loci (Bresnick et al., 2006; Kim and Bresnick, 2007), a modification might only affect a subset of the cognate target genes. GATA-1 (Evans and Felsenfeld, 1989; Tsai et al., 1989), which is crucial for controlling red blood cell, platelet, mast cell, and eosinophil production (Hirasawa et al., 2002; Migliaccio et al., 2003; Pevny et al., 1991; Shivdasani et al., 1997; Simon et al., 1992), activates or represses target genes with or without the cell type-specific coregulator FOG-1 (Crispino et al., 1999; Tsang et al., 1997), depending upon the locus (Johnson et al., 2007; Kim and Bresnick, 2007). As GATA-1 target genes regulated by FOG-1-dependent and -independent mechanisms are known (Kim and Bresnick, 2007), and the underlying mechanisms can be analyzed via genetic complementation in GATA-1-null cells (G1E) (Grass et al., 2003; Weiss et al., 1997), it is instructive to study GATA-1 as a model to understand how modifications affect distinct modes of activator function at endogenous loci.
The GATA-1 N-terminus contains multiple phosphorylation sites (Crossley and Orkin, 1994) and a single sumoylation site (K137) (Collavin et al., 2004). Deletion of amino acids 1–63, which removes two phosphorylation sites (Crossley and Orkin, 1994), is linked to the development of megakaryoblastic leukemia (Wechsler et al., 2002). Knock-in mice bearing Ser to Ala mutations of two sites within the N-terminus (S72 and S142) and also S310 (Zhao et al., 2006) exhibit normal steady-state erythropoiesis and responsiveness to acute stress in vivo (Rooke and Orkin, 2006). Deletion of amino acids 1–193 differentially affects target gene expression in G1E cells (Johnson et al., 2006). Since the 1–193 deletion eliminates K137, and protein-protein interactions requiring amino acids 1–193 are unknown, K137 might confer functionality to this important region.
The function of K137 sumoylation was initially assessed by expressing GATA-1 or GATA-1(K137R) in Xenopus embryos and scoring for globin and xGATA-1 mRNA as erythroid cell markers (Collavin et al., 2004). RT-PCR analysis revealed that GATA-1 and GATA-1(K137R) induced globin and xGATA-1 similarly. As GATA-1 and GATA-1(K137R) levels were not measured, and expression was assayed qualitatively at a single time, it is unclear whether GATA-1 and GATA-1(K137R) activities are similar or different. The SUMO ligase PIASy interacted with GATA-1 and increased K137 sumoylation upon overexpression of the factors in 293T cells (Collavin et al., 2004). In transiently transfected cells overexpressing GATA-1 and GATA-1(K137R) with PIASy, PIASy inhibited GATA-1- and GATA-1(K137R)-mediated transactivation of a luciferase reporter gene (Collavin et al., 2004).
Since GATA-1 is a critical regulator of hematopoiesis, it will be important to determine if K137 mediates endogenous target gene regulation, if similar levels of GATA-1 and K137 mutants function identically, and if K137 sumoylation accounts for the contribution of K137 to GATA-1 activity. Given the sophisticated knowledge of GATA-1 function (Kim and Bresnick, 2007), this is an attractive system for dissecting how sumoylation controls specific molecular steps instigated by a protein with dual activities to activate and repress genes. Using genetic complementation analysis, we demonstrate that K137 mediates GATA-1 function at certain, but not all, target genes, predominantly at FOG-1-dependent targets. Mutations of K137 and also V205 that mediates FOG-1 binding (Crispino et al., 1999) decreased chromatin occupancy at select sites, diminished FOG-1-dependent gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, strikingly, FOG-1- and SUMO-1-independent genes persisted at the periphery. These results illustrate a mechanism in which sumoylation of a master regulator of hematopoiesis selectively controls its function at specific loci and constitutes the first example in which differentially regulated members of a target gene ensemble reside in distinct subnuclear compartments.
Conditional activation of an estrogen receptor ligand-binding domain fused to GATA-1 in GATA-1-null G1E cells recapitulates a normal window of erythropoiesis (Welch et al., 2004) and therefore represents a powerful system for dissecting GATA-1 function. We engineered G1E clonal lines stably expressing ER-GATA-1 or ER-GATA-1 with point mutations of K137 (Fig. 1A) at comparable levels (Fig. 1B). Whereas ER-GATA-1 migrated as major and minor ~75 and ~105 kDa bands, respectively, the K137A and K137R mutants migrated solely as ~75 kDa bands. Thus, K137 is required for recovery of the ~105 kDa species, consistent with K137 sumoylation in 293T and mouse erythroleukemia cells (Collavin et al., 2004). Unlike ER-GATA-1, ER-GATA-1(K137R) was incapable of inducing erythroid maturation (Fig. 1C).
To determine how K137 contributes to GATA-1 function at endogenous loci, we quantitated GATA-1 target gene expression after estradiol-mediated activation of ER-GATA-1 or the K137 mutants. Estradiol lacks activity in G1E cells devoid of ER-GATA-1, and estradiol-induced transcription in G1E-ER-GATA-1 cells reflects GATA-1 activity to control its physiological target genes (Johnson et al., 2007; Johnson et al., 2006; Welch et al., 2004). Even though the K137 mutants and ER-GATA-1 were expressed at comparable levels (Fig. 1B), the mutants were considerably less active than ER-GATA-1 in inducing βmajor, α-globin, Ahsp, and Slc4a1 expression (Fig. 1D). Mutation of K137 also reduced the induction of Alas2, Hebp1, Ptdss2, Abcb10, and Tac2, (Fig. 1D). The mutants were at least as effective as ER-GATA-1 in inducing Csf2rβ2, Fog1, Epb4.9, and Eklf, (Fig. 1D). Thus, K137 differentially mediates GATA-1 activity at endogenous loci. We also analyzed the activity of GATA-1 and GATA-1(K137R) lacking ER after transient electroporation into G1E cells (data not shown). However, the protein levels were considerably lower than ER-GATA-1, and therefore the induction of βmajor and Slc4a1 was lower (~4–5 fold). The K137R mutant lacking ER exhibited a significantly lower activity than GATA-1 (p = 0.024) in this assay.
Of the four known FOG-1-independent, GATA-1-activated genes (Crispino et al., 1999; Kim et al., 2007), Fog1, Epb4.9, and Eklf do not require K137 (Fig. 1D). As ER-GATA-1 activates Tac2 in a FOG-1-independent manner and subsequently represses it in a FOG-1-dependent manner (Johnson et al., 2006), Tac2 differs from other FOG-1-independent genes. Of the FOG-1-dependent genes (βmajor, α-globin, Ahsp, Slc4a1, Alas2, Hebp1, Ptdss2, Abcb10, and Csf2rβ2), all except Csf2rβ2 require K137 (Fig. 1D). Although GATA-1 activates Csf2rβ2 in a FOG-1-dependent manner (Kim et al., 2007), nothing is known about its transcriptional mechanism that distinguishs it from other FOG-1- and K137-dependent genes.
GATA-1 also represses genes through FOG-1-dependent and -independent modes (Kim and Bresnick, 2007). We tested whether K137 is required to repress the FOG-1-dependent targets Gata2 (Grass et al., 2003; Pal et al., 2004) and c-kit, and the FOG-1-independent target Lyl1 (Johnson et al., 2007). K137A and K137R mutants were considerably less effective than ER-GATA-1 in repressing Gata2, c-kit, and Lyl1 (Fig. 1D), indicating that K137 is not solely required for activation.
Although GATA-1 is sumoylated at K137 (Collavin et al., 2004), lysines can also be acetylated and methylated (Yang and Seto, 2008). The K137 requirement for context-dependent GATA-1 activity might therefore reflect such modifications. To determine if ER-GATA-1 is sumoylated in G1E-ER-GATA-1 cells, we analyzed lysates under conditions that restrict SUMO protease activity. In estradiol-induced G1E-ER-GATA-1 cells, ER-GATA-1 migrated as ~75 and ~105 kDa species, with the ~75 kDa species being much more abundant (Fig. 2A). Immunoprecipitation with anti-SUMO-1 antibody enriched the ~105 kDa species, with its abundance exceeding that of the ~75 kDa species. Taken together with the report of K137 sumoylation (Collavin et al., 2004), these results indicate that SUMO-1 conjugation to ER-GATA-1 forms the ~105 kDa species.
FOG-1-dependent and -independent activated targets were largely sensitive and insensitive, respectively, to the K137 mutation. The preferential K137 requirement for GATA-1-mediated, FOG-1-dependent activation highlights an unexpected link between FOG-1 and K137. The GATA-1 N-terminal zinc finger contacts multiple FOG-1 zinc fingers (Cantor et al., 2002; Crispino et al., 1999; Liew et al., 2005), and no evidence exists that K137 and/or nearby sequences modulate FOG-1 binding. This link might reflect a FOG-1 requirement for K137 sumoylation or for sumoylated K137 function. As ER-GATA-1 is sumoylated in three clonal lines of FOG-1-null hematopoietic precursor cells (Cantor et al., 2002; Johnson et al., 2007) (Fig. 2B), FOG-1 is not essential for K137 sumoylation, and K137 is not required for ER-GATA-1 to bind endogenous FOG-1 in a co-immunoprecipitation assay (Fig. S1).
To determine if sumoylation underlies the differential K137 requirement for GATA-1 activity at distinct loci, we tested whether tethering SUMO-1 to ER-GATA-1(K137R) rescues its activity. We stably expressed a SUMO-1 fusion to ER-GATA-1(K137R) in G1E cells (Fig. 2C). Multiple clonal lines were generated to permit comparisons of clones expressing ER-GATA-1(SUMO-1/K137R) and ER-GATA-1(K137R) at comparable levels (Fig. 2C). Quantitative analysis of target gene expression indicated that ER-GATA-1(SUMO-1/K137R) activity far exceeded that of ER-GATA-1(K137R) at βmajor, α-globin, Ahsp, and Slc4a1 (Fig. 2D). By contrast, ER-GATA-1(SUMO-1/K137R) and ER-GATA-1(K137R) similarly induced Csf2rβ2, a K137-insensitive gene (Fig. 1D).
We developed an assay to measure the capacities of transiently expressed ER-GATA-1 to regulate endogenous targets in G1E cells. Using the Nucleofector system, ER-GATA-1, ER-GATA-1(K137R), and ER-GATA-1(SUMO-1/K137R) were readily expressed (Fig. 2E). Although ER-GATA-1(K137R) was slightly higher than that of ER-GATA-1 (Fig. 2E), it activated βmajor, α-globin, and Slc4a1 considerably less than ER-GATA-1 (Fig. 2F). ER-GATA-1(SUMO-1/K137R) activity was comparable to or greater than that of ER-GATA-1 (Fig. 2F), despite its expression being slightly lower than ER-GATA-1 (Fig. 2E). These results recapitulate conclusions from the stable assay (Figs. 1D and and2D).2D). ER-GATA-1 sumoylation at K137, and SUMO-1-mediated rescue of ER-GATA-1(K137R) activity indicate that sumoylation controls GATA-1 activity at select loci.
As an alternative approach to test whether sumoylation underlies the K137 requirement, we mutated E139 neighboring K137, which is a component of the sumoylation consensus. The E139A mutant was not sumoylated (Fig. 2G, top), and E139 resembled K137 in being required for ER-GATA-1-mediated activation (Fig. 2G, bottom). In aggregate, analyses with K137 and E139 mutants and the SUMO-1 fusion protein indicate that sumoylation of K137 explains the critical function of this residue. Even though the single major isoform of ER-GATA-1 is abrogated by mutating K137, anti-SUMO-1 antibody immunoprecipitated the isoform, tethering SUMO-1 rescued the K137 mutation, and mutating the sumoylation consensus dysregulated gene expression analogous to the K137 mutation, one cannot unequivocally rule out the possibility that K137 is subject to additional modifications in distinct contexts.
As certain GATA-1 target genes are insensitive to K137 mutations, presumably the K137 mutants localize to the nucleus. However, since GATA-1 target genes differ in the GATA-1 level required for regulation (Johnson et al., 2006), a change in nuclear or subnuclear localization might dysregulate select targets. Biochemical (Fig. 2H) and cell biological (Fig. 2I) analyses indicated that both ER-GATA-1 and ER-GATA-1(K137R) localized in the nucleus. ER-GATA-1 localized in speckles throughout G1E nuclei (92% of cells), analogous to endogenous GATA-1 (Elefanty et al., 1996). ER-GATA-1(K137R) localized in a ring-like pattern in 41% of the cells, with the least immunoreactivity centrally in the nucleus (Fig. 2I). Immunostaining with anti-nucleolin indicated that in certain cases nucleoli resided centrally in the ring (data not shown). The ring-like distribution was observed in only 8% of ER-GATA-1-expressing cells. Transiently expressed GATA-1 and GATA-1(K137R) lacking ER also localized differentially in G1E cells (Fig. S2).
The aberrant subnuclear localization of ER-GATA-1(K137R) might limit its availability for target gene occupancy. Quantitative ChIP analysis was conducted to compare chromatin occupancy by ER-GATA-1 and the K137 mutants at upstream regulatory regions of the β-globin (HS3 and HS2) (Johnson et al., 2002) and α-globin loci (HS-26) (Anguita et al., 2002; Pal et al., 2004), as well as the βmajor (Johnson et al., 2002), α-globin (Anguita et al., 2004), and Ahsp promoters (Pilon et al., 2006). The Ey promoter of the β-globin locus, which is hypoacetylated in definitive erythroid cells (Bulger et al., 2003; Forsberg et al., 2000) and not occupied by GATA-1 (Johnson et al., 2002), was used as a negative control. ER-GATA-1 and the mutants occupied HS3 indistinguishably (p = 0.178). ER-GATA-1(K137R) and ER-GATA-1(K137A) occupied HS2 29% and 39% less (p = 0.031) and the βmajor promoter 56% and 75% less (p = 0.011), respectively, than ER-GATA-1 (Fig. 3A). The mutants occupied HS-26 slightly less than ER-GATA-1 (p = 0.128), whereas this defect was greater at α-globin and Ahsp promoters (p = 0.011 and 0.010, respectively) (Fig. 3B). While K137 was not essential for ER-GATA-1 chromatin occupancy, it enhanced occupancy at select sites, resembling FOG-1 facilitation of GATA-1 occupancy (greatest at the βmajor promoter, less at HS2, and irrelevant at HS3) (Letting et al., 2004; Pal et al., 2004).
Submaximal levels of K137 mutants at certain chromatin sites might disrupt all or perhaps select GATA-1-instigated molecular steps at these sites. K137 might also function post-chromatin occupancy. As GATA-1 induces Pol II recruitment to promoters and distal regulatory sequences (Johnson et al., 2001; Johnson et al., 2003; Johnson et al., 2002), we tested whether K137 mediates Pol II recruitment to the βmajor, α-globin, and Ahsp promoters and also to the constitutively active RPII215 promoter (Im et al., 2005). K137 mutations strongly reduced ER-GATA-1-mediated Pol II recruitment at βmajor, α-globin, and Ahsp (p = 0.006, 0.007, and 0.001, respectively) but not at RPII215 (Fig. 3C).
GATA-1 binds and recruits multiple coregulators (Blobel et al., 1998; Kim et al., 2007; Stumpf et al., 2006; Tsang et al., 1997). We tested whether K137 mediates FOG-1, CBP, and TRAP220 recruitment to the β-globin locus, as well as the α-globin and Ahsp promoters. At HS3, which the K137 mutants occupied normally (Fig. 3B), ER-GATA-1 and the mutants similarly recruited FOG-1 (Fig. 4A). At HS2, in which occupancy by the K137 mutants was reduced modestly (Fig. 3A), ER-GATA-1 and the mutants also similarly recruited FOG-1 (Fig. 4A). The mutants recruited little to no FOG-1 to the βmajor promoter (Fig. 4A), the site at which their occupancy was most reduced (Fig. 3A). At the α-globin and Ahsp promoters, the K137 mutation had little to no impact on FOG-1 recruitment (Fig. 4B). Thus, modest decreases (27–54%) in K137 mutant occupancy did not compromise FOG-1 recruitment, whereas a larger decrease (66%) correlated with loss of FOG-1 recruitment.
GATA-1 induces CBP occupancy at HS3 and the βmajor promoter, but CBP resides at HS2 independent of GATA-1 (Im et al., 2005; Letting et al., 2003). GATA-1-mediated CBP recruitment to HS3 was K137-independent, whereas K137 enhanced CBP recruitment to the βmajor (Fig. 4A), α-globin, and Ahsp promoters (Fig. 4B). At the β-globin locus, TRAP220 recruitment was K137-independent at HS3, modestly enhanced by K137 at HS2, and K137-dependent at the βmajor promoter (Fig. 4A). TRAP220 recruitment to α-globin and Ahsp promoters also required K137 (Fig. 4B). These results indicate that K137 mutants have the capacity to recruit all the coregulators, but not at all sites. If K137 is absolutely required for GATA-1 binding to a coregulator in all contexts, one would expect defective recruitment at all sites.
Since K137 sumoylation is preferentially required for FOG-1-dependent gene activation, the sumoylation contribution to GATA-1 activity might utilize V205, which contacts FOG-1 and is required for maximal FOG-1 recruitment (model 1). Alternatively, sumoylation might function independent of the V205 requirement (model 2) (Fig. 5A). If model 1 is valid, tethering SUMO-1 to a V205G mutant would have no functional consequence. If sumoylation bypasses the V205 requirement, tethering SUMO-1 to the V205G mutant should yield a mutant capable of activating FOG-1-dependent genes, despite the absence of the key residue mediating FOG-1 binding.
We tested whether SUMO-1 rescues V205G activity to regulate FOG-1-dependent target genes in the transient complementation assay. Although ER-GATA-1 and ER-GATA-1(V205G) (Fig. 5B) were expressed similarly (Fig. 5C), only ER-GATA-1 strongly activated βmajor and α-globin (Fig. 5D). Tethering SUMO-1 to V205G (Fig. 5B) rescued activity to 116% and 70% of the ER-GATA-1 value, respectively (Fig. 5D). Tethering SUMO-1 to a C261P mutant (Fig. 5E), which disrupts the integrity of the C-finger, abrogating DNA binding, did not rescue activity (Fig. 5F).
Since K137 regulates subnuclear localization and mediates FOG-1-dependent gene activation, we tested whether V205 also regulates subnuclear localization. ER-GATA-1(V205G) localized in a ring-like pattern (71% of the cells) (Fig. 6A), similar to ER-GATA-1(K137R) (Fig. 2I), whereas ER-GATA-1(SUMO-1/V205G) resembled ER-GATA-1. A V205G/K137R mutant exhibited the ring-like pattern in 79% of the cells (data not shown).
We tested whether tethering SUMO-1 to ER-GATA-1(V205G) rescues activity via enhancing FOG-1 binding. Multiple co-IP analyses indicated that while the V205G mutation reduced FOG-1 binding to ER-GATA-1, tethering SUMO-1 did not increase binding (Fig. 6B). Thus, SUMO-1 endows the V205G mutant with the activity to regulate FOG-1-dependent genes at a level comparable to that of ER-GATA-1, despite reduced FOG-1 binding. We reasoned that SUMO-1 might restore the capacity of the V205G mutant to recruit FOG-1 to chromatin. To analyze molecular components at endogenous loci in cells expressing wild-type or mutant proteins, G1E clonal lines stably expressing V205G and SUMO-1/V205G mutants were generated. Similar to the transient assay (Fig. 5D), tethering SUMO-1 to ER-GATA-1(V205G) rescued ER-GATA-1-mediated activation of endogenous targets (Fig. 6D). Quantitative ChIP analysis revealed that ER-GATA-1(V205G) (clone 48), expressed ~2 fold higher than ER-GATA-1 (clone 11) (Fig. 6C), occupied HS2 similar to ER-GATA-1 but significantly less than ER-GATA-1 at the βmajor promoter (Fig. 6E). V205G recruited significantly less FOG-1 and Pol II to both sites. Tethering SUMO-1 to V205G rescued all defects, resulting in even greater occupancy than ER-GATA-1 at HS2. Thus, disruption of K137 sumoylation or FOG-1 binding reduces ER-GATA-1 chromatin occupancy at select sites (Fig. 6F), and tethering SUMO-1 overcomes the V205 requirement for GATA-1-mediated recruitment of FOG-1 to chromatin.
To establish the topographic relationship between GATA-1 and target genes regulated via distinct mechanisms, we conducted 3D immuno-FISH under conditions in which nuclear morphology is preserved. We measured the localization of FOG-1- and sumoylation-dependent (β-globin, α-globin, and Slc4a1) and –independent (Epb4.9, Fog1, and Eklf) targets relative to nuclear periphery-associated lamin and quantitated the percent of loci localizing at the periphery (outer 20% of the nuclear radius), as well as the inner 4 shells, each constituting 20% of the radius.
In uninduced G1E-ER-GATA-1 cells, ~50% of the β-globin (Fig. 7A,B) and α-globin (data not shown) loci localized at the periphery. In β-estradiol-treated G1E-ER-GATA-1 cells, β-globin, α-globin, and Slc4a1 were distributed throughout the 5 shells, whereas ~50% of these loci localized at the periphery in cells expressing ER-GATA-1(K137R) (Fig. 7A,B). As ER-GATA-1(K137R) is defective in inducing maturation (Fig. 1C), and locus positioning in cells expressing this mutant resembles that of immature cells, this difference might be related to the inability of ER-GATA-1(K137R) to induce maturation. Epb4.9, Fog1, and Eklf localized similarly in cells expressing ER-GATA-1 or ER-GATA-1(K137R), with ~50% of these loci residing at the periphery. Thus, ER-GATA-1 instigates the migration of certain loci away from the periphery upon maturation, consistent with the report that the β-globin locus in murine fetal liver erythroblasts resides at the periphery in immature cells and migrates inward upon maturation (Ragoczy et al., 2006). However, our results demonstrate that other GATA-1 targets reside at the periphery independent of maturation, and strikingly these loci do not require FOG-1 and GATA-1 sumoylation; only the loci that migrate inward are FOG-1- and sumoylation-dependent.
Activators often interact with multiple coregulators and are subject to diverse posttranslational modifications. Thus, it can be challenging to ascertain the contribution of individual regulatory events at endogenous loci. Herein, we used genetic complementation analysis to dissect the role of a posttranslational modification and a coregulator in the transcriptional control of various members of a target gene ensemble.
GATA-1 activates or represses target genes depending upon chromosomal context, but mechanisms underlying context-dependent activity are not established (Kim and Bresnick, 2007). Activation and repression occur via FOG-1-dependent and -independent mechanisms, but why only certain genes require FOG-1 is unknown. FOG-1 occupies FOG-1-independent loci, and therefore either FOG-1 functions redundantly at such loci or does not exert essential functions at all loci. Activities attributed to FOG-1 include facilitating GATA-1 chromatin occupancy (Letting et al., 2004; Pal et al., 2004), as well as binding the NuRD complex (Hong et al., 2005; Rodriquez et al., 2005), CtBP (Fox et al., 1999; Katz et al., 2002), and TACC3 (Garriga-Canut and Orkin, 2004; Simpson et al., 2004). The NuRD complex mediates GATA-1-dependent repression in certain contexts (Hong et al., 2005; Rodriquez et al., 2005). The significance of the CtBP interaction is unclear, since a mutant Fog1 allele encoding FOG-1 defective in CtBP binding lacks a phenotype (Katz et al., 2002). TACC3 can sequester cytoplasmic FOG-1, prevent GATA-1 binding to FOG-1, and suppress erythroid differentiation (Garriga-Canut and Orkin, 2004).
Although GATA factors recruit multiple coregulators to chromatin and are subject to diverse posttranslational modifications (Kim and Bresnick, 2007), no evidence exists that a single coregulator or modification dictates the precise transcriptional output. We demonstrate that GATA-1-mediated regulation of FOG-1-dependent target genes requires K137 sumoylation. Of the known FOG-1-independent activated target genes, K137 was not required for control of three of these four genes, demonstrating that it controls GATA-1 activity at select loci. As tethering SUMO-1 to the K137 mutant rescued activity, and disruption of the sumoylation consensus inhibited activity, similar to mutating the sumoylation site, K137 sumoylation is a crucial molecular switch to control GATA-1 function at an important subset of its target genes. Tethering SUMO-1 also rescued the V205 mutant, which was associated with restoration of FOG-1 recruitment to chromatin, although FOG-1 binding did not require K137, and K137 sumoylation did not require FOG-1. One cannot rule out the possibility, however, that FOG-1 modulates sumoylation in specific compartments in vivo. While trans-acting factor sumoylation is often implicated in transcriptional repression, and in a limited number of cases, in activation (Geiss-Friedlander and Melchior, 2007; Hay, 2005; Lyst and Stancheva, 2007), we are unaware of reports of sumoylation dictating distinct transcriptional outputs at different endogenous loci.
The control of subnuclear localization by sumoylation is established (Gill, 2004), and sumoylation can mediate nuclear pore docking (Mahajan et al., 1997; Matunis et al., 1996). Sumoylation of SATB2 (Dobreva et al., 2003) and Sp3 (Ross et al., 2002) confers a nuclear periphery localization, which is not the case with GATA-1 sumoylation. The ring-like pattern of K137 and V205 mutants resembles the localization of wild-type Sp3 (Ross et al., 2002). While SATB2 sumoylation reduces chromatin occupancy and promotes repression (Dobreva et al., 2003), and Sp3 sumoylation promotes repression (Ross et al., 2002), GATA-1 sumoylation facilitates occupancy, thereby conferring transcriptional control.
Since the molecular basis for FOG-1-dependency or independency is unknown, our demonstration that GATA-1 instigates the migration of FOG-1/sumoylation-dependent, but not -independent genes away from the nuclear periphery is of particular interest. The immobile, periphery-associated genes lack the coregulator and posttranslational modification requirements that are hallmarks of the genes that relocalize internally during maturation. Given the massive nuclear condensation during late erythropoiesis, genes residing in distinct subnuclear compartments might require different regulatory factors, e.g. genes at specific sites might require factors to counteract formidable repressive forces associated with the increasingly condensed chromatin. The organization of other genes in more hospitable nuclear subdomains would in effect buffer them against inhibitory influences. Given the differential subnuclear localization of two classes of GATA-1 target genes, it will be particularly instructive to define cis-elements at their loci and factors that confer specific subnuclear localizations and sensitivity or insensitivity of localization to maturation. Defining such sequences and factors that underlie nuclear compartment-specific transcriptional requirements has potential to be broadly important for understanding how complex gene networks are established and regulated during development.
G1E cell clonal lines, in which ER-GATA-1 and mutant derivatives were not overexpressed relative to endogenous GATA-1, and FOG-1-null cells were maintained as described in Supplemental Experimental Procedures.
G1E cells (3 × 106) were resuspended in 100 μL Nucleofector solution R (Amaxa Biosystems), and transfected with 24 μg endotoxin-free Maxi-prep DNA (Qiagen). Cells were transfected twice with the Nucleofector II system (Amaxa Biosystems) using the G-016 program, allowing 24 h between transfections. Cells were treated with 1 μM β-estradiol at 6 h after both transfections when pGD-ER-puro constructs were transfected. Cells were harvested at 24 h after the second transfection. Protein and RNA were prepared from 1 × 106 and 2 × 106 cells, respectively.
Wild-type and mutant GATA-1 were expressed as fusions with the ER ligand-binding domain using the pGD-G1ER-puro retroviral vector (Tsang et al., 1997) unless stated otherwise. Vector construction details are provided in Supplemental Experimental Procedures.
Clonal lines expressing mutant proteins at levels comparable to ER-GATA-1 were selected. To confirm that passages of cells retained comparable expression, lines were re-analyzed periodically as described in Supplemental Experimental Procedures.
GATA-1 sumoylation and endogenous FOG-1 binding were analyzed as described in Supplemental Experimental Procedures.
The immunofluorescence and 3D Immuno-FISH conditions are described in Supplemental Experimental Procedures.
Total RNA was purified with TRIzol (Invitrogen) from identical cultures used for Western blotting and ChIP, and gene expression was analyzed as described in Supplemental Experimental Procedures.
ChIP analyses were conducted as described (Im et al., 2004) and in Supplemental Experimental Procedures.
This work was funded by NIH grants DK68634 and DK50107 (EHB) and American Heart Association Predoctoral Fellowships (S-IK, H-YL). We thank Dr. Ken Young for assisting with Wright-Giemsa staining.
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This year, Starlight will brighten the lives of over 500,000 seriously and terminally ill children by granting their
wishes and by providing entertainment in hospitals and hospices throughout the UK.
All Starlight's activities are aimed at distracting children from the pain, fear and isolation caused by their illness. Starlight receives neither government nor lottery funding and relies entirely on the support of people like you.
For many children, the excitement and anticipation of their Starlight Wish gives them just the boost they need to carry on battling their illnesses. Six-year-old Harvey suffers from Duchenne Muscular Dystrophy, a condition which causes muscle weakness.
He loves Despicable Me and has lots of Minion toys and enjoys watching the film. Harvey has therefore asked for his Starlight Wish to go to Universal Studios to meet a minion, something which his parents told Starlight would mean the world to him.
Harvey's mum Sarah said, "We can't thank Starlight enough for sending us to Florida. Harvey was very, very tired on the trip but he pushed through it all and had the time of his life. When the minions came out especially to meet us his face lit up. At that moment we forgot all about Harvey's condition and the struggles he faces every day and we saw a normal six year old boy whose wish had just come true."
Upcoming wishes include a trip to meet the Strictly Come Dancing team for Emilia, aged 8, who is fighting acute lymphoblastic leukaemia; Joshua, who is 14 years old and has brittle bone disease, wishes to go on a Mediterranean cruise; and five-year-old Jasmine, who has severe epilepsy, wishes to be a princess for the day.
Your much appreciated contribution and support will help Starlight bring happy memories into the lives of many seriously ill children and their families, and for that I cannot thank you enough.
Very best wishes


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This eGift card may be used toward games, food, and more at the beloved family fun center
Traditional Italian dishes showcasing Napoletano cuisine, such as spaghetti carbonara with pancetta and brick-oven pizzas.
Hand-tossed pizzas with sweet or spicy tomato sauce; delicious wings in your choice of style, served with blue cheese or ranch dressing
From-scratch Italian specialities include lasagna, penne alla vodka, veal parmigiana, wood-oven baked pizzas, and tiramisu.
$16 for $30 worth of Italian-American cuisine.
Hand-tossed New York-style pizza, topped with fresh ingredients that include housemade sauce and your choice of meats and veggies
Seafood, chicken, and pizzas served alongside hearty pastas in a romantic eatery with warm-colored murals of Italian streets.
This brand new outpost of the beloved Italian restaurant serves up authentic pizzas and pastas inspired by the cuisine of naples.
Italian eatery serves classic and gourmet pasta dishes including steak and Seefood entrees .
From the merchant: Great traditional Italian hand-tossed pizza and fresh, made-to-order entrees.
Traditional pizzas, pastas, and sandwiches are served alongside homemade pigs in a blanket, habanero burgers, and a ghost-chili pizza
$20 for $35 worth of Italian-American cuisine.
Hand-tossed pizza and deli sandwiches served at casual eatery with DJ nights
Pizza, pasta and other Italian cuisines will be available for guests to tuck into, along with unlimited soft drinks or house beverages.
Flip flop friendly restaurant serves seafood cuisine and steak with a view of the ocean right over the beach
Greek dishes kissed with extra-virgin olive oil and served in a festive atmosphere with dancing and a terrace; free parking is available
Entrees such as poached lobster tails, fried flounder with sweet-corn bacon grits, and steaks from rib eyes to dry-aged porterhouses
Baked oysters flavored with sambuca, flounder stuffed with crabmeat, and surf 'n' turf beef tenderloin served with tuna and a crab cake
Neighborhood tavern offers various dishes, including Yuengling fried shrimp, swordfish tacos, and house-made crab cakes, and other pub fare
Enjoy the French-inspired cuisine, created by chefs using locally-sourced ingredients, in a relaxed yet elegant environment
National franchise features casual American food
More than 20 specialty maki rolls served alongside Japanese entrees such as shrimp tempura, chicken katsu, and vegetable yakisoba
Celebrate the 8th anniversary of The Route 58 Deli with mile-high corned beef and pastrami sandwiches, potato pancakes & New York cheesecake
Comforting favorites, such as juicy cheeseburgers, fresh fish tacos, signature salads served with a nautical pinup style twist
Traditional Japanese eatery offers up a wide selection of sushi and sashimi for customers to enjoy
A '50s-style diner serves cheeseburgers, cheesesteaks, and chicken sandwiches complemented by chocolate malts and fries
Enjoy Mexican cuisine filled with all the flavors of Central America
Neighborhood bar offers a large selection of appetizers and serves American cuisine such as burgers, country fried chicken, and pulled pork
Seafood dishes such as lump-crab cakes and steamed shrimp alongside classic American food such as Angus burgers and baby back ribs
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Hurricane Center To Issue Storm Surge Warnings During Hurricanes
The National Hurricane Center has enhanced their warning protocol to include life-threatening storm surge. This new product will help save lives during landfalling storms.
The National Hurricane Center has enhanced their warning protocol to include life-threatening storm surge. This new product will help save lives during landfalling storms.
The new system will give forecasters better information about developing storms across the planet.
Eunice pediatrician Dr. Nicole Michael travels to her native Haiti a few times a year to treat children there at a clinic in a small mountain village that lost its sources of food and income. She fears many will died of famine, and no access to food or clean water without aid. A non-profit mission organization is working to make sure that doesn't happen, and you can help.
The Atlantic tropical season is finally beginning to wind down as Hurricane Nicoles moves away from land.
A 55-year-old woman was worried about her Toyota Venza getting damaged by the storm last week. So she had a bright idea to keep it protected -- drive it straight into her house!
The U.S. National Hurricane Center says hurricane-force wind gusts are battering North Carolina's Outer Banks.
Current conditions show 12-15 foot seas with sustained winds of roughly 51 miles per hour.
Our friends at EarthCam have cameras set up all along the East Coast to watch the progression of Hurricane Matthew.
There is imminent danger for the Bahamas and Florida for the next few days. There is also a chance that the system could loop around and enter the Gulf by the middle of next week.
This weekend's inaugural 2016 Beach Town Music Festival in Vero Beach, Fla. has been postponed due to the predicted danger of Hurricane Matthew.


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SINGAPORE (AP) President Obama said Sunday that the United States and Russia would have a replacement treaty on reducing nuclear arms ready for approval by year's end, an announcement designed as an upbeat ending to a summit with Asia-Pacific leaders.
While publicizing progress with Russia on arms control — part of Obama's agenda to advance nuclear disarmament — the president and other leaders bowed to the obvious on climate change. They discussed a compromise agreement for a 192-nation gathering next month in Copenhagen, indirectly admitting that the meeting would not produce a new global treaty to reduce the heat-trapping carbon emissions warming the planet.
MORE ON THE SUMMIT: Obama talks climate, arms control
Nearing the end of his two days in Singapore, Obama attended a second summit with leaders of the 10 southeast Asian countries that make up the ASEAN group. Obama was the first U.S. president to sit in on the meetings, that included a senior leader of Burma— part of a shift in U.S. policy away from isolating the repressive military government.
Afterward, White House spokesman Robert Gibbs said Obama told the gathering, Burmese Gen. Thein Sein included, that his government must free long-detained democracy leader and Nobel laureate Aung San Suu Kyi and other political prisoners.
Obama "brought that up directly with that government," Gibbs said.
While Burma, also known as Myanmar, ranks high among nations that suppress human rights, a joint statement by the United States and the ASEAN group made no mention of Suu Kyi.
The whirlwind of summitry is part of Obama's first presidential trip to the region. Its emphasis on big issues such as climate change, disarmament and the economic crisis is part of Obama's approach to persuade emerging powers such as China to share in the burden of managing global challenges.
Obama arrived in Shanghai on Sunday night, launching a three-day visit to an important global U.S. partner and his first travels in China. His schedule called for a state visit to Beijing hosted by Chinese President Hu Jintao.
Obama is shifting relations to a more positive footing, away from disputes over human rights and the Chinese military buildup that have unsteadied ties. In Shanghai on Monday, Obama will address an audience of students from several universities and field questions from them and from submissions to the U.S. Embassy's website.
Obama and Russian President Dmitry Medvedev met on the sidelines of the Asia-Pacific summit of APEC nations to announce progress in negotiations on an updated pact to replace the START nuclear arms agreement that expires Dec. 5.
Obama said the pair discussed a successor to the 1991 Strategic Arms Reduction Treaty and described "excellent progress over the last several months."
"I'm confident that if we work hard and with a sense of urgency, we'll be able to get that done," Obama said, adding that technical issues remain.
Medvedev said he hoped negotiators would "finalize the text of the document by December."
Obama and Medvedev agreed in April to reach a nuclear arms reduction pact to replace and expand upon the one signed by President George H.W. Bush and Soviet leader Michael Gorbachev.
During a July summit in Moscow, Obama and Medvedev agreed to cut the number of nuclear warheads each nation possesses to 1,500-1,675 within seven years.
The treaty could be signed during Obama's travels to Europe in early December to accept the Nobel Peace Prize.
It was not clear whether Obama would use that same trip to attend the Copenhagen climate summit, given that any agreement reached on cutting greenhouse gas emissions would serve only as an interim, political document.
"There was an assessment by the leaders that it is unrealistic to expect a full internationally, legally binding agreement could be negotiated between now and Copenhagen which starts in 22 days," said Michael Froman, Obama's deputy national security adviser for international economic matters.
The prime minister of Denmark, Lars Loekke Rasmussen, the U.N.-sponsored climate conference's chairman, flew overnight to Singapore to present a proposal shifting the goal of the meeting to a "politically binding" agreement, in hopes of breathing life into the struggling process.
A fully binding legal agreement would be left to a second meeting next year in Mexico City, Froman said.
Obama backed the approach, cautioning the group not to let the "perfect be the enemy of the good," Froman said.
A major bill dealing with energy and climate in the U.S. Senate, a domestic priority of Obama's, is bogged down with scant hope of completion by next month. That would leave Obama little to show in Copenhagen.
During his Asia trip, Obama pushed for continued pressure on Iran and its nuclear program. Appearing with Medvedev, Obama said, "We are now running out of time."
"Unfortunately, so far it appears Iran has been unable to say yes" to a proposal on uranium reprocessing, Obama said.
Medvedev said, "We are prepared to work further, and I hope our joint work will reach a positive result. In case we fail, other options remain on the table." He has said further sanctions against Iran were possible if it did not open its nuclear program to inspections to prove it was not trying to build a bomb.
The five permanent members of the U.N. Security Council— the United States, Britain, France, Russia and China — along with Germany — have engaged Iran on its nuclear program, most recently with a deal for it to ship enriched uranium to Russia for further processing as fuel.
The United States and its allies say Iran is using its nuclear program as a cover for building a bomb. Tehran says it wants to build nuclear reactors only to generate electricity.
Obama wrapped his official schedule in Singapore late Sunday afternoon by meeting with Indonesia's Susilo Bambang Yudhoyono, president of the world's largest Muslim nation and Obama's home as a boy. Obama said he was excited about the prospect of improving relations with Indonesia and repeated his plan to visit next year.
He said the schedule would depend on his family; he wanted to plan a trip with his wife and daughters "so they can take a look at some of my old haunts."
Copyright 2009 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.
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Guide to San Diego Outdoors
Guide to San Diego for Families
One Day in San Diego
The walk and the surrounding area is a bit daunting, but SO glad we went! Wonderful, filling breakfasts! Many gluten free options. The french toast was awesome. We had to wait about 20 minutes, but well worth it!
We walked from our hotel and while there were numerous homeless people, that is just a part of downtown San Diego. We met a friend who is local who suggested it and we loved the place. We arrived at 9am on Saturday and managed to get there before the morning crowd.
- Fast friendly service
- Large portions
Walked to the mission from my hotel. A few interesting characters were encountered on the way. Arrived early on Sunday so got a table immediately. Had a Mexican style breakfast and coffee. The coffee was really good! The breakfast on both visits was fantastic. This is one of those hidden gems of San Diego. No wonder this place fills up...
Awesome breakfast and worth a short wait! Our party and the French toast and pancakes and subbed potatoes as a side. Presentation was a nice surprise! It deserves the high ratings!
Walk up or drive by and you might steer clear of this place if not for its stellar ratings. But grin and bear with it; the interior is cozy and inviting, and the experience continues from there.
I came on a Saturday morning expecting a crowd but finding none. The service was prompt and very friendly. Despite an extensive coffee...
Although our wait to get into The Mission was a tad lengthier than I would have preferred, that inconvenience was no fault of the restaurant. They are providing great breakfast options, and we did foolishly go there on Mother's Day. But even that made me feel good to see many mothers getting treated well on their special day. With tasty...
Spent a weekend in San Diego. Went to the Mission for breakfast on Saturday. Great spot! Fast and friendly service. Good coffee as well.
For an entree, you have to try the "Papas Locos" (sp?). They are fantastic! Well seasoned and plenty to eat. A must try.
The blueberry corn cakes are excellent as well. Give them a shot as...
We have family that lives in San Diego and ever since finding this place we go every morning we are in town. The staff are wonderful and the food is to die for! Go early because it does get busy and it's fairly small in there. The portion sizes and variety of food is top notch. Definitely a must if...
My fiancé and I had a great Sunday morning breakfast here. There was already a wait around 9am, but the line moved very quickly. Definitely was worth the wait. Try to Verde Rancheros or Papas Locos with eggs.
Go early or wait in line. In a bit of a sketchy neighborhood but thats downtown san diego. The food is so good and worth the wait. Love breakfast here. Service is good. Dog friendly patio. Would always return.
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New Plans For The New Year
It's fair to say that I did not post as much content as I would have liked in 2016.
As you can probably imagine, this is mainly an issue of time. Read more »
It's fair to say that I did not post as much content as I would have liked in 2016.
As you can probably imagine, this is mainly an issue of time. Read more »
Are you a struggling trader? Do you continue to lose money, even in a bull market?
If you answered yes to either question, you are not alone. Financial trading is difficult and statistics reveal that at least 90% of traders lose money. Read on for this road map for trading success. Read more »
Technical analysis refers to the study of past price action to predict future market moves. It makes use of charts, trading volume, and other statistical or mathematical indicators to gauge how the market might behave, based on the concept that the fundamentals of an asset are reflected in its historical price action. Read more »
This article contains a quick introduction to shorting shares then presents my favourite strategy for finding shorts. I then put this strategy to the test on historical data before finishing up with some final tips. Read more »
As a trader, there's an important lesson to learn early on, which is that trading losses are part and parcel of playing the game.
They are unavoidable, since no-one can predict the markets all the time and no-one can win on every single trade. In fact, many traders have found success by being right just 30-40% of the time. Read more »
Netflix and Google helped contribute to some big gains in the technology space last week. The Nasdaq had one of it's best weeks of the year, closing higher by 4%. It's tempting then, to take some cash off the table since the index is beginning to look extended on a short-term basis. Read more »
Most commentators tell you to cut your losses short and let your winners run. This is the 'golden rule' of trading. If you do this, only this, and nothing else, apparently, you are bound to make money. (So long as your risk settings are also right). Read more »
Lost some money last week by not following the rules. It happens. The important thing is to carry on. Over the weekend I have been working on some new strategies and fine-tuning existing ones. I have some exciting ideas to share when the time is right. Read more »


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Zatanna Zatara is a fictional character in the DC Comics universe. Created by writer Gardner Fox and artist Murphy Anderson, Zatanna first appeared in Hawkman #4. The character, both a stage magician and a real magician like her father Giovanni "John" Zatara, is known for her involvement with the Justice League, her retconned childhood association with Batman, and her crossing of the Vertigo line with characters such as romantic partner John Constantine an...... more on Wikipedia
Zatanna is ranked on...
Which superhero would you bring home to impress your parents? Do DC heroes or Marvel heroes make better girlfriends and boyfriends? We've all fantasized (right?) about bringing our favorite comic book characters home to meet the parental units and play with the dog before they whisk us away to their secret headquarters. The problem is, even some of our favor...
Zatanna videos
Injustice: Gods Among Us - Zatanna
Zatanna (1/5)
Zatanna's Spell (season 1)
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So, the lives of 120 babies would be prevented each year if only heedless, careless parents listened to those decisive, knowledgeable, consistent medical experts and stopped taking their babies to bed with them? OK, so I've put in the angry, sarcastic adjectives, but it feels like that's the basic thrust of this week's news about cot death – sudden infant death syndrome or SIDS.
Very little mention has been made of just who it was who advised the parents to sleep with their babies in the first place – none other than the medical profession.
Few people are more vulnerable to expert advice than anxious new parents, especially mothers. Of course they listen to and do what doctors, nurses, midwives, health visitors and the rest tell them is best. They are embarking on life's most challenging task and it's learn-on-the-job. There is no training.
In the 1970s when my children were born, cot death was terrifyingly common and no one knew what caused it. Everybody knew a family who had lost a child to SIDS and I knew three. Even as late as 1989 there were 1,545 such deaths in the UK according to Lullaby Trust statistics.
As young mothers we were firmly instructed to put our infants on their tummies to sleep. They told us that a child on its back could, if sick, choke on its own vomit and die – scary stuff so we did as we were told although, thinking about it now, babies had slept on their backs for millennia and it didn't seem to have endangered many lives.
In fact there was, as later research showed, a strong correlation between babies sleeping face down and cot death. Both ours survived, thank goodness, but we have lots of scary (now) photographs of them doing pram press ups as they lifted their heads to peer out like tortoises.
By the 1990s parents were being strictly instructed never to put a child to sleep belly-down because it can cause cot death. When I visited a new mother in a maternity wing in 1997 I was struck by the dictatorial tone of the notices around the ward – for all the world as if putting babies on their tummies was some bit of dangerous folly dreamed up by feckless mothers. There was no acknowledgement that advice had changed dramatically in the light of new information and understanding. What an insult, especially to all those families who had suffered the almost unimaginable tragedy of a dead child.
And now the same thing has happened again. My generation was told never to take a baby to bed in case you fell asleep and smothered him or her. Out of sheer fear I spent many hours on bitterly cold nights sitting on an upright chair in another room to breast feed to make absolutely sure I stayed awake.
Then about fifteen years ago the medical people started to advise parents that sleeping with a baby is sensible because the infant can feed easily almost at will without much disturbing the mother during the night - much like a puppy or kitten. This week's research from the London School of Hygiene and Tropical Medicine has shown that actually 40 per cent of the UK's 300 annual cot deaths would be prevented if babies slept in cots next to the mother's bed.
But I don't hear the medical professionals saying – or at least not very loudly – that this overturns what they said before. Instead they speak as if bed sharing were something dreamed up by remiss and stupid parents and that makes me very angry.
Of course medical research is ongoing and has been since before Hippocrates. Nobody knows all the answers and we never will. That's the nature of knowledge and the acquisition of it. New discoveries inevitably affect the way we do things and medical professionals no longer use blood-letting, blistering and leeches. Lives are saved every day as a result of new understanding about the causes of avoidable death – and, thank goodness, 'only' 300 babies a year now die in the UK in their cots although every individual case is one too many.
But it's hard to forgive the Nineteen Eighty-Four-style arrogance when suddenly advice changes dramatically and frightened parents are made to feel as if they are at fault because they are simply doing what they were authoritatively told the week before last – but now we're at war with Oceania and always have been, as Orwell would have put it.
Yes, I know that the medical profession is fearful of litigation, but it would be much easier to respect the official advice of health bodies if only more were humble enough openly to acknowledge that they, like the rest of us, are fallible and only able to offer informed advice based on the latest research - which may well be different from what earlier findings have suggested.
No one is wrong. We are all moving forward together in trying to stamp out devastating horrors such as cot death.
- More about:
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- Cycle the city streets to absorb the local atmosphere
- Family friendly
- Get inside tips from a local
- Great overview and orientation of the city
- Lunch included
- Perfect introduction for first-time visitors
Recent Photos of this Tour
Schedule details
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Pricing information
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Budapest Sightseeing Tour by Bike
- Local guide
- Use of hybrid, 24-speed bike
- Use of cycle helmet
- Cold refreshments during tour
- Bowl of Hungarian goulash
- Gratuities (optional)
- Food and drinks (unless specified)
- Hotel pickup and drop-off
- Confirmation will be received at time of booking
Cancellation Policy
If you cancel between 3 and 6 day(s) in advance of the scheduled departure, there is a 50 percent cancellation fee.
If you cancel within 2 day(s) of the scheduled departure, there is a 100 percent cancellation fee.
Traveler Reviews
Reviewed by: Keith L, November 2016
Our tour guide was really great, Anna. She was funny and knowledgeable, good english. I did this merely as a way to see some of the top sites and decide where to go back to later. Worked out great. Luckily had a really great/friendly tour group as well. Lunch was good, you pay for your own drinks, no biggie. Definitely worth it.
Reviewed by: Marta G C, October 2016
Everything was great including the food


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Against the Faith: Essays on Deists, Skeptics, and Atheists
Prometheus Books, 1985 - Religion - 250 pages
"The subject of this book is those who have placed themselves 'against the faith', in other words, those who have opposed the prevailing religious faith of their time. Such opponents adopt this position for a wide variety of reasons and in many different ways. They are sometimes fiery activists hammering against leaders and leading ideas and at other times are quiet, contemplative skeptics questioning all knowledge and all orthodoxy. They can be immersed in the politics of their time, like Bradlaugh or Thomas Paine. They can be poets like Heine and Shelley, historians like Gibbon, playwrights like Buchner, or novelists like George Eliot and Mark Twain. They may be scientists like Huxley, or philosophers like J.S. Mill. They may be most at home on the public platform, like Ingersoll, or in the study like Pierre Bayle. They can be relaxed men of the world like Hume or temperamental outsiders like d'Holbach. They may lead quiet and little known lives like the freethinker Collins or the clergyman Meslier, or they may be outstanding polymaths of their age, like Voltaire or Bertrand Russell.
This book covers deists, skeptics and atheists. Without attempting to be comprehensive, I have tried to show that there is a spectrum between the three. There has often been close contact between deists, who gently criticize the Christian faith, skeptics who questions all knowledge, and atheists, who detach themselves from any belief in God. Occasionally individuals have held all these positions at different periods of their lives. Furthermore the distinction sometimes made between the respectable philosophic skeptic and the disreputable agitating atheist is not clear-cut: philosophers sometimes agitate and frequently rub shoulders with activists, and reformers and campaigners often think quite deeply.
Since this book in the main covers Europe in the eighteenth and nineteenth centuries, the faith opposed is Christianity. A history of opponents to Hinduism, Islam and Buddhism would provide fascinating parallels, but that book has yet to be written. It is a mistake - and one to which opponents are particularly prone - to imagine the 'faith' as a monolithic entity, rather than an accumulation of various traditions. There can therefore be opposition to the faith from within as well as without and heresy and heterodoxy have sometimes been not far apart."
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Hometyre with Free Mobile tyre fitting - Chichester
Tyre dealers, tyre fitting, Mobile Tyre Fitting
My customer warned me that he had just spent £460 having his wheels specially painted, so he was worried about having tyres fitted as he didn't want them scratched.
I told him that as long as the wheels were painted 3 to 4 days ago, then the paint is fully hardened, plus we use plastic protectors on our equipment, he seemed happy but kept an eye on each wheel as I worked on it. He was having 20" Pirelli P Zero Rosso tyres fitted, I made sure that I used extra tyre grease to ensure the old tyres came off easily and the new ones slid back on. There were no scratches, the customer was happy and has since used me again.
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This customer wanted new tyres fitted to his newly painted alloys. He called out Hometyre and was so pleased with
Old or new vehicles, Hometyre can supply and fit tyres to them all.
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Kitchen Supplies
- Cooking Equipment
- Countertop Cookers and Warmers
- Hot Dog Roller Grills
- Nemco Food Equipment Silverstone Slanted Roller Grill 30 x 16 x 10 1/4 inch
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Nemco Food Equipment Silverstone Slanted Roller Grill 30 x 16 x 10 1/4 inch Product ID: EM8036SSLT Nemco Food Equipment
- "We are totally pleased with the shipment, very fast and when needed will order from your company again.
- "No issues. The product arrived in good shape and within a reasonable amount of time.
Nemco Food Equipment Silverstone Slanted Roller Grill 30 x 16 x 10 1/4 inch
Product Id: EM8036SSLT
Sold as: EachWeight: 55 lbs
List Price: $1,853.38Savings: $804.33
DescriptionNemco Food EquipmentMfg#: 8036S-SLT
A special tiered roller design adds an effective merchandising edge because it brings the deliciously tempting grilled hot dogs into better view for your customers. Rugged stainless steel and aluminum construction Simplest installation - plugs into any outlet with corresponding voltage Rollers available in either standard chrome or non-stick coating which cleans easily with just a damp sponge Two individual heat controls for front and rear rollers Temperature control with seven heat settings Roller grills slant 7 degree for effective merchandising Individual heating element in each roller with superior temperature consistency from end to end Sealed motor with ball bearings to prevent grease leakage out of motor Rollers rotate at a complete 360 degree turn by means of roller sprocket, motor sprocket and chain End bearing and grease barrier double protect against grease seepage Removable grease drip pan Preheat time is 20 minutes Full one year factory warranty NSF listed and UL listed.
Nemco Food Equipment Silverstone Slanted Roller Grill, 30 x 16 x 10 1/4 inch -- 1 each.
36 Hot Dog, 120 or 220 Volt, for Hot Dog Equipment
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Thanks for considering FoodServiceDirect.com as your source for Nemco Food Equipment Silverstone Slanted Roller Grill 30 x 16 x 10 1/4 inch! If you need help don't hesitate to email or call our Customer Care Representatives. At FoodServiceDirect.com, your satisfaction is our number 1 concern! We've been at it for over 15 years and we have satisfied thousands of customers like you with Hot Dog Roller Grills and our large selection of other restaurant supply items.


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Best Buy
Boy Genius Report is unearthing documents of a new service that Best Buy is presumably launching tomorrow, where it will begin to repurchase smartphones, laptops, tablets, and TVs.
If this were it -- and Best Buy were just setting up space within its massive stores to offer up a glorified pawnshop or the means for suppliers to get back parts or merchandise worth refurbishing -- it would simply be a puzzling move. Yes, Best Buy has followed GameStop
Unfortunately, this isn't just about the resale opportunities. Best Buy won't buy back your wares unless you paid the chain a premium for that right first.
Similar to its Geek Squad Black Tie Protection, where buyers pay for a set number of years of support and maintenance, the new Buy Back program is a premium service that Best Buy is pitching as protection against obsolescence on new purchases.
It may have been neat to know that Best Buy would repurchase any of the covered products during the first six months of ownership, at 50% of what customers shelled out, but folks have to pay a small premium for that coverage. For example, with a mobile phone, the scale slides quickly after the first six months -- all the way down to 20% during the last half of the second year -- so it's really more a confirmation of obsolescence than protection.
There may be a market here for serial early adopters, particularly for smartphones. Apple
We'll see tomorrow if Best Buy is really doing this, but it seems to be a flimsy solution to a problem that others are tackling more effectively without the upfront tollbooths.
It's easy to see why Best Buy is hungry for something new, as the superstore chain is coming off a horrendous preholiday quarter. I'm sure the margins on these plans opened up the eyes in the boardroom, but it just won't fly.
Online sales continue to eat at Best Buy's business. The failure of Circuit City is appearing to be more a case of foreshadowing than an opportunity. Best Buy shareholders appear to be the ones needing protection from obsolescence.
It stings, I know -- but it's true.
Would a premium buyback service work at Best Buy? Share your thoughts in the comment box below.


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Bet you didn't know about number 4
12 Pinterest recipes to get you in the mood for Bonfire Night
Seeing as social media is so huge, you'd think you know everything their is to know about the celebrities of this world. Right? Wrong - we bet you didn't know these 12 famous people had children.More
Matt LeBlanc married Melissa McKnight in 2003 and their daughter Marina Pearl was born in 2004. She suffered seizures from eight months old, though it later developed into a form of dysplasia by aged two. The couple later divorced in 2006.
Tom Hardy might play the badboy of your dreams most of the time, but what you might not know is he's a father of two - 8-year-old Louis, with ex-girlfriend Rachael Speed, and his second child, who was born in October 2015 with his wife Charlotte Riley.
14 of the craziest Airbnb listings you can stay in


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To avoid our own failed 'American Spring,' learn the region's lessons about effective opposition
That brutal history of suspicion and conflict between Jews and Catholics? It's a wash, according to 'The New York Times.'
Tired of ominous news updates and social media outrage? Here are classic Jewish texts to help revive the spirit and soothe the soul
A new book reminds us why Menachem Schneerson's genius for community is more vital today than ever
It's not about refugees, or security, or travel bans. It's about what America is—and what it ought to be.
The identity of America—unity—is under attack by Trump's administration, whose key goal is to divide. So stand up, shout out, and love thy neighbor.
Sure, it's about Portuguese Jesuits in Japan, but the movie's theological message is one we should all embrace
When our heroes are all simpering children, is it any wonder our president is, too?
If attacking Europe's Jews over the purported acts of Middle Eastern Jews isn't the definition of historical anti-Semitism, what is?
Is there anything behind repeated criminal allegations against the prime minister? Depends on your definition of 'anything.'
'The New Yorker' offers a master class in how not to respond to cheap alt-right provocations
Why the soldier's conviction is a moral outrage
A Colonialist state founded on the theft of Maori land blames Israel for its own crimes
Why did a prominent Israeli parliamentarian smuggle cell phones to a convicted murderer?
'The Last Guardian,' a new video game, is a masterly meditation on learning to care for, and communicate with, others
The Senate's ranking Democrat sees himself as a guardian of the Jews. So why does he insist on palling around with their detractors on the left and the right alike?
Israeli leftists are no strangers to sudden electoral defeats. Is there anything American liberals can learn from them?
Shalom, South Dakota!
Trump's victory may embolden some jerks—and a few real bigots—but now more than ever reasonable people need to stay reasonable
American Jewish groups need to broaden their Israeli coalition and work with sympathizers in the government, rather than engage in satisfying but counterproductive hard-sell tactics
Historical analogies are always flawed, but some moral principles shine eternal
In the midwest and the Middle East alike, cultures that favor outrage over outcome never get very far
The poet's greatest talent was his gift for healing
On my 40th birthday, contemplating life—and the value of compassion—in Trump's America
Bitter division after news reports call the greatest story of the Yom Kippur War into question
At 82, the prophet releases another profound and beautiful chapter in his manual for living with defeat
Not even Ewan McGregor can turn the author's narcissism into a relatable work of art in his adaptation of 'American Pastoral'
A new machine endangers a time-honored street food tradition
Unless we change the award's silly criteria, there's a good case to be made for the Israeli prime minister's significant contribution to regional stability
Freedom of worship, not appeasement, is the only just foundation for future peace


text:
Christian Friedel
photo by Thomas Schmidt
The German actor debuted in cinema in 2009 with The White
Ribbon by Michael Haneke, which won the Palme
d'Or at Cannes and is currently nominated for an Oscar as the best
foreign film. For years he performed in the local theaters of
Hanover and Dresden, but since Cannes everything has changed.
"Famous? I haven't yet noticed being so. Hankeke's rehearsals are
true life experiences, they last for days, over the corse of which
he teaches you to act in his own way. His obsession with detail
isn't oppressive, but, to the contrary, nourishes the actor. On the
set I felt all inhibition scatter, above all in front of the prying
eye of the camera." In the near future Friedel would like another
film project. "My dream? A Haneke 2! But also a film with
Tim Burton or David Lynch. Maybe
a more evil part, as an antagonist." He also has a knack for music
(a passion which scored him the part in the film), that he has
cultivated semi-professionally since childhood. "I hope to find the
time to become a true musician and develop a career as a theater
actor and cinematographer. I'm eclectic: doing only one thing has
always bored me."
(In the picture: suit and shirt, John Richmond; hat, Stetson; boots, Lottusse. Fashion editor Isa Petereit)
Michele Fossi, from L'Uomo Vogue, march 2010 (n. 409), p. 197
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text:
Crofty – so sorry to hear about your BFN – it always sucks. In answer to your questions...What type of luteal support are you on and how often?? Do they check your prog levels at 21 dpo?? Yes I have seen plenty of women on bubhub 35+ to conceive their 1st bubba – it can take longer than the younger ladies. Keep an eye on the 35+ thread – it is quiet there lately but there have been a few recent babies born and pregnancies. I hope your time is soon.
Paddlegirl – welcome & good luck! Our clinic doesn't do back to back stim cycles but you can do stim then FET back to back.
TTHC – glad to hear DS's birthday went well – he deserves to be spoiled. I often wonder the same thing with ob bookings - maybe its the "fertiles" who just know they will get pregnant when they try and book in early!!!!???
EO – How frustrating for you!!! I can't believe you have 40 follicles – wow and your oestrogen is sky high. How ru feeling, ru keeping up your fluids and high protein – they say its meant to help and rest up after EPU. I hope you get some great quality eggs. Go DH's super swimmers for some great ones!
We will gradually start telling people now. I really want to tell DD before too many people as she picks up on everything these days – she's too tired during the week, so will aim for the weekend.
Loulabelle – congratulations on your great news, glad bubba is healthy and all on track – how exciting! Gosh swine flu, you poor thing! And vomiting 10 times a day is just awful – i had hyper-emesis with DD and it is so draining. I hope you still have your helpers around.
Lindylou – I hope so, that would be great to be the first Sydney bubba! I hope your FS does go with the pred. Its worth a shot!
Hi to everyone else
AFM - thanks everyone for your lovley wishes, it all feels surreal, much more relaxed today though


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The Offering
July 29th, 2010
You ordered the baby.
(submitted by Teresa)
Are we sure this isn't one of those real doll fake babies?
Oh my. Unsure about saying this but one thought popped into my head immediately after seeing the praised child. "Rosemary's Baby". All those smiles and the blank stare. Please photographers, look, learn, nuh-uh.
Wow. Awkard. Family. Photo. Indeed.
"Light as a feather, stiff as a board…light as a feather, stiff as a board…"
at least turn the baby the other way-are they at a restaurant and the waiter took the picture? duh!
The boy on the right's face is priceless.
I wonder if this kid will end up in a mosh pit when he's older…
Is that baby a girl or boy?
The kid on the left is wondering why his hand is getting so much warmer. And wetter.
It's what's for dinner!!!!
"Take it! We don't mind." *Evil laughter* This one is downright creepy.
I love the expression of the boy on the right. :I
(singing)… "I bought it on Ebay."
i think without the title it would not have been nearly as creepy.
"The Offering"
Maybe it's from an ad for a Maternity Hospital…."Your Baby Delivered in 30 minutes,or,it's free."
The baby has a very just-lifted-off-the-cross jesus look on his face.
I think the boy on the left is so handsome it makes me wonder how old is he… I hope he's of age! >.<
I think the boy on the right looks like a young Ashton Kutcher.
Oh good, I'm not the only one who thought he's good looking. XD
This picture could be used for a sunscreen ad.
It takes a village of the Damned to raise a child.
You want fries with this?
Does anyone else notice how the dad literally looks like a larger version of the baby.
the oldest son looks thrilled…no doubt enjoying the baby screaming during his adolescent years.
Wait…they look familiar. Does anyone else notice this? Have they been on tv or on here in another pic?
If you look at the pic the mom is supporting the head. Besides the fact that the kids are old enough to know how to hold a baby.
There's no doubt who that baby's daddy is.
Human sacrifice to the God of Awkwardness
Win! =)
The two boys on the end look like they are ready to drop the kid and bolt to the nearest exit as soon as the camera clicks.
The older kids know what a new baby means and they don't look happy – the little girl doesn't know what this means for her YET this is why she is smiling
Oh, yeah. This was totally mom's idea. The rest of the family is just along for the ride.
You're absolutely correct. Ahahahahahaha!!!! The boys are groaning, the girl is giving a little embarrassed smile just like her dear ol' dad. And dad is just doin' what it takes to keep the peace.
And boy does dad have strong genes. The baby and the girl both have his mark on them.
The older kids will get their payback for this when they choose Mom & Dad's nursing home.
Also love how dad and sis have the same head tilt
The baby & dad have the same hair.
The Chosen One who will alert the Overseers.
Somehow playng "Stiff as a board, light as a feather" with a new born just doesn't have the same dramatic effect. Love the expressions on the boys!
Everybody has a hand in "Raising" this child.
And "crowd surfing" was born.
LOL Awesome.
I love how the one brother on our left seems to look like he is more protecting the baby than any of them… haha … good picture!
This reminded me of the "seances" we used to have at girl scout camp when somebody lies on the floor, everyone sits around them, you tell a scary story about them, and then lift them up using just your index fingers. That baby looks like, "What the….???!!!" and the older two kids are bored/embarrassed/jealous of all the attention the baby is getting.
LOL! "Stiff as a board, light as a feather."
Freaky! You get the sense it hatched out of a pod somehow.
The kids on both ends look so unamused..
I ordered this baby "well done". Geesh.
I asked for BABY BACK RIBS, you sick freaks…
Oh wow the two kids on either end are having the same thought. Something like this is NOT a good idea for a picture!
This is very disturbing. Looks either like a cover for a scary movie or an ad for a cult.
The best part is the look on the boys' faces.
I love that the boys on each end *get* how ridiculous this pose is. The expression on the younger boys face says it all.
My thoughts exactly.
I was thinking the same thing.
I'm a little bit creeped out by this one.
Hehe the older brothers did not seem to be so happy about the new-commer…
I love the brothers 'yes, we put up with this crazy s*** from our parents but one day we will leave the house forever' looks/attitudes
Exactly! LOL!
I agree
Those are the cutest kids I have ever seen on this awkward site!
That is a weird picture. The concept is great, the actual execution is very awkward.
Those younger kids were the perfect choice to support the baby's head!
The mother is supporting the head. Her arm comes up second from the right.
I like the brown paper bag taped together to make the backdrop…lol
that is kraft paper


text:
Doyle Entertainment Cairns
Corporate Functions, Kids Party, Kids Entertainment
Doyle Entertainment can supply :
1. Children's Entertainment - The Doyle Family Fun Show, Clowns, Magicians, Face Painters.
2. All types of 'Live' music bands, duos in ALL genres…… rock thru to Bush Music thru to original music acts.
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4. Production supplies ie. PA Systems, Lighting, Theming as well as operators and installers.
5. Event promotion, publicity and marketing.
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7. Mentoring of original artists.
8. Workshops catering for those wanting to make the entertainment industry their career.
Print this page
The total one stop shop for all 'Live' entertainment needs, no matter what the event or budget.
ALL entertainment live music, fun, comedy, fun original.
Forty-Five years in the Australian Music and Entertainment business brings huge benefits to you the client in the quality of the performers along with production and logistic aspects of your occasion ....... nothing can buy experience and I have certainly done my apprenticeship in ALL facets of the Australian entertainment business.
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Is Musical Knives Restaurant incorrect or missing key business information? Update your Hotfrog listing today.
Contact details
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Hotfrog NewZealand provides information regarding Musical Knives Restaurant in Auckland. Musical Knives Restaurant is located at 272 Ponsonby Road, Auckland Auckland and provides Food and Dining services. Contact them on 09-360 7440.
Opening hours
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The Definitive EMS Airway CourseDecember 1, 2, 2016Williamson EMS Public Safety Center304 Beasley DriveFranklin, TN 37064$180 Tuition• Learn the full range of airway management techniques — including use of video laryngoscopes & other advanced imaging devices, extraglottic devices, drug-assisted intubation and cricothyrotomy.• Work with an expert faculty who knows the unique challenges facing EMS providers.• Use all of the airway devices in small group sessions.• Practice decision-making and airway techniques in Code AirwayTM stations.• Face the most challenging patient scenarios in a no-risk environment. These include: elevated ICP, pulmonary edema, cardiogenic shock, status asthmaticus foreign body in the airway, direct airway trauma, multiple trauma with shock, and many more.Didactic and Hands-on Training• Prediction of the Difficult Airway• Video Laryngoscopy and other advanced imaging devices• Medication-assisted Intubation (including RSI and RSA)• Pediatric intubation• Endotracheal tube introducers (bougie)• Cricothyrotomy• Extraglottic devicesCE CreditsThis continuing education activity is approved by the CECBEMS.This continuing education is approved by First Airway, LLC, an organization accredited by the Continuing Education Coordinating Board for Emergency Medical Services (CECBEMS). CECBEMS Activity #14-FirstAir-F2-0001. CEH Number and Type: 16.0 Advanced.16.0 Contact Hours have been provided by the Air & Surface Transport Nurses Association (Certificate #2015-23). ASTNA is an approved provider for the California Board of Registered Nursing CEP 13575.
Group DiscountsIf your department is registering 5 or more individuals, please contact Kevin High, course director, for the group discount code. Please do this before you begin registration.
Thursday, December 1, 2016 - Friday, December 2, 2016 8:00 AM - 5:00 PMCentral Time
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Kevin High
Vanderbilt LifeFlightHome PageVanderbilt LifeFlight
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text:
PETER NICOL fought back from 5-1 down in the fifth game to defeat Jonathon Power, his great rival, and give England a flying start in their quarter-final against Canada in the World Team Championship in Vienna last night.
Nicol's win was the more remarkable because he has had flu and faces the toughest workload of any first string in the men's tournament. With Lee Beachill and James Willstrop still to face Graham Ryding and Shahier Razik to decide who would face Scotland or France in the semi- finals, England were elated but slightly unbelieving as Nicol, whom they had protected


text: Remnant 176,997pages on this wiki Add New Page Edit History Talk0 Share Frank EdwardsEarth-S(Earth-712) Alternate Reality Versions Alternate Reality Versions Humanity Nuked the Squadron Supreme(Earth-1121) References Retrieved from "http://marvel.wikia.com/wiki/Remnant?oldid=3140460" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Templated Articles Disambiguation Pages


text:
"Bad Santa" is the story of two conmen who go on a road trip to malls dressed as Santa and his elf. Rather than spreading good cheer, the duo's motive is to rob each establishment, a strategy that becomes complicated when they encounter an 8-year-old who teaches them the true meaning of Christmas.
IMDB rating 7.2/10
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ÒÇÉActivityÒÇæ The Santa Promises Teclast X70 R 3G Phablet Not Just Cheap - $49.99!!
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More About posterguyThey staring at walls and roofs as well into their lives. The same old, dull with some rare sighting of a lizard or a rat sneaking by, unmoved and unfazed by the dullness, they kept thinking what is wrong with their erros they found that although being a connoisseur of art, they have so little art around . Most of the people like art and appreciate it but still they keep their distance from it. posterguy»
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text: In recent years, natural gas has been considered a replacement for diesel fuel in large bore engines, due to its low cost, high heating value and widespread availability. Stoichiometric premixed spark-ignition (SI), defined as port-fuel injection (PFI) of natural gas (NG) followed by SI close to top dead center (TDC), has traditionally been used as the main fuel delivery and combustion method for light and medium duty engines. However, premixed SI of NG results in inefficiencies in the intake process and combustion that is knock limited as boost and load are increased. Traditionally, high knock is addressed by spark timing retard. Spark timing retard can lead to misfires and low brake mean effective pressures. Thus premixed SI has limited low load use in heavy duty where compression ignition of diesel fuel remains dominant. Spark-ignited direct injection (SIDI) of gasoline or GDI has been used for several years in light duty gasoline passenger car engines in combination with turbocharging to extend loads to higher BMEP levels (similar to diesel engines). SIDI method injects fuel during the compression stroke and as a result avoids excessive premixing of fuel and air. SIDI boosted engines can extend load to >20bar bmep and thus results in downsizing of engines to achieve similar power and torque as premixed SI with smaller displacement leading to reduced fuel consumption and reduced CO2 emissions. In this work the single cylinder engine testing was conducted of SIDI of NG using integrated spark injector igniter. The focus of this work is the application of spark ignited direct injection (SIDI) of natural gas for the conversion of heavy duty diesel engines. The influence of DI gaseous injector nozzle spreading angle will be studied. The impact of this parameters on thermal efficiency, specific fuel consumption, combustion stability, combustion phasing, combustion duration and emissions of NOx, HC, CO will be investigated. An optimal nozzle included angle of 100 degrees was determined based on the experimental results which led to the best thermal efficiency as well as good combustion stability.


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Want an alarm clock that automatically shuts off so you don't have to get out of bed? Does your alarm wake up the whole house? Then this is the alarm clock for you!Features:* Displays current… [more..]
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text:
6195 Back Forty Drive, Belmont 49306
See more homes for sale in Belmont, MI
Now is your chance to own this spectacular Custom Executive Home at the Very Top of Beautiful Dix Farms. With over 4700 square feet of finished living space, this Year Round Resort offers Updated Landscaping, a Tree Level Hot Tub, New Deck Addition, Main Level Master Suite w/ Large Closets, and New Bamboo Flooring on Main Level.Great Room with Wood Burning Fireplace and Beautiful 20'Angled Windows.Bird Watcher's Paradise, High Quality Outside Cedar Stain Done in 2015. Almost Every Room has Cable and Ethernet Connections. Dream Kitchen Features Granite Island,Second Prep Sink and New Appliances.Open and Spacious Loft Area. Lower Level Game Room with Wet Bar, Hobby Rooms/Bonus Rooms. Screened In Porch Has New Stairs, 3 Stall Extra Tall Garage. Call today
Property Information
- Status
- Active
- Address
- 6195 Back Forty Drive
- City
- Belmont
- Zip
- 49306
- County
- Kent
- Township
- Cannon Twp
- Possession
- 30 days after c
- Zoning
- Res
- Total Finished SqFt
- 4,794
- Above Grade SqFt
- 3,466
- Garage
- 3.0
- Garage Desc.
- 3, Attached
- Water
- Well
- Sewer
- Septic System
- Year Built
- 2003
School Information
- School District
- Rockford
- Elementary School
- Cannonsburg
- Middle School
- East Middle
- High School
- Rockford
- Taxes
- $7,690
- Association Fee
- $Quarterly
Rooms and Land
- MasterBedroom
- 17X14 1st Floor
- Bedroom2
- 13X14 2nd Floor
- Bedroom3
- 14X11 2nd Floor
- Bedroom4
- 16X14 2nd Floor
- Bedroom5
- 18X14 Lower Floor
- Bath
- 16X16 1st Floor
- Den
- 13X14 2nd Floor
- Dining
- 14X12 1st Floor
- Family
- 37X19 Lower Floor
- Kitchen
- 20X14 1st Floor
- Laundry
- 14X12 1st Floor
- Living
- 32X20 1st Floor
- Other
- Lower Floor
- Rec
- 19X17 Lower Floor
- Basement
- Walk Out
- Cooling
- Central
- Heating
- Forced Air, Natural Gas
- Acreage
- 0.58
- Lot Dimensions
- 195x295x185x71.42
- Appliances
- Built in Oven, Dishwasher, Dryer, Garbage Disposal, Microwave, Oven, Range, Refrigerator, Washer
- Fireplace Desc.
- Living, Wood
- Features
- All Window Treatments, Ceiling Fans, Ceramic Floor, Garage Door Opener, Generator, Hot Tub Spa, Security System, Wet Bar, Whirlpool Tub, Wood Floor
- Exterior Materials
- Vinyl, Wood
- Exterior Features
- Deck(s), Scrn Porch
Listing Video for 6195 Back Forty Drive, Belmont MI 49306
Mortgage Calculator
Listing Broker
Listing Courtesy of Keller Williams Gr North
Office Address 3237 Platinum St Ne
Listing Agent Steven Dykstra
Listings last updated: . Some properties that appear for sale on this web site may subsequently have been sold and may no longer be available.
Our Michigan real estate agents can answer all of your questions about 6195 Back Forty Drive, Belmont MI 49306. Real Estate One, Max Broock Realtors, and J&J Realtors are part of the Real Estate One Family of Companies and dominate the Belmont, Michigan real estate market. To sell or buy a home in Belmont, Michigan, contact our real estate agents as we know the Belmont, Michigan real estate market better than anyone with over 100 years of experience in Belmont, Michigan real estate for sale.
The data relating to real estate for sale on this web site appears in part from the IDX programs of our Multiple Listing Services. Real Estate listings held by brokerage firms other than Real Estate One includes the name and address of the listing broker where available.
IDX information is provided exclusively for consumers personal, non-commercial use and may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing.
All information deemed materially reliable but not guaranteed. Interested parties are encouraged to verify all information. Copyright© 2016 MichRIC LLC, All rights reserved.


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