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Transforming growth factor (TGF)‐βs are dimeric polypeptides that have vital roles in regulating cell growth and differentiation. They signal by assembling a receptor heterotetramer composed of two TβRI:TβRII heterodimers. To investigate whether the two heterodimers bind and signal autonomously, one of the TGF‐β protomers was substituted to block receptor binding. The substituted dimer, TGF‐β3 WD, bound the TβRII extracellular domain and recruited the TβRI with affinities indistinguishable from TGF‐β3, but with one‐half the stoichiometry. TGF‐β3 WD was further shown to retain one‐quarter to one‐half the signalling activity of TGF‐β3 in three established assays for TGF‐β function. Single‐molecule fluorescence imaging with GFP‐tagged receptors demonstrated a measurable increase in the proportion of TβRI and TβRII dimers upon treatment with TGF‐β3, but not with TGF‐β3 WD. These results provide evidence that the two TβRI:TβRII heterodimers bind and signal in an autonomous manner. They further underscore how the TGF‐βs diverged from the bone morphogenetic proteins, the ancestral ligands of the TGF‐β superfamily that signal through a RI:RII:RII heterotrimer.
Transforming growth factor‐β (TGF‐β) isoforms are secreted signal ligands that have vital roles in coordinating wound healing, modulating immune cell function, maintaining the extracellular matrix, and regulating epithelial and endothelial cell growth and differentiation (Massagué, 1998). The importance of the TGF‐βs is underscored by their conservation among vertebrates and their demonstrated roles in a variety of human diseases, including tissue fibrosis (Blobe et al, 2000) and cancer (Derynck et al, 2001). TGF‐βs are members of an extended signalling superfamily that arose in early metazoans (Kingsley, 1994). The superfamily has greatly diversified, with >30 known members in vertebrates, including the prototypical TGF‐βs, the bone morphogenetic proteins (BMPs), the closely related growth and differentiation factors (GDFs), and the activins and inhibins (Massagué, 1998).
TGF‐βs are disulphide‐linked dimers of identical 112‐residue protomers. The protomers include four disulphide bonds, three of which form a conserved structure known as a cystine knot (Sun and Davies, 1995). BMPs, GDFs, activins, and most other ligands of the TGF‐β superfamily share a similar structure, though the cysteine that forms the inter‐chain disulphide bond is lacking in three family members, GDF‐3, GDF‐9, and BMP‐15 (McPherron and Lee, 1993; Dube et al, 1998). The ligands of the superfamily signal by binding and bringing together two single pass transmembrane receptor kinases, known as receptor types I and II (Derynck, 1994). This initiates a transphosphorylation cascade where the type II kinase phosphorylates and activates the type I (Wrana et al, 1994). The type I kinase phosphorylates Smad proteins (Shi and Massagué, 2003) and other effectors (Zhang, 2009), which regulate the transcription of target genes (Massagué and Wotton, 2000).
TGF‐βs have been shown to assemble a receptor heterotetramer on the cell surface comprising two molecules of its type I receptor, TβRI, and two molecules of its type II receptor, TβRII, based on differential receptor tagging (Moustakas et al, 1993; Henis et al, 1994; Wells et al, 1999), two‐dimensional gel electrophoresis (Yamashita et al, 1994), and genetic complementation (Weis‐Garcia and Massagué, 1996). TβRI and TβRII have been further shown to form stable homodimers in the absence of TGF‐β (Chen and Derynck, 1994; Gilboa et al, 1998; Rechtman et al, 2009), suggesting a two‐step mechanism for assembly of a receptor heterotetramer.
The recently reported structures of TGF‐β1 and ‐β3 bound to the TβRI and TβRII extracellular domains support the binding stoichiometry deduced on the basis of the cell‐based experiments, with two molecules of each receptor symmetrically bound, TβRII at the 'fingertips', and TβRI directly adjacent on the underside of the 'fingers' (Groppe et al, 2008; Radaev et al, 2010) (Figure 1A). TβRI and TβRII directly contact one another in the complex and these receptor–receptor contacts are responsible for the pronounced stepwise manner with which TGF‐βs bind TβRII and recruit TβRI (Groppe et al, 2008) first determined based on genetic complementation studies with receptor‐deficient mink lung epithelial cells (Laiho et al, 1991). The additional constraint imposed by the receptor–receptor contact is thought to be further important for enhancing the specificity with which TβRI and TβRII bind TGF‐βs and preventing activation of TGF‐β responses by other ligands of the superfamily (Groppe et al, 2008; Massagué, 2008).
The binding of TGF‐β by two well‐separated TβRI:TβRII heterodimeric pairs suggests that the two heterodimers might bind and signal independently of one another. This is further suggested by the finding that low but measurable signalling was induced when TβRI and TβRII were artificially dimerized with small immunophilin domains (Stockwell and Schreiber, 1998) or when TGF‐β responsive cells are treated with monomeric TGF‐β1 or ‐β3 (Amatayakul‐Chantler et al, 1994; Zúñiga et al, 2005). Monomeric TGF‐β3, even though impaired 10–15‐fold in its affinity for binding and recruiting TβRI (Groppe et al, 2008), retains significant reporter gene activity with a reduction in potency of just 10‐fold relative to wild‐type homodimer (Zúñiga et al, 2005). Other studies, such as one in which the TβRI and TβRII kinases were fused to the extracellular domain of the erythropoieten receptor (Luo and Lodish, 1996) or another in which the TβRI kinase domain was fused to the TβRII extracellular domain (Okadome et al, 1994), do not however support independent signalling. Monomeric TGF‐β3 has been further shown to possess an intrinsic propensity to non‐covalently dimerize, especially in the presence of TβRI and TβRII (Zúñiga et al, 2005), suggesting that the retention of activity by the monomers might reflect their propensity to non‐covalently dimerize and assemble TβRI:TβRII heterotetramers, not assemble and signal through TβRI:TβRII heterodimers.
The objective of this study was to thoroughly investigate if TGF‐βs signal through two independently functioning TβRI:TβRII heterodimers. This was accomplished by isolating a disulphide‐linked TGF‐β3 dimer composed of a wild‐type protomer and a variant bearing substitutions of Arg25, Tyr90, Arg94, residues previously shown (De Crescenzo et al, 2006; Baardsnes et al, 2009) or implicated (Groppe et al, 2008) to be critical for binding of TβRI and TβRII. Using a series of complementary biochemical techniques, the substituted TGF‐β3 dimer was shown to bind the TβRII extracellular domain and recruit the TβRI with affinities indistinguishable from the wild‐type homodimer, but with one‐half the stoichiometry. Using three established assays for TGF‐β function, the substituted dimer was further shown to retain one‐quarter to one‐half the signalling activity of the wild‐type homodimer. Collectively, these results show that the two TβRI:TβRII heterodimers bind and signal nearly independently of one another.
Design and isolation of TGF‐β3 WD
The objective was to generate a form of TGF‐β that bound TβRII and recruited TβRI with affinities comparable to TGF‐β1 or ‐β3, but with one‐half the stoichiometry. This necessitated that a dimeric form of TGF‐β1 or ‐β3 be used as TβRI binds across the dimer interface and requires both protomers, as well as TβRII, to bind with high affinity (Zúñiga et al, 2005; Groppe et al, 2008; Radaev et al, 2010) (Figure 1B and C). This was accomplished by generating a heterodimer with one wild‐type protomer and one protomer in which Arg25 and Arg94 were substituted with glutamate and Tyr90 was substituted with alanine (Figure 1D). The importance of Arg25 and Arg94 for high affinity TβRII binding was first suggested based on the fact that these, along with Val92, are the only residues in the interface with TβRII that are substituted in TGF‐β2 (Hart et al, 2002), the isoform that binds TβRII weakly (Cheifetz et al, 1990). This was later confirmed by TGF‐β3–β2 and TGF‐β2–β3 chimeras in which swaps of these residues between isoforms, Arg25 and Arg94 in TGF‐β3 and Lys25 and Lys94 in TGF‐β2, decreased or increased affinity several hundred fold to that of the other isoform (De Crescenzo et al, 2006; Baardsnes et al, 2009). The hydrogen‐bonding arrangement of these arginines with the sidechain carboxylates of Asp32 and Glu119 on TβRII (Figure 1A, right), together with the dramatic decrease in affinity when conservatively replaced, led to the idea that substitution with glutamate would abolish TβRII binding altogether.
The blockade of TβRII binding would be expected to greatly impair the binding of TβRI due to the loss of receptor–receptor contacts essential for binding and recruiting TβRI (Groppe et al, 2008). This alone would probably be sufficient based on the weak apparent affinity of the TβRI extracellular domain for TGF‐β1, ‐β2, and ‐β3 (Kd∼30–100 μM) (Groppe et al, 2008; Radaev et al, 2010), though to further diminish binding, Tyr90 was substituted. This residue is centrally located within the TβRI interface and was replaced with a less bulky alanine sidechain, with the goal to reduce TβRI binding based on its substantial contact with TβRI (Groppe et al, 2008; Radaev et al, 2010).
The heterodimer was prepared by first producing wild type and R25E, Y90A, R94E triply substituted human TGF‐β3 monomers (designated, WT or W and dead or D, respectively) in bacteria. These were reconstituted from inclusion bodies, purified to near homogeneity in 8 M urea, and then diluted, in a 1:1 molar ratio, into refolding buffer (Cerletti, 2000). The folding mixture, which contained the desired heterodimer, TGF‐β3 WD, as well as wild‐type and substituted homodimers, TGF‐β3 WW and TGF‐β3 DD, respectively, was then fractionated using high‐resolution cation‐exchange chromatography at pH 4.0 (Figure 2A).
This separation yielded five major species, and as anticipated, three of these, b, d, and e, corresponded to reductant‐sensitive 25 kDa dimers (Figure 2B). The other two, a and c, corresponded to 12.5 kDa monomers (Figure 2B). The three dimers, as well as the two monomers were predicted to be positively charged under the experimental conditions, though reductions in the positive charge were expected for each arginine to glutamate substitution. Thus, peaks e, d, and b were predicted to correspond to the TGF‐β3 WW, WD, and DD dimers, respectively, while peaks c and a, the TGF‐β3 W and D monomers. To confirm this, TGF‐β3 W and TGF‐β3 D monomers were folded and fractionated under identical conditions. This yielded the anticipated chromatograms, with peaks e and c corresponding to dimeric and monomeric forms of wild‐type TGF‐β3, peaks b and a to the dimeric and monomeric forms of dead TGF‐β3, and peak d, the purported wild‐type–dead heterodimer, TGF‐β3 WD, with no matching counterpart. To confirm the identity of the TGF‐β3 WD, the protein was reduced and applied to a reverse phase C18 column. This led to two peaks, the ESI‐MS determined masses of which were within 1.0 Da of the predicted masses of the W and D monomers, 12722.5 and 12576.2 Da, respectively.
TGF‐β3 C77S, a variant of TGF‐β3 in which the cysteine residue that forms the inter‐chain disulphide has been substituted, was also produced. This variant is covalently monomeric (Figure 2B) and as shown previously binds TβRII with nearly the same affinity as the wild‐type dimer, but is impaired in its ability to bind and recruit TβRI (Ilangovan et al, 2004; Zúñiga et al, 2005; Groppe et al, 2008).
Qualitative analysis of receptor binding by native gel electrophoresis
The relative affinities and stoichiometries of receptor binding by the isolated ligands were assessed by analysing the complexes formed with the purified TβRI and TβRII extracellular domains using native gel electrophoresis. The initial experiments focused on TβRII binding and were performed by titrating a fixed amount of TβRII extracellular domain, or TβRII‐ED, with increasing molar amounts of the isolated TGF‐β3 WW, WD, and DD dimers and a TGF‐β3 WT dimer control (there in principle should be no difference between TGF‐β3 WW and WT, though these are designated differently to emphasize that TGF‐β3 WW was isolated from the folding reaction in which both wild‐type and dead monomers were added, whereas TGF‐β3 WT was isolated from a folding reaction in which only the wild‐type monomer was added). The results showed that TGF‐β3 WT, WW, and WD dimers formed detectable complexes with TβRII‐ED, whereas TGF‐β3 DD did not (Figure 3A). The fact that TGF‐β3 DD failed to yield a detectable complex was consistent with expectations regarding its reduced affinity for TβRII. Though less convincing, the results also support the anticipated stoichiometry, with the intensity of the complex bands maximizing in intensity at a 2:1 TβRII‐ED:TGF‐β dimer ratio for TGF‐β3 WT and WW, and a 2:2 ratio (same as 1:1) for TGF‐β3 WD.
The subsequent experiments focused on TβRI recruitment and were performed by titrating a fixed amount of TβRI extracellular domain, or TβRI‐ED, with increasing amounts of TβRII‐ED:TGF‐β dimer complex. The TβRII:TGF‐β complex was always added in a 2:1 molar ratio, regardless of whether the TβRII‐ED was needed or not, to ensure that binding of TβRI‐ED was not limited by TβRII‐ED. The ligands that bound TβRII, TGF‐β3 WT, WW, and WD, also bound and recruited TβRI‐ED. TGF‐β3 DD, which did not detectably bind TβRII, also failed to bind and recruit TβRI (Figure 3B). The stoichiometries in this case are more convincing, with the TβRI‐ED:TβRII‐ED:TGF‐β complex appearing neither undertitrated (i.e. excess TβRI present) nor overtitrated (i.e. excess TβRII:TGF‐β complex present) at a 2:1 TβRI‐ED:TGF‐β dimer ratio for TGF‐β3 WT and WW and a 2:2 (same as a 1:1) ratio for TGF‐β3 WD. These results also support the binding stoichiometry with TβRII as excess TβRII is present when TGF‐β3 WD, TβRII‐ED, and TβRI‐ED are combined in a ratio of 2:4:2 (same as 1:2:1), but not when TGF‐β3 WT (or WW), TβRII‐ED, and TβRI‐ED are combined in this same ratio. These results, though qualitative, indicate that TGF‐β3 WD binds and assembles a 1:1:1 TGF‐β3:TβRII:TβRI complex, not a 1:2:2 as does TGF‐β3 WT or TGF‐β3 WW.
Quantitative analysis of receptor‐binding affinities using SPR
TGF‐β3 WW, WD, and DD were quantitatively characterized in terms of their ability to bind TβRII‐ED and recruit TβRI‐ED using SPR. To accomplish this, the ligands were biotinylated in the presence of excess of TβRI‐ED and TβRII‐ED, separated away from the bound receptors by HPLC, and captured at moderate‐to‐low density, 100–150 resonance units (RU), on a streptavidin surface. Two sets of measurements were made, one in which increasing concentrations of TβRII‐ED was injected and another in which the running buffer was supplemented with a near‐saturating concentration of TβRII and increasing concentrations of TβRI‐ED were injected. The former provided information about TβRII binding, while the latter, TβRI recruitment (Groppe et al, 2008; Radaev et al, 2010).
The series of sensorgrams obtained from these two sets of measurements are presented in Figure 4 (left and right panels, respectively). Through visual inspection, the results are consistent with expectations: TGF‐β3 WW and WD robustly bind TβRII and recruit TβRI, while TGF‐β3 DD is neither capable of binding TβRII nor recruiting TβRI. The low surface density, together with the uniformity of the immobilized ligands, allowed the sensorgrams to be globally fit to a simple kinetic model (orange lines), yielding the association (ka) and disassociation (kd) rate constants as well as the dissociation constant (Kd) (Table I). These data show that TGF‐β3 WW and WD are indeed indistinguishable in their ability to bind TβRII and recruit TβRI, with Kds of 0.18±0.02 and 0.16±0.01 μM, respectively for binding TβRII, and Kds of 0.031±0.002 and 0.027±0.001 μM, respectively, for TβRI recruitment. These values are further shown to be equivalent to those of TGF‐β3 WT (Supplementary Figure S1; Table I). TGF‐β3 DD did not yield any detectable response, indicating it either binds TβRII and recruits TβRI very weakly or is non‐native. The reason for the systematic deviation in the kinetic fits during the dissociation phase for TβRII binding to TGF‐β3 WT, WW, and WD is not known, but does not alter our conclusions as near identical Kd values were obtained by fitting the equilibrium response, Req, as a function of receptor concentration to a standard binding isotherm (not shown).
TGF‐β3 C77S was reexamined in terms of its ability to bind TβRII‐ED and recruit TβRI‐ED (Figure 4D). The sensorgrams, together with the fitted parameters, confirmed that TGF‐β3 C77S bound TβRII with nearly the same affinity as TGF‐β3 WT, WW, and WD (Table I). TGF‐β3 C77S, in contrast, was significantly impaired in its ability to bind and recruit TβRI. The Kd in this case could not be obtained by kinetic analysis using a simple (1:1) model due to large systematic deviations in both the association and disassociation phases. This is likely because the TβRI‐binding site was partially modified during the biotinylation reaction. To derive the Kd, the data were therefore analysed by fitting the equilibrium response, Req, as a function of receptor concentration to a standard binding isotherm (not shown). This yielded a Kd nearly 100‐fold greater than TGF‐β3 WT, WW, and WD (Table I), consistent with the reduced affinity previously reported (Groppe et al, 2008). These data show that the TGF‐β3 WD dimer, unlike the TGF‐β3 C77S monomer, has not altered its affinity for the signalling receptors.
Quantitative analysis of receptor‐binding stoichiometries using SPR
To determine the stoichiometry with which TβRI and TβRII bind, the endoglin‐like domain of betaglycan, or BGe, was studied. BGe binds all three TGF‐β isoforms with high affinity (Mendoza et al, 2009) and its binding site does not overlap with TβRII (Verona et al, 2008). The rationale was that the maximal SPR response attained with BGe should reflect the amount of immobilized binding‐competent TGF‐β and allow one to infer stoichiometry based on the normalized maximal SPR response for binding of TβRI and TβRII.
The measurements were made using surfaces in which TGF‐β3 WW, WD, and DD were immobilized using standard carbodiimide‐based amine coupling. The rationale for this was to ensure that all three ligands were uniformly modified, which may not have been so with the biotinylated ligands described earlier since these were prepared in the presence of excess TβRI and TβRII and may have been differentially modified. The sensorgrams obtained upon injection of increasing concentrations of BGe over these surfaces are provided as Supplementary data (Supplementary Figure S2, panels A–C). To derive the dissociation constant, Kd, and maximal response, Rmax, the data were analysed by fitting the equilibrium response, Req, as a function of concentration to a simple binding model. The derived parameters show that the Kds are similar, with all three ligands binding in the low micromolar range (Table II). The same surface was used to assess TβRII binding and TβRI recruitment by injecting increasing concentrations of TβRII‐ED alone or TβRI‐ED in the presence of a near‐saturating concentration of TβRII‐ED. The sensorgrams show that TGF‐β3 WW and WD exhibit robust concentration‐dependent responses, but TGF‐β3 DD does not (Supplementary Figure S2, panels D–F and G–I, respectively). The fact that TGF‐β3 DD failed to bind TβRII and recruit TβRI, but bound BGe in a manner essentially indistinguishable from TGF‐β3 WW and WD, showed that its inability to bind TβRI and TβRII is a consequence of the R25E Y90A R94E substitutions, not conformational changes or misfolding.
The amplitudes of the responses at the highest concentration of injected receptor over the TGF‐β3 WW surface are each lower than BGe, which is expected, even for a 2:1 receptor:ligand stoichiometry, as BGe is 38 kDa in size whereas TβRII‐ED is 14 kDa and TβRI‐ED is 11 kDa. The responses at the highest receptor concentration over the TGF‐β3 WD surface are decreased even further relative to BGe, presumably due to the reduced stoichiometry. To quantify this effect, the equilibrium response as a function of concentration for TβRII binding and TβRI recruitment were normalized by the corresponding maximal response for BGe and then fitted to a standard binding equation as before. The fits for TβRII binding and TβRI recruitment yielded normalized Rmax values of 0.47±0.02 and 0.39±0.02 for TGF‐β3 WW and 0.19±0.02 and 0.15±0.01 for TGF‐β3 WD, respectively (Figure 5; Table II). The normalized Rmax values for TβRII binding and TβRI recruitment differ by a factor 2.47±0.37 and 2.05±0.21, respectively, providing the first quantitative demonstration of the reduced stoichiometry with which TGF‐β3 WD binds TβRII and recruits TβRI.
Isolation of ligand–receptor complexes and direct determination of stoichiometries
To directly demonstrate the reduced stoichiometry, an excess of TβRI‐ED and TβRII‐ED were added to TGF‐β3 WW and WD and the complexes were isolated using size exclusion chromotography. The elution profiles, and corresponding SDS gel, show that the TGF‐β3 WW complex elutes before the TGF‐β3 WD complex and both elute before the uncomplexed receptors (Figure 6A and B). The isolated complexes were analysed using native gel electrophoresis to ascertain that they were fully saturated with TβRI and TβRII. This was accomplished by challenging the isolated complexes with additional TβRII‐ED, TβRI‐ED, or both TβRII‐ED and TβRI‐ED (Figure 6C). This resulted in no apparent changes, indicating that the ligands were bound by their full complement of receptors.
To analyse the stoichiometry, the isolated complexes were separated using high‐resolution ion‐exchange chromotography in the presence of 8 M urea. The UV absorption profiles, recorded at 280 nm, included three components as anticipated (Figure 6D). The split TβRII peak is a consequence of deamidation of Asn19 and has no effect on TβRII's ability to bind TGF‐β (Hinck et al, 2000). The splitting of the TGF‐β3 WD peak is unexpected, but is not due to contamination of TGF‐β3 WD with either TGF‐β3 WW or TGF‐β3 DD as reanalysis of the TGF‐β3 WD peak from Figure 6D in the absence of urea yields a single peak well resolved from either TGF‐β3 WW or DD (Supplementary Figure S3, panel A). The splitting may instead arise from alternate slowly converting conformations under the conditions used to dissociate the complex, as reanalysis of material from the leading edge of the split peak in the presence of 8 M urea yields an identical split peak (Supplementary Figure S3, panel B).
To quantitate stoichiometries, the areas under the peaks were measured and compared with those for 2:2:1 and 1:1:1 TβRI:TβRII:TGF‐β3 dimer complex calculated from the corresponding molar extinction coefficients at 280 nm. The results show that the relative integrated HPLC peak areas uncorrected for differences in extinction coefficients for the TβRI:TβRII:TGF‐β3 WW complex, 0.099:0.45:1.00, closely match those expected for a 2:2:1 TβRI:TβRII:TGF‐β complex, 0.085:0.41:1.00, whereas those for TGF‐β3 WD complex, 0.043:0.16:1.00, match those expected for a 1:1:1 TβRI:TβRII:TGF‐β complex, 0.043:0.20:1.00 (Table III). These results unambiguously demonstrate that the TGF‐β3 WD heterodimer binds TβRII‐ED and recruits TβRI‐ED with an affinity indistinguishable from the TGF‐β3 WT homodimer, but with one‐half the stochiometry.
Biological activities of the heterodimer
The biological activity of the TGF‐β3 WD heterodimer was compared with TGF‐β3 WT and TGF‐β3 WW to determine the effect of its altered stoichiometry on signalling. The first assay involved measuring the induction of phospho‐Smad3, a direct downstream target of TβRI. MCF10A human breast epithelial cells were treated with 40 and 80 pM TGF‐βs for 30 min and the cell lysates were analysed by western blotting with a phospho‐Smad3 antibody (Figure 7A, upper panel). The results show that the activity of TGF‐β3 WD was partially compromised compared with TGF‐β3 WT and TGF‐β3 WW, whereas the other two variants tested, TGF‐β3 DD and TGF‐β3 C77S, were fully compromised. Time‐dependent Smad3 phosphorylation assays were performed to compare the rate and amplitude of signalling (Figure 7B). The appearance and disappearance of phospho‐Smad3 upon stimulation with TGF‐β3 WD, WW, and WT had similar overall kinetics, but the amplitude at all time points for TGF‐β3 WD was reduced by roughly a factor or four compared with TGF‐β3 WW and TGF‐β3 WT (Figure 7B, lower panel).
The ligands were further tested in a luciferase‐reporter gene assay by transfecting cultured mink lung epithelial (Mv1Lu) cells with a CAGA12 luciferase reporter and by treating them with the TGF‐βs over a range of concentrations for 18 h. The dose–response curves, presented in Figure 7C, show that TGF‐β3 WD is essentially indistinguishable from TGF‐β3 WW and TGF‐β3 WT, with EC50 values in the range of 10–16 pM (Table IV). TGF‐β3 C77S is ∼11‐fold less potent, with an EC50 of ∼140 pM, and TGF‐β3 DD is significantly less potent, with no detectable activity at 200 pM, the highest concentration tested (Table IV).
The ligands were also characterized in terms of their ability to growth inhibit mink lung epithelial cells by treating them with the TGF‐βs over a range of concentrations for 24 h. The dose–response curves, presented in Figure 7D, show that TGF‐β3 WD exhibits comparable potency to TGF‐β3 WW and TGF‐β3 WT, especially at lower concentrations, though at higher concentrations, TGF‐β3 WD appeared approximately two‐fold less potent, with an IC50 close to 0.8±0.2 pM versus 0.4±0.1 and 0.6±0.1 pM for TGF‐β3 WT and WW, respectively (Table IV). TGF‐β3 C77S, in contrast, weakly inhibited growth, with an IC50 close to 17±2 pM, and TGF‐β3 DD exhibited marginal inhibition, with an IC50 of ∼1300 pM (Table IV).
These results show that TGF‐β3 WD heterodimer possesses one‐quarter to one‐half the biological activity of the wild‐type homodimer, TGF‐β3 C77S is significantly compromised, and TGF‐β3 DD is severely compromised. These results, together with the studies presented above, show that the diminishment of receptor binding, either partially as in TGF‐β3 C77S or fully as in TGF‐β3 DD, attenuates biological activity, but eliminating the binding of one of the two TβRI:TβRII pairs reduces the activity by no more than a factor of four.
Receptor complex assembly on the cell surface
TGF‐β3 WD may not bind the cell surface receptors in the same overall manner as the purified receptor extracellular domains due to interactions between the transmembrane or cytoplasmic domains that promote assembly of a TβRI:TβRII heterotetramer. To investigate this possibility, single‐molecule TIRF‐based fluorescence imaging was used. This method measures the proportion of receptors that are monomeric or dimeric based on an analysis of the bleaching statistics, that is the fraction of molecules that photobleach in a single step versus those that bleach in two (Iino et al, 2001). This technique revealed that TGF‐β treatment leads to a significant increase in the proportion of dimeric receptors on the cell surface, with both TβRI and TβRII being about 90% monomeric and 10% dimeric in the absence of TGF‐β and about 65% monomeric and 35% dimeric in its presence (Zhang et al, 2009, 2010).
The same procedure was employed to determine whether TGF‐β3 WD led to any significant dimerization of TβRI or TβRII on the cell surface. This involved transiently transfecting cultured HeLa cells with either C‐terminally GFP‐tagged TβRI or TβRII, expressing these for a limited time to ensure expression at endogenous levels, treatment with TGF‐β3 WT or WD, and analysis of the fixed cells using single‐molecule TIRF‐based imaging. Typical TIRF images and bleaching patterns for cells transfected with TβRII‐GFP and TβRI‐GFP are shown in Figure 8 (panels A–C and E–G, respectively). These, as well as the corresponding bleaching statistics, are similar to those reported earlier, with TGF‐β3 treatment increasing the proportion of TβRI and TβRII dimers from 11.8±1.3 to 36.1±2.6% and 8.5±0.9 to 37.2±2.5%, respectively (Figure 8D and H). This readily measurable increase in dimers was not however apparent upon treatment with TGF‐β3 WD, with the proportion of TβRI and TβRII dimers essentially within the error limits of the control, 13.6±1.2 and 12.7±1.3%, respectively. These results show that the TGF‐β3 WD heterodimer is incapable of assembling a TβRI:TβRII heterotetramer on the cell surface.
The objective of this study was to thoroughly investigate if TGF‐βs signal through two independently functioning TβRI:TβRII heterodimers. This was accomplished by investigating a heterodimeric form of TGF‐β3 bearing substitutions in one of its protomers to block TβRII binding and TβRI recruitment. The heterodimer was shown using a series of complementary biochemical techniques to bind the TβRII extracellular domain and recruit the TβRI with affinities indistinguishable from the wild‐type homodimer but with one‐half the stoichiometry. TGF‐β3 C77S bound TβRII‐ED indistinguishably from the wild‐type homodimer, but was impaired nearly 100‐fold in its ability to bind and recruit TβRI‐ED. TGF‐β3 DD, though native, as shown by its ability to bind the betaglycan endoglin‐like domain, was reduced at least 200‐fold in its ability to bind TβRII‐ED and recruit TβRI‐ED.
The functional data showed that TGF‐β3 WD, which bound the receptor extracellular domains with affinities indistinguishable from wild‐type homodimer, but with one‐half the stoichiometry, had four‐fold lower activity compared with TGF‐β3 in the Smad phosphorylation assay, a two‐fold lower IC50 in the growth inhibition assay, and an indistinguishable EC50 in the reporter gene assay. TGF‐β3 C77S, which was significantly impaired in its ability to bind and recruit TβRI‐ED, had a nine‐fold higher EC50 in the reporter gene assay and a 43‐fold higher IC50 in the growth inhibition assay. TGF‐β3 DD, which did not detectably bind TβRII‐ED or recruit TβRI‐ED, had no detectable activity in the reporter gene assay and an IC50 three to four orders of magnitude higher than TGF‐β3 in the growth inhibition assay.
The fact that TGF‐β3 WD exhibits a small, but detectable decrease in activity compared with wild‐type dimer in the Smad phosphorylation assay and growth inhibition assay, but not the reporter gene assay is likely due to lower intrinsic sensitivity of this assay compared with the others. This is illustrated by the data of Amatayakul‐Chantler et al (1994) who showed that monomeric TGF‐β1 (TGF‐β1 C77S) was reduced in its potency eight‐fold compared with dimeric (WT) TGF‐β1 in a reporter gene assay, but >100‐fold in a growth inhibition assay. Thus, it is not surprising that TGF‐β3 WD, which is reduced in its growth inhibitory activity by no more than two‐fold, exhibits no detectable difference in its reporter gene activity.
The four‐fold reduction in Smad phosphorylation activity for the TGF‐β3 WD heterodimer shows that the two TβRI:TβRII pairs bind TGF‐β and function in a nearly autonomous manner. The diminishment in activity of the heterodimer compared with the wild‐type homodimer by an additional factor of two beyond that anticipated for independent binding and signalling may be a consequence of increased apparent affinity of the wild‐type homodimer for the cell surface receptors. This could occur by membrane‐localization effects, where the apparent affinity of the wild‐type homodimer for cell surface TβRII is increased after it binds TβRII through one of its two sites and becomes localized on the membrane surface. The other possible explanation is that the two TβRI:TβRII pairs functionally interact, but this seems unlikely given the limited magnitude of the effect.
The conclusion that the two TβRI:TβRII pairs bind and function in a near‐autonomous manner presumes that TGF‐β3 WD binds the cell surface receptors in the same manner as the purified receptor extracellular domains. This seems likely because the TIRF‐based single‐molecule fluorescence data obtained with C‐terminally GFP‐tagged TβRI and TβRII showed that treatment with TGF‐β3 WD leads to a negligible increase in the proportion of dimeric TβRI and TβRII on the cell surface, whereas treatment with TGF‐β3 leads to more than a three‐fold increase (Figure 8).
The current results indicate that receptor transactivation occurs exclusively within TβRI:TβRII heterodimers, not between. This is probably determined by the arrangement of the receptors as the last structurally ordered residue on the C‐terminus of TβRI and TβRII are separated by just 46 Å within a TβRI:TβRII heterodimer, but 80 Å between heterodimers (Figure 9A). The current results stand in contrast to results obtained in earlier studies in which the receptors were artificially dimerized. Two such studies employed TβRI and TβRII variants bearing dimerization domains, one employing the extracellular domain of the erythropoietin receptor (Luo and Lodish, 1996) and the other small immunophilin domains inserted between the kinase and transmembrane domains (Stockwell and Schreiber, 1998). The erythropoietin‐dimerized receptors led to TβRII‐TβRI transactivation, but not activation of downstream signalling, while the immunophilin‐dimerized receptors led to the activation of downstream signalling, though at a significantly reduced level compared with wild‐type homodimer. A third study employed TGF‐β as the dimerizer, but utilized a chimeric receptor comprising the TβRI kinase domain and the TβRII extracellular domain (Okadome et al, 1994). This construct, designated TβRII‐I, yielded no detectable signalling when transfected into a TβRI‐deficient cell line.
The discrepancy between these results and the previous emphasizes that the precise positioning of the receptors is important, with wild‐type‐like signalling when TβRI and TβRII are arranged natively, but diminished or no detectable signalling when arranged non‐natively. The spacing between the last structurally ordered residue on the C‐terminus of the two bound TβRIIs in the TGF‐β receptor complex is 104 Å (Figure 9A) and that between those of the two bound Epo receptors in the Epo receptor complex is 30 Å (Syed et al, 1998). The l04‐Å spacing, which would be expected for signalling with the combination of TβRII and TβRII‐I, is likely too large compared with the 46‐Å spacing when TβRI and TβRII are arranged within a TβRI:TβRII heterodimer. The 30‐Å spacing, which would be expected for the chimeric Epo‐TβRI and Epo‐TβRII receptors, enables efficient transactivation, but perhaps brings the TβRI and TβRII kinases too closely together so that Smad phosphorylation is inhibited. These observations suggest that the separation between the kinases is a critical factor for efficient signalling, though it should be emphasized that this conclusion is tentative given that orientation effects might also be important and that both TβRI and TβRII include 10 to 14 structurally disordered residues bridging the last structurally ordered residue of their extracellular domain and the first predicted residue of the transmembrane domain (Figure 9A). Thus, further studies are required to ascertain whether receptor activity is determined by distance alone, or whether orientation or other effects might also have a role.
The isolated TβRI and TβRII kinase domains have previously been shown to weakly interact using yeast two hybrid and other methods (Ventura et al, 1996). Thus, it seems likely that the close proximity between the TβRI and TβRII extracellular domains, as in TGF‐β3 or TGF‐β3 WD complexes, promotes this otherwise intrinsically weak interaction, and positions the kinases for optimal transactivation and signalling. The current results therefore emphasize that binding of TβRI and TβRII adjacent to one another and with direct contact in the TGF‐β receptor complex has roles beyond TβRI recruitment and enhancing specificity (Groppe et al, 2008), but also promoting transphosphorylation that leads to downstream signalling.
The finding that TGF‐βs signal through TβRI:TβRII heterodimers is of interest in light of recent single‐molecule fluorescence imaging studies that show that both TβRI and TβRII are predominantly monomeric in the absence of ligand (Zhang et al, 2009, 2010). The discrepancy between these findings and the previous findings that showed that the receptors are dimeric (Chen and Derynck, 1994; Gilboa et al, 1998; Rechtman et al, 2009) is likely a consequence of differences in expression levels, as the single‐molecule studies showed that TβRI and TβRII were predominantly monomeric when expressed at endogenous levels, but dimeric when overexpressed. The findings from the single‐molecule study, together with the current findings, therefore show that the TGF‐βs have adapted to bind and assemble TβRI:TβRII heterodimers, not TβRI:TβRII heterotetramers.
The data presented, even though they show that the two TβRI:TβRII pairs bind and function in an autonomous manner, does not imply that one of the two TβRI:TβRII pairs is dispensible. Thus, as explained above, one important function of the two pairs may be to increase potency by enhancing the apparent affinity for binding TGF‐β via membrane‐localization effects. The four‐fold increase in pSmad levels with the wild‐type homodimer versus the heterodimer might be vital in vivo where the local concentration of active dimer can lead to very different biological outcomes (McKarns et al, 2003).
The fact that TGF‐βs activate the Smad pathway through two near‐autonomously functioning TβRI:TβRII pairs stands in contrast to the BMPs, which are unable to activate the Smad pathway when one of the type II receptor‐binding sites is blocked (Knaus and Sebald, 2001; Isaacs et al, 2010). This suggests that BMPs have a minimal requirement for a type I:type II:type II heterotrimer. The type I and type II receptor extracellular domains do not contact one another in the BMP receptor complex (Figure 9B) and thus the requirement for a heterotrimer in the BMP system must be a consequence of direct or indirect interactions between the transmembrane or kinase domains of the receptors. One possible role for the type I:type II:type II heterotrimer is to promote efficient receptor transactivation and signalling. Another is to enhance potency via multivalent binding, perhaps overcoming the low intrinsic affinity that many BMPs have for their type II receptors (Nickel et al, 2009).
The requirement for a heterotrimer in the BMP system, but not the TGF‐β, may be related to differences in the manner by which these two subfamilies of ligands bind their receptors. The TGF‐βs bind the type I and type II receptors as two well‐separated heterodimeric pairs, whereas the BMPs bind their type I and type II receptors without any direct contact, but in much closer spatial proximity to one another (Figure 9). Thus, in analogy to the TGF‐βs, where direct contact between the extracellular domains promotes recruitment of the low affinity receptor and signalling, so too may the close proximity between the transmembrane and/or kinase domains of the receptors in the BMP system promote functions critical for ligand binding and signalling. Though speculative, it might be this function is related to enhancement of ligand binding by dimerization of the type II receptor, rather than signalling, as the type II receptors are somewhat closer together in the BMP system (84 Å) compared with the TGF‐β (104 Å) and the two type I—type II receptor distances are not that different from that in the TGF‐β system, with one 'short' distance compatible for transactivation, 35 Å, and one 'long' distance that is not, 72 Å.
Materials and methods
Protein preparation
Human TGF‐β3 was expressed, refolded, and purified as previously described (Cerletti, 2000) with modifications (Supplementary data). TGF‐β3 WW, WD, and DD were prepared by expressing and purifying the wild‐type (W) and R25E, Y90A, R94E (D) monomers separately and by combining them in a 1:1 ratio for refolding. The three dimers that formed, WW, WD, and DD, were separated from one another, as well as non‐dimerized W and D monomers, using cation‐exchange chromatography as described (Supplementary data). Other proteins were produced as described (Supplementary data).
Native gel assay
Native gel assays to assess the binding and stoichiometry of TGF‐β3 WT, WW, WD, and DD to the TβRI and TβRII extracellular domains, TβRI‐ED and TβRII‐ED, respectively, were performed as previously described (Zúñiga et al, 2005)
SPR‐binding assays
Binding studies were performed with BIAcore 3000 instrument (GE Healthcare) and were analysed using the software package Scrubber2 (Biologic Software). For kinetic experiments, TGF‐βs were biotinylated and captured on streptavidin chip surfaces (GE Healthcare). TGF‐β3 WT, WW, WD, and C77S were biotinylated in the presence of excess TβRI and TβRII, whereas TGF‐β3 DD was biotinylated in the presence of excess soluble betaglycan, using sulfo‐NHS‐LC‐LC‐Biotin (Pierce) at pH 7.5. Singly biotinylated TGF‐βs were separated from receptors and doubly and multiply biotinylated forms by Source S cation‐exchange column (GE Healthcare) in the presence of 30% isopropanol at pH 4.0. Surface densities of captured TGF‐βs were kept at 50–300 RU to minimize rebinding artifacts. For equilibrium experiments, TGF‐β3 DD, WD, and WW were covalently attached to flow cells using an amine coupling kit (GE Healthcare). Binding assays were performed by injecting two‐fold serial dilutions of the receptors in triplicate in HBS‐EP buffer (GE Healthcare) at a flow rate of either 10 μl per min (equilibrium experiments) or 50 μl per min (kinetic experiments). Surfaces were regenerated by a brief injection of 4 M guanidine hydrochloride (10 s contact time at a flow rate of 100 μl per min). Instrument noise was removed by double referencing. Equilibrium analyses were performed by averaging the equilibrium binding response near the end of the injection. Kinetic analyses were performed by global fitting with a simple 1:1 model. Standard errors were obtained from the variation among the derived parameters.
Stoichiometry by HPLC
TGF‐β3 WW and WD ternary complex with TβRI and TβRII were formed and purified as before (Groppe et al, 2008). Compositional analysis of the isolated complexes was determined by separating the components using high‐resolution ion‐exchange chromatography (Mono S HR 5/5 column, GE Healthcare) in the presence of 8 M urea and 50 mM sodium formate at pH 4.0 (0–400 mM NaCl in 50 mM sodium formate over 80 column volumes). Chromatograms were recorded at 280 nm and peaks areas of individual components were determined using the program Peakfit (Systat Software Inc., San Jose, CA).
Smad3 phosphorylation assays
MCF10A human breast epithelial cells were treated with 40 and 80 pM TGF‐β ligands for 30 min. Western blot was carried out as previously described (Zúñiga et al, 2005) using a rabbit polyclonal anti‐phospho‐Smad3 antibody (Upstate Biotechnology) or an anti‐actin antibody (Ambion). Time‐dependent Smad3 phosphorylation was performed in the same manner, but with different treatment times.
Growth inhibition assay
The effect of TGF‐β isoforms on the proliferation of cultured mink lung epithelial cells was tested as described (Cheifetz et al, 1990; Zúñiga et al, 2005).
Luciferase‐reporter gene assays
The induction of a TGF‐β CAGA12 luciferase‐reporter construct was performed as previously described (Dennler et al, 1998) with modifications (Supplementary data).
Single‐molecule fluorescence
HeLa cells were cultured and transfected with TβRII‐GFP or TβRI‐GFP plasmid and expressed at endogenous levels or lower as previously described (Zhang et al, 2009, 2010). For the ligand stimulation experiments, the transfected cells that were ready for fluorescence imaging, were added with 200 pM TGF‐β3 (WT) or TGF‐β3 (WD) in DMEM for 15 min at 4°C. Single‐molecule fluorescence imaging was performed and analysed as previously reported (Zhang et al, 2010). The experiments were performed three times and 200–300 spots were selected and analysed every time.
Supplementary data
Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org).
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplementary Information
Supplementary Information [emboj201154-sup-0001.pdf]
We acknowledge Christian Zwieb and Thomas Thompson for critically reading the manuscript. This project was supported by the NIH (GM58670 and RR13879 to AH; CA75253 to LS; CA54174 to the Macromolecular Interactions and Mass Spectrometry Shared Resources of the UTHSCSA CTRC), the Robert A Welch Foundation (AQ1431 to APH), the Consejo National de Ciencia y Tecnología (49828 to FL‐C), and the Chinese 973 project (2007CB935601 to XF).
- Copyright © 2011 European Molecular Biology Organization


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We used to jot down a friend's address and phone number in moleskin address books, erasing or crossing out old information whenever he or she moved.
The digital age made tracking our friends easier, but we still spend a lot of time manually updating our address books or we let Facebook become the guardian of our social network.
Why are address books so dumb when so many people carry smartphones? asks Silicon Valley entrepreneur Mrinal Desai. He and Jorge Ferreira want to –- ahem –- address that problem with a slick new iPhone app that keeps you connected to your friends because they are the ones who update their contact information when it changes.
Called Addappt (pronounced "adapt"), its creators are marketing it as an always up-to-date private address book that you don't have to update. Your friends do that. Change your contact information in the app and it automatically updates all of your friends' address books on mobile, desktop, iCloud, etc.

subset: commoncrawl


text:
Club Only
Skylite Cellars produces small quantities of distinguished handcrafted wines for wine lovers, from wine lovers! Our two locations can accommodate small and large groups. Space is available for private events. Tasting rooms are dog friendly!
25 S. Campbell Road Walla Walla, Washington 99362 Phone: 1.509.529.8000 Email: email@example.com
19255 Woodinville-Snohomish Road Woodinville, Washington 98034 Phone: 1.509.529.8000 Email: firstname.lastname@example.com


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I would ask you to recommend me the best English Bible scholar translation that would fit my needs. I'm quite acquainted with modern Spanish and to some degree modern German translations, but I'm not very updated about modern English translations (I use to read KJV).
I'm interested in the best and more accurate and updated philologically modern translation; A scholar translation, so to say, in order to use that always with the Hebrew (or Greek) originals side by side. Rather, to use it as a tool to read the originals and go more deeply into the pholological and historical questions. I would like to have it both in Accordance and in physical version (a study Bible with philological commentaries). I have a good level of ancient Greek (I'm teacher and philologist) and a intermediate level of Hebrew, but I want yo go more deeply into Hebrew and Aramaic. My interests are mainly philological/historical/history of ancient religions.
Thanks and regards.


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Here are my buy prices on common ingredients you will use for baking. All prices are after coupons are applied, and I am not brand-loyal. I have found the generic can be just as good as the name brand, so unless the name brand is cheaper, I do not buy it.
Sugar (brown, powder or granulated): 50 cents or less per pound
Flour: 50 cents or less per pound
Spices/flavorings: $1
Margarine sticks, four-pack: 75 cents
Oil: 3 cents per ounce ($1.44 or less for 48 ounces)
Evaporated milk: 10 cents per ounce (or less)
Sweetened condensed milk: 10 cents per ounce (or less)
12 ounces baking chips: $2 or less
Large eggs: 10 cents per egg
8 ounces cream cheese: $1.25 or less
Marshmallows: $1 or less
Sour cream: 10 cents or less per ounce
Peanut butter: 11 cents or less per ounce (no more than $2 for 18 ounces)
Shortening: $3 or less per can
You may not always be able to find these items on sale for my "buy price." When this happens, I do one of the following:
I will look for dollars-off store coupons to help reach my price. Example: A $5 off $30 Piggly Wiggly coupon that would help to get the price of my items down.
Purchase the items with Extra Bucks, Register Rewards, +UP Rewards or gift cards I get free with prescription transfers.
Clip those coupons
Combine coupons with those sales to score an even better deal. I found a $2 off two bags of Hershey Kisses. I snagged that coupon and was able to score a great deal on Hershey's Kisses at CVS. Don't forget that Kroger, Harveys and the Georgia Publix stores double coupons up to 50 cents with no minimum purchase required; Piggly Wiggly doubles coupons up to 99 cents with no minimum purchase required. Don't be in such a hurry to price-match; you may be missing out on the additional savings of double coupons.
Watch for store coupons, incentives
This year, I purchased my Hershey's Kisses at CVS. They were offering $3 in ExtraBucks if I bought four bags; after my $2 off two coupons (I had two of them) and the ExtraBucks, they were just 75 cents a bag. I also was able to snag free Ziploc containers at Publix when I purchased Nestle Toll House morsels. Because it was a store coupon, I was also able to combine a manufacturer's coupon on the Ziploc containers, making this a money-maker. I also had a coupon for the Nestle Toll House morsels that I printed online.
Watch for peelies on products
Thanks to Amie Ahlers, who shared her shopping trip to Publix on the Savannah Savvy Shopper Facebook page, I learned there were peelies for free candy on select boxes of Cocoa Krispies. I purchased several boxes with the peelies and was able to snag four bags of free M&M's that I will use for baking. I also found peelies on Gold Medal flour at Kroger. Please note, peelies must stay on the product and are not your property unless you are purchasing the item. Don't ever take peelies off of the item if you are not purchasing it.


text:
Well, here we are in the middle of the week,
in the middle of yet another month.
Seems every time I get out of bed,
another day has flown by.
What ever happened to the time
when a day dragged on and on
and on?
As fast as the days of May are passing
the stitching time is certainly not keeping up
and that in itself is disturbing.
Low and behold,
we are just as fast approaching
one of the most anticipated weekends
of the year.
So, at the end of this month,
The Stitching Sisters,
the Four From Forest
are off to spend a wonderful, relaxing,
stash packed, stitch full weekend
in a place where some refer to as
Heaven on the Huron.
Properly known as
With bags of stash, lamps and loads of laughter,
we will land in early Friday afternoon
to forget all that we have to deal with
and totally become enveloped in what else,,,, our stitching, and wonderful comradely.
The view is amazing
and with any luck
we will enjoy the late night fires
down by the lake.
In the meantime,
this evening we will gather
here @ Henley's Landing
and I am sure there will be a great deal of conversation
revealing which project everyone will be taking, which dish we will share,
and how much we are all looking forward
to out time together.
Not to mention sharing some time
with our Four From Forest.
For now,
I wish everyone a happy Wedstitchday
and also send our thoughts and prayers
to the Bosma family.


text:
This is a milk glass Paneled Grape pitcher and four tumblers made by Westmoreland Glass. Each of the five pieces is marked with the Westmoreland Glass logo. The pitcher measures 9" tall and is about 7 1/4" from the handle to the spout. Each tumbler is 5 7/8" tall and about 3" across. The set is in very good condition with no chips or cracks. Please email me with any questions.
4 other shoppers are interested in this item
Everyone collects something and we have something for you.
We Now Accept Layaway!!


text:
Studylog Web is designed to connect researchers within a lab, across multiple sites, and among collaborating organizations. Studylog Web saves collaborators hours per study by making critical data and information instantly available and accessible.
Where Studylog Desktop gives researchers the power, flexibility and in-depth functionality needed to conduct and manage all aspects of studies, Studylog Web provides a higher level view through a simple interface, and allows investigators and executives to propose study protocols and track changes, remotely follow study progress, view collected data, and generate meaningful reports.


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The reader was able to do voices so well it seemed more like listening to a play than just one reader.
Cold Sassy Tree by Olive Ann Burns
My review
rating: 4 of 5 stars
There is so much to love about this book. The characters are vibrant, the language is rich and there are good life's lessons throughout--but it's not preachy. This is a story told with lots of humor although it does have it's tear-jerker moments. Young Will Tweedy is 14 at the turn of the century and is living in the town of Cold Sassy, Georgia. His grandfather causes an uproar in this sleepy little town by eloping with the beautiful young employee in his store. That's bad enough but what really sets the tongues to wagging is that Grandpa's first wife just passed three weeks ago and is barely cold in the grave. The events subsequent to this scandalous event changes Will's outlook on life forever. He has an "old" grandpa who is made young again by the pretty young wife and he himself begins to grow up as he romanticizes about women, kisses someone for the first time, and drives around town in his grandfather's new automobile. Thumbs both up!
View all my reviews.
A beautifully written book full of rich substance. I give it 5 stars. I was hooked by the end of the first chapter when Grandpa Blakeslee announces to his family, only 3 weeks after the death of his wife, that he plans to marry 33 year old Miss Love Simpson. Is it for love, sex, convenience, cooking & cleaning, &/or companionship?
Burns makes these characters, who live in a small, religious, southern town in the early 1900s, come alive. It tells the story of the Georgia town Cold Sassy Tree, its inhabitants, and one particular family. The story centers around the transformation of 59 year old Grandpa Blakeslee and his 14 year old grandson Will Tweedy. It examines coming of age, love & death, spirituality and religious ideas and prejudices, morals and mores, and modernization.
I highly recommend this book!
It takes place in a small southern town in 1906. A widower of 3 weeks marries a woman half his age and the family and the rest of the town search for a way to deal with it....or not!
Hated to see this one end....


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It's dominos with all the bells and whistles in Domino Master®! Play solo or against friends and family in any of five exciting game variations including Mexican Train, All 3's, All 5's, Straight Dominos, or Bergen. See if you have what it takes to move up the ranks and score the coveted spot at the top of the Leaderboards or grab the exciting achievements in this great family game sure to please all ages.
By popular demand, TikGames has created a free title update that allows groups of players to continue playing from game to game without needing to return to the lobby and restart their match. This feature can only be allowed in Player Matches. Also, players who win a round get to choose the first domino placed in the next round, allowing skilled players to control suits to suit themselves.
New Game add-on content allows players to re-skin their Domino Master game to take on either a Hip Hop or a Winter theme with all new graphics, domino sets and music. Each downloadable content pack will be priced at 160 points.
"As an avid Xbox Live Arcade Domino Master player, I have had the opportunity to hear our customer feedback first hand on this game. We listened to their great suggestions and made improvements to the game to directly address their feedback," said Anatoly Tikhman, founder and CEO of TikGames. "With this new title update, finding and playing Domino Master against other players through Xbox Live® Arcade has been streamlined to get you into the game quickly and keep you there until you are done playing."
Key Features :
* Variable difficulty levels: The single player mode features three levels of difficulty for the A.I. players, including Beginner, Master, and Guru, to provide exciting game play for all ages.
* Five game modes: Play Straight, All 3's, All 5's, Bergen, or Mexican Train.
* Leaderboard support: See your name in lights as you check the leaderboards for each game type.
* Exciting multiplayer modes: Play Player Match or Ranked Matches. The ultimate party game comes to life when up to four players compete to see who can become the Domino Master.
* Custom rules: Modify the rules to create your own personal dominos game.
* Multiple domino sets: Choose from double sixes, double nines, or double twelve domino sets.
Domino Master, rated E for Everyone, is available for 800 MS Points.


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RSVP – Pandora is hosting an exclusive evening of performances from Pop Coast Hits artists: Meghan Trainor, Bebe Rexha, Why Don't We and a special performance by Madison Beer.
Ticketed – KIIS FM'S Wango Tango hosted by Ryan Seacrest, with performances by Ariana Grande, Shawn Mendes, Meghan Trainor, 5 Seconds of Summer, NF, Miguel, Janelle Monáe, Marshmello with a special guest performance by Logic, Backstreet Boys and Sabrina Carpenter at the all-new Banc of California Stadium in Los Angeles, adjacent to the LA Coliseum in Expo Park.
Ticketed – the ASCAP "I Create Music" EXPO – taking place May 7-9 at the Loews Hollywood Hotel in LA. Now in its 13th year, ASCAP EXPO features three days of panels, workshops, master classes, keynotes and one-on-one sessions with the industry's top hitmakers. This year features one of the most illustrious lineups to date, including discussions with St. Vincent, Meghan Trainor, Jason Mraz, JoJo, Billie Eilish and more. They'll
Continue reading…
Ticketed – There's no better way to celebrate the magic of the holiday season than with a show on the Jingle Ball tour. Various venues across the country will showcase a vast assortment of the year's hottest artists in an unforgettable night of performances including Bruno Mars, Justin Bieber, Britney Spears, Meghan Trainor, Fifth Harmony, Diplo, Alessia Cara, Lukas Graham, Daya, Special Guest Appearance from Niall Horan.
Ticketed – 97.1 AMP Radio presents #WeCanSurvive starring Bruno Mars, Ariana Grande, Charlie Puth, G-Eazy, Meghan Trainor, OneRepublic, and Pitbull, Saturday October 22nd at Hollywood Bowl!
Open – "Thank You" is the upcoming second major-label studio album by GRAMMY winning American singer-songwriter Meghan Trainor that includes her newest hit "No". Join us when Meghan visits to celebrates the release of "Thank You" with special CD signing Monday May 16th from 5pm to 8pm. This is a wristbanded event. Starting Friday, May 13th at 9am, purchase the new Meghan Trainor release "Thank You" at the Barnes &
Ticketed – The iHeartRadio Music Awards are officially BACK. The 2016 iHeartRadio Music Awards are set to take place on April 3rd, at the historic Forum in Los Angeles. In addition to this year's nominees, iHeartRadio is revealing the first two artists who will be performing live during this year's awards: Justin Bieber and Meghan Trainor.
To make it clear that Jennifer Lopez (since when does she not go by J.Lo?) is taking over Las Vegas for her residency at Planet Hollywood titled All I Have, she took over the American Music Awards by storm on Sunday November 22th just to give us a taste. At 46 years old, this woman has better moves and more energy than myself sipping on redbull vodka's. She opened up
Open – Join The Grove for our most spectacular Christmas event yet! On Saturday, November 14, we will ring in the 2015 holiday season with The Grove Christmas with Seth MacFarlane. Presented by Citi, the event will begin at 7:30 p.m. and will include MacFarlane singing some of the holiday's most cherished songs backed by a vibrant 55-piece orchestra. Grammy-nominated artist Meghan Trainor will join him, among others. Plus, experience


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Kampala- Uganda (PANA) -- A Southern Sudanese envoy said Tuesday President Omer E l Bashir's failure to attend the 15th African Union (AU) Summit in Kampala, Uganda, is a clear sign of his unwillingness to cooperat e in ending the war in Darfur and his party's deliberate steps to delay the January 2011 referendum.
Southern Sudanese Ambassador John Andruga Duku, leading an international campaig n for South Sudan's Referendum in 2011, said the failure by El Bashir to come personally to Kampala to discuss the pressing secur ity issues in Darfur and parts of Sudan must be sanctioned.
''What is the position of the African Union regarding its member states totally ignoring it when there are important decisions that touch on the security of an entire region,'' the Sudanese diplomat stated.
Prior to the summit, President Bashir visited Chad, a signatory to the Rome Stat ute, which created the permanent International Criminal Court (ICC) at The Hague , Netherlands but was not arrested, even though the court has issued a warrant of arrest for the Sudanese leader over alleged genocide charges.
The AU has called those warrants a threat to regional peace in Darfur and the en tire Sudan, which remains fragile.
African leaders meeting in Kampala, Uganda, were expected to make a declaration on the issue Tuesday, at the end of their three-day debate on various issues.
Duku said the failure by the AU to discuss the pre-referendum preparations ahead of 2011 meant that the entire Summit had lost an important moment in its histor y to tackle the subject, which is key to the stability of the entire East African region.
''The failure of the Comprehensive Peace Agreement (CPA) would have an impact on the peace in the Democratic Republic of Congo (DRC), Ethiopia, Kenya and Uganda.
Why are they (leaders) not talking about it.
Pressur e must be exerted on Bashir to stop undermining the CPA,'' he said.
The official said the ruling National Congress Party (NCP) was deliberately dela ying the referendum discussions by demanding to take entire control of the Referendum Commission, which is in the process of being created t o run the referendum in January, the last stage in a six-year plan for peace in Southern Sudan.
Duku said the recent efforts by the NCP to assume the control of the key positio ns in the referendum commission had already created suspicion within the Sudan People's Liberation Movement (SPLM).
''This is an illustration of the lack of faith.
It makes us very suspicious.
The re are deliberate delays in the referendum and the end result would be to postpone the referendum.
If this happens, we will revert back to war and this will affect the entire region,'' the official told PANA.
The Southern Sudanese official said President El Bashir could have assigned Vice President Salva Kiir to the AU Summit, if he was committed to its work and the overall peace in Sudan.


text:
Eliot Spitzer, who hopes to be New York City's next comptroller, likes to screw with his socks oncalf-length black hose, the atavistic shadow of sock garters visible in the mind's eye. That Spitzer paid for sex is not actually all that interesting. That he brought his nerdish Dudley Do-Right public persona to bed with him seems a window to his soul.
On the spectrum of sexual taste, partially clad intercourse between consenting adults hardly merits a mention. It's another jump to the writer Daphne Merkin, who confessed in print her predilection for being spanked, and Quentin Tarantino, who confesses his foot fetish somewhere in just about every movie. And these are miles from the 35-year-old law-enforcement officer who showed up in a psychiatrist's office wearing Winnie-the-Pooh bib-overall shorts and smelling strongly of talcum powder, explaining that he liked to wear diapers, that he defecated and urinated in them, and who would, according to the doctor's case study, often think about how I am a baby' and masturbate in his diapers several times a day.
Sexual self-discovery is a mysterious process, the only aspect of growing up that parents and teachers mostly leave children (if they're lucky) to sort out on their own. The word groping works here. It's you in the dark at first, maybe with props or pictures, and eventually with other humans, discovering over timesometimes over a very long timewhat gets you off, what turns you on. But how is it that you, being you, like this, not that? Why men, not women; why leather, not rubber; why dirty talk, but not dildos; why handcuffs, not horsewhips; why tongue, but not too much? And further, why do some people appear to be more sexually flexible, able to get off with men and women, orally and anally, while othersand this is true especially of menremain fixed in their erotic preferences, able to achieve orgasm only during vaginal intercourse, only inhaling the scent of someone's used bicycle seat, only looking at Internet pictures of children?
Freud would say that what he called perversions have their source in developmental stuckness: Something went wrong in the phases of childhood in which people learn to suck, poop, and get along with parents; psychoanalysis, the process of unsticking, would help them achieve something more like what once passed for normalcy (though even that normalcy, he thought, was an effort to recapture the satiated pleasure of breast-feeding lost after infancy). But in a world where Freud is bunk, what he called normal is now called vanilla, and it's complicated is a Facebook status, the why question sits out there like a sexual itch, begging for an answer.
Evolutionary psychology, the explanation du moment for all human mysteries, offers a partial clue. To start with, people are animals, programmed for sex in order to produce more people. The chemicals released in the brain during orgasmmainly dopamine and oxytocincreate a cycle in which hot sex begets more hot sex. The neuroscientist Jim Pfaus has discovered that if female rats have a long and satisfying mating session even in the most miserable setting (for them, a brightly lit box), they will continue to revisit the scene in the hopes of repeat action. If a researcher stops stroking a rat's tiny clitoris with a tiny brush, she will bite the researcher's sleeve, pleading for more. No woman who has ever continued to visit her hot boyfriend's filthy bed, even after he ceases to return text messages, can fail to recognize the comparison.
Diverse sexual interests may thus be evolution's way of helping humans to maximize the sheer amount of sex that goes on, matching desire for complementary desire and making sure we mix our genes up as much as possible. We are preprogrammed to need diversity, says Gregory Lehne, a psychologist at Johns Hopkins who studies human sexuality and gender identity (and the kind of person who is careful to use orientation to mean the people you love, taste to mean the things you like, and fetish to refer to an idiosyncratic preference that can turn compulsive and interfere with regular life). Imagine if every person was turned on by the same thing, if every woman was turned on by George Clooney. The diversity of the human species requires that we be turned on by a whole variety of things. Variety is a cosmic understatementhumans have a range of yearnings and cravings unmatched in the animal world. And when people are attracted to other people (or things) with no reproductive benefit, well, that's part of nature's plan, tooor at least an epiphenomenon of that plan. Evolution has never required that everybody breed, Lehne says.


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But the display info page for my motherboard - ASUS Proactive AI P5GDI Pro (plus info down the bottom "Press DEL to run Setup. Press TAB to display BIOS Post Message" and the Pentium sign on the right) - is showing so much longer everytime when I start up/after rebooting my computer.
The last time I started the computer, I'm sure the motherboard info display page stayed there for a good 3 mintues if not longer. As if it's a prolonged advertisment for ASUS's motherboard.
Could someone please help? Is there some time switch in the registry for this and probably got accidentally changed? I think the change took place after I uninstalled IE7 Beta 2 to put on the new Beta 3 but I don't think that would have much to do with the motherboard info display page???


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text: RED RIDING HOOD Once upon a time, there lived in a small cottage on the edge of a deep wood, a forester and his wife, and their dear little daughter. The little child was as lovely as a picture, and a great pet with everybody. Her mother liked to see her prettily dressed, and made … More RED RIDING HOOD


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MCC is the relief and development arm of the greater Mennonite church. Congregations are made aware of need for material aid worldwide through this agency. First Mennonite has responded by donating supplies for school kits, health kits, and relief kits. Comforters are pieced, knotted and donated as well.
MCC Opportunity - Feb - Mar 31, 21015 MCC HYGIENE KITS As the Syrian war continues and the group calling itself the Islamic State pushes its way into parts of Iraq and Syria, families often leave their homes and seek shelter in nearby towns. When the battle moves there, they flee again to another town or temporary shelters or neighboring country, usually arriving with only their official papers and the clothes on their backs. Wherever the displaced people and refugees go, they need safety, shelter, friendship, food and water, education and jobs. MCC works with partners in Iraq, Lebanon, Jordan and Syria to do all that we can with your gifts to meet those needs in the name of Christ. FMC has collected 63 hygiene kits which will be sent to the war torn area. Additional supplies will be accepted until March 1st. We have passed our reasonable goal of one for each family attending, how close can we come to our awesome goal of one for each person attending… 105
School Kits for MCC
Jean Oswald, Children's Education Superintendent
As a peace church, FMC reaches out to pain and suffering in the world. One avenue is through Mennonite Central Committee. During the month of August, school supplies were donated and bags sewn for school kits. Each kit includes 4 notebooks, 8 pencils, colored pencils, a ruler and an eraser. One Sunday after church, the youth of FMC gathered for lunch and stuffing of the bags. The task of "stringing" the bags was taken on by even the youngest children who attend Sunday school. All the bags were piled in the sanctuary for a prayer of dedication on Oct. 18. and 65 bags filled Jean's trunk. They were delivered to the 'Gift and Thrift" shop in Harrisonburg, and will be taken to Pennsylvania before being shipped to refugee camps around the world. The person receiving the bags in Harrisonburg said daily bags arrive from various churches.
First Mennonite Church
601 East Parham Road
Richmond, Virginia 23227
Pastor: Ryan Ahlgrim
Office Hours: Generally, 9-4 M-F
Secretary: Shelby Scattergood
Office Hours: Generally, 9-2 Tu & F
Directions to the church


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Lovely, spacious, move in ready 2 bedroom, 3 bath Townhome w/ attached garage, balcony, and fireplace. Great opportunity! The main floor features a cozy living room, which leads to a composite Trex balcony, providing natural light and fresh air on beautiful Colorado days. Stay comfortable all winter w/ your warm cozy fireplace. Head upstairs to your master bedroom w/ plush newer carpet. Upstairs features 2 nice size bedrooms and 2 bathrooms. Laundry is in the basement and washer/dryer are included. Stay cool in the summer w/ central A/C. Must see the adorable backyard off of the kitchen! This cheery townhome is an excellent investment opportunity. Located in a perfect location, on a quiet street. Just blocks from Summer Valley Park and Elementary School. Less than 5 minutes drive to the Valley Center Shopping Center. 15 minute walk to Quincy Reservoir. Cherry Creek school district! Smoke free and ready to move in! This home will go fast! Send your highest and best qualified offers!
The content relating to real estate for sale in this Web site comes in part from the Internet Data eXchange (IDX) program of METROLIST, INC., DBA RECOLORADO®. Real estate listings held by brokers other than RE/MAX Alliance are marked with the IDX Logo. This information is being provided for the consumers' personal, non-commercial use and may not be used for any other purpose. All information subject to change and should be independently verified. [more]
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Lelystad, Netherlands, News. 128kbps Nieuws, Weer, 70s 80s Musik
Little Hocking, OH USA, 80s. 128kbps All 80s Music - All The Time, Classic Rock
12 votes
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38 votes
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Montreal, QC Canada, 80s. 80s Hit Radio, New Wave
12 votes
Palm Springs, CA USA, Adult Contemporary . 128kbps Classic Hits From The 60s, 70s, And 80s
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30 votes
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22 votes
Berlin, Germany, 80s. 128kbps Der 80s Mix


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English Meaning
Of or pertaining to the rectum; in the region of the rectum.
- Of, relating to, or situated near the rectum.
This service is a free English - Malayalam Dictionary with English & Malayalam meaning of more than 125000 words. You can find out equivalent Malayalam meaning, definitions, Synonyms & more of any English word by using this service. The malayalam meaning is displayed with transliterated output (Manglish) as well & that will help people who doesn't know to read Malayalam language. Please support this free service by just sharing with your friends. If you find any bugs in this program please report me at email@example.com
Of or pertaining to the rectum; in the region of the rectum.
The Usage is actually taken from the Verse(s) of English+Malayalam Holy Bible.×


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Published Mar 18, 2015
If dum sum is your weekend tradition, tell us where to get the best shumai and pork buns in town.
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"…Everything on their dim sum menu is great, from har gows to sui mai, to deep fried taro balls, calamari and pork buns! Great flavor and quality, reas…"
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"…This place is always busy and excellent. With fast service, you can be out of the place in 30 minutes.…"
"…First they authentic; second, they are in Chinatown; third they are the best!…"
"…Such a great place in the Mission - good food and good prices!…"
"…can be a bit of a wait but the mango pudding is worth it…"
"…A little hole-in-the-wall that's been at the same location since the 1920s, and the best dim sum in San Francisco, hands down.…"
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Thursday, March 18, 2010
Insemination Day!!!
8 comments:
Nice they were proactive!ReplyDelete
Crossing my fingers :-).
Thanks Billy...BTW, I have been reading your blog (I started from the beginning & am only up to August, 2009 so far) but am really getting a lot of insight & inspiration from you!ReplyDelete
Thanks for the comment on my blog. I'll start reading yours from oldest first, as I have time. It's been interested reading others' experiences. Good luck!ReplyDelete
I hope the wait isn't making you too crazy! A Christmas baby would be wonderful :)ReplyDelete
I agree Mama, I have learned so much from the blogs I follow...ReplyDelete
This comment has been removed by the author.ReplyDelete
Thinking Sticky thoughts for you! I'll be reading through your two week wait....ReplyDelete
I have my first IUI next month so I'm very interested in your thoughts and will definitely be reading through all your older posts.
I am so excited for you! It sounds like it was all timed perfectly! I heard the tww is a nail biter, but hopefully it's gone by faster than you expected. Only a few more days! Good Luck!ReplyDelete


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This section contains 308 words
(approx. 1 page at 400 words per page)
In the Suburbs Summary & Study Guide Description
In the Suburbs Summary & Study Guide includes comprehensive information and analysis to help you understand the book. This study guide contains the following sections:
This detailed literature summary also contains Bibliography on In the Suburbs by Louis Simpson.
"In the Suburbs" can be considered a representative poem of Louis Simpson's, both in subject matter and style. Following in the footsteps of his literary idol, Anton Chekhov, Simpson has fashioned a career of chronicling the mundane lives of ordinary people. However, his descriptions of middle- class life are not without thorns. Undergirding his poems about suburbia and small talk lurks a pervasive sense of gloom and despair. The very collection in which the poem appears, At the End of the Open Road, published in 1963 by Wesleyan University Press, is itself an extended and complicated evaluation of American society in the middle of the twentieth century. The title is a response to Walt Whitman's vision of America as a place of endless possibility, described in his poem "Song of the Open Road." Simpson considers the country a hundred years after Whitman wrote, when its geographical, and by implication spiritual, frontiers have been exhausted. Simpson asks, what's next?
"In the Suburbs" is the second poem in the collection, following "In California," a dark piece about what happens when a dream has gone bad. "In the Suburbs" is only six lines long and comprised of just three sentences, each a separate statement about the emptiness of suburban life. Using the second person "you," Simpson pronounces both the meaninglessness of this existence and the futility of attempting to escape. In its evocation of a life that needs to be changed, it echoes both Rilke's poem "Archaic Torso of Apollo" and James Wright's poem "Lying in a Hammock on William Duffy's Farm in Pine Island, Minnesota." Statement poems like these are frequently anthologized because they are short and considered "easy" to understand. "In the Suburbs" is no exception, having appeared in a number of introductory poetry texts, including The Norton Anthology of Modern Poetry and Michael Meyer's Poetry: An Introduction.
Read more from the Study Guide
This section contains 308 words
(approx. 1 page at 400 words per page)


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Make a Donation
Index Page
About The Author
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Proverbs 10-12
Supplemental notes for the Daily Bible Study Bible Reading PlanProverbs Chapter 10
by Wayne Blank
King Solomon was one of the wisest people of Bible History until he began to disastrously (see The Division Of Israel) compromise with God's Wisdom (see Solomon's Compromise). Before his apostasy however (a lesson to everyone to never take righteous effort for granted), Solomon saw clearly how The Word of God is intricately a part of all things.
"The proverbs of Solomon.
Proverbs Chapter 11
Although the Proverbs deal very much with principles of simple "everyday life," they also consistently recognize the purpose for the physical stage of man's life - and how it can either be the end, or just the beginning, depending on one's free choice of accepting or rejecting The Lord's offer of salvation. The Proverbs are all based on obedience to at least one of the Ten Commandments (see also Do You Know Him?). Righteousness means upright before The Lord (see The Lord's Plumb Line).
"A false balance is an abomination to The Lord, but a just weight is his delight.
Proverbs Chapter 12
"Whoever loves discipline loves knowledge" (Proverbs 12:1 RSV) is obvious; discipline and disciple are from the same word which means to learn (see Disciples of Christ).
"Whoever loves discipline loves knowledge, but he who hates reproof is stupid.
Fact Finder: What does "temptation" mean?


text: LimaTek 93 Report post Posted June 1, 2016 Hello folks from Atari.io!Some of you may already know me from my previous forum post under the hardware section: http://forums.atari.io/index.php/topic/2062-limatek-1-a-new-pong-machine/ I thought I would introduce myself in a little bit more detail My name is Frank, I'm an embedded software engineer and computer engineer with a background in animation! I studied eight years at California State University Long Beach (yup... eight years - eesh!) With both of my backgrounds (artistic and technical) I like to design and create things, I've always had an affinity for technology in the 70's and 80's, and thus, retro games and computing. I've also had my work shown on television, such as Comedy Central and have worked on animated pilots (as I am an animator too!) Recently I decided to put both skill sets to work and started researching the architecture of the Atari systems and NES and came up with a pong machine prototype (as you will see in the link), and I thought to myself, "Huh! I think I may be onto something!" and decided to do some outreach regarding the pong machine or 'LimaTek 1' console to see what people thought. So far it's been very positive and hopefully I'd like to eventually sell these little guys as kits or completed systems - that would be awesome. Anyway - that's me! Just an artsy/eccentric technical guy who absolutely loves retro gaming! The Atari.io community has just been so awesome and I'd like to thank you all for the warm introductions and support 11 greenween, Clint Thompson, Ballblaɀer and 8 others reacted to this Share this post Link to post Share on other sites


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Big package wheels for all US-trucks
August 1, 2016 | Tuning | No Comments|
For all trucks!
This mod gives the player neovobrazimuyu imagination when choosing wheels and accesories to them, for more comfortable transportation of goods in his truck.
Wheels glow in the dark in zavisimomti from the chosen color of tires.
What is done:
– Wheels Collected in one large package.
– Mnogokratno Checked for all positions.
– Added Missing files.
– Added Icons in the gallery, the choice of tuning.
Version ATS: 1.3
Credits: The original concept of these wheels belong – Kyto! Wheels with tires for all trucks (Anatoly 7469) All other work on the package, assembly, debugging and adding the missing files and icons: losevo58 Manifesto: losevo58


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When we lived in Idaho and all our family would get together for Sunday dinner we would sit at the computer for at least an hour after dinner and watch video after video. Totally awesome. Since we don't get together with anyone to show off our great finds I have decided to make a verbal vomit section on my blog. Now here's the great thing. If you watch these and think, "I have something that is equally as awesome, or maybe even more awesome," send me a link and then I'll get to laugh and perhaps everyone else will too. Hope you enjoy the videos today.
This little baby is great. Noah rocks the song and I may like it better than the origional.
Now this is for teachers, and if you have a sense of humor. It was teachers appreciation week last week and this came out on Jimmy Kimmel. Now I dont usually feel this way, so please dont take offense and laugh.


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We'll help you decide what the best organization is for you.
We'll teach your company how to accurately keep records.
Our accountants are board certified and stay current with continuous certifications and seminars to ensure we are up-to-date on all of the newest information, laws, regulations, and techniques in our field.
Con nuestro servicio en su Idioma usted siempre estara seguro de lo que esta pasando con sus impuestos
Our accounting firm is here to help you and your business. From any accounting needs you might have to tax planning for your upcoming goals and dreams, we're here to help you achieve your dreams.
Aqui siempre estamos para ayurdales con sus cuentas y hacer sus taxes 100% confiables y que ustedes entiendan el processo de una manera sencilla
Need assistance with your tax planning? Don't we all. We've got you and your business goals and objectives in mind. You and all your tax planning needs are in expert hands. Leave it to us.
Si necesita ayuda para planear sus taxes para hoy o el futuro aqui estamos para ayudarle en su Idioma para explicarle de la manera mas sencilla para usted y familia
Plan for your taxes before your business is off the ground.
We'll help you apply for all the tax IDs and forms you need.
Copyright © El Jarocho Income Tax.


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Saturday, November 5, 2011
Havasupai Falls, Grand Canyon, AZ
Posted by Kristen at 8:13 PM
1 comment:
I can't believe you did Havasupai WITH kids!!! I remember when I did it my senior year of college and I noticed there were no kids or old people at the campground because of the hike in and out. Did you really hike the whole way, and did Cole do it himself? I am just so impressed. AND...it looks like you went down to Mooney Falls which is just ridiculous and I love the picture of you guys with the sign up in the air! Was the way down to Mooney Falls like sliding down poles on the exposed edge of the cliff and lowering yourself into caves where you couldn't see the bottom? I just remember being so scared! Great job, how fun...makes me want to go back!ReplyDelete
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Louvain Finance (LFIN), the finance center of the Université catholique de Louvain (UCL) has several openings as Ph.D. student (full time research position leading to a Ph.D. degree, with duties on financial data support) both at the campuses of Louvain la Neuve.
Candidate Profile
- Having a Master degree (min 90 ECTS) in finance, management, economics or any related fields by July 2018. For excellent candidates, September 2018 would be tolerated;
- average grades of 80% or more;
- excellent knowledge of English;
- Strong empirical and/or quantitative skills
- good knowledge or willingness to learn financial datasets such as Bloomberg, Bureau van Dijk, etc.
Position duties
- You will contribute to the development of the Financial data centre of Louvain Finance, in particular by being a point of contact for datasets such as Bloomberg.
- You will contribute to the development of the Louvain finance team, by for example managing the organization of important events or seminars
- You will participate to the research events organized by Louvain Finance
- You will perform a Ph.D. in Economics and management (with orientation finance)
Research areas
You will be performing a Ph.D. in the following areas (indicative):
- Macro Finance, Government and corporate term structure of interest rates, monetary policy
- Big data in finance and Mixed frequency analysis
We offer:
- competitive salary (1800 Euro net a month + social insurance) and a personal yearly budget
- outstanding, dynamic, international, and multidisciplinary research environment, at the highest level according to European standards
- possibility to co-operate with outstanding international scholars
- a contract of one year, which can be extended to 4 after positive evaluation
- possibility to spend part of the Ph.D. abroad (in agreement with the promoter)
- a full-time position starting in September 2018 (in exceptional cases earlier)
Recently graduated Ph.D. students from Louvain Finance are now working at National Banks (National Bank of Belgium and National Bank of Luxembourg), private institutions (such as KPMG) and universities (Rotterdam School of Management or IESEG business school). Outstanding candidates will be encouraged and helped to apply for additional funding in order to get scholarships.
Application instructions
- Send an email to firstname.lastname@example.com with object the vacancy reference;
- Include the PDF of your transcript of the grades obtained so far (even if you do not have the transcript from your January session it is fine);
- Include your CV with at least one contact reference and a motivation letter
- Deadline: March the 15th. The positions are open until they are filled, hence apply ASAP if interested.
- Only shortlisted candidates will be contacted.
The University
UCL is a world-class university offering the opportunity of cross-disciplinary research and teaching collaborations between its various research groups. Since its creation in 1425, UCL has both reached out to the world and invited it in. Through student exchanges, hosting international professors and researchers, interuniversity partnerships, participation in European and global networks, and a long tradition of cooperation in international development, UCL has exerted influence throughout the world for nearly 600 years. Today, UCL has 7 campuses in the French-speaking Community of Belgium. Its main campus is located in the city of Louvain-la-Neuve. The town offers a unique modern living environment inside a naturally preserved neighbourhood, conveniently connected at the heart of Europe.
Research environment
The selected candidates will be part of the institute for multidisciplinary research in quantitative modelling and analysis (IMMAQ). The IMMAQ was founded in 2009 as a research institute which combines fundamental and applied research in the fields of economics, statistics, finance, econometrics and operational research. It is composed by more than 200 researchers and consultants, of about 15 different nationalities. The common denominator of the IMMAQ is the use of a coherent set of tools and methods for quantitative modelling and analysis. The IMMAQ is made of five different entities: the CORE (Center for Operations Research and Econometrics), the IRES (Institute of Economic and Social Research), the ISBA (Institute of Statistics, Biostatistics and Actuarial Sciences), LFIN (Louvain Finance) and the CMCS (Statistical Methodology and Computing Service). These entities have strong international spirit and some of them have proven history of academic excellence. For example, the CORE has celebrated its 50th anniversary in 2016 and IRES will celebrate his 90th anniversary in 2018. Each of these entities are geographically very close, favouring the interaction among research members.
The successful candidates will be affiliated to Louvain Finance, the finance team of the IMMAQ. This is the youngest team of the IMMAQ, that has decided to extend its research excellences to the finance research. Louvain Finance is one of the biggest and most dynamic Belgian financial centres. It closely cooperates with many Belgian and European research entities such as the National Bank of Belgium and the European Central Bank. Thanks to these collaborations Louvain Finance has a prestigious seminar series, Ph.D. courses, and conferences, and organizes the European edition of the So.Fi.E. training school. This allows the team members to exchange research ideas and cooperates with world class researchers in finance. In the last years Louvain Finance has welcomed John Campbell, Viral Acharya, Frank Diebold, David Lando, John Cochrane, Kenneth Singleton, Marco Pagano, Allan Timmermann, Eric Ghysels, Lars Peter Hansen, Murillo Campello and Geert Bekaert among others. In the academic year 2017-2018, scholars such as Andrew Lo and Darrel Duffie will intervene in events co-organized by Louvain Finance.
Team members of Louvain Finance regularly cooperate with scholars such as Eric Ghysels, Murillo Campello, Damiano Brigo, etc.
Phd candidate
Louvain-la-Neuve , Belgium


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Attic Storage.
Central Air Conditioning, Cook Top Range, Dishwasher, Disposal, Dryer, Oven, Refrigerator, Washer & Dryer.
Driveway, Off Street, On Street.
To Be Built Berkley 4 Bed/3.5 Bath Model by Avid Award Winning HomeBuilder Mitchell & Best at Highland Reserve in Fulton. 4553 finished sq. ft. & open floor plan! Private owner's suite with spa bathroom. Lower level rec room & more! Photos not actual home. Stunning countryside views, rural setting & close to everything! Discounts & incentives valid with use of seller preferred lender/title co.
|HOWARD COUNTY PUBLIC SCHOOL SYSTEM School District
|POINTERS RUN Elementary , ATHOLTON High , Junior High , LIME KILN Middle School
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What is Xanax (Zanex)?
Xanax (Zanex) tablets are used for the management of anxiety disorder or the short-term relief of symptoms of anxiety. Anxiety or tension associated with the stress of everyday life usually does not require treatment with Xanax (Zanex). Anxiety associated with depression is responsive to Xanax (Zanex).
Xanax (Zanex) is also used for the treatment of panic disorder, with or without agoraphobia.
Xanax (Zanex) tablets are contraindicated in patients with known sensitivity to alprazolam or other benzodiazepines. Xanax (Zanex) may be used in patients with open angle glaucoma who are receiving appropriate therapy, but is contraindicated in patients with acute narrow angle glaucoma.
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Further information
Always consult your healthcare provider to ensure the information displayed on this page applies to your personal circumstances.