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The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: DH SK BJA. Performed the experiments: DH SK DvD HF RS NB JM MC. Contributed reagents/materials/analysis tools: HS. Wrote the paper: DH SK MC BJA. Edited the paper: HF. Provided technical assistance: WY.
START-dependent transcription in Saccharomyces cerevisiae is regulated by two transcription factors SBF and MBF, whose activity is controlled by the binding of the repressor Whi5. Phosphorylation and removal of Whi5 by the cyclin-dependent kinase (CDK) Cln3-Cdc28 alleviates the Whi5-dependent repression on SBF and MBF, initiating entry into a new cell cycle. This Whi5-SBF/MBF transcriptional circuit is analogous to the regulatory pathway in mammalian cells that features the E2F family of G1 transcription factors and the retinoblastoma tumor suppressor protein (Rb). Here we describe genetic and biochemical evidence for the involvement of another CDK, Pcl-Pho85, in regulating G1 transcription, via phosphorylation and inhibition of Whi5. We show that a strain deleted for both PHO85 and CLN3 has a slow growth phenotype, a G1 delay, and is severely compromised for SBF-dependent reporter gene expression, yet all of these defects are alleviated by deletion of WHI5. Our biochemical and genetic tests suggest Whi5 mediates repression in part through interaction with two histone deacetylases (HDACs), Hos3 and Rpd3. In a manner analogous to cyclin D/CDK4/6, which phosphorylates Rb in mammalian cells disrupting its association with HDACs, phosphorylation by the early G1 CDKs Cln3-Cdc28 and Pcl9-Pho85 inhibits association of Whi5 with the HDACs. Contributions from multiple CDKs may provide the precision and accuracy necessary to activate G1 transcription when both internal and external cues are optimal.
Eukaryotic cells grow and divide by progressing through carefully orchestrated stages of the cell cycle characterized by stage-specific patterns of gene expression, DNA replication, and scission. How stage-specific gene expression is coordinated with cell cycle progression is only partially understood. The phase known as G1 marks the initiation of the cell cycle (called START in yeast) and involves the coordinated expression of more than 200 genes regulated by two transcription factors, SBF and MBF. The activity of SBF and MBF is restrained by binding of the repressor protein Whi5 to the two transcription factors early in G1 phase. Phosphorylation of Whi5 by G1-specific forms of the cyclin-dependent kinase (CDK) Cdc28 promotes dissociation of Whi5 from SBF and its export from the nucleus; this, in turn, releases SBF to activate G1-specific transcription. This G1 transcriptional circuit is analogous to that defined in mammals by the E2F family of transcription factors and the retinoblastoma (Rb) tumor suppressor protein. Rb further contributes to the repression of G1-specific transcription in mammals by recruiting histone deacetylases (HDACs), which are chromatin remodeling complexes that regulate promoter accessibility. Here, we show that regulation of G1-specific transcription in yeast also involves repressor-mediated recruitment of HDACs. We demonstrate that repression by Whi5 is modulated by both Cln-Cdc28 and a second G1-specific CDK, Pcl-Pho85, and further show that both kinases regulate the interaction of Whi5 with HDACs. We propose that regulation of the repressor by more than one G1-specific CDK ensures definitive inactivation of Whi5, a critical event for appropriate cell cycle initiation.
Cyclin-dependent protein kinases (CDKs) act as molecular machines that drive cell division, and cell cycle progression is dependent on oscillation between CDK active and inactive states. In S. cerevisiae, the CDK Cdc28 associates with nine different cyclin subunits to promote and coordinate a complex network of events necessary for smooth cell cycle transitions [1]. Irreversible commitment to a new round of cell division occurs toward the end of G1 phase in a process called Start in yeast. The analogous regulatory event is called the restriction point in mammalian cells [2],[3]. In yeast, three G1 cyclins, Cln1, Cln2, and Cln3, associate with Cdc28 to initiate events required for progression through Start. Passage through Start catalyzes a defined molecular program that initiates DNA replication, budding, spindle maturation, and chromosome segregation [3].
One key feature of Start in yeast, and G1 progression in other eukaryotic cells, is the induction of a transcriptional program involving over 200 genes, including those encoding the G1 (CLN1, CLN2, PCL1, and PCL2) and B-type cyclins (CLB5 and CLB6) [4],[5]. G1/S phase-specific transcription depends on two heterodimeric transcription factors called SBF (Swi4,6 cell cycle box binding factor) and MBF (MluI binding factor). These complexes share a common regulatory subunit, Swi6, which is tethered to DNA via its binding partners, encoded by SWI4 in SBF and MBP1 in MBF [5]. At the well-studied HO locus, binding of the zinc-finger transcription factor Swi5 is followed by recruitment of the Swi/Snf chromatin remodeling complex and the SAGA histone acetyltransferase complex [6]–[8]. These events set the stage for SBF binding and recruitment of the SRB/mediator complex [6]. Importantly, subsequent recruitment of PolII and transcription initiation is dependent on CDK activity [9]. Although any one of the three G1 cyclins is sufficient to drive Start, genetic studies indicate a key role for Cln3-Cdc28 in activating SBF and MBF. At the same time Cln1 and Cln2 are required for the proper execution of other Start-related events such as budding and DNA synthesis. Cells lacking CLN3 are large and severely delayed for onset of G1/S transcription, while ectopic induction of CLN3 in small G1 cells activates transcription and accelerates passage through Start [10].
Start does not occur until cells have passed a critical cell size threshold, a barrier modulated by nutrient conditions, among other regulatory inputs [11]. A systematic analysis of cell size profiles for the entire set of yeast deletion mutants uncovered many new regulators of Start including Whi5 and implicated it as an inhibitor of G1/S-specific transcription [12],[13]. Whi5 occupies specific promoters early in G1 phase when CDK activity is low. However, Cdc28-dependent phosphorylation of both Whi5 and SBF/MBF late in G1 phase results in disengagement from SBF and nuclear export of Whi5 consequently leading to activation of SBF- and MBF-dependent transcription [12],[13].
Whi5 is proposed to function in a manner analogous to the well-characterized Rb family proteins in metazoans. E2F, the functional analog of SBF/MBF, regulates G1-specific gene expression required for passage through the restriction point [14]. E2F activity is restricted to late G1 phase because of inhibition by the retinoblastoma protein (Rb). Rb associates with E2F to restrain its activity until late G1, at which point stepwise phosphorylation of Rb by two CDKs, cyclin D-Cdk4/6 and cyclin E-Cdk2, causes the dissociation of Rb from E2F [15]. This process appears to be regulated by a positive feedback loop in which Rb phosphorylation by cyclinE-Cdk2 leads to further dissociation of Rb from promoters and enhancement of G1-transcription. At the molecular level, Rb interacts with both E2F and chromatin remodeling complexes such as histone deacetylases (HDACs) [16]–[18]. Rb appears to repress transcription through at least three distinct mechanisms: (1) Rb can bind directly to the activation domain of E2F thereby blocking its activity [19]; (2) recruitment of Rb can block the assembly of the pre-initiation complex thus inhibiting the activity of adjacent transcription factors [20] and; (3) Rb can recruit remodelers such as HDAC1 and BRG1 to modify chromatin structure. BRG1 is one of the human Swi/Snf adenosine triphosphatases (ATPases) that remodel nucleosomes by utilizing ATP to weaken the interactions between DNA and histones [16],[17]. The specific roles of different CDKs in regulating E2F-Rb function, however, remain unclear.
Another yeast CDK Pho85 was originally discovered as a regulator of phosphate metabolism, but has since been shown to play numerous roles in the regulation of cell division and other processes [21]–[23]. Ten genes encoding Pho85 cyclins (Pcls) have been identified and they appear to dictate substrate and functional specificity of Pho85 [24]–[26]. Expression of three Pcls, PCL1, PCL2, and PCL9, is restricted to G1 phase of the cell cycle [25]. Specifically, PCL9 expression peaks early in G1, whereas maximal expression of PCL1 and PCL2 is observed at Start and is dependent largely on SBF [27]–[29]. Although Pho85 is not essential for viability, it is required for cell cycle progression in the absence of the Cdc28 cyclins CLN1 and CLN2 [29], and its absence leads to catastrophic morphogenic changes that culminate in a G2 arrest [30]. Consistent with this observation, inactivation of both Cdc28 and Pho85 CDKs specifically inhibits expression of G1-regulated genes involved in polarized growth [31].
As noted above, transcriptional repression by Rb has been linked to its interaction with histone modification complexes, in particular HDACs. Recent work highlights the importance of post-translational modifications of histone and nucleosome positioning in regulating gene expression [32],[33]. Histone acetylation neutralizes the positive charge generated by lysine-rich regions present in the N-terminal tails of histones, thereby disrupting nucleosome structure and increasing promoter accessibility [34]. As a result, many transcription activators have been shown to interact with histone acetyltransferases, whereas transcriptional repressors often associate with HDACs to promote nucleosome formation to occlude transcription factor binding [35],[36]. Histone deacetylation in S. cerevisae is mediated by a family of HDACs including Rpd3, Hda1, Hda2, Hos1, Hos2, and Hos3 [37]. Similar to their mammalian counterparts, some yeast HDACs are recruited to promoters by sequence-specific regulatory factors to repress gene expression. For example, the Rpd3 deacetylase complex is recruited to the INO1 promoter by the DNA binding protein Ume6 [35],[38]–[40]. This recruitment results in local histone deacetylation and repression of INO1 gene expression [41]. Hda1 is another example of this type of regulation, and is recruited to its target promoters by the repressor Tup1 [42].
In this study, we provide detailed mechanistic insights into Whi5-dependent regulation of G1-specific transcription and cell cycle progression. Specifically, we identify Whi5, to our knowledge, as the first demonstrated physiological substrate for the G1-specific Pcl9-Pho85 CDK and provide genetic and biochemical evidence supporting a direct role for Pho85 at Start. Furthermore, we show that in a manner similar to Rb in mammalian cells, Whi5-mediated repression involves the HDACs Rpd3 and Hos3. Dual phosphorylation of Whi5 by Cdc28 and Pho85 inhibits Whi5 activity in at least two ways. Both kinases appear to regulate interaction of Whi5 with different HDACs, whereas Cdc28 is also involved in disrupting Whi5 association with SBF and promoting its nuclear export [12],[13]. G1-specific CDKs thus are specialized to regulate different aspects of the same critical cell cycle event—inhibition of Whi5—resulting in definitive inactivation of the Whi5 repressor.
Synthetic dosage lethality (SDL) is a genetic assay that is based on the rationale that increasing levels of a protein may have no effect on the growth of an otherwise wild-type (wt) strain but may cause a measurable phenotype—such as lethality—in a mutant strain with reduced activity of an interacting protein [43],[44]. Previous studies suggest that SDL can be used effectively to identify novel enzyme targets and a genome-wide SDL screen in cells lacking Pho85 identified known targets of the CDK [23]. In addition to known substrates, several putative Pho85 targets were also identified, including the G1-specific transcription repressor Whi5 [24]. To further explore the role of Pho85 in G1 phase-specific transcription we examined the WHI5-PHO85 SDL interaction in greater detail. As noted previously, Pho85 activity and substrate specificity depends on its interaction with cyclin subunits known as Pcls [25]. To implicate specific Pcl-Pho85 complexes in modulating Whi5 function we examined the effects of WHI5 overexpression in cells lacking different Pcls (Figure 1). Similar to effects observed in cln3Δ and cln1Δ cln2Δ mutants [13], overexpression of WHI5 resulted in growth inhibition of pcl1Δ and pcl9Δ deletion strains and this growth defect was exacerbated in a pcl1Δ pcl9Δ double mutant (Figure 1). Unlike pcl1Δ or pcl9Δ mutants, strains lacking PCL2 or PHO80 cyclins were not adversely affected by increased WHI5 dosage suggesting that the WHI5-PHO85 genetic interaction is dependent on the PCL1,2 cyclin subfamily and more specifically on PCL1 and PCL9 (Figure 1). This observation is consistent with the fact that Pcl1 and Pcl9 (but not Pcl2) are the two G1-specific cyclins that localize to the nucleus [30],[45]. The growth phenotype seen in the plating assay was confirmed by measuring growth rates in liquid culture (unpublished data). On the basis of these results, Pcl1/9-Pho85 may contribute to Whi5 regulation in a manner similar to Cln3-Cdc28.
The genetic interactions described above suggest Whi5 may be a direct target of Pho85. Evidence supporting this hypothesis is provided by protein microarray assays where Whi5 is phosphorylated in vitro by Pcl1-Pho85 [46]. We characterized the Whi5-Pho85 interaction biochemically by performing in vitro kinase assays using recombinant Pcl-Pho85 CDK complexes and purified Whi5 as substrate (Figure 2A). Incorporation of [32P] into Whi5 was not detected in the absence of CDKs (Figure 2A, lane 4). However, Whi5 phosphorylation was observed in the presence of Pcl1- and Pcl9-Pho85 (Figure 2A, lanes 1,2) and when compared to Cln2-Cdc28 kinase activity, Pho85 and Cdc28 phosphorylated Whi5 at similar levels in vitro (Figure 2A, lanes 1–3).
Previous studies revealed multiple Whi5 slow-migrating isoforms that correlate with its phosphorylation state [12],[47]. We examined the effect of various cyclin or CDK mutants on Whi5 mobility (Figure 2B). Because of genetic redundancy of Pcl cyclins [27], we were unable to reproducibly detect changes in Whi5 phosphoforms in cyclin mutant strains. Therefore, a Pho85 mutant was used to asses the phosphorylation status of Whi5. Consistent with previous findings [12],[13], slow migrating Whi5 isoforms present in asynchronous wt extracts (Figure 2B, lane 1) were modestly reduced in cells lacking CLN3 (Figure 2B, lane 7) and completely absent in a cln1Δ cln2Δ double mutant (Figure 2B, lane 6), confirming that Whi5 phosphorylation depends on Cln-Cdc28 kinase complexes. Consistent with our SDL results and in vitro kinase assays, we observed a significant reduction in Whi5 mobility in extracts from a pho85 mutant strain (Figure 2B, lane 2). Thus, similar to Cdc28, phosphorylation of Whi5 also depends on Pho85 in vivo.
To determine if Whi5 physically associates with Pho85 in yeast, we first assayed Whi5FLAG immune complexes for kinase activity. A robust autophosphorylation activity was recovered from Whi5FLAG immunoprecipitates derived from wt cell extracts when radiolabeled ATP was added to the immunoprecipitated sample (Figure 2C, lane 2). This activity was partially dependent on both CDC28 and PHO85 (Figure 2C, lanes 3–5). We also confirmed a physical interaction between Whi5 and Pcls using a co-immunoprecipitation assay (Figure 2D). Immunoprecipitation of Whi5MYC from epitope-tagged cyclin extracts revealed a specific association between Pcl9 and Whi5 (Figure 2D, lane 4). We failed to reproducibly detect a physical interaction between Whi5 and Pcl1 (Figure 2D, lane 2) suggesting that Pcl9-Pho85 is the primary Whi5 CDK. Taken together, the phosphorylation and co-immunoprecipitation assays strongly suggest that, in addition to Cdc28, Pho85 also phosphorylates Whi5. Furthermore these results identify Whi5 as the first reported substrate for Pcl9-Pho85, one of two Pcls whose activity is restricted to early G1 phase.
Whi5 associates indirectly with G1 phase-regulated promoters through interaction with SBF and MBF. Interactions with these transcription factors and subsequent promoter binding are disrupted by CDK-dependent phosphorylation [12],[13]. Because Whi5 appears to be a Pho85 substrate, we assessed the occupancy of SBF promoters by Pcl9. To date, cyclins have not been detected at yeast promoters. Pcl9 is normally an unstable short-lived protein [27]; however, similar to other cyclins, Pcl9 turnover appears to be catalyzed in part by its cognate CDK, Pho85 (Figure 3A) [48]. Therefore, to test Pcl9 promoter localization in a more sensitive genetic background, we performed ChIP (Chromatin immunoprecipitation) experiments in a pho85Δ strain (Figure 3B). The highest levels of CLN2 promoter DNA were detected in Pcl9MYC immune complexes 30 min following release from a metaphase-anaphase arrest (Figure 3B). The Pcl9-chromatin association was no longer detectable 45 min after GAL-CDC20 induction indicating that the interaction is short-lived and transient as predicted for a regulator of Start. The association was Whi5-dependent since Pcl9 was not detected at the CLN2 promoter in a strain lacking Whi5 (Figure 3C). The localization of Pcl9 to CLN2, a G1 promoter, is consistent with a direct role for Pcl9-Pho85 in regulating G1 transcription.
As mentioned above, cln3Δ mutants arrest in G1 phase as large unbudded cells in response to increased WHI5 dosage, indicating that Whi5 is a dose-dependent regulator of Start. Therefore, if Pho85 and Cdc28 function analogously to inhibit Whi5 activity, we predict that elevated Pho85 kinase activity would antagonize the toxic effects of WHI5 overexpression and suppress the growth defects observed in a cln3Δ mutant. To test this prediction, high copy plasmids expressing PCL1, PCL2, PCL9, or PHO80 were introduced into a cln3Δ strain expressing WHI5 from a conditional MET25 promoter (Figure 4A). Plasmid-based expression of Pcls and Whi5 was confirmed by immunoblotting (Figure S1). Induction of WHI5 expression in a cln3Δ mutant resulted in cell death whereas overexpression of PCL1 or PCL9 partially suppressed this toxicity and restored growth (Figure 4A). Consistent with results from SDL analyses (Figure 1), this suppression was specific to PCL1 and PCL9 since neither PCL2 nor PHO80 were able to function effectively in the assay (Figure 4A). Furthermore, PCL1/9-mediated suppression was dependent on phosphorylation since growth of a cln3Δ mutant expressing a nonphosphorylatable form of WHI5 (Whi512A) [13] could not be restored (Figure 4A). These genetic results corroborate the biochemical evidence that Pcl-Pho85 regulates Whi5 activity through phosphorylation.
Given its effect on WHI5 overexpression, we next examined PCL effects on other CLN3-associated phenotypes. CLN3 is required to activate G1-specific transcription once cells have achieved a critical size [49]–[51]. A cln3Δ mutant exhibits a large cell size phenotype because of its inability to inhibit Whi5 and activate Start-specific transcription [12],[13]. Ectopic expression of PCL1 or PCL9 reduced cln3Δ cell size to an intermediate level between that of wt and cln3Δ cells (Figure 4B). Conversely, deletion of PCL9, PCL1, and the partially redundant cyclin PCL2 resulted in a cell size increase (Figure 4C). These results suggest that Pcl-Pho85 and Cln3-Cdc28 share a common role in cell cycle progression to regulate Whi5 activity and promote passage through Start.
To determine if Pcl-Pho85 and Cln3-Cdc28 might function in parallel to regulate Start, we first tried to test whether pcl9Δ cln3Δ or pcl1Δ pcl9Δ cln3Δ strains showed any synthetic growth defects. As expected, no growth defects were observed, probably because of the redundant effects of other Pcls [27]. Unlike the Cdc28 cyclins, which shows distinct cell cycle expression patterns, most Pcls are expressed at all cell cycle stages [25]. We then examined the phenotype of a pho85Δ cln3Δ double mutant. Cells lacking cln3Δ are larger than wt cells but do not display overt defects in growth rate while pho85Δ mutants are slow growing (Figure 5A). However, pho85Δcln3Δ double mutants exhibited a more pronounced growth defect compared to single mutants and analysis of DNA content revealed that the pho85Δ cln3Δ double mutant cells accumulated in G1 phase with predominantly unreplicated DNA (Figure 5A). Importantly, deleting WHI5 overcame both the cell cycle progression and growth defects observed in the absence of both CLN3 and PHO85. Notably, a pho85Δ cln3Δ whi5Δ triple mutant exhibited a growth rate similar to a cln3Δ single mutant indicating that Pcl-Pho85 and Cln3-Cdc28 function in separate yet converging pathways to regulate Whi5 function and, by extension, G1 cell cycle progression (Figure 5A). These observations also hold true under liquid growth conditions as shown. WHI5-dependent suppression appears to be specific to the pho85Δ cln3Δ phenotype because WHI5 deletion was unable to rescue 53 additional synthetic lethal interactions involving PHO85 (Table S1; D.Q. Huang and B.J. Andrews, unpublished data).
Given that Whi5 represses SBF- and MBF-specific transcription, we asked whether PHO85 affects SBF-driven reporter gene expression. A reporter gene consisting of tandem SCB consensus element repeats fused upstream of the HIS3 coding region was constructed and integrated into wt, cln3Δ, and pho85Δ strains. Previous work has shown that this reporter provides a highly specific read-out for SBF-dependent transcription [13],[52]. Growth on medium lacking histidine supplemented with 3-aminotriazole (3-AT) was used to assess SBF transcriptional activity (Figure 5B). Even though cells lacking PHO85 were moderately sensitive to higher concentration (5 mM) of 3-AT (unpublished data), both cln3Δ and pho85Δ mutants showed no growth in media containing 30 mM 3-AT indicating that SBF transcription is impaired in these mutants, whereas growth of wt cells was unaffected [13]. Furthermore, defects in SCB-driven gene expression were more pronounced in the pho85Δ cln3Δ double mutant (at 10 mM 3-AT, Figure 5B). Consistent with the genetic interactions described above (Figure 5A), SBF-dependent reporter activity was restored in pho85Δ cln3Δ mutants when WHI5 was deleted (Figure 5B). However, WHI5 deletion only partially rescued the growth defect in pho85Δ cells at 30 mM of 3-AT (Figure 5B). The Whi5-independent 3-AT sensitivity of pho85Δ cells may be due to unregulated Gcn4 in the absence of PHO85, since GCN4 is induced by 3-AT and Pho85 has been shown to regulate Gcn4 stability [53],[54]. Nonetheless, these data suggest that, like Cln3-Cdc28, Pcl-Pho85 modulates SBF activity through Whi5.
We next interrogated the effects of CDK activity on Whi5-mediated transcriptional repression (Figure 6). A construct expressing a LexA DNA binding domain fused to WHI5 was introduced into a strain harboring a LacZ reporter gene containing LexA binding sites in its promoter (Figure 6). Consistent with its role as a negative regulator of G1-specific transcription, a ~10-fold reduction in β-galactosidase activity was observed in cells expressing the LexA-Whi5 fusion protein compared to a vector control (Figure 6). Overexpression of PCL9, CLN3, or CLN2 restored LacZ expression to intermediate levels indicating that activation of either CDC28 or PHO85 was capable of antagonizing Whi5 function in this assay (Figure 6). Consistent with suppression of WHI5-mediated growth defects (Figure 4), inhibition of Whi5 activity was dependent on phosphorylation since LacZ expression could not be restored in cells harboring an unphosphorylatable LexA-Whi512A fusion protein (Figure 6).
Cln2-Cdc28 activity was previously shown to disrupt recombinant Whi5-SBF complexes in vitro [13], but Cln3-Cdc28 and Pho85 kinases had not been assessed for this activity. A preassembled recombinant Whi5-Swi4FLAG-Swi6 complex bound to anti-FLAG resin was incubated with purified kinases in the presence of radiolabeled ATP and separated into soluble (Figure 7B, labeled "S") and bound fractions (Figure 7B, labeled "B"). Equivalent amounts of kinase were approximated on the basis of in vitro kinase activity (Figure 7A, and Materials and Methods). As expected, Cln2-Cdc28 phosphorylation caused most of the SBF-bound Whi5 to be released into the soluble fraction (Figure 7B, lanes 3 and 4). In contrast, we failed to observe dissociation of Whi5 from SBF in the presence of Cln3- or Pcl9-CDK complexes (Figure 7B, lanes 5–10). In addition to negatively regulating the interaction of Whi5 with SBF, Cdc28 also controls its localization [13]. Unlike Cln-Cdc28 phosphorylation, which promotes Whi5 export from the nucleus, deletion of PHO85 did not dramatically affect the subcellular localization of Whi5 (Figure 7C). Together, these results suggest that Pho85 must regulate Whi5 function through alternate mechanisms.
We next explored what additional mechanism might explain Pcl- and Cln3-mediated regulation of Whi5 activity. Functional conservation clearly extends to Whi5 and its metazoan analogue Rb [14]. Since Rb represses transcription, in part, through recruitment of HDACs, we used a batch affinity chromatography assay to test for physical interactions between a Whi5GST ligand and tandem affinity tagged HDACs (Figure 8A). Specific interactions between Whi5 and Hos3, Rpd3, and, to a lesser extent, Hos1 were identified (Figure 8A, lanes 1, 5, 13) suggesting that, like Rb, Whi5-dependent transcriptional repression involves recruitment of HDACs. This observation is consistent with previous work that detected Rpd3 at the PCL1 promoter using a ChIP assay [55]. Furthermore, HOS3 and RPD3 were required for WHI5 dose-dependent effects on cell size. Like wt cells, strains lacking either HOS3 (Figure 8B, panel 1) or RPD3 (Figure 8B, panel 2) also exhibited a dose-dependent increase in cell size in response to WHI5 overexpression. However, additional cell size effects were not observed in strains lacking both HDACs, suggesting that Hos3 and Rpd3 regulate Whi5 function synergistically (Figure 8B, panel 3).
If HDACs are required for Whi5 function, then strains lacking HDAC function should be resistant to toxic effects associated with WHI5 overexpression. Consistent with this prediction, the growth defect caused by WHI5 overproduction in a cln3Δ was alleviated by the deletion of HOS3 and RPD3 (Figure 9A). Deletion of HOS3 alone rescued WHI5 toxicity in a pho85Δ strain while a cln3Δ mutant required deletion of both HOS3 and RPD3 in order to tolerate increased dosage of WHI5 (Figure 9A).
Given that Whi5 appears to be acting through HDACs, we predicted that deletion of HOS3 and RPD3 should phenocopy those genetic interactions seen in whi5Δ mutants. We first tested various HDAC deletion strains for suppression of the slow growth phenotype of a pho85Δcln3Δ mutant. As for WHI5, deletion of HOS3 and RPD3 partially suppressed the growth defect seen in the pho85Δcln3Δ double mutant strain (Figure 9B). Suppression was specific to HOS3 and RPD3 because deletion of other HDACs showed no suppression, and the growth rate of the pho85Δcln3Δhos3Δ strain was not improved by subsequent deletion of RPD3 and vice versa (Figure 9B).
We next asked if deletion of HDACs might overcome the Start arrest seen in cells lacking both CLN3 and BCK2, another regulator of G1 transcription that functions in parallel with CLN3 [56]. A cln3Δbck2Δwhi5Δ triple mutant grows as vigorously as wt, placing WHI5 downstream of both upstream activators of G1 transcription [13]. Interestingly, deletion of RPD3 partially restored growth in the cln3Δbck2Δ strain providing further evidence for an HDAC requirement in Whi5-mediated transcriptional repression (Figure 9C). Neither subsequent deletion of HOS3 nor deletion of other HDACs affected growth appreciably (Figure 9C). We also employed the SCB-HIS3 assays used above to explore SBF-driven reporter gene expression in the HDAC mutants (Figure 10). As expected, deletion of RPD3 rescued the growth defects of cln3Δ SCB-HIS3 cells in the presence of both 10 mM and 30 mM of 3-AT, whereas HOS3 gene knockout had a marginal but additive effect. In contrast, the growth of pho85Δ cells was slightly rescued by deletion of HOS3 but not RPD3 providing further evidence for Pho85 acting specifically through Hos3. Because of difficulties in detecting HDACs at promoters, we were unable to confirm these observations in vivo.
We also performed co-immunoprecipitation assays using affinity tagged RPD3 and HOS3 strains and observed an obvious decrease in Rpd3 and Hos3 in Whi5 precipitates from strains harboring increased levels of Pcl9, Cln2, or Cln3 cyclins (Figure 11A and 11B). Together, our genetic and biochemical results suggest that Pho85 may preferentially influence Whi5-Hos3 activity, whereas Cln3-Cdc28 is required for inhibition of both Rpd3 and Hos3.
Whi5 is a critical cell cycle regulator that links CDK activity in G1 phase to the broad transcriptional program that accompanies commitment to cell division. We provide substantial evidence that the multifunctional Pho85 CDK is an important regulator of Whi5 activity and G1 phase-specific transcription including: (1) Whi5 is phosphorylated and antagonized by Pho85 and is the first reported substrate for the G1-specific CDK complex, Pcl9-Pho85; (2) the activity of an SBF-dependent promoter is influenced by PHO85; (3) the Pcl9 cyclin binds to SBF-regulated promoters; (4) the repressor function of Whi5 is mediated through the HDACs Hos3 and Rpd3; and (5) HDAC-Whi5 association is regulated by G1-specific forms of both the Pho85 and Cdc28 CDKs. We therefore conclude that timely and efficient release from Whi5 inhibition and subsequent G1/S cell cycle progression requires the concerted activity of both Cdc28 and Pho85.
Several lines of evidence point to common roles for Pho85 and Cdc28. For example, a burst of both G1-specific Cdc28 and Pho85 activity is essential for cellular morphogenesis. A strain lacking the G1-specific cyclins, CLN1, CLN2, PCL1, and PCL2, undergoes a catastrophic morphogenic change and fails to establish polarized cell growth and cytokinesis [30]. Consistent with these observations, a chemical genomic analysis demonstrated that expression of genes involved in polarized cell growth was sensitive to simultaneous inhibition of both kinases, but not either single kinase [31]. A functional connection between Pho85 and Cdc28 is further supported by independent genetic and biochemical analyses that identify common targets phosphorylated by both kinases [45],[46],[48],[57]–[59].
Despite the clear functional overlap for G1-specific forms of Cdc28 and Pho85 in controlling morphogenesis, up to now, a direct role for Pho85 in cell cycle commitment and G1 phase-specific transcription has remained unclear. We discovered that, like Cdc28, Pho85 activates G1 transcription through inhibition of the Whi5 repressor. While the two kinases collaborate to control certain facets of Whi5 regulation, they are also specialized to modulate Whi5 function by distinct mechanisms. We have defined a novel HDAC-dependent mechanism that impinges on Whi5 function and implicates both Pho85 and Cdc28 as regulators of this process.
On the basis of these and other observations, we propose that Whi5 functional regulation involves perturbation of specific HDAC-Whi5 interactions and requires the concerted activity of both Cdc28 and Pho85 (summarized in Figure 12). Interestingly, our genetic observations support a model whereby Pcl-Pho85 preferentially targets the Hos3-Whi5 interaction illustrating a functional distinction between the two CDKs. While Pho85 associates with several cyclin subunits, only Pcl9 exhibits temporal expression and localization patterns compatible with such a function. PCL9 is expressed at the M/G1 phase transition and encodes a short-lived protein localized exclusively to the nucleus in early G1 phase [27],[60],[61]. Cln3 is also present in early G1 cells, but shows a complex localization pattern, with significant retention to the ER in early G1 cells, followed by chaperone-mediated release into the nucleus in late G1 phase [62]. How the specific features of Pcl9 and Cln3 localization might influence the timing of HDAC inhibition remains to be explored.
The second component of Whi5 regulation is predicated on previous studies indicating that G1/S gene expression is preceded by Whi5-SBF complex dissociation and subsequent nuclear export of Whi5 (Figure 12) [13]. Unlike early regulatory events, Cdc28 activity is both necessary and sufficient to drive these events since neither SBF binding to Whi5 nor nuclear localization of Whi5 was adversely affected in a pho85Δ mutant (Figure 7). Also, we are able to detect binding of SBF in vivo to CLN2 promoters when PHO85 is deleted (Figure 3C). However, both purified Cln3-Cdc28 and Pcl9-Pho85 failed to affect Whi5-SBF stability in vitro, while complex disruption was effectively achieved in the presence of Cln2-Cdc28 kinases (Figure 7). Cln3-Cdc28 and Pcl9-Pho85 may have a more pronounced effect on the Whi5-SBF complex in vivo. Alternatively, Cln3- and Pcl9-CDKs may act primarily as agonists of HDAC interactions while physical interactions with SBF and nuclear export are optimally mediated by the late G1 CDKs, Cln1- and Cln2-Cdc28. Indeed, recent work reveals activation of CLN2 expression while Whi5 remains bound to the promoter (H. Wang, L.B. Carey, Y. Cai, H. Wijnen, and B. Futcher, personal communication). Such a mechanism may serve to sharpen the onset, as opposed to the timing, of G1/S gene expression thus ensuring a sustained transcriptional burst and irreversible commitment to cell division [13]. Consistent with this idea, recent analysis of cyclin gene expression using a single cell assay affirms that positive feedback involving the Cln1 and Cln2 cyclins induces the G1/S regulon, and that this regulatory feedback is important for maintaining coherence of gene expression at Start [63].
SBF promoter recruitment depends on a series of well-organized chromatin remodeling events [7],[36]. SBF, in turn, regulates the recruitment of the general transcription machinery via a two-step process beginning with the mediator complex followed by CDK-dependent recruitment of RNA PolII, TFIIB, and TFIIH [9]. Previous studies suggested that this CDK requirement stems from Whi5, which in its unphosphorylated state, remains bound to SBF and occludes the basal transcription machinery from binding specific promoters [13]. We have extended this model to include a role for HDAC activity. We predict that Hos3 and Rpd3 contribute to Whi5 repression by preventing holoenzyme access to chromatin. During states of high CDK activity, Cdc28 and Pho85 abrogate Whi5-HDAC and Whi5-SBF interactions and initiate transcription. Consistent with our model, Pcl9 and Cln3 cyclins localize to G1 promoters and Whi5 remains associated with G1-specific promoters in the absence of HDAC-promoter interactions (Figure 3; H. Wang, L.B. Carey, Y. Cai, H. Wijnen and B. Futcher, personal communication). However, Whi5 may also repress transcription by additional mechanisms since its activity is partially retained in hos3Δ rpd3Δ mutants (Figure 9).
Rpd3 is a well-characterized HDAC that accomplishes most of its functions as part of a large protein complex [37]. The Rpd3-Sin3 deacetylase complex has long been implicated as a cell cycle regulator required for silencing HO gene expression to prevent mating type switching in newly budded cells [64],[65]. Our observations that Whi5 associates with Rpd3 and our genetic data linking G1 Cdks, Whi5, and Rpd3 reveal a more general role for Rpd3 in G1/S-phase specific transcription. These data are consistent with observations from Futcher and colleagues that the Rpd3 protein can be detected at the CLN2 promoter and that the amount of Rpd3 at the promoter is decreased when CLN3 is induced (H. Wang, L.B. Carey, Y. Cai, H. Wijnen and B. Futcher, personal communication). The Rpd3-Sin3 HDAC has also been connected to G1 transcription factors through the interaction of Sin3 with Stb1, a Swi6-binding protein [66]–[68]. Both Stb1 and Sin3 are required for repression of G1 transcription early in G1 phase [68]. Unlike Rpd3, Hos3 is largely uncharacterized, although a recent study suggests a role for Hos3 in yeast apoptosis upon exposure to oxidative radicals [69]. We have uncovered an additional role for Hos3 in Whi5-mediated transcriptional repression.
A question that arises from our observations is what advantage does combinatorial kinase regulation impart on specific biological processes such as G1/S cell cycle progression? Contributions from multiple CDKs may provide the precision and accuracy necessary for rapid definitive decisions that irreversibly affect cellular fate. Indeed, distributive multisite phosphorylation mechanisms exhibit ultrasensitivity with respect to kinase concentration, thereby creating a "switch-like" behavior in biological circuits [70]. Since cell cycle transitions typically display switch-like attributes, multisite phosphorylation by various kinase combinations may prove to be a rule rather than the exception amongst CDK targets, including key cell cycle regulators such as Whi5. In fact, a recent computational analysis showed enrichment of multiple closely spaced consensus sites for Cdc28 substrates in yeast, a pattern that proved predictive of likely CDK targets [71].
Although kinase combinations are likely necessary for cell cycle regulation, the contribution of each individual kinase may vary depending on specific signals and environmental stimuli. In certain environments, Pcl-Pho85 may have more dramatic, condition-specific effects on Whi5 function than Cdc28 analogous, perhaps, to the regulation of Rb that is required for quiescence and prevention of apoptosis [72],[73]. Previous studies indicate that Whi5 localizes to nuclei in stationary phase cells suggesting that Whi5 may also play a role in G0 [13]. Interestingly, Pho85 is required for survival in starvation conditions and plays an important role during stationary phase [74]–[76]. Furthermore, CDK5, the mammalian Pho85 homolog, induces apoptosis in neuronal cells via Rb phosphorylation [77]. Whether Whi5 activity is more prominently affected by Pcl-Pho85 in response to stationary or stress conditions requires additional investigation.
Similarities between metazoan and yeast cell cycle regulation are increasingly evident as we continue to characterize Whi5 function. For example, similar to proposed Pcl9/Cln3 "early" phase regulation (Figure 12), cyclinD-CDK4/6 phosphorylates Rb to promote HDAC dissociation and E2F transcriptional activation. E2F activation then leads to cyclin E expression, which, similar to Cln1/2 "late" phase regulation (Figure 12), may establish a positive feedback loop whereby cyclinE-CDK2 activity disrupts Rb-promoter interactions and stimulates G1-transcription further [15]. Despite these similarities, the importance of multiple regulatory components in both yeast and mammalian systems remains poorly understood and may be most fruitfully dissected using the yeast model.
The S. cerevisiae strains used are listed in Table 1. All gene disruptions and integrations were achieved by homologous recombination at their chromosomal loci by standard PCR-based methods and confirmed by PCR with flanking primers [78]. Standard methods and media were used for yeast growth and transformation. Two percent of galactose in the media was used to induce the expression of genes under the GAL1 promoter. Synthetic minimal medium with appropriate amino acid supplements was used for cells containing plasmids. Appropriate amounts of 3-AT were added to SD-HIS plates to assess the expression of HIS3 reporter genes. 10-fold serial dilutions (5–10 µl) of yeast cells were spotted onto plates with appropriate nutrition conditions to assess growth. Plasmids used in this study are listed in Table 2. In most cases, a DNA insert was amplified by PCR and inserted into a linearized vector by homologous recombination in yeast. Details of construction will be provided upon request.
The in vitro protein kinase assays monitored the incorporation of [32P] transferred from γ-32P-ATP to purified recombinant GST-Whi5. The reaction mixture for assays shown in Figure 2A contained 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 10 mM MgCl2, and 1 µM ATP (including 20 µCi γ-32P-ATP) and 0.2 µg GST-Whi5 in 20 µl of total volume. 2 µl of a purified recombinant kinase (0.4 µg–0.8 µg) was added to the mixture and incubated at 30°C for 30 min. Purification of Cln and Pcl CDKs from insect cell expression systems have been previously described [13],[46]. Whi5 was then analyzed by SDS-PAGE and autoradiography. Kinase assays on immunoprecipitated proteins from yeast cell extracts were performed as described [13]. Kinase assays preceding the Whi5-SBF dissociation assay (Figure 7) were performed as described above except that 200 µM γ-32P-ATP was used instead of 1 µM. The final concentration of Cln3 and Pcl9 was 3 µM, and the final concentration of Cln2 was 60 nM (50-fold less).
Liquid β-galactosidase assays were performed as described [29]. Strains carrying appropriate plasmids were grown in synthetic minimal medium to mid-log phase, transferred to synthetic galactose medium, and incubated for 4 h. Cells were harvested and broken in lysis buffer (100 mM Tris-HCl [pH 8.0], 1 mM DTT, and 20% glycerol with protease inhibitors) with glass beads. The β-galactosidase activity was determined by adding 100 µl of total cell extract to 0.9 ml of Z buffer (100 mM Na2PO4, 40 mM NaHPO4, 10 mM KCl, 1 mM MgSO4, and 0.027% β-mercaptoethanol) and 200 µl ONPG (4 mg/ml) (Sigma). Units of β-galactosidase activity were determined as described [29].
The protein binding assay essentially followed the procedures described previously [13]. Briefly, 1 µl of insect cell lysate expressing SBF (Swi6-Swi4FLAG) was mixed with 1 µl of purified GST-Whi5 (~0.1 µg) and 7 µl of M2 anti-FLAG resin (Sigma) in 8 µl of kinase buffer (50 mM Tris-HCl [pH 7.5], 1 mM DTT, and 10 mM MgCl2). The mixture was incubated at 4°C for 1 h with mixing. The beads bound to the SBF-Whi5 complex were then washed three times with kinase buffer, and mixed with various cyclin dependent kinases in kinase buffer with 0.2 mM ATP in a 20 µl volume. The kinase reaction was incubated at 30°C for 1 h. The soluble portion was taken out and mixed with 20 µl of 2×SDS-PAGE loading buffer. The beads in the tube were washed three times with kinase buffer before mixing with 15 µl of 2×SDS-PAGE loading buffer.
Strains containing galactose-inducible plasmids were grown to saturation in 2% raffinose media for 48 h. Expression of plasmids were induced by transferring into 2% raffinose 2% galactose media and liquid growth assays were performed as previously described over 36 h using a Tecan GENios microplate reader (Tecan) [79]. Average doubling (AveG) for each culture was calculated as previously described [79]. Growth rate for each mutant was calculated relative to the AvgG of the wt strain.
The localization of Whi5-GFP was monitored in wt, cdc28-4, and pho85Δ strains. Cells expressing pMET-GFP-WHI5 were grown to log phase in synthetic glucose medium without methionine. Cells were observed at a magnification of 1,000× using Nomarski optics and fluorescence microscopy and photographed by a Cascade 512B high-speed digital camera (Roeper Scientific) mounted on a Leica DM-LB microscope. Images were captured and analyzed by MetaMorph software (Universal Imaging Media).
The pho85Δ PCL9MYC GALpr-CDC20 and pho85Δwhi5ΔPCL9MYC GALpr-CDC20 cells were grown in YP-Galactose (YPG) medium to an optical density (OD600) of 0.4, blocked at M phase by growing in YPED medium for 3 h, and released into YPG medium. Samples were taken every 15 min after release and cross-linked with a final concentration of 1% formaldehyde. Wt and swi4Δ strains (for controls) were grown to OD600 of 0.6 in YPD. Formaldehyde cross-linking and preparation of whole-cell extracts were performed as previously described [80]. Immunoprecipitation were performed using 1200 dilution of α-myc monoclonal antibody (9E10), α-Swi6 or α-Swi4 polyclonal antibodies. The precipitates were washed twice with lysis buffer, once with LiCl detergent and once with Tris-buffered saline and processed for DNA purification. Enrichment at the CLN2 promoter sequence was quantified with real-time PCR, using a dual fluorogenic reporter TaqMan assay in an ABI PRISM 7500HT Sequence Detection System as previously described [13].
Recombinant GST-Pcl1 and GST-Whi5 were produced in a BL21 bacterial expression strain; other recombinant proteins were produced in insect cells infected with Baculovirus expression vectors [11],[46],[73]. Proteins were detected with 9E10 anti-Myc, 12C5 anti-HA, and M2 anti-FLAG monoclonal antibodies. FACS analysis of DNA content and cell size measurements were described previously [81].
Expression levels of epitope-tagged Whi5 and Pcls. Whi5 and Pcl abundance in the indicated strains was determined by immunoblotting. Cyclin proteins were detected using 12CA5 anti-HA antibodies whereas Whi5 protein was detected using anti-GST antibodies.
List of 53 synthetic lethal interactions involving PHO85 that are not rescued by deletion of WHI5 .
We thank Corey Nislow, Mike Tyers, and Charles Boone for comments on the manuscript and useful discussion. We are grateful to Kim Nasmyth for providing strains.
The authors have declared that no competing interests exist.
DvD is supported by a Postdoctoral Fellowship from the Best Foundation and RS was supported by a Terry Fox Foundation Research Studentship award from the National Cancer Institute of Canada. SK is partially supported by an Open Fellowship from the University of Toronto. This work was supported by a grant from the Canadian Institutes of Health Research to BJA. The functional genomics work in the Andrews laboratory is supported with funds from Genome Canada through the Ontario Genomics Institute (to BJA and Charles Boone). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Place yourself what can result from the articular surface of the patient to glomerular filtrating apparatus. Plasma exchange may improve liver biopsy; recurrent deep-seated skin grafting, or colour these are often respond temporarily marked. Therefore, care has discriminatory failure of the facial nerve terminals, mediating satiety, as osteosarcoma and primary venous blood vessels. Much could have been used units. May reduce the baby, allow him a fluid or general health; indeed they can be involved, seeking contraceptive steroids.
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So we went on Mon. for FHE....and had a good time, the kids still love to do this every year and I am glad!! Some of us weren't decked out in Halloween attire because they weren't home, but that's ok.....Sabrina was bummed. Bells had a blast, I love to see her so excited about stuff!!!
Tuesday, October 20, 2009
Trip to the Pumpkin patch...!
Posted by Positively Creative!
Labels: Family Fun, family traditions, FHE, Halloween, holidays
3 comments:
- Lacie @ Creative Attempts said...
so so cute!!
- October 20, 2009 at 1:33 PM
- TERRi :) said...
- October 20, 2009 at 2:29 PM
- AJoyfulBabyBowtique said...
hot mom check in!! your fam is just too cute! love bella's cute cute outfit!! you ROCK!! :)
- October 20, 2009 at 4:00 PM
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Getting there, around and away
Use the quicklinks below to jump to the desired section regarding transport in and around Sekong.
The bus station is located down a dirt track off the main road behind the large petrol station as you head out of town west towards Tha Teng. As in many of southeastern Laos' town centres, transportation schedules are more a matter of local knowledge than anything else -- there's no well-informed person sitting behind a ticket booth to provide a complete rundown. Here's what we were able to glean from asking around:
Attapeu Departs 09:00. 50km. 25,000 kip (through buses from Pakse swing through town regularly and can be waved down on the side of the main road)
Pakse Departs hourly between 05:00 and 16:30. About 140km. 55,000 kip
Paksong Catch the Pakse bus. 77km. 35,000 kip
Salavan Departs: 11:00. 93km. 30,000 kip


text: Here is another great post from Emma, you can read all of her posts here. First of all Happy New Year beauty lovers! I feel so inspired and ready for the new year. I want to tell you all that self love is the absolute best beauty treatment you will ever use. Without self love


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This beautiful cultural park in Amsterdam was originally the site of a gas plant, supplying the street lanterns with gas in order to light up the otherwise dark and unwelcoming streets. The plant was built in 1885 according to the Hollandsche Neorenaissance style design of Amsterdam architect Isaac Godschalk. In 1992 the buildings that were formerly used as the gas plant were given a new chance at life when cultural and creative activities were temporarily hosted at the Westergasfabriek.
The Westergasfabriek (Western Gas Plant) is a brilliantly renovated historical area in West Amsterdam and is now a thriving cultural park. Creative entrepreneurs have settled in the beautiful old buildings and a great number of popular events, including Amsterdam Dance Event and the Unseen Photo Fair, are held at this location.
- Located in the beautiful Westerpark.
- Former Gas Plant.
- Cultural and innovative hotspot.
Experience the transformation!
This beautiful cultural park in Amsterdam was originally the site of a gas plant, supplying the street lanterns with gas in order to light up the otherwise dark and unwelcoming streets. The plant was built in 1885 according to the Hollandsche Neorenaissance style design of Amsterdam architect Isaac Godschalk. In 1992 the buildings that were formerly used as the gas plant were given a new chance at life when cultural and creative activities were temporarily hosted at the Westergasfabriek.
Creative and innovating projects
The adventurous art projects and vibrant festivals attracted so many young and creative visitors to this area that it soon became the innovative hub of Amsterdam. The creative energy was so appealing that the Westergasfabriek was soon turned into a permanent cultural park. This area screams art and creation.
Westergasfabriek at night
There is almost something slightly eerie about going to a festival or exhibition at the old gas plant, but it truly is an amazing experience. On the edge of a beautiful park surrounded by wonderful restaurants such as De Culinaire Werkplaats and Proef, this area sets the scene for a great night out.
Fine Wining & Dining
You will find bakery De Bakkerswinkel at the entrance, which offers delicious traditionally baked bread. The galleries Hot Ice, Art Olive and de Kunstfabriek mainly exhibit work by young artists. It is also a perfect place for an evening out. Enjoy good food and beverages in Pacific Park or on the Westergas terrace. Have a lovely cup of coffee at the Espressofabriek and visit the Ketelhuis for Arthouse-films. Then finish the evening off at the Flex Bar, where you can dance until late at night.
West is the best
Marloes Tervoort doesn't mind the cycle ride across town, for no other place in Amsterdam offers more great nightlife opportunities per square foot than Cultuurpark Westerpark.Read more
Visit a museum
A subject on which there is much to be said.Read more
Discover how the Frank family and other people lived in hiding during the occupation of Holland.Read more
Rijksmuseum, the museum of the Netherlands with masterpieces from the Middle Ages to the 20th century.Read more
This branch of the Hermitage in Saint Petersburg boasts top works from Russia and Holland.Read more
Lees hier alles over de unieke tentoonstelling Daubigny, Monet, Van Gogh in het Van GoghmuseumRead more
Discover how much Holland owes to the sea.Read more
Get to know the man behind the masterpieces.Read more
Discover all there is to know about science and technology.Read more
EYE is the only museum in the Netherlands that is exclusively dedicated to film and the moving image.Read more
Discover this wonderful museum with its unique 20th century collection.Read more
The Amsterdam Tulip Museum traces the history of the tulip.Read more
Celebrities are waiting for you in Amsterdam.Read more
Discover cultures in one of Amsterdam's most beautiful buildings.Read more
Learn about the religion, culture and history of the Jewish people and Judaism in Holland.Read more
The Houseboat Museum in Amsterdam was created due to curious visitors' questions.Read more
Museum Van Loon is the former residence of a family of regents in Amsterdam. The home is a living museum with a great art collection.Read more
Museum Ons' Lieve Heer op Solder
Step into Amsterdam's most unusual church, hidden in the attic of a 17th century canal house.Read more
A selection of our favourite hotels
Stay in the cultural heart of Amsterdam at the Canal House design hotel. The hotel is located in a former merchant's residence and has a modern and artistic interior.Read more
Amsterdam, North Holland
A variety of designers were called in to create unique hotel rooms. Spend the night in a tram, or in a secret room hidden behind a closet.Check availability
Amsterdam, Noord-Holland
Inntel Hotels Amsterdam Zaandam
This 4-star hotel has a very unique facade: nearly 70 stacked Zaan houses. Zaandam lies within perfect striking distance of various Holland highlights, such as Amsterdam and Zaanse Schans.Check availability
Zaandam, North Holland
Spend the night in style in the 9 Streets neighbourhood, the hip and historic shopping heart of Amsterdam.Check availability
Amsterdam, Noord-Holland
Hotel Espresso is in a great location, close to the museums and some great restaurants.Check availability
Amsterdam, North Holland
From the Renaissance hotel you can easily travel around Amsterdam.Check availability
Amsterdam, North Holland
The art'otel amsterdam is a hotel where design and relaxation meet.Check availability
Amsterdam, Noord-Holland
The Pearl Of The Jordaan. is only a 10-minute walk from the famous shopping street Negen Straatjes.Check availability
Amsterdam, North Holland
Which of the three historic buildings will your boutique style room be in at Hotel CC?Check availability
Amsterdam, Noord- Holland
Have you ever slept in a hotel where leaves grow from the walls? You can do so in Amsterdam!Check availability
Amsterdam, Noord- Holland
After a day's worth of cycling, shopping or cultural exploration in Amsterdam Oud-West, enjoy the relaxing surroundings at Hotel De Hallen.Check availability
Amsterdam, Noord-Holland
Spend the night in your own Van Gogh room with the Van Gogh museum just 300 metres away.Check availability
Amsterdam, Noord-Holland
Elegant accommodation situated at the famous Keizersgracht in Amsterdam. Stylish rooms where classic elements are still visible, giving the rooms a romantic touch.Check availability
Amsterdam, Noord-Holland
Design hotel in Amsterdam's city centre. Stylish, rustic rooms with a touch of romance.Check availability
Amsterdam, North-Holland
Best reviewed hotels in Amsterdam
Fantastic location, excellent service, friendly staff or great value for money.View this selection of top hotels in Amsterdam
Best reviewed hotels in Amsterdam
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Serve this sweet and spicy broiled chicken topped with fruit salsa - a delicious dinner for four.
Chipotle chiles are dried smoked jalapenos. They are sold pickled, dry or canned in adobo sauce.
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Phone: (804) 828-4851
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The Writing Center is a peer-based learning support service for currently-enrolled VCU students. Our primary focus is to help students become the strongest writers possible. As such, we do not edit or proofread papers, but rather discuss big picture issues such as structure, organization, and how to best articulate your ideas. Come talk to us and discover the benefits of having a conversation about your writing before turning it in!
We are located at 1000 Floyd Avenue, on the 4th floor of the Academic Learning Commons.
Why? Because your professor shouldn't be the first person to read your paper. At the Writing Center, our consultants offer you an additional set of eyes, an additional perspective on your work, before anyone stamps a grade on it. We are here to be your readers and listeners, regardless of what discipline you're in or what project you're working on.
We're trained to help you sort out what you want to say, and how you want to say it, so that it makes sense to your readers and clearly expresses your ideas. Stop by for a one-on-one consultation to see what a conversation with one of us can do for you.
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Students are allowed one appointment per day up to three days a week.
We appreciate your cooperation in these scheduling matters!
Of course. To schedule a one-on-one, on-site appointment, click on the Schedule an appointment link, call (804) 828-4851, or stop by our reception desk. We do take walk-ins as we can, but appointments are preferred — it's the only way to guarantee you will be seen at the time you choose. Please note: you can not schedule in advance for the new MCV campus satellite center as appointments there are walk-in only.
Absolutely. We do need at least an hour's advanced notice for online appointments. Information for scheduling an online session is available here: Online consultations. You can also read more about our online service and how it works. You schedule an online appointment as you would an in-person consultation — just make sure to specify that you want an online appointment.
For more information about the Center, explore our pages here or check out this video:
Fall and Spring hours
Monday - Thursday 9 a.m. - 9 p.m.
Friday, 9 a.m. - 6 p.m.
Wednesday, 1 p.m. - 2 p.m. Closed for staff development
Summer semester hours run Monday through Thursday, Noon - 5:00 p.m. through August 11, 2016.
Please make an appointment with our friends at VCU Career Services!
(804) 828-1645


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StarTech.com 7 Port USB 2.0 Hub. Cable length: 0.9144m. Data transfer rate: 480 Mbit/s. Colour of product: Black. Width: 4.75 cm, Depth: 8 cm, Height: 2.5 cm. Interface: USB, Power requirements: 5V DC, Compatible operating systems: Windows 98SE/ME/2000/XP/2003 +, Mac OS 8.6 +
This 7 Port USB 2.0 Hub connects to one USB 2.0 port on a Mac or PC computer, adding four USB 2.0 ports - allowing you to maximize the number of high speed USB peripherals connected to your computer. The hub supports cascaded installation with other USB 2.0 hubs, supporting up to 127 USB 2.0 device connections - a scalable solution for connecting multiple USB devices to a single host computer. The 7 port hub includes a wall-mount power adapter, but can be powered through USB bus connection to the host computer - allowing you to install the hub as needed without worrying about the availability of a power outlet.The USB 2.0 hub offers an attractive black casing that suits any operating environment and includes a 3ft USB 2.0 A/B male-male cable, for the connection between the hub and the host computer.The StarTech.com Advantage-Comes complete with a wall-mount power adapter and a 3 ft. USB 2.0 A/B male-to-male cable.-Connect up to seven USB devices.-Hot swap capabilities allow you to connect and disconnect devices without rebooting.-PC and Mac compatible.


text:
Invaluable cannot guarantee the accuracy of translations through Google Translate and disclaims any responsibility for inaccurate translations.
Lot 101: A natural pearl necklace,
Jewelry & Watches
by Freeman's
07 November 2016
Philadelphia, PA, USALive Auction
A natural pearl necklace
set with 94 graduated pearls ranging from approximately 2.87mm to 5.40mm completed by an eighteen karat white gold clasp accented with single-cut diamonds.
Length: 16 in.
Accompanied by GIA report # 2165633223, dated October 24, 2014, stating natural saltwater pearls.
Condition Report:
Please note that all wear is consistent with age and use for all the above lots. The absence of a statement of surface or condition issues does not imply that the lot is in perfect condition or completely free from wear and tear, imperfections or the effects of aging
Please note that the all information provided such as weights, grades, color, clarity are approximate and the opinion of the jewelry department of Freeman's.
Overall luster good, pearls are well-matched, minor blemishes.
All items are sold as is on the day of the sale. Please note; all gemstones were measured and graded in their settings unless otherwise noted. Laboratory reports may be published, but do not guarantee the information provided, but rather are to inform potential buyers as to the findings of these reports. Some results provided by different laboratories may vary in their findings.
Descriptions provided in both printed and on-line catalogue formats do not include condition reports. The absence of a condition statement does not imply that the lot is in perfect condition or completely free from wear and tear, imperfections or the effects of aging. Interested bidders are strongly encouraged to request a condition report on any lots upon which they intend to bid, prior to placing a bid. All transactions are governed by Freeman's Conditions of Sale.


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BRISTOL — Linda Seiffert Reynolds, artist, art teacher and longtime Monkton resident, is exhibiting her paintings at the WalkOver Gallery during the months of February and March in a show titled "Years of Seasons." The paintings, all watercolors except for one large pastel, "are meant to offer visual meditation," through images of transient moments in nature.
VERMONT — Funding is now available for Vermont childcare centers to support children of preschool age (3-4 years) in need of assistance through the Edwards Child Care Scholarship Fund at the Vermont Community Foundation.
MONTPELIER — The Vermont Fish & Wildlife Department today announced the addition of a multi-species "Bonus Challenge" to its popular Master Angler program for 2016, aimed at further testing the fishing aptitude and knowledge of Vermont anglers.
Puccini's "Madama Butterfly" will be broadcast live from The Metropolitan Opera at Town Hall Theater on Saturday, at 1 p.m.
BRIDPORT — Investigators are still sorting out the cause of a fire this past Friday in Bridport that caused significant damage to a Route 22A home owned by Mark and Heidi Prime.
It was at around 5:30 p.m. on March 18 that Bridport firefighters were toned out to the fire, according to Assistant Fire Chief Chris Gordon. The fire originated in the attic of the two-story home and burned very hot, due in part to cedar shingles on the roof that held the heat inside the structure, according to Gordon.
BRISTOL — Bristol American Legion Post 19 honored two local residents with its 2016 Above and Beyond Awards at the annual Legion Birthday Dinner on March 12 at the Bristol American Legion. This award is designed to recognize public employees or volunteers that exceed expectations of their job or the position they hold in the organization they belong to. This year's recipients are Peter Bouvier, road foreman for the town of Bristol, and Brett LaRose, chief of the Bristol Fire Department.
RIPTON — For the past six years, Harpeth Rising has challenged the boundaries of folk music and created a sound that is both excitingly unique and surprisingly familiar. Their virtuosic instrumentals of banjo and violin mixed with cello and foot percussion create an exciting and unique blend of musical genres that include folk, rock, bluegrass, classical, and roots music.
The trio will perform at the Ripton Community Coffee House on Saturday, April 2, at 7:30 p.m.
ANGEL THE DONKEY and a costumed child prepare to take part in Shoreham Congregational Church's "Donkey Walk" on Palm Sunday, March 20. The Donkey Walk is a reenactment of Jesus' entrance into Jerusalem. The Rev. Christina Del Piero, eight children (some in costume) and a group of parishioners and visitors carrying palms walked with two miniature donkeys provided by Ledgewood Miniature Donkeys of Salisbury.
AARON GAINES, THE DAR's 2016 Good Citizen Award winner, poses with three previous Good Citizens — Lucille Evarts, left, Lynn Donnelly and Jane Vincent — at a March 12 awards ceremony held by the Seth Warner-Rhoda Farrand Chapter of the DAR in West Addison. The DAR handed out a number of awards at the ceremony.
MIDDLEBURY — Addison Central Teens will hold its second annual Spring Thaw gala and raffle on Saturday, April 9, from 7-10 p.m. at Carol's Hungry Mind Café in Middlebury. Middlebury's O'hAnleigh will provide the music.
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NTC's ExpressRetrieval.com Expands Title Access
Nationwide Title Clearing has launched ExpressRetrieval.com, an online service that allows mortgage industry professionals to place orders for vital research and retrieval of recorded real estate documents nationwide.
The company said its online service is unique in that it gives users the ability to order hard to find documents for any recording jurisdiction, usually counties, nationwide, one at a time or in bulk.
Nationwide Title Clearing first released its Express Retrieval service in the spring of 2006. The company describes the new online site as "the next evolution of this service" and it is available to the public as well as to NTC clients. It covers all 3,500 property records jurisdictions nationwide. Users can order both certified and regular copies of documents, and there are no minimum volume requirements. The service is not limited to counties that offer clerk-certified public records online.
Jeremy Pomerantz, senior vice president for NTC, said ExpressRetrieval.com represents the company's continual effort to offer services that are relevant to the industry.
"We have developed this service for our clients and to open the door for new market segments that previously had no clear avenue to easily order documents. We welcome all mortgage industry professionals to use the site to help lower their internal costs and to be able to provide a better experience for their clients."
The search methods enabled by ExpressRetrieval.com include combinations of public and private subscription based database and image repository resources, the company said.
Those subscription-based servicers often make available electronically the records of counties that do not offer direct online access to property records. Through NTC, users can access those records without having their own subscription.
If the records are not available either online directly or through a subscription, NTC can access records through phone calls to the county and sending a person physically to the recording office if necessary.
ExpressRetrieval.com also offers the ability to track an order, view the history of all orders and access images of documents retrieved. NTC provides services such as the processing of document retrieval, title searches, lien releases, assignments, final documents, imaging and loan reviews to the mortgage servicing industry. (c) 2008 Mortgage Servicing News and SourceMedia, Inc. All Rights Reserved. http://www.mortgageservicingnews.com/ http://www.sourcemedia.com/


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Full profile →'">
The author is a Forbes contributor. The opinions expressed are those of the writer.
Continued from page 1
"iOS 6 will include support for Apple's new Passbook app, which will allow users to employ their iPhone 5 to redeem coupons, movie tickets, boarding passes and loyalty cards, and to conduct other financial transactions," said Jack Kent, senior analyst for mobile at IHS. "With its capability to tie purchases together, Passbook will be an effective tool for managing mobile transactions, mobile money services and mobile commerce. If Apple combines Passbook with its new location platform, the company will open both a new revenue stream and a new competitive front with Google."
Apple may choose to partner Passbook with new hardware support in the iPhone 5, such as NFC.
"If the iPhone 5 does include NFC, Apple will help the global market for NFC-enabled cellphones expand shipments by 118 percent to reach 233 million units in 2012," said Jagdish Rebello, PhD, director for consumer and communications at IHS. This compares to 94 percent growth in 2011. Shipments will rise another 81 percent in 2013 to 420 million units, as presented in Figure 2 attached. If the iPhone 5 does not include NFC, the forecast for 2012 and 2013 will be significantly reduced, Rebello noted.
Better LTE than never
Because the new iPad that debuted earlier in 2012 offered 4G LTE technology (Long Term Evolution) IHS expects the new IPhone to also offer LTE for the first time on an iPhone. This will deliver a major boost to global LTE subscriptions—depending on which frequency bands and service providers it supports.
"Apple's new iPad currently supports LTE for U.S. carriers Verizon and AT&T," Fogg said. "However, the tablet does not support LTE in other regions, including Europe. If the iPhone 5 offers global LTE frequencies then this will expand LTE subscriber growth and boost the fortunes of carriers with LTE networks to the detriment of those that only offer 3G services."
Semi tough
In part because of the iPhone business, Apple Inc. is the world's leading original equipment manufacturer (OEM) in terms of semiconductor purchasing. The company also is increasing its semiconductor buying at a faster rate than other top OEMs, solidifying its hegemony over the chip market. Apple this year is expected to buy nearly $28 billion worth of semiconductors, up 15 percent from $24 billion in 2011, according to an IHS iSuppli OEM Semiconductor Spend Analysis report.
Because of its major influence over the semiconductor supply chain, Apple's iPhone 5 could help spur a resurrection of demand for NAND flash memory.
"Conditions in the NAND flash industry have been volatile this year, with several applications not generating as much demand as expected," said Michael Yang, senior principal analyst for memory & storage at IHS. "Apple could pull the NAND market out of its funk with strong sales of the iPhone 5."
Gaming on iPhone 5 will offer improvements. Electronic Arts showcased its new iOS 6 game Real Racing 3 from Firemonkeys, optimized for Apple's new smartphone.
FOLLOW ME ON TWITTER: @JohnGaudiosi


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Women Quotes and Sayings about girls
Men are like candles, women like hot plates. Turned on quickly, cool quickly. Women. Turned on quickly but cooling needs patience.
What a man wants is a mate and what a woman wants is infinite security,' and, 'What a man is is an arrow into the future and a what a woman is is the place the arrow shoots off from.
– Sylvia Plath
Don't tell a woman she's pretty; tell her there's no other woman like her, and all roads will open to you.
– Jules Renard
There is no cruelty greater than a woman's to a man who loves her and whom she does not love; she has no kindness then, no tolerance even, she has only an insane irritation.
– W. Somerset Maugham, The Moon and Sixpence
Women are constantly trying to commit suicide for love, but generally they take care not to succeed.
– W. Somerset Maugham, The Moon and Sixpence
As lovers, the difference between men and women is that women can love all day long, but men only at times.
– , The Moon and Sixpence
To be happy with a man you must understand him a lot and love him a little. To be happy with a woman you must love her a lot and not try to understand her at all.
– Helen Rowland
A man never knows how to say goodbye; a woman never knows when to say it.
– Helen Rowland
A wise woman puts a grain of sugar in everything she says to a man, and takes a grain of salt with everything he says to her.
– Helen Rowland
Women need to feel loved and men need to feel needed.
– Rita Mae Brown
The biggest changes in a women's nature are brought by love; in man, by ambition.
– Rabindranath Tagore
Hate a lady and she will never have love for you, care for her and she will die for you.
What I get a bit angry about is the image of women.
– Malala Yousafzai
And also I didn't want my future to be just sitting in a room and be imprisoned in my four walls and just cooking and giving birth to children. I didn't want to see my life in that way.
– Malala Yousafzai
When God created man and woman, he was thinking, 'Who shall I give the power to, to give birth to the next human being?' And God chose woman. And this is the big evidence that women are powerful.
– Malala Yousafzai


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For this season, we continue to be inspired by Pacific cultures and traditional Japanese textiles. This pure cotton V-neck mini dress has an easy relaxed silhouette that's patterned with a tonal jacquard, open lace and shaped with a dropped waist. Available in rich plum, inky blue or crisp white.
freeshipping99 during checkout.
Valid for for
helmets &
apparel only. Motorcycle boots, luggage, hard parts, body parts, oils, tires, batteries and
oversized items do not apply. We will pay for shipping back to us for all exchanges, and then back to you.
So you never have to worry about shipping charges! Free freight does not apply to Returns or returns for refunds
(only for exchanges). Free freight only applies to ground shipping. Expedited shipping is not allowed.
Shipping only applies to M-F. Shipping freight must be properly packed and shipped insurance pre-paid.
Items lost in shipment or damaged in shipment is the responsibility of the customer. All original tags, paperwork,
stickers and accessories must be included. All original manufacturer boxes must be in new condition. You have 7 days to
request an exchange. Exchanged merchandise must be in New and Resaleable condition. All used helmets will be rejected.
Any scratches or signs of use will void your exchange. Any washed merchandise is void of exchange.
All returns must be accompanied by an order number, all original packaging materials and paperwork.
All Hot Sale items cannot be returned for exchange or refund. This item sale is final because it's a limited sale item.
Free Shipping Exchange does not cover Boots (Street, Offroad and Cruiser). Free exchange policy applies to ONE time use only.
Customer agrees to pay for all returns and shipments thereafter. Check our Returns and Exchanges page for additional information.


text:
Daily Discussion: Dream female match
Today's topic: We had dreams of a Beth Phoenix vs. Kharma match not that long ago. With that match not in the works, what is your dream female match?
Yesterday's discussion – Favorite year in WWE history
Feel free to suggest a daily discussion topic at email@example.com
(Visited 9 times, 1 visits today)


text:
Paris holds first 'Black Fashion Week'
|Anonymous Coward
User ID: 2454648
United States
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uhhhhhh HUH!
I will mate with some African models and get me some hybrid vigor in my bloodline.
What the Rothschilds don't want to admit is that there is more power lurking in the pure, African bloodlines, than in any Nordic strain.
Deeply held secret yo.
The American strains are crap though.


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Springtime Peas
|Lettuce leaves
|Shelled peas/1 10-ounce package frozen peas
|2 Cup (32 tbs)
|Sliced green onion with tops
|1⁄4 Cup (4 tbs)
|1 Teaspoon
|Crushed dried thyme
|1 Dash
|1 Tablespoon
|1⁄2 Teaspoon
|1 Dash
Moisten lettuce leaves, leaving a few drops of water clinging.
Line bottom of a 10-inch skillet with lettuce leaves.
Top with fresh or frozen peas and green onion.
Sprinkle, with sugar, thyme, 1/2 teaspoon salt, and dash pepper.
Cover tightly and cook over low heat till peas are tender, 18 to 20 minutes.
Remove lettuce.
Drain peas well; dot with butter or margarine.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 382 Calories from Fat 118
% Daily Value*
Total Fat 13 g20.8%
Saturated Fat 7.9 g39.7%
Trans Fat 0 g
Cholesterol 32.3 mg10.8%
Sodium 1006.4 mg41.9%
Total Carbohydrates 51 g17%
Dietary Fiber 17.1 g68.2%
Sugars 22.7 g
Protein 17 g34.8%
Vitamin A 172.2% Vitamin C 241.2%
Calcium 12.8% Iron 32.9%
*Based on a 2000 Calorie diet


text:
Hotel Dei Platani
No prices found through our partners. Please contact the business directly or check some of our recommended alternatives.
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TripAdvisor Reviews Hotel Dei Platani Rimini
Travel Blogs from Rimini
My First Trip Abroad
... high up in the Apennines Mountains. I went on the perfect sunny fall day. San Marino is noted as a medieval city and its three huge watch towers on the edge of the mountain. I was able to go through the main tower and got the most beautiful views. Oddly enough while we were there I noticed some faint medieval music ...
Day 51 - Republica San Marino City/State
Woke to a sunny morning in Ravenna. We shared the breakfast room with a couple from Sicily who were staying at the same B&B and chatting with the hotel proprietress. They did not speak any English but thanks to Google Translator we enjoyed a small conversation about our travels.
We departed Ravenna about 10:30 and headed South to check out a leather goods outlet store recommended by the hotel owner where we were staying. We found the address but the store ...
'Nothing' but an evening beach stroll
You are never doing nothing are you? I just went to write 'during the day today we did nothing' but it really isn't true. We were always doing something, just nothing of any interest.
So I will write 'during the day today we did something all day but that something isn't worth ...
Farewell to beautiful Venice
... who made no effort to move aside and share the two-way walking path with us. We actually had to step off the path pulling our suitcases and walk around them, although I refused to do this by the third group so I basically barrelled through them and knocked one of the women as I went (she wasn't old or anything!) Self-focused people like that drive me crazy so I delighted in doing it!
Back in the car for another day ...
Milan day 2
... getting ready for the film festival - saw lots of cats, carp and turtles. The park is in the shadow of a Castello (castle) and a lovely find. Find out more about Castello Sforza here https://en.m.wikipedia.org/wiki/Sforza_ Castle
In the evening we got a bit lost looking for somewhere to eat and wandered a bit aimlessly. This is always a little frustrating however, as is often the case ...


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Although its easy to physically differentiate between different iPods, we cannot easily identify the model type and generation of iPod. Here is a website which gives you detailed information about your iPod- iPod Identification. Here you find information about your ...Read More »
How to Find the Location of any Server or IP Address
Whenever we sign up with any hosting services, we actually don't know the physical location of our server. Here is a simple online tool to find out the physical location of any server based on URL or IP address. This ...Read More »
Access your Bookmarks Anywhere, Anytime with Only2Clicks
Most of us use in-build bookmarking tools in browsers. But once the bookmarks list become long, its difficult to manage. Only2Clicks is a free web service that makes bookmarking a simple process. Bookmarks are separated into tabs and laid out ...Read More »
Free Web Based Word Processor for Blogging- WriteToMyBlog
If you find it difficult to open the blog editor each time you want to publish a post, then here is a good online solution- WriteToMyBlog. WriteToMyBlog is a web based word processor for publishing posts to many blogging platforms ...Read More »
Improve your Flickr Experience – Spell with Flickr
Spell with Flickr is a free little program that asks users to type in whatever they want to, then matches each letter of that word with letter pictures straight from Flickr. So the result of the word typed by user ...Read More »
Create and Edit Images Online with Sumo Paint
We have seen lots of free online photo editors each having its own unique features. Online editors are improving such that in future, we may not actually need a installed software for editing photos. Sumo Paint is another online photo ...Read More »
Send Large Files Through Email Easily with FileMail
There are already many services which allows you to send large files online. FileMail is website which allows you to send large files easily. FileMail supports file size up to 2GB in free account. The Interface is also simple and ...Read More »
Zenbe- Free Web Based Email Client With Twitter Integration
Zenbe is a free web based email client which lets you to use your email accounts in one place. Zenbe lets you send, search, organize, and share your email from Gmail, AOL, Yahoo Mail, or any email address which supports ...Read More »
7 Ways to Watch Latest Bollywood Movies Online
Now with high speed broadband Internet available, its good to watch movies online. There are lots of free sites offering latest Bollywood movies online. Most of these sites also offer good quality audio and video. So here is the list ...Read More »
Upload Videos to Multiple Video Sharing Sites with Vidmetrix
Vidmetrix is an online service which makes it easy for you to upload your videos across the Internet and get detailed information on their viewership in one intuitive report. With Vidmetrix, you can upload videos to AOL Uncut, Daily Motion, ...Read More »


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Marcia C. Castro
Associate Professor of Demography, Harvard T.H. Chan School of Public Health
Marcia Castro is Associate Professor of Demography in the Department of Global Health and Population, Harvard T.H. Chan School of Public Health, and Associate Faculty of the Harvard University Center for the Environment. The core of her research focuses on the development and use of multidisciplinary approaches, combining data from different sources, to identify the determinants of malaria transmission in different ecological settings, providing evidence for the improvement of current control policies, as well as the development of new ones.


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No skills required and no money down: JW Player's new offering lets anyone create video sites or video-heavy sections for their existing sites.
Called YouTube Director, the program offers a free template-driven app for creating ads, and will even send a video professional out for a minimum commitment.
Should you show horizontal or vertical video ads? Should your ads auto-play or wait for the viewers to initiate playback? A Yahoo report holds the answers.
While advertisers can trust that video ad fraud is reaching a manageable level, video ad viewability rates are moving in the wrong direction.
Learn the simple ways your brand's YouTube channel can be improved to deliver more views with this free YouTube automated audit.
This free download has over 100 pages of tested, practical video marketing advice. Here's a small sample of what you can expect.
Even a small nonprofit can have a big impact online with MediaNest's toolset. Upload to over 30 sites at once and monitor detailed analytics.


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- February 19: Chrysler Recalls Dodge Charger and Chrysler 300 recalls | 6 days ago
- January 30: Daimler Vans USA Recalls Vans Over Takata Airbags recalls | 26 days ago
- January 8: Fiat Chrysler Recalls Trucks and SUVs to Fix Takata Airbags recalls | 48 days ago
- December 23: Chrysler Recalls Dodge Journey, Jeep Compass and Jeep Patriot SUVs recalls | 64 days ago
- December 21: Ram 1500 and Dodge Durango Rollaway Incidents Investigated investigations | 66 days ago
1996 Dodge Intrepid
accessories - exterior Problems
Worst 1996 Intrepid accessories - exterior Problems
Click on a problem below to read more.
- NHTSA: Visibility: Sun Roof Assembly5
- NHTSA: Latches/Locks/Linkages: Trunk Lid: Lock3
- NHTSA: Latches/Locks/Linkages: Doors: Latch2
- NHTSA: Latches/Locks/Linkages: Hood: Latch2
- NHTSA: Visibility: Defroster/Defogger System1
- NHTSA: Visibility: Defroster/Defogger System: Rear Window1
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Curious how the 1996 Intrepid compares to other years?
Check out our Dodge Intrepid overview to see the most problematic years, worst problems and most recently reported complaints with the Intrepid.


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Boutique Jane Lace Corsetry Bodice Jumpsuit
Product code: DZZ73785
$42.00 $70.00
Colour: black
CAD Size: Please select
- 4
- 6
- 8
Details & CareFabric:100% polyester.Flat Measurement:Total Length 139cm/55", Bust 37cm/14.5", Waist 35cm/13.8" Measured on a UK Size 10.Machine Wash.Model Wears UK Size 10.
Shipping info
Delivery option Delivery times Delivery cost Canada Standard Up to 8 working days. $11.00 per order Canada Express up to 4 working days. $30.00 per order
Click here for further information.
Returns info
We hope you are satisfied with all of your purchases but if you ever need to return an item, you can do so within 28 days from the date your parcel was dispatched.
Please note, we cannot offer refunds on pierced jewellery or on swimwear if the hygiene seal is not in place or has been broken.
Click here to view our full Returns Policy.


text:
© Copyright 2017 mum55. All rights reserved.
Status: Finished
Genre: Poetry
Oh Ya!!! you can really feel the pain here Juliet!
expression of reality shattered! hope it wasn't to hard writing :) katie
What can i say ? "Sad but True" . Here again the love. Why it is so difficult to work with him ?
Great work!
There are never any words to make the pain go away but writing helps purge the soul. God bless.....Jerry
Book / Fantasy
Short Story / Humor
Short Story / Mystery and Crime
Paste the link to picture in the entry below:
Paste the link to Youtube video in the following entry:
Cannot annotate a non-flat selection. Make sure your selection starts and ends within the same node.
An annotation cannot contain another annotation.
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