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Condensin I, which plays an essential role in mitotic chromosome assembly and segregation in vivo, constrains positive supercoils into DNA in the presence of adenosine triphosphate in vitro. Condensin I is constitutively present in a phosphorylated form throughout the HeLa cell cycle, but the sites at which it is phosphorylated in interphase cells differ from those recognized by Cdc2 during mitosis. Immunodepletion, in vitro phosphorylation, and immunoblot analysis using a phospho‐specific antibody suggested that the CK2 kinase is likely to be responsible for phosphorylation of condensin I during interphase. In contrast to the slight stimulatory effect of Cdc2‐induced phosphorylation of condensin I on supercoiling, phosphorylation by CK2 reduced the supercoiling activity of condensin I. CK2‐mediated phosphorylation of condensin I is spatially and temporally regulated in a manner different to that of Cdc2‐mediated phosphorylation: CK2‐dependent phosphorylation increases during interphase and decreases on chromosomes during mitosis. These findings are the first to demonstrate a negative regulatory mode for condensin I, a process that may influence chromatin structure during interphase and mitosis.
The packaging of long DNA molecules into compact rod‐like structures characteristic of mitotic chromosomes ensures faithful segregation of genomic material during mitosis and meiosis. Failure of this process may lead to aneuploidy, cancer, and cell death. Despite its importance, the molecular bases underlying dynamic changes in higher‐order chromosome structure during mitosis have remained enigmatic.
About a decade ago, studies using Xenopus egg extracts led to the identification of a five‐subunit protein complex, 13S condensin, now referred to as condensin I, which plays a central role in the assembly and maintenance of mitotic chromosome structure (Hirano and Mitchison, 1994; Hirano et al, 1997). Condensin I is the main component of mitotic chromosomes (Saitoh et al, 1994; Hudson et al, 2003; Maeshima and Laemmli, 2003). The two core subunits of condensin I, CAP‐C/SMC4 and CAP‐E/SMC2, belong to the SMC (structural maintenance of chromosomes) family of adenosine triphosphatases (ATPases), which are involved in many aspects of chromosome dynamics (Jessberger, 2002; Losada and Hirano, 2005; Nasmyth and Haering, 2005; Uhlmann and Hopfner, 2006). The SMC2–SMC4 heterodimer adopts a V‐shaped structure with an ATP‐binding 'head' domain and Walker A and Walker B motifs at the distal ends of each arm (Melby et al, 1998; Anderson et al, 2002). It is thought that ATP binding and hydrolysis of each 'head' domain modulates engagement and disengagement of the 'head' domain, a cycle that is presumed to reconfigure DNA and chromatin structure. Purified SMC2–SMC4 heterodimers possess the ability to reanneal complementary single‐stranded DNAs into double‐stranded DNA in an ATP‐independent manner (Sutani and Yanagida, 1997; Sakai et al, 2003). However, no ATP‐dependent activities have been detected for the SMC heterodimer. The remaining three subunits (CAP‐D2, ‐G and ‐H) are not related to SMC proteins and have possible roles in condensin regulation (Kimura and Hirano, 2000; Schleiffer et al, 2003). All five subunits of condensin I are highly conserved among eukaryotes ranging from yeast to the human (Hirano, 2000, 2005; Hagstrom and Meyer, 2003; Nasmyth and Haering, 2005).
The purified condensin I holo‐complex constrains positive superhelical tension into DNA in an ATP‐hydrolysis‐dependent manner, as has been shown using supercoiling and knotting assays (Kimura and Hirano, 1997; Kimura et al, 1999; Hagstrom et al, 2002). This activity is stimulated by the Cdc2‐dependent phosphorylation of the non‐SMC subunits during mitosis, suggesting that this process may contribute to compaction of chromatin fibers during mitosis (Kimura et al, 1998, 2001; Hagstrom et al, 2002).
More recently, a second condensin complex, condensin II, which has the same two SMC subunits as condensin I but a different set of non‐SMC subunits (CAP‐D3, ‐G2 and ‐H2), was found in vertebrate cells (Ono et al, 2003; Yeong et al, 2003). Condensin II contributes to mitotic chromosome assembly in a manner distinct from that of condensin I (Ono et al, 2003; Hirota et al, 2004; Gerlich et al, 2006), but its biochemical activities are poorly characterized. Condensin II associates with chromatin throughout the cell cycle, whereas condensin I targets mitotic chromosomes after nuclear envelope breakdown and dissociates from chromosomes during anaphase (Hirano, 2005). However, it has been reported that a small amount of condensin I persists within the nucleus during interphase (Schmiesing et al, 2000; Cabello et al, 2001). In addition to their mitotic functions, condensins I and II have been implicated in chromatin regulation during interphase. For example, condensin is required for transcriptional repression in Saccharomyces cerevisiae and Drosophila (Bhalla et al, 2002; Dej et al, 2004; Machin et al, 2004). In S. pombe, the condensin I subunit, Cnd2, is involved in the DNA checkpoint response and the DNA excision repair pathway (Aono et al, 2002). Human condensin I interacts with the PARP–1–XRCC1 complex and plays a role in the repair of DNA single‐strand breaks (Heale et al, 2006).
We investigated the regulation of the human condensin I complex during interphase. We previously reported that human condensin I is phosphorylated throughout the cell cycle (Takemoto et al, 2004). However, the sites at which condensin I is phosphorylated in interphase cells differ from those recognized by Cdc2 during mitosis (Takemoto et al, 2004). Now, we have found that CK2 is the main kinase that phosphorylates condensin I during interphase. CK2‐mediated phosphorylation during interphase greatly reduced its supercoiling activity, in contrast to the slight stimulatory effect of mitosis‐specific phosphorylation by Cdc2. In this report, we describe the effect of CK2‐mediated phosphorylation of condensin I on cell‐cycle regulation and discuss its biological significance.
Supercoiling activity of condensin I is suppressed by phosphorylation during interphase
We previously reported that condensin I is phosphorylated in logarithmically growing (log‐phase) cells and in mitotic HeLa cells, but specific sites detected by the phospho‐specific monoclonal antibody MPM‐2 are phosphorylated only during mitosis (Takemoto et al, 2004). Very little is known about the effects of interphase phosphorylation on the functions of condensin I, whereas mitotic Cdc2‐dependent phosphorylation stimulates the supercoiling activity of condensin I in Xenopus or human (Kimura et al, 1998, 2001) and is required for nuclear targeting of condensin I in S. pombe (Sutani et al, 1999). To characterize the roles of phosphorylation during interphase, we purified condensin I from log‐phase cells (in which more than 95% cells were in interphase) and mitotic HeLa cells and assessed its phosphorylation status and the effects of phosphorylation on its biochemical activities.
We first compared the phosphorylation levels of the interphase and mitotic forms of condensin I using Pro‐Q Diamond phosphoprotein gel stain, which detects overall phosphorylation (Schulenberg et al, 2003). Four of the five subunits (hCAP‐C, ‐D2, ‐G, and ‐H) were phosphorylated in both forms of condensin I, and their phosphorylation levels were indistinguishable (Figure 1A). However, two‐dimensional tryptic phosphopeptide mapping (Russo et al, 1992) revealed that, for each subunit, the sites that were phosphorylated during interphase differed from those that were phosphorylated during mitosis (Figure 1B).
To test the effect of interphase and mitotic phosphorylation on supercoiling activity, both forms of condensin I were dephosphorylated with lambda phosphatase (λ‐PPase) (Figure 1A, lanes 4 and 8). Removal of phosphates from the interphase form of condensin I resulted in a great increase in its supercoiling activity (see Figure 1C, upper panel), whereas dephosphorylation of mitotic condensin I resulted in a slight decrease in supercoiling activity (Figure 1C, upper panel). In contrast, the DNA‐binding activities of the interphase and mitotic forms of condensin I were almost unaffected by dephosphorylation (Figure 1C, lower panel).
To further investigate the effects of interphase phosphorylation, the supercoiling activity and phosphorylation status of condensin I were determined after incubation of dephosphorylated condensin I with an interphase Xenopus egg extract and subsequent repurification. This treatment resulted in a decrease in supercoiling activity to a level similar to that of interphase condensin I before dephosphorylation (Figure 1D) accompanied by subunit rephosphorylation. On the other hand, supercoiling activity was increased by treatment with a mitotic egg extract (data not shown). These results suggest that supercoiling activity is greatly suppressed by interphase‐specific phosphorylation. This is the first demonstration of negative regulation of condensin I.
Condensin I is phosphorylated by CK2 during interphase
In an attempt to identify the kinase or kinases that phosphorylate and inactivate condensin I during interphase, 4,5,6,7‐tetrabromobenzotriazole (TBB), an inhibitor of CK2, was added to the log‐phase HeLa cell culture because CK2 is abundant in eukaryotic cells, is constitutively active during the cell cycle, and has a wide spectrum of substrates (Litchfield, 2003; Meggio and Pinna, 2003). Condensin I in log‐phase HeLa cells was hardly phosphorylated in the presence of TBB (Figure 2A), suggesting that CK2 is a likely candidate for phosphorylation of condensin I during interphase.
To confirm that CK2 phosphorylates condensin I during interphase, we used a Xenopus egg extract because it faithfully reproduces cell cycle events and contains a large stockpile of structural proteins and regulatory factors. We first examined the effect of immunodepletion of CK2 from this extract. When dephosphorylated condensin I was incubated with an interphase egg extract, it was rephosphorylated to a level similar to that of the interphase complex, but it was almost unaffected by the CK2‐depleted interphase extract (Figure 2B, lane 6). When purified CK2 was added to the CK2‐depleted interphase extract, condensin I was rephosphorylated (Figure 2B, lane 7). In contrast, condensin I was phosphorylated in the mitotic extract depleted of CK2, even though the phosphorylation level was reduced (Figure 2B, lane 9). These results suggest that condensin I is phosphorylated by CK2 alone or by kinases activated by CK2 in the interphase extract, but is also phosphorylated by other kinases independent of CK2 in the mitotic extract, as has been reported for Cdc2 (Kimura et al, 1998, 2001), in addition to CK2.
As condensin I subunits have several consensus phosphorylation sites that are recognized by CK2, we examined whether purified CK2 could phosphorylate condensin I. Purified CK2 phosphorylated the hCAP‐C, ‐D2, ‐G and ‐H subunits of dephosphorylated condensin I, but not the sites that were recognized by MPM‐2, indicating that CK2 phosphorylates these subunits at sites other than those phosphorylated by Cdc2 (Figure 3A). Two‐dimensional tryptic phosphopeptide mapping indicated that the some spots of the phosphopeptides (Figure 3B, dashed circle) of the hCAP‐C, ‐D2, ‐G and ‐H subunits phosphorylated by CK2 resembled those labeled in log‐phase cells (compare Figure 3B with Figure 1B, upper panel). In contrast, the hCAP‐D2, ‐G and ‐H subunits of condensin I were phosphorylated by Cdc2, and the phosphopeptide patterns of the subunits phosphorylated by Cdc2 were similar to those labeled in mitotic cells (data not shown).
Next, we tested whether the supercoiling activity of condensin I was reduced by CK2 phosphorylation. When dephosphorylated condensin I was treated with CK2, its supercoiling activity was decreased to a level similar to that of interphase condensin I before dephosphorylation (Figure 3C). In contrast, the supercoiling activity of interphase condensin I was stimulated by Cdc2 treatment, but this activity was still weaker than that of the dephosphorylated form of condensin I (Figure 3C, lane 7). When dephosphorylated condensin I was phosphorylated by Cdc2, its supercoiling activity increased further (Figure 3C, lane 8) to a level similar to that of mitotic condensin I (data not shown), which suggests that both phosphorylation by Cdc2 and dephosphorylation of CK2‐specific sites are required for full activation of condensin I. Taken together, we consider that some sites are labeled by CK2 both in vivo and in vitro, and these sites are implicated in the inactivation of condensin I during interphase.
To test further the role of CK2‐mediated phosphorylation on the regulation of condensin I, dephosphorylated condensin I was treated by any combinations of CK2 and Cdc2 (Figure 3D). The Cdc2‐mediated phosphorylation level was not reduced by CK2‐mediated phosphorylation, as detected by immunoblotting using MPM‐2 that recognizes phosphorylation of Cdk‐consensus sequence (Figure 3D, upper). However, the stimulation of its supercoiling activity was weak when it was phosphorylated by CK2 and followed by Cdc2 (Figure 3D, lower, lanes 2 and 3). Alternatively, condensin I, which had been activated by Cdc2, was greatly inactivated by subsequent phosphorylation by CK2 (Figure 3D, lower, lanes 4 and 5). Thus, CK2‐mediated phosphorylation reduces the supercoiling activity of condensin I in opposition to its stimulation by Cdc2. The similar results were obtained as to another Cdk kinase, Cdk2/CyclinA, that are active during interphase (Supplementary Figure 1).
Dephosphorylation of CK2 consensus sites is insufficient for mitotic chromosome condensation
We then determined whether the dephosphorylated form of condensin I could support chromosome condensation in interphase Xenopus egg extracts lacking Cdc2 activity because the supercoiling activity of dephosphorylated condensin I was almost as strong as that of the mitotic form (Figure 1C). To manipulate the phosphorylation level of condensin I in extracts, we tested several protocols for differentially immunodepleting and restoring purified condensin I. The efficiency of immunodepletion was monitored using immunoblotting (Figure 4A). We did not expect the CK2‐specific sites of dephosphorylated condensin I to be phosphorylated after exposure to condensin‐ and CK2‐depleted extracts.
When sperm chromatin was incubated in a control mitotic extract as a positive control, it was converted into a cluster of mitotic chromosomes (Figure 4B, panel 1). Chromosome condensation was not observed in condensin‐depleted and condensin‐ and CK2‐depleted mitotic extracts (Figure 4B, panels 2 and 5). When purified condensin I from HeLa cell extracts in either the interphase or dephosphorylated forms was included with condensin‐depleted mitotic extracts, chromosome condensation was restored (Figure 4B, panels 3, 4, 6, and 7). Thus, CK2 depletion does not affect chromosome condensation in mitotic extracts. On the other hand, chromosome condensation did not occur in interphase extracts under any conditions (Figure 4B, panels 8–14), even when dephosphorylated condensin I was added to a condensin‐ and CK2‐depleted extract in which condensin I was not phosphorylated (Figure 4B, panel 14). We also examined time‐course experiments using 'interphase extract that was depleted of condensin I, and added back log phase condensin I', and 'interphase extract that was depleted of CK2 and condensin, and added back dephosphorylated condensin I'; however, chromosome condensation did not occur at any time points (Supplementary Figure 2). Therefore, we examined the association of condensin I with chromatin using quantitative immunoblotting. The amount of condensin I bound to sperm chromatin in interphase extracts was <10% of that of mitotic extracts under all conditions (Figure 4C). Depletion of Cdc2 from mitotic extract also resulted in the great reduction of chromosomal binding of condensin I (Supplementary Figure 3).
These results suggest that Cdc2 activity is required for most chromosomal binding activity of condensin I in Xenopus egg extract. Therefore, sperm chromatin is not converted into mitotic chromosome‐like structures in interphase extracts that lack Cdc2 activity, even when the supercoiling activity is stimulated by dephosphorylation. However, when dephosphorylated condensin I was included in a CK2‐depleted interphase extract, the amount of condensin I targeted to sperm chromatin was more than twice that observed when its phosphorylated interphase form was included (Figure 4C). Moreover, the supercoiling activity of dephosphorylated condensin I was more than four times stronger than that of interphase condensin I. Thus, it is tempting to speculate that CK2‐dependent phosphorylation of condensin I may control chromatin structure and function during interphase by regulating the chromatin‐binding and supercoiling activities of condensin I.
The CK2‐specific consensus sites in condensin I are dephosphorylated on mitotic chromosomes
To determine if the CK2‐specific consensus sites in condensin I subunits are phosphorylated in HeLa cells, we synthesized phosphopeptides containing single phosphoserine or phosphothreonine residues and prepared phospho‐specific antibodies. Among them, an antibody against the hCAP‐H sequence (PGLQAADphosphoS570DDEDLDD) was available for detection of phospho‐epitopes. The affinity‐purified antiphospho‐hCAP‐H antibody recognized hCAP‐H prepared from log‐phase cells (Figure 5A). Phosphorylation‐specific signals were blocked by the corresponding antigen peptide, but not by an unphosphorylated peptide of the same sequence (data not shown). λ‐PPase treatment resulted in a loss of phospho‐epitopes, and incubation of dephosphorylated condensin I with purified CK2 resulted in phosphorylation of the epitopes (Figure 5A). When dephosphorylated condensin I was treated with Cdc2 or Cdk2/CyclinA, this site was not phosphorylated (Figure 5B). Thus, it is likely that this phosphorylation site is specific for CK2.
We tested the phosphorylation level of this CK2‐specific site of condensin I purified from log‐phase or mitotic cells by immunoblotting using the antiphospho‐hCAP‐H antibody. The phosphorylation‐specific signal of the mitotic form of condensin I was about half as strong as that of interphase condensin I (Figure 5C). As a portion of condensin I is associated with chromosomes in mitotic cells (Schmiesing et al, 2000; Takemoto et al, 2004), we compared the extent of phosphorylation of this site in chromosome‐bound and ‐unbound condensin I in mitotic cells. The phosphorylation‐specific signal of condensin I associated with chromosomes was much weaker than that of chromosome‐unbound condensin I in mitotic cells (Figure 5D). This regulation is in contrast to that by Cdc2; chromosome‐bound condensin I has stronger phospho‐epitopes to MPM‐2, which recognizes the Cdc2‐phosphorylation sites, than chromosome‐unbound form (Takemoto et al, 2004). As a positive control for subcellular fractionation, we carried out immunoblot analyses with antibodies against histone H3 and lamin B1 (data not shown). In contrast, the CK2‐mediated phospho‐signal of condensin I purified from chromatin fraction was comparable to that of chromatin‐unbound condensin I in log‐phase cells (Supplementary Figure 4). Thus, this CK2‐mediated site was dephosphorylated efficiently only on mitotic chromosomes.
Because the CK2‐specific phosphorylation site present in the hCAP‐H subunit is also conserved in Xenopus, we determined whether this site is also phosphorylated in Xenopus egg extracts. The phospho‐hCAP‐H‐specific antibody also recognized phosphorylation of the corresponding site of the XCAP‐H subunit of Xenopus condensin I. Sperm chromatin was incubated in a mitotic Xenopus egg extract, and the CK2 phosphorylation status of chromatin‐bound and chromatin‐unbound condensin I was examined. Interestingly, the phosphorylation level of the CK2 site of chromatin‐bound condensin I was also much lower than that of the chromatin‐unbound form in the mitotic Xenopus egg extract (Figure 6A). When purified human condensin I was supplemented in the condensin I‐depleted mitotic Xenopus egg extract (Figure 4B), the CK2 site was also efficiently dephosphorylated (Supplementary Figure 5).
Taken together, these results suggest that the dephosphorylation of CK2‐specific phosphorylation sites on chromosomes may be a mitotic regulatory mechanism. Because dephosphorylation of CK2‐specific phosphorylation sites greatly increases the supercoiling activity of condensin I, this regulatory process may be required for mitotic chromosome condensation.
To test this hypothesis, a 10‐fold excess of purified CK2 (relative to the endogenous level) was added to a mitotic extract. The chromosomes that assembled in the CK2‐supplemented mitotic extract were longer, thinner, and more partially condensed than those that assembled in a normal mitotic extract (Figure 6B). To characterize and quantify the observed morphological defect caused by CK2‐addition, the length of the chromosomes assembled in the control and CK2‐supplemented extracts was measured as described (Maresca et al, 2005). On average, chromosome length in the CK2‐supplemented extract was about two‐fold longer than in the control extract, indicating that chromosomes assembled in the CK2‐supplemented extract were less condensed (Figure 6C). This effect was accompanied by an increase in the extent of phosphorylation of the CK2 site (Figure 6D). Quantitative immunoblotting indicated that about 50% of the condensin I on chromosomes assembled in the CK2‐supplemented extract was phosphorylated (Supplementary Figure 6). Time‐course experiment showed that the chromosomes assembled in the CK2‐supplemented extract exhibited morphological defects that were characterized by elongated chromosomes over the time, in contrast to the sperm chromatin being converted to compacted chromosomes within 2 h in the normal mitotic extract (–CK2) (Supplementary Figure 7). All condensin I and II subunits were properly targeted to the aberrant chromosomes (data not shown).
These results suggest that, in addition to phosphorylation mediated by Cdc2, dephosphorylation of the CK2‐specific sites of condensin I may be required for complete condensation of mitotic chromosomes.
Condensin I, a key protein that contributes to mitotic chromosome condensation in vivo, is constitutively present throughout the cell cycle. However, chromatin is relaxed during interphase and is converted into compact rod‐like structures (chromosomes) over a short period during mitosis. Thus, the function of condensin I must be tightly regulated during the cell cycle. The enzymatic activities of condensin I are positively regulated by Cdc2‐induced mitosis‐specific phosphorylation (Kimura et al, 1998, 1999, 2001; Hagstrom et al, 2002). In this study, we demonstrated that the negative regulatory mode of condensin I is induced by CK2‐mediated phosphorylation and that the positive regulatory mode of condensin I is induced by mitosis‐specific Cdc2‐mediated phosphorylation. An updated model for the regulation of condensin I during the cell cycle is shown in Figure 7. Translated condensin I is phosphorylated and inactivated by CK2 in interphase cells. At the onset of mitosis, condensin I is partially activated by Cdc2‐mediated phosphorylation and targeted to mitotic chromosomes, where it is activated further by dephosphorylation of CK2‐specific sites. Alternatively, it is also possible that the CK2 sites of condensin I was dephosphorylated in the cytosol and this form binds and functions on chromosomes (dashed line). Thus, together with Cdc2, CK2 may be a key kinase that regulates condensin I activity.
This is consistent with a previous report, in which the interphase form of condensin I was shown to be activated by Cdc2‐mediated phosphorylation, but the activity was still weaker than that of mitotic form (Kimura et al, 1999). It is possible that other mitotic kinases might be required for the complete activation of condensin I; however, we propose here an alternative model in which dephosphorylation of some sites is important. The result, in which dephosphorylation of mitotic condensin I has a subtle effect on its supercoiling activity, looks conflict with the previous report (Kimura et al, 1998). We consider the reason for the discrepancy is that the involvement of Cdc2‐ and CK2‐mediated phosphorylation in condensin I activity may vary during the development of an organism and between different organisms. When condensin I purified from mitotic HeLa cells is dephosphorylated, the reduction of supercoiling activity of condensin I by the dephosphorylation of Cdc2‐mediated sites may be offset by the stimulation by the dephosphorylation of CK2‐mediated sites; therefore, there is a little change in its activity. Whereas, the effect of Cdc2 may be more dominant in the Xenopus embryonic system, because the Cdc2‐dependent phosphorylation sites are almost fully phosphorylated in Xenopus mitotic egg extract (data not shown). The phosphatase(s) that dephosphorylate CK2‐mediated phosphorylation sites of condensin I on mitotic chromosomes remain to be identified. Okadaic acid, an inhibitor of type 1 and type 2A phosphatase, did not affect the phosphorylation level of the CK2‐dependent site in hCAP‐H attached to mitotic chromosomes (data not shown), suggesting that other types of phosphatase are implicated in this process.
In this study, we demonstrated that condensin I is negatively regulated by CK2. What is the physiological significance of the inhibition of condensin I during interphase? We consider two models: one is that it affects chromatin structure; the other is that it influences interaction of condensin I with other proteins. It is possible that CK2‐mediated phosphorylation contributes to the relaxation of chromatin because condensin I compacts chromatin during mitosis (Figure 7). It is believed that cellular processes, including transcription, repair, and replication, are tightly regulated by chromatin structure. Recently, several lines of evidence have indicated that condensins, in addition to their mitotic functions, play critical roles in interphase. Condensin I is required for gene silencing in S. cerevisiae and Drosophila (Bhalla et al, 2002; Dej et al, 2004; Machin et al, 2004). Condensin I is required for DNA repair and the DNA damage checkpoint response in S. pombe (Aono et al, 2002). Dosage‐compensation factors, which resemble the condensin complex, bind hermaphrodite X chromosomes and reduce gene expression in Caenorhabditis elegans (Chuang et al, 1994; Lieb et al, 1998; Hagstrom and Meyer, 2003). Interestingly, condensin negatively regulates gene expression via transcriptional control in many species. Because condensin I has activity to constrain DNA supercoiling and looping in vitro (Kimura and Hirano, 1997; Bazett‐Jones et al, 2002), it is possible that this activity is required to some extent for compacting chromatin during interphase and for repressing transcription. If so, it is tempting to speculate that CK2‐dependent phosphorylation of condensin I may relax the chromatin structure and stimulate transcription by suppressing supercoiling activity. Recently, it was reported that condensin I interacts with other proteins. For example, condensin I interacts with DNA methyltransferase (Geiman et al, 2004). The CAP‐G subunit of condensin I interacts with protein phosphatase 2A and is related to gene bookmarking (Xing et al, 2005). In Drosophila cells, the Barren (CAP‐H) subunit of condensin I and DNA topoisomerase II colocalize with Polycomb proteins and maintain gene silencing (Lupo et al, 2001). More recently, human condensin I was reported to interact with the poly(ADP‐ribose) polymerase I and XRCC1 complex, and was implicated in the single‐strand‐break repair pathway (Heale et al, 2006). Thus, it is also possible CK2‐mediated phosphorylation regulates the interaction of condensin I with other proteins and influences several chromatin functions.
Condensin I is abundant throughout the cell cycle, but exhibits dynamic cell‐cycle‐specific subcellular localization (Schmiesing et al, 2000; Cabello et al, 2001; Takemoto et al, 2004). Although a considerable amount of condensin I associates with mitotic chromosomes, most condensin I is present in the cytoplasm during interphase. Therefore, the concept that condensin I must be inactivated by the CK2‐mediated phosphorylation may be questioned. However, a subpopulation of condensin I is present in nuclei (Schmiesing et al, 2000; Cabello et al, 2001), suggesting that condensin I may play a role during interphase. This possibility is supported by evidence that condensin I has interphase roles, such as DNA damage checkpoint response, DNA excision repair, DNA single‐strand break repair, and transcriptional repression (Lupo et al, 2001; Aono et al, 2002; Bhalla et al, 2002; Dej et al, 2004; Heale et al, 2006). Thus, it is possible that the small amount of condensin I present in nuclei is sufficient for its interphase functions and that CK2‐mediated phosphorylation regulates these functions. In contrast to condensin I, condensin II is present in nuclei throughout the cell cycle, although the total protein level of condensin II is lower than that of condensin I (Ono et al, 2003, 2004; Yeong et al, 2003; Hirota et al, 2004). Further elucidation of the regulation of condensin II during interphase is warranted as its role may be more important than that of condensin I during this period. Our findings concerning the interphase modification of condensin I provide a framework for such studies.
Finally, it would be important to elucidate the physiological significance of the CK2‐mediated‐phosphorylation in the regulation of the chromatin structure and functions. To this end, identifying and mutagenesis of the physiological relevant targets of CK2 may be the best procedures. However, the work is difficult and will take a lot of time, because condensin I is very large protein complex (670 kDa) and contains a lot of CK2‐consensus sites, and moreover, expression and purification of the recombinant condensin I, especially Xenopus one, are difficult. We consider it as a long‐term goal.
Materials and methods
Rabbit polyclonal antisera were raised against synthetic peptides corresponding to the N‐terminal sequences of CK2α (MSGPVPSRARVYTDC), the C‐terminal sequence of Xenopus CK2β (CASNFKSPVKTMR), and human CK2β (CASNFKSPVKTIR), as described previously (Hirano et al, 1997; Kimura and Hirano, 1997). For phospho‐specific antibodies, phosphopeptides bearing the CK2‐specific consensus sequences of the hCAP‐C, ‐D2, ‐G, and ‐H subunits and the corresponding unmodified peptides were synthesized. The sequences of the phosphopeptides were as follows: CTEKEIKDphosphoTEKEVDDL for hCAP‐C, CVFSSDESphosphoSEEDLSAE for hCAP‐D2, CTAEADSEphosphoSDHEVPEP for hCAP‐G, and CPGLQAADphosphoSDDEDLDD for hCAP‐H. Phospho‐specific antibodies were affinity purified as described (Kimura et al, 1998). Among them, a phospho‐specific antibody against the hCAP‐H subunit was available for detecting CK2‐dependent phosphorylation.
Cell culture
HeLa S3 cells were cultured in suspension at 37°C in Dulbecco's modified Eagle's minimal essential medium containing 100 U/ml penicillin G potassium, 100 mg/ml streptomycin sulfate, and 5% calf serum. Mitotic cells were collected by adding 150 ng/ml TN‐16 (Wako) to logarithmically growing HeLa S3 cells (3 × 105 cells/ml) and incubating for 24 h, as described previously (Takemoto et al, 2004). 32P‐Labeling was performed as described (Takemoto et al, 2004).
Supercoiling and DNA‐binding assays
The supercoiling assay was performed as described elsewhere (Takemoto et al, 2004). For quantification, the relative amount of each topoisomer was measured by LAS‐1000 (FYJIFILM), and the mean superhelical tension was calculated. The DNA‐binding assay was conducted as described previously (Kimura and Hirano, 1997).
Phosphorylation of dephosphorylated condensin I in Xenopus egg extracts
Dephosphorylated condensin I was incubated with 100 μl interphase or mitotic Xenopus egg extract at 22°C for 30 min and purified using the anti‐hCAP‐G antibody coupled to protein A Sepharose beads, as described for purification of condensin I.
Detection of total phosphorylation
The total phosphorylation level of condensin I was determined using Pro‐Q Diamond phosphoprotein gel stain (Molecular Probes) as described (Schulenberg et al, 2003).
Immunodepletion and rescue
Condensin I was immunodepleted from Xenopus egg extracts as previously described (Hirano et al, 1997; Kimura and Hirano, 2000). For CK2 immunodepletion, 150 μg affinity‐purified anti‐CK2α and 65 μg anti‐CK2β was incubated with 10 μl protein A Sepharose beads (Amersham Biosciences) for 1 h. The antibody‐coupled beads were washed with XBE2 (50 mM HEPES (pH 7.7), 10 mM EGTA, 2 mM MgCl2, 0.1 mM CaCl2, and 1.7% sucrose), 100 μl Xenopus egg extracts was added, and the mixtures were incubated on a rotating wheel at 4°C for 1 h. The supernatants were recovered and incubated with a fresh batch of the same beads. After 1‐h incubation, the supernatants were recovered and used as depleted extracts. For rescue, purified condensin I or the CK2 fraction was prepared in XBE2 with 5 mM dithiothreitol and added to the depleted extracts at the same concentration as for the endogenous protein.
Supplementary data
Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org).
Supplementary Information
Supplementary Figure 1 [emboj7601394-sup-0001.pdf]
Supplementary Figure 2 [emboj7601394-sup-0002.pdf]
Supplementary Figure 3 [emboj7601394-sup-0003.pdf]
Supplementary Figure 4 [emboj7601394-sup-0004.pdf]
Supplementary Figure 5 [emboj7601394-sup-0005.pdf]
Supplementary Figure 6 [emboj7601394-sup-0006.pdf]
Supplementary Figure 7 [emboj7601394-sup-0007.pdf]
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and from the Solution Oriented Research for Science and Technology (SORST) from the Japan Science and Technology Agency. This work was also supported by the Bioarchitect II Research Project of RIKEN.
- Copyright © 2006 European Molecular Biology Organization


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Prel. Doc. No 3 of August 2003 - Succinct explanations in preparation of the Special Commission
|Practical operation documents
|Year of publication
|City of publication
|The Hague
|Description (or download in PDF, if available)
In August 2003, the Permanent Bureau issued a document to Member States and States Parties to the Apostille Convention providing succinct explanations relating to the Convention as well as a short questionnaire in preparation for the Special Commission on the practical operation of the Convention, which took place in October/November 2003.

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Strider Legolas Hamm, age 15, of Kokomo, crossed over the rainbow bridge on December 21, 2018.
Strider (AKA "Stri"), was born in February of 2003. When he was 6 weeks old he chose his owner, Christian, by jumping in his lap and showering him with kisses. At the time, Christian was 6 years old….and those moments were the beginning of their beautiful bond.
In Christian's words: "Strider was my best friend! I've spent the majority of my life having him around. He has shared every happy moment with me, as well as every heartbreak. And, in all the moments in between, he has proven his loving, gently and kind nature."
Strider was very special! He loved going for walks on any surface he could get his paws on. He was a snuggler, and really enjoyed laying around and having cuddle time with his family. Strider was also a bit of a ladies' man, and quickly made friends with every female who entered the home (furry or human). He loved to be fed ANY type of human food, and absolutely loved beggin strips.
Strider was sweet and happy, but he was also brave, tough and protective of his family! One day when Strider was about 8 years old, he and Christian were outside when all of the sudden a ferret showed up in their yard. Instinctively, Christian tried to scoop Strider up to protect him. However, Strider jumped off the porch before Christian could get to him! He quickly grabbed that ferret by the neck, showing him who was boss!
Strider showed his loyal and protective nature many times through the years. Last year this came to light in a very special way when he had the privilege of seeing Christian become a father. From day one, when Christian and Amelia brought little Emmeline home from the hospital, Strider was there by her side loving her and loyally protecting her….just as he had her father for so many years. This amazing animal will be so greatly missed by his family!
Strider's human survivors are his mom and dad, Christian & Amelia, his baby, Emmeline, his nana and pap, Mike & Coby, and his uncles Anthony and Daniel.
Strider is survived by furry brothers: Rocky, Ollie and Stripes; and one furry sister, Sophie.
Please feel free to share a special memory of Strider with his family on his tribute wall.


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About Blog – Urban Survival Skills is an information site on Urban and Suburban Survival topics such as Survival Planning and Preparation, Survival Skills, Survival Tools, Survial Training, Survival Skills and Knowledge, Survival Gear and Equipment, Survival Books, Survival Links.
All yer apps! Y IN HELL WOULD I BUY? YOU HAVENT FIXED THE LANDSCAPE MODE IN THE PRO VERSION! None of your apps do landscape IN ALL VIEWS, get on it! NOT JUST READING VIEW, that's FUBAR!! Then I will pay. Also, ALL pictures must be zoomable – currently only two sizes, and that doesnt work. Nobody would b able to read the tiny printing in illistrations – is thatstanding line? As in dead line, correct?
The "important" people who live on the east and west coasts of the USA like to refer to the rest of the country as "flyover country", because the only time they see it is when they fly over it while traveling from one coast to the other. That…
About Blog – The mission of the team behind Survival-Nation is to build a network of like-minded preppers to share resources, products and ideas. They want to continue to thrive even in the face of economic collapse whether it be by natural disaster or human destruction. They want your family to thrive and survive too.
I've taken the time to organize the gear into different categories for your convenience: to make browsing much easier. Check out the list and tell us if there's anything you think you should add to your own personal list of survival gear!
This 15 inch survival knife with drop point blade features a thick quality stainless steel blade with serrated top edge. Textured and ribbed solid metal handle and guard. Nylon sheath. Survival kit includes a hollow grip with a compass top to store items within the knife itself, as well as additional pouches on the sheath hold the rest. Complete survival kit.
As we discussed above, staying warm is your priority. Adding insulation to your clothing is always a welcome idea. Dry leaves on the floor of the woods make excellent insulators. All you need to do is stuff them into your jacket. They form an insulating layer between your body and the outside cold air, trapping your body heat.
Rather than MRE's, check out your local LDS/Mormon "Bishop's Warehouse" for cans & Mylar bags of bulk and dehydrated foods. The cans are designed for 30+ year storage and the Mylar bags are perfect for hiking, camping, and survival. They are a lot cheaper than MRE's. You can even buy some Mylar bags and make your own "meals in a bag" for even less money.
If you only remember one thing from this wilderness survival guide, it's this: The #1 wilderness survival priority is maintaining a calm center. Keeping your head on your shoulders. Staying cool. Sustaining a positive mental attitude.
Survival Power: Snare placement can be a key factor to whether or not you catch anything; your ability to know where to set snares relies on you learning and practicing basic trapping skills for capturing small game. Baiting traps with common game foods like nuts, seeds, and berries (and artificial baits) can help boost your snaring success.


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Pay Per Click ManagementOur PPC marketing strategies are 100% focused on ROI. Save money and time by working with our team of online advertising experts. Work With Us
PPC Management Agency In Miami
Pay-per-click Management (PPC) marketing is a type of digital marketing performed on platforms like Google Ads, Facebook, and Bing. You (as the advertiser) will only pay for clicks, or when a user clicks through to your desired landing page. It's a strategy of purchasing traffic to your website vs. earning visitors through organic methods, such as SEO.
One of the many ways we accomplish this growth strategy is by providing each of our PPC clients with a single point of contact to manage your project and your questions.
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- It is designed for recycled PSF and its material is clean and without label. It is used to recycle small capacity and stable quality raw material. The following is the process chart.
PET - 0.3 PET瓶片清洗线 (Material: Clean and without label)
No. Item Usage Quality Remark 1 Feeder Feeder 1 CS 2 Crusher Crush the bottle 1 CS 3 Screw Conveyer Feeder 1 SS or CS 4 Hot Washer Clean with hot water 1 SS or CS 5 Screw Conveyer Feeder 1 SS or CS 6 Friction Machine Clean 1 SS or CS 7 Floating Washer Clean with cooling water 2 SS or CS 8 Dryer Dry the PET flake 1 SS or CS


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Jeet Gayi Toh Piyaa Morre 21st June 2018 Written Episode Update: Varun Announces His Engagement With Dhani
Jeet Gayi Toh Piyaa Morre 21st June 2018 Written Episode, Written Update on TellyUpdates.com
Varun walks lifting Devika. Devika says he is so good to help maid. Varun says she is in pain. She says who is she. He reminisces their earlier meeting when he falls for her and she resists. He asks hum apke hain kaun. She says lucky no time phor/for luv, Inglisss/English, etc. He gets out of flashback. She asks why he helped her. He drops her on bed and says he would have done it to any human. She asks to stop worrying about her then. He shouts she is blabbering again and leaves. Varun punches sand bag reminiscing time spent with Devikaa. Ye Malal Ishq. He continues punching bag angrily.
Varun then sits for breakfast with family. He sees his favorite breakfast and excitedly thanks Dhani. Devika says she prepared it to celebration him getting a movie role. Varun angrily
Pandi comes and checks kundalis to fix muhurath. Devika while serving him water murmurs pandit will be finished if he finds mangalik girl's muhurath. Pandit gets afraid. Kesar yells what is she murmuring in Pandit's ears. Devikaa asks Pandit if she did. Pandit says muhurath is after 1 year. Varun enters and says engagement is tomorrow itself. Devika signals Pandit and revolves finger around her neck. Pandit says there will be big problems if engagement happens tomorrow. Varun says he does not care and says engagement is tomorrow itself.
Kesar keeps Varun's engagement suit in his room while Varun is busy speaking to film director. Devika enters and looks at suit. Varun enters and she slips. He holds her. She thanks him. He says she was about to fall. She suggests him not to get engaged. He asks what. She says fans don't like married hero, so he should wait till 2-3 more film releases. He angry. Their nok jhok continues. They both fall on bed. Dhani sees that and fumes in jealousy.
Preca: Devika tells Varun that Dhani is not opening door, guests have come, so she has solution for him, he can get engaged to her. Varun angrily says already she left him once, she will again. Devika says he lost his memory, then how does he remember this.
Update Credit to: MA


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INGRID 2011 is 6th edition of annual workshop devoted to e-Science and e-Infrastructure aspects.
In 2011 INGRID is hosted by Instituto de Fisica de Cantabria. It will take place in Santander, Spain in June 6-8, 2011.
Sensor Networks
Davide Adami Dipartimento di Ingegneria dell'InformazioneVia G. Caruso 16 - 56122 - Pisa, Italy
AbstractWill be available soon.
INGRID 2011 - June 6-8, 2011 valid xhtml
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This authentically-designed two-story Victorian is nestled in a neighborhood of stately homes with each generating its own unique personality. Our client called upon us to create a spectacular backyard worthy of this dramatic home.
The existing back porch was tripled in size to function as an indoor/outdoor space with special attention given to the integration of the same details a Victorian deserves. A raised colonnade was built to welcome family and friends to the pool house and summer kitchen.
An Old-Chicago Brick terrace and deck frame the oversized pool while your eye is drawn to the centerpiece of the backyard: A free-standing masonry outdoor fireplace with 15' chimney.


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- The Health System Transformation Space
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Assynt House
Assynt House is an exclusive use wedding venue in the Highlands of Scotland, weddings for up to 50 guests are accommodated in luxury, with fabulous food and canaps and a warm welcoming atmosphere.
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My celestial kits are here! Smudging is a natural way to cleanse energy and a lovely ritual to complete during the moon cycle or anytime you feel the need for a refresh. (You will not receive the exact pieces in the photo but they will be of comparable size.)
Included in my large sized kits are
1 Abalone Shell (6" - 8")
1 Organic Smudge Stick (4" - 6") OR 1 Palo Santo (3" - 4")
1 Box Wooden Matches
1 Jar of Sand (3")
1 Selenite Wand (4" - 5") OR 1 Rose Quartz (2" - 3")
1 Clay Marbled Moon
1 Instruction Kit Card with uses/meanings of each of the above pieces
Sage (can include rosemary and/or lavender) - organically grown in Central California
Rosemary ONLY - organically grown in Central California
Palo Santo - wild harvested from Ecuador
Sand - from the beach in Malibu, CA
If you would like both a smudge stick/palo santo or both selenite/rose quartz you can add them in the drop down menu for an extra $5 each.
To add on an amethyst or quartz crystal to your celestial kit use the listing below
*If you are looking for smaller or bigger sized shells, sage or crystals check out my Medium and Extra Large celestial kits below.
MEDIUM CELESTIAL KIT - https://www.etsy.com/listing/577754748/medium-celestial-kit?ref=shop_home_active_1
EXTRA LARGE CELESTIAL KIT - https://www.etsy.com/listing/591564679/extra-large-celestial-kit?ref=shop_home_active_1
3-7 business days
Buyers are responsible for any customs and import taxes that may apply. I'm not responsible for delays due to customs.
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Ship items back to me within: 14 days of delivery
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Contributed By Florent (Amsterdam - HQ)
See also 7.1 Types of Statutory Voluntary and Involuntary Insolvency and Liquidation Proceedings. The bankruptcy trustee will consider the various available alternatives for liquidation of the bankrupt estate, including the transfer of the business as a going concern by way of an asset sale. The bankruptcy trustee can be quite flexible in entering into an agreement with a potential purchaser, provided that the agreed transaction is in the best interest of the joint creditors of the bankrupt debtor. A sale of assets requires the prior authorisation of the supervisory judge. The bankruptcy trustee will also have to take into account the interests of secured creditors, who have a strong legal position in the process of selling assets over which they may have security. In practice, this means that the bankruptcy trustee needs the approval of a secured creditor before selling a secured asset to a purchaser. If the security rights of the creditor are recognised by the bankruptcy trustee, the creditor can claim the relevant part of the proceeds, while the bankruptcy trustee will request a fee from the secured creditor for the trustee's co-operation in facilitating the sale of the secured asset to the purchaser. Dutch law does not have any rules preventing an existing secured or unsecured creditor from participating in the sale process as a (potential) purchaser. As a rule, the bankruptcy trustee will not give any representations and warranties to a purchaser. Finally, a bankruptcy trustee is not bound by any pre-negotiated and pre-insolvency sales transaction of assets, except perhaps in pre-pack situations, to the extent that the prospective bankruptcy trustee (beoogd curator) made certain undertakings – which is generally unlikely.
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VLB (Techwell chip)
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VideoHive Doctor Writes a Prescription 12437508
Stock Footage \ Medical
Medical \ Prescription \ Doctor \ Background \ Clinic \ Uniform \ Hospital
Stock Footage - Doctor Writes a Prescription 12437508 by kycheryavuy @ VideoHive
Stock Footage \ Medical
A doctor sits at a table elite clinic and writes a prescription.
Video can be used for medical purposes.
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Alpha Channel: No; Looped Video: No; Frame Rate: 29.97; Resolution: 1920x1080; Video Encoding: H.264; File Size: 118mb; Number of Clips: 1; Total Clip(s) Length: 0:13; Source Audio: No; Color: Full Color; Setting: Indoors; Pace: Real Time; Composition: Mid Shot; No.of People: 1; Gender: Female; Age: Young Adult, Middle Aged.
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Baker's Twine - Mandarin Orange
Beautiful Baker's twine for all your craft projects. Use it to add that special touch to presents, attach gift tags, fancy decorations for parties and weddings and perfect for card making and scrapbooking. However you use it, the uniquely twisted twine in so many wonderful colours will add that touch of class you've been looking for!
Eco-friendly: 100% cotton and totally biodegradable.
Dimensions: approx 220 metres of twine, spool size - 10cm x 5cm
Colour: mandarin orange - orange and white cotton twine


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Implied Consent Law in Tennessee
Few statutes are as complex, and generate as much litigation, as the implied consent statute (Tennessee Code Annotated 55-10-406) in Tennessee. The purpose of the statute is to advise officers and citizens of the circumstances in which an officer can request, and sometimes require, a person to submit to a chemical test (blood or breath) to determine the amount of alcohol in their system at the time. In laymen's terms, "implied consent" means that when a person drives on a public road in Tennessee, that person is implicitly consenting to giving a breath test if an officer has "probable cause" to believe that the person has committed DUI or a related offense, such as vehicular assault or vehicular homicide.
In July of 2017, the Implied Consent Statute was the subject of sweeping changes that altered the procedure for chemical tests and the consequences for refusing them. In line with recent Tennessee and United States' Supreme Court decisions, the new Tennessee Implied Consent Statute now provides specific guidance for the administration of blood and breath tests.
In short, it the new Tennessee Implied Consent Statute makes it easier for an officer to administer a breath test to individuals that they believe are operating a vehicle, or have operated a vehicle, under the influence of an intoxicant. An "intoxicant", can include alcohol, narcotic drugs, or any substance that affects the central nervous system. If the person refuses the breath test, and the officer has properly advised that person of the consequences for refusing the test, then the refusal will result in the suspension of the person's license for one year, among other consequences.
Under the new Tennessee Implied Consent Statute, it is now a misdemeanor criminal offense to refuse to refuse a breath test in certain circumstances. Most notably, an individual may now be charged with a Class A Misdemeanor for refusing a breath test if:
- The individual was involved in an accident that resulted in the injury or death of another person.
- The officer has properly procured a lawful search warrant.
- The individual has a prior conviction for DUI or a related offense.
- The individual is travelling with a passenger younger than 16.
An individual may also be charged with a Class A Misdemeanor for refusing a blood test if the officer has procured a lawful search warrant or if "one (1) or more of the recognized exigent circumstances to the search warrant requirement exist." However the new statute does clarify that an officer may not rely on "implied consent" to justify a blood draw. Rather, the subject must give actual, voluntary consent, or the officer is required to get a warrant, unless there are "exigent circumstances" that make it nearly impossible for the officer to get a warrant.
What does all this mean to a person who just wants to know what to say when an officer asks if you will agree to a blood draw or breath test? The answer is complicated, and will vary on a case-by-case basis. Protecting yourself in these situations means hiring an attorney who understands Tennessee Implied Consent and DUI law, and can use that knowledge to limit the damage to your life personally and professionally. If you or a loved one has been charged with an alcohol related driving offense, contact the attorneys at the Law Office of James A.H. Bell as soon as possible at (865) 637-2900.


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Edited by Scott Morgan
#b#Climate , Cybersecurity At Princeton University#/b#
Princeton University's Carbon Mitigation Initiative (CMI), a program designed to study climate change that was launched in 2000 with the financial backing of BP, will get $11 million from BP as part of an extension of the partnership.
In 2000 BP gave the CMI $19 million for a 10-year research agreement. In 2008 a five-year extension of the partnership was announced, but no dollar figure had been decided upon. The extended partnership will run through 2015.
According to BP, the CMI is designed to clarify scientific and policy issues associated with supplying the world's increasing energy consumption as well as its need to lower carbon emissions. The research involves a combination of experimental and modeling investigations into such avenues as increasing ocean acidification and its effects on ocean life.
Also, the extension is expected to encourage a harder look at carbon dioxide capture and storage at coal-plus-biomass synthetic fuels plants. This is particularly important to the futures of the world's two largest coal users, the United States and China.
The CMI program includes 16 faculty and more than 70 research staff and students at Princeton.
Cybersecurity. The university also has been designated one of seven collegiate hubs for cybersecurity research through 2015. The National Security Agency and the federal Department of Homeland Security first announced the intended designation last summer as part of the federal government's bid to use engineering expertise to solve national security problems.
Princeton now is designated a National Center of Academic Excellence in Information Assurance Research, administered by the NSA and the DHS. The program allows Princeton faculty and students to apply to federal agencies for special fellowships, scholarships, internships, and research funding related to information assurance and the practice of protecting sensitive national security data.
#b#NRG's Electric Fuel#/b#
NRG Energy, based at 211 Carnegie Center, has announced plans to build a network of 50 to 150 charging stations for electric cars in Houston, Texas. Throughout 2010 the firm has expanded its westward reach by acquiring alternative and renewable energy concerns in Texas and California and is using Houston as a testing ground. It is also in talks with automakers to develop a consumer package ranging from $49 to $79 a month that would provide access to the network (branded under the name eVgo) and a home charger unit.
CEO David Crane says the network is the first of its kind and is designed to make electric and hybrid cars more practical and affordable. Crane, an outspoken proponent of nuclear power, has written and spoken extensively on the need to end carbon emissions from power generation. His company, he says, produces more carbon than Norway, and the main reason is that carbon-spewing fuel sources (mainly coal) are far cheaper than cleaner alternatives. If the world's energy emissions are to be cleaned, he says, clean-burning fuels need to be cheap and easy.
NRG's plans call for 50 charging stations to be installed by mid-2011. Each charging station would deliver about three minutes of drive time for every minute of charging time, and the stations are meant to be failsafes for customers who did not charge up enough at home. Home chargers will cost $1,500 to $2,000 and customers will sign a three-year subscription agreement to be added to their regular electric bills.
The home chargers run at 220 volts, the same as a clothes dryer or electric oven.
Crane hopes to round up 1,000 subscribers by the end of next year and see the numbers increase as more electric cars roll off the assembly lines. But there are a few bugs to work out of the system — chiefly what to do about non-subscribers who want to hit the pumps. A second question mark is nighttime refueling. At the outset, NRG plans to operate the recharging stations during the day, since demand for fuel diminishes at night. But as more electric cars hit America's roads, NRG would have to generate more power to meet the needs of night travelers.
If that becomes the case, NRG would consider a nuclear power solution, particularly since it is developing a nuclear plant in Texas.
NRG Energy Inc. (NRG), 211 Carnegie Center, Princeton 08540-6213; 609-524-4500; fax, 609-524-4501. David Crane, president and CEO. www.nrgenergy.com.
#b#Isles Becomes SBA Microlender#/b#
The U.S. Small Business Association has given $150,000 to Isles, a Trenton-based nonprofit that specializes in community development, to provide microloans to small businesses.
Businesses can apply for loans of up to $50,000, but Isles plans to offer amounts of $15,000 or less. According to Norma Diaz, Isles' asset-building coordinator, the agency will finance start-ups, expansions, and green enterprises. For additional information on SBA microloans from Isles Community Enterprises, contact Diaz at 609-341-4791 or visit www.isles.org.
#b#New in Town#/b#
York Risk Services Group, 3 AAA Drive, Suite 201, Robbinsville 08691; 609-807-9400; fax, 609-689-0632. Lorraine Blacknik, branch manager. firstname.lastname@example.com.
York Risk Services Group, which provides insurance, risk management, pool administration, and claims management for businesses, has opened an office in Robbinsville. The company is headquartered in Parsippany, but has 37 offices in 13 states.
Tyco International (TYC), 9 Roszel Road, Princeton 08540; 609-720-4200. Edward Breen, CEO. www.tyco.com.
Tyco International, maker of the ADT security system and parent company to valve manufacturer Tyco Flow Control, has acquired the assets of Australia-based Supavac Pty Limited and Supavac Chile SA, a manufacturer of air driven slurry management systems, for an undisclosed sum.
Supavac develops vacuum loading solids pumps, unique air transfer concepts, and slurry dewatering systems that are used for a variety of applications in the oil and gas, industrial manufacturing, mining, water, and commercial construction industries.
#b#Financing News#/b#
Cardinal Partners, 230 Nassau Street, Princeton 08540; 609-924-6452; fax, 609-683-0174. John K. Clarke, managing general partner. www.cardinalpartners.com.
Cardinal Partners, which provides venture capital for the healthcare and pharmaceutical industries, is part of a $16 million investment in Boston-based Verastem Inc., an early-stage biotech that aims to target cancer stem cells.
According to Verastem, cancer stem cells play an important role in the recurrence of cancer after treatment. Verastem is developing drugs to selectively kill cancer stem cells.
#b#Crosstown Moves#/b#
Workman & Skertic, 2642 Whitehorse-Hamilton Square Road, Hamilton 08619; 609-581-4682. Bob Workman, partner.
The accounting firm of Workman & Skertic has moved from 1200 Whitehorse-Mercerville Road to Whitehorse-Hamilton Square Road
Gennaro Costabile, president of Imagennaro's Catering, 36 Union Street, to six months of home confinement for allegedly filing a false corporate income tax return. According to the IRS, Costabile claimed he had made more than $493,000 in 2006, but later admitted that an additional $161,808 was not included in the return.
Ronald Brenner, 77, on November 18. A longtime executive in the pharmaceutical industry, he served as president of McNeil Laboratories, CEO of Cytogen, and managing partner at Hillman Medical Ventures. He also was a vice president at Johnson & Johnson. A memorial service will be held on Saturday, November 27, at 10 a.m. at Titusville Methodist Church.
Larry West, 62, on November 18. A former prosecutor in Middlesex County, he also was a flight instructor at Raritan Valley Flying School
Orlando Olaya, 47, on November 17. He was co-owner, with his brother Hector, of Princeton Diamond Exchange in the Princeton North Shopping Center. Services will be held on Saturday, November 27, at 11 a.m. at Abiding Presence Lutheran Church in Ewing.
Helen Boehm, 89, on November 15. She was the co-founder, along with her husband, Edward, of Boehm Porcelain in Trenton.
Robert Carroll Jr., 81, on November 15. He was CEO of R.E. Carroll, an industrial repackaging materials company in Trenton.
Shoichi Yoshikawa, 76, on November 4. He was a senior research physicist at Princeton Plasma Physics Lab.
Thomas Williams, 72, on September 30. He was a computer information specialist at Princeton University for 42 years.


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The best way to describe Gina, having only just met her is; bubbly, hilarious and full of vibes and character. Gina is a singer-songwriter whom has just graduated from Leeds College of Music studying Popular Music. It is not only Gina's love for music that has led her to release this EP named, "The Story" but also her love for helping others, especially those suffering from a mental illness.
This EP consists of three songs, containing jazz and soul elements in which Gina will be launching on the 23rd October in LS6 Café. All the money she raises from this is going to a local mental health charity, Cloth Cat.
Cloth Cat empowers people through music by providing musical training to improve the quality of people's lives, especially their employability. This often been one of the many causes for cases of mental health.
Gina is open to the fact she herself has never experienced any mental health issues but has been inspired by those around her that are going through some pretty difficult times and it is therefore amazing and so inspiring to see her using an incredible gift and sharing it with those in need.
If you live near Leeds or just want a night of celebrating the amazing work Gina is doing for Cloth Cat then please come along and hear her at 7pm (doors open)-11pm - LS6 Café and it's….. FREE ENTRY!! There will be a chance to buy her EP and also enter a raffle on the night!


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You will enjoy this well cared for 3 bedroom 2 bath year round home perfectly situated on an acre lot with 100 feet of shore line on Lake Ossawinnamakee. Home features an open floor plan, main floor master, vaulted ceilings, wood burning stone fireplace, main floor laundry, large wrap around deck, covered porch, walk out basement and two car attached garage. Property has southerly exposure, breath taking lake views, gentle elevation to a large level area at water's edge and sand lake bottom.
Listing ID#: 4980162
9305 Ossawinnamakee Road
Breezy Point, MN 56472


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Everybody is going to die sometime, but for some, serious medical conditions only prolong the wait upon their deathbed. From newborn infants with severe handicaps, to elderly men and women diagnosed with hopeless amnesia, euthanasia has found a place in society since society s creation., and a boundary is begging to be drawn.

Passive voluntary and active are the two mains forms of euthanasia. A…Read More
Category: Euthanasia

Euthanasia The modern legalization of Murder

Euthanasia (better known as murder) robs one of a natural death. If Euthanasia is legalized, then it should be carefully regulated and practiced by a competent individual, not of the medical society, in that person s home with family member s consent and presence. If Euthanasia is not regulated carefully, then medical institutions will abuse this practice and consider…Read More

Life or death

In my opinion, the beliefs and views of our country are somewhat hypocritical and unjust. As we grow from a young child to a mature adult, we are taught many things such as killing another human being is wrong, it is against the law and goes against most people's religious beliefs. Yet, there are some instances when this rule does not seem…Read More

The controversy over euthanasia is based on the same ethical and social platform from which the debates for abortion and capitol punishment stem. These social dilemmas all revolve around the sanctity of life and how far government can go in making choices involving individual rights. Although the debate over euthanasia is just heating up in the American judicial system, the practice of assisted suicide has…Read More

The word "euthanasia" comes from the greek–eu, "good", and thanatos, "death". Literally, "good death". The dictionary describes euthanasia as "a quiet and easy death, the means of procuring this or, the action of inducing a quiet and easy death." Euthanasia has a becomes a legal, medical, and ethical issue over which opinions are divided. I feel that if there is no hope for a cure…Read More

Webster's dictionary defines the term euthanasia as a painless, happy death. In recent years, a new term – assisted euthanasia has been introduced. This is when a terminally ill patient is assisted in committing suicide by their doctor or even by a friend or relative.

There is a story which I read of an Aids patient. As he approached his time of death, he decided…Read More

To start my essay on mercy killing, I must say, there are too many situations to just have one opinion on the matter.

Mercy killing, or the correct term Euthanasia , is currently illegal in Canada. Though activists keep trying to legalize it, it will be a long time before anything happens. Mercy killing, though prominent on recent CBC newscasts, has been around since before…Read More

Euthanasia nowadays, constitutes a moral issue that from time to time comes into view mainly from its supporters who, contrarily to more traditional opinions, believe that the man is the master of his life and that nobody else can force him to stay alive, especially when his life has become unbearable from the illness and the pain. This dilemma does not have an easy solution….Read More

Euthanasia in our society today

Euthanasia is a controversial subject, not only because there are many different moral dilemmas associated with it, but also in what constitutes its definition. At the extreme ends of disagreement, advocates say euthanasia (which in Greek means "easy death") is a good, or merciful, death. Opponents of euthanasia say it is a fancy word for murder.

Between the two extremes,…Read More

Euthanasia:

Is it "mercy killing" or just plain killing?

"In keeping with the root definition of 'euthanasia'- literally [meaning] 'good

death'- [supporters] of euthanasia insist they are talking about helping terminally ill

patients in insufferable pain die a dignified death- at the patient's request. But this

bears no resemblance to the true picture of the actual practice of euthanasia in the

United States" (Lyons np)….Read More


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I usually know when my period is about to show up because I pretty much turn into a raging b*tch—though of course I'll deny it if you ever try to call me on it, because duh. My cramps are BAD, you guys. And while I've never called in sick to work because of them, I've definitely thought about it. But get this: There's now a company in Mumbai called Culture Machine that's actually offering "First Day of Period" leave!
*googles flights to Mumbai*
The company—which is known for making viral videos—currently has 75 female staffers. In order to introduce them to the program, the powers-that-be filmed a video of the women talking about how much it basically sucks to work while surfing the Crimson Wave, then they kept the cameras rolling and filmed their reactions when they told them about the new initiative.
Pretty cool!
"The first day is obviously a not-so-comfortable day for most," President of Human Resource Devleena S. Majmuder explained in the clip. "It's time we face the reality. This is not an embarrassment. This is part of life."
Amen, sister!
While we're sure not everyone will be comfortable enough to actually use the policy—I know I would!—it's kind of cool to see a company being so progressive when it comes to implementing menstrual leave—and not just in their own office. Culture Machine is now trying to make FOP Leave happen all over India, and they've even launched a petition over on Change.org, which you should totally go sign right now, OK?
Hollee Actman Becker is a freelance writer, blogger, and mom of two who writes about parenting and pop culture. Check out her website holleeactmanbecker.com for more, and then follow her on Instagram.
Be the first to comment!


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This Cranberry Hummus is served with Sage Pita Chips and makes for the perfect holiday appetizer or solution to those Thanksgiving leftovers!
Pre-heat oven to 400 degrees.
In a small bowl, whisk together olive oil, chopped sage, and pepper.
Place pita triangles on a baking sheet and lightly brush with the oil mixture. Sprinkle with sea salt. Bake at 400 degrees for 6-9 minutes, until pitas are crispy.
While the chips are baking, whip up the hummus. Put tahini and lemon juice in a food processor and blend for 1-2 minutes, until tahini is nice and creamy.
Add in extra-virgin olive oil and salt and pepper to taste and mix a little bit more.
Add chickpeas to food processor and process for another 1-2 minutes. Add cranberry sauce and process for another minute, until hummus is well-blended and creamy.
Serve hummus with sage pita chips. Swirl a bit more extra-virgin olive oil into the hummus before serving, if desired.


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Watch Unlimited
Netflix Subscribers
This is an animated adventure movie for the entire family. It is about a zebra who is only half-striped. When he was born there was a drought. The herd was superstitious and decided Khumba was the reason the rains did not come that year. They made him an outcast and forced him to leave the herd. There was much danger for this half-striped zebra on his own in the wild. He had to find friends to help him survive.
This is a story of superstition and the way a group reacts when life gets hard for them. Khumba is blamed because he is different from the rest of his herd.
Khumba is currently available to watch and stream, download, buy on demand at Netflix, Amazon, Google Play, YouTube VOD online.


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Eva MendesThe sexy starlet made appearances in several music videos, movies, and commercials, before getting her big break with a minor role in the highly acclaimed film "Training Day", acting opposite Denzel Washington. It wasn't long before Holloywood took notice and she was soon cast in several box-office hits such as: "2 fast 2 Furious", "Hitch", "Out of Time", and "Stuck on You". Read More
Birth Day: Mar-05-1974
Height: 165cm
Celebrity Factor: 2.9
Eva Mendes Videos
Other Actresses (in the same age group):
Ari Meyers
Melinda Clarke
Anne Heche
Jennifer Lopez
Ashley Jensen


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Prepare to be educated as Kari describes her design process, shows beautiful quilts, and demonstrate useful tools. During this lecture, you will see several quilts from her current designs and sneak peeks at new projects.
Many of Kari's projects utilize over-sized piecing and subsequent trim-downs with rulers from Deb Tucker's Studio 180 Design. She will share tips for effectively using these rulers.
Visit her webiste: www.onpointquilter.com


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Can Steve Stricker Still Be A Ryder Cup Captain Too?/
On the news that Steve Stricker will captain the 2017 U.S. Presidents Cup team (with Nick Price captaining the Internationals at the loathsome Liberty National), this would seem to lessen the chances of Stricker also guiding the United States to 2020 Ryder Cup glory in his native Wisconsin.
Not since Jack Nicklaus has someone served as both Presidents Cup and Ryder Cup captain. And, well, Steve is no Jack.
But only the infamous PGA of America task force Task Force "Task Force" knows the answer (from their two meetings).


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She is pictured here with her granddaughter Angelia and son Christopher.
Nobel Prize-winning author Doris Lessing died at her London home on November 17, her publisher said.
Lou Scheimer, a pioneer in Saturday morning television cartoons with hit shows such as "Superman," "Fat Albert" and "He-Man," died October 17 at 84, according to his biographer.
Andy Mangels helped tell Scheimer's story in the book "Lou Scheimer: Creating the Filmation Generation."Pulitzer Prize-winning author Oscar Hijuelos died on October 12, his agent said.
By Stoyan Zaimov , Christian Post Reporter | Dec 1, 2017 AM The percentage of born again Christians in America continues declining, a major religious survey has found. Invokes Islamic text in response to Trump's Jerusalem move Published: 12 hours ago Art Moore President Obama praised Turkish President Recep Tayyip Erdogan as "a man of action" and one of the five world leaders with whom he had the strongest bond.
President Trump met with him at the White House last spring.
Singer Bobby "Blue" Bland, who helped create the modern soul-blues sound, died June 23 at age 83. Marc Rich, the commodities trader and Glencore founder whom President Bill Clinton pardoned on his final day in office, died June 26 at age 78 in Switzerland.
The legend was known for his phenomenal skills as a keyboardist, and his ability to bridge together jazz, rock, funk and R&B.
Erdogan even declared that "Jerusalem is the capital of the Muslim world". 1st Baal, now war goddess Athena as 'occult is soaring in popularity' Published: 17 hours ago They're doing it again!
The same group that recreated the Roman triumphal arch that once welcomed travelers to the …
Ossie Schectman, the former New York Knicks guard who scored the league's first basket, died Tuesday, July 30. NBA Commissioner David Stern called Schectman a pioneer, "Playing for the New York Knickerbockers in the 1946-47 season, Ossie scored the league's first basket, which placed him permanently in the annals of NBA history.
On behalf of the entire NBA family, our condolences go out to Ossie's family."Actress Eileen Brennan, who earned an Oscar nomination for her role as the exasperated drill captain in the movie "Private Benjamin," died Sunday, July 28, at her Burbank, California, home after a battle with bladder cancer. Actor Dennis Farina, a Chicago ex-cop whose tough-as-nails persona enlivened roles on either side of the law, died Monday, July 22. Above, Farina shoots a scene as Detective Joe Fontana in "Law & Order" in 2004.