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Messenger RNAs undergo 5' capping, splicing, 3'-end processing, and export before translation in the cytoplasm. It has become clear that these mRNA processing events are tightly coupled and have a profound effect on the fate of the resulting transcript. This processing is represented by modifications of the pre-mRNA and loading of various protein factors. The sum of protein factors that stay with the mRNA as a result of processing is modified over the life of the transcript, conferring significant regulation to its expression.
Messenger RNA (mRNA) transcripts are extensively processed before export. 5′ capping, splicing, and 3′-end processing represent nuclear processes that are large determinants of the fate of a transcript. As mRNA processing events involve different cellular machinery (Fig. 1), RNA sequences, and have different consequences for target mRNAs, these processes were long seen to be independent of one another. It has become clear over the last decade, however, that these events are integrated and coordinated in space and time (Schroeder et al. 2000; reviewed in Bentley 2002; reviewed in Moore and Proudfoot 2009). Nuclear processing steps require a large set of proteins, many of which are loaded onto the transcript as a result of processing, adding a layer of regulatory information that can affect export, localization, translation, and stability of the transcript. Examples of such proteins include the exon-junction complex (EJC), left behind from splicing, and the THO/TREX complex, loaded during elongation. Indeed, the highly integrated nature of nuclear mRNA processing adds a new level of complexity to our picture of gene regulation. The availability of genetic fluorescent tags and sophisticated microscopy technology adds a dynamic component to this picture, providing spatial and temporal information and highlighting how nuclear structure might regulate gene expression (reviewed in Gorski et al. 2006; reviewed in Moore and Proudfoot 2009).
Transcription, the major contributor to RNA biogenesis, takes place under constraints of an anisotropic nuclear landscape that is highly structured (chromatin, distinct nuclear bodies, etc.) and dynamic (gene mobility, diffusive factors, genomic reorganization during cell cycle progression, etc.) (Yao et al. 2008). The availability of increasingly sensitive equipment and fluorescent markers has made it possible to intensively interrogate transcriptional dynamics (Fig. 2) (Becker et al. 2002; Janicki et al. 2004; reviewed in Shav-Tal et al. 2004b; Darzacq et al. 2007; Yao et al. 2007). Such approaches provide a new perspective on mRNA metabolism that has been mostly based on biochemical data. For instance, whereas preformed transcription complexes are sufficient for in vitro transcription, live cell imaging experiments show that different transcription factors show a wide range of dwell times at the promoter and suggest a link between transcription complex assembly dynamics and transcriptional output, consistent with a subunit assembly model for transcription complex recruitment (Gorski et al. 2008; reviewed in Darzacq et al. 2009). Modulation of transcriptional speed and processivity is suggested to be a way of regulating gene expression (Darzacq et al. 2007; reviewed in Core and Lis 2008; Core et al. 2008). These techniques have revealed highly inefficient transcription by RNA polymerase II (Pol II) and stochastic assembly of transcription complexes (Darzacq et al. 2007; Gorski et al. 2008), and both assembly and processivity can be regarded as rate limiting steps of in vivo transcription (reviewed in Core and Lis 2008). A novel way to look at gene expression has been recently presented by analyzing transcript amounts in individual cells on the single molecule level. Here the mean expression level and its variation are accessible, leading to a detailed understanding of variability in gene expression within a population (Zenklusen et al. 2008). Time lapse experiments add further information concerning the expression mode of individual genes, providing insights into differences between constitutive expression and bursts in different species (reviewed in Larson et al. 2009). These experimental approaches allow for in depth characterization of transcriptional dynamics (reviewed in Darzacq et al. 2009; reviewed in Larson et al. 2009), and are likely to provide greater insight into downstream processes.
Transcription, particularly the carboxy-terminal domain (CTD) of RNA Pol II, contributes significantly to the integration of nuclear mRNA processing (Fig. 3). The CTD is an essential domain of the largest RNA Pol II subunit, composed of conserved YSPTSPS heptad repeats that are subject to reversible phosphorylation. It is well established that the CTD functions in transcription, and it has an equally important role in mRNA processing. The CTD interacts with a large number of protein factors, and among the protein domains shown to show preferential CTD binding are: CTD interacting domains (CIDs), WW domains, and FF domains (Verdecia et al. 2000; Smith et al. 2004; Noble et al. 2005). Serine 5 phosphorylation of the CTD occurs when RNA Pol II is at the 5′ end of the gene and is mediated by the TFIIH-associated kinase CDK7 (Kin28 in yeast) as transcription initiates. Serine 2 phosphorylation is mediated by PTEFb (CTDK1 in yeast) as the processive RNA Pol II elongates through the body of the gene (Komarnitsky et al. 2000; Peterlin and Price 2006). These phosphorylation marks are critical to the proper progression of transcription and are required for coordinating processing events (reviewed in Hirose and Manley 2000). Indeed, the CTD has been shown to bind over 100 different yeast proteins in its phosphorylated state (Phatnani et al. 2004). It can adopt different conformations depending on phosphorylation patterns, protein interactions, and proline isomerization via peptidyl-prolyl cis/trans isomerases (PPIases) (reviewed in Hirose and Ohkuma 2007). In this way, the CTD functions as a recruitment scaffold for different processing factors throughout transcription, thereby integrating processing events in time. Furthermore, changes in chromatin structure on transcriptional activation are likely to contribute to mRNA processing through gene positioning, or relocation at or near nuclear pores, and gene looping, or formation of DNA loops in which 5′ and 3′ ends make contact (reviewed in Moore and Proudfoot 2009). Recent evidence from studies using galactose genes in yeast showed that gene positioning can be a further regulatory step, and an interaction has been shown between transcription-dependent complexes (e.g TREX/TREX-2), chromatin remodeling complexes (e.g. SAGA), and nuclear pores (reviewed in Blobel 1985; Cabal et al. 2006; Klockner et al. 2009; reviewed in Moore and Proudfoot 2009). Export however, is likely to depend on successful maturation of the mRNA, and whereas in yeast gene gating to nuclear pores is suggested, mRNAs of other genes reach nuclear pores in a diffusive manner. Studies in mammalian cell systems have not supported gene gating, as fluorescence correlation spectroscopy (FCS), FRAP and single particle tracking consistently suggest diffusive behavior (Politz et al. 1998; Shav-Tal et al. 2004a; Grunwald et al. 2006; Politz et al. 2006; Braga et al. 2007; Siebrasse et al. 2008). In addition, high resolution studies of the nuclear periphery led to the conclusion that DNA is absent at the nuclear basket of nuclear pores (Schermelleh et al. 2008). Thus, transcription, and the associated machinery, serves to localize processing factors to the appropriate place on the nascent mRNA, stimulate and coordinate processing and possibly establish functionally significant chromatin conformations.
The first processing event an mRNA undergoes is 5′-end capping which requires three enzymatic activities: RNA triphosphatase, guanylyltransferase and 7-methyltransferase (reviewed in Shuman 2001). Occurring early in transcription after RNA Pol II has transcribed the first 25–30 nucleotides, the RNA triphosphatase first acts on the terminal nucleotide to remove the γ-phosphate. The guanylyltransferase then transfers GMP from GTP to form GpppN, which is subsequently methylated. In mammals, a bifunctional capping enzyme includes both amino-terminal RNA triphosphate activity and carboxy-terminal guanylyltransferase activity. In yeast, the RNA triphosphatase (Cet1) and separate guanylyltransferase (Ceg1) form a heterodimeric capping enzyme. In both cases, a separate methyltransferase (Abd1 in yeast) is required to methylate the guanine at the N7 position.
Mammalian capping enzyme binds directly to the elongating RNA Pol II with a phosphorylated CTD (termed RNA Pol II0) through its guanylyltransferase domain (Yue et al. 1997), thereby coupling capping to the early stage of transcription. Yeast capping enzyme subunits also bind directly and independently to RNA Pol II0, and this interaction is dependent on Kin28, the subunit of TFIIH responsible for serine 5 phosphorylation (Rodriguez et al. 2000). The loss of serine 5 phosphorylation during transcription correlates with the release of the capping enzyme, which is believed to occur before the nascent transcript is 500 nucleotides long (reviewed in Zorio and Bentley 2004). Ceg1 guanylylation activity is inhibited by the phosphorylated CTD but is restored and enhanced by Cet1, and this allosteric regulation may represent a means to temporally coordinate guanylylation and triphosphatase activities (Cho et al. 1998; Ho et al. 1998). In mammals, CTD binding to the guanylyltransferase has an allosteric affect, causing a twofold increase in affinity of guanylyltransferase for GTP (Ho and Shuman 1999). CTD phosphorylation that accompanies the transition from initiation to elongation has a clear impact on capping and allows communication between the transcriptional machinery and capping enzymes. It seems that capping also has a direct impact on transcription. Recent evidence indicates that capping enzymes can relieve transcriptional repression, suggesting an additional role in promoter clearance (Mandal et al. 2004). Temperature sensitive ceg1 yeast mutants are sensitive to drugs that disrupt elongation and show decreased transcription through promoter-proximal pause sites (Kim et al. 2004a). Furthermore, yeast capping enzyme subunits influence RNA Pol II occupancy at the 5′ end and may regulate transcription reinitiation (Myers et al. 2002; Schroeder et al. 2004). Taken together, it seems that transcription complexes are held at the promoter until capping occurs, after which the polymerase switches into an elongating mode.
Capping of transcripts confers stability. In yeast as well as mammals, capping helps protect the transcript from 5′→3′ exonucleases present in both the nucleus and cytoplasm (Hsu and Stevens 1993; Walther et al. 1998). The 5′→3′ degradation pathway involves deadenylation followed by rapid decapping by Dcp1/Dcp2, and degradation by the processive 5′→3′ exonuclease, Xrn1 (Hsu and Stevens 1993; Muhlrad et al. 1995). The cap is also important in mediating mRNA recruitment to ribosomes. The protein complex eIF4F recognizes the cap before translation, and facilitates circularization of mRNAs via an interaction with polyA-binding protein (PAB1), thereby aiding in translation reinitiation and enhancing protein synthesis (Tarun and Sachs 1996; Wakiyama et al. 2000). Transcripts engaged in translation are protected from degradation suggesting competition between translation and degradation (reviewed in Jacobson and Peltz 1996). Finally, depletion of CBC (cap-binding complex) from HeLa cell extracts inhibits the endonucleolytic cleavage step of 3′-end formation, reduces the stability of poly(A) cleavage complexes, and disrupts communication between 5′ and 3′ ends (Flaherty et al. 1997).
The precise removal of noncoding intervening sequences, or introns, from many pre-mRNAs is a required process for proper protein expression. In both yeast and mammals, this reaction is catalyzed by the spliceosome, consisting of the U1, U2, U4, U5 and U6 small nuclear RNPs (snRNPs) in conjunction with a large number of additional proteins (reviewed in Stark and Luhrmann 2006). Within the spliceosome, a series of RNA–RNA, RNA–protein, and protein–protein interactions is needed to identify and remove intronic regions and join exons, producing a mature transcript (reviewed in Collins and Guthrie 2000). The mature spliceosome carries out splicing through two transesterification reactions. First, the 2′-OH of a branch point nucleotide performs a nucleolytic attack on the first nucleotide of the intron, forming a lariat intermediate. Second, the 3′-OH from the free exon performs a nucleolytic attack on the last nucleotide of the intron, thereby joining exons and releasing the lariat intron. Intron identification relies on certain sequences, including the 5′ splice site, branch point (and downstream polypyrimidine tract) and 3′ splice site. In yeast, splice sites are easily identified, and although only 3% of genes contain single short introns, they account for more than 25% of cellular mRNAs (Ares et al. 1999; Lopez and Seraphin 1999; reviewed in Barrass and Beggs 2003). In mammals, however, splice sites are less clear, and many genes contains multiple introns that vary from a few hundred to hundreds of thousands of nucleotides (Lander et al. 2001). The presence of putative splice sites in higher eukaryotes does not necessarily lead to selection of these sites by the spliceosome. Flanking pre-mRNA regulatory elements, including intronic and exonic splicing enhancers or silencers, bind trans-acting regulatory factors that enhance or repress snRNP recruitment to splice sites. Generally, exonic splicing enhancers are bound by Serine/Arginine-rich (SR) proteins, whereas exonic splicing silencers are bound by heterogenous nuclear ribonucleoprotein (hnRNP) proteins (reviewed in Cartegni et al. 2002; reviewed in Singh and Valcarcel 2005). Therefore in higher eukaryotes, it is the cumulative effect of multiple factors that modulates splice site selection. As a result, 92%–94% of human transcripts are subject to alternative splicing, representing an important source of diversity in gene expression with serious implications for health and disease (reviewed in Nissim-Rafinia and Kerem 2005; Wang et al. 2008). Conversely, in yeast, SR proteins do not appear to have a significant role in splicing, consistent with the absence of exonic splicing enhancers (reviewed in Wahl et al. 2009).
The spliceosome is rich in proteins, containing approximately 125 different proteins (more than two-thirds of its mass), and spliceosome assembly is characterized by a remarkable exchange of components from one step to the next (reviewed in Wahl et al. 2009). Although RNA-RNA base pairing interactions are critical to the precise recognition of splice sites, they are generally weak and require additional proteins for enhanced stabilization. DExD/H-type RNA-dependent ATPases/helicases have long been implicated in rearrangements within the spliceosome, and many are conserved between yeast and humans. These proteins act at discrete stages of splicing including single-strand RNA translocation, strand annealing, and protein displacement (reviewed in Pyle 2008). Human spliceosomes also contain several PPIases that are absent in yeast, though the role of these proteins in splicing is not well understood. Similar to DExD/H-type RNA-dependent ATPases/helicases, they are recruited at discrete stages of splicing and are thought to be involved in at least one protein conformational switching event (reviewed in Wahl et al. 2009). Furthermore, post-translational modification of splicing factors and spliceosomal proteins may act as switches to allow fine tuning of the spliceosome (Bellare et al. 2008; reviewed in Wahl et al. 2009). The nature of interactions during such a tightly regulated protein-rich process is not very well documented and may be best studied using in vivo imaging techniques. For example, one recent study (Fig. 4) employed FRET-FLIM and revealed for the first time that different complexes of splicing factors show differential distributions in live cell nuclei (Ellis et al. 2008).
Mature mRNAs are occupied by a number of different proteins that determine their fate in many ways, and several of these associations are splice-dependent. As mentioned, various proteins associate cotranscriptionally and accompany the packaged mRNA into the cytoplasm where they can direct localization, translation and decay. Shuttling SR proteins specifically serve as mRNP binding sites for export factors, and the phosphorylation state of these proteins confers export competence (reviewed in Huang and Steitz 2005; reviewed in Moore and Proudfoot 2009). The THO/TREX complex, which functions in transcription and export, associates with spliced mRNAs at the 5′-most exon (Cheng et al. 2006). THO/TREX recruitment is enhanced by splicing, and promotes rapid export (Valencia et al. 2008). In mammals, REF/Aly and UAP56 (homologs of yeast Yra1 and Sub2), are recruited as a consequence of splicing and have a role in aiding export. Perhaps the most studied splice-dependent mark is the exon junction complex, or EJC. EJCs are stably deposited 20–24 nucleotides upstream of exon-exon junctions late in splicing (Le Hir et al. 2000). Interestingly, spliced mRNAs appear to have greater translational efficiency than their cDNA counterparts (reviewed in Le Hir et al. 2003). Aside from their role in nonsense mediated decay, EJCs appear to directly enhance translation initiation. Although there are several proposed mechanisms by which EJCs do this, they ultimately serve to promote the pioneer round of translation (reviewed in Moore and Proudfoot 2009). Finally, a number of DEAD-box proteins have recently been identified as associating with mRNAs in a splice-dependent manner, and these are believed to influence many aspects of mRNA metabolism (Merz et al. 2007; reviewed in Rosner and Rinkevich 2007). Taken together, it is clear that spliced mRNAs carry with them numerous protein marks that indicate their splicing history and have important downstream effects.
Initial work focusing on the link between transcription and splicing suggested that splicing occurs cotranscriptionally and factors involved in splicing colocalize with transcription sites (Beyer and Osheim 1988; Zhang et al. 1994). Similar to capping, the CTD of RNA Pol II has an important role in splicing. CTD truncation causes inefficient splicing in mammalian cells and inhibition of colocalization of splicing factors with transcription sites (McCracken et al. 1997; Misteli and Spector 1999). Furthermore, RNA Pol II0 has been shown to physically associate with splicing factors that do not bind RNA Pol IIA (RNA Pol II with an unphosphorylated CTD), suggesting this interaction depends on the phosphorylation state of the CTD. Both anti-CTD antibodies and CTD peptides can inhibit splicing in vitro, and expression of phosphorylated CTD peptides has a similar effect on mammalian cells in vivo (Yuryev et al. 1996; Du and Warren 1997). RNA Pol II0 enhances splicing in vitro, whereas RNA Pol IIA has an inhibitory effect (Hirose et al. 1999). This is believed to result from RNA Pol II0-dependent stimulation of the early steps of spliceosome assembly, possibly by facilitating the binding of snRNPs to the nascent transcript. Therefore, it seems that CTD phosphorylation can act to recruit splicing factors to the nascent transcript to ensure rapid and accurate splicing. Transcription is also linked to splicing in ways independent of the CTD. Promoter identity and expression levels of certain SR proteins are known to affect alternative splicing (Cramer et al. 1997). Furthermore, in both yeast and mammals, disruption of RNA Pol II elongation markedly shifts the balance of alternatively spliced isoforms (de la Mata et al. 2003; Howe et al. 2003). This is consistent with a "first come first served" model in which elongation rate regulates splice site selection, as 5′ splice sites are more likely to pair with newly transcribed 3′ splice sites. Additionally, the transcriptional coactivator, p52, is known to interact with SF2/ASF and stimulates splicing (Ge et al. 1998), suggesting that transcriptional machinery can modulate splicing factor recruitment.
Finally, splicing can also have an impact on transcription. The presence of introns can confer increased transcriptional efficiency, possibly through increase initiation rates (Brinster et al. 1988). Recruitment of snRNPs by TAT-SF1, an elongation factor that associates with P-TEFb, enhances elongation and this effect is dependent on the presence of functional splice signals (Fong and Zhou 2001; Kameoka et al. 2004). Reminiscent of the effect of capping on transcription, certain splicing factors have also been shown to promote elongation (Lin et al. 2008).
Capping also has a role in splicing. The 5′ cap structure increases splicing efficiency in mammalian cell extracts and in vivo (Konarska et al. 1984; Inoue et al. 1989). Depletion of CBC in cell extracts prevents spliceosome assembly at an early step in complex formation (Izaurralde et al. 1994). In yeast, CBC interacts with components of the earliest identified splicing complexes (Colot et al. 1996). Similarly, in mammals, successful capping and CBC recruitment is implicated in U1 small nuclear ribonucleoproteins (snRNP) recognition of the 5′ splice site, but this effect is specific to mRNAs with only one intron (Lewis et al. 1996).
Splicing defects can lead to potentially harmful protein variants. mRNAs are subject to quality control in the nucleus resulting in the prevention of export of splicing-defective transcripts. To date, several mutations in cis (conserved splice sites) and in trans (spliceosome components required for 1st or 2nd step catalysis) yield drastic reductions in mature mRNA without a corresponding increase in unspliced pre-mRNAs (reviewed in Staley and Guthrie 1998; Bousquet-Antonelli et al. 2000). Mature mRNA levels are largely restored in these same mutants when degradation is inhibited, suggesting that spliceosomes are able to act successfully on these substrates if they are not quickly destroyed. Thus, quality control acts on a number of different splice-defective pre-mRNAs, and degradation is in direct competition with productive splicing. This sort of kinetic competition is exemplified by the finding that decreased ATP hydrolysis of the DExD/H-type RNA-dependent ATPases, Prp16, leads to productive splicing of pre-mRNAs harboring mutant branch points that would normally be discarded (Burgess and Guthrie 1993). This same principal has been extended to other splice site mutations and members of the DExD/H-type RNA-dependent ATPases/helicases family (Mayas et al. 2006). By coupling spliceosomal rearrangements with irreversible ATP hydrolysis, these proteins ensure that splice-aberrant mRNAs which are unable to complete splicing within a time frame dictated by ATP hydrolysis rates, are discarded (reviewed in Villa et al. 2008). Quality control takes place in the nucleus, can act at numerous stages in the splicing process and serves to commit unspliced mRNAs to degradation pathways. One such pathway involves both 3′ → 5′ degradation by the exosome and 5′ → 3′ degradation by the nuclear exonuclease Rat1 (Bousquet-Antonelli et al. 2000). Other degradation pathways include Dbr1-mediated debranching of aberrant lariat-intermediates followed by export and cytoplasmic degradation, and Rnt1-mediated endonucleolytic cleavage of unspliced pre-mRNAs followed by nuclear degradation (Danin-Kreiselman et al. 2003; Hilleren and Parker 2003). An additional quality control mechanism involves spliceosome-dependent nuclear retention of unspliced transcripts. A number of different proteins seem to be involved in anchoring these transcripts at the nuclear side of the nuclear pore complex, and retention may be regulated by desumoylation (reviewed in Dziembowski et al. 2004; Galy et al. 2004; Palancade et al. 2005; Lewis et al. 2007). Furthermore, splice-defective mRNAs are also known to be retained at the site of transcription (Custodio et al. 1999; reviewed in Custodio and Carmo-Fonseca 2001). Thus, the cell has evolved various ways to ensure that unspliced transcripts do not leak into the cytoplasm. Finally, nonsense mediated decay (NMD) represents a highly specific form of quality control that extends to higher eukaryotes. The presence of an EJC downstream of a stop codon triggers degradation of translating mRNAs (reviewed in Stalder and Muhlemann 2008). Splice-dependent EJC deposition can increase the translational efficiency of normal mRNAs while ensuring rapid degradation of aberrant mRNAs.
The final step of transcription is endonucleolytic cleavage which occurs 10–30 nucleotides downstream of a signal sequence (conserved AAUAAA sequence in mammals or an AU-rich sequence in yeast), followed by poly(A) addition at the 3′ end (reviewed in Proudfoot 2004). The poly(A) tail is similar to the cap in that it is important for the stability and translational efficiency of the mRNA (Drummond et al. 1985). Cleavage requires multiple proteins, including cleavage/polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and two cleavage factors (CFIm and CFIIm) in mammals, or cleavage-polyadenylation factor (CPF) and two cleavage factors (CF1A and CF1B) in yeast. Poly(A) polymerase (PAP) then adds the poly(A) tail to the 3′-OH that is exposed on cleavage (reviewed in Proudfoot 2004).
CPSF and CstF are highly conserved between yeast and humans and are required for both cleavage and polyadenylation (reviewed in Shatkin and Manley 2000). CPSF recognizes RNA and facilitates PAP recruitment. The endonuclease responsible for cleavage in mammals is CPSF-73 and Ydh1 in yeast (Ryan et al. 2004; Mandel et al. 2006). CstF recognizes U/GU-rich elements found in the mRNA and is directly involved in polyadenylation. The conserved AAUAAA sequence and downstream U/GU site comprise the core poly(A) element, although additional auxiliary elements can influence polyadenylation efficiency (Gil and Proudfoot 1984; Sadofsky and Alwine 1984; Russnak 1991; Bagga et al. 1995). Cleavage is closely coupled to poly(A) tail synthesis, which also requires PAB1. A number of other factors also participate in 3′-end processing, and many do not share homologs in other systems (reviewed in Shatkin and Manley 2000).
As with splicing, transcripts can be alternatively polyadenylated, thereby altering stability, localization or transport. It is estimated that more than half of the genes in the human genome are subject to such alternative 3′-end processing, generating isoforms that differ in 3′ UTR length or encoding different proteins altogether (Tian et al. 2005). Alternative polyadenylation can be tissue specific, may be coupled to alternative splicing and can have implications for health and disease (Peterson and Perry 1989; Beaudoing and Gautheret 2001; Tian et al. 2005; Lu et al. 2007; reviewed in Danckwardt et al. 2008). In fact, many human genes contain multiple potential 3′-end cleavage sites, and appropriate site selection is achieved by alternate mechanisms, representing an additional layer of complexity in the regulation of gene expression (reviewed in Wilusz and Spector 2010). It is important to note that the mechanism and machinery responsible for alternative polyadenylation remain unclear.
Many factors involved in 3′-end processing have been shown to interact with the CTD, including CstF subunits. Purified CTD can stimulate the cleavage step and is needed for processing in reconstituted reactions (Hirose and Manley 1998). As with 5′ capping, the specific CTD phosphorylation pattern is important in 3′-end processing. In this case, loss of serine 2 phosphorylation in Ctk1 (yeast) or Cdk9 (Drosophila) mutants leads to a defect in 3′-end processing, likely resulting from improper or inefficient recruitment of processing factors (reviewed in Hirose and Ohkuma 2007). Furthermore, several yeast proteins involved in 3′-end processing preferentially bind the CTD phosphorylated at serine 2, which may ensure processing occurs as the polymerase reaches the end of a gene. In mammalian cells, unlike yeast, the CTD is also required for cleavage (Licatalosi et al. 2002). Once again, the CTD appears to mediate coupling between transcription and 3′-end processing.
Although transcription elongation continues for quite some distance after the poly(A) signal, transcription termination and 3′-end processing are intimately coupled. This was supported by the finding that termination requires functional poly(A) signals (reviewed in Hirose and Manley 2000). There are multiple ways in which 3′-end processing may be coupled to transcription termination:
Transcriptional pause sites positioned downstream of the poly(A) signal also seem to be important for 3′-end processing and termination of a number of mammalian genes, reestablishing the theme of kinetic coupling between transcription and processing (Yonaha and Proudfoot 2000).
Although 3′-end processing machinery is enriched at the 3′ end of genes, certain factors can be found at or near promoters toward the 5′ end. For example, CPSF can be recruited to promoters through an association with TFIID (Dantonel et al. 1997). Ssu72, a component of yeast CPF, seems to function at many stages, including transcription initiation, 3′-end processing and termination of certain mRNAs and snoRNAs (reviewed in Proudfoot et al. 2002). As described, CTD phosphorylation patterns are linked to processing events, and Ssu72, in conjunction with Pta1 (another component of yeast CPF), also appears to have phosphatase activity specific for serine 5 (Krishnamurthy et al. 2004). Additional genetic and physical interactions have been described between the transcriptional machinery (TFIIB and Sub1) and the 3′-end processing factors Ssu72 and Rna15 (Sun and Hampsey 1996; Wu et al. 1999; Calvo and Manley 2005). Interactions between factors located on opposite ends of genes is likely facilitated by the formation of gene loops, and TFIIB, Ssu72 and Pta1 all appear to have a role in this (Singh and Hampsey 2007). Overall, it seems that the transcriptional machinery, DNA template, nascent mRNA and 3′-end processing machinery are in constant communication for processing.
Evidence in yeast shows that transcripts undergoing aberrant 3′-end processing are disposed of. Defects associated with pap1-1 mutants are suppressed by deletion of the exosomal subunit Rrp6, which is known to interact with both PAP and the export factor Npl3 (Burkard and Butler 2000). Hypoadenylated mRNAs are retained in the nucleus at the site of transcription, and these transcripts are stabilized and exported in the absence of Rrp6 (Hilleren et al. 2001). In rna14 and rna15 mutant strains, defects in termination lead to readthrough transcripts and aberrant polyadenylation. Deletion of Rrp6 stabilizes the aberrantly polyadenylated population whereas depletion of Rrp41 stabilizes the population of long readthrough transcripts (Libri et al. 2002; Torchet et al. 2002). Therefore, different components of the exosome may have evolved specialized roles in mRNA surveillance, ensuring rapid degradation of transcripts that possess aberrant 3′ ends. Additionally, THO complex or sub2 mutants show defects in 3′-end formation, reduced mRNA levels and retention at the site of transcription. Codeletion of Rrp6 and TRAMP components restores mRNA levels, although the retention effect only requires Rrp6 (Libri et al. 2002; Rougemaille et al. 2007). These observations may suggest kinetic competition between 3′ end formation and degradation. Transcripts that do not quickly and efficiently undergo 3′-end processing are exposed to the exosome, and mutation of exosome components may allow more time for defective 3′-end processing machinery to function. Finally, recent evidence suggests that nuclear mRNP assembly factors are involved in releasing the 3′-end processing machinery from the transcript after polyadenylation (Qu et al. 2009). This may be a way to temporally coordinate 3′-end formation, mRNP maturation and export.
Transport through the nuclear pore complex (NPC) represents the link between the nucleus and cytoplasm. Several studies have investigated mRNA mobility in the nucleoplasm and have revealed probabilistic movement of mRNAs with diffusion coefficients between 0.03 µm2/s and 4 µm2/s (Politz et al. 1998; Shav-Tal et al. 2004a; Braga et al. 2007; Siebrasse et al. 2008). In pulse-chase experiments, mRNA was found in the cytoplasm within ~20 min after transcription (Lewin 1980). However, single-molecule tracking experiments suggest that transit through the NPC is significantly faster, on the order of fractions of a second. Different forms of RNA have been observed in close proximity to the nuclear envelope in electron micrographs (reviewed in Franke and Scheer 1974). Detailed resolution of individual mRNPs and how they move through nuclear pores is mainly derived from work using mRNPs of Balbiani Ring (BR) genes in salivary gland cells of Chironomus (Mehlin et al. 1992; Kiseleva et al. 1998). These large mRNPs, ~50 nm in diameter, have been visualized interacting with nuclear pores by electron microscopy. Because of their size, these mRNPs unfold at the NPC and show directional translocation through the NPC, beginning with the 5′ end (reviewed in Daneholt 2001). Hypothetical sequences for distinct steps in the export process have been assembled from EM series (Kiseleva et al. 1998). mRNA export likely involves distinct docking, translocation and release steps from the NPC, analogous to a ratchet model that includes a specific function for the mRNA associated DEAD box helicase DBP5 (reviewed in Stewart 2007). Direct interaction of DPB5 with Nup214, a cytoplasmic component of the NPC, has been shown (Napetschnig et al. 2009; von Moeller et al. 2009) and DPB5 is thought to promote export factor release and eventual reorganization of the mRNA (reviewed in Iglesias and Stutz 2008). DBP5 localization, the timing and location of loading onto the transcript, and how many export factors are actually attached to any individual transcript remains unclear (Zhao et al. 2002; Estruch and Cole 2003; Lund and Guthrie 2005; von Moeller et al. 2009). Discrepancy in the number of BR-mRNPs observed on the nuclear and cytoplasmic surfaces of nuclear pores in EM studies have been interpreted as uncoupled asynchronous functions of the export process, which should result in a waiting step during transport. Simultaneously, it was concluded that translocation through the central channel of nuclear pores is probably fast compared to the docking step on the nuclear surface of the pore (Kiseleva et al. 1998). Biochemical studies indicates that mRNA export competence is directly linked to transcription (reviewed in Kohler and Hurt 2007; reviewed in Hurt and Silver 2008; reviewed in Iglesias and Stutz 2008; reviewed in Carmody and Wente 2009; reviewed in Moore and Proudfoot 2009). Recently, a link between actin and transcription has been suggested. Actin (1) can be detected as a component of pre-mRNP complexes, (2) binds transcription factors, (3) is involved in chromatin remodeling, and (4) associates directly to RNA polymerases (reviewed in Miralles and Visa 2006). Interestingly, actin may also have a role in export, as it has been observed to associate cotranscriptionally with BR-mRNPs and remains associated throughout export (Percipalle et al. 2001). Furthermore, the nuclear export receptor exportin-6 shows specificity for profilin-actin, suggesting an additional role as an adaptor for export of certain mRNAs (Stuven et al. 2003; reviewed in Miralles and Visa 2006).
Export is mediated by protein factors associated with the mRNA, and mRNAs that do not carry the necessary adaptor and export factors are retained in the nucleus (reviewed in Iglesias and Stutz 2008). Most mRNAs seem to export via a TAP (Mex67 in yeast)-dependent pathway. TAP is not a member of the karyopherin family and does not rely on the GTPase Ran, which mediates nuclear import (Segref et al. 1997; reviewed in Macara 2001; reviewed in Dreyfuss et al. 2002). Export factors do not recognize the mRNA directly, but rather through adapters such as Aly/REF (Yra1 in yeast), which is necessary for export (Stutz et al. 2000; Zhou et al. 2000; Gatfield et al. 2001; Le Hir et al. 2001). However, CBP80, a component of the CBC, has been implicated in mediating contacts between transport receptors and mRNA (Hamm and Mattaj 1990; Cheng et al. 2006). Aly/REF has been identified to be loaded in a splice-dependent manner as part of the EJC (Le Hir et al. 2001). Complex regulation of Aly/REF links mRNA export to cell cycle progression (Zhou et al. 2000; Okada et al. 2008; reviewed in Okada and Ye 2009) and Yra1, has been linked to S-phase entry (Swaminathan et al. 2007). Conversely, in Drosophila and Caenorhabditis elegans, mRNA export is Aly/REF independent (Gatfield and Izaurralde 2002; Longman et al. 2003).
The transcription-export complex (TREX) exemplifies the tight coupling between transcription and export. Recruitment of the TREX complex is coupled to the transcription machinery in yeast but associated with the splicing machinery in metazoans (Masuda et al. 2005; Cheng et al. 2006). In metazoans, the TREX complex was initially thought to be part of the EJC (Gatfield et al. 2001; Le Hir et al. 2001) but is now known to be recruited to the 5′ end independently, in a splicing and 5′ cap-dependent manner (Masuda et al. 2005; Cheng et al. 2006). In yeast, molecular machinery serves to dock transcribing genes to nuclear pores, resulting in "gene gating." SAGA, involved in histone modification and DNA remodeling, has also been shown to interact with transcription factors (Grant et al. 1997; Larschan and Winston 2001; reviewed in Daniel and Grant 2007). Components of SAGA and TREX2 complexes are required for gene gating of the GAL locus following activation (Cabal et al. 2006; Wilmes and Guthrie 2009). Sus1, one such component, promotes NPC docking and export (reviewed in Blobel 1985; Jani et al. 2009; Klockner et al. 2009; Wilmes and Guthrie 2009). Sus1 is also involved in transcription elongation and may prevent harmful DNA:RNA hybrids during transcription (Klockner et al. 2009). Interactions have been shown for both TREX and TREX2 complexes with Swt1, an endonuclease that interacts with nuclear pores and is involved in mRNA quality control (Skruzny et al. 2009). Although major pathways for mRNA export have been identified, based on interspecies variations and the number of transport factors and cofactors involved, additional pathways are likely to exist. For example, export of certain viral and other mRNAs depend on CRM1 as an export factor (Ohno et al. 2000; reviewed in Dreyfuss et al. 2002; reviewed in Iglesias and Stutz 2008).
The extent to which processing and export might be regulated in a species- and differentiation-dependent manner currently remains unclear. Dynamic regulation of mRNA processing factors is a relatively new question, and it is still unclear to what degree mRNA export is regulated at the level of individual nuclear pores. A dynamic view of how mRNA transitions through the nuclear pore is lacking, but recent developments in mRNA labeling and imaging technology may provide the opportunity to fill this gap in the near future.
As discussed, a wealth of ensemble biochemical studies has provided great molecular and mechanistic insight into mRNA processing, leading to further questions that will require in vivo imaging approaches. Several recent studies highlight the importance of such techniques in gaining quantitative information on fundamental aspects of mRNA processing events, with spatial and temporal resolution. For example, the nucleus is the site of numerous essential cellular processes that are likely coordinated through highly organized and compartmentalized nuclear bodies with distinct functions, as observed using cellular imaging (reviewed in Misteli 2007). Nuclear bodies lack membranes and are highly dynamic yet steady-state structures. This represents a more advanced view from the original idea of nuclear factories for transcription and replication, in which core components localized to static discrete foci (reviewed in Iborra et al. 1996). This environment facilitates gene expression at multiple levels, including chromatin accessibility, transcriptional control, integration of processing events, stringent quality control, export of mature mRNPs to the cytoplasm and translation. Recent work suggests that Cajal body formation does not require specific gene loci and can initiate from any Cajal body protein, supporting a self-assembling model for nuclear bodies (Kaiser et al. 2008; reviewed in Misteli 2008). Additionally, the long-standing question of whether differentially spliced transcripts recruit distinct sets of basal pre-mRNA splicing factors has recently been addressed. Quantitative single-cell imaging has shown the first in vivo evidence of differential association of pre-mRNA splicing factors with alternatively spliced transcripts, supporting a stochastic model of alternative splicing which would predict that combinatorial sets of splicing factors contribute to splicing outcome (Mabon and Misteli 2005). FRET and FLIM techniques have been applied to investigate interactions between SR proteins and splicing components. Unlike biochemical methods, FRET can be used to study interactions in living cells, with minimal perturbation to the highly structured and dynamic nuclear environment (reviewed in Wouters et al. 2001; Ellis et al. 2008). Such an approach has revealed individual interactions that occur in the presence of RNA Pol II inhibitors, suggesting they are not exclusively cotranscriptional. FRAP analysis suggests that processing factors are highly dynamic and are exchanged between nuclear bodies and other nuclear locations in a matter of seconds (Phair and Misteli 2000; reviewed in Lamond and Spector 2003). Both FRET and FRAP have been used to study the localization and association of SF1 and U2AF. The mobility of these proteins is correlated with their ability to interact with each other, and they are believed to interact in what are described as extraspliceosomal complexes that form before and persist after spliceosome assembly (Rino et al. 2008). The development of real-time, single-molecule imaging techniques provides an especially exciting and promising opportunity to probe in vivo realities, reconciling molecular and mechanistic details within a kinetic and spatial context. One such example involves single particle tracking of U1 snRNP within the nucleus (see Fig. 5), revealing both mobile and transiently immobile states (Grunwald et al. 2006). Single molecule imaging makes the range of mobility states over a population of molecules immediately apparent, and changes in the behavior of any individual molecule during observation can be assessed. A major lesson from these studies is that "the mobility" of a given molecule is more likely a mixed population of different states.
This work was supported by National Institutes of Health grant EB2060 to R.H.S. and a DFG fellowship (DG 3388) to D.G. The authors would like to thank S. J. Orenstein, Drs. A. Joseph and V. de Turris for critical reading of the manuscript.
Editors: Tom Misteli and David L. Spector
Additional Perspectives on The Nucleus available at www.cshperspectives.org
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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Dzasokhov Criticizes Arab Countries and Turkey's stances on Syria
Dzasokhov Criticizes Arab Countries and Turkey's stances on Syria
MOSCOW, (SANA)- Chairman of the Syrian-Russian Friendship Association, Alexander Dzasokhov, expressed strong condemnation of the terrorist acts in Syria, mainly the recent terrorist attack which targeted Aleppo University on Tuesday.
Meeting a Syrian People's Assembly delegation headed by Chairman of the Press, Printing and Publishing Committee at the People's Assembly, Fayez al-Sayegh, in Moscow on Wednesday, Dzasokhov stressed the deep-rooted relations of friendship and solidarity linking the two Syrian and Russian people.
He dismissed some Arab countries' stances towards Syria as "a big mistake" they are to realize it later, criticizing also Turkish government stances which are harmful to the Syrian and Turkish peoples alike .
He called for activating the work with the international parliamentary, social and unionist organizations to support Syria and condemn the terrorist acts committed in it.
For his part, al-Sayegh expressed appreciation over Russia's firm and principled stance towards Syria, asserting the deep-rooted and historical relations between the two countries.
He indicated that the West, in cooperation with Qatar, Saudi Arabia and Turkey, work to mobilize all terrorists in the world and send them to Syria with the aim of weakening the Syrian state and getting rid of the terrorists themselves at the same time .
For his part, Syria's Ambassador to Russia, Riyad Haddad, thanked the Association as it played an important role in enhancing the relations between the two countries.
Haddad stressed that Russia is defending the principles of right and justice, the international law and UN charter.
Meanwhile, members of the Syrian delegation appreciated the efforts of the Association in conveying the facts regarding what is happening in Syria to the Russian public opinion.


text:
Greener Grass [05]Jan. 2nd, 2016 12:02 am
Title: Greener Grass [Part Five]
Pairing(s): YoonSic
Rating/Genre: PG-13 + Language; AU Healing Romance
Warning(s): Cyanide. :((
Disclaimer: I don't own Soshi. I don't own anyone, in fact. All Fiction.Author's Notes: HAPPY 2016, EVERYONE!!! I hope y'all had a better New Year than I did…ugh drama. So, please forgive any oddball errors in this bish. BUT BUT BUT, JESSICA'S 'GRAVITY' COVER HAS BEEN LOOPING ALL DAY. BLESS HER VOICE.
( miscreant )


text:
Listen to free music played by zoinx
Blip.fm is internet radio made social. It's easy to search for, play, and discover free music recommended by real people. Join today to create your own free station and start listening to @zoinx.
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text:
Security Job in Henderson, Nevada : NV 89014
Looking for Security Jobs? See currently available Security job openings on security.jobs.net . Browse the current listings and fill out job applications.
security.jobs.net is the starting point for a job search in any security job . We provide job resources for Security positions throughout the industry. security.jobs.net also provides Security jobs listings from around the United States. Job seekers can search and apply for jobs in Security by either city or state.
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Security Officer
Security Officer Job Description
Customer Service Experience and Professionalism Required
Armed Forces, Law Enforcement, Resort & Casino experience is preferred.
New licensees are welcome.
Sharp, Professional and Highly Motivated candidates are encouraged to apply.
Please read below and reply via email and include:
Your weekly availability
Your current resume and summary
This position is both physically and mentally demanding. It requires familiarity with computers and incident reporting, excellent verbal skills and the ability to walk/stand for long periods of time.
1. Sheriffs card, current unarmed Security Card or Provisional letter issued by the PILB (https://nevadapilb.glsuite.us). Applicants obtaining a Security Card must have a Provisional in order to be hired
2. Reliable transportation and a cell phone;
3. Fluency in English;
4. Be drug free. We are a Drug-Free Workplace;
5. Submit to a criminal background check;
6. Be at least 18 years old;
7. Eligible to work in the United States.
Patrol residential, industrial and commercial premises to prevent and detect signs of intrusion and ensure security of people and property.
Answer alarms and investigate disturbances.
Call police or fire departments in cases of emergency, such as fire or presence of unauthorized persons.
Operate detecting devices to screen individuals and prevent passage of prohibited articles or persons into restricted areas.
Answer telephone calls to take messages, answer questions and provide information.
Monitor and adjust controls that regulate building systems, such as air conditioning.
Escort individuals to specified locations and to provide protection.
Write reports of daily activities and irregularities, such as equipment or property damage, theft, presence of unauthorized persons or unusual occurrences.
Inspect and adjust security systems, equipment, and machinery to ensure operational use and to detect evidence of tampering.
Circulate among visitors, patrons and employees to preserve order and protect property.
Warn persons of rule infractions or violations and apprehend or evict violators from premises.
Monitor and authorize entrance and departure of employees, visitors and other persons to guard against theft and maintain security of premises.
Henderson Security Job
Security Officer Job Requirements
Public Safety and Security -- Knowledge of relevant equipment, policies, procedures and strategies to promote effective security operations for the protection of people, data, property and institutions.
English Language -- Knowledge of the structure and content of the English language including the meaning and spelling of words, rules of composition and grammar.
Customer and Personal Service -- Knowledge of principles and processes for providing customer and personal services. This includes customer needs assessment, meeting quality standards for services and evaluation of customer satisfaction.
Call security or police in the event of an emergency (performing this work is not part of the position responsibilities).
High school diploma or equivalency required. College level courses in business or hospitality preferred.
Must possess strong customer service background. Experience in similar position preferred.
Computer Literacy:
Command of information system hardware/software is preferred. Effective written and verbal communication skills. Multiple language fluency is desirable. Strong customer service, communication and interpersonal skills required.Special Requirements:
Standing, sitting, walking. The employee may be required to work at a personal computer for extended periods of times as well as talking on the phone for extended periods of time. Ability to work extended/flexible hours, weekends, and attend meetings as required.
The physical demands described here are representative of those that must be met by an employee to successfully perform the essential functions of the job. Reasonable accommodations may be made to enable individuals with disabilities to perform the essential functions.


text: 2010 Nissan GT-R $64,777 38,276 Miles GT-R Premium, AWD, Jotech Stage 2: 740 HP (600 whp), AAM Stainless Exhaust, HKS Stainless Resonated Midpipe, Greddy Stainless Downpipes, 3" Air Intakes, Powder Coated Intake Manifold and Piping,... Empire Exotic Motors Dallas, TX 197 mi (888)-677-4778 2016 Nissan GT-R $75,998 25,866 Miles Empire Exotic Motors has a wide selection of pre-owned luxury, and exotic vehicles with a dedication to delivering excellence as well . We pride ourselves with over 100 + years combined experience... Empire Exotic Motors Dallas, TX 197 mi (888)-677-4778 2012 Nissan GT-R $66,000 29,000 Miles 2012 Nissan GTr Black Ed. 3.8 L DOHC Twin Turbo V6 with 530 HP. Dual-clutch transmission. Jet black exterior, Black/ red Recaro leather interior. Owner 2009 Nissan GT-R $170,000 OBO 21,000 Miles T1 Race Development built 7 sec turn key drag car! 1650whp on E98! Show stopper of a car and also super fast. Best of both worlds! Hey guys I have decided it is time for me to sell my car and... Owner X Success Your e-mail has been sent. You will be sent a copy of this email to the email address you provided. Page 1 of 1 Show 10 Per Page 30 Per Page 50 Per Page 100 Per Page 1 - 4 of 4 listings Top Cities for Nissan Nissan in Philadelphia Nissan in Bridgeport Nissan in Orlando Nissan in Chicago Nissan in Columbus Nissan in Riverside Nissan in Dallas Nissan in Denver Nissan in San Francisco Nissan in Los Angeles Nissan in Greensboro Nissan in Seattle Nissan in Miami Nissan in Jackson Nissan in Tampa


text:
Wes Anderson Special - Part One
Join Penny Lane as she fills the hour with tracks featured in the movies of director Wes Anderson!
Hear tracks from the likes of Faces, Bobby Fuller Four, The Kinks, The Rolling Stones and more!
Penny Lane brings a little glitter to Punks in Parkas with a Glam Rock special!
Hear tracks from the likes of Brian Eno, New York Dolls, Jobriath, Gary Glitter and more!
Join Penny Lane as she plays you some hot tracks to ease you into Summer!
Hear music by the likes of Wilson Pickett, The Beatles, The Dramatics and more!
Penny Lane plays tribute to The Beatles seminal album, Sgt. Pepper's Lonely Hearts Club Band on it's 50th birthday by playing the masterpiece for you in its entirety!
Penny Lane opens the show with three Bond themes to honor Roger Moore, who died this past week at 89 years of age.
Penny then goes on to celebrate Punks in Parkas 12th Birthday party in style with tracks from the likes of The Bobbettes, Al Green, The Allah-las and more!
Penny Lane opens the show with a triple shot of Aretha Franklin and ends it with a tribute to Chris Cornell, who we lost today at age 52.
Between are tracks from the likes of Sam and Dave, Georgie Fame and the Sonics
Penny Lane brings you part two of her spotlight on British singer, Holly Golightly!
Hear tracks from Holly, along with Billy Childish and Thee Headcoatees!
It's May 4th - Audrey Hepburn's birthday! What better way to celebrate then with an whole hour of tracks by British songstress, Holly Golightly!!
Hear some of Holly Golightly's best tracks, including Run Cold, Eye For an Empty Heart and Wherever You Were!
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A great hour featuring tracks from the likes of Cannonball Adderley, Al Green and Serge Gainsbourg!
Penny Lane is binge watching The Get Down on netflix and brings you a show with some tracks inspired from the soundtrack!
Hear tracks from the likes of Baby Huey, Steve Marriot, Brian Wilson and more!


text:
Current Employment Opportunities
Supported Employment Coordinator
Salary: $30,759
- Function as Supported Employment Coordinator which will include, but not limited to: direct supervision of consumers, enclaves and work crews, supervision of all job coaches when the Supported Employment Manager is unavailable, completion of all rehabilitation paperwork for each consumer, coverage at all sites, preparation of consumers for career changes and supported employment placement.
- Complete Supported Employment paperwork as assigned by supervisor.
- Directly supervise assigned sites ensuring adherence to all specifications.
- Ensure quality control standards and maintained at all Supported Employment sites.
- Assist in completion of DDS attendance, waiver billing and contract company billing sheets.
- Cover at Supported Employment sites and Community Experience sites as needed.
- Orient new staff to Supported Employment and specific sites, including paperwork procedures and employer specifications.
- Chair/Co chair In House Committee as assigned.
- Associates Degree. Bachelors Degree Preferred.
- Five years experience working with persons with disabilities in supported employment.
- Valid Drivers License and ability to possess a state of CT Public Service License.
- You are required to be in possession of a car on site during your entire shift to transport yourself and/or consumers to community sites in all weather conditions and throughout all neighborhoods.
- Depending upon assignment, must be able to work in a variety of settings, weather conditions and locations.
- Must maintain CPI, CPR and First Aid certification.
- Knowledge and skills in Word, Excel and the internet.
- Work a flexible schedule that may include early mornings, evenings, weekends and/or holidays.
- Provide hands on personal care which may include toileting, feeding, cleaning and all other activities of daily living skills.
- Ability to transfer individuals requiring personal hygiene care.
- Have physical endurance to use physical crisis intervention techniques which may include restraining individuals.
- Provide physical interventions which may include lifting, mobility, first aid, and safety evacuations.
09/15/17 - 37.5 hrs.
Monday through Friday: 8:00 to 4:00 p.m.
Flexibility required for morning, evening and weekend hours.
Contact info:
Human Resources
Fax: 203-371-1479


text:
An agreement by BankBoston Corp. to tighten mutual fund sales practices in the wake of a regulatory probe underscores the need for banks to focus on investor suitability.
The agreement was confirmed Friday by the banking company and the Massachusetts Securities Division, and it ended a seven-month inquiry. BankBoston neither admitted nor denied wrongdoing.
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text:
Number Two
In this number two learning exercise, students learn to recognize, count, and print the number and word for 2. Students trace dotted examples and draw two objects in an empty jar.
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End of Unit 2 Assessment: Working with Two Texts - Reading, Listening, Summarizing, and Synthesizing
As a summative assessment for this unit on colonial trade, fourth graders listen to and read informational texts in order to demonstrate their ability to take notes, write summaries, and draw connections. Young scholars first listen as...
3rd - 5th English Language Arts CCSS: Designed
Daily Warm-Ups: Grammar and Usage
If grammar practice is anywhere in your curriculum, you must check out an extensive collection of warm-up activities for language arts! Each page focuses on a different concept, from parts of speech to verbals, and provides review...
3rd - 6th English Language Arts CCSS: Adaptable
Unit Conversions and Problem Solving with Metric Measurement
Convert young mathematicians' knowledge of place value into an understanding of the metric system with this five-lesson unit. Through a series of problem solving activities and practice exercises, students learn about the relationship...
3rd - 5th Math CCSS: Designed
Second Grade Adding and Subtracting
Reinforce addition and subtraction in your second grade class with a series of lessons that feature manipulatives, ten frames, spinners, and number lines. The unit is divided into three sections and includes concepts such as counting...
1st - 3rd Math CCSS: Designed
Grade 1 Supplement Set A4 Number & Operations: Equivalent Names
Engage young mathematicians in developing their basic arithmetic skills with these great hands-on activities. Using Unifix® cubes to model a variety of single digit addition and subtract problems, children build a basic understanding of...
K - 2nd Math CCSS: Adaptable
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text:
- 01:17 Submitted 9,300 word historical vampire story for antho. Am nervous. Experimented with style/voice. Hope it doesn't suck! #
- 14:46 @sadbhyl Dr. Who squee? What Dr. Who squee?! I want to squee! #
- 15:49 @sadbhyl Oooooooooh. Very very interesting. #
- 18:08 Watching Penny Serenade. Could Mr. Grant be more gorgeous? No. No, he could not be. #
- 19:39 Oh my god! Why did nobody warn me about Penny Serenade?! Just in case you felt like watching it, the little girl DIES and it's HORRIBLE. #
- 19:40 "Heart-warming study" my aching ass. Whoever wrote the movie summary, tricking me into watching the movie, can DIAF! #
- 19:51 The moral of Penny Serenade=One kid is as good as the other. If you lose one, adopt another! Two or three times if necessary! #
- 20:00 @eurydice72 If you did, I forgot your warning. #
- 20:24 Husband made this doll. Am proud but it's creepy as fuck, amirite? twitpic.com/2c86z #
- 20:30 @BillCorbett that sounds hot. #
- 20:32 Debating new series of photographs entitled "This is where Tara T. Cat sleeps." Since I have about 100 pics of my cat sleeping. #
- 22:44 @DakotaFlint Even the cats agree. They're scared of it and run any time they see it. #
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God, I hope they do.
God, I hope they do.


text:
School board approves sale of Educational Service Center
Space will be turned into apartments, town homes and accompanying parking areas
Members of the Cedar Rapids School Board have waved goodbye to the Educational Service Center.
During Monday's regular meeting, board members approved sale of the building to local real estate developer Steve Emerson. The $771,100 deal includes the structure, located at 346 Second Ave. SW and 353 Second Ave. SW, as well as vacant lots at 210 Fifth St. SW and the corner of Second Ave. and Fifth St. SW in Cedar Rapids.
According to Scott Olson, who is the Cedar Rapids Community School District's real estate representative in the transaction, this completes the district's disposition of its properties damaged in the 2008 flood.
No one spoke during the public hearing on the sale, which preceded board approval, during Monday's meeting.
Emerson is planning to turn the space into apartments, townhomes and accompanying parking areas. He had initially made an offer for a portion of the property in April 2012 and changed his offer once Maranatha Bible Church decided not to acquire the 210 Fifth St. SW lot, extending the process for Emerson and the district.Olson anticipated that the deal will close in mid-February, once additional paperwork is completed.
|Ernst blames Democrats for health care i ...
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|What's needed for autonomous cars to com ...


text:
All of The Elite Force went to a party, and two members did "it" and don't remember. One was Skylar Storm, and now she is pregnant, with one question.
"Is the baby Oliver's or Chase's?"
- Nov 04, 2016
Seriously? Justin Bieber? He is a trouble maker girl. Lol 😂 😂😂😂😂
Melody54000 Sep 18, 2016
Ooh! A love triangle! I've always seen a potential love triangle between Skylar, Oliver, and Chase. I'm Team Oliver. I think that Chase should end up with Sabrina. Or me. If that's possible. 😊
parkyoongimin Sep 15, 2016
Oh no. I feel like it's Oliver's baby because it isn't really like Chase to do something like that. This is really good so far!
liagibar Sep 15, 2016
Ah... I need to know! Please update soon, even though like you just published this 6 minutes ago!


text:
To order a fine art print of Hawaii, please contact me directly at firstname.lastname@example.com.
If you are an Art Consultant, Architect or Interior Designer and would like to request current wholesale pricing and available sizes, please contact me directly.
Fine Art Prints | Metal Prints | Canvas Gallery Wraps
The "Photography: Impact of a Medium" artist reception at 2nd City Council Art Gallery in Long Beach CA drew a large crowd despite the rainy weather. The fine art photography exhibit was jured by Robbert Flick. My friend and fine art photographer Mia DelCasino won the "Eye Opener" award for her piece "Passage Nine", which was taken in Hawaii. Congrats Mia!
The "Visual Expressions 2010" Artist Reception was a huge success. Even the rain did not keep the large crowd from attending the show this past Friday night. The Temecula Valley Arts Council's first art show of the year was held at The Mercantile Gallery in Old Town, Temecula and the show continues through Feb. 21st 2010.
The "California Visual Artists" opening at the 2nd City Council Art Gallery in Long Beach was a hit. I had such a good time meeting the other artists in the show and talking with so many interesting guests that night. The band Centrevol performed and rocked the crowd. A big "THANK YOU" to Mia DelCasino for taking these wonderful photos.


text:
OK, I'll admit it again, I am a sucker for well-made old-style games. The "Metal Slug" series is not especially old, but I love the way it has refused to adapt to the brave new world of 3-D gaming.
"Metal Slug 3," a new game for Xbox from SNK, is a rare return to the nonstop, two-dimensional, side-scrolling arcade action of the early 1990s. It is a game with a sense of humor and a sense of honor; the kind of game that challenges players without being hard simply for the sake of being hard.
In "Metal Slug 3," players lead one of four soldiers as they battle entire armies of enemies. Don't attempt to conserve ammunition in this game, the enemies come in all forms and they definitely keep coming.
You'll fight enemies of the human, monster, machine and bio-mechanical persuasions. Some die from a single shot, while others absorb bullets and grenades as if they were made of mochi.
The game begins with your soldier storming the beach. Remember, this is an old-fashioned side-scroller — almost the entire game is spent running toward the right edge of the screen. So you head right, and giant crabs attack you. Get through them, and you start to face choices.
You can commandeer a submarine and scroll through the terrors of the deep, or you can push on above sea level. On land you will encounter more crabs. Under the sea you deal with giant eels and jellyfish. Either way, the enemies keep coming.
So do the laughs.
The best part about these alternative routes is that they add a lot of replayability. "Metal Slug 3" is already the kind of game you play more than once — especially as it supports both single- and two-player action. The main arcade game only has five levels, but unlockable worlds and modes have been added, too.
Throughout the game, you will run into prisoners and victims whom you free. In exchange for freedom, these prisoners will give you bombs, grenades and other power-ups. My favorite power-up is a machinegun-toting orangutan that becomes your wingman for short snatches of time.
Perhaps the most popular power-up will be the "Zombie Mode," in which the player becomes temporarily undead and indestructible.
Along with power-ups, "Metal Slug 3" presents the player with a number of vehicles that add joy to the ride.
As mentioned earlier, there is a submarine. Fans of classic arcade games may recall "In the Hunt," in which players piloted a point-and-shoot submarine through perilous waters. Controlling the sub in "Metal Slug 3" is similar, but this game also includes a tank, robotic armor, a jet, a helicopter and an elephant.
All of this equipment is presented with exaggerated cartoon humor. So are the settings and the enemies. There's plenty of gore in the game, but it's silly cartoon gore. Everything in this game is highly stylized and fun.
When I first started playing "Metal Slug 3," I was astonished by how fun it was considering its old style of play. Age has not been kind to "Pac-Man," "Pong" or "Space Invaders" — all historic games that launched empires. In their original states, those games look like antiques, something you might play out of intellectual curiosity or a longing for nostalgia. Updates have not managed to restore their luster.
But "Metal Slug 3" is not as old or as classic as "Pong" or "Space Invaders." "Pong" was introduced 32 years ago; the first "Metal Slug" came out in 1996. I think that is the secret to "Metal Slug 3's" playability. It is not an old game or a remake of an old game; rather, it is a new game that deftly uses classic mechanics.


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Reliable and Long-Lasting
Space-Saving Design
Clean, Professional Appearance
Avalible as a Fire-Rated Door
Counter Doors work well in most service openings such as cafeterias, stadiums, hospitality, and educational facilities. Barton Overhead Door uses Cookson Counter Doors for their expectional reliability and compact design. Counter Doors are also availible UL labeled for up to 3 hours of fire protection. These doors can be operated by hand, crank, or electric operator.


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If you afraid of downloading the wrong ROMS, use this Crappy Tiny Reader (CTR) to check the 3DS ROM DATA. Make sure the Game Serial is the same with the info here. This tool is created by PsyKo who owns the http://scene.releases.free.fr/
DICASTIA 3ds ace assault combat english europe g-simulation horizon legacy venom 0067 - Ace Combat: Assault Horizon Legacy (E)
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AnonymousJanuary 23, 2012 at 9:05 PM delete
link no found lo puedes subir de nuevo al uploaking como antes pleaseReply
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£65.00 £30.00
Candy Stud Trainer
Shoes by Pieces Made from a leather-look fabric. Smooth matte finish. Spiked stud embellishment. Textured bumper panel. Lace-up closure. ABOUT PIECES. Encapsulating modern fashion trends with an eclectic range of accessories, Pieces brings forth a seasonal collection of jewellery, bags, and footwear. Interpreting Danish street style and catwalk trends, directional footwear sits alongside a playful jewellery line, with accessories benefitting from a seasonal colour palette updates.


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PRESS RELEASE FOR IMMEDIATE RELEASE JANUARY 11, 1997 (NASHVILLE, IND.) - MotorSports PhotoSource, a stock and assignment photography agency that specializes in motorsports images, has announced the creation of an online ...
(NASHVILLE, IND.) - MotorSports PhotoSource, a stock and assignment photography agency that specializes in motorsports images, has announced the creation of an online division.
MotorSports PhotoSource OnLine was created to provide the Internet community access to racing news, photographs, information and opinions through its web site.
Upon reaching the MSPS site at http://members.aol.com/msps01/msps01.htm, the user is greeted with an index of pages within the web site. By selecting the page of their choice a visitor can find viewable photographs, locate racing schedules, results and point standings, read an opinion column or subscribe to the free MotorSports PhotoSource OnLine News Service.
By subscribing to the news service a visitor will receive e-mailed news briefs regarding fast-breaking motorsports news as well as a regular newsletter.
In addition, the MSPS OnLine web site offers links to many other popular motorsports-related web sites and several top photography-related sites.
"Prior to now, MotorSports PhotoSource has dealt only with providing photographs to others in the racing and publishing industries," said Rodney A. Margison, owner of MotorSports PhotoSource. "Now we can provide that and more to a growing segment of the general population by way of the Internet."
In the future MotorSports PhotoSource OnLine intends to offer MotorSports PhotoSource racing apparel and licensed racing products for sale through their World Wide Web location.
For additional information regarding MotorSports PhotoSource OnLine or information on advertising on the MSPS OnLine web site or News Service, write to P.O. Box 1495, Nashville, Ind., 47448, or send e-mail to email@example.com.
RODNEY A. MARGIONS, OWNER MOTORSPORTS PHOTOSOURCE firstname.lastname@example.com


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KASEY KAHNE, No. 83 Red Bull Toyota Camry, Red Bull Racing Team Starting Position: 1st How was your qualifying lap? "We had a really good run. The Red Bull team did a nice job. The Toyota engine was good. We made some gains from practice. ...
KASEY KAHNE, No. 83 Red Bull Toyota Camry, Red Bull Racing Team
Starting Position: 1st
How was your qualifying lap? "We had a really good run. The Red Bull team did a nice job. The Toyota engine was good. We made some gains from practice. We were a little bit quicker there and hopefully we can do the same tomorrow and we'll be good for Sunday."
Are you surprised with your qualifying lap? "We were pretty decent in practice. The Red Bull Toyota was good and the guys have done a really nice job this week. Everything felt good, it was just I was too free so we tightened up there and I was actually pretty good right there, but I felt like I got a touch tight. It was a great lap for us. That was really good."
What will you work on during Saturday's practice? "Just keeping the car turning. The track will change a little bit Sunday, but we race during the day this year so it's going to be different than what we've had in the past. Just making sure you have grip, whether it's front grip or rear grip you need it here to go fast. You can definitely search at this track and find different spots on the track to go fast. It's a neat track so it's always nice to race at Homestead."
Are you looking forward to Sunday's race? "I'm looking forward to it. That was the best qualifying run by far since I've been at Red Bull. It was a little bit free in practice. A little bit too loose -- we ended up seventh. They made the right changes and the team did a really good job to get the pole. It was nice. I look forward to Sunday's race. It's gonna be exciting being up front now, being in the mix with Denny (Hamlin), (Kevin) Harvick and Jimmie (Johnson) -- they're all gonna be fast throughout the race. I'm just glad we are so far. Hopefully, we can stick with it tomorrow and have a good practice and be competitive and in the mix on Sunday."
DAVID REUTIMANN, No. 00 Aaron's Dream Machine Toyota Camry, Michael Waltrip Racing
Starting Position: 7th
Are you happy with your qualifying lap? "It was okay. We picked up a lot from practice so I'm happy with that. We were just a little bit loose, but overall hopefully it will hold up to be an okay lap. We have to work on making it turn better and stuff like that - normal stuff."
JOEY LOGANO, No. 20 Home Depot Toyota Camry, Joe Gibbs Racing
Starting Position: 19th
How was your qualifying lap? "The track picked up a lot of grip. I think we picked up what we were supposed to. We were just too tight. Just got under and plowed the nose which we knew the track was going to tighten up, but maybe a little bit more than what we thought it was going to there."
How was your car in race trim? "We were really loose in race trim to start today and we worked on it and got it pretty good by the end of the day so hopefully we can kind of start with what we had and fine-tune it tomorrow hopefully."
MARCOS AMBROSE, No. 47 Clorox/Kleenex Toyota Camry, JTG-Daugherty Racing
Starting Position: 20th
MARTIN TRUEX JR., No. 56 NAPA AUTO PARTS Toyota Camry, Michael Waltrip Racing
Starting Position: 25th
How was your qualifying lap? "It was okay. I was a little skeptical about going out early, but we picked up a little bit from practice, which was good. The guys did a good job on the NAPA Toyota today. We were off a little bit off the trailer and kind of ran out of time in practice, so that was a decent lap for us, for sure."
JOE NEMECHEK, No. 87 Young Eagles/HeatRedefined.com Toyota Camry, NEMCO Motorsports
Starting Position: 29th
MIKE BLISS, No. 66 Toyota Camry, PRISM Motorsports
Starting Position: 30th
KYLE BUSCH, No. 18 M&M's Toyota Camry, Joe Gibbs Racing
Starting Position: 33rd
CASEY MEARS, No. 13 GEICO Toyota Camry, Germain Racing
Starting Position: 34th
DENNY HAMLIN, No. 11 FedEx Express Toyota Camry, Joe Gibbs Racing
Starting Position: 38th
How was your qualifying lap? "It was tough. I told (the team), it looked just like Kyle's (Busch) lap, that's for sure. I got a feel for it, it was just too tight right there. The setup was quite a bit tighter than ours. It looked just like it. We didn't improve, but our car is gonna be fine tomorrow. We know that. This is a track you can pass. We're looking forward to that part of it. Friday has just never been our strong suit and so we gotta battle back again."
SCOTT SPEED, No. 82 Red Bull Toyota Camry, Red Bull Racing Team
Starting Position: 41st
What happened on your qualifying lap? "I wish I could say it was exciting, but when you're right next to the wall and touch it's not too exciting. It is what it is. We probably pushed it a little bit too hard. We knew going out first the track is not gonna be near as good right now as it will be in an hour-and-a-half. Just tried to get a little bit more than we probably should have."
KEVIN CONWAY, No. 7 Extenze Toyota Camry, Robby Gordon Motorsports
Starting Position: 42nd
LANDON CASSILL, No. 64 Empire Steel Toyota Camry, Gunselman Motorsports
Starting Position: 43rd
-source: toyota motorsports


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Listen Live to WEHC-FM Learn More About Emory & Henry College Make a Station Donation to WEHC-FM 90.7 radio
Racing Merchandise
Purchase a piece of WEHC-FM 90.7 and E&H History! Visit mmrstore.com
I wouldn't have been a race car driver or team owner if it weren't for my mass communications degree from Emory & Henry College.
~ Eric McClure, E&H Mass Comm., Class of 2000 and Martin–McClure Racing (MMR) Team Co-Owner
Eric McClure says he gained great benefit and confidence from his radio experience at Emory & Henry's WEHC-FM that led to his successful career as a NASCAR driver and team co-owner. As the station celebrates 25 years of operation, McClure wants to help ensure the financial security of the station so that future students can have the same opportunities that he did.
Purchase a piece of WEHC-FM 90.7 and E&H History! Visit mmrstore.com
Those who enjoy community, non-commerical radio are encouraged to Make a Gift to WEHC-FM. Simply choose WEHC from the "Designations" drop-down menu.
Martin-McClure Racing (MMR) — located just down the road from Emory & Henry College on Intersate-81 at Exit 22 — will donate proceeds to WEHC-FM, 90.7 in Emory, Virginia.
Eric McClure and Hal Martin launched Martin-McClure Racing (MMR) in 2015 and quickly found themselves in winner's circle for the 2016 K&N Series race at Bristol Motor Speedway, known as "the world's fastest half-mile." Prior to becoming a team co-owner, Eric drove a Toyota under the Hefty and Reynolds Wrap sponsorships in the highest levels of NASCAR racing. While Hal Martin and Eric McClure were both driving in the Xfinity series, they began planning to develop their own team.
Team co-owner Eric McClure wants to ensure that future Emory & Henry students gain the benefit of the radio experience that he and his classmates enjoyed. McClure graduated from Emory & Henry in 2000 and credits much of the success in his 14-year NASCAR driving career to his education. As a show of his support, MMR driver Chad Finchum will race the #39 Toyota for the benefit of WEHC-FM with both the 90.7 station logo and Emory & Henry logo on the car.


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Unmanned Aircraft Systems for Remote Sensing (UAS4RS) Conference 2016/
Getting Down and Dirty with UAS4RS: Searching for Solutions to Environmental Challenges
All doers, dabblers, and would-be dabblers are invited to join the inaugural UAS4RS 2016 (Unmanned Aircraft Systems for Remote Sensing) Conference to be held at the University of Queensland, Brisbane 17-18 February 2016. The aim of UAS4RS 2016 is to bring together the UAS community in Australia, and to present and discuss UAS technology for remote sensing applications and information extraction.
This conference is for you if:
- You're a 'doer': you already use UAS as part of your remote sensing workflow and would like to share your experiences and challenges;
- You're a 'dabbler': you've done some testing and think this technology might be the way forward for you; and
- You're a 'would-be dabbler': you've heard the hype and you're curious to know more.
The program is designed to be interactive and engaging! You will be actively linked with UAs researchers, developers, systems and service providers, as well as end-users. There will be presentations, posters, exhibitions, live demonstrations, and discussions to exchange ideas on the use of UAS for remote sensing applications.
So whether you have a story to share, equipment to demonstrate, or would simply like to come along and learn more about this exciting technology, you are welcomed to UAS4RS 2016!
For more information, abstract submission, and sponsorship and exhibition opportunities, please see the attached flyer and visit conf2016.uas4rs.org.au. Keep up to date by following on Twitter @UAS4RS.
The due date for abstract submission is very soon. Please submit your abstract before the 18th of December 2015.
Students are encouraged, in particular, to submit an abstract.


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Northwestern Michigan College physical therapy major
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Get the facts about Northwestern Michigan College physical therapy major, and registering for elective discussion sections and seminars. Which college degrees give you the best chances of finding employment after graduation? Qualifications for nursing programs vary widely. Learn about the various medical specializations available within technical training programs. Concentrating on Northwestern Michigan College physical therapy major, for example, you may gain an advantage in job recruiting on-campus. Check with the physical therapy department for course registration deadlines, and physical therapist program requirements. Please use the form above to request admissions info for Northwestern Michigan College.
Physical therapist assistants (PTA) and physical therapist aides work under the supervision of physical therapists, helping patients who are recovering from injuries regain movement and manage chronic pain. Further, they develop exercise programs that will help patients improve cardiovascular function, regain flexibility, and build muscle mass. All states require physical therapist assistants to have an associates degree from an accredited physical therapist assistant program. These programs typically last 2 years, and include courses in algebra, English, anatomy, physiology, and psychology. PTAs gain hands-on experience during supervised clinical work rotations, and may earn certification in cardiopulmonary resuscitation (CPR), and other first-aid skills.
Licensure typically requires graduation from an accredited physical therapist assistant program and passing the National Physical Therapy Exam, administered by the Federation of State Boards of Physical Therapy. Physical therapist aides, on the other hand, are not required to be licensed but earn less salary. Some states require that applicants pass additional state-administered exams, undergo a criminal background check, and be at least 18 years old. Employment of physical therapist assistants and aides is projected to grow 40 percent from 2017 to 2025, much faster than the average for all occupations. The median annual wage for physical therapist assistants and aides was $46,980 in May 2017. Beyond major requirements within the physical therapy department, you may consider Clarendon College food court hours as a complementary elective to your primary course of study.
Bon Secours Health System
Acts as liaison between unit and Nursing Services to assist in maintaining effective knowledge of nursing care practices that may be utilized in..
4 hours ago Charge Nurse Baylor Days 6a-6:30p
Lutheran Life Villages
Graduation from an accredited school of professional nursing. All nursing personnel assigned to the designated unit are responsible to this individual..
9 hours ago RN Staff Nurse - Rehab Services - Good Samaritan Medical Center
Legacy Health System
One year registered nursing experience or completion of either the Legacy Health RN Residency program or a nursing program that is equivalent in content..
5 hours ago Field Nurse Case Manager (RN) - Part-time
Experience in rehabilitation services industry, vocational/ occupational/ industrial nursing preferred. Diploma, Associate or bachelors degree in nursing or..
10 hours ago Hospice Nurse - RN Admissions
Seasons Hospice & Palliative Care
Minimum one year work experience in hospice, home health, geriatrics or general medical surgical nursing. Provides nursing assessment, initial plan of care and..
1 hour ago District Coordinator Health Services - RFP2995 AI048 Certificated
Central Unified School District
Child abuse reporting, community health, crisis planning, crisis intervention, nursing diagnosis and. CENTRAL UNIFIED SCHOOL DISTRICT POSITION.
1 hour ago


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Home Thankful Quotes Thankful Quites
Thankful Quites
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I was born and raised in New York City. And not so much in the big apple, but more consistently in the concrete jungle. This city literally birthed all that I am and intend to be. From everything that I've ever wanted, to everything I never had or once sacrificed, this city has been the foundation of all my ambitions, accomplishments and disappointments.
I made it my business, unknowingly perhaps, to conquer this city back in junior high school. I committed to that business in high school. Followed through with it in college. And sold everything for it, since then. I told myself in 2012 that I would conquer this city. First and foremost.
That was a major part of the struggle with moving to Los Angeles. In many ways, I felt like I hadn't yet conquered the city that raised me. I felt insecure and greatly guilty with the growing ambition, each day I spent in LA, to conquer Hollywood and what I consider to be, the land of the Lost and Found. And yet, I struggled even more with a realistic path of attempting, yet again, to conquer New York, with no money and only via means of going back to and through where I'd just came from. I just didn't see a way.
So, now having been back in New York for two months, and still wholeheartedly in the spirit of LA, I have finally conquered it. I had to find the rest of myself before I could expose and then reconstruct the city for what it is. That is, of course, the only way a person like me ever could conquer this city. A Black, woman, with no money, influence or tangible power.
To conquer New York City, I would have to expose that in fact, material and power were the only real ways to conquer the supposed city made for the dreamer. That the land of Lady Liberty, long ago, drowned her dream of easing the poor, weak and huddled masses, under millions of dollars and thousands of egos.
And so, I did have to come back. I first had to go find the rest of myself, or rather, go discover how to be the rest of myself. Then and only then was I allowed to come back to conquer the concrete jungle that raised me. Adorned proudly in all of my vibrant colors of beauty and bold flames of passion, I took claim on my land, my territory, my corner of the world. On my terms, solely under my authority, the way I saw fit.
Thus, as I said, I am more than a conqueror. So much more.
Ask New York City. Ask the Lower East Side. Ask Brooklyn. Ask the skyscrapers and the concrete. Go ahead. They'll tell you the truth. The way it really is. As it was, when it was. And how it is now.


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CAT IN HEAT: 17th August:
Our cat went into its first heat on 5th August for 30 hours. It was mild. Today it went into its second heat. This time I could see the symptoms from few days before. Her appetite was lost and she started eating dry food instead of raw chicken or fish. So I was careful to keep her indoors in time. Learning about acupressure for a cat in heat. It works but its effect reduces with time and the cat is craving for it, sort of like addicted. In the beginning she slept for 6 hours after acupressure, then 3, then 1, then none and wanted more. So stopping now (after doing it multiple times daily for 5 days). It did help her with the appetite; ate moderately after acupressure compared to almost nothing before acupressure.
Here are the steps from http://messybeast.com/catmassage.htm:
There are a number of specialised massage techniques e.g. TTouch and acupressure and books and videos describing these. Acupressure can be adapted for use on cats and if you take the time to learn it, is apparently enjoyed.
Acupressure can be used to bring a queen off call (to induce ovulation). For breeders, this has the advantage of avoiding hormones, vasectomised males or mechanical means (a cotton bud or glass rod). Pedigree cats are bred when the breeder decides, which is NOT every time they come into season. At other times is it necessary to stop the female from calling, usually by inducing ovulation is some way. The following procedure should be repeated every 10-15 minutes for an hour and then again 12 hours later (or easch time the oestrus queen is acting like a shameless hussy).
Step 1 is to pat the calling female in a way to mimic the footwork of the male during the initial stages of mating. A tomcat holds the female's scruff in his mouth and "treads" her hindquarters with his back feet. For the owner, this means patting or stroking at the bottom of the back to make her lift her rump. The female will lower her shoulders, raise her bottom and move her tail to one side. This is similar to the rump-lifting position cats adopt when being stroked on the lower back.
Step 2 is to gently stroke or tickle the area under her vulva. If she has not already done so, she will flick her tail to one side in readiness for "mating".
Step 3 is to use acupressure to induce ovulation. With one hand, scruff her to simulate the way a tomcat would hold her (or just lay your hand across her neck if she is very co-operative). With your other hand lying along her back pointing towards the tail, press your index or middle finger into the hollow between the tail head and pelvis opposite the side of the shifted-across tail. You will feel a small hollow near the backbone, just at the side of the tail where the tail ends and the back starts. Press downward and inwards at this point, possibly vibrating your finger slightly. It should trigger a post coital response – a long throaty growl and a huff after which she will pull away and roll over to wash under her tail.
And a video
TAIL INJURY: 15th August: Because of my carelessness that cat's tail was slammed heavily by the door in the winds. She shrieked so loud that my heart stopped. Then she ran away under the bed and did not let me come near. After 30 minutes she came into my room and I saw her bleeding at the tail. I was in panic and called a neighbor and she said she looks fine, nothing broken and that cats have amazing ability to heal. She healed in few hours, there is still like a dent in her tail which she allowed me to touch today and see by looking into her long hair. I am surprised how soon it healed on its own.
SOCCER: 1st August 2017: Discovered my cat in the potato crate playing with potatoes. So threw some potatoes on ground and it started playing soccer with it. After few days gave it tennis balls and now there is NO stopping her from becoming a soccer champion. She just loves tossing it all around the house, hiding from it, running away from it at marathon speed and then pouncing on it to continue playing soccer. We get to have hearty laugh watching her especially when she slips on the floor mats or bangs into the door while running at high speed.
RAISING PETS NATURALLY: 20th July 2017:
I never wanted to have pets as I did not like to have hair all over the house. My husband one day became very adamant about having a dog. I knew that I was the one who would have to take care of the pet so I insisted on no. When it became a recurring argument, I asked him to find out what all it takes to have a pet and he was reluctant for that. So I went to a neighbor who has dogs and cats and asked questions and made notes and was overwhelmed with the tasks, vaccinations, pet visits, cage, pet food, sterilization, walking time…
An year passed. One day suddenly a cat appeared in the porch, it was evading us so I did not bother. After 2 days I still saw it there, so gave it roti(flat wheat bread) pieces, brown rice. It ate some bread pieces and stayed there. I sat close by and called her. She came with inhibition and I started petting it. It starting making some sounds so I was not sure what was happening. I left the cat outside and went to sleep.
Next day went to store on my neighbors recommendation and bought pet food and the cat gobbled it as if it was hungry for days. She was very skinny. I went to my neighbors house and asked about the sound it made while I touched it and they to my delight said it is called purring and it means it is content. Then they told me to spay it, to vaccinate it and gave some food for it.
Since am a vegan, I asked my gardener to cook chicken for it and since then it dislikes dry pet food. My neighbor had suggested to give cooked chicken and fish.
Two days ago I googled "What is spaying" and was horrified to know that it means "taking out ovaries and uterus". Then I youtube some videos and came across these links and it disturbed me very much to know that the way humans are brainwashed into western medicine dependency and preventive organ removal (young adult humans get their tonsils and wisdom tooth removed on the advice of the doctors, breast feeding was discouraged and bottled food was encouraged, real food is made expensive and cheap processed disease causing food is made available every where), the same way the pet industry is.
My neighbor on my questions said "It is curious that we have had cats and dogs all through our lives of 70+ and never questioned what is spaying, what the alternatives are…". It was very honest of them to say so. I wish more people questioned the conventional manners in every aspect of life.
I asked my gardener of 50+, an innocent big heart village man, to bring raw meat the next day and he said "It will not eat it. I do not even feed my cat cooked meat, she only eats pet food". I said "let us try". And no-guesses here…my cat loved eating raw meat….but at the same time I felt so sad that why people do not question? A cat whose natural diet is raw mice, why do we cook meat for it?
So that is the update until now. Back to the question of if I like the cat having around? YES it feels nice to give love. It likes to cuddle in my lap close to my tummy and chest and is playful and yet independent. It is a quiet but playful loving presence at home.


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Buffett, Malone explore investment in Sprint: sources
SUN VALLEY, U.S. (Reuters) -- Warren Buffett's Berkshire Hathaway Inc and John Malone's Liberty Media Corp are exploring an investment of between $10 billion and $20 billion in U.S. wireless carrier Sprint Corp, people familiar with the matter said.
Masayoshi Son, the chief executive of Japan's SoftBank Group Corp, which controls Sprint, met Buffett and Malone separately this week at an annual gathering of CEOs in Sun Valley, Idaho, the sources said on Friday, confirming an earlier report in the Wall Street Journal.
Shares of Sprint were up 5 percent at $8.62 in afternoon trading on Friday.
Sources said last month that Sprint has entered into a two-month period of exclusive negotiations with Malone's Charter Communications Inc and Comcast Corp that has put its merger talks with U.S. wireless peer T-Mobile US Inc on hold till the end of July.
Unlike Malone, Comcast CEO Brian Roberts has shown little interest in his company investing in Sprint, the sources said.
Malone in January raised the possibility that major cable companies could get together and buy a wireless carrier.
Sprint, Berkshire Hathaway and Liberty Media did not immediately respond to Reuters requests for comment.
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