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The nuclear RNA and DNA helicase Sen1 is essential in the yeast Saccharomyces cerevisiae and is required for efficient termination of RNA polymerase II transcription of many short noncoding RNA genes. However, the mechanism of Sen1 function is not understood. We created a plasmid-based genetic system to study yeast Sen1 in vivo. Using this system, we show that (1) the minimal essential region of Sen1 corresponds to the helicase domain and one of two flanking nuclear localization sequences; (2) a previously isolated terminator readthrough mutation in the Sen1 helicase domain, E1597K, is rescued by a second mutation designed to restore a salt bridge within the first RecA domain; and (3) the human ortholog of yeast Sen1, Senataxin, cannot functionally replace Sen1 in yeast. Guided by sequence homology between the conserved helicase domains of Sen1 and Senataxin, we tested the effects of 13 missense mutations that cosegregate with the inherited disorder ataxia with oculomotor apraxia type 2 on Sen1 function. Ten of the disease mutations resulted in transcription readthrough of at least one of three Sen1-dependent termination elements tested. Our genetic system will facilitate the further investigation of structure–function relationships in yeast Sen1 and its orthologs.
TRANSCRIPTION termination by eukaryotic RNA polymerase II (Pol II) uses at least two pathways, one that is coupled to cleavage and polyadenylation of the nascent transcript [the poly(A)-dependent pathway] and one that involves the activity of the RNA/DNA helicase Sen1 (the Sen1-dependent pathway) (Kuehner et al. 2011). The Sen1-dependent pathway was first identified in the budding yeast Saccharomyces cerevisiae and is responsible for transcription termination of many short, noncoding RNA genes, including small nuclear (sn) and small nucleolar (sno) RNAs (Winey and Culbertson 1988; Steinmetz and Brow 1996; Rasmussen and Culbertson 1998; Steinmetz et al. 2001). It also restricts the elongation and accumulation of pervasive cryptic unstable transcripts (Arigo et al. 2006b; Thiebaut et al. 2006) and regulates transcription of some protein-coding genes by premature termination, i.e., attenuation (Steinmetz et al. 2001; Arigo et al. 2006a; Steinmetz et al. 2006b; Jenks et al. 2008; Kuehner and Brow 2008). A set of core factors distinguish the Sen1-dependent pathway from the poly(A)-dependent pathway, including Sen1 and the RNA-binding proteins Nrd1 and Nab3 (Steinmetz and Brow 1996, 1998; Conrad et al. 2000; Steinmetz et al. 2001; Carroll et al. 2007). However, some short messenger RNA (mRNA) genes, such as CYC1, may have hybrid terminators that require factors from both pathways (Steinmetz et al. 2006b).
S. cerevisiae Sen1 is a 252-kDa superfamily 1 helicase encoded by the essential SEN1 gene. Its helicase domain is located in the C-terminal half of the protein (Figure 1A), and the N-terminal 975 amino acids are dispensable for viability (DeMarini et al. 1992). The ortholog of Sen1 in the fission yeast Schizosaccharomyces pombe has ATP-dependent, 5′-to-3′ DNA and RNA unwinding activities in vitro (Kim et al. 1999). Two hypomorphic alleles of S. cerevisiae SEN1, sen1-1 and nrd2-1, have G1747D and E1597K substitutions in the helicase domain, respectively, and exhibit terminator readthrough on a subset of Pol II-transcribed genes (DeMarini et al. 1992; Steinmetz and Brow 1996; Steinmetz et al. 2001, 2006a; Kuehner and Brow 2008; Mischo et al. 2011; Hazelbaker et al. 2013). These findings indicate that the Sen1 helicase domain plays an important role in Pol II termination.
Two main models have been proposed for the mechanism of Sen1-dependent termination. In one model, Sen1 is proposed to be analogous to bacterial Rho helicase, translocating 5′ to 3′ along the nascent transcript until colliding with paused Pol II and terminating its elongation (Steinmetz and Brow 1996; Porrua and Libri 2013). In the other model, Sen1 unwinds RNA/DNA hybrids (R loops) formed between the nascent transcript and the template strand, thereby allowing recognition of termination elements in the nascent transcript by RNA-binding proteins (Mischo et al. 2011). Both models can account for an essential role of the Sen1 helicase domain in termination.
The human ortholog of yeast Sen1, called Senataxin, is encoded by the gene SETX. Mutations in SETX cosegregate with two progressive neurodegenerative disorders with juvenile onset: autosomal recessive ataxia oculomotor apraxia type 2 (AOA2) and autosomal dominant amyotrophic lateral sclerosis type 4 (ALS4) (Chen et al. 2004; Moreira et al. 2004; Lemmens et al. 2010). The mechanism by which the SETX mutations cause AOA2 and ALS4 has not been determined, but many of the missense disease mutations are located in the conserved helicase domain (Figure 1A and Supporting Information, Supporting References, and Table S1), suggesting that the diseases are associated with dysfunction of the helicase activity. Senataxin has been implicated in Pol II transcription termination on protein-coding genes (Skourti-Stathaki et al. 2011). Gene expression profiling showed that AOA2 mutations in SETX cause changes in gene expression profiles in patient fibroblasts, including genes involved in neurogenesis and neuronal functions (Fogel et al. 2014). Thus, it is possible that AOA2 and ALS4 result from defects in expression of genes essential for long-term neuron survival.
To better understand the role of Sen1 in the transcription termination process, we created a plasmid-based assay to characterize Sen1 function in vivo. We found that the essential region of Sen1 corresponds to the helicase domain and a flanking nuclear localization signal, but that efficient transcription termination by Sen1 requires its N-terminal domain as well. Using the crystal structure of the related helicase Upf1 as a guide, we determined that the growth and termination defects caused by nrd2-1 (sen1-E1597K) result from disruption of an intradomain salt bridge in the first RecA repeat. Examining the utility of Sen1 as a surrogate for Senataxin, we analyzed the effects of 13 human AOA2 mutations and showed that six are recessive lethal and that an additional five result in recessive termination defects. Our studies establish a facile genetic system for studying structure–function relationships in Sen1 and underscore the importance of the helicase domain in Sen1-dependent termination of Pol II transcription.
Materials and Methods
Plasmid construction
The SEN1 gene, including 434 bp upstream of the start codon and 212 bp downstream of the stop codon, was amplified from genomic DNA of the strain 46α by two PCR reactions. One created an upstream SalI site and an internal PstI site via a silent mutation in codon 965. The other created the same internal PstI site and a downstream SpeI site. (The sequences of all primers used in this study are available on request.) The PCR products were digested with the appropriate restriction enzymes and ligated into SalI/SpeI-cut pRS425 to create pRS425-SEN1(PstI). The SalI/SpeI insert from pRS425-SEN1(PstI) was subcloned into pRS313, pRS315, and pRS316. Point mutations in SEN1 were created by the QuikChange procedure (Stratagene) and subcloned into pRS313.
Deletions of SEN1 were created using pRS313-SEN1(PstI). The C-terminal sequences were deleted by the QuikChange procedure, leaving the stop codon intact. The initial N-terminal deletion was made by deleting codons 4–964 of the SEN1 ORF and adding a silent mutation to codon 3 to recreate the internal PstI site. In this construct, codons 3, 965, and 966 have the sequence TCT-GCA-GAA, with the PstI site underlined. In subsequent deletions, codons 1–3 and 965 were retained, and codon 966 was replaced with the indicated codon (e.g., 1004, 1089, etc.), which always begins with a G to retain the PstI site. The presence of this PstI site as well as one in the pRS313 vector sequence allowed construction of each plasmid by ligation of two PCR fragments. All deletion plasmids were sequenced across the deletion junction to confirm their identity. C-terminal deletions were added to N-terminal deletion constructs using the NcoI site at codons 1890–1892 and the downstream SpeI site.
Sen1-GFP fusion constructs were made from pRS315-SEN1(PstI). Using the QuikChange procedure, an internal AvrII site was created by silent mutation of codon 2230 to generate pRS315-SEN1(AvrII). Codons 2–238 of the GFP(S65T) variant were amplified from genomic DNA of a yeast strain (KES000) containing an IMD2-GFP gene fusion (derived from Life Technologies #95700). AvrII sites were created at both ends of the GFP(S65T) coding sequence to allow insertion into pRS315-SEN1(AvrII), generating pRS315-SEN1-GFP. N-terminal deletions of SEN1 in pRS313 were subcloned into pRS315-SEN1-GFP. To make GFP fusions of C-terminally deleted SEN1, sequences upstream of the AvrII site were deleted from pRS315-SEN1(AvrII) by the QuikChange procedure and the GFP(S65T) fragment was inserted into the AvrII site afterward. The only exception is that the C-terminal deletion after codon 1929 was made by inverse PCR from pRS315-SEN1-GFP and blunt-end ligation, removing the upstream AvrII site in the process.
The Senataxin/Sen1 chimeric constructs were made from pRS313-SEN1(PstI). For the entire SEN1-SETX ORF swap construct, SEN1 codons 4–2232(stop) were deleted and replaced with a GGA (Gly) codon to create a BspEI site by inverse PCR, digestion, and self-ligation. SETX codons 3–2678(stop) were PCR-amplified from pSETX (Suraweera et al. 2007) with a BspEI site at both ends. The PCR product was digested with BspEI and ligated into the BspEI-digested SEN1 ORF deletion construct to generate pRS313-SEN1/SETXORF. For the SEN1-SETX helicase domain swap construct, unique PmlI and NcoI sites were used to excise SEN1 codons 1175–1890 and replace them with SETX codons 1769–2484 to generate pRS313-SEN1/SETXhelicase.
pGAC24-CYC1 (Steinmetz and Brow 2003) and the pRS316-NRD1-CUP1 reporter plasmids (Kuehner and Brow 2008) have been described elsewhere. The pGAC24-SNR47 reporter plasmid was generated by amplifying the SNR47 terminator (Carroll et al. 2004) (positions +100 to +215, relative to +1 transcription start site) from DAB206 genomic DNA with an XhoI site introduced at each end. The PCR product was digested with XhoI and ligated to XhoI-cut pGAC24 (Lesser and Guthrie 1993).
Yeast strains
Plasmids were transformed into S. cerevisiae by the lithium acetate procedure (Schiestl and Gietz 1989). DAB206 [MATα cup1Δ ura3 his3 trp1 lys2 ade2 leu2 sen1Δ3::TRP1 (pRS316-SEN1)] is derived from 46α (MATα cup1Δ ura3 his3 trp1 lys2 ade2 leu2) (Lesser and Guthrie 1993). The sen1Δ3::TRP1 allele was PCR-amplified from the genomic DNA of DDY44-5B (MATα leu2-Δ1 ura3-52 trp1-Δ1 lys2-801 his3-Δ200 ade2-101 sen1-Δ3::TRP1 YCp50-SEN1C), which is a haploid spore of DD44-N3 (DeMarini et al. 1992). 46α was cotransformed with pRS316-SEN1 and the sen1-Δ3::TRP1 fragment and plated to synthetic complete medium lacking uracil and tryptophan, resulting in replacement of most of the SEN1-coding sequence with the TRP1 gene. Disruption of the chromosomal SEN1 allele in the resulting strain, DAB206, was confirmed by PCR. pRS316-SEN1 was replaced with pRS313-borne SEN1 alleles by standard plasmid shuffle techniques (Boeke et al. 1987). Loss of pRS316-SEN1 was selected for on synthetic complete medium containing 0.75 mg/ml of 5-fluoroorotic acid (5-FOA).
XCY361 (MATα his3-Δ1 leu2-Δ0 lys2-Δ0 ura3-Δ0 NHP6A-TagRFP-T:nat) was derived from BY4742 (Research Genetics, Inc.) by integrating the NHP6A gene fused with TagRFP-T followed by the nourseothricin resistance (nat) gene into the endogenous NHP6A locus. The NHP6A-TagRFP-T/nat DNA fragment for chromosomal integration was made as follows. The NHP6A-mRFP1.3/nat DNA was amplified from strain EY2426 (Kim and O'Shea 2008) with HindIII and XbaI sites added to the ends. The DNA fragment was digested with HindIII and XbaI and inserted into the cloning vector pRS315. To improve photo-stability for time-lapse imaging, the mRFP1.3 ORF was replaced with the TagRFP-T ORF (Shaner et al. 2008). Splicing by overlap extension PCR was used to join NHP6A with TagRFP-T (Warrens et al. 1997). This NHP6A-TagRFP-T fragment was then subcloned into the existing pRS315-NHP6A-mRFP1.3/nat plasmid with HindIII and AscI sites, leaving the nat gene in place but replacing mRFP1.3 with TagRFP-T. The linear NHP6A-TagRFP-T/nat DNA was produced by HindIII and XbaI digestion of the resulting plasmid and transformed into BY4742, followed by selection for resistance to 100 µg/ml nourseothricin (Enzo Life Sciences). Integration at the NHP6A locus was confirmed by PCR of genomic DNA with flanking oligos and sequencing.
Protein analysis
Rabbit antiserum against amino acids 1095–1876 of Sen1 purified from Escherichia coli was raised by Harlan Laboratories (Madison, WI). IgG was purified from antiserum using the Melon Gel IgG Spin Purification Kit (Thermo Scientific). For immunoblots of Sen1, 10 OD units of yeast cells growing logarithmically (OD600 of 0.5–1.0) at 30° in yeast extract/peptone/dextrose (YEPD) were pelleted by centrifugation and lysed by vortexing with glass beads (425–600 µm diameter; Sigma-Aldrich) in 100 µl phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) with Protease Inhibitor Cocktail P2714 (Sigma-Aldrich), and 100 µl of 2× SDS/PAGE loading buffer [100 mM Tris–Cl, pH 6.8, 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 200 mM β-mercaptoethanal] was added, and the mixture was heated at 100° for 5 min. Samples were electrophoresed using 4–15% Mini-PROTEAN TGX Precast Gels (Bio-Rad) at 160 V for 50 min and electroblotted onto Hybond-C extra nitrocellulose membranes (Amersham Biosciences) at 6 V/cm, 4° for 12 hr in 20% (v/v) methanol in Towbin buffer (25 mM Tris base, 192 mM glycine, pH 8.3). Blots were blocked with 5% dried milk in PBS buffer with 0.1% Tween-20 at 23° for 1 hr, incubated with purified Sen1 IgG (1:2000 dilution) for 1 hr and then with HRP-conjugated goat anti-rabbit IgG (Thermo Scientific) (1:50,000 dilution) for 1 hr. Blots were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and exposed to HyBlot CL Autoradiography Film (Denville Scientific). Immunoblots of Nrd1 were performed by the same protocol except the Nrd1 polyclonal antiserum (Steinmetz and Brow 1998) was diluted 1:1000.
XCY361 cells transformed with GFP-tagged SEN1 constructs in pRS315 were grown in synthetic complete medium lacking leucine at 30° to an OD600 of 0.5 and then diluted to an OD600 of 0.2. Two hundred microliters of culture was placed on the center of a glass coverslip precoated with 1 mg/mL Concanavalin A, Type IV (Sigma-Aldrich) solution, and viewed with a Nikon Eclipse Ti fluorescent microscope with a Nikon DS-Qi1Mc digital camera and NIS elements AR 4.12.01 software. Filter sets with excitation/emission wavelengths of 465–495/515–555 nm and 530–560/573–648 nm were used for the GFP images and red fluorescent images (RFP) images, respectively. Image files were processed and analyzed by ImageJ (National Institutes of Health) following the ImageJ User Guide (http://rsbweb.nih.gov/ij/docs/guide/146.html). For background subtraction, the command "Process>Subtract background>Create Background" was used to create the background, and the command "Process>Image Calculator" was used to subtract background from the original image. For contrast enhancement, the command "Image>Adjust>Brightness/Contrast" was used. In most cases, appropriate contrast was achieved by a single click on "Auto."
RNA analysis
Yeast cells were grown in YEPD at 30° to an OD600 of 0.8–1.0, quickly shifted to 37° by mixing with the same volume of 44° YEPD, continued growing for 1 hr, and were pelleted by centrifugation. Total cellular RNA was prepared by the glass bead-guanidinium isothiocyanate–hot phenol extraction method (Wise 1991). For Northern blots, 15–20 μg RNA was loaded in each lane, run on a 1% agarose/0.8 M formaldehyde gel, and blotted onto Zeta-probe membrane (Bio-Rad) by capillary transfer. The random-primed radioactive probes for NRD1 and SNR47 were generated using gel-purified PCR products as templates. The probe labeling protocol was adapted from the Prime-a-Gene Labeling System (Promega) using [α-32P]dTTP (PerkinElmer). After labeling, unincorporated nucleotides were removed by a G-50 Sephadex resin column (GE Healthcare). Probe hybridization was performed in Ultrahyb solution (Ambion) with probe concentration ∼106 cpm/ml. The blot was hybridized at 42° for >12 hr, washed in saline-sodium citrate (SSC) wash buffers (2× SSC, 0.1% SDS, 5 min per wash, twice, at 23°; 0.1× SSC, 0.1% SDS, 15 min per wash, twice, at 42°), and visualized with a Phosphorimager (Molecular Dynamics). Primers for PCR amplification of random-priming templates were the following: SNR47 fwd—5′-GGTCTCGAGATATATTTTCGCGTCATTCTTG-3′; SNR47 rev—5′-GGTCTCGAGCAGAAATAAAGAAAATGAAAGC-3′; NRD1 fwd—5′-CGAGTTACAGGAAAGGAACC-3′; and NRD1 rev—5′-TCTATTCCTTCTTGAGTTATATC-3′.
A plasmid-based system for characterizing yeast SEN1 function
Previous genetic studies of S. cerevisiae SEN1 used primarily chromosomal mutations and tested a small number of alleles (Winey and Culbertson 1988; Steinmetz and Brow 1996; Finkel et al. 2010). The essential chromosomal SEN1 gene was deleted previously (DeMarini et al. 1992), but the complementing plasmid used, although functional, was subsequently found to lack the SEN1 promoter, 5′-UTR, and part of the protein-coding region. To allow more facile analysis of mutant SEN1 alleles, we created a yeast strain, DAB206, that contains the sen1-Δ3 chromosomal gene disruption constructed by DeMarini et al. (1992), but the plasmid-borne wild-type SEN1 allele contains 434 bp upstream of the start codon and 212 bp downstream of the stop codon. Furthermore, the wild-type SEN1 allele is on a URA3-marked low-copy plasmid, pRS316, that can be selected against with 5-FOA to allow its replacement with a mutant allele (i.e., "plasmid shuffle"). The expression level of plasmid-borne SEN1 in strain DAB206 appears similar to the level of expression of chromosomal SEN1 in its parental strain, 46α (Figure 1B). We chose 46α as the parent of DAB206 because its chromosomal CUP1 genes are deleted (Lesser and Guthrie 1993), which allows the use of CUP1 reporter constructs to measure the extent of Sen1-dependent terminator readthrough (Figures 1, C–E). Although the CUP1 reporter assay itself cannot distinguish increased terminator readthrough from stabilization of a constitutive readthrough transcript, we previously used nuclear run-on and/or Pol II chromatin immunoprecipitation to show that Sen1-E1597K induces readthrough of all three terminators used in the CUP1 reporter assay (Steinmetz et al. 2006b).
Essential region of Sen1 corresponds closely to the helicase domain
We used the DAB206 strain to determine the minimal essential region of the Sen1 protein. Previously, a plasmid lacking the promoter, 5-UTR, and the first 975 codons of the SEN1 gene was found to complement a heat-sensitive mutation in SEN1 (DeMarini et al. 1992). We further delineated the essential region of Sen1 by systematically deleting its N- and C-terminal codons while leaving the first three codons, stop codon, and all flanking noncoding sequences intact. For most constructs, an additional alanine codon is present between codon 3 and the first downstream codon (see Materials and Methods). Mutant SEN1 alleles were introduced into DAB206 in a HIS3-marked, low-copy-number pRS313 vector (Figure 1C). Recessive viability of each mutant was determined by plating transformants onto medium containing 5-FOA (Figure 2A). For viable SEN1 mutants, their recessive growth phenotypes were scored at four temperatures: 16°, 23°, 30°, and 37° (Figure 2B).
Deletions of codons 4–964 and 4–1003 resulted in strong heat sensitivity and mild cold sensitivity. N-terminal deletion through codon 1088 strongly increased the cold sensitivity, whereas deletion through codon 1134 is recessive lethal at 30°. Sen1 1089-2231 appears to be overexpressed in vivo (Figure 2C), suggesting either that Sen1 levels are autoregulated or that the compromised function of the truncated protein at 30° selects for cells that have amplified the plasmid containing this allele. Deletion of the C-terminal 324 residues (codons 1908–2231) caused only weak heat sensitivity at 37° (Figure 2B) and no obvious change in expression level (Figure 2C). However, deletion of an additional 49 C-terminal codons (to codon 1858), including the extreme C terminus of the helicase domain, resulted in severe growth defects at all temperatures tested.
When N- and C-terminal codon deletions were combined, we identified the minimal length of the SEN1 ORF for viability, which includes codons 1004–1907 or 1089–1929 (Figure 2A). Both of these alleles confer strong cold and heat sensitivity (Figure 2B). Thus, the essential region of Sen1 is ∼900 amino acids long and corresponds closely to the helicase domain. However, a construct with only the 819 residues present in both viable constructs [codons 1089–1907; hereafter called "Sen1(HD)"] is not viable, so an essential function is supplied by either the N- or the C-terminal residues immediately flanking the helicase domain.
Analysis of copper sensitivity in the presence of CUP1-based termination reporter constructs showed that viable deletions of both the N- and the C-terminal regions of Sen1 result in terminator readthrough, although the N-terminal deletion exhibits stronger readthrough on all three terminators tested, consistent with its more severe high- and low-temperature growth defects (Figure 2D).
Nuclear localization of Sen1(HD) is required for cell viability
We sought to elucidate the essential function that is supplied by the residues flanking the helicase domain. Residues 1862–2093 of Sen1 were previously shown to contain a functional nuclear localization sequence (NLS) (Ursic et al. 1995), the N-terminal border of which was subsequently localized to residues 1890–1967 (Nedea et al. 2008). Moreover, a bipartite NLS-like sequence spans Sen1 residues 1908–1929 (DeMarini et al. 1992), which corresponds to the C-terminal essential flanking region (Figure 2A). We speculated that the flanking regions function redundantly in targeting the helicase domain to the nucleus. To test this hypothesis, we investigated localization of Sen1-GFP fusion proteins by fluorescent microscopy in the strain XCY361, which contains an intact chromosomal SEN1 gene and RFP-labeled nonhistone protein 6A (Nhp6A-RFP) as a nuclear marker (Figure 3).
Consistent with a previous study (Ursic et al. 1995), we found that wild-type Sen1-GFP predominantly localizes to the nucleus (Figure 3A). We further showed that the putative bipartite NLS is sufficient to direct nuclear localization, since GFP fused to Sen1 1907–1929 concentrates in the nucleus (Figure 3B). The subcellular distribution of Sen1(1089–1929)-GFP, which contains the bona fide NLS, resembles that of wild-type Sen1 (Figure 3C). Sen1(HD)-GFP, which contains only the helicase domain with no flanking sequences, displayed uniform distribution throughout the cell, with no nuclear enrichment (Figure 3D). However, Sen1(1004–1907)-GFP, which contains the helicase domain with N-terminal flanking sequence, exhibits weak nuclear localization (Figure 3E). This result indicates that the N-terminal flanking sequence has weaker NLS activity than the C-terminal NLS. Nevertheless, either the N- or the C-terminal flanking sequence is able to direct sufficient Sen1 helicase core to the nucleus to support cell viability at 30°.
To test if nuclear localization is the only essential function of the flanking sequences, we fused the SV40 NLS (PKKKRKVGIPQ) to the N-terminal end of Sen1(HD)-GFP. This fusion construct efficiently localized to the nucleus (Figure 3F). However, a strain containing SV40 NLS-Sen1(HD) grew extremely slowly even at 30° (Figure 2B). Therefore, while the SV40 NLS can rescue Sen1(HD) function, it does so much less efficiently than either the N-terminal or the C-terminal flanking sequences.
We noted that the Sen1(1004–1907)-GFP signal and the Nhp6A-RFP signal tend to segregate from one another within the nucleus, with the former exhibiting predominantly a crescent-shaped distribution (Figure 3G). Such a distribution pattern was not observed with any of the other Sen1-GFP constructs, suggesting that a subnuclear, perhaps nucleolar, localization signal exists in the N-terminal flanking region (see Discussion).
sen1-E1597K substitution disrupts an intradomain salt bridge
Using a positive selection for Sen1-dependent terminator readthrough, we previously isolated the nrd2-1 allele of SEN1, which harbors the missense mutation Glu1597 to Lys in the helicase domain (Steinmetz and Brow 1996). This residue is adjacent to motif II (Figure S1), which makes up part of the ATP-binding site, but the molecular defect of the E1597K mutation is unknown. Since no structural information is available for the Sen1 helicase domain, we used the yeast Upf1 helicase domain as a structure model (Chakrabarti et al. 2011). Based on sequence alignment (Figure S1), E1597 in Sen1 is equivalent to Upf1 E579, which forms a salt bridge with R623 in Upf1 that likely stabilizes the 1A domain (Figure 4A). Upf1 R623 aligns with R1641 in Sen1, so this salt bridge may be conserved in Sen1.
To test whether the molecular defect of sen1-E1597K is disruption of the putative salt bridge, we introduced a R1641E substitution. If the proposed salt bridge exists, sen1-R1641E and -E1597K should exhibit the same defects by breaking this salt bridge, but in combination they should suppress such defects by mutually restoring the salt bridge. As shown in Figure 4, B and C, R1641E alone conferred heat sensitivity and terminator readthrough phenotypes similar to those caused by E1597K. Moreover, the E1597K/R1641E double mutant was less heat-sensitive than either single mutant and exhibited no terminator readthrough. These results support our hypothesis that the nrd2-1 mutation disrupts a glutamate–arginine salt bridge between residues E1597 and R1641 that can be functionally replaced by a lysine–glutamate salt bridge. The success of this structure-guided mutational analysis demonstrates the utility of the Upf1 helicase domain structure as a predictive model for the Sen1 helicase domain structure, at least in the 1A and 2A domains, where the proteins are the most similar.
AOA2 disease mutations in yeast SEN1 cause growth defects and terminator readthrough
We next used our yeast genetic system to examine the effects of AOA2 disease mutations on Senataxin function. Initially, we attempted to complement the SEN1 deletion in DAB206 with pRS313-SEN1/SETXORF or pRS313-SEN1/SETXhelicase, in which the entire protein-coding region or just the helicase domain of SEN1, respectively, are replaced with the corresponding regions of the SETX cDNA (see Materials and Methods). However, the resulting strains did not grow on media containing 5-FOA, indicating that neither the entire SETX ORF nor its helicase domain can functionally replace their counterparts in SEN1. Therefore, we sought to characterize the disease mutations in the context of Sen1. This approach is restricted to the helicase domain as it is the only region where the sequence similarity is high enough to allow unambiguous identification of homologous residues. Among the 50 currently identified missense mutations that cosegregate with AOA2 or ALS4, 28 are located in the Senataxin helicase domain (Table S1). Most AOA2 mutations in the helicase domain are in residues conserved in Sen1. In contrast, the two ALS4 mutations in the Senataxin helicase domain (R2136C and R2136H) are in a residue that is not conserved in Sen1, so they cannot be tested in Sen1 (Figure S2).
We introduced 13 AOA2 mutations at analogous residues in Sen1 and characterized their effects in vivo (Table 1 and Figure S2). Wild-type Sen1 and the two previously isolated mutations, E1597K and G1747D, were included for comparison. Plasmid-borne mutant SEN1 alleles were transformed into the haploid yeast strain DAB206 to make merodiploid strains and observe their dominant effects. All 13 merodiploid strains were viable and grew normally at 30°, indicating that none of the disease alleles exhibit a dominant growth defect at this temperature. The merodiploid strains were then grown in medium containing uracil and plated on medium containing 5-FOA to select for clones that had lost the URA3-marked plasmid harboring the wild-type SEN1 allele. Six of 13 AOA2 mutants (Table 1 and Figure 5A) did not grow on medium containing 5-FOA, indicating that these AOA2 mutations render Sen1 inactive for an essential function. When these six alleles were tested for a dominant effect on readthrough of the SNR47 terminator, five exhibited snoRNA terminator readthrough, with sen1-I1370R being the only exception (Figure 5B). Thus, these five nonfunctional AOA2 mutant Sen1 proteins inhibit the function of the wild-type protein on the SNR47 terminator and therefore must accumulate to an appreciable level. In contrast, given that the I1370R substitution replaces a buried hydrophobic residue with a charged residue, it could potentially result in protein misfolding and degradation, which would preclude a dominant effect on wild-type Sen1.
All the viable haploid mutants grew similarly to the wild-type Sen1 strain at 16°, 23°, and 30°, but five of them displayed some degree of heat sensitivity at 37° (Figure 6, A and B). The W1166S substitution displays the strongest defect, followed by T1779P and then I1371F, N1413S, and K1788E with minor growth defects. F1767L and R1850H exhibited no temperature sensitivity. In summary, the severity of the AOA2 mutations in Sen1 is variable. Of the 13 mutants we tested, 6 of them are recessive lethal and another 5 cause detectable heat sensitivity.
We next tested if the viable AOA2 mutations cause defects in Sen1-dependent transcription termination. The haploid mutant strains were characterized in the copper resistance assay using the SNR47, CYC1, and NRD1 terminators. The viable mutants exhibited different levels of copper resistance (Table 1). Four of five heat-sensitive AOA2 mutants, sen1-W1166S, N1413S, T1779P, and K1788E, caused readthrough of one or more terminators used in our assay, and the rest of the mutants showed no readthrough effects. The terminators responded differently to the readthrough mutants. The NRD1 attenuator was responsive to all four AOA2 readthrough mutants, with sen1-N1413S showing less readthrough than the other three. Only sen1-T1779P caused SNR47 terminator readthrough, and no readthrough of the CYC1 terminator was observed in any of the AOA2 mutants. None of the AOA2 mutants caused readthrough of SNR47 or CYC1 terminators to the levels of the previously isolated readthrough mutant sen1-E1597K. In contrast, the founder sen1-1 mutation, G1747D, exhibits readthrough similar to the nrd2-1 mutant, consistent with its defect in pre-transfer RNA splicing being an indirect effect of transcriptional readthrough (Winey and Culbertson 1988; Steinmetz et al. 2001).
AOA2 mutations in Sen1 induce terminator readthrough at the NRD1 and SNR47 loci
Since the SNR47 terminator and NRD1 attenuator are in an artificial context in their respective CUP1 reporter genes, we sought to determine if termination defects also occur at the corresponding genomic loci in the presence of the AOA2 mutations. The strains carrying plasmid-borne wild-type SEN1, sen1-E1597K, -W1166S, -T1779P, or -K1788E were chosen for analysis by Northern blotting. All the cultures were shifted to 37° for 1 hr before RNA extraction to enhance the defects from the mutants.
The Northern blot results are shown in Figure 7A and quantified in Figure 7B. Previously, the chromosomal nrd2-1 mutant (sen1-E1597K) was found to accumulate excess NRD1 mRNA (due to readthrough of the attenuator) and extended SNR47 transcripts (Steinmetz et al. 2001). We observed similar results with the plasmid-borne sen1-E1597K allele. Terminator readthrough at these loci was also observed in some of the disease mutants. For the NRD1 gene (Figure 7, A and B, left), a small increase in transcripts in the sen1-W1166S and -T1779P strains implies decreased transcription attenuation. We did not observe excess NRD1 mRNA in the sen1-K1788E strain despite its strong copper resistance in the presence of the NRD1/CUP1 reporter gene (Table 1). This inconsistency could be explained by the fact that the endogenous NRD1 attenuator extends well into the Nrd1-coding region (Arigo et al. 2006a), which is not included in the NRD1/CUP1 fusion construct. K1788E may promote readthrough of the truncated NRD1 attenuator present in the reporter construct, but not the complete attenuator present at the endogenous NRD1 locus.
For the SNR47 locus (Figure 7, A and B, right), accumulation of SNR47-YDR042C readthrough transcripts was observed in the sen1-W1166S and T1779P mutants but not in sen1-K1788E. T1779P caused strong readthrough of SNR47 to a level similar to E1597K. Modestly elevated readthrough of SNR47 in the sen1-W1166S strain was detected in the Northern blot assay but not in the copper resistance assay, perhaps because this mutant protein is sensitive to increased temperature and RNA was prepared after a shift to 37°, while the copper resistance assay was done at 30°. In summary, our Northern blotting assays revealed termination defects caused by the AOA2 mutants at the endogenous loci, which in general agree with our CUP reporter results.
Several different functions in DNA and RNA transactions have been attributed the essential yeast Sen1 protein and its human homolog, Senataxin (Brow 2011). However, little is known about the natural substrates of its helicase activity and which are related to its essential function. We have developed a genetic system for in vivo analysis of structure–function relationships in Sen1 and have demonstrated the utility of this system by addressing several open questions. In particular, we determined that (1) the minimum essential region of Sen1 is the helicase domain plus a flanking nuclear localization signal; (2) the nrd2-1 mutation (E1597K) disrupts an intradomain ionic bond with R1641; and (3) AOA2 disease mutations in the Senataxin helicase domain have a broad range of effects when introduced into the homologous position of Sen1.
Multiple domains of Sen1 are involved in transcription termination
Only two conditional-lethal missense mutations in the SEN1 gene have previously been identified, and both are in conserved residues of the helicase domain (DeMarini et al. 1992; Steinmetz and Brow 1996). Both mutations are known to cause readthrough of certain transcription terminators by Pol II, indicating the important role of the helicase domain in transcription termination. Our deletion analysis shows that nuclear localization of the Sen1 helicase domain is sufficient for viability, but that efficient transcription termination and normal growth at a range of temperatures also requires the N- and C-terminal domains of Sen1 (Figure 2D). The strong readthrough effects of Sen1 965–2231 agree with the previous observation that the N-terminal part of Sen1 interacts with the Ser5-phosphorylated Pol II C-terminal domain (CTD) and that impairment of this interaction by the mutation R302W decreases Sen1 occupancy on noncoding RNA genes (Chinchilla et al. 2012). Therefore, deleting the N-terminal region could dissociate Sen1 from the Pol II CTD, rendering it unable to efficiently recognize terminators. The C-terminal deletion of Sen1 (Sen1 1–1907) resulted in readthrough of the NRD1 attenuator. This could be caused by disrupting its interaction with Nab3 or other termination factors, such as the phosphatase Glc7 (Nedea et al. 2008). However, we observed only moderate readthrough in this deletion mutant strain, suggesting that the interactions occurring in the C-terminal end are not critical for snoRNA termination or are functionally redundant.
By tracking Sen1-GFP fusion proteins in merodiploid strains (Figure 3), we showed that either the N- or the C-terminal flanking region can target the Sen1 helicase domain to the nucleus. The C-terminal flanking region contains a classical bipartite NLS that directs efficient import and is likely recognized by importin-α (Srp1) (Marfori et al. 2011). The N-terminal flanking region does not contain a classical NLS and provides weak yet site-specific subnuclear localization activity. The resulting distribution pattern of Sen1(1004–1907)-GFP resembles the shape of the nucleolus, which is not observed in the full-length protein, suggesting a different mechanism for the localization activity of the N-terminal flanking region. The importance of this region in Sen1 has been revealed by our deletion analysis, as removing it strongly increased cold sensitivity (Figure 2B). We hypothesize that the N-terminal flanking region interacts with snoRNP proteins, which serves to bring it into the nucleus and localize it to the nucleolus. Such an interaction is presumably more transient in full-length Sen1, where it may serve to recruit Sen1 to cotranscriptionally assembling snoRNPs, increasing the efficiency of transcription termination on snoRNA genes (Ballarino et al. 2005; Yang et al. 2005).
The heterologous SV40 monopartite NLS directs very efficient nuclear localization of the Sen1(HD), but results in very low viability. It is possible that a strong NLS at the N terminus of the helicase domain interferes with helicase function in some way.
Terminator readthrough is a common effect of the AOA2 mutations in Sen1
We tested 13 AOA2 mutations from the Senataxin helicase domain in Sen1, and 77% (10 of 13) caused dominant or recessive terminator readthrough (Figure 5B and Table 1), indicating that defects in transcription termination is a common feature of AOA2 mutations in yeast. The Upf1 helicase domain structure provides insights into the molecular processes disrupted by the AOA2 mutations (Figure 5A and Figure 6A). Some of the mutations that cause readthrough localize to the catalytic core and are expected to disrupt binding of nucleic acid (N1413S and T1779P) or ATP (D1616V and R1820Q) or the coupling of these binding events (P1622L) (Fairman-Williams et al. 2010). Other mutations are in the hydrophobic cores of the RecA domains (I1370R, I1371F, C1409Y, L1569W, and K1788E) and may alter the overall fold of the helicase domain. No growth or termination defects were observed with the F1767L and R1850H AOA2 mutations. These residues are on the exterior surface of the helicase domain and may be involved in Senataxin-specific interactions.
In this study we examined the effects of SEN1 mutations on Pol II transcription termination. Functions of Sen1 in other nuclear processes have been proposed, including Pol I transcription termination, DNA repair, RNA processing, and maintenance of genome stability (Ursic et al. 2004; Kawauchi et al. 2008; Finkel et al. 2010; Mischo et al. 2011; Alzu et al. 2012). Defects in these other processes may contribute to the growth defects that we observed in the presence of the AOA2 substitutions.
Potential of yeast for identifying Senataxin disease variants
Given that 2 of the 13 AOA2 mutations that we tested in Sen1 cause no growth or termination defect, our yeast genetic system has a significant false-negative rate in detecting disease-causing mutations in Senataxin. To determine the false-positive rate, we would need to test single nucleotide polymorphisms (SNPs) in Senataxin that are known not to cause disease in humans and can be aligned accurately with the Sen1 sequence. However, no such SNPs have been identified.
We further wondered if there is a correlation between the yeast growth phenotypes and the severity of AOA2 mutations in humans. As shown in Table 1, the tested AOA2 mutations were categorized into "lethal," "heat sensitive," and "normal" groups based on their growth phenotypes in yeast. Using published data, we determined the average age of onset and the average serum α-fetoprotein (AFP) level for patients with the three observed combinations of alleles from these three phenotypic groups, namely two normal alleles, two lethal alleles, and one each of the heat-sensitive and lethal alleles (Table 2). For comparison, we also calculated average values for patients who have two presumptive null alleles (nonsense mutation, frameshift mutation, or a large deletion in the helicase domain). If the yeast phenotype were predictive of the human disease severity, we would expect the individuals with two "normal" alleles to have a significantly later age of onset and/or a lower serum AFP level. However, this analysis yielded no significant difference (using a t-test with P < 0.05) in either the age of onset or AFP level between any two of the yeast phenotype groupings. Therefore, although our yeast genetic system provides a means for identifying potential disease-causing sequence variants in Senataxin, the severity of AOA2 mutations in yeast shows no apparent correlation with severity in the AOA2 population for which we could access clinical data.
Notably, we found that all but one of the AOA2 mutations that are lethal in Sen1 exhibit dominant readthrough of the SNR47 terminator (Figure 5B). Whether the corresponding SETX alleles cause any dominant effects in AOA2 carriers is not known. Although AOA2 is classified as a recessive disorder, our results in yeast suggest that a carrier trait may be detectable at the molecular level.
While the utility of our yeast genetic system for the characterization of the phenotypic consequences of human genetic variants is as yet uncertain, we have clearly established its utility for investigating structure–function relationships in yeast Sen1. More detailed analysis of the effects of the AOA2 mutations in Sen1, for example, by transcriptome studies, may yield insight into the nature of the molecular defects that lead to the symptoms of AOA2.
We thank Michael Culbertson, Christine Guthrie, Harold Kim, and Erin O'Shea for yeast strains; Jon Audhya and Martin Lavin for plasmids; Tom Gisel for plasmid construction; Elaine Brow and Christine Treba for creating mutant SEN1 alleles; Steve Martin-Tumasz for generating Sen1 antiserum; Lynn Weaver for technical assistance; Aaron Hoskins and David Wassarman for use of their microscopes; and Brow lab members for helpful discussions. This work was funded by grant R01 GM082956 from the National Institutes of Health (NIH) (to D.A.B.). K.E.S. was supported in part by NIH training grant T32 GM08349.
Supporting information is available online at http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.114.167585/-/DC1.
Communicating editor: A. Hinnebusch
- Received June 20, 2014.
- Accepted August 11, 2014.
- Copyright © 2014 by the Genetics Society of America


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Good stuff :) Sep 11
nice music Nov 25
Lovely songs Sep 19
Love the music Aug 25
Superb music Jul 16
Yo YO DUDE... Apr 28
OSUMM keep it up Apr 22
amazing work mate Apr 18
awesome music.. Sep 09
loved it Aug 05
I like it..) Jul 16
You and your songs, both rocked it..) Jul 16
love the like that track Mar 23
Superb ! :) Jan 03
Aye. I think.. well i like the Life is good (rugrats song)... the lyrics are good. but I think you could switch up beat after awhile.. and it would still be dec. Mar 25

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Sony asks gamers to sign new terms or face PSN ban
Sony is preparing to ban gamers from the PlayStation Network (PSN) unless they waive the right to collectively sue it over future security breaches.
The firm has amended PSN's terms and conditions and users have to agree to them next time they log in.
The move comes months after a string of hacking attacks compromised over 100 million accounts of the PlayStation Network subscribers.
It is, however, possible to opt out of the agreement within the next 30 days.
Gamers will now have to try to resolve any legal issues with an arbitrator picked by Sony, before being able to file a lawsuit.
Full Story here
Sony asks gamers to sign new terms or face PSN ban
Discussion in 'News' started by GiddyG, Sep 18, 2011.
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MOVIE SYNOPSIS [ General Audiences ] Movie of Paul Tanter. Was premiered in 2013, with production teams of UK. You can watch White Collar Hooligan 2: England Away online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2013.
Rating: 5 by 387 users./
Runtime: 98 minutes
Nick Nevern, Simon Phillips, Rita Ramnani, Peter Barrett, Vas Blackwood, Tom Benedict Knight, Tony Denham, Neil Large, Ewan Ross, Kelley Van Dilla, Pablo Olewski, Ashley Nicole Anderson, Leo Goodman, Daniel Attrill, Stuart Bailey and Juliette Bennett.
98-minute film (full movie) directed by Paul Tanter.
The original audio of White Collar Hooligan 2: England Away has dialogues in Dolby Digital, it was produced in UK.
It premiered in cinemas or television in 2013.
Screenplay written by Paul Tanter.
Haider Zafar.
Joe Aliberti, Matthew Helderman, Lyndon Baldock, Billy Murray, Adam Eldrett, Paul Tanter, Elana A. Mugdan, Simon Phillips, Alain Wildberger, Raheel Riaz and Toby Meredith.
This film was produced and / or financed by Runaway Features, Press On Features, Shivnath Productions, Chata Pictures and Templeheart Films.
Ascot Elite Home Entertainment, Momentum Pictures and Eagle Entertainment.
Without commercial breaks, the full movie White Collar Hooligan 2: England Away has a duration of 98 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, White Collar Hooligan 2: England Away in FULLTV Movies.


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Comments in our Visitors Book from guests this year - 2017
'Just what we hoped for in an Irish Cottage'
'We had everything we needed, and more. Loved the silence and the occasional visit from the collie'
'This is the most perfect place'
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'What a lovely peaceful place to stay. We felt very much at home'
'...a little corner of heaven. Can't wait to come back again in September'
Recommended By:
Reservations and enquiries to:
Sinead Connelly


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Screwtape letter for a mom of littles
Some of the links might be affiliate links. See the disclosure policy here. Some of you might have read The Screwtape Letters by C.S. Lewis. If you haven't, I highly recommend it. You can get your own copy of the... Continue reading →


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Ah found the source...
from http://www.cinemablend.com/new/Batma...Far-38745.html
Quote from the site (this interview was taken at Comic-Con):
When Warner Bros. announced at Comic-Con that they would be moving forward with a Superman/Batman movie, they didnt include a plot synopsis telling us what the movie is about but titles for the film mentioned by Goyer suggest that the film will have the two heroes at each others throats. During the panel celebrating the 75th Anniversary of Superman, the screenwriter told the audience, Were actually not sure whether the title is Superman vs. Batman or Batman vs. Superman but yes, itsthat rematch, that combination, the two guys onscreen and thats happening. Combined with Harry Lennixs aggressive Dark Knight Returns quote at Comic-Con and the fact that Warner Bros. has registered both BatmanvsSupermanMovie.com and Batmanvs.Superman.com and there appears to be a good amount of evidence suggesting that Superman and Batman will be enemies before they become friends.
By the way, a "versus" movie would be the absolute most boneheaded thing DC and Warner Bros. could ever do. It sounds like something you'd use in a fighting game, and we have one of those already. It's called "Injustice: Gods Among Us." As far as I know, Superman and Batman never got into a fight unless one of them were somehow mind-controlled, infected, or just flat out insane. The last one pretty much applied to Batman at the end of The Dark Knight Returns. In other words, the two fighting each other would just be out of character for both of them.
More: http://www.cinemablend.com/new/Watch...Con-38665.html
Director Zack Snyder and Man of Steel star Harry Lennix took to the stage at Comic-Con to announce Superman-Batman Movie and threw out this aggressive quote (according to them this quote is meant to help people UNDERSTAND better what the plot will be since they can't announce the official title yet):
"I want you to remember, Clark in all the years to come in your most private moments I want you to remember my hand at your throat I want you to remember the one man who beat you."
The quote is not a friendly one. It suggests SOMEONE fighting Clark (aka Superman) and then beating him.
As for your more serious post, I doubt it's going to be a straight "vs" film, as if they're actually going to be enemies the whole time. They'll probably be in conflict to start with, and then team up later. They're both heroes, they won't stay enemies for long. Also, I wouldn't bet on Bale or JGL being Batman. Batman is being rebooted, so it won't be either of them.
2. He said he will not adapt (of course he wouldn't want to adapt another mans work -- that would be plagiarism -- but he made it clear that it's gonna be inspired from that). He said the quote will help people understand more about how the movie will go. So in other words we're gonna see something of what inspired it, but with his own original artistic touch to it.
2) The most I could imagine would be borrowed from that story is the idea of Batman and Superman fighting at some point, and Batman being a little older. That story relied on the notion that both of them had been around for a long time, and it was an ending for Batman. These new films are supposed to be the BEGINNING of the DC Cinematic Universe, so a lot would have to be changed just because of that. Then there's the simple fact that the Superman in that story (who was portrayed rather out-of-character thanks to Miller's tantrum over being denied Superman in favor of John Byrne) was a very different character than the one we're left with at the end of Man of Steel. Can you see that Superman being a government stooge trying to hunt down Batman? Come on. That's not going to happen. So when you get right down to it, all we're left with is that Zack Snyder used a quote from the most famous Batman/Superman brawl to announce the film, and there's this little notion that the film will be inspired by it in some way, even though almost everything about that story would contradict what they want to do with this film, and who the characters are likely to be.
Nolan's Dark Knight trilogy needs to be left alone. I'm actually hoping that Christian Bale will not play Batman in this movie because although he was, in my opinion, the best actor to ever portray Batman, Snyder's version should be different. This addresses the lack of hints of Superman in TDK trilogy. Why would Nolan put any Superman easter eggs in his movies if he was never going to plan on bringing in Superman at all? He didn't even want his version of the Batman to be involved in a Justice League movie.
As I've said before, I'm not interested in a Batman vs Superman film. However, I'd like to see Batman teach Superman some Fighting skills. In Man of Steel Superman was getting his ass kicked by Kryptonians who were skilled fighters. Who better to teach Superman how to better kick ass than Batman?
"I won't be wronged, I won't be insulted, and I won't be laid a hand on. I don't do these things to other people and I expect the same from them."
-John Wayne (The Shootist)
Basically, we'll end up with one of these scenes. (Except replace Muhammad Ali with Batman. )
But weve also seen how Superman suffered from Lex Luthor (a mere human who is not even anywhere near as dangerous and skillful as Batman) in the Reeves Superman movies and in Superman Returns.
Supes fans most of the time would mention Lex Luthor this and Lex Luthor that, and how dangerously ingenious Lex is. But actually Batman is a more dangerous version of Lex Luthor.
Like Lex, Bruce Wayne is also a Billionaire who has the resources to spend on hi-tech weapons research. But unlike Lex who is unskilled in fighting, Bruce Wayne is a master combatant and is trained in ninja art. So were talking about somebody who has the resources and wealth of Lex and all the while having the skills of a master martial artist.
I think it would be interesting if Batman discovers Kryptonite as Supes weakness in Bats VS Supes. A man trained in the craft of ninja art wielding Kryptonite is a recipe for Supermans doom.
Batman casting will be announced within 30 days?!
2) Superman Returns is garbage.
3) You have it backwards. In the comics, Lex Luthor has been shown to be much more capable than what the Donner films may suggest. You have read the comics, right? Lex Luthor is almost like a more dangerous version of Bruce Wayne because he lacks Wayne's morals and his decency and his compassion and understanding. Luthor is a Bruce Wayne who doesn't care about collateral damage, and will hurt, or even kill innocent people to get what he wants, and what he wants is two things: a dead Superman, and total domination over Metropolis, if not America or the entire world.
4) Batman had trouble with Bane. A mere human with a fancy new brand of steroid addiction. Bane's not nearly as strong or as fast or as invulnerable as Superman is. Batman would not stand a chance in any unbiased fight. The only reason he does is because there's a portion of Batman fanboys who are so insecure about the worth of the character that they feel the need to put down every other character around him, and since Superman is the other half of the "big two" superheroes (meaning the two most popular), Superman is usually the target. So to make sure those fans aren't upset, Batman gets god-moded and Superman gets down-graded in wildly inconsistent ways, just to have that one splash page of Batman punching Superman. Which, as I've pointed out, usually never amounts to much because, as I've pointed out, Batman has never defeated Superman in any canonical published content. End of story.
2. That is subjective. So any book or movie that depicts Superman as a weakling is garbage?
3. I would rather have this world dominated by one of ours than be dominated by an alien.
4. Because Bane doesn't have Kryptonite weakness like Supes do, and Bane underwent the same ninja training as Batman.
Where you aiming at again you Blind fool?!
Attachment 6664
X-Ray Vision my a$$!! You can't even aim properly! Now here's what a precision hit feels like!
Attachment 6665
Do I make myself clear now you alien freak?! Now listen to me boy...
Attachment 6666
Torture is only beginning. Lets have a little fun here. YOU ARE TERRORIST and welcome to Guantanamo Bay Batman style!
Attachment 6667
Attachment 6668
I just Fked you up. There's handkerchief in the Batmobile. Go wipe your blood with it!
2) It's garbage for a lot of reasons. On top of being boring, overly reverential and unoriginal when it came to the Donner film, Superman was depicted as a whiny, self-centered stalker and a coward. I could go into it with much more depth but it's really just a pointless digression.
3) You sounded like Lex Luthor, saying that.
4) Bane doesn't have a Kryptonite weakness, but has an weakness to just about everything else, which Superman doesn't have.
5) Only the last one of the attached images worked for me, and that was just fan-art.
Superman is stronger than Bane. This is a Fact.
So the argument/discussion is whether a Human with Ninja Skills a Utility Belt full of Gadgets and defeat a being who can lift a continent into space (Superman Returns), is invulnerable to Bullets, Knives, Tanks, Nuclear Bombs, and is Faster than a Speeding Bullet.
The answer is Yes.
Now before anyone throws hate at me let me explain. Batman does not have the advantage over Superman in regards to Powers and abilities, so He'd have to outsmart him. Superman despite his powers is susceptible to Kryptonite and Magic, both of which Batman can use against him. Also if Batman could figure out how to simulate Kryptons Red Sun and Environment in a room and trick Superman into it the Man of Steel would be powerless.
Would Batman do this? Only if it were absolutely Necessary. Batman and Superman are Friends and there's a Reason why Superman gave Batman Luthor's Kryptonite ring. If Superman ever lost control and became a threat to Earth, he wanted to give Batman the means to stop him. I can't see them fighting in this movie, maybe at odds with one another at first but later a friendship will be formed between the two.
"I won't be wronged, I won't be insulted, and I won't be laid a hand on. I don't do these things to other people and I expect the same from them."
-John Wayne (The Shootist)
This is THE WORST NEWS OF ALL TIME....
Michael Bay: Speed Demon http://www.youtube.com/watch?v=cE1qfi_Avto
Michael Bay Tribute https://www.youtube.com/watch?v=qJes7Etbkzw
Yeah...I don't like this one bit.
I'll give him a chance.
I don't see what the problem is.
It's Daredevil, isn't it?
I really want to know what his Batman voice is going to sound like.


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The African Green Revolution aims to improve agriculture's productivity and profitability.
Investments across the value chain are considered key to increasing food production and improving food security. The AGR particularly targets smallholder farmers, who suffer from poor inclusion in value chains.
March 2013
Agriculture remains the economic backbone of most African countries.
Agriculture is the main sector generating jobs and income, contributing to economic growth, throughout Africa. However, agriculture often underperforms, with low yields and little profitability, largely due to shortage of agronomic inputs and efficient infrastructure connecting to markets.
In 2004 Kofi Annan called for a genuinely African Green Revolution.
Yara attended the conference in Ethiopia where the then Secretary-General of the United Nations urged stakeholders to rally behind his call. Yara became the first private sector actor to respond, establishing an Africa program.
The African Green Revolution Conferences called for public-private partnerships.
Yara initiated the Oslo series of conferences 2006–08, a multi-stakeholder platform to share ideas and forge partnerships. They were followed by the African Green Revolution Forum (Accra, Ghana 2010; Arusha, Tanzania 2012), of which Yara remains a co-sponsor and key partner.
Smallholders dominate agricultural production in most African countries.
Smallholders, many of them women, are more vulnerable to the risks of agriculture, like droughts. With low output and income, smallholders are generally poor, lacking the means to improve productivity and turn their farming into sustainable, profitable business.
Improving agricultural productivity is the key to increasing food security.
Productivity depends on the availability and application of inputs vital to modern agriculture, especially mineral fertilizers, hybrid seeds and crop protection agents. These inputs should ideally be delivered together with crop knowledge and agricultural tools.
Improving agricultural profitability is the key to reducing poverty.
Investing in agriculture is a strategy to create social and economic growth – for farming families, rural communities and national economies. Transforming smallholder farming from a way of living to viable business is central to sustainable agriculture.
Yara played a key role in establishing the Grow Africa partnership.
This public-private platform is designed to accelerate private sector investments and transformative change in African agriculture, building on previous initiatives. It was launched in 2011, with Yara as a member and co-chair of the Grow Africa Task Force.
Ghana was one of the first four countries to be included in the Grow Africa initiative.
The inclusion in the Grow Africa framework aims to trigger financial support and investments. Yara signed a Letter of Intent, signaling its plans to step up its investment and engagement in the country, e.g. by exploring a rice value chain including a smallholder perspective.
Yara has engaged in several food value chains in Africa.
The Ghana Grains Partnership (GGP) is one of these. Founded in 2008, it aims to increase smallholder productivity and food production amongst maize growers. Key interventions include the Masara N'Arziki Farmers Association, financing mechanisms and knowledge transfer.
Food value chains represent targeted interventions with great impact.
By engaging in value chain partnerships such as the GGP, delivering quality fertilizers and sharing its crop nutrition knowledge, Yara significantly helps increase productivity and profitability for smallholder growers, and the livelihood of their families.
Yara has made impact through its engagement in African agriculture.
By consistently supporting the African Green Revolution, Yara aims to make a significant and lasting impact on rural livelihoods by triggering capacity building and infrastructure development, making farming viable business opportunities also for small and medium size farms.
Maize for prosperity
Partnering for productivity
Prosperity in Ghana
Yara's Africa engagement
Ghana has taken a lead in African economic development, putting agriculture first. Yara has committed to the African Green Revolution – and to the agricultural transformation of Ghana. Engaging in the entire value chain and working in partnership is the key.
Oslo, March 24, 2013
Ghana Grains Partnership
"Yara gives me knowledge on how to plant and how to apply the fertilizer, how to do it."
Nindow Maize farmer, Northern Region, Ghana
"Yara gives me knowledge on how to plant and how to apply the fertilizer, how to do it."
In 2008 Yara, together with the local inputs provider Wienco, initiated the Ghana Grains Partnership (GGP), a public-private partnership working to improve the efficiency of the maize value chain in Northern Ghana.
The partners helped set up a farmers' association – the Masara N'Arziki ('Maize for Prosperity' in the local Hausa language). Towards the end of 2012 more than 8,000 farmers had joined, seeing maize yield levels triple compared to the average.
The Masara acts on behalf of its members, purchasing inputs and selling the crops, while the GGP provides seeds and fertilizers on affordable credit terms, as well as storage and transport facilities – helping to reduce losses and increase profits.
Ghanaian agriculture consists mainly of smallholder farmers, both in the staple crop sector as well as the economically vital cocoa production. Both the Government policies for agricultural development and the GGP target the crucially important smallholders.
Value chain engagement
The GGP is a model example of engaging throughout the food value chain, addressing major bottlenecks to improve productivity and profitability. Yara is a major provider of mineral fertilizer to Ghana, engaging both in cash crops such as cocoa and in staple crops such as maize.
Building on the experiences gained through the GGP, Yara – through the Grow Africa platform – will explore opportunities for a new partnership in the rice value chain.
Yara operates a fertilizer blending facility and a warehouse with a capacity of two million bags, distributing mineral fertilizer throughout Ghana. An important part of the business model is supplementing the product with knowledge, collaborating with farmers through crop clinics. Here, growers receive advice on how to best improve their yields.
The GGP is a prime example of how agricultural development can be done in an inclusive manner, raising yields and profitability both with and for the smallholders. Yara has committed itself to contributing its knowledge, products and solutions to promote an African Green Revolution.


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my dear 24mm tilt shift lens! :)
Ever since I've played with Bobby Earle's tilt shift lens, I've been wanting to get one. Coming into year 2010, I've decided that I should at least get one new lens for this year. I was actually debating between getting a 50 f/1.2L and a 24mm tilt shift lens. Since we've already have a 24mm L, the 24mm tilt shift seems unnecessary. Getting the 50 f/1.2 is more practical. However, I just love experimenting with different composition possibilities with a tilt shift lens. I love how it produces dreamy pictures. :) Hopefully somewhere down the line I'll have enough fund to purchase a 50 f/1.2L…then my collection will be complete. ha!
Here are some test shots that I took on this glorious morning.
Our front door looks so much nicer through the tilt shift lens. :)
I can't wait to bring it to Hong Kong with me in February! I will be going there with my sister and my cousin. I can't imagine the type of pictures I can get with this new baby!


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Worksop Rugby Club's junior players impressed a former British Lion so much that he's invited them to star at Headingley Carnegie Stadium.
John Bentley, who played for Great Britain in both rugby union and rugby league, paid a visit to Stubbing Lane last week to pass on his expertise to the club's youngsters.
And organiser of the special visit, Mark Walker, told the Guardian it was a successful evening.
"It was fantastic," he said.
"Going on from the coaching session, John has invited the kids up to Leeds Carnegie."
"They'll go up there one morning before Christmas, when Leeds are playing at home, and the players will train our kids."
"The kids will then go on the pitch at half-time to have a knock about."
Bentley's wisdom was not only picked up by the junior players last Thursday, but by Worksop's coaches as well.
Walker said: "We wanted our coaches Tim Lewis, Andrew Waddell and Justin Poxon to take note of his drills and the way he does things."
"John put an emphasis on taking command on the field and putting your name on the ball."
"I said to the coaches that they should take mental notes during the session and write it all down later on."
"John played rugby league for Great Britain and was a winning British Lion in 1997, so if he doesn't know what he's talking about, no one does."
And the youngsters in attendance were suitably impressed by their special guest.
"Quite a few of the kids came down and had done a bit of homework on him," Walker explained.
"They had a few things for him to sign, England shirts and books with pictures of him in."
"The majority of kids were our Under 15s, and there were some of the tag rugby kids as well, the six to eights."
"They can't wait to go up to Leeds."
"We took them to Rotherham Titans last year for a similar trip, so they know what to expect."


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Ashes Moments: Warne's Ball of the Century
Manchester has always had a reputation for being a spin bowler's paradise, with Jim Laker and Richie Benaud just two men who had previously profited at Old Trafford. In 1993, it was the site of Shane Warne's Ashes debut. His first ball on English soil became known as the 'Ball of the Century', as his heavily spun leg-spinner drifted outside Mike Gatting's leg stump, pitched and spun fiercely past the outside edge and into the top of off-stump. Gatting's look of utter surprise was almost as memorable as the ball that claimed him.
Source: ABC News | Duration: 1min 11sec


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Dustin Ashley
- 13 years old
- Date of birth: Jul 29, 1996
- Date of passing: Feb 20, 2010
|Let the memory of Dustin be with us forever
This memorial website was created in memory of our loved one, Dustin Ashley, 13, born on July 29, 1996 and passed away on February 20, 2010. We will remember him forever.
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Hot, Hot, Hot – 8/21/06
August 21, 2006
Hottest posts for today
- School Board District 1 race – Bergosh,
- Port Royal Cocktail Party Buzz
- Intelligent Design and Escambia Co. Public Schools
- David Letterman joins BSoc
- Evan Johns responds, 'I wrote that viewpoint'
- I am confused….Bilbo politics live in SOC
- Donovan emails revisited
- Hot, Hot, Hot – Weekend 8/20/06
- Donovan emails – 44 pages for your review
- Oops, another donation to the Maritime Museum
Intelligent Design and Escambia Co. Public Schools
August 21, 2006
Todd Leonard supporter and consistent blogger Tom has written that Intelligent Design is taught in the science classes of the Escambia County public schools. I asked the school district to send me the page or pages from the textbook. They emailed a page that has one paragraph on the subject.
Here is what is taught on the origins of life. I think it's fair and balanced and that ID doesn't need to be made into a campaign issue.
PDF File: Origins of Life
School Board District 1 race – Bergosh, Leonard, Waters
August 9, 2006
Todd Leonard's supporters have begun injecting their candidate into every education post. Others take the bait and the debate becomes a pro/con Leonard exchange. I'll take some responsibility because I used Leonard's campaign as way to debate Intelligent Design.
But it's time we move on. Therefore, I'm setting up this post on the Escambia County School Board District 1 race – Jeff Bergosh, Todd Leonard, Doug Waters and their supporters can go at it.
Any new "Vote Todd Leonard" comments to other postings will be deleted.
Hot, Hot, Hot – 8/8/06
August 9, 2006
Top 10 posts for 8/8/06
- Todd says Intelligent Design is not abou
- WCOA Pensacola speaks becomes Marty spea
- Stop Our City changes message
- Marty and Charlie road show
- SOC to handout yard signs tonight
- We thought Pensacola politics are rough
- What has WCOA become?
- Socialist candidate upset with Winners a
- Florida State paper purchase not loved b
- Oops, Marty got caught doing backroom de
Hot, Hot, Hot – 8/7/06
August 7, 2006
Top 10 Posts for today:
- Todd says Intelligent Design is not about
- CC craps out with letter
- Marty and Charlie road show
- Sorry, Todd – You are a monkey's uncle
- What has WCOA become?
- Hopkins wants City to Sale Trillium
- WCOA Pensacola speaks becomes Marty speaks
- SRIA quits posting board minutes
- Pensacola Sucks! Whatever
- Gannett buys FSU paper
Todd says Intelligent Design is not about religion
August 7, 2006
Escambia County School Board candidate Todd Leonard has modified his website Vote Todd Leonard to include an explanation page ( Todd explains ) for his position to bring Intelligent Design into the classrooms. It's a direct response to this blog – Oh, my God! or is it Todd's God?; Sorry, Todd – You are a monkey's uncle .
Leonard sees I.D. has just part of his reform agenda. I see it is a voodoo science, at best, and, at worse, as a backdoor attempt to bring religion in the classroom. Check out this view on the issue – Harvard professor weighs in
Hot, Hot, Hot – Weekend 8/5-8/6/06
August 7, 2006
Top 10 Posts
- Sorry, Todd – You are a monkey's uncle
- Weekend Escape – Edie Brickell
- Hopkins wants City to Sale Trillium
- County dumps on City again
- PNJ has shrunk
- Stidham stalls
- Can you pass Eighth Grade math?
- Rick's Analysis of Pensacola City races
- Stop the madness! Testing Pre-K
- Inside Emerald Coastkeeper Fundraiser
Hot, Hot, Hot – 8/4/06
August 5, 2006
Hot, Hot, Hot – 8/3/06
August 3, 2006
Blog moves to top 25 (#22). Thanks to Math Quiz and new SOC cartoon.
Here's today's top 10
Hot, Hot, Hot – 8/2/06
August 2, 2006
Another record. Most visitors to date. Top 25 blogs worldwide
- Sorry, Todd – You are a monkey's uncle
- Dammit, why cant we be first in something
- Walmart and Healthcare
- Charlie just doesn't get it
- Bye, Bye Raymond
- Oh, My God, Part II – Check out Tom Wells
- Private schools are not that great
- Healthcare for some, Part 2
- Who makes what?
- Letter from Parents of Lisa Dickson


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An interesting choice of Tory keynote speaker at the Russian Embassy
Guido Fawkes reports that John Whittingdale was the keynote speaker at the launch of Conservative Friends of Russia, which took place at the Russian Embassy last night.
Whittingdale is of course the Chair of the Culture, Media and Sport select committee, which is an interesting choice, considering how some aspects of Russian culture are being dealt with at the moment…
Back in 2010 Whittingdale said:
"I am fundamentally opposed to censorship. I believe that in a free society it is up to adults to choose what they wish to see"
Fair enough – but where does he stand on the censorship, arrest and imprisonment of Russian punk musicians?
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Q; How long will it take for delivery of this Monaco Walnut door?
A; We estimate 48 - 72 hours for our free delivery, the speed is dependant on the quantity of doors, 2 or more doors are direct from our supplier and a dedicated courier is used for single doors.
Q; What type of walnut is used?
A; We use sustainable walnut which is then fully decorated.... in laymans terms...fully varnished.
Q; What is the construction?
A; The Monaco Walnut Door has a solid core with the veneer on top to provide a 1st class pre-finished door and frosted safety glass as standard.
Q; What sizes are available?
A; The sizes are 1981x686mm, 1981x762mm and 1981x838mm, all 35mm thick.
These Monaco walnut flush veneer doors are beautifully veneered with a top quality walnut and pre-finished to a very high standard, the safety glass is known as "linea" and is mainly a clear effect with simple individual frosted lines which gives some privacy but not total.
See a full range of handles here and other door fittings here.
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Manny Pacquiao failed to deliver the knockout that he wanted, but his killer instinct will no longer be questioned.
Dominant Performance
Pacquiao, boxing's only eight division world champion, punished WBO light welterweight champion Chris Algieri for twelve full rounds to score a very lopsided unanimous decision win before a sold out crowd of 13,301 on Sunday noon at the Cotai Arena in the Venetian Macay, China. Pacquiao scored six knockdowns against the younger and taller Algieri in a dominant performance that erased all doubts about his lack of killer instinct in his recent bouts.
With the win, the only question left in the minds of boxing fans and experts is whether he will be finally able to face the undefeated Pound for Pound King and fellow welterweight champion Floyd Mayweather Jr. in a mega fight that has been talked about for five years now. Negotiations for that bout have reached a stalemate in the past because of disputes in blood and drug testing and purse sharing. The talks virtually died in 2012 when Pacquiao lost back to back fights to Timothy Bradley and Juan Manuel Marquez. But with the win on Sunday, Pacquiao has now won three in a row, including a payback unanimous decision win over Bradley last April.
Punching Power Gone?
Aside from his killer instinct, pundits questioned where Pacquiao's punching power had gone. The Filipino fighting congressman incredibly rose the weight classes with devastating knockout wins over Oscar De La Hoya, Ricky Hatton and Miguel Cotto. But after the Cotto fight in 2009 which was his first as a welterweight, Pacquaio hadn't scored a knockout win. That drought has now increased to nine fights after Algieri withstood Pacquiao's onslaught.
But it was a blowout as Pacquiao gave Algieri, who was a massive underdog in the fight, a beating he won't forget. In round two, Pacquiao scored on a massive right hook that swelled Algieri's left eye. He then landed a left hand that dropped an off-balanced Algieri. The Pacman continued to land at will on Algieri, connecting on solid head and body punches that began to soften Algieri. Algieri tried to put up some offense after round four, but it was obvious that Pacquiao was in a different league.
All Over But the Ending
By the middle rounds, Pacquiao had distanced himself from his challenger. The Pacman summoned his vaunted punching power and put Algieri down twice in round six. And instead of putting his foot off the gas pedal, Pacquiao kept looking for the kill . In the ninth, Pacquiao connected on his best punch of the night, a powerful left straight, that clearly hurt Algieri and seemed to keep him down for good. But the New York native got back to his feet, only to receive more punishment. Moments later, Algieri took a knee after a barrage of punches from the Filipino icon.
In the 10th, the same round when he backed off against Antonio Margarito, Pacquaio scored his sixth knockdown of the fight, and shutting down what was left of Algieri's will. Pacquiao continued to punish Algieri until the final bell but the American rode his bicycle to avoid the knockout. Despite failing to come up with a KO, Pacquiao said after the bout that he was satisfied with his performance because he did his best.
So now that Pacquaio has proven that he still has it, the ball is now in Mayweather's court. Floyd Mayweather finished his 2014 business last September with a second unanimous decision win over Marcos Maidana. The is no other opponent left for each man to face and there is no other fight the fans want to see from them. So will we finally see the fight next year?


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"That's great, thank you!"
The directors smiled as the eager first-year student flounced off stage and another took her place, ready to audition. I watched from the back of the small theater, where I sat with my director. She scribbled notes about each actor as they read from scripts clenched tightly in nervous hands. I still couldn't believe I was watching people audition to be in a play that I had written.
Okay, wait – maybe I should back up a bit.
This semester, I am taking the first of many writing workshops required for English majors in the Creative Writing concentration. Recently, we were asked by our professor to submit a short fiction piece, which our classmates would then workshop and give us feedback on, explaining what they liked, what they didn't like, and what could be improved. But, in my free time, I was also working on another project, a short play I was writing outside of class, simply for the pleasure of writing. And so, every time I sat down to get started on my homework assignment, the short play kept fighting for my attention.
UGH Just focus, Jamie, I grumbled to myself. Maybe you should just get the play done so you can focus on your homework.
Yes, okay, I agreed. I'll finish the play I'm working on first then start the assignment next week. After all, I still had ages before it was due.
When I finally scripted the last curtain at the end of my play, I couldn't help but smile, pleased with my work. And now it was time to roll up my sleeves and knock out that homework. But then the thought struck me: how strict was the term, fiction, exactly? Could I submit the short drama I had just penned for peer review? Did it have to be prose? To my delighted surprise, my professor gave me the green light when I asked him about this in our next class meeting.
Later, another student in the class pulled me aside.
"Do you know anything about Under Siege?" she asked me.
Under Siege? What was this new mystery? I inquired further, walking with her out of the library and into the chilly November air.
Apparently, Under Siege is a student organization open to actors from all disciplines in the university. And, as it turns out, they produce an entire showcase dedicated to short student works. After selecting their submissions, they cast and rehearse the plays before performing short readings at the end of the semester. My classmate was already involved in the club, and she asked me if I would be interested in submitting my work.
Um, let me think: YES.
I typed up an eager email and submitted my script. After a month of nail biting and frequent email checking, I was delighted to learn my piece – you know, the one I had written in my free time then turned in for class – had been chosen for the showcase my classmate had told me about. I was even invited to come and watch the auditions with my director. This was great. Now not only would I get to have my play critiqued by students in the workshop class, but I would also get to see it performed by Arcadia student actors. I couldn't believe it.
Now that we've casted the play and rehearsals are upon us, I am counting down the days until the night of the showcase. I'm not going to lie, I'm a little nervous about having my work performed in front of an audience. Originally, my audience had consisted of my fellow classmates. And because we have spent the past semester getting to know each other and becoming really close, I was comfortable with them. But now, my audience has grown into whoever decides to walk through the doors at the New Works Showcase December 12th in the Spruance Fine Arts Center Theater.
Nerves aside, more than anything else, I am extremely excited. Not only will my play be read, but a collection of five other works all written by Arcadia students will be performed as well, which I also can't wait to see. I took a play I was already writing in my spare time and pulled it into the worlds of both academia and student theater. Now I get to sit back and watch it grow both inside and outside the classroom.
How's that for extra credit?


text:
Most of you who have bought the Iron Man 3 DVD or scoured YouTube will have watched the special feature included entitled 'Agent Carter', a one-shot adventure set after the events of Captain America: The First Avenger (and if you haven't, go and bloody watch it!). In her mission, she encounters the Zodiac. No, not physically. It's more like a tiny vial with the symbol of the Zodiac holding a strange blue-coloured chemical. If you are not aware of who the Zodiac are, here is some background comic information for you which you better take a glance at before you read on;
The original Zodiac, funnily enough, debuted in the Avengers run. They were leaded by Cornelius van Lunt (Taurus), their identity based on astrology, with each member adopting the persona of a sign of the zodiac, being twelve in all. The group members share leadership of the organization, with the position rotating just as the astrological zodiac changes. When not united as a group, the members of Zodiac are all based in different cities throughout the United States. Dedicated to economic and political domination of the world, the group uses any means to accomplish this, including subversion; extortion and mass murder.
The fact that the team and/or the background information on the zodiac vial found by Carter in the short film were not seen or explored in this suggests that there is more to be learnt some time in the future, otherwise what would be the point in leaving it on a question mark like that? If you think about it, all the One-Shots have given us clues about the future of Marvel, and it appears to be that all these clues are relatively important. The Abomination being under custody of SHIELD, the nods to Roxxon and now the Zodiac, this is all starting to come together nicely. If the Avengers' next foe used the Abomination, it would be a perfect counter to Nick Fury's whole "yeah but we got the Hulk" attitude. Besides, Tim Roth has actually been contracted for several Marvel films in case he is needed so someone somewhere must be considering his character, no?
Of course, this is all speculation. But that doesn't change what we've seen here, and frankly the nature of the Zodiac would make their appearance perfectly timed given that the first Avengers movie had a little subplot showing the borderline corruption and trickster work performed by those within SHIELD, particularly Nick Fury's habit of covering things up even from the biggest names in the world. What do you think, people? Is it just a random storyline with no future, or do you expect to see the Zodiac at large within the next few years? Leave your thoughts as you please, but might I leave you with this last little note;
Thanos also had his own Zodiac once.


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As the countdown to Christmas officially begins, I have been thinking about what is important to Kyle and me and how we live that through this season. The holidays are the perfect time to celebrate what is the center of our life – to live creatively and to support others that do the same.
For the last couple of years, we have been able to successfully embody that in our gift giving by only giving things we or our friends have made. We try to take buying local one step further and "Buy Personal". We buy from people we know, that made something with their hands, thought something up in their brain, got together with friends, and said, "Hey I have a great idea." And when you know that person's name, know their story, know the money you pass to them will be used to continue to live their passion, it makes the whole world feel like a small town. These creators are the people that make life rich around us. These are the gifts we are giving this holiday season because when we buy from a creative, we are actually giving two gifts. We are telling two people, "I care about you and I really think you two should meet."
We are so lucky to have so many talented artists and creators in our life, and I want to share our riches with you! I would love for you to meet every one of our friends and then you too can buy personal! I assure you that we can introduce you someone that is making a gift for everyone on your list, from the smallest toddler to the trickiest recipient (hello Dad) and everyone in between.
Today, I would like you to meet Erin McGrane & Jeff Freling!
These beautiful people are our dearest friends and fantastic musicians. Jeff and Kyle began playing music together in 1987 and have played together ever since, even thru the decade Jeff spent in Chicago playing with the Blue Man Group. And I took promo photos of Erin's first band, Blue Museum, in 1990, well before her appearance alongside George Clooney in Up In the Air. And despite the scheduling complications of four busy lives, we have remained
great friends family and supporters of one another through all of our creative projects, including their newest. You need to know about it!
Victor and Penny
Performing as the duo Victor and Penny they are touring the country performing antique pop from coast to coast, performing over 150 dates in the last year. If we could get our travel dates to line up better it would be perfect, but at least we are lucky that we all come home to Kansas City. And this holiday season we are lucky to join them in the celebration of their new CD: Victor and Penny and their Loose Change Orchestra , Live at the Living Room Theatre
This is the perfect gift for anyone on your list!


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Like 20days now my bf wants a baby he been trying everyday sense my missed appointment. Now im having stomach pains feeling tired nausea feeling fat my question is could i be pregnant already or is it the depo symptoms?
I been on depo for 3years but Im tired of it so I missed my appointment on april 14,2015 its been?
- Asked
- 29 Apr 2015 by sweetymoore
- Active
- 30 Apr 2015
- Topics
- pain, nausea/vomiting, pregnancy, stomach pain, baby, stomach, depo
Responses (1)
by me being on the shot so long ppl were saying it could take uo to a year to get pregnant is that true or is depending on ur body
Things said by ""people" and "they say, " most likely are based only loosely on fact. It can take up to that long but you just as easily could get pregnant the day after your shot was expired.
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The much-anticipated musical being developed by Stephen Sondheim and playwright David Ives could well be on stage in the next year, according to the composer himself. At the recent Glimmerglass Festival in New York, Sondheim hinted that the show – a mash-up of two movies by surrealist Luis Buñuel – would premiere at New York's Public Theater in 2017.
In correspondence with The Stephen Sondheim Society, Mr Sondheim also revealed in passing that a reading of the show is scheduled for next month. Watch this space!


text:
Getting This
Getting This
Selling the Razer Hammerhead Pro V2 headphones with mic, in new and sealed condition. The headphone is going at a promotional price of $60 only (headphones retailing at $120). The set is perfect for both gaming and normal usage,with crisp clear mic for clear communications. Experience superior acoustic clarity and improved bass performance through the upgraded extra-large 10 mm dynamic drivers, over 20% larger than the original Razer Hammerhead, featuring an optimized inner acoustics chamber. The Razer Hammerhead Pro complements every style while retaining its signature durability factor. Designed with a high quality body to enable improved acoustics, the new flat-style cables also facilitate fuss-free mobility and easy storage while you're on the move. The discreet in-line microphone on the Razer Hammerhead Pro has been further enhanced with a 3-button remote control. Whether you need to control the volume or answer a call, the 3 Quick Action Control buttons give you greater flexibility with your mobile devices. INLINE REMOTE Frequency Response: 20 Hz – 20 KHz Impedance: 32 Ω ± 15% Sensitivity (@1 kHz): 102 ± 3 dB at 1 kHz max Input Power: 5 mW max Drivers: 10 mm, with Neodymium magnets Connector: 3.5 mm, angled gold-plated Cable length: 1.3 m / 4.27 ft Approximate weight: 19.6 g / 0.04 lbs MICROPHONE Frequency Response Signal-to-noise ratio Sensitivity (@1 kHz): 50 – 10,000 Hz ≥ 55 dB -45 ± 3 dB Pick-up pattern: Omnidirectional IN-LINE REMOTE Volume control buttons: Volume Up / Down, Changing tracks Center control button: Play / Pause, Accept / End call TAGS Razer Gaming Music PC Marshall Bose Shure Audiotechnica ATH Marshall Samsung Apple Naga Abyss Headphones Gaming Earpiece Sennheiser Mic Android
Postage at an additional $5. Door to door courier delivery services at an additional $10 :)


text:
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