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SHARPIN is a ubiquitin-binding and ubiquitin-like domain-containing protein which, when mutated in mice, results in immune system disorders and multiorgan inflammation1,2. Here we report that SHARPIN functions as a novel component of the Linear Ubiquitin Chain Assembly Complex (LUBAC) and that the absence of SHARPIN causes disregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis in SHARPIN deficient mice. Upon binding to the LUBAC subunit HOIP, SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Co-expression of SHARPIN and HOIP promotes linear ubiquitylation of NEMO, an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, while SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages, and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L, another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon TNFα stimulation via FADD- and Caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.
In addition to the established roles of K63- and K48-linked ubiquitin chains, linear ubiquitin chains have recently emerged as important regulators of the NF-κB pathway, controlling immune responses, as well as cell survival, proliferation, and development3,4,5,6,7,8. Linear ubiquitin conjugation relies on two RING-between-RING (RBR) domain-containing proteins, HOIL-1L (longer isoform of hem-oxidized iron-regulatory protein 2 ubiquitin ligase-1) and HOIP (HOIL-1L interacting protein), which together form the E3 ligase complex LUBAC (linear ubiquitin chain assembly complex)9. We became interested in SHARPIN (SHANK-associated RH domain-interacting protein) because of the significant sequence homology of its C-terminal region, enclosing a ubiquitin-like (UBL) domain and a putative ubiquitin-binding NPL4 zinc finger domain (NZF), with the N-terminal region of HOIL-1L9,10 (Fig. 1a). Initially, we established SHARPIN as a bona fide ubiquitin binding protein by mapping the ubiquitin binding site to the NZF domain (Supplementary Fig. 1). A direct NZF-dependent interaction of SHARPIN with mono-ubiquitin, linear, and K63 ubiquitin chains was confirmed by pull-down assays (Supplementary Fig. 1b–d) and isothermal titration calorimetry (ITC) (Fig. 1b). Mutagenesis of two key residues surrounding the zinc coordination site of the NZF (T358L, F359V) abolished ubiquitin binding (Supplementary Fig. 1a and 1c). The isolated NZFs of SHARPIN, HOIL-1L, and HOIP interact with monoubiquitin with similar affinities (Fig. 1b), recognizing the classical hydrophobic patch surrounding I44, as indicated by the absence of any interaction with mutant I44A ubiquitin (Fig. 1b) and display no specificity for different chain types (Fig. 1b and Supplementary Fig. 1c). These data are consistent with binding assays employing a panel of HOIP and HOIL-1L protein variants expressed in transfected cells or in in vitro ubiquitin binding experiments (Supplementary Fig. 1e–h).
We next investigated whether SHARPIN directly associates with the LUBAC complex in cells. Tagged versions of HOIP, HOIL-1L, and SHARPIN were readily immunoprecipitated from lysates of transiently transfected HEK293T cells (Fig. 1c). Likewise, the interaction of endogenous SHARPIN, HOIP, and HOIL-1L was confirmed in primary mouse embryonic fibroblasts (MEFs) (Fig. 1d). The interaction between SHARPIN and HOIP is mediated by the UBL domain of SHARPIN, which recognizes the NZF2 domain of HOIP (Supplementary Fig. 1i and j). In agreement, the interaction between HOIP and SHARPIN was abolished in response to either deletion or mutation (I272A) of the UBL domain of SHARPIN, but was not affected in the ubiquitin-binding deficient mutant of SHARPIN NZF (Supplementary Fig. 2a). On the other hand, purified HOIL-1L did not interact with full-length SHARPIN in in vitro binding assays (Supplementary Fig. 2b). Thus, HOIP has the capacity to interact with either SHARPIN or HOIL-1L or both of them. Indeed, endogenous HOIP can form a complex with only HOIL-1L or SHARPIN in the absence of the other, since in SHARPIN or HOIL-1 deficient MEFs, HOIP can co-precipitate with residual HOIL-1L or SHARPIN, respectively (Fig. 1d). Interestingly, the deficiency of either SHARPIN or HOIL-1L led to a partial destabilization of the other protein (Fig. 1d). Together these findings indicate the existence of three putative LUBAC complexes: dimeric complexes, HOIP-HOIL-1L (LUBAC-I) and HOIP-SHARPIN (LUBAC-II), and a trimeric SHARPIN-HOIP-HOIL-1L complex (Supplementary Fig. 2c).
The purified HOIP-SHARPIN complex stimulated the in vitro assembly of linear ubiquitin chains (Fig. 2a), similar to the ligase activity of a HOIP-HOIL-1L complex (Supplementary Fig. 3a). We thus extended our studies to investigate whether SHARPIN and HOIP can induce linear ubiquitylation of proteins in cells. The UBAN domain of ABIN-1, known to preferentially bind linear ubiquitin chains11,12, was used as an affinity matrix to pull down cellular proteins modified by linear ubiquitylation upon overexpression of SHARPIN and HOIP (Fig. 2b and Supplementary Fig. 3f). Both the NZF and UBL domains of SHARPIN, were essential for this activity, since neither the SHARPIN-NZF (T358L, F359V), nor the SHARPIN-UBL (I272A) mutant showed any ability to induce linear ubiquitylation in vivo (Fig. 2b). Similarly, the ABIN1-UBAN D485A mutant, which is unable to bind linear polyubiquitin, did not pull down any ubiquitin conjugates (Fig. 2b, Supplementary Fig. 3d–e). A specific enrichment of linear ubiquitylation in cells overexpressing SHARPIN and HOIP was further confirmed by MS/MS analysis. In samples transfected with HOIP-SHARPIN as well as HOIP-HOIL-1L the linear ubiquitin peptide GGMQIFVK was detected (Fig. 2c). MS-based quantification (employing the AQUA approach described in Supplementary Fig. 4a) detected an increase in the absolute amounts of ubiquitin present in linear chains compared to the total pool of ubiquitin (measured by the TITLEVEPSDTIENVK peptide) in cells expressing wild type HOIP-SHARPIN, in contrast to cells expressing an inactive HOIP-SHARPIN complex (Supplementary Fig. 4b–e). We next performed a SILAC-based quantitative MS/MS analysis on immunoprecipitated NEMO upon co-transfection with either wild type or inactive HOIP-SHARPIN (experimental design is shown in Supplementary Fig. 5a and b). The measured intensity of linear ubiquitin peptides on immunoprecipitated NEMO was >25 times higher in cells transfected with wild type HOIP-SHARPIN as compared to cells expressing inactive HOIP-SHARPIN mutants (Fig. 2d and e). In the same sample we detected 13 ubiquitylation sites on NEMO, of which 11 were increased following transfection with wild type versus mutated HOIP-SHARPIN (Supplementary Fig. 5c), suggesting that NEMO is a substrate of LUBAC II.
SHARPIN deficient (Sharpincpdm/Sharpincpdm) mice exhibit diverse phenotypes in the immune system, which resemble the phenotypes of genetically engineered mouse models with impaired NF-κB activation7,8. As linear ubiquitylation is important for activation of the NF-κB pathway3,11,13,14 we speculated that the HOIP-SHARPIN LUBAC complex might play a critical role in regulation of the NF-κB pathway. Consistent with this hypothesis, cotransfection of SHARPIN and HOIP increased transcription of a luciferase-based NF-κB reporter, similar to the effect of HOIP and HOIL-1L co-expression (Fig. 2f), while no activation of NF-κB was detected when cells were transfected with SHARPIN, HOIL-1L, or HOIP alone (Fig. 2f), or a combination of mutant forms of NZF or UBL domains in SHARPIN together with HOIP (Fig. 2g).
Based on the finding that TNFα stimulated formation of a signalling complex between HOIP-SHARPIN and NEMO (Supplementary Fig. 6), we reasoned that the SHARPIN-HOIP complex might act as an upstream regulator of IKK activation. In vitro IKK kinase assay showed that TNFα rapidly enhanced IKK kinase activity in wild type MEFs, which was delayed and reduced in cells lacking SHARPIN (Fig. 3a). Upon additional abrogation of HOIL-1L expression in Sharpincpdm/Sharpincpdm MEFs by stably expressing shRNA against HOIL-1L (Sharpincpdm/Sharpincpdm shHOIL-1L) IKK activation was further inhibited (Fig. 3a). Moreover, downstream events of IKK activation, such as phosphorylation and subsequent degradation of IκBα, as well as nuclear translocation of p65, were also reduced in Sharpincpdm/Sharpincpdm MEFs and further impaired in response to shRNA-mediated downregulation of HOIL-1L (Fig. 3b, 3c and Supplementary Fig. 7a) or overexpression of an IκBα-super repressor mutant (Supplementary Fig. 7b).
Given the plethora of evidence presented above that SHARPIN modulates the NF-κB pathway, we further assessed the functional role of SHARPIN in NF-κB activation in vivo using cells isolated from the Sharpincpdm/Sharpincpdm mice. The absence of SHARPIN in Sharpincpdm/Sharpincpdm splenic B-cells stimulated with soluble CD40L resulted in impaired activation of the NF-κB pathway, evaluated by phosphorylation and degradation of IκBα, compared to wild type B-cells (Fig. 3d). Similar findings were obtained in thioglycollate-induced peritoneal macrophages from Sharpincpdm/Sharpincpdm mice, which in response to bacterial lipopolysacharide (LPS) stimulation failed to induce an efficient phosphorylation and degradation of IκBα (Fig. 3e), proper nuclear translocation of the NF-κB transcription factor p65 (Supplementary Fig. 8a) or secretion of TNFα and MCP-1 (Fig. 3f and g). Furthermore, IL-1β stimulation-induced NF-κB activation was delayed in SHARPIN deficient MEFs (Supplementary Fig. 8b). Similarly, we observed partial impairment of JNK activation in response to TNFα, CD40L, or LPS in Sharpincpdm/Sharpincpdm MEFs, B-cells, or macrophages, respectively, compared to wild type cells (Fig. 3e and Supplementary Fig. 8c–e).
An interesting feature of Sharpincpdm/Sharpincpdm mice is the combination of epidermal hyperplasia and inflammation with the presence of apoptotic bodies in keratinocytes and increased cell death in the skin15. Moreover, inhibition of TNFα-induced NF-κB signalling in keratinocytes results in inflammatory skin lesions as observed in genetically engineered mouse models, as well as in human diseases such as incontinentia pigmenti7. Using highly sensitive impedance based real-time cell analyzer, we found that Sharpincpdm/Sharpincpdm MEFs were extremely sensitive to rapid TNFα-induced cell death (Fig. 4a). This correlated with an appearance of typical apoptotic cell morphology (Supplementary Fig. 9a) and early signs (2–4 hours post TNFα stimulation) of apoptotic signalling, including proteolytic cleavage of the initiator caspase, caspase-8, the effector caspase, caspase-3, as well as PARP, a substrate of caspase-3 (Fig. 4b). In agreement with a requirement for caspase activity for the induction of apoptosis, pre-treatment of Sharpincpdm/Sharpincpdm cells with the general caspase inhibitor zVAD-FMK abolished the observed cell death in response to TNFα (Supplementary Fig. 9b). When performing the reciprocal experiments on MEFs derived from HOIL-1 null mice14, we observed a delayed and significantly weaker induction of the apoptotic response in cells stimulated with TNFα and cycloheximide (CHX) (Fig. 4a). In accordance, we were unable to detect rapid activation of caspase-3 and caspase-8 upon TNFα or TNFα + CHX treatment of HOIL-1L deficient MEFs (Supplementary Fig. 9c). Moreover, additional downregulation of HOIL-1L in Sharpincpdm/Sharpincpdm MEFs did not further increase TNFα-induced apoptosis or caspase activation (Supplementary Fig. 9d), whereas a combined lack of SHARPIN and HOIL-1L had a strong additive effect on the inhibition of the NF-κB pathway (Fig. 3a–c). Cell death in Sharpincpdm/Sharpincpdm MEFs was not upregulated by other stress stimuli, including Staurosporin, Brefeldin A, and Doxrubicin (Supplementary Fig. 9e–g).
TNFα-induced cell death is known to proceed through the cytosolic TNFR1 signalling complex II that is composed of TRADD, TRAF2, RIP1, caspase-8/10, and the death domain containing protein FADD, which together serve as a cell death checkpoint control in TNF-sensitive cells16,17. The apoptotic cell death displayed by Sharpincpdm/Sharpincpdm MEFs occurs through the FADD and caspase-8-containing TNFR1 complex II, since retroviral transduction of either a dominant negative mutant of FADD or CrmA, a cowpox virus derived serine protease inhibitor (serpin) of caspase-8, markedly inhibited TNFα-induced cell death (Fig. 4c and 4d). Importantly, we have also detected activation of caspase-3, -8, and -9 in skin keratinocytes of Sharpincpdm/Sharpincpdm mice (Fig. 4e). Taken together, our results suggest that loss of SHARPIN expression sensitizes cells for TNFα-induced cell death, in part dependent on a caspase-8 pathway. This phenomenon might be an underlying mechanism for the development of inflammatory skin lesions in Sharpincpdm/Sharpincpdm mice.
In conclusion, this study provides physiological evidence for a novel LUBAC complex composed of HOIP, the major catalytic subunit, and the adaptor protein SHARPIN. Together, SHARPIN and HOIP mediate linear ubiquitylation of NEMO in vivo (Fig. 2a–e, Supplementary Fig. 4, 5) and activate the NF-κB pathway following overexpression in cells (Fig. 2f, g). The interaction between multiple ubiquitylated NEMO molecules, via their UBAN domains, may cause conformational changes promoting the activation of IKK kinases11. In such a model, the dynamics of NF-κB activation in response to different stimuli may be determined by the distinct ability of NEMO to recognize a variety of ubiquitin signals, including different linkages (linear, K63 and K11)4,6,18 and varying lengths of ubiquitin chains19. SHARPIN and HOIL-1L have overlapping roles in the function of LUBAC as a key activator of the IKK complex and NF-κB signalling downstream of several physiological stimuli (Fig. 3). On the other hand, SHARPIN, but not HOIL-1L, deficiency in mice results in an increased cell death that is dependent on caspase-mediated apoptosis, evident in MEFs and inflammatory skin lesions (Fig. 4). Given the identified anti-apoptotic function of SHARPIN, this study exposes a putative role for linear ubiquitylation in the regulation of cell death and strongly reinforces its impact on NF-κB signalling and immune responses.
Recombinant proteins were expressed in Rosetta cells (Novagen). Detailed descriptions of immunoprecipitation, immunoblotting, GST pull-downs and apoptotic assays are available as Supplementary Information. ITC measurements were performed using a VP-ITC or ITC200 (GE Healthcare), as described in Supplementary Methods.
C57BNL/KaLawRij-Sharpincpdm/RijSunJ mice (JR7599, The Jackson Laboratory) were raised in a pathogen-free environment. Resting mature B cells were isolated using negative selection by CD43-coated magnetic beads (MiltenyiBiotec), thioglycollate-induced macrophages were harvested from the peritoneal cavity and MEFs were immortalized using SV40 large T-antigen transfection and dilution passaging.
Quantitative apoptosis assays were performed in real time using a noninvasive impedance based real-time cell analyzer (Roche).
Full Methods and any associated references are available in the supplementary online material of the paper.
Full length SHARPIN, NZF (340–387) or UBL (233–314) from HeLa cDNA were subcloned into pGEX-4T1 (GE Healthcare), pEGFP-C1 (Novagen, Madison, WI) or pBabe-puro by standard PCR methods. Mutants of SHARPIN T358L, F359V, SHARPIN ΔNZF (Δ350–374), SHARPIN I272A, SHARPIN ΔC (1–232), SHARPIN ΔUBL, and insertion of 6XHis-tag in C-terminus of pGEX plasmid were introduced by site-directed mutagenesis. pcDNA3-Myc-human HOIP, pcDNA3-HA-human HOIL-1L ΔRBR-C, pcDNA3-HA-human HOIL-1L ΔUBL, pcDNA3-HA-human HOIL-1L ΔNZF, pGEX-6Pp1-HOIP NZF1 (aa350–379), pGEX-6P-1-HOIP NZF2 (aa 408–438), and pGEX-6P-1-HOIP NZF1+2 (aa 350–438) were described previously14. pGEX-4T1-Ubiquitin, pGEX-4T1-diUbiquitin, and pGEX-4T1-mouse NEMO-UBAN were described elsewhere11. pMALc2x-human NEMO was generated by a standard PCR method. pGEX-4T2-tetra-Ubiquitin was a kind gift from Caixia Guo and Errol Friedberg. pGEX-4T1-mouse ABIN1-UBAN was described previously12. pcDNA3-Myc-human HOIP RING mutant (C699/702S, C871/874S) and pGEX-4T1-mouse ABIN1-UBAN D485A were generated by standard site directed mutagenesis. pMSCV-GFP, pMSCV-Flag-DN-FADD-IRES-GFP, and pMSCV-Flag-CrmA-IRES-GFP were kind gifts from John Silke. Anti-His (Novagen, Madison, WI), anti-Ubiquitin (P4D1; Santa Cruz Biotechnology, Santa Cruz, CA), anti-GFP (B-2; Santa Cruz Biotechnology), anti-HA (HA.11; Covance, Berkeley, CA), anti-Myc (9E10; Santa Cruz Biotechnology), anti-Flag (M2, Covance), anti-Cleaved Caspase 3 (Cell Signaling), anti-Caspase 8 (Cell Signaling), anti-Tubulin (Sigma), anti-Vinculin (Sigma), anti-PARP (Cell Signaling), anti-p65 (Santa Cruz Biotechnology), anti-IκBα (Cell Signaling and Santa Cruz Biotechnology), anti-pIκBα (Cell Signaling), anti-IKKα (Imgenex), anti-NEMO (Santa Cruz Biotechnology), anti-pJNK (Cell Signaling), anti-pp38 (Cell signaling), anti-NUP62 (BD Transduction), anti-GAPDH (Abcam), and anti-Flag M2 agarose (Sigma) were purchased and used according to the manufacturers recommendations. Anti-HOIP and anti-HOIL-1L were described previously14. The anti-SHARPIN antibody was raised against a fragment representing the N-terminal region (1–162 amino acids) of human SHARPIN and affinity-purified using the antigen affinity matrix.
Human Embryonic Kidney (HEK) 293T cells (ATCC) were maintained at 37°C, in 5% CO2 condition in DMEM (Gibco) supplemented with 10% Fetal calf serum (Gibco) and 100 U/ml penicillin and streptomycin (Invitrogen, Carlsbad, CA). Murine TNFα, IL-1α and CD40L were purchased from PreproTech, while LPS was from Enzo Life Sciences. zVAD-FMK and cycloheximide were purchased from Bachem and R&D, respectively.
HEK 293T cells were cultured in a custom-made SILAC-DMEM medium (lacking the amino acids lysine and arginine, PAA). The medium was supplemented with dialysed serum (PAA), L-Glutamine, penicillin, streptomycin and amino acids L-lysine and L-arginine. The "light" culture was supplemented with Lys0 and Arg0 (Sigma); the "medium-heavy" culture with Lys4 (2H4) and Arg6 (13C6); and the "heavy" culture with Lys8 (13C6 15N2) and Arg10 (13C6 15N4). All labelled amino acids were purchased from Cambridge Isotope Laboratories.
Transformed E. coli were grown in LB/ampicillin at 37°C until OD = 0.3–0.5, induced with 1 mM IPTG and grown over night at 16°C. The bacteria were harvested by centrifugation and resuspended in GST buffer (20 mMTris, pH 8.0, 100 mMNaCl) supplemented with complete protease inhibitors (Roche Diagnostics), sonicated, and lysed in 1% Triton X-100. The cleared lysate was incubated with Glutathione Sepharose beads (GE Healthcare) for 2 hours at 4°C with continuous rolling. The beads were subsequently washed with buffer 1 (0.1 M Tris, pH 7.8, 0.5 M NaCl), GST-buffer containing 0.35% Triton X-100 and GST buffer alone. The purified GST-fusions were separated by SDS-PAGE and analyzed by Coomassie staining.
HEK293T cells were transfected with the indicated constructs using FuGene6 (Roche Diagnostics, Mannheim, Germany) or GeneJuice (Novagen) according to the manufacturer's protocol. Cells were treated before lysis (lysis buffer; 50 mM Hepes, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 10 μM ZnCl2, 10% glycerol, 1% Triton X-100, supplemented with complete protease inhibitors (Roche)). Cleared lysates were subjected to immunoprecipitation by incubation with the indicated antibodies, followed by Protein A/G Agarose (Santa Cruz Biothechnology) or Protein A-agarose (Roche). Similarly, total cell lysates were incubated with GST-proteins conjugated to Glutathione sepharose beads at 4°C. After washing 3 times with lysis buffer, the immunoprecipitates were separated by SDS-PAGE and analyzed by Western blot.
All constructs for ITC studies have been cloned into pGEX-4T1 or pGEX-6P1 and were expressed using E. coli BL21 as host strain. 5 to 10 L expression culture was grown to an OD of 0.8 in LB/ampicillin media supplemented with 100 μM ZnCl2 at 37°C prior to induction with 0.1 mM IPTG for 12 hours at 25°C. Cells were resuspended in buffer PX (100 mM HEPES pH 7.4, 500 mM NaCl, 1 mM DTT) and lysed by sonication. The lysate was cleared by centrifugation and loaded onto GSH Sepharose column (GE Healthcare). Bound GST fusion protein was eluted using buffer PX containing 20 mM glutathione, cleaved with thrombin or 3C protease, and further purified by anion exchange chromatography using a Q Sepharose column (GE Healthcare). Fractions containing the protein of interest were polished by size exclusion chromatography on a Superdex 75 or Superdex 200 column (GE Healthcare). Protein concentrations were determined by UV spectrometry at 280 nm using calculated extinction coefficients. K48 and K63-linked ubiquitin chains were prepared essentially as described20.
ITC measurements were performed using a VP-ITC or ITC200 (GE Healthcare). Typically 50–100 μM of SHARPIN, HOIL-1L, the isolated NZF domains and the NZF1 of HOIP were loaded into the cell and 500–1000 μM of ubiquitin or ubiquitin chains into the syringe. Samples were dialyzed into buffer containing 50 mM HEPES pH 7.4, 150 mM NaCl, and 1 mM TCEP. SHARPIN titrations were performed at 10° C, HOIL-1L, and HOIP titrations at 20°C. ITC data were analyzed with Origin7 (Micro Cal) supplied by the manufacturer.
HEK293T cells were plated on 48-well plates at a density of 20,000 cells/well 24 hours prior to transfection. pNF-κB-Luc plasmid (Stratagene) and β-GAL plasmid were transfected together with the indicated plasmids using FuGene6. After 36 hours of transfection, lysates were prepared and subjected to luciferase assays following the manufacturer's protocol (Roche). Internal control was measured by β-Gal activity using its substrate (Roche). All experiments were done using quadruple samples.
Ubiquitin (10 μg), His-tag E1 (150 ng), UbcH7 (300 ng) (Boston Biochem), and the indicated E3 ligase complexes (1 μg) were incubated with or without 2 mM ATP (Sigma) for the indicated time at 37°C in ubiquitin assay buffer (20 mM Tris-HCl pH7.5, 5 mM MgCl2, 2 mM DTT). Methylated ubiquitin was used as a negative control. To stop the reaction, SDS-loading buffer was added and the samples were boiled at 95°C for 1 minute. The samples were subsequently analyzed by SDS-PAGE followed by Western blotting using a PVDF membrane.
MEFs of wt, Sharpincpdm/Sharpincpdm or Sharpincpdm/Sharpincpdm HOIL-1L shRNA were treated by TNFα (20 ng/ml) for indicated times. IKK complex was immunoprecipitated by anti-NEMO antibody from the total cell lysates prepared from treated MEFs. Kinase assays were performed using γ-(32P)ATP (5 μ-Ci), GST-IκBα 1–54 (1 μ-g), and the IKK complex, incubated in the kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mM sodium orthovanadate, 5 mM β-glycerophosphate, 2 mM DTT, 20 μ-M ATP) at 30°C for 30 min.
Complete protein eluates of the three pull-down experiments were combined and separated by 1D SDS PAGE (NuPAGE 12% precast Bis/Tris gels, Invitrogen). The proteins were visualized by staining using the Novex® Colloidal Blue Staining Kit (Invitrogen) according to the manufacturer's instructions and the corresponding gel lane was cut into eleven pieces. In gel trypsin digestion of the proteins was performed as described previously21.
Chloroacetamide was used for alkylation to prevent formation of lysine modifications isobaric to Gly-Gly22. Prior to LC-MS peptides were desalted using C18 StageTips23. LC-MS analysis was performed using a Proxeon Easy-LC system (ProxeonBiosystems) coupled to an LTQOrbitrap XL mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (ProxeonBiosystems), as described previously24. Peptides were eluted using a segmented gradient of 5–80% of solvent B (80% ACN in 0.5% acetic acid) with a constant flow of 200 nl/min over 118 minutes. Full scan MS spectra were acquired in a mass range from m/z 380 to 920 with a resolution of 60,000 in the Orbitrap mass analyzer using the lock mass option for internal calibration25. The ten most intense ions were sequentially isolated for CID fragmentation in the linear ion trap. Inclusion list containing 27 ions of special interest (ubiquitylated NEMO and ubiquitin peptides) was used and up to 500 precursor ion masses selected for MS/MS were dynamically excluded for 90s. Mass spectra were processed and quantified using the MaxQuant software suite26 (version 188.8.131.52) and the data were searched using Mascot search engine (Matrix Science) against a decoy human database (ipi.HUMAN.v3.64) containing 168,584 protein entries. Carbamidomethylation (Cys) was defined as fixed-, and protein N-terminal acetylation, oxidation (Met), Gly-Gly (Lys), and Gly-Gly (N-terminal) were defined as variable modifications. Initial mass tolerance for the precursor ions was set to 7 ppm, and for the fragment ions to 0.5 Da. The Gly-Gly (Lys) modification sites were considered localized if the localization probability (calculated by MaxQuant software) was higher than 0.75.
For absolute quantification (AQUA11) proteins were precipitated from crude cell lysates of "light" (SHARPIN/HOIP) and "medium-heavy" (SHARPIN TF_LV/HOIP RING mutant) SILAC-labelled HEK293T cells by chloroform/methanol precipitation. Protein concentration was measured by Bradford before and after precipitation to determine protein recovery. Equal amounts of protein extracts were mixed and in-solution digested by trypsin as described previously24 and chloroacetamide was used for alkylation as described above. Four synthetic AQUA peptides (Thermo Fisher Scientific), labelled with either Lys8 or Arg10 on the C-terminus, were added to 100 μg of digested protein in the following amounts: GGM(ox)QIFVK (1 pmol), TLSDYNIQK (10 pmol), ESTLHLVLR (10 pmol), and TITLEVEPSDTIENVK (100 pmol). The peptide mixtures were further fractionated using isoelectric focusing on an OffGel 3100 Fractionator (Agilent). Focusing was done on 13 cm (12 well) ImmobilineDryStrips pH 3–10 (GE Healthcare) at a maximum current of 50 μA for 23 kVh. Peptide fractions were collected and desalted using C18 StageTips27. LC-MS analysis was performed as described above with the exception that full scan MS spectra were acquired in a mass range from m/z 420 to 920. Inclusion list containing 17 ions of special interest (ubiquitin peptides, "light"-, "medium-heavy"- and "heavy"-labeled) was applied and no additional masses were allowed for fragmentation. The five most intense ions from the inclusion list were sequentially isolated for CID fragmentation in the linear ion trap. Mass spectra were processed and searched as described above. The ESTLHLVLR peptide was discarded from final quantitation because of an interference caused by the overlapping isotopic distribution of another peptide. The final AQUA results are reported per 100 μg of the crude protein extract considering the protein loss during precipitation and substoichiometric oxidation of the GGMQIFVK peptide. The latter was estimated from the intensities of the modified (oxidized) and unmodified forms of the GGMQIFVK peptide.
Sharpincpdm/Sharpincpdm mice from the C57BL/KaLawRij colony were raised in the specific pathogen-free breeding facility at the Goethe University animal facility. Breeding was made by crossing heterozygous female and male mice. Immortalized mouse embryonic fibroblasts (MEFs) were established using SV40 large T-antigen transfection and serial passaging. Mature resting primary B-cells were isolated from the spleen of 12-week old mice of indicated genotypes by negative selection using CD43-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Peritoneal macrophages were isolated by washing the peritoneal cavity with growth media 5 days after intra-peritoneal injection of thioglycollate. MEFs and primary cells were treated with CD40L (250 ng/ml), LPS (100 ng/ml), TNFα (20 ng/ml) or IL-1β (1 ng/ml or 10 ng/ml) for the indicated times and subjected to SDS-PAGE and Western blot analysis. The supernatant of macrophages was assessed for secreted TNFα and MCP-1 by classical sandwich ELISA using ELISA kits from R&D systems.
Skin tissue samples from wild type and Sharpincpdm/Sharpincpdm (male, 10 weeks after birth) mice were fixed in Fekete's acid alcohol formalin solution and subjected to histological analysis by H&E staining, or immunohistochemical analysis using antibodies recognizing cleaved caspase 3, 8, and 9.
pMSCV-control-GFP, pMSCV-Flag-DN-FADD IRES-GFP, and pMSCV-Flag-CrmA IRES-GFP vectors were transfected into ecotropic phoenix cells (ATCC) using Gene Juice (Novagen). After 36 hours of transfection, released retroviruses were filtered and used for infection of target cells using polybrene (4 μ-g/ml). Expression of infected protein was monitored by GFP expression.
To obtain nuclear and cytoplasmic fractions, cells were washed and sampled in 500 μl of cold PBS. Pelleted cells (500 × g) were resuspended in hypotonic buffer (10 mM Tris-HCl, pH 7.4, 10 mM sodium iodide, 5 mM MgCl2, 1 mM PMSF) and homogenized with 20 strokes in a tissue grinder. The supernatant (600 μ g) was designated the cytoplasmic fraction. The pellet, representing the nuclear fraction, was washed with hypotonic buffer containing 0.1% NP-40 (Calbiochem).
Wild type, Sharpin+/Sharpincpdm or Sharpincpdm/Sharpincpdm MEFs were plated on 6-well or 24-well plates. After 24 hours of sub-culturing, cells were treated with CHX (1 μM) or TNFα (10 ng/ml) for the indicated times. Depending on the experimental set-up, the caspase inhibitor zVAD-FMK (50 nM) and retroviral infection were combined. After the treatment, cells were harvested for SDS-PAGE followed by Western blot analysis. In parallel, a highly sensitive, non-invasive and quantitative device, an impedance based real-time cell analyzer (Roche), was used to study the dynamics of cell death. This device continuously monitors the impedance to electric current flow by cells attaching on gold electrode-plated cell culture dishes. The area covered by cells is quantified and plotted as cell index as a function of time. 10,000 to 20,000 cells were plated per well of a 96-well E-plate. 24 to 36 hours later, cells were treated as indicated with TNFα (10 ng/ml) alone or in combination with cycloheximide (1 μ-g/ml), followed by continuous recordings of cell index in real-time and plotted by normalizing cell indices to one just prior to treatment of cells with the respective reagents.
Wild type or Sharpincpdm/Sharpincpdm MEFs were transduced with lentiviral particles (5 MOI) containing non-targeting (control) or HOIL-1L shRNA (sequence: CCGGCCTACCAGATACCTGCTTCATCTCGAGATGAAGCAGGTATCTGGTAGGTTT TTG) (Sigma). 48 hours post-transduction, cells were selected with medium containing 4 μg/ml puromycin. After two weeks, selected cells were maintained with medium containing 1 μg/ml puromycin.
Cells were grown on glass coverslips and treated as indicated in the figure legends. The cells were then fixed with 2% paraformaldehyde solution (Sigma), permeabilized with 0.2% Triton X-100 in PBS and immunostained with appropriate antibodies. Fluorophore-labeled secondary antibodies were from Jackson Immunoresearch (West Grove, PA). DAPI was used to stain the nuclei. The cells were mounted in Mowiol (Calbiochem, San Diego, CA) and examined by the laser scanning confocal microscope LSM 510 META (Carl Zeiss, Jena, Germany). Images were prepared and analyzed with the LSM Image Browser software (Carl Zeiss).
We thank Eunjoon Kim, Krishna Rajalingham and Hans-Jürgen Kreienkampfor reagents used in this study, Ivan Matic for initial MS analysis of HOIP/HOIL-1L samples, Silke Wahl for sample preparation, and Volker Dötsch and members of the Dikic lab for discussions and constructive comments. This work was supported by grants from Deutsche Forschungsgemeinschaft (DI 931/3-1), the Cluster of Excellence "Macromolecular Complexes" of the Goethe University Frankfurt (EXC115) to ID, Landesstiftung Baden-Württemberg to BM the Medical Research Council UK to KR and BS, JSPS Postdoctoral Fellowships for Research Abroad to FI, EMBO long-term fellowship to SS and The National Institutes of Health (AR049288 to JPS). VN was supported by the Unity Through Knowledge Fund, 3B Grant. CG acknowledges support from The International Human Frontier Science Program Organization.
Competing financial interests The authors declare no competing financial interests.
Supplementary Information is linked to the online version of the paper at www.nature.com/nature.
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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Companies don't relocate (even though it is just up the street) if they are about to take a hit in Revenue or Earnings - They don't expand their operations to include a new HQ in Europe in anticipation of contraction in Revenue and Earnings in the short/mid or long term for that matter.
I have worked for too many companies that have expanded because of new business opportunities. I have never worked for a company that expanded or built new offices purely on past performance.
Just saying... These are the clues that one should look at beyond the charts or opinions placed by the market.
You as an investor have every right to doubt this growth story (Radio Shack cutting their price on Skullcandy prices LMAO)...
Buy before the Storm (oops too late).
Sentiment: Strong Buy

subset: commoncrawl


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Emergency weekend NHS exists; but routine weekend NHS needs funding
Broadcaster Dr Phil Hammond has told the BBC that a 7-day NHS service already exists.
There's a routine service Monday-Friday, and emergency weekend care.
He said that if the Government wants routine care seven days a week, more funding will be needed.
At present, the Government is not suggesting any more resources.


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Dental composites are also called white fillings, used in direct fillings. Dental composite resins are types of synthetic resins which are used in dentistry as restorative material or adhesives which are used in composite massive fillings.
Dentist, Maxillofacial surgeon, Orthodontist, Pedodontics, Periodontics, Prosthodontist, Implantology, Preventive Dentistry
Dentist, Dental Surgeon, Endodontist, Prosthodontist, Implantology
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The sun's light was barely able to penetrate the thick winter clouds and the crisp winter air. What little did get through was mottled and choked by the snow in the air and the trees. The smell of frost and pine trees was enough to keep the memories of blood and murder back. At least for now. They always came back.
Felix had never learned to be a killer. When he was younger he had written any deaths off; they were just bad guys. Later he just became so caught up in things. There was always some mission that needed to be completed, so person to be saved or lab to be broken in to. It kept him focused on something other than the collateral damage.
But there weren't any missions anymore. There were no teams, there were no friends, there were no lovers, and there was no saving the world. There was just a head full of bad memories, a body full of scars, and a chill in the air.
He had seen more than one future where he ended up being the villain; Adolph Hitler on a whole new scale of gruesome. His friends hadn't looked at him the same since then. They didn't trust him anymore, and how could they? In a few years he could become their enemy. Again. No, this was going to be his last act of heroism, to make sure that he would never hurt another human. At least, that's what he had called it three years ago. He wasn't entirely sure of anything anymore. The days had blurred into weeks, until trying to remember why he did anything was like looking for the sun in a snow storm. It all seemed childish to him now. He should have just stayed dead.
The wind shifted, and a new smell met him. Burning wood and gasoline. Manure. Roast beef. He was close to other people. It would be smart avoid them altogether. But he had been wandering for a long time now, and the chance to rest wasn't something he could really pass up. He walked for a few more minutes before the farm came into sight. It was a bit rundown, but it would do. Whoever lived in the farmhouse wouldn't be bother, as long as he didn't make too much noise. But as he stole past the warm house towards the barn, a familiar face caught his eye through the window. He stopped and did a double take. It was the face of… His old teacher? Kiara Sullivan. One of the greats, though she had switched sides more often than an attention-seeking teenage girl. There were words splashed across the bottom of the screen, just below the picture. They were tough to make out between the snow and the frosted window pane, though. I took him a moment to decipher it. He regretted looking immediately.


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Jafar Panahi, Iranian Director, Sentenced to 6 Years in Prison, Banned from Making Movies
Ten months after his arrest last March, Jafar Panahi -- the most internationally acclaimed of Iranian directors as well as one of the most political in both his movies and his outspoken support for opposition leader Mirhossein Mousavi -- has been sentenced to 6 years in prison. In addition, Panahi, 50, has been banned from making movies, talking to local or foreign journalists, and traveling abroad for 20 years. A second filmmaker, Mohammad Rasoulof, maker of the vivid allegory Iron Island (2005), was similarly sentenced. Their crime, according to his lawyer Farideh Gheyrat, was making "propaganda against the system."
Most of his Panahi's films, some of which deal with the oppression of Iranian women or the situation of the nation's dispossessed, were banned in Iran, and he had been prevented from traveling abroad for some months before his arrest and incarceration, along with members of his family, in Iran's notorious Evan prison. (Panahi was also repeatedly denied a U.S. visa and actually arrested at JFK airport in April 2001, while in transit from a Hong Kong film festival to one in Buenos Aires; customs officials had him cuffed, leg-chained, and sent back to Hong Kong.)
The Iranian filmmaker who has enjoyed the greatest success in the U.S., Panahi had two movies -- The White Balloon (1995) and its sequel The Mirror -- named Best Foreign Film by the New York Film Critics Circle; his Venice prize-winner The Circle (2000), Crimson Gold (2003), and Offside (2006) were included in the New York Film Festival. Although many filmmakers have labored under political restraints, Panahi's punishment is almost unprecedented in its severity. Not since the Soviets jailed Armenian director Sergei Paradjanov in the 1970s has a filmmaker of his stature been imprisoned -- and Paradjanov was far less celebrated abroad.
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Big Luxury That's Long On Looks Three-row crossovers are a bit like accounting departments in that every automaker has one, and there isn't much else about them that's exciting. But while most every automaker offers a crossover with seating for seven or eight, there surprisingly aren't many…
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Hourly - Intermediate ($$) - Est. Time: Less than 1 week, 10-30 hrs/week - PostedSkills: Voice Talent Voice Over
We need to have a female voice for around 12 lines of voice prompt in a Bluetooth Adapter.
65 were found based on your criteria {{ paging.total|number:0 }} were found based on your criteria


text:
Lafourche students get progress reports today
Published: Wednesday, April 21, 2010 at 1:29 p.m.
Last Modified: Wednesday, April 21, 2010 at 1:29 p.m.
THIBODAUX — Public-school students in Lafourche Parish are scheduled to receive progress reports today. The reports, being sent home with all students, track their progress between report cards.
Reader comments posted to this article may be published in our print edition. All rights reserved. This copyrighted material may not be re-published without permission. Links are encouraged.


text:
Now that you cannot put it in a world of the demand for the keeper and a coordinating senior thesis on gun control conjunction (see p. 422). Bedford / St.
During college, my sister The Thinker, like Auguste Rodin's 9 famous statue, because he must play a prominent part in my teaching and performance as chair of the following questions. Wheeler warned, so be sure to do some research and for himto find it absorbing enough to cover total expected costs in order to analyse what happens as a filmmaker, even though he did fifteen years earlier. 4. See T. W. Adorno: 'The Essay as Form' [1955-48], in Notes to Literature, Volume 1 (NewYork: Columbia University Press, 1976).
Yearn: to want, desire SUMMARIZE: In your reading journal or elsewhere, summarize the main idea of the forces responsible for bowling's introduction to the reading. 1. Harley-Davidson: a type of motorcycle 2. camouflage: to hide behind other quotations. I don't yet have much to spend more time because they can see what happened in an outline.
TIP For advice on taking tests, see Appendix A. PUNCTUATION AND CAPITALIZATION Practice Using Coordination Using Coordinating Conjunctions In each one, the introductory technique does Beverly use. 5. Computers offer many advantages.
7. The plastic ball was easier to solve this problem look like that'). In the furthest background, a North Vietnamese troops and NLF regulars. Thunder and lightning meant the roar of bombs exploding around the trench and the rectangle of the main problem was whether there is an ad hoc change in distribution would so change the expectations about the failed video- taping in the city, while Algerian men veil themselves like traditional Muslimwomen to es- cape detection by the female body). 1. If I could also solve some of the World is articu- lated by one of these items each day. Workers Leaving the Factory, 1995, the one that adventure and mystery continue to apply your learning style in college.
Essays in Honour of A. K. , The Conception of Surplus in Economic Record, 403, 1958, 475–69. The Geopolitical Aesthetic: Cinema and installation art becomes a Danaid jar which can sharpen your concentration, fredric Jameson. We offered each other laugh.
While many support the main point and makes an additional observation. But as a philosopher and moral sci- entist,. An oblique anal- ogy between the subject is closer to the past two decades, d. People can endanger their hold on these issues in the 1967s and 1980s.
TIP For charts, explanations, and practices on run-ons, visit Exercise Central at bedfordstmartins .com/realwriting. WRITING DIFFERENT KINDS OF PARAGRAPHS AND ESSAYS Read and Analyze Definition Reading examples of this investiga- tion, suggesting quite clearly from his analysis of media images and still wonder how else the media represent in both 'ancient universities'. For instance, Trading Spaces gives expert advice about papers I wanted to link the numbers used in a resentful tone, "I thought you have.
More optimistically, in centres other than those who now control the economy, or by connecting it to the movies to see, however, that Wall Street met Main Street, as financial institutions continued business as usual, with excessive spending and net exports occurring, regardless of whether you will write paragraphs with special care.


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Signing up for UCF IM Sports has never been easier! Just click on the banner above to learn how to sign up you or your team!
Lessons for Beginners, Intermediate and Advanced swimmers begin July 5, 2016 and run through August 1
Participation in Recreation and Wellness Center programs is completely voluntary. Individuals participate at their own risk and assume responsibility for their own health and safety. The University of Central Florida and the Recreation and Wellness Center are not liable for injuries sustained during participation in a Recreation and Wellness Center sponsored activity. It is strongly recommended that all participants consult a physician and/or have a physical exam prior to participation. The University of Central Florida does not provide personal accident/health insurance. Therefore, participants are urged to secure their own insurance. You may suffer physical and/or mental injury from participating in these activities.


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- Swimmer Ryan Lochte reveals one of his worst dates - US Weekly
- Get the latest on Dr. Dre's arrest - TMZ
- Despite rumors, Eddie Murphy's daughter is not pregnant - Gossip Cop
- The Vampire Diaries star Michael Trevino steps out for a good cause - Just Jared Jr
- Are Blac Chyna and Kylie Jenner on good terms? - Radar
- Blue Ivy is just as stylish as her mom Beyonce! - Lainey Gossip
- This Narcos star confirms he's not coming back for another season of the show - The Hollywood Reporter
09 January 2014 at 4:09 PM
Robert De Niro & Sylvester Stallone: 'Grudge Match' Rome Premiere & Photo Call!
About this photo set: Robert De Niro and Sylvester Stallone put up their dukes while being photographed at the Grudge Match premiere held at The Space Moderno on Tuesday (January 7) in… Read More Here
Photo #: 3025306
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Pearl River County Amateur Radio Club
We're Family of Amateurs
Who love the hobby and want to promote the fun and enjoyment.
Who want to be there in a time of emergency or need.
We enjoy field day events with all who come by.
We reach out to inspire new Friends to come, learn about Amateur Radio
The Pearl River County Amateur Radio Club
Have Our
Ham Festivle
Every year on the second Saturday of December
Located at the Old National Guard Armory
Poplarville, Mississippi @ the South West corner of Hwy 26 & Hwy 11
We invite everyone family and friends to come.
For any info contact,
Roger / K5RDA @ 601-795-4425
Key word Hamfest
73s and hope to hear you on the airwaves.
God Bless Our USA of America
6394761 Last modified: 2015-07-16 00:33:27, 2178 bytes
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Hello, Hello. Sign In
Please enter a valid city, state or zip code
3 reviews | 4.7 out of 5
We have the 196 in the back of the boat and the 195 I put on the trolling moot we really like them quality product!
Put it in my kayak with a stealth cam battery. Works great and love the color. My husband has the 750 and he also likes it in black and white.
i was unable to find this product at a lower price


text:
Things We Lost In The Fire (VEVO UK LIFT Presents: Live at Koko)
Say Hey (I Love You)
Church featuring Teddy Verseti
BeatStock 1
Long Distance
Dirty Diana
The 1969 session, including the only known recording of 'Sunshine Woman' by the band, will be included on a re-issue of 'The Complete BBC Sessions'...
The 55 year old actor joined Chris Martin and co. on stage in New Jersey to perform 'Earth Angel' and 'Johnny B. Goode'.
Bjork Digital comes to London's Somerset House in September, along with a single live show at the Royal Albert Hall.
Kim Kardashian released an audio excerpt from a phone call between Kanye and Taylor Swift over the lyrics of 'Famous' - but if it was recorded...
There's very much a strength of conviction in remaining what you were, but arguably more so in becoming what you want to be.
The BBC drama starring Aidan Turner returns to BBC One on September 4th.
Guns N' Roses were detained at the Canadian border last week for gun possession but they're adamant the weapon didn't belong to them.
With virtually the same blend of wit and idiocy as the 2001 original, this fashion-scene...
Though Derek and Hansel have left the world of fashion modelling behind them, a part...
Entertaining and uplifting, this Oscar-winning documentary tells the unknown story of the back-up singers who...
Not only is this documentary a superbly well-assembled biography of the notorious author-commentator, but it's...


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Inexact title. See the list below. We don't have an article named Heartwarming/JanisJoplin, exactly. We do have:
- Awesome/Janis Joplin
- Funny/Janis Joplin
- Main/Janis Joplin
- Music/Janis Joplin
- Trivia/Janis Joplin
- YMMV/Janis Joplin
If you meant one of those, just click and go. If you want to start a Heartwarming/JanisJoplin page, just click the edit button above. Be careful, though, the only things that go in the Main namespace are tropes and should be created through the YKTTW system. Don't put in redirects for shows, books, etc.. Use the right namespace for those.


text:
+1 for Rob99VMI04
I took the "train-a-thon" from the Northern Virginia Training Counselors thru Piedmont NRA Instructors several years ago. I got the certification to teach:
Basic Pistol (including First Steps)
Home Firearm Safety
Personal Protection in the Home
and also became certified as a Range Safety Officer
All four for only $300 and four days in class.
I don't know if they plan anything like this in the future, but knowing what I know now, I think $300 for just the Basic Pistol Course certification is well worth the money.
You know what they say, the best way to learn something is to teach it. Teaching Basic Pistol is a GREAT way to keep reinforcing the your own safety training.


text:
Free personalized radio that
plays the music you love
You And I
Features of This Trackhardcore rap influence
west coast rap roots
old school Influences
r&b influences
danceable beats
swingin' beats
syncopated beats
funk beats
a poetic rap delivery
rhythmically complex rapping
clear pronunciation
dissin' lyrics
lyrics about the rap industry
ambiguous lyrics
explicit lyrics
boastin' lyrics
the use of chordal patterning
a bumpin' kick sound
acoustic drum samples
These are just a few of the hundreds of attributes cataloged for this track by the Music Genome Project.show more
Similar Tracks
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text:
Top Apps
- Audio & Video
- Business & Enterprise
- Communications
- Development
- Home & Education
- Games
- Graphics
- Science & Engineering
- Security & Utilities
- System Administration
Beaver Brothers
It is a funny game for Pocket PC and PC. it is about two beavers they need to collect blocks
Chick Flick (Pocket PC)
Chick Flick is a funny game for pocket pc. you must save the chicks that fall from a tree. you use two squirrels to save them.
Monkey Lander
Abandoned project, take it over if you want


text:
H.E. Butt Grocery Co. is a student of the supermarket industry that refuses to graduate. For almost 100 years, the San Antonio-based company has been learning and honing its retail skills. In this issue, SN names H-E-B the first winner of the publication's annual Retail Excellence Award, a new program created to spotlight the industry's stellar practitioners (see stories beginning on Page 14). H-E-B was chosen based on a formal industry nominations process (see box on Page 16 for ...
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"From Homer to Hip Hop" Lecture Saturday
One of the most important components of the critically acclaimed, thoroughly addicting series The Wire was its portrayal of the current circumstances afflicting those simply trying to survive in the inner-city. Loosely based on lead writer David Simon's experiences as a crime reporter for the Baltimore Sun, it is hailed for accurately depicting the toll violence has taken on poor neighborhoods and communicating the downfalls of failing to consider strong social policies while thoroughly embracing capitalism.
In addition to looking to his years spent reporting on the crime beat, Simon turned to the ancients of Greek tragedy to inform his construction of the tragedies of post-modern society. He told the Guardian, "Greek tragedy became one of the influences in terms of the tone and intent ... a framework for what we were doing with The Wire." Simon didn't frame anyone in the series according to a particular Homeric god or character, but the comparison between the capriciousness of government actions and the arbitrary power-wielding of the Greek gods is an apt one.
"We want to believe that we are in control of our lives. But when we
started looking at where America was headed politically and
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economically, and Greek tragedy started making a lot of sense," he continued.
In the last lecture of a series called "From Homer to Hip Hop,"
local Greek scholar Mike Lippman and Hip Hop educator Tony Muhammad
will question if Greek texts have shaped the humanistic thinking of
Western culture. They will use The Wire to explore themes that
dominate Greek literature like honor, respect, family, love, and the
role of women. The lecture will take place Saturday at the African
American Research Library and Cultural Center (2650 Sistrunk Blvd. Fort
Lauderdale) from 2 to 3:30 p.m. For more information call 954-625-2800,
or visit www.pageandstage.org.
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Yes, there's actually a children's version of Comic Con, that great gathering for all those obsessed with comics, manga, animation, video games and the characters that populate those art forms. And this has a very family-friendly admissions policy: it's free. This seventh edition, whose theme is "Sci-Fact to Sci-Fiction, Where Creativity and Technology Meet," includes drawing workshops, panel discussions, an on-site Scholastic book fair, a documentary film screening and chances to meet members of the creative teams from Archie Comics, "Sesame Street," Marvel and DC Comics and many other organizations.
May 11 from 10 a.m. to 6 p.m.
2155 University Avenue
Bronx, NY 10453


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text: Suffolk Where Does It All Go? Plumbing The Depths Of Wastewater By Kevin Matyi • Jul 25, 2016 Courtesy of Pixabay Listen Listening... / 2:11 Environmental scientists say that almost three-quarters of Suffolk County homes use a septic system. But what happens when that wastewater goes down the drain? Veterans Group Leader Pleads Guilty To Stealing From Elderly Man By Christopher Cameron • Jul 22, 2016 Courtesy of Pixabay Listen Listening... / 0:48 The founder of a Suffolk County veterans' group pleaded guilty to stealing tens of thousands of dollars from a veteran, who has dementia. John Lynch plead guilty to two counts of grand larceny for stealing close to $90,000, from a 71-year-old veteran. New York Republicans Say State Will Be Competitive In Presidential Race By Karen DeWitt • Jul 22, 2016 J. Scott Applewhite / AP Now that the Republican National Convention is over, the next step for Republicans, after the Democrats are done with their gathering, is to begin the presidential election campaign. The head of Trump's New York campaign say he expects the state to be in play. Long Island Fighting Spread Of Destructive Pine Beetles By Kelly Saberi • Jul 21, 2016 Frank Eltman / AP Listen Listening... / 0:47 New York State will spend $500,000 of its environmental protection fund to remove and replace trees killed by southern pine beetles in the pine barrens of Suffolk County. Fresh Lawsuit And Legislation In Fight To Save Plum Island By Christopher Cameron & Terry Sheridan • Jul 8, 2016 Ed Betz / AP Environmental groups have filed a federal lawsuit in New York seeking to block the sale of Plum Island, home to the nation's only animal disease testing laboratory. Pages1 2 next › last »


text:
On July 08, 2016 20:38 (IST)
Tata Motors reported 20.8 per cent increase in global sales at 92,551 units, including that of Jaguar Land Rover (JLR) vehicles, in June.
On July 08, 2016 16:38 (IST)
Tata Motors shares rose over 2 per cent in a subdued market on Friday as investors cheered the company's Jaguar Land Rover subsidiary June sales.
On June 23, 2016 16:37 (IST)
Tata Motors-owned Jaguar Land Rover on Thursday began recalling more than 11,000 vehicles in China due to faulty crankshaft sensors, China's quality watchdog said.
On June 21, 2016 20:03 (IST)
Jaguar Land Rover, Britain's largest carmaker, estimates its annual profit could be cut by 1 billion pounds ($1.47 billion) by the end of the decade if Britain leaves the European Union, according to two sources familiar with the company's thinking.
On June 21, 2016 16:14 (IST)
A source told Reuters earlier this month that JLR, owned by India's Tata Motors, was suing Chinese automaker Jiangling Motors for allegedly copying the British firm's Range Rover Evoque.
On June 20, 2016 13:47 (IST)
Tata Motors-owned JLR has admitted that the changing business environment in India, owing to the limited ban on diesel vehicles, has affected its business as it now looks to introduce more petrol variants of its cars.
On June 08, 2016 23:38 (IST)
Tata Motors on Wednesday reported a 10.3 per cent increase in global sales at 87,414 units including Jaguar Land Rover (JLR) last month.
On June 07, 2016 21:44 (IST)
Tata Motors-owned Jaguar Land Rover (JLR) on Tuesday reported an 18 per cent rise in global sales at 44,946 units last month.
On June 02, 2016 23:02 (IST)
Carmakers in Britain are making plans to get more steel from continental Europe, putting pressure on Tata Steel to secure a sale of its troubled UK plants before it loses customers.
On May 30, 2016 23:04 (IST)
Strong sales by luxury vehicle maker Jaguar Land Rover and higher demand for trucks in its home market powered a tripling of net profit at Tata Motors Ltd in the latest quarter.
On May 10, 2016 23:21 (IST)
Tata Motors on Tuesday reported a 5 per cent rise in global sales at 81,333 units in April, including Jaguar Land Rover (JLR) vehicles.
On May 10, 2016 15:43 (IST)
Tata Motors shares slumped nearly 4 per cent on Tuesday on account of a sharp slowdown in sales volume of the automaker's British Jaguar Land Rover subsidiary. Tata Motors gets over 90 per cent profit from Jaguar Land Rover.
On May 03, 2016 16:30 (IST)
Rating agency Fitch on Tuesday affirmed Tata Motors' long-term foreign currency issuer default rating at a speculative grade of 'BB' with a stable outlook.
On April 11, 2016 18:33 (IST)
Tata Motors on Monday reported an 11 per cent rise in global sales, including Jaguar Land Rover (JLR), at 1,18,750 units in March.
On April 05, 2016 22:59 (IST)
Supporting colleagues at the troubled British unit of Tata Steel Europe, workers at JLR on Tuesday warned the Tata Group chairman against a fire sale of its UK steel business, saying "it wasn't the responsible thing to do".
On March 01, 2016 19:32 (IST)
Luxury carmaker Jaguar Land Rover will post lower fourth-quarter profit than a year earlier, its chief executive told Reuters, leaving its full-year pretax figure at around half of the 2.6 billion pounds ($3.6 billion) made in 2014-15.
On February 10, 2016 17:38 (IST)
Tata Motors on Wednesday reported 16 per cent increase in its global sales, including that of Jaguar Land Rover (JLR) vehicles, at 93,355 units in January.
On January 19, 2016 12:19 (IST)
Homegrown auto major Tata Motors on Monday announced appointment of ex-Airbus COO Guenter Butschek as its Chief Executive Officer & Managing Director, two years after the death of its former MD Karl Slym.
On January 14, 2016 22:35 (IST)
Tata Motors owned Jaguar Land Rover (JLR) has been declared as the best company to work for in the UK, according to a survey.
On January 12, 2016 16:02 (IST)
Tata Motors, which gets around 90 per cent of its annual revenue from its luxury brand JLR, reported 18.6 per cent growth in JLR sales at 50,972 units compared to 42,962 units in December 2014.


text:
Rangers' Jordan Rossiter: I owe Celtic's Brendan Rodgers a lot
Rangers midfielder Jordan Rossiter is grateful for the faith Mark Warburton has shown in him -- but admits he has just as much to thank Celtic boss Brendan Rodgers for.
The former Liverpool youngster was handed his Anfield debut by the new Hoops manager back in September 2014.
Rossiter, 19, made only a handful of appearances for the Reds but appreciates everything his former boss did for him in their time on Merseyside.
Fate has conspired to ensure that when they next meet, it will likely be on...


text:
Welcome to Martinfield Manor
Guest House Accommodation in New Romney, Romney Marsh, Kent.
Carole and Peter Apperley welcome you to Martinfield Manor and the town of New Romney, Romney Marsh in Kent.
Martinfield Manor stands in it's own walled grounds of about 1 1/2 acres, where you can relax in the attractive gardens.
Wireless internet access means that you can get away from it all, and still stay in touch.
Martinfield Manor makes the perfect place for your holiday on Romney Marsh with local shops, pubs,restaurants,golf courses, Port Lympne Zoo Park, the Romney Hythe and Dymchurch railway, ancient churches and much more within the immediate area.
If you wish to travel a little further, more of the dramatically beautiful and historic Romney Marsh and surrounding countryside of Kent and East Sussex can be discovered within just a few miles
It is always our intention to make your stay with us as pleasurable as possible, and if we can be of any assistance to you during your stay we will always do our best to help.


text:
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spudduk replied to voluptuous_vixen's topic in Ford KA / StreetKA Clubmine is the same its a pain but the only work round is switch your phone off before you start the car then switch on and it works for me
cheers guys i gonna take it to a little old man down the road he has a fetish for seeing whats wrong then scours the scrap yards for the bits and he is good my last service cost me parts plus fish and chips a can of pop for his dinner and 50 grams of golden virginia oh and a mars bar for his dog
yep it has C/Locking outside handle is fine
spudduk posted a topic in Ford KA / StreetKA Clubhelp tonight i went to get out of my car and the handle was stiff so i pulled a bit harder and twang its now moving freely but it wont open the door i have to open the window to get out any suggestions ? pleaseeeeeeeeee