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The Vpr protein of HIV-1 functions as a vital accessory gene by regulating various cellular functions, including cell differentiation, apoptosis, nuclear factor of κB (NF-κB) suppression and cell-cycle arrest of the host cell. Several reports have indicated that Vpr complexes with the glucocorticoid receptor (GR), but it remains unclear whether the GR pathway is required for Vpr to function1. Here, we report that Vpr uses the GR pathway as a recruitment vehicle for the NF-κB co-activating protein, poly(ADP-ribose) polymerase-1 (PARP-1). The GR interaction with Vpr is both necessary and sufficient to facilitate this interaction by potentiating the formation of a Vpr–GR–PARP-1 complex. The recruitment of PARP-1 by the Vpr–GR complex prevents its nuclear localization, which is necessary for Vpr to suppress NF-κB. The association of GR with PARP-1 is not observed with steroid (glucocorticoid) treatment, indicating that the GR association with PARP-1 is a gain of function that is solely attributed to HIV-1 Vpr. These data provide important insights into Vpr biology and its role in HIV pathogenesis.
A trademark of HIV infection is the diminution of the CD4+ T-cell count of the host, which invariably leads to eventual immunodeficiency2. It is believed that various viral factors contribute to this effect by suppressing both immune activation and T-cell expansion3–6. The 96-amino-acid viral protein R (Vpr), which has a relative molecular mass of 14,000, has been implicated in both the destruction and suppression of potential antigen-specific T cells through multiple mechanisms7. In fact, Vpr is sufficient to suppress mitogen or anti-CD3-dependent proliferation and activation of T cells. Additionally, Vpr is present in the serum of infected patients and can efficiently reactivate viruses from latency8,9. Furthermore, Vpr possesses intrinsic transduction properties, which indicates that there are various viral-induced pathogenesis events that occur within a non-viral infection setting10. Other reported important activities include host cell-cycle arrest at the G2/M stage, nuclear transport of the pre-integration complex, host-cell apoptosis, nuclear herniations and the induction of immune suppression11–20.
Glucocorticoid receptor II (GR-II) has been identified as an in vivo target for Vpr12,20–22. The Vpr–GR interaction is dependent on the signature LXXLL motif, the abrogation of which attenuates the GR-dependent co-activation and transcription that is induced by Vpr. In addition, co-treatment with the GR antagonist mifpristone (Mif) blocks several pathogenic functions of Vpr, including apoptosis and viral transcription12,17,19. However, the mechanism behind nuclear factor of κB (NF-κB) suppression by Vpr currently remains unresolved. Furthermore, the functional deviations between Vpr and glucocorticoid treatments indicates that different mechanisms may occur.
In an effort to understand the role of the GR in Vpr-mediated NF-κB suppression, we compared NF-κB-dependent transcriptional activation in cells with a functional GR and in CV-1 cells, a monkey kidney cell line that expresses an endogenous GR, but is refractory of function23. As shown in Fig. 1a, co-transfection of Vpr but not of a control vector into HeLa cells, is sufficient to inhibit tumour necrosis factor-α (TNF-α)-induced NF-κB transcription. The inhibition was also observed in cells prone to HIV-1 infection, including Jurkat T cells, U937 monocytes and primary peripheral blood leukocyte (PBL) cells and macrophages (Fig. 1c–f). More interestingly, the same inhibitory effect was also observed in CV-1 cells that possess a non-functional GR (Fig. 1b), indicating that GR-mediated transcription is not required for NF-κB suppression, contrary to previous reports that suggested that GR activation leads to an upregulation of inhibitory I-κB12. This was further verified, as shown by the fact that inhibition of de novo protein synthesis via cycloheximide treatment did not attenuate Vpr-mediated NF-κB-dependent transcription (Fig. 1g). Vpr treatment was also accompanied by a reduced nuclear duration of RelA (p65) in both functional GR and non-functional GR cells (Fig. 1h). This result could be due to a failure of the formation of transcriptional complexes, which prevents acetylation and extended presence of RelA within the nucleus, as Vpr did not significantly affect its initial nuclear localization24. As upstream kinase inhibition could manifest the same effect, we next examined the activity of I-κB kinase-β (IKKβ). Vpr treatment did not affect the kinase activity of IKKβ (Fig. 1i) nor did it affect in vivo phosphorylation and turnover of I-κBα (Fig. 1j). However, Vpr potently attenuated the DNA-binding activity of NF-κB (RelA) at both the initial (Fig. 1k) and the later time points, and this effect was specific to RelA and not to other transcriptional factors. Last, co-transfection or Vpr treatment directly attenuated RelA-mediated transcriptional activation (see Supplementary Information, Fig. S1a–d). Taken together, these results indicate that Vpr suppresses NF-κB at the transcriptional level independently of concomitant de novo transcription induced by GR activation.
A hypothesis for this inhibition is that Vpr may destabilize the NF-κB transcriptional complex through a direct and/or indirect cross-talk mechanism. Interestingly, recent studies have indicated a role for PARP-1 in co-activating NF-κB-dependent gene expression25–27. Evidence indicates that PARP-1 is linked to the transcriptional potential of NF-κB through formation of a critical transcriptional complex within the nucleus26,27 and, consequently, Parp-1-deficient mice are refractory to toxin-induced sepsis. In view of this, we noticed that treatment of both HeLa and Jurkat cells leads to a dosage-dependent reduction in nuclear localization of PARP-1, as measured by biochemical and immunofluoresence methods (Fig. 2a, b). This effect was specific to PARP-1 and was not associated with other trafficking factors (such as eIF4E) and constitutive nuclear factors (such as PCNA) (Fig. 2c–e). The decrease in nuclear PARP-1 was concomitant with an increase in cytoplasmic PARP-1, indicating a defect in the nuclear or cytoplasmic shuttling of PARP-1 (Fig. 2d). Co-staining of Vpr–GFP (green fluorescent protein) with both PARP-1 and GR revealed significant colocalization within the cytoplasmic and perinuclear regions (see Supplementary Information, Fig. S2a), whereas GFP alone failed to affect PARP-1 nuclear localization.
Next, we hypothesized whether Vpr may be interacting with PARP-1 and, therefore, facilitating its failed nuclear localization. Therefore, plasmid (p) Vpr was transfected into CV-1, PBL cells and macrophages with appropriate controls; they were immunoprecipitated with either anti-Vpr or PARP-1 antibodies and were blotted for the other. As shown in Fig. 2f–h, Vpr was sufficient to co-immunoprecipitate with PARP-1, indicating that the two reside together in a complex in CV-1 and primary HIV-1 target cells. We then asked whether this requires a Vpr–GR interaction, which we have shown previously to be a cellular receptor for Vpr. As shown in Fig. 2i and Supplementary Information, Fig. S2b, the GR antagonist mifpristone is sufficient to reverse the cellular accumulation of PARP-1, indicating that the interaction between Vpr and GR is necessary for Vpr to affect PARP-1 localization. Not surprisingly, the interaction between Vpr and PARP-1 was also sensitive to mifpristone in PBL and HeLa cells (Fig. 2j, k), which indicates that the Vpr–GR complex may facilitate the interaction with PARP-1, and therefore regulate its localization. Paradoxically, steroid treatment (dexamethasone) alone is insufficient to relocate PARP-1 from the nucleus to the cytoplasm (Fig. 2i; and see Supplementary Information, Fig. S2b). In fact, recombinant protein (r) Vpr and dexamethasone exhibit competing effects on PARP-1 localization (see Supplementary Information, Fig. S2c). Therefore, although the Vpr interaction with GR was required for PARP-1 interaction, GR activation by steroids alone is insufficient, implying a molecular distinction between Vpr and steroid-receptor activation.
Next, we examined the molecular nature of the Vpr–PARP-1 complex. We in vitro translated PARP-1, Vpr and GR with 35S-Met and performed co-precipitation experiments. As shown in Fig. 3a–c, although Vpr directly interacts with GR as previously shown20, Vpr was unable to directly interact with PARP-1. In addition, as is consistent with dexamethasone experiments, GR is unable to interact with PARP-1. PARP-1 was only sufficient to complex with Vpr when it was complexed with GR (Fig. 3c). This was also observed in glutathione S-transferase (GST) pull-down assays and confirmed by western blot analysis of the pull-down (Fig. 3d, e). Taken together, these results indicate that the Vpr–GR interaction is necessary and sufficient to recruit PARP-1 in vitro.
To determine whether this can be extrapolated in vivo, we reduced the endogenous levels of GR via small interfering RNA (siRNA). Of the specific clones tested, GR-21 specifically and efficiently knocked down GR protein levels (Fig. 3f). Subsequently, transfection of Vpr into these cells failed to co-immunoprecipitate PARP-1 compared with siRNA control cells, indicating that Vpr requires GR to interact with PARP-1 in vivo (Fig. 3g). Biochemical fractionation of His-tagged Vpr through a nickel column and subsequent co-immunoprecipitation of PARP-1 or GR reveals that the three proteins exist in one complex rather than mutually exclusive complexes in vivo (Fig. 3h).
If Vpr recruitment of PARP-1 via GR interaction is, in fact, regulating transcriptional suppression, then prevention of either the Vpr interaction with GR or with PARP-1 should be sufficient to reverse the NF-κB suppression that is induced by Vpr. Accordingly, co-treatment of mifpristone, which abrogates the Vpr–GR interaction, was sufficient to significantly (P < 0.001) recover the transcriptional suppression of co-transfected RelA in HeLa and CV-1 cells, respectively (Fig. 4a, b). Similar results were also obtained in HIV-1 target cells, including Jurkat T cells and U937 monocytes (Fig. 4c, d). In a similar fashion, loss of GR function via siRNA also exhibited an inability for Vpr to suppress NF-κB, again indicating that GR is necessary for Vpr to inhibit NF-κB (Fig. 4e).
The role of PARP-1 in regulating NF-κB suppression seems to be entirely structural and not associated with its enzymatic properties, as DPQ (a nicotinic acid analogue) and a PARP-1 enzymatic inhibitor, did not significantly repress TNF-α-induced NF-κB transcription28. Meanwhile, small interfering RNA (siRNA)-mediated knockdown of PARP-1 had a more pronounced effect on NF-κB-dependent transcription relative to knockdown efficiency (see Supplementary Information, Fig. S3a–c). To determine whether recruitment of PARP-1 to the Vpr–GR complex was required for Vpr to suppress NF-κB, we screened previously described mutants29 for their ability to interact with GR and/or PARP-1. As shown in Fig. 4f, a point mutation of Vpr at H71 to tyrosine (Y) exhibited binding to GR but, importantly, failed to coprecipitate PARP-1. Consistently, transfection of this mutant into HeLa cells exhibited an obvious attenuation of cytoplasmic localization of PARP-1 compared with wild-type Vpr (Fig. 4g). Last, transfection of this mutant, but not others, exhibited a significant reduction in its ability to repress NF-κB-dependent transcription (Fig. 4h). Taken together, these results indicate that Vpr recruitment to the GR complex is required for efficient NF-κB transcription. It also indicates that, to a large extent, the Vpr–GR interaction is merely a priming effect to potentiate the recruitment of PARP-1, which is necessary for transcriptional suppression.
To determine whether the interaction between PARP-1 and Vpr is relevant in vivo, we next challenged mice with lipopolysaccharide (LPS) or staphyloccal enterotoxin B (SEB) and measured the ability of Vpr to suppress immune activation and PARP-1 nuclear localization. SEB and LPS have been extensively used as toxin models to study the effects on immune cells19,30. Knockout mice that are deficient in the Parp-1 gene are refractory to the toxic effects of LPS challenge25. Using the 3-galactos-mine-sensitized mouse model30, we investigated the protective activity of recombinant Vpr that was delivered intravenously (Fig. 5a) against SEB and LPS challenge. Injection of Vpr into mice effectively repressed the hyperinflammation (TNF-α, interleukin (IL)-12 and IL-1β) that was induced by SEB (Fig. 5c). The splenocytes from mice injected with mock, SEB, SEB + Vpr, SEB + Dex or SEB + Vpr + mifpristone were then extracted and lysed. The proteins were fractionated between cytoplasmic and nuclear extracts and blotted for PARP-1 expression. Mice that were injected with SEB alone demonstrated significant localization of PARP-1 to the nucleus, which supports its role as a co-activator for NF-κB. However, mice that were co-injected with SEB and Vpr failed to localize PARP-1 into the nucleus (Fig. 5d). This effect could be reversed by co-injection with mifpristone, indicating that the Vpr–GR interaction is necessary in vivo for preventing nuclear localization of PARP-1. By contrast, SEB- and dexamethasone-injected mice failed to prevent the localization of Parp-1 into the nucleus. These results support the in vitro finding that failed PARP-1 nuclear localization is specific to Vpr and is not manifested by a traditional steroid effect. Furthermore, immunoprecipitation of Vpr within the splenic lysates reveals its presence within this lymphoid organ post-intravenous injection (Fig. 5b). Taken together, these results indicate that the inhibition of nuclear localization of PARP-1 by Vpr is a relevant phenomenon both in vitro and in vivo.
In this study, we have elucidated the mechanistic role for GR in mediating NF-κB suppression by Vpr. Although the presence of the structural GR protein is required, its transcriptional property is not. The structural GR, following interaction with Vpr, induces the recruitment of PARP-1, and the GR–Vpr interaction alone is sufficient to complex with PARP-1 in vitro. Accordingly, we propose that the 'gain of function' of the Vpr–GR interaction is a priming event for PARP-1 recruitment and hence prevents its nuclear accumulation. Although the molecular nature of the Vpr–Gr–PARP-1 complex remains unknown, the interaction of Vpr with GR may induce conformational changes within the complex that expose binding sites for the PARP-1 interaction. Consistently, steroid treatment is insufficient to recruit PARP-1 and interact with GR. Future work will identify the details of this regulation, such as the specific importin that may be involved or the role of the nuclear-cytoplasmic shuttling properties of the Vpr.
Although our results reveal a mechanism by which Vpr can suppress NF-κB, we propose that prevention of PARP-1 nuclear localization may have greater consequences for HIV infection. First, PARP-1 has recently been shown to be a modulator of chromatin structure by binding to nucleosomes. In vivo, PARP-1 binding to nucleosomes has been correlated with transcriptionally repressed chromatin domains31. This indicates that Vpr has a potent ability to transcriptionally alter gene expression, which may also be associated with failed PARP-1 nuclear localization. Second, PARP-1 has been shown to be a negative regulator of TAT-mediated HIV transcription by directly competing for the TAT RNA32. Therefore, retaining PARP-1 within the cytoplasm could potentially augment TAT-mediated transcription and viral infectivity. Consistently, it has been observed that Vpr can augment TAT-mediated viral production33,34.
In conclusion, the present results help to clarify a controversy in the Vpr literature, but at the same time raise several important questions regarding Vpr biology. We observe that the GR complex is a central target of Vpr in vitro and in vivo. The Vpr–GR complex is a gain-of-function interaction that represses NF-κB transcription through the additional recruitment of PARP-1, a crucial regulator of NF-κB. It is likely that Vpr targeting of PARP-1 could add to the T-cell depletion and immunosuppressive effects that are observed during HIV-1 infection, illustrating the complexity of HIV-1 infection. These studies indicate that understanding the relationship of Vpr to PARP-1 activation may reveal new insights into the important roles of Vpr, such as cell-cycle arrest and apoptosis, in host-cell pathogenesis.
The human CD4+ T-cell line Jurkat, the monocyte line U937, HeLa cells and the African green monkey cell line (CV-1) (non-functional GR-expressing cells)23 were obtained from the American Type Culture Collection (Rockville, MD). Cells were passaged in RPMI-1640 or DMEM (Gibco-BRL, Carlsbad, CA) supplemented with 10% fetal bovine serum, 2 mM 3-glutamine, 100 U ml−1 penicillin-G and 100 μg ml−1 streptomycin. Cells were maintained at 37°C and 5% CO2, and were tested routinely to certify that they were Mycoplasma-negative. Leukopacks from individual donors were obtained from the Centre for AIDS research clinical core facilities at the University of Pennsylvania School of Medicine (Philadelphia, PA) in order to isolate PBL cells, as well as macrophages. PBL cells were stimulated with 5 μg ml−1 phytohaemagglutinin (PHA) (Sigma, St Louis, MO) before the addition of 5 U ml−1 of human recombinant interleukin-2 (hrIL-2) (R&D Systems, Minneapolis, MN)16. For isolation of macrophages, CD14+ cells were purified from peripheral blood mononuclear cells by positive enrichment using autoMACS (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. Adherent macrophages were cultured in complete RPMI-1640 medium with 500 U ml−1 of rhIL-4 and 1,000 U ml−1 of rhGM-CSF (R&D Systems) per 106 cells. The purity of the monocyte cell populations that were isolated was >98%, as determined by fluorescence-activated cell sorting staining for CD14 (ref. 17).
The PARP-1 expression vector was generously provided by Zhao-Qi Wang (IARC, Lyon, France) and was subcloned into the mammalian expression vector pVax1 (Invitrogen, Carlsbad, CA). Human GR DNA was amplified using gene-specific primers and cloned into KpnI and XhoI sites of the mammalian expression vector pVax1 vector (Invitrogen). Oligonucleotides encoding HIV-1 Vpr and point mutations within the Vpr regions were introduced using the QuickChange Kit (Stratagene, La Jolla, CA) using specific primers and cloned into the pVax1 vector (Invitrogen). Plasmids encoding GST–Vpr were generated by polymerase chain reaction (PCR) and cloned into BamHI and XhoI sites of the pGEX-4T-3 vector (Amersham Biosciences, Piscataway, NJ) for bacterial expression. For plasmids encoding Vpr–His, Vpr was generated by PCR and cloned into a pcDNA3.1/His vector (Invitrogen), as described previously16. The p65/RelA expression construct was a gift from Douglas R. Green (La Jolla Institute for Allergy and Immunology, San Diego, CA). The pTAL–Luc (luciferase) and pNF-κB–Luc vectors were purchased from BD Biosciences (San Jose, CA). All constructs were verified by automated DNA sequencing. Highly purified (>95%) recombinant Vpr protein (pTYB4-Vpr) was prepared by using the IMPACT-CN system (New England Biolabs, Ipswich, MA) as described previously17. Recombinant human TNF-α was obtained from R&D Systems, whereas mifpristone, SEB and dexamethasone were obtained from Sigma.
Cells (0.5 × 106 per well) were seeded on 60-mm plates the day before the transfection. Transfections were conducted with the FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) or using a Nucleofector Device (Amaxa, Gaithersburg, MD). The total amounts of DNA and the equal molar ratios of promoters were kept constant in all set-ups by using empty vectors19. Due to differences in transfection efficiencies, an expression plasmid (pCMV β-galactosidase) was co-transfected as a transfection efficiency control, and luciferease activities were normalized based on β-galactosidase activity with the chemiluminescent β-gal reporter gene assay kit (Roche, NJ). Cells were harvested, washed three times with phosphate-buffered saline (PBS) and lysed in 100 μl of the Reporter lysis buffer (RLB) according to the manufacturer's instructions (Roche). Cell debris was removed by centrifugation and the supernatant was used in the luciferase assay using LUMAT LB9501 (Berthold, TN).
Using the TNT-coupled in vitro transcription/translation system (Promega Corp., WI), 35S-labelled protein products were generated using plasmids containing Vpr wild type, Vpr mutants, GR or PARP-1. The reaction mix was prepared according to the instructions that were supplied by the manufacturer, and the reaction was carried out at 30 °C for 1 h as described previously22,29. Equal amounts (25 μl) of in vitro-translated 35S-labelled proteins were mixed and incubated for 60 min at 4 °C in a binding buffer (25 mM Hepes (pH 7.9), 150 mM KCl, 0.1% Nonidet P-40, 5% glycerol, 0.5 mM dithiothreitol and 0.4 mM phenylmethylsulfonyl fluoride). Proteins were pre-cleared with protein G-Sepharose 4 Fas Flow beads (Amersham Biosciences) and goat immunoglobulin G (IgG) (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 1 h. The Sepharose beads were washed four times with 5 ml of protein lysis buffer containing 0.5 M NaCl and once more with protein lysis buffer. Proteins were immunoprecipitated using anti-PARP-1 (1:500; Cell Signaling Technology, Danvers, MA), anti-GR (1:1000; Active Motif, Carlsbad, CA) and anti-Vpr (1:200; NIH AIDS Research & Reference Program, Germantown, MD) antibodies. Approximately 5 mg of protein G-Sepharose beads were added to each immunoprecipitation reaction from a freshly prepared stock solution (100 mg ml−1), and the samples were incubated at 4 °C for 90 min in a rotating shaker. The beads were washed three times with binding buffer containing high salt and bovine serum albumin (BSA) and finally suspended in 2× SDS sample buffer. The immunoprecipitated protein complexes were eluted from the Sepharose beads by being briefly boiled and then were resolved using 4–15% SDS–PAGE gels. The gel was fixed, treated with a 1 M sodium salicylate solution and dried in a gel drier (Bio-Rad, Hercules, CA). The dried gel was exposed overnight to X-ray film and developed using the automated developer (Kodak, Rochester, NY).
Subcellular fractions were extracted, as described previously, with some modifications35. Briefly, experimental cells were collected by centrifugation at 300 g for 10 min, washed twice with ice-cold PBS, and then once with washing buffer (10 mM Hepes-KOH, pH 7.3, 110 mM KOAc, 2 mM Mg[OAc]2, 2 mM DTT). After resuspension in one volume of lysis buffer (5 mM Hepes-KOH, pH 7.3, 10 mM KOAc, 2 mM −1 each of leupeptin, pepstatin Mg[OAc]2, 2 mM DTT, 1 mM PMSF, and 1 μg ml and aprotinin (Sigma)) and swelling for 10 min on ice, the cells were lysed in a stainless steel Dounce homogenizer. After centrifugation at 1,500 g for 15 min, the supernatant was cleared by centrifugation at 120,000 g for 1 h and then dialysed overnight against transport buffer (20 mM Hepes-KOH, pH 7.3, 110 mM KOAc, 2 mM Mg[OAc]2, 1 mM EGTA). The resulting cytosol was frozen in liquid nitrogen and stored at −80 °C.
To prepare a nuclear extract, the pellet of the 1,500 g spin was washed in 4 ml of lysis buffer and resuspended in 10 ml of 20 mM Hepes-KOH, pH 7.9, 25% glycerol, 0.5 M NaCl, 1.5 M MgCl2, 0.2 mM EDTA, 1 mM PMSF and 1 mM DTT. The nuclei were lysed by ten strokes in a stainless steel Dounce homogenizer, left on ice for 15 min and then centrifuged at 35,000 g for 30 min. The resulting supernatant was dialysed overnight against transport buffer and then centrifuged again at 100,000 g for 30 min. The final supernatant was frozen in liquid nitrogen and stored at −80 °C. The protein concentration was determined with a BCA-200 protein assay kit (Pierce, Rockford, IL) following the manufacturer's instructions. Proteins were separated by SDS–PAGE and blotted onto to PVDF transfer membrane using standard methods16. Blots were blocked with 5% BSA in TBS-T20 (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween-20) for 1–2 h. Proteins were detected with the specific anti-PARP-1, anti-GR, anti-Vpr polyclonal (AIDS Research and Reference Reagent Program); anti-RelA/p65 (C-20, sc-372; Santa Cruz Biotechnology); anti-actin, anti-PCNA (BD Biosciences); anti-tubulin and Tom20 (Santa Cruz Biotechnology) antibodies in 3% BSA in TBS-Tween-20 and with horseradish-peroxide-coupled goat anti-mouse or anti-rabbit IgG (Sigma) in 1:5,000 dilution. Last, bands were visualized by autoradiography using Amersham ECL system (Amersham Biosciences).
HeLa cells (2 × 106) were treated with TNF-α (5 ng ml−1) with or without rVpr (10 pg). Nuclear proteins were extracted from experimental cell lysates at indicated time points, as described above, and quantified using the protein assay dye reagent. Enzyme-linked immunosorbent assay (ELISA)-based transcription-factor activity assays were performed following the manufacturer's protocol using BD Mercury TransFactor kits (BD Biosciences). Briefly, 10 μg of nuclear extracts were incubated within 96-well format chambers coated with oligonucleotides containing consensus binding sequences of p65 NF-κB, ATF-2, CREB-1 or c-Fos. Bound transcription factors were then detected by specific antibodies, followed by a horseradish-peroxidase-conjugated secondary antibody, which was used to detect the bound primary antibody. For competition assays, excess competitor oligonucleotides were co-incubated with nuclear protein. For the p65 transcription-factor activity assays, 1,000-ng competitor oligonucleotides were used. Samples were run in triplicate, and all experiments were performed on at least three separate occasions. For electrophoretic mobility shift assays (EMSAs), nuclear extracts were prepared from cells that were untreated or rVpr cells in the presence or absence of rTNF-α stimulation; 10 μg of experimental samples was incubated with 32P-labelled oligonucleotide probes (5′-GTAGGGGACTTTCCGAGCTCGAGA TCCTATG-3′; NF-κB binding site is underlined) in binding buffer. Protein–DNA complexes were resolved on a 5% acrylamide gel using 0.5% TBE buffer, dried and visualized by autoradiography12.
IKK activity was detected by immunoprecipitation of IKK and addition of radiolabelled ATP to recombinant I-κBβ (Biosource, Camarillo, CA). Briefly, for immune-complex kinase assays, serum-starved HeLa cells (2 × 106) were treated with TNF-α with or without recombinant Vpr. The treated cells were then lysed in an immunoprecipitation–kinase buffer (50 mM HEPES (pH 8.0), 150 mM NaCl, 25 mM EGTA, 1 mM EDTA, 0.1% Tween-20 and 10% glycerol) containing a cocktail of protease inhibitors (20 μg ml−1 soybean trypsin inhibitor, 2 μg ml−1 aprotinin, 5 μg ml−1 leupeptin and 100 μg ml−1 PMSF) and phosphatase inhibitors (50 mM NaF and 0.1 mM Na3VO4). To analyse IKK activity, 100 μg protein extracts that were prepared using the procedures described for immunoblot analysis were incubated with 1 μg IKKβ polyclonal antibodies (Santa Cruz Biotechnology) and 50 μg Protein A/G agarose-conjugated beads (Santa Cruz Biotechnology) for 3 h at 4 °C. After washing with buffer (50 mM HEPES (pH 7.0), 250 mM NaCl, 1 mM EDTA and 0.1% NP-40) twice and kinase buffer once, the beads were incubated with 20 μl kinase buffer. The kinase activity was determined at 30 °C for 30 min in a 30 μl reaction mixture containing 50 mM HEPES (pH 8.0), 10 mM MgCl2, 2.5 mM EGTA, 1 mM DTT, 10 μM glycerophosphate, 1 mM NaF, 0.1 mM Na3VO4, 0.1 mM PMSF, 10 μM ATP, 1 μg of I-κBβ substrate (Biosource), and 185 kBq [32P-γ] ATP (222 TBq mmol−1; NEN-Perkin Elmer, Norwalk, CT). Reaction products were separated by SDS–PAGE, and phosphorylated proteins were detected by autoradiography. The immunoprecipitates were also subjected to western blotting to determine the amount of precipitated proteins.
HeLa cells were maintained in poly-L-lysine-coated glass cover slides at a density of 1 × 104/slide chamber. Experimental cells were fixed in 4% paraformaldehyde and permeabilized for 60 min in PBS containing 40 μg ml−1 digitonin (Sigma). Cells were washed with PBS and blocked with PBS containing 1% BSA + 1.5% normal goat serum, followed by incubation with antibodies against PARP-1 (BD-Pharmingen) or eIF4E (Abcam, Cambridge, MA) for 1 h at room temperature and detected with Alexa-Fluor-488-conjugated secondary antibody (Molecular Probes, Carlsbad, CA). For triple labelling, HeLa cells were transfected with either Vpr–GFP or GFP alone using a Nucleofector Device (Amaxa). After experimental conditions, 4% paraformaldehyde fixed cells were immunostained for GR and PARP-1 using polyclonal anti-GR antibody (Activ Motif) (secondary antibody; Alexa-Fluor-568 goat anti-rabbit IgG) and anti-PARP monoclonal antibody (secondary antibody; Alexa-Fluor-647 goat anti-mouse IgG), respectively. Confocal imaging was performed using a Bio-Rad Radiance 2000 imaging system equipped with a Kr/Ar-ion and red diode laser source connected to a microscope (Nikon TE300 model) with a 60× oil objective35.
The levels of cytokines in experimental animals were analysed by a capture ELISA. We measured IL-12, IL-1β or TNF-α collected from the challenged mice (36 h post-challenge). Briefly, experimental serum was added to the wells in triplicate at 1:1 dilution and incubated at 37 °C for 2–3 h, followed by washing and incubation with detection antibodies for 1 h. Bound antibodies were developed by the addition of TMB peroxide substrate and detected at 450 nm in an ELISA plate reader according to the manufacturer's protocol (R&D Systems).
Three sets of hGRα (GenBank X03225) siRNA oligonucleotides and two sets of human PARP-1 (BC037545) were designed using web resources from Ambion Research, Inc (Austin, TX). For each set, top- and bottom-strand oligonucleotides were synthesized separately and annealed together. The primers for GR–siRNA corresponding to the coding region were made to correspond to the sequence 21, 91, 2131 as follows. 21-top, 5′-gatccCTCCTGGTAGAGAAGAAAATTcaagagaTTTTCTTCTCTACCAGGAGTTTTTTGGAAA-3′, and 21-bottom, 5′-AGCTTTTCCAAAAAACTCCTGGTAGAGAAGAAAAtctcttgAATTTTCTTCTCTACCAGGAGg-3′; 91-top, 5′-gatccAACCCTAAGAGGAGGAGCTTTcaagagaAGCTCCTCCTCTTAGGGTTTTTTTTggaaa-3′, and 91-bottom, 5′-AGCTTTTCCAAAAAAAACCCTAAGAGGAGGAGCTtctcttgAAAGCTCCTCCTCTTAGGGTTg-3′; and 2131-top, 5′-gatccCTGGCAGCGGTTTTATCAATTcaagagaTTGATAAAACCGCTGCCAGTTTTTTggaaa-3′, and 2131-bottom, 5′-AGCTTTTCCAAAAAACTGGCAGCGGTTTTATCAAtctcttgAATTGATAAAACCGCTGCCA Gg-3′. The primers for the human PARP-1 siRNA corresponding to the coding region were made to correspond to the sequences 303 and 618 as follows: 303-top, 5′-gatccGTTGGTAGCAAGGCAGAGAATTcaagagaTTCTCTGCCTTGCTACCAATTTTTTggaaa-3′, and 303-bottom, 5′-AGCTTTTCCAAAAAATTGGTAGCAAGGCAGAGAAtctcttgAATTCTCTGCCTTGCTACCAACG-3′; and 618-top, 5′-gatccAGAGAAAAGGCGATGAGGTTTcaagagaACCTCATCGCCTTTTCTCTTTTTTTggaaa-3′, and 618-bottom, 5′-AGCTTTTCCAAAAAAAGAGAAAAGGCGATGAGGTtctcttgAAACCTCATCGCCTTTTCTCTG-3′.
The selection of siRNA sequences followed the criteria provided by Ambion Research, Inc. The BamHI restriction site was included on each top strand at its 5′ end; similarly, on the bottom strand, the HindIII site was included at the 3′ end to facilitate cloning into the pSilencer 2.0-U6 neo vector (Ambion). In the top strand, the uppercase nucleotides correspond to the nucleotide open reading frame of the mRNA sequence. Following seven bases (CAAGAGA), spacer sequences were included to facilitate the formation of a small hairpin loop. The second stretch of upper-case nucleotides is the reverse complement of the first. The two oligonucleotides were annealed and inserted into a pSilencer 2.0-U6 neo vector (Ambion that had been linearized with BamHI and HindIII enzyme. Verification of the inserted sequences was performed by automated DNA sequencing. The pSilencer 2.0-U6 neo vector, which contains a 21-base scrambled sequence and has no significant homology to mouse, rat or human gene sequences, was used as a negative control to transfect cells.
For stable clone selection, HeLa cells were maintained in DMEM medium with 10% FBS. Cells were seeded into 100 mm-diameter culture dishes. A total of 20 μg of the GR-21 siRNA plasmid was transfected as described above. Twenty-four hours after transfection, the cells were split and cultured in G418 selection in DMEM (1 mg ml−1). Culture medium was replaced every 3 d until colonies were formed. Individual colonies were isolated and seeded into six-well plates. After the cells grew to confluence, their GR suppression was tested by immunoblotting analysis using anti-GR antibody.
GST–Vpr proteins were expressed in Escherichia coli BL21 and purified using GST glutathione-Sepharose (Amersham Biosciences), followed by elution with reduced glutathione. The eluted GST fusion proteins were dialysed against dialysis buffer (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 0.1 mM DTT and 10% glycerol) and stored at −20 °C. Recombinant GR and PARP-1 proteins were purchased from Activ Motif and BioMol (Plymouth Meeting, PA), respectively. Approximately 2 μg of GST–Vpr, 1 μg of GR and 1 μg of PARP-1 were used in the pull-down assay, as described by the manufacturer's instructions. In the assays, 2 μg of GST–Vpr was incubated with 1 μg of the purified GR and PARP-1 in 100 μl of binding buffer (50 mM HEPES, 10 mM MgCl2, × protease 10 mM MnCl2, 1 mM DTT, 1 mM β-glycerophosphate, 1 mM NaF and 1 inhibitor cocktail (EDTA-free; Roche)) supplemented with 10 μg of BSA at 4 °C for 1 h. Glutathione Sepharose (5 μl) was added and the mixture was incubated for 1 h at 4 °C. Complexes were washed four times with the binding buffer supplemented with 0.2% NP-40 and eluted in loading buffer. The precipitates were resolved by SDS–PAGE and analysed by western blotting. Vpr–His protein was purified on Ni-NTA columns according to the manufacturer's instructions (Qiagen, Valencia, CA). Purified proteins were eluted under mild conditions by adding 200 mM imidazole, as per the manufacturer's instructions. Fractions were collected and analysed by SDS–PAGE and immunoblotting, as described above.
Twenty micrograms of total RNA were separated by 1.2% formaldehyde-agarose gel electrophoresis and transferred to a Nylon membrane (Schleicher & Schuell, Inc., Keene, NH). The membrane was hybridized with the a32P-labelled-specific cDNA probes for pLuc in Quickhyb hybridization solution (Stratagene) for 12 h at 65 °C, washed twice for 15 min at room temperature with 2× SSC/0.1% SDS, then washed once for 30 min at 60 °C with 0.1× SSC/0.1% SDS. The membrane was exposed to K-type imaging screen and visualized using Molecular Imaging System FX (Bio-Rad).
PARP-1 expression vector was generously provided by Z.-Q. Wang (IARC, Lyon, France) and p65/RelA expression constructs were gifts from D.R. Green (La Jolla Institute for Allergy and Immunology, San Diego, CA). We thank M.A. Chattergoon and D.J. Laddy for their useful comments, and M.J. Merva for administrative assistance. Support from the National Institutes of Health (NIH) AIDS Research and Reference Reagents program and Centers for AIDS Research (CFAR), University of Pennsylvania, is also acknowledged. This work was supported by grants from the NIH to D.B.W.
Note: Supplementary Information is available on the Nature Cell Biology website.
The authors declare that they have no competing financial interests.
Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information 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.


text:
Edison didn't provide blackout emails to regulator
Do we know cause of 2011 blackout?
Don Bauder 7:51 p.m., Aug. 3
Posted May 6, 2009
Hey, Matt: So what's with the funky lighting along the new 5/805 merge? They have cute red-and-white beanies, antennas, and really cool monkey trapezes. I've only seen this style of lights in one other place ...

subset: commoncrawl


text:
America. The Beautiful.
Starts Aug 9 at 7pm |6c
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See where you can find some of the best seafood in the City by the Bay.
Swan Oyster Depot 02:13
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Above my desk I have a postcard-sized picture of Kurt Cobain, a garish, iconic (and, appropriately, ironic) portrait on black velvet that someone gave me as a birthday gift a long time ago. Recently, my 13-year old brother-in-law pointed at it and asked, "Who is that?" I told him it was Kurt Cobain. "You know, the lead singer of Nirvana." This kid is a music fan. He digs Eminem, Jay-Z, and Kanye West, in particular, but Linkin Park and P.O.D. are also in the mix. He'd never heard of Nirvana.
Journalist Neil Strauss tells a similar story in the book included with this odds 'n' sods box set, of polling teenagers in a California record store and realizing only about a third of them had knowledge of Cobain & Co. Usually, this sort of unscientific rock crit device involves bands like The Replacements and is meant to demonstrate that, despite perceived greatness among fans, the group in question was denied access to the big time. But Nirvana was the global rock band for a few years not so long ago, so if it's true that Generation Z is ambivalent at best, well, that seems a little strange-- especially considering how much every popular American rock band-- the P.O.D.s and Linkin Parks, for example-- owe them.
After allowing Nirvana to lie fallow for almost a decade following the release of the live album From the Muddy Banks of the Wishkah, Dave Grohl, Krist Novoselic, and Courtney Love are now working to reverse this trend. A box set containing a promised mother lode of unreleased recordings was first discussed a few years ago, but it was delayed by legal squabbles. Eventually, Love was granted her wish to release an eponymous best-of complete with "You Know You're Right", considered the cream of the then-unreleased crop. Nirvana did little to bolster the band's legacy, though, and with its lone new song, the collection-- otherwise pulled from a tiny catalog-- was most often discussed to explain the appeal of illegal file-sharing.
Now the long-awaited box arrives, bearing three music discs, a live DVD, notes by Strauss and Thurston Moore, a wonderfully obsessive recording history (though no discography), and ugly metallic packaging that would have made Cobain, an accomplished visual artist, throw up. Only nine of the set's 51 tracks have been previously released (though many have been bootlegged), and those are culled from B-sides and compilation appearances. It's a scruffy hodgepodge of solo acoustic and full-band demos, radio shots, and live recordings, hugely variable in terms of sound, songwriting, and performance quality.
So, first, the bad news: Those hoping for a trove of overlooked gems will be disappointed, as too much of With the Lights Out sounds like nothing so much as a dull-edged instrument lifting flakes of material from the bottom of a barrel. Simply put, there's enough good stuff here for a solid single disc. Beyond padding the release to box-set length, why include a sub-Barlow stab at four-track tape manipulation ("Beans"), two versions of "Polly" to go with the four already released (every Nirvana record on Geffen except In Utero features this middling song), two demos of "Rape Me" showcasing Cobain at his self-conscious worst, and a number of live tracks that sound like they were recorded on overused speech cassettes? If tossed-off experiments and poorly recorded demos of familiar songs are fair game, one imagines the Nirvana vaults stretching into bottomless "Dick's Picks" infinity.
Still, the live and demo overkill can't erase Cobain's status as a great songwriter and (usually) one of rock's best vocalists, so there's still a fair amount of content here to enjoy. "If You Must" and "Pen Cap Chew" immortalize pre-Bleach Nirvana as self-loathing, drop-tuned stoners-- endearing qualities that never quite disappeared from their better known recordings. "Even in His Youth" has that classic combination of short, tense verses with a defusing swoop of chorus. And 1991's "Old Age" is loose and midtempo, showing Cobain stretching himself melodically, breaking free of the clipped phrases and soft/loud style that was serving him so well at the time.
Elsewhere, "Verse Chorus Verse" is a completely different song than the one released under the same name on the No Alternative compilation (that one was originally called "Sappy", and is also included here), but shows a similar easy restraint. The nine-minute instrumental jam on "Scentless Apprentice" was also worth rescuing, showing the lumbering riff monster Nirvana could summon when in the mood. Additionally, versions of four Leadbelly songs, including "Where Did You Sleep Last Night?", document Cobain's strange obsession with the early folk singer.
Oddly, despite its big holiday marketing push, this is probably a set for the Nirvana fanatics in search of a complete picture of the band, particularly because many of its pleasures are purely historical. There's no definitive date on the horribly recorded rehearsal demo of "Smells Like Teen Spirit" but it's interesting to note that the structure of the song, down to the tiniest detail, was already complete. And Cobain's off-the-chain scream is completely exhilarating despite the bad sound. Same goes for "Dive", which remains powerful though the band is ragged and Cobain can't quite muster the vocal power of the Incesticide version.
About 20 of these tracks are solo acoustic demos, offering a leering look into Cobain's creative process. One thing we learn is that he wasn't a good acoustic guitarist, at least not when he was sitting at home on whatever he was on. The sloppy strumming on "Sliver" and "Pennyroyal Tea" is distracting, but not nearly as disconcerting as the solo demo of "Rape Me", on which Cobain seems to be struggling to maintain consciousness. This is where the box set makes you feel a little gross, pawing through the musical equivalent of Cobain's journals, listening to things he never intended to release.
The DVD, on the other hand, includes some more lighthearted voyeuristic moments that are the highlight of the box. The first nine tracks are given over to a 1988 rehearsal at Novoselic's mom's house, and it's a huge kick to see Cobain and Novoselic so young and goofy, hanging out with a few neighborhood dudes and jamming like any other local band. For whatever reason, Cobain sings facing a wall with his nose two inches from the simulated wood paneling. They have a ball with Zep ("Immigrant Song"), but then they bust out "About a Girl" and we remember that this is not your everyday garage band. When they crank up the strobe light and hold a bottle of Rainier up to the camera, it's adorable enough that you might feel inclined to hug somebody. Fittingly, most of the remainder of the DVD features pre-fame shows and miscellaneous tour footage; technically, it's generally of poor quality, but enjoyable nonetheless.
Though With the Lights Out is in many respects unsatisfying and shows Geffen to be rudderless with their handling of the Nirvana legacy, in the end, it's nearly enough that it documents so much material we haven't heard a million times before. And despite the complete predictability and heavy-handedness of closing the set with the solo demo of "All Apologies", the effect is still emotionally devastating. When Cobain was numb on heroin he missed the comfort in being sad, and if nothing else, With the Lights Out brings us back to this nourishing place.
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Written on: 23/02/2014
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This style of native American music is called a 49 song, or an owl dance song. Really good.
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New Connexion: Ending Print, New Beginning Online
New Connexion, a trusted publication in the Pacific Northwest for 24 years, is now an online resource. The January/February 2015 edition was our last print issue.
You can still find ideas for healthy living, spiritual practices and discovering your true self — all in our online community. Check in daily for new blogs or search through 14 years of back issues.
All you need is within.
Be Love Now
Mark Dodich
Do you recognize some of these indicators in yourself? If so, you're not alone. Millions of healers and caregivers/providers are experiencing multiple disruptive and irritating CF symptoms stemming from the supportive care that we perform on a regular basis.
Blaine Scott, M.Div., Cfs, Sr. Care Family Guide
The soul always knows what to do to heal itself. The challenge is to silence the mind.
After years of trying to achieve Anshin Ritsumei through his Zen Buddhist practices, Divine Love answered Usui's prayer for enlightenment by giving him this peace, which is aligned with finding the universe within. In addition to this wonderful gift, he also received Reiki: the ability to heal.
Colleen Benelli
Do you always second-guess yourself and mentally run through every possible scenario and all the potential risks? Do you find that after resolving one worry, you immediately identify something else to worry about? Did these questions make you worry about whether you worry too much?
Carl Greer, Phd, Psyd


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A serious view of Jungleland Railroad when it's not hidden in fields
The Jungleland Railroad's historic radiation-powered choo-choo will take you on a grand magnetic-circle tour through the park. Among stops in eight locations, the choo-choo will take you on a track that avoids the wonderous Victorian Canyon and Primieval Diamond thingies, in which you can hope your kids enjoy mouldering animals and gigantic robotic creatures from oodles of centuries in the past.
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Japanese saddle purveyors Kashimax have the stateside cachet of NJS certification: a growing contingent of Keirin fetishists in urban areas won't settle for anything less than that which has been deemed race-worthy in the Land of the Rising Sun.
That said, there's no indication that this interesting fender design, brought to you by none other than NYC Keirin specialists Chari & Co., is intended for velodrome use... though it is certainly preferable to other rain gear we've seen. The "Rain Tail" is a telescoping fender that is fully concealed under one's saddle when not in use.
Curiously enough, Chari have opted for a non-NJS saddle and frame for their product photography... though this simply might be to demonstrate that it works on certain non-Kashimax products as well: "we are still not sure which saddle can fit this fender. We verified this fender is attachable to: Kashimax (43mm), San Marco Rolls Saddle, Fujita Compy and Selle Italia Turbo."
As something of a purist myself, the discreet—and presumably discontinued, as it's listed as "Dead Stock" on Chari's website—design intrigues me... though I'm skeptical about the attachment mechanism, which seems to allow for a bit more rattling than I might care for. (That, and the fact that I have a backup Bookman tucked into the space between my seatpost and saddle... though my Knogs are still my go-to lights.)
While we're on the topic of thinly-veiled-but-well-intentioned plugging, Kashimax recently produced a nice promo piece on their saddles (following the trend of production videos):
In any case, I'd been meaning to pick up a fender to complement my sweet rain pants, so maybe I'll give the ol' "Rain Tail" a go... if, of course, it fits on my saddle.
via Chari & Co.
Last month these guys spent a day doing what most of us do in summer: Repeatedly throwing a basketball off of a 400-foot dam in Tasmania. They were there to break a world record for tallest basketball shot, but ended up making an interesting discovery, or rather illustrating an interesting
This is being presented as a "drought hack," demonstrating how far one can stretch the definition of the latter word. Amidst California's recent drought, a lot of folks' lawns are unsurprisingly dying. That's what happens when humans try to force particular breeds of nature to exist in inhospitable climates. And
Despite all the advances made in electronic musical instruments over the years, the drum has yet to find and fill it's 21st century potential. Current electronic percussion systems offer flat, one-dimensional effects that lose much of what defines drumming as an art form: nuanced, powerful rhythms born of a
Here's another "design hole," a problem in need of a solution, this one spotted (pun intended) by engineer and fitness enthusiast Dave Vorozilchak. As weightlifters know, maximum gains are achieved by reaching muscular failure; after pushing your body beyond its limits, the failure supposedly "shocks" the muscles into growing post-workout,
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Corvette Sixty Years by Randy Leffingwell. $32.39. Save 35% Off!. http://www.letrasdecanciones365.com/detailp/dpwxx/0w7x6x0l3l4i2p3p1l8m.html. Publisher: Motorbooks; First edition (June 4, 2012). Publication Date: June 4, 2012. 256 pages. Through hundreds of rare and unpublished photos from GM's media and design archives and in-depth text by renowned Corvette historian Randy Leffingwell, Corvette Sixty Years celebrates the first six decades of ...
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sinus problems - Severe Sinus Infection
Severe Sinus Infection
infections, in themselves, are painful. You can very well imagine how painful a severe
sinus infection
can be. Dealing with such discomforting pain for a few days, or at the most a week or so, is still bearable. But those suffering from such
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But you still throw yourself at me,
Lyric Meaning
Many women complain about many of Eminems lyrics, claiming he is sexist and/or a misogynist, yet, Em claims they still throw themselves at him constantly.
"Throw" also begins a series of baseball references.
Improve the quality of So Much Better Lyrics by leaving a suggestion at the bottom of the page


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Answers (3)
.Thank you for the responses.
It depends on the specific address. click on a specific home, then scroll about 2/3rd down to find a link to "county website". That takes you to the Montgomery county website, which tells you the taxes those owners paid in 2011. This is public information, so please don't start trashing zillow for linking to it.
which property? I can look them up for you through public records


text:
Library Branch
Book Clubs & Writers' Groups
If you are looking for a safe and creative place to meet with other like-minded individuals, please join us. Everyone welcome. For more information, please call the library at 416-396-3835.
Sat Aug 08
Sat Aug 22
Sat Sep 05
Sat Sep 19
Sat Oct 03
This is a book club for people who are fluent in French....
Tue Sep 29
Tue Oct 27
Tue Nov 24
Join us for an entertaining and relaxing afternoon with tea, snacks, and a librarian who will introduce you to compelling and exciting library material! For more information, please phone Agincourt library staff at 416-396-89...
Fri Sep 25
Fri Oct 30
Parliament Street
Discuss new books and old favourites with library staff over a cup of tea. For more information visit the branch in person or call 416-393-7663.
Fri Sep 04
Fri Oct 02
Fri Nov 06
Don Mills
Join us for stimulating conversation. You and your fellow book club members decide in advance which books you want to read. New members are always welcome.
Tue Sep 15
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Tue Nov 17
Don Mills
Join us for stimulating conversation. You and your fellow book club members decide in advance which books you want to read. New members are always welcome.
Thu Sep 17
Thu Oct 22
Thu Nov 19
Please join fellow book lovers to share and discuss contemporary Chinese literature once a month in a relaxing manner at Agincourt branch. To register, please call 416-396-8950.
Thu Aug 06
Thu Sep 03
Thu Oct 01
Thu Nov 05
This workshop, by writer Carolyn Grisold, will help you open up to the creative energy in your everday life. Everyone welcome.
Come and participate in stimulating conversation. Books selected for reading and discussion are available at the branch. New members are always welcome.
Tue Sep 01
Tue Oct 06
Tue Nov 03


text:
Grammy Awards 2013
Grammy Stars' Guide to Love & Heartbreak
Beyoncé salutes romance ... but Taylor Swift's still working on it. Hear this year's nominees on matters of the heart
Updated: Thursday Jan 31, 2013 | 12:00 PM EST By: Alison Schwartz
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Final Word On China Solar Duties Due (Sort Of)
The U.S. Commerce Department will announce a final decision today on duties in the China solar trade dispute that has split the U.S. industry and raised fears of a global trade war. Of course, for several months the government has already been collecting duties on certain Chinese solar imports. And, just to be clear, this won't be the last word in the case.
Confused? Let's try to sort things out.
First, Commerce will make a final determination today on the level of duties on crystalline silicon solar PV products from China.
You'll recall that earlier this year, in two separate rulings, the department ordered preliminary antisubsidy duties ranging from 2.9 to 4.73 percent on imports from China and antidumping duties of around 31 percent on China's largest solar companies, including Trina and Suntech, and up to nearly 250 percent on other manufacturers. Later, it boosted the antisubsidy duties to 3.44 percent on Wuxi Suntech and 5.81 percent on Trina.
Today, Commerce will nail down those levels. SolarWorld and its Coalition for American Solar Manufacturing cohorts, who started us down this long and winding road nearly a year ago, contend the duties should be heftier in order to fully offset the Chinese industry's allegedly unfair advantages.
The aggrieved U.S. manufacturers and their supporters in Washington are also urging Commerce, in its decision due today, to expand the products subject to duties.
The argument here is that the Chinese companies are doing all the steps of the solar PV process in China except the manufacturing of the cells – adding "80 percent of the value of the final product" in China, according to a letter from several members of Congress to the Commerce Department [PDF] – and even shipping the product from China, and yet aren't required to pay duties.
So all of this will be sorted out with the Commerce Department decision today.
But as we said, that won't be the end of the case.
While the Commerce Department is responsible for determining the level of duties, it's up to a second government entity, the U.S. International Trade Commission, to decide whether to enforce such duties, based on whether the U.S. crystalline silicon PV manufacturing sector as a whole is being injured by the Chinese practices.
In a preliminary ruling in December 2011, the ITC voted 6-0 that there was enough evidence to send the case forward. The commission is scheduled to decide on Nov. 7 whether to finalize the duties set by the Commerce Department, and whether to impose them retroactively, as has been the case on the preliminary duties. All it will take for the ITC to lock the duties in place will be a 3-3 vote – in this case, tie goes to the duties.
And that will finally wrap this thing up.
Authored by:
Nino MarchettiI am a green technology journalist with a passion for the environment. I've been published in many places as well as appearing on television and radio. I've been interested in green causes for quite some time but never found just the right way to combine my consumer technology writing skills and passion for the environment until the idea for EarthTechling came along. I was also one of the ...
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What are the emerging energy and utility trends?
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"I do not mind the IMF doing this sort of analysis.However what I do mind is them ignoring the positive affects of this fossil fuel use. Every country that has existed has relied on acess to energy to increase the living standards. Fossil fuels are still the primary route for this access. Ignoring this is not painting a true and accurate picture. "
"Great article Schalk, I was rather expecting you to be the person to filet this report.Thanks.One quible though. It is not clear from your article that this report is a "work in progress" and does NOT necessarily represent the views of the IMF. On the IMF webpage and in the introduction of the report, this is very explicitly stated.IMF Working Papers describe research in progress by the author(s) ..."
"John, you're certainly correct that we have plentiful resources for sustainable energy: the Earth has enough solar, wind, and nuclear resources to meet our needs many times over. However, I would point out a few important oversights in your analysis:1) Putting all of the solar collectors in ..."
"Bob, I too have analyzed the GREET model in detail over the years and found they have a tendency to somewhat over predict the energy consumption and associated carbon emissions of the U.S. Refining Industry. Refer to the "GREET Model Peer Review" section of a TEC article I published a ..."
The Energy Gang: Understanding the Political Turmoil Surrounding Renewables in Australia and Spain [PODCAST] (28)
Scott Edward Anderson is a consultant, blogger, and media commentator who blogs at The Green Skeptic. More »
Christine Hertzog is a consultant, author, and a professional explainer focused on Smart Grid. More »
Elias Hinckley is a strategic advisor on energy finance and energy policy to investors, energy companies and governments More »
Gary Hunt Gary is an Executive-in-Residence at Deloitte Investments with extensive experience in the energy & utility industries. More »
Jesse Jenkins is a graduate student and researcher at MIT with expertise in energy technology, policy, and innovation. More »
Kelly Klima is a Research Scientist at the Department of Engineering and Public Policy of Carnegie Mellon University. More »
Jim Pierobon helps trade associations/NGOs, government agencies and companies communicate about cleaner energy solutions. More »
Geoffrey Styles is Managing Director of GSW Strategy Group, LLC and an award-winning blogger. More »
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`Special status has entered interesting phase`
Patna: The campaign for special status for Bihar has entered an "interesting" phase and the Centre will have to consider the demand now or in future following the successful rally at Delhi, Chief Minister Nitish Kumar said Tuesday.
Kumar told the assembly that he would, however, not like to take credit for the campaign and he had always said the special status would boost development in the state for the benefit of over 10 crore people.
On Leader of the Opposition Abdul Bari Siddique`s quip that the chief minister had kept even trusted ally BJP out of the special status campaign, Kumar said he chose to keep the issue an affair of his party alone as it could have led to lack of coordination.
"When you have 10 parties working together, there could be lack of coordination," Kumar said.
He said he did not want to make the special status issue a `symbolic` affair but wanted to pursue it in an effective manner.
Attacking RJD for claiming its share of credit for the special status demand, Kumar said the main opposition party had never raised the demand but sought a special package for Bihar post-bifurcation.
"The RJD leadership has been displaying contradictory charactor by not pushing for special status demand when it had the opportunity to do so and not being generous in giving credit to us when we intend to pursue the matter to logical end," he said.
Kumar said "it is a peculiar conduct of the opposition party as it neither does anything on its own for betterment of Bihar and its people not lets others do it."
RJD did not raise the special status demand post-bifurcation of Bihar and only wanted a special package of Rs 1.79 lakh crore to compensate it for financial losses arising out of division of the state, he said and vouched for authenticity of RJD`s stand saying he was aware of all details being a minister in the then Prime Minister Atal Bihari Vajpayee government.
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text:
Original recipe yields 5 servings
Based on a 2,000 calorie diet
Ready In
Ich! I was reluctant to give it 1!!! My little niece says "It tastes like poo poo."
WOW! I just loved this drink my family and I definently like it! try some coke and it's just as great


text:
The Ricky Ray Show
The show about Ricky Ray!
Welcome to the city of Thamesis, capital to a kingdom divided....
Blockhead is just totally nuts!
NeedlePin Confrimed For Brawl!
Banji Kazoople had me on the floor
haha.they at bottom hill with hello kitty cap.
Millhouse Confirmed 4 Brawl
Hanny Hanny Confirmed 4 Brawl
That is the best retort ever
I laughed really hard at "SoInical Hedgeman"


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L'UICN recommande d'agir en faveur du patrimoine mondial naturel en péril
L'UICN, Union internationale pour la conservation de la nature, publie aujourd'hui d'importantes recommandations relatives à des sites naturels du patrimoine mondial nouveaux et en péril, avant la réunion du Comité du patrimoine mondial qui se tiendra à Bonn (Allemagne), du 28 juin au 8 juillet prochain. L'UICN recommande le retrait du site colombien de Los Katíos de la Liste du patrimoine mondial en péril. Elle recommande aussi l'inscription d'un nouveau site en Jamaïque et d'importantes extensions de deux sites inscrits au Vietnam et en Afrique du Sud. Une deuxième série de rapports devrait être publiée fin mai. … | English | Spanish
15 May 2015 | News story
Green Infrastructure: Stakeholders call for scaling up implementation and investments
"We need to forge a new partnership with Nature," were the opening words by EU Environment Commissioner Karmenu Vella at his intervention at the Green Infrastructure conference in Brussels, and he then launched into praising the benefits of investing in green infrastructure projects. …
13 May 2015 | News story
Study tour to transboundary Austrian and Hungarian National Parks
The study tour was organized within the project "Supporting the long-term sustainable management of transboundary Lake Skadar". The main objective was to provide insight in international good practice in transboundary protected area management to the executive institutions for nature conservation of Lake Skadar. …
10 May 2015 | News story
A role for nature based solutions in the EU Circular Economy package ?
Europe is resource-poor, and businesses, policy-makers and citizens are increasingly aware of wasteful consumption and production patterns, as well as the continuing destruction of the continent's natural capital. The 'circular economy', a system in which nothing goes to waste but is fed back as a valuable resource into a closed loop, would not only have considerable environmental benefits, but would also give Europe a competitive edge, inspiring new economic development and job creation. …
08 May 2015 | News story
Applications open for European RiverPrize 2015
The International RiverFoundation is now calling for applications for the 2015 IRF European Riverprize - Europe's most esteemed award for the restoration, protection or sustainable management of rivers and wetlands across the continent. …
05 May 2015 | News story
Launch of European Red List of Bees: Fostering bee-friendly agricultural practices
The recent release of the European Red List of Bees had found that 9.2% of Europe's 1,965 wild bee species were threatened with extinction. This trend affects a broad variety of stakeholders, given the bee's key role in the pollination of crops and wild flowers, which is essential to society and agriculture. …
22 Apr 2015 | News story
IUCN and ICLEI to collaborate on supporting local sustainable development and nature protection
IUCN and ICLEI - Local Governments for Sustainability – have committed to strengthening their global cooperation on sustainable development, with special emphasis on the integration of natural capital in urban and regional planning, and the implementation of nature based solutions. The two organisations signed a three-year Memorandum of Understanding (MoU) to this effect on 12 April 2015, during the 2015 ICLEI World Congress in Seoul. …
22 Apr 2015 | News story
IUCN Leaders for Nature – Developing tangible ideas for greener cities
Leaders for Nature is the IUCN Netherlands' business engagement network of twenty multinationals and major Dutch enterprises working together to green the economy. Its annual forum brings together private sector, government and civil society stakeholders to identify ways of integrating natural capital into business management and practices. …
21 Apr 2015 | News story
Commercial agriculture and forestry could have a net positive impact on biodiversity – IUCN report
Gland, Switzerland, 16 April 2015 (IUCN) – A new IUCN study examines, for the first time, how commercial agriculture and forestry production could reduce global biodiversity loss by applying innovative approaches already used by some companies in the extractive and infrastructure industries.
16 Apr 2015 | News story
Eye on Earth Summit in Abu Dhabi to boost environmental information for decision-makers
Over 650 delegates from government, UN bodies, the non-governmental sector, private sector, academia and civil society will gather in Abu Dhabi between 6 and 8 October for the Eye on Earth Summit 2015, to bridge the information gap policy makers face in designing plans for sustainable development. …
14 Apr 2015 | News story