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In this study, we demonstrate that the lack of retinoic acid-related orphan receptor (ROR) γ or α expression in mice significantly reduced the peak expression level of Cry1, Bmal1, E4bp4, Rev-Erbα and Per2 in an ROR isotype- and tissue-selective manner without affecting the phase of their rhythmic expression. Analysis of RORγ/RORα double knockout mice indicated that in certain tissues RORγ and RORα exhibited a certain degree of redundancy in regulating clock gene expression. Reporter gene analysis showed that RORγ was able to induce reporter gene activity through the RORE-containing regulatory regions of Cry1, Bmal1, Rev-Erbα and E4bp4. Co-expression of Rev-Erbα or addition of a novel ROR antagonist repressed this activation. ChIP-Seq and ChIP–Quantitative real-time polymerase chain reaction (QPCR) analysis demonstrated that in vivo RORγ regulate these genes directly and in a Zeitgeber time (ZT)-dependent manner through these ROREs. This transcriptional activation by RORs was associated with changes in histone acetylation and chromatin accessibility. The rhythmic expression of RORγ1 by clock proteins may lead to the rhythmic expression of RORγ1 target genes. The presence of RORγ binding sites and its down-regulation in RORγ−/− liver suggest that the rhythmic expression of Avpr1a depends on RORγ consistent with the concept that RORγ1 provides a link between the clock machinery and its regulation of metabolic genes.
The retinoic acid-related orphan receptors α-γ (RORα-γ or NR1F1-3) constitute a subfamily of the nuclear receptor superfamily (1). Through alternative splicing and promoter usage, each ROR gene produces several isoforms that are expressed in a highly tissue-specific manner. RORs have been implicated in the regulation of embryonic development and in several metabolic and immunological processes (1–12).
RORs are among a number of nuclear receptors involved in the regulation of circadian behavior and clock gene expression (1,8,13–18). In mammals, the suprachiasmatic nucleus (SCN) functions as the central circadian pacemaker that integrates light–dark cycle input and synchronizes the autonomous oscillators in peripheral tissues (19–21). At the molecular level the clockwork consists of an integral network of several interlocking positive and negative transcriptional and translational feedback loops. The heterodimeric complex consisting of brain and muscle ARNT-like (Bmal1) and circadian locomotor output cycles kaput (Clock) or its paralog neuronal PAS domain protein 2 (Npas2), are involved in the positive control of the oscillator, while two cryptochrome (Cry) and three period proteins (Per) are part of the negative control mechanism. Several accessory pathways, in one of which the Rev-Erb nuclear receptors play a role (19–21), further regulate the core loop. Although several studies have provided evidence for a regulatory function of RORs, particularly roles for RORα and RORβ in the SCN, in the regulation of circadian rhythm and clock gene expression, their precise function and regulation are not yet fully understood (13,15,16,18,22–27). Less is known about the role of RORγ in the regulation of circadian rhythm. Recently, we reported that Npas2 is directly regulated by RORγ suggesting that it may be an important modulator of the circadian clock in peripheral tissues (25).
To obtain greater insights into the roles of RORs in the regulation of circadian rhythm, we examined the effects of the loss of RORα and/or RORγ on clock gene expression in several peripheral tissues of ROR knockout mice. Our data showed that particularly the loss of RORγ reduced the peak expression level of several clock genes in a tissue-selective manner without significantly affecting the phase of their rhythmic expression pattern. The effect of RORα deficiency on the expression of these clock genes was limited largely to the kidney. Loss of both RORα and RORγ in double knockout (DKO) mice had a greater impact on the peak expression levels of these clock genes than single knockouts suggesting a certain degree of redundancy between RORα and RORγ. We demonstrated that RORs regulate the transcription of Cry1, Bmal1, Rev-Erbα and E4bp4 directly as indicated by reporter gene and mutation analysis. This transcriptional activation was inhibited by ROR antagonists. Chromatin immunoprecipitation sequencing (ChIP-Seq) and ChIP-QPCR analyses indicated that RORs were associated with these ROR response elements (ROREs) in vivo supporting the conclusion that these clock genes are directly regulated by RORs. We further showed that this transcriptional regulation was ZT-dependent and associated with changes in histone acetylation and chromatin structure. Recent studies demonstrated that clock proteins and Rev-Erbs are important regulators of energy homeostasis and lipid/glucose metabolism indicating a connection between the controls of circadian clock and various metabolic pathways (14,20,28,29). Although RORs regulate the expression of several metabolic genes, whether and what role they play in this interrelationship has yet to be determined (4,14,30,31). The rhythmic expression of RORγ1 by Bmal1/Clock and Rev-Erb might lead to the rhythmic expression of RORγ1 target genes. The in-phase expression pattern of RORγ1 and the arginine vasopressin receptor 1a gene (Avpr1a), the dramatic decrease in Avpr1a expression in RORγ−/− liver, and the ChIP-Seq data are consistent with the hypothesis that RORs function as intermediate regulators, providing a link between clock proteins and their regulation of metabolic genes.
C57BL/6 staggerer (RORαsg/sg) mice, a natural mutant strain containing a 6.5kb deletion in RORα that prevents translation of the LBD, were purchased from Jackson Laboratories (Bar Harbor, ME, USA) (32). These mice exhibit a very similar phenotype as mice with a targeted disruption of RORα (1,33). C57BL/6 RORγ−/− and RORαsg/sgRORγ−/− DKO mice were described previously (4,10). Mice were supplied ad libitum with NIH-A31 formula and water and maintained at 25°C on a constant 12h light:12h dark cycle. Littermate wild-type (WT) mice were used as controls. All animal protocols followed the guidelines outlined by the NIH Guide for the Care and Use of Laboratory Animals were approved by the Institutional Animal Care and Use Committee at the NIEHS.
To study gene expression during circadian time, tissues were excised from WT, RORγ−/− and RORαsg/sg mice every 4h over a period of 24h, processed overnight in RNAlater® solution (Ambion, Austin, TX, USA) at 4°C, and then stored at –80°C until use. Tissues from DKO and littermate WT mice were collected every 6h over a period of 24h. Tissues were homogenized in RNeasy lysis buffer (RLT) (Qiagen, Valencia, CA, USA) with a Polytron PT-3000 (Brinkmann Instruments, Westbury, NY, USA). RNA was then extracted using a QIAshredder column and RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Hepa1-6 stable cell lines and brown adipocytes were directly lysed in RLT buffer and extracted as described for tissues. Cells were treated for 24h with and without 10µM of the inverse ROR agonist T0901317 (Sigma-Aldrich, St Louis, MO, USA) (34) or with 1µM the RORγ-selective antagonist 'A' (R)-N-(1-((4-methoxy-phenyl)sulfonyl)-4-methyl-1,2,3,4-tetrahydroquinolin-7-yl)-2,4,6-trimethylbenzene-sulfonamide provided by GlaxoSmithKline (V. Birault, in preparation) before RNA extraction.
QRT–PCR analysis was performed by SYBR Green I or the TaqMan system (Applied Biosystems, Foster City, CA, USA) to quantify gene expression. The RNA was reverse-transcribed using High-Capacity cDNA Archive Kit (Applied Biosystems). The reactions were carried out in triplicate in a 7300 Real Time PCR system (Applied Biosystems) using 20ng of cDNA and the following conditions: 2min at 45°C and 10min at 95°C, followed by 40 cycles of 15s at 95°C and 60s at 60°C. All the results were normalized by the amount of 18S rRNA or Gapdh mRNA. Product specificity was confirmed by melting curve analysis. All QRT–PCR primer and probe sequences are listed in Supplementary Table S1.
Brown adipocytes were isolated from postnatal day 2 WT and RORγ−/− mice by collagenase digestion as described previously (25). Preadipocytes were immortalized by infection with pBabe retrovirus encoding SV40-Large T antigen as described previously (25). Brown preadipocyte cell lines BAT(RORγ1), BAT(RORα4), BAT(RORγ1E502Q) and BAT(RORα4ΔAF2) stably expressing Flag-RORγ1, Flag-RORα4, mutant Flag-RORγ1(E502Q) or Flag-RORα4ΔAF2 (35) and Hepa1-6(RORα) and Hepa1-6(RORγ) stably expressing Flag-RORα, Flag-RORγ, were generated by infection with the respective pLXIN retrovirus (Clontech, Palo Alto, CA, USA) and subsequent selection in G418 (25). The stable cell lines BAT(E) and Hep1-6(E) containing the pLXIN empty vector were used as controls. The expression and the nuclear localization of ROR proteins were confirmed by western blot analysis and confocal microscopy, respectively. Cell lines established from three to five individual clones were examined in QPCR and ChIP analysis.
The ChIP assays were performed using a ChIP assay kit from Millipore (Billerica, MA, USA) according to the manufacturer's protocol with minor modifications. In short, livers isolated from WT, RORαsg/sg and RORγ−/− mice at the circadian time indicated, were homogenized with a polytron PT 3000 (Brinkmann Instruments) and cross-linked by 1% formaldehyde for 20min at room temperature. After a wash in phosphate buffered saline (PBS), an aliquot of the cross-linked chromatin was sonicated and incubated overnight with anti-RORα, anti-RORγ or anti-Clock antibody (sc-6062, sc-28559 or sc-6927, respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-acetylated histone H3K9Ac antibody (07-352; Millipore). After incubation with protein G agarose beads for 2h, and several washes, DNA–protein complexes were eluted. The cross-links were reversed by overnight incubation at 65°C in the presence of 25mM NaCl and subsequent digestion with RNase A and proteinase K, followed by purification of the ChIPed-DNA. The ChIPed-DNA was then amplified by QPCR; all reactions were carried out in triplicate. The procedure for ChIP analysis using Hepa1-6 cells was similar as described for tissues except that 2×106 cells were cross-linked with 4% formaldehyde for 10min and the immunoprecipitation was performed with anti-Flag M2 affinity gel (Sigma-Aldrich). The sequences of the primers used for ChIP-QPCR are listed in Supplementary Table S2.
ChIPed-DNA was prepared as described (36) using RORγ- and RORα-specific antibodies generated against amino acids 129–231 and 121–213 in mouse RORγ1 and RORα4, respectively. ChIP-Seq analysis was performed by the NIH Intramural Sequencing Center and data were analyzed as reported previously (36).
Formaldehyde-assisted isolation of regulatory elements (FAIRE) was performed as previously described (37). Briefly, mouse livers were cross-linked and sonicated as described for the ChIP assay. Samples were centrifuged and the DNA in the supernatants was isolated by three consecutive extractions with phenol–chloroform–isoamyl alcohol (25:24:1). After the final extraction, the FAIRE samples were reverse cross-linked as described for the ChIP assay. The enrichment of fragmented genomic DNA in the FAIRE samples relative to each input DNA was measured in triplicate by QPCR.
The RORγ1(–1338/–1) and RORγ1(–1338/–968) promoter regions, and the RORE-containing regulatory regions of Cry1 (+22976/+23214), E4bp4 (+5828/+6150), Bmal1 (–650/+105), Rev-Erbα (–657/+38) and Avpr1a (–68969/–67651), and Avpr1a (–63512/–61359) were inserted into the pGL4.10 or pGL4.27 reporter plasmids. Corresponding mutant pGL4.10/27 plasmids, containing mutations within the E-boxes (CACGTG into CAATTG) or ROREs were generated using a Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The sequences were verified by DNA sequencing. Human hepatoma Huh-7 cells were co-transfected with pCMVβ-Gal, pCMV-Sport6-Bmal1, pCMV-Sport6-Clock, pCMV-Sport6-Cry1 and the pGL4.10 RORγ1(–1338/–1) or pGL4.27-RORγ1(–1338/–968) reporter plasmid as indicated using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24h incubation, the luciferase and β-galactosidase were analyzed with a Luciferase Assay Substrate (Promega) and Luminescent β-galactosidase Detection Kit II (Clontech). All transfections were performed in triplicate, and each experiment was repeated at least twice. Except for Rev-Erbα, all ROREs were mutated from A/GGGTCA to A/GAATCA. The RORE1 and RORE2 within the Rev-Erbα promoter were mutated from GTGTCACTGGGGCA to ATATCACTGGGGCA and from GTGTCACTGGGGCA to GTGTCACTGAAGCA, respectively.
As shown in Figure 1, RORγ1 exhibited a strong oscillatory pattern of expression in mouse liver consistent with previous results with samples from a different set of mice (25). RORγ1 also exhibited a robust oscillatory pattern of expression in brown adipose tissue (BAT), kidney and small intestines (jejunum) with peak expression around ZT16 (Figure 1), a few hours earlier than in liver. RORα1 and RORα4 expression did not show a strong circadian regulation in liver, BAT, WAT or kidney (Figure 1). Because of its negligible level of expression, the circadian regulation of RORα was not analyzed in the small intestines. The oscillatory pattern of RORγ expression was not changed in BAT or kidney of RORαsg/sg mice. The loss of RORγ did not affect RORα4 expression in kidney and only slightly reduced its expression in BAT, while it had little effect on the expression of RORα1 (Figure 1). These data indicated that RORα and RORγ are regulated largely independently from each other and that RORγ1 rather than RORα1 or RORα4 exhibits a robust oscillatory pattern of expression in these peripheral tissues.
A number of studies have provided evidence for a role of RORs in the transcriptional regulation of several clock genes (13–18,21,23,24). Two recent studies reported on the transcriptional regulation of Npas2 in RORγ−/− and RORαsg/sg mice (24,25). However, information on the regulation of clock genes by RORs in vivo has remained rather limited. We, therefore, compared the circadian expression of several clock genes in more detail in a few peripheral tissues of WT, RORαsg/sg and RORγ−/– mice. In liver, BAT, WAT, kidney and small intestines of WT mice, Bmal1, E4bp4, Cry1 and Rev-Erbα mRNA reached an optimal level of expression at ZT0 (Bmal1 and E4bp4), ZT16-20 (Cry1) and ZT4-ZT8 (Rev-Erbα) in agreement with previous studies (Figure 2A and B) (13,18,38–40). In RORγ−/− mice, Cry1 mRNA expression was consistently suppressed up to 30–40% in all four tissues with the largest decrease seen in the small intestines (Figure 2A). These observations suggested that RORγ plays an important role in the regulation of Cry1 expression across several peripheral tissues. The oscillatory pattern of expression of Bmal1 in liver, BAT, kidney and small intestines from RORγ−/− mice was very similar to those in WT mice or slightly reduced at peak levels in some tissues (Figure 2A). A small shift in the circadian phase of Bmal1 expression was observed in the RORγ−/− small intestines. The peak expression of Rev-Erbα was reduced in the liver, BAT and small intestines of RORγ−/− mice, but was not significantly altered in RORγ−/− kidney. E4bp4 expression was selectively suppressed by about 40–50% at peak levels in RORγ−/− liver and kidney, but changed little in BAT and the small intestines of RORγ−/− mice. Peak expression of Per2 was somewhat elevated in RORγ−/− liver, slightly diminished in BAT and kidney, and very significantly reduced in the small intestines of RORγ−/− mice. These observations support the hypothesis that RORγ regulates the circadian expression of Cry1, Bmal1, Rev-Erbα, E4bp4 and Per2 in a tissue-selective manner.
We next examined the expression of these clock genes in liver, BAT, WAT and kidney of RORαsg/sg mice. As shown for RORγ−/− mice, the phase of the oscillatory pattern of expression of Bmal1, Cry1, Rev-Erbα, E4bp4 and Per2 was not significantly altered in any of the RORαsg/sg tissues analyzed (Figure 2B). In contrast, a significant reduction in the peak level of expression of Bmal1, E4bp4 (ZT18-20) and Per2 (ZT12) was observed in the kidneys of RORαsg/sg mice. Rev-Erbα peak expression at ZT8 was reduced in the kidney and WAT of RORαsg/sg mice, but was unaltered in the liver and BAT. No significant changes in the rhythmic expression pattern of Cry1 were observed in any of the four tissues of RORαsg/sg mice. These results demonstrated that the loss of RORα affected the expression of certain clock genes in a limited and tissue-selective manner. RORα regulated clock gene expression particularly in the kidney, a tissue in which it is relatively highly expressed (Y. Takeda, data not shown).
Previous studies have indicated that RORα and RORγ exhibited a certain degree of redundancy with respect to their effects on gene expression (4,7). To examine whether RORα and RORγ exhibited any redundancy in regulating clock gene expression, we examined the circadian expression of clock genes in liver, BAT and kidney collected from RORαsg/sgRORγ−/− DKO mice. As shown in Figure 3 and Supplementary Figure S1, the expression of several clock genes was reduced to a greater extent in certain tissues of DKO mice than those of RORγ−/− or RORαsg/sg single knockout mice. Particularly, Cry1 and Bmal1 mRNA expression were down-regulated to a significantly greater extent in the liver and BAT of DKO mice than in single knockout mice. The peak expression of Npas2 and Clock was also notably more reduced in DKO liver compared with their reported expression in single knockout mice (25). Little redundancy between RORα and RORγ was observed in the kidney. These results indicated that in some peripheral tissues RORα and RORγ exhibit a certain degree of redundancy regulating clock gene expression.
RORs bind as monomers to ROREs consisting of the consensus sequence AGGTCA preceded by a 6-bp A/T rich region, in the promoter regulatory region of target genes (1). ROREs have been identified in several clock genes; Cry1, Bmal1 and Rev-Erbα contain two putative ROREs, while the E4bp4 and Clock genes contain one site (Supplementary Figure S2A) (13,15,16,18,22,23,38,41,42). However, the significance of these ROREs and the role of RORγ1 in clock gene regulation in vivo are still far from understood. Both RORα and RORγ were able to induce the Luc reporter driven by the RORE-containing regulatory region of Cry1, E4bp4, Bmal1, Rev-Erbα and Clock in Huh-7 cells. RORα and RORγ induced promoter activity of Cry1, respectively, 24- and 8-fold, Bmal1 16- and 8-fold, Rev-Erbα 7- and 5-fold, 3- and 2-fold (Supplementary Figure S2B) and Clock 4- and 2-fold (Supplementary Figure S3A) compared with cells transfected with the empty vector. Except for the RORE2 in Rev-Erbα, point mutations in each of these ROREs greatly reduced the activation of the Luc reporter by either RORα or RORγ. Double mutations in RORE1 and RORE2 in Cry1 and Bmal1 almost totally abolished the activation by RORs. These observations suggest that both ROREs are important in the regulation of Cry1 and Bmal1 and that RORE1 is most critical for the ROR-mediated regulation of Rev-Erbα, E4bp4 and Clock consistent with previous observations (13,15,22). Similar results as for Huh-7 cells were obtained in HEK293 cells (Y. Takeda, data not shown).
Previous studies reported that the transcriptional repressor Rev-Erbα can compete with RORs for the binding to ROREs (14,15,38,43). Figure 4A shows that exogenous Rev-Erbα expression effectively inhibited the Cry1(RORE1/2), Bmal1(RORE1/2), Rev-Erbα(RORE1)-dependent activation by either RORγ or RORα in a dose-dependent manner, likely by competing with RORα and RORγ for the same ROREs. Rev-Erbα had only a small effect on the E4bp4(RORE)-dependent activation by RORs (Figure 4A).
Recently several ligands have been identified that function either as ROR agonists or antagonists (34,44–46). To examine the regulation of clock genes by RORs further, we examined the effect of the RORα and RORγ inverse agonist, T0901317, and the RORγ-selective antagonist 'A' on the activation of the RORE-containing regulatory region of several clock genes by RORs. T0901317 significantly inhibited Cry1(RORE1/2)-, Bmal1(RORE)-, Rev-Erbα(RORE)-, E4bp4(RORE)- and Clock(RORE)-dependent trans-activation of the reporter by both RORα and RORγ in Huh-7 cells (Figure 4B and Supplementary Figure S3B). In contrast, the RORγ-selective antagonist 'A' effectively inhibited the RORγ-induced trans-activation at concentrations 10× lower than T0901317, but had little effect on the activation mediated by RORα (Figure 4B).
To analyze the role of RORs and the effect of the RORγ-selective antagonist 'A', on the endogenous expression of Cry1, Bmal1, Rev-Erbα and E4bp4, we generated Hepa1-6 cells stably expressing the empty vector, Flag-RORα4 or Flag-RORγ1 (25). Expression of RORα and RORγ mRNA was greatly elevated in Hepa1-6(RORα4) and Hepa1-6(RORγ1), respectively (Figure 4C). Exogenous expression of RORα4 did not affect the levels of RORγ1 mRNA, neither did RORγ1 affect the expression of RORα, consistent with our conclusion that RORα and RORγ1 are regulated independently from each other. Most importantly, RORγ1 and RORα4 significantly increased the endogenous expression of Cry1, Bmal1, Rev-Erbα, E4bp4 and Clock compared with Hepa1-6(Empty) cells (Figure 4C and Supplementary Figure S3C). The addition of the RORγ-selective antagonist 'A' significantly reduced the expression of clock genes in Hepa1-6(RORγ1) cells, but had little effect in Hepa1-6(RORα4) cells (Figure 4D).
As shown for Hepa1-6 cells, Baml1, Cry1, Rev-Erbα and E4bp4 expression was also increased in brown preadipocyte cell lines, BAT(RORα4) and BAT(RORγ1), stably expressing Flag-RORα4 and Flag-RORγ1, respectively (Supplementary Figure S4). However, the expression of these clock genes was not changed in BAT(Flag-RORαΔAF2) and BAT(RORγE502Q), expressing Flag-RORαΔAF2 or Flag-RORγE502Q, mutants defective in their trans-activation function. Treatment of BAT(RORγ1) cells with the inverse agonist T0901317 inhibited the increased expression of Cry1, Bmal1, Rev-Erbα and E4bp4. All together, these observations are consistent with the conclusion that the expression of these clock genes is regulated by RORγ and RORα and is dependent on the activation domain of RORs.
To determine whether these clock genes were directly regulated by RORα/γ in vivo, we analyzed our genome-wide maps of RORα/γ binding sites derived from ChIP-Seq analysis using ChIPed-chromatin from ZT22 mouse liver, for clock gene-associated ROR binding sites. This analysis identified RORα and RORγ binding peaks at RORE-containing regions near or within the Cry1, Npas2, Bmal1 and E4bp4 genes (Figure 5A). The strongest peaks were associated with Cry1 and Npas2, while a moderate association was found with Bmal1 and E4bp4. Low associations were found with ROREs within Rev-Erbα and Clock (data not shown). Most importantly, the strong ROR-binding peaks (indicated by arrows) for Cry1, Bmal1 and E4bp4 corresponded to the RORE-containing regions studied in Supplementary Figure S2. In the case of Npas2, the strong ROR-binding peak corresponded to the Npas2-RORE described recently (24,25).
To examine the specificity of the recruitment of RORs to the RORE-containing regulatory region of the Bmal1, Cry1, Rev-Erbα and E4bp4 genes in vivo, ChIP-QPCR was carried out using ChIPed liver DNA from WT, RORαsg/sg and RORγ−/− mice (Figure 5B and Supplementary Figure S3D). Both RORα and RORγ were found to be associated with these RORE-containing regulatory regions in WT liver. As expected, ChIP analysis with the RORγ antibody did not show a significant increase in amplification of these regulatory regions in RORγ−/– liver; however, RORγ was still recruited to these RORE regions in RORαsg/sg liver in agreement with the specificity of the RORγ antibody. ChIP analysis with RORα antibody yielded the inverse results. Non-RORE containing regions within Gapdh and clock genes, which served as negative controls, showed low amplification.
Because of the strong oscillatory pattern of expression of RORγ1, we compared ChIP-QPCR analysis with liver tissues collected at ZT22, a time close to the peak expression of RORγ1 and Cry1 and Bmal1, with those isolated at ZT10, when these genes are expressed at lower levels. We hypothesized that RORγ1 would be associated with the Bmal1(RORE1/2) and Cry1(RORE1/2) regions particularly at the time when they are most highly expressed. Figure 5C confirmed that RORγ1 was more efficiently recruited to these regulatory regions in ZT22 liver than in ZT10 liver. No increased amplification was observed with samples from RORγ−/− liver at either ZT. RORγ1 showed a weaker association with the Rev-Erbα(RORE1) and E4bp4(RORE1) regions (Figure 5C). The recruitment of RORγ1 to the Rev-Erbα(RORE1) was higher at ZT22 than ZT10 and correlated inversely with the level of Rev-Erbα expression.
The association of RORα and RORγ with the RORE-containing regulatory regions of the Bmal1, Cry1, Rev-Erbα and E4bp4 was supported by ChIP analysis using an anti-Flag M2 antibody and chromatin isolated from Hepa1-6(Empty), Hepa1-6(RORα4) Hepa1-6(RORγ1) cells. As shown in Figure 5D, RORα and RORγ were strongly associated with ROREs in the Bmal1(RORE1/2) and Cry1(RORE1/2) regions and to a lesser extend to those of Rev-Erbα and E4bp4. No significant recruitment of RORs was observed in Hepa1-6(Empty) cells, to distal sites or the Gapdh promoter, which served as negative controls. Collectively, the ChIP-Seq and ChIP-QPCR data are in agreement with the concept that both RORα and RORγ regulate several clock genes directly through their interaction with specific ROREs.
Changes in gene expression are often associated with alterations in chromatin structure, histone methylation and acetylation. We, therefore, examined whether the association of RORs with the Bmal1(RORE1/2) and Cry1(RORE1/2) regions correlated with changes in histone acetylation and chromatin accessibility. Levels of histone acetylation, including H3K9Ac have been reported to correlate positively with actively transcribed genes (47). ChIP analysis with DNA samples from livers isolated at ZT10 and ZT22 showed that the level of H3K9Ac associated with the Cry1(RORE1/2) and Bmal1(RORE1/2) regions was considerably higher at ZT22 than at ZT10 (Figure 6A). This correlated with the high level of association of RORα and RORγ with these regions (Figure 5A and B) and the elevated expression of these clock genes at ZT22. The H3K9Ac signal was significantly reduced in RORγ−/− liver, not affected in RORαsg/sg liver, and further decreased in DKO mice. These observations indicate that the recruitment of particularly RORγ is associated with actively transcribed chromatin and elevated expression of Bmal1 and Cry1.
To assess chromatin accessibility, we performed FAIRE, which has been used as a tool to identify actively transcribed genes (37). FAIRE analysis on the Cry1(RORE1/2) and Bmal1(RORE1/2) regions with chromatin isolated from liver at ZT10 and ZT22 showed that the FAIRE signals were markedly higher at ZT22, a time at which Cry1 and Bmal1 are highly expressed, compared with that of ZT10 (Figure 6B). There was no significant change in FAIRE signal at the distal region of the Cry1 gene. Loss of RORγ or RORα expression led to a reduction in FAIRE signal at ZT22. This observation is consistent with our H3K9Ac data and suggests that the binding of RORs may promote a more open chromatin structure at these regulatory sites.
As shown in Figure 1, RORγ1 has a strong oscillatory pattern of expression. Previous studies reported that the RORγ1 promoter contains two successive E-boxes (E1 and E2) and is activated by Clock/Bmal1 heterodimers (18,48). Reporter gene analyses in human hepatoma Huh-7 cells confirmed that Clock/Bmal1 greatly enhanced RORγ1(–1338/–1) proximal promoter activity and that mutation of either E1 or E2 significantly reduced the activation, while the double mutation totally abolished the induction by Clock/Bmal1. Co-expression with the transcriptional repressor Cry1 almost completely blocked the activation of the RORγ1 promoter by Clock/Bmal1 (Figure 7A and B). To determine whether Clock protein was associated with the E-box-containing RORγ1(–1179/–1042), we performed ChIP analysis using chromatin isolated from mouse liver and an anti-Clock antibody. Our data showed that Clock protein was recruited to the E-box region of the proximal RORγ1(–1179/–1042) promoter region in vivo (Figure 7C). The recruitment of Clock protein to the Cry1 E-box (38) was used as a positive control. Relatively little association of Clock protein was observed with a distant region (at –6.2kb) of the RORγ1 promoter, which served as a negative control. The level of Clock recruitment to the RORγ1(–1179/–1042) region was not very different between ZT10 and ZT22 similar to what has been reported previously for the recruitment of Clock/Bmal1 to the Cry1(E-box) promoter region (38). These data strengthen the previous conclusions (18,48) that Clock/Bmal1 and Cry1 complexes regulate RORγ1 expression through their interaction with E-boxes in the RORγ1 promoter.
To assess chromatin accessibility in relation to the level of RORγ1 expression, we performed FAIRE on the RORγ1(–1179/–1042) promoter region with chromatin isolated from liver at ZT10 and ZT22. As shown in Figure 7D, the FAIRE-QPCR signal was markedly higher in samples isolated from liver at ZT22, a time at which RORγ1 is highly expressed, compared with ZT10 when RORγ1 expression is low. A low FAIRE-QPCR signal was obtained at a distal site. Our results are consistent with the concept that at ZT22 this region of the RORγ1 promoter is more accessible and associated with functionally active enhancers. This was supported by ChIP analysis using an H3K9Ac antibody. The level of H3K9Ac associated with the RORγ1(–160/–70) region was considerably higher at ZT20 than at ZT8 (Figure 7E). This is consistent with the high level of RORγ1 mRNA expression at ZT20 and a more open chromatin structure at this site.
We previously proposed that in addition to their modulation of clock gene expression, RORs might function as a link between the clock machinery and the circadian regulation of down-stream genes (1). The loss of RORs has been shown to affect the rhythmic expression of several genes involved in glucose and lipid metabolism, including perilipin 2 (Plin2 or Adfp) and arginine vasopressin receptor 1a (Avpr1a) (17,31). Figure 8A shows that as for Npas2 and Cry1, the peak expression of Avpr1a at ZT0, a time at which RORγ1 is most highly expressed, was greatly diminished in RORγ−/− liver. The loss of RORα had little effect on Avpr1a expression, while the pattern of Avpr1a expression in DKO liver was very similar to that in RORγ−/− liver. These observations indicate that Avpr1a expression is selectively regulated by RORγ1. Analysis of our genome-wide map of RORγ binding sites showed strong association of RORγ with two regions, A and B, upstream from the Avpr1a transcription start site (Figure 8B). RORγ was able to induce region A-dependent trans-activation, but not region B-mediated trans-activation (Figure 8C). ChIP analysis showed that RORγ was recruited to region A at ZT22 (Figure 8D), but not at ZT10, as observed for clock genes (Figure 5C). Although overexpression of RORα induced region A-dependent trans-activation (Figure 8C), RORα was only weakly associated with region A and B (Figure 8B and D). Together, these observations suggest that RORγ1, rather than RORα, is involved in the direct regulation of the rhythmic Avpr1a expression.
In this study, we demonstrate that RORα4 and RORα1 have a weak and that RORγ1 exhibits a robust oscillatory pattern of expression in BAT, WAT, kidney and jejunum as we reported previously for liver (25). We further show that the loss of RORγ did not significantly alter the expression pattern of RORα1 or RORα4, while the loss of RORα had little effect on the oscillatory expression pattern of RORγ1 in either BAT or kidney suggesting that RORα and RORγ are regulated independently from each other under the conditions tested. To obtain further insights into the roles of RORα and RORγ in circadian regulation in vivo, we compared the oscillatory expression pattern of Cry1, Baml1, E4bp4, Rev-Erbα and Per2 in several tissues from RORα and RORγ single knockout and RORα/RORγ DKO mice. The loss of either RORα or RORγ, or both had either a small or no effect on the phase of the rhythmic expression of these clock genes (Figures 2 and and3).3). However, the loss of RORα or RORγ affected the peak expression of several clock genes in a tissue- and ROR isotype-selective manner. Loss of RORγ significantly reduced the peak expression of Cry1, Rev-Erbα, E4bp4 and Per2 in most tissues tested and caused a small reduction in Bmal1 peak expression. The increased levels of Per2 in RORγ−/− liver might be related to the observed, reduced expression of E4bp4, which has been reported to function as a negative regulator of Per2 (49). The effect of the loss of RORα on the expression of Bmal1, Rev-Erbα, E4bp4 and Per2 was limited mainly to the kidney, a tissue in which RORα is relatively highly expressed, while Cry1 expression was little changed in RORαsg/sg kidney. We reported previously that the loss of RORγ, but not the loss of RORα, reduced peak expression of Npas2 and Clock (25). These observations indicated that both RORs, but particularly RORγ, are modulators of clock gene expression in several peripheral tissues. The tissue selectivity of clock gene regulation by RORα and RORγ might be due to differences in their level of expression and/or transcriptional activity.
We previously reported that RORα and RORγ exhibit a certain degree of functional redundancy in liver and Th17 cells. The hepatic expression of several Phase I and Phase II metabolic genes was affected to considerably greater extent in DKO than in single knockout mice (4) and both RORα and RORγ have been shown to promote Th17 differentiation and Il-17 expression (50). We, therefore, were interested in determining whether RORα and RORγ exhibited any redundancy in regulating clock gene expression in liver, kidney and BAT. Comparison of Cry1, Bmal1, Rev-Erbα, E4bp4, Per2, Npas2 and Clock expression between single knockout and DKO mice showed a complex picture. The expression of Cry1, Bmal1, Rev-Erbα, Npas2 and Clock in particular was reduced to a significantly greater degree in liver and BAT of DKO mice than in single knockout mice (Figure 3 and Supplementary Figure S1). A greater reduction in Cry1, Bmal1 and Rev-Erbα expression was also observed in DKO kidney; however, little redundancy was observed in the small intestines (jejunum), where RORα is not significantly expressed. Our data suggest that RORα and RORγ exhibit a degree of functional redundancy in their regulation of clock gene expression that explains some of the synergistic effects observed in some tissues of DKO mice. It is interesting to note that Rev-Erbα and Rev-Erbβ, which can compete with RORs for the same bindings sites, also work together in regulating clock gene expression (51,52).
The regulation of Cry1, E4bp4, Baml1 and Rev-Erbα by RORα4 and RORγ1 was supported by data showing that exogenous expression of RORα4 or RORγ1 in Hepa1-6 and BAT cells increased the expression of all four clock genes (Figure 4C and Supplementary Figure S4). Mutant ROR receptors lacking trans-activating activity were unable to enhance expression of these clock genes indicating that the induction of clock genes by RORs is dependent on the activation function of RORs. This was supported by data showing that ROR antagonists significantly reduced the induction of Bmal1, Cry1, E4bp4 and Rev-Erbα mRNA by RORα and RORγ.
RORs regulate gene transcription directly by binding ROREs in the regulatory region of target genes (1). Previous studies have identified putative ROREs in a number of clock genes (13,15,18,22–25,41). Reporter gene analysis showed that both RORα and RORγ were able to activate the Luc reporter under the control of the RORE-containing regions of Cry1, Bmal1, Rev-Erbα, E4bp4 or Clock (Supplementary Figures S2 and S3). Mutation of either the RORE1 or RORE2 in Bmal1 or Cry1 partially reduced this activation, whereas mutation of both ROREs totally abolished the activation of the Luc reporter suggesting that both RORE1 and RORE2 are required for the optimal activation of these genes by RORα and RORγ. In the case of E4bp4, Rev-Erbα and Clock the activation by RORα and RORγ was mediated through RORE1. The activation of these regulatory regions was inhibited by co-expression with Rev-Erbα, a transcriptional repressor that can compete with RORs for RORE binding (14,15,41,43). Moreover, treatment with the RORα and RORγ inverse agonist T0901317 significantly inhibited the activation by either RORα or RORγ, while the RORγ-selective antagonist 'A' inhibited the induction by RORγ, but not that by RORα (Figure 4 and Supplementary Figures S3 and S4). These observations are consistent with the concept that RORα and RORγ enhance the expression of clock genes directly by binding ROREs in their promoter regulatory region and further confirm that this induction requires the activation function of RORs. The mouse and human Cry1(RORE1/2) are distal from the transcription start site (TSS) and located within introns at +23086/23141 and +66014/66068, respectively. ROR activator complexes bound to distal ROREs might exert coordinated control of Cry1 expression via a physical interaction with protein complexes at the proximal promoter or TSS (53).
Our genome-wide maps of RORα/γ binding sites in liver identified major RORα and RORγ binding peaks near or within the Cry1, Npas2, Bmal1 and E4bp4 genes (Figure 5A). These sites corresponded to the ROREs studied in this article or reported previously (18,24,25). The recruitment of RORs to these regions was further supported by ChIP-QPCR analysis using ChIPed DNA from liver tissue and Hepa1-6(RORγ1) or Hepa1-6(RORα4) cells. The specificity of ROR binding was indicated by data showing that no significant recruitment of RORα or RORγ was observed in, respectively, RORαsg/sg and RORγ−/– liver or to non-RORE containing distal regions of the respective clock gene or Gapdh. Because RORγ1 exhibits a robust circadian pattern of expression, we determined whether the recruitment of RORγ1 to the regulatory regions was ZT-dependent. This was supported by ChIP-QPCR analysis showing higher levels of association of RORγ1 with the RORE-containing regulatory regions of Cry1, Bmal1 and E4bp4 at ZT22, when RORγ as well as these clock genes are relatively highly expressed, than at ZT10, when these genes are expressed at low levels. Moreover, increased competition for RORE binding by Rev-Erbα/β, which are highly expressed at ZT10, is likely a major factor in the reduced interaction of RORs with ROREs at this ZT. Unexpectedly, the recruitment of RORγ1 to the Rev-Erbα(RORE) was also higher at ZT22 than ZT10 despite the low expression of Rev-Erbα at ZT22 indicating that binding of RORγ is not necessarily sufficient to enhance transcription of Rev-Erbα. ChIP-Seq analysis suggests that this association is relatively weak suggesting that RORs may play a lesser role in regulating Rev-Erb expression. Collectively, our promoter analyses, and ChIP-Seq data support the concept that the up-regulation of Cry1, Npas2, Bmal1 and E4bp4 transcription by RORs in vivo occurs by a direct mechanism and involves recruitment of RORγ1 and RORα to ROREs within the regulatory regions of these genes. It is interesting to note that the Rev-Erb binding sites associated with Bmal1 and E4bp4 identified by ChIP-Seq analysis are the same as the ROR binding sites identified by our ChIP-Seq analysis (52) consistent with competition and regulation through the same binding sites. However, for Cry1 the major ROR binding sites were different from those described for Rev-Erb suggesting that RORs and Rev-Erbs may not always regulate clock gene expression through competition for the same response elements.
In many instances activation of transcription is accompanied by changes in chromatin structure. Actively transcribed regions are associated with an open chromatin structure and accompanied by changes in histone acetylation and methylation. ChIP analysis with an antibody against H3K9Ac, a marker for actively transcribed chromatin, showed that the level of H3K9Ac association with the Bmal1 and Cry1 regulatory regions in WT liver was higher at ZT22 than at ZT10 (Figure 6A). This association was diminished in RORγ−/– liver, but not altered in RORαsg/sg liver. FAIRE, which has been used as a tool to map open chromatin (37), revealed that the FAIRE signal was significantly increased at the Cry1(RORE1/2) and Bmal1(RORE1/2) regulatory region in liver at ZT22 compared with ZT10 consistent with increased chromatin accessibility at these sites. This correlates with the increased association of H3K9Ac and the relatively high level of Cry1 and Bmal1 expression at ZT22 (Figure 6B). The loss of RORγ caused a significant reduction in FAIRE signal at the Cry1(RORE1/2) and Bmal1(RORE1/2) region consistent with a positive relationship between RORγ binding, chromatin remodeling and transcriptional activation of these genes.
In addition to the circadian regulation of clock genes by RORs, RORγ itself exhibits a robust rhythmic expression in several peripheral tissues that is controlled by clock proteins (Figure 1) (13,15,18,25). The positive regulation of RORγ1 is partially mediated through the binding of Bmal1-Clock heterodimers to two E-boxes in the proximal promoter of RORγ1 consistent with a previous study (48). Moreover, one of the E-boxes (E2) within RORγ has been reported to be able to drive the oscillation of a reporter in cultured cells, indicating the importance of this E-box in the circadian regulation of RORγ by Clock/Bmal1 dimers (18). In this study, we show that the repressor Cry1 effectively blocked this activation (Figure 7). Cry1 likely mediates this repression through inhibition of the transcriptional activity of Bmal1/Clock heterodimers (54). ChIP analysis using an anti-Clock antibody showed that Clock protein was recruited to the (E1/2-box) sites in the RORγ1 promoter in liver consistent with the hypothesis that RORγ1 transcription is regulated directly by Bmal1-Clock heterodimers in vivo. Unexpectedly little difference was observed in Clock recruitment between ZT10 and ZT22, times of low and high RORγ expression, respectively. Similarly, a previous study demonstrated that the degree of Bmal1-Clock binding did not correlate with the level of activation of Cry1 transcription (38). These observations suggested that binding of Bmal1-Clock is not necessarily sufficient to activate transcription of RORγ1. The latter may be due to a repressor function of the Bmal1-Clock complex and the type of co-repressors or co-activators recruited to the heterodimer, or crosstalk with other transcriptional factors (55). In contrast to Clock binding, the association of H3K9Ac and the FAIRE signal was significantly increased at the RORγ1(E-box) promoter region in liver at ZT22 compared with ZT10 indicating that the transcriptional activation of RORγ1 correlated with increased chromatin accessibility consistent with an association between open chromatin structure and functionally active enhancers. In addition to its regulation by Bmal1/Clock, Rev-Erbs are recruited to the regulatory region of RORγ (52), consistent with the concept that they are also involved in the regulation of the rhythmic expression of RORγ.
In summary, study of RORα, RORγ and DKO mice showed that ROR receptors modulate the expression of several clock genes in an ROR isoform- and tissue-dependent manner. Particularly RORγ1 appears to affect peak expression of clock genes in several peripheral tissues. Study of DKO mice, ChIP-Seq and ChIP-QPCR analysis indicated a certain degree of redundancy between RORα and RORγ and demonstrated that in vivo RORα/γ regulate these genes directly and in a ZT-dependent manner through their interaction with ROREs. We provided evidence that this transcriptional regulation is associated with changes in chromatin structure. Our results are consistent with the concept of a reciprocal relationship between the regulation of clock genes and RORγ1 (Supplementary Figure S5). Recent studies demonstrated that clock proteins are important regulators of energy homeostasis and lipid/glucose metabolism suggesting a connection between the regulation of the circadian clock and various metabolic pathways (4,14,20,30,31). We previously proposed that RORs might function as a link between clock proteins and their regulation of metabolic genes (1). One might predict that the Bmal1/Clock/Rev-Erb-mediated rhythmic expression of RORγ1 leads to the rhythmic expression of RORγ1 target genes, including metabolic genes, such as Avpr1a. This concept is supported by observations showing that the oscillatory expression of RORγ1 and Avpr1a are in phase, that the loss of RORγ greatly reduces Avpr1a peak expression, and the presence of RORγ binding sites (Figure 7) (17). In this scenario, the rhythmic expression of downstream target genes, such as Avpr1a, depends on the rhythmic expression of RORγ1 and to a certain degree on Rev-Erb through its regulation of RORγ1 and/or its competition with RORγ1 for Avpr1a(RORE) binding (Supplementary Figure S5). Analysis of additional RORγ1 target genes is required to obtain further support for this hypothesis.
Supplementary Data are available at NAR Online: Supplementary Tables 1 and 2 and Supplementary Figures 1–5.
Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health [Z01-ES-101586]. Funding for open access charge: National Institute of Environmental Health Sciences, National Institutes of Health.
Conflict of interest statement. None declared.
The authors thank Drs Kristin Lichti-Kaiser and Gary ZeRuth for their comments on the manuscript and Laura Miller (NIEHS) for her great assistance with the mice.
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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Worlds Largest 3D Handcrafted Origami Peacock at MET
Mr. Vinod Chaudhary from MET Institute of Pharmacy has a different streak. It may sound little strange but this B Pharm student is interested in the origami as much as he is in the molecules and chemicals. He along with 42 student volunteers has created a three dimensional origami canvas measuring about 235 cubic feet, in which stands a uniquely hand crafted peacock using hand folded colourful paper sheets.
This gigantic peacock has 12180 paper sheets that have gone in its making. Passionate Vinod, began the work on 12th May at 10 am and finished it on 13th May 2015 afternoon, in effective 15 hours and 08 min. This 3-D origami peacock is magnificent, huge and yet beautiful. 93 x 112 x 39 cubic inches in size it is all set to make India proud with its entry into Limca Book of World Records. Believe it or not! See it to believe it.

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Sottini, the timeless bathroom brand built on a heritage of quality and innovation in design, unveils an exciting new addition to its portfolio at kbb Birmingham, 6th – 9th March 2016 with the launch of the new Mavone collection.


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Chefs to Defy Foie Gras Ban?
Rabbit albondigas, teriyaki foie gras, green garlic tempura, miso broth by chef Laurent Quenioux at Starry Kitchen.
Foie gras, the delicacy beloved by gourmands and protested by animal rights activists, may be off California menus by next summer. SB 1520, which bans force-feeding of ducks or geese to make foie gras and forbids selling foie gras produced that way, takes effect in July 2012. It was signed into law in 2004 by then-governor Arnold Schwarzeneger.
"When the ban comes in, we're going to serve it every day," chef Laurent Quenioux tells Bloomberg. "They can send me the foie gras police."
Some chefs, like Thomas Keller, are resigned to the ban, while others vow to band together and fight the ban via legal challenges in the courts. A similar foie gras ban was passed in Chicago then lifted in 2008 at the urging of Mayor Richard Daley.
In the meantime, cook it, if you got it. Here's a recipe for foie gras fingerling potato chips and a behind-the-stoves look at how chef Ludo Lefebvre makes his fabulous foie gras cupcake.
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A psychic attack is when someone sends their energy to you in a way that creates a unpleasant effect upon you. When one person sends their energy to another person and their energy, affects the other person in a detrimental way – this is a psychic attack.
One person sending their energy to another is not uncommon, in fact, it's completely natural in human interactions, as we 'send energy' to each other in nearly every thought, emotion or verbal communication that we have involving any other human being. You can send energy to someone by thinking about them, praying about them, talking about them in conversation, and each time you speak the name (or think it!) of another, you are calling in, and dialing up their energetic Spirit.
Think of yourself as two beings – a being with a physical body and a being with a spiritual body.
When your physical self wants to communicate with someone else in a physical body, you can call out to this other person with words and vocalizations, in-person, down the street, through an email or a telephone call, to grab their attention. And when your spiritual self – your emotive, thoughtful, and inner self – wants to get someone's attention – you can also send out your energy to connect with the inner self of this other person. This is how you can get the attention of someone's Spirit.
There are many ways to call up the energy of another.
Have you ever been really upset with someone, so upset, that you crafted an entire rant that you would lay on them, if ever given the chance?
Have you ever been so mad at someone, you were fuming with steam, and if you had been a volcano, you would have erupted?
Have you ever held an entire conversation in your head with someone, where you were 'letting' this someone really have it, but not to their face?
Well, if you have, then congratulations, because you've just psychically attacked someone. . . . on accident.
And, as you might imagine, it can happen so easily, that there is barely ever mal-intent involved. Most psychic attacks are non-intentional. We've all been angry with another person and thought about them in a way that was less than loving and full of compassion. We have all done this.
If you've ever been jealous of another, frustrated with another, wished you could have something that another person has, or been really furious with another, then you've psychically attacked them . . .at least once. Yes, even if was an accident. And someone has, likely done this to you. Probably more than once.
As individuals with physical, energetic, and spiritual bodies, we are able to operate within all three of these realms. Often times, if someone is feeling upset, angry, or frustrated with you (or other similar emotions), and they are not able, for whatever reason, to express and release those emotions freely or safely in the physical space, these feelings may still be directed at you at the level of spirit and energy.
Psychic attacks can have detrimental effects on the recipient of the attack.
Because we're all humans, even with the best intentions, we've all had the opportunity to think about someone in a kind and loving way, and therefore, send them positive pink-bubble gum energy. And conversely, we've all had the opportunity to have thoughts about someone else that were on the lower end of the love spectrum, and therefore, send them negative black tar energy. Or, sword energy. Or, punching in the stomach energy.
You can feel an emotional attack in the same way you can feel a physical attack.
Physical attacks – punches and slaps – can hurt emotionally.
Emotional attacks – word venom and swords – can hurt physically.
Psychic attacks occur at the level of emotion, energy and Spirit.
When you have something happening in one aspect of who you are (in your spiritual body or in your physical body) it often shows up in other areas of your life.
If you have been psychically attacked by another, you can feel it.
How do you know if you are being psychically attacked?
If you have just gotten into an argument, are having discord in a relationship with another or found yourself in an altercation with another human being just a few hours ago, and suddenly, you have:
A headache
Chest pains
Lower back pains
It's likely you are sensing the physical response of a spiritual issue, as psychic attacks can often feel like stabbing pains, punching in the stomach, squeezing of the head or crown or kicking in the back, they can account for many inexplicable and unpleasant sudden sensations. There are many ways to stop a psychic attack, through cutting cords, refusing the energy, or protecting your energetic body. Sometimes, though, an acknowledgement that it's happening and an intentional refusal of it is enough to stop the symptoms.
With love on your journey,
Want to learn about psychic attacks and how to stop them? Catch the recorded video class on this topic and learn how to prevent them, stop them, and deal with them – practically. Learn more here.
LAST UPDATED: October 16, 2014


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Articles Tagged 'Metro'
Photo Friday: The Skeleton of Syntagma
August 27, 2010 | Photo Friday | Read MoreAs asked by many readers of the article about the Wonderful, Marvelous, Metro of Athens, here is the star of the underground's archaeological exhibitions.
Photo courtesy of Eve Astrid Andersson
You need never feel alone in the Syntagma Station.
The Wonderful, Marvelous, Metro of Athens
August 26, 2010 | Travel Tips | Read MoreIn her essence, Athens is an adventurous city that thrives on spontaneity. It is my sweet and loving way of pointing out that it can, on occasion, feel somewhat disorganized. Chaotic, even. But surprise! The Athens Metro is one of the cleanest, safest, cheapest, most beautiful and reliable metro systems...


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I'll believe it when I read about it on the AP, but the site Hip Hop DX is saying the show has been pulled. Y'all know how internet rumors get started, but I am guardedly optimistic.
UPDATE (7/14): Still waiting. Latrice the young lady who has been running a petition since January to get Hot Ghetto Mess pulled indicated that she had also gotten an e-mail.
The cynic in my says that BET is just trying to fake us out to make it past the Television Critics Association panel on Sunday.
UPDATE(7/14): I just read an article that said we get 18000 hits a day. Dear Lord NO! That would be a nightmare! We have had a TOTAL of 18000 hits as of yesterday. I fully expect to go back to normal this time next week.


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Availability date: 06/27/2013
Please note the AEM cold air intake system will not work with most front mount intercooler kits. If you have or plan to have a FMIC please order the AEM short ram intake kit.
A cold air intake system relocates the filter outside of the engine compartment to deliver cool air inlet temperatures. Cooler air carries more oxygen, which translates into a more intense explosion in the combustion chamber to create more horsepower and torque. But that's only part of the power equation. Tuning the inlet pipe in length and diameter to match the engine's resonance helps move more air to the cylinders (think of your engine as a big air pump), and this tuning allows us to deliver large power gains. We also monitor fuel trim correction factors and all OBDII sensors during R&D to eliminate leaning the engine out and/or throwing a check engine light.
Every AEM cold air induction system features high standards of construction and we are so confident in their fit and performance that we guarantee them for the life of your vehicle. All AEM cold air systems include a lifetime DRYFLOW synthetic air filter and come with a limited lifetime warranty.
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Cate Le Bon & Tim Presley
February 3 @ 8:00 pm11:00 pm
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Witness The Tim and Cate Show, live in Detroit! Yes. Following their respective 2016 solo records (The WiNK and Crab Day – respectively!), Cate Le Bon and Tim Presley announce a co-headlining tour to bring their many great solo songs to life in a social eye.


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Informatica Qatar sees demand for newly launched Huawei devices
NewsTech April 5, 2012 Rushika Bhatia 0 Tell a friend
The Huawei MediaPad tablet has been an instant hit in Qatar with its powerful features that include a seven inch capacitive touchscreen, dual-core processor, Android 3.2 Honeycomb OS, and HD-capable front and rear cameras. The Honor has been one of Huawei's most popular smartphone devices with its stylish design, excellent display with a 4.0-inch TFT-LCD capacitive touchscreen, and Android 2.3 (Gingerbread) OS.
The U8500 is another feature-packed Android phone that comes with a 3.2-inch capacitive touchscreen, high-speed internet connectivity with HSDPA support, WLAN/WiFi 802.11 b/g support, Bluetooth 2.1 capability, and Android 2.2. The G2800s features FM Radio, Micro SD card support (up to 4GB), stereo headset interface and torch light.
Ramy Numair, Commercial Director – Devices, iQ, said: "Huawei has been expanding its share in Qatar's IT market. Informatica Qatar's extensive distribution network across the country has been a key factor that has allowed Huawei to penetrate various market segments. The new agreement we have signed with Royal Mobile is another important step that will further consolidate our stronghold in the market and bring Huawei products closer to IT consumers."
Rushika Bhatia Editor
Rushika Bhatia is one of the region's leading commentators on business and current affairs issues. She is the Editor of CPI Media Group's flagship title – SME Advisor magazine. In addition, she leads CPI Media Group's infographics division – with special emphasis on data, research and statistics. Rushika has a Bachelor's Degree from Indiana University, USA and is also CIMA qualified.
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Saturday, November 2, 2002 7 East 95th Street New York, NY
9:30 Registration and Refreshments 10:00 Introduction Patrick Quinn 10:15 Drawing from Life Sue Ferguson Gussow 11:00 Drawing with Light Larry Kagan 11:45 An ARC Discussion Does drawing have spiritual value? Initiator: Robert Rambusch 12:30 Luncheon 1:30 The Children of the Ark; drawing on the human spirit Jay Murnane and M.T. Streck 2:15 Drawing on Sacred Places Partick Quinn 3:00 A Conversation with the Speakers Initiator: Anik Pearson 4:00 Wine and cheese reception
Biographies of Speakers
Members/Fellows $45 Non-Members $50; Students $15 Fee includes continental breakfast, lunch and reception. ($5.00 extra at the door.)
For registration information, please contact Charles Henderson email@example.com or Tel: 212-870-2544
To discuss this or other ARC programs, please check our message board ARC Message Board
More about ARC including membership information
From time to time the Board of Directors elects as Fellows individuals it identifies as having made a distinguished contribution to their respective fields. The list of Fellows elected over a period of nearly four decades thus exemplifies what the Society understands as the necessary and vital connections between art, religion and culture. ARC Fellows
Spring 2002 MoMA's PAPA: Alfred Barr and the Religious Dimension of Modernism
Winter 2002 A Theology of Beauty
Fall 2001 Lifting the Veil
May 2001 Utopia/Dystopia
February 2001 Antigone Performance and Symposium
November 2000 Illuminations & Transformations: Cross-Cultural Spiritual Dynamics in Music, Text, Dance and Film
May 2000 Alternative Readings: Sacred Text Embodied in Visual Art
February 2000 The Meaning of Myth
November 1999 Myth, Ritual and the Mediation of Violence
May, 1999 Writers' Ways with Loving and Dying
February, 1999 The Divine Image Implications for a changing image of God.
October, 1998 Uneasy Constellations of Meaning Theological Perceptions and Visual Images in Sixteenth Century Europe & The Religious Art of Andy Warhol
May, 1998 Meeting AYNI: The Andean Concept of Reciprocity
Webpage design courtesy Cross Currents Charles Henderson, Executive Director


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Waves Introduces Sound Design Suite
Waves Audio, the world's leading developers of professional audio signal processing technologies, introduces the Sound Design Suite. With over 30 plugins hand-picked especially for sound designers and post production facilities, the Waves Sound Design Suite is a comprehensive and cost-effective collection of audio plugins that provides all the essentials without breaking the bank. In developing this collection, Waves worked closely with two of the hardest-working pros in the business: movie maven Scott Martin Gershin (Shrek, Braveheart, The Doors, JFK) and game guru Charles Deenen (Need for Speed, Fast and Furious).
From conventional effects to creative sonic textures, Sound Design Suite covers all the bases. With compression, EQ, pitch shifting, level maximization and more, users get everything they need to mold, modulate and mangle their sound. Whether for TV, movies or games, the Waves Sound Design Suite is the sound designer's all-in-one, off-the-shelf audio processing solution.
Scott Martin Gershin commented, "These are the basic building blocks that all Sound Designers should have. A lot of times, when you bought packages, you also ended up getting plugins that were kind of interesting but not very applicable because they weren't really focused on the needs of the Sound Designers. What I'm excited about is that now I can just go to a place I'm going to mix, or any other facility or rental company, and say, 'Give me this Pro Tools system and make sure it has the Sound Design Suite on it.' And that's all I need to say, because I know that within the Sound Design Suite, it's going to give me the building blocks."
Charles Deenen stated, "Since Sound Designers are not rich folks like some of our music brothers, the most valuable plugins at a good price-point was key. For the Sound Design Suite, we honed in what both Scott and I found to be the most useful plugs in the current Waves arsenal, at a reasonable price."
Waves Sound Design Suite includes the following Waves plug-ins: C4 Multiband Compressor; DeEsser; Doppler; Doubler; Enigma; H-Comp; H-Delay; L1 Ultramaximizer; L2 Ultramaximizer; L3 Ultramaximizer; L3-LL Ultramaximizer; Linear Phase EQ; Linear Phase Multiband; LoAir;MetaFlanger; MondoMod; Morphoder; PAZ Analyzer; Q10 Equalizer; PS22 Stereo Maker (TDM Only);Renaissance Axx; Renaissance Bass; Renaissance Compressor; Renaissance EQ; S1 Stereo Imager;SuperTap; SoundShifter; TransX; UltraPitch; and GTR3 (Software Only). Waves Sound Design Suite retails for USD $3200 TDM/$2300 Native.
For more information, please visit www.waves.com


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Jumbo Magnetic Numbers/Operations
Item #: 901-JV44132
Model #: LER0452
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- Education Subject Matter: English
- School Grade: Pre-K/Kindergarten
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Jumbo magnetic numbers is the perfect start to learning numbers and counting for pre-schoolers.
Learning Resources® Jumbo magnetic letter set for grades preK - K is bright and perfect for preschool teaching. Letter set includes lowercase letters of size 2 1/2" and is used for reading and learn spelling, sold as 36 per pack.
- Set of 36 numbers includes 6 operation signs
- Numbers measure 2-1/2"H
- Ages 3-6
- Small parts. Not for children under 3 years.
Education Subject Matter : English | School Grade : Pre-K/Kindergarten | Brand : Learning Resources | |
Weight : 0.80 lbs. per Pack |
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Developed as a follow-up project from Pycom's original WiPy Kickstarter feedback (where 60% of backers expressed their preference for a new WiFi and Bluetooth module and 20% for a new WiFi and LoRa module), the LoPy not only has all three, but it also doubles up as a LoRa Nano Gateway, able to connect up to 100 other LoPys in a 5km radius. The Amsterdam-based startup has formed partnerships with LoRa network operators like Senet and ThingsNetwork to give LoPy users the best possible network coverage.
LoPy uses MicroPython's open source scripting language to reduce microcontroller coding time, greatly decreasing the time to market for connected products. The company will make available ready to use coding templates all editable with the Pymakr IDE to be launched in March. Thanks to its multiple radios, the LoPy can bridge data across a smartphone and the LoRa network for versatile networking and messaging applications.
Visit the Kickstarter project at http://www.pycom.io/Kickstarter2
Visit Pycom at http://www.pycom.io


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This salad is perfect for Thanksgiving leftovers. But because it comes together so quickly, you'll want to make it more often. From Prevention® Healthy Cooking.
Walnuts are a good source of omega 3 fatty acids. Research shows that omega 3s may help reduce the risk of heart disease.
Stop Dieting and Lose Weight from Prevention® (2003) p. 131


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Great service from staff, casual, good hamburger, fries, casual dress, and casual simple dining area.
You get great food here and plenty of it! The breakfasts are lumberjack size and can fill the hungriest tummy. Highly recommend.
Dropped in for breakfast with my husband. We found the place to be clean, quiet, and adorable! The plates of food were big and sooooo very delicious! Prices were great and service was fast, friendly. We will definitely head back here!
I've eaten there quite a few times the food has always passed my expectations and the waitresses are very good at their job
jim bobs is real easy to access from two major roads. the food is ok the service could be improved on and the inside dining is just ok. the is a galley style restaurant to where you go up order your food pay for your food and they bring it to you. the place is never very busy which concerns... More
Our family of three at here for an average price. Being from the south BBQ is something we love and are still looking for the right place here in our new home. This was not bad but really reminded me of the chain shops of the south. Biggest problem is the food was all luke-warm when served. And there is... More
I love the selection they have for their sides and the BBQ is good...wish it wasn't so expensive though...they used to have a "special" on their menu that we loved to come for that was reasonably priced and was great portion sizes at a good price point...but then they took that away and coming there has become a lot more... More
great food, service, portions, and prices! we'll definitely come back even though we are about 30 mins away.
Jim Bob's is an order-at-the counter no frills BBQ restaurant which serves tasty food at a reasonable cost. All of us in our party of five enjoyed our choices. I had the brisket with sides of BBQ beans and potato salad. My wife enjoyed her bowl of chili. Jim Bob's is definitely worth checking out.
I was driving from Ashford to Forks and MapQuest sent me through a bunch of one-horse towns with nothing open (I left before 8:00 a.m.). I was so glad to see Jim Bob's. They have great breakfasts with pretty good coffee for WA (I'm a Dunkin' Donuts girl myself). You get a good meal for a pretty reasonable price.
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593 / 2802
The Mysterious UFO Device
What do you think this structure is? There are almost an infinite number of possibilities.
I was being driven around by a very nice driver with Tom and he took me through this technology park. Most of it was rather banal, but this one structure was so unusual, I had to get out and examine it. I walked around and underneath the structure to try to figure it out. I never did, so I just set up to take a photo.
- Trey Ratcliff
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Long Beach Public Library
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The Long Beach Public Library (LBPL) in Long Beach, California, operates one main library and 11 branch libraries.
Main Library[edit]
The Main Library's [1] first building was a Carnegie funded library. The Library opened in 1909 within Pacific Park, now known as Lincoln Park, downtown on Pacific Avenue at Broadway. When the building site was condemned for construction of a New Main Library and City Hall building in the late 1970s, the library was moved to a building located on Ximeno Avenue, for a period of about 18 months.
The current Main library building occupies a first floor and lower level at 101 Pacific Ave, at the intersection of Ocean Boulevard and Pacific Avenue, across the park from its former location.
The Long Beach Public Library operates the following branches:
Databases and downloadable media[edit]
- Databases are available to Long Beach Public Library cardholders.
- Overdrive Audio Books for Download are available free to LBPL card holders for work, school, or on the road.
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Coordinates: 33°46′04″N 118°11′41″W / 33.767808°N 118.194609°W


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Photo by Andrew Ottoson
Alex Koberstein takes a charge from Clearwater Valley's Dillon Fisher during a 61-32 GHS win Feb. 10.
GRANGEVILLE When Garrett Kennedy with a point blank jumper finished a nifty tic-tac-toe passing play that began with a Bulldogs defensive rebound and covered the entire length of the court in the blink of an eye, Clearwater Valley called an early timeout, already facing a 17-4 deficit to a Grangeville boys team playing in near-peak form. GHS went on to wipe out CV by a 61-32 final last Monday night, Jan. 3, the competition effectively ended on that play, which began with Michael Wilson taking off as a teammate nabbed the rebound, which he passed to Austin Parks, who fired it ahead to Wilson, who made a tough catch, then pivoted and zipped it to Kennedy for a layup.
Grangeville scored eight straight points to increase an early 3-2 lead to 11-2, then, after CV scored, broke a 6-0 run that included multiple baskets by Kennedy including the dagger layup.
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Customers are often under-utilized when it comes to link marketing. Even if your company isn't a Zappos when it comes to customer service, you can still use customers for link development. Here are some ideas, and insights to jumpstart your strategy.
Make it Exclusive
Make your customers feel special and like they belong by offering a special discount to all whom participate. This could be as simple as a special sale, or offering them access to new products before the general public.
Have items with a low cost of goods? Send everyone who participates a gift. Or send them custom T-shirts. They're low cost when ordered in volume.
If you're decide to offer T-shirts, create one that will start a conversation. That way you get your customers talking about your products and company. The right T-shirt could pay for this link marketing campaign.
Make it Interactive
Instead of just asking for a link, make it interactive. This could range from having customers take a picture of themselves with your product to an interactive contest.
Make it a Recurring Event
Create a game for your site. Each week or month, give a prize to the highest scorer.
Depending on your budget this could be a sophisticated game or something low-tech. What's important is it fits your customer demographics and keeps them coming back.
Make it Funny
It's no secret that funny things spread and go viral quickly. Use this to your advantage in mobilizing your customers. This works well for companies that are willing to make fun of their own industry or company.
Let's say you're in the wedding industry such as favors, rings, or even a florist. Run a contest for customers to submit the funniest wedding vows. Then have customer vote for the winners — that way it's more interactive.
Make it Drive Traffic
This one is simple. Create a contest for the person who drives the most traffic to your site during a given time period. Offer first, second, and third place prizes to get more people involved.
To add legitimacy, have people send traffic to a particular page you set up for this contest, then do a screenshot of the top refers. That way you can be transparent without revealing your normal top referrals to your competition. Depending on your analytics software, you may want to set up a separate account for this contest.
Make it Useful
Get your customers to contribute to a useful resource. Let's say you're in the travel industry. Have customers submit travel tips. Any tips published earns an entry to win a prize.
Make it Simple
Finally, make things as simple as possible. Don't make your customers have to think about it.
Let's say you're asking customers to blog about their favorite product. Make it as easy as possible. An example would be adding a copy-and-paste form for the image on each product page.
Once you find something that works for your business, find ways to expand it and incorporate it into your general marketing strategy.
Related reading
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Lens will be central to the fortunes of Pinterest's decidedly alternative entry to the digital advertising market, so we've taken a hands-on look at what it is, how it works, and just how effective it is.
7 quick ways to use content marketing to boost search ranking
When done well, content marketing can contribute to boosting a site's position in search rankings. And although this may not seem like the most obvious benefit to a successful content marketing strategy, it's an effect that no business can ignore.
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On February 28, 2017, ClickZ Intelligence presented the webinar 'Still using .com? Here's why 50% of all Fortune 500 companies are about to use .brand' in association with Neustar.
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With links continuing to reign supreme in SEO, but the difficulty of acquiring them increasing, influencer marketing can help you to generate authoritative links with the additional benefit of increasing your user engagement signals, traffic, and visibility.


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Diplomacy map quite amazing its like soviet enemy a bit but much different
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French Wine Guide
French wine guide > wine > health > wine and cancer (related page: french paradox)
Other wine pages : • Wine and health • French wine regions • French wine guide • Buy french wines
Top 5 Wine Books
French Wine to the Wines and Wine Regions of France Learn more
Price: $13.00
Wine Coolers French wine FAQWine Glossary
Can wine help cure cancer ? Laboratory-mice genetically inclined to develop carcinoma received food containing solid red wine extracts. The result was that it took much longer than normal for those mice to develop cancer and their life expectation increased by 40% in comparison with that of mice normally fed. This surprising study was carried out by a team of researchers from the University of Davis - California. Researchers attribute this result to the presence in red wine of polyphenols, in particular to catechine, and to their antioxidizing properties (see composition of wine). The same protection exists in tea and in a great number of fruits and vegetables. A recent article in the New York Times said that it seems that resveratol, present in the skin of grapes, inhibits the action of agents favourising cancer. It is the case in leukaeamious cells. But one must be very cautious in this matter.
Other wine and health pages : Wine and Alzheimer - Wine and Cancer - Composition of wine - Did you know this? - French paradox - Wine and infarct - Wine and health
french wine regions | grape varieties | winemaking | classification in France | wine tasting | wine and food | wine faq | legendary wines | wine shop | french wine guide
Recommend this page (to yourself or to a friend!)
French Wine Guide (english)
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Westerman v. DePuy Orthopaedics Inc et al
Alvinette Wheeler Westerman
DePuy Orthopaedics Inc, Johnson & Johnson Services Inc, Johnson & Johnson and John Does
Case Number:
October 11, 2011
Arkansas Eastern District Court
Batesville Office
Presiding Judge:
J. Leon Holmes
Nature of Suit:
Personal Injury- Product Liability
Cause of Action:
Jury Demanded By:
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The following documents for this case are available for you to view or download:
Date Filed | # | Document Text |
November 2, 2011 | 2 | CONDITIONAL TRANSFER ORDER (CTO-83) transferring this case to the USDC for the Northern District of Ohio to be included in MDL No. 2197 in re: DEPUY ORTHOPAEDICS, INC., ASR HIP IMPLANT PRODUCTS LIABILITY LITIGATION. Signed by Clerk of the Panel Jeffery N. Luthi. (vjt) |
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That would be more of an UI issue than an actual strong reason either way for a proposed reduction of stats. Personally, I don't have a dog in this fight; I have most of the stuff I want from older content and the few remaining pieces I do need I have friends and family who will gladly help me get them, but if you're going to tell me me the major reason for an item squish is "Coz big numbers are harder to add" then I'm going to suggest that a UI workaround would be just as effective and cause less issues while being easier to implement.
Because there's some idiot who thinks that the squish will be a nerf of the players.
They should be. Too many people feel so strongly about it and they will lose subs. I have a lots of casual friends that play, they spend a lot of time in old world content and doing other stuff solo and with 1-2 friends. By item squishing and then giving players a stat buff in old raids, they make old raids feel artificial. Lot of the reason I go back to solo old raids is nostalgia and "So last year I spent 50 hours wiping to this boss with 25 people.. can I solo it now?". By adding this "buff" in order to fix the issue the item squish will cause, they are ruining both of these reasons.
I don't want fake scaling in the game. That's a large reason why many people did not like GW2 and came back to WoW. 4 difficulties (not even including 10/25 differences) already makes raiding seem artificial enough.
GC already said it would affect players... What don't you understand about giving a stat buff to max level players when they are in old raid content so they can still solo it? That clearly means it does affect players and in order to fix it they are applying a cheap bandaid. The item squish is not as balanced as some people make it out to be, they know it, that's why they didn't apply it in MoP and even admitted many of their internal testers did not like it.
Last edited by tyrindor; 2013-08-01 at 01:41 PM.
It's not about "morons" or "kids these days who can't count above 10".
Everything is going to stay the same, but numbers will be easier to work with. No one pays attention to the last 3 digits of a 200 000 hit, might as well remove them.
I don't get it - how is this a bad thing?
"I like big numbers, so item squish is bad" is the same as saying "I don't like LFR, so LFR is bad".
Last I checked I don't need an artificial stat buff to solo old raids, so something is definitely changing.
Go ahead and post that number size doesn't matter on the Diablo forums and see the amount of crap you'll get, that's the whole point of an insanely popular genre. Try to understand the mentality of some gamers when it comes to number size. All nostalgia, the sense of strength, and the sense of progression over the last 9 years is all thrown out the window for people who think this way. You may not care strongly either way, but why ruin it for these people?
I totally get why some gamers don't understand or care about this... I'm not going to say their opinions are wrong because no one here is wrong. Different types of gamers, different mentalities, and different reasons for playing the game. By changing the numbers they are ruining a major aspect of why some people play the game in order to fix a problem no one was quitting the game over to begin with.
Soon as GC mentioned it, there was over a dozen threads.. with one of threads exceed 50 pages in a single day. That shows how strongly some people care that this doesn't happen.
Last edited by tyrindor; 2013-08-01 at 02:02 PM.
I am kind of on the fence,
One side I would be sad to have a "visual" drop in power (even though not a relative one), and I would be frustrated if they did not do something to allow soloing still.
On the Otherside, with Ilvl's getting squished the difference between say a Heroic dungeon weapon and a first tier raid weapon will not be as much of a DPS boost. For Example if they made ilvls only move up say 4-8 Ilvls between Difficulties instead of 13-17 weapons from previous tiers would be much more useful than they are now.
I remember being able to raid most of Black Temple with my Talon of Azshara/Merciless Glad Offhand from on my Rogue in TBC, and still did reasonable DPS within a mid-high tier raiding guild, and was rarely ever turned down for PuGs from gearchecks. Yet now If you try to get into a PuG ToT with only a 489 weapon, most of the time you will be scoffed at, or told you re not geared enough, with only 1 tier of difference.
I'm leaning more towards Yay, but will not say for it at the current moment until more information is released.
Bow down before our new furry overlords!
I think this is the biggest difference between the supporters and the opposition... When I have gone back and 5, 4, 3, 2 manned something I felt hugely letdown by how easy the raid had become and how inflated or stats had become. Thinking about how we used to take a big group of friends to this place and now my wife and I can do it together alone. This saddens me. It doesn't make me feel better. I feel like the super jock that decided to tackle the downs syndrome kid that was "supposed" to get a touchdown. It's just dumb and it's like the bully beating up the scrawny helpless kid to take his lunch money, his car, and his t-shirt.
They didn't admit that their testers didn't like it. The quote said that it felt weird, and if it felt weird to the guys that knew the reason for it then ppl that don't read forums would be down right confused and may quit without knowing what happened. I'm hoping that it's announced at Blizzcon and is plastered all over the launcher and every other thing that they can do to let ppl know when and why this is happening. That way no one has the ignorance excuse...
- - - Updated - - -
It's more than just the feelings that are evoked though. It's more than just easier workable numbers. It's about endgame character power and scaling as well. At the end of Wrath a fully geared ilvl 264 PvP character could one shot a character in PvP blues with all the right buffs lined up. That is a problem. This also happened in Cata and I'm sure it will happen in MoP. The numbers need to be reduced, and the amount of ilvls per expansion need to be reigned in. Our spells don't do %damage they do a static amount of damage for your character power. When our DPS and crits end up hitting for more than our health pools you run into a ton of issues. The ilvl squish will take care of that.
ILVL from heroic 5 man should match heroic gear from last content patch of previous expansion. Why Blizzard cant make it. Simply enough - you can fo to your first raid of current expansion with high end heroic gear of previous. Why not to allow this? Blizzard care about newcomers? Bullshit, people with full heroic gear will level up to the max in a matter of hours and will go to the raid, instead of ganking lowlvls. Blizzard worrying about too fast content clearing? No problem, make first raid harder, same with BoT\BWD. High raiders will farm gear for raids in a matter of days anyways. So what the problem? Why this jump like panda greens > HC Deathwing epics are nessesary?
I'm 100% up for item squish. Blizzard cannot undone all this nonscence, but at least next expansions wont be this silly.
- - - Updated - - -
Thats probably because of 100500 difficulties as it is now. SoO LFR, SoO Flex, SoO 10, SoO 25, SoO 10HC, SoO 25HC - 6 difficulties, OMG. Blizzard must be clinically insane.
50 ilvls above the top tier is actually enough, but they need to plan the ilvls out through the course of the expansion better. From BC to Wrath we went from 150 to 200/213. If Wrath had played out as planned it would have ended at 251 which ended up being the 10man normal ilvl. It was the experimentation that was for the better of the game that got us to where we are, and now for the better of the game they need to go back and fix the experimentation. People simply are more worried about their ego and thrill of squishing proverbial ants to see the bigger picture here though...
Last edited by Rizendragon; 2013-08-01 at 03:04 PM.
Using the term "Megadamage" worries me, as I am completely against this idea. It's disingenuous language where the letters "K" and "M," standing for thousand or million, would suffice. It makes it look like Blizzard is already in favor of this terrible change.
It's bad for a couple of reasons. First of all, when I level next expansion, I don't want to arrive where I've already been. Along the same lines, I want to do more dps, not less, when my character supposedly grows in power. Otherwise, they can not introduce a new level cap.
Even for people who claim to be bad at math or numbers, this change should be insulting. I'd not want to be treated like I'm in 6th grade by Blizzard. Most people can't admit they just don't like math, and pretend to be bad at it by not caring enough to try.
Last edited by Achtalon; 2013-08-01 at 03:31 PM.
This. Right now I just divide everything by 1,000. My mage has 33 sp, 500 hp, and does 150 dps. I do not see the issue here at all and why this is needed. Their argument for it being hard on computers is bs. . . they can just start rounding off the damage if there gets to be to many numbers. They laugh at the "mega" damage solution, but that solution seems alot more long term than a squish which they have to rebalance everything for where they could just go to doing kilo damage, and much much much later milo dmg.
nothing but empty space here bro. move along now.
Sure, I don't see why not if soloing old content remains a possibility.
People who are against the squish remind me of this. i(dot)imgur(dot)com/2yakM9Q.png
I think we are paying way to much attention to "ilvl". Item Level is just a mechanism to define stat budgets to keep things balanced. An increase in Item Level of 13 in and of itself means nothing, but is dependent on the logic that Blizzard applies behind the scenes for increase in stat budget. Blizzard could use 4 iLvl per tier and have the exact same effect on stats that using 40 iLvl's has, because they don't use all of the values in between. There is a LFR ilvl, Flex ilvl, Normal ilvl, and Heroic ilvl. Sure there is some valor upgrade stuff or thunderforged, but in the general sense, there are 4 main ilvl's actually used. Blizzard can define whatever they want for the backend stat budgets that are associated.
Another factor that can be used in an ilvl squish is a modifier based on relative level. If I'm level 90 and fighting a level 70, they can modify up my damage by 300% and the damage taken down by 300%, then cap the damage at the mobs max hp. That prevents stupid stuff like 2M dmg on a single hit since the mob can't literally take 2M dmg when it only has 2k hp. That keeps my relative power against old content intact, but when fighting current content, I don't have to do 200k-300k dps against a boss with 600M HP. Their relative power vs mine is what really matters, not the #'s that scroll by on the screen. If my dps against the current tier of content stays consistent, but grows with respect to older content, I'm progressing in power, but so is the content against which I'm playing.
An item squish is not just shifting from an exponential progression to a linear progression if done right.
too bad its not gonna happen
common sense tells us "don't fix what's not broken". Since the system is not gonna choke on our increasing amount of damage numbers any time soon in couple of years next, there is no point making full system wide change like this. A change made like this can't be reverted, and i m sure it will introduce a lot of bugs, which is very common with a system that's couple years old. which bliz understands pretty well.
So guess u guys have to deal with ur "mathophobia" on ur own :P
Originally Posted by Blizzard Entertainment