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Bacterial sepsis is characterized by a rapid increase in the expression of inflammatory mediators to initiate the acute phase response in liver. Inflammatory mediator release is counterbalanced by the coordinated expression of anti-inflammatory molecules such as interleukin 1 receptor antagonist (IL1-Ra) through time. This study determined whether activation of pregnane X receptor (PXR, NR1I2) alters the lipopolysaccharide (LPS)-inducible gene expression program in primary cultures of hepatocytes (PCHs). Preactivation of PXR for 24 hours in PCHs isolated from wild-type mice suppressed the subsequent LPS-inducible expression of the key inflammatory mediators interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNFα) but not in PCHs isolated from Pxr-null (PXR-knockout [KO]) mice. Basal expression of key inflammatory cytokines was elevated in PCHs from PXR-KO mice. Stimulation of PCHs from PXR-KO mice with LPS alone produced enhanced levels of IL-1β when compared with wild-type mice. Experiments performed using PCHs from both humanized-PXR transgenic mice as well as human donors indicate that prolonged activation of PXR produces an increased secretion of IL1-Ra from cells through time. Our data reveal a working model that describes a pivotal role for PXR in both inhibiting as well as in resolving the inflammatory response in hepatocytes. Understanding the molecular details of how PXR is converted from a positive regulator of drug-metabolizing enzymes into a transcriptional suppressor of inflammation in liver will provide new pharmacologic strategies for modulating inflammatory-related diseases in the liver and intestine.
Pregnane X receptor (PXR, NR1I2) is a ligand-activated nuclear receptor (NR) superfamily member expressed at high levels within the enterohepatic system of mammals. The biologic function of PXR is mediated together with its obligate partner retinoid X receptor α (Kliewer et al., 1998; Lehmann et al., 1998). To date, the ligands identified for PXR have been numerous, and they are structurally diverse as naturally occurring steroids (Kliewer et al., 1998), antibiotics (Lehmann et al., 1998), bile acids (Staudinger et al., 2001a; Xie et al., 2001; Goodwin et al., 2003), anticancer agents (Desai et al., 2002; Nallani et al., 2004), and the active ingredients in several herbal remedies (Moore et al., 2000; Brobst et al., 2004; Ding and Staudinger, 2005). Ligand-activated PXR positively regulates the drug-inducible expression of genes encoding key drug transporters and drug metabolizing enzymes that function coordinately to increase the uptake, metabolism, excretion, and efflux of xenobiotics from the body. In this way, PXR activation is associated with increased metabolism and clearance of a myriad of potentially toxic compounds, and is classically thought of as a protective response.
Clinical treatment with PXR activators can also lead to the repression or attenuation of other biochemical pathways in liver and intestine including both energy metabolism and the inflammatory response (Moreau et al., 2008). For example, it was demonstrated nearly 45 years ago that treatment with rifampicin (Rif), a prototypical ligand of human PXR, leads to a compromised ability to mount an effective immune response in cell-based assays (Păunescu, 1970). In vivo studies in rodents suggest that PXR activation suppresses inflammation and the acute phase response (APR) by attenuating the activity of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling (Shah et al., 2007). More recent studies using rodents indicate that PXR activation regulates intestinal barrier function through an interaction with Toll-like receptor 4 (TLR4), the molecular target of lipopolysaccharide (LPS) stimulation (Venkatesh et al., 2014). It is now widely accepted that the activation of PXR is associated with general suppression of the immune response, particularly in the intestine (Cheng et al., 2012; Dou et al., 2012, 2014). Currently, little is known regarding the molecular mechanism of this phenomenon in the liver or in hepatocytes.
Recent investigations indicate that ligand-mediated activation of liver-enriched nuclear receptors (NRs), liver receptor homolog-1, and liver X receptor β (LXRβ) initiate anti-inflammatory mechanisms and pathways that suppress the hepatic APR (Venteclef et al., 2010). These investigations reveal that post-translational modification of these two NRs by small-ubiquitin-related modifier (SUMO) is required for suppression of the hepatic expression of the acute phase protein and marker of the APR haptoglobin. A theme emerges in which metabolic NRs are modified by SUMO proteins to suppress the inflammatory response, particularly in hepatocyte and in macrophage cell types (reviewed in Treuter and Venteclef, 2011). Previous work from our laboratory revealed that PXR is also the target of SUMO proteins to suppress tumor necrosis factor α (TNFα)–mediated production of interleukin 1β (IL-1β) in primary cultured hepatocytes (PCHs) (Hu et al., 2010). Although much is known about LPS administration as an experimental model of Gram-negative bacterial sepsis in vivo in rodents, very little is known about PXR-mediated modulation of the LPS-inducible gene expression program in PCHs across species. We therefore wanted to using both mouse and human PCHs as model systems to further characterize the ability of ligand-mediated activation of PXR to suppress a broad array of LPS-inducible hepatic inflammatory response genes.
Administration of LPS, a glycolipid constituent of the outer membrane of Gram-negative bacteria, to live animals or cultured cells initiates a signaling cascade in cells through TLR4 receptor multiprotein complexes that include CD14, Myd88, and MD-2 as coreceptor proteins (Buer and Balling, 2003). Here, we perform concentration- and time-response analyses of the LPS-inducible gene expression program in both mouse and human PCHs. Our data reveal that PCHs respond to stimulation with LPS to rapidly induce the expression of key inflammatory mediators, including IL-1β and interleukin 6 (IL-6). Using a commercial gene array platform, we show that 24-hours of pretreatment of mouse PCHs with a strong rodent PXR activator, pregnenolone 16α carbonitrile (PCN), suppresses subsequent LPS-inducible inflammatory responses in PCHs. The follow-up experiments using PCHs isolated from Pxr-null (PXR-knockout [KO]) mice demonstrate that the diminution of LPS-inducible gene expression by PCN requires functional PXR in hepatocytes. Finally, using PCHs derived from both humanized-PXR transgenic mice (hPXRtg) and human donors, we indicate that activation of PXR enhances the secretion of interleukin 1 receptor antagonist (IL1-Ra), a key negative regulator of IL1 signaling, from hepatocytes. Taken together, these data shed new light on the molecular mechanisms that comprise the interface between PXR activation and resolution of the APR in liver in mammals.
Materials and Methods
Isolation and Culturing of Primary Hepatocytes.
PXR knockout (PXR-KO) mice were generated as previously described elsewhere (Staudinger et al., 2001b). The hPXRtg mice were previously described elsewhere (Lichti-Kaiser and Staudinger, 2008). Hepatocytes were isolated from male congenic (C57BL6) wild-type and PXR-KO mice aged 6 to 10 weeks using a standard collagenase perfusion method as described previously elsewhere (Staudinger et al., 2003). The primary cultures of human hepatocytes used in this study were derived from samples collected and provided by the University of Kansas Medical Center (KUMC) Department of Pharmacology, Toxicology and Therapeutics Hepatocyte Core Laboratory, and the KU Liver Center which is sponsored by the Department of Pharmacology, Toxicology and Therapeutics Biospecimen Core Laboratory and the Liver Center at the University of Kansas Medical Center.
Fresh isolated human hepatocytes were plated at a cell density of 0.5 × 106 cells/well in 12-well plates previously coated with 0.2 mg/ml type I collagen. The isolated hepatocytes (>80% viability) were maintained in Dulbecco's modified Eagle's medium supplemented with 100 nM dexamethasone, 100 nM insulin, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 10% bovine calf serum and kept at 37°C in a humidified incubator with 95% air/5% CO2. The hepatocytes were allowed to attach to the plate for 4 hours, and the medium was then replaced with serum-free Williams E medium, as described previously elsewhere (Staudinger et al., 2003).
Total RNA Isolation, Reverse Transcription, and Real-Time Quantitative-Polymerase Chain Reaction Analysis.
Real-time quantitative polymerase chain reaction (RT-qPCR) was performed as described in Ding and Staudinger (2005), according to the manufacturer's instructions (SABiosciences, Frederick, MD).
Analysis of Secreted IL1-Ra in PCHs.
A 200-μl aliquot of cell medium was removed and combined with 200-μl 2X Laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris-Cl pH 6.8, 10% 2-mercaptoethanol, 0.004% Bromphenol Blue) with 50 mM dithiothreitol. After removal of the culture medium, the cells were harvested by scraping them into 1X phosphate-buffered saline, and they were pelleted briefly in a microfuge. After the removal of the phosphate-buffered saline, the cells were lysed in 1X Laemmli buffer, and equal amounts were resolved using 10% SDS-PAGE. Western blot analysis was performed as described previously elsewhere (Xu et al., 2009) using a monoclonal antibody that recognizes human and mouse IL1-Ra (NBP1-96673; Novus Biologicals, Oakville, Ontario, Canada). Western blot images were quantitated by densitometry scanning of the X-ray films with the UVP Biodoc-It 220 image analysis system (UVP, LLC, Upland, CA) and 1D Gel Analysis Software (TotalLab, Newcastle upon Tyne, United Kingdom).
Statistical Analysis.
Where appropriate the statistical differences within an experimental group were determined using a one-way analysis of variance followed by the Duncan's multiple range post hoc test. Statistical differences between the experimental groups were determined using Student's t test.
LPS-Inducible Concentration- and Time-Response Analysis of Key Inflammatory Mediators in Mouse and Human PCHs.
Our primary goal was to determine the extent to which preactivation of PXR alters the subsequent LPS-inducible gene expression program in a PCHs. Therefore, we first examined LPS-inducible IL-1β gene expression in both mouse and human PCHs, performing concentration- and time-response analyses using RT-qPCR. Treatment of PCHs isolated from wild-type male mice aged 6 to 10 weeks with increasing amounts of LPS (0.01, 0.1, 1, 10, and 100 μg/ml of medium) for 12 hours produced robust induction of IL-1β messenger RNA expression at all concentrations examined (Fig. 1A). Based upon these results, and to provide a relatively strong inflammatory stimulus for our subsequent studies of possible PXR-mediated effects, we reasoned that LPS treatment should be performed using a relatively high concentrations (10 μg/ml) for initiation of the inflammatory response.
Although all the time points examined (1, 6, 12, 24, and 48 hours) exhibited significant increases in IL-1β gene expression levels in mouse PCHs, the 6- and 12-hour time points had the largest increases, showing an approximately 1,000- and 600-fold increase in IL-1β expression, respectively (Fig. 1B). When PCHs derived from human donors were used in identical analyses, a similar concentration- and time-dependent induction of IL-1β was observed (Fig. 2, A and B). Examination of the kinetics of LPS-inducible mouse and human IL-6 produced very similar results (Supplemental Figs. 1–4).
Taken together, these data indicate that treatment with LPS induces the expression of key inflammatory mediators in both mouse and human PCHs, even at very low concentrations (10 ng/ml). Our data are in agreement with other previously published investigations regarding the production of inflammatory cytokines in PCHs in response to treatment with LPS (Panesar et al., 1999; Liu et al., 2002). A time-response analysis of CYP3A gene expression using PCN (10 μM) as a prototypical rodent PXR activator and Rif (10 μM) as a prototypical human PXR activator indicated that 24-hours of treatment produces maximal CYP3A gene expression that is sustained through the 48-hour time point (Supplemental Fig. 5). Taken together, these data reveal that pretreatment with PXR activators for 24 hours and subsequent cotreatment of 12 hours together with LPS is expected to be useful treatment regimen in a broad assessment of the effect of preactivation of PXR on the subsequent LPS-inducible gene expression program in PCHs.
Preactivation of NR Superfamily Members PXR, FXR, and LXRα Suppresses LPS-inducible IL-1β Gene Expression in Mouse PCHs.
Together, IL-6 and IL-1β impart key aspects of the inflammatory response. IL-1β, in particular, is involved in regulating a variety of cellular activities including cell proliferation, differentiation, and apoptosis. Ligand-activation of NR superfamily members including PXR (Zhou et al., 2006), farnesoid x receptor (FXR, NR1H4) (Hollman et al., 2012), liver X receptor alpha (LXRα, NR1H3) (Ghisletti et al., 2007), and peroxisome proliferator antigen receptor α (PPARα, NR1C1) (Devchand et al., 1996) has been shown to suppress key aspects of the inflammatory response in several different cell types. Because expression of these four NR family members is highly enriched in hepatocytes, we sought to determine whether ligand-mediated activation of these receptors could alter LPS-inducible IL-1β gene expression in our PCHs (Fig. 3).
As expected, treatment of mouse PCHs with LPS alone for 12 hours increased IL-1β expression dramatically. Preactivation of PXR, FXR, and LXRα for 24 hours with their cognate ligands suppressed subsequent LPS-inducible IL-1β gene expression. In contrast, preactivation of PPARα had no effect on subsequent LPS-inducible IL-1β gene expression. Treatment of PCHs with each cognate ligand alone for 24 hours produced comparatively small but statistically significant increases in the expression of IL-1β. Treatment with each ligand also induced expression of their prototypical target genes Cyp3a11, bile salt excretory protein, Cyp7a1, and Cyp4a14 for PXR, FXR, LXRα, and PPARα, respectively (data not shown). These data indicate that some, but not all, liver-enriched NR superfamily members may exhibit anti-inflammatory properties in PCHs, namely, PXR, FXR, and LXRα. It is also possible that these three liver-enriched NRs share a common molecular mechanism of repression of LPS-inducible inflammatory responses in PCHs, likely through alterations in posttranslational modifications such as SUMOylation (Treuter and Venteclef, 2011).
Unexpectedly, treatment with NR ligands alone induced statistically significant but relatively low levels of IL-1β expression, which was observed consistently throughout our studies and those performed by others (Vavassori et al., 2009). Although the molecular mechanism of this phenomenon remains elusive, it could include the presence of cryptic or low-affinity NR response elements within the IL-1β promoter.
Preactivation of PXR Suppresses Key LPS-Inducible NF-κB Target Genes.
An important transcriptional mediator of LPS signaling in cells is NF-κB (Sen and Baltimore, 1986). Treatment of primary cultures of mouse hepatocytes with LPS for 12 hours induced expression of 16 well-known NF-κB target genes (Table 1). Treatment with PCN alone for 24 hours suppressed the basal expression of numerous NF-κB target genes (Table 2). When compared with LPS treatment alone, treatment with PCN for 24 hours and subsequent cotreatment together with LPS for an additional 12 hours produced significantly lower expression levels of several notable LPS-inducible NF-κB target genes, including IL-1β, IL-6, TNFα, and IL1-Ra (Table 3). These data suggest that PCN diminishes the APR by inhibiting the LPS-inducible gene expression program of key inflammatory mediators.
To further examine this PCN-mediated effect, we next chose to examine the LPS-inducible gene expression of IL-1β, IL-6, TNFα, and IL1-Ra in PCHs derived from wild-type and PXR-KO mice using independently designed RT-qPCR primer sets (Table 4). In wild-type PCHs, treatment with LPS alone for 12 hours induced the expression levels of all four genes examined; whereas preactivation of PXR with PCN for 24 hours and subsequent cotreatment together with LPS for an additional 12 hours produced approximately half that observed with LPS treatment alone (Table 5).
Remarkably, treatment with PCN alone for 36 hours produced comparatively small but statistically significant increases in the levels of all four genes examined in PCHs isolated from wild-type mice, but this effect was completely absent in the PXR-KO PCHs. All four genes examined were expressed at slightly higher levels in the vehicle-treated PXR-KO PCHs when compared with vehicle-treated wild-type PCHs. These data suggest a repressive role for PXR in regulating basal expression levels of key inflammatory mediators, and are also consistent with previous reports indicating that deletion of PXR in mice produces elevated levels of cytokine expression (Zhou et al., 2006; Wallace et al., 2010).
Treatment of PXR-KO PCHs with LPS alone for 12 hours significantly increased the expression levels of all four genes examined. Contrary to the results obtained with wild-type hepatocyte PCHs, pretreatment of PXR-KO PCHs with PCN for 24 hours and subsequent cotreatment together with LPS for an additional 12 hours did not suppress subsequent LPS-inducible expression of any of the genes examined. These data reveal that PXR is required for PCN-mediated decreases in LPS-inducible target gene expression.
Of note, the fold-induction of IL-6, TNFα, and IL1-Ra after 12 hours of LPS treatment was less robust in the PXR-KO cultures when compared with the 12-hour LPS-treated wild-type hepatocyte cultures. In stark contrast, LPS-inducible IL-1β expression levels were enhanced in PXR-KO PCHs when compared with those observed in wild-type PCHs (Table 5, note c). These data reveal that the absence of PXR produces a condition in which the expression of inflammatory mediators is heightened, and suggest that PXR is required for the effective resolution of the IL-1β inflammatory response through time. Closer inspection of the biologic function of the gene products identified in our gene expression analysis indicates that one gene product in particular, IL1-Ra, is intimately associated with resolution of the IL1-signaling pathway and is expressed at high levels in hepatocytes.
The expression of IL1-Ra messenger RNA, which is detected primarily in hepatocytes, is induced by several inflammatory mediators and encodes a secreted antagonist of IL1-signaling (Arend and Gabay, 2000; Arend and Guthridge, 2000). We therefore more closely examined the concentration- and time-dependent LPS-inducible expression of IL1-Ra messenger RNA in wild-type PCHs (Fig. 4A). Treatment with increasing concentrations of LPS (0.01, 0.1, 1, 10, and 100 μg/ml) for 12 hours produced significant increases in the expression level of IL1-Ra. We next treated wild-type cultures with high concentration LPS (10 μg/ml) for increasing amounts of time (1, 6, 12, 24, and 48 hours) to examine time-dependent LPS-inducible IL1-Ra expression levels (Fig. 4B). Significant levels of LPS-inducible IL1-Ra gene expression were observed at the 6-, 12-, 24-, and 48-hour time points. When compared with the time- and concentration-response analysis of IL-1β and IL-6, the detectable induction of IL1-Ra was delayed by several hours but remained relatively high all the way through the 48-hour time point (Fig. 4B vs Fig. 1B). Similar results were obtained using PCHs derived from a human donor to examine the concentration- and time-dependent induction of IL1-Ra messenger RNA (Fig. 5, A and B). These data indicate that the kinetics of LPS-inducible IL1-Ra gene expression are distinct from those observed for the inflammatory mediators IL-1β and IL-6, with expression levels of IL1-Ra increasing at later time points (6–12 hours) and exhibiting a longer period of sustained expression through the 48-hour time point.
There are two major isoforms of the IL1-Ra protein that arise from a single gene called IL1RN, and their expression is differentially regulated at the level of transcription by alternative promoters (Butcher et al., 1994). One form of IL1-Ra is a heavily glycosylated and secreted isoform (sIL1-Ra); the other is an intracellular isoform (icIL1-Ra). The primary function of sIL1-Ra is to competitively inhibit IL1-binding to cell surface receptors (Arend et al., 1990). The expression and secretion of sIL1-Ra is highly inducible in hepatocytes by inflammatory stimuli, whereas the expression of icIL1-Ra is not inducible and its biologic function remains largely unknown (Arend and Guthridge, 2000).
The observation that LPS-inducible IL-1β messenger RNA expression was enhanced in the PXR-KO PCHs when compared with LPS-treated wild-type PCHs (Table 5) prompted us to examine the PXR- and LPS-inducible level of sIL1-Ra and icIL1-Ra proteins in both medium and whole-cell lysate, respectively (Fig. 6). When treated with PCN or Rif alone for 36 hours, the level of sIL1-Ra protein increased in the medium approximately 2.5- and 3.5-fold in PCHs from wild-type and hPXRtg mice, respectively. When treated with LPS for 12 hours, the level of sIL1-Ra protein increased in the medium approximately 1.9- and 1.8-fold from wild-type and hPXRtg, respectively. When treated with PCN or Rif for 24 hours and then subsequently cotreated with LPS for an additional 12 hours, the level of sIL1-Ra protein increased in the medium approximately 1.7- and 3.1-fold from wild-type and hPXRtg, respectively. When PCHs from PXR-KO mice were used in identical experiments, PCN had no effect on induction of sIL1-Ra protein in the medium. In contrast, 12 hours of LPS treatment produced an enhanced effect in PXR-KO PCHs when compared with PXR-positive cultures. In addition, cotreatment with PCN and LPS together failed to diminish sIL1-Ra levels in the medium from the PXR-KO PCHs.
Taken together, our studies indicate that PXR activation has both an early negative regulatory role in the LPS-inducible expression of key inflammatory mediators such as IL-1β and a likely positive role in regulating ligand-inducible expression of the secreted form of the IL1-Ra protein at later time points. To more closely examine the potential positive role of PXR activation in regulating sIL1-Ra protein levels across species, we chose to perform a longer time-course study using primary cultures of human hepatocytes (Fig. 7). Treatment of PCHs from a human donor with Rif for 72 hours produced an approximately 3.9-fold increase in sIL1-Ra levels in the culture medium, whereas 24 hours of treatment with LPS increased levels by approximately 1.9-fold. Treatment of human hepatocyte cultures with Rif for 48 hours, followed by cotreatment with Rif and LPS together for an additional 24 hours produced an approximate 8.3-fold increase in sIL1-Ra in the culture medium. These data indicate that long-term preactivation of PXR in both rodent and human PCHs has a strong positive effect upon the secretion of sIL1-Ra from cells, an important and systemic negative regulator of IL1 signaling and key participant in the compensatory anti-inflammatory response in mammals.
The liver is a crucial organ that plays a central role in acute phase protein synthesis during bacterial sepsis. The resulting cytokine stimulation rapidly up-regulates expression of acute phase proteins, and simultaneously down-regulates key drug metabolism pathways in liver. Although proinflammatory cytokines initiate the APR through different cell surface receptors, they share a high level of redundancy with respect to the signal transduction pathways (e.g., Iκ-B kinase, p38, and JNK) by which they exert their influence in the nucleus (reviewed in Heinrich et al., 2003; Wajant et al., 2003; Lu et al., 2008; Weber et al., 2010). Initiation of the acute inflammatory response is mainly achieved through signal-dependent activation of NF-κB and activator protein 1 transcription factors through promoter response elements that regulate expression of genes encoding important proinflammatory cytokines.
Treatment of experimental models with LPS stimulates pattern recognition receptors, mainly TLR4, to induce the rapid expression and release of proinflammatory cytokines such as TNFα. Secreted TNFα further exerts its inflammatory function through binding to TNF receptor type 1 (TNFR1) and TNFR2 on various hepatic cell types, including hepatocytes. Separate receptor types recognize either IL-6 or IL-1β, respectively, to further initiate and amplify the acute inflammatory response in feed-forward loops. Importantly, treatment of liver with LPS leads to release of proinflammatory cytokines IL1β and TNFα from both nonparenchymal cells and hepatocytes. Stimulation with IL-1β induces TNFα secretion from rat hepatocytes, and stimulation of hepatocytes with either IL-1β or TNFα produces IL-6 secretion (Panesar et al., 1999; Yoshigai et al., 2014). Hence, there are multiple levels of interconnection and amplification that occur rapidly between and among inflammatory cytokines after bacterial sepsis.
From a historical perspective, hepatocytes were initially viewed as passive recipients of immune messages from nonparenchymal cells, including Kupffer cells (Volpes et al., 1992). However, more recent investigations have indicated that this is in fact not the case. Hepatocytes mount a robust response to challenge with either LPS or IL-1β to produce key inflammatory cytokines, including IL-6, TNFα, and IL-1β (Liu et al., 2002; Panesar et al., 1999; Spencer et al., 2013; Takano et al., 2012; Yoshigai et al., 2014). It is also now well known that hepatocytes express all the necessary machinery to respond to bacterial sepsis, including TLR4, CD14, Myd88, and MD-2 (Liu et al., 2002). A recent study has indicated that hepatocyte-specific knockout of the TLR4 receptor in mice significantly attenuates the systemic serum levels of the inflammatory mediators TNFα, IL-6, and IL-1β in response to a high-fat diet (Jia et al., 2014). Hepatocytes are therefore not merely passive recipients of immune signals from nonparenchymal cells but instead can be viewed as active participants in mediating an immune response to a variety of signals that include sepsis and morbid obesity.
Much attention has recently been given to the notion that PXR activation by Rif and its analogs may be beneficial in the treatment of inflammatory liver and bowel diseases (Wallace et al., 2010; Kakizaki et al., 2011; Cheng et al., 2012; Jonker et al., 2012). Additional research indicates a key role for PXR in maintaining the barrier function of the gut (Dou et al., 2012, 2014; Venkatesh et al., 2014). Thus, detailed knowledge of the molecular mechanisms governing PXR-mediated suppression of the APR in these tissues is vital but is currently lacking. It is well known that SUMOylation modifies the transactivation capacity of a myriad of transcription factors, and in most cases correlates with transcriptional suppression (reviewed in Gill, 2005). For example, the SUMO-modification of liver-enriched NR family members is implicated in suppression of NR-function and in modulation of the APR (Ghisletti et al., 2007; Venteclef et al., 2010; Treuter and Venteclef, 2011; Zhou et al., 2012; Balasubramaniyan et al., 2013). While several liver-enriched NRs are the molecular target of the SUMO signaling pathway, not all of them are. For example, constitutive androstane receptor, a close relative of PXR, is not SUMOylated (unpublished observation). We have previously shown that PXR is SUMOylated in response to TNFα treatment in both human and mouse PCHs (Hu et al., 2010). These observations suggest that shared molecular mechanisms exist to govern the conversion of the primary metabolic function of the liver during the nonseptic or noninflamed state to one that is involved in resolving the APR during sepsis or injury, which likely involves SUMOylation.
Here, we present a working model that defines a novel pathway for the feedback inhibition and resolution of the inflammatory response in hepatocytes through time (Fig. 8). After injury or infection, we propose that low stoichiometric amounts of SUMO-modified PXR function to directly suppress the proinflammatory mediators IL-1β, IL-6, and TNFα at the level of transcription, while the remainder of PXR protein is likely ubiquitinated and subsequently degraded by the 26S proteasome in a signal-dependent manner through time. Indeed, significantly lower levels of PXR protein are detected in endotoxin-treated mice (Teng and Piquette-Miller, 2005).
As the APR ensues through time, newly synthesized PXR protein becomes available for up-regulating ligand-dependent expression of novel or alternative PXR-target genes, including the negative regulator of IL1 signaling sIL1-Ra, possibly through cryptic or low-affinity PXR-response elements. Of note, a recent investigation indicates that peroxisome proliferator-activated receptor γ coactivator 1α, a strong PXR-coactivator protein, controls expression of IL1-Ra in liver (Buler et al., 2012). In this way, PXR activation gains a novel repressive anti-inflammatory function and plays an active role in the resolution of the inflammatory response through time. Future efforts should seek to determine whether this mechanism by which PXR is converted from a positive regulator of drug-metabolizing enzymes into a transcriptional suppressor of inflammation in the liver will provide new pharmacologic strategies for modulating inflammatory-related diseases in the liver and intestine.
In our current study, LPS was used to initiate the acute inflammatory response in an effort to determine the extent to which PXR-mediated suppression of the inflammatory response was dependent upon cell surface receptor types such as TLR4 receptor versus TNFR. Data presented here indicate that PXR activation negatively regulates the LPS-inducible gene expression program in hepatocytes similar to that observed with TNFα or phorbol ester stimulation (Zhou et al., 2006; Hu et al., 2010). Taken together, these data indicate that the negative regulatory role for PXR in inflammation is not specific to cell surface receptor type, and likely operates at the level of the promoters for key inflammatory cytokines TNFα, IL1β, and IL-6. In a symmetrical manner, stimulation of the inflammatory response is well known to suppress drug metabolism pathways in liver through the rapid and selective down-regulation of specific CYP enzymes. Several possible mechanisms have been proposed for this phenomenon including the reduction of PXR mRNA levels during sepsis (Beigneux et al., 2002). Another line of thought has postulated the disruption the association between PXR-RXRα heterodimer complex by a signal-dependent interaction of NF-κB with RXRα, thereby sequestering the active form of PXR that regulates drug-inducible gene expression (Gu et al., 2006). While plausible and not mutually exclusive, the precise signals and molecular mechanisms for inflammation-inducible repression of drug metabolism deserve further scrutiny.
Authorship Contributions
Participated in research design: Sun, Cui, Woody, Staudinger.
Conducted experiments: Sun, Cui, Woody.
Performed data analysis: Sun, Cui, Woody, Staudinger.
Wrote or contributed to the writing of the manuscript: Sun, Woody, Staudinger.
- Received July 21, 2014.
- Accepted December 12, 2014.
This work was supported by the National Institutes of Health National Institute Diabetes and Digestive and Kidney Diseases (NIDDK) [Grant R01 DK090558]. The human hepatocytes used in this study were derived from samples collected and provided by the University of Kansas Medical Center (KUMC) Department of Pharmacology, Toxicology and Therapeutics Hepatocyte Core Laboratory and the KU Liver Center.
↵This article has supplemental material available at dmd.aspetjournals.org.
- acute phase response
- farnesoid X receptor (NR1H4)
- 2-[3-[3-[[2-chloro-3-(trifluoromethyl)phenyl]methyl-(2, 2-diphenylethyl)amino]propoxy]phenyl]acetic acid
- 3-[(E)-2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-propan-2-yl-1,2-oxazol-4-yl]methoxy]phenyl]ethenyl]benzoic acid
- 2-[4-[2-[4-cyclohexylbutyl(cyclohexylcarbamoyl)amino]ethyl]phenyl]sulfanyl-2-methylpropanoic acid
- hPXRtg
- humanized-PXR transgenic mice
- interleukin
- IL1-Ra
- interleukin 1 receptor antagonist
- knockout
- LXRα
- liver X receptor α (NR1H3)
- LXRβ
- liver X receptor beta (NR1H2)
- lipopolysaccharide
- NF-κB
- nuclear factor κ-light-chain-enhancer of activated B cells
- nuclear receptor
- primary cultured hepatocyte
- pregnenolone 16α-carbonitrile
- PPARα
- peroxisome proliferator antigen receptor α (NR1C1)
- pregnane X receptor (NR1I2)
- Rif
- rifampicin
- RT-qPCR
- real-time quantitative polymerase chain reaction
- small-ubiquitin-related modifier
- tg
- transgenic
- Toll-like receptor 4
- TNFα
- tumor necrosis factor α.
- tumor necrosis factor receptor
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics


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06 December 2017
Blue Water Energy raises US$1.1 billion with second Guernsey fund
Carey Olsen's investment funds team in Guernsey has advised existing client Blue Water Energy LLP (Blue Water Energy) on the US$1.1 billion final closing of Blue Water Energy Fund II (BWE Fund II), surpassing its original fund raising target of US$1 billion.
BWE Fund II, which secured strong support from both existing and new investors, will support Blue Water Energy's longstanding buy and build strategy across the mid-market energy sector. The firm's inaugural fund, Blue Water Energy Fund I, which is also Guernsey-domiciled, closed in May 2013 with US$862 million of commitments.
Partner Andrew Boyce led the Carey Olsen team advising on the Guernsey legal aspects of BWE Fund II's structuring, establishment, regulation and closing, supported by senior associate Ruth Abernethy and associate Rachel de la Haye. International law firm Willkie Farr & Gallagher (UK) LLP provided the onshore UK advice.
Advocate Boyce said: "The final close of BWE Fund II parallels the remarkable success of Blue Water Energy's first fund, and their decision to structure through Guernsey once again, particularly with the successful inclusion of new investors, speaks volumes about the global view of Guernsey's highly regarded international regulatory regime, and of the Island and its fund industry as a whole.
He added: "Our congratulations on another extremely successful fund raise."
Founded in 2011 by Jerker Johansson, Tom Sikorski and Graeme Sword, London-based Blue Water Energy has US$2.5 billion under management and a global portfolio of 14 companies.

subset: commoncrawl


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Moving to a new state presents a unique set of challenges, even for well-equipped job seekers. When Sanique moved to Charlotte, N.C., from Alabama in 2014 after completing graduate school, she had a limited social network and an even smaller professional network. She was looking for a job in Human Resources – the field in which she has her master's degree – but ended up taking a position at a call center instead because that was the only job she could find. After working at the call center for a year, Sanique moved back to Alabama to regroup because she was unhappy with her career in Charlotte. A year later in 2016, Sanique moved back to Charlotte to give finding a career in Human Resources one more chance.
One day shortly after moving back to Charlotte, Sanique heard about a job fair that Goodwill was attending and decided to go. There, she met Craig Kalhagen, a member of Goodwill's Business Relationship Management team, who specializes in connecting qualified clients to hiring employers. Craig suggested that they set up a time to meet and talk about Sanique's experience and aspirations. Sanique never knew that Goodwill could help people find employment – she only knew about Goodwill's retail stores. However, she knew how important it was to meet new people and expand her network, so she decided to give it a try.
"I didn't have anything at the time," Sanique said. "I was open to trying out Goodwill because I was doing anything and everything to network and find the right fit."
Over the next few months, Sanique worked closely with Craig and Goodwill's Business Relationship Management team to try to find a career in Human Resources. Craig would send Sanique job openings that he thought would be a good fit for her, and Sanique would send Craig job openings that she thought she would enjoy. Craig would contact employers on Sanique's behalf, advocating her skills, capabilities and strong work ethic.
"It was such a great experience working with Craig and his team," Sanique said. "At the time, I was working with a couple of temporary staffing agencies, but working with Craig and his team was more personal. I felt valued, and they really took the time to help me find what I was looking for."
During her job search, Sanique felt frustrated at times, but she knew she couldn't give up. When Sanique was a child, she and three of her sisters were placed in the foster care system. Sanique's foster mom told her that foster kids rarely amount to anything, and Sanique has always used that as her driving force to succeed.
"I was only 11 when my foster mom told me that I would never make it out," Sanique said. "That's when I knew I had to make something of myself."
One day, Craig sent Sanique a job posting for which he thought she would be perfect. After reviewing the job description, Sanique also felt that it would be a great position for her. Craig soon connected Sanique to the hiring employer, and their hard work and perseverance paid off when Sanique was hired as the Human Resources Coordinator at a local home health agency in November 2016. Today, Sanique is thriving in her new career and enjoying the stability that full-time employment offers. She recently purchased her first home and spends her free time enjoying her new life with her family.
"My quality of life is so very great now," Sanique said. "I have a one-year-old daughter, and it feels great to be able to give her the life she deserves."
Despite Sanique's recent accomplishments, her journey is far from over. She has aspirations to continue growing in her career and making a name for herself.
"Right now, I'm more of an HR generalist," Sanique said. "In a few years, I'd like to focus on a Human Resources specialty, whether that be benefits, payroll or whatever. And ultimately, I would like to fall into a management position."
Sanique's advice to anyone who feels like giving up is to keep pushing through and searching for new opportunities. She stresses how important it is for job seekers to grow their social and professional networks, whether they can do so on their own or need the assistance of organizations like Goodwill.
"Stay focused and remain positive," Sanique said. "Continue to build your network. No matter if you're networking up, down or across levels, you never know who you'll run into. I've learned that a lot of job openings aren't posted online. Get out, meet new people and see what opportunities are out there."
Today, Sanique's future is looking brighter than ever, and she wants to pay it forward by sharing her story in hopes of inspiring others to persevere.
"I'm really glad I found out about Goodwill. I've been telling the world about it," Sanique said. "A lot of people with challenging childhoods feel like they'll never make it. I want to encourage people from all walks of life to do their best. You can succeed."


text:
Mitch Scorza
Mitch Scorza,
August 27, 2017
Salternative facts iAy que rico y sabor! Allow me to explain; in Latin American countries, this is a compliment mostly aimed at the food or "la comida." In Cuba, the food has a bounty of flavor, but the phrase is not meant to describe only the food. This phrase is meant to showcase a lifes...
Donnisha Brown
Donnisha Brown,
August 27, 2017
A Cuban is a Cuban In the United States, there are African Americans, Chinese Americans, Mexican Americans and many more Americans. Although it is not directly defined by race, wealth, livelihood and overall quality of life is differentiated culture to culture, neighborhood to neighborhood. In Hav...
Activists occupy Oakland, celebrate anniversary of movement
Kali Persall,
Managing Editor
October 12, 2016
It's been five years since activists with the Occupy movement made camp at the Frank H. Ogawa Plaza in Oakland to protest social inequality and wealth disparity. Today, the tents are long-gone and supporters say Occupy Oakland no longer exists. On Saturday evening, around 150 Occupy sympathizers gathered ...


text:
Dine in style while enjoying international and Austrian cuisine
At your hotel in Zurs at the Arlberg you will not only be spoilt when it comes to skiing, but also when it comes to your culinary wellbeing. The team around Renate & Guenther Wienerroither offer a variety of dishes – to suit any preference. Look forward to a tasty ski holiday in Lech-Zurs at the Arlberg!
Your Zurs hotel's restaurant is the location for your gorgeous breakfast buffet each morning, providing enough energy for a day filled with pristine skiing experiences in the Lech-Zurs ski resort.
Refreshments and snacks throughout the day: at Hotel Ulli at the Arlberg in Austria
Pleasant evenings: with a glass of fine wine at your Zurs hotel at the Arlberg
Have you built up an appetite for a dreamy ski holiday in Lech-Zurs at the Arlberg? Simply send a holiday enquiry to your Zurs hotel now. The Wienerroither family is looking forward to your visit!


text:
Zooey Deschanel sitcom New Girl has cast actress Brenda Song in a guest role.
The 24-year-old Suite Life of Zack and Cody star will play a love interest for Winston (Lamorne Morris) on the Fox series, Entertainment Weekly reports.
Daisy (Song) will fall for Winston when he gets knocked back by the attractive Holly - played by previously-announced guest star Brooklyn Decker.
Song's previous credits include a role on ABC drama Scandal and in 2010 film The Social Network.
Nate Corddry (Harry's Law, Studio 60) has also been cast on New Girl as a man who unsettles Jess (Deschanel) with his creepy behaviour.
The series continues on Tuesdays on Fox and will return to E4 in early 2013.


text:
Category: Abuse
Mom always said Daddy was a good man. They got married in the forest, built themselves a home and decided to have a baby. Daddy did a lot of building. He had very strong hands. She said they were beautiful hands, with one big vein that stood out on the back of each one. They were hairy, though. She said Daddy had a lot of hair.
When he died I guess she didn't know what to do. She loved him a lot, anybody could tell even if just from the way she remembered his details. Her memories painted my portrait of him more than any photograph ever did.
Claud was a different sort of man. The skin was thick on his neck and face, and he shaved it all twice a day to keep the prickles back. He had some white spots on his arms that he always said were from the sun, but I found them one day on the internet and learned they were a type of fungus: a mold, sort of, burrowed under the skin.
His arms were thick, usually crossed. His fingers were rough from dryness and daily use of a keyboard. His mind was sharp, he was good with numbers. An accountant. Loved golfing. I don't think he ever loved Mom as much as he loved golfing. Then again, I don't think he ever loved Mom much at all.
He loved me, though.
He told me so often, as he cupped his big hand over my entire shoulder, pressing his fingerpads into my arm. That hand would later cup an entire breast, and the first two fingers would reach all the way up inside me so I could feel them hit my pelvic bone. Then his cock did the same, from behind. It happened in my bedroom every night for years and years. It's how I knew that sleep would be coming soon along.
Mom forgot a lot of things very often. This was no different. Sometimes I would tell her the truth and she'd be shocked, infuriated. Sometimes she'd try to throw Claud out of the house and he would lock her in the hall closet with a key. I would sit on the carpet by the door and cry when she was in there for a long time. I never tried to ask for the key.
But there were many other days when Mom didn't remember any of it. She would keep saying, Honey, I don't understand. Honey, what are you saying? On those days she would tell me that none of it was true. Sometimes when Claud was away she would make a bed for me on the couch and put a hot towel on my forehead. She would sit by my feet and we'd watch Jeapordy or Wheel of Fortune. She never wanted to talk about Claud. I think for her it was easier to just pretend he wasn't there.
Then one day a man named Alan arrived at our home. Alan was a lawyer, and he said he had come to take me away. He said one of my teachers at school had contacted him on my behalf. He had a kind face. Mom was on her knees and crying and squeezing me tight in the entryway, her hands wrapped around and under my backpack. It held four of my school books and a change of clothes. Alan said they would have the rest of the clothes sent to me. It was best if Mom stayed away for a while, he said, so that I could orient myself.
Alan brought me to a hospital where the doctor's name was Dr. Lambert. He had glasses and a prickly white beard. His hands were warm. He pressed on my belly, listened to my breathing and shined a light into my eyes and ears. Then he put a mask over my nose and told me I would be very sleepy for a minute. A fog came and left me as I blinked up at the ceiling lights, cold and white. In the hospital almost everything was white.
My room was white, too. With a white bed and a white blanket. There was a little white table and chair and a window very high that let white light in. The door was white as well, and made of metal. It locked from the outside.
Alan the lawyer asked me many questions. He visited me once a day, except for Sundays. On Sundays there was a church service in the brunch room of the hospital, then we would go out to play in the gardens. They were simple gardens with bright green grass and shrubs that were pruned to have a curve. Sometimes there were yellow daffodils.
On the other days we would have school, classes in math and reading, sometimes science. The teachers often changed, as did the students. Some would come and some would go. It was normal. Sometimes we helped the cooks make lunch, at least those of us who were able. Some of the residents were unable to help much, so they stayed mostly in the break room. It had a large window, a record player and some books and toys for children. There were a few round tables, all white. The chairs were white, too.
Mostly Alan came to ask me questions about Claud. Alan was kind, but he didn't listen well. Many times he asked me what Claud looked like, so I had to repeat his description over and over again. It was not difficult for me to talk about what Claud did at night, or that Mom would often forget all about it. But it made me very sad to talk about Mom. I sometimes asked to see Mom, but Alan said it wasn't a good idea. He would just nod and write, nod and write and ask more questions.
Dr. Lambert said the same thing when I asked him about my mother later on. I began to see Dr. Lambert more and more after my first two years in the hospital. Like Alan, he asked me questions about Claud – but he was more forceful, his voice was louder, his look was more direct. He would fix my gaze and say, Honey, is that what you remember? I always said yes, because it was the truth. I had no reason to lie.
I was seeing Dr. Lambert twice a week when he told me he wanted me to start taking a small pill every morning. He explained that it was to help me experience my surroundings better. I didn't understand, as I felt that I experienced my surroundings perfectly well. But still I took it, every morning when the nurse brought it to my door with a paper cup of water. She made sure I took the pill, then she smiled and took back the paper cup. She told me not to worry, that lots of people take pills in the morning. "It's normal," she said.
At first the pills had no effect on me. I felt the same as I always had, neither happy nor sad. I went to my classes and daily activities. I contributed. I spoke with Dr. Lambert. Alan almost never came to visit me anymore.
Then I began to notice things, details I hadn't seen before. The paint was chipped in many places on the hospital walls, and often it was scratched or scuffed. The tables in the break room were sometimes dirty, before the nurse cleaned them with a cloth and some Windex. I always volunteered to help her, because I liked the smell of Windex and the blue splatters on the white tables and scrubbing the dirt away.
I hadn't seen Alan in three or four months, so one day I asked Dr. Lambert if he was too busy with his cases to come see me anymore. Dr. Lambert said I could
talk to Alan any time I wanted, and to just ask him myself. "Ask away," he said. He folded his hands and leaned forward, smiling. I suddenly felt very ill, so I asked to go to my room. "Of course," said Dr. Lambert. "I'll see you tomorrow, honey."
I had been in the hospital for two years or more. I had not seen Mom since I arrived, though Claud had visited a time or two. I shuddered to think of them alone together but I knew Claud wouldn't hurt her like he hurt me. He had told me many times he only wanted me that way, not her. He said it was different with me. Sometimes he put his fingers in my mouth and I tasted myself on them. Sometimes he left the lights on during. Sometimes he turned them off.
Dr. Lambert's questions came to be more direct. He began saying things like, "I want you to be more specific. I want you to be certain. Tell me more. Describe that again. Tell me more." He began to repeat himself. He was displeased with my answers. He'd lean in and gaze into my eyes. "You know the truth," he'd say. "You know."
One day he told me: "Claud does not exist. Claud never existed. You invented him. I'm sorry if this comes as a shock but deep down I believe you must know this." Dr. Lambert's eyes were clear. My stomach ached. "You know this, honey. You do."
I asked for my mother. He said she couldn't come. "Claud is real," I told him. Dr. Lambert disagreed.
In his office I sat in a special chair. A soft plastic clip was placed on each of my pointer fingers, which tightened and vibrated when Dr. Lambert pressed a small control button. He pressed the button every time I said it: "Claud is real." Buzz. "He exists." Buzz. It was uncomfortable, but it didn't hurt. I read the nameplate on the desk: Dr. Alan Lambert. He asked me again and again and again: Who is Claud? Is he real?
"Yes," I would say. "Claud is my stepfather. He is real." Buzz. Buzz.
As the months wore on my memories were crumbling, falling from my mind in chips and blocks. I remembered the taste of Claud's thick fingers in my mouth, pressing down on my tongue, as I remembered biting my own thumb so hard it drew thick bubbles of blood. I remembered being crouched by the closet with Mom locked inside and I remembered being inside the closet without her. I sometimes remembered her screaming and banging, but I wasn't sure from which side of the door.
Dr. Lambert's tone had changed. "This memory is falsified," he would say. "This memory is untrue. This is a projection. This did not happen in physical life."
All the white was still there on the walls, but now the scratches stood out to me. I knew the patients were disabled, mentally. I knew that I was one of them. I didn't know what I remembered and what I didn't, and I was always very tired. I missed my mother very much.
Who is Claud? Is he real?
"Claud is my stepfather." Buzz. "I… made him up." A pause.
"Good," Dr. Lambert said. "Very good, honey. Very good. You're progressing."
Claud does not exist. Claud never existed. Claud's not real. I made Claud up.
That's good, honey. Great, honey. Good, good, good.
The day finally came when Dr. Adam leaned to me and said the words: "Looks like you have a visitor." I turned around and there she was, like a ghost or a dream, in a dress and a robelike shawl, hands to her mouth, nervous. She looked at me through downturned eyes, wet with tears.
"Honey," she breathed, and she opened her arms.
Mom brought me back home in her red station wagon. It had been several years but my bedroom still looked exactly the same. For days we didn't speak to each other; there was too much and too little to be said. We settled into the house in silence.
It lasted weeks, perhaps, but time had lost meaning anyway.
The little house Daddy had built was chipped with wear and tear, but Mom had kept up good care of it on her own over the years. We spent entire seasons cleaning up the surrounding land and putting up fencing for goats, soft white lady goats for shearing and making wool yarn. Mom taught me to knit hats, then socks the next year, then gloves after that. We planted a vegetable garden and collected mushrooms in the refuse under the dry pine needles of the forest bed, saving them to cook later on.
In the kitchen one night the unspoken monster ripped itself out of me, sudden and ugly. "Was he real, Mom? Did Claud exist?" I felt weak, body heavy and collapsing, mouth wrenched and set in a silent sob from someplace deep down in my gut. She rushed over, embraced me, sat us down together backed up against the kitchen cabinets, humming to me as I cried into her lap, tears pooling on the linolelum. In low tones she told me, "Honey, whatever you remember is real. It's all real, honey." She held me that way for a long while, silently, pushing locks of hair back from my face.
Then, as if in a breath, she was gone. She was 47 and it was a Tuesday morning. She refused to wake up for much longer than usual. At first I was awed by her; she had achieved this brilliant, perfect stillness like what the soul longs for in life. I sat on her bed with my hand on her forehead until shadows stole over the room in late evening, as night darkened and bluish light filtered down through the open blinds.
While holding her there in the dark a desperate force suddenly tore through me, like a seizing orgasm of loss and rage, and I clutched tighter the limp head in my arms. How was I supposed to do this? The thought of relocating my mother's body terrified and overwhelmed me. A wild panic set in, my skin burned hot and I intensely feared my own being. Then suddenly, with a heavy presence, a thick- limbed, light white man occupied the bed beside me. With a large hand he reached out and cupped my kneecap, pressing his fingerpads lightly into my thigh. The other large hand tipped up my chin between thumb and forefinger and Claud murmured into my mouth, "Don't you worry, honey. I'll take care of everything now."
12 november
smearing black liner over my swollen eyelids, bloated from crying. must be out of the airbnb by 12. it is 11:55. he left at 7:30 to catch his flight, climbed into bed with me before he left, hiking boots and all, wrapped his big arms around my body and kissed me deeply as if it was our last kiss ever, which it would be.
i have a problem with love — too much of it to give out. a problem with timing — mine is terrible. a problem with pain — i like it too much.
love is a really ridiculous bond. almost more physical than mental. the mind doesn't choose who to love, the body does. the fact that i could sit at his side and listen to his rambling, all of which i find basic and relatively meaningless, contemplate his way of viewing the world and end up being disgusted, or disappointed, or just confused as to how he came to be this way — how could anyone? in conversations, feeling like i was constantly educating — giving out my knowledge and ideas like candy, most of which he accepted gladly, as he needed them — validating his existence over and over, which he told me so often nobody had ever done, while simultaneously he tore mine down.
according to enrique, i am
intelligente, lista, guapa, adventurera, diferente
dificil de entender, dificil de aguantar, rara, demasiado liberal, rápida a enfadarme, i push people, i like to get under their skin.
all true.
told him i wanted the feedback. i would listen. i did listen. i want to think about it. I want to improve. but so much of our incompatibility came from his direction and his blatant fear and intolerance for difference that it's difficult to know how i could have done better with this one. except for the obvious: to have left him immediately, or at least after the first intolerant attack on my being. when he called me a slut, when he behaved selfishly in bed, when he kicked me out of his car on the side of the highway. i held out. in the end mother nature brought us down — my period came before his final visit and it disturbed him. he wouldn't touch me after he realized.
i do know why i held out, but i don't know where all the love comes from, or why i have to feel all this for people who hurt me so badly. he never meant to. he's a good man with misogyniistic tendencies, like so many of them. nothing special. nothing unusual. i always think i'm just in it for the sex, then come to find out there's always been more to it. much more.
i feel destroyed. everything hurts. all i want to do is cry, i sound like a goddamn country song. lord help me, save me from the images that stick. his arms and his shoulders. he put a little weight on. his eyes, green. the warmth. cargo pants and hiking boots and two grey shirts. mountain boy stranded on an island; forever conflicted, forever torn in two. i love it, i love it, i love him. i shouldn't. there is better, there is more. jess, go and find it. jess, don't be like this. jes come on. please. raise your standards. don't give when you don't receive. jes, you're hard to handle. give credit where credit is due. listen more, listen better. stay calmer. retain control. control yourself.
this is nobody's fault. it ended well, with love. like pulling apart a peanut butter sandwich. messy, had bread in it?
because feeling pain is still feeling, and feeling is better than nothing. living is pain, and living is also better than nothing. loving is living, and loving is pain. pain means you're living. and so on and so forth. forever, or until you can't anymore.
itches i can't name
all over this broken
thing all tattered up
and covered in holes
the itch means healing
but i can't tell the new scars
from the old ones
every day i stain something
else with my blood,
the pale gaunt face full
of larvae like a nervous addict,
which i am, and was, and will be,
til sliver by sliver with dirty nails
i tear apart the rest of me and
pick to pieces the remains still
searching for something
of value
spit them out, these wasted days and wet-green nights rising up from your esophagus to greet against anyone's will your
lovers and your sisters and your friends and your parents make them
worry for you but never too much just enough to catch a whiff of the smoldering
human brains on stone tiled floors where
cold gets in so easy feel it creeping up the carnage contaminated by the time
it grabs your feet and legs to drag you under
i'm okay, i'm okay — you're shoveling shouting reaching out to grab hold of whatever's in reach
creamy rose pink with green sparkles dribbles thick makes you feel
safe watching feel the grip slip this is how we
fight our wars with pink with glitter with ooze like
crying all that bile from your eyes the sticky
worms running playground drills up and down your throat
red rover, red rover, why don't you come over?
red used to scare you always creeping in or up
more often out
that drip drip down your shaking knees that
seeping out the gashes in your stomach like a watermelon past its prime now just remember– don't eat the seeds, you can't afford for anything to grow inside you, and neither can the anything– that environment is uninhabitable
for living things
había un calvito con miedo de todo
con ojitos que brillaban al sol
quería irse lejos, pero temía estar sin la madre,
quería ver el mundo, pero temía viajar.
quería cocinar, pero temía aprender cómo,
quería amar, pero temía su corazón.
quería ser más sabio, pero temía irse al cole,
quería salir fuera, pero incluso temía el sol.
con suerte un día el calvito
se dejó caer de la cuna
y pasito a pasito
aprendió a caminar —
luego se puso pelo y
como dicta la naturaleza
tras su milagro mas asombroso
se atrevió de ser hombre
y el calvito se creció.
hey nacho
send some more pics of your
stumpy pink dick while you
hold it at the base with your
unwashed sheets and empty
walls in the background and
the the tv tuned to some
sports channel that
shit gets me so
wet i just
play rough with your pups. nip their skinny ankles, snap at the achilles. flip them on their backs and pin them down. you are their master, make them know it. twist their fur in your jaws and wrench it hard, when they've done wrong don't let them rest. back them into corners. goad them, make them snarl. draw their blood and shove their noses in the puddles. keep their errors current, no piss stain forgotten, no accidental bite and no ignored command. they will try to forget — don't let them. train them up to fighting dogs for back-alley snarling circles, gnashing teeth with larger beasts of less formal education. teach them to fight by fighting, only then will they survive.
read the back of the
label: it will tell you
your sins for the day
but there will be no
advice for repenting provided. do
it yourself with slimy digits
coughing over the toilet. be
discreet: the sound of a
splintering facade is harsh on
young ears and of course
apart from slim you must
also be strong.
i think
the most pain i felt
through all of that
were those two nights
where you slept
fully clothed,
in your sticky t-shirt and your
nylon shorts all clinging
to your hairy thighs and your
sweaty balls in the
sweltering heat
you laid face down with
your head turned away and
for those two days you didn't
you slept
with your arms wrapped
tight around your
chest and you wouldn't
come closer to
me so I laid there
near but not too near
staring at the
ceiling terrified
i'd bother you
i'd lose you
you were already
i want to
sha. ke you
I want to br eak that glass over your fac e
how much time you're w a s t i n g
thing just
knees jut ting in just
there's nowhere to run n ow
you know
I hate you more up
if you could
you stupid
cunt, you
so sick of you
so s i c k
we accept the love
we think
we know
pair of nothings
you and I


text:
I've wanted to create a Pharmacy for a while but it's taken a long time to get around to doing. Here it is at last built to match the large shops like Mrs Beetons and the Chocolate shop.
The pharmacy also combines a doctors office with a dentist chair as many doctors in Victorian times also practised dentistry.


text:
Grays Skinful Pro Hockey Glove - Black
The new Skinful Pro Gloves are full finger lycra gloves offering abrasion resistance. The new fleece lining provides greater warmth than previous Skinful. The elongated cuff design gives a more secure fit than before whilst the Climagrip palm provides great all-weather grip
- Full fingered lycra glove
- Elongated Cuff
- Climagrip Palm
- Fleece Lining
Standard UK Delivery (see below for Table Tennis delivery information)
Standard delivery to all address on the UK Mainland is FREE for orders £75.00 and over (£3.99 for orders under £75.00) and will take 2-3 days although please note some outlying areas are excluded (enter your postcode in your basket to check). For those who require a quicker delivery a NEXT DAY delivery service is available for £5.99 on most items when your order is placed before 1pm. Orders placed after 1pm will be sent on a next day service the day after. Please note that 'NEXT DAY' means the next working day, orders placed on a Friday will be delivered on Monday. Orders placed on a Saturday or Sunday will be delivered Tuesday.
On rare occasions we may not be able to provide a next day service for your order. If this is the case we will notify you within an hour of you placing your order.
Some areas of the UK are also not eligible for the next day service. Once you have entered your postcode in the checkout you will be offered next day delivery if it is available in your area.
Deliveries to Northern Ireland are £12.00. If you are located in Eire and wish to order we may be able to help but please call us for a carriage quotation.
Table Tennis Tables
Our table tennis tables are delivered by a 2 man crew on a day of your choice (please note that due to the unreliability of the service we do not offer a next day delivery service on table tennis tables). Once you have placed your order you will be phoned within 24 hours to arrange a suitable day for delivery (this will usually be within 5-7 days of your order date although can sometimes be quicker). Once this is arranged you will receive a text the night the delivery is due to advise you of the time slot you have been allocated for the next days delivery. Please feel free to order well in advance of when you need the table as you can arrange to have it delivered on any date that suits you no matter how far in advance (although please note they will only be deliver between Monday and Friday).
If you have decided not to keep your order it can be returned within 30 days for refund or exchange. If you have simply decided you do not wish to keep the order you will need to return the item at your own expense. Should the item be faulty or we have delivered an incorrect item postage will be reimbursed or we can arrange for collection of the item if you call us on 0800 783 5011.
For your convenience we absorb large shipping costs for some heavy items - for instance any type of goal, storage item or table tennis table. If you wish to return these and cannot do so by yourself we will need to work out a quote to have the item/s returned from you.
Products we are unable to refund or exchange
We are unable to offer a refund or exchange on personalised, made to order or special order goods unless they are faulty.
For further delivery and returns information please click here.


text:
231 Coffman St
Longmont, CO 80501
Telephone: (720) 204-1412
CATEGORY: Legal Services
For more than 10 years, I have helped families throughout Boulder, Weld and Larimer counties get through difficult legal matters, which has provided me with unique insights into the emotional and financial hardships associated with divorce, child support and custody.
At Shea L. Burchill, P.C., my law practice is focused 100 percent on family law matters. Unlike other attorneys who dabble in family law, I am committed to helping individuals pursue creative resolutions to complex family law issues. I devote necessary time, talent and resources to each case to ensure the highest level of client service. I listen to my clients and discuss all issues and options so that they can make informed decisions. I then tailor my litigation representation to each client's unique situation.
As a dedicated Longmont family law attorney, I possess sophisticated knowledge of Colorado's divorce codes. I leverage my knowledge and experience when I handle divorce litigation. I am dedicated to helping my clients receive a fair settlement, focused on the best interest of the children.


text: THE GREAT SPITALFIELDS HUGUENOT FEST…Don't even think of missing it.All the details are on the website! And very exciting – talks by Dan Cruickshank and Tessa Murdoch (and me!), walks around Sp;italfields every day (just turn up!). Or book if you're a group. Events at the V&A, Bishopsgate Institute, Guildhall. Walks are not only about the Huguenots but also the various immigrant groups who've settled in Spitalfields. Hear all about the fashionable silks the wealthy commissioned, how the weavers lived and died, the other industries the Huguenots excelled in. The choice is enormous with speakers such as Dan Cruickshank & Tessa Murdoch. But you can also just turn up for a walk without booking. I am doing a talk on the Spitalfields silkweavers at the Guildhall 2 pm, 9 April at the Guildhall, Aldermanbury, the City. No need to book for this one and free!


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Enterprise Account Executive
Remote - Minneapolis, MI /
Sales – Sales /
Full time
Have you heard of BlueCat?
We're one of those hidden gems that's been disrupting the market as a key player in the rapidly growing space of Adaptive DNS. We harness the power of DNS to provide secure, centralized network infrastructure to some of the world's biggest brands and enterprises like Facebook, Apple, Dell, and SAP so that employees can access the computing resources they need, when they need it. After years of experience, we moved into the security market, leveraging that goldmine of DNS data to help organizations protect their internal networks.
Right now you're thinking 'Great, so why is that interesting to me?' Here's why:
BlueCat occupies an incredibly strategic and valuable real estate on corporate networks – the 'first step' taken when any device, application or service connects to a network. We literally own the intersection of digital business and cybersecurity – and that translates into incalculable opportunities for innovation and growth.
BlueCat's next generation DDI solutions are perfectly positioned to meet the demands that cloud, virtualization, mobile and the explosive growth of connected devices have put on the entire enterprise network infrastructure and our customers are some of the biggest companies in the world. #MissionCritical
Here's what you need to know about our Sales team: The BlueCat sales team is a group of intelligent and market aware sales professionals who like to have fun and win together. We are a highly engaged team that loves bringing creative solutions to the table to help grow and transform our business. We know how to be competitive, learn and continue to support each other and our customers!
The Enterprise Account Executive opportunity at BlueCat is an exciting field based sales career opportunity developing a set of targeted named accounts, focused primarily on the Forbes 2000.
- Create, manage and execute a strategic territory coverage and sales plan that encourages multi-angle access into key accounts, with a focus on achieving revenue goals consistently on a quarterly and annual basis
- Meet and exceed established monthly and quarterly revenue objectives
- Plan and manage both account and deal strategy. Identify, qualify, orchestrate and close new business within net new and existing accounts
- Develop a detailed understanding of organizational structure, competitive landscape and technology heat map within entire account list.
- Prospect into targeted named accounts. Develop and execute field sales campaigns to create demand, work closely with Lead Generation and Sales Engineering resources to build and manage a robust pipeline of new business. Manage multiple complex sales processes simultaneously
- Articulate the BlueCat value proposition to senior IT leaders, effectively identify and quantify customer pains, highlight ROI and business value through a consultative sales approach
- Work within a team environment; manage internal and external resources to execute account and deal strategy and maximize sales efforts and success rate
- Establish & maintain strong and referancable relationships with existing client base
- Forecast accurately and maintain the system of record in Salesforce.com
Knowledge, Skills, Abilities & Experiences Required:
- Minimum of 3-5 years of technology sales experience, with a track record of consistent over-achievement against assigned quota and a high level of income attainment
- Bachelor Degree or equivalent experience required
- Demonstrated success in strategic account selling; strategic thinker with ability to create and execute account plan and strategy
- High intelligence and mental acuity
- Excellent communication skills
- Strong negotiation and closing skills
- Hunter mentality with proven experience in effectively building a regional territory
- Creative problem solver who can react quickly and multi-task
- Highly driven and self-motivated
- Strong leadership and people skills
- Strong business acumen; polished, and articulate, comfortable selling and presenting to senior level executives
- Tech savvy – Salesforce.com, MS Office, LinkedIn, Twitter, social media experience is a plus
Core Values and Overall Characteristics Sought in All BlueCat Employees:
Curiosity is fundamental to discovery.
Clarity is essential in effective communication.
Collaboration is optimal only with challenge and belief.
Transparency is critical to effective decision making.
Ownership ensures priorities are addressed.
BlueCat's Brand Attributes:
Courage is required to make our voice heard
Differentiation will build our brand
Discovery turns experiences into knowledge
What you'll get from us:
Fast growth for BlueCat means fast growth for you! With the world's biggest and hottest logos as our clients, we are a well-established business that still operates with the fun culture of a start-up. We get to work hard and still have all the fun without all the risk! Experienced leaders make sure we keep going to the moon and beyond.
While we're growing together, we'll have your back with solid compensation/bonuses, education budgets, record breaking sales quarters, team and company socials, choice of hardware, flexible hours, hackathons, and a company culture that values curiosity, clarity, collaboration, transparency and ownership.
Joining BlueCat means you'll be joining a company that's been certified as a Great Place to Work for the fifth year in a row! In addition to that, we've been included in the 2020 lists for the Best Workplaces for Inclusion and Best Workplaces for Women.
If you're as excited about the future of our company as we are, we would love to hear from you!
Accommodations will be provided throughout the recruitment, hiring and employment process in accordance with the AODA and the Ontario Human Rights Code.


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I met Olimpia for the first time at the 7th floor of the New York Times building. Tall, slender silhouette, dark brown long bob, and her signature round glasses: she clearly stood out from the crowd. Just landed from Italy, she swung by to say hi at the opening of "Townies", an exhibition on the … Continue reading
Category Archives: Illustration
Maira Kalman illustrates herself. A talk with an imaginative mind
A daydreamer who dances with words and images to find beauty in the mundane, Maira Kalman let her witty spirit splash in the tempera colors and come out with creative solutions while embarking in the discovery of the wonders of life. To an external observer, her prolific art looks like the immediate and fresh outcome … Continue reading
An art piece on paper: Soie by Tishina
Seta, Soie, Silk, 絹. If the person for whom you'd like to buy a present can read French, or has at home a version of "Silk", the novel by Alessandro Baricco, in her/his native language, then another good idea for a Christmas gift would be "Soie", the new edition of Baricco's 1996 masterpiece illustrated by … Continue reading
Plunging into Yayoi Kusama's wonderful dreamland
I have just started reading again, after around 20 years from my first time, "Alice's adventures in Wonderland". I specifically ordered on Amazon the special edition by Penguin Books, illustrated by Japanese artist Yayoi Kusama. I couldn't buy for myself a better present on Cyber Monday and, if you are looking for some inspiration for … Continue reading


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Independent Ford Specialist
MasterTech (Hull)
Click on image to enlarge
Welcome to MasterTech Just a few picture of Phil & Neil and our brand new fully equipped workshop.
and what the papers say about us.
We believe our premises,equipment & experience of Ford Vehicles are unrivalled in this area even by the main dealer. we have invested in all the latest equipment to repair both Cars & Transit.
If you do not own a ford we can still save you money we service & Mot all makes.


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Our blogs are a place for teachers, parents, and math geeks to reflect on and connect over their favorite learning techniques, tips, and tricks.
Recent Posts
Multiplication Story and Activity for 4X4=16Posted by Jen Wieber on November 20, 2015
This dizzingly fun activity will help you remember that 4X4=16!
Escape From Mr. Lemoncello's Library, Order of Operations, and Rebus PuzzlesPosted by Jen Wieber on November 17, 2015
Literature and Math come together! Tickle your Imagination with Escape From Mr. Lemoncello's Library, and then slueth your way through these challenging "Order of Operations" Rebus Puzzles!
Multiplication Story and Activity for 9x9=81Posted by Elicia Zahm on November 14, 2015
A silly story and fun activity to teach 9x9=81!
DIY Multiplication Velcro Dart Board GamePosted by Jen Wieber on November 11, 2015
Practice addition, subtraction and even multiplication with this DIY Velcro Dart Board Game!
Multiplication Story and Game for 7 X 9 = 63Posted by Jen Wieber on November 11, 2015
Throw the "sticky bee" at the "surfin' sign" when playing this fun DIY dart board game to learn that 7 X 9 = 63!


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Our dealerships sell all of the Continental range derivatives (GT V8 and V8 Convertible, GT & GTC W12, the Flying Spur and GT Speed), as well as Bentley's flagship Mulsanne.
Each vehicle in the current range offers the high-performance, styling and refinement that has always been the hallmark of Bentley automobiles. The manufacturer's use of high-quality materials and sophisticated technology means that drivers and passengers arrive at their destination refreshed and content, even after the longest journeys. Our bespoke programme also enables you to design your car in line with your specific desires, ensuring that you always receive maximum satisfaction from your new Bentley.
The sales executives at our dealerships are attentive to all the latest developments in the design and engineering of our vehicles, and will be able to tell you anything you need to know about the range. Select one of the links below for a detailed overview of the new Bentleys available from H.R. Owen and contact us today for the wide range of approved used Bentley models in stock and the Bentley finance options available to you.
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In today's highly competitive market, retailers are constantly competing to differentiate themselves. That's understandable; competition from bricks and mortar retailers and online operators has rarely been tougher. But the risk is they do so without knowing what their customers really want; something which should set alarm bells ringing in head offices up and down the country, given that customer expectations have also never been higher.
Launched today, our retail report Integrating the retail supply chain, suggests that only 39% of major retailers actually talk to customers to understand their needs, despite 77% believing that successful businesses put customer service first. Many retailers are making decisions based on gut instinct or their own perceptions of what customers want, rather than talking to them to understand their real likes and needs. This is particularly surprising in today's increasingly digitally enabled world where customer expectations are developing fast, often influenced by service experiences outside retail through companies such as AirBnB, Uber and Just Eat.
This can be extremely costly, because it means retailers can be focussing on the wrong areas. Many retailers continue to pursue a 'faster and freer' delivery mantra, often in the belief that they need to compete with online pureplay retailers like Amazon. In our experience, if they speak to their customers they may find that the customer doesn't actually need or value that level of service. For many customers, what is more important is that deliveries arrive when and where they are supposed to, and that the delivery window is as short as possible so they don't have to waste the whole day waiting for a parcel. There are, of course, examples of product sectors where next day or same day delivery is essential, but in the majority of cases, customers are making a planned purchase and it is more important to them to receive the delivery on terms that are convenient for them, rather than next day.
We recently worked with a high street retailer, who wanted to move to a standard next-day delivery offer because they felt this was what their customers wanted. The implications for this on their distribution operations would have been severe as all orders need to be received, picked, packed and dispatched in a short time window to enable next day delivery – with a consequential impact on the size of distribution facility needed. After some research, they discovered that their customers would be content with two or three-day delivery; their customers were making an informed decision to buy and what was more important to them was the assurance that it would arrive at a time and in a place of their choosing. Naturally, two or three-day lead times enable the smoothing of order peaks and troughs across a longer time period and result in a smaller, simpler, more cost effective distribution operation.
Such exercises can produce other insight, too. In the above example, customers revealed what was important to them, as well as what wasn't. They wanted returns-ready packaging – the ability to reuse packing for returning items without the customer having to source an alternative, and with a return-ready label included. They also revealed that all the additional special offers included in deliveries, which our client thought added value for the customer, were actually an annoyance and went straight into the bin. Essentially, the retailer had prioritised the wrong elements of the customer proposition, because it hadn't until that point found out what their real customers wanted.
Encouragingly, our report also found that leading retailers (those we define as omnichannel pioneers) are more than twice (76%) as likely as others (32%) to undertake in-depth customer data analysis, suggesting those who do take the time to find out what customers really want see benefits in both sales and bottom line profit. Critically, by combining historical sales data with real customer insights and an understanding of future trends they are able to ensure they deliver the right offer for their customers, and by neither under or over-servicing those needs, they are also delivering their proposition more cost effectively.
Retailers can also learn from trends outside their sector. Some of the online operators, such as Uber, demonstrate the potential that can be achieved with digital channels, including the ease with which transactions can be made and the benefits of adapting websites to mobile devices.
Those retailers which can use customer insight effectively will be able to stand out through offering differentiation to customers. It's a model John Lewis has used successfully for many years, driving loyalty through a singular focus on providing customers with what they want.
Businesses which can align their supply chains to deliver excellent, personalised, service will naturally differentiate themselves in the market and be more able to confidently move away from the race to offer ever faster and freer' delivery, focussing instead on improving customer satisfaction and retention, and ultimately generating higher profits.
The write is Stuart Higgins, retail partner at LCP Consulting. Click here to access the LCP retail report, Integrating the retail supply chain


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ATLANTA - Villa Christina invites you to enjoy a repast spanning from classic to exotic at Thanksgiving Brunch, featuring an abundance of traditional Thanksgiving favorites as well as many delightful Italian specialties. Feeling thankful about quality time with family and friends is much easier when you don't have to spend hours in the kitchen, preparing the dinner. Instead, enjoy each other's company and the delightful cuisine at Villa Christina. The festive brunch will be held on Thursday, November 24th, 2011, from 11:30AM - 3:00PM. Cost for brunch is $45.00 for adults, $25.00 for children ages 6 - 12, and free of charge for kids 5 and under.An incredible feast awaits you and your lucky guests at Villa Christina's tables. Whether you crave turkey or tortellini, pumpkin pie or creamy tiramisu, Villa Christina offers a full array of sumptuous selections. The bountiful menu features traditional tom turkey topped with fresh herbs and butter and roasted to perfection by our chefs, creamy mashed potatoes, smashed sweet potatoes topped with gooey, roasted marshmallows, fresh cranberry sauce and two types of scrumptious dressings. Executive Chef Andreas Georgakopoulos promises that fans of his inspiring Tuscan cuisine will be equally thrilled with the rustic Mediterranean dishes, including fresh seafood, assortment of salads, ravioli with lobster & ricotta cheese, sage dressing, and innovative pastas. Additionally, there will be a table featuring kid-friendly favorites, such as chicken fingers, macaroni and cheese, fresh, seasonal fruit, cookies and more. Restraint is admittedly difficult among all this bounty, but the decadence of Villa Christina's desserts cannot be overstated. Sweets, including warm bread pudding, chocolate mousse cake, classic pumpkin pie and bourbon pecan are outrageously decadent. This holiday is all about spending quality time with friends and family. Relax and allow yourself to indulge in the spectacular spread, while Villa Christina does all the work. Now, that's something to be truly thankful for!Thanksgiving Dinner To-GoBe a Guest at your Thanksgiving Feast, specially prepared by Villa Christina at Perimeter Summit's Award-Winning Culinary Team! Enjoy the holiday festivities and let Villa Christina at Perimeter Summit serve you!Thanksgiving To-Go MenuHerb-Roasted Turkey "12 lbs"Traditional Greek SaladChicken Fiesta SaladButtermilk Mashed PotatoesFresh Green Beans withRoasted Red PeppersSage StuffingRich Turkey GravyChoice of Pumpkin or Pecan PieTake out dinner for 6$120 + 7% Sales TaxAll orders must be made by 12:00 pm on Thursday, November 18, 2010Pick up on Thanksgiving Day 10:00am - 12:00pmThe Italian three-story, stacked-stone Villa Christina at Perimeter Summit features a gourmet restaurant, two elegant ballrooms, two private dining rooms, an exhibition kitchen, and an outdoor Event Pavilion. The main dining room boasts a breathtaking view of sprawling grounds, a beautiful stone terrace and a garden event pavilion surrounded by beautiful landscaping, hand-manicured rose gardens, meandering streams and waterfalls. Villa Christina serves lunch Monday through Friday from 11:30 a.m. to 2:30 p.m., and dinner Monday through Saturday from 6:00 until 10:00 p.m. Reservations are recommended for Thanksgiving. The restaurant, conveniently located just inside the Perimeter, at 4000 Summit Blvd, accepts all major credit cards and offers complimentary valet parking.For more information about the restaurant and its holiday happenings, visit www.villachristina.com, or call 404-303-0133.###
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Okemah is the largest city in and the county seat of Okfuskee County, Oklahoma, United States. It is the birthplace of folk music legend Woody Guthrie. Thlopthlocco Tribal Town, a federally recognized Muscogee Indian tribe, is headquartered in Okemah. The population was 3,223 at the 2010 census, a 6.1 percent increase from 3,038 in 2000. In that census, about 26.6 percent of the residents identified themselves as Native American.
Source: Wikipedia


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David City Public School
David City Public School in Butler County is accredited by Nebraska Department of Education and the North Central Association. Elementary school buildings are located in David City and Bellwood.
The maximum capacity of the public elementary school in David City is 450 and the Bellwood school has a capacity of 260. The public junior-senior high school was built in 1916 with additions in 1985 and 1993 and has a maximum capacity of 500.
Vocational courses offered in the high school include agriculture, business education, family and
consumer science, industrial technology, and industrial education. Adult education programs are available in the high school in cooperation with Central Community College. Distance learning programs are available via satellite.
St. Thomas Aquinas
St. Thomas Aquinas middle and high school and St. Mary's Catholic Grade Schools offer a Christian-centered educational environment. Aquinas High School, built in 1961, is accredited by the Nebraska Department of Education and North Central Association. In 1999, the Aquinas Chapel and building trades and construction classroom were completed. St. Mary's completed a 5,000 square foot building project in 2000. The addition includes two kindergarten classrooms and a resource room.
Academically, Aquinas and St. Mary's schools stress the basics of religion, reading, writing, and arithmetic. Standardized tests assist teachers in evaluating the students' progress on a regular basis. Several advanced placement courses are offered each year. Religion and Mass play a part of each day's activities.
The Catholic school system is financially supported by 13 parishes in Butler and Polk counties.
University of Nebraska-Lincoln Cooperative Extension in Butler County
University of Nebraska-Lincoln Cooperative Extension in Butler County delivers research-based education and information to citizens throughout the county. These educational programs, delivered via a variety of methods, are offered through a federal, state and county partnership arrangement. Our objective is to provide programs and other educational resources locally.
Our staff of extension educator and extension specialists, technicians, and support personnel specializes is providing programs throughout the county, and together with staff from all of Nebraska's 93 counties, we contribute to more than 1.5 million educational contacts each year with Nebraska citizens. Our 4-H Youth Development Program, for example, reaches more than 650 youth in Butler County alone. Statewide, 4-H touches the lives of more than 90,000 youth each year.
University of Nebraska–Lincoln Extension is built to provide practical education that focuses on issues which influence daily life that helps to keep Nebraska competitive in a global society. Extension Specialists and Extension Educators are committed to a theme of excellence with educational programs customized.


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|Michael Arrington / Uncrunched:
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|Matt McGee / Search Engine Land:
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|Groupon, Inc.:
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|Philip Elmer-DeWitt / Fortune:
|Ingrid Lunden / paidContent:
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|Erick Schonfeld / TechCrunch:
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PERM Ads for Dallas, TX
Dallas has quickly grown to become a commercial and cultural hub of the Texas region! It offers the largest urban arts district in the nation, plenty of unique historical sights, a diverse economy, and strong industrial sectors. This major inland port is home to a variety of businesses ranging from smaller operations to fast-growing corporations scattered throughout the city from the metro area to Preston Center, Uptown Village, and Koreatown.
Many growing businesses in the Dallas area are looking to hire immigrants, but must be sure to follow the strict guidelines set in place by the Department of Labor (DOL) to ensure that they are able to do this legally and without it coming at the cost of permanent Dallas residents not being able to find employment. Only approved recruiting firms like Ad Club are able to help your business obtain a PERM labor certification through an extensive series of PERM ads.
Why You Need Professional PERM Ads to Gain a Labor Certification in Dallas, TX
PERM ads are a major part of the process of legally hiring immigrant workers in Dallas. Requirements state that the ads must include the name of your Dallas business, instructions detailing where applicants should be sent, a general overview of the specific position, and possible mitigating factors like travel percentages. There are also stipulations regarding how long these ads must be placed along with various other posting requirements.
Quality, Creative PERMS Ads will Save Your Dallas Business Time & Money
The experienced team at Ad Club will coordinate with your legal team and your recruiters to make sure that each PERM ad is placed in accordance with legal requirements. Their team will also make sure that tear sheets of your ads accompany each invoice, and are delivered to you in a timely manner. When you work with Ad Club, you can expect high-quality ads with a creative flair for industry-leading prices.
Let Ad Club make your job a little easier if you are looking to post a job to hire immigrants and foreign nationals at your business in Dallas. Contact them today to learn more about the PERM labor certification and PERM ads!