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Nonalcoholic fatty liver disease (NAFLD), which occurs in approximately 17 to 40% of Americans, encompasses progressive stages of liver damage ranging from steatosis to nonalcoholic steatohepatitis (NASH). Inflammation and oxidative stress are known characteristics of NAFLD; however, the precise mechanisms occurring during disease progression remain unclear. The purpose of the current study was to determine whether the expression or function of enzymes involved in the antioxidant response, NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione transferase (GST), and glutamate cysteine ligase, are altered in the progression of human NAFLD. Human livers staged as normal, steatotic, NASH (fatty), and NASH (not fatty) were obtained from the Liver Tissue Cell Distribution System. NQO1 mRNA, protein, and activity tended to increase with disease progression. mRNA levels of the GST isoforms A1, A2, A4, M3, and P1 increased with NAFLD progression. Likewise, GST A and P protein increased with progression; however, GST M protein levels tended to decrease. Of interest, total GST activity toward the substrate 1-chloro-2,4-dinitrobenzene decreased with NAFLD progression. GSH synthesis does not seem to be significantly dysregulated in NAFLD progression; however, the GSH/oxidized glutathione redox ratio seemed to be reduced with disease severity, indicating the presence of oxidative stress and depletion of GSH throughout progression of NAFLD. Malondialdehyde concentrations were significantly increased with disease progression, further indicating the presence of oxidative stress. Nuclear immunohistochemical staining of nuclear factor E2-related factor 2 (Nrf2), an indicator of activation of the transcription factor, was evident in all stages of NAFLD. The current data suggest that Nrf2 activation occurs in response to disease progression followed by induction of specific Nrf2 targets, whereas functionality of specific antioxidant defense enzymes seems to be impaired as NAFLD progresses.
Nonalcoholic fatty liver disease (NAFLD) is recognized as an epidemic of modern, industrialized countries. Reports estimate that approximately 17 to 40% of the United States population is affected by NAFLD (McCullough, 2006; Ali and Cusi, 2009). It is a complex disease consisting of various stages characterized by progressive levels of hepatocellular damage. NAFLD originates as an infiltration of triglycerides within hepatocytes (Angulo, 2002) known as simple fatty liver (steatosis). Steatosis is generally considered a benign condition, although it renders the liver susceptible to further damage (Adams and Lindor, 2007; Marra et al., 2008). The initial accumulation of lipids within hepatocytes is described as the "first hit" in NAFLD pathogenesis and is believed to be associated with increased sensitivity to oxidative stress (Nagata et al., 2007). Steatosis can progress to the more severe nonalcoholic steatohepatitis (NASH), which is associated with an increase in inflammation, fibrosis, oxidative stress, and more widespread damage (Kleiner et al., 2005; Marra et al., 2008).
Although the mechanisms are not entirely clear, the presentation of oxidative stress is regarded as the "second hit" in NAFLD pathogenesis (Nagata et al., 2007). In addition, the development of inflammation and fibrosis, which cause impairments in liver function, are important factors in the differentiation of steatosis from NASH (Marra et al., 2008). Furthermore, activation of inflammatory cells and mitochondrial dysfunction are believed to be significant contributors to the perpetuation of reactive oxygen species (ROS) production in the progression from steatosis to NASH (Marra et al., 2008). NASH, which is considered to be the end stage of NAFLD, is estimated to affect 5.7 to 17% of the United States population and can further progress to cirrhosis and liver failure (McCullough, 2006). Approximately 15 to 25% of patients with NASH are estimated to progress to cirrhosis with 30 to 40% eventually dying of a liver disease-associated cause (McCullough, 2006).
Oxidative stress is a cellular state in which the generation of harmful molecules such as ROS, peroxidized lipids, reactive quinones, and electrophiles is disproportionate to the ability of the cell to remove these destructive agents. The capacity of a cell to manage oxidative stress is primarily mediated through antioxidant response elements (AREs), which are largely under the control of the transcription factor nuclear factor E2-related factor 2 (Nrf2) (Itoh et al., 1997; Jaiswal, 2004). Nrf2 is primarily a cytosolic protein due to proteasomal degradation regulated by Keap1 (Jaiswal, 2004; Zhang, 2006). When a cell is undergoing oxidative stress, Nrf2 escapes Keap1-mediated degradation, translocates to the nucleus, binds to ARE sequences within the promoters of target genes, and induces expression of phase I and II metabolizing enzymes and transporters pivotal to the maintenance of oxidative stress-inducing molecules (Jaiswal, 2004; Nakata et al., 2006; Zhang, 2006). Such enzymes include NAD(P)H:quinone oxidoreductase-1 (NQO1), glutathione transferase (GST) isoforms, and glutamate cysteine ligase (GCL) subunits (Itoh et al., 1997; Nguyen et al., 2000; Nakata et al., 2006).
NQO1 is a cytosolic protein that catalyzes the two-electron reduction of reactive quinones, which are capable of producing ROS and initiating redox cycling (Jaiswal, 2000; Ross and Siegel, 2004; Talalay and Dinkova-Kostova, 2004). Human GSTs are a large family of detoxifying enzymes that catalyze the conjugation of GSH to reactive electrophiles (Hayes et al., 2005). GSTs can be found in the cytosol, mitochondria, and microsomal membranes of most tissues throughout the body (Hayes et al., 2005). The cytosolic GST isoforms consist of several families, some of which are included in the Alpha, Mu, and Pi classes, each of which have multiple members (Pastore et al., 2003; Hayes et al., 2005). The Alpha and Mu classes have received significant attention as Nrf2 target genes, and specific Alpha isoforms are important for the inactivation of oxidative stress-inducing lipid peroxides. GCL is the rate-limiting step in the synthesis of GSH and is composed of two subunits, catalytic (GCLC) and modifier (GCLM) (Lu, 2009). Because GSH is a cofactor for GST isoforms, disruptions in GSH synthesis have the potential to severely disturb the detoxifying capacity of hepatic GSTs. However, reductions in GSH concentration do not always correlate with GCL expression or activity, e.g., during periods of significant depletion of GSH pools (Pastore et al., 2003; Lu, 2009).
Because of the increasing prevalence of NAFLD, elucidating the mechanisms of oxidative stress-induced injury within the liver is vital to understanding the pathogenesis of NAFLD and susceptibility to further damage. The aim of the current study was to evaluate the expression and activity of NQO1, GST isoforms, and GSH synthesis in the livers of adult patients with progressive stages of NAFLD.
Materials and Methods
Tris-HCl, EDTA, KCl, phenylmethanesulfonyl fluoride, 2,6-dichlorophenol-indophenol (DCPIP), NAD(P)H, 1-chloro-2,4-dinitrobenzene (CDNB), GSH, 3,3′diaminobenzidine tetrahydrochloride (DAB), and H2O2 were obtained from Sigma-Aldrich (St. Louis, MO).
Human Liver Samples and Tissue Preparations.
Frozen and formalin-fixed, paraffin-embedded adult human liver tissue was obtained from the Liver Tissue Cell Distribution System at the University of Minnesota, Virginia Commonwealth University, and the University of Pittsburgh. All samples were scored and categorized by a medical pathologist at the Liver Tissue Cell Distribution System according to the NAFLD Activity Score system developed by Kleiner et al. (2005) as part of the service provided by the agency to National Institutes of Health-funded investigators. Diagnosis was later confirmed by histological examination at the University of Arizona. Donor information, including age and gender, has been published previously (Fisher et al., 2009). The samples were diagnosed as normal (n = 20), steatotic (n = 12), NASH with fatty liver (NASH fatty, n = 11), and NASH without fatty liver (NASH not fatty, n = 11). Samples exhibiting >10% fatty infiltration of hepatocytes were considered steatotic. NASH (fatty) samples were diagnosed on the basis of >5% fatty infiltration of hepatocytes with significant inflammation and fibrosis. NASH (not fatty) samples were differentiated from NASH (fatty) samples by a reduction in fatty deposits within the hepatocytes (<5%). Furthermore, NASH (not fatty) samples presented more marked inflammation and fibrotic branching. Total RNA was isolated from human liver tissue using RNAzol B reagent (Tel-Test Inc., Friendswood, TX), according to the manufacturer's recommendations. RNA concentrations were determined by UV spectrophotometry. Integrity of the RNA was confirmed by ethidium bromide staining after agarose gel electrophoresis. Cytosolic fractions of human liver tissue were prepared by homogenization in buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 154 mM KCl with 0.5 ml/100 ml phenylmethanesulfonyl fluoride (100 mM) at 4°C. Homogenates were centrifuged at 10,000g for 30 min, and the supernatant was transferred to clean microcentrifuge tubes. Samples were then centrifuged at 100,000g for 70 min, and the supernatant was retained as the cytosolic fraction. Protein concentrations were determined using a Pierce BCA Protein Quantitation Assay (Thermo Fisher Scientific, Waltham, MA) per the manufacturer's protocol.
Branched DNA Assay.
Specific oligonucleotide probes (Supplemental Table 1) for NQO1, GCLC, and GCLM were diluted in lysis buffer supplied by the Quantigene HV Signal Amplification Kit (Genospectra, Fremont, CA). Substrate solution, lysis buffer, capture hybridization buffer, amplifier, and label probe buffer used in the analysis were all obtained from the Quantigene Discovery Kit (Genospectra). The assay was performed in a 96-well plate in which total RNA (1 μg/μl; 10 μl) was added to the capture hybridization buffer with 50 μl of the diluted probe set. The total RNA was then allowed to hybridize to the probe set overnight at 53°C. Hybridization steps were performed the following day per the manufacturer's protocol. Luminescence of the samples was measured with a Quantiplex 320 bDNA luminometer interfaced with Quantiplex Data Management Software (version 5.02).
Real-Time Reverse Transcriptase-Polymerase Chain Reaction.
cDNA was reverse-transcribed from total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Indianapolis, IN) per the manufacturer's protocol. Specific primers for GST isoforms A1, A2, A4, M1, M2, M3, M4, and P1 (Supplemental Table 2) were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Reactions were performed using the LightCycler 480 Probes Master Kit (Roche Diagnostics) and Universal ProbeLibrary Probes (Roche Diagnostics) according to the manufacturer's protocol. In brief, the reaction was performed as follows: one denaturation cycle (95°C for 2 min), 45 amplification cycles (95°C for 10 s and 60°C for 30 s), and one cooling period (40°C for 30 s). Relative mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase.
NQO1 Activity Assay.
NQO1 activity was determined by measuring the colorimetric reduction of DCPIP at 600 nm for an interval of 1 min as described previously (Aleksunes et al., 2006a). The reaction was performed in triplicate in a 1-ml format containing 200 μM NAD(P)H, 40 μM DCPIP, and liver cytosol in a Tris-HCl buffer (25 mM Tris, pH 7.4, and 0.7 mg/ml bovine serum albumin). Likewise, inhibition reactions were performed with the addition of 20 μM dicumarol to the reaction mixture. The activity was determined by the difference in absorbance rates of the uninhibited and inhibited reaction rates and was then normalized to total cytosolic protein content.
GST Activity Assay.
GST activity was quantified by spectrophotometry according to established methods using CDNB, which is a substrate for the GST isoforms A1, A2, A4, M1, M2, M3, M4, and P1 (Mannervik and Jemth, 1999). In particular, 1-ml reactions of 1 mM CDNB (prepared in 95% ethanol), 1 mM GSH, 0.1 M sodium phosphate buffer (pH 6.5), and liver cytosol were measured at 340 nm for a period of 1 min. All reactions were performed in triplicate. Enzyme activity was calculated as the difference of the rate of a sample reaction from a simultaneous blank reaction devoid of liver cytosol and was normalized to total cytosolic protein content.
Immunoblot Protein Analysis.
Cytosolic proteins (20 μg/well) were separated by SDS-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride membranes. The following mouse monoclonal antibodies were obtained from Abcam, Inc. (Cambridge, MA): 1:1000 NQO1 [A180], 1:500 GST A, and 1:500 GST P ([F2C2]. GCL protein was detected using mouse monoclonal antibodies from Abnova (Taipei City, Taiwan): 1:8000 GCLC (3H1) and 1:3000 GCLM (2B8). Levels of GST M (1:3000; Abcam, Inc.) and total ERK (1:1000 C-16 and C-14; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were measured using goat polyclonal antibodies. Quantification of relative protein expression was determined using image processing and analysis with ImageJ software (National Institutes of Health, Bethesda, MD) and normalized to total ERK.
GSH Assay.
GSH concentrations were determined using a Glutathione Assay Kit (BioVision Research Products, Mountain View, CA) per the manufacturer's recommendations in a 96-well format. In brief, ∼40 mg of tissue was homogenized and preserved with GSH buffer and 6 N perchloric acid. Samples were then precipitated with 3 N potassium hydroxide and centrifuged for 2 min at 13,000g. To detect reduced GSH, 5 μl of the neutralized sample was incubated with the o-phthalaldehyde probe and GSH buffer for 40 min. To detect oxidized glutathione (GSSG), 1 μl of neutralized sample was incubated with GSH buffer and 10 μl of GSH quencher for 10 min, followed by reducing agent and o-phthalaldehyde for 40 min. Fluorescence readings were captured using a SPECTRAmax GEMINI XS (Molecular Devices, Sunnyvale, CA) fluorescence reader.
TBARS Assay.
Malondialdehyde (MDA) concentrations were determined using the TBARS Assay Kit (Cayman Chemical, Ann Arbor, MI) per the manufacturer's recommendations for colorimetric measurement of MDA. In brief, ∼25 mg of tissue was sonicated on ice in radioimmunoprecipitation assay lysis buffer containing 1% NP-40, 1% w/v sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 8.0, 2 mM EDTA, and 1 Protease Inhibitor Cocktail Tablet per 25 ml (Roche Diagnostics). Samples were then centrifuged at 1600g for 10 min at 4°C, and the supernatant was preserved for analysis. To 100 μl of each sample and standard curve solutions, 100 μl of provided SDS solution was added followed by 4 ml of color reagent. Samples and standard curve solutions were boiled for 1 h, and the reaction was terminated by incubation on ice for 10 min followed by centrifugation at 1600g for 10 min at 4°C. Then 150 μl of samples and standard curve solutions were loaded in duplicate onto a 96-well plate, and absorbance was read at 540 nm in a Powerwave HT (BioTek Instruments, Winooski, VT) spectrophotometer.
Immunohistochemical staining for Nrf2 was performed on formalin-fixed paraffin-embedded human liver samples. In brief, tissue sections were deparaffinized in xylene and rehydrated in ethanol, followed by antigen retrieval in citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 0.3% (v/v) H2O2 in methanol for 20 min. All samples were incubated with a 1:75 solution of Nrf2 (C-20) antibody (Santa Cruz Biotechnology, Inc.) overnight at 4°C. Antibody binding was detected with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) and an avidin-biotin complex (Vector Laboratories). Color development was performed with a solution of 0.5% DAB in 0.015% H2O2 (0.01 M phosphate-buffered saline, pH 7.2). All slides were imaged with a Nikon Eclipse E4000 microscope and a Sony Exwave DXC-390 camera.
Quantification of Nrf2 Nuclear Translocation.
Hepatocyte nuclei positive for Nrf2 staining and hepatocyte nuclei negative for Nrf2 staining were quantified within a 20× field of vision with a total of 10 fields assessed for each sample. The number of total hepatocytes was calculated from the positive and negative nuclei counts and was used to determine the percentage of positive nuclei within a field. The average number of positive nuclei, negative nuclei, total nuclei, and percent positive nuclei was assessed for each diagnostic category.
Statistical Analysis.
The samples within this study can be categorized by disease progression/severity: normal < steatosis < NASH (fatty) < NASH (not fatty). All data collected in this study were continuous outcomes but exhibited a skewed distribution. Therefore, median values rather than mean values were compared, and the results are presented as box and whisker plots. Data were analyzed by a nonparametric trend analysis with a significance level of p ≤ 0.05 to determine significant increases or decreases with disease progression/severity. All analyses were performed with Stata10 software (Stata Corporation, College Station, TX).
Human NAFLD Histology.
Representative images of hematoxylin and eosin staining of human liver samples staged as normal, steatotic, NASH (fatty), and NASH (not fatty) are shown in Fig. 1. A diagnosis of steatosis was made for samples exhibiting >10% fatty infiltration of hepatocytes. Samples exhibiting >5% fatty infiltration of hepatocytes with significant inflammation and fibrosis were staged as NASH (fatty). Those samples presenting with a reduction in fatty deposits within the hepatocytes to <5% with increased inflammation and branching fibrosis were diagnosed as NASH (not fatty). Microvesicular lipid deposits are readily apparent in the steatotic samples, and sinusoidal structure remains somewhat intact. The NASH (fatty) stage is characterized by macrovesicular fatty deposits within hepatocytes and the development of inflammation and fibrosis. As the liver progresses from NASH (fatty) to NASH (not fatty), the infiltration of fatty deposits is greatly reduced; however, inflammation increases, as does fibrosis.
NQO1 Expression and Activity in Human NAFLD Progression.
Gene expression of antioxidant response enzymes is indicative of Nrf2 transcriptional activation of the ARE. NQO1 gene expression was measured in human liver samples by assessing the levels of NQO1 mRNA using the bDNA assay, shown in Fig. 2A. There was an increasing trend in NQO1 mRNA expression with progression of NAFLD. An immunoblot analysis of NQO1 protein levels in human cytosolic liver samples was conducted and normalized to total ERK. Representative immunoblots with two samples from each diagnostic category and densitometry results of NQO1 in each stage of NAFLD are shown in Fig. 2, B and A, respectively. Densitometric analysis of relative NQO1 protein levels in all 54 human liver samples revealed an increasing trend in NQO1 protein with disease progression. The enzymatic activity of NQO1 protein was measured in human liver cytosolic samples using DCPIP as the substrate. Enzymatic activity of NQO1 was significantly increased with progression of NAFLD.
GST Isoform Expression and Pan-GST Activity in Human NAFLD Progression.
The expression of various GST isoforms is also dependent on the ARE and, thus, on transcriptional activation by Nrf2. Because of significant homology among the GST isoforms, mRNA levels could not be measured by bDNA analysis, which requires the use of multiple oligonucleotides to identify specific mRNA molecules. Therefore, relative mRNA levels of the GST isoforms A1, A2, A4, M1, M2, M3, M4, and P1 were measured by reverse transcriptase-polymerase chain reaction in each stage of NAFLD. As shown in Fig. 3A, mRNA levels of the GST A4, A2, A4, M3, and P1 isoforms exhibited a significant increasing trend with disease progression. GST protein levels in cytosolic liver fractions were assessed for each isoform family (GST A, GST M, and GST P) and normalized to total ERK. Protein levels were measured for all 54 liver specimens in the study. Representative immunoblots depicting two samples from each diagnostic category and densitometry results are shown in Fig. 3, C and B, respectively. There were increasing trends in expression for both GST A and GST P protein. In contrast, GST M protein levels were decreased with NAFLD progression. The functional capability of the GST isoforms to effectively conjugate GSH to electrophilic substrates was determined spectrophotometrically in human liver cytosolic samples using CDNB as a pan-GST substrate. As shown in Fig. 3D, a significant decreasing trend in GST enzymatic activity was observed with NAFLD progression.
GCL Expression.
mRNA and protein levels of GCL, the rate-limiting enzyme in the synthesis of GSH, were assessed by the bDNA assay and immunoblot analysis in human liver samples. mRNA results of GCLC, the catalytic subunit of GCL, and GCLM, the modifier subunit, are shown in Fig. 4A. No significant alterations were observed in the mRNA levels of GCLC; however, GCLM mRNA levels exhibited an increasing trend as NAFLD severity increased. The densitometric results of GCLC and GCLM protein levels in all 54 human liver samples, along with representative immunoblots depicting two samples from each diagnostic category, are shown in Fig. 4, A and B, respectively. No significant alterations in GCL protein were observed.
GSH Concentration and Redox Status in Human NAFLD.
GSH and GSSG concentrations were determined fluorometrically using the BioVision Glutathione Assay Kit. The results are presented in Fig. 5, along with the GSH/GSSG ratio (redox status). There were decreasing trends in GSH and GSSG concentrations throughout disease progression. The redox status (GSH/GSSG ratio), an estimation of the oxidative state in cells and tissues, is shown in Fig. 5. The redox status exhibited a significant decreasing trend with disease progression.
MDA Concentration in Human NAFLD.
MDA is a product of lipid peroxidation and is often used as an indicator of oxidative stress in tissues and cells. MDA concentration was measured colorimetrically using the Cayman Chemical TBARS Assay Kit. The results, as shown in Fig. 5, reveal a significant increasing trend in MDA concentration with disease progression.
Nrf2 Activation in Human NAFLD.
Induction of ARE genes is dependent on the escape of Nrf2 from Keap1-mediated degradation in the cytosol, enabling its translocation to the nucleus where it may bind to ARE promoters. To determine whether Nrf2 had translocated into the nuclei of hepatocytes and was therefore activated, formalin-fixed, paraffin-embedded human liver samples were immunohistochemically stained for Nrf2. Representative images are shown in Fig. 6. Positive nuclear staining of Nrf2 (indicated by open arrows) can be seen in all stages of NAFLD. Nuclei negative for Nrf2 staining are indicated by closed arrows. Quantification of Nrf2 translocation revealed that in normal human liver samples an average of 2.06% of hepatocytes within a 20× field of vision exhibited positive Nrf2 nuclear staining. In contrast, steatotic, NASH (fatty), and NASH (not fatty) samples presented on average with 24.67, 22.60, and 22.34%, respectively, of hepatocytes bearing positive Nrf2 nuclear staining.
NAFLD, like obesity, is becoming an increasingly significant health concern. Accumulation of triglycerides within hepatocytes, insulin resistance, inflammation, chronic oxidative stress levels, and fibrosis in combination make this a complex disease. The specific mechanisms by which each of these parameters presents in the progression of NAFLD are not certain. With the exception of cytochrome P450 enzymes (Fisher et al., 2009), the metabolic capacity toward administered pharmaceutical agents in patients with NAFLD has not been well characterized. This is of serious concern as patients with NAFLD are often medicated for symptoms of the metabolic syndrome. Therefore, there is a great need for investigation into the pathogenic process of NAFLD as it relates to oxidative stress and the capacity to metabolize pharmaceutical agents.
The aim of the current study was to investigate the expression and functionality of enzymes, such as NQO1, GST, and GCL, involved in the antioxidant response. Of these enzymes, the GST isoforms, in particular, are known to play a significant role in the phase II metabolism of endogenous molecules and administered pharmaceutical agents, whereas NQO1 is primarily responsible for the reduction of oxidative stress-inducing quinones. Increased expression and/or activity of NQO1 has been shown to be an important protective mechanism against oxidative stress in cholestasis (Aleksunes et al., 2006b), coronary heart disease (Martin et al., 2009), acetaminophen-induced hepatotoxicity (Moffit et al., 2007), and primary biliary cirrhosis (Aleksunes et al., 2006a). Although not traditionally considered a drug-metabolizing enzyme, NQO1 is capable of reducing N-acetyl-p-benzoquinone imine, the toxic quinone metabolite of acetaminophen (Moffit et al., 2007). Furthermore, induction of NQO1 activity leads to protection against acetaminophen-induced hepatotoxicity (Moffit et al., 2007). We have observed an increase in NQO1 mRNA levels, protein, and activity with the progression of NAFLD. These findings are similar to observations made in animal models of steatosis and NASH. We previously found that Sprague-Dawley rats fed a methionine- and choline-deficient diet to induce NASH exhibited significant elevation of NQO1 mRNA and enzymatic activity (Lickteig et al., 2007). In a separate study, rats fed the methionine- and choline-deficient diet exhibited significant induction of NQO1 mRNA levels, and, when treated with oltipraz, an Nrf2 activator, were still able to induce NQO1 to an even greater level (Fisher et al., 2008). Together with our previous animal studies, the current data suggest that as NAFLD progresses, increased NQO1 expression and activity levels may confer a greater capacity to protect patients with NASH from oxidative stress-induced and pharmaceutical-induced quinone redox cycling.
In contrast, GSTs are responsible for protecting against electrophilic molecules such as those generated during oxidative stress or administration of pharmaceutical agents. The expression and activity of specific GST isoforms have been closely associated with the severity of multiple diseases. For instance, GST M1 and T1 polymorphisms imparting an impairment of enzyme functionality impose a greater risk for coronary artery disease in patients with type II diabetes (Manfredi et al., 2009). Furthermore, GST M1 functionality has been shown to be important in protection against oxidative stress-induced DNA damage in patients undergoing maintenance hemodialysis (Lin et al., 2009). Traditionally, the induction of GST isoforms has been associated with protective mechanisms because of their ability to catalyze the conjugation of GSH to various ROS molecules (Hayes et al., 2005). Although several GST isoforms are inducible by Nrf2, there is generally a low, constitutive expression of GST A3, A4, M1, M3, and M4 (Aleksunes and Manautou, 2007), whereas GST A1 and A2 are believed to be more highly expressed in liver (Coles and Kadlubar, 2005). Moreover, GST A4 expression is believed to be induced during periods of prolonged oxidative stress (Coles and Kadlubar, 2005). GST P genes, on the other hand, are considered to be inducible, but not constitutively expressed within the liver (Henderson and Wolf, 2005). At present, we have observed an increasing trend in GST A1, A2, A4, M3, and P1 mRNA expression throughout progression of NAFLD. The induction of GST P1 mRNA has previously been recognized as a marker for hepatocellular carcinogenesis in the rat but may or may not be associated with hepatocellular carcinoma in humans (Higashi et al., 2004; Sakai and Muramatsu, 2007). Therefore, the induction of GST P1 shown in the present study may be an indicator of a predisposition to hepatocellular carcinoma but is not definitive. We have observed increases in GST A and P protein with disease progression. In contrast with GST A and P protein, there was a significant downward trend in GST M protein levels. These data suggest that the stability of each GST protein family may be differentially regulated throughout progression of NAFLD.
Because of the increased expression of multiple GST isoforms and induction of protein levels of the GST A and P families, GST enzymatic activity could be expected to similarly increase with the progression of NAFLD. However, in the current study, we observed a gradual decline in GST activity levels. These results suggest that as NAFLD advances, patients may have a reduced capacity to combat the oxidative insults of electrophilic compounds. A similar phenomenon has been observed in a murine model of ulcerative colitis (Clapper and Szarka, 1998). Note that this downward trend in GST activity parallels the expression of GST M protein within our samples. GST A isoforms, on the other hand, are traditionally considered the dominant isoforms expressed in the liver (Coles and Kadlubar, 2005). These data indicate that there may be a more complicated mechanism of regulating GST isoforms during NAFLD progression.
Studies in human hepatic cirrhosis and hepatocellular carcinoma tissues have presented a complex interplay in GSH-dependent enzyme activities. These studies have shown that GST activity is significantly increased in cirrhotic livers compared with that of malignant hepatic tissue (Czeczot et al., 2006). Further analysis in this study identified a significant reduction in GSH within cirrhotic hepatic tissue compared with both malignant and normal tissues (Czeczot et al., 2006). In the current study, we observed a similar reduction in hepatic GSH levels during disease progression, and, although GSSG levels exhibited a significant downward trend, GSSG concentrations were much higher than those of GSH. Whereas this can sometimes be due to the insensitivity of the analytical method used or the labile nature of GSH in tissues, previous reports indicated that GSSG levels may indeed be much higher than GSH levels in NAFLD (Pastore et al., 2003). Using the GSH and GSSG concentrations measured in each sample, we calculated the GSH/GSSG redox ratio, which is an indicator of oxidative stress within the tissue. The reduction in the GSH/GSSG redox ratio observed during NAFLD progression indicates that oxidative stress is increasing with the severity of disease. An additional indication of the presence of oxidative stress during NAFLD progression was observed by the increasing concentration of MDA with disease severity. MDA is a product of lipid peroxidation that occurs under oxidative stress conditions. Increasing levels of MDA confirm the conclusion of increased oxidative stress occurring during NAFLD made during the measurement of GSH levels. Nevertheless, the decrease in GST activity seen with NAFLD progression cannot be simply attributed to the measured reductions in GSH concentration. In this activity assay, GSH is supplemented in the reaction, thereby reducing the confounding effect of depletion of GSH pools in the sample, therefore leading to the conclusion that additional factors must play a role in the conjugating capabilities of GST isoforms during NAFLD progression. One possible explanation is the adduction of 4-hydroxy-2-nonenal (HNE) to specific GST isoforms. Studies have identified GST P as a target of HNE adduction in human colorectal carcinoma cells (Codreanu et al., 2009). Inhibition studies of GST P1 have indicated that cysteine 47, which is associated with the active site of the enzyme, is the target of covalent binding by HNE (van Iersel et al., 1997). In the present study, we have observed a significant increase in expression of GST P protein that, if inhibited by HNE may, in conjunction with GST M protein expression, explain the observed depletion in GST enzymatic activity. Further investigations are necessary to identify the specific mechanism by which GST activity is inhibited in NAFLD.
To identify whether the observed reductions in GSH concentration are due to a depletion of GSH pools or impairment of GSH synthesis, we investigated expression of the GCLC and GCLM subunits of GCL. GCL is responsible for the initial catalysis of glutamate and cysteine to produce γ-glutamyl-l-cysteine (Lu, 2009). Further conjugation with glycine is catalyzed by GSH synthetase (Lu, 2009). GCL is regulated by feedback inhibition through GSH concentration, and induction of GCL expression does correlate with increased GSH synthesis; however, induction of GSH synthetase does not lead to a subsequent increase in GSH levels (Lu, 2009). In the current study, we have identified an increasing trend in GCLM mRNA levels; however, there was no significant alteration of GCLM protein levels. In addition, no alterations to GCLC expression were observed. Together, these data provide no indication of an impairment of GSH synthesis, thereby leading to the conclusion that the observed reductions in GSH levels are most likely due to a depletion of GSH pools.
As stated previously, activation of the transcription factor Nrf2 leads to nuclear accumulation of Nrf2 protein and induction of downstream target ARE genes (Zhang, 2006). In the current study, we have identified nuclear accumulation of Nrf2 in each of the stages of NAFLD. Quantification of hepatocytes exhibiting positive Nrf2 nuclear staining revealed a stark increase in the percentage of total hepatocytes in which Nrf2 was activated among each of the disease states compared with normal. Activation of Nrf2 is a viable explanation for the observed induction of ARE target genes. The increase in enzymatic activity of NQO1 implies an enhanced ability to diminish oxidative damage due to quinone redox cycling in NAFLD. However, the observed decrease in GST enzymatic activity suggests that patients with NAFLD have a limited capacity to manage electrophilic compounds. The current study provides novel insight to the mechanisms of oxidative stress as they occur in the progression of human NAFLD.
We thank the National Institutes of Health-funded Liver Tissue Cell Distribution System for continued assistance and procurement of liver tissue samples from patients with various stages of NAFLD. We also extend sincere appreciation to Marion Namenwirth (University of Minnesota), Melissa Thompson (Virginia Commonwealth University), and Dr. Stephen C. Strom and Kenneth Dorko (University of Pittsburgh).
This work was supported in part by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK068039]; the National Institutes of Health National Institute of Environmental Health Sciences [Grant ES006694]; and the National Institutes of Health National Center for Complementary and Alternative Medicine [Grant AT002842]. The Liver Tissue Cell Distribution System was sponsored by the National Institutes of Health [Contract N01-DK-7-0004/HHSN267200700004C].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
- nonalcoholic fatty liver disease
- nonalcoholic steatohepatitis
- reactive oxygen species
- antioxidant response element
- Nrf2
- nuclear factor E2-related factor 2
- NAD(P)H:quinone oxidoreductase 1
- glutathione transferase
- glutamate cysteine ligase
- glutamate cysteine ligase catalytic subunit
- glutamate cysteine ligase modifier subunit
- 2,6-dichlorophenol indophenol
- 1-chloro-2,4-dinitrobenzene
- 3,3′diaminobenzidine tetrahydrochloride
- extracellular signal-regulated kinase
- oxidized glutathione
- thiobarbituric acid reactive substances
- malondialdehyde
- 4-hydroxy-2-nonenal.
- Received June 15, 2010.
- Accepted August 30, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics


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BOSTON (AP) — New England's historic fishing industry doesn't spare much of its romance for the herring.
The tiny fish is, at most, a foot long. The price per pound often won't even buy 12 minutes at a Boston parking meter. Some people eat it pickled, but herring is mainly caught to become bait for more popular seafood, such as lobster.
The herring, though, is deeply important to fishermen and environmentalists, who are fighting to put greater restrictions on trawlers that pull up hundreds of thousands of pounds of herring at a time.
They argue that the large trawlers are depleting a species that's a critical food for just about every prized commercial fish in the region, from cod to striped bass. The herring's influence even extends to ocean tours, which depend on abundant herring to attract whales and birds to the ocean surface to feed and be seen.
"For many people who don't work on the water, make their money on the water, I think it's easy to underestimate the importance of herring," said Tom Dempsey of the Cape Cod Commercial Hook Fishermen's Association.
The New England Fishery Management Council is considering clamping down on midwater herring trawlers, named for the area of the water column they the pull their nets through. A series of eight public hearings around the Northeast wrapped up Thursday in Cape May, N.J., and council action is expected in June.
The proposals include tighter requirements on how the trawlers weigh their catch and bans on certain fishing areas.
A key proposal would force trawlers to carry independent observers on every trip, in part to stop suspected over-catching and dumping of protected species the herring boats snare unintentionally, such as cod and haddock.
"It's time for a change in that fishery," said Bob St. Pierre, who fishes for tuna, striped bass and groundfish out of Chatham.
But the herring industry says there's scant evidence their trawlers are the menaces they're portrayed as.
Mary Beth Tooley, a long-time herring industry member and also a Maine representative on the regional management council, said the stock is robust, and there's no research yet to contradict that. There's also no data to show the midwater trawlers are catching and killing huge amounts of other fish species.
Opponents complain scientists have simply been slow to collect the information. But Tooley said the herring industry is getting battered based on a faulty assumption that because the boats are big — up to 165 feet long — they're doing big damage.
"I think perception is everything," she said. "I don't think there's a lot we can do to overcome that perception."
Herring was the fifth-highest revenue fishery in New England in 2010, bringing in nearly $21 million on 140 million pounds of fish (15 cents per pound), according to the National Marine Fisheries Service. The bulk of the catch, about 80 percent, was bait for commercial and sport fishermen.
Herring are a schooling fish and can congregate in masses the size of Manhattan. Steve Weiner, a harpoon tuna fishermen, describes being atop colossal "vortexes" of herring, as whales, other fish and countless birds dove into them.
"It's scary even to be in it, because these animals are just drunk on herring," Weiner said.
But St. Pierre said he doesn't see any more huge herring shoals in local waters, and that's coincided with the appearance of the midwater trawlers.
The trawlers often work in pairs, pulling a football field-sized net between them, then sucking the herring from the net into ships' holds. The trawlers, with crews of about six, dominate the local herring catch and work with remarkable efficiency.
St. Pierre says he'll be working an area with abundant haddock, for instance, then the trawlers appear and the haddock are gone. Weiner said fishermen have also noticed the herring getting smaller, a possible sign that the older, larger herring have been fished to nearly nothing.
This evidence of a herring problem is mostly anecdotal, but Weiner says the decades of knowledge and observation from all corners of the industry can't be dismissed.
"The best available science in most of these fisheries is still pretty weak stuff," Weiner said.
The depletion of herring stocks could have numerous implications, fishermen and environmentalist say. Bait costs would rise for the region's lucrative lobster industry. Without herring to chase and eat, game and commercial fish could fade from inshore waters. Struggling species, such as cod, could fail to rebound without this key food.
"Herring are a vital food source for cod and many other species," said Jud Crawford of the Pew Environment Group. "It doesn't take a rocket scientist to get the idea that if you're pulling out one of the major food sources (for cod) ... you're at least decreasing the chances of recovery."
Regulators are now conducting a major assessment, expected to be completed this summer, which should answer key questions about herring's health.
In the meantime, the herring industry has anecdotal evidence of its own to counter critics. Tooley said reports of herring scarcity are just snapshots from people who don't track the fish for a living and see how plentiful it is.
Without hard evidence that shows otherwise, the industry isn't going to agree to being shut out of new areas, concede that long-used methods for weighing the catch are inaccurate or say the trawlers are harming other fish species.
But Tooley said the industry supports placing independent observers on every midwater trawler trip, to increase overall confidence in the fleet.
"We need to have a monitoring program that allows the fleet to operate without all this constant criticism," she said.

subset: commoncrawl


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NorthEast Carpet CleanningOxford, MA 01540ph: 508-987-3799fax: 508-987-3799alt: email@example.com
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Northeast Carpet Cleaning is locally owned and operated. We have been in business for over thirty years serving Massachusetts communities with the highest standards.
We are a family orientated business
Copyright 2013 NorthEast Carpet Cleanning. All rights reserved.
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I'm glad and sad Thanksgiving is over, I could do without the stress and rich eating. But miss the fun time with family and relaxing long weekend. And it means Christmas madness has officially begun! I have a pile of Amazon boxes waiting for me to open and wrap which will happen this weekend, maybe. Miko and Smokey have been enjoying cuddle couch time with hubby.
And Leela having her fireplace bed all to herself.
The holidays also bring some great vegan products like vegan nog, a simply amazing beverage for adding to coffee, making nog banana soft serve in the VitaMix, or guzzling straight from the carton. But the store bought stuff can contain a lot of sugar and preservative ingredients that aren't so awesome. Let's be real, something that disappears so fast in my fridge does not need preservatives. Here's a thick hemp seed-based nog recipe you can make anytime time of the year. It will add a creamy heft to your coffee with a festive flavor or make amazing nog banana soft serve ice cream in the blender.
Healthy Hemp Nog
- 1 1/2 cups shelled hemp seeds
- 1 cup pitted medjool dates
- 1 tsp. nutmeg
- 2 tsp. vanilla extract
- 1 tsp. cinnamon
- 4 cups water
Place all ingredients into a high speed blender and blend to a smooth and creamy texture. Serve and enjoy! Makes 6-8 servings.
Yeah, it's thick, creamy, hearty, and heavy as nog should be.
And stores in the fridge for up to 5 days, but you'll need to shake it up a bit before each serving.
The flavor is very nog like with a warming cinnamon and nutmeg blend.
Enjoy this rich holiday treat also loaded with protein, good fats, and fiber.


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- Corporate News
Victoria's Visitor Economy Hits $30 billion
Results from the latest National Visitor Survey have revealed Victoria's visitor economy has reached $30 billion, showing Visit Victoria is well on track to achieve a target of $36.5 billion by 2025 as set out in the Victorian Government's Visitor Economy Strategy.
The results of the National Visitor Survey for the year ending March 2019 show Victoria recorded double-digit growth across all key domestic tourism measures and was above the national average growth rates for number of visitors and nights.
Domestic overnight visitors to and within Victoria increased at a pace of 11.8 per cent, above the national average increase of 10.1 per cent, to 27.8 million total visitors. Domestic overnight expenditure grew by 11.9 per cent to reach $15.5 billion.
The results showed interstate tourism to Melbourne is strong, with visitation up by 10.9 per cent, visitor nights up by 14.6 per cent and expenditure up by 7.4 per cent. Melbourne's total domestic overnight growth in visitor numbers and nights was above the average rate for all capital cities, and interstate overnight expenditure in Melbourne increased by 7.4 per cent to reach $5.8 billion, $760 million ahead of interstate visitor spend in Sydney.
In regional Victoria, intrastate tourism is booming with increases in visitors (up 11.0 per cent), visitor nights (up 11.1 per cent) and expenditure (up 14.0 per cent). This growth is primarily coming from Melburnians, but there is still strong growth in the regional to regional market.
Domestic overnight expenditure for regional Victoria grew by 12.1 per cent to reach $6.8 billion, driven by high spend growth from intrastate visitors (up 14 per cent to $5.4 billion).
Visitor expenditure grew for eight out of Victoria's eleven tourism regions with the greatest increases seen in Daylesford and the Macedon Ranges (up 37 per cent), Mornington Peninsula (up 31.4 per cent), Victoria's High Country (up 24.5 per cent), Geelong and the Bellarine (up 18.2 per cent) and Murray (up 15 per cent).
Visit Victoria CEO Peter Bingeman said the results have been achieved with the assistance of interstate and intrastate marketing campaigns, alongside an array of major events and regional events.
Visit Victoria has a clear strategy to encourage Melburnians to visit regional Victoria, backed by the Your Happy Space campaign and more the 200 regional events over the past three years.Visit Victoria CEO Peter Bingeman
"The latest results show our strategy is working with an 11 per cent increase in intrastate visitors, an 11 per cent increase in visitor nights, and intrastate expenditure up by 14 per cent to reach $5.4 billion."
The strong performance from the intrastate market has driven Victoria's overall visitor economy to $30 billion when combined with international visitor expenditure.
"Visit Victoria has helped the state's visitor economy to reach $30 billion by backing local tourism operators, strategically marketing Victoria to a range of audiences and attracting the major events that make Victoria the sporting and cultural capital of the country," Peter Bingeman said.
"Visit Victoria will continue to work tirelessly to make Victoria's visitor economy the strongest in the country."


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April - May, 2016
Executive Board
Executive Board
Melissa Beaudoin
First Vice President & Programs
Second Vice President & Membership
Recording Secretary
Mikki Terry: 703-978-0205
Bobbi Green: 703-328-7260
Violeta Warner:
703 282-9779
Sitting Committees
Budget & Bylaws:
Bobbi Greene
Rosemary Espinosa
Ann Collins
Directory: Carol Ford
Lola LeCraw
Fundraising/Ways & Means: Lola LeCraw
Newsletter & Social Media: Violeta Warner
Parliamentarian: Jane Woods
Photographers: Mary Bell & Violeta Warner
2016 GMRW Schedule, Meetings & Conventions
Committee Needing Leadership
Campaign Activities
Current Issues
Homeland Security
Public Relations
State & Local Legislation
Young Women's Liason
Needed Now:
Precinct Captains
outnumbered by Dem EOs in Fairfax County, so please consider becoming
one. You learn a lot about the election process and you get paid!!for the Springfield District
"One Team - One Fight"
Contact Geraldine Davie at [email protected]
GMRW Membership Committee Report
By Patricia Martell
The GMRW Membership Committee is running a competition among our membership. The member that brings in the most new members during 2016 will be given a prize. There will be a mid-year and an end of the year recognition. If there is a tie, we will have a lottery at our meeting to award the prize. The end year total will be for the whole year while the mid-year is the total for 6 months. The prize which is pictured here is to be given in June.
Not Voting
By Ann M. Collins Chair, Constitution Committee
In our present chaotic political campaign environment, many people who support either party may be wondering whether "not voting is ok." Before making that final decision on whether or not to vote, just remember that your vote can make a difference. The power of the Federal government comes from the US Constitution--if it's not in the Constitution, it's not authorized. And 100 percent of that power is vested solely in 545 individuals. Thus, of over 300 million Americans, only 545 wield all of the power of the Federal government. Those individuals are 435 members of the US House of Representatives, 100 US Senators, one President, and nine Supreme Court justices. Other bureaucrats have limited or delegated power by the Congress. Thus, we can assume anything involving the Federal government that is wrong is 100 percent the fault of those 545 individuals. Those 545 individuals spend much of their energy convincing us that what they did is not their fault. They cooperate in this common con regardless of party. Therefore, Congress is the originator of all Federal government problems and Congress is also the only remedy available. That is why complaints and protests should be properly directed at Congress, not at the individual agencies.
Most importantly, the US Constitution gives Americans an opportunity to choose those 545 individuals by our election processes. Although the phrase "right to vote" is mentioned in more than one amendment to the US Constitution, the Fifteenth Amendment, however, does speak specifically of "the right of citizens of the United States to vote." It is a cherished right that should be exercised, seriously. When not exercised, it generally means the same as casting a vote for the opposition and many times allowing an unwanted individual to gain power.
Charley Reese (1937-2013), a former Orlando Sentinel columnist, was a popular conservative voice for decades arguing forcefully for less government and more personal freedom. The following excerpts from his March 1995 editorial (updated) gives some rationale for taking a stand to always exercise your right to vote. Think about it!
"...Politicans, as I have often said, are the only people in the world who create problems and then campaign against them....
"...It seems inconceivable to me that a nation of over 300 million cannot replace 545 people who stand convicted--by present facts--of incompetence and irresponsibility. I can't think of a single domestic problem that is not traceable directly to those 545 people. When you fully grasp the plain truth that 545 people exercise the power of the Federal government, then it must follow that what exists is what they want to exist...."
"...There are no insoluble government problems. Do not let these 545 people shift the blame to bureaucrats, whom they hire and whose jobs they can abolish; to lobbyists, whose gifts and advice they can reject; to regulators, to whom they give the power to regulate and from whom they can take this power. Above all, do not let them con you into the belief that there exists disembodied mystical forces like "the economy," "inflation," or "politics" that prevent them from doing what they take an oath to do....
"...They (545), and they alone, should be held accountable by the people who are their bosses. Provided the voters have the gumption to manage their own employees--we should vote all of them out of office and clean up their mess!"
The 2016 fall general election ballot will include presidential and congressional candidates. Also, Virginia allows absentee voting in person or by mail. Don't pass up this opportunity to help choose the next President and the individuals who will control the 115th Congress starting in January 2017. Encourage your family members and friends to vote, too. The future of our country depends on it!
Check out local Voting information:
Click here: Office of Elections - Fairfax County, Virginia
Dear George Mason Republican Women,
It has been a busy spring for us! George Mason Republican Women (GMRW) members played active roles in the VFRW, RPV, county, and district conventions. Thank you, all, for your hard work in making these events successes. Please join me in giving a special thank you and congratulations to our very own Linda Bartlett for faithfully serving as the VFRW President for two very successful terms. Thank you for the sacrifice of your time, your leadership, your vision, and your many accomplishments that have placed the VFRW in a position to thrive, Linda. We are proud to say that you are a member of the GMRW family!
With the state and local conventions now a memory, we look to RNC Convention in July and the final determination of our party's nominee. Once our Republican nominee is formally established, I join my voice with those who call for Republicans to unite behind our nominee. At the May GMRW meeting, I read an article written by the President of the Republican Women of Clifton, Bonnie Burkhardt. It eloquently explains why we need unite behind our nominee and ensure that a Republican is sworn into office in January. The article is included in this newsletter in the guest writer section. If you were not able to attend the May meeting, please take a moment and read this excellent article. Also, if you have friends and family that are hesitant to unite behind the Republican nominee, send them the newsletter and have them read Bonnie's article. It will help them unite behind the Republican nominee as it explains that a vote for president is more than a vote for just one me. It is also a vote for more than 3000 people that the president appoints.
At our May meeting, GMRW welcomed Dr. Milan Sturgis as our guest speaker. Dr. Sturgis spoke on Afghanistan and national security. He discussed the historic development of the state of Afghanistan, how it has impacted the entrenched conflict that it has become, the current conflict, and how modeling is used to anticipate actor behavior in the conflict. What an excellent and informative talk! Thank you for joining us, Dr. Sturgis!
Please mark your calendars and make your plans, now, to join us for our June meeting because this is a meeting you will not want to miss! First, we are excited to be hosting Milt Clary, who will be talking about women & fire arms and 2nd Amendment rights. Also, we will be presenting a resolution to the GMRW body for consideration. Fairfax County Public Schools Board Members Elizabeth Schultz and Jeanette Huff have been actively opposing the reassignment of public school bathrooms to gender neutral areas. We plan to present to the GMRW membership a resolution to Board Members Schultz and Huff formally supporting and thanking them for their efforts to protect the privacy and safety of our children. Please, join us on Saturday, June 18 at 12:30 PM, at King's Park Library in Burke for this important meeting.
Finally, I wish all of you a wonderful summer with your friends, families, and loved ones and I look forward to seeing you all again, soon.
Melissa Beaudoin, President
George Mason Republican Women
The Hilton McLean Tyson Corner
2015 President Geraldine Davie humbly accepted the awards for all the members of our club.
Photos from the VFRW Convention 2016
Guest Speaker: Dr. Milan Sturgis
Dr. Sturgis spoke on Afghanistan and national security. He discussed the historic development of the state of Afghanistan, how it has impacted the entrenched conflict that it has become, the current conflict, and how modeling is used to anticipate actor behavior in the conflict. What an excellent and informative talk! Thank you for joining us, Dr. Sturgis!
Bonnie Burkhardt, PresidentRepublican Women of Clifton
I voted for Ted Cruz in the primary. When people talk about party unity, they talk about the next President nominating between 3-5 new Supreme Court Justices. Only one percent of all the cases referred to the Supreme Court actually get heard. For the other ninety-nine percent, the ruling of the lower court stands and those justices are also appointed by the President. Presidential appointees control all the lower courts, appeals courts, etc.
There are between 3,100 and 3,500 political appointees granted positions from the Plum List, which is so named because it contained in the Plum Book which has a purple cover. Although the Civil Service may do all the paperwork for how to apply for and get a grant or how to shuffle papers for a law to be properly documented, the direction they get is from the political appointees. There is a six-page printout of just the list of departments within the federal government that get appointees. It starts with the Defense Department, which has been operating in spite of Obama's political appointees the last few years.
Other departments include Management and Budget, Economic Advisors, Environmental Quality, Trade, HUD, Science and Technology, Drug Control, Agriculture, Commerce, CIA, Education, Energy, Health, Housing, Labor, Justice, Transportation, Veterans Affairs, Battle Monuments, Arctic Research, Scholarships, Fellowships, Civil Rights, Fine Arts, Disabled, Product Safety, DC, Nuclear Anything, EPA, FDIC, Housing, Maritime, Mine Safety, Energy, Federal Reserve, Peace Corp, Federal Trade Commission, Medicare, NASA, Archives, Indian Affairs, Ethics, Alaska Natural Gas, Railroad, Securities & Exchange, Small Business Administration, Smithsonian, Social Security, Tennessee Valley Authority, Trade, Holocaust Memorial, International Trade Commission, and Postal Service, just to name a few. You are not just voting for President; you are voting for a slate of 3000 to 4000 people to run your government.
Although Obama may be smart, at least they say he is smart, there is no way he alone can come up with all the wacky policies that we have endured the last 7 years. Some examples include the recent Department of Education and the trans-gender bathroom issue, or redefining marriage. Then there is the destruction of the low-end used car market so poor people can't afford to buy cars anymore with the Cash for Clunkers program that cost millions. The Department of Energy wants to kill the coal mines and the Keystone XL Pipeline. Obamacare.
I have been a government contractor providing support to defend our country during the Cold War to the recent War on Terrorism. Some of the atrocities committed by the terrorists even outdo those of the Nazi's. I recently spoke with someone who had just come back from Oman and was assigned to deal with the Guantanamo detainees who had been sent to Oman. I asked him if they were now in prisons over there. He replied that they were just released to live with their families and could do whatever they wanted.
For those of you who voted for Mr. Trump, I know you are fired up and ready to take on the election in the fall. For the rest of you, I don't know if Mr. Trump was your second pick, your seventeenth, or your eighteenth. We do know that he has gumption and is willing to take on anything. Personally, I may prefer that he uses a few more words that have more than four letters. However, he will fight for what he believes in and he is willing to take the gloves off and go for it. We also know that he likes to surround himself with the best people to do the job. It is our responsibility and duty to support him and provide him the best advisors possible and the best people to execute his policies that we can. I think it is very notable that he announced Trey Gowdy as his pick for Attorney General. He is the Chairman of the House Select Committee investigating Benghazi and what Hillary Clinton did or didn't do. He named this pick for AG even before he named his Vice Presidential pick, which speaks volumes.
My ancestors didn't come over on the Mayflower, my grandfather came over on the Lusitania and he homesteaded this land. As near as we can tell, he had come over and homesteaded, then went back to find a woman to marry, and they both came over on the maiden voyage of the Lusitania. She took a job as a nanny and a maid to make sure she liked this new country, but eventually they did get married. When my grandfather homesteaded, he did not wait around for the government to put in water pipes, he dug his own well. He did not wait around for Section 8 housing, he built his own home. He helped build this country and make it great. My father was born and raised on that homestead, and I was raised there. Those traditions run deep in my family, and I will do whatever I can to make this country great again. I hope you will join me.
Esther Sparling Joan Hebrance
Mercedes Pfeffer
Trish Martell
Nancy Krakover Pam Heminger
Saturday, June 11, 2016
- Identify and describe all of the opportunities available to Republican volunteers in the county
- Provide sufficient information about each of these opportunities so Republican volunteers can pick those that suit their interests, time availabilities, and schedules
- Introduce volunteers to Republican county and district officials who can provide volunteer guidance and responsive assistance
- Help volunteers tap into established Republican networks in their neighborhoods and local areas
Fire Station #3
Please sign up NLT June 6 so we can prepare for you.
Saturday, September 10,2016
2:00 pm - 6:00 pm
2:00 pm - 6:00 pm


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The Chicago Dryer Vent Cleaning Wizard Partners with the American Red Cross Fundraising Event
Dryer Vent Wizard franchisees across the nation are donating one percent of their sales during the month of October to their local American Red Cross Chapter to raise funds for disaster relief efforts
By: Right Now Marketing Group, LLC
Fire is the biggest disaster threat to families and the number one disaster relief effort by the American Red Cross. According to the National Fire Prevention Association, over 17,000 home fires are caused by clothes dryers in the US each year.
An overheated clothes dryer caused a fire that destroyed a 24-unit apartment building in Chesterfield Twp, MI. The American Red Cross is assisting 9 families consisting of a total of about 30 people, including small children and a pregnant woman. "Dryer fires are most devastating when they occur in apartment buildings where many families' homes and lives are at risk," said Mory Matias, owner of Dryer Vent Wizard.
During National Fire Safety Month the American Red Cross is encouraging residents to take steps to minimize the risk of home fires by remembering two key fire safety steps: install smoke alarms and develop a fire escape plan. The Chicago Dryer Vent Cleaning Wizard adds a key point of his own, "Dryer vent cleaning is the number one way to prevent dryer fires," said Matias. "Clothes dryers are one of the most likely appliances to cause a fire."
"Fires can strike suddenly and spread quickly," according to a representative of the local Red Cross chapter. "It'
Mory Matias is happy to help a worthy cause by donating one percent of his sales during the month of October to the local American Red Cross chapter in support of disaster response efforts. "The American Red Cross does an excellent job helping families who have lost their home to fire," said Matias.
Dryer Vent Wizard is the largest chain of dryer vent cleaning, repair, replacement and alteration specialists in the nation with nearly 60 franchise locations in the U.S. Learn more about dryer fire prevention at http://www.dryerventwizard.com
Dryer Vent Wizard of Northshore Chicago
Mory Matias
518 W. Wrightwood Ave; Ste 3E
Chicago, IL 60614
Phone: 773-484-
Toll Free: 1-866-
Email: mmatias@
Accredited, Better Business Bureau of Chicago & Northern Illinois
Recipient, Angie's List '09 & '08 Super Service Award
Read So Many Dryer Fires at http://dryerventcleaningcookcountyill.blogspot.com/
This press release was submitted by Right Now Marketing Group, LLC
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We were inspired by The Transition Streets Challenge to create a vegie garden on the verge for kids in our Street. Cathy discussed the idea with Jasmine and Alexa, and they created invitations for kids in the streets.
We had our first working bee yesterday: 14 kids and nine parents were there for some or all of the afternoon. Cathy had done a lot of planning and preparation, which allowed us to get a lot done.
It was great to see everybody getting into it. The kids ranged in age from 3 (who didn't stay long) to 12, and worked really well. I think some of the parents were quite surprised! Quite a few kids stayed for the whole afternoon (1:00 – 5:00 pm). As with any kids' project there was plenty of playing as well as working – a pile of dirt is great for all sorts of games!
We weren't sure how well it would go down, but the kids were really positive. We kept hearing comments like "that was SOOOO much fun", "this was heaps more fun than I expected", "this was really fun and it was great to meet the other kids in the street".
The 11 school-aged kids from the street and a neighbouring street (there were a couple of friends of local kids there) went to three different schools, so they didn't all know each other. By the end of the day they were all great friends.
After a big day yesterday, most of them are walking down to our local Farmers Market (which is normally at the showgrounds but because of another event there is at an alternative venue nearer us) this morning to buy some seedlings, and most are coming back to do some more work this afternoon.
As we have a wide nature strip, our verge was over six metres wide which means that we have left a 2 metre strip for walking and .8 metre for getting out of cars. It is still a pretty big garden!
I'm sure the garden is going to make a difference to people in our street. The kids (and parents) are going to build stronger relationships (two of the parents realised they went to school together), even people without kids are going to take an interest and get to know the kids better (a few neighbours stopped by to say hi) and it is likely to lead to more social get-togethers.
Hopefully it will also encourage people to think about their food, develop an appreciation for fresh vegetables and move towards more locally grown organic food.
If you liked this post you might want to follow my blog (top right-hand corner of the blog), and you might like to look at:


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This week's luxury budget task Kabhi Ha Kabhi Na seems to have created a lot of differences among the housemates. Early morning, Sofia and Kamya decide patch up while Kamya swears that she will never forgive Andy.
Andy hints at the captaincy nominations while he is having a conversation with Tanishaa in the kitchen area while Gauahar chooses to ignore him. After Gauahar has left, Andy tells Tanishaa that the housemates will never vote for him to be the next captain as they want to see him out of the house. Tanishaa asks him to be optimistic and tries to calm him down. On the other hand, Armaan and Ajaz engage in a conversation about Sofia where they decide to maintain a distance from her because of her two faced attitude.
Later in the day, Bigg boss asks all the housemates to come up with two names who have been the best performers in the task. The selected people should also be ones who have actively participated in the house work. Kamya decides to play a trick and tries to influence the housemates to vote for Gauahar. She successfully manages to manipulate Sofia and Ajaz. The voting commences and there is a tie between Andy and Gauahar. Bigg Boss then decides to introduce another task as a tie- breaker.
For the task, Gauahar and Andy are required to take a plunge into the swimming pool wherein 65 bottles of chyawanprash of different flavours were placed on the floor of the swimming pool. Both Andy and Gauahar will be required to collect as many bottles as possible and throw them in the boxes that were kept near the pool. Each of them is allowed to choose one partner who will help them to put the bottles in the box. The person who collects the maximum number of the bottles will become the new captain of the house. While Kamya and team cheer for Gauahar, Andy gets support from Tanishaa and Sangram.
Later Bigg Boss announces another quick task for Gauahar, Andy, Ajaz and Kushal named Kisko Pyaar Karu wherein Ajaz and Kushal have to impress Gauahar with their charm. Andy is asked to be the host of the task and make it more interesting for the housemates. Ajaz is the first one to try his luck and sings a romantic song for Gauahar. After Ajaz its Kushal's turn who leaves everybody shell shocked by confessing his love for Gauahar and make it official in front of the world. He tells Gauahar that he wishes to grow old with her, listening to which Gauahar breaks into tears. In the second round, Ajaz fails to answer questions related to Gauahar's likes and dislikes, while it was a cake walk for Kushal.
As a part of the third and the last round, Kushal and Ajaz both get to go on date with Gauhar where she appreciates Ajaz for his loving nature. In the end, she announces Kushal to be the winner of the task and also adds that it's a wonderful feeling to be meeting a stranger on a show and falling in love with him. While Kushal and Gauahar are a happy couple now, Ajaz sulks over losing Gauahar to Kushal.
Now, enjoy reading koimoi.com on your Android Smartphone. Download the FREE App right here.


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Fort Collins swings but misses on second triple-digit weather prediction of 2018
So close.
Fort Collins fell a few degrees shy of forecaster predictions and failed to hit triple digits on Thursday. It would've been our second 100-degree day of 2018, which is a bit of an unusual feat for the Choice City.
Automated data from the Fort Collins weather station at Colorado State University showed highs in the mid-90s about 4 p.m., when the forecast said we'd hit 100. The official high temperature for Thursday wasn't available at press time.
It's still been mighty hot this summer. We logged our hottest day in six years on June 28, when temperatures topped out at 101 degrees.
The average high temperature for June was 86.4 degrees, compared with a 1981-2010 normal of 80.4 degrees. It was one of the city's 10 hottest Junes on record, according to Colorado Climate Center data.
The average high in July (through Wednesday) has been 90.1 degrees compared with the usual 86.5 degrees.
Triple-digit days are rare in Fort Collins. The city usually nets no more than two or three each year, if any at all. Our highest temperature on record is 103 degrees on July 21, 2005.
Hot weather will stick around through Saturday, according to the latest NWS forecast. Highs are expected to drop to the 80s on Sunday.


text: In mammalian embryonic stem cells, the acquisition of pluripotency depends upon Nanog, but the analysis of Nanog has been hampered by its requirement for early mouse development. of pluripotency (Boyer et al., 2005; Cole CCT007093 et al., 2008; Loh et al., 2006; Wang et al., 2006). In the early mouse embryo, Nanog expression marks the pluripotent epiblast and is essential for its establishment (Mitsui et al., 2003; Silva et al., 2009). A recent study has shown that activation of is required for acquisition of the pluripotent ground state in both embryonic development and somatic cell reprogramming (Silva et al., 2009). These studies raised the question of what function Nanog plays during embryonic development, and prompted us to investigate this issue in the zebrafish model system. The zebrafish blastula stage embryo is composed of three unique lineages: the extra-embryonic enveloping layer (EVL), the extra-embryonic yolk syncytial layer (YSL), and the intermediate deep cells, which form the entire embryo later. The YSL is unique to teleosts, and has been thought to share a common evolutionary origin with the mouse extra-embryonic primitive CCT007093 endoderm. Primitive endoderm markers such as are also expressed in the YSL (Brown et al., 2010; Fan et al., 2007; Ho et al., 1999; Sprague et al., 2006), and both tissues function as the signaling center to pattern the head mesoderm and endoderm (Chen and Kimelman, 2000; Fan et al., 2007; Ober and Schulte-Merker, 1999; Rodaway et al., 1999; Varlet et al., 1997). Between the 512 and 1K-cell stages, the collapse of marginal cells CCT007093 into the yolk cell results in the formation of the YSL precursor (Kimmel and Law, 1985b). Each YSL nucleus (YSN) undergoes usually three, but sometimes four or five, metasynchronous nuclear divisions, and becomes post-mitotic, just before the onset of epiboly (Kane et al., 1992; Kimmel and Legislation, 1985a). The YSL plays a critical role in generating the force to drive epiboly and inducing the ventrolateral mesoderm and endoderm (Chen and Kimelman, 2000; Mizuno et al., 1996; Ober Mouse monoclonal antibody to TBL1Y. The protein encoded by this gene has sequence similarity with members of the WD40 repeatcontainingprotein family. The WD40 group is a large family of proteins, which appear to have aregulatory function. It is believed that the WD40 repeats mediate protein-protein interactions andmembers of the family are involved in signal transduction, RNA processing, gene regulation,vesicular trafficking, cytoskeletal assembly and may play a role in the control of cytotypicdifferentiation. This gene is highly similar to TBL1X gene in nucleotide sequence and proteinsequence, but the TBL1X gene is located on chromosome X and this gene is on chromosome Y.This gene has three alternatively spliced transcript variants encoding the same protein and Schulte-Merker, 1999; Rodaway et al., 1999; Solnica-Krezel and Driever, 1994; Str?hle and Jesuthasan, 1993). During the mid-blastula stage, induction of endoderm requires the secreted Nodal proteins Ndr1 and Ndr2, which are expressed in the YSL and marginal blastomeres (Chen and Kimelman, 2000; Feldman et al., 1998; Ober and Schulte-Merker, 1999; Rodaway et al., 1999). In response, the transcription of the endoderm transcription factor is usually induced in marginal blastomere cells (Dickmeis et al., 2001; Kikuchi et al., 2001; Kikuchi et al., 2000; Poulain and Lepage, 2002; Reiter et al., 1999). Among them, plays a central role in endoderm induction, as its expression can autonomously induce the endodermal differentiation markers and in the lack of Nodal signaling (Dickmeis et al., 2001; Kikuchi et al., 2001). needs (Lunde et al., 2004; Reim et al., 2004). In order to understand the function of Nanog in early zebrafish advancement, the zebrafish was identified by us ortholog and discovered that it is necessary for YSL development. Knockdown of eliminates ventrolateral Nodal signaling, leading to an lack of ventrolateral endoderm development. By microarray evaluation, we discovered that mophants usually do not exhibit genes specific towards the YSL, where Nodal indicators are created for endoderm induction. Hereditary research and chromatin immunoprecipitation structured sequencing (ChIP-Seq) evaluation uncovered that Nanog-like straight activates the transcription from the homeodomain aspect Mxtx2, which specifies the YSL and promotes ventrolateral endoderm development. Our research illustrates a crucial function for Nanog-like in regulating endoderm development through the Mxtx2-Nodal pathway. Outcomes is necessary for early gastrulation To recognize the zebrafish ortholog, the mouse Nanog proteins sequence was employed for a great time search. This process led to the id of Zgc:193933 being a potential zebrafish Nanog ortholog. The homology between mouse Nanog and zebrafish Zgc:193933 is certainly noticeable in the homeodomain. Position of human,.


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Magellan MapSend
Streets Europe
31st August 2003
Review by
Dave Burrows
Magellan are a well known name in the handheld GPS
arena, along with Garmin they pretty much cover the majority of the handheld GPS
market. Magellan have a whole range of GPS receivers which you can load
MapSend (maps) onto which includes the Magellan Meridian range of GPS Receivers
and also the Magellan SporTrak range. In this review we won't cover the
GPS Receivers, but we will cover the MapSend product.
Magellan currently have a complete range of
European and North American maps. These are covered under the following
product sets:-
SporTrak, GPS 315 or GPS 320 receiver by downloading coordinates for over
500,000 points of interest from around the world.
DataSend Europe
Europe software allows you to download your routes and waypoints from your GPS
receiver to your PC, to easily record your journeys and edited them for future
use on a new adventure. For use with your Magellan SporTrak, GPS 315, GPS 320 or
SP24 receiver.
MapSend BlueNav Charts
Available on convenient preprogrammed Secure
Digital (SD) memory cards and CD-ROM, MapSend BlueNav charts offer a complete
range of detailed marine cartography for your Magellan
Meridian Gold, Marine, Platinum and Color,
Magellan FX324 MAP and FX324 MAP Color, or
Magellan SporTrak Pro (CD-ROM only),
SporTrak Pro Marine and SporTrak Color. Derived from Navionics
Gold charts, MapSend BlueNav charts are the most advanced marine cartography
available, providing large, highly detailed coverage areas and enhanced marine
navigation information, including marine currents, tides, port services and much
MapSend Streets & Destinations Canada
Detailed street-level maps of Canada make GPS
navigation in Canadian land and marine regions even easier with your Magellan
Meridian series, SporTrak Pro, SporTrak Pro
Marine, SporTrak Map, SporTrak Color, MAP 330 or MAP 330M handheld receiver.
MapSend Streets & Destinations in the US
Detailed maps make GPS navigation even easier with
your Magellan Meridian
series, SporTrak Pro, SporTrak Pro Marine,
SporTrak Map, SporTrak Color, MAP 330 or MAP 330M handheld receiver.
MapSend Streets Europe
Detailed street-level maps of Europe make GPS
navigation in the region even easier with your Magellan
Meridian series, SporTrak Pro, SporTrak Pro
Marine, SporTrak Map, SporTrak Color, or MAP 330 handheld receiver.
MapSend Streets in the US
Detailed street-level maps of the U.S. available
in regional files allow you to download just the maps you need, saving memory
while making GPS navigation even easier with your Magellan
Meridian series, SporTrak
Pro, SporTrak Pro Marine, SporTrak Map, SporTrak Color, or MAP 330 handheld
MapSend Topo in the US
The most advanced GPS topographic software
available, Magellan MapSend
Topo in the United States maximizes the navigation capabilities of your Meridian
series, SporTrak Pro, SporTrak Pro Marine, SporTrak Map, MAP 330 or MAP 330M
handheld receiver.
MapSend WorldWide Basemap
Extend your GPS basemap coverage to any region
around the globe by adding a MapSend
WorldWide Basemap to the built-in basemap of your Magellan Meridian
series, SporTrak Pro, SporTrak Pro Marine,
SporTrak Map, SporTrak Color, or MAP 330 GPS receiver.
In this review we will cover the MapSend Streets
Europe CD. MapSend price varies depending on the GPS Receiver you want to
use it for.
Installing MapSend is easy, just insert the CD and
follow the on-screen prompts. You'll be greeted with two types of install,
a minimal install which will copy around 60mb of data to your hard disk, or a
full install which is around 500mb of data copied to the hard disk.
If you select the full install, then you won't
have to use the CD again whilst it's installed onto your hard disk. Once
installed, running up the MapSend program will greet you with a mapping screen
like the one to the right..
Very similar to a screen you'll find in most
mapping programs, it will give you a complete map of the area of the earth.
MapSend comes with a fairly default toolbar.
You'll see from the screenshot below, that everything is in it's usual place,
you have a zoom in and zoom out option, a scale option, a
hand which allows you to drag the map around on screen (notice above that
there are no scroll bars!). You then have two icons, one is shown as EU
and one of the earth. This will zoom out/in to the area
depending on the button you select, which is just used for a quick zoom
also notice to the right of this there's a measuring tool, this will
allow you to measure two points on the map and find the distance between these
points. You also have a search by name (binoculars), Search by
address (house), the draw region tool and the cut region tool
(grayed out). Finally you have the place waypoint button, build
route, upload regions (to GPS), about and what's this.
pretty much covers the map and the toolbar, on the right hand side of the window
you have (top right of screenshot) the Routes where waypoints can be set, and
below this (bottom right of screenshot) you have the waypoints with latitude and
longitude co-ordinates. You can see these below.
Map Detail
Once you have the maps installed you'll find
there's quite a level of detail. Magellan have full street maps for Europe
in this product that take you right down to the smallest of roads, and is on par
with Microsoft AutoRoute, and Garmin's own mapping products for their GPS
Receivers. Both Garmin and Magellan use NavTech for the geodata provider
which is good to see as NavTech maps are more accurate compared with other
geodata providers across Europe.
Below you'll see a full zoomed in map right down
to street level. The roads may look a little basic, but do you really need
a high level of detail on a handheld GPS Receiver ?
Uploading Regions
you've zoomed in to the map area you want you can then use the draw region
tool to drag across an area of the map. This will then create a box
which can also be dragged around the map to place it over the area of the map
you wish to download to the GPS Receiver. If you then the cut region
tool then you will then cut this for upload and be asked to name the region.
A region is a section of map that can be highlighted and
cropped to the area you require, and uploaded to the GPS Receiver to cover
the area you will be passing when using the GPS Receiver.
MapSend Streets Europe you can also create routes.
allows you to add waypoints to the map at designated intervals, and then create
a route out of these waypoints.
When you
use the build route tool, it will plot a route as the crow flies rather
than using streets, so if you want to create a route then you will need to plot
quite a number of waypoints across a route to take this into account.
You can
see how the waypoints are set out on the screenshot to the left, and how the
route will go directly from one waypoint to another, e.g. WPT004 to WPT005.
route when finished can then be uploaded to the Magellan GPS Receiver and allow
you to use it on your journeys.
MapSend CD's
do a whole range of MapSend CD's. Here's just a small selection of what's
If you have
a Magellan Handheld GPS Receiver and you're looking to add map support
to it over and above the standard base map, then you'll need MapSend.
Magellan GPS Receivers can only accept a Magellan map, so you won't be
able to upload custom maps from other products, so make sure you
purchase MapSend for mapping support.
Comments ?
Have any comments about this review ? Post them here.
GPS Shopping


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Blood Orange & Neroli Thins - 200g
Blood Orange & Neroli Thins - 200g
These delicate thins, whose making is a jealously guarded secret in the Prestat house, are appreciated by everyone. Their lightness and sweetness make these delicacies suited for every occasion.
Delicate and delicious, Prestat has a secret way of making these literally 'wafer thin' chocolate discs. Each delicate thin is different from the other. Enjoyed by royalty and nobility, stars of stage and screen, indeed by anyone who delights in the pleasure that the finest chocolate brings.
Sweet Orange, lightly perfumed Neroli oil and the finest milk chocolate, a sure winner.
Ingredients: Milk Chocolate 99% (Sugar, Cocoa Butter, Milk Powder, Cocoa, Emulsifier: Soya Lecithin), Blood Orange Oil, Neroli Oil Flavour. For more information click here.
ALLERGENS: Milk and Soya. May contain traces of Tree Nuts, Egg, and Gluten.
Weight: 200g (32 thins).
Storage notes: the product must be stored in a cool, dry and odourless place and at a temperature between 15°C and 18°C.
Nutritional values per 100g:
|Kcal 577 - Kj 2403
|38.8 g
|- of which saturates
|17.9 g
|48.2 g
|- of which sugar
|44.8 g
|8.0 g
|0.25 g


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The '70s supernatural-cinema boom produced a number of provocative films that were more thoughtful than horrifying, and Audrey Rose is a good example. Adapted by Frank De Felitta from his own novel, the story concerns haunted Englishman Elliot Hoover (Anthony Hopkins), who believes a 10-year-old girl living in New York is the reincarnation of his daughter, who died in a horrific car accident. Hoover's obsession with the girl, Ivy Templeton (Susan Swift), traumatizes her parents, Janice (Marsha Mason) and Bill (John Beck). Complicating matters is the fact that Ivy keeps having seizures, creating the impression that she's "reliving" Audrey's death. While this might sound like a solid setup for a creepshow, the filmmakers have larger ambitions. Veteran director Robert Wise, whose résumé includes the restrained fright classic The Haunting (1963), methodically follows the story through a lengthy court trial and an epic hypnosis sequence, eschewing cheap jolts for intense discussions about the comparative values of eastern and western spirituality. So while the movie is mostly a bust as a thriller, it's interesting as an existential conversation piece. Mason is touchingly fraught as a mother in an impossible situation; her reaction shots during the hypnosis scene are especially potent. Hopkins' performance veers into strange directions, with flitting hand gestures and overly musical line deliveries, so it's hard to determine whether he succeeded at creating something otherworldly or failed at creating something believable. Either way, he's oddly entertaining. Beck is his usual stolid presence, supporting Mason without calling much attention to himself, and Swift is okay, doing a Linda Blair-lite routine. Slow and long but generally interesting, Audrey Rose gets points for trying to do something out of the ordinary.
Audrey Rose: FUNKY


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February 2, 2017
I think that the 1980 BMW 633 CSi is not only one of the best-looking cars to ever come out of Bavaria, but it just may be one of the best-looking large coupes, period! Very well-proportioned without a lot of unnecessary clutter, and just the right size for a long, comfortable drive with your luggage stowed safely out of sight. I saw this one while bicycling through a North Buffalo neighborhood last summer. I hope I didn't leave behind any drool.
Jim Corbran, RRofWNY
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Read Hawkeye vs. Deadpool
HAWKEYE VS DEADPOOL VS CRIME! Once upon a time, champions emerged to fight evil plaguing humanity. They fought for salvation, for all that is good in the world — and they won. They were kind, generous and self-sacrificing. They were heroes. These are not those heroes. Meet Hawkeye: Ladies man ("ladies man" because the ladies love to hate this man), crossbow crusader and, most importantly, Avenger. He's the only guy on the team without any powers, though, so cut him a little slack. Then there's Deadpool (MR. Deadpool to you, skippy) — the regeneratin' degenerate. You can shoot him, stab him, and make with the punchy-punchy right in his face, but nothing can keep the Merc with a Mouth down. What do they have in common? Halloween and a S.H.I.E.L.D. espionage mystery that has the both heroes racing against the clock while outshining one another. It's a Hawkeye & Deadpool comic 75 years in the making, give or take 75 years.


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Madera 3 Piece Queen Duvet Cover and Sham Set
Find amaxing comfort and style with this stylish queen madera duvet cover sham set.
Button Enclosure
Oeko-Tex certified
Wrinkle Resistant
- Incredibly Soft
- Expertly woven for breathability and year-round comfort
- Effortless Style - Colors compliment any decor
- It is highly recommended to wash before first use
- Always wash separately from other clothing
- Wash on normal medium-hot temperature
- Tumble dry and remove promptly
- Dry on low heat
- Do not iron.
Free Shipping
All orders are processed Monday-Friday and ship within 1-3 business days. Orders placed after 11 AM (Arizona Time) will process the following business day. Orders do not ship on weekends or holidays. All orders over $100 (excluding tax) in the Continental US ship FREE through FEDEX ground. Typical delivery time is 4-7 days.
Free Returns
Return your item FREE within 30 days of purchase for a full refund. After 30 days, you're eligible for an exchange or store credit for up to 365 days which is also FREE to ship back. See full return policy.
*All Summer Event, January White Sale & Last Chance Items are Final Sale


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"I always think: This could be so elegant. Then after I end up making it, it looks fucked up in this way."
Gina Beavers likens herself to an Impressionist painter capturing the everyday landscape; it's just that the world she's rendering is experienced mostly online, and is full of bad selfies, awesome manicures, and lots of junk food. The artist scavenges Instagram, Google, and stock-photo databases for imagery that she then translates into thick, sculptural paintings: a kimchi hotdog here, a make-up tutorial or novelty cake shaped like a male stripper there. "I always think: This could be so elegant," she tells GARAGE. "Then after I end up making it, it looks fucked up in this way. I can't help it—it's the process."
Sometimes fucked up isn't a bad thing—sometimes, it's the whole point. New Yorkers will have a chance to explore the past seven years of Beavers's career in "The Life I Deserve," opening at MoMA PS1 on March 31. Meanwhile, the artist herself has moved out of the city—she's now based in nearby Newark, New Jersey. Her studio shares a building with a hip-hop recording studio and a factory that produces garments for exotic dancers.
When I visited this week, several works were in various stages of completion. One is based on a found image of hands holding water glasses at various angles; Beavers reckons it's used as a visual reference for digital animation or video game design. She's replicating it in thick acrylic, creating the forms by layering inches of gel medium that she sculpts with a deli knife before painting. The piece is called Sober—Beavers has stopped drinking, and the titling is a way to sneak something personal into imagery that is essentially generic or anonymous. "I'm definitely picking things that are saying something, however superficially, about my life," she explains.
Elsewhere in the studio, a painting of a man's torso leans against the wall. The figure's massive pectoral muscles have collapsed, and are oozing down the surface of the work. She has kept the painting as a cautionary tale: Leave the medium time to dry, or bad things will happen. "The Life I Deserve" contains a number of similar works from a series Beavers has made based on Instagram selfies taken by bodybuilders and gym rats. The artist was intrigued by the formal possibilities—literally sculpting images of bodies that were themselves sculpted—but has found that there's not much of a market for the works. Most of the examples at PS1 are being loaned directly from her studio.
"For collectors in this era, to have in their homes—they're less interested in the male figure," she shrugs. "But I like making them. I guess we're all kind of self-fashioning, especially online—trying to make a version of ourselves." A similar thread of self-improvement or transformation runs through most of Beavers's subject matter. It's there in some of her most recognizable images, oversized depictions of women applying lipstick or mascara. The devil is in the details: the brittle cracks on lips, the spidery tendrils of eyelashes.
While Beavers finds most of her imagery online, the imposing, physical heft of her works doesn't always translate onto the screen. Her paintings bulge and flex, pushing their own limits. She's begun incorporating sports references as well—tennis balls, soccer balls—partly as an experiment to see how sculptural she can go. "What's the most three-dimensional form that I can make on a canvas?" she wondered, before crafting shapes out of papier-mâché that she then painted. All the macho athletic trappings also became a way to riff on gender. "Somebody had said, 'oh, your work is really feminine,'" she recalled. "I started making a bunch of lips with these balls on them, a dumb way to answer that: Is it masculine now?"
While Beavers seems reluctant to use her work to plumb her own psychology, the themes she's fascinated by are their own sort of diary. Sometimes that's very basic—an image of french fries and ketchup arranged to form a heart shape, because the artist loves french fries ("very deep," she laughs). But elsewhere, stock imagery—a cheesy picture of a woman who appears to be painting her own lips with an actual paintbrush—can be an oblique way to contemplate her own aspirations and anxieties.
The photographs she finds online are there to be appropriated and transformed into something idiosyncratic and new. Working that way ends up being more interesting than simply starting from scratch. "I don't think that anything that I come up with myself is that interesting," she admits. "It's the effect of being on Instagram—it makes you see how much you are just like everybody else. It follows that things that I come up with about my life, everybody has that—it's not really that distinct or that interesting. There's a flattening of identity. For some reason, I don't really want to delve deeply into my own shit." That might sound like the artist is hiding out, but the sum of all this strange, borrowed imagery ends up being almost achingly personal.


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Reminder: Please settle and pay your debt first before playing and buying your lottery ticket. Pay early to prevent loan interest. Save as much money into your private retirement scheme, because 75% people is not ready for their retirement nowdays and the kwsp, epf and cpf money will finish in just 5 years. Credit cards like visa and mastercard are easy to get, think carefully before you spend money and buy something with it. Do not play any online gambling or going to casino, because it is very addictive and hard to manage your budget. Clairvoyant and tarot psychic reading can help to increase chances of winning the lottery sometimes, but please be careful with scammer.
Msg #1
30-08-2011 12:40 am
this software give 90% chance to success?
Msg #2
30-08-2011 12:42 am
if really give 90% then it should be always hit....if fail also will be less...i did not try it...i still researching...


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Campus Candids
From left to right: Tim Utz ᾿18, Mary O'Sullivan, PhD, professor of chemistry and biochemistry, Matt Smardz ᾿19 and Marissa Rizzo ᾿20.
From left to right: Phil Sheridan, PhD, professor of chemistry and biochemistry, David Filion '19 and Ben Russ '19.
Mary O'Sullivan, PhD, and Phil Sheridan, PhD, professors of chemistry and biochemistry and five chemistry and biochemistry majors attended the 255th National Meeting of the American Chemical Society (ACS) in New Orleans, LA to present posters on their latest research results.
Tim Utz '18, Marissa Rizzo '20 and Matthew Smardz '19 presented two posters based on their research in Mary O'Sullivan's laboratory: "Synthesis of Guanidino Derivatives of Aryl-Substituted Polyamines as Inhibitors of Trypanosoma cruzi Trypanothione Reductase" and "N-Isopropyl Derivatives of Aryl-Substituted Polyamines: Synthesis and Inhibition of Trypanothione Reductase." David Filion '19 and Ben Russ '19 presented two posters based on their research in Phil Sheridan's laboratory: "Laser Ablation/Molecular Jet Laser Excitation Spectroscopy of CaNCO" and "Computational Study of Aluminum Monoisocyanate Isomers."
In addition, these students accepted the Commendable Student Chapter Award on behalf of the Canisius Student Chapter of the American Chemical Society (SCACS). This is the second highest award given by the national ACS to student chapters. This marks the 10th consecutive annual award that the Canisius SCACS has received.
Submitted by: Phil Sheridan, PhD, professor, Department of Chemistry and Biochemistry