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Dichlorodiphenyltrichloroethane (DDT) is a persistent estrogenic organochlorine pesticide that is a rodent hepatic tumor promoter, with inconclusive carcinogenicity in humans. We have previously reported that o, p'-DDT elicits primarily PXR/CAR-mediated activity, rather than ER-mediated hepatic responses, and suggested that CAR-mediated effects, as opposed to ER-mediated effects, may be more important in tumor promotion in the rat liver. To further characterize species-specific hepatic responses, gene expression analysis, with complementary histopathology and tissue level analyses were investigated in immature, ovariectomized C57BL/6 mice treated with 300 mg/kg o, p'-DDT, and compared to Sprague-Dawley rat data.
Rats and mice exhibited negligible histopathology with rapid o, p'-DDT metabolism. Gene expression profiles were also similar, exhibiting PXR/CAR regulation with the characteristic induction of Cyp2b10 and Cyp3a11. However, PXR-specific target genes such as Apoa4 or Insig2 exhibited more pronounced induction compared to CAR-specific genes in the mouse. In addition, mouse Car mRNA levels decreased, possibly contributing to the preferential activation of mouse PXR. ER-regulated genes Cyp17a1 and Cyp7b1 were also induced, suggesting o, p'-DDT also elicits ER-mediated gene expression in the mouse, while ER-mediated effects were negligible in the rat, possibly due to the inhibitory effects of CAR on ER activities. In addition, o, p'-DDT induced Gadd45a, Gadd45b and Cdkn1, suggesting DNA damage may be an additional risk factor. Furthermore, elevated blood DHEA-S levels at 12 h after treatment in the mouse may also contribute to the endocrine-related effects of o, p'-DDT.
Although DDT is known to cause rodent hepatic tumors, the marked species differences in PXR/CAR structure, expression patterns and ligand preference as well as significant species-specific differences in steroidogenesis, especially CYP17A1 expression and activity, confound the extrapolation of these results to humans. Nevertheless, the identification of potential modes of action as well as species-specific responses may assist in the selection and further development of more appropriate models for assessing the toxicity of DDT to humans and wildlife.
Dichlorodiphenyltrichloroethane (DDT) is a persistent organochlorine pesticide that is a hepatic tumor promoter in the mouse and rat [1], with inconclusive carcinogenicity in the human liver [2,3]. Technical grade DDT is a mixture of p,p'-DDT, o, p'-DDT and their metabolites such as 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE) and l,l-dichloro-2,2-bis(p,p-chlorophenylethane) (DDD) [4]. o, p'-DDT exerts estrogenic effects in responsive tissues such as the uterus [5] by binding to estrogen receptor (ER)α and β subtypes [6]. In addition, o, p'-DDT is an agonist for pregnane X receptor (PXR) and constitutive androstane receptor (CAR) [7,8]. Thus, it may elicit complex responses through multiple nuclear receptors.
Several structurally diverse estrogenic compounds elicit ER-mediated responses in the liver [9,10]. We have previously reported that o, p'-DDT elicits primarily PXR/CAR-mediated, rather than ER-mediated hepatic responses, and suggested that CAR-mediated effects, as opposed to ER-mediated effects, may be more important in DDT-induced tumor promotion in the rat liver [11].
Although the rat is a preferred toxicological model because of the accumulated background knowledge regarding chemical-induced toxicity compared to mice, several studies report significant species-specific differences in response to chemical exposures. For example, 1,4-bis-[2-(3,5,-dichloropyridyloxy)] benzene (TCPOBOP) acts as potent phenobarbital-type enzyme inducer in the mouse liver but not in the rat or human. This is due to a substitution of Thr350 in mice with Met in the rat and human CAR [12-14]. Alternatively, another phenobarbital-type enzyme inducer 2,4,6-triphenyldioxane-1,3 induces hepatic CYP2B in rats but not in mice [15]. Such differences may affect CAR-mediated hepatic drug metabolism and disposition following xenobiotic exposure. In addition, rat genome annotation lags behind the mouse and human in maturity, confounding a more comprehensive assessment of rat gene expression data. Furthermore, targeted gene-disruption can be used in mice to further elucidate the roles of PXR, CAR and ER in o, p'-DDT elicited effects. Thus, we were motivated to investigate hepatic gene expression in the mouse following o, p'-DDT treatment to identify species-specific and conserved responses. Overall, the hepatic gene expression profiles were comparable, however there were marked species-specific differences in genes regulated by ER, which may involve species-difference in CAR activation. Furthermore, blood levels of DHEA-S, a precursor of androgen and estrogen, exhibited mouse-specific elevation, which may also contribute to endocrine system disruption. Thus, the present study further elucidates the dynamics of o, p'-DDT elicited responses in the rodent, and identifies species-specific responses that may be important to o, p'-DDT exposure for humans and wildlife.
Female C57BL/6 mice, ovariectomized on postnatal day 20 were obtained from Charles River Laboratories (Raleigh, NC) on day 25. Mice were housed in polycarbonate cages containing cellulose fiber chip bedding (Aspen Chip Laboratory Bedding, Northeastern Products, Warrensberg, NY) and maintained at 40–60% humidity and 23°C in a room with a 12 h dark/light cycle (7 am-7 pm). Animals were allowed free access to de-ionized water and Harlan Teklad 22/5 Rodent Diet 8640 (Madison, WI), and acclimatized for 4 days prior to dosing.
Treatments and Necropsy
Mice (n = 5) were orally gavaged once or once daily for three consecutive days with 300 mg/kg b.w. o, p'-DDT (99.2% purity, Sigma-Aldrich, St Louis, MO) in 0.1 ml of sesame oil (Sigma-Aldrich) vehicle. An equal number of time-matched vehicle control animals (n = 5) were also treated in the same manner. Mice receiving one dose were sacrificed 2, 4, 8, 12, 18, and 24 h after treatment. Mice receiving three daily doses were sacrificed 24 h after the third treatment (72 h). All procedures were performed with the approval of the Michigan State University All-University Committee on Animal Use and Care. Animals were sacrificed by cervical dislocation and animal body weights were recorded. Whole liver weights were recorded and sections of the left lateral lobe (approximately 0.1 g) were snap-frozen in liquid nitrogen and stored at -80°C. The right lateral lobe was placed in 10% neutral buffered formalin (NBF, VWR International, West Chester, PA) for histopathology and stored at room temperature.
Following fixation of the right lateral lobe for at least 24 h in 10% NBF, the samples were embedded in paraffin according to standard techniques. Five μm sections were mounted on glass slides and stained with hematoxylin and eosin. All embedding, mounting and staining of tissues were performed at the Histology Laboratory, (Department of Physiology, Michigan State University). The histopathology of each liver section was scored according to the NTP Pathology guidelines.
Measurement of blood DHEA-S and androstenedione
Rat plasma samples were obtained from a previous study [11]. Mouse serum and rat plasma samples were used for determining dehydroepiandrosterone sulfate (DHEA-S) and androstenedione levels using a DHEA-S ELISA kit (Calbiotech Inc., Spring Valley, CA) and Androstenedione ELISA kit (Genway Biotech Inc., San Diego, CA), respectively. The detection limits, defined by the vendors, are 0.02 μg/ml of DHEA-S and 0.043 ng/ml of androstenedione, respectively.
Protein preparation and Western analysis
Total protein was extracted from liver samples from all the time points using the Total Protein Extraction kit (Chemicon International Inc., Temecula, CA). Protein concentrations were determined using Bicinchoninic Acid Protein Assay Kit (Sigma-Aldrich). Total protein (100 μg) was resolved on a 11% denaturing SDS-polyacrylamide gel, and transferred to Hybond-ECL membrane (GE Healthcare, Waukesha, WI). Blots were incubated with blocking buffer (0.1% Tris-buffered saline pH 7.4 containing 1% low-fat dry milk) for 10 min at room temperature. Goat anti-CYP17A1, goat anti-actin antibodies and donkey horseradish peroxidase-conjugated anti-goat IgG were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Immunochemical staining was performed as described previously [16], with dilution of 1:500 (anti-CYP17A1), 1:500 (anti-actin) and 1:10000 (anti-goat IgG) using blocking buffer, respectively. SuperSignal West Dura substrate (Thermo Fisher Scientific, Inc., Waltham, MA) was used for signal detection. The Western analysis was performed on three independent biological replicates.
RNA isolation
Total RNA was isolated from left lateral liver sections using TRIZOL Reagent (Invitrogen, Carlsbad, CA) and resuspended in The RNA Storage Solution (Ambion, Austin, TX). RNA concentrations were determined by spectrophotometry (A260) and purity was assessed by the A260:A280 ratio and by visual inspection of 3 μg on a denaturing gel.
Microarray analysis
Temporal changes in gene expression were assessed using an independent reference design in which o, p'-DDT-treated samples were co-hybridized with time-matched vehicle controls using 3 biological replicates and 2 independent labelings of each sample (i.e. dye swap) for each time point. Whole Mouse Genome 4 × 44 K Oligo Microarray Kit (Agilent Technologies, Inc, Santa Clara, CA) was used for global gene expression analysis. All the reagents and enzymes were provided by Agilent Technologies, and the microarray analysis was performed according to the vendor's protocol. The microarrays were scanned at 635 nm (Cy5) and 532 nm (Cy3) using a GenePix 4000B microarray scanner (Molecular Devices, Union City, CA). Images were analyzed for feature and background intensities using GenePix Pro 6.0 (Molecular Devices). All data were managed in the toxicogenomic information management system dbZach relational database [17].
Microarray data normalization and statistical analysis
Data were normalized using a semi-parametric approach [18]. Model-based t-values were calculated from normalized data, comparing treated and vehicle responses per time-point. Empirical Bayes analysis was used to calculate posterior probabilities (p1 [t]-value) of activity on a per gene and time-point basis using the model-based t-value [19]. Genes were filtered for activity based on the p1(t)-value. p1(t) values approaching one indicate changes in gene expression which are more robust. In this study, unique genes with a p1(t) > 0.999 for a minimum of two time points and absolute value fold change ≥ 1.5-fold compared to time-matched vehicle control for at least one time point were considered differentially expressed.
Hierarchical clustering
A total of 996 orthologous rat and mouse genes were in common on the rat cDNA, mouse cDNA, and Agilent mouse oligonucleotide microarrays based on HomoloGene http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene webcite. Differentially expressed orthologs (|fold change| ≥ 1.5 for at least one time point in either species) for ethynylestradiol (EE)-treated mouse liver, o, p'-DDT treated rat liver and o, p'-DDT-treated mouse liver, were hierarchical clustered using Cluster 3.0 and TreeView software http://rana.lbl.gov/eisen/ webcite. In addition, differentially expressed genes known to be regulated by PXR, CAR and ER agonists [20-23], were identified from the literature and their expression profiles were subjected to hierarchical clustering.
Correlation analysis
Correlation analysis was performed using relaxed criteria to allow for more inclusive comparison between mouse and rat data sets. Genes with a p1(t) > 0.99 and absolute fold change ≥ 1.5-fold at one or more time points in the o, p'-DDT-treated mouse liver samples were selected and used for correlation analysis. This analysis involved a multivariate correlation-based visualization application [24] that has been used to compare two independent gene expression profile datasets [11,25]. It calculates the temporal correlations between gene expression (fold change) and significance values (p1 [t]-value) for orthologous o, p'-DDT-treated mouse (this study) and o, p'-DDT-treated rat genes [11], and summarizes the results in a scatter plot.
Quantitative Real-Time PCR (QRT-PCR)
Expression levels of mouse Car (Nr1i3), Cyp17a1, Cyp2b10, Cyp3a11, Cyp7b1, Gadd45a, Gadd45b, Gapdh, Gclm, Pxr (Nr1i2), and Srebf1, and rat Gapdh and Cyp7b1 were measured by QRT-PCR. For each sample, 2 μg of total RNA was reverse transcribed by SuperScript II using an anchored oligo-dT primer as described by the manufacturer (Invitrogen). The resultant cDNA (1.0 μl) was used as the template in a 30 μl PCR reaction containing 0.1 μM each of forward and reverse gene-specific primers designed using Primer3 [26], 3 mM MgCl2, 1.0 mM dNTPs, 0.025 IU AmpliTaq Gold and 1 × SYBR Green PCR buffer (Applied Biosystems, Foster City, CA). PCR amplification was conducted in MicroAmp Optical 96-well reaction plates (Applied Biosystems) on an Applied Biosystems PRISM 7000 Sequence Detection System using the following conditions: initial denaturation and enzyme activation for 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. A dissociation protocol was performed to assess the specificity of the primers and the uniformity of the PCR generated products. Each plate contained duplicate standards of purified PCR products of known template concentration covering eight orders of magnitude to interpolate relative template concentrations of the samples from the standard curves of log copy number versus threshold cycle. The copy number of each unknown sample for each gene was standardized to that of Gapdh gene to control for differences in RNA loading, quality and cDNA synthesis. Primer sequences and amplicon sizes are provided as Additional file 1.
Additional File 1. Primer sequence.
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Statistical analysis
Body weight, relative liver weight, hepatic concentrations of o, p'-DDT, o, p'-DDD, and o, p'-DDE and QRT-PCR data are presented as the mean ± SE. Statistical analysis was performed with two-way ANOVA followed by pairwise comparisons using Tukey's Honestly Significant Difference post hoc test to control Type I error (α = 0.05). For QRT-PCR data, the relative expression levels of target genes were scaled such that the standardized expression level of the time-matched vehicle control group was equal to 1 for graphing purposes. All statistics were performed using SAS 9.1.3 software (SAS Institute Inc., Cary, NC).
Body Weight, Relative Liver Weight, and Histopathology
Relative liver weight was significantly (p < 0.05) increased at 72 h compared to vehicle-treated mice (Fig. 1A). No effect on body weight was observed. These results are similar to those previously observed in the rat o, p'-DDT study [11]. There were no significant histological changes by o, p'-DDT treatment, except for two animals exhibiting cell death of small numbers of hepatocytes at 4 h or 72 h, where the nuclei were shrunken and hyperchromatic.
Figure 1. Relative liver weight and tissue level analysis. (A) Relative liver weight. Immature ovariectomized C57BL/6 mice were orally administered 300 mg/kg o, p'-DDT or sesame oil vehicle at time 0, 24 and 48 h. Mice were sacrificed 2, 4, 8, 12, 18, 24 or 72 h after the initial dose. The relative liver weight was significantly increased at 72 h. (B) Hepatic concentration of o, p'-DDT and o, p'-DDD. (C) Hepatic concentration of o, p'-DDE. Hepatic tissue levels of o, p'-DDT, o, p'-DDD and o, p'-DDE were determined using high-resolution gas chromatograph/HRMS from three randomly selected mice orally gavaged with 300 mg/kg o, p'-DDT. The data are presented as mean ± SE. The asterisk (*) indicates a significant (p < 0.05) difference from the vehicle controls.
Hepatic o, p'-DDT, o, p'-DDD and o, p'-DDE concentration
Animals treated with o, p'-DDT showed significantly greater levels of o, p'-DDT, from 2 to 18 h compared to vehicle controls (Fig. 1B). The levels of the metabolites o, p'-DDD and o, p'-DDE were significantly greater than controls from 2 to 12 h, and at 72 h (Figs. 1B and 1C), indicating that o, p'-DDT is readily metabolized in the liver, and that much of the o, p'-DDT and its metabolites remain primarily distributed to the liver from 2 to 12 h, similar to the rat [11]. Note that there was a statistical outlier in the 72 h treated group, which lead to significant o, p'-DDE and o, p'-DDD levels, but was still included in the analysis as there was no technical or biological justification for its removal.
Microarray Data
Gene expression was assessed using the 4 × 44 K Mouse Agilent array containing approximately 44,000 oligonucleotide probes, representing 34,204 annotated genes including approximately 21,000 unique genes. Model-based t-values that compared treated and vehicle responses on a per time-point basis followed by Empirical Bayes analysis identified 1,206 differentially expressed genes across the time course based on a p1(t) > 0.999 for a minimum of two time points and absolute fold change ≥ 1.5-fold compared to time-matched vehicle control for at least one time point (Fig. 2A). Overall, 859 genes were induced while 382 genes were repressed. Changes in gene expression ranged from 36.4-fold induction for Cyp2c55 gene to -13.0-fold repression for gene Car3. The 12 h time point exhibited the greatest number of differentially expressed genes (Fig. 2B). All of the gene expression data as well as the identified differentially regulated genes are provided in Additional files 2 and 3, respectively.
Additional File 2. o, p'-DDT-treated mouse liver time-course study, full data set. Blank gene expression ratios or p1(t) values are due to lack of or inapproprate statistical data for computation.
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Additional File 3. Differentially regulated genes in the o, p'-DDT-treated mouse liver.a Highligted values indicate |fold change| > 1.5. b Highlighted values indicate p1(t) > 0.999.
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Figure 2. Microarray and QRT-PCR results. (A) Identification of differentially regulated genes. (B) Number of differentially expressed genes following o, p'-DDT treatment in the mouse liver. Differentially expressed genes were selected based on a p1(t) ≥ 0.999 at two or more time points and an absolute fold change ≥ 1.5 at one or more time points relative to time-matched vehicle controls. All differentially expressed genes are listed in Additional file 3. (C) Verification of microarray results by QRT-PCR. QRT-PCR results relative to time-matched vehicle controls are shown as bar and presented as mean ± SE. Microarray results are represented as lines. The dashed line indicates the expression level of the time-matched vehicle control. The asterisk (*) indicates a significant (p < 0.05) difference from the time-matched vehicle controls for QRT-PCR, n = 5. (D) Hepatic expression of PXR/CAR-target genes in o, p'-DDT-treated mouse. A heat map of o, p'-DDT elicited microarray expression profiles for selected PXR-, CAR-specific and PXR/CAR-shared target genes identified in the literature [20-23]. While some CAR-regulated genes such as Cyp1a1, Fmo5, Sult1d1 or Abcc2 were moderately induced, several PXR-target genes, including ApoA4, Ces2, Gstm2 or Insig2, exhibited strong induction. However, other PXR-target genes such as Hmgcs1 and Hmgcs2 were down-regulated.
A number of phase I drug metabolizing enzyme genes were induced such as Cyp1a1 (~2.2-fold), Cyp2b9 (~5.0-fold), Cyp2b10 (~18.4-fold), Cyp2b13 (~4.1-fold), Cyp2c39 (~2.1-fold), and Cyp2c55 (~36.4-fold) (Table 1). The induction of Cyp2b10 and Cyp3a11 were verified by QRT-PCR (Fig. 2C). The steroid metabolism genes, Cyp17a1 (~6.2-fold) and Cyp7b1 (~5.8-fold), were also induced, while Cyp51 and Cyp7a1 were down-regulated. Steroidogenic Cyp11a1 mRNA level was not detected by microarray or QRT-PCR (data not shown). Note that the hepatic induction of Cyp17a1 and Cyp7b1 by o, p'-DDT was observed in the mouse but not in the rat [11]. Several phase II genes (Gsta1 (~3.4-fold), Gstm2 (~3.2-fold), Ugt2b35 (~3.4-fold)) were also induced.
Table 1. Selected microarray results
A number of DNA damage-related genes such as Cdkn1a (~15.4-fold), Gadd45a (~13.5-fold), Gadd45b (~8.4-fold) and Tnfrsf19 (~5.9-fold) were induced following o, p'-DDT treatment, while Fas and Casp9 were down-regulated (~2.0-fold). The oxidative stress-responsive genes Gclm (~1.6-fold), Gsr (~2.5-fold), Hmox2 (~1.6-fold) and Nqo1 (~2.6-fold) exhibited induction. There was no clear induction for the cell proliferation-related genes such as Ccnd1, Ccnb2, Mdm2 or Stmn1, while their orthologs were induced by o, p'-DDT in the rat liver [11]. mRNA levels for Ahr and Pxr (Nr1i2) were slightly elevated, whereas Car (Nr1i3) mRNA levels decreased following o, p'-DDT treatment. Srebf1 which was previously shown to be down-regulated by o, p'-DDT in the rat liver [11], was also down-regulated in the mouse liver. Overall, the QRT-PCR results closely paralleled the expression pattern seen in the microarray data (Fig. 2C).
We also examined the expression of genes regulated by CAR, PXR or ER that were selected based on the null animal studies [20-23]. While CAR-specific targets such as Cyp1a1 (~2.2-fold), Fmo5 (~2.3-fold), Sult1d1 (~2.8-fold) and Abcc2 (~2.7-fold) showed relatively weak induction, several PXR-regulated genes were induced more strongly including ApoA4 (~5.8-fold), Ces2 (~3.4-fold), Gstm2 (~3.2-fold) or Insig2 (~5.0-fold) genes (Fig. 2D). However, other PXR genes, such as Hmgcs1 and Hmgcs2, were unexpectedly down-regulated.
Comparative Analysis of Gene Expression
Hepatic gene expression profiles of EE-treated mice, o, p'-DDT-treated rats and o, p'-DDT-treated mice were compared by hierarchical clustering focusing on the 538 orthologs represented on both microarray platforms that satisfied the |fold change| ≥ 1.5 for at least one time point in any data set. The filtering criteria used here were relaxed compared to those used in Figs. 2A and 2B to include more orthologs in the correlation analysis, and thus be more informative of the overall similarity between the two data sets. Despite the differences in microarray platform (i.e., cDNA vs. oligonucleotide), o, p'-DDT elicited gene expression profiles in the rat and mouse that were more similar to each other than to the EE-treated mouse profile (Fig. 3A).
Figure 3. Comparative analysis of global gene expression profiles elicited by o, p'-DDT. (A). Comparative gene expression analysis between EE-treated mouse, o, p'-DDT-treated rat and o, p'-DDT-treated mouse. A total of 996 orthologs were represented on the rat cDNA microarray, mouse cDNA microarray and mouse Agilent oligonucleotide microarrays determined by HomoloGene http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene webcite. 538 of these orthologs showed a |fold change| ≥ 1.5 for at least one time point in either species. These 538 differentially expressed orthologs were subjected to hierarchical clustering. The dendrogram illustrates that mouse o, p'-DDT gene expression profiles are more similar to rat o, p'-DDT gene expression profiles than the mouse EE gene expression profiles. (B) Correlation analysis using differentially expressed orthologous genes. The temporal profiles of o, p'-DDT-treated mouse liver (current study) and those of the o, p'-DDT-treated rat liver [11] were compared by determining the Pearson's correlation of the temporal gene expression (fold change) and significance (p1 [t] value) between orthologs. Both studies used comparable study designs and data analysis methods, although different platforms were used (i.e., rat cDNA microarray and mouse Agilent oligonucleotide microarray). 140 genes were identified as differentially expressed orthologs. (C) Scatter plot of the 140 differentially expressed orthologous genes. Correlations for gene expression and significance approaching 1.0 indicate that the behavior or the orthologous genes are similar and would fall in the upper right quadrant. Orthologs tended to localize in upper- or lower-right quadrant (32.9% and 47.9% of total number of spots, respectively), indicating that temporal gene expression changes for o, p'-DDT-treated mouse and rat liver are comparable. However, poor correlations between the temporal p1(t) values and gene expression fold changes would fall within the lower left quadrant. For example, Cyp17a1 fell into this quadrant suggesting that significant differences exist between the rat and mouse ortholog expression profiles.
Correlation Analysis
One hundred and forty orthologs were identified as differentially expressed on both rat cDNA and mouse oligo microarrays (Fig. 3B), which were further examined by correlation analysis to determine if their gene expression profiles were comparable. Again, the filtering criteria used here were relaxed compared to those used in Figs. 2A and 2B to include more orthologs in the correlation analysis. The scatter plot shows that a majority of the spots localized to either the upper-right (32.9%) or lower-right (47.9%) quadrants (Fig. 3C), indicating that the gene expression profiles between o, p'-DDT-treated rat and mouse were highly similar in terms of their gene expression pattern. Temporal correlation of the p1(t)-value (statistical significance) showed relatively low correlation between the two data sets, with 60% of spots located in the lower-right or lower-left quadrants. All correlation analyses results are provided in Additional file 4.
Additional File 4. Correlation analysis between microarray results of o, p'-DDT-mouse and rat.
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For example, Cyp17a1, a key enzyme gene for steroidogenesis (Fig. 4A) fell into the lower left quadrant (Fig. 3C) suggesting divergent gene expression profiles in the mouse and rat. Fig. 4B clearly demonstrates that Cyp17a1 is significantly induced in the mouse and is non-responsive in the rat in both microarray and QRT-PCR data. Cyp7b1, another steroid metabolism gene, exhibited a similar species-specific gene expression profile (Fig 4B). Moreover, hepatic CYP17A1 protein induction was evident at 18 and 24 h in the mouse liver (Fig. 4C).
Figure 4. Species-specific regulation of steroid hormone metabolism elicited by o, p'-DDT. (A) Overview of the role of CYP17A1 and CYP7B1 in steroid metabolism. CYP17A1 metabolizes pregnenolone and progesterone to produce DHEA and androstenedione, respectively. Hepatic CYP7B1 is involved in bile acid biosynthesis, and also responsible for 7α-hydroxylation of DHEA. (B) Hepatic Cyp17a1 and (C) Cyp7b1 mRNA levels in the o, p'-DDT-treated mouse and rat. QRT-PCR results relative to time-matched vehicle controls are shown as bars and presented as mean ± SE. Microarray results are represented as lines. o, p'-DDT induced Cyp17a1 and Cyp7b1 mRNAs in the mouse liver, while it did not affect in the rat liver [11]. The dashed line indicates the expression level of the time-matched vehicle control. The asterisk (*) indicates a significant (p < 0.05) difference from the time-matched vehicle controls for QRT-PCR, n = 5. (C) Representative Western analysis result for hepatic CYP17A1 protein in o, p'-DDT-treated mouse liver. CYP17A1 protein levels were induced at 18 and 24 h. Western analyses were performed on 3 independent biological replicates to verify the consistency of the results. C, control; T, 300 mg/kg o, p'-DDT. (D) Blood DHEA-S levels. DHEA-S level was significantly higher at 12 h following o, p'-DDT treatment compared to time-matched controls in the mouse, while it did not change in rats.
Effects on DHEA-S Levels
Species-specific induction of CYP17A1 mRNA and protein levels suggests that steroid metabolism may also be differentially affected. More specifically, since CYP17A1 metabolizes pregnenolone and 17α-hydroxypregnenolone to produce DHEA (Fig. 4A), blood levels of DHEA-S were measured using an enzyme-linked immunosorbent assay. Serum DHEA-S levels exhibited a significant (p < 0.05) increase relative to vehicle at 12 h in the mouse, (Fig. 4D). In contrast, plasma DHEA-S was not significantly different between treated and control rats. Mouse serum androstenedione levels were below the detection level (data not shown).
o, p'-DDT elicits phenobarbital-type activities in the mouse liver, characterized by the increase in relative liver weight or induction of Cyp2b10, Cyp3a11 and GST mRNA levels [27,28]. In the rat, we previously reported that o, p'-DDT elicits PXR/CAR-mediated responses, with negligible ER-mediated activity [11], despite the responsive of the liver to estrogens [9,29], and the association of estrogenic activity with hepatic tumorigenesis [30]. Consequently, PXR/CAR-mediated responses to DDT may have a more significant role in hepatic tumor promotion [11]. In order to further investigate o, p'-DDT elicited gene expression mediated by PXR/CAR, we investigated its hepatic expression profile using a comparable mouse model, study design and data analysis [11]. Comparative analysis indicated that the hepatic gene expression profiles were similar between rat and mouse. However, correlation analysis of orthologous genes revealed that hepatic Cyp17a1 mRNA and protein levels as well as serum DHEA-S were only elevated in the mouse following o, p'-DDT treatment.
Cyp17a1 exhibits dose-dependent induction by EE in the mouse liver [10], suggesting ER-mediated induction. In addition, induction of Cyp7b1 and down-regulation of Cyp7a1 by EE are abolished in ER alpha-null mice but not in ER beta-, Fxr-, Pxr- or Car-null mice [23]. Collectively, these results indicate that Cyp7a1 mRNA levels are regulated by ERα in the mouse liver. Considering the strong inductions of Cyp17a1 and Cyp7b1 observed only in the mouse liver as well as down-regulation of Cyp7a1 in the mouse liver, o, p'-DDT may elicit ERα-mediated activity in the mouse liver but not in the rat liver.
In addition, the mouse profile suggests that o, p'-DDT elicited gene expression was predominately PXR regulated based on genes known to be regulated by PXR-, CAR- or PXR/CAR [20-23]. In part, this could be due to the repression of mouse Car mRNA levels by o, p'-DDT compared to its induction in the rat liver [11]. Furthermore, several genes associated with cell proliferation (Ccnb1, Ccnb2, Mdm2 and Stmn1) were induced in the rat liver [11], but not in the mouse. Ccnd1, a known CAR-regulated cell proliferation-related gene [31], was not significantly induced in the mouse, further supporting the hypothesis that o, p'-DDT preferentially activated PXR.
CAR also has an inhibitory effect on ER-mediated gene expression [32]. Therefore, the availability of CAR may inhibit ER-mediated effects elicited by o, p'-DDT in the rat liver [11], while the down regulation of Car mRNA in the mouse and the preferential activation of PXR may facilitate more ER-mediated gene expression. Consequently, ER-mediated responses may be important when assessing DDT-elicited hepatic responses in the mouse in addition to PXR/CAR-mediated response when compared to the rat.
CYP17A1 is an important enzyme in steroid biosynthesis that metabolizes pregnenolone and progesterone to produce DHEA and androstenedione, respectively [33,34]. Therefore, induction of CYP17A1 mRNA and protein levels may affect DHEA metabolism. o, p'-DDT treatment increased DHEA-S levels in the mouse whereas no change was detected in the rat. Although the liver is usually not a major steroidogenesis organ, microsomes are capable of participating in steroidogenesis [35]. In addition, hepatic microsomal CYP17A1 exhibits age-dependent expression, with higher expression and activity in immature rats [35,36]. o, p'-DDT elicited changes in other steroidogenic enzymes (e.g., Cyp11a1) and metabolites (e.g., androstenedione) were below the level of detection. Collectively, alterations in steroid levels following o, p'-DDT treatment could affect steroid levels, since peripheral tissues can use circulating DHEA and DHEA-S to produce androgens and estrogens [37]. Furthermore, the 7α-hydroxylation of DHEA and pregnenolone by o, p'-DDT induced CYP7B1 could also facilitate elimination of DHEA or the synthesis of neuro-active hormones [38,39].
In addition to PXR-, CAR- or ER-mediated gene expression changes, o, p'-DDT induced Gadd45a, Gadd45b and Cdkn1, all of which are DNA damage-responsive genes [40,41]. Consistent with this are the reports of the DNA damaging potential of DDT [42-44]. Consequently, DNA damage may be an additional risk factor for tumor initiation/promotion following o, p'-DDT exposure in addition to PXR/CAR- and ER-mediated activities. Considering that the induction of DNA damage-responsive genes precedes Cyp2b10 or Cyp3a11 induction, the DNA damage may not be caused by oxidative stress derived from enzyme induction. Moreover, Gclm and Hmox, both known oxidative stress-responsive genes [45,46], exhibited relatively weak induction compared to rats [11], suggesting that oxidative stress was not strongly induced.
In conclusion, o, p'-DDT elicits a broad spectrum of species-conserved and specific effects. This includes PXR/CAR- and ER-mediated responses, altered steroidogenesis, oxidative stress, and DNA damage (Fig. 5). Although DDT is known to cause liver tumors in both mice and rats, the marked species differences in PXR/CAR structure, expression patterns and ligand preference as well as significant species-specific differences in steroidogenesis, especially CYP17A1 expression and activity, confound the extrapolation of these results to humans. Nevertheless, the identification of potential modes of action as well as species-specific responses may assist in the development or selection of more appropriate models for assessing the toxicity of DDT to humans and wildlife.
Figure 5. Potential species-specific responses to o, p'-DDT. In the rat liver, o, p'-DDT elicits PXR/CAR-mediated response, with negligible ER-mediated effects despite the established estrogenicity of o, p'-DDT in other target tissues (e.g., uterus). The absence of ER-mediated gene expression may be due, in part, to the inhibitory effects of CAR on ER. In contrast, o, p'-DDT elicits not only PXR/CAR-mediated responses but also ER-mediated effects such as the induction of Cyp17a1 and Cyp7b1 in the mouse liver. Gene expression profiling suggests that o, p'-DDT preferentially activates PXR in the mouse liver. Therefore, the inhibitory effects of CAR on ER-mediated signaling would be reduced in the mouse liver, allowing the activation of ER-mediated gene expression by o, p'-DDT. In addition, only mouse DHEA-S levels were increased, which could affect both endocrine and liver physiology. The induction of Cyp17a1 and Cyp7b1 may be involved in either the metabolism or disposition of DHEA, or the conversion of DHEA into other metabolites including neurosteroids.
CAR: constitutive androstane receptor (Nr1i3); Casp9: caspase 9; Ccn: cyclin; Cdkn1a: cyclin-dependent kinase inhibitor 1A; Cyp: cytochrome P450; DDT: dichlorodiphenyltrichloroethane; DHEA-S: dehydroepiandrosterone sulfate; EE: ethynylestradiol; ER: estrogen receptor; FXR: farnesoid X receptor; Gclm: glutamate-cysteine ligase modifier subunit; Gadd45: growth arrest and DNA-damage-inducible 45; Gsr: glutathione reductase; GST: glutathione S-transferase; Hmox2: heme oxygenase (decycling) 2; HMGCS: 3-hydroxy-3-methylglutaryl-Coenzyme A synthase; Hsd3b: hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase; Insig2: insulin induced gene 2; Mdm2: transformed mouse 3T3 cell double minute 2; Nqo1: NAD(P)H dehydrogenase, quinone 1; Stmn1: stathmin 1; PXR: pregnane X receptor (Nr1i2); QRT-PCR: Quantitative Real-Time PCR; Srebf1: sterol regulatory element binding factor 1; Stat: signal transducer and activator of transcription; TCPOBOP: 1,4-bis-[2-(3,5,-dichloropyridyloxy)] benzene; Tnfrsf19: tumor necrosis factor receptor superfamily member 19; UGT: UDP glycosyltransferase.
Authors' contributions
NK performed all microarray, ELISA and Western assays, conducted and statistically analyzed QRT-PCR assays, as well as compiled and interpreted the data collected and generated the primary draft manuscript. JCK designed/conducted animal studies and participated in interpretation of microarray data. LDB provided database support for the microarray data including quality control assessments and statistical analysis. ED participated in microarray data analysis. KJW carried out histology examination. CT and BC carried out tissue level analysis. TRZ conceived the study and its design and supervised its completion. All authors read and approved the final draft of the manuscript.
The authors thank Anna Kopec for helpful discussion and critical review of this manuscript. This study was supported by National Institute of General Medical Sciences (GM075838), U.S. Environmental Protection Agency (RD83184701). T.R.Z. is partially supported by the Michigan Agricultural Experiment Station
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text:
I am invoking multiple async calls of
thrift from my code. I would like to wait
for all of them to complete before going on with my next stage.
for (...) {
TNonblockingTransport transport = new TNonblockingSocket(host, port);
TAsyncClientManager clientManager = new TAsyncClientManager();
TProtocolFactory protocolFactory = new TBinaryProtocol.Factory();
AsyncClient c = new AsyncClient(protocolFactory, clientManager, transport);
c.function(params, callback);
// I would like to wait for all the calls to be complete here.
I can have a countdown in the callback like wait/notify and get this done. But does the
thrift system allow a way for me to wait on my async function call, preferably with a timeout ?
I didnt see any in the
TAsyncClientManager or in the
AsyncClient. Please help.

subset: commoncrawl


text:
SKIN PLAYERPRO V3 GOLD ELEGANT by Tak Team Studio NOTE: IF YOU HAVE PROBLEMS THAT MUSIC OFF THE SCREEN, THE SOLUTION IS STOPS: MENU CONFIGURATION LOCK SCREEN ENABLE DIS … 10+ downloads, Rating 5.00 (1) Free
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text:
- Filed Under
The readiness of Peyton Manning and the Indianapolis Colts offense for the start of the season were major question marks heading into Sunday night's prime-time matchup with the Chicago Bears at Lucas Oil Stadium.
The questions remain.
Manning only occasionally seemed to find his comfort zone and the offense only located the end zone once in a 29-13 loss to the Bears. ...


text:
NEP Broadcasting was the primary supplier of TV production technology to broadcasters delivering global coverage of the 107th U.S. Open from Oakmont Country Club in Pittsburgh last week.
NBC and ESPN coverage of the event was captured by NEP Supershooters' ND4, its newest HD truck. Coverage of the U.S. Open was the ND4's maiden voyage — only 2mi from where the unit was designed and built.
Outfitted with a virtual monitor wall within a spacious control room and furnished with a Thomson Grass Valley Kalypso switcher, Calrec Alpha audio console, Sony HDC1500 cameras and several EVS LSMs, the new ND4 joined NEP Supershooters' fleet of mobile trucks at the U.S. Open to form a massive broadcast compound.
For this year's Open Championship, NEP supplied more than $50 million worth of equipment and hardware to leading TV networks from around the globe, which, in addition to NBC and ESPN, included The Golf Channel, Sky Sports, TV Asahi and the USGA world feed.
For more information, visit www.nepinc.com.
An Inside Look at Four Virtual Reality Start-Ups
What Moves the Social TV Needle?
Mark Kudsi Directs 'Art of Patron Virtual Reality Experience'
BT Renews Intelsat Deal
Top Twitter TV Shows for 2014-15 Season Unveiled
comScore Introduces MobiLens Plus
North Korean Propoganda Art Inspires the Credits of 'The Interview'
Imaginary Forces Creates Kaleidoscopic Titles for 'Bosch'
Filmmaker Embraces Short Documentary Format with Oscar-Nominated 'Crisis Hotline'
North Metro Television Goes Wireless with Comrex LiveShot
Spectra Logic and HauteSpot Networks Introduce Body-Worn Camera Video Surveillance Solution
Huge Ultra HD Display Lights Up Times Square


text:
|By Kenrick Freemen
Article Rating:
|August 23, 2012 07:45 AM EDT
EPractize Labs is the first company that offered software study book and practice mock questions for Oracle's Java SE 7 Programmer II(1Z1-804, OCPJP 7) Exam to achieve Oracle Certified Professional, Java SE 7 Programmer. The software guide has been released in the early stage of OCPJP 7 Beta exam.
The product has been already reviewed by tons of exam takers and the current version of this exam simulator is 1.3. The good news from EPractize Labs is that they also offering Training Lab for Oracle's Upgrade to Java SE 7 Programmer II(1Z0-805, OCPJP 7 Upgrade) Exam to achieve Oracle Certified Professional, Java SE 7 Programmer Certification under upgrade path.
Set goals against each exam objectives for preparation - This helps the candidates to focus more on specific exam topics during study.
Set schedule for preparation - This helps the candidates to measure the progress over time.
The Training Lab comes with Pre Measurement Lab with limited number of questions to identify current skill level in Oracle Certified Professional, Java SE 7 Programmer exam objectives and also helps the candidates to familiarize with EPractize Labs exam environment.
The product offers theory guide in Basic Sessions with flash card like quiz exams to memorize the concepts.
To understand the exam objectives better, the kit comes with Advanced Sessions where practical examples/program examples are provided with flash card like quiz exams to memorize the concepts. I like the coding related questions with combo box selection answer type.
The above components improves candidate's knowledge level in exam objectives, the below components provide mock exam questions with brief explanations to get ready for OCPJP 7 exam.
You have quiz mode exam to check your answer instantly with timer for each question. This lab comes with customize option where topics, number of questions and time can be updated to suit your needs. Exam explanation windows have an option to add your own notes.
The Mock Exam Lab is the critical component of this preparation kit which has preset exams similar to real exam environment with time restricted mode. We can also have a customize option to select specific topic questions for practice.
The Final Exam offers a mock exam to judge your readiness to attend real exam. I recommend you to complete all the above component exams before appearing this test.
The product has an excellent component called Focus Lab to measure your weakness. Focus Lab stores all the wrongly answered questions in every lab. It classifies the questions based on the number of attempts. This helps you to select those questions that were attempted multiple times wrongly and focus your efforts to improvise on them. As this lab is an effort to track your short-comings in the practice, you might not be able to see any data at the start of the product usage and the respective buttons will be disabled.
The product also offers an option to save your exam resources (URLs or local files) for your exam preparation.
When you have completed any exam, you are shown a report detailing which answers you got right and which were wrong. From here you may view the questions and answers. Each question is shown along with the answer(s) you chose, and the correct answer(s). Except basic and advanced session exam questions, each question and answer also had details of why the correct answer(s) are correct and why the incorrect answer(s) are wrong.
The report component provides a summary of your certification preparation with OCPJP 7 Training Lab. The reports show you your progress of all the exams you had attempted so far. You can select any exam – Pre Measurement exam, Review Exam, Advanced Sessions, Session Rewind Quiz, Mock Exam and Final Exam and view the reports from Reports Lab. The kit also allows you to save the reports on your PC's local disk by using the "Publish Report" option.
The final component, assessment tracks your goals and schedules in four segments listed below.
Goals Tracker: It displays the tabular and graphical representation of your goals set versus goals achieved for every objective/topic.
Gantt chart: It displays the Gantt view of the lab-wise planned vs the actual schedule and the percentage obtained for the same.
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PPA Tracker: If you have successfully accomplished your goal, PPA Tracker greets you with a message that you have achieved your goal. Additionally you are also presented a Certification of Completion.
I strongly recommend this product for OCP Java 7 programmer exam preparation. The product is really worth for the price they are offering for your lifetime upgrade license. EPractize Labs is so confident that you will pass the exam after using their Training Lab that they offer an unconditional money back guarantee in the unlikely event that you fail. What else, go and download OCPJP 7 Training Lab trial at EPractize Labs website, explore the components, if you really like it purchase and unlock the software.
The 4th International Internet of @ThingsExpo, co-located with the 17th International Cloud Expo - to be held November 3-5, 2015, at the Santa Clara Convention Center in Santa Clara, CA - announces that its Call for Papers is open. The Internet of Things (IoT) is the biggest idea since the creation of the Worldwide Web more than
Jul. 5, 2015 08:00 PM EDT Reads: 745
The 17th International Cloud Expo has announced that its Call for Papers is open. 17th International Cloud Expo, to be held November 3-5, 2015, at the Santa Clara Convention Center in Santa Clara, CA, brings together Cloud Computing, APM, APIs, Microservices, Security, Big Data, Internet of Things, DevOps and WebRTC to one location. With cloud computing driving a higher percentage of enterprise IT budgets every year, it becomes increasingly important to plant your flag in this fast-expanding business opportunity. Submit your speaking proposal today!
Jul. 5, 2015 06:30 PM EDT Reads: 459
Explosive growth in connected devices. Enormous amounts of data for collection and analysis. Critical use of data for split-second decision making and actionable information. All three are factors in making the Internet of Things a reality. Yet, any one factor would have an IT organization pondering its infrastructure strategy. How should your organization enhance its IT framework to enable an Internet of Things implementation? In his session at @ThingsExpo, James Kirkland, Red Hat's Chief Architect for the Internet of Things and Intelligent Systems, described how to revolutionize your archit...
Jul. 5, 2015 06:00 PM EDT Reads: 537
The 5th International DevOps Summit, co-located with 17th International Cloud Expo – being held November 3-5, 2015, at the Santa Clara Convention Center in Santa Clara, CA – announces that its Call for Papers is open. Born out of proven success in agile development, cloud computing, and process automation, DevOps is a macro trend you cannot afford to miss. From showcase success stories from early adopters and web-scale businesses, DevOps is expanding to organizations of all sizes, including the world's largest enterprises – and delivering real results. Among the proven benefits, DevOps is corr...
Jul. 5, 2015 05:15 PM EDT Reads: 555
SYS-CON Events announced today that Secure Infrastructure & Services will exhibit at SYS-CON's 17th International Cloud Expo®, which will take place on November 3–5, 2015, at the Santa Clara Convention Center in Santa Clara, CA. Secure Infrastructure & Services (SIAS) is a managed services provider of cloud computing solutions for the IBM Power Systems market. The company helps mid-market firms built on IBM hardware platforms to deploy new levels of reliable and cost-effective computing and high availability solutions, leveraging the cloud and the benefits of Infrastructure-as-a-Service (IaaS...
Jul. 5, 2015 05:00 PM EDT Reads: 660
To many people, IoT is a buzzword whose value is not understood. Many people think IoT is all about wearables and home automation. In his session at @ThingsExpo, Mike Kavis, Vice President & Principal Cloud Architect at Cloud Technology Partners, discussed some incredible game-changing use cases and how they are transforming industries like agriculture, manufacturing, health care, and smart cities. He will discuss cool technologies like smart dust, robotics, smart labels, and much more. Prepare to be blown away with a glimpse of the future.
Jul. 5, 2015 04:45 PM EDT Reads: 552
SYS-CON Events announced today that ProfitBricks, the provider of painless cloud infrastructure, will exhibit at SYS-CON's 17th International Cloud Expo®, which will take place on November 3–5, 2015, at the Santa Clara Convention Center in Santa Clara, CA. ProfitBricks is the IaaS provider that offers a painless cloud experience for all IT users, with no learning curve. ProfitBricks boasts flexible cloud servers and networking, an integrated Data Center Designer tool for visual control over the cloud and the best price/performance value available. ProfitBricks was named one of the coolest Clo...
Jul. 5, 2015 04:30 PM EDT Reads: 1,408
17th Cloud Expo, taking place Nov 3-5, 2015, at the Santa Clara Convention Center in Santa Clara, CA, will feature technical sessions from a rock star conference faculty and the leading industry players in the world. Cloud computing is now being embraced by a majority of enterprises of all sizes. Yesterday's debate about public vs. private has transformed into the reality of hybrid cloud: a recent survey shows that 74% of enterprises have a hybrid cloud strategy. Meanwhile, 94% of enterprises are using some form of XaaS – software, platform, and infrastructure as a service.
Jul. 5, 2015 03:00 PM EDT Reads: 459
Internet of Things is moving from being a hype to a reality. Experts estimate that internet connected cars will grow to 152 million, while over 100 million internet connected wireless light bulbs and lamps will be operational by 2020. These and many other intriguing statistics highlight the importance of Internet powered devices and how market penetration is going to multiply many times over in the next few years.
Jul. 5, 2015 03:00 PM EDT Reads: 1,147
The basic integration architecture, as defined by ESBs, hasn't changed for more than a decade. Most cloud integration providers still rely on an ESB architecture and their proprietary connectors. As a result, enterprise integration projects suffer from constraints of availability and reliability of these connectors that are not re-usable across other integration vendors. However, the rapid adoption of APIs and almost ubiquitous availability of APIs amongst most SaaS and Cloud applications are rapidly redefining traditional integration approaches and their reliance on proprietary connectors. ...
Jul. 5, 2015 02:30 PM EDT Reads: 427
SYS-CON Events announced today that Dyn, the worldwide leader in Internet Performance, will exhibit at SYS-CON's 17th International Cloud Expo®, which will take place on November 3-5, 2015, at the Santa Clara Convention Center in Santa Clara, CA. Dyn is a cloud-based Internet Performance company. Dyn helps companies monitor, control, and optimize online infrastructure for an exceptional end-user experience. Through a world-class network and unrivaled, objective intelligence into Internet conditions, Dyn ensures traffic gets delivered faster, safer, and more reliably than ever.
Jul. 5, 2015 02:30 PM EDT Reads: 1,503
"We have a tagline - "Power in the API Economy." What that means is everything that is built in applications and connected applications is done through APIs," explained Roberto Medrano, Executive Vice President at Akana, in this SYS-CON.tv interview at 16th Cloud Expo, held June 9-11, 2015, at the Javits Center in New York City.
Jul. 5, 2015 01:30 PM EDT Reads: 451
WebRTC converts the entire network into a ubiquitous communications cloud thereby connecting anytime, anywhere through any point. In his session at WebRTC Summit,, Mark Castleman, EIR at Bell Labs and Head of Future X Labs, will discuss how the transformational nature of communications is achieved through the democratizing force of WebRTC. WebRTC is doing for voice what HTML did for web content.
Jul. 5, 2015 01:00 PM EDT Reads: 428
Today air travel is a minefield of delays, hassles and customer disappointment. Airlines struggle to revitalize the experience. GE and M2Mi will demonstrate practical examples of how IoT solutions are helping airlines bring back personalization, reduce trip time and improve reliability. In their session at @ThingsExpo, Shyam Varan Nath, Principal Architect with GE, and Dr. Sarah Cooper, M2Mi's VP Business Development and Engineering, will explore the IoT cloud-based platform technologies driving this change including privacy controls, data transparency and integration of real time context wi...
Jul. 5, 2015 12:00 PM EDT Reads: 390
Buzzword alert: Microservices and IoT at a DevOps conference? What could possibly go wrong? In this Power Panel at DevOps Summit, moderated by Jason Bloomberg, the leading expert on architecting agility for the enterprise and president of Intellyx, panelists peeled away the buzz and discuss the important architectural principles behind implementing IoT solutions for the enterprise. As remote IoT devices and sensors become increasingly intelligent, they become part of our distributed cloud environment, and we must architect and code accordingly. At the very least, you'll have no problem fillin...
Jul. 5, 2015 11:45 AM EDT Reads: 1,776
The Internet of Things is not only adding billions of sensors and billions of terabytes to the Internet. It is also forcing a fundamental change in the way we envision Information Technology. For the first time, more data is being created by devices at the edge of the Internet rather than from centralized systems. What does this mean for today's IT professional? In this Power Panel at @ThingsExpo, moderated by Conference Chair Roger Strukhoff, panelists addressed this very serious issue of profound change in the industry.
Jul. 5, 2015 09:30 AM EDT Reads: 556
Internet of Things (IoT) will be a hybrid ecosystem of diverse devices and sensors collaborating with operational and enterprise systems to create the next big application. In their session at @ThingsExpo, Bramh Gupta, founder and CEO of robomq.io, and Fred Yatzeck, principal architect leading product development at robomq.io, discussed how choosing the right middleware and integration strategy from the get-go will enable IoT solution developers to adapt and grow with the industry, while at the same time reduce Time to Market (TTM) by using plug and play capabilities offered by a robust IoT ...
Jul. 5, 2015 09:00 AM EDT Reads: 1,173
It is one thing to build single industrial IoT applications, but what will it take to build the Smart Cities and truly society-changing applications of the future? The technology won't be the problem, it will be the number of parties that need to work together and be aligned in their motivation to succeed. In his session at @ThingsExpo, Jason Mondanaro, Director, Product Management at Metanga, discussed how you can plan to cooperate, partner, and form lasting all-star teams to change the world and it starts with business models and monetization strategies.
Jul. 3, 2015 12:00 PM EDT Reads: 1,322
SYS-CON Events announced today that BMC will exhibit at SYS-CON's 16th International Cloud Expo®, which will take place on June 9-11, 2015, at the Javits Center in New York City, NY. BMC delivers software solutions that help IT transform digital enterprises for the ultimate competitive business advantage. BMC has worked with thousands of leading companies to create and deliver powerful IT management services. From mainframe to cloud to mobile, BMC pairs high-speed digital innovation with robust IT industrialization – allowing customers to provide amazing user experiences with optimized IT per...
Jun. 29, 2015 12:15 PM EDT Reads: 2,254
There will be 150 billion connected devices by 2020. New digital businesses have already disrupted value chains across every industry. APIs are at the center of the digital business. You need to understand what assets you have that can be exposed digitally, what their digital value chain is, and how to create an effective business model around that value chain to compete in this economy. No enterprise can be complacent and not engage in the digital economy. Learn how to be the disruptor and not the disruptee.
Jun. 29, 2015 11:00 AM EDT Reads: 1,586


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32 year old woman | Irving, TX, USA | Seeking men 25-35 within 20 miles
Friendship & future
Alright I figured I would give this 1 more shot... I really don't like to ramble on about myself. I would rather actually have a conversation with someone interested in getting to know me. With that said, I am a very outgoing and caring person. I try to give everything I do my all. My job is important to me. I do work a lot. I really don't have time to go out to bars or anywhere every night, for that matter, to find the perfect guy I am looking for. I am looking for someone who obviously I am attracted to. Someone who cares about what they look like and tries to stay active. I myself love just about any outdoor activities, although don't have a whole lot of time. I am looking for someone who's work is also important to them. Don't get me wrong. I am not all about work and no play! I love to take vacations also... Anywhere where there is a beach, is great! I am also looking for someone who likes animals. I am a huge animal lover and have a 1 yr old pit bull of my own. Friends and family are very important to me. My friends would probably describe me as always being the 1 making everyone laugh. I love to goof around. I am looking for someone I can be comfortable just being myself with.
Billiards / Pool, Bowling, Dancing, Football, Swimming, VolleyBall, Yoga
I have Dogs
1 yr old pit bull named Buddy
Music, time with friends & family, spending time with my dog, Buddy
Anywhere there is a beach, movies, going out for dinner, trying new bars/ places, volleyball, pool, darts, about anything outdoors
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'0" (152cms)
|5'7" (170cms) to 6'4" (193cms)
|Body type:
|About average, Athletic and toned
|No preference
|No preference
|No Way
|No Way, Occasionally
|Social Drinker
|No preference
|Retail / Food services
|No preference
|I'll tell you later
|No preference
|Never Married
|No preference
|Have kids:
|No preference
|Wants kids:
|Not sure
|No preference
|White / Caucasian
|Latino / Hispanic, Native American, Pacific Islander, White / Caucasian
|I'll tell you later
|Agnostic, Atheist, Christian / Catholic, Christian / Protestant, Spiritual but not religious, Other, Christian / Other
|Some college
|High school, Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral


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Come visit me, chaka's campaign story, Prayer Requests, Chaka#215291 ty 4 botd 062914, Botm Winners Circle-VOTE SPOT & CHAKA & CALY'S BUFFET & GROCERY.
Come visit me, Leroy, Little Bit & Chloe - Thank you for BOTD!, Gizmo - Thanks everybirdy for BOTD!, Max, Jasper - Thanks everybirdy for BOTD!, Micky - Thanks everybirdy for BOTD!, PEPPER - WOW! Thanks everybirdy for BOTD!, TiPaul and Cubird at the Derby Milonga, Floop - Thanks for BOTD!, Jockey Marley & Exchange Rate, Zak - Thank you everybirdy for BOTD! & Lee-Roial RIP - Thanks for remembering me..
Come visit me, Jedi luvs Cheyenne forever..., Snowflake luvs her Apache..., Tweety luvs Princess... & PANTHER...VOTE FOR ME FOR BOTM !!!.
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Come visit me, Bilou & Strawberry.
Come visit me, SKY, Little Angel, jesse & Queen Sweetie, Thank-you friends for BOTM.
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Come visit me, Dobby, Grey Bird, Angel Devynn (RIP), Peatree~ Vote 4 Panther #245623!, Bella, King Jester Bean ~ Thank you so much!, Honey, Angel Hermione (RIP), Angel Dudley (RIP), Pressie & Angel Weasley & Angel Jacob (RIP).
Come visit me, Gizmo and Divo, mom loves you both, Willie, Peppino & Falco.
Come visit me, 10K Hall of Fame, BOTD ARCHIVES, Sugar,Pippo, Mei Mei & Dolph~vote 4 Panther 245623 & The Calendar Page~Wonderful Kiwi for July.
Come visit me, Derby Farms, Chyna ~ Happy 4th of July & Bailey ♥ RIP.
Come visit me, Carter-Justice For Ozzi-TY 4 BOTD 8/28/14, Doo Diligence-Justice for Ozzi TY 4 BOTD!, Cookie-Justice For Ozzi-thank you for BOTD 7/31/14, Charlie-Justice 4 Ozzy-TY for BOTD 10/29/14, Tricky D- RIP 12/12/12-Thank you BOTD 12/12/14, Teal'c-Thank you for BOTD 10-14-14 Justice for Ozz, Elvis Thank you for BOTD 2/22/15, Joshua Chico Justice for Ozzi, Blue-Justice for Ozzi-TY 4 BOTD 9/7/14, Moggie -Justice for Ozzi, George (Gen George Hammond)RIP Gen 12/13/08, Cu-Bird -Justice for Ozzi, Nanday Security Services, LLC, Miranda -Justice for Ozzi-ty 4 botd 5/13/15, Captain Darling-RIP 5/19/2014 ty 4 BOTD 9/19/14 & Baldrick Thank you for BOTD 3/20/14.
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Come visit me, Callie*, * Mango*, GREEN* Today I am 10!!, Peetie Thank you for BOTD 3/26/15 :), Paulie* Today I am 9!! & Little Angel Peep meets Lola at the RB 6/8/14.
Come visit me, Hi everybirdie, i'm back!!!!.
Come visit me, ALEX, POPEYE, SAWYER, BLUE & JEWEL, GINGER & CALICO.
Come visit me, TAZ-.INDEPENDENCE DAY, SCOOBER: INDEPENDENCE DAY, CHICA ...4th of JULY, ROSIE....HAPPY 4TH OF JULY, NAPOLEON...HAPPY INDEPENDENCE DAY, SUZIQ...4th of JULY, THE GREY'T GREY WINGS - THANKS TEAM, The Proctors...GO HAWKS!, SIMON..HAPPY INDEPENDENCE DAY, Chloe-INDEPENDENCE DAY & JEWELS--INDEPENDENCE DAY.
Come visit me, INDIANA SAM.
Come visit me, The Ranbow Bridge, The Abused Birds, Sunny RIP 10-27-13, The Extinct Birds, Marble and Crystal- Silkie chickens, Mango-Wow, what a surprise! & Snowy Thanks for BOTD!.
Come visit me, Zip, Charlie, & Wilb~Time to get back to life!, Peekaboo says, "See my video!", ~~Peekaboo's Pasta Palace~~, ♥ BC Singles Page ♥, Charlie & the Lardfeathers Band & ~Zip's Photo Editing~CLOSED until April!~.
Come visit me, Jack Sparrow.
Come visit me, Kirby & Suzie.
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FlexWave AIP designed for BTS-to-DAS interface
Posted: 17 Jan 2014 Print Version
Keywords:BTS DAS system configuration flexibility remote monitoring[Summary of tips] TE Connectivity's FlexWave active integration panel (AIP) is designed to provide multi-carrier, multi-protocol connectivity between mobile operator base stations (BTS) and distributed antenna systems (DAS). The company says the FlexWave AIP reduces deployment time and costs of base station connections to DAS, improves system co......
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albertuszerk 25 Thomas Rossi Rassloff 16 gripspix (getting better now) Novowyr holospirit Ellenore56 marlenegonzalez84 web.werkraum andreas.klodt Rezvanova
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Posts Tagged 'sketch'
October was a wonderful month at Magnet! From a surprise Mike Myers show, to new ensembles, to Magnet performers in The NYTVF, a lot happened. To begin, Mike Myers dropped by for a special duo show with Magnet co-founder Ed Herbstman. After Jason Mantzoukas was unable to make the widely coveted Mantzoukas Brothers Show, the 3rd Mantzoukas Brother (Mike Myers) showed up to save the day. Herbstman and Myers had incredible chemistry, flawlessly weaving in and out of characters and scenes. Their show, just over an hour, was full of high energy sharp characters.
We had lots of new teams! Musical Megawatt (College, Wonderland), Megawatt (The Flood) and Magnet Sketch (Party., American Wormholes, Baby Shoes, Cash, The Misses) all had debuts this month! Congrats to the new players and be sure to check out these teams in November.
The 5th Annual NYMIF came and went with over 100 performers from as far as Toronoto and as close as Broadway! We had an incredible 5th Anniversary Festival which featured Baby Wants Candy, The Improvised Sondheim Project, and a closing night benefit for Gilda's Club NYC. Thank you to performers, audience members and our wonderful volunteers, we'll see you next year!
Happy Birthday to Armando Diaz! Our fearless leader in improv and sketch turned a whole year older this month. His birthday was kicked off with a little Halloween celebration with spooky juice, haunted candy and scary birthday candles. Cheers Armando- thanks for making us all better at what we love to do!
Magnet friends "All of Our Feelings at Once" are doing a sketch show that you should go and see! The Chicago-based sketch group is doing an Off-Broadway run at The American Theatre of Actors this Friday and Saturday (Oct. 25th & 26th) and are offering Magnet students/performers discounted tickets with the code: HAPPY.
The group was selected for participation in The Araca Project, which helps top sketch comedians bring their show to larger audiences. For tickets to Tonight or Tomorrow's 9pm show, CLICK HERE. For more information on All of Our Feeling at Once, check out their website HERE!
Congratulations to the new Magnet Sketch Teams! Thank you to all those who applied, we're excited to introduce these incredible teams! The first show is Sunday, September 29th at 9:00! Starting in October, Sketch Shows will run at 7:30 PM on Sundays. See you there!
1. Joe Lepore
2. Adam Twitchell
3. Rob Webber
4. Kate Emswiler
5. Matt Wassung
6. Ruby Marez
7. Alex Marino
8. Joe Miles
1. Jimmy O'Connell
2. Billy Bob Thompson
3. Todd Shaeffer
4. Sebastian Conelli
5. Becca Schall
6. Ingrid Ostby
7. Andy Mills
8. Jamaal Sedayao
1. Nivea Serrao
2. Megan Meadows
3. Andy Moskowitz
4. Jon Moisan
5. Ally Kornfeld
6. David Rynn
7. Bob Kern
8. Gretchen Poole
9. Matt Antonucci
1. Kevin Lalka
2. Gwen Mesco
3. Matt Alston
4. John Kitsis
5. David Sewell
6. Sean Taylor
7. Shalini Tripathi
8. Emily Johnson
9. Marcus Brunt
1. Marc Baller
2. Lane Kwederis
3. Mike Dwyer
4. Ari Miller
5. Matt Alspaugh
6. Jonathan Keller
7. Michael McLarnon
8. Matthew Pohlman Coonrod
9. Catherine Wing
We are now accepting applications for The Fall 2013 Circuit! Circuit Teams are made up of Magnet Theater students, graduates of our Training program and veteran performers. We believe that in order to get better at improv, you need to do it.
The deadline to apply is September 18th. Teams will be announced September 23rd. Shows will begin October 4th. Teams will alternate weeks through November 22nd. If you have any questions email firstname.lastname@example.com.
To Sign up, just click this link to fill out the form!
Thank you to everyone who applied and auditioned for Megawatt. We're honored to announce our fall line up:
New Team PAISLEY
Sam Rogal
Nick Jaramillo
Ali Fisher
Kelly Donahue
Branson Reese
Amanda Xeller
Evan Barden
Brendan Jordan
There will also be additions to The Wrath, Featherweight, Brick, The Music Industry and Chet Watkins
The Wrath
Andrew Yurman Glaser
Michael Kroll
Ross Taylor
Scott Lawrie
Rob Penty
Emily Shapiro
Mike Dwyer
Steven Slate
Alexis Lambright
Justin Moran
Elana Fishbein
Blake Merriman
Noel Dineen
Matt Shafeek
Lauren Ashley Smith
Frank Bonomo
Will Quinn
Rick Andrews
Julia Hynes
Tim Eberle
Joe Miles
Amie Roe
Jed Terres
Jamaal Sedayao
Caitlin Steitzer
The Music Industry
Kevin Cobbs
Sebastian Conelli
Nick Mykins
Dennis Pacheco
Charles Rogers
Phoebe Tyers
Matt Weir
Hannah Chase
Chet Watkins
Chris Camp
Karsten Cross
Russ Armstrong
Lauren Olson
John Roukis
Justin Peters
Andrew Vuilleumier
Along with returning groups Horses, All American and Call Tokyo.
The Magnet Theater is excited to announce that we are now accepting applications for the next round of MAGNET SKETCH TEAMS, which will run from September 29th through December.
Each sketch team will write and perform a show approximately every 3 weeks. Shows will take place Sunday nights at 7:30 pm. Completion of (or current enrollment in) Sketch Writing Level 2 is required to apply. Exceptions may be made for anyone currently performing on a Megawatt or Team Performance team.
Send an email with the subject line, "MAGNET SKETCH TEAM APPLICATION" and your name to email@example.com. Include your improv/sketch experience and at least 2 writing samples as attachments (please limit total to 10 pages). The deadline to apply is SEPTEMBER 13TH.
We're very excited to announce the next round of The Circuit, which will begin Friday, October 4th, 2013!
Applications for Circuit teams will open the first week of September and applicants will be chosen by lottery. If chosen, you will be placed on a team of 8 improvisers and assigned a coach. You will rehearse with your coach and team once a week, with rotating performances on Friday nights at 10PM at the Magnet Studio Theater.
If you have completed Level 3 of Magnet's Training Program and are not part of a Magnet house improv team, you are eligible to apply.
The Circuit is a great way to gain experience in being in an improv ensemble. We highly encourage you to apply.
Stay tuned for more information and announcements regarding applications in the next few weeks. Thank you and we are looking forward to an exciting new season of The Circuit!
If you have any questions, please email us at circuit [at] magnettheater [dot] com.
The Magnet Theater is proud to announce the newest members of Magnet Sketch Teams! Thank you to everyone who applied! Sketch teams perform every Sunday at 9 pm.
David Sewell
Chet Siegel
Becca Schall
Andrew Mills
Robert Ramsey
John Kitsis
Sebastian Conelli
Billy Bob Thompson
Megan Meadows
Ruby Marez
Alex Marino
Todd Shaeffer
Adam Twitchell
Ingrid Ostby
Robert Kern
Steph Garcia
Pat May
Emily Johnson
Marcus Brunt
Janie Stolar
Gretchen Poole
Jana Schmieding
Matt Antonucci
Lauren Olson
Matt Wassung
Rob Webber
Joe Lepore
Kevin Lalka
Shalini Tripathi
Alan Fessenden
Joe Miles
Sean Taylor
Kelly Warne
Gwen Mesco
James O'Connell
Kate Emswiler
Darrel Haynes
Matt Alston
Shacottha Fields is a bartender and performer at The Magnet Theater. Her one woman show, 1 Deep, was a Critics' Pick in Time Out Chicago. Shacottha performs weekly with her newly minted Megawatt Team, Call Tokyo and monthly with the all-female improv group, We Might Just Kiss. If you don't know and love Shacottha yet, read on…
*Questions: So Shacottha, what's your story?
I'm from Nacogdoches, Texas. A small place where everybody knows everybody, I graduated with 18 other people. I went to college for Business and right after I graduated I moved to New York City to pursue acting. I realize that my personality was not fit for a 9 to 5. I figured utilizing my gifts of listening and making people laugh was a better way for me to live.
*Question: When you got to New York, how did you get started?
I was taking an acting class from an agent that would come to class and yell at us, "If you want to be an actor, you have to take improv!" So I did. I have taken classes at The People's Improv Theater and Upright Citizens Brigade. I started taking classes at The Magnet Theater two years ago and I'm so happy I did. I loved the foundation of improv that I received, more so the wide variety of improv forms that I learned as well. It was like a whole world of improv that I never knew. Last year, I saw Ben Jones do his one person improv show "Just Ben." I was so inspired. The experience I had at that show was so amazing I just felt like I wanted to create something of my own to share with the world. Unsure of everything, I started coaching with Rick Andrews. Together we developed my own form for my one woman improv show. Shortly after, I gave birth to my own improv baby, 1 Deep, now traveling to different festivals. I am also proud to say that I am a part of a new house team at The Magnet Theater, "Call Tokyo".
*Question: If you could have one super power, what would it be?
My super power would be to fly. I've always felt birds had the best view of the world and all its beauty. I would fly all over the world and see all the different people, the way they live, the ocean and terrain. I would observe the change in its texture, tone and material. I would just fly all over to experience every nook and cranny of the world.
Fly with Ms. Fields and Call Tokyo every Wednesday at Megawatt!
"The Sleep Over" is the newest installment of The Director Series, a 4-week series of performances wherein a Director selects a cast and presents a different form. This month Kevin Cragg is directing "The Sleep Over." We conducted an interview with director Kevin Cragg via email about the show.
1. What is The Sleep Over?
The Sleep Over is largely a mono-character improv form that was developed in the early 2000s by then Harold Team Mother. Over the duration of the current Sleep Over incarnation, Kevin Cobbs, Megan Gray, Will Quinn, Amie Roe, Jamaal Sedayao, Emily Shapiro, Caitlin Steitzer, and Kristy Wesolowski introduce us to eight characters and take us on a journey through the world they inhabit.
2. Why are you directing it?
My interest in the Sleep Over stems from the long history I have had with the form. I was lucky enough to be present to watch Mother workshop the form at a tiny storefront theater and it has been one of my favorite forms ever since. By directing it, I have a reason to gather eight of my favorite improvisers and let them loose on one of my favorite forms.
3. What is your favorite type of improv?
Since I have performed improv for quite some time and I have watched improv even longer, I would have to say the type of improv that is my favorite is that which surprises me. For four Thursdays in April, I will be watching my favorite improv.
4. What is the future of improv?
Another realm for product placement.
The Sleep Over performs every Thursday in April at 10pm at The Magnet Theater. The cast features Kevin Cobbs, Megan Gray, Will Quinn, Amie Roe, Jamaal Sedayao, Emily Shapiro, Caitlin Steitzer, and Kristy Wesolowski.


text:
In 1920, a three-pronged Curse was unleashed upon America and the world, effecting the most dramatic and destructive changes to man since the Garden of Eden. This three-pronged Curse is: the women's rights movement; jazz, rock 'n' roll, and rap; and abortion and euthanasia. The Curse of 1920 chronicles the wholly destructive affects of this Curse, affording conclusive evidence from multiple sources, and critically gets to the very root of our nation's most compelling governmental, social, and religious problems. As this book clearly reveals, the root of this Curse lies in women, the black man, feminized men, the church, and is even rooted 3,726 years before in Abraham. But exposing these problems is only the beginning; for unless we take specific measures to reverse the Curse of 1920, it will continue to destroy us! These measures are clearly laid out here.
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