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The aryl hydrocarbon receptor (AhR) is targeted by ubiquitination for degradation by the proteasome shortly after its activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In silico screening identified p-anilinoaniline (pAA) as a putative inhibitor of an E2 ligase that partners with an E3 ligase implicated in AhR ubiquitination. We investigated whether pAA could modify AhR-dependent activation of its target gene CYP1A1. pAA (1–200 μM) alone did not affect AhR content, or stimulate CYP1A1 mRNA accumulation in human mammary epithelial MCF10A cultures. However, pretreatment with ≥100 μM pAA suppressed TCDD-induced CYP1A1 activation and AhR degradation via its functioning as an AhR antagonist. At a lower concentration (25 μM), pAA cotreatment increased TCDD-induced CYP1A1 mRNA accumulation, without inhibiting AhR turnover or altering CYP1A1 mRNA half-life. Whereas TCDD alone did not affect MCF10A proliferation, 25 μM pAA was cytostatic and induced a G1 arrest that lasted ∼7 h and induced an S phase arrest that peaked 5 to 8 h later. TCDD neither affected MCF10A cell cycle progression nor did it alter pAA effects on the cell cycle. The magnitude of CYP1A1 activation depended upon the time elapsed between pAA pretreatment and TCDD addition. Maximal AhR occupancy of the CYP1A1 promoter and accumulation of CYP1A1 heterogeneous nuclear RNA and mRNA occurred when pAA-pretreated cultures were exposed to TCDD in late G1 and early/mid S phase. TCDD-mediated induction of CYP2S1 was also cell cycle-dependent in MCF10A cultures. Similar studies with HepG2 cultures indicated that the cell cycle dependence of CYP1A1 induction is cell context-dependent.
In many cell types the aryl hydrocarbon receptor (AhR) undergoes proteolysis after the binding of agonist. For example, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding reduces AhR half-life from 28 to ∼3 h in murine hepatoma 1c1c7 cells (Ma and Baldwin, 2000). AhR degradation occurs as a consequence of its becoming polyubiquitinated, which targets it to the proteasome for proteolysis (Pollenz, 2002). Polyubiquitination is mediated by the sequential actions of three interacting but functionally distinct proteins: an E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin ligases (Pickart, 2001). There are approximately 50 known E2s and possibly hundreds of E3 proteins. The E3 proteins are responsible for substrate discrimination and the specificity of ubiquitination. A single E2 is able to associate with different E3s, and an individual E3 may associate with more than one E2 (Hershko and Ciechanover, 1998). Although the identities of the E2 and E3 proteins involved in ligand-induced AhR ubiquitination are not well characterized, some studies have implicated a role for the E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) (Lees et al., 2003; Morales and Perdew, 2007). CHIP contains a U-box domain, and it interacts with heat-shock protein 90 and hsc70 client proteins targeting them for degradation by the ubiquitin proteasome pathway (Connell et al., 2001). One of the E2-conjugating enzymes that partners with CHIP is UbcH5a (Jiang et al., 2001).
p-Anilinoaniline (pAA; also known as p-aminodiphenylamine, N-phenyl-1,4-benzenediamine) is an aromatic amine that is used in hair coloring products. It is also a major metabolite of the azo dye metanil yellow (Srivastava et al., 1982). The latter is extensively used in the textile, paper, lacquer/stain industries (Mittal et al., 2008), and in India as a food-coloring agent (Khanna et al., 1985). As a result of in silico screening, Banerjee (2006) identified pAA (identified as 05RB in the article) as a putative inhibitor of UbcH5a, an E2-conjugating enzyme that partners with the E3 ligase CHIP.
We initiated the current study with the intent of assessing pAA as a possible inhibitor of TCDD-induced AhR ubiquitination in the normal human mammary epithelial cell line MCF10A. We previously reported that TCDD induced AhR degradation and the expression of multiple AhR-responsive phase I and II metabolism-related genes (i.e., CYP1A1, CYP1A2, CYP1B1, and NQO1) in MCF10A cultures (Reiners et al., 1997; Gou et al., 2001; Joiakim et al., 2004). Unfortunately, MCF10A cultures ultimately proved to be an inappropriate model for the testing of our hypothesis because the line did not express UbcH5a. However, we found that pAA mediated a very reproducible time-dependent enhancement of CYP1A1 induction by TCDD that paralleled pAA-induced cell cycle arrest and release. In particular, our data indicate that TCDD-induced AhR occupancy of the CYP1A1 promoter and CYP1A1 transcriptional activation in MCF10A cells are maximal in late G1 and early/mid S phase cells.
Materials and Methods
TCDD was purchased from Midwest Research Institute (Kansas City, MO). TRIzol, trypsin/EDTA, epidermal growth factor, penicillin/streptomycin solution, horse serum, salmon sperm DNA, Taq DNA polymerase, phenol, Random Primers DNA Labeling System, PCRx Enhancer System, and PCRx Amplification mixture were purchased from Invitrogen (Carlsbad, CA). pAA, actinomycin D, dimethyl sulfoxide (DMSO), deoxynucleoside triphosphates, deoxyribonuclease 1, protease inhibitor cocktail, protein A agarose, and ribonuclease A were obtained from Sigma-Aldrich (St. Louis, MO). RNAqueous-4PCR kit was obtained from Ambion Inc. (Austin, TX). TaqMan Reverse Transcription Reagent, TaqMan Gene Expression Assays, and SYBR Green PCR master mix were from Applied Biosystems (Foster City, CA).
Cell Culture.
MCF10A human breast epithelial cells were obtained from the Cell Lines Resource, Karmanos Cancer Institute (Detroit, MI) and maintained as attached cultures in supplemented Dulbecco's modified Eagle medium/Ham's F-12 medium as described by Guo et al. (2001). HepG2 cells were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium supplemented with nonessential amino acids, 100 units/ml penicillin, 100 mg/ml streptomycin (Invitrogen), and 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA). Cultures were maintained at 37°C in a humidified atmosphere consisting of 95% air and 5% CO2. Cells were seeded at densities that assured exponential growth for at least 5 days. Cultures were treated on the second day of plating with either DMSO or different concentrations of pAA dissolved in DMSO. Solvent never exceeded 0.1%. For estimates of cell number and viability, cultures were trypsinized, washed with phosphate-buffered saline (PBS), suspended in PBS containing trypan blue, and counted with a hemocytometer. Viability was scored as the ability to exclude trypan blue.
Cell Cycle Analyses.
The procedure used for the determination of cell cycle phase by fluorescence-activated cell sorting has been described in detail (Reiners et al., 1999).
Western Blot Analyses.
The conditions used for the preparation of cell extracts, separation of proteins on SDS-polyacrylamide gels, and transfer of separated proteins onto nitrocellulose membranes have been described in detail (Guo et al., 2001). Nonspecific antibody binding to transferred proteins was blocked by preincubating membranes in PBS-T (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20) supplemented with 5% nonfat dry milk proteins. After washing with PBS-T, membranes were incubated with the appropriate horseradish peroxidase secondary antibody for 1.5 h at room temperature. Antibody detection was performed with an enhanced chemiluminescence reaction kit (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) and recorded on X-ray film.
RNA Preparation and Northern Blot Analyses.
The conditions used for RNA isolation, resolution of RNAs on agarose/formaldehyde gels, and transfer to nitrocellulose membranes have been described by Reiners et al. (1997). The probes and hybridization conditions used for the detection of human 7S, CYP1A1, and CYP1A2 RNAs have been described in detail (Reiners et al., 1997; Joiakim et al., 2004). Northern blot data were normalized by calculating CYP1A1 or CYP1A2 mRNA to 7S RNA signal strength ratios.
Real-Time Reverse Transcription-Polymerase Chain Reaction of CYP1A1 Heterogeneous Nuclear RNA.
Total RNA was isolated from MCF10A cultures using the RNAquous-4PCR kit according to the supplier's specifications. RNA was treated with DNase 1 according to the instructions provided by the supplier to remove trace amounts of DNA, and it was quantified using a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized from isolated RNA using TaqMan Reverse Transcription Reagents, as described by the supplier. For quantification of CYP1A1 heterogeneous nuclear (hnRNA), PCR amplification used the CYP1A1 forward primer 5′-TTGTGATCCCAGGCTCCAAGA-3′ and the reverse primer 5′-GGAGGCACCAAAATGTTCCTTT-3′. These primers amplify a 120-base pairs (bp) sequence that corresponds to a region spanning the first exon-intron (GenBank accession number AF253322). For glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA quantification, we used the GAPDH forward PCR primer 5′-AGAAAAACCTGCCAAATATGATGAC-3′ and the reverse PCR primer 5′-GCCCAGGATGCCCTTGA-3′. This amplifies a region 849 to 927 bp downstream of the transcription start site. PCR data were analyzed using the comparative cycle threshold (Ct) method. Relative quantification was based on relative expression of CYP1A1 normalized against GAPDH. Relative expression of the target gene was calculated as 2−ΔΔCt, where ΔΔCt was obtained by subtracting ΔCt of untreated cells from the ΔCt of treated cells. The ΔCt was calculated by subtracting the average Ct value for GAPDH from the average Ct value of CYP1A1. All PCRs were performed in triplicate (technical replicates).
Real-Time Reverse Transcription-PCR of CYP1A1 and CYP2S1 mRNAs.
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA) and reverse transcribed using the Omniscript RT kit (QIAGEN) and random primers (Invitrogen) according to the manufacturers' instructions. Human CYP1A1 and CYP2S1 mRNA contents were quantified as described previously (Duniec-Dmuchowski et al., 2007), using TaqMan Gene Expression Assays Hs00153120_m1 and Hs00258076_m1, respectively, in multiplex reactions with TaqMan 18S rRNA Endogenous Control (primer limited) to quantify endogenous 18S rRNA (Applied Biosystems). Cycle threshold values were used to normalize CYP1A1 or CYP2S1 levels to 18S RNA levels as described above.
Chromatin Immunoprecipitation Assay.
Culture medium was adjusted to 1% formaldehyde and incubated for 10 min at room temperature to cross-link protein-DNA complexes. Cultures were subsequently rinsed with ice-cold PBS and incubated for 5 min at room temperature with a 125 mM glycine/PBS solution. Cells were then washed with PBS, covered with PBS supplemented with 1× (v/v) protease inhibitor cocktail (Sigma-Aldrich), and detached by mechanical scraping. The cell suspensions were pelleted by centrifugation, quick frozen, and stored at −80°C until further processing. The cell pellets were subsequently mixed with SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) and sonicated using conditions that yielded DNA lengths of 200 to 1000 bp. The sonicated chromatin was diluted 1:10 with chromatin immunoprecipitation (ChIP) buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.0), and 167 mM NaCl] supplemented with protease inhibitors. Chromatin was precleared with salmon sperm DNA/protein A agarose (60 μl of a 50% slurry), 5 μg of salmon sperm DNA, and 1 μg of rabbit IgG with gentle agitation for 2 h at 4°C. Agarose beads were pelleted by centrifugation at 700g for 1 min. The supernatant fluid was transferred to a new tube, and a sample was put aside for determination of input DNA. One microgram of either AhR antibody or rabbit IgG was added to the remaining supernatant fluid. After overnight incubation at 4°C on a rotating platform, salmon sperm DNA/protein A agarose (40 μl of a 50% slurry) was added, and an additional incubation was performed for 1.5 h at 4°C before pelleting the agarose beads by centrifugation. The agarose beads were sequentially washed for 10 min each in 1 ml of low salt wash buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS], 1 ml of high salt wash buffer [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS], 1 ml of LiCl wash buffer [10 mM Tris-HCl (pH 8.0), 0.25 M LiCl, 1 mM EDTA, 1% IGEPAL, 1% sodium deoxycholate], and 2 × 1 ml of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Protein-DNA complexes were eluted from the beads by incubation with 250 μl of NaHCO3 (0.1 M) and 1% SDS for 30 min. Cross-linking was reversed by incubating the supernatant overnight at 65°C in 200 mM NaCl. DNA was subsequently purified by standard procedures.
AhR-DNA interactions were monitored using forward (5′-ACCCGCCACCCTTCGACAGTTC-3′) and reverse (5′-TGCCCAGGCGTTGCGTGAGAAG-3′) primers to amplify a region from −980 to −1125 of the CYP1A1 promoter (Matthews et al., 2005). This region contains two functional xenobiotic response elements (XREs) and yields a PCR product of 146 bp. The PCR mixture contained input or ChIP-isolated DNA, 1× PCRx amplification buffer, 1× PCRx enhancer solution, 0.2 mM deoxynucleoside triphosphate mix, 2.5 mM MgSO4, 0.4 μM each of forward and reverse primers, and 0.5 U of Taq DNA polymerase. The PCR used the following conditions and cycles: 95°C for 3 min, 35 cycles of 95°C for 45s, 58°C for 45s, and 70°C for 1 min, and then 70°C for 5 min. PCR products were separated on agarose gels and stained with ethidium bromide.
Electrophoretic Mobility Shift Assay.
The conditions used for TCDD-mediated AhR transformation in rat liver extracts, electrophoretic mobility shift assay (EMSA), and the sequence of the double-stranded radiolabeled oligonucleotide containing a consensus XRE have been published previously (Joiakim et al., 2004).
Image and Statistical Analyses.
EMSA, Western blot, and Northern blot films were scanned at 800 to 1000 dpi and saved as .tif images. Band intensities in the images were quantified with ImageJ software (National Institutes of Health, Bethesda, MD). Data were analyzed by one-way analysis of variance followed by Tukey's multiple comparison test, using GraphPad Prism version 4.02 for Windows (GraphPad Software Inc., San Diego, CA). P < 0.05 was considered statistically significant.
Effects of pAA on TCDD Induction of CYP1A1.
Exposure of MCF10A cultures to 10 nM TCDD induced the accumulation of CYP1A1 mRNA, whereas exposure to 1 to 200 μM pAA had no effect (Fig. 1A). However, depending on the concentration used, pAA pretreatment could either suppress or enhance dioxin-induced CYP1A1 mRNA accumulation (Fig. 1, A and B). In particular, pretreatment with concentrations of pAA ≥75 μM suppressed TCDD-induced CYP1A1 mRNA accumulation, whereas, concentrations of pAA ≥10 μM and ≤50 μM enhanced CYP1A1 mRNA accumulation. Kinetic analyses indicated that induced steady-state CYP1A1 mRNA levels were fairly constant after 5 to 18 h of TCDD treatment (Fig. 1C). In contrast, TCDD-induced CYP1A1 mRNA accumulation was notably bell-shaped in pAA-pretreated cultures, with maximal accumulation (e.g., ∼3–4-fold greater than dioxin alone) occurring 7.5 to 12 h after TCDD addition (Fig. 1C).
CYP1A2, like CYP1A1, is transcriptionally activated by TCDD in MCF10A cultures (Joiakim et al., 2004). pAA alone had no effect on CYP1A2 mRNA content in MCF10A cultures (A. Joiakim, unpublished data). However, pretreatment with 25 μM pAA enhanced dioxin-induced CYP1A2 mRNA contents by ∼2-fold (Fig. 1D). Note that maximal CYP1A1 and CYP1A2 mRNA accumulations occurred within a similar time period (compare Fig. 1, C and D).
The accumulation of CYP1A1 mRNA after TCDD treatment could reflect either enhanced CYP1A1 transcription or a stabilization of CYP1A1 mRNA. To examine the former, we made cDNA to total RNA and used PCR to amplify hnRNA sequences that encompassed the exon 1-intron 1 boundary of CYP1A1 (Fig. 2A). Analysis of hnRNA has been used as a surrogate assay for monitoring the transcriptional activation of CYP1A1 (Elferink and Reiners, 1996). Exposure to either DMSO or 25 μM pAA did not alter CYP1A1 hnRNA content over an 8-h time period, whereas TCDD treatment resulted in an ∼20-fold increase (Fig. 2B). Pretreatment with 25 μM pAA, followed by TCDD addition, increased CYP1A1 hnRNA content ∼2-fold above what accumulated after 6 h of just TCDD exposure (Fig. 2C).
To assess the effects of pAA on CYP1A1 mRNA stability, we pretreated cultures with TCDD ± pAA for 8 h before adding 5 μg/ml actinomycin D (ActD), a concentration that completely inhibits CYP1A1 transcription (Chen et al., 1995; Ciolino et al., 1999). Total RNA was subsequently collected at 2-h intervals for measurement of CYP1A1 mRNA (Fig. 3A). Decreases in mRNA content after ActD treatment became very apparent with passing time. Figure 3B depicts CYP1A1 mRNA half-life analyses based on data generated in four independent experiments. pAA pretreatment had no statistically significant effect on the rate of CYP1A1 mRNA turnover (Fig. 3B).
pAA Modulation of TCDD-Induced AhR Degradation.
A substantial reduction in AhR content occurred in MCF10A cultures within 6 h of dioxin addition (Fig. 4A). Pretreatment with pAA suppressed TCDD-induced AhR turnover in a concentration-dependent manner (Fig. 4A). A strong inhibition of TCDD-induced AhR loss was observed in cultures cotreated with 100 μM pAA (p < 0.05 for n = 4 independent experiments; Fig. 4A), whereas 10 μM pAA was without effect (Fig. 4A). Kinetic analyses of intermediate concentrations suggested that 50 μM pAA partially blocked TCDD-induced AhR degradation (p < 0.05 for n = 3 independent experiments; Fig. 4B). Although 25 μM pAA appeared to exhibit a small protective trend, the AhR contents of TCDD and TCDD + pAA-treated cultures were not significantly different (Fig. 4C).
Although the effect of pretreatment with 25 to 50 μM pAA on AhR content was small, we wondered whether it might be sufficient to mediate the enhanced accumulation of CYP1A1 mRNA after TCDD exposure. As an approach to the issue, we intended to knock down the putative E2 target of pAA (i.e., UbcH5a). Although we could easily detect UbcH5a in human embryonic kidney 293 cells by Western blotting, we were unable to detect it in MCF10A cultures (data not presented). Hence, it seemed unlikely that the effects of pAA on AhR content and CYP1A1 induction were related to pAA effects on the E2 UbcH5a.
AhR Agonist and Antagonist Activities of pAA.
The ability of pAA to suppress both TCDD-induced AhR proteolysis and CYP1A1 transcription at 100 μM is consistent with it functioning as an AhR antagonist. We used an EMSA to examine this issue. Incubation of rat liver cytosol with 10 nM TCDD effectively transformed the AhR into a species capable of binding to a radiolabeled oligo containing a xenobiotic-responsive element (Fig. 5A). In contrast, AhR transformation did not occur after incubation of cytosol with 1 to 200 μM pAA (Fig. 5A). However, cotreatment of rat liver cytosol with pAA suppressed the formation of TCDD-induced AhR-DNA complexes, in a concentration-dependent fashion (Fig. 5B). This suppression was observed at concentrations of pAA ≥50 μM. Hence, the EMSA assay suggests that pAA has no AhR agonist activity but can function as an AhR antagonist once a critical concentration is reached.
pAA Effects on CYP1A1 and CYP2S1 Induction Are Cell Cycle-Dependent.
We previously reported that pAA is cytostatic to MCF10A cultures in a concentration-dependent fashion over a range of 10 to 50 μΜ and causes an almost complete suppression of proliferation at 50 μM (Elliott and Reiners, 2008). Cell cycle analyses indicated that 25 μM pAA induced simultaneous G1 and S phase blocks in MCF10A cultures (Fig. 6A). During the first 7 h of pAA treatment, the proportion of cells in S phase held fairly constant, whereas the proportion of G1 cells increased due to the progression of G2/M cells. Thereafter, G1 cells began to transition into S phase where they accumulated for the next 8 h. Arrested S phase cells began to transition into G2/M ∼15 h after pAA addition (Fig. 6A). Notably, the onset of G1 cells transitioning into S phase, and the period of S phase cell accumulation, roughly correlate with when pAA cotreatment enhanced TCDD activation of CYP1A1 (compare Figs. 1C and 6A). This temporal relationship is significant because we previously reported that CYP1A1 induction may be cell cycle-regulated (Santini et al., 2001).
To examine the relationship between AhR function and cell cycle phase in detail, we quantified TCDD-mediated transcriptional activation of CYP1A1 at different times after pAA treatment. Before initiating this study, we determined whether TCDD had any effects on MCF10A cell cycle progression. A concentration of TCDD sufficient to activate CYP1A1 transcription neither altered cell cycle phase distributions over a 24-h treatment period when used singularly (Supplemental Fig. 1) nor altered the development and resolution of pAA-induced G1 and S phase arrest. The latter studies were performed in two ways. In one protocol, TCDD was added 1.5 h after pAA addition, and cell cycle analyses were performed throughout a 24-h period (Supplemental Fig. 1). In the second protocol, we treated cultures with TCDD at various times after pAA addition and harvested cultures 3 h after dioxin addition (Fig. 6B). This latter protocol facilitated very detailed analyses of TCDD effects on specific phases of the cell cycle. Overall, TCDD had no detectable effects on MCF10A cell cycle progression.
Cultures treated similarly to those in Fig. 6B were analyzed for CYP1A1 mRNA (Fig. 6C) and hnRNA (Fig. 6D) after the TCDD addition. In these studies, we harvested cultures within 3 h of TCDD addition to restrict the effects of dioxin to defined stages of the cell cycle. The length of time elapsed between pAA and dioxin additions influenced the extent to which CYP1A1 mRNA and hnRNA accumulated. CYP1A1 mRNA contents were maximally increased when dioxin was added to cultures in late G1 or when transitioning from G1 to S (i.e., time of harvest = 13 h), and in early/middle S phase (i.e., time of harvest = 15 h; Fig. 6C). Late S phase cells (i.e., time of harvest = 18 h) exhibited less CYP1A1 mRNA, and CYP1A1 mRNA content continued to drop as S phase cells transitioned into G2/M (Fig. 6C). The kinetics of CYP1A1 hnRNA accumulation and decline paralleled CYP1A1 mRNA content (compare Fig. 6, C and D). ChIP assays with AhR antibodies did not detect AhR occupancy of the CYP1A1 promoter in nontreated cultures, or cultures treated with only pAA for 1.5, 11, or 18 h (Fig. 6E). In pAA-pretreated cultures, AhR occupancy of the CYP1A1 promoter after TCDD addition qualitatively correlated with CYP1A1 hnRNA levels (compare Fig. 6, D and E). In particular, AhR occupancy of the CYP1A1 promoter was markedly less in cultures treated with TCDD 1.5 h after pAA addition, relative to cultures treated with TCDD 11 and 18 h after pAA addition.
CYP2S1 is transcriptionally activated by TCDD through an AhR-dependent mechanism (Saarikoski et al., 2005; Rivera et al., 2007). Thomas et al. (2006) previously reported that CYP2S1 mRNA accumulates in MCF10A cultures after the addition of dioxin. In asynchronous MCF10A cultures, we observed no accumulation of CYP2S1 mRNA within 3 h of TCDD addition (Fig. 7A). However, this time period was sufficient for CYP2S1 mRNA accumulation if the cultures were pretreated with pAA (Fig. 7A). Like CYP1A1, optimal accumulations of CYP2S1 mRNA occurred when cultures were treated with TCDD in late G1, during the G1/S transition and early/middle S phase (5–12 h after pAA addition; compare Fig. 7, A and B).
pAA Effects on CYP1A1 Induction in HepG2 Cultures.
We examined the effects of pAA in HepG2 cells to determine whether its effects on CYP1A1 induction were cell-type specific. pAA suppressed the proliferation of HepG2 in a concentration-dependent fashion (Fig. 8A) that mirrored the effects observed in MCF10A cells (Elliott and Reiners, 2008). Concentrations of pAA ≥25 μM were very cytostatic, with antiproliferative effects that persisted for at least 72 h. The cytostatic effects of 25 μM pAA reflected the induction of an early G1 and S phase arrest, with arrested G1 phase cells transitioning into S phase 8 to 16 h after pAA addition (Fig. 8B). Unlike MCF10A cultures, pAA-treated cultures remained arrested in S phase 28 h after treatment (Fig. 8B). Treatment of asynchronous HepG2 cultures with TCDD increased CYP1A1 mRNA content 11- to 19-fold within 3 h of dioxin addition (Fig. 8C). Pretreatment with pAA increased TCDD-induced CYP1A1 mRNA accumulation above that achieved with just TCDD (Fig. 8C). However, unlike what we observed with MCF10A cultures, the effects of pAA pretreatment on TCDD-mediated CYP1A1 induction were not cell cycle-dependent. Instead, pAA enhanced CYP1A1 mRNA accumulation irrespective of the cell cycle stage at which TCDD was added (Fig. 8C).
Although CYP2S1 mRNA was detected in the nontreated asynchronous HepG2 cultures reported in Fig. 8, we observed no induction of CYP2S1 in these cultures after a 3-h treatment with TCDD, or combined pAA and TCDD cotreatment (J. J. Reiners and Z. Duniec-Dmuchowski, unpublished data).
We initiated the current study to determine whether pAA was a modulator of agonist-activated AhR degradation. Unexpectedly, the culture model we used proved to be inappropriate because MCF10A cells failed to express the E2 ligase (i.e., UbcH5a) putatively targeted by pAA. Nevertheless, cotreatment with ≥50 μM pAA suppressed TCDD-induced AhR degradation in a concentration-dependent manner. Based on EMSA results, this activity most likely reflects the ability of pAA to function as an AhR antagonist and suppressor of AhR activation at such concentrations.
A second pAA-related activity became apparent at concentrations that ranged from 10 to 50 μM. In particular, treatment of MCF10A cultures with these concentrations before TCDD addition elevated CYP1A1 mRNA contents above that occurring with dioxin alone. We previously reported that this concentration range of pAA induces a graded cytostatic response in MCF10A cultures (Elliott and Reiners, 2008). The studies reported in Fig. 6A indicate that 25 μM pAA rapidly initiated simultaneous G1 and S phase arrest in MCF10A cultures. Arrested G1 cells begin to transition into S phase ∼7 to 10 h after pAA treatment and continued to accumulate in S phase for at least an additional 8 h. Thereafter, S phase cells transitioned into G2. By analyzing the effects of adding TCDD at different times after pAA treatment, we were able to assess the responsiveness of CYP1A1 in highly enriched phases of the cell cycle. The studies depicted in Figs. 6, B to E, indicate that dioxin induction of AhR occupancy of the CYP1A1 promoter and CYP1A1 transcription are optimal in late G1 MCF10A cultures, and as cells transition from G1 into S phase, and during early/middle S phase. This cell cycle phase dependence provides an explanation for the bell-shaped kinetics of CYP1A1 mRNA accumulation observed in Fig. 1C. In particular, the times at which pAA potentiated TCDD-induced CYP1A1 mRNA accumulation in MCF10A cultures corresponded to the period at which cultures contained the highest percentage of late stage G1 and S phase cells.
Our observation of a cell cycle dependence for AhR agonist induction of CYP1A1 is consistent with two other independent studies. In particular, we previously used centrifugal elutriation of asynchronous cycling U937 cell cultures to isolate populations in defined stages of the cell cycle (Santini et al., 2001). Subsequent treatment of these populations with TCDD showed a gradient of CYP1A1 mRNA accumulation, with maximal accumulation occurring in late G1 and early S phase cells. Jiao et al. (2007) used several approaches to generate MCF-7 cultures enriched in Go/G1, S, or G2/M phase cells. Maximal accumulation of CYP1A1 mRNA occurred in S phase-enriched cultures after short-term exposure to the AhR agonist benzo[a]pyrene. In addition to CYP1A1, the ligand-activated AhR regulates the transcription of numerous genes that have XREs in their promoters (Sun et al., 2004). In the current study, we observed that the induction of CYP2S1 by TCDD in MCF10A cultures was also cell cycle-dependent. Likewise, Jiao et al. (2007) reported maximal accumulations of CYP1A2 and CYP1B1 mRNA in S phase MCF-7 cultures after benzo[a]pyrene treatment. Hence, agonist induction of multiple AhR-responsive genes appears to be cell cycle-dependent in MCF10A and MCF-7 cultures. However, given that similar effects were not observed in HepG2 cultures, it appears that the cell cycle dependence of CYP1A1 induction may be cell-type specific. We do not know the basis for this cell context dependence, but it also extends to other aspects of CYP1A1 regulation in the three cell lines. Whereas pretreatment of MCF10A (Guo et al., 2001) and MCF-7 (Moore et al., 1993) cultures with 12-O-tetradecanoylphorbol-13-acetate initially suppresses TCDD-mediated induction of CYP1A1, a similar pretreatment enhances CYP1A1 transcription in HepG2 cultures (Chen and Tukey, 1996; Morgan et al., 1998).
Two timekeepers appear to regulate TCDD-mediated transcriptional activation of CYP1A1. The first entails the cell cycle and the second circadian rhythms. Regarding the latter, TCDD-mediated induction of CYP1A1 in the liver and mammary glands of mice is 23- to 40-fold greater at night than during daytime (Qu et al., 2007, 2010). This diurnal rhythm in CYP1A1 responsiveness to TCDD is inversely related to the expression of the protein Period 1 (Metz et al., 2006: Qu et al., 2010), a key protein regulator of the circadian clock (Reppert and Weaver, 2002). The occurrence of this cyclical rhythm for CYP1A1 responsiveness in a quiescent tissue (such as the liver) emphasizes the independence of the two timekeepers in some situations. However, several studies have demonstrated that circadian rhythms gate the expression/activation of cell cycle-related genes in proliferating tissues (Matsuo et al., 2003; Yang et al., 2009). For example, in normal human oral mucosa the maximal accumulation of Period 1 occurs in G1 before the expression of cyclin E, and the onset of S phase occurs after appreciable Period 1 loss (Bjarnason et al., 2001). A similar pattern is also observed in human skin (Bjarnason et al., 2001). Although speculative, it is conceivable that cell cycle- and circadian rhythm-dependent regulation of TCDD-induced CYP1A1 transcription may be linked in vivo, in some proliferating tissues.
Protocols that facilitate manipulation of cell density indicate that cell-cell contact also influences AhR activation and the transcription of several AhR-responsive genes (Cho et al., 2004; Ikuta et al., 2004; and references within). In particular, the expression of both CYP1A1 and CYP1B1 in cultured adherent cells, in the absence of any exogenous AhR ligand, is inversely related to cell density (Cho et al., 2004; Ikuta et al., 2004). In some instances, the level of CYP1A1 or CYP1B1 expression induced by culturing at low density or suspension culturing approximate what is achieved after treatment of near confluent, adherent cultures with AhR agonists (Cho et al., 2004; Ikuta et al., 2004; and references within). Studies with cultured keratinocytes (Ikuta et al., 2004) and fibroblasts (Cho et al., 2004) indicate that cell contact/density-mediated regulation of the AhR and CYP1A1/CYP1B1 is cell cycle phase-independent.
Agonist activation of the AhR can have diverse effects on the cell cycle. For example, contact inhibited, quiescent rat liver epithelial WB-F344 cultures reenter the cell cycle and proliferate after exposure to TCDD and several polychlorinated biphenyl and polycyclic aromatic hydrocarbon AhR agonists (Chramostová et al., 2004; Vondrácek et al., 2005). In contrast, TCDD induces a profound G1 or S phase arrest in a variety of cultured cell types (reviewed by Marlowe and Puga, 2005; Puga et al., 2009; Barhoover et al., 2010). In many cases, this arrest has been shown to be AhR-dependent (Marlowe and Puga, 2005). The MCF10A cells used in the current study appear to represent a third case. In particular, concentrations of dioxin sufficient to induce CYP1A1 transcription had no notable effect on MCF10A cell cycle progression. Taken together, these data indicate that dioxin effects on the cell cycle are cell context-dependent. However, it should be noted that even for a specific cell type, additional factors radically influence the effects of dioxin on the cell cycle. For example, whereas dioxin suppresses compensatory liver regeneration induced by partial hepatectomy, it enhances hepatocyte proliferation induced by the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxyl] benzene (Mitchell et al., 2010).
In summary, the current study demonstrates that dioxin-mediated transcriptional activation of CYP1A1 is optimal in late G1 and early/middle S phase MCF10A cells. Similar observations have been made in other cell lines (Santini et al., 2001; Jiao et al., 2007). These findings raise the broader issue of whether other genes associated with metabolic transformation are cell cycle-regulated. A recent report by Sugatani et al. (2010) demonstrated that UGT1A1 and CYP2B6 content in HepG2 cells inversely correlated with the activation state of cyclin-dependent kinase 2, a cell cycle-regulated protein. Knowledge of the cell cycle dependence of drug-metabolizing enzymes may be useful in the design of combinational therapeutic protocols in which one of the therapeutics is a cell cycle modulator.
Authorship Contributions
Participated in research design: Elliott, Joiakim, Kocarek, and Reiners Jr.
Conducted experiments: Elliott, Joiakim, Mathieu, Duniec-Dmuchowski, and Reiners Jr.
Contributed new reagents or analytic tools: Elliott, Joiakim, and Kocarek.
Performed data analysis: Elliott, Joiakim, Mathieu, Duniec-Dmuchowski, and Reiners Jr.
Wrote or contributed to the writing of the manuscript: Elliott and Reiners Jr.
This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL050710].
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.
- AhR
- aryl hydrocarbon receptor
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- ChIP
- chromatin immunoprecipitation
- carboxyl terminus of Hsc70-interacting protein
- pAA
- p-anilinoaniline
- dimethyl sulfoxide
- phosphate-buffered saline
- reverse transcription
- polymerase chain reaction
- hnRNA
- heterogeneous nuclear RNA
- bp
- base pair
- glyceraldehyde 3-phosphate dehydrogenase
- Ct
- comparative cycle threshold
- xenobiotic response element
- electrophoretic mobility shift assay
- ActD
- actinomycin D.
- Received August 26, 2011.
- Accepted February 16, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics


text:
Kansas is dotted with eight nearly new kidney dialysis clinics that sit mostly unused because the state is two-plus years behind on inspecting and certifying them.
Another four clinics want to expand services but can't as they await state inspections.
The delays are forcing Kansans like Marsha Voss of Olathe to travel past new facilities and get treatment for life-threatening kidney diseases at older facilities farther away.
Voss, who is legally blind and uses a motorized wheelchair, has to go to a treatment center in Lenexa three times a week instead of a new one in her neighborhood that would save her 1 1/2 hours each trip.
"The way it is now I leave at 10:30 (a.m.) and I don't get home until 5:30 (p.m.) and that's mainly because of the transportation," Voss said.
Federal funding for inspectors has been cut in years past and Kansas has not made up the difference. That's led to turnover in the health facility inspection force and a backlog for new dialysis centers which, by federal law, take a backseat to inspections of other types of facilities and existing dialysis centers with problems.
Kansas is not the only state with long delays. But some others have dipped into their own budgets to recruit and retain enough inspectors to keep from falling behind.
As of last week, the Missouri Department of Health and Senior Services had no dialysis centers awaiting their initial surveys.
The federal Centers for Medicare and Medicaid Services contracts with health departments in each state to do the inspections. Without them, the dialysis centers can't bill Medicare, which covers 85 percent of Americans in kidney failure.
Those are the patients who need new dialysis centers the most, said Syd Stevens, the administrator of Midwest Nephrology Consultants.
"It's really not fair that they have to wait to get in," Stevens said, "and it's really not fair if you're dialyzing farther away from you and this unit is ready and waiting three blocks down (the) street."
There's a specific provision within Medicare that extends eligibility to people on dialysis.
Even those who can continue working while their kidneys fail only use their employer-based coverage for their first 30 months of dialysis. After that, Medicare automatically becomes the primary coverage and the employer plan becomes secondary.
Stevens said dialysis centers roll out the red carpet for patients still under employer plans because commercial insurance pays more. Even at the certified facilities, Medicare patients are often relegated to less convenient time slots.
"Basically, as a Medicare patient you're left out … and those chairs can be coveted," Stevens said.
The clinic near Voss has been waiting for Medicare certification since January 2016. It has the capacity to see about 70 patients. Without certification, it sees four.
Facilities waiting across Kansas
There are two clinics awaiting certification in Topeka, including one that's been waiting since October 2015. The others are in Olathe, Garden City, Pittsburg, Manhattan, Lenexa and Kansas City.
Clinics waiting for the go-ahead to expand services are in Junction City, Manhattan, Chanute and Wichita.
"We are seeing delays across the board," said Kurt Koptish, a division vice president who oversees all the DaVita dialysis centers in the Kansas City area.
Matt Keith, a spokesman for KDHE, said the problem lies with CMS regulations that prioritize other inspections unless there are special circumstances, like a lack of other dialysis options anywhere nearby.
"Dialysis centers may petition CMS to have their initial surveys moved into a higher priority tier if they can make a clear and convincing case of need in their area to CMS," Keith said via email. "If CMS agrees with the petitioning facility, it will give KDHE clearance to move an initial survey of a dialysis center onto the schedule."
Koptish said DaVita also has long certification delays in Iowa, and a nephrology news website published a story last year citing two-year waits for initial inspections in New Mexico and waits of as long as five years in Oklahoma for facilities looking to expand services.
But Missouri provides a stark contrast.
"They typically survey within about 90 days," Koptish said.
Kansas Sen. Vicki Schmidt, a Republican from Topeka, said turnover within the state inspection force has been a problem. Five of the eight positions changed hands last year.
Schmidt said legislators should take a look at market pay rates for inspectors to make sure Kansas is competitive, because it's a waste for the state to train them only to see them go elsewhere.
"That kind of turnover has crippled this program," Schmidt said.
Jenkins proposes privatization option
The Lenexa facility is owned by Fresenius Medical Care, a German-based company. It and Colorado-based DaVita are the corporate giants of the kidney dialysis industry, with about 70 percent of the market share and almost 4,000 locations across the United States.
Their financial fortunes have grown as chronic conditions like diabetes and high blood pressure have pushed more and more Americans toward kidney failure. As of 2013 there were almost 500,000 Americans on dialysis, arranging their lives around thrice-weekly treatments, some waiting years for transplants that may never come.
Cameron Lynch, Fresenius' senior vice president of government affairs, said the Lenexa location has been awaiting an initial inspection since it opened nine months ago. He said it's treating patients on private insurance, but "currently serves less than 10 percent of the patient population it could serve if it had CMS certification."
Koptish said the four patients at the DaVita facility near Voss are also all on commercial insurance. A waiting list of Medicare patients has formed there, as slots at another DaVita location on the southwest edge of Olathe become increasingly scarce.
"It's running at 95 percent capacity, so it's pretty much full," Koptish said. "Right now there is a pretty strong need in the community."
The Kansas City, Kan., center awaiting inspection, which is also owned by DaVita, is at the bottom of KDHE's priority list. Koptish said that means it will probably be more than two years before it's certified.
Fresenius and DaVita are supporting a bill introduced by U.S. Rep. Lynn Jenkins, a Republican from Topeka, that would allow the federal government to contract with private sector companies to do the dialysis center inspections.
But even if the bill passes, the regulation-writing and contracting process would take months, so there's no quick fix for Kansans like Voss.
If the DaVita facility near her home were CMS certified, it would be within range of her assisted living facility's shuttle bus. But instead Voss rolled outside Wednesday to wait for the JO — Johnson County's bus service — to take her nine miles north to a DaVita facility in Lenexa.
Voss has been on dialysis for 3 1/2 years. She said she supports the Jenkins bill, but she'd also like to see Kansas invest more in inspectors.
"I'm not a rocket scientist but if it takes two weeks (to get certified) in Missouri and two years in Kansas you know it's got to do something with the money in Kansas," Voss said. "It really makes us look like we don't know what we're doing."
Andy Marso: 816-234-4055, @andymarso

subset: commoncrawl


text:
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Such input may be more tightly constrained in future studies using rare earth element and radiogenic isotopic data.
Future comminution studies would benefit from further consideration of the importance of 1) leaching lost 234U from source rock and bulk sediment samples, 2) wind deposition of fine-grained material and 3) the appropriateness and robustness of sample pre-treatment procedures.
Your access to the NCBI website at gov has been temporarily blocked due to a possible misuse/abuse situation involving your site.
This is not an indication of a security issue such as a virus or attack.
U-Th ages obtained by mass spectrometry in corals from Barbados: sea level during the past 130,000 years.
Calibration of the 14C timescale over the past 30,000 years using mass spectrometric U-Th ages from Barbados corals.
This result warrants concern for future studies using the comminution approach for which a secular equilibrium source (234U/238U) activity ratio is assumed.
Significant input of aeolian material may modify the measured (234U/238U) activity ratios.
Typically closed systems with high 234/238 ratios and little initial Th-230, these samples have yielded some of the most precise and accurate ages of the climate system over the last half million years.U at the time of sample formation must be known or calculated. With time, Thorium 230 accumulates in the sample through radiometric decay. The largest radioactive disequilibria are always found in the youngest materials.Over time, this signature goes away, eventually relaxing to a condition wherein the disequilibria are no longer detectable.
For instance, when crystals form in a magma, Th, U and Ra in the magma enter the different materials in different proportions, producing radioactive disequilibrium.


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The Period Following the End of the AE
So I've read and heard numerous times how after WMX7, when Austin turned heel, that the numbers in the ratings collapsed and house show numbers also dwindled.
I know from personal experience that the interest in wrestling was waning in the months leading up to WMX7. From a personal POV it seemed like there was only 2 or 3 of us at school who still watched it by early 2001, compared to a year previous when half of my year seemed to be watching the product.
However was it all over Austin turning heel? Or do people think that the cycle was simply over and people lost interest in the WWF following WMX7? Would it have happened even if Austin had stayed as a face? Was the boom period over regardless?
I think the death of WCW had a lot to do with the declining interest in professional wrestling by the early 2000's. I remember during the height of the Wars it was incredible to go back and forth and see which show was going to go over the top and pull out the stops to try and top the other show. When the NWO and Stone Cold/Rock was hot there was no time period hotter so naturally there was no where to go but down. I compare this time period to the period just after the Hulkamania era of the 80's where a lot of the superstars from era either left or transitioned.
But my main point is that WCW was completely unwatchable during the Russo era and turned ALOT of fans off the product. It was a no contest between RAW and Nitro. I don't think it all over Austin turning heel but a lot of storyline continuity was lost during this time period as well. Once creative went under the control of Stephanie McMahon all was lost. It was just absurd storyline and after absurd storyline
A few things came together.
One big issue was the end of the Monday Night Wars. PArt of the drama of pro wrestling in the 1990s was the WCW-WWF rivalry. WCW rose up with the NWO. WWF made a comeback with DX and Steve Austin. WCW has Goldberg. WWF has The Rock. WCW is reading out the taped RAW results. "Mick Foley Put My Ass In This Seat" beats live Nitro in the ratings.
WCW 2000 was a complete mess, but they could have come back and done something. But in 2001, WCW was gone.
If the Monday Night Wars were a story you were telling, that's the end of the story. Vince McMahon wins, Ted Turner Eric Bischoff and Vince Russo lose. The Invasion angle gets a lot of criticism, but the reality is that WCW as it was at the end just wasn't a threat. There's a reason that the only time the Invasion angle showed a pulse was when they added ECW and Steve Austin.
Sure, there were some guys on the shelf. But the Invasion wouldn't have been a success instead of a failure if Booker T and DDP had Scott Steiner and Jeff JArrett backing them up, much less Hugh Morrus and Lance Storm. (Although Lance Storm Canadianizing all of the WCW midcard titles was fun stuff).
"Who will be at Wrestlemania in 2014?" That question should drive everything WWE does.
"Will there be a Bound For Glory in 2011, post-Hogan?" That is my question about TNA.
January 4, 2010. Welcome to the TNApocalypse.
That also depends on when you consider the Attitude Era to have ended. A lot of fans consider WM17 the end of the Attitude Era, but there are just as many who feel WM19 was the end.
But I think Austin's heel turn could have been the beginning of something amazing, but WWE chickened out and turned him face again almost immediately. So we'll never know where that could have gone. Personally I feel Austin works better as a heel, and I was REALLY excited to see what they were going to do with him. Then he was face again. A few months later he turned heel again during the Invasion, but it was diluted because we'd literally just seen it. And then BOOM, he's face again. A total waste.
Attended SummerSlam 2007, Hell in a Cell 2009, No Way Out 2012, WrestleMania 29, Extreme Rules 2014, Royal Rumble 2015 (front row), and WWE Live from MSG.
I don't think Austin's heel turn had anything to do with the decline. Whether people want to admit it or not by that point The Rock was the ratings draw. His segments routinely outdrew Austins, so you take him away from the equation and that hurts things(he was gone for 6 months then). Plus on top of that HHH got hurt right after it so he was gone too.
And finally on top of both of them being gone, the biggest draws in WCW(Hogan, Flair, Sting, Goldberg, Hogan, Hall, Nash, Hart) were all gone, so any audience you got that would flip from WCW to watch WWE would tune out if they watched for any of those guys.
So the bottom line is between all the top guys from WCW being off TV and the two biggest non Austin attractions in WWE being on the sideline led to the decline in ratings. Not Austin turning heel.
I consider 19 the end of the attitude era if for no other reason that 18 gave us the ultimate attitude era match.
The rock vs hogan has to be considered attitude era, as does jericho /hunter.
the Five Moves of doom?
I think it was a combination of a few things like the end of the competition like WCW and ECW, Competition is good for business with no other mainstream companies all we had was WWE, Also If a Raw segment was dull you could switch over to Nitro,
This was also around the time of the anticipated WCW invasion storyline which I had high hopes for but just fizzled out dissapointingly and the Austin heel turn you mentioned at that time was in my opinion a horrible idea and Austin and Rocks full time careers were also winding down which couldn't have helped.
Looking back they did do a few good things but I just don't have the same fond memories for that era as I do for other eras even a couple of years later was an improvement
I have to say it was WCW going under that was the beginning of the end for the Attitude Era. The whole genesis from that period was that WCW was must see TV compared to RAW between '96 and '98. Stone Cold was on the rise in '96, and Vince wanted to make sure WWF stayed relevant. Keep in mind that you had ECW with the edgier product during the 90's, though they posed no real threat to WWF and WCW. Once there was no more competition, there wasn't much of a need for the shock value and T & A, and it gradually started to fade away.
WCW dying was one factor. But another factor was that Attitude had run it's course. 2002 saw "Billy and Chuck's wedding", Hot Lesbian Action, Katie Vick, and, in upstart TNA, masturbating midgets in the other semi-major US promotion.
I'm not saying HLA was as bad as the other three, but people forget there were strong reasons at the time that the sponsors pushed for the PG Era.
"Who will be at Wrestlemania in 2014?" That question should drive everything WWE does.
"Will there be a Bound For Glory in 2011, post-Hogan?" That is my question about TNA.
January 4, 2010. Welcome to the TNApocalypse.
That and things were not as interesting after the dream match of hogan/rock.
We had the wcw imports finally showing up as stars and not jobbers, and it was a weird year for headliners.
Hogan should not have been given a nostologia run with the title...just like taker should be nowhere near one today.
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text:
I have already recorded
What can be written,
What I am unable
To write remains hidden,
I have already said
What can be spoken,
What I am unable
To say remains silent.
In my love's smile,
And in her eyes,
Where music is played
But cannot be heard,
And a story is told,
Though not a
Word is uttered.
Do tell me, o love,
What my heart
Is saying to you.
For it refuses to
Reveal to me
The full extent of
Its plans.
THE CLIENT. "Listen and see with your heart.", my client earnestly tells me while gentling tapping my chest. I smiled and nodded. "I am advising you this because I treat you like my own son." He continued, then touching on my divorce and plans for the future. "Just because you have one misadventure, it doesn't mean that it is suppose to be for life... But before you proceed with any plan, I only ask you to do one thing. Please pray Istikharah! (Istikharah is a short, simple but important supererogatory prayers asking Divine guidance on specific issues - like changing jobs, moving house, choosing schools, migrating and of course, getting married) That way...," my client concluded, "God will give you guidance to make the right decision. Because you asked Him! He won't ignore you!"
THE HEART. Actually, I do try to listen to my heart. It is just that sometimes this life can be so darn noisy and distracting that I cannot hear my heart over the noise and static of this world. But once in a while, when everything FEELS just so, I do hear my heart, and this is what my heart is constantly saying, "Alhamdulillah, Alhamdulillah, Alhamdulillah..." Praise be to God, Praise be to God, Praise be to God... I cannot speak nor write this with total sincerity, but my heart recites this all the time... it is only I that am often deaf to my own heart. Silly and absurd but sadly true.
May you be granted the unimaginable joy and happiness to hear your heart, sunshine. There are secrets to be found out if you listen attentive enough.
Pax Taufiqa.
Hate has no place in Islam
Love will show the Way
Footnote: Cartoon was published in another earlier posting.


text:
It all started in the mid 80s ... an aluminum chassis, enameled wire, a lamp, some capacitors ... and construction basis of a draft from Italy begins .... some 24 hour later the manufacturing goes into operation .... the arrangements made .... and .... "WE ARE ON THE AIR"...!!!
Endless sleepless nights, was sitting in front of the lamps like love and to dawdle, to the smell, to tease them. I crush the variable capacitors, coils, all teasing .... Dont count the electric fuses... Let the securities then were burning at home. Truly endless hours in front of the microphone and telephone dedications, in a small area surrounded by vinyl records.
Truly endless hours of travel and the magic of FM, but really endless and times and adventures (and also the chase from the police authorities)!
Was really Madness, to do talks in the air until the morning to have ups and downs countless times all kinds of antennas from a simple dipol, 5/8, Ground Plane, Yagi, Collinear, what every you can think ... i spent "a tone" of money for transmitters, lamps, bases, and this dishonest varicap continue burned... emissions to pleasure both personal and my listeners, without any commercial exploitation, and music depending on the mood of the moment and not according to the "paid list" of the record companies.
Since that time .... madness dies not my friends ... and now vacant due to lack of frequency "welcoming band of FM" can I start again, "as ham pirate radio" on the web ... Although never digest condition "ham" always prefer and I prefer ... "FM radio lover."
As our old FM station, so our website will remain "amateur" for the whole world...
Always yours
"Dimitris - Studio 4.20"


text:
some canvases were used to illustrate other steps like how to glue down the image seamlessly. and of course how to paint so that the images looks "married" to the canvas. i love these little canvases, i keep them so that i don't have to start from scratch when i have a time crunch, when a project is due for publishing or entering a show, etc. i love them, love them.
but i have too many - i keep tripping over them - so i am selling them 2 to a pack for $6.50 plus s & h. or 4/pack for $12.00. their sizes vary from as big as 9" x 12" - to as small as 4" x 4".
use them to plunk down your project straight on top (like i do) or whatever you want. take the color that's on the canvas already as a clue as to what color project to adhere on top.
they are great for the artist on the go. (like me). these packs are a grab bag because i don't have time to sort unique orders.
and feel good, you are helping me - this is all part of a BIG effort to clear out my studio space so that i can continue creating! thanks for helping!!!!
get them at my etsy shop "pimpmyart" www.pimpmyart.etsy.com - they are a hot item! or email me at email@example.com


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Istanbul (AsiaNews / Agencies) - The Syrian Christian community in Turkey has received approval for the construction of its first church in turkish territory. The building should be built in the district of Yesilkoy, Istanbul, and should meet the needs of a community of about 17 thousand Syrians living in the metropolis. After years of discussion and caveats, the approval came from the Turkish Prime Minister and President. The leader of the Syriac community, Kenan Altınışık said that work will begin as soon as the location has been chosen.
"Half of our community live in the neighborhood of Yeşilköy, or the surrounding area. We rent churches for Sunday celebrations, but we can begin to celebrate only at 11.30, while, according to our tradition, the ceremony should end by 10.30 am, "says Kenan Altınışık.
The land for the construction of the church will be assigned to the ancient Christian community by the Istanbul Metropolitan Municipality, construction costs will be covered by the Syrians. A City Hall official said that they are already looking for a suitable land available for building the new church. The building will be built in a style with clear traces of the ancient Syriac culture. Contacts are ongoing between the community and officials to find a location that is satisfactory. In addition to the church a community center will also be built, the title given to the new place of worship will be "Church of Mother Mary ". The Syrians come mainly from the southeast of the country. Many of them migrated to Istanbul and abroad in the mid-80s, because of political instability in the region at the time.
- A low-budget film on the crucial role of fathers debuted as the number one new movie during its opening weekend in theaters across the U.S.
"There is an opportunity for fathers to step up in our culture and take more of a spiritual role and to affirm and love their kids and to prepare them for what should be a Godly legacy among families," Alex Kendrick, actor and director for "Courageous," told CNA on Oct. 6
"We see too many men accepting a 'good enough' mentality when their role is crucial to helping each child realize that God loves them."
"Courageous," has now ranked as the number four movie in the country, despite opening against six other movies with almost three times as many screens. The film opened on Sept. 30 to over 1,100 theaters across the U.S.
Kendrick, who works with the Sherwood Pictures production company that also produced the 2008 hit "Fireproof," explained that the film follows the lives of four police officers who grapple with their identity as men, fathers and societal leaders.
"We saw a correlation with the motto of law enforcement, 'to serve and protect,' with that of fathers and many ways he serves the family as spiritual leader protector to provide for them," Kendrick said.
"When a father is engaged and loving his children the way he should it's easier for them to believe that God loves them," he added, "if they're not engaged, it's more difficult for them to believe that God loves them."
"The father's role is crucial in that regard and we wanted to remind men of that and in a larger context, remind parents of their role to love and nurture their children."
On what served as the inspiration for the film, Kendrick said that between movies, the company goes through what they call a "season of prayer" to discern what their next step should be.
"We've learned that the more we seek God, the more we seek his favor, the more he tends to direct and bless us."
The group then "delved into the Scriptures and asked 'what does God say about fatherhood?' and then pulled those elements out and formed our plot around that."
Kendrick said that the movie is unique in that it was made in Albany, Ga. "(Y)ou can't get any further outside Hollywood," he laughed, adding that the actors were also selected based on their embodiment of the film's message.
"We look for people who resonate with the story, the purpose behind the movie," he said. "When the movie's over they don't look at it as a 'gig' or as just a job but they expect it to change culture and glorify God."
"Those are the kind of people we look for and we're happy to have found them. Who you see on screen in real life all believe what this movie is about and are speaking for it."
Kendrick observed that the success of the film can be traced to the hunger many Americans have to see basic virtues portrayed in movies – a rarity in today's cinematic culture.
"I think they respect the values presented on screen," he said. "For many, they want these values in their own life."
"It's really going after that part of us that wants that higher calling, that wants that more noble life and noble standard and a life that honors God."
With a modest budget of less than $2 million, and a cast of "no-name" actors, Sherwood Pictures has been thrilled with the unexpected response to the movie.
"We take joy in doing something that requires faith and gives God the opportunity to do only what he can do," Kendrick said. "We make the movies but God changes the heart."
He added that the personal feedback the company has gotten from moviegoers has been astounding.
"We're hearing so many testimonies from people all over the U.S. and Canada—hundreds and hundreds of emails and Facebook notes and things like that, even phone calls" from people relaying how touched they were by the film.http://www.catholicnewsagency.com/news/movie-on-importance-of-fatherhood-wows-at-box-office/
"It's been very, very exciting to see the response to this movie."
Anchor House director, Keith Fernett, says the charity decided to put itself under the microscope in the lead up to its 17 October £9.3 million appeal launch to prove to its supporters how much of a difference they are indeed making. "We are in the business of turning lives around and as a result of our work there are huge cost savings to society," Mr Fernett said.
"Because of our grass-roots support we saved society up to £3.2 million from lower crime, £388,000 through increased employment and £225,000 by hosting Alcoholics Anonymous."
The report also found that Anchor House saved the NHS £22,000 in hospital admissions, £14,000 in hospital outpatient treatments, £10,000 in acute mental health services and £5,000 in A&E treatment.
The award winning and nationally acclaimed charity accommodates up to 180 single homeless people each year, and addresses the root causes of homelessness through training, education, volunteering and personal rehabilitation.
Monsignor John Armitage, the charity's chairman and Vicar General of Brentwood Diocese, says despite Anchor House being a small charity with limited resources and operating in one of England's most deprived boroughs, the results achieved are "astounding".
"Last year 58 of our residents moved on to independent living, 32 were in employment and we trained more than 1,249 locals within the community," Monsignor John said. "We are foot soldiers of the Big Society initiative and this report proves that.
"Anchor House is a charity worthy of investment, not only because it succeeds and makes a difference, but because it is focused on helping society's most marginalised and vulnerable who are often over looked."
Andrew Logan from Oxford Economic says social return on investment (SROI) is a popular metric used to quantify the positive impacts charities and other types of institutions generate per £1 invested, however there is still very few charities taking advantage of it.
"To calculate the SROI a charity needs to monetarise the economic, social and environmental outcomes that its efforts produce over the time of investment and then divide it by the costs," Mr Logan said.
"This isn't an easy task and can be off-putting for charities with limited time and resources.
"But in comparison to other SROI studies of homeless projects which have been conducted in the UK, Anchor House was found to provide an exceptional high return to society."
To download a copy of the report or make a donation visit www.anchorhouseuk.org.
Anchor House is a project addressing the root causes of homelessness: deprivation; drug addiction; mental illness; unemployment; lack of skills/education and crime. Canning Town, in the London Borough of Newham, is the third most deprived borough in England and Wales.
CISA REPORT: CONAKRY, October 4, 2011 (CISA) -On the occasion of the celebrations for the 53rd anniversary of national independence, the President of Guinea, Alpha Conde, invitation the opposition to dialogue. This was after violent clashes on September 27 between the police and participants in an unauthorized demonstration.
"To the political parties and their militants I renew the offer to dialogue. No obstacle is insurmountable, no divergence can resist our common commitment to respect freedom and law", said the Head of State in a radio speech.
The clashes resulted in the deaths of two people and injury to 40 others. About 300 people were arrested in connection to the protests. The demonstration was organized by the opposition to protest on the modalities of convening the parliamentary elections on December 29.
Archbishop Vincent Coulibaly, Archbishop of Conakry and Co-President of the National Commission for Reconciliation (NCR), has appealed to the government and the opposition to dialogue "without conditions, in the name of God and on behalf of our Guinean brothers and sisters," he said this on September 28, during the day of prayer organized in 60 different places in the Country, by the government and the NCR.
Archbishop Coulibaly also said that the NCR is at God's and the people's service, and urged people "to remember all the victims of Guinea in your prayers every day, so that God opens the doors of Paradise to them".
The Archbishop of Conakry emphasized that "Guineans should live together in unity: God has endowed our Country with wealth and it is in harmony that we can enjoy it".
Catholic Communications, Sydney Archdiocese REPORT
7 Oct 2011
His Holiness Pope Benedict XVI will officially open Australia's first pilgrim centre in Rome, Domus Australia.
The opening will take place on Wednesday 19 October with many invited guests travelling from Australia for the special event.
Pope Benedict XVI will enter the beautifully restored St Peter Chanel Chapel where he will be escorted to the new altar by the Archbishop of Sydney, Cardinal George Pell, for the welcome and papal blessing.
Guests will include Australia's first resident Ambassador to the Holy See, Tim Fischer as well as Cardinals and Senior Clergy
of the Vatican, the Australian Bishops attending the Ad Limina in Rome, Australian Clergy and Religious living, working or studying in Rome
Australian seminarians in Rome as well as the Italian contractors who helped bring the project to completion and many of the supporters and sponsors who have supported the establishment of the pilgrim centre.
The St Mary's Cathedral Choir is also travelling to Rome to sing at the opening as well as other events during the week.
Nearly three years ago, a group of Australian Dioceses, led by the Archdiocese of Sydney and supported by each of the Australian Archdioceses finalised the process of buying the student house of the Marist Fathers located at Via Cernaia 14/B Rome, to establish a new pilgrim centre for visitors to Rome.
Continuing the long Church tradition of providing accommodation for pilgrims to holy places, Domus Australia is much more than just a place to visit or rest. It is a true religious and cultural centre for pilgrims.
As well as offering very comfortable accommodation for pilgrims, it provides a true Catholic pilgrimage experience, with daily Mass in English and an information centre providing the opportunity for visitors to Rome to learn more about the history of the Church and the many places of religious significance they can see.
"Domus Australia will provide Australians with a unique pilgrimage experience and further strengthen the links between the Church in Australia and the Church in Rome," Cardinal Pell said.
"It will be a real away from home for travelling Australians.
"However we are extremely honoured by the Holy Father's acceptance of our invitation to join us for the official opening and bless Domus Australia."
Pilgrims have already been staying at Domus for several weeks with the occupancy rate already running at above 80% . An investment which has to generate its own income that is financially good news.
A short walk from Stazione Termini (Rome's main railway station), Domus Australia has 32 rooms accommodating around 70 people. Each room as its own ensuite, air-conditioning, television, internet facilities and much more.
There is a lounge room and a very popular roof-top barbeque and bar area for relaxing after a day of discovery in the Eternal City.
Apart from the Chapel, underneath is a conference area for around 150 people.
A dining room for guests opens onto a courtyard and caters for around 80 people.
The official opening will take place at 5.30 in the afternoon, Rome time. It will be streamed live on the Archdiocese of Sydney's social network xt3.com and although in Sydney this will be in the early hours of the morning, the stream will be made available on demand later in the day.
Telstra is supporting the event by sponsoring the live webcast. More news and stories on the build-up to the official opening next week will also feature on xt3.com and the Archdiocese of Sydney websites.
Feast: October 8
Feast Day:
October 8
She was a tender virgin at Antioch, only fifteen years of age when she was apprehended by the persecutors in 311. Being alone in the house, and understanding that their errand was to carry her before the judge, where her chastity might be in danger, she desired leave of the soldiers to go up stairs and dress herself. But fearing to be an innocent occasion to others' sin, threw herself from the top of the house, and died on the spot by her fall: in which action, says St. Chrysostom, she had Jesus in her breast inspiring and exhorting her. She probably hoped to escape by that means; and might lawfully expose her life to some danger for the preservation of her chastity; but nothing will ever make it lawful for any one directly to procure his own death.
Whoever deliberately lays violent hands upon himself is guilty of a heinous injury against God, the Lord of his life, against the commonwealth, which he robs of a member, and of that comfort and assistance which he owes to it; also against his friends, children, and lastly against himself, both by destroying his corporeal life, and by the spiritual and eternal death of his soul; this crime being usually connected with final impenitence, and eternal enmity with God, and everlasting damnation. Nor can a name be found sufficiently to express the baseness of soul, and utmost excess of pusillanimity, impatience, and cowardice, which suicide implies. Strange that any nation should, by false prejudices, be able so far to extinguish the most evident principles of reason and the voice of nature, as to deem that an action of courage which springs from a total want of that heroic virtue of the soul. The same is to be said of the detestable practice of duels. True fortitude incites and enables a man to bear all manner of affronts, and to undergo all humiliations, dangers, hardships, and torments, for the sake of virtue and duty. What is more contrary to this heroic disposition, what can be imagined more dastardly, than not to be able to put up a petty affront and rather to offend against all laws divine and human, than to brook an injury or bear a misfortune with patience and constancy, than to observe the holy precept of Christ, who declares this to be his favorite commandment, the distinguishing mark of his followers, and the very soul of the divine law! Mention is made of a church at Antioch, and another at Constantinople, which bore the name of this saint in the fifth century.
SOURCE: http://www.ewtn.com/saintsHoly/saints/P/stpelagia.asp
|Luke 11: 27 - 28
|As he said this, a woman in the crowd raised her voice and said to him, "Blessed is the womb that bore you, and the breasts that you sucked!"
|But he said, "Blessed rather are those who hear the word of God and keep it!"


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Well, if I was going to the Open House, I needed a present, didn't I?
I found 3 big skeins of cotton yarn and decided on three dishcloths. But what pattern?
I have a thing about handknit cloths stretching out of shape. So when I found a round pattern, I figured that was the key. If it stretched, it should stretch equally in all directions.
Or at least that's the plan.
And I did mention that I was going to post another picture of the felted roving rug. Here it is.


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Each year, Palmetto Care Connections hosts an Annual Telehealth Summit of South Carolina. 2018 marks the sixth annual summit. The summit brings together providers, payers,...
Geneticist Offers a New Option for Families Seeking Answers
April 28, 2017 - Posted in Telehealth by Taylor Crouch
While patients at the Greenwood Genetic Center's (GGC) Florence site might wait to see a geneticist in person, they now have another choice—to see a geneticist over a secure video conference, or telehealth.
Telehealth gives patients more appointment options to choose from, which could allow them to see a geneticist sooner. In late 2016, GGC began offering telehealth appointments at their Florence site, which has no full-time geneticist. In fact, when it comes to clinical geneticists, South Carolina mirrors geneticist shortages nationwide.
GGC's plans are to expand and use telehealth beyond the Florence site, and to continue to increase access by meeting the patients wherever they are.


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E received a beautiful christmas present from her "Auntie" Laura – a lovely wee tea set decorated with fairy and flower designs. At 19 months, E loves to play with her dollies, putting them to bed and tucking them in, putting them in her toy high chair and feeding them, putting them on toy potties and generally fussing over them. So she has been having fun using the pretty tea set every day to feed her dolls :o)
Yesterday Mum gave E afternoon tea outside with her tea set. She put water in the tea pot and E had a lovely time pouring "tea" and generally getting very wet!


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You could go this way, the little girl says, pointing left.
Or you could go that way, an old woman croaks, gesturing right.
No! No! You have to go in that direction! the adventurer exclaims,
tipping her finger toward the sky.
I'd really rather stay right where I am, the hermit yawns and rubs her tired eyes.
There's a chance you might enjoy the view, if you take the risk and jump over there,
the dreamer smiles, motioning forward and straight ahead.
But you might find your answers here, a forlorn child begs,
pulling at you from behind.
I'm afraid you're lost my dear, feet frozen to the icy block of ground
Unable to move until the fear and confusion thaws –
But when that fiery day will come, it's hard to say,
And while you have the time to think –
You really ought to figure out where exactly you want to go.
– Written by Miss A on September 9, 2011


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MOXEE, Wash. - KNDO spoke to the family of one of the firefighters killed in the 30-mile fire on Wednesday afternoon to get their reaction on the charges.
Ken Weaver, the father of Devin Weaver, says he has mixed emotions about the charges against his son's former boss.
He says the charges will hopefully keep the forest service accountable for their actions.
He does feel sorry for Ellreese Daniels, who was the crew chief when his son passed away during the fire.
But he also says the charges should warn people about the dangers of getting into the fire service.
"Because by being charged like this, it strips away that blanket of invulnerability that the forest service and its employees have wrapped themselves in for the last 75 years," Weaver said.
Back in 2001, Weaver asked Okanogan County prosecutors to bring charges against Daniels. But they declined to pursue the case further because they couldn't come up with the money to fund the case.


text: 10" POS Android PCAP Touch Screen Overview Commercial Grade Tablet Integrated Android Media Player The integrated Android Media Player allows for super fast updates. If required you can also run third party Digital Signage software via the Android PC board. Designed for use in commercial applications this screen can run 24/7. Strengethened glass front and no external buttons or controls to avoid tampering. 10-Point PCAP Touch Technology Using the most responsive and sensitive commercial touch technology available. Not affected by dust or debris on the screen surface. Built in Wi-Fi Connect the screen to the CMS wirelessly via the integrated Wi-Fi, ethernet or by LAN Power Over Ethernet Power the screen using an Ethernet cable or the power cable provided. Plug and Play Plug and Play is the easiest and most simple way to upload content to the screen. Simply load images and videos on a USB stick, insert in the display. Wait for your content to copy over and then remove. Your images and videos will now play in a continuous loop RS-232 Control LED Backlight Improves brightness and contrast as well as improving their lifespan and reducing their power consumption. Unlike other forms of light this technology is mercury free; making it even more eco-friendly. The panel and backlight have a lifespan in excess of 50,000 hours. Control, scale and customise your display over long and short distances using RS-232, Integrated Mounting Solution Portrait/Landscape Orientation The screen can be used in either a landscape or portrait orientation depending on your requirement. The rear of the display includes an integrated and secure screw based mounting solution. PCAP Touch Screen State of the art Projected Capacitive interactive technology offers the most responsive and sensitive touch experience available. Designed for commercial applications, PCAP touch technology works by fixing a sensor grid between a layer of cover glass and the LCD panel, when a finger touches the glass the grid detects the coordinates of the touch point. Unlike other touch technologies PCAP is not affected by dust or debris on the screen surface, allows for up to 10 touch points and offers superior optical quality. Power over Ethernet Your screen can either be powered using the power cable provided or by using an Ethernet cable. This makes the display extremely flexible, allowing you to supply power to the screen using the more convenient of the two methods. RS-232 Control Once your screen is connected via RS-232 you can control, scale and customise your display remotely over short and long distances. Commercial Tablet Ideal for use in commercial applications these touch screens bring in a whole new level of audience engagement to your marketing campaigns. Unlike domestic touch tablets these screens can be in continuous use, thanks to their screwless design and enclosure made from light weight polymer these screens are ideal for shelf edge and POS displays. The screen comes with an integrated Android media player that means you can display content without the need for a dedicated PC. There is also a POS PCAP Touch Monitor version available without the built in media player but that allows you to attach external devices to the screen. Features 10 Point Touch 10 Touch Points Having up to 10 touch points allows for a wider 24/7 Usage Built with commercial grade panel and components variety of applications than these displays are designed ever before. This kind of to run 24/7 in constant use; functionality allows you to unlike domestic tablets. The manipulate zoom panel also has a lifespan in an out as well as perform of over 50,000 hours of many other touch gestures; continuous use. They can much like you would with also be used in landscape a domestic tablet. It also or allows images, for multiple portrait orientation. users interacting with the screen at one time. LED Backlight The is LED not but backlight only also display eco-friendly ensures has brightness used the enhanced and contrast. Android Media Player Their integrated Android media player network provides super-fast possessing speed improved for communication This technology increases with the CMS. It also allows the lifespan and reduces you to display live websites; the power consumption by this can be anything from a around 30%. This form of live web feed from a third light technology is mercury party to your social media free; this page. Should you need to friendliness. you can also run your own safeguarding screen's eco software have a if you CMS already solution. Technical Drawing: Display Size 10 inch Resolution 1280x800 Display Area (mm) 217.95x136.65 Aspect Ratio Panel 16:10 Brightness (cd/m2) 450 Colour 16.7M Colour Depth 10 Bit Viewing Angle 178° Contrast Ratio 800:1 Touch Technology Touch Projected Capacitive Touch Points 10 Glass Hardness Audio 7H minimum Speaker Type 1.5W, 8Ω Power Consumption (W) Power 18 Input Voltage AC110~240V (50Hz~60Hz) PoE Mode 24V Passive Unit Size (WxHxD mm) 239.9x169.2x26.8 VESA Holes (mm) Mechanical 50x50 Package Size (WxHxD mm) 333X278X75 Net Weight (kg) 1.5 Gross Weight (kg) 2.2 Working Temperature 0 °C to 50 °C Media Formats Media Resolution Internal Memory Feature Internal Media Player Optional 1920x1080/1080x1920 6GB CPU Quad-Core Cortex-A7 , up to 1.3GHz (RK3126) GPU ARM Mali-400MP2 RAM 1GB DDR3 ROM 6GB NAND USB USB2.0 HOST x1 Wi-Fi 802.11b/g/n OS Android 5.0.2 Graphic Engine Accessories Video (MPG, AVI, MP4, RM, RMVB, TS), Audio (MP3, WMA), Image (JPG, GIF, BMP, PNG) OpenGL ES 1.1/2.0 Cloud Network Software Errors and omissions excepted


text:
Grace Melarejo's e-book Cold Sore Freedom in 3 Days is really a gospel for people suffering from this enduring ache, which might be inferred from …
Grace Melarejo's e-book Cold Sore Freedom in 3 Days is really a gospel for people suffering from this enduring ache, which might be inferred from your 100, 000 pleased customers on her internet websites plus the several recommends from commentators. As a result for the authors 7 a long time struggling from this evil, this guide offers highly low cost, effortless and practical property cures which haven verified efficient by earlier readers.Grab A Copy Click here
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Search dating sites with email, 1. google image search
Get a Free Consultation
Due to hi5's shift in focus to social gaming, speed dating with lux reclassified hi5 as an online gaming site in early Your email is required. The user will then appear on the person's friend list and vice versa.
Get more of this in your inbox. We get it - hiring a private investigator can be confusing.
Is he or she always on the phone or computer? A unique, memorable username makes such a difference on an online dating profile. John reveals new social games network". Thankfully, many tools have surfaced to make the task of being able to find an email easier and more scalable. Sure enough, if you add "kimkomando" to the address for Twitter https: Is your partner always texting?
Reverse Email Address Search
It's really that simple. This can be a great was to build an online identity, but it can also make it trivial to tie our activity on various services together.
He or she will very likely check search engine caches for old pictures or bios that are easier to identify or contain embarrassing details.
You may not use SocialCatfish.
Archived from the original on March 2, Screenshot of Hi5's home page. Subscribe to our blog Thanks for subscribing!
You are commenting using your Facebook account. There are plenty of free and paid services which search and monitor social media and email accounts by username. This works best for one-owner websites without much reason to hide contact information, giving you a clear line-of-sight to their email — and the first and last name of that person.
The individual facts and conversations you post on dating sites might not give away your identity, but as a collective whole, they may. You can also filter down the valid and invalid emails too. You forgot that social engineering and catfishing happen, and can happen to you. Recently, Social Catfish users have reported an influx of or Nigerian scams.
The number one open source intelligence source that people with evil intent will try to use against search dating sites with email, or to identify you, is your social media profiles.
Since April 4th, 2016
Profile Searcher helps you decide who you can trust and who you can move forward with. The scan will take about 50 seconds.
San Francisco Business Times. Within a few minutes, you will know whether your partner has an online dating account or not. We continually update our algorithms to guarantee the integrity of the search results.
Somebody with malicious intent may use this to their advantage when trying to correlate your dating profile to other web content.


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I found a nice collection of Houdini tutorials from 3DBuzz. This one is very basic and covers few things we went through in class about selections of primitives, points, etc – something I'll need when I'm modeling the geometry objects that construct my character.
This one is quite interesting, they are talking about "box modeling" and creating as further as golem head from a box sop. Sound's like it will do the work for my little vector.


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[Abraham Lincoln]: Letter Signed By John Nicolay and Edwin Stanton. ...Click the image to load the highest resolution version.
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Arranging a Presidential Reception for Army Officers to Take Place Just Before the Signing of the Emancipation Proclamation[Abraham Lincoln]: Letter Signed By John Nicolay and Edwin Stanton. 5" x 8" letter on "Executive Mansion" letterhead, Washington, D.C., December 29, 1862 written and signed by Presidential Secretary John G. Nicolay and sent to Secretary of War Edwin Stanton. In full: "You will please notify the officers of the Army that the President will receive them on New Year's Day at half past eleven o'clock precisely." Stanton has endorsed below: "The Adjutant General will issue instructions in conformity with the foregoing notice. Edwin M. Stanton Sec of War". January 1, 1863 would be an important day as it was effective date for the Emancipation Proclamation. After the reception, the Cabinet met in a different room at the Executive Mansion where Lincoln signed the final draft of the Proclamation. Normal folds, bold signatures
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Elder B was insistent about cooking "gator". We cooked it. My comments? Pretty gamey. And it all gets stuck in your teeth. Honestly, not too big of a fan.
Pennsylvania Pittsburgh Mission July 31, 2013-2015. Email Isaac at: firstname.lastname@example.com Write Isaac at: 321 Maple Terrace # 1, Pittsburgh, PA 15211


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I felt a little sad as the holiday began. I missed family and the exciting celebrations of days gone by. To his credit, Jim had said earlier in the week that he was thinking about how we could celebrate the 4th. He came up with the idea of going out for breakfast.
So that was how our day started. And my almond-cranberry pancakes at LePeep were absolutely delicious! Jim enjoyed his "Eighteen Wheeler" breakfast too.
I told Jim we could just walk in our neighborhood for some much-needed exercise. But he thought it would be better to go somewhere else so we had a beautiful walk along the river in Mishawaka. We enjoyed the gardens, the kids on the splash pad, and the sculptures along the way.
We had a cookout of chicken thighs and veggies--some of which were pretty burned but still tasty. With rhubarb crisp for dessert, it was a good meal.
The Cubs beat the Cardinals after four losses to the Mets and that was worth celebrating!
We watched the Macy fireworks on TV and texted Dan who was there in person. The patriotic music was great and so were the fireworks from the four barges on the East River in New York City. It brought back memories of seeing them live several years ago from the 38th floor of the Corinthian where Laura and Michael lived. It also brought back memories of other firework displays--Lexington, Massachusetts in 1976 for the Bicentennial, Meredith College in Raleigh, NC when I was 9 months pregnant with Dan and supposed to be on bedrest, and the Rose Bowl fireworks in Pasadena when Jim was teaching two week stints at Fuller Seminary. All good memories--so many memories when you are of a certain age!
It was a quiet day but a good day after all.


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As featured in the New York Times "Modern Love" column. Fugitives from a man as alluring as he is violent, Andrea Jarrell and her mother develop a powerful, unusual bond. Once grown, Jarrell thinks she's put that chapter of her life behind her--until a woman she knows is murdered, and she suddenly sees that it's her mother's choices she's been trying to escape all along. Without preaching or prescribing, I'm the One Who Got Away is a life-affirming story of having the courage to become both safe enough and vulnerable enough to love and be loved. (She Writes Press)
Those wishing to get books signed will be asked to purchase at least one copy of the author's most recent title from Vroman's. For each purchased copy of the newest title, customers may bring up to three copies from home to be signed. This policy applies to all Vroman's Bookstore events unless otherwise noted. Save your Vroman's receipt; it will be checked when you enter the signing line.


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Airwheel H3S Automatic Folding Power ChairHas Caused a Sensation in the 79th CMEF(April 12, 2018)
Have interest and click the website to consult:
At each CMEF, almost 4,200 medical device manufacturers from over 28 countries, and about 120,000 visitors and buyers who are government procurement agencies, hospital buyers and dealers from over 100 countries and regions gather in CMEF for trading and exchanges, according to its official website. The attendance of CMEF is meaning for Airwheel, as it means it has extended its business line to the medical equipment. Its products will benefit more users. This time, Airwheel brings several electric wheelchairs, and let's get the first-hand detailed information of H3S automatic folding wheelchair.
Page: 1
Related Keywords:Electronics & Semiconductors,Health & Medicine,Living,Manufacturing & Industry,Marketing & Sales,


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Academic Catalog
Art Education
Term : Fall 2017
Catalog Year : 2017-2018
ARE 485 - Undergraduate ResearchReturn to search
Description: Individualized original research under the supervision of a research adviser. Pass-fail only. May be repeated for up to 6 units of credit. Course fee required.
Units: 1-6
Sections offered: Fall 2018 Spring 2019