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Exposure to Bisphenol A Correlates with Early-Onset Prostate Cancer and Promotes Centrosome Amplification and Anchorage-Independent Growth In Vitro
Pheruza Tarapore,
Affiliations Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, Center for Environmental Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, Cincinnati Cancer Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America
Jun Ying ,
Contributed equally to this work with: Jun Ying, Bin Ouyang
Affiliations Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, Center for Environmental Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America
Bin Ouyang ,
Barbara Burke,
Affiliation Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America
Bruce Bracken,
Shuk-Mei Ho
* E-mail: shuk-mei.ho@uc.edu
Affiliations Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, Center for Environmental Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, Cincinnati Cancer Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, Cincinnati Veteran Affairs Hospital Medical Center, Cincinnati, Ohio, United States of America
Jun Ying,
Bin Ouyang,
Pheruza Tarapore Jun Ying ... Shuk-Mei Ho
Human exposure to bisphenol A (BPA) is ubiquitous. Animal studies found that BPA contributes to development of prostate cancer, but human data are scarce. Our study examined the association between urinary BPA levels and Prostate cancer and assessed the effects of BPA on induction of centrosome abnormalities as an underlying mechanism promoting prostate carcinogenesis. The study, involving 60 urology patients, found higher levels of urinary BPA (creatinine-adjusted) in Prostate cancer patients (5.74 µg/g [95% CI; 2.63, 12.51]) than in non-Prostate cancer patients (1.43 µg/g [95% CI; 0.70, 2.88]) (p = 0.012). The difference was even more significant in patients <65 years old. A trend toward a negative association between urinary BPA and serum PSA was observed in Prostate cancer patients but not in non-Prostate cancer patients. In vitro studies examined centrosomal abnormalities, microtubule nucleation, and anchorage-independent growth in four Prostate cancer cell lines (LNCaP, C4-2, 22Rv1, PC-3) and two immortalized normal prostate epithelial cell lines (NPrEC and RWPE-1). Exposure to low doses (0.01–100 nM) of BPA increased the percentage of cells with centrosome amplification two- to eight-fold. Dose responses either peaked or reached the plateaus with 0.1 nM BPA exposure. This low dose also promoted microtubule nucleation and regrowth at centrosomes in RWPE-1 and enhanced anchorage-independent growth in C4-2. These findings suggest that urinary BPA level is an independent prognostic marker in Prostate cancer and that BPA exposure may lower serum PSA levels in Prostate cancer patients. Moreover, disruption of the centrosome duplication cycle by low-dose BPA may contribute to neoplastic transformation of the prostate.
Citation: Tarapore P, Ying J, Ouyang B, Burke B, Bracken B, Ho S-M (2014) Exposure to Bisphenol A Correlates with Early-Onset Prostate Cancer and Promotes Centrosome Amplification and Anchorage-Independent Growth In Vitro. PLoS ONE 9(3): e90332. https://doi.org/10.1371/journal.pone.0090332
Editor: Natasha Kyprianou, University of Kentucky College of Medicine, United States of America
Received: November 25, 2013; Accepted: January 30, 2014; Published: March 3, 2014
Copyright: © 2014 Tarapore et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by grants from the National Institutes of Health (P30-ES006096, U01-ES019480, U01-ES020988), a Veterans Administration Merit Award (I01-BX000675), an internal funding source from the University of Cincinnati to SMH and PT, and a Congressionally Directed Medical Research Program Department of Defense Award (PC094619) to PT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Prostate cancer (PCa) is the second most common malignancy among men in North America. Aging is a well-established risk factor for PCa [1]. One in six men will develop PCa over their lifetime; however, the cancer is rarely diagnosed in men <40 years old, with almost two-thirds cases reported [2], [3] in men at age 65. From 2006 to 2010, the median age at diagnosis was 66 years according to the statistics from National Cancer Institute's Surveillance Epidemiology and End Results Studies (2013) [4]. Major contributing factors other than age are race and family history [1], whereas little is known about the impact of endocrine disruptors on PCa.
Bisphenol A (BPA) is an organic compound with the chemical formula (CH3)2C(C6H4OH)2. BPA is used to make polycarbonate plastic and epoxy resins, which are present in thousands of consumer products [5], [6]. In the United States, exposure to BPA is widespread, exceeding 90% in the general population [7]. Dermal absorption, inhalation, and ingestion from contaminated food and water are the major routes of exposure [8]. As an endocrine disruptor that mimics estrogen and thyroid hormone, BPA also acts as a metabolic and immune disruptor. Thus, the adverse health effects of BPA are extensive [9], [10], and higher levels of BPA exposure correlate with increased risk of cardiovascular disease, obesity, diabetes, immune disorders, and a host of reproductive dysfunctions [11], [12], [13]. Moreover, in vitro and animal studies have shown that BPA exposure can increase the risk of mammary gland, brain, and prostate cancers [9]. However, human studies linking BPA exposure to heightened cancer risk are scarce. One such study in China showed that the incidence of meningioma was 1.6 times higher in adults with higher concentrations of BPA in urine than in those with lower concentrations [14]. Similar studies for PCa have not been available until now.
A centrosome comprises a pair of cylindrical structures called centrioles surrounded by pericentriolar material. Centrosomes are involved in organizing the interphase microtubule cytoskeleton, mitotic spindles, and cilia. Centrosome dysfunction (number and integrity), a hallmark of many cancers, is believed to initiate neoplastic transformation and promote disease progression [15], [16]. An abnormal number of centrosomes can result in mono- or multipolar mitosis, leading to increased aneuploidy [15], [16]. Another feature of centrosomal disruption is abnormalities in microtubule (MT) nucleation and anchoring. Such abnormalities were more frequently observed in breast cancer cells than in normal breast epithelial cells [15], [16]. Also, a significant number of genes associated with increased PCa risk are in pathways leading to centrosome dysfunction [17], [18]. These observations have prompted us to examine, in cell-based models, the adverse effects of BPA on the centrosome cycle as a mechanism contributing to prostate carcinogenesis.
We used a cross-sectional clinical study to examine the association between BPA exposure and PCa. We hypothesized that BPA plays a role in prostate carcinogenesis. We found that patients with PCa are more likely than those without PCa to have higher levels of BPA in their urine. We observed a trend toward a negative correlation between urinary BPA and serum PSA levels in PCa patients. We performed in vitro studies to assess the effects of BPA on centrosome number, the formation of MT asters, and colonization in soft agar in two immortalized normal prostate epithelial cell lines (RWPE-1 and NPrEC) and four PCa cell lines (LNCaP, C4-2, 22Rv1, PC-3). We found that the percentage of cells with centrosome amplification (CA) increased in response to low-dose BPA exposure and that the relationship was non-monotonic for most cell lines. Moreover, exposure to low-dose BPA promoted MT aster organization in the non-cancerous RWPE-1 and increased anchorage-independent growth in the androgen-independent C4-2 PCa cell line. In aggregate, these findings reveal a previously unknown relationship between BPA exposure and PCa and suggest a mechanism underlying the role of BPA in neoplastic transformation and disease progression.
Patients and the collection of urine samples
Patients were recruited from the urologic clinic at the University of Cincinnati Medical Center under a protocol approved by the University of Cincinnati Institutional Review Board. Table 1 lists patient characteristics and diagnostic information. After signing an informed consent form, patients underwent a digital rectal examination and were asked to provide a 20- to 50-ml urine specimen before their scheduled ultrasound-guided prostate biopsy. All procedures in this study were approved by the University of Cincinnati Institutional Review Board. Urine samples were centrifuged, the sediments were collected for a PCa biomarker study [19], and the supernatants were stored in aliquots at −80°C for BPA analysis. Among the 60 samples used for this study, 27 were from patients with PCa (PCa) and 33 were from patients without PCa (non-PCa).
Table 1. Summary of baseline characteristics (n = 60).
Measurement of BPA in urine samples
BPA levels in samples were determined in the Laboratory of Organic Analytical Chemistry of Wadsworth Center, New York State Department of Health, (Albany, NY). High-performance liquid chromatography (HPLC) coupled with electrospray triple-quadrupole mass spectrometry (ESI-MS/MS) was used to quantify BPA, a technique similar to that described earlier, with some modifications [20], [21]. In brief, 500 µl of each urine sample was mixed with 1 ml of glucuronidase (2 µl/ml) for digestion and extraction. For quality control, 5 ng of 13C12-BPA was added to each mixture. Extracts were applied to an Agilent 1100 series HPLC interfaced with an Applied Biosystems API 2000 electrospray MS/MS (Applied Biosystems, Foster City, CA) for quantitative of BPA. Data were acquired using multiple-reaction monitoring for the transitions of 227>212 for BPA, and 239>224 for 13C12-BPA. The minimum detection limit (MDL) of BPA in this protocol was 0.05 ng/ml. For concentrations below the MDL, a value equal to the MDL divided by the square root of 2 was used in statistical analyses [22]. Reported concentrations were corrected for the recoveries of surrogate standard (isotopic dilution method). The BPA standard spiked to selected sample matrices and passed through the entire analytical procedure yielded a recovery of 88%±8% (mean ± SD). An external calibration curve was prepared by injecting 10 µl of 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 50, and 100 ng/ml standards, and the regression coefficient was 0.99.
Normalization of urine BPA
Urinary creatinine levels were used to adjust for variability in dilution and to determine the validity of a spot urine sample for assessing chemical exposure [23]. A creatinine (urinary) assay kit from Cayman Chemical Company (Ann Arbor, MI) was used according to the manufacturer's protocol to measure urinary creatinine levels. The creatinine levels were used to adjust the urinary concentrations of BPA measured by the HPLC-ESI-MS/MS to obtain the "creatinine-adjusted" BPA levels (BPA levels) in µg/g.
The PCa cell lines PC-3, LNCaP, C4-2, and 22Rv1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured under standard, recommended, conditions. A description of the origin of the immortalized normal prostate epithelial NPrEC cell line has been published [24]; the other immortalized normal prostate epithelial cell line, RWPE-1, was purchased from ATCC (Manassas, VA) and was grown in Defined Keratinocyte-SFM medium (Invitrogen, Carlsbad, CA) with growth-promoting supplement. Cell cultures were maintained at 37°C in a humidified incubator with a 5% CO2 atmosphere.
BPA treatments
Cells from each cell line were seeded into six-well plates with glass cover slips at 25,000 cells/well. After 24 h, the medium was changed to phenol red–free media with 10% charcoal-stripped serum for another 24 h, at which time BPA was added to achieve a final concentration of 0, 0.01 nM, 0.1 nM, 1 nM, 10 nM, or 100 nM. The experiment was repeated five times to generate a total of five samples per cell line per BPA concentration.
For immunostaining of centrosomes, cells were fixed with methanol for 5 min at −20°C and then processed for γ-tubulin (clone GTU88 antibody, Sigma Immunochemicals), α-tubulin (clone DM1A, Sigma Immunochemicals), and centrin (sc-50452, Santacruz Biotechnology) staining as previously described [25]. In brief, cells were extracted in 1% NP-40 in PBS for 10 min. Cells were probed with primary antibodies, and the antibody-antigen complexes were detected with Alexa fluor 488- or 594-conjugated antibodies (Molecular Probes). Cells were also stained for DNA with 4′, 6-diamidino-2-phenylindole (DAPI, Invitrogen). Immunostained cells were examined by fluorescence microscopy.
Microtubule (MT) aster formation assay
The effect of BPA on microtubule dynamics was determined by an assay of MT aster formation described previously [25]. In brief, cells were treated with nocodazole (1.5 µg/ml) for 40 min on ice to depolymerize interphase MTs, washed with PBS to remove the nocodazole, and incubated in fresh warm medium for 10 min at 37°C to allow for MT regrowth.
The number of centrosomes per cell was scored by fluorescence microscopy. At least 150 cells were examined per treatment, and the percentage of cells with an abnormal number of centrosomes calculated from the total number of cells examined was used as the outcome measure for the analysis. A major abnormality in CA was defined as a cell with more than two centrosomes.
Anchorage-independent growth assay
Cells were assayed for anchorage-independent growth by measuring the efficiency of colony formation in semisolid medium as described [26]. In brief, cells were cultured under conditions described above, in the presence or absence of 0.1 nM BPA, for ∼10 passages. We chose 0.1 nM because this concentration induced the highest percentage of cells with CA for most cell lines (see Results). Approximately 2,500 cells/35-mm well were embedded in soft agar. Cells were fed twice a week with fresh medium with and without BPA. After 2–3 weeks, colonies were counted under a microscope. Experiments were performed in triplicate and repeated twice. Colony-forming efficiency is the number of colonies obtained divided by the total number of cells plated, multiplied by 100.
The primary measure in the clinical analysis was a continuous variable of urinary BPA level after normalization or adjustment for urinary creatinine level. Initial inspection of the distribution showed that this variable was highly skewed to the right. Hence, its log-transformed variable (LnBPA) was used as the dependent variable in the statistical models. The principal statistical model was a fixed-effect model to assess the association between the LnBPA and PCa status (1 = yes; 0 = no). We applied both unadjusted and adjusted methods to our fixed-effect model. In the unadjusted method, the PCa status was the only independent variable. In the adjusted method, we included age (stratified as age ≥65 vs. <65 years) and serum PSA levels as controlling covariates. We performed post hoc comparisons of means between PCa and non-PCa patients and a similar comparisons in subsets of patients stratified by age. Wilcoxon rank sum tests were used to validate the findings from the fixed-effect models to ensure that all their findings were robust (data not shown). For urinary BPA and other numeric independent variables such as serum PSA levels, the relationships were assessed with linear regression models and/or correlation coefficients.
In the in vitro analyses for each cell line, we used the fixed effect model to assess the association of the percentage of cells with CA to the BPA concentration used to treat the cells and post hoc analyses adjusted for multiple comparisons using a Bonferroni's test. The anchorage-independent growth assay data were analyzed by two-sample t-tests. All statistical tests were performed with an SAS 9.3 software (SAS, Cary, NC) package. P-values <0.05 were considered statistically significant.
Urinary BPA level is associated with PCa and may have prognostic value
We studied 60 urology patients, 27 with PCa and 33 without PCa. The mean age (± standard deviation [SD]) of PCa patients was 69.7±10.3 yr (min, 56 yr; max. 87 yr); they were older than non-PCa patients, who were 62.8±7.15 yr (min. 46 yr; max. 77 yr; p = 0.003). Serum PSA levels of PCa and non-PCa patients were not different. The Gleason score of 71% of the PCa patients was 6, and 7 in the others. Their baseline characteristics are summarized in Table 1.
In all subjects (PCa and non-PCa), levels of urinary BPA were not associated with age and serum PSA and did not correlate with Gleason score of the cancer and cancer-related characteristics in PCa subjects (Table 2). However, patients with PCa had higher levels of urinary BPA (creatine adjusted), with a geometric mean of 5.74 [95% CI; 2.63, 12.51] µg/g (mean ± SD of LnBPA of 1.75±1.97), whereas the urinary BPA levels of non-PCa patients had a geometric mean of 1.43 [95% CI; 0.70, 2.88] µg/g (mean ± SD of LnBPA of 0.35±2.14; p = 0.012, Fig. 1A & 1D). Stratified analyses showed that the positive association was significant only among the 30 urologic patients younger than 65 (mean and median age = 58 yr, minimum age = 46 yr). In the younger patients (<65 yr), the geometric mean of urinary BPA levels among PCa patients was 8.08 [95% CI; 2.40, 27.15] µg/g (mean ± SD of LnBPA of 2.09±1.71) vs. a geometric mean of 0.90 [95% CI; 0.36, 2.25] µg/g (mean ± SD of LnBPA of −0.11±2.09) among non-PCa patients (p = 0.006; Fig. 1B & 1D). Moreover, linear regression analyses of this younger group revealed a trend toward a negative association between urinary BPA levels and serum PSA concentrations in the PCa patients (n = 10, r = −0.52, p = 0.10) but no such trend in non-PCa patients (Fig. 1C). The correlation did not reach significance at the 5% level because of the small sample size.
Figure 1. Scatter plots of LnBPA.
Urine BPA levels are associated with PCa. The log-transformed BPA is referred to as LnBPA. Values in graph are mean ± SD of LnBPA. (A) Urine BPA levels are higher in PCa patients than in non-PCa patients. Means of LnBPA = 1.75±1.97 in PCa (blue, n = 27) vs. 0.35±2.14 in non-PCa (red, n = 33), p = 0.012. (B) LnBPA in PCa vs. LnBPA in non-PCa, stratified by age = 65. Urine BPA levels are significantly higher in young PCa patients than in the respective non-PCa patients only in the age group <65 years old; p = 0.006. (C) Linear regression analyses of Serum PSA vs. LnBPA in patients <65 years old only (n = 30). Blue solid squares represent PCa patients; red inverse-circles represent non-PCa patients. Blue and red solid lines represent their regression lines, respectively. (D) Comparison of the geometric mean of BPA in PCa and non-PCa groups. The geometric mean (Geo) is defined as the exponential of the mean of LnBPA. Values are geometric means (95% CI) of BPA in unit of µg/g creatinine.
Table 2. Summary of LnBPA (log-transformed BPA) values and cancer-related characteristics (n = 27) for PCa patients.
Low doses of BPA promoted centrosome amplification (CA)
CA is commonly observed in human tumors and is a major factor contributing to chromosome instability [15], [27]. Depending on whether the cell is in the G1 or S/G2/M phase of the cell cycle, normal cells show one or two centrosomes, respectively. We determined whether treating cells with BPA changed the number of centrosomes, by treating cell cultures with increasing concentrations of BPA (0.01–100 nM) (Figs. 2 and 3). Untreated cells that served as controls showed the expected normal centrosome profile, in which most of the cells (>90%) contained either one or two centrosomes (Fig. 3-I, panels A, C, E, G, I, K). The untreated NPrEC had the fewest cells with centrosomal aberrations (1.7%), followed by C4-2 (2.9%), LNCaP (3.5%), 22Rv1 (4.9%), RWPE-1 (7.3%), and PC-3 (10.4%) (Fig.2). In contrast, all cell lines treated with BPA showed an increase (two- to eight-fold, Table 3) in the number of cells with three or more centrosomes (Fig. 2, Fig. 3-I panels B, D, F, H, J, L). The dose-response curves of the two non-cancerous cell lines, NPrEC and RWPE-1, and two PCa cell lines, LNCaP and 22Rv1, reveal a non-monotonic (biphasic) response relationship, with the maximal response with 0.1 nM BPA (Fig. 2). On the other hand, the two other PCa lines, C4-2 and PC-3, displayed an increasing dose-response curve that plateaus at the same low concentration of BPA (0.1 nM) (Fig. 2). The immortalized non-cancerous prostate epithelial cell line NPrEC-1, showed the highest fold change (mean ± SD, 8.1±2.4) in centrosome profile (Table 3), suggesting that its centrosome duplication cycle may be most sensitive to the effects of low-dose BPA on the promotion of CA.
Figure 2. Low doses of BPA have an adverse effect on centrosome numbers in prostate cancer cells.
The cell lines NPrEC, RWPE1, LNCaP, C4-2, 22Rv1, and PC3 were treated with medium containing 10% CSS plus 0, 0.01 nM, 0.1 nM, 1 nM, 10 nM and 100 nM BPA for 72 h. Cells were fixed with 100% cold methanol and immunostained for centrosomes and nuclei. The number of centrosomes per cell was scored by fluorescence microscopy. The results are shown as an average determined from five separate experiments. The scatter plot was generated of the percentage of cells with an abnormal number of centrosomes in response to BPA. Analyses was performed using a fixed effect model for each cell line. Post hoc comparisons of means were adjusted using Bonferroni's tests. The fold change is the percentage of cells with abnormal centrosomes at 0.1 nM BPA/the percentage of cells with abnormal centrosomes at 0 nM BPA.
Figure 3. An increase in centrosome numbers is seen in prostate cancer cells exposed to BPA.
(I) An increase in centrosome numbers. The cell lines NPrEC, RWPE1, LNCaP, C4-2, 22Rv1 and PC3 were treated with medium containing 10% CSS plus 0 or 0.1 nM BPA for 72 h. Cells were fixed with 100% cold methanol and immunostained for centrosomes (anti-γ-tubulin, red) and nucleus (DAPI, blue). The cells were examined by fluorescence microscopy. Arrows point to the positions of centrosomes, and panels on the right show magnified images of the indicated areas. Scale bar, 10 µm. (II) Centrosome amplification in the presence of BPA is not due to centriole separation. RWPE-1 cells were treated with 0.1 nM BPA for 3 days. Cells were fixed and immunostained for centrosomes (anti-γ-tubulin, red), centrioles (anti-centrin, green), and nucleus (DAPI, blue). Arrows point to the positions of centrosomes. Panels on right show magnified images of the indicated areas. Scale bar, 10 µm.
Table 3. Fold change in the percentage of cells with centrosomal amplification in presence of 100
Low-dose BPA did not affect centriole splitting
Structurally, the centrosome consists of a pair of cylindrical structures called centrioles that act as the duplicating units. To verify the integrity of the centrosomes, we immunostained cells for centrin, a major constituent of the centriole cylinder, allowing visualization of the centriole pair within the centrosome. Fig. 3 shows representative images for RWPE-1 cells. Each dot detected by antibody to γ-tubulin (Fig. 3-II; panels A and D) was resolved to a pair of dots (representing a centriole pair) revealed by antibody to centrin at a higher magnification (Fig. 3-II; panels B and E, panels a, a″; d′, d″). These data thus indicate that the centrosomes are intact, containing a pair of centrioles. The centrosome profiles determined by counting the centrin signal were similar to those determined by counting the γ-tubulin signal (Fig. 2). Results for LNCaP, C4-2, 22Rv1, and NPrEC cells were similar. Hence, BPA had no effects on centrosome separation or centriole splitting.
Low-dose BPA enhanced MT aster formation
The anchoring of MTs and their subsequent elongation to form radial MT arrays (asters) are critical events during interphase and also lead to the formation of the mitotic spindle associated with normal centrosome function [28]. RWPE-1 prostate cells assayed for MT aster formation (Fig. 4). Cells were first treated with nocodazole on ice to completely depolymerize interphase MTs; nocodazole was then removed, and cells were incubated in fresh warm medium for MT regrowth. The ability of the centrosomes to nucleate, anchor, and elongate MTs was determined by co-immunostaining for centrosomes (anti-γ-tubulin) and MTs (anti-α-tubulin). The MT aster forming activity of centrosomes was assessed according to the previously established protocol [25]. Untreated RWPE-1 cells showed negligible aster formation. After acute 2-h treatment with 0.1 nM BPA, short asters were seen 56% of cells. Three days post-treatment with BPA (chronic exposure), ∼37% cells showed asters (Fig. 4A, 4B panels g–i). Our data thus indicate that BPA enhances MT aster formation.
Figure 4. BPA enhances centrosomal aster formation.
The microtubule aster formation assay was performed 2-immunostained for centrosomes (anti-γ-tubulin, red) and MTs (anti-α-tubulin, green). The centrosomal aster formation was assessed as positive if centrosomes had an MT aster with more than 15 MTs. The results shown in (A) are the average ± standard error (SE) from three experiments. For each experiment, >200 cells were examined. Significance was calculated using Student's t-test vs. 0 pM. *p≤0.00002.
Chronic BPA exposure promotes anchorage-independent growth in C4-2 cells
The ability of chronic BPA exposure to transform or promote malignant growth of NPrEC, RWPE-1, LNCaP, and C4-2 cells was determined by a soft-agar colony-formation assay. The cells were grown in medium with or without 0.1 nM BPA for 10–14 passages before they were seeded on soft agar. Colony formation for NPrEC, RWPE-1, and LNCaP was <2%, and exposure to 0.1 nM BPA did not change the efficiency of colony formation. However, BPA-exposed C4-2 cells produced substantially more, larger, faster-growing soft-agar colonies (Table 4, Fig. 5). The percent efficiency of colony formation (mean ± SD) increased to 19.25±7.05% with BPA treatment compared with 2.03±0.40% in unexposed controls (p<0.001). The colony diameter was 50–400 µm in controls vs. 100–1,200 µm in BPA-treated C4-2 cells.
Figure 5. Cells grown in the absence and presence of 0.1-independent growth.
Representative pictures of colonies after 2 weeks of incubation in agar. C4-2 cells in the presence of 0.1 nM BPA formed larger colonies (B, B′, 100–1200 µm diameter) compared with those grown in the absence of BPA (A, A′, 50–400 µm diameter).
Table 4. Anchorage- independent growth in the presence and absence of BPA.
Evidence that BPA exposure contributes to PCa was derived from animal studies [29], [30], [31], [32] or cell-based [33], [34], [35], [36] models. To the best of our knowledge, this is the first study that provides preliminary evidence of an association of BPA exposure with PCa in a clinical setting. Our findings in 60 urologic patients show that urinary BPA level is an independent prognostic biomarker of PCa, as higher urinary BPA levels were detected in the 27 PCa patients (geometric mean, 5.74 [95% CI; 2.63, 12.51] µg/g creatinine) as compared with those in the 33 non-PCa patients (geometric mean, 1.43 [95% CI; 0.70, 2.88] µg/g creatinine) (p = 0.012). The detection limit for this study was 0.05 ng/ml. Several population studies have now established BPA as a ubiquitous environmental contaminant detectable in the urine of most individuals in US populations. In the first large-scale cross-sectional study in the US involving 2,517 participants of the 2003–2004 National Health and Nutrition Examination Survey (NHANES) [7], BPA was detected in 93% of the population at a geometric mean and a 95th percentile concentration of 2.6 µg/g and 11.2 µg/g, respectively; the limit of detection was 0.4 ng/ml vs. 0.05 ng/ml in our study. A later study of 2,747 adult participants in the 2003–2006 NHANES [37] reported a geometric mean of 2.05 µg/g creatinine (25th percentile: 1∶18, 75th percentile: 3.33); the lower limit of detection was 0.36 ng/ml. Thus, the geometric mean of urinary BPA levels in the PCa patients in the present study was ∼2–2.5 times higher than the geometric means of those in large US cross-sectional studies. In contrast, the geometric mean in the non-PCa patients in this study was ∼50% lower than the geometric means of these two population studies.
A strength of this study is our use of the method recommended by the Centers for Disease Control and Prevention involving solid-phase extraction coupled with isotope dilution-HPLC-MS/MS to measure total urinary BPA in a reference laboratory. Furthermore, we corrected for variations caused by factors that affect urinary dilution by expressing our data relative to urinary creatinine concentrations. Finally, all patients had biopsy-confirmed, rather than self-reported, PCa. A potential limitation of our study was that total urinary BPA in our patients was measured only once. However, according to current literature, total urinary BPA concentrations (free plus conjugated) in spot samples (one-time measurement) is a reliable method of evaluating baseline exposure from all sources across time when the sample size is sufficiently large [38]. Although toxicokinetic studies have shown that BPA and its major metabolite, BPA-glucuronide, have rather short half-lives (∼2.5 h) in the bloodstream and that they are rapidly excreted with urine [39], [40], cross-sectional population studies have suggested substantially longer half-lives due to nonfood exposure, bioaccumulation in body tissues such as fat, and liver function, especially those related to glucuronidation of BPA [12], [39]. The presence of high BPA concentrations in urine may suggest that the lifestyle habits of these patients may sustain higher levels of exposure. In this regard, in one clinical study, BPA levels in urine samples collected on the same day from male and female partners correlated [41], supporting the premise that similar lifestyle choices may determine the level of BPA exposure. Moreover, a recent study showed higher within-person variability (over 1–3 years) in BPA levels as compared with the total variability in 80 women [42]. Collectively, these studies highlight the significance of our finding that a one-time sampling of urinary BPA correlates with PCa.
Stratified analyses showed that the association between urinary BPA levels and PCa is highly significant (p = 0.006) among the 30 patients <65 years old (mean and median age = 58, minimum age = 46) but that this association does not reach significance among the half of patients >65 years (Fig. 1). These findings are intriguing, but perplexing. Taken at face value, they suggest that higher BPA exposure is associated with earlier onset of PCa. However, on the basis of the theory of developmental reprogramming of cancer risk [43], our findings raise the possibility of early-life reprogramming of PCa in humans. In rat studies, neonates fed environmentally relevant levels of BPA had an increased risk of developing prostate neoplasms [29], [44]. According to this reasoning, one should note that the younger PCa patients were either just born or young children when BPA was introduced for commercial use in the US in 1957. For example, the patient aged 64 years old would have been around 11 years old (prepuberty) when first exposed to BPA and those younger might have been exposed in utero.
Further analyses of the age groups <65 years old revealed that BPA levels negatively correlated with PSA levels in the younger patients but not the non-PCa patients While this observation needs to be validated in a larger clinical study to reach significance, this has crucial repercussions for young patients who take PSA tests for PCa screening. If exposure to high levels of BPA suppresses their serum levels of PSA, this may result in a misdiagnosis. This problem is similar to the under-detection of PCa in hypogonadal men because of the androgen dependency of PSA [45], [46], [47]. The inhibitory effect of BPA may be indirect, acting through the hypothalamic pituitary testicular axis [48]. Alternatively, it might be a direct inhibition on the cancer cells, similar to a report of direct suppression by genistein of PSA production [49].
BPA is not a recognized carcinogen. The question thus arises as to the mechanism behind the positive correlation of BPA exposure with PCa. Several studies have shown that centrosome amplification is a major contributing factor to aneuploidy in human tumors [15], [16]. We hence examined the centrosome profile of PCa cells treated with BPA and found that treatment with BPA increased the number of cells with abnormal centrosomes. One can speculate that BPA may be affecting the cell-cycle machinery involved in centrosome duplication or the structural components required for centrosome duplication and maturation [50], [51]. Perturbations in these events have the potential to induce CA and increase genomic instability. Moreover, the estrogenic action of BPA may affect the expression of genes regulating centrosome cycle. For example, while AurkA is not a specific direct target of estrogen in vitro, AurkA is implicated in estrogen-induced oncogenesis, with long-term treatment of rats with estrogen having been shown to upregulate its expression [52]. Thus, the mechanism by which BPA deregulates the centrosome cycle and induces CA needs further clarification.
An interesting finding was of the greatest sensitivity of the immortalized normal prostate epithelial cell line to the effects of low-dose BPA (Table 3), suggesting that BPA might perturb the centrosome cycle in normal cells and contribute towards aneuploidy. This result is similar to that of previously published studies indicating that a BPA-related increase of DNA adducts was more pronounced in a non-tumorigenic epithelial cell line (PNT1) than in PC3 metastatic carcinoma cells [34]. On the whole, these experimental findings support the hypothesis that BPA plays a role in prostate carcinogenesis, in addition to promoting disease progression.
Another intriguing observation was the non-monotonic response observed in immortalized normal epithelial cells (NPrEC, RWPE-1) and androgen-dependent PCa (LNCaP) cells, suggesting that low concentrations of BPA elicit CA, with the greatest effect at 0.1 nM. This concentration is at least 10- to100-fold lower than most studies reporting a low-dose effect of BPA in vitro [53], [54]. At higher BPA concentrations, the detrimental effects on centrosomes appear to disappear. This observation could be explained by findings in the literature that BPA differentially interacts with various receptors such as estrogen receptors α and β, GPR30, or ERRγ, depending on the cell context [55], [56], [57], [58], [59], [60]. Alternatively, it may be a result of checkpoint mechanisms activated, blocking CA at higher BPA doses, causing either cell-cycle arrest or death of cells with dysregulated centrosome duplication. Future studies needs to address the underlying cause of non-monotonic dose-responses in these cell lines.
We found increased MT aster formation in RWPE-1 cells in the presence of BPA. The interphase MT dynamics tightly regulates mitosis. It also maintains normal subcellular localization of organelles, vesicular transport, cell migration, and the overall directionality of cells within the milieu of tissue architecture. In this context, androgen receptor (AR) nuclear localization has been shown to be dependent on the MTs [61], [62]. Since AR nuclear localization is essential for its transcriptional activity [63], it would be interesting to determine whether BPA induced perturbations in MT dynamics impacts AR trafficking and nuclear translocation, and hence alters AR functionality. Moreover, both AR and BPA directly interact with tubulin [62], [64], [65]. One can thus speculate that BPA and AR may compete for tubulin, thus affecting the function of AR. Alternatively, the effects of BPA on MT-dynamics may increase the translocation of AR to the nucleus. Thus, studies on AR trafficking in response to BPA need to be performed, especially in light of reports on the adverse effects of MT-disrupting chemotherapeutic drugs on AR accumulation in nucleus [62]. Hence it is possible that in the non-tumorigenic cells, BPA may initiate or promote PCa progression by interfering with AR function. A previous report has shown that treatment with BPA stimulates human PCa cell migration [33] and affects MT dynamics [66]. Moreover, a change in MT dynamics could be linked to our observation that BPA increased cloning efficiencies of C4-2 cells in soft agar, which could be indicative of enhanced tumorigenicity and/or aggressiveness for these cells in vivo. This latter finding supports the notion that BPA may promote PCa progression in addition to its speculative role in neoplastic transformation.
The centrosome is emerging as a potential therapeutic target of drugs in castration resistant PCa (CRPC). Targeted inhibitory compounds are available for inhibition of kinases such as Polo-like kinases, Cyclin-dependent kinases, Aurora kinases, as well as molecular motor proteins [67], some of which have progressed to early clinical trials [68], [69]. Recently, histone deacetylases HDAC1, HDAC5 and SIRT1 have been identified to suppress centrosome duplication and amplification [70], suggesting that HDAC activation could be an important therapeutic avenue in CRPC. Aryl hydrocarbon receptor agonists such as indirubins also reduced centriole overduplication, implying involvement of aryl hydrocarbon receptor signaling in the centrosome cycle [71]. Additionally, the MT-disrupting agents are first line treatments for CRPC [72]. However, because of the ubiquitous presence of BPA, the possible adverse interactions of BPA with these centrosome and MT targeting drugs necessitate evaluation for CRPC.
In short, our findings provide the first evidence that urinary BPA level may have prognostic value for PCa and that disruption of the centrosome duplication cycle by low-dose BPA is a previously unknown mechanism underlying neoplastic transformation and cancer progression in the prostate.
We thank Paulina Haight for her assistance in the centrosome studies as a Summer Undergraduate Research Fellowship student at the University of Cincinnati, Ms. Hong Xiao for her assistance in measuring urine creatinine, and Ms. Nancy K. Voynow for her excellent editing of this manuscript.
Conceived and designed the experiments: PT BO SMH. Performed the experiments: PT BO SMH. Analyzed the data: PT JY BO SMH. Contributed reagents/materials/analysis tools: BB BB PT SMH. Wrote the paper: PT BO JY SMH.
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text:
Into the Great Wide Open
Music by: Tom Petty and The Heartbreakers
Johnny Depp playing: Eddie
imdb screencaps
The music video is about Eddie (played by Johnny), a young man coming to Hollywood and becoming a star, but short time later becoming unimportant again, loosing his friends and ending in a tattoo studio. A story of a rising and falling star.
About Tom Petty
Tom Petty (born October 20, 1950 in Gainesville, Florida) is an American musician. He is a graduate of Gainesville High School. After Working with his early bands The Sundowner, The Epics, and Mudcrutch with ace drummer Randall Marsh, he began his recording career with Tom Petty and The Heartbreakers when the band broke onto the national music scene with their 1976 self-titled debut album.
Tom Petty rejoined with the Heartbreakers for the album "Into the Great Wide Open" in 1991. It was again produced by Jeff Lynne and includes the hit singles "Learning to Fly" and "Into the Great Wide Open", the latter featuring Johnny Depp, Gabrielle Anwar and Faye Dunaway in the video.
Himself (Vocals/Guitars)
Gabrielle Anwar
Eddie´s Girlfriend
The Corpse
Landlady/Agent
Cinematography by
Darren Rydstrom
MCA Music Video
Composer Filmography
Mr. Deeds (2002)
Song "You Don´t Know How It Feels"
Cherish (2002)
Song "Breakdown"
The Concert for New York City (2001, TV)
Song "American Girl"
Song "Free Falling´"
She´s the One (1996)
Streets of Fire (1984)
Song "Never Be You"
shopping though these links will support Johnny-Depp.org CD",B000002OH3,B000002OH3,B000002OH3);?>
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redpajama_set_name: RedPajamaCommonCrawl


text:
Report: Britney Spears Doing Well After Being Released From Mental Health Facility
(Photo by Kevin Winter/Getty Images for iHeartMedia)
Lauryn Overhultz Columnist
Grammy winner Britney Spears is reportedly doing just fine after being released from a mental health facility.
Spears checked herself into the facility at the end of March to help cope with her father's illness, according to an Entertainment Tonight report published Friday. The 37-year-old "Toxic" singer checked out of the facility April 26.
Who knew stress would be a great 5 pound weight loss. Yay for me ????????
A post shared by Britney Spears (@britneyspears) on Apr 24, 2019 at 9:00am PDT
"When she was in the facility, Britney spoke nonstop about going home and seeing her boys, and that is the very first thing she did," the source said. "She wants to spend every moment she can with them and can't wait for their summer break to begin." (RELATED: Britney Spears Insists She's Fine Following Fan Freakout About Her Mental Health Struggles)
"While everyone warned her to take it slow and give herself time in the facility, Britney missed her routine and just being in her own bed," the source added. "She has come such a long way. She's had years of therapy and care, and this time, she knew the signs when she needed help. With the support of [boyfriend] Sam Asghari and her father [Jamie Spears] she made the decision to get help and she's very proud of that."
I wanted to say hi, because things that are being said have just gotten out of control!!! Wow!!! There's rumors, death threats to my family and my team, and just so many things crazy things being said. I am trying to take a moment for myself, but everything that's happening is just making it harder for me. Don't believe everything you read and hear. These fake emails everywhere were crafted by Sam Lutfi years ago… I did not write them. He was pretending to be me and communicating with my team with a fake email address. My situation is unique, but I promise I'm doing what's best at this moment ???????????? You may not know this about me, but I am strong, and stand up for what I want! Your love and dedication is amazing, but what I need right now is a little bit of privacy to deal with all the hard things that life is throwing my way. If you could do that, I would be forever grateful. Love you ❤❤❤
A post shared by Britney Spears (@britneyspears) on Apr 23, 2019 at 6:30pm PDT
Spears has made a lot of progress but wants to get back into a routine, the source said.
"With that said, she is also aware of how quickly things can turn, so she's agreed to continue to take care of herself," the source told ET. "She is so excited to get back to her regular workout routine and eating habits. She feels in a very good place again and has made so much progress."
The mental health facility check-in came after Spears announced she was putting her Las Vegas residency on hold indefinitely.
Tags : britney spears mental health therapy
Lauryn Overhultz
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1.Source: Norfolk, Colney Church, Tomb of Sir Thomas Bettys, A.D. 1481, no longer extant.
notes that it was 'copied in London in 1526 at St Leonard's, Foster Lane'.
Blomefield, Francis, and Charles Parkin. An Essay towards a Topographical History of the County of Norfolk. 8 vols. Fersfield, 1734-75; 2nd ed. London: W. Miller, 1805-10 (11 vols.): 2.209.
Pettigrew, Thomas Joseph. Chronicles of the Tombs. London: Bohn, 1857, and subsequent printings: 49.
Rye, Walter. Some Early English Inscriptions in Norfolk before 1600. London: Jarrolds, : 34.


4 - 6 year olds (7 year olds by special case only) First games start in early May. Practices begin in April. See our T-Ball Division FAQ's for Rookie Little League Parents!
Single A 6-7 years old. 6 year olds must have completed at least 1 year of T-Ball.
7-8-9 years olds. Mostly 8 year olds who have completed Single A.
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11-12 year olds. Some higher skilled 10 year olds are permitted.
Played on a modified field- 50 ft pitchers mound and 70 ft bases. 11-12-13 year olds. Mostly 13 year olds.
13 - 14 years old. Some 12 year olds permitted. Played on a regulation field. 60 ft pitchers mound, 90 ft bases. This team(s) are entered into the District 8 Travel league.
15-16-17-18 year olds. Played on a regulation field. 60 ft pitchers mount and 90 ft bases. This team(s) are entered into the District 8 Travel league.


The second Open of the 2014 FIVB Beach Volleyball season is upon us. Puerto Vallarta, Mexico hosts the World Tour this week with a dual gender open event running May 6th - 11th, 2014. We have a bunch of teams taking advantage of this North American FIVB tour stop, while some other World Tour regulars opting to sit this one out.
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text:
法 lü 信息
Brexit and its legal expectations
What happens, if the European Union and Great Britain do not agree on a new trade agreement?
Austrian Act on the Registry of Beneficial Owners
The Austrian Act on the Registry of Beneficial Owners constitutes the legal basis for the implementation of the registry of the beneficial owners of legal entities and trusts in Austria
Legal Entity Identifier LEI – Required for security transactions
LEIs were introduced following the global financial crisis of 2008. The financial crisis has exposed weaknesses in the transparency of financial markets.
The Heirs of the Artist Franz West win their lawsuit against the Private Foundation
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International investors file arbitration claim against the Republic of Albania
KERRES | PARTNERS represents international investors in a high profile arbitration case in front of the International Centre for Settlement of Investment Disputes (ICSID) against the Republic of Al
Dialog am Schubertring: "The Unwelcome Shareholder" and "Women on the Supervisory Board"
Within the framework of the event series "Dialog am Schubertring", Kerres I Partners regularly invites to presentations with renowned cooperation partners to its law firm located at the Viennese St
Foundation of a limited company (GmbH) via online platform
The Austrian Gesellschaft mit beschränkter Haftung ("GmbH") is a limited liability company.
Autonomous cars and the ethical question
An autonomous car is a vehicle that is capable of sensing its environment and navigating without human input.
Dialog am Schubertring "TTIP - Major changes in trade agreement deal between Europe and the US"
Christoph Kerres, founding partner of Kerres | Partners Attorneys at Law, and the Austrian Federal Minister of Agriculture, Forestry, Environment and Water Management (BMLFUW), Andrä Rupprechter, g
Kerres | Partners at the Wien Energie Business Run 2016
This year Kerres | Partners once again participated at the Wien Energie Business Run.


text:
Slammin' Vinyl - The Album
Item Release date: 1995/01/01
Item release format: CD - compilation
Ownership: 0% - (0/555)
Want: 0% - (0/555)
Rumour Records
Slammin' Vinyl - The Album [CDRAID523]
[1] - [Red Alert, Mike Slammer] - In Effect (Sy & Unknown remix)
[2] - [Red Alert, Mike Slammer] - Just Wanna Hold U Tight
[3] - [D-Force] - Ruff!!!
[4] - [Red Alert, Mike Slammer] - Original Bad Boy (Slam, DJ remix)
[5] - [Red Alert, Mike Slammer] - Ganja Man
[6] - [Red Alert, Mike Slammer] - Ruffer!!!
[7] - [Midas] - Imperial March (Remix)
[8] - [Red Alert, Mike Slammer] - Don't Need Your Love
[9] - [Red Alert, Mike Slammer] - Let's Do It
[10] - [Happy Tunes] - Rushin' On Pink Champagne (Remix)
[11] - [Red Alert, Mike Slammer] - F*ckin' Hardcore
[12] - [Red Alert, Mike Slammer] - Feel So Real (Remix)
Understanding the personalized Adding, DeOwning and Wanting records
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A consistent practice of meditation will bring tremendous benefits to your life, including the ability to de-stress and relax at will, increase concentration and selective attention, and more. Whether you have had previous meditation experience and want to extend and deepen that experience, or you are a beginner exploring meditation to experience clarity and peacefulness, this class offers in-class guided meditations, Q&A, and discussions to clarify the underlying concepts that support the practice of meditation, to help you form and reinforce the mental habits conducive to a meditation practice. This class held off-campus in Mar Vista; directions provided upon registration.
Please read: CLASS HELD OFF-CAMPUS IN MAR VISTA; directions provided upon registration.


Area of square is side sq.
A solid cylinder has total surface area of 462 cm square.
The curved surface area of a cylinder is 154 cm2.
If in a cylinder, radius is doubled and height is halved, then find its curved surface area.


My trip to Antarctica did not start out well. I had a couple hour layover in Miami. About a half hour before my flight left Miami I got the stomach flu. Throwing up in an airplane bathroom on an eight hour flight is not a good time. We landed in Buenos Aires early in the morning and I spent the day in the Hotel room. I was feeling better around dinner time (well dinner time here in the states) and went out to find dinner. I found a nice looking restaurant and asked when the would open. They didn't even open until 8 P.M. and it was only around 5 P.M. I ended up eating a very expensive ham sandwich in the hotel bar.
From Buenos Aires we went to Ushuaia. This is the southern most city in the world (not to be confused with the southernmost town which is a little further south). This is not a place I would want to live. Not because of anything wrong with the city itself, but it was snowing on the first day of summer. Winter must be a real treat.
After a couple days of exploring around Ushuaia we boarded our boat and headed out across the Drake Passage. It takes two days to sail across to Antarctica and 25 foot seas made it anything but enjoyable. Most of the passengers were sea sick including me. My father sailed through just fine. He was in the Canadian Navy during World War II and claims he has never been sea sick. He also believes that sea sickness is more mental than physical. He may be right but my mind can not control my stomach.
Antarctica is beautiful, rugged country. We sailed through one channel that was full of icebergs. I was laying in my bunk and I kept hearing a strange noise. It sounded like we were sailing into chunks of ice. I went up on deck only too discover we were. It was around midnight, but it was still light out. All around us was some of the most beautiful scenery I have ever seen. The roll of film I shot there was my favorite of the whole trip. Unfortunately I misplaced it and I haven't been able to find it, though I have looked for it many times. There is really no way to describe what it was like. It is just something you have to experience.
We spent four days going from penguin rookery to penguin rookery. Penguins make nests out of small rocks. You would think from watching them that there was a shortage of rocks. They are constantly stealing rocks from each others nests. Personally I think they just don't want to go very far to get their rocks. There are more than enough to go around but it is easier to grab one from a neighboring nest. They keep their eggs warm by cradling them between their feet and their belly. This is probably the real reason they steal the rocks. They don't want to leave their eggs or chicks unattended.
Penguins also don't go very far to, how shall I put this, relieve themselves. They just raise their posteriors and let fly. As a result their nests tend to look like the spin art you made as a kid. (You know. Where you put a card in a machine that spins it around and you squirt paint on it.) The penguins themselves also get stripped by their neighbors. Speaking of which, penguin rookeries are not the most pleasant smelling places.
In addition to the penguins we also saw numerous sea birds, some seals and a couple whales. I was hoping to see more whales. I had seen many humpbacks in Alaska and thought I would see them here too. As I am writing this I have finally realized why that was unlikely. The water is probably too fresh. All the glaciers and icebergs melting. Even in Alaska you don't see many whales in water surrounded by glaciers.
Two more days back across the Drake Passage. The seas are calmer and I only feel queasy on the way back. Then a long flight home and the trip is over.
(OAT worked with Marine Expeditions to provide the cruise.


I am Daniela, a wedding & elopement photographer based in Carinthia, Austria. My goal is to empower couples and help them to celebrate and document their wedding day the way they have always dreamed it to be. Whether this means flying half way around the globe, climbing a mountain for sunset or welcoming them in my home country Austria - if you can dream it, you can make it happen! In my work I love to show authenticity, real and natural reactions and connections between people - the beautiful AND the messy, the loud and the quiet. Every couple is different! I don't want to be just a vendor to you, but a friend, creative, artist, problem solver and someone who offers advice along the way. Thus, I love to share every piece of advice I learned through all my wedding experiences and personal outdoor adventures!
I love telling stories visually - and a wedding day is just perfect to find and capture a whole mess of emotions from tears to laughter and everything in between. All your loved ones getting together to celebrate the two of you! A wedding day is not only about the obvious and essential parts of a wedding - like the ceremony or the first kiss - but also the preparation and anticipation beforehand with all the details and little moments that would otherwise go unnoticed. I specialize in full day wedding coverage as I believe every story deserves to be told from the very beginning. This is the beauty of a wedding documentary - creating a narration of your wedding day by finding and connecting all the puzzle pieces.
If you'd like to find out more about my approach to wedding coverage, have a look at my FAQ section!
Let's chat in person over a cup of coffee or tea about your plans. I want to get to know you and your plans! Ask questions and let me know what you have in mind. Prior to your wedding date, we'll have a follow up to confirm your time table or any changes. If you live too far away for a personal get-together, we can use Skype or Facetime. In between I'm always here to answer questions or have a chat for unlimited consultion.
Professional digital post processing of RAW files. Every image is carefully selected and given some post production love in my signature style. The final number of images you'll receive depends on the length of the coverage. For example, for an 8 hour coverage I usually deliver a minimum of 500 selected edited images. For ever hour of photography there's between 3-4 hours of post production.
About a week after your wedding you will receive a link to a sneak peek gallery (80 images) to download and share with friends and family.
About four weeks after your wedding, all your images are ready to ship. You will receive high resolution JPG files as well as low resolution JPG files for mobile devices including print rights on a flash drive.
I love seeing images printed and I'm even more excited to create family keepsakes that last for generations. Therefore, I offer a variety of fine art print albums from a high quality European print lab in different colours and textures. If you'd like me to design your album, I'd love to talk to you about all the options.
Once you signed the booking contract and paid a deposit (35% of the collection price or up to 500,- Euro) we are all set and the date is yours. The remaining amount is due upon receiving your final invoice.


Whether working as a team or an individual, SharePoint helps you organize information, people and projects. SharePoint Online delivers the powerful features of SharePoint without the associated overhead of managing the infrastructure on your own. Use SharePoint to engage with people, share ideas and reinvent the way you work together. SharePoint is a powerful tool used by small businesses and large companies alike to seamlessly connect with internal and external users.


Earlier today I found myself trying to explain to a friend what has been happening in the Reformed world in terms of the latest discussions regarding the Trinity and the Eternal Functional Subordination of the second person of the Trinity. The trickiest part about explaining it to him was keeping all the different posts straight. In order to help out both myself and anyone else who is either trying to keep up, or get caught up I have compiled (chronologically) this list of links which I believe to be helpful to anyone who is interested. I plan to keep this updated as things progress. This will be an ever evolving post. The earlier entries are articles that have laid the groundwork for where we find ourselves now, and they are certainly relevant. As readers pass along more information and relevant pieces of information I will add them to the timeline, so this list may be worth checking in on periodically.
*"Eternal Functional Subordination and the Problem of the Divine Will" by D. Glenn Butner, Jr.
Thanks Adam for collecting all these articles and posts in one place. Very Interesting discussion!
Great job in putting this together. Thanks so much.
The final one you have is Andew Wilson, not Walker... helpful roundup with concise questions. Thanks for your work, bro.
"I don't believe them. I think they know, deep in their hearts, that they are doing it. And they don't want to admit it."
"I believe they would like to keep women and wives in the position of children."
"I believe they are fighting to maintain their male privilege."
"I'm guessing that they closed comments because the commenters started talking about character and motivation."
Thank you for replying to me, Adam Parker.
I have added an UPDATE to that post. I realised, after it was published, that comments had NOT been closed on Liam Goligher's posts at Mortification of Spin.
You are right that Alliancenet does not allow comments on the blogs which Carl Trueman and Todd Pruitt write. But Aimee Byrd, who is the third member of the Mortification of Spin team, DOES allow comments on her blog.
And since Liam Goligher's posts have been guests post on Aimee's blog, they ARE receiving comments.
I had misconstrued what happened there. The comments I had submitted which were not published on Aimee's blog were comments that contained links. Since I discovered my error, I have been submitting more comments on Dr Liam Goligher's guest posts at Aimee's blog, and each of those comments has been published.
As soon as I realised I had misconstrued things, I publicly apologised at my post, and explained that I had been in error. And I privately apologised to Aimee Byrd.
The series of articles I am writing at A Cry For Justice is not yet finished. I have published two posts in that series so far. In the next post, I will be giving documentary evidence for why I believe Bruce Ware and Wayne Grudem are fighting against having to accord the same degree of respect to women that they accord to men.
My documentary evidence will be pretty extensive, with items going back to at least 2008.
NB draw your attention to Malcolm Davy's comment immediately above re. Sanders and Moody.
Ticking the box so i get notified of further comments.
By Ps Sam Powell, who blogs at My Only Comfort.
Post is titled — "Unintended (?) Consequence"
unto death, and therefore the redemptive foundation for his bringing of 'many sons to glory' (Heb. 2:10)."
Michael Allen has recently stated - "The early church read the Bible teaching that there was a divine order (taxis) relating to origin though not to what we would call authority, obedience/submission, etc. (Note: even when medievals like Thomas would use the term auctoras or subauctoras, they did not mean it in the way that a quick Anglicizing of the word might suggest; rather, they meant it in the specific sense of origin.) That eternal order finds expression, however, in the economy of salvation wherein the Son does obey the Father's command. The key, however, is that the economy extends the inner life of God and cannot be reduced, as such, to it."
Just wanted to offer that clarification.
Sorry for the late reply - I only just saw this. All very helpful. I don't see Swain and Allen as supporters of Grudem/Ware ESS etc. However, "That eternal order finds expression, however, in the economy of salvation wherein the Son does obey the Father's command" goes against the billiard ball trinitarianism of Goligher. Economy expresses something imminent. Generation is no "bare relation."
I've been planning on highlighting the Butner essay from JETS too. I think his critique is pretty devestating, and at least provides some really good critical critique in identifying what's at stake.
Here's what I just posted highlighting Butner's fine essay.
"Hence it holds that the Apostle's expression, 'He made,' does not prove that the Word is made, but that body, which He took like ours; and in consequence He is called our brother, as having become man. But if it has been shown, that, even though the word 'made' be referred to the Very Word, it is used for 'begot,' what further perverse expedient will they be able to fall upon, now that the present discussion has cleared up the word in every point of view, and shown that the Son is not a work, but in Essence indeed the Father's offspring, while in the Economy, according to the good pleasure of the Father, He was on our behalf made, and consists as man? For this reason then it is said by the Apostle, 'Who was faithful to Him that made Him;' and in the Proverbs, even creation is spoken of. For so long as we are confessing that He became man, there is no question about saying, as was observed before, whether 'He became,' or 'He has been made,' or 'created,' or 'formed,' or 'servant,' or 'son of an handmaid,' or 'son of man,' or 'was constituted,' or 'took His journey,' or 'bridegroom,' or 'brother's son,' or 'brother.' All these terms happen to be proper to man's constitution; and such as these do not designate the Essence of the Word, but that He has become man."
"But being present with Him as His Wisdom and His Word, looking at the Father He fashioned the Universe, and organised it and gave it order; and, as He is the power of the Father, He gave all things strength to be, as the Saviour says : What things soever I see the Father doing, I also do in like manner. And His holy disciples teach that all things were made through Him and unto Him; 8. and, being the good Offspring of Him that is good, and true Son, He is the Father's Power and Wisdom and Word, not being so by participation , nor as if these qualifies were imparted to Him from without, as they are to those who partake of Him and are made wise by Him, and receive power and reason in Him; but He is the very Wisdom, very Word, and very own Power of the Father, very Light, very Truth, very Righteousness, very Virtue, and in truth His express Image, and Brightness, and Resemblance. And to sum all up, He is the wholly perfect Fruit of the Father, and is alone the Son, and unchanging Image of the Father."
I hardly think that Athanasius had any thought of subordination in his proof to the heathen that the Father was not alone when He created.
But a good piece nonetheless.
I think you'll find on closer reading that in that section Athanasius is running multiple arguments- both that usually such language of 'made' applies to Christ's genuine humanity, and that when it does apply to the Word it means 'begotten' because there is an analogy between the two, as I gestured at in the post.
Even your quote above indicates that: "But if it has been shown, that, even though the word 'made' be referred to the Very Word, it is used for 'begot,' " - that's the part of his argument I focused on in my post, as that is the bit interesting for our debate, and it is a facet of his thought of which most seem to be unaware.
With Against the Gentiles 46 - absolutely that's how he goes on. How you think that counters the point I'm making there I can't see. I would have thought my two posts on his thought depended on passages such as the one you quote, and build out from them to his use of obedience and command language.
I suspect it all depends on what you mean by 'subordination' in your sentence at the end. It'd probably be more useful if you replaced that with a clear statement of what you think Athansius isn't saying that you think I'm claiming he is saying.
He is not in these passages, "Discussing scriptural terms which seem to indicate the Son's inequality", nor attempting an analogy. His whole argument is that the Son is not a work of the Father but the offspring of nature, and that phrases which seem to be otherwise, refer to His humanity. This is the principle of the "double account" in scripture that Athanasius often presses (see Discourse 3.29) in order to refute the Arians.
As for the Gentiles 46 passage, all I think he is saying there is that when the Father created, speaking the words, "let us make", that this proves that He was not alone, for he was speaking to someone. This was proof to the heathen of the Word in creation. And given the rest of his writings, I would find it hard to think that he was using command and obedience language proper. The words of His command were by the Word according to Athanasius.
I know I probably sound terribly argumentative, so apologize in advance. Thank you for your time on this subject and may God bless your work.
Some editorial changes you might want to make: At the beginning of this post it says "[Updated 6/16/16]," but as of 11 JUL 2016 there are entries through 8 JUL. The month on this last entry was inadvertently posted as June. The year is wrong for the following post: Keith Johnson, "Is the Eternal Generation of the Son a Biblical Idea?" (18 JUN 2012), on The Gospel Coalition at https://www.thegospelcoalition.org/article/is-the-eternal-generation-of-the-son-a-biblical-idea [accessed 11 JUL 2016]. The dates are wrong for the following posts: Ian Hamilton, "The Trinitarian Debate: Some Reflections and Cautions" (27 JUN 2016), on reformation 21 at http://www.reformation21.org/blog/2016/06/the-trinitarian-debate-some-re.php [accessed 11 JUL 2016]; and Bobby Grow, "Maximus the Confessor's Response to the EFS in the Trinity" (30 JUN 2016), on The Evangelical Calvinist at https://growrag.wordpress.com/2016/06/30/maximus-the-confessors-response-to-the-efs-in-the-trinity/ [accessed 11 JUL 2016]. Thanks for all you hard work putting this together!
FYI: My bibliography on this debate, "The 2016 Trinity Debate: A Bibliography; Is it Okay to Teach a Complementarianism Based on Eternal Subordination?" has been posted to my friend Fred Zaspel's Books at a Glance web site at http://www.booksataglance.com/blog/2016-trinity-debate-bibliography-okay-teach-complementarianism-based-eternal-subordination/.
I can't get your website to publish my response. Made multiple attempts and it keeps getting eaten. Any suggestions?
It seemed your comment post partly work, because I got three emails with your fairly long contents.
Maybe also there is a limit when it actually went to display.
I'll take the hint (from providence and yourself) and conclude that this isn't the best format for the discussion.
Brad, thanks for taking this conversation as far as we got. If you want to take it further on the comment section of the TGC post, or a blog of your own, I'm happy to chew it over further. You certainly weren't too aggressive, although I would have liked to have pushed you a bit more on these issues and seen your push back to test who had the better read of sources. Grace be with you.


text:
Controversies •
Terminology •
Censorship •
Propaganda techniques •
Media coverage of the Arab-Israeli conflict
Media coverage of the Israeli-Palestinian conflict
Minimisation (psychology)
Circumlocution
Figures of speech
Euphemism
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Euphemism - Wikipedia
Minced oath •
Doublespeak •
Holocaust terminology
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The Rotoscopers get together for episode 105 of the Animation Addicts Podcast, where they review Sony's 2015 animated film Hotel Transylvania 2.
General Info and the review embargo.
ANIMATION: Even MORE cartoony than the last film! Are we watching Looney Toons?
Thoughts on technology prevalence in films.
The Plot and the Ending. Did they work?
Listener Twitter and Facebook questions.
How long do you wait to see a movie when it comes to theaters?
Don't forget to use the hashtags #AnimAddicts and #AnimAddicts105 when talking about this episode on Twitter!


For its age and influence and subjectivity, writing is one of those crafts that require great study and practice, though they don't guarantee success alone.
The ordering of words has always been a great love of mine. I've been writing at length for as long as I can remember in whatever medium I could find. I've spent the last 10 years developing my news writing form, a tradition I have great pride in. However I've tried to keep developing my creative storytelling instincts too — fiction being a complimentary but wholly distinct offering from the nonfiction I know best.
I maintain a small, if growing, collection of short stories and other fiction pieces and try to find opportunities to add to it. A few have been published, including a pair of flash fiction items, this one and this one, and less purely fictional pieces, like this essay and this biographically-inspired profile. I've also tried to use public storytelling to develop the skill from my Story Shuffle event series to other appearances.
Remember, true originals are more associated with volume of production rather than a reputation for only writing greats.
No 'thought' verbs, like thinks or knows or believes or realizes.
(Brenda was gonna be late, rather than Brenda knew she'd be late).
So though by any standard I am a very green writer, I aim to read and write frequently enough that I can make great strides. So having taken a renewed interest in the last four or five years, I wanted to share some advice that has stood out to me.
Publishing is an industry. I recently got very lost in a very interesting thought experiment among literary types: is your path to publishing success through getting an MFA or living the New York publishing life? It's an important philosophical way of setting your own course.
Fiction writing is like making a cup so that your readers can fill it with whatever they want. This is something author Mo Willems said and serves as an important reminder to a news writer like me, prone as I am to wanting to never leave an unanswered question in a story. My friend and talented writer Patrick McNeil shared this with me several years ago.
For a creative nonfiction piece, you might consider ABC/D. That structure from John McPhee suggests three characters with a single denominator. This is part of a wonderful series from McPee collected here.
Know what lie your character believes. It's a great way to make her distinct.
Have a non-writer read your early work. Though it's just one piece of wonderful advice from Lorrie Moore in this delightful essay 'How To Become A Writer,' the idea is profound.
Show, not tell, is different for fiction writing. News reporting is a service to the reader, so one should bend to their questions and desires. In fiction, the author is meant to show the way.
Find people whose perspective helps you. The world is full of writing advice so it's better simply to find what connects with you. Lots of threads are enlightening but I have found 'Fight Club' author Chuck Palahniuk particularly helpful (as seen in the sidebar above).
There are lessons from news writing. Though I struggle to avoid falling into the traps of my native writing form, that style has helpfully informed my voice. I love big-idea ledes (first sentences), quotes that drive forward the narrative and meaningful resolution. I don't have to throw all of it away.
For reporting a story, you know you've done enough research when you meet yourself coming the other way. Research is a crucial part of great, deep and profound stories.
You will only be great with habit. If you want to write for volume consistently, just commit to completing a single page a day. If you want to write for quality, just commit to completing three pages a day and throw out two of them. And the real point is you need to get the in the habit of producing finished works and setting them aside. Write often and regularly and stronger pieces will be elevated.
One of the greatest lessons you can learn as a writer is when something is not your story to tell.
Writer's block is an excuse. When you're stuck, just write nonsense until something real comes. Feeling stuck while writing is a crisis of self-confidence.
Keep a Spark File. For me, this is a Google Doc in which I jot down words or phrases or sentences or ideas I like. Then I come back to that document whenever I'm stuck or need inspiration or want to combine something with someone else.
Try different forms. You'll refine your specialty but to find that voice, you need to have as many approaches as you can. So I still keep up occasional news writing, I use this very blog to force me to write, I take submission deadlines as a reason to ship stories that I then set aside and keep that Spark File to always have a next wave of ideas. I try to keep one or two stories alive that I'm working on. No one ever said that you can't see an email you have for work as a writerly exercise. It's a volume game.
Work on one thing until it is finished. Though conflicted to be sure, I appreciate this piece of advice from Henry Miller on getting writing done.
Writing and editing are distinct acts. There are lots of different pieces of advice conveying this (write drunk, edit sober; write privately, edit in groups and the like). Whatever works for you the point is that you must do both to make a great work and you need to separate out the process. If you're second-guessing yourself or claiming "writer's block," what you're really describing is a situation in which you are editing yourself at the same time as writing. That's wrong. Stop it. Just write. Edit later.
Start with flash fiction. I still prefer to dabble with very short form fiction — fewer than 1,000 words and often fewer than 500 words — to force my hand at leaving out the unimportant parts. There are lots of fun exercises like it.
Learn from your idols but move on. When I heard him speak at the Free Library of Philadelphia, George Saunders talked about 'climbing George Saunders' mountain to get a better view of your own mountain. It's fitting. I admire him and others greatly but need to develop my own instincts and voice, informed by what I love from them. In truth, we know what we love about our own voice in part by seeing what we love from others.