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MDM2 antagonists boost antitumor effect of androgen withdrawal: implications for therapy of prostate cancer
Molecular Cancer volume 10, Article number: 49 (2011)
Hormone therapy is the standard of care for newly diagnosed or recurrent prostate cancers. It uses anti-androgen agents, castration, or both to eliminate cancer promoting effect of testicular androgen. The p53 tumor suppressor controls a major pathway that can block cell proliferation or induce apoptosis in response to diverse forms of oncogenic stress. Activation of the p53 pathway in cancer cells expressing wild-type p53 has been proposed as a novel therapeutic strategy and recently developed MDM2 antagonists, the nutlins, have validated this in preclinical models of cancer. The crosstalk between p53 and androgen receptor (AR) signaling suggest that p53 activation could augment antitumor outcome of androgen ablation in prostate cancer. Here, we test this hypothesis in vitro and in vivo using the MDM2 antagonist, nutlin-3 and the p53 wild-type prostate cancer cell line, LNCaP.
Using charcoal-stripped serum as a cellular model of androgen deprivation, we show an increased apoptotic effect of p53 activation by nutlin-3a in the androgen-dependent LNCaP cells and to a lesser extent in androgen-independent but responsive 22Rv1 cell line. This effect is due, at least in part, to an enhanced downregulation of AR expression by activated p53. In vivo, androgen deprivation followed by two weeks of nutlin administration in LNCaP-bearing nude mice led to a greater tumor regression and dramatically increased survival.
Since majority of prostate tumors express wild-type p53, its activation by MDM2 antagonists in combination with androgen depletion may offer an efficacious new approach to prostate cancer therapy.
Despite advances in diagnostics and treatment, prostate cancer remains the second leading cause of cancer deaths in the US. Current treatments attempt to block cancer cell growth and induce cell death by removing or inhibiting the androgens that support tumor growth . Surgical (orchiectomy) or chemical (LHRH agonist/antagonist) castration to eliminate testicular- androgen can delay clinical progression . Anti-androgens such as flutamide or the more potent bicalutamide, which block the hormone-receptor interaction, have also been shown to improve survival [3–5]. Combined androgen blockade (CAB) applies both castration and anti-androgens, or estrogens to maximize the block on androgens including those produced from the adrenal gland. However, survival benefit from CAB is rather controversial and still under scrutiny . Unfortunately, the majority of prostate cancer patients will eventually become resistant to one or all of these therapeutic strategies.
The mechanisms behind the resistance to androgen deprivation are not well understood although existing experimental evidence suggest that androgen withdrawal predominantly induces a cessation of cell proliferation but not overt apoptosis. In vitro studies with LNCaP cells grown in charcoal-stripped serum to mimic androgen ablation show a decrease in proliferation without apoptosis . This is unlikely due to ineffective androgen removal because a recent study has indicated that tissue culture media supplemented with 10% fetal calf serum (FCS) contain castrate levels of testosterone and the level of androgen is well below serum levels of castrated males . Normal rat prostate (and likely normal human prostate gland) respond to androgen ablation with high levels of apoptosis leading to glandular involution [8–10]. However, in human prostate cancer cells, the apoptotic response to androgen deprivation is not as clearly evident. It has been shown that androgen deprivation induces cell cycle arrest rather than apoptosis in three well known androgen-dependent cell lines, LNCaP, CWR22, and LuCaP-35 in vitro and in vivo [6, 11, 12]. Eventually, cell proliferation resumes, leading to an androgen-independent state in these model systems in vivo. This makes them a good model to assess the ability of therapeutics to induce cell death in combination with androgen ablation. The molecular response to in vivo androgen withdrawal was studied closely in the human prostate cancer xenograft model CWR22 in nude mice. Androgen ablation induced a robust stress response with an apparent p53-mediated cell cycle arrest but no p53-dependent apoptosis. Additionally the increased expression of p53 was only transient [11, 13]. Lastly, studies of human tumor samples taken from patients that have undergone androgen deprivation show significant decreases in proliferation but minimal apoptotic index [9, 10, 14].
The p53 protein is a potent tumor suppressor that can induce cell cycle arrest or apoptosis in response to various forms of cellular stress . Under non-stressed conditions, p53 is tightly controlled by its negative regulator MDM2 via an autoregulatory feedback loop [16, 17]. p53 activates the transcription of the mdm2 gene and in turn MDM2 protein inhibits p53 transcriptional activity. In addition, MDM2 is a p53-specific E3 ligase which targets p53 for ubiquitination and degradation in the proteasome . As a result of proper functioning of this autoregulatory loop both p53 and MDM2 are kept at low levels. In response to stress, the cellular levels of p53 increase leading to activation of multiple target genes and the p53 pathway with its main functions: cell cycle arrest and apoptosis [15, 19]. These antitumor consequences make p53 a desirable target for pharmacological activation .
In addition to its role in cell cycle arrest and apoptosis, p53 has also been implicated in the regulation of AR . Although the mechanism by which p53 exerts its control over AR is not clearly understood, p53 over-expression has been shown to decrease androgen function apparently by reduction in the expression of androgen-dependent genes [22, 23]. However, this regulation is quite complex given that at physiological levels p53 may act to protect androgen signaling . Conversely, androgen signaling has been found attenuated in etoposide-treated LNCaP cells as the stabilized p53 binds to the AR gene promoter . Hence, p53 could facilitate the reduction of AR signaling by occupying and competing for AR promoter. A recent study has also implicated p53 negative regulator MDM2 in modulation of AR protein levels by targeting it for ubiquitin-dependent degradation .
We have demonstrated that a potent and selective small-molecule inhibitor of the p53-MDM2 binding, nutlin-3a, can stabilize p53 and activate the p53 pathway in a broad panel of wild-type tumor lines including prostate [26, 27]. By disrupting p53-MDM2 regulatory circuit, nutlin elevates not only p53 but also MDM2, a transcription target of p53. Although MDM2 is kept away from p53, nutlin-bound MDM2 retains its E3 ligase activity against itself and possibly other targets. It has been documented that elevated MDM2 can facilitate MDMX ubiquitination and degradation in the presence of nutlin . Therefore, one can expect that nutlin-induced elevated MDM2 could also facilitate AR degradation and further reduce AR levels when combined with androgen ablation. Here, we show that activation of the p53 pathway by nutlin-3a substantially augments the antitumor effect of androgen ablation. In vitro, combination of nutlin-3a with androgen deprivation in prostate cancer cell lines expressing wild-type p53 led to an increase in apoptosis over single agent treatments. More significantly, combination treatment of mice bearing LNCaP xenografts resulted in frequent complete tumor regressions and a dramatic increase in lifespan.
Nutlin-3a activates the p53 pathway in LNCaP prostate cancer cells
To study the combined effect of p53 activation and androgen deprivation, we chose the androgen-depenedent prostate cancer cell line LNCaP as a cellular model. We have shown previously that nutlin-3a activates p53 and inhibits the growth of LNCaP cells and xenograft tumors in nude mice . Incubation of proliferating LNCaP cells with nutlin-3a for 5 days showed a dose-dependent effect with EC50 of 0.5 μM (Figure 1A). Western blot analysis following 24 h nutlin treatment revealed p53 accumulation and concurrent increases in its transcriptional targets MDM2 and p21Waf1/CIP1 (Figure 1B). Additionally, BrdU cell cycle analysis showed a loss of S-phase and increase of G1 and G2/M phase fractions consistent with p53-mediated cell cycle block in G1 and G2 phases (Figure 1C). To assess the apoptotic activity of nutlin-3a we used the Annexin V assay. Nutlin showed both a dose-dependant and time-dependant increase in the apoptotic fraction (Figure 1D). These results confirm that MDM2 antagonists stabilize p53 protein in LNCaP cells and effectively activate p53 signaling and the main p53 functions, cell cycle arrest and apoptosis.
Nutlin treatment in combination with androgen deprivation further reduces AR levels and enhances apoptosis
In addition to the androgen-dependent LNCaP cells, two other prostate cancer cell lines were chosen to investigate the combination effect of androgen ablation and p53 activation: 22Rv1 and DU145. Wild-type p53 expressing 22Rv1 cells have an androgen responsive AR but can grow independent of androgen in vivo. DU145 cells that express mutant p53 were used as a negative control since MDM2 antagonists require wild-type p53 for their cellular activity . Cells were incubated in media containing either normal serum or charcoal-stripped serum (CSS) which has been shown to effectively eliminate androgen and thus mimic androgen ablation . A suboptimal nutlin-3a dose of 5 μM was used (Figure 1D). Cells were also treated with 5 μM of the anti-androgen hydroxy-flutamide (FLU) in the presence of complete or CSS. Western blot analysis of LNCaP cells revealed that CSS in combination with nutlin elicits further reduction of AR levels than CSS alone in LNCaP cells and to a lesser extent in 22Rv1 cells (Figure 2A-B). Both LNCaP and 22Rv1 expressed full- length AR (110 kDa and 114 kDa respectively), 22Rv1 also expressed a 80 kDa isoform. Analysis of the AR mRNA levels in LNCaP cells by quantitative PCR revealed a 2-fold decrease in the presence of nutlin or CSS but no significant change in the combination (data not shown). Stabilization of p53 and subsequent increases in p21 and MDM2 levels were only observed in the presence of nutlin but not CSS indicating that CSS does not cause stress-related p53 activation.
Then, we examined the apoptotic response after 48 hour drug treatment (Figure 2). While CSS did not induce apoptosis over the control level in LNCaP cells, the combination with nutlin enhanced the apoptosis observed with nutlin alone (Figure 2A). Flutamide did not show a significant change in apoptosis. However, the triple combination (Nutlin + CSS + FLU) further increased the apoptotic index in LNCaP but not in 22Rv1 cells (Figure 2B). As expected, the p53-mutant and AR-negative line DU145 did not show an increase in p21 and MDM2. AR levels were also undetectable and no significant change in apoptotic fractions observed (Figure 2C).
Bicalutamide (Casodex) enhances apoptotic activity of Nutlin-3a in LNCaP and 22Rv1 cells
Exponentially growing LNCaP, 22Rv1, and DU145 were incubated with 10 μM of another clinically used anti-androgen, Casodex (CDX), and/or 5 μM Nutlin-3a in the presence of complete serum or CSS. Percent apoptosis was determined by Annexin V staining after 48 hours (Figure 3). Data revealed that combined treatment with Nutlin-3a and CDX was more effective at inducing apoptosis than single agent treatments in both LNCaP and 22Rv1 cells. However, the combination of Nutlin-3a and CDX was less effective than Nutlin + CSS combination in LNCaP cells while triple treatment showed slight improvement over Nutlin + CSS combination. Nutlin + CDX combination showed a slightly better activity than Nutlin + CSS in 22Rv1 and triple treatment did not demonstrate greater effect over dual in this cell line.
AR reduction accounts for the increased apoptotic activity of nutlin-3a in CSS
To further investigate if the apoptosis enhancing effect of CSS is due to AR reduction, we depleted AR in LNCaP cells grown in normal serum using AR-specific siRNA. Western blotting confirmed the effective reduction of AR and showed that only in the presence of nutlin was there stabilization of p53 and activation of its transcription targets, p21 and MDM2 (Figure 4A). Addition of nutlin to AR-depleted LNCaP (siRNA-AR) cells led to a substantial increase in the apoptotic cell fraction over nutlin alone (Figure 4B). We then combined siRNA-AR and CSS with or without nutlin (Figure 4C). Apoptotic fraction of LNCaP cells in CSS + nutlin was nearly equivalent to the triple combination (CSS + siRNA-AR + nutlin), implying that CSS effectively reduces AR levels and that this is the main mechanistic cause for enhanced apoptotic response.
Nutlin-induced AR reduction does not involve MDM2-dependant degradation
MDM2 has been shown to serve as an E3 ubiquitin ligase for p53 and several other proteins, most notably MDMX . Moreover, AR degradation by Akt has been reported to require the E3 ligase activity of MDM2 . Since nutlin treatment leads to a dramatic elevation of MDM2 protein one can expect facilitated degradation of AR, if nutlin-bound MDM2 can retain its activity against AR as previously shown with another MDM2 target, MDMX . To test this possibility, we examined the protein levels of MDM2 and MDMX in the presence or absence of nutlin and/or CSS (Figure 5A). Nutlin treatment dramatically increased MDM2 levels and led to a significant reduction of MDMX but did not change AR protein levels. The reduction of MDMX was due to enhanced protein degradation since the proteasome inhibitor MG132 restored its levels. These results indicate that either MDM2 cannot facilitate AR degradation in LNCaP cells or that it has lost its activity against AR when in complex with the small-molecule inhibitor nutlin-3a. To distinguish between these possibilities, we generated a LNCaP cell clone stably expressing high levels of full length MDM2 protein under a CMV promoter. Western blot analysis showed significantly reduced levels of MDMX similar to nutlin-treatment. AR was also reduced but to a lesser extent. These experiments confirmed the previous observation that MDM2 protein can affect AR protein levels  and suggest that this ability is likely lost when nutlin-3a is bound to the MDM2 protein.
Androgen deprivation does not affect nutlin-induced miR-34 expression
Recently, Rokhin at al. reported that LNCaP cells cultured in androgen-free media and then treated with the DNA-damaging agent, doxorubicin, suppressed the expression of p53 transcription targets miR-34a and miR-34c, resulting in inhibition of apoptosis . It has been shown previously that nutlin activates miR-34(a-c) expression . Therefore, we examined if the combination of nutlin and CSS can affect miR-34 levels. Exponentially proliferating LNCaP cells were incubated in CSS, 5 μM nutlin-3a, or combination of both for 24 h, and miR-34(a, b and c) levels were measured by quantitive PCR. We found significant induction of all three miRNA species by nutlin (34a:4.4, 34b:35.8, 34c:36.4 fold average) and less than 2 × fold increase in the presence of CSS. However, the combination did not significantly change the levels of any of the three miR over nutlin alone (data not shown). These experiments suggest that androgen deprivation does not affect miR-34 induction by non-genotoxic p53 activation.
Nutlin-3a enhances antitumor activity of androgen deprivation in vivo and substantially increases lifespan of nude mice
Finally, we examined if androgen ablation combined with nutlin-3a could enhance anti-tumor activity against established human prostate tumor xenografts (Figure 6A). To this end, pre-castrated nude mice were implanted with sustained-release testosterone pellets 5 days prior to injection of LNCaP cells. Tumors were fully established with an average starting tumor volume of approximately 400 mm3 in each group. Mice were administered an optimal oral dose of nutlin-3a at 200 mg/kg twice a day (bid) for 14 days. Others were treated with removal of testosterone pellets as a model of androgen ablation. Additional mice were treated with a combination of ablation + nutlin-3a. Control mice were dosed with vehicle + sham pellet removal. Nutlin was well tolerated in all groups with no significant body weight loss in any group throughout the study. Tumor regressions were observed in all groups relative to vehicle. Nutlin-treated animals had 6/10 partial regressions, androgen ablation caused 20/20 partial regressions, and combination of nutlin + ablation yielded 13/20 partial regressions including 7/20 complete regressions (no complete regressions were observed in the other groups). At day 42 post tumor cell inoculation (last day of nutlin treatment), measurement of prostate specific androgen (PSA) in serum showed a 68% decrease with nutlin treatment alone compared to vehicle control (Figure 6B). Androgen ablation and combination treatment reduced serum PSA levels 94% and 98%, respectively.
Based on NCI criteria, an increase in lifespan (ILS) of ≥25% is biologically significant . Twice daily dosing of nutlin-3a produced a 57% ILS and androgen deprivation gave a 167% ILS (Figure 6C). Although the difference in tumor volume at day 42 between androgen deprivation and combination treatment was only 6-fold (Figure 6A), over time, nutlin + androgen deprivation resulted in a greater than 886% ILS. This was the result of the majority of regressions never re-growing and the mice therefore died of perceived natural causes (no gross signs of primary subcutaneous tumor or metastasis upon necropsy in all cases). Collectively, our results demonstrate a clear benefit of administering nutlin-3a in conjunction with androgen ablation.
Although androgen withdrawal/deprivation is the predominant course of treatment for advanced prostate cancer, eventually all patients will develop resistance to this therapy. Moreover, the mechanism underlying hormone refractory disease progression has yet to be elucidated. Our in vivo data supports the conclusion that ablation elicits an impressive yet transient antitumor effect in concordance with early clinical response. However, akin to long term clinical results, our xenograft data shows that over time androgen deprivation does not result in sustained regressions (Figure 6C). Disappointingly, clinical results also show that most if not all advanced prostate cancers become androgen independent after surgical or chemical castration . Taken together, these observations provide credence to the notion that ablation predominantly acts as an anti-proliferative rather than strong pro-apoptotic modality [11, 14, 32]. Further complicating matters is the apparent heterogeneity of advanced prostate tumors [33, 34]. Taking this into account, initial anti-androgen therapy may simply eliminate a modest portion of a genetically-diverse androgen-dependant tumor cell population and provide selective pressure to the remaining malignancy that favors growth of new or pre-existing androgen-independent cell populations [35–37].
Although the requirement for androgen is no longer necessary in refractory disease the AR can still potentiate tumor growth and survival, thus activity of the AR remains a focal point of therapy even in advanced stages of androgen independent prostate cancer [38–40]. It is also evident that there are numerous mechanisms by which aberrant androgen signaling occurs in the development and progression of prostate cancer. Examples include AR hypersensitivity via gene amplification, changes in ligand specificity via mutation, increases in AR gene expression or androgen biosynthesis, all of which permit growth advantages in low androgen environments [37, 40]. It is therefore imperative when using model systems to take into account the cellular milieu in which the AR exists. The LNCaP line used in this study for example, harbors the mutation T877A in the ligand binding domain. The consequence of this mutation is a loss of ligand specificity and promiscuous activation of AR with progestins, estrogens, adrenal androgens and surprisingly some anti-androgens.[37, 38, 41]. Consistent with published data, the androgen independent yet responsive 22Rv1 line show two mutant forms of the AR: a 114-kDa full-length form containing an exon-3 duplication and a truncated 80 kDa form which lacks the ligand binding domain ( and Figure 2B). In addition, the AR in 22Rv1 cells contains a His874 to Tyr substitution that also permits stimulation by adrenal androgens albeit likely through a different mechanism than the T877A substitution in LNCaP cells . Nevertheless, it is the AR that appears to be the principal accomplice in progressing towards complete androgen-independence [38–40].
Recent evidence suggests that the androgen receptor can confer cell survival by negatively regulating pro-apoptotic genes in the p53 pathway . Moreover, overexpression of wild-type AR in LAPC4 or mutant AR in LNCaP lines can promote cell survival by inhibiting p53 mediated apoptosis . Additionally, the potent androgen Dihydrotestosterone (DHT) has also been shown to decrease p53 levels and inhibit apoptosis in a dose dependent manner . Alternatively, AR expression can be diminished by increased levels of p53 . Highlighting the complex interplay between AR and p53 expression is the observation that physiological levels of p53 may be necessary to stabilize AR signaling . Interestingly, LNCaP cells cultured in steroid-free media decreased both p53 protein and mRNA levels equal to that of treatment with high concentration of DHT. However, p53 knockdown in LNCaP cells have shown no change in AR levels regardless of DHT . Taken together these findings suggest a mutual regulation of expression between p53 and AR. Therapeutic strategies aimed at disrupting the delicate cross-talk between AR and p53 must not only favor p53 expression, but also potentiate the apoptotic outcome of p53 activation.
Our published  and current data demonstrates that nutlin-3a alone has potent activity in the LNCaP cell line and in LNCaP xenograft models. As expected, nutlin-3a effectively activated the p53 pathway indicated by the increased expression of transcriptional targets, cell cycle arrest and apoptosis (Figure 1). These changes were not observed with CSS treatment and support the notion that androgen withdrawal by CSS does not cause stress-induced p53 activation. It is important to note that media supplemented with 10% fetal calf serum (FCS) does not represent normal physiological levels of testosterone but rather contain levels that are equivalent to that of serum in adult castrated males, nonetheless, LNCaP cells are able to maintain ~10 nM intracellular DHT concentration in 10% FCS by optimizing their AR and androgen metabolism . Thus LNCaP cultured in CSS is exposed to testosterone levels significantly lower than that measured in serum from castrated adult males . Unsurprisingly, we observed a discernible reduction in the expression of AR when LNCaP were cultured in CSS as opposed to FCS. Treatment with nutlin alone resulted in a relatively small decrease in AR protein levels (Figure 2B) likely reflecting the decrease of its mRNA levels. Greater reduction in AR protein occurred with concurrent nutlin and CSS treatment. This further reduction is not accounted for by the decrease in transcription or direct effect of elevated MDM2 on AR stability. This change may result from a complex set of events induced by activated p53 in an androgen-depleted milieu. Combination of nutlin and CSS enhaced the apoptotic response in LNCaP and to a lesser extent in 22Rv1 cells in vitro. (Figure 2). Presence of AR mutations in LNCaP and 22RV1 cells is likely responsible for the lack of robust in vitro response to flutamide as reported previously [38, 41]. However, the second generation AR antagonist, casidex, did show clear in vitro effect. The decrease in AR protein levels appear to play a key role in the enhanced apoptotic response since siRNA-mediated knockdown of AR augmented apoptotic effect of nutlin similar to the combination with CSS. The mechanism behind the enhanced apoptosis in an AR-depleted environment is not entirely clear. One experimentally supported possibility is that AR activation attenuates p53-dependent apoptotic signaling [41, 44] and efficient removal of AR expression leads to an overwhelming response in favor of cell death and not just cellular arrest.
In vivo, the combination of androgen ablation with nutlin treatment resulted in greater efficacy over corresponding monotherapy arms in established LNCaP xenografts and was the only group to show complete regressions in 7/20 mice despite the relatively high initial tumor volume (400 mm3). The greater degree of regression ultimately resulted in a dramatic increase of survival rates for the combination (Figure 6C). Taken together our data supports the notion that androgen withdrawal alone fails to induce a durable apoptotic response thereby allowing the eventual escape towards androgen independence.
The administration of nutlin with androgen deprivation offers a novel two-pronged approach to cancer therapy in which factors that sustain prostate tumor growth are effectively removed with a simultaneous unleashing of the powerful growth suppressive and pro-apoptotic activity of p53. As increasingly more potent anti-androgens are identified (e.g. abiraterone acetate and MDV3100 ), their combination with MDM2 antagonists, currently in Phase I clinical development, could offer a compelling strategy in future trials.
Cells, reagents and treatment
All cell lines used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and grown in the recommended media supplemented with 10% heat-inactivated FBS (Invitrogen, Carlsbad, CA) in a humidified environment with 5% CO2. Nutlin-3a was synthesized at Hoffmann-La Roche Inc, (Nutley, NJ). It was dissolved in DMSO and kept at -20°C as 10 mM stock solution. All other chemicals were purchased from Sigma (St. Louis, MO). Hydroxy-flutamide was dissolved in ethanol and maintained at 4°C at 10 mM stock concentration and used within 10 days. Bicalutamide was dissolved in DMSO and kept at 4°C as a 10 mM stock solution. MG132 was dissolved in DMSO and kept at -20°C in 10 mM aliquots. Cells were treated with 10 μM MG132 for 8 h prior to collection. For experiments in charcoal stripped serum (CSS) cells were washed with PBS and pre-incubated for 5 minutes in phenol free media with 10% CSS, washed again and replenished with phenol-free media containing 10% CSS with or without compound. The LNCaP-MDM2 overexpressing line was generated using the human full length MDM2 inserted into phCMV1 vector. The control is the empty phCMV1 and the selection marker is G418 at 250 μg/ml.
Cell proliferation/viability was evaluated by the tetrazolium dye (MTT) assay  as previously described . For cell cycle analysis, LNCaP cells (1 × 106 cells/T75 flask) were incubated with 10 μM nutlin-3a for 24 h. BrdU (20 μM; Sigma, St. Louis, MO) was added during the last 1 h prior to fixation and cells were processed and analyzed as previously described . For apoptosis assays, cells were seeded in 6-well tissue culture plates at 2.5 × 105 (22Rv1 & DU145) or 5 × 105 (LNCaP) per well and treated for 48 h or as indicated. The percentage of Annexin V-positive cells was determined as described . For qRT-PCR analysis, cells were seeded in 6-well plates, incubated overnight and treated with DMSO or 10 μM nutlin-3a for 24 h. Total RNA was isolated and analyzed as described previously . To quantify microRNA expression, total RNA was isolated using the TRIzol solution (Invitrogen) following manufacturer's instruction. 10 ng of RNA was converted to cDNA using the TaqMan® microRNA Transcription Kit and real-time PCR analysis was performed using TaqMan® microRNA assays (Applied Biosystems). For Western blotting, cells were grown in 75 cm2 flasks (106 cells per flask) or in duplicate in 6 well plates for siRNA experiments, lysed in 0.1 -0.2 ml RIPA buffer and analyzed as previously described . For siRNA experiments, human-specific AR SMARTpool siRNAs and non-targeting control siRNA were obtained from Dharmacon RNAi Technologies (Lafayette, CO) and used as previously described .
Precastrated male athymic nude mice (Crl:NU-Foxn1nu) were obtained from Charles River Laboratories (Wilmington, DE) and cared for as previously described . At 10 weeks of age, mice were implanted with a 12.5 mg sustained-release testosterone pellet (Innovative Research of America, Sarasota, FL) 5 days before the injection of tumor cells. A 1:1 mixture of human LNCaP prostate cancer cells suspended in phenol-free Matrigel and PBS were implanted in the right flank at a concentration of 1 × 107 cells in 0.2 ml total volume. The health of all animals was monitored daily by gross observation and analyses of blood samples of sentinel animals. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee in our AAALAC accredited facility.
Four weeks after inoculation, mice were assigned to different treatment groups with average tumor volume approximately 400 mm3. Mice were administered Nutlin-3a at 200 mg/kg bid (n = 10) orally for 14 days in 0.2 ml volume per injection or testosterone pellets were removed under ketamine/xylazine cocktail anesthesia to simulate androgen Ablation (AA) (n = 20). Other mice were treated with a combination of AA + Nutlin-3a at 200 mg/kg bid orally for 14 days (n = 20). Control mice were dosed with 0.2 ml vehicle bid orally for 14 days + sham pellet removal under ketamine/xylazine cocktail anesthesia. Tumor measurements and weights were taken 2-3 times per week. Tumor growth inhibition was calculated from percent change in mean tumor volume compared to the control group. Average percent weight change was used as a surrogate endpoint for tolerability in the experiment. Five random mice in each group were bled via the retrorbital sinus under anesthesia to obtain serum for PSA determination using a PSA ELISA (American Qualex, Inc., San Clemente, CA). Ten animals in each group were continuously followed beyond the last day of nutlin treatment to see if tumor re-growth would occur. In this second phase of analysis, survival was calculated using a cut-off individual tumor volume of 1000 mm3 as a surrogate for mortality. The increase in lifespan (ILS) was calculated as % using the formula: [(Median day of death in treated tumor-bearing mice) - (median day of death in control tumor-bearing mice)] × 100 Median day of death in control tumor-bearing mice. Statistical analysis was performed as previously described .
Sharifi N, Gulley JL, Dahut WL: Androgen deprivation therapy for prostate cancer. JAMA. 2005, 294: 238-244. 10.1001/jama.294.2.238
Huggins C HC: Studies on prostate cancer. 1. The effects of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res. 1941, 1: 293-297.
Schellhammer PF: An evaluation of bicalutamide in the treatment of prostate cancer. Expert Opin Pharmacother. 2002, 3: 1313-1328. 10.1517/146565188.8.131.523
Fradet Y: Bicalutamide (Casodex) in the treatment of prostate cancer. Expert Rev Anticancer Ther. 2004, 4: 37-48. 10.1586/14737184.108.40.206
See WA, Tyrrell CJ: The addition of bicalutamide 150 mg to radiotherapy significantly improves overall survival in men with locally advanced prostate cancer. J Cancer Res Clin Oncol. 2006, 132 (Suppl 1): S7-16.
Saeed B, Zhang H, Ng SC: Apoptotic program is initiated but not completed in LNCaP cells in response to growth in charcoal-stripped media. Prostate. 1997, 31: 145-152. 10.1002/(SICI)1097-0045(19970515)31:3<145::AID-PROS1>3.0.CO;2-K
Sedelaar JP, Isaacs JT: Tissue culture media supplemented with 10% fetal calf serum contains a castrate level of testosterone. Prostate. 2009, 69: 1724-1729. 10.1002/pros.21028
Kyprianou N, Isaacs JT: Activation of programmed cell death in the rat ventral prostate after castration. Endocrinology. 1988, 122: 552-562. 10.1210/endo-122-2-552
Westin P, Stattin P, Damber JE, Bergh A: Castration therapy rapidly induces apoptosis in a minority and decreases cell proliferation in a majority of human prostatic tumors. Am J Pathol. 1995, 146: 1368-1375.
Ohlson N, Wikstrom P, Stattin P, Bergh A: Cell proliferation and apoptosis in prostate tumors and adjacent non-malignant prostate tissue in patients at different time-points after castration treatment. Prostate. 2005, 62: 307-315. 10.1002/pros.20139
Agus DB, Cordon-Cardo C, Fox W, Drobnjak M, Koff A, Golde DW, Scher HI: Prostate cancer cell cycle regulators: response to androgen withdrawal and development of androgen independence. J Natl Cancer Inst. 1999, 91: 1869-1876. 10.1093/jnci/91.21.1869
Corey E, Quinn JE, Buhler KR, Nelson PS, Macoska JA, True LD, Vessella RL: LuCaP 35: a new model of prostate cancer progression to androgen independence. Prostate. 2003, 55: 239-246. 10.1002/pros.10198
Agus DB, Golde DW, Sgouros G, Ballangrud A, Cordon-Cardo C, Scher HI: Positron emission tomography of a human prostate cancer xenograft: association of changes in deoxyglucose accumulation with other measures of outcome following androgen withdrawal. Cancer Res. 1998, 58: 3009-3014.
Heinlein CA, Chang C: Androgen receptor in prostate cancer. Endocr Rev. 2004, 25: 276-308. 10.1210/er.2002-0032
Harris SL, Levine AJ: The p53 pathway: positive and negative feedback loops. Oncogene. 2005, 24: 2899-2908. 10.1038/sj.onc.1208615
Wu X, Bayle JH, Olson D, Levine AJ: The p53-mdm-2 autoregulatory feedback loop. Genes Dev. 1993, 7: 1126-1132. 10.1101/gad.7.7a.1126
Bond GL, Hu W, Levine AJ: MDM2 is a central node in the p53 pathway: 12 years and counting. Curr Cancer Drug Targets. 2005, 5: 3-8. 10.2174/1568009053332627
Honda R, Tanaka H, Yasuda H: Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 1997, 420: 25-27. 10.1016/S0014-5793(97)01480-4
Vogelstein B, Lane D, Levine AJ: Surfing the p53 network. Nature. 2000, 408: 307-310. 10.1038/35042675
Brown CJ, Lain S, Verma CS, Fersht AR, Lane DP: Awakening guardian angels: drugging the p53 pathway. Nat Rev Cancer. 2009, 9: 862-873. 10.1038/nrc2763
Cronauer MV, Schulz WA, Burchardt T, Ackermann R, Burchardt M: Inhibition of p53 function diminishes androgen receptor-mediated signaling in prostate cancer cell lines. Oncogene. 2004, 23: 3541-3549. 10.1038/sj.onc.1207346
Shenk JL, Fisher CJ, Chen SY, Zhou XF, Tillman K, Shemshedini L: p53 represses androgen-induced transactivation of prostate-specific antigen by disrupting hAR amino- to carboxyl-terminal interaction. J Biol Chem. 2001, 276: 38472-38479. 10.1074/jbc.M103652200
Nesslinger NJ, Shi XB, deVere White RW: Androgen-independent growth of LNCaP prostate cancer cells is mediated by gain-of-function mutant p53. Cancer Res. 2003, 63: 2228-2233.
Alimirah F, Panchanathan R, Chen J, Zhang X, Ho SM, Choubey D: Expression of androgen receptor is negatively regulated by p53. Neoplasia. 2007, 9: 1152-1159. 10.1593/neo.07769
Lin HK, Wang L, Hu YC, Altuwaijri S, Chang C: Phosphorylation-dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase. EMBO J. 2002, 21: 4037-4048. 10.1093/emboj/cdf406
Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C: In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science. 2004, 303: 844-848. 10.1126/science.1092472
Tovar C, Rosinski J, Filipovic Z, Higgins B, Kolinsky K, Hilton H, Zhao X, Vu BT, Qing W, Packman K: Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy. Proc Natl Acad Sci USA. 2006, 103: 1888-1893. 10.1073/pnas.0507493103
Xia M, Knezevic D, Tovar C, Huang B, Heimbrook DC, Vassilev LT: Elevated MDM2 boosts the apoptotic activity of p53-MDM2 binding inhibitors by facilitating MDMX degradation. Cell Cycle. 2008, 7: 1604-1612. 10.4161/cc.7.11.5929
Rokhlin OW, Scheinker VS, Taghiyev AF, Bumcrot D, Glover RA, Cohen MB: MicroRNA-34 mediates AR-dependent p53-induced apoptosis in prostate cancer. Cancer Biol Ther. 2008, 7: 1288-1296. 10.4161/cbt.7.8.6284
Kumamoto K, Spillare EA, Fujita K, Horikawa I, Yamashita T, Appella E, Nagashima M, Takenoshita S, Yokota J, Harris CC: Nutlin-3a activates p53 to both down-regulate inhibitor of growth 2 and up-regulate mir-34a, mir-34b, and mir-34c expression, and induce senescence. Cancer Res. 2008, 68: 3193-3203. 10.1158/0008-5472.CAN-07-2780
Johnson JI, Decker S, Zaharevitz D, Rubinstein LV, Venditti JM, Schepartz S, Kalyandrug S, Christian M, Arbuck S, Hollingshead M, Sausville EA: Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br J Cancer. 2001, 84: 1424-1431. 10.1054/bjoc.2001.1796
Mu Z, Hachem P, Agrawal S, Pollack A: Antisense MDM2 oligonucleotides restore the apoptotic response of prostate cancer cells to androgen deprivation. Prostate. 2004, 60: 187-196. 10.1002/pros.20044
Nwosu V, Carpten J, Trent JM, Sheridan R: Heterogeneity of genetic alterations in prostate cancer: evidence of the complex nature of the disease. Hum Mol Genet. 2001, 10: 2313-2318. 10.1093/hmg/10.20.2313
Shah RB, Mehra R, Chinnaiyan AM, Shen R, Ghosh D, Zhou M, Macvicar GR, Varambally S, Harwood J, Bismar TA: Androgen-independent prostate cancer is a heterogeneous group of diseases: lessons from a rapid autopsy program. Cancer Res. 2004, 64: 9209-9216. 10.1158/0008-5472.CAN-04-2442
Taplin ME, Bubley GJ, Ko YJ, Small EJ, Upton M, Rajeshkumar B, Balk SP: Selection for androgen receptor mutations in prostate cancers treated with androgen antagonist. Cancer Res. 1999, 59: 2511-2515.
Tso CL, McBride WH, Sun J, Patel B, Tsui KH, Paik SH, Gitlitz B, Caliliw R, van Ophoven A, Wu L: Androgen deprivation induces selective outgrowth of aggressive hormone-refractory prostate cancer clones expressing distinct cellular and molecular properties not present in parental androgen-dependent cancer cells. Cancer J. 2000, 6: 220-233.
Feldman BJ, Feldman D: The development of androgen-independent prostate cancer. Nat Rev Cancer. 2001, 1: 34-45. 10.1038/35094009
Veldscholte J, Berrevoets CA, Ris-Stalpers C, Kuiper GG, Jenster G, Trapman J, Brinkmann AO, Mulder E: The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens. J Steroid Biochem Mol Biol. 1992, 41: 665-669. 10.1016/0960-0760(92)90401-4
Agoulnik IU, Weigel NL: Androgen receptor action in hormone-dependent and recurrent prostate cancer. J Cell Biochem. 2006, 99: 362-372. 10.1002/jcb.20811
Yuan X, Balk SP: Mechanisms mediating androgen receptor reactivation after castration. Urol Oncol. 2009, 27: 36-41. 10.1016/j.urolonc.2008.03.021
Tan J, Sharief Y, Hamil KG, Gregory CW, Zang DY, Sar M, Gumerlock PH, deVere White RW, Pretlow TG, Harris SE: Dehydroepiandrosterone activates mutant androgen receptors expressed in the androgen-dependent human prostate cancer xenograft CWR22 and LNCaP cells. Mol Endocrinol. 1997, 11: 450-459. 10.1210/me.11.4.450
Dehm SM, Tindall DJ: Molecular regulation of androgen action in prostate cancer. J Cell Biochem. 2006, 99: 333-344. 10.1002/jcb.20794
McDonald S, Brive L, Agus DB, Scher HI, Ely KR: Ligand responsiveness in human prostate cancer: structural analysis of mutant androgen receptors from LNCaP and CWR22 tumors. Cancer Res. 2000, 60: 2317-2322.
Sun C, Shi Y, Xu LL, Nageswararao C, Davis LD, Segawa T, Dobi A, McLeod DG, Srivastava S: Androgen receptor mutation (T877A) promotes prostate cancer cell growth and cell survival. Oncogene. 2006, 25: 3905-3913. 10.1038/sj.onc.1209424
Rokhlin OW, Taghiyev AF, Guseva NV, Glover RA, Chumakov PM, Kravchenko JE, Cohen MB: Androgen regulates apoptosis induced by TNFR family ligands via multiple signaling pathways in LNCaP. Oncogene. 2005, 24: 6773-6784. 10.1038/sj.onc.1208833
Lassi K, Dawson NA: Update on castrate-resistant prostate cancer: 2010. Curr Opin Oncol. 2010, 22: 263-267. 10.1097/CCO.0b013e3283380939
Denizot F, Lang R: Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986, 89: 271-277. 10.1016/0022-1759(86)90368-6
We thank Baoying Huang for the experimental advice and critical discussion.
The authors declare that they have no competing interests.
CT participated in the design and execution of most in vitro studies and drafted the manuscript. BH designed and carried out the in vitro studies, analyzed data and participated in the preparation of the manuscript. KK participated in the in vivo studies and data analysis. MX generated the LNCaP cell clones. KP participated in the in vivo study design and data analysis. DH participated in the drafting and critical revision of the manuscript. LV participated in study design and coordination, data analysis and interpretation and prepared the manuscript.
All authors have read and approved the final manuscript.
Christian Tovar, Brian Higgins contributed equally to this work.
Authors’ original submitted files for images
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Cite this article
Tovar, C., Higgins, B., Kolinsky, K. et al. MDM2 antagonists boost antitumor effect of androgen withdrawal: implications for therapy of prostate cancer. Mol Cancer 10, 49 (2011). https://doi.org/10.1186/1476-4598-10-49

domain: medical


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Whether you're living abroad or just going on vacation this autumn, our guide on how to watch Strictly Come Dancing 2022 outside of the UK will tell you everything you need to know about enjoying the BBC reality series from overseas.
It's that time of year again!
Strictly Come Dancing 2022 returns to our screens this month for its 20th series, much to the delight of its devout fanbase. Last year's season was won by Eastenders star Rose Ayling-Ellis, who sailed her way into the finale with partner Giovanni Pernice after being tipped for victory early in the competition.
With just a couple of weeks to go until the first show of the 20th series, fanfare over the Strictly Come Dancing 2022's contestants has already been heating up. This year's cast includes a star-studded lineup of British celebrities, with the likes of Coronation Street's Kym Marsh, Loose Women's Kay Adams, and Paralympic swimmer Ellie Simmonds OBE all confirmed to take on the iconic dancefloor at the Elstree TV Studios in Hertfordshire this autumn.
Shirley Ballas, Craig Revel Horwood, and Motsi Mabuse will all return as Strictly judges 2022, while Bruno Tolioni has officially been replaced by Anton Du Beke. The panel's expert feedback will once again be mediated by Tess Daly and Claudia Winkleman, who have been co-hosting the live shows since Series 8.
In the aftermath of Queen Elizabeth II's death, the release date of Strictly Come Dancing 2022 has been postponed. Its pre-recorded first show was initially due to air on September 17, but will now premiere on Saturday, September 23. The first live show will be broadcast on Sunday, September 24, with the BBC now airing a Paddington movie on September 17 in honor of the late Queen.
HOW TO WATCH STRICTLY COME DANCING ONLINE IN THE UK
If you're in the UK and have a TV license, Strictly Come Dancing can be watched for free on BBC One or BBC iPlayer.
HOW TO WATCH STRICTLY COME DANCING ONLINE FROM ABROAD
If you're not in the UK while Strictly Come Dancing is airing, fear not – it's still possible to watch the glitzy show from abroad.
All you have to do is download a VPN, which will allow you to access shows usually only available when you are in the UK by changing your IP address to make your device think it is in a different location.
ExpressVPN (opens in new tab)(opens in new tab) is our VPN of choice and it also aced our sister site TechRadar's rigorous testing – meaning it is without a doubt the best VPN service out there.
Simply download ExpressVPN here (opens in new tab)(opens in new tab) to start streaming Strictly and you can also take advantage of the 30-day money back guarantee plus 3 months FREE (opens in new tab)(opens in new tab) when you subscribe for a full year.
Emma is a Lifestyle News Writer for woman&home. Hailing from the lovely city of Dublin, she mainly covers the Royal Family and the entertainment world, as well as the occasional health and wellness feature. Always up for a good conversation, she has a passion for interviewing everyone from A-list celebrities to the local GP - or just about anyone who will chat to her, really.
Emma holds an MA in International Journalism from City, University of London and a BA in English Literature from Trinity College Dublin.
The Queen once 'told off' this Prime Minister for trying to do the washing up at Balmoral
The former Prime Minister has shared touching memories of the late Queen Elizabeth II
By Robyn Morris • Published
Robbie Williams' bold Strictly outfit divides viewers, 'he may want to sack his stylist'
Robbie Williams' Strictly performance and outfit gained a lot of attention from viewers but not the nice kind of attention
By Aoife Hanna • Published

domain: entertainment


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Many of us were first exposed to Dead Poets Society in high school English class. But it doesn’t seem to age so it’s still an awesome film to re-watch. Much of that has to do with the charismatic Robin Williams, who plays a passionate English teacher aiming to inspire his students to view the world differently. Scenes from this gem of a film are still being referenced in pop culture today. And that infamous O Captain, My Captain scene makes us miss Robin more than ever. Remember the good old days with this Academy Award-winning movie this weekend.
Any time is a good time for a scary zombie flick, but now more-so than ever. Train to Busan is a South Korean horror film about a train going from Seoul to Busan. What starts off like any other normal train ride ends in a chaotic zombie apocalypse outbreak on the outside, as the passengers on the inside fight for survival. Stay home this weekend and take this terrifying ride that has redefined the zombie genre with its unique twist .
If you’re feeling like delving into an interesting piece of Americana, The Founder is for you. The movie delves into the story of Ray Kroc, who took two brothers’ idea for a fast-food burger joint and turned it into the biggest restaurant business in the world: McDonalds. This biographical drama stars Michael Keaton, Nick Offerman, and John Carroll Lynch. So pick up some burgers, kick back, and enjoy the show.
If you’re in the mood for something a little… unusual, Snake Outta Compton is your jam. The title pretty much speaks for itself. It’s about a giant mutant snake. And it’s attacking the city of Compton. Is this a thought-provoking, life-changing cinematic experience? No. Is it a fun time? Absolutely. Check out this funny and ridiculous monster flick today.
Swallow is a unique gem of a thriller you may not know about yet. But there’s lots of reasons you should. Critics are buzzing about Haley Bennett’s performance as a modern-day housewife, eagerly striving to be perfect in every role: wife, daughter-in-law, and soon-to-be mother. On the surface her life seems immaculate. But soon the immense pressure of these rigid expectations proves to be too much to handle. When the darkness of her past comes back to haunt her, the veil falls and reveals her true, vulnerable, imperfect self. Watch Bennett’s transformation in Swallow – just beware it may be a little hard to stomach.
Swallow shows us a glimpse at how far we will go when we’re pushed far enough. And there are other thought-provoking psychological thrillers that do the same in their own unique way. Whether we’re denying reality to the point of insanity, or striving for perfection to the point we become inhuman. The limits are endless and the consequences are dire. These films take a look at the fragility of the human psyche and the sinister places it can take us when we break.
Natalie Portman won a Golden Globe and Academy Award for her performance as Nina in Black Swan. The themes in the film parallel Swallow, as the story follows her journey to become a perfect ballet performer in Swan Lake. She needs to embody both innocence and sensuality as the White Swan and Black Swan, but Nina is timid and inexperienced, so she struggles with the latter. But she refuses to let that stop her. Hard work and perseverance take her to extreme places that are sometimes hard to stomach. Nina takes on an emotional, mental, and physical transformation as she relentlessly and mercilessly pushes herself to become the perfect dancer. And Portman took the role very seriously. She took dance lessons for a year and worked with a voice coach to give her a more child-like voice to fully embody Nina’s innocent character. Portman’s dedication really comes through in this visually stunning film, and its approach will make you question how far you’d be willing to go to achieve perfection.
The Devil’s Advocate
This star-studded film featuring Al Pacino, Keanu Reeves, and Charlize Theron is a must-see for any thriller lover. The Devil’s Advocate follows a successful lawyer from a small town who’s offered the opportunity of a lifetime to practice in New York City. Kevin, played by Reeves, quickly becomes massively successful and spends more time at work with his big-time boss John Milton, played by Pacino. Temptation and greed soon take over his psyche and we start to wonder who Kevin really is. We see him transform from a good, hard-working husband to a cold man with questionable character. But is that transformation worth the cost? The film takes an interesting look at morality and how fragile it becomes when you’re handed everything you’ve ever wanted. And it’s a definite must-see.
The Machinist is an ominous thriller starring the talented Christian Bale. The story follows Trevor, who suffers from insomnia and becomes emaciated as a result. He’s deemed an outsider by almost everyone he knows. Left to deal with his paranoid mind in isolation, Trevor slowly unravels and we’re taken along on the daunting trip to figure out why he can never sleep. The film can be uncomfortable to watch, especially with how malnourished Bale appears. He fully immersed himself in the role, going as far as crash-dieting and taking up smoking to lose 63 pounds. He was so weakened by the emaciation that he had difficulty running when filming action scenes for the film. His dedication is unparalleled, and the film is worth watching for his performance alone. This movie shows what our conscience will do to our psyche when guilt takes over and reality is too painful to bear. It’s an ominous journey worth taking.
Prisoners shows how far a father will go to protect his child. The star-studded cast in the film includes Hugh Jackman, Jake Gyllenhaal, Viola Davis and many more. On a seemingly normal day, Keller Dover’s six-year-old daughter and her friend suddenly go missing. Utter panic among the families ensues. Detective Loki, played by Gyllenhaal, is tasked with helping Dover, played by Jackman, to find the child and the kidnapper. When they finally home in on a suspect, they lack sufficient evidence to arrest him. This is where Keller takes justice into his own hands. Frantic and utterly desperate, Keller sees no limits when it comes to finding his daughter. The film makes us question our certainty about what’s right and wrong, as we perceive Keller go back and forth between a brave hero and a heartless monster. This intense journey will leave you wondering how far you would go to protect the ones you love.
The hilarious cast from Jumanji: Welcome to the Jungle is back again for some brand new adventures. Except, this time, the game has changed and anything can happen. Join the game and enjoy the crazy ride that is Jumanji: The Next Level – and get the full-length Get In The Game Mode included as a bonus!
We’ve even got some great tips on creating a family movie night complete with activities, a fun quiz, and everything you need to know about watching with younger viewers.
This Clint Eastwood directed drama is based on the true story of Richard Jewell, who saved thousands from an exploding bomb at the 1996 Olympics in Atlanta. When he’s falsely accused of being a terrorist by the press his life is ripped apart. Starring the talented Paul Walter Hauser, Sam Rockwell, Kathy Bates and more, Richard Jewell is a story you don’t want to miss. See why Clint Eastwood was inspired by this story in this exclusive clip:
This slasher re-make is every college girl’s nightmare. The story follows a group of sorority sisters who soon find out they have a terrifying stalker coming after them. Check out Black Christmas today.
NEW TO RENT
This legal thriller follows a defense attorney, played by Mark Ruffalo, and his arduous journey to expose a long history of environmental pollution by a massive chemical company. Praised by audiences and critics alike, Dark Waters is a must-see for serious drama lovers.
This much talked about drama follows an African American couple who go on the run after their first date goes horribly wrong. Queen & Slim is an unexpected ride you don’t want to miss.

domain: artistic


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The future of Lionel Messi remains uncertain as his contract with Paris Saint-Germain expires in June and he’s yet to reveal where he plans to play next season.
While it appears as though Saudi Arabia is out of the question, FC Barcelona remains lurking, hoping that a reunion is possible. Mundo Deportivo (h/t Canal Supporters) reports, citing sources, that Barcelona is exploring all solutions for Messi.
Even if they consider that the return of the Argentinian remains complicated, the Spanish club leaders have not thrown in the towel.
The Catalan side is studying a scenario in which Leo would adapt his salary to the reality of Barcelona’s wage bill in exchange for a percentage of the new income he would generate with a high-profile return.
After the player’s departure in 2021, Barcelona president Joan Laporta continues to dream of a Messi return. But in a challenging economic context, the La Liga club would seek the best way to make a competitive offer for the 2022 FIFA World Cup winner.

domain: news


Ember has released its latest limited edition app-enabled temperature controlled smart mug made of copper. Like the design? Let’s continue checking.
The limited edition Ember mug delivers a same design language with the original Ember mug, but unlike the previous solid white body, the new model shows off a shiny copper-tone appearance, and the wireless charging saucer also features the copper exterior to perfectly match with the mug.
Using built-in Bluetooth technology, the mug wirelessly connects with your smartphone so that you can set ideal temperatures for different beverages via its custom app on your smartphone, so it’s able to keep your drink at the perfect temperature. When reaching your chosen heat, it will notify you. And you can also remotely adjust temperature between 120°F and 145°F. Moreover, an integrated LED indicator is customizable by choosing your favorite color. The smart mug is IPX7 waterproof, which means you can fully submerge it in water for easy to clean.
The Ember limited edition copper temperature controlled smart mug is priced at $129.95 USD. If you’re interested, jump to Urban Outfitters for its more details.


When Cops Carried Wheelguns
In the beginning there were … uh … revolvers. And we paid for them out of our own pockets, along with most other stuff like uniforms, leather, ammo, handcuffs and raingear. But that was allright because we were making the princely sum of $505 a month.
I hired on a Northern California sheriff’s department in 1967 with a two-year LE degree and the feelings of invulnerability and invincibility only a 21-year old can possess. These two traits didn’t last all that long. I was issued a hat, a bat and a badge and given the keys to a ’67 Ford. I was actually on patrol for 17 months before being sent to the academy. Things were a lot different back then.
My first duty rig was purchased from a former city cop for a total of $65 and consisted of belt, holster, cuffs, drop pouch and an old-model 4″ Colt Trooper .357. That was an okay gun, aside from the fact the cylinder turned in the “wrong” direction, but wasn’t entirely satisfying. Being a “gun nut” (something that kinda continued on down the road), I wanted to carry one of two things: a .45 Auto or a S&W .44 Magnum. Elmer Keith was my hero.
The auto was outlawed by departmental policy. That same policy, however, allowed us to carry any revolver we desired so long as it was a minimum of .38 Special and had a 3 ½” or longer barrel. This left a lot of firearms-ground to be covered. Smith’s Model 29 had been issued a scant decade before and was very, very scarce, as in impossible to find. Undeterred, I learned one of our reserves had a 6 ½” 29 he was kind of afraid of and would part with for the modest sum of $185. To my wife of the moment, this was a new living room suite. To me, it was all my National Guard summer camp pay plus $35. The choice was simple.
I still have the gun.
Acquiring about a thousand rounds of assorted .44 Mag brass I began some serious reloading sessions on an RCBS C-press. My stock load was 21 grains of 2400 behind a 240-grain Keith hard-cast, gas-checked bullet, which I cast myself. Every service station in my various beats saved wheel weight lead for me and I’m forever grateful to them all. I loaded and fired that brass until the whole lot just about gave up the ghost, as did, eventually, the revolver. But it was all worth it.
Model 27 3 ½” with Super-Vel 110-grain JHP.
Mac and Sundance in 1973. Note the 6 ½” Hoyt holster carrying
Mac’s prized Model 29 .44, peeking out of his Ike jacket.
No Duty Reloads
Obviously, reloads weren’t going to cut it for duty use, so a small stock of factory ammunition was acquired and carried. My initial carry ammo was Remington’s 240-grain JHP, which was extremely hot. Recoil was excessive but controllable, particularly after much practice. My favorite round, however, was SuperVel’s 180-grain JHP. This scooted along at about 2,000 fps and had a magnificent muzzle flash, but little apparent recoil when compared to the Remingtons. I was able to conjure up a box or two of this stuff for my photo shoot. Long out of business and very hard to find. Lee Jurras was a genius.
Initially carried in an Earl Ginn swivel/snap holster, it soon occurred to me a long-barreled revolver in a swivel holster was not exactly conducive to getting in and out of a car, chasing crooks or even comfortably walking down the street. This holster was soon swapped for a then state-of-the-art Hoyt breakfront.
There are two types of cops who’ve carried pistols in Hoyt breakfront holsters: Those who’re going to lose their guns out of them and those who already have. I soon became one of the latter, on several occasions.
Going through the academy with a .44 Magnum was an experience for all. Everyone else was shooting .38 Special 148-grain wadcutters. Not many people liked to be near me on the line. Firing prone on the outdoor range at 60 yards (the old PPC course) involved seeing through a large haze of dust and gravel kicked up with each round. The saving grace was being the top gun of that class, which made it pretty hard to argue with my choice of weaponry.
Extra ammo was carried in both loops and — also cutting edge at the time — a Multi-Loader speedloader setup also made by Earl Ginn. This consisted of two 6-round rubber cups that snapped into a plastic tube fastened to the gunbelt. For quite awhile I was the only guy on the department who used speedloaders, since they almost hadn’t been invented yet. Everyone else was stuck with drop pouches and belt loops. Everyone else was also very slow.
Model 29 4″ with Ginn Multi-Loader cup. Cutting edge technology for the time!
Big Rifle Too
The big .44 belt loops also provided the ability to carry three .444 Marlin rifle rounds on my person. At the time my “trunk” gun was a Marlin lever gun in that caliber. Gave some people quite a start when they noticed those huge rounds. Kind of a “Josh Randall” effect. Look him up on the internet if you need to.
Speaking of “trunk” guns, for the longest time I carried a regular potpourri of long guns in the trunks of my assigned vehicles, both marked and unmarked. Some were authorized; some most definitely were not. Amongst them were an M-1 Carbine, a High Standard Model 10B shotgun, an H&R Reising Model 50, a Remington 721 .30-’06, an Armalite AR-180, an M3A1 Greasegun, a Colt 1921/28 Thompson and an honest–to-gawd 1918 Browning BAR.
Those were the days, eh?
Both the .44’s shown here are “Pre-29’s,” in that they were made just before S&W assigned numbers to their gun models. That first .44 6 ½” was a 4-screw gun made around 1958. It was joined by a 4″ version, also a 4-screw, when I moved to the detective bureau. The pictured Herrett grips were swapped onto whichever gun was being currently carried. This gun was acquired from a retiring deputy who’d carried it in uniform for years.
(Clockwise, L to R) Model 27 3 ½”, Model 29 6 ½”, Model 29 4″. Multi-Loader
in .44 Mag. Ammo is Super-Vel: .357 110-grain JPH and .44 Mag. 180-grain JHP.
A True Story
One of my most memorable occurences with this gun was the day, working detectives, my partner and I drove by a 7/11 store in a seedier part of the county. Riding shotgun, I looked over and saw a guy walking into the store with a pistol in his hand. We called in a “211 in progress” (robbery) and pulled into the store’s parking lot where, bigger than life, the guy lifted and pointed a Model 92 Beretta at the two female clerks behind the counter, both of whom raised their arms and backed away. I jumped out of the car, moved to where I had a direct shot at the crook through the closed glass door and yelled something original like “Sheriff’s Office. Drop the f**king gun!”
To the guy’s credit, just as I was starting to apply pressure to the single-action trigger (bad habit I developed), that’s exactly what he did. He dropped the gun. Which saved his life.
We both ran inside and I hooked the guy up while my partner secured the dropped gun, all the while hearing (but not really “hearing”) what the clerks were yelling at us. After the guy was safe I became aware they were saying “He works here! He works here!” Seems this fool was, in fact, a store employee (but not for much longer) who’d been policing up the parking lot and found a very real looking black plastic water pistol looking like it came from the Beretta factory. He thought it’d be fun to “play” with his fellow employees. Very bad idea.
Side Note: This 21 year old kid turned out to be an okay guy and somewhat of a gun nut. I’m sure his nightstand held copies of all the latest gun rags and he was fairly gun knowledgeable. After the dust settled and I’d unhooked him, he blew my mind a bit when he asked: “Sergeant, was that a Model 29 Smith you were pointing at me?” In fact, this guy later became an IPSC shooter and we competed against each other a few times. I noted, however, he always seemed to give me a very wide berth.
Mac and “Sundance” circa 1973. Note speedloader tube on belt.
My Special Enforcement Detail in 1972, consisting of one .44 Mag, one
.41 Mag, seven .357’s, one .38 Spec. — and one neurotic Doberman.
The third and final “N” frame in this roundup is an early-50’s 5-screw “Pre-27” S&W .357 Magnum with a 3 ½” barrel. To me this revolver is arguably one of the best looking and sexiest Smiths ever made. Just something about that barrel length sets the whole thing off, particularly in profile.
This particular gun was carried by a deputy sheriff in Northern California who later worked for my old department and retired as the chief of homicide. He was a resident deputy on the California coast back in the early 60’s when he received word a San Quentin escapee was enroute up the coast in a stolen vehicle. The bad guy ultimately showed up at the deputy’s one-car roadblock, bailed out of the car and capped off a shotgun round at the vehicle-shielded cop. Choosing his moment, the deputy fired one round just as the felon stuck his head above his vehicle’s passenger front fender. The .357 round glanced off the hood and ricocheted right between the bad guy’s running lights. He was pronounced dead at the scene.
All three of the revolvers discussed in this article have been fired extensively and, in a least one case, just about worn out. All were returned, many years ago, to S&W for refurbishment. This was a service Smith offered for a long time; sadly, it’s no longer an option. The 6 ½” went back first and was returned within several months, completely reblued, retuned and frigging beautiful. I think the total charge, including shipping was around $65.
When I retired from law enforcement I was carrying a 1911, which I think is a far more desirable pistol for that intended purpose. That having been said, I wouldn’t go back and change anything about those several years I carried those big guns around. If for no other reason, it’s fun writing nostalgic articles about them
By MacGregor “Mac” Scott
Let us know your own stories about favorite revolvers! Post a comment to share.


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Is there such a thing as having branches in two different MSAs?
Yes. We currently have branches in 2 MSAs.
And if so, do we need to report for both?
Wouldn't we just report for where the main branch is located?
All loans made in the MSAs where you are located will be reported on you financial institution's LAR with the appropriate census information. Loans outside the MSA you can report NA for the census information if you wish.

domain: economics


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TroonFIT Running on the Links Series 4
Music in the Garden Fall Concert Series
Junior Golf Program
Each session will have 3 levels; Beginners, Intermediates & Advanced. To attend the Advanced group, students will need to be preapproved by a Reid West Golf Academies Instructor.
Cowboy Country Motorcycle Tour
3 Day/ 2 Night guided motorcycle tour of cowboy country & Arizona frontier towns. From $850
Embassy Suites Phoenix Airport at 24th Street
Minutes from downtown...steps from everything. Located in the heart of Phoenix, Embassy Suites Airport at 24th Street is easily accessible to all Valley business districts, museums, cultural events and sports venues.
2333 E. Thomas Rd.
Phoenix, Arizona 85016
Toll Free: (800) 362-2779
Visit the Website

domain: travel


Dear Young Students In The State Of Iowa, With An Emphasis On Students Of Color
As a part of the series "Iowa Week 2020: Listening to Black Voices" on IPR's radio show and podcast "Talk of Iowa," IPR Classical's Jacqueline Halbloom sat down to talk with world-renowned opera star Simon Estes about his career and experiences with racism. He wrote this letter to young Iowans.
"Dear Young Students in the State of Iowa, with an emphasis on students of color,
My name is Simon Estes, and I was born in Centerville, Iowa.
I grew up in a family that was financially poor but rich in love and faith. We lived in a small house and didn’t have indoor water, electricity or toilet facilities. We did have an old upright piano, and we would stand around the piano while my mother, who was self-taught, played. And we would all sing along.
When I was in elementary school, I was first recognized as a good singer by my music teacher. In junior high I was asked to sing in the high school choir, but before that could happen, the teacher had to get permission from the principle of the junior high school.
I asked the high school choir director if he thought I could make a living by singing, and his reply was yes. Since then, I have sung 102 opera roles, at 84 different opera houses, with 115 orchestras and 90 of the world’s greatest conductors, all around the world. I have sung and have recorded in German, Italian, French, Russian, Spanish and English. I have had the pleasure and honor to sing for six presidents of the United States, kings and queens, spiritual leaders such as Billy Graham, Archbishop DesMond Tutu, two different popes and the Nobel Prize Committee.
All of these wonderful experiences happened ALL because of music and using the God given talent of voice. I would like to further encourage you young people to get involved with classical music, and perhaps YOUR lives will be transformed.
All students have a better chance with academic success when involved with music, and studies have proven this to be so. In these studies, the type of music that has inspired and improved academic grades is classical music.
I am encouraging all students to go to orchestra concerts or find an instrument of your choice and learn to play. Listen to classical music and become inspired, which in turn will round out your academic learning. Historically, students of color have not had the same opportunity to be involved. I strongly encourage you to talk with your choir or band/orchestra director if you are in high school to discuss possibilities for choices of instruments or music selections.
Watch Iowa Public Broadcasting, or listen to IPR Classical, Sirius XM radio, Pandora, ITunes or other venues online to hear symphonic works.
I would like you students to know that I didn’t know anything about classical music until I went to the University of Iowa. However, I did have three older sisters, with one sister playing classical music on our piano. I encourage you to listen to classical music, even in elementary school.
Composers are talented people who have been given a gift to write music. Take some time and listen to Bach, Brahms, Beethoven, Mozart, Schubert, Handel and Haydn. These composers wrote for orchestra and opera, and some started writing when they were five to seven years of age. Most classical music was composed by people from foreign countries such as; Germany, Austria, France, Russia, Italy and others. There are American composers such as: Copland, Rogers and Hammerstein, Gershwin and Burleigh.
I could write a whole book about classical music and the reasons why I feel it’s so important. I just want to encourage you to get involved in classical music. Share your desire for classical music, and ask your parents or teachers how to start to get involved.
I have emphasized classical music, but there are other forms of music that are also healthy to listen to, such as: Broadway Melodies, Jazz, Negro spirituals, Gospels, and Hymns. They will inspire you. They do that for me."
May God bless you,


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Year 8 Battlefields Trip
Earlier this week the History Department ran 2 trips to visit the Battlefields in Ypres, Belguim
Year 8 student Thea.S wrote this review of the trip:
As many parents/carers will know, a number of year 8 students have had the fantastic opportunity to go on a school trip to Belgium for 2 days, of which we have been able to experience the horrific scale of those who gave their lives in WW1. This was completed via a coach tour from England, through to France, and finally to Belgium. From cemeteries to indulgent Belgian Chocolate tasting - here’s what our History trip brought us:
To start with, the journey was taken via coach, until we reached the Euro Tunnel, where our coach was parked in the Euro Tunnel’s vehicle’s train. The journey was unbelievably quick - just 35 minutes and we were in France! It wasn’t long before we reached Belgium, and stopped at the Sanctuary Wood Museum to have lunch. The museum was a place where you could see just the type of experience those soldiers had during The Great World War - we were even allowed to explore the trenches and tunnels that those very men fought in - I found it extremely cramped and dark even with a torch… We then headed off to the Tyne Cot Memorial, the largest British memorial in the world - I was shocked at the extent of the number of graves placed here - almost 12,000 - with 8,369 of them unidentified.
After travelling for around 15 minutes, we briefly visited the Langemark German Cemetery, and yet again experienced the sheer number of lives lost in WW1, with hundreds of black square plaques remembering German lives. Visiting the Memorial Museum Passchendaele, however, provided us with a different approach to our learning. Here we learnt about the lifestyle of soldiers in the trenches: the medical care, the food they ate, and the artillery they used. There were some trenches here as well, a similar layout to the Sanctuary Wood Museum. I found this museum very informative and interesting.
As the sun faded into an evening light, we finished the day off with a filling dinner and a ceremony at the Menin Gate. This ceremony has taken place every evening at 20:00 since the 2nd of July 1928, it serves to remember those who fought in the war. This was followed by a trip to the local Belgium Chocolate shop, before heading off to bed. The next day, we set off to the Vimy Ridge, a Canadian warsite filled with huge bomb and mine craters, surrounded by masses of forest - again showing the awful number of lives lost. We later found out that every tree planted represented a Canadian death in the trenches. I personally found this tour particularly interesting, yet harrowing, as we were shown around the tunnels below and found out that they were actually carved out by hand - miners using their fingernails as digging tools in order to keep as quiet as possible. These were used to keep military lives safe and protect runners from getting killed while carrying messages to places. The tunnels lead to the trenches outside, with the Canadian offensive line just a couple of metres away from the German defence line. Finishing off that tour, we headed to Newfoundland Park, another Canadian remembrance site, where we had lunch.
After lunch, we visited Thiepval, a cemetery with names of fallen soldiers on every wall. Personally, this site hit me hard - the list of missing soldiers was never-ending, showing the true impact of war. We finished our trip at the Devonshire Cemetery, a small gravesite with a sad story behind it - lack of faith in British troops resulted in more lives being unnecessarily lost.
With this amazing experience behind us, we headed home with great pride of what our country has done to preserve our culture. No one could have prepared us for the impact of the Great World War and the heart-wrenching number of lives lost over the course of just 4 years. A big thank you to all of the teachers who gave up their time to support us on this trip.


Marantz is showcasing two networked audio/video systems, featuring four of its recently launched products at CES 2011. The two Marantz audio/video systems being showcased include one centered on the company's M-CR603 Network CD Receiver (SRP: $699), which shipped just last month, and another that features the innovative "game-changing" NA7004 Network Audio Player (SRP: $799). In addition to the M-CR603 and NA7004, Marantz will showcase its SR7005 A/V Receiver (SRP: $1,599) and AV7005 Preamplifier (SRP: $1,499). All four products are available.
• Explore more AV Receiver news from HomeTheaterReview.com.
• Read Dr. Ken Taraszka's review of the Marantz SR7005 AV Receiver.
• Learn more about AV Preamplifiers on HomeTheaterReview.com.
Marantz will demonstrate the new AirPlay music streaming capability with its compatible Marantz components at CES. AirPlay allows users to stream songs directly from their iTunes music libraries, as well as music and photos though their Wi-Fi networks from their iPhone, iPad or iPod Touch devices. The AirPlay upgrade, available for use with all of the new, IP-based Marantz products, including the Model SR7005 A/V Receiver, AV7005 A/V Preamplifier, NA7004 Network Audio Player and M-CR603 Networked CD Receiver, is priced at $49.99.
In addition to its 50-watt per channel digital amplifier and AM/FM radio, the component features a CD player, plus access to thousands of Internet radio stations, and allows for streaming of popular music services Rhapsody, Napster, and Pandora. The M-CR603 may be used as a connected zone player for users who already have a receiver-based home theater system in their family room, allowing for easy control of different zones of audio throughout the home. Features such as a front panel USB input for iPod add to the M-CR603's convenience and flexibility, and it can also be used as a D/A converter for those who have downloaded music on their PCs.
The Marantz NA7004 is a component that serves as a digital-to-analog converter for people with large collections of downloaded high-resolution music on their computers or other external storage devices who want to listen through their home audio systems.
The NA7004 incorporates a built-in Internet radio, and streams popular music sources such as Napster, Rhapsody, and Pandora. Among its key performance and convenience features are a USB front-panel input for digital connection to iPod and iPhone devices and a back panel M-XPort for wireless Bluetooth streaming with optional RX101 Bluetooth Receiver. The NA7004 also includes access to over 14,000 Internet Radio stations. File types supported include FLAC, 96/24FLAC, WAV, WMA, WMA Lossless, MP3, MPEG-4 and AAC. The USB Audio interface supports resolutions up to 24-bit/96 kHz; COAX and OPTICAL digital audio inputs are also supported.
The AV7005 Preamplifier takes the SR7005 feature set to the next sonic level with a regulated power supply featuring a custom wound transformer, and a dedicated preamplifier stage with symmetrical layout, gold-plated relays, and audiophile grade capacitors. Fully DNLA 1.5 compliant, the AV7005 integrates into the home network, and includes a built-in Internet radio, plus streaming of popular music sources such as Napster, Rhapsody, and Pandora as well as image streaming via Flickr. It features 6 HDMI 1.4(3D/Audio Return Channel) inputs, with 2 HDMI outputs, all ready for 3D. A front panel HDMI input allows for easy connectivity of camcorders and video game sources. The unit's M-XPort (Marantz eXpansion Port) delivers Bluetooth reception via the RX101 Bluetooth Receiver (sold separately).


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BEAUTIFUL GARDEN DECOR: This garden trellis brings a touch of European chic / Southern Charm to your garden with its delicate lines, patterns, and elegant scrollwork. Put it a few inches into the ground so as to keep it standing firmly. Display small hanging accents to create a. 2.1M/85 Metal Garden Arbor Arch with Gate, Backyard Decor Climbing Planter Frame.Create an elegant garden feature in your own backyard for special events or everyday use - Use as an attractive archway for climbing vines and other outdoor plants - Constructed of steel tubing and coated in weather-fighting black epoxy - Double gate design creates a sense of privacy and can be used to close off an area - Put it a few inches into the ground so as to keep it standing firmly - Versatile for wedding, ceremonies and outdoor venues - Easy to assembly with detailed instruction included. The classic design of this Outsunny garden trellis provides an easy and attractive way to add an elegant feature to your garden. Use it in your everyday life as an attractive garden arbor for climbing plants or decorate the arch with flowers or balloons for weddings and other special events. The latched, two-door gate on the garden arch features intricate scrollwork and 4-leaf clover for style and good wishes.
This makes it a perfect addition for weddings, ceremonies, and other outdoor events. Check out this garden gate from Outsunny, and add an awesome addition to your outdoor garden or backyard. Color: Black - Material: Metal Frame - Overall Dimension: 42.5" W x 17.75" D x 84.75" H - Single Door Panel Size: 41.25" W x 17.75"D x 83.5" H - Gate Vertical Bar Spacing: 4.3 - Net Weight: 24.2 lbs. We are committed to offering our customers value-oriented items in Outdoor & Patio, Home Goods, Health & Beauty, Sporting Goods, Pet Supplies, Toys & Hobbies.
We are always looking tobring new innovative items to our catalog so contact our Customer Service department with any suggestions you may have! Occasionally a warehouse is out of the product and the warehouse further from you will have to fulfill the order which could make the ETA up to 6 days. And you will be able to redirect your package(s). For items that are defective or damaged, Aosom may require pictures to help us determine the appropriate next step, which may be replacement parts, or a replacement unit. In this day and age, we understand people's time is valuable, so supplying pictures when you message us will help us assist you faster.The item "2.1M/85 Metal Garden Arbor Arch with Gate, Backyard Decor Climbing Planter" is in sale since Saturday, February 6, 2021. This item is in the category "Home & Garden\Yard, Garden & Outdoor Living\Garden Structures & Shade\Arbors & Arches". The seller is "mhcorp" and is located in Wilsonville, Oregon. This item can be shipped to United States.

domain: design


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Curious to see what your next product will be. The intuitive next step would be to go bigger, but I hope you will make another system in the travel market with 12V DC. Learn from the KiweeOne in use. Maybe target a much lower price and higher number of units.
About pricing luke, Crowdfunding price is around 400 USD, retail price will be around 1000 USD.
Did you mean that to you, Crowdfunding price is still too high or are you talking about current retail price?
I was talking about the market of your next product after KiweeOne.
For me personally, being grounded in a place without very sparse wind, any price is too high.^^ Even though it would be really nice to hold one in hand and test it.
Perhaps there are two mixable possibilities in time:
Lower the price year after year, increasing the production.
Keeping the price then increasing the power year after year until hundreds W.
An example for the second possibility: now a Kiwee is 100 W. Assuming a Kiwee will be 200 W in a year: it is now paid and delivered in one year.
This is also a way to involve partners as Kiwee scales. This way would mix buying and investment.
I would say collect feedback from customers before decidibg where to go next. Sell the exact same model for some time still.
You could go for some of these features:
- more power
- more usability
- higher altitude
- lower price
- lower weight
I would say that given the fact that you have already found out that the market is probably there, the most interesting would be to pursue lower price as the most important one. Even perhaps reduce altitude and power to get there (depending on feedback). My second one would be more usability so that your customers who are not kite interested are still happy with the product.
One way to reduce weight and cost would be to go single skin kite. Second perhaps would be a soft kite rotor, perhaps with rings like Rod is using.
I hope that you are not selling the crowdfunding product at a loss. So I’ll guess the cost to you for the product is between 200 and 300 euros. If you plan to sell it for a 1000 euros I expect the market from individual customers will be relatively small - but I’d be happy to be proven wrong. Businesses may see some value in it as an advertising platform.
I think that adds development time and with that cost. I would look at how the product is sourced and made, and how the business is run.
The cost of development is hard to quantify if you are a small company and the employees sometimes find time to test new stuff.
Having come so far, my guess is that @Kitewinder has a better guess about the next steps compared to us bystanders. Having an opinion is still fun though
Crowd price was around 60 % off @ 375 euros. So yes retail price is 900 euros not 1000 sorry.
Regarding benefit and production price, those questions are highly strategic sorry.
Just consider that average sales margin of any product is between 20 to 30 %.
Uses cases like phone charging and lighting are kiwee one’s use cases
I know. I was thinking about which the wisest steps are for scaling small awes. For example what demand would your 500W to 1kW System address? Would it be wiser to design it for 500W or 1kW? What can one do with that power?
@Tom, water treatment is a great use case for such product
I like how for water treatment a tank is enough to compensate for the intermittency of wind power.
Water treatment by electrolysis? What about desalination (by electrolysis)?
Olivier, for later do you envisage a crosswind version of kiwee, with automated take-off, flight by figure-eight or other, and landing?
All I know at the moment is that it will be a x5 to x 10 kiwee power with automatic launch and landing… Regarding crosswind flight, we might someday start to oscillates gently around zenith to slightly increase speed. Complete crosswind is more challenging. My guess is that crosswind awe will take at least 10 years to be fully operational, that leave us some time to test things.
Loyd’s patent looks to represent a crosswind kite with crank then reciprocating transmission between the plane and the ground station, with members within the tether. So it is not a belt or rope-drive transmission.
US4251040A.pdf (617.2 KB)
That sounds like a really nice package. Good to see you really forward leaning in terms of releasing bigger/better products
Automatic launch and landing would require a drastically different design, won’t it? To me it seems more difficult than crosswind flight.
Could also be possible to get more out of the current design, especially a higher capacity factor, by getting it to higher altitudes. Longer distance between lifter and turbine to have a higher windspeed for the lifter before it needs to lift the turbine and a longer rope drive are probably worth investigating.
Why not just scale up the current design? (with wings that can be disassembled maybe)
Maybe target the same consumers (phone, radio, laptop, cooler) up to 100W, but useable all day with awes operating only a short time. This would require a major battery pack. Nothing one would use when hiking or biking, but possible on a boat or a car. Electric car charging would also be interesting.
This consideration leads us back to this:
Kiwee one blades are already foldable.
We have been granted by ADEME, the French agency for climate environment and so on, to develop a 3 phases program, second phase is the product I described. So now we have to do it because we get money for that. As Luke said there is plenty possibility for kiwee. I don’t know If you have seen our latest videos but kiwee was outputting 250W. Best ever for us. Automatic launch will not be a major issue, you have to have the ability to inflate your soft kite, that pretty it… But we will see. Anyway I won’t start that product before kiwee success. I was thinking you guys will crucify me to says crosswind will be ready in 10 years


We all use hot water for many of our daily tasks. From showering to cooking, hot water is an important part of a comfortable life for families and employees. The moment your water heater stops working is also when your level of comfort decreases. If you're dealing with a broken water heater, call Woodbury Center Plumbing right away. We can complete water heater replacements and repairs for Woodbury Center CT area homes and businesses.
If you have a water heater, it's crucial to get it checked every so often by an experienced technician or repair guy. This is because an uncomfortable temperature may lead to illness in many instances. For instance, if the temperature is too cold, you may like to take a bath as opposed to use the washroom.
Some major problems that are a standard part of many Woodbury Center water heaters are the ones that need to be dealt with by a professional. If you realize that the burners are not properly installed, or they've been damaged somehow, you'll need to contact a/an Woodbury Center heating and air conditioning service company to come out and take a look at the device. Because there are such lots of heating and air conditioning services offered from the Woodbury Center area, you might find that it is hard to choose which one you wish to work with.
One of the best things which you can do when you are looking for a excellent hot water heater repair in Woodbury Center CT is to hire a company that has a lot of experience dealing with the different versions of hot water heaters. There are a few repair businesses that are actually specialize in certain makes of water heaters, like ones made by General Electric or Westinghouse. If you wish to manage a company that specializes in making repairs for these models of hot water heaters, then you may wish to take the time to research on the internet and find one that does have plenty of experience with them. These types of companies can help save you money and time when it comes to hot water heater repair in Woodbury Center CT since they know just how to work on these different models of water heaters.
Water heater repairs may be what most folks think of as the crucial part of having a home. If your hot water heater malfunctions, you can save hundreds of dollars on the price and hassle of replacing an old, outmoded water heater with a new gas or electric model. While water heaters are generally low maintenance appliances, they can and do break from time to time, sometimes needing hot water heater repair in Woodbury Center CT. In some cases, it is possible to have hot water heater repair in Woodbury Center CT repaired without buying a new unit.
Natural gas heaters and gas water heaters equally operate on natural gas or propane. They use a storage tank that has to be filled with fuel oil or natural gas. The two types of units must have their gas tank inspected periodically to make sure it is not leaking. Gas storage tanks may also experience leaks at any time. Leaks in these tanks must be repaired and the gas pump replaced whenever they cannot be repaired.
If you decide to go with a particular company due to their glowing reviews and satisfied customers, then inquire about their rates for Woodbury Center water heater installation. Make certain to ask them if they will be installing the device in your house or if they will come to it. If you hire a business that does both, you will be able to cut down on costs quite a bit.
The most frequent reason why people have difficulties with their hot water heaters is because of sediment build-up. This is something which you will want to keep in mind when considering an emergency repair in or Woodbury Center, Connecticut. You want to have your device checked out immediately if you discover sediment buildup in it. For those who have any sediment build-up in it, then you need water heater repair services straight away.
Moreover, for those who have a leaking hot water tank, it is a good idea to call in a professional immediately. A leaking hot water tank induces an immediate need to heat up the water from the tank, so it is very important that you do not wait before calling a plumber. If you are going to attempt to heat the tank by yourself, there's a chance that you will burn a few of the fittings or damage the tank beyond repair. Plus, the heat from a leaking hot water tank may actually spread itself throughout the home and really cause a fire in some cases.
The Woodbury Center electric or gas water heater repair companies generally offer free estimates on almost all types of repair works for all sorts of models. Whether you have an electric or gas heater, they will provide you a quote over the phone based on the time required to fix the problem. If the repair is minor, then you can always schedule another trip after some hours or days, according to the circumstances. Some companies even offer extra services like giving you tips on how best to maintain the equipment so that it lasts longer.
When you are considering water heater repair in Woodbury Center Connecticut, you want to consider the differences between gas and electric. Although both are powerful when it comes to heating your home, they are two distinct kinds of heaters. If your water heater is not functioning properly, you may simply have to have a professional come to repair it. While this may cost you more in the end, you can rest assured knowing that your house is secure and that the problem was taken care of.
Areas We Cover Include: 06798


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I have been suffering from my shoulders for the last three months.
I think it all started when I was going to the gym and working on my shoulders.
Now, when I roll my shoulders to the back, there are sounds coming from inside which I assume are from the bones in the joints.
If I continue to roll my shoulder faster or do other stretches with my arm I get a massive pain that affects the whole of the top of my back and waist.
I went to a doctor and he told me it's nothing important, but I believe there is something wrong. Who's right?
It is unlikely that you have injured any bones since you are able to roll your shoulders.
The noises you hear are caused by the tendons moving within the shoulder joints.
You may have damaged the soft-tissues (muscles and ligaments) if you were over-doing the gym exercises or lifting heavy weights.
Ease up on the upper body exercises for six to eight weeks to allow the tissues to recover.
You would do well to visit a physiotherapist to let a skilled professional assess your back and shoulders.
Your GP should be able to recommend one in your area and refer you.
The NetDoctor Medical Team
Other Qs & As


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Cricket: Donald Bradman’s debut baggy green auction result revealed
Winx sits comfortable alongside Australia’s greatest athletes
How a Bradman trait could be bring down Indian star Virat Kohli
Australia's position has been rattled by England on day two
A small but significant piece of history has been found in Gladdy.


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The 21st International Film Festival of Kerala began in Thiruvananthapuram on 09 December 2016. It opened with Navid Mahmoudi’s directorial debut ‘Raftan,’ which narrates the story of a couple attempting to migrate from Iran to Europe. The eight-day festival, hosted by the Kerala State Chalachitra Academy on behalf of the Kerala Cultural Affairs Department, is set to showcase 184 films from 62 countries.
So, let’s discuss some questions related to this post:
Q1. In which city The 21st International Film Festival of Kerala began?


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By John Hertz: It occurred to me, after mailing this thought in a letter of comment to another fanzine, that you might be interested also.
People talk – and quarrel – about what might be, or be candidates for, the great American novel.
Now and then some speculative fiction is proposed.
What about these (of course neither complete nor conclusive)?
- A Canticle for Leibowitz (Miller, 1960)
- A Connecticut Yankee in King Arthur’s Court (Twain, 1889; you can see a note by me via a set of links on the sidebar)
- The Wonderful Wizard of Oz (Baum, 1900)
- Alas, Babylon (Frank, 1959)
- The End of Eternity (Asimov, 1955 – see my note on it too)
- Space Cadet (Heinlein, 1948)
You might like to bear in mind a criterion I’ve used in another context.
A classic is an artwork that survives its time; after the currents which might have sustained it have changed, it remains, and is seen as worthwhile in itself.
Over a door at the central branch of the Pasadena Public Library is (adapted from Mary Carolyn Davies, in The Skyline Trail, 1924)
Be made whole by books as by great spaces and the stars.
“Some are born great, some achieve greatness, and some have greatness thrust upon ’em”, says Malvolio in Shakespeare’s Twelfth Night (Act II, sc. 5); others learn that, though he is ridiculous, they should not be harsh

domain: literature


CONTINUED FEDERAL SHUTDOWN WILL FORCE LOS ANGELES COUNTY FAMILIES INTO HOMELESSNESS
City of Alhambra – October 3, 2013 – (RealEstateRama) — With the second day of the Federal shutdown in effect, local governments are bracing for the worst. The Housing Authority of the County of Los Angeles (HACoLA) operates affordable housing programs, such as the Housing Choice Voucher Program (Section 8), which rely on the Federal government for their funding. Section 8 provides housing to over 23,000 households in Los Angeles County, including seniors, disabled, homeless, mentally ill and the working poor. Because the earlier sequester required public housing authorities to draw down reserves from the U.S. Department of Housing and Urban Development (HUD) earlier in the year, HACoLA funding is nearly exhausted.