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Research | Open | Published: 24 November 2008
Sensitization of ovarian cancer cells to cisplatin by genistein: the role of NF-kappaB
Leigh A Solomon1,
Shadan Ali2,
Sanjeev Banerjee3,
Adnan R Munkarah4,
Robert T Morris1 &
Fazlul H Sarkar3
Platinum-resistance (PR) continues to be a major problem in the management of epithelial ovarian cancer (EOC). Response to various chemotherapeutic agents is poor in patients deemed PR. Genistein, a soy isoflavone has been shown to enhance the effect of chemotherapy in prostate and pancreatic cancer cells in vitro and in vivo by reversing chemo-resistance phenotype. The goal of this study was to investigate the effects of combination therapy with genistein and cisplatin as well as other cytotoxic conventional chemotherapeutic agents in platinum-sensitive (PS) and resistant EOC cells.
The PS human ovarian cancer cell line A2780 and its PR clone C200 cells were pretreated with genistein, followed by the combination of genistein and either cisplatin, taxotere or gemcitabine. Cell survival and apoptosis was assessed by MTT and histone-DNA ELISA. Electrophoretic mobility shift assay (EMSA) was used to evaluate NF-κB DNA binding activity. Western blot analysis was performed with antibodies to Bcl-2, Bcl-xL, survivin, c-IAP and PARP.
Reduction in cell viability, and corresponding induction of apoptosis was observed with genistein pretreatment followed by combination treatment with each of the drugs in both cell lines. The PS cell line was pretreated for 24 hours; in contrast, the PR cell line required 48 hours pretreatment to achieve a response. The anti-apoptotic genes c-IAP1, Bcl-2, Bcl-xL, survivin and NF-κB DNA binding activity were all found to be down-regulated in the combination groups.
This study convincingly demonstrated that the current strategy can be translated in a pre-clinical animal model, and thus it should stimulate future clinical trial for the treatment of drug-resistant ovarian cancer.
There will be an estimated 15,520 deaths from ovarian carcinoma and 21,650 new cases diagnosed in 2008 [1]. Unfortunately, at the time of diagnosis the majority of patients will have disseminated disease. Resistance to platinum-containing regimens and tumor heterogeneity confers a poor prognosis in patients with epithelial ovarian cancer. Platinum-resistance is a complex issue and is currently believed to be associated with an unstable phenotype of ovarian cancer cells that are believed to be altered by tumor microenvironment and exposure to other drugs [2, 3]. Acquisition of chemo-resistance is one of the major limitations for the use of platinum complexes in cancer chemotherapy. Proposed mechanisms of cellular resistance include decreased cellular uptake of the toxic drug, increased cell efflux of the drug, improved cell DNA damage repair and the prevention of DNA cross-linking. These may be intrinsic properties of some cancer cells or acquired mechanisms due to exposure to chemotherapeutic agents.
Over the past few years it has been shown that a small portion of cancer cells known as "cancer stem cells" or "cancer stem-like cells" are responsible for the antagonism of the disease, resistance to therapy, self-renewal and unlimited proliferation in several cancers, including ovarian cancer [4–7]. Moreover, mutations may be one of the major factors contributing to the origin of ovarian cancer stem cells. Emerging evidence suggests that ovarian cancer stem cells are relatively resistant to conventional cytotoxic chemotherapeutic agents [8]. These therapies often cause severe toxicity because of their general effects on all rapidly dividing cells. It is important that we use targeted agents that discriminate between cancer stem cells and normal stem cells. One such agent which has been studied in our laboratory and by others is "genistein", a naturally occurring isoflavone present in soybeans has proven to have anti-tumor activity with minimal or no toxicity to nonmalignant human cells [9, 10]. Moreover, the incidence of ovarian cancer is approximately 10–50% lower in Asian countries compared to the United States [11], which could be associated with dietary factors. Asian women who migrate to the United States and their descendents seem to maintain the decreased risk [11]. In a case control study in Southeast China, Zhang, et al, found the odds ratio of developing ovarian cancer with a diet high in genistein to be half that of controls [12], and suggest that soy isoflavone may contribute to reduced cancer risk in Asian population [12].
Studies of various cancer cell lines, in our laboratory and others, have shown that treatment with the isoflavonoid genistein can inhibit cell proliferation. In the breast cancer cell line MDA-MB-231, treatment with genistein affected cell growth and apoptosis-related gene expression via a p53 pathway [13]. In some prostate cancer cell lines, genistein treatment leads to inactivation of the nuclear transcription factor Nuclear Factor-kappa B (NF-κB) via the Akt signaling pathway [14]. Other investigators have shown that the PTEN gene may also reverse chemo-resistance to cisplatin in ovarian cancer through inactivation of the PI3K/Akt cell survival pathway and can be a potential target for the treatment of chemo-resistant cancer [15]. Moreover, genistein also potentiated growth inhibition and apoptosis in certain pancreatic cancer cells by inhibiting Akt and NF-κB [16]. NF-κB is an important regulator of genes involved in cell survival and proliferation; it also plays an important role in the apoptotic pathway [16]. Additionally, tissue transglutaminase, an enzyme involved in protein cross-linking prevents apoptosis induced by cisplatin by activating the NF-κB survival pathway in ovarian tumors [17].
In a recent article it was reported that genistein induces apoptosis in ovarian cancer via different molecular pathways in both wild type and mutated BRCA1 estrogen receptor positive tumors [18]. Genistein also caused cell cycle arrest at G2/M phase in both dose- and time-dependent manner without causing any cytotoxicity [19]. Genistein can also induce both apoptosis and autophagic cell death in ovarian cancer cells [20]; however the role of genistein in chemo-resistant ovarian cancer cells has not been investigated. Therefore, the intent of this study was to evaluate the effect of genistein for sensitization of ovarian cancer cells to conventional cytotoxic chemotherapeutic agents by assessing the effects of combination treatments on cell growth, apoptosis and the DNA binding activity of NF-κB using a paired isogenic cisplatin-sensitive and a cisplatin-resistant ovarian cancer cell line.
Cells, Drugs and Reagents
Paired isogenic cisplatin-sensitive human ovarian cancer cell line A2780 and its cisplatin-resistant clone C200 were received as a generous gift from Dr. Thomas C. Hamilton of Fox Chase Cancer Center, Philadelphia, PA. They were maintained in RPMI media supplemented with 10% fetal bovine serum, insulin and penicillin and streptomycin. C200 cells were grown with cisplatin (3 μM) every 3 passages to maintain resistance. Cells were incubated at 37°C with 5% CO2. Taxotere (Aventis Pharmaceuticals, Bridgewater, NJ), was dissolved in DMSO to make a 4 μM stock solution. Cisplatin (Sigma) was dissolved in phosphate buffered saline to make a 1 mM stock solution. Genistein (Toronto Research Chemicals, Inc, ON, Canada) was dissolved in 0.1 M NaHCO3 to make a 10 mM stock solution.
Cell Viability Assay
Paired cells A2780 and C200 were chosen for this study. Cells (2–5 × 104) were seeded in a 96-well culture plate and incubated overnight. Cells were treated with varying concentrations of genistein (5–25 μM), cisplatin (100–2000 nM), taxotere (0.5 – 2 nM) and gemcitabine (10–100 nM) for 48–96 hours. Subsequent experiments were performed with doses that achieved a 40–60% decrease in cell viability in the platinum-sensitive cell line. Cells were pre-treated with genistein for 24 hours followed by combination treatment with genistein and either cisplatin, taxotere or gemcitabine for an additional 48 hours. After 72 hours of total treatment, the cells were incubated at 37°C with 1 mg/mL MTT reagent (Sigma, St. Louis, MO) for 2 hours. The formazan crystals were dissolved in isopropanol. Spectrophotometric absorbance of the samples was determined by the Ultra Multifunctional Microplate Reader (Tecan, Durham, NC USA) at 595 nm. When initial experiments with genistein and cisplatin did not show a significant effect in the resistant cells (C200), the pretreatment interval was increased to 48 hours with genistein alone, followed by 48 hours of combination treatment and the doses of genistein, gemcitabine and taxotere were increased in the C200 cell line.
Quantification of apoptosis by ELISA
The Cell Death Detection ELISA Kit (Roche, Palo Alto, CA USA) was used for assessing apoptosis in A2780 and C200 cells treated with genistein, cisplatin, taxotere, gemcitabine and their combinations according to the manufacturer's protocol. Briefly, A2780 and C200 cells were treated with 10–25 μM genistein, 250 nM cisplatin, 1–2 nM taxotere and 2–50 nM gemcitabine and the combination of these drugs for 72 to 96 hours. After treatment, the cells were trypsinized and 10,000 cells were added to lysis buffer. The cells were then centrifuged at 20,000 × g for 10 minutes and the supernatant was transferred into anti-histone-coated microtiter plates and incubated at room temperature for 90 minutes. This was followed by anti-DNA-peroxidase incubation for 90 minutes. After unbound antibodies were removed, the nucleosomes were quantified by color development with substrate. The optical densities of the samples were determined by the Ultra Multifunctional Microplate Reader (Tecan, Durham, NC USA) at 405 nm.
Protein extraction and Western blot analysis
A2780 and C200 cells were seeded in 100 mm dishes and allowed to attach for 24 hours. Cells were then treated with 10–25 μM genistein, 250 nM cisplatin, 1–2 nM taxotere and 2–50 nM gemcitabine and the combination of these drugs for 96 hours to evaluate the effects of treatment on expression levels of survivin, Bcl-2, Bcl-xL, c-IAP1, and was also used for assessing PARP cleavage, an indirect measure of apoptosis. The experiment was carried out for a minimum of three times. Cells were harvested by scraping from culture plates and collecting by centrifugation. Cells were resuspended in lysis buffer consisting of 250 mM NaCl, 50 mM Tris buffer (pH 7.5), 5 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM sodium fluoride, 1 mM sodium orthovandate, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 μg/ml pepstatin and a protein inhibitor which contain a broad spectrum of serine, cysteine and metalloproteases (Roche Applied Science, Indianapolis, IN) for 30 minutes on ice. Cell lysates were centrifuged for 20 min. Protein concentration was measured using BCA Protein Assay Kit (Pierce Rockford, IL). The samples were loaded on 7–12% SDS-PAGE for separation and electrophoretically transferred to a nitrocellulose membrane. Each membrane was incubated with monoclonal antibody against Survivin (R & D Systems, Inc. Minneapolis, MN), Bcl-2 (1:200, Calbiochem, San Diego, CA), Bcl-xL, c-IAP1 (Santa Cruz Biotechnology, Santa Cruz, CA), PARP (Biomol, Plymouth, CA), and β-actin (Sigma, St. Louis, MO). Blots were washed with phosphate buffer containing 0.05% Tween (PBST) and incubated with secondary antibodies conjugated with peroxidase. The signal intensity was then measured using chemiluminescent detection system (Pierce Rockford, IL).
Electrophoretic Mobility Shift Assay (EMSA) for NF-κB activation
EMSA was performed using the Odyssey Infrared Imaging System with NF-κB IRDye labelled oligonucleotide from LI-COR, INC. (Lincoln, NE). The DNA binding reaction included 5 μg of the nuclear extract mixed with oligonucleotide and gel shift binding buffer consisting of (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.25 mg/ml poly(dI): poly(dC). The reaction was incubated at room temperature in dark for 30 minutes. 2 μl of 10× Orange G loading dye was added to each sample and loaded on the pre-run 8% polyacrylamide gel and ran at 30 mA for 1 hour. NF-κB p65 antibody was used to confirm the super shift and the Rb antibody was used for assessing protein loading control.
Comparisons of survival, and apoptosis between the groups were undertaken by the Student t test. Statistical significance was assumed for a P value of ≤ 0.05.
Effects of genistein, cisplatin, gemcitabine and taxotere on the viability of A2780 and C200 ovarian cancer cells
The viability of A2780 and C200 cells treated with genistein (10–25 μM), cisplatin (250 nM), gemcitabine (2 – 50 nM) and taxotere (1–2 nM) were determined by the MTT assay. The platinum-sensitive A2780 cells were more sensitive to each drug than the platinum-resistant C200 cell line. Pretreatment of the A2780 cells for 24 hours with 10 μM of genistein followed by the combination treatment for 48 hours with each conventional chemotherapeutic agent resulted in a greater and significant inhibition of cell viability compared to each agent alone. On the other hand, C200 cells pretreated with higher than 10 μM genistein concentrations for more than 24 hours (48 hours) followed by combination treatment for additional 48 hours with higher concentration of each conventional chemotherapeutic agent than what was used for A2780 cells also resulted in a significant inhibition of cell viability (Figure 1). Our results showed that genistein can sensitize even the drug-resistant cell line and cause inhibition of cell viability. Further, to assess whether the loss of overall cell viability could also be due to the induction of apoptotic cell death, we examined the effects of genistein, cisplatin, gemcitabine and taxotere, and the combination treatments on apoptotic cell death.
Growth inhibition of human ovarian cancer cell lines A2780 (A) and C200 (B) treated with genistein (Gen), cisplatin (Cis), gemcitabine (Gem), taxotere (Tax) alone and the combination treatments were evaluated by the MTT assay. A2780 cells were treated with genistein (10 μM), cisplatin (250 nM), gemcitabine (2 nM) and taxotere (1 nM) and the combination treatment; and the C200 cells were treated with higher doses of genistein (25 μM), gemcitabine (50 nM) and doxetaxel (2 nM) as described under Materials and Methods. There was a significant reduction in the overall cell viability of A2780 and C200 cells treated with the drug combinations compared to cells treated with either drug alone. P values shown represent comparisons between each drug alone and the combination of both drugs using t-test.
Induction of apoptosis by genistein, cisplatin, gemcitabine and taxotere in A2780 and C200 ovarian cancer cells
Apoptosis assays were performed using the A2780 and C200 cell lines to evaluate the mechanism on the inhibition of cell viability using the Cell Death Detection ELISA. For the platinum-sensitive A2780 cell line, 24 hours pretreatment with 10 μM genistein followed by the combination treatment with 250 nM cisplatin, 2 nM gemcitabine and 1 nM taxotere for 48 hours showed a significant increase in apoptosis compared to either drug alone. Increasing the pretreatment interval to 48 hours with 25 μM genistein followed by 48 hours of combination treatment with 250 nM cisplatin, 50 nM gemcitabine and 2 nM taxotere for 48 hours also showed a significant increase in apoptosis compared to either drug alone in resistant cell line (Figure 2). Subsequently, we sought to find further evidence of apoptosis, as presented below.
Induction of apoptosis in human ovarian cancer cell lines A2780 (A) and C200 (B) treated with genistein (Gen), cisplatin (Cis), gemcitabine (Gem) and taxotere (Tax) alone and the combination treatments were evaluated by the ELISA assay. A2780 cells were treated with genistein (10 μM), cisplatin (250 nM), gemcitabine (2 nM) and taxotere (1 nM) and the combination treatment; and the C200 cells were treated with higher doses of genistein (25 μM), gemcitabine (50 nM) and doxetaxel (2 nM) as described under Materials and Methods. There was a significant potentiation in the induction of apoptosis observed in A2780 and C200 cells treated with both genistein and the other drugs as compared to cells treated with either agent alone.
The effects of genistein pretreatment and combination treatment on molecules related to apoptosis in A2780 and C200 cells
The mechanisms contributing to the potentiation of apoptosis by genistein pretreatment and combination treatment were evaluated in the A2780 and C200 cells. PARP cleavage was determined in A2780 and C200 cells that were treated with genistein (25 μM), cisplatin (250 nM), taxotere (2 nM), gemcitabine (50 nM) alone or the combination treatment of cells pretreated with genistein (Figure 3). We found significantly increased PARP protein cleavage product (85 kDa fragment) after 72 h treatment in A2780 cells (Figure 3). In contrast, C200 cells treated similarly showed comparatively less intense cleaved PARP with combination treatment only. The induction of apoptotic cell death could in part be due to inactivation of important survival genes; and therefore the expressions of pro-survival and anti-apoptotic molecules such as survivin, Bcl-2, Bcl-xL, and c-IAP1, which are transcriptionally regulated by NF-κB, were also evaluated. Expression of Bcl-2, Bcl-xL, survivin, and c-IAP1 proteins were significantly reduced in cells treated with the combination compared to either agent alone in both A2780 and C200 cells. These results suggest that genistein in combination with conventional therapeutics could down-regulate key survival proteins and, in turn, induced apoptotic cell death of both A2780 and C200 cells. Since we found a greater degree of down-regulation of survivin, c-IAP1, Bcl-2, Bcl-xL in cells treated with genistein in combination treatment compared to single agent treatment, we investigated the effect of each treatment on the DNA binding activity of NF-κB.
The expression of c-IAP1, Bcl-2, Bcl-xl, survivin and PARP in A2780 and C200 cells. Cells untreated or treated with 10 or 25 μM genistein (Gen), 250 nM cisplatin (Cis), the combination (Gen + Cis), 1 or 2 nM taxotere (Tax), the combination (Gen + Tax), 2 or 50 nM gemcitabine (Gem) and the combination (Gen + Gem). β-actin antibodies were used as internal controls for equal loading of proteins. Significant down-regulation of c-IAP1, Bcl-2, Bcl-xl, survivin and PARP was observed in A2780 and C200 cells treated with the combination of genistein and either cisplatin, gemcitabine or taxotere compared to cells treated with either drug alone.
Genistein inhibits NF-κB DNA binding activity
Specifically, the effects of pretreatment followed by the combination treatment were studied in the context of NF-κB activation. The treatment of cells with genistein alone significantly down-regulated the DNA binding activity of NF-κB in both the cell lines tested. Interestingly, the combination treatment groups demonstrated greater inhibition of NF-κB compared to the treatment of cells with any of the drugs alone (Figure 4). These results suggest that the pretreatment of cells with genistein sensitized ovarian cancer cells, especially the drug-resistant cells to cisplatin, taxotere, and gemcitabine induced growth inhibition and induction of apoptotic cell death, which is believed to be contributed by the inhibition of survival factors, and inactivation of the DNA binding activity of NF-κB.
NF-κB activation in A2780 (A) and C200 (B) human ovarian cancer cells. Supershift assay (C) showed the formation of bigger complex after addition of anti-NF-κB p65 antibody, resulting in the shift of NF-κB band. Cells untreated or treated with 10 or 25 μM genistein (Gen), 250 nM cisplatin (Cis), the combination (Gen + Cis), 1 or 2 nM taxotere (Tax), the combination (Gen + Tax), 2 or 50 nM gemcitabine (Gem) and the combination (Gen + Gem). Retinoblastoma antibodies were used as internal controls for nuclear protein loading as control. Significant inactivation of NF-κB was observed in A2780 and C200 cells treated with the combination of genistein and either cisplatin, gemcitabine or taxotere compared to cells treated with either drug alone.
Experimental drug resistance to platinum based chemotherapy is a major challenge for the treatment of human ovarian cancer. For ovarian cancer, the standard treatment includes aggressive surgical cytoreduction followed by combination chemotherapy with platinum and a taxane-containing regimen. Although the majority of patients will respond to this therapy, most will recur with chemo-resistant phenotype, which eventually kills patient. Overall, the survival of patients diagnosed with advanced stage disease remains poor, particularly if the tumor is "platinum-resistant" [21–23]. Platinum resistance is defined as tumor progression during initial treatment with a platinum-based combination chemotherapy regimen or recurrence within 6 months of completing front line therapy, and thus is considered intrinsic resistance behavior of ovarian cancer cells. Tumors and metastases may also acquire resistance over time mediated by various mechanisms. Platinum resistance in EOC remains a challenge to effectively treat using currently available second and third line therapies especially in patients having an overall response rate of less than 20%, suggesting that overcoming platinum resistance by novel approaches could be useful for improving the overall survival of patients.
The management of patients diagnosed with ovarian cancer has not changed significantly in the past two decades requiring development of additional treatment protocols for improving overall survival. Emerging evidence suggest that plant-derived non-toxic agents such as curcumin, and genistein show a variety of pharmacological effects [24, 25]. Genistein is a naturally occurring isoflavone present in soybeans that has anticancer properties [9, 13, 16, 26]. Therefore, we hypothesized that as in other cancer cell lines; platinum-resistance could be overcome in ovarian cancer cells by combining the conventional chemotherapeutic agents with a non-toxic flavonoid compound, such as genistein.
To test our hypothesis, we sought to assess the efficacy of genistein, a well tolerated naturally occurring substance, in combination with commonly used chemotherapeutic agents in a paired isogenic ovarian cancer cell lines (platinum-sensitive:A2780, platinum-resistant:C200). Though combination therapy was effective in inhibiting cell growth in the platinum-sensitive cell line, genistein pretreatment was required for a response in the platinum-resistant cell line. The observed inhibition of cell growth was subsequently correlated with an increase in the induction of apoptosis. We have further extended our observations to additional cytotoxic conventional chemotherapeutic agents such as taxotere and gemcitabine. We found that genistein pretreatment resulted in the appearance of cleaved PARP under all our experimental conditions, consistent with the increase in apoptosis. This finding suggests that genistein could sensitize ovarian cancer cells to platinum and other conventional chemotherapeutic agents-induced apoptotic cell death and these results are consistent with our previous findings in other cancer cell lines [9, 13, 16, 26].
Studies have shown that the underlying resistance to apoptosis is in part due to constitutive activation of NF-κB in pancreatic cancer [27]. Our results strongly suggest that the resistance of ovarian cancer cells treated with cisplatin could in part be also due to the activation of NF-κB and that the chemo-sensitization could be due to genistein-induced inactivation of NF-κB signaling, resulting in the inhibition of cell proliferation and induction of apoptosis (Figure. 5). Our hypothesis is also supported by the evidence of down-regulation of the important anti-apoptotic proteins such as Bcl-2, Bcl-xL. survivin and c-IAP2, which also happens to be the downstream genes of NF-κB. C-IAP-2 and survivin are members of the anti-apoptotic IAP (Inhibitors of Apoptosis) family of proteins suppressing apoptosis and their expression in tumors has been associated with poor prognosis and increased tumor recurrence in many tumors. Our findings reveal the expression of these anti-apoptotic proteins is decreased by genistein, and is probably driven by NF-κB activation suggesting another possibility for inhibiting tumor and that NK-κB, survivin and IAP'S may make an important contribution to the development of chemo-resistance.
Schematic diagram of potential mechanism of genistein induced chemo-sensitization of ovarian cancer cells to conventional chemo-therapeutic agents.
Multiple pathways linked with cisplatin resistance have been reported by many researchers. A synthetic triterpenoid inhibited IL-6-Stat-3 pathway which is one of the key pathway contributing to drug resistance in ovarian cancer [3]. Pretreatment of cisplatin resistant ovarian cancer cells with trichostatin, a histone deacetylase inhibitor overcomes mitochondria-dependent apoptosis by restoring p73 and Bax expression [22]. A small molecule inhibitor triethylenetetramine inhibited superoxide dismutase 1 which is over expressed in cisplatin resistant cells by enhancing the cisplatin sensitivity in the resistant cells [21]. These findings appear to lend support for our current observation and as such consistent with the role of genistein in the prevention of cisplatin-induced renal injury in mice [28], and its biological effects on breast [13], pancreas [27, 29], and melanoma cells [30], associated with the inhibition in the translocation of p65 subunit of NF-κB in the nucleus, which is otherwise increased by the cisplatin treatment.
In conclusion, the evidence provided by this study lend strong support for our hypothesis that genistein pre-treatment could overcome drug-resistance in ovarian cancer cells as documented by increased cell growth inhibition and the induction of apoptotic cell death. We have also shown that the genistein mediated chemo-sensitization of ovarian cancer cells to conventional chemotherapeutic agents was partly due to inactivation of the DNA binding activity of NF-κB and its downstream genes. Our results warrant further pre-clinical and clinical studies for assessing the value of genistein in overcoming drug-resistance in ovarian cancer in order to improve the overall survival of patients diagnosed with ovarian cancer especially those with drug-resistant characteristics.
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Division of Gynecologic Oncology, Karmanos Cancer Center, Wayne State University, Detroit, Michigan, USA
Leigh A Solomon
& Robert T Morris
Department of Internal Medicine, Karmanos Cancer Center, Wayne State University, Detroit, Michigan, USA
Shadan Ali
Department of Pathology, Karmanos Cancer Center, Wayne State University, Detroit, Michigan, USA
Sanjeev Banerjee
& Fazlul H Sarkar
Division of Gynecologic Oncology, Henry Ford Hospital, Detroit, Michigan, USA
Adnan R Munkarah
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Correspondence to Fazlul H Sarkar.
LAS and SA collected data for the study and prepared the original manuscripts. SB carried out the supershift assay; ARM and RTM supervised the project. FHS directed the research. All authors approved the final manuscript.
C200 Cell


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Wed 10th Oct 2012 - Bee Fields, Newchurch.
Wed 4th Oct 2012 - Corf Camp, Shalfleet.
Green Gym returned to a site that we haven't visited in a long time for the session this week, the meadow area along the cycle track between Newport and Shide. On previous visits, we had to deal with a very overgrown area but, because of improved access, this time it had been possible to mechanically flail the whole area prior to our arrival (phew!) Normally the cut material is left to rot into the ground which will enhance the soil - but not the case for meadows. Wild flowers are happy to grow in poor soil so the rotting material would only encourage weeds and bramble, which stops the wild flowers coming up through. Team GG were soon scattered across the whole area busily raking up into piles which were then bagged up and taken to the edges of the meadow. At the same time, other members were off along the banks of the river and surrounding areas doing the usual litter pick.
At the start of the session Team GG were presented, by Patricia Almond of the CPRE (Isle of Wight branch), with an award for our litter picking efforts across the Island and also a donation to help with the group's expenses. See top photograph.
This was a well attended session (perhaps because it was so central?) and the weather held good for us. It would be nice to return next summer and see how all our efforts paid off.
Photographs were supplied by Carrie & Mark.....many thanks!
Well here were are back again at Corfe Camp - unfortunately one of the main jobs - spreading some gravel on boggy areas - was abandoned, as instead of two very large bags of gravel, only one teeny tiny one was delivered - Ooops! However us Green Gymmers never say die, and we soon got stuck into more tasks. First a small team returned to the drainage ditch we dug out on our last visit to install a pipe to improve the water flow from said ditch, and a second team were tasked with clearing lots of bramble and tree growth from around one of the buildings to improve access. Other clearance work was also taking place, and after the break the final job was to clear away undergrowth from yet another ditch and clear away all the dead leaves to allow the water to flow away from one of the camping fields which of course with the recent rain was very waterlogged.
Thanks to Carrie for the editorial and to Carrie & Tony for the photographs this week.
Once again, this week's GG session was held at a new site for us - this time it was at the Bee Fields, Newchurch. The area we were working in is to the east of Newchurch village and is part of the nature reserve known as Martin's Wood. Originally this area was farmland but around 10 years ago it was planted with trees and shrubs to become the wonderful area for nature that it is today. Our task was to clear areas of grassland between the trees to provide open ground for some of the 70 types of bees that are established here. The bees are solitary - they do not live in swarms - and live in small holes burrowed into the soil. The light sandy soil here seems to suit their needs and it is amazing to see just how many bore holes there are. To encourage more to the area, we were lifting the top layer of grass and piling it on the edges of the cleared areas as apparently some bee species prefer to burrow into a bank rather that down into the soil.
Considering all the rain we have had recently, the weather held good for us and the sun even broke through a couple of times. With plenty of people attending this session, some were deployed to collect up the rabbit guards from the trees that had outgrown them.
Carrie's Nature Lesson.
Two finds this week - the first was a lovely Red Admiral (Vanessa Atalanta) - catching the sunshine on the top of someone's car. This stunning butterfly is a common sight in gardens during the summer. They love plants such as buddleias, michelmas daisies, ice plants, flowering ivy and even rotting fruit. There can be sightings as late as November on sunny days, and they have even been spotted during December, January and February, a time when few other butterfly species appear.
The second find was Centaury (Centaurium Erythraea) a low-growing biennial found on sand dunes, heaths, woodland rides, quarries and other dry, grassy areas. It is in bloom between June and September and, like other members of the gentian family, its pink flowers close during the afternoon. It has small, pink, five-petalled flowers held in clusters at the top of the stems, a rosette of oval leaves at the base of the stem and oval leaves appear in opposite pairs up the stem as well.
This week team GG were back at what is becoming a regular venue in our calendar - the Corfe scout camp. Although the weather has been pretty wet leading up to our session, we managed to stay dry which encouraged a good turnout. Our task was to improve the drainage along one of the main pathways so, after cutting back the overhanging growth, it was all hands to the spades and shovels! Excellent progress was made and as can be seen from the photograph above, a good section of ditching was completed.
Mark's Award - The Merlin Trophy 2012.
The two photographs above show the Merlin trophy and certificate that was presented to Mark by the Campaign to Protect Rural England. The wording on the certificate reads as follows....
This certificate is awarded to Mark Russell of Green Gym for
The work you do for the Island, for the inspiration you generate in others,
for the guidance and teaching you provide for people who could be considered to be disadvantaged but clearly blossom under your expert care in the countryside.
I am sure that all the members of Team GG will agree wholeheartedly with the above and would join with me in giving Mark our congratulations on winning such a prestigious award.
Many thanks to Carrie, Tony and Martin for the photographs this week.

redpajama_set_name: RedPajamaCommonCrawl


text:
<?php declare(strict_types=1);

namespace Tale\Reader\Text;

use Throwable;

final class ReadException extends \RuntimeException
{
    private Location $location;

    public function __construct(Location $location, string $message = '', int $code = 0, Throwable $previous = null)
    {
        parent::__construct("{$message} (at {$location})", $code, $previous);
        $this->location = $location;
    }

    /**
     * @return Location
     */
    public function getLocation(): Location
    {
        return $this->location;
    }
}

redpajama_set_name: RedPajamaGithub


text:
Q: Visualize array in a plot with colours in different styles I want to visualize an array with colour plots, so I can compare many arrays with each other and see where they are similar or not. An example array would be:
import numpy as np
import matplotlib.pyplot as plt

coef = np.array([7.84678e-06, -3.03161e-06, 4.1283e-06, -3.33764e-06, 8.18091e-07, 4.0515e-07, 5.03187e-06, 1.40076e-05, -0.240809, -0.0133863, 0.0479671, 0.0740471, 0.222519, 0.10136, 0.0594249, -0.103346])

plt.pcolor([coef,coef] , cmap = 'hsv' )
plt.show()

And get as plot:

Is there any other or better way to visualize arrays? Specially if I get a longer array with a length of 1000 or 2000 its not clearly arranged:

redpajama_set_name: RedPajamaStackExchange


text:
ICT sector report finds NZ industry thriving in 2017
ICT sector is delivering quality jobs and exports, new report finds.
By Rob O'Neill (New Zealand Reseller News) 28 July, 2017 02:05
The ICT product and service sector is an engine of jobs and export growth
The 2017 ICT Sector report demonstrates the strength of New Zealand's technology sector, said economic development minister Simon Bridges.
The report (pdf), which focuses on firms developing and exporting information technology services and software, was launched in Wellington yesterday alongside "ProjectR", an initiative focusing on virtual, augmented and mixed reality research and development.
IT product and services firms accounted for 41 per cent of ICT GDP in New Zealand in 2015, up from 28 per cent in 2007.
The report found there are 10,700 more jobs in computer system design today than in 2010 and 54 computer system design firms that employ more than 100 people – nine more than in 2015.
This is now about the same number of large firms as in the machinery and manufacturing sector.
Average salaries and wages of close to $100,000 in computer system design firms are twice the New Zealand average.
Employment in the ICT product and service sector by firm size.
"The IT services sector makes a significant contribution to our economy. In 2016, more than 11,000 IT firms, employed 29,700 people and contributed $3.6 billion dollars to New Zealand's GDP," Bridges said.
Firms in the sector invested $436 million in R&D in the last year, more than any other sector in the economy. Exports were close to a $1 billion.
Read more Te Papa incubator Mahuki pushes tech boundaries in the cultural sector
There are also more firms in the sector - both large and in start-up phase - and more local and international investors.
A challenge is to further develop training and career pathways to meet the demands of the sector, Bridges said.
Job vacancies in computer system design firms are high and harder to fill than in any other sector in the economy, the report found. Applicants with professional, technical and computer skills were cited by employers as the hardest to recruit.
Of all bachelors students enrolled in New Zealand, only eight per cent are studying information technology as their main subject.
Read more IRD reschedules transformation platform rollout to reduce risk
The number of graduates in computer science and information systems programs at tertiary level has, however, grown by eight per cent year-on-year since 2008.
'Born-digital' records pose a major challenge for government agencies
Google Glass is back, and it's eyeing up the enterprise
The most in-demand Kiwi IT skills for 2021 revealed
Christchurch developers produce "world-first" VR app for Ports of Auckland
Security Desperation Is Causing Businesses – Especially SMEs – To Look To Partners To Deliver Effective, Low-Resource Security Solutions
More from WatchGuard Technologies
Tags jobspolicygrowthaugmented realityvirtual realityICT sectoremploymen


text:
Act To Authorize the Secretary of the Interior To Provide for the Commemoration of the Efforts of Goodloe Byron To Protect the Appalachian Trail, and for Other Purposes
The concept Act To Authorize the Secretary of the Interior To Provide for the Commemoration of the Efforts of Goodloe Byron To Protect the Appalachian Trail, and for Other Purposes represents the subject, aboutness, idea or notion of resources found in Bates College.
The Resource Act To Authorize the Secretary of the Interior To Provide for the Commemoration of the Efforts of Goodloe Byron To Protect the Appalachian Trail, and for Other Purposes
Readex congressional thesaurus
3 Items that share the Concept Act To Authorize the Secretary of the Interior To Provide for the Commemoration of the Efforts of Goodloe Byron To Protect the Appalachian Trail, and for Other Purposes
Amending the Land and Water Conservation Fund Act of 1960, and for other purposes. March 27, 1987. -- Committed to the Committee of the Whole House on the State of the Union and ordered to be printed., (electronic resource)
Establishing a system for the collection and distribution of fees in the National Park System. October 1 (legislative day, September 24), 1986. -- Ordered to be printed., (electronic resource)
Recreation fee legislation. June 24, 1987. -- Ordered to be printed., (electronic resource)
Context of Act To Authorize the Secretary of the Interior To Provide for the Commemoration of the Efforts of Goodloe Byron To Protect the Appalachian Trail, and for Other Purposes
Amending the Land and Water Conservation Fund Act of 1960, and for other purposes. March 27, 1987. -- Committed to the Committee of the Whole House on the State of the Union and ordered to be printed.
Establishing a system for the collection and distribution of fees in the National Park System. October 1 (legislative day, September 24), 1986. -- Ordered to be printed.
Recreation fee legislation. June 24, 1987. -- Ordered to be printed.

 Data from Act To Authorize the Secretary of the Interior To Provide for the Commemoration of the Efforts of Goodloe Byron To Protect the Appalachian Trail, and for Other Purposes - Bates College

Data Citation of the Concept Act To Authorize the Secretary of the Interior To Provide for the Commemoration of the Efforts of Goodloe Byron To Protect the Appalachian Trail, and for Other Purposes


Bike To Work Challenge 2019: Melissa M.
#0 - Melissa M. has walked	0 miles over 0 trips.
#0 - Melissa M. has biked	0 miles over 0 trips.
#8 - Melissa M. has carpooled	150 miles over 12 trips.
Melissa M. has burned 0 calories and reduced their emissions by 0 lbs.


Top Children's Hospitals: Are Their Home Pages in the Same League?
Every couple of years CHILD Magazine surveys the nation's children' hospitals and ranks the top 25. Well, actually they rank the top 10 and then have 15 runners up, but since Seattle Children's (where I work) came in at #13 this year, I'm going to say they rank the top 25 – any problem with that?
Anyway, just for fun I thought it would be interesting to see whether the home pages (and by implication, the web sites) of these esteemed pediatric medical establishments were of a similar caliber in terms of the validity of their code.
I checked each of these sites using the W3C's validator using three simple criteria – did the page have a valid doctype, were any linked style sheet(s) valid and how many HTML errors were there on the page.
N/A = No character encoding; unable to validate page.
I dare say that most of these sites are powered by a CMS, which is a testiment to the quality of the code that most content management systems are still producing. However, 10 sites without even a doctype? That's a pretty basic omission.
I also recognize that some of the errors are produced by unencoded URLs, which can be tricky to avoid, and so for a few sites the errors are a little overstated. However, there are also plenty of basic HTML coding mistakes such as missing tags and so on.
Some of the CSS errors were rather entertaining too, including made up elements and some horribly basic mistakes (plus plenty of typos too).
Anyway, kudos to The University of Chicago Comer Children's Hospital for having a valid (almost – only one CSS error) site – it just goes to prove that it can be done.


text:
Browse committees
Overview & Scrutiny Committee
Dates of meetings for the Overview & Scrutiny Committee.
More information about Overview & Scrutiny Committee
Show meetings for the year: 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022
Earlier meetings.
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Information about Overview & Scrutiny Committee
The Committee is part of the Council's Overview and Scrutiny framework that helps to ensure that local public services are delivered effectively, efficiently and in the best interests of residents and people that live and work in the Epping Forest District. Overview and Scrutiny is an important mechanism through which public accountability can be exercised and all Councillors (except members of the Cabinet) can participate in the scrutiny of the Council and its partners. The primary role of overview and scrutiny is to hold local decision-makers to account and to help improve local services by:
(a) reviewing and challenging decisions taken by the Council and its partners;
(b) undertaking investigations into services or policy areas which are of interest or concern to people in the District; and
(c) making evidence-based recommendations to improve services provided by the Council and partner organisations;
Overview and Scrutiny has wide-ranging powers to investigate areas of local interest. Scrutiny activity is not restricted to services provided by the Council, as any area of interest to the local community may be investigated by Overview and Scrutiny. Councillors are also able to refer any local matter relating to local government, or affecting their ward or constituents, to Overview and Scrutiny. In this context, Overview and Scrutiny plays an important role in local democracy as it can help to:
(d) enhance the accountability of local services;
(e) improve the transparency of decision-making; and
(f) enable Councillors to represent the views of local people.


text:
Example Essays
Flash Card Generator
International Marketing Perspective What Are the Main&nbspCase Study
Sources: 12
Subject: Business - Advertising
Type: Case Study
Paper: #83483050
Related Topics: International Marketing, Beach, Rain Forest, Museum
Download this Case Study in word format (.doc)
Excerpt from Case Study :
International Marketing Perspective
What are the main factors that have contributed to Banyan Tree's success?
Banyan Tree's first success was finding a niche that the owners could exploit. As the case study says, they realized that there was an opening that they could use to gather guests who were above the level of the large hotel chains, but who did not want to spend the money required to spend a night in the most exclusive resorts. This price difference led to the owners forming the plan for their resort that was more reasonably priced than the large luxury resorts, but would give the customer more than the large hotel chains.
The next thing that they did was to realize what type of experience people were missing. First, people wanted a vacation experience that allowed them to be alone, like they had the space all to themselves, while they were positioned in the "lap of luxury." This experience was solidified by the idea if individual cabins that the guests would sleep in, and the other private amenities that they could have. The case study gives the example of a private villa with its own pool. This would allow the guest to have a private "skinny dip" while enjoying the exclusive services of the resort (Lovelock, Writz & Chew, 2008, 439). The second part of this was the design that the owners felt would take the guest away from the humdrum of their normal lives to the luxury that a resort should be. They wanted to make sure that people had as much of the native feel around them as possible. All of the resorts have a distinctly Asian feel because the company originates in Asia. This means that many of the villas resemble pagodas, or have koi ponds, or other features that made them feel more exotic to the guests. this ploy was actually a good method for taking the guests away from their normal lives and putting them in vacation mode.
The company also has a unique way of advertising. They have not invested in blanket advertising on television and radio the way that some resorts do. They have taken out advertisements in trade publications such as Conde Nast, but they primarily allow the advertising to be done by the people who have already visited one of the resorts (Roy, 2009). People who have already visited one of the resorts are able to tell their friends and acquaintances about the experience which gives the Banyan Tree all of the coverage that it needs.
Finally, the owners were able to enhance their brand. People were already raving about the sensation that is the resorts, but that just meant that other entrepreneurs and established chains were ready to capitalize on this new market. That meant that the owners of Banyan Tree had to think of a new way to generate revenue. They did this by opening up shops for local crafts people in the resorts and by extending the brand. They opened a new brand of resort and hotel and they came out with their own brand of products. In this way they were able to make the already valuable brand even more so.
2. Evaluate Banyan Tree's brand positioning and communications strategies. Can Banyan Tree maintain its unique positioning in an increasingly overcrowded resorts market?
The owners of the name Banyan Tree decided on the name because the tree is native to the area where they grew up. They said that the tree provided a canopy which seemed to shield people from the outside world, "like the canopy of a rain forest" (Lovelock, Writz & Chew, 2008, 439). The reason they wanted to use the tree is to give the customer the feeling that they were also covered by the luxury of the vacation site. The use of the Banyan Tree probably meant little to people from the West who patronized the resorts, but to people from that area of the world it most likely meant a great deal. The branding began with the choice of a name and a symbol that evoked peace and harmony. It was a brilliant strategy for an up and coming resort that was trying to make an inroad into a field that was crowded with entries. The group also wanted to establish a set of values that they knew their customers could get behind. They let it be known by their means of construction, use of products, and minimalist nature that they cared what happened to the environment. The owners also realized that of they treated their staff well then the employees would have a greater sense of ownership for both the brand and the welfare of the customers. This is borne out in the research that has been done on marketing. Wazir (2011) says that "you must understand the needs and wants of a business, its promoters, its clients and associates. Then you must recognize the role you play in the business and how you can influence its outcome positively." Banyan Tree realized from the beginning that for its employees to truly feel like they needed to make the business better, they would have to take some ownership for the brand. This meant a pride in doing what was needed in their own small corner of the enterprise to make the entire corporation run better. The employees also realized that if they did this, then they would be richly rewarded also.
The method of communication was mainly word of mouth, as was mentioned before, but the company also communicated its brand through the subtle advertising that it conducted. This was done in some of the trade publications specifically designed for the travel market, and in luxury travel magazines as well. Communication was also maintained through the position of the brand in other areas beside the resort community that they had established.
The fact that the owners of Banyan tree have been able to grow their brand into different products shows that the owners have the knowledge to further expand and grow. The best way that Banyan Tree can grow its existing sales while other brands are entering the market is to either develop its product further or to diversify into new products (The Product, 2012). They have done this with all of the new names and products that they have worked up since the marketplace became crowded. The company has diversified its product base to include a new hotel and spa name, and other product offerings that people know come with the same quality that they have come to expect in Banyan Tree.
3. Discuss whether the brand portfolio of Banyan Tree, Angsana, Colours of Angsana, and Allamanda, as well as the product portfolio of beach resorts and city hotels, spas, galleries, and museum shops fit as a family. What are your recommendations to Banyan Tree for managing these brands and products in the future?
The ownership of the original resort and hotel chain of Banyan Tree have tried to keep all of their offerings within the same window of products. The resort and hotel industry is not so overcrowded that they could not find room originally, and as they continue to open new markets, customers will flock to their new offerings. This is a lot like what Apple, Inc. did with their products just a few years ago. Originally, they were seen as just a computer company that dabbled a little bit in other venues. The Apple came up with a new way to listen to music with the iPod. They also created a proprietary system specifically designed to fuel the iPod with songs. Next, they remade the laptop computer. There next market invention was the iPhone. People were happy with their limited flip phones (like the ones from Star Trek) until Apple created a new desire. Then, Apple created another new market by envisioning and making the iPad. That is three completely new markets that Apple created in about a decade (Marketing Minds, 2012). Banyan Tree has used the same kind of strategy when it has come up with new ideas to help increase sales. Angsana was an idea that grew out of the fact that he company purchased a hotel in downtown Bangkok, and it was turned into a luxury hotel that would offer many of the same amenities that the resorts offered. They could not give the people in downtowns the same villa experience that people gained at their resorts, but they could give them the five star care and the spas. The Colors of Angsana was in response to another need that the hotel and resort guests probably did not know that they needed at the time. It was offered to give the guests the full experience of the culture of the surrounding city and region. All of these offerings fit well into the original idea from which the original spa was created.
The reason that all of this works so well and will continue to work…
Sources Used in Document:
Buchanan, R. (2011). Social responsibility and its effects on consumers. Retrieved March 14, 2012 from http://www.ehow.com/info_8207072_social-responsibility- its-effects-consumers.html
Lovelock, C.H., Writz, J., & Chew, P., 2008. Essentials of service marketing. New York: Prentice Hall PTR.
Marketing Minds, (2012). Apple's branding strategy. Retrieved March 12, 2012 from http://www.marketingminds.com.au/branding/apple_branding_strategy.html
Roy, B., 2009. Importance of word of mouth marketing. Retrieved March 11, 2012 from http://www.ehow.com/facts_5796882_importance-word-mouth-marketing.html
Cite This Case Study:
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International Marketing Is Really No
Topic: Business - Advertising
Paper #: 84141079
Technology is helping in the globalization of world economy in more ways than one with the effect that consumers almost everywhere want global products regardless of whether they live in Los Angeles or remote Africa. The forces driving globalization are (i) flow of information (ii) flow of people (iii) Technology: helping economies of scale with cheaper transportation makes global sourcing possible (iv) Globalization is helping attain lower cost as
International Marketing Management Part A
The argument of one-size-fits-all branding fits with institutionally-based branding strategies (Harris, Attour, 2003) yet on a product or service-specific basis the need for having highly specific, targeted, localized branding strategy that aligns with the cultural norms, values and expectations of potential customers is crucial for trust to be created and sustained (Cayla, Arnould, 2008). In many Islamic nations there is a strict code of dress and adherence to religious
International Marketing We Are a
Topic: Business
5 billion (a$28.2 billion) in 2008 (IKW, 2009). The Germans have a long history in the business, with "eau de Cologne" being invented in Koln 210 years ago (IKW, 2009). The German market is continuing to grow slowly, despite economic challenges, recording 10.5% growth since 2004 (Ibid.). There are approximately 525 firms in the industry in Germany, ranging from large international enterprises to small boutique cosmetic and fragrance houses. The industry
International Marketing Management A Overseas
However, the expatriate has the net advantage of significantly superior product knowledge and control, even if the costs of maintaining him are also higher. Foremost, the expatriate must be able to travel from the host country to the home country in order to conduct face-to-face meetings with his superiors and inform them of the progresses made. Traveling however consumes time and energy and the employee could become tired and
Topic: Economics
International Marketing Comparison of Spain and Czech Republic This paper is written out in the form of a comparison between the two countries of Spain and Czech Republic. In all the sub-heads, the situation in Spain is first discussed and that is followed by the relevant description about the Czech Republic. In all reasonableness, Spain has been a poor country with low employment, and this is being solved with temporary employment
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text:
Distributed 15 Local Producers
Distributed 15 is a new multi-artform work that explores way of distributing knowledge, action and shared responsibility for people and the natural world.
The work is created in five chapters across five natural sites and is delivered with multiple local and state partners representing a diverse range of cultural, community and ecological organisations and groups. The work will be performed in early 2023 by local community members and professional artists.
Annette Carmichael Projects is looking for a local producer in each of the following communities to work with the community and the creative team to deliver Distributed 15.
Margaret River/Busselton
Denmark/Albany (position filled)
Ravensthorpe/Hopetoun
Kalgoorlie/Norseman
To apply to be a local producer, please submit a cover letter addressing the selection criteria and CV to Annette Carmichael Projects producer@annettecarmichael.com.au before close of business on Friday 23 July 2021.
Click here to see the Distributed 15 Local Producer job description
updated-D15-Local-Producer-Job-DescriptionDownload
This project was made possible with support from Circuitwest and the Australian Government's Regional Arts Fund, which supports the arts in regional and remote Australia.


Save the dates! TFcon – America's largest fan-run Transformers convention will take place in the Washington DC area the weekend of October 25-27, 2019. TFcon DC will take place at the Hyatt Regency Reston in Reston, Virginia.
Discounted hotel block information will be available shortly. Dealer bookings and registration information will also be available in the near future.
We are very happy to have tracked down David Mendenhall and have him join the TFcon Los Angeles 2019 Transformers Generation 1 Reunion. Transformers fans will recognize him as the voice of Daniel Witwicky in Transformers The Movie as well as Generation 1 season 3 and 4 where he later became the headmaster component for Arcee. David will be taking part in an autograph session on Sunday starting at 10 am. David Mendenhall is presented by The Chosen Prime.
Unfortunately Melendy Britt has come down with the flu and will not be able to join us on Sunday. Her agent will have some signed photos at Morgan Lofting's table should anyone be interested. We hope she gets well soon.
Additionally Daran Norris and Laurie Faso will not be able to join us until 12pm today.
And for our final guest announcement we are happy to share that Transformers The Movie composer Vince Dicola will be able to join us for limited time at TFcon Los Angeles 2019 this weekend, being available to sign for fans on Saturday and Sunday at 3 pm on each day. Vince Dicola is presented by The Chosen Prime.
TFcon is happy to announce that Ian James Corlett will be a last minute guest addition to the 2019 convention in Los Angeles. Best known to Transformers fans as the voice of Cheetor in Beast Wars and Beast Machines he will be appearing for select signing windows during the weekend meeting with attendees.
Ian James Corlett is presented by The Chosen Prime.
TFcon sponsor The Chosen Prime is proud to announce the TFcon Los Angeles 2019 exclusive Make Toys MTRM-11G2 Screamer. This figure features fully painted wing camo, G2 Style paint and tampography, wing fillers and a few additional surprises not shown here. Designed by Jason Brehn from GetRightRobot this collectible represents the G2 iteration of our favorite treacherous Air Commander. This piece will be available at The Chosen Prime booth in limited quantities. The cost of the figure will be $145.
TFcon is very happy to welcome Transformers writer and producer Michael Charles Hill to the TFcon Los Angeles 2019 – Transformers Generation 1 Reunion. Michael wrote for the Transformers Generation 1 cartoon.
Hill will be appearing Sunday only at the world's largest fan-run Transformers convention. Michael Charles Hill is presented by The Chosen Prime.
TFcon is very happy to welcome Transformers writer Paul Davids to the TFcon Los Angeles 2019 – Transformers Generation 1 Reunion. Paul wrote for the Transformers Generation 1 cartoon and was the production coordinator.
Davids will be appearing Saturday and Sunday at the world's largest fan-run Transformers convention. Paul Davids is presented by The Chosen Prime.
TFcon is very happy to announce Marlene Aragon the voice of Elita-1 in Transformers Generation 1 will be joining the G1 Reunion event happening at TFcon Los Angeles 2019 for her first ever convention appearance.
Marlene will be taking part in autograph sessions with the attendees of America's largest fan-run Transformers convention on the Saturday of the convention. Please check the TFcon website for the full guest signing schedule prior to the event for exact times. Marlene Aragon is presented by The Chosen Prime.
TFcon is very happy to welcome Derrick J. Wyatt the art director, lead character designer, and a color stylist on Transformers Animated to TFcon Los Angeles 2019.
Derrick will be appearing be taking part in a Q&A panel with Eric Siebenaler and Marty Isenberg at the world's largest fan-run Transformers convention. Derrick J. Wyatt is presented by The Chosen Prime.


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Го́мфус () — род грибов семейства , включающий от двух видов. Ранее виды считались родственниками лисичек, но молекулярные исследования показали, что гомфусы — родственники весёлок и решёточников.

Представители
По данным EOL, род Гомфус включает следующие виды:

Ссылки

Роды грибов
Гомфовые

redpajama_set_name: RedPajamaWikipedia


text:
History, 08.11.2019 04:31, kaileyy06
The international monetary fund is a central bank for all nations of the world, but it is not involved in humanitarian relief or development projects.
The correct answer was given: imello51
This Answer Is False
The correct answer was given: deadlydemon0500
The statement is false.
First of all, the International Monetary Fund is not a central bank for all nations of the world; it is an international financial organization whose function is the monetary and financial assistance to nations whose economies are in a state of risk, through loans and other operations.
In addition, this organization is involved in social and sustainable development programs for the populations of these countries with financial problems, establishing these programs as requirements for many of their loans.
The correct answer was given: edjiejwi
The correct answer was given: homeowkrkk
The IMF routinely gathers funds for humanitarian and disaster relief all over the world. For example, the international institution matches donations for developing countries at 50 cents to the dollar.
They usually partner with relief agencies to get help to those who need it the most, such is the case of the victims of floods, earthquakes, disease, and famine, and different humanitarian needs.
The correct answer was given: BigGirlsTheBest
It is the correct answer on the test.
The correct answer was given: cee837
The answer is False.
International monetary bank is not a central bank for all the countries of the world. This organization has the aim and the purpose of giving financial or monetary assistance to the countries of the world who are in a need of that help.
The social and the environmental projects or the projects for the humanitarian relief are also taken up by the International Monetary fund for the people of the countries.
The correct answer was given: destinycolley728
The correct answer is false
The correct answer was given: AgentPangolin
True, it's headquartered in Washington, D.C., and its purpose is to foster global monetary cooperation, secure financial stability, facilitate international trade, promote high employment and sustainable economic growth, and reduce poverty around the world.
unbeknownst to the slave-ship captains, the daily dancing and exercise regime and vernacular dance are also derived from america's african-based slave legacy. both are african-american cuisines from the slavery era.
straight down to hell mate
Other questions on the subject: History
History, 21.06.2019 17:30, danielahchf
Adirect democracy is similar to a representative democracy because both forms of government a. have leaders with unlimited powers. b. are led by a single ruler. c. allow citizens to vote in elections. d. do not protect the rights of the citizens.
History, 22.06.2019 04:00, kingyogii
Jane determines x = 4 and y = 1 is the solution to the following system of equations.
History, 22.06.2019 04:30, laddy6433
In 1493, pope alexandrr vi settled the conflict betweenaspain & portugal about their rights to claim the lands they encountered during their explorations. explain why the pope settled the argument, how he settled the matter and the terms of the settlement
History, 22.06.2019 05:20, Abdirisack3250
All of the following changes typically happened in colonies as a result of imperialism, except: a. cultural practices were outlawed. b. they became more economically self-sufficient. c. european nations built modern infrastructure, including railways. d. the european language became the language of government and education.
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I was surprised, and actually a bit annoyed, when Google started trying to auto-complete my results as I typed them in. Has anyone noticed the new behavior of actually returning search results AS YOU TYPE? That's just downright disturbing.
I love it... sometimes it leads me down roads I didn't expect.
Yeah, they've got records of everything you type in now because of the autocomplete, too. Best I can tell with a packet sniffer watching my stuff on the outgo, it sends a pack of data and requests information back every single time you type a letter.
I am also not a fan. I'm sure all of the other search engines are doing the same thing, and they probably were before the autocomplete was as advanced.
You don't think they recorded search results before?
That's what I'm saying, that just because we're seeing the evidence of it now doesn't mean that they weren't collecting every letter we typed long before now (and not just google - a catalog of tracking cookies may not store keystroke data, but it tracks our movement to pipe ads to us). I didn't have a packet sniffer running 5 years ago to look, though, just now because the auto-complete made me wonder just how much data they're collecting.
When you check the tracking cookies and then go to their "opt out", it's funny that they tell you you'll be missing out on their special services if you don't allow them to track you.
Oh, I know they've been collecting data for a long time. The creepy part is watching this massive search engine, probably in a big room buried underground in Sunnyvale somewhere, trying to read my mind every time I type a letter. There's just something very Skynet about the whole thing.
It's ironic... a programmer came up with an implementation for Bing a year before and Microsoft, after careful consideration, said it was worthless.
That must be why my fingertips tingle.
We're not giving it away for free, anyway. We do get a streamlined presentation of like-kind content. I like it. It saves me a whole lot of time when I'm looking for something obscure, and I find value in that.
Turn on the ads on this forum and watch what comes up as you browse the different content categories.
All I do is move the cursor to the right and turn "instant off"!
You can go to "settings" and turn it off permanently there too.
It's so bad now that about the only way you can have any privacy is to pay some Albanian or Chezk rogue military guys put up a satellite just for you, so you can bounce encrypted communications off that.
Proxies are worthless because your ISP maintains eternal records.
Small ISPs who don't make a permanent record of everything are pretty much non existent any more.
Carnivore and Omnivore are ~ ~ ~ well they are almost everywhere.
Marketing people are almost everywhere buying up metadata and lists and pretty much everything else they can that connects yo to any imaginable possible market activity.
I recall the Creek was selling Disks with (I forget) was it images and project posts or just the whole ball of wax - and I don't recall if any one thought to remove all personally identifiable data?
It would be nice of the Creek could make the site non-searchable to Google and the like what with people using their real names.
One evening my home phone rang.
My wife picked it up.
There was a conversation going on the phone.
It was between a family member and a hotel reservation clerk.
My wife tried to speak into the dialog and no one noticed her - she was the fly on the wall.
Some time later I spoke to that other family member and asked him about it.
He told me that his conversation with the hotel reservation clerk had taken place Six-Months prior to my phone ringing that evening.
Someone recorded the whole conversation of a mundane banal boring hotel reservation, then some machine (or person?) somewhere made a connection to my home phone and played the recording.
You think the internet is scary?
Who has that sort of capability, to record and keep such useless material and to do what they did with it?
First guess would be "No Such Agency"
Back when ISP's still disconnected end user internet connections when there was no activity, even DSL (which was in its infancy), I think the Pres, or Vice Pres, (or something big), came here to the Portland area. A local ISP provided internet service to the office space the FBI had set up for the event (maybe it was secret service).
the ISP examined the activity for the connection and confirmed there was no data transfer taking place, so this is why the connection was dropping.
The FBI agent said something along the lines of "Trust us, there is data. We need your system to stop dropping the connection"
This comes directly from a friend of mine that worked at the ISP in middle management, so it is a reliable source.
The two of us discussed this over lunch one day and neither of us could figure out how they could possible be passing stealth packet streams across the connection.
Conversation ended with us agreeing it was pretty "spooky" and a weee bit concerning.
In any case, I think the suggested or previously used terms is nice. It helps you use terms that have been used before and likely are going to be successful.
You think the internet is scary? Who has that sort of capability, to record and keep such useless material and to do what they did with it?
If you came directly from a search engine, I can see the search engine's name and the search terms you used to get my site's URL on the screen prior to clicking on it.
I don't recall anyone telling me I was wondering around in the internet in total obscurity. Do you?
I like it too. It seems like they're giving you the top several searches with those letters. IIRC they used to post the search count next to each one, too. For laughs, or maybe tears, search "why do..." followed by a letter or two. For example, the top result for why do monkeys... yields, why do monkeys throw poop. Ah, the great questions of our society.
You can disable that feature. Why don't you do that???
The point is that it's creepy to have a computer somewhere guess at what you want before you even finish typing it in. I find it pretty creepy at any rate.


Med spas is a hybrid between a medical clinic and a day spa. They offer plastic and cosmetic surgery treatments. The medical spa is operating under the supervision of a Medical Doctor. The med spa is clinically driven compared to a day spa. They offer typical day spa treatments along with more clinical treatments requiring trained professionals in aesthetics. The med spa menu will offer laser hair removal, dermabrasion, photofacials, chemical peels, fillers or injectables like Botox and some natural wellness care. Acupuncture, nutritional counseling, and naturopathic doctor consultations are available as well.
Besides plastic surgery treatments, the med spa will have your day spa treatments like thai massage, hot stone massage, body wraps, salt scrubs, and facials. An esthetician will be on staff to recommend appropriate facial care for your skin. Medically driven treatments will be provided by a plastic or cosmetic surgeon, dermatologist, licensed esthetician or laser technician. They will perform consultations before starting any medical spa treatments. A combination of treatments may be recommended to provide the best results for your skin. Consecutive treatments may be required to complete your desired results if determined by the med spa professional.
The med spa services are designed to treat, resolve, and reduce symptoms from those diagnosed by your primary medical doctor. Offering massage techniques prior to a medical treatment will assist in the recovery or healing process. Med spas are some encompassing because they offer so many different services in one location with trained professionals. You will receive the pampering you need to relax, but also receive desired results in skin texture, a better complexion, youthful appearance, and overall feeling of well being.
In addition to standard cosmetic procedures, alternative therapies are a great addition to the med spa services because they can treat the mental as well as the physical. Nutritional counseling can assist you in maintaining healthier looking body and skin, so you can feel good about your image. Accupuncture works on the body's energy and improves circulation. A naturopathic doctor on staff can consult with you regarding certain deficiencies missing from diet or test which must be run to determine hormonal concerns. The list goes on to the variety of services which can be available to you at the med spa to improve your overall appearance physically, emotionally, and psychologically.


text:
United States Established: 1819 Scholars: 5
This ranking lists all the best researchers from the Mathematics discipline and affiliated with University of Virginia. There are a total of 5 researchers included with 3 of them also being included in the global ranking. The total sum for the D-index values for the best scientists in University of Virginia is 210 with a mean value for the h-index of 42.00. The total sum of publications for the best scientists in University of Virginia is 750 with the mean value for publications per scientist of 150.00.
The data for h-index, citations, awards, and publications presented in this ranking were last updated on Dec 6th, 2021.
This ranking lists all leading scientists affiliated with University of Virginia. There are a total of 5 scholars included with 3 featured in the ranking of leading scientists. A prominent scientist is one with a h-index >= 30 in the discipline of Mathematics.
The total sum for the H-index values for all prominent scholars in University of Virginia in 2021 is 210 with a mean value for the h-index of 42.00.
The total number of all publications by top researchers in University of Virginia in 2021 is 750 with a mean value for publications per researcher of 150.00.
The total number of citations by leading researchers at University of Virginia in 2021 is 35,973 with a mean value for citations per scientists of 7,194.60.
University of Virginia is at the moment ranked as no. 99 in our Mathematics university ranking. It is also ranked 50 in the university ranking for United States.
Researchers from University of Virginia have received a total of 6 awards and recognitions.
Please note that the research institution or university for a scientist is based on the affiliation information available on a variety of sources, such as their personal website, Google Scholar profile, MAG profile, and LinkedIn.
Biology and Biochemistry Business and Management Chemistry Computer Science Earth Science Ecology and Evolution Economics and Finance Electronics and Electrical Engineering Engineering and Technology Environmental Sciences Genetics and Molecular Biology Immunology Law and Political Science Materials Science Mathematics Mechanical and Aerospace Engineering Medicine Microbiology Neuroscience Physics Psychology Social Sciences and Humanities
532 World 269 National
Craig Huneke
University of Virginia, United States
Roberto Triggiani
Ken Ono
Weiqiang Wang
Brian Parshall


US Port and Rail Congestion Update | RIM logistics, ltd.
We would like to inform you that almost all of the US West Coast ocean terminal operations and most major US rail yards have been experiencing extreme congestion due to increased import volumes. Please know that RIM logistics, ltd. is doing everything possible to expedite your shipments. Based on the current port and rail conditions, all containers are subject to potential delays and possible demurrage and other related storage charges. Please be aware that we are working aggressively with the carriers, ocean terminals, the railroads and your/our truckers to minimize any potential demurrage and detention issues impacting your business. We sincerely appreciate your patience and understanding during this extremely challenging time.
There is significant chassis tightness in several key markets, as the heavy surge of imports that landed on the U.S. West Coast, prior to the New Year, continues to flow to the inland terminals. Currently, tightness of international chassis at our DIT (Dallas), Marion (Memphis), Global 4 (Chicago), Dupo (St. Louis), and Englewood (Houston) terminals are forcing us to ground and stack containers waiting for chassis. The circumstance at each terminal is slightly different and not each chassis pool is deficit; however, each terminal is being impacted.
There is still a considerable amount of import freight at the West Coast ports, so we expect heavy volumes to continue at the inland terminals for several weeks. Given that we expect to have to continue stacking some containers at various inland terminals over the next several weeks, the Union Pacific is making some changes to both improve your driver's experience as well as ensure that our terminals remain fluid.


Q: I'm trying to refinance my condo. My credit was approved but now the bank won't approve the loan because the condo association's fidelity bond is too low.
It's clear that I won't be able to sell the property if I can't refinance because of this issue. What should I do?
A: You've run smack into the top problem condo buyers and condo owners will have this year: If the condo building isn't approved by Fannie Mae, Freddie Mac, and FHA, buyers will have trouble getting a mortgage for a condo and condo owners won't be able to refinance.
You should work with your condo association to get it the condominium documentation up to the standards required by Fannie Mae and Freddie Mac. When it comes to FHA, you might consider getting the association approved by FHA. If you don't get your condo association approved, or your documentation does not comply with the guidelines required by Fannie Mae or Freddie Mac, I'm afraid your property values will tank because no one will be able to finance their purchase.
Insurance is an issue that has become a hot topic with Fannie Mae and Freddie Mac.
You should talk to a good mortgage lender in your area and assess what insurance coverages you need to conform to lending guidelines. While you do that, you should have that same lender review your budget and financials to determine whether there are any issues in those documents as well.
Another sticky issue is homeowner associations that have cash reserves that are too low or whose budgets do not allocate enough money on a year-to-year basis towards replenishing reserves. Some of these issues will arise in parts of the country where real estate values have declined quite a bit and whose values continue to fall.
Once you have an idea of any problems your association faces, the association can go to its insurance agent and update its insurance coverages and, if needed, update its budget to comply with newer requirement that may affect the association.
Don't work alone. Talk to your neighbors and alert them to the problem. Get their help in alerting all of the property owners and approaching the condo association with your concerns.
Your condo association board of directors may not even be aware there is a problem if you're the first person to try to buy in the building or refinance in the last year. They may not realize they have to apply to be approved by Fannie Mae, Freddie Mac and FHA and may not understand how long it will take to get that approval in place.
You should get moving sooner rather than later.


It's been a while since I showed you any progress on my Master Bedroom re-do. I really want to get the doors all painted before I show you the "after" pics and it's been raining every weekend here for what seems like a month! (I'm not complaining) But now that I am free from the constrains of going to work everyday, (I'm feeling much better by the way...thank you again for all of your wonderful comments) I have had the time to get some things done. Still no painted doors but I did make some pillows!
I have been seeing the ribbon and fabric roses around everywhere lately. The ribbon roses are very pretty but I LOVE the look of Gale Payne's shabby fabric roses. You can find her at The Wizard of Once Was. Gale's pillows were the inspiration for my own creations. I wanted to purchase some of her gorgeous pillows but since losing my job, making them was definitely more practical. So... Thanks Gayle for your wonderful inspiration!
This was made from some gorgeous white linen that I got at JoAnne for 50% off. Linen is a wonderful fabric to work with and I love the way it feels on the pillow. I coffee stained some white cheesecloth and made the roses by tearing strips of fabric. There is one rose made from a burlap ribbon that turned out really well too.
This one was made from natural cotton duck, a couple of vintage doilies, more roses and some wonderful gauzy fabric that I am now out of and really bummed. Time to search for something to replace it!!!
In addition to my pillows, I also have a new favorite art piece in the room. I did a swap with the extremely talented Lisa over at Tarnished and Tattered. I have coveted Lisa's beautiful altered bottles for a while now and she agree to do a swap with me!!! This little lovely arrived at my doorstep on Saturday. It is so cool, and she took the time to think about what I might like. I can honestly say... she was spot on!!
The rustic shabbiness of it is so beautiful. Thank you Lisa!!! I LOVE it!!!
Hopefully I will get the bedroom doors painted, and will be able to show you the rest of the bedroom very soon. Have a beautiful day my friends!!
the pillows are gorgeous..and I would expect that since you made them!
At least you are getting some things done...and you could always sell more of your fab creations...like those pillows!
Your pillows look great, Karen! I love pillows. Sophee is right, maybe you could sell your pillows. That style is so big right now! Love your altered bottle. Lisa did a lovely job! Is that a doorknob as the topper? Very creative!
the pillows are amazing! you are so talented :) i love the way they turned out!
Wow, Karen, those pillows are FABULOUS! YGG (You Go Girl)! I really love the weaving technique you showed and the use of different fabrics. I could just jump onto that bed and nuzzle in! I'm glad you're using your "free" time to focus on your wonderful creative talents. It's a good way to keep from being overwhelmed by a difficult situation. We'll keep thinking of and praying for you, Karen.
Fabulous pillows and I love the bottle...Lisa got your taste down to a T...stunning. I have a feeling that your new look bedroom is going to be wonderful.
Karen, your pillows are wonderful! Lisa is so talented, I love the bottle. Looks like your room is coming out gorgeous!!
The pillows are exquisite, beautifully designed and made.
WOW!!!! Your pillows are gorgeous and I love, love your bottle from Lisa!
Glad you are getting some creative time in with your free hours.
Wow! Your pillows turned out beautiful! Can't wait to see the end redo results!
Karen~ your pillows are fabulous.. and you created yet another perfect spot!
Glad to hear and see you are doing well..
Karen, Your pillows are fabulous!!! Really,really beautiful. You ought to sell them in your online shop.Seriously.
I Know! Isn't this rain so crazy! We have to finish painting the outside of our house and haven't been able to finish yet. UGH! Did you call the consignment shop? Your pillows are gorgeous!
Love the pillows you made and that we love the same colors, you always inspire me! That bottle is gorgeous and I'm sure what you made is so lovely too (I will have to go take a look this pm). I am so happy to hear you're feeling better and making the most being at home. I love coming here and get energized with your sharing what you're working on. I'll go check Gale's blog too, thanks for the link. Have a happy day... bet your dog's loving you home!
I believe "The Shop" should have some of those pillows! Lookin good girlfriend. I do LOVE the bottle from Lisa. Perfect for you! Thanks for you help yesterday. It's working perfectly! HUGS!
your pillows are so very beautifull, I LOVE the last one of them, well all 3, but that one is the best.......they are gorgeus.
And so is the bottle you exchanged with Lisa, I so love her art, and am happy to own a few pieces she did.
I saw what Lisa recieved from you, and it is so wonderfull and fabulous.
PS I love the new pic! You look so pretty.
I think Dawn first mentioned this last Fall...I went to Home Depot and they had the cotton canvas drop cloths on sale 2 6'x9' for $9.99.
Yes! I bought 2 sets of 2, brought them home, washed and dryed them, I chose not to iron...they came out soft, medium weight, totally neutral color and a crinkly finish to the fabric. They are finished on all four sides and squared - no worrying about the fabric running sideways! I made my new bedbroom curtains out of these drop cloths and I love them!
So good to hear you are doing better!
WOW...the roses that you placed on the pillows are just beautiful! The weaving and lace...just gorgeous! Love you altered bottle, I can see why you wanted to swap with her!!
Beautiful pillows Karen! I can't wait to see the rest of the room.
Your romantic pillows are lovely! Love all the different fabrics you used in them. And the roses are beautiful!
Amazing!!!!! Those pillows are spectacular Karen! Oh man, I want to make some like that!!!! 2die4!!
Karen, those are absolutely gorgeous!!! I love them! Tahnk you so much for ALWAYS inspiring me! Your artwork and craft is fabulous.
Can't wait to see the whole room.
AMA-ZING! The pillows are just the best. So, so pretty and they look great with all your re-doing!!
This is sooooo beautiful. You gave me the inspiration to try to make pillows like this. I am happy that you are feeling better.
Greetings from over the pond!
Wow! the pillows are really very stylish and covetable...if you get tired of them...I can give them a home LOL!
I too think that you are gifted and could possibly sell your wares.
Sorry to hear about your job. You are obviously a positive person and very creative.
Hi Karen, Your pillows are awesome! The ribbon roses turned out just gorgeous, you did such a great job! Lisa's bottle is very beautiful and I also love what you made for her! What a great swap!
I'm sorry to hear that you lost your job! Hopefully there will come a new chance for you soon!
Beautiful pillows girlfriend! I too totally love linen. That bottle is gorgeous and looks perfect with your style!
Your pillows - one word STUNNING !
Oh my gosh, your pillows are gorgeous!
Simply breathtaking pillows. You've got talent that makes people like me drool. I love everything you make. Keep it up!
Your pillows are so pretty. My favorite is the one with Mom's bracelet. The room must be gorgeous. Come for a visit.
I HAVE TO KEEP CHECKING to see what's new over here. And it's YOUR FAULT... I went to Staples to buy a new monitor (hate it when I miss out on pretties) but, they were out of stock on the one I wanted. Guess I'll place an order. HUGS!
The pillows are so gorgeous! I kive it! What a terrific look!
Oh Karen, I'm so loving your new bedroom makeover. Your pillows are just beautiful and you had a very awesome inpsiration in Gayle, she is totally amazing and so are her pillows. I love the way you added your sentimental items like your bracelet from your Mom...it's such a keepsake and one to look at and smile every morning!
And as for Lisa's swap...I'm just speechless. When she mentioned on her blog that she did a swap with you, only BEAUTIFUL things ran through my mind as could only imagine what the two of you could create...and I was right! Absolutely beautiful!!!
LOVE THOSE PILLOWS!!!cannot wait to try some myself. oh to have more hours in the day!!
your pillows are gorgeous!! you could sell them, ya know. they're that beautiful.
and the bottle from the exchange, oh, my gosh, so fabulous!!
Oh my Karen, these are absolutely gorgeous! I'd be so mad when my husband threw them on the floor at night to go to bed. Men...they just don't get it. Gorgeous bottle too. You always have some wonderful eye candy to view.