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Eur J Nucl Med Mol Imaging (2009) 36:1434–1442 DOI 10.1007/s00259-009-1117-x
Characterization of choline uptake in prostate cancer cells following bicalutamide and docetaxel treatment Sebastian A. Müller & Korbinian Holzapfel & Christof Seidl & Uwe Treiber & Bernd J. Krause & Reingard Senekowitsch-Schmidtke
Received: 23 October 2008 / Accepted: 2 March 2009 / Published online: 8 April 2009 # Springer-Verlag 2009
Abstract Purpose Choline derivatives labelled with positron emitters are successfully used for PET imaging of prostate cancer patients. Since little is known about uptake mechanisms, the aim of this study was to characterize choline uptake in prostate cancer cells, also following anti-androgen treatment or chemotherapy. Methods Choline uptake in prostate cancer cells (LNCaP, PC-3) and Michaelis-Menten kinetics were analysed using different concentrations of 3H-choline via liquid scintillation counting. Inhibition of 3H-choline uptake was assayed in the presence of hemicholinium-3 (HC-3), unlabelled choline, guanidine and tetraethylammonium (TEA), an inhibitor of the organic cation transporter (OCT). Changes in choline uptake triggered by bicalutamide and docetaxel were evaluated and choline transporters were detected via Western blotting. Results Michaelis-Menten kinetics yielded a saturable transport with Km values of 6.9 and 7.0 µmol/l choline for LNCaP and PC-3 cells, respectively. Treatment of cells with bicalutamide and docetaxel caused an increase in total choline uptake but had no significant effect on Km values. Uptake of 3H-choline was NaCl dependent and 4.5-fold higher in LNCaP cells than in PC-3 cells. 3H-Choline uptake was reduced by 92–96% using HC-3 and unlabelled S. A. Müller : K. Holzapfel : C. Seidl : B. J. Krause : R. Senekowitsch-Schmidtke (*) Department of Nuclear Medicine, Technische Universität München, Ismaninger Strasse 22, Munich 81675, Germany e-mail: [email protected]
U. Treiber Department of Urology, Technische Universität München, Munich, Germany
choline, by 63–69% using guanidine and by 20% using TEA. The high-affinity choline transporter was detected via Western blotting. Conclusion Choline uptake in prostate cancer cells is accomplished both by a transporter-mediated and a diffusion-like component. Results of inhibition experiments suggest that uptake is mediated by a selective choline transporter rather than by the OCT. Bicalutamide- and docetaxel-induced changes in total choline uptake could affect PET tumour imaging. Keywords [methyl-3H]Choline . Prostate cancer cell lines LNCaP and PC-3 . Anti-androgen bicalutamide . Chemotherapeutic agent docetaxel . 11C-Choline PET
Introduction Prostate cancer is the most common cancer in men and the second most common cancer type leading to death following lung cancer. In 2007, 218,890 new cases and 27,050 deaths from prostate cancer were estimated in the USA . The clinical appearance of prostate cancer is widely spread from low-grade indolent to aggressively growing and metastasizing cancers . Most prostate cancers show androgen dependence early in the course of disease. In many patients there is tumour progression from a slow-growing, androgen-dependent carcinoma toward a more and more aggressive, androgen-independent tumour [3, 4]. Therefore, approximately half of the patients relapse and there is a great variability concerning the period of response to an anti-androgen therapy. To achieve a better therapy response rate for patients with less androgen-dependent cancer or with advanced stages of disease, combination therapies with chemotherapy
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are a therapeutic option. Chemotherapy has not been shown to significantly improve survival rates in advanced prostate cancer in a number of studies [5–7]. However, in a recent study Berthold et al.  could demonstrate a significant survival benefit following therapy with docetaxel plus prednisone. Recent clinical trials have shown that chemotherapy can play an important role also in palliative therapy of prostate cancer, and thus docetaxel has become a standard chemotherapeutic agent for treatment of metastatic hormone-refractory prostate cancer [9–11]. In order to optimize the therapy regimen, it is important to determine the extent of disease. Unfortunately the value of clinical examination, ultrasound and current imaging techniques for staging and re-staging of advanced tumour stages is limited . PET and PET/CT studies using 11Cor 18F-labelled choline derivatives recently have shown promising results for re-staging prostate cancer in patients with biochemical recurrence [13–17] and in patients with advanced prostate cancer . The rationale for the use of choline for prostate cancer imaging is based on an increased content of phosphorylcholine and an increased phosphatidylcholine turnover in prostate cancer cells [19, 20]. Accordingly, prostate cancer tissue shows a characteristic increase in choline compared to normal prostate tissue, which can be detected by magnetic resonance spectroscopy (MRS) [21, 22]. Key enzymes of choline metabolism are upregulated [23, 24], and prostate cancer cells have shown an increased expression of choline transporters and an upregulated transport rate [25–28]. The quaternary amine choline as a charged hydrophilic cation needs specific transporters to pass the cell membrane. Mainly there are two groups of choline transporters: the high-affinity (Km <10 µM), NaCl-dependent choline transporter (CHT1) that provides choline for acetylcholine synthesis in the presynaptic cholinergic nerve terminal and the low-affinity (Km 30–100 µM), NaCl-independent choline transporter, which is ubiquitously located and provides choline for phospholipid synthesis . Also other choline transport systems have been described [30, 31]. Choline transport has been investigated in different tumour cells. Amongst these, little has been published on the choline transport in prostate cancer cells [28, 32]. Transport kinetics have been evaluated in the androgenindependent cell line PC-3, derived from human prostate cancer cells . In the cell lines PC-3 and LNCaP, [3H] choline uptake kinetics have been analysed with respect to irradiation . The results of these studies indicate that choline is transported through a system that in its kinetic properties is similar to the human choline transporter-like protein (hCTL1). Therapeutic approaches for the treatment of advanced prostate cancer include anti-androgen therapy, chemotherapy and targeted therapies. Anticancer therapies in prostate
cancer trigger inhibition of angiogenesis, proliferation and tumour growth involving regulation of the Ras signalling pathway activation. The Ras signalling pathway is also regulated through other modulators like those of androgen receptor signalling. Many of the therapeutic approaches in prostate cancer have in common that they modulate signalling pathways and therefore potentially modulate choline uptake and turnover in prostate cancer cells . The question to be addressed in this study was if choline can be used to characterize and quantify the modulation of choline uptake and transport caused by pharmacotherapy. The aims of these in vitro studies were: (1) to characterize the choline uptake and transport in androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cell lines with respect to uptake kinetics, competitive inhibition and sodium dependence, (2) to assess the influence of anti-androgen (bicalutamide) and cytostatic (docetaxel) therapy on choline uptake and transport and (3) to identify the CHT1 protein in prostate cancer cell lines by Western blotting using a polyclonal anti-CHT1 antibody.
Materials and methods Cell lines and cell culture The human prostate cancer cell lines LNCaP and PC-3 used for the uptake studies were provided by the Department of Urology of the Technische Universität München. For Western blot analysis of the CHT1 the following cell lines were additionally assayed for comparison: DU145 (human prostate cancer, provided by the Department of Urology, Technische Universität München), SW707 (human colon cancer, provided by the German Cancer Research Centre Heidelberg, Germany), PC-12 (rat pheochromocytoma, provided by the Department of Pathology, Technische Universität München, Germany), Kelly and Sy5y (human neuroblastoma, provided by the Department of Pediatrics, Universität Tübingen, Germany). Human erythrocytes, freshly prepared from volunteers, were provided by the blood bank from the Technische Universität München, Germany. All tumour cells were grown in RPMI medium (Biochrom, Berlin, Germany) supplemented with 10% fetal bovine serum and 1% non-essential amino acids in the cases of DU145, LNCaP and PC-3, 10% fetal bovine serum in the case of SW707, 5% fetal bovine serum in the case of Kelly and Sy5y and 10% horse serum in the case of PC-12. Supplements were obtained from Biochrom (Berlin, Germany). Cells were kept in an incubator at 37ºC in a humidified 5% CO2 atmosphere and subcultured every 7 days. For culture of PC-12 cells collagen-covered flasks were used (Greiner, Solingen, Germany).
Radiotracer uptake studies [methyl-3H]Choline chloride (specific activity 3.03 TBq/ mmol) was purchased from GE Healthcare (Freiburg, Germany). For uptake studies 1×105 subconfluent tumour cells in 100 μl uptake buffer (25 mM Tris/HCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, 140 mM NaCl, pH 7.4) were transferred to Eppendorf vials and preincubated for 10 min at 37ºC. To investigate NaCl dependence of the uptake, NaCl was replaced by 140 mM of N-methyl-D-glucamine (NMDG) . Uptake was initiated by addition of 100 μl [methyl- 3 H]choline (1.85 kBq, 0.61 pmol) diluted in uptake buffer together with unlabelled choline (10 μM). Uptake was allowed to proceed for 10 min at 37ºC in standard uptake experiments and between 1 and 240 min in experiments for determination of uptake kinetics. Uptake was stopped by addition of 1 ml of ice-cold phosphate-buffered saline (PBS) (Biochrom, Berlin, Germany). Subsequently, cells were washed three times with ice-cold PBS (1 ml each). Prior to determination of [methyl-3H]choline uptake, cells were lysed in 0.5 ml of 2% sodium dodecyl sulphate (SDS) overnight. 3H-Activity was then quantified by liquid scintillation counting using a beta-counter (Wallac, Turku, Finland). Cellular protein content was determined according to the method of Bradford (Bio-Rad, Munich, Germany). Uptake was expressed as pmol/mg cell protein. Inhibition of [methyl-3H]choline uptake Inhibitory effects on uptake of [methyl-3H]choline in LNCaP and PC-3 cells were assayed using increasing concentrations (0.01 μM–10 mM) of unlabelled choline and hemicholinium3 (HC-3), as well as guanidine and tetraethylammonium (TEA) (1 mM each). HC-3 is known as a specific competitive inhibitor of the CHT1  and TEA has been described to inhibit all enzymes from the organic cation transporter (OCT) family . After preincubation of cells (1×105) with inhibitors for 15 min at 37ºC, [methyl- 3 H]choline (1.85 kBq, 0.61 pmol) was added and uptake was allowed to proceed for 10 min. Termination of uptake was achieved by addition of 1 ml ice-cold PBS. Uptake was quantified via liquid scintillation counting as described above. Calculation of Michaelis-Menten kinetics A total of 1×105 cells (LNCaP or PC-3) in 100 μl uptake buffer per vial were preincubated for 15 min at 37ºC. Uptake kinetics were started by addition of 100 μl uptake buffer containing [methyl-3H]choline (1.85 kBq, 0.61 pmol) and different concentrations of unlabelled choline yielding final concentrations between 1 μM and 200 μM. Uptake was terminated after 10 min at 37ºC by 1 ml of ice-cold
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PBS. Liquid scintillation counting of 3H-activity was performed as described above. For determination of nonsaturable, unspecific uptake, experiments were carried out in the presence of an excess amount of unlabelled choline (10 mM). Specific, transporter-mediated choline uptake was calculated via subtraction of nonsaturable, unspecific uptake from total uptake. From the resulting graph the kinetic parameters Km (Michaelis-Menten constant) and vmax (maximum velocity) were calculated by non-linear regression using Prism 4 software (GraphPad Software, San Diego, CA, USA). Treatment of cells with bicalutamide and docetaxel To investigate the effects of the anti-androgen bicalutamide (Casodex®, AstraZeneca, Luton, UK) and the chemotherapeutic agent docetaxel (Taxotere®, Sanofi-Aventis, Frankfurt, Germany) on choline uptake, LNCaP and PC-3 cells were incubated with IC50 concentrations of the compounds for 5 days prior to analysis of uptake. For that purpose the following concentrations were applied as determined via clonogenic assays: bicalutamide, 243 μM (LNCaP) and 227 μM (PC-3); docetaxel, 0.75 ng/ml (LNCaP) and 1.2 ng/ml (PC-3). IC50 was chosen because the therapeutic window of both docetaxel and bicalutamide is relatively narrow. This means that the pharmacological efficacy of both compounds is low close below IC50 and high close above IC50. Uptake studies using [methyl-3H]choline (1.85 kBq, 0.61 pmol) and unlabelled choline in final concentrations varying from 1 μM to 10 mM per 1×105 cells and liquid scintillation counting of 3H-activity were carried out as described above. Detection of choline transporters via Western blotting Subconfluent tumour cells of the cell lines DU145, LNCaP, PC-3, SW707, PC-12, Kelly and Sy5y were harvested using 1 mM ethylenediaminetetraacetate (EDTA) in PBS. Total protein of the tumour cells and of human erythrocytes was extracted using a proteome extraction kit (ProteoExtract Complete, Calbiochem, Darmstadt, Germany) according to the instructions of the manufacturer. Extracts were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (8% acrylamide) and blotted onto a polyvinylidene difluoride (PVDF) membrane. Unspecific binding was blocked with 5% non-fat powdered milk and the membrane was probed with primary (polyclonal anti-CHT1 antibody from rabbit, Chemicon, Temecula, CA, USA) and secondary antibodies (goat anti-rabbit IgG coupled with alkaline phosphatase, Sigma, Taufkirchen, Germany) using standard techniques. Protein bands representing choline transporters were detected via colour reaction using nitroblue tetrazolium (NBT)/5-bromo-4chloro-3-indolyl phosphate (BCIP) as substrates.
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LNCaP cells as well as 17.3 μM (HC-3) and 7.3 μM (unlabelled choline) for PC-3 cells. At concentrations of 1 mM each, HC-3 and unlabelled choline reduced uptake of [methyl-3H]choline by 92% in LNCaP and by 96% in PC-3, respectively. At the same concentration (1 mM) the inhibitors guanidine and TEA were less efficient. While guanidine decreased uptake by 63% in LNCaP and 69% in PC-3, TEA only caused a reduction by approximately 20% in both LNCaP and PC-3 (Fig. 3).
Statistics Each value represents the mean ± standard error of the mean (SEM) of at least three measurements in quadruplicate. The significance level was determined by means of Student’s t test (p<0.05).
Results [methyl-3H]Choline uptake kinetics and NaCl dependence in prostate cancer cells
Determination of Michaelis-Menten kinetics As determined via [methyl-3H]choline, uptake of choline increased with increasing choline concentrations in the uptake buffer in both LNCaP and PC-3 prostate cancer cells. Choline uptake drastically increased from 1 μM to 10 μM, almost reached a plateau between 10 μM and 50 μM and moderately increased between 50 μM and 200 μM of choline (Fig. 4, total). Even at a choline concentration of 1 mM, saturation was not attained (not shown). Subtraction of the unspecific uptake—represented by the line through origin expressing choline uptake at 10 mM choline (Fig. 4, unspecific)—from the total, unsaturable uptake yielded the specific, transportermediated uptake (Fig. 4, specific). K m values for transporter-mediated uptake derived from the graphs in Fig. 4 using Prism 4 software were in the high-affinity range (< 10 μM choline)  for both LNCaP (6.9 μM) and PC-3 (7.0 μM) prostate cancer cells.
Uptake of [methyl- H]choline constantly increased up to 240 min in the prostate cancer cell lines LNCaP and PC-3 (Fig. 1). Within the first hour a linear increase in uptake was observed in both cell lines. Uptake of [methyl-3H] choline after 240 min was significantly higher in LNCaP cells (9.5 pmol/mg cell protein) compared to PC-3 cells (2.1 pmol/mg cell protein). In the presence of NaCl, [methyl-3H]choline uptake in LNCaP was up to 1.9 times higher and in PC-3 up to 2.7 times higher than uptake in NaCl-free, NMDG-substituted buffer (Fig. 1). Thus, choline uptake in both prostate cancer cell lines at least in part is driven by a NaCl-dependent component. Competitive inhibition of [methyl-3H]choline uptake For inhibition studies, [methyl-3H]choline uptake was allowed to proceed for 10 min and was subsequently stopped with ice-cold PBS. Competitive inhibition using HC-3 and unlabelled choline ranging from 0.01 μM to 10 mM resulted in sigmoid inhibition graphs (Fig. 2). As calculated using GraphPad Prism, IC50 values were 24.5 μM (HC-3) and 6.7 μM (unlabelled choline) for
Treatment of prostate cancer cells with IC50 concentrations of the anti-androgen bicalutamide and the chemotherapeutic
[ 3H]choline uptake [pmol/mg cell protein]
Fig. 1 Uptake of [3H]choline in LNCaP and PC-3 cells in the presence and absence of NaCl (140 mM) at different time points after addition of the tracer (1.85 MBq) to 1×105 cells. NaCl was substituted by 140 mM NMDG
Changes in choline uptake of prostate cancer cells after treatment with bicalutamide and docetaxel
LNCaP NaCl LNCaP NMDG PC-3 NaCl PC-3 NMDG
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a Choline uptake velocity [nmol/mg cell protein/10 min]
[ 3H]choline uptake [%]
Choline 80 60 40 20 0 0
HC-3/choline concentration [µM]
docetaxel increased Km in LNCaP cells to 8.6 μM and to 8.2 μM, respectively, but slightly decreased Km in PC-3 cells to approximately 6 μM with both compounds. Following docetaxel treatment of LNCaP, vmax increased almost threefold, whereas bicalutamide induced only a 100 80 60 40 20 0
unspecific specific 1.5
Fig. 2 Inhibition of [3H]choline uptake in LNCaP (a) and PC-3 (b) by increasing concentrations of HC-3 and unlabelled choline; 1×105 cells were incubated with [3H]choline (1.85 MBq) and HC-3 or unlabelled choline at concentrations of 0.01 µM to 10 mM
Choline uptake velocity [nmol/mg cell protein/10 min]
[ 3H]choline uptake [%]
[ 3 H]choline uptake [%]
HC-3/choline concentration [µM]
Fig. 3 Reduction of [3H]choline uptake (% of control) as induced by unlabelled choline, HC-3, TEA and guanidine (1 mM each)
Fig. 4 Michaelis-Menten kinetics for choline uptake in LNCaP (a) and PC-3 (b) prostate cancer cells. Specific, high-affinity uptake was calculated by subtracting unspecific uptake from total choline uptake
slight increase. In PC-3 cells both compounds caused negligible changes of vmax (Table 1). These results suggest that specific, transporter-mediated choline uptake is only faintly affected by bicalutamide and docetaxel. However, total choline uptake increased depending on choline concentration. In LNCaP cells choline uptake following bicalutamide treatment did not significantly differ from untreated controls at physiological choline concentrations of 5μM (p=0.581), 10 μM (p=0.066) and 20 μM (p=0.786). In contrast, bicalutamide significantly increased choline uptake at concentrations from 50 μM choline upwards, peaking at 355% of control at 10 mM choline (p<0.001 in each case). Docetaxel treatment of LNCaP cells significantly increased choline uptake at choline concentrations from 1 μM to 10 mM ranging from 146% (at 1 μM) to 210% (at 200 μM) of controls (p<0.001 in each case except for p= 0.008 at 500 μM and p=0.007 at 10 mM) (Fig. 5). In PC-3 cells bicalutamide significantly increased choline uptake at choline concentrations from 50 μM (159% of control) to 10 mM (296% of control) (p<0.001 in each case). Docetaxel treatment of PC-3 induced an average increase in choline
Eur J Nucl Med Mol Imaging (2009) 36:1434–1442 Table 1 Km and vmax values (± SD) for LNCaP and PC-3 cells following treatment with bicalutamide or docetaxel compared to untreated controls
Cell line LNCaP
Treatment Control (untreated) Bicalutamide Docetaxel Control (untreated) Bicalutamide Docetaxel
uptake of 127% of control at all choline concentrations assayed (Fig. 5). Increases ranging from 112% (at 20 μM choline) to 151% (at 1 mM choline) were statistically significant (p<0.001 to p=0,031) except for 20 μM choline (p=0.051) and 500 μM choline (p=0.062). Detection of CHT1s via Western blotting For detection of choline transporters, protein extracts of different tumour cell lines and erythrocytes were assayed
Choline uptake [%]
with a polyclonal antibody against the CHT1. In all tumour cell lines analysed, DU145, LNCaP and PC-3 prostate cancer cells, SW707 colon adenocarcinoma cells, PC-12 pheochromocytoma cells (rat), as well as Kelly and Sy5y neuroblastoma cells, however not in erythrocytes, a double band at 94 and 98 kDa could be detected, representing the CHT1 in these cells (Fig. 6). Additional CHT1-positive bands were observed in PC-12 cells (44 kDa) and Kelly cells (72 kDa) (not shown). In erythrocytes a double band at 186/200 kDa and a single band at 62 kDa could be demonstrated (not shown).
300,0 250,0 200,0 150,0 100,0 50,0 0,0 0
500 1000 10000
In the disease management of patients with prostate cancer, exact tumour staging at any time during therapy is of major importance because it implies important clinical consequences with respect to the therapy regimen. For PET imaging of prostate cancer 11C- or 18F-labelled choline derivatives have turned out to be superior to [18F]FDG [12, 34–40]. Anti-androgen therapy and chemotherapy potentially influence the uptake of [11C]choline and it has been reported that the choline uptake decreases after initiation of anti-hormonal therapy . Characterization of choline uptake
350,0 Bicalutamide Docetaxel
Choline uptake [%]
vmax [nmol choline/(mg protein · 10 min)] 1.7±0.14 2.2±0.32 4.8±0.23 0.9±0.04 1.0±0.06 1.1±0.04
Km (µM choline) 6.9±2.6 8.6±5.4 8.2±1.7 7.0±1.3 5.9±1.7 6.0±1.1
Choline uptake in prostate cancer cells was analysed in a buffer containing 140 mM NaCl. Using NaCl-free uptake buffer, choline uptake decreased to approximately 50% in LNCaP and PC-3 (Fig. 1). If the choline transporter in both prostate cancer cell lines is truly NaCl dependent, there
250,0 200,0 150,0 100,0
tes cy o 7 P 70 -12 ythr lly Ca -3 5y PC SW PC Er LN Sy Ke
500 1000 10000
Fig. 5 Choline uptake (% of control) in LNCaP (a) and PC-3 (b) after treatment with bicalutamide and docetaxel; 1 × 105 cells were incubated with [3H]choline (1.85 MBq) and unlabelled choline at concentrations of 1 µM to 10 mM in the presence of IC50 concentrations of bicalutamide or docetaxel
Fig. 6 Detection of the CHT1 via Western blotting. Protein extracts of different tumour cell lines and erythrocytes were separated via SDS-PAGE (8% polyacrylamide) and probed with a polyclonal antibody against the CHT1. A 94/98 kDa double band could be detected in all samples except for erythrocytes
should be only negligible choline uptake in NaCl-free uptake buffer. On the other hand, a choline transporter that functions independently of NaCl will not be inhibited by omission of NaCl. Thus, the results obtained suggest a combined transport system composed of two components: one being NaCl dependent and the other being NaCl independent . In Ehrlich-Lettré ascites tumour cells also two components of choline transport, one of which is NaCl dependent, have been described . Studying competitive inhibition of choline transport in both LNCaP and PC-3 prostate cancer cell lines revealed potent inhibition by a specific inhibitor of the choline transporter, HC-3, and by unlabelled choline and a somewhat less potent inhibition by guanidine, also a selective inhibitor of the choline transporter (at 1 mM each, Fig. 3). Hara et al.  also found that HC-3 inhibited the transporter-facilitated choline uptake for the PC-3 cell line. Inhibition of choline transport for the LNCaP prostate cancer cell line has not yet been described. We could show a potent inhibition of choline transport by HC-3, unlabelled choline and guanidine also for LNCaP cells. These results argue for similar mechanisms of choline transport in both prostate cancer cell lines analysed. IC50 values of 24.5 and 17.5 µM of HC-3 for LNCaP and PC-3, respectively (Fig. 2), are in between the respective values described for the CHT1 (0.001–0.1 µM) and low-affinity choline transporter CHT2 (~100 µM), again supporting a combined transport system . TEA only caused weak inhibition of choline uptake in the prostate cancer cell lines analysed (Fig. 3). Since TEA specifically inhibits OCT-mediated transport, an OCT-mediated choline uptake in prostate cancer cells can be excluded . Michaelis-Menten kinetics yielded an unsaturable total choline uptake with a drastic increase from 1 to 10 µM choline, an almost plateau between 10 and 50 µM and a moderate increase between 50 and 200 µM choline (Fig. 4). The two increases in choline uptake can be ascribed to the two components of choline transport in prostate cancer cells: the first increase is thought to be mediated by the high-affinity, transporter-mediated component with a Km value of 7.0 µM choline in the PC-3 cell line and the second increase by the low-affinity, unsaturable component. The Km value of 7.0 µM is in the same order of magnitude as the respective Km values already published by Holzapfel et al.  (7.5 µM) and Hara et al.  (9.7 µM). For the LNCaP prostate cancer cell line a Km value of 6.9 µM choline was calculated for the first increase which corresponds to the respective Km value recently published . Choline transport systems CHT1s have been characterized in different tissues including the cholinergic nervous system , fibroblasts , rat brain
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microvessel endothelial cells  and human keratinocytes . Moreover, malignant transformation of cells often is accompanied by elevation of choline uptake [44, 45]. Hara et al.  evaluated the transport kinetics of choline in the androgen-independent human prostate cancer cell line PC-3. The transport rate of PC-3 cells was found to be in between the one known for the human CHT1 (hCHT1) and the organic cationic transporter (hOCT1). Thus, it was in the range of the lower affinity human choline transporter-like protein (hCTL1) which is in line with our findings. Our Western blot results confirm the assumption that at least part of the choline uptake is mediated by the CHT1. Using the anti-CHT1 antibody we could detect specific bands of 94 and 98 kDa in the prostate cancer cells LNCaP and PC-3 as well as in other cancer cell lines known to express the choline transporter (Fig. 6). According to Apparsundaram et al.  who cloned the human, hemicholinium-3-sensitive choline transporter, the molecular weight of this transmembrane protein varies between 30 and 90 kDa. These findings correspond to our results. Treatment with cytostatic drugs and anti-androgens It is known that treatment of cells with cytostatic drugs induces changes in tracer uptake, for example of FDG, methionine or thymidine [47–49]. However, no data are available on the effects of chemotherapy and anti-androgen treatment on choline transport in prostate cancer cells. The chemotherapeutic docetaxel (Taxotere®) has been described to inhibit cell proliferation of PC-3 cells both in vitro and in vivo [50–52]. For evaluation of the effect of docetaxel on choline uptake, the prostate cancer cell lines PC-3 and LNCaP were treated with 227 µM and 243 µM docetaxel, respectively, resulting in approximately 50% survival as assayed via the clonogenic assay. In this study treatment with docetaxel induced an increase in total choline uptake but had no significant effect on Km values. Androgens are the primary regulators of prostate cancer cell growth and proliferation. Bicalutamide is a pure antiandrogen that inhibits gene expression and cell growth stimulated by androgens via blockage of the androgen receptor . The effect of bicalutamide on choline uptake in the androgen-dependent LNCaP cell line was evaluated following treatment with 0.75 ng/ml bicalutamide. This resulted in 50% survival. Though the androgen receptor in LNCaP cells reveals a point mutation in the steroid-binding domain, bicalutamide inhibited growth in LNCaP cells  as has been confirmed in our study. The effect of bicalutamide on choline uptake in the androgen-independent cell line PC-3 was assessed following treatment with 1.2 ng/ml bicalutamide. This resulted in reduction of survival by approximately 50%. This indicates that bicalutamide also significantly affects growth of
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androgen-independent prostate cancer cells. The findings by Floyd et al.  according to which bicalutamide induces apoptosis in PC-3 cells are in accordance with our results. Interestingly, docetaxel treatment of LNCaP and PC-3 cells induced a significant increase in choline uptake both at physiological and supraphysiological choline concentrations but had no significant effect on K m values. The bicalutamide-triggered increase of choline uptake was not significant at physiological but significant at supraphysiological concentrations in both cell lines. Thus, this increase should be due to an increase of unspecific choline transport. The increase in uptake of total choline might be due to the following reasons: (1) Drug treatment induces cellular stress which in turn results in an increase of cellular metabolism and thus raises the need for choline as used for synthesis of cell membranes. (2) Alterations in the cell membrane due to induction of apoptosis could finally result in an increased permeability for choline. This is corroborated by an increase of apoptotic nuclei and 3H-choline uptake following 2methoxyestradiol treatment of PC-3 cells . (3) Timing of choline uptake assessment might influence the choline uptake results. In our experiments the effects of docetaxel and bicalutamide on choline uptake were assessed at 10 min and thus might differ at other time points.
Conclusions Our results suggest that choline uptake in LNCaP and PC-3 prostate cancer cells is mediated by both a NaCl-dependent, saturable, specific, high-affinity transporter (CHT1) and a NaCl-independent, unsaturable, low-affinity component. Choline uptake is not mediated by the OCT -family of transporters. CHT1 could be identified via Western blotting both in LNCaP and PC-3 cells. Growth inhibition triggered by docetaxel and bicalutamide treatment had no significant effect on transport affinity in LNCaP and PC-3. However, total choline uptake was significantly elevated in both cell lines by docetaxel at both physiological and supraphysiological choline concentrations and by bicalutamide at supraphysiological choline concentrations. Acknowledgements We would like to thank A. Lehmer from the Department of Urology, Technische Universität München, for providing the human prostate cancer cell lines LNCaP, PC-3 and DU145. Conflicts of interest None.
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1441 3. Craft N, Chhor C, Tran C, Belldegrun A, DeKernion J, Witte ON, et al. Evidence for clonal outgrowth of androgen-independent prostate cancer cells from androgen-dependent tumors through a two-step process. Cancer Res 1999;59:5030–6. 4. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med 2004;10:33–9. 5. Beer TM, Garzotto M, Henner WD, Eilers KM, Wersinger EM. Multiple cycles of intermittent chemotherapy in metastatic androgen-independent prostate cancer. Br J Cancer 2004; 91:1425–7. 6. Petrylak DP, Tangen CM, Hussain MH, Lara PN Jr, Jones JA, Taplin ME, et al. Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med 2004;351:1513–20. 7. Tannock IF, de Wit R, Berry WR, Horti J, Pluzanska A, Chi KN, et al. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med 2004;351:1502–12. 8. Berthold DR, Pond GR, Soban F, de Wit R, Eisenberger M, Tannock IF. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer: updated survival in the TAX 327 study. J Clin Oncol 2008;26:242–5. 9. Anderson J, Abrahamsson PA, Crawford D, Miller K, Tombal B. Management of advanced prostate cancer: can we improve on androgen deprivation therapy? BJU Int 2008;101:1497–501. 10. Damber JE, Aus G. Prostate cancer. Lancet 2008;371:1710–21. 11. De Dosso S, Berthold DR. Docetaxel in the management of prostate cancer: current standard of care and future directions. Expert Opin Pharmacother 2008;9:1969–79. 12. Reske SN, Blumstein NM, Neumaier B, Gottfried HW, Finsterbusch F, Kocot D, et al. Imaging prostate cancer with 11C-choline PET/CT. J Nucl Med 2006;47:1249–54. 13. Cimitan M, Bortolus R, Morassut S, Canzonieri V, Garbeglio A, Baresic T, et al. [18F]fluorocholine PET/CT imaging for the detection of recurrent prostate cancer at PSA relapse: experience in 100 consecutive patients. Eur J Nucl Med Mol Imaging 2006;33:1387–98. 14. Rinnab L, Mottaghy FM, Blumstein NM, Reske SN, Hautmann RE, Hohl K, et al. Evaluation of [11C]-choline positron-emission/ computed tomography in patients with increasing prostate-specific antigen levels after primary treatment for prostate cancer. BJU Int 2007;100:786–93. 15. Husarik DB, Miralbell R, Dubs M, John H, Giger OT, Gelet A, et al. Evaluation of [18F]-choline PET/CT for staging and restaging of prostate cancer. Eur J Nucl Med Mol Imaging 2008;35:253–63. 16. Krause BJ, Souvatzoglou M, Tuncel M, Herrmann K, Buck AK, Praus C, et al. The detection rate of [11C]choline-PET/CT depends on the serum PSA-value in patients with biochemical recurrence of prostate cancer. Eur J Nucl Med Mol Imaging 2008;35:18–23. 17. Reske SN. [11C]Choline uptake with PET/CT for the initial diagnosis of prostate cancer: relation to PSA levels, tumour stage and anti-androgenic therapy. Eur J Nucl Med Mol Imaging 2008;35:1740–1. 18. Tuncel M, Souvatzoglou M, Herrmann K, Stollfuss J, Schuster T, Weirich G, et al. [11C]Choline positron emission tomography/ computed tomography for staging and restaging of patients with advanced prostate cancer. Nucl Med Biol 2008;35:689–95. 19. Ackerstaff E, Glunde K, Bhujwalla ZM. Choline phospholipid metabolism: a target in cancer cells? J Cell Biochem 2003;90:525–33. 20. Casciani E, Gualdi GF. Prostate cancer: value of magnetic resonance spectroscopy 3D chemical shift imaging. Abdom Imaging 2006;31:490–9. 21. Kurhanewicz J, Vigneron DB, Hricak H, Narayan P, Carroll P, Nelson SJ. Three-dimensional H-1 MR spectroscopic imaging of
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the in situ human prostate with high (0.24–0.7-cm3) spatial resolution. Radiology 1996;198:795–805. Kurhanewicz J, Vigneron DB, Nelson SJ. Three-dimensional magnetic resonance spectroscopic imaging of brain and prostate cancer. Neoplasia 2000;2:166–89. Ramírez de Molina A, Penalva V, Lucas L, Lacal JC. Regulation of choline kinase activity by Ras proteins involves Ral-GDS and PI3K. Oncogene 2002;21:937–46. Ramírez de Molina A, Rodríguez-González A, Gutiérrez R, Martínez-Piñeiro L, Sánchez J, Bonilla F, et al. Overexpression of choline kinase is a frequent feature in human tumor-derived cell lines and in lung, prostate, and colorectal human cancers. Biochem Biophys Res Commun 2002;296:580–3. Ratnam S, Kent C. Early increase in choline kinase activity upon induction of the H-ras oncogene in mouse fibroblast cell lines. Arch Biochem Biophys 1995;323:313–22. Katz-Brull R, Degani H. Kinetics of choline transport and phosphorylation in human breast cancer cells; NMR application of the zero trans method. Anticancer Res 1996;16:1375–80. Ackerstaff E, Pflug BR, Nelson JB, Bhujwalla ZM. Detection of increased choline compounds with proton nuclear magnetic resonance spectroscopy subsequent to malignant transformation of human prostatic epithelial cells. Cancer Res 2001;61:3599–603. Hara T, Bansal A, DeGrado TR. Choline transporter as a novel target for molecular imaging of cancer. Mol Imaging 2006;5:498–509. Lockman PR, Allen DD. The transport of choline. Drug Dev Ind Pharm 2002;28:749–71. Friedrich A, George RL, Bridges CC, Prasad PD, Ganapathy V. Transport of choline and its relationship to the expression of the organic cation transporters in a rat brain microvessel endothelial cell line (RBE4). Biochem Biophys Acta 2001;1512:299–307. Hoffmann K, Grafe F, Wohlrab W, Neubert RH, Brandsch M. Functional characterization of a high-affinity choline transport system in human keratinocytes. J Invest Dermatol 2002;119:118– 21. Holzapfel K, Müller SA, Seidl C, Grosu AL, Schwaiger M, Senekowitsch-Schmidtke R. Effects of irradiation on the [methyl3H]choline uptake in the human prostate cancer cell lines LNCaP and PC3. Strahlenther Onkol 2008;184:319–24. Gorboulev V, Ulzheimer JC, Akhoundova A, Ulzheimer-Teuber I, Karbach U, Quester S, et al. Cloning and characterization of two human polyspecific organic cation transporters. DNA Cell Biol 1997;16:871–81. Hara T, Kosaka N, Kishi H. PET imaging of prostate cancer using carbon-11-choline. J Nucl Med 1998;39:990–5. Hofer C, Laubenbacher C, Block T, Breul J, Hartung R, Schwaiger M. Fluorine-18-fluorodeoxyglucose positron emission tomography is useless for the detection of local recurrence after radical prostatectomy. Eur Urol 1999;36:31–5. de Jong IJ, Pruim J, Elsinga PH, Vaalburg W, Mensink HJ. [11C] choline positron emission tomography for the evaluation after treatment of localized prostate cancer. Eur Urol 2003;44:32–9. Picchio M, Messa C, Landoni C, Gianolli L, Sironi S, Brioschi M, et al. Value of [11C]choline-positron emission tomography for restaging prostate-cancer: a comparison with [18F]fluordeoxyglucose-positron emission tomography. J Urol 2003;169:1337–40. Sutinen E, Nurmi M, Roivainen A, Varpula M, Tolvanen T, Lehikoinen P, et al. Kinetics of [11C]choline uptake in prostate cancer: a PET study. Eur J Nucl Med Mol Imaging 2004;31:317–24. Jager PL, de Korte MA, Lub-de Hooge MN, van Waarde A, Koopmans KP, Perik PJ, et al. Molecular imaging: what can be used today. Cancer Imaging 2005;5:S27–32. Scher B, Seitz M, Albinger W, Tiling R, Scherr M, Becker HC, et al. Value of 11C-choline PET and PET/CT in patients with
Eur J Nucl Med Mol Imaging (2009) 36:1434–1442
suspected prostate cancer. Eur J Nucl Med Mol Imaging 2007;34:45–53. Giovacchini G, Picchio M, Coradeschi E, Scattoni V, Bettinardi V, Cozzarini C, et al. [11C]choline uptake with PET/CT for the initial diagnosis of prostate cancer: relation to PSA levels, tumour stage and anti-androgenic therapy. Eur J Nucl Med Mol Imaging 2008;35:1065–73. Haeffner EW. Studies on choline permeation through the plasma membrane and its incorporation into phosphatidyl choline of Ehrlich-Lettré-ascites tumor cells in vitro. Eur J Biochem 1975;51:219–28. Schloss P, Mayser W, Niehuis A, Betz H. Na(+)-dependent highaffinity uptake of choline into cultured fibroblasts. Biochem Biophys Res Commun 1994;199:1320–5. Janardhan S, Srivani P, Sastry GN. Choline kinase: an important target for cancer. Curr Med Chem 2006;13:1169–86. Kwee SA, DeGrado TR, Talbot JN, Gutman F, Coel MN. Cancer imaging with fluorine-18-labeled choline derivatives. Semin Nucl Med 2007;37:420–8. Apparsundaram S, Ferguson SM, George AL Jr, Blakely RD. Molecular cloning of a human, hemicholinium-3-sensitive choline transporter. Biochem Biophys Res Commun 2000;276:862–7. Haberkorn U, Reinhardt M, Strauss LG, Oberdorfer F, Berger MR, Altmann A, et al. Metabolic design of combination therapy: use of enhanced fluorodeoxyglucose uptake caused by chemotherapy. J Nucl Med 1992;33:1981–7. Haberkorn U, Morr I, Oberdorfer F, Bellemann ME, Blatter J, Altmann A, et al. Fluorodeoxyglucose uptake in vitro: aspects of method and effects of treatment with gemcitabine. J Nucl Med 1994;35:1842–50. Schaider H, Haberkorn U, Berger MR, Oberdorfer F, Morr I, van Kaick G. Application of alpha- aminoisobutyric acid, Lmethionine, thymidine and 2-fluoro-2-D-glucose to monitor effects of chemotherapy in a human colon carcinoma cell line. Eur J Nucl Med 1996;23:55–60. Li Y, Li X, Hussain M, Sarkar FH. Regulation of microtubule, apoptosis, and cell cycle-related genes by taxotere in prostate cancer cells analyzed by microarray. Neoplasia 2004;6:158–67. Li Y, Kucuk O, Hussain M, Abrams J, Cher ML, Sarkar FH. Antitumor and antimetastatic activities of docetaxel are enhanced by genistein through regulation of osteoprotegerin/receptor activator of nuclear factor-kappaB (RANK)/RANK ligand/MMP-9 signaling in prostate cancer. Cancer Res 2006;66:4816–25. Mantwill K, Köhler-Vargas N, Bernshausen A, Bieler A, Lage H, Kaszubiak A, et al. Inhibition of the multidrug-resistant phenotype by targeting YB-1 with a conditionally oncolytic adenovirus: implications for combinatorial treatment regimen with chemotherapeutic agents. Cancer Res 2006;66:7195–202. Agus DB, Cordon-Cardo C, Fox W, Drobnjak M, Koff A, Golde DW, et al. Prostate cancer cell cycle regulators: response to androgen withdrawal and development of androgen independence. J Natl Cancer Inst 1999;91:1869–76. Veldscholte J, Berrevoets CA, Brinkmann AO, Grootegoed JA, Mulder E. Anti-androgens and the mutated androgen receptor of LNCaP cells: differential effects on binding affinity, heat-shock protein interaction, and transcription activation. Biochemistry 1992;31:2393–9. St John Floyd M Jr, Teahan SJ, Fitzpatrick JM, Watson RW. Differential mechanisms of bicalutamide-induced apoptosis in prostate cell lines. Prostate Cancer Prostatic Dis 2009;12:25–33. Davoodpour P, Bergström M, Landström M. Effects of 2methoxyestradiol on proliferation, apoptosis and PET-tracer uptake in human prostate cancer cell aggregates. Nucl Med Biol 2004;31:867–74.

domain: biology


CADIZ - The plight of dogs being housed at the Harrison County Dog Pound during the cold winter weather with frostbite and Parvo problems reportedly arising drew the attention of some Hollywood celebrities through Facebook, resulting in a rescue involving all the remaining dogs in the facility.
The story that began when the Ohio Society for the Prevention of Cruelty to Animals sued the county for substandard conditions may have come to a conclusion Wednesday afternoon as the result of several county residents' efforts.
Cadiz resident Stacy Skinner posted photos she had taken of the dogs at the county pound on her Facebook page along with a plea for help a week ago. "It just spread like wildfire," said Skinner as she helped unload the rescued animals at the New Horizon Animal Hospital in St. Clairsville.
T-L Photo/MIKE PALMER
CADIZ resident Stacy Skinner, left, helps Jay Weiner of the The Gentle Barn foundation of Santa Clara, Calif., take a rescued dog, “Miss Waggs,” into the New Horizon Animal Hospital in St. Clairsville. See imagesat cu.timesleaderonline.com
The plea was noticed by Alyssa Milano who brought it to the attention of Ellen Degeneres who, in turn, contacted Ellie and Jay Weiner of the The Gentle Barn foundation in Santa Clara, Calif.
Jay Weiner said Gentle Barn received more than 10,000 complaints about the conditions at the pound, prompting him to take an overnight flight flight into Pittsburgh where he found that the process to adopt the dogs had began at Wednesday's meeting of the Harrison County Board of Commissioners.
A crowd of media was on hand as animal rights activists packed the meeting room in the courthouse in hopes of addressing the commissioners with their concerns about the conditions at the dog pound. The group spokesperson was Robin McClelland of the Appalachian Ohio Society for the Prevention of Cruelty to Animals, along with Harrison County Humane Society Officer Darla Smith.
The group was not permitted to address the board as Harrison County Commissioner Barbara Pincola explained that rules require any persons wishing to talk during the official meeting to meet the requirement of requesting to be placed on the agenda a minimum of nine days prior to the date.
Pincola issued a statement from commissioners, addressing the issues at the dog pound and admonished persons on Facebook for what she deemed unfounded attacks on the commissioners and for making her personal cell phone number available in the postings.
"We are a dog pound, not an animal shelter," Pincola said. "The shelter is required to hold stray dogs and cats for a minimum of 72 hours for owners to claim. After that, the dog is made available for adoption."
Harrison County Commissioner Mike Vinka added that the pound only euthanizes dogs if they are sick and that the pound has had to do so only four times this year.
Vinka added that he had inspected the facilities Tuesday accompanied by a veterinarian, and he found the conditions to be acceptable by legal standards.
Following the meeting, the Harrison County Sheriff's Office asked the Ohio Bureau of Criminal Identification and Investigation to investigate complaints at the county dog pound. Commissioners were quick to act as they met following the meeting and decided to place Harrison County Dog Warden Chris McMullion on unpaid suspension, effective immediately, and scheduling a hearing next week to discuss her possible termination.
In the meantime, Weiner rented a van in Wheeling and went to the New Horizon Animal Hospital to pick up crates to transport the animals from the pound.
"We were told by a lot of people that there were deplorable conditions here for dogs," said Jay Weiner. "What is happening over there is a real mark on how the officials see their facility."
McClelland said the animals at the pound were suffering, noting that three animals had been relocated prior to Wednesday's actions.
"Clearly the county is incapable of caring for these creatures," she said before adding that "substandard conditions had caused one dog to be taken due to frostbite on its belly and another female with a puppy had been brought to New Horizons the day before with severe internal bleeding due to hookworm infestation.
"One of the dogs has tested positive for Parvo, one will have to have an eye removed, "said Weiner, who rescued six dogs from the facility including Miss Waggs, a Rottweiler that has been at the facility in excess of one year.
"In addition to these dogs. a Lab is in the Cadiz Animal Hospital and two other dogs from the shelter are at Oak Park, a veterinary clinic in Dover.
"Each of these animals will be spayed or neutered," Weiner added, saying that it was required by law in California for all adopted animals, but not in Ohio. The question I want to pose is: 'If your intent is to control the animal population, then why would you take in dogs and not spay or neuter them before adopting them back out into the general populous?'
"The veterinarians and staff here are going to check each of these dogs," said Weiner, adding that Parvo is a highly contagious disease for canines. "They will be taken care of medically first and then we will work with them to evaluate them all emotionally.
"Our goal is to find them all homes locally," Weiner said, noting that he had already had nearly 100 inquiries from persons living within two hours of St. Clairsville. "We have volunteers all over the area and are very widely read on Facebook; these people are collecting donations to help fund the recovery effort which could be in excess of $10,000.
"As always, we will do a home check on all of the adoption applicants," Weiner said.
"Our goal is to prevent further animals from entering the shelter," said McClelland. "It needs to be taken away from the county."
The Guardians of Rescue organization out of New York City has contacted McClelland and asked that the AOSPCA send a formal request to build a shelter to serve the needs of the local area. McClelland would not say where the facility would be located, only that it would serve Harrison and surrounding counties.
Interim dog warden Tina DeWalt will be handling the duties during the suspension in Harrison County, and the dog pound will remain open to the public.
This news concerned McClelland and Weiner who said that she may not be properly qualified with one of them commenting, "When we were loading the animals today, she had no idea how to put a choke chain on a dog."
McClelland was informed that she has been placed on the agenda for next week's meeting of commissioners.
Operation Warm 'N' Cozy from New Philadelphia also rescued two of the dogs, a mother pit bull and her 8-week old pup. A relatively new shelter, owner Julie Tripodi said the shelter made room among its 17 dogs for the pair. More information can be found at www.operationwarmncozy.com
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Being snowed in all weekend had me dreaming of warm Spring days. I love the happiness that plants and flowers bring to a home, but I am admittedly awful at caring for foliage of any sort. This is why I love faux succulents. (I know, I know, I should be able to keep real ones alive.) I love being able to "plant" them in any container I wish, and then just forget about them. Today's post showcased some of our faux succluents in gorgeous glass bottles. It is a unique pairing with an unexpected coastal vibe, and the galvanized bucket adds a touch of farmhouse charm.
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domain: artistic


I recently purchased a Simmons SD7K electronic drum kit to teach myself to play the drums. I did a fair amount of shopping for an electronic drum kit before settling on the Simmons SD7K, weighing considerations of price and features. While the Simmons SD7K may not be the most advanced electronic drum set on the market today, I find that I am quite pleased with the balance of features and quality that Simmons has reached in its SD7K electronic drum set.
The Simmons SD7K electronic drum set has fifty preprogrammed drum kits in its control unit, which means that new drummers are free to experiment with, and learn, many different styles of drumming. These preprogrammed drum kit samples include modern rock drumming kits, retro 1960s drum kits, reggae kits, 1980s electronic synth kits, and many more.
In addition to the preprogrammed drum kits, the Simmons SD7K electronic drum set has fifty preprogrammed MIDI songs that you can use to practice your drumming chops with various music styles, again ranging from rock and reggae to jazz and swing. The MIDI songs can be played with or without preprogrammed sample drumming, so you can practice your improvisational drumming.
The Simmons SD7K electronic drum set has a total of five drum pads, including a snare drum pad that has an extra sensor for rim shots and a bass drum pad. The Simmons SD7K also has three cymbal pads, including a hi-hat, a crash, and a ride. I am quite satisfied with the sensitivity of the drum pads, but the cymbal pads seem to be less sensitive to quick drum rolls. The hi-hat includes a floor pedal trigger, but the bass drum pedal is not included with the Simmons SD7K electronic drum kit.
The Simmons SD7K control unit also has a MIDI interface to connect the SD7K to a computer for MIDI sampling. In addition, the Simmons SD7K also has quarter-inch input and output jacks to allow you to drum along to your favorite music. I often drum with a set of high quality stereo headphones so as not to annoy my neighbors, but you could also connect a drum amplifier if you wanted to use your Simmons SD7K in lieu of a traditional acoustic drum kit.
One thing to keep in mind when budgeting for your Simmons SD7K electronic drum kit is that there are a few accessories you will also need to get started. You will need to shop for a bass pedal, a drum throne, some good quality drum sticks, and either a set of headphones or a drum amplifier. I purchased my Simmons SD7K drum set from Guitar Center for $599, but I ended up spending just under $1000 after purchasing all the accessories I needed to get started as a beginning drummer.
All in all, I am extremely satisfied with my Simmons SD7K electronic drum kit. My SD7K has allowed me to learn the fundamentals of drumming while experimenting with many different styles of music and drum kits. The Simmons SD7K is a perfect starter electronic drum set for the beginning drummer or for the more experienced acoustic drummer who would like to get his feet wet with electronic drumming. If you have always wanted to learn to play the drums but are wary of buying a drum set for whatever reason, do yourself a favor and buy a Simmons SD7K electronic drum kit. My Simmons SD7K has made me into the fully-fledged drummer that I always wanted to be.


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Leaving Bristol at 10:15 returning at 8pm. We will be the guests of Salisbury Civic Society. Travelling from Temple Meads with lunch included.
Timetable (this is a guide, we will be in the hands of SCS).
10:00 Meet at Temple Meads station to catch the 10:20 train to Salisbury.
11:30 Arrive Salisbury, coffee.
12:00 In groups we will be taken around Salisbury by SCS guides finishing at the Rai d’Or for a two course lunch.
14:00 Cathedral where there are many attractions as well as the cathedral itself.
15:40 Cathedral refectory for tea.
16:00 Various guided tours available of Salisbury as provided by members of SCS
18:20 Meet Salisbury station.
18:40 Train back to Bristol.
19:50 Arrive Bristol.
Train fare and lunch will be £25 for members £30 for non-members.
Attractions at the Cathedral
(There is a suggested optional donation for admission to the cathedral.)
Cathedral guided tour
Cathedral Tower Tour (I will need to book this in advance at additional cost of £10.)
Attractions by the Cathedral, if you do not wish to explore the Cathedral
The Rifles Berkshire and Wiltshire Museum: £5, (£4 concessions)
Mompesson House: National Trust (non-members £6.50)
Arundels: Ted Heath’s house, guided tours £10
Salisbury Museum: Admission cost £8
Lunch at Rai d’Or
Thai restaurant. Two course meal It will be opened exclusively for us and we will be joined my members of Salisbury Civic Society. Cost
Train fare and lunch will be £25 for members £30 for non members.
You can book places by emailing me at firstname.lastname@example.org I will be taking bookings on a first come first served basis, priority to Members and their guests. Payment will be required after your place or places have been confirmed.
Please only pay when you have had confirmation of your confirmed place(s). If you wish to pay by cheque or bank transfer you will be sent payment details when you booking is confirmed. Please include £10 for each tower tour if you wish me to book this for you.

domain: travel


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ConcertWith.Me is a resale marketplace. Ticket prices may be above or below face value.
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About Buried Sleeper
Fuzzy droney space-rock from Glasgow, Scotland. Find us at Facebook, Twitter, YouTube and bandcamp. Contact: firstname.lastname@example.org As with any band straddling the genres of doom/stoner metal, Buried Sleeper began as a Black Sabbath-worshipping jam band, honing their sound into something far more unique and interesting. It is this sound that you hear now and has been critically acclaimed through live shows and album reviews. Buried Sleeper have been around for a long time in one form or another but are currently going strong with their anti-cliché brand of heaviness.


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If you appreciate the support coming from working in a group and would like to:
- ‘reset’ your health,
- reduce inflammation,
- eliminate certain foods from your diet to discover if they are the reason for your heal concerns
This may be an good option for you!
We are all on our health journeys and working in a group (online community) makes it more fun!
You can engage in the online group as much or as little as you want.
The advantage of this programme is that you will do it in a group of like-minded people.
You will be given the recipes, list of healthy foods and a lot of support from others.

domain: health


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Video Production & Post Production
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New from Van Guard, the ULTI Rack is a smart heavy duty builders rack that both looks the part and does the job. Made from lightweight aluminium, it is strong, built to last and fuel efficient. A roof rack for your van opens up a world of possibilities for business, whether it’s for extra cargo, or gear for your profession including accommodating 8 x 4 sheets.
Featuring the innovative Click & Lock mechanism that's designed to make fitting quicker and easier than any other van roof rack on the market. The aluminium sides of the Van Guard racks are closed, allowing for easy to securing of pipes, ladders and timber quickly & easily.
Van Guard roof racks are constructed with heavy duty aluminium cross bars with a T-Slot profile on the top surface. They are easily fitted by one person and the roof rack requires no drilling of the van. Fitting a Van Guard rack is a simple job, the side bars and the filling in cross bars simply clip into position and once fitted they are solid and won’t move.
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We are specialist roof rack providers, with a range of racks available includes commercial fleet market, industrial vehicles and other trades. Located in Miton Keynes, we are part of the MK PlyLining group, which was established in 1996 and have been providing an excellent service to our customers since. We supply a wide range of products, all available to buy online now, with fast delivery.


Reversible anionic redox reactions represent a transformational change for creating advanced high-energy-density positive-electrode materials for lithium-ion batteries. The activation mechanism of these reactions is frequently linked to ligand-to-metal charge transfer (LMCT) processes, which have not been fully validated experimentally due to the lack of suitable model materials. Here we show that the activation of anionic redox in cation-disordered rock-salt Li1.17Ti0.58Ni0.25O2 involves a long-lived intermediate Ni3+/4+ species, which can fully evolve to Ni2+ during relaxation. Combining electrochemical analysis and spectroscopic techniques, we quantitatively identified that the reduction of this Ni3+/4+ species goes through a dynamic LMCT process (Ni3+/4+–O2− → Ni2+–On−). Our findings provide experimental validation of previous theoretical hypotheses and help to rationalize several peculiarities associated with anionic redox, such as cationic–anionic redox inversion and voltage hysteresis. This work also provides additional guidance for designing high-capacity electrodes by screening appropriate cationic species for mediating LMCT.
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This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility, operated for the DOE Office of Science by Argonne National Laboratory under contract number DE-AC02-06CH11357. We are grateful to J. Freeland, T. Wu and G. Sterbinsky for their help during mail-in XAS measurements at the Advanced Photon Source. J.C. and I.R. acknowledge support from the National Science Foundation, under grant number DMR-1809372. K.K. acknowledges support from the National Science Foundation, under grant number CBET-1800357. A.M.A. and A.V.M. are grateful to the Russian Science Foundation for financial support (grant 20-13-00233). Access to TEM facilities was granted by the Advance Imaging Core Facility of Skoltech. HAXPES experiments (proposal no. 99210184) were performed on the GALAXIES beamline at the SOLEIL Synchrotron, France. The Ni K-edge XAS was collected on the ROCK beamline at the SOLEIL Synchrotron through a rapid access for urgent need. We are grateful to J. Sottmann and J.-P. Rueff for their assistance during the HAXPES experiments. J.-M.T and B.L. acknowledge funding from the European Research Council (ERC) (FP/2014)/ERC Grant-Project 670116-ARPEMA.
The authors declare no competing interests.
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Nature Materials thanks Kisuk Kang and Zhaoxiang Wang for their contribution to the peer review of this work.
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Li, B., Kumar, K., Roy, I. et al. Capturing dynamic ligand-to-metal charge transfer with a long-lived cationic intermediate for anionic redox. Nat. Mater. (2022). https://doi.org/10.1038/s41563-022-01278-2


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December 28, 2023Review Recipe
Table of Contents
Take Taco Tuesday up a notch with these flavorful taco bites. Delicate wonton wrappers are shaped into cups to create the perfect delivery method for tasty taco meat, melted cheese, and all your favorite taco toppings.
Taco Bites Ingredients
If you’re a fan of Mexican cuisine, then you have to try these mouthwatering taco bites. These delectable little morsels pack a big punch of flavor in every bite.
With a crispy shell filled with savory meat, flavorful spices, and your choice of toppings, taco bites are an explosion of flavor that your taste buds won’t soon forget.
- 24 wonton wrappers
- ½ pound of lean ground beef
- 3 tablespoons of water
- 1½ tablespoons of mild taco seasoning
- 1 cup of shredded fiesta blend cheese
- 1 cup of shredded lettuce
- ½ cup of guacamole
- ½ cup of sour cream
- ½ cup of pico de gallo
Substitutions And Additions for Mini Taco Bites
With all the irresistible taste of real tacos in bite-size, these fun mini taco recipe is a fun option for family dinner or your next party for the big game.
PICO DE GALLO: You can substitute chunky salsa for the pico de gallo for this great recipe. Salsa verde is another yummy choice.
BEANS: There are many bean choices that can work for these mini taco bites. Pinto beans, black beans, or refried beans would all be excellent. You could add them along with the meat or use them as the only protein.
SPICY: If you want a spicier alternative, you can substitute medium to hot taco seasoning.
You can also add hot sauce on top of the finished taco bites.
PROTEIN: You can substitute ground turkey, ground chicken, or even ground pork for ground beef in this easy recipe.
You could use a rotisserie chicken, slice-up baked chicken breasts, or baked chicken thighs.
You could also make them shrimp taco bites by making them with shrimp.
You could also use leftover taco meat which will make assembling your meal even faster.
CHEESE: Feel free to switch it up with colby, mild or sharp cheddar cheese, Mexican blend cheese, Monterey Jack, pepper Jack for a spicy kick, or a mild Mozzarella. Top with homemade white queso dip as well.
SEASONING: You can use a pre-made store-bought mix or make your own homemade taco seasoning. You could also add an extra dash of onion powder, chili powder, or garlic powder.
WONTON WRAPPERS: For a gluten-free option, try using gluten-free wonton wrappers.
Alternatively, thinly sliced zucchini or eggplant can be a unique, low-carb substitute.
You could also use corn or flour taco shells, phyllo dough, of puff pastry.
SOUR CREAM: Greek yogurt is a great substitute for sour cream, offering a similar texture and a tangy flavor, with the added benefit of extra protein.
OLIVES: Green or black olives are a classic topping choice.
PEPPERS: Try diced bell peppers for a crunchy, sweet element, or jalapeños if you want some heat.
ONIONS: A chopped onion is an excellent topping option. Consider using chives, white onions, green onions, or finely chopped red onion.
TOMATOES: Cherry or grape tomatoes can be a sweeter, juicier alternative to regular diced tomatoes.
LIME JUICE: Finish your tacos off with a squeeze of fresh citrus to enhance the other flavors.
How To Make This Taco Bites Recipe
There are two parts to making these taco bites.
First, you will make the wonton shells to serve your taco fixings.
Second, you will whip up the seasoned taco meat before assembling the tasty taco bites.
Follow our step-by-step instructions to make this fun twist on a classic.
OUR RECIPE DEVELOPER SAYS
You can bake the wonton cups the night before; just make sure to store them in an airtight container at room temperature.
You can also make the taco meat the night before. Simply heat the taco meat up just before you are ready to assemble the taco bites.
STEP ONE: Preheat the oven to 375°F. Lightly spray a regular-size muffin tin with non-stick cooking spray or olive oil.
You could also use a mini muffin pan for smaller bites if you’d like.
STEP TWO: Press one wonton wrapper into the prepared muffin tin opening. Overlay the second wrapper and press it into the first wrapper.
Be sure to press the wrappers against the walls of the cups as well as the bottoms.
Repeat for the remaining muffin cups.
Don’t press too hard on the wontons when placing them into the muffin tin, as you don’t want to tear them.
STEP THREE: Bake for eight to ten minutes or until the wrappers are a golden brown color.
The tips of the wonton wrappers may get a deeper golden color than the bottom.
Oven temperatures vary and may need to be recalibrated periodically to ensure they are accurate. Make sure to check your wontons at the lower end of the recommended baking time.
STEP FOUR: While the wontons are baking, add the half pound of ground beef to a medium skillet over medium-high heat.
Stir continuously, breaking the ground beef into small pieces, until the ground beef is cooked through and no longer pink.
You could easily double or triple this recipe. If you do this, be sure to use a large skillet when you brown the ground beef.
STEP FIVE: Reduce the heat to low. Drain the meat if necessary before proceeding.
Add the taco seasoning and the water. Stir to combine.
Allow the beef mixture to simmer for just a few minutes. Remove from the heat.
STEP SIX: Add one tablespoon of taco filling to each of the baked wonton cups.
STEP SEVEN: Sprinkle one tablespoon of the shredded cheese over the taco meat.
STEP EIGHT: Sprinkle one tablespoon of shredded lettuce over the cheese layer.
STEP NINE: Spoon one teaspoon of the guacamole over the lettuce.
STEP TEN: Spoon a one teaspoon dollop of the sour cream into the center of the guacamole.
STEP ELEVEN: Spoon one to two teaspoons of the pico de gallo on top of the sour cream. Serve immediately.
How To Serve Taco Bites
These would also make an easy dinner for your family. Serve with your favorite toppings and a side of homemade Spanish rice.
Storage Tips for Taco Bites
Proper storage ensures that your tasty bites stay fresh and delicious, ready for you to savor whenever the craving strikes.
MAKE AHEAD: Simply follow the recipe as directed and assemble the bites.
Once assembled, cover them tightly with plastic wrap or aluminum foil and store them in the refrigerator for up to 24 hours.
When you’re ready to serve, bake them according to the recipe instructions, and you’ll have freshly baked taco bites without the fuss.
IN THE FRIDGE: Store any leftover mini taco bites in an airtight container in the refrigerator for up to two days.
IN THE FREEZER: Add the cups to a baking sheet and freeze them for about an hour or until they are frozen solid.
Then, add the cups to a freezer-safe container. They can be frozen for up to two months.
REHEATING: Reheating taco bites is a breeze and can be done using multiple methods:
- OVEN: Preheat your oven to 350°F then place the taco bites on a baking sheet, and bake for ten to 15 minutes or until they’re heated through and the cheese is melty and bubbly.
- MICROWAVE: For a quick reheat, you can use the microwave. Place the taco bites on a microwave-safe plate and heat on high for 20-30 seconds per bite or until warm.
- AIR FRYER: Set your air fryer to 350°F. Arrange the taco bites in a single layer in the air fryer. Then, heat for three to four minutes.
Why We Love This Taco Appetizer Recipe
IRRESISTIBLE FLAVOR: These bites are bursting with the savory and zesty flavors of a classic taco. The seasoned ground beef, melted cheese, and diced tomatoes combine to create a mouthwatering taste that’s hard to resist.
PARTY SNACK: Taco bites are the ultimate appetizer. They’re bite-sized, easy to handle, and a crowd-pleaser at gatherings.
GAME DAY: The perfect party food for football season, the whole family will say this is one of their favorite recipes.
KID FRIENDLY: Their bite-sized nature and familiar taco flavors make them a fantastic option for family meals or kids’ parties.
It’s a fun way to introduce children to different flavors and textures while keeping the meal approachable and enjoyable. It’s also a great meal to have little helpers with since their tiny fingers can help layer toppings on their own mini tacos.
This delicious bite-size dish would make the perfect party treat. They are just the right size for picking up, and rather than dressing them up before serving, you could even allow your guests to add their own favorite toppings to their taco bites.
FREQUENTLY ASKED QUESTIONS
The cups and taco filling can both be made a day in advance, and you can assemble them right before you are ready to serve.
These mini taco bowls are a great option to freeze. Pop them in the freezer on a baking sheet and once they are frozen, add them to a Ziploc bag or container to store for up to two months.
There are plenty of ways to customize these little bites with chicken or beef filling and any taco toppings you like.
More New Recipes You’ll Love
- 24 wonton wrappers
- ½ pound lean ground beef
- 3 tablespoons water
- 1½ tablespoons mild taco seasoning
- 1 cup shredded fiesta blend cheese
- 1 cup shredded lettuce
- ½ cup guacamole
- ½ cup sour cream
- ½ cup pico de gallo
- Preheat the oven to 375°F. Lightly spray a regular-size muffin tin with non-stick cooking spray.
- Press 1 wonton wrapper into the prepared muffin tin opening. Overlay the second wrapper and press it into the first wrapper. Be sure to press the wrappers against the walls of the cups as well as the bottoms. Repeat for the remaining muffin cups.
- Bake for 8 to 10 minutes or until the wrappers are golden brown. (The tips of the wonton wrappers may get a deeper golden color than the bottom)
- While the wontons are baking, add the ½ pound of ground beef to a 1½ to 2-quart saucepan over medium-high heat. (You can also use a small nonstick skillet) Stir continuously, breaking the ground beef into small pieces, until the ground beef is cooked through and no longer pink.
- Reduce the heat to low. Add the taco seasoning and the water. Stir to combine. Allow the meat mixture to simmer for just a few minutes. Remove from the heat.
- Add 1 tablespoon of taco meat to each of the baked wonton cups.
- Sprinkle 1 tablespoon of the shredded cheese over the taco meat.
- Sprinkle ½ to 1 tablespoon of shredded lettuce over the cheese layer.
- Spoon 1 to 1½ teaspoons of the guacamole over the lettuce.
- Spoon a 1 teaspoon dollop of the sour cream into the center of the guacamole.
- Spoon 1 to 2 teaspoons of the pico de gallo on top of the sour cream. Serve immediately.
- You can bake the wonton cups the night before; just make sure to store them in an airtight container at room temperature. You can also make the taco meat the night before. Simply heat the taco meat up just before you are ready to assemble the taco bites.
- Don’t press too hard on the wontons when placing them into the muffin tin, as you don’t want to tear them.
- Oven temperatures vary and may need to be recalibrated periodically to ensure they are accurate. Make sure to check your wontons at the lower end of the recommended baking time.
Even More Recipes You’ll Love
- Taco Pie
- Chicken Taco Casserole
- Shrimp Tacos
- Taco Bell Frito Burrito
- Chicken and Shrimp Tacos
- Turkey Taco Ring
- Taco Ring
- Taco Soup
- Sheet Pan Tacos
- Fiesta Chicken
- Chicken Fajita Casserole
- Chicken and Dumplings
- White Chicken Enchiladas
- Crockpot Chicken Tortilla Soup

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After a 3-hour tooth extraction of a Monday Night RAW, it was refreshing to sit down on the couch tonight for an episode of Smackdown Live. With AJ Styles & Samoa Joe highlighting the show it was clear right off the bat that we were in for another entertaining Tuesday night.
The show opened up with the Miz TV set and Miz’s music, but it was R-Truth and Carmella that were standing in the ring. Truth and Carmella had essentially hijacked Miz’s show and used it as a way to interview Daniel Bryan about this match with the Miz at Super Showdown. This is where R-Truth is at his best. He’s a funny, charismatic guy who can elicit genuine laughter from the crowd without having to try too hard.
Oh, and the mandatory 7-second dance breaks? I’m into it.
— WWE (@WWE) September 26, 2018
This was a fun, light-hearted way to introduce the Bryan-Miz rivalry for this week. Miz would then take out his humiliation on R-Truth by taking him on in a singles match. Miz won the match, all the while taunting Daniel Bryan who was sitting at the commentary table. Miz is definitely gaining momentum heading towards WWE Super Showdown, but there’s still a ways to go. Don’t be surprised to see Daniel Bryan turn the tides in the coming weeks.
This was then followed up by a brief cut-scene featuring Charlotte Flair being attacked at a photo shoot by Becky Lynch. After the attack, Lynch demanded that the photographer take a picture of the champ.
— WWE (@WWE) September 26, 2018
I’ll say it again — All Hail Heel Becky.
Sheamus and Big-E had a singles match as the feud between The Bar and The New Day continues on the way to their Tag Team Championship match at Super Showdown. The match was pretty decent and was a nice back-and-forth between the two, but it was Sheamus who took the win after hitting the Brogue Kick on Big-E.
Rusev, accompanied by Lana, then came out to the ring to address Aiden English’s betrayal from the previous week. English came out and responded to Rusev by saying that everything was going well until Lana got involved. He also made an insinuation about, “that one night in Milwaukee” which drew a big reaction from the crowd.
I love that this seemingly scandalous night took place in Milwaukee, by the way. It doesn’t get more random than Milwaukee, Wisconsin for something like that to happen. We don’t know yet what English is hinting at but this certainly adds a new layer to his situation with Rusev. I’m still not sure what the endgame is here for either superstar, but I’m at least mildly intrigued so I guess I’ll just see where it goes from here.
We then got a women’s tag match featuring the team of Naomi & Asuka facing Sonya Deville and Mandy Rose. I still wish Asuka was in the title picture as she deserves to be, but pairing her with a legit talent like Naomi isn’t the worst thing either. I also still like Deville and Rose, but more as a tag team than anything else. I’m not sure how either would do as a singles competitor, but as a team, they have a strong dynamic.
Naomi & Asuka won without much trouble, as one would expect, but this served more to continue their feud with The Iconics who were on commentary during the match promoting the Super Showdown.
Shinsuke Nakamura took on Tye Dillinger, though that did nothing but provide the setting for a Randy Orton ambush. Orton interfered in the match and beat the hell out of Dillinger, who I guess he had targeted as the “next victim” that he alluded to last week.
Becky Lynch took on Lana in a singles match after the two of them had a brief exchange backstage following Lana & Rusev’s segment with Aiden English. The reasoning behind this match, despite their backstage exchange, was still a bit unclear. Corey Graves alluded to Lynch believing that Lana was, “bad for the Smackdown women’s division” but that was about it. Regardless, Lynch dominated the match and submitted Lana with the “Disarm Her” to pick up the victory.
This led to the show closer which featured the contract signing between AJ Styles and Samoa Joe for their match at WWE Super Showdown for the WWE Championship. Styles came out to the ring looking confident and ready to sign the contract. Samoa Joe? Nowehere to be found..
.. Until he appeared on the titantron. Joe revealed that he was at AJ Styles’ house. It felt like something like this was eventually going to happen. Joe had been hinting at going after Styles’ family for weeks now, and not only did he finally do that but he also showed the most sadistic side of him. He wanted AJ to feel helpless and enraged knowing there was nothing he could do to stop Joe from going to his house.
The show ended with Samoa Joe ringing the doorbell to AJ’s home and saying, “Daddy’s home” just as the screen went to black. That is a cliff-hanging ending like you read about, and it was an awesome way to enhance this rivalry. Samoa Joe is really rounding into form as the top heel on Smackdown Live, and this gives us something intriguing to look forward to next week.


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Dr. Raymond Topp practices Orthopaedic Surgery and Orthopaedic Surgery of the Spine near Brunswick, GA.
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"It’s been so much fun to hear from readers responding to my novel, As If Women Mattered. People of a certain age have expressed excitement because it brings them back to vivid times of the early 70’s and the women’s movement, and younger readers have expressed poignant wishes that they could have a women’s group like this. I’m excited that this book is finding its way into the world and before readers. I started this book a long time ago (I won’t divulge how long – but lets just say- I didn’t expect it to be a historical novel) The story was inspired by my own experiences with feminism and consciousness raising and I wanted to share the exhilaration of those times and the significance of women connecting with each other. I hope you enjoy it and would love hearing from you."
~ Virginia DeLuca


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Updated on August 18, 2022
Mi Honar Superstar is one of the famous singers in a television reality show on the Marathi television network. The TV show is coming up with its spin-off of show title does Mi Honar Superstar Chhote Ustad. The auditions of the show have already started. And the TV show will hit TV screens in a few days.
One of the famous singers and celebrities is going to be part of this TV shows. Here are complete details about the starting date and broadcasting schedule of the Star Pravah Mi Honar Superstar. Also, find out more information related to the judges and the host of the show.
Mi Honar Superstar Chhote Ustaad Starting Date
The exclusive promos of the shows are out. And they are receiving a massive amount of positive responses from fans. And the TV show will hit the screens on the 4 December. It will be broadcast every Thursday and Friday at 9:00 p.m.
The singing television reality show will replace the current tv show Mi Honor Superstar Jalosh Dance Cha.
Mi Honar Superstar Chhote Ustaad Judges and Host
Mi Honar Superstar Chhote Ustad will have one of the famous singers on the judging panel. None other than Sachin Pilgaonkar will be part of this TV show. At the same time, Adarsh Shinde and Vaishali Samant will also be on the judging panel. Both of them are big names in the music industry. The TV show is going to be presented by Siddharth Chandekar and child actress Avni Joshi.
The online broadcast of the show will be available at anytime and anywhere. Hotstar’s official website and the app will provide the online broadcast of the show. You can download the Hotstar app from the Google Play Store and app store.
Stay tuned with Audition Date and Place to get the latest updates from new TV shows. Also, don’t forget to check out audition updates of upcoming television reality shows.

domain: celebrity


In the run up the release of her Armor On EP, which is expected for release on March 27th, Dawn Richards, the former Dirty Money member, continues to impress. Having most recently released “Black Lipstick” and “Fly” the most recent cut to hit the interwebs is a 54 second preview of her forthcoming offering “Bombs”, along with its accompanying visuals.
“I wanted the art direction to be cohesive with the song and sound of the EP,” said Dawn. “Warriors fighting for the same cause. ‘Bombs’ is the ‘Hearts have arrived’ record.”
Check it out below.


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Transportation to Casa de Abuela
Casa de Abuela is a custom home built on a quarter-acre lot overlooking the rain forest. It has a large swimming pool and is just a five-minute walk from the beach. It is located in a neighborhood that overlooks Esterillos Oeste, a small village on the central Pacific coast. The neighborhood itself is situated on the top of a ridge and consists of 10 – 12 custom homes, most of which are owned by American or Canadian families that live there permanently.
The house features a bedroom wing and a great room separated by a two-story open-air hall. There are four bedrooms, each with its own private bath and air conditioning. Two of the bedrooms accommodate a family of four with a double bed and a set of bunk beds, all made by a local carpenter of Costa Rican cedar. The upstairs bedrooms also have external entrances for privacy.
The hall is over two stories high, providing cool breezes even on the hottest day. The great room has a cathedral ceiling and three sets of double doors for air circulation. One set leads out on to the large front porch, providing a perfect setting for morning coffee and bird watching. The kitchen area has a large island for cooking and serving. The dining area has a table that seats 12. Like the bedrooms, the great room is also air-conditioned.
ATTRACTIONS AROUND THE VACATION RENTAL
You can go to Jaco Beach just 15 minutes where you can find casinos, fine dining, sushi restaurants, and jazz bars. You can also get a massage or shop for souvenirs.
There are many exciting things to do from Oeste, all within an hour or less drive from the house. For example, you can go to Manuel Antonio, one of the finest national parks in CR, go white water rafting, ride the ‘zip’ lines over the rain forest, go deep-sea fishing, or take a monkey tour in a mangrove swamp.

domain: geography


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‘Murder Mystery’ dinner to benefit local cause in Jackson
JACKSON, Tenn. — The Jackson Elks Lodge #192 will host a murder mystery dinner benefiting a local cause.
“Dying For Chocolate” is the theme of the event, and the mystery begins as “Cherish Chocolates is looking for the face of their new product ‘To Die For.'”
Guests are invited to join the fun on Friday, February 25 or Saturday, February 26. Cocktails and mingling will be held at 6 p.m. each night with dinner to follow at 6:30.
Tickets are $25 a person. All proceeds will benefit the Hospice Home Fund of the West Tennessee Healthcare Foundation.
Jackson Elks Lodge #192 is located at 28 Executive Drive in Jackson.
For tickets and more information, click here or call (731) 668-9020.
For more local news, click here.

domain: news


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From Robin Williams to The Rock, it’s become common to find celebrities who play videogames, but it isn’t often that they will accomplish a major feat in their area of expertise, then go straight home and throw themselves into a competitive match of Counter-Strike: Global Offensive – but that’s just what football icon Neymar did.
A Reddit post by user ‘AbsoluteDealer’ brought our attention to Neymar’s journey from football field to first-person shooter, as an image taken from the star’s social media suggests Neymar went home after the recent football match between PSG and Clermont Foot and decided to play the FPS game as a way to unwind.
The image shows his gaming desk setup with CS:GO running, accompanied by the number “3” and a football emoji, a win emoji, then the letters “CS.” The only full word reads “bebado,” which according to Google Translate means “drunk” in Brazilian Portuguese. On his desk is a signed football, presumably from his recent match against Clermont where he scored a hat-trick, which would explain the emojis’ meaning.
Neymar has long been associated with CS:GO, even going so far as to stream himself playing the game on Twitch and even uses FACEIT for matchmaking. It’s not a gimmick, either, as Neymar clearly has the skill to play at high levels, being rank 3 on FACEIT and around Master Guardian 1 in normal ranked matchmaking.
While many gamers have to psyche themselves up to jump into the competitive CS:GO scene, getting their aim on-point in a practice warm up mode, Neymar seemingly doesn’t feel the pressure. It’s no wonder, because you have to have nerves of steel to score a hat trick and play for a club like PSG.