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Int. J. Mol. Sci. 2014, 15(5), 8773-8794; doi:10.3390/ijms15058773
: Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAFV600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1]. In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. Here, we treated two melanoma brain metastasis cell lines, H1_DL2, harboring a BRAFV600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss, with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1_DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1_DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1/2 and reduced the expression of pAKT and p-mTOR in H1_DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas.1. Introduction
Malignant melanomas arise from the transformation of melanocytes, and DNA damage of melanocytes from ultraviolet radiation is regarded as the most important environmental risk factor [3]. The tumors have the highest propensity to metastasize to the brain, seen in around 50% of the melanoma patients with advanced disease [4]. Current treatment strategies of melanoma brain metastasis involve surgery, chemotherapy, radiotherapy and radiosurgery. Treatment effects are, however, minimal, as multiple brain metastases are often found at the time of neurological presentation, which decreases the prognosis considerably [5]. Furthermore, the melanomas are regarded to be highly radioresistant [3]. Further, the blood-brain barrier (BBB) prevents effective drug delivery to the tumors [6], as most of the chemotherapeutics used today are either too large or hydrophilic to cross the BBB [4]. Thus, new therapeutic approaches are warranted, such as using small-sized targeted molecules that may cross the BBB more easily.
The mitogen activated protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K) signaling pathways are often activated in melanomas by genetic alterations [7]. The MAPK pathway plays a key role in modulating melanoma survival, proliferation, invasion and angiogenesis. In normal cells, this pathway is tightly regulated by extracellular signals from the cell membrane to the nucleus, mediated by a cascade of phosphorylation events. Frequent somatic point mutations have been found in the BRAF (66%) and N-RAS (15%) genes [8–10]. Around 80%–90% of the BRAF mutations display a valine to glutamic acid substitution (BRAFV600E) [11], although other activation mutations are also seen, such as BRAFV600K and BRAFV600R [12,13]. Around 69% of the BRAFV600E mutations are reported to be homozygous [14]. The activated RAF kinase phosphorylates and activates the MAPK/extracellular signal-regulated kinases (ERK1/2; MEK1/2), further phosphorylating and activating MEK, which, in turn, phosphorylates and activates ERK1/2. Gene expression, metabolism and cytoskeletal functions are then regulated by ERK1/2 [11,15,16].
The activity of the PI3K pathway is also increased in melanomas, due to the loss of function of the phosphatase and tensin homolog (PTEN), either through mutation, deletion or methylation. PTEN is a critical negative regulator of AKT by preventing its phosphorylation, with subsequent inhibition of the PI3K pathway [17]. Loss of PTEN function has been observed in 10% to 30% of melanomas and is commonly associated with the BRAFV600E mutation [13,18,19]. Increased activity in the PI3K pathway has further been related to an acquired resistance to BRAF and MEK inhibitors after therapy [20]. Thus, a combined inhibition of the MAPK and PI3K signaling pathways in treating melanoma could potentially increase the therapeutic efficacy.
Vemurafenib (PLX4032) is a serine threonine kinase inhibitor specifically targeting mutated BRAF and has recently been approved for treating BRAFV600E mutated melanomas by the US Food and Drug Administration (FDA) [12]. The penetrance of the drug into the human brain has, to our knowledge, not been investigated, but results from a study performed by Mittapalli and colleagues suggested the penetration of vemurafenib into the mouse brain [21]. The drug has shown encouraging results in clinical trials on advanced melanoma [12], although acquired treatment resistance has so far been a major issue [22,23]. Interestingly, there is a few reports in the literature on the treatment of brain metastases using vemurafenib [24–28] and also indicating increased patient survival [24,27] and tumor necrosis [28] after treatment.
Temsirolimus (CCI-779) is an ester derivative of sirolimus (rapamycin), inhibiting mTOR kinase activity. By binding to mTOR, which is downstream of PTEN, signal transduction in the PI3K pathway required for progression through the cell cycle is inhibited [2]. It has previously been reported that therapeutic concentrations of temsirolimus can be achieved in glioblastoma [29]. In a clinical phase II trial using temsirolimus in metastatic melanoma, only a minimal effect was seen, indicating that the drug may not be sufficiently active as a single agent [2]. However, in a murine model, temsirolimus enhanced the anti-tumor activity of cancer vaccines used to treat established renal cell carcinoma and melanoma [30]. This indicates that combined treatment with other drugs could be more effective.
The main purpose of the present work was to study whether combined treatment using vemurafenib and temsirolimus could inhibit the growth of human melanoma brain metastasis cells in vitro. To our knowledge, these two drugs have not been combined to treat melanoma brain metastasis cells before. In this study, we used cell lines previously derived from human melanoma brain metastases. Results in our lab suggest that these cells have the ability to specifically home to the mouse brain [31], thus making them a unique tool for studying the therapeutic effects of vemurafenib and temsirolimus on brain metastatic cells in vitro and, also, in later in vivo studies. Our in vitro results show that cell proliferation and migration may be inhibited when the two drugs are used in combination. Moreover, the combined treatment led to reduced pERK1/2, p-mTOR and pAKT activity. Global gene expression analysis indicated that several cellular functions were altered by combined treatment affecting the cell cycle, cell death and survival, cellular movement and DNA-replication, as well as DNA recombination and repair.
2. Results and Discussion
2.1. BRAF and PTEN Status of the H1_DL2 Melanoma Brain Metastasis Cell Line
Bidirectional DNA sequencing of the H1_DL2 cells showed that the BRAF mutation was heterozygous by a single mutation at exon 15 (nucleotide 1799) of the BRAF gene. This thymidine (T) to adenine (A) transversion mutation results in the substitution of valine with glutamate in codon 600 (V600E) (Figure S1A, red arrow). Furthermore, DNA copy number analysis showed that the H1_DL2 cell line had a homozygous deletion of PTEN on chromosome 10 (Figure S1B,C, red arrows). This indicates that both the MAPK and the PI3K pathways may be activated in these cells.
2.2. Treatment with Vemurafenib and Temsirolimus Induces Anti-Proliferative Effects in H1_DL2 and H3 Cell Lines Grown as Monolayers
The H1_DL2 cell line was effectively treated with vemurafenib, with an IC50 of 0.679 μM (Figure 1A, left). Treatment with temsirolimus alone was less effective, with an IC50 of 4.323 μM (Figure 1A, middle), while combined therapy was shown to be the most effective treatment (IC50 = 0.063 μM; Figure 1A, right).
A more detailed comparison of the drug effects on the H1_DL2 cells is shown in Figure 1B and Table 1. At a drug concentration of 0.05 μM, 82.8% of the H1_DL2 cells survived treatment when using vemurafenib, while 54.7% of the cells survived treatment with temsirolimus alone. However, only 31.0% of the cells survived a combined treatment, indicating a synergistic effect of combined therapy (co-efficient of drug interaction (CDI), 0.68; see Table 1). Single drug treatment with vemurafenib was effective at concentrations of 5 or 10 μM (29.5% and 24.4% survival, respectively), while treatment with temsirolimus showed a cell survival of 53.1% (5 μM) and 48.6% (10 μM). Combined treatment was the most effective, with cell survival of 21.0% at 5 μM (synergistic effect) and 15.9% at 10 μM. Pictures of cell survival after treatment with vemurafenib and temsirolimus are also seen in Figure S2. For a comparison, we assessed the treatment effects on the H3 melanoma brain metastasis cell line, which expresses WT BRAF and has a homozygous deletion of PTEN (data not shown). In general, the H3 cells were not as sensitive to therapy as the H1_DL2 cells, where vemurafenib treatment resulted in an IC50 of 5.105 μM (Figure 1C, left). Treatment with temsirolimus alone was even less effective, with an IC50 of 9.906 μM (Figure 1C, middle). An increased effect using a combined therapy was not seen (IC50 = 6.446 μM; Figure 1C, right).
A more detailed comparison of the drug effects on the H3 cells is shown in Figure 1D and Table 2. All H3 cells survived treatment with 0.05 of μM vemurafenib, while 87.6% of the cells survived treatment with temsirolimus alone.
The combined treatment did not increase cell death, as 85.6% of the cells survived treatment. At 5 μM, 64.0% of the cells survived after vemurafenib treatment, while 79.0% of the cells survived when temsirolimus was used alone. Combined therapy at this dose was not more effective (75.6% survival). Similar results were observed at 10 μM (42.3% survival with vemurafenib, 79.8% survival using temsirolimus and 46.8% survival after combined treatment).
2.3. Treatment with Vemurafenib Induces Anti-Migratory Effects in the H1_DL2 Cell Line Grown as Monolayers
A monolayer wound healing assay was applied to study the effects on cell migration during treatment. By using this assay, we could clearly observe the cell migration of individual tumor cells during a time course of 60 h (Figure 2, and Supplementary Movies 1–4). At 0 h, the width of the open area was around 500 μm (Figure 2A). The wound was closed after 60 h in untreated cells (Figure 2B) and cells treated with 5 μM temsirolimus (Figure 2C). When the H1_DL2 cells were treated with 5 μM of vemurafenib, around 40% of the wound was still open after 60 h (Figure 2D). The combined treatment (5 μM temsirolimus + 5 μM vemurafenib) did not increase the inhibition of cell migration compared to vemurafenib treatment alone (Figure 2E,F).
2.4. Treatment of H1_DL2 Spheroids with Vemurafenib and Temsirolimus Inhibits Spheroid Growth and Increases Cell Death
Spheroid growth was studied by measuring their diameters after Day 0 and 8 (Figure 3A, left, and Figure 3C). There was a 42.1% increase in mean diameter of untreated spheroids from Day 0 to Day 8. Treatment with 5 μM of temsirolimus inhibited spheroid growth compared to the untreated spheroids, as there was an increase in mean diameter of only 6.9% from Day 0 to Day 8. Single drug treatment with vemurafenib was more effective, leading to a 13.1% decrease in diameter from Day 0 to Day 8. Combined treatment reduced the mean diameter even more, as there was a decrease of 16.7% in mean diameter from Day 0 to Day 8, however not statistically significant when compared to vemurafenib treatment alone, p = 0.191.
After three days of treatment, the number of dead cells within the spheroids was studied by staining with ethidium homodimer, followed by confocal imaging (Figure 3A right, 3B,D). Vemurafenib (5 μM) increased cell death more effectively than temsirolimus (5 μM) when administered as a single drug, whereas the combined treatment (5 μM of each drug) did not increase the number of dead cells, compared to treatment with vemurafenib alone.
2.5. Western Blots Suggest Drug-Related Inhibition of the MAPK (Mitogen-Activated Protein Kinase) and PI3K (Phosphoinositide 3-Kinase) Pathways
Untreated H1_DL2 cells expressed pAKT, p-mTOR and pERK1/2, as shown by western blots (Figure 4). The expression of phosphorylated ERK1/2 disappeared after treatment with 15 μM vemurafenib, whereas no change was found in cells treated with temsirolimus. Treatment with 25 μM temsirolimus reduced the expression of p-mTOR, and the combined treatment (10 μM vemurafenib + 10 μM temsirolimus) inhibited the expression of phosphorylated ERK1/2 and reduced the expression of p-mTOR and pAKT.
2.6. Principal Component Analysis of Gene Expression Arrays Shows Alterations in Gene Clustering after Treatment
Principal component analysis (PCA) is an unsupervised global analysis approach and is useful for reducing the dimensionality of gene expression data. PCA allows for projecting multidimensional gene data onto just two or three new dimensions that explain most of its variance. As a result, it is possible to visually interpret major trends in the data, for example, in terms of the similarity between various data points [32]. The PCA of our gene expression data showed that the different samples separated well into tightly pooled clusters for each of the respective treatments, without any outliers (Figure 5).
2.7. Pathways Affecting Cell Cycle Checkpoint Regulation Are Affected by Combined Treatment
Functional analysis of negatively or positively regulated genes before and after combination treatment was performed using Ingenuity Pathway Analysis (IPA; [33]). Two of the top pathways were "Cell cycle: G1/S checkpoint regulation" (Figure 6A) and "Cell cycle: G2/M DNA damage checkpoint regulation" (Figure 6B), where a number of genes in the pathways were downregulated after combined treatment (5 μM vemurafenib and 5 μM temsirolimus).
2.8. Several Genes Related to Cell Survival and Invasion Are Affected by Treatment
Based on the z-score, showing whether potential downstream functions were "activated" (a z-score larger than two) or "inhibited" (a z-score less than −2), a list of 17 downstream functions could be selected (Table 3; p ≤ 0.005).
These functions were categorized into the following groups: cell cycle; cell death and survival; cellular movement and DNA replication; and DNA recombination and DNA repair (Figure 7A). Furthermore, to evaluate the effects of the different treatments (vemurafenib vs. control, temsirolimus vs. control and combination vs. control), five of the most activated functions from each of the major groups were studied in more detail (Figure 7B). Overall, the microarray analysis indicated that the inhibitory effects of treatment was related to major cellular functions within the H1_DL2 cells, such as the downregulation of genes associated with the cell cycle, cellular movement and DNA replication, DNA recombination and DNA repair and upregulation of genes associated with cell death.
2.9. Discussion
Melanoma patients with advanced disease have a high chance of developing brain metastases, which is associated with a poor prognosis. Apart from introducing Gamma Knife radiosurgery, conventional treatment of these metastases has not changed during the last two decades [3], necessitating the development of new therapeutic strategies. Several small molecule inhibitors targeting signaling pathways have been developed recently. However, with a few exceptions [24,27,28], they have not been studied clinically on patients with brain metastases from melanoma.
The two specific protein kinase inhibitors, vemurafenib [34–36] and temsirolimus [2,37,38], have previously been shown to target the MAPK and PI3K pathways, respectively. Lazar and colleagues showed that more than 10% of human melanomas display a loss of PTEN expression, in addition to BRAFV600E mutation [39]. PTEN is one of the critical negative regulators of AKT activity [40]. Thus, the loss of PTEN expression leads to increased PI3K/AKT signaling, particularly when BRAF is inhibited, which, in turn, may contribute to an intrinsic resistance of BRAFV600E mutated melanoma cells to vemurafenib treatment [17]. Thus, it is clear that single drug therapy targeting individual pathways may be insufficient to obtain therapeutic efficacy. The fact that melanomas develop resistance to vemurafenib emphasizes the importance of targeting multiple pathways simultaneously [9].
We set out to study whether the growth and proliferation of human melanoma brain metastasis cells could be inhibited more efficiently in vitro by combining therapies targeting two major pathways in melanoma progression. To our knowledge, this study is the first to demonstrate inhibitory effects on melanoma brain metastatic cell lines by combining vemurafenib and temsirolimus, and we show the regulation of genes after treatment that may be involved in reducing the invasive potential of melanoma cells.
The BBB has an important role in the treatment of brain metastasis. The delivery of chemotherapy is usually ineffective, due to an often intact vascular barrier in the brain with low passive transcellular permeability. Furthermore, the endothelial cells of the brain vasculature express active efflux drug transporters, which limit the uptake of most anticancer drugs. We have previously shown in an animal model of brain metastasis that the BBB is relatively leaky for small contrast agents (molecular weight (MW) below 300 Da) at early stages and permeable to magnetic resonance imaging (MRI) contrast agents (MW 0.566 and 2.066 kDa) at later stages of brain metastatic development [4]. These results suggest that small MW drugs, such as vemurafenib and temsirolimus, could potentially cross the BBB early in the development of brain metastases.
By targeting BRAF and mTOR simultaneously, we were able to inhibit the cell growth and proliferation (Figure 1) of the H1_DL2 cells in monolayers. Combined treatment resulted in a synergistic increase in cell death compared to single drug treatment (at concentrations of 0.05 and 5 μM), as shown by the resazurin assay (Table 1). However, temsirolimus alone was not able to decrease the migratory capacity of cells grown as monolayer cultures, compared to untreated cells. These results were supported by our gene analysis data, indicating minimal effects on genes related to invasive capacity after single dose treatment with temsirolimus (Figure 7B; Group 9). The gene analysis, however, indicated that combined therapy was more effective at inhibiting cell invasion, compared to single-dose treatment with vemurafenib.
Assays using tumor cells grown as multicellular spheroids are regarded as more relevant with respect to solid tumor growth seen in vivo [41]. In our study, vemurafenib was more effective than temsirolimus in inhibiting spheroid growth in vitro, and the combined therapy suggested an additional growth inhibition. The number of dead cells within the spheroids did not increase after combined treatment compared to vemurafenib treatment alone, which likely can be explained by the previously observed poor penetrance of ethidium homodimer into compact spheroid structures. Our results on monolayer cells and spheroids, taken together, indicate that combined therapy may inhibit single tumor cell growth, as well as when treating solid tumors, provided that the drugs would effectively cross the BBB. To study this further, upcoming animal studies will investigate the effects of single and combined treatment on brain metastasis, both at the single-cell level in the animal brain using iron oxide prelabeling of the tumor cells [31] and on solid tumor development.
The western blots showed that the expression of pMAPK was lost after combined treatment, and the expression of p-mTOR pAKT was reduced, suggesting that the MAPK and PI3K signaling pathways were inhibited.
Gene expression profiling is important for the identification of genes that may be involved in tumor progression and metastasis and also for studying how tumor cells respond to therapy. In addition, gene expression profiling has been important for a molecular sub-classification of tumors [42,43]. PCA is an exploratory visualization of gene expression data and discriminates between sample groups [32]. In our treatment study, PCA separated the sample groups for each respective treatment (Figure 5) into tightly pooled clusters well.
A functional analysis of negatively or positively regulated genes before and after combination treatment was performed using IPA. Interestingly, pathways representing cell cycle regulation were downregulated after combined treatment of our melanoma brain metastasis cells (Figure 6).
Further analysis of these pathways using the activation z-score (Figure 7A and Table 3) showed seventeen downstream functions that were either activated (z-score > 2) or inhibited (z-score < −2) by combined treatment. These functions could be grouped into four different categories: (1) cell cycle; (2) cell death and cell survival; (3) cellular movement; and (4) DNA replication, DNA recombination and DNA repair. We then performed a more detailed analysis of the downstream functions with the highest relative z-score, to study the differences in gene expression after single drug treatment and combined treatment. Gene sets related to G1/S phase, cell survival and cell invasion showed larger effects on gene expression after combined treatment, compared to the single drug treatments (Figure 7B). Interestingly, vemurafenib treatment did not show any effects on downstream functions related to the apoptosis of melanoma cell lines, while DNA repair mechanisms resulted in low action scores (<−2) irrespective of treatment. Based on our functional analysis, further work is warranted to verify key genes that may represent therapeutic targets. This was, however, beyond the scope of this study.
3. Materials and Methods
3.1. Cell Line and Cell Culture
The H1 and H3 cell lines were established in our laboratory from patient biopsies of human melanoma brain metastasis, as previously described [31,44]. Written consents were obtained from the patients before tumor material was collected. The Regional Ethical Committee (REC Number 013.09) and the Norwegian Directorate of Health (NSD Number 9634) approved the tissue collection and biobank storage of tumor biopsies and derived cell lines. The cells were authenticated in February, 2013, using the AmpFℓSTR® Profiler Plus® ID PCR Amplification Kit (Applied Biosystems Inc., Foster City, CA, USA), and short tandem repeat (STR) profiles were matched to the parent tumor and cross-checked with cell line profiles at [45].
The H1 cells were transduced with two lentiviral vectors, encoding Dendra (a green fluorescent protein (GFP) variant) and luciferase to obtain the H1_DL2 cell line. Flow cytometric isolation of cells by GFP expression was performed (BD FACSAria, Becton Dickinson, Franklin Lakes, NJ, USA).
The cell lines were grown in DMEM supplemented with 10% heat-inactivated newborn calf serum, four times the prescribed concentration of non-essential amino acids, 2% l-glutamine, penicillin (100 IU/mL) and streptomycin (100 μL/mL) (Cambrex, East Rutherford, NJ, USA). The cells were incubated in a standard tissue culture incubator at 37 °C, 100% humidity and 5% CO2.
3.2. BRAF Gene Mutation Status
The H1_DL2 and the H3 cell lines were checked for BRAFV600E mutation status by Sanger dideoxy sequencing of PCR amplicons from genomic DNA using primers, as described previously [1]. PCR amplicons were purified by ExoSapI (Affymetrix, Santa Clara, CA, USA) before sequencing, using the BigDye® Terminator v 3.1 Cycle Sequencing Kit (Invitrogen, Carlsbad, CA, USA).
3.3. PTEN Deletion Status
The PTEN deletion status in the H1_DL2 and H3 cell lines was studied by DNA copy number analysis. Genomic DNA was extracted from cell lines using the DNAeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's instructions. DNA was eluted in water, fragmented to an average size of 200–500 bp using DNAse1 (rDNAse1, Ambion, Life Technology Ltd., Grand Island, NY, USA) and labeled using the BioPrime aCGH Genomic Labeling Kit (Invitrogen, Carlsbad, CA, USA) and Cy3 and Cy5 dyes purchased from GE Healthcare (Chalfont St. Giles, UK), following standard protocols for Agilent aCGH. Commercially available female DNA pooled from multiple anonymous donors (Promega, Madison, WI, USA) was used as a reference. Labeled DNA was competitively hybridized to SurePrint G3 Human 2 × 400 K CGH microarrays (Agilent Technologies, Santa Clara, CA, USA) following standard Agilent protocols. The slides were scanned at 3-μm resolution using the Agilent High-Resolution Microarray scanner and the image data were extracted using feature extraction (Agilent Technologies). FE extraction files were imported into Genomic Workbench (Agilent Technologies) for visualization and analysis. Aberrations were called using the ADM2 algorithm with a threshold setting of 25, centralization on, with a threshold of 25 and an aberration filter min Probes = 5 and min AvgAbsLogRatio = 0.45.
3.4. In Vitro Drug Treatment
Vemurafenib was purchased from ChemieTek (Indianapolis, IN, USA) and temsirolimus from Sigma-Aldrich (St. Louis, MO, USA). Both drugs were dissolved in dimethylsulfoxide (DMSO) and stock concentrations of 50 mM were stored at −20 °C in aliquots until use. Final concentrations were made in growth medium (0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, 25, 50, 100 or 250 μM), before adding to the cell monolayers.
3.5. Resazurin Cell Proliferation Assay
Cell proliferation before and after treatment (vemurafenib, temsirolimus or combined treatment) was studied using a resazurin assay, as described previously [46]. Briefly described, 4000 H1_DL2 or H3 cells in 100 μL cell culture medium were seeded into each well of a 96-well plate and cultured for 24 h. The cell monolayers were then subsequently treated with drugs (at concentrations described in Section 3.4) for 72 h. After treatment, the culture medium containing drugs was removed, and fresh culture medium with 10 μg/mL resazurin (Sigma-Aldrich) diluted in phosphate buffered saline (PBS) was added. Following incubation for 4 h, the absorbance was measured at dual mode 560/590 nm with a scanning multiwell spectrophotometer (Victor 3 1420 multi-label counter, Perkin Elmer, Waltham, MA, USA), using WorkOut v2.0 software (Dazdaq Solutions Ltd., East Sussex, UK). The results were prepared in GraphPad Prism v6 software (GraphPad Software, Inc., La Jolla, CA, USA).
IC50 was defined as the drug concentration at which 50% of the cell growth was inhibited. The calculations of IC50 were performed in GraphPad Prism v6. The co-efficient of drug interaction (CDI) was calculated using the formula: CDI = AB/(A × B). According to the absorbance of each group, AB is the ratio of the combination group to the control in OD 560/590, and A or B is the ratio of the single drug group to the control group in OD 560/590. CDI < 1 indicates synergism; CDI < 0.7 indicates significantly synergistic effect; CDI = 1 indicates additivity; and CDI > 1 indicates antagonism, as described previously [47].
3.6. Cell Migration Assay
To study the effects of treatment on cell migration, a monolayer wound healing assay was performed. A silicone culture insert (Ibidi GmbH, Martinsried, Germany) was inserted into each well of a 4-well μ-Slide (Ibidi GmbH), and 70 μL of H1_DL2 cells (concentration: 9 × 105 cells/mL in cell culture medium) were added into each half of the culture inserts. The cells were allowed to grow to confluence for 24 h, before the culture insert was removed, resulting in a wound (a cell free gap) with a width of 500 μm within the culture. Drugs were then added into the wells (5 μM vemurafenib, 5 μM temsirolimus or 5 μM vemurafenib + 5 μM temsirolimus), and cell migration into the wound was studied by time lapse microscopy for 60 h, using a Nikon TE2000 inverted microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with an incubator holding 37 °C, 100% humidity and 5% CO2. Pictures were then processed and analyzed with Image-Pro Plus (MediaCybernetics, Warrendale, PA, USA). The experiments were performed in triplicate.
3.7. Spheroid Growth and Cytotoxicity
Multicellular spheroids from the H1_DL2 cells were prepared using a protocol modified from Ivascu and Kubbies [48]. Four thousands cells in 100 μL of cell culture medium were distributed per well in a Costar 96-well round bottom ultra-low attachment plate (Corning Inc., Corning, NY, USA). The plate was then centrifuged at 1000× g for 15 min and returned to the cell incubator for 5 days. At that time, the cells had formed spheroids with mean diameters of around 700 μm.
The spheroids were exposed to drugs for 8 days, either as single drugs (10 μM) or as a combined treatment (5 μM vemurafenib + 5 μM temsirolimus). Two orthogonal diameters were measured for each spheroid after 0 and 8 days with a Nikon TE2000 inverted microscope using the 4× objective, and the mean spheroid diameters (±SD) were calculated in GraphPad Prism v6.
The treatment effects on cells within the spheroids were evaluated at Day 3 using a Live/Dead Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR, USA), by staining the spheroids with 2 μM ethidium homodimer for 30 min at room temperature. The number of live cells (expressing GFP) and dead cells (stained red by ethidium homodimer) was visualized by confocal microscopy. For each treatment, three spheroids were studied, and both the acquisition of images and the analysis were performed with the exact same parameters for all spheroids studied. z-stacks of 50 optical sections through a total depth of 100 μm were acquired with an inverted Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) using a HCX PL APO CS 10 × 0.4 NA objective and further processed in Imaris version 7.6.3 (Bitplane AG, Zurich, Switzerland). Surface rendering was performed on both the red and green channel. Threshold levels for the red channel were set to visualize single cells, whereas the threshold level for the green channel was set to measure the total volume of the spheroid. Only red cells that were surrounded by the green volume were accounted for in the statistical analysis. The result is presented as the number of dead cells (red) per spheroid unit volume (green) and was prepared in GraphPad Prism v6.
3.8. Western Blot Analysis
H1_DL2 cell monolayers were grown in T75 culture flasks (Nunc, Roskilde, Denmark) until the monolayers were 70%–80% confluent. The cells were then treated with drugs (15 μM vemurafenib, 25 μM temsirolimus or 10 μM vemurafenib + 10 μM temsirolimus) and incubated for 72 h. The cells were washed twice with ice-cold phosphate buffered saline and lysed in 450 μL Kinexus lysis buffer (Table S1). The samples were harvested with a sterilized cell scraper, homogenized using a Kontes pellet pestle motor (Fisher Scientific), sonicated for 4 × 10 s (Bandelin Sonorex RK255S) and centrifuged at 10,000× g for 10 min at 4 °C. Aliquots of supernatant were stored at −80 °C until further analysis. Protein concentration of the cellular extracts was determined using the Bio-Rad Bradford assay (Bio-Rad Laboratories, Inc., Berkeley, CA, USA), as per the kit instructions, using bovine serum albumin as the standard. 20 mg of protein extract were electrophoresed on 4%–12% NuPage Bis-Tris-Glycine gels (Life Technologies, Carlsbad, CA, USA) for 50 min at 200 V. Proteins were blotted onto nitrocellulose membranes (Whatman, Germany) at 30 V for 90 min using a tank blot system. The membranes were blocked with 0.05 g Defco dry milk powder per mL of blocking solution (TBST buffer; 0.02 M Tris-HCl (pH 7.5), 0.15 M NaCl and 0.1% Tween 20) for 1 h at 4 °C. Immunostaining was performed using antibodies against p-AKT (Ser473) (dilution: 1:500), p-mTOR (Ser2448) (dilution: 1:500), p-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (dilution: 1:2,000) (Cell Signaling, Danvers, MA, USA) and GAPDH (dilution: 1:2,500) (Abcam, Cambridge, UK) diluted in blocking solution and incubated overnight at 4 °C. A glioblastoma xenograft, as described previously [49], was used as the positive control for pMAPK (noted as A in Figure 4), and the 293T cell line (ATCC # CRL-11268) served as a positive control for pAKT and p-mTOR (noted as B in Figure 4). The membranes were washed 4 times for 10 min each before being exposed to secondary antibody, anti-rabbit HRP (Beckman Coulter, Brea, CA, USA), diluted 1:20,000 in blocking buffer and incubated for 90 min at room temperature. Bound antibody was visualized by using a chemiluminescence detection system, SuperSignal West Femto, and imaged by LAS3000.
3.9. RNA Extraction after Drug Treatment of Monolayer Cultures
Total RNA was extracted from cell samples by the RNeasy Plus Kit (Qiagen). The H1_DL2 cells were grown in a 6-well plate (Nunc) with 5 parallels for each sample. All samples were treated and harvested at the same time. When confluent, cell monolayers were exposed to either 15 μM vemurafenib, 25 μM temsirolimus or a combination of 5 and 5 μM temsirolimus for 24 h. These cells were then washed once with PBS, followed by the addition of 350 μL RLT lysis buffer into each well. The cells were then removed using a cell scraper. The remaining procedure was followed according to the manufacturer's instructions, including DNase 1 solution treatment (Qiagen). RNA samples were stored at −80 °C until further analysis.
3.10. Illumina HT-12 Array; RNA Preparation, Labeling and Microarray Hybridization
All microarray experiments were performed using the Illumina iScan Reader (Illumina, Inc. San Diego, CA, USA), which is based upon fluorescence detection of biotin-labeled cRNA. 500 ng of total RNA from each sample were reversely transcribed, amplified and biotin-16-UTP-labeled, using the Illumina TotalPrep RNA Amplification Kit (Life Technologies, Carlsbad, CA, USA). The amount and quality of the Biotin-labeled cRNA was controlled using a NanoDrop spectrophotometer (Nanodrop Products, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). 750 ng of biotin-labeled cRNA were hybridized to the HumanHT-12 V4 Expression BeadChip (Illumina, San Diego, CA, USA), according to the manufacturer's instructions. The HumanHT-12 V4 BeadChip targets 47,231 probes derived primarily from genes in the NCBI RefSeq database (Rockville, MD, USA) (Release 38).
3.11. Microarray Analysis
Principal component analysis and analysis of variance were performed using Partek Genomics Suite (Partek Inc., St. Louis, MO, USA). The gene expression data was projected onto 3 principal components (PC 1, 2 and 3) and the projections were used for an exploratory visualization of the data. Results were significant if the absolute fold change was more than 1.6 or less than −1.6, with a q-value < 0.001. Ingenuity's Pathway Analysis (IPA) (Ingenuity®, [33]) was used for pathway and functional analysis. Plots were made using the R language v.3.0.1 (The R Foundation for Statistical Computing, Vienna, Austria).
3.12. Statistics
The statistical significance of differences was evaluated by an unpaired two-tailed t-test, using GraphPad Prism v6. Values presented in the figures represent means ± standard deviation. A two-tailed p ≤ 0.05 was considered to be statistically significant.
4. Conclusions
In summary, we show that treatment of BRAFV600E-mutated melanoma brain metastases cell lines may be effectively treated in vitro with vemurafenib combined with temsirolimus. Our results described here should be further verified in animal experiments, which could lead to new knowledge to be used in future clinical trials.
This study was funded by the University of Bergen, the Norwegian Cancer Society, the Norwegian Research Council, Elekta Instrument AB (Stockholm, Sweden), and Centre de Recherche Public de la Santé through a grant from the Ministry of Research and Higher Education in Luxembourg, the Fonds National de la Recherche of Luxembourg. We thank Bodil Hansen and Tove Johansen for technical assistance. We are also grateful to Kjell Petersen for assistance with the bioinformatics analysis.
Conflicts of Interest
The authors declare no conflict of interest.
- Author ContributionsI.D. performed monolayer cell growth experiments, in vitro drug treatments, western blots, spheroid growth assays, RNA extraction, data analysis, made figures and prepared the manuscript. S.H. did monolayer cell growth experiments, western blots, data analysis, made figures and contributed to the manuscript. D.S. performed DNA copy number analysis, made figures and contributed to the manuscript. J.K.V. performed microarray experiments, principal component analysis, functional analysis, made figures and contributed to the manuscript. E.S. did the wound healing assays, data analysis, contributed to making figures and movies and contributed to the manuscript. H.A.D. performed spheroid growth assays, including confocal imaging, data analysis, preparation of figures and contributed to the manuscript. K.O.S. transfected the cell lines with reporter genes, contributed to the resazurin assay and making figures and contributed to the manuscript. R.B. contributed to the design of this study, the data analysis and the writing of the manuscript. F.T. was responsible for the design of this study, contributed to all the experiments, including data analysis, made figures and finalized the manuscript.
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Concentration (M) | Temsirolimus (% Survival) | Vemurafenib (% Survival) | Combined (% Survival) | CDI |
0 | 100 ± 2.2 | 100 ± 2.2 | 100 ± 2.2 | - |
0.05 | 54.7 ± 1.5 | 82.8 ± 2.1 | 31.0 ± 0.8 | 0.68 |
5 | 53.1 ± 4.0 | 29.5 ± 1.5 | 21.0 ± 1.1 | 0.66 |
10 | 48.6 ± 4.2 | 24.4 ± 1.2 | 15.9 ± 1.1 | 1.34 |
Concentration (M) | Temsirolimus (% Survival) | Vemurafenib (% Survival) | Combined (% Survival) | CDI |
0 | 100 ± 4.7 | 100 ± 4.7 | 100 ± 4.7 | - |
0.05 | 87.6 ± 2.3 | 99.9 ± 6.5 | 85.6 ± 2.5 | 0.98 |
5 | 79.0 ± 3.4 | 64.0 ± 2.5 | 75.6 ± 4.3 | 1.50 |
10 | 79.8 ± 5.9 | 42.3 ± 1.2 | 46.8 ± 3.9 | 1.39 |
Bar Number | Downstream Functions |
1 | G1/S phase |
2 | S phase |
3 | M phase |
4 | G1 phase |
5 | G2/M phase |
6 | G2 phase |
7 | Cell survival |
8 | Apoptosis of melanoma cell lines |
9 | Invasion of tumor cell lines |
10 | Cell movement of tumor cell lines |
11 | Chemotaxis of PBMCs |
12 | Cell movement of carcinoma cell lines |
13 | Cytokinesis of tumor cell lines |
14 | Repair of DNA |
15 | Checkpoint control |
16 | Alignment of chromosomes |
17 | Chromosomal congression of chromosomes |
© 2014 by the authors; licensee MDPI, Basel, Switzerland This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).


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Question: Do the actors submit more or different scenes for the final round? It seems like that was the case last year, but I don't know if the rules have changed. (For instance, I thought I read Chandler Massey from Days submitted his break up scenes with Gabi for the pre-noms, and then almost coming out to Marlena for the final round). |
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gluconeogenesis from pyruvate--> labeling carbonModerator: BioTeam
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gluconeogenesis from pyruvate--> labeling carbonI have to predict the positions of the glucose molecule that would become labeled from the 1-14C pyruvate (i.e, the label in the carboxyl group of pyruvate).
a) Carbon-1 b) carbon-2 c) carbon-3 d) carbon-4 e) carbon-5 d) carbon-6 I first thought that carbons 1 and 6 were the correct answers, but they are not. Then I thought that 1,3,and 6 were correct but they are not. I know that pyruvate has 3 carbons and glucose has 6, but I know two how to approach this problem correctly. Any help will be appreciate it. Thank you and happy holidays!
look on this picture: http://upload.wikimedia.org/wikipedia/e ... enesis.png
and trace your atom! Just be carefull about symetrical molecules! http://www.biolib.cz/en/main/
Cis or trans? That's what matters.
Re: gluconeogenesis from pyruvate--> labeling carbon3,4... its a head to head bonding, not tail to tail, so 1,6 would be the reverse...
Re: gluconeogenesis from pyruvate--> labeling carbonWhen the two triosephosphates condense head-to-head to form a hexose, carbon 1 of the triose (originating from the carboxyl of pyruvate) forms carbons 3 and 4 of the hexose, carbon 2 of the triose forms carbons 2 and 5 of the hexose, and carbon 3 of the triose forms carbons 1 and 6 of the hexose.
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I don't think that the lying should matter . . . but if it does matter (I suppose as part of the character/integrity standard), I wouldn't place a whole lot of weight on their acquittals. They aren't *innocent* in the sense of having been proven to have not lied; the verdicts simply reflect conclusions that the prosecutions in those cases failed to prove beyond a reasonable doubt that they lied under oath.It could well be that the juries even felt that they had probably lied under oath, but they weren't certain enough to convict.The standard for enshrining someone in the Hall of Fame shouldn't be (if this is a relevant factor) they weren't proven beyond a reasonable doubt to have lied under oath. It should be much much higher. Tungster -- 1/10/2013 7:57 PM
It's the Hall of Fame, not the Hall of Morals. These are two of the greatest players of all time (probably both in the Top 10 of all time) despite their alleged transgressions. Regardless, the baseball writers should be removed from the equation. They have proven, through their own writings, that they can't be impartial. Myles Solomon -- 1/11/2013 11:36 AM
what happened to my comment? too close to the truth, or do you just post the ones you agree with? Anonymous -- 1/11/2013 6:45 PM
To Anonymous: your comment was inadvertently deleted as spam. Sorry about that - my fault, not the author's. I hope you can put it or something similar up again.Best,Mike McCann Michael McCann -- 1/12/2013 2:46 PM
Players who cheat to pad their stats and then lie about it don't deserve to be in the Hall of Fame. Their records are ill gotten gain. It also sends a direct and unmistakable message to kids and young players- cheat to get ahead. Who would want to support that? Anonymous -- 1/18/2013 10:24 AM
Why don't we just have another Hall of Fame of Cheaters and let it go? Then all the great names with all their records will be in a Hall of Fame and we can all go back to sleep. manotick -- 2/02/2013 6:22 AM
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Eric Eisenberg, Dean
Welcome to the Office of the Dean of the College of Arts and Sciences! It is my privilege to lead the College that is the intellectual heart of the University of South Florida. My leadership philosophy is to recruit the best faculty and staff in the world who are inspired by our mission, provide them with the tools they need to succeed, and stand back and celebrate their successes. I am so proud of my outstanding team of teachers, scholars, staff and administrators whose sole professional purpose is to enhance the lives of our students and our communities. Please explore all of the wonderful features of this site, join us at one of our many featured lectures and events, and contact us directly if there are other ways in which we can serve you. Go Bulls!
Eric M. Eisenberg, Ph.D. Dean Professor of Communication Email Curriculum Vitae


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In the National League, the Reds have the worst team ERA, over 5.00. The starting ERA is 4.92, 12th of 16 teams. Bullpen ERA is 5.23 -- by far the worst in the league, next teams are Philly and Houston at 4.65.
Is there any question how the Reds get better next year? Could the problem be any clearer?
Reds have three starting pitchers. Harang, Arroyo, Belisle. Team needs two starters.
Reds bullpen this year has included the following four pitchers, each of whom has received considerable pitching time -- Stanton (6.02 ERA in 62 outings), Coffey (6.50 ERA in 50 outings), Saarloos (7.39 ERA in 32 appearances), Majewski (8.68 in 26 games).
Team needs a two or three good relievers to add to Weathers, Burton and probably Coutlangus and Bray. Not that these four are all world beaters, but their numbers are within reason.
Two starters. Two or three relievers. Through some trades, promotions, free agency, that's what the team needs. It couldn't be any clearer. Krivsky's task is as simple as that. You win this division with around 90 wins, possibly fewer.


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Kilgore Trout stops Mason play dead in the water! DOUBLE OVERTIME . . .
Kilgore Trout stops Mason play dead in the water! DOUBLE OVERTIME . . . WHITEY CAN'T KEEP THE BROTHER DOWN . . . The President just . . . stood there, staring out vacantly at everybody. We didn't know what he was doing. He said it was a moment of silence. But for what? . . . An advanced challenge for the trolling master who has become bored. Not for n00bs! . . . Part 1-Lulz from Anons Anonymous is the go to collective of hackers for media outlets that want to drum up fear for internet freedom. The weapon of choice for Anonymous is a distributed denial of service attack, also known as DDoS. DDoS works by overloading a web server to the point where it can no . . . |
The chronicle.su is perfect in every way. Stop the Internet. |


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Author: T.J. Waters
Reader: Patrick G. Lawlor
Format: 9 CDs (Unabridged)
More than 100 students were admitted to the CIA's Clandestine Service to become Class 11, the most elite and secretive espionage training program in the country. Immediately following September 11, this was the largest, most diverse group in the agency's history. Go behind the scenes, where the spies-in-training learned subterfuge, mastering disguises, withstanding interrogations, and crossing into hostile territory without being detected.
Item #: E9X226 ISBN: 9781400102266
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Reader: Patrick Lawlor
Format: Playaway (Unabridged)
Item #: EP6268 ISBN: 9781608476268
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Friday, July 6, 2012
First July Color List...
21 comments:
The more I see... the more I like.
Love the color in your blog. Guess that is how you decided on the name.
I love the photos that flash in one spot from one to another. How do you do that?
Also I am one of seven sisters...
How did you end up living in Italy? (just curious)
ToOdLeS.Repliesoh my seven sisters! That is a lot of estrogen--:-) I loved growing up with four sisters but I guess I dont' know anything different?! The flashing photos are simply an animated gif file that you can do in photo software. :-) We actually don't live in Italy, we just visited there back in April for two weeks and I am still catching up on posting photos here and there!
I love the painting and the chocolate/ice cream treats. Yum! :)
Et tu, tutu?Lovely pictures! Also, the chocolate link to those brownie sundae bon bons... oh my gosh. YUM! I love Bakerella (and I still need to try to make her famous cake pops!!) Love it all!
neha madeSo happy for you that your sisters are visiting! That really is the best. When are you due?
ReplyDeleteWhat a cute family, absolutely adorable! :)
'The Wind of Inspiration' style blogBeautiful blog and inspiring photos and ideas - I took a look at your "Art", "Fashion", "Food" and "Family" post (I liked the "Naples" post a lot - Italy is wonderful), they are all wonderful and lovingly put together and your (growing) family looks so happy! I will make sure to visit your blog on a regular basis - thanks also for visiting my blog!


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Hoobastank - Crawling In The Dark Chords & Tabs |
Crawling In The Dark Chords & Tabs
If you want to play it acoustic it sounds good to me like this: Em C D Em I will dedicate and sacrifice my every C D C D Thing for just a second's worth of how my story's ending Em C D Em C And I wish I could know if the directions that I take D C D And all the choices that I make won't end up all for nothing [ Tab from: http://www.guitartabs.cc/tabs/h/hoobastank/crawling_in_the_dark_crd.html ] (Chorus) Em C D Em C D C Show me what it's for - make me understand it D C D I've been crawling in the dark, looking for the answer Em C D Em C D C Is there something more - than what I've been handed? D C D I've been crawling in the dark, looking for the answer Em C D Em Help me carry on - assure me it's okay to C D C D Use my heart and not my eyes to navigate the darkness Em C D Em C Will the ending be, ever coming suddenly? C D C D Will I ever get to see the ending to my story? (Chorus) *Break* Em Em Em Em Em D (x2) So when and how will I know? Em Em Em Em Em D (x2) C D Em How much further do I have to go? C D G And how much longer until I finally know? C D Em Cause I'm looking and I just can't see C D What's in front of me... in front of me (Chorus) Just my opinion.. Terry Cunningham Comments


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About keeping up with bugs, undocumented features and frequent updates of David
(batteries not included)
This is pretty much every project I've worked on:
And it accurately describes what having kids is all about. Whenever you think you've worked something out the landscape changes. You're back in the wild, it's getting dark and a low roar of the beast is heard in the distance...
Posted by Goran at 6:45 AM 0 comments Links to this post
Labels: diagram, management, project


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The Gardens of Pelham's retirement community provides an enhanced senior living environment focused on active aging - an approach that aims to maximize the quality of life and well being of seniors while fulfilling their individual needs and promoting purposeful living. Our care options and life enrichment programs allow our residents to live to their greatest potential in a community that is ideal for sharing past experiences and making new memories with neighbors, friends and loved ones.
2124 Old Montgomery Highway
Pelham, Alabama 35124
Office: 205.402.9922
Fax: 205.402.9943
Cell: 205.531.1379
Private suites and baths
Home cooked meals and snacks
Assistance with bathing and dining
Monitoring of medications
Emergency response system
Well-trained staff, available 24 hours
Housekeeping, laundry and linen service
In-house beauty and barber shop
Activities and social events
TV cable connections in all rooms


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No such thing as a small change | |
PerlMonks |
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Bravo, I just wanted to post the suggest for File::stat but you beat me to it. I would suggest you not return -1 for two things that could be a result of a vastly different failure, and I would instead die if I failed to stat the object. I also wouldn't import stat() as failing to include File::stat would surely result in a runtime error, and not the expected compile-time. (in addition two names for the same function has a potential to confuse, as does pollution is any form)
Evan Carroll www.EvanCarroll.com In reply to Re^3: Practical example of "Is Perl code maintainable"
by EvanCarroll


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At the Nov. 2 school board meeting, Mark Spurr was recognized for his years of service to the West St. Paul-Mendota Heights-Eagan School District. (submitted photo)
After eight busy years, Mark Spurr retired from the District 197 School Board.
Spurr served two terms on the West St. Paul-Mendota Heights-Eagan board, starting in 2008, and chaired his last meeting on Dec. 21.
Brenda Corbett
Nine candidates were on the ballot Nov. 3, each vying for one of the four-at-large seats on the ISD 197 School Board.
The board will see only one new face, as three of the four winners are incumbents.
ISD 197 school board elections: nine candidates, four seats On Nov. 3 residents of West St. Paul, Mendota Heights and part of Eagan will have the opportunity to select a majority of the ISD 197 school board. The terms of current school board members Brenda Corbett, Joanne Mansur, Byron Schwab and Mark Spurr are set to expire Dec. 31. All but Spurr are running for re-election.
This summer area school districts, including 197, 199, and 6, are participating in the Minnesota Department of Education's Summer Food Service Program.
Mark Quinn
School District 197 announced that Mark Quinn has been named the next principal at Moreland Arts and Health Sciences Magnet Elementary School in West St. Paul.
A handful of ISD 197's early childhood learning programs will be relocating to a building at 1970 Christensen Avenue in West St. Paul for the upcoming school year, to accommodate an increase in overall enrollment. (Review graphic)
As the current school year winds down with final exams, field trips and graduation ceremonies, District 197 is preparing for a bulge in incoming classes this fall — classes whose students may not graduate until 2029 or 2030.
Eric Friberg, a biology teacher at Henry Sibley High in School District 197, has been selected as a recipient of the 2015 WEM Outstanding Educator Award for Teacher Achievement, an honor that is accompanied by a $15,000 award.
Mary Philblad drops off her bus load of kids on Feb. 25 at Pilot Knob Elementary in Eagan. (Erin Hinrichs/Review)
From kindergarten to high school, many students start their school day before they enter the classroom. They begin their school day by stepping foot onto a school bus.
Gina Smethers and Micah Klotz of Inver Grove Heights Community Schools will be recognized as TIES Exceptional Teachers at the TIES 2014 Education Technology Conference at the Minneapolis Hyatt Regency on Dec. 9.
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Apparently, these days just laying your hands on the newest and coolest gadget is not enough. You also ought to tear it down to see for yourself what exactly are its hidden secrets. So now that we are done with the teardown of the new iPad, the guns are facing Apple TV. Folks at xmbc.org have run a teardown of Apple TV to see what does this new, 1080p-output supporting device contain.
According to this rather detailed operation of the device, it has been revealed that the new Apple TV device has a single-core A5 chip and 512MB of RAM. The flash storage of the device is 8GB. The A5 chip is identified as APL2498 whereas the RAM is apparently from Hynix.
The flash storage for the device has come from Toshiba. It is consistent with the flash storage size of the second generation Apple TV, so there isn't any change there. One significant difference between 2nd generation Apple TV and the current model is that the latter has an additional Wi-Fi antenna.
The major focus of the latest model of Apple TV is on being able to output 1080p content, in consistence with similar changes being introduced in the new iPad and iTunes content. The bump in the RAM for the device will be very supportive in ensuring it and the single-core A5 chip will be the key element in making it happen. You can head to the XMBC Forums page for a detailed discussion about this.
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Oh, nice, those do look better!The ToC shoulders do match the kilt really nicely, but I love the way the shoulders you're already using look in combination with the chestpiece and the hat.
awesome Nav :)
@Kamalia - much more druid like I think! I'm glad you approve. The only off thing about those ToC shoulders are the furry white/grey bits at the edge of the shoulders. If it didn't have those I might be tempted to change, but I do like these shoulders a lot more.@Tout - TY Toutie :)
I like that offhand :o
That mace looks wonderful, I wondered what you were doing in Borean Tundra. You look much more druidy now!
I agree with Ancient, much more druidy than before. Lookin' good! "Meee-oooow!" .....err I mean "Moooo-oooo!" ;)
@Ara - it's an easy to get one too!@TotA - I think it's a very good mace for a druid :) glad you approve!@Euphy - Moooooo! :D
I hope these comments work! Not sure why people can't comment lately, it makes me sad :(


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Lost root with the March, 2014 OTA update. Kingo, which worked so easily before, fails to root. None of the Odin-based or 1-Click methods will root it. Is anyone working on a method or prog that will root this phone?
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THE WORLD HAS BEEN INVADED BY ALIENS! It's up to a nerdy, lazy high school kid to save it!
3.81 / 5.00 7,646 ViewsRushed3! Originally, the concept of Rushed was to make myself animate as much as I can for one day. That's what the two previous Rushed animations were, renditions of what I had made in those one days. For the third entry, I invited some people to suffer with me on this rite of passage. The chance to become salty men of the seas. There were many casualties of staying all night animating but in turn, worked out all for the best. Right before you is the blood, sweat, and tears of members of FluidAnims.
Hope you enjoy our pain :D
unimaginably awesome
this is the best one you have made so far! of course im saying that because this is your newest one but still, all your work is amazing!
This animation has everything...
It even has a rape scene! But no, I would just like to point out that these guys never fail to impress. Jomm is once again the "Rushed" one, Endo is still kicking ass, and I totally noticed that nut shot by the way (when you pulled the triggers via scrotum), Alfa is of course always going to be a renowned veteran, and Tsang, I want to acknowledge that I found your skit awfully close to the hall scene in Old Boy, very sweet. Nonetheless, another great animation by the crew of FluidAnims. Keep up the awesome work, gents. And lady.~


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"Boa signed the new contract just before he went away for the international break. Boa has been here five years already and he wanted to sign for five more years,"
Chris Coleman
"♥² / x......................................................................... --------- = ∞ regret........Solve for x.................................. time²"
Andy Ronald Perez
"love squared divided by x over time equals infinite regret. Solve for x."
"Shaquille O'Neal, ... has signed a five-year, $100-million contract extension with the Miami Heat. ...Actually, they offered him $200 million if he would practice his free throws, but he said no."
Jay Leno
"The contract I signed wasn't for three or four years. It was for five years."
Joe Gibbs
"The job's not accepted until you sign the contract. I haven't signed anything yet. We're going down there to meet with them and take a look at Chapel Hill."
Frank Cignetti
"If mortgage brokers have nothing to hide, they have nothing to lose by signing this contract. If they don't sign the contract we will presume there is a violation of the law,"
Andrew Cuomo


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Thanks for helping us sort this video!
Thanks for helping us associate the correct Pornstars to this video!
Help Us: Do these Pornstars appear in this video?
Pornstar Name:
I think she needs mine for better and long multi orgasm. I need to identify her biodata first and then make a date..
nice tits with red nipples
She is vietnamese
She's Asian
I think they are Vietnamese
what's her name??
In 0:40 she say Vietnam and the language they talk aside english is NOT thai
she is vietnamese
Unless you want a conversation with her it doesn't matter if she's Viet or Thai!
she's from africa
who cares! xD
sexy long hair
she from pluto.
she is not thaigirl.
is from indonesian.
She says she's Vietnamese during the "interview"
What is her name
Who gives a shit she's hot love her hair .
their both viet
Can i teach you?
She says she's Vietnamese
Great sexy long hair Lady.
Would love to do her with that loverly long hair of hers and cute pussy oh yes she's great.
Does she have any other video?What's her name? The hair is pretty!!
she is well known viiet porn star
I let my sperm out off when I see this
What is her name
I would love a Vietnamese girlfriend.
Thai are brown
so handsome, what the boys name?
Little known fact viet girls give great anal, speaking from personal experience.
but she have thai nose :p
That's right !! They are Vietnamese :) They using Vietnameses I'm Vietnamese !!
vietnmese and so is the male ,pretty sure
She has nice pussy and nice breaths
they are both really cute. i LIKE
100% Vietnamese chick
She is thai
Vietnamese lolz
who cares where she's from! Given a chance I would simply fuck her.
I know she vietname girl.
They speaking vietnamese.
there're fake tits, aint bouncy cause plastic inside
The Vietnamese are soooooo good. Num num num. ForeplayBob
Viet of course, they speak vietnamese
no matter if viet or thai.... she looks pretty just like my viet girlfriend
seen this many yrs ago.. they both ABC.. it's from the asian fever series
what a nice pussy love to lick and fuck all day
shes not a vietkong. shes ah hot pinay oh ye..../
i think shes nipness
I guessed Vietnamese, and I was right. Yay.
what dvd is this from?
this is so hot
she is pilipina
Wherever she's from she is so fuckable!
we need more asians in europe ! That is for sure
I love young asian pussy. She is fucking hot. I would love to shoot cum all over her hairy little box.
She is'not Viet, but she 's beautiful.
fuckin crap
shes viet. i used have this porn on dvd, bunch of sexy veit. girl visit thailand, i'm sure it was called "Hello Bangkok" and something in vietanmese
dude, upload the full movie!
Doesn't matter what she is - all that matters is that she has pink in the right places.
She is vietnamese, she went to Goldenwest Junior College in Orange County, California. The music in the background is Caravan of Life (typical Vietnamese).
this one is dubbed,
the clip is edit, i have to said she had a nice rack but bad acting.
fuck off
she is a Vietnamese oversea live in US now
OOOHH MAN ! ! SHE... IS...... PERFECT..... I'd give all for her
she is vietnamese im am a vietnamese too she speaking vietnamese is 21 she born in vietnam
she is filipina
oh another hot asain man!
Germans are the best
shes not Vietnamese, even though the voice in this clip is Vietnamese. Just an audio edited.
I fuck my girl friend's sister who is a vitnamese.. And my GF doesnt know anything.. Sumtyms i fuck her sister and in the afternon i fuck mu GF not knowing that that i fuck both of them in the same bed.
Vietnam girl great pussy.
what a nice body ^_^ Tequilla..can i fuck you ^_^
i hate the guys that made this video! dont u ever think but us girls? we wanna see the guy... u only show that chick... but where is he... he has got a sooo cute face... damn i love asian guys... nd u ruined the whole video by not showing him!!! are there any more videos where this guy is in? i loooove hin... yummi
So long as she has a pussy ...why bother?
Who gives a fuck about her....what about him....MACHINE!!!!
Who cares if vietnamese or thai .. she is even hot and she enjoys smooth sex
she is a vietnamese. i am vietnamese, so i know it better. and her name is crystal. she said that in this vid.
is philippine ...because the song is a song philippine
the girl looks like an asian version of Uma Thurman
she is Viet and wow is she well put together. I thought at one point we were going to get a creampie finish. Ho that what I would do with that sweet little pussy blow my stack right up it and watch it seep out Mmmmmmgh
she is a lady boy, idiots!
Who cares where she is from, given a chance anyone would fuck her. My lady partner gives me a good hand job watching this as she loves to watch me cum loads
yea i agree with ABC... they're speaking vietnamese but its audio edited replacing orignal
I've fucked loads of both, but can hardly tell the difference. Prefer Vietnamese and Vietnam though. As someone above said, who cares where queens of fuck come from.
whichever....shes so hot!!!!
She looks chinese to me, maybe a crossbreed, but seriously who cares, she is beautiful either way.
they are not vietnamese thought, just some vietnamese guys edit audio, replace original voice
sure i think she is Viet
Some errors occurred, please try again later


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Jeff Robinson 1/28/2004 5:57:47 AM
I really like how you have coposed this! Great job! Jeff #91999Carolyn M. Fletcher 1/28/2004 6:00:49 AM
Nice one.. #351235Joy Brown 1/28/2004 6:27:25 AM
Beautiful, Dottie! Great colors! #351277Charlene Bayerle 1/28/2004 6:27:25 AM
Beautiful image Dottie!!!! #351278Judith G. Secco 1/28/2004 6:28:05 AM
This is really beautiful, Dottie. The colors are amazing. Would be very nice framed hanging on my wall!Dottie Shepherd 1/28/2004 6:30:46 AM
Thanks,Jeff, Carolyn, Joy, Charlene and Judy for your comments..!!! and Judy just need your shipping address and what size do you want!!! #351293Kasia Elser 1/28/2004 7:13:48 AM
Beautiful! #351434Barbara Bergeron 1/28/2004 7:39:28 AM
Beautiful colors and softness. Nicely done Dottie! #351486Leanne M.E. Boyd 1/28/2004 9:12:47 AM
I love the abstract and colour, Dottie - well done! #351592Patricia Marroquin 1/28/2004 9:56:20 AM
Soft and lovely, Dottie! :-) #351648Dottie Shepherd 1/28/2004 10:31:54 AM
Thanks, Kasia, Barbara, Leanne and Patricia for your comments..I really appreciate them!! #351688Jessica Hughes 1/28/2004 12:01:54 PM
Great DOF is this photo Dottie! The colors are beautiful! #351780Dottie Shepherd 1/28/2004 12:13:07 PM
Thanks, Jessica..for your comment..!!! #351796Colette M. Metcalf 1/28/2004 12:41:20 PM
Gorgoues, Dottie!! Love the colors!! #351835Dottie Shepherd 1/28/2004 6:30:51 PM
Thanks, Colette for your comment!! #352121Susan T. Evans 1/28/2004 9:06:04 PM
beautiful Dottie! #352217Carol Brill 1/28/2004 10:36:17 PM
So pretty with the colorful and soft detail here, Dottie! #352276Dottie Shepherd 1/29/2004 5:14:18 AM
Thanks, Susan and Carol for your comments..!! #352441Karen Engelbreth 1/29/2004 1:39:42 PM
Beautiful image, Dottie! #353224Dottie Shepherd 1/29/2004 3:40:23 PM
Thanks, Karen...appreciate your commentCarolyn M. Fletcher 2/15/2004 12:26:13 PM
Congrats, Dottie!!! #377754Claudia Kuhn 2/15/2004 12:54:34 PM
Wow Dottie - missed this one - but it sure is a winner!! Congrats!! #377926Murry Grigsby 2/15/2004 12:57:34 PM
Congrats on this excellent finalist Dottie! Good luck!! #377955Dottie Shepherd 2/15/2004 1:21:46 PM
Thanks Carolyn, Claudia and Murry..!!LOL #378143Kathleen Clemons 2/15/2004 1:27:55 PM
Congratulations to you, Dottie! #378186Dottie Shepherd 2/15/2004 1:32:49 PM
Thanks, Kathleen..!! LOL #378217Patricia Marroquin 2/15/2004 1:43:14 PM
A big congratulations, Dottie, on this lovely image! :-) #378293Leanne M.E. Boyd 2/15/2004 1:56:40 PM
Congratulations Patricia! #378393Susan T. Evans 2/15/2004 5:29:10 PM
Congrats Dottie! #379284Dottie Shepherd 2/15/2004 6:00:15 PM
Thanks everyone and LOL..!! #379383David and Carol Kelly 2/15/2004 7:33:47 PM
Beautiful, Dottie. Congratulations. #379558Dottie Shepherd 2/15/2004 8:00:10 PM
Thanks, David!! #379584Evy Johansen 2/15/2004 9:30:17 PM
Many congratulations, Dottie! Great image! #379754Judith G. Secco 2/16/2004 6:12:54 AM
Congratulations, Dottie. This has to be one of my favorites!Karen Engelbreth 2/16/2004 6:24:43 AM
Congratulations. This is a wonderful image, Dottie!#380613
Robin L. Wehrman 2/16/2004 7:36:28 AM
Congratulations Dottie! #381030Carol Brill 2/16/2004 1:16:28 PM
Congratulations and best of luck, Dottie! #381628Ellen Peach 2/16/2004 1:18:22 PM
Congrats, Dottie, well done! #381632Dottie Shepherd 2/16/2004 1:31:21 PM
Thanks, Evy, Judy, Karen, Robin, Carol and Ellen..!!! LOL #381668Susana Matos 2/17/2004 12:24:50 PM
Copyright for this gallery photo belongs solely to Dottie Shepherd. Images may not be copied, downloaded, or used in any way without the expressed, written permission of the photographer.
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11 hours ago
Off the Wall
The significant PR advantage the NRL has over the AFL when it comes to drug abuse by players is being rapidly undermined by a weak stance on the biggest challenge facing rugby league - alcohol.
And it is increasingly apparent that players, and their managers, are running the agenda when it comes to "dealing" with repeat offenders.
As a result, both the NRL, and the majority of clubs have been made to look weak and ineffective. And, as the Sydney Sunday Telegraph put it yesterday, league "is drowning in an ocean of booze".
The article detailed no less that four incidents in which NRL players were seen in public "as full as state school hat racks" in Sydney over a four day period last week. One of the game


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Friday, April 3, 2009
Children's book illustration
I'm so excited to have been signed by Guardian Angel Publishing to illustrate the latest book by Author Donna Shepherd. Here is a sneak peek at an illustration from Poodle and Doodle.
Here is a link to Donna's Oodles of fun blog.
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Sheng, Yong and Yang, Dongmin and Tan, Yuanqiang and Ye, Jianqiao (2010) Microstructure effects on transverse cracking in composite laminae by DEM. Composites Science and Technology, 70 (14). pp. 2093-2101. ISSN 0266-3538
Full text not available from this repository.Abstract
A particle discrete element method (DEM) was employed to simulate transverse cracking in laminated fiber reinforced composites. The microstructure of the laminates was modeled by a DEM model using different mechanical constitutive laws and materials parameters for different constituents, i.e. fiber, matrix and fiber/matrix interface. Rectangular, hexagonal and random fiber distributions were simulated to study the effect of fiber distribution on the transverse cracking. The initiation and dynamic propagation of transverse cracking and interfacial debonding were all captured by the DEM simulation, which showed similar patterns to those observed from experiments. The effect of fiber volume fraction was also studied for laminae with randomly distributed fibers. It was found that the distribution and volume fraction of fibers affected not only the transverse cracking path, but also the behavior of matrix plastic deformation and fiber/matrix interface yielding in the material.
Item Type: | Article |
Journal or Publication Title: | Composites Science and Technology |
Uncontrolled Keywords: | Discrete element method ; C. Transverse cracking ; B. Debonding ; B. Matrix cracking ; B. Microstructure ; A. Polymer-matrix composites (PMCs) |
Subjects: | T Technology > TA Engineering (General). Civil engineering (General) |
Departments: | Faculty of Science and Technology > Engineering |
ID Code: | 54213 |
Deposited By: | ep_importer_pure |
Deposited On: | 18 May 2012 16:56 |
Refereed?: | Yes |
Published?: | Published |
Last Modified: | 24 Mar 2017 04:15 |
Identification Number: | |
URI: | http://eprints.lancs.ac.uk/id/eprint/54213 |
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Forumwarz is the first "Massively Single-Player" online RPG completely built around Internet culture.
You are currently looking at Flamebate, our community forums. Players can discuss the game here, strategize, and role play as their characters.
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View Dum Cumpster's ProfileOnly public flamebate results are returned.
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Forumwarz Gameplay Test Squad: Sign Up Here!Knocking on wood, formica, and granite Log in to see images! (view post) |
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CONTEST: Help get me the Flamebate peen for 5 BP!BOOB THREAD NAO! Also, Kittiejenn rocks for making the epic mammary glands thread. that is all. (view post) |
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Thursday, September 14, 2006
Fasten Your Seat Belts
OPEC officially abandons production quotas
"[OPEC's President and Nigerian Oil Minister Edmond] Daukoru said: There is no quota, no, either up or down."
This is a significant move away from the previous 28 million barrel per day quota. While it is largely recognized that the quota has had no meaning for some time now, its formal abandonment is significant. The same action by the Texas Railroad Commission (the Texas oil & gas regulatory body) in the early '70's is viewed, with the benefit of historical hindsight, as the turning point signifying a peak in oil production.
In my opinion, interesting times are ahead. I don't see any catastrophic collapse or similar events in the next few years, but I do think that at current prices there now is a historical buying opportunity for oil. But that's a topic for a future note on post-peak investing strategies...
2 comments:
I can't afford seatbelts someone help me plz!!
EnergyBulletin.net comments:
In 1971 the Texas Railroad Commission (TRC) lifted the quotas on
oil production in Texas. Texas was the swing producer of U.S. oil
production at that time, being given the task of matching oil
production with demand many years earlier. The suspension of the TRC
quotas was the first signal that the U.S. had peaked. After 1971,
even with Texas producing at full capacity, aggregate U.S. production
clearly had peaked in 1970-71 and was to go into permanent decline,
never reaching that production level again.
The below article quotes an unidentified source reportedly close to
OPEC (a cartel of oil exporting countries closely modeled after the
TRC) that the suspension of OPEC quotas is an option being discussed
for next weeks OPEC meeting in Beirut. The fact that OPEC officials
are even considering the lifting of quotas should raise eyebrows as
it must mean that there's very little spare capacity left.
Nobody in OPEC would seriously contemplate suspending quotas if huge
amounts of spare capacity still existed because that would most
definately result in a total price collapse which would be the last
thing the oil sales dependent, heavily indebted OPEC countries
economies would need. Because members of OPEC are the swing producers
in oil production today, and if the suspension of quotas happens in
Beirut next week, I think it would then be almost certain that WORLD


text:
Christmas Poinsettia Beverage Napkins
Case Price: $19.95
Product Code: CPN1225
Christmas Poinsettia beverage napkins feature a large vibrant poinsettia printed on a white background, with gold lace accents and festive red borders. These elegant cocktail napkins are constructed from standard 2Ply (layer) tissue and coordinate nicely with our hunter green, gold and classic red solid colored tableware supplies. Host a memorable and budget friendly holiday dinner or cocktail party with our wholesale Christmas tableware products!
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Minimum Order: 1 Full Case. Not available in smaller quantities.
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text: JDK 1.2The source code for the JDK 1.2.2 is available from Sun here. You will need to obtain the Java 2 SDK 1.2.2 download, not the Java 2 SDK 1.2.2 for Linux. Once you have done this and agreed to the SCSL you can proceed to download the latest FreeBSD JDK 1.2.2 patchset. This contains patches, build instructions and other related information. |


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Your store is Leesburg VA. View Store Change Store
Your Material Cost:
2" 15.5ga. Staples 1000-Count
Heavy duty enough to keep flooring and planks in place. Includes 1000 staples.
Quick Highlights:- Lacquered collation - High carbon steel - Galvanized for rust and corrosion resistance - Coated with glue for superior holding power
Specs:- Length: 2" - Gauge: 15.5- Crown: 1/2"


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Q. I keep seeing what appears to be a radio-controlled Hummer. What is it? Gary Laster
A. Yes, it really is a remote-controlled car, used to return hammers, javelins and discuses to athletes. But they're not Hummers — they're Minis, supplied by Games sponsor BMW. It does sound incredibly lazy, but then each car covers the best part of four miles a day.
Q. Why do some athletes have gaffer tape all over them? Brian Atkinson
A. It's Kinesio tape, used for minor muscle injuries. It's stretchy and offers a little support, but mainly it lifts the skin away from the muscle, boosting circulation, which helps minimise the inflammation and pain from pulls and strains. Many scientists, on the other hand, believe it achieves precisely nothing.
Q. Is there an advantage to being in any particular lane in sprints? Cosmo Cooper
A. The middle is the place to be. On the inside, the bend is too tight to hit full speed — hence lane one is abandoned and lanes two to nine are used instead. In the outside lanes, it's hard to see where your rivals are (other than on the big screen) as they start behind you. Even in the 100m, no one likes lanes one, two and three: if wind blows into the stadium, the runners close to the centre are buffeted the most.
Please send your questions to email@example.com, or tweet using #indyolympics, live from the stadium — or your sofa.


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Diner Survey Results 7 responses
Brittany H
Insider Pages
September 28, 2012
The Chicken Was Rancid
Absolutely disgusting. I tried for the first time today (just moved to the area). I ordered General Tso's chicken all white meat with pork fried rice and an eggroll. Eggroll was so-so. However, the chicken was horrendous- the chicken…
Patti B
Insider Pages
December 9, 2010
No Thanks!
We have tried this place numerous times because it is in a convenient location. We were never wowed by the food but the last time we ordered was the last. After getting our food home and taking it all out there was a very disgusting bug in…
Insider Pages
July 26, 2009
Big Wing Wah
More Discount 10%off-----50%off for SUSHI\r
And a pretty good restaurant as well,The food always tastes so fresh and is served hot!\r
This chinese restraunt has great food and is very cute inside
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