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Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down‐regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.
An integrated analysis of proteomic and phospho‐proteomic data from BRAF inhibitor‐treated melanoma cells and a functional genomic screen for shRNAs sensitizing melanoma to BRAF inhibitor treatment identifies ROCK1 kinase as a combinatorial drug target.
Proteomic and phospho‐proteomic analysis on BRAF inhibitor‐treated melanoma cells identifies proteins involved in melanoma cell survival.
A synthetic lethality shRNA screen identifies shRNAs that sensitize melanoma to BRAF/ERK inhibitors.
Integrated analysis of the proteomic and genomic data reveals ROCK1 as potential companion target for BRAF mutant melanoma.
Melanoma is the most aggressive skin cancer and one of the top five most frequent malignancies in the USA (U.S. Cancer Statistics Working Group, 2013). Although treatment of metastatic melanoma has improved recently, no curative therapy is available. Clinically validated driver genes include the mutant oncoproteins BRAF (~50% of melanomas), c‐Kit (~15%) and NRAS (~20%) (Gray‐Schopfer et al, 2007; Hodis et al, 2012). The discovery of the common BRAFV600E mutation in melanoma (Davies et al, 2002) has resulted in the development of targeted therapies with significant clinical benefits. Until very recently, vemurafenib, a drug targeting BRAFV600E, has become the standard of care for patients diagnosed with mutant BRAF metastatic melanoma. However, although this compound or other drugs targeting other components of the MAPK pathway initially reduce tumor burden, eventually all melanomas become resistant and patients succumb to the disease (Flaherty et al, 2010; Chapman et al, 2011; Wagle et al, 2011).
Drug resistance in this setting is caused by a plethora of mechanisms, both MAP kinase dependent and independent, making it virtually impossible to design a single effective targeted therapy from which all patients would benefit. For example, several mutations located in the MAPK pathway have been detected in vemurafenib‐resistant cell lines or patient tumors, including activating mutations in MEK1 (Wagle et al, 2011; Trunzer et al, 2013), MEK2 (Wagle et al, 2014) and in NRAS (Nazarian et al, 2010). In addition, long‐term treatment with BRAF inhibitors has been shown to induce switching between RAF isoforms and amplification of BRAFV600E or expression of an alternative, 61‐kDa RAF splice variant lacking the RAS‐binding domain, causing constitutive activation of the MAP kinase pathway (Villanueva et al, 2010; Poulikakos et al, 2011; Shi et al, 2012). Furthermore, overexpression of CRAF is associated with BRAF resistance (Montagut et al, 2008). Other mechanisms, not involving the MAPK pathway, have been found as well, including up‐regulation of IGF‐1R (Villanueva et al, 2010), PDGFRβ (Nazarian et al, 2010), FOXD3 (Basile et al, 2012), EGFR (Girotti et al, 2013; Sun et al, 2014) or FGFR3 (Yadav et al, 2012) signaling. Overexpression of COT (Johannessen et al, 2010), Cyclin D1 (Smalley et al, 2008) and AEBP1 (Hu et al, 2013), amplification of MET and CTNNB1 (Vergani et al, 2011) and loss of NF1 (Whittaker et al, 2013) can also confer resistance to vemurafenib. The micro‐environment can play a role in resistance, as it was found that up‐regulation of HGF by the surrounding stromal cells occurs during resistance (Straussman et al, 2012). Lastly, BRAF inhibitor‐resistant tumors have increased levels of autophagy (Ma et al, 2014).
Since many resistance mechanisms are MAPK pathway dependent, clinical trials in which melanoma patients are treated simultaneously with BRAF and MEK inhibitor are ongoing (Flaherty et al, 2012). Although the clinical responses may improve as a result, resistance continues to be a major problem (Shi et al, 2014; Wagle et al, 2014). Therefore, it is important to identify novel therapeutic targets that can be used in the treatment of melanoma patients in combination with, or instead of, existing therapies. As vemurafenib is the best‐characterized drug for melanoma treatment, we pursued a multi‐angle approach, utilizing an integrated and unbiased proteomic and genetic screening platform to identify targets whose inhibition would increase the toxicity of vemurafenib toward melanoma cells.
Mass spectrometry (MS) analysis identifies induction of proteins involved in melanoma survival
To gain insight into the molecular mechanisms and signaling pathways underlying normal drug response and thereby screen for novel targets for drug sensitization, we combined (phospho)proteomic analysis and an shRNA library function‐based approach. We used a low‐passage human melanoma cell line, 04.01, which is sensitive to treatment with PLX4720, a preclinical vemurafenib analog. For the MS‐based proteomic approach, we performed a time course experiment, monitoring the changes of the proteome and phosphoproteome after 1 and 3 days of treatment with an IC50 dose of PLX4720 (Fig 1A). Alongside, we performed two shRNA screens on the same cell line to identify shRNAs that sensitize melanoma cells to targeted inhibition of either BRAF or one of its critical effectors, ERK (Fig 1B; described in detail below).
For the proteome and phosphoproteome analyses, cells were lysed, digested by Lys‐C/Trypsin, labeled using the dimethyl approach (Boersema et al, 2009), mixed 1:1:1 and fractionated by strong cation exchange (SCX) (Fig 1A). For the phosphoproteome analysis, SCX fractions were subsequently subjected to phosphopeptide enrichment by Ti4+‐IMAC (Zhou et al, 2013) and analyzed by LC/MS/MS on an LTQ‐Orbitrap Velos or Elite using a data‐dependent decision tree MS/MS method (ETD‐IT or HCD). Here, the most suitable fragmentation technique is automatically selected (according to the charge and m/z) to enhance the number of phosphopeptide identifications (Frese et al, 2011). For the whole proteome analysis, the SCX fractions were analyzed on an Orbitrap Velos, Elite or Q‐Exactive (Fig 1A). Overall, ~5,700 proteins and ~11,500 phosphosites (80% with a location probability ≥ 75%) were identified from the three biological replicates with a protein and peptide FDR ≤ 1%. At the protein level, we quantified ~3,800 proteins over all conditions and found 129, 406 and 313 proteins regulated significantly at 1 day/control, 3 days/control and 3 days/1 day, respectively, after performing a statistical assessment (P < 0.05), and choosing an arbitrary fold change cutoff of 1.5, corresponding to a total of 588 unique regulated proteins (Fig 2; Supplementary Table S1). Network analysis of the significantly changing proteins using Reactome as plugin in Cytoscape (Haw et al, 2011) revealed a high number of regulated protein–protein interactions at 3 days/control and a substantial up‐regulation of membrane proteins at 3 days/1 day (Supplementary Fig S1). A gene ontology (GO) analysis using Panther (http://www.pantherdb.org) (Mi et al, 2013) was performed on the proteins whose expression changed significantly. The most profound changes in protein expression levels were observed at 3 days, when receptors became particularly over‐represented (Supplementary Fig S2; Supplementary Table S2). A GO slim analysis of the proteome and phosphoproteome data using BiNGO as Cytoscape plugin (Maere et al, 2005) revealed enrichment for cytoskeleton organization (Supplementary Fig S3; Supplementary Table S3).
As expected, exposure to PLX4720 led to down‐regulation of the phosphorylation state of kinases within the MAPK pathway: phospho‐MEK, phospho‐ERK1/2 and phospho‐p90RSK (Fig 3A). Indeed also in our mass spectrometry analysis, we observed strong down‐regulation of the phosphorylation states of ERK1 (at 1 and 3 days) and ERK2 (at 3 days; Supplementary Table S1), which indicate a responsive state of the employed cell line. Moreover, the phosphorylation state of RPS6 was significantly down‐regulated especially at 1 day, indicative of an inactive state of the mTORC1 pathway (Fig 3B, Supplementary Table S1) (Roux et al, 2007).
Regarding the protein expression analysis, a number of proteins were differentially regulated (Supplementary Table S1); for simplicity, only a select set is depicted in Fig 2. This unbiased approach enabled us to observe also a number of changes upon PLX4720 treatment previously described in relation to drug resistance. For example, we detected up‐regulation of the transcription factor FOXD3 at 1 and 3 days (Fig 2) and one of its transcriptional targets, the receptor protein kinase Erbb3, at 1 day (Fig 2, left panel). This is consistent with previous findings, where FOXD3 has been found to confer resistance to PLX4720‐induced cell death by up‐regulation of Erbb3 (Basile et al, 2012; Abel et al, 2013). Moreover, we found several proteins that were changed upon PLX4720 treatment that play a role in lipid rafts. First, at 3 days, the lipid raft‐associated proteins, caveolin 1 and 2, and raftlin (Cav1, Cav2, Rftn1), were consistently up‐regulated (Fig 2, right panel; Supplementary Table S1). Interestingly, Cav1 has been reported to promote growth and invasion of melanoma cells (Felicetti et al, 2009). Second, at 3 days, we observed the up‐regulation of integrin alfa V (ItgaV) (Fig 2, right panel), whose expression has been reported to be positively regulated by Cav1 (Arpaia et al, 2012). Indeed, we found several other integrins to be up‐regulated such as Itga4, Itga1, Itga2, Itgb5 and Itgb1 (Supplementary Table S1). Third, Ptprf (or Lar) was up‐regulated in time (Fig 2), and this phosphatase has been reported to be localized in lipid rafts (Caselli et al, 2002). Fourth, we detected up‐regulation of Src at 3 days (Fig 2, right panel). Src membrane family proteins (Src, Fyn and Yes) are recruited for signal transduction in the cholesterol‐rich lipid raft membrane structures (Liang et al, 2001). Interestingly, Src kinase has been implicated in mechanisms of resistance to vemurafenib (Vergani et al, 2011; Girotti et al, 2013). The biological relevance of the lipid rafts in melanoma has been further suggested by the recent work of Zanfardino et al (2013) who demonstrated that simvastatin, a compound that can block cholesterol synthesis, reduces tumor growth in a melanoma xenograft. We also observed other proteins involved in PLX4720 resistance. At 3 days, we detected an increase of NRAS (Fig 2, right panel), whose up‐regulation is involved in vemurafenib resistance (Nazarian et al, 2010). At 1 and 3 days, we also observed a significant up‐regulation of RhoJ, which has been associated with melanoma invasion and chemoresistance (Ho et al, 2012, 2013) (Fig 2). In an independent experiment, we validated some of the identified proteins by Western blot analysis, which confirmed the upregulation of integrin beta 1, caveolin 1 and NRAS after 3 days (Fig 3C). Overall, we conclude from our MS‐based proteomics time course experiment that melanoma cells upon treatment with PLX4720 induce expression of multiple proteins that have been previously reported to contribute to vemurafenib resistance, as well as several novel proteins (Supplementary Table S1).
Parallel function‐based shRNA screens identify factors contributing to melanoma cell survival
Parallel to the proteomic analysis, we set out to perform an unbiased and function‐based genomic screen to identify potential drug target(s) that can be used in combination with BRAF inhibitors. Specifically, we performed a sensitizer screen for PLX4720 with a lentiviral shRNA library containing ~4,000 shRNAs targeting ~500 different kinases. We transduced the human melanoma cell line 04.01 with a multiplicity of infection (MOI) of 0.5 to limit the likelihood of multiple integrations per cell, avoiding competition for the RNAi silencing machinery. After pharmacologic selection, cells were treated for 1 week with either DMSO (control) or low‐concentration PLX4720 (IC20). Next‐generation sequencing was used to identify hairpins selectively depleted in the treated sample compared to the control, with the aim to identify drug enhancers (Fig 4A). As already described in the introduction, many resistance mechanisms to BRAF inhibition involve re‐activation of ERK. Also ERK inhibitors are currently being developed to treat melanoma (Morris et al, 2013). Therefore, we performed an ERK inhibitor (SCH772984) sensitization screen in parallel. For both screens, we selected the hairpins that were significantly depleted (P < 0.05) in the treated sample by at least 1/3 after 1 week of treatment. We excluded hairpins that were enriched after 4 days of treatment, to minimize the number of false‐positive hits. We compared the identified hairpins from both screens (PLX4720 and SCH772984) and found an overlap of 59 (Fig 4B, Supplementary Table S4). Selecting the genes that were silenced by at least two hairpins in both screens (to exclude potential off‐target effects and to increase the likelihood that the hits functionally interacted with the BRAF/MEK/ERK pathway) yielded five potential hits: AAK1, PLK4, IGF1R, MET and ROCK1.
Integration of proteomic and functional genomic analyses reveals regulation of ROCK signaling upon PLX4720 treatment
To select the hit from our sensitizer screens with the highest potential as drug companion target, we integrated our functional screening results with the proteomic data. Interestingly, we found several regulators of ROCK1 in our proteomic dataset. First, we detected significant down‐regulation of Rnd3 upon PLX4720 treatment (Fig 2, left panel). Rnd3 is a negative regulator of ROCK1 activity (Riento et al, 2003; Belgiovine et al, 2010) and fitting with our observation, Rnd3 is induced by RAF transformation in epithelial cells (Hansen et al, 2000). Furthermore, in melanoma cells that persist after BRAF inhibition, Rnd3 restoration decreased cell invasion (Klein & Higgins, 2011). Interestingly, FOXD3, also identified in our MS analysis and an established player in PLX4720 resistance (see above), is linked to ROCK1, as it down‐regulates Rnd3 in melanoma cells (Katiyar & Aplin, 2011). Notably, overstimulation of the MAPK pathway (resulting from transformation by RASV12) has been reported to negatively regulate ROCK1 expression in fibroblasts (Sahai et al, 2001; Pawlak & Helfman, 2002). ROCK1 is known to regulate the switch toward amoeboid motility (Sanz‐Moreno & Marshall, 2010; Sanz‐Moreno et al, 2011), and this round morphology has been associated to enhanced aggressiveness and motility of melanoma cells (Ramgolam et al, 2011). Second, after 24 h of PLX4720 treatment, we observed up‐regulation of Dock10 in our proteomic data (Fig 2, left panel), a guanine exchange factor (GEF) of Cdc42, which is required for amoeboid movement (Gadea et al, 2008). Third, as mentioned already, we found a number of integrins, including Itgb1, up‐regulated upon PLX4720 treatment. Integrins are involved in cell adhesion/movement, and Itgb1 has been shown to activate the RhoA‐ROCK pathway in colon carcinoma cells (Vial et al, 2003). Taking the data of both shRNA screens together, and integrating them with our proteomic analysis results, yielded the serine threonine kinase ROCK1 as an interesting and potential drug co‐target in melanoma therapy.
ROCK1 silencing sensitizes melanoma cells to BRAF and ERK inhibition
Four and two different hairpins were depleted for ROCK1 in the PLX4720 and SCH772984 sensitizer screens, respectively (Table 1). We next validated this in an independent experiment. Silencing of ROCK1 with three different hairpins resulted in decreased levels of ROCK1 (Fig 5A). Confirming our screen results, silencing of ROCK1 resulted in fewer viable cells upon treatment with either PLX4720 or SCH772984 as analyzed by dose response curves (Fig 5B and C). To determine whether, indeed, ROCK1 silencing renders the cells more sensitive to death upon PLX4720 treatment, we analyzed the levels of the pro‐apoptotic protein‐cleaved caspase 3 in treated cells. Treatment of PLX4720 showed increased cleaved caspase 3 levels, consistent with dose response analysis. Although silencing ROCK1 had no effect on cleaved caspase 3 levels under normal conditions, upon treatment with PLX4720, cells with silenced ROCK1 had a further increase in cleaved caspase 3 levels (Fig 5D).
To begin to translate these findings to a more clinically relevant setting, we turned to pharmacologic inhibition of ROCK. The ROCK inhibitor fasudil is being used for treatment of cerebral vasospasms (Olson, 2008). We examined whether inhibition of ROCK1 with a pharmacological inhibitor could have an additive effect on BRAF or ERK inhibition. For this purpose, we used GSK269962A, an inhibitor targeting ROCK1 and ROCK2, which has been shown to have vasodilatory effects in rats (Doe et al, 2007) and which is more specific than fasudil. We treated six different low‐passage melanoma cell lines with a dilution series of PLX4720 or SCH772984 with or without a fixed concentration of the ROCK inhibitor. Indeed, addition of the ROCK inhibitor profoundly enhanced the effect of either PLX4720 or SCH772984 to induce melanoma cell death (Fig 6A and B, Supplementary Fig S4). Furthermore, in the 93.03 and A375 melanoma cell lines, addition of ROCK inhibitor eliminated the population of residual cells that survived at the highest PLX4720 or SCH772984 concentration. This suggests that combined treatment with a ROCK inhibitor and a BRAF inhibitor may be beneficial for patients with BRAF mutant melanoma.
Here, we applied an unbiased multi‐angle approach to discover new potential targets that render melanoma more sensitive to clinically relevant inhibitors of the BRAF pathway, particularly those targeting BRAF and ERK. As a model system, we used a panel of BRAF mutant human melanoma cell lines sensitive to PLX4720 treatment. Interestingly, although we used a sensitive cell line in our proteomic platform, we detected elevated expression of several proteins that have previously been reported to be involved in resistance. They include NRAS, Erbb3 and Src, and the transcription factor FOXD3 (Fig 7). These observations raise the interesting possibility that melanoma cells activate multiple different pathways involved in resistance already very early on, as soon as they are exposed to targeted inhibition of driver oncoproteins. Our data provide a resource for future studies aiming to resolve the mechanism by which certain responses are selected in pathways leading to drug resistance and survival of the melanoma cells. Indeed, as we show here, the impairment of one of these drug‐regulated pathways can be effective in improving current targeted melanoma therapies.
In our function‐based and unbiased genomic approach, we employed two screens to identify factors whose targeted silencing sensitizes melanoma cells to BRAF pathway inhibition. Consistent with the fact that the inhibitors PLX4720 and SCH772984 target the same pathway, there are several overlapping hairpins between the two screens, increasing the robustness of this approach. Because of this overlap, we were able to stringently select for factors that are useful as targets in combination with inhibition of the BRAF/MAPK pathway in melanoma. The strength of this approach is illustrated by the finding that two out of the five hits, IGFR and MET, have already been implicated in resistance to BRAF inhibition (Villanueva et al, 2010; Vergani et al, 2011; Straussman et al, 2012).
By combining our genomic data with (phospho)proteomic analyses, we were able to identify a new target, ROCK1, whose inhibition rendered melanoma cells much more sensitive to BRAF/MAPK inhibition. While PLX4720 treatment influenced ROCK1 signaling, silencing of ROCK1 as well as the use of a pharmacologic ROCK inhibitor significantly increased the elimination of melanoma cells by PLX4720 and SCH772984 treatments. The use of ROCK1 as a potential target in cancer therapy has been suggested previously (Rath & Olson, 2012). Single ROCK1 inhibition reduces tumor outgrowth (Patel et al, 2012) and bone metastasis in breast cancer (Liu et al, 2009), while in prostate cancer, ROCK1 silencing reduces tumor growth (Zhang et al, 2013). However, ROCK inhibitors have not been extensively explored in melanoma. The only studies that show an effect of ROCK inhibition on melanoma growth have been performed on mouse cells (Nakajima et al, 2003; Routhier et al, 2010). To our knowledge, the use of ROCK inhibitors has never been reported in a combinatorial approach with the exception of one study combining ROCK inhibition and taxol on murine melanoma cells (Nakajima et al, 2003). Our results are consistent with and extend these findings, meriting further exploration of the use of ROCK inhibition in melanoma treatment.
Because resistance to single drug treatment is seen almost invariably in melanoma and other cancer types, combination treatments need to be developed and used as early as possible to keep the tumor burden limited at early stages. Indeed, the combination of vemurafenib with the MEK inhibitor trametinib is tested in a clinical trial (Flaherty et al, 2012). However, resistance still occurs upon this treatment, for example, owing to mutations in MEK (Wagle et al, 2014). Furthermore, also the subsequent treatment of MEK and BRAF inhibitors has been tested, but the differences compared to combined treatment remain small (Goldinger et al, 2014). A combination of a BRAF and PI3K inhibitor effectively eliminates BRAF inhibitor‐resistant melanoma cell line clones (Greger et al, 2012; Vredeveld et al, 2012); currently, a clinical trial using vemurafenib and BKM120, a PI3K inhibitor, is ongoing (ClinicalTrials.gov). Since resistance is caused by a multitude of events, targeting two or even more nodes simultaneously is likely to be beneficial, as many have suggested (Greger et al, 2012; Vredeveld et al, 2012; Kaplon et al, 2013; Villanueva et al, 2013). Unlike vemurafenib, ERK inhibitors are not yet beyond clinical trials for the treatment of melanoma. So far, it has been reported that ERK inhibitors have anti‐tumor effects in melanoma xenografts and that ERK inhibition can overcome MEK and BRAF inhibitor resistance (Morris et al, 2013). Furthermore, combination of ERK and PI3K/mTOR inhibition promotes cell death in resistant melanoma cells (Carlino et al, 2014). Our results warrant exploration of combinations of BRAF/ROCK inhibition or ERK/ROCK inhibition in further studies for the treatment of BRAF mutant melanoma.
Materials and Methods
Constructs and inhibitors
The shRNA kinome library contains hairpins targeting around 500 kinases. This library was assembled from The RNAi Consortium (TRC) human genomewide shRNA collection (TRC‐Hs1.0) (OpenBiosystems) in four different pools, each containing around 1,000 hairpins. pLKO‐SCR, pLKO‐shROCK1 #094 (ATTTACCTCTTGTTCTAACCG), #096 (TATGTCCAATACCATAGATGG) and #159 (TACTTTGTGTTCATTTACCTC) were from the TRC‐Hs1.0 library (OpenBiosystems). Inhibitors used were PLX4720 (Selleck), SCH772984 (Merck, via a MTA), GSK269962A (Axon Medchem) and metabolic poison phenyl arsine oxide (PAO) (Sigma).
Cell culture and viral transductions
BRAFV600E melanoma cells 04.01, 04.07, 93.03 and 00.08 were from Leiden University Medical Center, and the cell line identity was verified with STR profiling (PowerPlex 16 HS, Promega). These melanoma cells, mel888, A375 and HEK293T, were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 9% fetal calf serum (Sigma) and penicillin + streptomycin. For lentiviral transductions, HEK293T was transfected with 8 μg plasmid in medium containing 25 mM chloroquine, using the helper plasmids pMDLglpRRE, pHCMV‐G and pRSVrev, and refreshed after 6 and 24 h. After 48 h, virus supernatant was harvested and diluted for transduction. 04.01 melanoma cells were transduced with pLKO‐sh‐SCR or pLKO‐sh‐ROCK1 in the presence of 4 μg/ml polybrene and selected with 1.0 μg/ml puromycin. Four days after transduction, cells were set up for further experiments.
Kinome shRNA screen
04.01 melanoma cells were transduced with the four different pools of the shRNA kinome library at an MOI of 0.5. The next day, 1.0 μg/ml puromycin was added to the cells. Following 3 days of puromycin selection, cells were divided into three groups: a reference control, a DMSO‐treated sample and a sample treated with the IC20 concentration of either PLX4720 (0.15 μM, for the PLX4720 screen) or SCH772984 (0.015 μM, for the ERKi screen). Cells were harvested after 4 and 7 days of treatment with the respective inhibitors. Genomic DNA was isolated, and shRNAs were amplified by PCR using a FOR primer incorporating the Illumina sequencing primer and a 6‐bp index to allow discrimination of different samples (5′‐ACACTCTTTCCCTACACGACGCTCTTCCGATCTINDEXCTTGTGGAAAGGACGAAACACCGG‐3′), and a REV primer incorporating a P7 arm (5′‐CAAGCAGAAGACGGCATACGAGATTTCTTTCCCCTGCACTGTACCC‐3′). A second round of PCR was then performed using a FOR primer incorporating a P5 arm (5′‐AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT‐3′) and the P7 REV primer. The PCR products were purified and analyzed by next‐generation sequencing (Illumina HiSeq2000). The screens were performed independently three times, and results were analyzed with DESeq version 1.4.1 from R/Bioconductor (Gentleman et al, 2004; Anders & Huber, 2010). Hairpins were selected by comparing treated samples to DMSO‐treated samples at day 7 (fold change 0.66, P < 0.05) and treated samples to DMSO‐treated samples at day four (fold change < 1). Finally, hairpins from the two different screens (the PLX4720 and ERKi screen) were compared, and genes that were targeted by at least two hairpins in both screens were identified as hits for further analysis.
Dose response curves, drug treatments and mass spectrometry analysis
Equal cell numbers were plated on 96 wells, and the next day, cells were treated with different concentrations of indicated inhibitor(s). After 3 days, cell viability was determined with a cell titer blue assay (Promega), and fluorescence was quantified by a TECAN Infinite M200 scanner. Values were normalized to a negative control (no treatment, set at 100%) and a positive control for killing (PAO, set at 0%). For (phospho)proteomic analysis, 04.01 cells were plated on 10‐cm plate, and the next day, cells were treated with vehicle or 0.5 μM PLX4720. Cells were harvested the same day (control vehicle treated cells), after 24 h and after 3 days. Cells in the supernatant were not included in the analysis. For drug treatments for Western blot analysis, 04.01 melanoma cells expressing sh‐SCR or sh‐ROCK1 were treated with 0.15 μM PLX4720 for 3 days. Cells in the supernatant were included in the Western blot analysis.
Western blot analysis
Samples were lysed in RIPA (50 mM TRIS pH 8.0, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS) in the presence of a protease inhibitor cocktail (Roche) and phosphatase inhibitors (1 mM sodium pyrophosphate, 2 mM sodium fluoride, 10 mM β‐glycerophosphate, 2 mM orthovanadate). Protein concentration was determined using a Bradford assay (Bio‐Rad). Immunoblot analysis was performed using standard techniques on 4–12% bis‐tris precast gels (NuPAGE). Proteins were transferred on nitrocellulose membranes (Millipore). Primary antibodies were ROCK1(BD Transduction Laboratories), β‐actin (AC74, Sigma), cleaved caspase 3 (Asp175), caveolin 1, phopho‐MAPK (Thr202/Tyr204), MAPK, phopho‐MEK (41G9), MEK (L38C12), phospho‐p90RSK (Thr359/Ser363), RSK (against RSK1, 2, and 3) phospho‐RPS6 (Ser235/236), RPS6 (all Cell Signaling), integrin β1 (Bio‐Connect) and NRAS (F155, Santa Cruz). Protein detection was performed using ECL agent (Amersham), and developed films were scanned on an Epson Perfection 4990 Photo scanner.
Sample preparation for mass spectrometry analysis
The cellular pellets of control sample and cells exposed to 1 and 3 days of PLX4720 treatment were harvested and resuspended in lysis buffer [8 M Urea in 50 mM triethyl ammonium bicarbonate, pH 8.5, 1 mM sodium orthovanadate, 1 tablet of Complete mini EDTA‐free mixture (Roche Applied Science) and one tablet of PhosSTOP phosphatase inhibitor mixture per 10 ml of lysis buffer (Roche Applied Science)]. Cells were then lysed by 10 rapid passages through a 23‐gauge hypodermic syringe needle and by sonication on ice. After centrifugation (20,000 × g 30 min at 4°C), the protein concentration was determined by Bradford assay (Pierce). Proteins were reduced with 2 mM DTT at 56°C for 25 min, alkylated with 4 mM iodoacetamide at room temperature for 30 min in the dark and reduced again with 2 mM DTT at room temperature to prevent over‐alkylation. A first enzymatic digestion step was performed in 8 M urea lysis buffer using Lys‐C at 37°C for 4 h (enzyme/substrate ratio 1:50). The sample was diluted four times with 50 mM triethyl ammonium bicarbonate pH 8.5 and digested overnight at 37°C with Trypsin (enzyme/substrate ratio 1:50). Finally, the digestion was quenched with 5% formic acid. The resulting peptides were chemically labeled using stable isotope dimethyl labeling as described before (Boersema et al, 2009). The protein digests from the control sample, 1 day and 3 days of PLX4720 treatment were labeled as "Light" (L), "Medium" (M) and "Heavy" (H), respectively. An aliquot of each label was measured on a regular LC‐MS/MS run, and samples were mixed 1:1:1 (L:M:H) based on their peptide intensities and dried down. The procedure was repeated in three biological replicates.
Protein expression levels analysis
The labeled peptides were reconstituted in 10% formic acid prior to fractionation using strong cation exchange (SCX) as described previously (Helbig et al, 2010) for the protein expression levels analysis. The SCX system consisted of an Agilent 1100 HPLC system (Agilent Technologies, Waldbronn, Germany) with two C18 Opti‐Lynx (Optimized Technologies, OR) trapping cartridges and a polysulfoethyl A SCX column (PolyLC, Columbia, MD; 200 mm × 2.1 mm inner diameter, 5 μm, 200‐A). The peptides were dissolved in 10% FA and loaded onto the trap columns at 100 μl/min and subsequently eluted onto the SCX column with 80% acetonitrile (ACN; Biosolve, the Netherlands) and 0.05% FA. SCX buffer A was made of 5 mM KH2PO4 (Merck, Germany), 30% ACN and 0.05% FA, pH 2.7; SCX buffer B consisted of 350 mM KCl (Merck, Germany), 5 mM KH2PO4, 30% ACN and 0.05% FA, pH 2.7. The gradient was performed as follows: 0% B for 10 min, 0–85% B in 35 min, 85–100% B in 6 min and 100% B for 4 min. After injection of 200 μg of labeled lysate, a total of 45 fractions were collected, dried in a vacuum centrifuge and stored at −80°C.
Phosphoproteome analysis
The labeled peptides were reconstituted in 10% formic acid prior to fractionation using SCX as described previously (Hennrich et al, 2013). The experiments were performed on an Agilent 1100/1200 HPLC system (Agilent Technologies, Germany). Peptides corresponding to 3 mg of tryptic digested lysate were loaded onto a C18 trap column (strata‐x 33 μm Polymeric Reversed phase, 50 × 4.6 mm, Phenomenex, The Netherlands) for 5 min at 300 μl/min using aqueous 0.05% FA as solvent. Subsequently, peptides were eluted for 5 min from the trapping column with 80% ACN containing 0.05% FA onto a polysulfoethyl A column 200 × 2.1 mm, 5 μm particles and 200 Å pore size (PolyLC Inc., Columbia, MD) at the same flow rate. Separation was performed using a nonlinear 65 min gradient: isocratic for 2 min at 100% solvent A (5 mM KH2PO4, 30% ACN and 0.05% FA, pH 2.7); from 2 to 10 min at 3% solvent B (5 mM KH2PO4, 30% ACN, 350 mM KCl and 0.05% FA at pH 2.7); from 10 to 40 min a gradient to 35% solvent B; and from 40 to 45 min to 100% solvent B. The column was subsequently washed for 10 min with solvent B and finally equilibrated with 100% solvent A. Fractions were collected in 1‐min intervals for the first 40 min and for 3‐min intervals in the last 15 min. The fractions were desalted using Sep‐Pak Vac C18 cartridge (3 cc/200 mg, Waters), and the eluted peptides were dried down and stored at −80°C for phosphopeptide enrichment by Ti4+‐IMAC.
Phosphopeptide enrichment
Ti4+‐IMAC material was prepared and used essentially as previously described (Zhou et al, 2013). The prepared Ti4+‐IMAC beads were loaded onto GELoader tips (Eppendorf) using a C8 plug to approximately 1–2 cm length of material. The enrichment procedure for all SCX fractions was as follows: The Ti4+‐IMAC material was pre‐equilibrated two times with 50 μl of Ti4+‐IMAC loading buffer (80% ACN, 6% trifluoroacetic acid (TFA)). Next, each SCX fraction was resuspended in 50 μl of loading buffer and loaded onto the equilibrated GELoader tips. Then, the Ti4+‐IMAC material was washed with 50 μl wash buffer A (50% ACN, 0.5% TFA, 200 mM NaCl) and subsequently with 50 μl wash buffer B (50% ACN, 0.1% TFA). Bound peptides were first eluted by 30 μl of 10% ammonia into 30 μl of 10% FA. Finally, the remainder of the peptides was eluted with 2 μl of 80% ACN, 2% FA. The collected eluate was further acidified by adding 3 μl of 100% FA and subsequently stored at −80°C for LC‐MS/MS analysis.
LC/MS/MS analysis
The phosphopeptides and the 3+ SCX fractions of the whole proteome analysis were analyzed on LTQ‐Orbitrap Velos or Elite Mass Spectrometer equipped with an electron transfer dissociation (ETD) source (Thermo Fisher, Germany) and connected to an Easy UHPLC system (both Thermo Fisher Scientific, Germany). The columns were made in‐house from either Aqua™ C18 (5 μm, Phenomenex, Torrance, USA; 20 mm × 100 μm i.d.) for the trap column or Zorbax C18 (1.8 μm, Agilent; 38 cm × 50 μm i.d.) for the analytical column (Cristobal et al, 2012). Mobile‐phase buffers for nLC separation consisted of 0.1% formic acid in water (solvent A) and 100% ACN/0.1% formic acid (solvent B). The peptides were eluted during a 3‐h gradient and directly sprayed into the mass spectrometer. The flow rate was set at 100 nl/min, and the LC gradient was as follows: 7–30% solvent B within 151 min, 30–100% solvent B within 3 min, 100% solvent B for 2 min and 7% solvent B for 23 min. Nano spray was achieved with an in‐house pulled and gold‐coated fused silica capillary (360 mm o.d.; 20 mm i.d.; 10 mm tip i.d.) and an applied voltage of 1.7 kV. The mass spectrometer was programmed in a data‐dependent decision tree acquisition mode and was configured to perform a Fourier transform survey scan from 350 to 1,500 m/z (resolution 60,000) followed by higher collision energy dissociation (HCD; 32% normalized collision energy, resolution 15,000) or ETD fragmentation of the 20 most intense peaks depending on the charge state and m/z of the precursor as previously described (Frese et al, 2011). Supplemental activation was enabled for ETD. The 2+ SCX fractions of the whole proteome analysis were analyzed on a Q‐Exactive mass spectrometer connected to an Easy UHPLC system (both Thermo Fisher Scientific, Germany). The mass spectrometer was programmed in the data‐dependent acquisition mode and was configured to perform a Fourier transform survey scan from 350 to 1,500 m/z (resolution 35,000) followed by higher collision energy dissociation fragmentation of the 20 most intense peaks (25% normalized collision energy at a target value of 50,000 ions, resolution 17,500).
Data processing
Raw data were analyzed by MaxQuant (version 18.104.22.168) (Cox & Mann, 2008). Andromeda (Cox et al, 2011) was used to search the MS/MS data against the human UniProt database (20,247 entries, released 2012_02) complemented with a list of common contaminants and concatenated with the reversed version of all sequences. Trypsin/P was chosen as cleavage specificity allowing two missed cleavages. Carbamidomethylation (C) was set as a fixed modification, while oxidation (M) and phosphorylation of STY were used as variable modifications. For dimethyl labeling, DimethylLys0 and DimethylNter0 were set as light labels, DimethylLys4 and DimethylNter4 were set as medium labels, and DimethylLys8 and DimethylNter8 were set as heavy labels. Peptide identification was based on a search with a mass deviation of the precursor ion of up to 6 ppm, and the allowed fragment mass deviation was set to 0.05 Da for FTMS and 0.6 Da for ITMS. Data filtering was carried out using the following parameters: Peptide and protein FDRs were set to 1%, minimum peptide length was set to 6, and Andromeda minimum score was set to 60 [≅ Mascot score 20 (Cox et al, 2011)]. The reverse and common contaminant hits were removed from MaxQuant output. Protein quantification was performed by using only unmodified peptides and oxidation (M); the re‐quantify option was enabled. Only unique peptides with at least two ratio counts were used for protein quantification.
Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaino et al, 2013) with the dataset identifier PXD000497. PX reviewer account: username: review83857; password: 4G97r7 g3.
To filter for those proteins that show (or have) a consistent abundance level over three independent biological replicates (1 day/control and 3 days/control), we applied a one‐sample t‐test against 0 (no abundance change). Only those proteins that had a P‐value < 0.05 were considered. A two‐sample t‐test was performed to assess protein ratio differences between the two groups (3 days/control versus 1 day/control) and used as a filter to extract those proteins or phosphopeptides that show significant abundance differences (P‐value < 0.05). In addition to the statistical filters, only proteins and phosphopeptides with an arbitrary cutoff ratio ≥ 1.5 or ≤ −1.5 fold changes were considered. Furthermore, phosphopeptides were required to have a phosphosite location probability ≥ 0.75.
Reactome analysis
The significant entries at protein level (with a fold change ≥ 1.5 or ≤ −1.5) were analyzed by Cytoscape 2.8 (Smoot et al, 2011) using Reactome (Haw et al, 2011) and Cerebral as plugins (Barsky et al, 2007). Predicted interactions were removed from the analysis. Protein location was retrieved from UniProt database, and in case a protein had multiple locations, one was arbitrarily chosen to insert in Cerebral (Supplementary Table S4).
Gene ontology enrichment analysis by BiNGO
BiNGO Cytoscape plugin was used to perform gene ontology (GO) slim enrichment analysis (Maere et al, 2005). For the proteome analysis, the software was run using both target (significant regulated proteins with a fold change ≥ 1.5 or ≤ −1.5) and background list (the complete list of identified proteins and phosphoproteins), to calculate enrichment of biological processes across the target list. The same procedure was performed for the phosphoproteome. A hypergeometric test was performed to test for enrichment, and Benjamini & Hochberg false discovery rate correction was applied to correct for multiple testing. Enriched biological processes below the corrected P‐value threshold of 0.05 were considered.
We thank R. Kerkhoven and R. Beijersbergen for advice on the design and analysis of the shRNA screens, G. van Haaften and S. Huang for advice on the screening procedure, B, Gerritsen for statistical analysis of the screens, C.J. Vogel for STR profiling and N. Visser for excellent technical assistance. We thank E. M. Verdegaal and S. H. van der Burg for the 04.01, 93.03 and 00.08 melanoma cell lines. We thank Merck for financial support and for providing the ERK inhibitor. G.M. has been awarded an International postdoc grant from Vetenskapsrådet (VR), Sweden. K.G. was supported by an overseas training fellowship from the NHMRC Australia. A.F.M.A. and A.J.R.H. were supported by the Netherlands Proteomics Center, embedded in The Netherlands Genomics Initiative, and the PRIME‐XS Project, Grant Agreement 262067, funded by the European Union Seventh Framework Program. A.F.M.A. was supported by the Netherlands Organization for Scientific Research (NWO) with a VIDI grant (723.012.102). This work is part of the project Proteins At Work, financed by the Netherlands Organisation for Scientific Research (NWO) as part of the National Roadmap Large‐scale Research Facilities of the Netherlands (project number 184.032.201). D.S.P. and M.A.S. were supported by a Queen Wilhelmina award and a grant from KWF Kankerbestrijding.
Author contributions
MAS performed the validation of the screens and performed drug assay experiments, analyzed data, wrote the manuscript and approved the manuscript; GM performed the proteomic analyses, analyzed mass spectrometry data, analyzed data, wrote the manuscript and approved the final draft; KG performed the kinome shRNA genomic screens, analyzed the shRNA data and approved the final draft; LMR performed pathway enrichment analyses on proteomic data, analyzed the data and approved the manuscript; PAP performed the validation of the MS data by Western blot analysis and approved the manuscript; BvB performed the statistical analysis on the proteomic data, analyzed the data and approved the manuscript; SC performed the statistical analysis on the proteomic data, analyzed the data and approved the manuscript; AJRH analyzed the data and approved the final draft; AFMA designed the study, analyzed the data, wrote the manuscript and approved the final draft; DSP designed the study, analyzed the data, wrote the manuscript and approved the final draft.
Conflict of interest
A patent for the combination of MEK and ROCK inhibitors for the treatment of BRAF mutant melanoma has been filed with DSP, MAS and CJ Vogel as inventors.
Supplementary Information
Supplementary Figure S1 [msb145450-sup-0001-FigS1.pdf]
Supplementary Figure S2 [msb145450-sup-0002-FigS2.pdf]
Supplementary Figure S3 [msb145450-sup-0003-FigS3.pdf]
Supplementary Figure S4 [msb145450-sup-0004-FigS4.pdf]
Supplementary Table S1 [msb145450-sup-0005-TabS1.xlsx]
Supplementary Table S2 [msb145450-sup-0006-TabS2.xlsx]
Supplementary Table S3 [msb145450-sup-0007-TabS3.xls]
Supplementary Table S4 [msb145450-sup-0008-TabS4.xlsx]
Supplementary Table Legends [msb145450-sup-0009-Supp_tab_legends.docx]
This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
- © 2014 The Authors. Published under the terms of the CC BY 4.0 license


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Being aware is like breathing, it is fundamental to our everyday existence. Without awareness we are moving throughout life without breath, without direction, without connecting with ourselves and thus others.
Awareness is often mistaken as seeing outward, noticing what others are doing, how they are doing things, how they are stopping us from doing certain activities or stopping us from living our life.
Most of our notion of awareness is being aware of others and how they are affecting us. However, awareness is having an inward focus, not an outward focus.
(0:13) Don't mistake awareness of others with yourself
(0:34) Awareness of yourself is key
(1:15) A desire to learn about yourself
(1:30) Choosing not to learn leads to complaining
(2:06) Be your own leader
(2:32) Know thy self
(2:45) Tips to create more awareness
Know Thy Self
Awareness is knowing how we breath, knowing the patterns that we have in our everyday life, awareness is understanding our connection to the universe, awareness is knowing our beliefs, our mind, our spirit, our body. The first step to creating awareness is recognizing that you want to. Having awareness could be described as having a learning pathway for your life. Learning more about yourself is part of growing as a person.
When You Don't Know, You Stop Growing
It we choose not to gain self awareness then we stagnate in life and don't grow. This may sound very harsh but how many times have we looked at others and wondered why they have never moved on in life or grown. People like this usually complain about their life but are unwilling to do anything about it. Unfortunately this person who we are observing is a mirror of ourselves.
Becoming A Leader Through Self Awareness
A leader is a person who is leading their own life from strength to strength. Great leaders we see around us are great because they follow their own pathway, they know who they are, they have planned out their pathway for learning and they are on the journey of greater self awareness. They recognize that self awareness is something you must value and is then part of you for ever. Being a great leader is not following someone else but simply knowing yourself.
Ways to Develop Self Awareness
Training FAQ
Other questions that were asked in Coaching 101 this week.
- What is the fastest way to be a successful coach?
- What are the skills I must learn to be a good coach?
- What is it like to learn over the internet and on the phone?
Come along to an upcoming Coaching 101 class and learn more about coaching in general as well as programs offer here at ICA.

subset: commoncrawl


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Elementary Assessment Policy
9 November 2016
I am writing to update you about our new and improved methods of assessment here at Al Dhafra. In addition to the summary below, kindly find attached our updated Assessment Policy for Elementary.
Assessment in elementary / primary education is ongoing at Al Dhafra, as well as across Abu Dhabi schools. Children are being assessed throughout each lesson as well as by means of summative tasks in the middle and at the end of units. Continuous, ongoing assessment provides valuable information on children's development and learning and helps inform curriculum planning, teaching, and individualizing for each child.
As teachers assess students' work and performances, targets are set to help children improve their skills and understanding as well as their knowledge of the subject. A great emphasis is being placed on sharing these next steps so that students know how to move forward and make good progress. Teachers are implementing assessment for learning and providing meaningful feedback in line with the current, proven research on learning skills.
Formal mark entry on Digital Campus will take place twice a term: once at mid-term and a final grade will be entered at the end of each term. There will be a mark for Effort and also a mark in each subject for attainment.
Marks for mid-term will be available from mid-November. The levels A* to E are awarded to children based on their level of achievement against the Common Core Standards in English and Math and against the Virginia Standards of Learning in all other subject areas. Level descriptors will be available on the report card.
The assessment tasks vary. These are not simply 'Questions and Answers' tasks but often involve the students applying and explaining clearly their understanding and skills related to the subjects they have studied. Tasks include posters, role-plays, presentations, written tasks, comprehensions, ungraded quizzes, oral questioning, teacher observations, draft work, think-aloud, student-constructed concept maps, learning logs, portfolios, etc.
I hope you find all the information on our assessment and current policy informative and helpful. Please do not hesitate to contact us if you have any questions. We will ensure you are kept informed of all updates.
Kind regards,
Head of Elementary
Elementary Assessment Policy Arabic
Elementary Assessment Policy English
- Principal, Mr. Daniel Lee Bokelman:
- Vice Principal, Mr. David Evans:
- Head of Secondary, Mr. Darren Jones:
- Head of Primary, Mr. Duncan Silcock:
- Assistant Head Pastoral / SENCO / PODCO, Ms. Basma Iklil Sati:
- Assistant Head Curriculum and Assessment, Ms. Tracy Angela May:
- Head of ICT and Examinations, Mr. Ziyad Ashour:
- Student Guidance Counselor, Ms. Soha Hamze Bou Hamdan
Quick Links
Accreditation Reports
- ADEK Inspection Report, Published in 2018 ARABIC
- ADEK Inspection Report, Published in 2018 ENGLISH
- ADEK Inspection Report, Published in 2016 ARABIC
- ADEK Inspection Report, Published in 2016 ENGLISH
- ADEK Inspection Report, Published in 2014 ARABIC
- ADEK Inspection Report, Published in 2014 ENGLISH
- AdvancED Report
- Accreditation Certificates


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AL Central Battle - Twins Sox Odds
The top two teams in the American League Central meet on Tuesday as the Minnesota Twins host the Chicago White Sox in the opener of a critical three-game series. The Twins lead the Sox by three games so the Sox need to avoid getting swept. It is the first game of a six-game road trip for Chicago and they have struggled on the road this season.
[ Article courtesy of Bovada , where the current MLB line is Twins - 120, Total 8 Under -120 ]
The White Sox go with Jon Danks on Tuesday. He has been very solid all season with a 12-8 record and a 3.19 ERA. He went eight innings last time out against the Twins and allowed just one run while striking out seven. Danks has been about as good as you could ask for in his last three starts but he is just 1-1 although his ERA is 2.91. He has struggled a bit vs. the Twins in his career going 6-6 with a 4.96 ERA. The Twins send Scott Baker on Wednesday. He is 10-9 with a 4.76 ERA this baseball betting season. Last time out he gave up four runs on eight hits in six innings against the Sox. He is still 2-0 though in his last three starts with a 3.26 ERA. He has gotten some wins against the White Sox in his career even though his ERA is high. He is 6-3 with a 5.79 ERA against Chicago in his career and he gets slight chalk status here at Bovada. The Twins have simply owned the White Sox recently, winning 15 of the last 20 meetings. They took two of three at Chicago last week. None of the three betting games were close. The Twins won 6-1 and 12-6 while the White Sox win 6-1. Minnesota has really done well at home vs. the Sox winning eight of the last 11. Chicago has won seven of their last nine Tuesday games. They have struggled lately against right-handed pitching as they are 1-4 in their last 5 games vs. a right-handed starter. The White Sox are 5-2 in Danks' last 7 starts. The White Sox are 1-6 in Danks' last 7 road starts vs. the Twins. The Twins are 6-1 in their last 7 games vs. a left-handed starter. The Twins are 4-0 in Baker's last 4 starts and they are 14-5 in Baker's last 19 home starts. The Twins are 4-0 in Baker's last 4 starts vs. the White Sox. Looking at the total, the Under is 7-3 in the White Sox last 10 overall. The Under is 5-0 in the Twins last 5 overall. The Under is 9-3 in the last 12 meetings in Minnesota between the two teams.
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It's that time again .... our Design Team Challenge Time! This week our challenge is hosted by Sandye.
This time around Sandye has challenged the Design Team to create a School related card or project.
For her card this week, Sandye has used Riley and Company's Schoolgirl Sophie.
Don't forget to check out the Riley and Company Design Team's cards on their blogs:
- Frances Byrne
- Heather Bolli
- Jackie Rakoski-Diediker
- Joy Stagg
- Kelly Santi
- Kendra DeCota
- Monica Taylor
- Sandye Curry


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We don't feature that many cosplayers here in TAE, but we do appreciate how people like Regina, a.k.a. @picturresque, can transform themselves into practically any character they want. She got our attention when we found out how she was able to cosplay all of the major Sailor Moon characters in celebration of the animated TV show's 25 years.
She became quite the viral person of the week when countless blogs the world over started featuring her and her amazing copycat looks that obviously took a lot of effort and thought. It tugged on the heartstrings of many millennials who grew up watching the show, as well as anime lovers who simply adore all things Sailor Moon.
Recently, Sailor Moon fans from all over the world had major excitement when limited edition Sailor Moon makeup made its way into the scene. Triggering nostalgia among the kids and teens of yesteryear, the various makeup lines are available online and in-store from official distributors including Hot Topic, Amazon, and Disney World (Epcot).
However, HMU Regina took it upon herself to recreate the actual Sailor Senshis – all of them! Not just the canon ones like the titular Sailor Moon, and her cohorts Sailors Mercury, Mars, Jupiter, Venus, Uranus, Neptune, Pluto, Saturn, and the grown up Chibiusa also known as Black Lady. She even recreated a look for Sailor Sun, who was only created by fans for their fanfiction work. She did 3 looks for each character: human, sailor scout, and a modern/mature look inspired by the aforementioned characters.
Regina looks magical, no matter what she changes her look into. As a great makeup artist who's ultra-talented, this lady can do almost anything and have us believe she is that character. From simple yet classic looks like Audrey Hepburn's style in Breakfast at Tiffany's, to outrageous gothic looks like her Evil Queen and Red Riding Hood looks, and various simple and glam looks for all her catwalk and fashion show stints as a makeup artist, Regina truly knows how to make things pop.
She's not stingy with her information either. On her blog, she lists the products she uses, and on her Youtube channel she has tutorials. While she is German, she doesn't speak in her tutorials – she just shows you how to do it, detailing which products are used where. They're fun to watch, and we bet it's even more fun to actually learn from the stuff she posts!
Be sure to subscribe now to TAE Magazine, your ultimate source for worldly art inspiration!
All makeup done by Regina, photographs belong to her. For more information about the artist, visit the following:
Website/blog: picturresque.de
Instagram: @picturresque
Google Plus: PICTURRESQUE | Regina
Youtube channel: PICTURRESQUE Blog
Thanks so much for reading!
h/t: The Binge, Bustle, Pop Sugar, Preview Philippines, Revelist
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One little pill, so many questions
Whether the condom broke or you forgot to take your pill, it's a good idea to have a plan B—pun intended. But with all the different information circulating about emergency contraception—hearsay from your BFF or misinformed comments made by politicians—it can be tough to separate fact from fiction. To put your mind at ease, we tapped the minds of top experts to clear up some of the top myths about the day after's back up strategy: Plan B.
"The morning after pill does not literally need to be taken the morning after in order for it to work," says Jen Landa, M.D., an ob-gyn and hormone specialist. While there are several different types of emergency contraception pills, Plan B is the most commonly used. It should optimally be used in the first 72 hours after an episode of unprotected sex, says Landa. (Though she does note that if taken in the first 24 hours, Plan B is up to 95 percent effective.)
"Emergency contraception pills are completely different from medical abortion pills," says Sara Gottfried, M.D., author of The Hormone Cure. The difference boils down to the mechanism used to prevent or lose an unwanted pregnancy. Plan B will not affect an existing pregnancy or harm a developing fetus—it only has the ability to inhibit ovulation, Gottfried explains. As for medical abortion pills, those actually cause the fetus to stop developing and trigger the uterus to expel the pregnancy.
Repeated use of Plan B will not permanently mess up your fertility, but it's not something you want to get in the habit of doing, says Landa. First off, there are cheaper—and more effective!—methods of BC out there. What's more, if you use Plan B too often, your cycle could become very irregular making it difficult to know when you're fertile and whether or not you've become pregnant.
A dose of Plan B in the a.m. is not a hall pass for risky sex at night. While levonorgestrel—the active ingredient in Plan B—stays in your body a few days after taking it, the levels in your body decrease within hours of the dose, Gottfried says. That means the effectiveness of Plan B (against future sexual encounter) is greatly reduced. Also, sperm can stay alive in the body for up to seven days—far longer than the peak dose of Plan B—so that means an egg could become fertilized any time in that window, says Gottfried.


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|fedora / rope bracelet / espadrille / scarf / bikini top / sunglasses / flip flops / tote
With all this studying for the bar exam, I am looking forward to our honeymoon to Hawaii more than ever. Daydreaming about vacation had me pinning beachy styles this week and I wanted to share some of my favorites with you. If you have any suggestions for what to do and see in Kauai, I'd love to hear them!
Shop this post and more of my beach day picks here & find me on Pinterest at darahgabrielle >>


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Site News
www.uncovermichigan.com is Celebrating its 1st Birthday!!!
So today it is one year since i launched the first version of the www.uncovermichigan.com app sharing website.
It has been one hell of a journey the last 12 months, its been excitement, fun, laughter as well as a lot of damn hard work i might add!
In this past year www.uncovermichigan.com has grown into a massive project from when we first began, it has come way further than i had imagined it could do in this short space of time. There are a number of reasons fro this, sheer determination to provide the best app sharing experience, the team of staff i have the pleasure of working with & lastly but most importantly you guys the community and spirt of www.uncovermichigan.com! We have received masses of support over the past months, from encouraging comments, to offers of translations, to offers of modding etc..... Without the joint effort of the community no matter how good a service we create it would be worth nothing if it was not used and loved by its users. We do what we do to please and its as simple as that, if we have no one to please then there is no point!
To celebrate our Birthday we will be having a BIG BIRTHDAY GIVEAWAY!
Prizes to giveaway are
1 x iPad Mini 128GB Cellular
1 x £100 iTunes Voucher
2 x £50 iTunes Voucher
5 x £10 iTunes Vouchers
25 x www.uncovermichigan.com Premium Registrations
25 x iSignCloud Premium Registrations
I will announce the competition details in a further post this evening so be sure to look out for this!!!
I would like to say a few special thanks to a few certain individuals that make www.uncovermichigan.com what it is today a magnificent community service!
Well what a guy! An absolute genius at app development!
When i originally decided to launch www.uncovermichigan.com after the closure of AppTrackr, i had sought out an app developer who was supposedly working on the app as the site was been developed so we could launch both at the same time. About a week before the site launch i found out that no work had been done on the app and that the developer had no time to complete this. I began to panic and scoured the web looking for an app developer who would be able to work magic in a short timescale. This is when i stumbled across Zorro, i presented him with the project and he was confident that he would be able to get this done within a few weeks. At first i was doubtful it could be done in the timescale, but i was soon reassured when that evening he presented me with some prototypes of the App already pulling information from our old app database. From that moment on i have found myself constantly amazed at the work Zorro produces, he has provided us with www.uncovermichigan.com iOS App 1,2,3 & 4 (on the way), www.uncovermichigan.com+ & CrackAddict. Zorro i couldn't of got this far without you, thanks!
Tends to be in a grumpy mood (LOL)! But a legend when it comes to coding!
So Freddy joined the www.uncovermichigan.com team about 3 weeks after the initial launch, i was actually introduced to him via Zorro as they had worked together on some previous projects. It was at this point that www.uncovermichigan.com changed dramatically and we began to heavily implement new ideas and feature unseen before in the app sharing community. Finally there was a team of guys perfect for the job in hand, we all shared the same ideas and could all do something about it. The first thing Freddy did was practically bulldozer the old website and we basically started from fresh. I won't lie i was a bit gutted as i had spent weeks on www.uncovermichigan.com version 1 and i felt it was all been destroyed. Once Freddy had finished new site i was just amazed how well it looked, operated basically everything i loved. We started to add features like app submission approval, mass uploading, verified crackers, Hall of Fame, direct installer, OTA installer and much much more. All this was made possible by Freddy's amazing programming skills. Bro i couldn't of done this with out you either, thanks!
A very under rated Admin!!! Provides masses of background work for www.uncovermichigan.com!
So dkbame joined www.uncovermichigan.com in the early days, he was always a very active member who joined in all discussions. I had spoke to him privately on a number of occasions and we always got on like a house on fire. He would always offer his assistance and always stated that if there was anything i needed just to ask. As time went by he joined the cracking team and from there i really got to know him better, i liked what he was about a nice family man with the same ethos as myself. I asked dkbame if he would become a forum moderator, he did this and app moderating for sometime with no hassle or complaints ever. By this point i had been speaking to him on a daily basis and becoming good friends. June time last year i asked him if he would like to become an Admin, as i was getting busier in my life and www.uncovermichigan.com needed a reliable, well grounded Admin to be around more than i was able to. He has since started to help me with infrastructure, and is always keen to learn and do more things. Thanks mate for been part of the team!
Our very talented graphics designer and also forum GURU!!
Again Praiser has always been on the scene from the early days. He has always been involved heavily with the forums i think he lives there sometimes LOL! I wanted to say a specific thank you to Praiser as on many occasions at the very last minute i have contacted him stating that i urgently need some artwork making for a new project for example. Now i know that producing art is not just as simple as a few clicks it takes time to get right and also time to develop ideas for the intended work. He has never once complained at me, he has always just said ok bro, and then produced the highest quality graphics every single time without fail! So praiser thank you also for being part of the www.uncovermichigan.com team.
Please guys don't think i don't appreciate what every single member of staff does here on www.uncovermichigan.com that also includes www.uncovermichigan.com and also other crackers that submit here. If i was to thank each individual the post would be even longer than it is going to be. Please accept my biggest thanks to each and everyone of you that actively contributes to making www.uncovermichigan.com what it is today.
So many many thanks to
Now for some www.uncovermichigan.com History!!
www.uncovermichigan.com First Logo
www.uncovermichigan.com Website when it was born
www.uncovermichigan.com iOS App Version 1
www.uncovermichigan.com First iOS Icon
Now lets look at some great things www.uncovermichigan.com has achieved in its first year
www.uncovermichigan.com New Logo
www.uncovermichigan.com Current Website
(Top 100)
(Wishes System)
(Hall Of Fame)
(Crackers HOF)
As you can see the new website is just a million times better than the original www.uncovermichigan.com tragedy site.
Over the past year we have grown rapidly and have some impressive stats regarding registered users and also apps contained in our database
Forum Users & Stats
Account & App Stats from our Database
(Accounts show higher as originally we didn't register you on the forum at signup!)
So that means we have over 100,000 registered users on www.uncovermichigan.com! There are also many other unregistered users that use us on a daily basis.
www.uncovermichigan.com Current iOS App
Again the app is in a totally different league to the first version we saw above
www.uncovermichigan.com Current App Icon
Other Current Cool Apps from www.uncovermichigan.com
(www.uncovermichigan.com on NON JailBroken Devices available from http://RegMyUDiD.com)
(Powerful resign tool for OS X)
So guys that concludes my long post sorry about the length i wanted to get in as much info as i possibly could to show how far we have come in ONE YEAR, the sky is the limit and who know what new services we will have developed for you come our 2nd birthday!!
As mentioned above the BIG BIRTHDAY GIVEAWAY will be announced in a separate post later this evening with all details on how to enter!
Also i almost forgot in this year we also had some psychic powers as we predicted the fall of our departed freinds AppDora you guys all remember this one
Sorry i had to include this it was the highlight of my year ;)
Again many thanks to all you that support and continue to use www.uncovermichigan.com
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The number of Americans who are choosing to build a separate guest house when planning their new, custom homes, isContinue reading
Author: Lee Hicks
Want to Know The Real Cost of Installing a Surround Sound System? Read Here to Find Out How Much Money It'll Really Cost You
To boost enjoyment of music, television shows and movies, a surround sound system can be a fantastic investment. This allowsContinue reading
Basic Tools For Your First Kitchen. Read Here For The Perfect Checklist of Kitchen Essentials
If you or someone you know has either moved into their first apartment, or purchased their new house, here areContinue reading
Think You Don't Need a Security Alarm System? Think Again! Here Are The Top Reasons To Install an Alarm System in Your House
There are all too many reasons to install an alarm system in your home. One of the reasons that manyContinue reading


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Month: September 2016
Red Off The Shoulder Top in Paradise
Instagram Lately + A Nordstrom Giveaway
Surprise! You get two posts in one day today! I had to share two with you today since this one is SO GOOD! Scroll to the bottom to see what I mean! It's been a LONG time since I've done one… Continue Reading
Essential Jewelry Pieces for Fall with JTV.com
Thanks to JTV.com for sponsoring this essential jewelry pieces post and providing me with JTV.com jewelry pieces as compensation. Please remember that all comments and opinions are always 100% my own.
Friday Football Series – How Do You Wear Your Orange
Wednesday Wish List + Recent Purchases
Hey friends! I'm jetsetting off to somewhere fun for a long weekend, so be sure to follow me on Instagram and Snapchat! BTW, what do you all think of Instagram Stories? Are you pro-Snapchat or pro-Instagram? I've been trying to… Continue Reading
Can't Miss Labor Day Weekend Sales!
via Hey babes! Popping into your inbox on a Saturday with some great Labor Day sales that you need to know about this weekend! I promised you, Thursday that I'd let you know about all of the sales this weekend… Continue Reading
How To: Wear This Season's IT Camo Jacket + A Giveaway
*Be sure to scroll all the way to the bottom for an AWESOME giveaway, you won't want to miss!


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Sunday, November 26, 2006
UPDATE: Dynebolic 2.3
A new version of Dynebolic has been released, here are some changes, from the release notice:
Starting from this release dyne:II core runs efficiently on solid state devices, loading its system from a compact-flash or similar controller. It has been tested on some embedded setups with extremely good results in terms of speed and power saving. Moreover, this release significantly improves stability and performance, running on a brand new 2.6.18 kernel optimized for low latency realtime. New and updated software include: VNC for remote desktop operation and recording, MPlayer and ffmpeg audio/video codecs, DVD recording tools, Ksubtitle editor, FUSE and pcmcia card autodetection.


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- Categories
All categories • Pneumatics• How To Make Stuff• Fog, Fluids, Scents• Customer Service• Prop Making• Foam Filled Props• Electric Firecrackers• Customer Videos & Pics• Decorating Supplies• Video & Visual Effects• Other• Escape Room Tech• Controllers & Electronics• Electric Motors• Linear Actuators• Lighting• Sound & Audio• Talking Skulls
- Contact
- Other Places
Using a Double-Acting Cylinder as Single-Acting
You can use a double-acting cylinder as a single-acting. Simply use a 3-way solenoid and only supply air to the bottom of the cylinder. Leave the top air port of the cylinder open (or optionally install an exhaust muffler into open port as shown in the image).
The weight of the prop or mechanism is responsible for pushing the cylinder shaft back down.
Running a double-acting cylinder as a single-acting makes it very fast. This makes it a great option for simple pop up props.
Related Products
Related Entries
- How do I control the speed of an air cylinder?
- Video: Cylinder mounting
- Video: Double-Acting Hookup Kits
- Video: Single-Acting Hookup Kits
- Video: Pneumatic Cylinder Overview


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An intensely human documentary film that follows the lives of foreign women over 10 years. Foreign women workers in a Taiwan hospice for the elderly are there to provide for their families. They comfort themselves by singing a self-mocking song said 'No money, no honey'. Their greatest source of sadness is being foreign to their families in their own countries. Foreign women workers work for 3 years, the duration of a contract. During the 3 years they work with almost no days off. And what awaits these women is an awkward family reunion. After using all the money they've earned, these women head for foreign lands again by spending a fortune. They work for the family's future but can't see that future themselves, and can't share familial love with them. Money and Honey chronicles the women's hard work and family affairs, which are much harder than the work; their strength and unwillingness to giving up hope are affecting. (CHO Young-jung)
- Oct 23 Sun 2011 02:49
Program Note from Busan International Film Festival 2011


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While it is true that the real Deepthroat was Mark Felt and this was later revealed in 2005, this rather amusing comedy about how two teenage girls took down the president and all the president's men is surprisingly funny and smartly written.
What I also like about this film is the soundtrack. It has a lot of great songs from the 1970s on it.
The cast includes:
Kirsten Dunst as Betsy Jobs
Michelle Williams as Arlene Lorenzo
Dan Hedaya as President Richard M. Nixon
Bruce McCulloch as Carl Bernstein
Will Ferrell as Bob Woodward
Saul Rubinek as Henry Kissinger
Dave Foley as Bob Haldeman
And best of all you can watch it for free on YouTube.


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Animism: Drawing the Line
Aug 22, 2013
Para Site
4 Po Yan Street
Sheung Wan
Para Site has the pleasure to present Animism: Drawing the Line a talk by curator Anselm Franke. The lecture will draw from his research on the Animisim project and will explore the relation between the aesthetics and politics of animism, and their significance for reaching beyond the West / Non-West binary today. It will show that animism is a concept through which we can gain substantial insights into politics, namely through interrogating the practices of political inclusion and the acquisition of rights, and equally into exhibition practice, aesthetics and the question of "modernity".
"Animism" is a concept from 19th century European anthropology, where it designated humanity's "original religion" in which nature and the cosmos are being variously perceived as animated and populated by vital forces, spirits or different non-human persons. Animism was perceived as an essential "premodern" and "savage" worldview, and the discourse was intimately tied with colonialism. Animism acted as both an imaginary mirror of Otherness confirming the self-identity of Western modernity, and as a designation for ontological difference in various cultures. From a contemporary modern perspective, animism appears as a name for the making of the distinction between "objects" and "subjects" in an expanded field of environmental thought. But animism is also closely linked to "animation" in film and other artistic media.
The exhibition Animism will open on 24 August 2013, 17:30hrs at OCAT Shenzhen.
Anselm Franke is a curator and publicist based in Berlin, where he is Head of the Department of Visual Arts and Film at the House of World Cultures (HKW). He was the curator of the 2012 Taipei Biennial, and previously Director of Extra City Kunsthal Antwerpen, where he initiated the traveling exhibition Animism in 2010, which has since been realized in various collaborations in Bern, Vienna, Berlin, New York. After its presentation at OCAT Shenzhen, the exhibition will be shown at Ilmin Museum in Seoul.
Para Site is Hong Kong's leading contemporary art space and one of the oldest and most active independent art centres in Asia. It produces exhibitions, publications and discursive projects aimed at forging a critical understanding of local and international phenomena in art and society.


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Nearby Lodging
There are many hotels and places to stay between Fargo-Moorhead, Fergus Falls, and Detroit Lakes. We do not have any lodging specials to offer at this time, however, you can certainly check out the following links to help in planning a place to stay if you do not camp at our showgrounds.
For information on Motels and other area attractions:
- Fargo-Moorhead Conventions and Visitor's Bureau
- Detroit Lakes Visitor's Bureau
- Fergus Falls Visitor's Bureau
If you are in need of services, you are in luck, The closest convenience store with fuel and grocery staples is Lakes One Stop. Located at 12034 Co. Hwy 4 in Lake Park. If you need additional services, such as gas, added food items, auto-parts etc, you can visit Hawley, MN just 20 minutes to the north. The Co-Op in Lake Park has LP facilities.


text: Once upon a time, a lot of people viewed Microsoft as the epitome of corporate omnipresence, as many of us wrote our 11-part fantasy series in Word, checked our Hotmail accounts while surfing the web on Internet Explorer, probably on a computer running Windows. But now we have Google Docs, Gmail, and Chrome, and Microsoft is taking out full-page ads warning consumers of how this other company is the one to be reviled. [More]


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PittStart Registration
Log in here to register for your PittStart session or save your spot to spend the night on campus.
Welcome to the University of Pittsburgh, Class of 2023!
It's time for you to join us on campus for PittStart, your academic freshman orientation program. At your PittStart session, you will plan your fall classes, shape your unique academic path, and begin the transition to college.
Attendance at one summer PittStart session is mandatory for all incoming freshmen.
Parents/Guardians: You are not required to attend PittStart, but we do offer optional family programming. Take a look at our family agendas for more information!
Transfer students: PittStart programming is designed specifically for freshman transitioning to Pitt directly from high school. Transfer Students are invited to attend Pitt's New and Transfer Student Orientation. You can get more information here.
In order to register for PittStart you will need to do the following:
- Pay your enrollment fee online.
- Get your 7-digit student ID number from your email (if you paid your enrollment fee recently, we will send this to you in your PittStart registration email within 3-5 business days after paying).
Registration Form
Log in here to register for PittStart and/or sign up to spend the night in the Litchfield Towers on campus the night of your PittStart session. Please note that signing up for accommodations is a separate step and registering for PittStart doesn't automatically get you a spot in Towers!
Please choose a date wisely as you will not be able to change your PittStart date after you register.
Note: Your $300 enrollment fee includes a $100 tuition deposit and a $200 PittStart fee.


text: Home - News & Media - Past News Stories Lewis and Clark/Phillips66 Host NAPTA Expo Posted: March 13, 2014 Article by: Paige Allen, firstname.lastname@example.com - Dave Young, director of sales with Simtronics, shows Dale Hagan, facilities supervisor at Mallinckrodt Pharmaceuticals (foreground), a simulation program for process industries during the Lewis and Clark Community College/Phillips 66 North American Process Technology Alliance (NAPTA) Midwest Training Expo on the college's N.O. Nelson Campus in Edwardsville on Thursday. Industry professionals and educators from around the nation are attending the training expo designed to unite employers and educators in the Process Technology field. The North American Process Technology Alliance (NAPTA) is the standard–bearer of the Process Operations Technology (PTEC) curriculum and audits PTEC degree programs in North America and endorses those that meet its criteria. Lewis and Clark's PTEC Program has received the highest NAPTA endorsement. For more information or a complete schedule of the conference visit www.naptaonline.org.


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Related Ministry Sites
Andrew Wommack Ministries' list of Related Ministry Sites features other organizations that we consider like-minded in the Gospel of Jesus Christ. These links allow you to easily access their websites where you can learn more about them.
|Ambassadors To The NationsAmbassadors To the Nations has been taking mission teams, building churches, schools, medical clinics and raising up Bible Colleges in Nicaragua, Mexico, Guatemala, Nigeria, Cuba and many other countries.
|Andrew Wommack Ministries CanadaThis website was designed especially for Andrew's friends and partners in Canada.
|Andrew Wommack Ministries EuropeThis website was designed especially for Andrew's friends and partners in Europe, Asia and Africa. Here they can order products, make donations, and enroll in partnerships.
|Andrew Wommack Ministries GermanyThis website was designed especially for Andrew's friends and partners in Germany.
|Andrew Wommack Ministries IndiaThis website was designed especially for Andrew's friends and partners in India.
|Andrew Wommack Ministries of South AfricaThis website was designed especially for Andrew's friends and partners in South Africa.
|Bob Yandian MinistriesBob Yandian is the pastor of Grace Church in Tulsa, Oklahoma. He is also host of the Real Answers television broadcast and a weekly radio broadcast called Precepts with Bob Yandian. He is founder of Grace School of Ministry.
|Calvary CathedralPastor Bob Nichols and his wife, Joy, founded Calvary Cathedral in 1964. The church reaches people of all nationalities and ages with the message of how to triumph in God's best. They believe every person is valuable and precious to God.
|Charis Christian CenterPastor Lawson teaches the Bible in a practical way focusing on grace and faith. The church has a strong emphasis on missions and equipping believers to fulfill their God given destiny. Pastor Lawson is also an adjunct instructor at Charis Bible College.
|Christ in ActionChrist In Action (CIA) is a faith based nonprofit 501-(C)(3) organization founded by Dr. Denny & Sandy Nissley. At the request of The White House, they are currently developing a Faith Based First Responders Coalition to streamline a nationwide rapid response network in the event of future disasters. CIA has been responding to national disasters since 1998.
|Charlie & Jill LeBlancCharlie & Jill LeBlanc have pursued the call to teach, encourage and inspire by the Word of God through song, along with leading congregations all over the world into God's presence through praise & worship and anointed songs.
|City Church InternationalCity Church International is based in Hong Kong. They celebrate the grace of God knowing that it's what liberates us to become fully and uniquely what God intends us to be.
|Creation Evidence MuseumThe Creation Evidence Museum is a non-profit educational museum chartered in Texas in 1984 for the purpose of researching and displaying scientific evidence for creation.
|Crossroads MinistriesThe mission of Crossroads Ministries USA, Inc., a Christian, non-profit ministry, is to help meet the spiritual and emotional needs of the elderly who reside in our communities` care centers. This is a crucial and much needed ministry because 85 percent do not receive any visitors.
|David Hinton founded "Outreach For America" in 1988. It is a ministry designed to win the lost to Jesus Christ and to train others to do the same. David's unique way of mixing his music with the Word of God brings refreshing to God's people, hope to the hurting, and desire for God to those that are lost.
|Don Krow's Evangelistic OutreachThe heart of this website is to assist in world evangelism through tracts, booklets and the written Word. Here, you will find proven, hands on ministry gospel tracts (free!). Also check out Don's Discipleship Evangelism website.
|Duane Sheriff MinistriesDuane Sheriff is the pastor of Victory Life Family Worship Center in Durant, Oklahoma. He is gifted with the ability to communicate the simplicity of the Gospel. He began the free teaching tape/CD ministry in 1984. He desires to make the Word of God available to everyone.
|Kingdom Life Ministries InternationalKingdom Life Ministries International was born out of a desire to see leaders in the Christian as well as business world established in God's goodness.
|New Nature PublicationsNew Nature Publications was started by Ryan and Kylie Rufus in 2009 with the purpose of flooding the nations with numerous and diverse grace resources.
|The Solid Rock of AtlantaThe Solid Rock of Atlanta is a Spirit-filled, multicultural church where the love of Jesus breaks down man's walls of separation, including socio-economic, racial and denominational barriers.
|TMP Leadership Training InternationalLooking for a Spanish speaking Bible leadership training center? Check out this link!
|Terradez Ministries – Ashley & Carlie TerradezEmpowering believers in the promises of God! With an emphasis on grace and faith for healing, financial prosperity, and supernatural favor.TerradezMinistries.com
|Tricord GlobalBy providing training to those in poverty, Tricord Global affects individuals. We also teach social impact so that they turn around and teach others. Through the generations, cities and nations will rise out of poverty.
|Vida en CristoBarry and Betty Kay Bennett desire to bless the Hispanic world with the transforming power of the truth of Jesus Christ. They provide free Spanish articles and audio teachings.
|WallbuildersPresenting America's forgotten history and heroes with an emphasis on our moral, religious, and constitutional heritage.
|Watersprings RanchFounded by David and Carla Whatley, Watersprings Ranch is christian based, non-profit, long-term residential home for children who have been abused, neglected, or abandoned.
|Wealth BuildersFounded by Billy Epperhart, Wealthbuilders has a mission to coach, teach, train and mentor in the enterprise of wealth building for the purpose of transforming individuals, cities and nations


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Laurie Coombs is a follower of Christ, wife, mommy, author, public speaker, and the founding director of A New Song International. She is the author of Letters from My Father's Murderer: A Journey of Forgiveness, an incredible true story of grace, mercy, and the redemptive power of God. Her story was featured in Billy Graham's film, Heaven, as well as on many other national and regional radio and television programs. She is a contributor to Zondervan's NIV Bible for Women and writes at LaurieCoombs.org. Laurie and her husband, Travis, make their home in Nevada along with their three daughters.
To connect with Laurie, please visit LaurieCoombs.org or find her on Twitter, Facebook, and Pinterest.
I freely admit there have been times when I have given Satan entirely way too much power by focusing on him and his latest attack rather than on God.
It is true. Satan is real, he is our enemy, and he does attack God's people. But our attention should not be directed toward him.
Yes, we need to understand how Satan works and how to engage in spiritual warfare, for to completely ignore our enemy's presence would be foolish, but it seems to me that there's a difference between recognizing and protecting ourselves against attack and being consumed by a current or possible attack in the future.
The human mind is incapable of focusing on two things at the same time, so in all actuality, when we focus on Satan, we take our focus off Jesus. But this will never do. We will never be able to defeat Satan by focusing on Satan. It just won't happen. Darkness cannot drive out darkness, only light can do that (Martin Luther King, Jr.). And Jesus is that light. The Light. So our attention must remain on Him alone if we are to defeat Satan in our lives.
Any thoughts? Share in the comments.
Please note: I've intentionally kept this post short and sweet because Satan gets enough attention, as it is––he certainly doesn't need more. The more attention he gets, the more power we give him, so let's be sure to keep our thoughts of him in check and our eye on God.


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Trinity River Sanitary Survey
Description: This river survey is a report about the the health and sanitation of various portions of the Trinity River in Texas. The report includes accompanying photographs, diagrams, and graphs as well as statistical readings from the monitoring stations along the river. There is a table of contents following the title page and letter of transmittal.
Date: June 1925
Creator: Texas. State Dept. of Health.
Item Type: Refine your search to only Book


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Hell Pizza
This article contains content that is written like an advertisement. (August 2015) (Learn how and when to remove this template message)
|Fast food
|1996 in Kelburn, Wellington
|New Zealand
|Pizza, Pasta, Salads
Hell Pizza is a New Zealand-based pizza chain. Hell began in New Zealand in 1996 next to Victoria University, and has expanded within New Zealand and to the UK, Ireland, Canada, Korea and India. A store was previously open in Queensland, Australia though has since closed.
Hell Pizza was started by Callum Davies and Stuart McMullin.[1] In 2006, Hell sold the New Zealand Master Franchise rights to Tasman Pacific Foods (the NZ master franchisee for Burger King) for NZ$15,000,000. The NZ Master Franchise rights were purchased back from Tasman Pacific Foods in 2009. In August 2011, founder Warren Powell left Hell Pizza New Zealand and in July 2014, he left Hell Pizza International. There are 64 Hell stores nationwide in New Zealand, Australia had a store in South-east Queensland that has since closed and Canada has one store in North Vancouver.
The Hell theme is used in the menu. Seven standard pizzas are named after the seven deadly sins.
The company is notable for its controversial advertising and for being the first company in New Zealand to offer online pizza ordering, in 1996.[citation needed] They sponsored a segment on the television show Pulp Sport.
Mocking of Christianity[edit]
A radio advertisement that featured a humorous conversation about Hell attracted one complaint in New Zealand. The Advertising Standards Authority ASA did not uphold the complaint since "it did not reach the threshold to be likely to cause serious or widespread offence".[2]
"Selling your soul"[edit]
Hell Pizza used the expression "sell your soul" in mass advertising in 2013 in New Zealand. It received one complaint to the ASA that "This flippant attitude towards something that many New Zealand citizens ... believed to be significant and consequential..." The ASA ruled that there were no grounds to proceed, since "sell your soul" is a common saying and recognised as dark and satirical.[3] Hell also purchased the soul of a 24-year-old Wanganui man for $5001, after the online auction website Trademe withdrew his attempt to sell it there.[4]
Halloween dead celebrities[edit]
In November 2008, while under management from Tasman Foods, Hell Pizza New Zealand apologised for an advertisement featuring skeletal remains of Sir Edmund Hillary, Heath Ledger, and the Queen Mother, dancing on gravestones. The apology was to Hillary's family, which complained the ad was in "extremely poor taste". The ad was withdrawn from the company website on 3 November.[5]
Pizza Roulette[edit]
In 2012, Hell Pizza marketed 'Pizza Roulette', in which one unidentified slice of a pizza is doused with chili. Marketing called it the "hottest chili known to mankind".[6]
Real Rabbit Campaign[edit]
In April 2014 Hell Pizza released rabbit flavoured pizza for Easter and advertised it in New Zealand on billboards covered in rabbit pelts. The billboard has received both criticism and praise; the New Zealand Vegetarian Society criticised the billboard.[7]
Broken Charity Pledge[edit]
In 2009, Hell Pizza owner Warren Powell verbally pledged to pay all proceeds from a Hell takeaway van at the Big Night In Telethon to the charity KidsCan, amounting to about NZ$10,000. Julie Nelson, chief executive of the charity, said "after seeing reports that some of the money raised was failing to reach needy children, Powell reneged on the deal."[8] On 14 August 2011, the Hell Pizza Facebook page stated "We can confirm that we have now contributed $10,000 to the KidsCan Charitable Trust."[9]
In July 2010, after hackers attacked the Hell Pizza customer database, 230,000 customers were advised to change their passwords.[10]
|Wikinews has related news:
On 31 October 2006 Hell announced through a press release it would be promoting its "Lust" pizza by distributing 170,000 branded condoms.[11] The Family First Lobby said Hell has "crossed the line of what is decent and acceptable advertising material to be put in letterboxes of families".[12] It was later revealed that the condoms failed to comply with New Zealand labelling and packaging requirements, and remaining stocks were destroyed.[13]
- ^ "The history of Hell Pizza". Stuff. 11 April 2016. Retrieved 10 January 2019.
- ^ Advertising Standards Authority (2008-05-13). "08/231 - Hell Pizza Radio Advertisement". Advertising Standards Authority. Archived from the original on 2011-05-29. Retrieved 2008-10-06.
- ^ Advertising Standards Authority (2008-08-22). "08/396 - Hell Pizza Direct Mail Website and Television Advertisement". Advertising Standards Authority. Archived from the original on 2011-05-29. Retrieved 2008-10-06.
- ^ "Man sells his soul to Hell". Manawatu Standard. 4 July 2008. Retrieved 4 March 2013.
- ^ Tedmanson, Sophie (3 November 2008). "Pizza company apologies for ad featuring Hillary, Ledger and the Queen Mother". London: Times Online. Retrieved 2008-11-03.
- ^ "Pizza Roulette: One Slice From Pie Has 'Hottest Chili Known To Mankind' (VIDEO)". The Huffington Post. 13 March 2012. Retrieved 13 October 2014.
- ^ Guillaume, Jenna (11 April 2014). "A Pizza Chain In NZ Has Set Up A Billboard Made Of Dead Rabbits". BuzzFeed. Retrieved 13 October 2014.
- ^ Hurley, Bevan (24 July 2011). "Charity cops one Hell of a loss". The New Zealand Herald. Retrieved 13 October 2014.
- ^ KidsCan Update Hell Pizza Facebook page, "HELL regularly makes major contributions to local and national causes. One of the most recent was a donation of more than $175,000 to the Canterbury earthquake recovery/"
- ^ Barratt, Joseph (25 July 2010). "Police called over pizza hack". New Zealand Herald. Retrieved 29 July 2010.
- ^ "HELL distributes condoms to promote Lust". Scoop. Hell Pizza. 31 October 2006. Retrieved 4 March 2013.
- ^ "Hell's Pizza sinks to offensive advertising". Scoop. Family First Lobby. 2 November 2006. Retrieved 4 March 2013.
- ^ Hurley, Bevan (31 July 2011). "Free condoms weren't up to scratch". Herald on Sunday. Retrieved 25 September 2016.


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Download EBOOK Fantastical Fairies Fairy Ring Puzzle PDF for free
Category: Children's Books
The author of the book: Chronicle Books
Edition: CHRONICLE BOOKS
Date of issue: 24 March 2015
Format files: PDF, EPUB, TXT, DOCX
The size of the: 983 KB
Language: English
Description of the book "Fantastical Fairies Fairy Ring Puzzle":This vibrant double-sided puzzle features 26 magical fairies that join together in endless ways to create enchanting fairy rings.
Chronicle BooksRegrettably, at present we really do not have got info on the musician Chronicle Books. Nonetheless, we would value if you have virtually any specifics of it, and they are wanting to give the idea. Send it to us! We also have the many verify, and when everything are usually accurate, we'll post on our web site. It is very important for all of us that all accurate with regards to Chronicle Books. All of us thank you upfront to get able to head to fulfill people!
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