text:
- Research
- Open Access
Novel anti-glioblastoma agents and therapeutic combinations identified from a collection of FDA approved drugs
© Jiang et al.; licensee BioMed Central Ltd. 2014
- Received: 25 December 2013
- Accepted: 10 January 2014
- Published: 17 January 2014
The Erratum to this article has been published in Journal of Translational Medicine 2014 12:126
Glioblastoma (GBM) is a therapeutic challenge, associated with high mortality. More effective GBM therapeutic options are urgently needed. Hence, we screened a large multi-class drug panel comprising the NIH clinical collection (NCC) that includes 446 FDA-approved drugs, with the goal of identifying new GBM therapeutics for rapid entry into clinical trials for GBM.
Screens using human GBM cell lines revealed 22 drugs with potent anti-GBM activity, including serotonergic blockers, cholesterol-lowering agents (statins), antineoplastics, anti-infective, anti-inflammatories, and hormonal modulators. We tested the 8 most potent drugs using patient-derived GBM cancer stem cell-like lines. Notably, the statins were active in vitro; they inhibited GBM cell proliferation and induced cellular autophagy. Moreover, the statins enhanced, by 40-70 fold, the pro-apoptotic activity of irinotecan, a topoisomerase 1 inhibitor currently used to treat a variety of cancers including GBM. Our data suggest that the mechanism of action of statins was prevention of multi-drug resistance protein MDR-1 glycosylation. This drug combination was synergistic in inhibiting tumor growth in vivo. Compared to animals treated with high dose irinotecan, the drug combination showed significantly less toxicity.
Our data identifies a novel combination from among FDA-approved drugs. In addition, this combination is safer and well tolerated compared to single agent irinotecan.
Our study newly identifies several FDA-approved compounds that may potentially be useful in GBM treatment. Our findings provide the basis for the rational combination of statins and topoisomerase inhibitors in GBM.
- Glioblastoma
- Drug screening
- Patient-derived glioblastoma cell lines
- Rational combination
Novel therapeutic options are sorely needed to target glioblastoma (GBM), a notoriously treatment-resistant brain cancer. GBM is a leading cause of cancer-related death in the pediatric and adult populations, with most patients succumbing within 1-2 years[1, 2]. The standard therapies are inadequate, and their toxicities lead to severe life-long morbidity in the small number of patients that survive[2]. Despite this grim prognosis, GBM is an orphan disease that is in general not a priority for new drug development[3–5]. Moreover, the biology of GBM is complex and much remains to be learned about the putative key signaling pathways before they can be therapeutically exploited[6]. In view of the unmet and urgent clinical need, we were motivated to pursue recent data indicating that GBM may respond to some FDA-approved agents not previously identified as GBM therapeutics[7, 8]. The in vitro screening of a broad range of drugs already approved for other indications is attractive as in vivo toxicity and pharmacology are well defined, and such compounds can enter GBM clinical trials rapidly either as single agents or as combinations.
Accordingly, our goals were to identify and characterize single and combination agents having anti-GBM activity that we can potentially introduce into clinical trials quickly. To this end, using GBM cell lines and patient-derived GBM cell cultures, we screened a 446-compound NIH Clinical Collection (NCC) library comprising FDA-approved drugs. To further improve the anti-GBM potency of these drugs, we tested various drug combinations and compared them to single drug treatment.
Our screening strategy included multiple human GBM cell lines of different origins in order to provide cross-validation of findings. These cell lines included 4 established serum-grown, immortalized human GBM lines, 4 patient-derived stem cell like GBM primary cells grown as neurospheres, and 2 primary patient-derived GBM lines grown as adherent cultures[9–14].
We did not confine our screening to only adherent GBM stem cell lines despite reports claiming that such lines remain undifferentiated longer and constitute a simpler, less variable assay[11, 14]. It is not yet firmly established that the major therapeutic target in GBM is just the cancer stem cell tumor compartment and there are indications that other cell types within GBM may assume stem cell characteristics through genetic or epigenetic events[14–16]. In contrast to a single type or lineage of cells, neurospheres contain a mix of GBM stem cells and differentiated cells, which is more reflective of the composition of human GBM tumors[17]. Further, when dissociated neurospheres are implanted orthotopically, they are highly tumorigenic and authentically recapitulate the invasive natural history, composition, and histology of GBMs growing in humans[16]. Hence we report the identification of NCC active compounds through our screening approach on patient-derived stem cell-like GBM primary cells.
Our initial screening identified 22 compounds active against GBM (>50% cell death)[18] at pharmacological doses. These 22 compounds encompassed 11 drug classes. In particular, we found that the statin, pitavastatin, effectively induced cellular autophagy and suppressed tumor cell MDR-1 protein, to impressively enhance the potency of irinotecan, a topoisomerase 1 inhibitor used in cancer treatment[19–21]. This work newly identifies FDA approved drugs and drug combinations, notably pitavastatin and irinotecan, that may be useful for GBM treatment, and draws attention to the potential value of in vitro screening of approved compounds not currently used to treat GBM.
Materials and methods
Overview of cell-based (in vitro) screening for potential anti-GBM compounds
We acquired 446 small molecules that completed human clinical trials from the NIH Clinical Collection (NCC). The initial broad screen was performed on U87 cells plated at 2000 cell per well on 96-well plates incubated overnight. All compounds were added to the plates to attain a final concentration of 10 μM. After 72 hours of incubation with drugs, the inhibition of cell proliferation was quantified by the Alamar Blue viability assay. Briefly, after incubation, Alamar Blue (#BUF012B, AbD Serotec) was added directly to the culture medium, and the fluorescence measured at 560/90 to determine the number of viable cells (Infinite M200, Tecan Group Ltd.). The IC50 values were calculated using commercially available software (Prism®, Graphpad Software, La Jolla, CA). We defined active compounds as those eliciting a greater than 50% reduction of cell viability in three independent screens. The 15 most potent and available drugs or compounds were then re-screened with other established glioma cell lines (LN443, A172 and U118), with the four patient derived GBM stem cell like primary neurosphere lines (SK72, SK262, SK429 and SK660), and with 2 GBM stem cell like primary cells (SK72 and SK262) grown as adherent culture. Pitavastatin was also tested in combinations with the other 12 compounds. The IC50 values were determined with and without pitavastatin (2 μM), using the Alamar blue assay as described above.
Isolation, culture, and compound activity testing with patient derived GBM cells
Human GBM samples
Fresh human GBM material was acquired from 4 GBM surgical patients and cultured as previously reported[9, 13]. Briefly, the dissociated tissue was washed, filtered through a 30 μm mesh and plated onto ultra-low adherence flasks at a concentration of 500,000 to 1,500,000 viable cells/ml. The stem cell isolation medium included human recombinant EGF (20 ng/ml), human bFGF (10 ng/ml) and heparin (2 μg/ml). Sphere cultures were then passaged by dissociation, washed, resuspended in neural stem cell culture medium (#05750,Stemcell Technologies), and plated on ultra low-adherence 96 well plates at 2000 cells per well for all subsequent drug testing.
Alternatively, patient-derived dissociated GBM tissues were plated onto laminin-1 coated plates (Sigma, 3-5 μg/ml). Cell populations were dissociated using Acutase (Sigma) and expanded for 5-10 passages, then plated on flat bottom for drug testing.
Confirmation of stem cell marker expression
Primary neurospheres were cytospun onto glass slides. Adherent primary cultures were grown onto Permanox™ chamber slides (#70380,EMS). Cells were incubated with human Nestin antibody (M1259, R&D system) and then with fluorescein-labeled secondary antibodies, then stained with DAPI. The cells were visualized under a UV microscope (Olympus BX51).
Drug testing and survival assay
As explained above, cells were seeded onto either regular or ultra-low adherence 96 well plates and incubated for 18-24 hours and then treated with vehicle control or single drugs or drug combinations. After 96 hours of incubation, Alamar Blue was added directly to the culture medium, and the fluorescence measured at 560/90 after 4-12 hours to determine the number of viable cells. The IC50 was calculated.
Prediction of blood brain barrier (BBB) permeation by active compounds
Although ample evidence has demonstrated that drugs of virtually any size or chemotype can enter brain tumor via leaky tumor microvessels, the ability to penetrate the intact blood brain barrier (BBB) is reasonably hypothesized to be useful for treating tumor cells infiltrating normal brain tissue along fiber tracts[22–25]. Hence we estimated the capacity of active anti-GBM compounds to cross the BBB. We used standard software to calculate the Log BB value: Log BB = -0.0148 PSA + 0.152 CLogP + 0.139; PSA = polar surface area, p = octanol/water partition coefficient[26, 27].
Determination of cell cycle, autophagy, and apoptosis
Cell cycle analysis
GBM cells were seeded into 10 cm dishes at a density of 1 × 106, cultured overnight followed by the addition of 3 μM pitavastatin with 24 or 48 hours of incubation. Cells were trypsinized and fixed in 70% ethanol for 30 minutes, incubated with 25 μg/ml propidium iodide (PI) and 250 μg/ml RNAase in PBS for 1 hour at 50°C. After PI staining, cells were analyzed via flow cytometry (Canto, BD FACS), and the percentage of cells in G0/G1, S and G2 phases were calculated by ModFit LT software version 3.0.
Detection of caspase activity
Caspase-3 activity was measured with the Invitrogen Enzcheck caspase-3 assay kit #2 according to the manufacture's protocol. Briefly, 3 × 106 U118 cell were cultured and pitavastatin, irinotecan or the combination was added to the medium for 12 or 24 hours. Then 106 cells were lysed, DEVD-R110 fluorescence substrate was added, and the fluorescence signal was measured and compared with a standard curve (Infinite M200, Tecan). Caspase 3/7 activity was measured by the Apo-One caspase3/7 Kit (#G7790, Promega). 20,000 cells were seeded on to 24 well plates, pitavastatin and vehicle were added, followed by incubation (8, 12, 24 and 48 hours) and caspase 3/7 activity was measured using a fluorescence-based substrate.
Detection of autophagy markers-GFPLC3 punctuation
Retrovirus carrying the GFPLC3 was produced by transfecting the 293GP2 cells with the pVSV-G and pBABE-puro-GFPLC3 plasmids (GFP fused with LC3 gene at the N-terminal, Addgene #22405). Retroviral supernatants were harvested 48 hours later. U87, U118, U251 cells were seeded at a density of 2 × 105 in 6 well plates and infected 24 hr later with the VSV-G/GFPLC3 virus. Stable cell lines were selected for 1 week in 1 μg/ml puromycin. GFPLC3-expressing lines were seeded onto 24 well plates and treated with 1 μM pitavastatin for 48 hours. Presence of GFPLC3 punctuation, which is a marker of autophagy was detected by UV microscopy (Olympus IX81).
Western blot analysis for autophagy, apoptosis, and multidrug resistance protein
LC3 (autophagy), caspase-3 (apoptosis), and MDR-1 (multidrug resistance protein 1 or P-glycoprotein 1,ABCB1) and tubulin (as loading control) were detected by western blotting following drug treatment. Cell lysates (10-50 μg protein) were loaded on to either 14% SDS-PAGE gel (for LC3) or 4-12% gel (for caspase-3, and MDR-1), proteins transferred to PVDF membrane and probed with primary antibodies (L8918 Sigma, 9662 Cell signaling, Sc-8313 Santa Cruz, T4026 Sigma). The resultant protein bands were visualized by a supersignal kit (#1856136,Thermo Scientific) after incubation with HRP-labeled secondary antibodies.
Multi-drug resistance assay
A cell-based fluorescence assay kit (Cayman Chemical Company, MI, USA, #600370) was used to evaluate modulation of the MDR-1 protein by drugs. Calcein AM is a hydrophobic non-fluorescent dye that easily permeates living cells. The hydrolysis of Calcein AM by intracellular esterases produces calcein, a hydrophilic strongly fluorescent compound which is retained in the cell cytoplasm and can be measured using excitation and emission wavelengths at 485 nm and 535 nm, respectively. Calcein AM is a substrate of MDR-1 protein P-gp, which causes its rapid extrusion from the plasma membrane, preventing accumulation of the fluorescent calcein inside the cytoplasm. Therefore measurement of fluorescent calcein allows for detection of MDR activity in live cells. Hoechst Dye staining of nuclei measured using of excitation and emission wavelengths 355 nm and 465 nm respectively to normalize cell numbers in well. GBM cells were seeded at 5 × 104/well overnight, then pitavastatin was added to final concentration of 1, 3 and 10 μM. Twenty four hours after treatment, cells were incubated for Calcein AM/Hoechst Dye solution for 15 min, then fluorescent Calcein retention was measured 20 μM Verapamil or cyclosporine A treatments for 20-30 min as positive control of MDR-1 inhibition followed as the manufacturer's protocol. The results were expressed as ratio of Calcein AM/ Hoechst signal (C/H ratio). Photomicrographs were taken using fluorescence microscopy.
GBM patients' survival and free disease status relative to MDR-1 (ABCB1) expression
The GBM patient data were obtained from The Cancer Genome Atlas (TCGA) public data portal, and analyzed using the cBio Cancer Genomics Portal. This system is developed and maintained by the computational biology center of Memorial Sloan-Kettering Cancer Center. We investigated and regrouped GBM patients according their MDR-1 (ABCB1) expression. Firstly, we required the patients/case ID with the MDR-1 expression in all TCGA GBM provisional databases. The mRNA expression z-scores threshold were set as ±1 in our analysis. Then, we regrouped all the patients into 3 groups according to their MDR-1 expression as up-regulated, normal, and down-regulated. Finally, we inputted the down-regulated or up-regulated patients ID with normal expressed patients to select patient/case set to analyze patient survival and free disease status data. The Kaplan-Meier curves were drawn based on these analyses.
Animal studies
The in vivo studies were performed on nude mice to evaluate the drug effects on inhibition of tumor growth. 2 × 106 U87 cells were subcutaneously transplanted into the right and left flanks. Initial tumor growth was monitored every 3 days. Drug administration was initiated when the tumors reached a size of 100-120 mm3. Mice were regrouped into 5 groups of 6 mice each, without significant difference in tumor volume before drug treatment. The mice were treated with either PBS as control, low dose of pitavastatin (0.5 mg/kg), low dose of irinotecan (0.5 mg/kg), a combination of pitavastatin (0.5 mg/kg) and irinotecan (0.5 mg/kg), or high dose of irinotecan (5 mg/kg). All drugs were injected i.p. in 200 μl of PBS, once per day, on a 5-days-on, 2-days-off schedule. Tumors size and mice weight were measured 2 times per week. All mice were sacrificed after tumor sizes reach over 1 cm in diameter in the control group. Tumor volumes were calculated as (length*width*width/2). After sacrifice, all tumors were disserted and weighted. The animal protocol was approved by UCSD Institutional Animal Care and Use Committee (IACUC).
Statistical analysis
Activity against GBM cells was assessed by dividing the average number of viable cells (from three replicates per dose) by the average of three controls. At a type I error rate of 0.05, using a one-sided t-test, we calculated 80% power to evaluate whether a decrease in mean percent viable cells was significantly lower than 100%, if the observed mean percentage was 91.4%; we conservatively assumed the standard deviation of the percent viable cells was 15%. For significant difference by t-test (P < 0.05), labeled * at the bar graphs.
To quantify the synergism of drug combinations, the drug combination index (CI) was calculated as described by Chou[28]. ED50, ED75 and ED90 were defined as the drug dose able to inhibit cell growth 50%, 75% and 90%, respectively, for pitavastatin alone, irinotecan alone and mixture of two drugs (various ratios). A CI < 1 indicates synergy between the two drugs.
In vitro screening of drugs
U87 studies
The two serotonergic pathway inhibitors, sertraline and 5-nonyloxytryptamine also inhibited the survival of U87 cells, which agrees with previously published findings using an adherent GBM stem cell assay[11, 12].
A172, LN443 and U118 cells
To further characterize the most potent compounds identified in our initial screen, we re-screened, using the established cell lines A172, LN443, and U118, the 15 compounds that showed the highest potency with U87 cells (Figure 1C). We found that 8 drugs had greater potency than vincristine in all cell lines tested and 12 drugs had lower IC50 values than irinotecan. We selected 8 FDA approved drugs for further investigation using patient-derived GBM stem cell-like cells (D-Actinomycin, Liposome-encapsulated Doxorubicin/Doxil, Epirubicin, Irinotecan, Pitavastatin, Sertraline, Topotecan and Vincristine).
Stem cell-like GBM lines
The topoisomerase 1B (Topo 1) inhibitor topotecan exhibited potency that significantly surpassed the structurally related Topo 1 inhibitor irinotecan. Similarly, two statins exhibited good activity, which is promising as these drugs have low toxicity and owing to their target pathways may enhance the activity of currently used oncologic agents via synergism. The IC50 for pitavastatin was less than 10 μM in most of our cells tested (a range of 1.260 to 55.63 μM). Similarly, the IC50 of sertraline was in the range of 3.1 to 6.6 μM.
Predicted blood brain barrier permeation values of pitavastatin
The ability of pitavastatin to cross the BBB is predicted to be limited as the –log BB was calculated as -0.6499. However, the drug circulates freely in plasma and may enter the enhancing component of tumors via permeation through typically leaky tumor microvessels[25, 32].
Effect of pitavastatin on GBM cells
Considering the effectiveness of statins in our study, specifically pitavastatin in inducing cell death and owing to relatively fewer adverse effects, we decided to explore pitavastatin in detail.
Pitavastatin induces autophagy in GBM cells
We found that pitavastatin-treated GBM cells exhibited characteristics consistent with autophagy rather than apoptosis[20, 21]. After pitavastatin treatment, GBM cells showed extensive vacuolization, a key feature of cellular macroautophagy[33, 34]. Further, pitavastatin-treated cells stably expressing the GFP-LC3 fusion protein developed a punctate cytoplasmic pattern, suggesting that GFP-LC3 covalently linked to phosphatidylethanolamine (PE) and was inserted into double membrane autophagosomes (Figure 3C).
Morphological observations were confirmed by Western-blot analysis of LC3, which revealed a LC3-I to LC3-II transition, a hallmark of autophagy (Figure 3D)[34, 35]. The adherent versus sphere culture conditions did not influence the LC3 transition, which was observed in both U87, U251 adherent stable lines and in the stem cell-like SK72 cell spheres upon pitavastatin treatment. Furthermore, increasing concentrations of pitavastatin enhanced LC3-I to II transition (Figure 3D, right panel). In addition, Annexin staining failed to detect apoptosis after pitavastatin (data not shown) treatment. Caspase-3/7 activity was not detectable via fluorescence or by Western-blot analysis (Figure 3E). We could not entirely exclude the possibility that pitavastatin induced cell apoptosis by caspase independent pathways; however the cell cycle analysis shown in Figure 3B argued against this hypothesis, as it did not reveal a sub-G1 population, characteristic of apoptotic cells. The mechanism(s) of cell death induced by pitavastatin still needs more detailed investigation. Further, whether other statins can also trigger autophagy in human GBM cells remains to be determined, and this may depend, in part, on whether adherent cells or neurosphere cultures are assayed.
To elucidate the possible mechanisms by which pitavastatin decreases cell survival, we also used a virtual tumor cell technology. This is an in silico analysis using a comprehensive and dynamic representation of signaling and metabolic pathways underlying tumor physiology (based on CellWorks Inc. technology, Additional file2). Using this platform, we tested the effect of pitavastatin on two GBM cell lines (A172 and U251) using genomic profiles. In silico modeling data predicted a significantly increase in autophagy makers in both GBM cells following pitavastatin treatment (Additional file3).
Drug combinations
Enhancement of irinotecan via suppression of MDR-1 by pitavastatin
Irinotecan induces apoptosis, which is primarily responsible for its anti-tumor activity[36]. Although pitavastatin as a single agent did not induce apoptosis, in combination with irinotecan, it enhanced U87 caspase-3 activity as compared to irinotecan alone, both at 12 and 24 hours (Figure 4C). The major mechanism of drug resistance in GBM is the over-expression of the multi-drug resistance protein (MDR-1; p-glycoprotein 1 or ABCB1), seen in the BBB and neuroepithelial tumors such as GBM[37]. Multiple studies have established that MDR-1 is responsible for decreased drug accumulation in multidrug-resistant GBM cells[38, 39]. Interestingly, pitavastatin is a substrate of MDR-1[40]. We observed that MDR-1 gene transcription levels correlated directly with irinotecan concentration (Figure 4D). However, after combined pitavastatin and irinotecan treatment, the 140 KD MDR-1 band increased in intensity, suggesting MDR- glycosylation is suppressed, which attenuates the production of functional MDR-1 (Figure 4E)[41].
Pitavastatin inhibited MDR-1 function
As shown in Figure 4D and E, pitavastatin induced MDR-1 mRNA and decreased glycosylation of MDR-1 protein. To elucidate the effect of pitavastatin on MDR-1 function, we evaluated the drug exclusion capability directly, using the Calcein-AM assay. As showed in Figure 4F, after statin treatment, both U87 and SK72 GBM cells showed increased intracellular amounts of the MDR-1 substrate (Calcein AM), indicating that pitavastatin may inhibit drug-exclusion mediated by MDR-1. The MDR-1 inhibition was directly proportional to pitavastatin concentration. This result suggests that the increased caspase activity, observed in cells treated with irinotecan in combination with pitavastatin, may be due to its MDR-1 inhibitory effects, which in turn caused accumulation of irinotecan (Figure 4C).
Down-regulation of MDR-1 (ABCB1 gene) expression correlates with overall survival and longer disease-free status
Combination of Pitavastatin and Irinotecan enhances anti-tumor efficacy in vivo
In the present study, we sought to screen a library of FDA-approved compounds to rapidly identify new, non-GBM drugs that could be readily introduced into GBM clinical trials. Using a platform that employed a wide range of human GBM lines, including clinically relevant patient-derived primary GBM lines, our screening uncovered 22 compounds from different classes with anti-neoplastic activity in GBM. Among others, the cardiovascular drugs statins showed high efficacy in reducing tumor growth both in vitro and in vivo, drawing our attention to these relatively non-toxic cholesterol lowering drugs. The present study demonstrates the potency of pitavastatin relative to other statins. Importantly, our results demonstrated that co-administration of pitavastatin with low-dose chemotherapy, greatly increased the potency of the latter, lowering the IC50 values for irinotecan by 40- to 70-fold, with few adverse effects. Experimentally, we found that statins independently induced autophagy in GBM and that statins may potentiate chemotherapeutic agents by inhibiting MDR-1 function. This was consistent with in silico screening results using our virtual tumor cell technology, which suggested that pitavastatin affects cell viability by inducing autophagy.
Cholesterol has a key role in cell membranes, cell metabolism, cell signaling and has been implicated in tumor development and progression. Therefore, as cholesterol-lowering agents, questions about the anti-tumor effects of statins have been already posed[42, 43]. Statins decrease cholesterol levels by inhibiting the enzyme HMG-CoA reductase in the liver. In addition, mevalonate, and isoprenoid intermediates such as geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) in the cholesterol synthesis pathway are also depleted after statin treatment[44]. Another intermediate, dolichol, an essential substrate for protein N-glycosylation, is also blocked by statins[45, 46]. Considering that GBMs are highly proliferative taking up large quantities of cholesterol, potentially they may be vulnerable to statin treatment[47, 48]. However, the mechanism of sensitivity of GBM to statins has not been elucidated. Recent studies have shown that statins may have an anti-GBM effect in xenograft mouse models, by targeting the low-density lipoprotein receptor (LDLR), inducing apoptosis via ERK/AKT pathway[20, 47]. Other data hypothesize that statins may inhibit tumor growth by inducing autophagy via the NF-κB pathway in human colon cancer cell line[49]. Our data obtained in both stable cell lines and primary patient samples clearly demonstrated that pitavastatin induced macro-autophagy in GBM cells[20, 21]. Further experiments are now ongoing to investigate the signaling pathway(s) involved in this effect.
Importantly, we have shown that pitavastatin potentiated the anti-tumor effects of low-dose irinotecan, a topoisomerase inhibitor. Pitavastatin is know to be a substrate of the multi-drug resistance protein, MDR-1, which is overexpressed in GBM upon drug treatment and is partly responsible for the resistance of GBM to chemotherapy. Our data indicate that, in combination with irinotecan, pitavastatin suppressed glycosylation of MDR-1, thereby inhibiting its function and allowing irinotecan to accumulate intracellularly[37, 39, 40]. Accumulation of irinotecan is likely responsible for the increased apoptosis in the presence of pitavastatin. The MDR-1 expression in cancer cells can be a significant obstacle to the success of chemotherapy. Many MDR-1 inhibitors have been extensively tested in clinical trials but the results have been inconclusive. According to TCGA data, down-regulated ABCB1 (MDR-1) predicted better survival of GBM patients. Combining a statin with a chemotherapeutic agent represents a powerful, potential strategy for circumventing resistance and significantly enhancing efficacy. Here we have confirmed that pitavastatin may improve the therapeutic response to TOPO-1 inhibitors, by inhibiting MDR-1 function, and may be beneficial for GBM patients. It remains to be determined whether other statins exert a similar or a different anti-neoplastic mechanism as compared to pitavastatin, and whether different subtypes of GBM have different sensitivity to pitavastatin or display other mechanisms for statin actions. GBM is a complex and heterogeneous disease that likely accounts for the different results obtained across various studies.
Irinotecan is broadly used in solid cancer therapy, especially in combination with other drugs[50, 51]. In clinical use, the toxicity of irinotecan is generally manageable and reversible[52, 53]. However, in some patients it may lead to severe side effects, such as diarrhea and neutropenia that can be life threatening. In our animal model, co-administration of pitavastatin allowed for a reduced dosage of irinotecan and avoided drug toxicity at higher dosage. These data indicate a new strategy to develop better irinotecan-based drug combination.
Based on the promising results of our present study, we are now undertaking additional preclinical studies of GBM to optimize dosing and characterize efficacy, thus providing a solid basis for a clinical trial with pitavastatin and irinotecan for the treatment of glioblastoma patients.
The authors wish to thank Gabi Gomez for technical assistance in the laboratory.
Financial support
Grants from NIH (NIH 3P30CA023100-25S8), National Brain Tumor Society David Cook Chair of Research and from Barbara and Joseph Ajello trust fund, Tuttleman Family Foundation and Boston Fire Department/Kenney Foundation to S. Kesari.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.


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Scrap Car Hacklinge, Scrap your car for free
Scrap Car Hacklinge are your Hacklinge, Kent based free scrap car collection service. Scrap cars become a problem if you keep them for too long as you need to keep them taxed if they are on the road meaning you will be spending money on your scrap car. You don't have to pay to have your car collected. We will collect your old scrap car at no cost to you at all in Hacklinge.
Recent News in Hacklinge
As well as offering a completely free collection, we are also currently able to offer cash for scrap cars in Hacklinge depending upon the weight of the vehicle!
If you'd like us to make you an offer just ask when calling or add 'CASH QUOTE PLEASE' in the 'Additional Info' box when submitting our online form.
- DVLA notification
- Convenient collection times
- Completely free collection!
- Nationwide coverage
- Environment agency approved recycling facilities
Just ring 0800 043 5100 or simply fill out the form and we will be there!
Choose Scrap Car Hacklinge
We have built a reputation to be proud of after trading in the Kent area for over a decade. Scrap Car Hacklinge are very familiar with the local area and will be with you in no time at all. We will collect your car at the agreed time slot, you can rest assured. We can collect cars any time, day or night, seven days a week, and so work around your commitments.
We make sure that your car is disposed of the correct way. Unscrupulous companies may sell on your car rather than scrapping it, often without informing the DVLA of any change of ownership. This will leave you very vulnerable in the eyes of the law, and can easily result in you being fined a large sum of money. When we collect your old scrap car we always ensure that the DVLA are aware that the vehicle is in fact being scrapped, there's one less thing for you to worry about. Here at Scrap Car Hacklinge we make sure that we recycle everything, so as you can tell we always do our bit for the environment.
Ensuring that your car is disposed of correctly will in time make a difference to Kent in terms of keeping it clean and tidy. Having old broken down cars littering the streets makes the area look very run down and untidy, so scrapping your car will make your neighbours happy. Scrap car collection, call the team!
Reasons to scrap your car
There will be a lot of reasons for you to scrap your car. In our experience the car might have been written off and it is now down to you to get the car scrapped. If you have a car that is unusable you need to either declare it off road or scrap it, otherwise your will continue to need to pay road tax on the vehicle.
How to scrap your car
It couldn't be easier to scrap your car in Hacklinge. We have many lines into our offices and always answer the phone so call today. On the other hand just take a look at our very helpful guide to scrapping your car so you can see what the procedure is before you call or book.
The car scrappage scheme
The car scrappage scheme may seem like a splendid idea at first. However, you are in fact limited to what type of new car you can get. It is not possible to go out and buy any car you wish. This can also be a very frustrating issue for the people looking to get a new car as they have to be dictated to as to what car they can buy.
Just call us on 0800 043 5100 or fill out the following form:

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Latest Canon Offering Praised For Printer Ink Efficiency
Canon's newly-launched Pixma MX870 offers small business and home users the chance to cut back on their printer ink overheads, it has been claimed.
Reviewing the latest offering from the Japanese technology firm, PC Advisor praised the device's sleek looks, including its easy-to-use 2.5-inch colour LCD screen.
In addition, the website noted that, by printing at a rate of 6.2 pages per minute, the Pixma MX870 is more than fast enough for home or small-office users.
However, it is the praise reserved for the machine's printer ink cartridge efficiency which is likely to be of most interest to bosses looking to cut back on wastage and overhead costs.
By Canzil Ahmed


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How penang beaches can Save You Time, Stress, and Money.
Its intresting to read through as a result of many of the remarks and memories on Penang in this site! Effectively...i'm happy to convey I am a Penangite, a hundred% pure blood! ;) Born below and been remaining listed here all my lifestyle! And adore each individual minute of it.
I had been in Penang from 1975 to 1977, Dad was centered being a RAAF policeman While using the RAAF around the island, in addition to about the mainland occasionally. We lived at 80R Mt. Erskine Highway, throughout the highway from the substantial cemetery. We experienced somewhat pet monkey named Fred in addition to a German Shepherd named Larz. The hostie was about 10 mins walk absent from your home, lots of sports activities and functions to get entangled in. I attended the RAAF university there, a number of the greatest years of my life.
It had been a Frightening wanting spot, the fierce seeking dogs barked their heads off should you went close to and for many cause I assumed the spot was Just about mystical. I'm older and braver now, so the images my new wallpaper!!!
A short while ago revisited Penang with the RAAF Faculty reunion in Sept. What a marvellous time we had! My wife and two sons all want to return, and soon. Obviously the put has changed , targeted traffic and the other issues that come with the 21st century, But much on the character of instances past remains there. If you are a RAAF child check out RAAFSCHOOLPENANG.COM you are going to like it.Jumpe Lagi!
Ahh! the Reminiscences-----fishing off the rocks with a coke bottle being a reel-catching bloodworms and pippys to the Seashore-swimming every day-films and hot chips with tomato sauce with the Aust Hostel-a wonderful Amah, Chen,cooking curries, washing and cleansing anything in web page-makan carts with 25c foods and their late night cries-tambarans and durians-kites and spinning tops-bus rides to city-trishaws and Chilly Storage-cokes and haicuts in the Garrison Club-catching battling fish and chasing mud skippers with indian and chinese pals-swimming and diving at minden barracks pool-shoplifting from the naaffi-offering cheek to -pomme and aussie troopers (never to Significantly older than us )-movies and bafs education at minden barracks-prepare outings to KL and also the experiences of co-ed Bourne Grammar and great english ladies (You will find a e book there!
I lived in Penang 1968-1970 and once again 1973-1974. Went to your RAAF University website at Hillside to be a 12 year previous and have now produced an internet site on that college. I am looking for info for your new section around the Radio RAAF Butterworth, so if there is anyone who would like to add, remember to Call me. We want Publications for scanning, anecdotes from people who worked on the station, pictures, any historical or page factual info, stats etc.
To start with for Julian Derham - I lived in fourteen, Road four Vale of Tempe. From March 1967 right until March 1969. I suppose we must are up coming door neighbours. You wouldn't figure out the position now. All of the gardens are packed with luxuriant expansion, my area was just like a dust bowl (apart from when it rained) for the main two yrs. Most if not all the houses in the area have been "enhanced" not generally for the greater. In truth No 14 was Pretty much derelict, I am informed that the proprietor ran out of money. I generally don't forget the homes as awesome and airy. I had been the 1st occupant of my house. I think You may have been first in yours.
?? I'd love to know more about the great enjoy of my dad's existence, and would be interested to hear from anyone who can have information on the realm or the identify.
Very well what a sight ,with all These structures everywhere you go.I am unable to consider that it's the similar place that we lived in- Tanjong Bungah. we allso lived in Tanjong Gajah a number of years prior to. It positive was an fascinating the perfect time to live there. Roger and Ainsley
By using a historical past relationship again to 1800, it's the oldest neighborhood council in Malaysia plus the successor to your state's first city council - the George Town Metropolis Council.
Penang is a type of special islands that has pretty much every little thing at a moderate scale: beaches, lush forests for trekking, buying complexes for your here shopaholic, night spots plus position alternatives! Its an excellent island to reside in! What else am i able to say but....I like PENANG!! :) Neil Diack
I have some interesting (to me) slides of the temple which I think was at Alor Star - how memory fails us when we need it. If any individual would like some shots taken of precise sites, then I will probably be only far too pleased to try, and will deliver them by e-mail After i get again. Just allow me to know. I am still looking for members of Butterworth Basketball Workforce from 1968 once we went right down to Singapore with the FEAF Champs. Fall me a line.
Eighty Japanese fighters and bombers had flown more than Georgetown unopposed... A large number of people today had loaded the streets to look at the spectacle, which turned to tragedy once the bombs began to fall.


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Deep Wrinkle Remedy Serum
If you're serious about wanting to see a dramatic change in the appearance of your fine lines and wrinkles, Deep Wrinkle Remedy has you covered.
This breakthrough serum is the most potent anti-wrinkle formula available on the market today. Based on the recent DMAE research and it's many benefits, Deep Wrinkle Remedy utilizes our new hyaluronic acid technology to greatly boost the performance for long lasting, powerful effects.


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Integrated academic and social-emotional learning
NCS engages diverse and complex young people as whole human beings. Through socially- and academically-integrated instruction that is personalized and active, and through a challenging and arts-infused curricula, we develop knowledgeable and skillful leaders. New City students, becoming culturally sophisticated collaborators and problem-solvers, also become agents of change and citizens of what we hope to become a New, more just and sustainable, City. Academic and social-emotional learning go hand in hand. Integrated intentionally, they support each other, engaging students in inquiry and productivity gradually growing their intrinsic motivations. The New City approach targets academic gains while encouraging human development in all its variety.
Program Goals
- Teach an ethnic and socioeconomic mix of students in an urban setting.
- Focus on a core curriculum that students will address critically and with depth.
- Utilize instruction which is differentiated and challenging, active, arts-infused, interdisciplinary, and inquiry-based
- Model applied social and academic skills in collaboration with the larger community
- Launch students into the world as confident, competent, and socially responsible citizens.
- Share best practices with the education community by hosting workshops and trainings on the integration of social and academic learning, for pre and in-service educators.
Our Beliefs in Children
We believe that children want to be good and do their very best at school. To help children succeed, we must teach them how to behave at school and handle the variety of situations a school day presents. When working with children, we do not assume they know how to behave in every situation. However, we are committed to teaching children caring ways to treat themselves, the people around them and the materials they use. We know children need many opportunities to practice both academic and social skills. We see children as curious problem solvers. They want to actively engage with their world and be included in solving problems that involve them. Children want to put their hands on materials and their engagement is higher when they have the power to make choices about their learning. To learn deeply, children must learn to construct their knowledge. They need time and space to explore a concept and to build meaning from their experiences. We create lessons in which children connect prior knowledge to a concept or skill, gather new information, have an opportunity to meld their past experiences with this new information and then reflect on their learning. Children are social beings that learn best with others. To achieve and grow, children must feel safe. They need to feel a sense of significance and belonging in their learning community. Children want to experience joy in all that they do.


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My name is Muska, my pedigree name is Misty Heather Jacawaca but my friends call me Muska. I live in a house with my housemate, I enjoyed being an only cat because I had the whole house and garden to myself and I was always in charge. That has changed now because a young upstart has moved in, I am still the boss but I have to watch out for him all the time. He has only been here a week so I have not taught him the rules yet.
This was me when I was only a few weeks old, cute, I hear you say. I was three months old when I moved in with my housemate.
I did not go outside until after I had been neutered so when I first went in the garden it was snowing. I loved the snow, I enjoyed making footprints and trying to catch snowflakes. Each time I caught one it disappeared a great trick that I have nearly mastered myself, the only thing that lets me down is that when I disappear behind a chair or a door my tail gives me away.
Since I am a Maine Coon my tail is as long as my body, I am very proud of my tail.
I hope you enjoy my diary and musings on my pages,


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Great Pathfinder SUV, manufactured by Japanese automaker Nissan, goes to the next generation. Although it uses the same platform, the crossover will be totally different. New features and technological gadgets are ready. However, 2018 Nissan Pathfinder will retain its familiar look. The versatile crossover is more daring, with updates from the outside and inside. In addition, changes are expected in the engine that was used by the latest model. However, the powertrain is set, and not new. The balance of power, price and strong appearance is the main advantage of this SUV.
Exterior and Interior
This is probably the area where the new 2018 Nissan Pathfinder received the most attention. When you look at the new car of your profile, it still looks identical to the old model. However, now it has a new front-end. The V-movement design is a little more subtle. The grill is as big as before, but it has a new mesh in honeycomb. In addition, the headlights are much smaller than before, while the sticker features a new design with V-shaped accents as well. The result is a sports car look that is better suited to the current list of Nissan cars. Thanks to the modifications made to the front, the new Pathfinder is also more aerodynamic than before.
Most of the changes inside the 2018 Nissan Pathfinder cabin was made to groups of automotive equipment. Even the basic model now receives a new color of the 8-inch touch screen control the infotainment system. Options include adaptive cruise control, front collision warning, automatic emergency braking and even detecting moving objects. Fortunately, Nissan decided not to stop all physical buttons. This makes driving and adjusting climate controls much easier and less distracting.
We heard that 2018 Nissan Pathfinder will use the same transmission as its predecessor. Under the lap cover should be 3.5 – V6 powertrain. The power has been upgraded to 260 hp. Then, the updated transmission is adapted to the motor. A two-speed CVT gearbox is a modern system that reduces fuel consumption. At the same time, improving performance. On the other hand, the new Pathfinder V8 could use the device. However, moving 3.8 liters is less likely. This engine is far from the requirements of fuel economy, since the new crossover will be rated 25 mpg. The hybrid energy source is also possible, but no confirmation about it. However, we can expect.
Release and Price
All of these elements 2018 Nissan Pathfinder make a better overall package at the same price as before, which is always a win for the customer. The release date for the new car will start in September 2017 and its basic price but not yet announced it is likely to be just under $ 31,000, with higher specification cuts for just over $ 52,000.
You are viewing 2016 nissan pathfinder for 2018 update news, picture size 730x485 posted by Admin at February 24, 2017. Don't forget to browse another wallpaper in the related category or you can browse our other interesting wallpaper that we have. Please also read our Privacy Policy and DCMA for the copyright of the images.Back to 2018 Nissan Pathfinder Exterior and Interior


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Rise to Riches
Many of the Arabian Nights stories tell of men who rise from poverty to wealth and prosperity. This is evident through Aladdin of "Aladdin's Lamp," Ali Baba of "Ali Baba and the Forty Thieves," and Sinbad in "The Seven Voyages of Sinbad the Sailor," among others. In each of these stories, the protagonist begins with very little to show for himself, but manages to rise in society both through luck and perseverance. In fact, many of the characters experience good fortune, but must capitalize on that good fortune in order to succeed. Many characters fall back into poor luck after initially securing a fortune, but manage to recapture it through such perseverance. The idea of a common man rising in status was not only a popular concept during the Islamic Golden Age; the idea that we can overcome our societal standings and attain wealth is popular even today and will most likely remain popular long into the future.
Luck and Good Fortune
Usually, a character finds success in these stories through some rare stroke of good luck. Ali Baba just happened to be in the vicinity of the robbers when they opened the cave, so he knew how to access it and take treasure. The vizier Ja'far in "The Three Apples" happened to find the slave who was responsible, therefore avoiding his own execution. In these stories, the power of luck and fate is explored very often, because people everywhere love to believe that good fortune can befall anyone. And yet most of the stories add one twist to the theme - in most of these cases, the characters have to capitalize on the luck in order to succeed. For instance, Aladdin falls into good luck several times, but has to use his cleverness and perseverance to eventually come out on top. Luck (good or bad) affects us all, but the most successful of us know take advantage of whatever befalls us.
A common theme of adventure and daring is present throughout nearly all of these stories. In "The Seven Voyages of Sinbad the Sailor," Sinbad has a thirst for adventure on the open sea that cannot be quelled with merely a voyage or two; he goes out trading on a ship seven times before he finally decides to quit seeking excitement. Other major characters regularly face obstacles that test their wits, strength, and agility, all elements of adventure tales. Adventure stories such as these will always be appealing to readers, since we often like to imagine our lives as more exciting than they are. What is remarkable about The Arabian Nights is that the stories comment so poignantly on universal human nature even in the midst of such extraordinary adventures.
In a collection of stories that focuses so strongly on wealth and riches, greed naturally arises as a human vice that characters must counter in themselves and others. The antagonists in these tales are almost always evil, and their danger is usually attributed to their greed. Consider the magician in Aladdin, the forty thieves, or King Yunan's vizier - all characters who cause trouble in order to satiate their own greed. However, the stories are arguably most profound when the protagonists must counter their own greed. In some cases, like with Sinbad, their victory over greed secures their survival. In others, like with Ali Baba, the protagonists end up happy despite the vice. The main difference between the protagonists and antagonists overall that the protagonists seek wealth in order to better their lives; the antagonists continuously want more even when they have enough, and refuse to share. Overall, The Arabian Nights values the pursuit of wealth, but lays down its fair share of warnings along the way.
In many ancient and classical cultures, hospitality was considered sacred. Though this collection does not make many explicit comments on hospitality, it is nevertheless a recurrent and important theme. In particular, hospitality in these stories suggests an individual's connection to his greater community. For instance, Sinbad the Sailor feeds Sinbad the porter every single night as he tells his story, and gives him a large sum of money as he leaves. Further, his stories have an implicitly didactic purpose - he wants to teach the impoverished porter what he has learned about life. Similarly, Sinbad received wonderful hospitality from the many kings he encountered during his journeys.
The collection does not always present positive instances of hospitality, however. It can certainly be exploited, usually to the detriment of the exploiter. In "Ali Baba and the Forty Thieves," the thief captain takes advantage of Ali Baba's hospitality, and ultimately dies in the attempt. Without making an explicit comment on the theme, The Arabian Nights reflects its culture by presenting hospitality as sacred.
Contests and Competition
The protagonists of many of these stories take competition very seriously; the most prominent example is in "The Three Princes and the Princess Nouronnihar." All three princes universally accept the idea of competing for the princess's hand in marriage, despite their family connection. There are also instances of less healthy competition, as between Ali Baba and Cassim. (Often in these stories, poor men see themselves as in competition with richer men.) In most cases, however, a competitive spirits helps men to discover their inner strength and thereby prosper. Competition was considered one of the best ways for a man to prove his honor and strength, and this idea is very much reflected in the tales of The Arabian Nights.
The Power of Storytelling
Storytelling is arguably the collection's most poignant and lasting theme. In the frame story of The Arabian Nights, Scheherazade believes that the power and intrigue of her stories will be able to keep her husband from killing her or any more of his wives. This sentiment - that stories are not only entertaining but powerful, capable of changing people - is reflected throughout the many stories she tells. One can read the collection's stories as various attempts to convince Shahrayar to change his ways, and can see in the multiple framings the importance of constructing various narratives to help us understand ourselves.
For instance, Sinbad believes that his stories will help lead the impoverished porter to a better understanding of the world. Further, stories are very often used as a means of persuasion, such as in "The Three Apples" and "The Fisherman and the Jinni." The works in this collection emphasize the power behind words, and reflect how important storytelling was in Islamic society during this time period. Without them, hope would have been lost in a number of different cases.


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Download my Virtual Business
The Greater Clear Lake area offers a great quality
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by Claridata


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I've been wondering what to have for Christmas dinner. I don't want to eat too much flour and gluten, which is normal pretty predominant in fake meats. I want something nutritious and delicious with a mouthful of punch. This is it.
I remembered going to my brothers restaurant where he made me a pizza that was topped with walnuts. It gave the pizza and almost creamy feel. Walnuts are a great addition to savoury dishes. Walnuts and scouring the backyard for a bay leaves and rosemary. This dish is sorted.
Serves: About 10.
Time: 15 Minutes prep, 1 hour and 45 minutes total cooking time.
- 1 and a half cups of brown lentils.
- Half a cup of yellow split peas.
- One bay leaf.
- 2 Tablespoons of olive oil.
- 1 diced onion.
- One stick of celery, diced.
- One carrot, diced.
- 2 cloves of garlic, minced.
- 1 cup of chopped walnuts.
- Quarter of a cup of tomato paste plus 2 Tablespoons.
- 1 Tablespoon of chopped rosemary.
- 1 teaspoon chopped sage.
- 2 teaspoons of chopped dry basil.
- A few shakes of pepper.
- A good pinch of salt.
Place the lentils and the split peas in a saucepan and cover with water. Add the bay leaf and bring to the boil. Reduce the heat and simmer for 30 minutes. Drain. Keep the bay leaf.
While that is happening, you have time to prep the other ingredients.
Then, heat the olive oil in a separate pan over medium heat. Add the garlic, onion, celery and carrot and sauté for about 3 minutes.
Add the walnuts, tomato paste and herbs and mix together for a further 2 minutes.
Mix in the drained lentils and split peas. Add the salt and pepper. Taste it. Does it need more salt and pepper? Add to taste.
Pull out a loaf tin and grease well with olive oil. This will allow the loaf to slip out easily.
Stuff the mixture into the loaf tin. Once its all in, spread with the 2 Tablespoons of tomato paste. Place the bay leaf on top. It should look like the picture above.
Bake at 180C or 360F for 1 hour.
Place a dish over the top of the loaf tin. Being very careful because it's quite hot, turn the loaf tin with the dish attached so that the dish is now on the bottom. The loaf should slide right out.
Serve with mash potato.


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Yakushima Island
Enjoy the unspoiled richness of nature on a mysterious island where time stands still and giant prehistoric cedar trees that are thousands of years old reign supreme.
Yakushima is a round-shaped island situated approximately 60km south from the southern end of Osumi Peninsula in the southern part of Kagoshima prefecture. One fifth of the island is registered as a Natural Heritage Site. There are as many as six mountain peaks over 1,800 km high including Miyanoura-dake (1,935m above sea level), the highest mountain in Kyushu, and this is what gives the island its other name - the 'Alps on the ocean'.
The bedrock of the island is granite, and it is said that a ridge thrown up by movement in the earth's crust is what formed the island. The influence of the climate and air masses is very complex, and the main feature of this area is that temperature and seasonal changes are the largest on earth. It is possible to see plants from both subtropical zones and cold temperate zones on the island.
It rains so much in this area that it is said that it 'rains 35 days of the month', and we can see many rare animals and plants due to the abundance of water and the changes in temperature at different altitudes. The range of flora is very wide, from coral reefs to alpine plants, and among the animals, there are those that display traits unique to Yakushima such as the Yaku monkey and Yaku deer.
The term 'Yaku sugi (Japanese cedar)', the symbol of Yakushima, is used only for cedar trees over 1,000 years old. Yaku sugi growing in the area covered by clouds and mist sometimes extend roots even from the leaves and stalks and form themselves into strange shapes. A cedar discovered in 1996 and situated at an altitude of 1350m is assumed to be the largest and the oldest in the world, and is called 'Jomon sugi' (there are various theories as to its age, from 2,170 to 7,200 years old).
Apart from this, there are many other good sightseeing spots such as 'Shiratani Unsuikyo' where you can take a walk in the forest of Yaku sugi; 'Hirauchi Kaichu Onsen' where you can bathe in a spa right next to the sea; 'Ooko-no-taki' as the tallest waterfall with the largest amount of water in Yakushima and selected as one of the best 100 waterfalls of Japan; 'Senpiro-no-taki' where the water falls approx. 66m on to a gigantic granite monolith; 'Yakushima lighthouse' where you can see the deep blue ocean spreading out to the horizon and a beautiful sandy beach 'Sango-no-hama (coral shore)' that is known as a spot where you can collect star-shaped sand. You can also experience canoeing or forest bathing or take a tour of the whole island with a veteran guide.
Access: From Kagoshima City - approx. 2hrs 35mins to either Abou Port or Miyanoura Port by jet foil from the north pier of Kagoshima Honko, or approx. 4 hrs to Miyanoura Port by ferry. Or, approx. 40 mins to Yakushima Airport from Kagoshima Airport.
Further information: JNTO Travel Tips – Yakushima Island (PDF)


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Freelance writers wanted — paying job
Don't you get tired of reading the same over-published, randomly copied Craigslist jobs for freelance writers? These gigs are mostly trash, don't pay, and some are even out-right scams. However, there are work-at-home, telecommute jobs for writers — jobs that pay decent money. Here's one of my best finds.
A site called Cheapism hires freelancers to write short reviews of low-price, high-value consumer goods. Here's their ad:
Are you a thorough researcher, fluid writer, and cautious spender? If the answer to all three is yes, Cheapism.com would like to meet you. We are a new website that aims to be consumers' first stop on the way to finding the best inexpensive goods and services.
We are looking for freelance writers who will learn about a product category, identify the low-priced products, assess what the experts and users say about them, and write an article that tells consumers what they need to know and recommends the best budget buys.
In this time of economic stress, you can help put some lining back into consumers' pockets and cash into your wallet; last but not least, you'll have the good fortune of working with a collegial and easy-going crowd.
Contact us today at email@example.com
I went through the application process with these folks a while back. They are serious about their business and looking for long-term relationships with good writers who can produce quickly.
Cheapism pays adequately. You will be asked to submit samples and suggestions. You will undergo an in-depth telephone interview and the company principals will take a few days to decide if you're a good match. Feel free to ask questions.
Let us know how you fared with them and what you thought of the hiring process and the job.
More to read:
How to stop bitching and sell your writing


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Monday, February 13, 2012
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Wednesday, February 15, 2012
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Tuesday, February 14, 2012
Seeker's Rail Boss line
Seeker is making some big, progressive, refreshing moves, from its new lines of rods in both the fresh and salt, to adding members to its pro staff and creating a junior pro staff.
The rail rod phenomenon has taken over the long range scene, and it's bleeding more into local fishing. Even in this tough economy, rail rods are the big sellers, as most big fish long range guys are passing on the 6- and 6.5-foot roller guide rods and fishing the rail rods. But rail rods are big ticket, big price tag items, thanks to the cost of components being so high.
Coming up with a lower price point on a beefy, brutally tough rail rod line was part of the thought process of the Rail Boss line from Seeker.
"The design concept for the Rail Boss originated during a conversation
between a few of our team members," says Darin Dohi at Seeker. "The conversation began with the need for more affordable options on rods and reels for the long range fishermen."
At that point it was all about coming up with rods that fit the bill.
"This in turn sparked a debate about materials, actions, construction, how the rod could be configured to minimize the cost to the new long ranger just starting to build their rod quiver," added Dohi. "It was then the decision was made to go back to the company's long range roots and create a rod out of the traditional materials of years past but without sacrificing the action as well as durability."
And the Rail Boss was born.
The shots taken by Paul Sweeney above are from our trip on the
last month. We brought the 100- and 130-pound Rail Bosses out and the guys really liked them, as the heavy duty glass blanks really let them reef on the fish. While most rail rods are 7 footers, the Rail Boss rods are 6.5 feet long. It's been engrained in a lot of long range anglers that a rail rod HAS to be 7 feet--like a jig stick HAS to be 10 feet--but the 6.5-foot Rail Boss gives all the advantages of a rail rod, from the castability to the maneuverability around the props and rub rail and other anglers' lines.
The big things about the Rail Boss rods are that they are brutally strong and are built to hold up against the gnarly abuse rail rods take, from baking in the sun for two weeks to getting drug up and down the rails.
"The rods needed to perform," says Dohi, "but they also needed to last for those fishermen who may only fish long range every few years. The Rail Boss project incorporated all of the most important elements to building a suitable rail fishing rod."
Every aspect of building a rod that would meet the rigors of rail fishing went into Team Seeker's Rail Boss design process.
"The area of the blank where the rod would be levered against the rail has been reinforced," adds Dohi. "The foregrip took a little departure from the norm and only two small sections of EPDM foam was used for the base of the foregrip and the end of the foregrip. The cold shrink tubing is a major key as it allows the fisherman to drag the rod against the rail without destroying their foregrip. The tubing is highly tactile and will grab the rail even when wet. The rod is then finished off with a set of Fuji BHBNG ring guides and matching tip for castability and strength and wrapped with an all black 'custom rod' quality triple wrap detailed with metallic trim bands."
The whole Rail Boss line will be on display at the Fred Hall Show, which is just 23 days away. In the meantime, here is a shot of the first cow taken on a Rail Boss, a 230 that Matt Carleton took down on the
on the 130-pound Rail Boss
After seeing the Rail Boss 130- and 100-pound rods at work,
Captain Keith Denette ordered up a set for his anglers to fish cows with. Talk about speaking volumes...
Posted By Brandon On 02/14/2012 At 9:29 AM
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Applying from overseas: LOS ANGELES HARBOR COLLEGE
Applying from outside of the U.S. to Los Angeles Harbor College
Please be sure to visit the link below and begin your application process by completing the CCCapply application. If you have any questions regarding the application please make sure to contact us immediately and we will do our best to assist you.
Please note that the International Student Application requires submission of original documents and signatures.
We are now welcoming Fall 2018 applications, and the deadline to apply for the Spring semester is July 1st, 2018. Our Spring semester starts February 4th, 2018
Complete and sign the LAHC " International Student Program Application Form".
A passport size photo taken within the last six months.
Copy of your valid passport information page
Submit a completed "LAHC Affidavit of Support" accompanied by appropriate supporting financial documents. (The appropriate financial documents are documents showing that the sponsor has sufficient funds to cover one year of the student's educational and living expenses-- currently $22,800). Types of documents that may be submitted are a bank account or a letter from the sponsor's bank, or If an applicant is sponsoring himself or herself, he or she should write "Self" for the question "Student's Relationship to You." If the student's sponsor is a U.S. citizen, the sponsor will have to complete USCIS form I-134.
Evidence of English language proficiency:
TOEFL scores: PBT - 450 or above;
CPT - 133 or above
iBT - 45 or above
IELTS score: Band 5.0 or above
Step Eiken score: Level 2, pre-1, or 1
iTEP score: 3.5 or above
Other standardized tests may be used.
The test scores may be waived for students transferring from a U.S. college or university or from an international high school where English is the medium of instruction high school. Students in the Los Angeles area who are in an appropriate level in an English language program but do not have a TOEFL or IELTS score may be permitted to take the college's ESL assessment exam. Placement in the college's English 85 level will waive the TOEFL/IELTS requirement.
Official transcripts/certified copy of diploma from your high school/secondary school clearly stating that you graduating
A non-refundable $50.00 USD International Application fee in form of money order payable to LAHC INTERNATIONAL STUDENT PROGRAM
Once we receive all required original documents from you, your admission application will be processed. If all your documents are in order and we accept you as a student, we will provide you with an acceptance letter and your I-20 so you can begin to apply for your F-1 Visa in your own country.
Please note, applicants can submit original supplemental application forms in-person or via mail/courier. Our mailing address is listed at the bottom of the email.
1. Please begin the admission process with LAHC by completing all the required documents, and mail or courier by FED EX or DHL to our office as soon as possible.
2. Please be sure that all documents are original copy.
3. Please be sure to send cash or a money order of $50.00 USD with the documents. If you would like us to courier your acceptance letter and I-20 please be sure to let us know so we can provide you with courier fee information.
4. Once we receive all your required original documents, and required payments, we will proceed to grant you an acceptance letter and your I-20 from LAHC.
5. Once you receive the acceptance letter and I-20 from us, you may proceed to make an appointment with the Consulate/Embassy Office in your own country for your F-1 Student Visa interview.
6. In addition, you will need to complete the SEVIS form and send the payment directly to them, or you may complete the form and pay your fee on line. Follow the link and the instructions on the form.
Note: Please make a copy of everything you submit to us for yourself. You will need to bring a copy of what you submitted to us for your visa interview.
Mail your documents to:
Los Angeles Harbor College
International Student Office
1111 Figueroa Place
Wilmington, CA 90744


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Hello and welcome to MTI! You're probablly confused as to what exactly goes on here. Well, the truth is, it's whatever you make of it: all you have to remember is that the primary goal is to make the world a little less borring and simply a more fun place to live in. For more, click "About MTI" on the sidebar.
Today's find is from China. Someone did some creative littering and made things interesting for a tourist in Changzhou:
Show us how you made things interesting:


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The Coolidge greenhouse was construted as a part of the orginal construction of the school in 1938. Due to lack of maintenance and use, the greenhouse is in desperate need of rehabilitation. The ACE Coolidge Team, along with the school's Alumni association and other key stakeholders, not only plans to restore this historical keepsake, but also to create the first LEED certified school greenhouse in the area.
Renovation Project Plan
Rough Order of Magnitude estimate
Please send any donations and grants correspondence relating to the Coolidge greenhouse project to Frank Jones III (CCAA) at firstname.lastname@example.com or James Proctor (CCAA) at email@example.com
Donate to the Calvin Coolidge Greenhouse Restoration Project
Congratulations to our 2010 ACE Scholarship Awardees!
MICHIAS ZEWDU – Calvin Coolidge H.S., Civil Engineering / Award: DC Government Neighborhood Investment
Micky is the leader of the Men of Strength Club and President of the International Club, he is also co-founder and co-President of the National Society of Black Engineers (NSBE) junior chapter at Coolidge; Captain of the soccer team; Lead member of the S.M.A.R.T program (Student Multi-ethnic Action Research Team); Leader of the GEMS program (Gains Education Mathematics and Science) and a National Honor Society among his many activities
MATHEOS MESFIN – Calvin Coolidge H.S., Engineering / Award: DC Government Neighborhood Investment
Matheos is co-founder and co-President of the National Society of Black Engineers (NSBE) junior chapter at Coolidge; Founder and President of the S.M.A.R.T program (Student Multi-ethnic Action Research Team); Leader of Coolidge's College for Every Student; Member of the Men of Strength Club; and fluent in four languages (French, English, Amharic, Tigrigna). On Coolidge's "Principal's Highest GPA List", he achieved the National Achievement Award; Honor Roll, National Student Award; and is a Posse Scholar to Grinnell College.
Click here to read about all the ACE 2010 Scholarship Winners
ACE Team in the NewsThe ACE Mentor team at Coolidge continued it's second year with more endeavors and fun events. As part of National Mentoring month, ACE DC hosted an Open House at Coolidge in January 2010 with participants from the American Society of Landscape Architecture (ASLA), Takoma Park Association, American Forestry Service, Coolidge Alumni Association, and the Coolidge school administration.
Local DC nonprofit afterschool program adds diversity to engineering field
ACE Open House at Coolidge Rousing Success ! !
On behalf of the ACE Mentor Program in relationship with the Coolidge Senior High School administration, Coolidge Alumni Association, National Society of Black Engineers (NSBE), we would like to thank you for coming out and showing your support of the students at Coolidge SHS. Councilmember Bowser also sent us her wishes for the success of this event.
Click to view slide show of ACE Open House at Coolidge


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Contemporary Curves in a Family Garden
An elegant family garden in Oxfordshire, built around a redesigned deck platform.
The family had few exacting requirements, beyond somewhere to grow herbs, and a desire to use the garden as additional living space. Creating separate zones became something of a design direction when it became clear that the teenage children wanted areas where they could retreat with friends.
The main difficulties on site were an unattractive existing deck platform, which had to be retained to access the conservatory– though it was reshaped and the boards were replaced; and privacy issues from a nearby footpath. The existing garden lacked any sense of cohesion, and none of the areas worked in terms of useful outdoor space.
Curving pleached hedges helped create an instant sense of seclusion and maturity, as did a small grove of birch trees. Bespoke metal troughs filled with water reflect the changing clouds in the sky, adding to the tranquil feel, which is further enhanced with a soft, woodland planting scheme.
A custom designed, hand built timber 'chill-out zone' makes a secluded retreat; and a small terrace at the far end of the garden is the perfect spot to catch the last of the sun.


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By Keith RobinsonSeptember 12, 2013
Purdue's College of Agriculture will receive more than $20 million in university funding for plant sciences research and education to strengthen Purdue's leadership in developing new and novel ways to help feed a rapidly growing world population.
The plant sciences initiative, announced by President Mitch Daniels on Thursday (Sept. 12) during a President's Forum, is among 10 targeted programs designed to enhance research and educational opportunities for students and broaden Purdue's global impact.
"One of the critical questions we face is how to feed a growing world population," Daniels said. "This initiative can lead to answers to that question by helping to produce plants that have higher yields and can grow in a variety of environmental conditions."
The College of Agriculture plans to add 10 new faculty positions, some new facilities and remodeling of existing facilities, and programmatic support for research and student recruitment and education.
The investment will dramatically expand Purdue's capabilities in plant sciences, helping the university to move discoveries from the laboratory to commercialization or to the farm in innovative ways, said Jay Akridge, Glenn W. Sample Dean of Agriculture.
"In addition, we will be embedding educational opportunities for students throughout this process of discovery and innovation, creating a unique learning opportunity for the next generation of leaders in the plant sciences," Akridge said.
Gebisa Ejeta, distinguished professor of agronomy and 2009 World Food Prize laureate, said the funding reflects Purdue's leadership role in working to find ways to feed a world population expected to increase from about 7 billion now to 9 billion by 2050.
"Science, technology and innovation are key to feeding humanity sustainably," Ejeta said. "With this investment, Purdue University is making a commitment to the future of agriculture in Indiana and beyond and showing the collective resolve that we all share at this institution to remain among the very top tier of leading universities in the world."
Ben Hall, a Purdue graduate student in Agronomy, collects leaf reflectance data in a soybean field at Purdue's Agronomy Center for Research and Education. Hall was collecting data to determine variation in nitrogen content in the plants' leaves. Sensor technology has proven to be a precise and efficient method for phenotyping breeding populations. (Purdue Agricultural Communication photo/Tom Campbell)
Hi-Res Photo available
The plant sciences investment will be divided into these main areas, with student engagement a fundamental part of each:
The university initiatives were developed over several months of work that involved deans, faculty and others at the university. The university's trustees have reviewed and given support for the programs.
"This investment in the plant science research and education pipeline will catapult Purdue's efforts to enable faculty and students to translate their creativity into new products to help feed a hungry world," said Karen Plaut, senior associate dean for research and faculty affairs.
Related websites:
Department of Agricultural Communication, 615 West State Street, West Lafayette, IN 47907-2053 USA, (765) 494-8403
© | An equal access/equal opportunity university | Integrity Statement | Copyright Complaints | Maintained by Agricultural Communication
Trouble with this page? Disability-related accessibility issue? Please contact us at email@example.com so we can help.


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Estimated Up Time will be 14:00(2:00pm) (GMT+8)
1. Improvement of Trading House
Are you like me spamming left clicks whenever you try to buy an item from the trading house just to have someone beat you to it? Anyone changing mouse because of this reason?
I always thought it was needless to scroll through tons of bid pages just to find an item which i wanna buy instantly. My personal best is 80 pages for Horns, what about you? LOL! Anyway jokes aside, now we can categories how we want to see the items listed in the Trading House. Soon, you will be able to view them by either bid or buy now.
2. Enhanced Auto Party
Enhanced auto party system, now when you look for party at the Dragon Pillar the system will search do a search via the pillar and via the Party Window. The system will suggest you to enter Cross Server Party system if your party is incomplete after a long time.
3. Improved Weapon Feeding
There were people complaining that their gems were lost while evolving (gem slotted) weapons. Now, you will not be able to evolve or feed your weapon with a gem slotted weapon.
When feeding a weapon, players are usually blindly feeding it to reach Grade 10. Now an additional notification is added to your weapon to let you know the Grade 10 stats of the weapon. This allow players to make informed decision and decide for themselves whether the money and resources will be well spent.
4. Changes to Opening Treasure Chests
Ever wonder why you had 7 inventory slots but when you try to open a treasure chest the system forbids you? This problem has received tons of complains due to the fact that players need at least 8 empty slots to open a chest (regardless of the number of items the chest gives)
In the above picture, when a player tries to open a treasure chest, the player will receive 3 times with 1 item store in the 保管箱 Temporary Safe Deposit Box. Please take note that if the Temporary Safe Deposit Box is not emptied, the player will not be able to open new chests.
You can access the Temporary Safe Deposit Box from the footer menu below:
5. Adjustments to the World Chat windows
Remember the two irritating windows that take up extra space? Who cares about the World Shout anyway? I rather see the beautiful landscape of the environment. Well, now you can make the windows go invisible.
1st : Click on the chat gear here :
2nd : Drag the cursor to increase the transparency of the window. Just below there is an option 字体大小 to reduce the font size of the World Chat. Neat isn't it?
6. Strengthen the benefits of Member's Convenience Package
Now when you combine jewels, you can save 20% on the process costs.
7. Show off your achievements!
Now players are able to show off to other players their achievements by pressing Ctrl + Left Clicking on the Achievement Icon they unlocked.
8. Other in-game bugs resolved
- When players exit from party, QT chat suddenly gets muted.
- When doing cross server party, during instance party or PVP players will now be able to hear each other via QT
- When doing cross server party, during instance party or PVP players will now be able to hear each other via QT
- Some items that were unable to be used due to bug is resolved
- Some items were not able to use due to location has also been solved
- Some Sealed items did show the Break through and Evolve conditions has also been resolved
- When max 灵气 has been achieved the system still shows that you are able to receive 灵气
- Certain bugs while crossing between maps and areas
- Resolve the display of the PVP notification during PVP loading screen
- No notification when players receive the gifts from the Cash Shop
- Solve an issue on the Frog
- A missing teleportation point near the Frog Map in Dragon Forest.
SOURCE: Aspenach MMOSITE Blog
super thanks to aspenach for this useful information and allowing me to repost his blogs :D


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MP4 to MP3 Converter
- License:
- Shareware
- Price/Registration Fee:
- $24.95 USD Buy Now!
- Publisher:
- DVD-iPod.biz
- Last Updated:
- 05/09/07
- Version:
- 1.0
- Review:
- Read 3 Reviews
- User Rating:
Rate It
MP4 to MP3 Converter Description:
MP4 to MP3 Converter is designed to convert MP4 to MP3, it can also convert most popular video formats to MP3. Supported video formats include AVI Video (*.avi), MPEG Video (*.MPEG, *.MPG, *.MP4, *.M1V, *.M2V, *.M1P), DVD Video (*.VOB), Windows Media Video(*.ASF, *.WMV, *.ASX, *.WAX, *.WM, *.MWA, *.WMD, *.WMP, *.WMX, *.WVX, *.WPL).
Operating System Support: Win98, Windows2000, WinXP, Windows2003
Review for MP4 to MP3 Converter
MP4 to MP3 Converter based on 28 ratings. With 3 user reviews.
thanx a lot ya....
doesnt work for long files
good program but doesnt work for files which are longer than four or five hours
Great program. It was quick and the output was a great quality.


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Kayla has been creating art since she was old enough to walk. When she was 3, she created her first drawing on the bottom of our coffee table (which we still have) and laugh about today. It's one of the most precious drawings that we have. I knew then that she had something special inside of her that God was going to use one day. Now, she has moved into painting with watercolors and I am beyond amazed at the kind of art she creates with this paint set.
When she first started drawing, it was all about the colored pencils, but then she discovered the beautiful Watercolor sets that they have on Amazon and now, she cannot get enough.
How to Paint With Watercolor Sets
She paints everything these days. Her favorite though, is painting gifts for friends. She recently painted her best friend's Rottweiler dog from memory.
She is crazy talented and I am so glad that she has decided to attend art college. She has an amazing gift that I know is going to take her far in life.
Right after we got back from seeing the 7th installment of Star Wars, she painted her favorite character – R2D2. She loves him and many days, he sits and watches her while she paints.
Her favorite thing about these watercolor sets is their vibrant colors. These paint sets are not like the ones you get at the store. They are more vibrant because the paint is ground down into a fine powder which allows for a more vibrant brush stroke.
Regardless of how much you thin the paint out, the colors just pop on the page!
The set is offered in a compact carrying case that comes with everything you need to get started — brush, white paint, and your beautiful pigmented watercolors.
You can get these amazing sets for only $17.97 on Amazon. That is a STEAL! The first set she bought like this came from a local hobby store and she paid twice that amount for almost the same style of paint set.
Now, we can get them on Amazon and save her money so that she can still paint to her heart's content.
In fact, this is her most recent painting — and she made it for me.
She has really got an amazing talent and I cannot wait to see how she uses it in her adult life!


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Burkin hired at Sony
EmptySony Pictures Entertainment's International Motion Picture Production division has brought aboard Brian Burkin as senior vp of business affairs and operations.
Burkin will oversee the division's business affairs as well as negotiate deals and manage relationships with writers, directors and producers in film markets around the world.
He will work out of the New York office and report to Deborah Schindler, the division's president.
Burkin comes from Miramax Films, where he spent nine years, most recently as senior vp of business affairs.
He started his career in New York doing private practice in corporate law and commercial litigation.