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Intracellular trafficking as a determinant of AS-DACA cytotoxicity in rhabdomyosarcoma cells
Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma derived from skeletal muscle precursor cells, which accounts for 5-8% of all childhood malignancies. Disseminated RMS represents a major clinical obstacle, and the need for better treatment strategies for the clinically aggressive alveolar RMS subtype is particularly apparent. Previously, we have shown that the acridine-4-carboxamide derivative AS-DACA, a known topoisomerase II poison, is potently cytotoxic in the alveolar RMS cell line RH30, but is 190-fold less active in the embryonal RMS cell line RD. Here, we investigate the basis for this selectivity, and demonstrate in these RMS lines, and in an AS-DACA- resistant subclone of RH30, that AS-DACA-induced cytotoxicity correlates with the induction of DNA double strand breaks.
We show that inhibition of the multidrug-resistance associated protein (MRP1) has no effect on AS-DACA sensitivity. By exploiting the pH-dependent fluorescence properties of AS-DACA, we have characterized its intracellular distribution, and show that it concentrates in the cell nucleus, as well as in acidic vesicles of the membrane trafficking system. We show that fluorescence microscopy can be used to determine the localization of AS-DACA to the nuclear and cytoplasmic compartments of RMS cells grown as spheroids, penetrance being much greater in RH30 than RD spheroids, and that the vesicular signal leads the way into the spheroid mass. EEA1 and Rab5 proteins, molecular markers expressed on early-endosomal vesicles, are reduced by > 50% in the sensitive cell lines.
Taking the evidence as a whole, suggests that endosomal vesicle trafficking influences the toxicity of AS-DACA in RMS cells.
Rhabdomyosarcoma (RMS) is a malignant tumour derived from primitive rhabdomyeloblasts with differentiation towards skeletal muscle
Recently, we have shown that the acridine-4-carboxamide derivative AS-DACA (Figure
Figure 1
AS-DACA resistance in rhabdomyosarcoma cells
AS-DACA resistance in rhabdomyosarcoma cells. (A) Structure of AS- DACA. (B) MTT cell viability results presented as cytotoxicity curves for AS-DACA in RD and RH30. (C) Derivation of the AS-DACA-resistant RMS cell line, Res30. Dose- response curves from MTT cell viability assays performed at various stages in the development of AS-DACA-resistant RH30 cells. (D) Cytotoxicity curves of (i) 9-amino- DACA, (ii) doxorubicin, and (iii) mitoxantrone in Res30 cells. Error bars are SEM for independent experiments performed in triplicate.
Here we use the fluorescence properties of AS-DACA to permit direct visualization of its intracellular distribution. A novel situation is therefore created whereby the cytotoxic agent itself reveals the pH of the organelle in which it is localized. Our results show that AS-DACA not only accumulates in the nucleus, as expected, but also in acidic intracellular vesicles in RMS cells. However, we find that the membrane trafficking system differs between the two RMS subtypes, with the early endosome markers, Rab5 and EEA1, expressed to a lesser degree in the sensitive RH30 cell line compared to the resistant RD cell line. Generation of an AS-DACA-resistant RH30 subline resulted in an increase in expression of the early endosome markers, Rab5 and EEA1, suggesting that the membrane trafficking system plays a significant role in the determination of AS- DACA sensitivity. In addition, we show that cytotoxic sensitivity to AS-DACA correlates with the induction of DNA double strand breaks, but is un-perturbed by the inhibition of the multidrug-resistance associated protein MRP1. Lastly, we demonstrate that fluorescence microscopy can be used to examine the intracellular distribution of AS- DACA into both the nuclear and cytoplasmic compartments of RMS cells grown as spheroids, where penetrance is much greater in RH30 than RD spheroids, and that the vesicular signal leads the way into the spheroid mass.
In previous work we showed that the IC50 for AS-DACA in RD cells is 3800 ± 130 nM whereas in RH30 cells it is 20 ± 0.73 nM, indicating a 190-fold difference in sensitivity (Figure
Figure 2
Levels of phosphorylated H2A.X (γH2A.X) were determined using ELISA for the RMS cell lines RD, RH30, and Res30 following exposure to 20 nM of AS-DACA over time
Levels of phosphorylated H2A.X (γH2A.X) were determined using ELISA for the RMS cell lines RD, RH30, and Res30 following exposure to 20 nM of AS-DACA over time. Levels were calculated as percentages of γH2AX detected after 48 h-exposure to 4 μM AS-DACA. Error bars are SEM for two experiments performed in triplicate providing six independent readings per value.
Since AS-DACA is a known substrate of the ABC transport proteins
Figure 3
Assessment of Multidrug Resistance Associated Protein (MRP1) in RMS cell lines
Assessment of Multidrug Resistance Associated Protein (MRP1) in RMS cell lines. (A) MRP1 expression determined by indirect immunofluorescense microscopy showing intracellular distribution of MRP1 (green) in RMS cells following treatment with AS-DACA. Co-stained with the nuclear stain DAPI (blue). (B) Levels of MRP1 were determined by Western blot before (-), and after (+), exposure to AS-DACA (2 × IC50 for 16 h). Densitometry was performed on triplicate blots with band intensity normalized for both background noise and total protein loading differences as determined by Ponceau S staining. (C) Influence of (R)-verapamil (1 and 10 μM) on AS-DACA cytotoxicity. RD (top) and Res30 (bottom) cells were treated for 72 h with (R)-verapamil whilst being exposed to serially increasing concentrations of AS-DACA. Error bars in (A) and (C) represent the SEM for three independent experiments.
In contrast, although MRP1 is well-expressed in Res30 cells, its level decreases following exposure to the drug (Figure
Taking advantage of the fluorescent properties of AS-DACA
Figure 4
AS-DACA fluorescence properties
AS-DACA fluorescence properties. (A) Images of the intracellular distribution of AS-DACA in RD and RH30 cells captured using digital colour photography after a 4 h exposure to 2 μM drug. Images were captured at 600 × magnification. Scale bars represent 10 μm. Inset shows 4 h exposure to 2 μM doxorubicin. (B) Uncorrected fluorescence emission spectrum of AS-DACA in solution over the pH range 6.2 to 6.8. Excitation wavelength was 260 nm, λmax for absorption in the UV. (C) Fluorescent micrographs prepared using a deconvolution microscope for detection of AS-DACA. Cells were incubated for 10 min at 2 μM and show vesicle accumulation in RD (left), and RH30 (right) cells. Nuclei with reduced, or absent, staining are indicated (arrow).
We wished to determine whether the differences seen in the distribution of AS-DACA between the nucleus and vesicular structures in the two RMS cells lines is responsible for their differential sensitivity to AS-DACA toxicity. In addition, we were interested in discovering which of the cellular compartments is first to accumulate AS-DACA. Accordingly, we used a deconvolution microscope, equipped with computerized exposure control during imaging, for a more precise rendering of AS-DACA fluorescence. This was especially useful following the short treatment times designed to highlight the agents early accumulation throughout the cell. Figure
Figure 5
(A) Arrows indicate co-localization of AS-DACA and LysoTracker™ Red observed in the RD cells
(A) Arrows indicate co-localization of AS-DACA and LysoTracker™ Red observed in the RD cells. (a) Digitally magnified photograph with un-altered colour. (b) The green emission has been digitally removed highlighting the pink/purple colour that results from co-localization of AS-DACA and LysoTracker™ Red. (c) The red wavelengths are then additionally removed, further highlighting sites of co-localization. (B) Expression levels of Rab5, Early Endosome Antigen 1 (EEA1) Rab 7 and Lysosome- associated Membrane Protein 1 (LAMP1) were determined by Western blot before (-), and after (+), exposure to AS-DACA (2 × IC50) for 16 h. Tubulin was included as a loading control. Shown is a single representative blot for at least three independent experiments where the same result was found in each.
The expression levels of markers specific for vesicles active at various stages of the membrane trafficking process were examined in all RMS cells, after exposure to double their IC50 for AS-DACA for 16 h. The expression levels of Early Endosome Antigen 1 (EEA1), Rab5, Lysosomal-Associated Membrane Protein 1 (LAMP1), and Rab7, were assessed by Western blot analysis (Figure
We hypothesized that the decrease in the amount of early endosomes in RH30 cells contributes to their sensitivity to AS-DACA, due to a lack of vesicular sequestration, which would allow the drug to pass freely throughout the cell and into the nucleus and surrounding cells. To test this, we grew RD and RH30 cells as 3-dimensional spheroids, and assessed the ability of AS-DACA to pass through multiple cell layers. Unfortunately Res30 cells were unable to be grown as spheroids. After 4 h of incubation at 20 μM, the distribution of fluorescence at both emission wavelengths across the spheroids is equivalent for both cell lines, with the intensity of nuclear green fluorescence within 10 μm of the spheroid edge being 10% greater than that of vesicular blue fluorescence. After 24 h, however, the distribution of fluorescence across the spheroids is considerably altered. The blue fluorescence is now more intense than green at the outer edge (10 μm), the difference being the most pronounced in the RD cells where the peak is ~10% below that of the RH30 cells, indicating a larger AS-DACA signal in the vesicles. Furthermore, after 24 h, AS-DACA is seen to penetrate further into the RH30 spheroids, with equivalent levels of both green and blue fluorescence being found approximately twice as deep into the cell mass as observed at 4 h. By contrast, in RD cells, both fluorescence signals are below 30% relative fluorescent intensity beyond a penetration distance of 20 μm, indicating that AS-DACA is unable to penetrate far into the RD cell mass even after a lengthy incubation. Evidently, AS-DACA traverses packed RH30 cells much more readily than packed RD cells, and it is notable that the blue vesicular signal leads the way into the spheroid masses.
The sensitivity of RMS cell lines to new agents may help identify novel strategies in treatment for this often refractory sarcoma. Cytotoxins based on acridine-4-carboxamide chromophores, such as the methyl sulphone derivative of N-(2-(dimethylamino)- ethyl)acridine-4-carboxamide, known as AS-DACA (Figure
Active mechanisms that drive sequestration of weakly basic compounds into vesicles of the membrane trafficking system are not uncommon. Several of the ABC transport pumps have been implicated in such mechanisms
Classically, vesicle sequestration of the anthracycline antibiotics appears to be dictated by the "partitioning theory". The partitioning theory of drug sequestration, which was proposed not long after the discovery of the lysosome in the 1960's,
Like the anthracyclines, AS-DACA features a protonation site at the dimethylaminoethyl side chain (Figure
Figure 6
AS-DACA (1 μM) penetrates further into spheroids made from the RH30 sensitive cell line
AS-DACA (1 μM) penetrates further into spheroids made from the RH30 sensitive cell line. Confocal images of AS-DACA diffusion into spheroids made from the RD and RH30 cell lines after 4 and 24 h. Fluorescence intensity is shown as % Relative Fluorescence Intensity compared to distance penetrated into the spheroid for localization in the nucleus and in cytoplasmic vesicles.
Knockdown of the major vault protein (MVP) using siRNA disrupted lysosomal uptake of not only doxorubicin, but also the intracellular pH probe LysoSensor and the lysosomal specific antigen LAMP1. This study indicated that MVP was integral in redistributing the drug away from the nucleus to lysosomes
The implication from our findings suggests that reduced levels of early endosomal vesicles limit the sequestration and redistribution of AS-DACA away from the nucleus in RH30 cells and hence does not offer any protection from the effects of the drug with topoisomerase poisoning. Taking the evidence as a whole shows that endosomal vesicle trafficking influences the toxicity of AS-DACA in RMS cells and highlights potential new biological targets worthy of further study.
Interestingly, sequestration of AS-DACA was identified through the intracellular visualization of the drug. Most established molecules used to treat cancer however cannot be visualized and hence similar mechanisms may go undetected, only manifesting themselves through a poor tumour response to therapy. The unique fluorescence properties of AS-DACA may be exploited in new and alternative methods of assessing drug penetration, distribution and the overall pharmacokinetics of the drug. Hence, we believe that, regardless of the drugs fate in clinical development, it will provide an invaluable tool to cancer pharmacology researchers interested in understanding the pharmacodynamics and pharmacokinetics of weakly basic cytotoxic compounds.
AS-DACA was kindly provided by Professor W.A. Denny from the Auckland Cancer Society Research Centre, University of Auckland, New Zealand. ®-Verapamil, mitoxantrone, and doxorubicin were purchased from Sigma-Aldrich Pty Ltd.
Cell Culture, Cytotoxicity Assays and Development of an AS-DACA- Resistant Cell Line
RD and RH30 rhabdomyosarcoma cell lines were purchased from ATCC, Virginia, USA and were routinely tested for mycoplasma contamination as well as growth consistency prior to establishment and experimentation. The presence of the t[1:13] translocation was confirmed in the Rh30 lines. RD cells were maintained in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine (Sigma-Aldrich). RH30 cells were maintained in RPMI medium (Invitrogen) and supplemented with 10% FBS and 2 mM glutamine. All cell lines were grown at 37°C in humidified conditions and 5% CO2. Three-dimensional multicellular spheroids were cultured by adding 1.5 × 105 cells/ml to agarose-coated 96-well imaging plates (BD Biosciences), and allowed to aggregate for 96 h, resulting in the formation of a single spheroid per well
DNA Strand Breaks: Enzyme-Linked ImmunoSorbent Assay [ELISA]
AS-DACA-induced DNA double strand breaks were assessed by measuring the level of phosphorylated histone H2A.X
The influence of pH on the fluorescence properties of AS-DACA was investigated in phosphate buffers, at a concentration of 10 μM, using a Perkin Elmer LS-50B spectrofluorimeter equipped with a Starna Fluorimeter Micro Cell quartz cuvette. Fluorescence emission was measured from 280 to 800 nm with excitation at 260 nm, λmax for AS-DACA absorbance in the far UV.
Drug Distribution and Intracellular Probes
Cells were passaged at a 1:5 dilution from near-confluent plates onto glass coverslips placed in the bottom of the culture plate. They were subsequently grown for 16 h, and subsequently treated with high dose (1-2 μM) of AS-DACA for short exposures (10 m-4 h), to obtain optimal images with minimal quenching of fluorescence. When required, cells were further incubated for 45 min with LysoTrackerTM Red (Molecular Probes, Invitrogen) at a concentration of 100 nM. Cells were then washed once with ice cold PBS to minimize drug efflux, and immediately mounted onto glass microscope slides using FluorSave mounting media (Calbiochem).
Indirect Immunofluorescence - MRP1 Expression
Cells grown on glass coverslips were gently washed twice in PBS, fixed permanently by incubating with 4% paraformaldehyde for 20 min, and made permeable by treatment with 0.2% Triton X-100 for 10 min at room temperature. They were then washed 3 times with PBS, and incubated for 1 h at room temperature with MRP1 primary antibody diluted in PBS containing 0.1% BSA. Cells were washed and incubated with Alexa488-conjugated donkey anti-mouse (1:500) antibody in 0.1% BSA in PBS for 1 h in the dark. After washing, the cells were counterstained with 10 nM DAPI diluted in PBS, washed, and mounted onto microscope slides.
Fluorescence Microscopy
The majority of the fluorescent images were obtained using a Jenoptik ProgRes CF Scan digital camera on an Olympus BX50 epifluorescent microscope fitted with a UPlan FL 60 ×/corr 0.65-1.25 oil objective. A U-MWIBA3 filter was used for excitation of AS- DACA within the range 460 to 490 nm, and green emission detected between 510 and 550 nm. Red fluorescence, of doxorubicin and LysoTracker™ Red, was detected above 610 nm using a U-MWIY filter with excitation between 545 and 580 nm. UV excitation of AS-DACA at 330 to 385 nm was achieved using a U-MWV filter with all emissions above 420 nm detected. ProgRes® V2.5 software was used to capture the images. Image enhancement involving colour level adjustment as dictated by the RGB histogram, was carried out in Adobe Photoshop. No other computer enhancement was performed. Extensive photobleaching under continuous UV irradiation proved troublesome for fluorescence imaging of AS-DACA. To address this problem, we used a deconvolution microscope, Delta Vision OMX, which provided precise control of UV excitation exposure and image capture, allowing visualization of the AS-DACA intra-cellular distribution following short exposure times. AS-DACA fluoresced an intense green from excitation at wavelengths between 460 and 490 nm, and blue following excitation between 330 and 385 nm. Images were captured using true colour photography at 600 × magnification, and the displayed pictures are merged images of both filters with no enhancements performed.
Imaging Drug Penetration into Spheroids
Spheroids were incubated with 10 μM AS-DACA for 24 h and 12-bit confocal images collected on an Olympus FV1000 inverted microscope using an Olympus UPLAPO 10 ×/0.40 air objective lens. Scan rates of 4.0 μs/pixel, 1.5X zoom, Kalman averaging, and sequential collection of the green and blue channels, were applied for all images. Excitation and emission ranges for the 405 nm diode and 488 nm argon lasers, respectively, were as follows: Ex 405 nm, Em 425-475 nm; Ex 488 nm, Em 500-600 nm. Image planes were selected approximately 175 μm inside the spheroid to eliminate edge effects, and reflection imaging was used to ensure 100% laser penetration and emission at that depth within the spheroid. The images were not manipulated in any way, and were analysed using ImageJ software (NIH). A one pixel-wide line was drawn through the centre of the spheroid and the fluorescence intensity was measured. The values were normalised and the average intensity profiles for each wavelength were calculated
Western Blot Analysis
Western blot analysis was performed using established protocols as previously reported
Authors' contributions
SJW undertook the majority of the research procedures for this study as part of his postgraduate training. TH contributed the result from the deconvolution microscopy (Figure
This work was supported by funding from the Children's Oncology Foundation (SW, TH, DRC), and an Australian Postgraduate Research Award (SJW). SJW was a postgraduate student with the Faculty of Medicine, University of Sydney. TH was an Honours student with the School of Medical and Molecular Biosciences, The University of Technology Sydney.
Rhabdomyosarcoma and the undifferentiated sarcomas
Soft Tissue Sarcomas
Intergroup Rhabdomyosarcoma Study: Update for pathologists
Rhabdomyosarcoma: new windows of opportunity
The third Intergroup Rhabdomyosarcoma Study
Efficacy of ifosfamide and doxorubicin given as a phase II 'window' in children with newly diagnosed metastatic rhabdomyosarcoma: A Report from the Intergroup Rhabdomyosarcoma Study Group
Common musculoskeletal tumors of childhood and adolescence
In vitro assessment of novel transcription inhibitors and topoisomerase poisons in rhabdomyosarcoma cell lines
Experimental solid tumour activity of N-(2- (dimethylamino)ethyl)-acridine-4-carboxamide
Potential antitumor agents. 49. 5-substituted derivatives of N-(2-(dimethylamino)ethyl)-9-aminoacridine-4- carboxamide with in vivo solid-tumor activity
Bisintercalating Threading Diacridines: Relationships between DNA binding, cytotoxicity, and cell cycle arrest
Influence of substituent modifications on DNA binding energetics of acridine-based anticancer agents
From amsacrine to DACA (N-(2- (dimethylamino)ethyl)acridine-4-carboxamide): selectivity for topoisomerases I and II among acridine derivatives
Potential antitumor agents. 46. Structure-activity relationships for acridine monosubstituted derivatives of the antitumor agent N-2- [dimethylamino.ethyl]-9-aminoacridine-4-carboxamide
ATM phosphorylates Histone H2AX in response to DNA double-strand breaks
Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks
Cellular uptake of N-(2- (dimethylamino)ethyl)acridine-4-carboxamide (DACA)
Cytokinetic differences in the action of N-(2-(dimethylamino)ethyl)acridine-4-carboxamide as compared with that of amsacrine and doxorubicin
Topoisomerase I/II selectivity among derivatives of N-(2-(dimethylamino)ethyl)acridine-4-carboxamide (DACA)
Subcellular localization and activity of multidrug resistance proteins
Intracellular ABC transporter A3 confers multidrug resistance in leukemia cells by lysosomal drug sequestration
Separate roles for the Golgi apparatus and lysosomes in the sequestration of drugs in the multidrug-resistant human leukemic cell line HL-60
Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes
Commentary: Lysosomotropic agents
Lysosomal sequestration of amine-containing drugs: Analysis and therapeutic implications
Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient
Inhibition of endosomal sequestration of basic anticancer drugs: influence on cytotoxicity and tissue penetration
Accumulation of an anthraquinone and its platinum complexes in cancer cell spheroids: the effect of charge on drug distribution in solid tumour models
DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139


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Below is our collection of tidbits, odds 'n ends for Zula.
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With a nod to our favorite Gazetteers and using the location of where the historic<1> community of Zula once stood, the straight-line distance<2> beginning in Zula to:
We have found 3 communities that share the name Zula.
While the name Zula is unique within Alabama, we know of another 3 communities with the same name in the United States.
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Chattooga County
Our profile page for Zula, Chattooga County [Map]This community is historic.Distance: About 67 miles [107.8 km] to the Northeast
Wayne County
Our profile page for Zula, Wayne County [Map]Distance: About 216 miles [347.6 km] to the North Northeast
Woods County
Our profile page for Zula, Woods CountyWe found mention of this community, but have little information.<8>Although located in Woods County, we don't know the location of this community within the county.
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subset: commoncrawl


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One of the great tech non-events of the last few years involves Linux on PCs. Every so often, another wave of hype washes in about how companies are finally going to ditch their Windows machines in favour of the open-source operating system and productivity apps like Sun's StarOffice and (more recently) IBM's Symphony.
I suppose you can't blame IBM for trying to capitalise on the bad press of Windows Vista to try to give this story another spin. It has just agreed a deal with the three top Linux companies to distribute its own Notes and Symphony software alongside the operating system. The promise: a "turnkey" software package that, according to IBM, cuts 30 per cent or more from the cost of buying a new enterprise PC. Read more


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text: Ages of Titan Preceded byGolden Age Godswar Succeeded byAge of Darkness Retrieved from "http://scratchpad.wikia.com/wiki/Godswar?oldid=262854" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Titannica Time and Calendars of Titan


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|Family . Drama . Comedy
James' video-game skills get him on a professional team, but then he regrets going pro, so Stephanie and Charlie try to get him out of his contract.
|Episode Title:
|James Goes Pro
|2014-11-13 at 08:00 pm
It's a cookie-cutter comedy that's inoffensive, intermittently chuckle-worthy and totally predictable.New York Post Full Review
A warm, harmless, family-friendly show that offers a few sweetly amusing moments mixed in with the predictable twists. The kids are quite adorable, too.San Jose Mercury News/Contra Costa Times Full Review
Overall, Instant Mom is mild and unobjectionable.Variety Full Review


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Tuesday, December 15, 2009
The Mutants of Nightmare City - part 8
Wow, this guy drank way to much before he became a mutant! Or was it the "mutantism" that made him an old wino? Actually, during christmas he always wear those silly reindeer-horns on his head to show that has some sense of humor and can joke about it.
A legend in his own time! Professor Hagenbeck is the source of every mutant (I guess, because he was the expert and therefor he must mutate first). It's a pity that he just makes this appearance and then just leaves us alone for the rest of the movie...
The priest-mutant has always been a mystery because it's obvious that our heroes see his deformations first, because the nice part of the face is in our direction. But that's just a small detail. Even if I'm a devoted atheist, I admire that he's still praying even when he's a mutant. Probably one of those god damn liberal priests!
2 comments:
- Jocke Andersson said...
I love that priest. He looks like he got some extreme veneral disease in his face.
Well, I thought... would you sue me if I made a Zombies of Burial ground or something similar? ;)
- December 15, 2009 at 9:20 PM
- Ninja Dixon said...
Jocke: Of course! Go ahead, I'm looking forward to it already :)
- December 15, 2009 at 10:34 PM
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Question: Describe how a single plantwide allocation rate is used
Describe how a single plantwide allocation rate is used.
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Answer to relevant QuestionsWhy is the use of departmental allocation rates considered a refinement over the use of a single plantwide allocation rate?How can ABM be used by service companies?How many cost pools are in an activity-based costing system?Day Corp. is considering the use of activity-based costing. The following information is provided for the production of two product lines:Day plans to produce 400 units of Product A and 375 units of Product B. Compute the ...Fitzgerald makes handheld calculators in two models—basic and professional. Fitzgerald estimated $ 812,500 of manufacturing overhead and 625,000 machine hours for the year. The basic model actually consumed 250,000 machine ...
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Clara Bow
"People used to say that I had a feeling of closeness, a great warmth of loving everybody, that they could tell me their troubles."
Elaina Smith P.2
Clara Bow was a popular actress in the 1920's, often referred to as the "It" girl. She acted in silent films, often playing waitresses, manicurists, and salesgirls. Her characters usually had a main goal to win over a man, in which her vivaciousness, irresistible charm, and sensuousness always worked to her advantage in the roles. Clara was a well renowned sex symbol, being the first actress to visibly flaunt their sex appeal on the screen. This made her one of the most talked about actresses in Hollywood at the time. However, her childhood was not as glamorous. She was born in Brooklyn in 1905 during one of the worst heat waves New York had ever experienced. Her mother and grandmother both mentally ill, and her father wanted nothing to do with any of them. Her mother hoped that the heat would kill both her and Clara, who was then an infant, and didn't even bother to sign a birth certificate. Throughout her childhood, she would escape the tough aspects of her home life by imitating her favorite actresses from TV in the mirror.
After years of searching New York for talents contests and agents, she became discovered when she won the Fame and Fortune Contest in Motion Picture Magazine. They said even at the age of 16, she was full of confidence, ambition, and determination. A part of her prize was a role in a motion picture, which led to her stardom.
Some of Clara's most famous films include Black Oxen (1923), Dancing Mothers (1926), The Daring Years (1923), and Down to the Sea in Ships (1922).
Above is a scene from "Goes to Dinner".


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Recently, a story of supposed police brutality was in the news. The narrative was this: a 15-year-old black girl was innocently riding along on her bike, when Hagerstown police decided, for no reason at all, to detain her. After they arrested her, handcuffed her, and placed her in the backseat of their police car, they pepper-sprayed her. Sounds indefensible, right?
Well, unfortunately, the bodycam footage proves that entire story to be a complete lie.
Police were originally called when the girl hit a car with her bicycle. Just an accident, right? And that's all it would have been, except for the girl's behavior. She was combative to officers the entire time. She fought them, tried to leave the scene, swore at them, resisted arrest, kicked a police officer, and then used her legs to try to prevent the door of the cruiser from being closed. It was only after all of this had happened that an officer deployed the pepper spray.
You can see the entire video here:
Here's the thing: you can't leave the scene of an accident. Doing so is a crime. And this girl may think that because she can play the victim, she can do whatever she wants without any repercussions, but these police officers were wearing body cameras. She was telling her friends to "record this sh**, yo!", but she didn't realize that police already were. And that just ruined her entire effort. The sad thing is that the officers even warned her; while she was resisting them, they had her well-being in mind. They told her she was going to get herself hurt, and she did.
Now that the whole story has come out, it's clear that these officers are to be commended. She behaved despicably, and they reacted with an incredible of restraint.


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The Woes of Beta Software
There's always a "cool-factor" invovled with working with beta software and trying the latest products out before anyone else. It's definitely quite exciting to see some of the new features of Office 2007 in action and actually dig into Project Server 2007 and SharePoint Services 3.0.
At the same time, it's also incredibly frustrating, especially if you're the one that has to set up the beta environment :-S.
I've been hunched over my PC and laptop over the last 4-5 days trying to get Project Server 2007 to install on a VM image and it has been downright painful. From the dreaded "Thread was being aborted" error to the "Object reference not set to an instance of an object.." error. It has been slow and painful as each failed install took tons of time and there have been few answers on how to deal with these errors.
With regards to the first type of error, you may be able to solve it by following the warning in the documentation (starting the SharePoint Services Web Application service). In my case, I believe the process was actually timing out on me. Initially, I was trying to do the install over remote desktop from my laptop to my desktop. The documented fix did nothing for me after several tries. After switching to the desktop, I was able to work around this.
With regards to the second error, well, I'm still working on that, but it seems that to provision the PWA website, an Active Directory domain account is necessary. Don't ask my why as I have no clue why this should/would be the case. Apparently, it's fixed in Beta 2 Technical Refresh.
It was also fun coming across the superficial SQL Server collation error reported by SharePoint Services 3.0. I was on the brink of uninstalling and reinstalling SQL Server before I stumbled on the Microsoft Communities thread.
All in all, this has been brutal weekend. I'm simply drained from having to deal with this.
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I don't think there is anything wrong with Greenlight's website, Brian is the best resource for all your building needs. I get all my supplies from Greenlight. Brian and Kevin are the best, plus the new shop in Belmar is one town away from me so no shipping cost.
www.mahadysurfboards.com, www.mahadysurfboards.blogspot.com
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text: please i need to configure my xenforo software such that signature can show for smaller screen sizes. i have discovered that signature only shows on desktop and tablet. I want to be able to show signature on smaller mobile devices and all screen sizes generally.\n\nAnd please, i want to do same for the share link that appears on all threads and comments (e.g #1, #2, #3, etc)


text:
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Clairene Grillo's passing; thank you for heartwarming response
Every Moment is a Teaching Moment
Social media is a wonderful way to communicate and spread news. We had a very heartwarming response fm so many people when Clairene Grillo passed on that we decided to write one response to all and use it as a teaching moment for those who attend, have attended and will attend Minnesota Hockey Camps. First and foremost, we read every one of your comments and we realize there are more coming.. You, taking time to comment, show that you have the greatest intangible one can acquire. You sincerely care. In many cases, you were simply reciprocating for enjoying your experiences with Claire, some with us as a team, and Minnesota Hockey Camps. In all cases, this was deeply appreciated because this is her moment. Keep in mind, we know there are others who sincerely care and have their way of closure without saying or writing a word and we respect that.
Our daughter, Tracy, and I were talking about the word "care" yesterday simply because it comes to the forefront during moments like this. We felt it should be family taught @ a young age and it should be reinforced in the school system and co-curricular activities. Intangibles, to me, are like an elective in school. You have the Basic English, Math and Science, but none of this works for our children unless they acquire all of the intangibles required to succeed in life. Intangibles give our children a way to express their knowledge and skill set. They drive your mindset, which drives your skill-set. One cannot perform/work to the maximum without the other. They maximize each other. Take some time to teach them, "How life really works".
Unconditional care for others is easy to teach, especially if there are so many examples in the family. Teachers creating an awareness of the importance of caring are doing their students a huge favor. When athletes give back they will raise their game. Feeling good about yourself pumps your heart. When your heart is pumped you will see positive results. There is no better feeling than you feeling good about you. There is no better feeling than creating a better version of you. Life and performance will be greatly enhanced.
Do yourself a huge favor and write down all of the intangibles required for success; then rate yourself on each. Be sure to put the finishing touches on all of them with the following:
Skill to fit in.
Skill to express your skill.
Skill to figure it out when it's not in your parents or coach's manuals.
Rate your purpose, reason and substance levels while being ordinary and fun.
What is your intensity level and are you energy efficient?
Do you do everything hard and smart?
Do you get up each morning with the intention of doing something out of the ordinary; recognizing that it's human nature to get up and be who we are? What is your authenticity level?
What is your humor level; especially in adverse situations?
Are you interested in creating a better version of you?
How long does it take you to care? Is it unconditional or forced?
I was given a small box of three (3) chocolates the other night by a young squirt hockey player at a small gathering. The power of this gesture will do more for this young man than he can imagine. The joy in my eyes while receiving showed him there is an importance to giving. I will guarantee you he will play better the next time he goes on the ice; especially if he is aware of how this works and affects the human mind. He will also do better in school. Thanks, Gage.
I decided to leave the gathering @ my first opportunity and write this; thinking of all the people who took time to care; whether bringing something, writing something, calling, texting, praying or doing something in their own way. They will all do better @ what they do because of it and they certainly will see a better version of their self.
Simply put; this is how life works! Caring, giving and sharing have the power to change your life in a positive way. When you embolden those in your care, this is the highest level of caring and doesn't cost you a cent. Clairene Grillo was an expert quietly doing this when in her presence. I, and thousands of others, are the beneficiaries.
By the way, the chocolates didn't last very long in the heart shaped Valentine Day box.


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Every Time Danny Cleary
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Cranky Doodle Daddy
No quit in this one
Join Date: Feb 2004
Location: Royal Oak
Posts: 1,326
vCash: 500
Originally Posted by
Trust me, a lot of his friends there including myself, raised the eyebrow and asked what he was doing there...you think we just sit there in awe of danny cleary?LOL
Cleary got paid his money for his 20 goal season, and now look at him, Injuries have barred him for past couple years, but hes NOT hurt anymore. He has taken the time off, has done what he has done to get back to playing, yet still hasnt shown what he once was. Lets not also forget the times he was playing golf on one knee, thus applying more pressure to his knee than needed. The guy IMO and from what I have seen, just doesnt care anymore. Knows hes a Wing, gets paid like a top 6 winger and accepts just floating around....
I don't get your reasoning, nor what you are expecting from Cleary. Is he just to go to the rink, come back and be bubble-wrapped until it's time to go to the rink again? And, since when does physical inability equate to lack of desire?
The one thing that I've seen and that is consistently reflected in this thread is that Danny has heart. I can accept that his playing days may be at an end but at least he's still leaving it all on the ice every night.
Cranky Doodle Daddy
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Home →
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Regional Highlights
UPDATED November 2016: Where to see The Nutcracker ballet on Long Island, including performances in Nassau and Suffolk counties.
Ohman's New York Dance Theatre The Nutcracker: Hofstra UniversityWHEN: Dec. 17-18, Saturday-Sunday, 12pm and 5pmWHERE: John Cranford Adams Playhouse, HempsteadAGES: ALLWHAT: Ohman's The Nutcracker in its 35th season bringing joy to young and old alike with added music, scenes, dances, and the only one on Long Island performed in the tradition of New York City Ballet's legendary George Balanchine.WANT TO GO? $42; $32 seniors and children. 631-462-0964. ohmanballet.org.
The Nutcracker performed by Eglevsky BalletWHEN: Dec. 17, 6pm; Dec. 18, 1pm, and 5pmWHERE: Tilles Center for the Performing Arts, 720 Northern Boulevard, GreenvaleWHAT: The production will feature new and innovative choreography by the Eglevsky Ballet's Director, internationally renowned artist, Maurice Brandon Curry. The show promises to delight with visions of a Sugar Plum Fairy, her dashing Cavalier, angels, dolls, and a Christmas tree that grows right before your eyes!WANT TO GO? $48-$78. tillescenter.org.
Bolshoi Ballet: The Nutcracker Sensory-Friendly ScreeningWHEN: Monday, Dec. 19, see website for timeWHERE: Farmingdale Multiplex Cinemas, 1001 Broad Hollow Road, Farmingdale WHAT: Fathom Events and Autism Society of America present a sensory-friendly cinema event direct from Moscow. The lights will be turned up, the sound will be turned down, and guests are welcome to be themselves and get up, dance, walk, shout, or sing. The two-hour and 15 minute screening of Bolshoi Ballet's The Nutcracker will be held in four New York theaters, including one in Suffolk County. See website for more details and times.WANT TO GO? See website for price. 800-315.4000. showcasecinemas.com or fathomevents.com.
Max and Ruby in The Nutcracker SuiteWHEN: Dec. 4, 4:30pmWHERE: NYCB Theatre at Westbury, 960 Brush Hollow Road, WestburyAGES: Young elementary, TweenWHAT: Max and Ruby bring to life a magical performance to give a fun filled day out for all the family! Join the friendly bunny sibling characters as well as an incredible cast of talented dancers and catch Tchaikovsky's Nutcracker Suite live!WANT TO GO? $39 and up. 516-247-5200. thetheatreatwestbury.com.
Bolshoi Ballet: The Nutcracker Sensory-Friendly ScreeningWHEN: Monday, Dec. 19, 6pmWHERE: Island 16: Cinema de Lux, 185 Morris Ave., Holtsville AGES: 5 and olderWHAT: Fathom Events and Autism Society of America present a sensory-friendly cinema event direct from Moscow. The lights will be turned up, the sound will be turned down, and guests are welcome to be themselves and get up, dance, walk, shout, or sing. The two-hour and 15 minute screening of Bolshoi Ballet's The Nutcracker will be held in four New York theaters, including one in Nassau County. See website for more details and times.WANT TO GO? See website for price. 800-315.4000. showcasecinemas.com or fathomevents.com.
Ovations Dance Repertory Company's The Nutcracker Suite with Live OrchestraWHEN: Dec. 10-11, Saturday, 1pm and 7pm; Sunday, 1pm and 6pmWHERE: Patchogue Theater for the Performing Arts, 71 E. Main St., PatchogueAGES: 5 and olderWHAT: Ovations Dance Repertory Company presents The Nutcracker Suite with a live orchestra by The Atlantic Wind Symphony.WANT TO GO? Contact the box office at 631-207-1313 or call Ovations at 631-218-0315 for more information and tickets. ovationsdance.com.
The Nutcracker Presented by Ballet Long IslandWHEN: Dec. 4, 2-3:30pmWHERE: Brentwood Public Library, 34 Second Ave., Brentwood AGES: ALLWHAT: Enjoy this timeless classic loved by all. This program visits the enchanted "Land of Sweets "and the famous well known music that everyone loves and dances to. The Sugar Plum Fairy and her Prince also perform solos and a Grand Pas De Deux. WANT TO GO? 631-273-7883. brentwoodnylibrary.org.
Northport Nutcracker BalletWHEN: Dec. 10-11, Saturday-Sunday, 2pm and 4pm WHERE: Northport Middle School, 11 Middleville Road, NorthportAGES: ALLWHAT: Northport Nutcracker Ballet by Posey Dance Repertory Company. There will be a meet and greet with dancers and photo opportunity after performance.WANT TO GO? $20. 1-855-222-2849. tututix.com/poseyschool.
American Dance Theatre of Long Island Presents Clara's Dream: The NutcrackerWHEN: Dec. 2-4, times varyWHERE: Patchogue Theater for the Performing Arts, 71 E. Main St., PatchogueAGES: 5 and olderWHAT: Toys come to life, a Christmas tree grows before your eyes, flowers dance, and snowflakes waltz in this classic story.WANT TO GO? $17 and up. 631-207-1313. patchoguetheatre.com.
The NutcrackerWHEN: Dec. 28-29, 3pm and 7pmWHERE: Noel S. Ruiz Theater at CM Performing Arts Center, 931 Montauk Highway, OakdaleWHAT: This year's performance of The Nutcracker promises to deliver it's usual magic in a new theater with a cast of professional dancers all of whom are members of the Ballet Long Island joined by students of The Ballet Center. ?There will be a Nutcracker boutique in the lobby.WANT TO GO? $40. 631-737-1964. balletlongisland.com.
The Nutcracker Act IIWHEN: Nov. 30 and Dec. 21, 11am and 12:15pm; December 3, 1pmWHERE: Ballet Long Island, 1863 Pond Rd, RonkonkomaWHAT: Members of the Ballet Long Island joined by students of The Ballet Center present the second act of The Nutcracker. There will be an accompanying Nutcracker boutique in the lobby, offering a range of related memorabilia for fans of all ages.WANT TO GO? $18, $9 children and seniors. 631-737-1964. balletlongisland.com.
Seiskaya Ballet's NutcrackerWHEN: Dec. 16-19, times varyWHERE: Stony Brook University's Staller Center for the Arts, Stony Brook University/Staller Center, Stony BrookAGES: 9 and older WHAT: Hailed in its 1995 debut as Long Island's most lavish Nutcracker, the Seiskaya Ballet production of the classic holiday ballet returns to Stony Brook's Staller Center in a truly international collaboration, choreographed by world renowned Russian-born choreographer Valia Seiskaya.WANT TO GO? $40; $34 children and seniors. nutcrackerballet.com.
The NutcrackerWHEN: Dec. 17, 2pm and 4pm; Dec. 18, 12pm and 4pmWHERE: Huntington High School, 188 Oakwood Road, HuntingtonWANT TO GO? $30; $20 children and students. lynchballet.com.
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Kendrick Lamar - Ignorance Is Bliss + ON-SCREEN LYRICS MP3
Track #8 off of Kendrick Lamar's "(O)verly (D)edicated" Mixtape.
[HD] Ignorance is BLISS - Jellyfish MP3
Enjoy.. Comment, Rate, favorite.
Kendrick Lamar - Ignorance Is Bliss MP3
Kendrick Lamar O.D 9/15/10 Written by Kendrick Lamar Dir by dee.jay.dave & O.G Michael Mihail.
Kollektivet: Music Video - Ignorance is bliss MP3
Se Kollektivet hver fredag kl. 16:55 på TV 2! Programmet ser du også gratis på TV 2 SUMO: http://webtv.tv2.no/webtv/sumo/?treeId=3457 www.tv2.no/kollektivet ...
Ignorance is Bliss HD MP3
Animation music video featuring Bowser and Princess Toadstool. Soundtrack is "Ignorance is Bliss" by Jellyfish. Uploaded by request, I did not make this.
Ignorance is bliss? MP3
Is ignorance bliss? Real talk. Just wanted to have a conversation with you all about stuff that's been on my mind. Let me know your thoughts in the comments ...
jellyfish / ignorance is bliss MP3
Kendrick Lamar - Ignorance Is Bliss ft. Kendrick Lamar MP3
In this video, Kendrick Lamar writes a song based on a true story - "Ignorance Is Bliss." Pre-Order "good kid, m.A.A.d city" http://smarturl.it/gkmc.
Ignorance Is Bliss
Kendrick LamarLord forgive me [Verse 1:] Kill him where he stand or stand over him shake his hand Then jump back in that mini van, double back to his block And blam I ain't backing down for nothing I'm a back em down like Shaq with this black 2-2-3 in my hand Better pray that this chopper jam, like a radio single, man Police radio signals sayin' that a 187 land on your corner, Coroners comfort your mama, mama he's dead, The next morning hi[...]
3 Years Anniversary - Ignorance is Bliss [Re-Uploaded] MP3
Just because! reuploaded for pleasant seekers the pitch is just a little higher, it got better that way! JAG ÄLSKAR DEJ IDA.
Beartooth - Ignorance Is Bliss (Audio) MP3
Vote for Beartooth in the Alternative Press Music Awards! http://www.altpress.com/static/apmasvote.html "Ignorance Is Bliss" from Beartooth's album 'Disgusting' ...
Kendrick Lamar - Ignorance is Bliss [Lyrics] MP3
Off Kendrick Lamars O.D. mixtape [Lyrics] Lord forgive me [Verse 1] Kill him where he stand and stand over him shake his hand Then jump back in that mini van, ...
Living Colour - Ignorance Is Bliss (live) MP3
Living Colour - Ignorance is Bliss 1993 - David Letterman Show http://www.livingcolour.com http://www.facebook.com/livingcolour http://twitter.com/LivingColour ...
ROOM 94 - Ignorance Is Bliss (Official Music Video) MP3
Official Music Video for ROOM 94's single 'Ignorance Is Bliss' - OUT ON ITUNES NOW! Thanks! www.facebook.com/ROOM94 www.twitter.com/ROOM94 www.
Ignorance is Bliss - Scene from the Matrix MP3
In this scene Agent Smith talks to Cypher, who admits that he is willing to betray his friends outside of the Matrix, in order to get a life inside of the Matrix, ...
"Ignorance Is Bliss" By: Beartooth MP3
Disclaimer: I do not own any of the audio used in this video** Tumblr: http://www.tumblr.com/blog/scooby5250 Twitter: https://twitter.com/xxalex1021xx Instagram: ...
Ramones - Ignorance is Bliss MP3
Ignorance is Bliss by Ramones.
Face to Face | Ignorance Is Bliss (Full Studio Album) MP3
Ignorance Is Bliss was the fourth studio album by American band Face to Face. It was released under Beyond Records in 1999. Tracklist: 1. Overcome 0:00 2.
Flatbush Zombies - Ignorance is Bliss MP3
From the mix tape "Better Off Dead" I do not own the rights of this song. Enjoy! #zombiesforprez.
Kendrick Lamar - Ignorance Is Bliss (Braeden Bailey Remix) MP3
Vibe Life | Braeden Bailey - Ignorance is Bliss [Kendrick Lamar Flip] » SUBSCRIBE for more: http://bit.ly/vibelife » Like us on Facebook: ...
RZL DZL - "Ignorance is Bliss" Music Video MP3
Lockin Out Records Director: Sikander Khan.
The Alan Parsons Project - Ignorance Is Bliss MP3
Every once in a while in your life, a song will speak to you because it's happening right then and there. There was a time in my own life where I had tears ...
Rant! Bleach "Article" Ignorance Is Bliss! MP3
There are Problems in Bleach, Like in every Series. But its not an Abomination as you make it out to be. Get Hanson'd & Subscribe! :D http://tinyurl.com/nnacpgn ...
Ignorance is Bliss MP3
Ajahn Brahm muses about a famous old saying coined by Thomas Gray: ignorance is bliss.
[FR] Kendrick Lamar - Ignorance Is Bliss [Traduction / Sous-Titres] MP3
Dr. Dre à découvert Kendrick en écoutant ce son (sorti en 2010), et a décidé de signer un contrat avec lui. C'est ce morceau qui a propulsé la carrière du good ...
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ALICE LEWIS - IGNORANCE IS BLISS OUT MARCH 17/2014 Available on iTunes : https://itunes.apple.com/fr/album/ignorance-is-bliss-ep/id832376052 ...
Ignorance Is Bliss [Clean][Best Edit] - Kendrick Lamar MP3
Overly Dedicated No copyright infringement is intended - If you are the rightful owner of the content in this video and wish it to not be on my channel, message ...
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Follow Me: http://twitter.com/DJLobotomize Albums & Mixtapes:http://DJLobotomize.bandcamp.com Much appreciated if you subscribe to my channel.
Blue Bloods - Ignorance Is Bliss MP3
Frank makes a speech following a series of terrorist attacks before sitting down for dinner with the rest of the Reagan family. Subsribe to the "Blue Bloods" ...
Mario Kart Wii - Cheater Detection/Blocking System - Ignorance is Bliss (Work In Progress!) MP3
This video was made in a private room, so no complaints allowed! Compare the two videos side by side to get an idea of what's going on. Currently I am working ...
C2B3 - Ignorance Is Bliss MP3
C2B3 - Ignorance Is Bliss.


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Karmic Clouds is a amazing flavor, With 4 flavors together this blend is as delicious as it is complex yet tastes so simple. Just smoooth flavor
Flavor Profile:
Can I return if I don't like my order?
Yes you can. We have a 100% Satisfaction Guarantee
Do I need to Steep my juice?
Nope. Your juice will be delicious when you get it. With that said you will bring out more flavor by letting it sit in a dark, cool area for a while.
Will my Coils Gunk Up?
Nope, Our juice will not produce gunk. We do not use Sugar sweeteners. Thus your cotton, coils, and heads will last 5-10 X longer as a result. Check out this Infographic of a Month long E-juice Gunk test for the results.