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Manager, Publisher Services (Inside Sales)
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We spend a great deal of our time online. Whether it's for information, commerce, or entertainment, each of us has come to depend on what we research, discover, and share. Publishers – those who create and curate content – are what makes the Internet great. Yet these publishers practice their craft largely alone, in siloes – without reference points or insightful understanding about where they sit in the grand scheme of things. To add to the challenge, once a Publisher's content is in the wild, then the task of building engagement, growing a loyal following and enriching the engagement with that following can sometimes feel like shots in the dark or worse, a black box. Moreover, making money from their craft can be a complex task for any independent publisher who might prioritize generating content first and money second.
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Recently portable Raman probes have emerged along with a variety of applications, including carbon nanotube (CNT) characterization. Aqueous dispersed CNTs have shown promise for biomedical applications such as drug/gene delivery vectors, photo-thermal therapy, and photoacoustic imaging. In this study we report the simultaneous detection and irradiation of carbon nanotubes in 2D monolayers of cancer cells and in 3D spheroids using a portable Raman probe. A portable handheld Raman instrument was utilized for dual purposes: as a CNT detector and as an irradiating laser source. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) were dispersed aqueously using a lipid-polymer (LP) coating, which formed highly stable dispersions both in buffer and cell media. The LP coated SWCNT and MWCNT aqueous dispersions were characterized by atomic force microscopy, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy and Raman spectroscopy. The cellular uptake of the LP-dispersed SWCNTs and MWCNTs was observed using confocal microscopy, and fluorescein isothiocyanate (FITC)-nanotube conjugates were found to be internalized by ovarian cancer cells by using Z-stack fluorescence confocal imaging. Biocompatibility of SWCNTs and MWCNTs was assessed using a cell viability MTT assay, which showed that the nanotube dispersions did not hinder the proliferation of ovarian cancer cells at the dosage tested. Ovarian cancer cells treated with SWCNTs and MWCNTs were simultaneously detected and irradiated live in 2D layers of cancer cells and in 3D environments using the portable Raman probe. An apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay carried out after laser irradiation confirmed that cell death occurred only in the presence of nanotube dispersions. We show for the first time that both SWCNTs and MWCNTs can be selectively irradiated and detected in cancer cells using a simple handheld Raman instrument. This approach could potentially be used to treat various diseases, including cancer.
Portable Raman probes have made their way into the basic science laboratory through their use to detect and identify specific biological markers and organic molecules 1-2, but so far their usefulness has not been tested for pre-clinical theranostic applications. Herein we have studied the applicability of portable Raman probes in combination with nanomaterials for theranostics platforms. With the advent of nanotechnology, nanomaterials have provided a variety of options for biomedical applications 3-5. Carbon based nanomaterials, in particular, are of great interest internationally, because of their applicability to biomedicine 6. Among the carbon based nanomaterials, carbon nanotubes (CNTs) have excellent electro-optical properties, high tensile strength, and a high surface area-to volume ratio that facilitates surface functionalization 7. CNTs comprise single-walled carbon nanotubes (SWCNTs), double-walled carbon nanotubes, and multi-walled carbon nanotubes (MWCNTs) 8-10. The above mentioned physical properties have enabled tailor-made CNT conjugates, such as drug delivery vehicles 11 suitable for loading of various chemotherapeutic drugs (e.g. cisplatin 12, doxorubicin 13, paclitaxel 14), and gene delivery vectors 15-16 (such as siRNA) for gene therapy. The toxicity of pristine CNT aggregates can be ameliorated by adding a designer coat of biocompatible material to make the nanotubes highly dispersible in aqueous solution 17-18. Various oligonucleotide sequences, surfactants and polymers can be employed to render CNTs dispersible 19-21. The most common of these dispersants is polyethylene glycol (PEG), an FDA approved polymer used in the pharmaceutical industry 22-23. Several recent publications 24-26 have found PEGylated CNTs to be non-cytotoxic.
Raman spectroscopy, which determines the vibrational, rotational, and other lower frequency modes of a nanosystem, has been one of the key tools for characterizing CNTs 27-28. Raman spectra give unique signature peaks that can identify the CNTs, such as the radial breathing mode (RBM) in which all the carbon atoms are moving in-phase in the radial direction. Depending on the CNT diameter, the intensity could be strong or weak 29. The RBM band is detected at wave numbers of around 200 - 330 cm-1, and the Raman signal strength depends on the CNT diameter. In graphitic material, there is another specific spectral feature, known as the graphene peak or G band, which arises from neighboring atoms moving in opposite directions along the surface of the tube, as well as a second-order related harmonic G' band 30. The G band peak occurs around 1500 - 1600 cm-1 and the G' is visible around 2600 - 2700 cm-1. The synthesis of CNTs using metal catalysts gives rise to structural defects producing a dispersive disorder induced peak in the Raman spectrum. This defect peak or D band occurs at wave numbers around 1300 cm-1 31. These spectral features enable the identification of different types of CNTs. Recently Raman spectroscopy has also been used to track CNTs both in vitro and in vivo, giving rise to a new non-invasive imaging mode to track these nanomaterials 24, 32-34.
In this study we made combined use of the unique features of portable Raman probes and CNTs to create a theranostic platform for ovarian cancer 35. Ovarian cancer has a very high mortality rate for women around the world 36. Therapies based on CNTs have shown promising results in pre-clinical experiments by acting as vehicles to deliver drugs and genes to cancer tumor cells both in vitro and in vivo 37. CNTs have also been used for pre-clinical photothermal therapy of breast cancer 38-41. In this study we report a unique way of combining CNTs and portable Raman probes to effect simultaneous detection and photothermal ablation of ovarian cancer cells (Supplementary Material: Figure S1). Tissue exhibits a deep penetrability with very low absorption of near-infrared (NIR) photons in the wavelength range of 700 to 1100 nm 42-43. In our study we utilized SWCNTs and MWCNTs to kill ovarian cancer cells in vitro by selectively detecting and irradiating the CNTs both in 2D cell culture and 3D spheroids by deploying a portable Raman detector together with a handheld Raman laser probe. This study suggests that handheld portable devices used with specially designed theranostic nanomaterials could potentially lead to low cost therapies.
Functionalization of Carbon nanotubes. SWCNTs (Aldrich, MO) and MWCNTs (Helix, TX) were coated with phospholipid polyethylene glycol amine (LP) (1,2-distearoyl-sn-glycero-3-phosphoethanolmine-N-[amino(polyethyleneglycol)-2000] (ammonium salt) from Avanti Polar Lipids, Inc., AL) to render CNTs dispersible in aqueous medium. To accomplish this SWCNTs (1 mg) or MWCNTs (1 mg) were added to an aqueous solution of LP (1 mg ml-1) and sonication was carried out with the help of an ultra sonicator (Qsonica, CT) for 3 h. This process resulted in aqueous dispersed CNTs. To remove any uncoated SWCNTs or MWCNTs, the LP coated CNTs were centrifuged for 2 h at 10,000 rpm. After the centrifugation the bare uncoated nanotubes settled at the bottom of the vial. The supernatant was recovered, and unreacted LP was removed using a 5K MW centricon filter and stored for later use.
FTIR of Functionalized CNTs. Fourier transform infrared (FTIR) spectroscopy of LP coated SWCNT and MWCNT nanoconjugates, and of pristine SWCNT and MWCNT controls, was done using KBr pellets of each. The wave numbers of the transmittance of each sample were recorded using a Perkin-Elmer spectrum GX spectrophotometer.
Fluorescence Spectrometry. Fluorescence spectrometer was used to assess the presence of FITC on the LP coated SWCNTs or MWCNTs. For this fluorescence spectra of FITC conjugated to LP coated SWCNTs or MWCNTs (with pristine SWCNTs and MWCNTs as controls) were obtained using a Perkin-Elmer LS55 fluorescence spectrometer.
Atomic Force Microscope analysis of CNT nanoconjugates. Samples for AFM were prepared and analyzed as described with modifications 44-45. Briefly, biological AFM imaging of the carbon nanotube samples was carried out under a range of deposition conditions in fluid, rinsed with deionized water and dried under a gentle flow of N2 or Ar gas, using a gentle tapping-mode on a PicoForce Multimode AFM (Bruker, CA) consisting of a Nanoscope® V controller, a type E scanner head, and a sharpened TESP-SS (Bruker, CA), OMCL (Bruker, CA), or similar AFM cantilever. For either SWCNT or MWCNT sample visualization, suitable surface attachment of the respective sample was achieved readily by 5 min incubation of 20 μL of the fluid suspension on freshly-peeled mica disks of 12 mm diameters. The excess fluid was blown away by an inert gas gust, or blotted with filter paper, followed by two rinses each with 30 μL of deionized water to remove salt deposits and by a complete drying of the sample surface under gentler gas flow. The sample was then sealed into the instrument compartment (dehumidified by Drierite® particles) and imaged via standard optimizations. AFM images were evaluated within the Nanoscope software (version 7.2, Bruker), and exported to Image J (version 1.4x, NIH, Bethesda, MD) for further analyses and display.
Zeta Potential. To find the surface charge of LP coated SWCNTs and MWCNTs, zeta potential analysis was carried-out using a Zetasizer Nano series (Zen3600) from Malvern with Zetasizer software 6.0 as the interface.
Raman analysis of CNT nanoconjugates. Raman characterization was used for the analysis of the functionalized LP coated SWCNT and MWCNT samples for specific signature peaks along with controls consisting of pristine SWCNTs and MWCNTs. Raman characterization of aqueous dispersed CNTs was done by using characteristic SWCNT/MWCNT Raman signature peaks (G and D bands). Raman spectra were recorded on an iRaman 785 (B&W Tek, Inc, DE) spectrometer equipped with a microscope attachment, and laser spot size was focused to 1 μm diameter (×100 objective), with a power output of 35 mW. The excitation source was an argon ion laser, 785 nm.
2D and 3D cell culture. For 2D cell culture, OVCAR8 ovarian cancer cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (FBS) at 37°C in 95% air/5% CO2. Freshly plated cells were grown overnight, to 50-70% confluency prior to incubation for 3 hrs with 200 μL RPMI containing either LP coated SWCNTs or MWCNTs, and after 3 washes with PBS the treated cells were incubated further in fresh RPMI containing serum and carefully monitored microscopically (Axiovert 200m; Carl Zeiss). For 3D cell cultures, OVCAR8 cells were trypsinized, collected, mixed with matrigel, and plated in 8 well chambered cultured slides, which had been previously coated with a thin layer of matrigel. The matrigel mixed cells were observed for the spheroid formation.
Cell proliferation (MTT) assay. Cells were grown to 50-70% confluency overnight in 96 well plates. Next, the media was aspirated and the cells incubated with fresh media containing either LP coated SWCNTs or MWCNTs at various concentrations along with control cells for 48 h. Post treatment the cells were washed 2 times in PBS, and then incubated for an additional 24 h in fresh media. MTT was assessed using a CellTiter 96 AQ One Solution Cell Proliferation Assay kit (Promega, WI), and measured optically at 570 nm.
TUNEL assay. An APO-BrdU TUNEL (Terminal transferase dUTP nick end labeling) assay kit (Invitrogen, CA) was used to detect cells undergoing apoptosis. Briefly, cells were grown onto 8 well chambered cell culture slides until 50-70% confluent, followed by treatment with either SWCNTs or MWCNTs. Control cells were grown in the absence of CNTs. After treatment, the cells were washed in PBS (3x for 2 min each) and incubated in fresh media for 1 h. Next, cells were fixed in 3.5 % PBS-formaldehyde for 15 min at RT, rinsed in PBS (3x) and permeabilized in 0.5 % PBS-Tween 20 for 5 min at RT followed by DNA end-labeling for 1 h at 37 °C. Then, the labeled cells were washed, and incubated further with 95 μL antibody staining solution (AlexaFluor 488-conjugated anti-BrdU) for 30 min at 37 °C, followed by additional washes (3x). Fluorescence image acquisitions were performed with an Axioplan 2 (Carl Zeiss).
Confocal microscopy. OVCAR8 ovarian cancer cells were grown 50-70% confluent on 8-well chambered LabTek II coverglass, treated with either FITC or Qdot conjugated LP coated SWCNTs or MWCNTs, as described above and, incubated for 3 h. Cells were next washed with PBS (3x) and resuspended in fresh media. Live cell imaging was performed using an inverted Zeiss LSM 700 confocal microscope equipped with a CO2 module, heating unit and heating plate, using a 40x/0.75 M27 EC Plan-Neofluar objective. Imaging was carried out at 37°C in 5% CO2 with cells plated in LabTek II coverglass (Nalge Nunc International, NY). Images were acquired and processed with the Zeiss Zen 2009 image software. The fluorescence micrographs shown are representative of at least three independent experiments. Average fluorescence intensity was quantified using Zen 2009 software.
Transmission electron microscope imaging of SWCNTs/MWCNTs. A specimen of LP-coated SWCNTs or MWCNTs for TEM imaging was prepared by depositing a 3 μL droplet from the aqueous solution onto a Quantifoil grid and letting it dry in air. After adsorption for 3 min, the excess solution was blotted with filter paper, washed with a few 3-μl droplets of de-ionized water in order to remove any dirt, and left to dry. Transmission electron micrographs were recorded by means of a Tecnai TF30 TEM (FEI, Hillsboro, OR) equipped with a Gatan Ultrascan 1000 CCD camera (Gatan, Pleasaton, CA).
Preparation and characterization of CNT nanoconjugates. To enable CNTs to be used for photothermal therapy it is essential to preserve their structural integrity and, thus, the spectral response of the nanomaterial. The SWCNTs and MWCNTs were dispersed in a phospholipid-polyethyelene glycol (LP) containing a terminal amine functional group. LP-coated CNTs obtained by sonication in aqueous medium were a highly water dispersible CNT suspension without any strong visual aggregation. The CNT suspension was stable for at least two weeks, which can be attributed to the strong adsorption of the lipid moieties to the surface of the CNTs. The CNT nanoconjugates were characterized by atomic force microscope (AFM, Figure Figure11A&B) and transmission electron microscope (TEM, Figure Figure11C&D) for length and diameter distribution. AFM data showed that the SWCNTs were 50 - 500 nm in length with a few over 1000 nm and 0.5 - 1.5 nm in diameter, whereas MWCNTs were somewhat shorter in the similar range of the length but about 10 - 30 nm in diameter indicating a higher degree of CNT dispersion apart from a few smaller CNT bundles. Similar data resulted from the TEM analysis with SWCNTs having diameters of 0.5 - 1.5 nm and MWCNTs having diameters of 10 - 30 nm, supporting the fact that the LP coating yielded mostly debundled individual SWCNTs and MWCNTs. To make sure that the intrinsic electronic and spectroscopic properties of SWCNTs and MWCNTs were not significantly affected by the LP coating, a Raman spectroscopic analysis was carried out (Figure Figure11E&F). Raman spectra of LP coated SWCNTs and MWCNTs were recorded using a handheld portable iRaman 785 (B&W Tek, Inc, DE) laser probe (Supplementary Material: Figure S2). SWCNTs showed well resolved G and G' band peaks at 1590 cm-1 and ~2600 cm-1, respectively, and an RBM band was visible at 390 cm-1 together with a D band at ~1420 cm-1. The Raman spectra clearly show that the fundamental signature peaks of SWNCTs were preserved after the coating. MWCNTs also showed a G band at ~1590 cm-1 and G' band at ~2600 cm-1, although it should be noted that the Raman intensity was weak compared to that from SWCNTs at 785-nm laser excitation, as has been reported previously 46.
To evaluate the CNT nanoconjugates further with regard to their surface charges and functional groups, zeta potential analysis and Fourier transform infrared (FTIR) spectroscopy were carried out. FTIR spectra indicated the presence of the LP coat (Figure Figure22A). LP coated SWCNTs and MWCNTs were further conjugated with a fluorescent dye, fluorescein isothiocyanate (FITC), and characterized by spectrofluorometry (Figure Figure22B). The spectrum showed a peak at 521 nm, which is the emission maximum for the unconjugated control FITC; the dye-labeled SWCNTs and MWCNTs also showed the peak at 521 nm, whereas control SWCNTs and MWCNTs lacking the dye did not show any peak in this spectral region. The data clearly showed that CNTs were labeled with FITC, which was later used to observe the cellular uptake of the CNT nanoconjuagtes. The zeta potential analysis gave us an indication of the extent of positive charge on the surface of the SWCNTs (18.5 mV) and MWCNTs (5 mV) (Figure Figure22C). The data also point towards a 2-fold higher coating of LP on the SWCNTs compared to that on the MWCNTs, suggesting that a higher molecular weight LP coating could yield still better CNT dispersions, especially for MWCNTs.
Cellular uptake and cell proliferation studies of LP coated SWCNTs and MWCNTs in ovarian cancer cells. Most previous studies involving CNTs as delivery vehicles have utilized targeting moieties that could help take up nanoconjugates into cells. However, in the present studies we kept the nanoformulation in its simplest form and used dispersed, LP-coated CNT nanoconjugates directly to treat the cancer cells. We first tested cellular uptake of the CNT nanoconjugates by using confocal fluorescence microscopy. OVCAR8 47 ovarian cancer cells were first cultured in 8-well chambered culture slides, and after reaching 70% cell confluency the cells were treated with either LP-coated SWCNTs or LP-coated MWCNTs, both of which had been labeled with FITC for an hour, after which the cells were washed twice with PBS. Fresh medium was added after the washing, and confocal Z-stack imaging was performed (Figure Figure33A&B). The Z-stack confocal fluorescence imaging clearly showed cellular uptake of SWCNTs and MWCNTs as evidenced by the green emission from the FITC dye at a wavelength of 520 nm. This result indicated that SWCNTs and MWCNTs could diffuse through the cell membrane and that the positive charge on the CNT surface mediated their cellular uptake, as had been observed in a previous report 48.
Once the cellular uptake of LP-coated SWCNTs and MWCNTs was determined, the effect of CNT nanoconjugates on the proliferation of OVCAR8 cells was tested. Cells were cultured in 96-well plates in triplicates, and when the cells were 70% confluent, they were treated with a range of doses of LP-coated aqueous dispersed SWCNTs or MWCNTs. After 48 hrs of treatment the cells were analyzed by an MTT cell proliferation assay to compare the proliferation of the cells alone (as a control) to the proliferation of cells treated with various doses of SWCNTs and MWCNTs ranging from 2 to 100 µg/mL-1. As shown in the plot in Figure Figure33C, the LP-coated SWCNTs and MWCNTs did not hinder proliferation of the OVCAR8 cells at the tested dosages. Higher doses of CNTs were found to be toxic (data not shown). To investigate irradiation of the OVACAR ovarian cancer cells we employed the highest dosage of the SWCNTs and MWCNTs that did not hinder the cell proliferation.
Live cellular detection of CNTs nanoconjugates using a portable Raman probe. For live cellular detection of SWCNTs and MWCNTs, a handheld portable iRaman instrument with a 785-nm laser source was utilized. Cultured OVCAR8 cells were first probed with a 785-nm laser using the iRaman laser probe, and the resulting spectrum did not show any peaks attributable to CNTs (Figure Figure44A). Next the OVCAR8 cells were treated with either LP-coated SWCNTs or LP-coated MWCNTs and the laser irradiation was applied to the ovarian cancer cells. Distinct carbon nanotube peaks were observed in the Raman spectra (Figure Figure44B&C). The CNT Raman signals tended to decrease and disappear over time at certain spots in the culture well, whereas signals were retained for longer periods of time in other regions of the culture well. Possible explanations for this behavior are that the cells might be in dynamic motion within the culture well, or that SWCNTs/MWCNTs might be internalized by the cancer cells, thus giving more continuous signals than the signals from the non-internalized nanoparticles. To confirm this behavior we washed the cells with buffer, resuspended them in media, and probed them under the 785-nm laser light to see if we could obtain any CNT Raman signal. Indeed we observed Raman signals that were of lower intensity. Raman signals were also observed in the washing solution suggesting that unbound nanotubes that did not internalize were washed away. Clear MWCNT Raman peaks were hard to detect in vitro with the 785-nm laser light, because of the very low signal intensity. The observation that Raman signals were only detected when the laser probe traversed CNTs, and never in the absence of CNTs, showed the advantage of using a portable handheld device in combination with nanomaterials.
Live cellular simultaneous detection and irradiation of CNT nanoconjugates. For simultaneous detection and irradiation of the CNTs in vitro, a thermal profile of the nanotubes was first carried out (Supplementary Material: Figure S3). To achieve this, a k-type thermocouple was used to measure the temperature levels of the irradiated CNTs at different time intervals. SWCNTs/MWCNTs were introduced into the cell culture wells in the absence of cells. Raman laser light was shone onto the CNTs continuously using the 785-nm portable handheld iRaman laser probe. The temperature of the LP-coated SWCNTs/MWCNTs rose sharply to around 43 °C after 3 to 15 min, depending on the concentration of CNTs. We also found that the rise in temperature of MWCNTs was faster than that of SWCNTs. Cells treated with MWCNTs at the highest concentration that didn't show cytotoxicity in the cell viability assay reached a temperature of 32 °C after just 100 seconds of NIR irradiation. This compared to the 180 seconds that cells treated with SWCNTs took to reach the same temperature. OVCAR8 cells were treated for 3 hrs at this optimal dosage with LP-coated SWCNTs/MWCNTs and were then subjected to NIR laser irradiation in regions where the CNTs were traced with the 785-nm iRaman laser probe. Change in temperature of the cell media was recorded using the thermocouple which reached around 43 oC. After NIR laser irradiation the cells were fixed and stained for analysis using a TUNEL assay. Cells irradiated in the absence of CNTs showed neither the CNT peaks nor signs of apoptosis (Figure Figure55A&B), whereas LP-coated SWCNTs and MWCNTs both showed apoptosis induced by laser irradiation of the OVCAR8 ovarian cancer cells (Figure Figure55C-F). Interestingly, not only could we detect the CNTs in vitro, but we could also kill the cancer cells by prolonged irradiation of the SWCNTs/MWCNTs. TUNNEL staining of cells treated with CNTs in absence of laser did not show any signs of apoptosis (Supplementary Material: Figure S4). These results show promise for the use of portable handheld devices in combination with nanomaterials for detecting, thermally irradiating and killing cancer cells with minimal dosages of nanomaterials.
Simultaneous detection and irradiation of CNT nanoconjugates in a 3D environment. With the encouraging results from 2D cell cultures next we considered whether similar results could be obtained from 3D cell cultures, which would provide a more realistic model for the in vivo environment. OVCAR8 ovarian cancer cells were cultured in standard matrigel where the cancer cells grew in a three-dimensional geometry (Figure Figure66). 3D uptake of QSA580 (Ocean Nano Tech, LLC, AK)-labeled CNTs was assessed using Z-stack confocal imaging (Figure Figure66A). OVACAR8 cell spheroids were treated with LP-coated SWCNTs/MWCNTs for 3 hrs and then the spheroids were irradiated using the iRaman 785-nm laser probe. A strong SWCNT signal was detected, whereas the MWCNT signal was very weak and difficult to detect. Continuous irradiation showed a temperature rise that was detectable by a thermocouple. After irradiation cells were stained for TUNEL assay. Control spheroids that were irradiated in the absence of CNTs did not show green fluorescence. NIR laser on its own did not harm the cancer cells (Figure Figure77A&B). Cellular TUNEL staining indicated which cells were undergoing apoptosis (Figure Figure77C-F) following treatment with LP-coated SWCNT or MWCNTs and laser light. The intense signal of the SWCNTs could be due to more nanotubes being accumulated in the three-dimensional cellular structure than in the single layers of cells in the 2D culture. Although MWCNTs are also effective at killing cancer cells these nanoparticles are difficult to track with Raman probes. These results strongly suggest the advantage of using SWCNTs for simultaneous detection and killing of cancer cells using portable Raman probes. Addition of a targeting ligand onto the nanotubes would increase the target specificity.
Carbon based nanomaterials show a strong potential for biomedical applications, and carbon nanotubes are effective as drug and gene delivery vehicles. Nanotubes themselves can be used as "heat bombs" to kill cancer cells. Here, we have shown how the unique Raman properties of SWCNTs/MWCNTs in combination with a portable handheld device can be employed to kill cancer cells simultaneously by means of thermal irradiation. SWCNTs/MWCNTs can diffuse into and be taken up by cancer cells grown in a three-dimensional environment. Our results indicate that MWCNTs have a higher thermal generation capacity but are harder to detect because of the low Raman signal intensity at a wavelength of 785 nm. We have found that the SWCNTs provide a more suitable platform, because —in addition to being able to raise cellular temperatures—they can be detected easily.
Figure S1-S4.
This research was supported by the Intramural Research Program (IRP) of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH).
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Paul McCartney Plays Tribute to Jim Marshall (A Top Story)
Metallica's Kirk Hammett has also paid tribute to Marshall's amplifiers. "A Marshall amp had a clarity, a punch, a low end and had a type of distortion that wasn't around before Jim Marshall invented it," Kirk said in an interview with the New York Times.
The U.K's Download festival has also named its main stage for 2012 after Marshall. Organizer Andy Copping said, "The music industry lost an innovator and a legend with the sad passing of Jim Marshall. We had to remember him in some way and having the main stage dedicated to his name seemed the perfect tribute." - Check out the McCartney tribute here.
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They're massing in Virginia right now, preparing for a weekend assault on Washington, D.C. Who, you ask? The British, the Taliban, the Confederate Army?
No, it's America's truckers, or at least some of them. A loose confederation of independent truckers has vowed to run huge convoys of trucks around the Capital Beltway that circles Washington, D.C., but never penetrates it, shutting the region down during the Friday rush hour and throughout the weekend.
Of course, the region is usually pretty well shut down by traffic anyway, so there's some question whether anyone will notice. And then there's the little matter of Congress, which last time we checked convenes in the Capitol, which is smack in the middle of D.C., far from the noisy, dirty Beltway, which carries I-95 and I-495 through the area.
Most Congressmen bunk near the Capitol when they're in town and few ever have occasion to take the Beltway. So it's mostly furloughed federal workers and the few million other residents of Maryland and Virginia that will feel the brunt of the truckers' wrath, if any.
"Riding for the Constitution"
What's it all about? Why, it's about the Constitution, of course, something everyone talks about lately but which few have studied. Oh, a few people manage to read the thing but it takes quite a bit of non-ideological study of the actual history of the document and its interpretation by the courts over the years to really understand it.
Driving a truck -- or shouting loudly on TV, for that matter -- doesn't leave much time for studying constitutional law, so whatever argument is being advanced is likely to be rather basic.
It's surely not high fuel prices the truckers are mad about. Prices are relatively low right now, thanks to the economy once again heading for the tank as Congress diddles with the nation's wealth. Could it be the dread Obamacare that has driven the truckers to try extreme measures?
We're not sure, quite honestly. The truckers' website says they'll be "riding for the Constitution." Perhaps more significantly, it speculates the weekend of driving in a circle will spread into a nationwide truckers strike that will soon leave supermarket shelves empty.
Maybe that will happen about the same time the U.S. defaults on its debt. That will certainly get everything straightened out and back on track.


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June 20, 2007
DAVID MORA, PETITIONER,v.UNITED STATES OF AMERICA, DEFENDANT.
In 1995, pro se petitioner David Mora ("Mora"), together with four co-defendants, was convicted after a fourteen-week trial of several crimes related to organized narcotics trafficking. Following conviction, petitioner was sentenced to four terms of life imprisonment. His conviction was affirmed and certiori was denied. In an order dated September 28, 2001, the Court denied a petition filed by Mora under 28 U.S.C. § 2255. On December 26, 2001, the Court denied a certificate of appealability. On February 27, 2001, the Second Circuit also denied a motion for a certificate of appealability. On August 3, 2006, the Court denied petitioner's motion pursuant to Rule 60(b), seeking relief from the September 28, 2001 order denying the Section 2255 petition. The Court also denied petitioner's motion to amend his Rule 60(b) motion.
By letter dated December 29, 2006, petitioner inquired into whether a decision had been rendered on his Rule 60(b) motion and whether a decision had been made following an evidentiary hearing in a separate action brought by petitioner, with the case number CV-02-5973.*fn1 The Court responded by letter dated January 16, 2007, enclosing a copy of the August 3, 2006 order, as well as a copy of an order dated November 10, 2005 dismissing the action numbered CV-02-5973. Petitioner received the Court's letter on January 19, 2007. The next day, petitioner submitted a request to Bureau of Prisons staff to ascertain whether his institution's mail room had received a copy of the order around the time the order was originally issued. Mail room staff responded that no such copy was received at that time. Presently before the Court is petitioner's Motion to Reconsider and Resubmit New Judgment Securing the Right to Appeal Orders of November 10, 2005 and August 3, 2006 pursuant to Federal Rule of Civil Procedure 60(b). For the reasons set forth below, petitioner's motion with respect to the August 3, 2006 order, which I have construed as a motion to reopen his time to file an appeal*fn2 , is denied.
When the United States is a party to a civil case, notice of appeal "may be filed by any party within 60 days after the judgment or order appealed from is entered." Fed. R. App. P. 4(a)(1)(B). When a Section 2255 motion has been denied, "the 60-day time for appeal [runs] from the entry of the order denying [the] section 2255 motion." Williams v. United States, 984 F.2d 28, 30 (2d Cir. 1993) (finding that time runs from entry of order rather than entry of judgment "[s]ince no judgment was required to be entered"). In this case, the 60-day period during which petitioner could have appealed the August 3, 2006 decision lapsed on October 2, 2006.
Under Federal Rule of Appellate Procedure 4(a)(6)*fn3 , the district court may reopen the time to file an appeal for a period of 14 days after the date when its order to reopen is entered, but only if all the following conditions are satisfied:
(A) the court finds that the moving party did not receive notice under Federal Rule of Civil Procedure 77(d) of the entry of the judgment or order sought to be appealed within 21 days after entry;
(B) the motion is filed within 180 days after the judgment or order is entered or within 7 days after the moving party receives notice under Federal Rule of Civil Procedure 77(d) of the entry, whichever is earlier; and
(C) the court finds that no party would be prejudiced.*fn4
Fed. R. App. P. 4(a)(6). Federal Rule of Civil Procedure 77(d) provides in pertinent part that [i]mmediately upon the entry of an order or judgment the clerk shall serve a notice of the entry in the manner provided for in Rule 5(b) . . . , and shall make a note in the docket of the service . . . . Lack of notice of the entry by the clerk does not affect the time to appeal or relieve or authorize the court to relieve a party for failure to appeal within the time allowed, except as permitted in Rule 4(a) of the Federal Rules of Appellate Procedure.
Fed. R. Civ. Proc. 77(d).
The government does not dispute that petitioner has met prongs (A) and (C) under Rule 4(a)(6). The only issue is whether petitioner timely filed his motion according to the time limits under prong (B). To meet that requirement, petitioner had to have filed his motion within 180 days after entry of the order on August 3, 2006 or seven days after he received notice of the order, whichever is earlier. With respect to the first time period, 180 days had run by January 30, 2007. Regarding the second period, Mora received the January 16, 2007 letter from the Court on January 20, 2007, at the latest. On that date, Mora filed his Inmate Request to Staff form regarding his failure to receive the order in earlier mailings. Petitioner's Motion to Reconsider, p. 5. Based on this date, the most advantageous date for petitioner, the seven-day period after receiving notice lapsed on January 27, 2007. The date of January 20, 2007 governs because it is the earlier of the two operative dates under subpart B of Rule 4(a)(6). Under the "prison mailbox rule," a submission is considered to have been filed "at the moment of delivery to prison authorities for forwarding to the district court." Walker v. Jastremski, 430 F.3d 560, 562 (2d Cir. 2005); see also Fed. R. App. R. 4(b)(1). Mora's motion is dated February 17, 2007, the earliest date on which petitioner could have delivered his submission to prison authorities and 21 days after January 27, 2007. Therefore, Mora's motion to reopen is untimely. Because Mora cannot comply with the requirements of Rule 4(a)(6), his motion to reopen is denied.
For the foregoing reasons, petitioner's motion to reopen the time to file an appeal is denied. The clerk is directed to mail a copy of this decision to all parties.
Charles P. Sifton (electronically signed) United States District Judge
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Griffin debuts iPod flashlight, laser pointer add-ons
Griffin Technology has quietly introduced iBeam, a set of two small iPod attachments that turn the music player into a flashlight or a class IIIA laser pointer. The iBeam sells for $19.99 and will begin shipping by the end of October. Both accessories come with a protective snap-on cap that can be attached to any keychain.
"The iBeam flashlight is surprisingly functional when a quick beam of light is required in a dark car or doorstep. The iBeam laser pointer is an amazingly powerful red laser beam that is capable of being seen over a quarter of a mile away. Need we say how much fun this could be?"
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Powertrains Section Training Bench Fr - Chinaprice: 2718.00 Dollar US$
Company Contact:
- Posted By: FXB CO., LTD.
- Phone: 0086-0755-28077519
- Address: NO.5,ZHANGBEI INDUSTRIAL DISTRICT,ZHANGBEI RD.,AILIAN,LONGGANG AREA,SHENZHEN,P.R.CHINA, shenzhen, guangdong, China
- Email:
- Website: http://www.fxbgroup.cn
Published date: June 27, 2016 -
- Business Description: FXB was founded in 2003, committed to the development and production of automotive teaching aids products and software.
Settled in 13,000 square meters,it equips full equipment of modern industrial production plant and the independent industrial park of integrated office supporting.And our products have been sold to lots of countries and areas around the world.
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Common Rail Diesel Engine Training BenchEducation / Training - FXB CO., LTD. - China - June 27, 2016 - 10779.00 Dollar US$
i.About The Product The device is designed based on the Gonow 2.8TC electronically-controlled high-pressure common rail diesel engine (BOSCH common rail system) to simulate engine startup, speedup, slowdown and other actions, so as to illustrate the ...
Gasoline Engine Electronic Control System Training PanelEducation / Training - FXB CO., LTD. - China - June 27, 2016 - 3044.00 Dollar US$
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Ignition System Training PanelEducation / Training - FXB CO., LTD. - China - June 27, 2016 - 2817.00 Dollar US$
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text:
Nerja, Costa del Sol near Malaga - Suits 2 or 3 Families or Large Groups. Affords Individual Space to Each
Large villa with private swimming pool & parking spaces for 2 cars. Located on edge of Nerja, yet close enough to walk to the beach in a couple minutes and the town centre in 20 mins. Internet. Split into 3 self contained apartments, each with its own private sun terraces and dramatic sea and mountain views. Suitable for large groups of 2 or 3 families/generations etc, who want to go on holiday together, but can still have their own space at any time. Can be rented out on an individual basis to smaller groups of people. The large pool is surrounded by ample terrace and is sectioned off with toughened glass which, when in the pool, gives an 'infinity effect'. Almost every room has fantastic sea views and watching the sunrise/set over Nerja is unforgettable. Pristine condition throughout. Has all you need. Larger groups eg, sports clubs, wedding parties, etc can be catered for by combining the next door 'Orange Villa' to sleep 20 - 22 people but book early to ensure availability of both.
Top Traveller Review
Amazing place.
About the Owner
i am a business owner and qualified staff nurse, I'm 48 years old, married with one daughter and two lhasa apso dogs. I live in a small town called Holywood just outside Belfast.
i strive to keep my tenants as comfortable, cared for and as happy as possible. so far it's working according to the guest book comments!
what's really important is a top notch management company who keep everything spotless, well oiled and running smoothly. my tenants are always happy with how well they are looked after, and it took me a while to find the right people for the job. it's all very well and good having a beautiful villa but i believe the secret is in the running, cleaning and management of it.
Guestbook comments from the owner
What a fantastic holiday for adults & children! Casa Lola was just as described but even more beautifully located than we could imagine. The views from every room were amazing, you couldn't ask for better scenery. Layout of the villa was perfect for us as it was separated into 3 separate apartments which gave us all a chance to have our own space when needed especially to give the children all time to chill out & watch TV. Everything we needed for a lovely relaxing holiday was at hand, & Barry was at the end of the phone if we ever needed help. The transfers arranged for us were faultless and everyone that we met in Nerja was friendly. The taxi service to & from the supermarket was v helpful & easy to arrange with English speaking operators. We didn't really venture out that much, but we didn't feel the need to as we had it all! We will definitely be recommending it & returning to Casa Lola as it was the best holiday we've ever had! Thank you Mari for making it available!
1 Reviews
1 holidaymaker review
Amazing place.
We all enjoyed our stay at this lovely villa.All fantastic.The transfer people were also superb.The facilities are great everything you would need .The pool was particularly popular in the evening time when we returned from the beach or even for a quick evening swim.Thank you Marie so much for the ease of booking,clear information and above all such great facilities.
From the Owner
"I took about 3 years (Jan & Feb only) searching up and down the costas to find somewhere that ticked all the boxes as Nerja did eg, authentically spanish - not over-run with tourists - the real Spain on the coast (hard to find) - friendly with easy access - plenty to do - near a major airport - cultured and very charming - beautiful restaurants and shops. That's Nerja!!"
Activity and Interests
Special interest holidays:
Casa Lola also owns the next door 'Orange Villa' (Villa Naranja) ref no - s9126, which can sleep up to a further 8 people (total 22), meaning larger groups can be catered for eg, golf clubs, fishing clubs, walking clubs, wedding parties etc. Should you be interested in such a booking, contact the owner at your earliest convenience. These villas have proved to be very popular and large parties need to be booked well in advance to avoid disappointment.
It's only a 4 minute walk to a beautiful, quieter area of the beach, but where sun loungers etc, are usually available close by. A selection of restaurants, cafes and hotels can be accessed via a short stroll along the beach towards the town centre.
45 minute drive to Malaga airport. A taxi to the airport will cost approx 65 euros. Car hire is very cheap and petrol is considerably lower priced than in the UK. The infrastructure/motorways are superb and driving is much easier than some may think. Taxis are normally with you in minutes and cost 5 euros into town (5 min), so a car is not essential, especially if you enjoy walking 20/25 minutes to the town centre, however, I would recommend one for exploring the surrounding beauty of Nerja.
5 min drive to town centre or 20/25 min walk.
10 min drive to the famous caves of Nerja.
5/10 min drive to Torrox Costa (next town).
45 min to Malaga City and airport.
Approx 1 and a half hour drive to Marbella.
Further Details:
Nerja is a unique coastal town which still has an 80% Spanish population. Its laws are such that developers are not allowed to build over a certain height, keeping it non-commercialised and charmingly authentic. The whitewashed, pedestrianised and cobble-stoned streets meander their way past the many small craft, artistic and local produce shops, where you will thoroughly enjoy strolling at your leisure. Nerja is famous firstly for, The palm fringed,'Balcon de Europa' (balcony of Europe) which is apparently, the only balcony into the Mediterranean sea and has beautifully, breathtaking, panoramic views of the whole coastline and the mountains. It is located in the town of Nerja and is lined with cafes, restaurants, craft shops etc. Well worth a visit!
Secondly for, Cueva de Nerja (the caves of nerja) discovered many years ago by a local young boy while playing, these caves are like something you would imagine from Tolkiens, 'Lord of the Rings'. No visit to Nerja would be complete without seeing this amazing discovery! The month of August sees many performances in these caves including orchestra and opera. There are quite a few restaurants, pubs etc, within walking distance of this villa in punta lara, but the vast majority are in Nerja town centre.
Rental rates
|Rental Period
|Minimum Price
|Additional Nights
|Weekend Night
|Event Price
Low season
30 Oct 2016 - 21 Dec 2016
7 night minimum stay
for 7 nights
Christmas season
22 Dec 2016 - 4 Jan 2017
7 night minimum stay
for 7 nights
Low season
5 Jan 2017 - 19 Mar 2017
7 night minimum stay
for 7 nights
Mid season
20 Mar 2017 - 15 Apr 2017
7 night minimum stay
for 7 nights
Mid season
16 Apr 2017 - 25 May 2017
7 night minimum stay
for 7 nights
Mid season
26 May 2017 - 4 Jun 2017
7 night minimum stay
for 7 nights
High season
5 Jun 2017 - 20 Jul 2017
7 night minimum stay
for 7 nights
High season
21 Jul 2017 - 4 Sep 2017
7 night minimum stay
for 7 nights
Mid season
5 Sep 2017 - 18 Oct 2017
7 night minimum stay
for 7 nights
My Standard Rate
7 night minimum stay
for 7 nights
Additional pricing information
|No additional mandatory fees
Booking Notes:
20% non-refundable deposit payable at time of booking with remainder payable eight weeks prior to date of travel.
Casa Lola also owns the neighbouring Orange Villa (Villa Naranja) - property ref: S9126. It is therefore able to cater for even larger groups, eg, Golf Clubs, Wedding Parties, etc.
Changeover Day:
Flexible, but please contact owner for late arrivals which may incur a small call out charge for the key-keeper/management company.
Owner's Cancellation Policy
- 50% refund for cancellations more than 7 days before check-in date.
- 100% refund for cancellations more than 14 days before check-in date.
- 12
- Available arrival
- 12
- Available
- 12
- Unavailable
From the Owner
"The fantastic views from every room. Close proximity to the beach and town centre. Fantastic weather most of the year. The private pool. The security of the high gated walls. The residential area. The local food. The cleanliness and comfort of the villa - extremely well managed. All the mod cons."
- Air Conditioning
- Ceiling Fans
- Heating
- Internet
- Iron & Board
- Living Room ...
- Washing Machine
- Dishwasher
- Freezer
- Kitchen ...
- Microwave
- Oven
- Refrigerator
- Hob
- Child's Highchair
- Satellite / Cable
- Television
Pool / Spa
- Private Pool
- pets considered
Location Type
- beach
- near the sea
- town
- waterfront
- caves
- churches
- restaurants
Leisure Activities
- beachcombing
- bird watching
- eco tourism
- outlet shopping
- paddle boating
- scenic drives
- sight seeing
- walking
Local Services & Businesses
- ATM/bank
- fitness centre
- groceries
- hospital
- massage therapist
- medical services
Sports & Adventure Activities
- cycling
- deepsea fishing
- fishing
- golf
- jet skiing
- paragliding
- sailing
- diving
- bay fishing
- caving
- swimming
- tennis
- wind-surfing
- Bathroom 1 - Toilet, Shower Enclosure
- Bathroom 2 - Toilet, Shower Enclosure
- Bathroom 3 - Toilet, Shower Enclosure
- Bathroom 4 - Toilet, Shower Enclosure
- En-suite shower room to bedroom 1. Each apartment has good sized bathrooms and or shower rooms, all finished to a very high spec.
- 6 Bedrooms, Sleeps 14
- Bedroom 1 - 1 Cot
- Bedroom 2
- Bedroom 3
- Bedroom 4
- Bedroom 5
- Bedroom 6
- Description of sleeping capacity: Sleeps 14 (6+ Bedrooms) Notes on bedrooms: Apt 1 Bed 1 - Double bed, breakfast patio, Sea/mountain views. Bed 2 - twin beds, sea/mountain views. Apt 2 Bed 1 - Double bed, sea view. Bed 2 - Bunk beds, sea view. Apt 3. Bed 1 - twin beds. Bed 2 - twin beds, sea view. Apt 1 & 2 - Sofa beds.
- Golf ...
- Barbecue
- Ski & Snowboard ...
- Notes on accommodation: Intercom systems throughout. Securely gated all around. Outdoor lighting throughout inc, pool area. Smoke alarms throughout. High chairs/cots etc, can be obtained on request. Most windows have persianas. We can arrange food hampers/welcome packs, car hire and airport transfers as optional extras. A service technician is contactable every day should you discover any malfunctions. All air-con systems also supply hot air heating. Ample parking for up to 2 vehicles. Large BBQ and BBQ equipment on separate terrace. Outside: The swimming pool and surrounding terraced areas are not overlooked, making them really private so you are free to totally relax and unwind. An outdoor shower area is provided for your hygiene. Landscaped garden terraces are decked with a beautiful array of wonderfully coloured plants and flowers, each area designed specifically with that Mediterranean feel. A huge lower terrace lends itself to a fabulous BBQ area which is a must for gathering together in the warm summer evenings. Our large free-standing BBQ has served up many a treat and had plenty of compliments. Enjoy! There are numerous terraces to list them all individually. A seperate utility area houses two washing machines for your convenience and of course drying is never a problem in this sun drenched costa. Regular pool maintenence is carried out once weekly ensuring our pool is kept as clean as possible. Parking for 2 cars inc, carport. Utility Room: Washing machines x 2. Separate sink for hand washing. All linen baskets and clothes pegs supplied. Large separate terrace for washing line/drying area right beside utility room. Cleaning / Towels / Linen / Maid service: Towels and linen are supplied for each apartment, although you may wish to bring extra towels. The villa is cleaned for your arrival. If you want a mid stay clean (extra cost) please inform us in advance, but we won't disturb you, if it's what you prefer! Other: 1st apt has a 'snug area' (doubles as bedroom) with a log fire (or multi fuel), views across Nerja, beach and mountains. Small balcony with patio doors. This area is particularly cosy in the winter months when the temperature drops in the evening.


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$100.00 2 months ago
TOPS Brother of Bushcraft Knife - $100 (Hillsdlae)
Brand new, never used, factory edge. I bought this knife new and have never used or sharpened. This has the black coating with the black micarta handle scales. Kydex sheath with rotating clip. I also have the bag and accessories that came with this knife. Honestly, I wanted the tan model but could not find one in stock so bought this. It is just setting in my vault looking for a good home. $100 is
2 months ago
Battle Creek, MI
$100.00 3 months ago
Enzo Bushcraft Knife - $100 (Carmel)
Never used ENZO Bush craft knife. Brown Micarta Handle. Blade length 5 inches and a little over 1/8th thick and 1 inch wide. Over All length is 9 and 1/2 inches long. Nice new leather sheath with dangler. Priced at $100 firm. Please leave a contact number or I will not respond due to scammers.
3 months ago
Indianapolis, IN
Contact Us 6 months ago
Bushcraft knife (Winchester) hide this posting restore this posting
Handmade bush knife 1095 steel firesteel 550 cord very nice brand new
6 months ago
Winchester, VA
Contact Us
$160.00 7 months ago
Custom handmade bushcraft knife, matching ferro rod, leather sheath - $160 (Maben)
Custom handmade bushcraft knife made by me in my shop here in MS. Oal 8 3/4, 4" cutting edge. 1/8" 01 tool steel, HRC 58/59, tapered tang. Handle is cocobolo with green G10 liners, secured with carbon fiber pins and acraglas. Comes with leather sheath and matching ferro rod. Not taking orders at the moment, thanks for looking. $160, free shipping. Like my page on Facebook WW customs
7 months ago
North Mississippi, MS
$25.00 10 months ago
Condor Bushcraft Knife - $25
Very gently used, will be delivered razor sharp.
10 months ago
Twin Tiers, NY
$150.00 1 year ago
LT Wright Genesis Bushcraft 9" Knife - $150 (Paulden)
LT Wright, handcrafted Fixed blade, full tang knife, currently Out of stock on their site. It has the Green micarta handle, beautiful leather sheath, with dangler and a place for a ferro rod. "This nine inch overall Scandi ground bush knife really likes to work. We take the time to do the little things: making sure the epoxy isn't just on the scales, but around every bit of the hardware, including
1 year ago
Prescott, AZ


text:
How to peel tomatoes for canning
Step 1: Get a large pot of water boiling and next to that, place a large bowl or container 3/4 full of ice water.
Step 2: Carefully place the tomatoes into the boiling water and make sure they're completely submerged.
Step 3: Once the skin starts to split apart (appr. 2 minutes), remove from water and immediately submerge in the ice water.
Step 4: After the tomatoes have cooled down enough, remove from water and peel away the skin with your fingers and discard skins.
Step 5: Halve tomatoes and squeeze out seeds if desired. You're now ready to can.
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text:
In the Seventiesby Dennis O'Neil, Neil Adams (Illustrator), Dick Giordano (Illustrator)
Product Details
- 9781563895654
- Publisher:
- DC Comics
- Publication date:
- 01/28/2000
- Series:
- Batman Series
- Edition description:
- Graphic Novel
- Pages:
- 192
- Product dimensions:
- 6.54(w) x 10.20(h) x 0.35(d)
- Age Range:
- 12 - 18 Years
Customer Reviews
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Most Helpful Customer Reviews
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based on 0 ratings. 1 reviews.
In the early 70's Batman comics were overhauled to dispel the campy image created by the 60's TV show. The result was some of the finest work done in the history of comics as this volume will aptly display. The quality of story telling and dramatic artwork still remain a standard to be matched by comic book artists and writers three decades later.


text:
Online Class
Portraits in Watercolor
with Matt Rota
What type of piece is this? Painting Share a little about the materials, processes and techniques used to create this piece. The darkest colour, blue, was painted first and when completely dry, lighter, transparent colours were glazed over. Is the piece for sale? No


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United States District Court, E.D. Pennsylvania
April 24, 2015
SLAVKO PROPERTIES, INC. trading as 2536-38 N. BROAD STREET ASSOCIATES, L.P.; SLAVKO BRKICH; and VICTORIA BRKICH, Plaintiffs,v.T.D. BANK, N.A.; COBA PROPERTIES, INC.; ECP EAST LLC; and ECP RIDGE LLC, Defendants.
GERALD J. PAPPERT, District Judge.
AND NOW, this 24th day of April, 2015, upon consideration of TD Bank, N.A. and Coba, Inc.'s Motion to Dismiss, (Doc. No. 11), ECP East LLC and ECP Ridge LLC's Motion to Dismiss, (Doc. No. 25), Plaintiffs' responses thereto, (Doc. Nos. 19, 26), and TD Bank, N.A and Coba, Inc.'s Reply, (Doc. No. 24), IT IS ORDERED that:
1. The Motions to Dismiss (Doc Nos. 11, 25) are GRANTED;
2. Subparagraphs (b), (e), and (f) of Count I, Count II, and Counts III, IV, V, VI, VII, VIII, and IX are DISMISSED with prejudice;
3. Subparagraphs (a), (c), and (d) of Count I and Counts X and XI are DISMISSED without prejudice;
4. Plaintiffs are granted leave to file an amended complaint on or before May 8, 2015.
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Learn more about what you receive with purchase of this case.
September 28, 2011
JOEL HOLLEY, PLAINTIFF,v.GARY SWARTHOUT, ET AL., DEFENDANTS.
Plaintiff, a state prisoner proceeding pro se, has filed this civil rights action seeking relief under 42 U.S.C. § 1983. The matter was referred to a United States Magistrate Judge pursuant to 28 U.S.C. § 636(b)(1)(B) and Local Rule 302.
On September 1, 2011, the magistrate judge filed findings and recommendations herein which were served on all parties and which contained notice to all parties that any objections to the findings and recommendations were to be filed within fourteen days from the date the findings and recommendations were served. Defendants have filed objections to the findings and recommendations.
In accordance with the provisions of 28 U.S.C. § 636(b)(1)(C) and Local Rule 304, this Court has conducted a de novo review of this case.
Having carefully reviewed the entire file, the Court finds the findings and recommendations to be supported by the record and by proper analysis.
The Court notes for the record that, while Defendants appear, via their objections to the magistrate judge's findings and recommendations, to have attempted to rectify their failure to address whether Plaintiff's first appeal was sufficient to exhaust his administrative remedies as to all of his claims, the Court declines to address those belated arguments until they have been properly briefed before the magistrate judge in the first instance.
Accordingly, IT IS HEREBY ORDERED that:
1. The findings and recommendations filed September 1, 2011 (ECF No. 48), are ADOPTED in full; and
2. Defendants' January 24, 2011 motion to dismiss is DENIED without prejudice.
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Having unnecessary fat in various locations of your body can have a considerable influence on your health and self-esteem. While standard weight-loss through exercise and diet is a fantastic way to slim down in general, even the very best exercises can't target problem areas like the stomach, inner thighs, arms, and butts. Liposuction is a time checked treatment that is used to get rid of excess fat from specific areas of the body, permitting a specific to form and contour their body to their preference. Is liposuction right for you? Learn now.
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There are many benefits to this cosmetic treatment, consisting of:
• Instantly obvious changes. Unlike standard weight loss, liposuction produces changes that are right away visible in the body. Some difference is obvious right away, and the desired outcomes are typically attained in simply a few days.
• Proven and safe. This cosmetic treatment has been performed by knowledgeable cosmetic surgeons all over the world for years and the strategy has actually been refined over and once again to be safe and efficient.
• Healing time is typically fast. The downtime required after having this kind of treatment is typically much less than what is needed for other kinds of cosmetic procedures, including tummy tucks, breast reduction, and more. People who have had the treatment can often return to work far more rapidly than they prepared for and can return to living a healthy, active lifestyle.
• Weight reduction can be permanent. With the ideal maintenance methods, the fat that was eliminated throughout the liposuction procedure will not return.
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While there many benefits to liposuction, there are of course a couple of cautions that must be considered before making the final decision to move on with the procedure.
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Before having liposuction done, it is very important to analyze the potential drawbacks of the treatment and identify if the benefits outweigh the dangers in your certain case. Your cosmetic surgeon can help you find out more about the dangers connected with the treatment and can assist you choose if moving forward is the ideal thing for you.• Problems with general anesthesia. Due to the fact that liposuction is carried out under basic anesthesia, the treatment brings the very same risks as other kind of surgery where general anesthesia is used. Underlying medical conditions may increase these dangers.
• Negative responses. Bruising, bleeding, and pain are all to be expected, nevertheless, in unusual cases can cause more significant issues.
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There are dangers associated with liposuction, for numerous people, the benefits far outweigh them. Inform yourself about the treatment by having in-depth discussions with your specialist and think about how liposuction has the possible to affect you as an unique individual. Just you and your surgeon can identify if liposuction will provide you with the results you are looking for within your expectations.
Laser Liposuction procedure is a new non invasive procedure to loose unwanted bodyfat in Newark California
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Contact a Surgeon in your Newark California today.
If you're thinking about liposuction as a weight loss solution, it is necessary that you discuss your desires with a qualified plastic surgeon in your location. Your surgeon will perform a total exam and health history questionnaire to determine if liposuction can benefit you and assist you reach your physical and emotional goals. Call today for an examination and learn more about how liposuction can help you attain the body of your dreams.


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The glossy and transcendentally silly romantic melodrama ''Tattoo Bar,'' which opens today in Manhattan, is undeniably confident. The director, Jo Sol, imbues the film with a daffy, lurid quality that's as delectable as that last piece of candy rattling around in the bag that you can't keep away from. The actors' skins glisten, and there's plenty of skin to see because one of the characters, Simona (Mercedes Ortega), is an illustrated woman, the girlfriend of a tattoo artist and rambling philosopher, Francis (Miguel Molina).
Simona's body has many motifs, and her upper back has the phrase ''Francis Forever,'' one of the few bits of English in this Spanish-language film with English subtitles. And when the couple have a steamy embrace, they're a florid, living work of art. Francis and Simona return to Barcelona from their globe-trotting travels to open a bar, tattoo joint and piercing establishment called Tatawo. The place once belonged to Francis's best friend, Mariel (Alexis Valdés), also known as the Cuban, who is behind bars. There's much chatter about finding a way to get him out.
''When my brother's in jail, I feel this brick in my stomach,'' trills the sultry Francis, which only adds to the narrative confusion because we later learn that they are not related. Based on the convoluted and bewildering turns of plot, any effort to free the Cuban would call for the assistance of the weasel Enrique (Carlos Lasarte), who licks his upper lip and bats his eyes at young women. That is, when he's not exercising mysterious control over the Cuban's wife, Bug (Paulina Gálvez).
And hovering in the background is the angry, homophobic Fly (Juan Márquez), the Cuban's former partner in crime. He's a slender ice pick of a man, and it doesn't make sense that he's so thin, given his bent for scenery-chewing.
Eventually, the Cuban is released, living testament to Francis's concern: ''What made him the Cuban wasn't there anymore.'' Part of the Cuban's dolorous story is revealed in flashbacks throughout the picture, though it becomes hard to keep track of what's taking place as his beard appears and vanishes in both the past and the present. But bearded or not, Mr. Valdés's presence does finally give the picture constancy.
Francis's remark about the Cuban's being a shadow of himself is true; the Cuban wanders back to his old bar, a cold hostility glinting in his eyes. Even the ridiculous sight of Simona slipping off her silk gown in the bar's picture window -- a sight sure to increase tourist traffic on the Ramblas in Barcelona -- doesn't shake him out of his withdrawn state. In the flashbacks, before the arrest, Mr. Valdés makes the Cuban a boisterous, ursine man capable of outsize mirth.
And his melancholy registers, too. Though if you missed it, his wife, Bug, tells the Cuban: ''Jail takes all the soul away from you. You may have killed a man in Angola, but you had a soul.'' Perhaps his telling Bug, ''You're like embracing smoke,'' prompted this response from her. Eventually, Francis disappears, leaving a distraught Simona to weep and writhe in bed alone, losing herself in tattooing and piercing customers.
And with the Cuban's return -- and the uptick in the heartbroken Simona's business -- the tattoo bar explodes with popularity. With women swinging from trapezes on the ceiling and the apparent return of sexy lambada dancing, the bar is like a Barcelona Moulin Rouge, perhaps Cirque du Olé.
The Cuban returns to a life of crime because it seems that the bar doesn't make enough money to finance his grand desire: helping to pay for the surgical removal of Simona's tattoos, which are distracting reminders of Francis.
The noirish undercurrents of ''Tattoo Bar'' indicate that the decent Cuban is a man with a past who is drawn to tragedy -- a former cameraman, he lost an eye covering the civil war in Angola and has a milky white orb left in its place. The problem with the baroque and overripe ''Tattoo Bar'' is that everybody has a past. And there's so much crosscutting to those pasts in flashbacks, it's hard to keep track of whose past you're witnessing.
Written and directed by Jo Sol; in Spanish, with English subtitles; director of photography, Ángel Luis Fernández; edited by Julia Juaniz; music by Lol; production designer, Norbert Llaras; produced by Jordi Rediuc; released by ArtMattan Productions. At the Quad Cinema, 34 West 13th Street, Greenwich Village. Running time: 90 minutes. This film is not rated.
WITH: Alexis Valdés (Mariel), Mercedes Ortega (Simona), Paulina Gálvez (Bug), Miguel Molina (Francis), Elsa Pataky (Blanca), Juan Márquez (Fly), Gabriella Roy (Joany) and Carlos Lasarte (Enrique).Continue reading the main story


text:
Please note: We no longer feature this apartment in our portfolio
The Park View Apartments are conveniently located near to the railway station and the town centre, and provide spacious accommodation which is ideal for the business and leisure traveller alike.
Each of these modern apartments is furnished and decorated in an elegant contemporary style, and comes fully equipped with a range of modern conveniences including broadband internet access and plasma screen TV with the full Sky package of movies, news, music and sports channels.
The apartments are conveniently located within easy reach of the town centre. Maidenhead offers a range of local amenities, including shops, bars and restaurants, a leisure centre and the Norden Farm Centre for the Arts.
Maidenhead railway station is just a stone's throw away, offering main line services to a range of destinations including London, Oxford and Reading. There are local bus services, and the apartments offer easy access to the M4 motorway.
This apartment is equipped with two double beds and can sleep up to four people.
Sleeps 4 | Bathrooms 2 | Breakfast type: Room Only
If you would like to enquire about availability for this apartment, or to get more information please click here
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text: I was hoping there would be a price cut on the iPod Touch 2g after the 3.0 software update is released at the WWDC. But it didn't happen and with the Ipod Touch 3g releasing around September doesn't seem like a price drop would happen anytime soon. Am I correct? Should I buy the iTouch now? I cant wait until September.


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"You are certainly very versatile. From elegant, to high-concept, back to a soft & vulnerable bedroom look. Just the perfect model I'm looking for."
Think of me as your very versatile chameleon - ready to make your ideas come to life.
I have been modeling professionally for over two years - starting in Las Vegas, when a designer scouted me out.
I instantly fell in love with the industry. I love creating these images, love becoming the art. I am currently agency-represented (non-exclusive), but most of my (full-time) work is freelance, as I am good at managing myself, and creating my own schedules.
At the moment I am based in Cape Town, South Africa, but travel often, and work as I go. I have professional experience shooting for fashion, lingerie, art, bikini, and lifestyle. I also enjoy working as a promotional model/host.
I love meeting and collaborating with fellow artists, so please send me a message if you are interested in working with me!
You can see more of my work at:


text:
The Wanna Run Away With Me? Test
Their result for The Wanna Run Away With Me? Test ...
Let's Go.
You scored 86%!
Their Analysis (Vertical line = Average)
They scored 86% on Runaway-ability, higher than 100% of your peers.
All possible test results
Get Out of Here!
Well, hate to say it, but we're definitely not going on a journey together... we'd be at each others' throats by the first night. Read more
We Might Make It...
We'll probably be able to tolerate each other on our little adventure, but I don't know for how long... Read more
Let's Go.
Okay, let's go right now! I'm packing my bags! Read more
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