text:
|Home | About | Journals | Submit | Contact Us | Français
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma – mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOX™ Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.
The structural and electronic properties of SWNTs lend themselves to a variety of biomedical applications involving the detection and treatment of diseases, most notably cancer [1-6]. For example, the structural change in DNA upon shifting from the B to Z conformation sufficiently perturbs the electronic structure of SWNTs such that the change can be detected optically from living cells that have taken up DNA-SWNT complexes [7]. This and other works demonstrate how CNTs can be used as sensors within living cells [8,9]. In another example, exposing cells containing SWNTs to near infrared radiation kills the cells due to the efficient optical-to-thermal energy conversion of SWNTs, demonstrating that they can potentially be used in targeted cancer therapies to eliminate cancer cells [10]. Finally, there are a number of reports that CNTs facilitate the transport of bound oligonucleotides, peptides, and proteins across the plasma membrane [1,11-19]. However, despite these and other intracellular applications not listed here, there remain technical challenges towards realizing the potential benefits of CNTs in biomedicine. Namely, CNTs are extremely hydrophobic, bundle together, and are insoluble in water.
Two approaches have been used to modify the hydrophobic surface of CNTs to make them water soluble and biocompatible. The first has been to debundle and disperse CNTs in aqueous solution by covalently attaching water soluble substances to the CNT surface, and the second has involved the noncovalent association of material to the CNT surface [20-26]. In both approaches, a wide variety of organic adducts and biological materials have been used successfully including oligonucleotides [7,9,10,15,17,18,27-40], peptides [14,19,41-52], proteins [8,11-13,16,53-59] (most notably, bovine serum albumin (BSA) [60-63]), an assortment of polymers [64], and various cell culture media formulations [19,43,65-72]. While covalently attaching material to CNTs is advantageous for many applications, one serious drawback is that the covalent attachment introduces defects in the surface of the CNTs that often interfere with the electronic and optical properties that make CNTs so useful.
Beyond CNT dispersal, another challenge in the field is assessing whether CNTs are inherently cytotoxic [73-80]. At present, there are roughly as many publications reporting no apparent cytotoxicity [10,12-14,16-19,65-67,71,81-87], as there are reporting varying degrees of significant cytotoxicity [68-70,72,88-95]. Two major considerations in this area are how the CNTs are presented to the organism and the purity and concentration of the CNTs. For example, pulmonary toxicity of SWNTs has been established when large doses of dry, unpurified SWNTs have been blown into the lungs of rats [89,90,96]. This method of presentation is not relevant to the small measured doses of CNTs that would be used in chemotherapy and drug delivery. In fact, the biodistribution of chemically modified SWNTs injected into mice or rabbits was studied recently, and the CNTs were reported to be cleared rapidly with no evidence of toxicity [85,97,98]. CNT purity is also absolutely crucial. Many CNT syntheses use metal catalysts that are known to be toxic. Such impurities, and other carbonaceous impurities, must be removed from the samples in order to reach conclusions about inherent CNT toxicity, and it is not always clear from the published reports that they have been removed. Finally, many accounts of CNT toxicity have used MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) as a reporter of cell viability, and it was recently shown by Worle-Knirsch et al. that MTT itself binds to CNTs (quenching its fluorescence) and thereby introducing uncertainty in this assessment of toxicity [65]. In summary, while the question of whether CNTs have long-term toxicity in biomedical applications requires further research, early reports raising the alarm of toxicity in model cell culture systems have not been adequately verified.
Recently, our group reported that HiPco SWNTs, dispersed in a peptide solution or in media supplemented with serum, were taken up by HeLa cells in a time- and temperature-dependent fashion and did not affect the HeLa cell growth rate, evidence that the SWNTs inside cells were not toxic under these conditions [19]. This work also demonstrated that our dispersion preparation protocol (involving probe sonication and multiple centrifugations) was effective in removing metals from the raw, as-received SWNT-containing powder. Herein, we present the characterizations of an as-received CoMoCAT SWNT-containing powder using thermal gravimetric analysis (TGA) and scanning electron microscopy (SEM), and of SWNTs dispersed in Dulbecco's modified Eagle medium (DMEM) supplemented with fetal bovine serum (FBS) using atomic force microscopy (AFM), inductively coupled plasma – mass spectrometry (ICP-MS), and absorption and Raman spectroscopies. The resulting purified dispersions, termed DM-SWNTs, are next shown to have no effect upon the morphologies and growth rates of HeLa cells – a thoroughly characterized human epithelial-like cell line. Using confocal microRaman spectroscopy, it is shown that DM-SWNTs were taken up by cells in a time- and temperature-dependent fashion. Evaluation of the distribution of intracellular DM-SWNTs was performed using transmission electron microscopy (TEM) which revealed SWNT-like material in vacuoles. Finally, intracellular superoxide dynamics of cells exposed to DM-SWNTs were evaluated using fluorescence-based flow cytometry and MitoSOX™ Red – a selective indicator of superoxide in mitochondria. The MitoSOX™ Red fluorescence detected from control cells was statistically similar to that observed for cells incubated in DM-SWNT dispersions. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNTs are not inherently cytotoxic to HeLa cells.
The CoMoCAT method of SWNT synthesis involves a bimetallic Co-Mo catalyst supported on a silicon dioxide substrate [99-103]. The purification procedure includes removal of amorphous carbon by low-temperature oxidation, removal of the SiO2 substrate with HF, and removal of metals by HCl. The final product, a SWNT-containing powder, is rinsed with deionized water until its pH is neutral [104]. Visible microscopic examination of the lot used in this work revealed that the fine, black, fluffy powder comprised irregularly shaped particles with dimensions ranging from 5–50 μm. SEM revealed that the majority of these particles comprised tightly entangled networks of SWNTs, similar to those observed by Resasco and co-workers [105], and that these networks comprised small bundles of SWNTs with 5–20 nm diameters (Figure (Figure11).
TGA of the as-received SWNT-containing powder was performed to assess the powder's composition with respect to metals, SWNTs, and non-tubular carbon (NTC) species such as amorphous carbons, fullerenes, carbides, graphitic nanoparticles, etc. TGA measurements of the SWNT-containing powder were performed under the assumption that upon heating to 1000°C in O2, all carbon and metals were converted to their corresponding oxides, and that the presence of other trace elements could introduce small errors to calculated metal contents [106]. Figure Figure22 shows the weight percent decrease as a function of temperature (red trace) and the first derivative of the weight percent curve (blue trace) for the as-received SWNT-containing powder. The identities of the components corresponding to the three main peaks observed in the derivative plot were determined in experiments whereby the residues in the TGA pan were recovered and analyzed by Raman spectroscopy and/or XPS before, during, and after peak onset. In brief, peak 'a' at ~410°C was determined to comprise SWNTs based on the appearance of a strong G-band – a Raman resonance uniquely associated with SWNTs. The oxidation temperature of the SWNTs ranged between 375–450°C and was consistent with the oxidation temperature of CoMoCAT SWNTs observed by Resasco and co-workers [105]. Peak 'b' at ~505°C was determined to comprise NTCs based on the disappearance of the G-band and an increase of the D-band – a Raman resonance uniquely associated with miscellaneous forms of disordered carbon. Peak 'c' at ~700°C, 9% weight loss, was determined to comprise MoO3 by XPS and was supported by the ~700°C sublimation temperature of MoO3. XPS experiments also ruled out the presence of residual SiO2 in the as-received SWNT-containing powder. The remaining 5% mass at 1000°C (Figure (Figure2,2, red trace) was considered to be oxidized metals of Co and Mo, most likely CoMoO4 and MoO2. In summary, the oxidized SWNT-containing powder was classified as comprising ~70% SWNTs, ~7% NTC, and ~14% oxidized metals.
SWNT dispersions were prepared using a sonication and centrifugation protocol and DMEM supplemented with 5% FBS (DMEM/FBS). The resulting DM-SWNTs were homogeneous in appearance and could be stored for 30 d at 4°C before any SWNTs were observed to precipitate. The final concentration of SWNTs in DMEM/FBS was estimated to be ~50 μg/mL (Additional File 1) and SWNT lengths were estimated to be 100–400 nm (Additional File 2). Figure Figure33 shows the absorption spectrum of a representative DM-SWNT dispersion. The observed spectral profiles of DM-SWNTs were similar to the spectra of CoMoCAT SWNTs dispersed in sodium dodecyl sulfate (SDS) as prepared by Resasco and co-workers [103] and Stupp and co-workers [41], where the two predominant semi-conducting SWNT types present were (6,5) and (7,5) tubes with an average diameter of 0.8 nm. Specifically, the DM-SWNT peak observed at ~569 nm corresponds to the S22 optical transition of (6,5) tubes, the shoulder observed at ~587 nm corresponds to the S22 optical transition of (8,4) tubes, the peak observed at ~652 nm corresponds to the S22 optical transitions of (7,5) tubes at 644 nm and (7,6) tubes at 647 nm, the broad peak at ~1011 nm corresponds to S11 optical transitions of (6,5) tubes at 975 nm and (7,5) tubes at 1025, and the peak at ~1120 nm corresponds to the S11 optical transitions of (8,4) tubes at 1113 nm and (7,6) tubes at 1122 nm, which are all in accordance with spectroscopic assignments by Bachilo et al. [102]. In summary, the data indicate that CoMoCAT SWNTs dispersed in media supplemented with serum retain their optical transitions between van Hove singularities in the electronic density of states.
Confocal microRaman spectrometer acquisition methods and the interpretation of the Raman spectra of various SWNT dispersions prepared using our sonication and centrifugation protocol have been detailed previously [19,45,47,49]. A representative Raman spectrum for a DM-SWNT dispersion is shown in Figure Figure44 (blue spectrum; DMEM + 5% FBS). The spectrum clearly shows a number of well characterized SWNT resonances [100,107,108], in particular, two predominant radial breathing modes at ~281 and ~301 cm-1, the D-band at ~1303 cm-1, and the G-band in the 1550–1610 cm-1 region. Control spectra of DMEM/FBS without SWNTs did not display detectable resonances under these operating conditions (data not shown). Spectrometer stability was assessed by monitoring the reproducibility of the G-band peak intensity at ~1590 cm-1 since it is the most prominent Raman peak indicative of intrinsic SWNT features [109]. In brief, the relative standard deviation (RSD) of G-band peak intensities acquired from the same region of a SWNT dispersion was <1%, the RSD of G-band peak intensities acquired from four different regions of a SWNT dispersion was <10%, and the correlation coefficient for the linear relationship between the G-band peak intensity and relative SWNT concentration was 0.982 (Figures S3-S5 in Additional File 3). In summary, the data indicates that the FBS components coating the SWNTs did not significantly affect the G-band profile of SWNTs dispersed in this fashion, which is in agreement with previous reports using non-covalently modified SWNTs dispersed in aqueous solutions of peptides [19,41,45] and proteins [53,62].
While a variety of cell types have been cultured in pristine or functionalized CNTs solubilized in various growth media formulations [19,43,65-72], only a few of these reports have emphasized the important role that the added serum plays. The importance of FBS in dispersing SWNTs is evident in the series of Raman spectra shown in Figure Figure4.4. In brief, DMEM comprises inorganic salts, amino acids, buffers, vitamins, and minerals with the three major components being glucose (4500 mg/L), sodium bicarbonate (3700 mg/L), and sodium chloride (6400 mg/L). FBS is also a multi-component mixture comprising many low and high molecular weight substances. The major dissolved substances are proteins, lipids, steroid hormones, minerals, and metabolites. The most notable FBS components known to solubilize CNTs are BSA and phospholipids [10,53,58]. Using our sonication and centrifugation procedure, DMEM without FBS did not support SWNT dispersion as observed by the lack of detectable SWNT Raman resonances (Figure (Figure4;4; gold spectrum). Conversely, aqueous 5% FBS solutions (without DMEM) were quite effective in dispersing SWNTs (Figure (Figure4;4; red spectrum).
Previous elemental analyses of peptide-coated SWNT dispersions prepared using our sonication and centrifugation protocol revealed only trace amounts of metal catalysts even though the as-received HiPco SWNT-containing powder contained ~32% metals by weight [19]. Herein, we further characterize this protocol's ability to effectively remove toxic materials by analyzing CoMoCAT DM-SWNT dispersions. CoMoCAT SWNTs are made from a process that uses Co and Mo as catalysts rather than Fe, and thus, exposing CoMoCAT SWNTs to cells avoids the known cellular toxicity that Fe can impact to a CNT preparation [66,70]. The ICP-MS analyses of DM-SWNT dispersions revealed 6.64 ppm Mo and 1.55 ppm Co, and that the only Fe in our DM-SWNT dispersions was from the 0.10 ppm ferric nitrate in DMEM. For comparative purposes in the absence of EC50 values for dispersed SWNTs, the metal levels observed in the DM-SWNT dispersions were well below the 90 ppm EC50 of mammalian stem cells exposed to 30-nm MoO3 particles (as determined by MTS assays) [110], and the 19 ppm EC50 of murine fibroblasts exposed to Co (as determined by MTT assays) [111]. Since >99% of the Mo and Co present in the as-received SWNT-containing powder was not detected in the DM-SWNT dispersions (relative to oxidized metal levels from the TGA of the SWNT-containing powder), these data again demonstrate that our sonication and centrifugation protocol is an effective method for removing the heavier metal-containing SWNTs and bundles. Such results are important to note since it has not been made clear in all previous published reports of cells being exposed to CNTs if such metal-removing measures were implemented before the CNT cytotoxicity was assessed.
DM-SWNTs were additionally analyzed for the presence of Ti since it is possible that this metal could be introduced through the use of Ti-coated probe sonicator tips. ICP-MS analyses of DM-SWNT dispersions prepared using a probe tip that had been used for >20 non-continuous hours revealed 0.15 ppm Ti. For comparison, this level is well below the 250 ppm EC50 of rat liver cells exposed to 40-nm TiO2 particles (as determined by MTT assays) [112]. To our knowledge, this is the first report of such an analysis amongst the previous reports of cells exposed to SWNT dispersions prepared using probe tip sonication.
The main analytical approaches for assessing the presence of CNTs in cells and tissue have been optical [1,14,65,67,72,83,88,90,93,113], electron [11,15,17,37,68-70,89,114], and fluorescence [10,12-14,16,18,43,64,82,84,86] microscopies. While optical microscopy is ideally suited for live-cell analyses, this label-free technique lacks the specificity to unambiguously identify material observed in cells as CNTs. Electron microscopy offers high spatial resolution imaging of CNTs but is limited to slices of cells that have been fixed; multi-walled CNTs can be unmistakably identified in cells with this technique. In live-cell fluorescence microscopy, the detection of CNTs is indirect (i.e., it is through the detection of a visible fluorescent dye that is (non)covalently attached to the CNT or to molecules coating the CNT). Recently, direct and label-free mapping of CNTs inside living cells has been demonstrated using the intrinsic near-infrared fluorescence [7,9,81] or Raman scattering [9] of CNTs themselves.
Herein, the presence of CoMoCAT G-band intensities emanating from inside living cells incubated in DM-SWNT dispersions was evaluated using confocal microRaman spectroscopy. In the first series of experiments, cells were incubated in DM-SWNT dispersions for 60 h at 37°C. A representative transmitted white-light image of a single HeLa cell acquired through the Raman microscope is shown in Figure Figure5.5. Typical HeLa cells were observed to possess 10–30-μm widths and 40–70-μm lengths. The relatively large dimensions of HeLa cells, coupled with the 4-μm lateral resolution of the confocal microscope system, permitted Raman spectra to be acquired from distinct cellular regions [19]. For example, Figure Figure55 shows Raman spectra acquired from a cell that was incubated in a DM-SWNT dispersion. Intense G-band signals were observed from both cytoplasmic (Figure (Figure5A)5A) and nuclear (Figure (Figure5B)5B) regions. In the latter case, it should not be implied that SWNTs are in the nucleus because the detected G-band resonances could emanate from SWNTs located in the perinuclear region and/or in the cytoplasm immediately above or below the nucleus. Finally, control cells incubated in DMEM/FBS (without DM-SWNTs) had no detectable SWNT Raman signatures under these conditions (data not shown), and no SWNT resonances were detected from cell-free regions of the dish adjacent (≤5 μm) to cells (Figures (Figures5A5A and and5B,5B, dark blue spectra).
If the intense G-band signals emanated from DM-SWNTs inside cells, most likely the result of an active uptake process such as endocytosis, then the signals should be absent in cells exposed to DM-SWNTs at 4°C where energy-dependent uptake practically ceases. Figure Figure66 shows representative Raman spectra acquired from HeLa cells incubated in a DM-SWNT dispersion at 4°C. The peaks detected at ~1608 and 1651 cm-1 in the spectrum acquired from the cytoplasm are presumed to emanate from proteinaceous material, as denoted by the amide-I band at 1650–1659 cm-1 [115-117]. More importantly, the G-band intensities at ~1590 cm-1 recorded from cytoplasmic and nuclear regions were 99.9% less than those recorded from cells incubated at 37°C (Figure (Figure5).5). In summary, the lack of detectable G-band signals from HeLa cells incubated in DM-SWNT dispersion at 4°C indicates that HeLa cells do not uptake detectable levels of DM-SWNTs when their metabolic activity is low. In addition, the lack of G-band signals from cells incubated at 4°C indicates that there was negligible nonspecific adherence of SWNTs to HeLa cells (i.e., the rinsing procedures were sufficient to remove DM-SWNTs that were on the exterior surface of the plasma membrane).
In another series of experiments, the time-dependence of DM-SWNT uptake was evaluated. First, the heterogeneous distribution of DM-SWNTs was taken into consideration. As shown in Figure Figure5,5, the G-band intensities detected from the cytoplasm ranged from 20 to 500 a.u. and those for the nuclear region ranged from 10 to 350 a.u. It was therefore decided to perform all time-dependent studies with the Raman laser focused on the center of a cell's nuclear region. This selection was influenced by our previous observations of SWNT accumulation around the nuclear region as revealed through confocal fluorescence imaging of HeLa cells exposed to SWNTs dispersed with a fluorescent-labeled peptide [118], and by Strano and co-workers through Raman spectral mapping of 3T3 cells exposed to SWNTs dispersed with DNA [9]. Figure Figure77 shows Raman spectra from HeLa cells that were incubated at 37°C in DM-SWNT dispersions for 12, 24, 36, 48, and 60 h. In all cases, the number of cells displaying detectable G-band signals increased as the DM-SWNT incubation time increased. Typically, the G-band intensities acquired from HeLa cells incubated in DM-SWNTs for 60 h was 90% greater than those detected at 12 h. Specifically, <10% of the cells analyzed after 12 h incubation displayed detectable G-band signals, while >90% of cells analyzed after 60 h displayed significant G-band signals (n = 40 cells analyzed). In summary, the combined Raman evidence indicated that the observed G-band intensities emanate from DM-SWNTs inside HeLa cells, and that the uptake of DM-SWNTs by HeLa cells is a time- and temperature-dependent process. While complete elucidation of the mechanism(s) of SWNT uptake by cells still requires further investigation, our results are consistent with the work of Dai and co-workers [12] and Cherukuri et al. [81] who have demonstrated that CNTs are transported inside cells via a temperature-dependent mechanism, and contrast the work of Bianco and co-workers who provide evidence that CNT uptake follows a temperature- and endocytosis-independent mechanism [14,37,43].
TEM was used to examine the intracellular distribution of DM-SWNTs. Figures Figures88 and and99 show electron micrographs of HeLa cells incubated at 37°C for 60 h in DMEM/FBS (no SWNTs) or DM-SWNT dispersions, respectively. Colored arrows are used to denote the nucleolus and nucleus, vacuoles/vesicles, Golgi bodies, and mitochondria. In addition, it is important to note that all micrographs shown in Figures Figures88 and and99 were acquired from cells sliced in the plane of the nucleolus, as denoted by the low-magnification micrograph shown in Figure Figure8A.8A. The first observation from the comparison of control and DM-SWNT treated cells was the lack of any SWNT-like structures visible in or associated with Golgi bodies (compare Figure Figure8C8C with with9E)9E) and mitochondria (compare Figures 8D,E with with9B).9B). The most striking observations between control (n = 8) and DM-SWNT treated (n = 10) cells was the appearance of dense black aggregated material in the cytoplasmic vacuoles of the DM-SWNT treated cells (Figures 9A–D) that was not observed in control cell vacuoles (Figures 8D–F). In the highest magnification view of these material-filled vacuoles (Figure (Figure9D),9D), the observed material displays black features with 5–20 nm diameters and apparent lengths of 50–300 nm, which is similar to the dimensions of CoMoCAT SWNTs in our dispersions. Such observations are consistent with those of Dai and co-workers who used confocal fluorescence microscopy to image the co-localization of SWNTs coated with a dye conjugate of avidin and the fluorescent endocytosis marker FM 4–64 [12,13].
Conclusive evidence of SWNT-like structures in the nucleus was not observed (compare Figures 8A–E to Figures 9A,B,C,F). This is important to note since there is presently no consensus regarding the ability of SWNTs to enter the cellular nucleus or the mechanism for their entry. For example, data that SWNTs have crossed the nuclear membrane has been presented by Bianco and co-workers using TEM and 300–1000-nm long peptide functionalized multi-walled CNTs [15], and Lu et al. using radioactive labels and ~400-nm long RNA-modified SWNTs [18]. In contrast, Strano and co-workers used confocal Raman imaging to observe DNA-coated SWNTs in the perinuclear zone of 3T3 cells, but not in the nuclear envelope [9]. In summary, amongst reports presenting high-resolution TEM images of cultured cells and tissue exposed to CNTs [11,15,17,37,68-70,89,114], it is apparent that large multi-walled CNTs can be unmistakably identified in cells by visual observation. The situation is more difficult when cells have been exposed to SWNTs. In most cases, the purported SWNT material appears as a single, dense black mass of material and there are few structural features observable on-scale with the expected diameters of individual/bundled SWNTs (± coatings). In fact, when SWNTs have been observed to be densely internalized in cell vacuoles [69], there are no observable differences between those TEM images and TEM images of cells exposed to fullerenes, which also display vacuoles densely filled with black material [119]. Clearly, the development of complementary analyses capable of identifying SWNT and NTC species in such images is warranted.
A crucial question amongst reports concerning the adherence and/or uptake of CNTs by cultured cells [1,7,9-18,37,43,65-73,75,77,78,81-84,86,88,91-94,113,114] is whether CNTs are toxic. Previously, we observed that the growth rates of HeLa cells incubated for 4 d in ~100 μg/mL HiPco SWNTs dispersed in a peptide solution or in media supplemented with serum were statistically similar to controls [19]. The evaluation of CoMoCAT DM-SWNTs also involved monitoring growth rates over the course of 4 d. First, there were no discernable differences in the morphologies of HeLa cells incubated in DM-SWNTs for 60 h (Figures (Figures55 and 10B) relative to controls (Figure 10A; cells incubated in DMEM/FBS). Next, the growth rates of HeLa cells continuously exposed to DM-SWNTs were quantitated by calculating population double times (PDTs). A PDT is a measure of cell numbers at the early log growth phase and is used for comparisons of normal cell growth. PDTs were obtained from the slopes of the lines of a plot of the natural log of cell numbers versus time [120]. Figure Figure1111 shows such a plot over a time period of 4 d for cells cultured in DM-SWNTs and control cells (DMEM/FBS only). For both samples, the respective number of HeLa cells counted on days 1, 2, 3, and 4 were statistically similar at a 95% confidence level. The control HeLa cell PDT was 27 h and was statistically similar to the PDT of 29 h observed with HeLa cells cultured in DM-SWNTs. In summary, the data from this sensitive test argue that our preparations and concentrations of purified CoMoCAT DM-SWNT dispersions do not affect HeLa cell growth rates.
As recommended by Worle-Knirsch et al., the presentation of CNT cytotoxicity results should include at least two or more independent test systems [65]. Therefore, in conjunction with morphology and growth rate studies, fluorescence-based flow cytometry was utilized to investigate whether the uptake of DM-SWNTs by HeLa cells increased the production of reactive oxygen species (ROS). In these series of experiments, HeLa cells were incubated in DM-SWNT dispersions and incubated with MitoSOX™ Red – a novel fluorescent indicator for the selective measurement of superoxide (O2•-) production in cells [121-123]. MitoSOX™ Red is a non-fluorescent, cell permeable dye that forms a highly fluorescent product upon oxidation. Owing to its lipophilic triphenyl phosphonium cation, MitoSOX™ Red is selectively targeted to mitochondria – the major source of ROS in cells – where it can be oxidized by superoxide before exhibiting red fluorescence upon binding to nucleic acids [123].
In each fluorescence-based flow cytometry experiment, six different cell samples/controls were prepared and analyzed in triplicate with each individual trial representing the analysis of thousands of cells. Fluorescence microscopy was also used to validate that MitoSOX™ Red was distributed throughout the cytoplasms of cells, and that negligible dye leaked from the cells (data not shown). The first two flow cytometry control experiments involved measuring responses of cells incubated in DMEM/FBS without MitoSOX™ Red (± DM-SWNTs). These dye-free controls were prepared to establish background fluorescence levels of unstained HeLa cells (± DM-SWNTs) and are represented in the plot of events vs. MitoSOX™ Red fluorescence intensities as shown in Figures 12A &12B (and Figures S6A&B in Additional File 4). The means and standard deviations of fluorescence intensities from these two control experiments without MitoSOX™ Red were 3.07 ± 0.15 and 2.40 ± 0.44 a.u. for DMEM/FBS and DM-SWNT treated cells, respectively. Next, since it has recently been reported that binding of fluorescent viability dyes to CNTs can add uncertainty to cytotoxicity assessments [65], our series of experiments also included a comparison of responses from positive controls ± DM-SWNTs. Specifically, the responses of cells loaded with MitoSOX™ Red and exposed to 5 μmoles hydrogen peroxide were analyzed in the presence and absence of DM-SWNTs (Additional File 4; Figures S7A and S7B respectively). Both samples possessed statistically-similar fluorescence intensities indicating that SWNT quenching of the MitoSOX™ Red fluorescence was minimal.
Figures 12C &12D (and Figures S6C&D in Additional File 4) show representative responses of cells loaded with MitoSOX™ Red and incubated either in a DMEM/FBS control (no SWNTs) or in a DM-SWNT dispersion. The means and standard deviations of fluorescence intensities from these two experiments (51.0 ± 24.2 and 47.3 ± 22.1 a.u. for DMEM/FBS controls and DM-SWNT treated cells, respectively) were statistically similar. For comparison, the mean fluorescence intensities from the positive control shown in Figure 12E (and Figure S6E in Additional File 4) was ~7-fold greater (343 ± 101 a.u.). These results are akin to the results of Shvedova and co-workers who observed interesting relationships between the metal content of SWNTs and the iron-induced intracellular production of ROS. In brief, SWNTs containing 26.0 wt% Fe stimulated significant production of hydroxyl radicals by RAW 264.7 macrophages (vs. purified SWNTs containing 0.23 wt % Fe as detected by electron paramagnetic resonance spin-trapping assays), while fluorescence analyses with dihydroethidium incubated macrophages revealed similar superoxide and nitric oxides levels for both cells exposed to the Fe-containing SWNTs or purified SWNTs [66]. Nonetheless, while superoxide is just one of many potential reactive oxygen and nitrogen species, and while Co, Mo, Ti, and Fe are just four types of potential metal impurities, these data suggest that our preparations and concentrations of purified DM-SWNTs do not increase the concentrations of mitochondrial superoxide in HeLa cells under these culture conditions.
Herein, CoMoCAT SWNT-containing powders and DM-SWNT dispersions were characterized using AFM, ICP-MS, SEM, TGA, and absorption and Raman spectroscopies. Confocal micoRaman spectroscopy was utilized to determine that DM-SWNTs entered HeLa cells in a time- and temperature-dependent fashion. TEM revealed SWNT-like material in intracellular vacuoles. Flow cytometry showed that the fluorescence from MitoSOX™ Red, a selective indicator of superoxide in mitochondria, in control cells was statistically similar to that observed for cells incubated in DM-SWNTs. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. The combined results indicate that, using our sample preparation protocols (i.e., probe tip sonication followed by two centrifugations), and under our assay conditions (i.e., SWNT types, coatings, dimensions, concentrations, impurity types and amounts, and cellular exposure times), CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. Finally, the importance of thoroughly characterizing CNT materials before offering a CNT cytotoxicity assessment can not be over emphasized. We support the development of (i) standardized CNT sample preparation protocols, reference materials, and characterization methodologies, (ii) standardized methods for assessing whether CNTs are taken up by and/or adsorbed to cells, and (iii) a series of proven cell vitality assay conditions. Such measures are imperative to improve the accuracy and comparability of CNT cytotoxicity reports.
Dulbecco's modified Eagle medium (DMEM) was purchased from Irvine Scientific and was supplemented with 3700 mg/L sodium bicarbonate, 1% (v/v) penicillin, streptomycin, and amphotericin B (Sigma-Aldrich). Fetal bovine serum (FBS) was obtained from HyClone. Phosphate buffered saline (PBS; 8 mM phosphate, 150 mM NaCl, pH = 7.4) was sterilized by autoclaving at 120°C for 0.5 h. Deionized water (18.3 MΩ-cm) was obtained using a Nanopure Infinity water purification system (Barnstead). All other chemicals were of the highest quality available and were used as received.
All dispersions were prepared with CoMoCAT SWNTs (Product No. SP95-02-dry, Lot No. UT3-A001; SouthWest NanoTechnologies Inc.). The preparation of DM-SWNTs (i.e., SWNTs dispersed in DMEM supplemented with 5% (v/v) FBS (i.e., DMEM/FBS)) used a sonication/centrifugation protocol identical to that previously described by Chin et al. except that the centrifugation times were reduced [19]. Specifically, 1.0 mg of the as-received SWNT-containing powder was dispensed into an Eppendorf tube containing 1.0 mL of DMEM/FBS, vortexed for ~1 min, and probe sonicated for 10 min at 0°C. Probe-sonication was performed using a Branson 250 Sonifier, and the 2 mm diameter probe tip was placed one-third of the distance below the surface of the 1 mL suspension. The resulting black suspension was centrifuged in an Eppendorf 5417C centrifuge for 2 min at 16,000 g (14,000 RPM). The upper 75% of the supernatant was recovered without disturbing the sediment and placed in a clean tube before a second 2 min centrifugation at 16,000 g was performed. The upper 75% of the second supernatant was carefully recovered to afford a DM-SWNT dispersion. The preparation of aqueous dispersions in 0.15% (v/v) sodium dodecyl sulfate (SDS-SWNTs), 0.1% (v/v) TritonX-100 (TrX-SWNTs), or 5% (v/v) FBS (FBS-SWNTs) was identical to that described above except that DMEM/FBS was replaced by the corresponding surfactant or serum.
SEM was performed at 10 kV with a Zeiss-LEO Model 1530 variable pressure field effect scanning electron microscope. Samples of the as-received SWNT-containing powder were placed on a SEM mount with carbon black tape and analyzed without a conductive coating.
TGA was performed with a Perkin Elmer Pyris-1 thermal gravimetric analyzer equipped with a high temperature furnace and sample thermocouple. Samples (n = 3) of the as-received SWNT-containing powder were dried in air for 6 h at 100°C before being transferred into the platinum pan of the analyzer. The samples were heated from room temperature to 1000°C at 5°C/min in >99.9% O2 using a flow rate of 20 mL/min. A baseline was generated for each scan and baseline-subtracted thermograms were converted to weight percents. Thermal oxidation temperatures were identified by the peaks from the derivative of weight percent curve. Triplicate analyses yielded oxidation temperatures with a reproducibility of ± 2°C. The determination of a component's mass was performed by subtracting the weight percent lost between peak onset and end. In the case where two peaks overlapped (Figure (Figure2,2, peaks 'a' and 'b'), the weight percent lost for the non-overlapping half of each peak was calculated and doubled. Validation of this approach was performed through a Gaussian peak fitting routine to determine the weight percent loss (i.e., the peak area) of each component; the reported masses from the two methods matched within ± 1%. The total mass of oxidized metal was reported as the sum of the mass from MoO3 (peak 'c') and the mass remaining at 1000°C. Triplicate analyses demonstrated mass accuracies of ± 0.2%. The initial weight loss ≤300°C was ~5%. While additional error could be attributed to weight gain by the oxidation of metals, the major source of error in reported weight percentages emanated from the fitting of peaks with components displaying overlapping oxidation temperatures.
The absorption spectra of DM-SWNTs were acquired using a dual-beam Perkin Elmer Lambda 900 UV-VIS-NIR spectrophotometer and were background-corrected using DMEM/FBS. Scans were performed from 400–861 nm with a scan speed of 125.00 nm/min and a 0.44-s integration time and from 861–1350 nm with a scan speed of 125.00 nm/min and a 0.48-s integration time. The instrument was wavelength calibrated on a quarterly basis using Holmium standards.
Elemental analysis was performed using a ThermoElectron X-Series inductively coupled plasma mass spectrometer. Samples (100 μL of DMEM/FBS or DM-SWNTs) were acid digested using a protocol developed in association with PreciLab Inc. (Addison, TX). In brief, a solution of 25 μL of 37% HCl and 25 μL of 69% HNO3 was added to samples which were bath ultrasonicated for 20 min. Next, the samples were diluted with a 2% HNO3 blank to a total volume of 10 mL. All samples and standard solutions were sprayed into flowing argon and passed into the torch which was inductively heated to ~10,000°C. Ti and Co were calibrated using blank, 50-, 100-, and 250-ppt standard solutions, Mo was calibrated using blank, 250-, 1000-, and 5000-ppt standard solutions, and Fe was calibrated using blank, 0.25-, 1.0- and 5.0-ppb standard solutions.
Human epithelial-like HeLa cells were obtained from the American Type Culture Collection and were cultured in 100 mm diameter polystyrene tissue culture dishes (Sarstedt) in DMEM/FBS containing 15 mg/L phenol red in an incubator at 37°C with 90% air and 10% CO2. Aseptic conditions were maintained at all times and media was changed every 2 d. Cells were passaged 1:10 every 4 d upon achieving ~80% confluence.
HeLa cells were plated into standard 24-well plates (~1 × 104 cells/well; ~20% coverage) in DMEM/FBS (buffered with 10 mM HEPES; no bicarbonate) and incubated in air at 37°C. After 24 h, the media was removed and replaced by a 400-μL aliquot of freshly prepared DM-SWNTs or fresh media (control). Each group of cells was incubated further in air at 37°C for 1–4 d. On each day, some HeLa cells were washed twice with 400 μL of sterile PBS and harvested with 100 μL of trypsin-EDTA solution (Irvine Scientific) for Coulter cell counting. Population doubling times (PDTs) were determined using the equation PDT = ln (N/No)/t, where No represents the initial cell number, N represents the final cell number, and t represents the time interval between No and N [120]. Each group of cells was analyzed in triplicate; one-way ANOVA statistical analyses were performed at the 95% confidence level, where p < 0.05 was considered significant. Differential image contrast (DIC) images were acquired using a Nikon TE 2000-U inverted microscope and a 60×/1.4 NA APO-Plan oil-immersion objective.
All Raman spectra acquisition and sample preparation methods were similar to those described previously by Chin et al. [19]. Spectra were acquired utilizing a Horiba Jobin Yvon high-resolution LabRam Raman microscope system equipped with a 250-μm entrance slit and a 400-μm pinhole. The 633-nm laser excitation was provided by a Spectra-Physics model 127 helium-neon laser operating at 20 mW. The power density emanating from the 50×/0.5 NA LM-Plan objective was typically 3.4 mW as measured using a Newport model-1815C power meter with an 818 UV series photodetector. Wavenumber calibration was performed using the 520.5 cm-1 line of a silicon wafer; the spectral resolution was ~1 cm-1.
Raman spectra of SWNT dispersions were acquired by placing them into 35 mm polylysine-coated glass bottom "imaging" dishes (MatTek). The acquisition time for a 250-cm-1 spectral region was 10 s with a scan speed of 0.04 cm-1/s; all spectra were plotted as the average of three scans. For live-cell analyses, ~1 × 105 HeLa cells were seeded in imaging dishes with DMEM/FBS and incubated at 37°C in 90% air and 10% CO2. After 24 h, the media was removed and the HeLa cells were rinsed three times with sterile PBS. The cells were incubated further in air at 37°C (or 4°C) in 1 mL of either DMEM/FBS (control) or a freshly prepared DM-SWNT dispersion. Following the designated DM-SWNT incubation period (12–60 h), the cells were copiously rinsed at least three times with sterile PBS. After excess PBS was removed from the dish, 1 mL of fresh media was added and the dish was placed on the microscope stage for analysis at room temperature. Adherent cells were brought into focus by viewing transmitted white-light images obtained through a CCD video camera. The Raman acquisition time for a 250 cm-1 spectral region was 45 s with a scan speed of 0.18 cm-1/s; all spectra were plotted as the average of three scans.
Live cells were incubated in a DM-SWNT dispersion (or a DMEM/FBS control) for 60 h as described above. After the final PBS rinsing, the cells were fixed using 2.5% glutaraldehyde in 0.1 M cacodylate buffer and embedded in agarose. Cell pellets were cut into small pieces, post-fixed with 1% osmium tetroxide, en-bloc stained with 1% uranyl acetate, dehydrated in a graded ethanol series, and embedded in EMbed-812 resin. Ultrathin (~100 nm) sections were cut on a LEICA EM UC6 ultramicrotome, post-stained with uranyl acetate and lead citrate, and viewed using the JEOL JEM-1200EX II electron microscope at the Molecular and Cellular Imaging Facility at The University of Texas Southwestern Medical Center.
In all flow cytometry experiments, ~1.0 × 106 HeLa cells were seeded in imaging dishes with DMEM/FBS and incubated at 37°C in 90% air and 10% CO2. After 24 h, the media was removed, the cells were rinsed with sterile PBS, and the cells were incubated in fresh DMEM/FBS (control) or a DM-SWNT dispersion in air at 37°C for 60 h. In some cases, cells were rinsed at least three times with sterile PBS and loaded with a solution of MitoSOX™ Red (Invitrogen-Molecular Probes). Specifically, cells were incubated for 60 min at 37°C in a 10 μM MitoSOX™ Red solution prepared in 4:1 (v/v) DMEM/PBS. Next, cells were rinsed three times with PBS, harvested with 500 μL of trypsin-EDTA solution, centrifuged at 5000 RPM for 5 min, and resuspended in 3 mL of fresh 2% (v/v) FBS/PBS. Finally, cell suspensions were filtered through a 30-μm PreSeparation filter (Miltenyi Biotec). Fluorescence-based flow cytometry was performed using a Becton Dickinson FACSCalibur ® flow cytometer equipped with a 488 nm laser. MitoSOX™ Red fluorescence (λMax = 590 nm) was detected over the range of 564–606 nm and the background fluorescence was detected over the range of 515–545 nm. All quantitations were performed using CellQuest 7.5.3 software; in each experiment, well over 10,000 cells were analyzed.
The author(s) declare that they have no competing interests.
HNY performed the majority of the experiments and wrote the manuscript with PP. GRD, RKD, IHM, and PP designed the overall project and aided with data interpretations. CM ran the culturing facility and assisted with the interpretation of live cell data. EKW performed and interpreted the thermal gravimetric analyses. PB performed and interpreted the scanning probe analyses. MCD performed and interpreted the elemental analyses.
Supporting thermal gravimetric analysis data. Estimation of SWNT concentrations in DM-SWNT dispersions.
Supporting atomic force microscopy data. Atomic force microscopy of SWNT dispersions.
Supporting Raman spectroscopy data. Raman spectrometer reproducibility and calibration.
Supporting flow cytometry data. Event plots.
This work was supported by grants from the Robert A. Welch Foundation (PP; grant AT-1364 and IHM; grant AT-1326) and the Human Frontier Science Program (GRD; grant RGY0070/2005-C), by the gift of equipment from the von Ehr Foundation, and by funds from the State of Texas (RKD). We are grateful for assistance to this work by Chris Gilpin, Karis Hughes, Laurie Mueller, and Vicky Poenitzsch, and for insightful discussions with Ray Baughman, Alan Dalton, Radu Marches, and Ru-Hung Wang.
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.


text:
It was full of good idears.
Project Runway Finale.
Related Articles
Nina Dobrev Is a Guest Judge on Project Runway Season 15 (PHOTO)Jun 22, 2016 | 0
Want to comment on this? First, you must log in to your SideReel account!
With SideReel you can...
Track your favorite shows
Choose from 21,000 shows
Earn badges for watching

subset: commoncrawl


text:
Separate names with a comma.
Discussion in 'General Chat' started by jdp1962, Nov 10, 2005.
Whose been upsetting Mr Kipling then.
Must be a direct cause of global warming, everything else is,lol
....... or is it a car with one of them new 'bun-roofs'...... Oh dear *groan*
I wanted all the toppings with my chocolate chip muffin, but not literally!
That cars one serious **** in magnet! ok i'll leave now!
That'll teach him not to upset the waitress at the drive-thru!
Who said you can't have your cake and eat it.


text:
Group Booking Terms and Conditions
Because you are making a Group Booking, there are additional Terms and Conditions that apply. Please read them and click 'I Accept' below to continue with your booking
Hostel Specific Terms & Conditions
The remainder of the amount due will be charged to your credit card ???? Should numbers or room selection change, the per person deposit times the number of people who actually travelled will be deducted from the total accommodation charge levvied by the hostel.
The following group types are not accepted by this hostel: Holiday with Friends, Junior / Primary School, High / Secondary School, College / University, Business Trip, Stag/Hen/Bachelor Party, Sports Group, Cultural Group If you have misrepresented the type of group you are booking for, your booking may be cancelled by the hostel.
Any cancellations resulting from agreement to these terms and conditions under false pretences will result in loss of deposit.
General Group Booking Terms
By making a group reservation through Hostelworld.com you agree to the following terms and conditions as set by Hostelworld.com and as set by the hostel in question.
- In order to make a group reservation on Hostelworld.com it is necessary to pay a deposit. The balance is due directly to the hostel according to their payment conditions.
- The booking deposit is non-refundable
- Hostelworld.com are not responsible for changes or amendments to your reservation once it has been processed. All correspondence with regard to changes to your booking need to be dealt with directly with the hostel.
- Cancellations for reservations must be made directly with the hostel and must conform with the individual hostels cancellation policy (see below)
- It is your responsibility to pay the balance of your accommodation cost to the hostel in accordance with their payment conditions (see below). Failure to do so may result in your reservation being cancelled by the hostel.
- Individual hostels may have special booking conditions relating to group stays, please read the hostel booking conditions carefully before making your reservation.
Vous devez accepter les termes et conditions pour que nous puissions confirmer votre réservation
Bientot épuisé(es)
Description de l'établissement
Avis le plus récent
" Right on the river and a short walk from the bridge that takes you into Historic Bern. Getting here from the train station is easy and takes you right through the historic district. Though the facilities are 6 beds per room, each bunk is enclosed with its own temporary wall so you only share space with one other individual. Staff was extremely friendly and helpful. "- Anonyme
Les applications mobiles d'Hostelworld
Réservez facilement grâce à la nouvelle application mobile Hostelworld.


text:
Riad Noire D'Ivoire was the perfect place to be taken for birthday break in November. The decor of Riad itself is beautiful. Rebeca and Damian were extremely helpful and gave us lots of tips, and their staff will do everything to make your stay enjoyable and comfortable. We stayed in the Zebre Suite, which was luxurious - the highlight being the private roof top terrace and jacuzzi . We will never forget the champagne birthday breakfast - and the Hammam and massage in their Spa was so relaxing. We will be going back here without doubt!
- Official Description (provided by the hotel):
- Riad Noir d'Ivoire is a romantic destination composed of five suites, three of which have private roof terraces and jacuzzis, two junior suites and two double rooms. Services include Spa with traditional hammam, Boutique, fully equipped gym, library, restaurant and a heated pool. All suites have fireplaces and all rooms, have air conditioning, heating, mini-bars, hairdryers, wall safes and flat screen televisions. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Expedia, Orbitz, Booking.com, Travelocity and Odigeo so you can book your Riad Noir d'Ivoire reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Riad Noir d`Ivoire Hotel Marrakech
- Riad Noir d Ivoire Marrakech


text:
Gift Administration is responsible for administering the University's many gift funds. Gift funds are comprised of gifts that that may be spent in their entirety, including current use gifts and gifts for the construction, renovation and maintenance of the University's facilities (plant funds) and gifts to the endowment, which are meant to provide lasting support to the University. For endowment gifts, only the income earned by the gift from may be spent and the principal remains intact for perpetuity. Gift funds may be for the unrestricted use of the University or for restricted purposes as specified by the donor.
Gift Administration is responsible for:
- Setting up new funds and modifying existing funds, as necessary
- Allocating gifts to the University to the appropriate funds
- Maintaining ALICE, the University's funds database
- Financial stewardship of restricted funds
- Monitoring compliance with donor restrictions
- Preparing annual endowment income budget and gift administration year-end closing adjustments
- Preparing investor reports and performing the plant fund analysis; and
- Reconciling the central gift holding account
Gift Administration provides services in the following areas:
- Acts as an expert resource regarding the usage of restricted gifts
- Conducts training covering the administration of restricted funds for the Yale business community
- Serves as a permanent member of the University's Gift Implementation and Stewardship Committee
Treasury Services is responsible for maintaining the University's operating bank accounts, for monitoring the University's daily cash position, for processing all in-coming and out-going domestic and international wire transfers, the establishment of petty cash funds, and for the proper recognition in the general ledger of revenue transactions recorded through the Receipts Identification Form (RIF) system. It is also responsible for the establishment of merchant numbers for the acceptance of credit cards on campus and for monitoring the University's compliance with the Payment Card Industry Data Security Standards.
Reporting Cash Transactions
The Internal Revenue Service requires reporting of certain cash transactions in excess of $10,000 within 15 days of receipt of cash. Please visit Reporting Cash Transactions [1] or contact the University Tax Department at 2-5530.
Treasury Services advises the Yale community on domestic and international banking issues and provides the following services at its two Cashiers' Offices, which are located at 2 Whitney Avenue and 100 Sterling Hall of Medicine:
- Distribution of petty cash
- Issuance of cash and traveler's checks advances and reimbursements
- Delivery site for RIF (on–line deposit) tracking slips and funds
- Processing of returned checks
- Coin and change
- Night deposit safe (Yale Police must provide escort and Key for 2 Whitney)
- Processing of credit card transactions for departments that do not have their own credit card machines
- Pick up of accounts payable checks (2 Whitney Avenue only)
- Drop off for accounts payable documents (2 Whitney Avenue only)


text:
Boulder, COUSA
July 10, 2014
It makes all the things fun again.
I'm the Director of Development for a fresh startup in the Denver area. Our front end app uses AngularJS, and we've been loving it so far!
Meetup members, Log in
By clicking "Sign up" or "Sign up using Facebook", you confirm that you accept our Terms of Service & Privacy Policy


text:
I have to compile a simple list of the popular programming languages from the 70's, 80's, 90's and 2000's.
I've got it all except 2000. In your opinion what would constitute as the most popular programing language for 2000's? It's kind of hard of hard to narrow it down so I was hoping for some help.
Yes, this is a homework assignment, but it got me thinking this might make a good discussion on the forums.
What are some of your favorite languages over the decades? And why? (No that isn't part of the homework, just a question I'm interested in hearing the answer too.)


text:
Raiders Draft Illinois Defensive EndJihad Ward was Oakland's 2nd round choice, 44th pick overall
Raiders Owner Sets Sights On VegasOakland's NFL team could be on the move if stadium is built in Nevada
How Did The Rams Pull It Off?Sports business analyst Marc Ganis explains why Stan Kroenke prevailed in relocation
Report: Rams' Plan To Go To Inglewood Alone Off The TableThe 32 owners are expected to vote on all the possible relocation bids, and a final decision could be made today on what team will be in Los Angeles for the 2016 season.
DAHLBERG: Greed The Motivator In Rush To L.A.A.P. writer says loyalty to fans trumped by vast earning potential
NFL Confirms 3 Relocation FilingsAs anticipated, the Rams, Chargers & Raiders all formally asked for league approval to move to Los Angeles in 2016
Chiefs Owner: At Least One Team In L.A. Next SeasonClark Hunt made the prediction on the eve of the Rams, Raiders, and Chargers filing for relocation
NFL Hears From All 3 Cities Wanting To Keep TeamsSan Diego & St. Louis made plans public. Oakland has not.
Rams Staying Out West This WeekJeff Fisher's team practices at Raiders training camp in Napa, CA ahead of game in San Francisco
Fantasy Football: Chargers Vs. Raiders - Who To Play, Who To PassFor the final matchup on Thursday Night football, the Oakland Raiders host the San Diego Chargers. Both teams are already ruled out of the playoffs, but these players will have an impact on fantasy football playoffs.
CBS Sports' Boomer Esiason: Nothing Better To Belichick Than Beating JetsCBS Sports' "The NFL Today" studio analyst Boomer Esiason talks two big games in Week 16 and what's at stake for those in the playoffs.
Chargers And Raiders Battle In Final Thursday Night Game Of 2015The San Diego Chargers have one of the worst records in the NFL while the Oakland Raiders have a middle-of-the-road record at best.


text:
StartUpHire is the place to find Product Manager, Security jobs. Review the list of Product Manager, Security career opportunities to the left. These Product Manager, Security jobs represent some of the most rewarding careers available.
You can refine your job search for Product Manager, Security career opportunities by adding a job title, company name, or keyword to the search box above … or click the "Advanced Search" link for more options.
Don't see the employment opportunity you're looking for? Check back soon for more Product Manager, Security jobs, or sign up for an email alert (link below) to receive periodic updates on new Product Manager, Security jobs.
Do you believe that the future of IT Security will rely on machine learning and behavioral analytics? Does the convergence of data science and cyber security excite you? Do you want to work in an environment where your efforts will have a direct impact on customers, company,6/14/2016 Boston, MA Save This Job
Departments: Product Management Locations: San Francisco, CA Delphix is a rapidly growing data operations platform for the world?s largest enterprises. We?ve fundamentally changed the way that enterprises think about and use their data, but it?s still early days in terms of6/21/2016 San Francisco, CA Save This Job
Boulder, CO Overview LogRhythm is looking to hire a Product Manager that will own the product lifecycle of new features, products, and services, with a concentration on market research, pricing analysis and competitive analysis. You will partner with a small team tasked with6/2/2016 Boulder, CO Save This Job
Products (290) - Houston, TX - Full Time The Associate Product Manager will be responsible to drive technical research and investigation in order to help other product managers generate requirements for new product releases. The ideal individual will be able to install and use6/21/2016 Houston, TX Save This Job
Location: Austin, TX Job Description Do you want to be a technical Product Manager at a fast-growing mid-size company in the IT Security market? Do you want to help shape the future of IT Security? As a PM, conduct market research, listen to existing customers and prospects,7/14/2016 Austin, TX Save This Job
Location Burlington, MA Senior Product Manager Location: Burlington, MA Reports to: Vice President of Products Are you a high-energy, security savvy product management leader looking to join a fast-growing, market-leading company in the Burlington, MA area? Open7/5/2016 Burlington, MA Save This Job
Menlo Park AnchorFree?s mission is to provide secure and private access to the world?s information for every person on the planet. AnchorFree?s Hotspot Shield app is the world?s most popular freedom and privacy platform. Hotspot Shield is the starting point, for mobile and Web6/20/2016 Menlo Park, CA Save This Job
Danaher Company Description Arbor Networks is a leading provider of network security and monitoring solutions for global networks. Its customers include 90 percent of the world?s ISPs and large enterprises. Arbor solutions deliver best-in-class network security and visibility,4/20/2016 Ann Arbor, MI Save This Job
Product Management San Jose, California Apply Description Product Manager/Sr. Product Manager Location: San Jose, California Description Vormetric's mission is to secure world?s information. Vormetric is trusted by businesses and governments for over a decade. Vormetric?s5/29/2016 San Jose, CA Save This Job
Job Type: Full-Time Location: Nashville, TN Job Description: Are you seeking a position that offers you the opportunity to apply your technical and product management expertise? ENA may have the right position for your unique skills and abilities! ENA, the nation?s leading7/7/2016 Nashville, TN Save This Job
AnchorFree?s mission is to provide secure and private access to the world?s information for every person on the planet. AnchorFree?s Hotspot Shield app is the world?s most popular freedom and privacy platform. Hotspot Shield is the starting point, for mobile and Web access,6/20/2016 Menlo Park, CA Save This Job
Product Marketing & Management Austin, TX, United States SailPoint is the Worldwide Leader for Enterprise-Class IAM We minimize risk and maximize business growth by managing access to data and resources across your enterprise. We do it effectively and securely for every person6/19/2016 Austin, TX Save This Job
Location: Burlington, MA Product Manager Intern - Fall 2016 (September - December) The Product Manager Intern will work closely with Product Managers in product planning and execution throughout the product lifecycle, including: gathering and prioritizing product and customer6/5/2016 Burlington, MA Save This Job
New York City or Palo Alto MongoDB is looking for an outstanding and seasoned product manager for our cloud-based products. Our goal is to allow our customers to focus on innovation by removing the burden of operating, monitoring and scaling their MongoDB deployments. Candidates7/4/2016 New York, NY Save This Job
Description MarkMonitor?the world leader in brand protection and a Thomson Reuters Intellectual Property & Science business?is looking for a skilled Product Manager who has significant experience with ecommerce technology, IP protection, and scaling large technology programs;6/7/2016 San Francisco, CA Save This Job
? Location: Waltham, MA, United States ? Department: Products/Services Job Description: About the Job The Reporting and Analytics (Big Data) Sr. Product Manager will be responsible for defining and executing against the ?reporting & analytics? strategy for the Intralinks product5/24/2016 Waltham, MA Save This Job
Location:Atlanta Job Code:178 # of Openings:1 Description Product Manager Company Overview Bridgevine powers customer acquisition solutions through its next generation technology platform. We thrive in a digital market where product and service supply from leading service7/13/2016 Atlanta, GA Save This Job
Product Management Austin, TX JOB DESCRIPTION About Us At CloudFlare, we have our eyes set on an ambitious goal -- to build a better web. We believe that with our talented team, smart technology and engaged users we can solve some of the biggest problems on the web. We already6/11/2016 Austin, TX Save This Job
Job Description About Us: At CloudFlare, we have our eyes set on an ambitious goal -- to build a better web. We believe that with our talented team, smart technology and engaged users we can solve some of the biggest problems on the web. We already serve more web traffic than6/11/2016 San Francisco, CA Save This Job
at Twilio (View all jobs) San Francisco We are looking for a Product Manager to join our Messaging team to design and build our next generation of protection against SMS spam and phishing. As a PM, you will be responsible for defining the product roadmap, specing out the5/28/2016 San Francisco, CA Save This Job


text:
GOSEI GREAT MEGAZORD & ZORD VEHICLE COMBINATIONS! Megaforce Toys! (Nov 2012)
What do you think about this video?
Berenice De Hoyos: I love this video
Ahmed Jawad: Is the ranger white mighti mophan
Robert Fiedler: Love your videos Bruno and check out mine
Jonny Ly: I rather put the shark head and the hawk head on the side rather then the knees so the sea brother combination makes sense
jdzencelowcz: +MMPRtoys My only real gripe with it is, it's so small, like, smaller than the other megazords in that line; if they had made it a tiny bit bigger, they would've been able to keep some transformation functions.
Curtis Barton: I just got some of the remaining zord builder pieces from the Power Rangers Megaforce series to do an ultimate combination of black lion with all 3 bikes, ultra mode cycles, robo cycle, gosei jet, gosei ultimate megazord...I issue a challenge to all to see who can get closest to using all zord builder parts.
Leiyha Cash: Cool
Jake Lareno: You guys rock
Nhung Vu Thi: and red
Nhung Vu Thi: black
Nhung Vu Thi: i like the zord is blue
Paul McInnes: I have the gosie ground megazord
carlos quevedo: Hoye tonto porque noases bideos de power rangers spd
power ranger reveiws: Maryam Gaming it dosnt matter if your a kid or adult if you like them
Juliet Annett: last one was amazing
Lynsey McClung: Hxvx bhsfsbghdsgsheg a dafgavegghwgegegwgwgwva. Ggwffgwgqgwggegegwgehehhwhwhhsh P R R. p. M
Killopia Queen: Why are adults playing with kids toys? wtf?
Holiday: We don\t make alot, but our group do get to travel frequently. Just this year we went to Bangkok to get pictures and their holiday schedules. We are still in a growing phrase so best we can aim for is break even. I think everyone have to overcome the same type of challenge when starting something new. Don\t give up!
GOSEI GREAT MEGAZORD & ZORD VEHICLE COMBINATIONS! Megaforce Toys! (Nov 2012) 5 out of 5


text:
Written on: 24/01/2014 by callum_thomas1 (1 review written)
I didn't go for the full social media package as I already have a great Facebook following and a pretty ok Twitter following but I didn't have anything on Google Plus. I paid $150 for Wac Design to get me started on Google. They created all of my images for the background and avatar and they created and scheduled several weeks worth of content as well as giving me a prety solid following of nearly 200 people in my circle.
The biggest boost was linking my profile to my search results so I get my Plus page to the right of the results when people search my business but they also set it up so when I write content and blogs, my logo appears in the search results when that content is found which has given me a lot more exposure.
We did everything via email as I live in the States but they did have Skype if we needed it.
I would say very good value for money!
As rated by our community of reviewers
" Bullies " Read More
" Very unprofessional service..! " Read More
Written by kunaldesibar
" Beware! They can't install wordpress template but charge £££ " Read More
Written by plumber5971
" 6star Cellular Phone Products 6star.org - I would recommend you to... " Read More
Written by mdamin76
" Good company " Read More


text:
Related Searches: Face | Toys | Handbags & Accessories | Tops | Top Related Searches
as low as $1.79
as low as $1.77
as low as $1.64
as low as $25.00
as low as $39.98
as low as $1.81
as low as $4.09
as low as $6.00
as low as $3.97
as low as $4.99
as low as $2.25
as low as $1.59
as low as $1.87
sold by 2 Sellers
as low as $2.12
* Shipping costs are based on an estimate of UPS ground or equivalent carrier within the contiguous US, excluding Alaska and Hawaii. Please see Seller's website for actual shipping costs.
PriceGrabberAbout Us Jobs Advertise Sitemap Mobile Site
SupportMerchant Login Market Reporter Mobile Apps Help
Choose your Region:
Copyright ©2016 PriceGrabber.com, Inc. All Rights Reserved


text:
AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Mixing two singles with two walks, two wild pitches, an error, a stolen base, and two sacrifice flies, the AZL Cubs scored four in the 8th to break a 1-1 tie and defeat the AZL A's 5-1 at Dwight Patterson Field at HoHoKam Park Sunday night in Mesa.
With the game tied at one, Blair Springfield drew a lead-off walk on a 3-2 pitch to get the Cubs 8th inning started. Speedy Vismeldy Bieneme was sent into the game as a pinch-runner, and proceeded to steal 2nd base and advance to 3rd on the catcher's E-2 overthrow. He then scored on a Wild Pitch. Cody Shields followed by reaching base on a strike out/WP, beating the throw to 1st by a step. Pin-Chieh Chen looped a single down the LF line to move Shields to 2nd base, and Elliot Soto followed by drilling an RBI single into the 5.5 hole, scoring Shields, as Chen moved up to 3rd and Soto advanced to 2nd on the throw to the plate. After Ryan Cuneo was walked intentionally to load the bases and set-up a force at any base, Anthony Giansanti and Sergio Burruel hit consecutive F-7 sacrifice flies to plate Chen and Soto with the 3rd and 4th runs of the inning.
The Cubs had previously tied the game in the bottom of the 6th on consecutive two-out singles by Chen (a line drive sliced to LF), Soto (a high chopper that the 3rd baseman could not field cleanly), and Cuneo (a rocket ground ball through the box into CF).
Two more Cubs made their pro debut tonight, and both 15th round pick (and Algonquin, IL native) SS Elliot Soto (Creighton) and 23rd round selection RHP Matt Loosen (Jacksonville U.) had a hand in the victory.
Soto (considered one of the top defensive shortstops in college baseball) had two hits (including an RBI single) and made two flashy plays at shortstop, one a rangy stop and no-look back-handed flip that started a 6-4-3 DP, and another a full-steam running pick-up and off-balance throw to 1st to nail the batter and keep the go-ahead run off base in the 8th.
Loosen got credit for the victory, throwing 1.2 IP (20 pitches -15 strikes), allowing one hit (a single) while striking out one.
2009 16th round draft pick CF Cody Shields (AU - Montgomery) also made his "official" pro debut tonight (he missed the 2009 season recovering from TJS), although he had seen a lot of action in Extended Spring Training prior to breaking his hand in an intrasquad game about a month ago.
AA Tennessee LHP Casey Lambert (2009 TJS rehab) made his second AZL rehab start tonight, throwing two innings (25 pitches - 22 strikes) and allowing one run on three hits and no walks, while striking out three. RHP Eduardo Figueroa followed Lambert with five innings of one-hit shutout ball, walking one while striking out five (by far the best he's looked in his two years in Mesa), and RHP Jadel Mendez relieved Matt Loosen with a runner on base and two outs in the top of the 9th, before getting a strike out (swinging) to end the game.
In AZL Cubs roster news, RHP Manolin DeLeon (2010 knee surgery) has moved up to Peoria (he was at Daytona last season), RHP Aaron Kurcz (2010 10th round pick out of the College of Southern Nevada), LHP Eric Jokisch (2010 11th round pick out of Northwestern), and INF Pierre LePage (2010 13th round pick out of the University of Connecticut) have ben promoted to Boise, RHP Jesse Ginley (2009 NDFA) has been moved down from Boise to the AZL Cubs, and 3B-1B-C Bobby Wagner (2009 38th round pick out of Panola JC) has been released. (Wagner tried to make the conversion to catcher in Extended Spring Training, but it just didn't work out).
As for the three who got their tickets punched to Boise, Kurcz may not have the stamina or repertoire to be a starter, but he does have the earmarks of a closer or 8th inning set-up man (he throws mid-90's gas), Jokisch is a polished college lefty who (if he can stay healthy) could move quickly through the system, and LePage began his pro career last week like a house afire, getting seven hits and two SB in his first two games before the promotion to Boise. (LePage struck out just three times in 278 PA at U-Conn in 2010).
the trib...
Now that they don't own the team...actually telling fans to stop going to games if they want to send Ricketts a message.
Yeah, that really worked in the 50's...
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
I would target sending Zambrano to Texas, where he can get coaching from Mike Maddux and Nolan Ryan, and targeting Chris Davis in return.
Davis plays excellent defense, and you probably wont get a better talent like that in return. We don't plan on competing in the next few years with the roster we have so give Davis a shot over the next couple years, its better than watching the traveling sideshows of Ron Coomers try and fill out the position.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Texas has no money and are one of the teams that makes a big deal about "chemistry".
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Oswalt is a better fit, and they probably can't afford him.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
You're the best AZ Phil.
Draft rules question for you (or someone else with the knowledge). Now, if a player goes to a 4 yr. college, he isn't draftable until after his 3rd year or 21st birthday. Has that always been the rule? Or did they used to draft freshmen too?
I'll hang up and listen for my answer.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Az Phil -
With Ricketts in charge, the major league team being a total disaster this year, and a bunch of bad contracts looming for the coming 2 years do you envision the owner allowing an acutal "rebuilding process" to occur at Wrigley? I would envision the following: a) Trade all valuable commodities (some more valuable than others) such as Lilly, D.Lee, Nady, Silva, Theriot and b) Make an attempt to offload other players to save money (i.e. - we'll pay some/most of their salary if you take them) - Fukudome, Soriano (no chance), etc.
It seems to me that the best approach for this team would be to let the young kids play to see what we truly have and build towards 2012-13. This team needs to have Castro, Colvin, Cashner, Jay Jackson, HJ Lee and Brett Jackson (in 2 yrs) as the focal points of success if it is ever going to be good. May as well let Samardzija back up to see if he can hack it in the 2nd half of the season, right? Let Barney see if he can be a serviceable backup 2B/SS at the ML level. Let the kids play and then add 2-3 key FA's when we would have money to throw at them - I'd envision a 1B in 2012 (A.Gonzalez or Prince) to plug the middle of our lineup and a staff ace to support some young arms. Please give me your thoughts on the cubs best and likely approach this and the next 2 years. Thanks!
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Zambrano isn't a clubhouse cancer or a social outcast like Milton Bradley. He will fit in with any team in the major leagues.
The Rangers may be financially strapped but thats mainly because the Rangers have sucked since 1998, but nothing like a playoff run to bring people back to the ball park. You can credit Nolan Ryan for being an outstanding hands on president of baseball operations in Texas for starting that turn around.
Texas has a 70 million dollar payroll and Kinsler (28) and Young (33) aren't getting any younger. Josh Hamilton and Nelson Cruz are both 29. There is about a 3-4 year window of opportunity to win a WS with the Rangers current roster.
They will try and add a big money pitcher this year.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Thanks for the breakdown Phil - As a follow up to how they rebuild, do you have an updated top 5-10 list of guys still in the minors now that our big 2 are in the bigs (Castro and Cashner). I'm guessing B.Jackson, Lee, J.Jackson and Vitters are top 4 but I am interested in who has shown big jumps this season thus far. Thanks!
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
I'm really hoping McNutt and Jockisch are on the MLB Cubs at the same time at some point.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
I wonder if McNutt is on that list now. . . I also wonder if this year's current first rounder will make that list by the end of the year.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
I really like that you are so high on Jay Jackson....the Cubs will really need Cashner and Jackson to contribute (be front line starters) in the coming years to rebuild this thing. Do you see Castillo and Soto being able to platoon next season? There seems to be a significant drop off in top line talent after #6....it goes from core talent to possible contributors, is this an accurate assessment?
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Soriano to first base seems like a disaster to me. Do you have an opinion on Soriano's defensive potential at first base? I know he started his career in Japan as a 3B, but after watching him in LF for the past few years, I just see him losing his concentration and screwing up easy plays over there. But maybe being on the infield would help him?
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
no one ever likes putting a right handed guy on 1st because of how it effects the footwork, but it might be a necessity given what the cubs have vs. what new players need playing time...and the whole $$ thing restricting the movement of some players.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
It's not just footwork, it's also throwing to second and applying tags on pickoffs.
I am not sure I could get onto the "the Fielders can play 1st but Soriano couldn't" bandwagon. I am sure he'd be fine, if a bit short.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
yeah, it's more of a "i'd rather not do that" from a manager/GM/etc view than "this could be a disaster" thing.
you lose a bit of reach, you lose some of your angles, etc etc...but it's not like putting b.molina in CF or something. there's plenty of RH 1st who do it just fine.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Az -
Looking at the near term (the next 2-3 years), this is how I see this team taking shape:
C - Soto and Castillo
1B - Free Agent or Colvin (no in house options - probably need a big bat to help the young kids mature). Look for one year deal next year (LaRoche, Huff, even D.Lee again)
2B/SS - Castro/HJ Lee/Barney (may need a 2B FA for a year or two like Orlando Hudson due to his "Makeup" which is suddenly important for this team).
3B - Aramis / Vitters in a few years.
OF - Soriano, Byrd, Colvin, Brett Jackson
SP - Dempster, Cashner, Jay Jackson, Silva/Wells/Gorzallony. Most likely will add a FA starter at some point. Will need an ace but probably too soon to add Cliff Lee at year end. Will add someone in 2012 most likely.
RP - Marmol, Marshall and a bunch of young guys (Caridad, Guzman, Russell, Stevens, etc). We'll probably bargain hunt for veterans for the 8th inning role (Wood on the cheap next year). Cubs better lock up Marmol or they are a total mess in the pen.
Is this reasonable?
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Provocative speculation is always fun, but in reality, the likelihood of all of those young guys doing that well is pretty low. Some won't pan out. This post 2-3 years ago would show Felix Pie, Eric Patterson, etc. on this 2010 team, and probably wouldn't have had Colvin there. That's simply the nature of prospect development.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Been going through fangraphs for pitchers and seeing what pitchers throw and the average velocity.
Just for fun i looked at Kerry Wood, whose fastball velocity is about as good as its ever been but he has abandoned his slider which he used to throw about 25% of the time, also he doesn't use his cut fastball or changeup anymore. He is relying more on his curveball but any scouting report would suggest just sit fastball and tee off.
Maybe due to injuries he can't throw those anymore, but maybe he needs to go back to throwing his slider, which he did have a pretty good one.
Just found it interesting.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
kerry's still got his slider and it's rocking 87-90mph.
it doesn't have the movement it used to, but he throws it harder.
fangraphs used to be a lot more stable with info, but i think (because they're doing this with many players) they're seeing that high-80s slider and thinking it's something else.
Z update
should be back this week
"Probably a four- or five-game suspension would suffice," he said. "As much attention as the incident received in Chicago, you really can't justify a suspension of more than that for acting like a goof and arguing with a teammate."
Defending Big Z's big contract
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
It was just another example of Hendry having awful timing as to when he gave that deal.
If you remember, the offseason before he signed Z to the extension. Supposedly Z would have extended for 4/42 or something along those lines? Surely someone other than me can recall.
Whether it was Zell or Hendry, waiting 6 months to extend Z almost doubled the total contract value.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
You may have heard that somewhere, but the show me the list of guys who finished in the top 5 of Cy Young voting in two of three years at age 25 who were looking for a 4 year deals for a $2 million pay cut for the privalage of singing one.
At the time I said signing starting pitchers to long deals is always a bad idea, but there's no way in hell anyone who knew anything thought Z would take a contract close to what you mentioned there.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
It was a 5th year of service time contract.
Usually you can get a pretty good discount by buying out arbitration and the first couple years of free agency.
Unfortunately when the cubs signed him. He was in the middle of his free agency season. The Cubs leverage had already expired.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
List one comparable player. There's no way he was going to sign a contract that was that short for that cheap. In his sixth year he made $12.5 million...
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Longoria, and Braun and Nate McLouth off the top of my head.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
those guys were bought out before their first year of arbitration.
If I recall Oswalt is the best comparable...
# 5 years/$73M (2007-11), plus 2012 club option
* signed extension with Houston 8/06
* 07:$13M, 08:$13M, 09:$14M, 10:$15M, 11:$16M, 12:$16M club option ($2M buyout)
compared to
# 5 years/$91.5M (2008-12), plus 2013 vesting option
* signed extension with Cubs 8/17/07
* $5M signing bonus
* 08:$15M, 09:$17.75M, 10:$17.875M, 11:$17.875M, 12:$18M, 13:$19.25M vesting player option
Texas of course has no state tax so someone smarter than me can calculate what the take home pay actually is, on top of Z being younger when he signed the deal.
Re: Z
Speculations had been Zambrano could have gotten a seven-year, $140M deal in free agency, but Zambrano said, "Not everything is about the money. … I feel comfortable [in Chicago]." Ginnetti noted the deal is "somewhat greater than the estimated five-year package the sides were close to signing in spring training before the Cubs' sale was announced,"
and TCR reactions
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
I could be totally wrong. I just seem to remember that people were up in arms that Zell buying the club caused the Cubs to delay the extension. Which in turn drove up the price considerably.
My recollection was that before arbitration there was a 4 year 40 something contract on the table. Hendry had to pull the offer due to the Trib/Zell sale.
Dempster: Up to Zambrano to apologize
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
That team for 2011 does not excite me one bit.
What is the point in having Wells, Gorzo, Silva in the rotation and Cashner and Jackson out of it? Does Chicago think Cashner is going to stay in the pen or is that your general feel?
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
What's the word on Esmailin Caridad's injury?
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
damn...darin downs (former cub farmhand) pitching for AAA durham (TB)...i thought he was done after fracturing his skull last year.
he had a decent "comeback" in AA and was moved to AAA recently for the first time.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Jeez AZ PHIL - you had to remind us of another excellent management move with Patton? ugh.
Does Alessandro Maestri have a shot at making a MLB club down the road? Or, will he just punt and move to the "Pasta Circuit" in his own country?
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Phil ~
Is there some reason that Shafer was moved to the bullpen this year after anchoring the starting staff at Peoria last year?
He has put up some pretty spectacular numbers out of the pen so far, but I really thought his future was in the rotation. His '09 numbers were somewhat pedestrian, but he seemed to have the stuff and the stamina necessary for success as a starter.
Thanks in advance.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
So you're sayin that you see McNutt coming in to relieve Jockitch?
Curious if?
Is Carlos Silva pitching well enough that the cubs could deal him?
What if we sent along the 5.5 that Seattle sent to us? If we are really going into rebuild mode. It would seem smart to get out from his deal while the getting is good.
Just sayin
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
silva's 2011 being under contract for 11.5m might scare off people.
we see awesomeness keep happening, but not too many are buying into it yet. it just seems silly more of us aren't won over, but i know a lot of people (including me) that still don't quite believe what we're seeing.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
I don't believe it.
That is why I am advocating sending him out if possible.
Would the 5.5 Million that Seattle sent us, be enough to dump him on someone else?
Should be owed about 12-14 million for 1 1/2 Years if we included the money.
Re: Ship Silva
What's the point? Silva isn't a problem. If he slips as starter his control is still so good he should be useful in the pen where he and Z can fight over the pink bag.
The Cubs offense is what's killing us and given contract restrictions the only places it can be improved next year is at first, second, and the bench.
Fire Hendry.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Agreed on Fire Hendry,
I just hate to see us go into 2011 counting on Silva's a #3 Starter.
Better to use the good fortune to get out of 12-14 Million of bad contract and use even half that money in the draft or Latin America.
Even if we used that money on a LaRoche, Huff, or Encarnacion. It is likely better used there than on Silva going forward.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Why would we want to risk him regressing and have to put his $11.5 million salary in the pen? Deal him if you can. Maybe we get something useful for the next few years.
Nothing against Silva personally, I've been thrilled by his performance, but if we're rebuilding, and we have a fair amount of young starting pitching, it's time to start plugging those guys in.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
I would think that if he keeps pitching like this into next month, and we offered to pay a chunk of his 2011 salary, we should be able to get some prospect. If we're going to start rebuilding and one of the young pitchers might be ready but have no spot in the rotation, I say deal Silva. I don't want to see Atkins or Coleman or Jackson or whomever not get their innings in because of a veteran we don't necessarily need.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Kind of my point as well. Beyond the money he costs next year.
Zambrano can't stay in the pen for 2 more years. At worst he is our #5 until his contract expires.
Cashner, Jackson, Carpenter are all knocking on the door. Marshall and Gorzy are still in the Pen.
Lilly is likely (Hopefully) dealt at the deadline.
It's really time to accept our fate this year and build for a future beyond 2010 and really 2011.
Just my opinion.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
"Jon Heyman of SI.com reports that the Marlins are "strongly considering" D'Backs third base coach Bo Porter for their managerial vacancy."
that's ex-cub bo porter...of which i happen to have over 500 rookie cards for (don't ask, it was a bulk purchase for $5 back when he was still in A-ball).
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Not following too much, but I heard that Valentine had the gig.
Wha' happened?
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
The only thing anyone will say is "philosophical differences". I don't know if that means Valentine wanted more say so in front office moves or asked for free hookers at the post-game spread.
Philosophical Differences
German Nihilist.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
FLA probably doesn't want to pay him the money it's worth to him to put up with all the plane travel...dunno, though.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Hard to belive you've kept the cards this long.
Hoping he's the next Tommy Lasorda?
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
sadly, i just have storage space and keep them for who knows what reason. those cards (and many other failed player lots) don't take up much space or weight even if there's a few hundred.
in my "ebay days" when people had expendable money i'd buy lots like this from "box/case breakers" who'd keep the hot cards of the moment and sell the rest off so they can get to opening another few hundred packs of cards.
when you get lucky you can sell your 1-5 cent per-card investment into 10 cents to a buck+. i'd usually sell lots of 10 of a player for $1-$10 depending on how hot of a player they were at the moment. you could get some surprising turn-around on these "penny stock" card lots if you're ready to sell in smaller bulk when the stock is on the rise.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Good god. Have you figured the time spend into your equation though? Unless you just really like going through baseball cards...
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Not much time. I'd go to a card show and throw down $20-$100 on various card dealers or buy from "case breakers" online.
I'd have 50/100/200 etc. of something so it's not like i'm keeping track of 50/100/200 different things...one thing and a lot of it.
i put the cards into a shoe box with other cards...keep a list in the box and a master list of every box's list.
i'd check it every so often or when i hear someone's name blip on the sports shows and either sell or prepare to possibly sell.
i end up keeping 2/3rd of what i buy, but the 1/3rd that does sell gets a really nice premium. too bad the massive disposable income era of everyone living on massive debt is over.
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
What to do with Zambrano? MLB Trade Rumors is floating a few trade possiblities (not so much rumors as speculation). They include:
==> Zambrano to Houston for Carlos Lee
==> Zambrano to the Giants for Aaron Rowand and Edgar Renteria
==> Zambrano to Mets for Oliver Perez and Luis Castillo
==> Zambrano to Braves for Derek Lowe
==> Zambrano to Indians for Travis Hafner, Kerry Wood, and Jake Westbrook
==> Zambrano to Royals for Gil Meche, Jose Guillen, and Kyle Farnsworth
==> Zambrano to Yankees for A.J. Burnett
A commenter also floated the idea of:
==> Zambrano to Baltimore for Brian Roberts
Is trading Zambrano the best course of action? If so, do any of these trade scenarios make sense?
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Z for Burnett sounds like the only legitimate one, imo...not that i think it's gonna happen.
Z Update
Z to restricted list, out until after ASB, will undergo anger management treatment
Re: AZL Cubs Rally for Four in 8th to Defeat A's at HoHoKam
Chicago Tribune has an article up now:
"I think we all agree it's time he got help and address apologies later," Hendry said. "He will not meet formally or travel with the team before the (All-Star) break. "
Reliever Jeff Stevens will rejoin the team Wednesday."
Recent comments
Chapman shouldn't be reserved anymore on 40 man.
Interesting split on Heyward according to ESPN. As a CF, his slash line .292/.363/.375/.738. At RF: .212/.204/.300/.604. 21/72 as a CF, 58/273 as a RF.
He's also been better when batting 2nd, but he had a nice start in the 6 hole, but has slumped ever since. He was heating up before the All Star break, but is only hitting .108 in the 2nd half.
When we played the Reds with Chapman, I always thought of it as an eight-inning game. So now other teams have eight innings to try to get a lead against the Cubs. Should be a challenge, assuming three or four Cubs ever start hitting again.
I don't really try to get to know and like these players personally. I'm rooting for laundry, for the most part. Exceptions might be when a player makes trouble in the clubhouse or in the dugout. (Zambrano and Bradley come to mind. Also Papelbon.) But I don't think Chapman is one of those jerks.
Unfortunately, a pretty good summary. It looks like next year Heyward will be getting yet another batting stance adjustment.
The recent good news has been Baez. I'm afraid about the next league adjustment on him, though, which is probably right around the corner.
Bryant I don't worry about too much. Just not seeing the ball well right now. He'll turn it around. Russell's been good with men in scoring position all year and he's 22ish. He'll be fine but next year is likely to be his breakout year.
If Travis' back-to-back-to-back walks cost Hendricks the ERA title, that would really suck.
Edit: "A lifeless loss to a lousy Sox team."
This place is a real downer after a loss to the Sox.
I expect they will go 5-9 games above .500 the rest of the year. 96-98 wins will win the Division.
They should have one more 2-3 week hot streak in them.
However, several players are just "average" for the last month: Zobrist, Ross, Russell, Ceasar. Montero is terrible, plus he cannot throw anyone out. -WAR. Heyward is abysmal at the plate, but a plus in the OF. Still with RISP he has been terrible. KB has not been driving in runs as of late. But Apparently the team is still above average with RISP according to S Sahadev.
I came to that realization tonight. I kept expecting them to play better, but now I realize they aren't going to. They are a .500 team now.
- They have one reliable starting pitcher. Jake's magic is gone, and it doesn't look like it's coming back. Lester has been lousy recently. Lackey's ERA goes up every time he pitches.
- Heyward has been dead weight all year. I can't remember a single series where he was a significant offensive contributor. Not one. Great defense, but but if he were hitting .270 with 10 HR and played average defense, the Cubs would be better off.
new rule...no one's allowed to throw k.bryant a changeup
Team is .500 since early May and is playing like a .500 team. Lack of offense seems to be putting a lot of pressure on the pitchers...and they aren't handling it terribly well.
.500 the rest of the way still may win the division though.
...i hate espn.
nothing like settling into a cubs game to get a few minutes cutaway for an ortiz AB in the 6th inning of the det/bos game.
oh, at least they're doing split screen now...i guess.
I'm liking this rookie Nathan.
Richard DFA'd. Meh...
Throwbacks with fashionable cutouts would be a nice touch.
The next 2 games are nationally televised. I think we dominate tonight, hitting 3 HRs off Shields. Great night for KB to end HR drought facing HR prone pitcher in one of the most hitter friendly parks in the league.