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We obtained a global view of gene expression in both cell lines and pediatric acute lymphoblastic leukemia (ALL) samples that harbor one of several selected chromosomal abnormalities. When the cell lines were studied alone, we found that these chromosomal abnormalities were associated with the predominant variation in transcriptional programs across the set of cell lines studied. When cell lines and clinical samples were studied together, we found that each chromosomal abnormality (TEL/AML1, BCR/ABL, or MLL abnormalities) was associated with a characteristic gene expression signature that was shared by both cell lines and clinical samples. However, BCR/ABL was associated with a much more heterogeneous pattern of expression than were TEL/AML1 and MLL abnormalities. This observation has important implications for the study of BCR/ABL ALL. In addition, we systematically identified genes whose expression was associated with TEL/AML1, BCR/ABL, or MLL abnormalities in both clinical samples and cell lines. Although some of these genes have previously been described, many have not previously been reported to be associated with one of these chromosomal abnormalities. Notably, we found that the erythropoietin receptor (EPOR) is consistently highly expressed in TEL/AML1 ALL compared with BCR/ABL or MLL. (Blood. 2004;103:1043-1049)
Acute lymphoblastic leukemia (ALL) is diagnosed in approximately 4000 individuals each year in the United States1 and is the most common malignancy in childhood.2 Major advances have been made in the treatment of childhood ALL in the past several decades. In part, these advances have relied on risk-stratified treatment, guided by prognostic factors identified at diagnosis. In particular, important prognostic information is provided by the identification of recurrent chromosomal translocations. In addition to being prognostically informative, these translocations may also identify subtypes of ALL with distinct mechanisms of leukemogenesis. This notion is supported by recent studies that have identified gene expression patterns associated with particular chromosomal translocations in ALL. One study3 compared the gene expression patterns in clinical samples harboring an MLL abnormality with an otherwise uncharacterized collection of ALL and acute myelogenous leukemia (AML) samples. That study identified a number of genes associated with MLL ALL. Another study4,5 surveyed the gene expression patterns in a large collection of childhood ALL samples, including patients with a variety of different chromosomal translocations. The analysis of this data set focused primarily on identifying a relatively small number of genes that could be used to identify clinically important subtypes of childhood ALL.
We hypothesized that the distinct mechanisms of leukemogenesis that may be associated with different chromosomal translocations would be reflected in distinct patterns of gene expression that are shared by both clinical samples and cell lines with the same translocation. To test this hypothesis, we used cDNA microarrays to determine the gene expression patterns in both ALL samples and cell lines with selected chromosomal translocations. Our study expands on the already published studies in 3 ways: by studying both patient samples and cell lines, by using a different microarray platform that surveys approximately 43 000 features representing approximately 31 000 genes, and by using both unsupervised and supervised data analysis approaches that are different from those used in published studies of ALL.
Our study provides a global view of gene expression patterns in both cell lines and pediatric ALL samples. Our data confirm many of the genes that have been previously reported to be associated with TEL/AML1, BCR/ABL, or MLL abnormalities.3-5 More important, a number of features readily emerge from our analysis of the data that were not immediately apparent in previous studies of gene expression in ALL. We present 2 examples of such features. First, we find that BCR/ABL is associated with a more heterogeneous pattern of gene expression than are TEL/AML1 or MLL abnormalities. This observation has important implications for the future study of patients with BCR/ABL ALL. Second, we identify many genes associated with particular chromosomal abnormalities that were not readily identified in previous studies. An example of such a gene is the erythropoietin receptor (EPOR), which is consistently highly expressed in TEL/AML1-positive samples. We speculate that EPOR may play a critical role in the development of TEL/AML1- positive ALL.
Materials and methods
Patients and identification of chromosomal translocations
Informed consent was obtained from the guardians of all patients. Clinical samples were obtained from bone marrow aspirations from patients, prior to initiation of treatment, who were enrolled in Berlin-Frankfurt-Münster (BFM)-ALL protocols. Mononuclear cells were isolated from the bone marrow aspirate using Ficoll-Paque (Pharmacia, Uppsala, Sweden). The cells were placed in RPMI 1640 with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL), l-glutamine (2 mM) and 10% dimethyl sulfoxide (DMSO) and frozen in liquid nitrogen for long-term storage. A sample from each bone marrow aspiration was sent to a central cytogenetics laboratory (Giessen, Germany) where each sample was tested6 for the presence of BCR/ABL, TEL/AML1, and MLL/AF4 by polymerase chain reaction (PCR). Positive results were confirmed by fluorescence in situ hybridization (FISH). From the stored BFM samples, 38 samples were identified that contained more than 85% blasts and were positive for BCR/ABL, TEL/AML1,or MLL/AF4. Of these samples, we were successful in obtaining array data on 35 samples. Patient characteristics are shown in Table 1. In addition to the samples used for the microarray measurements, for Taqman PCR measurements, we identified an independent set of 20 TEL/AML1-positive samples and 20 samples negative for TEL/AML1, BCR/ABL, and MLL/AF4. These samples all had more than 80% blasts and a sufficient volume of cDNA to attempt Taqman PCR measurement. Relevant patient characteristics are shown in the Supplemental Materials on the Blood website; see the Supplemental Materials link at the top of the online article.
Cell lines
We selected human leukemia cell lines that contain a balanced chromosomal translocation that is recurrently found in ALL. The characteristics of the cell lines are shown in the Supplemental Materials. The cells were grown in complete RPMI 1640 (Cellgro, Herndon, VA), 15% heat inactivated fetal calf serum (Sigma, St Louis, MO) with amphotericin B (0.125 μg/mL; Sigma), gentamicin (200 μg/mL; Sigma), penicillin (100 U/mL), streptomycin (100 μg/mL), l-glutamine (2 mM) (supplied as penicillin, streptomycin, glutamate [PSG]; Omega Scientific, Tarzana, CA), insulin (5 μg/mL), transferrin (5 μg/mL), and selenium (5 ng/mL) (supplied as ITS; Gibco-BRL, Carlsbad, CA) at 37°C and 5% CO2.
RNA isolation
Total RNA was isolated from cell lines and clinical samples using Trireagent (Molecular Research Center, Cincinnati, OH) or Trizol (Gibco-BRL) according to the manufacturer's protocol. PolyA RNA was isolated from total RNA using FastTrack (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.
Gene expression measurements
All protocols are posted at http://cmgm.stanford.edu/pbrown/protocols/index.html. Microarrays were produced as previously described.7 We labeled the sample RNA and reference RNA with different fluorescent dyes (cyanin 5 deoxyuridine triphosphate [Cy5-dUTP] and Cy3-dUTP; Amersham, Piscataway, NJ) and comparatively hybridized them to an array. The reference RNA was a pooled mixture of RNA obtained from multiple different cell lines.
Two distinct microarray data sets were obtained. The first set consisted of microarray data for polyA RNA samples from each of the cell lines. For this set of samples, we used reference RNA that has been described previously8 and microarrays with approximately 24 000 elements representing approximately 19 000 genes (NCBI Unigene Build no. 160). The second data set consisted of microarray data for total RNA samples from all of the clinical samples and a representative set of 7 cell lines. Each of these total RNA samples was linearly amplified by using a protocol based on Wang et al.9 The reference RNA used for all of the arrays in this data set was Universal Human Reference total RNA (Stratagene, La Jolla, CA) that was linearly amplified using the same protocol. For this set of samples, we used microarrays containing approximately 43 000 features representing approximately 31 000 genes.
The fluorescence intensities of Cy5 and Cy3 on each array were measured by using a GenePix 4000 scanner (Axon Instruments, Foster City, CA). Images were analyzed by using GenePix Pro 3.0 software (Axon Instruments) to semiautomatically identify and quantify hybridization to the cDNA spots on the microarrays. Any areas of the microarrays with obvious blemishes were manually omitted from subsequent analysis. Spots were considered well measured only if the reference RNA fluorescent intensity was more than 3 times the local background and the regression correlation was more than 0.6. Any clone that was not well measured on at least 80% of the arrays was excluded from subsequent analysis. For each array, we used a scaling factor to set the mean sample-reference ratio for all well-measured spots to one. For all subsequent analysis, we used log2 of this normalized sample-reference ratio. We then centered the data for each clone so that its mean was zero. To analyze the data in an unsupervised manner, we used agglomerative hierarchical clustering.10 In the supervised analysis of the data, we used significance analysis of microarrays (SAM).11 We also used prediction analysis of microarrays (PAM)12 to identify genes that may be used to classify ALL samples as BCR/ABL, TEL/AML1,or MLL. We estimated the misclassification error for this classifier by using 10-fold cross-validation as described.12 Primary data are publicly available through the Stanford Microarray Database: (http://genome-www5.stanford.edu/cgi-bin/SMD/listMicroArrayData.pl?tableName=publication). Supplemental materials may be explored interactively at http://microarray-pubs.stanford.edu/all/.
Quantitative reverse transcription (RT)-PCR
We used random hexamer priming and either Superscript I (Invitrogen) or Taqman RT (Applied Biosystems, Foster City, CA) to generate cDNA. The Taqman probe and primers used to measure EPOR RNA abundance have been published.13 The forward primer was 5′-GCT CCC TTT GTC TCC TGC T-3′. The reverse primer was 5′-CTC CCA GAA ACA CAC CAA GTC CT-3′. The Taqman probe was 6FAM-AGC GGC CTT GCT GGC GG-TAMRA (Applied Biosystems). Cyclophilin A (CYCA; Applied Biosystems) was used as an endogenous control. The quantitative PCR reactions were performed in a total volume of 20 μL, containing 10μL TaqMan Universal PCR Master Mix (Applied Biosystems), 200 nM forward and reverse primers, 100 nM Taqman probe, and 5 μL cDNA. The reactions were carried out on an ABI Prism 7700 Sequence Detection System (Applied Biosystems), using the following temperature program: 50°C for 2 minutes, 95°C for 4 minutes then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. In the same reaction plate as the samples, quantitative PCR reactions were carried out on a dilution series of cDNA obtained from the cell line K562. The data from this dilution series were used to construct calibration curves for both EPOR and the endogenous control gene CYCA. These calibration curves were used to assign an expression level of EPOR and CYCA for each of the samples tested. Using these values, we calculated the EPOR/CYCA ratio.
Flow cytometry
A sample from each bone marrow aspiration was sent to a central laboratory (Oncogenetics Laboratory, Children's University Hospital, Berlin, Germany) where flow cytometry was performed as previously described.14,15 CD20 surface expression was considered positive if at least 20% of the leukemic cells had a fluorescence intensity more than 98% of the negative control cells.
Overview of gene expression patterns
First, we examined the gene expression patterns in a set of cell lines that each harbors a chromosomal translocation common in ALL. Figure 1 shows the result of hierarchical clustering of the cell lines on the basis of their similarity in gene expression. To confirm the reproducibility of the array measurements, we used duplicate arrays to determine the gene expression profile of each of 9 cell lines. Further description of these duplicate measurements is contained in the Supplemental Materials. For all cell lines tested, duplicate arrays cluster side by side, confirming the reproducibility of this method. This hierarchical clustering shows that cell lines cluster according to the chromosomal translocation that they contain. In particular, all t(9;22)-containing cell lines clustered together, and all cell lines that have an MLL abnormality clustered together. Smaller numbers of other cell lines also clustered in sets that shared a common translocation.
Next, we compared the gene expression patterns in a representative set of cell lines with those in pediatric ALL samples. Again, by hierarchical clustering, all duplicate arrays clustered side by side (data not shown). A description of these duplicate measurements is contained in the Supplemental Materials. In all subsequent analyses, we combined duplicate measurements, using their mean gene expression level. When the samples were analyzed by hierarchical clustering on the basis of their pattern of expression, the cell lines clustered closely together, distinct from the clinical samples as shown in Figure 2A. The cell line cluster is distinguished from the clinical samples principally by its relatively high expression of a large set of proliferation-related (mostly cell-cycle regulated) genes, as shown in Figure 2B. The result of hierarchical clustering of the clinical samples alone, without the inclusion of any cell lines, is shown in Figure 2C. Figures 2A and 2C both show the same general relationship among the clinical samples. Specifically, both figures show that the clinical samples fall into 2 major clusters. One cluster contains all of the TEL/AML1 samples and the other contains all of the MLL/AF4 samples. BCR/ABL samples, in contrast, are included in both large clusters, demonstrating that there is greater heterogeneity in the gene expression patterns in these leukemias compared with the TEL/AML1 and MLL/AF4 leukemias.
BCR/ABL ALL has a more heterogeneous pattern of gene expression than ALL with TEL/AML1 or MLL/AF4
To further investigate the heterogeneity in gene expression in the BCR/ABL samples, we applied hierarchical clustering to these samples alone. As shown in Figure 3A, this analysis identifies the same BCR/ABL clusters that are observed when all clinical samples and cell lines are included. In particular, we find 2 major clusters of samples. We used SAM11 to identify those genes whose expression was consistently different between the 2 clusters of BCR/ABL ALL samples. The sample BF122 could not be readily assigned to either of the 2 groups of BCR/ABL ALL and so it was excluded from this analysis. The results are shown in Figure 3B-C. Among the genes that we found to be statistically significantly differentially expressed in the 2 groups was CD20. Although CD20 was not the gene that most strongly distinguished the 2 clusters, it has the advantage of being routinely measured by flow cytometry on diagnostic ALL bone marrow samples.
We found reasonable concordance between the CD20 gene expression level as measured by microarrays and the CD20 protein expression level as measured by flow cytometry in the 10 patients for whom we had data (see Supplemental Materials). Because the median follow-up for the patients who contributed BCR/ABL samples was less than 3 years, we could not determine from these patients if the 2 clusters of BCR/ABL patients have different clinical outcomes. However, we were able to obtain outcome data for a set of childhood BCR/ABL patients with longer follow-up. We identified 65 BCR/ABL-positive patients who were enrolled in either BFM-ALL 90 (n = 26) or BFM-ALL 95 (n = 39) trials for whom CD20 was measured by flow cytometry. There was no difference in event-free survival (log-rank P = .7) or overall survival (log-rank P = .3) among those patients with CD20-negative leukemia (n = 28) compared with those with CD20-positive leukemia (n = 37).
Identification of genes whose expression is associated with TEL/AML1, BCR/ABL,or MLL abnormalities
To identify genes whose pattern of variation in expression in clinical samples parallels that seen among cell lines harboring the same translocations, we used SAM11 to analyze the results for all of the clinical samples and cell lines. This approach identified 901 clones representing 844 genes (1000 permutations, delta = 0.34, median false significant clones = 3.1) whose expression is consistently associated with either TEL/AML1 (363 clones representing 323 genes), MLL (449 clones representing 437 genes), or BCR/ABL (89 clones representing 84 genes). The results of this analysis are shown in Figure 4. That BCR/ABL leukemias show relatively few consistent features in their expression patterns further confirms the heterogeneous nature of gene expression in this subset of ALL.
The genes identified by this analysis may provide clues to the mechanisms of leukemogenesis that are associated with these different fusion proteins. The full list of genes identified by this analysis is available in the Supplemental Materials. As an example, Figure 4 shows some of the genes whose expression is increased in association with 1 of the 3 chromosomal abnormalities.
Another way to test whether cell lines and clinical samples with the same translocation share a common gene expression signature is to remove the consistent gene expression differences that distinguish cell lines from clinical samples and then to use unsupervised hierarchical clustering to organize the cell lines and clinical samples. To minimize the effect of this cell line-specific gene expression signature, we centered the expression measurements for each gene separately for the cell lines and for the clinical samples by subtracting the mean value for each group. As shown in the Supplemental Materials, after this transformation, hierarchical clustering shows that the general relationship among the clinical samples remains the same as shown in Figure 2. However, now the cell lines all cluster with clinical samples that share the same class of chromosomal abnormality, demonstrating that they also share a common gene expression signature.
PAM has been developed to identify a relatively small set of genes that define a distinctive molecular signature for distinct classes (eg, subtypes of cancer). Soft thresholding is used to adjust the number of genes that comprise the signatures. Then, using 10-fold cross-validation, the accuracy of the classifiers is tested over the range of possible thresholds. We used PAM to identify signature gene expression profiles for the 3 different subtypes of ALL included in our study. As shown in Figure 5, with cross-validation, 40 (95%) of the 42 samples are correctly classified over a broad range of thresholds. Over this range of thresholds, the BCR/ABL classifier has very few non-zero genes compared with TEL/AML1 or MLL. As an example, Figure 5C shows the classifiers obtained with threshold = 3.0. This indicates that BCR/ABL ALL has very few genes with a consistent pattern of expression, again confirming that BCR/ABL is a more heterogeneous subtype of ALL than TEL/AML1 or MLL. The genes that comprise these classifiers are listed in Supplemental Materials.
EPOR is consistently highly expressed in TEL/AML1 ALL
EPOR was identified by both SAM and PAM as characteristically expressed in association with TEL/AML1. To confirm that EPOR was more highly expressed in the TEL/AML1 subtype, we used the Taqman PCR assay to measure EPOR abundance in the available samples used to generate the array data and in an independent set of pre-B ALL samples. In the independent sample set, 16 TEL/AML1-positive and 10 TEL/AML1-negative samples had detectable levels of the endogenous control gene, CYCA, and were included in subsequent analysis. There was no sample that had detectable EPOR but not CYCA. Figure 6 shows the expression of EPOR (relative to CYCA) in both the original set of patients (Figure 6A-B) and in the independent set of samples (Figure 6C). There is a statistically significant difference in EPOR expression in TEL/AML1-positive samples compared with TEL/AML1-negative samples in both sample sets (t test P < .001).
We found a similar difference in expression of EPOR in the publicly available data from Yeoh et al.4 For the probe set 1087_at, representing human erythropoietin receptor mRNA, complete cds, the average intensity difference for TEL/AML1-positive samples (n = 79) is (1.9 ± 0.2 × 103 [mean ± SEM]), and for TEL/AML1-negative samples (n = 256) it is (-0.30 ± 0.08 ×103), a statistically significant difference (t test P = 4 × 10-15). This difference is confirmed by the Supplemental Information to Ross et al,5 in which EPOR appears in the list of 100 probe sets associated with TEL/AML1.
This study provides a global view of gene expression patterns in ALL cell lines and clinical samples that have selected chromosomal abnormalities. When the cell lines were hierarchically clustered on the basis of similarity in gene expression, they clustered according to their chromosomal translocation. This observation supports the idea that the presence of particular chromosomal translocations defines a distinct molecular subtype of ALL. It is interesting that the cell lines did not cluster primarily according to other well-recognized heterogeneous features. For example, the cell lines have heterogeneity with respect to immunophenotype and expression of myeloid markers, but neither of these features was more important than chromosomal translocation in determining the gene expression pattern.
In comparison to cell lines alone, the gene expression patterns observed when clinical samples and cell lines are studied together are somewhat more complex. We find that cell lines exhibit a characteristic pattern of gene expression that distinguishes them from their clinical counterparts, most notably the much higher level of expression of genes involved in cell proliferation. These differences between cell lines and clinical samples should be borne in mind when cell lines are used as models for ALL. Nevertheless, when this cell line-specific signature is minimized, it becomes clear that cell lines and clinical samples with the same chromosomal abnormality share a characteristic gene expression pattern.
Anotable feature in the global view of gene expression in the clinical samples was the relative heterogeneity in the gene expression patterns of the BCR/ABL samples. It is interesting to note that the BCR/ABL translocation results in the production of a novel tyrosine kinase, whereas the TEL/AML1 and MLL/AF4 translocations result in the production of novel transcription factors. Consequently, TEL/AML1 and MLL/AF4 may be expected to have a more direct effect on gene expression than BCR/ABL. The heterogeneous pattern of gene expression in BCR/ABL ALL may reflect the less direct, and perhaps more context dependent, effect of BCR/ABL on gene transcription programs. An alternative explanation is that TEL/AML1 and MLL/AF4 may each require a relatively restricted set of additional mutations for leukemia to arise. In contrast, a wider variety of additional mutations may be able to cooperate with BCR/ABL in leading to the development of leukemia. This wider variety of additional mutations would be expected to be associated with a more heterogeneous gene expression profile.
Although BCR/ABL is generally associated with a poor clinical outcome, it is well recognized that this subtype of ALL is a clinically heterogeneous disease. To date, the early feature of childhood BCR/ABL ALL that is most predictive of outcome is initial response to prednisone and intrathecal methotrexate.16,17 Although our study clearly demonstrates that BCR/ABL is associated with a much more heterogeneous pattern of gene expression than is either TEL/AML1 or MLL/AF4, the median follow-up of patients in this study is too short to determine if this heterogeneity has clinical significance.
The genes whose expression is heterogeneous in BCR/ABL ALL include several cell surface proteins (eg, CD20, CD73, and CD81). One of these proteins, CD20, is routinely measured on leukemic blasts by flow cytometry when ALL is diagnosed. Interestingly, all of the BCR/ABL cell lines (except BV-173, which was obtained from chronic myelogenous leukemia in lymphoid blast crisis) are CD20 negative. This finding may indicate that CD20- BCR/ABL ALL more readily adapts to in vitro culture conditions than does the CD20+ subtype. A recent Pediatric Oncology Group study identified CD20 expression (measured by flow cytometry) in ALL to be independently correlated with outcome, particularly in the poor risk subgroup (that includes BCR/ABL).18 However, we did not find that CD20 was associated with a difference in outcome in patients with BCR/ABL ALL treated on recent BFM clinical trials. A similar study of patients with BCR/ABL ALL treated by using other treatment regimens might reveal an association between CD20 and outcome.
Armstrong et al3 have proposed that, based on its distinct gene expression pattern, MLL leukemia is a distinct disease. Following this line of reasoning, our study supports the notion that BCR/ABL ALL is, in fact, more than one distinct disease. Consequently, we feel that the results presented here provide sufficient evidence to justify a future study of gene expression in a larger number of patients with BCR/ABL ALL to more fully characterize the molecular heterogeneity of this disease, ideally in both adults and children. It seems likely that such a study would at the very least be useful for identifying potential therapeutic targets tailored to specific subtypes of BCR/ABL ALL. For example, the use of anti-CD20 antibody has been proposed as a useful therapeutic strategy in those cases of BCR/ABL ALL that have high expression of cell surface CD20.19 A more comprehensive characterization of gene expression heterogeneity in BCR/ABL ALL would no doubt lead to even more potentially beneficial strategies for the treatment of distinct subtypes of this disease.
In this study, we identified genes that are characteristically expressed in cell lines and clinical samples with the same translocation. Although our study identifies many of the genes previously reported3,4 to be associated with TEL/AML1, BCR/ABL, or MLL abnormalities, we also identify a large number of genes that have not been previously reported.
Among the genes that have not previously been reported to be associated with the TEL/AML1 subtype is EPOR. EPOR is a member of the cytokine homology domain family of receptors20 and has been implicated in the development of erythroleukemia.21 EPOR expression has been investigated by flow cytometry in adult acute leukemia,22 although the association with cytogenetic subtype was not investigated.
A model for the pathogenesis of acute leukemia has recently been proposed23,24 in which 2 classes of cooperating mutations are required for the development of acute leukemia. One class, which includes TEL/AML1, impairs differentiation. The other class, which includes BCR/ABL, confers proliferation and/or survival. Interestingly, in EPOR-deficient progenitor cells, BCR/ABL can support healthy erythroid development,25 suggesting that EPOR and BCR/ABL may have overlapping functions in leukemogenesis. The results presented here thus lead us to speculate that high expression of EPOR provides the essential proliferation and survival signals in the TEL/AML1 subtype of ALL.
This work would not have been possible without the support and advice of Patrick O. Brown. We thank Drs Michael Cleary and Yoshinobu Matsuo for their gifts of cell lines and members of the Brown and Boxer lab for helpful discussions. We thank the Stanford Functional Genomics Facility for production of microarrays and the Stanford Microarray Database for providing a repository for the primary data and for hosting the companion website for this paper.
- Reprints:
Linda M. Boxer, Division of Hematology, 269 Campus Dr, CCSR 1155, Stanford University School of Medicine, Stanford, CA 94305-5156; e-mail: lboxer{at}stanford.edu.
Prepublished online as Blood First Edition Paper, October 2, 2003; DOI 10.1182/blood-2003-05-1518.
Supported by a grant from the National Institutes of Health (NIH) (CA92326) (L.M.B.). B.M.F. was supported by a Howard Hughes Postdoctoral Fellowship for Physicians and a Mentored Clinical Scientist Award from NIH (K08CA95563).
The online version of the article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
- Submitted May 13, 2003.
- Accepted August 28, 2003.
- Copyright © 2004 by The American Society of Hematology


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VIDEO: Check out New TV Spot for TEENAGE MUTANT NINJA TURTLES
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The city needs heroes. Darkness has settled over New York City as Shredder and his evil Foot Clan have an iron grip on everything from the police to the politicians. The future is grim until four unlikely outcast brothers rise from the sewers and discover their destiny as Teenage Mutant Ninja Turtles. The Turtles must work with fearless reporter April O'Neil (Megan Fox) and her wise-cracking cameraman Vern Fenwick (Will Arnett) to save the city and unravel Shredder's diabolical plan.


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JOB / Cleanroom Molding Lead
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Digital Storm Bolt Desktop Gaming PC Review
Cinebench 11.5
Version and / or Patch Used: R11.5 build CB25720DEMO
Developer Homepage: http://www.maxon.net
Product Homepage: http://www.maxon.net/products/cinebench/overview.html
CINEBENCH is a real-world cross platform test suite that evaluates your computer's performance capabilities. CINEBENCH is based on MAXON's award-winning animation software CINEMA 4D, which is used extensively by studios and production houses worldwide for 3D content creation. MAXON software has been used in blockbuster movies such as Spider-Man, Star Wars, The Chronicles of Narnia and many more.
Since both systems in the chart above feature the same Intel Core i7-3770K, performance is pretty close to identical. The Bolt performs just slightly worse than the iBUYPOWER Chimera 4SE.
Version and / or Patch Used: 2.09
Developer Homepage: http://www.wprime.net/
Product Homepage: http://www.wprime.net/
wPrime is a leading multithreaded benchmark for x86 processors that tests your processor performance by calculating square roots with a recursive call of Newton's method for estimating functions, with f(x)=x2-k, where k is the number we're sqrting, until Sgn(f(x)/f'(x)) does not equal that of the previous iteration, starting with an estimation of k/2. It then uses an iterative calling of the estimation method a set amount of times to increase the accuracy of the results. It then confirms that n(k)2=k to ensure the calculation was correct. It repeats this for all numbers from 1 to the requested maximum.
Again, the two systems perform pretty similarly due to the use of the same CPU. The Bolt again performs just slightly worse, producing a 32M time of 6.536 seconds and a 1024M time of 202.521 seconds.
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VOTD: Why Are Video Game Movies So Terrible?
Posted on Monday, July 18th, 2016 by Ethan Anderton
Even though this year brought the most successful video game movie of all time with Legendary Pictures bringing Warcraft to the big screen, there still hasn't really been a video game turned into a movie that has struck a chord with audiences and critics alike while also being a big hit at the box office. From the abysmal Super Mario Bros. in 1993 to more recent attempts such as Prince of Persia: The Sands of Time and Max Payne, video game movies have a terrible track record. Now a new video essay attempts to explain why. Read More »


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About Captiva House
Welcome to the charming and historic Captiva House at the legendary Tween Waters Inn Island resort and Spa. Known for Captiva's Gulf-front view of America's most romantic sunset.
Inspired by Executive Chef Jason Miller, our exciting "New Florida" menu is a colorful, wonderful exploration of island foods and flavors. Sometimes passionate. Sometimes mild. Sometimes fun. Yet all, truly great to admire and greater to enjoy — from appetizers, through entrees, to desserts.
Come, feast your eyes and appetite.
Dining Style:
Casual Elegant
American, Seafood, Steak
Hours of Operation:
Breakfast Daily: 7:00am - 10:30am; Dinner Nightly: 5:30pm-9:30pm
Cross Street:
2 miles north of Blind Pass Bridge
Dress Code:
Casual Dress
Price Range:
$31 to $50
Payment Options
AMEX, Diners Club, Discover, MasterCard, Visa
Phone Number:
(800) 223-5865
Enjoy nightly entertainment most nights at our Crow"s Nest Lounge adjacent to the Captiva House. Entertainment consists of the world famous hilarious NASCRAB Crab Racing interactive comedy show and crab race event that must be seen to believe. Two shows (children's 6PM and adult 9PM) most Monday and Thursday evenings throughout the year. Enjoy music stylings of a live and lively band every Friday and Saturday nights starting at 9PM. Plus, dance the night away to local singer Taylor Stokes, as he plays soft rock currents to classics most Tuesdays and Wednesdays starting at 9PM.
Parking Details:
Parking is open for all dining customers.
Executive Chef:
Jason Miller
Private Party Facilities:
The Old Captiva House:
Our historic restaurant that dates to the 1920s, beautifully appointed today with walls of white French doors and polished hardwood floors, yours to reserve in part or in total. Capacity 160.
Sunset Lounge:
This quiet, intimate room adjacent to the Old Captiva House is all windows and altogether charming. Beautiful views of Americas most romantic sunsets. Also opens to our Garden Terrace and the Old Captiva House. Capacity 50.
Private Party Contact:
Kerri Maw: 2394725161x404
Banquet, Bar/Lounge, Beer, BYO Wine, Corkage Fee, Entertainment, Full Bar, Non-Smoking, Outdoor dining, Private Room, Takeout, View, Wheelchair Access, Wine
Captiva House Ratings and Reviews
Service was quite slow. Felt as if they ...
Service was quite slow. Felt as if they were understaffed. The server took it upon himself to add an extra order of nigiri to our appetizers (and charged us for it) after letting us know they had no more tamago. Lobster spaghetti was very good, but the sea bass in puff pastry was bone dry and very fishy. The piano player was the best part.
We stayed at the Tween Waters Resort and ...
We stayed at the Tween Waters Resort and went to the restaurant for the convenience. We were pleasantly surprised at how good it was! The Oysters Alexander was the best baked oyster dish I've ever tasted! The sun set while we were dining making the view spectacular. The service was impeccable. It's a very small place so it was quiet and intimate. Reservations would be strongly recommended.
The dining room was beautiful and in a g ...
The dining room was beautiful and in a great location. The cocktail and wine list were great. The service was not exceptional but not terrible either. The sushi appetizer was good but my entree was horrible. I had the snapper wrap. The snapper was dry and fishy which I find completely unacceptable on an island where you can catch the fish fresh and there is a dock out back of the restaurant. When my husband and I tried to tell someone no one seemed interested. We didn't want a comp just wanted to let them know. It wasn't a great end to my birthday weekend and I would not go there again for a special occasion.
Our first visit to the Old Captiva House ...
Our first visit to the Old Captiva House. We do not usually go into places with expectations but always hope for a great meal. The food was delicious and the wine list was excellent. Our server was excellent, but they had him running all over the restaurant and that delayed service somewhat. All of our food was cooked well, and the taste was great. Gotta love a live piano man as well! We highly recommend and will return on our next visit to our home on Sanibel.
the fresh seafood was outstanding, the s ...
the fresh seafood was outstanding, the special Halibut and rice was my favorite! we also ordered almost every appetizer and loved them all (especially the lobster-cargot).
the cocktails were delicious as well (and with a heavy pour:)
the piano man played most of the night and you can't beat that!
We will definitely be back next time we stay on Captiva!
It's a lovely restaurant, and the servic ...
It's a lovely restaurant, and the service was good, although our waiter appeared a little nervous -- perhaps he was new. The grilled oysters were outstanding, however the lobster rolls and our entree's were to be honest, very average, and below expectations we anticipated, though the presentation was excellent. Having said that, I was at Thanksgiving buffet there a couple of years ago and I still remember how absolutely delicious everything I had was. Perhaps they simply had an off night, and I'd suggest you try it and form your own opinion. Thanks.
A favorite restaurant but the service ne ...
A favorite restaurant but the service needs to improve. It was too slow and waiter was out of room towards the end of the meal. The service doesn't match the delicious food and ambiance...it's good, but not quite good enough to make it an outstanding place to eat.
Fabulous dinner. We had not been to the ...
Fabulous dinner. We had not been to the old captiva house in a number of years and decided to try it again. It is as great as we remembered. Fish and steak were cooked to perfection. Wine list covers all tastes. Desserts are extremely good. The area is a resort and beautiful. Will be going the again in the future.
Disappointed no sunset view from dining ...
Disappointed no sunset view from dining area, so not romantic as described. Service was excellent. Nile and Gregory were very accommodating. Piano player Frank took request and was also excellent! Food was excellent!
My husband loved his filet mignon dinner ...
My husband loved his filet mignon dinner, but my grilled mahi mahi was not good at all. There was way too much seasoning on it, making it inedible. I just ate the vegetables instead. I didn't say anything to the waitress because I didn't want to spoil my husband's enjoyment. The ambience and view were quite nice, though.
We were a party of 4 and our waiter, Rob ...
We were a party of 4 and our waiter, Robert, was very attentive. Our meal was delicious. There was live music (piano) discreetly in the back ground. Drinks were awesome. Each time we visit Sanibel we make it a point to dine here.
My husband and I had an excellent meal h ...
My husband and I had an excellent meal here, as always. The restaurant is quaint and charming, and the service is excellent. The food is always quite good, especially the crab bisque, which is outstanding. Highly recommended!
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Process Development Jobs Still the Most...
Wall Street Decline Takes Its Toll...
Quadruple Helices Appear To Have Role...
As Immunotherapy Revolutionizes the Cancer Treatment...
Exagen Diagnostics will pay Cypress Bioscience $4 million to purchase its diagnostic business located in San Diego, CA. The initial fee will be split into two, 24 months apart, and Exagen could pay up to $4 million more in milestones as well as a 10% royalty on product sales. The deal covers all testing services, intellectual property rights, and equipment.
Cypress Bioscience is a pharmaceutical company developing drugs for patients suffering from a variety of disorders of the central nervous system. It also offers the Avise PG SM and Avise MCV SM therapeutic monitoring, diagnostic, and prognostic tests for rheumatoid arthritis. In February it picked up Cellatope's diagnostic technology for $2 million up front and $3 million in milestone fees. The platform uses cell-bound complement activation products to diagnose and monitor debilitating autoimmune disorders including systemic lupus erythematosus.
"The Cypress products and technology complement our genomic testing platform and the suite of diagnostic products being sold by Exagen today," remarks Scott Glenn, Exagen chairman and CEO. The firm's current emphasis is on gastroenterology, particularly inflammatory bowel disease and irritable bowel syndrome.
"We look forward to expanding our customer base from the current gastroenterology focus to include rheumatologists," Glenn continues. "Our next product out of our research group is in the area of therapeutic efficacy for the biologic agent anti-TNF. The initial test panel will provide the combined physician specialties with a diagnostic tool for identifying patients that will respond or not respond to this treatment."
Exagen Diagnostics leverages Coperna®, its algorithm-driven tool and search engine that runs on high-performance computational clusters, to identify relevant marker sets. The platform combines the ability to process massive amounts of data contained in genomic datasets with intelligent, rapid search methods, according to Exagen.
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Pre-submission Local Plan Part 1: Strategic Policies and Sites
Consultation on the Pre-submission Local Plan Part 1: Strategic Policies and Sites - Friday 19 August until 3 October 2016
Please note that there are now less than two weeks left until the end of the consultation.
The Pre-Submission Local Plan Part 1: Strategic Policies and Sites sets out the planning strategy for Waverley up to 2032 and includes specific policies to address the key issues and challenges facing the Borough. The plan has taken account of the comments made during the various consultations that have already taken place.
Waverley Borough Council plans to submit the Local Plan Part 1: Strategic Policies and Sites and associated documents to the Secretary of State for Communities and Local Government later this year, for examination. Documents are being published for a period of 6 weeks to give you the opportunity to formally comment. You can make general comments about the Plan at this stage, but the Council is asking for specific comments in relation to whether the Plan is legally compliant, sound and whether it has complied with the duty to co-operate. Guidance is available on what is meant by these terms.
Representations can be made during the publication period from Friday 19 August until 5.00pm on Monday 3 October 2016.
The following documents are available as part of the consultation:
- The Pre-Submission Local Plan Part 1: Strategic Policies and Sites
- A Sustainability Appraisal report
- Habitats Regulations Assessment
- The Statement of the Representations Procedure
In addition, there are a number of other supporting evidence documents which are available to view and download.
Responding to the Consultation
If you would like to respond you can do so in a number of ways:-
Online Consultation Website:
You can view the document, and comment online, via our consultation website. Please note that to submit your comments online you will need to register with the website, if you have not already done so.
If you have already taken part in a previous consultation, you will already be registered on the consultation portal. You can use your existing username and password and if you don't know these please do not register again; there are reminder prompts for both usernames and passwords.
Once the consultation is open you can then comment on different sections of the document. Please remember to submit these at the end of each set of questions. You can come back and amend them or finish going through the document throughout the consutlation period.
Download a Representation Form:
You can also download the document and representation forms (Parts A and B). These can be filled in electronically, saved by you and emailed back to us at email@example.com. Form A only needs to be completed once, but submit as many copies of Form B as you need to.
Print the representation form and post it back to us:
Although we would encourage all those intending to respond to use either of the online facilities as described above, you can also print the above forms to complete by hand and post them to us at:
- Planning Policy Team, Planning Services, Waverley Borough Council, The Burys, Godalming. GU7 1HR.
Form A only needs to be completed once, but submit as many copies of Form B as you need to.
View the documents and collect a paper copy of the representation forms:
The Waverley Borough Pre-Submission Local Plan Part 1, the Sustainability Appraisal Report and other associated submission documents are available for inspection at the following locations, where you can also collect paper copies of the representation forms:
- Planning Reception, Council Offices, The Burys, Godalming, Surrey, GU7 1HR. Monday-Thursday 9.00 am -5.00 pm, Friday 9.00 am - 4.00 pm. (Excluding Bank Holidays)
- Farnham Locality Office, South Street, Farnham, Surrey, GU9 7RN. Monday-Thursday 9am-5pm, Friday 9am-4.30pm (closed each day 12.30pm to 1.30pm) (Excluding Bank Holidays)
- Godalming, Farnham, Haslemere, Cranleigh and Bramley libraries. Please see www.surreycc.gov.uk/libraries for their opening times.
Form A only needs to be completed once, but submit as many copies of Form B as you need to.
Write or email
We would prefer you to use the representation form, but if you prefer to write or email, please reference your correspondence with the relevant paragraph or policy number.
All comments, however they are submitted, will be publicly available and cannot be treated as confidential (identifiable by name and organisation only). Please note that any other personal information provided will be used by Waverley Borough Council to monitor responses, and will not be used for any other purposes.
For more information or assistance on this consultation please contact the Planning Policy Team on 01483 523291 or email email@example.com.
Thank you in advance for your views on this important consultation.
Farnham Neighbourhood Plan
Please note that the Council is also consulting on the Farnham Neighbourhood Plan during this same consultation period. To find out more about this and how to make representations, please visit the the Farnham Neighbourhood Plan webpage.
Local Plan production timetable
A revised Local Development Scheme was approved by the Executive on 12 July 2016.
The Local Plan Newsletter
Local Plan newsletters give information about the work that has been carried out, the stage that we are currently at, and what will happen next.
Local Plan Newsletter - July 2016
More information
Please see the pages below for more information:
- Timetable and Local Development Scheme
- Call for Sites
- Core Strategy Withdrawn from Examination: October 2013
- Costs of the Local Plan / Core Strategy
- Potential Housing Scenarios and Other Issues for the Waverley Local Plan - Consultation 3 September - 17 October 2014
- Local Plan Part 2: Non Strategic Policies and Sites
You can also contact the Planning Policy Team on email@example.com or telephone 01483 523291.
Page owner: Jennie Falconer. Last updated: 19/09/2016 13:26


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Google+ is refining its search by adding real-time results and additional support for hashtags.
The first feature, real-time search results, allows users to click either the "Most recent" or "X more recent posts" to see the current real-time postings. Additionally, once you've switched to see real-time results, new posts relevant to your search will continue loading the moment they're posted. This lasts for the duration of your search session.
The second feature is additional support for hashtags. While Vic Gundotra, SVP of Engineering at Google, specifies that searches will still work just fine without hashtags, the tags are now more integrated with search results: Adding a hashtag will make the word link to a Google+ search on the topic.
This is likely to lead to the rise of Plus-specific hashtags, such as the various Twitter tags built by the community. Gundotra used the "#googleplusupdate," which has become popular on Plus in recent weeks, in the announcement of this product. Other tech, social, or completely random tags may follow soon.
The two new features are designed to "make it easier to follow and contribute to live events on Google+," according to Gundotra.
You might not see the feature yet because the rollout of both features is "gradual." No more specific timeline was provided.
This is the first major return of Google's Realtime search, which was shut down back in July when Google's access to Twitter's firehose of data expired. We knew previously that real-time would return with Google+, but most assumed it would be an external search that included Plus postings – not an embedded part of Plus itself.
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Re: The Father of Rueben Windham?????
In reply to:
Re: The Father of Rueben Windham?????
William Windham 1/15/10
Sorry I should have been clearer about that statement.It is an argument that has been made by some of our fellow researchers on this site.Since you were discussing Boddie...it was a relevant query
.... I personally have not spent time onReubin of Isle of Wight VA.I have spent my time on my direct line which I am sure about to Grainger TN.Past that...you would have to contact John B Windham. He has extensive material on the subject.
The argrument that has been posed but never clarified is the signature by Edward, the Burgess...where he signs his name Edward, Sen.Amasa, in his research, said it stood for Senior.Jim Windham says it does not, but neglects to state the information that he has that says what SEN after the signature means????


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Top Definition
Also referred to as the anus. Often characterized by 'striations' that are in a star pattern.
Wow, that girl has some nice striations on her brown star.
автор: Landon Howe 15 января 2005
Trending Now
Can you define these popular missing words?
An unwiped anus
"My brownstar is itching unforgivably"
автор: Anonymous 3 июня 2003
The anus when freshly browned.
Kyle forgot to wipe so his brown star bothered him (and everyone else around him) all day.
автор: Brownie the Clownie 25 ноября 2006
One's anus
As in brown star cowboy - a male of homosexual persuasion
автор: Anonymous 16 января 2003
Another term for an asshole
Frank became a brown star when he screwed my sister in the brown star.
автор: Jay Ess 29 декабря 2006
the mark left when one presses one's unclean anus against the skin of another.
She knew she'd had a bit too much to drink when she woke up with a brown star on her forehead and an empty box of Franzia at her feet.
автор: EB 18 августа 2003
asshole; balloon knot; red eye; one's donut
Homosexual men have loose brown stars.
Blow my balloon knot, slutty hoe-bag.
While in jail, PJ got his donut 'punched' in by Big Dick Black.
Blow my balloon knot, slutty hoe-bag.
While in jail, PJ got his donut 'punched' in by Big Dick Black.
автор: NYPD 9 февраля 2004


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struct cfg80211_ibss_params — IBSS parameters
struct cfg80211_ibss_params { const u8 * ssid; const u8 * bssid; struct cfg80211_chan_def chandef; const u8 * ie; u8 ssid_len; u8 ie_len; u16 beacon_interval; u32 basic_rates; bool channel_fixed; bool privacy; bool control_port; bool userspace_handles_dfs; int mcast_rate[NUM_NL80211_BANDS]; struct ieee80211_ht_cap ht_capa; struct ieee80211_ht_cap ht_capa_mask; };
The SSID, will always be non-null.
Fixed BSSID requested, maybe be
NULL, if set do not
search for IBSSs with a different BSSID.
defines the channel to use if no other IBSS to join can be found
information element(s) to include in the beacon
The length of the SSID, will always be non-zero.
length of that
beacon interval to use
bitmap of basic rates to use when creating the IBSS
The channel should be fixed -- do not search for IBSSs to join on other channels.
this is a protected network, keys will be configured after joining
whether user space controls IEEE 802.1X port, i.e.,
NL80211_STA_FLAG_AUTHORIZED. If true, the driver is
required to assume that the port is unauthorized until authorized by
user space. Otherwise, port is marked authorized by default.
whether user space controls DFS operation, i.e. changes the channel when a radar is detected. This is required to operate on DFS channels.
per-band multicast rate index + 1 (0: disabled)
HT Capabilities over-rides. Values set in ht_capa_mask will be used in ht_capa. Un-supported values will be ignored.
The bits of ht_capa which are to be used.


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Happy New Year everyone! I'm wrapping up the year with all of your favorite posts from 2013. It's exciting to look back over the year and see what's been accomplished, and it gives even more inspiration for what's to come in 2014! I've already been planning out my goals for the new year and I'll be sharing more on that later on but for now, let's see what inspired you the most in 2013!
Home Office Bookcases and Organization
Hey ya'll!! Today's a good day. You know why? Because it's Friday and because I finally have a BIG home…
Read More
Painted Hope Chest
One step closer to a completed room makeover this weekend! This hope chest has been in my family for years, and thankfully has been handed down to me. While these large hope chest might not be the most practical piece of furniture for a small room, the storage inside is priceless! I was determined to fit this hope chest into the functionality of the new home office space with a little paint and some trim to dress it up. [Please click to continue reading…]


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Its a Leica!!!... some says, and is a good camera, I like the quality, it works properly, I dont like the colors as on many Leica cameras, can be fixed on post prcessing, but mmm, in black and white are azom, I love how simple and sober the design are, I hate the colapsible type of lenses, why not a simple fixed lens, when i saw first time this camera off I loved it, but whe turn on and the colapsible lens were there pfff, never like this kind of lenses, dont combines with the rest of the ...View review in forums Leave a comment on this review
Leica X1
Well balanced in my hands. Reasonable bulk for its purpose. Relatively lightweight. Terrific lens. Resolution and color utterly satisfying. Autofocus a dissapointment despite firmware version 2.0, improved only M/F. For fast shootting, especially in low light situations, stick to manual focusing. Aperture and speed dials tend to turn quite easily. One must check. I only wish this camera had a faster AF and a 35-70 equivalent zoom. In all a star performer. S/N 3813xxx. Problems: Leatherette ...View review in forums Leave a comment on this review
I have grown to like this camera having initially thought that I had made an expensive mistake in buying it. Image quality is superb. Great colour, detail and texture. The lens is sharp. Build quality is very good - the camera feels good to hold. You get a free copy of Adobe Lightroom included. An optical viewfinder is essential and is very good though this is another cost. Overall an expensive camera with few "bells and whistles" but it does the important job of taking pictures very well. ...View review in forums Leave a comment on this review
I love this camera. Compact, excellent images, straight-forward controls. The best compact I have had. Only the Sigma DP1 came anywhere near it. I like it much better than the GF1, etc. etc.View review in forums Leave a comment on this review
- Canon EOS M58.8%
- Panasonic G85/G803.3%
- Panasonic FZ2500/FZ20001.9%
- Panasonic LX10/LX151.2%
- Panasonic GH5 development3.6%
- Sony a99 II15.9%
- Nikon KeyMission 170 and 801.0%
- Fujifilm GFX 50S development28.3%
- Olympus E-M1 II development18.7%
- Olympus E-PL80.1%
- Olympus 25mm F1.2 Pro1.5%
- Olympus 12-100mm F4 IS Pro1.9%
- Olympus 30mm F3.5 Macro0.1%
- Sigma 85mm F1.4 Art3.6%
- Sigma 12-24mm F4 Art2.6%
- Sigma 500mm F4 DG OS HSM Sport2.4%
- GoPro Hero50.8%
- GoPro Karma drone2.2%


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Gold Star Chili582 Mt. Carmel Pk., Cincinnati, OH 45245
Phone: (513) 528-0033
Please call the restaurant in advance to verify this information is still current. Please notify us if any of this information has changed. Restaurants have the right to reject this offer for any reason.
Deal DetailsKids eat free on:
Max Age: 10


text:
It's clean and close to the train station, making it convenient to do a quick 1 day, 1 night tour of Pisa or a few nights of doing train hops to surrounding destinations (Lucca, Cinque Terra...).
This is the ground floor of a residential house complete with fridge/sink/utensils.
They advertise "private bath" which is misleading. It's a shared bath. They accomodate 4 in bunks in one room and there's a Queen in a second bedroom. Then there's another bunk in an open area. If you're there with 8, the 1 bath could be a stretch however when we were there our schedules dovetailed and it wasn't a problem. If you're there with a larger group they had stow-away bed and foamies too.
The neighbour's courtyard backs onto the bedrooms but they were very considerate and shut things down around 10:30.
There's a gate up front. Best to let them know your time of arrival. We were lucky enough to have a cell so I don't know if there's a buzzer on the gate to call them. I think they live on the second floor.


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what is the value of a 1966 winchester model 94
Ordinarily I would ask you what the graded condition of the gun is, but in this case, it does not really matter that much. Used post-1963 production Model 94 Carbines manufactured up through the mid to late 1970s are seldom ever worth more than $250. Most are are found devoid of bluing on the receiver frame, and have lots of bumps & bruises. Those in that category are $125 - $175 guns.
Bert H.
you must buy cheap and sell HIGH


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Two weeks ago I wrote about writing for wireless devices. I said I'd written the first draft of the article on my personal digital assistant (PDA), using a stylus to poke an onscreen keyboard, to get into the "zone."
I said I thought the experience had made me a tighter writer, and I recommended it for other writers.
That didn't sit well with Robert Young, a writer in Geelong, Australia.
"I've agreed with nearly everything you've said in previous articles," he said, "and have a high regard for your expertise. But after reading your last column, I can only assume you've either slipped a cog or drunk too much of that Seattle coffee.
"Do you seriously think that drawing silly characters on a screen or poking at a keypad with a pointy stick will make me a better writer? Do you seriously expect me to deliberately take five times as long to write an article just to have a 'user experience'?"
But then he offered a lovely analogy: "A famous American actor once asked a famous Australian actor to shout at him and slap him around in order to help him get into character for a fight scene.
"The Australian replied, 'Why don't you just try acting?'
"So I say to you, Kathy, instead of going through these silly PDA machinations, why don't you just try writing?"
Due Diligence
Robert has a point. Being a good writer is like being a good actor. It's like being a good composer or a good golfer. That is, you're nowhere without talent, and to get anywhere at all you have to "do it" — act, compose, golf, or write — diligently. You have to practice your craft every possible moment.
But practicing your craft means more than writing melodies, playing three rounds of golf a day, or showing up for rehearsals seven days a week. Beethoven wouldn't have written the Ninth Symphony, Tiger Woods wouldn't have won every Major, and Julia Roberts wouldn't have gotten the Oscar for best actress without grueling, lonely hours of practicing different aspects of their craft day after day.
Here's my own analogy. Years ago I was a marathon runner. When I was training for a marathon, following the advice of running experts I would do two things: Some days I would run long miles at an aerobic pace, while other days I would run short wind sprints at an anaerobic pace.
Typing my article on a PDA reminded me of running wind sprints. It was painful, but it tightened my writing in ways that typing on a keyboard and looking at a big screen couldn't.
Having to Choose the Right Word the First Time
I also received email from Avanti Kumar, a British journalist living in Kuala Lumpur, Malaysia.
"I've planned and written whole articles on my PDA using on-screen graffiti," he wrote, "as it is the only way for me to write my columns while maintaining the responsibilities of my 'day job.' As you said, this is tedious!
"Even with the keyboard PDA, there is a forced economy when writing. So there is certainly an impact on style: It forces you to choose the right word even in the first draft."
Someone Had to Say It
A comment from Eric Miller, an advertising writer in Raleigh, NC, made me laugh:
"I used to write tight, but I couldn't handle the hangovers." Touchi.
"Composing articles on a PDA is an excellent idea," Eric continued. "I've used a folding keyboard with my PDA for about a year. I may have to give it up now!"
He cited other ways he imposes discipline on himself:
- Writing in haiku
- Writing longhand
- Writing longhand with his right hand (he's left-handed)
- Writing using a 300-word magnetic poetry set — "the child's version"
"The result of the last technique was so fresh, so concise, so free of 'adspeak,' that I decided to recreate the magnets as a Word file on my laptop (with the addition of a second set, it has grown to 600 words)."
Eric sent me the file, and I put it to the test with some short copy I was writing for a client site. (Don't worry, Robert, I didn't use it to write this article!) The client loved it.
With or without a keyboard, with or without cog slippage or too many Cherry Street Cafi double lattes, with or without a hangover or a full lexicon, there's more than one way to become a tighter writer.
Next time: when your copy never gets used.
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Seven ways to future-proof your digital strategy
Nurcin Erdogan Loeffler, head of strategy and innovation, Vizeum China, outlines the seven ways businesses can future proof their digital strategies.
How can chief marketing officers use disruption to stay ahead?
Chief marketing officers have shared their views on technology, innovation and how they see their roles transforming into the near future at an ... read more
The case of Skittles and how to react with real-time marketing
Every brand would love to see its hashtag trending on social media, but what if it's for the least expected reason? Should you ... read more
What does the modern marketer look like in a multichannel world?
In today's multichannel world how can marketers use data to ensure the experience a customer receives is relevant to them?


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We've gathered a few tweets from Oklahoma and national political figures in reaction to the Supreme Court's health care law decision.
Full CoverageBreaking News
Twitter reaction to Supreme Court health care decisionPublished 1:30 PM CDT Jun 28, 2012
CommentsThe views expressed are not those of this site, this station or its affiliated companies. By posting your comments you agree to accept our terms of use.blog comments powered by Disqus


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** NEW 6 Month Trial Membership Now Available**
Essential information to make informed decisions utilizing the power of the Association's collective knowledge & experience
Established over 50 years ago, FMCA is a cooperative information exchange serving credit professionals in the Home Furnishings industry. Members can access the best "bang for the buck" credit reporting available. Interchange meetings and direct member contacts support credit worthiness decisions under difficult circumstances.
FMCA participation enables:
FMCA News
FMCA Annual Meeting
September 21-23, 2016
Hotel Contessa
San Antonio, Texas


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Mark your calendar for November 5th and 6th and plan to attend "The Living Earth", Environmental Studies School, Course IV, Series IV. Offered by the Wisconsin Garden Club Federation, Milwaukee District, from 8 A.M. to 4 P.M. daily at Havenwoods Environmental Awareness Center, 6141 N. Hopkins Street, Milwaukee, WI 53209, we invite you to learn about "Water."
Sharon Morrisey, Horticulture Agent with Milwaukee County, is returning to discuss Organic Gardening and Michael Pauers with the Milwaukee Public Museum will instruct us on Aquatic Animals. Dr. Dale Olen will discuss Ecology and Effective Citizenship as it relates to our Earth. Paul Zamiatowski, a Certified Master Naturalist will guide us on our Field Trip at the Wehr Nature Center and speak to the topics of Wetlands and Aquatic Plants. Brenton Burger, from the "big island" down under and a guest lecturer at Marquette University, will share his knowledge on Sustainability. Judy Klippel, Superintendent at Havenwoods, will talk about Youth Programs in Environmental Education and Judy Newman, NGC ESS Chairman, will wrap-up the two days of activities on Saturday.
This school is open to the public and all NGC Garden Club Members. Required reading for Course IV is The National Gardener (1 year with 4 issues per year) and textbook Living in the Environment (7th edition or later) – G. Tyler Miller, Jr., available from NGC, Inc., the Internet and bookstores.
Course fee is $35.00 per day and lunch is available for an additional $8.00 per day. See WGCF website http://www.wisconsingardenclub.org/page/home.jsp to down load registration form or contact local chair, Carole Hopp (firstname.lastname@example.com) or call 414-771-7979 for more information. WGCF Website has further information on the vocabulary list, chapters of required reading, daily schedule, map and directions to Havenwoods and Instructor curriculum vitae. Area hotel information is also available upon request.
See you at Environmental School in November!
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- South Milwaukee Downtown Market temporarily moves for Evening on the Avenue Street Festival


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First attack on a cyborg
July 17, 2012 by Amara D. Angelica
UPDATE: McDonald's provided this statement to KurzweilAI on July 18, 2012:
"We share the concern regarding Dr. Mann's account of his July 1 visit to a McDonald's in Paris. McDonald's France was made aware of Dr. Mann's complaints on July 16, and immediately launched a thorough investigation. The McDonald's France team has contacted Dr. Mann and is awaiting further information from him.
In addition, several staff members involved… read more


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Added by Home Staging Channel Subscribe 1
Published 9 Feb 2009
The Kitchen includes much more than just walls, floor, and ceiling. There are many components in the kitche...
The Kitchen includes much more than just walls, floor, and ceiling. There are many components in the kitchen; most buyers will evaluate the condition of the house based upon this one room. A radiant, well organized and maintained kitchen speaks volumes to buyers that you have paid attention to every detail in the upkeep of your home. The first step in staging a kitchen is to get rid of unused clutter. Home buyers look for kitchens that are functional and spacious. The goal is to maximize all counter and cabinet space. Countertops should be clear of all items except for a few neutral decorative pieces displayed in the corners. Leaving appliances on your countertops gives buyers the impression that your kitchen has limited cabinet space. Also, discard of any items inside your cabinets and pantry that you do not use. Take everything out of your cabinets and drawers and clean and wipe down out all the cabinets and drawers. Throw away all of those products that you do not use. Put back items that you just can't live without. When putting items back into the cabinetry be sure to arrange them neatly and orderly. Remember that you want the kitchen to look as organized as possible.


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Because your health is important to you around the clock, Premier Health offers MyChart, a free, secure online connection to your doctor's office and your health information 24/7.
Barry McCorkle, MD provides an overview of MyChart. Press play below or read the transcript.
With MyChart you can log in from any computer to:
Premier Health strives to provide efficient, friendly care. We are pleased to offer MyChart as another way to improve our patients' overall experience.
Learn more:
MyChart is not for urgent medical matters. Please note that MyChart should not be used for urgent situations. Please call your physician if the situation requires immediate attention, or dial 911 if it is a medical emergency.
Source: Barry McCorkle, MD, Premier Infectious Disease
Content Updated: May 26, 2016
Our secure online Contact Us form is for non-medical questions, comments or suggestions. We will respond to your message via email within five business days.
If you would rather speak to a specific department representative, please view our online phone directory.
Patient Financial Services(937) 208-2777(855) 208-2777 Toll Free
Central Scheduling, Pre-Registration, Pre-Certification and Priceline(937) 499-7364(855) 887-7364 Toll Free
View all important numbers.
Financial Assistance
Help Us Touch Lives:
This site complies with the HONcode standard for trustworthy health information: verify here.


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Monitoring & Testing Companies in Greece
Current filtering criteriaYou can remove a filter by clicking on the x
Category: Monitoring & Testing ×
Employees number: 11-100 ×
Industry: Dry Cleaning ×
In: Europe ×
In: Greece ×
Scalewatcher North America Inc.
Office in GREECE
Scalewatcher: An electronic descaler friendly for the environment. Scalewatcher North America seeks confident, out-of-the-box thinking professionals interested in a technology and large ticket sales opportunity, acting as area manager for markets opening in all states. Scalewatcher is the original manufacturer of electronic, descaling technology used throughout Europe, ...
Distributor in GREECE
Industrial and Environmental Health and Safety Instrumentation: Air Sampling Pumps, Calibrators, Equipment, and Accessories. Colorimetric Gas Detector Tubes. Point Gas Monitors. Open Path Gas Detection. Sound, Noise, and Vibration Measurement Instruments. Real-time Dust Monitors. Flame Detectors.


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Satisfy Linda the Bride's picky order and sweet tooth! Use dozens of decorations to create the perfect wedding cake for her big day. A sample cake shows you what she wants, and you have to copy it exactly. Decorate quickly so you don't lose the valuable client!
Wedding Cake Contest is one of our selected Dress Up Games. Also very popular on this website right now are Yacht Wedding, Super Wedding Stylist, Draculaura Dreaming Wedding and Shopaholic Beach Models.
Report game as broken
Cancel report of broken game


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Top Definition
When you have overwhelming belly fat preventing you from being able to witness your own genitals. Also known as your spare tire.
That fattys got a bad case of dunlop disease
big willy mcClain tarafından 27 Nisan 2006, Perşembe
Trending Now
Can you define these popular missing words?


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3 for 2 on Selected Pre-owned Games*
Winter Sports 2012 gives you the opportunity to experience the thrill and excitement of some of the most high octane, adrenaline filled events on snow and ice!
Everybody knows that winter sports are for the true adrenaline junkies – nothing can compare to plummeting down a mountain side at over 120mph on nothing but a pair of skis or a snowboard.
Winter Sports 2012 captures that feeling of raw, stomach clenching excitement in extensive detail. Crammed with over 50 challenges spanning 6 winter sport and olympic events you have the chance to experience the rush in sub zero climates whilst on the go with your Nintendo 3DS!
Winter Sports 2012 on Nintendo 3DS Features:
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- Admissions
- Academics
- Research
- University Life
- Alumni
- About
Admissions & Info:
(703) 667-7921
Ph D Drexel University, Information Science and Technology
MBA George Washington University, Information Systems Technology
BA, University of North Carolina, Chapel Hill
Ruth is responsible for organizational training and for implementation and quality of the technical management, software development and service management processes for the Mission Solutions Group in QinetiQ North America. She has been with QinetiQ-NA and its legacy company, 3H Technology, for 5 years working on programs resulting in ISO, CMMI and ITIL credentials for several MSG business units. Prior to her experience with QinetiQ-NA, she held similar responsibilities for CMS Information Services which was acquired by CACI in 2004. Other professional work includes 4 years as an Associate Research Professor at George Mason University where she taught undergraduate and graduate courses in software and systems engineering. Prior to GMU, she held staff and management positions in systems and software engineering in several for-profit and not-for-profit companies including MITRE and Booz, Allen and Hamilton.
QinetiQ North America delivers world-class technology and responsive solutions to government agencies and commercial customers for many of their most urgent and complex challenges. QinetiQ North America is an independent, innovative technology provider operating with small company speed and agility while leveraging significant global resources. More than 6,400 QinetiQ North America engineers, scientists and other professionals have the mission knowledge and proven, reliable performance to meet the rapidly changing demands of national defense, homeland security and information assurance customers. QinetiQ North America is part of QinetiQ Group PLC, one of the world's leading defense and security technology companies.
For more information, please visit www.QinetiQ-NA.com.


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latest from Cebu Lifestyle
JAY Dilan is a photo enthusiast and a fine arts undergraduate who finished a woodworking and cabinet making vocational course. In December 2013, he took a picture of a bee which fascinated him.Read more
OUR ancestors called it common sense whenever they had to decide on things. Today, we call it discernment. We expect everyone to weigh things, analyze and listen to opinions of people we respect.Read more
Michelle: Vicky is engaged to Victor. She said their relationship has its share of highs and lows but it is strong and stable. However, there is one thing that bothers Vicky.Read more
PART of trying to lose weight is to control excessive insulin production in your body, and improve your insulin sensitivity. Just to review, the hormone insulin creates an environment wherein your body can utilize glucose for energy.Read more
"INDIA is too big." The owner of the biggest and hottest Indian restaurant in Cebu to date, explained right after dinner how "serving authentic Indian food" is a little bit more complicated than it seems.Read more
CEBU Grand Hotel's Cafe Lorenzo Bar and Restaurant launches their new food sets which officially started last Sept. 15 and will continue until Dec. 31.Read more
I WAS out of town for two weeks, staying with my cousins in Manila. I'm still on vacation mode, which affects my mood in a wonderful way every time I remember my cousin Mame's talent for home economics.Read more
MAC Cosmetics is a giant in the makeup industry due to its range of products ideal both for professional makeup artists and for those of us makeup junkies at home. There's a color and texture available for any color, any skin type, any age.Read more


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Brian Hartline is making his mark on Ohio without even suiting up.
The new Browns receiver, along with Cavaliers Anderson Varejao, Matthew Dellavedova, and Joe Harris attended "A Prom To Remember." The event was created to give teens with life-threatening illnesses the chance to attend prom just like their peers. Hartline was involved with it during his time in Fort Lauderdale.
MORE: NFL offseason stock watch | NFL mock draft | Salary cap casualties
"This feels like a dream," 15-year-old Amber told clevelandbrowns.com. Some patients weren't even sure if they would be well enough to attend, but those who felt the slightest bit up for it didn't regret their decision for one second.
Hartline signed with the Browns in the offseason, but he is already making a lasting impression. He made a two-hour drive just to attend the event, and says it won't be his last.
"I just really love seeing all the smiles on the kids' faces," Hartline said. "I plan on going to this event each and every year."
The residents of Ohio are happy to have Hartline as a part of the community.


text: After waiting a decent interval, nearly thirty-six hours, the Nobel committee announced today it planned to award Barack Obama another Nobel prize. There seems to be some dispute as to whether it should be the prize for peace or for economics. The committee did say they'd ruled out (prematurely, in my opinion) prizes for either physics or chemistry.


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Michigan State Names Football Captains
Spartan Digest Top Stories
Berk's Preview: Michigan State @ IndianaMichigan State is coming off an embarrassing loss to Wisconsin and will look to rebound against the surging Indiana Hoosiers on the road in prime time. Can the Spartans get back…
Spartan DigestYesterday at 8:44 PM
Site Predictions: Indiana HoosiersThe Spartan Digest staff and members of the site give their predictions on how Saturday's game against Indiana will go in Bloomington.
Spartan DigestYesterday at 7:35 PM
Know Your Foe: Indiana HoosiersSpartan Digest checks in with Peegs.com football analyst Matt Weaver to learn more about Michigan State's foe for this weekend, the Indiana Hoosiers.
Spartan DigestThursday at 10:13 AM
Podcast: Indiana Week & MoreSpartan Digest packs a lot in our latest podcast as Hannah Hultink joins Dave Berk to discuss the loss to Wisconsin, this week's match-up with Indiana and the loss of former…
Spartan DigestWednesday at 11:48 PM
Price Semi-Finalist William V Campbell TrophyMichigan State senior tight end Josiah Price has been named a semifinalist for the prestigious William V. Campbell Trophy, presented by Fidelity Investments.
Spartan DigestWednesday at 12:43 PM


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Whether you're looking to protect or improve your cell phone, there are a wide variety of accessories that can help. Kmart carries cell phone accessories, ranging from screen protectors to chargers, to keep your phone performing at its absolute best. You'll even find special equipment to make a device useable in a vehicle, so you can accept hands-free calls or properly use the phone's GPS.
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Aside from protectors and cases, chargers are one of the most vital cell phone accessories you can own. Choose from a wide selection of standard wall models or car chargers, so your device is always ready to go. Some cell phone chargers can even boost tablet, eReader and MP3 player batteries. Whether you own a traditional flip phone or smartphone, Kmart carries a wide variety of affordable cell phone accessories that fit your needs.


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Steve Allen was the first host of The Tonight Show, ... Allen pioneered the format of late night network TV talk shows, originating such talk show staples as an ... - read more
Aug 21, 2016 · In this brief history of the TV talk show, ... 'Late Show' talk show host David Letterman. ... When Did The First Talk Show Appear?: - read more
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|The University of Texas at Dallas | Office of Admission and Enrollment
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Catfish Soup
Newcastle, NSW, AU
Sunday, June 9th, 2013 at 12:00pm
All Ages
Great tunes, food & beer during the Blue Water Music Festival
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