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|Sylvia H Sardi1*#, Brian J Glassner2#, J Robert Chang1, Sun Hee Yim3
|1Laboratory for Innovative and Translational Technologies, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA
|2WaferGen Biosystems Inc., 7400 Paseo Padre Parkway, Fremont, CA 94555, USA
|3Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
|#These authors contributed equally to the manuscript
|Corresponding Author :
|Sylvia H Sardi
Harvard Catalyst-Laboratory for Innovative and Translational Technologies
Harvard Medical School, 77 Avenue Louis Pasteur
Boston, Massachusetts 02115, USA
Tel: 617-244-2964
E-mail: [email protected]
|Received September 18, 2014; AcceptedOctober 30, 2014; Published November 01, 2014
|Citation: Sardi SH, Glassner BJ, Chang JR, Yim SH (2014) Identification of Critical Biomarkers Responsive to Anti-Autophagy Therapies for Pancreatic Ductal Adenocarcinoma through a Performance Analysis of miRNA Platforms. J Bioanal Biomed S10:001. doi:10.4172/1948-593X.S10-001
|Copyright: © 2014 Sardi SH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Background: Autophagy is a fundamental catabolic pathway that involves the degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. The role of autophagy in cancer is complex, exhibiting tumor suppressive or tumor proliferative effects depending on the cancer type. Pancreatic ductal adenocarcinoma has constitutively activated autophagy that results in the selective proliferation of its tumor cells. Understanding the mechanisms by which the inhibition of autophagy occurs could shape our current therapeutic strategies for pancreatic cancer. Recent research has implicated microRNAs as important regulators of autophagy, but how miRNAs influence this inhibition is largely unexplored. Results: It is known that there exists inter-platform variability across miRNA platforms and therefore in order to identify bona fide miRNAs that are involved with the inhibition of autophagy we carried out a performance analysis of miRNA arrays from three vendors. In this paper we report our findings from this cross-platform analysis of miRNA arrays and the identity of several critical biomarkers of response to anti-autophagy therapies in the immortalized human pancreatic ductal adenocarcinoma cells 8988T as compared to normal human pancreatic ductal epithelial cells. Our cross-platform analysis of three different miRNA arrays revealed that RT-PCR-based technologies provide higher inter-platform reproducibility than hybridization-based technologies, a greater sensitivity indicated by larger dynamic ranges and reduced processing time. We also discovered two miRNAs, miRNA 720 and miRNA 29b to play a role in the inhibition of autophagy in 8988T cells and found miRNA 30a to be involved with the inhibition of autophagy in normal human pancreatic ductal epithelial cells. Conclusion: This cross-platform analysis of miRNA arrays contributed significant information about miRNA expression systems and led to the first report of miRNAs implicated in the inhibition of autophagy in pancreatic ductal adenocarcinoma. Our findings should provide new insights for developing therapeutic solutions for this extremely intractable disease.
|Inhibition of autophagy; microRNA array cross-platform analysis; Pancreatic ductal adenocarcinoma; Chloroquine therapeutic solutions; Qiagen miRNA arrays; NanoString miRNA arrays; WaferGen miRNA arrays
|HPDE: Human Pancreatic Ductal Epithelial Cells; PDAC: Pancreatic Ductal Adenocarcinoma Cells; miRNAs: microRNAs; RT-PCR: Real-Time PCR; Ct: Threshold value; R2: Coefficient of Determination; CV: Coefficient of Variation
|Autophagy is a cellular pathway that removes damaged organelles or unused or malformed proteins through the lysosomal machinery and plays a central role in maintaining cell homeostasis. Of the three different forms of autophagy, macro autophagy is the primary pathway and is characterized by the formation of the autophagosome that migrates through the cytoplasm until fusing with a lysosome. The contents of the autophagosome are degraded within the lysosome via acidic lysosomal hydrolases [1]. Autophagy has been shown to be associated with both tumor suppression and tumor cell survival, two opposing processes that can alter the state of an organism [2,3]. Most strategies used to combat cancer recommend inducing autophagy to enhance its tumor suppressive effects, followed by inhibiting autophagy and thereby inducing apoptosis and tumor cell removal. The most effective strategy would be to fine-tune these opposing controls based on the importance of autophagy in a particular cancer or genetic context.
|There are over 30,000 cases of pancreatic ductal adenocarcinoma in the US yearly; it is second only to the colon in cancers of the gastrointestinal tract, and is the fourth-leading cause of annual cancer-related deaths. Current treatments are profoundly ineffectiveincidence and mortality rates are essentially equal, and the five year relative survival rate for even local PDACs is only 15% [4]. PDACs are Ras-driven cancers that have constitutively activated basal autophagy that contributes to continued proliferation of its tumor cells [3]. Understanding how the inhibition of autophagy occurs is crucial for any successful therapeutic intervention in pancreatic cancer. One of the drugs currently available that has been shown to effectively inhibit autophagy in this type of cancer is chloroquine. This compound blocks lysosomal acidification and hence the last step in the autophagy pathway, autophagosome degradation [5]. The prevalence of autophagy in PDAC coupled with the effectiveness of chloroquine as an anti-autophagy inhibitor may provide a therapeutic solution for a very intractable disease. Chloroquine monotherapy, however, may not be effective [6] and other molecules may need to be combined with chloroquine in order to increase therapeutic efficacy.
|In recent years, microRNAs (miRNAs) have emerged as important regulators of a large number of fundamental cellular processes, including autophagy [1]. It has been reported, however, that interplatform variability exists across miRNA platforms [7] and therefore in order to identify bona fide and critical biomarkers responsive to antiautophagy therapies we carried out a performance analysis of miRNA arrays. We chose to utilize current and novel technologies to quantify levels of miRNAs in the inhibition of autophagy. Two platforms were Real-Time PCR (RT-PCR) based, Qiagen and WaferGen, whereas the third was hybridization-based, the NanoString platform. MiRNA levels in normal human pancreatic ductal epithelial cells (HPDE) and immortalized human PDAC cells (line 8988T) with and without chloroquine treatment were analyzed.
|Sample collection
|Four RNA samples were obtained from human cultured pancreatic cell lines provided by Dr. Alec Kimmelman's laboratory at Harvard Medical School. Samples consisted of a control sample, human pancreatic ductal epithelial cells (HPDE, "Sample A"), and three experimental samples ("Samples B, C, and D"). Sample B consisted of HPDE cells grown in culture media containing the autophagy inhibitor chloroquine (25 mM) for four hours. Samples C and D consisted of immortalized human pancreatic ductal adenocarcinoma cells (PDAC cells, cell line 8988T) grown in untreated media and cell culture media containing 25mM chloroquine for four hours, respectively [3].
|Total RNA extraction and quantification
|Total RNA was extracted from cells using an RNA Extraction Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. RNA sample quality was evaluated using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) that generates RIN quality scores. RNA concentration was determined using a NanoDrop (Thermo Scientific, Wilmington DE).
|Qiagen miRNA expression system
|Three micrograms total RNA from each sample were reverse transcribed with the RT2 miRNA First Strand Kit (Qiagen, Valencia, CA). The resulting cDNA was added to the 2X RT2 SYBR Green PCR Master Mix (Qiagen, Valencia, CA) and the mixture was aliquotted across the Genome Array Set 384-Well formatted RT2 miRNA PCR Array (Qiagen, Valencia, CA). This array system contains miRNA sequences annotated by the Sanger miRBase Release 14 [8-11]. A total of 376 human miRNA sequences in duplicate were represented in the array. The PCR plate was placed into the Light Cycler 480 (Roche, Indianapolis, IN) and the following cycling program was run – 10 minutes at 95°C and 40 cycles of 15 seconds at 95°C, 35 seconds at 60°C and 30 seconds at 72°C (Figures 1 and 3). In order to obtain statistically significant data, three technical replicates were run for each sample.
|WaferGen miRNA expression system
|The technology developed by WaferGen to analyze miRNAs is also RT-PCR based and has the capacity to screen 1,036 human miRNAs in quadruplicate during each run. Two reactions were performed before samples were loaded into the nanowell chip called the SmartChip. The first one was the polyadenylation of total RNA, and the second involved a reverse transcription step using a set of custom primers from WaferGen. Briefly, 1μg of total RNA was polyadenylated using the EpicentrePoly (A) Polymerase Tailing Kit (Epicentre, Madison, WI) and was reverse transcribed using the Applied Biosystems High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA) following the recommendations of the manufacturer, with the random primer supplied by the kit being replaced with a 20X SmartChip miRNA Reverse Transcription Primer (WaferGen Biosystems, Fremont, CA; final primer concentration 1μM). A quantitative PCR reaction using SYBR Green detection was subsequently performed on-chip with preloaded miRNA-specific primers that eliminated the need for pre amplification with complex miRNA primer pools. The cDNA pool was mixed with the Light Cycler 480 SYBR Green I Mix (Life Technologies, Carlsbad, CA) in a 1ml total final volume and then applied to the SmartChip Human miRNA Panel V3. The SmartChip Panel by WaferGen consists of 1,036 miRNAs from the miRBase version 18 sequence database [8-11], in quadruplicate.
|The final volume per reaction for the miRNA panel was 100 nL, with an equivalent of 100 pg of starting RNA loaded per reaction. Forty cycles of real-time PCR were performed on the SmartChip Cycler (WaferGen Biosystems, Fremont, CA), followed by a melt curve analysis. The SmartChip Cycler processes the qPCR assays in just over two hours (Figures 1 and 3).
|NanoString miRNA expression system
|Buffer and Code Set from the nCounter miRNA Sample Preparation Kit (NanoString, Seattle, WA) and 100ng of total RNA were added into a strip tube and hybridized overnight. This procedure was repeated for each total RNA sample and for three technical replicates. NanoString's hybridization platform for miRNA analysis allows for the screening of 800 human miRNAs with a maximum of forty-eight samples being processed during each run. For the NanoString platform, as miRNAs are released to the miRBase [8-11], new probes can be added to the Code Sets so the data obtained from the NanoString instrument is always current. Each target molecule is represented by a color-coded barcode. The hybridization of barcoded probes to a target molecule occurs through a multiplexed reaction directly in solution. Unique oligonucleotide tags, miR tags, are ligated to mature miRNAs using bridging oligonucleotide and other reagents from the nCounter miRNA Sample Preparation Kit. The bridges are removed using an enzymatic purification step. The mature miR tagged miRNAs are then hybridized to a probe pair – a Reporter Probe that carries the signal and a Capture Probe that allows the probe/target complexes to be immobilized for data collection (Figures 2 and 3).
|Following hybridization that occurs in a single overnight multiplexed reaction, the strip tube was placed onto the automated nCounter Prep Station (NanoString, Seattle, WA) and reagents from the nCounter Master Kits (NanoString, Seattle, WA) were added according to the nCounter miRNA Expression Assay Kit (NanoString, Seattle, WA) protocol. At this stage, sample processing occurs – excess probes are removed and probe/target complexes are bound, immobilized, and aligned on the nCounter Cartridge.
|Data analysis
|For the Qiagen platform, after manual collection of the threshold (Ct) values from the real-time PCR machine, the data was entered into the PCR Array Data Analysis Web Portal (Qiagen, Valencia, CA) and automatically analyzed. The fold change for each gene from the control group to the experimental group was calculated as 2-ΔΔCt [12].
|For the WaferGen platform, relative miRNA expression was determined by the data obtained with the real-time instrument and the ΔΔCt method as for the Qiagen system. Both raw Ctand Tm values for each assay and sample were collected for data analysis. A data quality screen based on amplification, Tm values from melt curves, and Ct and Tmvariability was performed to remove any outlier data before ΔΔCt calculations were used to determine fold change in miRNA levels.
|The mean Ct and Tm values determined from each of the 4 replicates on each SmartChip Panel were calculated by the SmartChip qPCR Software (WaferGen Biosystems, Fremont, CA) for each assay and reported (minus any discarded outlier assays). These replicate data were exported to Excel (Microsoft, Redmond, WA). For the sake of ease of computation, assays for which no Ct value was reported or for Ct values > 30.0, a Ct value of 30 was assigned (i.e., miRNAs absent or below the level of detection). ΔCt values were computed using the mean of all replicate Ct values for each sample/chip, not including Cts of 30 [13]. The average ΔCt for the experimental samples (B-D) and the control sample A for each assay were then computed. From these, ΔΔCts were computed (ΔCtExperimental – ΔCtControl).
|For the NanoString platform, sample nCounter cartridges (NanoString, Seattle, WA) were placed into the Digital Analyzer (NanoString, Seattle, WA), a fluorescent reader, for direct digital counting. The barcodes can be individually counted without the need for amplification providing very sensitive digital data. The nCounter data was imported and analyzed in nSolver Analysis Software (NanoString, Seattle, WA)
|For all three platforms, differences in the miRNA levels of samples B-D were expressed as fold-changes relative to the levels detected in sample A. Because all assays were reported in triplicate (except for the WaferGen assays which were reported in quadruplicate), mean values were computed prior to fold-change calculations. For the purpose of comparison between the different platforms, miRNA levels that differed by more than 2-fold (increased or decreased) relative to the control sample A levels were considered to be significant. In order to control the variability among experiments, a set of endogenous control miRNAs were incorporated into each miRNA expression system.
|Statistical analysis
|The coefficients of determination R2 were calculated for the intraplatform variation analysis. Individual hybridization values obtained from the NanoString platform and individual Ct values from the Qiagen and WaferGen platforms were compared between triplicate samples. The %CV is defined as the variation among multiple measurements in proportion to their mean. We calculated %CV for the inter-platform variation analysis. Average hybridization values obtained from the NanoString platform and average Ct values from the Qiagen and WaferGen platforms were compared among platforms for the 26 assays in common to all of them.
|Workflow comparison of miRNA expression systems from three vendors
|In our cross-platform analysis we first compared the workflow of the miRNA expression systems from three vendors. In the first sample processing step, the two real-time based assay systems from Qiagen and WaferGen followed very similar workflows (Figure 1). Both systems converted the miRNA species to cDNA with the aid of their respective reverse transcription kits. Samples containing the cDNA were then mixed with the validated miRNA assays and quantitative PCR reactions were run in the respective system instruments. Lastly, the data was analyzed with each vendor's proprietary software. For the hybridization based assay system from NanoString, the workflow was entirely different (Figure 2). Hybridization reactions were set up by adding sample, buffer and barcoded probes to a strip tube and hybridized overnight. The strip tube was then transferred to a station where sample processing occurs and probe/target complexes are bound to a cartridge. This cartridge was then placed into another instrument for direct digital counting. The resulting data was analyzed by NanoString's proprietary software.
|Several other parameters related to workflow were then compared including sample processing times, accessibility of the platforms and extent of high-throughput screening. A comparison of the three workflows revealed that the real-time based technologies from Qiagen and WaferGen have reduced sample processing times compared with NanoString's hybridization based platform which requires an overnight hybridization step (Figure 3). Also, Qiagen's miRNA expression system which can be run on a standard PCR machine is the most cost effective and therefore the most accessible of the three platforms. NanoString and WaferGen's miRNA expression systems require more elaborate technology platforms for sample processing and therefore can be inhibitive for many researchers. In regards to high-throughput screening, all three miRNA expression systems have this capability. WaferGen's miRNA platform offers the largest number of miRNAs, over 1,000 that can be screened during a single run. For the Qiagen platform, 376 human miRNAs can be screened during a single run. The NanoString platform offers an intermediate number of screenable miRNAs, approximately 800 compared to the other two platforms. The advantage of the NanoString platform compared with the other two platforms is that 48 experimental samples can be screened for miRNA expression during a single run instead of only a single sample.
|Comparison of fold-change data for two platforms
|In order to understand the meaning of the fold-change values better, comparison of the fold-change data common to the Qiagen and WaferGen platforms and the WaferGen and NanoString platforms were analyzed separately. A total of 50 miRNAs were common to the Qiagen and WaferGen platforms that displayed at least a 2-fold increase in miRNA levels as compared to the control sample A (Table 1). Approximately 20 miRNA assays that were common to the Qiagen and WaferGen platforms displayed at least a two-fold decrease in miRNA levels as compared to the control sample A (Table 1).
|When comparing the fold-data generated by the WaferGen platform to the fold-data generated by the NanoString platform, 23 miRNA assays displayed at least a 2-fold increase in miRNA levels as compared to the control sample A (Table 1). A two-fold decrease in miRNA levels as compared to the control sample A was observed for only 8 miRNA assays common to both WaferGen and NanoString (Table 1).
|Intra-platform variation analysis
|A comparison of individual data points among replicates for which there were at least three informative assays for the control sample A was performed for each platform. For the NanoString platform, coefficients of determination (R2) values were calculated for hybridization values from triplicate samples. A R2 value of 0.9804 was obtained for the NanoString platform from a variation analysis of 121 assays (Figure 4). The R2 value for triplicate samples from 744 assays run on the Qiagen platform was 0.9152 (Figure 4). A similar R2 value was obtained with WaferGen platform. A R2 value of 0.9116 was calculated for triplicate samples of 1004 assays analyzed with the WaferGen platform (Figure 4).
|Inter-platform variation analysis
|Among all of the assays employed, there were 26 miRNA assays overall that were common to all three platforms and displayed a significant difference relative to the control sample A (Table 2). Although the direction and magnitude of gene expression were similar for all 26 assays, the variability among the three platforms differed significantly. The NanoString platform exhibited the highest variability among the three, with a %CV at 17.1% (Table 3). The WaferGen platform demonstrated the best reproducibility with a %CV at 0.5% (Table 3). An intermediate reproducibility value was calculated for the Qiagen platform at 4.3% (Table 3).
|Dynamic range variations evident among the platforms
|The dynamic range for all three platforms was also significantly different. There were 46 assays from the NanoString platform that displayed a greater than 2-fold difference in miRNAs compared to the control sample. Fold changes for all assays on this platform ranged from a value of 0 to 6.5-fold (Table 4). WaferGen had the greatest number of assays, a total of 262, that displayed a greater than 2-fold difference in miRNA levels compared to the control sample. Fold changes for all assays on this platform extended from 0 to 10,420-fold (Table 4). The Qiagen platform had a total of 170 assays that displayed a greater than 2-fold difference in miRNA levels in the experimental samples relative to the control sample. The range of fold changes for all assays on this platform was intermediate to NanoString and WaferGen's, extending from 0 to 50.9-fold (Table 4).
|Cross-platform determination of miRNA levels in HPDE and 8988T cells treated with the autophagy inhibitor chloroquine
|In this study, we evaluated miRNA expression systems from three vendors by screening miRNAs involved with the inhibition of autophagy in immortalized human PDAC cells, cell line 8988T, as compared to normal human pancreatic ductal epithelial cells designated as HPDE cells. Analysis of the cross-platform miRNA data revealed two miRNAs to be significantly up-regulated in the 8988T cells grown in the presence of the autophagy inhibitor chloroquine. These miRNAs are miR-29b and the miR-720. Based on the NanoString platform miRNA data, miR-29b in 8988T cells exposed to chloroquine exhibited a 2.24 fold increase in expression relative to miR-29b levels exhibited by untreated 8988T cells (Table 5). Under the same experimental conditions but utilizing the Qiagen platform, a similar 2.46 fold induction of miR-29b expression was exhibited in chloroquine treated 8988T cells compared to untreated cells (Table 5). In contrast, no significant induction of miR-29b in 8988T cells was observed with the WaferGen miRNA platform following chloroquine treatment (Table 5).
|MiR-720 following chloroquine-autophagy inhibition exhibited a similar induction in expression levels to miR-29b, with all three platforms displaying enhanced miR-720 levels. According to the NanoString data, miR-720 was induced in HPDE cells grown in chloroquine. The fold induction was 1.71, slightly below the greater than 2-fold cut-off implemented to filter out likely significant miRNA expression changes (Table 5). Utilizing the Qiagen platform, however, miR-720 exhibited a 3.12 fold increase in 8988T cells exposed to chloroquine as compared with untreated 8988T cells (Table 5). With the WaferGen platform, a 2.18 fold induction of miR-720 was observed in 8988T cells exposed to chloroquine as compared with untreated 8988T cells (Table 5).
|We observed that miR-30a, a known and potent inhibitor of autophagy [1, 14], was down-regulated in HPDE cells treated with chloroquine for all three of the miRNA platforms (Table 6). This downregulation was not greater than 2-fold lower than control levels.
|A comparison of the NanoString, Qiagen, and WaferGen miRNA platforms showed that in general, for assays that were common to them, all three platforms behaved similarly in terms of identifying assays that had miRNA levels greater than 2-fold different in the experimental samples when compared to the control sample, both in magnitude and direction of induction. Of the three platforms, the WaferGen miRNA platform outperformed the other two in terms of reproducibility of the 26 assays in common to all of them, displaying the smallest % coefficient of variation (%CV). The Qiagen platform, although also RT-PCR based, displayed an intermediate %CV whereas the highest %CV and therefore the lowest reproducibility was exhibited by the hybridization-based platform, NanoString. From these experiments it can be concluded that RT-PCR based technologies provide higher inter-platform reproducibility than hybridization-based technologies.
|Our studies also demonstrated that the WaferGen miRNA platform displayed a dramatically broader dynamic range than the other two platforms for assays that presented a greater than 2-fold induction in miRNA levels relative to the control sample. Again the Qiagen platform, the other real-time PCR based platform, exhibited an intermediate dynamic range. The hybridization based NanoString platform had the smallest dynamic range. The number of assays that met the fold induction criteria and were therefore analyzed for dynamic range were greater for the WaferGen and Qiagen platforms than for the NanoString platform but this difference cannot account for the magnitude of induction observed with the WaferGen platform. Based on the dynamic range data, it can be concluded that the WaferGen platform provides the most sensitive miRNA expression data of the three platforms.
|Although the NanoString platform performed poorly compared to the other two platforms in all the inter-platform analyses, the NanoString platform scored highest in our intra-platform variation analysis. The R2 value calculated for the NanoString platform was the highest among all three platforms, reflecting a more linear response and the least variation between triplicate samples. For the Qiagen and WaferGen platforms the R2 values were similar, demonstrating their real-time PCR commonality.
|Our cross-platform analysis, in addition to providing valuable information about miRNA expression systems, resulted in several new scientific discoveries regarding miRNA expression and the inhibition of autophagy in normal HPDE cells versus immortalized human PDAC cells. In this manuscript we present the first evidence of miR-720 playing a role in pancreatic ductal adenocarcinoma. We observed a greater than two-fold increase in miR-720 expression in 8988T cells grown in the presence of the autophagy inhibitor chloroquine as compared with untreated 8988T cells. miR-720 has been shown to be deregulated in various cancers other than pancreatic cancer such as cutaneous malignant melanoma [15], multiple myeloma [16], esophageal cancer [17], vestibular schwannomas [18], and inflammatory breast cancer [19], among others. It has been observed previously that levels of miR-720 and three other miRNAs are significantly higher in biopsies from colorectal cancer patients than normal controls [20]. miR-720 levels are also higher in colorectal patients with mutated KRAS than in those with wild-type genotypes [20]. This could be a downstream effect of ERK pathway over-activation triggered by KRAS mutations. Kimmelman et al. have shown that glutamine supports pancreatic cancer growth through a Kras-regulated metabolic pathway [21,22].
|Additionally, we have confirmed previous reports of an association of miR-29b production with pancreatic cancer [23,24]. In 8988T cells treated with chloroquine, we observed a greater than two-fold induction of miR-29b as compared with untreated 8988T cells. It was shown previously that miR-29b was up-regulated greater than 2 fold in two human pancreatic cancer cell lines MiaPACA-2 and PANC-1 that were treated with two chromatin-modifying agents [23]. Previous studies also revealed that the knockdown of Dicer 1 expression in BxPC-3 cells from xenograft pancreatic tumor models and PANC-1 cells resulted in a significant decrease in miR-29b and other miRNAs. It was also shown in the same study, however, that the knockdown of Dicer expression in Capan-2 cells, also from xenograft pancreatic tumor models, had no effect on miR-29b expression but did decrease the expression of other miRNAs [24]. The observation that different pancreatic cancer cell lines exhibit obvious discrepancies in gene expression profiling implies that different molecular mechanisms are involved in the carcinogenesis of pancreatic cancer subclasses and highlights the importance of personalized therapy.
|MiR-30a has been shown to be a potent inhibitor of autophagy by down-regulating the expression of Beclin 1 and ATG5, two proteins important in the canonical starvation-induced macroautophagy pathway [25]. It has also been shown that the BCR-ABL tyrosine kinase inhibitor, imatinib, markedly inhibits the expression of miR-30a in human chronic myelogenous leukemia cells [26]. Our finding that miR-30a was down-regulated (although not greater than 2-fold lower than control levels) in the HPDE cells exposed to chloroquine and that this down-regulation was observed for all three platforms may indicate that miR-30a not only plays a role in the inhibition of autophagy but may also play a role in Ras-driven cancers, a correlation that has not been previously documented.
|Based on our findings, miRNA expression analysis can be accomplished using any of these three platforms in a very reproducible manner. The utilization of RT-PCR-based miRNA technologies from Qiagen and WaferGen, however, resulted in higher inter-platform reproducibility and greater sensitivity indicated by larger dynamic range values. The hybridization-based miRNA expression system from NanoString exhibited the best intra-platform reproducibility and had the capability of processing the largest number of samples during a single run, although the sample processing time was the longest of the three platforms, over twenty-four hours. In addition to supplying essential information about miRNA expression systems, our cross-platform analysis enabled us to present this first report of miRNAs involved in the inhibition of autophagy in pancreatic ductal adenocarcinomas. Additional validation and functional analysis can be performed to verify miR-720 and miR-29b as critical biomarkers of response to antiautophagy therapies in pancreatic ductal adenocarcinomas. Also, the exact role of miR-30a which plays a role in the inhibition of autophagy in normal human pancreatic ductal epithelial cells can be explored further. The combination of miRNA manipulation and anti-autophagy treatment with chloroquine should provide new therapeutic avenues for pancreatic ductal adenocarcinoma in the near future.
|The authors wish to thank Dr. Alec C. Kimmelman and Dr. Shenghong Yang from the Department of Radiation Oncology at Harvard Medical School for providing the RNA samples for this study. The authors would also like to thank Dr. Winston Patrick Kuo for providing access to the miRNA platforms. The authors also thank the vendors NanoString, Qiagen, and WaferGen for supplying the assays and reagents required for this study. The authors declare neither conflicts nor financial interests. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Harvard Catalyst, Harvard University and its affiliated academic health care centers.
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Repeal the Human Rights Act? I repeat again for good measure: Repeal. The. Human. Rights. Act?
I mean who doesn't want:
Right to life, right not to be tortured or subjected to inhumane treatment, right not to be held as a slave, right to liberty and security of the person, right to a fair trial, right not be retrospectively convicted for a crime, right to a private and family life, right to freedom of thought, conscience and religion, right to freedom of expression, right to freedom of assembly and association, right to marriage, right to an effective remedy, right not to be discriminated against, the right to the peaceful enjoyment of one's property, and the right to an education and finally free and fair elections….?
Seriously? Is this the face of modern Britain? By scraping the Act and essentially leaving the European Convention on Human Rights (formally Convention for the Protection of Human Rights and Fundamental Freedoms) we join an esteemed list of other countries that are not part of the Convention, which include: Belarus. Yup! Belarus, the standard bearer of democracy, civil liberty and freedom in Europe. Even Russia is a member, OK their human rights record can't really be shouted about, but they are still part of it!
Following the wreckage of the Second World War, the nations of Europe came together to define what they considered to be the fundamental rights that its citizens (or subjects in the case of the UK) should have guaranteed and enshrined in law. This was a remarkable watershed moment, and combined some of the basic ideals of rights and liberties. Yes, sounds of which still need to be expanded on, for example gender identity and sexual orientation – but it's always best to reform from within.
The principles of the European Convention of Human Rights is one the reasons I joined the LibDems, a party which in the affirms in the opening sentence of its constitution the fundamental values of liberty, equality and community. I spent most of my education at the Vienna International School, was founded in the principles of equality as per the Universal Declaration on Human Rights. Indeed the address of the school is Straße der Menschenrechte (Street of Human Rights). Growing up in a multilingual and multicultural environment has shaped my politics and made me the person I am now. We were taught from a young age the importance of human rights, of equality (regardless of race, religion, ethnicity, gender) and thus when it came to choosing the political party for me the LibDems were and are the natural fit.
It is the role of the LibDems to defend our Human Rights Act and to ensure that the fundamental principles of the European Convention on Human Rights are not eradicated in what will most certainly be a dark day for civil liberties on this country if the act is repealed.
We must ensure that we take this fight beyond Parliament and onto the doorsteps! As Liberal Democrats it is the fundamental principle of our party that the Human Rights Act remains and is not repealed!

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1 Corinthians 10:
12 Wherefore let him that thinketh he standeth take heed lest he fall.
13 There hath no temptation taken you but such as is common to man: but God is faithful, who will not suffer you to be tempted above that ye are able; but will with the temptation also make a way to escape, that ye may be able to bear it.
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17 For we being many are one bread, and one body: for we are all partakers of that one bread.
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Former super eaglect captain Kelechi Nwakali spotted frying garri.
As he took shots of his actions during the garri preparing process. the young striver who made to Europe through the eaglets quotes on his instagram post :
"when to the village today to do the things I know how to do the things I enjoy doing it was fun you will know if you have been in the village good to be back"


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Mexican food varies by region, because of local climate and geography and ethnic differences among the indigenous inhabitants and because these different populations were influenced by the Spaniards in varying degrees.
The north of Mexico is known for its beef, goat and ostrich production and meat dishes, in particular the well-known arrachera cut.
The six regions of Mexico differ greatly in their cuisines. In the Yucatán, for instance, a unique, natural sweetness (instead of spiciness) exists in the widely used local produce along with an unusual love for achiote seasoning. In contrast, the Oaxacan region is known for its savory tamales, celebratory moles, and simple tlayudas while the mountainous regions of the West (Jalisco, etc.) are known for goat birria (goat in a spicy tomato-based sauce).
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Who we are
The Nottingham and Nottinghamshire Sustainability and Transformation Partnership includes all the NHS organisations and local councils across the city and county.
We have also been working with a wider group of representatives from other organisations such as the independent patient champion HealthWatch and the community and voluntary sector to shape our Plan.
The STP - Our Local Plan
Every area of the country has produced a Sustainability and Transformation Plan looking at how local organisations will work together, over the next five years, to improve care, health and wellbeing for people in their area.
Our local plan (2016) covers Nottingham and Nottinghamshire - a population of just over one million. Alongside our full STP Plan we have produced a summary guide: "A healthier future for Nottingham and Nottinghamshire"
Update to the Plan
In July 2017 we published an Update to our five-year Plan for health and social care in Nottingham and Nottinghamshire, reflecting the comments about our Local Plan received from local people, staff and stakeholders at meetings, events and through written feedback.
There are specific sections on mental health and services for children and young people and carers as these were areas that people felt needed strengthening within the Plan.
Update to five-year health and social care plan published following public feedback
26 July 2017
Update published to joint, five-year Plan for health and social care in Nottingham and Nottinghamshire.
Nottingham and Nottinghamshire – 'advanced' in their five year planning for health and social care
21 July 2017
NHS England has today announced new ratings for the Sustainability and Transformation Partnerships in England.
Publication of feedback on the draft STP
9 June 2017
We have now published our report based on feedback from the public and stakeholders on our draft STP.
Click the map below to see the area in detail.


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Build Your CV
9635 Circle Way, Denver, Colorado 11111
E:ttennyson@email P: 555-222-8888
Experienced logistics professional looking to progress into supervisory role. Five years of experience working in warehousing and logistics, along with strong knowledge of key business principles. Often looked to as a leader by coworkers and as a mentor by new employees. Work history and educational background have helped in developing an excellent understanding all of the aspects of supply warehouse management, including safety and reporting requirements. Recognized by managers, shipping partners, and others in authority positions as one who would be an effective team leader.
- Maintain inventory database that catalogues all supplies and components available in regional warehouse for national home improvement store retailer.
- Coordinate with shipping partners to verify receipt and storage of new product orders, and to ensure that all invoices are transferred on to finance department.
- Prepare weekly reconciliation reports detailing all shipping and receiving activity to be shared with management team.
- Coordinated with the department managers at the eight stores in region to track in-store inventories.
- Prepared new purchase orders whenever inventory volumes met reorder criteria.
- Received shipment confirmations and invoices from clients and coordinated with their representatives to ensure payments were made in a timely fashion.
- Worked at the warehouse of regional mail and freight carrier.
- Prepared all packing materials for items as they came in to help ensure safe delivery.
- Loaded items on to trucks, planes, and railway containers in preparation to be shipped.
- Participated in internship program offered by regional freight carrier.
- Worked under logistics manager assisting with the management of shipping routes and weekly supply orders.
- Studied the processes used by company's web development department to maintain online tracking portal.
- Worked part-time for local grocer helping to maintain stock room operations while attending high school and college.
- Tasked with unloading grocery shipments and storing items in appropriate areas.
- Assisted the grocery manager in maintaining grocery inventory.
Avid University of Colorado sports fan who contributes football and basketball articles to local blog. Enjoy winter sports, especially snowmobiling and snowboarding. Travel to Montana with family every fall to go bow hunting and fishing.
Build Your CV
Logistics Assistant CV Must-Haves
What Does a Logistics Assistant Do?
The logistics assistant CV example provided here gives you a good idea of what such a document should look like as well as an understanding of the type of information it should contain. However, in order to complete your own, you must also have a general idea of what sorts of roles and responsibilities come with the position you're seeking. This knowledge will help you see what areas of your work history you should highlight in your CV. Logistics assistants usually work directly under a company or organization's logistics manager, performing support tasks such as tracking shipments and item receipts, monitoring inventory volumes, and working with financial and budgeting analysts on both purchase orders and client invoices. Another assigned task may be to manage a company's warehouse operations. A logistics assistant may also be given administrative duties, such as coordinating payroll and performing employee evaluations.
Tips for Creating a Great Logistics Assistant CV
Some may tell you that since your CV is all about you, there's no way that you can go wrong with it. This assumption fails to acknowledge that your CV is actually all about the job that you're seeking, and why you are the ideal fit for such a position. To keep your document focused on that message, remember the following tips when creating your CV:
– Tailor the message to the job. Read the posting multiple times and study the company's website to get an idea of its background. Use this information to customize whatever elements of your CV you can.
– List your work experience chronologically, and detail your educational history by listing the highest degree that you've obtained first (even if it might not directly apply to the job you're applying for).
– Don't use too many technical terms when describing your skills as these may not be familiar to human resource personnel.
– Don't include any information that could alienate a reader, such as subtle hints as to your political views or religious affiliations.


text: 4 1 2 3 4 4 Ben From Billy Talent Rapid Fire #JingleBallNorth - What's Your Favourite Holiday Song? Hedley Wraps Up 2016 & Looks Forward to the New Year Alessia Cara - What Did You Learn On Tour With Coldplay Alessia Cara's First Concert Niall Horan - FULL Interview from Jingle Ball North Niall Horan's Love for Canada Niall Horan - Who Hears Your New Music First? Kirk Hammett of Metallica Talks Music and Geek Culture Kardinal Offishall - The State of Hip Hop in Canada Kardinal Offishall - The State of Hip Hop in Canada Shaun Frank - Writing Closer Alx Veliz on his plans for 2017 Alx Veliz on his key Holiday Clothing choices Kardinal Offishall gives us tips on how to throw a killer Holiday party! Kardinal Offishall on the Key To Longevity Kirk Hammett of Metallica on Playing Small Venues If you were an Olympian, what sport would you participate in? How attractive is Prime Minister Justin Trudeau? 4 1 2 3 4 4


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Home » review » Tervis Review and Giveaway
Tervis Review and Giveaway
5 comments:
I love...love Tervis tumblers !! Matter of fact, I just bought my mom a cardinals one. I guess you can tell I live in the St. Louis area !! :) Gosh, hard to tell which one I would choose, I like them both for various reasons. The fish one is really neat, it would look like you had fish in your water while you were drinking. The Mum's one is just beautiful. Thanks for the great opportunity !! :)ReplyDelete
- AnonymousSeptember 26, 2013 at 2:55 PM
These are also for cold beverages, as they do not condensate.ReplyDelete
How much water do I drink? Not enough but I'm trying to improve.ReplyDelete
Those are really coolReplyDelete
- Chris - @nylonlover69 on TwitterOctober 3, 2013 at 8:45 AM
Bummer! Looks like I just missed this one!ReplyDelete
Please leave me a comment and let me know what you thought of this post! I read and appreciate all comments.


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By Jason Deign
Younicos, the battery system developer bought by Aggreko last year, launched an energy storage rental service on the first day of Energy Storage Europe yesterday.
Read more →
By Jason Deign
Younicos, the battery system developer bought by Aggreko last year, launched an energy storage rental service on the first day of Energy Storage Europe yesterday.
Read more →
By Jason Deign
Media reporting of Aggreko's purchase of Younicos this week highlighted increasing mainstream interest in energy storage players and markets.
Read more →
By Jason Deign
Combining battery storage with solar generation is already cheaper than using diesel in most microgrids, said experts at an Energy Storage Report webinar last week.
"As the key applications, we see islanded grids or microgrids, [where] PV and batteries are becoming cost-competitive with diesel," confirmed Valts Grintals from Delta Energy & Environment in the event organised alongside Ata Insights.
A case in point is France, where interest in solar plus storage is highest in islands and "non-interconnected zones," where around 60% of PV projects so far have been installed with batteries, Grintals said.
This interest is set to grow following the launch of a tender last year for almost 52MW more of island-based solar capacity, spread across 33 projects. "We expect to see more storage coming into these projects," said Grintals.
"In France, PV and storage is a great solution to stabilise the grid [and] help enable microgrid use, and it is already cost competitive."
Read more →
By Jason Deign
A project on Graciosa, Azores, has become key for the credibility of island-based storage following concerns over another plant more than 1,500km away.
The Younicos project on Graciosa is set to go live within weeks amid speculation that another attempt to power an island off renewables, in El Hierro, Canary Islands, has failed to meet expectations.
El Hierro's Gorona del Viento plant, which combines an 11.5MW wind farm with a pumped hydro storage system, was launched with much fanfare in 2014. Its initial aim was to replace 80% of diesel generation needed for the island grid.
Last month, the plant operator revealed the EUR€82m Gorona del Viento had allowed El Hierro to run continuously off nothing but renewable energy for 55 hours.
And last week Gorona del Viento said the plant supplied 67% of the island's power throughout July and had set a new record of 76 hours with 100% renewable production.
Read more →
By Jason Deign
Islands converting power supplies from diesel to renewables could be role models for the global transition to renewable energy, according to Younicos co-founder Clemens Triebel.
"If we're aiming for a high percentage of solar and wind energy, the expansion of renewables has to be matched with sufficient storage capacity from the start," said Triebel in an Energy Storage Europe press release.
In contrast, said Triebel, the German electricity system is still discriminating against energy storage in favour of conventional power plants.
"We still produce power according to a 19th century paradigm which holds that energy is best produced by large generators that rotate constantly," he commented.
"If we continue to cling to this notion, the grid will continue to be taken up by coal, gas and nuclear, blocking space for solar and wind energy more and more frequently."
Read more →
Recent battery management system advances are increasingly proving that the key to cost-effective energy storage might not be what you use… but how you use it.
Last year, for example, Energy Storage Report unveiled news of a French start-up called Enerstone, which is commercialising an active battery management system that can extend the lifespan of batteries by up to 30%.
The system monitors the health of each cell and redistributes loads so that "weaker ones work less than stronger ones, to extend the battery life to the benefit of the user," explained Enerstone's president and co-founder Alexander Chureau.
This month, meanwhile, a senior executive at Greensmith unveiled that its energy storage management systems had made it possible for lithium-ion batteries to beat lead-acid not just on performance but also on cost.
In one particular project, "the deployment of lithium-ion was half the price of lead-acid over the lifetime of the system, and it's entirely due to software," Leesa Lee, senior vice president, product management and marketing, told Energy Storage Report.
Read more →
Is investment in energy storage worth the effort? Didn't we find out last week that our industry is going nowhere because of the fundamental constraint of its energy return on investment (EROI)? Perhaps we had better take another look, just to be on the safe side.
First off: let's not panic. While EROI studies point to a possibly critical problem in relying too heavily on energy storage for renewable power generation, the effects, if real, are presumably only likely to kick in at relatively high levels of penetration.
We are a long way off that yet. Meanwhile, there is the fact that the science around EROI, while apparently robust, is still relatively immature and clearly evolving, as indeed are the technologies and manufacturing processes being described.
This potentially implies uncertainty around current EROI assertions and predictions.
Certainly, most sector professionals consulted by Energy Storage Report had few qualms about dismissing the research: "BS" and "hokum" were among the terms used by knowledgeable industry insiders.
Some observers question the impartiality of parts of the research to date.
Read more →
Battery storage is usually seen as being a handy adjunct to help renewable energy square up to traditional power plants. But on frequency regulation, at least, it seems batteries may actually be better than the generation sources they are helping to supplant.
That, at least, is the consensus emerging from early operating experience at Europe's largest commercial battery power plant, the 5MWh lithium-ion facility that WEMAG has commissioned from Younicos in Schwerin, Germany.
"Whereas coal-fired and other thermal plants typically take up to 30 seconds to adjust production up or down, and then just hit the neighbourhood of where they are supposed to be, batteries react within milliseconds," explains Philip Hiersemenzel of Younicos.
The fact that batteries might beat traditional power plants for frequency regulation has long been touted as a big selling point for battery storage and has already helped sell projects in the US.
Now the performance of WEMAG's installation is strengthening that business case.
Read more →


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Us: "Save 20% sitewide with code YUP20." You: ":D"
moon nature sky
wow… cloudless—how often does that happen? Great shot.
thank you. I took this picture on a very foggy day too! the fog was low and I had this great shot over it.
thank you
this is an amazing shot so peaceful
fantastic capture.. well done
Thank you Jeremy
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Give me for friend the man whose friends are few;
Who, though his heart be clean and stanch and good —
Though every fibre of his soul be true —
Is tactless, blunt, and seldom understood.
In such a drift God oft conceals a lode
Whose richness makes Golconda's wealth seem naught;
On such a one He ofttimes has bestowed
Large worth so hid it must be shrewdly sought.
So, while the rabble fawns on him whose friends
Are as the sands that rim the ocean's blue,
I choose the best of all that heaven sends —
Give me for friend the man whose friends are few.
— Strickland W. Gillilan.
Bassett's scrap book (Boston, Massachusetts), vol. 4 no. 1, March 1906, p. 1
Strickland W. Gillilan, Including Finnigin: A Book of Gillilan Verse, Philadelphia: Pearson Brothers, 1908, p. 93
Notes by Andrew Guild:
Strickland Gillilan was born in 1869 in Ohio (USA), and died in 1954. "Rediscovering Strickland Gillilan" by Kate Maynard has some more information about the poet.


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So I've been working on a video to upload on Youtube with some friends of mine! I'll post the link to it as soon as we are able to get it up and working! I curious to see if it gets any views or comments.
The following is a link to my channel: http://www.youtube.com/user/KriRip. Feel free to check it out and look at some of the videos I've liked and the other users I have subscribed to. This picture is a snapshot from my channel - it's a small sample of what it looks like!
I have currently been researching and looking into what the most famous videos on Youtube are. Most of them are music videos, babies/animals and surprising talent. For example, popstar Justin Beiber was found from a video of his on Youtube and now his music video of "Baby" has been the most viewed video on Youtube. Some other examples of extremely popular videos are ones such as "Charlie Bit my Finger" and the "Evolution of Dance" videos. What amazes me is how some videos become popular. Not to offend anyone who finds it hilarious, but I never understood why "David After Dentist" got the popularity that it did. I have attached the link here: http://www.youtube.com/watch?v=txqiwrbYGrs in case no one has seen it, however I'm sure you all have!
I am giving my presentation on Social Networking and Media tomorrow in class. My question for you all is: what do you think the effect on the music industry is because of the free access we have to watching music videos online? Do you think that there should be rules/regulations/fees to access these types of videos aside from the advertisements that we have to watch prior to viewing?


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okie people feel free to play the characters available..! ^^
those who have important roles please stay ACTIVE
[U][B]Characters Up For Grabs
People from hell
Enma Ai: Taken Norine07
Ichimoku Ren: Taken DiraChan669
Hone Onna: Taken Toya999
Wanyuudou: Taken Kokuyu
[U]Normal People
Hajime Shibata: Taken NeverEnd
Tsugumi Shibata: Taken Lionhearted911
Spider: Taken {Eziara}
Grandmother: Taken LilLaoRyo704
other people are welcome to join..! ^^ you can request for vengence..! ^^
people with small roles can play those who seek for vengence as well..! ^^


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The Official Bottomless Hole Game Awards Of 2018
2017 is over. Dead. Fully cremated. It's ashes are swirling in the breeze, getting in our eyes and blinding us. It has been quite a year to say the least. I, for example, bought a new cardigan. It's been mental. But when I haven't been ducking nuclear fire or tattooing 'Strong & Stable' on to various body parts I just about found time to play some video games. So, despite overwhelming public indifference, here is the Official Bottomless Hole Video Game Awards 2018:
Yes, technically speaking 'Hitman', was released last year. However as it got a psychical release in January 2017 I decided to include it in this list. Got a problem with that? What you gonna do? Dress up as a chef and poison my food? Attach explosives to my prized set of golf balls? Don the attire of my masseuse and snap my neck mid-massage? Do your worst.
Agent 47 is a bastard. Sure you could argue the people he kills deserve to die, but he clearly gets a sick kick out murdering his targets in the most darkly comic fashion possible. Like a sadistic Benny Hill. Maybe he is lashing out because of deep seated anger issues relating to his chronic alopecia and stupid neck barcode. I suppose if I looked like a waxed sex doll maybe I would want drop lighting rigs on top of people. Regardless of his hair situation, Agent 47 is a bastard. A glorious, glorious bastard.
Remember Prey? Remember when it was teased at E3 as some form of hairy-armed-man-drinking-coffee simulator? Remember when it looked vaguely intriguing? Unfortunately when 'Prey' released it became apparent quite quickly this was another standard 'choose how you play' semi-open world faux-rpg stealth shooter. A classic genre. The problem these games always have is creating levels and enemies that compliment multiple sets of play-styles and not making the player feel like they are being punished by the way they have chosen to play. If you want to see this done well then look no further than last year's Dishonored 2.
Prey fails on this level. My only vague memories of the games is a blur of spanner flailing while trying to hit endless amounts of spiders. Whichever style you play as, the game just isn't fun or engaging on a mechanical level. The combat is too cumbersome for it to work as a shooter, the environments are too narrow and cramped for it to work as stealth game and the hacking...well nobody ever actually plays these games with a hacking build anyway. All in all, no.
Ah 'Dark Souls', the main reason for my inevitable crippling RSI. This year saw the release of the final piece of DLC for the final game in the 'Dark Souls' series. The 'Demon Prince' isn't the best or even the hardest in the game, but it still made me angry enough to flay a live kitten.
Taking place halfway through the 'Ringed City DLC', you drop into a large arena to be greeted by an earth-shattering scream. Two health bars appear before you, one for the 'Demon of the Deep' and one for the 'Demon in Pain'. This pair, who sound like they were named after the two halves of 80's heavy metal double album, are pretty difficult to begin with, working in tandem with their wide-ranging attacks. But of course, 'Dark Souls' being 'Dark Souls', once they have both been slain their remnant transform into the 'Demon Prince'. Complete with fire breath, mouth lasers and stabby claws, this guy is an absolute prick. Swipe left.
Mario...in the real world...
There were a lot of weird goings on in 2017 gaming. The excellent 'Little Nightmares' concocted some suitable nightmarish creations including the twisted tv-loving Janitor and the gluttonous Chefs. The bizarre fates of the Finch family in 'What Remains Of Edith Finch' are both harrowing, heartbreaking and hilarious. However, these pale in comparison of the horror of Super Mario meeting regular human beings.
In the New Donk City level of 'Super Mario Odyssey' our titular Euro-plumber takes a break from turtle genocide to bounce and fling himself around Nintendo's version of New York City. Seeing Mario next to anatomically correct human beings is just...wrong. Any rational person upon seeing this mustachioed monstrosity would scream until their lungs exploded. In the real America it would take approximately 2.6 seconds before he was shot to death by the police. Trump would enact an Italian ban.
Luckily this fact is slightly negated by the fact that this level, much like the rest of the game, is excellent fun. It also means that in the Mario universe there is proud British county called Donkshire. I for one would like to live there.
Our cultural obsession with nostalgia reached a peak this year as a large section of the Western got all misty-eyed about the Nazis and decided that much like Jumanji they needed a 21st Century reboot. It's a sad indictment of our political discourse that making a game about murdering hoardes of Nazis is considered even remotely controversial. But here we are.
'Wolfenstein 2: New Colossus' is fairly run of the mill first-person shooter. It doesn't feature any dramatic twists to the formula that last year's 'Doom', 'Titanfall 2' & 'Overwatch' had. The shooting is satisfying, the stealth system is bare-bones but efficient, and the writing is surprisingly good. But that doesn't matter. In these traumatic times sometimes you need to channel your anger thoughtlessly plough-through hordes of fascists. Just like your Mum does. That is a sex joke. So if you want to do your bit for society and destroy and racing-hating oligarchy from the comfort of your own home this is the perfect solution. Sharpen your hatchets and charge up your laser beams it's Nazi hunting time.
So much for the tolerant left eh?
There absolutely no alternative option here. If you see any publication giving their GOTY to anything other than 'Breath Of The Wild' then you can perform a citizen's arrest on them as they are committing a hate crime. This is the pinnacle of open world gaming, a game so good I'm really going to struggle to make snide joke about it.
If you want a full break-through of why the game is so special you can find my previously blog entry on the subject. What I will say though is that the open-world is so fully and brilliantly realised that I haven't been able to bring myself to play another open-world game since. What's the point? Nothing will be as good. For me, this is the sign of a special game, and one which should studied like an artifact from history. It is up there with the Turin Shroud as one the holiest relics in human civilisation.
Hands down 'Breath Of The Wild' is the best game of the year. In fact, 'Breath Of The Wild' is not just the best game of 2017, it is the best thing of 2017. Better than the shock election result, better than Martin Shkreli getting sent to jail, better than when that kid flounced into their Dad's office while he was giving an interview about North Korea to BBC News. I didn't think I was capable of love until this game came along. I want to distill the game's essence and inject it into my veins. You could murder me now and I would mind because I realised that my life peaked the second that I had slain Calamity Ganon. It's good, that's my point.


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When this azalea begins to bloom each year, I confess to thinking it's a bit 'peely-wally' as they say in Scotland. Washed out. But as the days go by, the colour comes out, more and more vibrantly, and now, as you can see, it reflects the very glory of God. It is stunning.
What you can't see perhaps is that around its base there is a jungle of ground elder and buttercups and who knows what other insidious weeds, seeking to choke the life out of the azalea, or, at the very least, leach away its beauty. Despite the weeds (yes, I do intend to get out there and clear the space, one of these days ...) the azalea continues to amaze with its beauty.
Yesterday I had the privilege of visiting and praying with someone whose physical well-being is threatened. There is, however, no outward sign of the struggle going on within. Rather, she radiates the glory of God. Faith and love and grace drip from her every word. Peace emanates from deep within. We were there to minister to her, but she, in fact, ministered to us.
The heavens declare the glory of God. All creation declares his glory. And so do many believers who shine like beacons; as the darkness deepens, their inner light intensifies, leading others to safety and faith. May God bless this dear lady today, and continue to heal and bless her.


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Nothing inspires and motivates as much as a quote from someone who has been there and done that but what we love about writing quotes is that they are not always inspirational. Sometimes they are just intense and miserable… oh the life of a writer!
Favourite quotes by writers
"Writing today is like being stood stark naked in Trafalgar Square and being told to get an erection." – Louis de Bernieres, Captain Corelli's Mandolin
"This is the practice school of writing. Like running, the more you do it, the better you get at it." – Natalie Goldberg
"Everyone has talent. What is rare is the courage to follow the talent to the dark place where it leads." – Erica Jong, American Novelist and poet
"It is impossible to live without failing at something, unless you live so cautiously that you might as well not have lived at all – in which case, you fail by default." ? J.K. Rowling
"If sex and creativity are often seen by dictators as subversive activities, it's because they lead to the knowledge that you own your own body (and with it your own voice), and that's the most revelatory insight of all." – Erica Jong, American Novelist and poet
"I have nothing to offer anybody except my own confusion." – Jack (John) Kerouac, American novelist and poet
"I am a feminist, and what that means to me is the same as the meaning of the fact that I am black: it means that I must undertake to love myself as though my very life depends upon self love and self respect." – June Joran, American poet and essayist
"I therefore solemnly declare to all young men trying to be writers that they do not actually have to become drunkards first." – James Jones, American writer
"Pray inwardly, even though you do not enjoy it. It does good though you feel nothing even though you think you are doing nothing." – Julian of Norwich, Lady Julian
"For a life of sensations rather than of thoughts!" – John Keats, Poet
"When we write down our thoughts, they become our history" – Diane Corriette, English writer and poet
"Everything has its wonders, even darkness and silence, and I learn, whatever state I may be in, therein to be content."
– Helen Adams Keller (American Writer who became deaf and blind at 19 months but educated by Anne Sullivan she learned to speak, graduated in 1904 and became a distinguished lecturer and writer)
I prefer the absurdity of writing poems to the absurdity of not writing poems. – Wis?awa Szymborska
"Never anticipate tomorrow's sorry; live always in this paradisal now." – Omar Khayy'am, Perisan poet and scholar
"The deepest thing I know is that I am living and dying at once and my conviction is to report that dialogue."
– Stanley Jasspon Kumtz, American poet
"Two men look out from the same bars one sees the mud, and one the stars." – Fredreick Langbridge, English religious writer
"Always too eager for the future we pick up bad habits of expectancy." – Philip Arthur Larkin, English poet and novelist
"Powerful men often succeed through the help of their wives. Powerful women only succeed in spite of their husbands." – Linda Lee-Potter
"My imagination makes me human and makes me a fool; it gives me all the world and exiles me from it." – Ursula le Guin
"How sick one gets of being 'good' how much I should respect myself if I could burst out and make every one wretched for 24 hours." – Alice James, American diarist
"Money's a horrid thing to follow, but a charming thing to meet." – Henry James, American born British novelist
"To say, 'Well, I write when I really get into it' is a bunch of bull. Put the paper in the typewriter, stare at it a long time, get snowblindness if you have to, but write something." – Erma Bombeck
"It's better to entertain an idea than to take it now to live with you for the rest of your life." – Randall Jarrell, American Poet
"Time doesn't appear from nowhere, you have to make it." – Maeve Binchy, Irish novelist and playwright
"We write to taste life twice, in the moment and in retrospect." – Anaïs Nin
I hope you enjoyed these favourite quotes by writers and if you have any that inspire you it's a great idea to type them up and put them on your wall. They can really help with motivation.


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The project will use e. Go's electric cars and will be implemented in a new parking space on the RWTH Aachen university campus.
This MoU is for joint efforts in the development of electric vehicles (EV) through the exchange of information and technology.
Toyota is a conservative firm and the company will not change its stand on Android Auto until it is satisfied that user privacy was upheld.
The new car has received cosmetic upgrades to mimic the design of the recently launched LS sedan.
The car is effectively the upcoming SE Asian market BRio in a sporty body kit
Ducati will offering cornering ABS powered by Bosch on its complete range
The car is powered by a new K15B 1.5-litre petrol motor and has been redesigned from the ground up
Equipped with reworked exhaust, cold air intake, new high-flow fuel pump, performance-spec fuel injectors and a larger supercharger
The Malaysian market car only gets the 2.0-litre Ingenium petrol in 250PS/365Nm tune for now
Oriol Mena best placed Hero MotoSports Team Rally rider, ranked 16th overall
The rallies they will compete in, the machine Gill will drive, and MRF's future in the championship possibly as an official tyre supplier
The India Design Mark (I Mark) is conferred by the Indian Design Council that is an autonomous body of Indian government operating under the...
Is powered by the 3.0-litre twin-turbo straight six from the M3/M4.
The RS5 downsizes it's engine, but upsizes the fun quotient
The Japanese manufacturer enters the lucrative middleweight cruiser space
The newest addition to the list of good looking, 'chopped' motorcycles is, well, an American!


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HOTEL ASSIGNMENTS – Reservations for the three (3) program nights of September 19, 20 & 21, 2018 ONLY will be made for each Buyer (unless an option for hotel is not taken) by William H. Coleman, Inc. (WHC, Inc.) upon receipt of your registration form and payment.
Hotels will be assigned to Buyers in late June. If you do not know your assigned hotel, you can login to www.mywhcoleman.com and select "View Details" from the drop-down menu under the "Actions" column, or contact our Sales Department for your assigned hotel – E-Mail
NOTE: Accommodations/Rates DO NOT include breakfast (unless noted/advised by hotel), service fees, gratuities, taxes, or incidentals.
EXTENSION RATES FOR BUYERS – For accommodations prior to or after the program dates, Buyers must make reservations directly with their assigned hotel. The rates below are for all participating hotels and are valid 3 nights prior (September 16-18, 2018) and 3 nights after (September 22-24, 2018) the program dates – based on availability. At the hotel's request, you may be required to email a copy of your confirmation letter as verification of registration for the event.
We do not recommend to wait until the deadline to confirm your additional hotel nights since many hotels will fill up fast. The deadline for making additional night reservations at the rates below is August 1, 2018. Thereafter, reservations will be based on rate and space availability. Rates are in US Dollars.
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The picture shows Grandad Dale in 1951 (when I was nine) with my father and mother (Alf and Nell). I'm not sure where it was taken, but it looks like we were dressed up for the occasion!
All my best mates at that time were boys (boys and girls didn't often play together, in those days, apart from brothers and sisters). Girls were socialised to a different and lesser role in society, right from a very early age, and boys, in turn, were socialised to a more dominant and entitled role. Looking back on it, I realise that, although our parents were very advanced in much of their thinking, they conformed to the norm of the day at least in this respect. For example, my brother Max and I went to boarding school (to get a better education), but they didn't see this as necessary or appropriate for my sister Lynne.
So, you'll see below, that practically all of my mates (cobbers, as we sometimes called them) were boys.
Probably my very best mate was Robin Hargraves, who lived at 12(?) Whaka St. In those days, Whaka St was called "the old railway track", or something like that (I think it was originally a droving track). My older brother Max once tried to get all the way down the track to the railway station on horseback, but didn't make it. He gave up after riding through some bushes too quickly, straight into a clump of bush lawyer.
Now, bush lawyer is a vine with lots of thorns that hook onto your clothes. The old joke is that (like a lawyer) it won't let go of you until it has drawn blood. So, poor Max got entangled in the bush lawyer, the horse kept going, and Max ended up hanging from the bushes. When we heard the story, we thought it was very funny, even if he didn't!!!
The Stanaways lived in the house just past the Hargraves at 14(?) Whaka St. Their daughter, Marilyn, was a good friend of my sister's. One day, when they were quite young, I heard them chatting in the basement of our house, and Marilyn was telling Lynne that Santa Claus didn't really exist. I tell you, Lynne's face was literally white with shock. Fortunately I was able to explain that Marilyn was mistaken and that of course Santa existed. (I can't imagine where Marilyn got the silly idea from. Maybe she misunderstood something her parents said.)
To sidetrack for a moment, it was Whaka St I walked up on the day I ran away from home. I was about three or four, at the time, and had become seriously disenchanted with my mother. Mum did what good mothers throughout history have done when their toddler threatens to run away from home; she helped me pack my bag (with an apple and a soft toy, I think). Off I went, trudging resentfully into the twilight. Mum, of course, had rung ahead to "Auntie" Beryl (Robin Hargraves' mum), and by the time I'd gone the hundred metres to their house I'd calmed down enough to be coaxed home. I've no idea where I thought I was going, but it was a great adventure.
Another good mate was Terry McMahon who lived in a newly built house behind the Houghtons. If I remember right, the McMahons were catholics, which was something I hadn't come across before (our family wasn't religious, so being friends with a catholic was quite esoteric)! I remember they went to mass only once a month, because the nearest Catholic church was so far way away.
On one occasion, Terry and I had this ginormous game of monopoly that went on for days. At the end of each session we'd leave the board set up on their lounge floor and next day, after school, we'd resume battle. However, it all ended in extreme acrimony. I don't remember why, but we got so upset with each other that I set off home in a huff (yelling insults back at Terry) and he got so incensed that he threw a huge rock at my retreating figure. Well, maybe it was only a medium‑sized stone, but it did gash my head and there was blood everywhere. I can't remember whether that was the end of our friendship or not, but I imagine we got over it soon enough.
Another friend was the McMurchy boy (I can't remember his first name). His father owned a hardware(?) shop a few doors away from us (it might have been where the supermarket is now). Anyway, one day, when we were very young, he talked me into stealing a toy from his father's shop. (I swear it was his idea, not mine!!!) Anyway, we did the dastardly deed (we stole one of those "invisible ink" sets) and made a clean getaway, or so we thought. However, next day mum and dad started gently questioning me and eventually the whole truth came out. The toy was returned and apologies made to Mr McMurchy.
(Obviously, he had seen what was going on but, instead of nabbing us in the act, had kindly had a quiet word with my parents, who left me in no doubt that I'd done wrong. I don't know what he said to his own son!!!)
Most of my other mates were farmer's sons. Bradley McCrae lived on a sheep farm up on Griffin Road. He was a bit younger than me, but we both loved riding horses. It was on their farm that I first saw an aeroplane. It was a Tiger Moth and I think it may have been doing the first aerial topdressing in Maungaturoto.
The Snellings owned the sheep farm at the bottom of the Brynderwyn Hill (on the Maungaturoto side, just opposite where Atlas Quarries are now), and their son Doug was one of my very best mates. I had great adventures out there, and also fell hopelessly in love with his sister Una (ah, unrequited love). I'll tell you more about Doug and Una in later blog entries, when I talk about horse riding and dances.
The Dreadons were dairy farmers who were close family friends. They lived out on Mountain Road. Brian was more my sister Lynne's age, but I met him again many years later, in Wellington, and we became friends again. The photo (with Brian on the left) was taken much later, in 1983, when I was 41.
And, of course, there were the Houghtons, who lived across the road from us at 196(?) Hurndall Road. Their children (Len, Maureen and Ray) were good family friends, but none of them were my age, so they weren't really mates, but I was often across at their place.


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"Card sense is the ability to do the right thing at the wrong time or the wrong thing at the right time. It's something you can't buy and you can't find; you're born with it."
- Barry Crane (1927-1985)
The most successful matchpoint player of all time, the late Barry Crane was a master at pushing his opponents into bad decisions. His bidding style, described as "light, but disciplined", was to get in the auction early and often to direct leads and disrupt enemy communication. He was famous for his thin opening bids and overcalls, and his opponents would have had far more tops if they had doubled him more often.
Few have Crane's talent for sensing when it's right to make a "wrong" bid. If you hold a borderline hand, your decision depends largely on your estimation of the risks vs. rewards of bidding vs. passing. You can improve your results if you also consider which types of auctions tend to be dangerous and which offer the best odds for a gamble.
In general, the best conditions for aggressive overcalls are when you're not vulnerable, the opponents have a trump fit and they haven't yet shown their full strength. You're more likely to suffer a big penalty if you enter the auction late, after the opponents have exchanged information. They'll be more anxious to double if they've already determined that they don't have a good trump fit or game values.
One-level overcalls
These aren't often penalized, especially not vulnerable, and they can have obstructive value. Your opponents will usually have an easier auction if they can make natural, low-level bids, so you may gain an edge by forcing LHO to start with a negative double. A light overcall, though, can also mislead partner. It may not be worth the risk unless it also uses up bidding space.
After 1C on your right, consider overcalling 1S with as little as
When in doubt: Stretch to overcall 1S if your RHO opens a minor. Overcall a light 1D or 1H only if your suit is good enough that you want it led. Be more conservative when you're vulnerable.
Two-level overcalls
These are frequent problems for those who follow the "5 and 10" rule, which means they bid in direct seat with just about any 5-card suit and any 10+ points. A vulnerable two-level overcall is dangerous because doubling can be so profitable. Extracting a penalty is also fairly easy, since opener will stretch to reopen with a double if your LHO passes your overcall.
The minimum for a vulnerable two-level overcall should be a good 6-card suit
and at least 10-11 points or a very strong 5-card suit and 14+ points. After 1S
on your right, overcall 2H with
You can shade this down if not vulnerable, but it's still important to have a
good suit. At any vulnerability, a pass is the wisest choice over RHO's 1S
opening if you hold
When in doubt: Ask yourself what you would rebid if you overcall and partner advances with 2NT. If you'd have to pass 2NT -- that is, you don't have enough strength to raise to 3NT or enough length to rebid your suit -- don't make the overcall.
Overcalls of their 1NT
A strong 1NT used to effectively silence the opponents, but no more. Today's players are more willing to compete for the contract and force the opponents to alter their response structure.
For an overcall, length and strength in your suit are much more important than overall strength. If you're considering a penalty double, you need an attractive lead and a source of tricks, not just a lot of high-card points.
When in doubt:
Look for excuses to make a non-vulnerable overcall if you have a six-card suit or a two-suiter.
Be conservative with balanced patterns. Don't make a penalty double with a balanced hand unless you have a rock-solid 18 points or more.
Know your opponents' system. If they play negative or "stolen-bid" doubles after opening 1NT, you can be freer with your overcalls.
© 2012 Karen Walker


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Amy Winehouse
Mutya Buena
Mena Suvari
Mena Suvari has the phrase 'Word, Sound, Power' tattooed on his back and what looks like a squiggle on the front. Actually it is a '13' from '13 lucky ', apparently, rather than, for example, "third line of contact." We're pretty sure that "13" is an unlucky number, but what the hell. Simone's husband Sestito Mena has a match of '13', making the TATT deliciously romantic. Coincidentally, the Hell's Angels used to get the number "13" sewn on their jackets to signify his love of 'm' (the thirteenth letter of the alphabet) of 'marijuana'. Just a coincidence, however, we are sure.
Miley Cyrus
Miley might seem like a slight young thing nowadays, but, what with the tattoos and the new rock look, she'll soon morph into Lindsay and eventually one day go all Britney. And it'll be awful.
Tags : Tattoo Artist, Tattoo Artists


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The official Final Fantasy Youtube channel has updated with the launch trailer for Dissidia Final Fantasy NT. The trailer features a glimpse at some of the gameplay. Dissidia Final Fantasy NT is now available for the Playstation 4. Source: Final FantasyRead More
Dissidia Final Fantasy NT Intro Cinematic Streamed
The official Playstation Youtube channel has updated with intro cinematic for Dissidia Final Fantasy NT. The intro cinematic features Materia and Spiritus in a clash between the forces of good & evil. The channel also updated with a cutscene featuring Noctis from Final Fantasy XV. Dissidia Final Fantasy NT will be released in Japan on January 11 and wordwide on January 30, 2018. Source: PlaystationRead More
Golbez to Join Dissidia Final Fantasy Arcade Roster
Square Enix has announced that they will be adding Golbez to the roster for arcade version of Dissidia Final Fantasy. For those who are unaware Golbez is the one of the main antagonists from Final Fantasy IV. Players who match the conditions during the "Conflicts of Gods" event will be available to access the character early on November 16. The event will start on November 9 and the character will be available for all players late November. A new stage called the "Stellar Fulcrum" based on the location from Final Fantasy XI will be added to the November update to the arcade version of the game…Read More


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Dietitians believe in the power of food to enhance lives and improve health, without fads and gimmicks to deliver life-changing advice.
Dietitians empower their patients, clients, and communities to embrace food, to understand it, and to enjoy it. The advice and information they provide is tailored to their clients and patients personal needs and challenges, including taste and accessibility. They are regulated health professionals that undergo comprehensive training to allow them to translate the science of nutrition into terms everyone can understand.
Find out more here about dietitians and the difference between a dietitian and a nutritionist.
Food Portions Toolkit
My Menu Planner
Provides guidelines for parents and caregivers on breastfeeding and introducing solid foods to babies.
Breastfeeding is best for you and your baby. If you choose to use infant formula, here is some information on the different types available.
How To Use Different Oils When Cooking
Easy Meals for Great Leftovers e-book
Diabetes Menu Plan


text: tag:blogger.com,1999:blog-8767377518971394262.post2269435226544449524..comments2017-07-04T08:29:58.903-07:00Comments on Capes & Crowns: Capes & Crowns- 2013 Pottery Barn Mystery Box Challenge WINNER! Lauren firstname.lastname@example.com:blogger.com,1999:blog-8767377518971394262.post-6796029961037356212016-01-06T05:38:48.176-08:002016-01-06T05:38:48.176-08:00I'm reading this post again over two years lat...I'm reading this post again over two years later and it sinks in more than ever. At the end of the day you created all of this. With the love and support of family, friends and some talented vendors. I admire your honesty and dedication to remain true to your style and your vision. Haters gonna hate girl just keep brushing them off!Sandra Leehttps://email@example.com:blogger.com,1999:blog-8767377518971394262.post-30919822007762092462013-08-07T00:40:38.610-07:002013-08-07T00:40:38.610-07:00Congratulations Lauren- great tableCongratulations Lauren- great tableJenny Raulli-Bloom Designshttps://email@example.com:blogger.com,1999:blog-8767377518971394262.post-67556699009019959502013-07-05T20:46:27.036-07:002013-07-05T20:46:27.036-07:00Congratulations! Your work is gorgeous and I am ha...Congratulations! Your work is gorgeous and I am happy for you.Sandra Leehttps://firstname.lastname@example.com:blogger.com,1999:blog-8767377518971394262.post-80197980590875419082013-07-05T06:40:10.819-07:002013-07-05T06:40:10.819-07:00So excited for you Lauren and I love your {realnes...So excited for you Lauren and I love your {realness}!!! You are amazing at what you do and I don't know of a more deserving individual!!! I know the post may have seemed a bit repetitious to some, but the great Muhammad Ali once stated that it's the repetition of affirmations that leads to belief. And once that belief becomes a deep conviction, things begin to happen. You have a great fan base that affirm and believe in you and because of that you were able to make things happen!!! Thank you for being an excellent example of how to make {Big} things happen!!!destinyawakeshttp://firstname.lastname@example.com:blogger.com,1999:blog-8767377518971394262.post-81128134399654828012013-07-05T06:24:48.042-07:002013-07-05T06:24:48.042-07:00First of all...CONGRATS!!! I am so excited for you...First of all...CONGRATS!!! I am so excited for you and I can't imagine how you got by all those days being in limbo with that contest! As far as the other part, that is so unfortunate! It's amazing how you think people who are part of your reliable network disappear when you need them most! I came in late to the game and tried to help the most I could, but I'm so happy you won in the end! :) Wendy Bydehttps://email@example.com


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"Fargo" amassed a body count of trophies at Saturday's Independent Spirit Awards nearly matching the cinematic body count in the darkly comic tale of a kidnapping gone awry.
The film--which is also nominated for seven Oscars--won six statuettes: best feature, best director (Joel Coen), best screenplay (Joel and Ethan Coen), best actress (Frances McDormand), best actor (William H. Macy) and best cinematography (Roger Deakins).
Joel Coen, who had avoided several other awards ceremonies at which he was nominated, and McDormand (husband and wife in real life) swayed to a gospel choir and seemed relaxed and happy, though a tad sheepish over their embarrassment of riches.
McDormand won for her portrayal of the down-to-earth Marge Gunderson, a pregnant police chief in Brainard, Minn., who solves a grisly succession of murders with good-natured ease. While accepting her award, McDormand said she believed that the depth and unconventionality of her winning role would only be possible in the world of independent films.