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Whole-Genome Gene Expression Direct Hybridization Assay Guide
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1 Whole-Genome Gene Expression Direct Hybridization Assay Guide For Research Use Only 1, , ,000 ILLUMINA PROPRIETARY Catalog # BD Part # Rev. A
3 Notice This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document. The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY. ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USE OF SUCH PRODUCT(S) OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER'S ACQUISITION OF SUCH PRODUCT(S). FOR RESEARCH USE ONLY Illumina, Inc. All rights reserved. Illumina, illuminadx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iselect, CSPro, GenomeStudio, Genetic Energy, HiSeq, and HiScan are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Whole-Genome Gene Expression Direct Hybridization Assay Guide iii
5 Revision History Catalog # Part #, Revision Letter Catalog # BD Part # Rev. A Date June 2010 Part # Rev. A November 2007 Part # Rev. A November 2006 Whole-Genome Gene Expression Direct Hybridization Assay Guide v
7 Table of Contents Notice iii Revision History v Table of Contents vii List of Tables ix List of Figures xi Chapter 1 Overview Introduction Direct Hybridization Assay Direct Hybridization Assay Workflow BeadChips HiScan, iscan, BeadArray Reader, AutoLoader and AutoLoader GenomeStudio Integrated Informatics Platform Illumina Lab Protocols Technical Assistance Chapter 2 Standard Operating Procedures Introduction Acronyms Lab Setup Lab Maintenance Safety Precautions Best Practices Standard Lab Procedures Initializing the BeadArray Reader (Daily) Tracking Tools Illumina-Supplied Equipment and Consumables User-Supplied Equipment, Materials, and Reagents Chapter 3 Direct Hybridization Assay Protocols Introduction Sample Labeling (Optional) Quantitate RNA (Optional) Hybridize BeadChip Whole-Genome Gene Expression Direct Hybridization Assay Guide vii
8 viii Table of Contents Wash BeadChip Detect Signal Image BeadChip on the iscan System Image BeadChip on the BeadArray Reader GenomeStudio Integrated Informatics Platform Appendix A System Controls Introduction Controls for the Biological Specimen (Housekeeping Controls) Controls for Sample Labeling (RNA Spike) Array Hybridization Controls Signal Generation Controls (Biotin Control) Negative Controls Appendix B Troubleshooting Index Catalog # BD Part # Rev. A
9 List of Tables Table 1 AutoLoader and AutoLoader2 Features Table 2 Illumina General Contact Information Table 3 Illumina Customer Support Telephone Numbers Table 4 Direct Hybridization Assay Acronyms Table 5 Illumina BeadArray Reader Indicators Table 6 Sample Sheet Guidelines Table 7 Illumina-Supplied Equipment for Direct Hybridization Assay Table 8 Direct Hybridization Assay Consumables Table 9 User-Supplied Equipment Table 10 User-Supplied Materials Table 11 User-Supplied Reagents Table 12 [Optional] User-Supplied Labeling Control Reagents Table 13 Concentrations of Standard Ribosomal RNA Table 14 Volumes for RiboGreen Reagents Table 15 crna Masses Used Table 16 RNase-free Water Hyb Volumes Table 17 Hyb Mix Volumes Table 18 Sample Loading Table 19 System Controls Table 20 Troubleshooting Issues Whole-Genome Gene Expression Direct Hybridization Assay Guide ix
10 x List of Tables Catalog # BD Part # Rev. A
11 List of Figures Chapter 1 Overview Figure 1 Direct Hybridization Assay Workflow Figure 2 Hybridize Strand to BeadChip Figure 3 Wash BeadChip Figure 4 Bind Probes to BeadChip Figure 5 Imaging BeadChip Chapter 2 Standard Operating Procedures Figure 6 Illumina BeadArray Reader Figure 7 BeadScan Welcome Screen Figure 8 Lab Tracking Form Figure 9 Sample Sheet Example Chapter 3 Direct Hybridization Assay Protocols Figure 10 Dilution of Ribosomal RNA Standard Figure 11 Serial Dilutions of Ribosomal RNA Figure 12 Standard QRNA Plate with RiboGreen Figure 13 Sample QRNA Plate with RiboGreen Figure 14 Load the Illumina QRNA Protocol in SoftMax Pro Figure 15 Select the Standard RNA Screen Figure 16 Read the Standard QRNA Plate Figure 17 View Standard Curve Figure 18 Read the Sample QRNA Plate Figure 19 BeadChip Hyb Chamber Components Figure 20 Hyb Chamber and Gasket Figure 21 Place Gasket into Hyb Chamber Figure 22 BeadChip Hyb Chamber with Gasket in Place Figure 23 Dispense HCB into BeadChip Hyb Chamber Reservoir Figure 24 Seal the Hyb Chamber Figure 25 Place BeadChips into Hyb Chamber Inserts Figure 26 BeadChips in BeadChip Hyb Chamber Figure 27 Secure Lid Figure 28 Adding High-Temp Buffer to Hybex Waterbath Insert Figure 29 BeadChips Submerged Face Up in Beaker Figure 30 Removing the Coverseal Figure 31 Placing BeadChip into Diluted Wash E1BC Buffer Figure 32 Transfer Wash Rack to Waterbath Insert Figure 33 Static Incubation in High-Temp Wash Buffer Whole-Genome Gene Expression Direct Hybridization Assay Guide xi
12 xii List of Figures Figure 34 Washing BeadChip in Diluted Wash E1BC Buffer Figure 35 Washing Dish/BeadChip on Orbital Shaker Figure 36 BeadChip Wash Tray on Rocker Mixer Figure 37 Placing BeadChip into BeadChip Wash Tray Figure 38 Wash Tray Lid Figure 39 Slide Rack with BeadChip in Centrifuge Figure 40 Initialize iscan Reader Figure 41 Welcome Window Figure 42 Scan BeadChip Barcodes Figure 43 Deselect BeadChip Sections Figure 44 Review Window with Failed Stripes Figure 45 Monitor the Scan Figure 46 BeadScan Welcome Window Figure 47 Select BeadChip Docking Fixture Figure 48 Enter Data Repository and Decode Data Paths Figure 49 Place BeadChips into Illumina BeadArray Reader Tray Figure 50 BeadArray Reader Tilt Process Figure 51 BeadArray Reader Align Process Figure 52 BeadArray Reader Scan Process Figure 53 Scan Completed and Scan Completed with Warnings Screens Figure 54 Exit the BeadArray Reader Software Appendix A System Controls Figure 55 Cy3-Labeled Hyb Control Diagram Figure 56 Low Stringency Hyb Control Diagram Figure 57 Signal Generation Control Diagram Figure 58 Negative Control Diagram Appendix B Troubleshooting Catalog # BD Part # Rev. A
13 Chapter 1 Overview Topics 2 Introduction 2 Direct Hybridization Assay 3 Direct Hybridization Assay Workflow 5 BeadChips 6 HiScan, iscan, BeadArray Reader, AutoLoader and AutoLoader2 7 GenomeStudio Integrated Informatics Platform 7 Illumina Lab Protocols 8 Technical Assistance Whole-Genome Gene Expression Direct Hybridization Assay Guide 1
14 2 CHAPTER 1 Overview Introduction The Illumina Whole-Genome Gene Expression Direct Hybridization Assay system (Direct Hybridization Assay) integrates Illumina s proprietary BeadArray technology, a precise microarray scanning system (the Illumina HiScan or iscan System or the Illumina BeadArray Reader), hybridization equipment and accessories, and standard, off-the-shelf sample labeling protocols. BeadArray technology involves the preparation of quantitatively pooled bead libraries, and the self-assembly of those beads into precision microfabricated substrates to deliver a true high-density microarray platform. Gene Expression profiling assays performed on Illumina bead-based arrays offer numerous advantages over other array platforms, including high sensitivity, high reproducibility, and low sample input requirements. The Direct Hybridization Assay system offers: Choice of array platforms based on research species and sample throughput needs Consistent reproducibility from array to array Hybridization-based quality control of every feature in every array prior to shipment Compatibility with industry-standard sample labeling Reduced volume requirements, resulting in lower cost per sample Direct Hybridization Assay The Direct Hybridization Assay system uses a standard Eberwine protocol assay, in which gene-specific probes are used to detect labeled crnas. Each bead in the array contains a 50-mer, sequence-specific oligo probe synthesized in-house using Illumina s proprietary oligo manufacturing technology. Probe design incorporates a number of selection criteria including: Sequence context evaluation identifying regions of a transcript compatible with probe design, based on items such as splice-isoformspecific regions, sequence complexity, and uniqueness. Self-complementarity for hairpin structure prediction Melting temperature (Tm) Distance from the 3' end of the transcript A series of controls is incorporated into the assay, accomplishing these important tasks: Assessment of success of the major steps in the experiment Information on background and noise levels Catalog # BD Part # Rev. A
15 Direct Hybridization Assay Workflow 3 In the Illumina recommended sample labeling protocol, total RNA is converted to double-stranded cdna, followed by an amplification step [in vitro transcription (or IVT)] to generate labeled crna. This process can be carried out using standard third-party labeling kits (see Illumina-Supplied Equipment and Consumables on page 20). Direct Hybridization Assay Workflow The following sections describe the overall Direct Hybridization Assay workflow for Six-, Eight-, or Twelve-sample BeadChips. Detailed descriptions of each step are described later in this manual. Figure 1 illustrates the Illumina Whole-Genome Gene Expression Direct Hybridization Assay workflow. Day 1 or Earlier Sample Labeling Hands-on: 1 2 days Reagents Labeling Kit Output crna Day 1 Hyb BeadChip Hands-on: ~1 hour Incubation: hours Reagents HCB HYB Output BeadChip Day 2 Wash BeadChip Hands-on: ~30 min Reagents Block E1 E1BC EtOH HTW Output BeadChip Quantitate RNA Hands-on: ~30 min Fluorometer: 5 min Reagents RiboGreen RNA Kit Output Quantitated RNA Detect Signal Hands-on: ~30 min Reagents Block E1 E1BC SA-Cy3 Output BeadChip Optional Cold Storage Option Overnight Incubation Fill in the lab tracking form and the sample sheet as you perform the assay Image BeadChip Hands-on: ~15 min iscan System Scan Time 24 min/beadchip BeadArray Reader Scan Time 50 min 1.25 hours/beadchip Output Image and Data Files Figure 1 Direct Hybridization Assay Workflow Whole-Genome Gene Expression Direct Hybridization Assay Guide
16 4 CHAPTER 1 Overview Hybridize Labelled Strand The labelled RNA strand is hybridized to the bead on the BeadChip containing the complementary gene-specific sequence. Probe A Probe B Probe C Figure 2 Hybridize Strand to BeadChip See: Hybridize BeadChip on page 39 Wash BeadChip BeadChips are removed from the overnight hybridization and then washed. Figure 3 Wash BeadChip See: Wash BeadChip on page 48 Catalog # BD Part # Rev. A
17 BeadChips 5 Detect Signal Analytical probes are bound to the hybridized to the BeadChip, which allows for differential detection of signals when the BeadChips are scanned. (crna) Figure 4 Bind Probes to BeadChip See: Detect Signal on page 56 Image BeadChip The Illumina HiScan or iscan System or BeadArray Reader measures fluorescence intensity at each addressed bead location. The intensity of the signal corresponds to the quantity of the respective transcript in the original sample. Figure 5 Imaging BeadChip See: Image BeadChip on the iscan System on page 59 or Image BeadChip on the BeadArray Reader on page 67 BeadChips The BeadChip platform offers the following whole-genome formats: Six-sample Eight-sample Twelve-sample Each array in the matrix holds tens of thousands of different oligonucleotide probe sequences. These are attached to 3-micron beads that are assembled into the microwells of the BeadChip substrate. Because the microwells outnumber probe sequences, multiple copies of each bead type are present in the array. This built-in redundancy improves robustness and measurement precision. The BeadChip manufacturing process includes hybridization-based quality control of each array feature, allowing consistent production of highquality, reproducible arrays. Whole-Genome Gene Expression Direct Hybridization Assay Guide
18 6 CHAPTER 1 Overview HiScan, iscan, BeadArray Reader, AutoLoader and AutoLoader2 BeadChips are imaged using the Illumina HiScan or iscan System or BeadArray Reader. Each of these is a two-channel, high-resolution laser imagers that scan BeadChips at two wavelengths simultaneously and create an image file for each channel (for example, two per array). Only the green channel is used for the Direct Hybridization Assay. The HiScan or iscan System incorporate advanced optics and sensors to support higher throughput than the BeadArray Reader, while providing equally high data quality. The iscan Control Software, also known as GenomeScan (or BeadScan, for BeadArray Reader), determines intensity values for each bead type and creates data files for each channel. GenomeStudio uses this data file in conjunction with the individual manifest file (*.bgx) to analyze the data from the assay. Loading and unloading of BeadChips into the HiScan or iscan System or BeadArray Reader can be automated with the optional AutoLoader2 or AutoLoader respectively. Both AutoLoaders support unattended processing by placing BeadChips carriers in the imaging system s tray, so that it can scan the BeadChips. Features include: Table 1 AutoLoader and AutoLoader2 Features AutoLoader AutoLoader2 Integrated with iscan Control Software Integrated with BeadScan software Integrated with Illumina LIMS alert system Single-reader or dual-reader configuration Number of BeadChips supported per carrier 2 4 Number of carriers processed at a time Catalog # BD Part # Rev. A
19 GenomeStudio Integrated Informatics Platform 7 GenomeStudio Integrated Informatics Platform GenomeStudio, Illumina s new integrated data analysis software platform, provides a common environment for analyzing data obtained from microarray and sequencing technologies. Within this common environment, or framework, the GenomeStudio software modules allow you to perform application-specific analyses. The GenomeStudio Gene Expression Module is an application for analyzing gene expression data from scanned microarray images collected from systems such as the Illumina iscan System or BeadArray Reader. Experiment performance is based on built-in controls that accompany each experiment. GenomeStudio Gene Expression Module expression results can be exported and analyzed by most standard gene expression analysis programs. You can perform these analyses on individual arrays or on groups of arrays treated as replicates. Data analysis features of the GenomeStudio Gene Expression Module include: Choice of assay analysis within a single application Data tables for information management and manipulation Plotting and graphing tools Whole-genome display of sample data in the IGV (Illumina Genome Viewer) Data visualization of one or more samples in the ICB (Illumina Chromosome Browser) Data normalization Custom report file formats Gene expression and differential expression analysis Outlier removal for negative controls Ability to combine/merge methylation or genotyping data into a gene expression project Data imputation for missing probes on an array Assay-specific controls dashboards For feature descriptions and instructions on using the GenomeStudio platform to visualize and analyze mirna data, see the GenomeStudio Framework User Guide and the GenomeStudio Gene Expression Module User Guide. Illumina Lab Protocols Illumina lab protocols are designed to promote efficiency and minimize the risk of contamination. Chapter 2, Standard Operating Procedures, describes the standard operating procedures and tools for an Illumina assay lab and explains how to set up and maintain the lab area. For instructions on how to perform the Direct Hybridization Assay protocol, see Chapter 3, Direct Hybridization Assay Protocols. Whole-Genome Gene Expression Direct Hybridization Assay Guide
20 8 CHAPTER 1 Overview Technical Assistance For technical assistance, contact Illumina Customer Support. Table 2 Illumina General Contact Information Illumina Website Table 3 Region Illumina Customer Support Telephone Numbers Contact Number North America toll-free ILMN ( ) United Kingdom toll-free Germany toll-free Netherlands toll-free France toll-free Other European time zones Other regions and locations ILMN ( ) MSDSs Material safety data sheets (MSDSs) are available on the Illumina website at Product Documentation If you require additional product documentation, you can obtain PDFs from the Illumina website. Go to documentation.ilmn. When you click on a link, you will be asked to log in to icom. After you log in, you can view or save the PDF. To register for an icom account, please visit https://icom.illumina.com/account/register. Catalog # BD Part # Rev. A
21 Chapter 2 Standard Operating Procedures Topics 10 Introduction 10 Acronyms 12 Lab Maintenance 13 Safety Precautions 13 Best Practices 14 Standard Lab Procedures 15 Initializing the BeadArray Reader (Daily) 17 Tracking Tools 20 Illumina-Supplied Equipment and Consumables 23 User-Supplied Equipment, Materials, and Reagents Whole-Genome Gene Expression Direct Hybridization Assay Guide 9
22 10 CHAPTER 2 Standard Operating Procedures Introduction This chapter explains standard operating procedures and precautions for operating an Illumina assay lab. You will also find lists of standard equipment, materials, and reagents. The assay protocols described in the rest of this guide assume that you are familiar with the contents of this chapter, have implemented all the recommendations, and have obtained all of the requisite materials. Acronyms A number of acronyms and abbreviations are used in this protocol. Refer to the alphabetical list below for definitions. Table 4 Direct Hybridization Assay Acronyms Acronym Definition ATP Block E1 C CAP cdna crna CTP DNase dntp EB GTP HCB HTW HYB Hyb IVT MAGE-ML Adenosine Triphosphate Block Buffer Celsius Coverseal Alignment Plate Complementary Deoxyribonucleic Acid Complementary Ribonucleic Acid Cytidine Triphosphate Deoxyribonuclease Deoxynucleotide Triphosphate Elution Buffer Guanosine Triphosphate Humidity Control Buffer High Temperature Wash Buffer Hybridization Buffer Hybridize or Hybridization In Vitro Transcription MicroArray and Gene Expression Markup Language Catalog # BD Part # Rev. A
23 Acronyms 11 Table 4 Direct Hybridization Assay Acronyms (Continued) Acronym Definition ml mm ng NIST nm PBS PCR pm rcf RNA RNase SA-Cy3 T7(dT) 24 TE UTP UV Wash E1BC μl Milliliters Millimolar Nanograms National Institute of Standards and Technology Nanometers Phosphate Buffered Saline Polymerase Chain Reaction Picomolar Relative Centrifugal Force Ribonucleic Acid Ribonuclease (an enzyme that degrades RNA) Cy3-Streptavidin Poly-dT Primer with T7 Promoter Element Tris-EDTA (Ethylenediaminetetraacetic Acid) Uridine Triphosphate Ultraviolet Wash Buffer Microliter(s) Whole-Genome Gene Expression Direct Hybridization Assay Guide
24 12 CHAPTER 2 Standard Operating Procedures Lab Setup The following standard lab setup procedures should be performed for Direct Hybridization Assay labs. Prevent Product Contamination Unless sufficient caution is exercised, products may contaminate reagents, instrumentation, and samples, causing inaccurate and unreliable results. Product contamination can shut down lab processes and significantly delay resumption of normal operations. CAUTION It is imperative to establish procedures for preventing product contamination before working in the lab. FIFO It is important to keep a first in, first out or FIFO system for reagents. Rotating the stock of the remaining reagents will help to avoid accidentally using expired reagents. Lab Maintenance The following standard lab maintenance procedures should be performed for Direct Hybridization Assay labs. Daily and Weekly Cleaning CAUTION To prevent sample or reagent degradation, ensure all sodium hypochlorite (bleach) vapors have fully dissipated before starting any processes. Hot spots are areas in the lab that pose the highest risk of contamination Clean these items daily with a solution of 0.5% sodium hypochlorite (10% bleach). Typical hot spots include: Bench space Door handles Refrigerator/freezer door handles Computer mouse Keyboards Centrifuges Vortexers Thermal cyclers Once a week, thoroughly clean the entire lab area, including all of the bench tops and instruments that are not cleaned daily. Mop the floors with a 0.5% sodium hypochlorite (10% bleach) solution as well. Provide training for personnel responsible for cleaning the lab areas so that they know how to prevent product contamination. Catalog # BD Part # Rev. A
25 Safety Precautions 13 Safety Precautions CAUTION The protocols described in this guide should be performed by qualified laboratory personnel only. Exercise caution when handling biological samples to avoid crosscontamination. WARNING This protocol involves the use of an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. For more information, see the MSDS for this kit, which is available at References Please visit to see the latest material data safety sheets. CAUTION Please refer to governmental and facility safety standards applicable to your site. Best Practices To optimize your data and minimize errors and waste, read and follow these best practices whenever performing the Direct Hybridization Assay protocols. Items Falling to the Floor Treat anything falling to the floor in the lab area as if it were contaminated. Disposable items falling to the ground, such as empty tubes, pipette tips, gloves, or lab coat hangers should be thrown away at the end of the day or at the completion of the assay. During the assay, never touch any items that have fallen to the ground. Non-disposable items falling to the ground, such as pipettes or important sample containers, should be immediately and thoroughly cleaned with a 0.5% sodium hypochlorite (10% bleach) solution to remove product contamination. Use a 0.5% sodium hypochlorite (10% bleach) solution to clean any lab surface that has contacted the contaminated item. Individuals handling anything that has fallen to the floor, disposable or not, must throw away their lab gloves and put on a new pair. Whole-Genome Gene Expression Direct Hybridization Assay Guide
26 14 CHAPTER 2 Standard Operating Procedures Reagent Reuse Pipette Carefully BeadChip Handling RNase-Free Techniques Never reuse excess reagents. Discard according to your facility requirements. Perform all pipette dispenses carefully and slowly to avoid creating turbulence within the plate wells and Flow-Through Chambers. Avoid touching the BeadChip anywhere other than at the barcode end or on the edges. Take the following precautions while working with RNA: Wear gloves throughout experiments to prevent contamination from the RNase found on most human hands. Use a solution of 0.1% SDS and 0.1N NaOH to decontaminate surfaces that are potentially contaminated with RNase. Change gloves after touching skin (e.g., your face or hair), door knobs, common surfaces, or other surfaces that have not been decontaminated. Use a dedicated set of pipettes for RNA work. Use freshly opened aerosol filter tips and tubes that are tested and guaranteed to be RNase-free. Use RNase-free chemicals and reagents, and DEPC-treated water. Designate a low-traffic area of the lab that is away or shielded from air vents or open windows. Do not leave RNase-free containers open when engaged in conversation. Standard Lab Procedures Running the Direct Hybridization Assay protocols requires that you perform some basic setup and familiarize yourself with standard procedures. This section discusses the following topics: Balancing the Centrifuge Cleaning and Calibrating Pipettes Balancing the Centrifuge Cleaning and Calibrating Pipettes Whenever you centrifuge plates or BeadChips, place a balance plate or rack with BeadChips opposite each plate or BeadChip rack being centrifuged. The weights should be as similar as possible. Ensure that pipettes are properly calibrated, clean, and decontaminated. Where possible, use a multi-channel pipette to dispense reagents.\ To prevent evaporation and spills, which could lead to assay variability and cross-contamination, ensure that all 96 caps are securely seated in the wells. When you remove a cap mat, do so carefully and slowly, to avoid splashing the contents. Set the cap mat aside, upside down, in a safe location for use later in the protocol. When you place the cap mat back on the plate, be sure to match it to its original plate and orient it correctly. Catalog # BD Part # Rev. A
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Crown demerger: enhancing value?
In the strange world of corporate reorganisations, two plus two may well equal five.
Crown Resorts(CWN)
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Your maths teacher might not like it, but in the strange world of corporate reorganisations and spin-offs two plus two can potentially equal five. Herein lies the potential attraction of the proposed Crown Resorts restructure aimed at 'enhancing shareholder value'.
As noted in Crown spins off international ops and hotels on 16 Jun 16 (Buy — $12.75), Crown Resorts intends to demerge most of its international operations into a separate company....
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Crown ResortsQ.
Hi , Just looking for some comments from you guys about Crown Resorts in light of the recent speculation about Packer's possible privatisation moves. He has stepped down as director some say in preparation. I'm guessing you would still consider it...Read full Q&A +
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Hi Jon. Any updated thoughts on Crown - is the share price drift downwards just a function of poor sentiment follow the results (which you covered in your Read full Q&A +


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DOI: 10.1209/epl/i2003-10178-y
Sedimentation profiles of charged colloids: Entropic lift and charge separationA.-P. Hynninen1, R. van Roij2 and M. Dijkstra1
1 Debye Institute, Soft Condensed Matter, Utrecht University Princetonplein 5, 3584 CC Utrecht, The Netherlands
2 Institute for Theoretical Physics, Utrecht University Leuvenlaan 4, 3584 CE Utrecht, The Netherlands
(Received 31 October 2003; accepted in final form 5 January 2004)
We present Molecular Dynamics simulations and Poisson-Boltzmann theory of sedimentation equilibrium of suspensions of charged colloids, treated at the level of the primitive model, including the co- and counterions (microions) explicitly. The simulations provide the first direct confirmation of the theoretical low-salt predictions of i) a macroscopic separation of colloidal and microionic charge, ii) an almost homogeneous electric field in the suspension, iii) a highly non-barometric colloid density distribution. These effects, which cannot be explained within the usual effective one-component picture, should be measurable experimentally.
82.70.Dd - Colloids.
05.20.Jj - Statistical mechanics of classical fluids.
61.20.Ja - Computer simulation of liquid structure.
© EDP Sciences 2004


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Neuvoo India is the biggest job aggregator in the country with over 1548286 job opportunities available, 381 of which are Opening for Assistant Deputy Manager-related.Show more...
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Gurgaon, Haryana
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JOB for Assistant. Deputy Manager. Manager. Sales GurgaonAutomotive companyDepartment. Marketing ( Sales )a) Qualification BE BTECH in Mechanical ( Please note only mechanical is required )c...
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12 yrs Graduate Engineer (Mechanical. Production). Optional Post Graduate. Commodity Buyer. Desired Traits. Strategic Sourcing. Additional Notes. Reason for Opening. New position due to...
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We have an urgent opening with one of the reputed ITES BPO for the position of Deputy Manager Admin... Functional Area. ITES, BPO, KPO, LPO, Customer Service, Operations. Role Category. Assistant Manager...
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YEAR JOB CONSISTENCY. FOR MULTI NATIONAL LIFE INSURANCE LTD. HI. We have opening for RECRUITMENT... ON THREE YEARS COMPLETION CAREER PATH FOR BECOMING BRANCH MANAGER. Channel. Direct Marketing (loyalty...
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Excellent opportunity for the position of Assistant Manager Deputy Manager Personal Assistant for Leading MNC in Pune Designation Assistant Manager Deputy Manager Location Pune Shift General...
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September 7, 2011
Stephen Colbert gave the American Council on Exercise a shout out on The Colbert Report on Tuesday! Watch the clip below and have a good laugh:
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While the popularity of high-intensity interval training has been wonderful for our ability to get results from our fitness programs, it has also brought with it an increase in the prevalence of overtraining. ACE pro Jonathan Ross explains why you may be overtraining, yet...
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If you're a runner, one of the easiest ways to get hurt is to skip your warm-up. And no, a light jog doesn't count. Rather, your warm-up should address your individual needs and fire up your neuromuscular system. Here is a quick and easy routine that will prep your body for...


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In an evening that spent a lot of time on 75th-anniversary nostalgia, two-time Oscar winner Olivia de Havilland was a class act. The 86-year-old "Gone With the Wind" star, who looked smashing, made an oblique reference to the war – punctuated with a knowing look that telegraphed a lifetime's worth of experience. Then she introduced a class-photo sequence with about 60 Oscar-winning actors posing stiffly on stage, from Luise Rainer (the 93-year old actress who won back-to-back Oscars in 1936 and '37) to the freshly minted winners from 2003.
Olivia de Havilland had a memorable Oscar moment
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An Italian cameraman working for international news agency Associated Press was among at least five people killed in northern Gaza on Wednesday as sappers were dismantling an Israeli missile.
A spokesman for Gaza's interior ministry said all five had been killed in a blast which occurred as explosives experts were trying to dismantle an Israeli missile in Beit Lahiya.
Another six people were seriously wounded, an emergency services spokesman said.
The AP confirmed that one of its journalists had been killed, identifying him as Simone Camilli, a 35-year-old Italian who had worked for The Associated Press since 2005.
AP photographer Hatem Moussa was also badly wounded in the explosion, the agency said.
Camilli is the first foreign journalist to be killed in more than a month of bloodshed between Israel and Hamas militants which began on July 8.
He was at the scene with the photographer to cover the story of bomb experts dismantling unexploded ordnance, the AP said.
In a side room at the Kamal Adwan hospital, male relatives could be seen weeping and praying together, some people too shocked to talk.
In Rome, Italian Foreign Minister Federica Mogherini issued a statement expressing sorrow at Camilli's death.
"Simone Camilli's death is a tragedy for his family and for the country. Once more a reporter pays the price for a war that has lasted too many years," she said.
The blast occurred as Israel and Hamas militants were observing a 72-hour truce which expires at midnight local time as negotiators in Cairo tried to thrash out a more permanent ceasefire arrangement.
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Edin Dzeko hit his ninth and 10th league goals of the season as Roma stayed in hot pursuit of leaders Juventus with…
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Galactica Super Nova's sculptural staircase
Superyacht staircases are often a focal point for interior designers, giving them a chance to show off their command of flowing lines or use of precious materials.
The staircase on Heesen Yachts flagship Galactica Super Nova is a perfect case in point, with curved steps banded in steel strips that appear to spin out of the central support post, hug the treads and then continue out to wrap the walls of the curved stair surround.
Photo: David Churchill


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News MVM to Release Inu x Boku SS
posted on 2013-03-22 09:40 EDT
MVM has confirmed on twitter that it has acquired the Inu × Boku SS television anime series, though it gave no further release details.
The romantic comedy follows a mistress and her "servant" (bodyguard) in Maison de Ayakashi, a high-class building better known by the name "Demon House (Ayakashi-kan)." Rumors about it abound; in fact, the people who live in this mysterious building are all descendants of demons. Ririchiyo Shirakiin is troubled by her inability to interact with people, and comes to the building in order to change herself. There she meets Soushi Miketsukami, one of the Secret Service bodyguards assigned to each tenant. He tells her, "Please make me your dog!"
Naokatsu Tsuda (Shikabane Hime: Kuro, Squid Girl) directed the anime adaptation of Cocoa Fujiwara's original manga at David Production. Haruko Iizuka (The [email protected], Toaru Kagaku no Railgun) designed the characters and acted as chief animation director. Toshizo Nemoto (Towa no Quon, Phi Brain: Kami no Puzzle) oversaw the scripts, and Takuya Satō (Strawberry Marshmallow, Mayoi Neko Overrun!) supervised the scenario and setting.
Via UK Anime Network
Image © Cocoa Fujiwara/SQUARE ENIX ・ Inu x Boku SS Production Committee ・ MBS
This Week in Games - Nintendo Switch
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Available Now: Girlish Number - Fall 2016 Short Anime - Magic of Stella - March comes in like a lion - Occultic; Nine - Poco's Udon World - Long Riders - ClassicaLoid - Kiss Him, Not Me! - Haikyu! Season 3 - Drifters - Lostorage incited WIXOSS - ALL OUT!! - Keijo!!!!!!! - FLIP FLAPPERS - Brave Witches - and much more!


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FOR IMMEDIATE RELEASEPressnews.biz (Press Release) Jan 26, 2015 -- Formally BioOlympics Ltd. is permitted to attain a high level of superiority for most universal System Information and Diagnosis learn. With this acquisition, BioOlympics Ltd. becomes a global organizer in diagnostic molecular analysis images, offering a broad portfolio of conventional X-ray systems and digital to health professionals worldwide. BioOlympics Ltd. has the opportunity to sell high quality products from diagnostic imaging systems in international markets and efficient distribution networks BioOlympics arrive and serve more clients medical care. We are specialized in developing and manufacturing innovative Chemiluminescence Imaging System. But really, it is our mission to help turn our world into a better place, though the use of technology which really means we are here to save lives. BioOlympics Ltd is to better meet the needs of specialized chemiluminescence imager from community hospitals and clinics worldwide. Our band has done an outstanding job of customer service and creating a solid business, with steady growth in recent years even more long-term possible.
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Good lordy! i thought it was bigger, but the entire thing is smaller than a cent! Put me on your list to get one of these babies when you start cultivating them!
Good lordy! i thought it was bigger, but the entire thing is smaller than a cent! Put me on your list to get one of these babies when you start cultivating them!
what a cool plant!!!!
Hmmmm.... that's interesting, I never seen one like this before. Could this be a new variation?
Very nice Arie! That would be a big seller if it could be established in cultivation. Are the traps functional (for tiny bugs)?
I kinda like "Pinhead", Nep G. [img]http://www.**********.com/iBhtml/non-cgi/emoticons/smile.gif[/img] Or...'Tom Thumb'? or "Gnat Trap"?...or....name that plant!
yay, name that plant! How aobut Tiny Tim? lol, since it kind of is a handicap for the little bugger. Or springtrapper, because it sohuld be great at trapping spring hoppers.
How about D.Muscipula "smaller than a cent" [img]http://www.**********.com/iBhtml/non-cgi/emoticons/smile.gif[/img] Or D.Muscipula "Penny's worth" :biggrin: OR D.Muscipula " Teeny weeny" [img]http://www.**********.com/iBhtml/non-cgi/emoticons/smile.gif[/img].
i hope no one gone to add me to the list of the story tellers (my rajah is a small one :biggrin: ) so here is a link that could help you belive this mini vft is a true thing
(Edited by arie at 2:56 pm on May 28, 2002)
Arie wow..... thats amazing [img]http://www.**********.com/iBhtml/non-cgi/emoticons/smile.gif[/img]
WOWEE!!! It's smaller that a penny!!!!!! What the heck kind of insects could it possibly be able to eat?! [img]http://www.**********.com/iBhtml/non-cgi/emoticons/wow.gif[/img]
Liquid Plummer
Warning: Do not reuse the bottle to store beverages.
Awww thats just so darn cute!!!
I am the weirdo who sits next to you on the bus!


text:
It's slowly coming towards that time of year again folks. The days are getting shorter, there's a crisp chill in the air, the trees are beginning to change and most importantly of all: Black Friday is almost upon us. This year in the UK the Black Friday and Cyber Monday Vape Deals will be bigger than ever before. Yes that is correct, NamasteVapes is proud to bring to you the biggest online UK Vaporizer sale the world has ever seen: The Black Friday Vape Sale. You name it, we got it: Portable Vaporizers? Check. Desktop Vaporizers? Yes Sir. Vape Pens for sale? You bet. The biggest brand names? Oh but of course.
We here at NamasteVapes are ready to hit Black Friday with the biggest UK Black Friday and Cyber Monday Vape Deals unlike anything you have ever seen before! We're going to be having flash deals on both the Friday and the Monday but only up until stocks last. That's right folks, you best be prepared as it's fastest fingers first!
What makes this Black Friday Vape Sale even better?
The second half of 2016 we are set to see some of the biggest brands in the industry release a range of new portable vaporizers, including: the PAX 3, the Da Vinci IQ, the Firefly 2, the Sticky Brick and so many more awesome new and great vaporizers. So, what does this mean for you, you ask? Is it not obvious! NamasteVapes are going ALL out this November and we are so proud to be able to offer you your favourite Vapes with a massive reduction in price, in our unbeatable Black Friday Vape Sale.
We will have our UK Black Friday vaporizer sales on our Portable Vaporizers, our Vape Pens and Plenty of other great Vaping accessories and pens. Running from the Friday sales right through to our Cyber Monday Vape Sale. So no need to endanger yourself as wrestle with thousands of others through the stores or just to stand in a que for a ridiculous amount of hours, check back in here with us, for the best Vaporizer sale ALL year.
November 25th 2016 | NamasteVapes UK Black Friday Vape Sale
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A bicycle ride for two! Eco-friendly Leonardo DiCaprio takes to the road with friend Emily Mace
Leonardo DiCaprio has proved that he practices what he preaches by skipping the traffic jams and opting for a more environmentally-conscious mode of transport.
The Great Gatsby star was a dashing sight as he pedaled around Manhattan with friend Emily Mace on Thursday, avoiding the city's usual gridlock.
The couple were spotted bike-riding in New York City, and whether it was a romantic date or a friends' outing, the pair looking incredibly relaxed together behind the handlebars.
Green date: Leonardo DiCaprio went biking with friend Emily Mace in New York City on Thursday
Wearing a green T-shirt and a flat cap with slip-on shoes and jeans, The Departed star oozed with cool despite riding a basic simple-geared bicycle.
Brunette Emily was even more casual in black workout pants, trainers and a tank top as she rode a similar bike next to him.
A huge supporter of environmental efforts and one of the first celebrities to drive a Prius, Leo looked just as at home on two wheels as four.
Head down: Leo kept his eyes on the road as he rode
Tipping his hat: The Great Gatsby star avoided the sun with both a flat cap and shades as the pair enjoyed the summer day
Green living: DiCaprio is renown for his environmental issues and was one of the first stars to drive a Prius
Since getting his first electric car, Leo has bought a Tesla Roadster and a Fisker Karma plug-in, as well as adding solar panels to his house.
In 2007, he appeared at the Oscars with former Vice President Al Gore to announce that the Academy Awards were going green.
The 38-year-old has also cited global warming as 'the number one environmental challenge' and has been widely praised for his activism.
Day out: Whether a date or a pals' ride, the pair were enjoying the beautiful weather in Manhattan
As for his love life, that has been less stable as of late since his split with last serious girlfriend, model Bar Refaeli in May 2011.
Since then he has dated Gossip Girl actress Blake Lively and another model, Erin Heatherton, but that ended in October 2012.
He was most recently linked to 20-year-old German model Toni Garrn, who he whisked around some of Europe's most spectacular palaces in Venice and Monaco earlier this month.
Leo will next be seen in The Wolf Of Wall Street, where he lives life to excess in the 90s as New York stockbroker, Jordan Belfort.
Wide stride: Leo later ditched his bike and took to his own two feet instead to walk around the city
Bright colours: DiCaprio stuck with a similar casual style but swapped for a brighter teal T-shirt and shorts
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text:
Miyamoto Discusses Wii U Performance: "I'm Not At A Point Where I'm Concerned Yet"
Posted on Tuesday, March 12 @ 10:49:09 PST by Alex_Osborn
Nintendo's Shigeru Miyamoto recently spoke with the NY Times, providing his thoughts on online-connected game experiences, as well as the future of the Wii U.
"For a long time at Nintendo we didn't focus as much on online play because for many years doing so would have limited the size of the audience that could enjoy those features," Miyamoto told the outlet. "But certainly now we see that so many people are connected to the Internet. It opens up a tremendous amount of possibilities."
With regards to the Wii U's rather underwhelming start, Mr. Miyamoto went on to say, "We really view it as being the ideal device that families are going to want to have connected to that screen in the living room that everyone is going to gather around and watch. Certainly in the short term I would want to see it performing with probably a little more momentum. I think in the long term I'm not at a point where I'm concerned yet."
Do you think Nintendo should be "concerned yet" about the Wii U? Let us know in the comments below.
Minecraft: Story Mode
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Patch 248 Procedurally Generated Maps, Kaprosuchus, Diplocaulus, Chalicotherium and more. (1:38)
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Lady and the Painter trailer. (0:40)
Shadow Warrior 2
Behind the Schemes Flying Wild Hog. (16:19)
|More On GameRevolution


text:
Average Rating
Course Difficulty
Easy 32%
Medium 58%
Hard 11%
Top Course Tags
Lots of Writing
Great Discussions
Great Intro to the Subject
Not too easy. Not too difficult.
Course Overview:
I would recommend it! This professor was amazing and very nice. I see her every once in a while and she is very sweet.
Course highlights:
My writing skills were strengthened! I feel comfortable writing and actually use it for venting. I learn different writing tactics and also taught myself to finish projects way before due.
Hours per week:
3-5 hours
Advice for students:
Make sure to read the material! Get a planner and write all the due dates down.
Pretty easy, overall.
Course Overview:
I highly recommend this course because it's a stepping stone to learning the basics of proper grammar and language in different types of essays. This class can also be a big advantage by having the skills to write better if you want to become a writer, lawyer, mayor, etc.
Course highlights:
The highlights of this course is that there is only 4 essays to do for the entire term. From this class, I learned that different styles of writing must be achieved depending on the type of essay and my instructor also taught me about writing better in different points of view.
Hours per week:
3-5 hours
Advice for students:
Make sure to study, practice writing, and talk to your instructor if you have any questions. That way, you'll be fluent in writing properly by the time you finish the course.
Pretty easy, overall.
Course Overview:
I would highly recommend this course to anyone needing an English credit. Professor Goldstein was a phenomenal teacher and made the class enjoyable.
Course highlights:
There was a lot of writing in this course, which I thoroughly enjoyed. He implemented various works from history, which made the course interesting. I believe this course was my favorite in college.
Hours per week:
0-2 hours
Advice for students:
Go into the course expecting to write. Have an open ear to learn and to implement the teachings he gives. You will succeed if you put in the proper amount of work.


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The Raven Mysteries 5 : Magic and Mayhem
Written by Marcus Sedgwick, Pete Williamson
Part of the Raven Mysteries Series
Lovereading4kids Price £5.59
£6.99 saving £1.40 (20%)
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The Lovereading4Kids comment
Chaos strikes after a trip to the circus, and the castle is suddenly filled with thieves, bunnies, and lethal cabbages, in the fifth of Marcus Sedgwick's quirky mysteries.
The Raven Mysteries 5 : Magic and Mayhem by Marcus Sedgwick, Pete Williamson
The fifth quirky mystery by BLUE PETER-award winning team Marcus Sedgwick and Pete Williamson.
Join the wonderfully weird Otherhand family and their faithful guardian, Edgar the raven, and discover the dark secrets of Castle Otherhand. A trip to the circus has far-reaching consequences for the Otherhands when Fellah goes missing. Before long the Castle is plagued by a duck, a suspect fortune teller, and several cartloads of lethal cabbages. The family is preoccupied: Valevine is busy inventing a cabbage-counting machine; Minty is waiting to hear the secrets of the universe; and Solstice has discovered that Cudweed is concealing three hundred and forty-two rabbits in his bedroom. When chaos strikes and thieves infiltrate the Castle, it falls to Edgar to extract his family from a very fluffy predicament.
'Edgar is a wonderful creation' - The Daily Telegraph
About the Author
Other Formats
Book Info
256 pages
Interest Age: From 9
Marcus Sedgwick, Pete Williamson
More books by Marcus Sedgwick, Pete Williamson
Author's Website
Orion Children's Books (an Imprint of The Orion Publishing Group Ltd ) an imprint of Orion Publishing Co
Publication date
5th April 2012
Writing reviews help the children with their literacy skills and we always read the books together which gives us good quality family time!Cat Bisland (on behalf of the Bi
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text:
Travel Charme Bergresort Werfenweng, Werfenweng
- Swimming pool
Renovation work will only be conducted during business hours. Every effort will be made to minimize noise and disturbance.
- Free breakfast and free WiFi
Main amenities
- 166 guestrooms
- 2 restaurants
- Full-service spa
- Indoor and outdoor pool
- Free ski shuttle
- Ski rentals
- Health club
- Self parking
- Airport shuttle
- Terrace
- 24-hour front desk
- Daily housekeeping
For families
- Free cribs/infant beds
- Connecting/adjoining rooms available
- Private bathroom
- Free toiletries
- Garden
- Terrace
What's around
- IKARUS Cable Car (1 mi / 1.6 km)
- Hohenwerfen Castle (5.7 mi / 9.2 km)
- Burg Hohenwerfen (6 mi / 9.6 km)
- Werfen Ice Caves (8.1 mi / 13 km)
- Paul Ausserleitner Hill (8.1 mi / 13.1 km)
- Salzburg (SZG-W.A. Mozart) 37-min drive; 32.1 miles/51.7 km
- Werfen Station 11-min drive; 5.1 miles/8.2 km
- Bischofshofen Station 15-min drive; 7.5 miles/12 km
- Eben im Pongau Station 18-min drive; 14.1 miles/22.7 km
- Free parking nearby
- Self parking (surcharge)
- Airport shuttle (surcharge)
- Train station pick-up (surcharge)
- Free ski shuttle
Travel Charme Bergresort Werfenweng, Werfenweng
Hotel Info: 866-539-8117from $110
- Double Room, Mountainside
- Suite, Balcony, Mountain View
- Junior Suite, Balcony, Mountain View
- Double Room, Mountain View
- Panoramic Double Room, Mountain View
- Apartment (Type Two - Spa Access with surcharge)
- Apartment (Type One - Spa Access with surcharge)
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 166 rooms
- This hotel is arranged over 3 floors
- Check-in time 3 PM-2 AM
- Check-out time is 11 AM
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- One child (3 years old and younger) stays free when occupying the parent or guardian's room, using existing bedding
- Pets allowed *
- Specific rooms only, restrictions apply *
- Free WiFi in public areas
- Free WiFi in rooms
- Airport shuttle (available on request) *
- Train station pickup *
- Self parking *
- Free nearby parking
- Free ski shuttle
In the hotel
- Free breakfast
- 2 restaurants
- Bar/lounge
- Room service (during limited hours)
- Indoor pool
- Outdoor pool
- Free ski shuttle
- Ski storage
- Health club
- Full-service spa
- Spa treatment room(s)
- Ski equipment rentals on site
- Cross-country skiing on site
- Golfing nearby
- Downhill skiing nearby
- Sledding nearby
- Snowboarding nearby
- Ski passes available
- Spa tub
- Steam room
- Sauna
- One meeting room
- 24-hour front desk
- Concierge services
- Tours/ticket assistance
- Dry cleaning/laundry service
- Luggage storage
- Multilingual staff
- Number of buildings/towers - 4
- Year Built 2012
- Elevator/lift
- Designated smoking areas
- Garden
- Terrace
- Library
In the room
- Minibar
- Bathrobes
- Slippers
- Hypo-allergenic bedding available
- Pillow menu
- Premium bedding
- Balcony or patio
- Private bathroom
- Bathtub or shower
- Free toiletries
- Hair dryer
- Flat-screen TV
- Satellite TV channels
- Free WiFi
- Phone
- Daily housekeeping
- In-room safe
- Connecting/adjoining rooms available
Special features
Puria Premium Spa has 12 treatment rooms. Services include sports massages, massages, facials, and body scrubs. The spa is equipped with a sauna and a spa tub. A variety of treatment therapies are provided, including aromatherapy.
DiningFeinspitz - This restaurant specializes in international cuisine and serves breakfast and dinner. Guests can enjoy alfresco dining (weather permitting).
Alpenzeit - This fine-dining restaurant specializes in regional cuisine and serves dinner only. Guests can enjoy drinks at the bar. Open daily.
Lobbybar 902 - Onsite bar.
- Ski passes available
- Free ski shuttle
- Ski storage
- Ski lifts nearby
- Ski runs nearby
- Ski equipment rentals on site
- Cross-country skiing on site
- Snowboarding nearby
Travel Charme Bergresort Werfenweng, Werfenweng's small print
Also known as
- Travel Charme Bergresort
- Travel Charme Bergresort Hotel
- Travel Charme Bergresort Hotel Werfenweng
- Travel Charme Bergresort Werfenweng
- Travel Charme Bergresort Werfenweng Austria
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
Pet-friendly rooms can be requested by contacting the property at the number on the booking confirmation.
For transfers, guests must contact the property before arrival at the number on the booking confirmation.
Reservations are required for massage services and spa treatments and can be made by contacting the property before arrival at the number on the booking confirmation.
Mandatory fees
You'll be asked to pay the following charges at check in or check out:
- A tax is imposed by the city: EUR 1.10 per person, per night
The charges below are included in the total price displayed:
- Christmas Eve gala dinner fee for stays on 24 December
- New Year's Eve gala dinner fee for stays on 31 December
Optional extras
Self parking costs EUR 9 per night
Pets are allowed for an extra charge of EUR 25 per pet, per day
Airport shuttle service is offered for an extra charge of EUR 90 per vehicle (roundtrip)
Train station pick-up service is offered for an extra charge
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Is the description of this hotel not correct? Tell us


text:
Project Main Details
Please look at this video for reference:
Need hip young high energy fast paced.
Talent needs to be available on Monday 15th September for tweaks / finals record
Usage - Internal client video. NOT aired.
Thank you!!!! 2014-09-13 00:59:56 GMT 2014-09-14 18:00:00 (GMT -08:00) Pacific Time (US & Canada) Yes (click here to learn more about ) Closed 21 21 0 direct invitation(s) have been sent by the voice seeker resulting in 0 audition(s) and/or proposal(s) so far. Voice123 SmartCast is seeking 100 auditions and/or proposals for this project (approx.) Invitations sent by SmartCast have resulted in 21 audition(s) and/or proposal(s) so far.
Project Parameters
• Audio files must be delivered via FTP/Dropbox/Google Drive/cloud
Script Details
:66 ANTHEM (X1 script)
We see a montage of the highly motivated, highly capable people. We see the results of hard work and of projects being completed. A confident VO delivers a high-octane assessment of the ThinkPad user.
VO: Think for a moment about all that you do. To nail that interview. That meeting. That thought. That pitch.
You make it look easy–and look good doing it. (X1 shot) But it's not. Never is. It's hell. It's grueling. It's rewarding. You power through the day. You spill your coffee. No big deal. You get stuff done.
You step up. Move forward. Think big. Drop your computer. Drop it again. (X1 ideally)
Push on. Think bolder. Make strides. Make mistakes. Learn shortcuts. Get better. Make friends. And rivals.
You see shows. You know sushi. You love driving stick. Tick. Tick. Tick. You never stop thinking.
Business is brutal. But, hey, so are you. Dependable, powerful--like your team and your tools. (X1 Intel?) Light, quick, nimble and strong. Always moving. (X1 on the go)
You change jobs. Change careers. Change your mind. Make it work.
You take some time off. How about France. No, Costa Rica. You relax. You play. You unwind. You return.
Think about it all–where you've been and where you're going.
You already know how you're going to get there. (end with X1 shot)
SUPER: Think for yourself.
SUPER: ThinkPad logo.
Voice-Seeker Details
Voice123 Team Comments
Voice123 consultations with this voice seeker regarding this project and/or other projects by this voice seeker, via phone, chat, and/or email.
This project - phone.
Previous projects - phone.
This project - email or chat.
Previous projects - email or chat.
Corporate web site for this voice-seeker confirmed by Voice123
Note: Voice123 strives to establish the legitimacy of all projects posted. However, Voice123 subscribers and users are responsible for confirming information stated by prospective voice seekers, agents and/or clients. Voice123 subscribers and users assume all liability for use of any information found through Voice123, or any of its publications.
This page contains the most important details of this project. If you find the information on this project inaccurate or inappropriate, please let us know by contacting us.


text:
Bones & Pearl Studios
- Website www.facebook.com/bonesandpearl
Event times
Touch Me Baby!
Bad Art returns with its third installation - Touch Me Baby! For one night one, guests will be invited to freely interact with all artwork on show, without fear of alarms or getting told off for standing too close. By allowing physical interaction, Touch Me Baby hopes to integrate the art and public in a far more intimate…
Sat 19 Nov
- 17:00 – 21:00 Touch Me Baby! Bones & Pearl Studios
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text:
Bedrooms 1 Bathrooms 1 Receptions 1
Available From:Now
Stylish and interior designed one bedroom apartment, brand newly converted, situated in the heart of this popular historic market town. Offering easy access to Oxford (seven miles via A34) and London Paddington (45 minutes direct, Didcot Parkway). Short walk to local shops, parks and schools. Fully furnished. Council Tax Band B. Please contact our office for further information or to arrange a viewing.
£850 pcm + Fees
All rents are subject to additional fees, as detailed in our Guide to Charges:


text:
Hey guys I have a knight t bolt in 50 cal that has never been fired I am looking to sell. It has the stainless barrel, camo synthetic stock. It is in mint mint shape, I am looking to get $275 obo , thanks in advance and please feel free to ask any questions.
post #1 of 2
5/4/10 at 2:12pm
Thread Starter


text:
You missed it This great deal for Pardini's Café is over. But don't despair, we've got more great deals every day.
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Pardini's Café
Classic Local Restaurant with Modern Flavor
99% recommendedin LivingSocial customer ratings
Pardini's Café continues to bring Fresno-area diners back again and again for hearty lunch and breakfast fare in a casual setting -- grab something gourmet for half the price, with this deal:
• $10 for $20 to spend on breakfast or lunch, Monday through Friday
• Located on Shaw Avenue and open as early as 6 a.m.
Local Spot Satisfying Diners Since 1958
In 1958, Al Pardini began perfecting recipes for his catering business -- and though many of his classic recipes are presented at this café today, many have modern twists that delight diners old and young. Menu highlights include the half-pound hamburger, daily made-from-scratch soups, Linguiça and eggs, and the barbecue tri-tip sandwich -- regulars recommend ordering a side of thick-cut bacon or creamy potato salad. But don't just take our word for it -- peek at Paradini's mouthwatering gallery of menu items to see just how incredible this local eatery is.
Want more? Check out Pardini's Café on Facebook.
PAID VALUE EXPIRES ON October 27, 2021
PROMOTIONAL VALUE EXPIRES ON September 1, 2013


text:
The Video Devices PIX 240i features an IPS (In-Plane Switching) display. This new technology yields a brighter, sharper image with improved color accuracy and excellent off-axis performance. This attractive display provides immediate confirmation of recording, playback, audio metering, and Setup Menu selections. Monitor brightness, contrast and chroma adjustments are available for customized viewing needs.
The PIX 240i adds Quicktime recording using Apple ProRes or Avid DNxHD to any HDMI or HD-SDI equipped high-definition video camera. The portable PIX 240i records Quicktime files to CompactFlash cards or removable 2.5" solid-state hard drives.
Apple ProRes and Avid DNxHD video formats have emerged as industry standards. These I-frame codecs offer the best in picture quality, processing efficiency, and data storage requirements. They are ideal for editing, on-the-fly color grading, and video processing. Files recorded in the field on a PIX recorder are ready for the most popular editorial workflows.
NOTE: The PIX 240i now has 12-bit, 4:4:4 recording. This feature is ONLY available as a firmware update at: http://www.videodevices.com/products/portable-video-recorders/pix240i/downloads/
Video Devices PIX 240i Features:
Video Devices PIX 240i Specifications:
|Sound Devices PIX-CADDY-2
SSD Mount Accessory for PIX HD Recorders
The PIX-CADDY 2 provides a solid, removable connection for 2.5-inch solid-state drives (SSD) (not included) to attach to the PIX 220, PIX 240 and PIX 260 video recorders. Once secured to the caddy, SSD drives are quickly inserted and ...
In Stock
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|Sound Devices XL-WPH3
12VDC In-Line Power Supply
The XL-WPH3 is a universal, in-line AC-to-DC power supply with detachable IEC power cord for Sound Devices mixers and recorders. Input: 100–240, 50/60 Hz; output 12 VDC, 45 W on Hirose 4-pin connector.
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|Video Devices XL-B2
Battery for 7 Series recorders
Spare battery pack for 7-Series portable recorders. Compatible with Sony L-Series camcorder mount. 7.2 V, 4500 mAh.
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|Video Devices PIX-HC1A
Carrying Case for PIX 220(i) or PIX 240(i) and Accessories
The Video Devices PIX-HC1A is a rugged ATA -rated, waterproof, ultra high-strength polypropylene copolymer resin carry case with custom cut foam insert space for a PIX 220(i) or PIX 240(i), up to four 2.5" SSDs (with attached PIX-CADDY/2), ...
Usually ships in 7-14 business days.
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Mounting Arm for PIX 220 and PIX 240 Field Video Recorders
The Video Devices PIX ARM is a mounting arm for their PIX 220 and PIX 240 field video recorders. It mounts onto cameras with 1/4" or 3/8" screw connections and features a central knob that locks all joints.
Usually ships in 7-14 business days.
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|Video Devices PIX-HOOD
Sun Shield for PIX 220 & PIX 240
The Video Deivces PIX-HOOD improves screen visibility in full-sun shooting. The Hood snaps in place with four magnetic mounts and has no effect on button access.Note: PIX 220 and 240 sold separately.
Usually ships in 7-14 business days.
Need it sooner? Call us!
Our Price:$48.00


text:
Interpreting a national rule in such a way as to prevent comparisons between part-time and full-time workers during a selection process for redundancy did not contravene the Equal Treatment Directive, rules the European Court of Justice in Kachelmann v Bankhaus Hermann Lampe KG.
In Kachelmann v Bankhaus Hermann Lampe KG the European Court of Justice has ruled that it is not contrary to the EC Equal Treatment Directive for a redundancy selection process not to compare part-time workers with full-time workers.
© 2016 Reed Business Information Ltd
The materials and information included in the XpertHR service are provided for reference purposes only. They are not intended either as a substitute for professional advice or judgment or to provide legal or other advice with respect to particular circumstances. Use of the service is subject to our terms and conditions.
XpertHR is designed to work consistently across a range of browsers, including the latest Internet Explorer, Google Chrome, Firefox and Safari. XpertHR no longer supports Internet Explorer 6 or 7. Learn more.
Reed Business Information Limited trading as XpertHR is an Appointed Representative of Abbey Protection Group Limited which is authorised and regulated by the Financial Conduct Authority.
© 2016 Reed Business Information Ltd


text:
Playing the Spoons And Other Curious Instruments
Written by Liz Miles
Illustrated by
Part of the Culture in Action Series
Lovereading4kids Price £10.65
£12.99 saving £2.34 (18%)
when in stock
As soon as this book is back in stock we will send you an email.Compare book price We'd love you to buy your books from Lovereading - especially if we helped you to choose them! But as our mantra is guidance, and that includes guidance on price, please do use our 'Compare Book Price' service to discover if the best value deal is from Lovereading or another specialist bookstore.
Playing the Spoons And Other Curious Instruments by Liz MilesWith Culture in Action the arts are brought to life! The varied titles in the Culture in Action series build up into a comprehensive library from which readers can choose their favourite arts topics. Each book contains activities with easy-to-follow instructions.
About the Author
Other Formats
Book Info
32 pages
Interest Age: From 7
Liz Miles
More books by Liz Miles
Publication date
9th September 2010
I am so pleased to have signed my kids up as they are reading a much wider range of books and even choosing books out of their comfort zone.Angela East
I love Lovereading as it provides an honest opinion and showcases a range of fiction. Suited to both parents & kids alike, it's a must-use.Georgie Rowe – age 16
Lovereading is just a convenient way to find new books and hear others opinions on them.Sarah Murray – age 15
I love 'LoveReading 4 kids' because they let you read and learn things you'd never dreamed of learning before.Emily Horncastle – age 11
LoveReading4Kids is a modern and creative way of emphasising the value and importance of books in this digital age #booksforlifeAmrit Bunet – Teen
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I have told all my friends, family & teachers to see for themselves just how great the site is. Without fail, they are hugely impressed.Alexander Boxall – age 11
At @HHSHaringey we love @lovereading4kids because our pupils can practice reviewing & get free upcoming books before anyone else!Helen Swinyard – Heartlands High