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Disclosed is a process for amplifying DNA, a process for preparing and amplifying DNA, a kit of parts for DNA amplification and DNA preparation, and the use thereof, all of which are characterized by the use of tetraethylene glycol. Moreover, the use of tetraethylene glycol in a reaction solution by carrying out a DNA amplification and a reaction solution containing tetraethylene glycol for carrying out a DNA amplification are disclosed.
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The invention relates to a process for amplifying DNA or a process for preparing and amplifying DNA, a kit of parts for DNA amplification, and the use thereof, a reaction solution for carrying out DNA amplification, and the use of tetraethylene glycol in a reaction solution for carrying out DNA amplification.BACKGROUND
It is increasingly important to detect bioparticles such as bacteria, viruses, fungi, protozoa or the like in samples from various origins.
One option for doing so is the release of the nucleic acids of the cells contained in a sample, their subsequent specific amplification, and then detection thereof. The following steps are required for this purpose:
- Sampling of material (e.g. blood, sputum, urine, feces, liquor, as well as swab samples from the oral cavity, nasal region, genital tract; but also forensic samples, soil samples, etc.)
- Lysis (disruption of the cells)
- Nucleic acid preparation
- Nucleic acid amplification
- Detection of the amplified nucleic acids
This method is known in principle (e.g. Bowlen & Dürre, Nucleic Acids Isolation Methods, American Scientific Publishers, 2002).
In the known processes for preparing nucleic acids, ethanol is frequently used as a solvent. Today, ethanol is generally used, for example, as a washing buffer additive (e.g. DE-A 198 56 064, US 2005/0079535).
However, ethanol is classified as a hazardous material (HAZMAT, hazardous material) by IATA (International Air Transportation Association). Thus, additional fees and taxes apply in case of transport by plane. Moreover, complex steps for removing ethanol are required subsequent to the washing process because small quantities of ethanol may already strongly interfere with or completely block subsequent reactions such as PCR.
There are already efforts being taken to replace ethanol with other substances in processes for purifying nucleic acids.
For example, the earlier application EP 08163623.5 by the applicant proposes the use in the adsorption step of binding mediators, which are considered harmless according to the IATA guidelines. Among others, tetraethylene glycol is also mentioned as a binding mediator.
Another earlier application by the applicant, EP 08163624, proposes washing buffers that contain less than 24 vol %, particularly preferably no ethanol. Various solvents can be used as solvents; the washing buffer may, among other things, also contain tetraethylene glycol.
DE-A 102 53 351 points out that the use of alcohol or acetone in processes for purifying nucleic acids has great drawbacks. For example, even traces of alcohol affect the subsequent reactions to a very considerable extent. Therefore, customary purifying processes for nucleic acids always include an ethanol removal step. In order to avoid these issues, the document proposes avoiding the use of alcoholic components altogether. Among other things, an alcohol-free washing buffer is disclosed which contains NaCl, MgCl2 as well as Tris/HCl; no mention is made of tetraethylene glycol.
WO 2004/042058 describes buffer formulations for the isolation, purification and recovery of short-chain nucleic acids that contains monovalent and multivalent cations. An alcohol-free washing buffer containing NaCl, MgCl2 and Tris/HCl is also disclosed here.
WO 02/44400 discloses, as a reagent for extracting nucleic acids, an aqueous solution containing sodium metasilicate and a substituted ether. No mention is made of washing buffers.
U.S. Pat. No. 5,610,287 describes alcohol-free washing buffers containing Tris/HCl, NaCl as well as Tween 20.
WO 2008/071384 discloses the use of tetraethylene glycol dimethyl ether in various steps of DNA preparation and purification.
However, none of these methods have so far led to a substitute solution for ethanol that is satisfactory in every respect. In particular, it has not been cleared up, for any of the above-described substitute solvents, how the presence of the washing buffer constituents affects the subsequent DNA, amplification. For even small quantities of solvent usually affect the efficiency of PCR to a very considerable extent.
Therefore, the object of the invention is to provide an improvement of the preparation step, in particular in the preparation of DNA, in which the PCR carried out subsequently is not affected by the solvent used.DISCLOSURE OF THE INVENTION
Surprisingly, it was found that this object can be accomplished by using tetraethylene glycol, and that the presence of tetraethylene glycol does not affect the reactions steps carried out subsequent to the DNA preparation.
The invention therefore provides processes for amplifying DNA in which the reaction solution contains tetraethylene glycol.
In this case, the tetraethylene glycol in the reaction solution originates from the eluate of the DNA preparation on solid surfaces that was used for preparing the DNA.
Moreover, the invention relates to a process for the preparation and amplification of DNA, comprising the following steps:
- a) immobilizing DNA contained in a solution on a surface
- b) washing the surface with a washing buffer
- c) recovering the DNA by elution,
- d) carrying out a DNA amplification with the eluate obtained in step c), characterized in that the washing buffer comprises tetraethylene glycol.
Preferably in this case, the tetraethylene glycol quantity in the washing buffer is at least 10 vol %, preferably at least 48 vol %, and more, preferably at least 60 vol %.
Also preferably, the volume of the eluate in step d) is at least 10 vol %, preferably at least 30 vol %, more preferably at least 50 vol %, based on the entire reaction volume of the amplification reaction.
Furthermore preferred is a configuration of the process in which the tetraethylene glycol content in the entire reaction volume of the amplification is up to 20 vol %, preferably up to 12 vol %, more preferably up to 10 vol %.
Moreover, the washing buffer may contain one or more constituents selected from the group comprising chaotropic salts and buffering substances.
DNA amplification can be carried out as an end-point PCR, quantitative PCR or isothermal amplification.
Furthermore, the invention provides a process according to one or more of the preceding embodiments which is carried, out in a microfluidic device (lab on a chip, LoC).
Another aspect of the invention is a kit of parts for DNA amplification and DNA preparation according to the process of one or more of the above embodiments, comprising:
- a solution 1 as a washing buffer containing tetraethylene glycol, and
- reagents required for carrying out the DNA amplification.
Preferably, the kit of parts moreover comprises one or more of the following constituents, selected from
- a solution 2, comprising a binding mediator,
- a solution 3, comprising an elution agent,
- a solution 4 comprising a lysis buffer and optionally one or more proteases;
a microfluidic device (lab on a chip, LoC).
The invention further relates to the use of the above-mentioned kit of parts for the preparation and amplification of DNA obtained from biological materials, preferably from samples taken for molecular-diagnostic purposes (human and veterinary medicine), in particular from forensic samples and from viruses or bacteria contained in biological samples, such as, for example, blood.
Finally, the invention relates to the use of tetraethylene glycol in a reaction solution for carrying out a DNA amplification, in particular an end-point PCR, a quantitative PCR or an isothermal amplification; as well as a reaction solution for carrying out a DNA amplification, comprising reagents required for amplification and tetraethylene glycol, wherein the tetraethylene glycol content in the reaction solution is up to 20 vol %, preferably up to 12 vol %, more preferably up to 10 vol %.
According to the invention, DNA means genomic DNA, gDAN, plasmid DNA or artificially produced DNA, such as, for example, short DNA fragments. It can be obtained from any cell or virus or be artificially produced. Preferably, the DNA has a length of 12 or more base pairs. It is obtained, for example, from prokaryotic or eukaryotic cells, such as eubacteria (e.g. gram-positive or gram-negative bacteria), archaea, pathogenic bacteria, eukaryotic microorganisms or cells of a multi-cellular organism, e.g. blood cells, tissue cells, etc. It may originate from a natural, artificially modified or artificially produced virus, e.g. a virulent, attenuated or non-infectious virus. It may also originate from fungi, yeasts, parasites, protozoa or other microorganisms.
Those microorganisms or cells from which DNA is obtained according to the invention can be obtained from any sample, e.g. samples from humans, animals, plants or soil samples, e.g. blood, urine, tissue samples, hair, saliva, stool, serum, cerebrospinal fluid, forensic samples, soil samples, parts of plants such as stalks, leaves, seeds, blossoms or roots.
The process according to the invention is particularly advantageous for amplifying DNA from cells present in the sample in very small quantities, such as, for example, DNA from viruses present in low titers in whole blood, or DNA from forensic samples.
It was found that the process according to the invention is not suitable for the preparation of RNA. This reason for this is probably that the reverse transcriptase required for the transcription of the eluted RNA into DNA is considerably more susceptible to tetraethylene glycol than to the ethanol usually used as a washing buffer additive.
In its most general embodiment, the invention relates to carrying out a DNA amplification in particular by means of PCR or isothermal amplification, with tetraethylene glycol being contained in the reaction solution. This preferably stems from the washing buffer used in the preceding DNA preparation, i.e. from the eluate obtained subsequent to the DNA preparation. However, it may also be added to the reaction solution not until later. It was found that even larger quantities of tetraethylene glycol do not inhibit the PCR, which is very surprising in view of the fact that the usually used ethanol already leads to considerable interference in the PCR in very small quantities, and that, as experiments by the applicant have shown, even small quantities of triethylene glycol (4 vol % of the reaction solution for PCR) already lead to a complete inhibition of the PCR.
According to the invention, the reaction solution used for carrying out the DNA amplification can contain up to 20 vol % tetraethylene glycol, more preferably up to 12 vol % and still more preferably up to 10 vol % tetraethylene glycol. It was found that no inhibition of the PCR takes place even in case such large quantities tetraethylene glycol are present.
In principle, the preparation of the DNA may take place in any known way.
Preferably, the procedure according to the invention is as follows:
The first thing to take place is the lysis of the cells, i.e. the disruption and release of the DNA contained therein in the known manner by heating, ultrasound or grinding by means of beads, e.g. glass beads. Lysis takes place in a conventional lysis buffer. The lysis buffer is, for example, the buffer AL (QIAGEN GmbH; Hilden, Germany). Other lysis buffers are known to persons skilled in the art and are selected to be suitable for the microorganism to by lysed, as are the suitable lysis conditions. Several different lysis buffers may also be used. For example, PBS buffer (phosphate-buffered saline solution) containing proteinase K and lysozyme may also be used as a buffer. Optionally, an incubation process is carried out first and then another buffer is optionally added, such as, for example buffer G (GITC (guanidinium thiocyanate)/Nonidet® (nonylphenylethylene glycol)) and an incubation is carried out again. The suitable volume of the buffer(s) must be determined depending on the sample material and is, depending on the sample material, between 50 μl and 1 ml, for instance. The temperature and duration for the incubation is in each case suitably selected. Since some lysis processes are carried out by using enzymes, the selected temperature also depends on the activity of the enzymes. Zymolase, lyticase and lysozyme are used, as a rule, in a temperature range of 20° C.-37° C. Incubation with proteases, e.g. proteinase K or subtilisin, is usually carried out of 50-60° C. The duration of incubation is, as a rule, 5 to 20 minutes, preferably about 10 minutes. Incubation can be carried out, for example, in a thermomixer by means of shaking, e.g. at about 1400 r.p.m., or also on a Vortex® device (maximum level)), optionally with the addition of glass beads.
Subsequent to the lysis, the isolation of the nucleic acids released by lysis takes place by means of immobilization (adsorption) on solid surfaces.
In the purifying processes using adsorption, the selective binding, of the constituents of the contents of the lysate to or on a solid supporting or carrier material ("binding", immobilizing, step a)), the removal of undesired constituents from the solid supporting or carrier material ("washing", step b)), and the elution of the desired constituent ("elution", step c)). This process is also referred to as "bind-wash-elute".
The surfaces on which the DNA is immobilized according to the invention are conventional surfaces known in the prior art, such as membranes, plates or beads. They consist of a plastic material such as, for example polystyrene, mineral material, such as glass powder, diatomaceous earth, silica gels, magnetic particles, non-woven glass-fibers, diaphragm filters, metal oxides, latex particles. Silica-based particles are most frequently used. Magnetic silica particles provided in the MagAttract®-Kit (Qiagen. GmbH, Hilden, Germany) can be used particularly advantageously. The DNA is, immobilized in such a way that it is not detached from the carrier in any appreciable amounts by washing with water.
Usually, a washing step with a washing buffer follows the immobilization in order to remove undesired lysates also bound to the surface.
Generally, washing buffers are aqueous solutions. According to the invention, the washing buffers preferably contain tetraethylene glycol. Preferably, the tetraethylene glycol content in the washing buffer is at least 10 vol %, more preferably at least 48 vol %, and most preferred at least 60 vol %. Furthermore, the washing buffer comprises substances that are usually used for washing buffers, in particular (purified) water, salts such as, for example, guanidinium hydrochloride, sodium chloride, and buffers such as Tris/HCl. Particularly preferably, ethanol can be replaced with tetraethylene glycol in the known washing buffers AW1 and AW2 by the company Gingen, so that particularly preferred washing buffers according to the invention are TEG1 (48 vol % tetraethylene glycol, 2 M guanidinium hydrochloride) and TEG2 (60 vol % tetraethylene glycol, 100 mM NaCl, 10 mM Tris/HCl pH 7.5).
The pH value is preferably set to a desired value using conventional buffering substances. Buffering substances include, for example, Tris/HCl, sodium acetate, maleate, sodium citrate. The pH of the washing buffer used according to the invention is preferably 7 to 9.
Possible chaotropic salts that can be added to the washing buffer include, according to the invention, in particular barium salts, thiocyanates, perchlorates, guandinium salts and in particular sodium chloride, guandinium hydrochloride, sodium iodide, sodium perchlorate and guandinium thiocyanate.
After washing, a drying process may optionally be carried out, if required (e.g. drying in an open vessel in air, "air dry"), or a washing process may be carried out ("water rinse"). In the process, water is conducted over the solid-phase particles without homogenizing them in the process. This process reduces the quantity of binding mediator (e.g. ethanol) present without detaching the bound nucleic acid from the solid phase. Generally, however, this is not necessary if tetraethylene glycol is used in the washing buffer because it does not interfere with the subsequent reactions. Drying and/or washing with water is necessary for removing ethanol only in the case where the washing buffer contains no tetraethylene glycol but ethanol, i.e. if tetraethylene glycol is only added to the PCR reaction mixture.
Elution is subsequently carried out in order to detach the nucleic acid from the surface again. As a rule, this is done using a weakly saline, optionally buffered, aqueous, pH-stabilized solution. Suitable elution solutions are known to the person skilled in the art.
The DNA preparation thus obtained is then amplified, e.g. by PCR or isothermal amplification.
The preparation of DNA with the so-called MagAttract® technology, in which the DNA is adsorbed on magnetic silica particles (MagBeads), is particularly preferred according to the invention. These particles are available from Qiagen. The reason for this is that, when using these particles, substances from the washing buffer, such as, for example, ethanol or tetraethylene glycol, can be removed only with difficulty. This can be done successfully only to a partial extent by drying (air dry), and slightly better by washing. However, both processes mean complex additional steps in the purification protocol. However, residual ethanol present in the eluate interferes with the subsequent PCR to a considerable extent up to and including total failure, whereas, surprisingly, tetraethylene glycol does not interfere with it.
The PCR technique is comprehensively described in, for example, PCR Technology: Principles and Applications for DNA Amplificaition, Erlich, editor (1992); PCR Protocols: A Guide to Methods and Applications, Innis et al., Hrsg. (1990), R. K. Saiki et al., Science 230:1350 (1985) and U.S. Pat. No. 4,683,202.
PCR is an enzymatic method for the synthesis of specific nucleic acid sequences using two oligonucleotide primers (probes) that hybridize with opposite strands and flank a section of a target nucleic acid that is of interest. A series of repeated reaction steps comprising template denaturation, primer annealing and extension of the hybridized primer by DNA polymerase results in the exponential accumulation of the specific target fragment, whose ends are defined by the 5′-ends of the primer. The PCR method uses repeated cycles of DNA synthesis for the replication of the target nucleic acid. In the basic embodiment, PCR comprises the following steps:
- Adding specific primers to a sample which is assumed to contain the target nucleic acid. The primers are selected in such a way that they bind to complementary DNA strands present in opposite strands of the DNA. The target nucleic acid is localized within the primer binding sites and is a marker of the organism to be detected.
- Adding the four desoxynucleoside triphosphates, magnesium sulfate-containing buffer, polymerase enzymes and differrent additives and cosolvents, mixing with the sample and the primers
- Denaturing the sample by heating, separation of the DNA into two complementary strands
- Cooling, thus binding (hybridization) of the primers to the respective binding sites on the complementary DNA strands
- Primer extension on the DNA template by DNA polymerase.
These three steps, denaturing, hybridization and extension are repeated 40 to 50 times and result in an exponential amplification of the target sequences.
According to the invention, the PCR in step d) can be carried out as an end-point PCR or as a realtime PCR (quantitative PCR, qPCR). The procedure is known to the person skilled in the art. EP-A-0 512 34, EP-A-0 640 828, EP-A-0 519 338 (F. Hoffmann-La Roche AG) disclose, for example, qPCR. For this purpose, systems are on offer commercially, e.g. TadMan® (Roche Molecular Systems, Inc. Branchburg Township, N.J.).
According to the invention, all reagents customary for DNA PCR and kits available therefor can be used for carrying out the PCR. The primers, desoxynukleotide triphosphates, polymerases, the additives, cosolvents and marker probes are, as was described in the introduction, known to the person skilled in the art and are suitably selected. For example, appropriate PCR reagent sets by QIAGEN, such as QuantiTect® Probe PCR Mastermix or QuantiFast® Probe RT-PCR MasterMix, can be used for a TacqMan® analysis. For example, QuantiTect® PCR-Mastermix, QuantiTect®5× Virus-Mastermix or HotStarTaq®-Mastermix is, according to the invention, suitable for carrying out the PCR.
The primers are suitably selected and can be synthetically produced with methods known to persons skilled in the art, or are commercially available, for example from the companies Operon or MWG Biotech.
All markers known to persons skilled in the art can be used as markers. In particular, fluorescent markers such as 6-FAM, TET, Quasar® and/or HEX, are used for detection. Depending on the requirements, the person skilled in the art will also use other fluorophores.
Water is usually used as a solvent for the PCR reagents and primers.
According to the invention, the reaction solution for amplification contains tetraethylene glycol, preferably in an amount of up to 20 vol %, preferably up to 12 vol %, more preferably up to 10 vol %, based on the reaction solution. Surprisingly, it was found that the PCR proceeds very reliably and can be carried out successfully even in the presence of such large quantities of tetraethylene glycol.
In contrast, a reaction solution that contains already small amounts of tetraethylene glycol (e.g, from 1 vol %) is unsuitable for the amplification of RNA because the amplification efficiency deteriorates extremely in this case. Most frequently, no reaction can be observed anymore during RNA amplification at a content of 9 vol % TEG in the reaction batch.
The temperature cycles of the PCR are selected by the person skilled in the art in accordance with the PCR chemistry used, the underlying initial DNA and the primer selected and can be, for example, 15 min at 95° C. and 40× (15 seconds at 95° C., 1 minute at 60° C.). Temperature monitoring and the heating and cooling processes take place in the usual manner.
According to the invention, the DNA amplification can also be carried out as an isothermal amplification. Isothermal amplification processes are known in the prior art. They have in common that no temperature cycles are being used but that a certain reaction temperature is continually maintained. Examples for such processes that are suitable for DNA amplification and which therefore may be used according to the invention, include HDA (Helicase Dependent Amplification, see M. Vincent et al., EMBO, reports 2004, 795-800), RPA (Recombinase Polymerase Amplification, see O. Piepenburg et al., PloS Biology, 2006, 1115-1120), EXPAR (exponential amplification reaction, see e.g. J. Van Ness et al., PNAS 2002, 4504-4509].
A Hugl-PCR may also be carried out. Hugl is a target in human genomic DNA. The primer pair used for this PCR was developed by Qiagen some time ago. The PCR carried out therewith is very susceptible to interferences and is therefore used for demonstrating that preparations produced by DNA are particularly clean or contain no or almost no inhibitory contaminations.
The detection of the amplification products can take place in different ways, as was described in the introduction, depending on the markers used. This is known to the person skilled in the art. If fluorescent markers are used, detection is carried out with fluorescence spectrometry. In conventional PCR processes, up to 6 different fluorophores can be detected simultaneously (6 channel multiplexing). Then, the number of PCR cycles from which a significant drop of the fluorescence of the specific marker occurs (Ct, threshold cycle) is determined.
The classic PCR reaction is an end-point PCR. It is thus possible to detect the presence or absence of a target DNA; however, the method does not yield any information as to the initial concentration of the target DNA.
Real-Time PCR(RT-PCR; realtime polymerase chain reaction; also referred to as quantitative PCR (qPCR)) was developed in recent times. It enables the simultaneous observation of several nucleic acid amplification reactions by PCR, with the accumulated data being used for the quantitative determination of the initial concentration of a target nucleic acid. Therefore, this PCR reaction is also referred to as multiplex PCR. Real-Time PCR is disclosed, for example, in EP-A-0 512 34, EP-A-0 640 828, EP-A-0 519 338 (F. Hoffmann-La Roche AG). For this purpose, systems are on offer commercially, e.g. TadMan® (Roche Molecular Systems, Inc., Branchburg Township, N.J.). The method enables the rapid and precise quantification of the initial number of copies over a very broad concentration range. It is suitable for many applications such as gene expression studies, sequence and mutation analyses, testing of virus titers, the detection of pathogenic or genetically modified organisms. A Real-Time PCR kit with sequence-specific probes is available, for example, from Eppendorf AG, Hamburg (RealMasterMix Probe®) or from Qiagen GmbH, Hamburg (Artus® PCR Kits).
In the TagMan® PCR, the oligonucleotide probes are designed in such a way that they bind to the target nucleic acid sequence between the extension primers. Each oligonucleotide probe is marked on the 5′ end with a reporter molecule, such as a fluorophore, and on the 3′ end with a reporter molecule quencher. The marked probes are added to the PCR reaction mixture together with the primers and the sample. After denaturing, the reaction mixture is cooled off, wherein the marked probes preferably bind to the target nucleic acid sequence. Then, a cooling process is carried out down to the optimum temperature for primer binding and extension. During polymerization, when the DNA polymerase proceeds along the target nucleic acid strand from the 3′ end to the 5′ end with nucleotides being bound to the growing primer, the primer encounters the 5′ ends of the marked probes previously bound to the target nucleic acid strand. When the DNA polymerase encounters these marked probes, it exercises its 5′-3′-exonuclease activity and disintegrates the marked probes. Fluorophores and quenchers are released into the reaction mixture in the process. The TagMan® assay is designed in such a way that reporter molecules present within a predetermined vicinity of the quencher molecule emit almost no fluorescence. Therefore, no fluorescence due to the reporter molecule is initially detected in the PCR mixture, because the fluorescence of the reporter molecule is quenched by the quencher molecule located in spatial proximity. During PCR, the 5′ fluorescence-marked probes are released and separated in the process from the 3′ fluorescence quenchers. After the release, the fluorescent marker is quenched no longer and can therefore be detected by fluorescence spectrometry or other suitable means. Non-bound probes present in the reaction mixture are not an interference because they remain quenched. Marked probes which are unspecifically bound to nucleic acid sequence that have nothing to do with the target nucleic acid, also do not interfere because they are bound and therefore remain quenched. Therefore, every free reporter molecule detected in the reaction mixture is directly proportional to the quantity of the originally specifically bound marked probe, and therefore of the target nucleic acid. For detecting different target nucleic acids in a sample, probes with different fluorescence reporter molecules are used which fluoresce in different wavelength ranges and therefore enable the simultaneous detection of different target DNAs. 6-FAM, VIC, Bodipy-TMR, JOE and HEX are known and suitable as fluorescence reporters, TAMRA, MBG, Black Hole Quencher 1 (BHQ1), Black Hole Quencher 2 (BHQ2), Black Hole Quencher 3 (BHQ3), BodyPi 564/570 and Cy5 or the like are known and suitable as quenchers. The person skilled in the art will suitably select the reporters and quenchers preferably in such a way that a mutual superposition of the fluorescent emissions ("bleed through") is avoided. The combination 6-FAM/BHQ1, for example, is particularly suitable.
Apart from fluorescent markers, radioactive markers, chemiluminescent markers, paramagnetic markers, enzymes and enzyme substrates may also be used as markers.
Subsequent to amplification, the detection of the nucleic acids takes place by means of fluorescence spectrometry, as described above, or if other markers are used, by means of other detection methods.
The aforementioned nucleic acid preparation and amplification and detection methods can be carried out on a laboratory scale, i.e. on the micron or millimeter scale. Alternatively, microfluidic processes were developed in recent times. These may also be used according to the invention.
Microfluidics is understood to be handling and working with small quantities of liquid that have a volume that is lower by powers of ten than normal drops of liquid.
Devices for carrying out microfluidic methods that process a sample until the desired measurement result is obtained are also referred to as lab on a chip (LoC), i.e. disposable material as a laboratory on a chip of the approximate size of a credit card, with which molecular-biological determinations can be carried out, e.g. the determination of pathogens of an infection, foodstuff control, veterinary diagnostics, biological weapons analysis, environmental analysis. Analysis requires only few personnel and no special education or training for operation. LoC are inserted into an operator instrument having the size of a video cassette recorder, which then outputs the result. An LoC combines on a single chip microfluidic functions for sample extraction and enrichment of the analyte, for signal amplification and detection. The operation of the LoC and the operating unit is very simple, and analysis is extremely rapid compared with conventional analysis methods (30-60 minutes total process time, compared with at least 6 hours).EXAMPLES
Escherichla coli O157:H7 is available from the American Type Culture Collection under No. ATCC 700728.
Purified human gDNA was produced from human blood using the QIAamp® DNA Midi Kit.
Bacteriophage fr is available from the American Type Culture Collection under No. ATCC 15767-B1.
QA=QIAamp®, available from Qiagen
MasG=MagAttract® Suspension G, available from Qiagen
PBS=Phosphate buffered saline (solution)
AW=QIAGEN washing buffer AW (1/2), available from Qiagen
TEG=Tetraethylene glycol
SPE=Nucleic acid solid phase extraction (by means of QA o. MasG)
qPCR=Quantitative PCR
RT-qPCR=Reverse transcription-quantitative PCR
BHQ=Black hole quencher
The effects of various washing procedures on the efficiency of end-point and realtime PCR, respectively, are described in the following examples. The washing process with TEG described herein was respectively carried out with the buffers TEG1 and TEG2 as a substitute from AW1 and AW2. The buffers TEG1 and TEG2 are composed as follows:
TEG1: 48 vol % tetraethylene glycol, 2.5M guanidinium-HCl, remainder water
TEG2: 60 vol % tetraethylene glycol, 100 mM NaCl, 10 mM Tris/HCl pH 7.5, remainder water
40 μl of an overnight culture of E. coli O157:H7 was added to 800 μl whole blood or PBS, respectively. To this, 80 μl QIAGEN proteinase K and 800 μl buffer AL were added and this was mixed. This mixture was now incubated for 15 at 56° C. The lysate thus produced was used for all further purifications of the DNA obtained, DNA purification took place three times in each case, using the QIAamp® DNA Mini Kit or by means of a manual MagAttract® protocol, respectively. Purification using a QA DNA Mini Kit® was carried out in accordance with the instructions by the manufacturer and comprised 2 washing steps with AW1 and AW2, respectively, a short drying of the bound nucleic acid by air and elution with deionized water. The MasG® protocol comprised the following steps: 200 μl lysate was mixed with 200 μl isopropanol and 20 μl MasG® by means of short vortexing and then shaken for 5 min at 1400 r.p.m. on a thermomixer. Then, the MasG® beads were magnetically separated and the supernatant was removed and discarded. The MasG® beads were washed by iterative resuspension in the corresponding buffer, subsequent magnetic separation and removal of the supernatant four times with AW1 and AW2, respectively, in each case. Then, the separated beds were dried in air for 15 min in an open 1.5 ml reaction vessel and then resuspended in 100 μl water. For elution of the DNA, the beads distributed in the water were shaken for 2 min. Then, the beads were separated and the supernatant containing the DNA was removed. The DNA solutions thus obtained were added to a qPCR reaction. This was carried out by receiving 40 μl QuantiTect® PCR-Mastermix (Qiagen GmbH), 40 pmol forward primer (SEQ ID NO:1; 5′-CGATGATGCTACCCCTGAAAAACT-3′), 40 pmol reverse primer (SEQ ID NO:2; 5′TATTGTCGCTTGAACTGATTTCCTC-3′). 20 pmol probe oligo (SEQ ID NO:3; FAM-CGTTGTTAAGTCAATGGAAAACCTG-BHQ1-) in a 96-well PCR plate and drying the mixture overnight at 37° C. The dried-up reaction mixture was rehydrated with 80 μl eluate or 78 μl water plus 2 μl eluate, respectively, and shaken for 5 min at 1400 r.p.m. on a thermomixer. Then, the redissolved reaction mixture was subjected to the following temperature profile in a real-time cycler (MJ Research Opticon, Biorad): 15 min 95° C., 40×[15 sec 95° C., 1 min 60° C.]. The result is shown in
Result: While QA preparation permits the use of large quantities of eluate for the subsequent preparation, this is not possible for eluates obtained with the MasG® technology. One possible reason for this is the incomplete removal of ethanol from the MasG® beads prior to DNA elution.Example 2
As described in Example 1, a joint lysate of a mixture of 100 μl E. coli O157:H7, 3-ml PBS, 300 μl Qiagen proteinase K and 3 ml GIN3 [3M guanidinium isothiocyanate, 20% NP-40 (Nonidet P-40, octylphenoxypolyethoxyethanol)]. This joint lysate was carried out exactly as in Example 1, with the following exceptions. For washing the bound nucleic acid, TEG1/TEG2 was also used apart from AW1/AW2. Washing during the MasG®-based purification was carried out twice in each case with washing buffer 1 and 2. In the case of the samples treated with TEG-based washing buffer, the DNA-loaded MasG® beads were rinsed with 500 μl instead of being dried for 15 minutes. During this rinsing process, the beads were not resuspended, but the reaction vessel remained in the magnetic holder during the rinsing process, the holder ensuring that the beads were separated from the liquid as a compact pile. This step is necessary because TEG, in contrast to ethanol, virtually does not evaporate at room temperature due to its very high boiling point. The DNA solutions thus obtained were added to a qPCR reaction. This took place exactly as described in Example 1, by rehydration of a previously dried reaction mix with 65 μl eluate, or 63 μl water pülaus 2 μl eluate, respectively. The PCR conditions are also identical to those in Example 1. The result is shown in
Result: If large quantities of eluate from a MasG® DNA preparation are to be added to a subsequent qPCR, the use of ethanol-containing washing buffers (AW1/2) leads to inhibition effects that cause a total failure of the PCR. If TEG-containing washing buffers are used instead (TEG1/2), a previously dried PCR mastermix may be reconsitituted exclusively with this eluate and a qPCR can be carried out successfully.Example 3
A PCR was carried out in the tumor suppressor gene Hugl (abbreviation for human giant larvae) of 3 ng purified human gDNA with the HotStarTaq® mastermix (25 μl final volume; 10 pmol forward primer (SEQ ID NO:4; 5′-CACACAGCGATGGCAGCTATGC-3′, 10 pmol reverse primer [SEQ ID NO:5; 5′-CCCAGTGATGGGCCAGCTC-3′) in the presence of rising concentrations of TEG in the reaction batch with the temperature profile 15 min 95° C., 40×[30 sec 95° C., 30 sec 59° C., 1 min 72° C.], 5 min 72° C. It is known that this reaction is very susceptible to interference with the primers used, e.g. with regard to contaminations of the DNA template. The PCR product yield in the reaction batches was then examined in conclusion by agarose gel electrophoresis. The result is shown in
Result: The Hugl PCR carried out here can also be carried out sucessfully in the presence of large quantities of TEG (10 vol %).
Examples 1-3 demonstrate the superiority of TEG-containing washing buffers over ethanol-containing washing, buffers when using the MagAttract® technology for DNA purification. TEG washing buffers are particularly advantageous if large quantities of the produced DNA-containing eluate are to be used in enzyme-dependent amplification reactions (in this case quantitative PCR). This option is particularly important if the sample material used contains only minute nucleic acid quantities (viruses in low titers, single cells of forensic material, etc.).
The following examples demonstrate that TEG-containing washing buffers are unsuitable for RNA extraction from biological samples. Since TEG does not negatively affect the PCR reaction, as was demonstrated above, this observation is probably to be ascribed to a disruption of the activity of the reverse transcriptase by TEG.Example 4
A joint lysate of a mixture of 30 μl bacteriophage fr (a bacteria-specific virus with RNA-based genome), 3 ml blood, 300 μl Qiagen proteinase K and 3 ml GIN3 was incubated for 15 min at 56° C. SPE (solid phase extraction) was carried out solely by means of the MagAttract® method. Similar to the description in Example 1, the large lysate batch was divided into 600 μl portions and in each case 300 μl isopropanol and 30 μl MasG® was added. For washing the bound nucleic acid, either AW1/AW2 or TEG1/TEG2 was used again. A washing process was carried out once with 1 ml AW1 or TEG1, respectively, and twice with 0.5 ml AW2 or TEG2, respectively. This was followed by 5 minutes of air drying. Finally, the purified RNA was eluted with 100 μl water. All SPE steps (binding/washing/eluting) were carried out using the robot BioSprint® 15 (Qiagen, Hilden). The RNA thus obtained was subsequently used in an RT-qPCR. For this purpose 5 μl, 25 μl or 39 μl eluate, respectively, was mixed with 10 μl QuantiTect® 5× Virus mastermix (Qiagen, Hilden), 20 pmol primers in each case (forward: SEQ ID NO:6; 5′-CTTCTGATCCGCATAGTGACGAC-3′; reverse: SEQ ID NO:7; 5′-AACGGTCATTCGCCTCCAGCAG-3′) and 10 pmol probe (SEQ ID NO:8; 6-FAM-TAGGGGATGGTAACGACGAAGCA-BHQ1) against the fr genome, and 0.5 μl QuantiTect Virus PT Mix (final volume 50 μl). The following temperature profile served for the cDNA production and amplification: 20 min 50° C., 5 min 95° C., 40×[15 sec 95° C., 45 sec 60° C.]. The result is shown in
Result: Basically, the eluates washed with AW buffers show a significantly better RT-qPCR performance (tower Ct value) than the batches washed with the TEG buffers. Moreover, it is found that the use of large quantities of TEG eluate (39 ml to 50 μl RT-qPCR batch) not only leads to a poorer Ct value as compared to the use of smaller TEG eluate quantities (5 μl to 50 μl final volume), but may also lead to a total failure of the amplification reaction (only 1 of 4 batches with 39 ml per 50 μl reaction batch exhibited a successful amplification reaction).
One explanation of the poor performance of the tetraethylene glycol-washed samples is the fact that TEG depletion is not ensured due to the air drying process that was carried out. TEG has a boiling temperature of 306° C. and will therefore not transition into the gas phase during air drying.Example 5
Bacteriophage fr was also added to human blood and lysed, and the RNA was purified also in this example. All lysis and SPE steps were carried out in a manner identical to example 4. However, in order to remove TEG remaining after the washing steps as effectively as possible, the air drying process was in this case replaced with a water rinsing process. Again, 5 μl, 25 μl and 39 μl, respectively, were mixed for the subsequent RT-qPCRs with the same PCR components as in Example 4 to result in a final volume of 50 μl.
Result: As in Example 4, the eluates washed with AW buffers show a significantly better RT-qPCR performance than the batches washed with the TEG buffers. This time, the inhibition while using large quantities of TEG eluate (39 μl per 50 μl RT-qPCR batch) is much lower. This shows that washing with water removes residual TEG from the Mag-Beads® better than drying in air. Nevertheless, it remained inexplicable up to this point why the TEG eluates obtained poorer results as a matter of principle than the AW eluates.Example 6
The question that remained in Example 5, namely why the TEG eluates yielded poorer RT-qPCR results than the AW eluates as a matter of principle was pursued in the following experiment, A RT-qPCR (QuantiTect® Probe RTPCR Mastermix) of purified RNA from bacteriophage fr and a qPCR (QuantiTect® Probe PCR Mastermix) of purified human gDNA (forward primer: SEQ ID NO:9; 5′-GCCGCTAGAGGTGAAATTCTTG-3′, reverse primer SEQ ID NO:10; 5′-CATTCTTGGCAAATTTCG-3′, probe: SEQ ID NO:11; HEX-ACCGGCGCAAGACGGACCAGA-BHQ1), respectively, was carried out in the presence of rising concentrations of TEG in the reaction batch (4 replicates each). The temperature profile used was 30 min 50° C., 15 min 95° C., 4×[15 sec 95° C., 1 min 60° C.]. The result is shown in
Result: Even in larger concentrations, TEG does not change for the worse the performance of the PCR reaction in the case of DNA. This result confirms the data of the Hugl PCR presented in Example 3. In contrast, already small qunatities of TEG (from 1 vol %) lead to a dramatic deterioration of the RT-qPCR reaction efficiency in the case of RNA. At 9 vol % TEG in the batch, none of the 4 RT-qPCR reactions was successful (no amplification discernible).
1. A process for amplifying DNA, comprising amplifying DNS using a reaction solution comprising tetraethylene glycol.
2. The process according to claim 1, wherein the tetraethylene glycol in the reaction solution originates from eluate of a DNA preparation on a solid surface that was used for preparing the DNA.
3. A process for the preparation and amplification of DNA, comprising:
- a) immobilizing DNA contained in a solution on a surface
- b) washing the surface with a washing buffer
- c) recovering the DNA by elution,
- d) carrying out a DNA amplification with eluate obtained in step c),
- wherein the washing buffer comprises tetraethylene glycol.
4. The process according to claim 3, wherein the tetraethylene glycol in the washing buffer is optionally at least 10 vol %.
5. The process according to claim 3, wherein the volume of the eluate in step d) is at least 10 vol %, optionally at least 30 vol % based on the entire reaction volume of the amplification.
6. The process according to claim 1, wherein the tetraethylene glycol in the entire reaction volume of the amplification is up to 20 vol %.
7. The process according to claim 3, wherein the washing buffer moreover contains one or more constituents selected from the group consisting of chaotropic salts and buffering substances.
8. The process according to claim 1, wherein the DNA amplification is carried out as an end-point PCR, quantitative PCR or isothermal amplification.
9. The process according to claim 1, wherein said process is carried out in a microfluidic device optionally comprising lab on a chip, LoC.
10. A kit of parts for DNA amplification and DNA preparation according to the process according claim 3, comprising:
- a solution 1 as a washing buffer comprising tetraethylene glycol, and
- one or more reagents required for carrying out the DNA amplification.
11. A kit of parts according to claim 10, comprising one or more of the following constituents, selected from
- a solution 2, comprising a binding mediator,
- a solution 3, comprising an elution agent,
- a solution 4 comprising a lysis buffer and optionally one or more proteases;
- a microfluidic device optionally comprising lab on a chip, LoC.
12. A kit of parts according to claim 10, which is capable of being used for preparation and amplification of DNA obtained from bilogical materials.
13. A kit of parts according to claim 10, which is capable of being used for preparation and amplification of DNA obtained from samples taken for molecular-diagnostic purposes optionally comprising human and/or veterinary medicine, and optionally from forensic samples and/or from viruses or bacteria contained in blood.
14. Use of Tetraethylene glycol used in a reaction solution for carrying out a DNA amplification.
15. The tetraethylene glycol according to claim 14, wherein the DNA amplification is an end-point PCR, quantitative PCR or isothermal amplification.
16. A reaction solution for carrying out a DNA amplification, comprising one or more reagents required for amplification and tetraethylene glycol.
17. The reaction solution according to claim 13, wherein the tetraethylene glycol in the reaction solution is up to 20 vol %.
Type: Application
Filed: Jun 15, 2010
Publication Date: Jul 5, 2012
Applicant: QIAGEN GmbH (Hilden)
Inventors: Andy Wende (Hilden), Hans Robert Attig (Hilden), Ralf Himmelreich (Langenfeld)
Application Number: 13/380,002
International Classification: C12P 19/34 (20060101); C07C 43/11 (20060101); C12N 9/12 (20060101);


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Last Friday evening, I moderated a discussion at Chapman University. Speaking on the panel were Eugenie C. Scott, recently retired executive director of the National Center for Science Education, Ben Santer, research scientist at the Lawrence Livermore National Laboratory, and member of NCSE's board of directors, and Brian Alters, Professor at Chapman University in Orange, California, and president of the board of NCSE.
As I listened to the panelists talk about ongoing challenges to evolution and climate change education, I was struck by how important NCSE is, and what a privilege it is to be the new executive director.
Why does NCSE matter? Why do we care so passionately that students learn about evolution and climate science in a manner that is unmuddied by pseudoscience or false controversy? Here are just three of many reasons.
First, NCSE matters because we support the teaching of two of the thorniest topics in science education: evolution and climate change. Despite their importance, there is widespread resistance to these areas of science being taught well—or at all. The reasons for the resistance are somewhat different: resistance to the teaching of evolution is primarily religious in nature, while resistance to teaching about climate science is primarily political. Still, as with evolution, climate educators have to struggle with pernicious efforts to persuade people that the science is controversial. A further challenge for climate educators is the lack of a clear understanding of where this inherently multidisciplinary topic fits into the traditional science curriculum. In both cases, however, NCSE has the long and deep experience of working with educators necessary to help them address these issues.
Second, NCSE matters because the topics we focus on—evolution and climate change—not only face challenges in the classroom, but also are prime examples of how science denial is used to advance religious and political agendas. Casting doubt on science can be an effective weapon in the political arena, but it is an extraordinarily dangerous one for our society, which is facing complex challenges in the areas of health, food, energy, and environment. Hard choices and wise decisions will have to be made between competing priorities and where to invest scarce resources. Learning how to evaluate evidence and draw justified conclusions as scientists do are skills that our children need to develop, whether they become scientists themselves or not. Evolution and climate science both provide powerful case studies for students to learn about the nature of science and the sources of science denial.
Finally, we cannot afford to waste a single brilliant mind. Sitting in a classroom in Louisiana right now might be the girl who could grow up to discover the cure for Alzheimer's disease, or the boy who could grow up to develop drought-resistant wheat. Many of today's most respected scientists grew up in small towns with nary a research scientist role model in sight. J. Michael Bishop, for example, a winner of the Nobel Prize in Physiology or Medicine in 1989, grew up in a small rural town in Pennsylvania and received his first eight years of education in a two-room school. Phillip Sharp, a winner of the 1993 Nobel Prize in Physiology or Medicine, grew up in a rural community in northern Kentucky not far from where the Creation Museum now stands. Some science teacher, somewhere along the line, captured their imaginations and set their feet on the path to careers in science. Our lives have been enriched as a result. What if, instead, they had been taught that evolution is a flawed theory, climate change is a hoax, and scientific questions can be resolved by debate or reference to the Bible, rather than by evidence? Our world would be the poorer for it. NCSE knows this, and such miseducation is what we work tirelessly to combat.
NCSE's work is far from over. Educators still need our support to push back against misinformation and find the best resources possible to teach about evolution and climate change. I feel fortunate to be joining a team of scientists and advocates who work to ensure every child in America receives the best science education on topics that are challenging for many educators to address. Without NCSE, where would educators go when a student brings a creationist text to class or a parent demands that "both sides" of climate change be taught in her classroom? NCSE has been the go-to place for students, parents, educators, scientists, and community members for help dealing with science denial. I am thrilled to be taking on these challenges as the new executive director, because I don't want to imagine a world without NCSE. Do you?
(Photo credit: Rachel Fechser, The Panther)

subset: commoncrawl


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Several recent surveys reveal growth in DDRn, USB 3.0, PCIe, HDMI, SATA, and MIPI interfaces. Also, ethernet and ARM buses remain popular in specific areas.
By John Blyler, Editorial Director, IP Systems
Recently, I came upon a very brief chip design and functional verification survey conducted by Arrow Devices (see Figure 1). Although the survey size was fairly small – 56 participants – the data on interface protocol selections caught my eye.
Read the complete story at Chipestimate.com "IP Insider"


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A Verisk Business
Sign-in to our platforms to access our extensive research, our latest insight, data and analytics and to connect to our industry experts.
23 September 2019
China's electricity market is becoming cleaner, more efficient and more liberalised.
29 August 2019
Turkey's energy demand is expected to show strong growth throughout the forecast as the country's economy thrives
28 August 2019
This report analyses regularly the gas and power fundamentals and prices for the next six months in Argentina, Brazil, Chile and Bolivia.
02 August 2019
Despite integration challenges, green states are now challenging the industry to meet very deep decarbonization goals.
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In our office, we strive to give you the best possible dental service which you can see by reading some of the many patient reviews below. To help us spread the word please take a few minutes to post a testimony on one of the following websites. If by any chance we did not fully meet your expectations, please be kind to let us know and we will do our best to accommodate your needs.
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Aug 2013
4 Healthier Fast Food Picks
Who wouldn't love the luxury of always having a home-cooked meal, well, for every meal? In the end though even the best of us succumb to the drive-through from time to time. Just because that happens doesn't mean it has to sabotage your whole day. Now while I can't be there to give you the will power to side step ordering fries while the smell wafts into your car, I can give you some go to items so that choosing will be a no-brainer. Keep in mind that these may not be the best or optimal choice let's say compared to preparing something at home BUT they are BETTER choices when the alternative is skipping a meal or a double cheese burger , a shake and fries.
1. McDonald's: Premium Caesar Salad w/ Grilled Chicken
Packs 27 gms protein and 4 gms of fiber and surprisingly one of the lower sodium items for most sides/entrees (580 mg) it's ahead of the pack compared to other choices on the Golden Arches menu.
2. Starbucks: Chicken & Hummus
If you haven't tried hummus this is a good place to start. Starbucks offers more than caffeinated treats–you can get in 20 gms protein, 5 gms fiber and at 260 calories this is a great alternative to those 400+ calorie bakery items!
3. Whatburger: Scrambled Eggs
Whataburger may not conjure images for breakfast BUT their catch-phrase is "Just like you like it" so if your looking for a simple breakfast option on-the-go just ask for plain scrambled eggs and you'll get 14 gms protein, 160 calories–a much healthier option than most other breakfast fare.
4. Subway: Egg White and Cheese Egg Muffin
Well well well Subway has one of the healthiest breakfast options–especially if plain scrambled eggs just aren't going to cut it. This Egg white and Cheese Egg Muffin is 170 calories, 12 gms protein and 5 gms fiber.
For more nutrition information or to compare the items above to other menu items just click the links in the photo credit–one of the most important tips for any nutrition endeavor that I can recommend–AWARENESS!
13 08 2013
Very helpful! Thanks for the good read!


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Pot and Pan Hanger for Kitchen
Here are some considerations when using pot and pan hanger for the first time.
Here are some considerations when using pot and pan hanger for the first time.
Planning a small kitchen is not a joke. End up with a family meal
Copper cabinet pulls are one of those items that sometimes beg the question: is
If you're a budding chef, an accomplished cook or just a homeowner with beautiful
Kitchen tiles has been around for many years and has been used in various areas
Since you are likely to spend a lot of time in the kitchen, it
Kitchen Remodeling – Traditionally, the kitchen always been the center of family life and
Planning to give a whole new look to the existing kitchen in Denton, TX
Cabinets used to store almost all foods, pots and pans and cooking utensils. They
The kitchen is the heart of most homes. Here, the family pulled together to


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Secure Online Booking!
Book & pay online & get your lodge cheaper!
Silverdale Lodge
2 Bedrooms | Sleeps 4 | Free Wi-Fi | Hot Tub | No Pets Allowed
Silverdale Lodge is a stunning contemporary and newly built lodge. With the feel of a luxury beach house, with its wooden clad walls and bright colours, in a fantastic corner plot of the new Yealands development.
Accommodation details
The entrance door leads into the open plan living/kitchen/diner, with picture windows leading onto a secure and furnished veranda. Enjoy the use of the hot tub. The hallway leads to the rear of the lodge with a Twin bedded room, family bathroom and a Double bedroom with en-suite shower room.


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A 19-year-old woman and her 21-year-old husband were detained on the island of Lesbos on Wednesday after health workers became suspicious when they took the infant for emergency treatment at a local hospital.
Social workers raised the alarm after the couple first insisted the child was theirs but later claimed that in fact they had been looking after it for a family in Athens.
The man's 51-year-old mother, who lived with the pair at a Roma settlement in Mytilini on the island, was also detained pending further investigation.
A spokesman for General Police Command for the North Aegean area confirmed: "We have three people in custody and are trying to determine the true parentage of the infant." The arrests came just hours after a Greek state prosecutor warned that further cases of child abduction and fraudulent registration were likely to emerge as he ordered an emergency investigation into all birth certificates issues across Greece in the past six years.
Efterpi Koutzamani, ordered the probe following the discovery of Maria, the blonde girl claimed by a Roma couple in Farsala as their own, after DNA test proved she was not their biological daughter.
"The 'Maria' case may not be an isolated incident and this could have happened in other parts of the country," the Supreme Court prosecutor's order said.
It also emerged yesteday that just weeks ago a fair-haired 23-year-old man brought up in a Roma settlement in Thessaloniki, northern Greece, was DNA tested by police to determine whether he could be Ben Needham, who disappeared as a toddler on the island of Kos in 1991.
"I hope I am little Ben," the man told police before discovering that there was no DNA match with the Needham family.
Christos Salis, 39 and Eleftheria Dimopoulou, 40, the couple suspected of abducting the blonde child known as Maria, were charged with child abduction and sent to jail pending trial on Monday.
They deny snatching or buying the child but insist she was given willingly into their care by her destitute Bulgarian mother after being approached by a Bulgarian middleman, reportedly named Michalis.
Greek Police have been trying to locate the Bulgarian man and woman and have asked for assistance from Bulgarian police to trace the couple.
One theory being considered by investigators is that Maria, who is aged between five and six, may have been a victim of an extensive child trafficking network and was left at the settlement in Farsala, 200 miles north of Athens after plans to sell her into illegal adoption fell through.
The case has highlighted weaknesses in the country's birth registering system that are exploited by child traffickers.


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Customized Stock Plan Administration Manuals
To begin the creation of the Stock Plan Administration Manual, CompIntelligence will work in partnership with the Client to perform a comprehensive evaluation of the current internal equity compensation-related processes using industry subject matter experts (SMEs). CompIntelligence will spend the initial part of this phase with due diligence where we will interview key stakeholders, management, and vendors to understand the current process.
In the discovery and due diligence process, we will uncover the pain points and wish list items for all areas. CompIntelligence will develop documentation with data flows and system diagrams. We will also include recommendations for changes to the currently-used processes. We will create specifications for the ideal new processes based on what the Client stakeholders have shared with us, as well as our SME knowledge, and also create a roadmap of the steps to achieve the defined goals. Finally, we will create new, detailed process documentation incorporating new processes and newly adopted solutions if applicable.
Sample Project Scope:
- Project typically consists of both onsite and remote work
- Due diligence and process analysis
- Review of Client Stock Plans and current documentation
- Interviews with stakeholders, management and daily operations
- Process analysis and review with Client on equity platform procedures
- Data analysis and review with Client on transaction data and reporting
- Data flow analysis detailing integration/automation strategy
- Participant communication flow
- Provide guidance and recommendations based on analysis to incorporate into Client's current documentation or proceed with the Stock Plan Administration Manual as described below.
- Initial Deliverable: Executive Presentation of process review and recommendations
Overall Project Deliverable: Stock Plan Administration Manual
- More detailed identification of key recommendations not included in current process
- Document the equity administration and reporting business processes including, but not limited to:
- Business process mapping
- Data flows
- Tools / systems used in the process
- Control points
- Manual reconciliations performed
- System and data flow diagrams
- International processes and areas of concern
- Vendor interfaces and management
- The deliverable is a navigational PDF and Word version of the manual
- CompIntelligence will conduct a review meeting of the final deliverable with Client to explain all contents of the manual.
Need help finding the right solution?
Request a free consultation with one of our leading industry advisors to discuss the best solution for your organization's unique challenges and goals.
To request a free consultation, please contact us by filling out the form to the right


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Blog and Article Syndication SEO Link Building
[ANNOUNCEMENT FROM ADMIN] Anyone that requests to join Syndication Express must use their real name and put an acceptable profile picture up. This is a community for bloggers, article writers and authors. Your blog content will be shared on all the major social sites giving you massive exposure and branding. Additionally community members are encouraged to read, comment and share network members blog posts to the various social and bookmarking sites for SEO purposes. By doing this, members blog post will receive viral exposure, social signals, one way backlinks and targeted traffic from the search engines.
ATTN SE Members:
Invite your blogging friends to the Syndication Express community.
Put your 728x90 banner ad above for 5 days for $5.00.
After payment, message me with your banner details.
Puzzle 6: _ _ _ _ _ _ _ _ L L _ _ _ _ _ _
Clue: Event
Click here to solve the puzzle for banner advertising
Click each link to follow Syndication Express on Facebook, Twitter, Pinterest. Also follow the Syndication Express blog.
If YOU would like to post your banner ad below click here now to learn more. Your support will help to keep it free forever.
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OUR SPECIAL FEATURE: Syndication Automation - Will give your blog post massive syndication and exposure on all the major social sites and generate traffic to your blog content. CLICK HERE to learn about this UNIQUE feature on Syndication Express.
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Your banner ads will get exposure every time someone visits the Syndication Express site. Members and non-members are visiting the SE blogging platform.
Started this discussion. Last reply by Merle Gibbins Jun 22, 2014. 3 Replies 3 Likes
There is a growing interest once again in Bitcoins and they may one day become an accepted…Continue
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BE FULLY INFORMED ABOUT BITCOINShttp://qoinpro.com/2f0f00dde4e254a95896f766dbb96fb5There is a growing interest once again in Bitcoins…Continue
Started this discussion. Last reply by Merle Gibbins Jun 23, 2013. 6 Replies 1 Like
The very recently launched software…Continue
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There are so many people on line today who claim to be Gurus in the Network marketing industry that are really only there to entice unsuspecting newcomers to purchase training packages or software…Continue
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Posted on December 1, 2013 at 10:35am 1 Comment 1 Like
I have always been a lover of the sea and have been an enthusiastic SCUBA diver since 1972. Having spent my early diving years in the cold and murky waters surrounding England it was a fantastic experience for me when I moved to live in Asia in 1975 and discovered the untold pleasure of diving in the crystal clear tropical waters of the pacific.
It is impossible to adequately…Continue
Posted on November 6, 2013 at 11:30pm 0 Comments 0 Likes
In these trying times I am continually looking out for news that might bring some fun into the world and in so doing, brighten up your day. There is a farmer in Bathgate in Scotland who came up with a novel idea for brightening up the day for motorists who drive by his farm on the M8 motorway.…Continue
Posted on November 5, 2013 at 6:12am 0 Comments 0 Likes
Well we have just successfully survived another Halloween season when horrendous ghouls lurk around every corner and many house are decorated with terrifying figures. My own friend in Manila decorated her home with several scary creatures such as this one ~
Posted on November 4, 2013 at 3:18am 5 Comments 3 Likes
Posted by James Mc Combs on November 03, 2013
0 Comments…
My pleasure James !
I am delighted to be connected with you on Syndication Express. Your blog on the servere damage done to the Basilica of Santo Niño was news to me. I hope the cathedral can be restored, and it doesn't take a century to do it. God is Good and Works in Mysterious Ways.
Thanks for befriending me James. Great website.
I love your cover too James. Where did you get it?
Interesting cover for Syndication express. Nice to meet you and thanks for the connection.
Hello James I just love your page. I hope that your day and weekend is filled with blessings and continued success in all you do. Please check out my latest discussion and share with the various social sites. Appreciate it very much.
Thanks for the connection James. I enjoy connecting with like minded individuals. It will be nice to see your posts and to get to know you better. Have a prosperous New Year in 2013.
Hi James, thanks for the friend request. Nice to meet nice people !!
Hello James and welcome to Syndication Express. Please take time and read the Getting Started tab at the top of the page so you will know why I started this network. Glad to have you here and I look forward to seeing your content here. Have a fantastic year.


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Sexual health services are free and are available to everyone, regardless of sex, age, ethnic origin and sexual orientation.
They can help with:
Sexual health
- STI testing and treatment
- HIV testing and post sexual exposure treatment (PEP)
- pregnancy testing
- referral for termination of pregnancy (abortion)
- emergency contraception (morning after pill)
- on-going conception, including long acting reversible contraception (LARC)
- free condoms
Advice and signposting
- sexual assault
- relationship / domestic violence
This centre also offers more comprehensive sexual health and contraception services and manages complex sexual health services.
Please call the booking and information line to discuss an appointment.
There is no general walk in clinic at this centre, but there is a walk in clinic for young people (under 21):
- Monday 3:30 - 6:00pm
Who to contact
- Telephone
- 0333 300 2100 (booking and information line)
- Website
- shswl.nhs.uk/
- Parent Organisation
- Queen Mary's Hospital Roehampton
Where to go
- Address
Queen Mary's Hospital
Roehampton Lane
- Postcode
- Get directions
- Details
- Free
Local Offer
- Description
What is Wandsworth's Local Offer ?
To use this service you:
- can call the booking and information line.
How to complain, compliment or feedback about this service, activity or provider.


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Instrument Flying Refresher by Richard Collins. In this unique and instructive book, veteran IFR pilot Richard Collins takes the right seat beside Patrick Bradley, a relatively new IFR pilot.
The beginning of mastery of instrument flying comes when we develop the ability to be the equivalent of a right-seat critic of our own left-seat flying. (from the Preface to Instrument Flying Refresher)
In this unique and instructive book, veteran IFR pilot Richard L. Collins takes the right seat beside Patrick E. Bradley to demonstrate effective ways to grasp and solve IFR problems and eliminate uncertainties that may plague even current IFR airmen. Bradley, a relatively new IFR pilot, discusses the concerns and errors he has shared with most instrument fliers who are new or rusty. Collins then applies his own experiences and methods, including some lessons learned the hard way, and carefully examines airline and general aviation accidents to point out how threatening situations can be avoided or safely confronted.
IFR is a thinking game, and the authors emphasize ways of avoiding mental lapses that can turn simple difficulties into lethal crises:
Copyright Helicopters Only 1997 - 2020All Rights Reserved V120819GDHC-19310


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- Lakeville Journal
- Millerton News
- Compass A&E
- Obituaries
- Classifieds
- Regional
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Kent considers electing its Region One rep
KENT — At the Kent Board of Selectmen's regular montly meeting on Tuesday, Feb. 6, a parent requested that Kent change the way it chooses its representative to the Region One Board of Education.
In recent years, four of the six towns in the Region One School District have changed from an appointed representative to an elected representative. The town of Canaan/Falls Village was the first town to switch from having the members of its local board of education choose the Region One Board member.


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MADE BY MEN Exhibit during Quilt Show:
Each year we feature an exhibit of the quilts made and quilted by men. Over the years, this exhibit has grown in quantity of quilts and the number of men who submit them, from across the country.
Registration for the 2019 Sisters Outdoor Quilt Show will open on February 15th. Your quilt doesn't need to be completed at registration time. Registration closes when we've reached capacity or May 31st, whichever comes first.
LEFT: Hal of Finishing Touch Quilting, 2016
RIGHT: Love & Gillies B-17 by Collin Fellows Quilter, 2016 SOQS
Below: Mike Denton Quilting, our 2018 Machine Quilter Showcase Exhibit


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ENISA celebrates the European Data Protection Day
On 28 January 2018, ENISA joined the 47 countries of the Council of Europe, and the EU institutions, agencies and bodies to celebrate the 12th European Data Protection Day.
Published on January 29, 2018
© Copyright: Shutterstock
This date marks the anniversary of the Council of Europe Convention 108 on the protection of personal information, the landmark international law in the field of personal data protection.
Privacy by design and security of personal data processing
On this occasion, ENISA takes the opportunity to share some of its work in the fields of privacy by design and security of personal data processing.
With its latest report on 'Privacy and data protection in mobile apps', the Agency analyses the features of the app development environment that affect privacy and security. Additionally, this report discusses the promise that implementing privacy by design holds for data controllers and data subjects alike.
To this end, this report explicitly highlights the need for scalable methodologies and best practices that can help integrate privacy requirements into contemporary app design practices, while enhancing usability and addressing all parties of the complex app ecosystem.
Moreover, ENISA with its 'Handbook on security of personal data processing' complements its previously published guidelines on risk assessment and security measures for SMEs by providing specific use cases on pragmatic processing operations that are common for all SMEs.
For each use case, the handbook provides guidelines on the application of ENISA's approach in order to assess in practice the risks that affect the rights and freedoms of data subjects and to facilitate the adoption of the technical and organisational measures that are commensurate with the risk presented.
ENISA report on privacy and data protection in mobile apps: https://www.enisa.europa.eu/publications/privacy-and-data-protection-in-mobile-applications/
ENISA handbook on security of personal data processing: https://www.enisa.europa.eu/publications/handbook-on-security-of-personal-data-processing/
Stay updated - subscribe to RSS feeds of both ENISA news items & press releases!
News items:


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Regeneration of "Großes Torfmoor"
The raised bog "Großes Torfmoor" is located in the north-east of North Rhine-Westphalia in Germany. The importance for nature conservation is documented by the following national and international nature conservation categories:
pSCi according to the FFH Directive (609 ha)
Special Protected Area according to the EU Bird Directive (79/409/EWG)
(2496 ha)
Nature reserve (Naturschutzgebiet - 553 ha)
measures >>
measures >>
The Naturschutzbund Deutschland Kreisverband Minden-Lübbecke is the project leader. The project is co-ordinated with and supported by the Council Kreis Minden-Lübbecke, the regional administration Detmold, the Ministery of Environment and Nature Conservation. Belting Umweltplanung (company for environmental planning) is engaged for the project management.


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4 out of 5 stars
Read December 2, 2013
Pages: 49
Published: June 1, 2010
Publisher: Harlequin Teen
Another novella, another short review. This short story takes place when Meghan is going to the Unseelie Court with Ash. It fills the gap that happens between book one and two.
I got really annoyed with Ash. He was just all distant and acted like he didn't care about Meghan. He should have made it known that he cared about her since he wouldn't be able to show her feelings once they got to the Unseelie Court. Don't be a jerk doucheface! But Meghan was also kinda needy. I don't like needy girls. Then this wolf comes in and tries to kill Ash because Oberon told him to. But then Meghan tells the wolf that she's going with Ash by choice so it leaves. They also see Puck, who is in a coma thing in a tree, on the way.
Overall, this was a good little filler and I can't wait to read the last book of the series. This review also became longer than I planned it to!


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These galaxies keep spiraling
Towards an infinite number
Of impenetrable black holes—
Don't they know
They can only go in so deep.
new old kid on the blog, with an occasional old or new poem written off the old writer's block
That's the trouble, no warning sign on the Event Horizon. Tiptoe to the brink, and then... Shlup!ReplyDelete
I feel this way every time I read an Armantrout poem.
"Don't they know", indeed
How DARE you say that?!
What if Someone Important were to be eavesdropping?
Grounds for Expulsion from the Dead Tree Galaxy Academy!!
I'm a nobody with impeccable credentials--does that qualify me for exemption?ReplyDelete


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- Animals (1)
- Arts (38)
- Drama and dance (25)
- Gardening (15)
- Hobbies (20)
- Music (29)
- Reading and writing groups (27)
- Speakers (16)
- Yoga and Pilates (13)
Filter your list
- Animals (1)
- Arts (38)
- Drama and dance (25)
- Gardening (15)
- Hobbies (20)
- Music (29)
- Reading and writing groups (27)
- Speakers (16)
- Yoga and Pilates (13)
Filter your list
Ms Jude Warrender, Speaker
Any time or day considered
Speaker to local community and business organisations
Jude Warrender
0114 234 8202
Nick Robinson - Origami Artist
Nick has been folding paper since the early 80's and has been a member of the British Origami Society for over 30 years. He has served on their council for over 25 years. He is editor of their bimonthly magazine and maintains their web site.…
Nick Robinson
0751 686 1262
Over 50s needed for a study on reading and well-being
We are looking for volunteers aged 50+ to help with our exciting research on reading and well-being. Volunteers will get the chance to listen to a free audiobook (from a choice of 4) and will be provided with a £10 Love2Shop voucher as a thank…
Dr Giulia Poerio
0114 222 6544
Parson Cross Knit and Natter
Contact the library for more details
Penistone FM Community Radio
Not for profit, volunteer-led local Community Radio station broadcasting on 95.7FM and online to Penistone and the surrounding area. We promote local Community events and groups and play music from the 50's to the current day.
Pilates Plus - Pilates studio in Chapeltown S35
Pilates sessions with the benefit of a playgroup - you exercise while your child plays in the same room, alongside your mat. Pilates strengthens your core which in turn improves flexibility, gait and kinesthetic awareness, while reducing any lower back pain you may have. Adaptations available for injuries, pregnancy, and diastasis recti. Member of the Register for Exercise Professionals (REPs)
Linda Turner
Poetry Meet at Central Library
An informal group for anyone with an interest in poetry.
Raise The Roof
2nd Thursday in the month at 8.30pm (except Aug)
Raise the Roof is a folk singing club where everyone gets the opportunity to lead a song they know, regardless of experience. Raise the Roof is not a music session. Everyone is welcome. The songs are interesting with lots of choruses and harmonies to join…
Reading Group Hillsborough Library
Library based reading group for adults
Sue Taylor
Roger England, Speaker
Presentation aimed at the general public
Roger England
0114 268 0951
Royal Scottish Country Dance Society, Sheffield Branch
Monday 7.30-9.45pm (intermediate and beginners, term-time only, please contact for dates)
Come and join a friendly group who meet at 7.30 pm on Monday evenings at Silverdale School, Sheffield. Qualified RSCDS teachers. No partner required. Beginners especially welcome! For more information see our website.
0114 236 6723
The Royle Art Group
In late Autumn, Winter & Early Spring, the Group meet at St Chads' Church House, 56 Abbey Lane, S8 0BP. These meetings are held fortnightly on Monday evenings from 7.40pm - 9.40pm with a break for refreshments. Themes are programmed for each evening but…
SAGE (Support Arts Gardening Education), also known as SAGE Greenfingers
SAGE is a social therapeutic horticulture (STH) and an arts project for adults experiencing mental health difficulties
Louisa Bruce
0114 273 7718 07932 927153
SAGE Greenfingers - singing and arts
Women only singing group. The group is open to adults of any singing ability who are experiencing mental health challenges
0114 273 7718
Scott Engering, Speaker and field trip leader.
Illustrated talks and field trips for local history and nature conservation groups. I speak on geology, historic stone architecture and medieval churches in and around South Yorkshire and investigate their construction history.
Scott Engering
07908 371258
The Sentene Begins Here, a Course in Creative Writing
13th May-22nd July (10 weeks)
Taking inspiration from everything and anything, this friendly, and relaxed course makes creative writing accessible to everyone.
Shared Reading
Contact Darnall Wellbeing for the next date.
Darnall Wellbeing
0114 249 6315
Shared Reading
Every Tuesday
Come and listen to a story and poem read aloud and join in a relaxed chat about what we've read
Sue Taylor
0114 2039529
Sheffield 0 Gauge Group
Wednesday at 1pm & Friday at 7.30pm
Model Railway Club devoted to railway modelling in O Gauge.
Keith New or Paul Scott
0114 255 4898 01246 418608
Sheffield and District Family History Society
3rd Monday in the month 7.30-9pm
Sheffield and District Family History Society aims to promote the study of genealogy, family history and local history in the area for the benefit and education of the public. The society has a library held at Sheffield Archives. The society organises outings and lectures regarding researching local and family history. Meets at Cemetery Road Baptist Church, S11 (Monday).
01142873984 01142873984
Sheffield and District Orchid Society
2nd Sunday each month ( except for June and September in 2019 ) from 10.15am to 1.00pm.
Sheffield and District Orchid Society holds monthly meetings at the Sheffield Botanical Gardens Demonstration Centre normally in the morning of the second Sunday of the month from 10.15am to 1.00pm.
Peter Battle
0114 2745364
Sheffield Astronomical Society
Usually 2nd & 4th Tuesday in the month 7.30-10pm - please check Website or Facebook group for updates and outreach events.
Sheffield Astronomical Society is an amateur astronomical society which provides a place for everyone who is interested in astronomy and space to meet regularly. Also provides outreach educational visits to community groups and public observing sessions in the Sheffield area. A registered charity. Meets in…
07852 298 612
Sheffield Bach Society
Monday evenings from 7.30pm - 9.30pm
Sheffield Bach Choir was founded in1950. Around 60 strong, under our conductor Simon Lindley, we have built a reputation for musical excellence with dynamiic performances of modern classics and Baroque masterpieces. The choir regularly performs three of Bach's major choral masterpieces: Mass in B Minor, St Matthew Passion and St John Passion as well as Handel'sMessiah every December.
Liz Arnesen
07960 523 911
Sheffield Beekeepers Association (SBKA)
Sheffield Beekeepers Association exists to promote, support and further the craft of Amateur beekeeping. To raise awareness and educate the general public of the importance of bees and beekeeping to the environment.
Association Secretary
Sheffield City Opera
Wednesday 7:30-10pm (rehearsals, please see website for latest information)
Sheffield City Opera is Sheffield's only amateur operatic company. Over the past few years as well as performing concerts of staged scenes in local venues we have staged full performances of Massenet Cinderella, Gounod Faust, and Mozart Magic Flute. Next Production We are currently…
Marjorie Skidmore
0114 230 3204 07547 230632


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On March 5, 2015, at 1102 eastern standard time, Delta Air Lines flight 1086, a Boeing MD-88, N909DL, was landing on runway 13 at LaGuardia Airport (LGA), New York, New York, when it departed the left side of the runway, contacted the airport perimeter fence, and came to rest with the airplane's nose on an embankment next to Flushing Bay. The 2 pilots, 3 flight attendants, and 98 of the 127 passengers were not injured; the other 29 passengers received minor injuries. The airplane was substantially damaged. Flight 1086 was a regularly scheduled passenger flight from Hartsfield-Jackson Atlanta International Airport, Atlanta, Georgia, operating under the provisions of 14 Code of Federal Regulations Part 121. An instrument flight rules flight plan had been filed. Instrument meteorological conditions prevailed at the time of the accident.
The National Transportation Safety Board determines that the probable cause of this accident was the captain's inability to maintain directional control of the airplane due to his application of excessive reverse thrust, which degraded the effectiveness of the rudder in controlling the airplane's heading. Contributing to the accident were the captain's (1) situational stress resulting from his concern about stopping performance and (2) attentional limitations due to the high workload during the landing, which prevented him from immediately recognizing the use of excessive reverse thrust.


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Sorry, we couldn't find what you were looking for.
Just because...you inspire me!
When was the last time you surprised someone for no other reason than to see them smile? When it comes to giving, it's not just what we do, but why we do it. So go ahead, make every moment count and send a card "Just Because!"
©2016 pingg.com LLC | Disney Online Mom and Family Portfolio (Network Member)
Pingg.com owns and operates this website and collects the information you provide here, subject to its Privacy Policy.


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Hello My Name Is...
Ms. Amy Davis
(972) 230-0726 Ext.1113
Graduate of Justin F. Kimball High School Class of 2001
Bachelors of Science Degree in Chemistry from Sam Houston State University-2009
Bachelors of Science Degree in Biology from Sam Houston State University-2010
I enjoy teaching, reading, and participating in outreach programs!!!!
Class Schedule
1st Bell..................................................................7:20 am
1st Period AP Environmental Science/5th Period Chemistry 1..................... 7:30 am - 9:02 am
2nd Period Conference Period/6th AP Env. Science...................................9:12 am - 10:44 am
3rd Period Chemistry 1/7th Period Chemistry.......................................... 10:54 am - 1:18 pm
4th Period Chemistry 1/8th Period AP Env. Science.....................................1:22 pm - 2:54 pm
DeSoto Independent School District does not discriminate on the basis of race, color, national origin, sex, age, or disability in admission to its programs, services, or activities, in access to them, in treatment of individuals, or in any aspect of their operations. The lack of English language skills shall not be a barrier to admission or participation in the district's activities and programs. DeSoto Independent School District also does not discriminate in its hiring or employment practices.


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Compose Yourself is a digital music game where children of any ability can create, play and share world-class musical compositions in just minutes.Created by world-famous composer, cellist and advocate for music education, Philip Sheppard, as a way to inspire curiosity and interest in music, Compose Yourself offers a set of musical "building bricks" that encourage kids to play with music just as they would a construction kit. You simply flip, rotate, arrange and reorder cards with musical notes in a visual pattern, enter the codes on the website and instantly hear your piece played by a full symphony Orchestra at Abbey Road Studios. There are over 1 billion melodies you can create and then share with others.
Compose Yourself encourages kids to explore the world of music, triggers creative thinking, supports STEAM learning and brings out the musician in everyone whether they are 6 or 106. It's available exclusively on Amazon and ThinkFun.com.
Compose Yourself Videos
Bios & Resources
Product Photos
- Compose Yourself Spill Shot and Packaging
- Compose Yourself cards in play
Inventor Philip Sheppard Images
- Philip Sheppard Headshot (Photo Credit: Julian Macedo/Powder Photography)
- Philip Sheppard with Compose Yourself cards (Photo Credit: Tom Bowles)
- Philip Sheppard conducting the Compose Yourself orchestra (Photo Credit: Tom Bowles)
Percussionist Evelyn Glennie Images
- Evelyn Glennie playing the marimba for Compose Yourself (Photo Credit: Tom Bowles)
Philip Sheppard, Evelyn Glennie and Orchestra Images
- From the view of an orchestra member, Philip Sheppard conducting (Photo Credit: Tom Bowles)
- Philip Sheppard and Evelyn Glennie smiling (Photo Credit: Tom Bowles)
- Young girl conducts the orchestra (Photo Credit: Tom Bowles)
- Young boy conducts the orchestra (Photo Credit: Tom Bowles)
- Wide view of Philip Sheppard conducting the orchestra (Photo Credit: Tom Bowles)
Compose Yourself Game Play Images
- Boy composing with cards
- Girl entering card codes into the Compose Yourself website
- A boy and girl comparing cards together
- Two sets of hands comparing cards
- Three children using the Compose Yourself website together
- Girl holding a printout of her composition
- Girl playing her composition on a piano
- Boy playing his composition on a cello while two girls look on
All Images
- All Images – .zip file, 116mb


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The stylish nomadic renter: Millennials and Gen-Z
Oct 29, 2019 | By Maria Florencia Ameneiros
Jun 01, 2011
Cool-cat hoteliers, Morgans Hotel Group have teamed up with RCRD LBL, the music download website, to offer guests free playlists accessible through their hotel keycards.
A unique selection of songs has been specially curated to reflect the personalities of each of the each hotel sites in the Morgans chain, including the Delano, Mondrian Soho and Royalton in New York City. Playlists can be downloaded through the scannable QR code located on each hotel keycard using a mobile phone device. Additionally guests will have access throughout their stay via in-room iPads and the Morgans Tumblr blog.
Renowned for their boutique approach to hospitality this collaboration perfectly encapsulates the chains stylized design aesthetic and offers guests the opportunity to experience each of the hotel sites individual personality through free music, further keeping them ahead of the curve in the hotel sector.
Each twenty-tight compilation will be updated every three months, exclusively by RCRD LBL, and feature a fresh medley of up and coming artists including Best Coast, Wavves, Memory Tapes and The Drums to name but a few.
"Music has always been an important part of the experience at our hotels," notes Kim Walker, Creative Director for Morgans Hotel Group. "Delivering custom musical content unique to each of our hotels and making it available on a complimentary basis to our guests, is the next step in keeping our amenities fresh and our guests excited to see what's next from Morgans Hotel Group."
Know what's next. Become a WGSN member today to benefit from our daily trend intelligence, retail analytics, consumer insights and bespoke consultancy services.


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No Reports = No Information Latency
Sunnyvale, CA (PRWEB) March 25, 2014
VSSOD, an emerging provider of Operational Intelligence and Real Time Analytics Software, today announced that it had been selected to showcase its OpsVeda software at the DEMO Enterprise conference, on April 3rd, in San Francisco.
In addition, VSSOD will be presenting at the SAP Startup Focus Pavilion at the DEMO Conference as part of the DEMO CIO Council on the same day. The CIO Council, is a member-led executive organization of global business leaders working to drive value from the intersection of business, information technology and strategy.
OpsVeda, an operational intelligence application suite, leverages the SAP HANA® platform to enable real-time predictive visibility into business process exceptions as related business activities happen. Deployed on cloud or on premise, the Software enhances operational performance, in days & weeks.
"The opportunity to showcase at DEMO is a great honor. It gives OpsVeda the opportunity to demonstrate its unique data store and data acquisition model that enables transformation of enterprise reporting practices," said Sanjiv Gupta, CEO of VSSOD. "'No reports, no latency' is our mantra! OpsVeda provides continuous, real-time visibility into business activity data spanning systems and partners. This enables our customers to make critical business decisions on the fly."
About VSSOD Corporation
VSSOD is a software company focused on providing operational business insight and predictive analytics to enterprise customers. We enable real-time predictive visibility into process exceptions across execution processes spanning order fulfillment, supply management, shipment logistics and channel operations. VSSOD's OpsVeda Application Suite brings big data predictive analytics and deep business process expertise together to deliver outcomes for customers out of the box. VSSOD customers span the high-tech, consumer goods, apparel and footwear and life sciences industries.
See http://www.vssod.com for more information.
OpsVeda, VSSOD, VSS On Demand, VSSOD Business Agility Suite and Agile SalesOps are trademarks or servicemarks of VSSOD Corporation.
All other product and service names mentioned are the trademarks of their respective companies.


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Today I was flattered to have received an email from Blogger.com who was responding to my complaint about Blogger's problem. This is what I have said in my reply.
Dear Sxxxxxxe,
It's so warm that I get reply from a human.
I use firefox and my blog is one of the home pages that I would open. Every morning I start firefox at round 9:30am Hong Kong time. During the last 2 weeks perhaps I have only 1 day with my blog successfully opened.
Almost everytime I tried status.blogger.com but it disappointedly failed. At the time of this writing both my blog and your status page can't be read.
Interestingly in the last few days I have asked my sister in Malaysia to view my blog, it was OK in most days.
I think you may have a technical problem somewhere that your internet load balancing has put Hong Kong in low priority during your US night time. It's understandable that Blogger has scheduled shutdown and maintenance during those hours.
I have read newsgroups and others and found that I'm not the only one feeling this frustration. Blogger must put this right quick.
Tuesday, June 7, 2005
Blogger is talking
by sfong15 at 3:03 PM
Subscribe to: Post Comments (Atom)


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Pharma & Healthcare
Transport in secure refrigerated trailer, constantly monitored for both cooling and security.
High Value & Security
High value, theft targeted cargo are transported in accordance with TAPA-TSR 1,2 or 3 requirements. With our own fleet of trucks and highly trained drivers.
Fashion Logistics
Hanging garment and pallet-transport within Europe, the Balkans, Russia and Morocco.
Intermodal Solutions
Our intermodal solution yields a CO2 reduction of 70% and 35% transport cost savings. Delivered to your customer by Boekestijn Transport Service.


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a creative process
"Creativity is just connecting things. When you ask creative people how they did something, they feel a little guilty because they didn't really do it, they just saw something. It seemed obvious to them after a while. That's because they were able to connect experiences they've had and synthesize new things. And the reason they were able to do that was that they've had more experiences or they have thought more about their experiences than other people. Unfortunately, that's too rare a commodity. A lot of people in our industry haven't had very diverse experiences. So they don't have enough dots to connect, and they end up with very linear solutions without a broad perspective on the problem. The broader one's understanding of the human experience, the better design we will have."
Steve Jobs, Wired, February, 1996
Pretty great quote. An additional thing that came to mind after reading this was the importance of the question or the problem to solve. The quality of that is the foundation for good ideas. Sometimes it's a creative brief, or more purely, a desire to communicate a feeling or perspective. That is another discussion though, and the following thoughts, as they exist in this instant only, are what can be done if one possesses that thing. I have the problem, so what do can I do now?
Work with divine inspiration.
The best experience one can find. I think it is important to note, however, that this is illusive. It is contigent on what you got in your bag and how willing you are to cater to the Muse. Many a great songwriter fight here, and I thought this story was pretty indicative of real player's effort:
Ape something.
I remember Paul McCartney saying something about how many of his favorite songs were started by just trying to emulate a tune, and through the process of getting things he was comfortable playing and singing, it inevitably became something else. I think having a good client problem to solve before the pillaging, one that is well defined and intrinsictly their own, can allow for something new to form. You hold their issue in your mind and then get to finding inspiration in others work makes you pull elements together, rather than just mimicking something. Trouble occurs when you don't ask, "This is sweet, what if I found a different image and used my favorite font, etc." Shazam, mine now!
Find a process.
Ahhhhh. This is where the real freaks shine. I know this loops back up to letting something hit you, but it definitely feels like a different experience. This is the craft part, and can avoid a lot of tears. Doing some digging on your favorites is quite an experience. It is the method and thing one can do to get going. David Bowie has grocery sacks of words on torn up pieces of paper, which leads to my favorite: lists. Again, completely propped up by a good question…success demands it.
And here are some extra links on the foundation of these processes:
reference for more tricks as they apply to subjects: