text:
This invention provides improved components (e.g. array "pins", print head, substrate platen, print head platen, and the like) for microarray printing devices as well as microarray printing devices incorporating such components. In one embodiment, this invention provides a microarray print head comprising a plurality of glass or quartz spotting capillaries disposed in a support that maintains a fixed spacing between the spotting capillaries and that permits the spotting capillaries to move in a direction parallel to the long axis of the capillaries.
1. A microarray print head, said print head comprising:
a plurality of glass or quartz spotting capillary tubes disposed in a support that maintains a fixed spacing between said spotting capillaries and that permits the spotting capillary tubes to move in a direction parallel to the long axis of said capillary tubes, wherein said capillary tubes:
have an internal diameter ranging from about 20 μm to about 100 μm;
have an aperture ID or OD ranging from about 20 μm to about 75 μm;
have an outside diameter ranging from about 0.4 mm to about 1 mm;
have a center-to-center spacing ranging from about 1 mm to about 10 mm;
have a load volume ranging from about 0.05 μL to about 1 μL; and
range in number from about 2 to 256;
wherein said capillary tubes are in fluid communication with a manifold.
2. The print head of
3. The print head of
4. The print head of
5. The print head of
6. The print head of
7. The print head of
8. The print head of
9. The print head of
10. The print head of
11. The print head of
12. The print head of
13. The print head of
[Not Applicable]
[Not Applicable]
This invention pertains to the field of high-density microarray production. In particular, this invention provides methods and devices that permit high-density arrays to be printed with significantly smaller feature size and spacing and greatly improved reagent usage.
The immobilization of test molecules or "probes" on array supports has had a significant impact on drug discovery, medical diagnostic methods, and basic research. The use of high-density microarrays of organic molecules permits literally thousands of assays to be simultaneously performed on one or more samples. Using high-density microarrays, numerous analytes can be simultaneously detected and/or quantified permitting the rapid characterization of complex systems (e.g. complex assays for gene expression). High-density microarrays are also useful for "high-throughput" screening assays, diagnostics, and in many other contexts. The ability to manufacture microarrays in an efficient and cost-effective manner is of considerable interest to researchers worldwide and of significant commercial value.
In general, microarrays of greater density are preferred. A higher density array typically allows more assays to be performed simultaneously and/or, for lower sample volumes to be used for the same number of assays. In providing large, high-density arrays of molecules (e.g., probes or analytes) there are a number of considerations. The array elements (e.g. dots) should be substantially reproducible in size, particularly if one wishes to quantify an analyte. In addition, the array elements should be consistently and reliably positioned, and should be highly reproducible.
The basic approaches for generating arrays of test molecules such as nucleic acid, protein or other organic molecules fall into two general categories. In the first such approach the test molecules are directly synthesized onto the array support, while in the second such approach the test molecules are attached to the support post-synthetically. Each approach has its own limitations and drawbacks. For example, when an array is created by direct synthesis onto an array support, the efficiency of each synthetic step affects the quality and integrity of molecules forming the array. The magnitude of the problem increases with the complexity of the individual molecules, potentially resulting in an undesirable percentage of incorrectly synthesized molecules and incomplete sequences. Such contaminants can interfere with subsequent use of the array.
In addition, synthetic approaches (e.g. as described by Southern et al. (U.S. Pat. Nos. 5,770,367, 5,700,637, and 5,436,327), Pirrung et al. (U.S. Pat. No. 5,143,854), Fodor et al. (U.S. Pat. Nos. 5,744,305 and 5,800,992), and Winkler et al. (U.S. Pat. No. 5,384,261), are generally unable to construct microarrays of large macromolecules. Such technologies can also be expensive and difficult to implement.
In contrast, the second approach to array production allows the desired molecules to be synthesized and purified by conventional methods prior to their formation into an array. Consequently, the quality of the arrayed molecules, and thus the quality of the resultant array, is potentially greater than that produced by the direct synthesis approach.
Such "spotting" approaches include, but are not limited to inkjet, and direct surface contact printing. Inkjet devices require high reagent volumes and risk "probe" degradation during volatilization.
Direct surface contact printing (see, e.g., U.S. Pat. Nos. 4,981,783, 5,525,464, 5,770,151, and 5,807,522), are limited in their ability to reliably, reproducibly, and uniformly apply the array elements to the array substrate. Reagent usage is also relatively inefficient, and array density is limited.
The present invention provides improved components (e.g. array "pins", print head, substrate platen, print head platen, and the like) for microarray printing devices as well as microarray printing devices incorporating such components. In particular, methods and devices of this invention permit high-density arrays to be printed with significantly smaller feature size and spacing, and greatly improved reagent usage.
In one embodiment this invention provides a microarray print head, said print head comprising a plurality of glass or quartz (or other mineral), or ceramic, or porcelain, or ceramic spotting capillaries disposed in a support that maintains a fixed spacing between the spotting capillaries and that permits the spotting capillaries to move in a direction parallel to the long axis of the capillaries (i.e. the spotting capillaries can slide through the support). Preferred spotting capillaries are microcapillary tubes and particularly preferred spotting capillaries have a tapered tip (e.g. a ground, beveled tip). The capillaries can have any desirable cross-section (e.g. round, ovoid, square, triangular, irregular), however preferred capillaries are round in cross-section.
In certain preferred embodiments, the capillaries have a maximum load volume of about 0.5 mL. In certain preferred embodiments, the spotting capillaries have a load volume of about 0.2 mL.
Preferred print heads comprise at least 4 spotting capillaries, preferably least, 4, 16, 32, 64, or 128 spotting capillaries and in certain preferred embodiments, the spacing between two adjacent spotting capillaries is about 3 mm or less, center to center.
In certain preferred embodiments, the spotting capillaries have detents where the spotting capillaries have a rest position in which the detents contact a support stopping the movement of the spotting capillaries in a direction toward the substrate that is to be printed. The print head can also comprise a spring attached to a spotting capillary where, in the absence of a force against the printing tip of the spotting capillary the spring returns said spotting capillary to a rest position. The print head can be provided separately or can be found as a component in a microarray printing device.
In preferred embodiments, the spotting capillaries are in fluid communication (e.g. via flexible capillary tubing) with a manifold. In preferred embodiments, the manifold comprises a common port and a plurality of individual ports where an aperture into an individual port is disposed inward of the inside wall of the manifold. The manifold can be connected to a gas and/or vacuum source.
In another embodiment this invention provides a platen for positioning a substrate holder or a print head in a microarray printing device. A preferred platen comprises a support surface attached to a single guide rail such that the support surface can move along the guide rail, and motion of the support is constrained in a direction normal to the guide rail, and a flexible coupling to an actuator wherein the flexible coupling is rigid or stiff in a direction parallel to the guide rail, but is flexible in another direction. The platen also, optionally, comprises an encoder (e.g. optical encoder, magnetic encoder, electronic encoder, etc.) that encodes the position of said platen along said guide rail. In certain embodiments, the platen is attached to the rail by two bearings. Preferred flexible couplings include, but are not limited to a flexible sheet coupling (e.g. sheet metal, sheet plastic, etc.), a rod bearing, a ball bearing, a pin bearing and the like. Preferred actuators include, but are not limited to a stepping motor, a linear motor, a lead screw, and the like. In certain embodiments, the platen can further comprise a holder (e.g. a slide holder) for one or more microarray substrates. In certain embodiments, the platen is attached to a microarray print head (e.g. directly or through a movable stage). Preferred print heads in such cases include, but are not limited to any of the print heads described herein.
In still another embodiment, this invention provides a microarray printing device comprising a microarray print head (e.g. as described herein); and a microarray substrate holder attached to a platen (e.g. a platen as described herein). Preferred microarray printers can print at least 2,000, more preferably at least 5,000 array elements per spotting capillary per load. Preferred microarray printers can print array elements with a precision of at least 30 μm and/or with an average inter-element spacing of 130 μm or less. Preferred microarray printers can print 200 or more microarray substrates in a run. Particularly preferred microarray printers of this invention utilize pressure and/or vacuum to control reagent loading or dispensing. Certain microarray printers comprise the spotting capillaries are in fluid communication with a manifold. A preferred manifold comprises a common port and individual ports where an aperture into an individual port is disposed inward of the inside wall of the manifold. In certain preferred embodiments, the microarray printing device can loads reagents from a microtiter plate comprising at least about 864 wells.
This invention also provides a method of printing a microarray (e.g., a nucleic acid and/or protein and/or small organic molecule microarray). The methods involve providing an array substrate in a microarray printing device comprising one or more of the elements (e.g. spotting capillaries, print head, array substrate platen, print head platen, and the like) as described herein, providing a series of solutions comprising the reagents that will form features of the microarray; and operating said microarray printing device to print the microarray. In preferred methods the microarray printing device prints a microarray comprising at least 1,000 different array elements. In preferred methods the microarray printing device prints a microarray comprising having an average inter-feature spacing of about 130 μm or less. Preferred array substrates include, glass, quartz or other minerals, metals, ceramics, plastics, metal coated glass, metal coated plastic and the like. In preferred methods the microarray printing device applies negative pressure to load a spotting capillary and/or positive pressure to dispense from a spotting capillary. In preferred embodiments, the method involves loading feature-forming reagents from a microtiter plate comprising at least about 864 wells.
In still another embodiment, this invention provides (printed) microarrays. Preferred printed microarrays comprise at least about 1000 different array elements on an array substrate, where the array elements are separated by an average center to center spacing of about 130 μm or less, preferably about 100 μm or less, more preferably about 80 μm or less. Where the arrays are nucleic acid and/or protein arrays, the protein or said nucleic acid is preferably not a chemically synthesized protein or nucleic acid. Particularly preferred microarrays include nucleic acid nucleic acid microarrays. In certain embodiments, the nucleic acids comprising such microarrays have an average length greater than about 50, preferably greater than about 100, 200, or 500 nucleotides, more preferably greater than about 1000 nucleotides. The molecules comprising the array features are preferably adsorbed to the array substrate. In certain nucleic acid or protein arrays the nucleic acid or protein is not covalently coupled to the array substrate (e.g. not coupled directly or though a linker to a terminal nucleotide or amino acid). In particularly preferred microarrays, the features comprising the arrays are at an average density of about 40,000/cm2 or greater.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide.
The terms "nucleic acid" or "oligonucleotide" or grammatical equivalents herein refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10): 1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) Chemica Scripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111: 2321, O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566; Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acids include those with positive backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl. Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995), Chem. Soc. Rev. pp169-176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.
The terms "spotting capillary" and "pin" or "printing pin" are used synonymously to refer to the structure that is used to contact a microarray substrate and thereby deposit a reagent to form a microarray feature on that substrate. Unlike many printing pins, however, the spotting capillary is typically a tube and, while not limited to such, in certain preferred embodiments, display a round cross section.
An "array substrate" refers to the surface or support on which a microarray is printed. Array substrates include, but are not limited to glass, quartz or other minerals, ceramic, porcelain, metal, and metal-coated glass.
An "array feature" or "array spot" refers to a reagent or reagents deposited at a location on an array surface. Typically a feature is characterized by the presence of one or more specific molecules (e.g. particular proteins, nucleic acids, etc.).
A "guide rail" refers to a rail or other device that directs or orients the movement of a platen as described herein. In certain embodiments, guide rail can take any of a number of forms including, but not limited to T-shaped, round, triangular, square, ovoid, and the like. The guide rail is typically coupled to the platen through one or more bearings that permit motion of the platen in one direction (along one axis), but restrict motion in other directions.
A "print cycle" refers to the sequence of events involved in printing an array feature.
The term "microarray" refers to an array comprising at least about 10, preferably at least about 50, more preferably at least about 100, still more preferably at least about 500 or 1000, and most preferably at least about 10,000, 40,000, 100,000, or 1,000,000 different and distinct features. Preferred microarrays have an average feature density greater than about 100/cm2, more preferably greater than about 1000/cm2, still more preferably greater than about 5,000/cm2, even still more preferably greater than about 10,000/cm2, and most preferably greater than about 20,000/cm2, 40,000/cm2, 60,000/cm2, or even 80,000 cm2.
This invention pertains to a microarray printer that can be used to manufacture microarrays (e.g. of biochemical samples) by direct contact printing. The array printer of this invention is capable of printing microarrays at higher feature density, with greater speed and lower cost than previous microarray printing devices.
Without being bound to a particular theory, these efficiencies are achieved by the use of a combination of novel features. A novel printing pin permits vastly more efficient reagent usage and a greater number features to be printed per load. This reduces reagent costs, and because the pin does not require repeated refills during the printing process, the printing operation proceeds more rapidly.
In addition, positive control of reagent flow using pressure and vacuum, also improves reagent capture and delivery and reduces the incidents of mis-prints due to pin blockage during loads or print steps.
A novel design for the substrate support permits the use of a larger positioning substrate that can hold a greater number of array substrates (e.g. more than 10, preferably more than 20 or 50, still more preferably greater than 100, and most preferably greater than 150, 200, 250, 300, or even greater than about 500 standard slide-sized substrates) and position each array with greater accuracy and precision. The substrate support is kinematically more efficient permitting rapid accelerations and deceleration and thereby permitting a print run to proceed with greater rapidity, i.e., decreasing effective print time and reducing printing costs.
A related support design for the print head permit rapid acceleration and deceleration of the print head. The rapid positioning of the array substrate combined with the rapid positioning of the print head again, significantly reduces the time required for a print cycle thereby reducing costs.
These features combine to make possible the efficient printing of microarrays at extremely high efficiency with low reagent usage, and previously unobtainable feature spacing for printed microarrays.
I. Printing Pins and the Print Head
In one embodiment, this invention provides for print head 10 for printing microarrays and a microarray printer comprising such a print head. As illustrated in
The spotting capillaries 12 are preferably cylinders (e.g. capillary tubes) made of a rigid material such as glass, quartz or other mineral, ceramic, brittle plastic (e.g. acrylic), and the like. It was a surprising discovery of this invention that glass-like materials such as glass or quartz or ceramic could be effectively used as spotting capillaries. Moreover, particularly when fabricated and utilized as described herein, such glass, quartz or ceramic spotting capillaries have a useful lifetime vastly greater than that observed for the commonly utilized metal pins. Indeed, we have yet to determine the maximum lifetime of the spotting capillaries described herein, while it is believed that metal spotting are quite limited in their useful life.
The spotting capillaries used in this invention can be of any convenient size, however, in preferred embodiments (see, e.g., FIG. 2), the spotting capillaries are microcapillaries, with an inside diameter (bore width) of less than about 100 μm, preferably less than about 75 μm, more preferably less than about 50 μm, and most preferably less than about 30 μm, 25 μm or even less than about 20 μm. The load volume (loaded fluid volume) of the spotting capillary is typically 1 μL or less, preferably 0.5 μL or less, more preferably 0.25 μL or less, and most preferably 0.2 μL or less or even 0.1 μL or less.
The spotting capillaries need not have a constant internal diameter. The diameter at the aperture (spotting face) of the spotting capillary will, in part, determine the minimum feature size of the spotted microarray. Thus, smaller aperture diameters are preferred. Particularly preferred aperture diameters are less than about 75 μm, more preferably less than about 50 μm, and most preferably less than about 30 μm, 25 μm or even less than about 20 μm. In certain embodiments, the diameter of the internal channel expands to a maximum of about 75 μm, preferably about 100 μm, more preferably about 150 μm and most preferably about 200 μm. In one particularly preferred embodiment, illustrated in
The spotting capillary outer diameter determines the minimum inter-pin (inter-capillary) spacing, and the inter-pin spacing determines the minimum spacing of the reservoir(s) from which the print head can load reagents. Preferred spotting pins have an outside diameter of about 1 mm or less, more preferably of about 0.7 mm or less, and most preferably of about 0.4 mm or less. The 0.4 mm spotting capillaries can be mounted very close together and, with such close spacing, the print head can load reagents from standard 96 well, 384, well, 864 well, and 1536 well microtiter plates. In preferred embodiments, the center to center spacing of the spotting capillaries is about 10 mm or less, preferably about 5 mm or less, more preferably about 3 mm or less, and most preferably about 2 mm or even 1 mm or less. Such close spotting capillary spacing can be achieved, e.g. using the print head designs illustrated herein, allowing the use of even higher density sample reservoirs.
The support(s) for the spotting capillaries can take any of a number of a number of forms. For example, in one embodiment, the support can comprise a number of channels drilled, etched, or cast in a single metal or plastic piece. The channels then act as guides for the spotting capillaries. Alternatively, the support can be fabricated as by joining a collection of tubes e.g. metal tubes. The tubes can be glued or welded together to form a single support structure, each of the tubes acting as a channel for housing a spotting capillary.
In a particularly preferred embodiment, as illustrated in
The extended position of the spotting capillaries is limited by a detent 20. The detent can take any convenient form. For example, the detent could comprise a set screw (preferably plastic so as not to damage the spotting capillary), a drop of epoxy or other resin, and the like. In one particularly preferred embodiment, the detent is a disk attached to the spotting capillary. In one embodiment the detent stops up against the pin guide plate. The "downward" extent of the spotting capillary can be determined by the position of attachment of the detent to the spotting capillary. Alternatively, the guide plate can further comprise an adjustment means (e.g. a set screw, a shim, etc.) for each spotting capillary that can be used to adjust the downward travel for each spotting capillary.
In certain embodiments, the spring typically rests against a resisting surface, e.g. a spring capture plate 22.
It was also a discovery of this invention that spotting capillaries, particularly when fabricated of glass, quartz, other minerals, or ceramic or porcelain, show a dramatically improved lifetime, when the outer edges of the spotting tip of the spotting capillary, are not flush with the spotting face. Thus, in preferred embodiments, the outer edge of the spotting tip is beveled (see, e.g., 30 in FIG. 2).
The print head typically comprises a plurality of spotting capillaries. Preferred print heads comprise at least two spotting capillaries, more preferably at lest 4 spotting capillaries, still more preferably at least 8 or at least 16 spotting capillaries, and most preferably at least 32, 64, 128, or 256 spotting capillaries. Depending on the application, a print head can be configured to use fewer than the total number of available spotting capillaries.
The print heads of this invention can be fabricated using standard machining and glass handling techniques well known to those of skill in the art. The spotting capillaries are preferably fabricated by casting or by pulling a quartz or glass microcapillary tube using a commercially available microcapillary puller (e.g. Sutter Instrument P-2000 Capillary Puller). In particularly preferred embodiments, the microcapillary tip is then beveled using a glass grinder. Such spotting capillaries can be made to order by commercial glass production houses.
II. Platens for Array Substrate and/or Print Head Positioning
To reliably print an array at high feature density (spots/cm2) it is desirable to reliably and consistently position the spotting capillaries on the microarray substrate. The more precisely and consistently the print head can be positioned relative to the microarray substrate(s), the more possible it becomes to print arrays at a higher feature density.
Printing a larger number of arrays at a time, however, requires a larger array substrate support (platen). The larger the platen, the more difficult it becomes to reliably and consistently position it relative to print head. One approach to solve this problem is illustrated in FIG. 3. The platen illustrated on the left utilizes two guide rails to minimize torque and hysteresis of the platen introduced by the actuator which moves the platen, e.g. in the ±Y direction. To accurately print arrays, it is desirable to accurately position the platen to tolerances better than 50 μm, more preferably better than 25 μm. Such precise positioning requires extremely good bearing alignment for the four bearings and the guide rails must be extremely parallel. The rails and bearings must stay aligned throughout the printing operation. One of skill in the art will appreciate that such a device if will tend to jam and/or introduce positioning imprecision as the actuator drives the platen through jammed positions.
Such difficulties are solved with the platens of this invention. One embodiment of a platen 40 of this invention is illustrated by the platen on the right in FIG. 3 and in FIG. 4. This platen utilizes a single guide rail 42 to constrain the position of the support surface 44 that bears the array substrates 56. The support surface communicates with a guide rail 42 through one or more bearings 46. The support surface is coupled to an actuator 48 through a flexible coupling 50.
The bearing(s) 46 and the guide rail 42 prevent the support surface from rotating in response to a force created by the actuator. However, because there is only a single rail, there are no difficult alignment problems. Straightness of the guide rail is also not critical because any rail deformation will be constant and reproducible, i.e., repeated positioning of the array substrates will be consistent. The platen bears an encoder 58 that accurately encodes the location of the platen along the guide rail.
The support surface is coupled to the actuator through a flexible coupling 50 that is rigid in the direction of travel (±Y direction in FIG. 3), but compliant in other directions. This permits the actuator to accurately position the platen along the guide rail while not jamming or binding in other directions. Moreover, it is noted that when the moment arm from the actuator coupling to the array substrate(s) is equal to or preferably greater than the moment arm from the array substrate(s) to the guide rail error introduced at the coupling of the actuator to the support surface will be reduced at the array substrate(s).
The embodiment illustrated in
The flexible coupling 50 illustrated in
In certain embodiments, a pivot 52 is provided. In various embodiments, the pivot 52 includes, but is not limited to two points across the width (into the drawing in the side view), a cylinder etc. In this design, pitch motion will require the upper mounting plate to slide across the pivot, so this is preferably lubricated and/or fabricated of low-friction materials, etc.
Because the moment arm between the flexible motor coupling 50 and the print head is large compared to the moment arm between the print head and the guide rail 42, rotations or deflections at the motor coupling have minimal effect on the position of the print head.
Using the teachings provided herein, one of skill would recognize numerous embodiments for the flexible couplings used in the platens of this invention. In certain embodiments, as indicated above, the flexible couplings comprise flexible sheets (e.g. sheets of metal, plastic, or other flexible material). The sheets are selected of materials that are stiff in tension, but capable of bending in other directions. Other flexible couplings include, but are not limited to ball bearings, rod bearings, pin bearings, and the like.
A wide range of encoders can be used to encode the position of the platen/support surface. Encoders are well known to those of skill in the art and include, but are not limited to optical encoders, mechanical encoders, magnetic encoders, and electronic encoders. Various electronic encoders include, but are not limited to encoders that convert the change in resistance of a potentiometer or the change in capacitance of a capacitor into a movement or position. Optical encoders include, but are not limited to encoders that convert an optical signal, e.g. a bar code, an interferometric measurement, etc. into a movement or position. Similarly, magnetic encoders include encoders that a change in magnetic flux or field into a movement or a position. Suitable encoders (e.g. with a positional accuracy greater than about 50 μm, preferably with a positional accuracy greater than about 25 μm, more preferably with a positional accuracy greater than about 10 μm, and most preferably with a positional accuracy greater than about 5 μm, greater than about 2 μm, or greater than about 1 μm are commercially available.
The actuator can be any device or means capable applying a force to the platens of this invention and driving them in a plus or minus direction along the guide rail. Suitable actuators include, but are not limited to stepping motors, linear induction motors, pneumatic actuators, solenoids, piezo-electric actuators, lead screws, and the like. Suitable actuators, and associated motion control products are commercially available from a wide variety of companies (see, e.g., Biorobotics (U.K), Cartesian Technologies Inc. (U.S.A.). Virtek. (Canada), Intelligent Bio-Instruments a division of IAS Products, Inc. (USA), GeneMachines (U.S.A.), Genetix Ltd.(U.K.), Genomic Solutions Inc. (U.S.A.). Genpak Limited. (U.K.), and the like).
It is noted that in certain preferred embodiments, of the present invention, the slide support platen, with an encoder precision of about 2 μm achieves a spot (array element) precision of about 10 to about 20 μm at any array substrate location on the support surface. Particularly preferred platens achieve a spot (array element) precision of better than about 10 μm, more preferably better than about 8 μm and most preferably better than about 5 μm at any array substrate location on the support surface.
Similarly, the print head positioning platen achieves a precision of about ±10 μm or less, more preferably about ±5 μm or less, and most preferably about ±3 μm or less over the entire slide (array substrate) support surface.
III. Positive and Negative Pressure Control for Sample Loading and Dispensing
In a particularly preferred embodiment, the microarray printing devices of this invention utilize positive pressure and negative pressure (vacuum) to control sample loading and dispensing. Each "active" spotting capillary 12 is in fluid communication, e.g. via capillary tubing 70 with a manifold 64 (see, e.g.,
A preferred plumbing scheme is illustrated in FIG. 7. In preferred embodiments, the gas flow (e.g. air, nitrogen, argon, etc.) flows through the manifold always in the same direction for all operations. This assures that any liquid drops that may be left in the tubing always will be forced to move toward the waste bottle and will not be blown back into the manifold. If droplets do get into the manifold they may block the supply of printing pressure to one or more pins, thus reducing the reliability of the printing.
In general, the plumbing system comprises a pressure source 78, and a vacuum source 80. The pressure and vacuum sources are in fluid communication with a manifold 74, e.g. via gas and fluid tubing 84. The manifold is also in fluid communication with the spotting capillaries so that pressure or vacuum applied to the manifold is delivered to the channel (bore) in the capillaries. In preferred embodiments, there is a waste receptacle disposed between the low pressure/vacuum source 80 and the manifold 74.
In preferred embodiments, the system is designed so that the tubing from the waste bottle to the manifold preferably slopes downward to facilitate liquid flow, and we strive to minimize the volume of the tubing and waste receptacle so that the pressure changes are transmitted to the manifold quickly. Thus, while the tubing communicating the pressure and vacuum to the manifold and communicating the manifold to the waste receptacle can be essentially any convenient tubing (as long as it is resistant to the reagents employed), in preferred embodiments, the tubing is a low void volume tubing (e.g. a fine bore capillary tubing). Such tubings are well known to those of skill in the art.
In one preferred embodiment the pressure source 78 can apply two pressures, a blowout pressure (e.g. 15 PSI), and a positive pressure used during printing (e.g. from about 0.1 to 2, preferably from about 0.1 to 1, more preferably from about 0.1 to about 0.5, and most preferably about 0.3 inches of water). Similarly, in preferred embodiments, the vacuum source 80, can apply two pressures, a "high vacuum" for cleaning, and a "low vacuum" for printing operations. The waste receptacle is preferably a low volume waste receptacle (e.g. a 200 ml waste bottle).
A preferred manifold is illustrated in FIG. 8. This manifold comprises a common channel with an inlet port (manifold inlet) 66 and an outlet port (manifold outlet) 68. In fluid communication with the manifold are a number of capillary connectors 76 that each with an internal (manifold) capillary port 72, and an external capillary port 68. Each capillary connector is disposed to receive a connection (e.g. a tubing connection) to provide a fluid communication to a spotting capillary 12. The internal capillary port 72 is disposed inwards into the manifold so that the capillary port is not flush with the internal wall of the manifold. This prevents droplets from accumulating on the internal capillary port 72 which could interfere with reliable loading or delivery of samples.
The manifold can be made of any of a variety of materials and can take a number of different shapes. Preferred shapes however, permit the rapid distribution of pressure, permit the unidirectional flow of gas and waste, and permit the disposition of the internal ports 72 away from the internal surface of the manifold. Useful materials, include various plastics, glass, quartz, ceramic, and metals. In one preferred embodiment, the manifold is fabricated from stainless steel.
A plumbed print head is illustrated in FIG. 9. This figure illustrates the print head 10 comprising a plurality of spotting capillaries 12 (four visible in the figure). The spotting capillaries are in fluid communication with the manifold 64 via flexible capillary tubing 70. A pressure line 84 a can be seen at the upper right and a waste/vacuum line 84 b can be seen at the upper left.
IV. Preparation of a Microarray
The microarray printer of this invention can be used to print microarrays comprising essentially any molecules that can be suspended, dissolved, or otherwise placed in a solution. Preferred microarrays include, but are not limited to microarrays of biomolecules (e.g. sugars, carbohydrates, nucleic acid, proteins, and the like). Particularly preferred microarrays include nucleic acid and/or protein arrays. Methods of preparing and/or purifying biomolecules are well known to those of skill in the art (see, e.g., Berger and Kimmel (1989) Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif.; Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); U.S. Pat. No. 5,017,478; and European Patent No. 0,246,864.).
The materials that are to be printed (e.g. proteins, nucleic acids, etc.) are typically formulated in "printing solutions". Solutions for microarray printing are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 6,101,946, 5,958,342; and MacBeath and Schreiber (2000) Science 289: 1760-1763; Mark Schena (Ed.) (1999) Genes, Genomes and Chips. In DNA Microarrays: A Practical Approach, Oxford University Press, Oxford).
The microarrays can be fabricated on any of a wide variety of substrates well known to those of skill in the art. Such substrates include, but are not limited to glass, plastic, quartz and other minerals, metal, ceramic, porcelain, metal covered (e.g. sputtered) glass, and the like. A number of substrates, often derivatized to facilitate microarray printing are commercially available (see, e.g., silane slides from Sigma Chemical Co., the SuperClean™, SuperAmide™, and SuperAldehyde™ substrates from Telechem, International Inc., etc.).
In operation, the printing pins (spotting capillaries) are initially washed. In a preferred embodiment, this involves moving the print head over a wash bath and applying pressure (e.g., about 15 PSI) to the manifold to blow out any liquid remaining in the spotting capillaries. The spotting capillaries are dipped in and out of a cleaning solution (e.g. 0.001% Micro-90, Cole Palmer Inc.), in a sonicating bath at about 0.75 sec intervals for about 3 cycles while pressurized.
The device is switched from pressure to house ("high") vacuum and the spotting capillaries are dipped into the sonicated cleaning solution for 3 more cycles while drawing cleaning solution into the spotting capillaries. The spotting capillaries are then moved to a sonicating rinse bath that contains pure (e.g. double distilled) water.
The spotting capillaries are dipped in and out of water for about 3 cycles of about 0.75 sec each. This allows the formation of interspersed air bubbles and water in the tubing, assuring that the cleaning solution is more effectively removed from the walls of the tubes. Finally the spotting capillaries are dried by sucking air through them using house vacuum, blowing hot air over the spotting capillaries for about 4 sec, opening a vent to atmosphere and continuing vacuum for about 2 sec in order to clear liquid from the manifold and waste tubing (see FIG. 7).
The spotting capillaries are then filled by dipping the spotting capillaries into reagent reservoir(s) (e.g. a microtiter plate) containing the printing solution(s). Full house vacuum is applied to the manifold for about 0.15 sec in order to assure that the solutions enter the tips of the pins. The manifold is then vented to atmospheric pressure and the spotting capillaries are allowed to sit in the printing solutions for about an additional 0.75 sec so that capillary action fills the spotting capillaries. In one preferred embodiment, the spotting capillaries each contain approximately 0.2 ml when full. The spotting capillaries do not fill beyond their tops due to capillary action—the solutions do not enter the flexible tubing that connects the pins to the manifold. In some cases a slight vacuum of ˜0.2 inches of water is used to assist the filling. This is adjusted to be low enough so that the no liquid is drawn beyond the top of the pins. The entire wash, dry and fill functions take about 25 seconds.
To print an array feature or features, the print head is moved over the first printing substrate and lowered to make contact, and raised. In one preferred embodiment, in the upper position the tips of the printing pins are about 0.5 to 1.0 mm above the array substrate, and when printing the print head is lowered so that the pins would move about 0.2 mm below the substrate surface if no array substrate were present. When a substrate is present the spotting capillary tips contact it and the spring mounts allow the spotting capillaries to stop moving while the print head body continues its motion toward the substrate. In a preferred embodiment, the total time for the print head to move down, contact the slide and return to the upper position is about 0.05 to 0.2 sec. During the printing operation a constant pressure of 0.1 to 0.4 inches of water is applied to the manifold to keep the printing solutions at the tips of the spotting capillaries. This assures that the spotting capillaries are wet with the printing solutions so that liquid will be transferred to the substrate on contact. The pressure does not eject the printing solution. If this is not done, the solutions can pull away from the tips and the printing will stop. The small diameter of the tubing and the printing pins provides enough flow resistance to air so that the manifold pressure is maintained even if one or more of the pins does not contain printing solution. The cycle is repeated to print additional features or other array substrates.
The amount of printing solution that is deposited on the substrate depends on the interaction between the substrate and the printing solution, and the diameter of the tip of the printing pin. When printing salt solutions on glass, each fill of the printing pin (0.2 ml) can make at least 10,000 spots. When printing 20% DMSO solutions with DNA, the same load can print at least 2000 spots. This is many more spots than are possible with other printing systems. Thus the system described above can deposit less than 100 pL per spot in typical printing.
Use of the novel spotting capillaries and/or print heads of this invention provides extremely efficient reagent usage. In certain embodiments, the printer can print at least about 500 spots (features) per 0.2 μL load, more preferably at least about 1000 spots (features) per 0.2 μL load, most preferably at least about 1500 spots (features) per 0.2 μL load, at least about 2000 spots (features) per 0.2 μL load, at least about 5,000 spots (features) per 0.2 μL load, or at lest about 10,000 spots (features) per 0.2 μL load. Because the printing capacities are so high, the print head typically does not need to refill during a print run. This greatly decreases the duration of a print run.
V. Microarray Printing Device
The various elements described above, e.g. spotting capillaries, print head design, array support platen, print head support platen, vacuum and pressure system, manifold, sample loading unloading protocols, and the like can be incorporated individually, or in combination, into preexisting microarray printers or they can be assembled into a microarray printer built de novo.
Methods of designing and building microarray printing devices are generally known to those of skill in the art (see, e.g., U.S. Pat. Nos. 6,110,426, and 5,807,522, and publications of the Brown Laboratory at Stanford University (e.g., The McGuide. Version 2.0, available on the internet at http://cmgm.stanford.edu/pbrown/mguide/index.html, and from Cold Spring Harbor Laboratories).
In general, microarray printers of this invention will, in preferred embodiments, include a base adapted to hold reservoir(s) of printing solutions, a platen for supporting and positioning microarray substrates, and a platen for supporting and positioning a print head. The microarray printer will include actuators (e.g. motors) for driving/positioning the various platens and for vertically positioning the print head. The microarray printer, will typically include associated electronics to read encoded platen positions and/or to drive the various actuators to position the array substrates and print head. Typically such electronics will include a computer controller. The microarray printer can additionally comprise vacuum and pressure lines, reagent reservoirs, waste receptacles, cleaning baths and the like as described herein.
In a particularly preferred embodiment, the microarray printer will include the print head platen, the array substrate platen, a print head comprising spotting capillaries as described herein. The microarray printer will also preferably also include pressure and vacuum sources as described herein. While it is preferred that the microarray printer comprise all of the elements described herein, it is not required that all such elements be present. Thus, in certain embodiments, the microarray printer comprises one, two, or only a few of such elements. Thus, for example certain microarray printers may only comprise a print head according to this invention and/or the array substrate platen, and/or the print head support platen, and so forth.
VI. Microarrays
It is believed that the microarray printers of this invention permit the production of spotted microarrays with an accuracy, consistency, and array feature density previously unavailable. Thus, in certain embodiments, this invention provides high-density microarrays comprising a plurality of molecules, preferably biomolecules where the array comprises at least about 1,000 features (spots), preferably at least about 10,000 features (spots), more preferably at least about 40,000 features (spots), and most preferably at least about 100,000 features (spots), or at least about 1,000,000 features (spots). In particularly preferred embodiments, the features are present at an average center-to-center spacing of about 130 μm or less, preferably about 100 μm or less, more preferably about 80 μm or less, and most preferably about 65, 50, or 40 μm or less.
In preferred embodiments, the microarray is a protein and/or nucleic acid microarray. In nucleic acid arrays the nucleic acids preferably have an average length greater than about 100 nucleotides, preferably greater than about 500 nucleotides, more preferably greater than about 1,000 nucleotides, and most preferably greater than about 2,000, 3,000, 5,000, or even 10,000 nucleotides.
Preferred arrays of this invention have a feature (spot) density greater than about 20,000 features/cm2, preferably greater than about 30,000 features/cm2, more preferably greater than about 40,000 features/cm2, and most preferably greater than about greater than about 50,000 or 60,000 features/cm2.
Because the microarrays are spotted, the reagents are typically simply spotted, in preferred embodiments, the molecule(s) comprising the array features are simply adsorbed to said substrate. However, in certain embodiments the reagents and/or the array substrate can be derivatized so that the molecules comprising the features (spots) covalently couple to the substrate. Methods of so derivatizing macromolecules are well known to those of skill in the art. Thus, for example, the reagents can be derivatized with a sulfhydryl group (—SH) which will covalently couple to a gold surface (e.g. gold coated glass).
V. Kits
In still another embodiment, this invention provides kits comprising one or more containers containing the arrays described above. In certain embodiments, the arrays will comprise features representing nucleic acids from every chromosome in a subject organism (e.g. a human). In certain embodiments, the arrays will comprise features representing nucleic acids from every known expressed sequence tag (EST) for a given organism, or tissue, or whose expression is associated with a particular physiological state (e.g. a particular pathology).
These array constituents are merely illustrative. Numerous other arrays components will be recognized by one of ordinary skill in the art.
In certain embodiment, the kits can, optionally, additionally contain one or more of the following: detectable labels, hybridization reagents, software, buffers, and the like.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the purview of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
Citas de patentes
Citada por
Eventos legales
Imagen original


text:
It is an easy-to-build, stable, and cost-free or at least almost cost-free boat that can be divided into three parts, to be carried inside two small cars.
Step 1: Gathering the materials and the first steps
Materials :
- Palettes (ask hardware shops and/or trading companies)
- almost any kind of wood
- PVC skin ( like its used for ponds )
- Nails
( you can ask hardware shops for wood they do not need and are wanting to throw away - this way you can find good and unused wood)
Tools :
- Hammer
- Screwdriver
- Drill Machine
- Grinder
- Saw
We wanted to build a very cheap, easy to build and solid boat that could carry us four and also some additional luggage. Another important aspect was that we could transport it in an easy way, as we don't have a trailer. Therefore we have decided to build a boat that can be divided into three parts
We started by aligning three of the palettes in a row and checking whether we we would fit comfortably in it.
By building little "bridges" (as we called them) we started building a stable way to be able to connect the segments by pushing a large wooden strip trough them.
Be very careful when working as there are multiple possible dangers for example a lot of nails looking out of the pallets or the saws.
We do not take any responsibilities for possible injuries or damages that might occur to you.
Step 2: Setting up the side panels
We build the side panels by using some parts of the pallet.
They don't have to be very stable as there won't be too much force on it.
(The water will not reach higher than the pallets)
Step 3: Seats
When travelling the hardware store, and asking for unused wood that would be thrown away, we were offered some laminate for free.
We later on used this laminate for our noble seats.
Step 4: The helm
The back of the boat was built very strong so it can carry the helm.
When travelling we figured out that the helm is very useful on a lake, but it doesn't help too much on a river.
It is nice to see that our project inspired other people and i have to say that your boat looks really good. Regarding your question I got to say that our boat was longer than yours, what might be the cause for the additional stability, but it could be also the additional weight of 3 to 4 persons. If I remember correctly our boat had a flotation depth of approximately 15 cm.Making it heavier could help your boat, but i can not tell for sure.
now I'm regretting that fishing ship idea and thinking of a canoe plane (yes I did say that), fully made from spruce pallets (I think they make those)
It's like a Viking boat with that bird on the front....cool!
Hmm... someone should make a fishing ship out of pallets.
Well we already thought of taking a rod with us, but until now all we did was to chill a bit on the water. :)
As someone who also just posted a pallet transportation project...this is awesome! haha

subset: commoncrawl


text:
Lana Del Rey – Black Beauty (Night Crime Remix) : Deep House [Free Download]Posted in Chill by nick | Tags: Deep House, Free Download, Future house, Lana Del Rey, Remix
The incredible voice of Lana Del Rey has been remixed every which way in the past year; with noteworthy flips from ZHU, Stint, Village, and countless others. Today, we have a fresh new remix for 2015 from up and coming LA producers Night Crime of her song "Black Beauty" off Lana's Ultraviolence album. The duo have turned the song into a deep house masterpiece keeping the vocals in place and adding subtle bass plucks in the mix. Stream the full song below and grab a free download thanks to Night Crime. Enjoy!
Lana Del Rey – Black Beauty (Night Crime Remix) Free Download
ZHU Remixed Lana Del Rey "West Coast" : House / DiscoPosted in Chill by nick | Tags: disco, House, Lana Del Rey, Remix, ZHU
One of the most exciting new names in dance music ZHU has been enlisted to remix Lana Del Rey's newest irresistible single "West Coast". Being a producer from the west coast couldn't have been more fitting for ZHU to take on the track. He worked his now signature blend of dance all throughout the track incorporating elements of house, disco and electro with an indie spin. No idea on if we will see a release date here, but we can listen in full below. Enjoy!
ZHU x Lana Del Rey – West Coast | Stream Only
Lana Del Rey – West Coast (Stint Remix) : Unexpected Chill Electro [Free Download]Posted in Chill by Bryan | Tags: Chill House, Free Download, Lana Del Rey, Remix, Stint
The anticipation of Lana Del Rey's forthcoming third studio album Ultraviolence has reached its climax after a memorable performance at this years Coachella Music Festival and release of the debut single "West Coast" thats hit the radio waves by storm just before summer. We receive a remix that accentuates her vocals with beach-esque vibes from Vancouver newcomer Stint whose applied a beautifully creative electro spin complete with whistles and all. The original vocals are about the only thing left intact in this energetic completely new version of the song. Stream the remix below and grab an exclusive download on us. Enjoy
Lana Del Rey – West Coast (Stint Remix) | Free Download
Follow Stint through Twitter
Follow Thissongissick through Twitter
Lana Del Rey Previews Dark New Original Single "Shades Of Cool"Posted in Chill by nick | Tags: Dan Auerbach, Lana Del Rey, The Black keys
Lana Del Rey has been unveiling her more blues, soul focused upcoming album Ultraviolence executively produced by Dan Auerbach of The Black Keys. Two of her biggest hits "Summertime Sadness" and "Young & Beautiful" were co-written and produced by Rick Nowels and today we get the latest pairing of the duo with "Shades Of Cool" which is also the next single from the upcoming album. We get a drawn out emotion filled ballad having Lana stepping further away from the radio ready singer we grew to know. Listen in full below and enjoy!
Lana Del Rey – Shades Of Cool | Stream Only
Lana Del Rey Steps In The Right Direction On First Single "West Coast" From New AlbumPosted in Bangin, Chill by nick | Tags: Coachella, indie, Lana Del Rey
After performing the unreleased song "West Coast" in her set at Coachella this past weekend, Lana Del Rey has uploaded the full audio single online for our listening pleasure. This is actually the first single from her second artist album Ultraviolence, which is produced by Dan Auerback of The Black Keys. Having Dan on the album showcases a more matured Blues inspired Lana and leaves us anxiously wondering how the rest of the album will sound. Look out for more info in the coming weeks with a current tentative release date of May 18th.
Lana Del Rey – West Coast | Stream Only
[PREMIERE] Lana Del Rey – Gods and Monsters (ViLLAGE Remix) : Chill Trap / Future Bass [Free Download]Posted in Chill, Premieres by Bryan | Tags: Chill, Gods and Monsters, indie, Lana Del Rey, Trap, ViLLAGES
Not many would think the Southeastern European country of Romania would be home to an emerging electronic music producer, but thanks to advancements in technology we have discovered the talents of ViLLAGE via an incredibly well done remix. Taking the reigns of iconic singer / songwriter Lana Del Rey "Gods and Monsters", ViLLAGE adds a subtle dose of bass and chill trap vibes. The screeching synths accompany Lana's vocals perfectly and recapture the same emotions as the original. Stream the remix below and grab a free download as well! Enjoy
Lana Del Rey – Gods and Monsters (ViLLAGE Remix) | Free Download
Follow ViLLAGE through Twitter and Facebook
Follow Thissongissick through Twitter and Facebook
[PREMIERE] Lana Del Rey – Young & Beautiful (PatrickReza Remix) : Melodic Indie / Dubstep Remix [Free Download]Posted in Bangin, Premieres by nick | Tags: BANGER, Bass Music, Chill Dubstep, Dubstep, Free Download, indie, Lana Del Rey, PatrickReza, Remix
The unstoppable bass producer PatrickReza takes on the bone chilling single "Young & Beautiful" from Lana Del Rey and gives it a signature melody driven dubstep remix. He just recently released his all original The Woods EP, then Patrick in his words:
watched the Great Gatsby and heard this track and knew I had to remix it. Free Download because I love you. Sharing is caring! ♥ PatrickReza "
[PREMIERE] Lana Del Rey – Young & Beautiful (PatrickReza Remix) | Direct Download
Follow PatrickReza through Twitter and Facebook
Lana Del Rey – Summertime Sadness (Cedric Gervais RMX) {ETC!ETC! Trapped it! Remix} : Huge Trap / Indie Remix [Free Download]Posted in Bangin by nick | Tags: ETC!ETC!, indie, Lana Del Rey, Remix, Trap, Trap Music
Lana Del Rey got a huge remix to "Summertime Sadnesss" from Cedric Gervais which ETC!ETC! decided to flip into his own summer trap anthem. The song leaves Lana's vocals left in tact while turning it into a straight trap anthem filled with beefy snare rolls and all. The summer vibes pour out of this song that ETC!ETC! has nicely made available for free download. Enjoy!
Lana Del Rey – Summertime Sadness (Cedric Gervais RMX) {ETC!ETC! Trapped it! Remix} | Direct Download
Follow ETC!ETC! through Twitter
Lana Del Rey – Young and Beautiful (Kulkid Remix) + Original + DH Orchestral Remix : Chill House Remix [Free Download]Posted in Bangin, Chill by nick | Tags: Chill House, Kulkid, Lana Del Rey, Remix
Lana Del Rey's offering from the soundtrack to The Great Gastby is an incredible smooth ballad. From the emotional piano lines to powerful voice, "Young and Beautiful" is guaranteed to give you chills. We have fresh chilled out remix from unknown Kulkid adding some minor elements highlighting the track. Listen to the remix, Original, and the moving DH Orchestral Remix. You can purchase the original and Orchestral version through iTunes now. Grab the download of the Kulkid one. Enjoy!
Lana Del Rey – Young and Beautiful (Kulkid Remix) | Download | Facebook to Download
Lana Del Rey – Young and Beautiful (Original) | iTunes
Lana Del Rey – Young and Beautiful (DH Orchestral Remix) | iTunes
Follow Lana Del Rey through Twitter
Follow Kulkid through Twitter
Lana Del Rey – Bel Air (Music Video) : Smooth Video from Film "Tropico"Posted in Chill, Video by nick | Tags: Bel Air, Born to Die: Paradise Edition, indie, Lana Del Rey, Music Video
Lana Del Rey is in the final days before her double album, Born to Die: Paradise Edition. So fittingly we get a new music video and single. "Bel Air" is another powerful piano driven indie anthem that lets you get lost in Lana's powerful vocals. The video has a very simple theme, changing colors and showing her appear and disappear in clouds of smoke. The simplicity really accents how powerful her voice really is. You can now pre-order the new version of her album "Born to Die" Paradise Edition, including a handful of other new songs as well. Enjoy!
Lana Del Rey – Bel Air | Pre-Order iTunes
Lana Del Rey – Ride (Music Video) : Indie Short-Film Music VideoPosted in Chill, Video by nick | Tags: indie, Lana Del Rey, Music Video
Do you like Lana Del Rey? You'll love this, you don't like her? Still might enjoy this video. Here's her latest 10 minute music video / short film that was written by LDR herself. It's for "Ride", which we heard the audio with some other new tracks a few weeks back. This 10 minute long short film of a music video follows the story of Lana as a biker, story written by Lana. The editing and overall aesthetic of the video matches her past, with a nostalgic vintage 70′s wash. "Ride" is the lead single off The Paridse Edition of "Born to Die" coming out November 3rd (Pre-Order Here). Do you like when artists make short films like this? How do you feel about new Lana? Answer below. Enjoy!
Lana Del Rey – Ride | iTunes
Purchase all of Lana Del Rey through iTunes
Lana Del Rey – 5 Chill Unreleased SongsPosted in Chill by nick | Tags: Chill, Electronic, Folk, indie, Laid Back, Lana Del Rey
Lana Del Rey has been featured on the site through countless remixes, but we haven't given her original stuff nearly enough attention. She's been writing her next record, and has been leaking tracks through different soundcloud accounts. We've got 5 of the best tracks, including some of my favorite Lana tracks she's ever made. Downloads for these will be update soon. Purchase the available Lana Del Rey through iTunes here. Enjoy!
Lana Del Rey – Serial Killer | Download
Lana Del Rey – Ghetto Baby (Demo) | Download
The other 3 songs were removed at request of Lana's people.
A$AP Rocky – Ridin (Ft. Lana Del Rey) : Mellow Indie / Hip-Hop CollaborationPosted in Chill by nick | Tags: A$AP Rocky, ASAP Rocky, Hip-Hop, indie, Lana Del Rey, Rap
Never thought I would see buzzing rapper A$AP Rocky and indie singer Lana Del Rey on the same track.. but it's here, and it works pretty damn well. The unique contrasting styles form an incredibly catchy track with perfectly mellow bat carrying it out. Can't wait to see more from these two, enjoy!
A$AP Rocky – Ridin (Ft. Lana Del Rey) | Direct Download
Lana Del Rey – Diet Mountain Dew (Tommy Noble Remix) : Heavy Dubstep / Drumstep / Indie RemixPosted in Bangin by nick | Tags: BANGER, Bass Music, Drumstep, Dubstep, Electronic, Heavy, indie, Lana Del Rey, Remix, Tommy Noble
Today we get another remix to Lana Del Rey. We heard a hip hop and mellow dubstep remix to "Video Games". Well now we get a really heavy reworking of her track "Diet Mountain Dew" done by Tommy Noble, a two piece duo from the Central Coast of California. For this track, they flipped the very soft sung track by Lana Del Rey and really kick up the bass. The drop is HUGE and smooth. This is meant for some massive speakers, so don't ruin it with laptop speakers. Turn up the bass, enjoy!
Lana Del Rey – Diet Mountain Dew (Tommy Noble Remix) | Direct Download | Backup Download
Be Sure To Follow Tommy Noble through Official Facebook Here
My story has changed quite a lot since moving to Boulder, Colorado and starting Thissongissick back in 2010. My name is Nick Guarino and this is Thissongissick.com. We've grown a lot since, but the original goal has always stayed the same: making it easier for you to discover sick new music that you've never heard before. In high school I would make mix cd's for my friends to show them all the new music I was discovering. Now, through TSIS, we work with musicians and record labels from all over the world to showcase a variety of the freshest electronic, hip-hop, indie, and alternative music to a global following that is comprised of millions of music lovers in over 110 countries - every single month. So, make yourself at home while enjoying countless amazing musicians from a variety of genres and let the sick sounds fill your ears! - Nick


text:
- Join Date
- Dec 2010
- Posts
- 17
Posted On:
9/27/2011 11:45pm
Style: TKD--
How do we tell?
This is mainly a question for the LEOs on the forum here. A friend of mine was telling me a story that rather disturbed me today, and it disturbed me enough that I'm wanting the opinions and advice of some of the LEOs. I'm not going into the details of the story, as it was told in a private setting with the friend's trust, so this question is going to be somewhat vague. I would rather this stay in a PM, but I do not have the post count necessary to use the PM system. If this isn't the right forum to post this in then I ask that a moderator move it to the correct place.
Basically the long and the short is that there was an encounter at night with an unmarked police car out in the middle of rural Arkansas. However the two occupants of the supposed police vehicle were un-uniformed, armed men with no visible badges or identification. The only way to tell if they were LEOs (if they were at all) was the fact that they were driving an unmarked police vehicle (with the blue lights and everything) and they approached the pulled over vehicle in the manner that a legitimate cop normally would. The problem is that they were supposedly very aggressive.
And so arises a bunch of questions. First of all, are officers required to be in uniform and have a badge displayed in order to carry out duties? And how do we tell if an unmarked police car is a legitimate police vehicle? A quick search tonight on ebay showed that you can buy the LED lights to make a vehicle look like an unmarked car.
Also, considering the very unlikely event that I'm caught in the same situation, can I refuse to show them ID if they don't have a visible badge? Basically, what are we suppose to do in these type of situations?
Registered Member
- Join Date
- Aug 2011
- Location
- Atlanta, Ga.
- Posts
- 72
Posted On:
9/28/2011 12:32am
Style: None--
Very vague. Was it a traffic stop? Or did your friend just "happen upon" the unmarked car, or perhaps were the officers watching your pal?
I couldn't give advice without knowing more. I mean were they suits? Were they UC (vice/narc)?
In my experience, Narc UC's won't break cover unless it's absolutely necessary.
Detectives can stop cars. Their vehicles are usually equipped with blues in the grill, and prob dash and rear window mounted blue lights. Most likely though, they'll get a car stopped, and then call for a uniformed officer to meet with them.
Regardless, they should at least show badge and/or ID. If I'm approached by some guy claiming to be a cop, but shows me nada... I'm gonna tell him to piss off.
Regarding online sales of blue lights... I know any light mounted on a car that's blue or red is illegal in Georgia. PD here uses straight blues, not a combo of blue/red. FD is red only.
- Join Date
- Dec 2010
- Posts
- 17
Posted On:
9/28/2011 12:54am
Style: TKD--
The way it was described to me is that it was a traffic stop out on highway 94 in East Arkansas, which all farmland; there's nothing out there. Supposedly the unmarked vehicle had followed for some distance before lighting up the lights. They were supposedly wearing what sounded like business casual wear but had holstered weapons on their side. Sounded more like armed security guards than police officers. Again, no visible badge indicating either way. As for vice/narc UC, I don't know simply because I don't know the difference between the two.
Shoot it out, then check if they have badges, dead men can't deny you their wallets. If they do have badges, rob them, you've already killed two cops so a felony robbery charge isn't going to matter much, no matter how good your defence or your belief that legally they should have ID'd with badges, it doesn't matter, you're going down so you might as well enjoy your freedom. If they don't have badges, um, rob them, that's what they were trying to do to you.
Seriously, though, that's a messed up situation.GET A RED BELT OR DIE TRYIN'.
My dog is cuter and smarter than yours.
- Join Date
- Jun 2009
- Location
- Bonners Ferry, Idaho
- Posts
- 7,004
Posted On:
9/28/2011 2:32am--
If you friend is so bothered, he should file a complaint or at least make an inquiry to the local s/o or even to the ASP if he is worried about some sort of retribution.Falling for Judo since 1980
Registered Member
- Join Date
- Dec 2006
- Posts
- 559
Posted On:
9/28/2011 4:39am
Style: Karate--
I'm in the UK so may not be so helpful but as I understand it if you believe someone has impersonated a police officer you are duty bound to report it.
Sorry I can't be of any more help. It sounds like a terrifying situation to be in and if they were real officers it doesn't strike me as proper conduct not to properly identify themselves.
sorry double post - site weirdness today
Last edited by submessenger; 9/28/2011 10:56am at . Reason: dup
There was a spat of robberies in the Philadelphia area a few years back - perps pretending to be unmarked cars. The advice that came out was to signal your intention to pull over, then proceed to find a "safe" area in which to do so, preferably something well-lit and populated.
The last time I was pulled over, a few years back in Florida, I remembered the Philly happenings, and did exactly that. Officer had no problem with it.
I would hazard a guess that an LEO could not perform a legal arrest without properly identifying him/herself, and that possession of badge and other proper identification would be a requisite.
I think the missing piece of this puzzle is that the public is generally uninformed about such things. I look forward to what our resident LEOs have to say on the matter.
pro nonsense self defense
- Join Date
- Nov 2012
- Location
- San Diego
- Posts
- 13,050
Posted On:
9/28/2011 11:02am--
I've heard that in situations like this, you can request a uniformed patrol car to come pick you up if they're trying to detain you. If someone is impersonating an officer, they've gone through a good bit of effort and are doing so at substantial risk (impersonating a cop is a serious crime), so I figure if I'm dealing with a fake cop, I'm dealing with a real sinister asshole, so I'm not getting in his car.


text:
Finally I have a little bit of time to finish by much overdue Baselworld report. Well at least I won't have to compete for eyeballs now As usual I arrived the day before the fair is opened to the public. At this point the town is still quiet. There are some press around and the… Read More
Baselworld 2010
Kingston Non-dates are here!
The non-date Kingston C3 and BGW9 watches are here! This is the first batch of 100 watches (50 of each type). If you received the e-mail about submitting your parts kit orders you are in this first batch. We are sending out a second batch of the non-dates and the first batch of date dialed… Read More
Kingston Update
The parts kit ordering page is up. At the moment we are only taking orders from the first 100 customers that we will be shipping to first so we can concentrate on shipping rather than the paperwork for all of the customers. We have updated the ticket system to provide those customers with the ordering… Read More
Kingston Update
The watches are leaving Swtizerland some time this week. Our plan at this point is to begin shipments next week. We just finished updating the tickets for the first batch of BWG9 and C3 customers and I will be working on the parts ordering page later today. We will do our best to deliver as… Read More
Kingston Update
Mk II Restructuring
Industry shifts There are two major industry currents that are impacting us right now. The first is ETA's efforts to remove themselves as the movement supplier of necessity for most of the Swiss industry. In my opinion it will not be a matter of "if" but a matter of "when" this will take place. The… Read More
Kingston: Prep for Assembly
Here is what we do for each batch of components going out for assembly. It's far from just shoving a bunch of components into a different box and shipping them out to Switzerland. I apologize in advance for the picture quality. These were quick and dirty shots I took during different stages of the process…. Read More
Customer Service improvements
Similar to my stabilizing health the strides that we have made in improving customer service are only beginning to have an impact. Over the summer the time and effort that we made to improve our customer service infrastructure made the situation noticeably worse while we spent time and energy to integrate the software and built… Read More
Survey sent!
I have just sent out the e-mail update for the Project 300. If you have a deposit for the Project 300 please keep an eye out for it and take the survey that is included in the e-mail. If you do not receive the e-mail please check your SPAM folder first. If you still do… Read More
Kingston Update
The second batch of parts is almost ready to go to Switzerland. I am currently working on the C3 non-date Kingstons and getting them ready for assembly. I still have to post the pics I took from the BG W9 preparation which will give you a better idea of the scope of the process. In… Read More


text:
ISO: Cane Syrup
I'm trying to track down some cane syrup, like you'd find in Louisiana. Not golden syrup, but cane syrup. Any help would be great.
You haven't specified your syrup very well. Is it Karo? sucrose? fructose? Lyles?
How is it to be used?
With nothing to go on, I would make a syrup by boiling 2 parts water and 3 parts light brown sugar, stirring until there is a syrup. That will be a cane syrup because the light brown sugar is cane sugar.
re: jayt90
yes that might work but you must be carefull, a lot of brown sugars out there are made by adding molasses to refined white sugar, which is cheaper to manufacture, and usually the white sugar used is from sugar beets not sugar cane but the molasses is from the sugar cane though to give it a better flavor.
cane syrup is basically taking the sugar cane juice and reducing it down to a syrup, the best way to do that would be to find a carribean food vendor that sells fresh pressed cane juice and render it down yourself. there is a vendor at the busy bee flea market on hwy 27 that sells cane juice.
re: youdonut
Soon Lee has solid blocks of cane sugar, $3/lb
Soon Lee
629 Markham Rd, Toronto, ON M1J, CA
re: youdonut
how about using Demerara sugar instead of brown sugar and boiling water down using above procedure?
re: jayt90
It's just called Cane Syrup, it's its own product, not like a simple syrup or anything. I put not golden syrup, as in Lyle's. Cane syrup is similar to corn syrup (Karo), but not corn based. It's cane sugar based as the name would imply.
Try these guys, they are located in mississauga and can sell through thier website.
it seems that this may be available through some coffee shops maybe??
re: youdonut
Thanks, but that's more of a simple syrup made from cane sugar, this is more of a southern Louisiana/Mississippi product.
re: FoodJourno
I brought back some Steen's cane syrup from New Orleans, I was just there for Jazz Festival:) Man this stuff is sweet, but necessary in a lot of Southern baking.
I wonder if Whole Foods might have it? As an alternative, you could order it from:
www.cajungrocer.com which has all kinds of awesome products from Louisiana.
re: FoodJourno
I hunted high and low, to no avail.
I have ended up ordering a case every couple of years directly from Steen's.


text:
Skip to content
Drama News & Events
Box Office
About The Program
How To Apply
Student Center
Production Resource Office
Production Archive
High School Programs
Summer in Drama
Elizabeth Bradley
Arts Professor,
726 Broadway, Room 251
Arts Professor Elizabeth Bradley is an educator, theatrical producer, festival curator, presenter, and international cultural consultant. She was recruited in 2008 to the Tisch School of the Arts, New York University as Chair of Drama after heading the School of Drama at Carnegie Mellon University. Her appointment at Carnegie Mellon followed a wide-ranging career of almost thirty years in the professional performing arts in her native Canada. For a decade, Bradley was CEO of the Hummingbird Centre (now Sony Centre) for the Performing Arts in Toronto, the largest in Canada. The fourteen million dollar organization presents multi disciplinary attractions from around the world, including many of the major artists of the last two decades. Her theatrical producing credits include Tony-nominated Broadway productions, Edinburgh Festival premieres and extensive tours to major houses such as the Kennedy Center, and New York's City Center. She has worked directly for artists as manager and agent, and occupied a senior management position for the Stratford Shakespeare Festival of Canada. She is a recipient of the Queen's Jubilee Gold Medal for outstanding contribution to the arts in Canada. A past Chair of the International Society for the Performing Arts, an organization that links leaders in the arts from 51 countries, Bradley was instrumental in the creation of the Inaugural International Arts Forum in Beijing, China in 2001. She was the Founding Artistic Director of the first edition of the Pittsburgh International Festival of Firsts held in October of 2004. The Festival brought nine US premieres of cutting edge multi disciplinary performance work to Pittsburgh to complement the visual arts showcased in the Carnegie International. For the past five years, Bradley served as Senior Artistic Associate for the Stratford Shakespeare Festival, Canada under the Artistic Directorship of Des McAnuff. Currently she chairs the US board of the National Theatre of Scotland, and advises the Intiman Theatre in Seattle, WA. Her teaching and research interests focus on artistic entrepreneurship, which she currently teaches at New York University in a learning module initiated at Carnegie Mellon for graduate MFA students. The Self Start content has engaged students from Hong Kong to Glasgow. In addition, she teaches emerging arts leaders in a course sequence that focuses on global best practices in meeting contemporary sector challenges. This month she will serve on the jury for the Theatre Communications Group's Global Initiative Grant program. Her great passion is contributing to cultural collaboration and empowerment in whatever part of the world she is fortunate enough to be invited!
Undergraduate Admissions
Graduate Admissions
Academic Services
Student Life
Financial Aid and Scholarships
Career Development
Counseling Services
International Students
Incoming Students
What is Special Programs?
Study Abroad
Summer High School
Summer in New York City
Courses for Non-Majors
Programs for Visiting Students
Open Arts
Admin & Faculty
Contact Us
Tisch Events Calendar
Admin & Faculty Calendar
NYU Academic Deadlines Calendar
Many Ways to Give
Why Support Tisch
Dean's Council
Corporate Giving
Foundation Giving
Individual Giving
Tisch Gala
Who We Are
Institute of Performing Arts
Graduate Acting
Design for Stage & Film
Graduate Musical Theatre Writing
Performance Studies
Maurice Kanbar Institute of Film & Television
Undergraduate Film & Television
Graduate Film
Cinema Studies
Moving Image Archiving & Preservation (MIAP)
Rita & Burton Goldberg Department of Dramatic Writing
Emerging Media Group
Photography & Imaging
Interactive Telecommunications (ITP)
Clive Davis Institute of Recorded Music
NYU Game Center
Art & Public Policy / Arts Politics
Open Arts
Tisch Special Programs
Tisch Asia


text:
Aug. 23, 2012
The University of Georgia is participating in the inaugural Pledge Your Allegiance for College Colors Day spirit competition and is asking for fan support as a part the national College Colors Day Spirit Cup and $10,000.
By visiting www.espn.com/collegecolorsday, Bulldog fans can register to pledge their allegiance to Georgia as the school with the most college spirit and loyal fan base. All registrants are entered to win weekly $50 gift cards from ESPN Shop and one of two grand prizes including a $500 college shopping spree at ESPN Shop.
The website-based rivalry competition, which includes 165 participating colleges and universities and is an element of this year's 2012 College Colors Day celebration, is LIVE and will run through the entire month leading up to College Colors Day and the first Georgia Fan Up Friday on August 31. Throughout the competition, standings will be tracked nationally, as well as by conference.
College Colors Day is an annual celebration dedicated to promoting the traditions and spirit that embody the college experience by encouraging fans across America to wear their favorite college or university apparel throughout the day on Friday, August 31.
The inaugural national spirit competition is presented by The Collegiate Licensing Company (CLC), a division of IMG College, and NCAA Football..
Bulldog Fans are also encouraged to share the Pledge Your Allegiance for College Colors Day competition through Facebook.
Georgia fans can also show their Commitment to the G every Friday this fall by wearing your officially licensed Georgia gear. All members of the Bulldog Nation are encouraged to wear Georgia gear every Friday during football season to work, to play, on errands or hanging out with friends.
Fans can also win great prizes by participating in the Fan Up Friday Photo of the Day. Beginning on Friday, August 31, fans that submit photos in Georgia gear to firstname.lastname@example.com will have the chance to win great prizes. A different winner will be selected each week and the photos will be posted on the official UGA Athletics Facebook page at www.facebook.com/georgiadogs. Fans can also participate by sending photos to the @ugaathletics, the official UGA Athletics Twitter feed, with the hash tag #fanupfriday.


text:
To access members-only content on cta.org, please sign in below.
Register Now
April 16-17, 2013
CTA was joined by the California Federation of Teachers in filing a motion to intervene in a lawsuit aimed at overturning due process and other protections for educators. The lawsuit, known as Vergara v. State of California, was filed by a group of corporate special interests and billionaires with a catchy name who want to push their agenda on California public schools. The lawsuit by Students Matter alleges California Education Code provisions governing due process, dismissal and layoff are unconstitutional. The group admittedly took their battle to the courts after voters defeated an initiative and legislation failed in the state Capitol. The lawsuit is baseless and meritless, and hurts student learning. It's trying to legislate from the bench in an effort to keep educators and parents out of education policy decisions.
The California Democratic Party sent a clear and loud message in support of educators and proven education reforms this past weekend by approving a resolution, "Dispelling the Corporate 'Reform' Agenda." The resolution was sponsored by CTA, CFT and California Faculty Association delegates at the Democratic convention. Nearly 200 CTA members were delegates. The resolution calls out "the billionaires funding StudentsFirst and Democrats for Education Reform who are supporting candidates and local programs that would dismantle a free public education for every student and replace it with company run charter schools, non-credentialed teachers and unproven, untested so-called reforms."
The Democratic Party Resolution follows on the heels of the release of a confidential memo that shows StudentsFirst founder Michelle Rhee was aware of and took no action on the widespread cheating scandal that rocked Washington, DC public schools while she served as chancellor. Meanwhile, in contrast to Rhee, Bill Gates reversed course earlier this month by funding a new study that shows tying test scores to teacher evaluation does not improve teacher effectiveness. His letter in the Washington Post is worth the read.
Lawmakers have held a few hearings and have asked a lot of questions about the Governor's proposed state budget and Local Control Funding Formula (LCFF). The Department of Finance has released new reports that show how much money districts would receive under the LCFF compared to the current funding system. In some good news, state revenues continue to come in above estimates. Some are predicting that there will be an additional $4 billion for the revised budget in May. AB 375, the teacher dismissal bill by Assembly member Joan Buchanan that ensures student safety while protecting the rights of educators, was approved by the Assembly Education Committee. This CTA-supported bill streamlines the process and reduces costs, while protecting students and educators. The California Department of Education is getting ready to submit a new ESEA state waiver to the federal Department of Education. This state waiver counters a district waiver proposal submitted by a consulting group called CORE or California Office to Reform Education. CORE represents nine school districts and submitted a district waiver request without the support of any local teacher unions, which is something Secretary Duncan said is necessary. State and federal officials also question the legality of the district waiver.
Does your passion lie in quality instruction or being an advocate for teaching and learning? CTA is excited to announce the launch of a new program, called the Teacher Leadership Cohort. With a grant from NEA, the program is designed to develop a cohort of twenty-four teachers from across the state to assume new or expanded roles as strong advocates for the teaching profession. It is our opportunity to lead our profession. Cohort members will develop local projects around teacher effectiveness to share with colleagues. If you are interested, apply today. The application deadline is May 10. More information is also available at www.cta.org/ipd.
I want to take a moment to thank you for everything you do! Whether you are in Pre-K or higher education; working in the classroom, in the school office or in the cafeteria; YOU are making a difference for California students. I thank you for all of your hard work as we get ready to celebrate California Day of the Teacher and Education Support Professionals Day (ESP). Day of the Teacher is May 8, while ESP Day is May 22. Keeping with our sesquicentennial, the theme is "California Teachers: Honoring our Past, Guiding the Future." This year, I'm asking you to take a moment and breathe, and to think about some of your own accomplishments. Take a moment to give yourself a pat on the back for a job well done. It has been an incredible year and you deserve it. I also ask you to stop and thank a colleague or a mentor. It's what these days are meant for.
Every child deserves a chance to learn and no child succeeds alone.
© 1999-


text:
Zenerx have beat each one of other men supplement available in the market.
Therefore were referring to man supplement than there may be really many thins to discuss. Question were al busy sure we would like to choose the one which works effectively and bring bad negative effects or perhaps in other way it really is safe to use. Maybe you've attempted to look some information on some man supplement? When it yes there exists a big chance you have found one supplement called Zenerx. This is actually the supplement we are going to talk moe here. Why I chose Zenerx scamto discuss. Because i think here is the best man supplement among all around. You'd like to learn the main reason? Keep reading than.
Firs reason is that this man supplement go a long way like a charm. Comparing to customer reviews from this product I'm able to take a conclusion that Zenerx works for every man in a age. After searching precisely why it functions so well I do think this is because because the ingredients and in what way it really is blended. Ingredients is blended so well in order that it brings probably the most maximum effect on your stamina while having sex using your wife. Zenerx also is using safe natural elements. source. So you will never hear someone died using Zenerx.
If you decide to buy zenerx than just about you are having some issues while having sex. You could possibly wife is not happy with your performance right now or perhaps she wants more longer time while having sex together with you. (is zenerx working) Zenerxis able to assist you to since when it improve your stamina than you'll be much more stronger for a long time. make sure to finish all you need to do with your wife while having sex and leave her until the girl with satisfied. Zenerx is an solution for those man problem with the wife. Buy now and don't regret again.


text:
Hitting the road in time for spring 2013, the Fiat 500e puts an electric twist on this European micro car. The 500e features a 24-kWh lithium-ion battery that charges in roughly 4 hours with a level 2 charger. And with 111 horsepower and 147 lb.-ft. of torque, the zippy 500e will get you from 0 to 60 miles per hour in an estimated 9 seconds. Inside, the Fiat features a six-speaker Alpine Premium Audio System with SiriusXM satellite radio. Bluetooth and USB connectivity let users stream music and make hands-free phone calls. The Fiat Access smartphone app lets users remotely check the 500e's charging status and tire pressure, control HVAC settings and schedule the vehicle to charge at a certain time.
This list is missing the current king of electric vehicles. The Tesla S. Even their new crossover the Tesla X has better range and features than all of these vehicles.


text:
Europcar: Riga, Latvia
Related Airport: RIXCity: Riga View Location Map
Location: In Airport
Pickup Method: Shuttle to Pickup
Pickup Method: Shuttle to Pickup
Book with us today and take advantage of our low prices and cheap deals. And since our prices are always backed by our Best Price Guarantee, you can drive off the rental car lot knowing you have made the smartest choice. Make your Full Size rental car reservation at RIX's Riga Intl. confidently and enjoy your time in and around Old Town.


text:
Sticky Sheep » Communication » The Purpose Driven Mission Statement
The Purpose Driven Mission Statement
Oh, the mission statement. It's the statement that tells the world the role of your company. It's a statement that illustrates goals, purpose, and practice.
The problem is most mission statements don't define the company at all. Too many mission statements contain general, undefined words. They often contain those church words that people don't actually understand. They are safe, but yet meaningless.
Below are two examples of bad mission statement:
- By creating value for our customers, we create value for our shareholders. We use our expertise to create transport-related products and services of superior quality, safety and environmental care for demanding customers in selected segments. We work with energy, passion and respect for the individual. – Volvo
- Sony is committed to developing a wide range of innovative products and multimedia services that challenge the way consumers access and enjoy digital entertainment. By ensuring synergy between businesses within the organization, Sony is constantly striving to create exciting new worlds of entertainment that can be experienced on a variety of different products. – Sony
Seth Godin has his own take on mission statements:
"Mission statements used to have a purpose. The purpose was to force management to make hard decisions about what the company stood for. A hard decision means giving up one thing to get another. Along the way, when faced with something difficult, many managers just punted." – http://sethgodin.typepad.com
How does the church deal with mission statements?
Well, some are just as bad as businesses. However, many do it well, because they have a well defined purpose. The church knows who they are. They know their purpose, unlike many businesses.
Rick Warren, author and founding pastor of Saddleback Valley Community Church in Orange County, California, delves deeper in church creations. In his book, Purpose Driven Church, Warren takes on the steps of defining a church in four questions.
- Why does the church exist?
- What are we to be as a church?
- What are we to do as a church?
- How are we to do it?
Through the practice of answering the questions above, a church defines its purpose. In essence, it's mission statement. A clearly defined mission statement provides a purpose that can be used to initiate and refine a company's plans.
In Made to Stick, authors Chip and Dan Heath, touch on the mission statements and their common problems.
"We must explain our ideas in terms of human actions, in terms of sensory information. This is where so much business communication goes awry. Mission statements, synergies, strategies, visions — they are often ambiguous to the point of being meaningless."
Taking the Warren steps above can easily be translated into any business's mission statement:
- Why does the church exist? – Why does the company exist?
- What are we to be as a church? – What are the long-term goals?
- What are we to do as a church? – What are the short-term goals?
- How are we to do it? – How do we accomplish those goals?
The best mission statements are clear and concise. I know there are awful mission statements out there. Share your best and worst with me.
Filed under: Communication · Tags: business, church, communication, corporate, mission statement


text:
Site Security & Privacy
BrooklynPorkStore.com is the online shop that lets you order Italian specialty foods from Landi's Pork Store, a Brooklyn landmark for more than 80 years. Now run by the third generation of the Landi family, the store has expanded to offer Italian food online and deliver it throughout the United States, including Alaska and Hawaii. Finally, exceptional Italian food products are available to you no matter where you live.
The Brooklyn Pork Store offers an incredible range of Italian food products, including:
Now you can enjoy the flavor of fresh, authentic Italian specialty foods for special occasions, holidays, or just a delicious family meal. All of our Italian specialty foods are carefully packed to arrive at your home cold and fresh. We back our promise with a 100-percent satisfaction guarantee. Don't forget to add your name to our email mailing list to get special offers!
For more information about ordering Italian food online, please call 718.763.3230 or send an email to email@example.com.


text:
Below is the poem entitled Reaching (2006) which was written by poet
Kumari. Please feel free to comment on this poem. However, please remember, PoetrySoup is a place of encouragement and growth.
Read Poems by
R Kumari
Reaching to see what is in front of me
Why can't I see?
I have in my hand a gold band
Why do I see a pile of sand?
You have given me more and more and more
Why do I feel so unsure?
Like a rose half pulled out
My bed is not what I see about
Shifting and changing my heart leaps
Coming or going I have the creeps
A comment has not been posted for this poem. Encourage a poet by being the first to comment.