text:
|Publication number
|Publication type
|Application number
|PCT number
|Publication date
|Apr 20, 2010
|Filing date
|May 21, 2004
|Priority date
|May 22, 2003
|Fee status
|Also published as
|Publication number
|Jeffrey D. McBride, Francis Guy Gabriel, Peter J. Delves, John L. A. Fordham, Ian A. Cree, Keith Rawson
|Original Assignee
|Nalia Systems Ltd.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (13), Non-Patent Citations (2), Classifications (54), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention relates to an apparatus for use in assays for detecting and/or quantifying the presence of one or more analytes in a sample and to methods of performing such assays.
Numerous apparatus and methods for use in detecting and/or quantifying the presence of analytes in a sample have been developed. Such tests can be used, for example, to define the exact composition of a sample (qualitative assay) or to determine the precise amount or concentration of any given analyte in that sample (quantitative assay). Practical applications of such tests include environmental monitoring, medical research and analysis.
Common methods in the field of medical research and analysis include polypeptide and polynucleotide assays.
Examples of polypeptide assays include immunoassays, ELISA and Western blotting, all of which are based on the principle of antibody/antigen interaction. Immunoassays, for example, are usually carried out in multi-well plates. However, using such apparatus means that only a single analyte can be tested in each well. Assays for testing large numbers of compounds simultaneously have been developed. However, to date, these have mostly been limited to polynucleotide assays.
Polynucleotide assays, which are based on hybridisation between complementary nucleic acid sequences, include Northern and Southern blots, PCR and, more recently, micro-arrays. While micro-arrays allow up to several thousand compounds to be tested simultaneously, they have the disadvantage of generally requiring all analytes to be tested against a single sample.
Current polypeptide and polynucleotide assays are therefore restrictive in terms of the actual number of individual binding assays that can be performed at any one time. In addition, current methods are often not capable of detecting small amounts of a test substance. The most recognised explanation for this is the problem of background staining whereby irrelevant analytes bind non-specifically and particulate matter prevents analyte-specific binding and, after completion of an assay, often remains on the test surface thereby interfering with specific signalling and the clarity and reliability of any readouts.
There is therefore a need to develop improved methods for performing such assays. The present invention seeks to overcome at least some of the aforementioned problems, and seeks to provide an assay method, and apparatus for use therein, which is precise, reproducible, accurate, sensitive and specific.
The present invention relates to assay methods and apparatus for reliably and efficiently detecting and/or quantifying the presence (or absence) of one or more analytes in a sample.
In a first aspect of the present invention, there is provided an apparatus for use in an assay for an analyte comprising:
(a) at least one first receptacle comprising a fluid inlet and a fluid outlet;
(b) a porous membrane, such as a membrane positioned at the fluid outlet; and
(c) at least one analyte-specific binding agent,
characterised in that said at least one analyte-specific binding agent is immobilised in a spot on the underside of said porous membrane relative to the fluid inlet.
The porous membrane should be porous to the analyte of interest.
By immobilising the analyte-specific binding agents on the underside of a membrane positioned at the base of a receptacle, background staining that could otherwise interfere with the reliability and/or specificity of results obtained may be reduced. In addition, by moving the reaction zone to the outside of the receptacle, generation and analysis of images will be facilitated, allowing, in particular, detection and/or imaging means to be included as part of a compact apparatus (whereas they would normally have to be provided separately). The porous membrane also allows for partial removal of non-specific binding analytes on the upper surface of the porous membrane and the removal of particulate matter from a sample before it is brought into contact with any analyte-specific binding agents which could otherwise reduce non-specific binding. Similarly, subsequent reagents employed for detection of binding agent/analyte interaction may be filtered. The provision of filtering means other than the porous membrane is not excluded.
It will be appreciated from the previous paragraph that the porous membrane can be considered as having an upper surface or region for filtering a sample before it is brought into contact with the analyte-specific binding agent. This upper surface or region can be considered as being directly above the analyte-specific binding agent. Typically this upper surface or region contains no analyte-specific binding agent.
It is a preferred embodiment of the present invention that a plurality of analyte-specific binding agents be bound to the membrane (in the form of a microarray, for example). By placing the analyte-specific binding agents on the outside of the receptacle, the reaction zone will be more accessible to the array forming process, thereby allowing for a greater number of analyte-specific binding agents to be immobilised on each membrane, since, for example, the walls of the receptacle may partially obstruct the pins or jet device used for deposition of analyte-specific binding agents.
It should also be noted that, whereas microarrays have previously been largely limited to polynucleotide analyte-specific binding agents, the present invention allows for both polynucleotide and polypeptide analyte-specific binding agents to be employed (i.e. combining microarrays and general ligand binding assays, such as immunoassays).
In one embodiment, the apparatus of the present invention comprises a plurality of receptacles, each of which preferably comprises an array of analyte-specific binding agents. This will allow for a greater number of compounds to be tested in considerably less time than traditionally expected. In particular, the present invention provides a method of carrying out a large number of microarray assays simultaneously (i.e. one per well of a multi-well plate), preferably in a complete, automated process.
Preferably, the apparatus of the invention comprises a plurality of first receptacles in the form of a multi-well plate (e.g. a 96-, 384- or 1536-well plate). According to one embodiment, a plurality of analyte-specific binding agents is immobilised on the porous membrane of the or each well, preferably in the form of an array (such as a microarray). At least one control analyte-specific binding agent may be included amongst the array of binding agents.
Analyte-specific binding agents according to the invention may include polynucleotide probes (such as ESTs), polypeptide probes (such as antibodies or fragments thereof, cell surface receptors or enzymes) or any other type of chemical compound which has the ability to be associated with the target analyte. They may be immobilised directly or indirectly onto the porous membrane.
Further components of the apparatus of the invention may include:
1. One or more second receptacles whereby the or each first receptacle is positioned at least in part in a second receptacle such that said second receptacle may collect liquid from the fluid outlet of said first receptacle, the porous membrane of which may thereby be submerged.
2. One or more additional porous membranes positioned within said at least one first receptacle such that membrane pore size gradually decreases from fluid inlet to fluid outlet.
3. A means for drawing liquid from the at least one first receptacle through the fluid outlet. According to one embodiment, said means may comprise a vacuum pump. According to other embodiments, the means may comprise absorbence, positive displacement or centrifugal filtration.
4. A means for detecting binding of an analyte to the binding agent. According to one embodiment, said detecting means is selected from fluorimetric, chemiluminescent, radiochemical and colourimetric means. The detecting means preferably comprises a photometric device (such as a luminometer) capable of detecting and/or quantifying light emitted from porous membrane. Even more preferably, the detecting means may comprise imaging means, such as a camera or scanner. Imaging means can be included either in addition or alternatively to other detecting means.
According to a second aspect of the present invention, there is provided an apparatus for use in an assay comprising a plurality of first receptacles in the form of a multi-well plate (such as a 96-, 384- or 1536-well plate), characterised in that each receptacle comprises an array (preferably a microarray) of immobilised analyte-specific binding agents. As above, at least one control probe may be included amongst the array of binding agents.
The analyte-specific binding agents, according to the second aspect of the present invention, may be directly or indirectly immobilised onto a surface of said first receptacle. Preferably, said surface is a porous membrane which serves as a fluid outlet for the receptacle. Even more preferably, the analyte-specific binding agents are immobilised onto the underside of said porous membrane.
Further components of the apparatus according to the second aspect of the present invention may include any of (1)-(4) as listed above, adapted as necessary.
According to a third aspect of the present invention, there is provided a method of detecting an analyte comprising:
(a) providing an apparatus according the invention (and as described above);
(b) loading a sample to be tested into the or at least one of the receptacles; and
(c) detecting binding of the analyte to the analyte-specific binding agent(s).
According to one embodiment, said method may further comprise the step, between (b) and (c), of actively drawing liquid from the receptacle through the fluid outlet, with the use, for example, of a vacuum pump.
The sample may be, for example, a biological or environmental sample and may preferably include a detectable label, such as an enzyme, a fluorescent label or a radiolabel.
Detecting binding of the analyte(s) to the one or more analyte-specific binding agents in step (c) may be achieved using any one of fluorimetric means, chemiluminescent means, radiochemical means, colourimetric means, etc. depending of course on the nature of the label used. Preferably, binding is detected using a photometric device (such as a luminometer) capable of detecting and/or quantifying light emissions.
In a preferred embodiment, step (c) of the above described method is followed or replaced by imaging to individually detect and/or quantify light emissions from each area of binding agent immobilisation (said area being referred to herein as a "spot").
In a further embodiment, if the apparatus used in accordance with this method has more than one receptacle, a different sample is loaded to each receptacle.
In another aspect of the invention, there is provided a porous membrane having at least one analyte-specific binding agent immobilised on one side thereof. In use this side is the underside relative to a fluid inlet of the apparatus defined herein.
In a further aspect of the invention, there is provided a kit for use in a method of detecting an analyte, the kit comprising a porous membrane having at least one analyte-specific binding agent immobilised on one side thereof and at least one reagent for detecting binding of an analyte to the at least one analyte-specific binding agent.
In more detail:
In these
In FIG. 4—from left to right, top to bottom, thyroglobulin as probe spots were incubated with 100, 1000, 3000, 50, 750, 1500, 300, 1260, 7500 IU anti-thyroglobulin solutions. CCD-FDI exposure time of six seconds (FDI=fast digital imager).
FIGS. 5 and 6—are calibration curves generated by incubation of standard anti-thyroglobulin solutions with immobilised thyroglobulin as capture probe. Array signal is the mean density of the array signal following a six second exposure time.
FIG. 7—Analysis of arrays using colourimetric reagents. The lower half of
Within well zones, the spot rows comprise 12.5 μg, 25 μg, 100 μg, 50 μg/mL Tg and buffer as probes from top to bottom.
Wells in rows 1 and 3 have been incubated with standardised anti-thyroglobulin solutions. From left to right, each well has been incubated with 1000, 750, 500, 300, 200, 100, 50 and 0 IU anti-thyroglobulin.
Rows 2 and 4 have been incubated with standardised anti-thyroglobulin solutions or patient sera. From left to right, 1260, 3000, 5000, 7500 IU standardised anti-thyroglobulin, positive patient serum A (greater than 10,000 IU) diluted 1/800, 1/1600 and 1/3200, positive patient serum B (˜2560 IU) diluted 7.5 times.
Note, the upper image has been flipped such that wells correspond to the lower image. Standard curves given in
FIGS. 8 and 9—are anti-thyroglobulin calibration curve's generated using 4Cl—N/DAB as HRP substrate.
FIGS. 10 and 11—are anti-thyroglobulin calibration curves generated using MeDAB as HRP substrate. (correlation coefficient for
FIG. 12—representative positive sera at 1/1000 dilution. Consistent bright spots in columns 2 and 5 are standard positive controls. Bottom left, anti-RNP/Sm positive sera—Sm column slightly (Col 2) +ve c.f. RNP/Sm (Col 1), bottom right, anti-Scl 70 positive serum, middle left, anti-Sm positive serum, middle right, anti-Jo1, upper left, anti Ro (SS-A) serum, upper right, all 5 anti-sera combined (compare Example 3).
FIG. 13—representative images of array-wells incubated with sera or standard solutions. Top row, from left to right, Anti-sm serum from the National Health Service (NHS) diluted 1/800, anti-scl 70 serum (NHS) diluted 1/800, anti Jo1 (NHS) diluted 1/800, and Hashimoto's patient serum diluted 1/400. Middle row, anti-La (SS-B) serum (NHS) diluted 1/400, anti Sm-RNP serum (NHS) diluted 1/800, incubation buffer (PBST), and patient serum displaying anti-Ro activity. Bottom row, patient serum diluted 1/400 displaying anti Sm with or without anti-RNP activity (RNP is only present as a probe in complex with Sm), 750 IU anti-thyroglobulin standard, patient serum diluted 1/400 showing anti-Ro (SS-A) and La (SS-B) activity, and patient serum displaying anti sm with or without anti-RNP activity (note that the Sm probe signal is less than that of the Sm-RNP probe complex signal).
FIGS. 14 and 15—are calibration curves generated by incubation of standard anti-thyroglobulin solutions with immobilised thyroglobulin as capture probe, and performed in conjunction with a qualitative rheumatology auto-antigen array. Array signal is the mean density using a CCD exposure time of five seconds.
FIG. 16—probe sensitivity of colourimetric nanodot assay. Assay performed with 1000 IU anti-Tg Std. and 1/1250 anti-human IgG+1/250 Strep-HRP as secondary cocktail. Flat bed scanner as detector.
Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples and with reference to the accompanying drawings.
The present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.
The present invention relates to an apparatus for use in assays for detecting and/or quantifying the presence of one or more analytes in a sample and to methods of performing such assays.
Assays and Apparatus
In its simplest form, the apparatus of the present invention consists of a receptacle, the base of which consists of a porous membrane onto the underside of which is tethered (or "immobilised") a probe chosen for its ability to bind a target analyte. In use, a sample, in fluid form, is added to the receptacle and allowed to filter or pass through the membrane with or without positive or negative pressure applied from above or below the membrane to optimise binding kinetics. This, in turn, permits any analytes present in solution to bind to (or be bound by) the probe. Binding can then be detected and/or measured using standard methods (e.g. fluorescence labelling).
Embodiments of apparatus according to the present invention and useful in the method of the invention are shown in
In one embodiment of the present invention, as illustrated in
In more detail, in an embodiment of the present invention, microarrays comprising, for example 5�5 1-50 nl, or 5�5 12-20 nl, spots (which may be a mixture of probes and controls) may be printed upon the underside of a filter plate such as that shown in
Upon drying and blocking of non-spotted surfaces, the under-filter area may be re-enclosed with a flexible underdrain containing a fluid trap to present sample solution to the array.
Samples are added within the wells from above (as with traditional ELISA type assays). The sample is allowed to pass through the membrane for example by gravity, positive displacement or partial vacuum. The approach taken may depend upon whether a continuous or step-wise filtration process is required. Probe/analyte interaction occurs as the sample is drawn past the probe spots or incubated in the fluid trap.
The membrane may be subsequently washed (preferably more than once) and co-incubated with a labelling agent (e.g. a labelled antibody). Again, reagents may be added to the well from above, allowing the porous membrane to act as a filter. The washing process may then repeated to remove any unbound label.
Finally, detection of spots showing interaction is achieved by detection methods known in the art (e.g. detection methods used for microarrays such as colourimetric and/or chemiluminescent reagents, and imaging techniques). Images obtained in this manner are shown by way of illustration in
According to a preferred embodiment of the present invention, the apparatus comprises a plurality of receptacles (also referred to herein as "wells") forming a multi-well plate. The multi-well plate may be a 96-well (or microwell) plate, a 384-well plate or a 1536-well plate, allowing numerous samples to be analysed simultaneously (in e.g. high throughput screens). The plates will be of standard size and dimensions such that they can be used with existing robotics and automated systems. They are preferably formed of plastics materials such as polypropylene resin.
In more detail, 96-well plates of the present invention will preferably have a receptacle volume of approximately 0.4-0.5 mL. 384-well plates will preferably have a receptacle volume of approximately 10-100 μl with a standard 96-well footprint. 384-well plates are particularly suited to high volume DNA library manipulations and high throughput screening. 1536-well plates will preferably have a receptacle volume of approximately 1-15 μl, again with a standard 96-well footprint. 1536-well plates are particularly suited to automation and ultra high throughput screening.
The apparatus of the present invention is formed essentially from plastics materials including, but not limited to: polymethylmethacrylate, polystyrene, polyethylene, polypropylene and derivatives thereof. The material is chosen for its resistance to chemicals, solvents and alcohols. The plates may be clear when used for colourimetric reading, or coloured, e.g. white or black for luminescence and/or fluorescence reading.
As mentioned above, the base of each receptacle comprises a porous membrane. Preferably, the base will be flat-bottomed to maximise optical quality and imaging area.
Each receptacle comprises a fluid inlet and a fluid outlet. The fluid outlet comprises a porous membrane onto the underside of which at least one analyte-specific binding agent is tethered (this surface of the porous membrane is therefore also referred to as the "reaction zone"). The membrane will be permeable to the liquid portion of a sample and to the target analyte but impermeable to, and therefore capable of trapping, other (usually larger) components of the sample. It may be formed of a woven or non-woven fabric, paper, cellulose, glass fibre, polyester (or other polymer) or mixtures and derivatives thereof. Preferably, it will be formed of a mixed cellulose ester or nylon composition. Pore sizes will be in the range 0.01-1 μm, preferably in the range 0.1-0.5 μm, even more preferably in the range 0.4-0.5 μm. Ideally, the pore size will be about 0.45 μm. The membrane may be coated to create a hydrophobic surface, for example with poly-L-lysine, thereby improving stability and minimising spreading of the spots.
In one embodiment, one or more further porous membranes can be placed within each receptacle. Preferably, the membranes are positioned such that pore size gradually decreases from fluid inlet to fluid outlet. The pore sizes of the further membranes will vary depending on the sample to be filtered. Preferably, they will be in the range 0.01-10 μm, even more preferably in the range 0.1-5 μm, most preferably in the range 0.1-1.0 μm.
One or more of the membranes (including the final, reaction zone membrane) may comprise a "binder". The binder may be an antibody, ligand or other complementary molecule capable of binding unwanted cellular components or impurities, thereby improving the ability of these membranes to filter unwanted compounds from the sample. If a binder is included in the final membrane, it will preferably be positioned on the upper-side of said membrane (i.e. on the opposite surface to the reaction zone).
The apparatus of the invention may further comprise an "underdrain" or "collection tray" formed of one or more receptacles corresponding to the one or more wells of the apparatus such that, in use, each well is positioned at least in part in a corresponding receptacle. According to this embodiment, sample passing through the fluid outlet is collected in the receptacle allowing the membrane and any binding agents immobilised thereon to be bathed in the sample. This, in turn, increases interaction between the analyte and analyte-specific binding agent and therefore improves the sensitivity of the assay.
The apparatus may also comprise means for drawing liquid from the receptacle through the fluid outlet. Said means may consist of a vacuum pump, an air pressure pump, an adsorptive layer or any other standard means known in the art. Positive displacement can also be used for drawing liquid from the receptacle. Such means can be used to regulate the flow rate of the sample to achieve optimal reaction between the analyte and its binding partner.
Analytes and Analyte-Binding Agents
The term "analyte" as used herein refers to a compound or composition to be detected or measured in a sample. "Sample" refers to any desired material, usually of biological origin, to be tested for the presence of a target analyte. A sample may include, but is not limited to: blood or serum; saliva, sputum, tears, sweat or other secreted fluids; urine or faecal matter; biologically derived fluids such as cerebrospinal fluid, interstitial fluid, cellular extracts and the like; samples of environmental origin such as water or earth; food samples such as meat products, fruit and vegetables; test drugs etc.
As will be understood by the skilled person, the target analyte can be any specific substance or component that one is desirous of detecting and/or measuring in a chemical, physical, enzymatic or optical analysis. The analyte according to the present invention may be a polypeptide, polynucleotide or any other chemical compound or complex of chemical compounds.
In more detail, analytes that can be detected/quantified using the device or method of the present invention include, but are not limited to: antigens (such as antigens specific to bacterial, viral or protozoan organisms including, for example, Streptococcus, Hepatitis and HIV); antibodies (particularly those induced in response to infection, allergic reaction, or vaccination); hormones (such as human chorionic gonadotropin—hCG—used as a marker for pregnancy, oestrogen, progestin, testosterone or corticosteroids), proteins (such as circulating plasma proteins used as disease markers), nucleic acids (including circulating plasma nucleic acids, used as disease markers), and other physiological substances (for example, human growth factors, haemoglobin and cholesterol); a variety of enzymes (such as aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase and glutamate dehydrogenase); therapeutic compounds (such as steroids, antibiotics, tranquillisers and anticonvulsants); drugs (such as cocaine, heroin and marijuana); contaminants and environmental pollutants (such as pesticides, herbicides and aromatic hydrocarbons); vitamins; and any number of other natural or synthetic substances.
As will be appreciated by one skilled in the art, the number of natural and synthetic substances which can be detected by the assay devices and methods of the present invention is extensive, and includes, but is not limited to, the following groups of compounds: ACE inhibitors, alcohol deterrents (for example, disulfiram), anti-allergics, anti-anginals, anti-arthritics, anti-infectives (including but not limited to antibacterials, antibiotics, anti-fungals, anti-helminthics, anti-malarials and anti-viral agents), analgesics and analgesic combinations, local and systemic anesthetics, appetite suppressants, anti-oxidants, anxiolytics, anorexics, anti-arthritics, anti-asthmatic agents, anti-coagulants, anti-convulsants, anti-diabetic agents, anti-diarrheals, anti-emetics, anti-epileptics, anti-histamines, anti-inflammatory agents, anti-hypertensives, anti-migraines, anti-nauseants, anti-neoplastics, anti-oxidants, anti-parkinsonism drugs, anti-pruritics, anti-pyretics, anti-rheumatics, anti-spasmodics, anti-tussives, adrenergic receptor agonists and antagonists, cardiovascular preparations (including anti-arrhythmic agents, cardiotonics, cardiac depressants, calcium channel blockers and beta blockers), cholinergics and anti-cholinergics, contraceptives, diuretics, decongestants, growth stimulants, herbal preparations, hypnotics, immunizing agents, immunomodulators, immunosuppresives, muscle relaxants, neurologically-active agents including anti-anxiety preparations, antidepressants, anti-psychotics, psychostimulants, sedatives and tranquillisers, sore throat medicaments, sympathomimetics, vasodilators, vasoconstrictors, vitamins, xanthine derivatives, various combinations of these compounds, and the like.
Presence of the target analyte in a sample will be assessed by detecting binding of said analyte to an analyte-specific binding agent. The term "analyte-specific binding agent" (also referred to herein as "binding agent", "probe" or "binding partner") should interact with the analyte such that the presence of the analyte can be determined and/or measured, either directly or indirectly. Thus, the binding agent should show at least some specificity for the analyte of interest as opposed to other components of the sample under test. In one embodiment "binding agent" is used to describe a member of a binding pair which interact either chemically or physically to form a complex. Thus, an "analyte-specific binding agent" refers to an agent which, because of its three-dimensional structure, is capable of interacting specifically with the target analyte. An "immobilised" binding agent refers to a binding agent that is tethered, adsorbed, embedded or affixed, either permanently or semi-permanently, to the underside of the porous membrane of the present assay device. The binding agent will be immobilised in such a way that its affinity for the target analyte is not significantly altered.
The nature of the binding agent used in the present invention will depend on the nature of the analyte to be detected. Possible binding pairs include, but are not limited to: antibody and antigen; antibody and hapten; hormone and receptor; virus and cellular receptor; biotin and avidin; carbohydrate and lectin; effector and receptor molecules; enzyme and cofactor; enzyme and substrate; enzyme and inhibitor; complementary nucleotide sequences; biotin and streptavidin; biotin and avidin; protein and DNA; protein and RNA; DNA and RNA; and protein and protein. Wherein the binding agent is a nucleotide sequence, it will preferably be an EST (Expressed Sequence Tag). Binding agents for use according to the present invention will be prepared and immobilised using standard methods known in the art. Polynucleotide probes, for example, can be prepared by PCR amplification.
It should be noted that, while binding agents are generally described herein as the immobilised member of the analyte/analyte-specific binding agent pair, it is not intended that the reverse be excluded. It is indeed possible (and, in the case of DNA analysis, often preferred) that the analyte be bound and the probe added in solution. It is then the probe, rather than the analyte, which is labelled for detection and/or quantification Thus, for example, to test for the presence of a gene A of known sequence, genomic fragments from one or more patients are immobilised on a substrate. Labelled primers, corresponding to the sequence of A, are then added in solution. Binding of a primer to any of the immobilised fragments will indicate the presence of gene A in that patient's DNA.
The analyte-specific assay device and method of the present invention can easily be adapted for detection of further or different analytes simply by replacing the binding partner immobilised in the reaction zone. In a preferred embodiment, the device can be adapted simply by removing a used membrane from the base of a well and replacing it with a fresh membrane. The fresh membrane may be pre-prepared with one or more binding agents having been immobilised thereon or, alternatively, the one or more binding agents can be tethered to the membrane after it has been affixed to the well.
In a preferred embodiment of the present invention, a plurality of different binding agents is immobilised on the porous membrane of the or each receptacle in the form of an array, allowing more than one analyte to be detected simultaneously.
Arrays and Microarrays
Because the binding agent of the present invention is tethered to the underside of the porous membrane, a large amount of background staining can be avoided. This in turn means that sensitivity and accuracy is improved, allowing each well to be used to test for more than one analyte compound. Thus, in a particularly preferred embodiment, each well comprises a plurality of different binding agents.
The plurality of binding agents are preferably arranged in the form of an array. An array is an orderly arrangement of polynucleotide or polypeptide probes. Each probe in an array is immobilised in a defined area known as a "spot". Spots can be deposited manually or using any one of many automated printing process known in the art. Once the spots have been deposited, the membrane will be dried and the non-spotted areas blocked (with BSA or PVA, for example).
Depending on the size of the spots in an array, it will be referred to as a "macroarray" or a "microarray". Macroarrays contain spot sizes of about 300 microns or larger and can easily be imaged using standard gel and blot scanners. The spot sizes in microarrays are typically less than 300 microns in diameter. Microarrays usually contain thousands of spots.
The area of each spot for use in the present invention can be of any size and shape. For example, squares, ellipsoids, rectangles, triangles, circles, or portions thereof, along with irregular geometric shapes, may be used.
In one embodiment, each spot will have a surface area of between about 1 cm2 and 10−10 cm2. In some embodiments each spot will have a surface area of less than about 10−1 cm2, 10−2 cm2, 10−3 cm2, 10−4 cm, 10−5 cm2, 10−6 cm2, 10−7 cm2, 10−8 cm2 or 10−10 cm2. In a preferred embodiment, these regions are between about 10�10 μm and 500�100 μm. It is an advantage of the invention that, by "spotting" the underside (rather than the inside) of the well, a greater reaction surface becomes available. This is because the inside wall of the well interferes with the deposition of the spots on the periphery of the well. Thus, a greater number of spots per well can be included. Each reaction zone may comprise from one to several hundred thousand spots. Preferably, each reaction zone will comprise an array of 1, 5, 10 or more spots. In an even more preferred embodiment, each reaction zone will comprise an array of 50, 100, 500 or more spots. In a most preferred embodiment, each reaction zone will comprise an array of 4�4, 5�5 or 6�6 spots.
Together with the array of polynucleotide or polypeptide probes deposited onto the porous membrane of each well, it is preferable to also include at least one control probe against which results can be measured. Alternatively, when the apparatus comprises a plurality of wells, one or more of the wells can be used as controls. The sample added to the well(s) will contain a known concentration of each sample to be tested. Levels of binding in the control well(s) can then be used as a standard against which results can be measured.
According to an alternative aspect of the present invention, there is provided a multi-well plate (as defined above) wherein each well comprises an array of probes. The probes may be polynucleotide or polypeptide probes and may be tethered to the inside or to the outside of the well. If tethered to the inside of the well, the probes will preferably be positioned at its base. The base may be integrally formed with the rest of the well or it may be formed of a porous material for use in a filtration type assay. Preferably, the probes will be immobilised onto the underside of a porous membrane positioned at the base of the well (as described above).
In a preferred embodiment, the method of the present invention further includes a "washing" step comprising adding an aqueous solution to the or each well after the analyte/probe binding step in order to remove any unwanted matter (such as particulate or coloured material introduced by or with the sample solution, cellular components, etc.) from the reaction zone and to reduce any non-specific binding. A further washing step is preferably included after labelling to remove any unbound reagents and, again, to reduce any non-specific binding. Each washing step may include one or more (preferably a 4-6) washes. Inclusion of this step further improves the sensitivity and reliability of the present assay. In the present invention washing is greatly facilitated relative to conventional methods of aspiration by the filtration process.
Binding of the target analyte to an immobilised probe (or, conversely, binding of a probe to the immobilised target analyte) can be detected using any standard method known in the art.
Detection Methods
The assay methods of the present invention comprise a plurality of continuous or discontinuous steps. They can be described, in simple terms (and in accordance with certain preferred embodiments), as follows:
(a) providing an apparatus in accordance with the invention (e.g. a multi-well plate wherein each well comprises a porous membrane onto the underside of which is immobilised an array of probes);
(b) adding a sample to each well and allowing it to filter through the porous membrane such that analyte/probe binding occurs;
(c) optionally washing the membrane several times with an aqueous solution;
(d) adding labelling reagents to each well and allowing them to filter through the porous membrane such that analyte/label binding occurs (note: this step may be performed simultaneously with step b);
(e) optionally washing the membrane several times with an aqueous solution;
(f) detecting analyte/probe interaction and/or imaging.
Typical detection assays will use either a "competition" assay or a "sandwich" assay to detect/quantify binding of the target analyte. In a competition assay, the target analyte competes with a labelled analyte (or labelled analogue to the analyte) for an immobilized binding partner. A greater concentration of analyte in the sample results in a lower signal in the assay, as the labelled analytes are prevented from interacting with the immobilised binding partner. Thus, the signal produced during a competition assay decreases as the concentration of analyte in the sample increases.
In a sandwich assay, the target analyte is bound, or "sandwiched", between an immobilised, unlabelled first binding partner and a labelled second binding partner. Various further staining methods have evolved from this first and very simple method. For example, in the two-step indirect method, the target analyte is bound between two binding partners, one of which is immobilised. A labelled antibody, directed against the non-immobilised binding partner is then applied. This method is more sensitive than the direct method insofar as the labelled antibody is likely to react with a number of different epitopes on the non-immobilised binding partner, thus amplifying the signal and has greater specificity since more than one epitope on the analyte must be detected.
Further forms of detection will be apparent to the skilled person (e.g. from standard ELISA and DNA microarray assays). Further staining methods are described, for example, in the Handbook of Immunochemical Staining Methods, 3rd Edition (Thomas Boenisch, 2001, DAKO Corporation, California—see, in particular, pages 26-31), available at http://www.dakousa.com/ihcbook/hbcontent.htm.
The term "label" as used herein refers to any substance that is capable of producing a detectable signal. Labelling will be performed according to standard methods. Labels suitable for use in the present invention include, but are not limited to: chromatogens, fluorescent, chemiluminescent or bioluminescent compounds, radioisotopes or radionucleotides, catalysts, enzymes, enzyme substrates, cofactors, inhibitors or subunits, dyes, colloidal metallic and non-metallic particles, and organic polymer latex particles. Examples of specific labels include fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol, NADPH, α-β-galactosidase, horseradish peroxidase, etc.
Labels of use in the present invention can be divided into two types: those that can be detected by visual inspection (e.g. moieties which include or produce coloured elements such as colloidal metals, non-metals, and dye particles), and those which can only be detected with the aid of artificial detection systems (such as, for example, optical systems, spectroscopic systems and radiographic systems). Thus, according to a further embodiment of the present invention, the apparatus comprises means for detecting binding of an analyte to the analyte-specific binding agent.
The detecting means used will depend upon the nature of the label used. For example, if the label is a fluorescent label, the detecting means will be a fluorimeter. A large number of different fluorimeters are available in the art, any of which would be suitable for use in the present invention. With enzymes, either a fluorescent or a coloured product can be provided and determined fluorimetrically, spectrophotometrically or visually.
Preferably, the detecting means will be fluorimetric, chemiluminescent, radiochemical or colourimetric means. According to one embodiment, said means will be a photometric device capable of detecting and/or quantifying light emitted from the porous membrane such as a luminometer.
The detecting means may also or alternatively comprise imaging means capable of individually detecting and/or quantifying light emitted from each area of binding on the porous membrane (e.g. from each spot in an array). Possible imaging means include CCD (charge coupled device) apparatus and scanners. By placing the reaction zone on the underside of the apparatus, the present invention facilitates label detection and reading, thus allowing for more compact detection and/or imaging means to be used. Preferably, the detecting and/or imaging means will be formed integrally with the rest of the apparatus. Preferably, the detecting and/or imaging means will comprise a plurality of lenses, each lens corresponding to a well so that automatic reading of results can be performed.
Moving the reaction zone to the underside of the apparatus also means that interference caused by light reflecting off the receptacle walls is avoided and that levels of background interference from particulate matter can be reduced. Furthermore, a greater reaction surface is made available making it possible to include a greater number of spots in each array. This, in turn, means that the cost and time required for testing large numbers of analytes can be substantially reduced. The apparatus and methods of the present invention are therefore particularly suited to large scale genetic and proteomic studies and to ultra-high throughput screening.
Applications and Uses
The methods and devices according to the present invention can be used for both quantitative and qualitative assays. The most common applications of such methods will be for the determination of gene expression patterns (e.g. differential gene expression between identical cells subjected to different stimuli or between different cellular phenotypes or developmental stages), for immuno- and disease diagnostics and for studying protein function.
Such applications can be used, in particular, to gain a better understanding of disease pathology, i.e. what triggers the expression of a disease gene, its effect on the expression of other genes and on its host organism as a whole, its expression patterns across different populations, etc. This, in turn, will allow for diseases to be more easily diagnosed and treated. Many disease states are indeed characterised by differences in the expression levels of various genes either through changes in the copy number of the genetic DNA or through changes in levels of transcription of particular genes. Thus, changes in the expression levels of particular genes serve as signposts for the presence and progression of various disorders.
For example, the expression (or over-expression) of oncogenes can be an indicator for the onset of certain types of cancer, the detection of a specific genetic mutation can help to diagnose cystic fibrosis, etc. Similarly, viral infection is often characterised by the elevated expression of genes of the particular virus. Outbreaks of Herpes simplex virus, Epstein-Barr virus, cytomegalovirus, Varicella zoster virus, parvovirus and human papillomavirus, for example, are all characterised by elevated expression of various components of the respective viral genome. Thus, detection of elevated expression levels of characteristic viral genes provides an effective diagnostic of the disease state.
Expression levels can also be used to analyse the role of any given gene in the cell division cycle, development (cellular, embryonic, etc.), tissue differentiation and so on. Changes in levels of expression can be measured either in terms of mRNA transcription levels or in terms of protein levels. Thus, both the polynucleotide and polypeptide assays of the present invention can be used to study expression patterns and in diagnostic tests. The polypeptide assays can also be used to study protein interactions and, therefore, protein function.
Expression patterns are indeed insufficient to give a full picture of cellular and developmental mechanisms. While they may reveal which genes are involved in a particular process, it is also important to determine how they are implicated. Many proteins, for example, are involved in signalling pathways. These complex pathways provide a link between, for instance, the binding of a compound to a cell surface ligand and the downstream expression of a target gene. The present invention can be used, in this regard, to determine possible binding partners for any protein of interest (be they polypeptides, polynucleotides or any other type of chemical compound). For example, the assay of the present invention could be used to identify all compounds capable of interacting with the extracellular portion of a cell surface protein, which proteins are capable of binding a target DNA sequence, etc. Thus, the present invention can be used to provide a powerful insight into the exact function of any protein of interest and, on a larger scale, into the working of cellular mechanisms. It is a particular advantage of the present invention that many hundreds or thousands of compounds can be tested simultaneously, thereby substantially increasing the rate at which binding partners can be analysed.
Outside research and disease diagnosis, the present assay devices and methods can be used to detect and quantify toxins or other desirable or undesirable compounds in environmental and/or food samples. For example, they could be used to identify chemical compounds in a river which is suspected of being contaminated, for large scale drug screening, for determining levels of a given vitamin in a new food product and so on.
Reagents were obtained from the following sources:
PBS: 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4
PBST: 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, 0.05% v/v Tween-20
Human thyroglobulin was from Calbiochem (San Diego, Calif., USA)
Standardised anti-thyroglobulin solutions were provided by Cambridge Life Sciences (Cambridgeshire, U.K.).
Extractable nuclear antigens; proteinaceous La (SS-B), ribonucleoproteins-Smith complex (RNP/Sm), topoisomerase (Scl 70) and proteinaceous Ro (SS-A) from calf thymus and histidyl sRNA synthetase (Jo1) and the polypeptide Smith (Sm) and from bovine thymus are products of AroTec Diagnostics Ltd, New Zealand but were supplied by The Binding Site, Birmingham, U.K.
Reference, pooled human serum previously tested and shown to display anti-Sm, anti-Sm & RNP, anti-Jo1 & Scl 70, anti-Ro & La, anti-Jo1, or anti-Scl 70 activities from the National Health Service (NHS; UK) and Anti-human thyroglobulin were supplied by Cambridge Life Sciences, Cambridge, U.K.
Biotinylated Bovine serum albumin (Biotin-BSA) was a product of Vector Laboratories U.K., Peterborough, U.K.
Biotinylated goat polyclonal to human IgG H&L from Abcam, Cambridge, U.K.
Streptavidin-Horseradish peroxidase (HRP) polymer from Sigma (Poole, U.K).
Super Femto ELISA, 4-Chloro-1-napthol+3,3′-Diamino benzamidine (4Cl—N/DAB) and metal enhanced 3,3′-Diamino benzamidine (MeDAB), were products of Pierce (Rockford, Ill., U.S.A.).
Mixed cellulose ester membrane bottomed 96 well plates (Multiscreen) with a porosity of 0.45 μm, and a flexible, removable under-drain were a product of Millipore (Millipore, U.K. Watford, Millipore, U.K.).
Array Fabrication Prior to Bioassay.
Antigens were diluted in 50 mM Bicarbonate pH 9.5, 0.005% Bromophenol blue and 2.5% v/v glycerol to concentrations between 10-200 μg/ml and transferred into 96 well plates (precise concentrations given in the examples below). A robotic microarrayer, BioDot AD3200, with microsolenoid inkjet valve and ceramic tip (100 μm for 12 nl and 190 μm for 20 nl probe spots) was employed for non contact printing of spot-arrays (Bio-Dot, Cambridge, UK) onto the under-well surface of mixed cellulose ester microplates (the under-drain was first removed) in an ordered array as described in the examples (using the AxSys software supplied by the manufacturer). The spotted arrays were kept in a moist chamber at 4� C. overnight. Thereafter, inside the wells were washed 3�200 μl with PBS, 'flicking' out each time, followed by 3�100 μl on the under-surface. The under-drain was replaced, and the membrane blocked by the addition of 0.8% PVA in 0.1M phosphate buffer pH 7.2, shaking for one hour on a reciprocal shaker (Eppendorf 5436 thermomixer, Eppendorf, U.K.) before washing the plate using a vacuum (˜200 ul/well/30 secs) 3�200 μl PBST. Thereafter, the plates were ready for bioassay or alternatively allowed to air dry for storage at 4� C.
Scl 70
Jo 1
Scl 70
Jo 1
Jo 1
Scl 70
Scl 70
Jo 1
Where −ve = negative control, spotting buffer, +ve = positive control 1 μg/ml Biotin-BSA.
Rheumatology Array
Antibodies to Ro and La are often found in systemic lupus erythematosus (SLE) and Sjogren's syndrome (SS) patients. Anti-Ro antibodies in the mother are also strongly associated with congenital heart block. Anti-Sm antibodies are present in approximately 30% of patients with SLE and are widely regarded as a disease marker. Where a sample shows a negative Sm result, anti-RNP antibodies are present in 90% of mixed connective tissue disease (MCTD) patients. Anti-Jo-1 antibodies are classically found in patients with scleroderma.
Thyroglobulin Assay
Autoimmune thyroid gland disorders are characterised by the detection of anti-thyroid antibodies including thyroglobulin (Tg) and thyroid peroxidase (TPO). Both of these thyroid components play key roles in the biosynthesis of thyroid hormones. Circulating autoantibodies to Tg and TPO are present in up to 90% of patients with thyroiditis, idiopathic hypothyroidism, Graves' disease and subacute thyroiditis. Of all the autoimmune conditions, Graves' disease and Hashimoto's thyroiditis are perhaps the easiest to treat, provided that they are detected at an early stage. This requires sensitive detection of antibodies to Tg (and TPO), together with the ability to monitor the antibody response to drugs, radiotherapy or surgery.
−ve cont
+ve cont
−ve cont
Scl 70
+ve cont
Scl 70
−ve cont
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.
|Cited Patent
|Filing date
|Publication date
|Jul 28, 1983
|Jan 15, 1985
|Bio-Rad Laboratories, Inc.
|Supporting and filtering biochemical test plate assembly
|Nov 22, 1989
|Jun 11, 1991
|La Mina Ltd.
|Modular fluid testing device
|Sep 12, 1988
|Jul 9, 1991
|University Of Florida
|Membrane-strip reagent serodiagnostic apparatus and method
|Dec 18, 1989
|Sep 22, 1992
|Mclean Hospital Corporation
|Multiple antigen immunoassay
|Apr 2, 1997
|Aug 24, 1999
|The Perkin-Elmer Corporation
|Detection of low level analytes in samples using agglutination reaction capillary slide test and apparatus therefor
|Nov 5, 1996
|Nov 2, 1999
|Abbott Laboratories
|Method and apparatus for collecting a cell sample from a liquid specimen
|May 24, 2002
|Mar 13, 2003
|Lin He
|Microcolumn-based, high-throughput microfluidic device
|Feb 24, 1989
|Nov 2, 1989
|Costar Corporation
|Multi-well filter strip and composite assemblies
|Apr 10, 1991
|Oct 14, 1992
|La Mina Ltd.
|Biological fluids testing membrane module and method of conducting same
|Mar 31, 2000
|Oct 12, 2000
|Tropix Inc
|High throughput and high sensitivity detection assays
|Jul 17, 2001
|Jan 31, 2002
|Ey Lab Inc
|Reagent delivery device and method of use
|Jul 3, 2002
|Jan 16, 2003
|Georgia Tech Res Inst
|Filtration-based microarray chip
|Oct 15, 2002
|Apr 24, 2003
|Biocept Inc
|Microwell biochip
|International Search Report and Written Opinion; Application No. PCT/GB2004/002203; mailed Mar. 30, 2005.
|MultiScreen Methods: Guidelines for Bioassays on MultiScreen Plates: Protein Binding and Cell Loading; 1994, 1997.
|U.S. Classification
|436/518, 435/287.1, 436/810, 435/288.7, 436/805, 422/400, 435/810, 435/287.2
|International Classification
|Cooperative Classification
|European Classification
|Nov 22, 2006
Owner name: BIOCENTRE 5 LIMITED,UNITED KINGDOM
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MCBRIDE, JEFFREY D;GABRIEL, FRANCIS GUY;DELVES, PETER J;AND OTHERS;SIGNING DATES FROM 20051208 TO 20051220;REEL/FRAME:018544/0572
|Apr 8, 2008
Owner name: NALIA SYSTEMS LTD.,UNITED KINGDOM
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BIOCENTRE 5 LIMITED;REEL/FRAME:020768/0833
Effective date: 20080328
|Nov 29, 2013
|Maintenance fee reminder mailed
|Mar 5, 2014
|Fee payment
Year of fee payment: 4
|Mar 5, 2014
|Surcharge for late payment


text:
We were traveling around Greece and decided to spend a night in Leptokarya. We did not have prior reservations and we found by chance Hotel Dionisos. It was a really nice hotel. It looked new and modern. The rooms were spacious and cozy. They also had a big terrace and sea-view! The rooms were clean, the bed - comfortable. The... More
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subset: commoncrawl


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The following bibliography has been generated by bringing together all references provided by our content partners. There may be duplication.
- Crandall-Stotler, Barbara, Raymond E. Stotler, and D. G. Long / Goffinet, B., and A. J. Shaw, eds. 2008. Morphology and classification of the Marchantiophyta. Bryophyte Biology, Second Edition. 1-54
- Journal of the Hattori Botanical Laboratory 26: 244. 1963. (J. Hattori Bot. Lab.)
- Journal of the Hattori Botanical Laboratory 37: 252. 1973. (J. Hattori Bot. Lab.)
EOL content is automatically assembled from many different content providers. As a result, from time to time you may find pages on EOL that are confusing.
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Cream Cheese
Everything Spice–Coated Cheddar Cheese Ball
If you have leftover "everything" spice, stir it into Greek yogurt drizzled with olive oil for a savory breakfast.
Green Goddess, Pistachio, and Goat Cheese Ball
You want all of your ingredients at room temperature to make sure the mixture is smooth and homogenous.
Waffle with Cream Cheese and Smashed Berries
Use a frozen whole grain waffle, or freeze leftover homemade waffles between sheets of waxed paper.
Brown Bread and Boiled Eggs
Hard-boiled eggs travel well and can be boiled in bulk at the beginning of the week. Make egg salad with the egg-stras!
Horseradish-Dill Schmear
Throw this spread together a day in advance—the flavors will meld and intensify as it sits.
Horseradish-Cheddar Cheese Ball
This is part of BA's Best, a collection of our essential recipes.
Hot-Smoked Salmon with Caper Cream Cheese
Hot-smoked salmon, unlike cured, is fully cooked.


text:
Introducing Peppermint Six!
Say hello to the next iteration of our lightweight, stable, and super fast operating system. Peppermint Six is built on a long term support (LTS) code base, so as well as being customizable to your heart's content, will be "good to go" for quite some time. Peppermint Six is built on the Ubuntu 14.04.2 code base
File Management from Nemo
Fresh from the Cinnamon desktop environment the ultra smooth Nemo file manager allows easy connection to remote network shares, fine grained control of desktop icons, custom action scripts, and together with the new wallpaper manager easy configuration of desktop backgrounds
Web app integration with Ice
Peppermint's handy cloud & web application management tool 'Ice' continues to put web applications on an equal footing with locally installed apps by allowing them easy integration into system menus, and delivery to the desktop via SSB's so they mimic locally installed applications.
Latest Community News
Big "Thank You" to all involved
Team Peppermint would like to take this opportunity to thank all those involved in the planning, development, testing, and promotion of Peppermint Six. This includes all the forum moderators, trusted users, beta testers, website developers, graphic artists, and anyone else … ContinuedMay 29, 2015
Latest Corporate News
Peppermint 6 Respin with UEFI Support Released
Team Peppermint are pleased to announce a respin of our latest operating system Peppermint 6 with full UEFI / GPT / Secureboot support (64bit edition only), and a new version of Ice (our in house Site Specific Browser framework) that … ContinuedSeptember 6, 2015
Help Keep Peppermint Free For Everyone! Forever!
Peppermint is an Open Source project and uses all of its funds to provide its operating system and community services freely to the world, for everyone to to use. Consider making a donation today.


text:
Swine flu is all the rage these days, as you can't turn on the news without hearing of the latest diagnosed case in the United States or in other parts of the world. To be honest, I'm not even entirely sure what swine flu is exactly because I don't really care. I don't work with pigs, and I live inside a plastic bubble like John Travolta did in that one movie, so I'm not exactly worried.
("Touch me. I dare you.")
Still, there are the rest of you people who don't live in a bubble — I call you "world walkers" — who may be susceptible to the disease. Sucks for you. It also sucks for students at St. Francis Prep in New York, where an outbreak occurred amongst the schools' students last week. Everybody is fine and the school has undergone a pretty intense cleaning, but although the school is scheduled to reopen this week, that doesn't mean anybody wants to play their baseball team.
From the NEW YORK DAILY NEWS:
"I have heard from parents and they are extremely concerned," said coach Dennis Canale, whose CHSAA baseball champion Xaverian is scheduled to play St. Francis at Cunningham Park tomorrow and again on Thursday. "We're going to have a meeting with our principal (this) morning and find out what we're going to do. If it were just up to me I might take a chance, but this is 27 kids. I'm a parent, so I would feel the same way. You don't want to jeopardize the kids' health."
But speaking on behalf of the players, Xaverian pitcher Justin Giammarino said, "I don't think anybody's really worried about it."
St. Francis' baseball team already postponed Monday's game against Christ the King and today's versus St. Edmund Prepatory School. Before the outbreak of swine flu, it had to postpone several games due to the rainy weather.
St. Francis' AD Sal Fischetti (do they allow anybody who isn't Italian into these schools?) said that while some of the players on the school's team said they didn't feel well, upon being evaluated none of them were found to be infected. Still, it's too bad they're not going to be able to play because they could take advantage of this whole thing.
After all, who's going to want to pick up a baseball one of the St. Francis players touched or tag them out on the basepaths? They could put up 100 runs before the end of the first inning as opposing players did everything in their power to run away from the ball.


text:
We were first introduced to Astro on Fox's The X Factor season 1. Two years later the pint-sized MC has continued fine tuning his flow, acting and flexing his personality - his showing in the cypher is the best picture of the grind he has been on since The X Factor stage. Needless to say, when Premiere is spinning the record, Astro is dropping quotable lines.
(Photo: Maury Phillips/WireImage)
The Night's Big Winners10/14/2014
Cypher Revealed Remy Ma10/10/2014
10 Influential Marks Doug E. Fresh Left On Hip Hop10/02/2014
The Show: Brandy09/23/2014
Lyte Always Shines09/22/2014
Queen Latifah's 8 Best Stage Moments09/22/2014
Yo-Yo's Best Stage Moments09/22/2014
The Show: Young Thug09/19/2014
Cypher Revealed David Banner09/19/2014
Seat Check: Where Your Favorite MCs Are Sitting09/19/2014
The Warm Up: Go Inside Rehearsals09/19/2014
The Doug E. Fresh I Am Hip Hop Playlist09/19/2014
The Show: Jay Electronica09/19/2014
The Show: Rich Homie Quan09/19/2014
The Show: DJ Premier09/19/2014
The Show: YG09/19/2014
Common's 7 Best Stage Moments09/18/2014
Bobby Shmurda's Top Shmoney Dance-Worthy Performances09/18/2014
Rae Sremmurd Got What It Takes To Rock The Mic09/17/2014
Migos' 6 Most Trill Performances09/17/2014
The Show: Ty Dolla $ign09/17/2014
T.I. Swags Out At Every Show09/17/2014
The Show: Vince Staples09/16/2014
Meet The MCs Set Ignite The Cypher09/16/2014
Birdman Stays Fly on Stage09/15/2014
Emilio Rojas and Crew Bring the Buzz to The Cypher09/12/2014
Battle and Cypher Ready09/12/2014
Mustard on the Beat...and Stage09/10/2014
We Got MCs: Find Out Who's Performing09/09/2014
Cypher Revealed | Kevin Gates09/08/2014
Cypher Revealed Live Cypher09/08/2014
Cypher Revealed: Lil Mama09/08/2014
Cypher Revealed Taylor Gang09/08/2014
Uncle Snoop Returns: 10 Reasons Our Host Is The King of Cool09/04/2014
Browse The Full List Of Nominees08/28/2014
The Cypher Revealed - TDE10/15/2013


text:
Available in a larger size with a louder rattle chamber, the Jewel Bait Large Jig Rattles are designed to fit all Jewel Jigs, but were made especially for the new J Lock Flipín Jig. Give your jig a little extra fish attraction with the Jewel Bait Large Jig Rattles.
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Sony FS7 Water Housing Now AvailableSeptember 9, 2015 / 0 Comments
A-Series Canon 7D Mark II Waterhousing In StockNovember 22, 2014 / 0 Comments
SPL GP Series T-Shirt
July 1, 2014 / 0 Comments
Now Available
SPL Water Housings for GoPro Cameras— Now AvailableJune 30, 2014 / 0 Comments
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SPL Waterhousings Custom Camera Water Housings since 1996
We're proud to manufacture some of the toughest water housings for DSLR cameras and video cameras on the planet. Custom fabricated from aluminum, our A-SERIES Housings last a lifetime. Our SPLASH Series Housings, are a custom molded polyurethane to continue the next evolution of water photography. Tested by the world's best photographers in the most trying conditions and stood up to the beating every time. If you're looking to take photos of surfing, swimming, kayaking or any other water related activity contact us about SPL Waterhousings and shoot like a pro.


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About Us
This is what we believe here at Serrv. We believe disadvantaged artisans and farmers around the world should have the opportunity to break the cycle of poverty and support their families through fair payments and respectful relationships. Every basket, every tablecloth, every necklace we offer has a story behind it–of an empowered artisan or farmer who works in a safe environment, can send his or her children to school, and can save for the future with the reliable income he or she earns through fair trade.
We create direct, long-term relationships with small organizations and cooperatives in Asia, Africa, Latin America, the Middle East, and here in the United States, connecting you to the handmade products and foods their members make. We emphasize the word "partnership" in our work because that's really what these relationships are–we've created a system of creative collaboration based on mutual respect and trust, creating high-quality and well-designed items using sustainable materials and production techniques. Some of these partnerships have lasted for over 35 years!
The values of fair trade are central in everything we do. We collaborate with our partners in creating and maintaining programs for artisans and farmers focused on women's empowerment, educational support and skill trainings, sustainable community resource development, and more. Prepayments are always offered for orders, reasonable production timelines are agreed upon, and we provide grants so artisans can expand their businesses. And of course, we pay a fair wage.
In our 65th year of operation, we are dedicated to offering you, our customers, unique handcrafted products and superior service, along with the knowledge that you are helping to build a more sustainable world for us all.
Mission Statement
Serrv is a nonprofit organization with a mission to eradicate poverty wherever it resides by providing opportunity and support to artisans and farmers worldwide.
Our History
Serrv is recognized as a leader and pioneer in the fair trade movement, with origins in 1949 as one of the first fair trade organizations in the United States. As a founding member of both the World Fair Trade Organization and the Fair Trade Federation, Serrv has touched countless lives with opportunity to break the cycle of poverty.
Serrv began with a small group of Church of the Brethren relief workers helping refugees rebuild after World War II. The first product sold was a cuckoo clock from Germany! SERRV originally stood for "Sales Exchange for Refugee Rehabilitation and Vocation", but since our mission and work has expanded greatly since then, we now use Serrv as a word on its own. In 1999, Serrv incorporated as an independent nonprofit organization.
Over the decades we have steadily increased our impact through the number of partners we work with and the depth of our development work. From what began as a small catalog of a few items and one gift shop located in New Windsor, Maryland, we have grown to offer a large range of products through seasonal catalogs, three Serrv stores, and hundreds of stores and churches throughout the country.
Our Core Values
Our organizational values guide how we work and interact with partners, customers, staff, and volunteers. These include prioritizing improved quality of life for low-income artisans and farmers, collaboration, transparency and trust. See all of our Core Values here.
Introducing our New Brand Identity!
As we uphold Serrv's ongoing mission to eradicate poverty, we continuously challenge ourselves to improve the way we market products on behalf of our artisan and farmer partners. As part of our strategy, we've taken the step of rebranding the look and feel of our communications. We are excited to introduce our new brand identity this spring season!
The most important element of the new Serrv logo is what we call the "connections" mark. The eight links represent the core values of Serrv's global community. Our core values are what we stand for as a fair trade organization dedicated to lifting disadvantaged artisans and farmers out of poverty.
The mark also represents the close connections Serrv creates through these values. Together, our community (which includes our artisan and farmer partners, customers, donors, volunteers, fair trade allies, and staff) is creating endless stories of impact.
The new mark showcases what Serrv has created over the last 65 years and more clearly demonstrates how we are all working together to achieve our mission. We look forward to growing these connections even further in 2015 and beyond!
Read more about our new identity in our Spring 2015 newsletter.


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- 188Promote
Tote Bag
I'M A REASONABLE MAN...by Matthew Taylor Wilson
Our quality crafted Tote Bags are hand sewn in America using durable, yet lightweight, poly poplin fabric. All seams and stress points are double stitched for durability. Available in 13" x 13", 16" x 16" and 18" x 18" variations, the tote bags are washable, feature original artwork on both sides and a sturdy 1" wide cotton webbing strap for comfortably carrying over your shoulder.
Cool work ! Great choise of colors and typo !
Choice *
Awesome work!
amazing work, Matthew, I love the color palette :) and my fav word here is "reasonable", great lettering as usual!
Excellent :)
solid work man!
Rad design, bro!
I love this one Matthew!
Good one!
You know whats up with type. Nice piece!
Great design! I like the idea of it! Have a look of mine and see if you like it! http://society6.com/product/break-is-...
This is fantastic! I Love the graphic! I am also interested in this kind of design! check mine out if you like it too!http://society6.com/product/pm-051-32...
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Our story is unique and not what you would expect. This is not a giant coffee company. It has never been bought or sold, it has no shareholders, and our only partners are who we do business with. This story starts with the founder, Kevin Wiesehan, an Aeronautical Engineer at Boeing Aircraft who remains the original and sole owner of the business. While working in St. Louis, he was sent to Seattle, Washington to help a Boeing supplier who was having difficulties producing some aircraft parts. He fell in love with Seattle, the Mountains, the clean air and the stunning National Geographic scenery that Seattle has to offer and yes, the coffee. The Boeing supplier who he had come to help, offered him a position as the C-17 Program Manager, and the transplant to Seattle was complete. Kevin got Married and lived in Seattle for 3 years during the early 90's. He was working 100 hrs. a week. It is here our story begins as he witnessed the start of the coffee phenomenon in Seattle, he became a drive thru junkie and dreamt of leaving it all to bring this Coffee Craze home to St. Louis.
The Aircraft business instills a passion for quality in a person. Every process is documented, every error is defined and fixed, every nonconformance is rationalized. Each part of the process is over-thought, over-engineered, and 3 times stronger and better than the requirement. Money is no object for quality. It's not suprising that these ideas became the foundation of Brevé Coffee®.
In 1994 Mr. Wiesehan left Seattle, his job and his career in Aeronautics. He and his wife moved back home to start The Brevé Espresso Company® and bring something new to St. Louis. On March 5, 1995 they opened in 300 sq. feet of the Paul Brown Building in Downtown St. Louis with a walk-up window and became St. Louis' First Espresso Bar. This was Seven years before Starbucks came to the St. Louis area. The first day in business was terrible. There was an armed robbery in the building, it was was roped off and they did $58 in total sales and it went down hill from there. Nobody knew what a latté was in St. Louis in 1995, and he nor his wife could give them away. The shop was tiny but beautiful with restored 1906 wood floors and doors, mirrors and Syrup bottles that looked like wine to everyone else. But the owners never looked back.
Day after day the founder, his wife and his father struggled to make each day at least $1 better than the day before. They did, and very slowly the business improved. They introduced St. Louis to the first Panini Press Sandwich later in 1995, and business continued to grow slowly. Every night the founder would chart what little sales there were, planned advertising and developed a training manual. His wife would ask "Why are you making a training manual, you have no one to train". "But I will have" was the response, and when I do, I'll be ready. The 100 hr. work week had become the norm. By the end of the first year they had a whopping 3 employees and customers lined up around the building, but the competition was emanating and taking notes.
It wasn't long before coffee shops were popping up all over downtown St. Louis. Loyal customers who had been marveled by the success of Brevé were opening stores of their own. While frightening, their entrance into the coffee market seems to have little effect on Brevés business. With dreams of their own, they too launched head first into business. While many of them looked great upon opening, it became obvious that they lacked the structure to withstand the distructful test of time. Brevé continued to work as hard as ever inventing new products and recipes, systems and proceedures while roasting the finest coffee they could source. The long term engineering strenghts in the founder became evident as he defined what specialty coffee was. The words Specialty, Gourmet and all the other colorful words used to describe coffee were being overused and without any definition. It was though everyone was decribing themselves this way and still do.
Because Coffee is the #2 traded commodity in the world next to oil, Brevé created their own definition of "Specialty Coffee" nearly twenty years ago this way; It's is mountainous grown where heavy equipment is unable to be used. It is shade grown and truly hand picked and washed. It is sundried in shallow patios and hand raked. It is sorted by hand, bean by bean. It is screened in size to within 1/64th of an inch. Brevé then only pruchases these high grown, hand picked, sundried, washed, hand sorted, screened coffees at 17/64 and 18/64th of an inch with 0 defects per 1000 beans. There are no anomolies, no forigien objects, no under sized or oversized beans, no single or triple lobed beans and all at 12.5% moisture content. They are uniform in color, shape, size, density and moisture content. It is this underlying engineered quality blended from each growing region that quietly produces the most inticing, flavorful, smooth and robust coffee that then takes on its own romantic identy.
Brevé then took their uniquely blended top 1% coffees and coupled them with the expertise for long term success and produced business resources that extends far beyond coffee. A person can have a great passion for coffee, but without structure, that passion will only go so far. Today, to withstand a fiercly financed and competitive coffee market it takes more than passion and dedication, it takes educated know how.
Someone who makes a fantastic sandwich should not necessarly open a retail deli. You must have a truly great product, but to stay in business and compete, a business will need stratigies in Cost of Goods, Marketing, Labor, Store Management, Finance & Banking, Accounting, Legal, Point of Sale Systems, Advertising and the list continues.


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Cousins at Camm Corners Shirley Simon, Illustrated by Reisie Lonette When her father dies, Marcy would like to stay in New York with his fiancee, an actress, but her uncle sends her to live with her aunt and uncle in Ohio. She has a hard time adjusting to this family, and keeps planning to go back to New York. An interesting story. Lothrop, Lee & Shepard Company, 1963. For the Scott, Foresman Invitations to Personal Reading Program. Hard pictorial cover, 6 x 8-1/2 inches, 192 pages. School stamp inside front cover, slightly bumped corners. Otherwise clean with no tears. Sturdy binding. Under 2 lb.
Robin's Roost Treasures
Contact us!
PO Box 876Burns, OR 97720United States
Updated Thursday, February 11 2016©1995-2016 TIAS.com All rights reserved.


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$1.99Ever wondered where the aliens are? Bill is sure they don't exist, but after centuries searching the galaxy for any sign of intelligent life, I'm not about to give up yet.I've just received this hot tip from a gang of Luddites hiding out near M15. Hot enough that even Bill has chosen to join me for my final attempt to prove him wrong...SF short story, approx 4,000 words.
$1.99Vicious, congenitally bureaucratic, and proud of their resplendent flame-red uniforms which give them a five minute life expectancy in combat, the Flaming Space Weasels wiped out most of Dirk Beretta's fellow space marines at the battle of Din Bin Foo... then served them for lunch with a nice Merlot.In the aftermath, Dirk quits his job as poster boy for the Space Marines, to drown his sorrow in
$2.99How do you repair a dead planet?Maintenance Bot M-3 was always proud of the smooth and reliable operation of the starships it maintained, as they travelled the galaxy searching for life on unexplored worlds. It lived for the desperate struggle against the forces of chaos and decay, fighting against time to complete repairs before disaster struck.But this job may be beyond even its capabilities.A
$2.99Tired of healing wounded soldiers on the borders of the British Empire, Silas Cane returns to the heartlands of Victorian England as a rural doctor. The quiet Oxfordshire village is a welcome break from the chaos of war, until a smiling madman dies of a disease never seen before.After watching so many men die, Cane will do whatever it takes to keep the villagers alive.If they don't kill him first
$1.99After Dirk Beretta, ex- poster boy for the Space Marines, saves the Winterbotham Nachos heiress from a fate worse than death in the Space Weasel kibble factories, a romantic cruise around the galaxy with her looks like a well-earned rest.But Royalist revolutionaries on the Planet Of The Squid have other ideas. When they hatch a plan to replace the democratic government with a moronic monarch, Dirk
The One That Got Away: A Fishy Tale"You remember last summer, I told you about Frank's time machine?"Uncle Jim had a talent for tall tales over a pint of real ale, but this one was a whopper. Not content with finding his way a hundred years into the future, he just had to set out on the fishing trip of a lifetime.In a space suit. On Europa.A 4,000 word science fiction short story.
$3.99A stripper, a hitman, his target, and a monk with a severed head in his bag. It's a typical passenger manifest for the crew of the space freighter 'Big Momma'.Russ is hiding from 'Nam flashbacks. Alex may have met her dream guy. Max is tempted to throw them all out the airlock.They only have to survive a three week flight from the Heavenly Retreat habitat--The Sleaziest Place In The Solar System
$2.99What if every supernatural or conspiracy story you ever heard happened to be true?In 1952, when Ron Durston is called up into the army for National Service, all he wants is to be posted somewhere sunny like Tahiti or Weymouth where he can fraternise with the native girls and bring home a medal or two to impress his mates. Instead he finds himself standing guard at an army camp in Ass-End,
$1.99Frank has finally finished the time machine in his garage. But the expedition to the future with Uncle Jim, Fatso the cat, and a keg of real ale goes centuries off course. Can they solve one of the greatest mysteries in the history of beer before they find their way home, or will they become sizzling entertainment for a world with no TV or Internet?A 7,500 word science fiction short story.
$2.99A hundred light-year quarantine zone protects the primitive jungle tribes of the Platypus Planet from unauthorized interference. But not from BastadoCorp acquiring mining rights through bribery and fraud.When Dirk Beretta's girlfriend told him to get off the couch and look for a job, he found the perfect opportunity, thousands of light years from her, with free guns and beer, as security chief for
$1.99Darren's Robo-Rat project would have saved countless lives, allowing rescuers to go places no human could survive. If only the zombie apocalypse hadn't got in the way.Now he's trapped on a hotel roof in Saskatoon with his boss, while the girl he loves fights zombies far below. But Rob the zombie, a case of electronics, and a cordless drill may offer a chance to rescue her after all...A 7,500 word
$1.99No-one expects to find alien life in NGC 2419. But when the first probes encounter a dirty, industrial planet somewhere no human is known to have been, the race is on to be the first to welcome its inhabitants into the galactic community. Will it be two poker-playing starships, or The Future, for whom pretension is a vocation, not just a way of life?A 5,500 word science fiction short story.


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- Sheraton Burlington Hotel And Conference Center
- 870 Williston Road, Burlington (Map)
- yyyyy
- A Good, 3.85 / 5 Based on 171 reviews
z Promos Available
U0.5 miles from Higher Ground Burlington ( 5 mins )
&11 mins on foot
We hope you find our Higher Ground Burlington pages useful, with 86 nearby hotels to choose from and 1 visitor review to read.
For whatever reason you're visiting Higher Ground Burlington, we hope you have a fantastic time!
This venue is ranked #1 of 1 rated venue in Burlington
from 1 review
100% Recommend
BY: Liz B, West Fairlee, VT
This was the second Gogol Bordello show I have seen at Higher Ground and it was another great experience. The atmosphere is fantastic and there is never a long wait at the bar.
© ConcertHotels.com all rights reserved


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Zero Waste Bag
Written by Katie on April 23, 2010. Permalink
Add another reusable tote to your must-have list: this lightweight bag is made from a single sheet of #2 plastic and, just like the Ty Shower Curtain, you can write/draw on it as you wish. So, make notes, keep a travel log, or just keep it plain white (as I plan to do, at least at first). It's machine washable and can be recycled when it has worn out.
Purchase Information:Price: $45.00
Availability: Buy Zero Waste Bag here


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Tealeaf®, a leading provider of digital customer experience management and customer behavior analysis solutions, is now part of Customer Analytics at IBM®. Tealeaf customer experience management solutions provide critical visibility, insight and answers to help companies meet online conversion and customer retention objectives.
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|Quick links:
Latest Team Rankings
Free Rivals Alerts
Member Services
|ShopMobileRadio RSSRivals.com Yahoo! Sports
|College Teams
|High Schools
October 25, 2012
Hoyas and Jayhawks set to rumble
Premium Court.This is a press release. Discuss the series and its possible ramifications on
WASHINGTON - Georgetown University Head Men's Basketball Coach John Thompson III announced today that the Hoyas will play perennial national power Kansas in a four-year home-and-home series starting in the 2013-14 season.
Georgetown will visit historic Allen Fieldhouse on December 21, 2013, and also in the 2015-16 season. Kansas will make trips to Verizon Center to face the Hoyas during the 2014-15 and 2016-17 seasons.
The Hoyas and the Jayhawks are annually among the winningest programs in college basketball. Georgetown ranks 31st among the more than 340-plus NCAA Division I schools with 1,544 all-time wins, while Kansas ranks second with 2,070 wins.
Georgetown has played Kansas twice before, with the teams splitting a pair of games. The Hoyas beat the Jayhawks, 70-57, in the semifinal of the 1987 NCAA Southeast Regional in Louisville, Ky., with Reggie Williams scoring a game-high 34 points. The teams met last year in the EA Sports Maui Invitational in Hawaii, a 67-63 Kansas win.


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- Video
- Overview
- Information
- Reviews
- Help
Natural Kayak Gloves
Shopping Info
|NRS Natural Kayak Gloves with HydroCuff - When you're paddling in icy water you will want the NRS' warmest kayaking gloves ever, the Naturals. 3.5mm Terraprene is warmer, lighter, and more comfortable than traditional oil-based neoprenes. Pre-curved fingers and 4-way stretch material inside and out provide excellent flex and dexterity for a glove this thick. GripCote palms and fingers give you a great grip on your paddle and additional wear protection. The neoprene HydroCuff™ grips your arm, helping keep water out and warmth inside the glove. Naturally warm, naturally curved, naturally grippy, the NRS Natural gloves are just what you need when the cold settles in.
Sizing for NRS Natural Kayak Gloves:
I would recomend these gloves to anyone looking to stay warm and feel as if they were one with the paddle.
Joe K
Natural 3.5mm Wetsuit Neoprene Kayak Gloves with HydroCuff Wrist Closure by NRS Northwest River Supply for Kayaking
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The Essence of Asana: A Class Series
|High Desert Yoga - Yoga Workshop
|Yoga Workshop:
|The Essence of Asana: A Class Series
|Albuquerque NM USA
|Event is Over
|High Desert Yoga
|Kim Schwartz
$84 per series or $14 per class
This class series will focus on various groups of asanas. We will cover the essential aspects of organizing each group of asanas so that the similarities can be seen as well as the reasons for their differences. In this way we can see what each asana teaches about the other asanas in each group as well as how each group informs the other groups. It is recommended that the entire series be attended but each class will be self contained and available on a drop in basis. 1. Standing asanas 2. Forward bends 3. Twists 4. Back bends 5. Inversions 6. Restoratives and timed asanas
Kim was introduced to eastern philosophy as a child and began studying yoga in 1971 at age 18. In 1981 he met his Guru and began more formal studies in the yoga sutras and other aspects of yoga philosophy. Around that time he was also introduced to the Iyengar system of asana practice. In 1985 he was certified to teach yoga through the Temple of Kriya yoga. He was ordained a Swami in the Kriya lineage in 1986. He has taught ongoing yoga classes, teachers training programs and workshops here and abroad since that time. Though asana and pranayama have become primary disciplines, the philosophy of yoga is still at the heart of his practice and teaching.
Schedule Details
Tuesdays, July 23- August 27, 2013 7:30-8:45pm
Price Options
$84 for series.
$14 per class. You may use class packages for this series.
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I have tried to search for this, but have come up with nothing.
I am just curious as to why one would put the asterisk in the scenario below:
$var = *$self->{class_var};
What does the * do in this situation?
I took the above from the Net::Telnet module and I am simply trying to understand it.
The actual code is:
$s = *$self->{net_telnet};
I wonder, then, is net_telnet the package name?


text:
Average rating from 5 reviews.
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Fun for you and your dog
posted 8 years ago
Roy Hunter's book, "Fun Nosework for Dogs," gives some simple, yet fun games to play with your dog to utilize his sense of smell. It also provides exercises to train your dog for tracking and searching. One of our dogs has an exceptional nose. One of the book's games I like to play with her is to put my scent on an object, then intermix the object among about 30 other objects. It's so much fun to watch her "nose" through the pile and reject objects until she finds the correct one. And she's so happy and confident when she finds it.
Pros: Uses and develops a dog's sense of smell with games and exercises
Cons: Your dog never wants to stop
21 of 21 people found this review helpful.
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Pink is my favorite color these are wonderful.I will recommend a good website fou you: www.9footballshirt.org


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By BigSteve1983 0 Comments
Today I've got a job interview for a local estates agency (it's about an hour or so away via buses etc) for admin assistant.
Quite nervous however I'm just going to go for it and give it my best. After all that's all I can do isn't it.


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Kitty's stories were a verbal arcade of the brightest and best of Broadway. At 96, some might think her time had long ago passed her by. She once told me that she believed the golden era of the Broadway musical was long gone and would not make a comeback, except through the occasional revival, because there was neither the talent, nor the interest in such things today. I said I hoped she was wrong, but I feared she might be right.
Any journalist will tell you that the people you meet are the best part of this business. And I have been doubly blessed. Not only have I been fortunate to meet world leaders, but also talented performers from the Broadway stage — a place I dreamed of becoming a part of, before I entered the world of journalism.
I could live vicariously through people like Kitty Carlisle Hart, who knew the people I wished I knew, and some I actually came to know. That golden age — those magical years in the '30s and '40s when scores of shows ran simultaneously on and off-Broadway — will never live again except in the memory of those who were there and lived to tell and write about them. If those days are never to be again, at least we can know what they were like through books like Steven Bach's "Dazzler," a biography of Moss Hart, and through the stories Kitty told to those who wanted to hear them.
I wanted to hear them and to pass them on. She would have liked that. And I do so without a government grant.
Cal Thomas is co-author (with Bob Beckel) of the book, "Common Ground: How to Stop the Partisan War That is Destroying America".
TOWNHALL DAILY: Be the first to read Cal Thomas' column. Sign up today and receive Townhall.com daily lineup delivered each morning to your inbox.
Bush mocks Rubio and his fans: Stop whining that it's "unfair" that you're being attacked by another candidate's Super PAC | Allahpundit


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another ID - 27march by oomizuaoWatch
©2011-2016 oomizuao
I just wanted to draw my Dexter t-shirt...
art (C) Elise M. Syvertsen / oomizuao 2011
art (C) Elise M. Syvertsen / oomizuao 2011
Dexters Laboratory, I used to watch that show.
cool jacket
That show is incredible
so CUTE!!!!!! ^^
I love the shirt and the hoodie. You're getting a lot better with your human drawing! ^^
.. I need that shirt, or my life will be NOTHING
erm. can i has your shirt o:
Aww, you look really sweet here I love your expression The t-shirt is so awesome too! I miss Dexter
faved it for the epic t-shirt ^^
Hehe, that is a pretty awesome shirt I love your hair too.
I LOVE your hair colourr! DD
I want that T shirt! Where can I buy it ?
I love this pic, and the style too!
I love this pic, and the style too!
Haha, loving this ID (:
This picture looks excatly like my friend Bjørk. With the hair and clothes and EVERYTHING.
OMG! WIN! I can now wear my Dexter and Teen Titans and TMNT shirts in public, because I know I am not the only one!!
i bet youre really pretty also i think ive seen that shirt at hot topic here in the US haha.
hahaha nice gotta love dexter !! XD
Hahah nice
it's weird to see a drawing like this cause it's not in your usual style xD
My,arn't you adorable!Nice t-shirt too
this is soooo adorable i love it
love it!
Dexter rules! XD
OMG dexter ha! I remember. Wow.
YOU ARE A-MAZ-ING *sob* OH MAH GAWD! *sob* your just so amazing!..........well your art is really good is basically what im saying
Nice I love it
Oml, I haven't seen Dexter in FOREVER ^^'
ååå, vil ha!
amazing...LOVE the hair and pretty much all of it...yeah all of it, awesome.
I want her hoody.
I want her hoody.
I love your hair, and your shirt, so cute <3
Awesome shirt! Do want <3
And your hair.. amazing *w*
And your hair.. amazing *w*
huh we have the same haircut, except mine is longer on the other side
I love that t-shirt. Never got to buy it tho, it was out of stock when I could afford it :<
God I want that shirt *u* Great Job!
I love eet. :3
That shirt is epic.
Love that outfit haha <3
Awesome work : D
Awesome work : D
U look so cute haha and yeah, I remember Dexter. What happened to Cartoon Network? it's not the same anymore...
awww, you're so cute! and i adore the blue hair!
Indeed a fine day for science, Dexter.


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Re: where to travel this year?
Hello and welcome to VT.
I guess your question is way to general. It might be a good thing to describe yourself and what your interests are. Or, for example, tell us what kind of travel you prefer (adventures?).
Good luck,
Be the first to rate this answer!
Re: where to travel this year?
Why don't you travel along the Mekhong from the Vietnam Delta to the source? How about the Siberian Express to Moscow from Vladivostok? Kayak along the whole of the amazon!!!
Lots of ideas, so explain your interests!!
Be the first to rate this answer!
Would help if you gave us some idea of your interests
Be the first to rate this answer!
Re: where to travel this year?
thanks so much to everyone that has replied. i have done some further research and as i have completed 3 years of nursing and midwifery at university i am thinking about doing some volunteer work in the maternity area. i still have one more year to complete until i am fully qualified so am looking for volunteer work available to midwifery student.
At the moment i am concidering South Africa and somewhere in central or south america. i dont speak spanish but am considering doing an intersive spanish course before. however would want to go somewhere where english is commonly understood.
DOes anyone have any advise or organisations/clinics i could contact.
Be the first to rate this answer!
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text: I realise this a fundamental flaw in my understanding but I can't seem to get my head around it. I understand that bragg reflection occurs at the zone boundary and so electron wavevectors are diffracted back into the 1st brillouin zone, but what is the justification for only considering the 1st brillouin zone, for example, when considering energy gaps? This is also connected with my earlier question about why Umklapp processes are required, where large k is transmitted back into 1st brillouin zone by G: K1+K2=K3+G


text:
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Ringing in the New Year with Fresh Mochi
Full audio available here:
This holiday season, many families will gather to celebrate the Japanese New Year's tradition of making mochi.
You can pick up fresh or frozen mochi from many stores around the state at this time of year. But there's something special about making it the old fashioned way.
In the case of mochi pounding, too many cooks in the kitchen can actually be a good thing. Making mochi is hard work and involves using a wooden mallet to mash the steamed rice down to a smooth, sticky paste.
George Tanabe, a retired UH Manoa Professor Emeritus from Waialua, holds his own mochitsuki, or mochi pounding, every year. He describes the meaning behind the New Year's custom.
Sharon Fujino is a longtime Kaneohe resident. She anticipates more than a hundred family and friends will join her to ring in the New Year and help pound mochi. She remembers attending mochitsukis as a little girl and hopes the family tradition will continue for generations to come.
If you are interested in attending a public mochi pounding, contact the Japanese Cultural Center for more information.
Full audio available here:
(Molly Solomon / HPR News)