text:
Methods are provide for modifying a solid support, such as a glass slide, by silylating with an agent having the formula H2N—(CH2)n—SiX3 where n is between 1 and 10, and X is independently chosen from OMe, OEt, Cl, Br, or I, then activating with a crosslinking reagent, followed by reacting with an amine-containing polymer. The support can optionally be reacted with a crosslinking reagent again. The support thus modified may be used to make arrays and microarrays where a plurality of targets are stably associated with the support and arranged in a defined manner.
1. A method of attaching target polynucleotides to a surface of a solid support, the method comprising the ordered steps of:
a) silylating the surface with an agent having the formula H2N—(CH2)n—SiX3 where n is between 1 and 10, and X is independently chosen from OMe, OEt, Cl, Br, or I;
b) activating the surface with a first crosslinking reagent;
c) reacting the surface with an amine-containing polymer to form a modified solid support surface; and
d) attaching non-activated polynucleotide targets to the modified solid support surface, wherein the polynucleotide targets are arranged in a defined manner on the modified solid support surface thereby producing an array of attached polynucleotides.
2. The method of
3. The method of
4. The method of
5. The method of
6. The method of
7. The method of
8. The method of
9. The method of
10. The method of
11. The method of
12. The method of
13. The method of
14. An array of attached target polynucleotides, the array made by the method of
15. The array of
16. The array of
17. The array of
18. The array of
19. The array of
20. The array of
21. The array of
22. The array of
23. The array of
24. The array of
25. The array of
The present invention relates generally to the field of arrays and microarrays and, more particularly, to compositions and methods for modifying the solid supports of the arrays and microarrays.
Microarrays having a plurality of polymeric molecules spatially distributed over and stably associated with the surface of a solid support are becoming an increasingly important tool in bioanalysis and related fields. Microarrays of both polypeptides and polynucleotides have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microarrays find use is in gene expression analysis.
The current methods of manufacturing arrays and microarrays immobilize the polynucleotides at specific sites on solid supports by either synthesizing the polynucleotides at the desired position, or by presynthesizing the polynucleotides and then attaching them to the solid support. U.S. Pat. No. 5,445,934 discloses a method of on-chip synthesis. In this process, a glass slide is derivatized with a chemical species containing a photocleavable protecting group. Selected sites are deprotected by irradiation through a mask. The deprotected sites are then reacted with a DNA monomer containing a photoprotective group. The process of masking, deprotecting, and reacting is repeated for each monomer attached until an array of site-specific polynucleotide sequences is achieved.
Methods for immobilizing pre-synthesized polynucleotides onto solid supports include simple adsorption, ultra violet linking, and covalent attachment. In general, the attachment of unmodified polynucleotides to unmodified solid supports is inefficient. Therefore, the polynucleotides or the solid support has to be modified to enable attachment. Thus, polynucleotides modified with bovine serum albumin adsorb passively to microtiter plates (Southern, E. M. PCT 89/00460), and biotinylated polynucleotides bind tightly to plates or beads that are coated with avidin or streptavidin. In another method, Carrico, et al., U.S. Pat. No. 4,806,546, have described treatment of a nylon support with an alkylating agent to introduce amidine groups onto the surface of the nylon. The derived nylon surface possesses the capacity to noncovalently bind single stranded nucleic acids. The noncovalently bound nucleic acids are then used as probes to detect specific target nucleic acids in solution.
In a different approach, the solid support is modified with a suitable functional group and/or linker. Thus, the solid support is modified to carry an active group, such as hydroxyl, carboxyl, amine, aldehyde, hydrazine, epoxide, bromoacetyl, maleimide, and thiol groups (Lund et al., U.S. Pat. No. 5,474,895) on its surface to which oligonucleotide can be covalently or non-covalently linked. For example, U.S. Pat. No. 5,514,785 to Ness et al. discloses a process for covalently attaching an oligonucleotide to a nylon support. The nylon support is first treated with an amine-containing polymer thereby forming a reactive imidate ester on the surface of the nylon support. The imidate esters on the surface are then reacted with a primary or secondary amine-containing polymer to form amidine residues that are then conjugated with activated polynucleotides. In another method, disclosed in U.S. Pat. No. 6,013,789 to Rampal, polypropylene film is first aminated by a plasma discharge in the presence of ammonia gas, and then contacted with an oligonucleotide having a terminal phosphorimidazolide, whereupon the oligonucleotide becomes covalently linked to the polypropylene film via a phosphoramidate bond.
The immobilized polynucleotides can be used to construct arrays or microarrays for hybridization assays. A typical method of using microarrays involves contacting nucleotide sequences contained in a fluid with the sequences immobilized on the microarrays under hybridization conditions, and then detecting the hybridization complex. The resultant pattern of hybridized nucleic acids provides information regarding the profile of the nucleotide constituents in the sample tested. A widely used method for detecting the hybridization complex in microarrays is by fluorescence. In one method, probes derived from a biological sample are amplified in the presence of nucleotides that have been coupled to a fluorescent label (reporter) molecule so as to create labeled probes, and the labeled probes are then incubated with the microarray so that the probe sequences hybridize to the complementary sequences immobilized on the microarray. A scanner is then used to determine the levels and patterns of fluorescence.
The art methods of immobilizing polynucleotides on solid support usually result in low coupling yields. In addition, the polynucleotides are bound on the flat two-dimensional surface of the substrate, whereas it is thought that binding the polynucleotides within a three-dimensional polymer matrix would enable more efficient hybridization. Thus, there exists a need for methods and procedures for immobilizing polynucleotides to solid support for fabricating arrays and microarrays.
Methods are provided for modifying a solid support by silylating the support with an agent having the formula H2N—(CH2)n—SiX3 where n is between 1 and 10, and X is independently chosen from OMe, OEt, OPr, Cl, Br, or I, then activating with a crosslinking reagent, followed by reacting with an amine-containing polymer. The support can be further treated with a cross- linking reagent. A plurality of targets may be stably associated with the support and arranged in a defined manner.
Also provided are methods of attaching targets to a solid support. The support is silylating with an agent having the formula H2N—(CH2)n—SiX3 where n is between 1 and 10, and X is independently chosen from OMe, OEt, Cl, Br, or I, then activating with a crosslinking reagent, followed by reacting with an amine-containing polymer. The support can be further treated with a crosslinking reagent. The support is then contacted with the targets that can optionally have a spacer arm at the 5′-end or the 3′-end of the target.
Also provided is an array of polynucleotides made by modifying a glass slide by silylating with an agent having the formula H2N—(CH2)n—SiX3 where n is between 1 and 10, and X is independently chosen from OMe, OEt, Cl, Br, or I, then activating with a crosslinking reagent, followed by reacting with an amine-containing polymer. The support can optionally be reacted with crosslinking agent. A plurality of polynucleotides are stably associated with the support in a defined manner.
These and other objectives, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.
FIG. 1 provides a schematic representation of the five complimentary 59 mer oligonucleotide of SEQ ID NO.: 1-5 to YCF 23 and the five complimentary oligonucleotide of SEQ ID NO.: 6-10 to YCF.44.
FIG. 2 illustrates the relative Cy3 signal intensities corresponding to the binding of the YCF 44 polynucleotides onto derivatized glass slides: polyethylenimine (PEI), polylysine or polyhistidine. In this analysis, the signals corresponding to each type of polynucleotide (with or without an aminolinker) at a given concentration were averaged.
FIG. 3 illustrates the relative Cy5 signal intensities corresponding to the binding of polynucleotides onto derivatized glass slides: polyethylenimine (PEI), polylysine or polyhistidine. In this analysis, the signals corresponding to each type of polynucleotide (with or without an aminolinker) at a given concentration were averaged.
Before the present methods, arrays and microarrays are described, it is to be understood that this invention is not limited to particular methods and arrays described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a probe" means that more than one such probe can be present in the composition. Similarly, reference to "a microarray element" or "the microarray element" includes the possibility of one or more microarray elements, and the like.
The term "solid support" refers to any surface onto which targets, such as nucleic acids, may be immobilized for conducting assays and reactions.
The term "target", "DNA element" or "microarray element" refers to a molecule that has an affinity for a given sample. Targets may be naturally occurring or synthetic molecules, and may be attached, covalently or noncovalently, to a surface, either directly or via a specific binding substance. Examples of targets which can be employed by this invention include, but are not restricted to, DNA, RNA, oligonucleotides, oligosaccharides, polysaccharides, sugars, proteins, peptides, PNAs, monoclonal antibodies, toxins, viral epitopes, hormones, hormone receptors, enzymes, enzyme substrates, cofactors, and drugs including agonists and antogonists for cell surface receptors.
The term "microarray" refers to an array of targets synthesized or attached or deposited on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, beads, or any other suitable solid support, at high density.
In one embodiment, polynucleotides and activated polynucleotides are attached to an amine treated solid support, such as a glass slide. The attachment can be covalent, noncovalent, or a combination of the two. The array or the microarray thus manufactured may be contacted, under hybridization conditions, with a fluid sample containing labeled nucleic acid probes complementary to the polynucleotide target. The hybridization pattern from the probe may be detected to obtain information about the genetic profile of the labeled nucleic acid sample.
In general, the invention comprises the steps of treating a solid support with a silylating agent to give aminoalkylsilated solid support; reacting the treated solid support with a crosslinking reagent, preferably the multifunctional reagent, cyanuric chloride (i.e., 2,4,6-trichlorotriazine), followed by treatment with an amine-containing polymer, preferably polyethylenimine (PEI); optionally activating the PEI-modified surface with a crosslinking reagent; and then contacting the solid support with targets, such as polynucleotides that may be optionally modified to contain a linker group.
In the present invention, a solid support is treated initially with a silylating agent. The solid support may be composed of any material that is compatible with the fluids with which the surface comes in contact, such as, for example, glass, gel, silicon, fused silica, plastic, ceramic, paper, metal, or other polymers such as nylon. The substrate may be rigid or flexible, and may define a shape that is substantially planar in the shape of a circle, an ellipse, a square, a rectangle, a triangle, or any other convenient substantially planar shape. In the preferred embodiment, a rectangular glass slide is used as the substrate. The silylating agents are chosen such that they react with the reactive groups present at the surface of the substrate to form a primary amine. For example, the silylating agent NR′2—R—SiX3, where R′ is independently chosen from hydrogen, methyl, ethyl, or propyl, R is (CH2)n, where n is 1 to 10, and X is independently chosen from hydrogen, alkyl, Oalkyl, F, Cl, Br, or I, where alkyl is preferably loweralkyl of from 1 to 6 carbon atoms with the proviso that all three X groups together cannot be alkyl, upon reaction with the Si—OH groups present at or near the surface of the glass slide yields aminoalkysilanated glass slides. In the preferred embodiment, the rectangular glass slide is reacted with 3-aminopropyl-trimethoxysilane to yield aminopropylsilanated glass slides.
The aminoalkylsilanated glass slide may be treated with a multifunctional crosslinking reagent. In one embodiment, the crosslinking reagent contains a reactive group at one end that can react with the nitrogen atom of an amine group to form a nitrogen-carbon bond. Such reactive groups are well known in the art, and include halides, esters, epoxides, and the like. The crosslinking agent additionally contains a protected reactive group at the opposite end that is capable of being deprotected and undergoing further reaction with the amine-containing polymer. Crosslinking reagents include, but are not limited to, N-succinimidyl-4-(iodoacetamido)-benzoate (SIAB), disuccinimidyl suberate, 1-ethyl-3-(dimethylaminopropyl)carbodiimide and 2,4,6-trichlorotriazine (cyanuric chloride). The crosslinking reagent is preferably cyanuric chloride.
The aminoalkylsilanated substrate treated with the crosslinking reagent may then be reacted with an amine-containing polymer. Any primary, secondary, or tertiary amine-containing polymer may be employed. The amine-containing polymer may be polyethylenimine, polyallylamine, polyvinylamine, polyhistidine, polyomithine, polylysine, and polyarginine. In the preferred embodiment, the solid substrate, having been silylated and activated with the crosslinking reagent, is coated and modified with polyethylenimine (PEI). The resulting PEI-modified surface of the solid surface may optionally be treated with a crosslinking reagent again, such as cyanuric chloride.
The PEI-coated glass slides may be used for manufacturing arrays and microarrays. In the preferred embodiment, targets are immobilized on the PEI-coated glass slides. The targets may include polynucleotides, oligonucleotides, haptens, cytokines, proteins, peptides, saccharides, and the like. The polynucleotides include nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phosphorothioate, methylimino, methylphosphonate, phosphoramidate, guanidine and the like; nucleic acids in which the ribose subunit has been substituted, e.g. hexose phosphodiester; peptide nucleic acids, and the like. The polynucleotides may be single or double stranded, and may be PCR fragments amplified from cDNA. The polynucleotides may cover the known 5′ or 3′ nucleotide sequence, may be the full length sequence, or may be unique polynucleotides selected from particular areas along the length of the sequence. The polynucleotides used may be polynucleotides that are specific to a gene or genes of interest in which at least a fragment of the sequence is known, or that are specific to one or more unidentified cDNAs which are common to a particular cell type, developmental or disease state. The polynucleotides for use in the present invention may be synthetic polynucleotides or PCR fragments from about 2 to about 50,000 consecutive bases, preferably about 5 to about 500 consecutive bases in length, more preferably about 40-70 bases in length, most preferably about 50 to 65 bases in length. A spacer (linker) arm, i.e., a chemical moiety that extends or links other chemical groups, and preferably is a carbon chain containing from about 2 to about 12 carbon atoms, more preferably about 6 carbon atoms, containing a blocked amine group can be coupled during synthesis using conventional chemistry to the 5′-hydroxyl group of a polynucleotide. Alternatively, a polynucleotide can be modified at the 3′-end with a spacer arm containing a blocked amine group by methods well known in the art.
Preferably, a polynucleotide possessing a 5′-linker arm or a 3′-linker arm such as:
is employed wherein, as a spacer arm, n is 2-12 inclusive, preferably 6; Y is an amine or a thiol, preferably a primary amine; and A is an polynucleotide, ranging from between about 2-50000 consecutive bases, preferably about 5 to about 500 consecutive nucleotides, with only the 5′-hydroxyl or the 3′-hydroxy group requiring modification for attachment.
The selected polynucleotides are then associated with the polymer coated glass slides for manufacturing arrays and microarrays. The arrays may be produced according to any convenient methodology, and a number of different array configurations and methods for their production are known to those of skill in the art and disclosed in U.S. Pat. Nos. 5,445,934, 5,532,128; 5,384,261; and 5,700,637; the disclosure of which are herein incorporated in their entirety by reference. For example, the polynucleotides may be stably associated with the substrate through non-covalent means, such as by interacting with a positively charged surface of the substrate. Alternatively, the polynucleotides possessing the 5′- or the 3′-terminal linker arm may be bound to the substrate through co vaient means, such as via an alkylamino-linker group. In another embodiment of the invention, the polynucleotides may be associated with the substrate through both non-covalent and covalent means.
In order to conduct sample analysis using the arrays and microarrays of the invention, the RNA or DNA from a biological sample is made into hybridization probes, as is well known in the art. The hybridization probes are then contacted with the arrays and microarrays of the invention under hybridization conditions to produce a hybridization pattern. Suitable hybridization conditions are well known to those of skill in the art and reviewed in WO 95/21944 to Maniatis et al. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used for detection or visualization to determine the levels and patterns of fluorescence. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously.
The hybridization pattern can be used to determine quantitative information about the genetic profile of the nucleic acids in the sample that was contacted with the array to generate the hybridization pattern, as well as the physiological source from which the labeled sample nucleic acid was derived. The data provides information about the physiological source from which the sample nucleic acid were derived, such as the types of genes expressed in the tissue or cell which is the physiological source, as well as the levels of expression of each gene, particularly in quantitative terms.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperatures, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
The cDNA microarrays were made according to U.S. Pat. No. 5,807,522 to Brown et al. The polynucleotides were synthesized and purified by Operon Technologies, and used without further purification. The chemical reagents were obtained from Aldrich Chemical Company, and used without further purification. The organic solvents were of HPLC grade.
This example serves to demonstrate a method of silylating a glass slide. Ten glass slides, visually inspected to be free of scratches, were selected. The selected glass slides were immersed in 1 L solution of 0.1% SDS in distilled water (w/v) and subjected to ultrasonication for approximately 30 minutes. The glass slides were then dried, treated to ultrasonication treatment in acetone for approximately 30 minutes, and air dried.
A 2.0% (w/v) silane solution was prepared by dissolving 3-aminopropyltrimethoxysilane in deionized water with gentle stirring. The dried glass slides were dipped in the silane solution for 2 minutes. The glass slides were rinsed free of excess silane solution by ultrasonication in distilled water for about two minutes, and rinsing with distilled water. The washed slides were cured at about 120� C. for about 30 minutes, and stored in a dustless environment until used.
This example serves to demonstrate a method for making 60 PEI coated glass slides. All reactions were done at about 4� C., unless otherwise indicated. Cyanuric chloride (12.7 g) was dissolved in 1 L of n-hexane and sodium carbonate. (25.0 g). Separately, polyethylenimine (1.0 g, MW 25,000 Da) was dissolved in 1 L distilled water at room temperature. The solutions, the glass slides from Example 1, 2 L of n-hexanes, and additional 1 L of distilled water were separately stored for at least 2 hours in a refrigerator capable of controlling the temperature at less than about 4� C. The glass slides were placed in two slide racks and the racks were place in the cyanuric chloride solution, with vigorous stirring of the solution. After about 1 hr., the glass slides were washed in 1 L of n-hexanes for about 10 minutes in an ultrasound cleaner, and air-dried. The slides were then placed in the polyethylenimine solution for about 1 hr. The slides were then washed with 1 L of distilled water for 10 minutes in an ultrasound cleaner, air dried, centrifuged to 1000 rpm, washed in 1 L of n-hexanes for about 10 minutes in an ultrasound cleaner, placed back in the cyanuric chloride solution for about 1 h., washed in 1 L of n-hexanes for about 10 minutes in an ultrasound cleaner, and air dried. The slides were then washed with about 1 L acetone for about 20 minutes in an ultrasound cleaner at room temperature twice, and air dried. The slides were then stored desiccated at about 2 to 25� C.
This example serves to demonstrate a method for making 60 polylysine coated glass slides. The procedure of Example 2 was repeated, except 1 g of polylysine (Sigma) was used.
This example serves to demonstrate a method for making 60 polyhistidine coated glass slides. The procedure of Example 2 was repeated, except 1 g of polyhistidine (Sigma) was used.
The following example describes the application of polynucleotides onto a PEI-modified glass slide prepared in Example 2.
Synthetic oligodeoxyribonucleotides (59 bases in length with 3′-aminoalkyl linker modifications) were purchased from Operon Technologies (Alameda, Calif.). Seq. ID. No.: 1-5, shown in FIG. 1, each targeting a yeast control fragment (YCF 23) target sequence, were used in this experiment. Dilutions of these oligodeoxyribonucleotides (6.25-100 micromolar) were made in 2�SSC buffer (pH 7.4) and transferred to 96-well microtiter plates. The resulting samples were spotted onto polyethylenimine-modified glass slides in the Incyte Microarray Systems production facility. After arraying, the slides were washed in 0.2% SDS solution for 2 min. Next, the slides were rinsed in deionized water for 1 min and immersed in a 2% casein solution in phosphate buffered saline at 60� C. for 30 min. Then, the slides were rinsed in 0.2% SDS for 5 min, rinsed in deionized water for 1 min, and spun dry.
The following example describes the use of an array generated according to Example 5 in a hybridization experiment. A synthetic RNA transcript corresponding to the yeast control fragment (YCF 3) target sequence was prepared as follows. A clone containing the YCF 3 target sequence was provided by the Incyte Microarray Systems production facility. The clone was amplified with vector-specific primers to generate the corresponding PCR amplicon. This material was further amplified with PCR primers specific to the 3′- and 5′-ends of the target sequence. The forward primer contained a T7 promoter at its 3′-end (NNNNN NTAAT ACGAC TCACT ATAGG GAG) and the reverse primer contained a 30-base poly(dT) sequence at its 5′-end. The PCR products were purified using gel filtration. The purified T7 product was used as a template to generate RNA through in vitro transcription using Ambion's MEGAscribe� kit. (Austin, Tex.). The RNA was quantitated by UV spectroscopy.
Cy5-labeled cDNA probe was transcribed from 1 ng of RNA template by reverse transcription using 5′ Cy5 labeled random 9-mers (Operon Technologies). Reactions were incubated for 2 hrs at 37� C. with 200 Units MMLV reverse transcriptase (Life Technologies, Gaithersburg, Md.), 4 mM DTT, 1 unit RNase Inhibitor (Ambion, Austin, Tex.), 0.5 mM dNTPs, and 2 μg of the Cy5-labeled random 9-mer in a buffer provided by the manufacturer (25 μL final volume). The reactions were terminated by incubation at 85� C. for 5 min. The reactions were then purified with a TE-30 column (Clontech, Palo Alto, Calif.), made up to a volume of 90 μL with deionized water, and precipitated with 2 μL 1 mg/mL glycogen, 60 μL 5M NH4OAc, and 300 μL ethanol. The pellets were resuspended in 24 μL of hybridization solution: 5�SSC, 0.2% SDS, 1 mM DTT.
The Cy5-labeled cDNA probe was applied to the array, covered with a 22-mm2 glass cover slip, placed in a sealed chamber to prevent evaporation, and incubated at 60� C. for six hrs. After the hybridization, the arrays were washed for 10 minutes in 1�SSC/0.1% SDS at 45� C. then for 3 minutes in 0.1�SSC/0.2% SDS at 25� C. The arrays were imaged by confocal laser scanning using GenePix™ scanners (Foster City, Calif.) with a 10 μm resolution. The signal was converted into 16-bits-per-pixel resolution, yielding a 65,536 count dynamic range. Incyte GEMtools™ software (Incyte Pharmaceuticals, Inc., Palo Alto, Calif.) was used for image analysis. Signals were corrected for local background. The example demonstrates the use of this invention for detection of a target sequence in a microarray format using immobilized polynucleotides.
The following example compares the performance of glass slides coated with polyethlenimine, polylysine and polyhistidine.
In order to compare their ability to bind polynucleotides, and to examine the hybridization performance of the corresponding arrays, the following experiment was conducted. Polynucleotide sequences (Seq. ID. No.: 6-10, shown in FIG. 1) were designed against an intronic yeast control fragment sequence, YCF 44. The polynucleotides were purchased from Operon Technologies. Each sequence was synthesized as four different derivatives: (1) unmodified; (2) 3′- aminolinker modified (unlabeled); 5′-dye-labeled (cyanine 3, no aminolinker); and 5′-dye labeled (cyanine 3) plus 3′-aminolinker modified. Polynucleotides of the same sequence plus a 3′-aminolinker were mixed in a 10:90 molar ratio (dye labeled: unlabeled). Polynucleotides without a 3′-aminolinker were likewise mixed in a 10:90 molar ratio (dye labeled:unlabeled). Each mixture was reconstituted in 2�SSC buffer and distributed into a 96-well plate at 1 μM, 10 μM and 100 μM concentrations. Each of the resulting samples was then printed onto the modified glass substrates four times to enable signal averaging. Next, the slides were washed, blocked, and dried as described in Example 6. Then, the slides were scanned using a two-color laser-scanning device (Axon Instruments) to obtain a relative measure of the amount of Cy3 dye deposited at each spot. Representative data is shown in FIG. 2. In each case, the aminolinker-modified polynucleotides bound in greater abundance compared to the corresponding sequences without aminolinkers. Nevertheless, the data indicates that the polynucleotides bind via a mixture of covalent and noncovalent interactions.
In order to measure the hybridization activity of polynucleotides bound onto the coatings according to this example, a synthetic RNA transcript was prepared from YCF 44 as described in the previous example. The generation of Cy5-labeled cDNA probe, hybridization and data analysis were conducted as described in the previous example. The resulting hybridized arrays were then scanned in the Cy5 channel to provide a relative measure of hybridized probe. Representative data is shown in FIG. 3.
Patent Citations
Non-Patent Citations
Referenced by
Legal Events
Original Image


text:
What is multiple sclerosis?
Multiple sclerosis is a progressive autoimmune disease and the most common neurological disease diagnosed in young adults. It is believed that multiple sclerosis occurs when the body's own immune system attacks the central nervous system. Commonly called MS, the disease generally gets worse with time and can cause significant nerve damage.
The progression and severity of multiple sclerosis varies greatly among individuals. The severity of multiple sclerosis ranges from mild to severe and disabling, and it can result in muscle weakness, loss of balance, and difficulty walking.
In some cases, multiple sclerosis can lead to serious complications, such as choking and paralysis. Early diagnosis and medical care can help manage and control symptoms and minimize complications of multiple sclerosis.
What are the symptoms of multiple sclerosis?
The symptoms of multiple sclerosis result from inflammation, swelling, and lesions on the myelin, a fatty material that covers and insulates the nerve cells in the central nervous system. Myelin greatly improves the transmission speed of nerve impulses without decay of the nerve signal. Loss of myelin leads to abnormal transmission of electrical impulses through the central nervous system an... Read more about multiple sclerosissymptoms
What causes multiple sclerosis?
The causes of multiple sclerosis are not well understood. Researchers believe that the body's immune system begins to attack the nervous system, specifically the myelin. The myelin is a fatty material that covers and insulates the nerve cells in the central nervous system. Healthy myelin is vital to the normal, rapid movement of electrical impulses through the nerve pathways.
In m... Read more about multiple sclerosiscauses
How is multiple sclerosis treated?
There is no cure for multiple sclerosis. However, the disease can be managed and the symptoms controlled to various degrees of success with an individualized, multifaceted approach that includes medications and other therapies. You can help manage multiple sclerosis by consistently following your treatment plan.
Treatment plans are geared toward managing your symptoms and suppress... Read more about multiple sclerosistreatments

subset: commoncrawl


text:
By Clyde Hughes |
Monday, 03 Feb 2014 09:01 AM
© 2015 Newsmax. All rights reserved.
Thursday, 30 Jul 2015 07:39 AM
A giant Burmese python was captured in Florida's Everglades National Park earlier this month, and it was so long it took . . .
Wednesday, 29 Jul 2015 18:56 PM
Nick Gordon is still wearing the wedding ring commemorating his relationship with Bobbi Kristina Brown, even after her d . . .
Wednesday, 29 Jul 2015 18:12 PM
A tree fell on a group of kids at a summer camp outside Los Angeles on Tuesday afternoon, injuring eight, including two . . .
Newsmax, Moneynews, Newsmax Health, and Independent. American. are registered trademarks of Newsmax Media, Inc. Newsmax TV, and Newsmax World are trademarks of Newsmax Media, Inc.


text: The RVCA ANP Coaches Jacket is a nylon coaches jacket with lining, snap closures down front, and double welt pockets at front. The jacket has elastic at the cuffs and drawstring at the waist and a RVCA ANP patch at the left chest. Constructed of 100% nylon shell and 100% polyester lining. RVCA detailing throughout. Available in colorways as seen below. Model wears a medium.


text:
procmailex - procmail rcfile examples
$HOME/.procmailrc examples
For a description of the rcfile format see procmailrc(5).
The weighted scoring technique is described in detail in the procmailsc(5) man page.
This man page shows several example recipes. For examples of complete rcfiles you can check the NOTES section in procmail(1), or look at the example rcfiles part of the procmail source distribution (procmail*/examples/?procmailrc).
Sort out all mail coming from the scuba-dive mailing list into the mailfolder scubafile (uses the locallockfile scubafile.lock).
scubafile Forward all mail from peter about compilers to william (and keep a copy of it here in petcompil).
{ :0 c
:0 petcompil }
:0 c
:0 A petcompil
:0 c
petcompil If you are fairly new to procmail and plan to experiment a little bit it often helps to have a safety net of some sort. Inserting the following two recipes above all other recipes will make sure that of all arriving mail always the last 32 messages will be preserved. In order for it to work as intended, you have to create a directory named `backup' in $MAILDIR prior to inserting these two recipes.
:0 c
backup :0 ic | cd backup If your system doesn't generate or generates incorrect leading `From ' lines on every mail, you can fix this by calling up procmail with the -f- option. To fix the same problem by different means, you could have inserted the following two recipes above all other recipes in your rcfile. They will filter the header of any mail through formail which will strip any leading `From ', and automatically regenerates it subsequently.
:0 fhw
| formail -I Add the headers of all messages that didn't come from the postmaster to your private header collection (for statistics or mail debugging); and use the lockfile `headc.lock'. In order to make sure the lockfile is not removed until the pipe has finished, you have to specify option `w'; otherwise the lockfile would be removed as soon as the pipe has accepted the mail.
:0 hwc:
| uncompress headc.Z; cat
:0 hwc:
| gzip Forward all mails shorter than 1000 bytes to my home address (no lockfile needed on this recipe).
* Split up incoming digests from the surfing mailing list into their individual messages, and store them into surfing, using surfing.lock as the locallockfile.
| formail +1 -ds Store everything coming from the postmaster or mailer-daemon (like bounced mail) into the file postm, using postm.lock as the locallockfile.
postm A simple autoreply recipe. It makes sure that neither mail from any daemon (like bouncing mail or mail from mailing-lists), nor autoreplies coming from yourself will be autoreplied to. If this precaution would not be taken, disaster could result (`ringing' mail). In order for this recipe to autoreply to all the incoming mail, you should of course insert it before all other recipes in your rcfile. However, it is advisable to put it after any recipes that process the mails from subscribed mailinglists; it generally is not a good idea to generate autoreplies to mailinglists (yes, the !^FROM_DAEMON regexp should already catch those, but if the mailinglist doesn't follow accepted conventions, this might not be enough).
:0 h c
| (formail -r -I A more complicated autoreply recipe that implements the functional equivalent of the well known vacation(1)? program. This recipe is based on the same principles as the last one (prevent `ringing' mail). In addition to that however, it maintains a vacation database by extracting the name of the sender and inserting it in the vacation.cache file if the name was new (the vacation.cache file is maintained by formail which will make sure that it always contains the most recent names, the size of the file is limited to a maximum of approximately 8192 bytes). If the name was new, an autoreply will be sent.
As you can see, the following recipe has comments between the conditions. This is allowed. Do not put comments on the same line as a condition though.
SHELL=/bin/sh # for other shells, this might need adjustment
:0 Whc: vacation.lock
Store all messages concerning TeX in separate, unique filenames, in a directory named texmail (this directory has to exist); there is no need to use lockfiles in this case, so we won't.
texmail The same as above, except now we store the mails in numbered files (MH mail folder).
texmail/. Or you could file the mail in several directory folders at the same time. The following recipe will deliver the mail to two MH-folders and one directory folder. It is actually only one file with two extra hardlinks.
texmail/. wordprocessing dtp/. Store all the messages about meetings in a folder that is in a directory that changes every month. E.g. if it were January 1994, the folder would have the name `94-01/meeting' and the locallockfile would be `94-01/meeting.lock'.
`date +%y-%m`/meeting
MONTHFOLDER=`date +%y-%m`
:0 Wic
| mkdir $MONTHFOLDER :0:
MONTHFOLDER=`date +%y-%m`
DUMMY=`test -d $MONTHFOLDER || mkdir $MONTHFOLDER` :0:
${MONTHFOLDER}/meeting If you are subscribed to several mailinglists and people cross-post to some of them, you usually receive several duplicate mails (one from every list). The following simple recipe eliminates duplicate mails. It tells formail to keep an 8KB cache file in which it will store the Message-IDs of the most recent mails you received. Since Message-IDs are guaranteed to be unique for every new mail, they are ideally suited to weed out duplicate mails. Simply put the following recipe at the top of your rcfile, and no duplicate mail will get past it.
:0 Wh: msgid.lock
| formail -D 8192 msgid.cache Beware if you have delivery problems in recipes below this one and procmail tries to requeue the mail, then on the next queue run, this mail will be considered a duplicate and will be thrown away. For those not quite so confident in their own scripting capabilities, you can use the following recipe instead. It puts duplicates in a separate folder instead of throwing them away. It is up to you to periodically empty the folder of course.
:0 Whc: msgid.lock
| formail -D 8192 msgid.cache :0 a: duplicates Procmail can deliver to MH folders directly, but, it does not update the unseen sequences the real MH manages. If you want procmail to update those as well, use a recipe like the following which will file everything that contains the word spam in the body of the mail into an MH folder called spamfold. Note the local lockfile, which is needed because MH programs do not lock the sequences file. Asynchronous invocations of MH programs that change the sequences file may therefore corrupt it or silently lose changes. Unfortunately, the lockfile doesn't completely solve the problem as rcvstore could be invoked while `show' or `mark' or some other MH program is running. This problem is expected to be fixed in some future version of MH, but until then, you'll have to balance the risk of lost or corrupt sequences against the benefits of the unseen sequence.
:0 :spamfold/$LOCKEXT
| rcvstore +spamfold When delivering to emacs folders (i.e., mailfolders managed by any emacs mail package, e.g., RMAIL or VM) directly, you should use emacs-compatible lockfiles. The emacs mailers are a bit braindamaged in that respect, they get very upset if someone delivers to mailfolders which they already have in their internal buffers. The following recipe assumes that $HOME equals /home/john.
mailbox Alternatively, you can have procmail deliver into its own set of mailboxes, which you then periodically empty and copy over to your emacs files using movemail. Movemail uses mailbox.lock local lockfiles per mailbox. This actually is the preferred mode of operation in conjunction with procmail.
To extract certain headers from a mail and put them into environment variables you can use any of the following
SUBJECT=`formail -xSubject:` # regular field
FROM=`formail -rt -xTo:` # special case :0 h # alternate method KEYWORDS=| formail -xKeywords:
The following recipe prints every incoming mail that looks like a postscript file.
:0 Bb
| lpr The following recipe does the same, but is a bit more selective. It only prints the postscript file if it comes from the print-server. The first condition matches only if it is found in the header. The second condition only matches at the start of the body.
:0 b
| lpr
{ :0 B b
| lpr }
:0 HB b
| lpr Suppose you have two accounts, you use both accounts regularly, but they are in very distinct places (i.e., you can only read mail that arrived at either one of the accounts). You would like to forward mail arriving at account one to account two, and the other way around. The first thing that comes to mind is using .forward files at both sites; this won't work of course, since you will be creating a mail loop. This mail loop can be avoided by inserting the following recipe in front of all other recipes in the $HOME/.procmailrc files on both sites. If you make sure that you add the same X-Loop: field at both sites, mail can now safely be forwarded to the other account from either of them.
:0 c
| formail -A If someone sends you a mail with the word `retrieve' in the subject, the following will automatically send back the contents of info_file to the sender. Like in all recipes where we send mail, we watch out for mail loops.
| (formail -r ; cat info_file) | $SENDMAIL -oi -t Now follows an example for a very simple fileserver accessible by mail. For more demanding applications, I suggest you take a look at !SmartList? (available from the same place as the procmail distribution). As listed, this fileserver sends back at most one file per request, it ignores the body of incoming mails, the Subject: line has to look like __
{ MAILDIR=$HOME/fileserver # chdir to the fileserver directory :0 fhw # reverse mailheader and extract name
| formail -rA The following example preconverts all plain-text mail arriving in certain encoded MIME formats into a more compact 8-bit format which can be used and displayed more easily by most programs. The mimencode(1)? program is part of Nathaniel Borenstein's metamail package.
{ :0 fbw
| mimencode -u -q :0 Afhw | formail -I The following one is rather exotic, but it only serves to demonstrate a feature. Suppose you have a file in your HOME directory called
URGMATCH=`cat $HOME/.urgent`
cat newtext | procmail ./mimeconvert | mail email@example.com
DEFAULT=| # pipe to stdout instead of
:0 Bfbw
| mimencode -q :0 Afhw | formail -I 'MIME-Version: 1.0' \
procmail(1), procmailrc(5), procmailsc(5), sh(1), csh(1), mail(1), mailx(1), binmail(1)?, uucp(1)?, aliases(5), sendmail(8), egrep(1), grep(1), biff(1), comsat(8), mimencode(1)?, lockfile(1), formail(1)
Stephen R. van den Berg
Philip A. Guenther
5 pages link to procmailex(5):


text:
THE BLACKLIST (2013) TV Show
THE BLACKLIST (2013) TV Show is a High Resolution Wallpaper free for download. You able to download THE BLACKLIST (2013) TV Show widescreen wallpaper free for your desktop background, tablet, and smartphone device for free in variety size below.
THE BLACKLIST (2013) TV Show - HD Background
- Credit to original photo uploader
THE BLACKLIST (2013) TV Show - HD Wallpapers
Tags :
Categories : movies
Added : Tuesday, September 24th, 2013
Download THE BLACKLIST (2013) TV Show in high resolution for free. Get THE BLACKLIST (2013) TV Show and make this THE BLACKLIST (2013) TV Show for your desktop, tablet, and smartphone device.To set this THE BLACKLIST (2013) TV Show as wallpaper background on your desktop, click on the 'Set as Wallpaper' link below to save HD Wallpaper on your desktop computer and then right click on the Wallpaper Image and select the option "Save image as" or "Set As Background".THE BLACKLIST (2013) TV Show
Related Posts
DISCLAIMER: All wallpapers and backgrounds found here are believed to be in the "public domain". All of the images displayed are of unknown origin. We do not intend to infringe any legitimate intellectual right, artistic rights or copyright. If you are the rightful owner of any of the pictures/wallpapers posted here, and you do not want it to be displayed or if you require a suitable credit, then please contact us and we will immediately do whatever is needed either for the image to be removed or provide credit where it is due. All the content of this site are free of charge and therefore we do not gain any financial benefit from the display or downloads of any images/wallpaper.


text:
Main News Page
Monday, January 2, 2012
More items for
Environmental Controls
Dozens of older U.S. power plants will have to clean up or shut down under the first-ever national standards aimed at reducing their toxic air pollutant emissions.
New Mercury and Air Toxics Standards (MATS), just released by the Environmental Protection Agency, are designed to slash mercury, arsenic, metals, arsenic, acid gas, nickel, selenium, cyanide and other toxic air pollutants emitted by coal- and oil-fired power plants.
Wikimedia Commons
The plants are the largest remaining source of uncontrolled toxic air pollution in the United States.
EPA issued the long-awaited rule Dec. 21, saying it would "slash emissions of these dangerous pollutants by relying on widely available, proven pollution controls that are already in use at more than half of the nation's coal-fired power plants." Overdue Rule
The agency called the rule long overdue, noting that 11 years had passed since EPA issued a scientific and legal determination that mercury emissions from power plants should be controlled—and that more than 20 years had passed since Congress passed the 1990 Clean Air Act amendments authorizing EPA to control toxic air pollutants from plant smokestacks.
EPA was also bound by a Consent Decree deadline issued by the D.C. Court of Appeals, which struck down an effort by the administration of President George W. Bush to exempt the power plants from toxic air pollutant controls.
Public Health Imperative
EPA Administrator Lisa Jackson announced the new rule at a press conference at Children's National Medical Center in Washington, D.C., underscoring the agency's view that the issue is a public health imperative.
When fully implemented in 2016, the rule will reduce emissions from coal by 90 percent, acid gases by 88 percent, and soot-producing sulfur dioxide by 41 percent.
EPA estimates that the new standards will prevent up 11,000 premature deaths, 4,700 heart attacks, 130,000 cases of childhood asthma symptoms, and about 6,300 fewer cases of acute childhood bronchitis each year.
"By cutting emissions that are linked to developmental disorders and respiratory illnesses like asthma, these standards represent a major victory for clean air and public health—and especially for the health of our children," said Jackson, who called the rule President Obama's "biggest clean air action yet."
"The Mercury and Air Toxics Standards will protect millions of families and children from harmful and costly air pollution and provide the American people with health benefits that far outweigh the costs of compliance." Clean Up or Shut Down
Power plants are responsible for half of the mercury and over 75 percent of the acid gas emissions in the United States.
The new rule will force plant operators to install pollution control equipment, switch to cleaner-burning natural gas, or shut down. The rule is expected to hit hardest in the Midwest and in the coal belt—Kentucky, West Virginia and Virginia—where dozens of units probably will be mothballed, the Associated Press reported.
More than 32 mostly coal-fired power plants in a dozen states will likely shut down, and 36 might have to close, said the AP, which surveyed electricity generators and EPA officials on the rule's impact.
On the other hand, although hundreds of the nation's 1,200 coal- and oil-fired units lack modern pollution controls, most will find ways to comply and continue to run, the AP's analysis showed.
At $9.6 billion a year, the rule will be one of the costliest ever issued by EPA. However, the agency contends that the American public will see up to $9 in health benefits for every dollar spent on implementation.
Jobs Impact
EPA received more than 900,000 public comments during the rule-making process—ensuring, the agency said, that the resulting standards focus on "readily available and widely deployed pollution control technologies" manufactured by U.S. companies.
Sensitive to chronic political criticism that equates government standards with job loss, EPA insisted that manufacturing, engineering, installing and maintaining the pollution controls to meet the standards would "provide employment for thousands" of people, "potentially including 46,000 short-term construction jobs and 8,000 long-term utility jobs." 4 Years to Comply
The rule sets technology-based emissions limit standards for mercury and other toxic air pollutants, reflecting levels achieved by the best-performing sources now in operation.
The final rule sets standards for all Hazardous Air Pollutants emitted by coal- and oil-fired Electric Generating Units (EGUs) with a capacity of 25 megawatts or greater. All regulated EGUs are considered major under the final rule.
Existing sources generally will have up to four years to comply with MATS. Additional implementation time may be granted on a case-by-case basis.
Cheers: 'The Right Thing'
The rule drew cheers from urban and public-health officials.
Albert A. Rizzo, MD, of the American Lung Association, called the standards "a huge victory for public health."
New York City Mayor Michael Bloomberg said Obama had "done the right thing by ignoring the false claims of a narrow special interest and siding with the public health and the public good. The new EPA mercury standards will save countless lives and improve the quality of life for millions."
Alan Baker, of the American Public Health Association, said the "critically needed standards could mean the difference between a chronic debilitating, expensive illness or healthy life for hundreds of thousands of American children and adults."
He added: "The dangerous health risks associated with coal-burning power plants is no longer an elusive, distant threat." Jeers: 'Overly Aggressive'
Manufacturing and conservative groups blasted the rule, predicting energy price spikes and shortages.
The National Association of Manufacturers accused EPA of pursuing "an overly aggressive agenda that is harming manufacturers' ability to compete."
"Electricity prices will go up with this regulation, impacting many consumers nationwide," NAM said in a statement.
"Manufacturers use one-third of our nation's energy supply, so a jump in energy prices will have a devastating impact on companies of all sizes, harming their ability to create jobs, invest and grow."
The American Coatings Association had no immediate comment.
Rep. Darrell Issa, R-CA, a leading Obama critic, said the rule could "unintentionally jeopardize the reliability of our electric grid," while the U.S. Chamber of Commerce released an ad saying the rule "could threaten America's energy supply."
The conservative National Center for Public Policy Research was livid, saying Obama had given "hard-working families higher electricity prices for Christmas."
Keeping Coal
Like it or not, however, the rule is unlikely to unseat coal as the most common electricity source.
"In the industry, we retire units. That is part of our business," John Moura, manager of reliability assessment at the North American Electric Reliability Corp., which represents the nation's electrical grid operators.
On the other hand, the rule could accelerate the retirement crunch, and hundreds of units will need to be idled temporarily to install controls, officials note. NERC is requesting more time to comply with the rules, to avoid too many simultaneous shutdowns.
"We are getting a little hammered here, because we see multiple requests," Moura told the AP.
Comment from Tom Schwerdt, (1/9/2012, 2:18 PM)
The industry has been allowed to hang onto old, highly polluting plants for decades without any remediation - sounds like it is finally time to address the issue.
Comment from James Johnson, (1/11/2012, 10:58 AM)
When one checks the information from the National Atmospheric Deposition Program it is readily seen the power industry year by year has been reducing emmissions for quite some time. In fact there was a 2020 target set for emmissions over a decade ago and the current day emmissions have already met that target level, but we do not hear of that because it does not suit the political agenda. As obama stated when campaigning for office - coal will not be outlawed but I am going to make it too costly to use to force the nation to green energy. This new regulation is right in line with the political agenda. Perhaps he should have checked out how the green energy agenda is working in France. It is bankrupting the nation there.
Join the Conversation:
The one way to control a tank: DRYCO
Temporary climate control equipment.
Dehumidifiers, Heaters, A/C, Power, More…
Call: 866-379-2600
Join SSPC and Enhance Your Career !
Profession Overspray Removal Services
The Finest Name in Safe Overspray Removal. We specialize in removing all contaminants from any surface. Family Owned and Operated for 33 years! Fully insured. Satisfaction is guaranteed.
Built from the ground up.
Dust Collectors Available!
We deliver to your Jobsite!
Built Tough! Priced Right!
Sherwin-Williams Protective & Marine Coatings
With 4,000 distribution points and 3,700+ years of experience, Sherwin-Williams delivers the products, support and expertise you need, right where you need it.
From Detection to Correction
Corrosion Probe, Inc. has the Most Concrete Coatings Experience and Expertise. Contact us today at
860-767-4402, www.cpiengineering.com
BLACK BEAUTY® App
Download the free app
for BLACK BEAUTY® Abrasives.
Explore all the features from
the palm of your hand.
Toll Free: 1-888-733-3646
SIMPLY the BEST In Wet Abrasive Blasting
Automated controls simplify operation. 100lb. media capacity & enclosed water tank, for hours of continuous blasting. Dry weight of 350 lbs. & 150 psi capable.
Number 1 Crushed Glass Abrasive in America!
Manufactured from 100% Recycled Glass. NO Crystalline Silica, Beryllium & Arsenic
Great White Metal Finish! 4 plants on Navy QPL
Call us at 866-775-6226
US Minerals - Black Magnum
With nationwide production and distribution capabilities, U.S. Minerals supplies a complete range of coal slag and other abrasives to meet all of your blasting needs.
The Technology Publishing Network
© Copyright 2000-2015,
Technology Publishing / PaintSquare, All rights reserved
2100 Wharton Street, Suite 310, Pittsburgh PA 15203-1951; Tel 1-412-431-8300; Fax 1-412-431-5428;
E-mail firstname.lastname@example.com


text:
If you like this, Harlequin recommends
- The Bridegroom's Bargain
- by
- Jan 2012
- Francesca
- by
- Aug 2009
- Governess Brides Bundle
- by
- Apr 2008
- Reawakening Miss Calverley
- by
- Oct 2014
The Rake's Proposal
No society beauty had been able to tempt Lord Ivo Trenchard to give up the single life. None, that was, until he met an enchanting innocent--in boy's clothes--who challenged him to teach her how to kiss!
He watched in amazement as the wild tomboy transformed into a stunningly elegant lady who took all of London by storm. His offer of marriage took them both by surprise.Was he merely protecting Joscelin Morley from a threatening scandal--or was she his true choice, his only love?


text:
Complete this form to request that a news article be published on the History website. Fields with asterisks are required:
History in
The College of Liberal Arts at UT
Art Credits | Information on Copyright | Privacy | Web Accessibility | Send CommentsPDF files require Adobe Acrobat Reader software to view.
All architectural drawings are from the Alexander Architectural Archive at the University of Texas at Austin, sheets 6, 13, 14, and 17. The logo was inspired by a detail from the drawings. Images are either copyrighted by the University or used under fair use laws. Any unauthorized use or duplication is prohibited.


text:
Christina Cooks
Family Faves MakeoverSeries Website »
Episode Guide »
Mashed potatoes, baked beans, French toast...guaranteed to get the family to sit down together. But these 'sit down' foods are usually loaded with calories, fat and simple carbs. I'll show you how to lighten 'em up and still have the family run to the table!


text:
What The British Say And What They Mean
Photo: Today I Learned Something New
I checked the office twice, no one here is British so I can't confirm exactly how accurate this is. That being said, I am picturing all my favorite English movies and characters and it plays out pretty well. Like Simon Pegg from Shaun of the Dead or Stanley Kubrick from A Clockwork Orange or even Harry Potter from.. well you know. I don't know about you guys but I read this out loud (without thinking) and in my best British accent. My favorite line is probably "could we consider some other options."
Any English followers out there that can confirm this guide?
Newest 5 Comments
Yeah, I would say the first two columns are accurate... the third worries me slightly. I may have to re-evaluate how I speak to some people
Yes, this list is fairly accurate - the tone of voice is the important thing, British people use a sort of dry, understated humour in, uh, well, most conversations really. If the tone of voice sounds ever so slightly like it might be being said by Batman's Butler Alfred, it will usually mean the opposite of the literal meaning. Having lived with Americans, I always found this was the thing that perplexed them, not the superficial linguistics differences (pants vs trousers, lift vs elevator, etc).
For an excellent guide on this kind of thing, have a leaf through Kate Fox's book 'Watching The English' - it's readable anthropology - not bad at all!
For an excellent guide on this kind of thing, have a leaf through Kate Fox's book 'Watching The English' - it's readable anthropology - not bad at all!
It's usually best if people just say what they mean.
I'm British and I say most of this list is utter bollocks. What I mean is, most of this list is utter bollocks. What others should undertand is, most of this list is utter bollocks.
An Australian version of this is needed including "Your Shout", "Yer nar" and the reversal of "Mate' and "Cu#t".


text:
www.toubalondon.comThe TramperyStudio 12125-127 Mare StreetE8 3RH
Touba London (Men's) Biography
Touba London (Men's) Lookbook
Touba London (Men's) Press Release 1
Representation, Distribution and Public Relations activities are the three main areas of Touba London.
Touba London provides a showcase for International and British designers & artists to display their creativity and talent in all areas of fashion, photography and artwork by curating Showroom presentation and photographic and artwork exhibition in London, Paris and Tokyo.
Touba London is also a representative, principal distributor and agent to established and exciting new International and British designers.
Public Relation activities are also one main activities in Touba London increasingly eclectic, vibrant and wide ranging endeavour.
Yuko firstname.lastname@example.com: +44 (0) 20 8541 5447Mobile: +44 (0) 7973 429177
25 September - 05 October 201309:00 - 21:00


text:
Devils win streak reaches fourThe Associated Press | February 10,2013NEWARK, N.J. Adam Henrique and Bobby Butler scored power-play goals 2:12 apart in the third period, and the New Jersey Devils extended their winning streak to four games with a 3-1 victory over Pittsburgh on Saturday that snapped the Penguins five-game winning streak.
Ilya Kovalchuk assisted on the late goals, and defenseman Andy Greene had two assists, including the primary one on rookie Stefan Matteaus first NHL goal.
Martin Brodeur made 24 saves and provided a little humor by chasing his stick late in the second period of his 1,200th career game. He became the first NHL goalie to reach the milestone.
Penguins captain Sidney Crosby had a six-game point streak snapped, his longest since a career-high, 25-game run in 2010.
Brandon Sutter scored early for the Penguins, who gave the Devils 10 power-play chances. The teams will play again Sunday in Pittsburgh.
PHILADELPHIA Danny Briere scored 1:47 into overtime to lift Philadelphia over Carolina.
Jakub Voracek, Matt Read and Brayden Schenn also scored for Philadelphia, which went 3-0-1 on a four-game homestand.
Joe Corvo scored the tying goal in the third period, and Jiri Tlusty and Jeff Skinner also had goals for the Hurricanes, who had won two in a row.
The Flyers have gone 24-4-4 against the Hurricanes since Nov. 28, 2003. Philadelphia is 5-6-1 at the quarter point of the lockout-shortened 48-game schedule. Carolina fell to 5-4-1.
Ilya Bryzgalov made 30 saves in the victory and earned his third assist of the season.
DETROIT Niklas Kronwalls third-period goal broke a tie, and Pavel Datsyuk had a goal and an assist in Detroits narrow win over Edmonton.
Henrik Zetterberg had two assists, and Jimmy Howard made 23 saves for Detroit (6-4-1).
Rookie defenseman Justin Schultz scored the lone goal for Edmonton (4-4-3), and 40-year-old Nikolai Khabibulin stopped 28 shots in his first game of the season
The Red Wings killed off a two-man disadvantage that lasted 1:07 early in the third period.
OTTAWA Al Montoya made 33 saves, and Alex Ponikarovsky scored the only goal in Winnipegs win over Ottawa.
Ponikarovsky broke the scoreless deadlock 5:57 into the third period. The Senators had no answer against Montoya, who replaced an ailing Ondrej Pavelec (flu) and posted his first shutout of the season and third of his NHL career.
The Jets (5-5-1) won for the second time in six games and snapped a three-game road skid.
Ottawa backup Ben Bishop made his second start of the season and was solid in stopping 36 shots. The Senators (6-4-2) have scored just seven goals in past five games, including four in a 4-3 win over the Buffalo Sabres on Tuesday.
The Senators have lost a pair of 1-0 decisions in that span. Ottawa has allowed only 15 goals in eight games.
BOSTON The game between the Tampa Bay Lightning and the host Bruins was postponed because of the blizzard that dumped more than 2 feet of snow on the Boston area.
No makeup date had been scheduled yet. Originally scheduled for 1 p.m., the game was pushed back until 7 p.m. to allow the storm to pass. But public transportation was not expected to resume on Saturday, and roads were still being cleared.
SAN JOSE, Calif. Mike Smith made 33 saves through overtime and then turned aside two more shots in the shootout to carry Phoenix past San Jose.
The only offense in the entire game was provided by Phoenixs Mikkel Boedker and Radium Vrbata, who both netted shootout goals against Sharks goalie Antii Niemi.
Smith, who recorded his fourth shutout of the season, made several acrobatic saves, including one in the shootout. Smith has 21 career NHL shutouts.
Niemi stopped 21 shots before the shootout to earn his first shutout of the season and 20th in the NHL.
Smith improved to 14-19 in shootouts, and Niemi fell to 19-13.
San Jose, which has gone to a shootout in four of six games, dropped its second straight tiebreaker.
Smith, who shut out the Sharks three times last season, improved to 6-3-1 in his career against them.MORE IN Wire SportsBOSTON The Boston Red Sox will have to wait a little longer to start rebuilding their team. Full StoryGAINESVILLE, Va. Full StoryTURNBERRY, Scotland If the first day of the Womens British Open was all about Donald Trump,... Full Story
- Most Popular
- Most Emailed
- RICHARD'S POOR ALMANACK: On this day 1739, 'Richard Palmer' identified in prison at York Castle as the notorious outlaw DICK TURPIN; IN 1836, Battle of the Alamo begins near San Antonio de Bexar, Texas; 1896, the Tootsie Roll invented by LEO HIRSCHFELD.
- RICHARD'S POOR ALMANACK: On this day in 1472, Orkney, Shetland islands put up as collateral by Norway to Scotland in lieu of dowry for MARGARET OF DENMARK on her marriage with JAMES III, king of Scotland; 1962, JOHN GLENN first American to orbit Earth.
- TOMORROW'S HEADLINES TODAY: City mayoral candidates debate campaign issues; Hartford, Conn., woman still missing; Neal Goswami reports attempts to legislate suicide; local woman loses 100 pounds through TOPS program.
- RICHARD'S POOR ALMANACK: On this day in 1878, JOHN TUNSTALL murdered near Lincoln, New Mexico, by the outlaw JESSE EVANS; in 1930, ELM FARM OLLIE first cow to fly in aircraft, first to be milked airborne; 1955, nuke test WASP; '79, snow in Sahara.
- TOMORROW'S HEADLINES TODAY: Rutland Herald News Editor Alan J. Keays and staff writer Gordon Dritschilo discuss stories planned for the February 18, 2015, edition of the newspaper: Winter budgets maxed, legal marijuana, Springfield bank job, USPS slowdown
- RICHARD'S POOR ALMANACK: On this day in 1249 AD, ANDRE of LONGJUMEAU is dispatched by LOUIS IX of France to meet the KHAGAN, ruler of the Mongol Empire; in 1804, during 1st Barbary War, STEPHEN DECATUR scuttles the pirate-held USS Philadelphia in Tripoli.


text:
DuPont introduced nylon to the world at the 1939 New York World's Fair. Now, over 70 years later, DuPont has commercialized the most advanced fiber platform since that breakthrough moment. Sorona® renewably sourced fiber is made, in part, with renewably sourced (biobased) ingredients* instead of petrochemicals, thereby helping reduce our dependency on oil. From durable carpets to high-performance fabrics, the unique properties of Sorona® make it ideal for a range of applications. Learn more at sorona.dupont.com
Together with The Goodyear Tire & Rubber Company, we are co-developing an integrated process to manufacture an alternative to petroleum-based isoprene at industrial scale. BioIsoprene™ monomer will enable Goodyear to produce high quality synthetic rubber for tires without dependence on petroleum-based feedstock.
As BioIsoprene™ monomer is produced from renewable raw materials, manufacturers in many industries will be able to count on a reliable, consistent supply that is not subject to fluctuating oil prices. This creates tremendous opportunities for growth across a wide range of industrial applications – from adhesives and specialty elastomers and surgical gloves, to advanced transportation and jet fuels.
* Sorona® contains 37% annually renewable plant based ingredients by weight (28% biobased carbon)


text:
MINK Baseball League Expands to Branson
It may be Major League Baseball Playoff time, but there was big news from the MINK League this weekend. The MINK League held their annual meeting last weekend in St. Joseph and announced that the Branson Nationals will be a new team coming to the league. This is the first time a new team has come into the league since the Sedalia Bombers and St. Joseph Mustangs came on board in 2009.


text:
Gear Review
RalphSSter From St. louis
I have had these brakes on my single speed for about 7 months now and I won't install any other brake on my bikes. The stopping power and overall weight of them is second to none. I highly recommend them!
View More Details on Shimano XTR BR-985 Race Brake


text:
Now Playing
Loading streams...
Connect with Us
Most Active Stories
Reporter & Morning Edition Host
Reporter & All Things Considered Host
Programming & Operations Director, Here & Now Host
Host, Folktales & Meet the Music
Host, Matinee and Con Brio
Alice Fordham
Alice Fordham is an NPR International Correspondent based in Beirut, Lebanon.
In this role, she reports on Lebanon, Syria and many of the countries throughout the Middle East.
Before joining NPR in 2014, Fordham covered the Middle East for five years, reporting for The Washington Post, the Economist, The Times and other publications. She has worked in wars and political turmoil but also amid beauty, resilience and fun.
In 2011, Fordham was a Stern Fellow at the Washington Post. That same year she won the Next Century Foundation's Breakaway award, in part for an investigation into Iraqi prisons.
Fordham graduated from Cambridge University with a Bachelor of Arts in Classics.


text:
Students are looking for jobs. Employers are looking for students.
Make the connection with the Summer Jobs Service Program!
This Employment Ontario service is funded by the Ontario government.
Domtar Inc., Dryden Operations, is currently seeking an
experienced individual to become part of one of our multi
skilled maintenance teams while contributing to our ongoing
success. A progressive community set between Winnipeg and
Thunder Bay, Dryden offers excellent medical, educational and
recreational facilities.
As a qualified Pipefitter, you have successfully completed Grade 12 or community College Certification, and will have Pipefitter certification with at least 5 years of experience. Experience in the Pulp and Paper industry will be considered an asset. Welding accreditation would be advantageous.
Reporting to the Maintenance Supervisor, your responsibilities
• You have a safety-oriented mindset with the focus on mill safety and environmental policies and procedures.
• Layout, fabricate and construct industrial process and pressure piping including hydraulic, pneumatic in both stainless and mild steel.
• Troubleshoot and perform repairs to hydraulic and pneumatic
• Must be able to interpret schematics as well as machine detail drawings
If you are interested in an opportunity to work with an organization that is an Equal Opportunity Employer and offers a full range of employee benefits and a competitive wage package, consider joining our Team. Please forward your resume, in confidence, by December 14, 2012 to:
Erika Pouru, Domtar Inc.,
Duke Street, Dryden, Ontario P8N 2Z7
or e-mail: firstname.lastname@example.com.
We would like to thank all applicants for their interest; however, only those candidates selected for interviews will be contacted.


text:
Head Coach Andy Fleming
Aug 16, 2013
CINCINNATI – The No. 22 Xavier men's soccer team, led by Preseason All-BIG EAST selections Nick Hagglund (Cincinnati, Ohio/Lakota West HS) and Matt Walker (Batavia, Ohio/Batavia HS), opens the fall season with an exhibition match against Belmont on Sunday night (7 p.m.) at the XU Soccer Complex. The Musketeers then head to Fort Wayne, Ind. for their final exhibition match, where they will face Loyola (IL) in the Shindigz National Soccer Festival on Aug. 23 at 8 p.m.
"It will be a nice change of pace to play against another team and to get a gauge on where we are at in all facets of our program," said head coach Andy Fleming. "For me an exhibition game covers everything from the warmup, to the pregame meal, our game day operations and so many other small factors that the naked eye doesn't see."
"Like most teams, we will come away with some things to work on and some areas that we might have previously addressed," Fleming said. "We will be missing several key players due to nagging injuries and will look at several different combinations. Our new guys will get plenty of playing time as we use this game to develop our depth and make a small step forward towards our opener."
Xavier is coming off a banner 2012 season (14-3-5 overall), which included the program's first NCAA Tournament at-large bid, tourney victory and third-straight appearance into the 48-team field. Additionally, the Musketeers posted a program-record 14 victories and set new marks for assists and total points. XU's final loss of the season came against Indiana, the eventual 2012 national champion. Individually, Luke Spencer became program's the first MLS Draft Pick (23rd pick, Second Round; New England Revolution) and Hagglund earned XU's first NSCAA All-American award. Outscoring the opposition 31-8 with four shutouts during the 2012 campaign, Xavier finished with a 9-0-1 record on their home pitch. Dating back to the 2011 season, the Musketeers has gone 14-1-2 over their last 17 home contests and are currently riding a 13-match home unbeaten streak (11-0-2).
Xavier's returning 17 players, including eight starters from last season, along with nine newcomers will navigate a 2013 slate that features matchups against six 2012 NCAA Tournament teams, including two that advanced to the Final Four. The Blue and White open the regular season on Friday, Aug. 30 (5 p.m.) when they battle Furman on day one of the College of Charleston tournament.


text:
Basketball Tournament
Cheverus fends off Portland's upset bid
PORTLAND—The Portland boys' basketball team, heavy underdogs after losing twice to Cheverus this regular season by an average of 24 points, had the mighty, undefeated, top-ranked, defending state champion Stags on the ropes Wednesday evening in a Western Class A semifinal at the Cumberland County Civic Center, but couldn't finish the job.
The fifth-ranked Bulldogs, who overcame a dreadful start and an early 11-0 deficit, actually led by five points with just over five minutes to go, but sloppy play and a renewed commitment by Cheverus turned things around.
The Stags, led by senior Griffin Brady and junior Louie DiStasio, showed their championship heart and went on a 10-0 run to seize control, then managed to hold on for a 45-41 victory to improve to 20-0, send Portland home at 12-8 and set up a regional final showdown with No. 2 Bonny Eagle (17-3) in the Western A Final Saturday at 9 p.m., at the Civic Center.
"I think that's the worst we've played in a long time, but I thought we showed an immense amount of courage at the end," said Cheverus coach Bob Brown. "It's a 32-minute ballgame, thank God."
Surprise, surprise
Cheverus, the defending Class A state champion, was dominant in the regular season, winning all 18 games with varying degrees of ease. In the quarterfinals Friday, however, the top-ranked Stags were pushed before eliminating No. 8 Marshwood, 52-40.
Portland completely turned what appeared to be a lost season around, winning its final five games to make the playoffs as the No. 5 seed. Friday, the Bulldogs led No. 4 South Portland virtually the whole way in a stunning 69-45 rout.
The Stags had no trouble with the Bulldogs this year, winning, 49-28, at the Expo Dec. 23 and 70-43 at home Jan. 29. Cheverus had beaten Portland seven straight times dating to Feb. 5, 2008 (a 37-31 Bulldogs' triumph at the Expo).
The teams had met 21 times in the postseason dating back to 1932, with the Bulldogs holding an 11-10 edge. The last meeting was in the 2008 semifinals, a 63-49 Cheverus victory.
Wednesday, it looked initially as if the Stags would cruise again, but in a microcosm of its season, Portland dug out of a hole and dazzled everyone on hand with its resilience and will.
Just 15 seconds in, Cheverus sophomore Matt Cimino, who seemed no worse for wear after returning from Friday's knee injury, made a layup. While the Bulldogs were turning the ball over left and right, the Stags raced to an 8-0 lead behind a long jumper from Cimino. two DiStasio free throws and a layup by senior Peter Gwilym (from senior Joey Savino), forcing Bulldogs coach Joe Russo to call timeout.
At that juncture, with 3:48 still to play in the first quarter, Portland had turned the ball over seven times.
It didn't get much better the rest of the period as the Stags took an 11-0 advantage as Gwilym fed Cimino for a layup and junior Cam Olson made a free throw.
Finally, with 56.1 seconds showing in the opening stanza, the Bulldogs got on the board when junior Peter Donato made two free throws.
While Portland shot itself in the foot repeatedly in the first eight minutes, Cheverus could have had a much bigger lead.
"The start didn't bother us a bit," Russo said. "We talked how well we played defense. I said, 'Boys, it's 11-2. That's all they have is 11 points for eight minutes. Stick with your game plan and keep your composure.' They did and we got back."
Portland turned things around in the second quarter and made it a game by halftime.
After freshman Jayvon Pitts-Young, who for the second straight playoff game gave his team a tremendous spark off the bench, hit two foul shots, senior Connor O'Neil answered with one for the Stags to make it 12-4.
The Bulldogs then rattled off six straight points as Donato scored on a putback, sophomore Nate Smart scored on a driving layup and Pitts-Young hit two free throws to make it a 12-10 game. DiStasio answered with two foul shots to end a 4 minute, 20 second drought.
Then, with 1:36 to go before halftime, a strange situation developed.
Pitts-Young was fouled by Savino (his third) and went to the line for two shots. He missed the first, Cheverus grabbed the rebound and raced down for a layup by Gwilym.
Problem was, it was supposed to be a two-shot foul.
By rule, since the clock didn't start and the basket was scored, the officials had to count it and the Stags, even though no time had elapsed, had extended their lead to 16-10.
"That was a weird thing," Russo said. "We were yelling for two shots. Because Cheverus made the hoop, they couldn't take it away. That's how they had to administer it. It was a miscue on their part, but we got over it."
Pitts-Young then got his second free throw and made it. Junior Mike Herrick then got on the board for Portland to pull the Bulldogs within 16-13 at the break.
"We had a rough second period," DiStasio said. "We put them on the line."
Brown was frustrated with the stagnation of his offense in that period.
"I think we executed offensively as badly as you can," Brown said. "Nothing that we were supposed to do got done."
When Herrick opened the third quarter with two free throws and freshman Justin Zukowski drained a 3 with 5:19 to play, the Bulldogs had a stunning 18-16 lead.
Cheverus fought back to go up, 20-18, on a layup from senior Griffin Brady (after a DiStasio steal) and a pair of Brady free throws. Portland tied the score on a leaner from Herrick, but Brady converted an old-fashioned three-point play (layup, foul, free throw) to make it 23-20 Stags.
Again, Portland fought back.
After Cheverus senior Nick Burns was called for a technical foul, Herrick made two free throws. Then, with 1:56 remaining, Donato added two foul shots for a 24-23 advantage.
DiStasio put the Stags back on top with two shots from the line, but Smart answered with two of his own. Cheverus went up, 27-26, on a Cimino hook shot, but a free throw from Pitts-Young tied the score and with 10.8 seconds left in the third period, Pitts-Young fed Donato for a layup to give the Bulldogs a 29-27 lead heading for the final quarter.
There, Portland had its chances to spring an epic upset, but the Stags snatched victory from the jaws of defeat.
The Bulldogs took their biggest lead of the night behind a foul shot from senior Dylan Goodman (DiStasio picked up his fourth foul on the play, but remained in the game) and a Donato leaner, which made it 32-27 with 5:17 to go.
Then, Cheverus' offense finally awakened.
DiStasio got the rally started with a pretty left-handed layup on the drive. Gwilym then stole a pass under the Portland basket, was fouled and made both free throws. With 4:43 to play, Gwilym stole the ball again and fed DiStasio for a short jumper that put the Stags ahead for good, 33-32.
Twelve seconds later, Cimino made a layup after DiStasio broke the press and on the next Bulldogs' possession, Gwilym forced yet another turnover which led to DiStasio feeding Cimino for a layup and a 37-32 advantage.
"We always thrive off our defense," said DiStasio. "We played great defense all game. Our offense comes from our defense. At the end, it sparked us and we got some easy baskets. If I'm not hitting (shots), I have to find guys open to hit them."
"(Louie) did a great job at the end," Brown said. "His heart and his determination. He made plays when we needed them."
Russo blamed that stretch for costing his team the win.
"(Cheverus) pressed us like they did all game, but we handed the game over," he said. "It was a gift. I'd be lying if I didn't say I was disappointed. We thought in the fourth quarter, we had just as good a chance as they did. Those steals were devastating because of how they were created and where we gave them the ball. Turnovers long or on the sidelines aren't as bad. I can't even call them steals. They were miscues. They were assists. When you lay the ball under their basket and a guy picks it up and lays it in, it's an assist. That's why it's frustrating."
Portland fought back within two, 37-35, on a Donato three-point play with 2:46 remaining, but 10 seconds later, at the other end, Brady hit a leaner, was fouled by Zukowski and hit the ensuing free throw to make it 40-35.
With 1:49 to go, sophomore Nick Volger made one of two free throws to pull the Bulldogs within four, but DiStasio fed O'Neil for a layup with 1:29 to go to make it 42-36.
A 3-pointer banked home by Herrick with 1:14 showing again pulled Portland within a possession, 42-39, but after a Brady miss and a Pitts-Young rebound, the Bulldogs gave the ball away for 33rd and final time.
With 47 seconds left, Gwilym made the second of two free throws for a four-point lead, but Volger hit a jumper with 32.8 seconds remaining to make it 43-41 and keep the pressure on.
With 12.5 seconds to go, O'Neil went to the line for two clutch free throws and he sank them both to put Portland on the brink. Herrick was short at the other end and that did the trick.
Cheverus 45 Portland 41.
"We thought we had it the whole time," said DiStasio. "We were confident. It was a tough game. After halftime, we just wanted to move the ball. We were in foul trouble and we didn't want to put them on the line. We wanted to get the ball in transition."
Cimino led the Stags with 12 points. He added six blocks, three rebounds and a steal. Brady and DiStasio (three steals) each had 10 points. Gwilym had his usual stellar sparkplug effort, despite foul trouble, scoring seven points, to go with four steals and three boards. O'Neil had five points and four rebounds and Olson added a point and three rebounds, two steals and a block. Cheverus turned the ball over an uncharacteristic 16 times, but only gave it away four times in the second half. For the game, the Stags were 17 for 22 from the free throw line.
Even in victory, Brown had plenty of concerns following the game.
"We played perimeter offense and we had no rebounds," he said. "We had maybe two passes, then we forced shots. I have a 6-9 guy and a 6-7 guy and we didn't go inside. We did at the end. Finally.We're a good basketball team that didn't execute tonight. We even had bad decisions at the end. We did not play anywhere near an 18-0 team plays. Why? I don't know. Probably lousy coaching, but it didn't get done. We weren't disciplined enough to do what we were supposed to do. I can tell you right now, if we play that way Saturday night, we don't stand a chance against (Bonny Eagle)."
Portland was paced by Donato's 13 points, 10 rebounds and two blocks. Herrick, who didn't score until just before halftime, finished with 11 points, five boards and a steal. Pitts-Young had six points and two steals, Smart four points, seven rebounds and two steals, Volger and Zukowski three points apiece and Goodman one. Senior Matt McInnis didn't score, but grabbed four boards. The Bulldogs' 33 turnovers were too much to overcome. They shot 17 of 23 from the line.
"The issue was the last two minutes of the game," Russo said. "We played for 30 minutes. Unfortunately, it's a 32-minute game. It came down to two minutes. Three weeks ago, we didn't even think we were going to get in the tournament, but we came here to win tonight. I'd lie if I said I wasn't disappointed in the last two minutes."
While Portland was crestfallen after the loss and rightfully so, there's no overstating just how marvelous this team's resurgence was. The Bulldogs weren't even close to resembling a playoff squad when they were 6-7 and Russo came close to throwing in the towel when he spoke about playing hard in the final games to make the experience pleasant for the seniors.
In the final accounting, he did one the finest coaching jobs of his storied career, doing what he does without peer in molding a team as a season progresses and having it peak in the biggest games.
Rest assured, the Bulldogs' experience was more than pleasant once the calendar flipped to February.
"What we accomplished this year epitomizes how to get better every game," Russo said. "It was probably one of my most enjoyable groups to coach. They responded. That's why it hurts. My guys worked their butts off every day. I'm more disappointed and hurting for them. They gave it their best shot."
With only Goodman and McInnis graduating, it's safe to say Portland will be one of the preseason favorites in 2011-12.
"Most of them will be back if they can deal with me," Russo said. "We have to get better."
Two steps away
As for Cheverus, which appears more mortal by the game, it survived and advanced and is now just two victories from repeating as Class A champion for the first time since 1981-82.
Standing in its way is a dangerous and very, very confident Bonny Eagle squad. The Scots held off Deering, 41-33, in their semifinal. On Jan. 13, the Stags raced to a 25-1 lead after one quarter and humbled host Bonny Eagle, 63-34, behind 18 points from O'Neil.
The teams last met in the playoffs in 1997 regional final, 63-52 win for Cheverus.
The Stags know they have their hands full as they seek to extend their win streak to 25.
"(Bonny Eagle has) gotten a lot better since we played them," DiStasio said. "We'll have to come out much harder. We need to want it a lot more. We have to come back and play our basketball."
"They're looking very good," Brown added. "They play within themselves. They get the right people the shots. If we don't play better, we'll go home."
The Class A state final is Saturday, March 5, at the Augusta Civic Center. Game time is 7 p.m. Bangor and Edward Little are meeting in the Eastern A Final Saturday.
Sports Editor Michael Hoffer can be reached at firstname.lastname@example.com and followed on Twitter @foresports