text:
In a homogeneous immunoassay, fluorophore-conjugated lipopolysaccharide derived bacterial antigens are reacted with antibodies specific for the antigens. Quantitative detection of the formation of an immune complex is obtained by measuring the change in fluorescence polarization after complex formation. The reaction occurs quickly (less than two minutes to equilibrium), and involves the addition of only one reagent to a diluted serum specimen. The absence of a solid phase separation step eliminates false positive results and increases throughput.
1. A purified fluorophore-conjugated bacterial antigen comprising an oligosaccharide fragment of an O-antigen polysaccharide from the lipopolysaccharide of a Gram negative bacterial cell wall and a fluorophore conjugated to said oligosaccharide fragment, wherein said fluorphore-conjugated oligosaccharide fragment is able to bind to antibodies specific to said bacterial antigen and said binding produces a detectable change in fluorescence polarization of the flurophore-conjugated oligosaccharide conjugate.
2. The fluorophore-conjugated oligosaccharide fragment of
3. The fluorophore-conjugated oligosaccharide fragment of
4. The fluorophore-conjugated oligosaccharide fragment of
5. The fluorophore-conjugated oligosaccharide fragment of
6. The fluorophore-conjugated oligosaccharide fragment of
7. The fluorophore-conjugated oligosaccharide fragment of
8. The fluorophore-conjugated oligosaccharide fragment of
This application is a continuation of U.S. application Ser. No. 08/520,313, filed on Aug. 28, 1995, which issued as U.S. Pat. No. 5,976,820 on Nov. 2, 1999.
The presence of a foreign antigenic substance in the body typically gives rise to an immune response. Antibodies specific for the substance are secreted into the blood stream. Diagnostic tests revealing exposure of an animal to an infectious agent rely upon in vitro detection of antibodies specific for antigens expressed by such an infectious agent. In a typical immunoassay, antibodies contained in a serum sample are brought into contact with an antigen preparation. The resulting immune complex is detected in a variety of assay formats including enzyme-linked immunosorbant assay (ELISA), agglutination tests, complement fixation assay, etc.
Many of the assay formats involve adsorption of antigens into a solid phase of such as a plastic bead or particle, a plastic well, or the surface of a plastic or glass tube. The serum containing antigen-specific antibodies are contacted within antigen coated onto solid phase, and washed to remove unreacted serum components. The solid phase-bound antibodies are then detected, for example, by reacting with a species-specific antibody bearing a reporter molecule, washing to remove unreacted reagent, and finally adding a reagent to activate the reporter.
It will be apparent that such assays involve complex binding relationships. There is a tendency for nonspecific antibody to stick to the solid phase, giving rise to false positive tests. Even the best ELISA assays will produce a low level of false positive results. For some of the agglutination assays, specificities of less than 70 percent are obtained.
There have been attempts to eliminate the solid phase artifacts referred to above by detection of analyte antibodies entirely in liquid phase. Termed homogeneous assays, these systems rely upon measuring some changed physico-chemical parameter brought about by molecular interactions. For a review of homogeneous assays systems, see Jenkins, S. H., J. Immunol. Meth., 150: 90 (1992). For applications to macromolecules, see Hosoda, et al., J. Immunol. Meth., 121: 121 (1989). For review of the theory and practice of fluorescence polarization, a particularly elegant homogeneous system, see M. E. Jolley, J. Anal. Toxicol., 5: 236 (1981).
In the field of infectious disease diagnostics, there is a strong need for assays which have extremely high specificities. In particular, in veterinary diagnostics there are the so-called "program" diseases, which official agencies such as the United States Dept. of Agriculture or Agriculture and Agri-Food Canada administer, and whose eradication is part of their official mandate. The regulations require that a very large number of animals be tested to ensure that new infection is promptly detected and eliminated, and to prove absence of infection.
One of the most important program diseases of livestock is bovine brucellosis. This disease is caused by Brucella abortus, a Gram negative bacterium inhabiting the genital tract of cattle. Infected heifers are highly susceptible to abortion, and the economic loss to the cattle industry has been prodigious. Through concerted international efforts to screen for infected animals, and disposal of test positive animals at slaughter, the disease is essentially eradicated in Canada, and the incidence in the U.S. has diminished to less than 150 infected herds. However, Mexico is a persistent reservoir of infection, and movement of cattle in commerce to the U.S. poses a continual risk of new disease. Also, wild animals such as deer and elk are a reservoir of potential new disease in domestic livestock.
As more states become brucellosis-free, and as the overall incidence of disease continues to decline there will be a new emphasis on surveillance programs. In a surveillance situation sensitivity of the assay becomes less important than specificity (so long as the test is sufficiently sensitive to detect a typical reactor). In surveillance testing, false positive tests are troublesome, since any positive test cannot be ignored. For any such positive test, it is official procedure to trace the animal, retest it, and subject it to quarantine. This is extremely costly, and a waste of limited resources.
A large number of cattle in the United States are vaccinated for brucellosis in calfhood, with about 73 percent gaining protective immunity from superinfection with field strains of B. abortus. Unfortunately, none of the currently available tests distinguish with high accuracy between true field strain infected animals and vaccinated ones. Specificity is as low as about 50 percent for agglutination tests and up to as high as 97 percent for a C-ELISA assay. In a surveillance program, 3 percent false positives for calfhood vaccinates is a large number in view of the several million cattle tested annually in the U.S. In fact, the high incidence of positive tests for vaccinates, has led some states to repeal their mandatory calfhood vaccination laws, thus depriving the industry of one otherwise valuable tool in the eradication of the disease.
Conventional assays for detection of brucellosis include the ubiquitous agglutination card test in the U.S., the BPAT test in Canada, various versions of a complement fixation test, the Rivanol test, milk ring test (for dairy specimens), and several forms of ELISA including a highly sensitive and specific competitive ELISA developed by Agriculture and Agri-Food Canada. A solid phase bead-based test known as PCFIA is a high throughput automated test used in several official laboratories in the U.S. Many of these tests are commonly used together on the same samples because no one of the conventional, generally available tests detect all the antibody isotypes typically found in field specimens. For a general review of conventional brucellosis tests, see Nielsen, K. and Duncan, J. R., eds., Animal Brucellosis, CRC Press: 1990, Chapt. 8.
Brucellosis is but one example of veterinary disease for which a need exists for a sensitive yet highly specific assay approaching 100 percent. Other diseases include tuberculosis (M. bovis), bovine leukosis, leptospirosis, foot and mouth disease (Africa and South America), and salmonellosis.
In the present invention, a fluorophore-conjugated antigen dissolved freely in solution reacts with antibodies contained in a diluted serum sample to form a immune complex. The immune complex is detected by a change in fluorescence polarization.
It is an object of the invention to provide a homogeneous assay for detecting antibody to selected antigens, thereby eliminating complex, time-consuming adsorption, washing, and detection steps. The assay involves only a single reagent, and a single step reaction in which an immune complex is detectable by a simple fluorescence polarization measurement.
It is a further object of the invention to provide an assay which has extremely high specificity, with less than 0.1 percent down to 0 percent false positive tests, while maintaining a sensitivity of greater than 98 percent.
It is a still further object to provide an assay in which the antigen reagent can be prepared in large reproducible lots of highly uniform composition and performance.
The antigen reagent is also very stable with virtually indefinite lifetime when stored at refrigerated temperatures in the presence of a microbial inhibitor such as sodium azide.
The present method of the invention detects antibodies to bacterial antigens present in fluid comprising combining fluid which may contain antibodies to a bacterial antigen with a fluorophore-conjugated oligosaccharide antigen derived from the lipopolysaccharide antigen derived from the lipopolysaccharide fraction of a bacterial cell wall to form a mixture, incubating the mixture for a time sufficient to form an immune complex, and measuring the extent of formation of the immune complex by comparing the fluorescence polarization after complex formation to an unreacted control value.
The invention provides fluorophore-conjugated antigens comprising oligosaccharides derived from the lipopolysaccharide fraction of a bacterial cell wall. The fluorophore-conjugated oligosaccharide is one embodiment derived from the O-antigen chain of the cell wall lipopolysaccharide fraction from bacteria, such as Gram negative bacteria, having us O-antigen chain.
The invention similarly comprises a homogeneous immunoassay in which antibodies to an antigen are reacted with an antigen to form an immune complex, the improvement to previous technology being a fluorophore-conjugated oligosaccharide antigen of bacterial cell wall forming a fluorescence polarization detectable immune complex with antibodies specific thereto.
The fluorophore-conjugated oligosaccharide antigens may be derived from any bacterium having a lipopolysaccharide cell wall fraction, as antigens derived from Gram negative bacteria such as B. abortus, Leptospira spp, or Salmonella spp. The fluorophore will have an emission life of 1 to 10 nanoseconds, and its emission may vary over a wave length of 400 to 800 nanometers. In the present invention, the fluorophore fluorescein is efficacious.
FIG. 1 is a frequency distribution of positive and negative serum samples from a study of 9230 known positive or negative sera.
FIG. 2 is a frequency distribution of 8669 negative sera and 561 positive sera from culture confirmed infected cattle.
FIG. 3 is a rectilinear plot of sensitivity vs. specificity.
FIGS. 4A and 4B are plots of four control sera included with each 100 tests.
The assay of the present invention takes advantage of the well-known highly specific binding of antibodies to their corresponding antigens. When a bacterium invades an immunocompetent animal, certain structural/chemical moieties present on its surface are recognized as foreign by the immune system. Secretion of antigen-specific antibody ensues. The population of antibodies is generally very heterogenous, with individual antibody molecules recognizing and binding different epitopes. In preparing useful antigens as targets in immunoassays, it is therefore important to select antigenic structures which contain the immunodominant epitopes, so that there is a strong likelihood of correctly identifying the largest possible number of positive sera.
Gram negative bacteria have cell walls that characteristically contain a diverse family of specialized glycolipids in which the saccharide forms repeating O-antigen chains, attached through non-repeating core glycan to lipid A, which is integrated into the outer membrane. In Salmonella, as a typical model system, synthesis of the entire core sequence occurs by glycosyl transfers from sugar nucleotides, with addition at the nonreducing terminal and catalysis by glycosyl transferases. The O-antigens generally act as surface antigens, and contain many of the immunodominant epitopes. These antigens also, when shed, behave as bacterial toxins. The O-antigen chains are synthesized separately as modular glycans. The modules are spliced together before becoming linked to the core sequence.
In many bacterial species, the O-antigen chains are simpler than in Salmonella. In one species of great interest from a diagnostics standpoint, B. abortus, the O-antigen polysaccharides have 1-2 linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl repeating units. The O-polysaccharide is extractible as an approximately 15,000-30,000 molecular weight molecule. There are many extraction methods for obtaining purified O-chain antigen reported in the literature for Gram negative bacteria generally. For Brucella abortus or Yersinia enterocolitica 0:9, phenol-water extraction, followed by dialysis and centrifugation may be used, as described in U.S. Pat. No. 4,831,126 or 5,006,463, hereby incorporated by reference. This method has been modified somewhat to improve purity and yield, as described in Nielsen, et al., Am. J. Vet Res. 50: 5 (1989).
In extracting and purifying a lipid-free arabinomannan antigen preparation from Mycobacterium species, the lipid fraction may be removed by extraction with acetone, ether-methanol, and chloroform successively, and then releasing the antigen fraction by alkaline hydrolysis. Further purification may be effected through ion exchange chromatography and gel filtration.
Extraction and characterization procedures for Gram negative bacterial polysaccharide antigens are well-known in the literature. Satisfactory methods for the preparation of the intact polysaccharide antigens of the present invention are available for all significant Gram negative pathogens, or may be readily adapted from existing methods. Libraries of the general structural characteristics of families of polysaccharides of diverse origin and of the antigens of pathogens in particular are available. For example, see Griffith, et al., "Type-specific carbohydrate antigens of pathogenic bacteria," Carbohydrate Polymers, vol. 14, p.339. Also a detailed review is available in Vols. 235 and 236 of Methods in Enzymoloy, Academic Press: 1988. Another source of protocols for extraction of antigenic polysaccharides from bacteria for extraction of antigenic polysaccharides from bacteria is Methods in Carbohydrate Chemistry, vols. 4, 8, 28, 50, 179, and 230 (Academic Press: various years).
In preparing the antigens of the present invention, it is necessary to reduce the antigen to a small size, but not so small as to lose its immunodominant epitopes. In the case of many of the O-antigen repeating subunits, the length of polymer estimated to fill an antibody combining site is on the order of 4-6 sugar residues. The O-antigen may be conveniently reduced in size by several methods, including acid or base hydrolysis, enzymes, oxidative or reductive processes, or by techniques known to result in selective cleavage at specific glycosidic linkages. The general protocol for preparation of bacteria-derived polysaccharide antigen fragments useful in the present invention is:
(1) Separate polysaccharide antigen from other cellular components. (Conventional method by one of the published techniques).
(2) Selective precipitation of the polysaccharides or of their salts or complexes.
(3) Resolubilization and optional chromatographic separation based on charge, size, or ligand affinity, or combination thereof.
(4) Partial hydrolysis to reduce the size (or by the specific cleavage or oxidative/reductive methods referred to above). Base hydrolysis in 0.1 to 1.5N NaCl is preferred for any of the bacterial O-antigens.
(5) Labeling with a fluorescent moiety.
(6) Purification by gel filtration (size) and ion exchange chromatography (separation by charge).
It will be recognized that the conditions of extraction and fragmentation of the O-antigens will differ somewhat from the one genus or species of Gram negative bacteria to another. While the above general protocol is generally applicable to any given bacterium, there will be variations in procedure. Some experimentation will be necessary to achieve a satisfactory antigen in a given situation. The ultimate test is performance of the labeled antigen against actual blinded serum panels of known infected and disease-free animals. Use of monoclonal antibodies to screen suitable tracers is remarkably, and surprisingly, unreliable. However, if the general protocol set forth herein is followed for the fluorescence polarization-based assay disclosed hereinafter, a suitable reagent can be identified with very limited experimentation. For example, complete identification and preliminary validation of a fluorescein-conjugated reagent obtained from an alkali hydrolysate of B. abortus O-antigen was developed in about three weeks of testing. The generic Mycobacterium arabinomannan-derived reagent took even less time to develop. In general, a suitable antigen will produce greater than 99 percent and upwardly 100 percent specificity for the target organism, with greater than 98 percent sensitivity utilizing sufficient positive and negative sera to give statistical significance levels having p values of less than 0.05.
The chemistry of carbohydrate fluorescence labeling is very complex, and the number of fluorescent derivatives possible is virtually unlimited. For a detailed review, see Ginsburg, V., "Complex Carbohydrates", in Methods of Enzymology, vol. 138, Academic Press: 1987. In the Examples, fluorescein was conjugated to the O-antigen through coupling fluorescein isothiocyanate. Many other coupling mechanisms are known in the art. Some aspects of the coupling rationale are as follows:
Most carbohydrates do not possess amino or carboxyl groups. The only groups available for attachment of fluorophores are hydroxyl groups and the terminal reducing aldehyde group. Therefore, conventional protein labeling reagents may not be appropriate for the labeling of carbohydrates with fluorochromes. Fluorescein isothiocyanate (FITC), however, will react slowly with hydroxyl groups. Indeed, FITC-labeled dextran and FITC-labeled inulin have been used for many years in permeability studies in cells and tissues [Rutili, G. and Arfors, K. E., Microvasc. Res. 12:221 (1976); Sohtell, M., et al., Acta Physiol. Scand. 119:313 (1983)].
Dichlorotriazinyl-aminofluorescein (DTAF) [Blakeslee, D., J. Immunol. Methods 13:305 (1976)]; [Blakeslee, D., J. Immunol. Methods 17:361 (1977)] reacts readily with alcohols in the presence of water [Chayen, R., et al., Anal. Biochem. 39:533 (1971)]; DeBelder, A.N., et al., Carbohyd. Res. 30:375 (1973)]. DTAF was used to successfully label the 6-position of the sugar moiety in muramyl depeptide when all efforts to employ FITC had failed [Hiebert, C. K., et al., J. Med. Chem. 26:1729 (1983)].
Reductive amination of the terminal reducing sugar residue has been used to label carbohydrates with a variety of fluorophores including pyridylamine [Hase, S., et al., J. Biochem. 95:197 (1984)] and fluoresceinyl-ethylene diamine [Baynes, J. W., et al., Anal. Biochem. 170:382 (1988)]. The carbohydrate and fluorophore, possessing an amino group, are mixed together and the Schiff base formed is reduced to a stable amino linkage with sodium cyanoborohyride. Alternatively the terminal aldehyde may be converted to an amino group by incubation with ammonium bicarbonate and then reacted with fluorophores such as carboxyfluorescein or dansyl chloride [Manger, I. D., et al., Biochemistry 31:10724 (1992)]
Aldehydes may be generated in carbohydrates by periodate oxidation of vicinal hydroxyl groups [Rothfus, J. A. and Smith, E. L., J. Biol. Chem. 238:1402 (1963)]. RNA was labeled at its 3′-terminal ribose in this way with 5[[[2(carbohydrazino)methyl]thio]acetyl]-aminofluorescein [Janiak, F., et al., Biochemistry 31:5830 (1992)] and with rhodamine and fluorescein thiosemicarbazide [Bauman, J. G., J. Histochem. Cytochem. 29:227 (1981)]. The carbohydrate moiety of human alpha thrombin was labeled with aminoethyl and aminohexyl thioureal fluorescein [Carney, D. H., J. Supramol. Struc. 13:467 (1980)] and the cercarial glycocalyx of Schistosoma mansoni by fluorescein semicarbazide [Samuelson, J. C. and Caulfield, J. P., J. Cell Biol. 100:1423 (1985)].
Aldehydes may be generated and labeled at specific residues by combining enzymatic and chemical reactions. For example, galactose oxidase may be used to form the C-6 aldehyde on terminal D-galactose or N-acetyl-D-galactosamine residues [Avigad, E., et al., J. Biol. Chem. 237:2736 (1962)]. The same enzyme, in combination with neuraminidase, was used to modify galactosyl residues penultimate to sialic acids and subsequently reacted with fluorescein β-alanine hydrazide [Wilchek, M. and Bayer, E. A., Methods Enzymol. 138:429 (1987)].
The choice of fluorophore is to a large extent a matter of convenience once a coupling chemistry has been selected. Virtually any fluorophore having a fluorescence life of between 1 and 10 nanoseconds, preferably 4 to 7 nanoseconds, and having a wavelength of 400 to 800 nanometers will have efficacy in the present invention. The choice of fluorophores will influence the configuration and requirements of the optics of the instrument used to read the assays. It will also impact the optics technology utilized. For a detailed listing and discussion of a variety of interesting, commercially available fluorophores, see Handbook of Fluorescent Probes and Research Chemicals, ed. Karen Larison, by Richard P. Haugland, Ph.D., 5th ed., 1992, published by Molecular Probes, Inc. Preferred fluorophores in the labeled antigens of the present invention are fluorescein, rhodamine, BODIPY™, Texas Red™ and Lucifer yellow. Fluorescein is most preferred for its convenience in screening O-antigen fragments for good assay candidates.
In the assays of the present invention, the fluorophore-conjugated oligosaccharide antigens are mixed with a serum sample diluted in a conventionally buffered solution, allowed to react for 1 to 3 minutes, and read in a fluorescence polarization instrument. Typically a background value is obtained prior to addition of the antigen reagent. The assay is homogeneous, in that no further steps are required, as in heterogeneous assays in which antibody contained in the sample must be bound to antigen coated onto a solid surface, with subsequent washing and detection steps. The assay is carried out at temperatures of 15� C. to 37� C., but the assay may be carried out under any temperature and reaction conditions which permit specific immune complex formation.
In fluorescence polarization (FP), a molecule conjugated to a fluorophore is present in solution. The molecular rotation of the molecule in solution is a function of its size and the viscosity of the solution. If the fluorophore-conjugated molecule is relatively small compared to its size when bound to a larger molecule, such as an antibody, the fluorescence emitted by such small molecule will be relatively less polarized than when it is bound. This is because the complaced ligand target molecule is at relatively large, and rotates more slowly. Hence, more of the emitted light is directed in one geometric plane, and the fluorescence polarization value increases. The application of FP to quantitative detection of drugs in an immunoassay is disclosed in U.S. Pat. No. 5,427,960, hereby incorporated by reference. For reference to some of the key developmental papers in the literature of this technology, see Perrin, F., J. Phys. Radium 7:390-401 (1926), Weber, G., Adv. Protein Chem. 8:415-59 (1953), Dandliker, W. B. and Feigen, G. A., Biochem. Biophys. Res. Commun. 5:299-304 (1961), Dandliker, W. B. and de Saussure, V. A., Immunochemistry 7:799-828 (1970), Dandliker, W. B., et al., Immunochemistry 10:219-27 (1973), Jolley, M. E., J. Anal. Toxicol. 5:236-240 (1981).
FP has the following important characteristics: it detects change in size of molecules, it detects change in viscosity of the medium, it may be used to detect and measure binding of small molecules to big molecules. In addition, it has broad application in detection and measurement of the conversion of large molecules to small molecules. The FP assay is homogeneous, (reactions take seconds to minutes), utilizes stable reagents, (no enzymes, substrates or radioisotopes) and is highly automatable, (single incubation, a single reagent is possible, and no washing steps are required).
Further advantages of the antigens and immunoassays of the present invention will be apparent from the Examples which follow:
Preparation of Brucella Tracer. O-polysaccharide (3.0 mg) was dissolved in 600 μL of 0.1 N NaOH and incubated at 37� C. for 1 hour. 300 μL of a freshly prepared solution of FITC isomer I (Sigma Cat. No. F-7250) in DMSO (Mallinkrodt Cat. No. 4948) 100 mg/mL) was added, mixed well, and incubated 1 hour at 37� C. The reaction mixture was then applied to a DEAE-Sephadex A25 (Signa Cat. No. A25-120) column (20 mL packed volume in a 20 mL syringe, equilibrated with 0.01 M phosphate buffer pH 7.4). The column was eluted with 0.01 M phosphate buffer, pH 7.4. Two fractions were collected. The first was buffer (7 mL) and the second, a bright green fluorescent fraction (tracer I10-1; 7 mL). The elution buffer was then changed to 0.1 M phosphate, pH 7.4. Two fractions were collected. The first was buffer (10 mL) and the second, a bright green fluorescent fraction (tracer I10-2; 25 mL). The latter fraction contained the best tracer.
Therefore, the yield is approximately 5000 assays per mg.
Brucellosis Assay. The following is the general protocol for performing the B. abortus assays in the examples which follow.
1. All steps of the assay were performed at ambient temperature.
2. To 2 mL of phosphate buffered saline, containing 0.05% lithium dodecyl sulfate and 0.1% sodium azide (PBSA), was added 20 μL of serum sample.
3. A blank subtraction was performed on an FPM-1™ (Jolley Consulting and Research Inc., Grayslake, Ill.).
4. Tracer (10 μL, diluted in PBSA to yield approximately 1.5 nM fluorescein equivalents in the final assay mixture, was added.
5. After an incubation of greater than 2 minutes the fluorescence polarization of the tracer was determined.
Extraction and purification of Lipid-free Arabinomannan Antigens of Mycobacterium paratuberculosis or Mycobacterium bovis. The protocol for preparation of the antigen is as follows. Also refer to Misaki, A., et al., J. Biochem. 82:701 (1977) and Sugden E. A., et al., Infect. Immun. 55:762 (1987).
1. Grow a laboratory-adapted strain of M. paratuberculosis (Animal Diseases Research Institute strain V) in modified Long's synthetic medium [Landi, S. Bull Internat. Union against Tuberc 49:112 (1973)] for 3 months at 37� C. Alternatively, grow M. bovis AN-4 in modified Reid's synthetic medium [Angus, R. D., J. Biol. Stand. 6:221 (1978)] for 10 weeks at 37� C.
2. Kill M. paratuberculosis in 3% phenol and kill M. bovis AN-5 by autoclaving at 121� C. for 45 minutes.
3. Wash M. paratuberculosis or M. bovis killed bacilli with water and drain dry. Store frozen at 20� C.
4. Extract lipid from bacilli at 4-7� C. with acetone, ether-ethanol (2:1), and chloroform successively for 4 hours for each solvent, and with chloroform-methanol (2:1) for 18 hours (i.e., overnight). Wash with acetone for 1 hour to displace the chloroform-methanol. Use enough solvent volume in each case to give approximately 10 fold that of the settled bacillary mass. After each extraction filter through Whatman No. 1 filter paper. Perform in fume hood.
5. Air-dry bacilli in a fume hood and store at 4-7� C. in a polyethylene container.
6. Extract 100 g of dried bacilli with 100 ml of 0.5 N NaOH at 70� C. for 8 hours in a glass beaker placed over a submersible magnetic stirrer in a water bath. Cover lightly with parafilm and purge with a low stream of N2 gas from a compressed tank. Stir constantly.
7. Cool extract and neutralize to pH 7.0-7.5 with glacial acetic acid using a pH meter at room temperature.
8. Centrifuge extract at 30,000�g for 30 minutes at 5� C. and collect extract supernatant. Discard the pellet.
9. Dialyse extract supernatant against tap water for 2 days in 3,500 Molecular Weight Cutoff dialysis tubing, and for 2 days with 2 volumes of each with at least a 20 fold excess of distilled water for each volume.
10. Add 100% Ethanol with stirring in a glass beaker at room temperature to give a final concentration of 30% ethanol. Centrifuge at low speed at room temperature and collect supernatant. Discard precipitate.
11. Add a further quantity of 100% ethanol with stirring in a glass beaker at room temperature to give a final concentration of 66% ethanol. Centrifuge at low speed at room temperature and collect supernatant. Discard precipitate.
12. Add a further quantity of 100% ethanol with stirring in a glass beaker at room temperature to give a final concentration of 80% ethanol. Centrifuge at low speed at room temperature and collect supernatant.
13. Suspend the 80% ethanol gummy precipitate in approximately 100 ml water and dialyse for 2 days with 2 volumes of each with at least a 20 fold excess of distilled water. Lyophilize and store in a desiccator at 4-7� C.
14. Resuspend in a minimum volume of 1.0M NaCl, 0.1M Tris, pH 8.0, 0.02k NaN3 (Tris-NaCl) and stir to completely dissolve. Estimate carbohydrate concentration by the phenol-sulfuric acid test using 10 mg % glucose in Tris-NaCl as a standard. To 0.2 ml sample, add 0.2 ml 4.5% phenol and mix well. Add 1 ml of concentrated sulfuric acid rapidly to the mixture. After mixing further and cooling, read at 490 nm in a spectrophotometer. The recovery of carbohydrate in the 80% gummy precipitate was approximately 10 mg/g (on a dry-weight basis) of dried solvent extracted bacilli.
15. Apply up to 1000 mg of 80% ethanol precipitated carbohydrate material in no more than 30 ml to a 5.0�89.5 cm column of Sephacryl S-200 (Pharmacia Fine Chemicals) pre-equilibrated with Tris-NaCl and run at 81.9 ml/h and 4� C., collecting 25.2 ml fractions. Monitor fractions with the phenol-sulfuric acid test (step 14) and collect pools (e.g. fractions 36-48, 49-60, 61-70). The later fractions have lower molecular weights. Dialyse with tap and distilled water as before (step 9) and lyophilize. Store at 4-7� C. in a desiccator.
16. Chromatograph selected pools reconstituted in 30 ml of 0.05M Tris, pH 8.0, 0.02% NaN3 (Tris) on a 1.6�69.5 cm column of DEAE-Sephacel A25 (Pharmacia) pre-equilibrated with Tris, and run at 25 ml/h at 5� C., collecting 14 ml fractions. Elute with 300 ml of Tris and collect the non-retained carbohydrate material, monitoring with the phenol-sulfuric acid test (step 14).
17. Dialyse the pooled non-retained carbohydrate from DEAE-Sephacel chromatography as before (step 9), determine the carbohydrate concentration with the phenol-sulfuric acid test (step 14) and lyophilize in 10 mg aliquots (Note: 10 mg vials are based on the phenol-sulfuric acid definition of mass and not on a dry-weight basis). Store at 4-7� C. in a desiccator.
18. The recovery of the Sephacryl S-200 pool of 36-48 (step 15) from DEAE Sephacel (step 16) is approximately 5 mg/g (on a dry-weight basis) of dried solvent extracted bacilli and is defined as alkaline-extracted lipid-free arabinomannan.
1. To each of two vials of lyophilized arabinomannan in 5 ml serum bottles labeled M PARA STR V AZ-AM ANTIGEN 10 mg 20-11-86 add 1 ml of 0.1 N NaOH freshly prepared. Stopper with grey lyophilization stoppers and incubate in a 37� C. incubator oven for 1 hour.
2. Add the two 1 ml aliquots of arabinomannan in 0.1 N. NaOH to a 16�100 mm disposable (Baxter T1290-6) test tube containing 2.2 mg of freshly weighed 5-Fluorescein Isothiocyanate (Eastman Kodak Cat. No. C10868, lot H3F, Dye content 76%, 11/73). Incubate 1 hour at 37� C.
3. Apply reaction mixture to a 29�1 cm column of Sephadex G-25 (which had been slurried and had had the fines removed) pre-equilibrated with 0.1M solidum phosphate pH 7.0. Flow rate was approximately 0.25 ml/ml. Collect 1 ml fractions and monitor at 492 nm.
Observed clear separation of leading yellow void peak from following yellow orangish material.
4. Apply Sephadex G-25 pooled fractions 10-14 to an 18�1 cm column of DEAE-Sephadex A25 equilibrated with 0.1M sodium phosphate pH 7.0 and elute with same buffer at 0.25 ml/min. Collect 1 ml fractions and collect pools 11-15 (tracer 3), 16-20 (tracer 1), and 21-15 (tracer 2).
Monoclonal antibodies cannot be used to screen tracers for reactivity to antibodies generated by natural infection in bovines, as shown in Table 2. Tracer 94082502, for example, reacts strongly with mAB ST9, specific for the o-polysaccharide from B. abortus. However, on testing against a panel of five known positive bovine sera, it failed to detect two of them (POS1, POS3) and reacted weakly with a strong positive (POS4). Tracer 9408201, however, reacted strongly with all of these samples, but poorly with mAb YST9.
A fluorescence polarization assay for the detection of antibodies to M. paratuberculosis and M. bovis was compared to a reference ELISA (ADRI). Results compared favorably with some exceptions as seen in Table 3. Negative samples 54 was positive in ELISA (high cutoff) but negative by FP. Positive samples 52C8, 27A9 and 194F9 were negative by ELISA (high cutoff) and positive by FP (105 mP cutoff). Using the low cutoff ELISA was positive on positive samples 36-33 and FP was negative; samples 52C8 and 27A9 became positive. However, using the low cutoff ELISA, negative samples 103E9, 106E9, 54 and 56 became positive.
The fluorescence polarization assay for B. abortus antibodies was tested twice on a panel of bovine serum samples which gave disparate results on reference assays. See Table 4.
The FP results were consistent with the test interpretations on all samples. Note especially samples OW-1 (CARD false negative), IC88 (CARD and CF false positive), TO-1� (CARD false positive), and CV25 (CARD false positive).
In a blind study, a total of 9480 bovine sera were tested in addition to sets of four controls, included with every 100 samples tested. The controls were a strong positive, a weak positive, a negative and a serum derived from a B. abortus strain 19 vaccinated animal. Test sera included 8669 sera from Canadian cattle, previously tested negative by routine serological tests, 561 sera from animals from with B. abortus has been isolated, either from tissues or milk and 250 sera from animals vaccinated with B. abortusstrain 19 at various times previously. One lot of O-polysaccharide tracer was used for all tests, and was prepared as disclosed in Example 1.
Assay sensitivity and specificity for the FPIA before and after decoding of the serum samples are as follows. Using a preliminary cutoff of 107.2 mP, the point estimates of sensitivity and the specificity of the FPA after initially testing all samples were 98.2+/−1.1% and 99.8+/−0.09%, respectively. After decoding and retesting false positive and false negative samples, the values increased to 98.5+/−1% and 100%, respectively.
FIGS. 1 and 2 represent histograms of the distribution of results obtained with the positive and negative sera in the FPA before and after decoding of the samples. While the cutoff from FIG. 1 is difficult to determine because of the overlap between positive and negative samples in the 90 to 140 mP region, it is clear that the cutoff should be above 100 mP and a 107.2 value as calculated using the 100th percentile of the negative sera is not unreasonable. From FIG. 2, however, after retesting 24 false positive samples, it is apparent that the cutoff should be set at a lower level. If a value of 90 mP is selected, three false negative results and one false positive are evident. These findings were confirmed using ROC analysis (FIG. 3). Thus, if a cutoff of 90 mP is selected, the sensitivity and specificity values are 99.02 and 99.96%, respectively.
Of the eight serum samples from animals with confirmed brucellosis that remained negative in the FPIA, 1 originated from a cow infected with the vaccine strain of B. abortus and was negative on all serological tests. Of the remaining, three animals were infected with field strains of B. abortus and two were serologically negative on all tests while two sera were positive in all tests including the PFIA. In addition to these 8 serum samples, a sample from another strain 19 infected animal was positive in the FPA but negative in other serological tests. These data are presented in Table 5.
The four control sera, a strong positive, a weak positive, a negative and a serum from a vaccinated animal, included in each 100 samples tested were plotted in FIGS. 4A, B, C and D. Each point represents an individual determination. The upper and lower lines for each set of points indicate +/−2 standard deviations of the mean. Of the sera obtained from B. abortus strain 19 vaccinated cattle, 2 gave positive reactions in the FPIA using cutoff of 90 mP, giving a point specificity value of 99.2%. For comparison, the specificity in the BPAT was 48.6%, the CFT was 49.0% (87 of 250 sera were anticomplementary and no diagnosis could be made), the I-ELISA was 56.3% and the C-ELISA was 97.2%. These data are presented in Table 6.
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Nelvana Enterprises
Battle of the Planets blasts into the 21st century
Corus Entertainment's Nelvana and Japan's d-rights are rebooting the cult anime series for a new generation of boys.
Deals: Pacific Northwest, eOne, Nelvana, Breakthrough
Pacific Northwest Pictures picks up the Canadian rights to Liv Ullman's Miss Julie (pictured), while eOne takes the North American rights to Saar Klein's After the Fall.
Nelvana, Spin Master pact on girl-skewing preschool series
Nelvana Enterprises and Spin Master are developing Little Charmers, a new girl-skewing CGI preschool series set to debut on Treehouse and Nickelodeon in 2015.
BBC acquires Sidekick animated series
The deal with Nelvana Enterprises will see the first two seasons of the Canadian cartoon head to the U.K.
Deals: Children's entertainment
New global markets for Doki (pictured), Beywarriors Beyraiderz and kaboom! secures further rights to Zack & Quack.
Nelvana Enterprises names co-heads to lead global business
On the heels of Corus Entertainment revealing management changes that put Colin Bohm at the helm of the Corus Kids vertical, Nelvana has named two co-heads who will jointly manage the company's global distribution and licensing portfolio.
KidsCo sends Syfy Kids to Asia
The channel will launch in Asia with around 120 hours of programing, including Matt Hatter Chronicles (pictured), dubbed in local languages like Mandarin, Cantonese, Malaysian and Indonesian.
Mike the Knight rides to Latin America
Discover Kids Latin America has snapped up the CG-animated preschool series (pictured) from Toronto's Nelvana Enterprises.
Nelvana names new toy partner for Babar
The Toronto-based children's entertainment prodco is joining forces with NY-based Yottoy Productions to develop plush toys based on its classic kids series.
Nelvana expands with digital releases
The kids entertainment production company is this year launching new apps, webisodes and eBooks featuring its popular brands Franklin the Turtle, Max and Ruby and Scaredy Squirrel.

subset: commoncrawl


text:
Hello there, I made a new app
Startup Sound Enabler for Windows 8 will allow you to have a startup sound in Windows 8.x with just one click.
As you might be knowing, Microsoft removed the startup sound from Windows 8 and above COMPLETELY.
This app is my try to revive it.
If you are interested, there is nothing special under the hood. I have an article on how to achieve the same manually. The application does exactly the same, but bloody saves your time.
Download Startup Sound Enabler for Windows 8


text:
DJ Pauly D Wants To Be Godfather To Snookis Baby
Now that Nicole "Snooki" Polizzi has finally confirmed her pregnancy, some of her co-stars reached out to the diminutive star to congratulate her on Twitter while others… have stayed mum.
Both DJ Pauly D and Deena Cortese gave their Jersey Shore co-star a special shout out on their Twitter pages, with Pauly D hoping Snooks and fiancé Jionni will pick him as a Godfather while Deena can't wait to meet "the little meatball" in her oven.
Both Pauly D and Deena both wrote:
"Congrats To My Girl @snooki So Happy For Her And Jionni!! I Better Be The Godfather #UncleDJPaulyD"
"Congrats @snooki my Lil meatball!! Can't believe we're gonna have a little baby meatball running around soon.. Your gonna be a great mommy"
Strangely both Mike "The Situation" Sorrentino, Sammi Sweetheart, Ronnie Magro and Vinny Guadaicantspellhisname kept mum about the Snooki news even though everyone else in the Twitterverse were talking about her pregnancy. Snooki's best friend, Jenni "JWoww" Farley also didn't give any special shout outs to her girl, but we're sure she congratulated her way before everyone else as the two are filming a reality television show together.
Tell us, do you think DJ Pauly D should be baby Snooki's Godfather? We think he's be a great choice!
Photo via Twitter.com


text:
Raven Sorvino
Song & Album Downloads
Language Artz Records
Featuring: Kris Kasanova
Song: Mona Li$a
On her new paper-chasing anthem Raven Sorvino is joined by Doley Bernays and Kris Kasanova, who together pool their talents in hopes that it earns them enough coin to buy the famous Da Vinci painting,... More
Posted 2 years ago
Featuring: El Prez
Song: Homegrown
NY has Nicki Minaj and Miami has Trina, so why can't California have their resident female spitter in the mainstream mix as well? We think that they can, and she goes by the name of Raven Sorvino.... More
Posted about 3 years ago
Featuring: Dave East
Song: Hands Up
Previously introduced to our pages via Who Gwan Test Me, Golden State rhymesayer Raven Sorvino has joined forces with NYC-repping Booth newcomer Dave East to craft a new collaborative EP. On the... More
Posted 3 years ago
Song: Who Gwan Test Me
Introduced to the Booth as a guest contributor to Don't Say Goodbye, a Rapper Big Pooh-fronted leak off Booth-sponsored compilation album Executive Suite, Raven Sorvino returns to make her solo... More
Posted 3 years ago
Artist: Rapper Big Pooh
Featuring: Donny Goines | Raven Sorvino
Song: Don't Say Goodbye
All Out, the reader-acclaimed inaugural leak off Kareem Fort's forthcoming, Booth-hosted compilation album, is a damn hard act to follow, but the HiPNOTT Records C.O.O. may just have outdone... More
Posted 3 years ago
Sample Text - Sample Link


text:
Hi everyone
Here are some pictures of Disneys Halloween Party 2008.
This party was fantastic!! I had a lot of fun. I hope you will like the pics.
Slideshow of pictures, photos, and videos, from webshots
Dailymotion - Halloween Party 2008 Ã* Disneyland Paris - Jouer avec le feu, a video from Rystall. disneyland, halloween, feu, spectacle
Dailymotion - Halloween Party 2008 Ã* Disneyland Paris, a video from Rystall. Halloween, Disneyland, Paris, Dingo, Goofy


text:
Tigers hope to turn corner
Carroll needs win vs. Southwest to control its destiny
The Carroll football team is coming off a tough loss to in-town rival Ray, but the Tigers don't have any time to weep. Their playoff lives are at stake.
Carroll plays San Antonio Southwest tonight at Cabaniss Field in a game that could have a big bearing on the District 27-5A playoff race. A win allows the Tigers to control their own destiny and would mean they could clinch a playoff berth next week with a win over Victoria Memorial.
A loss would likely drop the Tigers to fifth place and mean they would have to beat Victoria next week and get help from other teams to get that final playoff berth.
Tonight's matchup is equally big for Southwest, which hasn't been to the playoffs since 2003.
The Dragons (6-2 overall, 3-1 district) can clinch a playoff spot with a win. Since losing its district opener to Ray, Southwest has reeled off three straight wins against Victoria, King and San Antonio East Central.
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text:
Prosecutors in Incheon said Yang Hoe-jung turned himself in at their office, which is investigating the role of businessman Yoo Byung-un in the sinking of the ferry Sewol.
Mr Yoo's body was found by a farmer in an orchard on June 12.
The structurally defective and heavily overloaded ferry capsized and sank on a routine journey on April 16, killing 304 people, 250 of them teenagers from the same school on a class field trip. Twelve of their teachers also died in the incident, the country's worst maritime disaster in 44 years.
The driver was the last among a group of people close to Mr Yoo who had been wanted for allegedly helping him elude South Korea's biggest manhunt.


text:
Schmalfeldt said that the book was also meant to highlight the importance of clinical trials in the search for cures, as well as new and better treatments for diseases. In 2007, Schmalfeldt volunteered for a clinical trial at Vanderbilt University Medical Center in Nashville to test the safety and tolerabilty of deep brain stimulation in early PD.
"In my clinical trial, we started out with 30 people. Fifteen of us were randomized to the surgical group, 15 were randomized to the control group, meaning they would continue to take their regular medication and then be compared at various intervals with those of us who had the surgery to see if our rates of progression differed," he said. "The theory this clinical trial is trying to prove is that doing deep brain stimulation (DBS) surgery earlier in the course of PD will be beneficial to patients, and may even slow down or reverse the progression of the disease."
Schmalfeldt noted that the study is currently seeking the funds to expand to medical centers around the country, enrolling hundreds of new Parkinson's patients. Schmalfeldt said 100 percent of the author proceeds from sale of his book will be donated to the National Parkinson Foundation as well as the Vanderbilt University Medical Center's DBS Research Fund.
"Deep Brain Diary" is available as a 249-page hardback at Lulu.com as well as from online booksellers such as '/>"/>
|Source: PRWeb
Copyright©2010 Vocus, Inc.
All rights reserved


text:
Additional file 4.
Phylogenetic tree from Bayesian analysis of nucleotide sequences for 12 protein-coding genes with third codon positions excluded (7,856 characters). The best-fit substitution model for each of 12 genes was estimated using the AIC criterion implemented in MrModeltest 2.3. The resulting best-fit model for each of 12 genes was then used for Bayesian analysis. Bayesian posterior probability values (BPP), shown above the nodes, were estimated after the initial 200 trees (the first 2x105 generations) was discarded as burn-in (see methods for analysis details).
Format: PDF Size: 455KB Download file
This file can be viewed with: Adobe Acrobat Reader
Sultana et al. BMC Evolutionary Biology 2013 13:12 doi:10.1186/1471-2148-13-12


text:
Tokyo — Long before the ghostly images of Fukushima's nuclear workers in white suits and gas masks appeared in newspapers and magazines around the world, photographer Kenji Higuchi was recording the lives and risks of the industry's front-line laborers.
The 74-year-old, with longish gray hair, published some of the first images of nuclear workers toiling inside a reactor in 1977. He documented the struggles of radiation victims and, over a half-century, wrote 19 books, including "The Truth About Nuclear Plants" and "Erased Victims." But in this energy-hungry nation, his no-nukes message did not carry very far. "I was the least popular photographer in Japan," he said.


text:
Influence your business outcome in real-time and extract more value from your Big Data – with predictive analytics from SAP. Our solutions can help you bring predictive insight to everyone in your organization – across applications and mobile devices.
Siemens AG is a global powerhouse in electrical engineering and electronics.
The company develops and manufactures products, designs and installs complex systems and projects, and tailors a wide range of services for individual requirements.
Empower your entire organization with easy access to predictive insight. Uncover new business opportunities, predict trends, and proactively respond to change.
Put predictive analytics in the hands of a broad spectrum of users– not just data scientists – with SAP InfiniteInsight. This software automates predictive modeling, so users at all levels of the business can self-service their predictive needs to uncover customer insights and make forward-looking decisions with ease.
Run big data analytics on terabytes of data – and harness the power of real-time predictive analytics – with SAP HANA, our ultra-fast in-memory platform.
Let our seasoned consultants help your organization evolve from sense-and-respond to predict-and-act capabilities – for a sharper competitive edge. Services include:
Find more SAP solution information. Industries › Lines of Business ›


text:
Interpretation Services for Police
As the population of non-English speakers grows, demand for high quality interpretation services increases. Global Voices can help you bridge that gap at any Police station throughout Scotland, England and Wales.
At Global Voices, our interpreters are tested and trained to specifically cope with police practices and procedures: arrest, detention and statement taking. They possess an up-to-date knowledge of the terminology and glossaries used in criminal and civil cases. Their absolute impartiality guarantees that they deliver precise, calm and coherent messages, even under strenuous circumstances.
As an ISO 9001:2008 certified interpretation company, we make sure that our police interpreters are fully trained, fully disclosure verified and have experience in interpreting in similar situations. We have many years of experience in providing the Police with qualified interpreters covering all community languages.
Interpreting for the Prison Service
As the inmate population of non-English speakers grows, the need for high quality legal interpretation services also increases. As well as our services for the Police, Global Voices also offers translation services for the Prison Service to ensure that inmates understand the information and services appropriate for them. Global Voices can provide this service with the same high quality, accuracy and security.
Reducing Interpretation Costs
Global Voices has developed an innovative video interpretation system for the Police, a secure, reliable, web-based, on-demand video interpretation service that enables the Police to have 24/7 access to any community language or Sign language 365 days of the year from any computer.
This secure, online interpretation service means that the Police have on-demand access to interpretation services whenever it is required. All that is required is a computer that is able to access the internet and a webcam. Video Interpretation is the perfect system for the Police to reduce interpretation service costs, while maintaining quality and reliability of interpretation.
To find out how Video Interpretation works, request a live demo or to find out more about our interpretation services, contact us today or complete our free quote request form online.


text:
Mother Shot Holding Baby
By Associated Press | April 30, 2013
Last Updated: April 30, 2013 10:10 am
Mother shot holding baby: Police officers in Alabama are looking for a person who shot a woman holding a baby.
BIRMINGHAM, Ala.—Police in Alabama are asking for the public's help in finding the gunman who fatally shot a woman who was holding her 10-day old son.
Birmingham Police Sgt. Johnny Williams says the victim, identified as 24-year-old Sherri Williams, was holding her baby and standing in the doorway of an apartment in the Gate City neighborhood when shots rang out Monday afternoon.
Officials say two men at a nearby store got into a fight. One of the men chased the other down the street and fired a gun as he ran.
Authorities say Williams was hit by a stray bullet and was pronounced dead at the scene.


text:
Viewers: in countries Watching now:
Good typography can add tremendous power to your design and your message, whether it is a print- or screen-based project, a still or motion graphic, a 3D or 2D graphic. This course explains good typographic practices, so that you can develop an "eye" for type and understand how to effectively use it. Author Ina Saltz explains type classifications (serif vs. sans serif, display type vs. text type), how type is measured, sized, and organized, and how spacing and alignment affect your design. She also explains how to use kerning, tracking, leading, and line length, and covers the history and current trends in typography. The course teaches the principles of legibility, readability, and compatibility, and how they should be considered when you're selecting and designing with type.
Perhaps at this point you're thinking that typography has so many rules and standardized practices and principles. Well, you're right, but any body of knowledge has its rules. If you were learning to play the piano, you would first have to learn to read the musical notation. Then you would have to practice at a very basic level, playing scales over and over again until they were exactly right. Eventually, you might learn to play a complex piece of music, and you'd have to work to hit every note until the piece was perfect.
Then and only then would you know enough to begin to interpret that piece of music by allowing it to become more expressive, seen through the prism of your own experience. Like any other field, typography has its conventions and rules. You must know and learn to follow those rules. Yet, some of the most wonderful and effective designs completely violate those rules. One of my teachers Donald Jackson put it this way, all rules can be broken in divinely successful ways.
You can learn from those who have successfully broken the rules, but it does no good to try to copy them. Understand that the reason they have been able to break the rules successfully is that they know the rules inside and out. So my advice to you is learn the rules, but keep your eyes open for examples of wonderful designs that have successfully broken those rules. Look at them critically to understand why they have been able to make that leap. And when you are ready, don't be afraid to be daring. Remember, it's not brain surgery, and there is rarely only one solution to a design problem, so be bold.
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Lane Medical Library
Reference Desk
Google Health News
- Sniff test may help detect autism earlier - NEWS.com.au
- US: First measles death in 12 years - Independent Online
- Cystic fibrosis hope as new gene therapy improves condition - Telegraph.co.uk
- Could insulin pills prevent diabetes? Big study seeks answer - Washington Post
- FDA to evaluate risk of codeine cough and cold meds for children - CNN
- Thailand's First MERS Patient to Leave Hospital - NDTV
- New Brain Research Could Solve Memory Loss Issues - Pioneer News
- The Truth About Cubas HIV Breakthrough - Daily Beast
- Treatment for cystic fibrosis approved - Columbus Dispatch
- Brain-Eating Amoeba Kills California Woman - Huffington Post
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UpToDate: A point-of-care clinical information resource containing succinct and aggressively updated clinical topic reviews.PubMed: provides fulltext access to Lane's resources. Includes the MEDLINE database, which contains coverage of over 5000 journals and more than 16 million citations for biomedical articles, including, but not limited to, clinical trials, systematic reviews, case reports, and clinical practice guidelines.Lane RSS feeds (Really Simple Syndication)
Limit your PubMed search to Cochrane Reviews, AHRQ Evidence Reports, BMJ Clinical Evidence topics, FPIN Clinical Inquiries, and ACP Journal Club article reviews. Limit your PubMed search to systematic reviews, meta-analyses, reviews of clinical trials, evidence-based medicine, consensus development name conferences, guidelines and citations to articles from journals specializing in review studies of value to clinicians.The National Guideline Clearinghouse (NGC): A comprehensive database of evidence-based clinical practice guidelines and related documents.PubMed Guidelines: PubMed search restricted to articles identified as "guidelines"PubMed Reviews: Reviews limited to selected pediatric journals, core clinical journals, past 5-years, and English-language.eMedicine: A point-of-care clinical information resource containing articles on 7,000 diseases and disorders. The evidence-based content, updated regularly, provides the latest practice guidelines in 59 medical specialtiesMedlinePlus: A repository of health information from the National Library of Medicine. Links are from trusted sites. No advertising, no endorsement of commercial companies or productsLPCH CareNotes via MicroMedex: Patient education handouts customized by LPCH clinical staffBMJ Clinical Evidence. A clinical information tool built around systematic reviews summarizing the current state of knowledge about prevention and treatment of clinical conditionsMicroMedex: Premier pharmaceutical information source containing multiple databases and drug reference tools. Of particular value is DRUGDEX Evaluations, one of the most comprehensive drug sources available.Micromedex Lab Advisor: Evidence based laboratory test information


text:
Pronunciation: Coolah
Added on: 2009-09-03 Listened: 79 timesin: Australia, Village, place names, drink
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Can you pick a slightly different hue out of a lookalike lineup?
A New Bathroom With Vintage Charm
Pattern, pattern everywhere — Persian Pear wallpaper by Joanna Rock envelops the master bedroom in a Lake Michigan house designed by Martin Horner of Soucie Horner. The Simla sleigh bed from Robert Lighton is accented with throw pillows from Lynda O'Connor and Max & Company on top of embroidered pillow shams by Jed Johnson. Wisteria's Moorish chest doubles as a night table. Rug by Oscar Isberian.
In the master bedroom of an Birmingham, Alabama, apartment decorated by Doug Davis and Hannon Kirk Doody, a pair of chairs by Lee Industries is covered in the company's Serengeti with leather piping.
Your Favorite Rooms of the Week


text: I want to buy a Carradice Camper Longflap saddle bag for commuting and light weekend touring. My saddle is a specialized something or other (forgot the model) without bag loops. I know I'll need something to attach the straps too but I'm sure what to get. There is this thing, but will I need some kind of support as well? I can buy this but will I still need the other thing to attach the bag too? It looks like it has a place to attach the straps already intergrated into the frame but I'm not sure. What do you all think?


text:
question about banding on 5d Mark II
Started May 20, 2012 | Discussions thread
Regular Member • Posts: 117
Processing everything in 16 Bit solved all of my problems with that a long time ago.
I never shoot JPEG.
Save to JPEG when you're done, convert back to 8 bit and sRGB.
You should not see banding.
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Stock Market & Financial Investment News
News Breaks
February 21, 2013
PBF Energy announces board approval for rail expansion project
The Board of Directors approved the recently announced expansion project to double the company's heavy crude unloading capability to 80,000 barrels per day by the fourth quarter of this year. Upon completion, PBF Energy will be able to deliver more than 150,000 barrels per day of crude-by-rail directly into the Delaware City refinery.
News For PBF From The Last 14 Days
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July 1, 2015
BofA/Merrill's Top 10 US Ideas for Q3
BofA/Merrill published its Top 10 U.S. Ideas Quarterly for Q3 that identifies companies it sees having significant catalysts in the next three months. The list includes eight Buys and two Underperforms. The Buys are Applied Materials (AMAT), Boise Cascade (BCC), Burlington Stores (BURL), Comcast (CMCSA), Comerica (CMA), Eli Lilly (LLY), Facebook (FB), and International Game (IGT). The Underperforms are BlackBerry (BBRY) and PBF Energy (PBF).
June 18, 2015
PBF Energy acquisition positive for PBF Logistics, says Wells Fargo
After PBF Energy (PBF) announced a deal to acquire the Chalmette Refinery and associated midstream logistics assets, Wells Fargo said the transaction should be positive for PBF Logistics (PBFX) since it increases the parent company's potential drop-down inventory. The firm reiterated its Outperform rating on PBF Logistics.
On The Fly: Top stock stories at midday
Stocks on Wall Street opened in positive territory and continued to tack on gains throughout the morning. The move has been broad based, with each of the major averages gaining at least 1%. The move may be reflecting investors feeling a sense of relief following yesterday's FOMC meeting, after which the central bank repeated its promise to be gradual in raising rates, without specifying when rate "lift-off" will commence. ECONOMIC EVENTS: In the U.S., the consumer prices index rose 0.4% in the month of May, versus expectations for a 0.5% increase. The core reading, which removes food and energy prices, rose 0.1%, versus the expected increase of 0.2%. Initial jobless claims dipped 12,000 to 267,000 in the week ended June 13, versus expectations for 277,000 first-time claims. The Philadelphia Fed manufacturing survey climbed 8.5 points to 15.2 in June, topping expectations for a reading of 8. The leading indicators rose 0.7% to 123.1 in May, topping expectations for a rise of 0.4%. In Europe, International Monetary Fund chief Christine Lagarde reportedly said Greece won't be given a grace period if it fails to make its scheduled payment to the fund on June 30. Meanwhile, finance ministers of euro area member states are set to meet today, but expectations for progress on Greece are low. Also, the Norwegian central bank cut interest rates to a record low, while the Swiss National Bank kept its deposit rate at record lows as well. In Asia, the Shanghai composite index tumbled 3.7% overnight, doubling its weekly decline to 7.4% and increasing fears that a bubble in the nation's stock market may be preparing to burst. COMPANY NEWS: Shares of enterprise software, hardware and IT services provider Oracle (ORCL) dropped 7% after the company reported lower than expected fourth quarter earnings per share and gave a lower than expected forecast for profit in its new quarter. Analysts at several firms, including Susquehanna, Wells Fargo and Cantor, suggested the post-earnings drop may be a buying opportunity, citing progress in the company's cloud efforts... Fitness tracker maker Fitbit (FIT) surged nearly 50% in its first day of public trading. The company priced its initial public offering at $20 per share, topping expectations, then opened at $30.40 per share, giving it a market value near $6.3B in early trading. Near noon, the stock had pulled back a bit to trade just below $30 per share. MAJOR MOVERS: Among the notable gainers was PBF Energy (PBF), which gained 18% after announcing that its subsidiary has signed a definitive agreement to purchase Chalmette Refining, a joint venture between affiliates of Petroleos de Venezuela and ExxonMobil (XOM). Also higher was BioMarin (BMRN), which rose 13% after the company reported results from a study of BMN-111 in dwarfism and several analysts hiked their price targets on the stock to reflect the increased probability of success for the drug. Among the noteworthy losers following their earnings reports were Bankrate (RATE), which fell 18%, and Jabil Circuit (JBL), which dropped 7%. INDEXES: Near midday, the Dow was up 218.44, or 1.22%, to 18,154.18, the Nasdaq was up 74.96, or 1.48%, to 5,139.84, and the S&P 500 was up 23.57, or 1.12%, to 2,124.01.
Exxon Mobil to sell 50% interest in Chalmette Refining to PBF Energy
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PBF Energy to acquire Chamlette refinery, related logistics assets for $322M
PBF Energy (PBF) announced that its subsidiary has signed a definitive agreement to purchase Chalmette Refining, LLC, consisting of the 189,000 barrel per day Chalmette Refinery and related logistics assets, from ExxonMobil (XOM) and PDV Chalmette, LLC. With the acquisition PBF will increase its total throughput capacity to over 725,000 barrels per day. The purchase price for the assets is $322M, plus working capital including inventory to be valued at closing. PBF Energy does not expect to issue equity to finance any portion of this transaction. The transaction is expected to be approximately 20% accretive to 2016 consensus earnings in the first year of operation and is expected to close prior to year-end 2015, subject to customary closing conditions and regulatory approvals. The Chalmette Refinery, located outside of New Orleans, Louisiana, is a 189,000 barrel per day, dual-train coking refinery with a Nelson Complexity of 12.7 and is capable of processing both light and heavy crude oil. The facility is strategically positioned on the Gulf Coast with strong logistics connectivity that offers flexible raw material sourcing and product distribution opportunities, including the potential to export products. In addition to refining assets, the transaction includes 100% ownership of the MOEM Pipeline, providing access to the Empire Terminal, as well as the CAM Connection Pipeline, providing access to the LOOP facility through a third party pipeline. The transaction also includes 80% ownership in each of the Collins Pipeline Company and T&M Terminal Company, both located in Collins, Mississippi, which provide a significant clean products outlet for the refinery via the Plantation and Colonial Pipelines. Also included in the transaction are a marine terminal capable of importing waterborne feedstocks and loading or exporting finished products; a clean products truck rack which provides access to local markets; and a crude and product storage facility with approximately 7.5 million barrels of shell capacity. PBF estimates the acquired MLP-qualifying pre-tax earnings from the logistics assets of Chalmette Refining, LLC, to be at least $30M.
PBF Energy to acquire Chamlette refinery, related logistics assets
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text:
Commanding magnificent views across Carmarthen Bay from its stunning cliff-top setting between award-winning Saundersfoot and Amroth beaches, this peaceful park offers couples and families a perfectly located base for exploring Pembrokeshire's majestic National Park.A superb indoor pool, adventure playground and bar number among an array of on-site facilities. While all the traditional seaside delights of eternally popular Tenby await nearby.
This park has:Indoor heated poolWi-Fi accessBarSnooker/poolOutdoor adventure playground
Real reviews, from customers like you.
Up to
10th - 26th APRIL
All breaks for all arrivals
Clean comfortable caravan in pleasant surroundings, very close to lovely walks to nearby beaches, very friendly and helpful staff
The accomodation was excellent we could not have asked for better and the views over the sea to Caldey Island were superb
We had a lovely long weekend stay at Meadow House Park. The caravan was warm, clean and comfortable. The area is beautiful even in wet weather. the staff were vry friendly and helpful. we intend to go back there.
Brilliant holiday we were blessed with sunny warm weather. People we're so helpful and polite, clean beach, ideal location for a family holiday.
The park was clean and tidy, again people we're welcoming, children could play safely
We had a wonderful holiday, spacious, beautiful and spotlessly clean caravan. Very helpful staff, great location
Lovely site and friendly staff. Caravan was in excellent condition and very clean. Perfect for a weekend away.
This is a very well kept park, spotless accommodation and in a quiet, relaxed setting. It was nice to come back to after a day in the busier areas. It has a lovely 15 min walk down to Amroth beach which was uncrowded and has a store and family restaurants. There is also the Colby Woodlands Garden very close by which is a great.
It would have been helpful to have had a clothes airer or washing line provided for drying wet towels etc. Also the internet connection was non-existent in our caravan.
Brilliant holiday, very family friendly and staff were extremely accommodating when we enquired about staying another night
loved it so relaxing and location great


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Secure Air Parks Edinburgh is just a one-minute transfer away from the airport terminal. It's technically classed as an off-airport car park and it offers off-airport parking prices. But because it's so close to the airport grounds, it's ultimately convenient too. The car park's name doesn't lie - it's a fully secured place to store your car and has all the facilities to ensure it's kept safe while you're away.
|Car Park
|Transfer Time
|Keep Keys
|8 Day Price
|Edinburgh Secure Air Parks
|3 mins
Transfers:Transfers run 24 hours a day, every five to 10 minutes and take just one minute.
Security: The car park holds the Park Mark award for safer parking. It has 24-hour CCTV surveillance and perimeter fencing.
On arrival at Secure Air Parks: On arrival at the car park, please report to reception and present your printed email booking confirmation or quote your reference.
On your return to Secure Air Parks: Full return instructions will be advised when you arrive at the car park.
Important information:
There are allocated disabled parking spaces
Trailers are not permitted
Directions are supplied with your booking confirmation.
Directions from postcode:
Park and Fly Edinburgh Very easy and straight forward. Car was parked for us on departure and waiting for us on arrival. Mrs Dill (5 out of 5)
Start your holiday early and relax the night before you fly
If you are driving to the airport, an airport hotel with parking is a great way to travel. Relax in comfort at the hotel and simply park on-site, or at a nearby location.
We're proud to be working with...
© Airparks.co.uk 2015
Airparks Services Limited, registered in England and Wales, No 2652781, registered office 100 Mackadown Lane, Birmingham, B33 0JD.


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Welcome to the home of the most dedicated autoclicker in the world :-)
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text:
When you upgrade Custom Category Fields from 0.1 to 1.0, 1.0.1 or 1.2 (current) any custom data you added in 0.1 will disappear. You can recover it by running these two SQL commands:
insert into wp_ccf_Fields(field_name,field_type,category_type) select distinct kkeys,'text','Category' from wp_cat_fields;
insert into wp_ccf_Value(field_name,field_value,term_id) select kkeys,kval,cat_id from wp_cat_fields;
Note that this doesn't take advantage of any of the new field types.
Also, you'll have to replace all calls to
get_cat_fields() with their 1.x analogues, e.g.
get_cat_fields($cat, $field) is replaced with
categoryCustomFields_GetCustomFieldByTaxonomyIdAndFieldName($cat, $field).


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Hostelworld "meet the world" by Lucky Generals
Rating: 5.0
By Matt Williams, campaignlive.co.uk, Thursday, 10 March 2011 12:11AM
The agency won the business after a pitch and will now handle Organix's marketing activity across all channels.
Stephanie Hildon, the marketing director of Organix, made the appointment.
DLKW Lowe's first campaign is due to launch later this year and work is expected to focus heavily on social media activity.
Previously, the incumbent on Organix's advertising account was TMW, which picked up the business in February last year.
During its time working on the business, TMW launched a series of digital campaigns for the brand and developed its CRM activity.
Organix is the bestselling baby-snack manufacturer in the UK, producing a range of cereal, fruit purees and steam-cooked meals. It also produces snacks for toddlers under the Organix Goodies banner.
This article was first published on campaignlive.co.uk
Before commenting please read our rules for commenting on articles.
If you see a comment you find offensive, you can flag it as inappropriate. In the top right-hand corner of an individual comment, you will see 'flag as inappropriate'. Clicking this prompts us to review the comment. For further information see our rules for commenting on articles.
Rating: 5.0
Agency: Grey London
Rating: 5.0


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AFRICA/CENTRAL AFRICA - "The whole country is terrorized": new testimonies on the drama of Central Africa
Bangui (Agenzia Fides) - Fides received further dramatic testimony from the Central African Republic, gripped by clashes between former Seleka rebels and "anti Balaka" self-defense groups, while the French troops of the African Union are doing their best to ensure a minimum of security.
"The whole country is terrorised" refers to Caritas Internationalis Fr. Anicet Assingambi of the parish of St. Carlo of Lwanga, in the north of Bangui, the capital. Tens of thousands of people are seeking shelter in Bangui's church buildings and church compounds throughout the country.
"There are five thousand people hiding in our parish buildings. A woman told me she'd seen her brother gunned down. All men are shot at if they go outside. There is a young man dead on the street outside, but it's too dangerous to go outside to remove his body", says Fr. Assingambi.
"We have nothing to give them" - says Fr . Assingambi. "Their homes have been looted. They're mourning their dead. When they arrived in the church, the children were crying. We sang hymns and said prayers and that has helped calm people down. We have never seen anything as bad as this before. We're at the mercy of God, please pray for us".
The statement sent to Fides says that at the moment it is impossible for Caritas workers to reach out to the people in need.
In Bossangoa in the north of the Country , Caritas fears for the safety of over 40,000 people camped out in the Catholic mission compound and 1,600 people who are hiding in a school. Yesterday, December 6, a rocket landed in the mission miraculously not killing anyone.
The Catholic Church in Central African Republic is calling for an immediate cease-fire and for humanitarian aid to be allowed. (L.M.) (Agenzia Fides 07/12/2013 )
Receive News from Fides Service in your email
- 2015-05-18 - "We have to be humble to have the courage to forgive one another", said the Archbishop of Bangui
- 2015-03-26 - Caritas Bangui distributes aid to the displaced Muslims in Mbaiki
- 2015-03-03 - Archbishop of Bangui visits two militia leaders in prison
- 2015-02-24 - "Our country can be compared to a desert, where we must combat the temptations of the devil" says Archbishop Nzapalainga
- 2015-02-06 - "The conflict is political not religious" say three participants
- 2015-01-28 - Caritas: more funds needed for refugees from the Central African Republic
- 2015-01-14 - "Signs of hope but still too much violence, also due to the strong movement of weapons", say the Bishops
- 2015-01-13 - The situation is improving, the Bishops launch a campaign for social cohesion
- 2015-01-07 - Archbishop of Bangui threatened with death while bringing assistance to the displaced
- 2014-12-30 - Archbishop of Bangui pays a visit to former Seleka rebels again


text:
Does anyone have suggestions for a PHP, Python, or J2EE-based web forum that has a good API for programmatically creating users and forum topics?
Take the 2-minute tour ×
Stack Overflow is a question and answer site for professional and enthusiast programmers. It's 100% free, no registration required.
phpBB would be the first that comes to mind as open-source, simply because it's free.
In reality almost all forum platforms have some sort of 'api' in that you can do whatever you need programatically, it just may not be as simple as 'add_user(bob)'. A few lines of code and a SQL query or two and you can usually achieve everything you need.
Out of personal preference I would recommend vBulletin, however it does have a fee. The benefit of this is that it has a very strong modding community that have probably already accomplished everything you need at http://vbulletin.org.


text:
Unsupervised AI makes up some pretty funny jokes
Unsupervised joke generation from big data [PDF], a paper by University of Edinburgh researchers Sasa Petrovic and David Matthews, describes an ingenious and successful method for teaching a computer to make up jokes like "I like my relationships like I like my source, open;" "I like my coffee like I like my war, cold;" and "I like my boys like I like my sectors, bad." The researchers wrote code that called on Google's n-gram database to find noun-attribute pairs, zero in on nouns with ambiguous meaning, and automatically generate jokes.
Read the rest
Read the rest


text:
We Want Our Gay TV: Showtime and MTV Partner to Create a Gay Cable Channel-But Will They Be First across the Finish Line? (Television)
Meers, Erik, The Advocate (The national gay & lesbian newsmagazine)
While studying the feelings of gays and lesbians about the idea of creating a gay cable TV channel, Showtime senior vice president Gene Falk and MTV consultant Matt Farber got some surprising results. "It blew our researchers away," recalls Falk, describing the data collected for their parent company, media powerhouse Viacom. "The magnitude of the response was the best they have ever seen for a premium service. I'm out. Matt's out. So it wasn't like we were in the dark about this. The gay community is a vastly underserved market in mainstream media."
Viacom agreed. In mid January the conglomerate announced that it is planning a 24-hour premium gay-oriented cable channel, a joint venture between Showtime and MTV--two networks already known for their gay-friendly fare. "The gay market is a large, diverse market, and it's willing to pay for programs, for a voice of its own," says Farber. "Will & Grace is wonderful, but it still needs to conform to make sure it's a top five show. By having a dedicated channel, you can show a diversity of story lines. But there's no question we wouldn't be here without Queer as Folk being a success, without Will & Grace being in the [coveted] Seinfeld time slot."
There's no scheduled launch date (although published reports have speculated at early 2003), but Falk and Farber are hard at work developing a business plan for the yet-unnamed channel. The model calls for charging a modest flat monthly fee for the service, with additional revenue generated through PBS-style program sponsorships.
"The gay community probably represents something north of 10 million adults," says Farber. "That represents significant buying power. Whenever you connect with and give a voice to people, it's always a good business plan."
Predictably, the right wing has come out swinging against the very concept of a gay TV channel. In a recent appearance on Fox News, conservative battleax the Rev. Jerry Falwell commented, "If we are going to go that route, then when are we going to create a channel for those who enjoy kiddie porn? When are we going to do a channel--a legal channel--for those involved in or submitting to bestiality?"
Gay activists have been quick to join the fray, countering that because the channel would require users to subscribe and pay for the service, no one who doesn't actively seek out the channel will see it. After all, cable already works on the principle of niche marketing, as Lifetime TV for women and Black Entertainment Television attest.
Nevertheless, observes Joan Garry, executive director of the Gay and Lesbian Alliance Against Defamation, "it's not surprising to me that the Right has come out against this. They've been loudly opposed to any images of gay people." Garry, who has worked for Showtime and MTV, adds, "[Both networks] have done groundbreaking work before, and it has proved to be both smart business and the right thing to do."
Though no specific programs have yet been selected, planners have plenty of ideas on how to fill the 24-hour slate. "We're going to acquire movies and documentaries that aren't available in video stores," says Farber. "There are potential series. There will be news, talk shows, relationship shows. No doubt, it's going to be a challenge."
Part of the difficulty will be in appealing to all the elements of the wildly diverse gay community in a single channel. "I don't think it's possible for an. organization to represent everyone all the time," says Falk. …


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One thing you shouldn't do is put pressure on the caregiver, Weeks said, as it just adds to their load.
"The phrases 'Call me if you need anything' or 'Call me back' can be stressful," she said. "Better: 'You don't have to call me back. I just wanted you to know we are thinking of you.' Listening is one of the greatest gifts."
Weeks has been on both sides of this equation, having cared for her daughter, Heather, while she battled colon cancer. The foundation was started in her name after she passed away in 2006 to carry on her goals; Heather was employed by the Ovarian Cancer Research Fund before her death.
"When our daughter was seriously ill with cancer, we were so touched by those who reached out to help us with meals, cards, a night out or just that call to tell us they were there for us," Weeks said. "Just showing up and being a good friend is key. Remember — if you are afraid to talk to your friend who's caregiving for a terminal patient, imagine what they are feeling."
To find a support group to fit your needs, visit caregiver.com/regionalresources/states/NY/support/onandaga.htm or call the Onondaga County Department of Aging and Youth at 435-2362.
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text:
Six Nations: France beat England with late converted try in Paris
Last Updated: 02/02/14 2:10pm
Yoann Huget of France dives in to score his second try
Gael Fickou scored a try four minutes from time as France beat England 26-24 in a Six Nations thriller at the Stade de France.
France went ahead after just 30 seconds through Yoann Huget and the winger added a second try midway through the first half as the hosts built up a 16-3 lead.
England hit back through Mike Brown's try just before the half and Luther Burrell's debut try and a drop-goal from Danny Care saw the visitors edge 21-16 ahead.
But Maxine Machenaud's penalty 10 minutes from time set up a thrilling finale and a superb multi-phase move was finished by Toulouse centre Fickou under the posts, with Machenaud kicking the match-winning conversion.
France produced the best half-hour of the Philippe Saint-André era to start the game but progressively ran out of steam against an England side that went from strength to strength.
France dominated at scrum time but England will harbour a number of regrets having seen their pack take the game by the scruff of the neck and dominate most of the second half.
France got off to the perfect start when Huget latched onto a grubber from debutant fly-half Jules Plisson, and raced in after just 32 seconds.
Jean-Marc Doussain missed the conversion and Owen Farrell pulled a penalty back for England before Doussain hit the target with his second kick at goal to leave France 8-3 ahead at the end of a gripping opening 10 minutes.
The frantic pace continued as Huget bagged his second try of the first quarter. The French wing eluded a poor tackle from Alex Goode before passing inside to Brice Dulin, whose chip ahead bounced favourably for his flying team-mate.
Again Doussain missed the conversion but found the mark shortly afterwards as France's superiority at the scrum earned a penalty that gave the hosts a 16-3 lead.
Bold decision
England finished the half strongly however, and Brown's try just before the break kept his team alive. Danny Care took the bold decision to run a very kickable penalty but he linked with Billy Vunipola and Brown beat three defenders, stretching an arm out over the whitewash in the corner.
Farrell missed the conversion but England came out of the dressing room after the interval breathing fire.
Care was denied a try when he was held inches short of the line but Farrell reduced the gap to five points with a penalty.
England drew level after the irrepressible Vunipola bust through the French defence before offloading to Burrell, who could cruise in untouched under the sticks.
Farrell's conversion gave England the lead for the first time as France's fast start seemed like a distant memory.
A cheeky drop-goal from Care - with a penalty coming - ensured England got something for their sustained pressure as the hosts' composure disintegrated.
Machenaud and Goode traded penalties but although England clearly held the upper hand, they were unable to land the killer blow as Tom Youngs failed to find his jumpers at a line-out five metres out, letting a golden opportunity slip by.
With the game hanging in the balance and France finally finding some attacking poise, Dimitri Szarzewski bolted into space, held his line before finding Fickou, who dummied past Goode to score under the posts and leave Machenaud with an easy conversion.
Six Nations 2014: France v England
Praise for England
Dewi Morris praises Billy Vunipola, Danny Care and Owen Farrell despite England's loss to France.
Lancaster defends strategy
England head coach Stuart Lancaster has defended his use of replacements during their 26-24 defeat by France in Paris.
Robshaw: We must start better
England captain Chris Robshaw admits his side cannot afford any more slow starts in the Six Nations this season.
- Lancaster proud in defeat
- Catt: Wings have free rein
- Backing for England new boys
- Backing Les Bleus
- France v England: Teams
- Pressure to perform
- Pre-match nerves for Burrell
- Lancaster: Debutants to cope
- The next level
- England name Nowell, Burrell
- Plisson to start for France
- Robshaw: Beating France a must
- England ponder prop selection
- Vunipola relishing French test
- Ashton axed from England squad
- Lawes: Keep your discipline
- Rowntree: England pack a punch
- Baptism of Fire


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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
39 year old woman | Houston, TX, USA | Seeking men 34-46 within 25 miles
Lust is easy. Love is hard. Like is most important. ~Carl Reiner~
Hello!! I have been in Houston, Texas for over a year now and I love the diversity here and all the events catered to the different cultures. The suburban lifestyle is something I am adapting to but it is great that the city life is approximately 30 minutes away. On the weekends I am usually out and about, exploring an art exhibit/festival, concert or taking a short road trip to Galveston or a long road trip to Austin or San Antonio. I like to maintain active but I also do enjoy my weekends at home cleaning and watching movies. I focus on being a positive person and like to be around positive energy as well, life is too short to live negatively. I believe in embracing the moments that made you smile and learn from the ones that didn't. What I am looking for on Match is to meet my best friend and life partner. That person to call when something funny, scary, serious or exciting has just happened to you, that someone that knows just how you take your coffee, the one you take road trips with and make up corny games to play along the way, have belly aching laughs with, knows how to compliment a lady, adventurous and that someone you feel safe in their arms. The character qualities I seek in my partner are humor, integrity, wit, attentive, confident, benevolence, family oriented, pet enthusiast, easygoing, non-pretentious, articulate and a good listener. Have a great day!
Camping , Coffee and conversation , Dining out , Hobbies and crafts , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Nightclubs/Dancing , Performing arts , Playing cards , Political interests , Religion/Spiritual , Travel/Sightseeing , Volunteering , Wine tasting
Billiards / Pool, Other types of exercise, Tennis / Racquet sports, Walking / Hiking, Yoga
I have Dogs
My brat is my love, yes I do humanize my dog :)
Middle of the Road
New venues with a unique ambiance, Paradise Island, Hamptons, Central Park
John Jay College of Criminal Justice of the City University of New York, New York, NY
True Blood, Sopranos, So you think you can dance, decorating, reading, road trips, lavender, sunsets
Concerts, Museum, The theater, movies, park, beach, bars, lounges, camping, festivals
Aleph by Paulo Coehlo and one of favorites is Tuesdays with Morrie..
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'0" (152cms)
|5'9" (175cms) to 6'2" (187cms)
|Body type:
|About average
|Slender, About average, Athletic and toned, A few extra pounds
|No preference
|Auburn / Red
|No preference
|No Way
|No Way, Yes, but trying to quit
|Social Drinker
|No preference
|Financial / Accounting / Real Estate
|No preference
|I'll tell you later
|No preference
|Never Married
|Never Married, Divorced
|Have kids:
|No, Yes, and they live away from home, Yes, and they live at home
|Wants kids:
|Latino / Hispanic
|Asian, Latino / Hispanic, Middle Eastern, Pacific Islander, White / Caucasian
|Christian / Other
|No preference
|English, Spanish
|Associates degree
|Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral


text:
Welcome to Smyrna Soccer Club! Originally founded in 1988 as Smyrna West Soccer Association, we decided to simplify our name and changed it to Smyrna Soccer Club several seasons ago. That first season we had roughly only 40 players. Now in our 24th year, we have over 1,200 members and are continuing to grow each season as the wonderful sport of soccer continues to gain in popularity, not only here in Smyrna but across the entire globe.
It is important to know that the board members of this league are all volunteers. We take tremendous pride in providing the youth of our community with a program that is instructional, educational and fun at an extremely affordable price to our members, especially compared to other leagues in the metro-Atlanta area. Please understand that the three to four weeks prior to the season starting are our busiest time, so if it seems we're a little frazzled it is only because we are! Can we do things better? Sure, but as volunteers this league depends on help from you. We will be a little better or a little worse depending on the amount of help you give. If you would like to help, please contact us.
Eric Homansky
John Robinson
2688 Atlanta Road Smyrna, Georgia 30080