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|Publication number
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|Application number
|Publication date
|Feb 21, 1989
|Filing date
|Sep 30, 1985
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|Sep 30, 1985
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|Robert J. Carrico, William L. Patterson
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|Miles Inc.
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|Patent Citations (6), Non-Patent Citations (11), Referenced by (71), Classifications (18), Legal Events (6)
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This invention relates to means of immobilizing nucleic acids such as DNA and RNA on solid supports. Immobilized nucleic acids find particular use as probes for determining the presence of particular polynucleotide sequences by hybridization with complementary single stranded polynucleotides. Nucleic acid hybridization is useful as an analytical method in the fields of human and veterinary medicine, agriculture, and food science, among others. In particular, the method can be used to detect and identify etiological agents such as bacteria and viruses, to screen microbes for antibiotic resistance, and to detect malignant cells.
Hybridization assays commonly involve the immobilization of either the nucleic acids present in the test sample or the probe nucleic acid. Such solid-phase techniques are concluded with the detection of hybrids formed on the immobilized phase between the probe and complementary sample polynucleotides. By far the most commonly used matrix for immobilization of nucleic acids in these methods has been microporous nitrocellulose membranes. More recently, microporous nylon membranes have become popular because they have better mechanical strength than nitrocellulose. Some manufacturers have introduced positive ionic groups such as quaternary ammonium ions into nylon membranes to improve their wetting proprieties. All of the known nitrocellulose and nylon membranes used to immobilize nucleic acids require high salt to adsorb the polynucleotides to their surface and baking at around 80� C. to permanently fix the adsorbed DNA or RNA.
Detection of resulting immobilized hybrids formed on the solid matrix is conveniently accomplished by the addition of a detectable protein reagent that binds specifically to the hybrids. Normally such protein reagent will comprise an antibody or other binding protein that is specific for binding to a ligand moiety on the probe nucleic acid or to the unique configuration of the hybrid itself. Examples of the former are the detection of probe nucleic acids bearing a biotin or a hapten group by binding of avidin or anti-hapten antibody. Examples of the latter are the use of antibodies selective for DNA.RNA, or RNA.RNA duplexes or intercalated or otherwise antigenically modified duplexes. The specifically binding protein reagent is labeled with a detectable component, commonly an enzyme.
The problem with use of enzyme-labeled, or otherwise detectable, protein reagents to determine hybridization on the conventionally known solid matrices is nonspecific adsorption of such reagent. This nonspecific binding limits the sensitivity of the overall assay procedure. Accordingly, there is a need for better solid matrices for immobilizing nucleic acids, matrices which do not require high salt or baking in order to obtain efficient and stable immobilization. Further, such new matrices are needed particularly for use in hybridization assays, and particularly where resulting hybrids are detected with labeled protein reagents. Also, since hybridization procedures typically require several incubation and washing steps, the known microporous membranes are not amenable to rapid processing because they are fragile and difficult to handle. A solid, more rigid support material would overcome these problems.
It has now been found that nucleic acids can be efficiently and stably immobilized on a solid support or matrix comprised of nylon having amide groups which have been derivatized to amidine residues. Such derivatization is conveniently accomplished by anhydrous activation of the amide groups of nylon by treatment with an alkylating agent such as a trialkyloxonium salt, followed by anhydrous reaction of the alkylated amide groups with an appropriate amine. The precise nature of the interaction between the nucleic acid and the amidine derivatized nylon support is not fully understood, however, it is believed that electrostatic and perhaps other noncovalent forces are principally involved.
The present method of immobilizing nucleic acids is characterized by several advantages. There is no need for the presence of high salt concentrations to attain adsorption of nucleic acids to the modified nylon surface. Further, there is no need to bake the adsorbed nucleic acids in order to obtain a stable immobilization to the solid support. A particular advantage where proteinaceous reagents such as enzyme-labeled conjugates are to be used in the detection of hybrids formed on the support is that the modified nylon surface exhibits a very low nonspecific binding of such reagents, enabling more sensitive detection limits to be achieved.
The extent of derivatization of amide groups in the nylon support necessary or desired for a particular nucleic acid hybridization will normally be determined through routine experimentation. Thus, reaction conditions can vary widely depending on the particular needs for the immobilized nucleic acid. Using a bead form of the nylon support as an example, in the normal case it is anticipated that anywhere between 100 and 400 nanomoles of the available amide groups exposed on the surface of a 4.8 mm diameter bead become derivatized. This gives 140 to 560 nanomoles of amidine groups per square centimeter assuming the surface to be smooth. Derivatization of between about 200 and 250 nanomoles of exposed amide groups on such a 4.8 mm diameter bead has been found to be particularly useful in immobilizing nucleic acids.
The first step in the derivatization process involves activation of the amide group with an appropriate alkylating agent under anhydrous conditions. Activating agents will serve to -alkylate the amide to form an imidate salt. Upon subsequent reaction with an amine, amidine residues are formed. Useful alkylating agents include dialkyl sulfates, alkyl triflates, alkyldiphenyl sulfonium salts, alkyl perchlorates, and particularly the trialkyloxonium salts such as the lower alkyl salts, preferably trimethyloxonium and triethyloxonium salts. The salt counteranion will generally be selected from hexachloroantimonate, hexafluorophosphate, and tetrafluoroborate, with the last named counterion being preferred.
The amine employed can be selected from primary and secondary aliphatic and aromatic amines. They can have one or multiple primary and/or secondary amine groups. For example, diaminoethane, diaminohexane, spermidine, spermine and polyethyleneimine are useful. The amine should have at least one primary or secondary amine group to react with the activated amide but, in addition, it can contain cationic or potentially cationic groups such as tertiary and quartenary amines and guanido residues. Particularly useful amines are diamines and polyamines. Good results have been obtained with diamines which result in the formation of aminoamidine residues on the nylon support. Such diamines include alkane diamines, particularly α,ω-alkane diamines having between 2 and 12 carbon atoms, such as 1,6-hexane diamine, ethylenediamine, putresceine, and cadoverine. The resulting amidine residues have the formula: ##STR1## wherein Q is the residue from the amine used to react with the alkylated nylon and X is a counter anion. Where diamines are used, Q will be --R--NH2 where R is preferably alkylene, phenylene, alkyphenylene, or phenalkylene.
A particularly useful general method for derivatizing the nylon support according to the present invention begins with treatment of the support with a solution of trialkyloxonium salt, e.g., trimethyloxonium tetrafluoroborate, in a nonaqueous solvent such as methylene chloride, carbon tetrachloride or diethyl ether. The incubation will proceed for between about 11/2 and about 5 hours, preferably with agitation, with about 3 hours being optimal. The temperature of the reaction can vary widely and is primarily a matter of choice. Room temperature treatments are normally used. The activated support is removed from the nonaqueous solution, optionally followed by a series of washings with the nonaqueous solvent. The activated nylon is then reacted with the selected amine, e.g., an alkane diamine such as a 1,6-hexane diamine, in a nonaqueous solvent such as those mentioned above. This reaction is allowed to proceed for about 1 to about 5 hours under ambient conditions, with a reaction time of at least about 3 hours being preferred. Preferably, the derivatized support will then be washed extensively, first with a nonaqueous solvent and finally by water or buffer overnight or longer as desired. The end product can be stored under water or buffer or in dried form.
The nylon support is generally contemplated to comprise any polyamide, including those composed of αω-aminocarboxyl acid monomers as well as those condensed from diamine and dicarbocylic acid monomers. The activation procedure can be applied to any polyamide, regardless of the length of the monomeric units. Aromatic, alkyl, or alkenyl backbones will all give aminocarbalkoxy-substituted nylon. Similarly, the backbone and amide groups can be widely substituted, however, certain functional groups, if present, such as carboxyl, hydroxyl, phenol, and amines, may be modified during alkylation. This can be tolerated so long as the ultimately derivatized nylon serves a substantial function to adsorb nucleic acids.
The conformation and general composition of the support can vary as desired for its application to nucleic acid immobilization provided that there are nylon amide groups exposed at its surface for derivatization and interaction with nucleic acids. The support can be in the form of beads, strips, microtiter wells, test tubes, microporous membranes, and the like. Beads have been found to be particularly advantageous due to their manipulability and high surface area. Particular use has been made of nylon beads having diameters in the range of 1 μM to about 1 cm. Supports comprised uniformly or nonuniformly of nylons can be used, or one can use nylon coated on a non-nylon core or base.
The derivatized nylon support of the present invention can be used to immobilize nucleic acids in general, including DNA, RNA, and derivatives or modifications thereof, comprising any desired number of bases. Genomic and plasmid nucleic acids as well as restriction fragments thereof and synthetic oligonucleotides can be immobilized according to the present invention.
Typically, a desired nucleic acid or population of nucleic acids will be immobilized on the derivatization nylon support by incubation of the support in a buffered solution or dispersion of the nucleic acid. The buffer will preferably be of low ionic strength, normally having a salt concentration of about 0.5M or less and a pH between about 4 and about 10, with about 7 being preferred. Useful buffers include phosphate, carbonate, Tris, and the like, and will contain nuclease inhibitors such as ethylenediamine tetraacetic acid (EDTA), sodium dodecylsulfate, or aurin tricarboxylic acid. The incubation will proceed for between about 1 and 4 hours to obtain saturation of the binding sites. Shorter incubation times can be used where adsorption of less than saturating amounts of the nucleic acid is acceptable. The temperature of the incubation will be preferably between 20� and 60� C., with slightly elevated temperature being preferred, e.g., 50� C. Upon completion of immobilization, the support will then preferably be incubated in a solution of nonspecific nucleic acid, salmon sperm DNA being well known for this purpose, to saturate unoccupied nucleic acid binding sites.
The immobilized nucleic acid can be used in affinity chromatography and purification methods using single- or double-stranded nucleic acids as the affinity ligand, and will find particular application to hybridization assays to detect a particular polynucleotide sequence in a test sample such as biological fluid. In general, the present means for immobilizing nucleic acids can be used in any hybridization protocol involving the use or formation of a solid-phase polynucleotide.
In typical hybridization assays either the sample nucleic acid or a probe nucleic acid is in an immobilized form when brought into contact with the other for purpose of determining complementarity. The probe will have at least one single stranded base sequence substantially complementary to the sequence of interest. The present method for immobilization will in general be more useful for providing immobilized probe to the assay because extended incubation times can be used in manufacturing or preparing this element without increasing the time necessary for the actual assay.
The formation of resulting hybrids, indicating the presence of the sequence of interest in the sample, can be detected in a number of ways. As is known in the art, the one of the sample nucleic acid and the probe which is not immobilized can be labeled with a detectable marker, such as a radioisotope, fluorescer, chemiluminescer, enzyme, or specifically bindable ligand. The amount of such label that becomes associated with the nylon support is thus directly related to the degree of hybridization. Alternatively, dual hybridization methods are known wherein a first portion of the sequence of interest hybridizes with immobilized first probe and is detected by hybridization of a mutually exclusive second portion of the sequence of interest with a labeled or otherwise detectable second probe.
A particularly attractive approach to labeling the probe is to incorporate a binding site for a specific binding substance into the probe molecule either by selection of specific nucleotide sequences or by chemical modification of the probe. Examples of binding sites existing in the nucleotide sequence are where the probe comprises a promoter sequence (e.g., lac-promoter, trp-promoter) which is bindable by a promoter protein (e.g., bacteriophage promoters, RNA polymerase), or comprises an operator sequence (e.g., lac operator) which is bindable by a repressor protein (e.g., lac repressor), or comprises rare, antigenic nucleotides or sequences (e.g., 5-bromo or 5-iododeoxyuridine, Z-DNA) which are bindable by specific antibodies (see British Patent Specification No. 2,125,964). Binding sites introduced by chemical modification of the probe are particularly useful and normally involve linking one member of a specific binding pair to the probe nucleic acid. Useful binding pairs from which to choose include biotin/avidin, haptens and antigens/antibodies, carbohydrates/lectins, enzymes/inhibitors, and the like. Where the binding pair consists of a proteinaceous member and a nonproteinaceous member, it will be preferred to link the nonproteinaceous member to the probe since the proteinaceous member may be unstable under the denaturing conditions of hybridization of the probe. Preferable systems involve linking the probe with a ligand such as biotin or hapten and employing labeled avidin or anti-hapten antibody, respectively. Preparation of useful ligand-labeled probes is known, for example, see European Patent Publication Nos. 63,879 and 97,373; and PCT Publication No. 83-002,286.
Hybridization formats which are particularly useful with the present invention are those involving the use of an immobilized polynucleotide probe and determination of the resulting hybrids by binding of a labeled or otherwise detectable hybrid binding reagent, usually a specific binding protein such as an antibody selective for the hybrid. Antibodies which have selectivity for particular duplexes compared to single stranded nucleic acids and duplexes of other types include antibodies to DNA.RNA, and RNA RNA, and to a limited extent DNA.DNA, as well as antibodies to intercalated duplexes.
Antibodies specific for DNA.RNA hybrids can be stimulated by immunogens comprising homopolymeric or heteropolymeric polynucleotide duplexes. Among the possible homopolymer duplexes, particularly preferred is poly(rA).poly(dT) (Kitagawa and Stollar, Mol. Immunol., 19, 413 [1982]). However, in general, heteropolymer duplexes will be preferably used and can be prepared in a variety of ways including transcription of φX174 virion DNA with RNA polymerase (Nakazato, Biochem., 19, 2835 [1980]). The selected DNA.RNA duplexes are adsorbed to a methylated protein, or otherwise linked to a conventional immunogenic carrier material, such as bovine serum albumin, and injected with a desired host animal [see Stollar Meth. Enzymol., 70:70 (1980)].
Antibodies to RNA.RNA duplexes can be raised against double stranded RNAs from viruses such as reovirus or Fiji disease virus which infects sugar cane, among others. Also, homopolymer duplexes such as poly(rI).poly(rC) or poly(rA).poly(rU), among others, can be used for immunization as above.
Monoclonal antibodies selective for DNA.DNA duplexes are reported in European Patent Publication No. 135,139.
Antibodies to intercalated duplexes are raised against an immunogen which normally comprises an ionic complex between a cationic protein or protein derivative (e.g., methylated bovine serum albumin) and an anionic intercalated nucleic acid complex. Alternatively, the intercalated complex can be covalently coupled to a carrier protein.
The anti-hybrid antibodies described above will be normally labeled with a detectable group as described above to enable ready determination of hybrids formed on the support of the present invention. Alternatively, the antibody reagent can be detected based on a native property such as its own antigenicity. A labeled anti-(antibody reagent) or protein A will bind to the primary antibody reagent to enable its detection.
The present invention will now be illustrated, but is not intended to be limited, by the following examples.
A single stranded DNA probe with a sequence complementary to E. coli 23s ribosomal RNA was adsorbed onto aminoamidine derivatized nylon beads. The probe was hybridized with E. coli 23s RNA and the hybrid was detected with antibody to DNA.RNA hybrids.
Burnished nylon beads (4.8 mm in diameter, available from Precision Plastic Ball, Co., Chicago, IL, USA) were placed in methylene chloride, approximately 20 ml per 100 beads. Trimethyloxonium tetrafluoroborate at 0.3 g per 100 beads was added and the solution vigorously stirred for 30 minutes. The beads were removed and quickly washed with methylene chloride, and shaken for 4 hrs in a fresh solution of 1,6-hexanediamine in methylene chloride at 18 mg/ml, 20 ml per 100 beads. The beads were washed with methylene chloride and then with generous amounts of deionized water overnight. They were dried in vacuo at 40�-50� C.
The beads were assayed for amine groups using the TNBS assay. One bead was shaken at room temperature for 3 hours with 0.85 ml of 0.1M Na2 B4 O7 buffer, pH 9.3 containing 1.17 mM trinitrobenzene sulfonate (TNBS) which reacts with amines on the surface and stains the bead yellow-orange. Then 150 μL of 30 mM glycine was added to react with excess TNBS for 30 minutes. A 40 μL aliquot of the reaction mixture was diluted into 0.96 mL of 0.1M sodium phosphate buffer, pH 7.0 containing 1.5 mM sodium sulfite and the absorbance at 414 nm was recorded. The total TNBS employed in these reactions was determined with a control prepared as described above except a bead was not included.
A calibration curve is prepared by reacting various levels of glycine (from 0 to 0.8 mM) with 1.6 mM TNBS in the Na2 B4 O7 buffer for 30 minutes. Then 40 μL aliquots of these reaction mixtures are diluted into 0.96 ml of the phosphate/sulfite solution and the absorbances are recorded. The difference between the total TNBS measured without a bead and the amount of excess TNBS after reaction with a bead gives the molar quantity of amine groups on the bead. Usually each bead contained between 200 and 250 nmoles amine groups.
Twenty-five beads were added to 3.0 mL of 50 mM sodium phosphate buffer, pH 7.4, containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 50 μg of probe DNA. The mixture was shaken at 50� C. for 8.5 hours and 0.22 ml of alkali treated salmon sperm DNA (5.2 mg/mL) was added and the shaking was continued for 4 hours. The beads were transferred into 5.0 ml of hybridization solution and they were shaken at 55� C. for 11 hours. The beads were rinsed twice with 2 to 3 mL of 1 X SSPE, 0.1% SDS and used for hybridization (1 X SSPE is 10 mM sodium phosphate buffer, pH 7.7, 0.18M NaCl, 1 mM EDTA).
Control beads were prepared as follows. Fifty-two aminoamidine nylon beads were added to 6.0 ml of sodium phosphate buffer, pH 7.4, containing 0.5 ml of alkali treated salmon sperm DNA (5.2 mg/mL). The mixture was shaken at 50� C. for 17 hours and the beads were transferred to 6 mL of hybridization solution and shaken at 55� C. for 6 hours. They were rinsed twice with 1 X SSPE, 0.1% SDS.
Preparation of DNA.RNA hybrid - The hybrid was prepared.by transcription of φX174 virion DNA with DNA dependent RNA polymerase from E. coli. The procedure was described by Nakazato (1980) Biochem. 19:2835.
Preparation of methylated thyroglobulin Bovine thyroglobulin (Sigma Chemical Co., St. Louis, MO, USA), 100 mg, was combined with 10 ml of anhydrous methanol and 400 μL of 2.5M HCl in methanol. The mixture was allowed to react for 5 days on a rotary mixer at room temperature. The precipitate was collected by centrifugation and washed twice with methanol and twice with ethanol. Then it was dried under vacuum overnight.
Immunization of mice--One hundred fifty micrograms of DNA.RNA hybrid in 250 μl of 20 mM Tris-hydrochloride buffer, pH 7.4, 1 mM EDTA was combined with 150 μg of methylated thyroglobulin in 250 μl water. A precipitate formed and 2.5 ml of the Tris buffer was added. The entire mixture was emulsified with an equal volume of Freunds adjuvant. BALB/c mice were each immunized with 0.5 ml of the suspension. Booster immunizations were given 3 weeks later and at one week intervals thereafter. Blood was taken at two week intervals beginning one week after the first boost.
Antibody titers in the serums were measured by an enzyme labeled immunosorbent assay method. Immulon II (Dynateck, Alexandria, VA, USA) microtiter wells were coated with DNA.RNA by placing 50 μl of a 5 μg/ml solution in each well. The DNA.RNA was in 0.015M sodium citrate buffer, pH 6.8, containing 0.15M NaCl. When the solutions had stood at room temperature for 2 hours, the wells were washed with 0.02M sodium phosphate buffer, pH 7.4, containing 5 mg bovine serum albumin/mL and 0.5% Tween 20 detergent (v/v). Appropriate dilutions of antiserums were added to the wells to allow binding of the antibodies to the immobilized DNA.RNA. Then the bound antibodies were detected with enzyme labeled anti-mouse IgG by well-known procedures. Spleen cells from a mouse with a high serum titer to DNA.RNA but low titer to single stranded DNA were fused with myeloma cells to produce hybridomas (Stuart et al [1981]Proc. Natl. Acad. Sci. USA, 78:3751; Galfre and Milstein [1981]Meth. in Enzymol., 73:1;) e.g., the cell line deposited with the American Type Culture Collection, Rockville, MD, USA as ATCC HB 8730.
Cloned hybridomas are grown intraperitoneally in mice to produce adequate quantities of antibody for further work. Antibody was isolated from the ascites fluid by anion-exchange high pressure liquid chromatography.
Each bead was placed in 150 μL of hybridization solutioncontaining the indicated amount of 23s RNA. They were incubated at 55� C. for 30 minutes in 0.5 ml of this solution and finally rinsed once more.
RNA.DNA hybrids formed on the beads were measured by immunoassay. Each bead was shaken at room temperture for 30 minutes in 50 μL of 0.02M sodium phosphate, pH 7.4, 0.15M NaCl, 0.5% (w/v) bovine serum albumin, 0.5% (v/v) Tween 20, 1 mM EDTA (PBS/BSA/Tween/EDTA) and then 100 μL of this solution containing 0.1 μg antibody to RNA.DNA was added. The shaking was continued for 30 minutes and the beads were rinsed twice (0.5 ml each) with 50 mM sodium phosphate buffer, pH 7.4, 0.5% (v/v) Tween, 0.5% (w/v) BSA, 1.0 mM MgCl2 (phosphate/BSA/Tween/MgCl2) One hundred-fifty microliters of phosphate/BSA/Tween/MgCl2 containing β-galactosidase antimouse IgG (diluted 500-fold, Amersham, Chicago, IL, USA) was added to each bead and shaken for 1 hour. The beads were rinsed with 0.5 mL of the phosphate/BSA/Tween/MgCl2 and washed twice for 5 minutes each with shaking and then rinsed once more.
The amount of β-galactosidase label bound to each bead was measured by adding 0.45 mL of 50 mM sodium phosphate buffer, pH 7.4 containing 5 mM MgCl2 and 0.8 mM 7-β-galactosyl-3-[3-dimethyl-aminopropylcarboxamide]coumarin (Worah et al [1981] Clin. Chem., 27:673) and incubating the mixture at 25� C. for 30 minutes. Then 1.5 ml of the 50 mM sodium phosphate buffer, pH 7.4, 1 mM MgCl2 was added and the fluorescence was recorded.
A substrate control containing only the substrate and buffer was run to correct the results for background fluorescence. Also, the activity of 20 μL of the β-galactosidase conjugate diluted 20,000-fold was measured for calculation of nonspecific binding to the control beads.
The results were as follows:
______________________________________ 23s RNA added to hybridization NetDNA on Bead (pg/assay) Fluorescence______________________________________Probe DNA 0 1220" 16.5 1395" 50 1452" 165 1750" 495 2560Salmon sperm (control) 0 1620 μL of β-galactosidase antimouse IgG 76(1:20,000)______________________________________
The amount of RNA.DNA hybrid detected increased in proportion to the amount of 23s RNA added to the hybridization mixture.
The nonspecific binding of β-galactosidase antimouse IgG to the control bead was 0.035% of the total enzyme conjugate exposed to the bead.
A study was made of nonspecific binding of β-galactosidase-streptavidin to aminoamidine beads coated with DNA.
Sulfhydryl residues on β-galactosidase were exposed by reduction with dithiothreitol. β-Galactosidase (30,000 units, grade VIII, Sigma Chemical Co.) in 2 mL of 0.1M. N-2-hydroxyethyl-piperazine-N'-2-ethane sulfonate buffer (HEPES), pH 7.0, 0.09M NaCl, was combined with 3.5 μmol of dithiothreitol and allowed to stand at room temperature for 4 hours. The dithiothreitol was removed by chromatography on a 2.5�80 cm column of Sepharose Cl-6B (Pharmacia Fine Chemicals, Piscataway, NJ, USA) in the buffer described above. Fractions containing protein were combined into a pool. The number of moles of sulfhydryl groups per mole of enzyme were measured by the method of Ellman (1959) Arch. Biochem Biophys., 82:70.
Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (Pierce Chemical Co., Rockford, IL, USA), 5.3 mg, was dissolved in 250 μL of anhydrous N,N-dimethylformamide and a 40 μL aliquot was combined with 3 mL of 0.1M HEPES buffer, pH 7.0, 0.15M NaCl. A 25 μL aliquot o this aqueous solution was added to 825 μL of HEPES/NaCl buffer and 100 μL of 1 mM glutathione (reduced form). When this reaction mixture had stood at room temperature for 15 minutes, the unreacted glutathione was determined by Ellman's method. The results were used to calculate the SMCC concentration.
Streptavidin (obtained from Bethseda Research Laboratories, Gaithersburg, MD, USA) was exchanged into 0.1M HEPES buffer, pH 7.0, 0.15M NaCl by gel exclusion chromatography in Biogel P-6DG (Bio-Rad Laboratories, Richmond, CA, USA). Following exchange, 1.75 ml of 3.7 mg/mL streptavidin was combined with 17.6 μL of 61 mM SMCC and allowed to react for one hour at 30� C. The reaction mixture was chromatographed on a 1�25 cm column of Biogel P-6DG in the HEPES buffer. The fractions corresponding to the first effluent peak with absorbance at 280 nm were pooled and assayed for maleimide content by back titration of glutathione as outlined above.
Since the maleimide was subject to hydrolysis, coupling to β-galactosidase was initiated as soon as possible. Activated streptavidin, 3.9 mg, in 3.3 ml of the HEPES buffer was added to 32 mg of reduced β-galactosidase to give a reaction volume of 9.3 ml. After 4 hours at 25� C. the reaction was quenched by adding 800 μL of 1 mM glutathione and incubated an additional 30 minutes t 25� C. Then the reaction mixture was chromatographed on a 1.5�110 cm column of Biogel A-1.5 m (Bio-Rad Laboratories) and developed with 0.1M HEPES buffer, pH 7.0, 0.15M NaCl. Two milliliter fractions were collected and absorbances at 280 nm were recorded. The first peak was pooled for further study.
Beads prepared essentially by the method described for the control beads in Example 1 above were shaken for 30 minutes in 225 μL of 0.1M Tris-hydrochloride buffer, pH 8.0 containing 0.5% BSA, 0.5% Tween 20 and 1 mM MgCl2. Then 25 μL of the β-galactosidase-streptavidin conjugate diluted 100, 500 or 5000-fold was added to appropriate beads and shaken at room temperature for 1 hour. The beads were washed twice by shaking for 5 minutes with 0.5 ml of the Tris-buffer solution containing 0.5M NaCl.
Enzyme adsorbed nonspecifically to the beads was measured by the fluorescent assay described in Example 1. Also, the enzyme activity of the diluted β-galactosidase-streptavidin conjugate was measured and the nonspecifically bound enzyme was calculated.relative to the total enzyme exposed to the bead. The results were as follows:
______________________________________Dilution of Nonspecificβ-galactosidase-streptavidin Binding (%)______________________________________1:100 0.00491:500 0.0039 1:5000 0.0062______________________________________
The nonspecific binding was virtually proportional to the level of β-galactosidase-streptavidin exposed to the beads.
For comparative purposes, a study was made of nonspecific binding of β-galactosidase conjugates to prior art microporous nylon membranes.
Pall Biodyne A (available from Pall Corporation, Glen Cove, NY, USA) is a nylon membrane with 1.2 μm pores and appended positive and negative ionic groups. A 15 cm2 piece of the membrane was exposed for 1 hour at room temperature to 2 mL of 14 X SSPE containing 50 μg/ml of alkali treated salmon sperm DNA. Then the membrane was air dried and baked at 80� C. in vacuo for 5 hours. The membrane was incubated at 55� C. for 17 hours in 3 mL of hybridization solution. Then it was incubated for 30 minutes in 5 mL of 1 X SSPE, 0.1% SDS.
Nytran (manufactured by Schleicher and Schuell, Keene, NH, USA) is a modified nylon-66 membrane used as a transfer medium for both DNA and protein. It has appended cationic groups. A sample was prepared for a mock hybridization by wetting in distilled water and then soaking in 6 X SSPE for 30 minutes. The membrane was baked at 65� C. in vacuo for 2 hours and incubated in hybridization solution at 55� C. for 17 hours.
The membranes were cut into 0.5 cm squares and each square was shaken at room temperature for 60 minutes with 150 μL of PBS/BSA/Tween/EDTA. Then the membranes were rinsed twice with phosphate/BSA/Tween/MgCl2 (0.5 mL each). They were shaken for 1 hour with 150 μL of phosphate/BSA/Tween/MgCl2 containing the indicated dilutions of β-galactosidase-antimouse IgG or β-galactosidase-streptavidin conjugates. The membranes were rinsed once with 0.5 ml of phosphate/BSA/Tween/MgCl2 containing 0.5M NaCl and then washed twice for 5 minutes each with shaking.
Enzyme bound to the membranes was assayed by incubating them at 25� C. for 30 minutes in 250 μL of 50 mM sodium phosphate buffer, pH 7.4, containing 1 mM MgCl2 and 0.8 mM 7-β-galactosyl-3-[3-dimethyl-aminopropylacarboxamide]coumarin. At the end of the incubation 1.5 ml of 50 mM sodium phosphate buffer, pH 7.4, 1 mM MgCl2 was added and the fluorescence was recorded. The enzyme activities of the diluted β-galactosidase conjugates were also measured and the nonspecific binding to the membranes was calculated relative to the amount of enzyme exposed to the membrane. The results were as follows:
For binding of β-galactosidase-antimouse IgG:
______________________________________Galactosidasebeta. NonspecificMembrane conjugate dilution Binding (%)______________________________________Biodyne A 1:500 1.1" 1:2000 1.2" 1:5000 1.1Nytran 1:500 0.88" 1:2000 0.61" 1:5000 1.0______________________________________
For binding of β-galactosidase-streptavidin:
______________________________________ β-Galactosidase NonspecificMembrane conjugate dilution Binding (%)______________________________________Biodyne A 1:500 off scale" 1:2000 1.2" 1:5000 1.6Nytran 1:500 0.29" 1:2000 0.69" 1:5000 0.76______________________________________
The nonspecific binding increased almost in proportion to the β-galactosidase conjugate concentration.
The present invention has been particularly described and exemplified above. Obviously, many other variations and modifications of the invention may be made without departing from the spirit and scope hereof.
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|National Research Development Corporation
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|U.S. Classification
|536/25.3, 435/179, 536/22.1, 435/803, 435/6.16
|International Classification
|Cooperative Classification
|European Classification
|Nov 4, 1985
Owner name: MILES LABORATORIES, INC., ELKHART, IN., A CORP. OF
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:PATTERSON, WILLIAM L.;CARRICO, ROBERT J.;REEL/FRAME:004478/0165
Effective date: 19851031
|Nov 27, 1987
Owner name: MILES INC.
Free format text: MERGER;ASSIGNOR:MILES LABORATORIES, INC.;REEL/FRAME:004797/0813
Effective date: 19870901
Owner name: MILES INC.,INDIANA
Free format text: MERGER;ASSIGNOR:MILES LABORATORIES, INC.;REEL/FRAME:004797/0813
Effective date: 19870901
|Aug 6, 1992
|Fee payment
Year of fee payment: 4
|Oct 1, 1996
|Maintenance fee reminder mailed
|Feb 23, 1997
|Lapse for failure to pay maintenance fees
|May 6, 1997
|Expired due to failure to pay maintenance fee
Effective date: 19970226


text:
After Melanie finished her studies in Ulm/Germany in 2004, she was part-time employed by the Centre of Military and Veteran's Health (CMVH), to assist with the research into veteran quality of life, and by the UQ Social Research Centre to analyse longitudinal survey data.
From 2005-2009 Melanie worked for the Australian Longitudinal Study on Women's Health (ALSWH) as a Research Statistician. Her main tasks included data preparation, data management, statistical analysis, preparation of scientific papers, contribution to technical reports and reports prepared for the Australian Government Department of Health and Ageing.
Since May 2009 Melanie has been employed by the Institute of Social Science Research (ISSR) as a data archivist. In this position she is responsible for archiving social science data for the Australian Social Science Data Archive (ASSDA), which includes thorough data checking and preparation. Furthermore, she conducts research into archiving (longitudinal) data and DDI.3, and she is ASSDA's contact person for clients in Queensland.
Melanie has strong skills in data management and statistical analysis with survey data.
Berecki-Gisolf J, Spallek M, Hockey R, Dobson A. Height loss in elderly women is preceded by osteoporosis and is associated with digestive problems and urinary incontinence. Osteoporos Int. 2009 Jul 14.
Ford J, Spallek M, Dobson A. Self-rated health and a healthy lifestyle are the most important predictors of survival in elderly women. Age Ageing. 2008 Mar;37(2):194-200. Epub 2007 Dec 6.
Lucke J, Waters B, Hockey R, Spallek M, Gibson R, Byles J, Dobson A. Trends in women's risk factors and chronic conditions: findings from the Australian Longitudinal Study on Women's Health. Womens Health (Lond Engl). 2007 Jul;3(4):423-32.
Spallek M, Turner C, Spinks A, Bain C, McClure R. Walking to school: distribution by age, sex and socio-economic status. Health Promot J Austr. 2006 Aug;17(2):134-8.
Turner JV, Spallek M, Najman JM, Bain C, Purdie DM, Nixon J, Scott D, McClure R. Socio-economic distribution of environmental risk factors for childhood injury. Aust N Z J Public Health. 2006 Dec;30(6):514-8.
Bain C, McClure R, Najman J, Nixon J, Purdie D, Scott D, Spallek M, Turner J. Socio-economic Distribution of Environmental Risk Factors for Childhood Injury. Australian and New Zealand Journal of Public Health, 2006, 30(6). pp. 514-518.
Staff Details
Research Fellow in Longitudinal and Life Course Methods
Masters equivalent degree in Medical Documentation and Information Technology, University of Applied Science, Ulm, Germany

subset: commoncrawl


text:
» Double-knotted fringes
» Half-lining
» Sidebands
Note: If you would like to have a different atara sewn on, go to our Atara page and add an atara to your cart.
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Posted by Hope on 9th Oct 2017
This Tallit is amazing and I really like it I also got another Tallit a while back that one is awesome too and I am quite happy with the Tallitot that I own and I am definitely going to use this site for any thing I may need. (Replacements and/or gifts to friends). I was surprised that Ben sold to me with my first order but I am grateful, the closest Judaica store that sells the Tallit katan is a Chabad store in Houston. If I could rate this 1,000,000,000 stars if I could!
Posted by J. Rogge on 3rd Jan 2017
What I wanted in the beginning was the Hamefoar Prestige tallit based on the look, but I wanted the weight of the Echt Turkish, but with single fringes and a Traditional Atara. In the end I decided for the Echt Turkish because i want to feel the weight of the commandments and G-ds will when I pray. The Echt Turkish tallit is beautiful. It is nice and heavy, stays on and doesn't slip and the Traditional Atara is fantastic. I cried when I held the tallit in front of me for the blessing, this was probably more spiritual in nature, but i am so grateful for this very beautiful tallit to clothe me in prayer. When first inspecting the tallit there were some stitching threads that i had to trim, but everything else was great. Very minimal adjustments needed when being worn, once its on it stays on, depending on how actively you daven. I am 6 feet tall and got the 70 size, if i could i would get the 80 because I am a bigger man. I also got the thick Ptil Tekhelet which I am very happy with too. Overall I am very impressed and will continue to shop at Ben's Tallit Shop for my Judaic needs. Thank you
Posted by Jonathan K on 1st Jul 2016
Beautifully made and gorgeous looking tallit. Ben provides great advice, asks the important questions and assures a total quality experience.
Posted by N.R. Davies on 9th Nov 2015
This is a beautifully made tallis and is a great hiddur mitsvah to use it....certainly feels like it is "there," which is a wonderful aid to my kavanah. Thank you also, Ben, for your excellent personalised service!
Posted by Dominic P. on 13th Jun 2014
The Echt Tallit you sent is not just a quality product, it´s real craftmanship, and it feels great when I wear it.
Me and my family are the only Jews in the place where I live, so I wear it just in my home. For me is not to show it, but to fulfill the mitzvah with kavanah, and this tallit helps me in that.
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text: The why aspect of a living sacrifice Romans 12:1) I beseech you therefore, brethren, by the mercies of God, that you present your bodies a living sacrifice, holy, acceptable to God, which is your reasonable service. Why? God before time, at the beginning of time stood to declare words that have come to pass, that shall not return unto Him empty or void. With the lips of God, the earth, the heavens, the land, the sky, and the seas, and all life came to be. Had it not been for our loving Creator, we would not be here today, none of us would be here at all. He is the source of creation, of all that came to be. He is glorious and arrayed with Holiness. He is flawless and perfect in every way. His beauty and His pureness exceeds and excels all things in heaven and on Earth. The magnitude of His knowledge is unsearchable, as deep as the sea, as vast as the skies. His mercy is exceedingly great despite the grave erroneous ways of mankind, and of our ways Psalm 103:10) He hath not dealt with us after our sins; nor rewarded us according to our iniquities. Even though we all deserve hell and to spend all eternity there burning away, melting like wax figurines, God (Ephesians 2:5) "Even when we were dead in sins, hath quickened us together with Christ , (by grace ye are saved" and (Romans 6:23) "the gift of God is eternal life through Jesus Christ our Lord." God did not say, everyone who wants to be saved, pay such and such amount, why because what our Lord did for us on that cross giving His life for us, while we were in sins, while we were yet sinners, worthy of damnation, is priceless and holds more value than the finest rubies or the twinkling and glitter of the greatest diamonds. All riches imported, gathered in one collective mass accumulate to nothing when compared to the sacrifice in which of Lord gave. Romans 5:7) For scarcely for a righteous man will one die: yet peradventure for a good man some would even dare to die. Romans 5:8) But God commendeth his love toward us, in that, while we were yet sinners, Christ died for us. We have all fallen tremendously short of the glory of God. Before a perfectly righteous and just God, even one sin is a grave and deadly thing. There is not one of us who can honestly say, I deserve eternal life. Romans 3:23) For all have sinned, and come short of the glory of God Now ponder the magnitude, because we have all broken every law that God has made and has spoken as it is written (James 2:10) "For whosoever shall keep the whole law, and yet offend in one point, he is guilty of all." And despite our huge short coming, we have been brought into a relationship with Christ. Again, this precious gift was offered to us freely and without cost. Why? God's love wherewith He loved us was so great, that He was willing to crush His only begotten son Jesus Christ for us. Isaiah 53:10) "But the LORD was pleased To crush Him". If God were to put a price on the death of His son, we would all not be able to buy it; salvation, nor for that matter is something we can earn with any amount of works. Ephesians 2: 8) For by grace are ye saved through faith; and that not of yourselves: it is the gift of God: 9) Not of works, lest any man should boast. It is priceless, with such immense and richness in value, He … died … for…us. Liberated now through Christ Jesus, shall we live unto ourselves perpetually and constantly thinking, me, me, me, me? Or shall we live rather continually to please the Lord our God, Jesus Christ, who died for us, resurrected, and is seated in glory at the right hand of God the Father? This is the why aspect, a drop of the immense reasons why we should live for God, to present our bodies, our being, unto Him in a manner holy and acceptable. He deserves every fiber of our being.


text:
Environmental Geosciences
A considerable amount of detailed information on storms and hurricanes and their resulting impact upon landfall has been compiled over the past 15 years. These data have allowed for the development of detailed and successful prediction methodologies. However, there is also a need for generalized or more simply applicable tools for predicting coastal impact from extreme meteorological events. Two such pragmatic tools have been presented. In the first, mean and maximum beach and coast erosion quantities have been correlated to the Saffir-Simpson hurricane damage potential scale, resulting in an amended Saffir-Simpson scale. In addition, two figures have been produced, one of which relates storm surge to storm tide rise time, and the other which relates storm surge to forward speed of a storm or hurricane. These can be used as nomographs to assess the erosion damage potential in real time as an event is approaching the coast. The second tool is based on the binomial probability theorem. It allows one to assess, for instance, encounter probabilities for known return periods and encounter periods, and is of valuable assistance in the design phase of a coastal project.
James H. Balsillie received his B.S. in geology from the Department of Earth Sciences, Portland State University, Portland, Oregon, in 1970 and conducted graduate studies and research at Florida State University, Department of Geology, Tallahassee, Florida. Professional interests include structural geology, sedimentology, statistics in geology, coastal engineering geology, and numerical computer modeling. He served with the Coastal Engineering Research Center as a principal investigator for 5 years, with the Bureau of Beaches and Coastal Systems of the Florida Department of Environmental Protection as a coastal engineer for 18 years (including chief of the analysis/research section), and has served with the Florida Geological Survey as a coastal engineering geologist and sedimentologist for the past 7 years. He has written over 90 published papers and numerous computer programs and numerical models.


text:
Tonight I'll show you how to add a touch screen for less than 50 € to your precious. This may be an EeePC or another laptop or even an LCD screen, the possibilities are endless. The only challenge is:
– the ability to disassemble the entire case out of the computer. And when I say completely, you'll see that I'm serious here soon!
– To have enough room in the case to house the controller. It is a small 52 * 23mm electronic card, a little less than 5mm thick. In an EeePC 1000H, it goes very well, as you'll see.
Basically, if you are able to disassemble your machine without breaking anything, well let's go!
The kit:
I bought mine on eBay, here: Visual Touch World. For Europe, allow between two to four weeks to receive your precious. They have many different models, regardless of your screen you should find what you need.
Once ordered, you should receive a nice black box with this in it:
On the left: the controller. It is no more than a small USB hub, slightly improved. It comes with a piece of copper tape, which I did not used. In the center the touch panel still covered and above, a small plastic tool that you will need to access the LCD panel.
Above all, I recommend you not to lose/mix the screws you remove from the case. There ales loads of them, so be methodical. My method:
Now we're going to begin the disassembly. Once you've removed the battery and the power cord, we will have to open access door to the hard drive/RAM/WiFi card:
Beware not to lose the small plastic washer on each screw from the trap:
Once the trap removed, you will find the hard drive, wireless card and its two antennas and the RAM module. Remove the screws on both and gently pull out the RAM module. The antennas should pop out without forcing.
Once everything is removed, we will start to open the casing. Remove the following screws:
Now we will remove the keyboard. Be gentle to avoid damaging anything. Use a flat and thin plastic tool if possible. There are four tenons:
Gently lift the keyboard, you should find the connector. To disconnect it, push each side of the black part gently. Beware not to scratch the motherboard if you use a tool, this might make your computer completely unusable!
Remove the second connector (touchpad):
Then the fan and shortcut keys connectors:
You can now open the case by removing the upper side (the one that receives the keyboard). Take it easy, use the blue plastic tool if necessary. You should get this:
Some other connectors to remove (camera and its ground, microphones, speakers and fan, if you have not already done it):
Then the LCD connector and its ground
So we have left: two screws for the left hinge, 2+1 for the right one and two screws on the motherboard. Once removed you can remove the motherboard (mind the speakers connector). You should get this:
In red on the last image, the place where we will fit the controller.
Now, the screen. Remove the small rubber pads with a small screwdriver, remove screws:
Now we will go to the really tricky part, the opening of the casing of the screen. Here more than elsewhere GO GENTLY to avoid breaking everything!You should obtain this:
Now remove the screws that hold the screen, on the left:
Same on the right, you can now remove the LCD. You can also remove the two metallic rods on the screen's sides. That is not compulsory thought.
At this point, clean the screen THOROUGHLY. Remember it is the last time that you have access to it! Once the touch screen is attached, it is very difficult to go back! Also, pay attention to not let dust, hair or anything, the screen should be absolutely perfect! If in doubt, here is a trick to get a clean room. Go into your bathroom, turn on the shower (cold water) and flush a few minutes. The water will absorb the dust in the air and, by convection, the air will be somewhat cleaner. Well, I did not need that, but when in doubt …
Now, get ready to place the touch screen, with its connector ON THE RIGHT. Peel off the protective film, the adhesive tapes protections and go, put it in place. Be careful!
Once properly positioned, rub the adhesive tape a bit with a piece of clothe. Reassemble the screen.
Still a delicate step, close the screen housing. Touch panel connector is very long, but the operation requires some adjustment.
Now, the controller board. It will fit here:
To stick it, I used double-sided tape, the one commonly used to stick mirrors. This type of double-face fits perfectly in shape and is very strong, which guarantees that the card does not wander into the PC (which might be annoying: D).
Cover it with a simple piece of tape to insulate it from the motherboard, we never know …
Now, replace the motherboard by passing wires through the hole nearby. For touch panel wire, I chose to pass from the top. See by yourself, the goal is that the assembly does not create extra thickness, annoyong when closing the case. Connect it all, tape it, make sure the screw holes are free:
Note for the touch panel connector: no matter which direction you insert it, the controller will handle it correctly after calibration.
Now reassemble everything carefuly! Follow this tutorial in the opposite direction, you will find everything you need.
Once everything is reassembled, turn on your computer. If it starts, good for you! Now install the driver that you will find here. Reboot, the utility is located in the taskbar near the clock. You will find everything you need to calibrate your screen. 4 points are are, but 9 points is good too;). For those like me that use, the driver under Ubuntu 10.04 + is not necessary. Perform the calibration on windows, it will be stored in the controller. So no need to calibrate Ubuntu.
Voila! A short video:


text:
Ten Easy Ways To Help You Stay Young
Release Date: 23 February 2009 | Certificate: Exempt
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Falkland Islands
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This inspiring programme offers top tips and advice on staving off the ravages of time, and improving overall health and wellbeing for body and mind. Topics covered include relaxation, exercise, healthy eating, striking a work/life balance, happiness, fashion, beauty, and much more.
Release Date: 23 February 2009
Certificate: E - Exempt from Certification
Region 2
Sound: Dolby Digital
Running Time: 55 minutes
Production Year: 2009
Main Language: English
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text:
From $399,950 - $509,667
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The Price Range displayed reflects the base price of the homes built in this community.
You get to select from the many different types of homes built by this Builder and personalize your New Home with options and upgrades.
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Carriage Hills at Crystal Valley
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text:
U.S. Rep. Timothy V. Johnson issued the following statement today in response to President Obama's speech to the United Nations General Assembly.
"I am disappointed that the White House balked in its belated response to the Hamas-backed Palestinian Authority's attempt to gain statehood through the United Nations," Rep. Johnson said. "This attempt contradicts earlier promises and represents a direct threat to the state of Israel.
"The White House has since pledged to block the Palestinian statehood scheme in the Security Council, but this administration missed a golden opportunity to hold Palestine responsible for its guile and to place blame where it belongs.
"I remain steadfastly committed to protecting Israel's safety and sovereignty, as I have throughout my career."
Rep. Johnson also today signed on to a Dear Colleague letter from House Foreign Affairs Subcommittee Chair Steve Chabot opposing Palestinian unilateral efforts at the UN.
Rep. Johnson also joined as a cosponsor to H.R. 394, a resolution that states Congressional support for Israel's right to annex Judea and Samaria in the event the Palestinian Authority continues to press the United Nations to unilaterally declare a Palestinian state.
Source: http://timjohnson.house.gov/index.cfm?sectionid=29&parentid=7§iontree=7,29&itemid=477


text:
Arsene Wenger confused why Alexis Sanchez took Arsenal penalty at Hull (video)
Santi Cazorla is Arsenal's first choice penalty taker
Arsenal bagged an impressive win at Hull on Saturday and the Gunners are surely getting into their groove now.
Arsene Wenger's side have recovered from their slow start to the campaign with three wins on the bounce in the Premier League.
There were a huge amount of positives to take from Arsenal's 4-1 win at Hull, not least the performance of Granit Xhaka.
Related: An explanation of Granit Xhaka's celebration after his first Arsenal goal v Hull.
Why did Alexis Sanchez take Arsenal's penalty at Hull?
Arsenal were already 1-0 up at Hull thanks to Alexis Sanchez's opener when they won a penalty just before the break.
Alexis Sanchez took it and saw Hull goalkeeper Eldin Jakupovic dive to his right for an excellent save.
This is not the first time Alexis Sanchez has missed a penalty for Arsenal in the Premier League.
The fact Sanchez took the penalty was a real surprise to many Arsenal fans, since Santi Cazorla is the Gunners main spot kick taker.
For example, a week ago, Cazorla took the Arsenal penalty to bag a late win over Southampton.
When questioned after the game, Arsenal boss Arsene Wenger was as much at a loss as anyone else about why Sanchez took it – see video below.
Ultimately, Arsenal won 4-1 and Sanchez scored two, so we doubt anyone is losing any sleep about it…


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Egyptian anti-Mubarak protesters watch from defensive positions on a rooftop along an area where clashes have taken place the past several dayse on the "Day of Departure" February 04, 2011 near Tahrir Square in Cairo, Egypt. The Egyptian military and demonstration organizers enforced a several hundred meter buffer zone between anti and pro Mubarak factions of protesters and were able to prevent large scale clashes between the two sides that had taken place the past several days. .Add to Cart Add to Lightbox Download
- Filename
- Egypt Revolution Day 11_012.JPG
- Copyright
- 2011 Scott Nelson
- Image Size
- 5616x3744 / 2.5MB
- Contained in galleries
- 18 Days of Egypt's Revolution


text: so i'm thinking about getting my first tatoo. I want it to be on my right upper arm so it cannot be seen when i'm wearing a tshirt and want it to say "No regrets". However, i have no idea for a design for it.. something other than just the words would be cool but i have no idea. Thanks for any input


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Very helpful and always have the part or can get it soon...excellent at keeping track of warranty and past sales transactions right in the computer any time you need to know. Neve...
All reviews seem positive
Well-known chain offers basics like batteries and spark plugs, and novelty items including wheel covers, racing seats and motorized razor scooters.
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Limitations of Science
It is astonishing and ridiculous to say that any thing which is not explainable according to the present knowledge and tools of science is not real or is not true. Nonetheless, un-real never exists, and what is real, does not necessitates any support to prove its existence or validity. It is a truth that what is real, it never cease to exist without someone's approval or validation.
Science counts and recognizes its findings in terms of "Quantity" i.e., a phenomenon that can be explained or observed by way of measurement or quantification is scientific. This approach of science is purely materialistic one. How the science can justify the phenomenon of "love and hate", which are purely qualitative in nature, on its terms of quantification. Though, most of the devastations happened in the history of men kind due to these "love and hate" considerations. One may say these are emotions and are governed by psychologists. But science don't believes and qualifies the well researched findings of renowned psychiatrists - parapsychologists e.g. telepathy, past life regression and hypnotism.
We find that science does not have any considerations to cognize the facts in terms of "Quality".
Any phenomenon in science is normally traced from external to internal (without to within) or outside to inside. One of the rationalists' may say that tree or plant is a pre-requisite for its seed and other may say that a seed is the pre-requisite of a tree or plant. Similarly, an egg is pre-requisite for a hen's being and conversely a hen is pre-requisite for an egg. But a spiritualist declares that both egg and hen are part and parcel of the same one. What science defined in 19th and 20th centuary is now not true. Such findings of science has now been redfined. The Science is subject to change it's verdicts and theories with time. There is nothing universally true in science.
Nothing can be produced from no-thing is scientific. Discoveries in astronomy and physics have shown beyond a reasonable doubt that our universe did in fact have a beginning. Prior to that moment there was nothing; during and after that moment there was something: our universe. The big bang theory is an effort to explain what happened during and after that moment. If something so much big the size, the infinite cosmos, which is still expanding and expanding, sprang out of "nothingness" (expanding universe that began as an infinitesimal singularity, which appeared out of nowhere for reasons unknown. This is the Big Bang theory), Scientists have no explanation for the sudden explosion of light and matter.
Einstein is probably the best known and most highly revered scientist of the twentieth century, and is associated with major revolutions in our thinking about time, gravity, and the conversion of matter to energy (E=mc2). Although never believe in personal God, he recognized the impossibility of a non-created universe. He once remarked to a young physicist: "I want to know how God created this world, I am not interested in this or that phenomenon, in the spectrum of this or that element. I want to know His thoughts, the rest are details." Einstein's famous epithet on the "uncertainty principle" was "God does not play dice" - and to him this was a real statement about a God in whom he believes. A famous saying of his was "Science without religion is lame, religion without science is blind."
Drawing upon the Holographic Principle, the premise behind the Fermilab project is that space is two dimensional, and that the third dimension is inextricably linked with time. If that's the case, our 3D world is merely an approximate illusion. Assuming that's true, the illusion is likely imperfect and blurry, just as photographs and videos are, especially when viewed on a granular level.
Scientists aren't even sure if life could have evolved naturally via unintelligent processes. If life did not arise by chance, how did it arise?
The universe is ordered by natural laws. Where did these laws come from and what purpose do they serve?
People of every race, creed, color, and culture, both men and women, young and old, wise and foolish, from the educated to the ignorant, claim to have personally experienced something of the supernatural kind. So what are we supposed to do with these prodigious accounts of divine healing, prophetic revelation, answered prayers, and other miraculous phenomena? Ignorance and imagination may have played a part, but all these lot of people world vide can not be classed as ignorant. Is there something more, which needs to be considered?
Lord Sri Krishna says in Bhagvat Gita, "I am every where, in every being of the universes, nor less not more, and every thing including planets, stars and galaxies are within me still I am beyond it. Is it possible for the science to testify this statement of God? Certainly not, if it can be then it would be an effort of science to finite the God that is infinite.
In the worlds of Swamy Vivekanand:- "Religion deals the truth of the metaphysical world, just as chemistry, physics and other material sciences deal with the truth of the physical world."
Science addresses itself to things within space-time-causality whereas spirituality addresses to things beyond the barrior of space-time-causality. The mastery over the art of self control means mastery over the senses and mind that enables man to know and see things beyond time, space and causality One can not love without the knowledge of beloved. So, one can not love God without knowing Him.
I intend to highlight through this post that the limitations of science may be removed if both science and spirituality move with hand-in -hand together.
Er Rameshwara Gupta


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Most mission statements are full of blah truisms and are anything but inspirational. A company's employees don't really need to be told that, 'The mission of XYZ Widgets is to make the best widgets in the world while providing excellent service.' They must think, 'As opposed to what? Making the worst widgets and offering the lousiest service?'
Such statements show that management lacks imagination, and perhaps in some cases, direction.
At the opposite end of the scale is the statement that fails through flowery waffling. An example: 'Yahoo powers and delights our communities of users, advertisers and publishers – all of us united in creating indispensable experiences, and fueled by trust.' That sounds wonderful, but what does it mean?
Whoever wrote it should try listening to the company's CEO, Marissa Mayer, who said: "Yahoo is about making the world's daily habits inspiring and entertaining." It's not perfect, but it would be a step in the right direction.
Would it fit a coat of arms?
Some companies are not actually able to carry out their mission. The reasons can range from a disruption in the markets to a merger or acquisition, and then there are cases like Enron's: Before the giant energy company went bankrupt in 2001, ruining the lives of tens of thousands of employees and investors, its vision and values statement was, 'Respect, integrity, communication and excellence'. Say no more!
While some mission statements consist of one vague statement, others are too long, which may reflect a lack of understanding of what a company does. Still other companies don't know what differentiates them from their competition.
The mission statement for the pharmaceutical giant Bristol-Myers reads, 'To discover, develop and deliver innovative medicines that help patients prevail over serious diseases.' Well, you can't argue with that, but surely this can be said of every drug company. Why would a person buy Bristol-Myers' products or invest in its stock, rather than its competitors'?
So that's what not to do. If you need to write a mission statement, I think you should try for something closer to a heraldic motto than a speech. They were often simple because they had to fit across the bottom of a coat of arms, and they were long-lasting because they reflected a group's deeper values.
When I was a boy, I was fascinated by such mottos. One of my childhood heroes was the pilot Douglas Bader, who lost both his legs in a crash early in his career, but went on to fly fighter planes for the Royal Air Force during WWII.
After seeing the movie Reach for the Sky, which told his story, I remember asking my father about the RAF motto, 'Per ardua ad astra.' When he told me that it meant 'Through adversity to the stars', I thought the idea of battling one's way to the stars at all costs was the most inspiring thing I'd ever heard.
Related: Why You Should Think Like a Big Business
Screw it, let's do it!
A few years later, at Stowe School, I was taught the school's motto, 'Persto et praesto', which means 'I stand firm and I stand first'. This motto caused a lot of giggling among our group of adolescent schoolboys, but it was nevertheless excellent for guiding us forward into adult life. Brevity is certainly key, so try using Twitter's 140-character template when you're drafting your inspirational message.
You need to explain your company's purpose and outline expectations for internal and external clients alike. Make it unique to your company, make it memorable, keep it real and, just for fun, imagine it on the bottom of a coat of arms.
If we had to put ours on a coat of arms, Virgin's would probably say something like, 'Ipsum sine timore, consector', which very loosely translated means, 'Screw it, let's do it!'
Related: 3…2…1 Business Lift-Off


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Bangkok is amongst the greatest metropolitan areas in the full of Asia which is also believed to become South East Asia's main enterprise facilities. This can be a cosmopolitan town the place men and women from all nations pay a visit to. The city can be very crowded and there is certainly numerous traffic jam in this town. It's as a result nearly all of attendees who make their strategies for Thailand vacations must be informed about the general public transportation process. These general public transport helps you access your vacation spot promptly for that reason are also very convenient. Aside from general public transportation, there are other modes of transportation also. Let us focus on them.
While looking for low cost flights to Bangkok, you furthermore recommended evade the monsoon time to go there. You'll be able to visit Bangkok any time in remaining portion of the calendar year. Also you may head over to Bangkok again then which coincides with gatherings in your neighborhood. You can enjoy the King's birthday, Bangkok Jazz Pageant and Chinese New 12 months. All these events depend the visits.
Just one definitely well known districts of London is Notting Hill. Uncovered insights on details of lebua hotel bangkok. Some time located in close proximity to Chelsea and Kensington locations, while in the north-western component of these. An appartment in Notting Hill just several minutes' walk to the well known Portobello Road Marketplace, and will change in line with Notting Hill Carnival held each year in July. A single might come across terrific streets in conjunction with very household places in Notting Pile. It can be an excellent space for residents who love their peace of suburbia as well as the close proximity of your bangkok town resorts.
A further Gaudi monument well worth seeing might be the Park Guell. Joan Guell, an incredibly loaded Barcelona businessman, gave Gaudi the to be able to layout the full park just the way in which he wanted. It feels correct an attractive and extravagant spectacle you not miss out on.
Should you are seeking arts and tradition, the centre has some thing to existing you. Music within the Philharmonic Orchestra can be employed. Following a extensive working day the particular earth city, browsing and examining out many vacationer attractions, means to end time is having a overall performance inside philharmonic to relaxation your is captivated to. Verify out the box business with regard to splendid night at any present. Lose kilos listed here is surely clean as well as the philharmonic showcases abilities from both equally the local and international scene. In addition to the box place of work what exhibits really need to make a reserving previously for the reason that exhibit tickets do sell ever more!
To start organizing in this particular journey, Favourite myself, an volume be what exactly is crucial to settle to start with? Produced by rather apparent that i will get there and stay somewhere once i am there, so I began to look out for your top rated deals on flights and lodging for Chiangmai and Bangkok.
Driving in Bangkok is often done although not recommended, somebody happen to be below for around 6 months to inside of a year's time. I'd being to put a calendar year prior to I began to travel in town. Imagine me it is dislike driving in Brooklyn or Palm Coastline. Most avenue indications are in Thai they usually may very well be recycled perfectly positioned at most corners. Site visitors routing could distinctive on a daily basis hurry hour as opposed to usual afternoon duration. And soon after 20 odd several years this i hardly ever push in the old segment of the city. It is best you stay with taxi and subway.


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Hello, my sweeties! I hope you had an amazing weekend! Here is another photo session from my stay in Seoul. This shoot was done with photographer Olga Lian. We thought to create something elegant and at same time fun. A beautiful and luxurious pearl necklace from Susanna Galanis worked pretty well for this photo shoot. Red lipstick and red sandals captured the strong sexuality of the formless oversized jumpsuit. Daniel Wellington watches are the best way to stay super classy.
Home Unlabelled Spring 2015: Elegance Overloaded
Spring 2015: Elegance Overloaded
By Diana Horsfall 6:10 AM
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Boston Market (in the US) has buy one get one free dinners until 4/18 for our Tax Day.
Boston Market Tax Day Coupon
Print it out, head on in.
That and some Smirnoff Ice Wine Coolers and, yeah, let's trade.
what to eat when trading
psychology is the key to success in trading. are you match tough?
Moderator: moderators
- TygerKrane
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Tax Day Break
**Krane catches Tyger** !>I'm here to chew bubble gum and make major pips...and I'm all out of bubble gum.<!


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Block or Allow robots using the robots.txt file
How to Block or Allow search engines and other robots, including a list of robot names.
Edited: 2011-07-24 18:49
To only Block or allow access for certain crawlers or robots, you would need to know how the robot identifies it self. The best way to figure that out, would be to either look at their websites, or search for this information on Google.
This article shows how to exclude a few different well-known search engines. Note. It might be a better option to totally exclude all other search engines, then the ones that you specifically allow.
Below would prevent Google from indexing images placed in a directory called images, as well as any other files located in the images Directory
User-agent: Google
Microsofts Bing identifies it self as msnbot, and can be blocked with robots.txt using the below code.
User-agent: msnbot
Yahoo uses the name "Slurp" for their search engine, perhaps reflecting the sound you make when you eat? Anyway, the way to block Yahoo is shown below:
User-agent: Slurp
Alexa and the Internet Archive identifies it self as ia_archiver. If you don't want past versions of your website to be archived, then you might want to consider blocking the Internet Archive.
User-agent: ia_archiver


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- Follow The Tree of Mamre on WordPress.com
Follow me on TwitterMy Tweets
Blogs, blogs, blogs ...
News and Punditry
Reliable Sources
Tag Archives: Bund
Photo of the day: The Bund at Night, Shanghai
It is such an iconic scene. In the old days, the Bund was the financial center of Asia–like modern-day Hong Kong, Singapore, and Tokyo combined. However, while Shanghai is now reascending, the Bund today is little more than a museum … Continue reading
Posted in China Tagged Asia, Bund, Cathay Hotel, Empire of the Sun, HSBC Building, Huangpu River, Peace Hotel, Pudong, Shanghai Customs House Leave a comment
China In 1932 (Film)
The Youtube page advertises this as a travelogue of Shanghai in 1932. Not quite. It begins with the arrival by boat at Shanghai, and then quickly moves to Beijing, with some shots of what looks to be boats on the … Continue reading


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Engage, Enthuse, Inspire: How I've used simple experiments to bring science to life on TV, on YouTube & on Stage.
Looking for some demonstrations to take back to the classroom? In this interactive talk Greg will give you the heads up on lots of ways to inspire children and adults alike with science. Greg will introduce you to some of the experiments he's performed, the lessons he's learnt along the way, and encourage you to put down the textbook and get hands on with science.


text: Can anyone verify the various editions of the 1933 King Kong on laserdisc? So far I am aware of a few versions, Criterion, Kong/son of kong double feature from Image, and another Image entertainment release, perhaps with different commentary. Does anybody know about the various releases and the full details on their extras? I have the criterion edition, but I have heard of other editons containing different extras.


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- Holds natural styles longer
- Optimum shine and softness
- Great for smoothing and holding hair
often Natural Hair For Styling: For best results use BE total body shampoo. Apply creamy natural hair butter to clean hair while styling. Overnight Styling On Dry Hair: Locs:While hair is damp add natural hair butter to hair closest to the scalp. Use finger tips, palm or comb rolling technique to twist new growth clockwise. Use a clip to hold section in place. Continue until complete, sit under the dryers until hair is dry. Remove clips and style. follow up with shea coco condition spray on your scalp and on your locs. Natural Hair Styles: Scoop out a liberal amount of Natural Hair Butter will hold natural styles longer with optimum shine and softness and without build up or sticky feel. Use for natural hair while wet to set in curls by twisting sections of the hair.


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Simple And Effective Stop Smoking Strategies That Are Successful
It can be really hard for many people to quit smoking, people still struggle to quit. If you're one of these people, use these tips to help in breaking free from tobacco. Apply as many as necessary so that you can get on the way to stopping smoking.
Simple And Effective Stop Smoking Strategies That Are Successful,- Writing it down can change your whole mental outlook. This can help you stay motivated, and it may be easier because you might be more focused to see it through.
These people can offer tips, guidance, and guidance for quitting. Support groups can be found at recreational centers, even on the Internet, so take some time to research what's available to you.
Tell yourself you need to go for a walk first, or try to drink a glass of water prior to smoking. If you still feel you need that cigarette, the delay may at least reduce your smoking for that day.
Make a list of what methods you can use to help you quit smoking. Each person has a unique way to taking care of things their own way. It is very important to find what your best for you. Making a list will accomplish this.
Simple And Effective Stop Smoking Strategies That Are Successful,- Make sure that you get sufficient sleep as you attempt to stop smoking. For many individuals, staying up late during the night gives them increased cigarette cravings. You may get tempted to sneak a cigarette while no one else is with you. If you go to bed and get up on a schedule, you will be focused and able to stop any cravings.
By telling yourself to wait 10 minutes, you will usually avoid the craving. If the craving is still there, repeat this step until it does.
Try changing your diet habit by eating more veggies in fruits and vegetables instead of sugary snacks when you are quitting smoking. This will help help prevent any weight gain that you might experience.
You may want to look into therapy to help with nicotine replacement. Nicotine withdrawal can lead you feeling restless, irritable, and becoming frustrated or irritable. The constant cravings can be overwhelming. You can help alleviate these feelings by using nicotine replacement therapy. It is very dangerous to smoke while using these products; therefore, though.
Talk to a doctor about quitting smoking. Your doctor may have additional resources for quitting that you did not have.
Motivation and positive attitude are key points when you quit smoking. Try to imagine how much better your life is going to be after you quit. Consider that your teeth are going to be brighter and cleaner, your teeth will look whiter, and your residence will no longer have a smoke odor. While knowing the dangers of smoking may scare some into putting the tobacco down, a positive outlook can also be very powerful.
The above advice should be of assistance to you as you begin to taper down on your tobacco usage. Your life and your health are certainly worth the effort. Put yourself first, and realize that you deserve a healthier life without the risks and problems that smoking causes. You will see many benefits in your health and quality of life after you give up smoking.


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Hoping to find a recipe for a really wonderful carrot cake for Mothers Day. Mom definitely prefers something with grated carrots and a lot of real flavor.
Does anyone have a great recipe to share? I'd prfere something that would work as two 9 inch layers or a bundt since these are the only baking pans I own!
Thank you!


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KEY TO THE CITY - USA City Directory Top USA City Resource Guide
Gulf Beaches of Tampa Bay Chamber of Commerce
501 150th Ave
Madeira Beach, FL 33708
Phone: 727-391-7373
Toll-free:800-944-1847 Web:
| Search Key to the City
Custom Search
| Search All the Web
Custom Search
To learn how to submit USA City information to Key to the City, go to Publication Information Page
Use your back button to return to the previous page
To go to a new state, choose the States page
Return to the USA City Directory - Top USA City Resource Guide
Thanks for coming! Don't forget to come back soon!!


text:
|» Products
|» Operation Theater Light
|» Ceiling Operation Theatre Light
Ceiling Operation Theatre Light
Product Code : 09
Product Description
Ceiling Operation Theatre Light:
Ceiling shadowless operating light with 19" inches' dome,with an in-spring mechanism for free movement. Natural daylight and cool light with special heat absorbing & color correcting filter glasses. Sterlizable handle for easy feather touch movement.
Technical Specifications
Room height: 10ft
Dome dia: 521mm
Halogen bulb: 24vx150w
Reflector: Parabolic
Colour temp: 4200k
Height adjustment: 914mm
Temp. increase: 4oCto 5oC
Control panel: on transformer
Power: 220v AC/DC
Intensity(lux): 50000 appx.
Other Products in 'Operation Theater Light' category


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You're So Vain
Posted by Michelle Hirsch,
Dimitri Shvorob just made my day, without knowing it. He just posted VANITY, clearly one of the most brilliant submissions ever to see the light of day on MATLAB Central. In Dimitri's words: "When called, the function displays your FileExchange number-of-downloads and rank statistics, adds them to a history file, 'FileExchangeHistory.mat' in work directory, and plays Handel's Hallelujah Chorus if rank has improved since last time." This function has made me so much more efficient, and again lets me do one more thing without leaving MATLAB (see last week's pick). I can now check my File Exchange status several times a day without interrupting my work. Also, I couldn't help but notice that my POTW-mate Doug has recently crossed 10,000 downloads. Kudos, Doug! I remember my first 10,000 downloads fondly. ;)
Comments are closed.


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Links and News
Club Links
|Rotary International
|Rotary International in Great Britain and Ireland
|RIBI on YouTube
|District 1090
|Twitter Newbury Rotary
|Facebook Newbury Rotary Boules In The Square
|Facebook Newbury Rotary Club
|Facebook West Berks Young Photographer
From the local Rotary District
Children in Need Collection at London Heathrow
Always a great day as Rotarians and friends join together to raise funds for Children in Need. With wonderful support from our partners at LHR, last year over £43000 was collected! Be part of the action!details
Winners of the Purple4Polio Art Award
Announcing the winners of the art competition open to primary and secondary schools in Rotary in the Thames Valley Districtdetails
Turning Purple for Polio at Oxfordshire Science Festival
To celebrate 100 Years of the Rotary Foundation "doing good in the World", we were privileged to participate in the Explorazone at the Oxford Town Hall, part of the prestigious Oxfordshire Science Festival in Junedetails


text:
All results
1 - 1 of 1 listing
Newly listed Sort
Select sortingNewly listed Lower price first Higher price first
Brand new iphone 6,6s,6s plus and samsung s6,s6 edge,s7 s7edge
Place Your Order Now. NOTE: These phones are 100% UNLOCKED and can be used with any SIM card. No activation required (Insert Your SIM CARD and start using the phone, many more in stock) Shipping and delivery to you is done with FedEx / DHL / Ups / USPS courier service within 72hrs. ------- WHY BUY F...
Calicut in Kerala (India)
300.00 Dollar US$view Cell Phones - Accessories published 1 year ago viewed 24x
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GraphicRiver Ladybird Cartoon 10270251
Stock Vector \ Characters \ Animals
Cartoon \ Ladybird \ Shadow \ Ladybug \ Nature \ Single \ WildlifeIllustration
Stock Vector - Ladybird Cartoon 10270251 by rubynurbaidi @ GraphicRiver
Stock Vector \ Characters \ Animals
Illustration of cute cartoon ladybird.
Layered: No; Graphics Files Included: JPG Image, Vector EPS; Minimum Adobe CS Version: CS.
Starfish Cartoon; Octopus Cartoon; Sea Horse Cartoon; Lobster Cartoon; Birds; Turtle Cartoon; Crab Cartoon.
Keywords: animal, beetle, black, bright, bug, cartoon, color, cute, design, detail, fauna, fly, glossy, icon, illustration, insect, isolated, ladybird, ladybug, nature, one, play, red, shadow, single, small, spot, vector, wildlife.


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The article on La Paz, Mexico ["Serenity city," Nov. 4], had grossly misleading information about crime rates and safety. The author cited the fact that La Paz had "only" 16 murders in 2011 and proceeded to compare that statistic to the number of murders in San Diego, San Francisco and Los Angeles, implying that La Paz is much safer than those cities. Even the most novice statistician knows that the total number of murders (or any crime) is a meaningless statistic. What counts is the murder rate per unit of population, normally calculated as murders per 1,000 residents. When you do that math, here is how the cities cited in the article rate:
San Diego, .028 murders per 1,000 people
San Francisco, .062
La Paz, .063
Los Angeles, .077
Now which city looks safest to visit? If the murder rates were for the counties of San Diego and L.A. rather than the cities, the distortion in the article is even worse.
John Williams
Falls Church
I rarely write to newspapers but felt that I had to write to congratulate you on the article on urban Africa ["Going urban," Nov. 11]. Africa is quickly urbanizing, so it's appropriate to focus on the cities — the music scene, the visual arts scene, the markets and the people. This is an important way to help change Westerners' conceptions of what Africa is and what it has to offer. I have lived and worked in Africa for several decades, and it is an amazing place on so many levels.
Susan Igras
James Lee's article on York Barbell caught my eye immediately ["In Pa., a museum with real weight," Nov. 11]. I started working out with weights while serving in the U.S. Air Force (1962-1966) and have continued to do so at one level or another during these past 40-odd years. Bob Hoffman and York Barbell became icons for my workout programs.
I met Hoffman in 1970 during a contest in Harrisburg, Pa. He was a titan in the world of bodybuilding and physical fitness. A large portion of what is done in the fitness culture today is the evolutionary result of what Hoffman created more than a half-century ago.
Rev. M. Vincent Turner
Silver Spring
Thank you so much for the article on turbulence ["Roughing it: A worrywart's guide to turbulence," Nov. 11]. It was great. Recently I was on a British Airways flight to London. There was some bounce, and being the nervous flier that I am, I told the flight attendant that I was a little nervous. After things settled down, she came back and told me that the captain wanted a word with me. She brought me to the galley and I met the captain, who was charming and explained about turbulence and what is involved.
About 15 minutes before we landed, the attendant came back and said that the captain wanted to know whether I'd like to see the flight deck after we landed. So I was treated to an explanation of the deck and the flight maps.
It was really terrific that he took the time to explain this to me. On my flight back, I was just fine. I hope that the article helped a lot of people like me.
Amy Ballard
Thank you for the incredibly informative article on air turbulence. I've always had an unnatural fear of flying solely because of turbulence. I will no longer be bound by that fear.
Bob Ward