text:
|Publication number||US7858385 B2|
|Application number||US 09/859,701|
|Publication date||Dec 28, 2010|
|Filing date||May 16, 2001|
|Priority date||May 16, 2001|
|Also published as||US20030027129|
|Publication number||09859701, 859701, US 7858385 B2, US 7858385B2, US-B2-7858385, US7858385 B2, US7858385B2|
|Inventors||Benjamin P. Warner, George J. Havrilla, Grace Mann|
|Original Assignee||Los Alamos National Security, Llc|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (17), Non-Patent Citations (11), Referenced by (10), Classifications (18), Legal Events (11)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This invention was made with government support under Contract No. W-7405-ENG-36 awarded by the U.S. Department of Energy. The government has certain rights in the invention.
The present invention relates generally to binding events and more particularly, to a method for detecting binding events between receptors arrayed onto a substrate and at least one potential binder using micro-X-ray fluorescence spectroscopy.
The desire to hasten the identification of potentially important polymers, drugs, catalysts, ceramic superconductors, phosphors, chemical and biological sensors, and other materials is a constant challenge that has prompted the use of combinatorial synthetic and screening strategies for synthesizing these materials and screening them for desirable properties. Combinatorial synthesis involves assembling a “library”, i.e. a very large number of chemically related compounds and mixtures, usually in the form of an array on a substrate surface. Combinatorial screening involves identifying which members of the array, if any, have the desirable property or properties. The array form facilitates the identification of a particular material on the substrate.
The synthesis of a surface-bound array of oligopeptides, short chain products of the condensation of amino acids, has been described in PCT Publication No. WO 90/15070 to M. C. Pirrung et al. entitled “Very Large Scale Immobilized Peptide Synthesis”, incorporated herein by reference. Oligopepeptides were chosen because they exhibit the types of binding specificity of their longer-chain polypeptide counterparts, such as proteins. The chemical properties and in particular, the binding properties of a protein depend almost entirely on the exposed surface amino acid residues of the polypeptide chain. These residues can form weak noncovalent bonds with other molecules. An effective binding between the protein, one example of a group of materials herein referred to as “receptors”, and the material that binds to the receptor, referred to herein as “binder”, generally requires that many weak bonds form simultaneously between the protein receptor and the binder. Binders include organic molecules, inorganic molecules, salts, metal ions, and the like. The bonds between the protein and the binder form at the “binding site” of the protein. The binding site is usually a cavity in the protein that is formed by a specific arrangement of amino acids that often belong to widely separated regions of the polypeptide chain and represent only a minor fraction of the total number of amino acids present in the chain. Binders must fit precisely into the binding site for effective binding to occur. The shape of these binding sites can differ greatly among different proteins, and even among different conformations of the same protein. Even slightly different conformations of the same protein may differ greatly in their binding abilities. For further discussion of the structure and function of proteins, see: Bruce Alberts et al., “Molecular Biology of the Cell”, 2nd edition, Garland Publishing, Inc., New York, 1989; and H. Lodish et al., “Molecular Cell Biology”, 4th edition, W. H. Freeman and Company, 2000.
After a receptor array is prepared, it is screened to determine which members have the desirable property or properties. U.S. Pat. No. 5,143,854 to M. C. Pirrung et al. entitled “Large Scale Photolithographic Solid Phase Synthesis of Polypeptides and Receptor Binding Screening Thereof”, which issued Sep. 1, 1992, hereby incorporated by reference, describes one such screening method. A polypeptide array is exposed to a ligand (an example of a binder) to determine which members of the array bind to the ligand. The ligands described are radioactive, or are “tagged”, i.e. attached via one or more chemical bonds to a chemical portion that fluoresces when exposed to non-ionizing, ultraviolet radiation. Thus, the attached portion, i.e. the tag, makes the binder visible by interrogation with ultraviolet radiation. Tagged molecules have also been used to aid in sequencing immobilized polypeptides as described, for example, in U.S. Pat. No. 5,902,723 to W. J. Dower et al. entitled “Analysis of Surface Immobilized Polymers Utilizing Microfluorescence Detection,” which issued May 11, 1999. Immobilized polypeptides are exposed to molecules labeled with fluorescent tags. The tagged molecules bind to the terminal monomer of a polypeptide, which is then cleaved and its identity determined. The process is repeated to determine the complete sequence of the polypeptide.
It is generally assumed that the attachment of a fluorescent tag to a potential binder only serves to make visible the otherwise invisible potential binder, and does not alter its binding properties. Since it is well known that even small changes to the structure of a molecule could affect its function, this assumption that a tagged binder, i.e. a “surrogate”, has the same binding affinity as the untagged binder may not be a valid one. Small structural changes that accompany even a conformational change of a receptor have been known to affect the binding affinity of the receptor. The tagged surrogates are structurally different from their untagged counterparts, and these structural differences could affect their binding affinities. Since binding affinities derived using tagged surrogates are suspect, the binding properties of receptors and binders should be evaluated using the untagged binder or receptor and not with a tagged surrogate.
Therefore, an object of the present invention is to provide an efficient, combinatorial method of evaluating the binding properties of untagged potential binders with receptors.
Another object of the present invention is to provide a combinatorial screening method for directly comparing the binding properties of receptors/binders with their tagged surrogates.
Additional objects, advantages and novel features of the invention will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
In accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention includes a method for detecting a binding event between at least one binder and members of a receptor array. The method comprises the steps of exposing a plurality of receptors to at least one potential binder; arraying the receptors onto a substrate; exposing each member of the array that has already been exposed to potential binders to X-ray radiation to induce an X-ray fluorescence signal from each member of the array now bound to at least one binder, thereby indicating that a binding event has occurred; and detecting an X-ray fluorescence signal resulting from exposure to the X-ray radiation from any member of the array where a binding event has occurred.
Briefly, the invention includes a method for detecting a binding event between members of a surface-bound receptor array and at least one potential binder using micro-X-ray fluorescence spectroscopy. A plurality of bead-supported receptors were exposed to at least one potential binder for a period of time sufficient for binding to occur, and then immobilized as an array onto a surface. Each member of the array was exposed to X-ray radiation. The detection of an X-ray fluorescence signal from a member of the array indicated that a binding event had occurred between that receptor and a binder. The intensity of the signal indicated the extent of the binding event, and could also be used to determine the binding affinity of the receptor to the binder.
The method of the invention uses X-ray fluorescence as a probe to detect binding events. X-ray fluorescence spectrometry is a powerful spectroscopic technique that has been used to determine the chemical elements that are present in a chemical sample, and to determine the quantity of those elements in the sample. The underlying physical principle of the method is that when an atom of a particular element is irradiated with X-ray radiation, the atom ejects a core electron such as a K shell electron. The resulting atom is in an excited state, and it can return to the ground state by replacing the ejected electron with an electron from a higher energy orbital. This is accompanied by the emission of a photon, i.e. X-ray fluorescence, and the photon energy is equal to the difference in the energies of the two electrons. Each element has a characteristic set of orbital energies and therefore, a characteristic X-ray fluorescence spectrum.
An X-ray fluorescence spectrometer is an apparatus capable of irradiating a sample with an X-ray beam, detecting the X-ray fluorescence from the sample, and using the X-ray fluorescence to determine which elements are present in the sample and providing the quantity of these elements. The X-ray fluorescence spectrometer used to demonstrate the invention was the commercially available EDAX Eagle XPL energy dispersive X-ray fluorescence spectrometer, equipped with a microfocus X-ray tube, lithium drifted silicon solid state detector, processing electronics, and vendor supplied operating software. The EDAX Eagle XPL spectrometer can be used to determine the quantity in a sample of any element with an atomic number equal to or greater than the atomic number of sodium (sodium has atomic number 11). The vendor software used to operate the spectrometer allowed the simultaneous quantitation of 15 different elements for each member of the array.
The method of the present invention was demonstrated with a polymer bead-supported oligopeptide library purchased from Biopeptide Co., a commercial vendor. The library consisted of 625 unique 11-mer oligopeptides, i.e. each oligopeptide had a unique sequence of 11 amino acids. The oligopeptides used had the following general formula: XaaGlyGlyXaaGlyGlyXaaGlyGlyXaaPhe (SEQ ID NO:1). In this formula, the phenylalanine (Phe) amino acid of the oligopeptide is chemically bonded to the polymer bead support. The abbreviations for the amino acids in this formula are standard three-letter abbreviations used for the α-amino acids found in proteins and can be found in many textbooks (for example, see table 29.1 in F. A. Carey, Organic Chemistry, McGraw-Hill, 1987, pp. 1086-1087). In the formula: Phe is phenylalanine; Gly is glycine, and Xaa is any one of the following five amino acids: histidine (His); arginine (Arg); serine (Ser); tryptophan (Trp); and tyrosine (Tyr). Permutation of these amino acids among the four ‘X’ positions generates a library of 625 unique 11-mer oligopeptides.
The bead support was a polymer made from Wang polystyrene resin. About 2 micrograms (μg) of each unique oligopeptide (corresponding to about 0.06 nanomoles, nmol) was supported on each bead. That is, each bead contains only one kind of 11-mer oligopeptide. The beads were about 80-120 micrometers in diameter, with an average diameter of about 100 micrometers.
The array format facilitated the identification of the particular oligopeptide-supported beads that exhibited the most significant X-ray fluorescence and therefore, the strongest binding effects. Once identified, these beads were then removed from the array and the amino acid sequence of the bound oligopeptides was determined by Edman degradation analysis. These oligopeptides were also characterized by MALDI-TOF MS, a mass spectroscopic technique. All other reagents used were commercially available and used without further purification.
The exposure of bead-supported receptors to potential binders such as organophosphates, organosulfonates, halides, metals, and the like, generally involved incubating the library of beads in an aqueous solution (pH 5.2-5.6) containing potential binder (about 0.5-100 μmol) for about 5-48 h at room temperature. In some cases, multiple treatments with a binder or binders resulted in an enhanced binding effect. After the incubation period, the beads were removed from the solution, washed with copious amounts of water, air-dried, and immobilized either on glass microscope slides with double-stick tape, or on Tacky Dot™ plates, to provide an oligopeptide receptor array. Tacky Dot™ plates have arrays of adhesive dots on the surface and were used without any chemical modification. An X-ray fluorescence spectrometer was then used to determine which beads of the array had a measurable amount of binder attached. Each member of the array was exposed to spatially restricted X-ray radiation of a nominal beam size of 50 micrometers, and the X-ray fluorescence of particular elements were detected and quantified. If, for example, an organophosphate was tested as a potential binder, the element phosphorus was chosen as a detected element. For potential organosulfonate binders, sulfur was chosen. For potential halide binders (as halogenated organic molecules), the particular halides (bromide for brominated organic compounds, chloride for chlorinated organic compounds, etc.) were chosen. For potential metal ion binders, the particular metal ion was chosen, etc. The X-ray fluorescence intensity was used to detect a binding event, and can also be used to determine the binding affinity of the binder to the receptor. The amount of receptor present on a bead can be verified from the X-ray fluorescence of the oligopeptide-supported bead prior to incubation in the binder solution. Quantification of the binder content was used to determine which of the substrate bound oligopeptide receptors were the most efficient receptors for binding a particular binder.
Particular examples of phosphates and phosphonic acids that were tested as potential binders included 4-chloro-5-bromo-indolyl phosphate, diethyl thiophosphate, diethyl dithiophosphate, diethyl cyanophosphonate, and methylphosphonic acid. Examples of organosulfates, sulfonic acids, and sulfides that were tested as binders include indolylsulfate, hydroxyethyl-piperazinepropanesulfonic acid, toluenesulfonic acid, L-cysteine, and N,N-diethylaminoethanethiol. Halide in the form of hydrogen chloride was also tested as a binder. Metal ions, in the form of soluble halide and nitrate salts, were also tested as binders; those tested include the following: ZrCl4, La(NO3)3, Ce(NO3)3, Pr(NO3)3, Nd(NO3)3, Sm(NO3)3, Eu(NO3)3, Gd(NO3)3, Tb(NO3)3, Dy(NO3)3, Ho(NO3)3, Er(NO3)3, Tm(NO3)3, Yb(NO3)3, and Lu(NO3)3.
The method of the invention, in particular, was used to detect binding of N,N-diethylaminoethanethiol and/or methylphosphonic acid with members of the 11-mer oligopeptide library. An aqueous solution of these materials was prepared by combining 0.2 mL of a 200 mM solution of methylphosphonic acid (40 μmol) with 0.2 mL of a 210 mM solution of the hydrogen chloride salt of N,N-diethylaminoethanethiol (42 μmol). About 2500 of the oligopeptide-supported beads (corresponding to 5 mg total weight with about 1.6 μmol total oligopeptide) were incubated in the solution at room temperature for 2 days. The beads were then removed from solution, washed with 10 mL of water, air dried, and immobilized on a Tacky Dot™ plate as an array. The beads of the array were then analyzed for binding using micro-X-ray fluorescence spectrometry using the EDAX micro-fluorescence instrument. Two beads in particular displayed a strong binding effect with methylphosphonic acid. Their amino acid sequences, as determined by Edman degradation analysis, were the following (the amine end group belongs to the amino acid at the end of the chain): 1) HisGlyGlyHisGlyGlyHisGlyGlyArgPhe (SEQ ID NO:2); and 2) TyrGlyGlyTyrGlyGlyTrpGlyGlyTyrPhe (SEQ ID NO:3). Two different beads displayed a strong binding effect with the thiol. Their amino acid sequences, as determined by Edman degradation analysis, were the following: 3) SerGlyGlyArgGlyGlyHisGlyGlyHisPhe (SEQ ID NO:4); and 4) TrpGlyGlyHisGlyGlyHisGlyGlyTrpPhe (SEQ ID NO:5).
The invention was also used to screen potential metal catalyst binders. The chemical procedure employed was similar to one described by A. Berkessel and D. A. Herault in “Discovery of Peptide-Zirconium Complexes That Mediate Phosphate Hydrolysis by Batch Screening of a Combinatorial Undecapeptide Library”, Angew. Chem.-Int. Ed. 1999, vol. 38, p. 102, hereby incorporated by reference. Berkessel et al. exposed a combinatorial array of bead-supported oligopeptides to zirconium tetrachloride. The resulting zirconium complex binds to some of the peptides, and some of the peptides with bound zirconium catalyze the hydrolytic cleavage of a phosphate ester. The catalytic activity of each exposed bead was determined by exposure to 4-chloro-5-bromo-indolyl phosphate, which undergoes phosphate ester cleavage to form 4-chloro-5-bromo-indoxyl and rapidly oxidizes to 4,4′-dichloro-5,5′-dibromo-indigo, an insoluble blue dye. The relative intensity of the blue color indicates the relative amount of blue dye for a particular bead, and was assumed to be proportional to the activity of the catalyst on that bead; a more intensely blue colored bead indicates a more active catalyst.
With regard to an example of the present invention, about 12,500 oligopeptide-supported beads weighing a total of about 25 milligrams, corresponding to about 8.2 total micromoles of oligopeptide, were pretreated by incubation in a room-temperature, aqueous solution of zirconium tetrachloride, ZrCl4 (4.0 mg, 17 μmol in 0.6 mL water), for about 3 days. The beads were removed from solution, washed with about 10 mL of water, and air-dried. A solution of EPPS buffer (EPPS=hydroxyethyl-piperazinepropanesulfonic acid) at a pH of 5.3 was prepared. A buffered solution was prepared by combining about 0.3 mL of a 320 millimolar (mM) solution of the EPPS buffer solution with 0.3 mL of a5.4 millimolar (mM) solution of 4-chloro-5-bromo-indolyl phosphate (about 1.6 μmol) and 0.3 mL of a 8.6 mM solution of zirconium tetrachloride (about 2.6 μmol). About 2600 of the pretreated beads, weighing 5.3 mg were incubated in the buffered solution at room temperature for about 15 h. They were removed from the buffered solution, washed with about 10 mL water, air dried, and immobilized on a Tacky Dot™ plate in the form of an array. Each member of the array was analyzed by X-ray fluorescence spectrometry. The Tacky Dot™ plate was placed in the EDAX MXRF instrument under vacuum. The instrument was operated at 35 kV and 500 μA using a rhodium X-ray tube. The area scanned was 14×7.4 mm using a step size of 27 μm in the x-direction and 19 μm in the y-direction. The pixel area was 512×400 with a 200 μsec dwell time per point. The point spectra were acquired with an acquisition time of 100 live seconds, the amount of time that the detector is actively obtaining counts. The single point spectra provided the elemental intensities for the beads, several of which are listed in the Table below.
TABLE Bead Color Zr (counts) Br (counts) Br/Zr P (counts) 1 Yellow 93.68 303.49 3.24 545 2 Amber 34.92 118.74 3.40 82.21 3 Clear 13.75 115.2 8.38 184.41 4 Dark gray/blue 7.92 299.68 37.84 104.29 5 Yellow 37.69 330.98 8.78 264.62 6 Dark yellow 4 157.93 39.48 50.16 7 Clear 6.06 131.88 21.76 32.21 8 Gray 0 81.9 0.00 29.72 9 Dark yellow 10.27 262.58 25.57 116.86 10 Gray/blue 19.56 105.71 5.40 21.87 11 Yellow 15.56 259.2 16.66 191.59 12 Yellow 11.35 241.71 21.30 79.2 13 Blue/gray 47.93 276.95 5.78 86.24 14 Clear 15.12 178.57 11.81 185.84 15 Yellow 11.15 119.19 10.69 100.88
The most effective bead-supported catalyst produces the most product per unit of catalyst, and is a blue colored bead with a high Br count and a low P count and large Br/Zr ratio. Of the fifteen beads listed in the above Table, bead 4 includes all of these features. Blue beads 10 and 13 have significantly smaller Zr/Br ratios than bead 4, indicating poorer catalyst activity. In addition, those beads with a high Zr content bind strongly to Zr, indicating that the corresponding oligopeptides could be used as a separation agent specific for Zr. Analysis by MALDI-TOF Mass spectrometry, or by Edman degradation as previously described, can provide the amino acid sequences for the oligopeptides.
The examples described involve monitoring the elements P, Br, and S, which are present in the binder but not in any of the receptors. The X-ray fluorescence signal due to these elements indicates that the particular binder with these elements is present, making the determination of a binding effect straightforward. It should be understood that the X-ray fluorescence spectrometer can determine whether a particular element is present and also the quantity of that element. Therefore, the analysis may include a determination of the quantity of an element common to both the binder and the receptor. If a binding event occurs between a binder and receptor, and both the binder and receptor include the elements carbon and oxygen, for example, an X-ray spectrometer capable of analyzing for these elements can distinguish between the amount of these elements present in the receptor and the amount present in a binder-receptor complex. For binders that only include elements that are also common to the receptor, the difference in the signal intensity between the receptor and the binder-receptor complex for these elements provides an indication that a binding event has occurred.
Other metal ions, purchased as standard ICP solutions (100 μg/mL in 2% HNO3), were also tested as binders; they include ions of Be, Ca, Cd, Co, Cr, Cu, Fe, Li, Mg, Mn, Mo, Ni, Pb, Sb, Se, Sr, Ti, TI, V, and Zn. Metal ions of Ce, Ag, Ba and Hg, available as a standard solution, 1000 μg/mL in 2% HNO3 solution, were also tested as potential binders.
Oligopeptides are only one type of receptor that can be used with the present invention, and clearly many other types of receptors can also be used. Esters, amines, imines, aldehydes, ketones, amides, ethers, olefins, halogenated organic molecules, antibodies, drugs, steroids, amino acids, and nucleotides can be used. Other types of oligomers such as oligonucleotides, oligosaccharides, and oligopeptides can be used. Polymers such as polyolefins, polyurethanes, polyesters, polycarbonates, polyamines, polyamides, halogenated polymers, polypeptides, polynucleotides, and polysaccharides, and nucleic acids, to name a few, can also be used as receptors. Both naturally occurring and man-made materials can be used. Also, complex structures of molecules can be used as receptors that include cell membrane receptors, viruses, cells, cellular membranes, organelles, and the like.
Similarly, many different materials can be tested as potential binders. These materials include organic molecules such as esters, amines, imines, aldehydes, ketones, amides, ethers, olefins, and halogenated organic molecules. Antibodies, drugs, hormones (e.g. steroids and the like), amino acids, and nucleic acids can also be tested as potential binders. Oligomers such as oligonucleotides, oligosaccharides, and oligopeptides, and polymers such as polyolefins, polyurethanes, polyesters, polycarbonates, polyamines, polyamides, halogenated polymers, polypeptides, polynucleotides, polysaccharides, nucleic acids, to name a few, can be also be tested as binders.
Both naturally occurring and man-made materials can be used as binders. Metal ions such as calcium ion, barium ion, sodium ion, potassium ion, iron ion, palladium ion, silver ion, and strontium ion can be used. Anions such as bromide, chloride, iodide, sulfide, and selenide, for example, can be tested as potential binders. Complex ions such as oxyanions, polyoxoanions, phosphate, organophosphates, sulfate, organosulfates, zirconate, molybdate, tungstate, chromate, for example, can be tested. Also, agonists and antagonists for cell membrane receptors, toxins, enzymes, enzyme substrates, cofactors, and monoclonal antibodies can also be tested.
The invention can also be used to determine the binding affinity (BA) between a binder and a receptor. The BA is defined as the concentration of the binder-receptor complex divided by the product of the concentrations of the binder and the receptor. To determine the binding affinity (BA), the concentrations of the binder and receptor, and the concentration of the binder-receptor complex must be determined. There are several ways, both non-destructive and destructive (e.g. removal of the receptor from the support bead), of determining the concentration of the receptor. The concentration of the receptor can be determined directly and non-destructively (i.e. the receptor remaining on the bead) by a spectroscopic technique such as by X-ray fluorescence. The receptors can also be cleaved from the bead, collected, and quantified using high performance liquid chromatography. The concentration of the receptor can also be estimated indirectly from parameters related to the support beads, such as the size and surface area of the beads.
Similarly, the concentration of the binder can be determined by quantifying the amount of binder remaining in solution after the bead-supported receptors have been submerged in the binder solution for the time period allowed for binding to occur.
It is generally assumed, particularly in the development of new catalysts on ligand (e.g. a peptide ligand) coated beads, that the distribution of ligand on each bead is similar. This often unstated assumption is illustrated by the methods used to determine catalyst efficiency. These methods as exemplified in the aforementioned Berkessel et al. publication rely on quantifying the product of the catalytic reaction without quantifying the amount of catalyst that is formed. Product quantification methods that ignore the quantification of the catalyst lead to faulty conclusions about catalyst efficiency since catalyst efficiencies cannot be determined without knowledge of the quantity of the catalysticatalysts used.
The present invention uses micro-X-ray fluorescence to determine the presence and relative amounts of elements. These elements can be in the form of metal ions such as calcium ion, chloride, bromide, iodide, phosphorus, and sulfur, the latter two being important constituents of polypeptides such as enzymes, RNA, and DNA. Thus, the invention provides a non-destructive method of screening the binding of a receptor array to a potential binder, and for quantifying the binding affinity. A commercially available micro-X-ray fluorescence spectrometer has been used to evaluate the binding of materials to an array of oligopeptides, each oligopeptide bound to a polystyrene bead substrate.
In summary, the present invention provides a method for detecting binding events between arrayed receptors and potential binders. The invention provides significant advantages over known methods for measuring binding affinities since known methods often require either radioactive binders, or binders that include a covalently attached label that fluoresces upon exposure to ultraviolet excitation radiation. Since the invention does not require radioactive or chemically tagged materials, the problems dealing with handling of radioactive materials and the disposal of radioactively contaminated waste are avoided. Importantly, since the use of artificially tagged materials is not required, there can be no interference from the tag in the evaluation of the binding affinity of the corresponding desired untagged material. Further, in contrast to methods that require tags, the method of the present invention can be used to evaluate the binding affinity of materials that do not fluoresce while exposed to ultraviolet radiation. It should be understood that although tagged materials are not required, they could also be used and this aspect of the invention offers a distinct advantage in that the invention can provide a direct comparison of binding affinity of the untagged binder with that of the corresponding tagged surrogate. This comparison could validate or invalidate the assumption that a particular untagged binder and its tagged surrogate have the same binding affinity to a particular substrate.
The foregoing description of the invention has been presented for purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching.
The embodiment(s) were chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.
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|U.S. Classification||436/172, 436/119, 430/966, 436/518, 436/124, 436/103|
|International Classification||G01N33/58, G01N33/543, G01N21/76, G01N33/00|
|Cooperative Classification||Y10T436/18, Y10T436/16, G01N33/582, G01N33/54373, Y10T436/19, Y10S430/167|
|European Classification||G01N33/58D, G01N33/543K2|
|Sep 20, 2001||AS||Assignment|
Owner name: REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE, NEW
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WARNER, BENJAMIN P.;HAVRILLA, GEORGE J.;MANN, GRACE;REEL/FRAME:012201/0014;SIGNING DATES FROM 20010629 TO 20010705
Owner name: REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE, NEW
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WARNER, BENJAMIN P.;HAVRILLA, GEORGE J.;MANN, GRACE;SIGNING DATES FROM 20010629 TO 20010705;REEL/FRAME:012201/0014
|May 15, 2003||AS||Assignment|
Owner name: ENERGY, U.S. DEPARTMENT OF, DISTRICT OF COLUMBIA
Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF CALIFORNIA;REEL/FRAME:014073/0108
Effective date: 20020103
|May 18, 2006||AS||Assignment|
Owner name: LOS ALAMOS NATIONAL SECURITY, LLC, NEW MEXICO
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE;REEL/FRAME:017913/0867
Effective date: 20060501
|Jul 7, 2014||FPAY||Fee payment|
Year of fee payment: 4
|Jul 7, 2014||SULP||Surcharge for late payment|
|Sep 17, 2014||SULP||Surcharge for late payment|
|Sep 19, 2014||SULP||Surcharge for late payment|
|Oct 16, 2014||AS||Assignment|
Owner name: CALDERA PHARMACEUTICALS, INC., NEW MEXICO
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:LOS ALAMOS NATIONAL SECURITY, LLC;REEL/FRAME:033961/0617
Effective date: 20141015
|Apr 9, 2015||AS||Assignment|
Owner name: XRPRO SCIENCES, INC., NEW MEXICO
Free format text: CHANGE OF NAME;ASSIGNOR:CALDERA PHARMACEUTICALS, INC;REEL/FRAME:035401/0627
Effective date: 20141202
|Jan 28, 2016||AS||Assignment|
Owner name: ICAGEN, INC., NORTH CAROLINA
Free format text: CHANGE OF NAME;ASSIGNOR:XRPRO SCIENCES, INC.;REEL/FRAME:037638/0543
Effective date: 20150828
|May 15, 2017||AS||Assignment|
Owner name: GPB DEBT HOLDINGS II, LLC, NEW YORK
Free format text: SECURITY INTEREST;ASSIGNORS:ICAGEN, INC.;ICAGEN CORP.;CALDERA DISCOVERY, INC.;AND OTHERS;REEL/FRAME:042379/0067
Effective date: 20170515
Owner name: GPB DEBT HOLDINGS II, LLC, NEW YORK
Free format text: SECURITY INTEREST;ASSIGNORS:ICAGEN, INC.;ICAGEN CORP.;ICAGEN-T, INC.;AND OTHERS;REEL/FRAME:042378/0935
Effective date: 20170515

domain: chemistry


text: Walter Abish was born in Vienna, Austria. Much of his childhood was spent in China. He became an American citizen in 1960. Abish's fascination with human communication led him to write works focused on the use of language. His first novel, Alphabetical Africa (1974), was an experiment in alliteration, moving forward and backward through the alphabet while telling the story. Throughout the 1970s, he wrote short stories that demonstrated a variety of unique writing formats. His second novel, How German Is It (1980), a more conventionally written book, received the 1981 PEN/Faulkner award, an honor bestowed by his peers. In Eclipse Fever (1993), Abish continues to play with language, this time within the context of a suspense story about Mexico's social and intellectual elite. Abish lives in New York where he is a lecturer in English and Comparative Literature at Columbia University.

domain: library


text:
Offering 110 rooms, the comfortable Mercure Edinburgh Haymarket Hotel features a tennis court and a sauna available on site. Mercure Edinburgh Haymarket Hotel welcomes guests to its contemporary rooms since 2015.
Nestled in the center of Edinburgh and a 20-minute walk from Scottish National Gallery of Modern Art train station. The city center is 2 km away from the hotel.
Mercure Edinburgh Haymarket Hotel welcomes guests to take advantage of individual climate control, an in-room safe, high-speed internet, a Nespresso coffee machine and a sitting area supplied in the rooms. Room facilities also include an electric kettle, a dishwasher and a stove for self-catering.
Guests are offered a buffet breakfast served in the bar. There is a restaurant serving international dishes on site. The snack bar serves soft drinks and tea. Offering a variety of dishes, Indaba and Passorn are about 400 meters away.
Turnhouse airport is about 15 minutes by car.
Mercure Edinburgh Haymarket Hotel offers 24-hour front desk, laundry and dry cleaning.
Active travelers will appreciate a gym and a fitness centre available on site.

domain: travel


Kristaps Porzingis sat out his second straight game with Achilles tendinitis and could miss the Martin Luther King Day matinee vs. Atlanta. He has missed five games total with the lingering issue, and now the medical staff’s edict is for him not to come back until he’s 100 percent. Hence, Hornacek said Porzingis “may be doubtful” vs. Atlanta but added the 7-foot-3 Latvian “hopes to play.’’
Porzingis said it’s not his call and will listen to the medical staff. “No problema,” Porzingis said, using his Spanish. “I didn’t feel any pain. I hope [to be ready]. The way I feel right now, yes, I feel I’ll be available [Monday]. It all depends if the doctors let me go.”
Source: Marc Berman of the New York Post
Sources: Jimmy Butler Prefers Trade to Clippers, Interested in Knicks, Nets
After registering a trade request with the Minnesota Timberwolves, All-Star Jimmy Butler is most interested in pursuing a future with the Los Angeles Clippers, league sources told ESPN.
Butler’s ability to partner on the Clippers with a second star player in a deep summer free agency market holds a strong appeal to Butler, league sources said.
The Clippers have two max contract slots available in July, and are emerging as a front-runner for Toronto’s All-NBA forward Kawhi Leonard when he becomes a free agent in July, league sources said.
Butler told Minnesota president of basketball operations and coach Tom Thibodeau that he would like to be traded before reporting for Timberwolves media day on Monday and prefers the possibility of the Clippers — as well as the New York Knicks, league sources said. Butler told the Timberwolves that he planned to leave the franchise in free agency next summer, sources said.
The Brooklyn Nets’ ability to sign two max free agents in the summer will keep them in consideration for Butler, but he has prioritized the Clippers and Knicks over them, league sources said.
Source: Adrian Wojnarowski of ESPN
Sources: Brandon Knight Had ‘clean up’ Procedure on Left Knee
On @theathleticnba @watchstadium: Houston’s Brandon Knight underwent surgery on his left knee, considered a “clean up,” and will miss some time, per league sources.
— Shams Charania (@ShamsCharania) September 20, 2018
Source: Shams Charania on Twitter
Report: Pelicans Waive Emeka Okafor
The New Orleans Pelicans announced today that the team has requested waivers on center Emeka Okafor.
Okafor, 6-10, 252, appeared in 26 regular season games last season with New Orleans after signing with the team on February 3, 2018 and finished the season with averages of 4.4 points, 4.6 rebounds and 1.0 blocks per game.
Source: New Orleans Pelicans


One of the things you'll notice quickly is that by default, Windows Vista will not map samba shares - nor can you use Samba as a PDC (Primary Domain Controller). This is due to the default security policy of Windows Vista only using NTLMv2 for authentication - something Samba doesn't support as yet.
To fix this, run (Win+R) "secpol.msc"
Navicate to Local Policies -> Security Options
Change the value of the policy "Network Security: LAN Manager authentication level" from "NTVLM2 responses only" to "LM and NTLM - use NTLMv2 session security if negotiated"
This should now allow Windows Vista to both map network drives based on Samba servers, and use Samba as a PDC.


text:
When Joseph first became aware of the impact of music against images, he was inspired to create. But growing up in a place without a film program, he turned to the stage, composing incidental music for plays throughout high school and college. He began his musical study on piano and later to playing trumpet and French horn in the school band, and singing in the choir. He discovered a passion for oboe and earned a degree in oboe performance from Boise State University.
Joseph now focuses his talents in the film music industry in Los Angeles. His work can be heard in numerous features, commercials, web series, and short films.
Joseph has also prepared scores and parts, served as orchestrator and arranger for other industry colleagues, including Emile Mosseri (Last Black Man in San Francisco), Miriam Cutler (Dark Money, RBG, Love, Gilda), and Penka Kouneva (Life Link, Blue). Additionally, he has performed oboe parts, whistling, vocals, voice-over and narration.

domain: artistic


According to numerous industry experts and journalists, the Acer Iconia Windows 7 dual screen tablet and the Motorola Atrix 4G are some of the hottest things to have come out of this year’s CES 2011, and are the most loved stars of the show. While we have already spoken about the innovative Acer Iconia and its dual-screen laptop form factor, the Motorola Atrix 4G is certainly not lacking in innovation. Currently running Froyo, the Atrix will get an upgrade to Gingerbread soon.
[RELATED_ARTICLE]While it is just a phone (though some would beg to differ), the Atrix 4G is supremely well-endowed, and, comes with exciting accessories in the form of docks, which convert the phone into something more. Let us start with the specifications: 4-inch (960×540) Gorilla Glass capacitive touchscreen with qHD rating and 24-bit colour, 1GHz dual core Nvidia Tegra 2 CPU/GPU, 1GB RAM, 16GB internal storage (expandable to 48GB with microSD card), 5MP rear camera capable of 720p HD and front-facing VGA camera for video calls, 2.4GHz and 5GHz Wi-Fi 802.11n support, dual-microphones for active noise cancellation, and a massive 1930 mAh battery. All this madness, squeezed into less than 11mm of thickness.
The phone’s docks include:
1) Motorola HD Multimedia Dock, which gives users the flexibility of docking their phones with three USB ports and one HDMI, perfect for connecting it up to a TV, keyboard, mouse, speakers, all at the same time.
2) Motorola Laptop Dock, the more revolutionary one of the two, and perhaps what makes the Motorola Atrix’s form factor totally ridiculous…the dock is basically an ultralight and ultrathin laptop without a CPU. It has an 11.6-inch screen, and a full-sized keyboard, along with stereo speakers, and a 36Wh three-cell battery which is rated to delivers up to eight hours of battery life. Total weight, including phone, is approximately 1kg. Many tech blogs who have had a chance to use the device in this dock have gone away giddy with excitement, claiming superlative response times with no perceptible lag, as well as a great environment for web-browsing.
Announced on AT&T currently, there is no news of a global release anytime soon, but hey, we can hope can’t we?


text:
Designed especially to mark the 95th birthday of Queen Elizabeth II, this commemorative flowerpot features a delicately hand-carved Bourbon rose, 'La Reine Victoria', on the front and a celebratory inscription on the reverse that reads:
Her Majesty The Queen's 95th Birthday
Free Delivery Offer (saving £29.50)
The Small Print:
Free delivery applies to mainland UK only. Pots will be delivered to the same address. Offer subject to availabilty. Items will arrive within four to six weeks of placing the order unless we contact you directly. Offer ends 30 April 2021. For full T&Cs click here.
|Her Majesty The Queen's 95th Birthday Pot (996)||22.00cm - 8 1/2"||34.00cm - 13"||
|Pair of Her Majesty The Queen's 95th Birthday Pot (996x2)||0.00cm - --"||0.00cm - --"||
Meet the Maker
Debs has been working as a Head Decorator at the pottery since 2015. She really enjoys the creativity and diversity involved in working with clay and loves the friendly team she is in. Before joining us Debs studied a Masters in Sound Art and creates her own work in her spare time.


text:
126 Newark Avenue
No hours of operations available
Pizza Prantha is located at 126 Newark Ave, Jersey City, NJ. This location is in the Historic Downtown neighborhood. This business specializes in Pizza.
by merchantcircle on April 28, 2015 from
Privacy & Terms

domain: geography


text:
Gambian hip-hopper Shuga has been on the go since the start of her career and there seem to be no stopping in sight.
Born Sarah Sillah, Shuga is a British-born Gambian singer-songwriter who began her music career at a very young age. She started out as a dancer before joining a hip-hop dance group in London.
Her breakthrough moment came with the release of her anthem The Smiling Coast featuring Gambian rap ace Gee.
The song became a radio and TV hit.
Over the years, she has performed in concerts in Africa and Europe.
Last year, she starred at the annual Open Mic fest, where she delivered a superb set including a remix of Smiling Coast alongside Gee.
Shuga said the song was inspired by her experiences when she visits family and friends in The Gambia.
The video of the song was voted most-watched Gambian music video of 2014.
Shuga has worked on numerous projects which include her EP and collaborated on score in the scene. She hopes to make gains in the global music scene someday.


text:
I wholeheartedly believe that we have the right to speak our minds, however, when the content of our beliefs spews only hatred and judgement, there is no place for it on a college campus. Recently, pro-life campaigners have taken to the UNC campus, holding signs of graphic images and yelling words filled with anger and hate.
Luckily, many students feel so passionately that they have been willing to stand in front of the images to ensure that no other students see them, and holding signs that say things like “trigger warning.” Personally, I believe that as women, we, individually, are the only ones that can make decisions surrounding our bodies. Not other women, and definitely not other men. My beliefs aside, I understand that not everyone is going to agree with me. That’s fine. Every person is entitled to their own beliefs, whatever they may be.
However, shaming other women for not agreeing with your beliefs is not okay. Doesn’t the Bible say love thy neighbor? I’m no expert, but I’m pretty sure that includes all neighbors, regardless of their personal beliefs. If you expect other people to value and respect your opinions, then it is crucial that you do the same. Holding graphic signs and screaming at vulnerable college students that might be experiencing the very thing you are protesting against does not make you a martyr, fighting for your cause, only an ignorant bigot spreading anger and hate.
Grace Louise Murtha

domain: sociology


- Part 2 of Chris Jericho’s “Talk Is Jericho” podcast interview with WWE Hall of Famer Edge is available at this link.
– While last night’s Christmas RAW was taped a week ago, JBL and Michael Cole did voice-over work live from WWE headquarters in Stamford.
– If WWE does reach an agreement for Bill Goldberg to return at WrestleMania XXX, he will be in great shape. Goldberg noted on Twitter that he still trains 6 days a week.
|Seth Rollins On Brock Lesnar's Title Reign - "I Think It's Bull Crap, I Don't Appreciate It One Bit"|
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