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Patent US7101621 - Azlactone-functional hydrophilic coatings and hydrogels - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsSurface coatings including azlactone-functional hydrogels and articles with the coatings disposed thereon are disclosed. Methods of making the coating and controlling the gellation time of the hydrogels are also disclosed....http://www.google.com/patents/US7101621?utm_source=gb-gplus-sharePatent US7101621 - Azlactone-functional hydrophilic coatings and hydrogelsAdvanced Patent SearchPublication numberUS7101621 B2Publication typeGrantApplication numberUS 10/896,664Publication dateSep 5, 2006Filing dateJul 22, 2004Priority dateMay 18, 2001Fee statusPaidAlso published asCA2445758A1, CA2445758C, EP1387857A1, US6794458, US20030049435, US20050003198, WO2002094890A1Publication number10896664, 896664, US 7101621 B2, US 7101621B2, US-B2-7101621, US7101621 B2, US7101621B2InventorsLouis C. Haddad, James I. Hembre, Jerald K. Rasmussen, Daniel SarpongOriginal Assignee3M Innovative Properties CompanyExport CitationBiBTeX, EndNote, RefManPatent Citations (21), Referenced by (6), Classifications (79), Legal Events (3) External Links: USPTO, USPTO Assignment, EspacenetAzlactone-functional hydrophilic coatings and hydrogels
US 7101621 B2Abstract
Surface coatings including azlactone-functional hydrogels and articles with the coatings disposed thereon are disclosed. Methods of making the coating and controlling the gellation time of the hydrogels are also disclosed.
a material disposed upon the substrate, the material comprising
i) at least one azlactone-functional copolymer comprising a plurality of azlactone moieties and a plurality of azlactone functional groups, wherein the copolymer is derived from monomers comprising at least one alkenyl azlactone monomer and at least one hydrophilic or water soluble comonomer;
ii) a first crosslinker comprising a first nucleophilic moiety and a second thermally reactive moiety; and
iii) optionally, a second crosslinker comprising a first nucleophilic moiety and a second thermally reactive moiety;
a) the first moiety of a first crosslinker is covalently bound to a first azlactone moiety; and
b) the second moiety of the first crosslinker is covalently bound to i) a second azlactone moiety or ii) the second moiety of the second crosslinker, with the first moiety of the second crosslinker being bound to the second azlactone moiety; and
wherein the material is a crosslinked hydrogel.
2. The article of claim 1 wherein the substrate comprises a plate, a film, a particle, a fiber, a column, a bead, a web, or a membrane.
3. The article of claim 1 further comprising a primer.
4. The article of claim 3 wherein the primer is polyethylenimine, polyvinylidene chloride, a colloidal dispersion of metal oxides, or an ambifunctional silane.
5. The article of claim 1 wherein the first crosslinker and the second crosslinker have the same chemical structure.
6. The article of claim 1 wherein the first crosslinker and the second crosslinker have different chemical structures.
7. The article of claim 1 wherein the chemical structure of the second moiety of the first crosslinker is the same as the chemical structure of the first moiety of the first crosslinker.
8. The article of claim 1 wherein the chemical structure of the second moiety of the first crosslinker is different than the chemical structure of the first moiety of the first crosslinker.
9. The article of claim 1 wherein the chemical structure of the second moiety of the second crosslinker is the same as the chemical structure of the first moiety of the second crosslinker.
10. The article of claim 1 wherein the chemical structure of the second moiety of the second crosslinker is different than the chemical structure of the first moiety of the second crosslinker.
(b) a hydrogel material disposed upon the substrate
wherein the hydrogel material comprises:
a) the first moiety of the first crosslinker is covalently bound to a first azlactone moiety; and
wherein the hydrogel material is a crosslinked hydrogel; and
(c) a functional material comprising (i) a nucleophilic group reacted with a third azlactone moiety of the azlactone-functional copolymer and (ii) another reactive site.
12. The composite of claim 11 wherein the substrate comprises a plate, a film, a particle, a fiber, a column, a bead, a web, or a membrane.
13. The composite of claim 11 further comprising a primer.
14. The composite of claim 13 wherein the primer is polyethylenimine, polyvinylidene chloride, a colloidal dispersion of metal oxides, or an ambifunctional silane.
15. The composite of claim 11 wherein the functional material is a polypeptide, a polynucleotide, a polysaccharide, or any combination thereof.
16. The composite of claim 11 wherein the first crosslinker and the second crosslinker have the same chemical structure.
17. The composite of claim 11 wherein the first crosslinker and the second crosslinker have different chemical structures.
18. The composite of claim 11 wherein the chemical structure of the second moiety of the first crosslinker is the same as the chemical structure of the first moiety of the first crosslinker.
19. The composite of claim 11 wherein the chemical structure of the second moiety of the first crosslinker is different than the chemical structure of the first moiety of the first crosslinker.
20. The composite of claim 11 wherein the chemical structure of the second moiety of the second crosslinker is the same as the chemical structure of the first moiety of the second crosslinker.
21. The composite of claim 11 wherein the chemical structure of the second moiety of the second crosslinker is different than the chemical structure of the first moiety of the second crosslinker.
22. The composite of claim 11 wherein the functional material comprises a biologically active material.
This application is a division of U.S. patent application Ser. No. 09/860,944, filed May 18, 2001, now U.S. Pat. No. 6,794,458.
This invention relates to porous, hydrogel coatings useful for the immobilization of biologically active molecules and, particularly, to crosslinked, azlactone-functional hydrogel coatings that are useful for the preparation of DNA and protein arrays, diagnostic devices and materials for the separation of biological species. More particularly, this invention relates to porous hydrophilic, crosslinked, azlactone-functional coatings and gels.
Coatings derived from copolymers of polymerizable azlactones and olefinically unsaturated monomers are known. Such coatings are derived, in general, from rigid, high glass transition temperature (Tg), hydrophobic copolymers. Crosslinking is accomplished by dispersing or dissolving the azlactone copolymer with a crosslinking agent, generally in an approximately stoichiometric amount to the azlactone, in a volatile organic liquid, applying the mixture to a substrate, then allowing the coating to crosslink via azlactone ring-opening reactions with the crosslinking agent. Suitable crosslinkers are polyols and polyamines. Polyamines, such as ethylene diamine, react with azlactones at room temperature, thereby forming crosslinks. Because of the rapid reaction between azlactones and primary amines, incorporation of a ketone solvent in the coating mixture is desirable. Polyols react much slower with azlactones and generally require a catalyst, such as a strongly acidic or basic catalyst, to promote crosslinking.
Various coatings derived from azlactone copolymers are known. For example, known coatings include copolymers of 2-alkenylazlactones with acrylic acid esters and copolymerizable vinylidene compounds having at least one hydroxyl group that crosslink on drying or mild heating. Such polymers crosslink by reaction of the hydroxyl groups on one chain of the polymer with azlactone groups on other chains. In general, an acidic or basic catalyst is again needed to facilitate the crosslinking reaction. Coatings derived from azlactone copolymers that are crosslinkable by exposure to radiation are useful in imaging applications. Uncrosslinked azlactone copolymers may be used to coat a variety of substrates. These coated substrates can be used for the immobilization of functional materials, including biologically active species such as proteins. Crosslinked azlactone-functional moieties may be included in a coating over the surfaces of chemically reactive, porous supports. These reactive supports can, in turn, be reacted with biologically active materials to produce adduct supports.
While there are a variety of methods for producing coatings derived from azlactone-functional materials, some of which provide azlactone-functional coatings useful for the immobilization of other species, there remains a need for additional or improved methods for providing coated materials for use in the immobilization of biologically active materials.
This invention relates to the preparation of reactive hydrophilic coatings and hydrogels that can be applied to various substrates for the purpose of covalently attaching a functional material to the substrate. In particular, the invention provides a crosslinked hydrogel for coating a substrate comprising at least one azlactone-functional copolymer comprising a plurality of azlactone moieties, a plurality of azlactone functional groups, and at least one comonomer, and at least one crosslinker comprising a first moiety and a second moiety, wherein the first moiety of a first crosslinker is covalently bound to a first azlactone moiety and the second moiety of the first crosslinker is covalently bound to a second azlactone moiety or a second crosslinker.
In some embodiments of the crosslinked hydrogel of the present invention, the second moiety of the first crosslinker is covalently bound to a second azlactone moiety. In such embodiments, the first crosslinker may be a primary polyamine, a polyether polyamine, a compound containing both a primary and a secondary amine, or any other suitable crosslinker. In other embodiments, the second moiety of the first crosslinker is covalently bound to a second crosslinker molecule. In such embodiments, the first crosslinker may be bound to a second crosslinker molecule having the same chemical structure as the first crosslinker. Alternatively, the first crosslinker may be bound to a second crosslinker having a different chemical structure than the first crosslinker. In either embodiment described above, the first crosslinker, the second crosslinker, or both may be a heterobifunctional crosslinker such as an aminoalkylalkoxysilane.
In some embodiments of the present invention, the crosslinked hydrogel includes polymers made from ionic or non-hydrophilic comonomers.
This invention relates to the preparation of reactive hydrophilic coatings and hydrogels that can be placed on the surfaces of various substrates or within the structures of various structured (i.e., macro- or microstructured) substrates for the purpose of covalently attaching a functional material to the substrate. More specifically, the present invention provides compositions and processes for applying coatings including azlactone functionality onto substrate surfaces. The coatings may include thin films, thick gels, or any intermediate thickness. These coatings may provide for the attachment of functional materials to the substrate. A “functional material” is any chemical species having (a) a nucleophilic group that can react with an azlactone and (b) another reactive site, which is desired to be attached to the substrate to accomplish a specific purpose. In certain embodiments of the present invention, the functional material includes a biologically active material.
For the purposes of this invention, the following definitions shall have the meanings set forth.
“1�/2� amine-containing compound” as used herein shall mean any compound, molecule, composition or complex having one primary amine-containing functional group and at least one secondary amine-containing functional group.
“Azlactone functional group” shall mean a functional group having the structure:
wherein R1 and R2 are, independently, an alkyl group having 1–14 carbon atoms, a cycloalkyl group having 3–14 carbon atoms, an aryl group having 5–12 ring atoms, an arenyl group having 6–26 carbon atoms and 0–3 S, N, or nonperoxidic O atoms, or R1 and R2 taken together with the carbon to which they are both joined form a carbocyclic ring having 4–12 carbons, and n is the integer 0 or 1.
“Functional group” as used herein shall mean a combination of atoms in a molecule, compound, composition or complex that tends to function as a single chemical entity. Examples of functional groups include, but are not limited to, —NH2 (amine), —COOH (carboxyl), siloxane, —OH (hydroxyl), and azlactone. For example, prior to reaction, certain crosslinkers may contain one or more amine functional groups and certain copolymers may contain one or more azlactone functional groups.
“Heterobifunctional” as used herein shall mean, with respect to any molecule, compound, composition or complex, having more than one functional group and having at least two functional groups that are different from one another. For example, an amino acid is heterobifunctional because it contains two functional groups, the amino group and the carboxyl group, that are different than one another.
“Hydrogel” means a water-containing gel, i.e., a polymer that is hydrophilic and will absorb water, yet is insoluble in water.
“Ionic,” with respect to monomers, shall be construed broadly to refer to monomers that inherently have a formal charge as well as monomers that are acidic or basic enough that they can acquire a formal charge when in contact with an aqueous medium.
“Moiety” as used herein shall mean the portion of a functional group from a first reactant that combines with a functional group of a second reactant to form a covalent bond in the reaction product. For example, in a peptide bond, the —NH— that participates in the peptide bond remains from the amine functional group of one amino acid and is therefore considered, herein, to be an amine moiety in the peptide product. The —C═O that participates in the peptide bond remains from the carboxylic acid functional group of the second amino acid and is therefore considered a carboxyl moiety in the peptide product.
“Non-hydrophilic” as used herein shall refer, with respect to any molecule, compound, composition or complex, to any material that has a Hydrophilicty Index of less than about 40.
“Pot life” shall mean the length of time during which a coating formulation remains soluble and homogeneous with low attendant viscosity.
“Primer” shall mean any suitable material that promotes or improves adhesion between the copolymer and the substrate. “Primer” shall include both inert primers and reactive primers. Inert primers act as an adhesion-promoting interlayer between the copolymer and the substrate. Reactive primers form covalent bonds between the copolymer and the substrate to improve adhesion.
The azlactone-functional hydrogel coatings of the present invention are produced by first preparing a solution of a hydrophilic, azlactone-functional copolymer. This copolymer is then formulated with an appropriate crosslinker, and the mixture is then coated on or applied to an appropriate substrate. The crosslinker reacts with a portion of the azlactone groups of the copolymer, thereby forming the porous, crosslinked hydrogel. Unreacted azlactone groups in the hydrogel coating are then available for the attachment of fictional materials for the appropriate end uses.
Azlactone-functional copolymers may be prepared by a variety of free radical polymerization processes in which alkenyl azlactone monomers are copolymerized with comonomers. Typical solution polymerization processes have been reported, for example, in U.S. Pat. No. 4,304,705, issued to Heilmann et al. and U.S. Pat. No. 3,583,950, issued to Kollinsky et al. For the purposes of this invention, suitable comonomers include, without limitation, hydrophilic or water-soluble monomers such as acrylamide, methacrylamide, N-mono- and N,N-disubstituted acrylamides and methacrylamides, N-vinylamides such as N-vinylformamide and N-vinylpyrrolidinone, and hydroxyalkylacrylates and acrylamides such as 2-hydroxyethylmethacrylate and N-acryloyl-trishydroxymethylaminomethane. For many applications, uncharged copolymers may be desirable in order to reduce the possibility for nonspecific binding of biological macromolecules to the coatings. For specific applications, however, ionic comonomers may also be incorporated into the copolymers. Ionic monomers may be anionic or cationic. Anionic monomers include unsaturated acids and their metal salts, such as acrylic, methacrylic, maleic, fumaric, and itaconic acids, vinyl phosphoric and phosphonic acids, styrenesulfonic acid, and 2-acrylamido-2-methyl-1-propanesulfonic acid; cationic monomers include amine-containing monomers such as 2-, 3- or 4-vinylpyridine, (3-acrylamidopropyl)trimethylammonium chloride, 2-diethylaminoethylacrylate and methacrylate, 3-dimethylaminopropylacrylate and methacrylate, and similarly substituted acrylamides and methacrylamides.
Copolymers within the scope of this invention also may include non-hydrophilic comonomers. As used herein, non-hydrophilic comonomers include any comonomer that has a Hydrophilicity Index of less than about 40. The Hydrophilicity Index (or “H.I.”) is an empirical concept that may be useful for describing the hydrophilic character of monomers suitable for use in the present invention. H.I. is defined as:
H.I.=total molecular weight of all hydrophilic groups in the monomer/molecular weight of the monomer�100. molecular weight of the monomer
Hydrophilic groups are generally those that are functionally capable of forming hydrogen bonds with water. Examples of hydrophilic groups include, but are not limited to, —N—, —NH—, —NH2—, —O—, —OH, —COOH, —C═O, —OC═O, —CO2 −M+ (wherein M+ is an alkali or alkaline earth metal cation), —SH, SO3H, —SO3 −M+, —NHCONH—, and other ionic functional groups.
Non-hydrophilic comonomers may be incorporated at less than about 50% by weight and still maintain sufficient hydrophilicity of the coatings and help minimize nonspecific binding. Certain embodiments incorporate non-hydrophilic comonomers at less than about 30% by weight. Suitable non-hydrophilic comonomers include, without limitation, known acrylate and methacrylate esters, styrene, and other free radically polymerizable monomers.
Once the appropriate azlactone-functional copolymer has been prepared, coating mixtures are formulated by adding crosslinkers to the copolymer. This is conveniently done in an appropriate organic solvent that is nonreactive with azlactone functional groups. The copolymer may be diluted with solvent to a concentration of about 5% by weight or less prior to the addition of crosslinker. In other embodiments, the copolymer may be diluted with solvent to concentrations of about 10% by weight or about 20% by weight prior to the addition of crosslinker. The solvent used for dilution may be the same solvent in which the copolymer was prepared or may be one or more different solvents. Crosslinking, or gellation time, is conveniently controlled by copolymer concentration and the amount of crosslinker added, thereby allowing adequate time for coating or for filling structures, followed by rapid cure time to provide finished product. In general, the lower the copolymer concentration or the lower the amount of crosslinker, the longer it will take for the crosslinking/gellation to occur.
Crosslinkers useful for the purposes of the present invention include, without limitation, materials that include nucleophilic groups that will undergo ring-opening reactions with azlactone functional groups. Suitable crosslinkers include primary polyamines, such as ethylenediamine, 1,3-propanediamine, 1,3-diamino-2-hydroxypropane, 1,6-hexanediamine, tris-(2-aminoethyl)amine, and the like; and polyetherpolyamines, such as 4,7,10-trioxa-1,13-tridecanediamine, 3,6-dioxa-1,8-diaminooctane, amine-terminated polyethyleneglycol and polypropyleneglycol homopolymers and copolymers, and the like. To achieve the purposes of the invention, the stoichiometry between the nucleophilic groups of the crosslinker and the azlactone functional groups of the copolymer should be less that 1:1 so that the final crosslinked hydrogel still contains reactive azlactone functionality. Thus, the azlactone content in the original copolymer will provide an upper limit on the amount of crosslinker that may be added to the coating formulation. The intended final use of the hydrogel may also dictate, to a certain degree, the amount of crosslinker used in the formulation. The amount of crosslinking will influence the swelling and porosity of the hydrogel, thus affecting the rate of diffusion of reagents or target molecules into and out of the hydrogel. Generally, less crosslinking provides a hydrogel having larger pores, thereby allowing diffusion of larger biological macromolecules through the hydrogel.
As indicated above, gellation time can be controlled to a certain extent by controlling the concentrations of copolymer and crosslinker. As used herein, gellation time refers to the amount of time necessary for a solution that can form a gel to become no longer fluid. In many instances, these parameters provide adequate control to allow placing the coating solution into the proper configuration prior to the occurrence of gellation. For some applications or product concepts, however, these parameters by themselves do not allow long enough gel times for use in manufacturing. Through the use of some novel crosslinking schemes, the present invention now provides coating formulations with extended pot lives; that is, the coating formulations remain soluble and homogeneous with low attendant viscosities for extended periods of time. Upon evaporation of the solvent and/or raising the temperature of the coated substrate, the coating formulations crosslink to produce the hydrogels of the present invention. These novel crosslinking schemes are achieved by using heterobifunctional crosslinkers, i.e., crosslinkers that have one nucleophilic functional group that reacts with the azlactone group at ambient temperature in solution (e.g., a primary amine) and at least one other functional group that can lead to a crosslinking reaction upon removal of the solvent or upon raising the temperature. One class of crosslinkers that may be used in this manner is the aminoalkylalkoxysilanes such as, for example, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N-[3-(trimethoxysilyl)propyl]ethylenediamine, or other aminoalkylmono-, di- and tri-alkoxysilanes. The amino group undergoes a ring-opening addition reaction with an azlactone group, providing a pendant alkoxysilane group on the copolymer. Upon dry-down, the alkoxy groups may be hydrolyzed and subsequently form siloxane crosslinks between polymer chains. Depending upon the nature of the substrate, covalent bonds may simultaneously be made with functional groups on the surface of the substrate (for example, if the substrate is siliceous, siloxane linkages to the substrate may be formed).
Another class of crosslinking agents useful for prolonging pot life or gellation times includes primary/secondary (1�/2�) amine-containing compounds. In these materials, the primary amine provides rapid reaction with an azlactone group on the copolymer at room temperature, while the secondary amine is relatively slow to react. Removing the solvent, raising the temperature of the coated article, or both allows the secondary amine to react to form the hydrogel. Suitable 1�/2� amine-containing compounds include, without limitation, N-methyl-1,2-ethanediamine, N-ethyl-1,2-ethanediamine, N-isopropyl-1,2-ethanediamine, and other N-alkyldiaminoalkanes. Increasing the steric bulk of the N-alkyl substituent provides a greater barrier to reaction of the secondary amino group, thus necessitating a higher temperature to produce crosslinking.
Once formulated, the coating solutions can be applied to desired substrates and dried (optionally with the application of heat) to produce the hydrogels of the present invention. Coating methods can vary widely depending upon the particular substrate, and may be selected from methods known in the art. These include, for example, extrusion coating, die coating, dip coating, air-knife coating, gravure coating, curtain coating, spray coating, use of wire-wound coating rods, and the like.
With certain substrates, the hydrophilic azlactone-functional polymer will exhibit fairly good adhesion. Crosslinking to produce a hydrogel insolubilizes the coating and reduces the likelihood of the coating coming off of the substrate in subsequent manipulations. Adhesion of the coating to the substrate may be improved, if desired, by any known method. Such methods include, but are not limited to, various pre-treatments to or coatings on the surface of the substrate, such as corona or plasma treatments, or by the application of primers. Suitable primers include, without limitation, polyethylenimine, polyvinylidenechloride, primers such as those reported in U.S. Pat. No. 5,602,202, issued to Groves, and colloidal dispersions of inorganic metal oxides in combination with ambifunctional silanes such as those reported in U.S. Pat. No. 5,204,219, issued to Van Ooij et al., 5,464,900, issued to Stofko et al., and 5,639,546, issued to Bilkadi. Other methods of increasing adhesion to polyolefin substrates are reported in U.S. Pat. No. 5,500,251, issued to Burgoyne et al.
The hydrophilic coatings and hydrogels of the invention may be applied to a wide variety of substrates. The substrates may be natural or synthetic, organic or inorganic, porous or nonporous, flat and substantially featureless or highly structured. The substrates may be film-like, particulate-like, or molded plastic articles. Suitable substrates include, without limitation, standard 96-, 384-, or 1536-well plastic microtiter plates, including filtration plates; grooved, microreplicated films; microfluidic channels in microfluidic devices; embossed or microstructured films; tubes or capillaries; spin tubes or spin columns; glass, ceramic, or metal particles or fibers, including porous particles or fibers; porous or nonporous polymeric fibers or particles, such as chromatographic particles; oriented or non-oriented polymeric films; woven or nonwoven webs (such as fibrous webs); porous or microporous membranes; and the like.
The substrate chosen will depend upon the intended application or device. Those applications include, without limitation, devices such as DNA or protein arrays; biological assay or diagnostic devices; capillary electrophoresis, electrochromatography, or other separation devices; chromatographic supports for affinity, ion exchange, hydrophobic interaction, or other types of separations and purifications; cell selection or separation devices; and the like. For example, when using oriented polymeric films as the substrate, the coatings of the invention are advantageously used to prepare high-density, miniaturized arrays as described in International Publication Number WO 99/53319.
Once the reactive coatings or hydrogels are applied to the substrates, the residual azlactone functionality is available for reaction with the functional material. Again, the intended application will dictate the identity of the functional material. Preferred functional materials are biologically active materials such as proteins, enzymes, oligonucleotides, or any other species that may interact with biological species. Derivatization may be conducted in aqueous, buffered media, as is well known for reactions of azlactone-functional substrates, although other media such as organic solvents may be used.
Gel Formation in Microtiter Tray Format
40% Solids solutions in methyl ethyl ketone (MEK) of 90:10 w/w and 70:30 w/w copolymers of dimethylacrylamide (DMA) and vinyldimethylazlactone (VDM) were prepared by standard free radical polymerization techniques. Portions of each were diluted with 20:1 v/v isopropanol:MEK to give solutions of 2.5% and 10% solids of each copolymer. Portions of each of these dilute solutions were then formulated with enough aminopropyltrimethoxysilane to react with and provide crosslink densities of 2.5% and 10% by weight, thus providing a total of 8 different formulations. These solutions were then deposited, using a micropipette, into the wells of a 384-well polypropylene microtiter tray. Some of the wells were left exposed to the ambient atmosphere while others were covered with Scotch Brand Magic Mending Tape (Minnesota Mining and Manufacturing Co., St. Paul, Minn.). The mixtures in the wells were observed to thicken and become gelled (crosslinked). Shrinkage was observed in the untaped wells due to solvent loss, however, swelling reoccurred upon addition of solvent or water. Upon removal of the tape from the taped wells, the gels were smoothly pulled out of the wells and remained tightly adhered to the tape in a raised gel pattern. Except for the gels prepared from 90:10 poly(DMA/VDM)/10% crosslinker, all have residual azlactone functionality which can be used for covalent attachment of biological or other functional materials.
Formation of Gels in 96-Well Plate
The formulations of Example 1 were used to fill wells (250 microliters each) in a commercial polystyrene 96-well microtitration plate. Because of the larger volume, longer gellation times were observed. Some of the gels could be pulled from the wells with tape as described in Example 1. Alternatively, addition of smaller amounts of solution, or of lower % solids solutions, allowed formation of thin coatings of azlactone-functional hydrogels on the interior surfaces of the microwells.
Formation of Reactive Coating within Wells of a 1536-Well Plate
A 40% solids solution (MEK) of 80:20 w/w poly(DMA/VDM) was diluted to 20% solids with isopropanol (IPA), formulated with enough ethylenediamine to provide a crosslink density of about 10% by weight, then applied to a commercial 1536-well plate. A windshield wiper blade was used to coat and force the solution into the wells. Upon drying, a reactive, azlactone-functional polymeric coating was obtained within the wells.
Formation of Thin, Reactive Coating in a Microtiter Tray
A portion of polypropylene 384-well microtiter tray was cleaned by rinsing with methanol and isopropanol, then dried in an oven at 50� C. for 5 minutes. 1 μl, 2 μl, 3 μl, 4 μl, 5 μl or 10 μl of 0.1% w/w polyethylenimine in deionized water were added to adjacent wells in each of two separate rows. A third row of wells was left untreated. The tray was dried in the oven again for 5 minutes. 2 microliters of 5% solids 70:30 poly(DMA/VDM) copolymer, prepared as in Example 1, was added to each well. The tray was again oven-dried for 5 minutes. A solution of 5-((5-aminopentyl)thioureidyl) fluorescein cadaverine (1 mg/ml in dimethylformamide, Molecular Probes, Inc., Eugene, Oreg.) was diluted to a concentration of 20 micrograms/ml using a buffer consisting of 1 M sodium sulfate in 50 mM 3-[(1,1-dimethyl-2-hydroxyethyl)amino-2-hydroxypropanesulfonic acid (AMPSO), pH 9.0, in deionized water. 2 microliters of this dilute dye solution was added to each of the coated wells and allowed to dry. The tray was then washed thoroughly with deionized water, wetted with some of the above AMPSO buffer, and observed under short wavelength (254 nm) UV irradiation. All wells were examined for fluorescence. Green fluorescence within a well indicated that the fluorescein cadaverine had reacted with the azlactone copolymer coating and had become covalently bound within the well. The tray was then washed with the AMPSO buffer and reexamined. No fluorescence was observed in the untreated wells, indicating that the coating had not adhered to the copolymer and had been washed off with the AMPSO buffer. However, all wells primed with polyethylenimine still showed fluorescence, with the intensity increasing with increasing PEI level. The tray subsequently was washed with 1% w/w SDS (sodium dodecylsulfate) in deionized water and reexamined. No change was seen, indicating good adhesion and/or the PEI priming accomplished crosslinking.
The above experiment was repeated using a microtiter tray containing larger (ca. 8 mm�8 mm square) wells, and larger volumes of the various solutions. Similar results were observed.
Effect of Solvent, Concentration, and Crosslinker on Gel Time
The 70:30 w/w p(DMA/VDM) copolymer from Example 1 (Polymer A), prepared at 40% solids in MEK, was determined by gel permeation chromatography (GPC) to have a weight average molecular weight (Mw) of 600,000. A similar copolymer (Polymer B) was prepared at 40% solids in toluene, and determined to have a Mw of 702,000. These two copolymer solutions were diluted with either methyl ethyl ketone (MEK) or isopropanol (IPA) to concentrations varying from 5–15% solids, then formulated with enough ethylenediamine to react with 1–10% by weight of the total monomer units in the copolymer. Gellation times (time at which the solution was no longer fluid) were observed. Results are listed in Table 1.
TABLE 1 Hydrogel Formation Polymer % Solids Diluent % ED Gel Time A 10 IPA 1 84 min A 10 IPA 2.5 50 min A 10 IPA 5 27 min A 10 IPA 10 25 min A 15 IPA 1 30 min A 15 IPA 2.5 18 min A 15 IPA 5 16 min A 15 IPA 10 13 min B 5 IPA 10 30 min B 10 IPA 10 2.25 min B 5 MEK 10 >48 hrs B 10 MEK 10 60 min These results illustrate control of gellation times and also demonstrate the inhibitory effect of MEK on the formation of crosslinked hydrogels.
Copolymer B was diluted with IPA to give polymer concentrations of 50 mg/ml and 100 mg/ml of solution, respectively. Samples of each solution (3 ml each) were formulated with enough diamine to provide 10% and 20% crosslinking. Diamines utilized were N-ethyl-1,2-ethanediamine, N-propyl-1,2-ethanediamine, and N-isopropyl-1,2-ethanediamine (N-EED, N-PED, and N-IED, respectively). Gellation times were observed at room temperature and at 65� C. No gellation was observed at room temperature over several days. At 65� C., gel times occurred in the following order: N-EED (fastest)>N-PED>N-IED. In a separate experiment, an IPA solution of copolymer B/10% N-EED was dried down at room temperature; the dry polymer readily re-dissolved upon addition of solvent. By contrast, a sample dried at 65� C. would not redissolve, indicating that crosslinking had occurred at the elevated temperature. Infrared spectroscopy verified the extent of reaction with various crosslinkers by comparing the intensity of the azlactone carbonyl absorption band at about 1820 cm−1 to the amide carbonyl band at about 1640 cm−1. All of these results illustrate that secondary amines can be used to control the rate of crosslinking reactions with azlactone functional groups.
Additional Experiments on Gel Formation
Numerous experiments were conducted on the formation of gels using microcentrifuge tubes as containers. Various polymer solutions were prepared varying from 1.0% solids to 20% solids, then these were reacted with a variety of crosslinkers (using varying amounts of each separate crosslinker), and the gellation times noted. Copolymer solutions used included:
a) 95:5, 90:10, and 70:30 w/w p(Acrylamide/VDM) in water;
b) 80:20 w/w poly(DMA/VDM) in isopropanol
Crosslinkers Included:
a) Aminopropyltrimethoxysilane
b) 1.0 M ethylenediamine in ethanol
c) 10% w/w PEI in methanol
d) Polylysine in water
Gellation times of minutes to hours after mixing were obtained, and could be controlled by varying polymer % solids and amount of crosslinker added. These same variables affected the stiffness of the gel that formed.
Coating of Microreplicated Films
A microreplicated polypropylene film approximately 5.5 cm wide, containing 25 channels approximately 1 mm�1 mm in cross-sectional dimensions, spaced 1 mm apart, and running the length of the film, was used as the substrate. Several coating solutions of Polymer A similar to those described in Example 5 were prepared (varying from 1–15% solids and containing 1–10% crosslinker), and were applied by syringe into channels of the substrate. After the coating was crosslinked and dried, the coated substrate was placed in deionized water to rehydrate the gels. In many instances, the gels were observed to swell and pop out of the channels, thereby indicating poor adhesion to the substrate. To minimize this problem, the substrate was primed by dip coating in a 1% w/w solution of polyethylenimine (PEI) in methanol followed by drying at ambient temperature overnight. New coating solutions at 5% and 10% solids with 2.5%, 5%, and 10% crosslinker were applied and allowed to gel. The gels were observed to remain in the channels when challenged with deionized water, even though they did swell somewhat.
Immobilization of Proteins on Microreplicated Films
The PEI-primed substrate of Example 7 was used, and channels were filled with gels derived from 5% solids copolymer formulated with 5% ethylenediamine as crosslinker. Separate pieces of coated substrate were individually dipped in the following protein coupling solutions for 30 minutes to 1 hour: (a) 5 mg/ml bovine serum albumin in phosphate buffered saline (PBS), pH 7.5, containing 1.0 M sodium sulfate; (b) 5 mg/ml myoglobin in 0.1 M 2-[N-cyclohexylamino]ethanesulfonic acid (CHES), pH 9.0, containing 1.0 M citrate; (c) 1 mg/ml Protein A in PBS, pH 7.5, containing 0.9 M sodium sulfate. After coupling, the substrates were washed thoroughly with 1.0 M sodium chloride in PBS to remove any noncovalently bound protein.
Protein Coupling was Evaluated as Follows:
(a) A coated substrate was reacted with myoglobin, coupled as described above, and then observed with an optical microscope. The coated substrate contained reddish-brown stained gels in the channels, indicative of coupled myoglobin.
(b) A coated substrate was reacted with myoglobin and placed in a solution of BCA protein assay reagent (Pierce Chemical Co., Rockford, Ill.). The formation of a deep purple color indicated the presence of coupled protein.
(c) A coated substrate that had been reacted with Protein A and a coated substrate that had been reated with albumin were separately soaked in dilute solutions of rabbit IgG FITC (fluorescein isothiocyanate) conjugate (Sigma Chemical Co., St. Louis. Mo.) in PBS. The coated substrates were washed with PBS, then observed microscopically for bound fluorescence. The coated substrate that had been reacted with Protein A exhibited a strong fluorescent signal, thereby indicating that the IgGs had bound to the Protein A. The coated substrate that had been reacted with albumin exhibited no fluorescence. When the fluorescence intensity of the Protein A coupled gel was measured using a raster scanning device equipped with a 488 nanometer laser, fluorescein filters, and a photomultiplier tube, the intensity was found to be 74,300 RLU (relative light units) above background (uncoated plastic). Control samples that had not been exposed to proteins also exhibited no bound fluorescence.
Similar results were obtained when these experiments were repeated on a microreplicated film having much smaller “V groove” channels. These microchannels had a triangular cross section with a base of approximately 0.3 mm and a height of approximately 0.35 mm.
Coating Glass Slides with Azlactone Polymers
Azlactone/dimethylacrylamide copolymer was premixed with 3-aminopropyltrimethoxysilane (APTMS) and then coated onto glass slides. The amino group of the APTMS is able to react with an azlactone group on the copolymer and the trimethoxysilyl (TMS) group of the APTMS is able to react with the glass. Also some of the TMS groups will react with other TMS groups, thereby resulting in crosslinking.
Glass Slide Preparation:
Frosted-ended glass slides were placed in 1 M sodium hydroxide and allowed to soak for 10 minutes. The slides were removed with forceps and washed under a stream of distilled water to remove all excess sodium hydroxide. The slides were then dipped in 1M HCl and soaked for at least 10 minutes with gentle rocking or occasional stirring. Finally, the slides were washed in a stream of distilled water to remove all of the HCl, rinsed with methanol, and placed in an oven to dry (50�–70� C.).
1. Polymer Solvent: 20:1 IPA:MEK (200 mL IPA and 10 mL MEK)
2. Polymer solutions: A stock solution of 50:50 w/w DMA/VDM copolymer, prepared at 40% solids in MEK, was diluted to 2%, 5% and 10% solids using the Polymer Solvent.
3. APTMS: This reagent was used undiluted or as a 1:10 dilution in Polymer Solvent. Just before use, 1 ml of APTMS was mixed with 9 ml of the Polymer Solvent and kept in a sealed glass vial.
4. Fluorescein cadaverine: 100 μg/mL of fluorescein cadaverine in a solution of 1M sodium sulfate in 50 mM AMPSO buffer at pH 9.5.
Slide Coating:
The coating solutions were prepared, as shown in Table 2, just before coating. Two slides were coated with each of the listed solutions, and one cleaned slide was kept as a blank (13 slides in all). Coating was accomplished with a number 14 wire-wound coating rod. Coated slides were dried in the oven at 50� C. for 10 minutes or until completely dry.
TABLE 2 1:10 2% co- 5% co- 10% Undiluted diluted polymer polymer copolymer APTMS APTMS Coating Solutions (ml) (ml) (ml) (μl) (μl) B1: 2% copolymer, 10 140 5% crosslinker B2: 2% copolymer, 10 280 10% crosslinker B3: 5% copolymer, 10 350 5% crosslinker B4: 5% copolymer, 10 70 10% crosslinker B5: 10% copolymer, 10 70 5% crosslinker B6: 10% copolymer, 10 140 10% crosslinker Reaction with the Fluorescein Cadaverine:
To verify the presence of reactive azlactone functionality, coated slides were evaluated by reaction with fluorescein cadaverine. A water-soaked piece of filter paper was placed in the bottom of each of 13 petri dishes. Each coated slide was placed in a separate petri dish on top of the wet filter paper with the coated side up. Onto each slide were placed two spots each of 2 μl, 5 μl, and 7 μl of the fluorescein cadaverine solution, being careful to keep the spots separated. Each of the petri plates were then covered and allowed to stand overnight at ambient temperature. Each slide was washed well with deionized water, followed by a solution of 5% SDS, deionized water, and finally with AMPSO buffer at pH 9.5. After washing, all slides were examined under the microscope, noting intensity of the fluorescence and quality of the adhesion to the substrate. All formulations exhibited good binding of the fluorescent amine, however B1, B3, and B5 did not display as good adhesion as the other formulations. The blank showed no fluorescence.
Example 9 was repeated using the 70:30 w/w poly(DMA/VDM) copolymer of Example 1. In this case, all formulations exhibited excellent adhesion and dye binding, while the blank again showed no fluorescence.
Example 10 was repeated using polycarbonate film as the substrate instead of glass slides. Results were similar to those observed in Example 10.
Coating of Particulate Substrates
A 70:30 w/w copolymer of DMA and VDM similar to Polymer B of Example 5 (0.9 g of a 22.3% solids solution in isopropanol/toluene) was diluted to 25.5 ml with IPA to give a 1% solids solution. Ethylenediamine (4.8 μl) was added to provide 10% crosslinking. The resulting solution was mixed in a round bottomed flask with 5.0 g POROS 50-R2 (porous, crosslinked styrene-divinylbenzene chromatographic beads from PerSeptive Biosystems, Inc., Framingham, Mass.). The mixture was placed on a rotary evaporator and concentrated to dryness under vacuum. Diffuse reflectance infrared analysis of the coated particles showed absorbances at about 1650 and 1820 cm−1, indicative of the amide and azlactone carbonyl stretches of the copolymer, respectively.
A protein coupling solution consisting of 5 mg/ml myoglobin in 0.1 M CHES, pH 9.0, containing 1.0 M citrate and 0.1% by weight TWEEN 20 detergent was prepared. 30 mg of coated beads were mixed with 5 ml of protein solution. In a separate reaction, 30 mg of coated beads were pre-wet with 0.1 ml of methanol prior to the addition of protein solution. Beads were allowed to react with the protein coupling solution with end-over-end mixing for 1 hour. 1% SDS solution (3 ml) was added to each reaction and mixing was continued for an additional 30 minutes. The mixtures were centrifuged and the absorbance of the supernates was measured at 532 nm and compared to a standard prepared by mixing 5 ml of protein solution with 3 ml SDS solution. From the supernate absorbances, the beads in the first experiment were calculated to have coupled 27.9 mg myoglobin per gram of bead, whereas beads in the presence of methanol coupled 38.0 mg myoglobin per gram of bead. A control, uncoated bead exhibited no coupling of myoglobin.
In analogous experiments, porous and nonporous silica beads were coated with azlactone functional copolymers using either ethylenediamine or APTMS as crosslinkers.
Coating of Nonwoven Webs
A variety of nonwoven webs were coated with hydrogels derived from a 70:30 w/w poly(DMA/VDM) prepared by free radical polymerization in IPA and crosslinked at the 10 wt. % level with either ethylenediamine (ED) or N-ethylethylenediamine (N-EED). The copolymer was diluted to either 1.5% or 3.0% solids with IPA prior to formulation with the crosslinker. Nonwovens used were commercial samples of spunbond nylon (Nylon), polybutyleneterephthalate (PBT), polyethyleneterephthalate (PET), or polypropylene (PP). Samples of the nonwoven were soaked in coating solution for 30 minutes, patted dry with a wipe (Texwipe Co., Upper Saddle River, N.J.), and dried in an oven at 50� C. for 30 minutes. Following coating, the amount of available azlactone functional groups was determined by converting the azlactones to primary amines, then measuring the amine content using a commercial ninhydrin assay kit (Applied Biosystems Corp., Foster City, Calif.) according to the manufacturer's instructions. For this assay, a weighed sample of the coated nonwoven was allowed to react with a 0.5 M solution of ED in IPA for 30 minutes, then washed successively with 3–15 ml portions each of dimethylformamide, deionized water, and ethanol prior to beginning the ninhydrin assay. Results are listed in Table 3.
Functionality of Coated Nonwovens
(nmol/mg)
Coating of Oriented Polymer Films
Coating of the compositions of the present invention on biaxially oriented polyethylene film for the purposes of preparing miniaturized arrays is reported in International Publication Number WO 99/53319.
A substrate film was corona treated according to known techniques prior to coating. The substrate was coated by extrusion die coating with 70:30 w/w poly(DMA/VDM, with 10% 1,2-ethylenediamine (Aldrich Chemical Co., Milwaukee, Wis.) by weight as crosslinker, prepared and diluted with isopropanol. Another substrate was treated and coated similarly, except that the coating was prepared and diluted in methyl ethyl ketone. Each substrate was tested for stability as follows.
A 50 mM sodium phosphate buffer was prepared at pH 8.38 with 1% (w/w) sodium dodecyl sulfate (SDS) in deionized water. The substrates were immersed in the buffer solution at 80� C. for 5 hours. The substrates were analyzed using attenuated total reflectance (ATR) IR spectroscopy to detect presence of the coating.
The coating prepared in isopropanol provided better adhesion than the coating prepared in methyl ethyl ketone. Even better stability, even prior to relaxation of the substrate, is obtained by applying IPA-based coatings to metal coated oriented films such as those described in International Publication Number WO 01/16370.
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS3488327Jun 30, 1967Jan 6, 1970Roehm & Haas GmbhPreparation of coating materialsUS3583950Nov 27, 1968Jun 8, 1971Roehm & Haas GmbhAzlactone copolymersUS3598790Jan 5, 1970Aug 10, 1971Roehm & Haas GmbhAzlactone copolymersUS4304705Jan 2, 1980Dec 8, 1981Minnesota Mining And Manufacturing CompanyRadiation-curable polymers containing pendant unsaturated peptide groups derived from azlactone polymersUS4451619Sep 30, 1982May 29, 1984Minnesota Mining And Manufacturing CompanyMethod of hydrophilizing or hydrophobizing polymersUS4485236Sep 27, 1982Nov 27, 1984Minnesota Mining And Manufacturing CompanyAzlactone-functional compoundsUS5149806Mar 28, 1990Sep 22, 1992Minnesota Mining And Manufacturing CompanyAzlactone michael adductsUS5204219Jan 21, 1992Apr 20, 1993Minnesota Mining And Manufacturing CompanyPhotographic element with novel subbing layerUS5292840Apr 10, 1989Mar 8, 1994Minnesota Mining And Manufacturing CompanyPolymeric supportsUS5344701Jun 9, 1992Sep 6, 1994Minnesota Mining And Manufacturing CompanyPorous supports having azlactone-functional surfacesUS5436147Jun 16, 1993Jul 25, 1995Nucleic Assays CorporationHeterobifunctional crosslinked agents for immobilizing molecules on plastic substratesUS5464900Oct 19, 1993Nov 7, 1995Minnesota Mining And Manufacturing CompanyWater soluble organosiloxane compoundsUS5500251Jun 17, 1994Mar 19, 1996Air Products And Chemicals, Inc.Process for coating low energy surfacesUS5602202Jun 22, 1995Feb 11, 1997Minnesota Mining And Manufacturing CompanyMethods of using acrylate-containing polymer blendsUS5639546Sep 3, 1991Jun 17, 1997Minnesota Mining And Manufacturing CompanyCoated article having improved adhesion to organic coatingsUS6156478 *Oct 30, 1998Dec 5, 20003M Innovative Properties CompanyPhotocurable and photopatternable hydrogel matrix based on azlactone copolymersDE1936155A1Jul 16, 1969Feb 25, 1971Roehm & Haas GmbhCross-linked coating compositionsWO1998028026A1Dec 19, 1997Jul 2, 1998Ciba Geigy AgReactive coatingsWO1999053319A2Apr 9, 1999Oct 21, 19993M Innovative Properties CoHigh-density, miniaturized arrays and methods of manufacturing sameWO2000026725A1Mar 8, 1999May 11, 2000Minnesota Mining & MfgPhotocurable and photopatternable hydrogel matrix based on azlactone copolymersWO2001016370A1Aug 21, 2000Mar 8, 20013M Innovative Properties CoArrays with mask layers and methods of manufacturing same* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS7556858 *Sep 30, 2004Jul 7, 20093M Innovative Properties CompanySubstrate with attached dendrimersUS8367198Nov 17, 2008Feb 5, 20133M Innovative Properties CompanyPrecisely-shaped porous particlesUS8524368Dec 14, 2011Sep 3, 2013Wisconsin Alumni Research FoundationCharge-dynamic polymers and delivery of anionic compoundsUS8574420 *Nov 21, 2011Nov 5, 2013Wisconsin Alumni Research FoundationUltrathin multilayered films for controlled release of anionic reagentsUS20120065616 *Nov 21, 2011Mar 15, 2012Lynn David MUltrathin Multilayered Films for Controlled Release of Anionic ReagentsWO2011109151A1Feb 11, 2011Sep 9, 20113M Innovative Properties CompanyLigand guanidinyl functionalized polymers* Cited by examinerClassifications U.S. Classification428/357, 428/689, 428/364, 428/500, 428/447, 428/402, 526/260, 428/411.1International ClassificationC08F226/06, B32B27/00, C08F8/30, B32B27/02, C40B40/10, C09D201/06, C09D133/26, C12N11/08, B32B27/08, C40B60/14, C08K5/17, C09D157/10, C07B61/00Cooperative ClassificationC08F220/54, C08F2810/20, Y10T428/31504, C08F2810/50, C08F2800/20, C08K5/544, Y10T428/31663, C08J3/075, C08F8/42, C08F8/32, C08J7/047, Y10T428/31855, Y10T428/31935, C08J3/244, C09D139/04, C08J3/246, C40B40/10, Y10T428/2907, Y10T428/2913, C08K5/17, B01J2219/00659, Y10T428/2924, B01J2219/00511, B82Y30/00, B01J2219/00637, Y10T428/2998, B01J2219/0061, B01J2219/00641, B01J2219/00677, C09D157/10, Y10T428/2991, Y10T428/2982, B01J2219/00612, B01J2219/00725, B01J2219/00527, B01J2219/00644, B01J2219/00596, C12N11/08, C40B60/14, B01J2219/00315, C07B2200/11, C09D201/06, Y10T428/256, B01J2219/00621, C09D133/26, B01J2219/00626, B01J2219/00497, B01J2219/00605, Y10T428/29, B01J2219/00317, B01J2219/00585European ClassificationB82Y30/00, C08K5/17, C09D133/26, C09D157/10, C12N11/08, C08F8/30, C09D201/06Legal EventsDateCodeEventDescriptionJun 12, 2007CCCertificate of correctionMar 5, 2010FPAYFee paymentYear of fee payment: 4Feb 6, 2014FPAYFee paymentYear of fee payment: 8RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services

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Find out if your remodeling dreams fit your budget reality.Try the Real Cost Finder 5 sunbrella fabric Storage & Closets Design Photos
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Acronafplia Pension B&D: 2017 Room Prices, Deals & Reviews | Expedia
1-800-238-0767 Acronafplia Pension B&D
Points of Interest War Museum - 0 miles
Get an overview of this hotel3-star boardwalk Nafplio guesthouse in Nafplion City Centre
Coffee/tea in a common area, tour/ticket assistance, and a garden are available at this guesthouse. WiFi in public areas is free. Other amenities include a TV in a common area. Room Amenities
All 8 rooms provide free WiFi, refrigerators, and flat-screen TVs. Room service, Egyptian cotton sheets, and hair dryers are among the other amenities available to guests. Information missing or incorrect? Tell us!Opens in a new window Select to view moreAbout the Guest house Tab 1 of 2 selectedSelect to viewGuest Reviews Tab 2 of 2 Acronafplia Pension B&D
6 Agiou Spiridonos square
Acronafplia Pension B&D features tour/ticket assistance, a garden, and coffee/tea in a common area. Public areas are equipped with complimentary wireless Internet access. A total renovation of this property was completed in February 2015. Total number of rooms - 8 Number of floors - 2 Coffee/tea in common areas Front desk (limited hours) Tours/ticket assistance Free WiFi Garden Luggage storage Free parking nearby Year Built 2015 Number of buildings/towers - 2 Television in common areas Show all guest house amenities
Air conditioning In-room climate control (air conditioning) Private bathroom Bathrobes Free toiletries Hair dryer Shower only Full-sized refrigerator/freezer Flat-panel TV Free WiFi Blackout drapes/curtains Room service Rollaway/extra beds (surcharge) Egyptian-cotton sheets Show all room amenities
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cron job replace (to restore) a database - Stack Overflow
cron job replace (to restore) a database
i have looked around and i've only found a partial solution to what i REALLY need to achieve. Basically i am setting up a test site to allow users to make changes and i need to restore the database every hour
I have found this script for the cron that works fine but just for the first time...basically this script will not restore an existing database because of duplicate entries, so is not good for my test site
mysql -u user -ppassword databasename < /path/to/backup.sql
basically all i want to do is to restore my dump database (and possibly all the files as well) but i can't find how to do it...
This is the error that i receive via email when i run this script ERROR 1062 (23000) at line 62: Duplicate entry '1' for key 'PRIMARY'
So i guess i should have a way of deleting the database first and immediately after restore the original dump....I am not good at all with this so please bare with me!...
database replace cron jobs share|improve this question asked Jun 23 '11 at 15:52
There are two options that I can think of here:
You can create another backup of your original database with an option that DELETEs the tables before restoring the database. This effectively adds DELETE lines to the generated .sql file. The command to do this is:
mysqldump --add-drop-table -u [username] -p [password] [database] > backup.sql
Use mysqlimport to import a backup for a database that already exists like so:
mysqlimport -u [username] -p [password] [database] backup.sql
share|improve this answer answered Jun 23 '11 at 16:15
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Latest revision as of 16:20, 13 June 2017 (view source)
''[[Canada]]  [[Image:Gotoarrow.png]]  [[Quebec]]  [[Image:Gotoarrow.png]]  [[Quebec_Land_and_Property|Land and Property]]''
| link5=[[Quebec_Land_and_Property|Land and Property]]
[[Image:1024px-Ville de Québec01.jpg|thumb|right|300x250px|1024px-Ville de Québec01.jpg]]
[[Image:Northern Quebec Map.jpg|center|600px|Northern Quebec Map.jpg]]

[[Image:Southern Quebec Map.jpg|center|600px|Southern Quebec Map.jpg]]

[http://familysearch.org/ FamilySearch]
Québec Notarial Records - 558738 images from the judicial districts of Montreal, Québec and Terrebonne from 1800-1900 are available to browse. At the time of printing there was no searchable-by-name database.
[http://www.ancestry.ca/ Ancestry.ca]
Québec Notarial Records (Drouin Collection) 1647-1942 - These are available on this subscription site. Most of the records are in French although there are some in English.
Library and Archives Canada- [http://www.collectionscanada.gc.ca/databases/lower-canada/index-e.html Lower Canada Land Petitions ]
Library and Archives Canada have an index database for Upper Canada Land Petitions searchable by name and place. The results provide the necessary information to access the petition by microfilm.
*[http://www.qfhs.ca/ Québec Family History Society]
*[http://www.sgcf.com/ Société généalogique canadienne-francaise]
*[http://pages.infinit.net/pbenoit/list_ah.htm Société de recherché historique Archiv-Histo]
*[http://www.banq.qc.ca/accueil/ Bibliothèque et Archives nationales du Québec]
== The Seigneuries
 ==
A [http://fr.wikipedia.org/wiki/Liste_des_seigneuries_de_la_Nouvelle-France list of all of the seigneuries of New France], with the dates of their foundation is found at [http://www.Wikipedia.fr Wikipedia.fr]. A [http://www.memoireduquebec.com/wiki/index.php?title=Liste_des_seigneuries_%28seigneurie%29 list of the seigneuries of Quebec] is found at [http://www.Memoireduquebec.com memoireduquebec.com]. A [http://www.memoireduquebec.com/wiki/images/3/39/MdQCarte_des_seigneuries.jpg map of the seigneuries of Quebec], made by A. E. B. Courchesne in 1923, accompanies the list. Most seigneuries had a frontage of several miles along the Saint Lawrence river and estuary (Le fleuve Saint Laurent), or along a major river, although some seigneuries surrounded large lakes.

{{Tip|In [[Quebec Genealogy|Quebec]], deeds and other documents about land transfers between individuals are in the notarial records. (See also [[Quebec Notarial Records]].)}}
In Québec, deeds and other documents about land transfers are in the notarial records. Notaries ''(notaires)'' have registered all types of contracts since 1626. These deeds, wills, marriage contracts and other records were recorded and the originals given to the parties involved with the notary keeping a copy known as “minutes.”
These records are not normally indexed by the names of the persons involved in the contract; instead they are collected by the name of the notary. These notarial records are sent to the ''protonotaire'' of the local judicial district when the notary involved no longer is employed as a notary. Notarial records before 1900 have been deposited in the branches of the Archives Nationales du Québec (see contact information at end of this module).
Notarial records after 1900 are only available to the person involved or the person’s legal representative who may request copies from the judicial district office that has the records. Addresses of the judicial offices are given in Marthe Faribault-Beauregard’s ''La Généalogie: Retrouver ses ancêtres''. Current addresses of the district offices are listed in annual editions of ''the Canadian Almanac and Directory''.
*Quinton, Robert J., ''The Notaries of French Canada, 1626-1900: Alphabetical Chronologically, by Area Served.'' Pawtucket, Rhode Island: R.J. Quintin, 1994. {{FHL|34401|item|disp=FHL book 971.4 N3n; film 1750788 item 120}}
Since 1997 the ''Chambres des notaires'' has collaborated with the Archiv-Histo Society to produce the Parchemin Project (''Banque Parchemin''), an index to the files ''(greffes)'' of deceased Quebec notaries. The database is searchable online (in French) or by a set of CD-ROMs held by major research repositories. You can search the database by key word (''mot-clé'') such as your ancestor’s surname, or by the name of a local notary. There is also a map-related feature for determining the name of a notary who worked in your ancestor’s locale. The database includes other types of papers prepared by notaries, besides land-related documents. Presently the period of the documents in the database is 1635 to 1800, but the Society’s own data bank holds millions more, and they will assist you:
:'''Société de recherche historique Archiv-Histo'''
2320, rue des Carrière
Montréal, Québec H2G 3G9
Telephone: 514-763-6347
Library and Archives Canada holds Lower Canada Land Petitions 1764-1841 (RG 1, L3L) and records of the Gaspé Land Commission (RG 1, L 7, vols. 79-80) which are nominally indexed in their finding aid 1801. Both the records and the index are microfilmed. The microfilm shelf list is posted on Library and Archives Canada website. The petitions may have information on the petitioner, his family parentage, military service and time of settling the land making them very valuable genealogically. The patents that were granted gave the name of the grantee, a description of the land and the date of the grant, also very useful.

=== Land Transfers after the First Grant ===
Subsequent sales, gifts, bequests, or other transfers of land have been a matters of '''local''' record since about 1831, under the responsibility of the ''Ministère de la Justice'' and registered at the ''Bureau d’enregistrement'' [Legal Registry Office] of the various Judicial Districts. In 1996 there were 55 different land registry offices. Current addresses are given in the ''Canadian Almanac'' or in a current Québec Government Telephone Directory, or Québec Legal Telephone Directory, and probably other legal directories. Ask your librarian. You must know the street address or Range and lot number and it helps to know the Ward or Parish.
Most land transfer documents will have been drawn up by the local Notary, and if over 100 years old, may be in his ''greffe'' in the ANQ. That is a less expensive way to search for such records, so if you can find one land transfer document and get the name of the Notary, you can search the ''greffe'' for other papers and probably track other sales backward or forward.<ref>Douglas, Althea. "Québec Land, Judicial, Tax Records (National Institute)," ''The National Institute for Genealogical Studies'' (2012), https://familysearch.org/learn/wiki/en/Qu%C3%A9bec_Land,_Judicial,_Tax_Records_%28National_Institute%29.</ref>
==== Jésuit Estates ====
==== Archives nationales du Québec ====
Throughout Québec there are regional Provincial Archives centers known as Les centres d’Archives nationales du Québec:

:'''Abitibi-Témiscamingue et Nord-du-Québec'''
27, rue du Terminus Ouest
Rouyn-Noranda, Québec J9X 293
Telephone: 819-763-3484
Email: [mailto:archives.rouyn@banq.qc.ca archives.rouyn@banq.qc.ca]

:'''Bas-Saint-Laurent et Gaspésie,Iles-de-la-Madeleine'''
337, rue Moreault
Rimouski, Québec G5L 1P4
Telephone: 418-727-3500
Email: [mailto:archives.rimouski@banq.qc.ca archives.rimouski@banq.qc.ca]
:'''Côte-Nord'''
700, boulevard Laure, Bureau 190
Sept-Iles, Québec G4R 1Y1
Telephone: 418-964-8434
Email: [mailto:archives.sept-iles@banq.qc.ca archives.sept-iles@banq.qc.ca]

:'''Estrie'''
225, rue Frontenac, bureau 410
Sherbrooke, Québec J1H 1K1
Telephone: 819-820-3010
Email: [mailto:archives.sherbrooke@banq.qc.ca archives.sherbrooke@banq.qc.ca]
:'''Gaspé'''
80, boulevard de Gaspé
Gaspé, Québec G4X 1A9
Telephone: 418-727-3500 ext. 6573
Email: [mailto:archives.gaspe@banq.qc.ca archives.gaspe@banq.qc.ca]
:'''Mauricie et Centre-du-Québec'''
225, rue des Forges, bureau 208
Trois-Rivières, Québec G9A 2G7
Telephone: 819-371-6015
Email: [mailto:archives.trois-rivieres@banq.qc.ca archives.trois-rivieres@banq.qc.ca]
:'''Montréal'''
535 avenue Viger est
Montréal, Québec H2L 2P3
Telephone: 514-873-1100 option 4
Email: [mailto:archives.montreal@banq.qc.ca archives.montreal@banq.qc.ca]
:'''Outaouais'''
855, boulevard de la Gappe
Gatineau, Québec J8T 8H9
Telephone: 819-568-8798
Email: [mailto:archives.gatineau@banq.qc.ca archives.gatineau@banq.qc.ca]
:'''Québec'''
Campus de l’Université Laval
Pavillon Louis-Jacques-Casault
1055, avenue du Séminaire
Case postale 10450, succursale Sainte-Foy
Québec, Québec G1V 4N1
Telephone: 418-643-8904
Email: [mailto:archives.quebec@banq.qc.ca archives.quebec@banq.qc.ca]
:'''Saguenay - Lac-Saint-Jean'''
930, rue Jacques-Cartier est, bureau C-103
Chicoutimi, Québec G7H 2A9
Telephone: 418-698-3516
Email: [mailto:archives.saguenay@banq.qc.ca archives.saguenay@banq.qc.ca]
==== Québec Genealogical Societies ====
:'''Quebec Family History Society'''
15 Donegani Avenue, P.O. Box 715
Pointe-Claire - Dorval Postal Station
Pointe-Claire, Québec H9R 4S8
Telephone: 514-695-1502
Email: [mailto: qfhs@bellnet.ca qfhs@bellnet.ca]

 The leading English-language society, they sell maps and the full index to Quebec Crown land grants, sorted alphabetically by surname, available online to members only.
:'''Société généalogique canadienne-française'''
3440, rue Davidson
Montréal, Québec H1W 2Z5
Telephone: 514-527-1010
Email: [mailto:info@sgcf.com info@sgcf.com]

Both societies have many links to other resources.<ref>Murphy, Sharon L., Brenda Dougall Merriman, and Frances Coe. "Québec Additional Land Records (National Institute)," ''National Institute for Genealogical Studies'' (2012), https://familysearch.org/learn/wiki/en/Québec_Additional_Land_Records_(National_Institute).</ref>
[[Category:Quebec Land and Property Records]]
Latest revision as of 16:20, 13 June 2017
Retrieved from "https://www.familysearch.org/wiki/en/index.php?title=Quebec_Land_and_Property&oldid=2888625"

value: This website is a nonprofit organization and website offering genealogical records, education, and software. It is operated by The Church of Jesus Christ of Latter-day Saints , and is closely connected with the church's Family History Department.


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bradlei comments on Who is your favorite Seahawks player of all time?jump to contentmy subredditsannouncementsArtAskRedditaskscienceawwblogbookscreepydataisbeautifulDIYDocumentariesEarthPornexplainlikeimfiveFitnessfoodfunnyFuturologygadgetsgamingGetMotivatedgifshistoryIAmAInternetIsBeautifulJokesLifeProTipslistentothismildlyinterestingmoviesMusicnewsnosleepnottheonionOldSchoolCoolpersonalfinancephilosophyphotoshopbattlespicsscienceShowerthoughtsspacesportstelevisiontifutodayilearnedTwoXChromosomesUpliftingNewsvideosworldnewsWritingPromptsedit subscriptionsfront-all-random | AskReddit-funny-pics-todayilearned-videos-news-movies-gifs-worldnews-gaming-aww-Showerthoughts-explainlikeimfive-mildlyinteresting-Jokes-tifu-television-IAmA-sports-Music-photoshopbattles-creepy-science-space-food-nottheonion-TwoXChromosomes-OldSchoolCool-Fitness-LifeProTips-DIY-history-askscience-personalfinance-dataisbeautiful-Futurology-UpliftingNews-gadgets-WritingPrompts-books-Art-Documentaries-EarthPorn-nosleep-InternetIsBeautiful-listentothis-GetMotivated-philosophy-announcements-blogmore » SeahawkscommentsrelatedWant to join? Log in or sign up in seconds.|Englishlimit my search to /r/Seahawksuse the following search parameters to narrow your results:subreddit:subredditfind submissions in "subreddit"author:usernamefind submissions by "username"site:example.comfind submissions from "example.com"url:textsearch for "text" in urlselftext:textsearch for "text" in self post contentsself:yes (or self:no)include (or exclude) self postsnsfw:yes (or nsfw:no)include (or exclude) results marked as NSFWe.g. subreddit:aww site:imgur.com dogsee the search faq for details.advanced search: by author, subreddit...this post was submitted on 18 Feb 201321 points (80% upvoted)shortlink: remember mereset passwordloginSubmit a new linkSubmit a new text postSeahawkssubscribeunsubscribe33,998 readers146 users here now
created by Measure76a community for 5 yearsmessage the moderatorsMODERATORSMeasure76easyanticBrian BosworthliquilifeAutoModeratorSeahawks_ModApexeaedeosmr_ssercKleeziE...and 3 more »discussions in /r/Seahawks<>X18 points · 2 comments The 2016 Seattle Seahawks: No Points for Degree of Difficulty8 points Russell Wilson is a referee in the offseason· 5 comments Tarvaris Jackson won’t re-sign with Seahawks before free agency5 points Treyarch Pays Tribute To Seahawks RB Marshawn Lynch On His Retirement52 points · 7 comments Need a Valentine template? Seahawks have your back.2 points · 46 comments Who are some current players that you would like to see retire as Seahawks?Reddit Community Mock Draft - Monday (2/15) @ 6pm EST. I would be very happy if we come together and make some awesome picks cause I am the GM right now for us.4 points [Russell_Bot] Seahawks.com Content from 2016-02-1314 points · 55 comments Who do you guys think will be selected this year by the Seahawks from this years Draft and why?5 points · 44 comments Sherm and Gronk "on set" together, best friends forever confirmedThis is an archived post. You won't be able to vote or comment.
202122Who is your favorite Seahawks player of all time? (self.Seahawks)submitted 2 years ago by [deleted]124 commentsshareloading...sorted by: besttopnewcontroversialoldrandomq&ayou are viewing a single comment's thread.view the rest of the comments →[–]bradlei 4 points5 points6 points 2 years ago (0 children)Best tackle in the history of the league. permalinkembedsaveparentgive goldPosts are automatically archived after 6 months.Got Itaboutblogaboutvaluesteamsource codeadvertisejobshelpsite rulesFAQwikireddiquettetransparencycontact usapps & toolsAlien Blue iOS appmobile betabuttons<3reddit goldreddit storeredditgiftsUse of this site constitutes acceptance of our User Agreement and Privacy Policy (updated). © 2016 reddit inc. All rights reserved.REDDIT and the ALIEN Logo are registered trademarks of reddit inc.Advertise - sportscreate a new accountusername:password:verify password:email: (optional)remember meget the best of reddit emailed to you once a week. learn moresign uplog inusername:password:remember mereset passwordlog inBy signing up, you agree to our Terms and that you have read our Privacy Policy and Content Policy.English [en]العربية [ar]Беларуская мова [be]български език [bg]Bosanski [bs]català [ca]česky [cs]dansk [da]Deutsch [de]Ελληνικά [el]English (Australia) [en-au]English (Canadian) [en-ca]English (Great Britain) [en-gb]English [en-us]Esperanto [eo]español [es]español [es-ar]eesti keel [et]Euskara [eu]فارسی [fa]suomi [fi]français [fr]Gàidhlig [gd]עברית [he]मानक हिन्दी [hi]hrvatski [hr]Magyar [hu]Հայերեն լեզու [hy]Bahasa Indonesia [id]íslenska [is]italiano (Italy) [it]日本語 [ja]ಕನ್ನಡ [kn_IN]한국어 [ko]Latin [la]1337 [leet]LOL [lol]lietuvių kalba [lt]latviešu valoda [lv]Nederlands [nl]Nynorsk [nn]Norsk [no]Arrrrrrrr! [pir]polski [pl]português [pt] (*)português [pt-pt]português brasileiro [pt_BR]română [ro]русский [ru]slovenčina [sk]slovenščina [sl]српски језик [sr]Srpski [sr-la]Svenska [sv]தமிழ் [ta]ภาษาไทย [th]Türkçe [tr]українська мова [uk]Tiếng Việt [vi]中文 [zh] (*) incomplete volunteer to translateπ Rendered by PID 22056 on app-15 at 2016-02-14 23:30:26.237444+00:00 running fb6d7b9 country code: US.

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Play X-Wing - Charleston X-Wing Meetup (Charleston, SC)
This month we'll be trying out the first of the new large ships, the GR-75 Medium Transport. This baby is big, about the length of 4 star fighters, and moves like the space whale that she is. Be sure not to get in her way because she'll simple run you over!
We'll be running training sessions, dogfights, and scenario games. Even if you don't own any ships we have some we can let you test pilot. We're open to everyone so be sure to invite your friends.
And as always, please be respectful of our host The Green Dragon by cleaning up after yourselves and supporting their business. Outside food is not allowed but snacks and drinks are available for purchase. There's also a $2 room fee to cover our table time. Join or login to comment.
Jeff W. I'm going to be late, because of stupidity in my family 0 · May 17, 2014 A former member No worries, I'll be here all day ;) 0 · May 17, 2014 Jeff W. I'm going to be late, because of stupidity in my family 0 · May 17, 2014 A former member I'll be there all day. There's some board gaming going on in the morning and we'll probably play The Resistance or something after X-Wing. So don't sweat it if you can't make 1pm. 0 · May 14, 2014 5 went
We are a group dedicated to playing and teaching the Star Wars Miniatures X-Wing Game here in Charleston, ensuring it is a fun and rewarding experience for all skill levels. (Waves fingers) THIS IS the group you're looking for. It's for anyone looking to the play or learn the Star Wars X-Wing Miniatures Game. We play on Thursday evenings and some Saturdays at the Green Dragon in North Charleston, SC. We are open to all ages and skill levels, including you Rookie!

value: This website is a service used to organize online groups that host in-person and virtual events for people with similar interests. It was founded in 2002 by Chairman Scott Heiferman and four co-founders.


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Petroleum Minister Dharmendra Pradhan will raise India's concerns about rising crude oil prices at the meeting of the Organisation of Petroleum Exporting Countries (OPEC) to be held in Vienna later this month, the Minister said on Monday ... OPEC and non-OPEC countries are also expected to decide on extending the output cuts, which, along with the geopolitical tensions in the Middle East, have helped push up crude prices....
India will pitch for "responsible" crude pricing at the upcoming conference organised by oil producers' cartel OPEC, Union minister Dharmendra Pradhan said on Monday ... whenever we have met OPEC members, we have told them that crude oil prices should be controlled, reasonable, responsible and should meet demand ... We do not want crude at $25 per barrel....
New Delhi, Jun 18 () India will pitch for 'responsible' crude pricing at the upcoming conference organised by oil producers' cartel OPEC, Union Minister Dharmendra Pradhan said today ... "There will be an OPEC conference in Vienna on June 22...Whenever we have met OPEC members, we have told them that crude oil prices should be controlled, reasonable, responsible and should meet demand ... We do not want crude at USD 25 per barrel....
"There will be an OPEC conference in Vienna on June 22...Whenever we have met OPEC members, we have told them that crude oil prices should be controlled, reasonable, responsible and should meet demand ... India will pitch for 'responsible' crude pricing at the upcoming conference organised by oil producers' cartel OPEC, Union Minister Dharmendra Pradhan said today ... We do not want crude at USD 25 per barrel....
India, the world’s fastest growing oil consumer is guzzling down Iranian crude before US sanctions start squeezing supplies from the Persian Gulf nation. India imported 771,000 barrels of crude oil a day from Iran in May, a 35 per cent increase from the previous month, tanker tracking and shipping data compiled by Bloomberg show....
Aviation and oil marketing companies' share prices gained between 2.5 percent and 6 percent after sharp fall in crude oil prices in the international markets ... Brent crude futures, the international benchmark for oil prices, has been falling sharply ahead of likely increase in the output by OPEC, Russia and its allies in the meeting in Vienna later in the week ... The fall came after crude futures fell around 3 percent on Friday....
Angel Commodities' report on Crude Oil ... The base import price of crude palm oil and RBD palmolein was cut by $1 4 each and $17 to $6 44 per tn and $6 73 per tn, respectively ... For all commodities report, click here. Disclaimer ... ....

value: This website is a news aggregator founded in March 1995 and launched online in 1998. In 2003, Search Engine Watch praised the service for its "Special Reports", and called it "an interesting alternative" to other news aggregation services.


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Upgraded Chipotley - Review of Carrburritos, Davidson, NC - TripAdvisor
All Davidson Restaurants Restaurants near Carrburritos
All things to do in Davidson Things to do near Carrburritos
Upgraded Chipotley - Carrburritos
United States North Carolina (NC) Davidson Davidson Restaurants Carrburritos
“Upgraded Chipotley”
Carrburritos 445 S Main St, Davidson, NC 28036-8112 +1 704-237-3040
Website Improve this listing Ranked #11 of 35 Restaurants in Davidson
Restaurant details Good for: Bar scene Description: Funky, fresh and flavorful California Mexican Cuisine. Indoor and Outdoor seating. Comfortable full bar featuring award-winning,specialty margaritas, Mexican beer, as well as imports and domestics. Catering for large and small groups. There are newer reviews for this restaurant
119 helpful votes “Upgraded Chipotley”
This is an upscale take on the Chipotley model of serving casual Mexican food and SO MUCH BETTER! Burritos and tacos and quesadillas made to order....fish tacos excellent.....home made salsa....margarita menu (excellent) and full bar/beers....everything fresh. The atmosphere is a bit funky, casual, clean with limited outdoor seating. Definitely a better choice to the fast food Mexican chain restaurants. Good addition to,the Davidson dining scene. We hope they do well and continue to thrive.
Helpful? Thank JMac28046 Report Ask JMac28046 about Carrburritos
English first Any Melhughes
6 helpful votes “Great, fresh Mexican”
Casual "fast food"...food was excellent & very fresh! Great service, great value, great atmosphere. A few patio tables too. Good addition to Davidson!
Helpful? Thank Melhughes Report Ask Melhughes about Carrburritos
Mexican food at its best: fresh ingredients, made to order, large servings. Comfortable, clean and roomy area. Awesome margarita menu. Great value for the money.
Helpful? Thank Andi M Report Ask Andi M about Carrburritos
106 helpful votes “Great, Fresh, Tasty Burritos...”
Here's the 1,2,3s...1. Like Moe's or Chipoltle, you order and pay, but then someone brings you your order. Not just burritos and tacos, but also a full bar, cheap beers, good house margaritas. 2. Fresh tasting burritos with fresh ingredients. Burritos are large enough to share if you're not too hungry. Had pescado and beef. Both were very tasty. Burritos are different than others in that they don't contain rice, but I didn't miss it. 3. Salsa, Salsa, Salsa! There are several different kinds, all made in house. Make sure and try then all.
Helpful? Thank Pack-Less Report Ask Pack-Less about Carrburritos
50 helpful votes “Fresh, fast, and friendly!”
My husband and I stopped in for a light dinner on our way to a party. We split a chicken quesadilla and a $2 Nattie Boh. The quesadilla was fresh and cheesy and the fresh salsa really made it, so be sure to ask for that on the side. The staff was very friendly and the place had a fun, casual vibe. With food that fresh, tasty, and cheap, what's not to like? We would definitely return for a more substantial meal.
Helpful? Thank MDtravelertoo Report Ask MDtravelertoo about Carrburritos
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Nieuwjaarsborrel 2010 – New Years Drink - The Den Bosch Dutch Language Meetup! ('s-Hertogenbosch)
January 8, 2010 · 9:00 PM
david ik heb van er heel genoten. 0 · January 9, 2010 A former member Veel genoten! 0 · January 9, 2010 A former member Loved it! Hope we can make the next gathering! 0 · January 9, 2010 A former member Look at all these beautiful people going to the meetup! You guys are going to have a great time. 0 · January 7, 2010 15 attended
david jorg schroder
107 international ladies
367 Friends of Culti Group
147 Socializers


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Keisel, Beachum may return for Steelers793dScott BrownMike Tomlin is the AFC North coach of the year23mJamison HensleyWhy the Steelers keep giving players signing bonuses they already have2hJeremy FowlerSteelers' Shaun Suisham kicks U.S. citizenship through the uprights18hJeremy FowlerDuke Johnson is the AFC North rookie of the year1dPat McManamonFrom Eric Weddle to Eli Apple, Steelers will explore all options at DB1dJeremy FowlerOutside expectations for Pittsburgh Steelers highest since 20112dJeremy FowlerMartavis Bryant to train in L.A., talk with counselor once a week this offseason2dJeremy FowlerGeno Atkins is the AFC North defensive player of the year2dColey HarveySteelers can't emulate Broncos' D because there's no Von Miller on the roster3dJeremy FowlerAntonio Brown is the AFC North offensive player of the year2dJeremy FowlerKeisel, Beachum may return for SteelersEmailcommentDec 10, 2013Scott BrownESPN Pittsburgh Steelers reporter CloseCovered Steelers for six years for the Pittsburgh Tribune-Review
Author of five books, including "Heaven Sent: The Heather Miller Story," which highlights the friendship a young girl with cancer developed with several Steelers playersFollow on TwitterEmailprintcommentPITTSBURGH -- The Steelers' playoff hopes can be listed as doubtful but at least they are relatively healthy.Defensive end Brett Keisel (foot) and left tackle Kelvin Beachum (knee) will be limited in practice this week but each could play Sunday night against the visiting Bengals."We anticipate having most if not all of our guys available," coach Mike Tomlin said Tuesday at his weekly news conference.Keisel has played in just one of the Steelers' past four games -- and sparingly at that -- because of plantar fasciitis. Tomlin said the 12th-year veteran underwent an extensive workout on Monday and the Steelers will continue to monitor his progress.Beachum, who sprained his knee in a 22-20 loss Baltimore on Thanksgiving night, will also push to return to action this week. The second-year man will likely be limited during the early part of the week, and even if Beachum is able to play he may not return to the starting lineup.Tomlin said a decision on whether Beachum or Mike Adams starts at left tackle will hinge on how much the former is able to practice this week. Adams played his best game of the season in the Steelers’ 34-28 loss to the Dolphins after making his first start at left tackle since late September.In other injury news, defensive end Ziggy Hood (ankle) and wide receiver Jerricho Cotchery (shoulder) will be limited in practice but both are expected to play Sunday.

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Oh! by Scolohofo | 724354208126 | CD | Barnes & Noble®
by ScolohofoScolohofo
ScoLoHoFo -- guitarist John Scofield, saxophonist Joe Lovano, bassist Dave Holland, and drummer Al Foster -- is the modern-jazz answer to the “supergroup,” but unlike a fly-by-night all-star concoction dreamed up by publicists and management, this musicians’ collective is an actual working outfit that toured together twice before entering the studio. The payoff is an
ScoLoHoFo -- guitarist John Scofield, saxophonist Joe Lovano, bassist Dave Holland, and drummer Al Foster -- is the modern-jazz answer to the “supergroup,” but unlike a fly-by-night all-star concoction dreamed up by publicists and management, this musicians’ collective is an actual working outfit that toured together twice before entering the studio. The payoff is an album that succeeds on every level -- great original material, a groove that won’t stop from Holland and Foster (is there another drummer in the world who gets a better sound from his set?), and career-peak soloing from Scofield and Lovano. There isn’t a piece of filler in the entire 77 minutes. The opening title cut sets the group off on a tight, relaxed groove, with superb blowing from Lovano on the tenor. Scofield has never sounded better than on the sometimes intricate, sometimes punchy riffs he offers in response to Lovano’s throaty meditations on “New Amsterdam.” However, simply highlighting a few examples of the inspired musicianship doesn't convey the depth and consistency of the project; you can dive in anywhere to find breathtaking playing. Let’s hope the realities of the contemporary jazz scene will nurture the continued existence of an “all-leaders” group like this one.
All Music Guide - Matt Collar
Combining the talents of tenor saxophonist Joe Lovano, guitarist John Scofield, bassist Dave Holland, and drummer Al Foster, there is an uncredited fifth member on jazz supergroup Scolohofo's debut recording, Oh! -- Miles Davis. Every one of these musicians, except for Lovano, gained their first real success with the legendary trumpeter -- an experience that informs their careers to the present. The aesthetic on Oh! is resolutely Milesian -- impressionistic, spare, soft, funky, progressive, but always with an ear to the blues. Scolohofoda? His sound is almost literally present, an "implied tone" whenever Scofield's dissonant chord clusters and Lovano's whispery throat tones collide. These guys played with Davis in his later fusion period during the '70s and '80s when Davis' "group" aesthetic came to the fore and became perhaps even more important than his individual contribution. And, while everyone gets their featured solo spot, the overall effect is one of intensely soft and layered patches of sound. Scofield's trademark "chicken scratch" lines match perfectly with Lovano's fuzzy spittle tone and the rhythm section of Holland and Foster offers its own wryly propulsive counterpoint. Musically, the goal is resolutely post-bop, but with an acoustic, folky underpining that allows for some interestingly arranged melodic moments. This contrasts nicely with the free-flowing, loose improvisation informed by '60s free jazz, fusion, and modern progressive styles. Longtime fans of the work of these individual musicians will find much to enjoy here, but there is the sense of new or at least rediscovery on Oh! -- another Davis trademark -- which bodes well for future collaborations.
Embedded in this comfort zone...is sparkling playing and ironclad unity from all.
0724354208126
Shorter Form
Scolohofo Primary Artist
Joe Lovano Soprano Saxophone,Tenor Saxophone,Sax (Curved Soprano)
Foster Drums
Dave Holland Producer
Joe Lovano Producer
John Scofield Producer
Foster Producer
Eli Wolf Producer,Art Direction

value: This website is an American bookseller. It is a Fortune 1000 company and the bookseller with the largest number of retail outlets in the United States.


text:
L. Mabel Birdslow (1878-1898) - Find A Grave Memorial
L. Mabel Birdslow
22 Dec 1898 (aged 19–20)
138641994 · View Source
Find A Grave Memorial 138641994
Find A Grave, database and images (https://www.findagrave.com : accessed ), memorial page for L. Mabel Birdslow (1878–22 Dec 1898), Find A Grave Memorial no. 138641994, citing Woodlawn Cemetery, Sandy Creek, Oswego County, New York, USA ; Maintained by Charlene Cole (contributor 49062115) .
Add Photos for L. Mabel Birdslow
Fulfill Photo Request for L. Mabel Birdslow
your computer for L. Mabel Birdslow memorial.
I thought you might like to see a memorial for L. Mabel Birdslow I found on Findagrave.com.

value: This website is a website that allows the public to search and add to an online database of cemetery records. It is owned by Ancestry.com.


text:
Los Angeles, CA $90,000 Jobs, Employment
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Jobs 1 to 10 of 41,603
Can be located in Mountain View, CA or in Tempe, AZ. *This is a direct-hire or contract-to-hire opportunity....
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StaffRehab is in need of a qualified Licensed Marriage and Family Therapist (LMFT or LMFTI) for a position in Victorville, CA....
CA IdentityMinder and SiteMinder
CA SiteMinder and CA IdentityMinder. Project Engineer 6 - CA IdentityMinder and SiteMinder. Familiarity with CA SOA gateway for web services security....
View all Deloitte jobs in Portland, OR - Portland jobsSalary Search: CA Identityminder Siteminder salaries in Portland, ORLearn more about working at DeloitteRelated forums: Portland, Oregon - Deloitte
Sr. Solution Architect - 44920 Description Our company At Adobe, we’re changing the world. How? We give people the tools to bring their ideas to life and
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Direct client is looking for a CA Unified Infrastructure Management (UIM) Nimsoft Subject matter expert for a long term role....
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text:
 Das Seabrook 308 in Margate buchen
Details zum Seabrook 308
Seabrook 308 is an apartment in Margate with access to barbecue facilities. Guests staying at this apartment have access to free WiFi. The apartment includes a kitchen with an oven and a microwave, as well as a kettle. A flat-screen TV is offered. Shoprite (Margate) is 200 metres from the apartment.

value: This website is a Singaporean online travel agency and metasearch engine for hotels, vacation rentals, flights, and airport transfer. It is owned by Agoda Company Pte.


text:
BBB Business Profile | Durham Regional Financial Center
> Durham Regional Financial Center
(919) 806-4596
2634 Durham Chapel Hill Blvd STE 212 Durham, NC 27707-2831
Principal: Ms. Glyndola Beasley, CEO
Durham Regional Financial Center provides community financial education and homebuyer education as well as credit and debt counseling.
Durham Regional Development Group
Durham Regional Financial Center has received 0 out of 5 stars based on 0 Customer Reviews and a BBB Rating of A+.

value: This website is a private, nonprofit organization whose self-described mission is to focus on advancing marketplace trust, consisting of 106 independently incorporated local BBB organizations in the United States and Canada, coordinated under the International Association of Better Business Bureaus  in Arlington, Virginia.The Better Business Bureau is not affiliated with any governmental agency. Businesses that affiliate with the BBB and adhere to its standards do so through industry self-regulation.


text:
Adams County Colorado Mission, Benefits, and Work Culture | Indeed.com
Learn more about working for Adams County!
Brighton, CO(10)
Adams County, CO(6)
Commerce City, CO(4)
Strasburg, CO(1)
See all 31 Adams County Colorado jobs
Adams County is located in northeastern Colorado. The county is 18 miles wide and 72 miles long, ranging from urban uses in the western portions of the county, to crop and grazing land in the central and eastern portions.
Agricultural activities are the single largest land use throughout the county, accounting for more than – more... three quarters of the land area. Most irrigated farm land is located in the northwest section of the county, where it is supported by an extensive network of canals. The central and eastern portions of Adams County, including the Town of Bennett, are agriculturally based.
The county manager is appointed by the board to carry out policy directions and to supervise and coordinate the work of the staff in the departments that fall under direct control of the board.
The county attorney is appointed by the board and provides legal advice and representation for the board. The County Attorney, with the approval of the board, also advises and represents all other Adams County elected officials, the county manager, department and division heads, and appointed commissions, boards and committees.
The other constitutional officers elected to four-year terms are the Assessor, Clerk & Recorder, Coroner, District Attorney, Treasurer, Sheriff and Surveyor. Their powers and duties are prescribed by state statute. The county commissioners have no direct authority over the other elected officials in the county except that commissioners approve budgets for all other elected officials’ departments. – less
Adams County Colorado website