text:
|Publication number
|Publication type
|Application number
|Publication date
|Feb 27, 2001
|Filing date
|Feb 18, 1998
|Priority date
|Feb 18, 1998
|Fee status
|Also published as
|Publication number
|Richard A. Clark, Doris Greiling
|Original Assignee
|The Research Foundation Of State University Of New York
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (19), Non-Patent Citations (25), Referenced by (8), Classifications (14), Legal Events (9)
|External Links: USPTO, USPTO Assignment, Espacenet
The subject matter of this application was made with support from the United States Government under National Institutes of Health Grant No. AG 101143-12.
The subject invention is directed to an extracellular matrix for wound healing and to a method of enhancing wound healing using the extracellular matrix.
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
It is estimated that in 1992 (US), 35.2 million wounds required major therapeutic intervention (Medical Data International, Inc. 1993). Surgical incisional wounds are performed with aseptic technique, and are closed by primary intention. Most repair and heal uneventfully. Many traumatic wounds and cancer extirpations, however, must be left open to heal by secondary intention. Furthermore, chronic wounds have significant tissue necrosis and fail to heal by secondary intention. It is estimated that 5.5 million people in the US have chronic, nonhealing wounds and that their prevalence is increasing secondary to the increase in age-related diseases, the increase in Acquired-immune Deficiency Syndrome (AIDS), and the increase of radiation wounds secondary to cancer intervention. In the US approximately 1.5-2.5 million people have venous leg ulcers; 300,000-500,000, diabetic ulcers; and 2.5-3.5 million, pressure ulcers (Callam et al. 1987; Phillips and Dover 1991; Lees and Lambert 1992; Lindholm et al. 1992). These acute and chronic open wounds require long-term care and procedures that include skin grafting and tissue flaps, debridement, frequent dressing changes and administration of pain medications. This care is costly and labor intensive. Furthermore, these wounds have a severe impact on the patients' quality of life. The chronic dermal ulcerations can cost as much as $40,000 each to heal and more disappointing is that 50% reappear within 18 months of healing. Chronic dermal ulcers are also associated with mortality. As many as 21% of patients in intermediate-care facilities with pressure ulcers die (Bergstrom et al. 1994).
Although multiple millions of dollars have been spent on the development of numerous recombinant growth factors (Abraham and Klagsbrun 1996; Heldin and Westermark 1996; Nanney and King 1996; Roberts and Sporn 1996) and organotypic skin replacements (Boyce et al. 1995) for use in open wounds over the past decade, the evidence of cost-effective benefit is meager thus far (Brown et al. 1989; Robson et al. 1992a; Robson et al. 1992b; Phillips et al. 1993).
Many attempts have been made to produce a composition which can be used to facilitate wound repair. Many of these compositions involve collagen as a component. U.S. Pat. Nos. 4,950,483 and 5,024,841 each discuss the usefulness of collagen implants as wound healing matrices. U.S. Pat. No. 4,453,939 discusses a wound healing composition of collagen with a fibrinogen component and a thrombin component, and optionally fibronectin. U.S. Pat. No. 4,970,298 discusses the usefulness of a biodegradable collagen matrix (of collagen, hyaluronic acid, and fibronectin) for wound healing. Yamada et al. (1995) disclose an allogeneic cultured dermal substitute that is prepared by plating fibroblasts onto a spongy collagen matrix and then culturing for 7 to 10 days. Devries et al. (1995) disclose a collagen/alpha-elastin hydrolysate matrix that can be seeded with a stromal-vascular-fraction of adipose tissue. Lamme et al. (1996) disclose a dermal matrix substitute of collagen coated with elastin hydrolysate. U.S. Pat. No. 5,489,304 and Ellis and Yannas (1996) each disclose a collagen-glycosaminoglycan matrix.
There are also numerous compositions which involve hyaluronic acid (HA) as a component. Ortonne (1996), Borgognoni et al. (1996), and Nakamura et al. (1997) each discuss the usefulness of HA for wound healing. In Nakamura et al. (1997), the HA was combined with chondroitin sulfate in one series of experiments. In U.S. Pat. No. 5,604,200, medical grade HA and tissue culture grade plasma fibronectin were used in combination with calcium, phosphate, uric acid, urea, sodium, potassium, chloride and magnesium to create a moist healing environment that simulates the fetal in utero wound healing matrix. U.S. Pat. No. 5,631,011 discloses a composition of HA and fibrin or fibrinogen.
Various other compositions have also been explored for their wound healing capabilities. Kratz et al. (1997) used a gel of heparin ionically linked to chitosan. Bartold and Raben (1996) studied platelet-derived growth factor (PDGF). Henke et al. (1996) disclosed that chondroitin sulfate proteoglycan mediated cell migration on fibrinogen and invasion into a fibrin matrix, while Nakamura et al. (1997) concluded that chondroitin sulfate did not affect wound closure in a corneal epithelial wound. Henke et al. (1996) also disclosed that an anti-CD44 antibody blocked endothelial cell migration on fibrinogen. U.S. Pat. No. 5,641,483 discloses topical gel and cream formulations containing human plasma fibronectin for healing of cutaneous wounds. Schultz et al. (1992) disclose a composition of epidermal growth factor (EGF), fibronectin, a synthetic collagenase inhibitor, and Aprotinin.
Various studies involving fibronectin (FN) and/or particular fibronectin peptides and wound healing have also been reported. Many of these studies involve the RGD sequence, part of the cell binding domain of FN (see Schor et al. 1996; Steed et al. 1995; Sponsel et al. 1994; Kartha and Toback 1992; Kishida et al. 1992). Schor et al. (1996) disclose that only the gelatin binding domain of FN (GBD) stimulates fibroblast migration into a 3-D matrix of native type I collagen fibrils at femtomolar concentrations; whereas peptides of the other FN functional domains do not stimulate fibroblast migration in this assay at femtomolar to nanomolar concentrations. Schor et al. (1996) also disclose that the RGDS-containing cell binding domain of FN does, however, stimulate fibroblast migration in the transmembrane (or "Boyden chamber") assay. Steed et al. (1995) disclose that the RGD peptide matrix (known as Argidene Gel™ or as Telio-Derm Gel™) promoted wound healing. On the contrary, Sponsel et al. (1994) disclose that an RGD peptide impaired healing of a mechanical wound made in a confluent monolayer of one epithelial cell line. Kartha and Toback (1992) also concluded that an RGDS peptide completely inhibited cell migration into a wound area. Kishida et al. (1992), however, disclose that an RGD-albumin conjugate adsorbed onto a polyurethane sponge exhibited tissue ingrowth-promoting activity.
Other portions of FN have also been studied for wound healing activity. U.S. Pat. No. 5,198,423 studied the effects of a polypeptide containing a cell binding domain and a heparin binding domain of FN on wound healing. U.S. Pat. No. 4,589,881 studied the effects of a 108 aa polypeptide fragment of FN on wound healing, as well as a biologically active fragment thereof. Sponsel et al. (1994) studied the effect of the tetrapeptide REDV and the peptide LDVPS on wound healing.
The severity of the problem of chronic, nonhealing wounds dictates that continual efforts be made to define new and more effective matrices and methods for facilitating wound healing.
This need is met by the subject invention which provides an extracellular matrix for enhancing wound healing. The extracellular matrix comprises peptides from two or more fibronectin domains in a backbone matrix. The extracellular matrix facilitates wound healing by providing an environment that intrinsically recruits new tissue cells to the wound site.
A method of enhancing wound healing is also provided by the subject invention. The method comprises applying the extracellular matrix as so defined to a wound.
Using an in vitro model of cell movement across 3-dimensional extracellular matrix boundaries that simulates early wound repair (Greiling and Clark 1997), the experiments related to the subject invention established that multiple domains of FN were required for fibroblast movement. In one embodiment, the subject invention provides a hyaluronic acid backbone derivatized with the minimal FN sequences that are optimal for tissue cell recruitment. These constructs can be used to accelerate the healing of acute gaping cutaneous wounds and chronic cutaneous ulcers.
These and other features and advantages of this invention will be evident from the following detailed description of preferred embodiments when read in conjunction with the accompanying drawings in which:
FIG. 1 illustrates the in vitro model for assaying cell transmigration from a collagen gel into a fibrin gel;
FIG. 2 illustrates the general structure of fibronectin, showing the number and relative positions of the basic functional domains;
FIG. 3 illustrates the effect on cell migration of FN and FN-120 with or without PDGF;
FIG. 4 illustrates the relation of the various recombinant FN proteins to the domains of FN;
FIGS. 5A-5D illustrate the effect on cell migration of various recombinant FN proteins;
FIG. 6 illustrates the levels of fibroblast adhesion for various recombinant FN proteins;
FIG. 7 illustrates the relation of various deletion mutants of the Hep II domain to the Hep II domain of FN;
FIG. 8 illustrates the relation of various synthetic peptides to the Hep II and IIICS domains of FN;
FIGS. 9A-9H illustrate the effect on cell migration of various synthetic peptides; and
FIGS. 10A-10B illustrate the requirement of fibronectin for transmigration.
The subject invention provides an extracellular matrix for wound healing which comprises peptides from two or more fibronectin domains in a backbone matrix. As used herein, an "extracellular matrix" refers to a scaffold in the cell's external environment with which the cells may interact via specific cell surface receptors. As further used herein, a "wound" is intended to include both acute and chronic dermal wounds including, for example, surgical incisional wounds, traumatic wounds, cancer extirpations, radiation wounds, venous leg ulcers, diabetic ulcers, and pressure ulcers.
The extracellular matrix according to the subject invention comprises peptides from two or more fibronectin domains in a backbone matrix. These components are necessary for the subject extracellular matrix to enhance (e.g. improve, increase) wound healing, although additional components may also be included in the extracellular matrix. These additional components, such as platelet-derived growth factor as discussed below, may further enhance the beneficial effects of the extracellular matrix on wound healing.
Enhancement (e.g. improvement, increasing) of wound healing refers to the traditional sense of wound healing where clean closure of the wound occurs. Since naturally occurring wound healing involves the movement of fibroblasts into the wound site, enhancement of wound healing can be assayed in vitro using the model for cell transmigration provided in copending, co-assigned U.S. Ser. No. 08/723,789, filed Sep. 30, 1996 (the contents of which are incorporated by reference herein). Briefly, the model provides a contracted collagen gel containing fibroblasts surrounded by a fibrin gel (see FIG. 1). When the extracellular matrix of the subject invention replaces or is added to the fibrin gel, fibroblast movement from the collagen gel into the extracellular matrix or modified fibrin gel is enhanced compared to movement into the "gold standard" fibrin gel.
The extracellular matrix of the subject invention comprises peptides from two or more fibronectin domains in a backbone matrix. Fibronectin is a multi-domain, multifunctional cell adhesion protein found in blood and in a variety of tissue extracellular matrices (Yamada and Clark 1996). Although encoded by only a single gene, FNs exist in a number of variant forms that differ in sequence at three general regions of alternative splicing of its precursor mRNA. Some of this alternative splicing involves cell adhesion sequences, thereby providing a post-transcriptional mechanism for potentially regulating cell interaction. Nevertheless, all FN molecules appear to consist of the same basic functional domains. As shown in FIG. 2, these domains include two heparin binding domains, Hep I and Hep II; two fibrin binding domains, Fib I and Fib II; a collagen or gelatin binding domain; a cell-binding domain; and a variably splice IIICS domain, which contains within it CS1 and CS5 subdomains. Each domain is composed of FN repeats denoted as thin rectangles for the type 1 repeats, ovals for the type 2 repeats, and wide rectangles for the type 3 repeats.
The peptides from two or more fibronectin domains are therefore selected from the domains indicated above. Suitable peptides include those from the cell binding domain (such as peptides including the amino acid sequence SEQ ID NO:1: and peptides including the amino acid sequence SEQ ID NO:2:), the IIICS domain (such as peptides including the amino acid sequence SEQ ID NO:3 which is a truncated CS1 peptide; or the peptide designated CS1 and having an amino acid sequence as shown in SEQ ID NO:4; or the peptide designated CS5 and having an amino acid sequence as shown in SEQ ID NO:5), and the heparin II binding domain (such as the peptide designated H-I and having an amino acid sequence as shown in SEQ ID NO:6; or the peptide designated H-II and having an amino acid sequence as shown in SEQ ID NO:7; or the peptide designated H-III and having an amino acid sequence as shown in SEQ ID NO:8; or the peptide designated H-IV and having an amino acid sequence as shown in SEQ ID NO:9; or the peptide designated H-V and having an amino acid sequence as shown in SEQ ID NO:10; or the peptide having an amino acid sequence as shown in SEQ ID No:11 {a COOH-terminal HepII peptide that binds α4β1}). Preferably, the two or more fibronectin domains according to the subject invention are selected from the type III repeats (the wide rectangles in FIG. 2) or the IIICS region.
Having identified the peptides from two or more fibronectin domains that will be added to a backbone matrix, it should be readily apparent that these peptides can be made by any suitable means; for example, the peptides can be constructed using peptide synthesis technology (synthetic peptides), or cut out of a naturally occurring fibronectin molecule using proteolytic enzymes. Small peptides will probably need to be conjugated (covalently attached) to the backbone matrix but non-covalent delivery systems may also be appropriate (for example, the peptides may be held within the backbone matrix by affinity interactions).
As further used herein, a "backbone matrix" refers to natural extracellular matrices as well as biocompatible synthetic polymers. These backbone matrices provide the scaffold of the extracellular matrix and when the peptides of two or more fibronectin domains are in the backbone matrix, cells can move around on the scaffold. According to this invention, the peptides are provided as relatively small molecules and therefore must be held within to the backbone matrix or else the peptides would diffuse away. The backbone matrix thus provides the structure to which the peptides can be conjugated or non-covalently retained.
There are numerous examples of backbone matrices suitable for use in the subject invention. These examples include fibrin, hyaluronic acid, polyethylene glycol, poly-L-glycol, and poly-L-lactate. Hyaluronic acid is commercially available as a dry (for example, lyophilized) powder, and can be reconstituted to a hyaluronic acid gel (in accordance with manufacturer's suggestions) for use in the subject invention. Depending upon the viscosity desired, a hyaluronic acid gel having about 5 milligrams to about 50 milligrams of hyaluronic acid per milliliter of reconstituting solution can be used. At 5 milligrams/milliliter, the hyaluronic acid gel will be more liquid, and at 50 milligrams/milliliter the hyaluronic acid gel will become more viscous and less easy to manipulate. The use of the gel will, in part, dictate the desired viscosity. If the extracellular matrix can be "poured" into and contained in a wound area, then a more liquid form of the hyaluronic acid gel will be satisfactory. If the extracellular matrix is "spread" over and/or into a wound area, then a more viscous form of the hyaluronic acid gel will be desirable. In either case, a dressing of some form will often cover the applied extracellular matrix to help prevent contamination and infection of the wound. It should be readily apparent that the extracellular matrix itself (and each of its components) must be sterile (free of biological and/or chemical contamination) to also prevent contamination and infection of the wound.
Preferably, the hyaluronic acid gel is provided as a gel having about 20 milligrams of dry hyaluronic acid per milliliter of reconstituting solution. Suitable reconstituting solutions include, for example, sterile distilled water, sterile phosphate buffered saline (PBS), or a cell culture medium.
As used herein, "hyaluronic acid" is intended to include the various forms of hyaluronic acid (HA) known in the art. These various forms include HA chemically modified (such as by cross-linking) to vary its resorbtion capacity and/or its ability to be degraded. Optimal HA formulations will be resorbable in a few days to a week.
The peptides can be conjugated to the backbone matrix using techniques known in the art. Such techniques include the use of a heterobifunctional crosslinking reagent. In some embodiments, it may be desirable to alter the amino terminal amino acid of the peptide to allow conjugation to the backbone matrix. U.S. Pat. Nos. 5,652,347 and 5,616,568 disclose methodology for the functional derivatization of hyaluronic acid using dihydrazide. The contents of each of these patents is incorporated herein by reference.
The extracellular matrix can further comprise other components which further enhance the cell migration effect of the matrix. Such additional components include, for example, platelet derived growth factor.
The invention further provides a method of enhancing wound healing which comprises applying the extracellular matrix (as described herein) to a wound. As discussed above, the method of applying the extracellular matrix to the wound may vary depending on the type and location of the wound as well as the viscosity of the extracellular matrix. Preferably, the extracellular matrix is viscous enough to be "spread" over the wound and will not run off after application.
Normal Human Dermal Fibroblasts
Primary cultures of human adult dermal fibroblasts, acquired from Marcia Simon (Living Skin Bank, SUNY at Stony Brook), the ATCC (Bethesda, Md.), or the NIA (Bethesda, Md.), are cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) containing 42 mM sodium bicarbonate and supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS, HyClone, Logan, Utah), at 37� C. and 5% CO2/95% air in a humidified atmosphere. The cells are used between passages 4 and 12.
Fibroblast Migration Assays: Transmigration from Organotypic Collagen Gel Constructs into Fibrin/Fibronectin Gels or Outmigration over Protein Coated Surfaces
Preparation of Floating, Contracted Collagen Gels
Fibroblast cultures at 80% confluence are harvested by treatment with 0.05% trypsin/0.01% EDTA. Trypsin is inactivated by addition of soy bean trypsin inhibitor in PBS containing 0.2% BSA. The cells are washed twice with DMEM+2% BSA and resuspended at a concentration of 1�106 cells/ml. The fibroblasts are mixed with neutralized collagen (Vitrogen 100, Celtrix Labs., Santa Clara, Calif.), 2% BSA, 30 ng/ml PDGF-BB, 30 μg/ml fibronectin, and concentrated DMEM so that the final concentration of DMEM and sodium bicarbonate is 1x. 600 μl of the cell mixture is added to the wells of a 24-well tissue culture plate, which has been precoated with 2% BSA. The collagen is allowed to polymerize at 37� C. The final concentration of collagen is 1.8 mg/ml and each gel contains 6�104 cells. After two hours incubation, the gels are gently detached from the plastic surface to allow contraction with the addition of 0.5 ml DMEM+2% BSA and 30 ng/ml PDGF-BB per well. The gels are incubated overnight at 37� C. in 100% humidity, 5% CO2 and 95% air.
Preparation of Protein Coated Wells
Fibronectin, its fragments or recombinant domains were diluted with concentrated DMEM to the appropriate concentration. Aliquots of 450 μl protein solution are added to the wells of 24-well tissue culture plates (Becton-Dickinson, Lincoln Park, N.J.). After a 2 hour incubation at 37� C. in 5% CO2, plates were dried overnight at room temperature under sterile conditions.
Preparation of Two-dimensional Outmigration Model
Plates coated with dried proteins were washed once with PBS and incubated with 2% BSA for 1 hour at 37� C. to block nonspecific binding sites. After washing the plates three times with PBS, contracted-collagen gel organotypic constructs were attached to the coated plates. DMEM, 2% BSA and 30 ng/ml PDGF-BB was added to assay plates so that the medium was level with the top of the collagen gel.
Preparation of Three-dimensional Transmigration Model
For preparation of "gold standard" transmigration assays containing a dermal organotypic construct surrounded by a fibrin clot as previously described (Greiling and Clark 1997), dried fibrin fibril-coated dishes are washed once with PBS and fibroblast-contracted collagen gels are placed on the surface. Fibrinogen, at a final concentration of 300 μg/ml, is mixed with DMEM and 1.0 U/ml thrombin, added to the wells so that the solution is level with the top of the collagen gel, and allowed to clot at room temperature for 30 min. When needed, other supplements such as 30 ng/ml PDGF-BB are added to the mixture. For HA 3-dimensional transmigration, wells are coated overnight at 37� C. with an appropriate solution of HA. The next day a fibroblast-contracted collagen gel is placed on the HA-coated well in DMEM, with or without 30 ng/ml PDGF-BB, is added so that the solution is level with the top of the collagen gels. All migration assays are quantified after a 24 hour incubation at 37� C. in 100% humidity, 5% CO2 and 95% air.
Evaluation of Cell Migration
The number of migrated cells was quantified under a Nikon inverted phase microscope by visually counting identifiable cell nuclei located outside of the contracted collagen gel in the fibrin gel (transmigration assay) or on the matrix (outmigration assay). Within a given experiment each condition was run in triplicate and means �SD calculated. All experiments were repeated at least three times. Statistical differences among conditions can be determined by ANOVA.
Fibroblast Adhesion Assay
Assay plates are prepared as described under fibroblast migration assays. The assay for measuring fibroblast adhesion to matrix proteins are performed essentially as described (Gailit et al. 1993) except that the cell concentration is lowered to 100,000 cells/ml or 10,000 cells/well. Cells are allowed to attach for 60 min at 37� C. before the unattached cells are washed away and the attached cells fixed with 2% glutaraldehyde. After fixation, attached cells are air dried at room temperature and then 100 ml of 0.1% crystal violet in 0.2 M boric acid, pH 9, is added to each well and the microtiter plate shaken at 600 rpm on a plate mixer for 20 minutes. (The staining solution is prepared fresh from a stock solution of 5% crystal violet in 20% methanol.) Excess stain is removed by three washes with water. The stained cells are again air dried before the crystal violet is solubilized by adding 100 ml of 10% acetic acid to each well and then shaking the plate at 600 rpm for 20 minutes. The absorbance at 590 nm is measured with a dual wavelength microtiter plate reader (THERMOmax, Molecular Devices, Menlo Park, Calif.) and the reading corrected for light scattering by subtraction of the absorbance at 450 nm.
Assay of Wound Healing
The extracellular matrix of the present invention was tested by use of the in vitro model as described in U.S. patent application Ser. No. 08/723,789, which is hereby incorporated by reference. The basis of the in vitro model is a contracted collagen gel containing fibroblasts which acquire a tissue-like phenotype within the collagen matrix. Surrounding the collagen gel, or dermal equivalent, with a fibrin clot produces a simple inside-outside model of the early cutaneous wound (FIG. 1). Without an added stimulus, no more than a few of the normal adult human dermal fibroblasts within the collagen gel would migrate into the fibrin gel. However, the transmigration of fibroblasts from the collagen gel into the fibrin gel is enhanced by the replacement of the fibrin gel with the extracellular matrix of the subject invention or by the addition of the extracellular matrix to the fibrin gel, since the extracellular matrix facilitates cell movement thereby enhancing wound healing.
Fibronectin (FN) is required for fibroblast migration through both fibrin clots and hyaluronic acid (HA) gels. Initially, experiments were conducted to determine whether FN, either in a fibrin gel or in a collagen gel, is required for fibroblast transmigration. To do this, FN was selectively removed from each matrix material. First, residual FN was removed from the fibrinogen preparation by affinity chromatography on gelatin. After removal of FN, fibroblast transmigration into the fibrin clot was decreased by about 80% (FIG. 10A). Transmigration could be restored by the addition of FN to the fibrin gel. Optimal cell movement was observed with 30 μg/ml, a FN:fibrinogen ratio of 1:10, the physiological plasma ratio. In FIG. 10A, migration induced by 30 ng/ml PDGF-BB (shaded bars; open bars: 0 ng/ml PDGF) was measured under the usual assay conditions. The fibrinogen preparation used to form the fibrin gel was untreated (left), treated with gelatin-Sepharose to remove FN (center), or treated with gelatin-Sepharose and then supplemented with 30 μg/ml FN (right).
Second, exogenous FN was excluded from the collagen gel by omitting serum and substituting PDGF-BB, which is equally effective at stimulating fibroblast-driven collagen gel contraction. In experiments with FN-free collagen gels no transmigration occurred (FIG. 10B). Transmigration was only observed when FN was present in both the collagen gel and the fibrin gel; 30 μg/ml FN in each gel seemed the most effective. In FIG. 10B, migration induced by 30 ng/ml PDGF-BB (shaded bars) was measured under modified assay conditions. Contraction of the collagen gel was stimulated with serum as usual (FBS) or with 30 ng/ml PDGF-BB (PDGF). The fibrinogen preparation used to form the fibrin gel was untreated (Fb), treated with gelatin-Sepharose to remove FN (Fb-FN), or treated with gelatin-Sepharose and then supplemented with 10, 30, or 100 μg/ml FN. The inclusion of 30 μg/ml FN in the collagen gel and in the fibrin gel restored transmigration to a normal level.
Having established that FN is required for fibroblast migration, the FN domains necessary for migration were next examined. In the first experiment focused on the specific sequences of the FN molecule necessary for migration of fibroblasts, assay wells were coated with 120 nmol/l of either intact FN (FN) or the 120 kDa fragment of FN (FN-120). The FN-120 fragment was isolated from a chymotrypsin digest as previously described (Wikner and Clark 1988). FN-120 contains the RGDS cell-binding domain, but lacks the Hep II and IIICS domains (see FIG. 4). Surprisingly, although FN-120 promotes 70% adhesion of fibroblasts compared to intact FN, it does not allow migration (FIG. 3). Reduction and alkylation of FN dimer to the monomeric state did not affect its ability to mediate either adhesion or migration.
Next, the Hep II domain was examined for its ability to support migration. For this purpose a variety of recombinant FN proteins were obtained from Jean Schwartzbauer at Princeton University (Barkalow and Schwarzbauer 1991)(FIG. 4). Referring to FIG. 4, recombinant proteins are indicated by MBP. Assay plates were coated with 4 to 400 nmol/l of FN, FN-120 or recombinant proteins by drying the protein solution overnight at room temperature. The coating efficiency of all proteins was essentially the same as judged by the bicinchoninic acid protein assay (Tuszynski and Murphy 1990). A recombinant Hep II domain (HV0) supported migration at a level less than 20% of the migration observed on intact FN (FIG. 5A). Addition of FN-120 and HV0 on these plates, which presented both the RGD cell-binding and heparin-binding domains, respectively, in a non contiguous array, enhanced fibroblast migration to approximately 45% of the maximum level seen with intact FN (FIG. 5A). When recombinant FN protein CHV0, which contains both the RGD cell- and the heparin-binding domains in one molecule (FIG. 4), was used to coat the plates approximately 55% of the maximum level of migration was attained (FIG. 5B). Thus, about the same level of migration occurred whether the RGD cell- and heparin-binding domains were available to the cells in contiguous, or non contiguous, arrangments. Neither the FN-120 fragment alone, nor a control recombinant peptide III1-6, containing the 1st through the 6th FN type III repeats without known cell-binding sites (FIG. 4), allowed any migration (FIG. 5A and 5B).
Next, experiments were conducted to examine whether the Hep II domain combined with the IIICS domain, which contains classic α4β1 binding sites, supports more migration than the Hep II domain alone. To this end, a recombinant protein HV120 containing both these domains (FIG. 4) was added to the surface of assay plates. As shown in FIG. 5C, HV120 supported 40% of the maximal migration observed with intact FN. When plates were coated with HV120, containing the heparin-binding and IIICS domains, and the FN-120 FN fragment, containing the RGD cell-binding domain, migration comparable to that on intact FN was observed (FIG. 5C). When a recombinant protein CHV120, which contains the RGD-cell binding domain, the heparin-binding domain and the IIICS domain within one molecule (FIG. 4), was used to coat assay plates, essentially the same result was obtained; that is, maximal levels of migration were seen compared to migration on intact FN (FIG. 5D).
Migration supported with CHV0 (about 55% maximum, FIG. 5B) was completely abolished if the arginine-pair XX in the 13th type III FN repeat of the Hep II domain was mutated to uncharged amino acids (CHV0-RR-TM) as previously described (Barkalow and Schwarzbauer 1991). In contrast, CHV0-RR-TM supported fibroblast adhesion at levels comparable to intact FN, CHV120 and CHV0 (FIG. 6).
These results lead to the conclusion that short, specific sequences within the Hep II domain are absolutely necessary for cell motility. To address this, deletion mutants of the Hep II domain were investigated (HV0-13a, HV0-13b14a, and HV0-14b, which were missing selected sequences of amino acids in the 13th and 14th type III repeats as shown in FIG. 7 and previously described (Barkalow and Schwarzbauer 1991; Barkalow and Schwarzbauer 1994)). HV0 and its mutants supported little migration by themselves at concentrations up to 400 nmol/l (Table 1). Furthermore, the addition of FN-120 along with the Hep II deletion mutants to the assay plates did not enhance migration (Table 1). In contrast, when FN-120 was added to the assay plates in combination with the entire recombinant Hep II domain (HV0) migration was enhanced (Table 1 and FIG. 5A). These results confirm the conclusion that subdomains within the Hep II domain are required for optimal cell migration.
To further define which Hep II and IIICS subdomains are involved in fibroblast migration on FN, synthetic peptides previously shown to be active in cell adhesion were manufactured from sequences in the 12th, 13th, and 14th FN type III repeats (peptides I, II, III, IV, and V)(McCarthy et al. 1988; McCarthy et al. 1990; Drake et al. 1993; Mooradian et al. 1993) and the IIICS segment (CS1 and CS5)(Humphries et al. 1987; Komoriya et al. 1991; Mould et al. 1991)(FIG. 8). FN peptide I (FN-C/HI; SEQ ID NO:6:), peptide V (FN-C/HV; SEQ ID NO:10:), CS1 (SEQ ID NO:3:), and CS1i (i=inactive, SEQ ID NO:12:) were purchased from Peninsula Laboratories Inc. (Belmont, Calif.). FN peptide II (FN-C/HII; SEQ ID NO:7:), peptide III (FN-C/HIII; SEQ ID NO:8:), peptide IV (FN-C/HIV); SEQ ID NO:9:), and CS5 (SEQ ID NO:5:) were synthesized by SynPep (Dublin, Calif.). The purity of all peptides was higher than 97%.
When added to the outmigration assay, peptides III (FIG. 9A), IV (FIG. 9B), V (FIG. 9C), I (FIG. 9D), and II (FIG. 9E) inhibited the migration of fibroblasts onto dried FN in a dose-dependent manner. CS1 (FIG. 9F) had a slightly enhancing effect in the higher concentrations while CS5 did not influence the migration at the tested concentrations (FIG. 9G). A control scrambled peptide of CS1 (CS1i), did not influence migration (FIG. 9H). To determine whether the observed effect of synthetic peptides on the migration was specific for FN, collagen coated assay plates were substituted for FN coated plates. Peptides III (FIG. 9A), IV (FIG. 9B), and V (FIG. 9C) had no effect on migration over collagen while peptides I and II (FIG. 9D and 9E, respectively) had an inhibitory effect in a dose-dependent manner. CS5 enhanced the migration at higher concentrations (FIG. 9G) while CS1i did not influence the activity (FIG. 9H).
To determine whether the results obtained from the two-dimensional outmigration assay related to more complex, 3-dimensional transmigration, the same synthetic peptides were added to the fibrin/FN gel in the transmigration assay. Most peptides gave essentially the same results. In aggregate these data demonstrate that 3 major domains of FN are required for fibroblast migration.
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.
no additional
protein added to
100 � 2.7b
15.3 � 1.1c
45.4 � 1.1
23.3 � 1.7
23.3 � 2.2
6.3 � 2.4
8.7 � 4.8
20.1 � 2.0
15 � 3.7
aAll proteins and peptides were assayed at concentrations from 3 to 400 nmol/l, however, maximum fibroblast migration was observed when 120 nmol/l protein was added to assay plates. Therefore, the data shown were acquired from plates coated with 120 nmol/l FN, recombinant peptides or FN-120.
bFibroblast migration on fibronectin (FN) was normalized to 100%.
cData are presented as mean � SD percent migration of that observed on FN. All conditions were run in triplicate.
Abraham, J. A., and Klagsbrun, M., in "The Molecular and Cellular Biology of Wound Repair", 2d edition, Clark, R. A. F., ed, Plenum Press, New York, N.Y. (1996).
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|U.S. Classification
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|International Classification
|Cooperative Classification
|European Classification
|May 24, 1998
Owner name: RESEARCH FOUNDATION OF STATE UNIVERSITY OF NEW YOR
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:CLARK, RICHARD A.;GREILING, DORIS;REEL/FRAME:009228/0136;SIGNING DATES FROM 19980416 TO 19980518
|May 27, 1998
Owner name: NATIONAL INSTITUTES OF HEALTH, THE, MARYLAND
Free format text: CONFIRMATORY LICENSE;ASSIGNOR:SUNY AT STONY BROOK, NY;REEL/FRAME:009253/0087
Effective date: 19980402
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Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF
Free format text: EXECUTIVE ORDER 9424, CONFIRMATORY LICENSE;ASSIGNOR:THE RESEARCH FOUNDATION OF STATE UNIVERSITY OF NEW YORK;REEL/FRAME:020961/0297
Effective date: 19980501
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Year of fee payment: 8
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|Maintenance fee reminder mailed
|Oct 8, 2012
|Maintenance fee reminder mailed
|Feb 27, 2013
|Lapse for failure to pay maintenance fees
|Apr 16, 2013
|Expired due to failure to pay maintenance fee
Effective date: 20130227


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NEWS & TUTORIALS on After Effects, Final Cut Pro, AVID and much more.
A blog by Premiumbeat
The 'Magic' of Disney? New Software Automatically Edits Video Footage
This is the beginning of the end: Disney has created a software that can intelligently edit video without human intervention.
5 Tips for Jewelry Photography
Learn how to shoot high-end jewelry photography using these 5 steps.
A Compressed Explanation of Video Compression
Compression doesn't have to be confusing. The following video explains video compression quickly and clearly!
The New Blackmagic URSA Footage is Gorgeous
The Blackmagic URSA is officially out in the world and the test footage is amazing!
3 Free Color Grading Tools
Discover these free color grading tools to give your video projects a professional, stylized look.
An Overview of Apple Motion's New Features
Apple's latest update adds an improved contrast filter and range selector for text. The following video tutorial shows us how to use them.
Microsoft Unveils Their Own Hyperlapse 'Warp Stabilizer'
Microsofts latest algorithm will blow your mind and stabilize your shaky footage.
Proper Set Etiquette Will Get You Hired Again and Again
One of the most important things you can do when starting out in the film industry, is learn to abide by the unwritten rules of the set.
After Effects Video Tutorial: Texturized Space Scene
Learn how to create a realistic space scene in this Adobe After Effects video tutorial.
30 Days of After Effects: The Perfect Way to End the Summer
Get over your post-summer slump with this awesome After Effects tutorial series created by School of Motion.
After Effects Video Tutorial: Electric Type
Learn how to create an awesome electric type effect in this video tutorial from the Creative Dojo.
Why All Directors Need to Take Up Acting
Indie film directors today have largely become more concerned with gear, effects, and post-production than with story and acting, which is one reason so many indie films fail to have any success. While the technical side of filmmaking is critical in today's filmmaking environment, it is raw performance that will always be what captivates audiences most.

subset: commoncrawl


text:
Relevant offers
Christchurch Earthquake 2011
A big quake could leave Wellington reliant on barges and helicopters for survival, new disaster predictions show.
Massive landslides would cut off Porirua, the Hutt Valley and Wellington from the rest of New Zealand and from one another, with inbound roads taking up to four months to clear.
All three areas would rely on barges and helicopters to ferry in food, clean water and vital medical supplies.
The would be no power, water or gas for at least three weeks, and for more than two months in some Wellington suburbs.
Wellington city would be cut off for the longest, with no power for at least two months and no gas for three.
It would take at least 55 days to open State Highway 2 between the Hutt Valley and Wellington, and three weeks to connect the capital and Porirua.
The grim predictions are contained in a report by the Wellington Lifelines Group, which includes 20 of the region's major infrastructure companies and civil defence authorities.
It is based on a "worst-case scenario" of a 7.5 or higher magnitude quake, which has a less than 10 per cent chance of striking within the next century.
The report also reveals just how vulnerable the region's basic services would be in a big quake.
Its hills funnel vital roads, powerlines and water mains into bottlenecks, often crossing Wellington's major fault line, meaning it would take considerably longer to restore services in Wellington than it did in Christchurch after the February earthquake last year.
It would also be harder to keep the city supplied, with the report noting Wellington's supermarkets have no major backup storage in the region.
Regional Emergency Management Group co-ordinator Bruce Pepperell said road access would be the biggest priority. "Parts of the region will be completely cut off from others."
The stretch of State Highway 1 sandwiched between the coast and cliffs along Centennial Highway would probably be the most difficult to clear.
"If it is necessary to blow the top of the hill into the ocean [to open a road], that is what will happen."
Greater Wellington regional council chairwoman Fran Wilde, who also chairs the lifelines group, said the report was part of a wider resilience review by infrastructure firms after the Canterbury quakes.
However, many improvements would take years, and the public would need to decide on the tradeoffs between cost and quake resilience, she said. "Unless you have a limitless budget, there is no quick fix."
Wellington Electricity Lines has already said it will need to charge more for electricity to strengthen its infrastructure.
The lines company has 300 substations in potentially quake-prone buildings, and the mostly underground power network is vulnerable to quake damage.
Chief executive Greg Skelton said the company would seek "customer feedback" on a price hike, which would require Commerce Commission approval.
The regional council is also considering new emergency water reservoirs, and briefly entertained building a desalination plant on the south coast.
Both Pepperell and Wilde stressed the latest predictions were for the worst possible event, and a smaller less-damaging quake was far more likely.
"It is not a cause for everyone to go pick up their bags and leave," Wilde said.
The report says that, whatever strengthening is built into services, it is equally important that people prepare personally for a big disaster.
- The Dominion Post
Sponsored links
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Which memorial design do you like most?Related story: Christchurch earthquake memorial designs unveiled


text:
Heat Wave Prompts 2nd Flex Alert In A Row Called For CaliforniaA Flex Alert called by the Operators of California's electricity grid continues Thursday as states across the West swelter.
Storms Knock Out Power To Thousands Up And Down CaliforniaEmergency crews are working to restore power to thousands of customers throughout California after heavy wind knocked down trees and power lines.
Eucalyptus Tree Takes Out Power Lines, Blocks Access To Homes In BerkeleyA large eucalyptus tree took out power lines, causing an outage to hundreds of homes in Berkeley during Tuesday morning's rainstorm, PG&E said.
Garbage Truck Hits Utility Pole, Knocking Power To Nearly 1K San Jose HomesAbout 850 homes are without power Thursday evening in East San Jose after a garbage truck struck a utility pole, bringing down power lines earlier in the day, according to PG&E officials.
PG&E Transformer Explodes Injuring 2 In Bernal HeightsPG&E is trying to figure what caused a transformer to explode in San Francisco injuring 2 people.
Geothermal, Solar Power Facilities May Have Been Damaged In 62,000 Acre Valley FireAlameda Municipal Power reported Monday that geothermal and solar power facilities near Middletown may have been damaged in the 62,000-acre Valley Fire, currently burning through Lake, Napa, and Sonoma counties.
Sweltering Bay Area Heat Putting Strain On Power Grid; Residents Urged To Conserve EnergyCalifornia power grid operators are calling for another "flex alert" Wednesday, urging residents to conserve power as temperatures peek into triple digits for a second day.
PG&E Still Trying To Explain How Squirrel Managed To Cause Huge East Bay Power OutagePG&E is still trying to explain just how a squirrel managed to cause a widespread power outage after tens of thousands of people in the South Bay and East Bay were plunged into darkness Monday night because the system couldn't handle the heat in the south, and a squirrel in the East Bay.
PG&E Says Squirrel Caused Outage That Impacted 45,000 In East BayOfficials say a number of power outages have hit thousands of Pacific Gas & Electric Co. customers Monday, including more than 40,000 in the East Bay and 10,000 in San Jose.
Balloon Causes San Francisco Power Outage That Affected 9,000 People In Fisherman's Wharf, North Beach NeighborhoodsA helium balloon is to blame for thousands of residents and businesses losing power in San Francisco's Fisherman's Wharf and North Beach neighborhoods Monday afternoon, according to PG&E.
Tesla 'Powerwall' Battery Technology Unveiled, Set To Cost Around $3KElectric car maker Tesla Motors wants to park its battery technology in homes and businesses across the U.S.
Tesla's Mystery Product May Be Battery Power For Your HomeTesla shares are getting a charge from a Bloomberg report that the company will unveil a home battery and a large-scale utility battery, at an event at its Hawthorne design studio next week.


text:
Relevance Ranking for Vertical Search Engines
1st Edition
- eBook ISBN 9780124072022
- Print ISBN 9780124071711
Print book
Print book
In plain, uncomplicated language, and using detailed examples to explain the key concepts, models, and algorithms in vertical search ranking, Relevance Ranking for Vertical Search Engines teaches readers how to manipulate ranking algorithms to achieve better results in real-world applications.
This reference book for professionals covers concepts and theories from the fundamental to the advanced, such as relevance, query intention, location-based relevance ranking, and cross-property ranking. It covers the most recent developments in vertical search ranking applications, such as freshness-based relevance theory for new search applications, location-based relevance theory for local search applications, and cross-property ranking theory for applications involving multiple verticals.
Software Engineers, Computer Scientists, Academic Researchers, Applied Scientists Web professionals and Researchers.
"The authors of this book are active researchers in vertical search technology. This book provides researchers and application developers a comprehensive overview of the general concepts, techniques, and applications in vertical search."
-Prabhakar Raghavan, Vice President of Engineering at Google
"This is an excellent book that gives the first and comprehensive introduction and overview on vertical search, an emerging and important field!
Researchers and practitioners will find this book provides a comprehensive overview and systematic treatment on theories, methodologies and practices for vertical search ranking, covering several very promising topics, such as search on news and medical information, entity search, mobile search, as well as multi-aspect ranking, aggregating vertical search ranking and cross vertical ranking.
I found it a great pleasure to read!"
-Jiawei Han, Abel Bliss Professor, Department of Computer Science, Univ. of Illinois at Urbana-Champaign


text:
AVG Technologies News
News on AVG Technologies continually updated from thousands of sources around the net.
Lifeboat to Distribute AVG Business Managed Workplace | @CloudExpo @LifeboatVAD #SaaS #Cloud #Storage
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APA Names Juliette Wolf-Robin to Be Executive Director
Posted by Holly Hughes on Monday September 9, 2013 | Business
Posted by Holly Hughes on Monday September 9, 2013 | Business
American Photographic Artists (APA), the not-for-profit trade association and advocacy group, has selected Juliette Wolf-Robin to be its new executive director. She will report to the APA board and work with directors of local APA chapters.
Wolf-Robin was previously business development and marketing director for The Brand Union NY. She has worked at FoundFolios, Creativity's Spare* and Alternative Pick and has been a frequent speaker at PhotoPlus Expo and APA events. In announcing the selection, APA president Theresa Raffetto said, "Juliette understands the ever-changing playing field the photo world has become and the importance for photographers to not only understand their value but to evolve and stay relevant." Raffetto also said, "Throughout her career she has put an emphasis on education and knowledge for both the photographer and the user and we are excited to see where we can take APA with Juliette by our side."
"Her industry experience combined with the fact that she's married to a photographer [David Robin] has given her a unique understanding of the business end of our industry as well as an emotional investment in its success. She'll be a potent advocate for not only APA but for all professional photographers," notes APA Executive Vice President Ric Kasnoff.
Posted by David Walker on Friday August 19, 2016 | Business
California-based Brooks Institute and Massachusetts-based Hallmark Institute of Photography have cancelled classes for the fall and announced plans to close down. The two schools, both private for-profit visual arts colleges, have struggled over the past decade with declining enrollments, financial stress, and management shake-ups. Administrators are also blaming new regulations regarding for-profit schools. "[R]ecent changes... More ›
Posted by Conor Risch on Thursday August 11, 2016 | Business, Copyright/Legal
When we were researching our story "What Lawyers See When They Look at Editorial Photography Contracts," which appeared in the June issue of PDN, we asked photographers to tell us about editorial contracts they feel are unfair to photographers. We received a copy of a Condé Nast contract sent to a photographer in 2013 as... More ›
Posted by Conor Risch on Wednesday July 20, 2016 | Business, Media
In the current editorial photography market, budgets are shrinking as contract terms become less favorable for photographers. As a follow-up to our story "What Lawyers See When They Look at Editorial Photography Contracts," we surveyed photographers who shoot editorial assignments about the financial challenges of the editorial photography market. A total of 142 photographers responded.... More ›


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Native Plants
Q. Who is Mr. Smarty Plants?
A: There are those who suspect Wildflower Center volunteers are the culpable and capable culprits. Yet, others think staff members play some, albeit small, role. You can torture us with your plant questions, but we will never reveal the Green Guru's secret identity.
Did you know you can access the Native Plant Information Network with your web-enabled smartphone?
Ask Mr. Smarty Plants is a free service provided by the staff and volunteers at the Lady Bird Johnson Wildflower Center.
rate this answer
Wednesday - July 27, 2011
From: Hernando, FL
Region: Southeast
Topic: Plant Identification
Title: Plant identification of chenille-like plant in Florida
Answered by: Nan Hampton
QUESTION:I live in Central Florida. I have a small, 8-10 inch plant that grows wild in the yard and has a 1 to 1-1/2 inch, bright red, feathery flower on it. I can't seem to find it on the internet and I'm wondering what it is. Some say it's chenille but the description of chenille doesn't fit since it's not a bush and doesn't get any bigger than about 10 inches. Thanks.
Acalypha hispida (chenille plant) is the ornamental plant you refer to and it is a native of New Guinea and Malaysia. There are several other species, both native and non-native, of the Acalypha that grow in Florida and one of these is likely to be the plant you describe.
The most likely one is Acalypha chamaedrifolia (bastard copperleaf). It is a native (Florida and the West Indies) herbaceous plant that grows only few inches tall. Here are more photos and information and cultivation information. There is a cultivar called red cat's tail that has flowers that are redder than the native version.
If that doesn't look like your plant, here are some other possibilities in the Genus Acalypha that grow in Florida:
Acalypha alopecuroidea (foxtail copperleaf) is a non-native herbaceous plant about 30 cm high and here is a photo.
Acalypha amentacea subsp. wilkesiana (Wilkes' copperleaf) is a non-native woody shrub about 1.5 m tall.
Acalypha arvensis (field copperleaf) is a non-native herbaceous plant growing 30 to 50 cm high. Here are more photos and more information.
Acalypha gracilens (slender threeseed mercury) is a native herbaceous plant that grows to less than a meter tall. Here are more photos and information form our Native Plant Database.
Acalypha ostryifolia (pineland threeseed mercury) is a small native herbaceous plant. Here are photos and more information from our Native Plant Database and the University of Missouri Extension.
Acalypha rhomboidea (diamond threeseed mercury) is a native herbaceous plant that grows to about 0.3 m. Here are photos.
Acalypha setosa (Cuban copperleaf) is a non-native herbaceous plant about 0.3 m high. Here are more photos.
Finally, if none of the above appears to be your plant, please visit our Plant Identification page where you will find links to plant identification forums where you can submit photos for identification.
More Plant Identification Questions
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November 10, 2009 - I bought a tree at the 2008 LBJ Wildflower plant sale, it is growing great. I would like to plant it in the proper location/soil but lost the name tag and can't identify it. It has very fine leaves...
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May 29, 2009 - Please,I have a green houseplant w/5-6 inch wide heartshaped leaves that grows small,green fingerling pods. Very long,zig-zaggy stems on this plant. No florist/gardener here can identify.Pretty and ge...
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April 20, 2011 - I'm doing a Flower Project for my Biology class. My partner and I have found a flower that we cannot identify and neither can our teacher. I found it on Interstate 35 going through Ft. Worth, Texas. ...
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Plant identification for Westcave Preserve
June 16, 2011 - Green greetings! In the canyon at Westcave Preserve last month, I saw a plant I don't know: low, broadish leaves (with rounded edges as I recall); very tall, delicate stems with tiny white flowers sp...
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UK police arrest six in £1m phishing scam
Police have arrested six people in connection with a £1m online phishing scam in which money was stolen from hundreds of students.
Police in the UK have arrested six people in connection with a £1m online phishing scam in which money was stolen from hundreds of students.
The phishing scam was designed to trick students on government loan schemes into revealing their bank account details. The information was then used to withdraw cash amounts of up to £5,000 a time.
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The victims received e-mails asking them to update details using a link which connected to a phishing web page hosted by the cyber criminals.
The Metropolitan Police Central e-crime Unit (PCeU) seized computers and other equipment from addresses in London, Manchester and Bolton after investigating the scam since August.
A 26 year-old man and a 25 year-old woman were arrested in Manchester, a 25 year-old man was arrested in Deptford, south London, a 49 year-old woman and a 31 year-old man were arrested in Stratford, north-east London, and a 38 year-old man was arrested in Bolton.
The PCeU said a great deal of personal information was compromised and cleverly exploited for substantial profit.
The suspects, who were arrested on suspicion of conspiracy to defraud, money laundering and Computer Misuse Act offences, are being held at police stations in London, Manchester and Bolton.
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Concha Alborg was ready for a new start. Her daughters were grown and married, and she'd retired five years ago as a professor at St. Joseph's University, after a long career.
So last fall, she sold the grand townhouse in Society Hill she'd owned with her late husband. And in January, she purchased a high-rise condo at Hopkinson House on Washington Square, kicking off a new chapter in her life.
Alborg, who grew up in Spain, came to Philadelphia 37 years ago to pursue a doctorate at Temple University. She fell in love with the vibrancy of the city and stayed.


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JD Sports Forum! / Re: Super Bowl XXXVIII New England vs Carolina« on: January 31, 2004, 09:02 AM »
but why the hatred for the Pats?
Their not flashy, they just get the job done.
I think that answers it for most people.
I think it also might be that the Pats fans are more vocal and have been about the team and you how people in forums like to be contrary.
I do have a question though for Mikey D. I have been so busy this week the only thing I have seen about the whole Bowl is some BS coverage of the teams eating or parties or something...so here is my question:
Is Bruschi gonna play? I haven't heard a word about THE GAME and it isn't on the front page of ESPN or anything.
Also, is Kliff Kingsbury healed up too? I mean just in case something happens to Brady? I'd like the Pats to win if it meant he got a ring.
As of right now, I am still going to root for the Panthers though. Being a Dallas fan, I have to root for the NFC. Unless Denver or the Texans were in it, they are my AFC teams.


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$7.19The constant noise of contemporary life makes it easy to miss the most profound message the world has ever known: Jesus loves you. Innovative teachers Craig Gross and Jason Harper will separate the religious from the real as they show how this simple truth is worth our undivided attention. The authors weave Jesus' narrative with their own stories of serving among the "least of these" in this
Eyes of Integrity (XXXChurch.com Resource)
Living Free in a World of Sexual TemptationAs a young pastor, Craig Gross became overwhelmed with the sheer number of people he found himself counseling who were mired in the destructive world of online pornography. Their lives were broken, their hearts and minds corrupted. How, he wondered, could he minister to the many hundreds of thousands, perhaps millions, of people trapped by sexual addiction?Through accountability software and an
Kauffman Amish Bakery Series (Book 1)Take a trip to Bird-in-Hand, Pennsylvania, where you'll meet the women of the Kauffman Amish Bakery in Lancaster County. As each woman's story unfolds, you will share in her heartaches, trials, joys, dreams … and secrets. You'll discover how the simplicity of the Amish lifestyle can clash with the "English" way of life—and the decisions and consequences that follow. Most importantly, you will be
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When You Believe in God But Live as if He Doesn't ExistGrowing up, all my friends would have described my family as a Christian family. I assumed all my friends were Christians as well. We all believed in God. We occasionally attended church. We were good people. Even though we believed in God, we didn't know his word, didn't understand the gospel, and didn't pursue his will. We believed in God, but we lived as if he didn't exist. After pastoring for
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Hearing God, Having the Guts to Respond"Without a hint of exaggeration," says Bill Hybels, "the ability to discern divine direction has saved me from a life of sure boredom and self-destruction. God's well-timed words have redirected my path, rescued me from temptation and re-energized me during some of my deepest moments of despair." In The Power of a Whisper, vision is cast for what life can look like when God's followers choose to
Field-Tested Strategy for the 360° LeaderThe book you hold resonates with this conviction: that leaders such as you have the potential to be the most influential forces on planet Earth. Yours is the staggering responsibility and the matchless privilege of rallying believers and mobilizing their spiritual gifts in order to help people who are far from God become fully devoted followers of Christ. Life transformation and the eternal
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Why He Is So Much Better Than Trying Harder, Doing More, and Being Good EnoughAbandon dead, dry, rule-keeping andembrace the promise of being truly known and deeply loved. JeffersonBethke burst into the cultural conversation in 2012 with a passionate,provocative poem titled "Why I Hate Religion, But Love Jesus." The 4-minutevideo literally became an overnight sensation, with 7 million YouTube views inits first 48 hours (and 23+ million in a year). The message blew up
Connecting with Your Husband Mind Body Heart SpiritMaximize the sexual and emotional potential in your marriage! With down-to-earth wisdom based on the experiences of the thousands of women she's counseled, Shannon Ethridge–author of the million-plus-selling Every Woman's Battle series–shows women how to create the healthy, exhilarating sex lives they (and their husbands) desire.Every woman deserves to enjoy great sex with her husband, without


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Calendar of Events
- Saturday, 22 October, 2016
- APIEL - Appalachian Public Interest Environmental Law Conference 9:00 AM
- Sunday, 23 October, 2016
- APIEL - Appalachian Public Interest Environmental Law Conference 9:00 AM
- Thursday, 06 October, 2016
- Southeastern Medieval Association Conference 12:30 PM
- Wednesday, 26 October, 2016
- Trunk or Treat 6:00 PM
- Tuesday, 04 October, 2016
- Poverty, Inc. 7:00 PM
- Wednesday, 19 October, 2016
- A Night at the Museum: Careers in Museums, Archives, and Libraries 4:00 PM
- Tuesday, 18 October, 2016
- Physics Open House for High School Students 9:00 AM
- Wednesday, 16 November, 2016
- 13th Annual Riggsby Lecture on Medieval Mediterranean History and Culture 5:30 PM
- Tuesday, 18 October, 2016
- Anthropology Lecture on Disease: Ingesting Sacred Soils: Beyond 'dirt-eating' as disease or geophagia as biological instinct in Caribbean Studies 8:10 AM
- Wednesday, 26 October, 2016
- Class in Session: Careers in Education through Non-Traditional Means 4:00 PM
- Thursday, 13 October, 2016
- Chemistry 501/ ETS-ACS S.C. Lind Lecture - UT/ORNL Presentations 5:00 PM
- Thursday, 20 October, 2016
- Information Session: Learn about Degrees, Careers in Design 12:30 PM
- Saturday, 08 October, 2016
- Fabulous Fall Plant Sale 9:00 AM
- Wednesday, 05 October, 2016
- Child and Family Studies Fall Colloquium Series 3:00 PM
- Friday, 28 October, 2016
- The SPS 2nd Annual Pumpkin Drop 4:30 PM
- Tuesday, 04 October, 2016
- Science, Policy, and the Election 6:30 PM
- Friday, 07 October, 2016
- Fabulous Fall Plant Sale Preview 4:00 PM
- Friday, 14 October, 2016
- Wednesday, 16 November, 2016
- 13th Annual Riggsby Lecture on Medieval Mediterranean History and Culture 5:30 PM
- Tuesday, 21 February, 2017
- Haslam College of Business Employer of the Day - PYA 10:00 AM
Tours of high-performance computing facility for UT faculty and students
The National Institute for Computational Sciences (NICS) would like to offer tours of our High Performance Computing (HPC) resources located at the Oak Ridge National Laboratory.
NICS is one of the leading HPC centers for excellence in the United States. NICS is co-located on the campus of UT as well as at ORNL, hosting the world's most powerful computing complex. The center's mission is to expand the boundaries of human understanding while ensuring the United States' continued leadership in science, technology, engineering, and mathematics. NICS strives to accomplish this mission by facilitating transformational scientific discoveries, and by providing scientists and researchers from around the world with leadership-class HPC resources, facilities, and support.
Transportation to ORNL will be available from UTK campus. You will get the chance to see some of the most powerful supercomputers in the world, talk with HPC Consultants, and get more information, including applying for HPC resources, using the computers, data analysis, and scientific computing assistance. If you are looking for resources promptly for your research applications or your big data analysis, you surely want to join us in this event!
Seats are limited. If you are interested, please register online
https://www.surveymonkey.com/s/UTK-NICS .
Hong Liu
HPC Consultant
National Institute for Computational Sciences
Friday, 31 October, 2014
Other Dates For This Event:
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Pooling assets from KSU
Could the pools be kept open longer?
By The Mercury
It struck us this weekend in City Park that two terrific Manhattan assets really ought to be paired together: K-State students and City Pool.
Read More ...
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Show Summary Details
Journal of the International Association for Semiotic Studies / Revue de l'Association Internationale de Sémiotique
Editor-in-Chief: Danesi, Marcel
SCImago Journal Rank (SJR) 2015: 0.275
Source Normalized Impact per Paper (SNIP) 2015: 0.661
Impact per Publication (IPP) 2015: 0.191
- Online
- 1613-3692
On the Spatial and Cultural Characteristics of Courtly Romance
Citation Information: Semiotica. Volume 20, Issue 1-2, Pages 1–38, ISSN (Online) 1613-3692, ISSN (Print) 0037-1998, DOI: 10.1515/semi.1977.20.1-2.1, September 2009
Publication History
- Published Online:
- 2009-09-29
Citing Articles
Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the "Citation Alert" on the top of this page.
Cesare Segre
Russian Literature, 1997, Volume 41, Number 3, Page 385
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Joe Bonamassa You & Me
LOW STOCK: Only 2 copies left
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CD Details
- Released: January 26, 2009
- Originally Released: 2009
- Label: Premier Artists
Song previews provided courtesy of iTunes
Product Description:
Personnel: Joe Bonamassa (vocals, guitars); Rick Melick (piano, organ, tambourine); Carmine Rojas (bass instrument); Jason Bonham (drums).
Additional personnel: Doug Henthorn (vocals); Pat Thrall (guitar); Jeff Bova (programming).
Despite his statement in the liner notes that "In an era where it is best to play it safe, I chose to take a risk...," there isn't much surprising or risky about young guitarist Joe Bonamassa's fifth studio album. Most of his previous releases have mixed blues covers with his own originals, all played with a rocker's attitude, volume and less-than-subtle approach. This one follows suit and even though he goes on to say that he "wanted to make a blues album, not a rock album that has blues on it," as in the past; it's impossible to claim that he has succeeded with You & Me. That doesn't make this a bad or disappointing disc; quite the contrary, it's a solid blues-rock release and arguably his best work to date. But as early as the second track, an original rocker titled "Bridge to Better Days," Bonamassa takes off on an early Free/Savoy Brown-styled stomper. Things settle down and get more rootsy on the following two slow blues tracks, although a lovely Bonamassa original, "Asking Around for You," adds strings, not exactly a touch most would associate with pure blues. Regardless, it's extremely effective and when the strings return on a nine-and-a-half-minute cover of Led Zeppelin's "Tea for One," it is a spine-tingling experience and possibly this album's finest moment. Drummer Jason Bonham, who is excellent throughout, brings additional authenticity to the song his dad first played on. Bonamassa unplugs for a few mid-disc tracks, including a cover of "Tamp 'Em Up Solid" (oddly credited to Ry Cooder but typically known as a traditional piece, even on Cooder's version). Twelve-year-old harmonica whiz L.D. Miller does his best John Popper imitation on a hyperactive version of Sonny Boy Williamson's "Your Funeral and My Trial" (someone needs to inform the kid that playing lots of notes really fast doesn't mean he has soul), and the instrumental titled "Django" shows that Bonamassa has been listening to Gary Moore's "Parisienne Walkways." It adds up to a quality Bonamassa disc that will please existing fans and might bring some new ones into the fold, but it's also one that doesn't take the chances that he claims might push the guitarist into uncharted territory. ~ Hal Horowitz
Joe BonamassaThe Ballad of John Henry
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Precipice Trail is a 1.5 mile heavily trafficked out and back trail located near Bar Harbor, ME that features beautiful wild flowers and is rated as difficult. The trail offers a number of activity options and is accessible from March until November.
nature trips
rock climbing
trail running
wild flowers
no dogs
The Precipice Trail is a difficult and very steep out and back (or loop) trail using iron rungs and ladders on exposed cliffs providing spectacular views of Acadia National Park. The trail opens after nesting Peregrine Falcons leave the nest. Do not recommend this hike during wet conditions, or if you experience vertigo; also not recommended for small children (no pets). The trail starts up steps and through some boulder scrambles. While officially a non-technical hike due to strategically placed iron rungs, ladders and rock ledges, this is really a climb that requires you to be able to navigate some tricky spots by pulling yourself up (e.g. the very first example of this might even be the hardest). If you are careful and pay attention, this will be a great and thrilling experience. Leave hiking poles behind, as you won't need them and they'll just get in the way. One of the very best short hikes in Acadia, similar to what you get on Katahdin's Knife Edge.
This is a partly technical trail that does parallel ridge edge traverses and ladder and rung climbing. I highly recommend this trail for anyone not afraid of heights. There are rock scrambles and beautiful views.
You need to be able to lift yourself up, climb cliff face ladders and have an adventurous spirit.
Don't let the descriptions scare you off. You need to be careful. Beyond that it is a blast.
Go for it!
Our group are all in their late sixties. No probs!
You get such a feeling of accomplishment from completing this trail, and get some beautiful views.
Absolutely the most fun hike my family and I have ever attempted. The views along the way are amazing. The iron rung sections are challenging, but certainly well placed. My 14 and 11 year olds scrambled up here with no problem, but it is not for everyone. Just be aware of your party's limitations (the 'warning' rock is very useful in helping to determine comfort level). Step carefully and enjoy the view!
Seems a little harder than moderate.
Amazing hike! Rated as moderate but should definitely be rated as hard.
Awesome hiking spot, awesome view .
Do not go if you have fear of heights
Fantastic!! Went with our teenagers. We all loved it.
This is the closest "hiking" you'll get to mountain climbing. The app shows this hike as moderate but it should be rated hard. This is not for the average couch potato. Some people turned around but my wife and I enjoyed it a lot. Spectacular must do trail.
Amazing and terrifying! I'm afraid of heights and some of the areas (especially near the top) almost put me in tears, but it was so worth it and so rewarding. Take it slow. It's not a hard climb, just a hairy one.
Awesome views! Very challenging hike with intense rung sections.
Whew...that last quarter mile to the top is quite the hair raising experience....walking along the ledge with just the iron hand rail. It had my daughter (15 yrs) nervous! We made it and took the easy way down. Lots of climbing!
It currently is open. I liked the trail a lot!


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A Houston-area man is suing Harris County over the evidence dump investigation at the Harris County Precinct 4 Constable's Office. David Bellamy spoke exclusively to KHOU 11 News Tuesday night.
Bellamy's case was dismissed and a lot of people might think it's a victory for him, but he spent time in jail based on evidence the Harris County District Attorney's Office didn't have.
"We were picking a jury that day," said Bellamy. "I thought my life was over. It pretty much ruined the whole family."
MORE: Attorney calls for federal investigation of Pct. 4, DA's office
It was his case that cracked the investigation into missing evidence wide open. It was later discovered in March that a deputy constable destroyed more than 21,000 pieces of evidence while cleaning out Precinct 4's evidence room. Precinct 4 Constable Mark Herman said that deputy constable has been fired.
"That should never happen, man, not to a legal system that's supposed to be just," he said.
Bellamy doesn't try to hide his past, he's a convicted felon.
"I got out in 2012, I haven't been in trouble since," he said.
The district attorney's office pushed for a plea deal in the case, but Bellamy maintained his innocence. Last year, he was pulled over and arrested on drug charges.
"I can't get a bond for months, lost my job, my wife had no way of even supporting her, kids left out there," he said.
Bellamy says prosecutors kept offering plea deal after plea deal, but when his attorney finally asked to see the evidence, there was nothing there.
"Why should the government be held to a lower standard than ordinary people? I can't try to induce you to do something that I know is based on false information. That's called fraud," said Joe Jones, the attorney now representing Bellamy in a civil lawsuit.
The lawsuit holds Harris County and District Attorney Devon Anderson responsible.
"They knew they couldn't try the case, they knew the evidence didn't exist and they pursued prosecution against this man for months," said Jones.
Now, Bellamy is a free man, but left wondering how many others took plea deals based on evidence that was dumped.
"They shouldn't take the law into their own hands and take action against people just because they think they're vulnerable or in a bad spot," said Bellamy.
We are still waiting to hear exactly how many cases were impacted. Bellamy's attorneys believe they never would have known if they hadn't pressed to see this evidence.


text:
Curious George sitting among cakes! The cake in front of George is a 10" round. He is about 20" tall. There are two medium size cakes behind him plus cupcakes. George's torso is cake.
CakeCentral.com is the world's largest cake community for cake decorating professionals and enthusiasts.
(if this is a private computer)
Yes please, register now!


text:
President Obama Visits Baton Rouge After Criticism That He's Late
President Obama tours Louisiana's communities affected by the flood that damaged over 60,000 homes and killed 13 people.
It's hard to imagine things getting any worse for Jimmy "Henchman" Rosemond, but charges against the fugitive mogul keep piling up.
After an imprisoned man came forward earlier this week claiming responsibility for the 1994 shooting of Tupac Shakur and fingering Rosemond as the one who put out the hit, new allegations have surfaced that Rosemond orchestrated the 2009 murder of another man who was a known associate of 50 Cent's G-Unit rap crew. Rosemond, the president of Czar Entertainment and manager of Game and Sean Kingston, is currently a fugitive from federal marshals—he went on the run in May after being indicted for leading a cocaine distribution ring.
Rosemond associates Rodney Johnson and Brian McCleod were recently charged with the murder of the G-Unit affiliate, Lowell Fletcher. In 2009 Fletcher had recently been released from prison for assaulting Rosemond's 14-year-old son when he was shot to death while walking down the street in the Bronx. At the time, G-Unit was involved in a beef with Czar Entertainment, which represented their rival, Game.
Rosemond's lawyer, Jeffrey Lichtman, denies his client's involvement in the Fletcher murder, releasing the following statement to MTV News: "The only 'evidence' tying that murder to Jimmy Rosemond is the uncorroborated word of a criminal seeking to get out from under a lengthy jail sentence. Prosecution witnesses have been bribed and threatened, solely in an effort to convict Jimmy, a target of the US Attorney's office for years."
Recently, Lichtman called allegations by Dextar Isaac that he was given $2,500 from Rosemond to shoot Tupac a "flat-out lie." The embattled executive has not been heard from since May.
(Photo: Johnny Nunez/WireImage)
President Obama tours Louisiana's communities affected by the flood that damaged over 60,000 homes and killed 13 people.
The only person that can send Gatlin home without the gold medal? A young man from Trelawney, Jamaica, whose last name literally means to run.


text:
Three more names to Nebraska injury list
Big Red Report Top Stories
Cotton recaps Oregon visitSacramento (Calif.) Inderkum defensive back Trajon Cotton, a Nebraska prospect, talked about his official visit to Oregon and his plans for his next few visits..
Scout Football12:15 PM
Former Nebraska assistant passes awayFormer longtime Nebraska assistant coach Milt Tenopir has passed away, according to multiple tweets from those inside the Nebraska football program.
Big Red ReportYesterday at 7:45 PM
Updated Scout 300 Trend Meter For 2017The updated Scout 300 Trend Meter for the 2017 class takes a look at which schools are trending for each uncommitted member in the Scout 300.
Scout FootballYesterday at 11:15 AM
Nebraska live presser updates (week 5)Live Nebraska Monday morning press conference updates.
Big Red ReportYesterday at 10:44 AM
Farmer and Moore doubtful for SaturdayNebraska wide receiver Alonzo Moore and offensive lineman Tanner Farmer are doubtful for this Saturday against Illinois.
Big Red ReportYesterday at 10:35 AM