text:
|Publication number
|Publication type
|Application number
|Publication date
|Feb 14, 1984
|Filing date
|Dec 17, 1982
|Priority date
|Dec 14, 1981
|Fee status
|Publication number
|Kurt S. Stenn
|Original Assignee
|Research Corporation
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (3), Non-Patent Citations (8), Referenced by (16), Classifications (15), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
______________________________________ No. of amino acids No. of amino acidAmino per protein molecule molecules per 1000Acid 62,752 daltons amino acids______________________________________Asp 62 111Thr 40 72Ser 46 81Glu 67 119Pro 19 35Gly 26 46Ala 43 77Val 33 59Met 15 26Ile 24 44Leu 69 123Tyr 12 22Phe 31 56His 12 21Lys 34 60Arg 21 37Trp 4 7Cys 2 4;______________________________________
This application is a continuation-in-part of application Ser. No. 330,396, filed Dec. 14, 1981, now abandoned which is a continuation of application Ser. No. 244,963, filed Mar. 18, 1981, now abandoned which is a continuation of application Ser. No. 135,631, filed Mar. 31, 1980, now abandoned.
It has long been suspected, and earlier studies have confirmed, that there is a specific factor or activity in mammalian serum and plasma which supports epidermal cell movement. [Stenn, British Journal of Dermatology, Vol. 98, pages 411 (1978); Stenn et al., Arch. Dermatol. Res., Vol. 264, pages 3-15 (1979); U.S. Pat. No. 3,948,875].
Recently efforts have been directed toward the characterization of the epidermal cell migration promotion activity in serum. See Levene, M., Br. J. Dermatol., Volume 86, pages 481-490 (1972); Coombs, V. A., Nissen, B. K. and Marks, R., Arch. Dermatol. Forsch, Vol. 249, pages 367-372 (1974); Stenn, K. S. and Dvoretzky, I., Arch. Dermatol. Res., Vol. 264, pages 3-15 (1979); Mitrani, E. and Marks, R., Br. J. Dermatol., Vol. 99, pages 513-518 (1978); Griffiths, G., Jones, H. M., Cryer, J., Kay, J. and Marks, R., J. Invest. Dermatol., Vol. 46, 430 (abstr.) (19781); and Ooka, H., Yamauto, K., Okuma, Y., Suga, S. and Wakasugi, M., Exp. Gerontol., Vol. 10, pages 79-83 (1975). Mitrani et al., for example, describe a method for fractionating serum by combining ammonium sulfate precipitation and molecular sieve chromatography.
Other spreading factors have been described [Lipton, A., Klinger, II, Paul, D. and Holley, R., Proc. Natl. Acad. Sci. U.S.A., Vol. 68, pages 2799-2801 (1971); Wolf, L. and Lipton, A., Exp. Cell. Res., Vol. 80, pages 499-502 (1973); Burk, R. R., Proc. Natl. Acad. Sci. U.S.A., Vol. 70, pages 369-372 (1973); R. R. Burk, Exp. Cell. Res., Vol. 101, pages 293-298 (1976); Wall, R. T., Harker, L. A. and Striker, G. E., Lab Invest., Vol. 39, pages 523-529 (1978); Zetter, B. R., Nature (London), Vol. 285, pages 41-43 (1980); and Leonard, E. T. and Skeel, A., Exp. Cell. Res., Vol. 102, pages 434-438 (1976)].
Recently a serum spreading factor for fibroblasts was isolated by Knox et al. (Exp. Cell. Res., Vol. 123, pages 421-424 (1979)) and Whateley et al. (Biochem. J. Vol. 185, pages 349-354 (1980)) from fetal calf serum. The protein differs from the protein of the present invention by origin, purification and chemical properties and activity.
It is an object of the present invention to provide an epithelial cell movement enhancing protein and a method for its preparation.
The present invention provides a novel protein which enhances epithelial cell movement, in vitro and in vivo. The protein may be derived from human serum or plasma.
The protein is a single-chained, glycosylated, trypsin digestible, α2 serum globulin of apparent molecular weight 62,000 daltons, and an N-terminal amino acid sequence of ASN-SER-PRO-LEU-ASP-GLU. It is resistant to the protease inhibitors, diisopropyl fluorophosphate and phenyl methyl sulfonyl fluoride. It has an isoelectric point of 5.1-5.2, and an extinction coefficient of about 1. Antibody to the purified protein blocks all the epidermal cell spreading activity of human plasma (or serum); in addition, immunoelectrophoresis of the antisera against whole plasma (serum) reveals a single protein arc.
The protein may be prepared from human plasma or serum by ammonium sulfate fractionation from human serum or plasma and purified by ion-exchange and gel filtration chromatography, isoelectric precipitation and preparative polyacrylamide gel electrophoresis.
The present invention also provides an additional protein derived from human plasma or serum which does not itself enhance epithelial cell movement but does synergistically enhance more than 10-fold the epithelial cell movement activity of the above-described protein. The epithelial cell movement enhancing protein of the present invention will be referred to hereinafter as the "protein". The protein which enhances the epithelial cell movement activity of the "protein" will be referred to hereinafter as the "co-factor."
The protein of the present invention supports: (a) dissociated guinea pig epidermal cell spreading in vitro; (b) mouse ear skin epidermal sheet spreading in vitro; and (c) swordtail fish epidermal sheet spreading in vitro.
The purified protein will support cell spreading at a concentration of 10-20 μgm/ml if no other protein is present. If bovine serum albumin is present at 0.5 mg/ml, it will support spreading at 1 μgm/ml.
That the active factor is a protein is further substantiated by the following facts: (a) its spreading activity co-migrates on electrophoresis with Coomassie Blue-protein staining band; (b) its activity is destroyed by trypsin; (c) its amino acid composition is typical of proteins; (d) its activity corresponds to a molecule with the amino terminal sequence of NH2 -ASN-SER-PRO-LEU-ASP-GLU.
The protein is glycosylated as demonstrated by its periodic acid-Schiff staining on sodium dodecyl polyacrylamide gel electrophoresis and its binding to sugar-affinity lectin (Concanavalin B) resin. The protein is a single chain protein having an apparent molecular weight of approximately 62,000 daltons.
The protein also has the following electrophoretic properties; (a) it has an isoelectric point between 5.1-5.2; (b) compared to other human serum proteins it travels on immunoelectrophoresis with the α2 globulins.
The protein has the following amino acid content:
______________________________________ Residues per 1000 Residues perAmino Acid amino acids 62,752 daltons______________________________________CYS 4 2ASP 111 62THR 72 62SER 81 46GLU 119 67PRO 35 19GLY 46 26ALA 77 43VAL 59 33MET 26 15ILE 44 24LEU 123 69TYR 22 12PHE 56 31HIS 21 12LYS 60 34ARG 37 21TRP 7 4______________________________________
Standard procedures for automated amino acid analysis with a DURRUM D-500 amino acid analyzer were used to determine the amino acid composition. The protein was hydrolyzed in 6 N HCL under vacuum for 20, 48, 72 and 110 hours. Cysteine residues were determined by performing acid oxidation according to the method of Moore (J. Biol. Chem. Vol. 238, pages 235 (1962)). Tryptophan residues were determined by mercaptoethanesulfonic acid hydrolysis according to the procedure of Penke, B., Fesencz, R., and Kovacs, K. (Anal. Biochem., Vol. 60, page 45 (1974)).
The amino acid sequence of the N-terminal of the protein was determined automatically using the Beckman 890C sequence with a Sequemat P-6 autoconverter. Derivative analysis was conducted using a Water Associates HPLC with an automatic injector and programmed gradient elution. Norleucine was used as the internal standard, with the following results:
______________________________________ PredominantCycle Number Amino Acid Concentration______________________________________1 Asparagine 1.57 μmoles2 Serine 0.37 μmoles3 Proline 1.81 μmoles4 Leucine 2.62 μmoles5 Aspartic 1.27 μmoles6 GLutamic 1.73 μmoles______________________________________
The determined amino terminal amino acid sequence is then NH2 -ASN-SER-PRO-LEU-ASP-GLU.
The extinction coefficient of the purified 75 kd protein was measured experimentally as about 1.0 using the Lowry assay (J. Biol. Chem., Vol. 193, page 265 (1951) with bovine serum albumin as the standard. The determination indicates that 1 mg/mg solution of the purified protein has an absorbance of about 1.0 when using an ultraviolet wave length of 280 nm. The protein is prepared by:
(1) diluting mammalian plasma or serum with water, adjusting the pH thereof and dissolving therein sufficient ammonium sulfate to produce an approximately 33.3% saturated solution and removing the thus formed precipitates;
(2) dissolving in the supernatant sufficient ammonium sulfate to produce an approximately 66.7% saturated solution;
(3) removing the resultant precipitate from said solution, suspending it in water and dyalyzing the suspension against a buffered solution having a pH of about 6.0;
(4) placing the dialyzed fraction in a buffer solution and subjecting the solution to anionic chromatographic separation;
(5) eluting the chromatographically adherent fraction with an aqueous buffered solution having a pH of about 6.0 and an ionic strength of about 12 mmhos;
(6) dialyzing the eluted fraction against an aqueous buffer solution having a pH of about 6.0;
(7) subjecting the dialysand to isoelectric precipitation; and
(8) subjecting the separated fraction to electrophoresis.
The "protein" enhances epithelial cell movement, in vivo and in vitro, and is useful in such applications as wound-healing, etc.
The "co-factor" increases the epithelial cell movement enhancement activity of the "protein" more than 10-fold, when combined with the "protein.
The invention is further illustrated by the following non-limiting examples wherein: All buffers were made 0.02% in NaN3 and 0.1 mM in phenylmethylsulfonyl fluoride. Dialysis bags were prepared as described [Stenn, K. S. and Blout, E. R., Biochemistry, Vol. 11, page 4502 (1972)]. In all tissue culture experiments Dulbecco's modified Eagle's medium (DME medium) (GIBCO) containing antibiotics was used. The cultures were incubated at 35�-38� C. in 95� air/5% CO2, in a water vapor-saturated environment. In a typical experiment the fractions to be tested were dialyzed against 0.1 mM Tris.HCl(pH 8.0) plus 100 mM NaCl then added to an appropriate volume of 2�DME medium and sterilized by Millipore filtration (0.45 μm pore diameter).
Human Plasma from normal donors 1-2 weeks after bleeding was cleared by centrifuging at 5000�g for 20 min. at 4� C. At 4� C., saturated solution of (NH4)2 SO4 was added to 1 liter of plasma. The precipitate occurring at 33.3% saturation was discarded after centrifugation (5000�g, 20 min., 4� C.). The 66.7% (NH2)2 SO4 precipitate was retained, solubilized in 400 ml of distilled water, and dialyzed against four changes of 4 liters of 50 mM Na2 HPO4 /NaH2 PO4 (pH 6.0) for 24 hrs. The dialysand was added to a DEAE Bio-Gel A (100-200 mesh; Bio-Rad) column (8�8�23 cm) equilibrated with the dialysis buffer and the column was washed until the effluent had an absorbance at 280 nm of less than 0.05. The product of this washing step is the above noted "cofactor." The active fraction was eluted stepwise with 50 mM NaH2 PO4 /Na2 HPO4 (pH 6.0) plus 100 mM NaCl, pooled, and dialyzed against three changes of 4 liters of 0.5 mM NaH2 PO4 /Na2 HPO4 (pH 6.0) for 24 hrs. The dialysand was cleared by centrifugation and titrated to pH 5.3 with 0.1 mHCl with slow stirring. The supernatant was cleared by centrifugation and again titrated to pH 4.6. The precipitate was collected by centrifugation and then dissolved in 24 ml of 50 mM NaH2 PO4 /Na2 HPO4 (pH 6.0) plus 100 nM NaCl. This isoelectric precipitation step was then repeated identically. The isolation precipitate solution was loaded onto a gel filtration column (Sephacryl S 200, Pharmacia 5�90 cm, equilibrated with 50 mM NaH2 PO4 /Na2 HPO4 pH 6.0 plus 100 mM NaCl). The active fraction from gel filtration (approximately 4 mg protein) was applied to a polyacrylamide large slab gel (0.5�12�16 cm) (Davis, Ann. N.Y. Acad. Sci. Vol. 121, page 404 (1964)) across which a potential of 110 V was established. The active fraction ran with an Rf value of 0.5-0.57. The active fraction was cut out and run on a second identical gel but following the conditions of Laemmli [Nature, Vol. 227, pages 680-685 (1970) 15% acrylamide]. The protein travelled as the only diffuse band on this gel. The protein was eluted from this gel electrophoretically at 100 volts using an apparatus described by Doly and Petek [J. Chromatography, Vol. 137, pages 69-81 (1977)]. The protein yield of this elution step was 2%. The eluate was washed with 50 mM NaH2 PO4 /Na2 HPO4 (pH 6.0) plus 100 mM NaCl, concentrated (Amicon filter PM-30), and stored at room temperature.
Using the method of Laemmli (Nature, Vol. 227, page 680 (1970)) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of 12.5% acrylamide the purified preparation shows a single, but somewhat diffuse, Coomassie-Blue staining band. After reduction with mercaptoethanol the mobility does not change. Compared to the electrophoretic mobility of standard proteins (phosphorylase b, 94 kd; bovine serum albumin 67 kd, ovalbumin 43 kd, carbonic anhydrase 30 kd, soybean trypsin inhibitor 20.1 kd; lactalbumin 14.4 kd) the cell spreading protein has a molecular weight of about 62 kd.
Amino-terminal amino acids analysis of the protein conducted by Edman degradation using an automated amino acid sequenator revealed one N-terminal amino acid, asparagine.
Exposing the purified fraction to a 0.5% trypsin solution for 15 min. at 37� C. led to complete destruction of spreading activity, thereby supporting the conclusion that the activity is a protein.
The yield of Example 1 is given in Table 1. Virtually all the spreading activity was present in the 66.7% (NH4)2 SO4 precipitate. In step 3 the dialyzed active ammonium sulfate fraction was placed on an anion-exchange resin (DEAE-Bio-Gel), washed copiously, and step eluted with 0.1 M NaCl. This fraction, when assayed alone, had no greater specific activity than the material placed on the column, even though considerable inactive protein had been removed. This observation suggested that a second component had been removed in the wash. Indeed, as shown in Table 1, adding the wash which contains the "co-factor" from the ion-exchange step (at a protein concentration of 0.5 mg/ml) to the assay enhanced specific activity more than 10-fold. The action fraction, purified further by isoelectric precipitation at pH 4.6, was poured over a gel filtration column and the active fractions were pooled. Finally, this concentrated preparation was separated in a preparative electrophoresis step. In this system the spreading activity travelled with an Rf value of about 0.50-0.57.
TABLE 1______________________________________ Specific Activity Assayed Assayed with Yield*Step alone cofactor %______________________________________1. Human plasma 195 220 1002. 66.7% saturated (NH4)2 SO4 946 1,357 112 precipitate3. Ion-exchange chromatography 684 11,765 73 fraction4. Isoelectric precipatate 1167 16,250 32 (pH 4.6)5. Gel-filtration fraction 1510 37,302 106. Preparative electrophoresis -- 60,000 2 fraction______________________________________ The dissociated epidermal cell spreading assay was used. *Yield assaus were determined in the presence of cofactor.
Three different epidermal cell spreading assays were employed: dissociated epidermal cell, explant outgrowth, and epiboly. For the dissociated epidermal cell assay 0.4-mm-thick sheets of truncal skin were removed from deeply anesthetized (ether) 200-250-g Hartley male guinea pig by using a dermatome after clipping the fur and treating with a depilatory cream. Epidermis (without dermal contamination, as judged by light microscopy) was stripped from this preparation after incubation at 37� C. for 30 min. to trypsin (GIBCO no. 7010) at 0.5 mg/ml in calcium and magnesium-free Tyrode's solution. Epidermal cells were shaken and scraped from the epidermis, centrifuged, suspended in DME medium, and counted (hemocytometer). Approximately 2�105 cells suspended in DME medium were placed in each Petri dish (34�10 mm, Falcon), and incubated at 37� C. After 2 hr the supernatant medium was discarded and replaced by test medium. At this time 10% of the cells were adherent but none were spread. After incubating 4-20 hrs in the test medium the number of spread cells per 100 attached nonkeratinized cells counted was determined by staining the cells with Giemsa stain, Stenn et al., supra. The number of spread cells divided by the protein concentration (mg) of the spreading fraction in the growth medium was expressed as spreading units. A cell was considered spread if its cytoplasm was distributed on the substratum in a radial or polar pattern, Vasiliev, J. M. and Gelfand, I. M., Int. Rev. Cytol. Vol. 50, pages 159-274 (1977). For the epidermal outgrowth assay and the epiboly studies the procedures of Stenn and Dvoretzky, supra, were followed.
Briefly, for the epidermal cell spreading assay mouse ear skin disks were laid onto plastic tissue culture Petri dishes and held down by a fibrin clot. The explants were gently overlaid with culture medium (DME plus the test fraction) and incubated for 3 days. At this time the maximal radial epidermal outgrowth from the explant edge was measured. For the epiboly experiments mouse ear skin explants were floated upon tissue culture medium (DME plus the test fraction) for 2 days and then Bouins, fixed, embedded, sectioned and stained histologically as previously described by Stenn et al., supra. The extent of epidermal movement about the lower surface of the explant was measured and expressed as a percent of lower surface covered by epithelium.
That spreading activity is not a property of all fractions in plasma is apparent from FIG. 1 which depicts the results of a gel filtration experiment. Virtually all spreading activity (with all three assays) was associated with a retained, smaller molecular weight, fraction. FIG. 1 represents an elution profile of human plasma by gel filtration and the identification of spreading activity. Five milliliters of whole human plasma was filtered through a 5�90 cm Bio-Gel P-200 column in a buffer of 0.01 M NaH2 PO4 /Na2 HPO4 (pH 8.0)+0.1 M NaCl. Five-milliliter fractions were collected at a rate of 12 ml/hr. Each 10 tubes were pooled and tested by each of the three spreading assays dissociated cell assay (open bars) nonquantitated explant spreading (+), and epiboly (++), , A280. The abscissa represents fraction number minus 160.
FIG. 2 depicts the gel filtration assay data of the active isoelectric precipitate. The gel (Sephacryl S-200, Pharmacia) was packed into a 5�90 cm column and equilibrated with 50 mM NaH2 PO4 /Na2 HPO4 (pH 6.0)+100 mM NaCl. The 3-ml sample contained 50-60 mg of protein. The column was developed at the rate of 36 ml/hr and 3-ml fractions were collected. Protein elution was measured by absorbance at 280 nm () and spreading activity by the guinea pig dissociated epidermal cell assay ().
FIG. 3 depicts the polyacrylamide gel electrophoretic pattern of the ultimately purified protein and comigrating spreading activity. The Davis gel of the final step material is shown along the abscissa with the corresponding spreading activity recorded on the ordinate.
On SDS-PAGE electrophoresis the purified protein stains positive with the periodic acid-Schiff procedure following the method of Fairbanks et al. (Biochemistry, Vol. 10, page 2606 (1971) indicating that the protein is glycosylated. Additional evidence that the protein is glycosylated is that the epidermal spreading activity of serum is completely adsorbed by a sugar-binding lectin column (Concanavalin B-Sepharose Pharmacia, 50 mM NaH2 PO4 /Na2 HPO4 (pH 6.0) and 100 mM NaCl).
Antibody to the protein was produced in rabbits by injecting subcutaneously the purest preparation suspended in Freund's adjuvant. Protein (500 μgm/animal) was injected every 2 weeks over a 6 weeks period. Two weeks after the final injection blood was collected and serum prepared by allowing the blood to clot. The serum was made 40% saturated in ammonium sulfate and centrifuged. This immunoglobulin-rich precipitate fraction was dialyzed against phosphate buffered saline.
This antiserum was analyzed by immunoelectrophoresis using standard procedures. Briefly a 1% agarose gel in sodium barbital buffer pH 8.6 was layered over a glass slide. On each side of the slide a 2 mm diameter well was made and proteins were added. The preparation was placed across an electric potential for 1 hour at 4� C. Afterwards a long trough was made between the wells into which antiserum was added. This preparation was allowed to diffuse overnight at 24� C.
By immunoelectrophoresis the antiserum gave a single arc when run against the purified protein or whole human plasma or human serum. The antiserum was mixed with antihuman albumin and the immunoelectrophoresis repeated. The antiserum was also run with anti α1 antitrypsin or anti α2 macroglobulin.
By immunoelectrophoresis, the protein was shown to travel slower than albumin and α1 anti trypsin and slightly faster than α2 macroglobulin. By this technique its electrophoretic mobility was determined to be in the α2 globulin region of human serum protein.
Using the techniques of immunodiffusion and immunoelectrophoresis with purified protein, antibody, and serum proteins and their antibodies, no cross-reactivity (i.e., immunological identity) was found with human albumin, fibronectin, laminin, α2 macroglobulin, α1 antitrypsin, β2 microglobulin, and collagens (Type I, III, IV, V).
Adding the immunoglobulin fraction of the anti-serum to whole human plasma blocked the epidermal spreading activity of plasma. The immunoglobulin fraction of the antiserum was isolated by ammonia sulfate precipitation at 30% saturation. This fraction was then diluted and added to the culture media with various concentrations of human plasma (HP). The spreading of dissociated guinea pig epidermal cells was measured (Table 2):
TABLE 2______________________________________ Dissociated Epidermal Cell SpreadingMedium (% � 1 S.D.)______________________________________Medium 199 alone 0 � 0 0 � 0Medium 199 � antisera (1:10) 0 � 0 0 � 0Medium 199 + HP (1:500) 46 � 7 46 � 7Medium 199 + HP (1:500) + antisera (1:10) 0 � 0 0 � 0Medium 199 + HP (1:500) + antisera (1:100) 14 � 10 14 � 0Medium 199 + HP (1:500) + antisera (1:1000) 45 � 7 45 � 7______________________________________
The above experiments indicate that there exists in human blood a single soluble protein which supports epithelial cell spreading and that the latter property is not a function of the totality of serum or plasma but corresponds to a specific protein. This protein supports single cell spreading as well as epithelial sheet movement. It is active at concentrations as low as 1.3 μgm/ml and, although it is active alone, it requires a second non-dialyzable component for maximal activity. The augmenting "co-factor" enhances the spreading activity by about 13�.
A theory as to the mechanism of action of this factor may be formulated from an interpretation of the three assays. In one assay individual cell cytoplasm spreading is measured; in the other two epithelial sheet movement is measured. The significant difference between the latter two assays is the substratum upon which the cell sheets travel. Earlier studies (Stenn, Brit. J. Derm., Vol. 98, pages 411-416 (1978); Stenn et al. (Biochemistry, Vol. 11, pages 4502-4515 (1972) had indicated that epithelial cell movement in the outgrowth and epiboly assays occurred, at least for the first three days, even after lethal irradiation. The above experiments indicate that the phenomena in these systems is cell movement, and to a lesser extent, cell division. The aspect of cell movement involved (adhesion, cytoskeleton structuring, metabolism, etc.) and how the effect occurs is, at present, unknown. Because the factor supports maximal spreading in all three systems, it is believed that the same phenomenon occurs in the three assays.
Factors supporting the movement of other types of cells have been described. The epidermal growth factor of Cohen, J. Biol. Chem., Vol. 237, page 1555 (1962), is a small, heat-stable molecule unlike the factor or protein of the invention. Fibronectin has been reported to support the spreading of many different cell types (Ali et al., Cell, Vol. 14, pages 439-446 (1978)), but in the mature epithelial systems used here fibronectin is not active. Other workers have studied serum factors which stimulate non-epithelial cell movements in vitro (Burk, Proc. Nat. Acad. Sci., Vol. 70, page 369 (1973); Burk, Exp. Cell Res., Vol. 101, page 293 (1976); and Lipton et al., Proc. Nat. Acad. Sci., Vol. 68, page 2799 (1971)). These studies, however, have been phenomenological so that a molecular comparison is not yet possible.
In view of the fact that the earliest stages of all types of wound repair involve covering the wound by means of active epithelial cell movement such a protein plays an important role in the process of wound closure.
|Cited Patent
|Filing date
|Publication date
|Jun 21, 1960
|Feb 25, 1964
|Hyland Lab
|Tissue culture medium
|Nov 27, 1973
|Apr 6, 1976
|Stanley Cohen
|Process for the preparation of epidermal growth factor and new derivative using cross-linked polyacrylamide gel at a pH of 1-3
|Jan 22, 1975
|Aug 3, 1976
|Solco Basel Ag
|Tissue cell stimulating blood extracts
|Arch Dermatol. Res. vol. 264, pp. 3 15 (1979), Stenn et al.
|Arch-Dermatol. Res. vol. 264, pp. 3-15 (1979), Stenn et al.
|Biochem Journal, vol. 185, No. 2, Feb. 1980, Whateley et al.
|Biochem-Journal, vol. 185, No. 2, Feb. 1980, Whateley et al.
|Brit. J. of Dermatology (1978), 99 pp. 513 518, Mitrani et al.
|Brit. J. of Dermatology (1978), 99 pp. 513-518, Mitrani et al.
|British J. of Dermatology, vol. 98, pp. 411 416 (1978), Stenn.
|British J. of Dermatology, vol. 98, pp. 411-416 (1978), Stenn.
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|U.S. Classification
|530/380, 530/395, 530/416, 424/531, 530/830, 424/530
|International Classification
|Cooperative Classification
|European Classification
|Dec 17, 1982
Owner name: RESEARCH CORPORATION 405 LEXINGTON AVE., NEW YORK
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:STENN, KURT S.;REEL/FRAME:004080/0461
Effective date: 19821216
|Dec 4, 1984
|Certificate of correction
|Sep 15, 1987
|Maintenance fee reminder mailed
|Feb 14, 1988
|Lapse for failure to pay maintenance fees
|May 3, 1988
|Expired due to failure to pay maintenance fee
Effective date: 19880214


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Company owner of the Prismatic Studios. Prismatic Studios is a game company, bent on trying to change the game industry. Create game to prove to people that you should send much for time on a game then just through it out to the the public full of bugs. We want to expand more then just into the game industry. We want go into the technology business too, and space. We are very new company.
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subset: commoncrawl


text:
"This seafood restaurant boasts two bar areas — an inside bar shaped like the bow of a boat with three beers on tap, and an oyster bar toward the back, decorated with fishing gear."
Actually, it has three bars. The third is the only dock bar on Shem Creek (as in, it's built on pilings over the creek).
This place has been in business in 1982. The food is good, including the steaks, but service can be a inconsistent.
My wife and I went to the beach with our dog. As we left we wondered if Red's was dog friendly. They were more than dog friendly, they gave us a great seat on the deck and brought our big dog a bowl of water. Almost every customer and server stopped by our table to pay him some attention.
The service and food were really good and the staff were super friendly.
We've been back twice since then for the all-you-can-eat crab legs and loved it. The wait during summer was pretty bad (it never crossed my mind that it was tourist season until we got there), during the off-season we never had to wait for a table.
Comparing the Seabrook location to the Mt Pleasant location it's hard to believe it's the same restaurant, the vibe is so different. The Mt Pleasant Red's is much more "rustic" and like a bar that happens to serve seafood. If that's your thing great, I prefer the more open and airy Seabrook location for it's laid back atmosphere. It's mid July as I write this and I can hardly wait for September, when I plan to be back at Red's.
Our meal was so horrible that I cannot even rate it with 1 star. Meal took 40 minutes to arrive at the table, food was ice cold. Burger arrived 10 minutes later - cooked well done vs med rare as ordered. The lettuce in the grilled tuna salad was rancid and rotting. Manager comped entree portion of the meal - but worse was the attitude - she really did not care. I'm local and posting on every blog I can find.
This is my new favorite restaurant in the city. The food was amazing - unique, extremely well thought out and done, and an overall gastronomic delight. To top it off, the staff was knowledgeable, friendly and personable bringing together the total presentation in a way you just don't find very often. I was with a group from various locations across the country. There were numerous comments that this meal ranked at or near the very top of all meals they'd ever had - both here and abroad I couldn't have been prouder of this local gem. Can't wait to go back.
Warning: please do not go to this place. I have been here three times and each time there has been some kind of drama surrounding the bar area. I have never been in any kind of altercation at a bar and unfortunately the general manager at the bar was completely rude and classless.. Please do not waste your time and energy coming to this place, Downtown Summerville has a lot more to offer than the terrible service given here. Overall, very displeased and appalled by the low level of professionalism and service at this place.
Never ate there and probably never will but you ignorant people who write reviews really need to edit before hitting "send'. Your spelling and grammar is atrocious. I hope that if you can afford a meal out that you all have at least a 2nd grade education......
Philly born and raised. This place takes a backseat to no one. I have never had a better cheesesteak. Period.
Philly's is amazing! I've never had a problem anytime I've gone and the food is super delicious! The wait staff are always friendly and the wait time can be long depending on when you go, but it's so worth it!
Disappointing in one word is the experience we just had at this highly over-rated restaurant. We are locals (26 and 36 years) and very familiar with downtown dining options. We walked over from the Dock Street after a late afternoon Spoleto performance. We did not order "cocktails" or a first course (which seemed to personally affront our waitress). Long story short we waited over an hour for our entrees after being told by the waitress that she "forgot" to "hit send" on our order. The entrees finally arrived; I ordered swordfish which tasted more fishy than what my husband brings home straight off the boat. I sent it back and declined to order another entree because of the long wait.
The manager came over to our table and did comp my husband's entree. He then proceeded to very arrogantly and condescendingly inform me that the crab roe was responsible for the fishy taste and further enlightened me by telling me it was an acquired taste. Of course the implication being that I am not nearly sophisticated enough to appreciate fishy crab roe. I understand that the staff and chef do not care about this bad experience because they do not need or want our business. We will not return.
I have always had excellent service here. The ambiance is appropriate and the food is great. It is one of my favorite restaurants, and I'm so glad I don't have to leave West Ashley to eat there!
absolutely beautiful food and well prepared my only caveat is a little salty but i got everything fried so thats probably my problem.
Ok first this is a dive..however this is not a negative. Draft beer not ice cold, bologna sandwich is great but too expensive and to small..i mean two slices of bologna, really! Jerky is great..i ordered the Chili with meat and sausage. I thought they gave me the vegan one but they said no. So good luck finding the meat in it. It was also the WORST chili I ever had. They are lucky I gave them a 3.. a good place to drink though.
We have often visited over quite a few years. I can say,that fir the past 3.5 years,there has been zero disappointment . All ordered items were just exquisite . Matters not if fried,broiled,etc.,they know what they are doing. The salads are first class too,of course. I particularly love the crab cakes. Reservations are smart on weekends.
I see people still love to hate Halls. It is a great Restaurant and I wish I could afford it more.
I live in NYC and here and have to tell you this place has the best service I have seen in a steak house. The food is great and the owner and his two sons make sure that everything is taken care of. I go their at least once a week when Im in Charleston, its almost impossible to get a reservation after 8PM and the place is packed every night. This is my favorite place in town.
Awful, son at the door is rude and they treat you like they dont want your business. Come to find out they have bought other restaurants downtown and they all are failing!
Absolutely my favorite seafood restaurant in Charleston!! Great food, excellent service and owned by a local family. Go try it and get hooked.
Luv this place and the outdoor bar!
A "health food" place with No calorie, fat or sugar information available. Pistachios in my no nuts on the ingredients list, Baja style salad. Lemonade was water - not watery, just water. Green tea was also just water! We paid for combos which means a side and a drink, and we all had just water. Counter help was on her phone entire time and made no offer to make anything right. We won't be back!!
Thanks for the warning. I just don't have time, or money, to waste on some place that is full of themselves. Too many nice bars in town to bother with this one.


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Heh. Another day with no deal.
No worries. Stock futures are higher. At least for now, noo
post-TARP deal-inducing crash.
Sorry, we're not getting a deal-inducing crash.
Welcome to Digital Industry Insider, our collaboration with GE to cover the 'Industrial Internet'
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THE SILICON VALLEY 100: The most amazing and inspiring people in tech right now
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There are a few segments of the auto industry that are growing rapidly. Weirdly, though, one of the most notable is the compact cargo van market. What use to be the sole terrain of the Ford Transit Connect and the occasional Dodge Grand Caravan-based Ram C/V Tradesman is becoming a notable battleground. Nissan has dove headfirst into the market with its NV200, which will also be sold as a Chevrolet City Express and Ford recently released a heavily redesigned, more user friendly Transit Connect.
2015 Ram Promaster City Tradesman Cargo
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- Volvo shoots for self-drivers by 2021
- Jeep spends $1 billion on factories
- Find Parts & Accessories for your vehicle!
- Obama rolls out new EV plan
- Infiniti dealers ranked best, Tesla worst
- Compare Volvo XC90 and Lincoln MKX


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Structural differences between a clean, reconstructed Pt(100) surface and one exhibiting chemical or structural irregularities have been identified by means of a scanning tunneling microscope. The (temperature-dependent) defect structure of a surface which had undergone a phase transition involving mass transport was characterized and compared to results obtained using other techniques. The catalytic activity of surface step sites was probed by the thermal decomposition of ethylene. The resulting surface roughening and buildup of carbon, which could be resolved in STM images, clearly showed that the decomposition proceeds from terrace edges.
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StartUpHire is the place to find SENIOR MECHANICAL ENGINEERING jobs. Review the list of SENIOR MECHANICAL ENGINEERING career opportunities to the left. These SENIOR MECHANICAL ENGINEERING jobs represent some of the most rewarding careers available.
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We are seeking a versatile Senior Mechanical engineer to join our engineering team in Watertown, MA. The successful candidate will focus on performing theoretical and FEA analysis for thermal and structural studies. S/he must have the ability to calculate and predict forces,6/18/2016 Watertown, MA Save This Job
SpaceX was founded under the belief that a future where humanity is out exploring the stars is fundamentally more exciting than one where we are not. Today SpaceX is actively developing the technologies to make this possible, with the ultimate goal of enabling human life on7/16/2016 Seattle, WA Save This Job
Job Duties Act as project lead for a cross-functional team focused on designing and developing technology for new and existing respiratory products. Collaborate with and manage input from marketing, industrial design, engineering, quality and regulatory affairs and manufacturing6/12/2016 Irvine, CA Save This Job
DEPARTMENT Engineering CITY Los Angeles, CA TYPE Full-Time What you?ll do As a Senior Mechanical Engineer, you will be responsible in managing material development projects related to polymer/plastics/glass and adhesives for consumer electronic device screen protection6/19/2016 Los Angeles, CA Save This Job
Job Description Xplore is seeking an energetic, highly motivated individual with excellent mechanical engineering skills for a challenging and rewording position designing rugged tablet products. The successful candidate will create and document mechanical designs, interface4/11/2016 Austin, TX Save This Job
Scope of Position Under the guidance of the Sr. Mechanical Engineer, the Mechanical Engineer will provide technical support for a team dedicated to designing and developing new products for the Company. Essential Function / Responsibilities Creation of product design6/12/2016 Fremont, CA Save This Job
Location:Mt. Pleasant, PA Job Code:204 # of Openings:1 Description Senior Process Engineer- Mechanical Mt. Pleasant, Pa Summary Description: The Senior Process Engineer will be responsible for process improvement programs to support manufacturing operations. The position6/26/2016 Mt. Pleasant, PA Save This Job
Engineering Kent, WA, United States More experience. More success. More breakthroughs. Novinium is the world leader in technology to rejuvenate and extend the life of underground electrical cables, with a success rate that provides a better-than-new warranty. Our culture of6/7/2016 Kent, WA Save This Job
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Job Tracking ID: 512170-526269 Job Location: Fremont, CA Job Level: Mid Career (2+ years) Level of Education: Masters Degree Job Type: Full-Time/Regular Date Updated: March 15, 2016 Years of Experience: 5 - 7 Years Job Description: Soraa Laser Diode SLD has an opening for a6/22/2016 Fremont, CA Save This Job
Essential Duties and Responsibilities include the following. (Other duties may be assigned) Develops prototypes for new concepts using rapid prototyping and creative methods for proof of concept and feasibility. Designs and tests components, processes and assemblies. Testing6/13/2016 San Jose, CA Save This Job
Primary Location: Boston, MA Oasys Water seeks a motivated and disciplined Senior Manufacturing Engineer to join the team at our Boston, MA facility. The Senior Manufacturing Engineer has responsibility for the manufacturing and continuous improvement of our proprietary6/1/2016 Boston, MA Save This Job
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Job Function:Intern Department:Research Location:Boston, MA Date Posted:5/24/2016 Share Email this job Lux Research is seeking summer intern candidates for its Market Intelligence team! The best qualified intern candidates will be rising juniors or seniors pursuing a degree in5/30/2016 Boston, MA Save This Job
To support its continued exciting growth, Avo is seeking an individual that can fill the role of Mechanical Program Manager. This is a senior engineering position that reports directly to the CTO. Responsibilities Manage multiple photonic-based programs, provide system6/7/2016 Horsham, PA Save This Job
DEPARTMENT Engineering CITY Los Angeles, CA TYPE Full-Time What you?ll do As a Senior Product Quality Engineer you will be responsible for driving the execution of quality goals and objectives with a strong emphasis on New Product Introduction (NPI). The ability to leverage6/19/2016 Los Angeles, CA Save This Job
Job Description Sr Mechanical Engineer Unchained Labs is committed to building the first cool biologics tools company. Our mantra? Create big value through great products that let scientists do big things. We take the unconventional route of buying commercial businesses,7/26/2016 Pleasanton, CA Save This Job
DEPARTMENT Product Management CITY Irvine, CA TYPE Full-Time What you?ll do As a Senior Product Development Manager, you will be responsible for product development and maintenance for key product lines in the Commercial Networking business. While the day-to-day job has7/19/2016 Irvine, CA Save This Job
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Princeton Lightwave seeks a Senior Optoelectronic Package Design Engineer. Responsibilities include the concept development, detailed design, prototyping and transfer to manufacturing of active devices like high power laser diodes, discrete and arrayed photodetectors, and high5/30/2016 Cranbury, NJ Save This Job


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Welcome to Digital Photo Training, Basic to Pro
June 11 · 10:00 AMHISTOGRAM: NEED TO KNOW AND, DON'T NEED TO KNOWARTIST'S SHORTCUT TO TECHNICAL PERFECTION YOUR CAMERA CAN SHOW YOU A HISTOGRAM OF YOUR PICTURE. You may have seen it. It looks like a graph. It is a graph, and it is the... Learn more
June 9 · 7:00 PMADD A FLASH THE CHEAP WAYAll: Most digital cameras have built-in flashes. Many photographers become aware of the pros and cons, the advantages and disadvantages of the built-in flash. Some... Learn more
June 7 · 7:00 PMBACKGROUND: DREAMY BLUR OR PINE-NEEDLE SHARP?BACKGROUND BLUR AND SHARPNESS -- THAT'S DEPTH OF FIELD! But you knew that. Maybe you'd like to know HOW TO CONTROL IT! This session will clearly, decisively,... Learn more
June 5 · 10:00 AMSpot Metering: The Ansel Adams GiftAll: Digital cameras all have built-in light meters. Many of these light meters include a "spot" metering mode. The spot meter is one of the most SATISFYING of all... Learn more
June 2 · 7:00 PMKNOW YOUR CAMERA (BASICS)KNOW YOUR CAMERA! Learn what M-A-S-P or M-Av-Tv-P mean. Learn precisely WHY you would use those settings. There are EASY ways to use your camera. There are even EASY... Learn more
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188 search results for Berlin Film Festival.
No subtitles needed to be seduced by trailer for Berlin sensation 'Tabu'
Type: Post | Date: Saturday, Mar 24, 2012Fledgling distributor Adopt Films has U.S. rights to Miguel Gomes's film
'Definitive' family-authorized Bob Marley documentary slated for April release
Type: Article | Date: Tuesday, Feb 7, 2012Magnolia Pictures acquires rights to feature film about life of the reggae legend
Chile's 'Gloria,' Australia's 'The Rocket' among recent entries in foreign Oscar race
Type: Post | Date: Tuesday, Sep 10, 2013Where do we currently stand in this wide-open category?
Roundup: Gerwig, Waltz, Schamus tapped for Berlinale jury
Type: Post | Date: Tuesday, Jan 14, 2014Also: On White's eviction from the NYFCC, and Cannes' new president
Romania's 'Child's Pose' takes Golden Bear at Berlin, David Gordon Green gets Best Director
Type: Post | Date: Saturday, Feb 16, 2013Danis Tanovic takes Grand Jury Prize, 'Gloria' star Paulina Garcia is Best Actress
Berlinale: Jeremy Irons derailed in 'Night Train to Lisbon,' but Arvin Chen charms again
Type: Post | Date: Thursday, Feb 14, 2013Chen's 'Will You Still Love Me Tomorrow' a worthy follow-up to 'Au Revoir, Taipei'
Roundup: Tim Robbins and Susanne Bier on Berlinale jury
Type: Post | Date: Monday, Jan 28, 2013Also: Soderbergh talks not-quite-retirement, and Foster champions Wallis
Roundup: On Leonardo DiCaprio's odd Oscar history
Type: Post | Date: Tuesday, Jan 15, 2013Also: The genius of Jodie Foster's speech, and the oldest living Oscar nominees
Berlinale Diary: 'Aujourd'hui,' 'Coming Home,' 'The Outraged'
Type: Post | Date: Friday, Feb 10, 2012Hip-hop icon Saul Williams an unlikely lead of Competition highlight
Berlinale Diary: 'Farewell, My Queen,' 'The Delay'
Type: Post | Date: Thursday, Feb 9, 2012Fest opens with Diane Kruger in lavish but tart take on the Marie Antoinette story


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Kichler 300119NI
Brushed Nickel 52" Outdoor Ceiling Fan with Blades, Light Kit and Pull Chain
Item # bci816238 | Model # 300119NI | Windham Collection
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Discover a selection of top forums networking overrun on the internet. Come to share your passion on forums networking overrun and meet other fans of networking overrun. Then, why don't you create your own forum of networking overrun ?
The Sabbat have overrun Dallas for too long. The inner circle has chosen you to take backt he city. After you have fought for your right to survive, how will you continue to grow the city?
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text: Barack Obama Celebrated Health Care Reform Alone TonightAdrian Chen3/24/10 2:00amFiled to: SadBarack Obamamichelle obamaMarcus Samuelsson30EditInvite manuallyPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink On Barack's big night, guess where Michelle was? Dining at Aquavit in New York, which is owned by the White House's favorite hunky chef, Marcus Samuelsson. (Above) Michelle ate lobster salad while probably sending Obama dirty texts under the table.Reply30 repliesLeave a reply


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24 Cats Posing Like Pin Up Girls
25 Most Dangerous Cities In The World
You Won't Believe What This Shadow in the Sea Is!
What Were These People Thinking? 16 Hilarious Tattoo Fails
20 Terrifying Animals You Didn't Know Were Real
Jay Leno Invites A Perfectly Resto-Modded Porsche 356 Into His Garage
24 Of The Most Hilarious Moments Captured At The Perfect Time
Apparently Gravity Has A Weird Effect On Girls
Five Winter Essentials for Short Haired Dogs
This Cats vs Zombies Short Film Will Make Your Day
What Happens When Your Dog Is Over Protective
McDonald's Manager Confirms A 'Secret Menu' Exists
14 Crazy But True Facts About Kung Fu
10 Healthiest Dog Breeds
What You Do When Your Crush Is Coming
10 Celebrities Who Almost Died While Filming
Daily Dose of Cute: 10 Most Squeal-Worthy Baby Animals
The Real Secret to Online Dating
38 Adorable Puppy Bed Hogs
The Top 5 Most Insanely Ridiculous Cat Videos On YouTube
Want a Job On the World's Most Isolated Island?
Did a Native American Have a Bobcat as a Pet?
The Happiest Animal In The World: The Quokka
Breaking Bad: Fans Continue to Throw Pizzas on Walter White's Roof
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How to Avoid Being Attacked by a Shark
Try Not to Smile When You See This
10 Rad Inventions You're Gonna Want to Buy Right This Second
17 Rare Animals You Had No Idea Existed
Have You Ever Seen A Forever Puppy?


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Hi Maryanna Novak,
This is Loftek Customer Service. So sorry to hear that you have a problem on night version, actually few of our camera have such issue. If possible , plesae help return back the defective camera to us for inspection. We will sent you a replacement.
Any problem , feel free contact our Customer Service or Tech Suport. Thanks .


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Donald Trump rescued from hotel elevator
Donald Trump was trapped inside a hotel elevator before taking the stage at a Colorado Springs town hall, where he blasted the Fire Marshal…
Denver Golf Course to close temporarily
Denver7 reporter Eric Luther tells us this is part of a controversial flood prevention project.
Drivers wait 12+ hours for cars after shooting
Denver7 reporter Amanda del Castillo tells us a person was killed and another was injured during a shootout in LoDo early Saturday.
Downtown shooting leaves 1 man dead
Denver7 reporter Amanda del Castillo tells us more about this shooting where another victim is expected to survive.
Hailstorm devastates Wyoming town
Denver7 reporter Lance Hernandez traveled to Pine Bluffs to get a look at the damage first-hand.
Dog rampage in City Park neighborhood
Denver7 Investagor Ryan Luby tells us more about the problems facing this Denver neighborhood.
Afternoon thunderstorm chances increase Sunday
First Alert Weather Meteorologist Stacey Donaldson tells us it will be hot and sunny, with a slight chance for isolated storms in the…
Guns missing inside Lincoln County Sheriff's Office
Denver7 Investigator Tony Kovaleski tells us more about the sentencing of Zane Nestor.
Little girl accidentally locked inside hot…
Denver7 reporter Jennifer Kovaleski spoke to fire officials who offered some tips for parents.
Firefighters rescue child locked inside hot…
Denver7 reporter Jennifer Kovaleski tells us more about the horrifying moments a mother went through after her child was accidentally locked…
Driver who hit 8-year-old Longmont girl…
Kyle Couch, 20, told police he thought he hit a curb until he saw 8-year-old Peyton Knowlton's stepfather waiving at him.
Anti-Trump protesters gather outside Trump rally
Denver7 reporter Marc Stewart tells us more about what went outside as Trump was speaking to supporters.
Donald Trump makes stops in Colorado
Denver7 reporter Marshall Zelinger tells us what he saw inside the Trump rallies in Colorado Springs and Denver.
Cupcake the horse doing better after daring rescue
Denver7 reporter Sally Mamdooh talked with Cupcake's owner a day after a rescue with a happy ending.
Cupcake the Horse gets rescued by South…
Denver7 was there at the exact time Cupcake the Horse was rescued by South Metro Fire crews.
Mixed reactions for new bike lane on South Broadway
Denver7 reporter Brendaliss Gonzalez tells us some major changes are coming to south Broadway starting next week.
Hillary Clinton to speak in Denver next Wednesday
A source confirmed with Denver7 the Democratic presidential nominee will be in Denver next week.
3 people hospitalized after hit-and-run
Police are looking for 1990s green Mustang after a hit-and-run in Denver
Search for missing Lakewood paramedic
Dozens of volunteers search the area near Green Mountain for Eric Pracht, a missing paramedic who was last seen Friday night.
Medical breakthrough could help detect melanoma
Breakthrough possible at Colorado State University in skin cancer detection
Hail pounds parts of Colorado
Golf-ball sized hail pounds Colorado Springs and other areas
Police investigating shooting
Police are investigating a shooting at a motel on Colfax Avenue in Aurora.
Donald Trump visits Colorado Friday
The Republican presidential nominee is holding a town hall at the University of Colorado, Colorado Springs on Friday at 2 p.m. and a rally in…
Cabin, outbuildings burned at Beaver Creek Fire
The largest wildfire currently burning in Colorado has scorched more than 43 square miles and destroyed a cabin and several outbuildings.
2 injured in shooting involving bail bondsman
Police are investigating a shooting involving a bail bondsman near Kipling and Morrison.
Wings Over the Rockies cancels field trips…
Denver7 reporter Marshall Zelinger tells us the venue is hosting Donald Trump on Friday.
Researchers bracing for West Nile's ugly cousin
Denver7 reporter Russell Haythorn tells us more about the Rift Valley Fever -- and how Colorado is preparing for it.
Firefighters help save Cupcake the Horse
Denver7 reporter Marc Stewart tell us more about the rescue in Cherry Creek State Park.
Horse rescued after getting stuck in the mud
Denver7 reporter Brendaliss Gonzalez tells us how crews worked to rescue Cupcake, a 13-year-old horse stuck on Cherry Creek State Park.
Buttercup rescue
Buttercup rescue
Is DPD delivering on security at the 16th St. Mall?
Denver7 reporter Liz Gelardi sat down with the Denver Police Chief to get some answers.
Commerce City asks feds to review police department
Denver7 reporter Jennifer Kovaleski tells us Commerce City officials are asking the Department of Justice to review its long-embattled police…
Bear sneaks into car in Evergreen and destroys it
Denver7 reporter Lance Hernandez tells us more about an Evergreen couple who captured their neighbor using pots and pans to get a bear out of…
Debate over 16th St. Mall pavers
Denver7 reporter Brendaliss Gonzalez tells us a major change could be coming to the 16th St. Mall -- and you can help decide the outcome.
Excitement in Broncos Country as Training…
Denver7 reporter Sally Mamdooh tells us us the excitement fans felt as Training Camp began for the Denver Broncos.


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C.O.W. #216 the painter_Voting!
Topic: the painter
Deadline for the voting: June 22nd
this weeks Requirements...
- the creature can be any color, but the fluid it produces must be blue. The shade of blue is up to you.
- you must show how the creature makes the fluid in your design. As well as how they apply it.
- You need to keep in mind why it marks its territory, but its acceptable to only include that in your description.


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Deutsche Telekom outruns market with 13% jump in earnings
Deutsche Telekom beat its projected gains in 2015, posting adjusted EBITDA of €19.9 billion, a leap of 13.3 percent.
Deutsche Telekom beat its projected gains in 2015, posting adjusted EBITDA of €19.9 billion, a leap of 13.3 percent.
BlackBerry has snapped up UK cybersecurity firm Encription, and launched a new cybersecurity practice to offer organisations new consulting services, tools and strategies.
A Mobile World Congress panel discussion about digital operators took an unexpected turn when differences emerged between Telefónica and MTS.
The joint start-up incubator formed late last year by the innovation arms of European operators Deutsche Telekom, Orange and Telefónica, and Singapore-based SingTel, has been rebranded as Go Ignite.
Bouygues Telecom said its aggressive transformation had started to pay off in the French mobile market, after it announced a seven percent jump in subscriptions and a return to profitability in 2015.
Orange's Head of Strategic Partnerships has said she hopes to explore more opportunities with Google after the two companies announced a joint project in Africa at Mobile World Congress.
Ericsson and Amazon Web Services (AWS) are poised to form an alliance to create a number of new 'cloud innovation centres' with mobile operators and aid their digital transformation.
Deutsche Telekom will launch a new operator-managed OTT mobile communications service in the second half of 2016, it said at Mobile World Congress in Barcelona.
Ericsson is in talks with US operator AT&T to bring its Digital Life wireless home security and automation system to international customers.
Consumer trust in mobile operators is at an all-time low, according to a new poll. The study of over 8,000 people in eight countries found 71 percent don't trust mobile operators to take care of their data, with their trust in other brands only just lower at 75 percent.
Deutsche Telekom has joined with service providers CenturyLink, Reliance and SK Telecom to form a global alliance to deliver large scale network services to enterprise customers in international markets.
Telefónica has revealed it is rolling out big data analytics tools in an effort to anticipate outages and black spots on its global network.
Nokia Growth Partners (NGP) has closed a new US$350 million fund for investments in the burgeoning internet of things (IoT) market, taking its total available for new investments to $500 million.
Ericsson has announced two major content initiatives at Mobile World Congress, including the establishment of a global content delivery alliance and membership of 20th Century Fox's Innovation Lab.


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Campus Housing:
I only stayed in an on campus dorm freshman year. Everyone on the floor was pretty friendly towards each other, and I know plenty of people that met their best friends because they were on the same floor.
Campus Housing:
The dormitory rooms are just about what you would expect. Single, double, and triple rooms are offered at slightly high prices. They provide you adequate space for your belongings and for your personal comfort. The community bathrooms (shared by an entire hallway) are cleaned every morning while suite bathrooms (shared by two rooms) are cleaned once per week. The Resident Assistants do a great job of organizing activities for your hallway and entire dorm to promote some level of social activity. Most of the freshman hill dorms (except for Barton, which is relatively new) could use a touch of renovation. However, the buildings are still in fine condition and serve as good living spaces. Freshman hill is also very close to the academic buildings on campus. No student should have to walk more than 10 minutes to a class.
Campus Housing:
Living on campus is exceptional. No problems at all.
Campus Housing:
Campus life at RPI is quite nice. I lived in BARH my first year which wasn't the best because it is not a part of the freshmen five dorms where most of my friends lived. However, my friends that lived on freshmen hill had good rooms and a nice environment close to everyone their year and to commons dinning hall. Barton was the nicest hall with air conditioning. My second year I lived at Quad which was super convenient because it was about a five minute walk to all of my classes. When I lived in Quad the rooms where not the best. They were really dark and small and the furniture was kind of old. However, these dorms were renovated last year so they seem very promising now.
Campus Housing:
Pretty much all the housing is nice that I've seen. Some of the buildings are definitely nicer than others. For freshmen: Barton, Nugent and Davison are definitely the nicest options. Hall and Cary are decent. Crockett and Nason are pretty meh. Idk about Bray, but probably similar to Cary. They're all basically the same distance to the academic buildings. BarH is farther but apparently pretty nice to live in.
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Freshmen are, with few exceptions, required to live on campus. Everyone agrees that the nicest dorm is Barton Hall, and there is little room for argument. However, the other freshman dorms on the Hill have all been renovated in the past years, so none are horrible anymore. The quad is more convenient because it is closer to main campus, and BARH is nice because you can stay inside to go to the dining hall. The dorms are expensive, which is why many move off campus after freshman year.
The housing on campus is comparable to that at most colleges. Dorms provide excellent opportunities to meet new people and offer social outlets for the residents. Renovations to Freshman Hill and Cary Hall going coed means there is no longer a clear-cut "Worst Dorm On Campus" award winner, so freshmen don't have to worry about getting stuck in a complete dungeon. Whether you want to live in BARH or the Quad is really a personal preference. The Quad is closer to main campus but is broken up into smaller units and thus is a more isolated environment than the other dorms. BARH is an extra 10-minute walk to main campus compared to the dorms on Freshman Hill, but on cold days having a dining hall just outside your door can be wonderful.
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The case for DavidDecember 6th, 2011 at 10:22 am by David Farrar
In my blog at Stuff, I make the case for David – in fact for both Davids, weighing up their respective strengths.
In my blog at Stuff, I make the case for David – in fact for both Davids, weighing up their respective strengths.
What is going on with ipredict? Shearer's price for winning the contest is still high, but his probability of being Prime Minister by 2015 has dropped by more than half.
Shearer may be making a mistake by "annointing" H3Robertson as deputy. Labour have to get away from the obsessive Clark Simpson era. Robertson will be the tail wagging as Shearer will not be his own man. Robertson is a more powerful person, who is used to getting it his way.
Grant Robertson will be Shearer's Brute. An "accident" waiting to happen.
Despite of all the hype aound Shearer (even Silent T considers himself the underdog), I reckon his opponent will win the debates and could become the chosen one.
The final decision will be made by the old guard of Goff, King and the rest of the deadwood, whom do not like the smarmy, arrogant Cunliffe. Interesting times await the socialists.
We have to get away from the long shadow of the UN. When did it become a graveyard and training ground for our leaders?
Shearer did OK out of his years there – not too much danger, a fat pension and a lot of gooey-eyed stuff about his valiant service. You only have to see these guys in action to understand what a corrupt, feather-bedded organisation the UN is. And let's think about what exactly a 'Somali war lord' would look like. Certainly not a fearsome Samurai.
And now according to Whaleoil he has two trusts, the UN pension and a portfolio of properties. Why don't we see his affluence as part of the total picture?
I am so tired of being fed crap by Labour.
I must admit, I'm struggling to care.
After senior Labour member Trev Mallard's gutless smearing of Brethren women ("chinless scarf-wearers") revealed the party's queer prejudices, it's obvious that the chinless wonder-boy David Cunliffe doesn't really stand a chance…Shearer will be the chosen one.
what Rich Prick said – either about who it will be or anyone's opinion about who it will be.
And let's think about what exactly a 'Somali war lord' would look like. Certainly not a fearsome Samurai.
Quite right… because obviously a Somali warlord is an African, while a Samurai comes form… oh, never mind.
Just say ten "I hate socialists" and begone with you.
re BeaB "I am so tired of being fed crap by Labour."
Lord, aren't we all – but an amazing feature of this "struggle" is that it's cleared the smoke obscuring just how tense are relations between Labour MPs. Undercurrents and divisions show clearly that parliamentary Labour is a very divided ship – and how could it not be, when all remaining incumbents seem focused first and foremost on the self-interest, way before they consider anything else. The fact Labour types are always spouting "unity and brotherhood" underlines this – so it's even more surprising that the media has never once attempted to focus on what's, transparently, a blatant lie.
Again. how many times has it been reported that Cunliffe isn't liked? Yet neither the manifold implications of that, nor especially it's lack of relevance to the rendering of effective leadership, have been remotely examined. Our media spend far too much time on the trivial – and too often completely fail to seek what is indeed substantial. I don't care whom Labour elects – it's a poisoned chalice for either one. However it's certain that with either one they're not going to score in the next six years – but they'd do far less poorly under one style of leadership than they will do under the other.
I sure hope they choose Cunners.
David Prosser


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Tuesday 10.20.15 my new book IF THE RAINDROPS UNITED goes on sale at book stores.
For much of October & November, I will be doing special stand-up shows where the ticket includes the show and the book!
Also in October & November I will be doing book signing events at book stores. Each book store appearance will include a talk about my new book, a Q&A, and a book signing.
|06.29.16 - 07.30.16 http://judahfriedlander.com/judah-tour/
|Vermont Comedy Club
|Burlington, VT
|TIME: 7:00pm | Age restrictions: No Minors. Address: 101 Main St..
|08.05.16 http://judahfriedlander.com/judah-tour/
|Guild Hall
|East Hampton, NY
|TIME: 9:30pm | Age restrictions: No Minors. Address: 158 Main St..


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It's packed full of some really useful Tax Tables, Calculators and important dates
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Ensure you are complying with the requirements which, although law from April 2016, introduce new filing requirements from 30 June 2016. Under the rules, companies are required to maintain a register of people with significant control (PSC register). In addition, the details of people with significant control (PSCs) have to be filed with Companies House from 30 June 2016.
A PSC is defined as an individual that:
The details of the individuals which need to be entered on the PSC register include:
Failure to comply with the requirements of the PSC regime could lead to the company or directors, or identified PSCs committing a criminal offence. The company and its directors could face a fine or imprisonment or both.
Further guidance can be found on the Companies House website or please contact us for more help and support in this area.
Internet link: Companies House
Our FREE monthly eNEWS email will keep you up to date with the latest news in accounting.


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Strategies from K-12 schools, districts, and programs that are dramatically improving the learning process.
We share evidence- and practitioner-based learning strategies that empower you to improve K-12 education.
I have been living in South Korea for the last 10 years teaching international students.
5 years 1 month ago to Teacher Leadership


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25% off lightweight hoodies? Heavy. Use HOODSRGOOD.
This old tree has suffered the ravages of time and nature out in the open of one of the back paddocks of the Charlemont farm property.
australia charlemont clouds farms geelong hdr landscape rust storm trees
Nice abstract tree,what a stunning photo
what a great fined Ohil,this tree looks amazing,I love the sky,wonderful dof and comp.well done my friend
OOPS make that Phil,
My gosh, this is gorgeous… ;-)
great find and interesting shapes also well composed with the extra point of interest in the background.
Wow, what a find. Fantastic!
Love the angle – and your processing is so good – well done.
Poor old tree, I think it needs a walking stick to support it!.Makes a great image!.
Excellent HDR. Well found …
Gorgeous work, as always Phil. The tree has a very Salvador Dali quality about it.
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you are viewing a single comment's thread.
I would take a .25 cookie tomorrow and treat it like fillet minion.
It's adding time, no retreat no surrender and no offering
up a nickel with 5 minutes to go.......So much or all your predictions of $29.50 or better! roflmao
How great it is to cost average. One makes lots of money hahaha. August is the date, and now if we get higher and higher like we did yesterday, all is nice. 28.50 will be a 33% win. Gotta love it
I'm right there with you, just hoping for table scraps.
If I excercise a call tomorrow would I get the dividend?


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The same populations of brain cells encode memories that occur close together in time, according to new research.
Speaking of Science
February 1, 2016|
February 2016's selection of notable quotes
February 1, 2016|
February 2016's selection of notable quotes
January 26, 2016|
Jordan Kurland, associate general secretary of the American Association of University Professors, has passed away at age 87.
January 1, 2016|
January 2016's selection of notable quotes
December 31, 2015|
A year's worth of noteworthy quotes
December 1, 2015|
Welcome to the Microbiome, The Paradox of Evolution, Newton's Apple, and Dawn of the Neuron.
December 1, 2015|
See the soldier whose recovery from a debilitating muscle injury was greatly aided by a cellular therapy plus physical therapy.
December 1, 2015|
What to do when you realize your publication is fatally flawed
December 1, 2015|
Physical therapy and exercise are critical to the success of cell therapies approaching the clinic.
November 24, 2015|
One large provider says the shortfall should clear up by early 2016.
November 4, 2015|
Researchers use network theory to estimate the importance of relationships among researchers and institutions in attracting grant money.


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Amazon Pets is your one stop shop for all your pet supplies. Looking for dog feeding and dog watering supplies? Need an extensive selection of dog products and supplies? Well look no further, you'll find everything you need right here at Amazon Pets. From automatic feeders to dog nursing supplies, from dog travel bowls to dog fountains, we're your online pet store.
Keeping healthy, clean and efficient feeding and watering supplies for your dog is very important to their long term health and happiness. Our products encompass some of the best dog feeders and dog watering products available. Dog bowls come in every size, material and function you can imagine. You'll find large elevated feeders, as well as dog bowls that encourage slow feeding. Or, if you just want the prettiest dog bowl available, you'll find it here.
Automatic pet feeders take the guess work out of feeding fido and make sure you never miss a feeding. The dog fountains also provide a continual flow of clean, filtered, healthy water. No more messy, dirty dog water bowls! Of course we have all the accessories for cleaning the automatic dog feeders and pet fountains, too.
Raised bowls and feeding stations are a real necessity for larger breeds as well as senior dogs, so be sure to look over the selection here at Amazon Pets. And don't feel you have to leave Fido home when you hit the road. Our dog travel bowls make it easy to bring your pooch along on the adventure. Road trip! It's all here at Amazon Pets.


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Hi there,
I'm trying to tweak TDH's terrific Notes Blog Core theme (http://wordpress.org/extend/themes/notes-blog-core-theme) with the dropdown menu described at Webdesigndev (since the site seems to be down at the moment, I'm giving you the google cached site: http://tinyurl.com/yzs9vfz).
Although I'm but a newbie at CSS and not familiar with JavaScript/jQuery I think it would be managable for me to do it (on top of that I really suck at php so I'm working in strong headwind). But - there is a but - I have large troubles finding the automatic attributes that are given the page lists when using the page widget and without them I'm totally lost. I've searched the codex without finding anything that gives a clue about what I should do, so my last hope is you here at the forums. :-)
Can anyone give me the automatic page widget list attributes (if such a thing even exists) or maybe even guide me with the specifics for the theme I'm about to use together with the meny.
Thanks in beforehand!
My test site is here: http://test.ackerfors.se/1/


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The RRPC is collecting flood resiliency data.March 26,2014RRPC will gather
regional flood data
The Rutland Regional Planning Commission will begin to collect flood data for the Rutland region.
The work will focus on Act 16, which requires a "Flood Resilience Element" to be included municipal and regional plans.
The RRPC will consult with the state Rivers Program to research flood hazards in Vermont to develop a flood resilience element for its regional plan. The RRPC will also work with all 27 municipalities in the region to complete a flood resilience plan checklist.
This preliminary checklist provides a menu of steps that could be taken to improve flood resiliency and is intended to help a municipality get started in a self-assessment. The RRPC will also hold a training workshop to inform local officials about flood resiliency.
Towns may use the study results in identifying vulnerable areas and to enhance their basic emergency plans, hazard mitigation plans and town plans. The RRPC will integrate the results into the regional plan.
The project is funded by federal Clean Water Act.MORE IN This Just InNORTH BENNINGTON Two public meetings, in North Bennington and Shaftsbury, have been set for... Full StoryThe Board of Aldermen has endorsed a major jump in city pension funding. Full StoryHUBBARDTON The annual celebration of Hubbardton has been moved to Sunday due to the weather... Full Story
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Diamond Hotel Philippines Opens the Lobby Lounge
Diamond Hotel Philippines opens a new dining attraction, the Lobby Lounge, serving a wide variety of treats, from full meals to desserts.
Meet with colleagues over steaming hot coffee, catch up with friends during afternoon tea, or spend time with loved ones over a leisurely meal. It is the perfect place to be seen or to unwind after a busy day, while enjoying a favorite drink or indulging in a late night dessert with friends.
The Lobby Lounge offers Diamond Hotel's bestselling cakes, pastries and an updated ala carte menu selection. Diamond's signature delicacies abound, including no less than the Ube Ensaymada, Supermoist Chocolate Cake, Le Royale and Baked Cheesecake. Experience Paris with Ispahan (raspberry and lychee) and Mont Blanc (chestnut and rum), as these specialty cakes will please the most discriminating tastes. Other bestsellers are also available: Cecilia, a coconut mousse pie; Vanessa, made from pistachio macaroon and fresh strawberries;
Set to satisfy food connoisseurs is a sublime selection of savory dishes. Starters include Zucchini Cream Soup, Truffle Fries and the much-loved GM's Salad. The Slow Roasted Beef Belly, Porcini Mushroom Crepe and Spinach and Ricotta Ravioli are sure-fire palate-pleasers. Then, cap the meal with a scoop of Diamond Hotel's homemade ice cream, which has proven to be an instant favorite. In delightfully unique flavors like Black Sesame, Genmaicha (Green Tea with roasted brown rice), Azuki (Japanese Red Bean), Taro and Jackfruit, Apple Pie, and more, diners definitely keep coming back for seconds. The different ice cream variants are also available in one-pint and half-gallon containers for guests to take home and share.
Savor a cup of Kohikan Coffee, Nespresso Coffee, or Hediard Tea while dining. And for those who want to relax and while away their time during the evening, the Lobby Lounge also features an extensive selection of wines and cocktail drinks.
The Lobby Lounge is open daily from 7am until 11pm. For reservations please call (632) 528-3000 ext. 1121.
Of course, all these are only a part of the Diamond Hotel experience. To enjoy everything that this Manila hotel has to offer, proceed to its homepage at http://www.diamondhotel.com/
For more information, call Diamond Hotel Philippines at (632) 528-3000 or visit the hotel's website at http://www.diamondhotel.com/
Diamond Hotel Philippines
Roxas Boulevard cor.
Dr. J. Quintos St.,
Manila, Philippines, 1000
Phone Number: (632)528-3000
Melanie Samonte-Pallorina
PR Manager
Phone Number: 528-3000 ext. 1525
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Cute and simple outfit ideas. You wake up, go to your wardrobe, and freeze. What are you going to wear today? It's all staring you in the face, but nothing is making sense. Finding what to wear can be a…
Michel Canetti illustrations
Michel Canetti is a French painter and artist. He is one of the leading fashion illustrators. He has completed many projects for advertising and magazines, working for the most famous names in fashion and luxury products.
Absurd Shoes
Finding the perfect pair of shoes?!? A good pair of dressy sandals allows women to walk around all day long and look stylish. When comfort and high style come together in colorful straps or sculpture-like heels, it is a winning combination. 😀…
Not Necessarily Wearable
These are works of art and they are not necessarily wearable. Stay chic! 😉 They can't ignore you!
Christmas Tree Brooches
Brooch – ornamental pin with a clasp to attach it to a garment. Brooches developed from the Greek and Roman fibula, which resembled a decorative safety pin and was used as a fastening for cloaks and tunics. Brooches have been…
Apex Predators or Teeth Shoes
These shoes are made from the finest leather and over one thousand teeth dentures. Perfect for Halloween! Creepy! source: http://fantichandyoung.co.uk/ This work addresses parallels between social evolution and evolution in the natural world. Nature as model or nature as a threat.
Dogs in Wigs
Wigs are not just for humans! And don't forget your camera for lots of photo opportunities!!!
Weird Bra Designs
These look like some of the most uncomfortable bras ever made. Half art, half underwear, not very sexy and even scary.


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The optic disc is an important feature in the retina. We propose a method for the detection of the optic disc based on a supervised learning scheme. The method employs pixel and local neighbourhood features extracted from the ROI of a digital retinal fundus photograph. A support vector machine based classification mechanism is used to classify each image point as belonging to the cup and retina. The proposed method is evaluated on a sample image set of 68 retinal fundus images. The results show a high correlation (r>0.9) with the ground truth segmentation, with an overlap error of 6.02%, and found to be comparable to the inter-observer variability based on an independent second observer segmentation of the same data set.
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Book your room at Circle K Ranch
Escape with us to the beauty of the majestic San Juan Mountains Find the quiet serenity and adventure that youve been longing for CIRCLE K GUEST RANCH AND HUNTING OUTFITTERS.Would you like to give us feedback? Share it! Opens in a new window
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Arizona Cardinals
Two Cardinals land on USA Today 'All-Joe Team'
It's certainly not the sexiest of postseason honors, but it's an honor nonetheless.
USA Today released it's 21st annual All-Joe Team, a squad made up of hard workers that weren't selected to Pro Bowl teams. And the Arizona Cardinals, despite their 5-11 record, had two representatives on this year's All-Joe Team.
Defensive end Calais Campbell, who many feel should have received a Pro Bowl nod, landed on the All-Joe Team.
Says USA Today:
He's not quite J.J. Watt, but Campbell is the rare 3-4 defensive end who can wreck a game. He's averaged seven sacks over the past four seasons, and his 6-8 frame plays havoc with passers and kickers.
The other Cardinal honored was punter Dave Zastudil, who enjoyed a fantastic season for Arizona.
Dropped 46 punts inside opponents' 20-yard line, a new NFL record. Despite the horrid field position Arizona often had, Zastudil didn't merely bomb away and instead maintained an excellent net (41.4 yards) while forcing a league-high 36 fair catches. And he did a lot of work, his 112 punts 21 more than anyone else last season.
Former Sun Devil Vontaze Burfict of the Cincinnati Bengals, who went undrafted last year but worked his way into a starting spot, also landed All-Joe honors.
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The Rundown - Cincinnati Bengals
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Off the Edge previews the Cincinnati Bengals. #OTERundown
The Rundown - Indianapolis Colts
Thursday July 28
Off the Edge previews the Indianapolis Colts. #OTERundown
Big Deal...or No Deal?
Thursday July 28
Athletes keeping their mouth shut, a luxury car bet and just a little shark bite!
Gregg Rosenthal - NFL.com & NFL Network
Thursday July 28
Gregg and B-Train talk Cards position battles and QB questions in the NFC West


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How can I get a curved TextBox like the Facebook Status TextBox with html and css only?
Can Anybody illustrate me how to do that ??
We started with Q&A. Technical documentation is next, and we need your help.
Whether you're a beginner or an experienced developer, you can contribute.
How can I get a curved TextBox like the Facebook Status TextBox with html and css only?
Can Anybody illustrate me how to do that ??
My Cross-Browser, CSS-only version of the Facebook-style textbox:
I've used
According to this question,
Then, for coloring the border in a cross-browser way, i used
At this point, i've used
This created the gray border to the arrow.
(*) I've chosen a color a bit darker than the textbox's borders one, because of the "brightening" effect generated by mixing the two triangles. With #888 instead of #bbb, the result is acceptable and more similar to the original color than using the original color itself.
Here is the commented code and the demo:
Hope that helps...
quite a few possibilities:
My prefered one would be to get an triangle image that fit's your needs then put it via css above the input field.
You could fix it giving the input field the class triangled_input, setting it to relative. And giving the triangle image the attribute absolute. Changing the offset till it's at the place you wanted.
Of course you'll have to adjust the offsets for the triangle to fit your needs.