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Biofilms are composed of bacterial cells embedded in an extracellular polysaccharide matrix. A major component of the Escherichia coli biofilm matrix is PGA, a linear polymer of N-acetyl-d-glucosamine residues in β(1,6) linkage. PGA mediates intercellular adhesion and attachment of cells to abiotic surfaces. In this report, we present genetic and biochemical evidence that PGA is also a major matrix component of biofilms produced by the human periodontopathogen Actinobacillus actinomycetemcomitans and the porcine respiratory pathogen Actinobacillus pleuropneumoniae. We also show that PGA is a substrate for dispersin B, a biofilm-releasing glycosyl hydrolase produced by A. actinomycetemcomitans, and that an orthologous dispersin B enzyme is produced by A. pleuropneumoniae. We further show that A. actinomycetemcomitans PGA cross-reacts with antiserum raised against polysaccharide intercellular adhesin, a staphylococcal biofilm matrix polysaccharide that is genetically and structurally related to PGA. Our findings confirm that PGA functions as a biofilm matrix polysaccharide in phylogenetically diverse bacterial species and suggest that PGA may play a role in intercellular adhesion and cellular detachment and dispersal in A. actinomycetemcomitans and A. pleuropneumoniae biofilms.
Bacterial cells in a biofilm are surrounded by a self-synthesized, three-dimensional matrix that holds the cells together in a mass and firmly attaches the bacterial mass to the underlying surface (3). This matrix, referred to as the slime layer, glycocalyx, or extracellular polymeric substance (EPS) matrix, can comprise up to 90% of the biofilm biomass (8). In addition to its structural role, the EPS matrix provides biofilm cells with a protected microenvironment containing dissolved nutrients and secreted enzymes, as well as other biological molecules and abiotic substances originating from outside the biofilm. The EPS matrix may also contribute to the increased resistance to antibiotics and host defenses exhibited by biofilm cells (27, 32).
Polysaccharide is a major component of the EPS matrix in most bacterial biofilms (34). One of the best-characterized matrix polysaccharides is a hexosamine-containing polymer produced by several staphylococcal species, including Staphylococcus epidermidis and Staphylococcus aureus, and by Escherichia coli. This polymer, called PIA (also PNAG, PS/A, or SAA) in Staphylococcus spp. (2, 24, 28-30) and PGA in E. coli (36), consists of a linear chain of N-acetyl-d-glucosamine (GlcNAc) residues in β(1,6) linkage. Various forms of this GlcNAc polymer appear to differ in molecular weight, in the degree of deacetylation of the GlcNAc residues, and in the presence of O-acetyl substituents (24, 28, 30). PIA and PGA have been shown to play roles in abiotic surface attachment and intercellular adhesion (13, 30, 37), and PIA has been shown to protect S. epidermidis biofilm cells from host innate defenses, including phagocytosis and antimicrobial peptides (36).
In S. epidermidis, the production of PIA is encoded by a cluster of four tightly linked genes named icaADBC (10, 13, 30). IcaA is a transmembrane glycosyltransferase that synthesizes the GlcNAc polymer backbone (10). Sole expression of icaA induces only low enzymatic activity, but coexpression of icaA with icaD, a small gene that is translationally coupled to icaA, leads to a significant increase in enzymatic activity and is related to full phenotypic expression of PIA (10). IcaB is a secreted protein that is homologous to several polysaccharide N-deacetylases, including rhizobial NodB chito-oligosaccharide deacetylase, peptidoglycan GlcNAc deacetylase, and chitin deacetylase, suggesting that IcaB may be a deacetylase. IcaC is a predicted transmembrane protein with unknown function. In E. coli, the production of PGA is encoded by a cluster of four tightly linked genes named pgaABCD (37). PgaC exhibits 40% amino acid identity to S. epidermidis IcaA, suggesting that PgaC is also a glycosyltransferase. Although PgaD exhibits no primary amino acid sequence similarity to IcaD, both proteins are small (137 and 101 amino acid residues, respectively), and both contain two predicted transmembrane segments, which suggests that PgaD and IcaD may be functionally related. PgaA and PgaB are predicted to be outer membrane or secreted proteins (37). The N-terminal 300 amino acid residues of PgaB exhibit 25% identity to IcaB, suggesting that PgaB may also be a deacetylase. PgaA and the C-terminal 370 amino acid residues of PgaB are not homologous to any proteins in the National Center for Biotechnology Information (NCBI) public database.
A glycosyl hydrolase that degrades S. epidermidis PIA was recently identified (21). This enzyme, named dispersin B (DspB), is produced by the gram-negative oral bacterium Actinobacillus actinomycetemcomitans, the causative agent of a severe form of periodontal disease that affects adolescents (38). Treatment of S. epidermidis biofilms with recombinant A. actinomycetemcomitans dispersin B protein causes dissolution of the EPS matrix and detachment of biofilm cells from the surface (21). Treatment of A. actinomycetemcomitans biofilms with dispersin B also causes the detachment of cells from the surface (20), suggesting that A. actinomycetemcomitans biofilms contain a matrix polysaccharide that is structurally related to PIA. In this report, we present evidence that a cluster of four A. actinomycetemcomitans genes that is homologous to E. coli pgaABCD (19, 38, 40, and 12% amino acid identity, respectively) encodes the production of a hexosamine-containing exopolysaccharide that is structurally related to E. coli PGA and that this polysaccharide is a substrate for dispersin B. We also show that the A. actinomycetemcomitans PGA-like polysaccharide is not required for the attachment of cells to surfaces but may play a role in the aggregation of cells inside the biofilm colony and the detachment of cells from the colony. We further show that a similar hexosamine-containing polysaccharide constitutes a biofilm matrix component in the closely related bacterium Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia (11).
Bacterial strains, media, and growth conditions.The bacterial strains used in this study are listed in Table 1. A. actinomycetemcomitans and S. epidermidis strains were grown in Trypticase soy broth (Becton Dickinson) supplemented with 6 g of yeast extract and 8 g of glucose per liter. Media were supplemented with 3 μg of chloramphenicol/ml, 40 μg of kanamycin/ml, 20 μg of nalidixic acid/ml, 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), 0.01% Congo red dye, or 1.5% agar when appropriate. A. pleuropneumoniae strains, which were isolated from the lungs of pigs with porcine pleuropneumonia, were cultured in Mueller-Hinton broth supplemented with 6 g of yeast extract, 8 g of glucose, and 5 mg of NAD per liter. E. coli strains were grown in Luria-Bertani broth supplemented with ampicillin or chloramphenicol (50 μg/ml each) or 1 mM IPTG when appropriate. E. coli cultures were incubated in tubes with agitation. All other bacteria were incubated statically. A. actinomycetemcomitans was incubated in the presence of 10% CO2. All cultures were incubated at 37°C.
Targeted mutagenesis of A. actinomycetemcomitans pgaC.The pgaCD region of A. actinomycetemcomitans strain CU1000 was amplified by PCR using the forward primer 5′-TGACCGGATCCTCAAGCAGGTAAACCATAG-3′, which introduced a BamHI site (underlined) 27 bp upstream from the pgaC start codon, and the reverse primer 5′-TGACCGCTGCAGttaTTTTTTCTTTTTTCTCC-3′, which introduced a PstI site (underlined) immediately downstream from the pgaD stop codon (lowercase). PCRs were carried out as previously described (19). The PCR product was digested with BamHI and PstI and ligated into the BamHI/PstI sites of plasmid LITMUS28, resulting in plasmid pVK80 (Table 1). The DNA sequence of the insert from pVK80 (1,532 bp) was 99.3% identical (with six silent changes and five replacement changes) to that of the pgaCD region from strain HK1651 (Actinobacillus Genome Sequencing Project [http://www.genome.ou.edu/act.html ]). Plasmid pVK80 was mutagenized by random in vitro insertion of the kanamycin resistance transposon R6Kγori/KAN by using an EZ-TN transposon insertion kit (Epicentre). Two mutant plasmids, pVK81-1 and pVK81-5, which contained transposon insertions in the middle and at the end of pgaC, respectively, were used to transform strain IDH781N to kanamycin resistance using a natural transformation protocol provided by Mrinal Bhattacharjee and David Figurski (Columbia University). Recombination of the transposons into the chromosomes of the transformants (designated JK1048 and JK1049) (Fig. 1) was confirmed by PCR using the forward primer 5′-GACGGTGATGCGGTATTGG-3′ and the reverse primer 5′-CCTCTATCCGGGCTGGTCCATAC-3′ (corresponding to bp 412 to 430 and 1207 to 1229 in the sequence with GenBank accession no. AY438014 , respectively), which flanked the transposon insertion sites, and by DNA sequence analysis of the transposon junctions.
Genetic complementation of A. actinomycetemcomitans pgaC.The BamHI/PstI insert from pVK80 was ligated into the BamHI/PstI sites of the broad-host-range plasmid pJAK16, which placed pgaCD under the control of an IPTG-inducible tac promoter. The resulting plasmid (pVK82), or pJAK16 as a control, was mobilized into A. actinomycetemcomitans strain JK1048 by using the RK2 oriT-defective mutant plasmid pRK21761 as previously described (35). Plasmid-harboring strains were grown in media supplemented with chloramphenicol and IPTG.
Expression of A. actinomycetemcomitans pgaCD in E. coli.Plasmid pVK82, or plasmid pJAK16 as a control, was transformed into E. coli strain DH5α. Transformants were incubated in broth for 16 h, diluted 1:50 in 3 ml of fresh broth, and incubated for an additional 5 h until the optical density of the culture (at 590 nm) reached 0.5. IPTG was added to a final concentration of 1 mM, and the cultures were incubated for an additional 2 h. Cells were harvested by centrifugation, washed three times with phosphate-buffered saline (PBS), and resuspended in 1 ml of PBS. The cells were treated with dispersin B as described below.
Expression of A. pleuropneumoniae pgaCD in E. coli.Genomic DNA isolated from A. pleuropneumoniae strain IA1 was amplified by PCR using the forward primer 5′-TGACCGGATCCTCAAGCAGGTAAACCATAGGGATTGGTatgATTCTAGAAATATTCAG-3′, which inserts a BamHI restriction site (underlined) 27 bp upstream from the pgaC start codon (lowercase), and the reverse primer 5′-TGACCGCTGCAGttaAGGTTTTTTATGGCGAC-3′, which inserts a PstI restriction site (underlined) downstream from the pgaD stop codon (lowercase). The PCR product was digested with BamHI and PstI and ligated into the BamHI/PstI sites of pJAK16, resulting in plasmid pVK93. The DNA sequence of the insert from pVK93 was 99.6 to 100% identical to pgaCD homologues from four other A. pleuropneumoniae strains (representing serotypes 1, 5, and 7) in the NCBI public database. Plasmid pVK93 was transformed into E. coli strain DH5α, and transformants were cultured and induced with IPTG as described above.
Expression of S. epidermidis icaAD in E. coli.Genomic DNA isolated from S. epidermidis strain NJ9709 was amplified by PCR using the forward primer5′-TGACCGGATCCTCAAGCAGGTAAACCATAGGGATTGGTatgCATATTTTTAACTTTTTAC-3′, which inserts a BamHI restriction site (underlined) 27 bp upstream from the icaA start codon (lowercase), and the reverse primer 5′-TGACCGCTGCAGtcaTATGTCACGACCTTTC-3′, which inserts a PstI restriction site (underlined) downstream from the icaD stop codon (lowercase). The PCR product was digested with BamHI and PstI and ligated into the BamHI/PstI sites of pJAK16, resulting in plasmid pVK91. Plasmid pVK91 was transformed into E. coli strain DH5α, and transformants were cultured and induced with IPTG as described above.
A. actinomycetemcomitans dispersin B purification and treatment. A. actinomycetemcomitans dispersin B was purified as previously described (20). The protein concentration was determined by using a Bio-Rad Protein Assay kit. The specific activity of the enzyme was 970 U per mg of protein, where 1 U of enzyme activity was defined as the amount of enzyme needed to hydrolyze 1 μmol of 4-nitrophenyl-β-d-N-acetylglucosaminide to 4-nitrophenol and N-acetylglucosamine per min at 25°C in 50 mM sodium phosphate buffer (pH 4.5)-100 mM NaCl (20).
Bacterial cells were treated with 40 μg of dispersin B/ml in PBS for 30 min at 30°C. The cells were pelleted by centrifugation, and the supernatant was assayed for total hexosamine as described below. Crude polysaccharide samples were treated with 40 μg of dispersin B/ml in PBS for 30 min at room temperature. The samples were reprecipitated with sodium dodecyl sulfate (SDS) as described below, incubated at 100°C for 3 min, and then centrifuged. The supernatants were analyzed for total hexosamine as described below.
Expression and purification of A. pleuropneumoniae dispersin B.Genomic DNA isolated from A. pleuropneumoniae strain IA1 was amplified by PCR using the forward primer 5′-GCTAGTCCATGGACTTACCTAAAAAAGAAAGCG-3′, which inserts an NcoI restriction site (underlined) at codon 26 of dspB, and the reverse primer 5′-GCAGGGATCCctaGTGGTGGTGGTGGTGGTGATGCGATTTCGGATCATTAG-3′, which inserts a BamHI restriction site (underlined) and six His codons (italics) flanking the dspB stop codon (lowercase). The PCR product was digested with NcoI and BamHI and ligated into the NcoI/BamHI sites of the T7 expression plasmid pET-28a, resulting in plasmid pRC2. The DNA sequence of the insert from pRC2 was 99.7 to 100% identical to sequences of dspB homologues from four other A. pleuropneumoniae strains in the NCBI public database. Plasmid pRC2, which encoded dispersin B (minus its leader peptide) fused to a hexahistidine metal-binding site at its C terminus, was transformed into E. coli strain BL21(DE3), and the fusion protein was purified by using Ni affinity chromatography as previously described (20).
Surface attachment and biofilm formation assays.To quantitate surface attachment, 100-μl aliquots of serial twofold dilutions of inocula were transferred to the wells of a 96-well polystyrene microtiter plate (model 324662; Falcon) and incubated for 4 h. The wells were then washed with water and stained with crystal violet as previously described (16). The bound dye was solubilized in 100 μl of 100% ethanol, and the optical density of the ethanol-dye solution was measured using a Bio-Rad Benchmark microtiter plate reader set to 590 nm. To measure biofilm formation, cells were grown in 96-well microtiter plates for 16 h and then stained and quantitated as described above. Surface attachment and biofilm formation assays were performed three times with similar results.
Biofilm detachment assays.Biofilms were grown for 16 h in the wells of a 96-well microtiter plate as described above. The biofilms were washed with water and then treated for 2 h at 37°C with 10 mM sodium metaperiodate in 50 mM sodium acetate buffer (pH 4.5) or with 100 μg of proteinase K (Sigma)/ml in 20 mM Tris (pH 7.5)-100 mM NaCl. After treatment, the biofilms were washed with water, stained with crystal violet, and quantitated as described above. Biofilm detachment assays were performed three times with similar results.
Autoaggregation assay.One well of a six-well microtiter plate (model 353046; Falcon) was filled with 4 ml of fresh broth containing 107 CFU of bacteria/ml, and the plate was incubated for 16 h. The biofilm that formed on the surface of the well was rinsed with PBS and then scraped from the surface into 1 ml of PBS by using a cell scraper. A total of 200 μl of the cell suspension was transferred to a 0.5-ml polypropylene tube (model 6530; Corning), and the tube was vortexed at high speed for 10 s, incubated statically for 5 min, and photographed.
Immunofluorescence microscopy.Aliquots of bacterial-cell suspensions (15 μl each) were heat fixed onto printed glass microscope slides, soaked in ice-cold methanol for 2 min, and then air dried. The fixed cells were incubated with 15 μl of anti-S. epidermidis PIA antiserum or the corresponding preimmune serum (diluted 1:50 in PBS) for 30 min at 37°C in a wet chamber (23). The slides were washed three times for 2 min each time with PBS and then air dried. Bound antibodies were detected using a fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G antibody (Sigma) diluted 1:80 in PBS. After incubation for 30 min at 37°C in a wet chamber, the slides were washed twice with PBS and once with double-distilled water for 2 min. The slides were then air dried and viewed under a Zeiss fluorescence microscope at a magnification of ×1,000. PIA-producing S. epidermidis strain 1457 and its PIA-negative icaA insertion mutant, 1457-M11, were used as positive and negative controls, respectively (23).
Isolation of crude polysaccharide.Confluent biofilms were grown in 100-mm-diameter tissue-culture-treated polystyrene petri dishes (model 430167; Corning) as previously described (17). The biofilms were washed with PBS, scraped from the surface into 5 ml of PBS with a cell scraper, and then transferred to a 15-ml conical centrifuge tube. The cells were pelleted by centrifugation for 10 min at 4,000 rpm in a Sorvall model RT7 centrifuge. The cell pellet was resuspended in 0.5 ml of 50 mM sodium acetate buffer (pH 5.8)-100 mM NaCl and then transferred to a 1.5-ml microcentrifuge tube. An equal volume of 10 mM Tris-HCl (pH 8.0)-5 mM EDTA-0.5% SDS was added, and the tube was vortexed briefly and incubated at 100°C for 3 min. The tube was centrifuged for 10 min at 15,000 rpm in a Sorvall MC12V microcentrifuge, and the supernatant was decanted. The pellet contained the crude polysaccharide. The yield was ∼300 μg of crude polysaccharide per mg (wet weight) of cells.
SDS-polyacrylamide gel electrophoresis analysis.Polyacrylamide gels consisted of a 10-mm-long stacking gel (5% acrylamide) and a 35-mm-long running gel (15% acrylamide). The buffer system of Laemmli was employed (22). Prior to electrophoresis, crude polysaccharide samples (10 μg each) were resuspended in 25 mM Tris-HCl (pH 8.0)-5 mM β-mercaptoethanol-0.5% SDS and incubated at 100°C for 10 min. Samples were loaded directly onto the gel without centrifugation. After electrophoresis, the polysaccharides were stained with silver using a Bio-Rad Silver Stain kit.
Total hexosamine assay.After centrifugation, total hexosamine was measured in the supernatant by using the Morgan-Elson colorimetric assay (33). All assays were performed at least three times with similar results.
Nucleotide sequence accession numbers.The DNA sequence of pgaCD from A. actinomycetemcomitans strain CU1000 was deposited in GenBank under accession no. AY438014 . The DNA sequences of pgaCD and dspB from A. pleuropneumoniae strain IA1 were deposited under accession no. AY618480 and AY618481 , respectively.
A. actinomycetemcomitans pgaC mutants are defective in exopolysaccharide production.We constructed two isogenic mutants of A. actinomycetemcomitans strain IDH781N that contained transposon insertions in pgaC by using in vitro transposon mutagenesis, natural transformation, and homologous recombination (Fig. 1). The mutant strain JK1048, which contained a transposon insertion in the middle of pgaC, bound to polystyrene with the same avidity as the wild-type strain (Fig. 2A). Strain JK1047, which contains a transposon insertion in a gene (flp-1) required for type IV pilus production and biofilm formation (16), failed to bind to polystyrene. Unlike biofilms produced by the wild-type strain IDH781N, JK1048 biofilms were resistant to removal by the carbohydrate-modifying agent sodium metaperiodate and sensitive to detachment by proteinase K (Fig. 2B). These results are consistent with the hypotheses that JK1048 was deficient in exopolysaccharide production and that expression of pgaC was not required for surface attachment. Mutant JK1049, which contained a transposon insertion near the 3′ end of pgaC (Fig. 1), displayed the same adherence and autoaggregation phenotypes displayed by strain JK1048 (data not shown).
To test whether pgaC was involved in intercellular adhesion, we grew biofilms in polystyrene petri dishes and then scraped the biofilm cells from the polystyrene surface and transferred them to a tube (Fig. 2C, left). Five minutes after being vortexed, cells of the wild-type strain had settled to the bottom of the tube, whereas JK1048 cells remained in suspension. After 15 min, JK1048 cells had begun to settle, whereas cells of the flp-1 mutant JK1047 remained in suspension for long periods of time (data not shown). A plasmid carrying the wild-type A. actinomycetemcomitans pgaCD genes (pVK82) restored the ability of JK1048 cells to rapidly autoaggregate (Fig. 2C, right). These findings indicate that strain JK1048 exhibited a reduced autoaggregation phenotype.
To demonstrate that the pgaC mutant was deficient in exopolysaccharide synthesis, we grew wild-type strain IDH781N and mutant strain JK1048 on nutrient agar containing Congo red dye, which has an affinity for bacterial exopolysaccharides (1, 9, 39). Strain IDH781N produced red colonies on Congo red agar, whereas strain JK1048 produced white colonies (Fig. 2D). Plasmid pVK82 restored the ability of JK1048 colonies to bind Congo red dye.
To confirm that the pgaC mutant was deficient in exopolysaccharide production, we analyzed crude polysaccharide isolated from the wild-type strain IDH781N and the mutant JK1048 by polyacrylamide gel electrophoresis (Fig. 3). Strain IDH781N produced an abundant, high-molecular-weight material that was retained in the stacking gel during electrophoresis (lane A). This material was absent from cells of strain JK1048 (lane B), and its production was restored by transformation of JK1048 with the complementary plasmid pVK82 (lanes C and D). This material was resistant to extraction by phenol (lanes E and F) and to digestion by proteinase K (data not shown), consistent with the hypothesis that it contained primarily polysaccharide. The material was degraded by dispersin B (lanes G and H) and was absent from IDH781N cells that were pretreated with dispersin B (lanes I and J). Cells of a different serotype of A. actinomycetemcomitans (strain CU1000) also produced the high-molecular-weight material (lane K), which was degraded by dispersin B (lane L). These findings indicate that A. actinomycetemcomitans pgaC encodes the production of a high-molecular-weight extracellular polysaccharide that is a substrate for dispersin B.
Enzymatic treatment of exopolysaccharide suggests the presence of a hexosamine-containing component.We treated A. actinomycetemcomitans cells with dispersin B and analyzed the cell supernatants for the presence of total hexosamine (Fig. 4A). Supernatants from mock-treated IDH781N cells contained no detectable hexosamine (<10 nmol per mg [wet weight] of cells), whereas supernatants from dispersin B-treated IDH781N contained high levels. Treatment of crude polysaccharide isolated from strain IDH781N resulted in the release of a similar amount of total hexosamine (126 ± 12 μmol per mg of polysaccharide). Cells of the pgaC mutant strain JK1048 released no detectable levels of total hexosamine, even after treatment with dispersin B (Fig. 4A). The complementary plasmid pVK82 restored the ability of JK1048 cells to release hexosamine after treatment with dispersin B. These data indicate that the pgaC-dependent exopolysaccharide produced by A. actinomycetemcomitans contains a hexosamine component.
Evidence that the A. actinomycetemcomitans pgaC-dependent polysaccharide is structurally related to E. coli PGA and S. epidermidis PIA. E. coli strains DH5α and TRXWEC (pgaC mutant) were transformed with plasmid pPGA372, which contains the E. coli pgaABCD locus and promoter region (37). The transformants released high levels of hexosamine when treated with dispersin B (Fig. 4B). The levels of hexosamine released by strains DH5α and TRXWEC harboring the vector plasmid pUC19 were similar, suggesting that pgaC is absent or not expressed in strain DH5α. The level of hexosamine released by DH5α harboring pPGA372 was similar to the level released by IPTG-induced DH5α cells harboring pVK82 (containing A. actinomycetemcomitans pgaCD) or pVK91 (containing S. epidermidis icaAD) (Fig. 4C). These findings suggest that E. coli pgaC, A. actinomycetemcomitans pgaC, and S. epidermidis icaA encode the production of similar hexosamine-containing polysaccharide substrates for dispersin B. In addition, wild-type A. actinomycetemcomitans IDH781N cells, but not cells of the pgaC mutant JK1048, cross-reacted with antiserum raised against S. epidermidis PIA (Fig. 5). Taken together, these data suggest that the pgaC-dependent polysaccharide produced by A. actinomycetemcomitans is structurally related to E. coli PGA and S. epidermidis PIA.
Evidence that A. pleuropneumoniae biofilms produce a hexosamine-containing extracellular polysaccharide.Two different wild-type strains of A. pleuropneumoniae released high levels of total hexosamine when treated with A. actinomycetemcomitans dispersin B (Fig. 4D), suggesting that A. pleuropneumoniae cells produce a hexosamine-containing extracellular polysaccharide similar to that produced by A. actinomycetemcomitans. The genome of A. pleuropneumoniae contains a locus that is homologous to A. actinomycetemcomitans pgaABCD (46, 46, 68, and 31% amino acid identity, respectively). When E. coli DH5α cells carrying the A. pleuropneumoniae pgaCD genes on a plasmid (pVK93) were treated with dispersin B, high levels of total hexosamine were released (Fig. 4D). These data suggest that the A. pleuropneumoniae pgaCD genes are orthologous to E. coli and A. actinomycetemcomitans pgaCD. The genome of A. pleuropneumoniae also contains a homologue of A. actinomycetemcomitans dspB (59% amino acid identity). Purified recombinant A. pleuropneumoniae DspB protein caused the detachment of A. pleuropneumoniae biofilms from surfaces (Fig. 6A). A. actinomycetemcomitans dispersin B also caused the detachment of A. pleuropneumoniae biofilms from surfaces (Fig. 6A), and A. pleuropneumoniae DspB caused the detachment of A. actinomycetemcomitans and S. epidermidis biofilms from surfaces (data not shown). These data suggest that A. pleuropneumoniae DspB exhibits the same substrate specificity as A. actinomycetemcomitans dispersin B. Like A. actinomycetemcomitans biofilms, biofilms produced by wild-type strains of A. pleuropneumoniae were sensitive to detachment by sodium metaperiodate and resistant to removal by proteinase K (Fig. 6B and C). Taken together, these data suggest that A. pleuropneumoniae biofilm cells produce a hexosamine-containing polysaccharide intercellular adhesin that is structurally and functionally related to the pgaC-dependent polysaccharide produced by A. actinomycetemcomitans.
Our findings demonstrate that the pgaABCD locus of A. actinomycetemcomitans encodes the production of a high-molecular-weight, hexosamine-containing extracellular polysaccharide adhesin that is a substrate for dispersin B. This polysaccharide is structurally and functionally related to E. coli PGA and S. epidermidis PIA, suggesting that it is composed of a linear polymer of GlcNAc residues in β(1,6) linkage and that this linkage is the natural substrate for dispersin B. Because of the genetic similarity between the pgaABCD loci of A. actinomycetemcomitans and those of E. coli, this polysaccharide is referred to here as A. actinomycetemcomitans PGA.
Our data indicate that PGA is a component of A. actinomycetemcomitans biofilm colonies. Our findings suggest that PGA may play a role in intercellular adhesion within the biofilm colony and detachment of cells from the colony (17, 18, 20). PGA does not appear to play a major role in A. actinomycetemcomitans surface attachment (Fig. 2A) or biofilm colony formation (J. B. Kaplan, unpublished data), phenotypes that have been shown to be mediated by proteinaceous, adhesive type IV pili (5, 6, 14-16). The fact that pgaC mutants were sensitive to detachment by proteinase K (Fig. 2B) suggests that PGA may also function as a physical and/or chemical barrier which prevents the access of external agents to the bacterial cells and adhesive pili within the biofilm (3). PGA may account for the increased resistance to antimicrobial agents exhibited by A. actinomycetemcomitans biofilm cells compared to that exhibited by cells grown in planktonic form (4). A similar protective function was ascribed to S. epidermidis PIA (36).
Our findings constitute the first report of biofilm formation in A. pleuropneumoniae. Our data suggest that the A. pleuropneumoniae pgaCD and dspB genes are orthologues of A. actinomycetemcomitans pgaCD and dspB, respectively. The similar detachment phenotypes exhibited by biofilms produced by wild-type A. pleuropneumoniae and A. actinomycetemcomitans strains (Fig. 2B and 6B and C) further suggest that A. pleuropneumoniae forms biofilms by mechanisms analogous to those employed by A. actinomycetemcomitans. Biofilm formation has been shown to play an important role in the colonization and pathogenesis of A. actinomycetemcomitans (7, 31). Biofilm formation may also have relevance to the colonization, pathogenesis, and transmission of A. pleuropneumoniae.
Homologues of the pgaABCD locus are present in the genomes of several other beta and gamma Proteobacteria, including Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Burkholderia cepacia, Chromobacterium violaceum, Photobacterium profundum, Pseudomonas fluorescens, Ralstonia solanacearum, Xanthomonas axonopodis, Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis. Our findings support the hypothesis that these homologous loci encode the production of hexosamine-containing exopolysaccharides that stabilize biofilms in these species (37).
We thank Mrinal Bhattacharjee and David Figurski (Columbia University) for providing the protocols and accompanying bacterial strains and plasmids used for natural transformation of A. actinomycetemcomitans; Xin Wang and Tony Romeo (Emory University) for providing E. coli strains and plasmids; Lorraine Hoffman and Tim Klinefelder (Iowa State University) for providing A. pleuropneumoniae strains; David Furgang for technical assistance; Bruce Roe, Fares Najar, Allison Gillaspy, Sandra Clifton, Tom Ducey, Lisa Lewis, and Dave Dyer of the Actinobacillus Genome Sequencing Project for providing sequence data prior to publication; and Daniel Fine for helpful assistance throughout the course of the project.
This work was supported in part by Public Health Service awards DE15124 (to J.B.K.) and DE12585 (to N.R.) and a grant from the Deutsche Forschungsgemeinschaft (SFB 470 Teilprojekt C10) to D.M.
- Received 7 May 2004.
- Accepted 7 September 2004.
- Copyright © 2004 American Society for Microbiology


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Abstract – 2017 ESCAP Congress, Geneva
Original state of the art abstract, full audio lecture and original slide presentation by Raquel E. Gur (Lifespan Brain Institute / University of Pennsylvania, USA) on Early course and intervention in youth at-risk for psychosis: results from longitudinal studies (ESCAP 2017 Congress in Geneva, Switzerland).
The increased focus on early identification of subthreshold psychotic symptoms provides an opportunity to examine the emergence of psychosis, follow individuals longitudinally and evaluate the effects of early intervention on functional outcome. Most studies of youth at risk for psychosis have capitalized on help-seeking individuals and examining predictors of transition to schizophrenia spectrum disorders. Several pre-presentation environmental variables are emerging as potential predictors of early course of psychotic illness. Interventions vary and include pharmacological and behavioural approaches. While there is no evidence that current pharmacological approaches to the treatment of schizophrenia are effective in impacting clinical risk, cognitive remediation and other behavioural treatment are increasingly used.
The presentation will highlight complementary approaches we have applied to a community sample of youths at risk for psychosis and to help seeking individuals early in the psychotic illness. The early presentation is a dynamic process characterized by anxiety, depression and subthreshold positive and negative symptoms. There is evidence for cognitive deficits and neuroimaging abnormalities indicative of neurodevelopmental aberrations. The main neurocognitive deficits associated with risk for psychosis included complex cognition and social cognition.
The importance of negative symptoms in the early course of psychosis and the adverse effects of neurocognitive deficits on functioning led us to examine the feasibility of remedial programs that target complex cognition and social cognition. To ameliorate cognitive deficits we are currently examining a Cognitive Remediation intervention consisting of eight 120-minute sessions in a small group format (BrainHQ modules, Posit Science Inc). To address negative symptoms and deficits in social cognition we have implemented Theater Improvisation Program to improve Social Cognition (TIPS), an 18 session CBT-informed theater training provided by professional Theater Directors. We will present preliminary results from these interventions and plans to disseminate them into publicly available intervention "toolkits" for clinicians and investigators.
Listen to the full lecture, or download the audio file.
View Raquel Gur's slide presentation (pdf, 48 pages).
Read more about Raquel Gur (pdf, 10 pages).
Subscribers to the ESCAP News Alert service will receive our updates on child and adolescent mental health issues and event news. Note that ESCAP carries no conflicts of interest – we never send commercial messages.

subset: commoncrawl


text:
Prof. Nazif M. SHAHRANI
Indiana University, USA
He was born in Afghanistan- Bedehşan. He finished his license in 1967 at Kabil University, Faculty of Education. He completed his post graduate at Hawaii University, his master and doctor's degree at Washington University in anthropology field. He made anthropologic studies in Wakhan district which is between pastoral nomad Kirghiz and Wakhi community. He became the consultant of "The Kirghiz of Afghanistan" named ethnographic movie. He worked with Nevada University, Stanford University and UCLA. He made field researches on Afghanistan, Turkey, Pakistan and Uzbekistan. He has been a lecturer at Indiana University Departments of Anthropology, Middle Eurasia Studies, Near East Languages and Cultures since 1990.


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Found search engine
Original recipe serves 4
3/8 cup sugar 0.1665 cup cocoa powder 1 tablespoon all-purpose flour 0.1665 cup milk 1/8 cup butter ; lightly beaten 1 egg(s) 4 ounces cherries ; fresh or canned cherry pie filling 0.5 pie shell
You will also need: saucepan
Black Forest Pie
Submitted by Laura1
Cherries, chocolate and flaky crust
Original recipe serves 4
½ 2x 3x Reset
Ingredients (serves 2)
You will also need: saucepan
In a saucepan, combine sugar, cocoa, flour and milk until smooth. Add butter. Bring to a boil - cook and stir for 2 mins. Remove from heat. Stir a small amount of hot mixture into eggs. Return to pan. fold half of cherry pie filling in. Pour into pastry shell. Bake at 350 degrees for 35-40 mins. Cool completely on wire rack. Before serving top with remaining pie filling.


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Tipp step on the gas in extra-time
Tipperary 2-22 Cork 0-25
By Fintan O'Toole
Thursday, June 04, 2009
AFTER an enthralling 80-minute saga, Tipperary eventually edged past Cork in last night's Bord Gáis Energy Munster U21HC quarter-final at Semple Stadium.
In a match that crackled with electricity throughout, Tipperary conjured up two key factors to enable them to breast the tape in first place. Plundering the only two goals of the game was of immeasurable value, while their big names came to the fore as the action trundled into extra-time.
Defensive lynchpins Padraig Maher and Thomas Stapleton generated storming finishes, while Noel McGrath hurled a world of ball when switched to midfield. In attack, Seamus Callanan made several lung-bursting runs deep into the Cork defence, while Pa Bourke's gilded attacking play registered a hefty 1-9 total against the backdrop of a team total of 17 wides.
It was rough justice on Cork to wind up with the losers' garlands. They fought vigorously from the start with a robust defence to base their challenge on. Paudie O'Sullivan was immense at centre back with Aidan Walsh and Conor O'Sullivan putting in great shifts elsewhere in the backline.
In attack Patrick Horgan was in luminous form but struggled lacked of a supporting cast. Subs Luke O'Farrell and Padraig Gould did enough when introduced to suggest they should have started, while Lorcan McLoughlin did swing over three fine points.
Normal time was a gripping affair with the sides sticking close to each other from the off. In all there was equality on the scoreboard on 13 occasions during the 60 minutes.
The first half was peppered with goal opportunities that Tipperary missed but before half-time they got the move and execution just right.
An errant handpass in the Cork defence was seized by Pa Bourke who transferred to Callanan, and the centre-forward eschewed the option of tapping over the bar to dispatch the ball towards Shane Bourke. The JK Brackens man turned neatly and lashed a drive to the far corner.
In the scoring stakes the match could have been distilled down to a shootout between the side's respective free-takers in the first half, Patrick Horgan and Pa Bourke displaying laser-like accuracy from dead balls.
Horgan also sparkled from open play with a beautiful brace of points to complement his three frees, while Bourke arrowed over six points in the first half. Midfielder Leigh Desmond chipped in with an important pair for Cork as well, but it was Shane Bourke's late salvo that saw Tipperary lead 1-8 to 0-9 at the interval.
Cork composed a splendid start to the second half reeling off points from Horgan, 3) and Andy Walsh to nudge themselves 0-13 to 1-9 in front.
But then Tipperary pounced for a critical score. Callanan made a lung-bursting run on the wing, passed inside to Patrick Maher who was fouled and Pa Bourke crashed the resultant penalty to the net.
That score nourished Tipperary's hopes and they stormed on looking far more assured. McGrath's switch to midfield paid a handsome dividend as he dictated plays, corner-backs Donagh Maher and Kevin Maher attacked the ball with great success and when McGrath clipped over a 49th minute point, the tide of momentum seemed to lay behind Tipperary as they led 2-12 to 0-15.
But Cork summoned up a wonderful recovery and created a gripping finale. They strung together a sequence of points from Gould, O'Farrell, 2) and McLoughlin to creep ahead by one. Tipperary's retort consisted of Bourke and Callanan points to restore them to the ascendancy, but an exquisite sideline cut by Desmond tied it up again.
Substitute John O'Neill, who had an enterprising cameo when introduced, appeared to have nailed victory for Tipperary only for a rousing score by McLoughlin to deliver the spectre of extra-time, at 2-15 to 0-21.
Tipperary made a clinical burst early on in extra-time and it proved essential to the outcome. Callanan started to weave incisive runs and his brace helped send them 2-18 to 0-22 clear. Cork replied forcefully with a Horgan free but the Glen Rovers dangerman was then denied a goal by a point-blank save by Bill McCormack.
It proved Cork's best chance at getting the goal their challenge badly required and Tipperary were in no mood to relinquish superiority from thereon in. Callanan and Bourke teamed up to insert daylight between the sides late on, and O'Neill applied the coup de grâce to seal their passage through to a semi-final date with Waterford on July 15.
Scorers for Tipperary: P Bourke 1-9, 0-5f, 1-0 pen, 0-1 '65); S Callanan 0-6, 0-1f, 0-1 '65); S Bourke 1-1; J O'Neill 0-3; N McGrath 0-2; M Gleeson 0-1.
Scorers for Cork: P Horgan 0-12, 0-9f; L Desmond, 0-1 sideline; L McLoughlin 0-3 each, L O'Farrell, P Gould 0-2 each, W Egan, S White, Andy Walsh 0-1 each.
TIPPERARY: B McCormack; D Maher, Padraig Maher, K Maher; B Maher, T Stapleton, T McGrath; S Hennessy, G Ryan; M Gleeson, S Callanan, N McGrath; P Bourke, Patrick Maher, S Bourke.
Subs: B Fox for T McGrath, 28; S Carey for Gleeson, 40; J O'Neill for Hennessy, 51; S Quinlan for Carey, 58; J Coghlan for K Maher, 70; S Hennessy for S Bourke, 77.
CORK: C Cronin; K Murphy, J Herlihy, C O'Sullivan; Aidan Walsh, P O'Sullivan, S White; L Desmond, W Egan; B Corry, L McLoughlin, R White; R O'Driscoll, P Horgan, Andy Walsh.
Subs: L O'Farrell for O'Driscoll, 34; C O'Driscoll for Murphy, P Gould for White, both 40; R Clifford for Andy Walsh, 48; E Keane for O'Herlihy, C McCarthy for Clifford, both 70; G O'Connor for Egan, 77.
Referee: Pat Casey (Waterford).
Prolific Horgan not enough to stop Tipperary advancing
Thursday June 04 2009
HOLDERS Tipperary clinched a dramatic extra-time victory against Cork at Semple Stadium last night in front of 4,138 spectators.
The Premier County now travel to Walsh Park to face Waterford in the semi-final on July 15.
Lorcan McLoughlin's 59th-minute point for Cork sent the tie into extra-time but both sides missed chances to win it before two additional periods of 10 minutes.
This incredible tie was level on 13 occasions, including eight times in the first half before Shane Bourke's goal on the stroke of half time handed Tipp a 1-8 to 0-9 lead.
Tipp grabbed a second goal when Pa Bourke netted a penalty eight minutes into the second half.
Last year's beaten All-Ireland finalists Tipp, with nine senior panellists in their starting team, registered 17 wides and struggled to shake off a dogged Cork, inspired by 12 points from Patrick Horgan.
However, leading by 2-18 to 0-23 after the first period of extra-time, a late surge sealed it for Tipp.
Scorers — Tipperary: P Bourke 1-9 (1-0 pen, 5f, 1 65), S Callanan 0-6 (1f, 1 65), S Bourke 1-1, J O'Neill 0-3, N McGrath 0-2, M Gleeson 0-1. Cork: P Horgan 0-12 (9f), L McLoughlin, L Desmond 0-3 each, P Gould, L O'Farrell 0-2 each, S White, W Egan, A Walsh 0-1 each.
TIPPERARY — B McCormack; D Maher, Padraic Maher, K Maher; B Maher, T Stapleton, T McGrath; S Hennessy, G Ryan; M Gleeson, S Callanan, N McGrath; P Bourke, Patrick Maher, S Bourke. Subs: B Fox for T McGrath (28), S Carey for Gleeson (41), J O'Neill for Hennessy (51), S Quinlan for Carey (58), J Coghlan for K Maher (70+1), S Hennessy for S Bourke (77).
CORK — C Cronin; K Murphy, J Herlihy, C O'Sullivan; A Walsh, P O'Sullivan, S White; L Desmond, W Egan; B Corry, L McLoughlin, R White; R O'Driscoll, P Horgan, A Walsh.
Subs: L O'Farrell for R O'Driscoll (34), P Gould for R White (42), C O'Driscoll for Murphy (42), R Clifford for Walsh (48), E Keane for Herlihy (70), C McCarthy for Clifford (70), G O'Connor for Egan (77).
Ref — P Casey (Waterford).


text:
or 4 payments of
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1981-88 Cutlass Front License Mount Bracket Cover. We have sold a ton of front tag brackets and only recently had a few requests for the delete. So if you want that clean look with out the bracket we have reproduced the new DELETE Bracket in Polished stainless steel.
This part replaces the original GM part number #22511203.


text:
From: Lisa Guillemin <firstname.lastname@example.com>
Greetings fellow class of 76 falcons! Hoping this finds everyonewell and in good spirits. As happens every year, March has now becomeApril (no fooling.) Before long, April turns to May. Yes, springrepresents renewal, starting fresh and, of course, graduation. Have40 years passed since we tossed our caps in the air the evening ofJune 17, 1976? Yep!
So much has happened since then. We have married/found oursoulmate, started and raised families, graduated college, earnedcertification or professional license, began and changed careers.When we speak of graduation today, far more recent and meaningfulgraduation memories come to mind than from our high school.
While each passing decade takes us further from 1976, there issomething that remains and unites us still. Yes, we graduated highschool together but it is the hundreds and hundreds of days beforethen that we have shared. We were young together. That alone is worthcelebration. Our reunion committee members, Sue Baker Putman, KathyAllen, Nancy DeGange-Shannon, Jim Hazelton and Lisa Guillemin are soexcited to be planning this celebration
Who: the committee members listed. What: C.V.H.S. Class of 76,40-year reunion.
Where: La Cañada/Flintridge Country Club.
When: Saturday August 20, 2016, reception at 7:00, buffet dinnerat 8:00. Any why? One of our classmates gave the best answer &emdash;because.
The committee checked out several venues for our 40-year reunioncelebration and found the La Cañada Country Club to be themost economical as well as closest to our old "stomping grounds"where we made many of the memories we now share. If circumstancesprevent your attending our reunion, we understand. You are with us inspirit. For all others, our evening together promises to be greatfun!
Please RSVP soon. firstname.lastname@example.com
From: William Birney <firstname.lastname@example.com>
Date: April 7, 2016 at 11:37:03 AM PDT
To: firstname.lastname@example.com
Subject: Class of 1976 update
Hello, I'm Bill A. Birney from CV class of 76 and I'm happy toannounce a Facebook-created event to coincide with our upcoming40-year class reunion. Friday night 8/19/2016 the night before ourbig high school reunion in La Cañada, I've organized apre-reunion dinner.
To all alumni of 1976 and surrounding years as well, pleasecome join all of us Falcons to help kick off what will be an awesomeclass of 76 reunion weekend :-) Feel free to look me up on Facebookfor more info
"Bill A Birney" see you there!!!!


text:
In this article, we will talk about the amazing uses of rhenium. Rhenium is a rare refractory metal that not only has good plastic, mechanical and creeps properties, but also has good wear and corrosion resistance. Rhenium can be processed into rhenium plates, rhenium rods, sheets, as well as wires, and are widely used in aerospace, electronic industry, petrochemical industry, and other fields.
According to data released by the United States Geological Survey in 2013, superalloys are the largest consumer of rhenium, accounting for about 80% of the total, and catalysts are the second largest.
The global reserves of rhenium are very small and the price is relatively expensive. Therefore, rhenium is mostly used in alloy form. Rhenium, as an alloy additive, can greatly improve the properties of the alloys.
Rhenium can form a series of alloys with tungsten, molybdenum, platinum, nickel, thorium, iron, copper and other metals. Rhenium tungsten, rhenium molybdenum, and rhenium nickel superalloys are the most important rhenium alloys, which are widely used in the aerospace and electronics industries.
Tungsten-rhenium alloy (W-Re alloy) is an alloy composed of tungsten and rhenium. One type is low-rhenium alloys with rhenium content of less than 5%. The other is per-rhenium alloys, which contain 20% to 30% rhenium.
The rolling deformation of molybdenum with 35% rhenium can reach 90% at room temperature, and the tensile strength, ductility, and resistivity of molybdenum-rhenium alloys increase with the increase of rhenium content at room temperature. Mo-5%Re and mo-41% Re are commonly used as thermocouple wire materials for aerospace structural materials.
Tungsten-rhenium alloy and molybdenum-rhenium alloy made by adding the appropriate amount of rhenium into tungsten and molybdenum thermocouple wire not only has good plasticity, but also can be processed into various structural materials, and have high hardness, high strength, and high-temperature resistance.
Molybdenum-rhenium alloy is often used to manufacture high-speed rotating X-ray tube target material, long life grid plate for microwave communication, space reactor core heating tube, high-temperature furnace heater, and high-temperature thermocouple.
Tungsten-rhenium alloy is widely used in high-temperature technology, electric vacuum industry, bulb industry, atomic energy industry, analysis technology, medical treatment and chemical industry and other fields, and is often used in the manufacture of special electronic tube and color picture tube filament, high-temperature components, thermocouple and so on.
The uses of rhenium in engines can improve the creep strength of nickel superalloys, which can be used in the manufacture of combustion chambers, turbine blades and exhaust nozzles of jet engines.
Rhenium nickel alloy is the core material of the modern jet engine blade, turbine disc, and other important structural components. The uses of rhenium can not only improve the performance of nickel-based superalloys but also produce alloys for single crystal blades. Both effects can enable turbines (especially high-pressure turbines) to work at higher temperatures.
In addition, by keeping operating temperatures low, the difference between the actual operating temperature and the turbine's maximum permissible temperature can be widened, thus extending service life.
Rhenium can improve the strength and plasticity of tungsten, molybdenum, and chromium at the same time, which is known as the rhenium effect.
For example, if a small amount of rhenium (3% ~ 5%) is added, the starting temperature of the recrystallization of tungsten can be increased by 300℃ ~ 500℃. W-Re and Mo-Re alloys, used in aerospace, aviation high-temperature structural parts (nozzle, nozzle, heat shield, etc.), elastic components and electronic components.
Rhenium and its compounds have excellent catalytic activity (rhenium has the highly selective catalytic function for many chemical reactions), so rhenium is often used as a catalyst in the petrochemical industry and other fields.
Catalytic reforming is one of the petroleum refining processes and an important means to improve gasoline quality and produce petrochemical raw materials. Rhenium platinum alloy, as a catalyst in the process of catalytic reforming, can improve the depth of reforming reaction and increase the yield of gasoline, aromatics, and hydrogen.
In addition, rhenium can be used as a catalyst for automobile exhaust purification, while rhenium sulfide can be used as a hydrogenation catalyst for cresol and lignin.
Thank you for reading our article and hope you enjoyed it. If you want to know more about rhenium and the amazing uses of rhenium, you can visit Advanced Refractory Metals (ARM) for more information, which supplies people with high-quality refractory metals all over the world.
Copyright © 1994-2019 Stanford Advanced Materials, All Rights Reserved.


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Technical Progress Report July, 1st - July, 15th
Development Report July 1st – July 15th
Hello, and welcome to the Stegos Development report for our 20th Sprint, covering the period from July 1st through July 15th.
The entire development team has been firing on all pistons in preparation for the launch of our mainnet. With the launch just a few weeks away, most of our hard work has been geared towards testing the blockchain and putting finishing touches to the Stegos app.
App improvements
We've added several features to the Stegos wallet app, including support for multiple accounts within a single wallet. We've also implemented transaction history and transaction status tracking to make sending and receive STG a breeze.
Verifiable delay functions
Verifiable delay functions (VDFs) have been added to the blockchain core code. VDFs make block times more predictable, which is particularly important in the early stages of our chain when usage patterns are likely to be irregular. Keeping block times stable will help us to manage the annual inflation of STG tokens.
Stress testing
It's important to check that the Stegos blockchain is robust before we launch. Using an automated transaction generator, we stress-tested all the vital blockchain subsystems to ensure they can handle heavy transaction loads.
Upcoming Tasks
Sprint 21 will hopefully be the final sprint before the mainnet launch. We'll be continuing with stress testing and adding more VDFs, including replacing Hashcash in the network authorization protocol.
We'll also be putting the finishing touches to the wallet app, to ensure that everyone has a smooth experience with Stegos right out of the gate.
As always, if you want to help with testing, contact us in the Telegram group to request some test tokens.
Please join us on Telegram Chat to get test tokens and let us know your thoughts. Subscribe to official Telegram Channel for the latest news.


text: This Masters Hat is exclusively available to members only and we were able to purchase a few at Berckmans Place for you! This Masters Berckman's hat features a raised leather circle with a gold Masters logo on the front. The strap on the back is leather and is fully adjustable. It gives you a classic look with great comfort.


text:
Create a Ning Network!
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Met dank overgenomen van EUobserver (EUOBSERVER), gepubliceerd op maandag 12 november 2012, 9:25.
An independent Scotland would be welcomed into the European Free Trade Area (EFTA) if it could not secure EU membership. EFTA countries adopt most EU single market legislation without voting rights or contributions to the EU budget. EFTA spokesman, Tom Gronningsater, said that EFTA membership offered "several advantages" to Scotland.
Tip. Klik hier om u te abonneren op de RSS-feed van EUobserver
Meer over ...


text: Puma mixes up earth tones with vivid accent colors for the Puma Easy Rider Trek model. Ever since its debut, the Easy Rider has become one of Puma's most popular sneakers. The combination suede and leather upper with a carbonized multi-level sole made it revolutionary for its time, and people still love the Easy Rider today. The Trek model is a fresh take on an original and perfect for the spring. They sport a unique colorway that won't be denied.


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There will be a series of exclusive events at LEGOLAND® Discovery Centre Manchester taking place from Friday 10th February to Sunday 12th March.
Visitors can come meet Gotham City's caped crusader in LEGO form and have a picture taken with the scene-stealing star. Activities also include a themed scavenger hunt and LEGO Batman™-inspired LEGO builds with our Master Model Builder, Alex. No extra ticket required!
Other facilities at LEGOLAND® Discovery Centre, including the 4D cinema, rides and creative play sessions in the builder workshops, are also available for visitors.
Spend This Valentine's With Your One True Love – LEGO®
As LEGOLAND® Discovery Centre Manchester hosts AFOL Valentine's Evening
Description: Production:Clients M-O:Merlin Attractions:LDC:Images:heart.pngIf LEGOLAND® Discovery Centre's Adult Fans of LEGO® (AFOL) nights are the highlight of your calendar, then the wait is over as LEGOLAND® Discovery Centre Manchester, at intu Trafford Centre Manchester, is hosting a Valentine's special on Tuesday 14th February, for one night only.
LEGO® lovers shouldn't delay in booking this AFOL singles night, where budding builders can reignite their imaginations and enjoy an exciting evening with hundreds and thousands of LEGO® bricks and, of course, like-minded people that share the same passion!
A fun-filled Valentine's alternative, LEGOLAND® Discovery Centre Manchester's AFOL night starts at 7pm and is priced at £20 per person, wine and canapés are also included. Make full use of the facilities, including 4D cinema, rides and enjoy some creative play sessions in the builder workshops.
Some of the centre will be sectioned off, as LEGOLAND® Discovery Centre gears up for an all-new makeover. Launching in Spring, the new-look LEGOLAND® Discovery Centre will include a brand new LEGO® Ninjago City Adventure play feature, new floor-to-ceiling window café, a new bigger and better Creative Workshop where LEGO® fans can watch the LEGOLAND® Discovery Centre Master Model Builder at work, a newly located LEGO® Racers Build & Test Zone and new Birthday Party rooms with three brand new Birthday Party experiences.


text:
... the Table Lands of South Africa...don't bother... get it all done at your one stop Province: Newfoundland.
These formations were once part of the Earth's Mantle!
Here is the Earth's mantle exposed. There is very little growth as the rock is mostly Peridote which lacks the nutrients to support most plant life.


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This summer I had the pleasure of judging the very well organized Oregon State Fair wine competition. Elaine Lisle, Desa Swaim and their crew did a tremendous job and I truly enjoyed getting to better know my fellow judges. Last week I received the results and my judge's notes in the mail.
Below is a list of my favorite wines including the medal won (if available):
Phelps Creek Chardonnay
2007 - Nice toasty notes with lemon cream and a little baking spice. Integrated with a long finish. Gold Medal.
Amity Vineyards Pinot Blanc
2007 - Pretty fruit salad, melon and floral aromas with a touch of white pepper. Nicely balanced with acid and finishes clean -- very refreshing.Silver Medal.
Orchard Heights Gewürtztraminer
2008 - Beautiful aromas of slate, rain, sweet basil and honeysuckle. A little pétillance on the palate with floral and pear flavors. Nice acid balance with a clean finish. Silver Medal.
Chateau Lorane Viognier
2006 - I'd never heard of this winery before but I also liked its Tempranillo (see below). Lavender, floral and spicy aromas lead to a very pretty, ripe and round mouth-feel. Finishes lush and clean. Silver Medal.
Firesteed Dry Rose
2008 - watermelon, cranberry and herbal notes. Nice bright acidity leads to mouth-watering refreshment -- needs a good sunset. Bronze Medal.
Coelho Pinot Noir
2006 - Aromas of raspberry, sour cherry and pretty baking spices. Balanced with excellent structure. Gold Medal and Best of Red.
Stangeland Winery Pinot Noir
2006 - Cranberry, raspberry and nutmeg aromas with hints of dark chocolate. Bright, mouth-filling fruit with a solid finish. Bronze Medal.
Chateau Lorane Tempranillo
2004 - A huge nose filled with deep black cherry, raspberry and milk chocolate. Plus notes of fresh leather and curing tobacco. Ripe and round in the mouth with good tannic structure. One of my top three.
Devitt Merlot
2004 - Another new winery for me. Nose filled with plum, sage, fresh leather, cigar box -- it's a touch of old world mixed with ripe fruit. Nice attack and lingering finish. Silver Medal.
Zerba Cellars Red Blend
2006- Green peppercorn, pine cone and leather aromas matched by ripe blueberry, blackberry and raspberry. A hint of mint on the palate with nice balance between fruit and acid; great length. Gold Medal.
Devitt Cabernet Sauvignon
2005 - Nose of cigar box, cedar, black fruit and new leather saddle. Big, juicy black fruit with a silky mouth feel. Great lingering finish.
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December 2017


text:
Posted February 6, 2019
Fastenal January sales climb 13 percent
Fastenal recorded sales of $446.9 million in January, a 13 percent increase from $394.4 million in January 2018.
Daily sales of $20.3 million compared to $17.9 million last year.
Sales to manufacturing customers grew by 13.8 percent, while sales to non-residential construction customers increased by 16.7 percent.
Employee headcount of 21,841 grew by 4.9 percent from 20,814 in January of last year.


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Download and extract the IVPN .ovpn config files to your computer system.
In the QNAP web interface, open the AppCenter and search for qvpn.
Click the + Install button to add the QVPN Service app to your device if it is not installed already.
Open the QVPN Service app and under the the VPN Client area, click VPN Connection Profiles. Click the Add button and choose OpenVPN.
Use the file picker dialogue box to choose the VPN server location you prefer and click OK.
Enter your account ID that begins with 'ivpn' and any password in the credential fields and (optionally) change the Profile Name to something different. Click the Apply button.Only your account ID is used for authentication. The password field can be left empty or set to anything if your client software requires a non-blank password.
Note: You are free to choose whichever server location you prefer. Our Japan server is used here only as an example.
Add as many VPN server profiles as you would like.
Click the Connect icon under the Actions column. The connection may take 10 to 30 seconds and the Status indicator will turn green when the connection is successful.
Click the Use VPN as NAS Default Gateway button for extra settings related to the default gateway and failover.
If you check the Allow other network devices in the same subnet to connect to the VPN through the NAS option, change the default gateway on your computer system to the local network IP address of the QNAP device to route all of the Internet traffic on your computer through the NAS VPN.
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Instead of using servers separated by a great distance, you might prefer to use a location with multiple servers. This may offer a more seemless and familiar network experience if failover occurs:
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Gummy Smile treated with Gum Lift and Porcelain Veneers
Doctor's Comments
Excessive gum display can cause people to feel embarrassed to smile, such was the case in this young lady. Here the problem was an over-development of the maxillary jawbone, which caused a gummy smile and the teeth appearing too long.
In certain situations it is possible to provide improvement with braces and orthognathic (jaw-correcting) surgery. However in this case the patient declined this course of treatment and wanted a quicker makeover. This was achieved with a gum lift procedure, followed by porcelain veneers to the upper front 6 teeth.


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Camp Worksop
Xplore's ultimate teenage sports summer camp
for 12 to 16 year olds.
Located on the edge of the Sherwood Forest, is our largest and one of the longest standing boarding schools in the area. Renowned for it's excellent sports facilities, fantastic activity provisions and all-round exciting atmosphere makes Camp Worksop one of the best language camps for social teenagers who love being active, outdoors and surrounded by others.
We are able to offer a varied and extensive multi-activity programme at Camp Worksop due to the number of students we have on site. This gives more activities to choose from each day than any other Xplore camp so there is always something to have a go at!
London: 3 hours Manchester: 1 hour 45 minutes Lincoln: 45 minutes
I think Xplore is really fun! I usually just stay in my house, but at summer camp I can run with my friends and go outside, that is very good.
What is included?
Here is a list of the main elements included in the camp, please use our table below for more detail:
- Return airport transfers (from/to London Heathrow International)
- 14 nights accommodation
- 3 meals a day (specific dietary requirements can be catered for)
- English language classes (inc. course materials)
- Full activity programme
- Excursions
- 24-hour care and supervision
- Endsleigh travel insurance
At Camp Worksop, the activities available are endless and we are able to offer a varied and extensive multi-activity programme due to the number of teenagers we have on site. The great sports facilities include a sports hall, an indoor swimming pool, and tennis /squash courts. There is woodland for team games, bushfire and campfires as well as adventure facilities such as a climbing and abseiling wall. There is even a floodlit artificial turf pitch on-site to use.
English Levels: A2 - B2 CEFR Pathway Table
There are a number of English courses available at Camp Worksop and are best suited to teenagers who have studied English for at approximately two years and want to speak as much English as possible.
Following an initial placement test, our English teachers will place all learners into classes dependent on their test results, this camp offers Elementary (A2), Intermediate (B1) and Advanced (B2) courses in English.
As a teenage summer camp, Worksop provides engaging English courses with interesting topics and focal points to keep the course vibrant and different to traditional school lessons. We understand that improving language skills comes from engaging students with interesting debates and teaching methods.
At the end of the 2-week course, learners will complete an examination for the level they have been learning at. We will then receive a certificate from Anglia Examinations, which confirms your English level in accordance with the Common European Framework of Reference (link). Classroom learning time per week is 20 hours and our class sizes are small with a maximum of 16 students per teacher.
For students achieving a pass grade or higher for B1 and B2, they will receive an Ofqual approved certificate through Anglia Examinations partnership with AIM Awards.
|Dates & Prices
|4th July 2020 - 18th July 2020
|2 Weeks: £1,815
|18th July 2020 - 1st August 2020
|1st August 2020 - 15th August 2020
Amazing Excursions
|Included in the Price (For more information click on the icon below)
Return transfers from and to London Heathrow International Airport (LHR) on arrival and departure dates are included. Please ensure your arrival flight arrives at LHR between 10:00 – 15:00 and your departure flight leaves LHR between 12:00 – 17:00. Unfortunately, flights outside of these times or to/from a different airport will ensure an additional cost – please check with our bookings team first.
Worksop has the capacity to hold up to 300 students and is our largest residential centre over the summer. Campers will stay in 1 to 6 bedded rooms within the huge Manor House and a brand new stand alone accommodation block. Rooms are furnished to a basic but comfortable standard. We always have separate quarters for boys and girls and where possible place campers of a similar age in a room together. If you have any special requirements or would like to request to share with a friend, we will try to accommodate you, just let us know in advance.
Imagine eating every meal inside the Great Hall of Hogwarts – that's what mealtimes are like at Camp Worksop.
Sit with friends on huge banquet tables and enjoy a selection of freshly prepared, nutritious food with both hot and cold options. Dietary requirements can be met when we are aware in advance.
During the 2-week programme, our staff will take all our campers on 3 organised day trip excursions. Spend the day off-campus with new friends and see the sights of London, Liverpool, Manchester, Sheffield, York and Lincoln. (3 excursions during 2-week programme – Wednesday, Saturday, Wednesday)
Care and Supervision
Our Camp Leaders are responsible for the pastoral care of all those in our care and ensure that everyone is sleeping well, eating well, joining in, making friends and having a great time. During free time, our teenagers are able to explore the campus with friends, whilst being supervised by their house parents. Being located in a town, we offer campers the ability to go off-site as an alternative to an activity session; however a member of Xplore staff will always accompany them.
International Camp
Our camps are available to children from all over the world. Integration and immersion is central to how we operate as an organisation, and by encouraging lots of social interaction between different cultures and creating a diverse international community at camp, it can really develop every child's self-confidence in communicating with others
Travel Insurance
Xplore has partnered with Endsleigh Insurance to provide invaluable cover specifically designed for Xplore students. You will receive a summary of insurance cover after booking, however key benefits include:
|English Plus
|English Plus Football


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City Council looking to stiffen civil penalties for hit-run drivers. (credit: Peter Haskell/WCBS 880)
NEW YORK (CBSNewYork) —The City Council is considering imposing tougher civil penalties for drivers involved in fatal hit-and-run accidents.
City Councilman Jimmy Van Bramer, of Queens, said there have been three fatal hit-and-runs in his district in the last 18 months.
He has introduced a bill that would impose a maximum penalty of $5,000 for any driver who hits someone and takes off, CBS 2's Janelle Burrell reported.


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LONDON — "Hoa Hakananai'a" — "Lost or Stolen Friend" in English — is one of the British Museum's most popular exhibits. Standing 7 feet, 9 inches tall, the statue's prominent brow and deep-set eyes are the backdrop for thousands of tourist selfies every year.
But now the government of Chile has announced that it will form a committee to try to recover the 1,000-year-old statue for the Rapa Nui, the indigenous people of Easter Island. Chile annexed the island, which lies more than 2,200 miles from its coast, in 1888.
The Chilean Foreign Ministry said in a statement released on Tuesday that "Hoa Hakananai'a" was a "tangible link" to the island's history. It is "sculpted in basalt — stone that stands out for its hardness and elegance," the ministry said, and also has unique carvings on its back.
A spokeswoman from the Chilean Embassy in London would not provide further details on any formal request for the statue's return.
Chile's taking on the Rapa Nui cause comes as European museums are facing growing pressure to repatriate objects to their countries of origin. President Emmanuel Macron of France, having declared that "African heritage cannot be a prisoner of European museums," has appointed two advisers to plan how to return African objects.
The British Museum is no stranger to pressure to return items from its collection — Greece has waged a long campaign for the restitution of the so-called Elgin marbles. "We believe that there is great value in presenting objects from across the world, alongside the stories of other cultures at the British Museum," a spokeswoman for the museum said in an emailed statement.
Jo Anne Van Tilburg, an archaeologist and director of the Easter Island Statue Project, the longest-running research project into the statues, said by telephone interview that British sailors were taken to a sacred area of the island in 1868, and found "Hoa Hakananai'a" inside a building, buried up to its torso. They bartered for it, and dragged it to their boat in a procession led by a dancing chief, Ms. Van Tilburg said.
"However, this was done within a context where the Rapa Nui people were suffering a great deal of deprivation," she added.
Ms. Van Tilburg said that she had excavated two statues on Easter Island with carvings on their backs — the only others with such decoration among some 1,000 statues. But "Hoa Hakananai'a's" carvings are unique, she added, as is the type of rock it is carved from.
"Hoa Hakananai'a" represents "the entire culture that inspired it," Ms. Van Tilburg said, adding that she understood why the Rapa Nui want it back.
But asked whether the statue should be returned, given that it is an ambassador of sorts for the Rapa Nui culture, Ms. Van Tilburg replied, "I am in two minds."
"If I was asked as a professional researcher to come down on one or the other point of view, it would be that of the Rapa Nui people," she said.
At the British Museum on Wednesday, many tourists were unaware of the controversy, but, when it was explained to them, they held equally mixed views.
Astrid Allard and Ines Oukkate, both 20 and visiting from France, were taking a photo of the statue with their fingers posed in such a way that it looked like they were picking its nose. "I think both sides can have arguments, but it's been here for a very long time, right?" Ms. Allard said. "I think more people can see it here."
But Ms. Oukkate disagreed. "If they want it back, I think it's better for them to have it," she said. "Museums are here to show respect to other countries by showing things to the public."
If a country asks for its items back, and the museum refuses, the respect is gone, she added.
"True," Ms. Allard replied, adding, "Argh — it's a tough debate."
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Legal Blog
The Weekly Scenario: Criteria for Social Security Disability Insurance
Question: Can my wife (soon to be ex-wife) continue to receive SSD (Social Security Disability benefits) after we are divorced? Answer: Yes, assuming certain criteria are met. Social Security Disability Insurance (SSD) provides a benefit to individuals who are considered (i) disabled and (ii) have paid enough money into the Social Security system. Dependent family members of SSD beneficiaries, such as spouses or children, may also receive dependent Social Security benefits. Even after a divorce, an ex-spouse may still be entitled to collect SSD benefits. Keep in mind that for SSD, there is no minimum threshold earnings record necessary to support an ex-spouse's benefits. However, these conditions must be met:
- The ex-spouse is at least 62 years old.
- The spouses were married for over 10 years.
- The ex-spouse is not eligible to collect a larger benefit based on a personal earnings record or the earnings record of someone else.
- The ex-spouse has not remarried.
Comment: The rules are more complicated if the disabled spouse does not currently receive SSD benefits at the time of divorce. In certain circumstances however the disabled spouse can still file a claim for SSD benefits. If you have any questions or would like more information please contact Steve Shane at: email@example.com | 301.575.0313.
Steve Shane provides strategic counseling to clients in need of estate administration, charitable giving and business continuity planning while minimizing estate, gift, and generation-skipping transfer tax exposure. He offers legal guidance to clients on asset protection and the proper disposition of assets in accordance with the client's objectives, while employing tax planning techniques such as the use of irrevocable trusts, life insurance planning, lifetime gifts and charitable trust. He is also experienced with drafting documents for business planning, the incorporation and application for exemption for Private Foundations and the administration of decedents' estates. You can also connect with Offit Kurman via Facebook, Twitter, Google+, YouTube, and LinkedIn. WASHINGTON | BALTIMORE | FREDERICK | PHILADELPHIA | WILMINGTON | VIRGINIA


text:
Napsa Stars continued their good run as they thrashed Kabwe Youth 3-0 while Green Eagles came from a goal down to beat Mighty Mufulira Wanderers 2-1 at Nshinde Stadium.
The MTN/FAZ Super League is currently on recess because of the international break but three rescheduled matches, however, they went ahead. Napsa Stars welcomed newcomers KYSA, Green Eagles traveled to Nshinde to face Mighty Mufulira Wanderers while struggling Power dynamos hosted Buildcon.
Napsa Stars went into their match against KYSA knowing a win would take them to second place while their opponents were hoping to move from the bottom of the table. The Pensioners started strong and took the lead just after five minutes, Collins Sikombe getting on the end of a Doisy Solo pass to slot home from close range and put Napsa Stars in the lead.
In the second half Burundian striker Laudit Mavugo stole the show scoring his first goal of the season eleven minutes after the start of the second stanza before adding his second and Napsa's third four minuted later to complete an emphatic 3-0 win and send Napsa Stars to second place with 21 points leapfrogging Red Arrows.
At Shinde Stadium, Green Eagles came from a goal down to beat Mighty Mufulira Wanderers 2-1. Badidi Katende put Wanderers in the lead seven minutes into the second half but goals from Gozon Mutale and Hosea Silwimba turned things around and gave the visitors a well deserved 2-1 win to see then move to 6th place on the log.
At Arthur Davies Stadium, Power dynamos woes have continued as they gave up a 1-0 lead to draw one-all with Buildcon. Christian Ntouba Epoupa put Power dynamos in the lead while Eric Kabulo leveled matters with his first goal in Buildcon colors 1-1 the match finished.
MTN/FAZ Super League Rescheduled games
Saturday, November 9th
Power Dynamos 1-1 Buildcon
(C.E.Ntouba 53'| E. Kabulo 66')
Mighty Mufulira Wanderers 1-2 Green Eagles
(Katende 52' |G. Mutale 71, H.Silwimba 75')
Napsa Stars FC 3-0 Kabwe Youth Soccer Academy
(C. Sikombe 5', L. Mavugo 56' 60')


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During the 2020 Liberal National Convention in Ottawa, elections will be held for the LPC National Board and the National Executive of each Commission.
The National Board is a team of volunteers from across the country who are passionate about promoting Liberal values and improving Canada's future. They include community leaders, parents, and professionals who volunteer their time to participate in this role. The board works together to provide oversight and guidance to the Party in organizational, financial and strategic matters.
The four commissions are the Indigenous Peoples' Commission, the National Women's Liberal Commission, the Senior Liberals' Commission, and the Young Liberals of Canada. Commissions add vitality and diverse perspectives to the Liberal Party and provide a forum for engagement and representation of a demographic group of Registered Liberals.
More details will be available soon regarding the eligibility and election of officers.
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DRIVING: Your Day in Traffic Court
–Constable Tim Schewe (retired), DriveSmart
"I'll see you in court!" This hollow threat often ended conversations at the roadside after a driver was issued a traffic ticket for a violation. I knew that few of them would actually carry out their intention and if they did, there would probably be no coherent defence made.
Like many things in life, success often depends on preparation as much as it does on the delivery. Traffic court is one of those occasions.
I wrote the Q&A: How to Deal With a Traffic Ticket to assist in the process starting with being pulled over and ending with the conclusion of the court case. Links at the bottom of the article refer you to other reliable sources of information appropriate for courts in British Columbia.
If you are really serious about defending yourself well, probably the best $25 (plus tax, of course!) and 30 minutes you can spend is by taking advantage of the Canadian Bar Association's Lawyer Referral Service. This will help you decide if you want to represent yourself or hire the lawyer to defend you. Should you decide to manage your own defense you will very likely receive valuable advice.
The officer is required to disclose information on the case against you if you request it. Disclosure is essentially a summary of the evidence that the officer intends to present to the court. Should you require specific information, ask for it. Do this well in advance of the trial and I would suggest that you do so in writing and either mail or deliver it to the officer's workplace.
This step is where people often take advice from the internet to make outlandish requests and become upset when it is refused. The case of R v Wong comes out of a B.C. traffic court and may be considered as a guide to reasonable requests that the presiding judicial justice will support.
Speaking of case law, there is a fairly extensive collection on the DriveSmartBC web site. Searching "case law" and the offence you are interested in may turn up relevant examples of how the courts view a particular situation.
You can also do your own research on the Canadian Legal Information Institute's website.
The DriveSmartBC Forum contains two sections, Traffic Court and Traffic Tickets that may be of some assistance.
If you feel that your Charter Rights have been violated, most commonly your right to a trial within a reasonable length of time, you cannot do this in traffic court. You must notify the court registry and arrange for a hearing in provincial court instead. The judicial justice does not have the authority to preside over these cases.
You can learn a lot through the experience of others. Contact your local court registry and find out when traffic court is being held. It may even be possible to find out if the officer that issued your ticket will testify. Sit in on a morning or afternoon session as a spectator. On the day of your trial, you will already know what to expect.
This advice is valid until our provincial government replaces the traffic court system with an adjudication process. The progress of that began in 2012 with amendments to legislation and as far as I am aware, we are only now preparing to test e-ticketing in some areas of B.C.
Additional Resources
- Story URL: http://www.drivesmartbc.ca/traffic-tickets/your-day-traffic-court
- DriveSmartBC: Where better than average drivers satisfy their curiosity.
- Sign up for Tim's weekly DriveSmart newsletter!
- How is your driving knowledge? Take the Drive Smart Refresher test
- It's motorcycle season again: Here's how cagers can aim to not kill a rider
- Driving (Video): Remember this one word, Reißverschlussverfahren (zipper driving)
- "It's still drive-able" and other excuses, and what enforcement can result
- You'll be surprised to learn these are the greatest school zone offenders (plus a rules refresher)
- DRIVING: Your Day in Traffic Court
- DRIVING: BC Drivers put roadside workers' lives at risk
- Wet roads: Please slow down and increase your following distance
- DRIVING: Keep your eye out for fawns and deer families
- DRIVING: Slow Down, Move Over … It's the law!


text: Article | January 11, 2018 | Advanced ICU Care Advanced ICU Care's 2017 Year in Review With a mission to collaborate with our partner hospitals to deliver improved clinical outcomes, Advanced ICU Care works each day with bedside care teams to save lives. In this interactive newsletter, we are proud to share 2017's noteworthy accomplishments, highlight key contributors, discuss the impact of our multiple integrated care hubs and, most importantly, celebrate the mutual successes achieved working with our partners. Please click here to view the exciting happenings from 2017.


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The Michigan based design firm ASC Creative Services, has released images of a Pontiac Firebird that can be yours if you provide a 2010 Chevrolet Camaro and a fat bank account. The body kit includes Firebird decals and other styling cues that make it hard to miss.
Pontiac Firebird returns!
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Are you on the lookout for the newest style for a celebration, business or leisure? There's a large selection of trend shops in Rotterdam. Big outlets of main vogue retailers like Zara, H&M , C&A and Peek & Cloppenburg, Vero Moda, Jack & Jones, Claudia Sträter, River Island, Replay, Coolcat, SOLELY, Steps and Bershka all have a department in Rotterdam. The division store Bijenkorf has massive retail areas with the newest style as effectively. There are also some one-of-a-kind vogue shops in Rotterdam, that appeal to lots of visitors from outside Rotterdam.
Subsequent, you'll want a high with a powerful vertical line. I think button-down shirts are at all times a sensible choice—they're traditional and adjustable, and just about flattering any manner you wear them. That is the shirt I bought on my last Madewell purchasing trip (see, I promised I might function it in an outfit submit!), and a lighter coloration is essential right here, as a result of it helps draw the attention up (i.e. look at my face, not at my intestine). I have two guidelines when I shop. Only purchase what I like and what I will truly put on. That might sound silly and like frequent sense, but when I ask myself these two questions earlier than I hit the register, it helps me with these compulsive purchases.


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It was last October when we shared the news that the Garrison Art Center had announced a new executive director, Katie Schmidt-Feder.
Now, it's Schmidt-Feder who shares a bit of news:
"We are excited to announce that Kit Burke-Smith has joined the Garrison Art Center staff as education coordinator.
"Becky Gordon, who has done a phenomenal job serving as both education and exhibitions coordinator for the last year, will continue as exhibitions coordinator, giving her more flexibility for other career pursuits.
"Kit has been an arts educator for over 12 years. After attending the prestigious Interlochen Arts Academy, she earned a BFA from Rhode Island School of Design and an MFA from SUNY New Paltz. She has taught studio art classes and workshops in metalsmithing, fiber arts, drawing, design, color theory and art history at SUNY New Paltz, Interlochen Center for the Arts, Dutchess Community College, Mill Street Loft, Manitoga Design Center, Beacon Art Studio, Beetle & Fred, Compass Arts, Rivertown Homeschool Hub and The Culinary Institute of America. Kit has also served as a site educator and docent for Manitoga: Russel Wright Design Center. In addition to being an educator she maintains a studio practice in Beacon, and runs an art jewelry business, Kit Burke-Smith Jewelry. Kit has also volunteered to organize and work many community events, including art auctions, fundraisers, after-school programs, libraries and city-wide art and business interfacing experiences."
We at WAG welcome Burke-Smith and also remind art lovers of the Center's annual Holiday Pottery Show and Sale. This year's edition of the popular event will run Nov. 17-26.
For more, visit garrisonartcenter.org.
– Mary Shustack


text:
The Flooded Plain
September 12, 2013
Comments on: "The Flooded Plain" (2)
Good to see this post Tom!
Hope you are doing well! Xx
Hi Tom
Jeremy was just saying that he had seen your post 'Hanging on in there' and at the same time i was looking at my emails and saw that you had just posted another one! I am often wondering how you are getting on Tom and looking forward to coming to see you in November sometime. Lots of love, as always, and hope things are improving,albeit i imagine gradually.