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The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α2-antiplasmin (α2AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α2AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α2AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.
Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H6NQEQVSPLTLLAG4Y (designated XL1); H6DQMMLPWAVTLG4Y (XL2); H6WQHKIDLPYNGAG4Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α2AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α2AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA.
Fusion protein XL5-HSA (DQMMLPWAVTLG4Y-HSAH6) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in vitro. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into in vivo clots formed in thrombosis models in both mice and rabbits.
Recombinant serum albumins provide an attractive scaffold for the delivery of attached therapeutic peptides or proteins [1,2]. They are the most abundant proteins in the plasma of mammals, reaching concentrations of 40-80 mg/mL (0.6-1.0 mM) in humans [3]. They are slowly cleared plasma proteins [3,4] whose longevity in the circulation derives from a well-characterized mechanism of recycling via the major histocompatibility complex-related Fc receptor for immunoglobulin G (FcRn) [5-7]. Serum albumin is also unusual among plasma proteins in that it is not glycosylated [3], a property that simplifies its recombinant expression and production [8]. If candidate therapeutic peptides or proteins can be fused to albumin in a manner that allows them to express their endogenous activity, the resulting fusion protein often demonstrates improved pharmacokinetic and pharmacodynamic properties due to acquisition of albumin-like circulatory characteristics [1]. For these reasons, albumin has been fused to numerous proteins such as interferon [9], interleukin-2 [10], butrylcholinesterase [11], coagulation factors VII [12] and IX [13,14], hirudin [15,16], and barbourin [17], as well as to smaller peptides [18].
In order to address unmet clinical needs in the area of thrombosis, the pathological formation of clots within intact blood vessels, our laboratory previously investigated novel recombinant proteins comprised of portions of α2-antiplasmin fused to the N-terminus of human serum albumin [19]. α2AP is cross-linked to fibrin clots by activated factor XIII (FXIIIa), where it promotes clot stability by inhibiting the fibrinolytic enzyme plasmin [20,21]. We [19] and others [22,23] have demonstrated that competing α2AP-fibrin crosslinking results in fibrin clots that are more easily dissolved in vitro, suggesting potential applications of such competitors as adjuncts to thrombolytic therapy.
Previously, we attempted expression of three α2AP-HSA fusion proteins in Pichia pastoris, a methylotropic yeast shown to produce recombinant HSA in high yield and with indistinguishable biophysical properties to its plasma-derived counterpart [24]. Our strategy was to retain the N-terminal sites of α2AP-fibrin crosslinking, especially Q14 [25], but to delete the C-terminal plasmin-binding and plasmin-trapping reactive centre loop of this serpin-type inhibitor. We sought to provide as native a protein environment as possible for the α2AP cross-linking motif by maximizing the portion of α2AP fused to HSA. However, only a chimeric protein comprised of α2AP residues 13-42 fused to HSA was secreted by the yeast; the two longer fusions involving residues 13-73 and 13-109 were not stably expressed. Moreover, α2AP(13-42)-HSA was partially proteolyzed within its α2AP moiety. While we demonstrated that α2AP(13-42)-HSA became a substrate of FXIIIa and competed for α2AP crosslinking to both small and macromolecular substrates, we could not demonstrate cross-linking to clots in vivo above background levels seen with recombinant HSA [19].
In the present study, we sought to improve the ability of HSA fusion proteins to compete with α2AP for FXIIIa-mediated cross-linking. The α2AP moiety was reduced to 12 residues (α2AP 13-23 followed by Lys24Ala), to avoid previously observed sites of proteolysis by P. pastoris proteases, and compared to two 12-residue artificial FXIIIa substrate peptides selected by phage display for high reactivity [26]. These motifs were expressed with or without N-terminal His tags, to allow for purification of full length proteins away from partially proteolyzed products. We hypothesized that replacing α2AP 13-42 with shorter, potentially more active FXIIIa substrate motifs would improve the stability of fibrin cross-linkable HSA proteins and improve in vivo clot retention.
DNA Manipulations
All oligonucleotides employed in this study were synthesized at MOBIX Lab, a McMaster University core facility. The sequences of all plasmids generated in this study were validated by DNA sequencing, also performed by MOBIX Lab, prior to use for protein expression. Standard molecular biological protocols for DNA restriction and ligation, plasmid mini-DNA preparation, purification of DNA from agarose gels, and transformation of E. coli Top10 (Invitrogen) to ampicillin resistance, were employed [19]. Three plasmids encoding fusion proteins XL4-, XL5-, and XL6-HSA were first prepared, by analogous means. Synthetic oligonucleotides, whose sequences are shown in Table 1, were first annealed by heating to 95°C and slow cooling to room temperature, in pairs, for each construct, respectively: ML-08-5116 and ML-08-5117; ML-08-5118 and ML-08-5119; and ML-08-5120 and ML-ML-08-5121. Annealed oligonucleotides were then separately ligated to the 6325 bp XhoI-KpnI restriction digestion fragment of previously described plasmid pPICZ9ss-α2AP(13-73)-HSAH6 [19], replacing the α2AP(13-73) codons, forming plasmids pPICZ9ss-XL#-HSAH6, where # corresponds to 4, 5, or 6, respectively. In order to construct XL-HSA constructs in which the His tag was not present on the C-terminus of the HSA moiety, an intermediate construct was prepared. A full-length preproHSA cDNA in pCDNA3.1 (Invitrogen), pC3ppHSA, was restricted with XbaI and ApaI, and the 804 bp minor fragment was ligated to the major 5786 bp XbaI-ApaI fragment of pPICZ9ss-α2AP(13-73)-HSAH6, yielding plasmid pPICZ9ss-α2AP(13-73)-HSA, in which the HSA open reading frame terminated without hexahistidine codons. The 5768 bp XhoI-KpnI restriction fragment of pPICZ9ss-α2AP(13-73)-HSA was then combined with the following pair-wise annealed oligonucleotides: ML-08-5104 and ML-08-5105; ML-08-5106 and ML-08-5107; and ML-08-5108 and ML-08-5109. This approach yielded plasmids pPICZ9ss-XL#-HSAH6, where # corresponds to 1, 2, or 3, respectively. A final expression plasmid was constructed to reverse the orientation of the His tag of fusion protein α2AP(13-73)-HSAH6 [19]. Plasmid pBAD-H6-α2AP [27] was PCR-amplified using oligonucleotides ML 09-3874 and 17225 [27], and the 125 bp XhoI-KpnI restriction fragment of the resulting PCR product was inserted between the corresponding sites of pPICZ9ss-α2AP(13-73)-HSA to yield pPICZ9ss-H6α2AP(13-42)-HSA. Each completed plasmid listed above was used to transform Pichia pastoris strain X33 to Zeocin (Invitrogen) resistance as previously described [18].
Table 1. Oligonucleotides employed in this study
Fusion protein expression, purification, and characterization
HSA fusion proteins were purified from media conditioned by transformed Pichia pastoris cells and induced with 0.5% vol/vol methanol, as previously described [18,19,28], except that the methanol induction phase of protein production was extended to 96 hours. At that point, the conditioned media was neutralized and clarified by centrifugation [1], treated with protease inhibitors (5 mM benzamidine and 0.1 mM phenylmethylsulfonyl fluoride), and purified using Ni-NTA agarose chromatography (Qiagen). Purified proteins were analyzed by SDS-PAGE and immunoblotting using polyclonal affinity-purified goat anti-human α2AP antibodies (Affinity Biologicals) and murine monoclonal anti-HSA antibodies (Genway Biotech). They were also characterized by automated Edman degradation at the Advanced Protein Technology Centre of the Hospital for Sick Children, Toronto, Canada.
Transglutamination assays with biotinylated pentylamine
In order to quantify FXIIIa-catalyzed transglutamination of plasma-derived α2AP and recombinant HSA fusion proteins, a microtiter plate (Immulon 4 HBX, Thermo Scientific) protocol was developed. Test HSA-related proteins (1.7 μM) were first incubated for varying times in Tris-buffered saline (50 mM Tris-Cl pH 7.5, 150 mM NaCl) containing 5 mM CaCl2 and 5 mM biotinylated pentylamine (BPA; EZ-Link pentylamine-biotin, Pierce) supplemented with 20 nM human FXIII (Enzyme Research Labs, ERL) and 1.0 IU/mL human thrombin (ERL). Reactions (0.1 mL) were stopped by addition of an equal volume of 20 mM EDTA pH 8.00 and transferred to microtiter plates. Microtiter plates were prepared for use by coating with 2.5 μg/mL mouse anti-HSA monoclonal antibody (Genway) in 50 mM sodium carbonate pH 9.6 overnight at 4°C. All other incubations were at room temperature. All washes were performed with Tris-buffered saline supplemented with 0.05% (vol/vol) Tween 20 (TBST), and repeated three times. Anti-HSA-coated plates were washed and then blocked for one hour in TBST, then washed again. Following transfer of the stopped transglutamination reaction mixtures to the plate, it was incubated with shaking for one hour and washed, prior to reaction of captured proteins with a 1:2500 dilution of alkaline phosphatase-conjugated streptavidin (Jackson Labs) in TBST for one hour. Following a final wash step, colour was developed by addition of 1.0 mg/ml p-nitrophenyl phosphate disodium salt (PNPP) in diethanolamine buffer (1.02 M diethanolamine pH 9.8) (Thermo Scientific) and absorbance was quantified on an ELx808 plate reader (BioTek Instruments) at 405 nm for up to 15 minutes. In some reactions, the protocol was modified to measure transglutamination of α2AP by substituting anti-α2AP antibodies for anti-HSA antibodies, and purified human plasma-derived α2AP (ERL) for HSA-related proteins.
Transglutamination of fibrin(ogen)
FXIIIa-catalyzed transglutamination of the natural substrate fibrinogen was assessed using SDS-PAGE and immunoblotting of cross-linking reactions as previously described [19]. Briefly, 100 nM FXIII was first activated to FXIIIa by reaction with 5.0 IU/ml thrombin for 5 minutes at 37°C; thrombin was then inactivated using Phe-Pro-Arg chloromethylketone to 10 μM final concentration, to prevent clotting on addition to fibrinogen. The resulting FXIIIa (50 nM) was reacted with 6.0 μM human fibrinogen (specifically depleted of plasminogen, von Willebrand factor, and fibronectin, ERL), and 1.0 μM substrate proteins in TBS containing 10 mM CaCl2.
Reactions were stopped by addition of SDS and analyzed on 8% SDS-polyacrylamide gels under reducing conditions, with immunoblotting and chemiluminescent development of antibody-decorated blots, as described [19].
In vitro clot lysis
As previously described [19], the formation and dissolution of human plasma clots was followed by recording changes in turbidity, using an ELx808 plate reader (BioTek Instruments) set to take absorbance readings at 340 nm every 30 seconds for 4 hours, and quantified as the area under the curve.
Retention of iodinated proteins in mouse vena cava treated with ferric chloride
Purified human fibrinogen (Sigma), human α2AP (ERL), or XL5-HSA recombinant proteins were iodinated using the Iodogen method as described by the manufacturer (Pierce), using either sodium 125I or 131I. Unincorporated radioactivity was removed by exhaustive dialysis versus phosphate-buffered saline. Specific activities of labelling exceeded 1 × 109 cpm/mg. Radioiodinated proteins were then tested in the mouse ferric chloride vena cava thrombosis model, modified from [29,30]. CD-1 mice were anaesthetized using gaseous anaesthesia (3% isofluorane) at all times during this procedure, and a heating pad was employed to ensure maintenance of normal body temperature. A mid-line incision was made, first through the skin and secondly through the muscle layer. Viscera were gently displaced and haemostatic clamps employed to keep the incision open. At this time radioiodinated proteins (5 × 106 cpm in 0.1 ml sterile saline) were injected via the tail vein. The vena cava was then exposed and a 2 × 4 mm piece of Whatman paper soaked in 10% (w/vol) ferric chloride was applied. The viscera were covered with gauze soaked in warm saline during this time. The time from radioactive protein injection to application of ferric chloride was fixed at 5 minutes. Three minutes after its application, the filter paper was removed and viscera replaced. Thirty minutes later, the vena cava was re-exposed, the vessel was excised, and the clot was transferred to a tared tube for weighing, and then subjected to γ counting using either an Auto Gamma 5530 Minaxi γ counter (Perkin Elmer) or a Cobra II γ counter (Packard) to quantify incorporated radioactivity.
Retention of iodinated proteins in rabbit jugular vein thrombi
New Zealand White rabbits were subjected to a modified Wessler procedure [31] as previously described by this laboratory [19]. Briefly, animals were initially anesthetized with 100 mg of ketamine, then maintained in the anesthetized state with 1.5% isoflurane. They were then cannulated via the carotid artery and the jugular veins isolated. One ml of whole blood was freshly drawn and anticoagulated with 1/9th volume of 3.8% w/vol sodium citrate. Two centimeter long sections of the right and left jugular veins were isolated, emptied of blood, and isolated using bulldog clamps. Clotting of the anticoagulated, autologous whole blood was initiated by combining it with warm (37°) human thromboplastin reagent Thromborel S (Dade Behring) in a 1:4 (vol:vol) ratio, supplementing it to 3.3 × 106 cpm/ml 131I- fibrinogen and 125I-recombinant protein, and re-introducing 0.15 ml of the clotting blood into the isolated segments. This was done for both isolated jugular veins and blood flow was held in stasis for 30 minutes, at which time the clamps were removed, and blood flow restored. Clots were recovered 60 minutes later, following jugular vein opening by incision, and removed, weighed, and γ-counted as described above for murine clots. In some experiments clots were extracted overnight in 5.0 M urea, microcentrifuged, and the supernatant removed prior to re-counting. The proportion of urea-stable protein incorporation was then calculated, adjusted for radioactive decay between the recorded times of the first and second γ-count. All animal experiments were carried out under the terms of an approved Animal Utilization Protocol reviewed, approved, and monitored by the Animal Research Ethics Board of the Faculty of Health Sciences, McMaster University.
Statistical analysis
Statistical tests were performed using GraphPad Instat version 4 (GraphPad Software). Multiple comparisons used one-way parametric analysis of variation (ANOVA) with Tukey-Kramer post-tests where data were normally distributed, and nonparametic ANOVA with Dunn's post-tests where they were not.
Expression and characterization of fusion proteins
Media conditioned by Pichia pastoris cultures transformed with expression plasmids encoding fusion proteins XL(1-6)-HSA (see Figure 1) were first screened for recombinant expression of HSA-related polypeptides by gel analysis. As shown in Figure 2A, although there were some differences in the time course of expression, five of six of the yeast cell lines secreted a major methanol-dependent polypeptide of the expected size of the HSA fusion proteins, of 70-80 kDa. The exception was the cell line programmed to express XL3-HSA, which secreted no detectable methanol-dependent polypeptides.
Figure 1. HSA fusion protein design, expression, and characterization. (A) Schematic diagram of proteins. Relevant polypeptides are shown in linear form, oriented from N-terminus to C-terminus, with different regions labelled on the bar diagram. HSA residues are shown in black, while candidate FXIIIa substrate motifs are shown in grey. White bars correspond either to hexahistidine tags (H6) or to tetraglycine-tyrosyl spacer segments (G4Y). Polypeptide names and predicted length in amino acids (a.a.) is shown at right. (B) Amino acid sequence of candidate FXIIIa substrate motifs. The amino-terminal portion of α2AP previously fused to HSA [19] is shown, including putative sites of proteolysis deduced by amino acid sequencing, on the top line (α2AP(13-42)). The three candidate FXIIIa motifs fused to albumin with or without adjacent His tags, are shown below (XL1,2, or 3).
Figure 2. Expression, purification and characterization of HSA-related recombinant proteins. (A) Electrophoresis of conditioned media from P. pastoris transformed with different expression plasmids. Conditioned media samples taken 24, 48, 72, or 96 hours after the start of methanol induction are shown on 10% reducing SDS-polyacrylamide gels stained with Coomassie Blue. The protein product encoded by the expression plasmid is shown above each gel. The position of molecular mass markers (200, 150, 120,. 100, 85, 70, 60, 50, 40, and 30 kDa) is shown to the left of each panel and labelled (XL4-HSA) in one instance as an example. (B) Upper panel, gel profile of purified recombinant proteins on 10% SDS-polyacrylamide gels electrophoresed under reducing conditions and stained with Coomassie Blue. Proteins (400 ng) are identified above each lane as in Figure 1A, plus GST (E.coli-expressed glutathione sulfotransferase); M, molecular mass markers identical to those used in Figure 1A. Middle and lower panel, replicate gels differing only in the amounts of protein loaded per lane, to avoid overloading with antibodies of different affinity, were immunoblotted and probed with Anti-HSA antibodies (middle pane, 100 ng/lane) or anti-hexahistidine antibodies (lower panel, Anti-His, 500 ng/lane).
The five putative HSA fusion proteins were purified from conditioned media induced with methanol for 96 hours, using nickel chelate affinity chromatography, and reacted both with anti-HSA and anti-hexahistidine antibodies, as shown in Figure 2B. The same properties were observed for H6α2AP(13-42)-HSA and previously described α2AP(13-42)-HSA [19] (Figure 2). Similar results were obtained when an additional two independent P. pastoris Zeocin-resistant cell lines were examined, in addition to the ones shown in Figure 2 (data not shown).
Because the minor, 2-4 kDa difference between the shortest (HSAH6) and longest (H6α2AP(13-42)-HSA) putative expression proteins was not resolvable on the gel system we employed, we sought independent confirmation of the identity and integrity of the expressed protein products by amino acid sequencing. For XL(4-6)-HSA, a single N-terminal sequence was detected by Edman degradation: NQEQVS; DQMMLP; and WQHKID, respectively. For XL1-HSA and XL2-HSA, Edman degradation showed intact hexahistidine amino-termini. For H6α2AP(13-42)-HSA, two sequences were detected: the major sequence arising from the expected hexahistidine tag, and the other a mixture of amino acids suggestive of a similar pattern of partial proteolysis we previously reported for α2AP(13-42)-HSA [19]. In that regard, batches of the latter protein were prepared using 96 hours of methanol induction, as employed for all other proteins in this study, for consistency and for comparative purposes. Edman degradation showed a mixture of termini similar to those previously observed from cultures induced for lesser periods of time [19]. These included termini commencing with Leu23 (LKLGNQ), Leu25 (LGNQEP), and Ser38 (SPPGVC) but no detectable full-length product. Three separate batches of such protein preparations returned similar results (data not shown).
While the amino acid sequencing results showed that positioning of a hexahistidine tag on the N-terminus of α2AP(13-42)-HSA improved the integrity of the resulting purified protein product, the yield was reduced from 40-50 mg/l for AP(13-42)-HSA to 5-6 mg/l for H6α2AP(13-42)-HSA. Yields for the purified XL-HSA proteins varied from 10-12 mg/l (XL1- and XL2-HSA) to 20-35 mg/l ( XL4- and XL5-HSA) to 80-85 mg/l (XL6-HSA).
Characterization of recombinant fusion proteins as fXIIIa substrates for cross-linking to lysine donors
Having demonstrated that all recombinant fusion proteins with the exception of XL3-HSA had been produced, we next asked whether the addition of the candidate FXIIIa sequences had actually converted the proteins into substrates for cross-linking by FXIIIa. Cross-linking reactions were performed in solution, using the chemical amine donor biotinylated pentylamine, and reaction products were captured using antibodies immobilized on microtiter plate walls and quantified colorimetrically using alkaline-phosphatase-linked streptavidin. As shown in Figure 3A, while there was evidence of some cross-linking for all proteins tested, the highest levels were observed for fusion proteins XL5-HSA and XL2-HSA, in that order. While the results shown in Figure 3A were limited to initial (2.5 minute) reactions, by five minutes the cross-linking of XL2-HSA and XL5-HSA had become comparable (data not shown).
Figure 3. FXIIIa-mediated cross-linking of recombinant proteins and plasma-derived α2AP to biotinylated pentylamine. Panel A shows transglutamination reactions, in which 20 nM FXIIIa cross-linked 5 mM BPA to 1.7 μM HSA fusion proteins (identified below the × axis), were stopped after 2.5 minutes and the relative BPA incorporation was quantified using anti-HSA capture and alkaline phosphatase-conjugated streptavidin detection as outlined in "Methods". Colour generation was quantified as the optical density at 405 nm (OD (405 nm)). The mean ± standard deviation (SD) of 6 determinations is shown, with background (reactions lacking FXIIIa) subtracted. Panel B shows a time course of FXIIIa-mediated cross-linking of BPA to 1.7 μM α2AP, using the same conditions as employed in Panel A except that anti-α2AP antibodies were used to capture biotinylated α2AP after reaction termination at various time points. The mean ± the standard deviation (SD) of 3 determinations is shown, with background (reactions lacking FXIIIa) subtracted. Panel C shows BPA-α2AP crosslinking reactions of 5 minutes' duration, as shown in panel B, except that competitor proteins identified on the × axis were included at 12 μM. The mean ± the SD of 5-14 determinations is shown, with background (reactions lacking FXIIIa) subtracted.
Similar results were obtained when HSA fusion proteins were employed as competitors of the FXIIIa-dependent transfer of biotin from BPA to α2AP, which exhibited time-dependent crosslinking on the time scale employed (see Figure 3B). As shown in Figure 3C, of seven proteins tested using a seven-fold excess of competitor over α2AP, only XL5-HSA, XL2-HSA, and H6α2AP(13-42)-HSA significantly reduced cross-linking of α2AP; the relative efficacy of the proteins was in the order listed. To minimize the possibility that reactivity with BPA was an artefact that did not accurately predict reactivity with macromolecular donors, the ability of XL5-HSA to be cross-linked to fibrinogen was tested. As shown in Figure 4, XL5-HSA and α2AP exhibited similar kinetics in forming high molecular weight products with fibrinogen that were dependent on added FXIIIa, although the fact that different antibodies, with potentially different binding efficiency, had to be employed limited the comparison to a qualitative assessment. A similar pattern was observed for both proteins in that three higher molecular weight bands of 140 kDa and greater formed in a factor XIIIa- and fibrinogen-dependent manner; with respect to plasma-derived α2AP, these bands have been suggested to represent α2AP-fibrinogen Aα chain, α2AP-fibrinogen Aα dimer, and α2AP-fibrinogen Aα polymers [32].
Figure 4. FXIIIa-mediated cross-linking of α2AP and XL5-HSA to fibrinogen. FXIII was pre-activated to fXIIIa by thrombin, the thrombin inactivated with FPRck, and the fXIIIa then combined with fibrinogen and α2AP (A) or XL5-HSA (B) in transglutamination reactions probed, after reducing SDS-polyacrylamide electrophoresis, with either (A) anti-α2AP or (B) anti-HSA antibodies. The position of pre-stained molecular mass markers is indicated to the left of the panels. Reactions containing only FXIII, only fibrinogen, or only α2AP are also shown.
Comparison of fusion proteins as competitors of α2AP-mediated in vitro clot protection
α2AP attenuates the ability of tPA to lyse fibrin clots by inhibiting plasmin generated by tPA-mediated activation of plasminogen. This property can be demonstrated in vitro using α2AP-deficient plasma. As shown in Figure 5A, clots formed in the absence of tPA and α2AP were stable for > 4 hours, whereas the addition of tPA lead to complete lysis within 60 minutes. Restoration of α2AP to its physiological concentration of 1 μM restored approximately 70% of the area under the turbidity curve. The ability of the HSA-related fusion proteins to compete for the α2AP-mediated lysis attenuation was tested by addition of 20 μM fusion proteins. Although at this excess, some changes in the shape of the turbidity plot in the presence of fusion protein and the absence of fusion protein could be noted (Figure 5A), the area under the curve (AUC) of the turbidity curve did not differ from baseline, as exemplified for XL5-HSA and XL6-HSA in Figure 5B. XL5-HSA significantly reduced the α2AP-mediated attenuation of clot lysis, shifting the AUC from 70.8 ± 14% of maximal clotting to 40.5 ± 2.9% (Figure 5B); in contrast XL6-HSA did not compete for the α2AP effect. Extension of these comparisons to the complete set of recombinant fusion proteins showed that only XL5-HSA and H6α2AP(13-42)-HSA, under the conditions employed, reduced the α2AP effect, and that only the XL5-HSA reduction was statistically significant.
Figure 5. Effects of α2AP and recombinant HSA-related proteins on plasma clot formation and lysis. Clot formation and lysis were followed by monitoring turbidity (absorbance at 340 nm) every 30 seconds for 4 hours using a plate reader, of clots formed using diluted α2AP-deficient plasma, recalcified with 5 mM CaCl2 and supplemented with both 5 nM thrombin and 0.125 nM tPA, and taking the area under the turbidity versus time curve (AUC). Reactions were carried out with or without addition of 1 μM α2AP and/or 20 μM fusion protein. (A) shows results of a single representative experiment. (B) shows quantification of turbidity plots for reactions similar to those shown in A, in the presence or absence of XL5-HSA or XL6-HSA competitor proteins; reaction components are indicated (+ or -) below the graph. The mean ± SD of 5-14 determinations is shown. AUC values were normalized, taking the no tPA, no α2AP condition as 100% (grey bar); white (open) bars correspond to tPA only condition. (C) shows similar results to panel B for additional competitor proteins identified below the graph. As in panel B, the mean ± SD of 5-14 determinations is shown.
Fusion protein XL5-HSA is retained in experimental thrombi in mice and rabbits
Having obtained several lines of evidence that fusion protein XL5-HSA was the most effective FXIIIa substrate of the recombinant proteins tested, we next asked whether, like α2AP, it was capable of being retained within whole blood clots in vivo. As shown in Figure 6A, when equal doses of radiolabeled α2AP, XL5-HSA, and HSAH6 were injected into mice that formed experimental thrombi in the vena cava due to ferric chloride administration, both α2AP and XL5-HSA were retained within clots to a significantly greater extent than HSAH6. The retention of α2AP on a clot weight basis, was also significantly greater than that of XL5-HSA.
Figure 6. Retention of radiolabeled proteins in experimental thrombi formed in mice or rabbits. (A) The radioactivity remaining in clots induced by surgical, topical application of 10% ferric chloride to the vena cava of anesthetized mice is shown, as a fraction of the total radioactive protein dose injected, divided by the weight of the clot. Injected radiolabeled proteins are identified on the × axis. The mean ± SD of 12 determinations is shown. (B) The radioactivity remaining in rabbit jugular vein clots formed in clamped-off vessels in situ for 30 minutes in the anesthetized animal, then exposed to circulating blood for 60 minutes, prior to clot recovery and γ-counting is shown, expressed as in Figure 6A. The extent (p < 0.001, p < 0.05) of statistical significance is shown for the data sets linked by horizontal lines.
Similar results were obtained using a larger, rabbit experimental model in which the radiolabeled proteins were introduced, with a coagulation activator, into a whole blood segment within the isolated rabbit jugular vein. As shown in Figure 6B, the three proteins were found to become incorporated into the clot with the same relative efficacy as in the murine model; both α2AP and XL5-HSA were incorporated to a greater extent than HSAH6. However, in common with the murine results, the XL5-HSA tracer incorporation was significantly less than that of α2AP.
Fusion protein XL5-HSA is retained in a urea-sensitive manner in experimental thrombi in rabbits
In the larger animal model, the rabbit, the greater physical size of the clot permitted its manipulation, unlike with the much smaller murine thrombi. Prolonged stasis and the direct introduction of tissue factor activator into the isolated vessel strongly favoured thrombus formation in the rabbit model, as compared to the localized application of indirectly procoagulant ferric chloride to a localized area of a much smaller vessel in the mouse. Accordingly, clot weights from rabbits ranged from 21.4 to 54.1 mg versus 5.1 to 11.4 mg in mice. This greater size permitted clot extraction in 5.0 M urea and determination of the fraction of clot-bound radioactivity that was insensitive to urea, a hallmark of cross-linked protein clot incorporation. In these experiments, the three 125I-labeled proteins were each separately co-administered with 131I-labeled fibrinogen. As shown in Figure 7A, approximately 50% of injected fibrinogen was clot-associated in a urea-insensitive manner; there were no differences between treatment groups, as expected because tracer-level doses of the recombinant proteins were employed. While significantly greater amounts of α2AP remained clot-bound after urea extraction than XL5-HSA (15.8 ± 2.8% of injected dose versus 7.52 ± 1.7%) (Figure 7B), this represented 15- and 7.5-fold greater urea-insensitive retention than for HSAH6 (1.00 ± 0.43%).
Figure 7. Retention of radiolabeled proteins in experimental thrombi formed in rabbits following extraction in 5.0 M urea. Rabbits were co-injected with 131I-fibrinogen and 125I-proteins identified in Figure 6 and jugular vein clots were formed as described in "Methods" Clots were γ-counted at the close of the experiment, and then extracted overnight with 5.0 M urea, the supernatant aspirated, and the clots recounted. The fraction of the total injected radiolabeled protein dose found in the clot after urea extraction is shown for 131I-fibrinogen, identified by the co-injected 125I protein on the × axis in (A) and for 125I-labeled proteins in (B). Differences between 131I-fibrinogen among the three groups in (A) are not statistically significant; the extent of statistically significant differences between groups is shown for the data sets linked by horizontal lines in (B). The mean ± SD of 12 determinations is shown.
In this study, we sought to increase the stability and activity of HSA fusion proteins containing short N-terminal extensions that are substrates for transglutamination by FXIIIa. Our long-term goal was to produce well-tolerated therapeutic proteins for injection, with the long circulatory half-life of albumin, and the ability to compete the cross-linking of α2AP to fibrin in thrombi. Replacing active α2AP in the clot with a protein incapable of inhibiting plasmin could render pathological clots easier to dissolve. Others have suggested that chemically or mutationally inactivated α2AP could serve this purpose [22,23], while we have focused on the albumin fusion strategy [19], due to the ease of large-scale production of recombinant albumin [8]. In this study, we built chimeric albumin fusion proteins modelled on α2AP(13-42)-HSA, seeking to eliminate the partial proteolysis of this prototype, but retain and enhance its ability to be cross-linked by FXIIIa. Our approaches included shortening the natural α2AP-derived cross-linking site, replacing it with either of two FXIIIa highly active substrate sequences identified by phage display, and capping all three novel chimerae with hexahistidine tags.
Substituting the sequence DQMMLPWPAVTL for α2AP(13-42), in fusion protein XL5-HSA, was found to be the most effective approach. This sequence had been reported to be a highly favourable substrate for FXIIIa-mediated transglutamination, when expressed fused to a phage coat protein or to glutathione sulfotransferase (GST) (designated F11 in [26]). When expressed fused to HSA, it yielded the candidate protein most rapidly and efficiently cross-linked to BPA, and the most effective competitor of α2AP crosslinking, both with small substrates and in competition of α2AP's in vitro antifibrinolytic effect. The molecular context of the fusion partner was relevant, since sequence WQHKIDLPYNGA, identical to sequence F28 in [26], except for substitution of protease-susceptible R for P at position 8, appeared to be much less active in fusion protein XL6-HSA than in the GST context. Similarly, α2AP(13-23)K24A, although shown to be effectively crosslinked by FXIIIa as α2AP(13-23) or α2AP(13-24) free peptides [33-35], was ineffective at competing α2AP crosslinking to fibrin in fusion protein XL4-HSA. Although our avoidance of Lys or Arg residues in the fusion motifs was successful in avoiding degradation in each case, only XL5-HSA was clearly superior not only to XL4-HSA or XL6-HSA but also to α2AP(13-42)-HSA.
Although the requirements for optimal substrates of FXIIIa have not been precisely defined, they are thought to involve positioning of a reactive glutamine residue within a highly flexible sequence [36]. Fusion proteins XL4-, XL5-, and XL6-HSA all contained a reactive glutamine at position 2, analogous to Q14, the most reactive glutamine in the native α2AP sequence. Q14 is more effectively cross-linked by FXIIIa in the major form of α2AP that circulates in plasma, Asn-α2AP, than in the minor precursor form, Met-α2AP, which retains residues 1-12 [37]. Our finding that positioning a hexahistidine tag N-terminal to the FXIIIa substrate motif reduced its activity in fusion proteins XL1- and XL2-HSA was consistent both with this natural example and with the general concept of flexibility of the reactive glutamine.
Previously, we found that α2AP(13-42)-HSA was no more likely to be present in vivo in the rabbit jugular vein model of thrombosis than recombinant HSA [19]. In contrast, XL5-HSA was retained in thrombi in vivo to a significantly greater extent than recombinant HSA in both rabbit and murine models. Its degree of retention was less than that of human α2AP in both cases, suggesting that there is still room for improvement in defining an optimal sequence to render HSA efficiently cross-linked by factor XIIIa. Nevertheless, this is the first demonstration of targeting of an inactive carrier protein to in vivo clots by an attached transamidation substrate motif, although, as described below, others have shown in vivo clot association of transamidation substrate motif peptides [35] and peptide-contrast agent conjugates [33,34]
The insolubility of cross-linked fibrin in 5.0 M urea is a property that has been known for many years, and indeed is used clinically to diagnose FXIII deficiency [38]. Using this property, we showed that in vivo clot-associated XL5-HSA is urea-insoluble, like the majority of bound fibrin or α2AP, but unlike the majority of bound HSA. Although much indirect or in vitro evidence supports the importance of α2AP cross-linking to fibrin with respect to clot resistance to fibrinolysis [22,23,39,40], this is to our knowledge the first demonstration of cross-linking of either α2AP protein or an engineered polypeptide into in vivo thrombi. Other groups have used peptides in this regard, but predominantly in an analytical or imaging mode distinct from long-term goal of reducing thrombus size using cross-linkable proteins. Robinson et al. showed that biotinylated α2AP (13-24) peptide became cross-linked to human thrombi embolized into the lungs of ferrets and mice when infused in vivo [35]. Jaffer et al. employed an α2AP (13-24) peptide linked to a near infrared fluorochrome to image murine thrombi formed following ferric chloride treatment of the femoral vessels [34], while Miserus et al., used a similar α2AP (13-23) peptide conjugated to a gadolinium-containing contrast agent to visualize murine carotid artery ferric chloride-induced thrombi [33]. While these peptides and peptide-chemical conjugates show great promise for future improved detection of thrombi and stratification of patients, peptides are typically cleared from the circulation with extremely rapid pharmacokinetics. That fusion protein XL5-HSA was found to localize in in vivo thrombi to a greater extent than unmodified recombinant HSA suggests not only its superiority over our previously described α2AP (13-42)-HSA chimera, but also its potential utility in being incorporated as a "Trojan horse" into thrombi, and making them more susceptible to natural or pharmacological thrombolysis.
Recombinant HSA with a DQMMLPWPAVTLG4Y N-terminal extension (XL5-HSA) was more active as a substrate for FXIIIa-mediated cross-linking to either artificial or natural transglutamination partners than other candidate motifs of identical or longer length, with or without an N-terminal His tag. XL5-HSA, unlike α2AP(13-42)-HSA or H6α2AP(13-42)-HSA, was not subject to detectable proteolysis by P. pastoris. Of the proteins tested, XL5-HSA was the most effective competitor of α2AP-mediated resistance of fibrin clots to lysis. In contrast to previous results with α2AP(13-42)-HSA, radiolabeled tracer XL5-HSA was found to a greater extent in experimentally-induced intravascular clots in both murine and rabbit in vivo venous thrombosis models; importantly, the majority of clot-associated XL5-HSA and plasma-derived α2AP, but not recombinant HSA, was shown to be insoluble to 5.0 M urea, suggesting covalent cross-linking. Our results suggest that fusion protein XL5-HSA has been sufficiently optimized over prototype α2AP(13-42)-HSA to warrant in vivo testing as a potential protein drug rendering thrombi more susceptible to natural or pharmacological clot lysis.
Authors' contributions
WPS conceived of the study, secured competitive funding, directed experiments, and wrote the manuscript. LJE-S performed all in vitro and in vivo experiments and developed and refined experimental protocols. Both authors participated in editing and revising the manuscript. Both authors read and approved the final manuscript.
The authors thank Sharon Gataiance and Varsha Bhakta for expert technical assistance, and are grateful to Dr. Edward Przydial, University of British Columbia and Canadian Blood Services, for the generous gift of tPA. This study was made possible by a Grant-In-Aid from the Heart and Stroke Foundation of Ontario (award number T6588) to WPS.
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Sheffield WP, Mamdani A, Hortelano G, Gataiance S, Eltringham-Smith L, Begbie ME, Leyva RA, Liaw PS, Ofosu FA: Effects of genetic fusion of factor IX to albumin on in vivo clearance in mice and rabbits.
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text:
Mon-Fri: 7am-1am EST / Sat-Sun: 7am-11pm EST
ApplianceHotpoint Range
Level of DifficultyReally easy
Time to do repair15 - 30 mins
Age of Appliance5 - 10 years
Screw driversWrench(Adjustable)
6 out of 7 people found this instruction helpful
While I was waiting on another part to arrive, I decided it would be a good time to do a thorough cleaning. When I lifted the top of the range, I lifted it to far and cracked both front burner tubes. The top of the range should not be lifted more than 4 inches without removing the 3 screws that hold each burner in place. When the new lines came in, I removed both front burners, knobs, and then removed the front plate behind the knobs using a Phillips screwdriver. The screws are located on the bottom of the plate. Using my adjustable wrench I replaced the lines. Then I CAREFULLY bent the gas tubes and remounted the burners. Over all an easy install. Just be careful, the gas tubes are aluminum and will break very easy. This is the second time I used PartSelect, and I am very satisfied.
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Part Number: PS240685
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Female English bull dogs. . Unaltered will be 2 in September. . House broken. .. Good with kids other animals cats and other dogs. . AKC papers available. . Contact me with any questions
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Beautiful english bull dog pup 3 weeks old ready new home may 7. Will have shot in good health. Mom and dad,on site to see. One boy and one girl please call 4132197519
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"Elizabeth has written an absolutely critical book for social entrepreneurs. She provides a unique perspective on how different kinds of capital can be blended and sequenced. If you are not familiar with how to attract grants, low-interest loans, equity, loan guarantees, etc., to grow your business, you'll find this to be a hugely valuable investment in yourself. Read it, use it!"—Don Shaffer, president and CEO, RSF Social Finance
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Wireless Mouse
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2014 Ford Mustang Shelby GT500
Hand built with pride
This is the end of the line for this Mustang body style. The 2015 restyling will be slimmed down for aerodynamics and fuel efficiency, but with some traditional Mustang elements. The new car is expected to debut sometime around mid-April next year for the 50th anniversary of the Mustang. Will there be another GT500?
Ford isn't talking about the new model or possible variants, but there has been a Special Vehicle Team since the 1993 SVT Cobra. And a V8 is at the core of Mustang history, said my Mustang mole in Dearborn, Mich.
The engineers, designers and planners own Mustangs and are immersed in the culture, said my source. "They live in the (car) community, they own the cars and do the things that the enthusiast community does.
"Bottom line, we are not going to disappoint our fans."
2014 Ford Mustang Shelby GT500
--Body style: 4-seat, rear-wheel drive coupe
--Engine: aluminum, supercharged four-valve DOHC 5.8-liter V8 with over-rev capability
--Transmission: Tremec TR6060 6-spd manual
--Fuel economy: 15/24 mpg city/hwy.; premium fuel required
--Fuel tank: 16 gal.
--Length/wheelbase: 188.2/107.2 in.
--Curb weight: 3,850 lbs.
--Standard equipment includes: carbon-fiber driveshaft, LeMans top stripes with Gt500 side rocker stripes, HID projector headlights, quad rear-exit stainless steel exhaust with 3.5-in. tips, sport buckets seats with racing stripe inserts, Shaker audio system, air conditioning, 4.2-in. LCD screen
--Safety features include: 4-channel ABS, electronic brake force distribution, traction control
--Brakes: 4-wheel power discs; front 15-inch Brembo discs with six-piston calipers; 13.8-inch single piston rear
--Steering: electric power-assisted rack and pinion; 37-foot turning circle
--Suspension: independent MacPherson struts front, with reverse-L lower control arm, standard twin-tube dampers, 33.2 x 5.0-mm tubular stabilizer bar; 34.6 x 5.5-mm tubular stabilizer bar (on coupe); rear, 3-link solid axle with coil springs, Panhard rod, standard twin-tube dampers, 23-mm solid stabilizer bar, 25-mm solid stabilizer bar
--Tires and wheels: Goodyear Eagle F1 SuperCar G: 2 P265/40ZR 19-in. front; P285/35ZR-20 rear; Performance Package, 16-spoke 19x9.5-in. forged-aluminum front, 20x9.5-in. rear
--Base price: $55,445, including $795 freight charge; price as tested $65,470
--Options on test vehicle: Ruby Red paint $395; SVT Track Package $2,995; electronics package with navigation $2,340; Shelby leather Recaro seats $1,595
--Where assembled: Flat Rock, Mich.
--Warranty: 3-year/36,000-miles bumper to bumper; 5-years/60,000-miles powertrain with roadside assistance


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David Krejci, Anton Khudobin return
|01.11.13 at 9:31 am ET
WILMINGTON — David Krejci and Anton Khudobin were in town and skating with Bruins teammates Friday morning, leaving Dougie Hamilton as the only member of the projected opening-night roster not not practicing with the team this week. Hamilton can't technically make the leap to the NHL until the new collective bargaining agreement is ratified Saturday. Tyler Seguin, who has been skating with fellow Bruins since returning from Switzerland, was absent Friday, as was Johnny Boychuk.
Krejci played for Pardubice HC of the Czech Extraliga during the lockout, scoring 16 goals and adding 11 assists in 24 games. Khudobin played in the KHL and said recently that he would consider going back after his contract with the Bruins expires.
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The Berkley Gulp! Euro Larvae is the next generation in freshwater soft bait. It is biodegradable, made of natural ingredients, and has 400 times more scent dispersion than your ordinary soft bait. The Berkley Gulp! Euro Larvae has life-like detail that imitates natural prey and comes in a string for multiple rigging options.
The Berkley Gulp! Euro Larvae comes in a 150 count convenient re-closable zipper bag and in an assortment of different colors.
The Berkley Gulp! Euro Larvae is made in the U.S.A.
Enter each item individually, then click the "+" or press enter; or type multiple items with a "," between each one.


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The Additional Chief Metropolitan Magistrate, Egmore, on Tuesday convicted three persons for running an illegal telecom set-up in Triplicane that was raided by the CBI in 2005.
An official release stated that the Economic Offences wing of the CBI framed charges that three persons were running an illegal telecom set-up from a building on Triplicane High Road. Incoming ISD calls were received on Internet using Voice Over Internet Protocol technology and the voice calls were delivered through local telephones with false caller line identification.
The internet connections obtained in the name of an employee of the accused and the telecom set-up were in operation in violation of the Indian Telegraph Act. The operation caused loss to the tune of Rs.57.5 lakh to the government by way of evasion of prescribed charges on ISD calls.
The accused were convicted for six months imprisonment on two counts and a total fine of Rs.3,150 imposed, the release added.
Keywords: Indian Telegraph Act, Voice Over Internet Protocol


text: Minister for International Development Cooperation Gunilla Carlsson yesterday met Kofi Annan during his visit to the Ministry for Foreign Affairs. They spoke generally about developments in Africa and about the situation in the Horn of Africa and in Kenya. Mr Annan's mission as the Joint Special Envoy of the United Nations and the League of Arab States to Syria was also discussed.


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Andrew Meyer
|Andrew Meyer
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|Candidate for
|Ohio House of Representatives District 16
|Campaign website
|The information about this individual is current as of when his or her last campaign ended. See anything that needs updating? Send a correction to our editors
Meyer's professional experience includes working as an attorney.
Campaign themes
The following campaign themes were highlighted on Meyer's campaign website:
- Excerpt:"Our regional economy needs a new dynamism, new focus, and innovative ideas necessary to retake our place as an economic leader and powerhouse. We have a powerful foothold in the two tremendous growth areas of biomedical research and technology and green jobs. I believe our legislature should provide incentives to cultivate these strengths for the benefit of our larger economy, and help make us the "Silicon Valley" of these fields. We need to focus on the real issue of job creation, not the smoke screen of blaming our teachers and safety forces for our economic problems."
- Excerpt:"As a former substitute public school teacher, I know a child's education is a long term investment in our future. Our community can't afford the reckless and extreme cuts to education funding forced on our schools by the current administration. With your help, I will make sure parents, students and educators have the resources they need to prepare our young people for the future. In addition, I support the right of teachers to negotiate in good faith for the supplies, smaller class sizes, and benefits necessary to ensure schools in our District continue to attract the talented professionals we expect in our children's classrooms."
- Excerpt:"It's time for our state government to realize that a healthy environment means a healthy Ohio. The current administration has sacrificed our long term future for short term gain all too often. For example, permitting the dangerous method of drilling known as "fracking" in our state parks today will permanently damage one of our most precious resources to the detriment of generations to come. And, green jobs are a true growth area of the international economy in which we have already have an advantage. We can't afford to ignore the opportunity."
Meyer ran in the 2012 election for Ohio House District 16. Meyer defeated Todd LeVeck in the March 6 Democratic primary and was defeated by incumbent Nan Baker (R) in the general election which took place on November 6, 2012.[2][3][4]
|Ohio House of Representatives, District 16, General Election, 2012
|Vote %
|Nan Baker Incumbent
|Andrew Meyer
|Total Votes
|Ohio House of Representatives District 16 Primary Democratic Primary, 2012
|Vote %
|Andrew Meyer
|Todd LeVeck
|Total Votes
Recent news
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- All stories may not be relevant to this legislator due to the nature of the search engine.
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External links
- Official campaign website
- Biography from Project Vote Smart
- Legislative profile from Project Vote Smart
- ↑ West Life, "Baker seeking third term on same platform – jobs," December 22, 2011
- ↑ Ohio Chamber of Commerce, "2012 General Assembly Primary Candidates," January 17, 2012
- ↑ Ohio Secretary of State, "2012 Democratic Primary Results," accessed June 12, 2014
- ↑ Ohio Secretary of State, "Ohio official results for 2012 General Election," accessed June 10, 2014
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Pakistan on Wednesday hinted at the possibility of participating in the coming Bonn Conference on Afghanistan but ruled out any high-level representation on the ground that Afghan soil had been used by North Atlantic Treaty Organisation (NATO) to attack the country in what the Army calls a "deliberate" act of aggression.
Agreeing to consider German Chancellor Angela Merkel's repeated requests for Islamabad's participation, Prime Minister Syed Yusuf Raza Gilani said he would refer the suggestion of having Pakistan's Ambassador in Germany attend the deliberations to the Parliamentary Committee on National Security.
Ms. Merkel called Mr. Gilani to impress upon him the importance of Pakistan's participation at the meeting to make it meaningful. As Mr. Gilani was unwilling to budge on high-level participation, she suggested the Ambassador be permitted to represent Pakistan so that its seat at the table was not left vacant.
In view of bilateral relations and the fact that the German Foreign Minister was among the first to personally call his Pakistani counterpart to express solidarity with Pakistan and condole the death of 24 Pakistan Army soldiers in the NATO firing at Pakistani outposts on Saturday morning, Mr. Gilani agreed to refer the request to the Parliamentary Committee.
Meanwhile, the formal communication to the U.S. asking it to vacate the Shamsi airbase has been sent with December 11 set as the deadline.
Pakistan has released footage of two posts which came under fire from helicopters of the coalition forces in Afghanistan and wanted to know where the NATO casualties were in case there was firing from the Pakistani side.
Keywords: Bonn Conference on Afghanistan, NATO attack


text:
Glitzy & GLAMorous Kate Spade Inspired Pool Party
- posted in:
- Birthday Party Ideas
- Parties for Girls
Attention, party-loving fashionistas: this one's got chic STYLE and girlie FUN all rolled into one! Jen Carver of Banner Events (and her fabulous team of on-trend vendors) dreamed up a fabulously glitzy & glam Kate Spade Inspired Pool Party for her daughter's 10th birthday. And oh my stripes, polka dots and gold glitter confetti – is it alright that I just want this party for ME!? Pretty sure I'm not going to be the only one either, because this "retro glam meets modern chic" vibe would work SO well for all sorts of ages and occasions.
Scroll down to check out all the cheeky fun… like clever chocolate bar "purses", black & white striped bathing suit cookies that might just be my new favorite thing, and classic Kate Spade style patterns and icons dressing up every pretty little detail. ;)
PARTY HIGHLIGHTS to look out for:
– Bold black & white striped towels, bathing suit cookies & mod table runner
– Glam mini donuts, confetti dusted cake, and black/white Rice Krispie spades
– Water bottles and striped straws adorned with DIY paper bow ties
– "PARTY" marquee letters + jumbo balloons with matte & metallic tissue tassels
– Step-and-repeat inspired photo booth backdrop + matching beach ball props
– Black sequin linen, gold glitter confetti plates & sparkly spade centerpiece topper
– Heart sunglasses + custom black & white striped cover ups for the guests
- May 21, 2015 by jennifer
- tags:
- no tags


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Young Pirates dig deep
So much for logic.
When the University Interscholastic League realigned Granbury with six Fort Worth schools and Aledo last February, some eyebrows were raised.
Fort Worth schools specialize in basketball – fast-paced basketball.
Aledo, while playing a more deliberate style, was a playoff team a year ago and features arguably the best player in the district with Taylor Johnson.
And there was Granbury, a team with hardly enough varsity experience to fill the eye of a needle.
But also with more determination and fortitude than any other team in the league, apparently.
Despite their lack of experience, the Pirates did what no other Granbury team did since before this current crop of players was in kindergarten.
They advanced to bidistrict. Last night, Granbury (17-14) was facing Waco University in Waxahachie.
"(Brandon) Fichter moving in helped. He was on the varsity at Grand Prairie, and he's been a good leader for us," said Granbury coach David Breazeale. "Collin McCann has also been a vocal leader."
Both are seniors, two of six on the roster. Fichter played a major role in the 62-58 win over O.D. Wyatt that clinched the postseason berth.
Wyatt finished second in district and led the league for most of the season.
The Pirates, while inexperienced, have a lot of players – talented players. Breazeale used the depth to help the team get experience while on the job.
"Our depth helped us a lot," Breazeale said. "You've got to have depth to win at the 4A and 5A level.
"I played a lot of young kids, but the young kids I played were talented."
Breazeale also credited the players for playing in a lot of select league games – staying together for cohesiveness.
"They played about 110 games since the end of last year," he said. "It's not the same, I know, but it gives them a lot of experience going against the quickness."
That depth, led by Ficther's clutch play, led to one of the biggest wins in school history against Wyatt, a team that had beaten Granbury 21 points in Fort Worth.
"They just played out of their minds," said Breazeale of his squad. "That was as hard as any team's played since I've been at Godley."
Among his coaching success, Breazeale led Godley to four regional berths in seven seasons and took Graford to the 2000 Class A State Tournament.
At Mineral Wells, his last stop before Granbury, Breazeale led an overachieving Rams team to the postseason. And now he has the Pirates playing to a level the program hasn't achieved since they advanced three rounds in the postseason to conclude the 1999-2000 season under Brett White.
Whatever the outcome against Wyatt, the Pirates were guaranteed at least one more game. They would have played Aledo in a tiebreaker to determine fourth place in district.
Breazeale didn't want to get in that way, and neither did his team.
"We didn't back in," he said.
Instead, the Pirates displayed intestinal fortitude. Mired in a three-game losing streak, one that included blowing a late 10-point lead at Aledo, they found themselves tied at the half against lowly South Hills in the next-to-last regular season game.
Granbury came out and scored the first seven points of the third quarter and went on to win by 17.
[email protected] | 817-573-7066, ext. 256
Category: Page One News Archived


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DMV to Internet-less New Yorkers: Get in Line
By Jared Chausow
By Katie Toth
By Elizabeth Flock
City Hid Facts From Feds About Rikers
By Albert Samaha
Flood Wall Street Gets Pepper Sprayed and Arrested
By Anna Merlan
By Jon Campbell
Report Sheds Light on Anti-Muslim Bias
By Jon Campbell
Rikers Island Lawsuits Over Diabetic Care
By Albert Samaha
SAGITTARIUS (Nov. 22Dec. 21): Here are a few of the fine improvements I expect you to have accomplished by the end of June: tips on how to live well in two worlds; an addition to the reasons why people find you attractive; a crash course that helps you become more fluent in the language of intimacy; richer, more interesting feelings than you've experienced in a long time; and practical insights into how to avoid being flustered by paradoxes that have driven you crazy in the past.
CAPRICORN (Dec. 22Jan. 19): "If you make people think they're thinking," said author Don Marquis, "they'll love you; but if you really make them think, they'll hate you." My objective in this week's horoscope is to prove him wrong: I want you to love me for making you really think. In the hope of accomplishing this goal, I'm giving you the assignment of revising two of your long-standing opinions or theories about the way the world works. As you aggressively seek out the information that will help you change your mind, try to feel tender compassion for me, the wise guy who's asking you to undertake such an arduous and potentially rewarding task.
AQUARIUS (Jan. 20Feb. 18): When my friend Keith and I were in college in the early 1980s, we were extravagant ambassadors for poetry. On weekends we'd roam from party to party, reciting Neruda poems to audiences of drunk punks and declaiming Ginsberg verses as we teetered on the tops of cars. On occasion we'd scrawl our own poems on the walls of strange living rooms or improvise surrealistic spoken-word rants in the streets, begging for alms. Years later, I write a syndicated astrology column that might be described as a stealth poetry invasion, and Keith is a producer for a national news broadcast, onto which he sometimes brings noted poets to close the show with a lyrical splash. So now I ask you, Aquarius: What raw passion would you like to turn into a polished gig in the future? Now is a good time to make a deep commitment to it.
PISCES (Feb. 19March 20): "I usually solve problems by letting them devour me," wrote Franz Kafka. That's an interesting approach, I guess, and though it might work for a fire sign or air sign, it's not a wise policy for you Pisceans. In fact, I urge you to fervently resist any temptation you might have to allow your problems to gobble you up. On the contrary, be like a gargantuan sea monster in the midst of the perfect storm. Rise up as high as the dark sky and growl back at the thunder. Shoot flames from your mouth at the lightning. Become too big and ancient and wild to ever be devoured.
Homework What does this mean to you: "In the same way that you judge others, you will be judged"? Comment by going to realastrology.com and clicking on "Email Rob."
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Telstar 28
Join Date: Mar 2006
Location: New England
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Definitely sounds like water has gotten into the deck.. that's a bad thing. While potting the holes as described by Paul is a good idea... you'll probably have to remove the stanchions and pot the holes for them as well. However, you need to find out whether the core has rotted or is just wet. If it is just wet... you can dry it out by drilling holes to let air in and water out...
Did you get a survey on the boat??? If not, this is a good example of why you get a survey... and if you did, ask your surveyor about it and why he missed it... Wet core is definitely something that should have caused a price adjustment in the purchase price. It can be an expensive repair. However, from your description I would guess that the deck is cored with end-grain balsa and that the damage is fairly limited at the moment. The reason I'm guessing end-grain balsa rather than a foam or marine plywood, is that water tends to migrate more freely when it has been cored with the latter two materials. End-grain balsa tends to keep water confined due to the way it wicks water.
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Telstar 28
New England
You know what the first rule of sailing is? ...Love. You can learn all the math in the 'verse, but you take
a boat to the sea you don't love, she'll shake you off just as sure as the turning of the worlds. Love keeps
her going when she oughta fall down, tells you she's hurting 'fore she keens. Makes her a home.
Cpt. Mal Reynolds, Serenity
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Just 100 metres from St. Mark's Square, this small guest house is set in a 16th century town house. All air-conditioned rooms come with a flat-screen satellite TV and free Wi-Fi. The Rialto Bridge is a 5-minute walk away. With classic furnishings and tiled floors, rooms at Maison De Charme Balbi come with a modern private bathroom. Breakfast is self-service, and consists of coffee and croissants. Guests can have it in the breakfast room with kitchenette. Offering a very central location, the Balbi is served by water-bus lines 1 and 2. The Calle Vallaresso Waterbus Stop is 500 metres away, and it is a 7-minute walk to the San Zaccaria Stop.Please let Maison De Charme Balbi know your expected arrival time in advance. You can use the Special Requests box when booking or contact the property. A surcharge applies for arrivals outside check-in hours. All requests for late arrival must be confirmed by the property. Please note full payment is due at check-in. The guest house occupies the ground floor and 1st floor of a building with no lift.Ostrovokv2
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The security task force led by Sannidhanam police special officer, A.K.Venugopal, has conducted a combing operation in the forests surrounding Paandithavalom on Wednesday.
According to police, as many as 50 persons were taken into custody in the combing operation. All the 50 persons were wayside vendors and a majority of them reportedly hail from various North Indian States, police said.
The security squad have also conducted surprise inspections at various pilgrim shelters and donor houses at the Sannidhanam, later.
Mr Venugopal said security would be further tightened at Sabarimala in the coming days.
Five hotels fined
Meanwhile, another squad led by executive magistrate, V.Balakrishnan, have imposed a fine to the tune of Rs 20,000 on five hotels at Sannidhanam for violating the guidelines prescribed by the District Collector on Wednesday.
The squad also seized stale food from certain eateries at the Sannidhanam in the raid and have registered cases against the licensees, official sources said.
Rs 12 crore revenue
The Devaswom authorities at Sabarimala said the total revenue collection at the Ayyappa temple during the Makaravilakku festival has crossed Rs 12 crore on Tuesday evening against the Rs 10.4 crore
revenue at the pilgrim centre during the same period in the previous year.
The Aravana sales alone have fetched revenue worth Rs 4.06 crore, besides the Hundi collection of Rs 5.29 crore, sources said.
Keywords: combing drive, Paandithavalom, Sabarimala, Makaravilakku festival


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Get all the latest in MMA at http://MMAWeekly.com -- Bart Palaszewski talks about his UFC 137 victory over Tyson Griffin and how it cost him a new puppy. watch video >>
Bart Palaszewski destroyed Tyson Griffin at UFC 137, and we talked to him after the fight. Check out the exclusive interview. watch video >>
MMA H.E.A.T.'s Karyn Bryant talks with Bart Palaszewski minutes after his win at UFC 137 and hears what he has to say about his KO victory over Tyson Griffin. Bart lets us know his strategy leading watch video >>
MMA Plus speak to Jimi Manuwa ahead of his co-main event fight with Jan Blachowicz at UFC Fight Night: Krakow on April 11 watch video >>
MMA Plus spoke with TUF China veteran Rocky Lee as he prepares to make his UFC debut at UFC Fight Night in Krakow, Poland. watch video >>
Tune in this Friday, as Inside MMA correspondent Ron Kruck sits down with UFC Middleweight Champion Chris Weidman for an exclusive interview. AXS TV is Your Home for MMA every Friday Night! Find AXS watch video >>
UFC Bantamweight Champion T.J. Dillashaw joins AXS TV's INSIDE MMA for an interview with series hosts Kenny Rice and Bas Rutten. During the interview, Dillashaw gave an update on his recently announc watch video >>
UFC President Dana White joined AXS TV's INSIDE MMA for a one-on-one interview with correspondent Ron Kruck. During the interview, White shares his thoughts on former UFC welterweight champion George watch video >>
The MMA media spoke to Russia's Albert Tumenov following a hard fought decision victory over Nico Musoke in Stockholm, Sweden. watch video >>
The MMA media speak to UFC featherweight contender Sam Sicilia after the American knocked Akira Corassani out cold at the Tele2 Arena in Stockholm, Sweden. watch video >>
Chechnya's Mairbek Tausimov spoke to the MMA media following a dominant second round knockout victory over Greece's Anthony Christodoulou at UFC on Fox in Stockholm, Sweden. watch video >>
The MMA media speak to Nikita Krylov following his first round submission victory at UFC on Fox 14 watch video >>
MMA Plus caught up with tiger muay thai's Mairbek Taisumov a head of his fight Anthony Christodoulou. watch video >>
SUBSCRIBE - https://twitter.com/SubmissionAus Bill Goldberg makes a big appearance on Submission Radio to talk about many topics, including his NFL career, his pro wrestling career, and his love for watch video >>
GSP talks everything MMA, UFC, and more in this 45 minute sit-donn interview with "Showdown" Joe Ferraro at The Gentleman's Expo in Toronto. watch video >>


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The dual attack by terrorists on Karachi International Airport has forced the Mumbai Police to review their security apparatus at places witnessing high footfall and at vital installations. The police, in a review carried out in March this year, had identified around 600 spots in the city where maximum damage can be caused in a terrorist attack.
On Sunday night, Tehreek-e-Taliban had launched a deadly attack on Karachi airport, killing at least 35. The terrorists had posed as security force personnel and launched an attack using grenades and rocket launchers.
Following this, Mumbai Police sources said central intelligence agency sounded an alert across the state, suggesting them to step up security at vital installations and places witnessing high footfall. The alert had a "specific" mention of Karachi attack, sources added.
"An alert has been sounded at airports, railway stations and places that figure in past intelligence inputs. We have initiated fresh audits and a review of security. We are conducting meetings with those responsible for security at important places and doing a general hardening of target," said additional director general of police, state Anti-Terrorism Squad, Himanshu Roy.
Some of the places that have high footfall are Siddhivinayak temple, Mumba Devi temple, Zaveri Bazaar, Gateway of India, malls, multiplexes, stock exchange and ports.
"These places have figured in previous attacks or reconnaissance conducted by terrorists. All possible measures will be taken to ensure there is intensified monitoring of movements of suspected persons at these places. We also take measures such as checking lodges, restaurants and getting information about stolen vehicles and SIM cards procured through bogus documents as it has been seen in the past that terrorists conceal their identity and stay in the city to conduct recce," said a senior police officer.
"In the audit, we will identify the loopholes, whether redeployment of men is required, whether there is any laxity in checks, functioning of CCTV cameras, etc," said joint commissioner of police, law and order, Dhananjay Kamlakar.
"We regularly carry out Sagar Kavach and Mumbai Kavach operations in the city to check our preparedness of coastal security and other places in the city. In Mumbai Kavach alone, over 100 manned and unmanned points are covered," said additional commissioner of police, protection and security, Kiran Shelar.


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Advice on Cal 34 MK III Purchase
Hello all. I'm new to the board but not to sailing. Grew up sailing on my family's 40ft-ish boats, mostly coastal cruising.
I'm ready to take the next step and buy my own boat. I want something for coastal cruising, and perhaps someday offshore. Not interested in racing; mainly just taking it easy and exploring.
I am looking at a 1978 Cal 34 mk III. It looks to be in good shape for a boat of its vintage. Has new AP, GPS, chartplotter, and transmission. 24 HP Westerbeke engine with around 1300 hours. The sellers have sailed the West Coast on it (several thousand mile trip) recently.
The asking price is $29,000. That seems higher than a lot of other Cal 34s I've seen online, but it does have brand-new electronics and transmission.
Any input on the price, the boat, etc? All comments are appreciated!
What kind of shape is the boat you're looking at compared to the others. If it is in significantly better maintained shape, it may be worth the price difference. I'd highly recommend you read the Boat Inspection Trip Tips thread I started, as it will help you determine whether this boat is worth looking at further.
I think you are starting at the right place - a boat well above the average asking price. If you can establish that it is worth a premium, IMHO you are far more likely to end up adead of the cost curve, than if you were bottom feeding. Don't be afraid to spend the extra dollars for a premium boat, the seller probably is still taking a hosing.
Thanks for the replies so far. It's in pretty nice condition. Paint looks good, wood on the deck could use some work and the inside looks clean and well-maintained. The VHF is ancient, and the stove is alcohol (no oven, like I've seen online on some of them) but other than that it looks in good condition. The engine is spotless.
remember to look at the sails. A new genoa, main, storm jib, and spin, will cost about half the asking price of the boat you're looking at. Maybe more, maybe a little less depending on material, etc...
Hows the rusty steel mast beam as that issue follows them right up to the Cal 40
Cal 34 steel beam
Is the keel encapsulated?
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Mon-Fri: 7am-1am EST / Sat-Sun: 7am-11pm EST
ApplianceWhirlpool Freezer
Level of DifficultyReally easy
Time to do repair15 - 30 mins
Age of Appliance5 - 10 years
NutdriverScrew drivers
0 out of 1 people found this instruction helpful
removed ice bin from freezer then removed front from ice bin then all i had to do was remove e clamp from end of shaft that made it possible for me to remove the ice crusher parts then slide off the drum. all that was left was to put back together.
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