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Hudson Valley Shakespeare Festival has announced the lineup for its popular new works series, HVSF2. (more...)
BEDLAM presents the sold-out, award-winning and critically acclaimed production of SENSE & SENSIBILITY, returning to THE GYM AT JUDSON (243 Thompson Street) for an encore run beginning this Friday, June 17, 2016, through Sunday, October 2, 2016. (more...)
BWW Review: George Bernard Shaw's SAINT JOAN Is Honest And Immersive (more...)
?BEDLAM has announced the sold-out, award-winning and critically acclaimed production of SENSE & SENSIBILITY will continue its run at THE GYM AT JUDSON (243 Thompson Street) from Friday, June 17, 2016, through Sunday, October 2, 2016. Following a 2 month hiatus, The Dashwood Sisters are back from their spring holiday and regular performances will resume. (more...)
The Nora Theatre Company is proud to present Bedlam performing their two version of TWELFTH NIGHT: TWELFTH NIGHT (or WHAT YOU WILL) and WHAT YOU WILL (or TWELFTH NIGHT). Both plays are the text of TWELFTH NIGHT written by William Shakespeare. (more...)
The Drama League (Executive Artistic Director, Gabriel Shelian-Shanks) gathers today for the 82nd Annual Drama League Awards Ceremony, hosted by 2016 Tony® Award-nominees, Zachary Levi (She Loves Me) and Megan Hilty (Noises Off), in the Broadway Ballroom at the Marriott Marquis Times Square (1535 Broadway). (more...)
The Pearl Theatre Company has announced programming for the 2016-17 season. All four productions will take place at The Pearl Theater (555 West 42nd Street.) (more...)
The 2016 Lucille Lortel Awards for Outstanding Achievement Off-Broadway were handed out last night to recipients in 18 categories, with two special awards and two honorary awards also bestowed. The Lortel Awards were distributed in a ceremony at NYU Skirball Center hosted by Zachary Levi, star of Broadway's She Loves Me. This year's event was once again a benefit for The Actors Fund. BroadwayWorld is taking you to the red carpet below! (more...)
The Off-Broadway League tonight presents the 31st Annual Lucille Lortel Awards for Outstanding Achievement Off-Broadway at NYU Skirball Center. BroadwayWorld will be bringing you the full list of winners below. Stay tuned to find out who will win big! (more...)
Abingdon Theatre Company will present a special one-night only engagement of THE DORK KNIGHT, written and performed by Jason O'Connell, on Monday, May 16 at 7pm in the June Havoc Theatre (312 West 36th Street). Eric Tucker is set to direct. (more...)
The 2016 Drama League Award Nominees for Outstanding Play, Outstanding Revival of a Play, Outstanding Musical, Outstanding Revival of a Musical, and the much-coveted Distinguished Performance Award were just announced this morning at Sardi's, by Aladdin stars Adam Jacobs, Courtney Reed and James Monroe Iglehart.
Check out the full list of nominees below! (more...)
McCarter Theatre Center Artistic Director and Resident Playwright Emily Mann is pleased to announce the lineup for the upcoming 2016-2017 Theater Series. (more...)
Having just completed successful and critically acclaimed runs of Pericles and A Midsummer Night's Dream, Artistic Producer Janet Alexander Griffin today announced the program for Folger Theatre's upcoming twenty-fifth anniversary season. (more...)
The Off-Broadway League today announced nominations in 18 categories for the 31st Annual Lucille Lortel Awards for Outstanding Achievement Off-Broadway. The Lortel Awards will be presented on Sunday, May 1, 2016 at NYU Skirball Center beginning at 7:00 PM EST. This year's event will once again benefit The Actors Fund. (more...)
Two River Theater, under the leadership of Artistic Director John Dias and Managing Director Michael Hurst, announces the lineup of productions for its 2016/17 Season, which will launch in September with August Wilson's Ma Rainey's Black Bottom, directed by Tony Award-winner Ruben Santiago-Hudson. Full-season 6-play subscriptions are on sale now from 732.345.1400 or 21 Bridge Avenue, Red Bank. Subscribers save 25% on tickets, receive prime seating and a number of additional benefits. Visit tworivertheater.org for more details. Single tickets will go on sale in August 2016. (more...)
Titan Theatre Company opens the final production of its 2015-16 season this week, Shakespeare's epic political drama, JULIUS CEASAR. Performances will begin March 25th at the Queens Theatre, and BroadwayWorld has a behind-the-scenes look at rehearsal below! (more...)
The Oregon Shakespeare Festival (OSF) Artistic Director Bill Rauch announced the Oregon Shakespeare Festival's 2017 playbill today. (more...)
BEDLAM and BURNT UMBER PRODUCTIONS are pleased to announce the sold-out, critically acclaimed production of SENSE & SENSIBILITY which is currently playing through Sunday, April 17, 2016 at The Gym at Judson (243 Thompson Street), will return to The Gym at Judson for an encore run from Friday, June 17, 2016, through Sunday, October 2, 2016. (more...)
Actors' Shakespeare Project leadership team Allyn Burrows, Artistic Director, Kimberly Dawson, Interim Executive Producer and Mara Sidmore, Director of Education announce today the four plays, dates and directors that will comprise the lineup for the company's 13th season. Subscriptions are now on sale, and single tickets will go on sale this summer. For subscriptions and more information visit actorsshakespeareproject.org or call 617.776.2200 ext. 225. (more...)
'Dead Dog Park' is on stage at 59E59 now through Sunday, March 6th. This is one to see, an excellent, pointed play that deals with racial tensions as a white officer is accused of pushing an African-American youth out of a four-story window. (more...)
1 2 3 4 … 10 next »
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Jon Jones retains UFC crown
TORONTO - Jon Jones retained his Ultimate Fighting Championship light-heavyweight crown with a unanimous decision over Alexander Gustafsson in the UFC 165 headliner on Saturday at Air Canada Centre.
In what was perhaps the frontrunner for "Fight of the Year," all three judges scored it for Jones (19-1, 13-1 UFC): 48-47, 48-47 and 49-46.
Gustafsson gave the champion all he could handle. He cut Jones over the right eye in the first round with a glancing blow, and the cut grew into a gash over time, the Swede scoring with repeated multi-punch combinations to the head and body.
After 25 minutes, Jones was bloodied and swollen, but he answered the first serious challenge of his career.
"I've been asking for a dogfight for a long time, and I finally got that dogfight I was looking for," he said. "Tonight was a blessing in so many ways. I got the victory, and I got to prove a lot to myself. I'm not satisfied. I've got to do a lot of work in the gym to improve my game."
In the co-main event, Renan Barao retained his interim bantamweight champion in spectacular fashion, as he stopped Eddie Wineland with a spinning back-kick to the face and follow-up punches in the first round.
Wineland (20-9-1, 2-3 UFC) succumbed to the blows 35 seconds into Round 2 as he was put away by strikes for the first time in his career.
The two world-ranked bantamweights battled to a relative stalemate through the first five minutes, though Barao started to zero in on his foe late in Round 1.
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Lady Gaga: 'Mary Jane Holland' Snippet - Listen Now!
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Conceived and designed the experiments: VSP JR CL. Performed the experiments: JR CL MI ML PN. Wrote the paper: JR CL NK PN VSP.
Enhanced production of a 42-residue beta amyloid peptide (Aβ42) in affected parts of the brain has been suggested to be the main causative factor for the development of Alzheimer's Disease (AD). The severity of the disease depends not only on the amount of the peptide but also its conformational transition leading to the formation of oligomeric amyloid-derived diffusible ligands (ADDLs) in the brain of AD patients. Despite being significant to the understanding of AD mechanism, no atomic-resolution structures are available for these species due to the evanescent nature of ADDLs that hinders most structural biophysical investigations. Based on our molecular modeling and computational studies, we have designed Met35Nle and G37p mutations in the Aβ42 peptide (Aβ42Nle35p37) that appear to organize Aβ42 into stable oligomers. 2D NMR on the Aβ42Nle35p37 peptide revealed the occurrence of two β-turns in the V24-N27 and V36-V39 stretches that could be the possible cause for the oligomer stability. We did not observe corresponding NOEs for the V24-N27 turn in the Aβ21–43Nle35p37 fragment suggesting the need for the longer length amyloid peptide to form the stable oligomer promoting conformation. Because of the presence of two turns in the mutant peptide which were absent in solid state NMR structures for the fibrils, we propose, fibril formation might be hindered. The biophysical information obtained in this work could aid in the development of structural models for toxic oligomer formation that could facilitate the development of therapeutic approaches to AD.
The molecular pathology of Alzheimer's disease (AD) is characterized by increased accumulation of 39 to 43 residue long beta-amyloid peptides (Aβ) in plaques in the brains of Alzheimer's disease (AD) patients. Interestingly, individuals of nearly all ages have moderate amounts of matured amyloid peptide fibrils present in their brains, but only a certain percentage of them develop AD. Previous work in this area has indicated that the presence of oligomeric forms (amyloid-derived diffusible ligands, ADDL) of the 42 residue beta-amyloid peptides, rather than fibrils, are responsible for the neuronal damage and synaptic plasticity in the central nervous system in AD [1], [2]. It has also been shown that the severity of neuronal damage is well correlated with the ADDL content of Aβ peptides in the Alzheimer's diseased brain [3].
Monomers of the wild type form of Aβ42 (Aβ42WT) associate into unstructured assemblies with variable aggregation numbers [4], [5]. Both oligomers and fibrils originate from these unstructured intermediates. Ultimately, ADDLs are unstructured intermediates leaving only matured fibrils as the most stable entities [6], [7]. Much effort has been undertaken to probe the Aβ fibril state utilizing solid-state NMR [8], X-ray [9], Cryo EM [10], electron microscopy [11], neutron scattering [12], atomic force microscopy [13] and other spectroscopic methods [14], [15].
Very little is known about the structure of the ADDL form of the beta-amyloid peptide. The inherent formation of higher order aggregates by Aβ is a great challenge for the experimental characterization of ADDLs. Researchers working in this area are hampered by the difficulties of obtaining sufficient concentrations of ADDL for spectroscopic measurements [16]. Efforts have been made to search for means to stabilize the oligomeric, ADDL, species. For example, Wetzel et al. and others have identified small molecules that stabilize the Aβ42WT in the proto-fibrillar forms [17], [18]. The work of Selkoe et al. [19] comparing neurotoxic properties of Aβ42WT and the Arctic mutant E22G Aβs, proposed that the stability of ADDLs is inversely correlated to the nucleation rate of the formation of fibrillar aggregates. Using photo-induced cross-linking and gel electrophoresis, Bitan et al. suggested the predominant ADDL species of Aβ42WT are monomers, dimers, trimers, pentamers, and hexamers [20]. They have also observed that tetramers are rare and thought to be unstable intermediate species in the oligomerization pathway. A predominantly ADDL forming mutant of Aβ is yet to be known in the literature.
Based on our earlier in silico modeling studies, and supported by other work suggesting the formation of β–strand character in the C-terminus of Aβ, we propose an Aβ variant with two point mutations (Met35 to norleucine, and Gly37 to D-proline) to form stable, soluble oligomers [21], [22], [23]. Previous work has demonstrated the ability of single substitutions in the Aβ sequence to alter stability and morphology of Aβ assemblies [24]. First, we substituted the isosteric norleucine in place of Met35 to act as a non-perturbing replacement which would remove issues that could result from the variability of the oxidation state of Met35. Secondly, we substituted a D-proline for G37 in order to stabilize the oligomeric structure seen in simulations, i.e. a C-terminal beta hairpin with a turn at residues 37–38 [23]. In this paper, we probe structural properties of Aβ42Nle35p37 and its truncated version Aβ21–43Nle35p37 using different biophysical techniques. In particular, we have used Nuclear Magnetic Resonance (NMR) spectroscopy to determine structural properties of ADDL form, and finally use this data in molecular modeling to refine observed structural features.
In a previous computational study, Kelley et al hypothesized that a turn promoting mutation such as G37p would stabilize trimeric Aβ oligomers [23]. In agreement with this prediction, the mutant peptide, Aβ42Nle35p37, was found to adopt a significantly higher yield of soluble oligomer than that of Aβ42WT. This is seen in the one-dimensional 1H NMR spectra of Aβ42WT (Figure 1A) and Aβ42Nle35p37 (Figure 1B) for samples prepared in the same manner. The Aβ42WT spectrum has very little observable signal consistent with the peptide having precipitated from solution (no signal to detect) and/or formed very large molecular weight aggregates (significant line-broadening). Whereas the same spectral region for Aβ42Nle35p37 shows amide and aromatic resonances characteristic of soluble, non-aggregating peptides/proteins.
Conformational analyses of the mutant and Aβ42WT peptides were also carried out by Circular Dichroism (CD) spectroscopy. A representative CD trace of Aβ42WT aggregate is observed to develop into β-sheet rich, mature fibrils over a period of 12 hours with the characteristic strong absorbance at ~220 nm (Figure 2, Curve WT). However, in the case of the Aβ42Nle35p37 mutant, no change was observed in the CD spectrum after one week. Figure 2, Curve Mut shows the CD spectrum of Aβ42Nle35p37 having a strong absorbance around 197 nm typical of random-coil, disordered states in solution.
Thioflavin T fluorescence was used to assess aggregation and mature fibril formation as β-sheet content is directly correlated with fluorescence intensity of the dye. We observed about four times more Thioflavin T fluorescence in the Aβ42WT peptide when compared to Aβ42Nle35p37 (Figure 2 inset) confirming the aggregating and non-aggregating nature of the WT and mutant peptides, respectively.
High-resolution analysis of the peptide solution preparations were carried out using atomic force microscopy (AFM). Figure 3 shows AFM images of the soluble form of Aβ42WT and Aβ42Nle35p37 peptide preparations deposited onto clean silicon wafers. The wafers for Aβ42WT wildtype preparations showed particles with a mean globular structure height of 4.32 nm but with a variation of particle sizes ranging from 2.06 to 14.79 nm. Notably, particles appeared to be adhered to each other or connected by thin fibrils suggesting they were in the process of forming larger aggregates. The wafers for the Aβ42Nle35p37 mutant preparations showed particles with a similar mean globular structure height of 4.00 nm compared to wildtype, however the distribution of heights was much smaller at 2.29 to 4.84 nm. This, along with the relatively uniform density of globular structures observed, suggests the mutant peptide is stabilized in smaller oligomers.
Having demonstrated that the full-length mutant Aβ42Nle35p37 peptide forms assumes a stable, low-molecular weight form in solution we proceeded with multi-dimensional NMR experiments on the mutant peptide. The quality of two-dimensional 1H-1H TOCSY and NOESY spectra were marginal due to signal overlap and line-broadening (Figure 4ABC) likely caused by a combination of low-level aggregation, conformational heterogeneity, and conformational averaging between transiently structured conformations.
In order to aid the assignment problem, we built on previous work suggestive that the N-terminal residues of the Aβ sequence are flexible and unstructured by working with a truncated form of the mutant peptide, Aβ21–43Nle35p37 [23], [25]. When HFIP film of this truncated mutant peptide was dissolved, we observed higher solubility (>4 mg/ml) compared to the full-length mutant peptide (~1 mg/ml). This truncated peptide remained in solution without any fibril formation, similar to the full-length mutant, for a period of more than 6 months (data not shown).
The TOCSY and NOESY spectra of the truncated peptide gave slightly better quality data (Figure 4DEF) when compared to the full-length mutant peptide. Assignments, though not trivial, were more tractable on this construct. Working with the information gleaned from the truncated form allowed a degree of validation for the full-length mutant assignments (the spectral profiles showed similarities though not one-for-one overlays). Interestingly, save for Y10 and K16, the N-terminal residues in the full-length mutant peptide, Aβ42Nle35p37, were not readily assigned, consistent with the N-terminus being flexible and unstructured. Assignments are given in Tables 1 and and22 for Aβ42Nle35p37 and Aβ21–43Nle35p37, respectively.
A wealth of significant NOE crosspeaks was not expected from these peptides but the few that we observed were rather interesting. Comparative intensities of NOEs for Hα-NH (i, i+1) and Hα-NH (i, i) are indicative of turn conformation [26], [27], [28]. Specifically, residues that have extended conformation show higher Hα-NH (i, i+1) NOE intensity compared to that of Hα-NH (i, i), while residues involved in turn conformation show higher Hα-NH (i, i) intensity than the corresponding Hα-NH (i, i+1) intensity. For the full length mutant Aβ42Nle35p37 peptide, Hα-NH (i,i) NOE cross peaks are observed for G25-S26 and G38-V39. Also observed are NH-NH (V24-G25) and NH-NH (G25-S26) NOEs suggesting a type I turn in the region of V24-N27. Hα-NH (p37-G38) and NH-NH (G38-V39) NOEs suggest that the p37-G38 segment forms a type II′ β-turn. The Hα-Hδ (V36-p37) cross peak observed between V36 and p37 indicates that p37 is in the trans conformation. Earlier work on Aβ42WT monomers suggests turns around D7-Y10 and V24-N27 [29], [30]. Our data supports the existence of this second turn. Though we do not observe the first turn, we cannot discount the possibility of its presence being very transient in nature given the difference in spectral properties observed between the full-length and truncated mutant peptides.
For the truncated mutant Aβ21–43Nle35p37 peptide, as in the full-length mutant, we observed Hα-NH (p37-G38) and NH-NH (G38-V39) NOE cross peaks suggesting a type II′ β-turn involving residues V36-V39. Similarly, an Hα-Hδ (V36, p37) cross peak suggests the presence of trans conformation for p37. We do not observe NH-NH NOEs for the turn around V24-N27, but this may be the result of N-terminal flexibility in this truncated construct.
Refinement via molecular dynamics using the NMR constraints was used to produce an ensemble of structures for the NOE containing regions of the full length mutant Aβ42Nle35p37 peptide. (Figure 5, PDB S1 and PDB S2) We see the existence of two definite turns – the D-proline induced beta-hairpin (residues V36-V39) and a more N-terminal turn consisting of residues V24-N27, which is in agreement with the available SS-NMR fibril studies and complimentary unconstrained MD simulations [6], [31], [32], [33].
Aβ42WT was found to adopt a soluble oligomeric form to a much greater extent when mixed with the mutant. By co-solubilizing Aβ42WT with Aβ42Nle35p37 in 14 and 41 ratios, the mixtures resulted in one-dimensional 1H NMR spectra very similar to that acquired on Aβ42Nle35p37 alone (Figure 6AB). To ensure the spectra observed were not simply the result of all Aβ42WT precipitating, oligomerizing, or aggregating to itself leaving only mutant Aβ42Nle35p37 peptide observable by NMR, we also produced the same WT: mutant peptide mixtures using uniformly 15N-labeled Aβ42WT. We were able to observe resolved 1H{15N} HSQC spectra (Figure 6C) verifying the ability of the mutant peptide stabilizing the WT in solution, and suggesting that the mutant peptide could act as an inhibitor to fibril formation of Aβ42WT. Acquiring two-dimensional TOCSY experiments on the mixture samples found similarities in CαH chemical shifts to the mutant peptide alone (Figure 6D) suggestive that the Aβ42WT adopts a similar conformation to the Aβ42Nle35p37 mutant when the two are mixed.
In our effort to design a soluble oligomer-forming mutant, we have previously predicted that the mutation of G37p will result in enhanced β hairpin formation and increased oligomer stability. Two-turn structures were obtained using NOE refinement. The occurrence of the first turn at V24-N27 is in agreement with the available SS-NMR fibril studies. The second turn consisting of residues V36-V39, absent in previous studies, is introduced here by the mutation. In the nucleated polymerization model fibrils are likely to grow by monomer addition [34]. The two-turn structure of the mutant possibly depletes monomer content by diverting peptide into a stable oligomer formation.
Earlier NMR studies showed that Aβ monomers adopts a collapsed coil (mostly random) with a well-defined central hydrophobic cluster (L17-A21) and turn- or bend-like structures (D7-E11 and A21-S26) [35]. The data presented here are consistent with these previous observations. Additionally, we have shown a β-turn in the C-terminal region of the peptide. Wuthrich et al. have also studied the structure of the oxidized form of Met35ox Aβ40 and Aβ42 peptides in aqueous Tris-HCl buffered solutions at pH 6.4–8.2 [36]. They showed unstructured peptide strands punctuated by turns around S8-V12 and F20-V24 regions. Their 15N{1H} NOE data showed that the Aβ42Met35ox has reduced flexibility at the C-terminus relative to the Aβ40 Met35ox suggesting insipient structure around this region, consistent with our hypothesis of a beta hairpin in the same region.
How does this structural data compare to previous SS-NMR data studies on fibril structure? Almost all fibrillar studies show a hairpin turn forming somewhere between residues 24 and 30. Examining three of the most recent and notable studies, we find three different sets of amino acids forming the turn location, although it is arguable that this may be attributed to the differences in information content for the various methods and their corresponding sensitivity to structural disorder [6]. Ohman's 2006 study of residues 1–42 predict a turn consisting of residues 25–28 or GSNK in the sequence [31]. Riek's predicted turn of the same chain is shifted two amino acids towards the N-terminus at residues 27–30 and sequence NKGA [32]. Tycko's recent work concerning Aβ40WT has found a turn at residues V24-N27 with sequence VGSN [6]. This turn location is in agreement with our oligomer data. Moreover, our ensemble highly resembles an unconstrained MD study of the same chain, in which the ensemble was clustered and the most populated node was presented [33].
Our refined ensemble shows a less static ensemble than the SS-NMR fibril studies. The d-Pro induced turn at residues V36-V39 disrupts the inter-chain contacts present in the fibril models by changing the monomer topology. This leaves the VGS turn sequence to stabilize itself exclusively via intra-chain contacts, and we suggest this to be the reason we see greater flexibility in this region.
The AFM data for the mutant Aβ42Nle35p37 peptide suggest that it forms predominantly low molecular weight species in solution. The 4 nm mean AFM particle height would be in the range of five- to eight-mer complexes by a statistical analysis performed by Lobanov et al. on the radius of gyration of >3500 protein domains in the SCOP database [37]. But notably, the domains in the Lobanov study were compact, folded, α and/or β containing proteins. The likelihood of the Aβ mutant peptide being in a loose, predominantly undefined structure, would possibly reduce the number of monomer units present per complex.
The data presented in this paper indicate the structurally disordered oligomeric assemblages of Aβ42WT and mutant differ in their propensity to form oligomers and fibrils. Aβ42WT peptide formed fibrils at the concentration of 0.4 mM at 10% DMSO/ PBS. The mutant preparation resulted entirely in low molecular weight entities. NMR studies on Aβ42Nle35p37 showed occurrence of two β-turns in the stretches V24-N27 and V36-V39.
Upon mixing Aβ42Nle35p37 mutant with Aβ42WT, Aβ42WT peptide is stabilized in solution suggesting a significant reduction in fibril formation. Presumably such reduced fibril formation is due to the engineered β-turn of the mutant (V36-V39) hindering the formation of the C-terminal β-turn (V24-A30) found in the fibril SS-NMR structure. Although our finding implies the existence of a stabilizing structure for the ADDLs in the mutant peptide, we were not able to detect any known secondary structure stretches, other than the two β-turns, by 1H-NMR and CD spectroscopy. This suggests that β-sheet or α-helix formation is not required for the ADDL stability. Finally, the ability of this mutant to inhibit the aggregation of WT Aβ peptide opens a door to another use for this mutant peptide, since a variant of this peptide or a small molecule peptide mimic could potentially serve as a means to inhibit Aβ aggregation.
How are these results useful in gaining insight into the nature of WT Aβ? While our NMR structural data of the mutant does not directly give structural data regarding the WT, the fact that the mutant mixed with the WT has slowed aggregation suggests that the C terminal beta hairpin presumably stabilized by the mutant does have structural relevance for understanding the nature of the aggregation of WT Aβ. Future work could either use the C terminal beta hairpin motif for small molecule drug discovery in order to find novel small molecule inhibitors of Aβ aggregation.
Synthetic peptide Aβ42Nle35p37 of the sequence DAEFRHDSGY10EVHHQKLVFF20AEDVGSNKGA30IIGLNLVDpGVV40IA was synthesized and purified by Anaspec (San Jose, CA). AEDVGSNKGA30IIGLNLVDpGVV40IAT (Aβ21–43Nle35p37) was synthesized and purified by the Stanford Protein and Nucleic Acid Facility. Recombinant purified Aβ42WT, both unlabeled and 15N-labeled, peptides (>95%) were obtained from rPeptide (Bogart, GA). All peptides were used as supplied. Solution samples of the peptides were prepared as follows. Peptides were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to concentrations of 1 mM HFIP, evaporated over nitrogen, and then dried in a Savant Speed Vac for 1 hr. The resultant peptide films were further kept under vacuum for a few hours to remove solvent traces. The films were stored at −80°C until use. Peptide films were dissolved to ~0.5 mM concentrations in 10% d6-DMSO/10 mM PBS buffer to a final pH of 7.2. The solutions were incubated at 4°C for 12 hours, and then transferred to 37°C for 4 hours. The resulting solutions were centrifuged at 13000 rpm in a desktop centrifuge (Eppendorf) at 4°C for 30 min to remove any precipitated gel-form of the peptides. After centrifugation, the peptide concentrations were adjusted to 0.4 mM (concentrations were checked by UV visible spectroscopy), 0.05% sodium azide added as a bacteriostat and transferred to 5 mm NMR tubes. Mixtures (41 and 14) of Aβ42Nle35p37Aβ42WT (total peptide concentration of 1 mM) were prepared in HFIP. The solvent was evaporated using speed-vac and resultant peptide films were dissolved in 10% d6-DMSO/10 mM PBS buffer and processed as above. d6-DMSO was purchased from Cambridge Isotope Laboratories (Cambridge, MA). Tris(2,2′-bipyridyl)dichloro ruthenium(II) (Ru(Bpy)) and ammonium persulfate (APS) were purchased from Sigma.
Aβ42Nle35p37, having been dissolved and processed above (10% DMSO/PBS buffer), was passed through a 10/30 Superdex 75 HR column to remove DMSO and immediately analyzed by CD. An Aβ42WT sample was prepared into PBS buffer in order to demonstrate a representative beta-sheet rich CD trace. The fibrils from the Aβ42WT preparation were centrifuged at 12000 rpm for 15 min to separate from any remaining monomer and the pellet was re-suspended in 10 mM phosphate pH 7.2 for CD measurement. CD measurements were performed using a 0.1 mm path length quartz cell in an Aviv 62A DS Circular Dichroism spectrometer (Aviv Associates, Lakewood, NJ) at 22°C.
Mean residue ellipticity (θ) was determined according to the equation θ=θObs • MRW/(10 • l • c); where θObs is observed ellipticity, MRW is mean residue weight of the peptide, c is peptide concentration (g/L), and l is optical path length (cm). The thioflavin T (ThT) binding studies of Aβ42WT and mutant peptides were carried out by incubating precipitated fibrils of the Aβ42WT or eluted oligomer preparation from the SEC column. The 25 µM amyloid fibrils (Aβ42WT) or oligomers of mutant were mixed with 5 µM of ThT dye concentration. After 2 h incubation, the fluorescence of the sample solutions were recorded by exciting at 440 nm and the intensity at 480 nm is monitored using Perkin-Elmer LC 55B spectrofluorimeter. The bandwidths of excitation/emission wavelengths were 5 nm/each.
AFM topography images were acquired in the light tapping mode regime using a Multimode AFM (Veeco, USA). Resonant frequencies of the uncoated silicon tips (MikroMasch, USA) were roughly 150 kHz, with scan rates around 5–8 µm/s. Peptide samples were prepared using the protocol described above and diluted to 25 µM before application. Peptide solutions were adsorbed by incubating onto Pirahna cleaned silicon wafers with molecular smoothness (~1 Å RMS roughness) as determined by AFM. After incubating for 10 min, the wafers are gently washed repeatededly with MilliQ water.
NMR spectra were acquired at the Stanford Magnetic Resonance Laboratory on a Bruker Avance 500 MHz spectrometer running TopSpin v1.3 and equipped with a 5 mm H{CN} Z-axis gradient CryoProbe. Peptide samples were prepared as described above in 10% d6-DMSO/10 mM PBS buffer of pH 5.5 or 7.2. One-dimensional 1H experiments were acquired with 16384 total data points, 12 ppm spectral window, and number of scans ranging from 64 to 256. Two-dimensional 1H-1H TOCSY (TOtal Correlation SpectroscopY) experiments were acquired with 2048 total data points, 11 ppm spectral windows, 60 or 80 ms dipsi2 mixing times, for 256 to 512 increments of 32 to 128 scans per increment. Two-dimensional 1H-1H NOESY (Nuclear Overhauser Effect SpectroscopY) experiments were acquired with 2048 total data points, 11 ppm spectral windows, 100, 150, 200 or 400 ms mixing times, for 128 increments of 128 scans per increment. The two-dimensional 1H-15N HSQC (Heteronuclear Single Quantum Coherence) experiment was acquired with 1024 total data points, 12 ppm (1H) and 40 ppm (15N) spectral windows, for 128 increments of 256 scans per increment. Sample temperatures were regulated from 15 to 30°C. Water suppression was accomplished with WATERGATE in each experiment. The data was processed in TopSpin and analyzed with SPARKY [38].
Atomic level structures for the 2 predicted turns were obtained using molecular dynamics. Chains of eight amino acids long at the site of each turn were created in an extended coil conformation. Each chain was capped with an acetyl group on the N-terminus and a N-methyl on the C-terminus to avoid any termini association due to charge effects. Chains were then solvated with tip3p water in a dodecahedral box, with dimensions allowing a 1.5 nm separation between peptide atoms and the edge of the box. After an energy minimization, a harmonic potential was added based on the NOE data. The spring constant for this potential was set to 1250 kJ/mol, and applied outside of the accepted range of 0.5 nm, with a truncation creating a constant maximum force beyond 0.6 nm. NOE distances were allowed to fluctuate and updated every 10 ps, allowing the capability for multiple constraints on each atom. Forces contributing to the hydrogen-bond distances were applied at every step for a more rigid network. The peptides were simulated using Gromacs and the Amber 2003 force field [39], [40], [41], [42]. After 3 ns equilibration time, each system was run for 5 ns with structures taken every 1 ns, producing an ensemble.
PDB file of five snapshots of the V24-N27 turn from NMR refinement, as displayed in Figure 5a.
PDB file of five snapshots of the V36-V38 turn from NMR refinement, as displayed in Figure 5b.
We thank Muralidhar Reddy Yerramadha and Zhenan Bao for technical help and assistance.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The authors acknowledge support from the National Institutes of Health via an R01 grant to VSP (NIH R01-GM062868) and the National Institutes of Health NDC Nanomedicine Center (PN1 EY016525). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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ChelseaFebruary 07, 2012No commentsPPC Advertising broad match, broad match modifier, exact match, keyword match types, Negative Keywords, phrase match
Setting keyword match types is an important step in creating a successful PPC campaign. You have the ability to designate these match types in both Google AdWords and Microsoft adCenter. However, if you are using them simultaneously, you should do so with caution. There are differences with the way these functions work in AdWords and in adCenter. It is...Read more
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Elizabeth Rage takes you through the steps to create the Enchantress look f...
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Following our column last week, which encouraged event promoters and organisers to run a waste-conscious event this summer, we were very pleased to see that this month's New Zealand Festival of Tennis set a great example by doing just that.
The high profile festival, held in the first two weeks of January, reduced its environmental footprint by working with the Glass Packaging Forum and LOVENZ to actively ensure that the event would be sustainable.
The waste minimisation action plan included encouraging spectators to recycle their containers and packaging, and the results were fantastic.
Overall, a massive 26 tonnes were recycled, which is a 57 per cent recycling rate across both tennis tournaments.
This recycling consisted of:
14.5 tonnes of mixed recyclables.
7.9 tonnes of glass bottles.
3.8 tonnes of cardboard.
One of the most encouraging aspects of the recycling programme at the tennis was that the event's title sponsor, Heineken, was a key proponent in ensuring that the event would be sustainable. Committing to sustainability as part of their sponsorship activities is important to their brand and provides a great example for other event sponsors.
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Click here to print the obituary below
Thomas HYSER
True to himself and his beliefs. Age 79 of St. Paul. Survived by children Kyra Longfellow (David) and Courtland (Kristin); granddaughters Marin and Lily; sister Grace Hyser; brothers Wally (Kathleen) and Ray (Betty); sisters-in-law Jane, Florence and Marcy and extended family and friends. He served in the Navy, retired from Burlington Northern Railroad, was an avid reader and lived simply. He will be missed. Memorial Service Saturday (4/5) at 2:00 PM at TWIN CITIES BIBLE CHURCH, 2555 Hazelwood Street, Maplewood with visitation one hour before the service. Private interment Fort Snelling National Cemetery. 651-777-2600
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The Utah Story presenting Sorta Great Moments with Mary Blair attraction, January 8, 1987, 8:03 p.m.
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gilt noun (SURFACE)
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gilts [ plural ] also gilt-edged securities UK specialized
› a type of investment offered by the government that pays a fixed rate of interest and is considered low-risk
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text: Kenny Wait has been a member of The Black Rose Band for the past 4 years and is now booking solo shows while the band is on hiatus. His solo sets include many fan favorite Black Rose Band songs and never before heard songs reserved just for these shows! To celebrate the new Black Rose Band album coming out in the next couple weeks a brand new song entitled "Thanksgiving Day" is available for download, FREE!!!!


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Hmm the Uzar's are related to the Coppola's?
That was my connection between the Scire's and the Lanza's, the "Barbara Lanza-Coppola" wedlock.
Cassandra Scire -> Phylis Byington -> Christopher Rodia -> Ty Scire Skarupa -> Barbara Lanza-Coppola -> Rest of Lanza family..
So now we have the Scire's, Rodia's, Uzar's. and Lanza's all being officially related. What a happy little family.
Alison M Uzar is related to Frank Coppola, who is 74 years old and lives in Bronxville, NY.
Alison M Uzar is also related to Mary Belkin, who is 45 years old and lives in Seymour, CT.
Alison M Uzar is also related to Jennifer Piccione, who is 37 years old and lives in Holmdel, NJ.
Alison M Uzar is also related to Marylou Coppola, who is 72 years old and lives in Bronxville, NY.
Alison M Uzar is also related to Frank Coppola, who is 55 years old and lives in Yonkers, NY."
Alison M Uzar is related to George E Uzar by marriage. Alison M Uzar was a Coppola prior to marriage to Uzar.
Alison M Uzar
Jennifer A Piccione
Lenore C Greene
Marylou Lou Coppola
Frank J Coppola
Barbara Lanza-Coppola is the same Coppola...
Michael J Coppola
Michael John Coppola
Jeanne P Coppola
Dacia L Coppola
Barbara A Coppola
Others of interest:
Peter G Lanza
Nancy E Lanza
Joseph A Lanza
Frances Lanza
Debra A Lanza
Anonymous Coward 17949712
Another name to put into this tapestry is the mothers maiden name CHAMPION.
James Champion being the retired police captain from Kingston, NH...
Anonymous Coward 9977516
The drama that goes on in these small towns is ridiculous. Nothing would surprise me.
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18 Terrible Examples Of The "Good Sport Plus Size Woman" Meme
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Beautifully summer inspired, this little mint lovely is day time chic at its most stylish. The grey and black florals on the minty backdrop are pretty and vibrant, and the swing design is perfect for a super SS14 finish. Style with a pair of chunky ankle boots, a fedora and a long strappy satchel.
Approx length 85cm/33.5" (Based on a UK size 8 sample)
95% Viscose 5% Elastane
Kyla wears a UK size 8 and her height is 5'7"


text:
List all words that contain cleavers
Only the word cleavers was found, nothing longer.
Some random words: if veal ohs einkorn jeez thack eager
This is not a dictionary, it's a word game wordfinder.
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Dogs and Puppies for Sale and Adoption(1 - 15 of 330,148)
$1,500 AKC Mastiff PuppiesMastiff · Nicholasville, KYAKC Mastiff puppies, born 7/4/16. Three Males, 1 apricot 2 fawn. Dam has champions in bloodlineShana H. · 12 minutes ago
$600 AKC Registered female English Bulldog PuppyBulldog · Baltimore, MD… . Shots, De-wormed, Health Guaranteed and Vet- Checked. This Puppy grew up with kids and love ...Noevelin J. · 38 minutes ago
$1,200 CKC Registered Bull Terrier PuppiesBull Terrier · Knoxville, TNCKC registered puppies 6 days old.Have two girls one brindle and one white with little black mark and 3 boys.One all white, then with wit...Maria S. · 48 minutes ago
$800 Boston Terrier PupsBoston Terrier · Copley, OHBeautiful Boston Terrier Puppies. Born May 13th are ready for new homes. 1 male 1 female. Also 1 brindle female born May 2nd. Very lov...Kelly D. · 1 hour ago
$800 AKC German Shepherd Puppy- Yellow MaleGerman Shepherd · Seneca, MOAKC German Shepherd Puppies3 males and 1 female left Born: 6/15/16 Ready to join your family: 8/10/16 (8 weeks old) Shots, wormed and vet...Kristina D. · 1 hour ago
$400 AKC Yorkies - 2 litters to choose fromYorkshire Terrier · Baltimore, MDYappy Yorkies has 2 litters to chose from! Traditional colors & rare chocolate sable. get more pics & pricing by contacting 651x560x8961 ...Noevelin J. · 34 minutes ago
$1,500 English Crème Golden Retriever PuppyGolden Retriever · Atlanta, GAI have a English Crème Golden Retriever puppy, 8 weeks old, Champion bloodline. Grandparents were imported and were registered with the ...Jiayue D. · 11 hours ago
$700 Belgian Malinois PuppiesBelgian Shepherd Malinois · Chandler, AZBeautiful Belgian Malinois puppies, 8 weeks old, current vaccines (5in1), dewormed, very good with kids. No AKC. Purebred. We're looking...Veronica E. · 12 hours ago
$850 Sweet Mini PoodlePoodle · Jersey City, NJSimba is a sweet 7 month old miniature poodle looking for a loving home. She has had all of her shots and unfortunately I can not care f...Dumebi E. · 12 hours ago
$750 huskySiberian Husky · Arvada, COhi i have a beautiful male husky he is 9 weeks old very playful text me or call me seven two zero three one six four five six eightEduardo D. · 12 hours ago
$1,000 F1 Standard GoldendoodleGoldendoodle · Rochester, NY3 Gold Males, 1 Gold Female, & 2 Jet Black Goldendoodles are left! Black Females: $1000, Gold Males: $1500, Gold Female: $2000. Ready for...Alicia M. · 13 hours ago
$1,200 AKC Yorkshire Terrier PuppiesYorkshire Terrier · Baltimore, MDThree beautiful baby yorkshire terriers for sale. They were born on June 9th and will be ready August 9th. They come with their shots up ...Casey M. · 13 hours ago
$300 Yorkie FemaleYorkshire Terrier · Kansas City, MOAlmost 4 months old up to date on shots, very playful. Goes to the potty outside. Loves rides in the car and squeaky toys, comes with foo...Jazmin Y. · 13 hours ago
$600 Goldendoodle puppiesGoldendoodle · Diamond, MOMom is F1b Goldendoodle, dad is first generation Goldendoodle. The pups are 62.5 percent Standard Poodle and 32.5 percent Golden Retrieve...Laura C. · 14 hours ago
$1,800 AKC Great Dane Merle Mantle Female PuppyGreat Dane · Ocala, FLAKC Great Dane Puppies for sale. Born June 14th, 2016 and will be ready for their forever home August 12th. Nora had 7 healthy pups! 4 bo...Kristen H. ·2 listings · 16 hours ago
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Our florists in St Petersburg or Palm Harbor flower shops can help deliver beautiful flower arrangements right to your front door. Pinellas Park florists and flower shops in Clearwater that are located right in the neighborhood can guarantee the freshest flowers available. You can trust real Largo local florists and Safety Harbor flower shops for reliable florist-delivered flowers.
The flower is the poetry of reproduction. It is an example of the eternal seductiveness of life.- Jean Giraudoux
When bright flowers bloomParchment crumbles, my words fadeThe pen has dropped ...- Morpheus
I didn't know the names of the flowersnow my garden is gone.- Allen Ginsberg
In the hope of reaching the moonthat blossom at their feet.- Albert Schweitzer
Find florists from nearby cities:
Summer is
You make Me Smile
Your Local florist will create a unique gift, floral arrangement or basket. Artfully designed, hand created and delivered! That's the local florist difference.
727-725-5817 |
store hours | view map101 Main St Ste F Safety Harbor, FL 34695-3656
Family Owned Since 1955! Large selection of fresh flowers, plants & gift line Shop online or call our shop.
800-876-1186 |
store hours | view map1919 Drew St. Clearwater, FL 33765
Custom arrangements and candy delivered by a CLOWN! In business since 1975. Delivery available county-wide.
800-333-7415 |
store hours | view map1140 Main St Ste 1 Dunedin, FL 34698-5331
Serving the Dunedin area for 16 years. Master designers on staff. Full service florist.
727-791-6068 |
store hours | view map2645 Sunset Point Rd Clearwater, FL 33759-1513
"You Name It We Do It". A Full Service Florist. Fruit & Gourmet Baskets, Fresh Cuts and Plants.100% Satisfaction GTD.
0 mi. from center of Dunedin
Dunedin Florists on Larger Map
Copyright © 2016 Teleflora – Fresh Flowers Hand Delivered Daily. All rights reserved.


text:
List all words starting with unproblematic
Only the word unproblematic was found, nothing longer.
Some random words: gained eld nyala ne chablis ami eide
This is not a dictionary, it's a word game wordfinder.
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text:
Separate names with a comma.
Man, those Banners of yours are frickin amazing.
That is Amazing!
If you want a real Banner that doesn't suck, use this:
I tried again.
This is one of my older banners. I'd like the same thing, except instead of the pic of Max Payne and Mona on the right, i want this pic, in black and...
Sorry, but I accidentally saved the image as 1 layer, so I cant choose a new background.
What do you mean by Beserk?
Hey, that's actually good. Nice!
Yeah, I just completed a DMC banner for my profile.
Are you seriously interested in starting a DS devision?
how do you get the image to go to 972x_____
MAN. GIMP is really confusing. I don't even know how to add/do anything.
yeah, a few. I'm not sure if you'd know them or not.
I woudn't trust Mm if I were you. He, if you didn't already know, likes to make stuff up, and piss people off.
But Hell, It's kinda Funny.
Oh, and I looked at your blog before.
Try ForumCircle. Free Hosting and the domain name is not too long. Oh, and did I mention the forums actually...
Sweet! Thanks A lot. I need to make a new Banner (Along with a Flash game) for my clan FAST.
Oh, and B4 I forget WUL'd.
This Craptastic Banner Maker Pro. I need a better program. All I can do really right now is move images, add text, fade, and bevel. That's pretty...
What program do you guys use?
I'd like a header with a Family Guy theme. I'd like Flash1103 to be put in somwhere too. I will WUL the person who makes the one I'll use. If you...
May someone do me 2 header's, I want the first 4 pics together and the other 4 on another header without my name. I will WUL 3 times to whoever does...
Do you want both with out your name?
Look in my profile.
Hey guys,
So, I went in to best buy yesterday, the one I used to work at, and I was browsing the video game and music section, although I usually...
I'm soooooo glad Jaffe moved on to God Of War. TM2 was the only one that was great to me.
Hey guys,
I finally went to go see PoTC a couple of days ago, and although I felt it was entertaining, and that the characters were interesting...
I liked Majora's mask so much better.
I took a vacation once to Burbank, and I came by Insomniac's building, and I saw Ted Price (Really nice guy by the way, and absolutley Loves every...
Great Album though, and Thunderthief is still worse.
Well they definitely know what to advertise. It's good they showed more of the controller than, "Hey look at this fancy box!"
Hey, guys.
I was just browsing over in ol' ebay looking for some classic rock albums I might have missed, and some ps2 controllers. (after...
Nuetral milk sucks.
Beatles and CCR are very good though.
I actually saw Fogerty in concert about 3 weeks ago.
Zeppelin is by far the greatest band of all time.
Who do you like, Keane?
Zidane? FF9? Why is Zidane playing soccer?
Ok, I will.
But promise me one thing.
Since I won't post in this thread any more, I don't want any Outfit members posting in my thread like a...
I should leave? Why?
Sure, That's why my thread was locked for Spamming, when you said SPAM SPAM SPAM.
If a mod is reading this, please ban him.
[face_dancing] Yeah, and do you guys have a website?[face_dancing] [face_Gloat]
Same Here, but some total ass-mongrel mentioned it.
I'm sorry there was so little people wwho responded.
And yes, that guy who suggested Zeppelin was a total idiot.
Nice job.
Thanks for bieng the one person to try to remain nuetral with the console wars.
That's cool.
But quite a departure for square.
I'm suprised it's just for the 360.
I think everyone here can agree, besides you, that zeppelin and Elvis wrote some of the best songs of all time and they changed the way music was...
That's what I said in the previous blog.
Good. A man who knows what he is talking about.
Advice: Don't EVER eat deep fried twinkies. INSTANT DIARRHEA. Not kidding, and this isn't regular "Pepto Bismol" diarrhea, this is like toilet seat...


text:
Today's mortgage rates are lower.
This morning, June Durable Orders fell 4.0% from May, much weaker than the consensus for a decline of just 1.0%. June Pending Home Sales rose 0.2% from May, below the consensus for an increase of 1.0%. Investors are waiting for today's Fed announcement.
As a mortgage rate shopper, it's important to know when today's rates are changing. This is because, when mortgage rates change, mortgage lenders will not honor rate quotes which have not been previously "locked".
To lock today's mortgage rates, then, be sure to commit with your lender before current rates begin to move. Whether you're trying to lock a purchase or a refinance loan, the market waits for no one.Click to see today's rates (Jul 28th, 2016)
Today's mortgage rate analysis is based on live mortgage-backed securities (MBS) pricing provided by MBSQuoteline, a real-time mortgage market data service available to loan officers, real estate agents, and other finance professionals.
The MBS data supplied by MBSQuoteline is the same market data used to formulate current mortgage rates by the nation's mortgage lenders.
The chart at top depicts today's Fannie Mae mortgage bond pricing. Fannie Mae bonds are linked to conventional mortgage rates which include mortgage rates for programs such as the Home Affordable Refinance Program (HARP 2.0), the 3-percent down Conventional 97 loan, the HomePath mortgage program, and others.
MBS prices are inversely related to today's mortgage rates. When bond prices rise, mortgage rates sink. In general, a twenty-five basis point change in MBS pricing -- up or down -- leads to a 0.125 percentage point change in mortgage rates.
Note that the chart above does not depict the path of today's Ginnie Mae mortgage bonds, although Ginnie Mae bonds and Fannie Mae bonds tend to move in similar directions.
Ginnie Mae bonds correlate to today's mortgage rates for FHA loans, such as the FHA Streamline Refinance, which are insured by the Federal Housing Administration; VA loans guaranteed by the Department of Veterans Affairs; and USDA loans guaranteed by the U.S. Department of Agriculture.
Mortgage rates change all day, every day. The mortgage rates you get from your bank "now" won't be the same rates you get from your bank in an hour. Be smart when you shop. Compare multiple lenders and get your best deal.
Get today's real-time mortgage rates now. Your social security number is not required to get started, and all quotes come with instant access to your live credit scores.Click to see today's rates (Jul 28th, 2016)
The information contained on The Mortgage Reports website is for informational purposes only and is not an advertisement for products offered by Full Beaker. The views and opinions expressed herein are those of the author and do not reflect the policy or position of Full Beaker, its officers, parent, or affiliates.
Deborah C. Television Crewer
The Mortgage Reports is part of my morning routine. As I read, I learn more, and have come to understand the mortgage industry. I can't thank you enough!
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I am an aspiring homeowner and The Mortgage Reports helps me daily. Thank you for your excellent information.
2016 Conforming, FHA, & VA Loan Limits
Mortgage loan limits for every U.S. county, as published by Fannie Mae & Freddie Mac, the Federal Housing Administration (FHA), and the Department of Veterans Affairs (VA)


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Jobs in South Africa
9 Jobs Found: Showing 1 - 9
Treasury Manager - AfricaNational Oilwell - South AfricaPosted 21 JulJob Title: Treasury Manager Africa Reports to: Treasury Manager MEAIP Function: The Treasury Manager for Africa is responsible for executing the tr
After Sales ManagerTangent International - South AfricaPosted 10 JulGrow sales and profitability of Services offering, adapt regional services strategy to his/her territory, direct and implement action plans Ensure t
L2 Customer Support Engineer (English and French) - $30KCrossover - South AfricaPosted 30 JunAre you a results-driven professional motivated by a drive to bring your customers success? Are you an effective communicator capable of collaboratinFeatured Job
Technology Services Specialist (TSS ) AssociatePosted 9 JunA reputable logistics company is currently recruiting for a Technology Services Specialist (TSS )Associate to be based in Cape Town-South Africa
System & Network Administrator-AssociatePosted 9 JunA reputable logistics company is currently recruiting for a System & Network Administrator-Associate to be based in Johannesburg-South Africa Job Pu
Senior Network Solutions ArchitectPosted 9 JunA reputable logistics company is currently recruiting for a Senior Network Solutions Architect . This position will be based in Johannesburg-South Af
Sales ExecutiveAdvanced International Employment Services - South AfricaPosted 1 JunAdvanced International Employment Services (part of Bin Butti International Holdings Group) is one of the leading Executive Search Firms and Manpower
Senior Pre-SalesIST Networks - South AfricaPosted 3 JulUnderstand and analyze customer contact center requirements, Develop a solid contact center design from a set of customer business requirements, Devel8
Senior SalesIST Networks - South AfricaPosted 3 JulIdentify business issues and goals to be addressed by the product, Work with the product strategy team to define the product roadmap, Work with custom9
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user interface artist jobs
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Music Sites Seek Senior Front-End Developer: Wordpress, PHP, Ecommerce
Country Rebel - San Diego, CA
Create simple, efficient site layout/user interface by using standard HTML/CSS practices. We work with celebrities, music artists and our own creative site...
Rocket Gaming Systems - Las Vegas, NV
Experience creating eye-catching animations for logos, characters and user interfaces. Position Responsibilities Reporting to the Lead Artist, you will work...
Lead Unity Developer
DoubleDown Interactive - Seattle, WA
Ability to develop compelling user experiences in a mobile environment. Work closely with game designers, producers, artists and developers to expand our...
Graphic & Web Designer / Production Artist
ZDCA Design & Development - Santa Rosa, CA
You thoroughly understand the intricacies, extent, and limitations of designing an intuitive website user interface....
Digital Designer/Web Production Artist
Brighton Collectibles - City Industry, CA
Email design, feature pages, user experience, video content, social, mobile, apps. The Digital Designer will have extensive knowledge of Adobe Creative Suite...
Senior Interaction Designer
EMD - Billerica, MA
As part of the team you will sketch and design user interfaces that empower our business users. Translate research insights into adequate user experience...
Web Designer
Aureus Group - Des Moines, IA
Must have understanding of site structure, navigation, UI principles, wireframes, and user flows. Ability to successfully communicate conceptual ideas, design...
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DoubleDown Interactive - Seattle, WA
Ability to develop compelling user experiences in a mobile environment. Work closely with game designers, producers, artists and developers to expand our...
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MisterArt.com - Houston, TX
Developing front-end and user interface code:. 3-5 years developing front-end and user interface code for Web sites....
Junior UX designer
BayOne Solutions - Redwood City, CA
Role Scenario We create magic for users every single day. Help share pur story with the world visually – entertaining, engaging and exciting our users with your...


text: 0600 0830 Oscar-winning depiction of the events surrounding Japan's devastating attack on Pearl Harbor during World War II, with Joseph Cotten and Jason Robards. Flashing images. (1970)(138 mins) Also in HD Film Tora! Tora! Tora! Film National Lampoon's Animal House 0830 The worst frat house on campus fights back against the fiendish Dean Wormer in the original gross-out comedy. John Belushi stars. Strong language/sex. (1978)(102 mins) Also in HD 1030 El Dorado Film Gun fighter John Wayne and sheriff Robert Mitchum take on ruthless cattle baron Ed Asner in Howard Hawks' outstanding Western. James Caan also stars. (1966)(122 mins) Also in HD 1245 1030 1455 CIA man Brad Pitt faces death in a Chinese jail; veteran agent Robert Redford is his only shot at freedom. A pacy action thriller from director Tony Scott. (2001)(127 mins) Also in HD 1245 Spy Game Film 1645 Alfred Hitchcock's suspense classic. Swapping stories of difficult dames, two guys admit to having murder in mind. But only one of them is serious. (1951)(98 mins) Also in HD 1455 Strangers On A Train Film Black Snake Moan Film 1845 God-fearing Samuel L Jackson chains trampy Christina Ricci to a radiator to try and save her soul. Controversial drama with Justin Timberlake. Contains strong language/sex. (2006)(111 mins) Also in HD 1645 Jonah Hill's disgraced journalist discovers that an accused killer has assumed his identity. True-life drama with James Franco and Felicity Jones. Strong language. (2015)(95 mins) Also in HD 2030 1845 True Story Film Mistress America Film A college student has her life turned upside down when she meets her ultra-hip soon-to-be-stepsister. Screwball farce with Greta Gerwig and Lola Kirke. Strong language. (2015)(81 mins) Also in HD 2200 2030 Film Things To Do In Denver When... 2200 ...You're Dead: Thriller about an ex-con who is forced back into his old ways by a business debt. Starring Christopher Walken, Andy Garcia and Steve Buscemi. (1995)(111 mins) Also in HD 0000 Theeb Film Oscar-nominated coming-of-age tale about a young Bedouin boy fighting for survival in the desert during the First World War. Foreign language with subtitles. (2014)(96 mins) Also in HD 0150 0000 Black Snake Moan Film God-fearing Samuel L Jackson chains trampy Christina Ricci to a radiator to try and save her soul. Controversial drama with Justin Timberlake. Contains strong language/sex. (2006)(111 mins) Also in HD 0350 0150 A Dirty Shame Film Provocative comedy starring Tracey Ullman as a repressed, puritanical woman who becomes a sex addict after a blow to the head. With Johnny Knoxville. Strong sex references. (2005)(85 mins) Also in HD 0530 0350 Sky Cinema Preview - July 2016 1 Film 0600 Previews of the great films now playing on Sky Movies plus other highlights showing on Sky. Also in HD 0530 0600 Martin Scorsese's first feature film is a rehearsal for Mean Streets, with Harvey Keitel as a streetwise young buck in New York's Little Italy. Violence/sex/mature themes. (1968)(85 mins) 0745 Film Who's That Knocking At My Door


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LOT #6242 |Sold for: Sign-in or Join (free & quick)
1878 $1 Goloid Dollar, Judd-1562, Pollock-1753, R.6-7, PR67 Red and Brown NGC....Click the image to load the highest resolution version.
Sold for: Sign-in or Join (free & quick)
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Red and Brown Superb Gem Proof 1878
1878 $1 Goloid Dollar, Judd-1562, Pollock-1753, R.6-7, PR67 Red
and Brown NGC. Ex: Simpson. Goloid dollar that features Liberty
on the obverse, E PLURIBUS UNUM above and the date below.
Description of the composition within a solid wreath on the
reverse: GOLOID./ 1 GOLD./ 24 SILVER./ .9 FINE./ 258 GRAMS. Around
the wreath are the legends UNITED STATES OF AMERICA and ONE DOLLAR.
Struck in copper with a reeded edge. This highly reflective Superb
Gem displays two-toned color, with bright blue in the fields and
original red around the devices and peripheries. The surfaces are
essentially flawless except for a few tiny, almost invisible spots
on the obverse. (NGC ID# 27M7, PCGS# 71924)
Judd-1562 Goloid Dollar in Copper
Fees, Shipping, and Handling Description: Coins & Currency (view shipping information)
Sales Tax information | NGC Coin Grading Guarantee | Terms and Conditions
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Auction Info
Apr-May, 2011
27th-1st Wednesday-Sunday
Bids + Registered Phone Bidders: 7
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Five classical music clips in the movies
Some lesser known classical music appearances in the movies
Garrett Harris 7 p.m., July 25
Big-berry Manzanita (Arctostaphylos glauca), Mount Woodson, Poway, California, June 2012. A beautiful California native with the largest population growing in San Diego County.
Image posted by chendri887
June 19, 2012
Garrett Harris 7 p.m., July 25
Ian Anderson 5 p.m., July 25
Dorian Hargrove 4 p.m., July 25
Jeff Smith 2:55 p.m., July 25
Matt Potter 1:30 p.m., July 25
Vincent Farnsworth 11:30 a.m., July 25
Walter Mencken 11 a.m., July 25
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text:
List all words starting with kabala
2 words found.
List all words starting with kabala sorted by length or by how common the words are.
Some random words: of ecocatastrophe eigenmode emerald dakoit engender wo
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This is a SAGE Creation, using the powerful graphics improving by <Brightness of Non-Bloom>(Made in China). The Skirmish and Netwrok is saluting to <Red Alert2>, the 3 official factions such as Allied, Soviet and Yuri in <Ra2 - Yuri' Revenge> are reproduced to 3D, I also added another 3 new factions into, such as alien faction known as Bacterian, every faction has different heroes who can using a lot of kind of hero skill to help your armies, like <War Craft>, or <Dota>. In the Missions plot, I bring a fan fiction of <Gradius> and <Salamander> series,it tells a story that the Earth is invaded by Bacterian in 2013 A.D, Russia, EU, America, China and other countries are fallen one by one, you are one of the survivors, you must act as the savior,combine with other heroes scattered all over the country, lead the last armies to defeat them and save your mother planet, write down the history of the Heroes Legend at the end.
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