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A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication
Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved September 12, 2008 (received for review May 29, 2008)
We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC50 value of 20 μM, which was improved to 600 nM via synthetic optimization. The resulting compound, GRL0617, inhibited SARS-CoV viral replication in Vero E6 cells with an EC50 of 15 μM and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs.
Proteolytic enzymes are key regulators of physiological processes in humans and are also essential for the replication of pathogenic viruses, parasites, and bacteria that cause infectious disease. Their importance in such fundamental processes has been widely recognized, and as a result, since the mid-1990s, >30 new protease inhibitors have entered the marketplace for the treatment of a wide spectrum of diseases including HIV/AIDS (1). These drugs target at least 10 structurally diverse proteases representing every class of protease (metallo, aspartic, serine, and threonine), with the exception of the cysteine proteases.
Historically, the development of cysteine protease inhibitors with drug-like properties has been plagued with a number of challenges, most notable being their toxicity and lack of specificity due to covalent modification of untargeted cysteine residues. As a result, only a small number have entered into late-phase clinical trials thus far. Despite such challenges, cysteine proteases hold significant promise as drug targets, because they are involved in many disease-related processes and as such, a number of compounds have entered into preclinical evaluation or development (2). To capitalize on the success of targeting proteases as therapeutics, especially in the antiviral drug discovery area, we report here the discovery and optimization of a noncovalent lead inhibitor of the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS), SARS-CoV.
Although the spread of SARS-CoV, which caused the pandemic of 2002–2003, was effectively halted within a few months after the initial outbreaks, the recent isolation of strains from zoonotic origins thought to be the reservoir for SARS-CoV (3, 4) accentuates the possibility of future retransmissions of SARS-CoV, or related coronaviruses, from animals to humans. The development of novel antivirals against SARS-CoV is therefore an important safeguard against future outbreaks and pandemics, but so far potent antivirals against SARS-CoV with efficacy in animal models have not yet been developed.
Because of the complex nature of SARS-CoV replication, a number of processes are considered essential to the coronaviral lifecycle and therefore provide a significant number of targets for inhibiting viral replication. An early and essential process is the cleavage of a multidomain viral polyprotein into 16 mature components termed nonstructural proteins (nsps), which assemble into complexes to execute viral RNA synthesis (reviewed in refs. 5 and 6). Two cysteine proteases that reside within the polyprotein, a PLpro and a 3C-like protease (3CLpro), catalyze their own release and that of the other nsps from the polyprotein, thereby initiating virus-mediated RNA replication (Fig. 1A). Despite numerous biochemical, structural, and inhibitor-development studies directed at 3CLpro (reviewed in ref. 7), potent antivirals that directly target 3CLpro have yet to be developed. In contrast, structural and functional studies directed at PLpro are far less numerous but have established important roles for PLpro beyond viral peptide cleavage including deubiquitination, deISGylation, and involvement in virus evasion of the innate immune response (8–12). Recent studies have also shown that an enzyme homologous to PLpro from the human coronavirus 229E, PLP2, is essential for viral replication (13).
Results and Discussion
Identification of a SARS-CoV PLpro Inhibitor.
The numerous functions and requisite roles of PLpro in viral replication and pathogenesis suggest that PLpro may serve as an attractive target for antiviral drugs. Therefore, to identify potential inhibitors of PLpro, we developed a sensitive fluorescence-based high-throughput screen based on our initial work, which showed that PLpro is more catalytically active toward ubiquitin-derived substrates relative to polyprotein-based peptide substrates (11). We used a commercially available peptide substrate representing the 5 C-terminal residues of ubiquitin derivatized with a C-terminal 7-amido-4-methylcoumarin (AMC) fluorogenic reporter group (Fig. 1B). To validate the assay, we performed a prescreen of 10,000 diverse compounds in the absence of reducing agent to assess the reactivity of PLpro's active site cysteine with electrophiles common to many diverse compound libraries. The vast majority of hits displaying >60% inhibition were determined either to be known electrophiles or to exhibit no inhibitory activity in the presence of reducing agent during follow-up analysis (data not shown). Although the majority of cysteine protease inhibitors described in the literature act covalently, the inherent electrophilic nature of these compounds often leads to nonspecific reactivity with unintended nucleophiles, resulting in adverse side effects (14). In the interest of discovering and developing only noncovalent inhibitors against PLpro, 5 mM DTT was incorporated into all subsequent primary high-throughput screens.
A primary screen of 50,080 diverse, lead-like, and drug-like compounds was performed in 384-well plates, in duplicate, which resulted in a Z′-factor of 0.8 [supporting information (SI) Fig. S1). Only a small number of compounds, 17 total (0.04%), were found to have >35% inhibitory activity toward PLpro (Fig. 1C). These 17 compounds were subjected to a series of confirmatory and secondary assays to test for interference of AMC fluorescence, dose-dependent inhibition of PLpro, and inhibition of the enzyme in the presence of Triton-X, a test to eliminate promiscuous inhibitors (15). Of the original 17 hits, 9 compounds were found to interfere with the fluorescence of the AMC reporter group, and of the remaining 8 compounds, only 2 reproducibly inhibited PLpro in a dose-dependent manner, both in the absence and presence of Triton-X. Compound 7724772, a racemic mix of 2-methyl-N-[1-(2-naphthyl)ethyl]benzamide (Fig. 1C), inhibited PLpro with an IC50 value of 20.1 ± 1.1 μM and was chosen for further development (Fig. 2A).
Because compound 7724772 contains a stereogenic center adjacent to the carboxamide moiety, both the (S) enantiomer, 1, and the (R) enantiomer, 2, were synthesized to determine whether PLpro exhibits any stereospecificity. At 100 μM, the (S) enantiomer was found to have only slight inhibitory activity (14%), whereas the (R) enantiomer inhibited PLpro activity >90% with an IC50 value of 8.7 ± 0.7 μM (Fig. 2A). The stereochemical preference for the (R) over the (S) enantiomer established 2 as a lead compound and is consistent for a protein that utilizes the 4-location model for stereospecific recognition (16). Therefore, compound 2 served as the basis for further rounds of synthetic optimization.
Synthetic Optimization of Lead Increases Potency.
The preference for a methyl group over hydrogen at the R1 position of 7724772 was found to be required for potency based on a structure–activity analysis of the primary high-throughput screen (HTS) data and on the follow-up studies of the primary screen. Therefore, other substituents at the R1 position were explored. The substitution of a chlorine atom at R1, 3, resulted in a 2-fold decrease in inhibitory potency (IC50 = 14.5 ± 0.9 μM) compared with 2, and the substitution of a larger ethyl group, 4, almost abolished activity (IC50 > 100 μM), suggesting that the optimum size of a substituent at the R1 position is a methyl group. Compound 2 was further optimized by probing the effect of changing the orientation of the relatively bulky naphthalene group (R2 in Fig. 2A). Replacing the 2-naphthyl group of 2 with a 1-naphthyl to form compound 5 resulted in a 4-fold increase in inhibitory potency (IC50 = 2.3 μM). Finally, the addition of a second functional group to the R3 position of the ortho-methyl benzene ring of 5 was explored. Addition of an NHAc group, 6, was found to have little effect on the IC50 value (2.6 ± 0.1 μM) compared with 5, whereas the addition of a nitro group, 7, decreased activity nearly 3-fold. In contrast, the addition of an amino group at the same position increased the inhibitory potency almost 4-fold (IC50 = 0.6 ± 0.1 μM), suggesting that an additional hydrogen bond may be formed in the enzyme–inhibitor complex. The resulting compound, designated GRL0617, is the most potent inhibitor in the series (Fig. 2A) and was therefore subjected to more extensive mechanistic and structural analyses.
Mechanism of Inhibition of Most Potent Lead-GRL0617.
To characterize the mechanism of inhibition by the GRL0617 class of compounds, kinetic and biochemical studies of the enzyme–inhibitor complexes were performed. A kinetic study of PLpro activity, in which the concentration of its optimal substrate, ISG15-AMC, was varied relative to fixed concentrations of inhibitor, reveals that GRL0617 is a potent competitive inhibitor of PLpro with a Ki value of 0.49 ± 0.08 μM (Fig. S2A). Progress curve analysis also suggested that the inhibitor is noncovalent. To further probe for noncovalent inhibition of PLpro by GRL0617, PLpro was incubated for 1 h with 12 μM GRL0617 (>20-fold the Ki value), and the resulting complex was dialyzed to allow the inhibitor to diffuse away and therefore restore enzymatic activity (Fig. S2B). Approximately 25% of the PLpro activity was recovered after 3 h of dialysis compared with a recovery of 100% for the enzyme without inhibitor. The inability to fully recover PLpro activity after 3 h could be either a result of a slow off-rate of the inhibitor from the PLpro-GRL0617 complex or a result of covalent modification of the active-site cysteine by a direct reaction with the inhibitor or by indirect oxidation. Because GRL0617 has no apparent thiol-reactive groups, the inability to recover enzymatic activity is likely a result of both mechanisms, a slow off-rate of inhibitor and oxidation of the cysteine, despite the use of reducing agents throughout all studies. Evidence for cysteine oxidation is revealed in the structural studies described below.
SARS-CoV Antiviral Activity.
To probe the antiviral activity of the PLpro inhibitors, several compounds were assayed for their ability to rescue cell culture from SARS-CoV infection. The viability of virus-infected Vero E6 cells as a function of inhibitor concentration was measured relative to mock-infected cells by using a luminescence assay, which allows for the evaluation of both inhibitor efficacy and cytotoxicity (Fig. 2B). Most significantly, GRL0617 and compounds 5 and 6 display significant antiviral activity with EC50 values ranging from 10 to 15 μM without toxicity up to the highest concentration tested (Fig. 2 A and B). Notably, the increasing antiviral potency correlates with the in vitro inhibition of PLpro, suggesting that the compounds work directly on the enzyme in cells.
Structural Basis for Potent Inhibition of SARS-CoV PLpro Revealed by X-Ray Crystallography.
To better understand the molecular basis for inhibition of PLpro by GRL0617 and the structure–activity relationship described above, we determined the X-ray structure of the PLpro-GRL0617 complex to a resolution of 2.5 Å (Table S1). The structure reveals unambiguous electron density for the inhibitor, which binds in a cleft leading to the active site (Fig. 3A). The inhibitor is well-removed from the catalytic triad and instead bound within the S3 and S4 subsites of PLpro (Fig. 3B). The interaction between GRL0617 and PLpro is stabilized through a pair of hydrogen bonds and a series of hydrophobic interactions stemming from residues lining the pocket. Specifically, the amide group of the inhibitor forms hydrogen bonds with the side chain of D165 and the backbone nitrogen of Q270 (Fig. 3C). D165 is highly conserved among the ubiquitin-specific protease (USP) family of deubiquitinating enzymes (17) and among most coronaviral papain-like proteases (11, 18). Several structural studies of USPs have revealed that this aspartic acid residue hydrogen bonds with the backbone of ubiquitin molecules at the P4 position, an interaction presumed to be important for ligand stabilization (19–21).
Aside from the 2 aforementioned hydrogen bonds, the majority of contacts between PLpro and inhibitor GRL0617 are hydrophobic in nature. The 1-naphthyl group is partly solvent-exposed but forms hydrophobic interactions with the aromatic rings of Y265 and Y269 and with the side chains of P248 and P249 (Fig. 3C). These residues line the pocket and accommodate the leucine at the P4 position of PLpro substrates (10) (Fig. 3B). The (R)-methyl group, attached to the stereocenter of the inhibitor, points directly into the interior of the protein between Y265 and T302, where it is accommodated by a cavity that is mostly polar in nature. The positions of 3 bound water molecules in this cleft suggest the potential for extending the (R)-methyl group further into the pocket by the addition of polar substituents.
The di-substituted benzene ring at the opposite end of the inhibitor occupies the putative P3 position of bound substrate. The benzene ring stacks against the aliphatic portions of G164, D165 and Q270, whereas the ortho-methyl substituent at the R1 position points into the floor of the cavity, which is lined by the side chains of Y265, Y274 and L163 (Fig. 3C). The other ring substituent, -NH2 at the R3 position of GRL0617, extends from the opening of the cleft where it is surrounded by a series of polar groups, including the side chain oxygens of Q270 and E168 and the hydroxyl of Y269, any of which could serve as a hydrogen bond acceptor (Fig. 3 C and D).
Comparison of the unbound and inhibitor-bound structures reveals 2 significant conformational differences, both presumed to be induced by inhibitor binding. In the apoenzyme structure, a highly mobile loop hinged by 2 glycine residues (G267-G272) is positioned in different conformations in each of the 3 monomers of the asymmetric unit (10). Movements of homologous loops in the deubiquitinating enzymes USP14 and HAUSP upon substrate binding have been observed (19, 20). With PLpro, inhibitor-binding induces closure of this loop such that it clamps the inhibitor to the body of the protein (Fig. 3D). The side chains of Y269 and Q270 become well-defined and reorient to close over the inhibitor, whereas the mainchain of the loop moves to within hydrogen bonding distance of the carbonyl at the center of the inhibitor. Additional movements are observed upon inhibitor binding whereby the side chain of L163 moves to cradle the ortho-methyl of the benzene ring while simultaneously blocking access to the catalytic triad. The plasticity of this region, especially the G267-G272 loop, which is a highly variable region both in length and sequence among papain-like proteases, may account for the range of substrates recognized by these enzymes.
In contrast to the motions observed outside of the catalytic center, the residues of the catalytic triad of PLpro (C112, H273, D287) undergo limited movement between the bound and unbound conformations. Interestingly, a significant amount of residual electron density surrounding the sulfur atom of the catalytic cysteine was observed. Modeling and refinement of this density against a fully oxidized sulfur atom was consistent with a sulfonic acid moiety, vs. sulfinic or sulfenic acids (Fig. S3). This observation likely explains the inability of PLpro to regain full activity after incubation with inhibitor over extended periods of time. The presence of reducing agents in solution most likely helps to maintain the active site cysteine of PLpro in a reduced state. However, upon inhibitor binding, loop closure may restrict access to the cysteine by reducing agents but still allow for oxidation, thereby generating an inactive enzyme. A similar mechanism has been proposed for protein tyrosine phosphatase 1B inhibitors (22).
Inhibitor Specificity.
Structural and functional studies have revealed that PLpro is homologous to human deubiquitinating enzymes and is capable of cleaving ubiquitin and ubiquitin-like modifiers such as ISG15 (9–12, 23). Because there are >50 putative deubiquitinating enzymes in humans that are also cysteine proteases (24), it is important that any inhibitors being developed be selective for PLpro. To test the selectivity of the lead inhibitor against PLpro, the inhibitory activities of GRL0617 against a series of cysteine proteases, including the human deubiquitinating enzymes HAUSP, USP18, UCH-L1, UCH-L3, and a papain-like protease (PLP2) from the human coronavirus NL63, were tested (Fig. 4A). Although the tested enzymes share similar active site architectures to PLpro, it is significant that none of these DUB-like enzymes were inhibited by GRL0617. Structural alignment of PLpro with 1 of its closest structural neighbors, HAUSP, reveals that at least 2 residues of HAUSP, F409 and K420, sterically clash with the inhibitor-binding site (Fig. 4B). Based on a structural alignment of 54 human ubiquitin-specific proteases (USPs), these 2 residues are >80% identical among family members (17, 21), suggesting that GRL0617 is unlikely to inhibit other human USPs.
To further explore the specificity of inhibitor GRL0617 for PLpro over human DUBs, we probed the ability of human deubiquitinating enzymes from cellular lysates to be modified by the active-site-directed probe HA-Ub-vinyl sulfone (VS) in the presence and absence of GRL0617. HA-Ub-VS and similar Ub-derived covalent modifiers have been invaluable tools in identifying and studying various novel cellular and viral deubiquitinating enzymes (25–27). Numerous cellular DUBs become modified when treated with HA-Ub-VS and can therefore be visualized by Western blot analysis by using an anti-HA antibody. If these DUBs are potentially inhibited by compound GRL0617, concurrent treatment with the compound should reduce the extent of VS modification. When lysed cells were treated with HA-Ub-VS in the absence and presence of GRL0617, no change was noted in the immunoblot pattern (Fig. 4C). When PLpro was added to the lysate, it too underwent modification by the HA-Ub-VS, but unlike the cellular DUBs, its modification by the VS was almost completely eliminated in the presence of GRL0617S (Fig. 4D).
This work validates PLpro as a candidate drug target for the development of therapeutics against SARS-CoV. We discovered and optimized the potency of a first generation of lead compounds that act as noncovalent competitive inhibitors of PLpro by binding to the S4-S3 subsites and inducing a conformational change that renders the active site nonfunctional. Although the in vivo potency of this first generation of compounds is likely not yet sufficient to make them therapeutics, they will serve as an important basis for further optimization of in vivo potency and ADMET properties. Together, these results hold significant promise for the development of noncovalent inhibitors directed against virally encoded cysteine proteases that also share significant structural homology with host-encoded deubiquitinating enzymes. A number of viral DUBs are predicted to exist (28), and in addition to SARS-CoV PLpro (11), the deubiquitinating activity and X-ray structures of herpesvirus UL36USP (29) and adenovirus proteinase (30) have been reported. Although our goal has not been to discover inhibitors of human deubiquitinating enzymes, recent studies have begun to evaluate this group of enzymes as potential anticancer drug targets (31). However, because of the high active-site sequence homology among >50 USPs, selective targeting of 1 USP vs. another remains an unresolved issue (25). Here, we demonstrate that selective noncovalent targeting of an enzyme highly homologous to human USPs can be achieved, which engenders new opportunities and potential for selectively targeting this class of enzymes.
Materials and Methods
PLpro Purification and Kinetic Assays.
Untagged native SARS-CoV PLpro (polyprotein residues 1541–1855) was expressed and purified to >99% purity, as described (11). Kinetic assay development was first optimized in a 96-well plate format to establish suitable assay conditions and incubation times. The fluorogenic peptide substrate, Arg-Leu-Arg-Gly-Gly-AMC (RLRGG-AMC), was purchased from Bachem Bioscience. PLpro activity as a function of substrate concentration was measured to determine a suitable subsaturating substrate concentration for HTS. Enzyme concentration and incubation time with substrate were optimized to yield a linear response in a 6-min time frame. BSA was included in the assay to stabilize PLpro, to prevent the adsorption of PLpro to the assay plate, and to reduce the effects of promiscuous inhibitors. Reducing agent, 5 mM DTT in this case, was included in all assays to eliminate cysteine-reactive compounds.
Primary HTS Screening.
A compound library consisting of 50,080 structurally diverse small molecules was purchased from ChemBridge and maintained as 10 mM stock solutions dissolved in DMSO and stored desiccated at −20°C. The automated primary screen was performed on a Tecan Freedom EVO 200 robot equipped with a Tecan 3 × 3 mounted 96-well dispenser and a 384-pin stainless steel pin tool (V&P Scientific) with a 100-nL capillary capacity. Fluorescence values were measured on an integrated Tecan Genios Pro microplate reader. All assays were performed in duplicate at room temperature, in black flat-bottom 384-well plates (Matrix Technologies) containing a final reaction volume of 50 μL. The assays were assembled as follows: 40 μL of 142 nM PLpro in Buffer A (50 mM Hepes, pH 7.5; 0.1 mg/ml BSA; and 5 mM DTT) was dispensed into wells and then incubated with 100 nL of 10 mM inhibitor (20 μM final concentration) for ≈5 min. Reactions were then initiated with 10 μL of 250 μM RLRGG-AMC in Buffer A, shaken vigorously for 30 s, and then incubated for 6 min. Reactions were subsequently quenched with 10 μL of 0.5 M acetic acid, shaken for 30 s, and measured for fluorescence emission intensity (excitation λ: 360 nm; emission λ: 460 nm). Each 384-well plate contained 32 positive control wells (100 nL of DMSO replacing 100 nL of inhibitor in DMSO) and 32 negative control wells (assay components lacking PLpro). Because of the low hit rate of compounds displaying significant PLpro inhibition, compounds that showed >35% inhibition were selected for further analysis. Secondary screening methods are included in SI Text.
Synthesis of GRL0617 and Related Compounds.
1H-NMR and 13C-NMR spectra were recorded on Varian Oxford 300, Bruker Avance 400 spectrometers. Optical rotations were recorded on Perkin-Elmer 341 polarimeter. Anhydrous solvent was obtained by distillation of dichloromethane from CaH2. All other solvents were reagent grade. Column chromatography was performed with Whatman 240–400 mesh silica gel under low pressure of 3–5 psi. TLC was carried out with E. Merck silica gel 60-F-254 plates. The detailed protocols for synthesis of compounds 1–7 are included in SI Text.
IC50 Value Determination.
IC50 measurements were performed by hand, in duplicate, in a 96-well plate format. Buffer, enzyme, and substrate conditions matched those of the primary screen. Reactions containing 50 μM substrate, 2% DMSO, and varying concentrations of inhibitor (0–200 μM) were initiated with the addition of enzyme. Reaction progress was monitored continuously on a Tecan Genios Pro microplate reader (excitation λ: 360 nm; emission λ: 460 nm). Data were fit to the equation : vi = vo/(1 + [I]/IC50) by using the Enzyme Kinetics module of SigmaPlot (v. 9.01 Systat) where vi is the reaction rate in the presence of inhibitor, vo is the reaction rate in the absence of inhibitor, and [I] is the inhibitor concentration.
Reversibility of Inhibition.
To test the reversibility of inhibition, 50 nM PLpro was incubated with and without inhibitor (at 20-fold the inhibitor IC50 concentration) in buffer containing 0.05 mg/ml BSA; 50 mM Hepes, pH 7.5; 5 mM DTT; and 1% DMSO in a final volume of 3 mL, for 1 h at room temperature; 1.5 mL of each sample was then dialyzed against 1 L of dialysis buffer (50 mM Hepes, pH 7.5; 5 mM DTT) for 3 h at room temperature by using 10,000 molecular weight cutoff Slide-A-Lyzer dialysis cassettes (Pierce). Samples were transferred to 1 L of fresh dialysis buffer each hour. The other 1.5 mL of each sample (undialyzed samples) were excluded from dialysis but remained at room temperature for the 3-h time period. All samples were assayed for activity after the 3-h incubation in the same manner as used for IC50 measurements.
PLpro de-ISGylating Assays.
PLpro activity with ISG15-AMC (Boston Biochem) was measured in 96-well half-volume plates at 25°C in buffer containing 50 mM Hepes, pH 7.5; 0.1 mg/ml BSA; 5 mM DTT; 2% DMSO; and fixed inhibitor concentrations of 0, 0.1, 1, and 3 μM. Substrate concentration was varied from 0 to 16 μM, and release of AMC was measured in the same manner as for the IC50 measurements described above. The Ki and mode of inhibition of inhibitor GRL0617 were determined through Lineweaver–Burk analysis of the above data by using the Enzyme Kinetics module of SigmaPlot.
Inhibitor Specificity Assays.
The specificity of inhibitors 2, 5, and GRL0617 was tested against 2 human ubiquitin C-terminal hydrolases, UCH-L1 and UCH-L3, the human deubiquitinating enzyme HAUSP, the human de-ISGylating enzyme USP-18, and a coronaviral papain-like protease from HCoV NL63, PLP2. Full-length UCH-L1 and full-length UCH-L3 were purchased from Biomol International; the catalytic domain of HAUSP (residues 213–548) and full-length USP-18 were purchased from Boston Biochem; and PLP2 was purified as described (32). All kinetic assays were performed at 25°C in 50 mM Hepes, pH 7.5; 0.1 mg/ml BSA; and 5–10 mM DTT in a 96-well plate format. Enzymes were assayed in the absence and presence of 100 μM inhibitor, with 100 nM ubiquitin-AMC (Boston Biochem) as substrate (excitation λ: 360 nm; emission λ: 460 nm), with the exception of USP-18, which was assayed with 1 μM ISG15-AMC (Boston Biochem) as substrate. PLpro was assayed under the same conditions, as a control.
Electrophilic Labeling and Detection of DUB Profile in Vero E6 Cells.
For each labeling reaction, 50 μg of Vero E6 lysate was preincubated with 0–40 μM GRL0617 for 30 min in 60 μL of homogenization buffer (50 mM Tris, pH 7.5; 5 mM MgCl2; 0.5 mM EDTA; 2 mM DTT; 2 mM ATP; and 250 mM sucrose). After the incubation, 0.8 μg of the probe HA-Ub-VS (Boston Biochem) was added and then incubated for an additional 30 min at room temperature. Samples were subjected to 10% SDS/PAGE, and the proteins were then transferred to nylon membrane and immunoblotted with mouse monoclonal antibody specific to HA followed by goat anti-mouse IgG-HRP. For samples containing PLpro, 0.1 μg of purified enzyme was added to cell lysate samples and analyzed as above. The DUB profile approach was modeled from the experiments of Kattenhorn et al. (33) and detailed materials and methods of lysis, labeling, and detection are included in SI Text.
SARS-CoVAntiviral Activity Assays.
Vero E6 cells were maintained in Minimal Essential Media (MEM) (Gibco) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Gibco), and 10% FCS (Gemini Bio-Products). The SARS-CoV Urbani strain used in this study was provided by the Centers for Disease Control and Prevention (34). All experiments using SARS-CoV were carried out in a Biosafety Level 3 facility by using approved biosafety protocols.
Vero E6 cells were seeded onto flat-bottom, 96-well plates at a density of 9 × 103 cells per well. Cells were either mock-infected with serum-free MEM or infected with 100-fold the median tissue culture infective dose of SARS-CoV Urbani per well in 100 μL of serum-free MEM and incubated for 1 h at 37°C with 5% CO2. After the 1-h incubation period, the viral inoculum was removed and, 100 μL of MEM supplemented with 2% FCS and containing the inhibitor compound of interest at the desired concentration (serial 2-fold dilutions from 50 to 0.1 μM) was added. Cells were incubated for a period of 48 h at 37°C with 5% CO2. Each condition was set up in triplicate, and antiviral assays were performed independently on at least 2 separate occasions. Cell viability was measured 48 h after infection by using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), according to the manufacturer's recommendations. Cell viability for the CellTiter-Glo Luminescent Cell Viability Assay was measured as luminescence and output expressed as relative luciferase units (RLU).
Crystallization, X-Ray Data Collection, and Structure Refinement.
The complex of inhibitor GRL0617 with PLpro was crystallized by vapor diffusion in a sitting-drop format after a 16-h incubation of 8 mg/ml PLpro (in 20 mM Tris, pH 7.5; 10 mM DTT) with 2 mM inhibitor at 4°C. Immediately before crystallization, the sample was clarified by centrifugation. A 1-μL volume of the enzyme-inhibitor solution was then mixed with an equal volume of well solution containing 1 M LiCl; 0.1 M Mes, pH 6.0; and 30% PEG 6,000 and equilibrated against well solution at 20°C. Before data collection, crystals were soaked in a cryosolution containing well solution, 400 μM inhibitor, and 16% glycerol. Details of X-ray data collection and structure refinement are included in SI Text. Final X-ray data collection and refinement statistics are given in Table S1.
We thank S. Forrester for help with crystallization condition screening, Y. M. Baez for assistance with kinetic specificity assays, B. D. Santarsiero for crystal structure data collection support, D. C. Mulhearn for computational modeling support, and T. E. O'Brien for assistance with statistical analysis of antiviral activity. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. This work was supported by Public Health Service Research Grant P01 AI060915 ("Development of Novel Protease Inhibitors as SARS Therapeutics").
- 1To whom correspondence may be addressed. E-mail: akghosh{at}purdue.edu or mesecar{at}uic.edu
Author contributions: K.R., M.E.J., S.C.B., A.K.G., and A.D.M. designed research; K.R., S.P., J.T., K.S., M.C., S.B., R.C., and W.F. performed research; J.T., B.S.P., and A.K.G. contributed new reagents/analytic tools; K.R., S.P., K.S., M.E.J., S.C.B., A.K.G., and A.D.M. analyzed data; and K.R., J.T., S.C.B., A.K.G., and A.D.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates for the crystal structure of the PLpro-inhibitor complex have been deposited with the Research Collaboratory for Structural Bioinformatics Protein Data Bank (accession nos. 3E9S and RCSB049054).
This article contains supporting information online at www.pnas.org/cgi/content/full/0805240105/DCSupplemental.
- © 2008 by The National Academy of Sciences of the USA
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text:
ISM releases fourth report into the effects of Brexit on the music profession
The Incorporated Society of Musicians (ISM), the professional body for the UK's musicians has today, Tuesday 7 May 2019, published its fourth report into the effects of Brexit on the music profession, titled Impact of Brexit on Musicians.
This unique research, conducted in February 2019, builds on previous surveys of musicians and reveals the concerns of more than 2,000 musicians in areas such as future work, mobility and visas, transportation of instruments and equipment, and health and social security.
The headlines from the report include:
- Almost 50% of respondents identified an impact on their professional work since the EU referendum result in 2016 – 95%
of whom said it was negative (from 19% in 2016, to 26% in 2017, to 40% in 2018, and to 50% in 2019)
- 63% of respondents cited difficulty in securing future work in EU27/EEA countries as the biggest issue they face due to Brexit – and more than 1 in 10 respondents reported that offers of work have been withdrawn or cancelled with Brexit given as a reason.
- 85% of survey respondents visit the EU27 for work at least once a year, 22% visit the EU27/EEA more than 11 times per year and more than a third (35%) spend at least a month per year working in EU27/EEA countries.
- One in seven musicians have less than a week's notice between being offered work and having to take it.
- 64% of survey respondents said a two-year, multi-entry visa would allay their concerns about their future ability to work in the EU27/EEA if freedom of movement rights were lost
- 95% of respondents preferred the two-year visa over an 'extension of the Permitted Paid Engagement (PPE) visa.
- 83% of respondents said it would be beneficial for a government department (e.g. BEIS) to provide a dedicated hotline for musicians to offer guidance on mobility issues
- More than half of respondents (58%) reported that they were concerned about the transportation of instruments and/or equipment in the EU27 & EEA in the future.
- Amongst other vital recommendations for Government, the report calls for freedom of movement to be protected for musicians, or a two-year working visa to be introduced.
Lord Black of Brentwood, endorsing the ISM's report, said:
'Music is an essential part of our national identity, and can play an increasingly important role in the UK's soft power. If and when the UK leaves the EU, it is our prosperous music industry (now valued at £4.5bn a year to the economy), our musical heritage, and our worldwide reputation for musical excellence which must inevitably be one of the most secure engines for prosperity in post-Brexit Britain. If musicians cannot travel easily to the EU27, this will all be put at risk.
This new report from the ISM clearly demonstrates the reliance musicians place on freedom of movement to work and tour in the EU27 at short notice, and I echo their call for a multi-entry visa.'
Lord Jay of Ewelme, endorsing the ISM's report, said:
'This new report from the ISM clearly demonstrates the reliance musicians place on freedom of movement to work and tour in the EU27 at short notice, without the need to demonstrate the level of their earnings or their qualifications, and I echo the ISM's call for a multi-entry visa. The value of the music industry is growing year upon year – now worth £4.5bn to the UK economy – and deserves protection.'
Baroness Neville-Rolfe DBE CMG, endorsing the ISM's report, said:
'This is a valuable and detailed report and the recommendations must be considered rapidly by the government. Given the scale of its economic and artistic contribution, appropriate action needs to be taken to protect the position of the UK music industry following Brexit'
Deborah Annetts, Chief Executive of the Incorporated Society of Musicians said:
'Impact of Brexit on Musicians demonstrates how much the music workforce depends on EU27/EEA countries for professional work, and reveals a profession who are deeply concerned about the future as the UK prepares to leave the EU.
Musicians' livelihoods depend on the ability to travel easily and cheaply around multiple countries for work in a short period of time. If freedom of movement is to end, the Government must ensure that free movement rights are maintained for musicians, or introduce a two-year multi-entry visa for British musicians working in the EU27 – which 95% of respondents preferred over the Permitted Paid Engagement (PPE). PPE is not the answer.
This report also demonstrates how much the music workforce relies on UK-EU mechanisms - for example, the EHIC scheme and A1 certificate - to support and enable them to work in the EU27/EEA.
At a time of great uncertainty, musicians need to know their jobs in EU27/EEA will be secure once the UK leaves the EU. Therefore we call for the Government to take action, using the recommendations outlined in this report, to protect musicians' livelihood and the all important music and wider creative industries.'
A musician responding to the survey discussed the impact of Brexit on their current work:
'I've had ensembles questioning me as to whether it's feasible for them to employ me post-Brexit. They're turning to me for guidance and there is nothing I can offer them. I'm about to pull out of a project starting on the 1 April because it is a recording and if we discover there are problems, it'll be difficult to replace me at short notice.'
Another, concerned about the transportation of instruments and/or equipment in the EU27/EEA in the future and how restriction on free movement of goods could lead to the introduction of carnets, said:
'Carnets that have to be filled in for tours outside the EEA are immensely time-consuming to fill in, but almost more importantly delay the travel process immensely and create huge extra costs for orchestras.'
The recommendations in the report include:
- The Government must maintain freedom of movement under existing rules for the music profession.
- If freedom of movement rights cease, the Government must introduce a two-year, cheap and admin-light, multi-entry touring visa.
- A Government Department (e.g. BEIS) must set up a dedicated hotline to offer guidance on mobility issues.
- The Government (DEFRA) must expand the list of approved CITES-designated ports for entry and exit, to include Dover-Calais and the Eurotunnel at the very least, so that musicians can travel more easily with their instruments.
- DEFRA must clarify post-Brexit CITES regulations, providing clear guidance that covers all the possible Brexit outcomes (e.g. after the transition period).
- The Government must maintain European Health Insurance as provided by the EHIC system, or provide an equivalent scheme.
- HMRC must maintain the A1 certificate system or provide a suitable equivalent.
- The Government must engage with the music sector to provide a roadmap on all issues relating to mobility rights, customs regulations, health insurance provision and social security.
Notes for editors
About the report
This survey had 506 respondents from a cross-section of the music workforce including but not limited to performers, composers, directors, teachers, academics, and sound engineers. This adds to the 1600 musicians who have responded to previous ISM research on Brexit. Respondents covered every genre, from classical to thrash metal, folk to funk, jazz to film music, and so on. The majority of respondents were UK-based.
The full report, plus the appendix of full results, can be found at ism.org/reports
About Save Music
The Save Music campaign, launched in October 2018 by The Incorporated Society of Musicians, is calling for freedom of movement to be maintained for musicians after Brexit – or failing that, a two-year working visa which allows musicians to tour easily in EU and EEA.
Artist supporters of Save Music
SK Shlomo
'I am totally behind the ISM's campaign to promote freedom of movement for musicians, it is so important for artists like me where touring is our livelihood. We need to be able to work and requiring a visa every time we need to enter Europe would make touring unfeasible.'
Hannah V
'I moved to London from Berlin over a decade ago and have been fortunate enough to tour the world with global superstars and travel regularly as a producer and songwriter. Ultimately, the best music comes from collaboration and borders should not hinder that process. Hannah V: producer, songwriter and artist.'
'I've just been selected to tour European festivals in 2019 as part of the EU funded (potentially for the last time) INES Talent programme. Travelling to perform live for people is part and parcel of what we do as musicians, for some of us, it's the reason we do it. Additional costs and barriers, especially to independent artists, could be the difference between being able to grow internationally or not. Art knows no boundaries, so artists should be able to share their music & go to their fans in Europe without restrictions. #SaveMusic'
Riot Jazz
'We'd love to be ambassadors of the Save Music campaign. It's something that means a lot to us. We love to travel the world introducing our music to other cultures and we love discovering music from other countries. There's no better way to learn about other styles and genres of music than travelling and experiencing it first-hand. Not to mention being able to meet new musicians. Travel is what keeps us inspired and it helps to keep our music fresh. This year alone we've travelled to Russia, Croatia, France, Germany. And over the last few years we've played shows in Italy, Bulgaria, Andorra and Finland.'
Ayse from Savages/Kitebase
'The impact of music in our daily lives is so important - we need to protect that. Art should be for everyone, to price musicians out of being able to tour is to our determent. Free movement for musicians is essential. #SaveMusic'
Anna Patalong
'Freedom of movement is of course essential for our jobs, enabling us to move freely around the continent and work without restrictions. But even more importantly it allows artists of every kind in Europe to interact and share our languages, cultures and ideas. Collaboration is fundamental to Opera, which is an amalgamation of many art forms, and the opportunity to, as Oscar Wilde put it, 'play gracefully with ideas' is vital to keep us producing great work. The further apart we move, the harder that becomes.'
'Europe is our life-blood as a band, always has been. Which is why we support the ISM campaign to preserve freedom of movement for musicians 100%. Not as much for ourselves but especially for the many artists and bands who are starting out. Playing in Europe may end up being impossible for them financially, which is completely unjustifiable on every level. Please help by raising your concerns with your local MP and sharing this campaign.'
Bushra El-Turk
'I back the ISM's Save Music campaign because my career as a composer has mostly benefited from being a member of the EU; through the work I have received, through the European cities I have travelled to so I can work with orchestras and festivals, and the wealth of opportunities I have been exposed to without which would not have happened had I not been an EU citizen. The music industry is one of the UK's most vital, important and successful exports. The UK will suffer without it. And art, like the wind, does not understand borders and, ultimately, should not need a visa.'
Dom James, The Dixie Ticklers
'Freedom of movement means so much to The Dixie Ticklers, both as a band and on behalf of my guys who are all busy on the world class European jazz and pop scenes. We need to rely on our music making, and the skills involved in that, not bureaucracy to score gigs. If we lost freedom of movement, European promoters would definitely be put off from checking out the UK scene, and it'd make things impossible for gigs that come in last minute. Vice-versa, during my time running Jazz Nursery on the Southbank, we often booked European artists and bands, but would have been totally unable to without this freedom. We need to stay part of the European music scene, and not exist as some impenetrable curiosity across the channel. Music is a universal language, it knows no borders, so let's not impose them.'
'It's quite simple. Without freedom of movement through the EU the consequences will only be negative for touring artists. There's no upside. It doesn't help educationally, it doesn't aid cultural enrichment, it doesn't help artists develop their work and will only hamper the world's inclusivity. But if things are going to change then the proposed 2 year visa that the ISM are approving seems as good a way forward as we can see. But this is not just a personal gripe on behalf of musicians and artists wanting to make the world a more nurtured place. The real strangeness of it is that the creative 'industries' (after the finance services) are the UK's second largest exporting sector. So in a world where economics is unfortunately top dog you'd imagine this argument might hold more sway than it appears to. There's no upside.'
Madeleina Kay
'Music is about individuals and communities sharing ideas, creativity, inspiration and passion. Physical barriers to travel limits the ability of musicians to flourish and grow as artists, to the detriment of our cultural diversity. In a digital age where content is shared freely online, it is increasingly hard for musicians to make their way. Freedom of movement is imperative to the freeflow of imagination and talent, and for safeguarding artist's livelihoods.'
Help Musicians UK
'At Help Musicians UK we want a world where musicians thrive and freedom of movement for musicians is vital for a healthy music industry. We therefore share ISM's concerns on the impact of leaving the EU on working musicians and wholeheartedly endorse their #SaveMusic campaign. It is crucial that artists can continue touring Europe easily, as live music and touring are the lifeblood of the UK's music economy.'
About the ISM
The Incorporated Society of Musicians (ISM) is the UK's professional body for musicians and a nationally recognised subject association for music education. Since 1882, we have been dedicated to promoting the importance of music and protecting the rights of those working in the music profession. We support almost 9,500 musicians across the UK and Ireland with our unrivalled legal advice and representation, comprehensive insurance and specialist services. Our members come from all areas of the music profession and from a wide variety of genres and musical backgrounds. As well as working musicians, our membership also includes recent graduates, part-time and full-time music students, and retired musicians. We campaign tirelessly in support of musicians' rights, music education and the profession as a whole. We are a financially independent not-for-profit organisation with no political affiliation. This independence allows us the freedom to campaign on any issue affecting musicians.
Download the report
- ISM Brexit report May 2019 (1.69MB PDF)

subset: commoncrawl


text:
|Up a level
Henig, S., Tsolacos, S. and Nanda, A. (2019). Which sentiment indicators matter? An analysis of the European commercial real estate market. Journal of Real Estate Research,
Yin, C., Ward, C. and Tsolacos, S. (2018). Motivated monitoring: The importance of the institutional investment horizon. International Review of Financial Analysis, 60, pp. 197-212. doi: 10.1016/j.irfa.2018.08.011
Kim, K-M., Kim, G. and Tsolacos, S. (2018). How does liquidity in the financial market affect the real estate market yields?. Journal of Property Investment & Finance, doi: 10.1108/JPIF-03-2018-0020


text:
by Jill Berry @JillBerry102 – originally posted on Staffrm
This is the workshop session I led at the WomenEd unconference in Reading on Saturday 8th October 2016. It was a brilliant day and a celebration of how far the WomenEd initiative had come since the first national unconference in London the year before.
I talked about leadership generally, so whether participants were aspiring middle leaders, senior leaders or heads I hoped they would find something in the presentation which was relevant and useful. Since finishing as a head in 2010 I've completed a doctorate, researching the transition from deputy to head. I've also written a book about this research and my own experience, published by Crown House in November 2016, entitled 'Making the leap – moving from deputy head': https://www.crownhouse.co.uk/publications/making-the-leap Unlike my doctoral thesis, the book summarises my research findings for a professional, rather than an academic, audience.
I was aware, when writing the thesis and when working on the book, that much of what I was saying was equally relevant to those making transitions at other levels. It reinforced my conviction that leadership at different stages is not different in nature, but simply in scope. What you learn from being a successful middle leader, or how effectively you manage the transition from middle to senior leadership, will serve you well if you choose to apply for headship in due course.
The workshop considered the different stages in the leadership application process and how you can give yourself the best possible chance of success at each stage. Appointments are certainly not an exact science, and selection panels, in my experience, don't always get it right. However, you want to emerge at the other end, even if unsuccessful and disappointed, recognising that you did your best and gave a good account of yourself. So how can you do that?
1. Prepare carefully. Do your research and be thorough, so that you feel confident that you are a good match to the school and the role. The job has to be right for you, as well as the reverse.
2. Use what you have learnt to craft your application so you clearly demonstrate the fit between what you have to offer and what the school/post requires. Focus on your potential and what you could do in the future, given the opportunity. Don't just talk about the past and what you HAVE done and leave it to the selection panel to make the connections.
3. Go into the interview feeling confident that if you have done 1 and 2 successfully you are giving yourself the best possible chance of convincing them that not only are you capable of doing this job, and doing it well, you are actually the BEST candidate of all those they will consider. Prepare as fully you can without seeming over-rehearsed and stilted.
If you do all this and feel at the end of the process that you couldn't have done any more, that should give you confidence in the future. The right job is out there. Don't stop looking!
Photo: My network of new heads who all started their headships, as I did, in the year 2000. We don't look TOO bad 17 years later! Photo credit: Hilary Moriarty
Jill Berry, October 2017


text:
- A 23-year-old barber from Texas started handing out condoms along with his business cards.
- But he was stapling the two items together – puncturing holes in the condoms.
- He shared his business plan on Twitter, but users were quick to point out his mistake.
A 23-year-old barber from Texas thought he was on to something when he started attaching condoms to his business cards – but there was a major flaw.
On Sunday, Twitter user Jake Gamez shared photos of one of the cards for FadeAway Barbershop stapled to a condom, saying: "Gotta include one of these with every card! You're going to need it after I cut your hair. Trust me."
Gotta include one of these with every card! You're going to need it after I cut your hair ????????Trust me ???????? pic.twitter.com/V8XEusCgoe
— jake gamez???????? (@gamezjakee) March 10, 2019
Twitter users, however, were quick to point out his mistake – by stapling the card to the condom, he had poked holes in them, rendering them useless.
Read more: An Instagram fitness model with 13 million followers was bitten by a pig while trying to take photos with them in the Bahamas
"ummm are we just gonna ignore the staple in the middle of the condom orrr," one user tweeted.
"But… the hole… from the staple…???" another wrote.
But… the hole… from the staple…??? https://t.co/7sgaQXlEPw
— manda (@GirlYeetsW0rld) March 10, 2019
One simply wrote: "Wait."
One Twitter user said, "This main ain't dumb, he's gonna be cutting their kids hair too and doubling his profits."
This man ain't dumb, he's gonna be cutting their kids hair too and doubling his profits https://t.co/JVbqwznVil
— Hun (@anouskamoss_) March 10, 2019
Another asked whether somebody was going to tell him – but if he didn't know in the beginning, he definitely does now.
Somebody gonna tell him? https://t.co/1CAPoGNUf0
— Justin Herbert Stan Account (@Michael2Wallace) March 10, 2019
A Twitter moment asked, "Were these free condoms a mistake or a cheeky business plan?"
Mistake or not, the tweet has certainly drawn attention to his business.
Gamez retweeted some of the responses to his intial tweet and shared a photo of some of his work to his new fans.
"Thank you all for being here," he wrote. "Truly an honour."
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text:
CVS Health Q2 revenue $65.3 bln, up 3%; FactSet consensus $64.0 bln
(END) Dow Jones Newswires
August 05, 2020 06:36 ET (10:36 GMT)
Copyright (c) 2020 Dow Jones & Company, Inc.
Tesla, Inc., formerly Tesla Motors, Inc., designs, develops, manufactures and sells fully electric vehicles, and energy storage systems, as well as installs, operates and maintains solar and energy storage products. The Company operates through two segments: Automotive, and Energy generation and storage. The Automotive segment includes the design, development, manufacturing, and sales of electric vehicles. The Energy generation and storage segment includes the design, manufacture, installation, and sale or lease of stationary energy storage products and solar energy systems to residential and commercial customers, or sale of electricity generated by its solar energy systems to customers. The Company produces and distributes two fully electric vehicles, the Model S sedan and the Model X sport utility vehicle (SUV). It also offers Model 3, a sedan designed for the mass market. It develops energy storage products for use in homes, commercial facilities and utility sites.
(END) Dow Jones Newswires
August 05, 2020 06:36 ET (10:36 GMT)
Copyright (c) 2020 Dow Jones & Company, Inc.
Earnings Calendar and Events Data provided by |Terms of Use| © 2020 Wall Street Horizon, Inc.
Options are not suitable for all investors as the special risks inherent to options trading may
expose investors to potentially rapid and substantial losses.
Please read Characteristics and Risks of Standard Options before investing in options.
Copyright © 2020. All rights reserved.


text:
Isn't it irritating to know someone who never had any trouble with pimples! Or that unfortunate friend who has too much trouble with their acne. Well here are some answers to that question that actually lie with the bacteria's residing in our skin.
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text:
15'x30'x54" Preference Oval Pool
What's Included:
- 20 Gauge Blue Liner
- Thru-Wall Skimmer & Return Fitting
Original Price:
$5,456.00Save $1,539.00 (22% OFF)
This item is currently out of stock
Receive an email when it's back in stock:
- Includes skimmer and blue liner. For liner upgrades or accessories click Customize this Pool
- Top seats (7" width, 11 1/2" height) are printed with a brushed pattern - Vertical supports offer a grey steel look with metallic highlights
- All Structural Parts Are Treated With A Double Coating Of A Heat-Setting Polyester That Ensures A Great Look For A Long Time
- 54" high wall finished Steel Core Is Pretreated With Crystalline Zinc Phosphate To Improve Resistance To Corrosion
- Reinforced Steel Core - Anti-Corrosion Coating Of Magnesium, Copper & Aluminum Increases Rust Protection by 72%
- Beautiful Printed Patterns - Liquid Inks & Embossing Powders - Very Detailed And Provides A Finished Look
- Polypropylene Connecting Parts Offer A Grey Color
- Oval Yardmore space-saving system saves much needed yard space and looks more attractive
- Optional LED lighting strips hidden behind the upright covers to enhance your backyard experience
- 20 gauge blue liner included with pool price with the option to upgrade by clicking the Customize tab
- Widemouth thru-wall skimmer and water return fitting included
- Wall Color/Design: Grey
- Warranty: Manufacturer's 60-year limited warranty
- Installs above ground or semi-inground (installation not included)
- Warrantied for semi-inground installation up to halfway (27 inches) in the ground
- Approximate inside (water) dimensions - 15' X 30'
- Approximate outside dimensions (space required) - 18' 2" X 31'
- Approximate Water Capacity - 10,500 Gallons
Unique Design
Influenced by contemporary trends reflective of today's new urban spaces, Preference is sure to satisfy the expectations of those looking for something different. The Preference is a totally different and impressive class of pool.
Strength and Value
The Preference introduces textures, colors, angles and even proportions with the introduction of patterns through new printing technology. The Preference structural parts are treated with a double coating of heat setting polyester that ensures a great look for a long time. The top seats are printed with a pattern and offers a grey steel look with Metallic Highlights. It's reinforced steel core with anti-corrosion coating of Magnesium, Copper & Aluminum increases rust protection by 72%. Combined with the wall's graphic, Preference achieves an unparalleled level of quality.
Sleek Oval Design
The Preference oval features a Yardmore space saving support system that incorporates superior strength and an attractive overall experience. This oval system is supported with heavy steel beams and straps that run underneath the pool, eliminating the need for poles or braces protruding at each side of the pool. This sleek oval design allows more pool size while occupying less space, giving a clean and uncluttered presence.
Shipping Estimates
Order Times for Above Ground Pools
Above ground pools are too large to be shipped through UPS, Federal Express, or the USPS. We typically ship all pool orders out 24-48 hours after you place your order unfortunately due to recent circumstances surrounding COVID-19, please expect shipping delays of 2-4 business days. Transit time can vary depending on your location but generally takes 4-8 business days to most areas.(AZ, CA, ID, MT, ND, NM, NV, OK, OR, SD, UT, WA, WI, WY will generally take 7-12 business days). Business Days are Monday thru Friday. When the shipment arrives at your local terminal, the carrier will call you to make an appointment before making the delivery to your home.
Important! An adult must be at the delivery location to receive and sign for an above ground pool and will be required to move the boxes from the truck at the time of delivery (we recommend at least 2 people to unload cartons from the truck). For your convenience, Liftgate Service can be added for $39 at checkout. With Liftgate Service, the delivery will come on a special truck with a hydraulic lift. The driver will use the lift to place the pallet on the ground curbside. (The truck driver is not responsible in moving your pallet to your desired location). This will make receiving the shipment much easier, especially if only one person is home to receive it.
Support / Downloads
Review by TG Evans, FL on Jul 19, 2020
This pool has been wonderful, we put a center drain in and our pool stays cool all year round with no trees nearby in Florida. Everything came as stated and was packed very well. We order extra protection stuff such as ground cover, padded sides, edge wedges. This pool is a perfect size, not to big and not to small. Will keep doing business with Pool Factory in the future.
Review by Michelle H. PA on Nov 1, 2019 Verified Purchase
We purchased a partial inground pool. Quality is very good. I love the liner I chose that looks like colorful tile. Quick delivery as well.
Review by William F. NC on Oct 21, 2019 Verified Purchase
Ordered Online and it was delivered to my house all bundled up and everything just as ordered. I recommend getting the tailgate service since it was dropped just outside my door. Well worth the extra money. A++ to Pool Factory.
Review by Nathan NJ on Nov 29, 2018 Verified Purchase
I purchased a new 18' x 33' x 54" above ground pool here because the price was superior compared to my local stores and others online. It was shipped to my home and arrived plastic wrapped on one giant skid. I opted to install it myself and I had the experience and assistance. I did realize that I had some of the wrong post caps for my style pool and after calling customer service, the correct parts were overnighted to me without a second thought. The pool was well described on the website and am very pleased with my purchase.


text:
Pebblefestive @ Flat iron square
WHEN: Saturday 30 November 11AM – 5:30PM
WHERE: Flat Iron Square, 53b Southwark St, SE1 1RU
COST: £10 (10% donation to Marine Conservation Society). Get your tickets here.
Kick off your conscious Christmas in style at Pebblefestive a sustainable and ethical market for gift-shopping and workshops.
- 45+ brands for zero waste Christmas gifts
- #EverydayActivism ideas to make 2020 your greenest year yet
- Free talks & workshops (see schedule below)
- Kids' activities including plant pot painting & hulahooping
Talks Programme
12PM-12:45PM – How to make it a leftover Christmas
1PM-1:45PM – What if we harness the power of positive advertising?
2:30PM – 3:15PM – How to make this winter of your discontent
3:45PM – 4: 15PM – Freedom Brewery beer tasting
4:30PM – 5:30PM – What is / What if – Transition Towns founder Rob Hopkins in conversation with Georgina Wilson-Powell
Workshop Programme
11:30AM – 12:15PM Conscious Christmas Crafts
12:30PM – 1:30PM – Make festive tipples with Abel & Cole
2PM – 4PM – Learn to fabric wrap your gifts
Kids' Activities
11:30PM – Hulahooping & juggling with Playtone
All Day – Win a prize with our colouring competition in the Cantina
2PM – 4PM – Get the kids decorating plant pots for presents with Urban Growth along with some winter bulb planting
To find out more about Elephant Park's festive events, click here.
To see a list of other events, click here.


text:
Villavej 25
8300 Odder
Telefoon:+45 87 81 79 99
11 new holiday houses in the best Northern style, located right by the beach. A 150 metre bathing jetty, a renovated shipyard, a large fleet of sailboats, dinghies, surfboards and the flagship Princess Alexandra is setting the stage for a holiday or a weekend getaway filled with experiences. There is a ten minute walk to the ferry harbour in Hou, with departures to the islands of Tunø and Samsø.
Hou Sea Sport Center is suitable for everyone and handicap-friendly. Wheelchair users can enjoy the nature, the ocean and the beach first hand here. The houses are 100 square metres each with eight beds - all handicap-friendly with lots of space and no skirtings or edges.
Hou Sea Sport Center has been awarded the accessibility label.
Villavej 25
8300 Odder
Meeting facilities
Free parking
Tennis court
Bicycles for hire
Capacity: Beds (88)
Easy access


text:
For everything noob electronics -
Get news, products, tutorials for free!
arduino uno setup, arduino ide, arduino software install, arduino usb driver, test arduino setup, how to install arduino ide, how to program arduino
Express Delivery
Order is shipped within 1-2 business days from Maharashtra, India.
All shipments to major cities are express delivered by Air using Fedex / BlueDart / Delhivery and delivered within 1-4 business days
All India delivery supported using Speed Post
All shipments are Tracked online
Standard Delivery Charges Applicable
This guide will go through each steps to ensure that your Computer Setup is ready for Getting Started on Arduino.
If you bought arduino for first time, it will also help you to check whether your development environment is all set to get started on Arduino preferably Arduino UNO.
If Green LED is on, Arduino is working.
Arduino IDE is software package that acts as Code Editor / Compiler / Uploader for Arduino programming language. It can be used to program Arduino Uno, Mega, Due, Zero, Nano, STM32, ESP32, ESP8266 and many more micro controllers.
You can install IDE in any OS like Windows 10, Ubuntu, Mac.
To install Arduino IDE software, visit below links -
Arduino Board is a Hardware. To connect any hardware to a computer, you need to install its driver. So which driver we need to install depends on what board you have.
The goal here is to specify IDE which USB Serial Port is Arduino connected to -
This Port belongs to any hardware that is connected to your computer. To confirm the Serial Port of Arduino, disconnect the USB Cable and check if the Port list changes. If it changes, then the missing port belongs to the Arduino. Connect back the USB Cable to same USB port and it will appear again.
Important Note for FIrst Time Users - If you don't see port change in above step, it means that you did not install the USB Driver applicable for the board.
For boards bought from https://noobtronics.ltd, we use CH340G as USB to UART converter for which drivers can be found on below link -
Once you confirm the port of Arduino, lets keep it selected (tickmarked) in the Tools menu.
The goal here is to specify IDE which Arduino board you want to program.
100s of different boards from Arduino or Third Party vendors (like us 😜) can be programmed via Arduino IDE, so it is important that we specify which board that you want to program.
If it is Arduino UNO board running on ATmega328P MCU, you can specify it by selecting in
If it is Smartduino UNO board running on ATmega328PB MCU, you can specify it by selecting in
Tools -> Board -> "Smartduino Uno 16Mhz"
Tools -> Processor -> ATmega328PB
The goal here is to specify IDE which Arduino Code that you want to program Arduino with.
As the blog title suggest, we will be uploading Blink Example code to the Arduino.
Open the Blink code by going to File -> Examples -> 01.Basics -> Blink
The code in Blink Example will look as follows -
void setup() {
// initialize digital pin LED_BUILTIN as an output.
pinMode(LED_BUILTIN, OUTPUT);
// the loop function runs over and over again forever
void loop() {
digitalWrite(LED_BUILTIN, HIGH); // turn the LED on (HIGH is the voltage level)
delay(1000); // wait for a second
digitalWrite(LED_BUILTIN, LOW); // turn the LED off by making the voltage LOW
delay(1000); // wait for a second
The goal here is to command IDE to upload the Code to Arduino.
Its simple, Just Click the Upload icon.
Once you click it,
IDE will compile the code into binary (1s and 0s) and the binary will be uploaded to Arduino.
If it results in 'Done Uploading' it means that Mission is Accomplished. Congratulations !
Your Arduino LED should blink as follows -
If somehow, 'Done Uploading' does not appears or it say 'Uploading Failed' then something must be wrong.
You can turn on the Debug Logs in Arduino IDE by following steps -
Now retry the above test and read the debug logs to identify the issue.
Common Problems for 'Uploading Failed'
Since so many things can go wrong here, at https://noobtronics.ltd we check our Arduino Boards first before they are delivered to you and provide Assured Quality by doing following extensive tests -
Hope you like this post 😊


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All the men and woman of: DCF, DSS, DHS,CPS
[Child ] Human Trafficking American Style
For goodness sake let's hope not . . . Please if you agree with what's here let Donald Trump Know so that he is not going to be a hypocrite. I write this because i learned today that The Trump administration will make combating human trafficking a priority . . .
CLICK HERE to TELL Donald Trump "We are all hypocrites when we cannot see ourselves or judge ourselves the way we see and judge others."
When my eyes opened 5 am 6/28/2017 i realized the TV was on. It's unusual because i am not likely to watch TV often but i must have fallen asleep with it on. Oddly, on the TV as i gained consciousness was the following:
The Trump administration will make combating human trafficking a priority, Ivanka Trump said on Tuesday: http://www.politico.com/story/2017/06/27/ivanka-trump-human-trafficking-priority-240005
That's when i knew i was going to write this: what is Human, Child, trafficking? What is it to Traffic?
Trafficking in persons(child, human beings, man, woman) means:
"the recruitment, transportation, transfer, harbouring or receipt of persons, by means of the threat or use of force or other forms of coercion, of abduction, of fraud, of deception, of the abuse of power or of a position of vulnerability or of the giving or receiving of payments or benefits to achieve the consent of a person having control over another person, for the purpose of exploitation. Exploitation shall include, at a minimum, the exploitation of the prostitution of others or other forms of sexual exploitation, forced labour or services, slavery or practices similar to slavery, servitude or the removal of organs…"
Ivanka Trump I admire your priority to combat this. Americans need someone with your status and power to crusade as you are. My aim with this post is to help you see clear that your, United States, State of Massachusetts people running that corporate organization and others all throughout America are participating and GUILTY of the Human Traffic. I am sad to tell you, i take not joy in what I am compelled to write. I feel obligated because i have been shown this information and now i show you, all of you.
Maybe You are unaware of the facts so i am going to take time today to share this with you and everyone one else who supports your crusade. My conclusions are not only my own but so many Americans are speaking up. I've drawn from my research and the many examples, my first hand experience and observation to bring this factual and empirical evidence forward and hopefully i will draw light to this sad, disturbing truth.
i challenge & invite you or anyone prove me wrong. Please let us know: [email protected],
Ivanka Trump, help us bring Dylan home and Stop human trafficking on America. Thank you in advance – David
Let's Begin with this video: Nancy Schaefer exposes the EVIL CPS. A speech given by Nancy Schaefer on Child Protection Services The System Can Not be trusted It does not serve The People of USA
THIS VIDEO SPEAKS FOR ITSELF. . . . https://www.facebook.com/NoTruthNoJustice/
[email protected] Let her know: "We are all hypocrites when We cannot see ourselves or judge ourselves the way we see and judge others."
KIDS FOR CASH | Official Trailer | FilmBuff
Child Trafficking American Style!
CAN ANYONE DISAGREE LET ME KNOW: [email protected]
Ivanka, I'd like to know why Non of this was mentioned today on TV today?
All I can think of is that you don't know Yet.
https://www.facebook.com/DonaldTrump LET HIM KNOW
#PizzaGate Norway Exposes Horrifying Child Sex Trafficking Network
This next one makes my skin curl . . .STILL IT WAS NOT REPORTED IN YOUR UNITED STATES . . .IGNORED
i challenge & invite you or anyone to prove me wrong. Please let us know: [email protected],
STATE of OREGON MAKES a KIDDIE PORN VIDEO with Gaston's CPS kidnapped Daughter
Part 1 of a grusome TRUE Story documented in the Court Records of Oregon. Wilbur Gaston, a man with grown, children in his mid 60's fathers a daughter with with a unmarried woman, who soon loses all her children to CPS. Wilbur Finds out, and fights to get his daughter, Melissa Gaston, OUT of the Foster – CPS system, and with help from Pam, a local Artist, WINS. It takes 6 months for 4 year old Melissa to stop
screaming spontaneously after Wilbur gets custody back. Wilbur marries 40+ yr old Pam Gaston, 20 years his junior. After 25 months of custody, Melissa is feeling safe with her Bio Dad, Wilbur Gaston,
Then CPS thugs, send local police to kidnap Melissa playing in the front yard with a friend. Pam & Wilbur fight in court for years to win his daughter Melissa back. The State of Oregon, apparentlyl under
the direct order by CPS agent "Larry Lawson" according to Wilbur, orders Oregon School teacher, Hazel S. to make a porno film on video. the Idea is to get liitle Melissa to accuse her own innocent dad
of making her dance naked and "touch herself" where it feels "good". Then use the video against
Wilbur , after editing, to permanently "REMOVE" and "TERMINATE" his parental Rights, and put him in
Jail so he can NEVER have his daughter back – – for the crime of rescuing her from CPS abuse in Foster homes. Wilbur tells us in a later clip how at age 15, Melissa "found" her dad via the internet, and they got together for a afternoon, and talked and Melisssa said when she turns 18 she wants to come live with
her Dad. BECAUSE the only time she felt "Safe" was when she was those 25 months as a little girl
living with her Dad , Wilbur Gaston, who rather resembles Santa Claus out of uniform. Wilbur sued those who were prosecuting him for "stalking" etc. and in the end… either Wilbur would be found guilty or the STATE of OREGON would owe Wilbur $500 million dollars. Wilbur WON.
Oregon has a judgement against it rendered by a JURY, for $500 million they owe Mr. Gaston, and inspite of the 12000 Oregon State Bar members of Oregon, none has been willing to Perfect the Judgement and start collecting the $$ owed . Wilbur struggles on Soc. Secutity. and Pam got murdered before she could start the collection on the Judgement, . . . by aerial Poisoning by a Helicopter that flew over and dropped a mist on their trailer in a woods, after Pam was forced out of
her home in Mt. Angel , Oregon. Police refused to investigate. Ask yourself. Can this be true. Can the State of Oregon kidnap a mans daughter, use her for a porno film, send it around to CPS agents as a case example of "child Pornography" and then be caught, ordered to pay up, and after a murder of a woman, Pam, simple lock up the tape in a Judges
"safe", so the father can NOT show anyone else how awful the Court system and CPS is?
Research, and start with VOICE for the CHILDREN web site, where you can find Teacher Hazel S.
confession. Then call your CongressCritter and demand Federal Prosecution of the guilty judges, CPS agents, police, and the abolishment of CPS in total. Note, Wilbur's sister calls it NAZISM.
In the Interest of the Children" CPS Exposed- Freedom Forum
Foster Care and Adoption = Governments Stealing Children for Profit – How it Works
This is all time I have today and let me just say that i am only scratching the surface on just how many people are speaking up about this and why its happening and how the United States Government, protector of freedom and rights and property is fully engaged in and GUILTY of Human (persons) Child Traffic via CPS, DCF, DSS and so on. . . . I hope my time today was time well spent LET HIM KNOW if you agree: https://www.facebook.com/DonaldTrump


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House of Voltaire sells unique artworks, limited edition prints, homewares, fashion, ceramics, books and objects by leading contemporary artists and designers. Drawing inspiration from influential artist's shops including Keith Haring's Pop Shop and the Bloomsbury Group's Omega Workshop,
Whether online or at one of our special intermittent presentations, every purchase from House of Voltaire directly supports Studio Voltaire, one of the UK's leading independent arts organisations. Our pioneering public programmes of exhibitions, participation projects, live events and offsite commissions have gained an international reputation for excellence.
House of Voltaire can be shopped year–round online and at special intermittent presentations.


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Modern society has well and truly embraced social media. It's rare to find anyone these days without a facebook account, and it is not at all unusual for individuals to maintain multiple accounts with social media providers like twitter, snapchat and instagram.
We live in an age of over-sharing.
Photos, videos, status updates, online diaries or vlogs; every single aspect of your life, the highs and the lows can be shared with the world online. Certainly the saying that "it didn't happen unless you posted it," is increasingly becoming true. Only last week an image appeared on facebook with the picture of a news reader, the caption below citing "Mt Lofty Tragedy: Young Lady 'Smashes Lofty' and forgets to check-in at the summit." We all laughed. It reminded us of how ridiculous our lives online have become.
But should everything be shared? Are there times when we should hold back from sharing? The simple answer is absolutely. Everything you post online should be curated with care. Everything. Under normal circumstances.
Circumstances are not always normal and there may be times when you need to exercise even greater care with regards to this. For example, you may be in the midst of a separation and court proceedings for a Family Law matter could be underway. With tensions already at fever pitch it makes sense to avoid doing anything that might throw fuel to the flame, or provide ammunition to be used against you in court. In particular we would specifically advise our clients to avoid:
- Posts which comment on your relationship status, particularly those which denigrate your ex with a comparison to a new relationship.
- Sharing of information in relation to purchases made with a joint credit card. This includes holiday snaps.
- Sharing of images or videos of yourself partying, consuming alcohol or taking drugs. Be extra cautious of anything shared that occurs while you are the parent caring for children of the relationship.
- Posting denigrating comments about your ex-spouse, their family, or friends.
- Discussing publicly in any way private details about Court proceedings.
You see the problem with social media, and what you post on it, is the way it can easily be taken out of context. Images can suggest something where there is nothing, text can also be subversive. The best way to protect yourself is to exercise extreme caution in what you post, when you post and how you post it.
"But my ex is blocked from my account." I hear you say.
Sure, but unfortunately you can't stop a mutual friend from sharing a screen shot of what has been posted with your ex outside the site. It happens more than you might think.
While you may think that you have control over what you post online, in truth you don't. Anything appearing online, no matter how high you set your privacy, is really only as private as those who see it will allow. If they can see it they can share it and it has the potential to sting you when it really matters in Court.
In summary, when going through Family Law proceedings we strongly advise, no matter how inflamed you may feel, that you err on the side of caution when it comes to online sharing. The internet is not the best forum for counselling and certainly no place to air your dirty laundry.


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2 Definitions of Lanes
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- n. - A narrow way or road
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37 words found using the letters in "Lanes"
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Managing Creativity and Innovation is an almost perfect primer for the classic view of innovation and the role of the manager to influence innovation in this model. The book is organized into eight chapters and, within the paradigm chosen by the anonymous authors, is clear and easy to read Each chapter includes background information, tips for the manager on how to consider and use the information, and a concluding summary.
The eight chapters of Managing Creativity and Innovation focus on the following:
The very qualities that might make Managing Creativity and Innovation attractive to a manager looking for the basics of innovation are also its major weaknesses. The classic, reductionist approach – the absence of concepts associated with complexity and emergence – misleads managers. It fixes their attention on what appear to be dualities associated with innovation and, thereby, encourages a false sense of predictability and controllability.
In the first chapter, for example a figure depicts "An Industry Timeline of Radical and Incremental Improvement." First, an underlying assumption of this chapter and the one that follows is that radical and incremental improvement are discrete entities. As Carlisle and McMillan (2006) have pointed out, the complex adaptive system does not differentiate between radical and incremental or short-term and long-term; it adapts to its changing environment in a way which appears to benefit it most in any current fitness landscape. Second, the "timeline" itself recognizes only two dimensions to be measured in distinguishing between or choosing radical or incremental innovation – the dimensions of time and performance or cost improvements. These variables are far too limited when searching for the patterns or attractors that underlie innovation. There is no attention paid to the power of small changes to bring about emergence. Consider some currently familiar innovations such as the near-mythic story of the invention of "post-its" or the discovery of the currently popular sweetener sucralose, commercially known as Splenda. Splenda emerged from experiments designed to develop a new insecticide to take the place of DDT. When Phadnis, the chemist, was told to test the product, he heard taste instead of test (Bilger, 2006). And he did so despite the potentially toxic nature of the product. Indeed, a small event – a misheard vowel – brought about a change in customer buying habits, people's eating choices, and the creation of many food products based on the new sweetener. This book ignores the implications of these well known stories of innovation.
The story of Splenda illustrates another problem with the classic view. In Managing Creativity and Innovation, managers are urged to consider the greater expenditure of resources of time or money associated with radical innovation over incremental innovation. This is illustrated in the text with the figure of "The S-Curve: An Established Technology and a New Rival." In this view, the judgment of whether an innovation is to be considered radical or incremental is made by the organization before the fact, that is an organization decides on its intention: to create an incremental or radical innovation. However, from a complexity perspective, the difference between radical and incremental, if such a distinction is worth making, is actually in the effect of the innovation on the marketplace, indeed on society. One could characterize, for example, all recent additions to cell phone technology as incremental. However, Rheingold ( 2002) and others writing for the popular press have documented the effect of just one innovation, "texting," on social networks, particularly those of adolescents. Instead of identifying innovations as radical or incremental, it might prove more useful to consider grouping innovations by patterns of self-similarity, that is to see what could be learned by considering them through a process of fractal interpretation.
Managing Creativity and Innovation has some value in its discussions of opening opportunities for creativity to many people in the organization. However, the authors present these ideas as a series of controllable steps rather than an acknowledgement of how innovation can emerge from truly broad-scale engagement (Taylor, 2006) in which the manger or leader is both open to the narrative of innovation and willing to allow the organization to move to the creative edge of chaos.
- Bilger, B. (2006). "The search for sweet: Building a better sugar substitute," The New Yorker, ISSN 0028-792X, May 22, 40-46.
- Carlisle, Y. and McMillan, E. (2006). "Innovation in organizations from a complex adaptive systems perspective," Emergence: Complexity & Organization, ISSN 1521-3250, 8(1): 2-9.
- Rheingold, H. (2002). Smart mobs: The next social revolution, Cambridge, MA: Perseus, ISBN 0738206083.
- Taylor, W. C. (2006). "Here's an idea: Let everyone have ideas," New York Times, ISSN 0362-4331, March 26, Sec. 3, p. 3, Col. 1.


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"No Man Only Needs a Little Salary"
Charley in Arthur Miller's Death of a Salesman.
"No Man Only Needs a Little Salary"
Latest Papers
The working papers collection comprises historical papers as well as current ideas and works in progress on some of the major issues and topics of our times.
The Fire of Mata Mata (released 2 October 2014)» more
A discussion with Batumbil Burarrwanga, traditional owner, leader, master artist and weaver at her spiritual home, Mata Mata, on the Arafura Sea, North East Arnhem Land
Wonders of the World (released 28 September 2014)» more
Tribute to one of the great men of Lake Evala
The Greatest Show on Earth (released 15 September 2014)» more
Stephen Pigram, Australia's greatest contemporary troubadour, is the artistic director of a show that will take not only Australia, but the world by storm.
Work With What You Have Got (released 4 August 2014)» more
An Independent Review of Andrew Forrest's Creating Parity
The Good Sock (released 28 July 2014)» more
A story about the value of good socks for a working man.
Inside Words (released 20 June 2014)» more
Reviving the Ancestral Ties, Friendship and Trade between Cape York Peninsula and Timor Leste
Tough Times, Tough Questions (released 14 June 2014)» more
Nyunggai Warren Mundine calls an extraordinary meeting of his Indigenous Advisory Council with Prime MInister Abbott and says the Commonwealth government must earn the trust of the Aboriginal community.
Twelve Things You Need to Know About Timor-Leste (released 25 May 2014)» more
We Australians need to act quickly to understand the new Timor-Leste.
A Submission to the Joint Select Committee on the Constitutional Recognition of Aboriginal and Torres Strait Islander Peoples
Submission to Senator Brandis in the light of the WA Senate election and other considerations


text: This high-end jewelry boutique, located in an unusual office space on Turtle Creek, actually feels like a cozy living room. Owners and siblings Kim Burgan and Darin Kunz aim for a comfortable ambiance where customers can sit, relax, and have a drink while shopping for stylish new jewels or future family heirlooms. If you're shopping for an engagement ring, rest assured. The two will prep you on the ins and outs of diamonds, so you know you're working with honest experts.


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Free delivery on orders over £65
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Established 25+ years
Ideal365 is the one stop shop for all your Housekeeping Supplies needs. We have vast array of Housekeeping products, multi purpose cleaners, stainless steel cleaners, wipes and cloths to name a few.
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text: Many providers are available, including Theplanet.com, Rackspace.com, and Hostway.com. Which pages and posts are most important? These should have other pages and posts linking to them. Make sure to link to the most important content. Keeping broad-based objectives in mind is a good way to keep your e-mail marketing strategy focused on the big picture. The length of search phrases continues to grow. Back when the Internet was just an upstart, single keywords were the only thing you needed.


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Welcome to the LINCOLN's School Room.
Within this school room are smaller teacher rooms created by students to exchange homework help and classroom related information. If you want to add a new teacher room please do so. All rooms are FREE to join and create. Invite other classmates to join, post and chat.Radcliffe College was a women's liberal arts college in Cambridge, Massachusetts, and was the coordinate college for Harvard University. It was also one of the Seven Sisters colleges. Radcliffe College conferred joint Harvard-Radcliffe diplomas beginning in 1963 and a formal merger agreement with Harvard was signed in 1977, with full integration with Harvard completed in 1999.
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SAG-AFTRA represents voiceover talent in all contracts areas, including animation, video games, promos, trailers, commercials, documentaries, audiobooks and more.
Safety Resources
Voiceover performers are covered under several SAG-AFTRA collective bargaining agreements to guarantee fair wages, health and retirement contributions, residuals and additional compensation, and advocate for vocal health.
For Members
SAG-AFTRA contracts provide voiceover performers the following benefits:
Guaranteed Minimum Rates: These rates are tailored to the particular type of work. Some of the rate sheets are below, but please check with staff for details about your upcoming job.
Health Insurance: By working under SAG-AFTRA agreements, you can earn credits towards quality, affordable health coverage (including wellness care, medical, hospitalization, prescription drug, mental health, chemical dependency and dental benefits). These benefits are provided by the SAG-AFTRA Health Plan; for more information visit www.sagaftraplans.org.
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Additional Benefits: There are additional benefits available exclusively for members of SAG-AFTRA through programs such as the AFL-CIO's Union Plus. Benefits ranging from mortgage and refinancing savings to auto insurance to discounts on flowers and more are available at no cost to the union or its members. Visit www.unionplus.org for more information. Click here to visit the SAG-AFTRA benefits page.
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My Burberry Black perfume for Women by Burberry
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7.8 out of 10 based on 6 ratings
|My Burberry Black
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Popularity Total Hits: 12468
Burberry My Burberry Black In stock: Prices starting from $5.49 to $136.44 based on 24+ offers.
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My Burberry Black Burberry - What our experts say:
Fragrance My Burberry Black perfume for Women by Burberry was released in 2016. Who Is It Best For? Well it is an aroma highlighted by a bouquet of fragrant fruity, sweet and powdery scented tones that will bring a sensual, romantic and calming perfumed sensation. Examining it closer once applied you will notice a lingering quality of softer fragranced woody, balsamic and animal wildlife hints that hide an essence of elegant, uplifting and bright feelings.
Based on the combination of notes Burberry My Burberry Black is classified as Oriental Floral fragrance to the nose. The fragrance composition is a creation of Francis Kurkdjian. The emanation of flavors and scent notes for My Burberry Black unfurl in a whiff of white flowers opening top notes including jasmine. The floral and fruity heart center notes blend in with candy while the deep amber and woody base notes leave an impression of amber and patchouli.
Before buying My Burberry Black, sample it, and see if it blends with your body chemistry and character, the more perfume reviews you read on Burberry My Burberry Black from different people and especially experts who have the nose the better prepared for making your choice. If My Burberry Black seems like a pleasant smell for your taste or maybe special occasion then the best is to aromatize yourself and give it a try!
Designer fragrance My Burberry Black was released in Eau de Toilette concentration.
My Burberry Black perfume for Women by Burberry gets a rating of 7.8 out of 10 based on 6 ratings.
Burberry's version of My Burberry Black is now in stock: Prices starting from $5.49 to $136.44 based on 24+ offers. Find the latest pricing from the best online stores, compare today's offers and shop online here!
Burberry My Burberry Black - Product Description Overview
The exciting collection of My Burberry perfumes welcomes another member with My Burberry Black fragrance, drawing inspiration from the black Burberry Heritage trench coat.
The stunning new fragrance brings together the smell of sun drenched jasmine flower and ripened peach nectar smoothed out with elegant candied rose while the base brings the warmth of amber and patchouli resulting in a pleasantly potent and highly sophisticated aroma.
My Burberry Black Eau de Toilette is expected to initially role out in the Middle East from May 2016 before worldwide launch in September 2016.
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Jump start your San Diego vacation plan with this printable 7-day itinerary. This San Diego Itinerary includes San Diego's top attractions for families, including
- SeaWorld San Diego
- San Diego Zoo
- LEGOLAND California
- U.S.S. Midway Museum
- Beach Day
- Balboa Park
- Cabrillo National Monument
You'll get a list of dozens of alternate activity options with price, age and accessibility guides. It's easy to quickly customize this itinerary to fit your family's interests, schedule and budget.
This San Diego itinerary also includes tips for saving money on your vacation. If you pay full price for San Diego's top attractions, you're paying too much! I'll show you several easy ways to find deals and discounts. This itinerary can quickly pay for itself.
Your 15-page itinerary has been designed with bold, full-color fonts and photos on a white background, which makes it easy to view on your mobile device or print. Print every page, or just the pages you need.
You will be emailed a downloadable file immediately after purchase. The download link will be active for 14 days. Once you download the PDF itinerary to your device, it's yours to keep.
Want a less expensive alternative? My San Diego Itinerary – Basic Edition includes the same itinerary and money-saving tips as the In Depth version, with fewer tips and recommendations. You'll get loads of helpful information to jump start your San Diego vacation planning in just three pages.
Happy travels!
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