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Thursday, August 12, 2004
My kids this year are so much higher than my kids last year...not in the drug sense-- they are a lot less high in that regard. I mean in the reading and writing regard.
I had no idea that all eighth graders were not, in fact, incapable of forming a complete sentence. I really believed that all kids that age (except for one or two waspy teens in private school somewhere in Maine or New Hampshire, maybe) would simply refuse to do homework.
Not true. It really really matters where you work. I stumbled into a good district. These kids don't have a lot of money, but the school culture is intense. Parents are really involved. Teachers spend real time doing planning and not whining about it. The administration trusts its teachers. The teachers are willing to work with the administration. Wow.
When I give my students homework, they groan good-naturedly and take out their planners to write it down. When they get upset about something in class, maybe they make a little noise and need a talking to, but mostly they do what they need to do. I am blown away. Blown away and so happy I could spit. It makes me want to just settle right down in Graham, North Carolina. Except not really. But now I know what to look for in a school when I go where I do want to settle.
In other news, today I called in "tropical storm bonnie" at the bookstore. It was raining so hard and the wind was so awful, I didn't know if I could make it to work. So I decided to take a much needed rest and read young adult books in bed. Heavenly.
North Caroliners-- mark your calendars! Next Karaoke date at Bub O'Malleys: August 19! Don't miss out!
Tuesday, October 20, 2009
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12 Flags for Straws, Sucker or Lollipop Sticks, Cake Pop Sticks, Aqua Blue Color Scheme, Wedding, Bridal Shower, Baby Shower, Birthday
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Conceived and designed the experiments: MAA NZ JEL BAA SV WRG MBA KT KS GR HW FW MC AJB JCA SCB HEH. Performed the experiments: MAA JEL BAA TJT MG LH SV PH JZZ FB SL HW MBA KT GR LLF HW. Analyzed the data: MAA NZ JEL BAA SV KT KNR HW FW AJB JCA SCB HEH. Contributed reagents/materials/analysis tools: NZ BAA TJT LH SV KTV PH JZZ SL WRG ET MBA FW JCA SCB. Wrote the paper: MAA BAA KS JCA SCB HEH. Edited and approved final manuscript: MAA FW LPA JCA SCB HEH.
Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.
Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC50 values as low as 5±3 nM and 0.13±0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.
Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.
Notch signals normally participate in a variety of cellular processes, including cell fate specification, differentiation, proliferation, apoptosis, migration, and angiogenesis [1]. The four mammalian Notch receptors (Notch1-4) all have a similar modular domain organization. The extracellular domain contains a series of epidermal growth factor (EGF)-like repeats that participate in binding to ligands [2], followed by a negative regulatory domain (NRR) that, in the absence of ligand, maintains the receptor in a protease-resistant conformation [3], [4]. During trafficking to the cell surface, the NRR is clipped by a furin-like protease at a site called S1 [5], dividing Notch into two subunits that are held together by contacts in the N-terminal and C-terminal portions of the NRR. The intracellular portion of Notch (ICN) contains RAM [6] and ankyrin-repeat domains that both participate in binding to the DNA-binding factor CSL [for CBF-1/Su(H)/Lag1] [7], as well as nuclear localization sequences and a C-terminal PEST degron [8].
Activation of Notch receptors is normally induced by binding of Jagged [9], [10] or Delta-like [11]–[13] ligands expressed on neighboring cells, which initiates a series of additional proteolytic cleavages. The first is catalyzed by a metalloprotease of the ADAM (a disintegrin and metalloprotease) family [14], [15] and occurs at a site called S2, which lies within the NRR just external to the transmembrane domain. This primes Notch for additional cleavages within the transmembrane domain that are carried out by the multiprotein membrane complex γ-secretase [16]. The final cleavage liberates ICN from the membrane, allowing it to enter the nucleus and activate the transcription of Notch-responsive genes (e.g., HES1, HES5, NRARP, Deltex1 (DTX1), c-MYC). This depends on binding of ICN to the transcription factor CSL [7], [17], [18] and recruitment of Mastermind-like coactivators [19]–[22]. Post-translational modification events, such as glycosylation of the extracellular domains of both receptor and ligands also play an important role in Notch-ligand interactions [23], [24], and such modifications may play a part in tissue-specific responses to various ligands [25].
In addition to its developmental roles, dysregulation of Notch signaling is associated with a number of different cancers. The clearest example is T-cell acute lymphoblastic leukemia/lymphoma (T-ALL, see below), in which activating mutations in the NRR and/or the PEST domain of Notch1 are found in over 50% of cases. Increases in Notch signaling, perhaps induced by ligand-mediated activation, have also been associated with breast, colon, ovarian, and lung cancer [26]–[30]. For example, co-expression of Notch1 and Jag1 has been associated with poor outcomes in patients with breast cancer [31]. Delta-like-4 signaling through Notch1 regulates the formation of tip cells during angiogenesis [32] and is also likely to play an important role in pathological angiogenesis [33], making it a promising therapeutic target [34].
The discovery of gain-of-function Notch1 mutations in 55–60% of human primary T-ALL samples [26], including all of the major T-ALL subtypes, greatly expanded the known role of Notch1 in this disease, moving it to the center of T-ALL pathogenesis. The most common leukemogenic Notch1 mutations (35–40% of tumors) lie in the "heterodimerization domain" (HD) of the NRR and lead to ligand-independent Notch signaling activity [35], [36]. Mutations that result in deletion of the PEST degron (20–30% of tumors) are also frequent in T-ALL and cause a synergistic increase in Notch signaling when aligned in cis with HD mutations in the same Notch1 allele [35]–[37]. Notch1 signaling drives the growth of T-ALL cells [38], [39], making it an attractive target for rational pharmacological intervention.
A number of different strategies [34] are in development to inhibit Notch signaling for therapeutic purposes. One approach is to block the proteolytic release of intracellular Notch from the membrane by treatment with inhibitors of gamma secretase (GSIs). In a number of tumor cell lines carrying HD domain mutations, blocking proteolytic activation with GSIs triggers cell-cycle arrest and variable degrees of apoptosis [40], [41]. However, the poor selectivity of GSIs, which inhibit the proteolysis of all four Notch receptors, and the processing of an expanding list of other substrates by gamma secretase [16], [42], [43], constitute significant potential limitations for this class of anti-tumor agents. Studies in animal models using the GSI LY 411,575 have shown significant dose-limiting toxicity in the intestine [44]. The toxic effects of GSIs in mice appear to result from simultaneous inhibition of Notch1 and Notch2 [29], [45], which leads to the accumulation of secretory cells at the expense of absorptive enterocytes. Clinical trials with the GSI LY450139 in Alzheimer's disease patients also identified diarrhea as the most frequent adverse effect in human phase I studies [46].
An alternative route that may overcome the toxicity associated with GSIs is selective targeting of Notch1 with inhibitory antibodies. In support of this approach, antibodies capable of selectively modulating Notch3 signaling have been reported recently [47]. The most potent inhibitory antibodies are directed against the NRR and are proposed to stabilize the autoinhibited form of the receptor [47].
In this study, we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two different classes of antibodies were identified. One class is ligand-competitive, being directed against the EGF-repeat region of the receptor that encompasses the ligand-binding domain (LBD), and the second is allosteric, being directed against the NRR region. Both classes of antibodies are selective for Notch1, bind Notch1 on the surface of human tumor cell lines, and inhibit ligand-induced expression of Notch target genes in cell lines expressing wild-type Notch1 receptors. NRR-targeting antibodies are also capable of recognizing and inhibiting Notch1 receptors bearing "class 1" NRR mutations, but are less effective in inhibiting Notch1 activation in T-ALL cells than GSIs. These findings have implications for selective targeting of normal and mutated Notch1 receptors with antibodies as well as our understanding of Notch1 receptor activation in T-ALL cells.
Cancer cell lines (LS-1034, BxPC3, Colo_205, and TALL-1) purchased from ATCC (Manassas, VA) were maintained at 37°C under 5% CO2 in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated (HI) FBS (Hyclone, Logan, Utah), 2 mM L-glutamine (Invitrogen) and 1× Pen-Strep (Mediatech, Herndon, VA). T-REX™-293 and Flp-In™ -3T3 cell lines purchased from Invitrogen were maintained at 37°C under 5% CO2 in Dulbecco modified Eagle medium (DMEM) with high glucose (Invitrogen) supplemented with 10% HI FBS (Hyclone), 2 mM L-glutamine (Invitrogen), and 1× Pen-Strep (Mediatech). For the ligand stimulation assays, cells were resuspended in DMEM high Glucose medium without phenol red and supplemented only with 10% HI FBS (Hyclone).
Cell lines stably expressing either full-length wild-type or chimeric Notch receptors or Notch ligands were generated to test the binding and potency of Notch antibodies. The human and mouse (only Notch1) full-length cDNA sequences coding for Notch1, 2, and 3, Jag1, and DLL1 were chemically synthesized by DNA2.0 Technologies (Menlo Park, CA). The cDNA encoding DLL4 was amplified by RT-PCR from Colo_205 cells following described protocols [48]. Chimeric human Notch receptors (Notch1, 2, and 3) were created by inserting the sequence encoding the DNA binding domain of Gal4 into a Notch cDNA previously deleted of the sequence encoding most of the RAM domain and the ankyrin repeat domain as described [35]. Each of these cDNAs were cloned into the pcDNA5/FRT/TO vector (Invitrogen) and co-transfected with pOG44 (Invitrogen), a plasmid encoding Flp recombinase, into T-REX-293 cells (human wild-type Notch receptors), T-REX-U2OS cells (mouse wild-type Notch1, chimeric receptors), or 3T3 Flp-In cells (Notch ligands) using Fugene6 (Roche, Indianapolis, IN) following the manufacturer's protocols. Cell lines with stably integrated cDNAs were selected with hygromycin (100 µg/ml) (Mediatech). Expression levels of the Notch proteins were assessed by Western blot and flow cytometry. The T-REX-U2OS and -293 cells express the Tet repressor (for tight regulation of gene expression), and also contain a single genomic FRT (Flp recombinase target) site, which permits creation of isogenic recombinants containing a single transgene under the control of tetracycline.
Assays were performed as described [35]. Stable Flp-In U2OS cells bearing isogenic transgenes encoding Notch1-, 2-, or 3-GAL4 chimeric receptors were transiently transfected with a pFR-Luc reporter plasmid (Stratagene, Cedar Crest, TX) containing five copies of the Gal4 binding site (5X-upstream activation sequence (UAS)). After 24 hr, cells were treated with doxycycline (2 µg/ml) (Sigma-Aldrich, St. Louis, MO) in the presence or absence of Notch antibodies and IgG control, and overlaid at a 11 ratio onto Flp-In-3T3 cells stably expressing Notch ligand or parental Flp-In-3T3 cells seeded in 96-well plates. Luciferase reporter activities were measured after an additional 24 hr in whole cell lysates using the Bright-Glo assay kit (Promega, Madison, WI) and a VictorLight luminometer (Perkin-Elmer, Waltham, MA). The potency of the antibodies was determined with serial dilutions that generate sigmoid curves. IC50 values were determined using a 3-parameter logistic curve fit with maximum values arbitrarily fixed at 100%. In the mouse Notch1 reporter assay, Notch activity in cells stably expressing the mouse full-length wild-type Notch1 receptor was determined as described above using a luciferase reporter gene containing four copies of the CSL binding site (4XCSL-luciferase reporter gene). To test ligand-independent activation of Notch1 signaling generated by receptors bearing specific T-ALL mutations, T-REX-U2OS cells were transiently transfected with full-length Notch1 DNA constructs bearing those mutations and the 4XCSL-luciferase reporter gene. Cells were then treated with antibodies (10 µg/ml) and luciferase activity measured after 24 h as described above. In these experiments, T-REX-U2OS cells transiently transfected with the full-length wild-type Notch1 receptor were used as the baseline control.
Ligand-competitive binding of antibodies was measured by Notch1 extracellular domain (ECD) displacement in a dissociation-enhanced time resolved fluorometric assay (DELFIA). Briefly, Maxisorp 96-well plates were coated with Notch ligand DLL4 (100 µl/well, R&D Systems, Minneapolis, MN) at 2 µg/ml in D-PBS (GIBCO-14080) and incubated at 4°C overnight. The coated plates were blocked with 5% BSA in D-PBS. Notch1 ECD-Fc fusion protein (Notch1 amino acids Ala19-Gln526) (R&D Systems) was pre-labeled with europium (Eu) reagent according to the manufacturer's procedure (PerkinElmer). Serially diluted Notch1 monoclonal antibodies were preincubated with a fixed amount (0.5 µg/ml) of Eu-labeled Notch1 ECD-Fc for 2 hr with shaking, followed by addition to the DLL4 ligand-coated plates. After incubation at room temperature for 1 hour with slow shaking, the plates were washed and DELFIA® Enhancement solution (Perkin Elmer) (100 µl/well) added. Fluorescence signals were read after a 5-min incubation in a Victor3-V plate reader (Perkin Elmer) at excitation/emission wavelengths of 340/615nm.
Flow cytometric detection of Notch1 was performed with doxycycline-inducible T-REX-293 cells stably expressing the full-length Notch1, -2, or -3 receptors, respectively; or the human cancer cell lines LS-1034, BxPC3, Colo_205, and TALL-1. T-REX-293 cells were treated with 2 µg/ml doxycycline (Sigma-Aldrich) for two days to induce Notch expression before collecting them for staining. Flow cytometric detection of Notch ligands was performed in Flp-In-3T3 cells stably expressing Jag1, Jag2, and DLL1 ligands, respectively, using primary antibodies purchased from R&D Systems. Cells were harvested from tissue culture flasks using trypsin (Mediatech) and resuspended in PBS with 2% fetal bovine serum (FBS; Hyclone) (FBS/PBS). Cells (1×106) were incubated with 1 µg of each antibody for 40 minutes at 4°C, followed by washing and resuspension in 0.1 ml of FBS/PBS containing 1 µg of phycoerythrin-conjugated secondary antibodies for 30 minutes at 4°C. Cells were then washed, resuspended in 0.34 ml PBS with 1% formaldehyde (Polysciences, Inc., Warrington, PA) and analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA). The secondary antibodies used were goat anti-human Fcγ specific F(ab')2 fragments (Jackson ImmunoResearch, West Grove, PA) to detect the primary anti-Notch1 Abs, donkey anti-goat IgG (H+L) specific F(ab')2 fragments (Jackson ImmunoResearch) to detect anti-Jag1 and anti-Jag2, and rat anti-mouse IgG κ light chain antibody to detect anti-DLL1 (BD Pharmingen, San Diego, CA).
Parental Flp-In 3T3 cells or Jag2-expressing 3T3 cells were co-cultured at a ratio 11 with LS-1034 or TALL-1 cells in the presence of 20 µg/ml of each antibody or 5 µM GSI for 19 hr. Cells were harvested and RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was synthesized from 0.5 µg of purified RNA using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. qPCR was performed in triplicate using 2 µl cDNA sample or control, Brilliant II QPCR Master Mix with ROX (2X) (Stratagene, Cedar Creek, TX), and the inventoried probes and primers (Applied Biosystems Assays on Demand) for human HES1, HES5, DTX1 (Deltex1), and GAPDH. PCR cycling was performed at 95°C for 10 minutes to allow enzyme activation, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute using the Mx3005P QPCR System (Stratagene). Analysis was performed using MxPro QPCR Software version 3.0 (Stratagene).
Human T-ALL cell lines (TALL-1, DND-41, KOPT-K1) were cultured at 37°C under 5% CO2 in RPMI 1640 (Invitrogen) supplemented with 10% FBS (Hyclone), 2 mM L-glutamine (Invitrogen), 100 UI/ml penicillin G, and 100 µg/ml streptomycin (Invitrogen). For growth assays, 30 µl of cell suspension (9×104 cells/ml) was seeded in 384-well white plates (Corning, Lowell, MA). Drugs and/or antibodies were added after 4 hr of pre-culture. Drugs were dissolved in DMSO and antibodies in sterile PBS. Assessment of the cell growth after 72, 96, and 120 hr of treatment was carried out by the Cell-Titer Glo luminescence assay (Promega) in an Envision Multi-label plate reader (Perkin-Elmer, Waltham, Massachusetts). The assay was done in triplicate and each experiment was repeated twice. The significance of differences in cell viability was assessed by the Student t-test. Synergism was determined by isobologram analysis of dose-response curves for cells exposed to varying concentrations of GSI (compound E, Axxora, San Diego, CA), dexamethasone (Sigma), and/or Notch1 inhibitory antibodies. When used in combination, the ratio of the two drugs was 11, while the ratio of antibody to drug was 401. The degree of synergism was determined by the combination index (CI) method [49], which was computed from the dose-response curves with Calcusyn version 2 software (Biosoft, Cambridge, UK). Significant synergism was defined as a CI<0.7.
In order to determine the effects of Notch1 antibodies on the Notch1-dependent gene expression signature in T-ALL cells, we first selected a set of genes that defined the Notch1 on versus off state from Affymetrix microarray expression profiling of 7 Notch1-mutated T-ALL cell lines treated in duplicate with vehicle versus the GSI compound E (500 nM) for 24 hr [39]. From a set of ~500 genes with differences of p<0.01 by 2-sided Student's t-test, 16 sentinel genes were selected to define the Notch1 off signature based on mean fold changes >1.5 between the Notch1 on versus off states. Four control genes with stable expression across the two states were selected to control for well-to-well variability in total RNA: GAPDH, NFX1, NISCH, and GTF. We next adapted this signature to an assay that uses ligation-mediated amplification (LMA) and a Luminex FlexMAP fluorescent bead-based detection system. Full details of this methodology have been described elsewhere [50]. Briefly, the 20 genes were subjected to 34 cycles of amplification by LMA, yielding biotinylated PCR products containing molecular barcode sequences. These PCR products were hybridized in solution to beads dyed with unique fluorescent colors containing complementary barcode sequence. Following hybridization and staining with streptavidin-phycoerythrin (SA-PE), the beads were analyzed by dual-color flow cytometry, in which the bead color identifies the gene of interest and PE intensity the quantity of transcript. DND-41 cells were treated with control antibody (10 µg/ml) (6 replicates), NRR WC75 Notch1 antibody (10 µg/ml) (7 replicates), DMSO (0.08%) (6 replicates), or GSI (1 µM) (14 replicates) for 72 hr and then analyzed for gene expression. To normalize measurements within each experiment, expression of Notch marker genes was expressed relative to the average expression of the four control genes. We also evaluated the overall performance of the signature by calculating two summary scores combining information about all of the signature genes: the summed score and the weighted summed score. The summed score metric combined expression ratios by summing them with a sign determined by the expected direction of regulation as determined from the positive controls (GSI-treated). The weighted summed score metric is a variant of the summed score metric that combines expression ratios by summing them with a weight and sign determined by the signal-to-noise ratio of the positive control (GSI-treated) and negative controls (DMSO-treated). Signal-to-noise ratio is defined by:
where μi1 represents the mean expression of samples from class 1 for feature i and σi1 represents the standard deviation of class 1 for feature i.
Notch ectodomains were cloned into the pLEXm mammalian expression vector with C-terminal His6 tags, expressed in HEK-293T cells using a PEI-based transfection protocol and harvested after 3 days. Whole cell extracts were prepared by lysis with RIPA buffer containing 1250 protease inhibitors followed by centrifugation to remove cellular debris. Lysates were mixed with 5–10 µg primary antibody overnight at 4°C, then with 50 µL Protein A-agarose suspension for an additional 2 hours. Beads were then collected by centrifugation, washed 3 times with PBS, and resuspended in 50 µL 2× SDS sample buffer containing 100mM DTT. Samples were boiled for 5 minutes and run on a 5–20% Tris-Glycine gel, then transferred to a nitrocellulose membrane at 15V for 1 hour. Blots were washed twice with Tris-buffered saline, pH 7.5 (TBS) and blocked in TBS +3% BSA for 1 hour. Incubation with Penta-His antibody (11000) was performed overnight in TBS +3% BSA, followed by incubation with goat α–mouse antibody (110,000) in TBS +10% milk for 1 hour. Three wash steps were done following the incubation with primary antibody (two with TBS-Tween-0.2% Triton-X (TBST) and one with TBS), and four washes with TBST were done following the incubation with secondary antibody. Blots were exposed for 10–20 minutes and analyzed using an Alpha Innotech gel documentation system.
A plasmid encoding the human Notch1 NRR (residues E1446-Q1733; Genbank ID 148833507) was modified to contain a N-terminal hexahistidine tag followed by a TEV cleavage site. The Notch1 NRR precursor was prepared essentially as described for the loopout form of the Notch1 NRR [37]. The purified protein was labeled with EZ-Link NHS-PEG4-Biotin (Pierce-Thermo, Rockford, IL) according to the manufacturer's instructions. Biotinylated Notch1 NRR was then captured onto neutravidin-coated 96-well plates. Binding of the anti-NRR antibodies was allowed to proceed for one hour in Tris buffer (25 mM, pH 7.4), containing NaCl (150 mM), CaCl2 (5 mM), 0.05% Tween, and 0.5% BSA. Antibody binding was detected with a goat anti-human antibody conjugated to horseradish peroxidase using the fluorogenic substrate quantaBlu (Pierce-Thermo).
A total of 16 high-affinity antibodies against Notch1 were selected from a phage library, using both cell-based and recombinant protein panning approaches. The details of the panning strategy and the identification, cloning and ranking of hits by affinity will be described elsewhere. All antibodies were shown by flow cytometry to bind to full-length Notch1 overexpressed in HEK cells (Table 1 and data not shown). Since antibodies against the Notch1 extracellular domain were likely to interfere with receptor-ligand interactions, we first evaluated all antibodies in a ligand competition assay with recombinant DLL4 and the Notch1 ectodomain (EGF repeats 1–13), which includes the ligand binding domain (LBD; EGF repeats 11–13). As shown for a subset of antibodies in Figure 1, most, but not all, antibodies inhibited binding of the Notch1 ectodomain to immobilized DLL4. Fixed dilutions of antibodies were used for this initial characterization, as the intent was to classify the antibodies by mechanism rather than to establish their relative potencies. Antibodies that failed to compete with DLL4 binding, such as WC75 and WC629 (Figure 1), did not recognize EGF repeats 1–13. Instead, these antibodies were found to bind to the negative regulatory region (NRR) of Notch1 (supplemental Figure S1). In contrast, none of the ligand-competitive antibodies bound to recombinant NRR (data not shown). A total of seven antibodies (listed in Table 1) were selected for further characterization, five ligand-competitive antibodies (referred to as LBD antibodies) and two antibodies recognizing the NRR (referred to as NRR antibodies).
To evaluate the functional activity of the LBD and NRR antibodies, we generated a panel of stable T-REX-U2OS cell lines expressing Notch1-, Notch2-, or Notch3-Gal4 fusion receptors. Notch signaling in these reporter cell lines was monitored by transient transfection of a Gal4-luciferase reporter plasmid. To activate Notch signaling, reporter cell lines were co-cultured with 3T3 cell lines stably overexpressing various Notch ligands.
The five LBD and two NRR antibodies were characterized for their effect on Jag2-induced Notch signaling, using a well-characterized NIH 3T3 cell line expressing Jag-2 [51]–[52]. The ligand-competitive LBD antibodies were able to block Jag2-stimulated Notch1 activity completely (Figure 2A and Table 1). The basal reporter activity in the presence of parental Flp-In-3T3 cells (no ligand expression) was neither significantly inhibited nor stimulated by these antibodies (data not shown), indicating that, despite their potential to crosslink Notch1, free LBD antibodies do not have agonistic activity. The NRR-specific antibodies were also potent antagonists of Jag2-dependent Notch1 signaling (Figure 2B and Table 1), presumably through allosteric stabilization of the NRR domain in a metalloprotease-resistant autoinhibited conformation. The generally greater potency of the NRR antibodies as compared to the LBD antibodies correlated with a higher affinity for Notch1-expressing cells, as assessed by flow cytometry (Table 1). However, at saturating concentrations, the NRR antibodies maximally inhibited Notch1 signaling by 70 to 80%, whereas LBD antibodies were able to completely inhibit the Notch1 activation by Jag2. As seen with the LBD antibodies, the NRR-binders did not exhibit detectable agonist activity in co-culture assays using parental 3T3 Flp-in cells.
The specificity of both classes of antibodies for Notch1 as opposed to Notch2 or Notch3 was evaluated in reporter assays with Notch-Gal4 fusion receptors. Neither LBD nor NRR antibodies significantly inhibited ligand-stimulated Notch2-Gal4 and Notch3-Gal4 signaling (Figure 2C). Species cross-reactivity was tested in T-REX-U2OS cells stably expressing murine Notch1 and transiently transfected with a CSL-luciferase reporter. In co-culture experiments with 3T3 cells expressing human Jag2, greater than 50% inhibition of mouse Notch1 was seen with several of the antibodies at 167 nM (Figure 2D), suggesting that future efficacy and tolerability studies of these antibodies can be conducted in mouse models.
Because we have been unable to create Notch4 reporter lines that generate a luciferase signal in response to ligand stimulation, we tested the antibodies for specificity toward Notch1 as opposed to Notch4 by comparing the ability of representative LBD and NRR antibodies to immunoprecipitate the Notch1 and Notch4 extracellular domains. Western blot analysis (supplemental Figure S2) showed that the allosteric antibody WC75 and the ligand competitive antibody WC613 both immunoprecipitated Notch1 but not Notch4.
Based on current models of Notch receptor activation, the inhibitory activities of LBD antibodies were expected to show a stronger dependence on the activating Notch1 ligand than NRR antibodies, which are not ligand-competitive (Figure 1). We therefore compared the ability of these two classes of antibodies to inhibit Notch1 signaling in co-culture assays with 3T3 cells stably expressing Jag1, Jag2, DLL1 or DLL4. Expression of Jag1, Jag2, and DLL1 was confirmed by flow cytometry using specific antibodies in each of the stable 3T3 cell lines (data not shown). It was not possible to determine the levels of DLL4 by flow cytometry due to lack of an appropriate antibody, but luciferase reporter assays using 3T3-DLL4 cells confirmed the ability of DLL4 to strongly activate Notch signaling. As expected, the antagonistic activity of LBD antibody WC613 was strongly dependent on the particular ligand-expressing cell line used to activate Notch1 (Figure 3A). 3T3-Jag1 mediated signaling was most sensitive to inhibition, while 3T3-DLL4 signaling was most resistant. The same rank order was observed with the other LBD antibodies (Table 2). While it is tempting to speculate, the variation in EC50 values cannot be solely attributed to the identity of the particular ligand since we were not able to quantify the ligand levels on the various 3T3 cell lines in absolute terms. In contrast to the LBD antibodies, the activities of both NRR antibodies were minimally dependent on the nature of the ligand-expressing cell line and both antibodies inhibited DLL4 signaling, albeit to a maximum of 50% to 60% (Figure 3B, Table 2). These data are consistent with the current models of ligand-dependent Notch receptor activation, i.e., that LBD antibodies compete with ligands for access to the Notch1 LBD, whereas NRR antibodies allosterically inhibit ligand-induced conformation changes in the NRR.
Notch1 signaling is increased in a variety of cancers and activates downstream target genes, including HES1, HES5, DTX1, NRARP, and c-MYC [7], [18], [38], [53]. Two human lines expressing wild-type Notch1 receptors, the colorectal carcinoma cell line LS-1034 and the T-ALL cell line TALL-1, were used to test the ability of the LBD and NRR antibodies to modulate Notch activity. Notch signaling was induced by co-culture of these cell lines with 3T3 cells expressing Jag2. Although other Notch receptors are expressed in these cells (i.e., Notch2 and 3 was detected in LS-1034 cell lysates by Western blot, and Notch3 on the surface of TALL-1 cells by flow cytometry; data not shown), the specificity of the antibodies (unlike GSI) allows one to assess the effects of Notch1 inhibition per se. In LS-1034 cells, ligand-dependent transactivation of Hes1 transcription was inhibited significantly by each of the antibodies tested at saturating antibody concentrations. The LBD antibodies (e.g., WC613, WC133) almost completely inhibited Hes1 transactivation, while the NRR antibodies (WC75, WC629) were partially inhibitory (Figure 4). A similar correlation was observed in TALL-1 cells, in which the ligand-dependent transactivation of both HES5 and DTX1 was also inhibited by all of the antibodies tested at saturating antibody concentrations, with the LBD antibodies being more effective than the NRR antibodies (Figure 4). These results correlate with the ability of the LBD and NRR antibodies to totally or partially, respectively, inhibit ligand-dependent Notch1 activation in the reporter assays. Because other Notch family members besides Notch1 may contribute to basal and induced Notch signaling in these cells, we compared the effects of the antibodies with a "pan-Notch inhibitor", the gamma-secretase inhibitor (GSI). At a concentration of 5 µM, GSI inhibited ligand-dependent activation, and suppressed expression below the basal level observed in co-culture with the parental 3T3 Flp-in cells, as might be expected based on the ability of GSIs to block the activity of all Notch family members expressed on the tumor cells (Figure 4).
As described above, the potency of the antibodies in the reporter assays correlated with their affinity for Notch1, as assessed by flow cytometry conducted on cells engineered to express Notch1 stably (Table 1). To establish a correlation between phenotypic response and the binding affinity of the LBD and NRR antibodies to Notch1 expressed on the surface of cancer cells, flow cytometry was performed with LS-1034, BxPC3, Colo_205, and TALL-1 cells using saturating concentrations of the WC75 (NRR) and WC613 (LBD) antibodies. All the cell lines showed detectable levels of Notch1 on the cell surface (Figure 5) that correlated with levels of Notch1 detected in Western blots of whole cell lysates with an antibody directed against intracellular Notch1 (not shown). The relative binding affinities of NRR and LBD antibodies for Notch1 varied among cancer cell lines. With LS-1034 cells, NRR antibodies showed greater binding than LBD antibodies, while the converse was true for TALL-1 cells (Figure 5). The explanation for this cell line-dependent variation in the stoichiometry of binding of NRR and LBD antibodies is not readily apparent, and likely to be complex. It is possible, for example, that expression of various competing ligands, epitope masking by cell-type specific glycosylation, or other post-translational modifications of Notch1 [54] may differentially affect the binding of antibodies to their respective epitopes.
Notch1-dependent proliferation has been previously reported in some cancer cell lines [55]–[57]. To evaluate the anti-proliferative effect of Notch1 antibodies on a cell-line derived from a solid tumor, LS-1034 cells were grown in monolayer culture in the presence or absence of the Notch1 antibodies for up to 96 hr. Although these cell lines express wild-type Notch1 on their cell surface (Figure 5), treatment with anti-Notch1 antibodies at saturating concentrations (0.1 µM) did not affect their proliferative capacity (data not shown).
Leukemogenic point mutations in the NRR of Notch1 cause conformational changes that lead to ligand-independent S2 cleavage [35], suggesting that LBD antibodies should have little effect on the activation of receptors bearing such mutations. In contrast, NRR antibodies raised against wild-type receptor might be able to inhibit such mutated receptors if conformational changes induced by the mutations are not so great as to preclude antibody binding and if antibody binding prevents the adoption of conformations that are permissive for metalloprotease cleavage. To initially test this idea, Notch1 receptors bearing diverse NRR mutations were transiently expressed in U2OS cells, and antibodies were scored for their effect on activation of a Notch-dependent luciferase reporter gene. Mutations tested included six class I mutations, which destabilize the NRR; one class II mutation (from the T-ALL cell line P12-Ichikawa) consisting of a direct repeat in exon 27 of Notch1 that duplicates a 14 amino acid sequence containing the S2 cleavage site [26]; one juxtamembrane class III mutation (from the T-ALL cell line Jurkat) consisting of a direct repeat in exon 28 of Notch1 that inserts a 17 amino acid sequence [58]; and VSV, an artificial mutation that inserts 14 amino acids into the juxtamembrane region [58].
All class I mutations tested (L1594P, L1597H, R1599P, L1601P, L1679P, V1677D) were inhibited by the NRR antibody WC75 at 10 µg/ml (Figure 6A), whereas LBD antibodies generally had little effect on these mutated forms of Notch1 (data not shown). In contrast, juxtamembrane insertional mutations (Jurkat, P12-Ichikawa, and VSV) were completely refractory to inhibition by both NRR and LBD antibodies (Figure 6B and data not shown). These data indicate that NRR antibodies are capable of recognizing and stabilizing Notch1 receptors bearing common class I mutations, and provide additional support for the idea that juxtamembranous insertional mutations activate Notch1 through a mechanism distinct from that of class I mutations [35].
We next asked whether the WC75 antibody could inhibit the expression of Notch1 target genes in the T-ALL cell line DND-41 which expresses Notch1 receptors bearing the compound class I mutation L1594P/D1610V. To look at the effects of WC75 on the Notch signature, we used a luminex bead-based assay that depends on ligation-mediated amplification of mRNAs captured by oligonucleotides on beads [50]. The pattern of gene expression changes induced by WC75 resembled that produced by the GSI compound E (Figure 7A), indicating that WC75 is capable of inhibiting this particular form of mutated Notch1, but the extent of inhibition by WC75 across the entire Notch1 signature was less than that produced by GSI. The relatively weak inhibitory effect of WC75 on Notch1 target gene expression was confirmed by qRT-PCR analysis of two well-characterized Notch-dependent transcripts, DTX1 and c-MYC (Figure 7B).
Growth assays were also conducted to compare the effects of WC75 and GSI on T-ALL cell growth (Figure 8). WC75 reduced the growth of DND-41 cells and KOPT-K1 cells (which bear a L1601P class I NRR mutation), but to a significantly lesser degree than GSI. Isobologram studies showed that WC75 (10 µg/ml) has weakly synergistic antiproliferative effects on KOPT-K1 cells when used in combination with dexamethasone (combination index=0.45, not shown), whereas GSI produced stronger synergistic effects (combination index=0.1, not shown). Taken together, these studies show that in T-ALL cells, signals generated by Notch1 receptors bearing class I NRR mutations are not inhibited as effectively by NRR antibodies as they are by GSI.
Notch pathway inhibitors represent an opportunity for targeted treatment of several different human cancers. Tumors for which inhibition of Notch signaling may be particularly desirable include breast cancer, where high levels of Notch1 signaling are associated with poorer prognosis [28], [31], and T-ALL, in which activating mutations in Notch1 are found frequently and where treatment with Notch pathway inhibitors, such as GSIs, arrests growth [34], [40], [41].
In the studies reported here, we characterize the in vitro activity of Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Antibodies could be grouped into two mechanistically distinct classes, ligand-competitive antibodies targeting the EGF repeat 1–13 region and allosteric, NRR-binding antibodies. Antibodies in both groups have potencies in the nanomolar to picomolar range and are highly specific for Notch1. The antibodies recognize endogenous receptors on tumor cell lines, inhibit the expression of Notch target genes in some tumor cell lines, and block Notch-dependent transcription in transfected cells. Ligand-competitive antibodies bind to the EGF-repeat 1–13 region (LBD) of the receptor and show a strong dependence on the particular ligand-expressing cell line used for co-culture. The variation in antagonist potency as a function of activating ligand might arise for a number of different reasons. Possibilities include not only differences in the intrinsic affinity of Notch1 for various ligands, but also variation in ligand expression level, differential modulation of ligand affinity by glycosyltransferase modification of Notch1, variable ligand-mediated cis-inhibition in Notch-expressing cells, etc. Additional mechanistic studies will be required to evaluate the potential therapeutic use of these ligand-competitive antibodies. For instance, DLL4-dependent events, such as tumor neoangiogenesis [33], may be relatively insensitive to the LBD antibodies reported here. On the other hand, cancers in which over-expression of Jag1, Jag2, and DLL1 are associated with poor survival, such as prostate [60] and breast [61]–[63] carcinomas, CNS tumors [57], and multiple myeloma [62], may be tractable targets.
The second group of inhibitory antibodies binds to the NRR, the activation switch of the receptor located ~1000 residues C-terminal to the ligand-binding EGF repeats. The mechanism of inhibition of NRR antibodies with respect to ligands appears to be allosteric, with little dependence on the type of ligand used for transactivation. However, the NRR antibodies were incapable of completely inhibiting ligand-dependent Notch1 activation; whether this stems from masking of the binding epitope in a subset of receptors, residual intrinsic responsiveness of antibody-bound receptors, or some other mechanism remains to be determined. Of note, binding of the WC75 and WC629 NRR antibodies is abrogated by EDTA (Supplemental Figure S1), which relaxes the structure of the NRR [3], [59]. Together these data indicate that NRR antibodies bind to a conformational epitope on the auto-inhibited conformation of the NRR and prevent adoption of the open, protease-accessible conformation upon ligand interaction.
Cell culture studies with human solid tumor cell lines, including LS-1034, showed that the LBD and NRR antibodies have no significant anti-proliferative effect. The lack of anti-proliferative activity in monolayer culture is not unexpected, as even GSIs lack activity against many solid tumor cells in culture, despite their activity in in vivo models (unpublished data). Growth inhibition and apoptosis have been reported following siRNA mediated knock-down of Notch1 [55], [57], [65]. It is possible that down-regulation of Notch protein levels may have a greater impact than inhibition of ICN1 production on cross-talk with other pathways that drive cancer growth [66], [67], as well as the expression of key factors involved in cell cycle progression [68]. Cell culture models of physiologically relevant Notch-ligand interactions have been reported [55], [57]; however, in vivo models will be required to conclusively evaluate the therapeutic potential of Notch1 antibodies.
Of interest, the NRR antibodies bind and inhibit ligand-independent activation of Notch1 receptors harboring T-ALL associated mutations, while LBD antibodies generally do not. Nevertheless, it appears that in contrast to GSIs, the ability of NRR antibodies to inhibit growth is likely to be limited to T-ALL lines bearing class I Notch1 mutations, as receptors harboring unusual juxtamembrane insertional mutations [35], [58] were completely resistant to the inhibition by NRR antibodies. In addition, even Notch1 receptors harboring class I mutations appear to be partially resistant to inhibition, particularly in T-ALL cells. In part, this may be due to the allosteric mechanism of inhibition by NRR antibodies which, as shown for wild-type Notch1 signaling, results in incomplete inhibition. An additional possibility is that aberrant trafficking of such receptors in T-ALL results in intracellular proteolysis and activation in vesicular compartments that are not accessible to antibody, but can be reached by membrane-permeable GSIs.
Similar to the results with human solid tumor cell lines expressing wild-type Notch1, the proliferation of T-ALL cell lines was minimally affected by NRR antibodies. However, T-ALL cell lines are significantly more sensitive to GSI-mediated inhibition of Notch1 activation. Together these data suggest that the therapeutic potential of NRR antibodies is higher in tumors that have intact extracellular Notch1 and depend on ligand for Notch1 activation; breast cancer is one such tumor. It is also possible that such anti-Notch antibodies may have value as inhibitors of stromal activities that support tumor cell growth, such as angiogenesis, which depends on a DLL4-Notch1 signaling axis [32], [69]. In addition to their therapeutic potential, these antibodies may find utility as biomarker tools, for detection of Notch1 on the surface of tumor cells, and as probes of Notch1 function and signaling mechanisms.
Calcium-dependence of epitope binding by anti-NRR antibodies. Biotinylated Notch1 NRR was captured onto neutravidin-coated 96-well plates. Binding of the NRR antibodies was allowed to proceed for one hour in Tris buffer (25 mM, pH 7.4), containing NaCl (150 mM), CaCl2 (5 mM), 0.05% Tween, and 0.5% BSA. The (−) column for each condition indicates the absence of EDTA, and the (+) column indicates the presence of EDTA (10 mM). Antibody binding was detected with a goat anti-human antibody conjugated to horseradish peroxidase using the fluorogenic substrate quantaBlu (Pierce-Thermo). The three control experiments were performed by omitting the Notch1 NRR antigen (no Notch1 NRR), the anti-Notch1 NRR (no 1-Ab), or the secondary anti-human antibody (no 2-Ab).
Antibodies WC75 (A) and WC613 (B) immunoprecipitate Notch1 but not Notch4. 293T cells were transfected with plasmids expressing the complete ectodomains of Notch1 or Notch4 containing His6-tags at their C-terminal ends. Immunoprecipitation was performed after lysis of the transfected cells. WCE: whole cell extracts; FT: supernatant remaining after immunoprecipitation; IP: WC75 (A) or WC613 (B) immunoprecipitate. Detection was performed with an anti-His6 antibody. His6-tagged molecular weight markers are loaded in the leftmost lane.
We thank Philip Bennett for statistic analyses.
Competing Interests: MAA, FW, NZ, AJB, JCA, and SCB are co-inventors of a patent application related to the antibodies reported in the study. The patent is entitled: "Generation and Characterization of anti-Notch Monoclonal Antibodies" (Application Serial No 61/199,753, filed on 11/20/2008). MAA, NZ, JEL, BAA, TJT, MG, LH, SV, KTV, PH, JZZ, ET, KTV, LLF, HW, FW, MC, AJB, LPA, and HEH are employees of Merck & Co., Inc. None of the authors from Merck & Co., Inc. received funding from any of the organizations (National Institutes of Health, Leukemia and Lymphoma Society, and the William Lawrence and Blanche Hughes Foundation) listed in the Financial Disclosure.
Funding: This work was supported in part by NIH (National Institutes of Health) grants P01 119070 and R01 CA092433 (JCA, SCB), a SCOR (Specialized Center of Research) award from the Leukemia and Lymphoma Society (JCA, SCB, KS), and the William Lawrence and Blanche Hughes Foundation (JCA, SCB, KS). WRG is a Leukemia and Lymphoma Society special fellow. The funding organizations had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Milton Friedman Quotes About TaxesQuotes about: Taxes
Whenever you try to do good with someone else's money, you are committed to using force. How can you do good with somebody else's money, unless you first take it away from them? The only way you can take it away from them is the threat of force: you have a policeman, tax collector, who comes and takes it from them.
I am in favor of cutting taxes under any circumstances and for any excuse, for any reason, whenever it's possible.
The reason I am is because I believe the big problem is not taxes, the big problem is spending. The question is, 'How do you hold down government spending? The only effective way I think to hold it down, is to hold down the amount of income the government has. The way to do that is to cut taxes
You could not possibly maintain the current level of government taxation without the taxes being hidden, and they are hidden in two very different ways. They are hidden through withholding, but they are also hidden by being imposed on business, supposedly on business, when really, of course, business can't pay taxes, only people can pay taxes.
What works for Sweden wouldn't work for France or Germany or Italy. In a small state, you can reach outside for many of your activities. In a homogeneous culture, they are willing to pay higher taxes in order to achieve commonly held goals. But "common goals" are much harder to come by in larger, more heterogeneous populations.
The long-range sloution to high unemployment is to increase the incentive for ordinary people to save, invest, work, and employ others. We make it costly for employers to employ people; we subsidize people not to go to work We have a system that increasingly taxes work and subsidizes nonwork.
Business corporations in general are not defenders of free enterprise. On the contrary, they are one of the
chief sources of danger....Every businessman is in favor of freedom for everybody else, but when it comes to himself that's a different question. We have to have that tariff to protect us against competition from abroad. We have to have that special provision in the tax code. We have to have that subsidy.
If the US government spends 40 percent of the nation's income, as it does through either borrowing or taxes, that income is not available for people to spend. The deficit is an indirect method of taxation. Of course, politicians prefer to borrow instead of tax because then someone down the road has to deal with the consequences.
Milton Friedman
- Born: July 31, 1912
- Died: November 16, 2006
- Occupation: Economist
- Cite this Page: Citation


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Kathy Lien - BK Asset Management
Last quote by Kathy Lien
Kathy Lien quotes
The euro shot higher because Draghi made it very clear that it is not a question of if, but a question of when they would start tapering asset purchases.feedback
If [President Donald] Trump responds with more than harsh words for North Korea, we could see dollar/yen drop as low as 108.feedback
The disappointment from (U.S. Federal Reserve Chair Janet) Yellen at Jackson Hole on Friday has carried over to trading this week.feedback
Due to the uncertainty in the global markets and recent sell-off in U.S. stocks, there's no better reason than the now for Yellen and Draghi to stick to script.feedback
Aug 17 2017
Investors are getting nervous because there seems to be no clarity in sight. We could see the U.S. dollar index continue to fall, possibly get below 93 to 92.50 or so. That reflects the business environment that we could be looking forward to in the coming months, because if businesses don't have confidence in the president, they're not going to necessarily have confidence in the economy going forward.feedback
Aug 17 2017
The disillusion of parts of Donald Trump's administration is causing big headaches; it's really affected the stock market, but also the U.S. dollar.feedback
The market does not truly believe the U.S.-North Korea showdown is over.feedback
The setback for Trump is a setback for the U.S. dollar. I think that really casts doubt on the Trump administration's broader strategies.feedback
That's why you have not seen much in the way of additional follow through in dollar demand.feedback
Investors have been skeptical of the Fed's hawkishness in the days leading up to Janet Yellen's testimony and when she failed to sufficiently emphasize the improvements in the economy, the dollar U-turned as the bulls abandoned their trades quickly.feedback
No Eurozone economic reports were released today so this was purely a function of anti-dollar flows. The email correspondence between Trump Jr. and a Kremlin-connected Russian lawyer makes investors nervous and they are turning to the euro, with its steady data improvements and hawkish central bank for safety.feedback
Jun 30 2017
The shifting monetary policy trajectories of other central banks is making other currencies more attractive relative to the U.S. dollar.feedback
May 01 2017
If they remain cautious, the currency pair could slip back down towards 0.7450.feedback
Apr 19 2017
The Beige Book is scheduled for release on Wednesday and while the U.S. economy in general continues to recover, if Fed districts report a slowdown, the dollar could extend its losses quickly.feedback
The U.S. dollar took its cue from yields, weaving in and out of negative territory. President Trump has been flexing his muscles, which makes investors nervous. The situation in North Korea is still uncertain but after Syria and ISIS, North Korea could be Trump's next target.feedback
Apr 07 2017
We didn't have a completely, unambiguously negative report. I think that brought some investors relief.feedback
The weakness in sterling was directly related to all the uncertainty and questions that we know have, after the U.K. began these proceedings. There's a lot more questions than answers, I do believe that we could see further sterling weakness.feedback
Feb 28 2017
Fed speak has been very consistent and it's only a matter of time before the dollar responds appropriately.feedback
Feb 27 2017
Investors were initially disappointed by the delay of the tax plan but looked past that by the end of the day.feedback
Feb 23 2017
We would not be surprised to see GBP/USD retrace to 1.25 but breakouts after long periods of consolidation tend to have continuation.feedback
Feb 21 2017
With no major U.S. economic reports on the calendar this week, the main question before us is whether Wednesday's FOMC minutes will give the dollar the kick it needs to propel higher. The fear is that Europe will become even more fractured in the coming years, as anti-EU, populist sentiment spreads across the region.feedback
Feb 15 2017
Investors latched onto these words even though she spent more of her Congressional testimony talking about solid consumer spending, and the pickup in business and consumer confidence.feedback
Feb 09 2017
It's been a broad-based dollar rally driven by the headlines that Trump plans to announce something phenomenal on taxes in the next few weeks, in his words. That was really the crux of the dollar rally shortly after his election and I think investors are getting really excited about that again.feedback
Jan 26 2017
The problem that the greenback is having right now is two fold - first Trump has been talking down the currency and second, his policies make foreign investors nervous. Until the market comes to terms with the risk/benefits of Trump policy, the dollar may have a tough time mimicking the one way moves in stocks and bonds.feedback
Jan 25 2017
Over the past few months we have seen strong correlations between the U.S. dollar, U.S. yields and U.S. stocks but today, as the Dow Jones Industrial Average climbed above 20,000 for the first time, the greenback barely budged.feedback
Jan 19 2017
Politics will overshadow economics.feedback
Jan 19 2017
With less than 24 hours to go before Donald Trump becomes the President of the United States, investors are dumping dollars ahead of the inauguration as they worry about the new President's trade and currency policies.feedback
Unless Donald Trump attacks the dollar again on Friday, we have seen the end to a month of losses in the greenback.feedback
The dollar is marking time before U.S. President-elect Donald Trump's press conference on Wednesday. Trump will most likely repeat everything that he previously vowed to achieve while giving little details on specific policy actions. The question then becomes how the markets will react - will they be disappointed by the lack of specificity or encouraged by his pledge to spend.feedback
Dec 28 2016
Until data starts to turn negative or the headlines suggest that (U.S. president-elect) Trump's stimulus program could fall short of expectations, the dips in the dollar will be shallow with the currency aiming for new highs. But at the first sign of bad news there could be massive correction in what is quickly becoming a crowded long dollar trade.feedback
Dec 27 2016
The prospect of Fed tightening next year is keeping bonds under pressure, yields up and the dollar bid, and obviously the Case-Shiller data is helping that.feedback
Dec 22 2016
What I'm immediately worried about is those earnings. We always see earnings miss as a result of a strong currency, regardless of how much hedging a lot of these companies are supposed to do, regardless of how many times this happens, and I think that that could really start to pull back stocks.feedback
Dec 22 2016
January always brings us significant volatility, and I think it really depends upon the presidency and how quickly they come out guns blazing with the stimulus package. I think in the start of the year there definitely could be some profit-taking.feedback
Dec 22 2016
We won't get a lot of that stimulus until the second half of the year at the earliest, and I think at the onset, it's really going to be more pain than gain. We still have six months to go before we get any real boost, in my opinion.feedback
Dec 16 2016
Emerging market countries have been hit the hardest by capital leaving in search of higher yields and return along with the growing cost of paying back dollar denominated debt.feedback
Dec 07 2016
The big question is whether there will be an ECB surprise that triggers the same 4-cent (400 basis point) move in euro/dollar that we saw in December 2015.feedback
Nov 30 2016
These improvements confirm that a rate hike is coming on December 14th. They also boost the chance of further tightening in 2017 but with Fed fund futures only pricing in a 30 percent chance of another hike by May, investors see a hike followed by a long pause from the Fed, which is the biggest problem for the dollar.feedback
Nov 22 2016
Don't be mistaken by the mixed performance of the U.S. dollar today because dollar bulls remain in control.feedback
Nov 21 2016
(Investors) unwound positions immediately after his victory and laid on fresh long dollar, long stock trades.feedback
Nov 10 2016
It would her last decision before President Trump can pressure the central bank and the move would show that she isn't buckling on future political pressure.feedback
Nov 10 2016
Instead of mourning, investors cheered a Trump victory as they hoped he will be positive for the economy. He ran on a campaign of aggressive spending and this is the first time in eight years that there's prospect of a sizable fiscal stimulus package.feedback
Nov 09 2016
Based on the strength of the U.S. dollar and the rally in U.S. stocks, investors are banking on a Clinton victory.feedback
Nov 04 2016
Taking a look at the leading indicators for non-farm payrolls, the service sector reported slower job growth, jobless claims rose to a three-month high, driving the four-week average up to 257,000. ADP reported a smaller increase in corporate payroll growth and confidence is down all around.feedback
Oct 26 2016
These reports are not expected to have a dramatic impact on the dollar but with USD/JPY eyeing 105, stronger reports could give the pair the push that it needs to make a run for this key level.feedback
Oct 18 2016
Hotter inflation does not equal a hotter dollar.feedback
Oct 12 2016
We're still looking at the dollar remaining strong.feedback
Oct 07 2016
It's a low liquidity sell-off. Typically when we see this, the reversal is violent but with fundamental support, the pound could find a new range between 1.22 and 1.25 per dollar.feedback
Oct 05 2016
The U.S. dollar should continue to outperform but after six straight days of gains, traders should beware of a correction in USD/JPY ahead of Friday's non-farm payrolls report.feedback
With profits being squeezed the battle for market share can't go on and this deal ushers in a new period of cooperation between OPEC nations and specifically between Saudi Arabia and Iran. While we wouldn't be surprised by some back-pedalling between now and November, this is a historic moment and one that should have a lasting impact on the Canadian dollar.feedback
Sep 27 2016
So I do fear that we could see a deeper correction in stocks.feedback
While investors are biding time ahead of Wednesday's Federal Reserve announcement, they are also weary of holding dollars given the unlikely chance of a rate hike this week.feedback
Sep 16 2016
These disappointing reports should have driven the U.S. dollar sharply lower, but instead the greenback ended the day higher versus the British pound and unchanged against the euro and the Japanese yen.feedback
Sep 09 2016
The main takeaway from all of these comments is that despite slower job growth and weaker manufacturing and service sector activity, U.S. policymakers still believe rates should rise and this consistent message has not been lost on investors.feedback
Sep 02 2016
Investors took profits on their long dollar positions ahead of Friday's nonfarm payrolls report.feedback
Aug 31 2016
While the latest string of Japanese data has been decent with the jobless rate improving and retail sales rising strongly in July, Japanese officials are clearly still frustrated with the weak growth in the economy.feedback
Aug 18 2016
When we did not receive the unambiguous hawkishness from the FOMC minutes, that kind of opened up the door to additional (dollar) selling.feedback
Aug 17 2016
Dollar traders were not impressed. Given the recent weakness of U.S. data and the strong downtrend in dollar/yen ... dollar traders were looking for unquestionable hawkishness. They received no additional clarity on whether rates will rise by December and were simply told that the Fed is still in wait-and-see mode.feedback
Aug 16 2016
Investors dumped U.S. dollars today, pushing the greenback lower against all of the major currencies. Fed Presidents [William] Dudley and [Dennis] Lockhart warned that the market is underpricing tightening and rates could rise this year ... yet based on the price action of the dollar, investors are not buying what [they] are saying. The market was looking instead at soft U.S.feedback
Aug 10 2016
The strong housing market is one of the main factors preventing a more aggressive move from the RBNZ.feedback
Aug 10 2016
If the RBNZ wants to be proactive, they could send a strong message to the market with a 50 basis points (bps) cut, but we believe they will go for a 25bps move to avoid invigorating the housing market, with a warning of more [easing] to come.feedback
Aug 03 2016
The U.S. dollar traded higher against most of the major currencies ... dollar/yen appears to be finding a bottom above 101.feedback
Aug 02 2016
The majority of economists surveyed expect the RBA to cut interest rates by 25 basis points but we feel that a rate cut is not a done deal.feedback
Jul 21 2016
The weakness of the euro provides automatic stimulus to the economy, which means the ECB can afford to wait. So the potential for an initial short squeeze is high if the central bank stands pat and the outlook thereafter will depend on how strong of a message the ECB sends.feedback
Jul 20 2016
The sell-off in the euro ahead of monetary policy meeting tells us that some traders are hoping for immediate action in the form of support for Italian banks or an extension of the end date for bond purchases.feedback
Jul 15 2016
Sterling's reaction today to BOE is a classic example of misplaced expectations. Economists were calling for a cut, investors believed them and when the BoE kept policy unchanged, sterling jumped from 1.3250 to 1.3475. The rally faded as the day progressed because the main takeaway from BoE is simple - rates will be lowered in three weeks and [Gertjan] Vlieghe [a member of the BoE's monetary policy committee] wanted the move to happen today.feedback
Jul 06 2016
This is just the beginning.feedback
Jul 06 2016
We saw investors flock into the safety of U.S. dollars as the liquidation spread to stocks, commodities, the euro, Australian and Canadian dollars.feedback
Jul 01 2016
Carney has always been nervous about how Brexit would impact the U.K. economy and now that it is a reality, he is preparing for the worst, agrees with the doom and gloom scenario and wants to assure the market that the BoE will be proactive.feedback
Jun 30 2016
Sterling dropped like a hard rock after Bank of England (BOE) Governor Carney said another rate cut is coming this summer. Carney also warned that the BOE can react more rapidly than other institutions and the central bank will have its first full projections in August - which means we'll be looking for a move around that time.feedback
Jun 28 2016
It was your typical turnaround Tuesday in the financial markets. Currencies and equities traded higher across the board at the start of the North American trading session, but as the day progressed, the move lost momentum when investors realized that the fundamental story hasn't changed.feedback
Jun 28 2016
The odds may be against them but investors are hoping that the worse is over for currencies and equities and the gaps on Friday will be filled.feedback
Jun 28 2016
In the near term, risk aversion and market uncertainty makes the euro less attractive to investors. In the long run, Brexit also raises questions about the Eurozone' s viability because if major countries like Britain start dropping out the EU, nationalism could drive smaller Eurozone nations to exit out of the euro. Expects the euro to "make another run" for the $1.0900 level.feedback
Britain needs to invoke Article 50 to redefine its relationship with the EU but leaders of the leave campaign refuse to act quickly. The longer they wait, the worse it will be for sterling.feedback
Jun 24 2016
I don't think the sterling has completely priced in the damage that this will have on the economy, in the short term.feedback
Central banks around the world, especially the Japanese, are sitting on their hands waiting to see how global developments over the next week or two are going to unfold.feedback
Jun 09 2016
The smaller-than-expected increase in U.S. jobless claims helped [the turnaround in the dollar], but it has been a while since this report had any meaningful impact on the greenback. So the question now is whether today's reversals will turn into a ... bottom for the dollar.feedback
May 23 2016
The shift in market expectations for Fed tightening and the confirmation from Fed presidents is a significant development that has shifted the market's appetite for dollars. The dollar lost some of its momentum towards at the end of the week and with a relatively light U.S. calendar next week devoid of major market moving economic releases, we could see choppy trading.feedback
May 20 2016
If one of the most dovish members of the central bank thinks that a rate hike is imminent, then perhaps investors really need to re-think and re-price their expectations for tightening this year.feedback
May 16 2016
The strongest increase in retail sales in more than a year drove the U.S. dollar sharply higher against all of the major currencies, but the gains did not last.feedback
May 16 2016
Growth is expected to turn positive after a negative quarter and given the market's focus on the timing of BOJ easing, stronger or weaker growth could have an unusually significant impact on the yen.feedback
May 10 2016
Short covering is the primary reason for the strong gains in dollar/yen. With everyone from the Bank of Japan governor to the finance minister and prime minister of Japan threatening to intervene if foreign exchange moves become too rapid, speculators are finding fewer reason to be remain short dollar/yen.feedback
May 10 2016
Don't underestimate the power of short covering.feedback
Apr 28 2016
Global troubles no longer worry the Fed, putting them one step closer to raising interest rates.feedback
Apr 22 2016
This reversal caught many investors by surprise because the main takeaway from today's meeting is the ECB has no immediate plans to add stimulus nor did they feel that the currency was high enough to renew concerns about its impact on the economy. The ECB is still dovish, they see euro area outlook risks tilted to the downside and expect rates to remain at present or lower levels for an extended period of time.feedback
Investors were relieved that oil did not fall 10 percent on the back of the Doha meeting and they were quick to reward risk currencies like the Australian and New Zealand dollars with gains.feedback
Mar 18 2016
At 113, the BoJ has leeway to wait but at 110-111 with the risk of further losses, they may not be able to forestall easing for much longer.feedback
Mar 18 2016
If USD/JPY drifts lower again, we expect more aggressive action from the Bank of Japan.feedback
Mar 18 2016
In a matter of minutes right around lunchtime in the U.K., USD/JPY jumped nearly 100 pips from its low of 110.67. This is the third time in two months that the BOJ stepped in to buy USD/JPY below 111 as they clearly don't want to see the currency pair trading on the 110 handle.feedback
Mar 01 2016
The general effects of the RRR reduction are questionable but China's government would not have taken this move if they were not concerned about the economy. Chinese data is notoriously difficult to handicap but this gives us reasons to believe that manufacturing and service sector activity slowed in the month of February.feedback
Feb 18 2016
While there has been some confusion as to whether 'support' equals action, oil traders are simply relieved that the world's fourth largest holder of oil reserves is willing to cooperate.feedback
The main reason why investors are selling dollars is because they no longer believe that the Federal Reserve will raise interest rates in 2016, What's interesting is that (Janet) Yellen did not say anything in her testimony over the past two days to suggest that would be the case. Nonetheless, Fed fund futures are no longer pricing in a rate hike this year, validating the decline in the dollar.feedback
Feb 10 2016
By the end of her testimony, sentiment completely shifted.feedback
Feb 10 2016
For the most part, dovish undertones of (Yellen's) testimony were enough to reinforce what the market had been thinking all along, which is that the Fed is probably not going to go through with a rate increase next month.feedback
Many economists believed he would avoid making specific comments about more policy action but as we pointed out, the 3-cent rise in the euro and decline in oil prices since December encourages the ECB head to be characteristically dovish.feedback
Jan 05 2016
It is important to remember that if Chinese stocks continue to fall, the Chinese government will not hesitate to step in to turn the tide around. There's no question that the People's Bank of China (PBOC) will increase stimulus this year especially given recent developments.feedback
Jan 05 2016
It is too soon to tell whether the selloff will turn into a deeper correction because on one hand, part of the breakdown can be blamed on panic selling after circuit breakers kicked in and on the other China is experiencing a real slowdown.feedback
Sep 14 2012
The retail sales and consumer price reports give the Federal Reserve very little reason to doubt their decision to increase monetary stimulus.feedback


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The Shrine Bowl of the Carolinas is already busy putting together the Game Day Program for the 79th Shrine Bowl of the Carolinas. The game will be played on Saturday, December 19th, at Gibbs Stadium in Spartanburg, South Carolina.
The Shrine Bowl of the Carolinas is America's original High School Football All-Star Game and has been played every year since 1937. Advertising in the Game Day Program is a great way to show your support for High School Football in your state and help support the children who are patients in the Shriners Hospitals.
To create your ad for the game day program, click here to go directly to our ad page with Josten's to make your own player recognition ad or business support ad.
Contact firstname.lastname@example.com for more information.
Stay tuned and check back as we start the 2015 game day program ad promotions. Your ad will be completed and submitted online to the publisher.


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Getting closer to the limit of my seven day homemade bread recipe post challenge.
I made something quite unique for day 6th. The taste and texture of this specific bread may remind of pizza crust ,focaccia or something you may have tested in Italian restaurants.I used Kalamata olives, something I had on hand at the moment, you can use any spicy or salty olives.I also used some sliced cherry tomatoes to give the bread a nice design.That's an optional thing for my homemade bread recipe ,you can skip it . Let's just get to the recipe and hope you'll take time and try another one of my
homemade bread recipe experiment results 🙂
Happy baking!
1 Cup warm water
2 tbs olive oil
⅔ Cup whole wheat flour
1 ½ Cup all purpose flour
2 tsp instant dry yeast (if using active dry, activate in warm water before adding)
¼ Cup semolina
2 tsp sea salt
1 tbs brown sugar,firmly packed
½ Cup sliced olives
1-2 tbs cornmeal for dusting the baking pan (or use semolina to dust)
Combine all ingredients in a mixing bowl and mix until smooth and sticky dough forms
Transfer onto lightly floured clean working surface and knead a few more minutes
The dough will be sticky but don't be tempted to add too much flour
Use a bench scraper or spatula to help you knead and bring the dough together
Drizzle a large bowl with olive oil and place the dough in it
Cover with plastic wrap or damp towel
Let it rest in a warm place until doubled in size (1-1½ hours,depending on temperature )
Turn it onto working surface and knead gently using a bench scraper or flat spatula if needed
Again: try not to add too much flour
Return back to greased bowl for second rise (50-60 minutes)
After second rise place the dough on a working surface
Knead for another 2-3 minutes
Using olive oil and cornmeal generously grease and dust baking pan (I used 9"x6" loaf pan)
Place the dough in the prepared baking pan
Let it rest and rise another 30-50 minutes lightly covered with a towel or plastic wrap
At this point you can preheat the oven to 475° F/250° C degree
Bake 25-30 minutes or until the top has bright golden color
Allow the bread to cool down at least 30 minutes before slicing


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Pröwess have fearlessly accepted the challenge of keeping classic rock 'n' roll alive, and with their debut EP release Headfirst, they've proven they've got what it takes. The five-man lineup boasting dynamic vocals from Dalton Bowes and the driven guitars from Scott Roby and Kip Wilson strike a chord that'll teleport listeners back to the heart of 80's rock 'n' roll. Pröwess stems from another generation and their in-you-face rock 'n' roll is a breath of fresh air in genre that's evolved over the years.
Headfirst opens with "No Survivors," the band's first single and most representative track. Stemming from Pröwess' "experiences on the road," the leadoff anthem offers a slow build up that creates a great deal of anticipation for the introduction of Bowes' vocals.
The vocalist's shrieking tone is reminiscent of Joe Elliot and Def Leppard – an artistry that impeccably meshes with the band's style. "Show Me" and "Killing a Giant" follow and showcase Pröwess' diversity – the latter of which unveils an upbeat, pleasing melody with an undeniably catchy chorus.
Throughout the record, Pröwess stays true to their roots, incorporating extended guitar solos, synonymous with classic rock 'n' roll. There's elegance amidst the heavy tones. Whether it's the squealing or just the quintessential shredding from the axe wielding shredders, Pröwess never reaches for something they're not. The quintet excels from working within their wheelhouse and ultimately creates a powerful five-track record with high replay value.
The most successful bands are those that leave their listeners wanting more. Pröwess adopts this approach with Headfirst and teases its listeners with five songs that'll leave any classic rock fan wanting more. While Headfirst is top-notch, Pröwess leaves the impression that they've only scratched the surface on their rock 'n' roll journey and that there's plenty more to come from these five courageous rock warriors.


text: I'm wondering, MMS app seems to convert hire pics to somewhere around 100KB. Is there a way to send picture messaging with losing quality? Like if I send 2 MB pic, the recipient get 2 MB pic as well. Instead, No matter what size you send, they get a pixelated picture. Any ideas?


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New Stanton, Pennsylvania (PressExposure) October 17, 2008 -- Find it cumbersome to go out to the store and buy a baby stroller with your newborn in tow? You donât need to worry anymore. You can get your stroller delivered home. Stroller.com offers a free shipping facility for all orders above $50. Most orders are usually shipped by the end of the next business day. The company ships orders to all US States, US Territories, and to Canada only. You can check out the various products on the companyâs website and then place the order and make your payment online. With varied types of strollers on offer from some of the best baby stroller manufacturers, you have a wide choice. The strollers include standard strollers, jogging strollers, umbrella strollers, lightweight strollers, and other versatile strollers. For both infants and toddlers, you could choose either single or double strollers.
A part of the OnlineStores Group Inc, stroller.com has always laid emphasis on customer service. Following the credo of âyou are a person and not a number when you callâ, the company offers an efficient telephone response service. Useful information and specialist product knowledge can be had online on the buying guides and stroller safety tips. With stroller reviews and other stroller information on the website, the customer can make an informed decision about the purchase.
Stroller.com stocks strollers from top brands such as Jeep, Schwinn, Joovy, Kolcraft, BabyPlanet, Zooper, Englacha, InStep, Baby Jogger, and Protect-a-Bub, among many others. With years of experience in the manufacture of strollers, these firms are known to release into the market top quality strollers that combine good looks with proper maneuverability and adequate safety features such as a five-point harness and efficient brakes. Stroller accessories can also be acquired online such as rain covers, sun protection fabrics, stroller organizers, and stroller travel systems.
About stroller.com: www.stroller.com is an online baby stroller outlet that offers a wide array of strollers to suit all tastes. It is part of the Online Stores, Inc group.
About Online Stores, Inc: A Pittsburgh, PA based corporation launched in 2002, this organization includes six online retail stores. The Pittsburgh Business Times has rated this organization in the âtop 500 retailerâ listing.


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Hunger Games Signed Movie Script X21 Jennifer Lawrence Hemsworth Josh Hutcherson
Sold for:
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- Sold Date:
- Source: eBay
This auction is for a color copy of the entire movie Hunger Games s igned script !! This script has the signatures of these 21 stars:Stanley Tucci Wes Bentley Jennifer Lawrence Willow Shields Liam Hemsworth Elizabeth Banks Paula Malcomson Gary Ross Josh Hutcherson Woody Harrelson Toby Jones Lenny Kravitz Amandla Stenberg Dayo Okeniyi Leven Rambia Jack Quaid Donald Sutherland Alexander Ludwig Isabelle Fuhrman Jacqueline Emerson Amber Chaney Script comes professionally bound with industry standard 3 hole punch and brass fasteners. This awesome brand new first draft movie script has 127 pages and is filled with all of the memorable lines from this blockbuster film. This script is dated August 7, 2010. Be one of the few to own such a epic script that will certainly go down in history. Check out my other auction for scripts and other great stuff. I offer combined shipping on multiple items. No rights are given or implied since this is sold from collector to collector. Please note the autographs on the cover are reprints. Thanks for looking and have a great day. Scripts make wonderful gifts:) In a dystopian future, the totalitarian ... read more


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Debater of the Week: Glasscandie.
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November 12th, 2010, 05:23 PM
Hey... Where's Perry?
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text: Im new here so i don't know if this is the right section or not. Sorry if its not. But anyways i've had my droid since tuesday and today my camera button quit working. It worked fine yesterday but for some reason it just crapped out on me. It doest even go to the camera page when you hold it and i've tried all the settings Anyone know what's up? I thought maybe just a loose conection but when i took the battery cover of it had torq he's screws so i can't take it apart to see Thanks.


text:
- Description
DaVinci Resolve 12 Studio Software for Education 25-49 Licences (each)
Blackmagic Designe DaVinci Resolve 12 Studio is a video software that is the world's most advanced editing and color grading system for independent users working on SD, HD and Ultra HD projects. The studio version of this software has many key features that the free version doesn't have like the noise reduction plugin and the 3D stereoscopic grading.
DaVinci Resolve 12 studio redefines post production workflows by allowing assistant editors to prepare footage while editors cut the picture, colorists grade the shots, and sound editors mix and finish audio, all in the same project at the same time. You no longer have to waste time conforming and you no longer have to wait for a locked edit before starting color and audio work! That means picture editors, colorists, and sound editors can now work in parallel, leaving more time for everyone to be creative.
DaVinci Resolve 12 studio makes importing, syncing and organising footage fast. Whether you're going out to the web, tape, or theatrical distribution, DaVinci Resolve features everything you need to deliver projects in virtually any format! You'll be able to get to work quickly and output files fast, so you'll never miss a deadline.
This price if for a order between a minimum of 25 and a maximum of 49 copies of this software. If you want more of less of the software see our other options.
- Slick New Interface that's Cleaner, Easier on the Eyes
- Multicam Editing with a Host of Sync Options
- Improved Trimming including Advanced Simultaneous Multi-Track Trimming
- Nesting Timelines for Working on Large Projects with Multiple Complex Timelines
- On-Screen Controls for Manipulating and Animating Motion paths of Graphics
- Highly Customizable Transitions Using Curves Editor with Bezier Handles
- Real Time Audio Mixing by Recording Fader Moves
- VST and Audio Unit Plugins for Full Control of Audio
- Export to Avid Pro Tools for Professional Audio Mix
- Improved Media Management and Bins
- Enhanced 3D Tracker
- Brand New 3D Keyer for Color Correction and Compositing
- Additional Information
Additional Information
Manufacturer Blackmagic Design SKU SBLMDVRESSTUD2549 Model BMD-DV/RESSTUD/25-49 GTIN NA
Blackmagic Design DV/STUFUS/25-49 - F...£612.00 (inc. vat) £510.00 (ex. vat)
Blackmagic Design DV/STUFUS - Fusion ...£942.00 (inc. vat) £785.00 (ex. vat)
Blackmagic Design DV/RESSTUD/5-24 - D...£706.80 (inc. vat) £589.00 (ex. vat)
Blackmagic Design DV/RESSTUD/50+ - Da...£471.60 (inc. vat) £393.00 (ex. vat)


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Picture: Soulja Boy arrives to perform at the Kick-off of the Macy's 'mstyle lab' concert tour at Macy's in Ft Lauderdale, Florida Ft Lauderdale, Florida...
U.S. officials have threatened to ban Soulja Boy's upcoming album from military base stores if it includes his anti-army song Let's Be Real.
Bosses at the Army and Air Force Exchange Service (Aafes), which oversees merchandise sold on military bases, will not stock the rapper's Respect My Hustle on their shelves if the controversial track makes the final cut.
The rapper came under fire earlier this week (beg05Sep11) after releasing a promo for Let's Be Real, which contains the line, "F**k the Fbi and the army troops... fighting for what? Be your own man..."
Soulja Boy has since apologised, but Ltc Thomas Shrader from the Aafes tells Tmz.com the lyrics "are unacceptable and not the kind of thing we would want to market".
Aafes bosses will re-evaluate the boycott if Soulja Boy decides not to feature the song on his album.


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Helen Langehanenberg works with an enthusiastic young mare showing the benefit of a great warm up. The total running time of this two part video is 22:00.
Video Description
Helen rides a six year old going M level, after having walked for 15 minutes. She warms up by allowing the tendons and joints to warm up and get ready for work while searching for the contact. She makes sure she keeps her balance off the forehand and doesn't roll her neck in the canter. The thorough warm up leads to relaxation in the mare to prepare her for the work.


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Clearly defining your service quality standards enables you to demonstrate your commitment to your customers. However, your customers expect more than just promises. They expect independent, validated proof that your service pledges are being consistently delivered. That is why you need third-party service certification from a trusted provider to confirm that your service quality standards have been verified, measured and controlled.
As a leading provider of certification, inspection, verification and testing in France, we can help you to make a transparent commitment to service delivery with our independent Qualicert certification.
We offer you unrivaled expertise and proven experience, developing over 130 validated services. Plus, we provide you with expertise that stretches across all economic sectors, including logistics, education, hospitality, real estate, automotive and franchise networks.
We can help you:
To find out how our Qualicert service certification can help you make a commitment towards service excellence, contact us today.
published 06/27/14
published 04/19/17
published 09/18/17
published 07/01/16
published 06/04/15
published 07/16/15
published 12/05/16


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Conor C.
Canyon, TX, 79015
Joined on September, 2013
Gender: Male
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EducationSenior Mechanical Engineering Major at West Texas A&M University
ExperienceI have taken College Algebra, Calculus I & II, Differential Equations, Calculus Physics I & II.
I would like to be a tutor because I can usually help people by explaining in ways that are easier to comprehend than what the teacher may teach. I am very patient and enjoy working with people. I have always been good at math and science and enjoy solving problems and understanding how and why things work the way they do.
HobbiesI am pretty laid back and easy going, but I get the job done when it's time to work. School is my top priority in life, but I also like to have fun. I enjoy video games, hunting, fishing, anything outdoors. I am very good with electronics and computers and such.
Preferred Subjects
Algebra, Geometry, Trigonometry, Calculus, Physics, , Differential Equations, Statics, Dynamics, ANSYS, MATLAB
Inquire about this tutor
The tutor will respond if they are available/interested.


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NICOSIA, June 7 (Reuters) - Cyprus's Supreme Court said on Friday it could not rule on lawsuits against the central bank seizing control of two of the largest commercial banks, which triggered big losses in depositors' savings as part of an international rescue deal.
Complaints had been brought by about 50 customers of the two banks who lost money when the banks were seized.
In a decision taken by majority, the court ruled it did not have jurisdiction to handle recourses over the resolution of Popular Bank, which is being wound down, or over Bank of Cyprus, where large depositors have been forced to forfeit savings for equity stakes in the bank.
It ruled that lawsuits against the seizure of the banks came under the remit of private civil cases in other courts.
The Supreme Court, the most senior legal authority on the island which handles recourses against state institutions, was called to address whether it was competent to rule on the case, not to decide on the merits of the complaints.
Thousands of depositors lost millions when the central bank took control of the two commercial banks in March, forcing losses on uninsured deposits exceeding 100,000 euros. The losses were conditional on Cyprus receiving 10 billion euros in aid from the International Monetary Fund and the EU.
One Nicosia lawyer said he did not consider the Supreme Court case would have any effect for the hundreds of civil cases pending against the raiding of big depositors' bank accounts to plug bank recapitalisation shortfalls.
"Having read the judgement I can say with respect it is not the best that legal science could produce. In all probability the principles it invokes will be confined to its own facts rather than become a precedent," said Costas Velaris, a barrister who is assisting affected depositors.
Customers in Popular, also known as Laiki, have lost deposits exceeding 100,000 euros, while Bank of Cyprus clients are forced to see 37.5 percent of cash over the insurable threshold converted to equity, with the possibility that an additional 22.5 percent of their savings could also be converted. (Reporting By Michele Kambas; Editing by Pravin Char)


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Sugar, we all know, is a silent killer. There is hardly any health benefit that sugar offers to us, apart from giving a whole lot of energy.
Yet, sugar is such an integral part of our daily intake that it is almost impossible to overlook it either. We have made it a rule to add sugar in whatever food we consume.
So, the only way forward is to find alternatives to sugar that cause our health little harm in terms of diabetes, obesity and other severe ailments.
But hunting for alternatives to sugar is not an easy task either. Several sugar substitutes are actually synthetics and not better than the thing we are looking to avoid. And some of the substitutes are even linked to cancer.
Reducing the intake of sugar also leaves a positive impact on the environment, as growing the water-intensive sugarcane from which sugar is obtained causes a big loss to the biodiversity than any other crop.
In this article, we've discussed about some of the natural substitutes for sugar that are not harmful as the synthetics:
1. Honey:
A great substitute for sugar. Pick raw honey over processed ones to make full use of the original nutrition. They are also antibacterial. The greater sweet flavour of honey makes you use less of it, which means it harms you less despite having more calories than sugar.
2. Fruit Juice:
This is a common substitute for sugar. But, it is very important to drink juice which doesn't have added sugar and is original in form.
3. Molasses:
Although sugar is made from the same sugarcane, which also leaves the molasses as a byproduct, we tend to ignore the more nutritional molasses and go for the less-healthy sugar crystals. Molasses is not only sweeter than sugar but it is also a good source of iron and calcium, making it a perfect substitute for sugar.
4. Coconut Sugar:
Coconut sugar is gaining popularity because it has low glycaemic index. It can be easily used as a substitute for ordinary sugar. It is made after the sap from the coconut palm is heated to evaporate the water content and turn it into usable granules.
5. Xylitol:
Found in chewing gum, xylitol is a five-carbon sugar, which means it prevents the growth of bacteria. It is produced by our body, besides being found in fibrous fruits and vegetables, corn cobs, etc.
6. Date Sugar:
This is produced from dried dates by dehydrating them. The nutritional benefits of dates and the sweet flavour makes the date sugar an ideal alternative to brown sugar. Date sugars are unsuitable to be used in drinks and baked items though.
7. Brown-rice Syrup:
Made from boiling brown rice, this gluten syrup has many of the benefits of the healthy brown rice and can be used as a substitute for sugar in pancakes and salads.
8. Stevia:
Three hundred times more sweet than sugar, stevia is a herb found in South America; and is used as a dietary supplement to sugar. It has no calories and neither does it leave any glycaemic impact, which makes it suitable for diabetic patients and those who are trying to keep their weight under check.