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CNS (central nervous system) adrenaline (epinephrine) is implicated in a wide range of physiological and pathological conditions. PNMT (phenylethanolamine N-methyltransferase) catalyses the final step in the biosynthesis of adrenaline, the conversion of noradrenaline (norepinephrine) to adrenaline by methylation. To help elucidate the role of CNS adrenaline, and to develop potential drug leads, potent, selective and CNS-active inhibitors are required. The fragment screening approach has advantages over other lead discovery methods including high hit rates, more efficient hits and the ability to sample chemical diversity more easily. In the present study we applied fragment-based screening approaches to the enzyme PNMT. We used crystallography as the primary screen and identified 12 hits from a small commercial library of 384 drug-like fragments. The hits include nine chemicals with two fused rings and three single-ring chemical systems. Eight of the hits come from three chemical classes: benzimidazoles (a known class of PNMT inhibitor), purines and quinolines. Nine of the hits have measurable binding affinities (~5–700 μM) as determined by isothermal titration calorimetry and all nine have ligand efficiencies of 0.39 kcal/mol per heavy atom or better (1 kcal≈4.184 kJ). We synthesized five elaborated benzimidazole compounds and characterized their binding to PNMT, showing for the first time how this class of inhibitors interact with the noradrenaline-binding site. Finally, we performed a pilot study with PNMT for fragment-based screening by MS showing that this approach could be used as a fast and efficient first-pass screening method prior to characterization of binding mode and affinity of hits.
- benzimidazole
- catecholamine
- drug discovery
- enzyme inhibition
- fragment-based screening
- phenylethanolamine N-methyltransferase (PNMT)
The identification of suitable starting compounds for development into drugs is a major challenge for drug discovery and development programmes. FBS (fragment-based screening) has been developed as a method for the rapid discovery and subsequent elaboration of hits into quality lead compounds [1,2]. This approach is based on the principle of screening simple low-molecular-mass compounds that make a few high-quality interactions with the target site. Resulting hits tend to bind to the targets efficiently yet with low affinity and, with a practically designed library, are readily tractable for follow up chemistry [3]. These fragment hits are used as 'anchors' that can be elaborated to fill additional regions of the target site. This results in potent compounds with minimal chemical complexity that can then be developed into drugs. A major challenge in establishing FBS has been the development of sensitive methods to detect the weak binding of hits (with typically milli- to micro-molar binding affinities) [4]. Methods currently used include ITC (isothermal titration calorimetry), NMR, surface plasmon resonance, MS and X-ray crystallography [1,5–7].
FBS-X (FBS by X-ray crystallography) provides the greatest level of information for the elaboration of hit compounds [8]. However, costly infrastructure for crystallization, crystal handling and data collection is required for screening hundreds of crystals in a high-throughput manner. Although many different FBS methods are now used, there have been few reports comparing hits identified against the same specific target by orthogonal methods. NMR is frequently employed as a first-pass screen before detailed structural characterization of hits by X-ray crystallography, and MS has the potential to be applied in a similar manner [9]. Comparative FBS-X and FBS-MS campaigns have not been reported, as far as we are aware, yet there are potentially significant advantages to a combined MS/X-ray screening approach due to their complementary attributes.
Pharmaceutical companies have developed and applied FBS-X with success [10]. An ideal target for FBS-X has an accessible active site, can be crystallized in the apo form and requires only minor conformational changes in the crystal form to accommodate ligand binding. However, many drug targets do not conform to these requirements, including the human enzyme PNMT (phenylethanolamine N-methyltransferase; EC 188.8.131.52; 30.7 kDa), which catalyses the final step in adrenaline (epinephrine) biosynthesis, the methylation of noradrenaline (norepinephrine) to form adrenaline (Figure 1A). PNMT has a potential role in a wide range of disease-relevant processes including the central control of blood pressure [11], pituitary hormone secretion [12], ethanol intoxication [13], Parkinson's disease [14] and the neurodegeneration observed in Alzheimer's disease [15]. Structures of recombinant hPNMT (human PNMT) in complex with inhibitors and substrates (e.g. PDB code 1HNN) show that hPNMT has an enclosed active site (Figure 1B) suggesting that enzyme conformational changes may be needed for ligands to bind [16]. Apo crystals are not readily available because we were unable to crystallize the enzyme in the absence of AdoHcy (S-adenosyl-L-homocysteine), which binds to the cofactor-binding site. Furthermore, when crystals are grown in the absence of an inhibitor or substrate at the noradrenaline-binding site, this site is occupied by a phosphate molecule [17]. However, we were encouraged to attempt FBS-X because we solved the crystal structure of hPNMT (PDB code 3HCD) in complex with the physiological substrate R-noradrenaline by using rapid soaking methods on hPNMT–AdoHcy–PO4 crystals [17]. We therefore proposed to apply this same soaking method for FBS-X screening of hPNMT, with the aim of identifying novel chemical frameworks for inhibitor design. We also performed an FBS-MS pilot study to determine whether this approach could be used as a more efficient first-pass screen prior to crystallographic analysis of selected hits.
Overall, we showed that FBS-X can be successfully implemented within an academic environment. Our crystallographic screening of the non-ideal enzyme target hPNMT identified 12 hits, including three benzimidazoles (a known class of PNMT inhibitors), and nine of the hits could be characterized by ITC. Five compounds were synthesized to further characterize the benzimidazole class. Our pilot study showed that the orthogonal screening method of FBS-MS could be used as an even more efficient first step in an FBS campaign.
The FBS library was purchased from ActiveSight (a Rigaku company), and comprised 384 compounds with a mean molecular mass of 142 Da and of predominantly rigid low-complexity (see the Supplementary Table Library at http://www.BiochemJ.org/bj/431/bj4310051add.htm); all compounds were provided as 200 mM stock solutions in 100% DMSO and as cocktails of four fragments each at 50 mM in 100% DMSO. All the compounds are also available for purchase directly from Sigma–Aldrich. For chemical syntheses, all reagents and solvents used were reagent grade and of the highest purity commercially available. 4-Methoxybenzene-1,2-diamine was sourced from Alfa Aesar, all other compounds and reagents were sourced from Sigma–Aldrich.
Crystallization of hPNMT
C-terminally His6-tagged hPNMT was expressed and purified as described previously [18]. The protein was concentrated to 50 mg/ml and mixed with AdoHcy (to a final concentration of 2 mM) taking the final concentration of protein to 40 mg/ml or 1.25 mM. The protein mixture was crystallized by hanging drop vapour diffusion using QIAGEN EasyXtal Tool crystallization trays for ease of soaking. Drops comprised 1.5 μl of protein and 1.5 μl of precipitant equilibrated over 400 μl of precipitant solution {4–8% PEG [poly(ethylene glycol)] 6000, 0.25 M LiCl and 0.1 M sodium cacodylate, pH 5.5–6.0}.
FBS by X-ray crystallography
Library screening was conducted blind, in that we made no assumptions about the contents of the library, or what compounds might be expected to bind PNMT. Prior to screening, control experiments were performed using known hPNMT inhibitors to confirm that ligands could be soaked into hPNMT–AdoHcy–PO4 crystals and that the solvents used for fragment screening and cryoprotection did not interfere with binding. Crystals (~0.25 mm in each dimension) were transferred into 1 μl of soaking solution. Crystal soak solutions were prepared by adding 1 μl of fragment cocktail to 9 μl of stabilizing solution, taking the final concentrations to 6.6% PEG 6000, 0.3 M LiCl, 0.1 M sodium cacodylate, pH 5.7, 4× 5 mM fragments and 10% (v/v) DMSO. Crystals were soaked for 15 min.
Crystals were cryoprotected by transferring directly from the soak solution into mother liquor supplemented with 25% ethylene glycol for 10–15 s before being flash-cooled. Data for hPNMT–AdoHcy–PO4 were measured from a crystal transferred directly into the cryoprotectant solution without undergoing a soak step. X-ray diffraction data from hPNMT crystals were measured using the UQ ROCX Diffraction Facility or the Australian Synchrotron. At the UQ ROCX Diffraction Facility, crystals were irradiated with X-rays of wavelength 1.542 Å (1 Å=0.1 nm) generated from a Rigaku FR-E Superbright copper rotating anode generator operating at 45 kV, 45 mA and data were measured using one of two alternate methods: (i) crystals were flash-cooled by plunging into liquid nitrogen and placed on the camera using a Rigaku ACTOR™ crystal mounting robot, X-rays were focused with Osmic Vari-Max HF optics and diffraction data measured on a Rigaku Saturn 944 CCD (charge-coupled-device) detector; or (ii) crystals were flash-cooled on the camera in the gaseous nitrogen stream at 100K, X-rays were focused with Osmic HiRes2 Confocal Max-Flux™ optics and diffraction data measured with a Rigaku R-AXIS IV++ imaging plate area detector. Data collected at UQ ROCX Diffraction Facility were processed using Crystal Clear (Rigaku). Data collected at the Australian Synchrotron were measured at 100K with X-rays of wavelength 0.95667 Å on beamline 3BM1 [19] using a MAR 165 CCD detector, and processed using HKL2000 [20]. Crystals diffracting to better than 2.7 Å resolution were used for data collection.
Phasing and automatic structure solution were carried out using MIfit (ActiveSight). The structures were solved by molecular replacement using the structure of hPNMT–AdoHcy–SK&F29661 (PDB code 1HNN), with the inhibitor and waters removed, as the starting model. Active-site density was individually evaluated and assessed for the presence or absence of a ligand. If density indicating a bound fragment was present, ligands were modelled manually using COOT [21] and co-ordinates and refinement parameters were generated by PRODRG [22] and PHENIX [23]. Further refinement was carried out using PHENIX [23]. Cross-validation was done by R-free analysis from 5% of the reflections set aside from refinement.
Where density was present at the active site but did not unambiguously identify the bound ligand from the four compounds in the cocktail, a deconvolution step was performed. This involved collecting data from crystals soaked in solutions containing the individual compounds (rather than the cocktail of four compounds). In these cases, the final concentration of the compounds in the soak solution was increased to 20 mM rather than the 5 mM concentration used in the cocktail screen. The higher concentration generally resulted in improved density. Deconvolution also allowed identification of the binding of more than one fragment per cocktail.
Structure determination of hPNMT in complex with elaborated benzimidazoles
Compounds 13–17 were designed and synthesized (see below for details of synthesis) to further characterize the binding of benzimidazoles to hPNMT. The crystal structures of these compounds in complex with hPNMT were determined as follows. Crystals of hPNMT–AdoHcy–PO4 were soaked in a solution of the synthesized compounds as described above for fragment screening. In this case, compounds were solubilized at 100 mM in 50% DMSO/water and used to prepare soak solutions of final concentrations 6.6% PEG 6000, 0.3 M LiCl, 0.1 M sodium cacodylate, pH 5.7, 5–10 mM compounds and 2.5–5% DMSO.
Crystals were cryoprotected, diffraction data collected and processed, and structures solved and refined at the UQ ROCX diffraction facility using method (ii) described above, with the exception that phasing was carried out using PHENIX [23] and structures were solved by difference Fourier methods. The procedure in this case was to model and refine the structure of the protein first, followed by addition of water molecules, then AdoHcy and finally the ligand. The criteria used to include a water molecule were: the presence of 2Fo−Fc density at 1σ and Fo−Fc density at 3σ and at least one possible hydrogen bond within 3.2 Å. Cross-validation was performed as described above. All of the crystal structure electron density maps revealed density indicative of AdoHcy at the cofactor-binding site and density consistent with a benzimidazole bound at the noradrenaline-binding site.
FBS by MS
MS was performed on an APEX® III 4.7 Tesla FTICR mass spectrometer (Bruker Daltonics) fitted with an Apollo™ ESI (electrospray ionization) source operated in positive ion mode. XMASS NT V6.1.2 MS software on a PC platform was used for data acquisition. Broadband excitation was used to analyse a mass range from m/z 50–6000 and each spectrum was an average of 64 transients (scans) with 512000 data points acquired in low-resolution mode with an acquisition time of approx. 4 min/sample. Samples were infused into the ESI source at 2 μl/min. Nitrogen was used as both the drying gas (125 °C) and nebulizing gas. Relevant parameters include a 104-fold pressure reduction between source and analyser regions with an ESI source pressure (10−7 kPa) and high-vacuum analyser region pressure (6×10−11 kPa). Agilent ESI tuning mix (Santa Clara) was used for an external three-point calibration. The hexapole ion accumulation time was 3 s.
To confirm correct ESI-MS parameter adjustment, parameters were optimized using a known hPNMT ligand (7-aminosulfonyl-3-ethyl-1,2,3,4-tetrahydroisoquinoline; Ki of 1.8 μM) [24]. Fragments analysed (Supplementary Figure S1 at http://www.BiochemJ.org/bj/431/bj4310051add.htm) were selected to include a range of FBS-X-positive and -negative hits. A total of 12 fragments were provided with no indication as to which compounds were hits by FBS-X. A stock solution of 31.5 μM hPNMT was prepared in 10 mM ammonium acetate (pH 7); stock solutions of AdoHcy and each of the 12 fragments (representing three of the FBS-X cocktails) were prepared in methanol. Aliquots of all solutions were used immediately or stored at −30 °C. Samples for MS analysis were prepared as follows: 3 μM hPNMT, 0 or 30 μM AdoHcy and 300 μM (100 equiv.) fragment in a total volume of 100 μl. Samples were incubated at room temperature (25 °C) for 30 min prior to MS analysis. A total of 24 MS datasets were acquired to analyse the 12 fragments (i.e. with and without added AdoHcy cofactor). The protein consumption per 100 μl sample was 0.01 mg, however, this sample volume was prepared for convenience and could be reduced as the consumed sample volume was 8 μl per MS acquisition (4 min acquisition at 2 μl/min).
If a hPNMT–fragment or hPNMT–AdoHcy– fragment non-covalent complex is formed in solution then the complex was observed in the ESI mass spectrum. The mass difference between the peaks for the unbound hPNMT and the hPNMT–fragment (Δm/z) can be multiplied by the charge state to give directly the molecular mass of the binding fragment i.e. Mr fragment=Δm/z×z to provide confirmation of the identity of the hit fragment. Complexes with AdoHcy were similarly treated to identify and confirm the fragment hits. ESI-FT (Fourier transform) MS analysis of a solution containing 3 μM hPNMT yielded the ESI positive ion mass spectrum shown in Supplementary Figure S2(A) at http://www.BiochemJ.org/bj/431/bj4310051add.htm. Peaks corresponding to the 13+ to 10+ charge states of hPNMT were observed. The ESI-FTMS spectrum of 3 μM hPNMT with added 30 μM AdoHcy yielded the ESI positive ion mass spectrum shown in Supplementary Figure S2(B) and, as an example, the ESI-FTMS mass spectrum of a hPNMT–AdoHcy–hit (compound 6) solution is shown in Supplementary Figure S2(C). In this spectrum each charge state consists of a peak corresponding to hPNMT–AdoHcy and a peak corresponding to the hPNMT–AdoHcy–fragment non-covalent complex. Actual mass values of the peaks observed in Supplementary Figure S2(C) and calculation of the mass of the fragment hit with compound 6 are given in Supplementary Table S1 at http://www.BiochemJ.org/bj/431/bj4310051add.htm.
Compounds found to bind hPNMT by FBS-X or FBS-MS were subsequently analysed by ITC. ITC was carried out using a high-sensitivity MicroCal (GE Healthcare) iTC200 system at 25 °C. Purified protein at a concentration of 200 μM plus 600 μM AdoMet (S-adenosyl-L-methionine) in 20 mM Tris/HCl, pH 7.2, 1 mM EDTA and 4.3% DMSO were loaded into the sample cell (Vcell, reaction cell volume of ≈200 μl). DMSO in the sample was calibrated to match the residual DMSO after fragment dilutions. The fragment hit at 1.5–3 mM in the same buffer was then titrated into the sample cell with 20×2.5 μl injections. The heat released was measured and integrated using MicroCal Origin 7.0 software. The association constant (Ka; 1/Kd), enthalpy (ΔH) and stoichiometry (N) were calculated using a single-site binding model. Measurements were repeated 2–4 times for each ligand and errors calculated as the S.D. from replicate experiments. In each case the protein concentration was fixed as a known quantity and the apparent ligand concentration was adjusted slightly during curve fitting so that the binding stoichiometry was approx. 1 (i.e. an assumption of 1:1 binding was made).
The energies of interaction were calculated from Ka values using the Gibbs free energy equation: ΔG=−RT(ln Ka), where R is the gas constant (1.9872 kcal/mol; 1 kcal≈4.184 kJ), T is the temperature of the experiment (298 K) and Ka is the association constant for the compound. LE (ligand-binding efficiency) values were calculated according to LE=−ΔG/HA, where ΔG is the free energy of binding (kcal/mol) and HA is the number of heavy atoms (non-hydrogen atoms) in the compound.
Compound synthesis
Benzimidazoles (compounds 13–17, see Figure 5) were synthesized using procedures published previously with minor modifications as indicated (see the Supplementary Experimental section at http://www.BiochemJ.org/bj/431/bj4310051add.htm). Generally, an appropriately substituted nitroaniline was reduced by palladium-catalysed hydrogenation to the corresponding phenylenediamine. The phenylenediamine derivative was condensed with cyanogen bromide to form the desired 2-aminobenzimidazole as the hydrobromide salt. Methoxy-substituted 2-aminobenzimidazoles were converted to their corresponding phenol derivatives using 48% hydrogen bromide. Compounds 13 and 17 have not been reported previously and details of their synthesis are reported below. Compounds 14–16 are known compounds and the details of our syntheses of them are given in the Supplementary Experimental section.
The m.p. (melting point) was determined in open capillary tubes on a Thomas–Hoover melting point apparatus calibrated with known compounds, but are uncorrected. Proton (1H-NMR) and carbon (13C-NMR) NMR spectra were taken on a Bruker DRX-500 spectrophotometer. Proton chemical shifts are reported in p.p.m. relative to TMS (tetramethylsilane; 0.00 p.p.m.) or to methanol-d4 (3.31 p.p.m.). Carbon chemical shifts are reported in p.p.m. relative to C2HCl3 (77.2 p.p.m.) or methanol-d4 (49.1 p.p.m.). HRMS (high-resolution MS) was performed on a Ribermag R 10-10 mass spectrophotometer. Hexane refers to the mixture of hexane isomers (boiling point of 68–70 °C).
2-Amino-4(7)-hydroxybenzimidazole·hydrobromide (compound 13·HBr)
Ether-mediated cleavage [25] of 2-amino-4(7)-methoxy-benzimidazole [25] (compound 18) was performed by dissolving compound 18 (0.40 g, 2.4 mmol) in 20 ml of 48% hydrogen bromide. The solution was refluxed at 100 °C for 16 h. The reaction was washed with diethyl ether and evaporated under reduced pressure to yield the crude compound 13·HBr as a brown solid. Trituration from 2-propanol/hexane yielded a viscous brown liquid that was dried in vacuo to yield 13·HBr as a brown solid (0.24 g, 66%), m.p. >250 °C: 1H-NMR (500 MHz, MeOD) δ 7.07 (t, J=8.1, 1H), 6.84 (d, J=7.5, 1H), 6.70 (dd, J=0.6, 8.1, 1H); 13C-NMR (126 MHz, MeOD) δ 151.8, 145.0, 132.7, 125.9, 119.7, 110.8, 103.6; HRMS (m/z): [M+H] calculated for C7H7N3OBr, 227.9772, found 227.9775.
2-Amino-5(6)-chloro-7(4)-hydroxybenzimidazole·hydrobromide (compound 17·HBr)
Reduction [26] of 4-chloro-2-methoxy-6-nitroaniline [27] (compound 19) was performed by dissolving compound 19 (0.30 g, 1.8 mmol) in methanol (40 ml) and adding 30 mg of 10% palladium on carbon. The mixture was stirred under hydrogen for 16 h. The palladium was removed by filtration over Celite®. The Celite® was washed with methanol and ethyl acetate. The combined organic washes were concentrated under reduced pressure to a viscous brown liquid. Column chromatography (silica gel, ethyl acetate/hexanes of 3:7) yielded 5-chloro-3-methoxybenzene-1,2-diamine (compound 20) as a brown solid (0.18 g, 58%) that was used for the next synthetic step without further purification: m.p., 97–99 °C; 1H-NMR (500 MHz, CDCl3) δ 6.39 (dd, J=2.1, 4.4, 2H), 3.82 (s, 3H), 3.42 (s, 4H); 13C-NMR (126 MHz, C2HCl3) δ 149.0, 136.4, 124.5, 122.0, 109.7, 103.3, 56.1; HRMS (m/z): [M+H] calculated for C7H10ClN2O, 173.0482; found 173.0373.
Diamine compound 20 (0.18 g, 1.0 mmol) was dissolved in acetonitrile (20 ml) and water (5 ml) and cooled to 0 °C. Cyanogen bromide in acetonitrile (5M, 0.20 ml, 1.0 mmol) was added dropwise over 5 min. The reaction mixture was warmed to room temperature and stirred overnight. The solvents were evaporated under reduced pressure to give 2-amino-5(6)-chloro-7(4)-methoxybenzimidazole·hydrobromide (compound 21·HBr) as a brown solid (0.30 g), which was used for the next synthetic step without further purification, m.p., 68–70 °C: 1H-NMR (500 MHz, MeOD) δ 7.01 (d, J=1.6, 1H), 6.94 (d, J=1.6, 1H), 3.98 (s, 3H); 13C-NMR (126 MHz, MeOD) δ 152.6, 147.5, 132.8, 131.2, 119.4, 107.9, 105.6, 57.1; HRMS (m/z): [M+H] calculated for C8H9ClN3O, 198.0434; found 198.0383.
Ether cleavage of compound 21·HBr was carried out using the procedure given for the synthesis of compound 13·HBr above, yielding compound 17·HBr as a brown solid (0.10 g, 36%), m.p. >250 °C: 1H-NMR (500 MHz, MeOD) δ 6.88 (d, J=1.7, 1H), 6.72 (d, J=1.7, 1H); 13C-NMR (126 MHz, MeOD) δ 152.3, 145.5, 133.1, 130.7, 118.8, 111.2, 104.0; HRMS (m/z): [M+H] calculated for C7H7N3OCl, 184.0278; found 184.0266.
For the FBS-X experiments, we used crystals of hPNMT grown in the absence of inhibitor or ligand for the noradrenaline-binding site. The crystal structure resulting from such crystals reveals a phosphate molecule bound in the noradrenaline-binding site (Figure 2); the phosphate apparently co-purifies with the enzyme during an affinity purification step. The phosphate forms favourable interactions with the side chains of Lys57 and Asn39. For a hit to be detected in the FBS-X experiments, one or more of the four fragments of the cocktail must displace the phosphate during the 15 min soaking step. Data collection and refinement statistics for a representative crystal structure of hPNMT–AdoHcy–PO4 are included in Supplementary Table S2 at http://www.BiochemJ.org/bj/431/bj4310051add.htm.
Of the 96 cocktails containing four compounds (384 compounds in total) in the library, X-ray diffraction data were collected for 94 cocktails (Table 1). Two of the 96 cocktails caused crystal cracking during the soak, preventing high-resolution diffraction data measurement. Owing to the high-throughput nature of the screen, these two cocktails were not considered further. Evaluation of the eight compounds in these two cocktails will be investigated in another study. A total of 16 of the 94 cocktail soaks gave electron density suggestive of hits, i.e. binding of a chemical to the noradrenaline-binding site. The chemical identity of two of the hits was obvious from the initial X-ray data. An additional 51 datasets were measured to deconvolute hits where it was not clear which compound of the cocktail had bound or to measure higher resolution data for confirmed hits. During this process, four of the initially identified hits were found to be false positives. In summary, a total of 12 compounds (out of a possible 376) were identified as binding in the target site of hPNMT, giving a hit rate of 3.2% for the FBS-X process. The 2Fo−Fc maps showing the ligand density for these 12 hits are provided as Supplementary Figure S3 at http://www.BiochemJ.org/bj/431/bj4310051add.htm. Summary statistics for the X-ray data are given in Table 1 and data collection and refinement statistics for the 12 hit crystal structures are included as Supplementary Table S2. The structures of the 12 hits and their binding mode to hPNMT are shown in Figure 3.
Analysis of fragment hits
The 12 fragment hits all bind the noradrenaline-binding pocket of hPNMT, the same site as other structurally characterized hPNMT inhibitors. Furthermore, all 12 fragments bind within the same region of the pocket (Figure 4A). All 12 fragment hits contain either or both a five- or six-membered aromatic ring, sandwiched between the side chains of Phe182 and Asn39. In this position, the rings form π–π stacking interactions with the side chain of Phe182. The importance of this interaction is evident in the structure of 6-chloro-oxindole (compound 4) bound to hPNMT (Figure 3). Unlike other ligand-bound hPNMT structures, there are no direct hydrogen-bond interactions formed between compound 4 and hPNMT. In addition to the π–π stacking interactions between the benzene ring and Phe182, the only other interaction that might contribute to binding of compound 4 is a hydrophobic interaction between the chlorine atom and the aromatic ring of Tyr222.
Three chemical frameworks are present in more than one fragment hit: (i) the benzimidazole core (compounds 6, 7, and 9); (ii) the purine core (compounds 2 and 10); and (iii) the quinoline core (compounds 1, 3 and 11). Despite different substituents, the benzimidazole-based fragments all bind in the same orientation within the active site. In contrast, the binding modes of the two purine-based fragments and the three quinoline fragments vary. This variation allows different functional groups to make favourable interactions (e.g. see compounds 2 and 10, in Figure 3) demonstrating that the presence of substituents can alter the binding pose of a core fragment. It should also be noted that the library used for FBS-X screening contains a further ten benzimidazole compounds, none of which were found to bind the noradrenaline site. Similarly, the screening library also contained other quinolines and purines that did not bind to the target site under the conditions used for FBS-X. The observation that different substituents affect binding of compounds to PNMT provides further validation for the specificity of hits identified by FBS-X and may be useful for structure–activity relationship analysis in the development of more potent inhibitors.
Superposition of the fragment hits demonstrates how opposite ends of the noradrenaline pocket bind different chemical functionalities (Figures 4A and 4B). Hydrophilic nitrogen and oxygen atoms of hit compounds are clustered at one end of the pocket, forming direct and water-mediated interactions with the active-site residues Glu185, Glu219, Asp267 and the main chain of Phe182. In contrast, carbon and chlorine atoms of hit compounds are clustered at a hydrophobic region of the pocket consisting of the side chains of Val53, Met258, Val269 and Val272. These same amino acid residues are involved in binding characterized previously between inhibitors and substrates [28–30].
In some cases, we found unfavourable contacts between the fragments and hPNMT residues. Many unfavourable interactions resulted from clashes with the hydroxy group of Tyr35, which is close to the hydrophobic residues Val53, Met258, Val269 and Val272. We also observed cases where the electron density for all or part of the fragment was weak. For example, density for the flexible chain of formanilide (compound 8) is weak. In two other cases, 1-aminoisoquinoline (compound 3) and 2-hydroxynicotinic acid (compound 12), the fragments adopt different binding poses in the two hPNMT molecules in the asymmetric unit. This may be due to a lower affinity of these ligands compared with other hits. Finally, for 4-bromo-1H-imidazole (compound 5), it was difficult to determine from the electron density alone the correct position of the ring nitrogen atoms because the compound fits the density equally well in two orientations, rotated 180 ° around the long axis. Compound 5 was modelled in one orientation on the basis of predicted favourable interactions (see Figure 3).
Water molecules in the active site are important because they can indicate potential areas for favourable elaboration of fragments. We found that many water molecules are conserved across the 12 enzyme–fragment structures (Figure 4C). There are five major water-binding sites, four of which are located in the hydrophilic region of the binding pocket described above. These four sites, show the greatest level of conservation across the 12 structures, and mediate interactions with Glu185, Glu219 and Asp267. The presence of defined water-binding pockets suggests that these regions favour ligand interactions and would probably be amenable to interactions with elaborated inhibitors.
FBS by MS
MS has high sensitivity and a wide dynamic range and can be used to detect non-covalent complexes with a Kd in the range from 10 nM to 1 mM [9]. We were interested in using MS as a first-pass FBS method, so we performed a pilot study on hPNMT to ascertain whether MS could identify the same hits as FBS-X. For proof-of-concept, an ESI-FTMS analysis was conducted on a subset of 12 of the 384 fragments from the library. The 12 fragments were intentionally chosen to include several FBS-X hits. ESI-FTMS screening was conducted blind in that the researcher collecting and analysing the data did not know which of the 12 compounds were FBS-X hits. Of the 12 fragments tested, FBS-X identified four fragments as hits (compounds 1, 6, 11 and 12; Figure 3), three of which were detected by ESI-FTMS on first inspection of the data (compounds 1, 6 and 12). The fourth FBS-X hit, compound 11, was detected upon re-evaluation of the ESI-FTMS spectra, although it was identified as a weak complex. In addition, ESI-FTMS identified two other hits (Supplementary Figure S3); these two fragments were not identified by FBS-X.
ITC evaluation of fragment hits
To confirm and characterize further the 14 fragment hits (12 identified by FBS-X and the two identified by ESI-FTMS), and to prioritize the fragment hits for follow up chemistry and optimization, ITC was used to determine their dissociation constants (Kd) for hPNMT, and thermodynamic parameters of binding. All of the nine fragment hits detected by ITC demonstrated favourable enthalpic contributions (Figure 3 and Table 2). Of the 12 FBS-X hits, the measured binding affinities for nine fragments ranged from approx. 5 to 700 μM. Of these, two fragments (compounds 1 and 11) show low-affinity binding (below 200 μM) that results in sub-optimal Wiseman c-values (<1) for accurate affinity determination by ITC and this is reflected in the relatively large errors [31]. Nonetheless the data do allow prioritization of these compounds for follow-up. Binding of the remaining three FBS-X hits (compounds 4, 8 and 12) could not be detected by ITC. The two additional hits identified by ESI-FTMS were also not detected by ITC. Furthermore, whereas the binding of 4-quinolinol (compound 1) was detected in ITC experiments, and a Kd of 0.7 mM was calculated from the ITC measurements, the signal was weak. The two tightest binding fragments with Kd values of approx. 5 μM (2-aminobenzimidazole, compound 6, and 2-amino-1-methybenzimidazole, compound 7), are both based on the benzimidazole core. As discussed above, benzimidazoles have been shown previously to inhibit PNMT [32]. The third fragment hit with a benzimidazole core (5-chlorobenzimidazole, compound 9), had a 20-fold lower binding affinity (Kd of 97 μM), suggesting that direct and water-mediated hydrogen bonds to the 2-amino groups of compounds 6 and 7 contribute considerably to ligand affinity for hPNMT. The dissociation constants were used to calculate their LE values (Figure 3 and Table 2). The LE values ranged from 0.39 kcal/mol per HA for 4-quinolinol to 0.86 kcal/mol per HA for 4-bromo-1H-imidazole (compound 5). The two tightest binding benzimidazole-based fragments, compounds 6 and 7, had high LEs of ~0.7 kcal/mol per HA.
Elaborated benzimidazole compounds
Three of the identified hits (compounds 6, 7 and 9) contain a benzimidazole ring system. The electron density for these three hits was excellent, and ITC measurements confirmed that all three bind to hPNMT, two of them with very high LE values. Benzimidazoles have been investigated previously as inhibitors of PNMT, although the earlier work was performed on the bovine enzyme [32]. To further characterize the interaction of benzimidazole-based fragments with the human enzyme, a series of compounds (compounds 13–17; Figure 5) closely related to the FBS-X benzimidazole hits were synthesized. The 2-amino group was retained in this series because in compounds 6 and 7 it forms direct and water-mediated hydrogen bonds to the enzyme. The binding of compounds 13–17 to hPNMT was evaluated by ITC and X-ray crystallography. The results of these analyses are shown in Figure 5 and data collection and refinement statistics for the structures are included as Supplementary Table S2. ITC curves are shown in Supplementary Figure S4 at http://www.BiochemJ.org/bj/431/bj4310051add.htm, and the ITC-derived thermodynamic parameters of the 12 fragment hits and five elaborated compounds are given in Table 2.
Hydroxy-substituted 2-aminobenzimidazole compounds (compounds 13 and 14)
Superposition of the benzimidazole-based fragments with the fragment hit 4-quinolinol (compound 1), suggested that a hydroxy group added to the 7- or 6- position of compound 6 would interact with Lys57 and potentially with Tyr40. To test this, compounds 13 and 14 were synthesized and their binding characterized (Figure 5 and Table 2). The crystal structures of hPNMT–AdoHcy–compound 13 and hPNMT–AdoHcy–compound 14 showed that both these elaborated compounds bound in a flipped binding mode compared with the predicted binding mode, i.e. instead of forming an interaction with Lys57, the hydroxy group is oriented away from Lys57. In hPNMT–AdoHcy–compound 13 the hydroxy group makes no favourable interactions, and sits in a hydrophobic pocket comprising the side chains of Met258 and Val269. This same position is occupied by the chloro substituent of compound 9 when it forms a complex with hPNMT (Figure 3). In addition, the benzimidazole core of compound 13 is shifted away from the hydrophobic pocket compared with the original hit fragment, compound 6. This movement results in an unfavourable interaction between the six-membered aromatic ring and Lys57. These changes result in a reduced enthalpic binding contribution of compound 13 compared with compound 6; this is balanced by a smaller entropy loss upon binding with overall a small net decrease in the free energy of binding. This is reflected in a decreased binding affinity and LE of the elaborated compound compared with the original hit (compound 13, Kd of 15 μM and LE of 0.60 kcal/mol per HA; compound 6, Kd of 6.3 μM and LE of 0.71 kcal/mol per HA), indicating that the hydroxy modification to compound 6 is not favourable for binding.
Similarly, the hydroxy group of compound 14 is not favourable for binding to hPNMT. In the crystal structure of the hPNMT complex, the hydroxy group is unfavourably close to Met258 and waters are displaced from the hydrogen-bonding network. These changes result in a reduced enthalpic contribution, a reduced binding affinity and a reduced LE compared with the original hit, compound 6 (compound 14, Kd of 20 μM and LE of 0.58 kcal/mol/HA).
Chloro- and fluoro-substituted 2-aminobenzimidazole compounds (compounds 15 and 16)
Superposition of the fragment hit compounds 6 and 9 bound to hPNMT indicated that a hydrophobic group at the 5-position of the benzimidazole system of compound 6 might contribute additional favourable interactions. To investigate this further, two more compounds were designed: compound 15, combining the 2-amino substituent of compound 6 and the 5-chloro substituent of compound 9; and compound 16 combining the 2-amino substituent of compound 6 and the smaller fluoro substituent at the 5-position (Figure 5). Both compounds 15 and 16 bound as predicted and the 2-chloro derivative compound 15 demonstrated the most favourable binding affinity (Kd of 1.8 μM and LE 0.72 kcal/mol per HA) of this series. The binding affinity of the fluoro-substituted compound 16 (Kd of 7.2 μM), was similar to the parent compound 6, although the enthalpic and entropic contributions were slightly altered, with a less favourable enthalpy but more favourable entropy.
Hydroxy- and chloro-di-substituted 2-aminobenzimidazole (compound 17)
Di-substitution of the 2-aminobenzimidazole core with the 5-chloro and 7-hydroxy substituents in compound 17, did not significantly improve affinity (Kd of 14 μM) compared with the hydroxy substitution alone (compound 13, Kd of 15 μM). As a result, the LE was reduced compared with the original, unsubstituted fragment compound 6, and the separately substituted compounds 13 and 15. Despite extensive trials we were unable to measure X-ray diffraction data for hPNMT–AdoHcy–compound 17. Crystals soaked in this compound cracked during cryoprotection or gave very weak diffraction, indicating that binding of the compound induces crystal disorder or conformational changes to accommodate compound 17. As no improvement in binding affinity or enthalpic contribution was observed, we did not investigate compound 17 further.
Using FBS-X we found that 12 compounds from a library of 374 compounds bound to the noradrenaline-binding site of hPNMT by displacing phosphate from the crystal structure. Our hit rate of 3.2% is similar to that of other FBS-X campaigns [33–38], despite the fact that our target crystal structure was not in the apo form. We also investigated the use of high-sensitivity ESI-FTMS, which has been used previously to screen natural product and combinatorial chemistry libraries against other enzymes [39,40]. For our proof-of-concept study, we used a subset of the fragment library. ESI-FTMS identified the same four hits found by FBS-X for this subset. FBS-MS data acquisition took only 4 min, so an automated approach could process a 384 compound library in just over 24 h using 0.4 mg of protein. By comparison, our successful FBS-X campaign took 2–3 months and used ~20 mg of protein. These results suggest that ESI-FTMS could be used as a filter to generate a short-list for subsequent characterization by X-ray crystallography. This could in theory reduce the number of crystal structures to be measured from ~150 to ~20, thereby saving time, human resources and protein, and making the technique accessible to many more academic laboratories.
Using ITC we determined the dissociation constants of nine of the 12 FBS-X fragment hits. The results showed that the fragments bind with affinities in the high to low micromolar range and all nine hits for which affinity data were measured had LE values≥0.4 kcal/mol per HA. Generally, LE values above 0.4 kcal/mol per HA are desirable through the process of optimization [38,41]. Many of our hits had considerably higher LE values, emphasizing their high quality.
Several of the FBS-X hits are benzimidazoles, a known class of PNMT inhibitor. In 1970, researchers at the MSD (Merck Sharp and Dohme Research Laboratories) showed that benzimidazoles (including 2-aminobenzimidazole, compound 6, and 5-chlorobenzimidazole, compound 9) inhibit bovine PNMT. Thus at 28 μg/ml compound 6 inhibits binding of 0.57 mM DL-normetanephrine to bovine PNMT by 30% and compound 9 inhibits binding by 57% [32]. Our FBS-X results identified that these compounds also interact with human PNMT and show for the first time how they bind in the noradrenaline pocket. The MSD study also showed that unsubstituted benzimidazole is an inhibitor of bovine PNMT (28% inhibition under the conditions described above) [32]. Benzimidazole is one of the 376 compounds in the library used in the present study, but was not identified as a hit by FBS-X. This finding could be explained by one or more of the following: (i) benzimidazole does not inhibit PNMT by binding the noradrenaline-binding site; (ii) the noradrenaline-binding site of the human and bovine enzymes differ sufficiently that they recognize benzimidazole differently; or (iii) the screening method we employed is less sensitive than the assay used to detect inhibition in the bovine enzyme.
Several PNMT inhibitors have been shown to be competitive for noradrenaline binding [42–44], although it has been shown that a dichloro-substituted benzimidazole PNMT inhibitor was non-competitive with noradrenaline and uncompetitive with AdoMet [45]. These early results indicate the possibility of an additional binding site for benzimidazoles. Our data clearly show that the three benzimidazole hits and the elaborated compounds bind at the noradrenaline site. However, the hPNMT crystals used for our studies have AdoHcy (as a cofactor product) bound at the AdoMet site. Furthermore, ITC analysis was performed in the presence of excess AdoHcy. Consequently, if the benzimidazole compounds bind to the hPNMT AdoMet site this would not be identified under the conditions of our experiments.
The improved binding affinity of the designed benzimidazole compound 15 compared with the original compound 6, is largely due to an increased entropic contribution to binding (Table 2). Enthalpic optimization is more desirable than entropic opimization in progressing from hit to lead [46]. Typically, enthalpically optimized leads more closely mimic the thermodynamic characteristics of natural protein–ligand interactions, are more soluble and often represent best-in-class compounds in drug discovery programmes [46,47]. Our results therefore indicate that although compound 15 shows higher affinity than compound 6 this improvement may not necessarily correlate with an improved drug lead.
In summary, we have shown that FBS-X can be implemented successfully within an academic environment on a non-ideal protein target. Using FBS-X we identified 12 hits that bind hPNMT at the noradrenaline site. Our pilot study suggests the screening process could be streamlined further by performing FBS-MS first. Three of the FBS-X hits are from a chemical class of known PNMT inhibitors that provide an unanticipated internal control for the screen. Overall, we structurally and thermodynamically characterized the binding of 17 small molecules to hPNMT, including the first such characterization of the benzimidazole class of compounds bound to the noradrenaline-binding site.
Nyssa Drinkwater and Jennifer Martin conceived the idea. Nyssa Drinkwater performed the majority of the research and Jennifer Martin co-ordinated the study. Hoan Vu and Sally-Ann Poulsen designed and performed the MS experiments. Kimberly Lovell, Thomas Prisinzano, Kevin Criscione and Gary Grunewald designed and supervized compound synthesis and contributed to compound characterization. Michael McLeish provided the plasmid for the enzyme and contributed to data interpretation. Brett Collins assisted in measurement and analysis of ITC data. Nyssa Drinkwater prepared the first draft of the paper. All authors contributed to discussions on the study and provided intellectual input to the manuscript.
This work was supported by an University of Queensland Graduate School Research Travel Grant and an Australian Postgraduate Award (to N.D.); an Australian Research Council Discovery Project award [grant number DP0664564 (to J.L.M.)]; an Australian National Health and Medical Research Council Biomedical RD Wright Career Development Award (to B.M.C.) and Senior Research Fellowship [grant number 455829 (to J.L.M.)]; an Australian Research Council Linkage Equipment and Infrastructure Award [grant numbers LE0668382 (to J.L.M.), LE237908 (to S.-A.P.)]; and by the National Institutes of Health [grant number NIH HL034193 (to G.L.G.)].
We are grateful for access to the UQ ROCX Diffraction Facility and thank Karl Byriel and Gordon King for their assistance. We thank staff at the MX1 beamline of the Australian Synchrotron for their invaluable advice. The MS data could not have been measured without access to the Eskitis Institute FTMS facility. We thank Dr F. Anthony Romero for helpful preliminary discussions and Duncan McRee for advice and information regarding the ActiveSight library.
The co-ordinates and structure factors for all 17 crystal structures have been deposited with the PDB under accession codes 3KPJ, 3KPU, 3KPV, 3KPW, 3KPY, 3KQM, 3KQS, 3KQT, 3KQV, 3KQW, 3KQO, 3KQP, 3KQQ, 3KQY, 3KR0, 3KR1 and 3KR2.
Abbreviations: AdoHcy, S-adenosyl-L-homocysteine; AdoMet, S-adenosyl-L-methionine; CCD, charge-coupled-device; CNS, central nervous system; ESI, electrospray ionization; FBS, fragment-based screening; FBS-X, FBS by X-ray crystallography; FTMS, Fourier transform MS; HA, heavy atom; HRMS, high-resolution MS; ITC, isothermal titration calorimetry; LE, ligand-binding efficiency; m.p., melting point; MSD, Merck Sharp and Dohme Research Laboratories; PNMT, phenylethanolamine N-methyltransferase; hPNMT, human PNMT
- © The Authors Journal compilation © 2010 Biochemical Society


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Multiple ChecklistAsked by DC3Church on December 14, 2016 at 11:51 AM
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We want to select the minstry name and when we select the ministry name, then the checklist will appear and they can submit.
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About: Royal Lancaster Infirmary / Cardiology Royal Lancaster Infirmary Cardiology LA1 4RP
Posted by jim nelson
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Response from Royal Lancaster Infirmary 10 months ago
Dear Mr Nelson, thanks very much for taking the trouble to contact us and let us know about your recent experience at the RLI. We're delighted your initial treatment here was good and will pass on your kind comments to those involved. We wish you all the best for your next phase of treatment in Blackpool and hope you continue to receive good care.
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Category: Codecanyon | Mobile
Tags: Graphicriver | Logo Templates | nulled | free download | theme | script | rip | CityTour
In archive: 2016 | April 2016 | 20.April 2016
CityTour is full application by using Titanium's Alloy for Mobile App and CodeIgniter for Backend. Moreover, it supports JSON API also to link App and Backend. Our app will support for iOS(iPhone & iPad) and Android with portrait orientation.
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For iOS, Please provide your device''s UDID and invite email. I will push from TestFlight.
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In a sport that has been affected by technology more than most others, manufacturers have again changed the game and again may be helping to rewrite the record books.
Swimsuit manufacturers have made their latest move to create a water-shedding super suit with cap and goggles, launching a three-piece ensemble Wednesday in New York.
It's the latest bid to advance the battle of sport technology past the now-banned polyurethane suits that came out before the 2008 Olympics. Those inflexible suits – which could take 45 minutes and a helping hand to get into – made the body buoyant and created a flurry of records. It was not something swimming's governing body, FINA, wanted to see when the record book was rewritten more than 40 times at the Beijing Games in 2008 and at the 2009 world championships in Rome. The high-tech suit was banned and engineers went back to the drawing board.
Wednesday, they unveiled the future.
And with new technology that's supplanting the polyurethane suit, 14-time Olympic gold medalist Michael Phelps of the United States predicts records will fall, again, next summer at the 2012 London Games.
"People have got to get out of their heads that these records are untouchable," Phelps told Reuters. "Everything is possible. That's something I have learned in my career. You're going to see a lot of fast swims and hopefully we're going to see a lot of new records in the next year and I think we will."
The new technology is based on three parts working together, from the cap down to the knees, not just the suit itself.
"The discussion wasn't just about the suit but also the cap – which is almost a bowl, worn with an under-cap to keep the hair in place – and the goggles, which were the most comfortable I've worn," said Swimming Canada chief executive officer Pierre Lafontaine, who was in New York to see manufacturer Speedo launch its latest generation Fastskin3 system.
The new system's cap is designed using three-dimensional head mapping to fit head and face exactly. The goggles, Lafontaine said, allow a swimmer to see the lane beside him or her a little more clearly. They are leak-resistant, have an improved outer profile and offer 180-degree peripheral vision. The suit has compression fabric to stop muscle vibration, flexible fabric in the shoulders to offer high stretch and a flat-seamed body stability web which makes for a 3-per-cent improvement in starts and turns, the maker says. Men's swim shorts – or jammers – have no drawstring, making for a flatter fit; and women's suits have a unique armhole entry system.
Swimming has been affected by technological changes more than most sports. In other sports, technology has been geared toward competition. In mountain biking, for example, four women with helmet-mounted cameras went around the course to be used at London's 2012 Olympics, so that Canadian riders have a feel for what lies ahead and can groom for the Games. But with polyurethane suits, the idea was to find a loophole in the regulations and gain an advantage – a technological kind of doping. It rendered the record book and historic performances meaningless.
Officials hope that the new system will result in records falling at the 2012 Olympics in London. When FINA banned the polyurethane suits and swimmers went back to a hodge-podge of old technologies, swimmers went about 20 months without a record-setting performance.
"It helps to have technology with you," Lafontaine said. "You're not racing to be second on the podium."
U.S. backstroker and world record holder Ryan Lochte pronounced himself "unstoppable" with the new technology. Canadian freestyler Ryan Cochrane, an Olympic medalist at Beijing, was also among the stable of athletes the manufacturer brought in for the reveal.
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The claim for the system is that it will produce an 11-per-cent improvement in oxygen economy and reduced drag, which will help a distance swimmer cut through the water stronger for longer periods.
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New Delhi: The death of a young boy from Arunachal Pradesh, Nido Taniam, in Delhi has led to a furore among the Northeast Indians. The Northeast community has decided to hold a protest march on Saturday in front of the same sweet shop where he was allegedly beaten to death. The Ministry of Home Affairs has sought a report from the Delhi Police on the death of the young student, who was the son of an Arunachal Pradesh MLA. Sources in the Home Ministry say three people have been detained. Delhi Chief Minister Arvind Kejriwal has condemned the death and has assured a probe. "The Delhi government strongly condemns the murderous assault on a college student from Arunachal Pradesh in the Lajpat Nagar Market on Wednesday, which has resulted in his death. Such gruesome and inhuman incidents must not be tolerated and the Delhi government is working on ordering a probe into how the innocent life was lost and what was the response of the law enforcing agencies. The government is contemplating a magisterial inquiry into this death, which is not only tragic but raises serious questions on the alleged beating reportedly in public view," Kejriwal said in a statement. The Delhi Police has registered a murder case and has begun probe into the incident. "A case has been registered..we are questioning all the people involved in the fight," said P Karunakarn, DCP, South East Delhi. Taniam's family has alleged that he was beaten to death. The incident took place on January 29 and the victim's friends say there was a fight with a shopkeeper which escalated. The victim succumbed to injuries on Thursday. Sources saying that prima facie death appears to be due to internal injuries. Some say the shopkeepers and a few others made some obscene remarks on his blonde hair. It led to the fight. The police picked up the boy and later dropped him back in the same locality. After that the shopkeeper and his friends allegedly beat him up again. Activist Binalakshmi Nepram has tweeted, "Shopkeepers #LajpatNagar made fun of victim's blonde hair.Argument broke out. 6-8 locals came out and thrashed victim with Iron rods & sticks (sic)." "Police arrived/settled case with fine Rs. 10,000 & took deceased to PS but later policemen dropped him at same place where fight took place (sic)," she added. The people from the North East have demanded the strictest action against both police and the attackers. Arunachal Pradesh Resident Commissioner Avinash Kumar said, "The police must act and arrest those behind this. It's a heinous crime. The Northeast students are very agitated. We will ensure that the culprits are booked under the law. We will send the body to his native place on Saturday morning." According to eye witnesses the altercation began when he broke the window of a sweet shop. He broke the window after they made comments about his hair (he had blonde hair), said his friends. However, student union leader JT Tagam has said that the incident involving the AP student does not seem to be a racist incident. Union Minister for Minority Affairs Ninong Ering, who also hails from Arunachal Pradesh, has expressed shock over the incident. He has demanded a thorough probe and punishment.
from in.com
Turkey President, Erdoğan, spreading conservative propaganda by replacing jihad in school curriculum with Darwin's theory of evolution.
from in.com
Forthcoming Budget will have Stipulations for Simplified Direct and Indirect Taxation - MOS Finance
Minister of State for Finance, Mr. Arjun Ram Meghwal on Wednesday promised that the forthcoming budget would have stipulation and provisions that would lead to further simplification of tax administration and tax dispensation of both direct and indirect taxes to enable business community to render their transaction smoothly and effectively.
from in.com
Social Initiative Help India Learn Expands Reach to over 120 Schools with over 50 Volunteer Teachers On-Board
Develops smartphone based Maths videos in Hindi for underprivileged students
from in.com
UN Supports Better Welfare for Farm Animals Worldwide
For the first time ever in the history of the United Nations (UN), farm animals worldwide will be better protected following the adoption of groundbreaking global policy recommendations on animal welfare in farming at the UN Committee on World Food Security meeting in Rome.
from in.com
SevaChild India, Local Rishikesh and Dehradun NGOs Join Hands to Prevent Blindness in Children of Uttarakhand
Mumbai-based SevaChild India, along with the Rural Development Institute (RDI), Divine Shakti Foundation and Him Jyoti School for Girls came together to conduct a health, hygiene and sanitation awareness day on 17th October 2016 at the Mayakund slum. It was combined with the distribution of Vitamin A and Albendazole to children below the age of five.
from firstpost
AAP MLA Somnath Bharti arrested for allegedly assaulting Aiims security guard
In fresh trouble for Aam Aadmi Party (AAP), MLA Somnath Bharti was on Thursday arrested by Delhi Police for allegedly assaulting a security guard of the All India Institute of Medical Sciences (Aiims)...
from firstpost
United Nations: A happy shouting ground for Pakistan, it was India's turn to holler back
Pakistan loves the United Nations, especially its annual General Assembly in New York. This provides it with an international platform to project its positions, often born of paranoia. Pakistan Prime ...


text: Jenna Lea Rosen is a vocalist who performs multiple roles on the Emmy Award-winning Disney Junior series Sofia the First and the Disney Channel series Elena of Avalor, in which she sings for a number of characters. She is also the speaking and singing voice for Shelly in "Cool Hand Fluke". She is also the voice of Ciela in "Flight of the Jaquins".


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Getting Lucky
Getting Lucky
By DC Brod
Tyrus Books, Hardcover, 9781440531989, 332pp.
Publication Date: December 18, 2011
List Price: $24.95*
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When a young reporter is killed in a hit and run accident, freelance writer Robyn Guthrie agrees to finish one of the stories the reporter had been writing for the local newspaper. But nothing is as simple as it seems when she finds out about shady land deals, an old high school nemesis, and Robyn's aging mother.


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I spent the last 90 minutes hacking away with Kenny and Jim from WeFarm and we managed to get internet and FrontlineSMS working concurrently…Continue
Started this discussion. Last reply by kiwanja Dec 4, 2011.
Hi everyone,We're currently reviewing some of the functionality of FrontlineSMS.Question: Do you use multi-word Keywords, or do you solely use single words?Examples of multi-word keywords:"FRONTLINE…Continue
Tags: action, keywords, keyword
Started Feb 17, 2010
User Rick encountered the following issue when trying to run FrontlineSMS on Ubuntu 8.10 (Intrepid Ibex) using an AMD64 processor:Problem 1: Serial Drivers Incorrect:java.lang.UnsatisfiedLinkError:…Continue
Tags: drivers, ubuntu, 64-bit, sqlite, rxtx
Started this discussion. Last reply by Alex Anderson Oct 28, 2011.
Alex Anderson has not received any gifts yet
|Sender phone number
|The phone number that the message came from
|Sender's name
|The name of the contact that this message came from. This will not work if the contact is not saved in FrontlineSMS.
|Recipient's phone number
|The phone…
Posted on March 19, 2009 at 6:00am
Posted on January 27, 2009 at 8:30am — 18 Comments
Good Day,
How is everything with you, I picked interest on you after going through your short profile and deemed it necessary to write you immediately. I have something very vital to disclose to you, but I found it difficult to express myself here, since it's a public site.Could you please get back to me on:( email@example.com) ) for the full details.
Have a nice day
Thanks God bless.
Mrs Stella.
Hi! Mr. Anderson I real appreciate your contribution you are giving to people about technical support in Using and configuring Frontline sms Big up Brother I hope we shall be cooperating together.
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Get all 50 Shötgun Piñata releases available on Bandcamp and save 25%.
Includes unlimited streaming via the free Bandcamp app, plus high-quality downloads of Soul Blender single, Sour Grapes & Spilled Milk, The Bunny Milk EP, Octubre, Clique Cliches EP, Strange - The Bootleg, Dystopiate, The Hell's Lounge Houseband EP, and 42 more. , and , .
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Shötgun Piñata is D Mojo Brown, born in Newark DE. Although the concept of the music project began in Cincinnati, Ohio. He is also a a spoken word artist, a writer and artist. Heavily influenced by Golden era hip-hop, 80's Brit Synth pop, Industrial Dance Metal, Jazz, and Sci-fi movie scores(among others). Shotgun Pinata records for Fantastic Planet Industries record label in Berkley, CA. ... more
Streaming and Download help


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2016-17 FOCUS New Leadership Team - Working together, everyone achieves more!
The time has come for stakeholders of FOCUS Academies to refocus on FOCUS. We must institute a sense of urgency to improve student learning. We are a transformed organization, a laser beam focus on increased accountability, communication, and training designed to ensure students will learn and not "just be taught".
This school year, FOCUS Academies' staff will use reflective tools, which enable leaders to enact three learning agendas: improving student learning, enhancing professional learning, and building system-wide supports for all participants' learning. The framework tools help leaders clarify the mission and core values, promote professional community, and highlight the need for inquiry and engage all stakeholders in the school's improvement process.
Based on school transformation research, FOCUS Academies will concentrate on the following initiatives:
We will no longer settle for mediocrity, excuses and lack of buy-in. We believe in VISION, ACTION, BUY-IN, and MORE ACTION. I believe that capacity building is making the most of people and then out of those resources making the most of the people resources and systems to deliver results. We will be a high performing team that produces high performing students.
Leroy McClure, Jr., CALT
FOCUS Academies
Parent Letter


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Quince and Cranberry SaucePublished: October 2008
IngredientsMakes about 3 1/2 cups Servings
- 5 quinces (2 to 2 1/4 pounds), peeled, cored, cut into 1-inch chunks
- 3 cups water
- 1 1/2 cups sugar
- 3 1/2 tablespoons fresh lemon juice
- 2 teaspoons finely grated lemon peel
- 1
- 1 1/4 cups fresh or frozen cranberries (about 4 ounces)
- 1/4 teaspoon coarse kosher salt
Bring first 5 ingredients to boil in heavy large saucepan over medium-high heat, stirring until sugar dissolves. Reduce heat to medium-low. Cover and simmer until quinces are soft, stirring occasionally, about 20 minutes. Pour mixture into large strainer set over bowl; reserve juices.
Return quince mixture to same saucepan; mash with potato masher. Add cranberries; cook over medium heat until most of berries burst, stirring frequently, about 8 minutes. Stir in salt. Transfer sauce to bowl. Cover and refrigerate sauce and reserved juices separately until cold, about 3 hours. DO AHEAD Can be made 2 days ahead. Keep chilled. Before serving, stir enough reserved juices into sauce to thin to desired consistency. Serve sauce cold or at room temperature.
Peeling & Coring
A quince's peel is thin like an apple's and usually removed before cooking (it adds an unpleasant bitterness). To peel a quince, simple use a vegetable peeler on the whole (uncut) fruit. To core a quince safely, first peel it, then cut it lengthwise (through the core) into six wedges with a large chef's knife. Carefully remove the core with a sturdy small paring knife. Quinces oxidize quickly: To prevent browning, immediately drop the cut pieces into lemon water.Recipe by Jill Silverman Hough


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Latest Issue of CARING Focuses on Health IT for Home Care & Hospice
The latest issue of CARING magazine will soon be available. The in-depth subject this month is heath information technology and how it is changing the home care and hospice landscape.
For the issue, Richard Brennan, Executive Director of the Home Care Technology Association of America (HCTAA) – a NAHC affiliate - served as Guest Editor. Below are his introductory notes to the issue reprinted in their entirety.
What if we could envision the future of health care delivery and determine the fundamental components of the new standards of care that will be needed to support patient-centered, personalized, empowered health care in America? What technologies will drive the delivery of health care to the home? What business and clinical practices will be leveraged to support value-based longitudinal care coordination? And how will technologically sophisticated home care agencies interact with other providers in the spectrum of care to coordinate care and support new models of care? In this issue of CARING, we provide readers with a series of articles and case studies – stepping stones to innovation – to determine a path forward.
Chris McGurn starts our technology coverage with our cover story. Several members of Congress got a hands-on telehealth demonstration when representatives from NAHC and its affiliate Home Care Technology Association of America (HCTAA) visited them in their offices on Capital Hill. NAHC President Val J. Halamandaris stressed the benefits and cost-saving potential. "Once you see how this technology works, we hope you will become an advocate of it," said Halamandaris. "There's a real premium in keeping people out of the hospitals, which these devices help to do."
:: Read
Full Article
Senate Majority Leader Harry Reid (D-NV) Confirmed as a General Session Speaker at NAHC's Annual Conference & Exposition
Senate Majority Leader Harry Reid has earned the respect of colleagues from both sides of the political aisle. Every day, he delivers results for all Nevadans, including hardworking families like those he grew up with in the small mining town of Searchlight, Nevada.
Despite his leadership position, he still lives in Searchlight, and he is still guided by the lessons and values he learned there. The Las Vegas Sun newspaper summarized Senator Reid's story by saying that he "has gone from underdog to Senate's top dog."
Since Nevadans first elected him to the Senate in 1986, Senator Reid has developed a reputation as a consensus builder and skillful legislator. His Republican colleagues have praised his reasoned, balanced approach. Senator Orrin Hatch, Republican from Utah, has said, "We all respect Senator Reid. He is one of the moderate voices around here who tries to get things to work."
To register for this year's Annual Meeting, and to hear Majority Leader Harry Reid in person, please click here.
Home Delivered Meals Promote Rebalancing, Save States Money, Says Health Affairs
Earlier this month, Health Affairs released a study titled "Providing More Home-Delivered Meals is One Way to Keep Older Adults with Low Care Needs out of Nursing Homes" (the Study). In the Study, authors Kali S. Thomas and Vincent Mor of Brown University demonstrated how programs that deliver meals to seniors can transfer "low-care" senior out of nursing homes into the home and community. The Study showed 26 out of 48 states seeing savings, which would approach $110 million. Highlights of the Study are discussed below.
Background Data
The Study gave 2011 expenditures on home delivered meals as totaling approximately $769 million nationally. Baseline data from 2009 showed that approximately 3 percent of senior citizens nationwide received home-delivered meals, with state percentages varying from a high of 8 percent in Wyoming to a low of less than 1 percent in Maryland.
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Phone: (202) 547-7424Fax:
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Heavy snow piles up in central Alberta, frigid temperatures gradually ease
Friday, November 22, 2013, 7:35 AM -
The classic battle between fall and winter continues across Alberta, with winter maintaining an upper hand.
A frontal system in British Columbia moved into northwestern Alberta on Thursday, prompting snowfall warnings for parts of the region.
"General amounts of 10 to 20 cm of snow are expected, however heavy bands of snow are likely to develop, which may locally produce higher amounts of snow up to 30 cm," Environment Canada said in a statement.
The snowfall warning remained in place for the City of Edmonton early Friday, with an additional 5 cm of snow forecast.
"All snowfall warnings will likely end later this morning, as the system weakens," EC adds.
This latest blast of snow comes after frigid temperatures settled into the province on Wednesday.
Morning lows dipped to -30 in places like Edmonton, feeling closer to -40 with the wind chill.
Fortunately, these bone chilling conditions are expected to gradually ease through the weekend.
"The core of the Arctic air is sliding off to the east, bringing some relief from these frigid and extreme temperatures," says Doug Gillham, a meteorologist at The Weather Network.
Will the temperature roller coaster continue into December?
Dramatic fluctuations in the weather are typical of autumn in Canada as we transition from summer to winter, but with less than two weeks to go until the start of December, will the temperature roller coaster continue?
Gillham has those answers in his latest Insider Insight.
Be sure to also check back next Monday, November 25 as we release the 2013/14 Winter Outlook.
HELP MAKE THE WEATHER NETWORK BETTER FOR YOU: We're looking for users that are interested in providing us valuable insight to help us enhance our products and services. Would you like to share your opinion? If so, please CLICK HERE TO PARTICIPATE.


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Ring and Pinion, 3.73 Ratio; 96-98 Grand Cherokee ZJ, for Dana 44
Ring/Pinion for Dana 44, 3.73 1996-1998 Jeep Grand Cherokee (ZJ) By Omix-ADA
Pricing: $840.99
Have you added taller tires? But now your torque is gone? You can regear to restore the missing performance. Omix-ADA has a full range of ring and pinion gear sets for a variety of Jeep axles.
- 16514.21
- 16514.21
- 804314032296
- Omix-ADA
- New
Shipping Information
- Item Requires Shipping
- 18.4 lbs.
- W11.2500" x H6.0000" x L11.5000"
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Red vs white meat, which is healthier?
Meat is one important food source for the body. Protein, minerals, and the high iron in it is very beneficial for the formation of cells, tissues, and make your limbs function properly.
According to healthmeup.com, there are two main types of meat that you can consume. Namely red meat and white meat. Actually, between red and white meat, which is healthier?
White meat
White meat consists of chicken, fish, poultry and other meats.
Nutrition, for example every 100 grams of chicken breast contains 172 calories,
21 g protein, 9 g fat, 0 g carbohydrate, 2.7 g saturated fat, 64 mg cholesterol, and 63 mg of sodium.
Benefits ,white meat is a source of protein, and is very low in calories.
Nutrition, one serving of chicken breast contains vitamin B, B6, B12, C, D and E, niacin, and magnesium.
Shortage, chicken breasts tend to have a drier texture, which can make it boring to eat.
In conclusion if you need a lot of protein but low in calories, then you should have white meat. That's why white meat is incorporated into a lot of healthy diets.


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Sign up for exclusive email offers from Air Savings to destinations around the world and save even more off the next flight you book with us.
Now all ye desis can have your favorite caffeine fix Starbucks just before boarding the flight or mere moments after landing in Mera Bharat Mahaan.
Tata Starbucks, the U.S. coffee chain's Indian joint venture, has opened a store in Terminal 3 (domestic terminal) of the Indira Gandhi International Airport in New Delhi.
A second Starbucks coffeeshop will come up soon in the international terminal of the New Delhi airport.
Tata Starbucks CEO Avani Saglani Davda described the store's launch at the New Delhi Indira Gandhi International airport as "consistent with our commitment to work thoughtfully to open stores in locations where our customers want and expect us to be."
Like in the U.S., Starbucks has identified the travel segment as a major growth opportunity in India and intends to aggressively go after that market.
In the U.S., Starbucks coffeeshops are a common sight at rest areas on major interstate highways like New Jersey Turnpike. Most big U.S. airports including JFK, LaGuardia and Newark, NJ have Starbucks stores.
As part of its expansion in the Indian travel segment, Starbucks will launch coffee shops at the Chhatrapati Shivaji International Airport in Mumbai by the end of March 2013.
Don't be surprised to see Starbucks kapi stores at Chennai and other South Indian airports later this year.
Starbucks' Indian airport stores offer Arabica coffee from the region as well as breakfast items like vegetarian and non-vegetarian sandwiches including Mediterranean Foccasia with Roast Vegetables, Wild Mushroom Pockets and Chicken Ham and Roasted Peppers Ciabata.
Also on Starbucks airport shops' menu are cakes, muffins, pastries, breads and wraps.
Starbucks entered India in October 2012 with three stores in Mumbai, at the historic Elphinstone Building in Horniman Circle, at the Oberoi Mall and the Taj Mahal Palace Annexe.


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I was shocked last night
So last night i did bed time routines like i do every night.
And my husband is always joking that vincent hates him and is always screaming and crying at him.
So as bed time last night approches i fed vince, and went and told haiden and logan to say good night to daddy abd they did. I went up and brushed haiden and logans teeth and read each a story. As i was reading a story i could hear vincent crying. So after i finished story i went back down to my husband and vincent watching my husband trying to calm down.
As soon as vince saw me he calmed down, so we played a game, my husband held him and turned him away from me and he screamed and cried. We did this repetedly, so i took vince and was about to read to him. My husband came back and as soon as he got close to vincent, he would cry and burry his head into me. My husband kept doing it and vince did the same thing. Then my husband said good night, and i read vince a story, tucked him in his crib and said good night.
But cant beleive how vincent acted last night. He really does hate his father.


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Property: Le RihaniAddress: 52 Derb El Arsa | Kennaria, Marrakech 40000, Morocco
Phone Number:
Location: Morocco > Marrakech-Tensift-El Haouz Region > Marrakech
Bar / Lounge Beverage Selection Free Breakfast Free High Speed Internet ( WiFi ) Restaurant Room Service Spa Suites Swimming Pool Airport Transportation
#50 Spa Hotel in Marrakech
#77 Romantic Hotel in Marrakech
#96 Family Hotel in Marrakech
Price Range (Based on Average Rates): $$
Number of rooms: 8
- Official Description (provided by the hotel):
- The luxury of a palace, the warmth of a familyhome, simplicity under the stars... ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Priceline and Agoda so you can book your Le Rihani reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Le Rihani Marrakech
- Le Rihani Hotel Marrakech


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Target: Jim Butler, Chair of the Ohio House of Representatives Judiciary Committee
Goal: Stop domestic homicide by charging harsher punishments for non-fatal strangulation.
Andre Jeter allegedly stabbed his wife, Monica Weber-Jeter, 28 times. Weber-Jeter spent 32 days in the ICU before succumbing to her injuries. Her murder was preempted by a string of escalating attacks by her husband, including a near-fatal strangulation. Now, her family is pushing for stricter penalties for non-fatal strangulation. Demand that non-fatal strangulation be made a felony in Ohio and help prevent domestic homicide.
Strangulation is one of most accurate predictors of future homicide in domestic partnerships. It is also one of the most common forms of physical abuse. Studies suggest that up to 68 percent of women in abusive relationships have been strangled by their partner at least once. These attacks are red flags for an escalation of violence in the partnership. Weber-Jeter endured at least one strangulation attempt by her husband prior to her murder and she nearly died. Jeter admitted to strangling his wife and served only 11 days of a 180-day sentence. If he had been convicted of a felony instead of a misdemeanor, then Weber-Jeter might still be alive.
Weber-Jeter's family initiated 'Monica's Law' after her death. This bill identifies strangulation as lethal force and classifies it as a third-degree felony. Thirty-eight states already prosecute strangulation as a felony. It is time that Ohio joined them and took a stand against domestic violence. Sign below to urge the Ohio House of Representatives Judiciary Committee to pass this bill and take a step towards ending domestic homicide.
Dear Chairman Butler,
Monica Weber-Jeter died after her husband allegedly stabbed her 28 times. This wasn't the first time Andre Jeter attacked his wife, though. Early in the year, Andre confessed to strangling his wife until she was nearly unconscious. He served 11 days of his 180-day sentence and was released on probation. Nine months later, his wife was dead.
Strangulation is one of the most accurate predictors of homicide in domestic partnerships. Nearly 68 percent of women in abusive partnerships have reported being strangled by their partner at least once. Strangulation is a form of lethal force and often leads to domestic homicide.
In Ohio, non-fatal strangulation is considered a misdemeanor and carries a light punishment. Thanks to 'Monica's Law', that could change. If passed, this bill will make non-fatal strangulation a third-degree felony. We urge you to pass this bill and take a stand against domestic violence.
[Your Name Here]
Photo Credit: holder


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Kid Dynamite - 3 O'Clock MP3
Amazing song from the now seminal debut release from Kid Dynamite. Lyrics: I know you asked for rain, but I brought you fire. I didn't think you'd mind. I know it's ...
[Just Dance 3] Dynamite - Taio Cruz MP3
[HD Link] http://www.youtube.com/watch?v=VevE4v065sA&hd=1 It is recommended to watch the video in HD. • Song: "Dynamite" by Taio Cruz [Full version] ...
Kid Dynamite live at the Fest 10 - 1/3 MP3
Battlefield 3 - Kid Dynamite MP3
It's a hell of a thing, killing a man. You take away all he's got and all he's ever gonna have. Songs: Jim Noir - My Patch Jerry Reed - I'm Gonna Write a Song Me ...
Kid Dynamite - Kid Dynamite (Full Album) MP3
1. Pause 2. K05-0564 3. Sweet Shop Syndicate 4. Table 19 5. Ph. Decontrol 6. Showoff 7. Bookworm 8. Scarysmurf 9. The Ronald Miller Story 10.
Kid Dynamite - 3 o'clock MP3
released 01 October 1998 JT1039 Jason Shevchuk: Vocals Dan Yemin: Guitar Steve Farrell: Bass David Wagenschutz: Drums Recorded September 1998 ...
Kid Dynamite - Living Daylights & Bookworm Live @ FYF Fest 9-3-11 in HD MP3
First show in l.a. in 12 years, so amazing.
Just Dance 3 Dynamite Taio Cruz YouTube MP3
Jacques Rougeau vs. Dynamite Kid FIGHT story (Part 3) MP3
JACQUES ROUGEAU VS DYNAMITE KID FIGHT STORY - PART 3 This is the 3rd and final part of the most detailed interview ever with "The Mountie" Jacques ...
Kid Dynamite - Shorter, Faster, Louder [ FULL ALBUM ] MP3
Shorter, Faster, Louder LP (Jade Tree, 2000) TRACK LISTING: 1. Pits & Poisoned Apples 2. Death and Taxes 3. Cheap Shot Youth Anthem 4. Copout 5. Handy ...
TFDA SHOWDOWN 2012 vol 3. Poppin Quarterfinal 1. Kid Dynamite vs Funky Mo MP3
Poppin Quarterfinal 1 / Kid Dynamite(Winner) vs Funky Mo at TFDA SHOWDOWN 2012 vol 3 This a battle and show competition that has been taking place now ...
This Time Next Year - 3 o'clock (Kid Dynamite) MP3
no one had this uploaded so i did the world a favor.
Kid Dynamite - Shorter, Faster, Louder (Full Album) MP3
1. Pits & Poisoned Apples 2. Death and Taxes 3. Cheap Shot Youth Anthem 4. Copout 5. Handy with the Tongue Sword 6. Living Daylights 7. Introduction to the ...
Kid Dynamite - Live at the Fest 10 - Part 2/3 MP3
Kid Dynamite - Self Titled (Full Album - 1998) MP3
Banda: Kid Dynamite Album: Self Titled Año: 1998 Sello: Jade Tree Genero: Hardcore/Punk Tracklist: 01. Pause 02. K05-0564 03. Sweet Shop Syndicate 04.
Kid Dynamite - Live at the Fest 10 - part 3/3 MP3
Kid Dynamite VS Michael Wagner (Part 3) MP3
29 March 2014.
Mike Tyson's Punch Out #3 - Kid Dynamite Himself MP3
and that marks the end of Mike Tyson's Punch Out. see ya next playthrough :)
KidDynamite vs pangstrom pt.3 MP3
quick basement fight ufc rules third and final fight of the night.
Tiger Mask vs Dynamite Kid- Match 3 part 1 MP3
Tiger Mask vs Dynamite Kid- Match 3 (1-28-82)
Kid Dynamite @ Groezrock 2013 (Club Show), FULL SET - part III MP3
Kid Dynamite @ Groezrock 2013 (Club Show). Friday 26 April 2013. FULL SET footage. Awesome gig in a 350 people capacity venue. It was amazing!
XBTW-Mr.Flex V.S Kid Dynamite (best 2 out of 3) MP3
If you leave negative comments about a little kid, I will not reply to it and it will be deleted. Troll elsewhere) Instagram: Diesel_True Brah Brah having fun riding in ...
Paul Bley Kid Dynamite MP3
Disclaimer: I don't own the music or the pictures, they belong to their rightful owners. Buy Paul Bley music on i Tunes. Paul Bley - Piano Gary Peacock - Bass ...
Kid Dynamite - Hateful MP3
JT1085 KID DYNAMITE was: Jason Shevchuk: Vocals David Wagenschutz: Drums Dan Yemin: Guitar, Bass on tracks 1-3 & 5 Steve Ferrell: Bass on tracks 4 ...
LIGHTYEAR - Kid Dynamite MP3
Legendary Derby ska punkers Lightyear! This is the third song from their 3 track self-released CD-R 'Just Another Demonstration'. Recorded at Premier Studios ...
Kid Dynamite and The Blast - Live at Long Wongs Tempe, AZ 2/4/14 MP3
Kid Dynamite & The Blast - Live at Long Wongs Tempe, AZ 2/4/14 All songs included in this video will be on the upcoming 2014 LP "Year of the Blast".[excluding ...
Kid Dynamite - Bookworm MP3
JT1085 KID DYNAMITE was: Jason Shevchuk: Vocals David Wagenschutz: Drums Dan Yemin: Guitar, Bass on tracks 1-3 & 5 Steve Ferrell: Bass on tracks 4 ...
Cus D'Amato TIGHT DEFENCE THE D'amato Style Number System... 7 Jab,,, 1 Lead Hook,,, 2 Rear Straight Punch,,, 3 Lead Uppercut,,,, 4 Rear Uppercut,,,, ...
Tiger Mask, Great Sasuke and Mil Mascaras vs Dynamite Kid, Dos Caras and Kuniaki Kobayashi - 3/3 MP3
Part 1/3 (end of the match, in-ring promos) The Original Tiger Mask, The Great Sasuke and Mil Mascaras vs Dynamite Kid, Dos Caras and Kuniaki Kobayashi on ...


text: WSI Industries, Inc. (Nasdaq: WSCI) today reported net sales of $4,657,000 for the third quarter of fiscal 2010 ended May 30, 2010, which were comparable to the prior fiscal 2009 third quarter of $4,736,000. The Company's net income improved significantly to $158,000 or $.06 per diluted share versus the prior year quarter of $12,000 or break-even per diluted share. Year-to-date sales in fiscal 2010 were $12,970,000 as compared to $14,773,000 a year ago. Year-to-date diluted earnings per share for fiscal 2010 were at $.10 as compared to a loss of $.07 in the prior year. Michael J. Pudil, chief executive officer, commented: "As reflected in our fiscal 2010 third quarter results, we have continued our steady growth out of the recession with our earnings. At $.06 per diluted share, our third quarter net income is at its highest level in 7 quarters." Pudil continued: "Our ATV and motorcycle markets continue to lead the way with an increase in sales over the prior year quarter. In addition, and for the first time in several quarters, we have seen simultaneous growth in other parts of our core business as we experienced sales increases in our aerospace, defense and biosciences markets over the prior year fiscal third quarter. We anticipate that our fiscal 2010 fourth quarter will show additional improvements." President and COO Benjamin Rashleger stated: "We are very pleased with our results for the quarter considering the continued suppression of the manufacturing segment. These results again show WSI's success in managing our business and costs in parallel with our current business levels. We have begun to see some industries show signs of improvement in their demand, and we will continue to cautiously control our expenses and make the necessary investments and staffing adjustments as we find them necessary to support our customer's needs." Rashleger continued: "We are finalizing our branding and marketing strategy, as well as our web site redesign project, and we are optimistic that these tools will provide support to our goal of expanding and diversifying our customer base. We are excited about the opportunities we are working on, and we continue to remain focused on managing our business to provide the highest value of service to our customers – existing and new."