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The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas' disease chemotherapy is sterol 14α-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols.
Methodology/Principal Finding
In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51Mt), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51Mt. Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51Tc, demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site.
CYP51Mt-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51Tc.
Author Summary
Enzyme sterol 14α-demethylase (CYP51) is a well-established target for anti-fungal therapy and is a prospective target for Chagas' disease therapy. We previously identified a chemical scaffold capable of delivering a variety of chemical structures into the CYP51 active site. In this work the binding modes of several second generation compounds carrying this scaffold were determined in high-resolution co-crystal structures with CYP51 of Mycobacterium tuberculosis. Subsequent assays against CYP51 in Trypanosoma cruzi, the agent of Chagas' disease, demonstrated that two of the compounds bound tightly to the enzyme. Both were tested for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. This compound is currently being evaluated in animal models of Chagas' disease. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability of a single amino acid residue at a critical position in the active site. Our work is aimed at rational design of potent and highly selective CYP51 inhibitors with potential to become therapeutic drugs. Drug selectivity to prevent host–pathogen cross-reactivity is pharmacologically important, because CYP51 is present in human host.
Citation: Chen C-K, Doyle PS, Yermalitskaya LV, Mackey ZB, Ang KKH, McKerrow JH, et al. (2009) Trypanosoma cruzi CYP51 Inhibitor Derived from a Mycobacterium tuberculosis Screen Hit. PLoS Negl Trop Dis 3(2): e372. https://doi.org/10.1371/journal.pntd.0000372
Editor: Greg Matlashewski, McGill University, Canada
Received: September 5, 2008; Accepted: January 6, 2009; Published: February 3, 2009
Copyright: © 2009 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH RO1 grant GM078553 and by the Vanderbilt-Meharry Center for AIDS Research (to L.M.P.), NIAID-TDRU (Tropical Disease Research Unit) grant AI35707 (to JHM), and the Sandler Family Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The drug development pipeline targeting diseases caused by trypanosome parasites is sparse [1]. Despite significant advances in its control over the last 15 years [2], Chagas' disease, caused by the parasitic protozoan Trypanosoma cruzi [3], remains a major public health concern in Latin America, with an estimated total of 8 million people infected [4]. Nifurtimox and benznidazole, the two principal drugs for treatment of Chagas' disease, were launched in 1967 and 1972 respectively, and suffer from the twin liabilities of serious side-effects and reduced efficacy in chronic T. cruzi infections [2]. A potential new target for Chagas' disease chemotherapy is sterol 14α-demethylase (CYP51) [5], a cytochrome P450 heme thiolate-containing enzyme which is involved in biosynthesis of membrane sterols in all biological kingdoms from bacteria to animals [6]. T. cruzi sterols are similar in composition to those in fungi, with ergosterol and ergosterol-like sterols the major membrane components [7]. Clinically employed antifungal azoles [8],[9] inhibit ergosterol biosynthesis in fungi and are partially effective against Leishmania and Trypanosoma parasites [10]–[12]. Azoles block CYP51 activity, resulting in decline of the normal complement of endogenous sterols and accumulation of various 14α-methyl sterols with cytostatic or cytoxic consequences [11]. Aside from the compounds optimized for antifungal therapy, other CYP51 inhibitors with strong anti-T. cruzi activity have also been reported [13]–[15].
Mammalian CYP51 shares relatively modest overall sequence identity – below 30% – with its fungal and protozoan counterparts, but within the active site the amino acid residues are far more conserved. Based upon crystal structures of CYP51 of M. tuberculosis (CYP51Mt) [16]–[20], three of the thirteen active site residues, Y76, F83, and H259 (numbering according to CYP51Mt), are invariant throughout the cyp51 gene family. Two residues, F78 and F255, are specific to the methylation status of the C-4 atom in the sterol nucleus [18],[21], and amino acid identities of seven other positions strongly overlap across phyla [19],[20]. Of the thirteen residues, only one, R96, seems to be phylum-specific. This similarity confines design of selective CYP51 inhibitors to a species-specific cavity in the active site defined by the hydrophobic residues F78, L321, I322, I323, M433, and V434.
To discover novel inhibitors, we previously screened a library of small synthetic molecules against the CYP51Mt target [19]. The N-[4-pyridyl]-formamide moiety of the top hit, α-ethyl-N-4-pyridinyl-benzeneacetamide (EPBA), was found to bind unvaryingly in the CYP51 active site with Y76, H259, and the heme prosthetic group. The uniformity of interactions with CYP51 suggested that this scaffold could be used to target a variety of chemotypes to the active site. To verify this assumption, we determined the binding modes of second generation compounds containing the N-[4-pyridyl]-formamide moiety by determining their co-crystal structures with CYP51Mt. We also spectrally characterized binding of these compounds to CYP51 of both T. cruzi (CYP51Tc) and the related protozoan parasite T. brucei, the causative agent of African sleeping sickness, (CYP51Tb). Two compounds were selected based on their nanomolar binding affinities toward CYP51Tc and subsequently tested in vitro for inhibitory effects against both pathogens. One of the two compounds revealed potent and selective inhibitory effect against T. cruzi infection in mouse macrophage cells.
Preparation of CYP51Mt
CYP51Mt double C37L/C442A and triple C37L/F78L/C442A mutants were prepared as described elsewhere [19]. The surface exposed cystein residues C37 and C442 were removed via replacement with leucine and alanine, respectively, to improve protein homogeneity and aid crystallization [18]. The functionally important F78 in the active site was replaced in the triple mutant by leucine, which invariantly occupies this position in the mammalian CYP51 isoforms.
Preparation of CYP51Tc
Design of the CYP51Tc expression vector was based on an entity in the NCBI data bank (ID AY283022 [22]), which was modified by replacing the first 31 residues upstream of Pro32 with the fragment MAKKTSSKGKL from the CYP2C3 sequence [23] (CYP2C3 residues marked in bold) to improve protein solubility, and by inserting a His6-tag at the C-terminus to facilitate purification. This coding sequence (kindly provided by M. Waterman in the form of the pET vector) was subsequently sub-cloned into pCWori vector [24] between the NdeI and HindIII restriction sites and in this form used to transform Escherichia coli strain HMS174(DE3).
Transformants were grown for 5 h at 37°C and 250 rpm agitation in Terrific Broth medium supplemented with 1 mM thiamine, 50 µg/ml ampicillin, and trace elements. CYP51Tc expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration 0.2 mM) and δ-aminolevulinic acid, a precursor of heme biosynthesis (final concentration 1 mM). Following induction, temperature was decreased to 25°C and agitation to 180 rpm. After 30 hours the cells were harvested and lysed by sonication. Insoluble material was removed from crude extract by centrifugation (30 min at 35,000 rpm). The supernatant was subjected to a series of chromatographic steps, including nickel-nitrilotriacetic acid (Ni-NTA) agarose (QIAGEN), followed by Q-Sepharose (Amersham Biosciences) in the flow-through regime, and then by S-Sepharose (Amersham Biosciences). From the S-Sepharose, protein was eluted in a 0.2 to 1.0 M NaCl gradient and observed by means of a 12% SDS-PAGE to be virtually homogeneous. Fractions containing P450 were combined, concentrated using a Centriprep concentrating device (Millipore), and stored at −80°C. Twenty mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, and 1 mM DTT were maintained throughout all chromatographic steps. Spectral characteristics of CYP51Tc are shown in Figure 1A.
Main panel shows the absolute protein spectrum, while insert shows CO-bound reduced difference spectrum.
Preparation of CYP51Tb
The expression vector for CYP51Tb (ID EAN79583) was generated using T. brucei genomic DNA and upsteam GCGCGCATATGGCTCTTGAAGTTGCC and downstream CGCAAGCTTCTAGTGATGGTGATGGTGATGGTGATGAGCAGCTGCCGCCTTCC primers. The underlining denotes an NdeI restriction cloning site in the upstream primer and the HindIII restriction cloning site in the downstream primer followed by the stop codon. The bold sequence in the upstream primer highlights second codon replaced with alanine to optimize expression in E. coli cells [24]. The boldface in the downstream primer indicates the His8 tag. The original genomic DNA contained internal NdeI site at 345 base pair which was removed by introducing a silent mutation via the quick-change mutagenesis protocol (Stratagene). DNA amplification reaction was carried out as follows: 5 min at 94°C, annealing for 1 min at 55°C, and extension for 1 min at 72°C, for 30 cycles, followed by extension for 10 min at 72°C. The purified 1.5 kb PCR product was ligated into the pCR 2.1 TA cloning vector (Invitrogen). Insert was subsequently cleaved with NdeI and HindIII and ligated into pCWori vector digested with the same restriction enzymes and treated with alkaline phosphatase. The identity of the resulting vector was confirmed by DNA sequencing.
E. coli HMS174(DE3) strain was co-transformed with this vector and the pGro7 plasmid (Takara) encoding the E. coli chaperones GroES and GroEL. Double transformants were selected on agar plates containing both ampicilin and chloramphenicol. One liter of Terrific Broth medium supplemented with 1 mM thiamine, 100 µg/ml ampicillin, 40 µg/ml chloramphenicol, and trace elements was inoculated with 10 ml of overnight culture and growth continued at 37°C and 250 rpm agitation until OD600 reached 0.3. At that point expression of chaperones was induced with 0.2% arabinose. Growth continued at 27°C and 180 rpm until OD reached 0.6. Then CYP51Tb expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration 0.3 mM), and δ-aminolevulinic acid (1 mM). Following induction, temperature was decreased to 15°C. After 48 hours the cells were harvested and lysed by sonication. Purification was conducted similarly to as described above for CYP51Tc, with the qualification that S-Sepharose was used in the flow-through regime, while the protein was bound to and eluted from the Q-Sepharose column. Spectral characteristics of CYP51Tb are shown in Figure 1B.
Crystallization, Data Collection, and Determination of Crystal Structure
Five compounds (Fig. 2), purchased from ChemDiv (San Diego, California) were used for co-crystallization with the CYP51Mt C37L/C442A double mutant. Compared to the wild type, this construct has superior propensity for crystallization. Compound numbering is according to the order in which they were received in our laboratory, with number 7 being the first used in the current work. Ligands were dissolved in Me2SO at ≤100 mM stock concentration, and brought to final concentrations ranging from 1 to 5 mM in the crystallization mix, depending on ligand solubility. Protein concentration was 0.2 mM. A narrow crystallization screening grid (15–30% PEG 4000, 2–12% isopropanol, 0.1 M HEPES, pH 7.5), previously devised to obtain CYP51Mt crystals [16],[18],[19] was utilized for co-crystallization of complexes by the vapor diffusion hanging drop method. Four co-crystal forms were obtained, all diffracted to resolutions between 1.56 to 1.60 Å. Diffraction data were collected at 100–110 K at the Southeast Regional Collaborative Access Team (SER-CAT) 22ID beamline, Advanced Photon Source, Argonne National Laboratory using SER-CAT mail-in data collection program (Table 1). The images were integrated and the intensities merged with the HKL2000 software suite [25]. The structures were determined by molecular replacement using coordinates of estriol-bound CYP51Mt (Protein Data Bank ID 1X8V) as a search model. The final atomic models were obtained after a few iterations of refinement using REFMAC5 [26] and model-building using the COOT graphics modeling program [27]. The quality of the structures was assessed by the program PROCHECK [28]. One residue, A46, was found in the generously allowed region of the Ramachandran plot in all structures where, together with the adjacent G47, it enables a sharp turn between two β strands.
1Compounds are identified by the numbers in the ChemDiv, Inc., product library catalog.
Spectroscopic Binding Assays
Spectroscopic binding assays were performed at room temperature in 1-ml quartz cuvette containing 1 µM or 2 µM CYP51 in 50 mM Tris-HCl, pH 7.5, and 10% glycerol using a Cary UV-visible scanning spectrophotometer (Varian). Concentration of CYP51 was determined at 450 nm from the difference spectra between the carbon monoxide-bound ferrous and water-bound ferric forms, with an extinction coefficient of 91,000 M−1 cm−1 [29]. In the first round, compounds dissolved in Me2SO at 10 mM concentration were added to the 2 µM protein solution in 0.5 µl aliquots, resulting in concentration increases from 5 µM to 50 µM in 5 µM increments. The same amounts of Me2SO alone were added to the protein in the reference cuvette, followed by recording the difference spectra. In the second round, compounds with high affinities were diluted to 100 µM by Me2SO and titrated into 1 µM protein solution in 1 µl aliquots to increase compound concentration from 0.1 µM to 2 µM in 0.1 µM increments. To determine the KD, we used the GraphPad PRISM software (Graphpad Software Inc.) to fit titration data to either rectangular or quadratic hyperbolas to correct for the bound ligand fraction, according to the functions ΔA = (Amax(S/KD+S) or ΔA = (Amax/2[E])((KD+[E]+[L])−((KD+[E]+[L])2−4[E][L])1/2), respectively, where E is total enzyme and L total ligand concentration, Amax the maximal absorption shift at saturation, and KD the apparent dissociation constant for the enzyme-ligand complex.
T. cruzi Assay
Irradiated (1000 rads) J774 mouse macrophages were plated in 12-well tissue culture plates 24 h prior to infection with 105 T. cruzi Y strain trypomastigotes for 2 h at 37°C. Cultures were maintained in RPMI-1640 medium with 5% heat-inactivated fetal calf serum and 5% CO2 with the addition of 10 µM compound 8 or 10. Untreated controls, controls treated with the inhibitor K11777 (10 µM) [30],[31], and uninfected macrophage controls were also included. All cultures were in triplicate and medium was replaced every 48 h. Treatment with CYP51 inhibitors continued for up to 27 days. Subsequently, treated cultures were maintained without inhibitor for an additional 13–15 days to confirm inhibitor effectiveness and cure of infected cells. Cultures were monitored daily by contrast phase microscopy to determine presence of T. cruzi infected cells and free infectious trypomastigotes (Table 2).
To determine IC50, mouse J774 macrophages were irradiated (1000 rads) to deter growth and plated onto 12-well tissue culture plates. Cells were infected with 105 tissue culture trypomastigotes of the Y strain of T. cruzi for 2 h at 37°C, as described above. Next, medium was replaced with the addition of compound 10 at 0, 1 nM, 10 nM, 100 nM, 500 nM, 1 µM, 5 µM, and 10 µM; these cultures were incubated for 52 h at 37°C. Controls with 10 µM K11777 and 10 µM compound 8 were also included. All treatments were performed in triplicate to ensure statistical validity. Cultures were then fixed in 4% paraformaldehyde in PBS for 2 h at room temperature and stained with DAPI (10 nM) in PBS. One hundred cells and their intracellular parasites were quantified as previously described to estimate the mean number of parasites/cell [32]. Mean P/cell data were plotted against compound concentration to estimate the IC50.
Mammalian Cell Toxicity Assay
Toxicity was evaluated in bovine muscle cells (BESM), mouse J774 macrophages, and human Huh7 hepatocytes against compound 10 at 10 µM, 50 µM and 100 µM concentrations. After 48 h in culture at 37°C, cells were stained with 10% Tripan Blue and the number of live versus dead cells was quantified (Table 3).
T. brucei Assay
Trypanosomes were grown in complete HMI-9 medium containing 10% FBS, 10% Serum Plus medium (Sigma Inc. St. Louis Mo. USA) and 1× penicillin/streptomycin. Trypanosomes were diluted to 1.0×105/ml in complete HMI-9 medium. Diluted trypanosomes were aliquoted in Greiner sterile 96-well flat white opaque culture plates using a WellMate cell dispenser (Matrix Tech., Hudson, NH, USA). Compounds 8 and 10 were serially diluted in Me2SO. Trypanosomes were incubated with the compounds for 48 h at 37°C with 5% CO2 before monitoring viability. Trypanosomes were then lysed in the wells by adding 50 µl of CellTiter-GloTM (Promega Inc., Madison, WI, USA). Lysed trypanosomes were placed on an orbital shaker at room temperature for 2 min. The resulting ATP-bioluminescence of the trypanosomes in the 96-well plates was measured at room temperature using an Analyst HT plate reader (Molecular Devices, Sunnyvale, CA, USA).
Crystal Structures of CYP51Mt-Inhibitor Complexes
Co-crystals were obtained for compounds 8, 9 and 11. Compound 10 failed to generate any crystals with CYP51Mt. Compound 7 was not found in the CYP51Mt active site in the crystal, which is consistent with lack of spectrally detectable binding (Fig. 2). Compounds 8 (3-{[(4-methylphenyl)sulfonyl]amino}propylpyridin-4-ylcarbamate), 9 (cis-4-methyl-N-[(1S)-3-(methylsulfanyl)-1-(pyridin-4-ylcarbamoyl)propyl]cyclohexanecarboxamide), and 11 (N-[(1S)-2-methyl-1-(pyridin-4-ylcarbamoyl)propyl]cyclohexanecarboxamide), were observed bound in the CYP51Mt active site as predicted, through the coordination of the heme iron via a lone pair of aromatic nitrogen electrons of the N-[4-pyridyl]-formamide moiety (highlighted in gray in Fig. 2) and interactions with the invariant residues Y76 and H259 (Fig. 3). Functional groups other than the N-[4-pyridyl]-formamide moiety in compounds 9 and 11 either were accommodated in the species-specific cavity or else protruded through the opening of the active site toward bulk solvent. H259 hydrogen-bonded to the carbonyl oxygen in both compounds, while interactions with Y76 were mediated by two similarly positioned water molecules (Figs. 3A and 3B). The residual Fo-Fc electron density map suggested two alternative conformations for compounds 11 and 9, designated by pink and cyan respectively in Figures 4A and 4B. In the CYP51Mt-compound 11 complex, the cyclohexane ring protruded toward the bulk solvent (Fig. 3A), barely interacting with the protein in two alternative conformations (Fig. 4A). Together with the limited interactions of the isopropyl moiety, this lack of contact explains the low binding affinity of 11. In the CYP51Mt-compound 9 complex, the methylcyclohexane moiety protruded toward bulk solvent, while the methylsulfanyl group loosely bound in the species-specific cavity (Fig. 3B) in two alternative conformations (Fig. 4B). The side chain of M433 also adopted two alternative conformations. In both complexes, a portion of the BC-region was disordered and missing from the electron density map. Although racemic mixtures were used for co-crystallization, only one enantiomer of each compound was found in the active site.
Compounds 11 (A), 9 (B), and 8 (C) (highlighted in pink) bound in the active site of CYP51Mt are shown looking in from the active site opening. For clarity only one conformation of each compound is shown. Protein is represented by the semitransparent accessible surface (gray). The ordered BC-loop obstructs the view in the CYP51Mt-8 complex in (C). The invariable elements of the CYP51 active site, Y76, H259 (yellow), and heme (green), are in a stick mode. Water molecules are shown as red spheres. Oxygen atoms are red, nitrogen blue, sulfur yellow. Images were generated using the VMD program [35].
Compounds 11 (A), 9 (B), and 8 (C) are shown surrounded by the CYP51 active site residues. The fragments of the electron density 2Fo-Fc map (gray mesh) are cut at 1.2 σ. Different conformers in (A) and (B) are highlighted in pink and cyan. Images were generated using PYMOL [36].
A different binding mode was revealed for compound 8. Its flexible backbone allowed it to fold head-to-tail over the heme plane to bring the methylphenylsulfonamide group into intramolecular stacking interactions with the pyridinyl moiety and also with the heme macrocycle (Fig. 3C, 4C). Folding minimized the nonpolar surface of compound 8 by exposing the sulfonamide group to interactions with Q72, K97, and the heme propionate side chain. The hydrophobic side chain of K97 aligned along the methylphenyl moiety. A similar folding of the benzothiadiazolsulfonamide group has been observed in previous work for 2-[(2,1,3-benzothiadiazol-4-sulfonamide]-2-phenyl-N-pyridin-4-acetamide (BSPPA) [19]. Mutually stabilizing protein-ligand interactions involving the BC-loop residues including F78 result in increased binding affinity of the CYP51Mt-compound 8 complex and in unambiguous electron density both for compound 8 (Fig. 3C) and for the entire BC-region. In the CYP51Mt-compound 8 complex, H259 directly H-bound to the amide nitrogen of compound 8, whereas Y76 interacted hydrophobically with the compound's flexible backbone (Fig. 3C).
Binding Affinities of CYP51 Ligands
Binding affinities of all five compounds were examined against both wild type and a 'humanized' F78L mutant form of CYP51Mt, CYP51Tc, and CYP51Tb using spectroscopic assays (Fig. 5). These assays utilize the property of P450 enzymes to shift the ferric heme iron Soret band following replacement of a weak ligand, the water molecule, with a stronger one, the nitrogen-containing aromatic pyridinyl group (Fig. 5A). All compounds had markedly reduced or no binding affinity toward CYP51Mt, compared to the parental EPBA (Fig. 2). No binding was observed for compound 7, while the KD for compound 11 exceeded 100 µM, indicating weak binding.
(A) Type II spectral responses of CYP51Tc to increasing concentrations of compound 10. The concentration dependence of compound 10, fluconazole (B), and compound 8 (C) binding were deduced from the difference absorption changes obtained from the titration of CYP51Tc with increasing concentrations of the inhibitor. The concentration dependence of fluconazole (D) was deduced from the difference absorption changes obtained from the titration of CYP51Tb.
However, the binding affinity of all compounds examined, including compound 7, was significantly higher to CYP51Tc than to CYP51Mt. Remarkably, the binding affinities of compounds 8 and 10 to CYP51Tc were 300- and at least 500-fold respectively higher, equaling or exceeding that of the antifungal CYP51 inhibitor fluconazole, which was used as a reference (Fig. 5B, C). A KD of at least 40 nM was estimated for compound 10 by spectral assays, with the binding curve reaching a plateau at about a 1∶1 protein to ligand ratio. This value strongly suggests that the KD must be notably higher, although further dilution of protein in an attempt to obtain a more accurate value significantly decreased the quality of the spectra and this effort was thus abandoned. The IC50 of ∼1 nM for T. cruzi intracellular growth inhibition, determined for compound 10 as described below, may better reflect true KD value. Compounds 8 and 10, which had highest binding affinity to CYP51Tc, were spectrally silent toward CYP51Tb, indicating no binding in the active site (Fig. 2). As expected, CYP51Tb had nanomolar affinity for fluconazole (Fig. 5D), but again, the plateau was reached at a 1∶1 protein to inhibitor ratio, so the binding constant could not be determined more accurately. Compound 9 bound both CYP51Tc and CYP51Tb with the same affinity.
The KD values for compounds 8 and 10 slightly decreased for the F78L CYP51Mt mutant compared to the wild type, while the KD values for the other compounds increased (Fig. 2).
Inhibitory Effects against T. cruzi
With submicromolar affinities toward CYP51Tc of 160 nM and <40 nM respectively, compounds 8 and 10 were examined in vitro for inhibitory effects against both T. cruzi and T. brucei. In a mouse macrophage assay, T. cruzi completed its intracellular development in 5 days in untreated controls, resulting in death of host macrophages and abundant trypomastigotes in culture supernatant (Table 2). As anticipated, the control compound K11777 [30] cured T. cruzi infection. No parasites survived a treatment regime of 27 days with compound 10. Cure of host cells was confirmed by incubation of the cultures for an additional 15 days in the absence of inhibitor. In contrast, and similarly to untreated controls, T. cruzi completed its development in 5 days in cultures treated with compound 8.
An IC50 of ∼1 nM concentration for compound 10 (Fig. 6) was estimated for T. cruzi intracellular amastigotes. T. cruzi developed well intracellularly in untreated macrophages with a final mean number of 3.57±0.5 P/cell (0% inhibition). As determined previously, 10 µM compound 10 was deleterious for T. cruzi, with a mean of 0.25±0.01 P/cell (100% growth inhibition). Ten µM of control compound K11777 was also parasiticidal for T. cruzi with a mean of 0.25±0.01 P/cell (IC100) [30], while compound 8 was not parasiticidal at this concentration with a mean of 1.22±0.1 P/cell (data not shown).
T. cruzi intracellular multiplication was evaluated at 52 hr of incubation at several concentrations of the inhibitor by determining the number of parasites/cell. Intracellular parasites were counted per one hundred cells to estimate a mean number of parasites per cell. Approximation of concentration dependence of mean P/cell±SD data with a smooth curve highlights the 50% drop in parasite count at ∼1 nM compound 10. SD did not exceed 14% of the mean.
Toxicity for mammalian cells was addressed by treating the three different cell types with increasing concentrations of compound 10 (Table 3). No toxicity was observed at 10 µM compound 10, while 50 µM was mildly toxic for muscle cells. One hundred µM compound 10 was toxic for all mammalian cells tested, especially muscle cells.
Consistent with the spectral binding assays, neither compound 8 nor 10 had any inhibitory effects against cultured T. brucei even at the highest tested concentration of 10 µM.
Protein Data Bank Accession Numbers
The atomic coordinates and structure factors determined in this study (Protein Data Bank IDs 2W09, 2W0A, and 2W0B) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
We explored sterol 14α-demethylase (CYP51) as a potential target for trypanosomiasis chemotherapy by probing CYP51Mt, CYP51Tc, and CYP51Tc with second generation compounds that contain a universal building block, the N-[4-pyridyl]-formamide moiety, which is capable of delivering small molecule compounds to the CYP51 active site. The affinities of the N-[4-pyridyl]-formamide-derivative compounds that we tested against CYP51Mt were lower than that of EPBA (Fig. 2), from which the formamide building block was derived. Affinities of all compounds examined were much higher toward CYP51Tc than to CYP51Mt. Strikingly large increases in binding affinities – 300 and 500 fold – were observed for compounds 8 and 10. Although compound 10 did not produce crystals with CYP51Mt, based on the binding modes of compounds 9 and 11, we reason that the methylcyclohexanecarboxamide moiety of compound 10 protrudes toward the BC-loop, suggesting that the indole ring binds in the species-specific cavity, including the space occupied in CYP51Mt by the F78 aromatic ring, which is absent from CYP51Tc but present in CYP51Tb and CYP51Mt. Consistent with this hypothesis, compound 10 selectively bound CYP51Tc, inhibited T. cruzi growth with the IC50 value close to the KD estimated in the spectral binding assays, and cured mouse macrophages infected with T. cruzi Y strain at 10 µM concentration without harming them.
In contrast, compound 10 failed to bind CYP51Tb despite the identity of 12 of the 13 active site substrate binding residues, and 83% overall sequence identity between T. cruzi and T. brucei CYP51 orthologues. This result is a striking indication of the sensitivity of CYP51 to alterations of the topography of its active site at position 78. The difference in position 78 is of functional importance, because phenylalanine at this site is strictly specific to protozoa and plant species metabolizing 4α-methylated sterols [18]. Interestingly, T. cruzi is the only protozoan where the corresponding position (position 105 according to T. cruzi numbering) is occupied by isoleucine. Consistent with this observation, CYP51Tc is catalytically more closely related to its fungal and animal orthologues, preferentially converting 4α,β-dimethylated sterol substrates [21], whereas T. brucei CYP51 is strictly specific to 4α-methylated obtusifoliol and norlanosterol [33]. The proteobacterium Methylococcus capsulatus, known to synthesize sterols from squalene [34], is the only other known organism having isoleucine in the CYP51 position corresponding to F78. Not surprising, compound 10 was inactive against T. brucei in inhibitory assays in vitro.
In humans and animals metabolizing 4α,β-dimethylated 24,25-dihydrolanosterol, position 78 is always occupied by leucine. Therefore, the F78L substitution in the CYP51Mt binding site was examined and found to slightly increase binding affinities toward compounds 8 and 10, as opposed to the rest of the compounds whose binding affinities decreased (Fig. 2). Although a single amino acid substitution does not by any means convert bacterial protein into its mammalian counterpart, this finding is consistent with lack of toxicity in mammalian cells at inhibitory concentrations, and supports the possibility of rational design of highly selective anti-protozoan CYP51 inhibitors. The latter is of particular pharmacological importance as far as host-pathogen cross-reactivity is concerned, since CYP51 is present in human host.
The increased binding affinities toward CYP51Tc of all the compounds we tested may indicate more extensive involvement of the BC-loop and C helix in protein-inhibitor interactions in CYP51Tc than in CYP51Mt. Assuming that compound 8 binds CYP51Tc in a similarly compact donut-like shape that fills the space adjacent to the porphyrin ring, its 300-fold increase in binding affinity could be achieved solely by stabilization of the BC-region of CYP51Tc without engaging the species-specific cavity. This possibility opens the door to a rational design effort in which the beneficial features of both compounds 8 and 10 would be combined to yield third generation compounds that would more potently and selectively inhibit CYP51Tc. Toward this end compound 10 is currently being evaluated in animal models of Chagas' disease.
We thank Potter Wickware for critical reading of the manuscript, Prof. Paul Ortiz de Montellano for valuable contributions, Prof. M. Waterman for the chimeric CYP51Tc-CYP2C3 coding sequence and support of the project at its initial stage, and the Southeast Regional Collaborative Access Team (SER-CAT) Argonne National Laboratory (mail-in program, Jhongmin Jin) for assistance with data collection.
Author Contributions
Conceived and designed the experiments: PSD JHM LMP. Performed the experiments: CKC PSD LVY ZBM KKHA LMP. Analyzed the data: PSD ZBM LMP. Contributed reagents/materials/analysis tools: PSD JHM LMP. Wrote the paper: LMP.
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- 8. Georgopapadakou NH (1998) Antifungals: mechanism of action and resistance, established and novel drugs. Curr Opin Microbiol 1: 547–557.
- 9. Sheehan DJ, Hitchcoch CA, Sibley CM (1999) Current and emerging azole antifungal agents. Clin Microbiol Rev 12: 40–79.
- 10. Mishra J, Saxena A, Singh S (2007) Chemotherapy of leishmaniasis: past, present and future. Curr Med Chem 14: 1153–1169.
- 11. Roberts CW, McLeod R, Rice DW, Ginger M, Chance ML, et al. (2003) Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa. Mol Biochem Parasitol 126: 129–142.
- 12. Maya JD, Cassels BK, Iturriaga-Vasquez P, Ferreira J, Faundez M, et al. (2007) Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their interaction with the mammalian host. Comp Biochem Physiol A Mol Integr Physiol 146: 601–620.
- 13. Urbina JA, Payares G, Molina J, Sanoja C, Liendo A, et al. (1996) Cure of short- and long-term experimental Chagas' disease using D0870. Science 273: 969–971.
- 14. Buckner F, Yokoyama K, Lockman J, Aikenhead K, Ohkanda J, et al. (2003) A class of sterol 14-demethylase inhibitors as anti-Trypanosoma cruzi agents. Proc Natl Acad Sci U S A 100: 15149–15153.
- 15. Urbina JA, Payares G, Sanoja C, Molina J, Lira R, et al. (2003) Parasitological cure of acute and chronic experimental Chagas disease using the long-acting experimental triazole TAK-187. Activity against drug-resistant Trypanosoma cruzi strains. Int J Antimicrob Agents 21: 39–48.
- 16. Podust LM, Poulos TL, Waterman MR (2001) Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proc Natl Acad Sci USA 98: 3068–3073.
- 17. Podust LM, Stojan J, Poulos TL, Waterman MR (2001) Substrate recognition sites in 14α-sterol demethylase from comparative analysis of amino acid sequences and X-ray structure of Mycobacterium tuberculosis CYP51. J Inorg Biochem 87: 227–235.
- 18. Podust LM, Yermalitskaya LV, Lepesheva GI, Podust VN, Dalmasso EA, et al. (2004) Estriol bound and ligand-free structures of sterol 14α-demethylase. Structure 12: 1937–1945.
- 19. Podust LM, von Kries JP, Nasser Eddine A, Kim Y, Yermalitskaya LV, et al. (2007) Small molecule scaffolds for CYP51 inhibitors identified by high-throughput screening and defined by x-ray crystallography. Antimicrob Agents Chemother 51: 3915–3923.
- 20. Nasser Eddine A, von Kries JP, Podust MV, Warrier T, Kaufmann SH, et al. (2008) X-ray structure of 4,4′-dihydroxybenzophenone mimicking sterol substrate in the active site of sterol 14alpha-demethylase (CYP51). J Biol Chem 283: 15152–15159.
- 21. Lepesheva GI, Zaitseva NG, Nes WD, Zhou W, Arase M, et al. (2006) CYP51 from Trypanosoma cruzi: a phyla-specific residue in the B′ helix defines substrate preferences of sterol 14α-demethylase. J Biol Chem 281: 3577–3585.
- 22. Buckner FS, Joubert BM, Boyle SM, Eastman RT, Verlinde CL, et al. (2003) Cloning and analysis of Trypanosoma cruzi lanosterol 14alpha-demethylase. Mol Biochem Parasitol 132: 75–81.
- 23. von Wachenfeldt C, Richardson TH, Cosme J, Johnson EF (1997) Microsomal P450 2C3 is expressed as a soluble dimer in Escherichia coli following modification of its N-terminus. Arch Biochem Biophys 339: 107–114.
- 24. Barnes HJ, Arlotto MP, Waterman MR (1991) Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli. Proc Natl Acad Sci U S A 88: 5597–5601.
- 25. Otwinowski Z, Minor W (1997) Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol 276: 307–326.
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DeLano WL (2002) The PyMOL molecular graphics system. San Carlos, CA, USA: DeLano Scientific.


text:
The Andis AGC professional two speed grooming clippers feature detachable blades, a powerful rotary motor, a locking switch button to prevent accidental shut off and improved durability. Read More
Freight charges apply
AGC Super 2-Speed Professional Clipper
Strokes per minute: 3,400/4,400
The AGC Clippers feature:
- detachable blades
- a powerful rotary motor
- new contoured design
- locking switch button prevents unit from accidentally shutting off
- removable drive-cap for easy cleaning
- locking blade hinge keeps blade secure
- sealed motor for a cool, quiet, long life.
The design provides:
- improved durability
- resists harsh chemicals
- break-resistant
- maintenance free - no oiling or greasing of internal parts is needed
Compatible with Oster A5 blades.
The normal speed is equal to the high speed on the AGC 2-speed, the high speed is 25% higher.
- Clipper
- Size 10 UltraEdge high-carbon steel blade
- 4 oz oil
- Blade drive
- Blade chart
- Brand: Andis®
- Type: Corded
- Color: Maroon
- Strokes Per Minute: 3,400/4,400
- Speed(s): 2-Speed
- Brand Compatibility: Andis & Oster
Reviews (1)
"Very sharp/fast but hot."
I got these to save on the bi-monthly summer shaving for my chow mix. The clipper just mows right through her VERY thick/fluffy fur. I shaved her 3 times this year and it's as sharp as day one. I have to break it up into 2-3 fifteen minute sittings because the blade gets hot, but it seems that this is the case for all good clippers so all-in-all I am very satisfied with this product.

subset: commoncrawl


text:
By Sgt. Sara MooreApril 8, 2008
WASHINGTON (AFIS, April 8, 2008) - The United States should take 45 days after the last "surge" brigade leaves Iraq in July to evaluate the situation before deciding future troop levels, the coalition's top military commander said here today.
Gen. David H. Petraeus reported his recommendation in testimony before the Senate Armed Services Committee. He testified along with U.S. Ambassador to Iraq Ryan C. Crocker about the situation in Iraq and progress made since September, when the two leaders last delivered an update to Congress and the president.
Petraeus and Crocker both said that while Iraq has made significant security, economic and political gains, the situation remains fragile and the progress is reversible if the United States does not stay the course.
As the United States draws its forces down to the pre-surge level of 15 brigade combat teams, leaders must ensure that the security gains made so far are not jeopardized, Petraeus said. The 45-day evaluation period would give commanders time to assess conditions on the ground and determine when they can make recommendations for further troop reductions, he said.
"This process will be continuous, with recommendations for further reductions made as conditions permit," Petraeus said. "This approach does not allow establishment of a set withdrawal timetable; however, it does provide the flexibility those of us on the ground need to preserve the still fragile security gains our troopers have fought so hard and sacrificed so much to achieve."
Petraeus cited the operational and strategic considerations he took into account when forming his recommendation, including:
-- The military surge has achieved progress, but that progress is reversible;
-- Iraqi security forces have strengthened their capabilities, but still must grow further;
-- The provincial elections scheduled in the fall, refugee returns, detainee releases, and efforts to resolve provincial boundary disputes will pose challenges;
-- The transition of "Sons of Iraq" -- organized groups of local citizens helping with security -- into the Iraqi security forces or other pursuits will require time and careful monitoring;
-- Withdrawing too many forces too quickly could jeopardize the progress of the past year;
--Performing the necessary tasks in Iraq will require sizable conventional forces as well as special operations forces and advisor teams;
-- The strain on the U.S. military has been considerable;
-- A number of security challenges inside Iraq also are related to significant regional and global threats; and
-- A failed state in Iraq would pose serious consequences for the greater fight against al-Qaida, for regional stability, for the already existing humanitarian crisis in Iraq, and for the effort to counter Iran influence.
Since September, there has been "significant but uneven security progress in Iraq," Petraeus told the committee. Levels of violence and civilian deaths have been reduced substantially, al-Qaida and other extremists have been dealt serious blows, the capabilities of Iraqi security forces have grown, and there has been involvement of local Iraqis in security, he said.
"Nonetheless, the situation in certain areas is still unsatisfactory, and innumerable challenges remain," he said. "Moreover, as events in the past two weeks have reminded us, and as I have repeatedly cautioned, the progress made since last spring is fragile and reversible."
Several factors have contributed to the progress made in Iraq, Petraeus said. Iraq added more than 100,000 additional soldiers and police to its security forces ranks in 2007. Counterinsurgency operations across the country have pursued al-Qaida, fought criminals and extremists, fostered local reconciliation and enabled political and economic progress. The country also experienced a shift in attitude among the Iraqi population, he said.
"Since the first Sunni 'awakening' in late 2006, Sunni communities in Iraq increasingly have rejected [al-Qaida in Iraq's] indiscriminate violence and extremist ideology," he said. "These communities also recognized that they could not share in Iraq's bounty if they didn't participate in the political arena."
More than 91,000 Sons of Iraq local security volunteers are under contract to help coalition and Iraqi forces protect neighborhoods and secure infrastructure and roads, Petraeus said. These volunteers have helped to reduce violence and contributed to the discovery of improvised explosive devices and weapons caches, he said. The Sons of Iraq have been directly responsible for many lives and vehicles saved, and their value far outweighs the cost of the contracts to pay them, he said.
"Given the importance of the Sons of Iraq, we are working closely with the Iraqi government to transition them into the Iraqi security forces or other forms of employment, and over 21,000 have already been accepted into the police or army or other government jobs," Petraeus said. "This process has been slow, but it is taking place, and we will continue to monitor it carefully."
For nearly six months, security incidents in Iraq have been at a level not seen since early-to-mid 2005, Petraeus reported. Also, the level of civilian deaths has decreased to a level not seen since the February 2006 Samarra mosque bombing. Deaths due to ethno-sectarian violence have fallen since September, and the number of high-profile attacks is far below what it was a year ago, the general said.
While this progress is significant, al-Qaida is still capable of lethal attacks, and the coalition must maintain pressure on the organization and the resources that sustain it, Petraeus said. Defeating al-Qaida will require actions by elite counter-terrorist forces, major operations by coalition and Iraqi conventional forces, a sophisticated intelligence effort, political reconciliation, economic and social programs, information operations initiatives, diplomatic activity and many other actions, he said.
Iraqi security forces have grown considerably and continued to develop since September, Petraeus said. More than 540,000 people now serve in the Iraqi forces, and half of Iraq's 18 provinces are under Iraqi provincial control. Additionally, Iraqi's training base has become more robust and is expected to generate another 50,000 Iraqi soldiers and 16 army and special operations battalions through the rest of 2008, he said.
Coalition officials expect that Iraq will spend more than $8 billion on security this year and $11 billion next year, he said, allowing the United States to reduce its Iraqi security forces fund for fiscal 2009 from $5.1 billion to $2.8 billion.
Recent operations in Basra, where Iraqi forces responded to a spike in violence by Shiia extremists, highlighted improvements in the Iraqi forces' ability to deploy units, supplies and replacements on very short notice, Petraeus said. However, they also underscored the considerable work still needed in the areas of logistics, force enablers, staff development, and command and control.
Crocker, the U.S. ambassador to Iraq, cited Iraq's political gains in the past months. Iraq's parliament has formulated, debated and passed legislation dealing with issues of reconciliation and nation building, he said. The parliament has passed such important laws as a pension law, de-Baathification reform, a provincial powers law, and a vote to change the design of the Iraqi flag.
Crocker also noted the gains made in Iraq's economy and in improving governance and services. The most obvious indicator of these gains has been the revival of marketplaces and long-shuttered businesses, he said.
Iraq is now earning the financial resources it needs for reconstruction through oil production and export, and so the coalition's focus has shifted to capacity development through the provincial reconstruction teams, Crocker said. The 25 PRTs throughout Iraq have been working to improve provincial and local governance capabilities and to establish links between provincial and federal governments.
"We are seeking to ensure that our assistance, in partnership with the Iraqis, leverages Iraq's own resources," Crocker said.
Looking at the progress in the political and economic arenas, as well as the security gains, it is clear the strategy that began with the U.S. troop surge is working, Crocker said. However, it does not mean U.S. support should be open-ended, he said. In this vein, the United States and Iraq have begun negotiating a bilateral relationship that will include economic, political, diplomatic and security cooperation, he said.
This relationship will be a legal framework for the presence of American troops in Iraq, but it will not establish permanent bases there and will not specify troop levels, Crocker said.
"Our aim is to ensure that the next president arrives in office with a stable foundation upon which to base policy decisions, and that is precisely what this agreement will do," he said.


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Souped-up Coraid aims to change its spots
From pussycat to panther?
Comment Coraid used to be a downmarket beast, selling its Ethernet storage wares off a barrow on the street - now it's got shiny new management and has new aspirations. Can it make the transition and become a creature to be reckoned with?
Coraid sells storage using AoE, ATA-over-Ethernet, protocol which is very lightweight, making iSCSI look complicated and demanding of network resources. The company has been selling product through the channel with little marketing. It decided it needed more business "oomph" and the venture capital backers brought in new management.
CEO Kevin Brown, on a tour of Europe to boost the company's prospects with channel partners and the press, was operating in full boost mode. He said this about the situation he found when he arrived: "Coraid got to 1,000 customers in the Linux market with two part time support people… [the product] just worked… We came in and saw wet dynamite. Let's dry it out and light the fuse. The channel is hungry - they used to have alternatives like Compellent and 3PAR... No more. So there is a channel opportunity."
"We raised $35m through Menlo Ventures and others. [Now] we're now up to 1,400 customers… We have 130 universities as customers. We're getting pulled up into a lot of mid-range enterprises.
"Revenues in the last calendar quarter were five times higher than five quarters ago. 2010 revenues were nearly three times 2009 revenues. That's without concerted EMEA effort. We had Linux-based partners so it it was a foundation to grow on."
"We quintupled our ASP (average system price) since the new management team arrived 18 months ago… The company pre-us didn't have business people to take the company forward."
Brown and the Coraid backers see an opportunity to slip the thin end of a low cost AoE wedge into the storage array market and capture a lot of low-end and maybe mid-range business. They are hammering away at this wedge for all they are worth - and they will be worth a whole lot more if Coraid succeeds.
Storage pricing
Brown sees a transition opportunity opening up in storage because of mainstream storage vendor pricing; he refers to these vendors as "the cartel".
"Change has to happen in storage because storage costs too much. There's a fundamental mismatch between raw HDD costs and the price points the cartel is offering. There is a generational shift from Fibre Channel (FC) to Ethernet, from big iron to commodity scale-out."
"We're bringing in flat Layer 2 Ethernet, [with] jumbo frames, and take full advantage of the switches. Ten gig ports are dropping below $500… We start at less than $600/TB for our SRX line… and [with] 4U and 36 drives our price/performance story is a 5-10X advantage over FC arrays. We're faster than FC and about 1/5th price. It's typically $3,000-$10,000/TB for a FC array and we're starting at under 1/5th of that."
"We can set up an array in 60 seconds; three commands; it's done. After that it's self service in the hypervisor. You configure storage as if it's the C: drive… With us multi-pathing goes away, port zoning and bonding goes away. The whole idea of a fixed connection network goes away.
Sponsored: Becoming a Pragmatic Security Leader


text: Gear MSM's new "Stealth Compact Pouch" David Reeder October 14, 2013 0 COMMENT MilSpec Monkey (who probably needs no introduction here) has released a new, smaller version of the Stealth pouch – the Stealth Compact. Designed as an organizational/utility style pouch with a narrower width to improve mounting options, the Stealth Compact is built in the USA for MSM by Tactical Tailor. I've had many conversations with the Monkey and have always been impressed with his ideas about load carriage, modularity and the efficient of use of space. The development of this particular breed of utility (not quite admin) pouch is no great surprise. The interior has less elastic than its predecessor, instead using billowed pockets to retain load flexibility while maintaining ease of access. The billowed pockets, along with relatively large primary compartment and sleeve pockets seem to give this pouch good versatility; the primary pocket opens via double zippers with an intentional limited opening, while the front tray pouch drops open almost all the way. It will mount via 2 MALICE clips to packs, armor, chest rigs, etc. as needed. Dimensions are approximately 7 in. x 4.5″ x 3″ when filled. Note: if this isn't quite compact enough to fit your real estate constraints, stand by. He's working on an even tighter "tactical organizer." More on that to come. Retail is $30.00 – available in MultiCam, Coyote, Black, Foliage and Ranger Green. Purchase yours here. Explore RECOILweb:TNVC TNV/PVS-14 Night Vision MonocularReloading: Frankford Arsenal's Hand PrimerHot damn - Brownells now sells gunsSHOT Show Gallery Three


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Taking the stage for the first and largest election forum, candidates competing in three citywide races on Wednesday made what could be their highest profile pitches to the voting public before the Feb. 26 primary.
At the forum at Burbank City Hall, which was sponsored by the League of Women Voters Glendale/Burbank, City Council candidates were asked about the projected $1.3-million budget shortfall, climbing utility rates, a $252 million unfunded pension liability and a list of infrastructure needs.
"We need to look at the size of the city workforce, and streamline the way the city works," said candidate Bob Frutos, a Los Angeles police officer.
Incumbent David Gordon touted his history of fiscal responsibility, noting he was the sole dissenting vote against pay raises for top utility officials in 2010 — a time when the rest of the city was "trying to tighten our belts."
Still, incumbent Dave Golonski — who is currently serving as mayor — said Burbank's utility rates are among the lowest in the area.
"You're not going to see lower utility rates anywhere — that's because costs are going up," he said.
Part of that is due to the infrastructure needs in the Sacramento-San Joaquin Delta, a major water source for Southern California, said incumbent Jess Talamantes.
"I do everything I can locally, but I can't control what happens to the Delta," Talamantes said.
But rates are only expected to increase, partly because of the state's renewable energy mandate, which cost Burbank an extra $17.8 million just this year.
Political newcomer and former Marine Juan Guillen advocated fighting against the state's "inefficiency."
"Our hands are not tied," Guillen said. "Why are we passing the buck off, making residents pay for energy we don't need?"
David Nos also focused his comments on the city's budgetary concerns and unfunded pension liability.
"We have to figure a way to bring in more revenues to fix shortfalls," Nos said. "It's going to take tough negotiations."
Among the topics covered by the candidates for city clerk were ideas to increase voter registration and community outreach, tackle pending budget cuts and addressing online voting.
Gloria Salas, an office administrator at a law firm, said she'd initiate low-cost outreach efforts, such as speaking at community meetings and senior centers, sending voter information home with children after school and handing out fliers at local shopping centers.
In terms of online voting, Salas said the process has not yet proved to be immune to technological snafus or hacking.
Interim City Clerk Zizette Mullins said Burbank's currently focused on increasing voter registration with the mail-in ballot process and social media.
"I believe [online voting] will happen in the future, but not any time soon," Mullins said, noting that she recently launched a Facebook and Twitter page to keep voters in the loop on election information.
Mullins said she'd bring a wealth of experience to the position, with 25 years working for the city of Glendale and six years operating a small business in Burbank.
A 15-year city employee — currently an executive assistant in the finance department — Nonna von Sonn touted her community involvement. She participates in Burbank's Relay for Life, Habitat for Humanity and the National Management Assn.
"I would like to be the face of the city," von Sonn said. "People will feel comfortable with me."
If a city clerk candidate gets more than half the votes in the primary, she will be appointed. If not, the top two vote-getters will square off in the April 9 election, while the third candidate will be eliminated.
Any City Council candidate that gets more than half the total votes in the February primary secures a seat, and the lowest vote-getter will be eliminated. If no candidate gets more than half the votes, all six candidates will move forward to the April election.
Candidates for the Burbank Unified school board also participated in the event on Wednesday, but were slated for a more targeted election forum on education issues Thursday night.
For both the primary and general election, residents can only vote via mail-in ballots. Feb. 11 is the last day to register to vote for the primary.
The ballots can be mailed in or dropped off at the City Clerk's office.
-- Alene Tchekmedyian, Times Community News
Follow Alene Tchekmedyian on Google+ and on Twitter: @atchek.


text:
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Greg Beno has not received any gifts yet
hey! lets play sometime. Im Cory text me at 7752238948
Hi, I'm Mark, the founder of Tennisopolis, the world's largest and most active tennis network. Please share Tennisopolis with your friends by supporting our Facebook page. Click here for a Guide to Meet Tennis Partners.
Have a look at our introduction page which will help you navigate the massive network which now has over 37,000 members and 500 local tennis groups.
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text:
Show us how Pro-Expert toothpaste gives you the confidence of the expert, whatever the situation!
2014년 06월 29일 (일)에 공모전 마감
동영상 - 애니메이션
Have you ever felt shy, incompetent or uncertain about yourself? Isn't it terrible when lack of confidence stops us from doing things? That joke not told, that proposal not made… How sad! Oral-B Pro-Expert is no ordinary toothpaste. It boosts your confidence, the confidence of the expert. We need your help to show to the world, in an inventive and witty way, that Pro-Expert is the toothpaste you need to get the confidence of the expert!
Create a witty, unexpected and shareable video or animation that shows that regardless of the situation, Oral-B Pro-Expert gives you the confidence of the expert.
FORMAT: Videos or animations; maximum length:1 minute.


text:
Urban Harvest's Farmers Market welcomes SNAP Benefits!
50 vendors now accepting SNAP/EBT!
What is SNAP?
The USDA's Supplemental Nutrition Assistance Program (SNAP) offers nutrition assistance to millions of low-income individuals and families. Through benefits distributed by the Food and Nutrition Service, SNAP aims to increase food accessibility to communities in need.
How to use SNAP at the Farmers Market:
Stop by the green Urban Harvest tent to exchange your SNAP benefits for $1 or $5 coupons to spend on eligible products. These coupons do not expire, so you can spend them the same day or save for another time!
What can I buy with my SNAP coupons?
- Fruits, vegetables, and herbs
- Cheese, eggs, and other dairy products
- Meat products (red meat, seafood, poultry)
- Bread and other baked goods
- Honey, maple syrup, jams, and sauces
- Seeds and plants that produce food (e.g. basil seeds or tomato plants)
For more about our SNAP vendors, check out the interactive map below, and visit the Meet Our Vendors page!


text:
Non-prescription Drugs and Their Side Effects
Grosset & Dunlap, 1977 - Drugs, Nonprescription - 128 pages
From inside the book
18 pages matching antacid in this book
Where's the rest of this book?
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Acetaminophen acid Advisory Review Panel agents Alcohol allergy and/or Anesthetic antacid Anticholinergics antihistamines aspirin aspirin substitute asthma Astringent avoid Benzalkonium Benzocaine Brand Manufacturer burn Caffeine carbonate cause chloride Chlorpheniramine chronic common cold compounds congestion constipation consult your doctor cough suppressant Cream Delivery Form Dextromethorphan diarrhea discomfort disease dose FDA Advisory Review fever guaiacolate headache hemorrhoids hydrochloride hydroxide infection irritation itching judged safe Label laxatives lngredients Magnesium medically active ingredients Methapyrilene mg 5 gr mg Caffeine mg Phenylephrine mg/2 tbl mg/tab or tsp mg/tsp mild to moderate Moderate 1-2 Tab mucus nasal nausea non-prescription drugs non-prescription medications non-prescription products Oint pain Pectin pharmacist phenacetin Pheniramine Phenylephrine Phenylpropanolamine Powder prescription PRODUCT CHART products containing Pyrilamine Rapid recommended dosages relieve runny nose safe and effective Salicylamide salicylates scopolamine Selected List self-medication SELF-TREATMENT Self-Treatment side effects simethicone skin Sodium Sodium bicarbonate Spray stomach stools throat tissue treat Undecylenic vitamins Zinc oxide


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Tuesday, April 7, 2009
Peeps Mash Ups - Savory
I will preface this post with the following: I may have gone off the deep end.
In my joy of experimentation with Peeps and mashing their sticky innards into other foodstuffs, I've jumped from fun & tasty to the illogical conclusion of savory & disgusting.
(Okay, well, they're not all disgusting.)
I like marshmallow Peeps as an idea, but not much as a confection. They're fun to look at, pose & contemplate. But I don't get much pleasure out of eating them. Pairing them with savory items was supposed to be a way to moderate their grainy over-sweetness.
I picked up a few things to try with my Peeps for 2009: Goldfish Crackers, Cheetos, Pepperoni (turkey for me) and Nacho Cheese Sauce.
Scroll slowly, otherwise you'll overwhelm yourself.
Pepperidge Farms has come out with some new flavors & variations of the classic Goldfish Crackers. As a kid the Pizza flavored ones were my favorite but now I prefer the Cheddar. (Though given a choice I'd probably still opt for Cheezits.)
The new variation that I picked up is in their whole grains line. This one is Toasted Corn.
After reading over the ingredients, corn is pretty far down on the list. And they're not really corn flavored, they taste more like those Chik'n Biskit crackers I used to like to drink with flat 7-UP in a friend's basement as a pre-teen while we played with Troll Dolls.
The crunch and salt actually goes well with the sweet fluffy marshmallow and grainy sugar coating. It's the dusting of garlic that the crackers seem to have that ruins it for me. A plainer cracker, like Saltines or Oyster Crackers is probably a much better and actually realistic Mash Up idea.
I could have gone decadent and upscale:
Nope, I went the other way:
I had trouble finding my favorite brand of plastic cheese. I usually buy Frito Lay, the stuff that comes in a squat can and probably has a shelf life of three years. All I could find is this Mission Cheddar Cheese Dip.
I'm sorry friends, I wussed out. I didn't eat this. I thought it was fun to construct. In the future I think I might use different color Peeps than the traditional yellow. I think the green to simulate jalape?os and maybe the purple & pink to really make it jarring would have the best impact.
I did try Peeps on corn chips ... which is actually rather nice. The salty grains of the chips and the sweet sugar granules played nicely.
There's probably a great idea in there somewhere for S'Mores Nachos using chocolate tortilla chips, chocolate chips, caramel sauce & Peeps. (All toasted under the broiler, natch.)
To be fair to my readers, I did eat this.
This is a Peep in that Mission Cheddar Cheese Dip.
The Mission Cheddar Cheese Dip is a bit too soupy and watery for me. I like a thicker, more gelatinous cheese sauce (or just real gooey, stringy cheese).
The cheese was tangy and salty and had a cheddar "flavor" to it. The combination with the Peeps wasn't so much bad, but the tangy yogurt quality of the dip didn't go well with the fake vanilla flavor of the Peeps. I don't recommend you try this at home.
One of the original items I was interested in smashing into my Peeps was Cheetos. And why not, they're both some ghastly colors.
Cheetos now come in some insane flavors and textures. I like both the Crunchy Cheetos and the classic Cheese Puffs. (And some crazy sweet versions in other lands.)
Peeps have a definite advantage over Cheetos in several ways. Though Peeps are brightly colored, they don't make a mess on your hands. And Peeps may actually be better when they're stale.
It was funny how much more I tasted the corn when combined with the sweetness of the marshmallow. Corn (Grits) is a great breakfast cereal so this was like mixing my savory grits with my sweet grits. The crunchy texture was a good offset to the silky smooth marshmallow and the micro crunch of the granulated sugar.
The Flaming Hot Cheetos might be a fun experiment.
And here it is, the pi?ce de r?sistance is futile. A while back someone suggested bacon or jerky with Peeps.
But really, when it comes to preserved meats and mashing names together, there's nothing that can come close to Peeperoni. (It's hilarious, I know!)
It took me a while to find turkey-based Pepperoni (as I don't eat mammals, except for gelatin because I can't possibly give up gummis), which I know is nothing like real pepperoni except that it comes in little disks and contains insane amounts of salt.
This Hormel Turkey Pepperoni smells like feet. I didn't know if that was normal or not.
First, like the cheese sauce experiment, there is no "mashing the sticky Peeps into the pepperoni" action. Peeps don't stick to pepperoni. It's like pushing to north ends of magnets together.
So I broke out the toothpicks.
The salty and chewy texture of the pepperoni was a nice complement to the sweet fluffy chew of the marshmallow. I think part of the failure of this is the abject lack of fat in this turkey version.
As an alternative to this, I think a sweet Lebanon Bologna might actually be interesting, especially if fried and put together on some slices of sweet egg bread.
As a bit of a palate cleanser, I did mix up something tasty. I had some Chocolate Mousse Peeps which went fantastically well with the crunchy, sweet and nutty All Natural Sunbursts.
For even more Peepitude, here's a list of some other Peep occurrences on the internet ... some you've probably already seen, but some you might not have. Serious Eats is also devoting this week to Peeps Week.
Here are my previous Mash Ups.
Related Candies
Next entry: Manhattan Chocolates Dipped Mint Cremes
Previous entry: Russell Stover Eggs (2009 edition)
Meticulously photographed and documented reviews of candy from around the world. And the occasional other sweet adventures. Open your mouth, expand your mind.


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This is Key Stage 4 Physics ~ GCSE.
Welcome to my new look Gondar Design Physics website. This is just one of five sections at Gondar Design Science. All my old HTML pages have been copied to this WordPress content managed system: the originals have been reduced to automated transfers to the appropriate page on this site now. Here you can post comments or ask questions on almost every page. Please register first.
We do NOT use your email for any purpose other than
to send you a password or password reminder.
Use the links at the bottom right of this page to change subjects.
The photograph at the top of each physics page was taken in an old Somerset quarry.


text: This course improves students' vocabulary, comprehension and study skills. Application of these skills will enable a student to read his/her college textbooks more effectively and efficiently. Topics include topic, main idea, details, writing patterns, critical reading and note taking. Four Equivalent Hours (EH) awarded for successful completion. Successful completion meets the reading proficiency required for enrollment in credit courses. NOTE: Students enrolled in DVR-0061 must take PAS-1010 in the same semester. (Formerly DVR-0060. Students who have successfully completed DVR-0060 should not take DVR-0061.) 3 class/2 lab hours. Academic Level Undergraduate Level Subject Developmental Reading Department LEARNING FOUNDATIONS Credits 4 Prerequisites DVR-0051 : Developmental Reading DVR-P005 : DVR-0051 Proficiency Corequisites None Return to Paralegal/Legal Assistant, Assoc. of Applied Science Academic Calendar Catalog and Class Schedules Online Learning Bookstore Register For Classes Request Information


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text:
T Spa Infinity Pool
The indoor swimming pool, in the sanctum of the T Spa, is a strip of azure, ultra pure energy. Double infinity effect on Lake Como inside and out and a breathtaking view of the lake from the outdoor Jacuzzi and lounge area. 5 different types of Jacuzzi, including those hidden away in the private grotto plus an exclusive swim-against-the-current workout.


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this is something I have been doing a long time but I really don't know if this is the right path.
I'll give a basic example.
if I have a stock table, and I want the row to have a yellow background color code warning when an item reaches 2 in quantity, and to turn red when the item reaches 0 in quantity.
What I did was something like this:
I have also used this method but extended a bit, like creating switches on db which is nothing more than a state which is either yes/no (or true/false), I can then present certain html elements acording to those switches states. I used that for instance to make a check box default state to checked according to what the user chose earlier.
Code: Select all
$stock= new Product();
if($stock-data > 0){
foreach($stock->data as $st){
if($st['qty'] <= 2){
echo "". $st['id']. "	";
echo "". $st['name']. "	";
echo "". $st['price']. "	";
echo "". $st['qty']. "	";
}elseif($st['qty'] == 0){
echo "". $st['qty']. "	";
echo "". $st['name']. "	";
echo "". $st['price']. "	";
echo "". $st['qty']. "	";
echo "". $st['qty']. "	";
echo "". $st['name']. "	";
echo "". $st['price']. "	";
echo "". $st['qty']. "	";
I need your opinion on this, if this is awkward, if it should really be done in client side using javascript.
Any feedback will be appreciated.


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You are here: Home ‹ Board index ‹ Movie Discussion ‹ All Other/Older Movies ‹ (2009) Under The Sea 3D
Release: Blu-Ray/DVD/Digital Copy combo pack
Discuss this movie. In Theaters and available on DVD and Blu-ray.
2 posts • Page 1 of 1
Release: Blu-Ray/DVD/Digital Copy combo pack
Warner Home Video has announced IMAX: Under the Sea for release on a BD/DVD/Digital Copy combo pack on March 30. This documentary on marine life in the Pacific, narrated by Jim Carrey, was released in IMAX 3-D theaters in 2009.
Jim Carrey Movies/Series/Books in 2018:
True Crimes, I'm Dying Up Here (Season 2), The Zen Diaries of Garry Shandling (documentary), Kidding (Season 1), Homey Don't Play That! (Book) and Untitled novel
Join Jim Carrey Online on Facebook, Twitter, YouTube, Instagram, VK and Snapchat
That's something worth buying! The question is, will they release it in 3D?
"Snakes! Why it had to be snakes?"
-Raiders of the Lost Ark (1981)
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text:
Event description
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Why not come along as a guest one Thursday and meet some new contacts in a great location?


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All Items : Vintage Arts : Regional Art : Asian : Korean : Paintings : Pre 1980 item #935565 (stock #0414)
Korean Art and Antiques
Shaman's Feast Woodblock Print on Traditional Hanji Paper by Hong Sung Dam in 1982 (21 x 15 inches, 53.5 x 38 cm, including margins). Hong Sung Dam is a woodblock print artist who was a leader of the minjung, the peoples' political movement that arose from the ashes of the Gwangju Massacre where the Korean government slaughtered hundreds (some witnesses say thousands) of their own citizens in the streets in response to demonstrations in Gwangju. He was arrested in 1989, and suffered brutal torture in prison. He was released in 1992 after a three-year-long campaign by Amnesty International. After the subsequent democratization of Korea, Hong was commissioned by the government in 1996 to create the large (120 feet) Minjung Mural in Gwangju. Early minjung art is much sought after, and Hong Sung Dam is one of the most revered and acclaimed minjung artists. He donated much of his early work, so opportunities to buy are very infrequent (practically non-existent, actually). So we are very pleased to offer this rare collection of woodblock prints on traditional hanji paper by Hong Sung Dam that were created before his imprisonment, hidden from authorities by his friends, and donated to Amnesty International after their successful effort to get him released from prison. Own a piece of history and a beautiful work of art from a man who grew up in poverty (on the island of Haui) and illness (severe tuberculosis as a youth) to become an inspiring artist who gave his life to his art and his people.
All Items : Vintage Arts : Regional Art : Asian : Korean : Paintings : Pre 1950 item #838636 (stock #0298)
Oil Painting by Park Deuk Soon (1910 - 1990), signed and dated 1949, with original frame. Park Deuk Soon studied in Tokyo with Ryohei Koizo and graduated from the Pacific Fine Arts School in 1938. He exhibited often at the National Art Exhibition and the Chosun Art Exhibition. He was also an educator and an active member of the Kukjeon, and served for a time as President of the Korean Painters Association. Frame: 23.5 x 19 inches, 59 x 48 cm; Painting: 17 x 12.25 inches, 43 x 31 cm.


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In July, I'm going to become an aunt. My sister-in-law and brother are expecting their first baby, Isaac, and we're all very excited to welcome him to our family. My other sister-in-law, Lisa, and I planned a baby shower a couple of weeks ago. We decided on the popular "She's About to Pop!" theme and scoured Pinterest for ideas.
We used shades of blue, turquoise, and green along with lots of circles in our decor to stick with the pop theme. There are a ton of fabulous ideas out there for this theme, so I'll just highlight a few of the things that we added our unique twist to.
I think that the cousins-to-be might be more excited than anyone!
I decided that the large space above our mantel needed some baby shower love, so I got an engineer's print made at Staples with a picture of the mommy and daddy. I used spray adhesive to mount it to a large scrap piece of plywood. The print was less than $3, so it was an inexpensive way to add a big impact piece. I also pulled an old skid out of my dad's burn pile. I put it there a few months back after I was experimenting with ways to take the wood apart. I gave up on that, but resurrected the skid when I needed a way to hang up letters for Isaac's name. The letters are a combination of salvaged sign letters and wooden letters from the craft store that I painted and distressed. I borrowed my brother's old teddy bear and tiny baseball cap from my mom's cedar chest for some sweet personalization from Isaac's daddy.
Our shower was in the afternoon, so we didn't serve a complete meal. We tried to make each food work with the POP theme. However, I neglected to take a picture of the actual food. Our menu consisted of popcorn chicken (from Chick Fil A), Popeye's spinach dip, fruit pops (fruit on bamboo skewers), and jalapeno poppers (from the frozen food section at the grocery). On the dessert table, we had cake pops, push pops, and Snap, Crackle, Pop! Rice Krispie Treats. Lisa made all of the cake pops and push pops and they were the definite hit of the shower. She did a great job!
I was trying to come up with a way to serve a large number of cake pops and still make it look nice. I was playing around with the idea of putting styrofoam in a box or container when my mom suggested pool noodles. Have I mentioned that she is a genius??? I ended up finding blue and green pool noodles at the Dollar Tree and cut them with a serrated knife to fit just inside the opening in my buffet cabinet. We stacked them and hot glued them together and added a scrap board on the bottom for some extra weight. Then, we took a small screwdriver and punched holes for the cake pop sticks. The noodles held them perfectly!
Behind this display, I added the bottom section of the skid from the mantel that I cut off with a jigsaw. These letters are also a combo of salvaged sign letters and painted/distressed wooden craft store finds.
We enjoyed some yummy popcorn from Cornival Popcorn Shop and guests took some home as favors. We used six different flavors of popcorn and put them in chalkboard paint labeled buckets from the dollar store. I found the red and white scoops at Dollar Tree, as well.
Instead of playing games, we had all the supplies for our guests to decorate a onesie for baby Isaac. My mom prepared fabric iron-ons ahead of time and we had plenty of buttons, paint, fabric markers, etc. available so people could be creative.
Before we know it, we will be seeing a cute, squishy little baby in those clothes! I can't wait!


text:
World Council Supports Revisions to Global Anti-money Laundering Regulations
Financial Action Task Force rules prove scalable to credit unions
April 04, 2012
PARIS — World Council of Credit Unions yesterday came out in strong support of revisions to regulations developed by the Financial Action Task Force (FATF) designed to combat money laundering and terrorist financing, and supported the FATF's revised due diligence recommendations that can be scaled to acknowledge credit union members' relative lack of risk in these areas. World Council representatives spoke to this issue at an April 2 meeting held by FATF at the Paris headquarters of the Organization for Economic Cooperation and Development, where the inter-governmental regulatory body is domiciled.
More than 100 participants attended the two-day meeting to discuss revisions to FATF's International Standards on Combatting Money Laundering and the Financing of Terrorism and Proliferation, better known as the 40 recommendations. World Council supports the recommendations overall, and especially the FATF's revised customer due diligence (CDD) recommendations that should apply to the relatively low-risk scenarios offered through credit unions, according to Michael Edwards, World Council chief counsel and vice president for advocacy and government affairs.
"World Council strongly supports the FATF's recommendations and interpretive notes regarding simplified CDD measures for lower risk customers and transactions," said Edwards, who represented the global credit union trade organization with David Phillips, president and CEO of Credit Union Central of Canada, a World Council member organization. "We asked the FATF to consider issuing additional guidance on this issue to help reduce regulatory burdens on credit unions when there is no indication of money laundering or terrorist financing."
The revisions World Council supports are those recognizing the relative lack of risk generally offered by credit union members and their transactions. As natural-person financial cooperatives, credit unions do not serve corporate entities with opaque ownership structures that might easily hide criminal money laundering or terrorist activities. Credit unions generally know their members, making such activities harder to mask, Edwards said.
Language already exists within the CDD recommendations offering a simplified approach, which World Council believes will reduce regulatory burdens to levels appropriate to the general risk found in credit unions, Edwards explained. FATF reacted positively to World Council's comments, noting that while a simplified CDD could not uniformly apply to all credit unions on an institution-wide basis, national or provincial regulators could reduce regulatory burdens on credit unions in cases where members were likely to be low-risk.
"The renewed emphasis on a risk-based approach in the revised FATF standards is a welcome change," said Phillips. "This opens new opportunities for the design of anti-money laundering regulation that is appropriate for credit unions."
FATF's revisions also indicate further recognition that credit unions generally pose less risk overall due to their cooperative nature and the role played by their member-owners, according to Brian Branch, World Council president and CEO.
"We urge FATF to recognize the value of financial cooperatives' relationships with their members through the simplified customer due diligence revisions," said Branch. "Credit unions continue to be a positive global economic force and recognition of that role by global regulatory bodies is necessary to maintain and protect that positive role that financial cooperatives fulfill."
World Council of Credit Unions is the global trade association and development platform for credit unions. World Council promotes the sustainable development of credit unions and other financial cooperatives around the world to empower people through access to high quality and affordable financial services. World Council advocates on behalf of the global credit union system before international organizations and works with national governments to improve legislation and regulation. Its technical assistance programs introduce new tools and technologies to strengthen credit unions' financial performance and increase their outreach.
World Council has implemented 300+ technical assistance programs in 89 countries. Worldwide, 89,026 credit unions in 117 countries serve 260 million people. Learn more about World Council's impact around the world at www.woccu.org.


text:
|2008-04-08, 06:01
|Link #22
Holy Beast ~Wuff!~
...or maybe the real reason is that we should just buy pizza and collect token for cheese-kun and limited edition CG merchandise.
yes. i have a strong gut feeling this is the underlining philosophical message. eat well from a 18" cheese stuff deep base pizza will stimulate a healthy well-being for all.
being hungry while musing deep ideas is bad for you.
pizza hut is your friend. call now.
*logs online*
|2008-04-08, 06:15
|Link #23
The Traitor
I don't think we should bring up the Emperor...
He hasn't been on that much to even define his personality or how he even thinks. He's made speeches(Could have been easily biased because...duh, he needs to rev up the Nobles and Barons)
I also doubt he would be using Lelouch as a Successor, but the fact he married a Commoner such as Marianne due to her being an excellent Pilot could be a sign that he was trying to aim for a stronger Son -FAILURE, Lelouch can't even dig a hole to save his life(seriously, I could see him dying without food in the island arc cause he failed to dig a hole)
We also have no idea what the King Desires, or what Rangarok/Ragnares or whatever he's searching for. Obviously he wishes to find it first using his Geass Ruins, Clovis himself wished to gain power using C.C. and Schneizel attempted to use the ruins at Kamenija, whoever does Succeed the Emperor will obviously take his place with the 'Ragnarok' plan or whatever.
He only watches Lelouch because he is about the only son(Game chars don't count) Who have received Geass. He's the best candidate to succeed him if he does get beaten, simply due to the fact Lelouch's power and he also assumes his mind is evil(He laughs during Euphie's scene and says " That fool actually did it! "; Somehow I think Lelouch moved up on his book due to it, simply because he didn't see it coming and is proud of his idiotic son killing off his siblings)
|2008-04-08, 06:18
|Link #24
Join Date: Dec 2007
Age: 28
I'd honestly say that the whole thing with Lelouch would be secondary in his mind with the plan he has been working on. If his master plan involved making Lelouch the successor than that would actually be pretty disappointing.
|2008-04-08, 12:41
|Link #25
Senior Member
Join Date: Feb 2008
And pretty pointless to bring in Geass at all. There's something deeper here, I can feel it, you don't go saying stuff like 'it's tied to the very existence of mankind' or 'it has the power to either create or destroy the world' for something as tangible and mortal as a mere throne.
|2008-04-08, 12:44
|Link #26
The Dark Knight
Join Date: Dec 2005
Location: From the deepest abyss in the world, where you think?
Age: 33
Most of the misery was bought on by unknown forces (Which Marianne and C2 may have been involved in) and the Emperor simply stuck by to his policy. That and you really can't expect him to show any weakness either.
For all intents and purposes his ultimate opponent could be Schenzeil(Which I am thinking he will be), C2 or even Marianne
|2008-04-08, 13:45
|Link #27
Join Date: Dec 2007
Age: 28
Well, Schneizal was created with the thought of making Lelouch a nemisis, so he will probably be the person that Lelouch clashes with the most.
C.C. and Marianne actually being somewhat sinister seems quite possible as well. It would be incredibly cruel, but that makes it all the more likely. They may not be on the Emperor's side, but they may not be much better than him in what they are willing to do.
|2008-04-08, 18:26
|Link #28
Holy Beast ~Wuff!~
personally i think the Emperor before did not think much of him at all, it was only after his attempt as Zero that is now making him interested in him.
i think the Emperor will play around with him, i think the current situation was left to the authorities.
They were ones to use him as bait for C.C. obviously the Emperor did not even tell them the truth about C.C. the target they were aiming for.
the fact is, if lulu/zero survives the attempts on his life by the Emperor then i think he will seriously consider him an heir.
I'm certain he holds true to what he preaches, and that he would never hand his throne over to anyone who cannot kill him.
its the ultimate Darwinism. Law of the jungle.
|2008-04-29, 17:06
|Link #29
Kalulu YES WE CAN
Join Date: Aug 2006
Location: CALIFORNIA!!
Age: 32
Well I had no idea where to post this thus I posted in philosophy since philosophy kind of incorporates everything :P. This post will look in the different ideas and thought process of different characters in this series.
First off Lelouch. In order to understand what lelouch is doing is to understand why he is starting rebellion to start off. I am not trying to Justify what he does or saying what he is doing is okay. I am simply stating his goals. Goal 1a. is to make a peaceful world for Nunally. Goal 1b. to take revenge for his mother Death. It is obvious at this point to say Goal 1a. has completely taken over 1b. Example 1 will be his willingness to abandon his plan for vengeance to get Nunally. His self reflection in Season 1 last episode is evidence to this. He realizes why is he fighting this lonely war if he is not with Nunnally at the end. The next step in the Analysis is how he plans to achieve this goal.
Destruction or Deconstruction (Path of Carnage). Lelouch finds the current form of Government oppressive and a terrible system. I believe we can agree that Brittanian is not the ideal form of Government. It oppresses the weak which is one of the main reasons he dislikes it. Oppression of the weak makes people Nunnally not worth very much in the world as she is weak. So Lelouch plan from the very beginning and reiterates with Urabe in Episode 2 R2 is to destroy britannian. Specifically the way he wishes to achieve this is by taking down the Emperor. This is proven by the Fact that constantly talks about Emperor and wishes to take him down.
This Destruction turns the world into Chaos. The Chaos that is war. This motif is not new. We can go back to the times Babylon and talk about Myth of Marduk. As Marduk destroys Kingu and out of the Ashes he creates Mankind. This idea is called Combat Myth, and it is talk about throughout antiquity. The reason I drawing from here is because Lelouch is Marduk. Marduk tries to free the other gods from serving. Thus Out of the chaos of Kingu he creates humanity. This idea of Chaos turning into creation is also found in the Hebrew Text of Genesis 1:1-2. The point being Lelouch way of getting his goal is thrusting the world into Chaos and rebuilding from that chaos at the end.
Since we talk about what his goal is and how he plans to get there one finally comes to idea of is it right. As said earlier there is no right answer to this question as this deals with a lot of grey. One must not put our own ideals onto him. For instance His priorities are wrong. Well for you maybe. Lelouch priorties are as he sees fits. Do I wish for him to change? Yes! I would want him to embrace his comrades more (Kallen ). This is Lelouch Rebellions not yours. Hopefully I put some perspective into why he does what he does. Again this can change as the story progresses but for right now it is like this. Next up is the Foil Suzaku but it is for another time.
|2008-04-29, 17:22
|Link #30
Has a life IRL
Join Date: Nov 2007
Location: Somewhere in the Anglo-Saxon Sphere
Personally, I feel that the most unspoken philosophical point in Code Geass is how Zero is the perfect embodiment of Britannian ideals. Power, cunning, rule by virtue of results, the not so occasional back stabbing, everything.
|2008-04-29, 17:42
|Link #32
Has a life IRL
Join Date: Nov 2007
Location: Somewhere in the Anglo-Saxon Sphere
|2008-04-29, 17:55
|Link #34
Has a life IRL
Join Date: Nov 2007
Location: Somewhere in the Anglo-Saxon Sphere
The United States of Japan's claims to justice are, at best, tenous. One of the original Black Knights prepares to shoot the student council for having the nerve to say that Zero would protect them. Zero holds the exact same Student Council members hostage as a tool against Suzaku. Where has the USJ shown that it has any more justice than Britania? That it accepts a handful of Britanians? Britannia takes nonbritannians as well when they follow the ideals of Britannia as expressed by the Emperor.
And that's not even getting into Zero's hypocracies as to what he's done while spouting off that USJ bs...
|2008-05-02, 07:49
|Link #35
I disagree with you all.
Join Date: Dec 2005
Well, the USJ are mostly there to gather people around him. If he thought being openly "evil" would bring him more power than pretending to serve justive, he'd be kicking puppies and raping kittens on live TV.
I don't think Lelouch even is that much philosphically opposed to Britania's way of doing things. It's personal. Britania's destroyed his happiness twice, and is a threat to his sister, so he wants to destroy it. Unlike his father, he sympathises with the weak, but if not for Marianne's death and his fall from grace, he wouldn't care enough to do much about it.
|2008-05-02, 08:31
|Link #36
Holy Beast ~Wuff!~
Zero was the one who pledge the student council safety, but it was Lulouch who held them hostage against Suzuku.
You might say it's the same thing, but it's not.
Zero's agenda is to free Japan, protect the weak and up hold justice. So far he has done this.
However lulu is prepared to do anything to topple the Empire and to protect his sister with whatever means possible.
In the eyes of the people they are two separate individuals, thus what they see of each (or not see, as is the case for one)
they both have largely remind true to there goals.
And definitely have not done anything to meet the same level of hypocrisy as Suzuku has managed to do.
Obviously, when you know the man behind the mask, you tend to associate the action of one, to the other personality, thus creating the illusion of hypocrisy.
|2008-05-02, 23:04
|Link #37
Reality Blender
Join Date: Feb 2007
|2008-05-03, 07:45
|Link #38
Logician and Romantic
Join Date: Nov 2004
Location: Within my mind
Age: 38
|2008-05-03, 12:28
|Link #39
Join Date: May 2004
Lelouch doesn't really give a shit about freeing Japan he is just using the Japanese to his advantage so he can accomplish his goals. He is only appearing as a hero of Justice to get more and more support from the community. He is freeing Japan not because he really cares about freeing the Japanese but so that he has a loyal force he can use to achieve his own goals.
Also, yea he does care about his friends at school he is not a heartless bastard but he does follow the exact ideals of the Emperor. Oh, but he does hate when the strong pick on the weak because of his sister's condition.
|2008-05-03, 12:48
|Link #40
no worries
Join Date: Apr 2008
Location: Israel
Age: 36
he thinks he knows what he wants but that might change when he grows up a bit...
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