text:
|Home | About | Journals | Submit | Contact Us | Français
Conceived and designed the experiments: TK CH MN. Performed the experiments: TK CH. Analyzed the data: TK CH MN. Wrote the paper: TK CH MN MF.
17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reduction of estrone to estradiol, which is the most potent estrogen in humans. Inhibition of 17β-HSD1 and thereby reducing the intracellular estradiol concentration is thus a promising approach for the treatment of estrogen dependent diseases. In the past, several steroidal and non-steroidal inhibitors of 17β-HSD1 have been described but so far there is no cocrystal structure of the latter in complex with 17β-HSD1. However, a distinct knowledge of active site topologies and protein-ligand interactions is a prerequisite for structure-based drug design and optimization. An elegant strategy to enhance this knowledge is to compare inhibition values obtained for one compound toward ortholog proteins from various species, which are highly conserved in sequence and differ only in few residues. In this study the inhibitory potencies of selected members of different non-steroidal inhibitor classes toward marmoset 17β-HSD1 were determined and the data were compared with the values obtained for the human enzyme. A species specific inhibition profile was observed in the class of the (hydroxyphenyl)naphthols. Using a combination of computational methods, including homology modelling, molecular docking, MD simulation, and binding energy calculation, a reasonable model of the three-dimensional structure of marmoset 17β-HSD1 was developed and inhibition data were rationalized on the structural basis. In marmoset 17β-HSD1, residues 190 to 196 form a small α-helix, which induces conformational changes compared to the human enzyme. The docking poses suggest these conformational changes as determinants for species specificity and energy decomposition analysis highlighted the outstanding role of Asn152 as interaction partner for inhibitor binding. In summary, this strategy of comparing the biological activities of inhibitors toward highly conserved ortholog proteins might be an alternative to laborious x-ray or site-directed mutagenesis experiments in certain cases. Additionally, it facilitates inhibitor design and optimization by offering new information on protein-ligand interactions.
Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the NAD(P)H dependent reduction of the weak estrogen estrone (E1) to the biologically most active estrogen estradiol (E2; Fig. 1) [1]. This reaction, which represents the last step in E2 biosynthesis, takes place in target cells where the estrogens exert their effects via the estrogen receptors α and β. Besides their physiological effects, estrogens are involved in the development and the progression of estrogen dependent diseases (EDDs) like breast cancer, endometriosis and endometrial hyperplasia [2]–[4]. In the past few years, aromatase inhibitors have been intensively investigated for the treatment of EDDs [5]–[7] but they lead to unwanted side effects due to their strong reduction of estrogen levels in the whole body. Therefore reducing local E2 levels by inhibition of 17β-HSD1 is a promising therapeutic approach for the treatment of EDDs. An analogous intracrine concept has already been proved successful for the treatment of androgen dependent diseases such as benign prostatic hyperplasia and alopecia by using 5α-reductase inhibitors [8]–[11]. 17β-HSD2 catalyzes the reverse reaction (oxidation of E2 to E1; Fig. 1) and inhibition of this enzyme must be avoided for the therapeutic concept to work. However, specific inhibition of 17β-HSD2 in bone cells may provide a novel approach to prevent osteoporosis [12].
17β-HSD1 is a cytosolic enzyme that belongs to the superfamiliy of short-chain dehydrogenases/reductases (SDRs) [13]. It consists of 327 amino acid residues (34.9 kDa) and the active form exists as homodimer [14]. 17β-HSD1 comprises a Rossmann fold, associated with cofactor binding, and a steroid-binding cleft [15]. The latter is described as a hydrophobic tunnel with polar residues at each end: His221/Glu282 on the C-terminal side, and Ser142/Tyr155, belonging to the catalytic tetrad, which is present in the majority of characterized SDRs [16], on the other side [17]. To date 22 crystal structures of 17β-HSD1 are available as apoform, binary or ternary complexes [18]–[20]. All crystal structures show an overall identical tertiary structure, while major differences have been identified only for the highly flexible βFαG'-loop. It is not resolved in ten crystal structures, while the remaining twelve showed high b-factor values for this area, which is an additional hint for the flexibility of the βFαG'-loop. In some crystal structures a short α-helix was observed in the loop region but its occurrence seems not to be dependent on the presence of steroidal ligands, cofactor or inhibitor. However, the position and length of the α-helix changes: in the apoform (PDB entry 1bhs) the helix is limited to the beginning of the loop while in presence of steroidal ligands and/or cofactor it is shifted to the end (PDB entries 1dht, 1equ, and 1iol). Further, dependent on the presence of cofactor and ligands, the βFαG'-loop can occupy three possible orientations: an opened, semi-opened, and closed enzyme conformation [21].
Several steroidal and non-steroidal inhibitors of 17β-HSD1 have been described [18], [22]–[37], but only for the former cocrystal structures exist. While several computational studies have been performed in order to elucidate the interactions of non-steroidal inhibitors with 17β-HSD1 [26], [27], [33], [37], [38], structural data confirming the results are still missing.
However, a distinct knowledge of active site topologies and protein-ligand interactions is a prerequisite for structure-based drug design and optimization. To further increase this knowledge, inhibition values obtained for one compound toward proteins, differing only in few residues might be advantageous. For this purpose, wild type proteins and their mutants carrying a set of point mutations can be used. As alternative, proteins from various species, which are highly conserved in sequence and differ only in few residues, might be considered.
This latter approach was applied in the present study employing human and marmoset monkey (callithrix jacchus) 17β-HSD1. Selected human 17β-HSD1 inhibitors, representative of our structurally diverse inhibitor classes, were tested toward the marmoset 17β-HSD1. The resulting inhibitory potencies were compared with those obtained for the human enzyme and remarkable differences were only observed in the class of the (hydroxyphenyl)naphthols. In order to rationalize the species-specific inhibition profiles at the structural basis, a homology model of the marmoset enzyme was built using a human 17β-HSD1 x-ray structure as template. Further, the docking poses of selected compounds into both the human crystal structure and the modelled marmoset 17β-HSD1 were considered. Notably, the marmoset homology model and the docking poses of the inhibitors presented herein were validated by their ability to explain inhibition data. Subsequently, the complexes of two representative inhibitors, docked into the marmoset model and the human crystal structure, respectively, were subjected to MD simulations to investigate their conformational equilibrium. In addition, binding energy calculations as well as energy decomposition analysis were performed, with the aim to investigate the influence of the marmoset amino acid variations on the inhibitory potencies. The current work provides new insights into the marmoset 17β-HSD1 active site topology, reveals probable inhibitor binding modes in human and marmoset 17β-HSD1, and identifies amino acids responsible for species specificity in 17β-HSD1 inhibition.
To identify regions that are conserved through several species, the sequences of rodent, cynomolgus, marmoset and human 17β-HSDs 1 were aligned (Fig. 2A). The N-terminal region (residues 1–190), which constitutes the common Rossmann fold as well as the catalytic tetrad, is highly conserved for the analyzed species. Remarkable differences were observed in the F/G segment (residues 191–230), which is lining the steroid-binding site, and the C-terminal region (residues 231–285). Sequence alignment of human and marmoset 17β-HSD1 revealed that they share 80% sequence identity and 85% similarity (Fig. 2A). Focusing on the steroid-binding site (residues 94 to 196 and 214 to 284), the identity increases to 87%, with five major amino acid variations observed in the marmoset enzyme: A191P, E194Q, S222N, V225I and E282N. In contrast, comparing cynomolgus and human 17β-HSD1, which show even 91% identity, only one of the aforementioned amino acid variations can be found (E282H; Fig. 2A). With the exception of marmoset 17β-HSD1, Ala191 and Glu194 are conserved through the analyzed species (Fig. 2A) indicating the significance of the observed variations between human and marmoset. 17β-HSDs 1 of mouse and rat are 83% similar to the human enzyme in the first 287 amino acids. In both analyzed rodent enzymes His221, which is involved in steroid binding [17], is mutated into a glycine (H221G). Moreover, several other amino acids of the substrate-binding site are replaced by more bulky residues (L96F, N152H, M193Y/H, S222Y). Interestingly, rat and mouse 17β-HSDs 1 are significantly less sensitive to inhibition by steroidal inhibitors compared to the human ortholog [39] and different classes of non-steroidal potent human 17β-HSD1 inhibitors turned out to be only weak inhibitors of E2 formation in rat liver preparations [40]. This might be partially explained by the absence of His221 as interaction partner as well as by the reduced volume of the active site (M193Y/H, S222Y).
Sequence identities between human 17β-HSD2 and 17β-HSD2 of the selected species range from 61% (mouse) to 93% (cynomolgus) while the F/G segment and the C-terminal region show the most pronounced variability (Fig. 2B). The comparison of 17β-HSD1 with the correspondent type 2 enzymes of the analyzed species revealed, that the type 2 enzymes have about 80 additional N-terminal residues relative to the type 1 enzymes. Sequence comparison showed that they share a very low overall sequence identity (≤25%) with major differences in the F/G segment and the C-terminal domain, which constitute the active site. However, some amino acid motifs, characteristic for SDR enzymes [18], are highly conserved: the T-G-xxx-G-x-G motif, the Y-xxx-K sequence and the N-A-G motif (Fig. 2B). For marmoset 17β-HSD2 only a fragment of the primary sequence is available, which is 143 amino acids in length and constitutes the Rossman fold whereas the F/G segment and the C-terminal region are missing (Fig. 2B). This segment is 29% identical to marmoset 17β-HSD1.
As human and marmoset 17β-HSD1 differ in few residues of the active site, comparison of the inhibitory potencies of selected inhibitors, observed toward both enzymes, is suitable to further increase the knowledge of active site topologies and protein-ligand interactions. In particular, the amino acid variations in the flexible βFαG'-loop, which is suggested to play a crucial role in ligand binding, will be used to elucidate the function of the loop in more detail.
Marmoset placental tissue was used as enzyme source and the proteins were partially purified following a described procedure [41]. Tritiated E1 was incubated with 17β-HSD1, cofactor and inhibitor. The separation of substrate and product was performed by HPLC. In an assay similar to the 17β-HSD1 test, marmoset placental microsomes containing 17β-HSD2 were incubated with tritiated E2 in the presence of NAD+ and inhibitor. Labelled product was quantified after HPLC separation.
The inhibition values of compounds 1–20 (Fig. 3) are shown in Table 1. The bis(hydroxyphenyl) substituted arenes 1–11 showed comparable or even higher inhibitory potencies toward marmoset 17β-HSD1 compared to the human enzyme. For example, compound 8, which is a potent inhibitor of human 17β-HSD1 (IC50=151 nM), showed a stronger inhibitory potency toward marmoset 17β-HSD1 (IC50=4 nM). Comparable inhibition data for human and marmoset 17β-HSD1 were also measured for the bicyclic substituted hydroxyphenylmethanones 12 and 13. This indicates that the binding of the two aforementioned inhibitor classes is reinforced or at least not affected by the amino acid variations in marmoset 17β-HSD1. In the class of the (hydroxyphenyl)naphthols, the human 17β-HSD1 inhibitor 14 (IC50=116 nM) showed also a good inhibitory potency toward marmoset 17β-HSD1 (IC50>50 nM). The introduction of space filling substituents in position 1 of the naphthol core of 14 is beneficial for the inhibition of the human enzyme (16, IC50=26 nM), but for marmoset 17β-HSD1 no increase in inhibitory potency was observed (16, IC50>50 nM) compared to the unsubstituted 14. Interestingly, further enlargement of the substituents in 1-position of 14 led to highly active human 17β-HSD1 inhibitors (19, IC50=15 nM) while a reduced potency toward marmoset 17β-HSD1 (19, IC50>50 nM) was found.
The inhibitory potencies of compounds 1–20 toward marmoset 17β-HSD2 were also determined to prove whether marmoset monkey is a suitable species for in vivo evaluation of 17β-HSD1 inhibitors (Table 1). Remarkably, lower selectivity of compounds toward non-target marmoset 17β-HSD2 was observed, when comparing to human 17β-HSD2.
In order to obtain a more precise picture of the three-dimensional structure of the marmoset 17β-HSD1, a homology model was generated. A set of 100 models was built with MODELLER 9v7 [42] using the ternary complex E1-NADPH-human 17β-HSD1 as template. This complex was obtained by docking E1 into human 17β-HSD1 employing the Protein Data Bank (PDB) entry 1fdt with conformation B for residues 187–200 (in the following determined as 1fdtB). This 3D structure was chosen as it represents a ternary complex with NADP+ and E2 in the closed enzyme conformation, as the βFαG'-loop is resolved, and as this protein structure was already successfully used in previous docking studies with the investigated compound classes [30], [36]. The best model was chosen according to internal DOPE-score [43] and PROCHECK [44] tests. Notably, it presents a short α-helix (residues 190–196) in the region between the βF-sheet (residues 178–186) and the αG'-helix (residues 209–227), with Pro191 in the first turn of the helix (N1 position; Fig. 4), thus differing from the secondary structure of the template.
The selected model was further refined both as holoenzyme with NADPH and as ternary complex with NADPH and E1 by MD simulations. The trajectories of the two MD simulations were stable with a α-carbon root-mean-square deviation (RMSD) to the starting structure below 3.0 Å (Fig. 5). No major structural differences were observed when comparing the simulated holoenzyme and the ternary complex. Therefore, the following results, based on analysis of the ternary complex, also apply to the holoform.
After an initial inward rotation in the MD simulation, the short α-helix (residues 190–196) remained stable for the last 5 ns of the MD simulation, with an average backbone RMSD to the final conformation of 1.03 Å. Remarkably, a similar behaviour cannot be expected for the region between the βF-sheet and the αG'-helix in the human enzyme. The differences in the crystal structures suggest a high flexibility for the βFαG'-loop: it is not resolved in ten crystal structures and the remaining twelve show high b-factor values for this area. Some crystal structures present a short α-helix in the loop region whose position and length varies dependent on the presence of steroidal ligands, cofactor or inhibitor. In the apoform (PDB entry 1bhs) the helix is limited to the beginning of the loop, whereas in presence of steroidal ligands and/or cofactor it is shifted to the end (PDB entries 1dht, 1equ, and 1iol). Further, in the human enzyme, the βFαG'-loop axis occupies different orientations dependent on the presence of cofactor and ligands. In the marmoset however, both the holoform and the ternary complex show a helix starting already at the beginning of the βFαG'-loop with its axis in only one conformation.
The presence of the newly formed α-helix (residues 190–196) induced a different orientation of the side chain of Met193. Compared to the template structure, Met193 protrudes deeper into the substrate-binding site and stabilizes E1 by hydrophobic interactions (Fig. 4). Furthermore, during MD simulation a kink in the loop between the βD-sheet and the αE-helix was observed. Thereby the side chain of Leu96 was brought closer to E1 allowing Van der Waals contacts (Fig. 4). Summarizing, in the final part of the MD simulation E1 was stabilized by both lipophilic interactions and hydrogen bonds: Leu96, Leu149, Met193, and Phe259 constrained the steroidal scaffold while Ser142/Tyr155 and His221/Asn282 interacted with the carbonyl oxygen in 17-position and the 3 OH-group, respectively. The latter residue took over the H-bond acceptor abilities of Glu282, which is involved in forming an H-bond with the 3 OH-group of E1 in the human enzyme.
Employing CASTp [45], the active site volumes of human and marmoset 17β-HSD1 were calculated. The above-described conformational changes of Leu96 and Met193 as well as the S222N and V225I mutations resulted in a reduced volume of the marmoset 17β-HSD1 active site (478 Å3) compared to that of the human ortholog (627 Å3).
The stereochemical quality of the holoenzyme and the ternary complex models obtained from MD simulations was checked with PROCHECK [44]. The majority of the residues of the investigated structures were found to occupy the most favoured regions of the Ramachandran plots, while the other residues occupied the additional allowed regions. In detail, in the ternary complex 78.1% of the residues were placed in the most favoured region, 20.2% in the additional allowed region, 1.2% in the generously allowed region, and only 0.4% in the disallowed region (for the holoform: 81.8%, 17.4%, 0.8% and 0%).
As the potent inhibitors of human 17β-HSD1 12 and 19 are structurally diverse and exhibit different potencies toward marmoset 17β-HSD1 they were chosen as representatives to rationalize the observed species-specific inhibition profiles. Employing AutoDock 4 [46], both compounds were docked into the active sites of marmoset and human 17β-HSD1 using the equilibrated, ternary complex after 5537 ps and the x-ray structure 1fdtB (see experimental part), respectively. The inhibitor poses used for further investigation were selected considering binding energy and statistical representativity (cluster population; Table S1) and are shown in Figure 6.
In the human enzyme both inhibitors are placed in the substrate-binding site and they occupy an apolar subpocket consisting of the following amino acids: Gly94, Leu95, Leu96, Asn152, Tyr155, and Phe192 (Fig. 6A). While the carbonyl group of compound 12 mimics the D-ring keto function of E1, forming H-bonds with Ser142 and Tyr155, its para-OH group resembles the 3-OH of E1, which interacts with His221 via an H-bond. The meta-hydroxyphenyl moiety is projecting into the subpocket, where it forms an additional H-bond with Asn152 and is stabilized by π-π-interactions with Tyr155 and Phe192.
The (hydroxyphenyl)naphthol-core of compound 19 occupies the substrate-binding site and is stabilized by three H-bonds: the 2-OH group interacts with Ser142 as well as with Tyr155 and the OH group in meta position of the phenyl ring in 6-position interacts with His221. In this case, the sulfonamide substituted phenyl ring in 1-position of the naphthol core protrudes into the subpocket, where it is stabilized by H-bonds with Asn152 and with the -NH- of the backbone of Leu95 (Fig. 6A).
Also in the marmoset enzyme both compounds occupy the substrate-binding site, but only 12 protrudes into the apolar subpocket (Fig. 6B). Due to the altered side chain conformation of Leu96 in the marmoset enzyme, compound 12 is slightly displaced toward the C-terminus (Fig. 6C). Regarding the interaction pattern, only minor changes were observed: the carbonyl group forms only an H-bond with Ser142 but for the para-OH group a second H-bond with Asn282 was observed (Fig. 6C).
Interestingly, compound 19 resulted in a completely different binding mode when docked into the homology model of marmoset 17β-HSD1 with respect to its position in the human crystal structure 1fdtB (Fig. 6D). The OH-group in meta position of the phenyl ring makes an H-bond with the backbone carbonyl oxygen of Cys185 and the 2-OH function forms H-bonds with His221 and Asn282 in a bifurcated fashion. The sulfonamide substituted phenyl ring is located in the C-terminal gate and might be stabilized by π-π-interactions with Phe259 and an H-bond with the backbone -NH- of Leu262.
With the aim to validate the docking results and to unravel possible induced-fit mechanisms, different MD simulations were run in explicit aqueous solution. Distance restraints were applied to inhibitors only in the first ns of the MD simulations with the aim of maintaining their proper orientation. For the rest of the MD simulation no restraints were used and the whole complexes were left free to move. This was done in order to avoid trapping the inhibitor in an unstable conformation, which could bias the results. The RMSD values of the heavy atoms of the inhibitors and of the Cαatoms of the enzymes were analyzed as a function of time to assess the degree of conformational drift, as shown in Figure 7.
In the simulation of 12 bound to human 17β-HSD1 the CαRMSD of the protein as well as the RMSD of the heavy atoms of 12 showed a stable plateau (~2.2 Å) from 1.0 to 2.5 ns (Fig. 7A). After 2.5 ns the CαRMSD of the protein increased and a minor fluctuation of the heavy atom RMSD of 12 was observed. The latter finding could be related to a slight shift of the inhibitor toward the C-terminal end of the enzyme. Notably, the hydrogen bonds between 12 and 17β-HSD1, which were observed in the initial structure, were conserved during the 4 ns simulation suggesting that both protein and 12 fluctuations do not impact the inhibitor binding.
Both human 17β-HSD1 and compound 19 were stable in the simulation of their complex (Fig. 7B). During the simulation (after 1.5 ns) the hydrogen bond of the meta-OH group with His221 is replaced by an H-bond with Glu282. However, after 2.1 ns, this hydrogen bond is interrupted for 0.2 ns allowing the hydroxyphenyl ring to rotate freely around the axis of the bond to the naphthol core thereby inducing a minor fluctuation of the heavy atom RMSD of 19. After this short interruption the H-bond interaction with Glu282 is re-established. On the other side, the hydrogen bonds between the 2-OH group and Ser142/Tyr155 as well as between the sulfonamide moiety and Asn152/Leu95 endured constant.
During the MD simulation of 12 in complex with marmoset 17β-HSD1, the meta-hydroxyphenyl moiety of 12 moved further out of the subpocket. This was reflected by the minor fluctuation of the RMSD of the heavy atoms of 12 after 1.4 ns (Fig. 7C). While this motion caused the break of the H-bond with Asn152, it also placed the meta-OH group in an appropriate distance to Asn222, thus allowing a new H-bond formation.
The analysis of the MD simulation of the marmoset 17β-HSD1-19 complex revealed an overall stable CαRMSD of the protein. However, after 1.8 ns, a ~0.5 Å fluctuation of the heavy atom RMSD of 19 was observed (Fig. 7D) corresponding to the rotation around the axis of the bond between the hydroxyphenyl ring and the naphthol core. Thereby the H-bond with the backbone amino group of Leu262 was lost.
Furthermore, for each of the four MD trajectories absolute free energy (ΔG) and relative binding affinity (ΔGbind) were calculated applying MM/PBSA methods and NMODE analysis (Table 2). This was done for the following stable sectors: 1000 to 2500 ps for 12 in complex with human 17β-HSD1 (Fig. 7A), 2300 to 4160 ps for 19 in complex with human 17β-HSD1 (Fig. 7B), 1500 to 4160 ps for 12 in complex with marmoset 17β-HSD1 (Fig. 7C) and 1800 to 4160 ps for 19 in complex with marmoset 17β-HSD1 (Fig. 7D). All four complexes showed favourable ΔG values, ranging from −7.3 kcal mol−1 to −3.2 kcal mol−1. The free energies observed for compound 12 in complex with human (ΔG=−4.7 kcal mol−1) and marmoset 17β-HSD1 (ΔG=−5.8 kcal mol−1) are in the same range. This is in accordance with the inhibitory activities of 12, which are comparable for both species (see Table 1). Regarding compound 19, the complex with human 17β-HSD1 shows a more favourable free energy (ΔG=−7.3 kcal mol−1) than the one with marmoset 17β-HSD1 (ΔG=−3.2 kcal mol−1). This is mainly due to poor entropic contributions in the latter case. Remarkably, this finding is in concert with the experimentally determined inhibition data: compound 19 is a highly potent human 17β-HSD1 inhibitor (IC50=15 nM) with reduced activity toward the marmoset enzyme (IC50>50 nM).
Focusing on the specific interactions, which mediate the binding of 12 and 19 to human and marmoset 17β-HSD1, we have analyzed the interaction energies of both inhibitors with the residues of the binding sites, employing a pairwise per-residue energy decomposition analysis.
Inspection of the interaction energies with human 17β-HSD1 (Table 3) showed that the hydrogen bonds of 12 (−4.1 kcal mol−1) and 19 (−5.7 kcal mol−1) with Asn152 contributed most to the interaction energies. Besides Asn152, further residues of the subpocket (Gly94, Leu95, Leu96) interact with both inhibitors revealing energies from −0.3 kcal mol−1 to −1.8 kcal mol−1. The energies of the hydrogen bonds between 12 and the catalytic residues Ser142 as well as Tyr155 are −1.3 kcal mol−1 and −1.5 kcal mol−1, respectively. In case of compound 19 the interaction energies are −1.3 kcal mol−1 for the H-bond with Ser142 and −1.3 kcal mol−1 for the H-bond with Tyr155. Further binding site residues, which significantly contribute to the binding of compounds 12 and 19 (Leu149, Pro187, Phe192, Met193, Val225, and Phe259) show interaction energies from −0.9 kcal mol−1 to −2.2 kcal mol−1. Regarding the polar amino acids at the C-terminal end of the binding site, His221 takes primarily part in the binding of 12 (−1.8 kcal mol−1 for 12 vs. −0.2 kcal mol−1 for 19) while Glu282 is mainly involved in binding compound 19 (−0.1 kcal mol−1 for 12 vs. −4.3 kcal mol−1 for 19).
The interaction energies of 12 and 19 with marmoset 17β-HSD1 are listed in Table 4. Remarkably, for inhibitor 12, the energy contribution of the H-bond with the marmoset Asn152 (−1.6 kcal mol−1) is 2.6 fold reduced compared to the human enzyme, while for compound 19 it is almost lost (−0.2 kcal mol−1). Both for 12 and 19 reduced energies were also observed for interactions with Gly94, Leu95, and Leu96. Interestingly, inhibitor 12 showed an interaction with Asn222 in the marmoset enzyme (−3.3 kcal mol−1), which was not observed for Ser222 in human 17β-HSD1 (−0.8 kcal mol−1). This is not the case for 19, as the interaction with Asn222 (−0.3 kcal mol−1) does not contribute significantly to the interaction energy. In addition to Leu149, Pro187, Met193, Ile225, and Phe259, which interact with both compounds in the human enzyme, Gly186, His221, and Asn282 significantly contribute to the binding of 12 and 19 to marmoset 17β-HSD1 with interaction energies in the range from −0.4 kcal mol−1 to −3.6 kcal mol−1. Interestingly, in marmoset 17β-HSD1 the interaction energy between 12 and Asn282 (−3.2 kcal mol−1) is 32 fold increased compared to that with Glu282 in the human enzyme (−0.1 kcal mol−1). In marmoset 17β-HSD1 an increased energy is also observed for the interaction of compound 19 with His221 (−2.6 kcal mol−1) compared to the human enzyme (−0.2 kcal mol−1).
When the three-dimensional structures of marmoset and human 17β-HSD1 are compared, one of the most striking features is the small α-helix including the residues 190 to 196. It is formed in the segment between the βF-sheet and the αG'-helix starting from the interface residue Thr190, which is half in and half out of the helix (N-cap position). In contrast to the human enzyme, where this region is highly flexible, as suggested by the different crystal structures, the α-helix stayed stable during the MD simulation in both the holoform and the ternary complex. The observed conformational stability might be explained by the presence of a proline in position 191 instead of an alanine. Proline is a favourable candidate for N1 position because of its own conformational properties: with only one rotatable angle it loses less entropy than other amino acids in forming an α-helix and thereby it should have some stabilizing influence [47]. Furthermore, in an analysis of sequence-structural characteristics in protein crystal structures, proline was found to be a favoured residue at N1 position. Especially the residue pair involving threonine at N-cap and proline at N1 position, which is observed for marmoset 17β-HSD1, has a high prevalence [48].
In order to analyse the influence of the conformational changes in marmoset 17β-HSD1 on ligand binding, docking studies with subsequent MD simulations, free energy calculations, and energy decomposition analyses were carried out. While the conformational differences between the marmoset and the human enzyme did not affect the binding mode of 12 remarkably, the suggested binding mode of 19 differed strongly in 17β-HSD1 of both species. One possible explanation for that might be the lower sterical demand of 12 compared to 19. However, the energy contribution of the interaction between 12 and the marmoset Asn152 is reduced, whereas it was outstanding in complex with the human enzyme. Due to the minimal shift of 12 in the marmoset binding pocket, the geometric parameters for the H-bond with Asn152 are no longer optimal. Interestingly, in the marmoset enzyme an additional interaction of 12 with Asn222 is observed, which seems to compensate the deficit in interaction energy due to the absent interaction with Asn152 resulting in comparable binding energies for 12 in complex with human and marmoset 17β-HSD1. The latter finding is in accordance with the inhibition data observed for compound 12 and validates the marmoset 17β-HSD1 model.
Considering compound 19, no particular interactions with the subpocket residues of the marmoset enzyme exist. Although weak interactions between 19 and the C-terminal region of marmoset 17β-HSD1 are observed, the binding free energy is less favourable compared to that calculated for the human 17β-HSD1-19 complex. As the C-terminal part of the enzyme has already been discussed as a potential product exit gate of the enzyme [21], inhibitor 19 might be solvent exposed. This is consistent with the unfavourable entropy term of this complex resulting in the least favourable free energy.
Obviously, the presence of a proline in the flexible loop region and the thereby induced conformational changes in marmoset 17β-HSD1 are decisive for the species specific inhibition of 19. On one hand interactions with subpocket residues like Asn152, recently discussed as relevant interaction partner [49], are prevented and on the other hand the inhibitor is forced in an unfavourable solvent exposed conformation.
The bis(hydroxyphenyl) substituted arenes (compounds 1–11) show similar or increased inhibitory potencies toward marmoset 17β-HSD1 when comparing to human 17β-HSD1. Recently performed docking experiments proposed a steroidal binding mode when the human crystal structure 1fdtB was used [33]. The high inhibitory potencies toward marmoset 17β-HSD1 are in concert with the modelled structure of marmoset 17β-HSD1 as steroid-like binding is not affected by the proposed conformational changes. Obviously, they even stabilize the bis(hydroxyphenyl) substituted arenes in the marmoset 17β-HSD1 binding pocket as indicated by the observed inhibitory potencies.
Differing inhibitory potencies toward human 17β-HSD1 and 17β-HSD2 may arise from sequence variations in the regions 94–196 and 214–284 (numbering according to 17β-HSD1), which might lead to differences in the active sites of the two human subtypes. A lower selectivity of compounds toward non-target marmoset 17β-HSD2 was observed, when comparing to human 17β-HSD2. Obviously, the differences in the active sites of marmoset 17β-HSD1 and 17β-HSD2 are less pronounced compared to the human orthologs. However, as the available marmoset 17β-HSD2 sequence is missing the F/G segment and the C-terminal part this hypothesis cannot be proved.
The validity of the presented homology model is further substantiated by its ability to explain the reduced inhibitory potency of C-15 substituted estrone derivatives toward marmoset 17β-HSD1 [39]. The substituents in 15-position of the steroid were designed to occupy the hole between the flexible βFαG'-loop and the αG'-helix in the human enzyme [50]. Together with the helix formation and the conformational changes in the βD/αE-segment, the S222N mutation limits the size of the hole in marmoset 17β-HSD1 and thereby might reduce the inhibitory potency toward the marmoset enzyme.
An elegant strategy to gain more knowledge of active site topologies and, in particular, of protein-ligand interactions is to compare inhibition values obtained for one compound toward ortholog proteins from various species, which are highly conserved in sequence and differ only in few residues. Thereby, such an approach can be a valid alternative to site-directed mutagenesis. As human and marmoset 17β-HSD1 enzymes meet these criteria, selected human 17β-HSD1 inhibitors were assessed for their inhibitory potencies toward marmoset 17β-HSD1. While a species specific inhibition profile was observed in the class of the (hydroxyphenyl)naphthols, representatives of the other evaluated compound classes showed similar or even higher inhibition compared to those observed for the human enzyme. Using a combination of computational methods, including homology modeling, molecular docking, MD simulation, and binding energy calculation, a reasonable model of the three-dimensional structure of marmoset 17β-HSD1 was developed and inhibition data were rationalized on the structural basis. In the marmoset 17β-HSD1, residues 190 to 196 form a small α-helix, which is obviously stabilized by the presence of a proline in N-cap position (residue 191) and induces conformational changes that affect ligand binding. Furthermore energy decomposition analysis highlighted the important role of Asn152 as interaction partner for inhibitor binding.
This work could not only offer a better understanding of the active site topologies and of the protein-ligand interactions, but also provides novel structural clues that will help to design and optimize potent human 17β-HSD1 inhibitors with improved inhibitory potency toward marmoset 17β-HSD1. This is an important step to turn compounds, which show a promising pharmaceutical profile, into candidates for in vivo evaluation. Thus, our combined computational approach could also be considered as a valuable tool to achieve this goal.
The amino acid sequences of rat (accession number P51657), mouse (P51656) cynomolgus (Q4JK77) and marmoset 17β-HSD1 (Q9GME2) as well as human (P37059), cynomolgus (Q4JK76), marmoset (Q9GME5), mouse (P51658) and rat (Q62730) 17β-HSD2 were obtained from the uniprot webpage. These sequences were pairwise aligned with human 17β-HSD1 (PDB code: 1fdt) using MAFFT version 5 [51]. Using this alignment, a set of 100 comparative models of marmoset 17β-HSD1 was built employing Modeller9v7 [42], with the ternary complex E1-NADPH-human 17β-HSD1 as template. This complex resulted from docking of E1 to human 17β-HSD1 (PDB code: 1fdtB) (see below). The best homology model was then selected according to the Modeller energy score, DOPE score [43] and PROCHECK [44] tests. The reliability of the built homology models was checked by Prosa2003 [52] (Fig. S1), ERRAT [53], and Verify3D [54] (Fig. S2).
MD simulations were performed using the AMBER 9.0 suite program [55]. The partial atomic charges for E1 and the inhibitors were derived from the molecular electrostatic potential (MEP) previously calculated using GAMESS [56], according to the RESP methodology [57]. For the protein, partial atomic charges were read from the AMBER 9.0 libraries. The AMBER99SB force field [58] was employed to define atom types and potentials for the protein, while the general AMBER force field (gaff) [59] was used to define all needed atom types and parameters for E1 and the inhibitors. For NADPH (charge −4), the parameters previously reported by Ulf Ryde were applied (http://www.teokem.lu.se/~ulf/).
The input files for the MD simulation were prepared with the xLEaP module of AMBER. Each system was solvated with an octahedral box of TIP3P water molecules of 10 Å radius and neutralized by the addition of Na+ ions. Finally, for each complex the topology and the coordinate files were written and used in the MD simulations.
Before starting the production-run phase, the following equilibration protocol was applied to all systems. At the beginning the system was energy-minimized in two stages: firstly, the solvent was relaxed while all the solute atoms were harmonically restrained to their original positions with a force constant of 100 kcal mol−1 Å−2 for 1000 steps; and secondly, the whole molecular system was minimized for 2500 steps by conjugate gradient. Subsequently, the system was heated during 60 ps from 0 to 300 K at constant volume conditions (NTV, PBC conditions), and then equilibrated keeping both temperature and pressure constant (NTP, PBC conditions, 300 K, 1 atm) during 100 ps. Electrostatic interactions were computed using the Particle Mesh Ewald method [60], and the SHAKE [61] algorithm was employed to keep all bonds involving hydrogen atoms rigid. NADPH, E1 and the inhibitors were constrained during the equilibration with a force constant of 20 kcal mol−1 Å−2. After equilibration, a MD production stage (NTP, PBC conditions, 300 K, 1 atm) was performed. The total simulation length differed for the various complexes ranging from 4 to 6 ns. Distance restraints were applied to substrate/inhibitor with the aim of maintaining their proper orientation at the beginning (first ns) of production stage.
For the ternary complex of marmoset 17β-HSD1 with E1 and NADPH, two additional distance restraints were used: between the keto-oxygen of E1 and the side chain oxygen of Ser142 (d=2.40–3.00 Å; force constant: 10 kcal mol−1 Å−2) and between the oxygen of the OH-group in 17-position of E1 and the NE2 nitrogen of the His221 side chain (d=2.70–3.40 Å; force constant: 10 kcal mol−1 Å−2).
For the ternary complex of human 17β-HSD1 with 12 and NADPH, three additional distance restraints were used: between the keto-oxygen of 12 and the side chain oxygen of Tyr155 (d=2.80–3.40 Å; force constant: 10 kcal mol−1 Å−2), between the oxygen of the OH-group in meta-position of 12 and the OD1 oxygen of the Asn152 side chain (d=2.70–3.30 Å; force constant: 10 kcal mol−1 Å−2), and between the oxygen of the para OH-group of 12 and the NE2 nitrogen of the His221 side chain (d=2.70–3.30 Å; force constant: 10 kcal mol−1 Å−2).
For the ternary complex of marmoset 17β-HSD1 with 12 and NADPH three additional distance restraints were used: between the keto-oxygen of 12 and the side chain oxygen of Ser142 (d=2.50–3.10 Å; force constant: 10 kcal mol−1 Å−2), between the oxygen of the OH-group in meta-position of 1 and the OD1 oxygen of the Asn152 side chain (d=2.40–3.00 Å; force constant: 10 kcal mol−1 Å−2), and between the oxygen of the para OH-group of 12 and the OD1 oxygen of the Asn282 side chain (d=2.50–3.10 Å; force constant: 10 kcal mol−1 Å−2).
For the ternary complex of human 17β-HSD1 with 19 and NADPH three additional distance restraints were used: between the oxygen of the OH-group in 2-position of the naphthol core and the side chain oxygen of Tyr155 (d=2.80–3.40 Å; force constant: 10 kcal mol−1 Å−2), between the oxygen of the OH-group in meta-position of the phenyl ring and the NE2 nitrogen of the His221 side chain (d=3.00–4.20 Å; force constant: 10 kcal mol−1 Å−2), and between the nitrogen of the sulfonamide moiety of 19 and the OD1 oxygen of the Asn152 side chain (d=2.70–3.40 Å; force constant: 10 kcal mol−1 Å−2).
For the ternary complex of marmoset 17β-HSD1 with 19 and NADPH two additional distance restraints were used: between the oxygen of the OH-group in 2-position of the naphthol core and the NE2 nitrogen of the His221 side chain (d=2.70–3.40 Å; force constant: 10 kcal mol−1 Å−2) and between the oxygen of the OH-group in meta-position of the phenyl ring of 19 and the backbone carbonyl oxygen of Cys185 (d=2.40–3.00 Å; force constant: 10 kcal mol−1 Å−2).
Trajectories were analyzed using the AMBER ptraj module, the MMTSB toolset [62] and the molecular visualization program VMD (Visual Molecular Dynamics) [63]. The resulting low energy structures were extracted for the homology model (apoform and ternary complex) and subjected to a subsequent minimization of 1000 steps (500 steps of steepest descent followed by 500 steps of conjugate gradient), using the sander module of AMBER. The modified generalized Born solvation model (IGB=2) [64] was used. Active site volumes of low energy structures were calculated using the CASTp [45].
The three-dimensional structures used for docking studies were either retrieved from the PDB (1fdt, conformation B for residues 187–200) or from the homology modelling with subsequent MD simulation (equilibrated ternary complex after 5537 ps). The cocrystallized E2 and water molecules were removed from the PDB file. Hydrogen atoms and neutral end groups were added, NADP+ was turned into NADPH and correct atom types were set. Ionization states and hydrogen positions were assigned using the Protonate 3D utility of MOE2009.10 (Chemical Computing Group Inc., Montreal, Canada). Ligand structures were built in MOE and RESP charges were assigned as described above. The 17β-HSD1 three-dimensional structures and ligand structures were prepared for docking studies through the graphical user interface AutoDockTools4 [46]. For the ligands, non-polar hydrogen atoms were deleted, rotatable bonds were defined and RESP charges were kept. For the protein, non-polar hydrogen atoms were deleted and charges were added to the structure. Autodock4.2 [46] was used to dock the ligands in the steroidal binding site of the processed protein structures. A box, centered on the steroid-binding site, was set to define the docking area. Grid points of 90×90×90 with 0.250 Å spacing were calculated around the docking area for all the ligand atom types using AutoGrid4.2. For each inhibitor, 50 separate docking calculations were performed. Each docking calculation consisted of 25×105 energy evaluations using the Lamarckian genetic algorithm local search (GALS) method. Each docking run was performed with a population size of 250. A mutation rate of 0.02 and a crossover rate of 0.8 were used to generate new docking trials for subsequent generations. The docking results from each of the 50 calculations were clustered on the basis of root-mean-square deviation (RMSD=2.0 Å) between the Cartesian coordinates of the ligand atoms and were ranked on the basis of the free binding energy.
The calculation of binding free energy was evaluated using the MM/PBSA (Molecular Mechanics/Poisson Boltzmann Surface Area) method as implemented in AMBER11 [65]. The electronic and Van der Waals energies were calculated using the sander module in AMBER11. The solvation free energy contributions may be further decomposed into an electrostatic and hydrophobic contribution. The electrostatic portion is calculated using the linearized PB equation. The hydrophobic contribution is approximated by the LCPO method [66] implemented within sander. The changes in entropy upon ligand association ΔS are estimated by normal mode analysis. For stable plateaus of the MD trajectories, snapshots were collected every 20th frame (every 20 ps) and used to calculate relative binding affinity (ΔGbind) and absolute free energy (ΔG).
A free energy decomposition of the protein ligand complexes was performed on a pairwise per-residue basis using the MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) method as implemented in AMBER11. The GBSA implicit-solvent solvation model was used in order to avoid the retarding effect of the PBSA method.
[2, 4, 6, 7-3H]-E1 and [2, 4, 6, 7-3H]-E2 were bought from Perkin Elmer, Boston. Quickszint Flow 302 scintillator fluid was bought from Zinsser Analytic, Frankfurt. Marmoset 17β-HSD1 and 17β-HSD2 were obtained from marmoset placenta according to previously described procedures [41]. Fresh marmoset placenta was homogenized and cytosolic and microsomal fractions were separated by centrifugation. For the partial purification of 17β-HSD1, the cytosolic fraction was precipitated with ammonium sulfate. 17β-HSD2 was obtained from the microsomal fraction.
Inhibitory activities were evaluated by an established method with minor modifications [41]. Briefly, the enzyme preparation was incubated with NADH [500 µM] in the presence of potential inhibitors at 37°C in a phosphate buffer (50 mM) supplemented with 20% of glycerol and EDTA (1 mM). Inhibitor stock solutions were prepared in DMSO. The final concentration of DMSO was adjusted to 1% in all samples. The enzymatic reaction was started by addition of a mixture of unlabelled- and [2, 4, 6, 7-3H]-E1 (final concentration: 500 nM, 0.15 µCi). After 10 min, the reaction was stopped by the addition of HgCl2 (10 mM) and the mixture was extracted with diethylether. After evaporation, the steroids were dissolved in acetonitrile. E1 and E2 were separated using acetonitrile/water (4555) as mobile phase in a C18 reverse phase chromatography column (Nucleodur C18 Gravity, 3 µm, Macherey-Nagel, Düren) connected to an HPLC-system (Agilent 1200 Series, Agilent Technologies, Waldbronn). Detection and quantification of the steroids were performed using a radioflow detector (Agilent 1200 Series, Agilent Technologies, Waldbronn). The conversion rate was calculated after analysis of the resulting chromatograms according to the following equation: . Each value was calculated from at least three independent experiments.
The 17β-HSD2 inhibition assay was performed similarly to the 17β-HSD1 procedure. The microsomal fraction was incubated with NAD+ [1500 µM], test compound and a mixture of unlabelled- and [2, 4, 6, 7-3H]-E2 (final concentration: 500 nM, 0.11 µCi) for 20 min at 37°C. Further treatment of the samples and HPLC separation was carried out as mentioned above. The conversion rate was calculated after analysis of the resulting chromatograms according to the following equation: .
Energy profile drawn for the marmoset 17β-HSD1 model using PROSA. Energy profiles of marmoset 17β-HSD1 in complex with NADPH (orange) and of marmoset 17β-HSD1 in complex with NADPH and E1 (green).
Verify3D results for the marmoset 17β-HSD1 model. Verify-3D results are shown for the secondary complex of marmoset 17β-HSD1 (orange) with NADPH and for ternary complex (green) with NADPH and E1; residues with positive score are reasonably folded.
Cluster analysis of molecular docking results. All energies are expressed in kcal mol−1. The lowest energy conformation of each cluster, which is marked in bold was used for further investigation.
We thank Prof. Dr. Rolf W. Hartmann for helpful discussion and Prof. Dr. Almuth Einspanier for providing marmoset placenta.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The study was founded by financial resources of Saarland University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
UNIT4's Anwen Robinson's message to tier 1 ERP players, such as Oracle and SAP, is 'bring it on', after it was recently revealed that the company's Agresso platform was selected to underpin the government's first independently run shared services centre.
Working with its partner Arvato, Agresso will replace the SAP's ERP platform in the government's ISSC1 shared service centre, which will provide back office services to the Department for Transport and its executive agencies.
UNIT4 has typically been branded as a tier 2 player in the ERP market and Robinson said that the contract win is a "milestone moment" for the company.
"I am thrilled. There's been a lot of work that's gone into this and I think it is a culmination of our efforts over the years. We were in the right place, at the right time, with the right solutions," she told Computerworld UK at the UNIT4's customer conference this week in Birmingham.
"The flexibility that we have shown over the years is playing big time to what the market wants right now - especially from a public sector perspective."
She added: "Budgets are being slashed and the government has been looking for an alternative solution."
When asked if Robinson thought they had won the contract because of a lower price point, she said that this came into play, but also was down to being able to prove with a back catalogue of customer references that Agresso could work for a major central government project.
"I think our systems are proven. If you look at where our Agresso system originated from, in Scandinavia, it underpins about 95 percent of government over there. That's the pedigree," she said.
"If you look at our customer base in the UK, we are the leading provider of finance solutions in local government and the leading provider to post-16 education. So we have got a very strong reference base and track record of delivery, despite having been below the radar."
She added: "Our solutions also give choice. If you want it wholly in the cloud, that's fine, it's the same Agresso solution that can be delivered on premise. Or a hybrid of the two. We are ticking the boxes in terms of direction of travel that government wanted to go in. For far too long the only option has been tier 1 vendors, when actually there are others."
UNIT4 was also recently selected, along with its partner BT, to provide a shared services platform to the tri-boroughs area in London. This was another significant contract win for the company. Robinson said that these successes are down to the fact that UNIT4 recognised a change in the public sector where it was no longer looking for software companies to overhaul ERP estates, but for strategic partners that could guarantee efficiencies and savings.
"Three years ago we typically gained our success by doing it all ourselves. But when I took over I recognised that the key focus at the start of the recession in the public sector was to deliver shared services with a strategic partner. Because of this we had to raise the profile of UNIT4 and of Agresso, because traditionally those partners (such as Arvato and BT) would have worked with SAP and Oracle," said Robinson.
"This is a step change for us, it reinforces our partner policy in terms of working with key strategic plays that recognise what our key differentiators are, and that it's not more of the same. What's underpinning these two deals is that they have both been with partners."
She added: "We are moving away from doing it ourselves and are very much focused on supporting the partner."

subset: commoncrawl


text:
Files complaint with U.S. Department of Justice
Compiled by MCJ Staff
Coinciding with the state's celebration of the emancipation of slavery in the United States, The Milwaukee Branch of the NAACP filed on Tuesday a complaint with the U.S. Department of Justice against the City of Milwaukee for its continuous noncompliance with civil rights and equal opportunity laws.
"The DOJ should fully investigate the complaint and direct the City of Milwaukee to discontinue practices and institute changes that will end both disparate impact on and the disparate treatment of minority and women contractors," said NAACP Milwaukee Branch President James Hall in a press statement.
The civil rights organization is also calling for a community-driven external audit of the city's administrative application of civil rights and equal opportunity laws and policies as it relates to the city's procurement system.
The audit would help the city to determine the following:
• If procurement coordination between departments is compliant with local, state, and federal civil/equal rights laws and policies;
• If procurement coordination between the city's subcontractors and their subcontractors is compliant with local, state, and federal civil/equal rights laws and policies;
• If the city has operated its procurement system in violation of the Federal False Claims Act;
• If skill training is required in local, state, and federal civil/equal rights laws and policies for staff charged with administering the city's procurement system and
• What actions the city will need to take to address oversight, coordination, and administrative operations of its procurement system to ensure compliance and local, state, and federal civil/equal rights laws and policies and to mitigate disparities.
Hall called on Mayor Tom Barrett and Common Council President Willie Hines to establish a working group or task force of community members to oversee the audit of the city's administrative application of the civil rights laws and policies pertaining to the procurement system.
In a letter to the mayor and council president, Hall said the audit should cover the time frame used for the D. Wilson Consulting Services' disparity study (2005-2010).
"We believe this audit should be conducted primarily to determine if there were administrative issues with applying local, state, and federal civil/equal rights laws and policies that precipitated the disparities in contracting shown by the recent D. Wilson Consulting Service report and issues related to the same in the Mason Tillman 2005 study of the Emerging Business Enterprise (EBE) program."
The Tillman, Wilson and EBE reports show conflicting dollar amounts procured by the city. When carefully studied, the conflicting amounts could call into question the validity of the city procurement system's transparency. The Mason Tillman Study reveals the deficiencies in the city's administrative and oversight approach to contract procurement.
The Wilson and Tillman studies collected anecdotal evidence of discrimination compiled from 62 one-on-one personal interviews with business owners who have done business with or attempted to do business with the city and/or MMSD as a prime contractor or as a subcontractor.
The interviews supports the belief the city's approach to contract procurement does not address the underutilization of specific race/ethnic and gender groups in the areas of construction and goods and services.
According to the Wilson report, some minority business owners reported being confronted with racial slurs, never offered opportunity and did not get paid for work performed, etc. causing economic hardships on their small businesses.
"The city does not have uniform policies or procedures for all departments within the city that ensure adequate protection for subcontractors, which are typically small, minority and women-owned businesses, from business practices that can be detrimental to the survival of those businesses," according to Hall.
Hall noted the city has spent $1 million over 18 years on four disparities in contracting studies and found large disparities, as well as poor administration of equal opportunity in all city contracting opportunities between Majority and Minority/Women Business Enterprises.
Yet, despite the city's documented knowledge of these problems and despite having specific recommendations on how to reduce or eliminate them, the complaints persist.
"The NAACP's 102-year mission aims to ensure that the fundamental rights of all Americans are protected and advanced," Hall said. "Our request is being made out of that historical focus and concern.
"Consequently, we emphasize that it is the responsibility of each political leader in government and all community stakeholders to uphold the federal and state constitutional rights of citizens and ensure that these rights are fully protected at all times."


text:
Today, I am here to support the effort of the Timken Company and the United Steelworkers Union to maintain the order -- and limit further harm to the domestic industry and its workers.
The Timken Company, which is headquartered in Canton, Ohio, is a major U.S. producer of tapered roller bearings (TRB).
It has several of its TRB facilities in my state, as well as facilities that produce bearing quality steel.
In Ohio, there are nearly 4,700 Timken workers, including more than 2,500 whose jobs are directly dependent upon continuation of this order.
Since the second sunset review, there have been several plant closings and layoffs in the domestic industry.
Without the restraining effects of this order, I feel certain that greater harm would have been suffered in my state during your current period of investigation.
In January 2009, Timken laid off 60 workers at its Bucyrus, Ohio tapered roller bearing factory. In 2009, it closed the Canton Bearing facility and Gambrinus Bearing facility.
Fortunately, the Gambrinus roller plant remains open.
Some foreign competitors have proven time and time again that they are willing to do anything -- even cheat to gain an economic advantage over American innovation.
And too many American companies like Timken have been undermined by countries that game the system.
Fortunately, because of some of the wise decisions you've made -- and new efforts to enforce trade law -- we're seeing some improvements.
U.S. companies are beginning to bring production back to the United States.
When a company decides to re-shore, U.S. production often increases -- which means new jobs are created here at home.
But the viability of repatriated production depends on the ability of domestic producers to defend against unfairly traded imports.
Too often foreign producers, aided and abetted by their governments, refuse to abide by internationally-agreed upon rules for international trade.
They engage in anti-competitive tactics such as dumping and improper subsidies to gain market share in export markets -- no matter the impact on the industries in those markets.
China is one of the worst offenders.
Dozens of cases have been filed in recent years on products from China -- across a wide spectrum of industries including products like steel, solar panels and wind energy structures to name but a few.
Distortions in the Chinese economy -- including granting loans to favored industries; price controls in various areas; state ownership of sectors; and restrictions on key inputs to bearings such as steel -- create an environment in which export prices often bear no relationship to underlying actual costs.
It is these market distortions driven by China's state capitalism system that so severely disrupts the American market place.
And it is these distortions that our trade remedies must correct.
That is why it is so very important that the existing antidumping order on Chinese tapered roller bearings remain in place.
If the order is revoked, imports of tapered roller bearings from China will be dumped on the U.S. market at artificially distorted prices.
Companies, such as Timken, which operate in the free market, will be virtually unable to compete domestically, and will struggle to and remain a viable company.
History has shown that foreign producers of tapered roller bearings are drawn to the U.S. market and who can blame them?
The U.S. market is highly attractive with a very large customer base, well-developed infrastructure and channels of trade, and easy and open access.
You can be sure that Chinese producers find the U.S. market no less attractive than producers in other countries.
Indeed, the U.S. has been, and continues to be by far, China's single largest export market for tapered roller bearings.
If the order is removed, it is a certainty that China will resort to the same tactics of dumping that they have used successfully in countless other markets -- to seize market share away from Timken and other domestic producers.
The impact on manufacturers in my state will be nothing short of devastating.
You have the power and the authority to prevent that from happening.
The work of this Commission is important to workers and producers in my state and across our nation, and I urge you to exercise your authority to once again do the right thing and defend them in this review.
Thank you.
Source: http://www.brown.senate.gov/newsroom/press/release/in-testimony-before-international-trace-commission-brown-goes-to-bat-for-timken-workers-by-urging-panel-to-uphold-duties-and-preserve-jobs


text:
LONDON, Dec. 30 (UPI) -- Richard Boston, founder of one of Britain's first environmental magazines and a proponent of real ale, has died after a short illness.
He was 67.
Boston became a feature writer and regular columnist for The Guardian newspaper in 1972 and was well known for his column, "Boston on Beer," which noted the highlights of drinking true British-brewed ale at a time when large breweries were attempting to impose mass-production on the nation.
Boston was also known as an early environmentalist. He went on in 1977 to found "The Vole," one of the first environmental publications in Britain. He had the backing of the Dartington Trust and Monty Python star, Terry Jones.
The Guardian noted that the magazine started out with a bang with a circulation of about 20,000 before it ceased publication some three years later. Boston once told an interviewer, "Although ideologically I'm democratic, temperamentally I'm dictatorial."
Boston returned to The Guardian where for several years he wrote features, reviews and columns.


text:
Trot like a turkey and learn about honey badgers
Things to do over Thanksgiving weekend
Scott Ellis 7 a.m., Nov. 22
Norsemen capture league title in defensive struggle
When Steele Canyon and Valhalla meet each season, the Jamacha Helmet trophy is on the line. Friday night in Spring Valley the stakes were higher and the Norsemen won a 14-7 defensive battle to capture both the helmet and the Grossmont South League title.
"It feels awesome," said Valhalla quarterback Pete Thomas. "We've been working for this since January."
Valhalla's defense stopped Steele Canyon twice in the final two minutes. On each occasion, the Cougars had the ball in Norsemen territory.
"Coach gives us the philosophy 'bend, don't break,'" said Valhalla defensive lineman Leonard Pannek. "We don't get nervous, we just play our hardest, get low and dig for every play."
With just more than two minutes left, Steele Canyon (5-3-1, 2-1-1 Grossmont South) had the ball inside the Valhalla 10-yard line and appeared headed for a tying score. But a botched handoff between Cougars quarterback Brad Boehmke and tailback Alex Perlin left the ball on the turf, which Pannek pounced on.
"I was like, 'Oh! There's the ball!'" Pannek said. "I just jumped on it and held on for dear life while my teammates got in front of me."
Following a Valhalla punt, Steele Canyon had the ball at the Norsemen 43-yard line with 1:26 left. But after an incomplete pass and a holding penalty, Valhalla defensive back Kweishi Brown picked off Boehmke to seal the win for the Norsemen.
"It means a whole lot. We always love beating Steele Canyon – it's always a great feeling," Pannek said.
Valhalla (7-2, 4-0) went ahead 14-7 in the third quarter on a 65-yard touchdown pass from Thomas to receiver Jevon Hasten. Hasten caught the ball in stride and sped past the Steele Canyon secondary for the score.
"The guy was on me and Pete threw a perfect pass and I was there," Hasten said.
Defense dominated from the start of the game and after a scoreless first quarter, Steele Canyon broke the ice in the second quarter. The Cougars had the ball for nearly 11 minutes on a scoring drive capped with a Boehmke one-yard plunge.
It took Valhalla only a minute to drive 62 yards and tie things up. Traivonne Brown scored a nine-yard touchdown on a direct snap and the teams remained tied going into halftime.
"We're two really evenly matched teams," Thomas said. "We just happened to pull this one out tonight."
With the win, Valhalla clinches the Grossmont South League title outright. Thomas, who finished 12-for-20 for 208 yards, now has his sights set returning to Qualcomm Stadium next month.
"Last year we were a big team and I think it surprised some people," Thomas said. "Some people thought it was a fluke."
The Norsemen will look to finish league play undefeated when they face Granite Hills (3-6, 0-4). Steele Canyon gets Monte Vista (2-7, 1-3) in their regular season finale.
Valhalla players celebrate their win over Steele Canyon


text:
How To Eliminate Negative Thoughts & Emotions
Today I wanna riff about shitty thoughts and how to deal with them… First off, I'll start by saying that a negative thought is any thought that you have that produces an undesirable/damaging emotional response inside your body. For example; stress, anxiety, unease, anger, frustration, depression, fear, guilt, regret. Essentially, any thought that becomes a […]Continue reading


text:
Once again colette presents a new series of wall drawings in the Domestic series. Graphic designers from around the world created wall drawings for colette. This time they worked with Geneviève Gauckler, Tania et Vincent, Ich & Kar, Sophie Toporkoff, Kustaa Saksi and Furi Furi.
The new series of Colette Domestic Wall Drawings is now available online.
More images follow after the jump.


text:
Female, 16, Toronto
I was sixteen, he was seventeen. We had been dating long-distance (his town was about an hour drive away from my city) for a couple of months at the beginning of the school year. We would meet up during the weekends at a mall in a town in between, then hang out around a nearby Japanese zen garden, making out and feeling each other up and down until my mom came to pick me up and he had to drive back home.
During either our first or second date, he had performed oral sex for the first time, and I was a first-time recipient. We found a clearing in the bushes where he laid down his hoodie and I went down on top of it. That was the most fun I'd had under the still-high September sun.
When we had chatted about it over IM, he let me know that if I wanted him to do that again, I should shave completely. I wasn't unruly or anything, just neatly trim, but he preferred completely bald. Those were fair terms, I thought.
Unfortunately, this chat history was saved, my compliance recorded. (This was before I learned to put a password on everything.) My mother discovered the dialogue, and in her frenzied, recently laid-off state, she went mental. She thought this meant I would end up just like her: a single mother with a kid who had the uncanny ability to disappoint her at the most inopportune times. She forbade me to see him again, to prevent my (in her mind) otherwise inevitable unwanted pregnancy, which she was now in no position to support financially.
But our puppy love was stronger than that. We still talked for hours each day, trying to figure out how to get him back on my mother's good side until we came up with our brilliant plan: have a Meet the Parent dinner so my mom could see that our love was real, that our oral lovemaking wasn't in vain.
She had just gotten a lead on a new job, and consequently was feeling less like we'd end up like every other broken welfare family out there, and more like my boyfriend could be a decent human being who cared about me. It was a date.
Mid-day that Saturday, she dropped me off at the suburban shopping center (as per usual) with the plan to reconnect with us in the evening after our day of window-shopping and her day at her new office near the mall. My boyfriend and I did a lap or so around the mall before heading to the zen garden around sunset.
With the days ending earlier, and the cold Canadian nights coming earlier than preferable, we'd had the foresight to pack a comforter; laying down a hoodie would no longer suffice. It was nice, nuzzling in the bushes under the stars. Making out under the stars. Groping each other under the stars. Having him push his pants down enough to take his dick out and slip it in me for a pump and a half before an emergency pullout and ejaculation in his boxers, under the stars.
"Oh shit," he said.
He looked around for materials to clean up the mess, and opted to use the comforter to sponge his soiled silk skivvies. "We're meeting my mom in an hour," I offered.
He continued to air-dry his undies until it was time to head to the restaurant to greet my mother. He went to the restroom before shaking her hand. While our twilight activities had been over in a flash, time flowed viscously during dinner. Over the course of our courses, my boyfriend managed to keep a straight face while being grilled about his aspirations of being a rock star, playing hockey, his family, and everything else a mother would want to know about the boy who she knew to be orally corrupting her little girl.
When we finally finished dessert and settled the tab, my mom seemed content with my boyfriend's performance during the interview. As we said goodbye, she even gave him a hug. I imagine if she knew my first time had happened just moments earlier, their meeting would have ended in more of a stranglehold.


text:
Enjoy an ad free experience by logging in. Not a member yet? Register.
Results 1 to 2 of 2
Thread: PHP AVG Query
- Join Date
- Jan 2013
- Posts
- 8
- Thanks
- 0
- Thanked 0 Times in 0 Posts
PHP AVG Query
Hi everyone
First of all, thanks to everyone who advised me and pointed me in the direction of a MySQL tut on my last post, it has helped enormously.
Just wondering if anyone could help me out on the below:
I have a MySQL table that has data from surveys and is identified by an ID per review.
I'm trying to get the PHP script to calculate the average of 10 columns across th row - is this possibe? The code I'm trying to use is:
$avgtocalc = mysql_real_escape_string($_GET['ad1']); $average = mysql_query("SELECT * FROM reviewdata WHERE address='$avgtocalc' AND published LIKE 'Live' AVG ('livedthere' + 'parking' + 'crime' + 'noiseneighbours' + 'noisetraffic' + 'distanceshops' + 'distanceschools' + 'conditionpavements' + 'conditionroads' + 'nightlighting')/(10) ") or die(mysql_error());
Does anyone have any ideas? I've Googled this to death but can't find what I need.
Thanks in advance
- Join Date
- Sep 2002
- Location
- Saskatoon, Saskatchewan
- Posts
- 17,025
- Thanks
- 4
- Thanked 2,668 Times in 2,637 Posts
Using quotations are literal in SQL. That will try to add the strings, use either the accent grave (`) or preferably nothing if you don't use reserved words or spaces.
The location itself is probably wrong. If you want to select that, you need to place it within the columns to select of the query. This is sitting somewhere in the WHERE clause. On the other hand, if you want to use it for a where clause, you would then need to use a group and a HAVING in order to do it.
I would suggest aliasing it as well, otherwise you'll need to use a fetch_row command in order to offset it numerically, iterate the resultset with a foreach, or use the associative value of what that expression is.
Also this has nothing to do with PHP, moving to MySQL forum.PHP Code:
header('HTTP/1.1 420 Enhance Your Calm');


text:
Over 1.8 million nonprofits and charities for donors, volunteers and funders
Causes: Emergency Assistance, Human Services
Mission: Soupmobile, inc. Is a non-profit mobile soup kitchen feeding, clothing, and caring for the needy and homeless in the dallas area. Soupmobile also provides housing assistance for qualifing individuals.
Programs: Feeding: the soupmobile serves 200,000 meals per year to the homeless and hungry.
christmas: the soupmobile takes 500 homeless people to a high end hotel for christmas in our group homes.
housing: the soupmobile houses the homeless in our group homes.


text:
Sign In
Contact Us
Create your custom Crayola product in just minutes. Visit the Crayola My Way factory to get started now. Tip the crayon will guide you through each simple step!
Turn any art into a real stuffed animal! Super easy to order!
Add To Favorites
Celebrate birthdays in a big way! Write your greetings with Crayola® Window Markers. What a great surprise!
To help a favorite friend or relative celebrate a birthday, surprise them with a giant-size birthday greeting.
Find a window or mirror your friend will notice. Use Crayola Windows Markers to write your Happy Birth-Day greeting.
Illustrate the greeting with a drawing just for them. Show something special they'll do that day or about the person. If you draw a birthday cake, include the correct number of candles!
If you draw on a window, close the curtains until the morning of your friend's birthday. Open the curtains for a birthday morning surprise!
Let's make something!
Be creative and put your special touch on a personal notecard for family and friends.
Add To Favorites
Do you like to fish? Make a 3-D triarama of your favorite fishing spot.
Get inspired by the sweet-smelling scents and rich colors of Crayola® Power Lines Washable Markers with Scents and write
Designing t-shirts together at a family reunion or summer barbecue is not only fun, but also makes a great keepsake to c
Invite the whole neighborhood to join your scavenger hunt. Leave a trail of clues painted on the sidewalk. Who will find
What happens on the 12th day of Christmas? Three kings arrive on camels with candy and gifts. Fill a box with hay (for t
Explore celebrations across the globe! Capture a celebratory moment of your own by designing a colorful fabric banner!
Celebrate a graduation, good grades, or a homecoming with pizzazz. Use Crayola® Sidewalk Paint to craft a sign that anno


text:
Heidi Klum revealed she is more than just a pretty face in a new interview. In fact, the X Factor judge is a normal mom who secretly tosses some of her kids' artwork so it won't overwhelm the house.
"I think that it's important for kids to see their own art on the wall — it makes them feel important. To be honest, I just hold onto the best things. A lot of times, I open the drawers, and when they're not looking, I get rid of a few things. . . . You have to do it; otherwise it would take up the whole house!" Klum admitted to Lil Sugar.
As a mom who has condemned a few scribbles to the garbage bin, I had to laugh reading the German beauty's comments. Although every piece of art my daughters create is precious to me, I can't very well display each coloring page they grace with a crayon.
That being said, of course, I feel a tiny bit guilty when I toss a paper plate dog or a toilet paper roll with dozens of buttons glued to it. But like Klum, I proudly show off plenty of creations my kiddos make at home and school. We have a bulletin board in the kitchen that displays their latest masterpieces. Our dishwasher is covered with coloring book creations. And both of my girls have bulletin boards in their rooms tacked full of artwork.
Meanwhile, Heidi Klum also talked about her instincts as a mom.
"You always have to do what your gut tells you. It's about doing what you think is the right thing to do in all situations, and I think as moms, we're always trying to just do our best," the mom-of-four told Lil Sugar.
How do you determine which mini masterpieces to save and which ones to toss?
Photo credit: Flickr


text:
MEMPHIS, Tenn. — Fred's Inc. today announced that its board of directors has declared a quarterly cash dividend of $0.06 per share.
The dividend is payable on June 17, 2013, to shareholders of record as of June 3, 2013.
Fred's Inc. operates 715 discount general merchandise stores, including 21 franchised Fred's stores, in the southeastern United States. For more information about the company, visit Fred's website at www.fredsinc.com.
Get connected and follow us on LinkedIn for the most in-depth coverage of drug store news. Join the conversation.


text:
HUNTINGTON PARK, Calif. (AP) — A fire feeding on soybean oil at a frozen food plant sent flames soaring through the roof Monday and created a towering plume of smoke that was seen for miles around the eastern Los Angeles suburb.
About 150 firefighters from several departments fought the blaze, which was reported by employees at about 5:30 a.m., Los Angeles County Fire Inspector Don Kunitomi said.
Employees told reporters that they heard an explosion and then made a futile effort to douse the flames with fire extinguishers.
More than four hours after the fire began at Windsor Quality Food Co., it was still too dangerous for firefighters to go inside. Instead, they poured streams of water on it from outside.
Huge flames and smoke surged from the top of the building just before dawn.
"Anytime a fire burns through the roof, it's extremely dangerous for firefighters" because of the threat of collapse, Kunitomi said.
The fire was stubborn and was being fueled by soybean oil used for cooking inside the plant, he said. However, a firewall prevented the flames from spreading to nearby buildings.
Employees fled the building. The only reported injury was to an employee of a neighboring business who hurt an elbow after falling down while running away, Kunitomi said.
The cause of the fire was not immediately known.
Huntington Park is about 5 miles southeast of downtown Los Angeles.


text:
The following stories have been hand-selected by the Assignment Desk at News4:
Stink Bugs Wreaking Havoc on Farmers, Gardners
Stink bugs are devastating Frederick County's crops. Scenic View Orchards in Sabillasville has lost 30 percent of its raspberries, 25 percent of pears, 15 percent of apples and 10 to 15 percent of peaches and nectarines. "And we're the lucky ones," said Richard Masser, whose family owns and operates the orchards. "Other people have lost most of their peach crop and sweet corn." (Frederick News Post)
Washingtonians Love to Tweet
Washington ranks No. 12 on a list of the nation's most social media-savvy cities, but the region ranks third in the nation for Twitter use by its business people. The study, conducted by NetProspex, based its Twitter-savvy rankings on the average number of Twitter accounts, tweets and followers. New York and San Francisco ranked as the top two Twitter towns in the nation. Sacramento and Phoenix round out the top five. The report also ranked cities on overall social media use by companies. Washington's No. 12 ranking put companies here behind those in cities that include Boston, Seattle and Denver in use of social networking for business promotion. (Washington Business Journal)
College Students Targeted in Rash of Attacks
The fall semester has gotten off to a troubling start for several universities in Richmond and across the state as police investigate assaults and robberies of students on and off campus. One rash of assaults occurred over Labor Day weekend at the University of Richmond, where authorities are investigating three campus assaults in one hour in which students were punched while walking alone; another assault in which a man was hit so hard that his nose was broken and he lost a tooth; a bizarre break-in at a campus apartment; and a report of a sexual assault. (Richmon Times-Dispatch)
Stolen Dog returned
A dog stolen outside a Fredericksburg 7-Eleven late Friday has been returned to its owner. City police spokeswoman Natatia Bledsoe said the 2-year-old wire-haired terrier ended up at an animal shelter in Spotsylvania County. The woman who took the animal to the facility initially tried to sell the dog on Craigslist, Bledsoe said, but later pulled the ad. The dog's owner, a 52-year-old Charles Street resident, tied the dog to a pole late Friday when he went into the 7-Eleven on Amaret Street to purchase extra minutes for his phone. The man saw three people petting the dog while he was in the store, but he wasn't concerned because the dog is friendly and often attracts strangers. (fredericksburg.com)


text:
I knew that title would get your attention.
So, I saw this 'review' of the Wave Jet (all 40 secs of it):
And was wondering: has anyone even seen one of these operating in surf? Or better yet, anyone rode the Wave Jet? Are there any online reviews for it?
The only time I seem to 'see' the Wave Jet is in the ads with GMac on the thing.
Maybe I'll use some of my Social Security bennies & purchase a Wave Jet when I turn 68. Just to go out every single day in any blown out conditions. Until then, just curious as to where the technology took this in terms of performance.
Results 1 to 10 of 69
Thread: Wave Jet Review
Apr 14, 2013, 07:54 PM #1
Wave Jet Review
Last edited by yankee; Apr 14, 2013 at 08:02 PM.
Apr 14, 2013, 08:20 PM #2
ask greg long, he saw him last.
Apr 15, 2013, 12:23 AM #3
It might be fun for what it is but it isnt surfing. Surfing is generating speed from the wave, without the motor pushing you that thing would sit there like a waterlogged soft top.
Apr 15, 2013, 04:34 AM #5Member
- Join Date
- Apr 2011
- Posts
- 57
so ****ing gay
Apr 15, 2013, 01:21 PM #6
Wouldn't mind having that to cruise around the intercoastal. No way I would want to be seen on one in the surf.
I'd like an SUP wave jet to use for fishing.
Apr 15, 2013, 03:10 PM #8
still inferior to the Roy Stuart. Simple physics.
still inferior to the Roy Stuart. Simple physics.
Last edited by Zippy; Apr 15, 2013 at 06:03 PM.
Apr 15, 2013, 06:37 PM #10
they were demoing these things in vb last year around ecsc , I didn't try it out but from what I saw its not worth the price, they are fairly big and heavy and the board really limits what you can do performance wise, good luck getting it on rail.


text:
DFE Click here for Course Outline
Course Description:
This course was developed to provide an introduction to the exciting and growing field computer crime investigations and computer forensics. It is designed to train investigators in electronic discovery and the fundamentals of conducting an effective computer forensic examination.
Digital Forensic Essentials is ideal for someone new to the computer forensic field. However, this course is also valuable to someone who has been in the field and would like to brush up on a few topics. The attendee will learn sound forensic imaging procedures, how to conduct examinations, and validate forensic operations as well as how to report findings in a clear and concise manner.
The "Digital Forensic Essentials" course is specifically designed for corporate, government and military personnel who, in the performance of their duties may be asked to conduct a basic digital forensic examination. This course is essential to information security, risk management, loss prevention, corporate security and law enforcement personnel who encounter digital evidence while conducting an investigation.
Participants must be familiar with common computer functions. This workshop caters to those with no previous experience in digital forensics; however, if you are just beginning in information security, then this course is not appropriate for you. Ideally, participants should be responsible for locating, collecting, analyzing, and testifying to evidence of malicious activity on a computer or network of computers.
Key Content:
Methodology for conducting a computer forensic examination.
The workshop provides an introduction to the field of computer forensics and the basis for gathering electronic digital artifacts. Participants are introduced to the concepts, situations and personalities they may encounter while investigating an incident.
NetRunner Training Center at Dare Corporate Center275 Slater StreetSuite 900Ottawa, Ontario K1P 5H9613-748-6222
Valerie Yersh
For assistance please call (613) 748-6222 or e-mail Valerie Yersh, at firstname.lastname@example.com


text:
Evan Goldberg Thinks 'This is The End' Sequel is Entirely Possible
by Ethan Anderton
June 26, 2013
Source: ScreenDaily
If you haven't seen the apocalyptic comedy This is The End from writers and directors Seth Rogen and Evan Goldberg, you'll probably want to stop reading right now, because there's no avoiding spoilers when talking about sequel potential. ScreenDaily recently sat down with Goldberg and struck up a conversation about a sequel to the film which has proven to be quite the R-rated comedy success after pulling in just over $60 million after a little over two weeks in theaters, and that's on a $31 million film. So when asked about the possibility of follow-up, Goldberg said, "If you ask me, I'd say there's a good chance of sequel." Read on!
However, the co-writer and co-director followed up, "If you ask Seth [Rogen], he'd say no. So, we'll see." A sequel seems unlikely only because at the end of the film, nearly all of the main characters are dead, Seth Rogen and Jay Baruchel finally gain entry into heaven, meet up with their friend Craig Robinson, and partake in a jam session with The Backstreet Boys. It's hilariously ridiculous, and it makes for some hearty laughter. So if Goldberg thinks a sequel is a likely possibility, just where could the story go from there?
Well, in what sounds like a really interesting approach, the story that features Rogen, Baruchel and Robinson playing versions of themselves alongside James Franco, Jonah Hill and Danny McBride would get a whole lot more meta. Goldberg explains, "We actually talked about doing a sequel where it starts at the premiere of This is the End. Seth's a cokehead in this version, Michael Cera is a calm dude with a boyfriend, Rihanna and The Backstreet Boys are back." So This is The End would actually be a feature film as opposed to a "real" story, but that's just the start. We're not sure where things would go next.
Goldberg also says, "We have a lot of ideas: a heaven and hell, for example, and a garden of Eden version where Danny [McBride] is Adam." That's pretty vague, but the fact that Rogen and Goldberg discussed some potential plotlines sounds promising. Though they could have just talked about it while they were smoking weed one day. The real hurdle is getting that kind of cast together again and Goldberg mentions, "We lucked out that they were all available. I honestly don't know if we could get the guys together." We won't hold our breath, but a This is The End sequel could be pretty fun. Thoughts?
Follow Alex's main profile on :


text:
2009-06-25 / Letters/Opinion
Thanks To Our Crossing Guards
Dear Editor:
Now that schools are closing, a word of thanks to all our School Crossing Guards for doing a wonderful job getting kids safely back and forth to their schools.
It can be a dangerous job and they are out there — rain or shine! William Cassata Cassata Fuel Oil


text:
Detroit Lakes Girls Hockey Opener
Some of the growing pains for the young Detroit Lakes girls' hockey team showed through in its 7-1 season opening loss in Fergus Falls Friday.
The Lakers did play up to the Otters in the first period, trailing only 2-1, but Fergus Falls went on a 5-0 run in the final two periods.
Sophomore forward Emily Seebold scored DL's lone goal, which came 7:54 into the first period, with assists coming from senior defensive player Callie Johnson and freshman Chandra Johnson.
But the Otters set the tone early, scoring 26 seconds into the game off of junior defensive player Lauren Norgren's stick.
A two-goal second period put Fergus Falls in control 4-1, and then the Otters finished the game off with three more in the third.
Norgren finished with a season-opening hat trick.
DL sophomore goalie Veronica Badurek had 21 saves, while the Lakers had 18 shots on goal.
Jordie Salwei gained the victory in net for the Otters with 17 saves.
The Lakers will travel to Alexandria Thursday, for a 7:30 p.m. start.
Laker results
Detroit Lakes at Fergus Falls, Friday, Nov. 9.
Lakers 1 0 0 - 1
Otters 2 2 3 - 7
1st Period -- Scoring: FERGUS - Norgren Goal 0:26; DL - Seebold Goal (PP) (Johnson, Johnson) 7:54; FERGUS - Kolstad Goal (Norgren) 10:45. Penalties: None. Goalie Changes: None.
2nd Period -- Scoring: FERGUS - Norgren Goal (Niblock, Kolstad) 16:09; FERGUS - Odland Goal 16:56. Penalties: None. Goalie Changes: None.
3rd Period -- Scoring: FERGUS - Norgren Goal 3:42; FERGUS - Kuehl Goal (Kolstad, Norgren) 10:38; FERGUS - Kuehl Goal (Niblock) 11:27. Penalties: None. Goalie Changes: None.
Saves - DL (Veronica Badurek) 21....FF (Jordie Salwei) 17.
Shots on goal - DL 7 4 7 - 18...FF 8 10 10 - 28.


text:
Eliza Doolittle, fictional character, a Cockney flower girl who is transformed into a woman of poise and polish in George Bernard Shaw's play Pygmalion (performed 1913; filmed 1938; adapted as the stage musical My Fair Lady, 1956; filmed 1964).
Keep Exploring Britannica
We welcome suggested improvements to any of our articles. You can make it easier for us to review and, hopefully, publish your contribution by keeping a few points in mind.
- Encyclopædia Britannica articles are written in a neutral objective tone for a general audience.
- You may find it helpful to search within the site to see how similar or related subjects are covered.
- Any text you add should be original, not copied from other sources.
- At the bottom of the article, feel free to list any sources that support your changes, so that we can fully understand their context. (Internet URLs are the best.)
Your contribution may be further edited by our staff, and its publication is subject to our final approval. Unfortunately, our editorial approach may not be able to accommodate all contributions.
Our editors will review what you've submitted, and if it meets our criteria, we'll add it to the article.
Please note that our editors may make some formatting changes or correct spelling or grammatical errors, and may also contact you if any clarifications are needed.
There was a problem with your submission. Please try again later.


text:
Written on: 06/01/2012 by lisa_green (1 review written)
Wahl Series 800 Hairclipper is really an excellent hairclipper. In the past, I have used a few hairclippers but this cuts far better without any kind of uneveness.
It also easy to hold and the functioning too is excellent. Now its been 8 months since I brought this clipper and have to say that this clipper is definitely worth the money.


text:
There is a press box area in Miami that honours the undefeated 1972 Dolphins team and every year Don Shula and Bob Griese and some other players of that era gather to crack a bottle of champagne when the last unbeaten team of that NFL season is no longer perfect.
Bobby Ackles remembers the strangest celebration of all.
"We were playing the Broncos on Monday night and they were unbeaten late in the season," said the B.C. Lions president and former Dolphins executive. "It was (John) Elway against (Dan) Marino, as good as it gets. We weren't supposed to win but we beat the Broncos that night.
"And afterwards Griese is quoted as saying: 'Great, we keep our record intact' and his kid (Brian) was one of the quarterbacks on the Broncos. And I'm thinking: 'Boy, that's kind of selfish, isn't it?' If my kid had a chance to break one of my records, I'd want him to do it.
"When you worked for the Dolphins, you couldn't help but hear about the undefeated season. It was everywhere."
Ackles is relating the story on a long-distance line trying to avoid talking about his own undefeated team. The B.C. Lions are 11-and-oh. The whispers of an unbeaten season already have begun. It's the sixth inning and a no-hitter is being pitched and nobody on the field or the bench will talk about it.
"You don't," Ackles said, "want to jinx yourself."
The Lions have not done a whole lot of jinxing this season. They have not lost since last year's Grey Cup. They have won two pre-season games, 11 consecutive regular season games, and with first place in the West all but guaranteed and the Grey Cup being played in Vancouver this year, well, you can see how this story looks to unfold.
There is magic in the air and on the field and even Wally Buono, who was a rookie linebacker with the Montreal Alouettes when the Dolphins went undefeated, knows how historical this season can be.
"History happens," Buono, the Lions' hugely successful coach, said in a telephone interview yesterday. "The focus can't be on the history, it has to be on the task at hand.
"We've not done anything yet that warrants anything. We're not going out trying to make history. We just want to continue on the road we're on."
A road toward history. The 1948 Calgary Stampeders are the only unbeaten team in Canadian Football League history. They went 12-and-oh in the regular season, won a two-game playoff (although tying the first game) and won the Grey Cup.
Now the season is 18 games. There are seven more obstacles for the Lions. Miami won 17 in a row, including Super Bowl VII. For the Lions to pull off a perfect season, the challenge is overwhelming. They would have to go 20-and-oh.
"Is it possible?" Buono asked rhetorically. "Everything is possible. You buy a lottery ticket because you think you've got a chance. Somebody is going to win. The person who wins the lottery probably didn't have a chance, but it's possible, right?
"There's always an exception to the rule. Why can't it be us?"
It's strange how things just seem meant to be: In 1972, sports psychologist Frank Lodato was working with Larry Little and Bob Matheson and other key members of the championship Dolphins. He wrote a book about the '72 team. Only 33 years later, he happens to be Buono's team psychologist. There are no books planned: Only he feels something that you always explain.
"The Dolphins lost their first Super Bowl to the Cowboys and, in fact, were beaten badly," Lodato said. "In the next training camp, Shula told them their goal was to get back to the Super Bowl and win it. They never lost sight of that goal."
The Lions lost to the Argos in their first Grey Cup with Buono in charge. "I sensed the same kind of feel in camp," Lodato said. "I keep hearing: 'We're so close. We can't miss out on this opportunity.' "
It isn't only the Lions' record that seems astounding. Dave Dickenson, the impish-looking quarterback, is passing at a 76% completion rate. That isn't logical.
Neither is an undefeated season.
"You can't sit around and say you're making history," Buono said, "because as soon as you say it, you probably won't be."
A look at the remaining games on the B.C. Lions' schedule:
Sept. 24, at Edmonton
Oct. 1, vs. Saskatchewan
Oct. 10, at Winnipeg
Oct. 16, at Montreal
Oct. 22, vs. Winnipeg
Oct. 28, at Edmonton
Nov. 5, vs. Saskatchewan


text:
Rob Cimmarusti has brought vitality and life to countless area musicians' recordings. Now, area musicians are helping to bring energy and health back to the longtime Quad-City producer and engineer.
A benefit concert to help with the costs of Cimmarusti's medical bills from his battle with some health issues will begin at 8 p.m. Friday, Jan. 11, at the Rock Island Brewing Company.
The lineup offers an array current and former rock acts — The Dawn, Three Years Hollow, and reunions of Einstein's Sister and Tripmaster Monkey — all of whom have collaborated with Cimmarusti and all of whom consider him a terrific friend and influential talent.
"It's a pretty amazing event," said Matthew Clemens, an author and longtime friend of Cimmarusti's. "The band lineup is like a who's who of the last two decades, and what's most amazing is that (Cimmarusti) worked with all of them over all that time. He's had a pretty impressive career."
"It's going to be an incredible show," said Kerry Tucker, guitarist and vocalist for Einstein's Sister and a co-organizer of the event along with Sean Ryan of The Dawn.
"Three Years Hollow is a huge draw on the local music scene," Tucker added. "The Dawn are, too, and this is going to be their last show for a while because their drummer is going into the military."
Tucker said it would be the first Einstein's Sister show in eight years at which the band plays its original material.
"And I think this is the first Tripmaster Monkey show with all four of the original lineup since at least the '90s," he said. "So it's a pretty big deal, and I think it's going to be a great show and a real tribute to Rob."
Cimmarusti has served as a producer and engineer for each of the bands, as well as several others on the local scene, helping to capture, create and fine-tune the sounds that have captivated their fans.
He's also worked as a sound engineer for a plethora of regional events, concerts and church gatherings.
"He's helped so many people, and we all wanted to do something to help him," Ryan said. "He's just a really friendly, helpful guy. He's not just a guy you go into the studio with and record and pay your bill. You remain friends with him."
Ryan said The Dawn has recorded four albums with him, and all of the bands on the bill have recorded multiple albums under his guidance.
"He's been a Quad-Cities musician's best friend over the last 20-something years, and he's a terrific guy," Tucker said. "He's incredibly dedicated, takes great pride in what he does and he's the most generous and fun engineer I've ever worked with."
Tucker also called Cimmarusti a "very funny and warm-hearted guy."
"The best thing I can say about him is that you always had a great time working with him and that always showed up in your work," Tucker added. "You can hear that sense of creativity and fun in his recording, and that's a tribute to him."


text:
Credit Advice
Have a question? Have advice to share? The combined knowledge and experience of everyone in the Credit Karma community can help you. Enter your question or help others below to get started!
QuestionPosted in Auto Loans
Question By
0 People Helped
All Responses
3945 People Helped
Depends on your score.
with 2K down and an interest rate below 5% you'd be at just under 500 a month for 7 years.
Rather than risk what "might be" I suggest that you go to your bank or credit union and pre-qualify for the loan with them. Generally cheaper that auto finance companies and you'll be able to talk to them about your monthly payment and ither peramitors of the loan in advance. Plus it's like negotiating with cash at the dealer!
Good luck
Reply to this Question
Write your response:
The Credit Advice pages of the Site may contain messages submitted by users over whom Credit Karma has no control. Credit Karma cannot guarantee the accuracy, integrity or quality of any such messages. Some users may post messages that are misleading, untrue or offensive. You must bear all risk associated with your use of the Credit Advice pages and should not rely on messages in making (or refraining from making) any specific financial or other decisions.