text:
Interleukin 18 (IL18) is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP) that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV) IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV) IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding site on IL18.
Author Summary
Interleukin 18 (IL18) is an important cytokine in inflammation and immunity. Mammals and poxviruses encode homologous inhibitory proteins of IL18, named IL18BPs, which regulate IL18 activity and, in the case of the viral proteins, contribute to virulence. Previous structural and functional studies revealed residues at IL18:IL18BP interface that are critical for the high-affinity binding, including a phenylalanine on IL18BPs, which is conserved among nearly all IL18BPs with the notable exception of yatapoxvirus IL18BPs. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the high-resolution crystal structure of the Yaba-Like Disease Virus (YLDV) IL18BP:IL18 complex. The structure revealed a 2∶2 bivalent binding complex, which has not been observed in any other IL18BPs. Through mutagenesis and functional studies, we found a set of interacting residues that are unique for the association of YLDV-IL18BP and IL18, likely compensating for the lack of the interactions involving the conserved phenylalanine. Despite this extensive divergence, however, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs. Our study suggests that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding site on IL18 but the interface on IL18 is malleable by a broad and diverse family of mammalian and poxvirus IL18BPs.
Citation: Krumm B, Meng X, Wang Z, Xiang Y, Deng J (2012) A Unique Bivalent Binding and Inhibition Mechanism by the Yatapoxvirus Interleukin 18 Binding Protein. PLoS Pathog 8(8): e1002876. https://doi.org/10.1371/journal.ppat.1002876
Editor: R. Mark L. Buller, Saint Louis University, United States of America
Received: May 25, 2012; Accepted: July 10, 2012; Published: August 23, 2012
Copyright: © Krumm et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by NIH grants AI081928 (JD), AI079217 (YX), and by Oklahoma Agricultural Experiment Station at Oklahoma State University under Project OKL02618 (JD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Poxviruses are a family of large, complex DNA viruses, infecting a variety of organisms including insects, reptiles, birds and mammals [1]. The poxvirus family is further subdivided into genera based on shared characteristics such as host range, morphology, antigenicity, and sequence similarity [2]. Four genera of poxviruses are known to be pathogenic to humans, including molluscipoxvirus, orthopoxvirus, parapoxvirus, and yatapoxvirus. As an immune evasion strategy, poxviruses encode an assortment of decoy receptors for chemokines and cytokines [3]. One such strategy for evasion of the host immune response is through modulation of the interleukin 18 (IL18) signaling pathway. IL18 is a pro-inflammatory cytokine belonging to the interleukin 1 superfamily and plays an important role in both innate and acquired immune responses by inducing interferon-γ (IFN-γ) production from T lymphocytes and macrophages while also enhancing the cytotoxicity of natural killer cells [4]. IL18 activity is modulated in vivo by a negative feedback mechanism involving a naturally occurring IL18 inhibitor, the IL18 binding protein (IL18BP) [5]. Homologues of IL18BPs are also encoded by many poxviruses including molluscum contagiosum virus and orthopoxviruses [6], [7] such as variola virus, the causative agent of smallpox.
Yaba-Like Disease Virus (YLDV) along with Yaba Monkey Tumor Virus (YMTV) are members of the yatapoxvirus genus of poxviruses. These viruses produce a very distinct disease in primates that is characterized by epidermal histiocytomas and vesicular lesions of the head and limbs [8]–[10]. Although their exact host reservoir is not well established, it is presumed that the immunomodulatory proteins expressed by these viruses can at least partially cope with the primate/human immune system. Analysis of YLDV and YMTV genome revealed yatapoxviruses encode a predicted IL18BP family member, designated as 14L [11], [12]. YLDV and YMTV 14L proteins share approximately 54% sequence identity between each other but less than 14% with IL18BPs of the orthopoxviruses such as the variola virus and the mousepox ectromelia virus (ECTV). Despite this low sequence similarity, YMTV 14L was previously shown to be a functional IL18 inhibitory protein with comparable affinity as orthopoxvirus IL18BPs [13].
The high-resolution crystal structure of the ECTV-IL18BP in complex with human IL18 revealed the structural basis by which orthopoxvirus IL18BPs antagonize IL18 signaling through direct competition with IL18 cognate receptor for binding [14]. The crystal structure along with mutagenesis studies identified a set of conserved residues from IL18 and IL18BPs as key to complex formation. In particular, a phenylalanine (F67 in ECTV-IL18BP) residue that is highly conserved in IL18BPs was found indispensible for IL18 binding [14]–[17]. Mutations of this site in all IL18BPs examined to date significantly decreased or even completely abolished the binding to IL18. In addition, a residue on IL18, K53, was shown as a 'hot spot' for binding IL18BPs, since mutations at this site drastically decreased binding affinity [18]. A strong π-cation interaction between IL18 K53 and the conserved phenylalanine residue (F67) of ECTV-IL18BP was revealed in the structure of ECTV-IL18BP:IL18 complex, explaining its important role in binding. Surprisingly, phylogenetic analysis and sequence alignment revealed the presence of a threonine (T64) in yatapoxvirus IL18BPs at the position equivalent to the conserved phenylalanine (Figure 1) [19]. Furthermore, mutation of K53 on IL18 only modestly affected the binding with YMTV 14L [13]. To understand how yatapoxvirus IL18BPs bind IL18, we determined the high-resolution crystal structure of YLDV-IL18BP:IL18 complex. This structure along with the functional analysis through mutagenesis and Surface Plasmon Resonance (SPR) provide new insights into the mechanism by which IL18BPs inhibit IL18. The result provided here could be helpful for developing inhibitors for IL18 or IL18BP.
Structure based sequence alignment of various IL18BPs was created using the crystal structure of YLDV-IL18BP as the template. Lettering and numbering above alignment correspond to YLDV-IL18BP topology and numbering scheme. Colored stars above residues indicate the three binding sites on IL18 with which they interact, red: site A, orange: site B, cyan: site C. Solid black circles above residues indicate resides involved in homo-dimerization. The two intra-chain disulfide bonds are indicated with the green letters. The cysteine residue forming the unique inter-chain disulfide bond is indicated with a pink triangle. The sequence alignment was performed with the FATCAT server [44] and ClustalX [45], and the figure was created with ESPript [46].
Architecture of the YLDV-IL18BP:IL18 Complex: Bivalent Binding
Recombinant human IL18 and mature YLDV-IL18BP (residue 20–136) proteins were individually purified from E. coli, and were subsequently used to reconstitute a complex of IL18:YLDV-IL18BP. Initially, wild-type (WT) IL18 and YLDV-IL18BP were used, but no quality crystals were obtained. In efforts to improve crystallization, we mutated some non-essential cysteines and additional surface residues in IL18. Substitution of four cysteines with serines in IL18 was previously shown to increase the stability of IL18 without affecting IL18 activities [20]. We found that the use of an IL18 mutant [IL18 (8S), see Materials and Methods], containing substitutions of the four-cysteines with serines and substitutions of four surface residues opposite to the IL18BP binding interface with alanines, greatly improved crystal quality and reproducibility. The crystal structure of IL18 (8S) in complex with YLDV-IL18BP was determined to 2.7 Å. Furthermore, a crystal from the complex of IL18 (8S) and a three-cysteine mutant of YLDV-IL18BP (ΔC21, C87S, C132S) diffracted to 1.75 Å (see Materials and Methods, Table 1). The two structures are essentially identical to each other with a root mean square deviation (r.m.s.d) of less than 0.4 Å, and the three mutated cysteines in YLDV-IL18BP are distant from the IL18 binding interface. Therefore, for discussion of protein:protein interactions between YLDV-IL18BP and IL18, we will mainly focus on the higher resolution structure of the mutant YLDV-IL18BP.
The structure of the complex shows that the YLDV-IL18BP forms a homo-dimer in a back-to-back fashion, with each protomer binding to a molecule of IL18, forming a hetero-tetramer complex with a stoichiometry of 2∶2 (Figure 2A). The complex displays an elongated v-shaped architecture with the IL18BP homo-dimer at the center and IL18 at the ends.
A). YLDV-IL18BP displays a homo-dimer (green and cyan), each adopting a β-sandwiched Ig-fold structure. Each protomer binds one molecular of IL18 (yellow and magenta), forming a 2∶2 complex. The intra-chain disulfide bonds (SS) in YLDV-IL18BPs are shown as yellow sticks. The inter-chain SS bond at the C-terminus of YLDV-IL18BPs is shown as orange spheres. B). Stereoview of superimposition of the monomer complexes of YLDV-IL18BP (green):IL18 (magenta) and ECTV-IL18BP (blue):IL18 (orange). The N- and C-terminus of YLDV-IL18BP are indicated. The intra-chain SS bonds are shown as in A. Residue C132 is shown as yellow spheres. The β-strands A, B, E and F in YLDV-IL18BP are labeled.
As seen in the ECTV-IL18BP:IL18 complex [14], IL18 adopts the same β-trefoil fold, which is comprised of 12 β-strands (β1–β12) with one short α-helix and one 310-helix. The IL18 molecules in the two structures show very little conformational changes, with an r.m.s.d. of only 0.5 Å from 134 aligned IL18 Cα backbones (Figure 2B). The two IL18 molecules in the current complex structure are also nearly identical, having only a 0.1 Å r.m.s.d from 150 aligned cα backbone residues.
Each protomer of YLDV-IL18BP adopts a canonical h-type immunoglobulin (Ig) fold [21] comprised of mainly β-sheets as observed previously for ECTV-IL18BP (Figure 3A). However, there is an r.m.s.d. of 2.9 Å over the 106 aligned cα backbone residues between the two viral IL18BPs. ECTV-IL18BP has an extended β-sheet architecture with predominantly shorter loops connecting the β-sheets and β-strands, while YLDV-IL18BP has comparatively shorter β-sheets with extended connecting loops. The two IL18BP structures differ mostly at one sheet of the β-sandwich fold (βA, βB, βE and βF) that is not involved in binding IL18 (Figure 2B). There is a major rigid-body movement on this β-sheet, especially at strands βA, βE and βF where twists are estimated at about 30 to 45 degrees. Compared to ECTV-IL18BP, there are two additional α-helices located between β-strands F and G (H2), and at the C-terminus (H3). YLDV-IL18BP contains five cysteine residues, forming two intra-molecular disulfide bonds (SS) (C21–C87, C43–C111) and one inter-molecular SS bond (C132–C132), in contrast to only two intra-molecular SS bonds in ECTV-IL18BP. C43–C111 is the conserved SS bond among many Ig-fold proteins, which connects the two β-sheets and plays a key role in maintaining overall integrity of the Ig-fold structure. C21–C87 SS bond connects the very N-terminus prior to βA with the loop between βE–βF. In contrast, the very N-terminus prior to βA of ECTV-IL18BP is SS bonded to the neighboring βB. In the triple-cysteine mutant YLDV-IL18BP:IL18 structure, the βE–βF loop is not visible in the electron density map, suggesting that C21–C87 SS bond stabilizes the local structure, particularly the βE–βF loop (Figures 2, 3A). However, the C21–C87 SS bond is not critical for the overall structure of YLDV-IL18BP or its binding with IL18 (shown later).
A). YLDV-IL18BP dimerizes back-to-back, abutting on one edge of the Ig-fold through extensive hydrophobic interactions. The secondary structures are labeled. The inter-chain C132-C132 SS bond is shown as orange spheres. B). The E42 from one protomer appears to be protonated, forming a hydrogen bond with E42 from the other protomer. E42 also forms both intra-chain and inter-chain hydrogen bonds with R44. E42 and R44 residues are shown as sticks. The 2mFo-DFc electron density map is shown in blue. C). Hydrophobic stacking of residues H30 and P32 of one YLDV-IL18BP protomer with their counterparts of the other protomer. D) Residue V33 of one protomer is inserted into the hydrophobic platform comprised of I29, V31, V124, and I129 of the second protomer.
Back-to-Back Dimer of YLDV-IL18BP
YLDV-IL18BP dimerizes back-to-back, abutting on one edge of the β sandwich, exposing the opposite edge for binding IL18 (Figures 2,3). The homo-dimer interface involves mainly βA, βB, βH and the C-terminal helix H3. It involves extensive hydrophobic interactions in the center (I29, H30, V31, P32 and V33 from βA, the aliphatic side chain of E122, V124 from βH and I129 from H3) flanked by hydrogen bonding and charge-charge interactions, burying approximately 1,700 Å2 and 1,465 Å2 solvent accessible surface area (ASA) for the wild-type complex and the triple-cysteine mutant complex, respectively (Figure 3). The lack of the C132-C132 SS bond in the triple-cysteine mutant complex caused the disorder of four residues at the C-terminus beyond S132, resulting in a slightly smaller ASA. At the dimer interface, the imidazole ring of H30 from one protomer stacks on the P32 from the other (Figure 3C), while V33 is forming favorable van der Waals interactions with a hydrophobic platform comprised of I29, V31, V124 and I129 from the other protomer (Figure 3D). E42 from one protomer appears to be protonated, forming favorable hydrogen bonds with E42 from the other protomer. E42 also forms both intra-chain and inter-chain hydrogen bonds with R44 (Figure 3B). The inter-chain C132-C132 SS bond covalently links the two promoters. Indeed, a non-reducing SDS-PAGE showed that YLDV-IL18BP proteins expressed in E. coli or in mammalian cells formed disulfide-bonded dimers (Figure 4). However, the triple-cysteine mutant of YLDV-IL18BP displays nearly identical structure as the WT protein, and it exists as a dimer in solution judging by size exclusion chromatography (Figure 5) and dynamic light scattering analysis (data not shown). Therefore, the dimerization is not solely dependent on the C132-C132 inter-chain SS bond.
SDS-PAGE analysis of purified YLDV-IL18BP proteins in the presence (+) or absence (−) of 2-mercaptoethanol (2-ME). A). Purified YLDV-IL18BP:IL18 protein complex that was expressed in E. coli. IL18 and YLDV-IL18BP are indicated by black and red arrows, respectively. B). Purified YLDV-IL18BP that was expressed and secreted from mammalian cells. The YLDV-IL18BP was engineered with a C-terminal site-specific biotinylation site, which allows its detection in Western blot with streptavidin. Two N-glycosylation sites were predicted for YLDV-IL18BP. Panel B also shows the SDS-PAGE of YLDV-IL18BP that was untreated (−) or treated (+) with endoglycosidase PNGase F.
Various SUMO fusion proteins containing WT, triple-cysteine (ΔC21, C87S, C132S) mutant and the HVEC (H30A/V33R/E42R/C132S) mutant YLDV-IL18BPs were purified and analyzed on a Superdex S200 sizing column. Both WT (Green) and triple-cysteine mutant (blue) display as a dimer, with a calculated MW about 65 kDa estimated from the retention volumes. The HVEC mutant (red) displays predominantly as a monomer, with an estimated MW about 31 kDa. The minor peaks were identified from SDS-PAGE analysis as impurities unrelated to YLDV-IL18BP.
We performed additional mutagenesis studies to verify the importance of the residues at the homo-dimerization interface. E42R/C132S (EC) mutant remained a dimer in solution as the WT (data not shown), while mutants bearing H30A/V33R/C132S (HVC) substitutions or H30A/V33R/E42R/C132S substitutions (HVEC) appeared as monomers in solution based on size exclusion chromatography (Figure 5) and dynamic light scattering analysis (data not shown). Therefore, hydrophobic interactions as well as the disulfide bonding together contribute to the dimerization. When measuring the binding affinity with IL18 by Surface Plasmon Resonance (SPR), we found that the monomeric YLDV-IL18BP (HVEC) had a 3-fold decrease (student t-test P-value<0.05) in binding affinity than the dimeric WT YLDV-IL18BP (Table 2).
Sequence analysis shows that the residues key to YLDV-IL18BP homo-dimerization (H30, P32, V33, C132) are conserved only in yatapoxvirus IL18BPs but not in other IL18BPs (Figure 1). Therefore, dimer formation and bivalent binding of IL18 seems to be unique to yatapoxvirus IL18BPs. In fact, ECTV-IL18BP and human IL18BP were reported to be monomers in solution [14], [22].
YLDV-IL18BP:IL18 Complex Interface
YLDV-IL18BP binds IL18 by using the same edge of the β sandwich as observed in ECTV-IL18BP:IL18 complex structure. Specifically, the following regions on YLDV-IL18BP are observed at the interface: loop connecting βB–βC, βC, the short βD, helix H1, βG, and loop connecting βG–βH (Figures 2,3). Similar to what was observed in the ECTV-IL18BP:IL18 complex structure, YLDV-IL18BP molecule sits atop the opening of the IL18 β-barrel and binds the cytokine through extensive hydrophobic and hydrogen bonding interactions (Figure 6), covering about 1,957 Å2 of ASA, which is comparable to the ECTV-IL18BP:IL18 complex at 1,930 Å2 ASA as identified by the program AreaIMol of the CCP4 suite [23]. The numbers of residues involved at binding interfaces are also comparable between the two inhibitory complexes. YLDV-IL18BP contributes mainly 19 residues to the complex interface while IL18 contributes 23 residues, in comparison to 17 residues from ECTV-IL18BP and 25 residues from IL18 using NCont of the CCP4 suite [23]. To assess the energetic contributions to binding by residues at the binding interface, we performed site-directed mutagenesis on both IL18 and the monomeric YLDV-IL18BP (HVEC) and assessed the effects of the mutations on the binding affinity by SPR (Figures 7,8,9,10). In addition, to probe the difference in IL18 binding by IL18BPs of YLDV and ECTV, we performed binding studies of various IL18 mutants with the two IL18BPs simultaneously (Figures 9,10). We will describe the results from these functional studies in context of our depiction of the YLDV-IL18BP:IL18 complex interface. As we described in the previous ECTV-IL18BP:IL18 complex structure, we will continue to use the three identified binding sites, labeled as A, B and C on IL18 here (Figure 6).
A). Key residues of YLDV-IL18BP at the interface. YLDV-IL18BP binds nearly identical surface of IL18 as previously observed in ECTV-IL18BP inhibitory complex. IL18 is shown as surface representation and colored grey. YLDV-IL18BP is drawn as a ribbon diagram with β-sheets colored in yellow. Binding sites A, B and C on IL18 surface are colored red, orange and cyan respectively. YLDV-IL18BP residues involved in binding IL18 are shown as stick representations. Each insert details the interactions involved in the respective binding site between YLDV-IL18BP and IL18. B). Unique interactions at binding site A. Carbon atoms of YLDV-IL18BP and IL18 are colored in yellow and pink, respectively. The secondary structures of YLDV-IL18BP and IL18 are colored in cyan and green, respectively. Red dashed lines indicate H-bonds. C). Unique interactions at binding site C. YLDV-IL18BP P116 is involved in favorable hydrophobic interactions with IL18. Polar interactions at site C involve S105 and D110 residues of IL18. The coloring scheme is the same as in B.
Biotinylated IL18 was captured on a BIAcore streptavidin-coated CM5 chip, and its binding with IL18BP was monitored with a BIAcore 3000 sensor. All the YLDV-IL18BP proteins were expressed in E. coli as SUMO fusion and purified to near homogeneity. The IL18BP mutants were derived from the monomeric form of the protein with the HVEC mutation. The injection of IL18BP started at ∼150 s and stopped at 900 s. The colored lines are the responses obtained with different IL18BP mutants and normalized to a maximum of 100 resonance units (RU) (except for Y56A) for ease of comparison.
SPR analysis was performed as described in Figure 7 with YLDV-IL18BP at 5 different concentrations. The binding curves were globally fitted with BiaEvaluation software to a 1∶1 binding model. The colored and black lines are the actual responses in RU and globally fitted curves, respectively.
Biotinylated YLDV-IL18BP and ECTV-IL18BP were captured on two different flow cells in a BIAcore streptavidin-coated CM5 chip, and their binding with IL18 was monitored simultaneously with a BIAcore 3000 sensor. The injection of IL18 started at ∼150 s and stopped at 900 s. The colored lines are the responses obtained with different IL18 mutants and normalized to a maximum of 100 RU (except for P57R, S105R for YLDV-IL18BP) for ease of comparison. YLDV-IL18BP and ECTV-IL18BP were expressed and secreted from mammalian cells and underwent in vitro biotinylation as described in Materials and Methods.
SPR analysis was performed as described in Figure 9 with IL18 at 5 different concentrations. The binding curves were globally fitted with BiaEvaluation software to a 1∶1 binding model. The colored and black lines are the actual responses in RU and globally fitted curves, respectively.
Site A of IL18 contains key residues Y1, L5, K53, D54, S55 and P57, making extensive interactions with YLDV-IL18BP (Figure 6A,B). The detailed interactions are similar to those observed in the previous ECTV-IL18BP:IL18 complex structure except for the loss of one of the 'hot spot' interactions involving a phenylalanine (F67 in ECTV-IL18BP, described below). Substitution of S55 of IL18 with alanine was previously shown to decrease its binding affinity to orthopoxvirus IL18BP by 7-fold [18]. As observed in the ECTV-IL18BP:IL18 complex, the side chain hydroxyl of S55 is tethered to the main chain amino group of Y1 via a hydrogen bond in the YLDV-IL18BP:IL18 complex. Y56 on βC of YLDV-IL18BP occupies nearly identical position as observed in the ECTV-IL18BP complex without any conformational changes. Its phenolic group is tethered to the D54 main chain of IL18, while its aromatic side chain together with the methyl group of T64 from YLDV-IL18BP, and L5 of IL18 constructs a hydrophobic wall, entrenching the aliphatic side chain of K53 from IL18 (Figure 6B). We found mutation of Y56A on YLDV-IL18BP completely abolished its binding to IL18 (Figures 7,8, Table 2), which is consistent with previous functional analysis of other IL18BPs [16].Therefore, a tyrosine residue at this location in IL18BPs seems to be a conserved 'hot spot', as the most important point to anchor the inhibitory protein to IL18.
As predicted, T64 of YLDV-IL18BP, located on the tip of βD, indeed occupies the same location of F67 from ECTV-IL18BP. This phenylalanine residue is highly conserved in IL18BPs of various species including human, all orthopoxviruses and MCV. Mutations at this location of various IL18BPs were shown to dramatically reduce the binding with IL18 [15]–[17]. In the ECTV-IL18BP:IL18 complex, F67 is inserted into an induced hydrophobic pocket, forming strong interactions with IL18 residues located on the surface while forming a strong π-cation interaction with the charged head group from the side chain of K53 of IL18 [14]. These interactions are absent in YLDV-IL18BP complex due to the presence of a threonine instead of phenylalanine. Interestingly, T64F substitution in YLDV-IL18BP did not increase the binding of IL18 (Table 2, Figures 7,8), while T64A substitution only caused a 2.5-fold decrease (student t-test P-value<0.05) in binding affinity. Consistent with the lack of π-cation interaction, IL18 K53 contributes less to the binding with YLDV-IL18BP than with the orthopoxvirus IL18BPs. While K53A mutation drastically reduced the binding affinity to orthopoxvirus IL18BPs [i.e., more than 100-fold decrease for variola IL18BP [18], Figures 9 and 10], this mutation had less impact on binding affinity with YLDV-IL18BP (about 30-fold decrease, Table 3, Figures 9, 10). K53 of IL18 nevertheless remains important for binding to YLDV-IL18BP, because polar interactions involving K53 are preserved in the current structure. Specifically, the positively charged amino head group on the side chain of K53 forms salt bridges with D66 and E76 of YLDV-IL18BP, similar to the interactions of K53 with two glutamate residues of ECTV-IL18BP in the ECTV-IL18BP:IL18 complex structure.
Site A differences also include the side chain rotation and repositioning of Q67 on YLDV-IL18BP (equivalent to H70 of ECTV-IL18BP) and Y1 on IL18, creating a novel interaction that was absent in the ETCV-IL18BP:IL18 structure. Q67 is located in close vicinity to T64 and rotates about 90 degree (vs. H70 of ECTV-IL18BP) forming bifurcated hydrogen bonds with the hydroxyl group and the main chain amide nitrogen of T64. Y1 of IL18 rotates about 80 degrees and stacks on the aliphatic portion of the Q67 side chain, forming favorable van der Waals interactions (Figure 6B). Y1A substitution of IL18 and Q67A substitution of YLDV-IL18BP decreased the binding affinity by 20- and 4-fold, respectively (student t-test P-value<0.05, Table 2, 3, Figures 7,8,9,10). In contrast, Y1A mutation did not affect the affinity with ECTV-IL18BP (Table 3, Figures 9,10). Since T64A substitution of YLDV-IL18BP showed very little effect on binding to IL18 (Table 2, Figures 7,8), the hydrogen bond between the side chain of Q67 and the main chain of T64 seems to be more significant than its interaction with the side chain. It is likely Q67 further stabilizes the local structure, including helix H1 where D66 locates, allowing correct positioning of this acidic residue for interacting with K53 of IL18.
Site B is a large, predominantly hydrophobic cavity spatially adjacent to Site A on the surface of IL18 (Figure 6A). As observed in the ECTV-IL18BP complex, three non-contiguous residues, Y54, I114 and P119 (Y51, T113 and V118 in ECTV-IL18BP) from YLDV-IL18BP βC, βG and G-H loop reside but do not fully occupy the pocket. Y54A substitution in YLDV-IL18BP had negligible effects on IL18 binding, while I114A mutation caused only a 1.8-fold decrease in binding affinity (statistically not significant, student t-test P-value>0.05) (Figures 7,8, Table 2), similar to substitutions of the equivalent positions of other IL18BPs [16], [17]. Therefore, site B interactions appear not essential for binding of YLDV-IL18BP, similar to observations for other IL18BPs [14].
Site C of IL18 is next to Site B and mainly comprised of 10 IL18 surface residues involving a mixture of charged and hydrophobic interactions. Similar to what was observed in the ECTV-IL18BP complex, the loops connecting βB–βC and βG–βH of YLDV-IL18BP interact with Site C on IL18 predominantly through hydrophobic interactions. YLDV-IL18BP F52 adopts nearly identical conformation as ECTV-IL18BP F49, which is inserted into the large hydrophobic pocket of Site C [14]. Surprisingly, F52A mutation of YLDV-IL18BP had negligible effect on binding with IL18 (Figures 7,8), in contrast to the mutation at this location in other IL18BPs significantly decreasing binding affinity to IL18 (83–fold decrease for human IL-18BP:human IL18, 8-fold decrease for ECTV-IL18BP:human IL18 and 138-fold decrease for ECTV-IL18BP:murine IL18) [16], [17]. This difference can be explained by several unique interactions of YLDV-IL18BP at Site C. Residue P116 of YLDV-IL18BP is situated in a hydrophobic groove formed by aliphatic side chains from M60, Q103 and M113 of IL18 and is stabilized by a stair-wise hydrophobic stacking by side chains from F49, Y48 and K23 of YLDV-IL18BP (Figure 6A,C). In addition, P116 main chain is hydrogen bonded with the hydroxyl group from the side chain of IL18 S105, further stabilizing the complex interface. Indeed, P116A substitution reduced binding of YLDV-IL18BP to IL18 by approximately 6-fold (student t-test P-value<0.005, Figures 7,8, Table 2). Mutation of the equivalent residue in human IL18BP (P153) showed negligible effect on its binding affinity to IL18 [15], so P116 appears to be specific for YLDV-IL18BP in binding to IL18. Y48 and K23 of YLDV-IL18BP are hydrogen bonded with IL18 D110 through side chain to side chain interactions. K117 of YLDV-IL18BP forms a bifurcated hydrogen bond with side chains from IL18 S105 and D110 (Figure 6C). D110A of IL18 decreased affinity with YLDV-IL18BP by 6-fold (student t-test P-value<0.05) but had no effect on binding with ECTV-IL18BP (Figures 9,10). Similarly, S105R of IL18 caused more than 30-fold decrease (student t-test P-value<0.005) in binding affinity with YLDV-IL18BP but had no impact on binding with ECTV-IL18BP (Figures 9,10). Therefore, D110 and S105 of IL18 are specifically required for the binding of IL18 with YLDV-IL18BP but not for ECTV-IL18BP. The specificity of S105 towards binding of YLDV-IL18BP is further signified by a double mutant, S105R/P57R of IL18. This double mutant completely abolished the binding with YLDV-IL18BP, while it had a much smaller impact on binding with ECTV-IL18BP (Table 3, Figures 9,10).
We previously determined the crystal structure of the ectromelia virus IL18BP in complex with IL18, revealing the structural basis for the binding and inhibition of IL18 by the IL18BPs [14]. Despite an overall low sequence homology between the diverse viral and host IL18BPs, the key residues of ECTV IL18BPs at the IL18 binding interface are highly conserved. Mutagenesis studies on human and viral IL18BPs also showed that these key residues are almost universally critical for the binding of IL18BPs to IL18. Thus it was enigmatic that functional yatapoxvirus IL18BPs lack a key phenylalanine residue (Figure 1) that has been identified to be essential for many other IL18BPs to bind IL18 [14]–[17]. It is similarly puzzling that a residue of IL18 (K53) that is critical for binding orthopoxvirus IL18BPs only played a modest role in binding with the YMTV-IL18BP [13]. In this report, we resolved these questions by determining the crystal structure of YLDV-IL18BP:IL18 complex and by performing extensive mutagenesis and SPR studies. We revealed two unique signature features of YLDV-IL18BP that distinguish yatapoxvirus IL18BPs from the rest of IL18BP family members. First, YLDV-IL18BP forms a homo-dimer and interacts with IL18 in a 2∶2 binding mode. Second, the binding of YLDV-IL18BP and IL18 does not rely on two of the 'hot spot' interactions that were shown to be essential for the binding of all previously studied IL18BPs, including a phenylalanine (F67 in ECTV-IL18BP) at site A and another phenylalanine at site C (F49 in ECTV-IL18BP). Instead, yatapoxvirus IL18BPs evolved interactions with some IL18 residues (Y1, D110, S105) that are specifically important for binding with YLDV-IL18BP. It appears that YLDV-IL18BP shifts and disperses the binding energy across the IL18-binding interface rather than concentrating the binding energy on a few hot spots as is the case for all other IL18BPs examined to date.
It was previously reported that ECTV-IL18BP and human IL18BP are monomeric in solution [14], [22]. In contrast, YLDV-IL18BP forms a disulfide-bonded dimer, which was demonstrated not only in the crystal structure but also in solution by non-reducing SDS-PAGE and gel filtration analysis. The dimer interface is quite large (about 1,700 Å2) and involves extensive hydrophobic interactions in addition to the intermolecular disulfide bond, indicating that the YLDV-IL18BP dimer is intrinsically stable in solution. The dimer could only be separated into monomers by mutations that disrupt both the hydrophobic interactions as well as the inter-chain SS bond. Analysis of the monomeric YLDV-IL18BP (HVEC) showed that the dimerization was not essential for binding IL18 but enhanced the binding affinity by 3-fold in our in vitro assay. Although this enhancement in binding affinity as measured by SPR is modest, it is possible that the dimerization may be more important for the function of YLDV-IL18BP during infection of the host, perhaps by increasing the half-life of the protein in the infected tissue or by increasing the avidity of binding to IL18 at low protein concentration. In fact, divalent or multivalent binding is an important, inherent feature of many biological systems to enhance the effectiveness of binding of ligands to receptors and of antibodies to antigens [24]–[28]. More specifically, this has been a feature for quite a few poxvirus cytokine binding proteins. For example, ectromelia virus IFN-γ binding protein forms a tetramer, which is required for efficient IFN-γ antagonism [29]. Myxoma virus T2 protein, a Tumor Necrosis Factor (TNF) Receptor homolog, is secreted as both monomer and dimer, and the dimeric T2 is a more potent TNF inhibitor [30]. Because residues of YLDV-IL18BP involved in dimer formation are only conserved in yatapoxviruses, yatapoxviruses IL18BPs may be unique among IL18BPs in that they use bivalent binding to increase the affinity and avidity for IL18.
Another difference between YLDV-IL18BP and all other IL18BPs is the lack of two of the 'hot spot' interactions at the binding sites A and C on the surface of IL18. The structure of ECTV-IL18BP:IL18 complex showed that a conserved phenylalanine (F67) is engaged in hydrophobic and strong π-cation interactions with K53 of IL18 at binding site A [14]. Alanine substitutions of K53 of IL18 significant decreased binding with orthopoxvirus IL18BPs [18], while alanine substitutions of the conserved phenylalanine (equivalent to ECTV-IL18BP F67) in human, MCV and orthopoxviruses IL18BPs significantly decreased or completely abolished binding of IL18 [15]–[17]. The current structure of YLDV-IL18BP:IL18 complex showed that a threonine residue (T64) is present at the position equivalent to the phenylalanine, indicating the π-cation interaction with K53 is not important for YLDV-IL18BP: IL18 complex. Indeed, T64F or T64A substitution of YLDV-IL18BP had negligible or minor (2.5-fold decrease in affinity for T64A, student t-test P-value<0.05) effect on the binding with IL18, while K53A of IL18 had a more modest effect on binding with YLDV-IL18BP than with orthopoxvirus IL18BPs. A similar loss of 'hot spot' interaction was also observed at binding site C. A phenylalanine residue on IL18BPs that binds to site C of IL18 was previously shown to be important for binding IL18 in orthopoxvirus, MCV and human IL18BPs [15]–[17]. Although a phenylalanine (F52) is present at the equivalent position in YLDV-IL18BP, it is not important for binding to IL18 (Table 2, Figures 7,8). Through structural and mutagenesis studies, we have identified contact residues that are unique to the YLDV-IL18BP:IL18 binding interface. This includes Q67 of YLDV-IL18BP and Y1 of IL18 at site A, P116 of YLDV-IL18BP and S105 and D110 of IL18 at site C. Our data are in agreement with the conclusion of a more delocalized energy distribution for binding of IL18 to YMTV-IL18BP [13]. The structural and functional studies of two different IL18BP complexes suggest that there is a degree of plasticity in the IL18BP:IL18 interface that could accommodate certain mutations in IL18BPs without compromising their binding affinity to IL18.
Despite the differences in several key residues for binding IL18, the current YLDV-IL18BP:IL18 complex structure showed that YLDV-IL18BP targets the same surface of IL18 as ECTV-IL18BP does in the previous complex structure. This suggests that all IL18BPs inhibit IL18 function by blocking a putative receptor-binding site on the surface of IL18. Similar to previous findings on human and poxvirus IL18BPs, Y56 of YLDV-IL18BP (interacting with site A of IL18) was found to be absolutely essential for binding to IL18, indicating that this conserved tyrosine residue is an obligatory 'anchor' for binding of all IL18BPs to IL18. The conservation and variation in functional residues and their specific interactions with IL18 suggest that IL18BPs share a common ancestor but may have undergone significant evolution through different selection pressures, resulting in a conserved inhibitory mechanism albeit with mutations of interface residues.
The biological activity of IL18 is determined in part by its relative affinities for IL18 receptors and IL18BP. The binding of IL18 to its receptors triggers multiple cellular responses vital to immunity, but excessive IL18 activities are associated with many autoimmune and inflammatory diseases [4], [31]–[37]. Functional IL18BPs are present in many poxviruses including variola virus and vaccinia virus, providing a key strategy of poxvirus immune evasion by inhibition of IL18 cytokine activity. Therefore the studies on IL18BP:IL18 inhibitory complexes could serve dual purposes by providing important clues on how to develop functional inhibitors targeting either IL18 or poxvirus IL18BP. These inhibitors could potentially modulate IL18 and poxvirus IL18BP activities, which may benefit efforts in developing treatments against some autoimmune and inflammatory diseases and in developing treatments for potential pathogenic outbreaks associated with poxvirus infections.
Materials and Methods
Protein Purification and Crystallization
Mature IL18 and YLDV-IL18BP (residues 20–136) were individually cloned into a modified pET vector as SUMO fusion proteins with N-terminal 6×His tags and expressed in E. coli BL21 (DE3) gold (Stratagene) or Rosetta-Gami 2 (Invitrogen) strains, respectively. An IL18 mutant (C38S, C68S, C76S, C127S, K67A, E69A, K70A, I71A) with substitutions of four nonessential cysteines [20] and four additional surface residues opposite to the IL18BP binding interface, IL18 (8S), and the triple-cysteine mutant of YLDV-IL18BP (residues 22–136, C87S, C132S) were cloned and expressed in the same way as WT proteins. The individual proteins were purified using the similar double Ni-nitrilotriacetic acid (Ni-NTA) procedure as described [14]. Briefly, the his-tagged fusion proteins were first purified from cell lysate by Ni-NTA affinity column (Qiagen) and then co-dialyzed with ULP1 protease to remove the SUMO moiety, exposing the authentic N-terminus for both proteins. The cleaved protein mixtures were subsequently passed through a second subtracting Ni-NTA column and further purified by size exclusion chromatography on a Superdex s200 column. The YLDV-IL18BP and IL18 (8S) proteins were mixed together and the complexes were subsequently purified from size exclusion chromatography and each concentrated to 9 mg/ml. The complex of wild-type YLDV-IL18BP:IL18 (8S) crystallized in a condition containing 18% PEG3350, 0.1 M Bis-Tris Propane/Citric Acid, pH 6.5, while the crystallization condition for the complex of triple-cysteine mutant YLDV-IL18BP:IL18 (8S) is 12% PEG3350, 0.1 M Tris, pH 8.0. 25% ethylene glycol was added step-wise to the mother liquid as cryoprotectant.
To make biotinylated IL18, mature human IL18 (residues 37–193) was cloned into a modified pET vector containing a C-terminal 6×His tag along with the coding sequence (GLNDIFEAQKIEWHE) for biotinylation. This plasmid and a plasmid encoding biotin ligase (Avidity) were co-transformed into E. coli BL21 (DE3) gold (Stratagene) for expression. One step Ni-NTA affinity purification was used to purify biotinylated and his-tagged IL18.
Mammalian Expression, Purification and In Vitro Biotinylation of IL18BPs
For mammalian expression of YLDV-IL18BP, a mammalian expression plasmid for YLDV 14L was constructed as described previously for the construction of expression vector for human IL18BP [16]. Briefly, YLDV 14L was amplified by PCR from genomic DNA of YLDV and cloned into pYX45 with NheI and BamHI sites, so that 14L ORF was appended with a C-terminal biotinylation tag and 6-His tag. 293T cells were transfected with the expression plasmid and then infected with vTF7.3, a vaccinia virus expressing T7 polymerase. 3 days later, the medium was harvested and incubated with Ni-NTA resin (Qiagen). The resin was then washed and added with E. coli biotin holoenzyme synthetase (Avidity). After the biotinylation reaction, the protein was eluted with phosphate-buffered saline containing 300 mM imidazole. The ECTV-IL18BP was expressed in HEK293T cells, purified from the culture medium and biotinylated essentially as described previously [18].
Structure Determination
An initial set of data for the tripe-cysteine mutant YLDV-IL18BP:IL18 (8S) was collected at beamline X29 of National Synchrotron Light Source, Brookhaven National Laboratory. Initial phases were determined by molecular replacement using Phaser [38] of the CCP4 suite [23] and a search model containing IL18 along with a trimmed poly-alanine model of the ECTV-IL18BP (PDB ID 3F62). A subsequent data set from a crystal of WT YLDV-IL18BP:IL18 (8S) complex was collect at beamline 19-ID of the Advanced Photon Source, Argonne National Laboratory. The structure of the complex containing WT YLDV-IL18BP was solved similarly as the structure of the complex containing the triple-cysteine mutant YLDV-IL18BP. All datasets were processed with HKL3000 [39]. PHENIX [40] was used for refinement and Coot [41] was used for iterative manual model building. Translation, libration and screw-rotation displacement (TLS) groups used in the refinement were defined by the TLMSD server [42]. The structure of the triple-cysteine mutant YLDV-IL18BP:IL18 (8S) complex was refined to 1.75 Å resolution with Rwork and Rfree of 19.0% and 23.1% respectively. The structure of the WT YLDV-IL18BP:IL18 (8S) complex was refined to 2.7 Å resolution with Rwork and Rfree of 21.9% and 27.0% respectively. The final models are of good refinement statistics for both complexes as shown in Table 1. All molecular graphic figures were generated with PYMOL [43].
Surface Plasmon Resonance
The SPR analysis was done essential as described previously [15], [16]. Briefly, biotinylated IL18BP [∼300 resonance units (RU)] or IL18 (∼250 RU) was captured onto a BIAcore CM5 chip coated with streptavidin. Various concentrations of IL18 (from ∼1 nM to ∼40 nM) or IL18BP (from ∼2 nM to ∼60 nM) were injected at a flow rate of 20 µl/min. The chip coated with IL18BP was regenerated with a 10-µl injection of 1 M NaCl, 50 mM NaOH, while the chip coated with IL18 was regenerated with a 10-µl injection of 10 mM glycine (pH 2.5). The sensorgrams were analyzed with BIAEVALUATION software (BIACORE). The binding data from the injection of at least five different concentrations of analyte were globally fitted to a 1∶1 binding model. Analyses with the same concentration series were done twice.
We thank the staff of beamline 19ID at the Advanced Photon Source along with the staff and organizers of RAPIDATA 2011 of Brookhaven National Laboratory for their generous support.
Author Contributions
Conceived and designed the experiments: YX JD. Performed the experiments: BK XM ZW YX JD. Analyzed the data: BK YX JD. Wrote the paper: BK YX JD.
- 1.
Moss B (2007) Poxviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields Virology. Philadelphia: Lippincott Williams & Wilkins. pp. 2905–2946.
- 2.
Damon I (2007) Poxviruses. In: Knipe DM, Howley PM, editors. Fields Virology. 5th edition. Philadelphia: Lippincott Williams & Wilkins. pp. 2947–2976.
- 3. Johnston JB, McFadden G (2003) Poxvirus Immunomodulatory Strategies: Current Perspectives. J Virol 77: 6093–6100.
- 4. Dinarello CA (1999) IL-18: A TH1-inducing, proinflammatory cytokine and new member of the IL-1 family. J Allergy Clin Immunol 103: 11–24.
- 5. Novick D, Kim SH, Fantuzzi G, Reznikov LL, Dinarello CA, et al. (1999) Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response. Immunity 10: 127–136.
- 6. Xiang Y, Moss B (1999) IL-18 binding and inhibition of interferon gamma induction by human poxvirus-encoded proteins. Proc Natl Acad Sci U S A 96: 11537–11542.
- 7. Born TL, Morrison LA, Esteban DJ, VandenBos T, Thebeau LG, et al. (2000) A poxvirus protein that binds to and inactivates IL-18, and inhibits NK cell response. J Immunol 164: 3246–3254.
- 8. Knight JC, Novembre FJ, Brown DR, Goldsmith CS, Esposito JJ (1989) Studies on Tanapox virus. Virology 172: 116–124.
- 9. Downie AW, Espana C (1973) A comparative study of Tanapox and Yaba viruses. J Gen Virol 19: 37–49.
- 10. Downie AW, Espana C (1972) Comparison of Tanapox virus and Yaba-like viruses causing epidemic disease in monkeys. J Hyg (Lond) 70: 23–32.
- 11. Brunetti CR, Amano H, Ueda Y, Qin J, Miyamura T, et al. (2003) Complete genomic sequence and comparative analysis of the tumorigenic poxvirus Yaba monkey tumor virus. J Virol 77: 13335–13347.
- 12. Lee HJ, Essani K, Smith GL (2001) The genome sequence of Yaba-like disease virus, a yatapoxvirus. Virology 281: 170–192.
- 13. Nazarian SH, Rahman MM, Werden SJ, Villeneuve D, Meng X, et al. (2008) Yaba monkey tumor virus encodes a functional inhibitor of interleukin-18. J Virol 82: 522–528.
- 14. Krumm B, Meng X, Li Y, Xiang Y, Deng J (2008) Structural basis for antagonism of human interleukin 18 by poxvirus interleukin 18-binding protein. Proc Natl Acad Sci U S A 105: 20711–20715.
- 15. Xiang Y, Moss B (2001) Correspondence of the functional epitopes of poxvirus and human interleukin-18-binding proteins. J Virol 75: 9947–9954.
- 16. Xiang Y, Moss B (2001) Determination of the functional epitopes of human interleukin-18-binding protein by site-directed mutagenesis. J Biol Chem 276: 17380–17386.
- 17. Esteban DJ, Buller RM (2004) Identification of residues in an orthopoxvirus interleukin-18 binding protein involved in ligand binding and species specificity. Virology 323: 197–207.
- 18. Meng X, Leman M, Xiang Y (2007) Variola virus IL-18 binding protein interacts with three human IL-18 residues that are part of a binding site for human IL-18 receptor alpha subunit. Virology 358: 211–220.
- 19. Watanabe M, Goto N, Watanabe Y, Nishiguchi S, Shimada K, et al. (2005) Evolution of interleukin-18 binding proteins and interleukin-1 receptor, type II proteins. Int J Mol Med 15: 561–566.
- 20. Yamamoto Y, Kato Z, Matsukuma E, Li A, Omoya K, et al. (2004) Generation of highly stable IL-18 based on a ligand-receptor complex structure. Biochem Biophys Res Commun 317: 181–186.
- 21. Bork P, Holm L, Sander C (1994) The immunoglobulin fold. Structural classification, sequence patterns and common core. J Mol Biol 242: 309–320.
- 22. Kimura T, Kato Z, Ohnishi H, Tochio H, Shirakawa M, et al. (2008) Expression, purification and structural analysis of human IL-18 binding protein: a potent therapeutic molecule for allergy. Allergol Int 57: 367–376.
- 23. Collaborative Computational Project N (1994) The CCP4 Suite: Programs for Protein Crystallography. Acta Crystallogr D Biol Crystallogr 50: 760–763.
- 24. Cheong HS, Chang JS, Park JM, Byun SM (1990) Affinity enhancement of bispecific antibody against two different epitopes in the same antigen. Biochem Biophys Res Commun 173: 795–800.
- 25. Neri D, Momo M, Prospero T, Winter G (1995) High-affinity antigen binding by chelating recombinant antibodies (CRAbs). J Mol Biol 246: 367–373.
- 26. Huang BC, Davern S, Kennel SJ (2006) Mono and bivalent binding of a scFv and covalent diabody to murine laminin-1 using radioiodinated proteins and SPR measurements: effects on tissue retention in vivo. J Immunol Methods 313: 149–160.
- 27. Muller KM, Arndt KM, Pluckthun A (1998) Model and simulation of multivalent binding to fixed ligands. Anal Biochem 261: 149–158.
- 28. Zhou HX (2003) Association and dissociation kinetics of colicin E3 and immunity protein 3: convergence of theory and experiment. Protein Sci 12: 2379–2382.
- 29. Nuara AA, Walter LJ, Logsdon NJ, Yoon SI, Jones BC, et al. (2008) Structure and mechanism of IFN-gamma antagonism by an orthopoxvirus IFN-gamma-binding protein. Proc Natl Acad Sci U S A 105: 1861–1866.
- 30. Schreiber M, Rajarathnam K, McFadden G (1996) Myxoma virus T2 protein, a tumor necrosis factor (TNF) receptor homolog, is secreted as a monomer and dimer that each bind rabbit TNFalpha, but the dimer is a more potent TNF inhibitor. J Biol Chem 271: 13333–13341.
- 31. Tarallo V, Hirano Y, Gelfand BD, Dridi S, Kerur N, et al. (2012) DICER1 Loss and Alu RNA Induce Age-Related Macular Degeneration via the NLRP3 Inflammasome and MyD88. Cell 149: 847–859.
- 32. Dinarello CA (2000) Targeting interleukin 18 with interleukin 18 binding protein. Ann Rheum Dis 59 Suppl 1: i17–20.
- 33. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H (2001) Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu. Cytokine Growth Factor Rev 12: 53–72.
- 34. Konishi H, Tsutsui H, Murakami T, Yumikura-Futatsugi S, Yamanaka K, et al. (2002) IL-18 contributes to the spontaneous development of atopic dermatitis-like inflammatory skin lesion independently of IgE/stat6 under specific pathogen-free conditions. Proc Natl Acad Sci U S A 99: 11340–11345.
- 35. Dinarello CA (2009) Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 27: 519–550.
- 36. Dinarello CA (2003) Anti-cytokine therapeutics and infections. Vaccine 21 Suppl 2: S24–34.
- 37. Dinarello CA (1999) Interleukin-18. Methods 19: 121–132.
- 38. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, et al. (2007) Phaser crystallographic software. J Appl Crystallogr 40: 658–674.
- 39.
Otwinowski ZaM, W. (1997) Processing of X-ray Diffraction Data Collected in Oscillation Mode. In: C.W. Carter JRMS, editor. Methods in Enzymology: Macromolecular Crystallography, part A. Academic Press. pp. 307–326.
- 40. Adams PD, Grosse-Kunstleve RW, Hung L-W, Loerger TR, McCoy AJ, et al. (2002) PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr D58: 1948–1954.
- 41. Emsley PaC K, (2004) Coot: model building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132.
- 42. Painter JaM EA, (2006) Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr D Biol Crystallogr 62: 439–450.
- 43.
DeLano WL (2002) The PyMOL Molecular Graphics System. Available: http://www.pymol.org.
- 44. Ye Y, Godzik A (2003) Flexible structure alignment by chaining aligned fragment pairs allowing twists. Bioinformatics 19 Suppl 2: ii246–255.
- 45.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al.. (2007) Clustal W and Clustal X version 2.0. Bioinformatics: 2947–2948.
- 46. Gouet P, Robert X, Courcelle E (2003) ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res 31: 3320–3323.


text:
I have recently developed a range of brightly-coloured ceramic bowls.
First of all I gave a lot of thought to the exact shape I wanted them to be and then I experimented with many different colour combinations and patterns before choosing the ones that I believe work best together. I add a fine line of slip around the inside of the bowl, where it is likely to be filled to, allowing for extra interest here.
The larger the bowl, the more lines it has, hence they can be collected in different sizes or different colours. I am now working on some larger statement pieces to go with them.
I make my ceramic sculptures from slabs of stoneware clay which I mark first with a variety of tools and found artefacts. I then tear off sections of the clay slab twisting and joining them at random. I like the glaze on these to look weathered so I either use high temperature dry glazes that look a bit like peeling paint or low temperature crackle glazes which allow the surface details to show through whilst still maintaining an aged appearance.
I often fire a piece of work several times using different temperatures until I am happy with the look and feel of it. Occasionally I add images onto the surface using ceramic decals which may reveal more about the meaning of the piece. I may also give it a smoke firing and, occasionally, add a touch of gold leaf.
September 2011: Merton Arts Trail
October 2010: The London Potters at the Morley Gallery
July 2010: Art in Clay at Hatfield House
November 2009: Demonstration of slab building with impressed slabs at the V&A
October 2009: The London Potters at the Morley Gallery
July 2009: Dulwich Craft Fair
May 2009: Battersea Art Fair
May 2008 Battersea Art Fair.
April 2006: Rock Lobster Gallery, Cowes, Isle of Wight.
Aug 2005: Touché Gallery, Stroud, Gloucestershire.
Nov 2004: Gallery Charlick, London WC1.
Sept 2004: Dulwich Craft Fair, London.
Sept 2002: Private commission of large wall-piece to represent a wedding anniversary.
July 1999: Sept '02 - Several private commisions for small wall-pieces.
June 1999: Candover Gallery, Alresford, Hampshire.
January 1999: INDE Gallery, Carlton Mill, Nottingham.
Sept 1998: Exhibition of 'Faith', Fisherton Mill, Salisbury.
March 1998: Battersea Contemporary Art Fair, London SW11.
Oct 1997: Wandsworth Artists Open Exhibition, London SW18.
Sept 1997: Solo exhibition - South Hill Park Arts Centre, Bracknell, Berkshire.
June 1996: The Tabernacle Gallery, Powis Square, London W11.

subset: commoncrawl


text:
Jay Rockefeller to retire
Democratic Sen. Jay Rockefeller will not run for reelection in 2014, passing up a bid for a sixth term and putting in play a Senate seat in deep red West Virginia.
In an interview with POLITICO, Rockefeller — the chairman of the Commerce, Science and Transportation Committee and one of the most senior Senate Democrats — said he had been wrestling with the question of whether to run again since October but had not made up his mind to retire until very recently.
Story Continued Below
"I'm going to serve out my term," the 75-year-old Rockefeller said. "It was a very hard decision for me. Once it's made, like any hard decision, it eases up. But it was a very tough decision for me."
( PHOTOS: Jay Rockefeller's career)
Rockefeller is scheduled to make a formal announcement at 11 a.m. back home in West Virginia.
Rockefeller said he decided to go public with his retirement now — one that is sure to shake up the 2014 Senate landscape — because it felt like the right move and because he didn't want months of public speculation over his political future.
"It's part of my nature," Rockefeller said. "One is, I think that always in me, I want to do things — if I have a statement to make or an announcement that has at least some consequence to it — I want to do it in the way that I want to do it, at the time that I want to do it, with the words that I want to use.
"I do it now because I'd just rather clear the air and just get on with things," he added.
Rockefeller acknowledged that Democrats will have a hard time holding onto his West Virginia Senate seat, but he insisted that he wasn't leaving because of fear of any GOP challenger or fear that he wouldn't win next year.
"Political opposition, I never worried about that," Rockefeller insisted. "It's not a part of this. It was easy enough not to have to worry about that."
The great-grandson of oil tycoon John D. Rockefeller, Jay Rockefeller was born in New York City in 1937. After graduating from Harvard University in 1961 with a bachelor's degree in Far Eastern languages and history, he traveled to West Virginia in 1964 as a VISTA volunteer. The experience changed his life, Rockefeller said, giving him a mission that John Davison Rockefeller IV knew would become his career. In fact, it became his life's work.
"I came to West Virginia looking for a mission and a cause that would possess me for my professional life," Rockefeller said. "When I went, I wasn't particularly welcomed there… But at that end of that first year in that community [Emmons], it was just so clear that … was what I was looking for. I was looking for a cause, I was looking for a mission, I was looking for something that would possess me, which would make really proud, something that was hard every day and was uphill, and it wasn't going to be on Wall Street."
In 1966, Rockefeller was elected to the West Virginia House of Delegates. Two years later, he became West Virginia secretary of state.
The 1960s and early '70s were heady times for the Rockefeller family in politics. Nelson Rockefeller, Jay's uncle, was the Republican governor of New York, later becoming vice president in 1974. Another uncle, Winthrop, in 1966 became the first Republican elected governor of Arkansas in nearly 100 years.


text:
Are there any Easter eggs left at your house?! There are a couple on the top shelf in the kitchen and they seem to keep catching my eye...must resist!
So I promised a mammoth bank holiday beauty blitz post and I hope not to disappoint you. As I had the house to myself it seemed like Saturday evening was the perfect time for the blitz (I had also worked all day so felt a little tired and in need of relaxation!)
Firstly I applied Ojon Restorative Hair Treatment and tied my hair back whilst having a bath. This hair treaty regenerates and restores hair making it smooth, silky and shiny. As someone whose hair is on the oily side, I find I can get away without conditioner for several days after this treatment. The nutty smell isn't my favourite fragrance but it does grow on you!
Following the bath, it was pedicure time. I'd exfoliated my feet with Body Shop Peppermint Smoothing Pumice Foot Scrub (the only foot scrub I will ever use!) and the applied cuticle remover round my nails. I rubbed on a little olive oil to moisturise then painted with Rimmel Lycra Pro Nail Polish in Purple Addict. The long lasting nature of this Polish is great if you exercise regularly as it stays put for a good week without chipping.
Facial wise, I slathered on a thick layer of Decleor Prolagene Gel. This is probably the most expensive thing I own! But you can use it for pretty much everything. Blemishes, sunburn, stretch marks, acne scars, dry skin and even under the eyes. I let it sink in before applying a few drops of A'Kin Pure Radiance Rose Hip Oil.
Incase you hadn't heard, Rose Hip Oil is incredibly gentle and popular with those who have dry/sensitive skin. The bottle lasts a very long time and you can use it alone or under moisturiser.
Next up was a recommendation from my lovely friend Helen. She's very pale like me but has a gorgeous natural glow thanks to Palmers Cocoa Butter Formula Natural Bronze Body Lotion which builds a gradual tan. I applied to my arms and legs and they are now brown though my legs are slightly streaky so I am going to give it a few more applications then let you know what I really think!
Last but not least....hands! Sally Hansen Cuticle Remover was applied then I used a 4-sided buffer to file, remove ridges and smooth. The purple theme continued with Essie Nail Lacquer in Bond With Whomever and then Leighton Denny Miracle Drops were applied to speed up drying. I then finished with Leighton Denny Cuticle Oil and Crystal Tips Top Coat.
My final bank holiday pampering will be an early night, I don't care that I haven't filled the dishwasher, there is a pile of ironing to do and toys all over the place! Lights out everyone!


text:
All items > Knife Accessories > Paracord
- Rating:550 lb.
- Length:100 ft.
- Color:OD Green, Tan
- Model Number:RG1051H
- Brand:Atwood Rope
- Product Type:Paracord
- Country of Origin:USA
- Weight:0.50 oz.
A must have for all your outdoor adventures, this 550 paracord from Atwood Rope Mfg. is excellent for everything from survival bracelets and camping tie downs to survival kits and bug out bags. Paracord use is limited only by your imagination. Use it to tie down gear, hang a hammock, or keep backpacking supplies out of the reach of bears and other creatures. This 550 lb., 7 strand core comes in a 100 foot length so you'll have plenty of slack to handle you tasks.


text:
Macon County may be one step closer to streamlining mental health services for citizens who need those resources.
At their April meeting the board of commissioners approved a grant application through Macon County Public Health to create a new position to coordinate the No Wrong Door program.
"We held a community forum about two months ago, and from that forum we took all the data that was collected and synthesized it before coming to the conclusion that we need a mental health system navigator," MCPH Director Jim Bruckner said. "We want to apply for an Evergreen Foundation grant for $60,000 to hire that person. There are some county matching funds needed, but we have been able to find those funds within our current budget."
Bruckner explained that the new person would act similarly to a veteran's services coordinator, in that the system navigator would not necessarily provide a mental health service but would connect a citizen to the proper place to access the service they need.
Bruckner added that Macon County has received a grant from East Carolina University to provide tele-psychiatry through the health department. ECU will provide all of the equipment needed to do psychiatric evaluations along with up to four hours per week of psychiatrist clinical time.
"Part of this new position we are looking to create would be to coordinate that time and make sure that the people who need the service most have access to it," Bruckner said. "The other piece of this is the No Wrong Door piece, and that is to connect people with resources that are available."
No Wrong Door is an initiative that started with the N.C. Department of Commerce in 2016. The Macon County mental health task force has adapted the concept to hopefully better connect potential clients with the mental health and substance abuse treatment services that they need, no matter where they turn for help initially.
In March, commissioner Ronnie Beale hosted a No Wrong Door community forum in Franklin to procure data to help build the program locally. The forum brought together elected officials, business owners, healthcare providers, church leaders, law enforcement officers and educators to talk about potential solutions to the area's lack of mental health assets.
"That forum was just the first step in implementing the No Wrong Door program," Beale, who serves on the state mental health task force, said. "We are trying to be proactive with mental health in Macon County rather than be reactive. We hope that these efforts will lead to less people spending days in the ER waiting for services because they have a mental health or substance use issue."
According to MCPH statistics, Macon County's suicide rate and rate of overdose were above the state average from 2012-2016. Along with the higher-than-average rate of suicides and overdoses, the Macon County Community Health Assessment for 2016 noted that 70 percent of all patients seen for a mental health issue also had some form of physical medical condition.
The MCPH statistics noted that approximately 26 percent of all Americans over the age of 18 suffer from a diagnosable mental illness. In Macon County, roughly 6 percent of citizens who have a diagnosable mental illness suffer from what is considered a "serious mental illness," meaning that they require long-term treatment and care.
A motion to seek the Evergreen Foundation grant and subsequently hire a mental health systems navigator for Macon County, if the grant is awarded, passed unanimously.
Stronger as One: Community leaders rally for suicide prevention and mental health wellness
Art exhibit to help celebrate Mental Health Awareness Month
News Archive
To improve your search results use quotation marks when searching for a specific phrase.
For example, if you search for substance use, a search WITHOUT quotation marks would find listings that include the words
"substance" OR "use." A search WITH quotation marks ("substance use") would find listings only with the whole phrase "substance use."
Are you sure you want to delete this item?


text:
to connect to outgoing mail server - ISPs blocking port 25 traffic
The recent increase in viruses and SPAM has led certain popular UK ISPs
to take drastic action by blocking port 25 (outgoing email) traffic from
any third party mail server. Unfortunately this action inevitably results
in the blocking of legitimate outgoing email traffic.
The solution is to modify the port with which your e-mail program communicates
with our server from the default port 25 to port 587 (smtp submission). This can be done be accessing the
advanced properties of the e-mail account in your e-mail client program.
Further information on this issue can be found at the following web sites:
Unable to send mail - Relay access denied
If you are getting this error then you are either trying to use the wrong
outgoing mail server for your account, or you have not configured outgoing
mail authentication correctly.
If we are providing your email account then you should use our outgoing mail
server - relay.netzoomi.net. However to use this server you must either use
outgoing mail authentication or be using our dial-up Internet connection.
For details on how to set-up outgoing mail authentication see our Email
Account Set-up page.


text:
The houseboat is what you make of it, and the possibilities are endless. That's good or bad, depending on whether you've addressed these key issues regarding your houseboating future.
Slip Availability
Do you know where you can get a slip? In some parts of the country, dockage isn't available at any price and, in other spots, it is priced out of sight. Make sure you know the whole story about the marina's rules, costs and so on. Some marinas, for example, require you to remove your boat during a hurricane warning. Some require that you carry a large liability limit in case your boat catches fire and damages other boats. The marina's location may also dictate your controlling height, width, length or depth.
To The Limit
Some lakes have horsepower limits. Even if you choose the largest houseboat, you may have to settle for a small kicker. Know your waterway's discharge requirements, so you'll buy a houseboat that is equipped for suitable pump-out or sewage treatment. Also check the size restriction for houseboats on the body of water you plan to keep your boat on. Some lakes may surprise you by their restrictions.
Fresh Approach
Have an idea of what facilities are available on the waters you'll use. In some regions, you can't find a diesel station or a holding tank pump-out for miles around. If you plan long periods of boondocking, plan for the right sized tank that you'll need to supply fresh water for your family for a week or two, and blackwater and graywater tanks to match.
The Tall & Short Of It
Does the river or lake where you will use the boat have controlling heights (usually dictated by bridges) or depths? In some waterways, water levels vary so greatly by seasons, droughts, snow melt, tides, water management, mosquito control and other factors, a houseboat that is too deep may not be floatable all year.
Parking Spaces
If you are buying a trailerable houseboat, know where you'll keep it. Your homeowner association may not let you park it on your own property. If you'll be using a storage lot, know the costs (insurance extra?) and rules (can you get the boat out at 4 a.m. to go fishing?). If you'll store it in your garage, know the maximum height and width, including the trailer that can be accommodated.
Elbow Room
Every brochure tells you how many a boat will sleep, but consider how many the boat will actually accommodate. People don't just sleep. They use the bathroom, eat at the dining table, stow their gear and must find daytime storage for all the pillows and bed linen used on the dinette and sofa bed each night. Look not just at how many couples can be bedded, but where singles will sleep and where small fry can be safely bunked.
Purchase With Purpose
There's little point in devoting money and space to a luxurious bathroom with tub, spa and Nautilus equipment if you'll spend most of your time at resort marinas that have all this and more. On the other hand, you'll need at least one and a half baths, preferably two or more, if you'll do a lot of boondocking with a full house.
Ground Rules
Have a heart-to-heart with your spouse and family to make sure you are all on the same wavelength. The cook may see the houseboat as a great place to entertain, envisioning a grand galley, spacious deck and snazzy guest quarters, while you picture it as a getaway where you'll do nothing but fish or read. Or, your spouse may see the houseboat as an escape from household drudgery and plan on docking at swank, waterfront restaurants for dinner each night. If you see yourself enjoying candlelight dinners on deck and breakfast in bed, and your spouse ends up working even harder on the boat than at home, you'll soon be dead in the water.
Sold On Safety
Your children may not want to be packed off on cruises unless they can take a friend or two with them. If you have very small children, you may have to do a selling job on your spouse about how safe houseboating can be. Communicate, and wrangle it all out ahead of time.
Do The Math
Cost has to be addressed at some point. Call a lending agency and supply some ballpark figures to get an idea of what monthly payments would be for a houseboat priced at whatever you think your dream boat will be. Make discreet inquiries to learn if you can qualify for a loan of that size. Then call an insurance agency, describe your imaginary boat, and get a rough estimate of yearly costs. (Whether or not you want insurance, the lender and/or the marina will most-likely require it.) The agent will probably want to know where you'll use the boat, whether it's diesel or gasoline, the hull material and other details.
Uncover The Costs
Add in a monthly cushion for maintenance. Even if you'll do all routine maintenance yourself, there will be yearly hauling, replacements, spares and the cost of work you'll have to hire someone to work on specialty items such as engines and electronics. If you're buying a new boat, you'll also have hefty bill for all the new gear you'll have to add: the required safety equipment, charts and guidebooks, canvas covers and enclosures, anchors, lines, bed linens, galley furnishings, etc. Even without adding the goodies you'll want eventually (dinghy, PWC, davits, more electronics, satellite TV, solar, outriggers, deck furniture, etc.) you will probably run up a pretty good bill. Don't get in over your head with the boat payment alone. That is just part of the cost of boat ownership.
No-Worry Warranties
Warranties are the bugaboos of houseboat ownership. Early in the bargaining process, know exactly what guarantees you have, and who will honor them. It's likely you'll have a shipload of separate warranties for everything from the radios to the refrigerator in addition to the hull and superstructure, engines, head and so on. The biggest problems occur when a boat owner is whipsawed between a supplier and a boat manufacturer, with the toilet manufacturer blaming the plumber who installed it and the boat manufacturer bouncing you back to the company that built the head.
Know, too, where the warranties will be honored. If an accessory has to be removed from the boat and shipped to China, the warranty loses its luster. It goes without saying that you bear much of the burden, too, so read owner manuals thoroughly so you don't void a warranty by using the wrong oil, hooking up the polarity backwards or waiting too long to grease the wildcat. Here's where it pays to buy from a manufacturer or dealer who has a sterling reputation, one who will do everything possible to deliver a great houseboat and go to bat for you if something goes wrong.
Like what you read?
Want to know when we have important news, updates or interviews?
Join our newsletter today!
Send to your friends!


text:
Exploring other food blogs is always a source of inspiration for me. I love tap-tapping on my laptop late into the night, or early in the morning when I have my coffee and all is still, surfing the ever-changing WWW-landscape, uncovering treasures at ever turn. The recipes file on my hard drive is bursting with deliciousness (and a tremendous back-log!) from all the picture perfect dishes I have picked up here and there.
But I wonder how many of the not-so-perfect ones make it out here? Should we even bother putting out our less-than-stellar attempts? I think so. Once in a while I don't mind reading about how this didn't turn out quite the way it was expected to; or how that just seemed to go wrong at every turn. Why? Because the thing I like best about blog dishes is human on the other end – yup, that's you! Yes, you without the test kitchen nor legions of assistants. You that sometimes produces moments of culinary brilliance, and sometimes just needs to get dinner on the table in 20 minutes after a stressful day at work. You who would love to buy organic and free-range everything, but sometimes has to make do with pre-packed flim-flam and parmesan cheese in that bright green container.
Here's a less-than-stellar moment (one of many I tell you!), from my kitchen to yours…
My best friend K brought me back a hefty bag of Pancit Bato from her trip to Camarines Sur. After our first trip to Bicol, the magazine sent her back to do a more in-depth feature on the water sports complex at Camarines Sur. In between whipping around the cable park attached to a board, and exploring the gorgeous islands of Caramoan, she managed to get me some fresh Pancit Bato noodles. Bato in Tagalog (the Filipino dialect where I live) means stone (or rock). These aren't noodles of stone tough…they are named after the town of Bato in Bicol, where they are made. After the noodles are prepared, they are baked for a bit in a pugon (wood-burning oven) giving it its special toasty flavor. The fresh noodles will only last 7 days at room temp – as K was advised not to place them in the fridge. So after their journey back with K, I was all excited to use them!
K dropped them off at my place with instructions and tips, and I got to them right away. The instructions called for cooking them in water and Knorr Meaty Seasoning, then adding veggies and maybe some kind of meat or seafood. K said the one she ate had bits of pork and kikiam (the Chinese que-kiam – made of ground meat and vegetables and wrapped in thin bean curd sheets). C wanted chicken and shrimp. So I just tossed everything in – bits of pork belly, slices of chicken fillet, shrimp, and kikiam – along with some garlic and onion, and thinly sliced carrots and cabbage.
Perhaps the amount of my improvisation confused my poor noggin into forgetting some basic rules: Do not over-cook noodles. Do not over-cook shrimp. Do not over-cook veggies.
Sometimes we have to learn the hard way!
The dish as a whole was tasty…definitely not inedible (thank goodness!). I had it for lunch and dinner. C had it for dinner. Our building's whole staff had it for lunch and dinner too – it was a big batch! I could taste the slightly smoky flavor from toasted noodles, I really liked that it had a lot of sahog (that means the meat and veggies that you add in), and I rediscovered my love for kikiam. The noodles were too soft though and the shrimp too hard…ack! Had I failed K's noodles? Oh dear. I am definitely doing a number of things differently if I come by these special noodles again:
- I will use the specified amount of liquid.
- I will cook it al dente and not turn part of it into noodle-mush.
- I will add the veggies later rather than sooner.
- I will add the shrimp last.
Anyhoo, let's not sweat the small things. Things may not always go smoothly in our collective human kitchen but that's no reason to fret. After all:
- I have a best friend who would drag a bag full of fresh noodles around for me 🙂
- I have now been baptized in the ways of pancit bato
- I have a building full of guards and administration staff that will not bite the hand that feeds them 😉
- I have extra kikiam to eat!
- Despite the over-cooking, it still made for a half-way decent photo 😉


text:
17 Lovely Floor Plan Web App - If you wish to offer your house in the immediate future, it may make sense to just attempt to create the payments while you've got the house available on the market. An open home is a means to invite the community into the program to find out what you do. Thus it's possible that even in the event you owe more than your residence is worth, you could qualify. Therefore, be certain your home is kept neat and tidy. Buy everything you'll need to clean out the house, based on how big it is, the cleaning procedure may take as much as a week. For instance, if you're interested in constructing a residential house, it's vital that you work with a professional who's experienced enough in the region.
Just simply set your existing debts into the system, and you'll have a strategy to pay them off in the right order. An electrical plan will be essential to demonstrate the location of all outlets required to operate equipment. By entering some basic numbers into the application, you'll have a personal plan to receive completely out of debt, including your mortgage, in under 10 years. If you'll go by way of them, you're going to be certain that the log residence plans are reliable and you'll probably be assured your log property cabins will endure the test of time.
If you're planning of constructing a home, you should look for the help of a designer. If you are aware that you need to offer your property, entering into financing modification may not make sense on your part. Ultimately, it's your decision how your house will turn out. The house ought to be precisely modeled so the deck can be accurately designed. Finding an entire house painted is a huge task. So it isn't excellent for envisioning a complete house (but it is named Floorplanner not Houseplanner).
If proper planning isn't done for the painting job, a lot of issues can occur when the actual painting of the home is being done. Employing fantastic planning makes it less probable that changes might have to be made when the program is finished. Usually, Medicaid planning is a part of the services supplied by Elder Law attorneys. The main reason for Medicaid planning is straightforward.
There are several ways to extend your house. You wouldn't build a house on a faulty foundationand exactly the same logic is at play in regards to your basement. For quite a few, the home constitutes much or the majority of their life savings. Take a look at our reviews of the very best interior design computer software alternatives to observe the way that it will be able to help you renovate and remodel your current home, whether you wish to knock out a wall or simply rearrange your furniture. It's also valuable if you've got your current home's plans and aspire to design an addition. For instance, if you've got a present home and just aspire to recreate an idea you saw on a popular home renovation show, you likely don't require a complete software suite. Funeral homes throughout the country are discovering the worth of producing funeral programs in-house by utilizing funeral home program.
The Master Plan sets a development direction and tone that is utilised to maintain consistency once the plan is implemented over a long time period. As soon as your floor program is finished, it's simple to share it. Floor plans are reasonably simple for the layperson to understand because most individuals are acquainted with the drawings representing geometrical objects.
In 1986, it expanded to bring a program that supplies loans with interest rates which are below the market to eligible veterans to fix and increase their present homes. The program is known as second chance leasing, because it provides tenants a second opportunity to rent a house, to boost their credit score, and most significantly, to become more responsible and law-abiding citizens. It also comes with a nutrition plan. It then generates a basic plan that you can customize to your liking. Such programs can truly be a huge aid for veterans who would like to purchase their own houses from the available Weatherford houses for sale. An amazing workout program will use the very best portion of each.


text:
TOP Samson Ps01 Pop Results
TrendShopio.com is a new shopping comparison website in United States. On TrendShopio you'll find a broad range of products from USA's top online shops. TrendShopio allows you to compare Samson Ps01 Pop prices and products, read reviews and shop online for deals and discounts. You can save both money and time by using the TrendShopio service. And best of all: it's a free service for consumers in United States. The price and Samson Ps01 Pop information displayed on TrendShopio are provided by merchants. TrendShopio is an independent company, and our main objective is to provide American shoppers with a fair comparison of prices and products like Creamy Corn, Building Center, 3.75" Clip Point.
TrendShopio only lists qualified and reliable retailers that you can trust. We welcome your feedback, so please add your product reviews and comments.


text:
Shock Absorber - 1ASSP003341ASSP00334
Frequently bought together
Part Details
- Quantity: 4 Piece Set
Our direct fit gas-charged shock absorbers help to restore your vehicle's handling and ride quality to just like new.Direct Fit
Fits and functions correctly. Competitors combine applications to reduce the amount of parts they carry, resulting in: incorrect vehicle height, poor response and an uncomfortable ride.Rigorously Tested
Made from higher quality materials than the competition. Built to have a longer life in any environment or road conditions.Matched Parts
Not all shocks and struts are manufactured the same. Mixing and matching can result in ride quality issues and decrease the life of the parts.Saves Time & Money
Over buying the parts individually, or having to search different parts that are correct for your vehicle (because we did it for you!)
Item Condition:
Attention California Customers:
WARNING: Cancer and Reproductive Harm - www.P65Warnings.ca.gov.
Lifetime Warranty
This item is backed by a Limited Lifetime Warranty. In the event this item should fail due to manufacturing defects during intended use, we will exchange the part free of charge (excludes shipping charges) for the original purchaser.
|Eagle Talon Front Wheel Drive Front & Rear Shock Absorber 4 Piece Set
|Mitsubishi Eclipse Front Wheel Drive Front & Rear Shock Absorber 4 Piece Set
Part #: 1ASSP00334
Order must be placed by 4pm ET
Get it in: 2 - 4 Business Days
Want it sooner? Choose Next Day Air at Checkout


text:
Pay per minute, Pay per day, and Unlimited minutes per month. Some companies offer a combination of these options with their different plans.
Let's Compare Prepaid-Cell-Phones and get better Cellular Phone service.
Some of the prepaid plans are part of major contract plan vendors like Verizon, Alltel U, and T-Mobile. Other plans like Tracfone Prepaid and Net10 Cellular Virgin Mobile buy minutes from the major carriers and resell them to you.
Many Prepaid Cell Phone Plans have a fifteen or thirty day money back guarantee if the phone doesn't work well in your area or you are not happy with it.
In this sale you get 600 minutes and 120 days of service. This is the best deal I have ever seen for a prepaid cell phone. Only 5 cents a minute with a free phone. Check out Net10's latest phone specials! Offers start at $29.99.
***April 25, 2008***
Compare Prepaid-Cell-Phones from T-mobile, Alltel U Prepaid, Virgin-Cell, Verizon-Prepaid, Net10 Cellular, Boost Prepaid, Simple Freedom, Walmart Prepaid phones, Cricket, Tracfone and others that may come along.


text:
Guest Blog: The Webber Family Tours Italy By Bike!
Hey everyone, another adventure is about to begin... this time a family bike tour to Italy! We'll send updates to our favorite website mudandcowbells.com and maybe it will inspire some fresh adventures for you readers!
The packing is almost complete and we're headed to the airport in a few hours. We'll fly Denver-Frankfurt-Florence and then comes the fun part: ride town-to-town for 8-days thru the bicycling nirvana of central Tuscany!!
Our plan is to make a loop south of Florence. We don't have a specific route or towns picked out, but we have a good list of recommendations and plenty of guidebook info. Tuscany is a huge tourist destination, and perhaps the most touted bike touring place in the world, so there is plenty of info available.
We are going super-light, with a credit card and one change of clothes. After a bunch of tours over the past 15 years, we've got a system pretty dialed. Travelling light allows us to ride normal bikes, cover some miles, and not suffer too much with extra-heavy loads. If you choose the right gear, you really don't need much. Our packing list is pretty short: one set of riding kit, one set of casual clothes, and various nic-nacs, toiletries, maps, and of course a bottle opener. All our stuff, for 3 people, will fit in just one set of panniers and one rear trunk. I think it is like 15 or 20 pounds total.
Our last big tour was 2008, two years ago, in the Netherlands. We did 12 days, hitting some cool places and enjoying the fantastic bike trail systems. In that tour, I rode my cross bike with Ella on the tag-a-long behind me, and Sally on her road bike. Ella was only 6, so being on mostly flat bike paths away from busy roads was perfect. We saw the height of spring flowers, the North Sea, plenty of canals, and even took in the Amstel Gold classic and sportive ride.
Ella is now 8 years old, and she'll be riding on the back of our tandem road bike with me up front. She's too short to reach the cranks, so I installed a "kid back" or "kiddie cranks". This is an additional set of cranks mounted on the stoker seat tube, above the normal cranks, with a chain linking them together. I also installed a kid's saddle and handlebars, so it is pretty comfortable and easy to ride. Using a tandem is much more stable and easier than a tag-a-long. It is lighter, faster, and we can actually work as a team. The downside is that it is kinda tricky to set up, and travelling with a tandem is difficult.
That's all I've got time for now, gotta catch a plane!
Pete, Sally & Ella


text: David; It sounds to me as though you are seeking low power beowuf as a solution. There have been a few people build such machines, and it is possible to build a fast, useful beowulf cluster that uses very little electrical power and has sufficient muscle to do some serious work. I have a small 14 node cluster which I built a year ago. It uses very little power , and runs so cool that no room air conditioning is needed. In fact, my p4 machine makes more noise and heat than the beowulf cluster in my apartment. http://mini-itx.com/projects/cluster/ The above link shows the original 12 node configuration. At present, there are some motherboards available which give a very nice combination of cost, performance and low power use. The trick is to "right-size" everything for your needs and available resources. The down side is that the small low-power go-fast stuff is a little more pricey than the plain vanilla pc hardware a beowulf is usually buit from , but not insanely so. Transmetta has some nice boards, and the minit-itx boards are not bad at all for the cost. Also, there are some rather nice small form factor motherboards that use AMD's geode cpu. When I start comparing cost, power use, and performance, so far the most attractive motherboards seem to be the mini-itx boards with the nemiah core cpu. However with some low power geode boards now running at up to 1500 MHz, that may change. The Transmeta boards are probably the fastest of the low power boards, but the power use per MIPS is not as good as other boards if you believe the Transmeta printed specifications. Glen PS: I have also made a few comments below. David Mathog wrote: >At Wed, 16 Feb 2005 19:08:05 +0100 Vincent Diepeveen wrote: > > > >>Date: Wed, 16 Feb 2005 19:08:05 +0100 >>From: Vincent Diepeveen <diep at xs4all.nl> >>Subject: Re: [Beowulf] Academic sites: who pays for the electricity? >>To: "David Mathog" <mathog at mendel.bio.caltech.edu>, >> beowulf at beowulf.org >>Message-ID: <184.108.40.20650216190804.0106fcc0 at pop.xs4all.nl> >>Content-Type: text/plain; charset="us-ascii" >> >>At 08:16 16-2-2005 -0800, David Mathog wrote: >> >> >>>In most universities services like electricity, water, and >>>A/C are paid for by the school. To do so they take "overhead" >>>out of every grant. Partially as a consequence of this they >>>typically have a very poor ability to meter usage on a room >>>by room basis. >>> >>>Now somewhere between the 10 node Pentium II beowulf sitting on >>>a lab bench and the 1000 node dual P4 Xeon beowulf in a machine >>>room that takes up half the basement the cost of the electricity >>>(both for power and A/C) goes from a minor expense to a major >>>one. Really major. For instance, in that hypothetical large machine, >>>at 10 cents per kilowatt hour (a round number), assuming 100 watts >>>per CPU (another round number) that's: >>> >>> For a dual p4 xeon machine at full throttle, it comes out to about 250 watts per node (or a little less) including the network adapters and switching. >>> 1000 (nodes) * >>> 2 (cpus/node) * >>> .1 (kilowatts/cpu) * >>> .1 (dollars/kilowatt-hour) * >>> 365 (days /year) * >>> 24 (hours/day) = >>>----------------------- >>> 175200 dollars/year >>> >>> >>Complete academic nonsense calculation. If you use quite some electricity >>the electricity gets up to factor 20-40 cheaper. Getting a factor 10 >>reduction in usage bill is pretty easy if you negotiate properly. >> >> > >Well, it isn't complete nonsense, unless you care to dispute the >number of days in a year, hours in a day, or cpus in a dual node >computer! > >The only term you're complaining about is the price of >electricity. I'm not privy to the electrical rates that our >school pays, they may well be an order of magnitude lower. My >home rates certainly aren't, but then, I don't buy as much >power as the campus. It's also not at all clear that the >campus would sell power to the end users at the same rate >which it pays the utility. > > You are forgetting the cost of cooling the cluster. Big machines make a lot of heat, and need a lot of cooling. >I don't really understand your point about keeping the units >running versus restarting them. Sure, it would be really bad >to try to boot all 1000 nodes simultaneously, in all likelihood >it wouldn't work. That's why they are typically started at N >second intervals, where N depends on your hardware. >Surely there is some N large enough so that the peak current >draw during the restart never exceeds the random fluctuations >observed when all units are running normally. Or is your >point that the electricity company doesn't want the facility >to draw _less_ current than it uses normally at >steady state? > > It is important to keep the cluster up and running, and only cycle the power when you must. The inrush currents at turnon stress components and shorten the life of the nodes considerably. Also, thermal cycling puts mechanical stresses on boards and components that can cause components and connections to fail. In a large cluster that is middle aged (@ 2 years old), you can reasonably expect to lose a couple of nodes every time you power down and come back up. After a while, this can be expensive. Shutting down a big machine is not a trivial thing. >On a somewhat related note, it would be nice if rack nodes >had some graceful way to conserve electricity. For instance, >something along the lines of: if the CPU utilization goes >below 5% for 10 seconds ratchet the clock down by a factor of 10. >When CPU usage goes above 90% ratchet for 2 seconds move it back >up again. Notebooks can do this sort of thing, but it seems not >to be a "feature" of most full size motherboards. This should >also lower the average temperature in the case, at the expense >of increased thermal cycling. Hard to say off hand if that's >a plus or a minus as far as hardware longevity goes. Certainly >it would be a plus in terms of energy conservation. > > A lot of modern cpu's have the ability to actually shut off unused internal circuitry. VIA CPU's, AMD's geode, Transmeta, and some intel cpus have these features. >Regards, > >David Mathog >mathog at caltech.edu >Manager, Sequence Analysis Facility, Biology Division, Caltech >_______________________________________________ >Beowulf mailing list, Beowulf at beowulf.org >To change your subscription (digest mode or unsubscribe) visit http://www.beowulf.org/mailman/listinfo/beowulf > > > -- Glen E. Gardner, Jr. AA8C AMSAT MEMBER 10593 Glen.Gardner at verizon.net http://members.bellatlantic.net/~vze24qhw/index.html -------------- next part -------------- An HTML attachment was scrubbed... URL: <http://www.beowulf.org/pipermail/beowulf/attachments/20050217/be311034/attachment.html>


text:
The Jaunty Springs Health Centre is the Doctors surgery serving the Sheffield area. Healthcare and wellbeing are forefront in most peoples mind.
Here you can find all the relevant contact info for Sheffield based Doctors Surgery, Jaunty Springs Health Centre, as well as links to pages for all other Sheffield based doctors.
You can also find local NHS Clinics, Hospitals and other health services by using the category links in the breadcrumb at the top of this page.
DISQUS is open to all our users and can be used to share comments & pictures about any business, letting others know what you think.
Please note that there are terms of fair usage and all comments are subject to moderation. Comments which include offensive content of any kind will not be accepted. Furthermore comments are not to be used to vent personal grievances, post links or discuss unrelated topics.
A frequent business type near Jaunty Springs Health Centre is Take Away Restaurants .
The local Parking Garages offer a safe and secure place to leave your vehicle. For freshly baked bread and cakes, roll on down to the Bakery. Rest assured, there are a couple of doctors in the area. Conduct research and borrow literature from the Local library.
Roses are red, violets are blue. If you are in need of a florist there's one near you. Looking for insurance? There is an insurance agent closeby lend assistance.