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Apaf-1 co-assembles with cytochrome c to form the apoptosome, which then binds and activates procaspase-9. We removed pc-9 catalytic domains from the holo-apoptosome by site-directed thrombinolysis. A structure of the resulting apoptosome-pc-9 CARD complex was then determined at ~9.5Å resolution. In our model, the central hub is constructed like other AAA+ protein rings but also contains novel features. At higher radius, the regulatory region of each Apaf-1 is comprised of tandem 7- and 8-blade β-propellers with cytochrome c docked between them. Remarkably, Apaf-1 CARDs are disordered in the ground state. During activation, each Apaf-1 CARD interacts with a pc-9 CARD and these heterodimers form a flexibly-tethered "disk" that sits above the central hub. When taken together, the data reveal conformational changes during Apaf-1 assembly that allow pc-9 activation. The model also provides a plausible explanation for the effects of NOD mutations that have been mapped onto the central hub.
Programmed cell death provides a method to sculpt tissues in developing metazoans and maintain tissue homeostasis (Ferraro et al., 2003). Apoptosis is also used by cells to defend against pathogens and hyper-proliferative cells. Thus, apoptotic pathways are down-regulated in certain cancers and autoimmunity diseases (Song & Steller, 1999; Salvesen & Dixit, 1997; Green & Evan, 2002). Conversely, apoptosis is up-regulated in diseases that destroy cells, such as AIDS, neuro-degeneration and ischemic stroke (Thompson, 1995; Danial & Korsmeyer, 2004).
In chordates, programmed cell death requires cytochrome c release from mitochondria in the intrinsic pathway. This occurs in response to developmental cues, cytotoxic damage or genomic stress (Wang, 2001; Newmeyer & Ferguson-Miller 2003). Cytochrome c binds to the inactive Apaf-1 monomer and triggers apoptosome assembly. This process probably requires conformational changes coupled with nucleotide exchange, while the requirement for nucleotide hydrolysis remains controversial (Reubold et al., 2009; Liu et al., 1996; Hu et al., 1999; Jiang & Wang, 2000; Kim et al., 2005; Yu et al., 2005). The apoptosome binds to procaspase-9 (pc-9) and activates the zymogen to create a platform that mediates proteolytic processing of down-stream procaspases (Li et al., 1997; Srinivasula et al., 1998). Executioner caspases, such as pc-3 and pc-7 (Zou et al., 1997), target certain intra-cellular proteins for proteolysis which leads to an orderly cell death (Danial & Korsmeyer, 2004).
The nucleotide oligomerization domain (NOD) family contains ~23 proteins in human cells that participate in apoptosis, innate immunity and other processes. NOD proteins are members of the AAA+ super-family of ATPases (Inohara and Nunez; 2003; Leipe et al., 2004) and contain a conserved nucleotide binding domain (NBD) and a small helical domain (HD1) that bind nucleotide (Figure 1A, DeLaBarre and Brunger, 2003; Danot et al., 2009; Zhang et al., 2000). In addition, NOD proteins contain a novel winged helix domain (WHD) that is involved in oligomerization, which is followed by a second helical domain (HD2; Figures 1A, 1B; Riedl et al., 2005; Yan et al. 2005). Based on electron cryo-microscopy, the NOD of Apaf-1 is used to assemble a central hub. Seven arms radiate from the hub and are formed by HD2. A regulatory region is located at the end of each arm and contains two β-propellers with cytochrome c sandwiched between them (Yu et al., 2005; Acehan et al., 2002). However, the architecture of the human apoptosome is not clear because a previous map at ~12.8Å resolution did not allow automated and unbiased docking of Apaf-1 domains. Indeed, computer modeling suggested that Apaf-1 may follow a classic AAA+ paradigm for ring formation (Diemand and Lupas, 2006; Propell et. al., 2008), but this remains to be shown experimentally.
Procaspase-9 binding to Apaf-1 is mediated by homotypic interactions between CAspase Recognition Domains in each protein (CARDs; Qin et al., 1999). While precise details of pc-9 activation are not known (Rodriguez, J. and Lazebnik, 1999; Zou et al., 1999), it appears that pc-9 zymogens may be disordered when bound to the apoptosome (Acehan et al., 2002). Apical procaspases are monomers in solution (Boatright et al., 2003, Chao et al., 2005; Li and Yuan, 2008), and executioner caspases are constitutive dimers (Riedl et al. 2001). Thus, pc-9 activation may require dimerization mediated by the C-terminal p10 domain (Yin et al., 2006; Pop et al., 2006, Boatright et al., 2003). The resulting pc-9 dimer is functionally asymmetric and contains one active catalytic site and a second, non-functional catalytic site (Renatus et al. 2001). When taken together, these data are consistent with a proximity-induced dimerization model for apical procaspase activation (Boatright et al., 2003; Renatus et al. 2001).
In this paper, we report the structure of a human apoptosome with bound pc-9 CARDs at ~9.5Å resolution. Molecular modeling revealed novel domain interactions within the central hub and provided insights into apoptosome assembly. We also find that the architecture of the heptameric platform in "active" and ground state apoptosomes is similar. In particular, the arrangement of NBDs within the central hub is reminiscent of their packing in AAA+ rings. However, important differences in the NODs may reflect apoptosome function. We also modeled tandem β-propellers in the regulatory region of Apaf-1 and found that they contain 7- and 8-blades, respectively. Remarkably, Apaf-1 CARDs are flexibly tethered to NBDs in the ground state. In the presence of initiator procaspase, Apaf-1 CARDs interact with pc-9 CARDs to form a flexibly-tethered disk that sits above the central hub. This disk could facilitate the proximity-induced dimerization of pc-9. Finally, our model provides a scaffold upon which known NOD mutations can be mapped and understood.
Previously, we found that apoptosome-pc-9 complexes tended to aggregate during sample preparation for electron cryo-microscopy (Acehan et al., 2002). This may have been due, in part, to high concentrations required to force particles into holes in the carbon support film. In addition, pc-9 molecules were largely disordered on the apoptosome, with the exception of a dome of density located above the central hub. This suggested that few if any interactions would exist between pc-9 and the apoptosome in low salt buffer (LSB), except for the Apaf-1 CARD-pc-9 CARD complex (Qin et al., 1999). To prepare a sample for imaging, we inserted a thrombin site in the linker between the N-terminal CARD and p20 domain of pc-9(D315A) to create pc-9t. We titrated the ratio of pc-9t to Apaf-1 with glycerol gradients to find the saturation point (Figure 1C, left panel). When thrombin was added to the complexes the resulting products included pc-9t catalytic domains, which ran at the top of gradient, and the apoptosome-pc-9 CARD complex (Figure 1C, middle panel). However, pc-9t was susceptible to self proteolysis during over-expression. Hence, we engineered a pc-9t mutant with two additional cleavage sites mutated to alanine (E306A, D330A). The resulting apoptosome pc-9t complex migrated normally on glycerol gradients (Figure 1C, right). Complexes with the pc-9t mutant were assembled and used for structure determination, after releasing the catalytic domains with thrombin. We will refer to the resulting particle as the apoptosome-pc-9 CARD complex.
After modifying the pc-9 linker, we tested the pc-9t zymogen for proteolyic activity using Ac-LEHD-AFC at 100 uM. Cleavage of the susceptible bond following aspartate removes AFC fluorescence quenching. In a time course, pc-9t by itself has appreciable proteolytic activity that may be due to self-aggregation mediated by the pc-9 CARD or the longer linker, as thrombin cleavage brought the activity down to background levels (Figure 1D, compare green and red curves). We then added pc-9t to the apoptosome such that binding was just saturated. Under these conditions, proteolytic activity increased and the true activation level was obtained by comparing apoptosome-pc-9t and apoptosome-pc-9 CARD complexes (Figure 1D, blue and black curves). Complex formation with pc-9t at 100 nM Apaf-1 led to at least a 10-fold activation. We conclude that a single clip in the pc-9 CARD linker is sufficient to release catalytic domains from the apoptosome. This argues against extensive interactions between pc-9 catalytic domains and Apaf-1 in LSB (Acehan et al., 2002). Since pc-9t apoptosomes are active in LSB, the data favor a proximity-induced dimerization model for pc-9 activation (Boatright et al., 2003).
Images of ~42,000 apoptosome-pc-9 CARD complexes in frozen LSB were collected from ~400 micrographs. After refinement with C7 symmetry (EMAN 1.8; Ludtke et al., 1999), we obtained a final map from ~34,000 particles. FSC and Rmeasure curves (Sousa and Grigorieff, 2007) gave 0.5 estimates of 8.3 and 8.9Å resolution, respectively (Figure S1). However, inspection of the map and crystal structure docking with Chimera (see next section) suggests that the actual resolution is ~9–10Å, since individual α-helices are only partly resolved. Thus, we truncated the resolution of the map at 9.5Å resolution. We observed a disk-like density sitting above the central hub of the apoptosome in class averages (not shown). In the final map, this disk was at a lower resolution than the platform and there were no strong connections between these two features. For clarity, we truncated the disk to ~18Å resolution. Top and side views of the map are shown with the platform and disk in blue and magenta, respectively (Figures 2A, 2B). The disk in the original map is shown in the inset (Figure 2B). Disk formation is reminiscent of dome-like density observed in a lower resolution map of the apoptosome-pc-9 complex (Acehan et al., 2002). After removing the disk, top and bottom views reveal a wealth of detail in the central hub, arm and regulatory region (Figures 2C, 2D).
We used a crystal structure of Apaf 1-591 with bound ADP (Figure 1B, PDB code: 1Z6T) to dock NBD, HD1, WHD and HD2 domains into the map, as well as homology models of the C-terminal β-propellers and a crystal structure of bovine cytochrome c (2B4Z). In this work, Apaf-1 domains are color-coded (Figures 1A, 1B), and the nomenclature is based on a structure-based sequence alignment (Figure S2, Riedl et al., 2005). All domains were initially docked as rigid bodies using Chimera (Goddard et al., 2005). They were then re-connected within the map and their fit was refined using Flex-EM (Topf et al, 2008; Supplementary Methods). At this resolution, we did not model all movements of helices within domains. We started with HD2, since its position in the arm was known. Based on the quality of the docking, we were able to ascertain the correct handedness of the 3D map. This was borne out by the fit of domains in the density which gave cross-correlation scores between 0.85–0.95 (Table S1). A close-up of an arm with docked HD2 is shown in Figure 3A, as viewed from below the apoptosome. An icon view of this region is also shown. The fit of HD2 in the arm is shown within a semi-transparent map in Figure 3B and the front surface is cut-away, to reveal the docking of helices and loops within high density features (Figure 3C). A mesh-work has been drawn on the inside of the iso-surface, which allows one to differentiate between inner and outer surfaces. Finally, a side view of the HD2 arm highlights the excellent fit of helices in the density (Figures 3D, 3E).
In the next step, we docked WHD-HD2 as a rigid body since there is an extensive interface and a short linker between these domains in the crystal structure (Riedl et al., 2005). We also docked the NBD-HD1 pair as a rigid body, since they form the nucleotide binding pocket and the linker between them is also short (~5 residues). We then relaxed these restraints to dock domains separately within the map. A small rotation was apparent between domains in each of the pairs. The WHD in the context of its neighboring HD1 is shown in a bottom view (Figures S3A, S3B). The β-hairpin "wing" in the winged helix domain is easily recognizable in side and top views (Figures S3B and S3D–S3F). In addition, helix α18 from HD1 packs against the β-hairpin and the HD1-WHD linker may interact with α23 of WHD. There is good density for the central β-sheet and dATP/ATP in the NBD, but local conformational changes were not modeled. Finally, the 4 helix bundle of HD1 fits snugly between adjacent WHDs.
The regulatory region was constructed by docking homology models of 7- and 8-blade β-propellers (Supplmentary Methods) and cytochrome c (Mirkin et al., 2008, 2B4Z) into the density. Ramifications of our modeling of the regulatory region will be discussed in the next Section. After refinement of the entire Apaf-1/cytochrome c model, a pdb with a complete subunit was docked automatically into 7 equivalent positions in the map without major clashes between chains. The quality of the final fit is indicated by the high cross-correlation (0.91; Table S1). An over-view of the final platform model is shown in top and bottom views (Figures 4A, 4B), with the model viewed within a semi-transparent map.
At this stage, there was no room to fit seven Apaf-1 CARDs within the platform. Hence, the disk must contain Apaf-1 CARD-pc-9 CARD complexes (Qin et al., 1999). However, the size and low resolution prevented us from modeling 7 CARD-CARD heterodimers within the disk. We surmise that Apaf-1 CARDs are flexibly-linked to their respective NBDs within the platform, which leads to a blurring of the disk. This idea is consistent with the location of linker helix α8 (Figure S3E), as the N-terminus of this helix points up towards the disk. Intriguingly, one Apaf-1 isoform with a shorter CARD-NBD linker is still functional (aa99-109 missing in Apaf-1L; Saleh et al., 1999). This suggests that a longer link between the disk and platform may not be required for full function.
We then took the final platform model with 49 domains and docked it as a rigid body into a density map of the ground state apoptosome (Yu et al., 2005). The overall fit was excellent with no significant unfilled regions (Figures 4C, 4D). Hence, the platform probably does not change significantly when pc-9 is bound, with the possible exception of CARD-NBD linker movements.
A ribbon diagram of structural elements in the apoptosome is shown in a top view (Figure 5A), and a single Apaf-1 monomer is surrounded by a dashed boundary line. Additional views and subunit contacts are shown in Figures S4 and S5, respectively. The close-packing of domains is shown in Figures 5B–5D, with a transparent surface overlayed onto the apoptosome model to give a perception of depth. The hub is comprised of two rings: an inner ring containing NBDs encircled by an outer ring that contains alternating HD1 and WHDs (Figures 5B, 5C). The critical unit for assembling the hub is comprised of an NBD-HD1 pair and a WHD.
The hub model differs significantly from a previously published domain model that was based on a lower resolution map (Yu et al., 2005). In the earlier work, it was thought that Apaf-1 CARDs may play an integral role in apoptosome assembly, since these domains tended to self-aggregate and CARD-CARD interactions seemed likely (Yu et al., 2006; Shiozaki et al., 2002). In addition, the resolution of the previous map was not sufficient to allow unambiguous, computational docking of small domains, so manual methods were used. When these factors were combined, it resulted in a local minimum being chosen in which NBD and HD1 positions were flipped, because a ring of Apaf-1 CARDs was modeled erroneously at the center of the hub. It is now clear that Apaf-1 CARDs are flexibly-linked to the platform. In addition, higher resolution coupled with more robust docking programs has allowed an unambiguous solution.
In general, the hub has an architecture that is reminiscent of rings formed by other AAA+ ATPases (Diemand and Lupas, 2006; Riedl et al., 2005). However, significant differences arise from the presence of an initiator specific motif in the NBD and from the packing of WHDs in the hub. These differences may reflect the fact that Apaf-1 forms a stable platform instead of performing mechanical work like many AAA+ ATPase rings. This difference is also reflected in the low intrinsic ATPase activity of the apoptosome (Jiang and Wang, 2000; Kim et al., 2005; Yu et al., 2005).
The NBD plays a critical structural role because it is centrally located in the apoptosome and may nucleate ring assembly. Two major changes within the NBD are apparent at this resolution. First, the initiator-specific motif (ISM, helix α12) is present in members of the initiator clade, which are most similar to NOD proteins in the AAA+ superfamily. The ISM of Apaf-1 is inserted between β2 and α13 in the NBD. Together, α12 and α13 form a helix-loop-helix motif. Seven of these paired α-helical motifs align in a nearly vertical orientation to form a ring that encircles the central pore of the apoptosome (Figure 6A; ISM ring). Second, the α8 linker helix in one NBD packs against an adjacent NBD. Nucleotide triphosphate is bound near the subunit interface in a deep pocket between the NBD-HD1 pair and there is no obvious entrance or exit path (Figures S3E, S3F). Density for the "arginine finger" in the NBD (actually K277 at the end of β5) of the adjacent subunit is weak, so it is hard to judge whether NTP hydrolysis could occur in the apoptosome. Neither the vertical packing of the α12-α13 pair to form the ISM ring (Figure S6), nor the re-arrrangement of linker helix α8 could have been predicted using previous AAA+ ring structures (Diemand and Lupas, 2006).
The AAA+ proteins do not contain a winged helix domain; instead NBD-HD1 pairs interact with neighbors to form rings and oligomers (Danot et al., 2009; Hanson and Whiteheart, 2005). Hence, the WHD is a major addition to NOD proteins. In the central hub, the WHD interacts with a neighboring NBD-HD1 pair (Figure 6B). This generates an outer ring with alternating WHD and HD1s within the hub. The HD1-WHD linker was not modeled explicitly, but density for this feature is present between HD1 and WHD (Figures 3B, 3C). Within an Apaf-1 monomer, the end of helix α19 in HD1 and the HD1-WHD linker interact with α24 in the WHD. In the formation of the HD1-WHD ring, the loop between α18 and α19 of HD1 contacts the β-hairpin of WHD (Figures 6B, S3D). The WHD also interacts strongly with the HD2 arm which positions the regulatory region of each subunit at higher radius (Figure 5C).
A more detailed sequence analysis of the regulatory region revealed 15 WD40 motifs rather than the 13 repeats identified previously (Figure S7, Experimental Procedures). This allowed us to model tandem 7- and 8-blade β-propellers within the regulatory region. The resulting model is reminiscent of the crystal structure of actin interacting protein 1 (1PGU; Voegtli et al., 2003), except that an 8-blade propeller in Apaf-1 replaces the second 7-blade propeller in Aip1. The docking of β-propellers within the map shows that the V-shape of the regulatory region is supported at the base by helices α30, α31 and α32 in HD2 (Figures 6C, S8). The model is strongly constrained by the length of the HD2-propeller linker and by links between the two β-propellers. A novel feature is that the HD2-linker forms a d-strand in the last blade of the 8-blade propeller before continuing on to form the 7-blade propeller and the rest of the 8-blade propeller (Figures 6D, S7). This prediction remains to be verified with higher resolution data.
We then docked a crystal structure of cytochrome c into the map (2B4Z). However, several related orientations were found with similar docking scores. This was due to the paucity of longer helices and the presence of large loops in cytochrome c, which are not well defined at the current resolution. Therefore, we chose the orientation with the highest score, but this has to be verified. Even so, each docking placed the heme group in a protected environment between the two β-propellers, consistent with data which shows that the heme is not solvent accessible (Purring-Koch & McLendon, 2000). Several residues on cytochrome c were identified previously through mutations which interfere with Apaf-1 mediated procaspase activation (Yu et al., 2001; Hao et al., 2005). In general, our fit for cytocrhome c places most of these residues, including lysines, in orientations where they could interact with the β-propellers.
In this work, we determined a structure of the apoptosome-pc-9 CARD complex at ~9.5Å resolution. We then used molecular docking with crystal structures to create a model for Apaf-1 subunits within the active apoptosome. Our model shows how the central hub is constructed by the NOD and provides insights into conformational changes that occur during assembly. The model also provides a rationale for the roles of cytochrome c and ATP/dATP in assembly and helps to explain phenotypes that arise in certain NOD mutations. Importantly, we find that flexibly-linked Apaf-1 CARDs in the active apoptosome interact with pc-9 CARDs to form a disk that may play a role in procaspase-9 activation. Finally, the model provides a blueprint for other NOD proteins which form activation platforms.
We determined the structure of an apoptosome-pc-9 CARD complex after removing the pc-9 catalytic domains with a single thrombin clip in each subunit. Hence, pc-9 catalytic domains are not tightly bound to Apaf-1 in LSB. We also find that pc-9 is flexibly-tethered to the apoptosome by Apaf-1 CARD-pc-9 CARD heterodimers, which form a "disk" that sits above the platform. Thus, our model is representative of the heptameric platform at ~9.5Å resolution. Based on the model, we provide insights into construction of the central hub, arm and regulatory region. In particular, the central hub is reminiscent of other AAA+ rings in which the NBD-HD1 pair mediates assembly through extensive lateral interactions between adjacent subunits. In addition, nucleotide triphosphate is located at the interface between adjacent NBDs. However, NBD-NBD interactions are modified in the apoptosome by the ISM helix (α12, which partners with helix α13 to form a helical pair motif. This helix pair associates with 6 copies of itself to form a ring at the center of the hub that encircles the central pore. At the same time, linker helix α8 between the CARD and NBD is repositioned between neighboring NBDs, near bound ATP/dATP. In addition, helix domain 1 in the NBD-HD1 pair rotates a bit, while maintaining contact with the nucleotide and interacts with the WHD from an adjacent Apaf-1 during assembly. This novel HD1-WHD interaction forms an outer ring within the hub.
Formation of the HD1-WHD ring positions seven HD2 arms and their attached regulatory regions, which extend from the central hub. While we have achieved an accurate model of the central hub and arm, the regulatory region remains somewhat blurred because we don't have atomic structures for Apaf-1 β-propellers. Even so, we were able to model tandem 7- and 8-blade β-propellers using extensive sequence information and pdb structures, including Aip1, which has two β-propellers with a similar V-like configuration (Voegtli et al., 2003). Finally, we placed cytochrome c between the two β-propellers in an orientation that agrees with known data.
Our model is consistent with functional and mutation studies of Apaf-1 (summarized in Table S2 and Figure S9). For example: Apaf-1-ALT in a prostate cancer cell line is not functional (Ogawa et al., 2003). This isoform is terminated after HD1 and thus, is missing the WHD, arm and regulatory regions. WHD loss could weaken or prevent the assembly of an Apaf-1-ALT ring. Additional studies revealed two loss of function mutants, Apaf-1 (L415P) and 437ter (Table S2; Harlan et al. 2006). When leucine 415 is replaced by proline, helix α23 in WHD would be disrupted (Figure 6B). This mutation may change the conformation of this important region and prevent assembly. For Apaf-1 437ter, termination occurs before a conserved histidine (His438) in the WHD, which may partly destabilize the inactive monomer (Danot et al., 2009). However, the greatest effect of this mutation would be the elimination of helix α24, which forms an important contact to HD1 in the ring. The loss of α24 would also destabilize α20 and α21 in the WHD. Hence, the 437ter mutation is predicted to prevent assembly due to loss of the HD1-WHD ring. Alternatively, incomplete assembly might lead to aggregation and block pc-9 activation.
Our model can be extended to other NOD proteins (Inohara and Nunez, 2003). However, specific details will vary as NOD proteins contain different N-terminal effector and C-terminal regulatory regions. For example, Nod2 is associated with Crohn's disease, Blau syndrome and early on-set sarcoidosis (Hugot et al., 2001; Miceli-Richard et al., 2001; Kanazawa et al., 2005; van Duist et al., 2005). Crohn's disease may be due to loss of function mutants whereas gain of function mutations may be responsible for Blau syndrome and early on-set sarcoidosis (Wilmanski et al., 2008). Several loss of function mutants, including C333Y and S344T, along with Crohn's disease mutants L348V and H352R, are located in the ISM helix region (Table S2 and Figure S9). These mutations may disrupt interactions that are critical to nucleation and assembly of the central hub. Another loss of function mutant, V492E, is located in sensor-2 helix, which is equivalent to helix α18 in Apaf-1. This region mediates interactions between HD1 and a WHD from an adjacent subunit. Mutations in this feature could disrupt assembly or lead to an unstable complex.
Platform assembly in the apoptosome is the first step in pc-9 activation (Hill et al., 2004; Zou et al., 1997, 1999; Rodriguez and Lazebnik, 1999; Acehan et al., 2002; Yu et al., 2005). Apaf-1 exists in solution as an inactive monomer and binds cytochrome c, in a series of steps that leads to an open conformation. The open form then promotes apoptosome assembly (Acehan et al., 2002). We do not have a crystal structure of the complete Apaf-1 monomer; instead we have a model with bound ADP that is missing the C-terminal regulatory region (Apaf 1-591; Riedl et al., 2005). The compact nature of the N-terminal region suggests that it may be a good first approximation for a closed, inactive Apaf-1. However, some uncertainty remains about the nature of bound nucleotide in the inactive monomer. Thus, Apaf-1 purified from sf21 cells contained dATP and hydrolysis of the γ-phosphate occurred upon cytochrome c binding (Jiang and Wang, 2000; Kim et al., 2005). Conversely, Apaf-1 purified from Hi-Five/sf21 insect cells and from bacteria contained bound ADP (Bao et al., 2007; Reubold et al., 2009; Riedl et al., 2005). In this case, ATP hydrolysis was not required for apoptosome assembly (Bao et al., 2007; Reubold et al., 2009). However, cytochrome c binding and nucleotide exchange were necessary to stabilize the monomer in an assembly competent form.
Beta propellers in the regulatory region are thought to lock Apaf-1 in an inactive conformation. To emulate this state, we placed "cartoon" β-propellers at the C-terminus of folded Apaf 1-591. This created a compact monomer while retaining the rough geometry of the regulatory region (Figure 7A, left panel). Note that we kept the NBD in a fixed position throughout this analysis. In the first stage, cytochrome c may bind between two β-propellers with a geometry like that observed in the apoptosome. This would trigger an opening of the compact Apaf-1 monomer with the regulatory region moving away from the NOD region. During this step, the WHD and regulatory region would swing ~120° about a pivot point located between HD1 and WHD. In the crystal structure of Apaf 1-591 (Riedl et al., 2005; Danot et al., 2009), the WHD is bent back towards ADP and a conserved histidine (His438) forms a hydrogen bond with ADP to help stabilize the inactive monomer. This hydrogen bond would be broken when the Apaf-1 monomer opens up.
In the second stage, nucleotide diphosphate is exchanged for dATP/ATP to presumably lock the Apaf-1 monomer in an open and assembly competent form (Figure 7A, right panel; Acehan et al., 2002). The overall effect of these large domain movements is two-fold. The rearrangements would expose the α12-α13 helix pair along with the rest of the NBD, so that the NBD ring in the central hub could assemble. Changes in the vicinity of bound ATP/dATP must be responsible for the transition from a closed to an open form, as shown in a bottom view of the NBD that compares closed and open conformations (Figure 7B). In our model, there is room in the binding pocket to accommodate ATP. However, nucleotide exchange and divalent cation binding probably alter interactions with the CARD-NBD linker, which may trigger a repositioning of helix α8 during assembly. At the same time, HD1 rotates by ~12° relative to the NBD and the HD1-WHD linker flips through an angle of ~50–60° to help form a contact with an adjacent Apaf-1 subunit. Although we could not accurately model the HD1-WHD linker (see dashed line, Figure 7B, right panel), there is clear density for this feature in the 3D map. Linker movement is coupled with a rotation of the WHD by ~120° (for clarity, only helices α20 and α21 are shown). This would position WHDs and HD1s to form the outermost ring in the hub, as NBDs in each Apaf-1 associate to form the inner ring. The relative geometry of the WHD-HD2 pair remains fixed during this large conformational change and creates the extended arm which supports the regulatory region. Clearly, more precise details of nucleotide exchange and its function as a trigger for assembly will require a higher resolution structure of the apoptosome.
The major goal of Apaf-1 assembly is the formation of a stable platform while releasing CARDs from interactions with their regulatory regions. This frees up seven Apaf-1 CARDs that remain flexibly-linked to the platform through CARD-NBD linkers which are ~17 residues long. Indeed, the CARD-NBD linker is also disordered in the crystal structure of Apaf1-591 (Riedl et al., 2005). There is also a precedent for CARD flexibility in a crystal structure of the CED4-CED9 complex (Yan et al., 2005). In this case, a CED4 CARD was missing because it did not make stabilizing contacts in the crystal.
We tested the idea of CARD flexibility by constructing Apaf-1 with a thrombin site in the CARD-NBD linker. After apoptosome assembly, Apaf-1 CARDs were readily removed by thrombin (data not shown), consistent with these domains being flexibly-tethered to the platform. Thus, flexibly-linked CARDs in the apoptosome are free to interact with pc-9 CARDs. This allows the formation of a disk that is likely comprised of Apaf-1 CARD-pc-9 CARD heterodimers (Qin et al., 1999). Disk assembly may be driven by the high local concentration of CARDs in the region above the central hub. The importance of this disk will be described in a forthcoming paper.
Apoptosomes are present in chordates and arthropods while a smaller protein, CED4, is present in C. elegans. Conservation of the NOD suggests that apoptosomes will have a similar architecture for the central hub. However, in Drosophila the apoptosome contains 8 subunits per ring, rather than the seven subunits found in the human complex. Even so, the general arrangement of protein domains appears to be similar in human and Drosophila apoptosomes (data not shown; Yu et al., 2006).
A crystal structure of an inhibited CED4-CED9 complex revealed a non-symmetric CED4 dimer (Yan et al., 2005). We compared the CED4 dimer to a pair of Apaf-1 subunits extracted from our apoptosome model. This revealed an excellent correspondence between structures when they were aligned on the NBD of the leftmost subunit (Figure 8). In the CED4 dimer, there are three major interfaces. In the first site, the ISM helix (α11) and α11a (or a loop) are packed against α12 (equivalent to α13 in Apaf-1) of the adjacent molecule. In addition, linker helix α8 is packed between the NBDs, although its length and position differs from the equivalent helix in the human apoptosome. At the second site, the HD1-WHD linker from one subunit interacts with the adjacent NBD (asterisk in Figure 8, right panel). At the third site, HD1 interacts with a WHD from the adjacent molecule (arrow in Figure 8, right panel). These three contact sites are similar, though not identical, in a lateral Apaf-1 dimer extracted from the apoptosome (Figure 8, left panel). Hence, the CED4 dimer could represent a building block of the C. elegans apoptosome in which CED9 blocks the oligomerization interface of one CED4 molecule. This predicts that a CED4 apoptosome may contain 7 or 8 subunits. However, the CED4 subunits have a closer lateral spacing which arises in part, from differences in the HD1-WHD linker (see asterisk in Figure 8) and linker helix α8. This might favor a CED4 apoptosome with 8 subunits when the dimeric nature of the putative building block is factored in. Intriguingly, CED4 binds ATP and does not have ATPase activity so nucleotide exchange may not drive assembly. Instead, the removal of CED9 by EGL1 may be sufficient to promote assembly of this apoptosome from dimeric CED4 building blocks (Yan et al., 2005).
In summary, we have presented a comprehensive model of the human apoptosome based on molecular docking into a density map at ~9.5Å resolution. The new model of the platform is consistent with known biochemical properties and can explain the phenotypes of critical mutations in NOD family members. When combined with sequence information, the model provides a blueprint for the structure and assembly of other NOD family members. This work also sets the stage to develop a deeper understanding of the mechanism of pc-9 activation.
Recombinant Apaf-1 was expressed and purified from sf21 insect cells (Acehan et al., 2002; Yu et al., 2005). Procasepase-9 mutants were generated with the Quickchange kit (Strategene) and a pc-9t linker mutant was created by inserting a thrombin site (LVPRGS) after Lys100. Procaspase-9 proteins were expressed and purified as described (Chao et al., 2005). Bovine heart cytochrome c (Sigma) was purified by cation-exchange chromatography (HiTrap S, Amersham). To assemble the ground state apoptosome, Apaf-1 in LSB (20 mM HEPES-KOH [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol) was incubated with cytochrome c (in a 1:1 molar ratio) and 0.1 mM dATP at 30 °C for 30 min. For the apoptosome pc-9 complex, Apaf-1 was incubated with cytochrome c, procaspase-9 and 0.1 mM dATP at 30 °C for 30 min. To obtain the apoptosome-pc-9 CARD complex, 3 units of thrombin (Sigma) were added per 50 ug Apaf-1 and the solution was incubated at 30 °C for an additional 30 min. To monitor assembly, apoptosome samples were applied onto a 10–40% glycerol gradient (2.1 ml) volume in LSB and centrifuged at 17°C for 2.5 hrs in an RP55S swinging bucket rotor (55,000 rpm; Sorval). Fractions were collected manually from the top of the tube (14 fractions, 150 ul per fraction), separated on an 8–18% gradient gel by SDS-PAGE and visualized with Coomassie blue.
A fluorogenic substrate, Ac-LEHD-AFC (Calbiochem), was used for pc-9 proteolytic activity assays. Fluorescence experiments were carried out on a Fluoro-Max2 fluorimeter (Jobin Yvon–SPEX, Instruments SA, Inc.) at room temperature using a 600 μl cuvette with a path length of 1 cm. Samples containing pc-9 or the apoptosome pc-9 complex were diluted with LSB to 600 μl. The ratio of pc-9 to Apaf-1 was obtained from titration experiments with glycerol gradients to insure that no free pc-9 was present. Cleavage reactions were started by adding Ac-LEHD-AFC (100 uM) to the sample. Release of the AFC fluorescent moiety was monitored at 505 nm with an excitation wavelength of 400 nm.
After assembly, the apoptosome pc-9 CARD complex was obtained by thrombin treatment in LSB and concentrated to ~3 mg/ml using YM-30 Microcons (Millipore) in the presence of Nonidet P40 (~0.15% final concentration). Aliquots of the concentrated sample (2 μl) were pipetted onto Quantifoil R1.2/1.3 holey grids, which were then blotted and plunged into liquid ethane, cooled with liquid nitrogen. Plunging was done at 100% humidity and room temperature using a Vitrobot Mark 3 (FEI). Data were collected on film at 62,000X with a Tecnai F20 microscope operated at 120 kV for optimal contrast. Electron micrographs were scanned using a Zeiss scanner at a step size of 7 um and binned to 2.26 Å/pixel. Particles were selected using BOXER (Ludtke et al., 1999) and a hybrid structure factor was generated (Yu et al., 2005). Since the complex was prone to aggregation, only ~100 particles could be selected from each micrograph, which gave a weak signal for CTF fitting. Thus, CTF parameters were determined from the adjacent carbon film with FITCTF, and these values were then used to manually adjust the CTF of the summed particles with CTFIT in EMAN 1.8 (Ludtke et al., 1999). For 3D reconstruction, a ground state apoptosome map was used as a starting model (Yu et al., 2005). About 42,000 particles were selected and after refinement ~34,000 particles were included in the final map with ~238,000 subunits. Map filtration was accomplished by computing a hybrid 1-D structure factor during CTF determination, then applying it to the map during refinement with the setsf option. This was combined with a low-pass filter based on the resolution determined for the final map.
For docking experiments, the four domains (NBD, HD1, WHD and HD2) and helix α8 were taken from Apaf-1 (1-591) (1Z6T; Riedl, et al., 2005). A crystal structure of bovine heart cytochrome c (2B4Z; Mirkin et al., 2008) was also used. Docking was carried out in UCSF Chimera (Goddard et al., 2005) and refinement with Flex-EM (Topf et al, 2008). Further details on the modeling of the two β-propellers and the docking procedures are described in Supplementary Methods. Symmetry related subunits were generated in Moleman2 using 7-fold symmetry by rotating the model of the first subunit about the z-axis by multiples of 51.4° (Kleywegt et al. 2004). Figures were made using Chimera and Adobe Photoshop.
We thank Yigong Shi for providing a pc-9 clone, Fenghe Du for sf21 insect cells and helpful discussions on Apaf-1 purification, and Niko Grigorieff for use of the Zeiss scanner. Computations were done on a Linux cluster at the Scientific Computing and Visualization Group at Boston University. Coordinates for the apoptosome model and the density map have been deposited with the RCSB (xxxx) and EMD (EMD-5186), respectively. The Topf laboratory is supported by an MRC Career Development Award (MT) and the Human Frontier Science Program (RGY0079/2009-C). Wang and Akey laboratories are supported by NIH grants; Dr. Xiaodong Wang was supported by the Welch Foundation and HHMI.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text: I stay here 2 nights a week for work. The rooms ARE old but are being refurbished. The beds are comfortable and plenty of space to move. I was originally concerned about the highway traffic but after many nights I can honestly say it doesnt keep me awake! Towels are clean, electric blankets for warmth, the onsite restuarant has some... More

subset: commoncrawl


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The company has added a new HD receiver to the prototype transmitter displayed at NAB2005. It is now ready to accept orders for the complete HD wireless camera system. Deliveries are planned for the first quarter of 2006.
LinkHD maintains the full detail and resolution of HD format TV pictures throughout transmission. Link Research has achieved this by using its MPEG compression and modulation techniques. The result? A high definition wireless camera system with low transmission delay, a low-power requirement and a robust diversity signal.
For more information, visit www.linkres.co.uk.
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Huge Ultra HD Display Lights Up Times Square


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Old Dominion 45
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Robins Stadium, Richmond, VA
Top Performers
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Rushing: J. Laub (RICH) - 14 CAR, 24 YDS, 1 TD
Receiving: B. Edwards (RICH) - 11 REC, 182 YDS, 1 TD
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BusinessWeek "Best of the Web" Winner 3 Years Running
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- Signal Processors(108)
Results 1 - 20 of 108 matches
Top RatedLexicon MX400 Dual Stereo/Surround Reverb Effects Processor
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Single channel compressor/limiter. The dbx 160A is the latest generation of the legendary dbx 160 family of...
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Neutrik 48-Point TRS Patchbay
Extremely sleek design with grounding option and 2 rows of balanced sockets for a total of 24 front and 24...
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StartUpHire is the place to find Business Development Representatives jobs. Review the list of Business Development Representatives career opportunities to the left. These Business Development Representatives jobs represent some of the most rewarding careers available.
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Description SpringCM is looking to quickly grow its business development team. In this position, you will collaborate with talented people in both marketing and sales roles. Day-to-day goals are focused on creating and nurturing sales opportunities for the outside sales teams.5/4/2015 Chicago, IL Save This Job
Allegro is a leading provider of energy trading and risk management (?ETRM?) software solutions. Our ETRM software solutions support some of the largest energy trading companies worldwide, encompassing all energy commodities. Allegro provides transparency and efficiencies across5/3/2015 Dallas, TX Save This Job
HubSpot seeks a Business Development Representative (BDR) to join our highly successful Latin America sales team. This is a great opportunity to position yourself for excellent opportunities for career growth. We're looking for hard-working, competi6/20/2015 Cambridge, MA Save This Job
Business Development Representatives (BDRs) identify and create new qualified opportunities for Sales Directors at eMarketer. Successful BDRs are highly effective communicators, door openers and opportunity builders. eMarketer BDRs are able to access senior-level executives,5/24/2015 New York, NY Save This Job
More information about this job: Overview: Founded in 2000, Logi Analytics provides businesses the fastest and easiest way to deliver business analytics, dashboards, and reports to a broad range of users, as well as to embed them into existing applications ? all for a fraction5/18/2015 McLean, VA Save This Job
Join the 2014 Stevie Awards winner of the ?Sales Department of the Year?! ZoomInfo is looking for our next generation of sales leaders. We hire young professionals who have a history of success in school and in their limited work experience to become Business Development5/17/2015 Waltham, MA Save This Job
Department: Sales Location: Boston, MA Are you looking to jump-start your career with one of the fastest growing technology companies in America? Are you comfortable being surrounded by incredibly smart and driven people that push you to be better? Are you hard working,5/29/2015 Boston, MA Save This Job
ABOUT PEOPLELINX PeopleLinx makes social selling easy. Founded by early LinkedIn employees, PeopleLinx guides sales professionals to build relationships, attract qualified leads, and drive upsells using online social networks. Our technology maps to your sales process,5/26/2015 Philadelphia, PA Save This Job
POSITION SUMMARY The Business Development Representative is responsible for lead nurturing, generating quality leads and appointment setting for Calabrio sales representatives. This position will coordinate ongoing communications and lifecycle of existing Calabrio leads5/13/2015 Minneapolis, MN Save This Job
According to Inc. Magazine and Forbes, TapInfluence is redefining the marketing equation with intelligent software that gives brands and agencies access to a proven, measurable path to consumer influence and impact. Our powerful marketplace of content creators and social media6/17/2015 Boulder, CO Save This Job
The Sales Development Representative is responsible for identifying and qualifying sales opportunities for Bit9's sales organization. This is accomplished through outbound calling and e-mailing. With plenty of leads provided, the Sales Development R5/17/2015 Waltham, MA Save This Job
Job Description BetterCloud is looking for a highly motivated Sales Development Representative to join our rapidly growing team. We value hard work and attitude over experience, and are looking for an individual who wants to roll-up their sleeves and make an immediate impact on5/9/2015 New York, NY Save This Job
KnowledgeTree seeks Sales Development Representatives (SDR) to join our growing sales team. Are you competitive and self-motivated? Do you have previous sales experience or a high degree of interest in sales? Do you want to join a high producing team in a fast-paced5/25/2015 Raleigh, NC Save This Job
AtTask, Inc is a leading provider of Work management and portfolio management software We are a growing company with great opportunity, and we are looking for qualified and competitive Sales Development Representatives. This is a team-oriented position that offers a clear career5/23/2015 Lehi, UT Save This Job
Austin, TX We're looking for talented Sales Development Representatives who will be responsible for identifying and qualifying sales opportunities in North America. The role focuses on identifying the business need while also conducting information-gathering to help pre-qualify5/18/2015 Austin, TX Save This Job
About Influitive Influitive is a rapidly-growing innovator in the marketing software space. Backed by top-tier venture capitalists including Hummer Winblad, First Round Capital, New Enterprise Associates, Lightspeed Ventures and Relay Ventures, Influitive helps B2B marketers4/29/2015 Boston, MA Save This Job
About Apigee, The API Company Apps are changing the way we live, and APIs are the secret ingredient that makes apps work. Apigee gives businesses and developers everything they need to be successful in the app economy. Hundreds of companies including AT&T, eBay, Pearson, Gilt6/25/2015 San Jose, CA Save This Job
Description Work with the next frontier of virtualized, bleeding edge technology and enterprise level clients Solve big business problems with a team that truly values innovation Be part of a growing company with opportunities for career advancement! Skytap, Inc. delivers the6/9/2015 Boston, MA Save This Job
Sales Development Representative - Mountain View Mountain View, CA Sales and Business Development Elasticsearch is on a mission to make real-time data exploration easy and available to anyone. We spend our days engineering the world?s most advanced open source search and5/18/2015 Mountain View, CA Save This Job
Sales Development Representative - Mountain View Mountain View, CA Sales and Business Development Elasticsearch is on a mission to make real-time data exploration easy and available to anyone. We spend our days engineering the world?s most advanced open source search and5/18/2015 Mountain View, CA Save This Job


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Fernando PereiraShared publicly -
Growing up in Europe, I didn't pay much attention to the construction of the Euro, and whatever little I remember has nothing to do with the economics of it. Now, older, having lived in the US for a while, with a Greek wife, I'm looking at the way the Euro is unraveling and I've been using the opportunity to try to figure out how it works (or, rather, why it doesn't).
The core mechanism that allows multiple states to share the same currency is pretty simple: since the weaker states can't devalue their currency to compensate for their trade deficit with the stronger ones, money has to flow from the stronger economies to the weaker ones in order to maintain the balance.
We see that in the US: as measured in GDP per capita, there's about a 2:1 ratio between the strongest states and the weakest ones. To compensate for that, a lot of money flows between states, through the federal government. Most taxes in the US are federal taxes, i.e. about 75%, and the federal government doesn't necessarily spend the money it collects in the exact states where it collects them. As an example, about 130 billion dollars paid by California in federal taxes don't make it back into California. Texas and New York are the two other states that have a negative balance of more than 100 billion each. For those 3 states, that outflow on money represents 5.7%, 7.2% and 7.4% of their respective GDPs. California is literally sending money to other states so that those states can buy California stuff. The same is true for Texas, New York, and about 20 of the 50 states that are sending money to the other 30.
Looking back in history, the Marshall Plan followed a somewhat similar logic: the US sent aid to Europe, to allow Europeans to buy US goods, which was both a stabilizing mechanism for European currencies that otherwise were in a devaluation spiral, and an outlet for the huge US industrial production. For reference, the Marshall Plan amounted to 120 billion dollars (in today's dollars) over 4 years, which is tiny compared to the amount of money that the federal government now redistributes across state lines.
We can compare that to the situation in the Eurozone/EU, where the GDP per capita varies by a factor of about 2.3:1. Germany's balance in the EU budget is negative by less than 9 billion Euros. France's and Italy's follow at approximately 6.5 billion and 6 billion. Germany's 9 billion Euros is tiny compared to California's 130 billion dollars, especially since Germany's GDP is 60% larger than that of California. Since the US and EU economies have approximately the same size, that's a reasonably apples-to-apples comparison. The biggest negative balance that a Eurozone country has with the EU is about 0.41% of its GDP. The biggest positive balance is 1.3%. Within the US, only 4 states out of 50 fall within that range.
That's the problem right there: Germany is not flowing enough money out to other Eurozone countries to compensate for its own very strong economy. That's true of other rich European countries as well, e.g. Netherlands, Austria, France.
From the Greek point of view, the only way to get that money to flow in order to maintain balance had been for the government to borrow. That wasn't irresponsible borrowing. That was mechanical, predictable. Greece's poor historical discipline around government finances only accelerated an unavoidable process, but it's not a root cause.
In fact, predictably, pushing Greece into austerity made things worse, much worse: with the root cause being Greece's relatively weak economy compared to the rest of the Eurozone, an austerity approach can only put Greece in a position where it needs even more money to flow in in order to maintain balance.
Even if we assume that all of Greece's debts get somehow forgiven with no further constraints and that Greece manages to run a balanced government budget, it would still be in an unsustainable position in the current Eurozone as its weaker economy would force additional money to flow in. Unless the Eurozone very significantly increases the amount of money that it redistributes across borders, Greece should get out of the Euro at the first opportunity, i.e. literally Monday morning, July 6.
Worse, with Greece out, it's only a matter of time for another weak country to find itself in the same position: that might be Portugal, Spain, Italy, or if Bulgaria, Romania or even Hungary join quickly enough that might go through that same death spiral quickly enough to see the Eurozone as a revolving door, with barely enough time to come in before being back out.
Once that first batch of weak countries is out, there'll always be more that'll be at the bottom of the scale and will find themselves in the same position. France is comfortably in the middle of the pack within Europe today, but attrition will eventually push it toward the bottom, and France having to leave the Euro is a true nightmare scenario for everyone.
In order for the Eurozone to survive, its rich members will need to send a lot more money to the poorer ones: the rich ones literally can't continue reaping benefits from a currency based on the European average without sharing those benefits with the poorer ones that bring that European average down. Otherwise, the Euro will consume country after country until it hits a country that is literally too big to fail.


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Jets bringing back Burmistrov, Pardy and Halischuk
Alex Burmistrov is officially back with the Winnipeg Jets.
Alex Burmistrov is officially back with the Winnipeg Jets.
One small stride for Connor McDavid, one giant leap for the Edmonton Oilers.
The biggest deal of the day had nothing to do with anything that happened on the NHL's free agent market.
The Edmonton Oilers have signed Mark Letestu and Andrej Sekera, according to TSN.
Until Brendan Shanahan removed a prominent nameplate from the depressing Maple Leafs dressing room, his promise to change team culture for incoming coach Mike Babcock was ringing hollow.
The hot dog vendor who parks daily at Front and John Sts. just lost his most reliable customer.
The best free agent acquisition of 2015: Peter Chiarelli.
The Winnipeg Jets' season was ended by the Anaheim Ducks. For Michael Frolik, that meant tuning into the Stanley Cup playoff series between the Ducks and Calgary Flames.
The Senators quickly found out they erred last summer by signing veteran David Legwand just in case Jean-Gabriel Pageau wasn't quite ready for prime time.
It's not often a 28th-place team that spent the better part of a decade with a tin cup in its hand waiting for draft lottery handouts is considered the place to be in the NHL, but Edmonton is heading in that direction.


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Specialty practice has eye on vision therapy
Some children suffer from a disconnection between the brain and the eyes.
Dan Fortenbacher changed the name of his optometry practice in 2000 because he wanted it to reflect how his patients felt after receiving treatment. Read More
Summit encourages West Michigan to rethink dementia
National thought leaders gather in GR to discuss innovative programming.
More than 120 community members and leaders from health care, business, philanthropy, education and research gathered at a day-long inaugural summit recently to envision and foster a collaborative and dementia-friendly culture in West Michigan. Read More
Children's Healing Center provides germ-free space
First-of-its-kind concept is in the midst of a capital campaign and hopes to open in September.
West Michigan will welcome a new addition to the field of health care this fall when the first recreational center for children with weak immune systems opens its doors. Read More
The welfare queen myth
David Graham recently wrote for The Atlantic in "The True Face of Medicare Fraud" (June 19, 2015) that the people most likely to bilk the system are doctors and medical providers, not "welfare queens." Read More
First & Main brings small town vibe to senior communities
The assisted living and memory care complex in Wyoming is the first of many that are planned.
An assisted living and memory care community anticipated to open this fall will not only provide clinical care, but also main street amenities and technology. Read More
Research center seen as catalyst
Economic impact will create ripple effect lasting for years.
As the second research center in the downtown Grand Rapids area, the impact of Michigan State University College of Human Medicine's new facility could create a ripple effect of economic activity. Read More
Enriching older adult lives through technology
Programs aim to help seniors interact, communicate and learn.
Organizations serving older adults are leveraging technology and innovative programs to change how people age and provide life-enriching experiences to seniors. Read More
Provider reduces burden of family caregiving
Studies indicate caregivers are suffering from lost productivity and wages.
As the economic impact of unpaid family caregivers increases in the United States, an in-home provider serving the Greater Grand Rapids area is looking to reduce the burden and stress of providing care to family members. Read More
Primary care practice joins health system
A health system is expanding its presence in the region by opening a new primary care practice with a team of doctors moving from another system. Read More
Health system breaks ground on $24.5M project
A health system has broken ground on a $24.5-million project to expand and renovate a local center. Read More


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32 egypt 31 hutect albadawy ahmed 2010 30 shots 29 architecture cairo 27 islamic 24 05 21 مسجد 20 masjid 16 28 13 السلطان 12 sultan ومدرسة 11 alhambrapalace 10 hassan حسن spain 7 granada mamluks mamluk the 6 badawy ibn 5 tulunids قصرالحمراء أحمد بن 04 pattern tulun طولون 4 08 hutectshotsahmedalbadawy al 3 citadel 29 الرفاعي hutectshots 14 elmansoura eldin h5alhambrapalaceقصرالحمراءgranadaspain river قلعة صلاح الأيوبي ahmedalbadawyandalusiaandalucia 16 قصرالحمراءgranada hutectshotsahmedbadawyislamicarchitecture nile محمد ahmedalbadawyandalusiaislamicarchitecturehutectshots ومسجد الدين rifai salah علي minaret wall emir muhammad مدرسة madrasa ali mosquemadrasa قايتباي sarghatmish 17 with mosque dome الأمير street madrassa qalawun le elmuiz قلاوون hutectshotsahmedalbadawyislamicarchitectureandalusia سرغتمش one din allah
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Ahmed Al.Badawy 50
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text: How Should Sexual Offenders Be Punished? Dodai Stewart and 47 othersEditDelete Invite manuallyPromoteXDismissXUndismissBlock for jezebelHide Yesterday, a rapist in Florida voluntarily underwent surgical castration to avoid a sentence of life in prison, reports ABC News. Bobby James Allen, 35, received 25 years in prison with 10 years probation, and requested castration in exchange for the reduced sentence. » 9/21/07 3:30pm 9/21/07 3:30pm


text: My family and I have driven past the Red Rabbit countless times and finally decided to stop for lunch. It is a drive-in style experience where they hang the try from your window. The waitress greeted us quickly and told us how to order. We had the Bunny Burger which was good and the French fries... read more


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Web / UI / UX & Graphic Designer
Slovakia - Last active: 4 days ago - Tests: 2 - Portfolio: 11
I'm a passionate user experience and graphic designer who helps individuals and companies take their ideas and projects into beautiful visual prototypes ready to be pushed to development. I'm not a blind designer who cares only about the visual aspect, but rather tries to get the designs right for the audience the business is targeting, and create products which are usable and help the customers. The projects I like working on the most are mobile applications and complex web based applications as well as company/personal websites. On such projects, I can help you with designing the customer flow, user experience and the user interface or other related graphics for the brand. Plus, I can advise on your online presence and how to improve conversions based on multiple years of running my own online business in Internet Marketing niche selling digital and information products. Things I've designed before: - Brochures - Box/Book/PDF covers - Websites / Website themes (WordPress) - Mobile apps (iOS and Android) - Sales pages (for iPhone app, information product launch...) - Web based applications from scratch - Brand identities - UI - user interfaces used in websites/applications The software I use during projects: - Adobe Photoshop CC - Adobe Illustrator CC - Adobe InDesign CC - InVision, Skype... I'd be more than happy to help your business grow and work on some interesting and serious projects.
Freelancer Creative: Art direction & Design
Italy - Last active: 2 days ago - Tests: 4 - Portfolio: 41
Branding / Editorial / Promotion / Advertising Motivated and curious Creative with 12+ years' experience in Design & Advertising. I am hardworking and a fast learner with good communication skills and a strong design background. Graduated in Advertising in Sao Paulo (Brazil) I also hold a Master on Graphic Design by the London College of Communication, part of the acclaimed University of the Arts London, graduating with Merit honours with a design research about the use of graphic signifiers to sell tradition into product labels. My most recently works include the launching of Clinique's concept store in Brazil, the book design for the acclaimed author Mark Bittman who writes the weekly column "The Minimalist" for the New York Times, MAC Cosmetics catalog, AOC campaign and 22 book covers launched on 2012/2013. Beyond those clients I have also worked to Novartis, GlaxoSmithKline, Estee Lauder, SmashBox Los Angeles, Haagen Dazs, Danone, Pillsbury, Warner Bros, HTH and many other brands.
Creative design and graphic strategies
Mexico - Last active: 1 day ago - Tests: 8 - Portfolio: 10
http://be.net/fergarza Print design, web concepts and wireframing, corporate identity, color theory, photography and stationery products + accessories design for retail, plus digital ad design and its development. Ample experience in building web layouts, with full development of all graphic and visual details. First-rate quality, a fresh perspective and a ROI mentality give me the push to bring the desired result for complete client satisfaction.
Branding Expert / Graphic design and other creative solutions.
Croatia - Last active: 1 day ago - Tests: 4 - Portfolio: 95
I am a visual identity creator and overall graphic/web designer with over 12 years of experience. I have created a wide range of visual identities, logotypes, graphic & web design projects including graphic design for startup companies and small businesses across the globe. I can deliver professional designs in a timely manner with a very keen eye to detail and excellent project management skills. My main advantage lies in the fact that I enjoy my job. I have enormous passion for my work and I give 100% to every project, till the client and I are both satisfied with the final version. Looking forward to work with you and to contribute to your success.
- Adobe Photoshop
- Adobe Illustrator
- Adobe InDesign
- Adobe Acrobat
- Adobe FreeHand
- Photo Editing
- Photograph Color Correction
- Photography
- Logo Design
- Web design
- Online Community Management
- Facebook Marketing
- Print design
- Print Layout Design
- VectorWorks
- Typography
- Poster Design
- Flyer Design
- Adobe Creative Suite
- Book Cover Design
- Desktop Publishing
Book Cover Designer, for Create Space and Kindle, Graphic Design
Serbia - Last active: 1 day ago - Tests: 5 - Portfolio: 112
Hello, I am Nada. In the last 9 years, I have worked both for advertising agencies and as a freelancer. I have designed a wide range of book covers, magazines, logos, catalogs, posters and Illustrations. I earned my Master Degree in Graphic Design 2005. My postgraduate studies were oriented toward designing book cover, layout interioir, typography and illustration.
Graphic Designer, Photographer & Marketing Consultant
United States - Last active: 1 day ago - Tests: 4 - Portfolio: 1
Portfolio : http://www.astarkdesign.com/ Design - Any print or web job, big or small is welcome. I have a passion for publications and have had experience in many areas of design. If your project requires a new area to explore, I will excitably research new solutions and work with you to come up with the best design possible. Photography - Well experienced in newborn photography. Also experienced in head shots, events, and architecture. Aside from for-hire photo shoots, my photography in my portfolio can also be purchased for print or for use on marketing materials. Marketing - My marketing process begins by evaluating your current marketing tactics, if any, and identifying the opportunities for optimization. From there, I create an outline of your top competitors and what my recommended steps are that can be taken to differentiate your brand and set you above the rest. Other marketing services include: Branding, User Interface Optimization, Consulting Services, Conversion Optimization, and Advertising. About - My name is Alexandra. I am a well rounded artist, living in the 'burbs of Chicago. My main focus over the years has been graphic design and photography, as well as a side of marketing. Every aspect of a creative field appeals to me, though my creative side has always been balanced out by my curiosity of the business world. And that's how I'd like to think I ended up where I am. I love to create beautiful but functional pieces of art. Whether that function is to be hung on a wall or to drive customers to a website and connect them with a brand and product. I love it all. Philosophy - The world is full of visual messages and lately the media has been on a mission to deliver a sensory overload. I feel it is my mission as an artist to create visually appealing work to give back a little to the world around me. Design, photography and marketing are not simply putting words and imagery together, nor are they a way to shout information at a consumer until they listen. They are a way of expressing information that is both useful and appealing to a viewer and the sender. When someone comes across information that visually captures their attention amongst the noise of every day life, that information is retained. And in my book, that is a successful piece of art. Credentials - BFA – Graphic Design & Photography – Elmhurst College BA Communication Studies – Elmhurst College Freelancing for 8 years 3 Years Full Time Agency Designer 1 Year Professional Event Photographer and Column Author 1 Year Professional Newborn Photographer for Area Hospitals 4 Years Gallery Assistant 4 Years Student Publication Designer, 2 Years Art Director
Logos, corporate identity. UI/UX Design. Mobile and web.
Ukraine - Last active: 8 days ago - Tests: 4 - Portfolio: 37
Some designers tend to possess a strong belief that they are artists and their creative ideas are not subject to criticism. It is a mistake to take design craft for an art or consider whatever is done valuable just because it has been forced out. It can be called "creativity". I don't think so. The designer's task is doing a job that will earn the customer an income. The point is getting the people the customer works with to understand what was done for them. Everything that designers should make is to help their client's goods and skills be attractive, interesting and well sold. I'm not the exception. So let's try to create chances for your good sales together.
Professional Illustrator, concept artist and designer
United States - Last active: 7 days ago - Tests: 2 - Portfolio: 16
Andrew Gaia has been working in design and illustration for over ten years. He spent seven years working in the design/marketing department in the medical field before pursuing his MFA in illustration. His goal was always to produce art that would inspire and motivate other to see the world in a new way. Andrew has a deep love for fantasy and imaginative works as well as traditional subjects found throughout history. Andrew has worked as a freelancer for personal commissions, facebook applications and editorial projects. You can find a collection of Andrews work at www.andrewgaia.com. If their are any question about him or his work feel free to contact him as he would be delighted to hear from you.
Illustrator / Animator / Graphic Artist
Canada - Last active: 8 days ago - Tests: 2 - Portfolio: 1
"Every artist dips his brush in his own soul, and paints his own nature into his pictures." Henry Ward Beecher 1813 My creations are the reflection of a delicate and precarious balance between traditional and modern, orthodoxy and new technologies. Mostly based on illustrative and time-based techniques, it is the result of a whirl of different methods that converge in the attempt to represent rhythm and timing. My work is trying to capture and embed life in an elapsed fraction of time into a representtion that baulks at the confinement of the stationary nature of the canvas .
Graphic Design, Books, eReports, Web Graphicss
United States - Last active: 13 days ago - Tests: 5 - Portfolio: 4
I have 20+ years of experience in all aspects of creative graphic design, and specialize in distinctive book design and production--especially Print-On-Demand books with CreateSpace and IngramSpark. I also design e-Books, eBook covers, and eReports--which are ideal for lead generation. I strive for perfection, and work with integrity and efficiency to help my clients make their vision come to life—communicating every step of the way. Please take a look at my portfolio.


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Legacy post new track
Cool song
They went from sounding like a really forgettable rise clone to just forgettable. At least their guitarist ended up in blessthefall.
Not bad.
I really hope the next song they put out is much better than this one
Oh cool, another Rise band. Artery Recordings doesn't have any good bands at all other than maybe Vanna.
MAYBE Vanna....
Yeah I'm not a huge fan of that type of music bro, Vanna is cool I guess but it's whatever really.
Carnifex go listen to this band EP its pretty damn good i got it back in feburary or something and I still keep spinning it
I'll give it a go.
Yeah we want some more generic metalcore. lol
Just listened to the ep, did we listen to a different ep or something because all I heard was the same song posted here 3 times over.
Digging: Thaw - Earth Ground
I guess we did
• Brand New release new song as a f..• Tool + Mastodon supergroup• Slint guitarist survives suicide ..• Muse unleash new single• Scarlett Johansson forms band• Nickelback get groovy • New Muse song• Steven Wilson premieres new tune• B.B. King Dead• Brand New premiere new song
• The Getaway Plan LP Stream• Lamb of God unveil new video• Atlas Genius drop new track• Soulfly sell their souls to metal• Myrkur releases new video• Geoff Tate resurfaces• 808s & Dark Grapes III stream• Khost stream two new albums• Dan Tompkins leaves Skyharbor• The Wonder Years detail new LP• Cradle debut third new song• Shinedown debut new video• Eliwagar stream new LP• Polyenso reveal album title• Chris Squire passes away» see all news
Bands: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z


text: We went on a very busy evening and had to sit at the bar right near the door and the cold air coming in. It did not dampen our experience one little bit. (The staff were always right there to close the door when someone came in.) In fact, sitting at the bar was a plus, because the woman taking care of us was exceptional. She clearly loved her job, the restaurant and the food and her enthusiasm was contagious. But it was easy to be enthusiastic because the food was just wonderful. The wild boar ragu with gnocchi was, in our waitress' words, "insane". Every bite was delightful. The chard stem gratinata is one of those dishes for which the whole is way more than the sum of it's parts. Incredible. I loved that we could purchase "wine flights", 3 (almost) half glasses, thereby experiencing different Italian wines with our meal. We were too full for dessert, but we were reassured we could come for coffee and dessert at any time. I just loved this place (so did my husband.)


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Select Local Merchants
Segway of RichmondRead MoreThe streets and buildings of Richmond breathe with history. Patrick Henry's famous words, "Give me liberty or give me death," echo in St. John's Church, where the statesman gave his infamous speech; the ghost of Poe wanders the streets of the Church Hill district, where he lived and wrote; and the bones of presidents James Monroe and John Tyler lie peacefully under the lush grass of the Hollywood Cemetery, a National Historic Landmark. These are just a few of the locations that segway riders glide through during educational tours organized by Segway of Richmond, which has been an authorized dealer and tour operator in Richmond, VA since 2008 and has served 43,000 happy customers during that time. The company's most popular jaunt, the two-hour Landmark Tour, takes groups to nearly 20 historic buildings, churches, and museums, whereas other ventures focus on specific aspects of the city's past, such as the Black History Tour or the Edgar Allan Poe Tour. In addition to learning about the men and women who shaped the culture of Richmond and practicing their preferred mode of transportation, tour-goers can explore the architecture of Victorian and Edwardian homes during a Fan District Tour or think about murals, mosaics, and sculptures during a Public Art Tour.1301 East Cary StreetRichmond, VA US
A P C O A Standard ParkingRead MoreRiverfront Canal Cruises traces Richmond's roots back to George Washington, who first lobbied the Virginia General Assembly to create the city's canal system as a means of linking the coast to trade opportunities in the west. In 40-minute tours, covered boats glide down the James River and Kanawha Canal, granting passengers an up-close view of the waterways without having to mount a webcam on a duck. Throughout the trip, guides share old stories about the canal and the Canal Walk, broadening guests' knowledge of the area's rich heritage.501 N 7th StRichmond, VA US
Rail and Public TransportationRead MoreLike cuss words in Mandarin, flowers can convey more meanings than can possibly be documented here, including "Happy Holidays," "Thank You For Letting Me Be Myself Again," and "I Told You Krakow Was Once Poland's Capital." Today's deal lets you formulate a floral communique to anyone in the country, and help people who need it the most in the process. For $30, you get $50 worth of plant and floral arrangements from Touched by Flowers. TBF will give a percentage of the proceeds to a charity of your choice from more than 30 different nonprofit organizations. Your Groupon also covers the $9.95 service and transmission fee, for a $60 total value. Shipping is included.Follow @Groupon_Says on Twitter.600 E Main StRichmond, VA US
Harlem GlobetrottersRead MoreIn their 85th season, the Harlem Globetrotters have entertained millions of parents, children, and general basketball admirers with a unique brand of athletic precision and showmanship. For their latest 4 Times the Fun North American tour, the Globetrotters will add new 4-point shot spots located 35 feet from the basket, which is 12 feet farther than the official 3-point line but several thousand miles closer than the prime meridian.601 E Leigh St.Richmond, VA US
Leete Tire and Auto CenterRead MoreSince 1942, Leete Tire & Auto Center, Inc. has been fitting autos with new tires by brands including Michelin, BFGoodrich, Uniroyal, Continental, Toyo, and Goodyear. Though the family-owned tire shop's been around for well over half a century, it stays up-to-date by equipping its 10-bay garage with modern technologies, such as the Space Needle, which sews up cars or the moon after meteor collisions. In 1995, Leete Tire & Auto Center spawned another location in Richmond that features six bays capable of servicing passenger vehicles as well as light trucks. As NAPA AutoCare centers, both tire shops also enlist ASE-certified technicians trained to keep rides healthy. These skilled pros render maintenance services including state inspections, computerized alignments, and brake inspections.12 S 2nd StRichmond, VA US


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Press release from PR Newswire
Neogen Corporation Announces 2nd Quarter Results Conference Call
Wednesday, December 21, 2011
Neogen Corporation Announces 2nd Quarter Results Conference Call10:43 EST Wednesday, December 21, 2011LANSING, Mich., Dec. 21, 2011 /PRNewswire/ -- Neogen Corporation (NASDAQ: NEOG) announces the following Webcast:What: Neogen Corporation 2nd Quarter Results WebcastWhen: Thursday, December 22, 2011 @ 11:00 a.m. ETWhere: http://www.videonewswire.com/event.asp?id=84094 How: Live over the Internet -- Simply log on to the web at the address above. Contact: Terry Maynard of Neogen Corporation, +1-800-234-5333.If you are unable to participate during the live webcast, the call will be archived on the Web site www.neogen.com.Neogen Corporation develops and markets products and services dedicated to food and animal safety. The company's Food Safety Division markets dehydrated culture media, and diagnostic test kits to detect foodborne bacteria, natural toxins, genetic modifications, food allergens, unique proteins, drug residues, plant diseases and sanitation concerns. Neogen's Animal Safety Division markets a complete line of diagnostics, veterinary instruments, veterinary pharmaceuticals, nutritional supplements, disinfectants, and rodenticides.SOURCE Neogen Corporation


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Commercial Services and Supplies
Company Overview of AlphaGraphics, Inc.
Executive Profile
Kevin K. Cushing
Chief Executive Officer, AlphaGraphics, Inc.
|Total Calculated Compensation
|This person is connected to 0 Board Members in 0 different organizations across 2 different industries.
Mr. Kevin K. Cushing served as Chief Executive Officer of AlphaGraphics, Inc. at Pindar PLC and was responsible for driving overall business operations to pursue opportunities and increase global business. Mr. Cushing has more than 25 years of experience in operations, finance, sales and marketing, real estate development and managing large and small organizations. Prior to joining AlphaGraphics, Inc., Mr. Cushing created Progressive Partners in Minneapolis, MN, with ...
Corporate Headquarters
215 South State StreetPhone: 801-595-7270
Salt Lake City, Utah 84111
United States
Fax: 801-595-7271
Board Members Memberships
Former Chief Executive officer of Alphagraphics and Director
The University of Chicago
Saint Mary's University of Minnesota
Other Affiliations
Annual CompensationThere is no Annual Compensation data available.
Stocks OptionsThere is no Stock Options data available.
Total CompensationThere is no Total Compensation data available.
|There is no Competitor Compensation data available.
Sponsored Financial Commentaries
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To contact AlphaGraphics, Inc., please visit . Company data is provided by Capital IQ. Please use this form to report any data issues.
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Several customers will showcase solutions
AMD and Sapphire, which is the exclusive global distribution partner for AMD FirePro graphics, will be joining up with several customers in order to demonstrate a range of hardware and software solutions based on AMD FirePro graphics during Hannover Messe Industrial Show (HMI).
Do stop mucking about
Several key Alteria shareholders have forced the company back to the negotiating table to discuss the merger with Intel.
One cable to rule them, one cable to bind them
It seems that everyone is wanting to copy Apple in putting a USB Type-C port onto their hardware.
Planning a spoiler
AMD is expected to show off its latest Radeon card at Computex, but the dark satanic rumour mill suggests that Nvidia has a GeForce GTX 980 Ti spoiler up its sleeve.
12 inch wafers
Powerchip is to set up a joint-venture fab in the capital city of China's Anhui Province with the Hefei city government the big idea is to build 12-inch wafers and provide foundry services mainly for driver ICs.
Sharp on the cutting edge
Sharp announced that it's going to manufacture the world's sharpest display.
Game-ready driver for GTA V
Nvidia has released its latest WHQL-certified Geforce driver, which is also a Game Ready driver for the highly anticipated PC version of Rockstar's Grand Theft Auto V game.
Thanks but no thanks
Qualcomm has issued a statement in response to an idea floated by Jana Partners a couple of days ago.
No Android Wear, all about fitness and health
Asus has launched a smartwatch designed for fitness buffs, but it might appeal to the average geek due to its great battery life.
The price of being China's Apple
Chinese smartphone giant Xiaomi is complaining about the ever-growing number of faked products.


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July 2, 2015 | Glenn Johnson and Robert Morales
June 29, 2015 | Robert Rusu
June 25, 2015 | Jim Tressel
June 24, 2015 | Ray "Boom Boom" Mancini
June 17, 2015 | Mayor McNally
June 13, 2013 | Download
Rev. Lewis Macklin visits the newsroom today for a special Father's Day edition of Vindy Radio.
The show will air from 8 a.m. to noon live from the Vindicator. Macklin will be on at 10 a.m. with host Louie Free and Vindy Editor Todd Franko.
Former Ohio Democratic Party chairman and state Senate president Harry Meshel of Youngstown will be live on air at 9 a.m.
Also, Youngstown State University head football coach Eric Wolford will visit at 11:20 a.m.
Tune in to our Father's Day themed show.
Use the comment form below to begin a discussion about this content.


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Poll: Who shoulders most of the blame for the shutdown of the federal government?
The House 41%
The Senate 4%
Both chambers 8%
President Obama 27%
There's enough blame to go around 20%
Votes Cast: 1296
This survey is not a scientific sampling, but offers a quick view of what readers are thinking
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Studying abroad should not only be about what you can learn in the classroom, but what you can learn about the people through culture and conversation. Granada provided this opportunity through home stays and so many other cultural excursions.
Go with the best
Central is affiliated with some of the finest schools in the world. Through our study abroad programs, you, too, can experience the best. A little about our schools:


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Storage Containers & Baskets
- Addison Wall System Rattan Baskets Sugg. Price: $59.95 – $74.95 Our Price: $47.96 – $59.96
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Professional Resources
Advance your career and enhance your opportunities
How to Achieve Classroom-family Balance—Guilt-free
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A story about Marlena in Redding
The folks at Blendtec are a blessing to me, and to lots of others, too. They give me a $435 blender to give to one lucky attendee at each of my classes. Once, to an audience of 400 in Dallas, I said: "This amazing machine is a game-changer for your health. I really wish I […]4 Comments • Continue Reading →
More…..about scars on the human body
Nikki wrote me about my last blog post. She said she was pondering the scar conversation we had, and about how scars point to some other things: She said, isn't it amazing how the autonomic nervous system does things like breathing, creating white blood cells to fight infection, starting self-healing processes immediately upon being wounded. […]1 Comment • Continue Reading →
And what to do about SCARS?
On the long drive home from Tijuana to Utah earlier this month, I stopped at a McD's because they have the cleanest bathrooms. And, well, because I'd drunk the 32 oz green juice I swerved off the freeway to get, at the Henderson Whole Foods Market. Inside the stall, a pink Post-It Note was taped […]4 Comments • Continue Reading →
thoughts on service
I don't know about you-all, but I have a lot of sick people in my life right now. A couple of my tennis-club girlfriends are having serious issues affecting their ability to play–these being women in their 30's and 40's–like ovarian cancer and blurry vision suspected to be a brain tumor. Another of my good […]13 Comments • Continue Reading →


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Aereo Presses the Pause Button
World Cup fans must find alternatives to watch noon match
By Lisa Granatstein on Jun. 28, 2014 - 11:01 AM EDT
With the Supreme Court on Wednesday effectively putting the kibosh on Aereo's streaming service, CEO Chet Kanojia announced that at 11:30 a.m. today Aereo would "pause operations temporarily" until it…
Kevin Byrnes, VP of Ad Sales, on the Launch of Newsmax
Fledgling news net taps Golf Channel vet
By Sam Thielman on Jun. 5, 2014 - 11:08 AM EDT
Newsmax, a conservative news site that specializes in media watchdogging, is turning into a full-blown cable network—or rather, a satellite network. The new net launches June 16 in DirecTV's 20…
DirecTV Turns to Fans for 'Cable Effects' Ad
Satellite TV provider asks public for their stories
By Michelle Castillo on Apr. 14, 2014 - 9:00 AM EDT
Fed up with cable? DirecTV wants to hear your frustration. The satellite TV provider is asking fans for suggestions for its new "Cable Effects" storyline. A panel of judges will review…
Diller: Aereo Is 'Finished' If It Loses Supreme Court Battle
Should it prevail, the company will expand to top U.S. markets
By Anthony Crupi on Apr. 2, 2014 - 12:44 PM EDT
Should Aereo lose its impending U.S. Supreme Court case, backer Barry Diller believes it'll be game over for the upstart distributor. Speaking to anchor Erik Schatzker during an appearance on Bloomberg…
Dish-Disney Carriage Deal Disables the Hopper
Many concessions in long-term distribution pact
By Anthony Crupi on Mar. 4, 2014 - 12:08 PM EST
Six months after the expiration of their joint carriage deal, the Walt Disney Co. and Dish Network have agreed to a long-term pact that will not only keep ABC, ESPN…
DirecTV Keeps Plugging Away at NFL Sunday Ticket Renewal
Satellite TV giant looks to retain exclusive rights to package
By Anthony Crupi on Feb. 20, 2014 - 7:21 PM EST
DirecTV continues to plug away at a deal that would allow it to retain the exclusive rights to the NFL Sunday Ticket package, but the satellite-TV giant said it has…
The Number of Blacked-Out NFL Games Dropped From 16 Last Season to Just 2
Threat of FCC, legislative action seem to be working
By Katy Bachman on Jan. 27, 2014 - 9:37 PM EST
Fans and policy advocates fighting to end the NFL's sports blackout rule may have already won—even if the rule is still on the books. The number of blacked-out football games went…
Ad of the Day: Life Gets Even Worse for Cable Customers in DirecTV's New Horror Stories
Campaign's third year
By David Griner on Jan. 8, 2014 - 1:20 PM EST
It's been two years since DirecTV and Grey New York launched their "Cable Effects" ad campaign, detailing the strange and terrible fates that can befall cable customers. In that time,…
FTC Approves Nielsen Acquisition of Arbitron
Deal subject to conditions
By Anthony Crupi on Sep. 20, 2013 - 7:04 PM EDT
The Federal Trade Commission on Friday gave Nielsen Holdings the necessary clearance to acquire Arbitron. Subject to standard closing conditions, the deal is now expected to close on Sept. 30. Per…
Editor's Letter: Heat Seekers
Adweek's Hot List continues to evolve and expand to reflect the industry
By James Cooper on Sep. 1, 2013 - 10:13 PM EDT
Invariably, like the change of seasons or perhaps a migration of birds south, the calls start coming in May: "Have you started to work on the Hot List yet?" In covering…