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We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms), Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor "dyads." E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B+/− cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.
Citation: Mohler PJ, Davis JQ, Bennett V (2005) Ankyrin-B Coordinates the Na/K ATPase, Na/Ca Exchanger, and InsP3 Receptor in a Cardiac T-Tubule/SR Microdomain. PLoS Biol 3(12): e423. doi:10.1371/journal.pbio.0030423
Academic Editor: Ivor Benjamin, University of Utah Health Sciences Center, United States of America
Received: July 5, 2005; Accepted: October 12, 2005; Published: November 29, 2005
Copyright: © 2005 Mohler 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.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: 3D, three-dimensional; AEBSF, 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride; DHPR, dihydropyridine receptor; GFP, green fluorescent protein; InsP3R, inositol 1,4,5-trisphosphate receptor; NCX1, Na/Ca exchanger 1; NKA, Na/K ATPase; RyR, ryanodine receptor; SERCA2, sarcoplasmic reticulum Ca2+ ATPase; SR, sarcoplasmic reticulum; T-tubule, transverse tubule
Defects in Ca2+ homeostasis underlie major diseases of the heart including congestive heart failure, cardiac hypertrophy, and fatal cardiac arrhythmias [1,2]. Ca2+ ions enter cardiomyocytes through voltage-sensitive Ca2+ channels (dihydropyridine receptor [DHPR]) located in invaginations of the plasma membrane known as transverse tubules (T-tubules). DHPR is localized in a microdomain of the T-tubule that is synapsed with sites in the sarcoplasmic reticulum (SR) that are enriched in Ca2+-release channels (ryanodine receptor [RyR]; [3,4]). Ca2+ that enters through DHPR must be balanced in each contraction cycle (~100 ms in mouse) by Ca2+ export. Ca2+ export is accomplished primarily by the Na/Ca exchanger 1 (NCX1), which is driven by the transmembrane Na+ gradient provided by the Na/K ATPase (NKA) [5]. The requirement for rapid export of Ca2+ is a specialized feature of heart that is not present in skeletal muscle, where DHPR directly activates RyR without Ca2+ import.
Ca2+ export has historically been an important therapeutic target in the management of heart failure. Cardiac glycosides increase [Ca2+]i by inhibiting NKA, thus elevating [Na+]i and indirectly inhibiting Ca2+ efflux through NCX1 [6]. Several considerations suggest that NCX1 and NKA operate together in diffusion-limited physiological spaces. Cardiac glycosides do not elevate averaged cytoplasmic [Na+] to levels sufficient to inhibit NCX1, suggesting NKA and NCX are coupled [6,7]. Arrhythmia caused by cardiac glycoside toxicity is believed to result from a transient inward current carried by NCX1 [8]. However, NCX1 would not be expected to generate inward flux of Na+ if averaged [Na+] was elevated sufficiently to inhibit Ca+2 efflux. Numerous studies support a tight functional coupling between NCX1, NKA, and intracellular Ca2+ stores in heart [9–13]. However, while co-localization between NCX1, NKA, and intracellular Ca2+ stores is described in smooth muscle [14], the relative localizations of these proteins in heart are undefined. Additionally, no biochemical evidence exists for a direct link between NKA, NCX1, and other SR proteins in cardiomyocytes.
Ankyrin-B is a multivalent adapter present in cardiomyocytes that binds individually to NCX1, NKA, and inositol 1,4,5-trisphosphate receptors (InsP3Rs), and potentially could play a role in functional coupling of these proteins [15–17]. Loss-of-function mutations in ankyrin-B cause a dominantly inherited human cardiac arrhythmia syndrome associated with sudden cardiac death [15,18]. Mice heterozygous for a null mutation in ankyrin-B (ankyrin-B+/− mice) are haploinsufficient and have a similar cardiac phenotype as humans heterozygous for loss-of-function mutations of ankyrin-B [15,18]. Adult ankyrin-B+/− cardiomyocytes exhibit elevated [Ca2+]i transients and, in the presence of beta-catecholamines, exhibit delayed and early afterdepolarization events and extrasystoles [15]. In contrast, action potential duration, inward Ca2+ current, and diastolic [Ca2+] are normal in ankyrin-B+/− cardiomyocytes [15].
We report here that NKA (alpha 1 and alpha 2 isoforms), NCX1, and InsP3R are complexed with ankyrin-B within a microdomain of cardiomyocyte T-tubules, and that the complex is deficient in ankyrin-B+/− cardiomyocytes. Additionally, we present evidence that loss of the ankyrin-B-based complex is the molecular defect in cardiac arrhythmia due to ankyrin-B mutation in humans and mice. The ankyrin-B complex is not present in skeletal muscle, smooth muscle, or brain, and may have evolved in the context of specialized requirements for cytosolic Ca2+ regulation in cardiomyocytes.
Ankyrin-B Is Required for T-Tubule Localization of NKA, NCX1, and InsP3R in Cardiomyocytes
Ankyrin-B as well as NKA, NCX1, and InsP3R are selectively lost from Z-line/T-tubule sites in haploinsufficient ankyrin-B+/− cardiomyocytes [15]. The localization of these proteins was further resolved by three-dimensional rendering of consecutive confocal Z-sections of cardiomyocytes labeled by immunofluorescence (Figure 1). Wild-type ankyrin-B is organized in an intracellular tubular lattice in parallel with both the M-line and Z-line/T-tubules, but also including perpendicular axial branches that connect M-line and Z-line staining (Figure 1C). Z-line ankyrin-B staining is co-linear with the dyad marker DHPR in two-dimensional images, but is distinct from the DHPR in three dimensions (<2% of DHPR-positive voxels [three-dimensional (3D) pixels] overlap with ankyrin-B-positive voxels; not shown). Z-line ankyrin-B staining significantly overlaps in three dimensions with InsP3R (~45% of InsP3R-positive voxels co-localize with ankyrin-B-positive voxels; Figure 1D) as well as T-tubule-associated NCX1 (~53%; Figure 1E) and NKA (~51%; Figure 1F).
Boxes in differential interference contrast images (A and B) represent sites that were imaged in (C–F). Immunolocalization of ankyrin-B (red) and (C) DHPR (green), (D) InsP3R (green), (E) NCX1 (green), and (F) NKA (green) in wild-type (left) and ankyrin-B+/− cells (right). Ankyrin-B+/− myocytes were labeled and imaged using identical protocols. M-lines (middle of A-band) are denoted by arrowheads; T-tubules over I-bands are denoted by arrows. Scale bar = 2 μm. Distance between arrowheads is ~1.8 μm. Images represent at least twenty Z-sections at 0.18-μm intervals. In (C), perpendicular axial branches are denoted by white arrowheads.
In contrast to wild-type cardiomyocytes, ankyrin-B+/− cells lack Z-line staining as well as the axial lattice of ankyrin-B that connects Z-line- and M-line-associated populations (Figure 1C–1F, right panels). DHPR staining and organization is unaffected in ankyrin-B+/− cardiomyocytes. InsP3R immunofluorescence (Figure 1D), as well as NCX1 and NKA isoform staining (Figure 1E and 1F) are markedly reduced at T-tubule sites. Residual InsP3R, NKA, and NCX1 rarely co-localize with ankyrin-B (levels of InsP3R, NKA, and NCX1 are reduced to levels too low for accurate determination of overlap).
Ankyrin-B-Coupled NKA, NCX1, and InsP3R Are Co-Localized
The Na/Ca exchanger has been localized at sites on cardiac T-tubules distinct from DHPR and RyR [19]. Moreover, NKA isoforms, NCX1, and InsP3R have been individually localized at T-tubule sites in cardiomyocytes [15,19–21]. However, localizations of all three proteins relative to each other have not been addressed. The relative localization of NCX1 with NKA alpha 1 and alpha 2 isoforms, InsP3R, DHPR, and RyR was evaluated using multiple combinations of double-labeled 3D images of mouse cardiomyocytes generated using confocal Z-stacks (Figure 2; Table 1). As expected, DHPR and RyR are co-localized, with ~55% voxel overlap of DHPR with RyR and ~42% overlap of RyR with DHPR (Figure 2B; Table 1), values comparable to voxel overlaps observed previously [19]. T-tubule NCX1 immunofluorescence (also localized at the sarcolemma [21]) is distinct from DHPR staining, with a voxel overlap of less than 5%, as reported previously [19] (Figure 2C; Table 1).
Adult cardiomyocytes were labeled with indicated antibodies. 3D reconstructions of each cell (A, differential interference contrast) are shown for each combination. Staining pairs include (B) DHPR (red) and RyR (green), (C) DHPR (red) and NCX (green), (D) NCX (red) and NKA alpha1 (green), (E) NCX (red) and NKA alpha 2 (green), (F) NKA alpha 1 (red) and NKA alpha 2 (green), (G) InsP3R (red) and RyR (green), and (H) InsP3R (red) and NCX (green). Voxel co-localization was performed for non-sarcolemmal voxels. T-tubule/Z-lines are indicated by white arrows. Scale bar = 5 μm.
A new finding is that T-tubule NCX1 immunofluorescence co-localizes in submicron-sized domains with the T-tubule population of both NKA alpha 1 (Figure 2D; Table 1) and alpha 2 isoforms (Figure 2E; Table 1), with 55% (alpha 1) and 50% (alpha 2) voxel overlap. T-tubule NCX1 also co-localizes with InsP3R (Figure 2H; Table 1), with 56% of InsP3R voxels overlapping NCX1 and 50% of NCX1 voxels overlapping InsP3R. In contrast, InsP3R and RyR are not co-localized at T-tubule sites, with less than 5% voxel overlap (Figure 2G; Table 1). This is the first direct evidence to our knowledge that RyR and InsP3R are localized in spatially defined compartments of the endoplasmic reticulum and SR of ventricular cardiomyocytes.
NKA alpha 1 and alpha 2 isoforms were similarly distributed over the T-tubule (Figure 2F; Table 1) and sarcolemma (not shown). The T-tubule co-localization occurred in three dimensions as voxels with alpha 1 signal containing alpha 2 signal ~52% of the time, while voxels containing alpha 2 signal contained alpha 1 signal ~46% of the time. NKA isoforms have been proposed to have unique functions based on differences in localization and/or affinity for cardiac glycosides [22]. These differences may depend on the species and cell type, and have not been reported in mouse cardiomyocytes [23]. Our results in mouse ventricular cardiomyocytes suggest no major differences in NKA alpha 1 and alpha 2 localization at T-tubules (Figure 2) or sarcolemma (not shown). These results demonstrate co-clustering of NCX1, NKA, and InsP3R within microdomains along the T-tubule/SR that are distinct from classic T-tubule/SR junctions populated by DHPR and RyR. Moreover, the clusters of these proteins as well as ankyrin-B are reduced or absent in ankyrin-B+/− cardiomyocytes.
T-Tubule-Associated Ankyrin-B Is a Specialized Adaptation of Cardiomyocytes
The expression of 220-kDa ankyrin-B in skeletal muscle is nearly 10-fold lower than in heart (Figure 3A). Moreover, in contrast to ankyrin-B localization in cardiomyocytes (Figure 3B), ankyrin-B is not present over T-tubules of skeletal muscle, but instead is concentrated at punctate sites on the sarcolemma, over the A-band, and at costameres (Figure 3B). Additionally, in contrast to cardiac muscle, NCX1 and NKA isoforms are nearly undetectable over T-tubules of skeletal muscle, but are instead concentrated at the sarcolemma (Figure 3C). Finally, ankyrin-B expression is nearly absent from smooth muscle (Figure S1), and ankyrin-B-based complexes of NCX1, NKA isoforms, and InsP3R are not detectable in brain (see below). Therefore, the ankyrin-B-based complex of NKA, NCX1, and InsP3R is a specialized feature of cardiac myocytes.
(A) Relative expression of 220-kDa ankyrin-B in adult mouse heart and skeletal muscle (n = 3, p < 0.05). For the immunoblot in the left panel, 50 μg of total lysate was analyzed.
(B) Ankyrin-B (AnkB) immunostaining (red) in frozen sections of heart (H) and skeletal muscle (Sk Mus, SkM). The Z-lines (arrowheads, also asterisks in right skeletal muscle panel) overlap with alpha-actinin (green). Arrows indicate A-bands. Ankyrin-B is localized at costameres in skeletal muscle, visualized as staining continuous with alpha-actinin near the sarcolemma, but does not extend into the interior. Ankyrin-B localizes over the A-band in both heart and skeletal muscle. Scale bars = 5 μm.
(C) NCX1 (left panel), NKA alpha 1 (middle panel), and NKA alpha 2 immunostaining (right panel) in skeletal muscle. Scale bars = 18 μm.
Ankyrin-B Coordinates NKA, NCX1, and InsP3R in a Macromolecular Protein Complex in Cardiomyocytes
The finding that ankyrin-B is co-localized with NKA, NCX1, and InsP3R in cardiac T-tubule microdomains and that all of these proteins are coordinately reduced in ankyrin-B+/− cardiomyocytes raises the question of their molecular organization. Given previous evidence that ankyrin-R can form heterocomplexes between two ankyrin-binding proteins [24], we wondered whether ankyrin-B could form a multi-protein complex involving NKA, NCX1, and InsP3R in cardiomyocytes. We therefore performed a series of immunoprecipitations from detergent extracts of mouse heart with antibodies against NCX1, alpha 1 and alpha 2 isoforms of NKA, and InsP3R, followed by immunoblots to detect associated proteins (Figure 4). As reported previously, ankyrin-B antibody co-immunoprecipitated NKA isoforms, NCX1, and InsP3R, but not DHPR, SR Ca2+ ATPase (SERCA2), or calsequestrin (Figure 4A) [15]. NCX1 antibody co-immunoprecipitated 220-kDa ankyrin-B as well as NKA alpha 1 and alpha 2 isoforms and InsP3R; DHPR, SERCA2, and calsequestrin were not co-immunoprecipitated (Figure 4). Moreover, NKA alpha 1– and alpha 2–specific antibodies co-immunoprecipitated 220-kDa ankyrin-B as well as NCX1 and InsP3R, but, again, not DHPR, SERCA2, or calsequestrin (Figure 4A). Finally, antibody specific for InsP3R also co-immunoprecipitated 220-kDa ankyrin-B along with NKA alpha 1 and alpha 2 and NCX1, but not DHPR, SERCA2, or calsequestrin. DHPR-, SERCA2-, and calsequestrin-specific antibodies did not co-immunoprecipitate 220-kDa ankyrin-B, NCX1, InsP3R, or NKA isoforms. These mutual co-immunoprecipitations provide evidence for a macromolecular protein complex in heart containing ankyrin-B coupled to alpha 1 and alpha 2 isoforms of NKA, NCX1, and InsP3R. While other proteins may be in this protein complex, components of the classic T-tubule/SR junction (DHPR [also RyR; not shown]) as well as components of the SR (SERCA2 and calsequestrin) are not included.
(A) Immunoprecipitations and co-immunoprecipitations from detergent-soluble extracts from adult mouse heart.
(B and C) Detergent-soluble lysates from wild-type or ankyrin-B+/− mouse hearts were used for immunoprecipitations with indicated antibodies (IB, immunoblot; IP, immunoprecipitation). Immunoprecipitations of ankyrin-B+/− extracts employed doubled amounts of input lysate to compensate for 50% reduction of ankyrin-B.
(D) InsP3R co-immunoprecipitates 220-kDa ankyrin-B, NCX1, and NKA from detergent-soluble heart lysates (Input = 10%). In contrast, InsP3R co-immunoprecipitates 220-kDa ankyrin-B, but not NCX1 or NKA, from detergent-soluble lysates of mouse brain (Input = 10%).
ANKB, ankyrin-B; C. IG, control Ig.
We next asked whether ankyrin-B was required for mutual co-immunoprecipitation of NKA, NCX1, and InsP3R by comparing wild-type hearts and ankyrin-B+/− hearts, which are deficient in ankyrin-B (Figure 4B and 4C). Ankyrin-B+/− hearts express reduced levels of 220-kDa ankyrin-B (decreased ~50%), NKA alpha 1 and alpha 2 (both reduced ~15%), NCX1 (reduced ~16%), and InsP3R (reduced ~33%) [15]. Strikingly, ankyrin-B+/− heart lysates exhibited over 60% loss of the ability of ankyrin-B antibody to co-immunoprecipitate NKA, InsP3R, or NCX1, even when the quantity of lysate was doubled to equalize the starting amount of ankyrin-B (Figure 4B). Moreover, a similar reduction in NCX1 co-immunoprecipitation of Na/K pump isoforms and InsP3R occurred using doubled ankyrin-B+/− lysates (Figure 4C). NKA alpha 1 and alpha 2 antibody also failed to co-immunoprecipitate a significant fraction of NCX1 or InsP3R from ankyrin-B+/− doubled lysates. Finally, InsP3R antibody immunoprecipitated minimal levels of NCX1 or NKA isoforms from ankyrin-B+/− heart (Figure 4C). These results demonstrate that a specialized population of ankyrin-B, which is reduced in ankyrin-B+/− heart, is critical for ankyrin-B interactions with NKA alpha 1 and alpha 2, NCX1, and InsP3R.
Ankyrin-B, NKA, InsP3R, and NCX1 are all expressed in brain at levels comparable to those in heart tissue. However, while immunoprecipitation of 100,000g detergent extracts of brain tissue with antibody against the InsP3R co-immunoprecipitated ankyrin-B, NKA and NCX1 were not present (Figure 4D). Moreover, NKA and NCX1 were also missing when the immunoprecipitation was performed with antibody against ankyrin-B (not shown). These results are in contrast to the report that NCX1, NKA, InsP3R, and ankyrin-B co-immunoprecipitate along with several other proteins from 27,000g supernatants of detergent extracts from brain [25]. The difference could result from use of a 27,000g supernatant in the other study and a 100,000g supernatant in our experimental protocol. A major complication with a lower speed supernatant is the likely presence of large complexes such as those connected by short actin filaments that would be removed with more centrifugation. Our results demonstrate that co-expression of ankyrin-B with NCX1 and NKA in the same tissue is not sufficient for formation of a complex from a 100,000g supernatant.
Reconstitution of an Ankyrin-B-Based Complex of NKA, NCX1, and InsP3R
Co-immunoprecipitation experiments as presented in Figure 4 provide evidence for interactions between ankyrin-B and its partners in vivo. We next evaluated whether ankyrin-B could form a complex with the NKA, NCX1, and InsP3R in vitro using purified proteins (See Materials and Methods). We first confirmed, using 125I-labeled proteins and immobilized ankyrin-B membrane-binding domain, that purified ankyrin-B membrane-binding domain directly interacts in vitro with purified NCX1 expressed in Sf9 cells (Kd = 5 nM), purified NKA from kidney (Kd = 50 nM), and purified InsP3R from cerebellum (Kd = 3 nM) (Figure 5).
Saturation binding of purified (A) 125I-labeled NCX1, (B) 125I-labeled NKA (NKA), and (C) 125I-labeled InsP3R with glutathione Sepharose–immobilized GST-ankyrin-B membrane-binding domain or GST control Sepharose. Inset for each panel represents Scatchard analysis of ankyrin-B interactions following subtraction of nonspecific binding to GST-Sepharose. Proteins were isolated as described in Materials and Methods.
We next asked whether ankyrin-B could form a multivalent complex with these proteins. Association of InsP3R with NKA and NCX1 in the presence or absence of soluble ankyrin-B membrane-binding domain (purified as a GST-fusion protein and then cleaved from the GST-tag; Figure 6A) was assessed using biotinylated InsP3R bound to neutravidin-Dynabeads (Figure 6B) and 125I-labeled NCX1 and NKA. 125I-labeled NCX1 and 125I-labeled NKA associated with InsP3R-coated beads only in the presence of ankyrin-B membrane-binding domain (Figure 6C). In fact, while the intensity of the NCX1 band is ~50% of the band intensity of NKA (not shown), the picomoles of each protein bound to the InsP3R-coated beads was approximately equal (Figure 6C; specific activity of 125I-NKA ~504,000 cpm; 125I-NCX1 ~270,000 cpm). In contrast, 125I-labeled NCX1 and 125I-labeled NKA failed to bind to InsP3R-coated beads in the absence of ankyrin-B (Figure 6C). Additionally, in the presence of ankyrin-B, InsP3R-coated beads simultaneously associated with both 125I-labeled NCX1 and 125I-labeled NKA with no decrease in binding capacity compared to reactions where only one labeled protein was used (Figure 6C). These results demonstrate that interaction of InsP3R with either NCX1 or NKA is ankyrin-B-dependent and that these proteins can assemble in vitro in the absence of additional co-factors or regulatory proteins.
(A) Purified NKA (α/β subunits), InsP3R, NCX1, and ankyrin-B membrane-binding domain (ANKB MBD) were examined by SDS-PAGE and Coomassie blue.
(B) Stained gel of control beads and beads plus purified InsP3R.
(C) NKA and NCX1 were 125I-labeled and incubated with control Dynabeads or Dynabeads coated with InsP3R with or without ankyrin-B membrane-binding domain. Bound protein was analyzed by a gamma-counter (C) and by SDS-PAGE and phosphorimaging (not shown; see Materials and Methods for protein measurement, n = 3, p < 0.05).
Human E1425G Mutation Abolishes Ankyrin-B Association with NCX1, NKA, and InsP3R
One test of the physiological importance of the ankyrin-B-based complex is whether mutations in ankyrin-B resulting in loss of the complex also cause arrhythmia. E1425G mutation of ankyrin-B causes human cardiac arrhythmia and loss of activity of ankyrin-B in restoring normal Ca2+ waves to ankyrin-B+/− neonatal cardiomyocytes [15]. The mechanism for loss of function due to the E1425G mutation, which is located close to the C-terminal regulatory domain and distant from the membrane-binding domain (Figure 7A), is not known. However, the regulatory domains of ankyrins (Figure 7A) can modulate activities of N-terminal membrane- and spectrin-binding domains [26,27].
(A) 220-kDa ankyrin-B domain organization. The human LQT4 E1425G mutation is marked.
(B) Immunoblot of expressed wild-type and mutant GFP-ankyrin-B proteins.
(C–E) Relative binding of rat heart lysate (C) InsP3R, (D) NCX1, and (E) NKA to wild-type and mutant GFP-ankyrin-B proteins. Bound NCX1, NKA, and InsP3R were evaluated following quantitative immunoblot (pan-InsP3R, pan-NKA, and NCX1 Ig) and phosphorimaging (n = 3, p < 0.05).
The effect of the E1425G mutation on the ability of ankyrin-B to bind to NKA, NCX1, and InsP3R was evaluated using detergent extracts of heart tissue (not shown) and using purified proteins isolated as in Figure 6A. Evaluation of the binding properties of the E1425G mutant protein requires full-length 220-kDa ankyrin-B. We have not yet successfully generated full-length 220-kDa ankyrin-B in bacteria. Therefore, we used mammalian HEK293 cells to generate full-length wild-type and mutant ankyrin-B polypeptides for our binding studies. Recombinant green fluorescent protein (GFP)–220-kDa ankyrin-B that was either wild-type, with the E1425G mutation, or with a E1425D mutation was expressed and immuno-isolated from HEK293 cells using an affinity-purified antibody against GFP immobilized on Protein A agarose.
The levels of immobilized GFP-ankyrins were all equivalent in these assays (Figure 7B). E1425G ankyrin-B exhibited a 60%–70% loss of association with NCX1, NKA alpha 1 and alpha 2, and InsP3R from cardiac lysates (not shown), and as pure proteins (Figure 7C–7E). The conservative E1425D mutation at this site had no effect on binding of NCX1, NKA, or InsP3R (Figure 7C–7E). The finding that the E1425G mutation abolishes the ability of ankyrin-B to bind to NCX1, NKA, and InsP3R (Figure 7) suggests that interaction of ankyrin-B with either all three or some combination of these proteins is required for its function. It is possible that the E1425G mutation affects other protein interactions of ankyrin-B, although these remain to be identified.
This study presents the discovery of an ankyrin-B-based macromolecular complex of NKA (alpha 1 and alpha 2 isoforms), NCX1, and InsP3R in cardiomyocytes. The complex is localized in a microdomain along cardiomyocyte T-tubules resolved by 3D confocal microscopy as distinct from the classic dyad formed by DHPR and RyR. This microdomain was first described by Moore and colleagues, who also distinguished the T-tubule NCX1 from dyad proteins RyR and DHPR and from voltage-gated sodium channels by light microscopy using image deconvolution and wide-field epifluorescence microscopy [19]. T-tubule-associated ankyrin-B is a specialized adaptation of cardiomyocytes and is not evident in smooth muscle, which does not express significant ankyrin-B levels, or in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. The T-tubule domain containing ankyrin-B-coupled NKA, NCX1, and InsP3R thus is a specialized adaptation of cardiac cells that is not present in other types of muscle cells.
We propose a scale model for the ankyrin-B-based complex (Figure 8) based on previous structural reports and on evidence from this study that ankyrin-B can promote association between purified NKA, NCX1 and InsP3R (see Figure 6). In this scheme, the extended ankyrin-B membrane-binding domain adapts the NKA and NCX1 to the InsP3R in a configuration that would allow for regulation of cytosolic Ca2+ in a spatially privileged domain (Figure 8). It is likely that all participants in such an assembly have mutually interacting surfaces. In this case, the role of ankyrin-B could be to stabilize the assembly and/or possibly direct its cellular localization. The resulting macromolecular complex capable of coupled transport would accomplish the intended purpose of "restricted space" previously invoked to explain the action of cardiac glycosides [28]. However, the dimensions of a complex would be on the order of 10–20 nm, while an anatomical space or "synapse" between the endoplasmic reticulum and plasma membrane is 500–1,000 nm in size and would not provide a effective barrier to diffusion of small ions with radii less than 1 nm. A test of the idea of coupled transport by ankyrin-B-complexed proteins would be to selectively interfere with participation of individual members of the complex by knocking in mutants lacking ankyrin-B-binding activity.
(Left) Model of ankyrin-B-dependent complex of NKA, NCX1, and InsP3R at T-tubule/SR sites distinct from the classic "dyad."
(Right) Scale model of ankyrin-B complex based on approximate dimensions of represented proteins. The protein ratios are not representative for in vivo couplings as there are likely 50–100 RyR2 for each InsP3R in a ventricular cardiomyocyte [32].
While binding and localization data are consistent with simultaneous interaction of a single ankyrin-B molecule with NCX1, NKA, and InsP3 receptor, it also is possible that only one or two ankyrin-B-associated proteins are bound at a given time. It will be important in future experiments to isolate ankyrin-B-based macromolecular assemblies and directly determine stoichiometries of component proteins. A current challenge is that ankyrins also associate with spectrin and spectrin/actin complexes (reviewed in [17]), as well as proteins such as obscurin [29,30].
A role for InsP3R in heart is unknown. InsP3R-dependent Ca2+ signaling has been proposed to regulate excitation–contraction coupling in atrial myocytes by modulation of the activity (priming) of juxtaposed RyR [31]. However, based on the low ratio of InsP3R to RyR [32], the high Ca2+ buffering capacity of the cytosol [33], and now the distinct localizations of these Ca2+-release channels, it is unlikely that InsP3R Ca2+ release could affect the activity of RyR-mediated Ca2+-induced Ca2+ release in ventricular cardiomyocytes. Also, a role of InsP3R in Ca2+ signaling is difficult to reconcile with an environment where [Ca2+]i transients occur continuously [2]. Our model suggests a counterintuitive role for InsP3R as a "Ca2+ pressure valve" for export of excess SR Ca2+ from the cell (Figure 8). Consistent with this idea is experimental evidence for functional coupling of SR Ca2+ stores with Ca2+ efflux [9,11].
Loss of the ankyrin-B-based complex may provide an explanation for the cardiac arrhythmia syndrome due to ankyrin-B mutations in humans and mice. The E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components (NCX1, NKA, and InsP3R) of the complex (see Figure 7). Moreover, ankyrin-B+/− mice have a related cardiac arrhythmia, and ankyrin-B+/− cardiomyocytes are also deficient in the complex, while the expression and subcellular localization of other cardiac ion channels and transporters (e.g., Nav channels, which associate with a second ankyrin gene product, ankyrin-G) remain normal [15,18,34]. The electrical basis for ankyrin-B-dependent cardiac arrhythmia has been proposed, based on observations with ankyrin-B+/− cardiomyocytes, to be due to elevated Ca2+ transients that provoke afterdepolarizations and extrasystoles following catecholamine-induced stress [15]. These predictions of a calcium-based phenotype are also supported by absence of abnormalities in the localization or expression of Nav channels (also normal cardiac action potentials) and K channels in ankyrin-B+/− and ankyrin-B−/− cardiomyocytes [15,18]. Absence of the ankyrin-B complex would be predicted to result in less efficient export of calcium from the SR and could result in elevated calcium transients.
Ankyrin-B+/− cardiomyocytes display preferential loss of ankyrin-B immunoreactivity at Z-line/T-tubule domains compared with M-line staining (see Figure 1). Potential explanations for this preferential loss may include reduced T-tubule ankyrin-B protein stability (half-life), increased T-tubule/SR membrane turnover, or differences in the association of each ankyrin-B population with the underlying cytoskeleton. Alternatively, reduced expression of the T-tubule/SR population of ankyrin-B in ankyrin-B+/− cardiomyocytes may result from differences in the molecular identities of ankyrin-B polypeptides at each domain. For example, ankyrin-B immunoreactivity at the M-line may represent an ankyrin-B splice form that lacks Ank2 exon 23 (exon targeted in the ankyrin-B knock-out mouse) but still reacts with ankyrin-B Ig.
Interaction between InsP3R and ouabain-associated Na/K pump has been reported to be responsible for slow Ca2+ oscillations in cultured renal proximal tubule and Cos7 cells [35]. Our results with pure proteins suggest that InsP3R and the NKA do not interact directly, at least not with high affinity (see Figure 7). Thus, it will be of interest to evaluate possible participation of ankyrin-B or possibly other adaptor proteins in this system. More generally, determinants of cellular localization and partnerships with physiologically related proteins likely are an essential aspect of function for all ion channels and transporters. Ankyrins are ubiquitously expressed and display diversity in protein interactions. Based on the findings of this study, and previous findings that ankyrin-G is required for coordinating voltage-gated Na channels and L1CAM cell adhesion molecules at axon initial segments [36,37], we predict that ankyrins are likely to contribute to higher order organization of multiple channels and transporters in a variety of tissues.
Materials and Methods
Mice used in these studies were adult WT C57BL/6 mice and ankyrin-B+/− littermates (C57BL/6), 3–6 mo of age and weighing 30–40 g. Animals were handled according to approved protocols and animal welfare regulations of the Institutional Review Board of Duke University Medical Center. Mouse ventricular cardiomyocytes were isolated as described in [15].
Antibodies not described in [15] include NCX1 (Affinity Bioreagents, Golden, Colorado, United States; Swant, Bellinzona, Switzerland), alpha 1 and alpha 2 ATPase (Transduction), DHPR (ABR, Alomone), affinity-purified GFP polyclonal Ig, and affinity-purified pan-InsP3R polyclonal Ig generated against the C-terminus of mouse InsP3R (residues 2592 −2750). When unavoidable, mouse cells were immunostained with monoclonal antibodies that had been first affinity-purified. For these monoclonal antibodies, we confirmed that our staining was specific by control experiments in rat cells. Additionally, Alexa anti-mouse secondary antibodies were examined for background immunoreactivity. Adult cardiomyocytes were stained as described [15]. Isolated ventricular mouse cardiomyocytes were double-labeled and imaged in three dimensions by rendering of confocal Z-scans obtained at 0.18-μm increments near the center of isolated cells using a 100 power/1.45 NA objective (LSM 510, Zeiss, Oberkochen, Germany). LSM Z-stacks were transferred to Volocity software (Improvision, Lexington, Massachusetts, United States), and identical protocols were used for 3D rendering of WT and ankyrin-B+/− cells. Volocity Classification software or LSM 510 software was used to measure voxel or pixel co-localization. Data represent at least three separate experiments with at least five areas measured for each experiment. Areas measured do not include sarcolemmal membrane voxels. Using monoclonal and polyclonal antibody directed against the same protein, cardiac double-labeling, and voxel co-localization revealed that the maximal co-localization for the same protein was ~65% consistent with previous studies [19].
Ankyrin-B mutagenesis.
GFP–220-kDa ankyrin-B mutants E1425G and E1425D were created using site-directed mutagenesis. The mutated region was subcloned into a native GFP–220-kDa ankyrin-B plasmid, and the plasmid was completely sequenced to verify that no additional mutations were introduced.
When appropriate, data were analyzed using a two-tailed Student's t-test, and values less than p < 0.05 were considered significant. Values are expressed as the mean ± standard deviation.
Protein modeling
3D protein structures for the model in Figure 8 were approximated based on published structures [38–43].
Immunoprecipitation and solubilization of heart proteins
Adult heart immunoprecipitations and quantitative immunoblotting were performed as described [15]. Briefly, adult mouse heart and brain were dissected and rinsed in PBS plus 0.32 M sucrose and 2 mM Na EDTA, flash frozen in liquid nitrogen, and ground into a fine powder. The powder was resuspended in 4 volumes of 50 mM Tris HCl (pH 7.35), 10 mM NaCl, 0.32 M sucrose, 5 mM Na EDTA, 2.5 mM Na EGTA, 1 mM PMSF, 1 mM 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF), 10 μg/ml leupeptin, and 10 μg/ml pepstatin using a Dounce homogenizer (Kimble/Kontes, Vineland, New Jersey, United States). The homogenate was centrifuged at 1,000g to remove nuclei. Triton X-100 and deoxycholate were added to the post-nuclear supernatant for final concentrations of 1.5% Triton X-100 and 0.75% deoxycholate. The lysate was pelleted at 100,000g for 1 h at 4 °C, and the supernatant was re-cleared at 100,000g for 1 h to remove residual large membranes or vesicles. The resulting supernatant was used for immunoprecipitation (see Figure 4) as described [16], or for binding experiments.
Binding studies
GFP–220-kDa ankyrin-B and mutants (E1425G and E1425D) were expressed in HEK293 cells and purified using affinity-purified GFP Ig coupled to Protein A agarose beads. Briefly, cells were lysed in above homogenization buffer plus 1.0% Triton X-100 and 0.5% deoxycholate. The extract was centrifuged at 100,000g, and the supernatant was incubated with GFP affinity-purified Ig coupled to Protein A sepharose. The beads were washed with homogenization buffer plus 1.0% Triton X-100. Purified proteins were incubated with 10 μg of affinity-purified GFP Ig or control Ig coupled to Protein A sepharose beads for 4 h at 4 °C. The beads were washed four times with homogenization buffer plus 1.0% Triton X-100. Protein bound to each mutant GFP–220-kDa ankyrin-B was eluted, analyzed by quantitative 125I-labeled Protein A immunoblot, normalized for relative GFP–ankyrin-B expression, and then compared to WT GFP–220-kDa ankyrin-B binding.
For in vitro complex reconstitution experiments, 0.5 ml of Dynabeads M-270 Epoxy (1 × 109) beads (Dynal Biotech, Brown Deer, Wisconsin, United States) were washed in PBS and incubated with 5 mg of neutravidin in PBS in a final volume of 1 ml for 48 h at 25 °C. Beads were then washed in BSA binding buffer (20 mM Hepes [pH 7.3], 50 mM NaCl, 1 mM Na EDTA, 0.1% Triton X-100, 1 mM sodium azide, and 5 mg/ml BSA). Purified InsP3R (0.5 ml; 200 μg/ml) was incubated with a 20-fold molar excess of NHS-LC-biotin (Pierce Biotechnology, Rockford, Illinois, United States) overnight at 4 °C. For control biotin, the same biotin was incubated overnight in PBS. Biotin-InsP3R was then dialyzed against binding buffer without BSA to remove unbound biotin. Then 50% of the neutravidin Dynabeads were pre-incubated with the control biotin for 2 h at 4 °C, washed, and resuspended in BSA binding buffer (control neutravidin Dynabeads). Neutravidin Dynabeads were incubated with the dialyzed biotin-InsP3R while control neutravidin Dynabeads were incubated with the same concentration of unlabeled InsP3R for 2 h at 4 °C. Dynabeads were then washed 3× in BSA binding buffer and used for binding experiments. Coated Dynabeads (InsP3R at 20 nM) were incubated with 125I-labeled NCX1 (20 nM final concentration; specific activity 274,000 cpm/pmol) and/or 125I-labeled NKA (20 nM final concentration; specific activity ~504,000 cpm/pmol) in the presence or absence of a pre-incubation with ankyrin-B membrane-binding domain (20 nM final concentration) in a final volume of 50 μl of BSA binding buffer (20 mM Hepes [pH 7.3]; 50 mM NaCl, 1 mM Na EDTA, 0.2% Triton X-100, 1 mM NaN3, and 5 mg/ml BSA). Following 4 h, the beads were washed in binding buffer minus BSA, and both pellet and supernatant samples were assayed for 125I in a gamma counter (n = 3). The samples were then examined by SDS-PAGE and phosphorimaging (n = 3). Values for picomoles bound of 125I-labeled NCX1 or 125I-labeled NKA in experiments where only one labeled ligand was used were calculated from counts of 125I-labeled protein bound and specific activity. Values for picomoles bound of 125I-labeled NCX1 and 125I-labeled NKA when two labeled proteins were incubated together (125I-labeled NCX1 + 125I-labeled NKA) were calculated by first determining a ratio of counts of 125I-labeled protein bound/band intensity for unique bands of 125I-labeled NCX or 125I-labeled NKA in single protein binding experiments (i.e., 125I-labeled NCX + ankyrin-B + InsP3R beads). The intensity of these same bands was measured in the gel lanes where the two proteins (i.e., 125I-labeled NCX + 125I-labeled NKA + ankyrin-B + InsP3R beads) were combined to determine the number of picomoles bound of each protein. We observed approximately equal picomoles of 125I-labeled NCX1 and 125I-labeled NKA bound to InsP3R beads when ankyrin-B was included in the binding reaction. However, because of the lower specific activity of 125I-labeled NCX1, the band intensity on the gel was approximately 50% of that of 125I-labeled NKA. Saturation binding was performed essentially as described in [16] but using glutathione beads. Briefly, ankyrin-B membrane-binding domain was purified as described in [16]. Increasing concentrations of 125I-labeled InsP3R, 125I-labeled NCX1, or 125I-labeled NKA were incubated for 2 h at 25 °C with glutathione Sepharose-immobilized GST-ankyrin-B membrane-binding domain or GST. The beads were washed and counted in a gamma counter. The data were corrected for nonspecific binding at each concentration by subtracting values obtained with GST-coated beads.
Protein purification
Full-length human NCX1 was cloned from a human heart library (Clontech, Palo Alto, California, United States) into pBacPak9 (Clontech) using standard molecular techniques. For purification of NCX1, a His-tag was engineered to the C-terminus. NCX1 was expressed in SF21 insect cells using a generated recombinant baculovirus. Cells were infected in monolayer cultures with a MOI of ten for 72 h at 27 °C. Cells were harvested and washed in PBS, and cell pellets were snap frozen and stored at −80 °C. All subsequent procedures were performed at 4 °C in the presence of protease inhibitors (100 μg/ml AEBSF, 100 μg/ml benzamidine, 30 μg/ml leupeptin, and 10 μg/ml pepstatin). Cells were syringed and sonicated in cell homogenization buffer (PBS, 1 mM Na EDTA, 1 mM DTT, and 1 mM sodium azide) to break the cell membranes, then centrifuged at 100,000g for 30 min to collect membranes. Cell membranes were pre-extracted with 20 mM CHAPS (pH 12) for 30 min followed by 20 mM PB (pH 7.3), 0.5 M NaCl, 0.5 M urea, 0.5% Triton X-100, and 0.5 mM beta mercaptoethanol. The cell residue was resuspended in extraction buffer (50 mM PB (pH 8.0), 0.3 M NaCl, 10 mM imidazole, 0.2% Triton X-100, 1 mM beta mercaptoethanol, 1 mM sodium azide, and 2% Sarkosyl) for 20 min. The extract was centrifuged at 100,000g for 1 h, and the supernatant collected and diluted 10-fold in buffer lacking Sarkosyl. The diluted extract was applied to a column of Ni-NTA Sepharose, washed with 10–20 column volumes of dilution buffer, and eluted with buffer plus 0.3 M imidazole. Peak fractions were pooled, adjusted to 10% glycerol, snap frozen, and stored at −80 °C. Sheep kidney NKA was isolated in membrane-bound form from outer medulla as previously described [44]. The NKA was extracted and purified as previously described [45]. The InsP3R was purified from frozen bovine brain cerebellum by a modification of published procedures [16,46]. All procedures were carried out at 4 °C in the presence of protease inhibitors (100 μg/ml AEBSF, 100 μg/ml benzamidine, 30 μg/ml leupeptin, and 10 μg/ml pepstatin). Cerebellum was homogenized using a polytron in five volumes (weight/volume) of homogenization buffer (10 mM Hepes [pH 7.3], 0.32 M sucrose, 2 mM EGTA, 1 mm DTT, and 1 mM sodium azide), and centrifuged at 2,000 rpm for 10 min. Membranes were then collected at 30,000g for 1 h. Membranes were prewashed in wash buffer (50 mM Tris HCl [pH 8.0], 1 mM Na EGTA, 1 mM DTT, and 1 mM Na azide), then resuspended to the homogenization volume with the wash buffer. InsP3R was extracted from the membranes by the addition of 2% final Triton X-100 for 30 min, and supernatants collected after centrifugation at 30,000g for 1 h. The extract was adjusted to 0.25 M NaCl and applied to a 50-ml heparin Sepharose column equilibrated in 0.25 M NaCl and 0.2% Triton X-100 extraction buffer. The heparin Sepharose was washed with ten column volumes of equilibration buffer, and then eluted with 0.5 M NaCl buffer. Peak fractions were pooled and dialyzed against ten volumes of column buffer lacking NaCl and 20 mM Tris HCl (pH 8.0). A precipitate formed after dialysis and was collected by centrifugation at 100,000g for 20 min. The pellet was resuspended in column buffer with the addition of 1.0 M NaCl and was re-centrifuged as above. The InsP3R released into the supernatant was then adjusted to 0.2 mM CaCl2 and 0.2 mM MnCl2, and applied to a 4-ml ConA Sepharose column. The column was washed in 20 column volumes of buffer, the elution started with the addition of 1 M mannose, the elution stopped, and the column allowed to sit in elution buffer overnight. The elution was continued the following day and fractions collected, aliquoted, snap frozen, and stored at −80 °C.
Supporting Information
Figure S1. Ankyrin-B-Based Complex Is a Specialized Feature of Cardiac Myocytes
Ankyrin-B is expressed in ventricular cardiomyocytes but not in smooth muscle lining large arteries. Image represents adult mouse heart immunostained with ankyrin-B-specific Ig.
Accession Number
The NCBI (http://www.ncbi.nlm.nih.gov/) accession number for ankyrin-B is NM_020977.
We gratefully acknowledge the following research support and grants: the Howard Hughes Medical Institute and a focused giving grant from Johnson and Johnson to VB. PJM is supported by a National Scientist Development Award from the American Heart Association.
Author Contributions
PJM, JQD, and VB conceived and designed the experiments. PJM and JQD performed the experiments. PJM, JQD, and VB analyzed the data and contributed reagents/materials/analysis tools. PJM and VB wrote the paper.
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text:
© Copyright 2017 Kimmay. All rights reserved.
Status: Finished
Genre: Romance
AW! that's so adorably sweet!! -gushes like a fangirl- xD KMU??=]
YAY finally they go out! Love it by the way KMU
It seems kind of rushed. I mean I love the idea of them together, really I do. Yet, it seems like all of a sudden their all "lovey-dovey".
I don't mean to critisize, because the chappy was really sweet :) I can't wait to read more
AAWWW!!! So cute!! I love it!! Update me?-Ariel
The perfect couple! ;) I love them both and I think they are cuteee! Please kmuuuuuu
Awww so adorable!! They are too cute together. I wonder how the troupe will react to Ryan and Kimmie Being together when only a day or so before they wanted to tear each others heads off :) kmu!! And please update soon!
Caaann you feel the loovvee toonightt??
The peace the evening brringss??
AAwwww, too cute! Great chappie Kimmie! :)
Awh (": Soo cute! Kmu?
My heart is doing kickflips right now!!! Awesome chappie, lil sis! :) and i can't believe there's no more chapters to click next! This is seriously gonna kill me! I know it will be awhile before you uodate again, but Good luck with your exams, sweets! :) study hard ans hope you ace all your exams!! We're here waiting for the next update ok. :)
Kimmie, this is such a wonderful chapter! So magical! They finally kissed!!! Eeeeeppp!! :) yaaaayyy!! They are just too cute for words, i truly love them, they have great chemistry! Great job with your characters, kimmie! :)
Glad the date was a success. And oooo she's gonna dream that their bodies are intertwined... :) nice...
Ok, they're finally together... But i have a feeling there will be some bad stuff coming and it'll put their relationship in jeopardy. I can feel it.... (sigh)
KMU, ms kimmie! :) :) :) love you, girl!
kmu?? love it :)
they're soooo cute!
plz plz plz update !!!!
They went on their first date!!! I'm so happy right now!!! Amazing chapter!! Please keep me updated :D
omg yay finally i love it
that was really good!
wtf does kmu mean? but anyway i love it!!!!perfect it couldnt of been done any other way
Ummmm im totally jealous of Kimmie! She is sooo lucky...hahaha great chapter and please keep me updated!
I love it! i just read all chapters today, and i really cant wait for you to update!!(:
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Sofia Vergara Loses It When She Hears Her Voice On Helium!This is hilarious!
Sofia Vergara Feels Social Media Puts Pressure On Women; Posts Sexy, Messy Selfie (Photo)Modern Family star Sofia Vergara is currently filming a new movie The Brits Are Coming, but that did not stop her from posting a ridiculous sexy pic on her Instagram page.
VIDEO: Watch Bill Cosby's Creepy Interview With Sofia Vergara From 2003
Sofia Vergara's Ex Suing For Control Of Her Eggs! What!?Sofia has moved on, this dude clearly has not.
Sofia Vergara Lists The Three Biggest Problems For Girls With Big Breasts
Sofia Vergara & Joe Manganiello Are Engaged
Kory: The Freaky Sofia Vergara/Emma Watson Vine Explained!So that explains it!
Kory: I Don't Know What This Video Is, But I Can't Stop Watching ItIs this vine of Emma Watson, or Sofia Vergara? Either way, I am so confused! Mind BLOWN!
Sofia Vergara Sings Carols To Her "Modern Family" Co-StarsYes, it's bad...but SO good!
Red Carpet Fashion At The 64th Annual Primetime EmmysGlamor and glitz as only Hollywood can do it, celebs modeled their fashions on the red carpet at the 64th Annual Primetime Emmys last night. Who was the best and worst dressed?
Pink Is The New Face Of Covergirl
Happy Feet Two In 3D


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Basic Web Application for Adding Pop-up Comments on User-Added Text
- Status Closed
- Budget $250 - $750 USD
- Total Bids 26
Project Description
We are hiring a developer to create only the beginning of an application. If this milestone is completed well, there will be more work immediately. For completion of this project, please have this up and running on a development webserver we can access to test its functionality as described. The requirements are as follows:
"Carbonara" will be a site that lets users create text-only web pages that can be commented on by other users. Users can add comments by selecting parts of the text and writing them into a pop-up box; when a comment has been added, the related text becomes underlined. The comments can be viewed in a JavaScript pop-up window by clicking on the text that has been commented on. Think of it as a place where writers can post their work (let's just call all their work "stories" to keep it simple) and have other writers/users critique the stories in detail. Any word, phrase, sentence or paragraph can receive a comment. All comments, once added, can be accessed by all other visitors (none are private). The homepage consists of a list of the 10 most recent pages that have been added, and a link to an "Archive" provides a page with an alphabetical list of all stories.
Build a basic website that will allow the following:
1. Homepage / Viewing Stories
A visitor to the site can either click to 1) add a new story or 2) just browse the existing stories. The main content displayed on the homepage is a center column that displays "Recently Added Stories", with just the title of the 10 most recently added stories displayed. A link to an "Archive" provides a page with an alphabetical list of all stories. Each listed title links to the corresponding story's page.
2. Adding Stories
From the homepage, a visitor clicks an "Add New Story" button which takes him to a new page with a series of blank text fields: "Title", "Topic", "Source URL", "Author(s)", and the main field, which is very large, for "Text". The user pastes his story into the "Text" field and fills in the other fields. He clicks submit. A new page with his story has been created. Its Title, and a link to it, will now appear on the homepage under "Recently Added Stories".
3. Adding Comments
A user navigates to a story, and begins reading. When the user wants to make a comment, he can highlight any word, phrase, sentence or paragraph (whichever his comment relates to). Once the text is highlighted, a small pop-up button that says "Add a comment" pops up next to the highlighted text. The user clicks on this pop-up button. A much larger pop-up screen with a text field now appears. He types his comments into the text field, and then clicks on a "Submit" button. His comment is now connected to that piece of text, and the text that he has commented on becomes underlined on the page. Only text that has comment(s) connected to it is underlined.
4. Viewing Comments
Comments don't appear unless the users clicks somewhere on the text that is underlined. When this happens, a large pop-up window appears that shows the full comment. Below the comment, the user can click an "Add new comment" button to enter another comment about the exact same text and submit it. If there are multiple comments on a particular part of text, a scroll bar on the pop-up window can allow users to see other comments.
That's all we need for now. If you are selected and the project is completed successfully, more work will follow immediately.
This is important: To separate you from the spammers, please write VERIFYING I AM REAL as the first line of your bid. We will delete all bids that do not start with this phrase, since most bidders never read the requirements. Thank you for being one who does.Get free quotes for a project like this
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Opinion + International Women's Day
To mark International Women's Day, we want to hear from female readers around the world about the barriers to equality they face
Relax, guys – it's International Men's Day (the official one)Richard HerringEvery year on International Women's Day, people ask: when's our big day? Well, anti-sexist warriors, I hope you're celebrating now
Women are the future of cities – and New York City gives them the keys to empowermentChirlane McCrayLeaders of our world's cities cannot afford to be so indifferent to the injustices faced by women, as 80% of women and girls will live in urban areas by 2050
Even if 'bossy' could be banned, there are far better ways to boost girls' self-esteemHadley Freeman
Hadley Freeman: Facebook's Sheryl Sandberg and Beyoncé want to ban the word 'bossy' as disparaging to women. It's a good start, but there's so much more to be done. Here are my top tips
Ellie Cosgrave: A year after being sexually abused on a tube train I returned to dance for all women who have been assaulted. Project Guardian will help make us safer
Poverty matters blog Hugo Chávez knew that his revolution depended on womenSelma James and Nina López
Selma James and Nina López: And he wasn't the only one. Presidents of Tanzania and Haiti have both benefited from making women central to progress
Suzanne Moore: IWD feels like a cosy exercise for a coterie of women, with too much new age babbling. I'd rather have a holiday
What Welsh women can teach the worldEllie Mae O'HaganEllie Mae O'Hagan: We're not 'country girls' as the media would have it, but creative, passionate, open and free. Just ask Bonnie Tyler
Sayeeda Warsi: Other nations are showing us up. On International Women's Day, consider the great women of Thailand, India and Brazil
Helen Grant: True equality won't come from throwing women scraps of state help, as Polly Toynbee seems to think
Kevin Powell: Sadly, we have to confront the fact that Rush Limbaugh is no aberration. It's up to us men to challenge men's abuse of women
Catalina May: International Women's Day: Maternity leave is now longer and abortion is on the agenda, but there is still a way to go
Lijia Zhang: International Women's Day: But Chinese women's participation in politics is seriously lagging behind other countries
This year let's celebrate … the US backlash against sexismJessica Valenti
Jessica Valenti: International Women's Day: The Republican primaries have thrown up shocking misogyny, but the response is heartening
International Women's Day marks the first era in living memory that the equality drive has gone into reverse, writes Polly Toynbee


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EVGA Shows Off New High Bandwidth "Pro SLI Bridge HB" Bridges
Subject: Graphics Cards | June 22, 2016 - 06:40 AM | Tim Verry
Tagged: SLI HB, nvidia, EVGA SLI HB
Earlier this month we reported that EVGA would be producing its own version of Nvidia's SLI High Bandwidth bridges (aka SLI HB). Today, the company unveiled all the details on its new bridges that we did not know previously, particularly pricing and what the connectors look like.
EVGA is calling the new SLI HB bridges the EVGA Pro SLI HB Bridge and it will be available in several sizes to accommodate your particular card spacing. Note that the 0 slot, 1 slot, 2 slot, and 4 slot spacing bridges are all for two graphics card setups; you will not be able to use these bridges for Tri SLI or Quad SLI setups. While Nvidia did not show the underside of the HB bridges when it first announced them alongside the GTX 1080 graphics card, thanks to EVGA you can finally see what the connectors look like.
As many surmised, the new high bandwidth bridges use both fingers of the SLI connectors on each card to connect the two cards together. Previously (using the old-style SLI bridges), it was possible to connect card A to card B using one set of connectors and Card B to Card C using the second set of connectors for example. Now, you are limited to two card multi-GPU setups. That is the downside; however, the upside is that the HB bridges promise to deliver all of the necessary bandwidth to allow for high speed 4K and NVIDIA Surround display setups. While you will not necessarily see higher frame rates, the HB bridges should allow for improved frame times which will mean smoother gameplay on those very high resolution monitors!
The new SLI bridges are all black with an EVGA logo in the middle that is backlit by an LED. Users are able to use a switch along the bottom edge of the pcb to select from red, green, blue, and white LED colors. In my opinion these bridges look a lot better than the Nvidia SLI HB bridge renders from our computex story (hehe).
Now, as for pricing: EVGA is pricing its SLI HB bridges at $39.99 with the 2 slot spacing and 4 slot spacing bridges available now and the 0 slot and 1 slot spaced bridges set to be available soon (you can sign up to be notified when they are available for purchase). Hopefully reviews will be updated shortly around the net with the new bridges to see what impact they really have on multi-GPU gaming performance (or if they will just be better looking alternatives to the older LED bridges or ribbon bridges)!
Also read:
- GeForce GTX 1080 and 1070 3-Way and 4-Way SLI will not be enabled for games
- EVGA Forum Discussion on Pro SLI HB Bridges
Whoops! AMD Radeon RX 480 Specifications on Newegg
Subject: Graphics Cards | June 22, 2016 - 12:49 AM | Scott Michaud
Tagged: rx 480, Radeon RX 480, polaris 10, Polaris, amd
The AMD Radeon RX 480 is set to launch on June 29th, but a VisionTek model was published a little early (now unpublished -- thanks to our long-time reader, Arbiter, for the heads up). Basically all specifications were already shared, and Ryan wrote about them on June 1st, but the final clock rates were unknown. The VisionTek one, on the other hand, has it listed as 1120 MHz (5.16 TFLOPs) with a boost of 1266 MHz (5.83 TFLOPs).
Granted, it's possible that the VisionTek model could be overclocked, even though the box and product page doesn't mark it as a factory-overclocked SKU. Also, 5.16 TFLOPs and 5.83 TFLOPs align pretty close to AMD's ">5 TFLOPs" rating, so it's unlikely that the canonical specifications slide underneath this one. Also, TFLOP ratings are basically a theoretical maximum performance, so real-world benchmarks need to be considered for a true measure of performance. That said, this would put the stock RX 480 in the range of a GTX 980 (somewhere above its listed boost clock, and slightly below its expected TFLOP rating when overclocked).
There is no price listed for the 8GB model, but the 4GB version will be $199 USD.
Fermi, Kepler, Maxwell, and Pascal Comparison Benchmarks
Subject: Graphics Cards | June 21, 2016 - 09:22 PM | Scott Michaud
Tagged: nvidia, fermi, kepler, maxwell, pascal, gf100, gf110, GK104, gk110, GM204, gm200, GP104
Techspot published an article that compared eight GPUs across six, high-end dies in NVIDIA's last four architectures: Fermi to Pascal. Average frame rates were listed across nine games, each measured at three resolutions:1366x768 (~720p HD), 1920x1080 (1080p FHD), and 2560x1600 (~1440p QHD).
The results are interesting. Comparing GP104 to GF100, mainstream Pascal is typically on the order of four times faster than big Fermi. Over that time, we've had three full generational leaps in fabrication technology, leading to over twice the number of transistors packed into a die that is almost half the size. It does, however, show that prices have remained relatively constant, except that the GTX 1080 is sort-of priced in the x80 Ti category despite the die size placing it in the non-Ti class. (They list the 1080 at $600, but you can't really find anything outside the $650-700 USD range).
It would be interesting to see this data set compared against AMD. It's informative for an NVIDIA-only article, though.
Windows 10 versus Ubuntu 16.04 versus NVIDIA versus AMD
Subject: Graphics Cards | June 20, 2016 - 08:11 PM | Jeremy Hellstrom
Tagged: windows 10, ubuntu, R9 Fury, nvidia, linux, GTX1070, amd
Phoronix wanted to test out how the new GTX 1070 and the R9 Fury compare on Ubuntu with new drivers and patches, as well as contrasting how they perform on Windows 10. There are two separate articles as the focus is not old silicon versus new but the performance comparison between the two operating systems. AMD was tested with the Crimson Edition 16.6.1 driver, AMDGPU-PRO Beta 2 (16.20.3) driver as well as Mesa 12.1-dev. There were interesting differences between the tested games as some would only support one of the two Linux drivers. The performance also varies based on the game engine, with some coming out in ties, others seeing Windows 10 pull ahead and even some cases where your performance on Linux was significantly better.
NVIDIA's GTX 1080 and 1070 were tested using the 368.39 driver release for Windows and the 367.27 driver for Ubuntu. Again we see mixed results, depending on the game Linux performance might actually beat out Windows, especially if OpenGL is an option.
Check out both reviews to see what performance you can expect from your GPU when gaming under Linux.
"Yesterday I published some Windows 10 vs. Ubuntu 16.04 Linux gaming benchmarks using the GeForce GTX 1070 and GTX 1080 graphics cards. Those numbers were interesting with the NVIDIA proprietary driver but for benchmarking this weekend are Windows 10 results with Radeon Software compared to Ubuntu 16.04 running the new AMDGPU-PRO hybrid driver as well as the latest Git code for a pure open-source driver stack."
Here are some more Graphics Card articles from around the web:
- GeForce GTX 1070 and GTX 1080 FE Overclocking @ [H]ard|OCP
- DX11 vs DX12 Intel 4770K vs 5960X Framerate Scaling @ [H]ard|OCP
- MSI GTX 1080 & GTX 1070 Gaming X 8G Overclocking Review @ OCC
- EVGA GeForce GTX 1080 FTW Gaming ACX 3.0 Review @HiTech Legion
- Gigabyte GTX 1080 G1 Gaming RGB @ Kitguru
- ASUS GTX 1080 Strix Gaming 8 GB @ techPowerUp
- HIS Radeon R7 360 GREEN iCooler OC 2GB Graphics Card Review @ NikKTech
NVIDIA Announces PCIe Versions of Tesla P100
Subject: Graphics Cards | June 20, 2016 - 05:57 PM | Scott Michaud
Tagged: tesla, pascal, nvidia, GP100
GP100, the "Big Pascal" chip that was announced at GTC, will be coming to PCIe for enterprise and supercomputer customers in Q4 2016. Previously, it was only announced using NVIDIA's proprietary connection. In fact, they also gave themselves some lead time with their first-party DGX-1 system, which retails for $129,000 USD, although we expect that was more for yield reasons. Josh calculated that each GPU in that system is worth more than the full wafer that its die was manufactured on.
This brings us to the PCIe versions. Interestingly, they have been down-binned from the NVLink version. The boost clock has been dropped to 1300 MHz, from 1480 MHz, although that is matched with a slightly lower TDP (250W versus the NVLink's 300W). This lowers the FP16 performance to 18.7 TFLOPs, down from 21.2, FP32 performance to 9.3 TFLOPs, down from 10.6, and FP64 performance to 4.7 TFLOPs, down from 5.3. This is where we get to the question: did NVIDIA reduce the clocks to hit a 250W TDP and be compatible with the passive cooling technology that previous Tesla cards utilize, or were the clocks dropped to increase yield?
They are also providing a 12GB version of the PCIe Tesla P100. I didn't realize that GPU vendors could selectively disable HBM2 stacks, but NVIDIA disabled 4GB of memory, which also dropped the bus width to 3072-bit. You would think that the simplicity of the circuit would want to divide work in a power-of-two fashion, but, knowing that they can, it makes me wonder why they did. Again, my first reaction is to question GP100 yield, but you wouldn't think that HBM, being such a small part of the die, is something that they can reclaim a lot of chips by disabling a chunk, right? That is, unless the HBM2 stacks themselves have yield issues -- which would be interesting.
There is also still no word on a 32GB version. Samsung claimed the memory technology, 8GB stacks of HBM2, would be ready for products in Q4 2016 or early 2017. We'll need to wait and see where, when, and why it will appear.
NVIDIA Releases 368.51 Hotfix Driver
Subject: Graphics Cards | June 19, 2016 - 02:37 AM | Scott Michaud
Tagged: nvidia, graphics drivers
GeForce Hotfix 368.51 drivers have been released by NVIDIA through their support website. This version only officially addresses flickering at high refresh rates, although its number has been incremented quite a bit since the last official release (368.39) so it's possible that it rolls in other changes, too. That said, I haven't heard too many specific issues with 368.39, so I'm not quite sure what that would be.
As always with a hotfix driver, NVIDIA pushed it out with minimal testing. It should pretty much only be installed if you have a specific issue (particularly the listed one(s)) and you don't want to wait until one is released that both NVIDIA and Microsoft looked over (although Microsoft's WHQL certification has been pretty lax since Windows 10).
Oddly enough, they only seem to list 64-bit links for Windows 8.1 and Windows 10. I'm not sure whether this issue doesn't affect Windows 7 and 32-bit versions of 8.1 and 10, or if they just didn't want to push the hotfix out to them for some reason.
How far can a GTX 1070 Founders Edition go?
Subject: Graphics Cards | June 14, 2016 - 05:46 PM | Jeremy Hellstrom
Tagged: GTX1070, nvidia, overclocking
Overclocking the new Pascal GPUs can be accomplished with the EVGA Precision X tool as it allows you to bump up the power, temperature target and fan speed as well as the frequencies for the GPU and memory easily and effectively. [H]ard|OCP set out to push the 1070 as far as it would go with this software in a recent review. The power target can only be increased to 112%, which they implemented along with setting the fan to 100% as this is about the maximum performance, not about peace and quiet. After quite a bit of testing they settled on 2062MHz GPU and 4252MHz RAM clocks as the highest stable frequency this particular card could manage. The results show a card which leaves the TITAN X in the dirt and this card does not even have a custom cooler; we anxiously await the non-Founders Edition releases to see what they can accomplish.
"In our overclocking review of the NVIDIA GeForce GTX 1070 Founders Edition we will see how far we can overclock the GPU and memory and then compare performance with GeForce GTX TITAN X and GeForce GTX 980 Ti. How high will she go? Can the $449 GTX 1070 outperform a $1000 GTX TITAN X? The answer is exciting."
Here are some more Graphics Card articles from around the web:
- NVIDIA GeForce GTX 1070 On Linux: Testing With OpenGL, OpenCL, CUDA & Vulkan @ Phoronix
- MSI GeForce GTX 1070 Gaming X Review - It's RGB! @ HiTech Legion
- NVIDIA GeForce GTX 1070 8 GB @ techPowerUp
- MSI GTX 1080 & GTX 1070 Gaming X 8G Review @ OCC
- Deep Learning & CUDA Benchmarks On The GeForce GTX 1080 Under Linux @ Phoronix
- Gigabyte GTX 1080 G1 Gaming 8 GB @ techPowerUp
- Gigabyte GeForce GTX 1080 G1 GAMING @ Guru of 3D
- MSI GTX 1080 Gaming X 8 GB @ techPowerUp
AMD "Sneak Peek" at RX Series (RX 480, RX 470, RX 460)
Subject: Graphics Cards, Processors | June 13, 2016 - 07:51 PM | Scott Michaud
Tagged: amd, Polaris, Zen, Summit Ridge, rx 480, rx 470, rx 460
AMD has just unveiled their entire RX line of graphics cards at E3 2016's PC Gaming Show. It was a fairly short segment, but it had a few interesting points in it. At the end, they also gave another teaser of Summit Ridge, which uses the Zen architecture.
First, Polaris. As we know, the RX 480 was going to bring >5 TFLOPs at a $199 price point. They elaborated that this will apply to the 4GB version, which likely means that another version with more VRAM will be available, and that implies 8GB. Beyond the RX 480, AMD has also announced the RX 470 and RX 460. Little is known about the 470, but they mentioned that the 460 will have a <75W TDP. This is interesting because the PCIe bus provides 75W of power. This implies that it will not require any external power, and thus could be a cheap and powerful (in terms of esports titles) addition to an existing desktop. This is an interesting way to use the power savings of the die shrink to 14nm!
They also showed off a backpack VR rig. They didn't really elaborate, but it's here.
As for Zen? AMD showed the new architecture running DOOM, and added the circle-with-Zen branding to a 3D model of a CPU. Zen will be coming first to the enthusiast category with (up to?) eight cores, two threads per core (16 threads total).
The AMD Radeon RX 480 will launch on June 29th for $199 USD (4GB). None of the other products have a specific release date.
Subject: Graphics Cards | June 9, 2016 - 01:45 AM | Scott Michaud
That wasn't even capslock. That was pure shift key.
A little after Computex, GIGABYTE announced their GTX 1080 XTREME GAMING graphics card, which should be their flagship of the GeForce GTX 1080 line. It is a three-fan design, although the center fan overlaps with the two edge ones. It will also accept two, eight-pin PCIe power connectors, which gives a theoretical maximum draw of 375W.
Also, taking a cue from EVGA's recent VR-Edition 980 Ti, GIGABYTE includes a I/O front panel for cases with an extra 5.25" bay. This contains two HDMI ports and two USB 3.0 ports to allow users to quickly connect VR headsets to and from their PC. When connected, it disables two DisplayPort outputs on the card, routing them to the front-panel's HDMI instead. I'm not exactly clear on why you would need two HDMI connections in the front, but okay.
They are also releasing their own SLI HB connector, which they claim will support speeds up to 1080 MHz. The SLI HB standard, from NVIDIA, clocks up to 650 MHz. We assume that this is a typo on GIGABYTE's part (having 1080 on the brain for some reason...) but we've contacted NVIDIA to see what's up.
Currently no pricing or availability information. It comes with a three year warranty that can be upgraded to a four-year warranty by registering your product and signing up to their "XTREME GAMING Club".
GeForce GTX 1080 and 1070 3-Way and 4-Way SLI will not be enabled for games
Subject: Graphics Cards | June 9, 2016 - 12:44 AM | Ryan Shrout
Tagged: sli, pascal, nvidia, GTX 1080, GP104, geforce, 4-way sli, 3-way sli
IMPORTANT UPDATE: After writing this story, but before publication, we went to NVIDIA for comment. As we were getting ready to publish, the company updated me with a shift in its stance on multi-GPU configurations. NVIDIA will no longer require an "enthusiast key" to enable SLI on more than two GPUs. However, NVIDIA will also only be enabling 3-Way and 4-Way SLI for a select few applications. More details are at the bottom of the story!
You'll likely recall that during our initial review of the GeForce GTX 1080 Founders Edition graphics card, we mentioned that NVIDIA was going to be moving people towards the idea that "only 2-Way SLI will be supported" and promoted. There would still be a path for users that wanted 3 and 4 GPU configurations anyway, and it would be called the Enthusiast Key.
As it turns out, after returning from an AMD event focused on its upcoming Polaris GPUs, I happen to have amassed a total of four GeForce GTX 1080 cards.
Courtesy of some friends at EVGA and two readers that were awesome enough to let me open up their brand new hardware for a day or so, I was able to go through the 3-Way and 4-Way SLI configuration process. Once all four were installed, and I must point out how great it is that each card only required a single 8-pin power connector, I installed the latest NVIDIA driver I had on hand, 368.19.
Knowing about the need for the Enthusiast Key, and also knowing that I did not yet have one and that the website that was supposed to be live to enable me to get one is still not live, I thought I might have stumbled upon some magic. The driver appeared to let me enable SLI anyway.
Enthusiasts will note however that the green marker under the four GPUs with the "SLI" text is clearly only pointing at two of the GTX 1080s, leaving the remaining two...unused. Crap.
At this point, if you have purchased more than two GeForce GTX 1080 cards are simply out of luck and are waiting on NVIDIA to make good on it's promise to allow for 3-Way and 4-Way configurations via the Enthusiast Key. Or some other way. It's way too late now to simply say "we aren't supporting it at all."
While I wait...what is there for a gamer with four GeForce GTX 1080 cards to do? Well, you could run Ashes of the Singularity. It's multi-GPU mode uses MDA mode, which means the game engine itself accesses each GPU on its own, without the need for the driver to handle anything regarding GPU load balancing. Unfortunately, Ashes only supports two GPUs today.
Well...you could run an OpenCL based benchmark like LuxMark that access all the GPUs independently as well.
I did so, and the result is an impressive score of 17,127!!
How does that compare to some other products?
The four GTX 1080 cards produce a score that is 2.57x the result provided by the AMD Radeon Pro Duo and 2.29x the score of SLI GeForce GTX 980 Ti cards. Nice!
So there you go! We are just as eager to get our hands on the ability to test 3-Way and 4-Way SLI with new Pascal GPUs as some of the most extreme and dedicated enthusiasts out there are. With any luck, NVIDIA will finally figure out a way to allow it - no matter how it finally takes place.
IMPORTANT UPDATE: Before going to press with this story I asked NVIDIA for comment directly: when was the community finally going to get the Enthusiast Key website to unlock 3-Way and 4-Way SLI for those people crazy enough to have purchased that many GTX 1080s? The answer was quite surprising: NVIDIA is backing away from the idea of an "Enthusiast Key" and will no longer require it for enabling 3-Way and 4-Way SLI.
Here is the official NVIDIA statement given to PC Perspective on the subject:
With the GeForce 10-series we're investing heavily in 2-way SLI with our new High Bandwidth bridge (which doubles the SLI bandwidth for faster, smoother gaming at ultra-high resolutions and refresh rates) and NVIDIA Game Ready Driver SLI profiles. To ensure the best possible gaming experience on our GeForce 10-series GPUs, we're focusing our efforts on 2-way SLI only and will continue to include 2-way SLI profiles in our Game Ready Drivers.
DX12 and NVIDIA VR Works SLI technology also allows developers to directly implement and control multi-GPU support within their games. If a developer chooses to use these technologies then their game will not need SLI profiles. Some developers may also decide to support more than 2 GPUs in their games. We continue to work with all developers creating games and VR applications that take advantage of 2 or more GPUs to make sure they'll work great on GeForce 10-series GPUs.
For our overclocking community, our Game Ready Drivers will also include SLI profiles for 3- and 4-way configurations for specific OC applications only, including Fire Strike, Unigine and Catzilla.
NVIDIA clearly wants to reiterate that only 2-Way SLI will get the attention that we have come to expect from the GeForce driver dev team. As DX12 and Vulkan next-generation APIs become more prolific, the game developers will still have the ability to directly access more than two GeForce GTX 10-series GPUs, though I expect that be a very narrow window of games simply due to development costs and time.
NVIDIA will enable support for three and four card configurations in future drivers (without a key) for specific overclocking/benchmarking tools only, as a way to make sure the GeForce brand doesn't fall off the 3DMark charts. Only those specific applications will be able operate in the 3-Way and 4-Way SLI configurations that you have come to know. There are no profiles to change manually and even the rare games that might have "just worked" with three or four GPUs will not take advantage of more than two GTX 10-series cards. It's fair to say at this point that except for the benchmarking crowd, NVIDIA 3-Way and 4-Way SLI is over.
We expect the "benchmark only" mode of 3-Way and 4-Way SLI to be ready for consumers with the next "Game Ready" driver release. If you happened to get your hands on more than two GTX 1080s but aren't into benchmarking, then find those receipts and send a couple back.
So there you have it. Honestly, this is what I was expecting from NVIDIA with the initial launch of Pascal and the GeForce GTX 1080/1070 and I was surprised when I first heard about the idea of the "enthusiast key." It took a bit longer than expected, and NVIDIA will get more flak for the iterated dismissal of this very niche, but still pretty cool, technology. In the end, this won't have much impact on the company's bottom line as the quantity of users that were buying 3+ GTX GPUs for a single system was understandably small.
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Show support for Team Canada by wearing the Men's Adidas COC Ultimate Contrast Hoodie. It is made of ClimaWarm® lightweight and breathable insulating brushed fabric. The design details include handwarmer pocket, thumb holes and drawcord hood for superior comfort.
Please note that store inventory is updated online every 24 hours. As a result, do not hesitate to contact the store before going to confirm product availability.


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June restaurant openings and closings in metro Phoenix
Fifteen restaurants opened in June in metro Phoenix, including Pomo Pizzeria Napoletana, Virtu and Joyride Taco House. But more than half as many closed, including the Scottsdale and Phoenix branches of Scratch Pastries and the Mesa branch of Tilted Kilt Pub & Eatery.
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English heavyweight, Frank Bruno
English heavyweight, Frank Bruno
...and anyone else think pro bodybuilder Brandon Curry looks like a jacked up version of Coach Tomlin??
Originally Posted by rpmpit
Yeah, looks like coach, but he also looks like Tracy Morgan
Doesn't that mean he also looks like Omar Epps??
its brian fellow on roids
We all know the Omar Epps thing. I was trying someone newOriginally Posted by ikestops85
thats exactly what i thought when i saw the pic....Brian Fellows on roids.Originally Posted by frankthetank1
I almost forgot about the weekly look-alike contests we used to do. I'll have to start some advanced scouting of the Houston Texans' team bio roster pictures to prepare for week one.
The Rams' offense featuring weapons such as Marshall Faulk, Torrey Holt, and Isaac Bruce were known as "The Greatest Show on Turf"
The Steelers' offense featuring weapons such as Le'Veon Bell and Martavis Bryant should be known as "The Greatest Show on Grass"
This has nothing at all to do with respective playing surfaces at the Edward Jones Dome vs. Heinz Field.
2015 MNF Executive Champion!
those were my favorite!!Originally Posted by RuthlessBurgher


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Thank you to those that joined us at One Marylebone for the event
On Wednesday 25th November, we held our event "A Culture of Innovation" at One Marylebone. Watch the video to see some of the best moments from the night.
Is a culture of innovation something you can create or are organisations simply born with it? This controversial question is one many executives are grappling with as they face losing market share to more agile and innovative competitors.
The event brought together a selection of influential and inspiring speakers to give their thoughts on innovation and answer the question, are some of us just born to innovate?
You can also check our event hashtag #PAInnovate15 for key insights from the night.
The audience heard from a series of influential speakers on the evening and were inspired by their original and inventive approaches to innovation in their industry.
Sir Ben Ainslie, Land Rover BAR's team principal and skipper and four times Olympic sailing gold medallist
Laurence Bret-Stern, Senior Marketing Director, EMEA & LATAM, LinkedIn
Marc Koska OBE, Inventor and founder of Safepoint
Yvonne Rodgers, COO, Barclays Global Financial Crime division
Jonathon Hogg, PA expert in people and talent
Sir Ben Ainslie, Land Rover BAR's team principal and skipper and four times Olympic sailing gold medallist Sir Ben Ainslie has set out to achieve something that has never been done before; to bring the America's Cup home to Britain. To do so, he has brought together and inspired a world class sailing, technology and business team, with innovation at its heart.
Jonathon Hogg, PA expert in people and talent Jonathon leads our people and talent team, who help our clients build high-performing leadership teams and attract and develop the right people to grow their business. His current passion lies in helping organisations become more open to creativity and innovation – something he believes is essential for future economic prosperity. Marc Koska OBE, Inventor and founder of Safepoint Marc Koska OBE is the inventor of the K1 and following Auto-Disable syringes, the founder of Safepoint Trust (a UK-registered charity) and a highly regarded advocate around the world for needle safety in both immunisation and therapeutic sectors. His mission in life is the mandatory use of auto-disable syringes globally, to reduce cross-infection through dirty needles and needle-stick injuries, the world's ninth biggest killer annually.
Yvonne Rodgers, COO, Barclays Global Financial Crime division Yvonne is widely experienced executive with strong strategic execution focus. Yvonne has in-depth experience of global markets, having spent several years living and working in US, Thailand, Singapore, China. She has led and implemented several multi-country Merger/Acquisition transactions involving both revenue development and restructuring. Yvonne is currently the Chief Operating Officer for Barclays Global Financial Crime division. Laurence Bret-Stern, Senior Marketing Director, EMEA & LATAM, LinkedIn As Senior Marketing Director for the EMEA and LATAM Regions, Laurence Bret is responsible for supporting LinkedIn largest line of business – Talent Solutions, through lead generation, lifecycle nurturing, sales enablement and thought leadership programs. Prior to LinkedIn, Laurence worked in the US, the Netherlands and France where she held senior marketing positions at Orange and AOL and participated to the launch of Silentale, an enterprise data management company. She is now based in London and heads up an international team located in 10 different countries. Laurence holds a Post-graduate degree from Dauphine University.
Is innovation in your organisation broken? Our latest research, which surveys 750 senior business and government executives across 15 countries, reveals many organisations are wasting their best ideas. We found that 47% of senior executives describe their innovation activity as a 'costly failure' and 50% have seen a brilliant idea fail for avoidable reasons. Our research raises a number of vital questions for leaders responsible for delivering innovation.
Download the report
At PA we don't just believe in making a difference. We believe in making the difference.
We define success as achieving exceptional results that have a lasting impact on businesses, communities and individuals worldwide. This principle has remained the cornerstone of our ethos since 1943 - and it continues to underpin everything we do.
Find out how we make the difference and see some examples of innovation in practice by reading some recent client stories.
Register here
To find out more about our recent Innovation event, contact our speakers below:
Alan Young PA IT transformation expert
Email | LinkedIn
Jonathon Hogg PA people and change expert
What are your thoughts?
Please note this event is suitable for senior professionals with the following job titles: Head of, Director, CxO or equivalent.
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Acai berry
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Acai Benefits: Acai is a fruit that is only grown in South America. It has 500% more antioxidants than blueberries, which is why it also gives you radiant skin. It contains omegas, vitamin C, vitamin E, potassium, calcium and fiber! Make an Acai bowl or add it to your smoothies.
Acai berry powder benefits the body in hundreds of ways and has been shown to improve overall health and wellness
There are many benefits to eating blueberries, acai berries, and goji berries (also called wolfberry). #HealthyBerries
Acai berry has a wide array of phytonutrients. Its range of ANTIOXIDANTS, amino acids & OMEGA FATTY ACIDS can help protect the tissues & cells of the body & make this fruit an ANTI-AGING, immune boosting superfood. It is also rich in B VITAMINS, ELECTROLYTES & TRACE MINERALS that are helpful for reducing inflammation & improving energy. The acai berry's ability to eliminate free radicals & reduce oxidative stress also assist your body with DISEASE PREVENTION, WEIGHT LOSS, & METABOLIC…
Add the superfood acai berry to your breakfast with this healthy smoothie bowl recipe.
This beautiful, colorful, fruity triple berry coconut acai bowl is the perfect way to start your day! It's vegan, healthy, and it has freeze-dried acai powder in it, a superfood loaded with antioxidants and essential amino acids.
Acai-Eistorte. | Foodlovin'
Acai berries
Antioxidant packed acai bowl recipe + how to use less acai berry powder but keep the taste and health benefits.
Top 10 Super Foods: coconut oil, Goji Berries, acai, kelp, maca root, bee pollen, chia seeds, spirulina, manuka honey, cacao | @andwhatelse