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Ionotropic glutamate receptors (iGluRs) mediate the majority of excitatory neurotransmission in the central nervous system and function by opening a transmembrane ion channel upon binding of glutamate. Despite their crucial role in neurobiology, the architecture and atomic structure of an intact iGluR is unknown. Here we report the crystal structure of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive, homotetrameric, rat GluA2 receptor at 3.6 Å resolution in complex with a competitive antagonist. The receptor harbors an overall axis of 2-fold symmetry with the extracellular domains organized as pairs of local dimers and with the ion channel domain exhibiting 4-fold symmetry. A symmetry mismatch between the extracellular and ion channel domains is mediated by two pairs of conformationally distinct subunits, A/C and B/D. Therefore, the stereochemical manner in which the A/C subunits are coupled to the ion channel gate is different from the B/D subunits. Guided by the GluA2 structure and site-directed cysteine mutagenesis we suggest that GluN1/GluN2A NMDA (N-methyl-d-aspartate) receptors have a similar architecture with subunits arranged in a 1-2-1-2 pattern. We exploit the GluA2 structure to develop mechanisms of ion channel activation, desensitization and inhibition by non competitive antagonists and pore blockers.
The development and function of the human brain, and its remarkable capacity for experience dependent change, hinges on the organization and dynamics of chemical synapses, communication 'contact zones' between neurons. At these specialized sites, chemical transmitters released from presynaptic terminals diffuse across the synaptic cleft and activate receptors localized primarily on the postsynaptic cell1, thereby transmitting the flow of information from one neuron to another. Glutamate is the chemical transmitter of excitatory synapses in the central nervous system2, 3 and receptors for this ubiquitous neurotransmitter are of two classes: metabotropic and ionotropic4. Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels fundamental to neurotransmission at excitatory synapses and are implicated in nearly all aspects of nervous system development and function5. iGluRs are also involved in chronic neurodegenerative conditions, in psychiatric disorders and in acute injury or trauma6-9.
Comprising the iGluR receptor family are α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; GluA1-4), kainate (GluK1-5) and N-methyl-D-aspartate (NMDA; GluN1; GluN2A-D; GluN3A-B) receptors10-14. Whereas NMDA receptors are obligate heterotetramers14, AMPA and kainate subunits form functional homotetramers10-12, 15, although native receptors are almost exclusively heterotetramers16-18. Each subunit has a modular composition19 and includes a large extracellular amino-terminal domain (ATD)20 that participates in subtype-specific receptor assembly, trafficking and modulation, a ligand-binding domain (LBD) central to agonist/competitive antagonist binding and to activation gating21, a transmembrane domain (TMD) that forms the membrane-spanning ion channel22 and a cytoplasmic C-terminal domain involved in receptor localization and regulation23.
AMPA, kainate and NMDA receptors are related in amino acid sequence yet they diverge with respect to function5, 34. Whereas non NMDA receptors exhibit kinetics of activation, deactivation and desensitization on a millisecond time-scale35, NMDA receptors are slower, with corresponding molecular processes occurring on a time-scale of tens to hundreds of milliseconds36. Furthermore, AMPA and kainate receptors only demand glutamate for activation, while NMDA receptors function as coincidence detectors, requiring membrane depolarization to relieve magnesium block37 together with binding of glycine38 and glutamate. AMPA receptors sojourn through multiple sub conductance states contingent upon agonist concentration15, 39, suggesting independent LBDs and a sequential mechanism of activation40. AMPA and kainate receptors undergo profound desensitization, while NMDA receptors desensitize by way of glycine-dependent and glycine-independent mechanisms41.
The pharmacology of iGluR family members is distinct. AMPA receptors, for example, are non competitively antagonized by small molecule binding to the juxta membrane region42. By contrast, the ATDs of NMDA receptors harbor binding sites for polyamines, protons, zinc ions and ifenprodil36, 43. In AMPA receptors, the LBD possesses binding sites for modulators of receptor desensitization and deactivation26, 44. All iGluR subtypes, however, possess binding sites for pore blockers within the transmembrane ion channel45.
Despite divergent functional properties, iGluR family members have a common structural design. Clues to the symmetry and architecture of iGluRs derive from studies of isolated domains, demonstrating that ATDs and LBDs assemble as dimeric entities24-28, from electron microscopy on intact receptors, showing an overall 2-fold symmetry29, 30 and from amino acid sequence analysis and biophysical studies suggesting a ~4-fold symmetric ion channel19, 31. Absent from our understanding, however, is an accurate, atomic resolution description of iGluR architecture and symmetry, a definition of subunit arrangement in homomeric AMPA and heteromeric NMDA receptors32, 33, and proof of the symmetry mismatch between the 2-fold symmetric extracellular domains and the presumably 4-fold symmetric ion channel26. To answer these fundamental questions, we embarked on crystallographic and functional studies of a full length eukaryotic iGluR.
We exploited fluorescence-detection size exclusion chromatography (FSEC) 46 to discover that the rat GluA2 receptor10, 11, expressed as the unedited47, 'flip' variant48, was a promising candidate for structural studies (Supplementary Fig. 3a). We further harnessed FSEC to find that n-undecyl-β-D-thiomaltoside, the competitive antagonist ZK 20077549 and a modified receptor polypeptide, deemed GluA2cryst, were the optimal detergent, ligand and protein construct for crystallization trials, respectively (Supplementary Figs. 1, 2, 3b-d, 4-6). GluA2cryst binds 3H AMPA with a Kd of 3.5 ± 0.5 nM and yields glutamate-gated currents similar to the wild-type receptor (Supplementary Figs. 7 and 8). Crystals of GluA2cryst belong to the P1 space group, contain one tetrameric receptor in the unit cell and diffract to 3.6 Å resolution (Supplementary Fig. 9; Supplementary Table 1).
We solved the GluA2cryst structure by molecular replacement, using the high resolution structures of the isolated GluA2 ATD27 and the antagonist-bound form of the isolated GluA2 LBD 25 as search probes. Phases were improved by multidomain non crystallographic symmetry (NCS) averaging, solvent flattening and histogram matching. The resulting electron density maps were of sufficient clarity to build the transmembrane helices comprising the ion channel domain and the linking polypeptides connecting the LBD to the ion channel and the ATD to the LBD (Supplementary Figs. 10-12). The weakest density was observed for the S2-M4 linker connecting the LBD and the M4 transmembrane domain. Residues on the cytoplasmic side of the membrane connecting membrane helices M1 to M2, and M2 to M3, were not visible in electron density maps.
We probed the veracity of the polypeptide trace by preparing selenomethionine (SeMet)-labeled receptor of GluA2cryst and of 4 methionine point mutants at Leu542 (M1), Gln586 (Q/R site, M2)47, Ile612 (M3) and Val800 (M4) and by inspecting the corresponding anomalous difference Fourier maps. We formed crystals with a mercury-labeled variant of ZK200775, measured diffraction data at the Hg LIII peak, mapped the antagonist binding site in the LBDs, and found additional mercury binding sites, presumably due to residual methyl mercury chloride (Supplementary Table 1; Supplementary Figs. 13-16). Taken together, the diffraction experiments support the experimental structure, which was refined to good crystallographic statistics and stereochemistry (Supplementary Table 2).
The GluA2 receptor has a shape like the capital letter 'Y' where the three major domains are arranged in layers (Fig. 1a, b). The TMDs form the ion channel and define the narrow 'base', the ATDs are splayed outward, like diverging prongs, at the 'top' of the 'Y', and the LBDs, complexed with antagonist molecules, are sandwiched in between the ion channel and ATDs. In this closed, antagonist-bound state of the receptor there are no prominent vestibules or cavities near the ion channel domain and instead the LBD layer rests like a thick slab on top of the ion channel pore (Supplementary Fig. 17).
The symmetry and subunit arrangement of the tetrameric GluA2 receptor is without precedent. The receptor has an overall yet approximate 2-fold axis of molecular symmetry oriented perpendicular to the membrane plane (Fig. 1c, d; Supplementary Fig. 16). This 2-fold axis of symmetry relates one ATD dimer27 to another, one LBD dimer26 to the second, and half of the pore-forming TMDs to the other half. The ion channel domain has an approximate 4-fold axis of rotational symmetry (Fig. 1c, d).
Each prong of the receptor 'Y' is defined by an ATD dimer in which the 'local' dimer 2-fold axis is oriented ~24° off of the overall 2-fold axis (Fig. 1c, d). 'Below' the ATDs are the LBDs, also organized as a pair of dimers in which the local 2-fold axes within each dimer are also tipped off the overall 2-fold axis of symmetry by ~19° and are thus not aligned with the 'local' ATD 2-folds. "Below" the LBDs the 4-fold axis of the ion channel is approximately aligned with the overall 2-fold axis of symmetry. The multiple, non aligned axes of local symmetry, together with the 2-fold symmetry of the LBDs and the 4-fold symmetry of the ion channel, result in symmetry mismatches between the ATDs, LBDs and TMDs.
The ATD, implicated in receptor assembly, trafficking and localization, forms two distinct types of subunit-subunit contacts (Fig. 2). On the one hand, within each ATD 'dimer', there are extensive subunit - subunit contacts (A-B or C-D) that are indistinguishable from the contacts seen in the high resolution crystal structures of the isolated GluA2 ATDs27 (Supplementary Fig. 18a). On the other hand there is an interface between ATD dimers (~330 Å2), located on the overall axis of 2-fold symmetry, that is mediated by residues on the L2 lobes of the B and D subunits (Fig. 2b). Within the context of the ATD 'layer', we define the B/D and A/C subunits as 'proximal' and 'distal' to the overall 2-fold axis of symmetry. The B-D, dimer-dimer interface, while small, is observed in the packing of GluA2 ATD dimers in crystals of the isolated ATD27 (Supplementary Fig. 18b). We suggest that the ATD B-D interface, together with the subunit 'crossover' between the ATD and LBD layers, provides a molecular explanation for the role that the ATD plays in the assembly and stability of the tetrameric receptor.
At the LBD layer each agonist binding domain is also a partner in readily identifiable 'dimers' and these dimers, in turn, interact across the overall 2-fold axis (Fig. 2c). As a consequence of the subunit crossover between the ATD and LBD layers, the local LBD dimers are formed by the A-D and B-C subunits, with the A and C subunits and the B and D subunits proximal and distal to the overall 2-fold axis, respectively. Within a LBD dimer there are multiple contacts between domain 1 of each subunit, recapitulating the interactions seen in the high resolution crystal structures of the isolated, water-soluble GluA2 LBD25. In the GluA2cryst structure, which corresponds to an antagonist-bound, non desensitized state, the domain 1 - domain 1 interface is 'intact', as visualized in the wild-type25, the Leu483 to Tyr mutant or the cyclothiazide26 and aniracetam-bound 44 structures of the isolated LBD. Domain 2, by contrast, does not participate in significant intersubunit interactions within a LBD dimer, a finding that is also in harmony with high resolution studies of the isolated LBD50. Lodged in the 'clamshell' of each LBD is a bound antagonist, thus proving that the agonist/competitive antagonist binding site is located within and not between subunits.
There is a small (~224 Å2) interface between LBD dimers, an area consistent with weak dimer - dimer interactions26, 51. Like the ATD dimer - dimer interface, the LBD dimer interface is located on the overall 2-fold axis of symmetry and is composed primarily of residues at the 'bend' between helices F and G, with the α-carbon atoms of residues Lys 663, Ile 664 and Ala 665 of subunits A and C 8-13 Å apart. The relationship between LBD dimers is not perfectly 2-fold symmetric, however, and whereas residues at the end of helices G and K of the A and B subunits (Ser 676 and Lys 770) are in van der Waals contact (Cα-Cα distance of 6.1 Å), the equivalent residues in subunits C and D are not in contact (Cα-Cα distance of 9.7 Å).
Viewed from the extracellular side of the membrane down the overall 2-fold axis of symmetry, four GluA2cryst subunits arrange their transmembrane domains around an axis of ~4-fold rotational symmetry (Fig. 2d). In harmony with topology studies52, 53, each subunit has 3 transmembrane helices (M1, M3 and M4), a central pore-like helix (M2)54 and a polypeptide pore-lining loop that is disordered in our electron density maps (Fig. 3a, b). Leading from the LBD, the S1-M1 polypeptide segment adopts an extended conformation until reaching the TMD, at which point the polypeptide forms a ~90° turn and initiates a short helix (pre-M1) oriented nearly parallel to the membrane. The pre-M1 helix acts like a cuff around the 'top' of the ion channel domain, making contacts with carboxyl and amino terminal ends of helices M3 and M4, respectively. M1 is the first bona fide transmembrane segment and it resides on the exterior of the ion channel domain (Fig. 3c). Within the pore lies the M2 helix, positioned largely on the basis of tube-shaped electron density and the anomalous difference density peak from the SeMet-labeled Gln 586 to Met (Q/R site) mutant. The M3 helices line the inside of the ion channel domain, are ~52 Å in length and, in the present, antagonist bound structure, cross at the level of the pre-M1 'cuff' helices, near the membrane - aqueous solution boundary, forming a ~12 Å occlusion of the putative ion permeation pathway. Residing on the exterior of ion channel domain is the M4 helix, connected to the S2 segment of the LBD by two turns of helix and a short extended region of polypeptide. There are extensive subunit-subunit interactions between the transmembrane segments with the M4 segment of one subunit making interactions primarily with the transmembrane domains of an adjacent subunit (Fig. 3d). These interactions provide a molecular basis for the crucial role of the M4 helix in receptor assembly and function22.
Cognizant that the GluA2cryst structure (Fig. 4a) possesses an unorthodox subunit arrangement and molecular symmetry, we tested whether the subunit arrangement and domain-domain contacts in the crystal structure reflect interactions adopted by the receptor in a non crystalline environment. To accomplish this, we introduced cysteine residues into the three unexplored interfaces at sites that should result in spontaneous disulfide bond formation (Fig. 4b-d). We purified the mutant, tetrameric receptors to homogeneity and probed the extent of spontaneous subunit crosslinking by gel electrophoresis under reducing and non reducing conditions (Fig. 4e-g; Supplementary Fig. 19).
At the ATD, dimer-dimer, B to D subunit interface, we introduced a cysteine at Val 209 (Fig. 4b). For the wild-type-like receptor, in the presence or absence of reducing agent, the GluA2 subunit migrates at a position consistent with its calculated molecular mass. By contrast, for the Val209 to Cys mutant, we observe reducing agent dependent dimer formation, thus supporting the presence of this dimer-dimer interface in the intact receptor under native conditions (Fig. 4e). In the LBD layer, there are two distinct subunit - subunit interfaces. One interface is within a LBD dimer and is formed by extensive contacts between domain 1 of 2-fold related subunits, faithfully mirroring the thoroughly documented dimer interface observed in the isolated LBDs of AMPA 25, 26, 51, kainate55 and NMDA56, 57 receptors. The second interface, between subunits (A and C) proximal to the overall 2-fold axis, is composed of only a handful of intersubunit contacts (Fig. 4c). We therefore tested whether residues in this interface could form inter-dimer disulfide crosslinks. At both Lys 663 and Ile 664 cysteine mutants formed redox dependent dimers (Fig. 4f), supporting the presence of this interface in the intact receptor. This LBD dimer-dimer interface is also important for agonist dependent gating because steady state currents of the Ile 664 to Cys mutant are potentiated ~5-fold following receptor reduction58 (Supplementary Fig. 20).
The apex of the ion channel domain, defined by the C-terminal ends of M3, provides an important test of the GluA2cryst structure, not only because residues at the end of M3 define the gate of the ion channel in this antagonist bound state, but also because the end of M3 and the M3-S2 linker span the transition between the 4-fold symmetry of the ion channel and the 2-fold symmetry of the extracellular domains. In two subunits (A/C), the Met 629 side chains point toward each other and the α-carbons are separated by ~12 Å whereas the corresponding α-carbon atoms in subunits B/D are 30 Å apart (Fig. 4d). In satisfying agreement with the GluA2cryst structure, we find that cysteines introduced at position 629 form redox dependent crosslinks (Fig. 4g), consistent with the proximity of Met 629 residues and the structure of the ion channel domain. We may not see complete crosslinking to a dimer position because of the overall 2-fold symmetry of the extracellular domains or because some of the cysteine residues may have suffered chemical modification during expression or purification.
We propose that the architecture of kainate receptors is similar to that of the GluA2cryst AMPA receptor based on the remarkable observation that the isolated GluR6 ATD dimer forms a similar dimer-of-dimers arrangement in the crystal lattice, yielding a 'tetrameric' complex28 like that in the GluA2cryst structure (Supplementary Fig. 21). Superposition of α-carbon atoms for 292 residues per subunit in the structurally conserved regions of GluA2cryst and isolated GluR6 ATD dimer-of-dimers yields a r.m.s. deviation of 3.2 Å. This observation, in combination with the fact that LBDs of AMPA and kainate receptors form similar local dimers55, demonstrates that principles of architecture and symmetry are conserved between AMPA and kainate receptors.
NMDA receptors are the most complex subfamily of ionotropic glutamate receptors, not only because they are obligate heterotetramers, requiring a glycine-binding GluN1 or GluN3 subunit together with a glutamate binding GluN2 subunit, but also because their ATDs bind ions and molecules that modulate receptor activity36. Even though the structure of the GluN1/GluN2A LBD heterodimer is known56, there is no conclusive experimental knowledge of how subunits are arranged in heterotetrameric NMDA receptors. To determine whether the GluA2cryst structure provides a paradigm for understanding NMDA receptor architecture, we carried out cysteine-directed crosslinking experiments on the rat GluN1-GluN2A NMDA receptor.
Guided by superpositions of the GluN1-GluN2A LBD heterodimer56 onto the GluA2cryst structure, we designed cysteine substitutions at putative interdimer interfaces. There are three possible arrangements of the GluN1-GluN2A LBD heterodimers within a heterotetrameric receptor and we can distinguish between these models depending on whether redox dependent crosslinking requires cysteine substitutions in only GluN1, in only GluN2A or in both GluN1 and GluN2A subunits (Supplementary Figs. 22 and 23). Remarkably, only a single cysteine substitution in GluN1 at Glu 699 is required to crosslink NMDA receptor subunits at the LBD level (Fig. 5a). Indeed, none of the 6 cysteine substitutions in GluN2A promoted crosslinking, while simultaneous substitutions in both GluN1 and GluN2A resulted in crosslinking similar to substitutions in GluN1 alone. Within the LBD layer of the NMDA receptor, therefore, we propose that GluN1 and GluN2A subunits form diagonal pairs with the GluN1 subunit proximal to the overall 2-fold axis of symmetry (Fig. 5b).
What is the arrangement of NMDA receptors within their TMD? Here we exploit the GluN1 and GluN2A equivalents of GluA2 residue Met 629. Because the GluN1 subunit is proximal to the overall 2-fold axis of symmetry at the LBD layer, we predict that the GluN1 residue, Pro 661, will be proximal to the overall 2-fold axis at the TMD layer and that the GluN2A residue, Phe 631, will be too far away to form a crosslink (Fig. 5c). In concordance with this prediction, GluN1 (Pro 661 to Cys)-GluN2A (wild-type) but not GluN1 (wild-type) - GluN2A (Phe 631 to Cys) receptors demonstrate redox dependent crosslinking (Fig. 5a). Even though we have not explored crosslinking of NMDA receptors ATDs, based on our observation that subunits switch proximity to the overall 2-fold axis between the ATD and LBD layers, we predict that the ATDs are assembled as 'local' heterodimers and that NR2 ATDs mediate the dimer-dimer contacts, proximal to an overall 2-fold axis of symmetry (Fig. 5d). This arrangement would appropriately position the L2 lobe of the GluN2A ATD to alter receptor structure and function by either modulating ATD dimer-dimer contacts or ATD-LBD interactions, ultimately influencing ion channel gating via perturbation of the LBD36, 57.
There is a remarkable swapping of domains, involved in 'local' dimers between subunits, that is illustrated by tracing neighboring polypeptide chains through the receptor. Within the ATD layer, subunits A and B interact with each other to form a local ATD 'dimer' (red dimer, Fig. 6a, d). When the polypeptide chains of subunits A and B pass to the LBD layer, however, subunit A forms a LBD dimer with the corresponding domain of subunit D (blue dimer, Fig. 6a), while subunit B associates with subunit C (orange dimer, Fig. 6d). Within the TMD, the transmembrane helices of subunits A and B form extensive contacts with each other as well as with subunits C and D (Fig. 3c, d).
The swapping of extensive local dimer interactions between subunits and the symmetry mismatch between the LBDs and TMDs mean that within this homotetrameric receptor, where each subunit is chemically identical, there are two conformationally different subunit pairs related by the overall axis of 2-fold molecular symmetry: subunit A is equivalent to C and subunit B is equivalent to D yet the A/C pair is distinct, in conformation, from the B/D couple (Fig. 6c, f). These differences in conformation can be illustrated by superimposing the LBDs of subunits A and B (Fig. 6b, e), showing the large differences in orientation of the flanking ATDs and TMDs. In subunit A or C, for example, there is a substantial interface between the ATD and LBD (~315 Å2) whereas in subunit B or D there are no similar contacts (Fig. 6b, e). Another fundamental difference is that the LBD of the B/D subunits is ~6 Å closer to the putative membrane plane than the A/C subunit pair.
The conformational difference between the two types of subunits is defined by polypeptides linking the ATDs, LBDs and TMDs that have the same amino acid sequence but adopt different conformations. These polypeptides also mediate symmetry transitions between domain layers. The symmetry mismatch between the ATDs and the LBDs, characterized by different orientations of their local two-fold axes of symmetry (Fig. 1c,d) and domain swapping (Fig. 6a,d), is mediated by the ATD-S1 linkers (Thr 377 - Lys 393). On the one hand, the ATDs of the A and C subunits interact with their LBDs via an ATD-LBD interface (Fig. 6a-c) and the ATD-S1 linkers adopt a more compact conformation. On the other hand, the ATDs of the B and D subunits are suspended between LBD dimers and the ATD-S1 linker nearly spans a LBD dimer, taking on an extended conformation (Fig. 6d-f).
On the extracellular side of the membrane, both the ATD dimers and the LBD dimers are arranged with an overall 2-fold symmetry. By contrast, the GluA2cryst TMD has 4-fold rotational symmetry (Fig. 3c). Where is the 2-fold to 4-fold symmetry mismatch structurally reconciled? We can answer this question by examining selenium and mercury positions derived from SeMet-labeled receptor and from crystals grown with the mercury-containing antagonist. Selenium sites at Met 629, a residue within the highly conserved 'GluArgMetVal' sequence at the end of M3 immediately preceding the S2 segment, clearly show 2-fold symmetry (Fig. 7a,b). Mercury sites at Cys 528, near the extracellular end of M1, however, demonstrate unambiguous 4-fold symmetry (Fig. 7a,b). By viewing the receptor perpendicular to the overall 2-fold axis, and defining the approximate membrane boundary based on residue polarity, we see that Met 629 is just 'outside' of the membrane-spanning region whereas Cys 528 resides within the membrane bilayer. The 2-fold to 4-fold symmetry transition occurs within this region, a ~10 Å thick slab between the LBDs and TMDs (Fig. 7c).
To precisely define where the transition occurs, we applied the 4-fold rotational transformation associated with the TMDs to one of the receptor subunits and calculated the root-mean-square (r.m.s.) deviation of α-carbon atom positions between the superimposed subunits. Where the receptor conforms to 4-fold symmetry, the r.m.s. deviations in atom positions are on the order of the coordinate uncertainty. In 2-fold symmetric regions, however, the r.m.s. deviations in atom positions are much greater. Thus, by plotting the r.m.s. deviations in atom positions as a function of distance along the overall 2-fold axis, we can follow the 2-fold to 4-fold symmetry transition (Fig. 7d). This analysis demonstrates that the transition is abrupt and is located at the boundary of the extracellular leaflet of the membrane bilayer, within the region of the ion channel domain encircled by the pre-M1 'cuff' helical segments.
The symmetry mismatch between the LBDs and the TMDs is resolved by 3 linking peptides - the S1-M1 (Lys 506 - Gly 513), M3-S2 (Val 626 - Glu 634), and S2-M4 (Gly 774 - Ser 788) linkers - making the transition from the 2-fold, parallelogram-like symmetry of the LBDs, to the 4-fold symmetric, square geometry of the TMDs (Fig. 8a). Indeed, these are the central elements of the iGluR gating machinery that transform ligand-induced structural changes in the LBD dimers into the movement of the transmembrane domains that opens and closes the central pore of the 4-fold symmetric ion channel. In bridging 2-fold to 4-fold symmetry transition, the peptide segments linking the LBDs and TMDs can be grouped into two pairs that belong to diagonal subunits A/C or B/D; within each group, the linkers adopt approximately similar conformations whereas between the groups the conformations are clearly distinct.
Accommodation of the 2-fold to 4-fold symmetry mismatch is illustrated by the different conformations of the S1-M1 linkers which, when passing from the β7 strand of the LBD to M1 (TMD), either come from inside (A/C) or outside (B/D) of the 'M1 circle' (Fig. 8b). The M3-S2 linker provides a particularly striking example of how the conformations of the A/C and B/D linking peptides differ. In the A and C subunits, the M3-S2 linker adopts a helical conformation to Met 629 (Fig. 4d). For the B and D subunits, by contrast, the helical conformation is broken at Val 626 and following this residue, the peptide adopts an extended conformation. The difference in main chain conformation means that the α-carbon atoms of Pro 632 in the A and C subunits are ~27 Å apart whereas the corresponding atoms in the B and D subunits are separated by ~50 Å (Fig. 8c). A third striking illustration is provided by Cys 773, a conserved residue at the end of helix K of the LBD. For Cys 773, in subunits B and D, the α-carbons are 33 Å apart and in the A and C subunits, the span is much larger, 69 Å. To reach the M4 in TMD, which possesses 4-fold symmetry and where the distances between the same atom in the A/C and B/D subunits are necessarily the same, the A/C and B/D S2-M4 linkers take on different conformations and different orientations relative to the overall 2-fold axis (Fig. 8d).
The 4-fold rotationally symmetric GluA2cryst ion channel is shaped like a Mayan temple with a broad cytoplasmic base, ~42 Å on a side, and with a bluntly pointed extracellular 'top'. In the present, competitive antagonist-bound state, the ion channel unambiguously adopts a closed conformation by the crossing of the M3 helices (Fig. 9). The crossing of the helices occurs near the highly conserved Ser-Tyr-Thr-Ala-Asn-Leu-Ala-Ala-Phe (SYTANLAAF) motif, with Thr 617, Ala 621 and Thr 625 occluding the ion channel permeation pathway22, 59. In the Lurcher mouse60 there is a substitution of Ala 636 by Thr in the GluD2 subunit, leading to spontaneously opening ion channels61. The equivalent residue in GluA2 (Ala 622) participates in close contacts with the M3 helix of a neighboring subunit, suggesting that introduction of bulky residues can directly destabilize the tight helix crossing associated with the resting, closed state of the receptor, leading to constitutively open ion channels. The ion channel permeation pathway is also occluded above the SYTANLAAF motif by a pair of Met 629 residues on the A/C subunits protruding their side chains towards the center of the pore. Remarkably, these methionines are adjacent to the Glu 627-Arg 628 motif that, when mutated, strongly perturbs receptor desensitization62.
Stimulated by hypotheses19 and experimental data31, 63 proposing a common architecture for the ion channel pores of iGluRs and K+ channels, we superimposed the transmembrane domains of GluA2cryst onto the bacterial K+ channel KcsA54 (Fig. 10, Supplementary Fig. 24). Despite the low pairwise identity of ~20% between the aligned amino acid sequences of the rat GluA2 receptor channel and KcsA (Supplementary Fig. 25), the M1, M2, and M3 segments of GluA2cryst overlap remarkably well with structurally equivalent portions of KcsA (Fig. 10a). In fact, superposition of α-carbon atoms for 64 residues per subunit in GluA2cryst and KcsA yields a r.m.s. deviation of 2.2 Å. Not included in this comparison was transmembrane segment M4, which is absent in KcsA and bacterial glutamate receptors64. In addition to the overall similarity, the structural alignment demonstrates that the occlusion of the ion conductive pathway, i.e. the regions of these channels involved in gating and defined by the carboxyl terminal region of M3 in GluA2cryst and TM2 in KcsA, is remarkably similar (Fig. 10b). By contrast, the region of KcsA that confers ion selectivity - the extended Thr-Val-Gly-Tyr-Gly selectivity filter - is completely different in amino acid sequence in iGluRs and is disordered in the GluA2cryst structure, observations consistent with the fact that K+ channels are highly selective and iGluRs are not.
The pre-M1 'cuff' helix is oriented nearly parallel to the membrane, at the interface between the membrane and extracellular solution and is a structural feature that underscores similarities and differences between iGluRs and K+ channels65. Appearing as a 'collar' around the TMD bundle, the pre-M1 helices may restrain the mobility of M3 in the closed state and, by virtue of its connection, via the S1-M1 linker, to the LBD, promote opening of the ion channel gate upon agonist binding to the LBD. Whereas some K+ channels have a similar element of structure65, the length of the helical segment as well as its conformation relative to the ion channel diverge from the GluA2cryst pre-M1 helix. In fact, Pro 520 in the 'elbow' of the GluA2cryst pre-M1 is highly conserved across iGluR subtypes yet is missing in K+ channels (Supplementary Fig. 25), possibly suggesting a distinct role of the pre-M1 element in iGluR structure and gating.
Immediately 'above' the Q/R site, clearly within the transmembrane portion of the ion channel domain, is a central cavity (Fig. 9a) similar to that observed in potassium channels54. In the GluA2cryst structure there are conspicuous 'gaps' between the transmembrane domains that result in a portal between the central cavity, within the ion channel, and the membrane environment. We speculate that this portal may be occupied by residues projecting from the transmembrane domains of AMPA receptor auxiliary subunits66, 67, thus providing a mechanism by which auxiliary subunits modulate ion channel properties, such as the extent of block by polyamines68.
The amino acid sequence and structural relationships between GluA2cryst and K+ ion channel pores allow us to speculate on the conformation of an iGluR in an open channel state (Supplementary Fig. 25 and 26). On the basis of superimposing the ion channels of GluA2cryst and Shaker65, we suggest that the transmembrane helices of iGluR will bend and splay away from the central axis of the channel, mimicking the iris-like opening of K+ channels. Although roughly similar in overall nature, the TMD movements during activation gating of iGluRs and K+ channels may be different in molecular detail69. In fact, the residues lining the ion permeation pathway or the amino acids that perturb gating via genetic or chemical modification, together with the types of non-competitive antagonists and channel blockers that bind to the TMD, both in AMPA (Supplementary Fig. 27) and NMDA (Supplementary Fig. 28) receptors, are highly specific to iGluRs, thus providing further evidence of distinction with potassium channels.
The GluA2cryst structure allows us to interpret decades of studies in the context of an intact receptor. Most fundamentally, the GluA2cryst structure proves that agonist binding sites are located within individual subunit 'clamshells' and not between subunits, as is the case with trimeric P2X70 and pentameric Cys-loop71 receptors. This architectural principle is consistent with the observations that binding of multiple agonist molecules and subsequent ion channel activation are largely stepwise, sequential processes15. Indeed, the independence of LBD function is further supported by the fact that they can be genetically excised and studied as isolated soluble domains72. Together, these properties justify exploiting the wealth of structural and biophysical experiments on soluble LBDs of AMPA25, 58, 73, kainate55, 74 and NMDA receptors56, 75, 76 to illuminate principles of gating in full length iGluRs.
The GluA2cryst structure is a complex with the high-affinity competitive antagonist ZK 200775. Antagonist binding stabilizes the binding domain 'clamshell' in an open conformation (Supplementary Fig. 29)25 and, in the context of the LBD dimer, places the transmembrane associated linker regions closest together (Supplementary Fig. 30). The conformation of the LBD trapped by ZK 200775 while similar to the apo, resting state, reported for the isolated LBD25, is nevertheless more open or overextended (Supplementary Fig. 31). Binding of full agonists, such as glutamate, quisqualate or AMPA result in closure of the clamshell by movement of domain 2 closer to domain 1 by a ~25° rotation (Supplementary Figs. 30,31)25, 77. In the context of the water-soluble LBD dimer, closure of both clamshells increases the separation of the regions linking the binding domains to the transmembrane domains by ~20 Å, using the α-carbon of Pro 632 (M3-S2 linker) as a reference (Supplementary Fig. 30). This movement, therefore, 'pulls apart' the M3 helices at the bundle crossing, opens the ion channel, and is the fundamental conformational change, within the binding domains, that transmits the energy associated with agonist binding to the work required to open the ion channel.
Superposition of two 'dimer' structures of the soluble LBD glutamate complex25 onto the LBDs of the GluA2cryst structure allows us to visualize activation-related movements in the context of the tetrameric receptor (Fig. 11, Supplementary Fig. 32). Using α-carbon atoms of residues Lys 506, Pro 632, and Cys 773 as reference points for the S1-M1, M3-S2, and S2-M4 linkers, respectively (Supplementary Fig. 33), the most significant movement involves the M3-S2 linker, an observation in agreement with the critical role of the M3 helix in channel gating. As a consequence of the overall 2-fold symmetry relating one ligand binding domain dimer to the other, the movements of the M3-S2 linkers are ~2-fold symmetric, i.e. there are large conformational changes within dimers and smaller changes between dimers (Fig. 11c, d), consistent with chemical modification studies suggesting a breakdown in the 4-fold symmetry of the ion channel upon receptor activation78. Augmenting the intradimer motions of this simple model, there is almost certainly a perpendicular component of movement, between LBD dimers, to facilitate opening of the ~4-fold symmetric pore. A component of movement between dimers is supported by the observation that reduction of the Ile 664 disulfide bridge potentiates glutamate-induced GluA2 currents58, 79 (Supplementary Fig. 20).
The differences in position and conformations of the LBD - TMD 'linkers' between the A/C (proximal) and B/D (distal) subunit pairs also mean that the consequences of agonist binding and LBD clamshell closure on the TMD must necessarily be distinct (Fig. 11c, d). We can see that by measuring the distance between a reference residue, such as Pro 632 in the M3-S2 linker, and the overall 2-fold axis of symmetry, an axis which is approximately coaxial with the TMD 4-fold axis (Fig. 11c, d). In doing this, we see that the α-carbon of Pro 632 is ~13 Å and 25 Å distal from the 2-fold axis for the A/C and the B/D subunits, respectively. Upon agonist binding and LBD closure, we predict that the α-carbons of Pro 632 will move away from the 2-fold axis by ~4 Å and 7 Å for the A/C and B/D subunits, respectively. Thus, the extent of conformational movement for the proximal (A/C) and distal (B/D) subunits is substantially different and is greater for the distal (B/D) subunits. We therefore hypothesize that not only will the consequence of agonist binding to ion channel gating be different for the proximal (A/C) and distal (B/D) subunits, but that the agonist-induced conformational changes in the distal subunits may be more important to activation gating, simply because the extent of the predicted conformational change is larger. Conversely, we suggest that agonist-induced conformational changes in the proximal subunits may play a comparatively smaller role in activation gating. This explains, at least in part, why in NMDA receptors, glycine binding to the GluN1 subunit, which we predict occupies the proximal position in the LBD layer and thus transmits a smaller conformational displacement to the TMD in comparison to the distal subunits, does not result in significant ion channel opening in the absence of glutamate.
A hallmark of AMPA and kainate receptors is rapid and profound desensitization, or ion channel closure, following receptor activation12, 48. The molecular principles of desensitization and the structural relationships between the resting/closed and desensitized states of the ion channel pore are not yet understood for an intact receptor. To address these questions we can exploit the desensitized-like structure of the Ser 729 to Cys (S729C) mutant of the isolated LBD dimer58. By superimposing the desensitized-like S729C dimer onto the GluA2cryst structure we find that both D2 lobes of the isolated dimer superimpose well on the corresponding lobes of the tetrameric receptor, even though the isolated domains are bound with glutamate (closed clamshell) and in GluA2cryst, the LBDs are 'open' and bound with antagonist. This analysis demonstrates that peptide linkers connecting the LBDs to the TMDs can adopt a similar separation in the antagonist-bound GluA2cryst state and in the glutamate-bound S729C desensitized-like dimer form (Fig. 12 and Supplementary Fig. 30). Desensitization therefore simply involves rupture of the LBD D1-D1 interface and rotation of entire binding domain subunits to allow for the D2 domains and the linkers to the ion channel to adopt a closed state-like conformation.
In the context of the GluA2cryst structure, rearrangement of D1-D1 LBD interface during desensitization (Fig. 12c) demands movement of the ATDs and the ATD-LBD linkers (Fig. 12b). Upon receptor desensitization, we predict changes in both the distances between and within ATD dimers. These observations provide mechanisms, grounded in three-dimensional structure, by which binding of ions and small molecules to the ATDs of NMDA receptors can modulate receptor function80. Ligands alter the conformation of the ATD 'clamshell' and propagate these conformational changes throughout the receptor either directly, through the ATD - LBD linkers, or indirectly, via changes in the ATD dimer-dimer contacts across the overall 2-fold axis of symmetry.
The GluA2cryst structure uncovers domain organization and molecular symmetry of iGluRs. Crystallographic and site-directed chemical modification data demonstrate that AMPA, kainate and NMDA receptors have ~2-fold symmetric 'dimeric' extracellular domains, a ~4-fold symmetric ion channel, and a symmetry mismatch that renders diagonally related subunit pairs distinct. In heterotetrameric GluN1/GluN2A NMDA receptors, subunits are positioned in a GluN1-GluN2A-GluN1-GluN2A pattern. Activation gating of iGluRs originates within individual LBDs with agonist binding inducing closure of the clamshell, separation of the transmembrane domain linkers, and opening of the ion channel gate. Desensitization (inactivation) results from rupture of the agonist-bound LBD dimer D1-D1 interface, leading to rigid body rotation of individual LBD domains within the dimer and allowing for D2 domains, linkers to the ion channel and the ion channel gate to adopt a closed state-like conformation. Gating can be perturbed at many sites by small molecule inhibitors,and modulators and auxiliary protein subunits (Supplementary Fig. 34). The underlying architecture and symmetry of iGluRs, as revealed by the GluA2cryst structure, has implications for understanding how these molecules perturb receptor function.
The GluA2cryst – green fluorescent protein (GFP) fusion (see Supplementary Materials) was expressed in baculovirus-infected Sf9 insect cells and was purified using metal-affinity chromatography in buffers supplemented with 1.0 mM n-dodecyl-β-D-maltoside. Following cleavage by thrombin to remove GFP, the receptor was further purified by size-exclusion chromatography in a buffer supplemented with 1 mM n-undecyl-β-D-thiomaltoside and synthetic lipids. Crystallization was performed under paraffin oil, at 4° C, using a precipitating solution composed of 7-11% (w/v) PEG 20,000, 0.1 M 2-(N-morpholino)-ethanesulfonic acid (MES; pH 6.0-6.5) and 300 μM [[3,4-dihydro-7-(4-morpholinyl)-2,3-dioxo-6-(trifluoromethyl)-1(2H)-quinoxalinyl]methyl]phosphonic acid (ZK 200775). Selenomethionine-labeled receptor was prepared and crystallized using similar conditions. Diffraction data sets were indexed, integrated and scaled using HKL2000. Initial phase information was obtained by molecular replacement using the isolated amino terminal domains and ligand-binding domains as search probes. The ion channel and interdomain polypeptides were iteratively built using the computer graphics program COOT and the structure was refined using the computer program Phenix. The ion channel functional activity of the GluA2cryst construct was measured by whole-cell or outside-out patch clamp experiments. Ligand binding activity was evaluated by 3H-AMPA saturation and competition assays. NMDA receptor crosslinking experiments were done by transiently transfecting human embryonic kidney tsA 201 cells with wild type or mutant GluN1 and GluN2A plasmid DNA and analyzing oligomeric behavior of protein on gradient SDS PAGE gels by Western blot. Structure superpositions were done using Superpose (CCP4).
We thank the personnel at beamlines 5.0.2, 8.2.1, and 8.2.2 of the Advanced Light Source and at beamline 24-ID-E of the Advanced Photon Source. We also thank T. Homrichhausen for help with cloning and FSEC screening, L. Vaskalis for assistance with illustrations, and Gouaux lab members for discussion. M.P.R. was supported by an individual NIH National Research Service Award. This work was supported by the NIH. E.G. is an investigator with the Howard Hughes Medical Institute.
Supplementary Information is linked to the online version of the paper at www.nature.com/nature.
The authors declare no competing financial interests.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Alternative Title: acoustical horn
Learn about this topic in these articles:
loudspeaker systems
A horn enclosure uses a flared tube to obtain the best acoustic coupling between the loudspeaker cone and the outside, thereby radiating the best possible coherent wave from the speaker cone. Such a system is extremely efficient and is therefore used in public-address systems, open-air theatres, or other places in which great acoustic power is desired. Because a good quality bass horn enclosure...

subset: commoncrawl


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TALLAHASSEE (AP) -- One winner of the "Fantasy 5" game will collect $262,687, the Florida Lottery said Sunday.
The winning ticket was bought in Port Charlotte, lottery officials reported.
The 605 tickets matching four numbers won $70 each.
The numbers drawn Saturday night were 1-4-5-8-24.
© 2017. All Rights Reserved. Contact Us


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Gleeks, we're mourning with you. And so are Cory's friends and co-stars. RIP Cory Monteith.
Your first job or internship can be an eye-opening experience. Here are some realistic tips to help you succeed.
J.K. Rowling published a mystery novel under a pseudonym! We're just waiting for her to reveal her Figment username so we can read her HP fic. IT COULD HAPPEN.
Jay-Z claims to have 99 problems. One artist is on a mission to playfully illustrate all of them.
Need the perfect place to do your summer reading? We suggest The Reading Nest at the Cleveland Public Library.
Even corgis need to cool off! This adorable dog beats the heat in his own pool.


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THOUSANDS Testimonials from REAL PERSON who already received their orders : CLICK HERE
We sell ORIGINAL CIGARETTES for Cheapest price,
thats why we have more than 10.000+ SATISFIED customers...
Pall Mall brand cigarettes were introduced in 1899 by the Butler & Butler Company, in an attempt to cater to the upper class with the first \"premium\" cigarette. It is named after Pall Mall, a well-known street in London.
In 1907, Pall Mall was acquired by American Tobacco with the sale of Butler & Butler. The new owners used the premium brand to test innovations in cigarette design, such as the \"king-size\" (now the standard size for cigarettes at 85mm), and a new way of stuffing tobacco that supposedly made cigarettes easier on the throat.


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CLEARANCE: Nalu Blue and Hibiscus Reversible Kai Bottoms
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Gemlike colors will have you mesmerized. This kai is sure to turn heads and it's reversible!
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Roasted Red Kuri Squash Soup
Red kuri squash is a popular ingredient in France, where it is known as potimarron because its flesh has a slightly nutty quality that is reminiscent of the chestnut (marron, in French). In this recipe shared by The Cook's Atelier, a cooking school and epicurean center in Beaune, France, it is roasted to bring out its sweet, nutty flavor before it is pureed into a velvety cold soup with a beautiful color. For an extra-decadent dish, you can thinly shave some truffles on top.
- 3 to 3 1/2 lb. (1.5 to 1.75 kg) red kuri squash
- 2 Tbs. extra-virgin olive oil, plus more for brushing
- Sea salt and freshly ground pepper
- Leaves from 2 fresh sage sprigs
- 1 cup (3 oz./90 g) thinly sliced leeks, white and light green parts only
- 1/2 cup (2 oz./60 g) thinly sliced carrots
- 1/2 cup (2 oz.60 g) thinly sliced shallots
- 1/2 cup (2 oz./60 g) thinly sliced onions
- 6 garlic cloves, peeled and smashed
- 2 Tbs. honey
- 4 sprigs fresh flat-leaf parsley
- 3 sprigs fresh thyme
- 1 bay leaf
- 6 cups (48 fl. oz./1.5 l) vegetable stock
- 4 Tbs. (1/2 stick) (2 oz./60 g) unsalted butter
- 1/4 cup (2 oz./60 g) crème fraîche
- Freshly grated nutmeg for garnish
- Minced fresh chives for garnish
Preheat an oven to 350°F (180°C). Line a baking sheet with aluminum foil.
Cut the squash in half and scoop out and discard the seeds. Brush the inside of each squash half with olive oil and sprinkle with salt and pepper. Tuck a few sage leaves in into each squash half. Place the squash, cut side down, on the baking sheet and roast until completely tender, about 1 hour. Remove from the oven and set aside until the squash is cool enough to handle. Discard the sage leaves, then scoop the squash flesh into a bowl. Discard the peels.
In a stockpot over medium-high heat, warm the 2 Tbs. olive oil. Add the leeks, carrots, shallots and onions, and cook, stirring often, until the vegetables are tender, about 6 minutes. Add the garlic, 1 1/2 tsp. salt and 1/2 tsp. pepper and cook gently for 3 minutes, reducing the heat as necessary to keep the garlic from coloring. Stir in the honey and cook, stirring, for 2 to 3 minutes.
Meanwhile, make the bouquet garni: Place the parsley, thyme and bay leaf on a square of cheesecloth and tie closed with twine.
Add the bouquet garni and the vegetable stock to the stockpot. Bring to a simmer over medium-high heat and cook for 10 to 14 minutes. Add the roasted squash to the pot and continue to simmer gently until the flavors are blended, about 30 minutes. Remove the pot from the heat and discard the bouquet garni. Let the soup cool slightly.
Transfer the soup, in batches as necessary, to a blender and carefully puree until smooth. (Be very careful, as the hot soup may splash out of the blender.) Strain the soup through a fine-mesh strainer into a bowl, tapping the side of the strainer if necessary until the soup passes through. Taste the soup and season to taste with salt and pepper. Let cool to room temperature, then refrigerate until ready to serve.
When you're ready to serve the soup, in a saucepan over medium heat, warm the butter, stirring frequently, until all of the milk solids turn golden and the butter has a nutty fragrance. Be careful not to let the butter burn. Remove from the heat.
To serve, ladle the soup into serving bowls. Top each with a dollop of crème fraîche and a drizzle of the browned butter. Grate some nutmeg over the top, garnish with chives and serve immediately. Serves 6 to 8.
The Cook's Atelier, Beaune, France


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Afterschool Program
The YMCA Afterschool program is a value driven program that puts a strong emphasis on our YMCA core values: Caring, Honesty, Respect and Responsibility. We strive to provide every child with activities that encourage a healthy spirit, mind and body.
Please visit the Ryan Family YMCA to learn more about the Y's Afterschool Program.
The YMCA is one of the largest providers of child care in Tarrant County and serves children from infancy through pre-school. Click Here to learn more.


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[fin-lee, find-] /ˈfɪn li, ˈfɪnd-/
a city in NW Ohio.
Tagged: f
Read Also:
- Find the lady
noun 1. another name for three-card trick
- Find true north
Get on the right course, proceed in the right direction, as in We'd better find true north before our competitors do. This term alludes to locating the position of the North Pole from a particular point. [ ; late 1900s ]
- Fine
[fahyn] /faɪn/ adjective, finer, finest. 1. of superior or best quality; of high or highest grade: fine wine. 2. choice, excellent, or admirable: a fine painting. 3. consisting of minute particles: fine sand; a fine purée. 4. very thin or slender: fine thread. 5. keen or sharp, as a tool: Is the knife fine enough […]
- Fineable
[fahy-nuh-buh l] /ˈfaɪ nə bəl/ adjective 1. . [fahy-nuh-buh l] /ˈfaɪ nə bəl/ adjective 1. subject to a ; punishable by a . /ˈfaɪnəbəl/ adjective 1. a variant spelling of finable /ˈfaɪnəbəl/ adjective 1. liable to a fine


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Sherzai says in talks with rivals on alliance
Sherzai says in talks with rivals on alliance
JALALABAD (Pajhwok): Presidential contender Gul Agha Sherzai on Monday said he was in talks with a number of rival candidates on forming an election alliance and the outcome would be known soon.
"We are in negotiations with Abdul Rahim Wardak, Hidayat Amin Arsala, Qutbuddin Hilal and others to make an alliance. I will not quit in anyone's favour," Sherzai told Pajhwok Afghan News during an exclusive interview.
Sherzai, who arrived in eastern Nangarhar province as part of his campaign, came hard on President Hamid Karzai for his unstable domestic and foreign policies.
"Karzai sometimes calls Pakistan as a brotherly country and sometimes as an enemy," he said, adding corrupt individuals and warlords held key government positions during Karzai's government.
"Criminal groups trained during the incumbent president's tenure will create many problems after foreign troops are left," he said.
He viewed the ongoing peace process as a futile practice, saying the High Peace Council (HPC) was comprised of individuals, who had earlier fought against the Taliban.
He said the appointment of Salahudin Rabbani as the HPC chief after his father's assassination was part of the government's political compromises.
After winning elections, Sherzai plans to invite impartial tribal elders and religious scholars from across the country to a jirga which will be tasked with advancing the dialogue process with the Taliban and other armed opposition groups.
The former Nangarhar and Kandahar governor said Afghanistan's future was linked to transparent elections and urged the masses to exercise their right to vote.
Related Article
Download "Pajhwok" mobile App, on your smartphone to read and access latest news, features, interviews, videos and photos about Afghanistan.


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The purpose of iBelieve.com is to help women wrestle with the deeper issues of how their faith relates to the world around them. It seems that Christian women today are facing a set of challenges unique to their generation. With an emphasis on personal experiences, our goal is to create a team of writers and bloggers looking to share how they have seen the struggles and triumphs of life through the lens of the Christian faith. We hope to create a place where our audience can feel uplifted by authentic examination of what it means to be a Christian woman in today's culture.Read daily devotions for women, blogs by your favorite Christian female bloggers, articles on faith, relationships, health and beauty, food and home, and motherhood. iBelieve.com also provides Christian ecards and inspirations to encourage and uplift your sisters in Christ!
A Prayer of Blessing for St. Patrick's Day
By Debbie McDaniel
"For the Lord God is a sun and shield; the Lord bestows favor and honor; no good thing does He withhold from those who walk uprightly." Ps. 84:11
In 433 A.D., history tells us that St. Patrick was praying for God's divine protection in his work and ministry in Ireland while facing powerful enemies. He wrote this prayer called St. Patrick's Breastplate, which is still widely known and recited today.
God soon opened the door for him to share Christ with the Irish King Laoghaire and his subjects, and allowed him to be a part of bringing them out paganism's dark hold to the saving power of Jesus Christ. St. Patrick was a man who knew much about darkness, hardship, and enemy territory. He had spent years in slavery after being kidnapped by pirates at age 16. And it was in those years that he later tells how he learned about the Mighty power of God's Presence and prayer.
The life of St. Patrick is such an example to us still today, that even in the darkest of times, God's Sovereignty shines over His people. He can't help but to come to our aid. He can't help butto bless us and let His favor surround our lives. Because He's a good and loving God. And nothing escapes His notice. He watches over all, and we can trust that our lives and circumstances are held securely in His hands.
A Prayer of St. Patrick (from St. Patrick's Breastplate):
"As I arise today,
may the strength of God pilot me,
the power of God uphold me,
the wisdom of God guide me.
May the eye of God look before me,
the ear of God hear me,
the word of God speak for me.
May the hand of God protect me,
the way of God lie before me,
the shield of God defend me,
the host of God save me.
May Christ shield me today.
Christ with me, Christ before me,
Christ behind me,
Christ in me, Christ beneath me,
Christ above me,
Christ on my right, Christ on my left,
Christ when I lie down, Christ when I sit,
Christ when I stand,
Christ in the heart of everyone who thinks of me,
Christ in the mouth of everyone who speaks of me,
Christ in every eye that sees me,
Christ in every ear that hears me.
Dear God,
We thank you that your presence is powerful and real even today. We thank that your Word reminds us over and over again, that you are always with us. Thank you that you go before us, and that you cover us from behind. Thank you for the gift of your Spirit, that you live within us, bringing life and peace. Thank you for your protection over us, that you surround our lives as a shield. Thank you for your grace and favor, for your blessings and love that you so graciously shine over your people. We look to you today, our Lord and Savior, our Rock and Redeemer. Our trust and security are in you alone. Though things may often feel uncertain around us, though struggles may mount up and we feel like we're facing enemy ground, though darkness tries hard to steal our joy away, we choose to rest in you. Thank you Lord that you are our firm foundation. We walk in your strength and victory, as you pave out our paths, and lead our way. We praise you that you bless us and keep us, that you make your face shine over us. We love you Lord. We hold on to your truth, and rely on your presence in us, and around us.
In Jesus' Name,
Happy St. Patrick's Day, may God's favor and blessing cover you this day, and every day.
Find more by Debbie at www.debbiemcdaniel.com, Facebook, or Twitter.


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8/5/2008 (12:14 am) by Russell Jones
I recently installed an airport card (new) into my G4 Quicksilver Power Mac. When I play Itunes and send signal to my airport express (new), it plays for a few seconds the pauses for a couple of seconds. This continues. I've swapped airport cards, but nothing works. Any suggestions...anybody?


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Picture: Daft Punk - The 2013 MTV Video Music Awards - New York, NY, United States - Monday 26th August 2013
Daft Punk bassist Nathan East has recruited pals Stevie Wonder, Sara Bareilles and Michael Mcdonald to work on his first solo album.
The acclaimed musician has worked with popular artists such as Michael Jackson, Beyonce and Andrea Bocelli during his 30 years in the music industry, and now he's heading into the studio for an album of his own.
East will be releasing a yet-to-be-titled record next year (14), which will contain both original songs and covers.
He has already tapped Wonder, MCDonald and Bareilles for the record, but is still hoping to enlist Eric Clapton and Anita Baker, who he's also worked with in the past.
East is also in talks for creating an accompanying documentary about the process of making the album.


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Miss Teacher is a story of Nemphomeniac woman who is suffered by hypersexual disorder. It is also a story of Tanevsh and Sehar. Miss Rose, who has joine a college as a teacher, soon traps Rahul and attracts him with her beauty. After that she also disturbs Tanvesh's relation with Sehar by doing the same with him. Rose keeps physical relation with her students. But why she has become so? What is her past? Why she can't control her lust? Watch out the full movie that also comments on this serious sexsual disorder.
Watch out the full movie.
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- 43 years old
- Date of birth: Mar 23, 1971
- Date of passing: Jan 15, 2015
|Let the memory of Jennifer be with us forever
"Today marks a year since we lost you. The pain is still here every day, but the memories get a little easier. I miss and love you my sister!"
"Babe I'm missing you so much today. My heart hurts so bad. I want to be with you. I love you and miss you. Keith"
"Happy Birthday sis. It's our first one without you, and losing you is still so fresh. I miss you and love you!!"
"Happy Birthday babe. I love you and miss you. Keith."
"I would like to send my deepest condolences the family of Mrs. Fisher. I hope you find comfort in the many memories you have shared with Mrs. Fisher. I can tell she will be missed by many. There is something comforting that I would like to share. It is in the bible at Acts 24:15 "There is going to be a resurrection." And also 1 Corinthians 15:26 "And the last enemy death will be brought to nothing." In the future Jehovah God will resurrect all those in the grave and reunite them with there families on the earth. He will also destory death forever. God yearns to do this because he views death as a enemy and He can't wait to resurrect all those sleeping in death. If you would like to find more comforting thoughts please go to www.jw.org"
"I miss you so much that I hurt. I want to see you so bad."
"I love and miss you, sis."
"I love you babe. Your loving husband Keith Fisher."
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Hello and welcome to my personal bio page. I look forward to sharing my story with you and how I got started along my path in life through sport. I grew up in a sports family in the sports town of Peterborough, Ontario. Regardless of the time of year I was always playing something. Being Canadian and having a dad that played professional hockey I was on skates at a very young age. I was then introduced to lacrosse by a couple of Peterborough Lakers who were running workshops at schools in Peterborough when I was 6. For the next 24 years I played hockey every winter and lacrosse every summer. Year after year I would put one set of equipment away and pull the other one out. I loved both hockey and lacrosse and have been very fortunate to have played both sports professionally. I am a big believer in playing more than one sport. Not only did I play hockey and lacrosse but I also played soccer, basketball, golf, ran track and cross-country. The skills you learn from each and every sport will only make you a better athlete and player.
Over the years I have worked in many different hockey and lacrosse camps .I am not only passionate about playing the game of lacrosse but also about teaching the game to the next generation of players. My whole life I have been involved in sport and I'm thankful for the knowledge and sacrifice my family and coaches have made to help me become the person and player I am today. I want to especially thank my wife, parents, grandparents and brothers for the sacrifices they made and for their never-ending support. I look forward to meeting you and sharing my passion and knowledge with you at one of our events.
Sincerely, Brad Self
World Championships
- 1 Gold Medal for Canada (2015)
NLL Career
- 3 NLL Championships (2012-2014)
- Rochester Knighthawks (2012-Present)
- Chicago Shamrox (2008)
- Philadelphia Wings (2006)
- Colorado Mammoth (2004-2005)
- Ottawa Rebel (2003)
- Drafted Buffalo Bandits (2001)
MSL Career
- Peterborough Lakers (2003-present)
- 2 Mann Cups (2004, 2012)
- 6 Ontario Championships
- 1 Rookie of the Year (2003)
OLA Career
- Peterborough Lakers (1998-2002)
- 1 Bobby Allan Award for the Top Scorer (2002)
- 1 MVP in All-Star game (2002)
- 1 Jim Veltman Award for Most Outstanding Player (2002)
Hockey Career
- Fischtown Pinguins German Bundesliga (2010-2011)
- SC Riessersee German Bundesliga (2006-2010)
- Saint Mary's University (2002-2006)
- Peterborough Petes (1998-2002)
- Drafted Buffalo Sabres (1999)
Download & View Brad's Stats:


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I'm a pretty decent artist when I'm trying to save a penny, which is always. Wait. That came out wrong… I just mean I'm always thrifty. Anyway, I created this fabric vine collage to hang above the bed in our first apartment, and it was easy enough, I made another in black and silver for a friend. The only tools you need are a staple gun, and a needle and thread.
I just wrapped a canvas with fabric by stapling it to the back of the frame. Then I pinned the stem of the vine in place and sewed it by hand, all the way through the canvas. For the leaves, I ironed interfacing to the back of my gold satin fabric for stiffness, cut out the shapes, and used spray adhesive to attach the leaves. Though, in my most recent fabric collage I used mod podge with better results (I'd recommend matte).
This would also make a great paper collage; try it with scrapbook or magazine paper, and you won't even need the needle and thread. Then show me what you make! Share pics on Flickr.
Instructions for download: Use the four "layout" pages as a grid to map the vine, and the two pages of leaves as patterns to cut them out.
Vine pattern download (pdf): Free Vine Collage Download


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Lynette, thanks for joining us here today! What time period does the book take place? Why did you select that particular era; what about it appealed to you?
I agree about the fascination. It's a period in history full of interest and human-ness, I know that's not a word, but it fits.
Thank you for hosting
Mary, the word that pops into my mind is raw. But human-ness works!
Also want to thank our host for having us.
Lynette Willows will be here this morning. I'll be in later. Unfortunately, I have a funeral this morning, and don't expect to be back until mid-afternoon.
Thank you both for joining us here today! Was it difficult for you to collaborate on a book or did you feel that the book was better because you both worked on it? Best of luck with your book tour!
This novel sounds great! I love reading about horses. Cheers, Jody Jodyakessler(at)hotmail(dot)com
I love the premise of the story. I'm looking forward to reading the book.
It will be fun to explore the era!
I want to thank our host for having us here.
PM, the book was definitely better with both of us. We each have our strengths and weaknesses,and we both make up for what the other may be missing. We toss ideas back and forth and we have the best working relationship ever! We also both have an addiction to research, and that worked out beautifully too.
Jody, thanks for visiting. Both Carley and I have a love of horses, so even though it was a tobacco plantation, we wanted to add the element of equine management into the plot and characters too.
Ingeborg, if you decide to give it a read, I hope you enjoy it as much as we did writing it. Thanks for visiting.
Anonymous, as a Canadian, I had my doubts about it, but when Carley convinced me to write with her in this era, I soon became fascinated. I also learned there were Canadian ties to this time period, which only increased my interest. Thanks for popping in.
As a reader, I find it admirable when authors diligently research into whatever topic that makes up the story. Like horses. I'm impressed that Lynette researched thoroughly using authentic resources. Thanks to both!
Nice background about the book
Nice background about the book
bn100candg at hotmail dot com
Was it hard working as a team to create one cohesive story?
andralynn7 AT gmail DOT com
Thank you for posting this, I am really excited about the book and also the giveaway is fantastic!
hense1kk(at)cmich(dot) edu
Sorry for the late post. I'm playing catch-up here so I'm just popping in to say HI and sorry I missed visiting with you on party day! Hope you all had a good time!
kareninnc at gmail dot com


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Fu Hua Guang Dong Hotel
No. 98-102 Rua Francisco Xavier Pereira, Macau, MO
Select your stay dates to check availability
Fu Hua Guang Dong Hotel
About the hotel
Property Location
With a stay at Fu Hua Guang Dong Hotel in Macau, you'll be minutes from Fortaleza do Monte and Ruins of St. Paul's Cathedral. This hotel is close to Senado Square and A-Ma Temple.
Make yourself at home in one of the 156 guestrooms.
Rec, Spa, Premium Amenities
Make use of convenient amenities, which include complimentary wireless Internet access and tour/ticket assistance.
Enjoy a satisfying meal at a restaurant serving guests of Fu Hua Guang Dong Hotel.
Hotel Facilities
- Number of floors - 22
- Restaurant
- Free WiFi
- Elevator/lift
- Tours/ticket assistance
- Total number of rooms - 156
Room Facilities
- Private bathroom


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Mens and womens handbags, purses, shoe bags for travel and travelling bags
Business in Sherman Oaks, CA


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Project comments (0) | Write a comment
Happy Easter Card
|LeNae Gerig
Get all the major supplies needed to make this project with the Happy Easter Bunny Card, or you can order them separately.
Happy Easter Card
By LeNae Gerig
Click to Enlarge
Designer Tip: When using the squeegee to scrape off the excess paint, don't scrape too hard. Glide the squeegee across the surface of the stencil without putting too much pressure on the stencil. Too much pressure will case the paint to squeeze under the stencil and bleed. Altering stencil images: If you don't want to use a portion of the stencils, tape (or mask) that portion off before applying paint.
- Icy Rainbow Creative Pack
- Bunny Love Stencil
- Bunny Border Stencil
- Sizzling Dimensional Paints
- Floral Ribbon Set
- Easter Dazzles™ Gold
- White Cards & Envelopes
- 3-D Foam Squares Variety Pack
- Stencil Tape
- White cardstock
- Cut the green/dot border paper to 5" square. Place the border at the top and use stencil tape to secure the Bunny border stencil at the bottom. Tape over the carrots. Apply purple to the 3 bunnies and aqua to the grass. Remove the tape before the paint dries. Use the tip of a pencil to apply a dot of pink paint to the bunny noses. Let dry.
- Place the card fold at the side and glue the paper to the card front. Glue a 5"x2 ½" strip of coordinating dot paper to the bottom of the card. Glue a length of pink rick rack across the paper seam. Place the "Happy Easter" Dazzles( over dot paper at the bottom.
- Stencil the Love Bunny stencil, using purple paint for the bunnies and dessert for the heart, onto white cardstock. Remove the stencil, dot pink noses and let dry. Cut into a 3"x1 ¾" rectangle and mat with both pink paper and purple vellum, leaving 1/8" wide borders. Foam tape above the bunny border and glue a sheer pink dot bow to the top left corner.


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All of our online classes happen live, real time, there are no recordings to listen to, these are live class only.
Click on the link below for a short instructional video on how to participate online using the Adobe Online Classroom:
Before attending, please click the link below to test your system and make sure that you have all the necessary updates to participate in Adobe Online Classroom:
Once you have completed the above test, click on the appropriate link below to attend your class:
- Meditation Class
- Healing Class
- Women's Intuition
- Men's Class
- Novice Aura Reading
- Graduate Aura Reading
- Update Adobe Flash on your computer.
- Use a headset to talk with us online.
- Mute your MIC when you are not talking.
- Once you've started your webcam, freeze your screen using the pause button.
- If your mic isn't working, use the chat window to type questions or to share.


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California is the ideal utopia for ruling class liberals. The "richerals" as Victor David Hansen describes wealthy progressives have carved out a niche of prosperity for themselves while impoverishing the citizenry.
Mr. Hansen has published a series of essays describing the degeneration of a once prosperous state into a medieval society. Here is an excerpt from WND:
Hanson dubbed it "the weirdest place in the world" in his new e-book, a collection of PJ Media essays titled "The Decline and Fall of California." And who could argue the point when he calls out absurdities like Tiburcio Vasquez Elementary School – an institution of learning named after a 19th century robber and murderer?
That just scratches the surface of his thesis, though. Hanson demonstrates the sweeping impact of liberal elitism on the state's culture. "California is both more poorly managed than any time in its past, more divided between rich and poor, more fragmented by opportunistic ethnic identity politics, more impoverished by massive illegal immigration – and never more naturally wealthy."
California is naturally wealthy, yet is impoverished by socialist policies. Doesn't that remind you of Venezuela?


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Spurs-Cavaliers Preview
The last time the San Antonio Spurs had a winning streak this long, coach Gregg Popovich gave two of his best players the night off and the result was the most lopsided loss of the season.
If Popovich decides to rest some regulars this time, the result could be far different as the Southwest Division-leading Spurs face the Cleveland Cavaliers - though the slumping last-place team should have Kyrie Irving back in the lineup.
San Antonio goes for an eighth consecutive victory Tuesday night as the host Cavs try to end their season-high seven-game skid.
With the Spurs (36-14) playing for the third time in four nights, Tim Duncan and Tony Parker didn't play as the team's 11-0 run - the league's longest surge this season - ended with a 137-97 loss at Portland on Feb. 21.
San Antonio split its next six games, but has won 10 of 11 to build a comfortable division lead over second-place Dallas.
The Spurs have showcased some great depth during their winning streak with seven players averaging at least 10.0 points. San Antonio's bench outscored Indiana's reserves 52-29 in Saturday's 112-103 victory.
"The more practice we get, the more we play together, I think we're realizing how deep this team is,'' said Stephen Jackson, who has averaged 9.7 points in seven contests since being acquired from Golden State.
The Spurs could continue to benefit from that depth as they play their final 16 regular-season games in the next 24 days. With a game at Boston on Wednesday, Popovich could give his backups extended playing time at Cleveland.
The Cavaliers (17-33), meanwhile, need all the healthy bodies they can get. Daniel Gibson (foot) is likely done for the season, center Anderson Varejao (wrist) has been out since Feb. 10 and Irving missed Saturday's 91-74 loss in New York due to a sprained right shoulder he suffered a night earlier.
It was originally believed Irving, who leads all rookies with 18.9 points per game, would sit out a second straight game after he didn't participate in any contact drills during Monday's practice. However, coach Byron Scott said his starting point guard participated in Tuesday morning's shootaround without any problems and that Irving told him he "feels great."
The Rookie of the Year favorite will wear a protective brace on his shoulder, Scott says.
Undrafted rookie Donald Sloan got his first career start Saturday in place of Irving, finishing with 10 points, seven rebounds and four assists.
"I thought Donald did a real great job running the show, getting guys the ball when they needed it," Scott told the Cavs' official website. "Being able to break down defenders and get to the basket."
Cleveland, though, will have to take better care of the ball as it tries to avoid its first eight-game losing streak since a league-record 26-game slide Dec. 20, 2010-Feb. 9, 2011. The Cavaliers committed 20 turnovers Saturday and have given the ball away 17.0 times per game since their last victory.
Irving has averaged 5.3 turnovers in his last four games.
San Antonio has had 8.9 steals per game during its winning streak.
The Spurs' 109-99 win March 2, 2011, was their third consecutive victory over the Cavs and ended a two-game skid in Cleveland.
These teams finish the season series April 22 in San Antonio.


text: Nuggets 102, Warriors 91Player of the game: Denvers Andre Iguodala finished with 29 points and five rebounds but more importantly helped shut down Klay Thompson in the second half.Thompson had 16 points in the first half but went without a point in the second half largely because of Iguodalas defense.Key stretch: The Warriors couldnt get anything to go down early in the second half and it set the tone for the remainder of the game.The Warriors missed their first nine shots of the third quarter, and the Nuggets scored the first 15 points of the period to turn a 53-47 halftime deficit to a 62-53 lead.Making matters worse during that stretch was that Harrison Barnes picked up his fifth foul and Stephen Curry his fourth.We didnt help the cause by turning over the basketball and allow them to get transition points, Warriors coach Mark Jackson said. So, they got it going but we didnt try to stop it. We actually helped them keep it going.Danilo Gallinari finished with 20 points for the Nuggets (7-6), and Ty Lawson had 18 points and nine assists. David Lee led the Warriors (7-6) with 21 points and 10 rebounds.Currys tough half: After a couple of real nice games, Curry was a little more passive against the Nuggets, failing to get off a shot in the first 13 minutes. Curry, who had 56 points in the previous two games, was scoreless in the first half an picked up three fouls.Curry finished with just six points, four turnovers and three assists.They didnt do much in the first half, Jackson said of Curry and Barnes. And we found ourselves with the lead -- so we dont get caught up in individual nights.Bench strength: The Warriors bench was instrumental in keeping them in the game in the first half, getting contributions from plenty of substitutes.Charles Jenkins, who has been used sparingly this season, had six points in five minutes; Andris Biedrins had six rebounds in nine minutes; Jarrett Jack had eight points in 12 minutes; and Carl Landry had six points and four rebounds in 12 minutes.One of the reasons that Warriors coach Mark Jackson had to go to his bench a good bit in the half was because starting small forward Harrison Barnes picked up four fouls in six minutes.Landry finished with 19 points and Jack 17 for Golden State.


text: Hey all, sorry if this topic has been covered extensively but I did search high and low and couldn't really find any concrete answers to my question, so hopefully some one will be able to help I graduated this year in Graphic Design, and since then have been doing internships at several design studios around London. The place i'm currently interning has expressed an interest in taking me on, but they have concerns over the amount of work coming in at the moment. Their solution to this was to take me on, on a freelance basis, minimum 3 days a week (with the possibility of doing 5, depending on work load) for 3 months at a time, which they said will just roll on until they feel safe/expand the business. However being a recent graduate, I have no idea how much I should expect as an hourly/daily rate as a junior. I've done research into salaries, but there isn't much information out there at a junior freelance level. I spoke to my cousin who is a Senior Designer, and used to freelance, and he said he would expect it to be around £15 ph maybe even up to £20 in London, (although £20 sounds a a bit too high!) but he is slightly out of the loop with these things now he isn't freelancing. I'm aware of the other factors that I need to take into consideration, sick days, holiday, travel, living expenses, the fact I may not be as experienced in certain areas ect, but I was hoping someone could give me a rough figure to go with. Thanks! Phil.


text:
Once in a Blue Moon
Vicki Covington
John F. Blair, Publisher
$26.95 hardcover
6 x 9
256 pages
April 2017
Abraham Kasir has properties scattered across Birmingham, but the ones on Cullom Street hold a special place in his heart. So does his newest tenant, Landon Cooper, a transplant from one of the city's ritzier suburbs. Landon's a psychologist managing her own mental illness in the face of divorce and downsizing. Her upstairs neighbor, Abi, is a country girl trying to shake her rural roots even as her father's illness pulls her back home. Across the street, Jet is a former prostitute reeling from an uncovered truth about her birth mother. Jet has a crush on her neighbor Sam, a weed dealer paying his way through college and wrestling with his dreams for the future. Mr. Kasir acts not just as a landlord but as a father figure to each of them—and most of all to his grandson, Jason. Fresh out of high school, Jason is trying to follow in his grandfather's footsteps while his own father struggles with drug addiction.
These are the voices in Once in a Blue Moon, Vicki Covington's new novel set during Barack Obama's first presidential campaign. Change is in the air as readers follow this community through one transformative year. In league with other great Southern novelists including Anne Tyler and Fannie Flagg, Covington writes with tenderness and humor while asking important questions about family, faith, race, class, and—ultimately—hope.
"It's wonderful to have a new Vicki Covington novel to recommend. Once in a Blue Moon tells the story of a little community of lost souls who find each other in a season when hope and change seem like real possibilities. This is a lovely book, full of delight and real feeling. I can't think of another quite like it.
Mark Childress, author of Crazy in Alabama and Georgia Bottoms
"Vicki Covington is one of the most gifted and talented writers of the New South. In her latest novel, Once in a Blue Moon, she has created a most interesting group of diverse and unconventional characters, thrown together by chance, who soon come to discover the true meaning of friendship, family, and community."
Fannie Flagg, author of The Whole Town's Talking
"I wondered if anyone could ever capture the excitement so many of us felt at Obama's first election. Vicki Covington, an expert at conveying emotion, has. She also describes the intricate and specific world of Southside Birmingham, which conveniently has counterparts in every Southern city—small pockets of progressive and diverse life where people go along and get along. I'll keep this tender book on my shelf."
Rheta Grimsley Johnson, author of The Dogs Buried Over the Bridge


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September 7, 2017
Pika are small, cute mammals that live in broken rock habitats or talus fields high in the mountains above treeline. Adorable as they are, these critters might have a serious story to tell about the...
April 5, 2017
Climate researchers say the the weekly climb to gather air samples has become more of a mission than a job.
March 9, 2017
Thanks to a $6.8 million renewal grant to CU Boulder's Institute of Arctic and Alpine Research (INSTAAR) from the National Science Foundation (NSF), research at the Niwot Ridge study area – one...
November 15, 2016
Information on Niwot Ridge Long-term Ecological Research Program 2017 request for proposals.
October 26, 2016
NWT LTER invites proposals from early-career investigators for a 2-year postdoctoral fellowship.
September 27, 2016
NWT's annual meeting is on Wednesday, October 5, 11:00 - 2:00, in the SEEC Sievers Conference Room. NWT scientists will give presentations on current research and we will discuss plans for NWT VII....
January 14, 2016
Let It Snow! The Science of Winter
January 14, 2016
Researchers say Arikaree Glacier in Boulder County could melt in 20 years
January 14, 2016
Colorado high peaks losing glaciers as climate warms
January 11, 2016
December 14, 2015
Special Issue: Monographs in long-term alpine research at Niwot Ridge Long Term Ecological Research site, Colorado Front Range, 1995-2015
May 29, 2015
November 2, 2014
New study: Rising temperatures challenge Salt Lake City's water supply - Sensitivity study helps the city, others in the Intermountain West, plan for the future
October 28, 2014
Have you ever wondered what a Research Experience for Undergraduates (REU) with the Long Term Ecological Research (LTER) Network can do for a student? Well, Adam Formica can tell you. Now a Kellett...
This material is based upon work supported by the National Science Foundation under Cooperative Agreement #DEB-1637686. Any opinions, findings, conclusions, or recommendations expressed in the material are those of the author(s) and do not necesarily reflect the views of the National Science Foundation.
Please contact firstname.lastname@example.com with questions, comments, or for technical assistance regarding this website.


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Welcome to Monson Westside Ministries, Inc.
Friday, November 24, 2017
Helping our community.
Church Forms
Click on the form(s) you need. Each form is a PDF, You can download Adobe Acrobat Reader here
Once the prompt displays, you will need to save the selected file to to your local PC.
Print the final form and submit to the church office:
Church Usage Request (PDF)
Building Usage Guidelines and Fees (pdf)
Funding Ministry Request (pdf)
Purchase Order Form (PDF)
Effective June 2009 all Church purchases must be approved before purchases can be made. All purchases are placed through the church office. Please feel out the following form and submit to the Church Office.
The steps for making a purchase:
1. Complete a Purchase Request Form. Forms will be available by the church mailboxes and will also be available on the Monson Web Page in the Forms Section.


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West Wing Briefing
Bill Clinton content to cheer Obama on
At a highly unusual news conference more than two weeks ago, President Obama introduced former President Clinton, then left the briefing room to go to a Christmas party while Clinton touted a tax agreement the White House was pushing.
But for most of the last two years, the opposite has happened: Obama has campaigned to enact goals Clinton failed to achieve.
On Wednesday morning, Obama signed into law a repeal of the ban on people who are openly gay from serving in the military - a ban that Clinton tried to get rid of in the early 1990's before settling on "don't ask, don' tell'" as a compromise.
In the afternoon, the Senate is likely to give final approval to a major nuclear weapons treaty with Russia that the Clinton administration unsuccessfully pursued more than 10 years ago.
And most famously, Obama earlier this year signed a massive health care expansion that had eluded his Democratic predecessor.
These policies and priorities are of course not unique to either man; most Democratic presidents would likely have pushed them. And their successful passage during Obama's second year in office has as much to do with timing as either president's legislative skill.
Support for gay rights, for example, has increased dramatically since Clinton tried to change the military's policy on gays almost two decades ago. Many of the people who helped push Obama's health-care law through Congress, such as then-chief of staff Rahm Emanuel, personally participated in and learned from the 1993-94 experience.
The former president does not sound envious when he talks about his successor's achievements. Tensions from the 2008 campaign have cooled, as the two have praised each other publicly and Obama has sought the advice of both Clinton and his wife, the highly regarded Secretary of State Hillary Rodham Clinton.
"I was in the Oval Office talking to the president about a number of things," Bill Clinton said in an interview last week on Larry King Live, recalling his meeting with Obama earlier this month.
"I said, 'would you like me to call some Democrats in the House?' He said no, I want you to go the briefing room and tell the press. I said 'well, I'm out of practice.' He said, you'll be fine, it's like riding a bicycle."
"So he dragged me in there and the rest is history," said Clinton.


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To wash A Refrigerator Plant Engineering Projects :Monday, January 25th, 2016
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The Honeysuckle
The stunning honeysuckle (Lonicera periclymenum) blossoms appear in early summer and provide a heady scent around the garden at Midnight Apothecary, as well as in the neighbouring parks, railroad sidings, and streets. I have used some vodka here to maintain the strength of the cocktail but not drown out the taste of honeysuckle. The rowan in the foam provides bitter and sour notes to counteract the sweetness of the honey and sourness of the lemon. And rowan works beautifully with brandy. The sweet, sharp mint opens up the palate to make the drink balanced and refreshing.
5 spearmint (Mentha spicata) leaves
1oz (30ml) cognac (or mid-range brandy)
½ oz (15ml) vodka
¾ oz (22ml) honeysuckle syrup
¾ oz (22ml) freshly squeezed lemon juice
Tools: Cocktail shaker with strainer
Glass: Martini
Ice: Cubes
Garnish: Honeysuckle blossom
Serves 1
Chill the glass thoroughly in the freezer or refrigerator for 2 or 4 hours respectively. Alternatively, fill the glass with ice.
Smack the mint leaves between the palms of your hands to release the essential oils and drop them into the cocktail shaker. Add the cognac, vodka, honeysuckle syrup, and lemon juice. Fill the shaker two-thirds of the way up with ice, cover, and shake hard for 20 seconds. If you used ice to chill your glass, empty it out. Strain the cocktail into the chilled glass. Remove the whipper containing the
Rowan, Honey, and Lemon Foam from the refrigerator, shake it a few times, and, holding it vertically, gently discharge the foam until it reaches the level of the rim of the glass. Wait a few seconds for the foam to settle. Garnish with the honeysuckle blossom and serve.
The blossom heads are the perfect size to fill a champagne coupe or martini glass as a garnish on top of the egg white foam. Encourage your guests to try to suck the nectar out of the blossoms, although the bees will usually have got there first.
Honeysuckle syrup
8 large handfuls of unsprayed
honeysuckle flowers, leaves and
stems removed
Approximately 2 cups (400g) caster sugar
Juice of ½ lemon
Sealable presentation bottle(s), sterilised
Makes approximately 1 pint (500ml)
Place the honeysuckle flowers in a nonreactive bowl and cover with cold water, then leave to steep for 12 hours, or at least overnight, at room temperature. Make sure the flowers are completely covered by the water.
Strain the mixture into a measuring cup, discarding the flowers. Pour the liquid into a nonreactive pan. Measure an equal amount of sugar to the liquid and add to the pan. Bring to a boil, and let simmer for 5 minutes. Feel free to replace half the sugar with a handful of chopped sweet cicely leaves, but bear in mind that this will adjust the colour.
Remove from the heat, let cool, add the lemon juice, and funnel into the sterilised presentation bottle(s).


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The TSX Information Tech index is up 18% over the past year, which is relatively steep in comparison to the lagging 11% drop in the TSX Composite. However it's the Financial Technology sector that's grabbing all the headlines, putting the phrase "FinTech" into almost every investor's mind. Today, we roundup some of our favorite FinTech companies that might sail through the clouds.
Alex Cutulenco | November 23, 2015:
Financial technologies, or FinTech, is somewhat of a nascent industry, which is combining the power of technology and bringing it into the long-standing, brick and mortar financial services industry. FinTech can range from anything such as online payment processing and commercial lending, to online customer onboarding. Technology is revolutionizing the way consumers and businesses transact, by moving it to the Cloud. This has had a major impact on the current state of the financial services industry, as it has the power to lower fees and commissions banks charge, providing for faster processing of lending requests, and making transactions more efficient. The change is making large financial institutions reorganize (as seen with TD Bank Group announcing management changes to its TD Ameritrade division – TD's online brokerage arm), and possibly think about acquiring FinTech companies, which are stepping on their toes. Today, we roundup some of our favorite FinTech companies that might sail through the clouds.
VersaPay (TSXV: VPY)
Price: $1.25 | Market Cap: $32.2 million
VersaPay is financial technology company that has developed a proprietary SaaS solution, VersaPay ARC, focusing on accounts receivable management designed for mid-sized businesses. The group also offers business and consumer customers a cloud-based credit card and electronic funds transfer service with PayPort and similar services to third-party technology partners via the VersaPay Gateway. Company has generated $5.2 million in revenue over the past 12 months.
IOU Financial (TSXV: IOU)
Price: $0.55 | Market Cap: $33.9 million
IOU Financial is a developer of a proprietary online software that allows small businesses to apply for a working capital loan, via an online process and, if accepted, have the funds transferred to their account within a matter of 24-48 hours. The Company developed an algorithm to access the lender and their ability to pay back the loan, effectively making sure not to give out bad loans to unworthy clients. IOU Financial has been lending to small businesses since 2008 and has funded over $270 million in loans.
Related: See Frank Holmes' insight into IOU Financial by watching his interview with SmallCapPower >>
TIO Networks (TSXV: TNC)
Price: $1.72 | Market Cap: $102.6 million
TIO Networks is the leading multi-channel expedited bill payment processor serving the largest telecom, wireless, cable and utility bill issuers in North America. TIO integrates its Transaction Processing System to the accounting back-office of the biller partners (such as AT&T for telephone/WiFi bill payments), which allows billers to post payments accurately and quickly to the appropriate customer accounts. Once integrated with a biller, TIO is able to offer to the billers' customers the ability to pay their accounts through multiple access points, including self-service kiosks, POS/clerk-assisted locations, smartphone applications and more. The Company has generated $68.3 million in revenues over the past 12 months.
Alex can be reach at: firstname.lastname@example.com