Insulin receptor (IR) signaling plays a critical role in the regulation of metabolism and growth in multicellular organisms. IRs are unique among receptor tyrosine kinases in that they exist exclusively as covalent (αβ)2 homodimers at the cell surface. Transmembrane signaling by the IR can therefore not be based on ligand-induced dimerization as such but must involve structural changes within the existing receptor dimer. In this study, using glycosylated full-length human IR reconstituted into lipid nanodiscs, we show by single-particle electron microscopy that insulin binding to the dimeric receptor converts its ectodomain from an inverted U-shaped conformation to a T-shaped conformation. This structural rearrangement of the ectodomain propagates to the transmembrane domains, which are well separated in the inactive conformation but come close together upon insulin binding, facilitating autophosphorylation of the cytoplasmic kinase domains.
The insulin receptor (IR) is a ubiquitously expressed receptor tyrosine kinase (RTK) that is of fundamental importance for the regulation of glucose, protein, and lipid metabolism and growth (Saltiel and Kahn, 2001). Its dysfunction has been linked to severe pathologies including diabetes mellitus, cancer, and Alzheimer's disease (Haeusler et al., 2018).
In the canonical model of RTK signaling, receptor activation is mediated by ligand-induced dimerization and oligomerization (Ullrich and Schlessinger, 1990). However, a subset of RTKs can form noncovalent but inactive dimers before ligand binding (Maruyama, 2014; Freed et al., 2015). IRs are unique among RTKs in that they exclusively exist as covalently linked (αβ)2 homodimers at the cell surface (Fig. 1 A). Independent of their oligomeric state, all RTKs are activated by their cognate ligands, arguing that distinct allosteric changes within the dimeric receptor structure may contribute to receptor activation and signaling outcome (Maruyama, 2014; Freed et al., 2017).
IR reconstitution into nanodiscs and activity assay. (A) Left: Schematic cartoon showing the ectodomain (ECD), transmembrane domain (TMD), and tyrosine kinase domain (TKD). Right: Structural model of the full-length human (αβ)2 IR assembled from available crystal structures of the tyrosine kinase (Hubbard et al., 1994) and extracellular domain (Croll et al., 2016) as well as the nuclear magnetic resonance solution structure of the transmembrane domain (Li et al., 2014). (B) Silver-stained native PAGE of glycosylated full-length human IR reconstituted into MSP1E3D1 nanodiscs. (C) SDS-PAGE of nanodisc-embedded IRs under nonreducing (−DTT) and reducing conditions (+DTT). (D) Activity assay showing that both CHAPS-solubilized and nanodisc-embedded IRs are autophosphorylated upon exposure to insulin. IRα, IR α subunit; IRβ, IR β subunit; IR(αβ)2, mature IR; pIRβ, phosphorylated IRβ; pro-IRαβ, unprocessed intracellular form of IR. Experimental details are provided in the In vitro phosphorylation assay and SDS-PAGE and Western blots sections in Materials and methods as well as Figs. S1 and S2.
Our structural understanding of IRs derives largely from crystallographic and nuclear magnetic resonance studies of receptor fragments (Hubbard et al., 1994; McKern et al., 2006; Menting et al., 2013; Li et al., 2014; Cabail et al., 2015; Croll et al., 2016), most notably those of an unliganded ectodomain (Croll et al., 2016) and a truncated ectodomain in complex with insulin (Menting et al., 2013). Attempts to image full-length IRs by EM yielded inconclusive results and did not reveal ligand-dependent structural changes (De Meyts and Whittaker, 2002), leaving the structural basis for transmembrane signaling to be elucidated (De Meyts, 2015; Tatulian, 2015).
To obtain insights into IR activation, we reconstituted recombinant full-length IR into lipid nanodiscs and visualized them by single-particle EM. In the absence of insulin, the IR ectodomain adopted the symmetric inverted U-shaped conformation described in previous studies of the ectodomain (Tulloch et al., 1999; McKern et al., 2006; Croll et al., 2016). Upon insulin binding, however, the receptor ectodomain converted into a T-shaped conformation that brought the transmembrane domains together, presumably facilitating autophosphorylation of the tyrosine kinase domains.
To elucidate the mechanism underlying transmembrane IR signaling, we produced recombinant full-length human (αβ)2 IRs (Fig. 1 A) in freestyle HEK293F cells, purified them in CHAPS to near-homogeneity by a single affinity chromatography step, and used membrane scaffold proteins (MSPs; Denisov et al., 2004; Schuler et al., 2013) to reconstitute them into lipid nanodiscs (Fig. 1, B and C; and Fig. S1). For reconstitution, we used a ternary lipid mixture consisting of phosphatidylcholine, sphingomyelin, and cholesterol at a ratio that forms homogeneous membranes in the liquid-disordered phase (Tumaneng et al., 2010). Assaying kinase activity upon exposure to insulin demonstrated increased autophosphorylation activity over basal levels for both the detergent-solubilized and the nanodisc-embedded IR (Figs. 1 D and S2), although the latter exhibited considerably higher background activity.
Full-length IRs were first reconstituted with MSP1E3D1, an MSP variant that forms nanodiscs of ∼12 nm in diameter (Denisov et al., 2007), and the resulting structures were imaged by negative-stain EM. Although heterogeneous, many particles showed circular shapes representing nanodiscs, from which additional densities extended (Fig. S3 A). About 10,000 interactively selected particles were subjected to K-means classification into 200 classes (Fig. S3 B) and to iterative stable alignment and clustering (ISAC; Fig. S3 C; for classification details, see Table S1). K-means classification assigns every particle to one of the specified number of classes and can therefore be used to quantify particle populations (see numbers throughout the study). ISAC only uses a subset of the particles, namely those that fall into stable and reproducible classes. ISAC is thus not suitable to quantify particle populations, but it produces averages with very reliable and crisp features (for more details, see Yang et al., 2012). Some ISAC averages revealed an L-shaped density extending from a nanodisc (Fig. S3 C), presumably representing unprocessed monomeric IRs (see Fig. 2 for an interpretation of the 2D class averages in terms of the 3D structures they likely represent). Because monomeric IRs do not exist at the cell surface under physiological conditions (Hedo et al., 1983), they will not be discussed further. Many ISAC averages showed an inverted U-shaped density that either extended from a single nanodisc (20%; see the Image processing section of Materials and methods for estimation of particle populations; Fig. 3 A, top) or connected two nanodiscs (80%; Fig. 3 A, bottom). The width of the U-shaped density (∼8–11 nm) was consistent with the respective IR ectodomain dimension seen in a previous negative-stain EM study (Tulloch et al., 1999) and in crystal structures (McKern et al., 2006; Croll et al., 2016). The unliganded ectodomain extended 8.5–10 nm from the edge of the nanodiscs, which presumably is an underestimation of the total length of the ectodomain because the nanodisc density makes it impossible to identify the precise position where the protein emerges from the membrane. However, as most IR dimers reconstituted into two separate nanodiscs, the two transmembrane domains must generally be well separated from each other in unliganded receptors.
Schematic illustration of the conformations assigned to IRs in nanodiscs. (A and B) Representative class averages (A) and schematic drawings (B) of IRs in nanodiscs adopting different conformations: U-shaped dimer (ectodomain in red) in one (1U) or two nanodiscs (2U), T-shaped dimer (ectodomain in blue) in one (1T and II) or two nanodiscs (2U), and L-shaped monomer (ectodomain in dark gray) in one nanodisc (L). (C) Models of how the nanodisc-embedded IRs may look in solution. Because the tyrosine kinase domains are flexibly tethered to the transmembrane domains, their locations are somewhat speculative, especially in the 2T conformation. Note, however, that extra density in the class averages of the 1T and II conformations may represent tyrosine kinase domain dimers.
Selected EM averages of IRs reconstituted into MSP1E3D1 nanodiscs and ligand-induced IR activation. (A) In the absence of insulin, IRs reconstituted into one nanodisc (1U; top) or two nanodiscs (2U; bottom) and adopted a U-shaped conformation. (B) Upon insulin addition, all IRs in a single nanodisc adopted the T-shaped conformation (1T; middle), whereas IRs in two nanodiscs adopted either the U-shaped (top) or T-shaped conformation (2T; bottom). (C) In the presence of insulin, all IRs reconstituted into a single nanodisc. Most of them adopted a T-shaped conformation (top), whereas some showed an II-shaped conformation (II), likely representing a different view of IRs in the T-shaped conformation (bottom). The first three averages show a globular density that may represent dimerized tyrosine kinase domains (TKDs; arrows). (D) Upon insulin addition, all IRs showed a T-shaped conformation. The side length of individual panels is 47.7 nm. (E) Quantification of IR populations with a particular ectodomain (ECD) conformation at given insulin concentrations. (F) Cartoon illustrating the ligand (green) binding–induced conformational change in the ectodomain and its coupling to the transmembrane domains (TMDs) with concomitant activation of the tyrosine kinase domains by autophosphorylation (red asterisks). Our experimental data imply that binding of one ligand is sufficient to induce the transition of the IR ectodomain from the U-shaped to the T-shaped conformation.
Ligand binding by IRs displays negative cooperativity. The first ligand binds with high affinity and is suggested to suffice for receptor activation under physiological ligand concentrations (Lee and Pilch, 1994). At concentrations of ∼1–100 nM, a second insulin molecule can bind with lower affinity but accelerates dissociation of the first ligand. At ligand concentrations >1 µM, three insulin molecules may occupy the receptor (De Meyts and Whittaker, 2002; Kiselyov et al., 2009).
To understand the consequences of insulin binding, IRs were first incubated with a saturating insulin concentration of 1 µM before they were reconstituted into nanodiscs. EM images of this IR preparation showed that all receptors were incorporated into a single nanodisc (Fig. 3 C). All averages showed a dramatic conformational change in the ectodomains from the U-shaped conformation predominantly to a T-shaped conformation (∼72%; Fig. 3 C, top). A smaller fraction showed II-shaped ectodomains (26%), presumably representing a different view of the T-shaped conformation (Fig. 3 C, bottom). Additional exposure to 1 µM insulin after reconstitution showed that all dimeric IR ectodomains adopted the T-shaped conformation (Fig. 3 D). Importantly, because all ligand-bound IR dimers were incorporated into a single nanodisc, the two transmembrane domains must be in close proximity and presumably interact with each other.
To confirm that the conformational change also occurs in membrane-embedded IRs, we used IRs reconstituted into nanodiscs in the absence of insulin (Fig. 3 A) and exposed them to 1 µM insulin. Averages still revealed IRs in the U-shaped conformation connecting two nanodiscs (Fig. 3 B, top), but these constituted a smaller fraction (∼53%). Strikingly, all IRs that were reconstituted into a single nanodisc appear to have undergone the conversion from the U-shaped to the T-shaped conformation upon insulin binding (Fig. 3 B, middle). The T-shaped receptors had a wingspan of ∼12.5–14.5 nm and extended ∼11–13.5 nm from the edge of the nanodiscs. In addition, averages now also showed T-shaped IRs extending from two nanodiscs (∼16%; Fig. 3 B, bottom). From these results, ligand binding to IRs reconstituted into two nanodiscs should convert their ectodomains into a T-shaped conformation, which, in turn, would exert a driving force to bring the transmembrane domains together. This force will tend to bring the two nanodiscs together, which was indeed seen in some of the averages (Fig. 3 B, bottom). Furthermore, nanodiscs connected by a T-shaped IR in general appeared to be closer to each other than those connected by a U-shaped IR (Fig. 3 B, compare top with bottom). The resulting juxtaposition of the two nanodiscs may only occasionally lead to fusion, allowing the transmembrane domains to interact, whereas in most cases, the two nanodiscs would likely remain separate, preventing the transmembrane domains from interacting.
To test whether the transition into the T-shaped conformation also occurs at nonsaturating ligand conditions, we exposed IRs embedded in nanodiscs to an insulin concentration of 12 nM as well as to the physiologically relevant and highly substoichiometric insulin concentration of 0.8 nM. The quantification of the receptors in different conformations (Fig. 3 E and Table S3) implied that one ligand is sufficient to induce the conversion of the IR ectodomain from the U- to the T-shaped conformation.
Crystallographic studies established that in unliganded IRs, the membrane-proximal segments of the ectodomains are ∼12 nm apart (McKern et al., 2006; Croll et al., 2016). We therefore hypothesized that reconstituting unliganded IRs into smaller nanodiscs should increase the number of IRs that incorporated into two nanodiscs. To test this hypothesis, we repeated the above experiments with MSP1D1, an MSP that forms smaller nanodiscs of ∼10 nm diameter (Denisov et al., 2004). As expected, in the absence of insulin, all receptors adopted the U-shaped conformation. More importantly, with the smaller nanodiscs, the two transmembrane domains of the dimeric IRs incorporated exclusively into separate nanodiscs, strengthening the notion that the transmembrane domains do not interact with each other in unliganded receptors (Fig. S4 A). In the presence of insulin, however, even with the smaller nanodiscs, all IRs reconstituted into single nanodiscs (Fig. S4 C). In summary, all results were consistent with those obtained with IRs reconstituted into the larger nanodiscs (Fig. S4).
The T-shaped conformation of the activated IR described in this study resembles what has been seen in previous EM images of IRs in detergent and reconstituted into liposomes (Christiansen et al., 1991; Tranum-Jensen et al., 1994; Woldin et al., 1999). These studies, however, did not reveal conformational changes in the receptor upon exposure to insulin, whereas our data now provide direct structural evidence for a ligand-driven conformational change in the IR ectodomain and its importance for signal propagation across the membrane. In the absence of such data, mutually exclusive models have been proposed for IR activation (De Meyts and Whittaker, 2002; Ward et al., 2013; Kavran et al., 2014; Lee et al., 2014; De Meyts, 2015; Tatulian, 2015). In one model, IR transmembrane domains interact with each other in the inactive state, and insulin binding then pries them apart (Lee et al., 2014). In an opposing model proposed for the homologous insulin-like growth factor-1 receptor, the ectodomains keep the transmembrane domains separated in the inactive state. Ligand binding would then release this inhibition and allow the transmembrane domains to interact (Kavran et al., 2014). An inhibitory role of the ectodomain has also been suggested for inactive preformed dimers of the EGF receptor in which the unliganded ectodomain exerts steric constraints to prevent the association of the N-terminal segments of the transmembrane helices (Zhu et al., 2003; Arkhipov et al., 2013). In line with this model, tryptic removal of the IR ectodomain was shown to result in constitutively active kinase domains (Shoelson et al., 1988). Our results unambiguously support the latter model also for IR regulation (Fig. 3 F): insulin binding induced a large conformational change in the IR ectodomain, releasing the constraint on the transmembrane domains and concomitantly facilitating autophosphorylation of the kinase domains. Indeed, some averages of the receptor in the T-shaped conformation showed a strong globular density (arrows in Fig. 3 C) that was not seen in averages of the receptor in the U-shaped conformation, suggesting that the tyrosine kinase domains in these averages may be more restricted in their localization and dimerized.
Given the importance of the rearrangements in the IR transmembrane domains, we envision that the lipid environment will have an effect on receptor activation and regulation, a notion that can be tested further using the nanodisc system. Our results also provide novel structural insights that answer longstanding questions concerning the mechanism of how insulin activates the receptor, thus improving our understanding of the receptor and providing new perspectives for pharmacological intervention strategies.
cDNA for human IR isoform A was cloned into a pTT6 vector as described previously (Gerl et al., 2014), resulting in a construct consisting of the human IR isoform A followed by a C-terminal human rhinovirus 3C protease cleavage site and a tandem affinity purification tag.
The receptor was produced in suspension-adapted FreeStyle HEK293F cells (RRID: CVCL_D603; Thermo Fisher Scientific) that were grown at 37°C in protein-free FreeStyle 293 expression medium (Thermo Fisher Scientific) supplemented with 1× penicillin/streptavidin at 90 rpm and 8% CO2. Cells at 2 × 106 cells/ml in antibiotics-free medium were transiently transfected with 1 mg/liter endotoxin-free plasmid DNA (precomplexed with polythelenimine at a 1:8 [wt/wt] ratio; Gerl et al., 2014). The cells were maintained for 64 h at 31°C, 8% CO2, and 90 rpm. After harvesting the cells by centrifugation at 1,000 g for 10 min, the cell pellet from 1 liter of cell culture was resuspended in 100 ml ice-cold detergent-free HBS (50 mM Hepes, pH 7.5, and 150 mM NaCl, supplemented with 8 mM Pefabloc, 10 µM E64, and two tablets of complete protease inhibitor cocktail [Roche]). Resuspended cells were disrupted using a tip sonicator (Ultrasonic Homogenizer Sonopuls HD 3100 with an SH70 G horn and MS 73 microtip [3 mm]; Bandelin Electronic) in the absence of detergent by four to six sonication cycles (1 s pulse and 1 s pause for 30 s total followed by 1 min pause at 17% amplitude). Protein purification was performed at 4°C as described previously for EGF receptor (Coskun et al., 2011), except that the pH of all purification buffers was 7.5 and that after the washing step with 20% (vol/vol) glycerol-containing running buffer, the column was equilibrated with HBS supplemented with 1% (wt/vol) CHAPS and 8% (vol/vol) glycerol for overnight incubation with human rhinovirus 3C protease and protein elution.
IR concentration was estimated using a molar extinction coefficient of 377,585 M−1 cm−1 as calculated by ProtParam (ExPASy), assuming 42 of the total 94 cysteines are forming disulfide bonds in the dimeric receptor (Croll et al., 2016). The typical yield of purified IR was ∼0.5 mg from 1 liter of cell culture.
For quality controls, analytical size-exclusion chromatography was performed using a Superose 6 3.2/300 column (GE Healthcare) with IR elution buffer at a flow rate of 0.05 ml/min at room temperature followed by SDS-PAGE of the collected fractions and silver staining. Purified IR was immediately reconstituted into nanodiscs.
Plasmids encoding MSP pMSP1E3D1 (20066) and pMSP1D1 (20061) were purchased from Addgene. Both MSP variants were produced in E. coli BL21Gold(DE) (Agilent Technologies) and purified as described previously (Inagaki et al., 2012) with the exceptions that prepacked His-Trap HP columns (GE Healthcare) were used for immobilized metal ion affinity chromatography and that an additional wash step with cholate was introduced (50 mM Tris, pH 7.4, 200 mM NaCl, and 32 mM cholate). The His tag was not removed.
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-stearoyl-d-erythro-sphingosyl-phosphorylcholine (SM), and cholesterol were purchased from Avanti Polar Lipids. All lipid stocks were quantified on a regular basis to correct for solvent evaporation by a phosphate assay for phospholipids (Rouser et al., 1966) or an Amplex red cholesterol assay kit (Invitrogen) and were controlled for their stability by thin-layer chromatography. DOPC, SM, and cholesterol were mixed at a ratio of 80:15:5 mol%, dried under a stream of nitrogen, and left under vacuum overnight to remove residual solvent. To better solubilize the lipid mixture containing cholesterol and sphingomyelin and to improve reproducibility, the dried film was first used to form liposomes by addition of reconstitution buffer (20 mM Hepes, pH 7.5, and 100 mM NaCl) with vigorous shaking at 54°C followed by 10 freeze-thaw cycles and 21 extrusions through a 100-nm pore-size polycarbonate filter. The resulting unilamellar liposomes were then solubilized by addition of cholate (Alfa Aesar) to a final concentration of 18 mM and subsequent sonication in a water bath.
IRs were reconstituted into nanodiscs in the presence or absence of human insulin (I2643; Sigma-Aldrich). For reconstitutions in the presence of insulin, 0.3 µM IR in 50 mM Hepes, pH 7.5, 150 mM NaCl, 1% (wt/vol) CHAPS, and 8% (vol/vol) glycerol was incubated with 1 µM insulin for 1 h on ice. Before usage, insulin was dissolved to a concentration of 180 µM in 5 mM HCl. Insulin was added to the IR reconstitution mix to a final concentration of 1 µM. Even though the pH of the solution did not change, the same amount of 5 mM HCl was added to the unliganded receptor to rule out potential effects of HCl on IR. For nanodisc formation, 0.3 nmol liganded or unliganded IR was incubated with 200 nmol cholate-solubilized lipids and 10 nmol MSP1E3D1 or MSP1D1 for 30 min in the presence of 13.2 mM CHAPS at 25°C with gentle shaking at 300 rpm. The reconstitution mix was centrifuged at 20,000 g for 20 min at 4°C, and the supernatant was extensively dialyzed against at least a 1,000× excess of 20 mM Hepes, pH 7.5, and 100 mM NaCl for 26 h at 4°C with three buffer exchanges followed by a fourth exchange to 25 mM Hepes, pH 7.5, and 150 mM NaCl for 18 h. Dialysis was performed in Spectra/Por 7 standard regenerated cellulose pretreated dialysis tubings (Spectrum Labs) with a molecular weight cutoff of 10 kD (for the first 18 h) and 50 kD (until the end of the dialysis). Because of the aggregation propensity of nonreconstituted MSPs, aggregates were removed by centrifugation at 150,000 g for 45 min at 4°C. The supernatant was snap-frozen in liquid nitrogen and stored at −80°C. IR-containing nanodiscs were separated from empty nanodiscs by size-exclusion chromatography using a Superose 6 10/300 GL column (GE Healthcare) with 25 mM Hepes, pH 7.5, and 150 mM NaCl, and only the peak fraction was collected.
For EM experiments of IR reconstituted into nanodiscs with MSP1E3D1, the peak fraction was split into four aliquots. One aliquot was imaged directly by negative-stain EM, and the other aliquots were incubated with varying amounts of insulin (final concentrations of 0.8 nM, 12 nM, or 1 µM) for 1 h at 4°C before imaging. For IR reconstituted into nanodiscs with MSP1D1, the peak fraction was split into halves. One half was imaged directly by negative-stain EM, whereas the other half was incubated with 1 µM insulin (final concentration) for 1 h at 4°C before imaging.
The concentration of IR in nanodiscs was estimated by UV spectroscopy at 280 nm with a molar extinction coefficient of 438,030 M−1 cm−1 for samples reconstituted with MSP1E3D1 and 420,445 M−1 cm−1 for samples reconstituted with MSP1D1 as calculated with ProtParam (ExPASy) assuming that 42 of the 94 cysteines formed disulfide bonds.
A saturating insulin concentration (200 nM) was added to purified CHAPS-solubilized IRs or nanodisc-embedded IRs and allowed to bind for 1 h on ice. Phosphorylation was performed with 0.25 mM ATP in 25 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM MgCl2, and 1 mM MnCl2 in a final volume of 25 µl and incubated for 12 min at 25°C. For solubilized IRs, the CHAPS concentration was kept at the critical micellar concentration of 0.6% (wt/vol). The reaction was stopped by addition of SDS sample buffer supplemented with 2.5 mM EDTA. The reaction mixtures were subjected to SDS-PAGE, and IR tyrosine kinase domain phosphorylation was assessed by Western blotting.
Electrophoresis was performed with precast 4–12% Bis-Tris gels with MOPS SDS running buffer or 3–8% Tris-acetate gels or NativePAGE 3–12% Bis-Tris gels (Invitrogen) using the corresponding commercial running buffers (Invitrogen). Proteins were stained with Coomassie blue or silver stain or were transferred to polyvinylidene difluoride membranes for immunodetection. Membranes were blocked overnight with 5% (wt/vol) nonfat dry milk powder in TBST (0.1% [vol/vol] Tween-20 in 20 mM Tris, pH 7.4, and 150 mM NaCl) supplemented with phosphatase inhibitors (1 µM NaVO4 and 20 µM β-glycerophosphate). Membranes were probed with antibodies against phospho-IRβ (Tyr-1150/1151; 1:1,000; 3024; RRID, AB_331253; Cell Signaling Technology) or against total IRβ C terminus (1:2,000; 3020; RRID, AB_2249166; Cell Signaling Technology). The secondary antibodies goat anti–rabbit IgG (H+L; 170-6515; RRID, AB_11125142; Bio-Rad Laboratories) or goat anti–mouse IgG (H+L; 170-6516; RRID, AB_11125547; Bio-Rad Laboratories) conjugated to horseradish peroxidase were used at a 1:10,000 dilution, and detection was performed using SuperSignal West Femto maximum-sensitivity substrate electrochemiluminescence substrate (Thermo Fisher Scientific) with a charge-coupled device imager (Imager 600; GE Healthcare).
An aliquot (3.5 µl) of full-length IR reconstituted into nanodiscs was adsorbed to a glow-discharged 200-mesh copper grid covered with a thin carbon-coated plastic film. After adsorbing the sample for 1 min, the grid was blotted with a filter paper and washed twice with buffer (25 mM Hepes, pH 7.5, and 150 mM NaCl), once with deionized water, and once with a 0.75% (wt/vol) uranyl formate solution, blotting the grid with filter paper between each drop, and then it was stained for 30 s with the uranyl formate solution. After blotting the grid again with filter paper, the remaining solution was removed by vacuum aspiration (see Ohi et al.  for further details). Images were collected using an XR16L-ActiveVu charge-coupled device camera (AMT) on a Philips CM10 electron microscope (FEI) operated at an acceleration voltage of 100 kV. The calibrated magnification was 41,513× (nominal magnification of 52,000×), yielding a pixel size of 2.65 Å at the specimen level. The defocus was set to –1.5 µm.
Images of nanodisc-embedded IRs showed a very heterogeneous particle population. Therefore, for further image processing, particle selection focused on particles that showed one nanodisc with an additional density extending from them or two nanodiscs connected by a density. For each sample, ∼10,000 particles (the exact numbers of images and selected particles are summarized in Table S1) were manually selected using the e2boxer.py command of the EMAN2 software package (Tang et al., 2007) and windowed into 180 × 180–pixel images. After image normalization and particle centering, the particle images were classified into 200 groups using K-means classification procedures implemented in SPIDER (Frank et al., 1996). For the ISAC algorithm (Yang et al., 2012) implemented in SPARX software (Hohn et al., 2007), the particle images were reduced to 76 × 76 pixels and subjected to several ISAC generations specifying 50 images per group and a pixel error threshold of 0.7 (the number of ISAC generations and resulting number of averages for each sample are summarized in Table S1).
To estimate the approximate number of receptors adopting a particular conformation, we used the K-means classification into 200 groups and visually assigned the averages to show (a) a U-shaped IR reconstituted into one nanodisc (1U), (b) a U-shaped IR reconstituted into two nanodiscs (2U), (c) a T-shaped IR reconstituted into one nanodisc (1T), (d) a T-shaped IR reconstituted into two nanodiscs (2T), (e) a II-shaped IR (II; we only observed IRs in this conformation associated with one nanodisc), and (f) an L-shaped IR (L; we only observed L-shaped IRs associated with one nanodisc, consistent with the assumption that these particles represent IR monomers). For each set of averages, the particles in the classes assigned to one of these six classes were summed up and are listed in Table S2. Many of the averages could not be unambiguously assigned to one of the six classes. These classes (X) were considered uninterpretable and were excluded from the quantification. The assignments of the classes for each experimental condition are denoted in Fig. S3 C and dataviewer content showing the 200 SPIDER class averages. In addition, the particles showing the L-shaped nonphysiological monomeric IR were not considered when calculating the percentage of dimeric receptors in each conformation (1U, 2U, 1T, 2T, or II) as listed in Table S3.
Fig. S1 illustrates the biochemical analyses of purified IR and IR reconstitution into nanodiscs. Fig. S2 shows autophosphorylation assays for IRs in detergent and in nanodiscs. Fig. S3 presents EM analysis of IR reconstituted into large nanodiscs with MSP1E3D1 in the absence of insulin, showing an EM image and the class averages obtained by ISAC and K-means classification. Fig. S4 shows the conformation of IR reconstituted into small nanodiscs with MSP1D1 under different insulin exposure conditions. Table S1 provides statistics for EM image collection and ISAC classification. Table S2 provides the numbers of particles assigned to specific IR conformations, and Table S3 provides the corresponding percentages of the dimer population.
The authors acknowledge Karim Fahmy and Jana Oertel for initial support with nanodisc production.
Financial support was provided by the Deutsche Forschungsgemeinschaft TRR83 TP18 and FOR 2682 (to Ü. Coskun), the Deutsche Forschungsgemeinschaft–funded Dresden International Graduate School for Biomedicine and Bioengineering (GS97 to T. Gutmann), the German Federal Ministry of Education and Research grant to the German Center for Diabetes Research (to Ü. Coskun), and a Christiane Nüsslein-Volhard Foundation grant (to T. Gutmann). This research was also supported by a postdoctoral fellowship from the Canadian Institutes of Health Research (to K.H. Kim) and a Helmsley Postdoctoral Fellowship from The Rockefeller University (to K.H. Kim).
Author contributions: Ü. Coskun and T. Walz designed the experiments. T. Gutmann and M. Grzybek performed biochemistry, and K.H. Kim performed EM experiments. T. Gutmann, K.H. Kim, M. Grzybek, T. Walz, and Ü. Coskun analyzed the data and wrote the manuscript.
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© 2018 Gutmann et al. https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).


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Women's Health Connecticut (WHC), one of the nation's largest and most successful single-specialty groups, announced today that New Britain OB/GYN, has joined its overall network. The move further solidifies WHC's position as Connecticut's premier ObGyn practice.
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About Women's Health Connecticut
Founded in 1997, Physicians for Women's Health (d/b/a Women's Health Connecticut) is currently one of the largest and most respected women's health practices in the nation, with more than 270 highly-trained and devoted healthcare providers. By focusing solely on women's health, WHC provides unsurpassed expertise and devotion to addressing the changing needs of women across Connecticut, and more recently into Massachusetts. Its commitment and united voice for women has been influential in the women's health sector for more than 2 decades.
About New Britain OB/GYN
New Britain OB/GYN has been an ObGyn physician practice committed to promoting the health and well-being of women in a nurturing environment through supportive, compassionate and committed care. Its six providers will continue their reputation for offering a highly patient-centered environment, a caring experienced staff, comprehensive on-site services and a fully integrated team approach. New Britain OB/GYN currently has two offices located throughout central Connecticut in Southington and New Britain.
© 2019 Women's Health USA. All Rights Reserved.
We're growing across the U.S. and we're looking for provider talent in AZ, CT, MA, NV and TX. Find out more here.

redpajama_set_name: RedPajamaCommonCrawl


Items where School is "Huddersfield Business School > Emerging Markets Research Group"
Al-Shaghroud, Maha and Ng, Wilson (2015) Collective sensing as a micro-foundation of dynamic managerial capability:A situated cognition approach to identifying strategic opportunities. In: BAM2015 Conference Proceedings. BAM.
Anchor, J.R (2014) Earnings Expectations of EU Business School Students. In: SRHE Conference 2014, 10th-12th December 2014, Newport, South Wales.
Anchor, J.R (2000) Non-metallic mineral products. Industry and the British Economy : Cambridge Econometrics. pp. 98-100.
Anchor, J.R (2000) Non-metallic mineral products. Industry and the British Economy : Cambridge Econometrics. pp. 110-112.
Anchor, J.R (2001) Non-metallic mineral products. Industry and the British Economy : Cambridge Econometrics. pp. 96-98.
Anchor, J.R (2001) Non-metallic mineral products. Industry and the British Economy : Cambridge Econometrics. pp. 97-99.
Anchor, J.R (2002) Non-metallic mineral products. Industry and the British Economy : Cambridge Econometrics. pp. 97-99.
Anchor, J.R (2002) Non-metallic mineral products. Industry and the British Economy : Cambridge Econometrics. pp. 95-97.
Anchor, J.R (2003) Non-metallic mineral products. Industry and the British Economy : Cambridge Econometrics. 8.1-8.3.
Anchor, J.R (2003) Non-metallic mineral products. Industry and the British Economy : Cambridge Econometrics. 8.1-8.4.
Anchor, J.R (2005) Non-metallic mineral products. Industry and the British Economy : Cambridge Econometrics. 8.1-8.4.
Anchor, J.R (2006) Non-metallic mineral products. Industry and the British Economy : Cambridge Econometrics. 8.1-8.5.
Anchor, J.R (2000) Other mining. Industry and the British Economy : Cambridge Econometrics. pp. 62-65.
Anchor, J.R (2000) Other mining. Industry and the British Economy : Cambridge Econometrics. pp. 74-77.
Anchor, J.R (2001) Other mining. Industry and the British Economy : Cambridge Econometrics. pp. 60-63.
Anchor, J.R (2001) Other mining. Industry and the British Economy : Cambridge Econometrics. pp. 60-62.
Anchor, J.R (2002) Other mining. Industry and the British Economy : Cambridge Econometrics. pp. 60-63.
Anchor, J.R (2002) Other mining. Industry and the British Economy : Cambridge Econometrics. pp. 58-60.
Anchor, J.R (2003) Other mining. Industry and the British Economy : Cambridge Econometrics. 2.8-2.11.
Anchor, J.R (2003) Other mining. Industry and the British Economy : Cambridge Econometrics. 2.7-2.10.
Anchor, J.R (2005) Other mining. Industry and the British Economy : Cambridge Econometrics. 2.8-2.11.
Anchor, J.R (2006) Other mining. Industry and the British Economy : Cambridge Econometrics. 2.11-2.15.
Anchor, J.R (2007) Other mining. Industry and the British Economy : Cambridge Econometrics. 4.11-4.15.
Anchor, J.R (2007) Other mining. Industry and the British Economy : Cambridge Econometrics. 4.1-4.5.
Anchor, J.R (2008) Other mining. Industry and the British Economy : Cambridge Econometrics. 4.1-4.6.
Anchor, J.R (2009) Other mining. Industry and the British Economy : Cambridge Econometrics. 4.1-4.6.
Anchor, J.R (2010) Other mining. Industry and the British Economy : Cambridge Econometrics. 4.1-4.6.
Day, John and Rahman, Hafiz (2011) The Potential Influence of Entrepreneurial Role Models in the Teaching of Entrepreneurial Marketing: I want to be Like ... ? In: Research at the Marketing/ Entrepreneurship Interface. Global Research Symposium on Marketing and Entrepreneurship, USA, pp. 37-49.
Feather, Denis, Anchor, J.R and Cowton, Christopher J. (2011) The Value of the Undergraduate Dissertation: Perceptions of Supervisors. In: Paper presentations of the 2010 University of Huddersfield Annual Learning and Teaching Conference. University of Huddersfield, Huddersfield, pp. 41-56.
Siddiqui, Kalim (2008) Free-Market Illusion and Global Financial Crisis. Mainstream weekly, XLVI (49).
Siddiqui, Kalim (2011) Political Economy of Development in Pakistan. ZNet.
Siddiqui, Kalim (2009) Politics and Religion in Modern India. ZNet.
Yasin, Naveed (2012) The Enterprise Academy at the University of Strathclyde: A Case Study. Case Study. University of Huddersfield.
Yasin, Naveed (2012) The Enterprise Summer School, Modules and Events at the University of Manchester: A Case Study. Case Study. University of Huddersfield.
Yasin, Naveed (2012) Essential Enterprise Skills for Early Career Researchers. LERU Doctoral Summer School at University College London: A Case Study. Case Study. University of Huddersfield.
Yasin, Naveed (2012) The Talent Pool & Summer School at the University of Birmingham: A Case Study. Case Study. University of Huddersfield.
Yasin, Naveed (2012) iSolve at Cardiff University: A Case Study. Case Study. University of Huddersfield.
Yasin, Naveed, Williams, Dina and Pitchford, Ian (2012) Enterprise and Entrepreneurship for Postgraduate Research Students : Report 1 : Desk Exercise: Enterprise Skills Development Programmes. Project Report. University of Huddersfield.
Yasin, Naveed, Williams, Dina and Pitchford, Ian (2012) Enterprise and Entrepreneurship for Postgraduate Research Students : Report 2 : Enterprise and Entrepreneurship at Five UK Universities. Project Report. University of Huddersfield.
Yasin, Naveed, Williams, Dina and Pitchford, Ian (2012) Enterprise and Entrepreneurship for Postgraduate Research Students : Report 3 : Training Needs Analysis. Project Report. University of Huddersfield.
This list was generated on Sat Apr 20 06:49:35 2019 UTC.


text:
Q: failure to post PHP update to MySQL i have a button i am trying to get where if i click it it updates the column 'gorg' in the table users to giver according to the current user (session) logged in. Everytime i click the button i get
Access denied for user 'root'@'localhost' (using password: NO)

Here is the top of the php page (BTW i am 100% positive my DB Connection info is correct)
<?php
session_start();
include('src/sql_handler.php'); //this is where my DB Connection info is located
include('src/facebook_handler_core.php');
if(isset($_POST['submitgiver'])) {
    $query = "UPDATE users SET gorg='giver' WHERE email='".mysql_real_escape_string($_SESSION['email'])."'";
    $result = mysql_query($query) or die(mysql_error());

}

{
  if(isset($_SESSION['gorg'])=="Giver")
  {
    header('Location: picktreetype.php');
  }
  else if(isset($_SESSION['gorg'])=="Gatherer")
  {
    header('Location: gatherermap.php');
  }

}

?>

and now for the html
<form method="post" action="<?php echo $PHP_SELF;?>">

<input type="submit" class="button orange" name="submitgiver" value="Giver">

</form>

UPDATE:
heres the SQL_HANDLER
<?php

class MySQL_Con {

    private $host = 'localhost',

            $user = 'fruitfo1_admin',

            $pass = 'password',

            $db = 'fruitfo1_fruitforest',

            $_CON;

    function MySQL_Con() {

        $this->_CON = mysql_connect($this->host, $this->user, $this->pass);

        if(!$this->_CON)

            die(mysql_error());

        else {

            $select_db = mysql_select_db($this->db);

            if(!$select_db)

                die('Error Connecting To Database'.mysql_error());

        }

    }

    function End_Con() {

        mysql_close($this->_CON);

    }

}

?>

A: Apparently your connection setup doesn't include a password. Please post the sql_handler (WITH OBFUSCATED password) to be able to debug further.
If you're 100% positive it's correct, as you're saying, you can try explicitly passing sql handle to mysql_query.
Another note, mysql_* are deprecated, you really should consider switching to either mysqli or PDO.
Also, using root user for ANY kinds of web-applications is a no-no.

A: Your DB doesn't have a password. Try this
  private $host = 'localhost',

        $user = 'fruitfo1_admin',

        $pass = '',

        $db = 'fruitfo1_fruitforest',

        $_CON;

A: i found my issue, it did have to do with exactly what i thought it was. heres my updated code
if(isset($_POST['submitgiver'])) {
$mysql_hostname = "localhost";
$mysql_user = "fruitfo1_admin";
$mysql_password = "password";
$mysql_database = "fruitfo1_fruitforest";
$bd = mysql_connect($mysql_hostname, $mysql_user, $mysql_password) or die("Could not connect database");
mysql_select_db($mysql_database, $bd) or die("Could not select database");
$query = "UPDATE users SET gorg='giver' WHERE email='".mysql_real_escape_string($_SESSION['email'])."'";
$result = mysql_query($query) or die(mysql_error());
}

basically that IF statement that was attempting to POST wasnt linking back to my handler, so i placed the SAME db connection from the handler and carried it over inside the if isset statement and it worked!

redpajama_set_name: RedPajamaStackExchange


Nakia is a multi-media creative and lawyer based in New York City. Demonstrating an interest in the arts from an early age in her native Jamaica, Nakia began her creative career styling photo shoots for Kesha Lambert Photography, and later documenting styling projects and fashion industry news and opinion as founding editor of web magazine, StyleChile.
Nakia continues to provide creative direction and styling services on ad campaigns for StyleChile; for editorials in publications such as UPTOWN Magazine; and as commissioned by brands and individuals.
Additionally, Nakia is founding principal of Stelle Legal, a firm that provides legal and advisory services to clients in the fashion, art and entertainment industries. Nakia also practices corporate transactional law for a Fortune 500 company, and appears in WETV's Money.Power.Respect. -- a docuseries focusing on lawyers in New York City.
Nakia served on an advisory board for Moore College of Art & Design, and is a member of the Board of Directors of Bronx Legal Services.


Extraterrestrial objects plunge into the sea, releasing the seeds of life on Earth. That's the event depicted in this image, by John Brackenbury of the University of Cambridge, which recently won a Visions of Science Photographic Award in the UK. The concept isn't as outlandish as it seems. Panspermia - the notion that the basic building blocks of life float through the universe and occasionally take root in a hospitable environment - for years has been an alternative theory to how protocells formed. As beautiful as this visualization is, panspermia isn't really about eggs. Most proponents (including Nobel Prize winner Svante Arrhenius) figure that random grains of biological material from outer space set evolution into motion. But for Brackenbury, eggs embody panspermia's unconventional recipe for primordial soup.
How Can I Swap Safely?


text:
import os
from flask import Flask
from raven.contrib.flask import Sentry

env_config = [
    ('SEARCH_INDEX_PATH', '/tmp/testindex'),
    ('SEARCH_INDEX_SECRET', 'supersecretnotreallythough'),
]

sentry = Sentry()

def prime_config(config):
    for key, default in env_config:
        val = os.environ.get('RIGIDSEARCH_' + key)
        if val is not None:
            config[key] = val
        elif default is not None:
            config[key] = default

def create_app(config_filename=None, config=None):
    app = Flask(__name__.split('.')[0])
    sentry.init_app(app)
    prime_config(app.config)

    if config:
        app.config.update(config)
    if config_filename:
        app.config.from_pyfile(config_filename)

    from rigidsearch.api import bp as api_bp
    app.register_blueprint(api_bp)

    return app

def make_production_server(app, options):
    import logging
    from gunicorn.app.base import Application

    log_handler = logging.StreamHandler()
    log_handler.setLevel(logging.WARNING)
    app.logger.addHandler(log_handler)

    class RigidsearchServer(Application):

        def __init__(self, app, options):
            # Non-optional gunicorn attributes
            self.usage = None
            self.prog = None
            self.cfg = None
            self.callable = None

            self._app = app
            self._options = options
            self.do_load_config()

        def init(self, *args):
            options = self._options.copy()
            options['worker_class'] = 'gevent'
            options['proc_name'] = 'rigidsearch'
            options['timeout'] = 300
            return options

        def load(self):
            return self._app

    return RigidsearchServer(app, options)

redpajama_set_name: RedPajamaGithub


Application letter addressed to the Chief Executive, which should contain information about the applicant and details of the establishment applying for tourism utility.
If the application is made by a company, the relevant application letter must be signed by the company's lawful representative(s) according to the signature on the identity document(s). A copy of the representative(s)' identity document(s) (front and reverse side on the same page) as well as the company chop must be appended to the application.
If the application is made by an individual, he/she should sign the relevant application letter according to the signature on the identity document, with a copy of the identity document (front and reverse side on same page) attached.
Past and coming five years financial data (For newly established enterprise, only the financial data of the coming five years is needed. For enterprise established less than 5 years, the financial data from newly established till now and the coming five years is needed.).
Number of customers visitation (please list out the percentage by hotels, restaurants, retails and others).
The menu(s) and alcoholic beverage menu(s) must have the name of the restaurant and the price list.
Remarks: Applicant must submit all menus and alcoholic beverage menus that are in-use at the relevant restaurant for client's selection.
If the application is made by a company, an original certificate of company registration issued by the Conservatory of Commercial and Movable Property within the past three months must be submitted.
4. Original identity document of the person submitting the application must be produced.
NB: After the relevant application is approved, it will be published in the Official Bulletin of the Macao Special Administrative Region and take effect on the day of publication.
Any other factors benefiting local tourism.


text:
Foreign Competitors Set Up Fabs in China and Compel Country's Domestic Foundries to Expedite Their 28nm Production This Year, Says TrendForce
10 April 2017 Semiconductors / Telecommunications TrendForce
United Semiconductor Xiamen (USCXM), China-based subsidiary of major foundry UMC, is reported to begin production on the 28nm technology licensed from its parent company this second quarter. As international semiconductor companies set up fabrication plants in China, the country's domestic foundries will face intensifying competition in terms of technology, market share and recruitment of technical personnel. According to the global market intelligence firm TrendForce, Semiconductor Manufacturing International Corporation (SMIC) and Shanghai Huali Microelectronics Corporation (HMLC) are the two leaders among Chinese foundries in the development of the 28nm processing. The existence of foreign-owned fabs in China will pressure these two domestic manufacturers to further accelerate their timetables for this technology.
TrendForce's research finds that the most advanced node currently available for China's foundry industry is the 28nm. SMIC's wafer capacity for 28nm devices represented less than 1% of the global total for 2016. The world's largest foundry TSMC accounted for 66.7% of the global 28nm capacity for the same year, while the second-largest GlobalFoundries had 16.1% and the third-place UMC had 8.4%.
Data from SMIC's quarterly earnings report reveals that the share of 28nm devices in the company's total revenue in the fourth quarter grew to 3.5%. At a road show held on February 15, SMIC's CEO Dr. Tzu-Yin Chiu stated that the 28nm node will be a major revenue growth generator for 2017. Chiu expects that 7~9% of the company's total revenue by the end of this year will come from products made with 28nm or more advanced technologies. SMIC's 2016 annual results announcement also projects that 28nm products will make up nearly 10% of the total revenue at the conclusion of 2017.
HLMC is the other major domestic foundry that is working on the 28nm processing. Though the company announced that its collaboration with MediaTek has led to successful tape-out of 28nm mobile chips, there is still some progress to be made before mass production can be realized. In terms of capacity planning, HLMC started the construction of its second fab at the end of 2016. This facility, which will be initially responsible for 28nm production, is scheduled for completion and trial runs in 2018. HLMC is also considering to have its second fab transition to more advanced nodes (the 20nm and the 14nm) and adopt FD-SOI processing in the future.
TrendForce notes that currently China's IC industry is in its high-growth period. With support from various levels of government and local funds, domestic IC manufacturers are making huge investments to expand the scales of their operations. The take-off growth of China's IC market is also drawing foreign companies and capital. Major international semiconductor enterprises want to establish themselves in the country during this critical time so that they will be in a more competitive position later on.
From the aspect of technology transfer, foreign semiconductor companies that have set up shop in China generally have licensed technologies that are at least a generation behind from what they currently have in their headquartering countries. This practice puts domestic manufacturers at a disadvantage because they are competing in the home market with products made from similar technologies.
As the more advanced foreign-owned fabs in China begin production, domestic manufacturers will be under increasing pressure to catch up
Within the foundry sector, international companies so far have undertaken several fab projects in China to compete against the production capabilities developed by domestic manufacturers. TSMC's Nanjing fab, which will begin to operate in 2018, is assigned to produce devices on a 16nm FinFET process. This will put pressure on SMIC's timetable for its 14nm FinFET process that is expected to be ready for mass production at the start of 2019. In Chengdu, GlobalFoundries is also expediting a fab project that will be completed in 2019 and feature 22nm FD-SOI manufacturing. This plant can be seen as attempt to overtake HLMC's development schedule for the same technology.
More recently, UMC announced that it will license its 28nm technology to its China-based subsidiary United Semiconductor Xiamen (USCXM). This move followed UMC's success in mass production for its 14nm FinFET technology. USCXM was at the early stage of adopting 50/40nm technology when it began operation at the end of last year. The subsidiary foundry will now undergo a dramatic upgrade and at the earliest will begin making devices on a 28nm Poly/SiON process this second quarter. At end of 2017, USCXM monthly wafer capacity is estimated to reach around 16,000 pieces, of which 10,000 pieces are expected to come from its 28nm process. This would make USXCM the leading 28nm producer that is located in China.
SMIC, which is also currently producing on a 28nm technology, will be impacted by UMC's plan. SMIC has been making low-end and mid-range IC products using the 28nm Poly/SiON process. The company is inexperienced with HKMG, which is the more advanced manufacturing solution. Additionally, UMC is ahead of SMIC by two years in the mass production on the 28nm technology. USCXM will thus gain access to a more mature 28nm technology that can offer better and stable yields. The licensing deal will ultimately affect SMIC's plan for its 28nm manufacturing.
In sum, TrendForce believes China's foundry industry will face a very heated and direct competition for technical personnel, resources and market shares once foreign-own fabs located within the country start operating. Domestic IC manufacturers will now have to really fast track their technology roadmaps so that they are prepared for the coming struggle.
TrendForce Reports 1Q17 Global Smartphone Production Volume Fell 23% From Prior Quarter Due to Seasonality; Demand to Remain Weak in Second Quarter
TrendForce Says Potential Scale of Micro-LED Market Could Reach US$30~40 Billion If It Was to Supplant the Current Display Chain
Attribution Guide
In-Depth Analyses
Value of 5G Market Will Reach US$37 Billion in 2026 as Applications Related to Metaverse Act as Notable Demand Driver, Says TrendForce
Foundry Revenue Is Forecasted to Drop by 4% YoY for 2023 Due to Slow Inventory Consumption and Falling Wafer Input from Customers, Says TrendForce
QoQ Decline in DRAM ASP Will Moderate to Around 13~18% for 1Q23, but Slump Will Continue, Says TrendForce
1Q23 Consumer DRAM Datasheet
Server Market Bulletin_20230119
DRAM Market Bulletin_20230118


79.94 % / 10802 votes.
I walked past a homeless guy with a sign that read, "One day, this could be you." I put my money back in my pocket, just in case he's right.
72.79 % / 3047 votes.
The video from the ring infects people with airborne pathogens. The video went viral.
52.54 % / 38 votes.
Your phone screen is brighter than your future.


text:
Devastating Blow To Feminism: This Woman Sucks
Although the past few years have been an incredibly powerful time for women's rights, feminism has just suffered a pretty crushing setback: Tragically, this woman sucks.
Devastating. This is not good news for the feminist cause.
The notion that all women are powerful queens who deserve the world sadly doesn't even come close to applying to 28-year-old Sarah Baylor, whose boring hobbies, grating personality traits, and overall lack of positive attributes prove that some women can (and do) absolutely suck. Between the below-average work she does at her totally inconsequential job, the meandering anecdotes about owning a ferret she foists on unsuspecting acquaintances, and the fact that she honestly smells kind of bad, Sarah unfortunately does not bring a single thing to the table that would compel someone to fight for women's rights on her behalf. It's tough to imagine anyone getting behind a movement intended to empower someone as unpleasant to be around as this god-awful woman.
Unfortunately, it's difficult to extend any sisterly support to someone who sucks this much ass.
There's no telling how far Sarah will set back the feminist cause over the course of her life, but her apparent commitment to using her bad personality to totally blow every personal and professional opportunity that comes her way suggests that she will continue giving women a bad name for the foreseeable future. Had Sojourner Truth known that one day a woman like Sarah would go through life without ever doing anything even marginally impressive—seriously, not even winning some kind of bogus "citizenship" award in elementary school—would she have bothered to fight for women's rights in the first place? It's hard to say.
Yikes. We can only imagine what kind of damage Sarah already done to feminism just by being her selfish, slovenly, and frankly horrible self. Here's hoping the feminist agenda finds a way to prevail despite such an awful blight on the movement.
More posts in News »


Dual Power Connectors - WiringProducts. Ltd.
Dual high power connectors come in three sizes with each size rated for 50 amp, 175 amp, and 350 amp current capacity. They are all rated for use up to to 600 volts continuous AC or DC operation. Each dual power connector allows you to connect two power cables together creating a highly reliable and high current power connector. For 12 to 6 AWG wire, use the 50 amp connector pins and housings, for 4 to 1/O AWG cable, use the 175 amp connector pins and housings, and for 2/O to 4/O AWG cable, use the 350 amp pins and housings. The power connectors are unisex, and will mate together by simply rotating one of the connectors 180 degrees. Like colors will only mate together. For example, two red connectors will mate together, but a red and a black connector will not. Connector pins and housings are sold separately. The pins are tin plated and can be either crimped or soldered to your cables. Housings feature retention spring clips which will hold the cable and pin securely inside the housing when inserted properly, but it can easily be removed using a small flat screwdriver to release the retention spring. PLEASE NOTE: Two (2) housings and four (4) contacts are required for a complete connection.


text:
Elizabethan names
Finding the right name for a character is one of the most important stages in writing a book, particularly when it comes to historical fiction when names must feel authentic. I'm often asked where I found the name Hawise (pronounced Ha-wees-a) and the answer is in the 15th-century will of Hawise Aske which I read for a seminar long before I even thought about writing Time's Echo. It would have been an usual name by the late 16th-century, when Time's Echo is set, but for some reason it stuck in my mind and it never occurred to me to call Hawise anything else. However, so many people puzzled over the pronunciation that I have called the heroine of The Memory of Midnight Nell, a name I don't see causing similar problems.
A page from the Wardmote Court Book*
For other names, I unashamedly pillaged the wardmote court records which I've been working on for so long. I didn't want to name my main characters after real individuals but several of the secondary characters, or those who make fleeting appearances in the story, were real people who lived in York in the 1570s and 1580s: William Beckwith, Lancelot Sawthell, John Harper, Christopher Milner, Richard Lydon, Anne Ampleforth, Miles Fell and Nicholas Ellis.
In some ways it feels uncomfortable to use real people's names and make up stories about them, but I like to think that most of them would have enjoyed the idea that they have not been completely forgotten. Miles Fell would probably have objected, of course, but there's always one …
Originally Francis was called William Bewlay, until I found a real William Bewlay in the records and decided to change the name (which was fine until I did a search and replace for Will and William and ended up with all sorts of odd sentences beginning 'Francis you go to market?' and so on). It was important that Ned Hilliard came from outside York, so I deliberately picked a name that wasn't in the records … or so I thought until I came across a 'Mr Hilyerd' recently. It gave me quite a turn.
For other characters, I tend to pick a surname from the records at random. For men, there are a surprising number of unusual Christian names to choose from. Besides the ubiquitous William, Robert, John and Thomas, the wardmote records contain a number of names that feel anachronistic – Brian, Martin, Gregory, Anthony, Miles – as well as several that clearly stem from the popularity of courtly romances: Lancelot, Arthur, Tristram, Percival and a smith called Gawain Bensdine. Other unusual names include Sir Valentine Brown, Marmaduke Wyman, Samson Percival, Ambrose Awne, Augustine Dockam and the wonderfully named Hercules Welbourne. If I didn't think readers would think I was pulling their leg, I'd like to put them all in a book one day!
Female characters for the Elizabethan period are much harder to name. There are far fewer women named in York's civic records, and when they are presented for an offence, they are usually referred to by their title (Lady, Mrs or 'uxor') or simply as a someone's wife. Occasionally a prostitute or scold is named but the range is limited: Anne, Isabel, Joan, Margery.
I had better luck looking through the Calendar of Assize Records for the second half of the 16th century, which gives Christian names for women indicted or mentioned as witnesses. Here I found Elizabeth, Agnes, Alice, Ellen, Joan, Jocosa, Martha, Cecily, Mary, Margaret, Margery, Catherine, Juliana, Benedicta, Jane, Silvestra, Constance, Anne, Thomasine, Rebecca, Isabel, Emma, Charity, Christine, Petronella, Sarah, Dorothy, Avis, Mabel, and Edith.
Interestingly (or something else!), I found no Eleanor, which is Nell's full name, although Ellen and Helen are closely related. Perhaps it will turn out that Nell, like Hawise, is named for a grandmother, although it was more normal at the time to call a child after a godparent or, in the case of girls at least, the midwife.
* A list of parish constables and churchwardens appointed in the Walmgate wardmote court, April 1581. YCA E31, p.87. By kind permission of City of York Council Archives and Local History. www.york.gov.uk/archives
Tags: 16th century, assize courts, Elizabethan names, historical fiction, naming characters, time slips, Time's Echo, wardmote courts
Posted on: January 31st, 2013 | 16 Comments
Thanks for the post, I really enjoy learning about all sorts of interesting names.
I love the Welsh name for Ambrose, which is Emrys. The female name that I love in your list is Juliana, because my husband's name is Julian.
Besides having the name be historically correct, do you ever choose names because of their meanings?
Pamela Hartshorne says:
I can't say I think about a name's meaning, Tess. I tend to go more on its feel and the personal associations it has for me. I want to call my next Elizabethan heroine Jane, because its plainness suits her, but I'm thinking of calling her flighty sister Juliana …
And thanks for the snippet about Emrys/Ambrose – am filing that little fact away for the future! I know an Ambrose, in fact, but it sounds better in Welsh, don't you think?
I think Emrys is a lot more romantic and mysterious than Ambrose..plus it's Welsh! My husband is Welsh. I love it so much that when we were contemplating having a baby my name choice for a boy was Kai Emrys, but my husband's choice was Rowan (no children for us though).
Also, There was an historical Emrys whow was known as Merlin, but he wasn't the historical Merlin of course. I'm sure that's why in the BBC show Merlin they sometimes call him Emrys.
Julie Parker says:
Interesting post thanks Pamela. I would really like to know how you came up with the name 'Hap' for the dog. I remember in the book there was a mastiff which bit someones leg and that this incident had actually been recorded. That was just fascinating!
Thanks, Julie. In my first draft, I called Hawise's dog Lucky, but a medievalist friend queried whether lucky would have been used in the same way at that period. She suggested hap, meaning chance or fortune (good or bad) as an alternative, and I actually like it much better as a name, so Hap he became.
Miles Fell did indeed have a mastiff that bit Nicholas Ellis on the leg in York in 1577. That was the incident that inspired the whole book, in fact. Sadly we don't know that dog's name!
Oh yes, Hap is a great name! I loved little Hap and when he was killed it was dreadful! (Sorry, I'm so used to writing on FB or texting I am finding it hard to control my use of emoticons and may be overusing!) How many drafts do you write? Do you write the whole thing as it comes, or plan the whole story first then write it? Your work as Jessica Hart is very different isn't it – is it difficult to separate your writing styles?
I was fond of Hap too and I got quite emotional when he was killed as writing the scene reminded me so much of when my own old Westie, Mungo, had to be put to sleep.
As for writing process … I try to plan before I start, but in practice I never stick to it, and most of the plotting happens as I write. I do a rough draft (a shitty first draft) which is more or less stream of consciousness, and then go back and start a proper draft. I never look at the rough draft again and always wish I could skip that part of the process, but it seems to be important! The proper draft is usually pretty agonizing too, and I would expect to go back and rework a lot of it before submitting anything.
I go through exactly the same process when I'm writing as Jessica Hart, in fact, although the stories and the style are quite different. I couldn't write two books at the same time as some authors do as my mind needs to be in quite a different mode for each genre. It is a struggle sometimes, yes!
Megan Reddaway says:
Do you mean it would have been an *un*usual name by the 16th century (first paragraph)? It's a lovely name, and a great story.
P.S. I see the name derived from Hedwig (like Harry Potter's owl), which morphed into Havoise in Normandy, then Hawise in Norman England, and then Avis or Avice.
Interesting! I didn't know origin of the name, Megan, so thanks for that. I've come across Avis quite a few times in the 16th century records but didn't realise the name was related to Hawise.
I'm away from home at the moment so can't access (or rather, don't know how to!) the original post to answer your query about whether Hawise usual name in the 16th century but it sounds as if I misstyped. I certainly meant that it was *un*usual by the 16th century, but whether I actually said that or not is another matter! I love the name Hawise too, but won't make the mistake of giving my character an unusual name again!
Lyn Millman says:
There are quite a few recorded Hawises throughout medieval history, usually interchangeable with Avice, sometimes Avis. As you will know, names and spellings weren't set in stone back then – look at all the Mauds/Matildas/Mallts who often turn out to be one and the same – and there are usually French (Avice) and English (Hawise) versions of every name.
Av-iss/How-iss: this seems to imply that it's pronounced 'How-iss' and not 'Ha-wees-a'.
You may be right about the pronunciation, Lyn. I just took the pronunciation I've heard medievalists using, and not being a linguist struggled to find a way to convey that sort of catch of breath after the 's' – maybe that last 'a' in Ha-wees-a is misleading as I've written it. I intended it to be a swallowed sound rather than a drawn out 'a'; really just wanted to indicate the name wasn't pronounced Haw-wise, as my first editor read it!
I hadn't registered the connection between Avis and Hawise before, I must say, so thank you for that.
Geoffrey Tobin says:
Names often skip a generation, for instance a man may name a daughter after his mother, which, curiously, is genetically sound, X-chromosomally. Duchess Hawise of Brittany was named after her grandmother Hawise of Normandy, a sister of Duke Richard II and of Emma, twice Queen of England.
"Hawise" is a Germanic name said to translate literally as "battle fight": many German women's names are martial in character. The modern form, Avis, is a homonym for the Latin word for "bird". Puns and allusions like this often reflect Breton influence.
Emrys Wledig is a Welsh name for the early-to-mid sixth century polemicist Gildas's historic British military leader and hero Ambrosius Aurelianus: "Wledig" rather grandly means "emperor", though if he is identical with his contemporary Riothamus (flourished 468-471) whom the Romans called High King of the Britons (the literal meaning of "Riothamus"), then he was an emperor of sorts, with extensive territory on both sides of the Channel.
Ambrosius's ancestry may be alluded to by the 11th century epitaph of Alan Rufus, leader of the Bretons in England. The Latin adjective "rutilans", meaning brilliant red-gold, is used to describe Alan. Gold suggested to me a reference to the name Aurelius. When I looked up the Aurelius gens (clan) of Ancient Rome, I learnt that the dictator Julius Caesar's mother was named Aurelia Cotta and her mother was Rutilia. The Breton dukes claimed a familial connection with Caesar, so this may be it
The Aurelii are fascinating because they were plebeian, i.e. commoners, but nonetheless provided many consuls and emperors. The Hypogeum of the Aurelii in Rome tells us more about them: they were early adopters of Christianity, and they weren't fussy about name order: the apparently garbled form "Aurelius Ambrosius" is a perfectly standard variation of the name Ambrosius Aurelianus.
Coupling this with Gildas's statement that his hero's parents "wore the purple" strongly suggests what Ambrosius's ancestry was, and why Riothamus was a close and loyal ally of the Romans despite Britain's (and Brittany's) declared independence.
Donna Graham says:
I have just started reading "Time's Echo", yesterday, and cannot put it down. I love the way you move from the present to the past, flawlessly. I love the story and the names of the characters you have found. Doing a bit of family history it is surprising to see unusual names, and from not that far back really, Providence, Sampson/Samson and Jabez have been found, and side-tracking the family lines, because you hit a brick wall – found Snowball – a first name of a gentleman, all these are from families around 20-60 miles from York. I just love the book and although interested in how names are pronounced, not going to take anything to literally. I have an Aunt, we live Yorkshire called Doreen – pronounced "Dooreen", but a work colleague who came from Cumbria pronounced her name "Dureen". So, as all things, it depends where you come from and where you live as to how you pronounce it.
This is the first book of yours I have read and an definitely going to look out for more. I love a good book and not all authors can produce page turners you can't put down (C J Sansom is another favourite).
Thank you so much for your kind comments, Donna. I am so pleased that you're enjoying Time's Echo. Am envious too of your Yorkshire connections and those interesting family names. I have never heard of a Snowball before – am sure there must be a story there! Happy reading – and researching!
You can use these tags:   <acronym title="">
      <q cite=""> <s>
Please type "3153f5":


text:
Crosby Kitchens in liquidation affects NTP Kitchens and Kutchenhaus (Nobilia) franchise
Industry NewsKBB News September 19, 2013 - Comments Off on Crosby Kitchens in liquidation affects NTP Kitchens and Kutchenhaus (Nobilia) franchise
Rotherham-based Crosby Kitchens has confirmed that it is has gone into liquidation, while its sister company Aberdeen-based NTP Kitchens has been placed into receivership.
Gordon MacLure and Ewen Alexander from Johnston Carmichael have been appointed receivers and have already commenced talks with staff, key suppliers and customers. Three of Crosby Kitchens's six staff members have been made redundant with immediate effect, while the remaining employees have been retained to assist with a closing down sale and other administrative matters.
It is thought that some 60 to 70 customers with outstanding orders are being consulted.
NTP, which designs, builds and installs kitchens, employs 23 members of staff at its Bridge of Don headquarters. The company also operates the Kutchenhaus franchise in Scotland on behalf of Nobilia, employing six staff members at its Union Square store.
A spokesman for Crosby Kitchens and NTP Kitchens said: "It is with deep regret that we confirm today that NTP Kitchens has been placed into receivership, whilst Crosby Kitchens has entered liquidation.
"These developments stem from the recent recession, which had a hugely negative impact on retail sales, with customers proving to be extremely cautious when it came to discretionary purchases such as kitchens.
"It was hoped that the market would have picked up significantly by this point – and demand for kitchens would have returned to pre-credit crunch levels – but unfortunately this has not materialised, leading us to today's decisions."
Samsung Canada Solves Dinner (and Design) Dilemmas for Fresh Cooking Series
Kitchen & Bath Industry Show Las Vegas Feb 2014


text:
Q: TypeScript type checking JSX children My TypeScript Version is 2.3.2
According to this Type checking JSX children
The following code should work:
import * as React from 'react';
import * as ReactDOM from 'react-dom';

interface TestProps {
    children: string | JSX.Element;
}

const Foo = (props: TestProps) =>
{props.children}
;

// Error on Foo
ReactDOM.render(
    <Foo>

Test

    </Foo>,
    document.getElementById('content'),
);

But i get the following compilation error:
TestTsxChildren> tsc --version
Version 2.3.2
TestTsxChildren> tsc
main.tsx(11,5): error TS2322: Type '{}' is not assignable to type 'IntrinsicAttributes & TestProps'.
  Type '{}' is not assignable to type 'TestProps'.
    Property 'children' is missing in type '{}'.

What did i do wrong? Or did i not understand what the issue was trying to fix?

A: This is because the definition file for React is not updated.
The definition file needs to include interface ElementChildrenAttribute { children: {}; } so that TypeScript compiler known the name of the "children" property.
PR to update the definition file: https://github.com/DefinitelyTyped/DefinitelyTyped/pull/16327)


You MUST have a prior business relationship with the recipient, relating to the contents of the advertising email before you send them advertising email.
DO ask your customers to provide email addresses when communicating with them. At the same time ask them to indicate whether or not they are prepared to receive advertising and/or information messages at those addresses.
DO respect the wishes of those customers who provide addresses but ask not to receive marketing email messages.
DO keep records of documentation relating to the above "opt in" process.
NEVER purchase a list of email addresses.
NEVER send messages to addresses which were "harvested" from web sites.
If you have access to an email database which was collected for one business or product, NEVER use it for marketing another unrelated business or product. (Eg clients whose details were collected while involved in banking, should never be sent email advertising insurance).
Always provide full identification and contact details of the party sending the advertising emails.
Ensure that the "from" address is valid and will accept non-delivery reports (bounce messages).
Limit the size of advertising messages. Large unsolicited messages are a cause of irritation to the recipient, even when you do have a business relationship with them. Typically messages should be kept under 300kB. Note that when a file or graphic is attached to an email, it is encoded which increases its size, so attachments should be smaller than this.
If you wish to include large items like graphics or price-lists in your messages, rather host these on a web site and just put hyperlinks in the message referring to the external material. If the documents are very large, indicate in the message how large they are so that people can chose not to download them if they are on a slow or expensive link.
Mail returned to the "From" address listed in the message must be accepted and processed carefully.
Ensure (ie run tests and verify) that your own anti-spam filters (and those of your upstream service provider) do not filter out responses and bounce messages to your advertising messages.
Invalid addresses which generate "bounce" messages should be removed immediately from the database. Ideally this should be done automatically by software.
Any response which indicates that the message caused offence to the recipient should result in that email address being removed from the database.
Any response which requests information as provided for in the ECT ACT should be dealt with in a responsible manner. Information supplied back to the correspondent should be correct.
Every response which requests a follow-up with respect to the service or product advertised should be afforded a personal reply.
Ensure that when addresses are removed from the list, that they are removed from all copies of the list.
Do not configure an "auto-responder" on the mailbox to which returned messages are delivered. Sending an automated response in reply to every bounce message or reply would itself be a form of spamming.