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Two potential calmodulin-binding sequences in the ryanodine receptor contribute to a mobile, intra-subunit calmodulin-binding domain
Xiaojun Huang, Ying Liu, Ruiwu Wang, Xiaowei Zhong, Yingjie Liu, Andrea Koop, S. R. Wayne Chen, Terence Wagenknecht, Zheng Liu
Journal of Cell Science 2013 126: 4527-4535; doi: 10.1242/jcs.133454
Xiaojun Huang
Wadsworth Center, New York State Department of Health, Albany, New York 12201, USADepartment of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, New York, 12201, USA
Ying Liu
Wadsworth Center, New York State Department of Health, Albany, New York 12201, USA
Ruiwu Wang
Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology, and of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Xiaowei Zhong
Yingjie Liu
Andrea Koop
S. R. Wayne Chen
Terence Wagenknecht
Zheng Liu
For correspondence: liuz@wadsworth.org
Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction between R3595–CFP and K4269–YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating.
Calmodulin (CaM) is a 16 kDa ubiquitous calcium-sensing protein. It can bind to more than 30 proteins and enzymes that are involved in growth, apoptosis, motility, mitosis and development (Bähler and Rhoads, 2002). CaM consists of four EF-hand calcium-binding motifs, which give CaM the ability of binding four Ca2+ in total. Two of these EF-hand motifs form the N-terminal lobe of CaM, while the other two form the C-terminal lobe. In muscle excitation–contraction coupling, CaM modulates the major participating transporters, including the ryanodine receptor (RyR) in sarcoplasmic reticulum (SR) membrane and the L-type calcium channel (DHPR) in the transverse tubule (Aracena et al., 2005).
RyR is a calcium release channel that controls calcium flow from the lumen of the SR to the cytosol. It plays a key role in excitation–contraction coupling, during which an electrical stimulus (from a neuron) triggers muscle contraction. The electrical stimulus induces depolarization of the muscle plasma membrane, and activation of the DHPR. RyR is the next target to be activated, resulting in an increase in the concentration of Ca2+ in the cytosol. The increased cytosolic Ca2+ concentration triggers sliding filament movements within the myofibril and results in the contraction of muscle. Mammalian RyRs mainly exist in three isoforms: RyR1 and RyR2 are predominantly distributed in skeletal and cardiac muscle, respectively; RyR3 was first cloned from brain, but is also expressed in the diaphragm, smooth muscle and several abdominal organs (Kushnir et al., 2005).
CaM regulates the RyRs by a direct interaction and by an indirect phosphorylation through CaM-dependent kinase II (Ai et al., 2005). Both apo-CaM (no Ca2+ bound) and Ca2+-CaM (four Ca2+ bound) can bind to all three RyR isoforms with high affinity (Kd value in the nanomolar range) and regulate RyR channel functions. At low (resting) Ca2+ concentration (∼100 nM), CaM exists mainly as apo-CaM, and apo-CaM activates RyR1 (Buratti et al., 1995; Damiani and Margreth, 2000; Tripathy et al., 1995) and RyR3 (Chen et al., 1997; Yamaguchi et al., 2005), but inhibits RyR2 (Balshaw et al., 2001; Xu and Meissner, 2004). At high Ca2+ concentration (∼1 µM), most CaM exists as Ca2+-CaM, and Ca2+-CaM inhibits all three RyR isoforms (Buratti et al., 1995; Chen et al., 1997; Xu and Meissner, 2004; Yamaguchi et al., 2005). Interestingly, apo and Ca2+ forms of CaM bind at distinct but overlapping locations in RyR1 according to three-dimensional (3D) cryo-electron microscopy (cryo-EM) results from our laboratory (Samso and Wagenknecht, 2002; Wagenknecht et al., 1997), implying that CaM moves on the surface of RyR1 by ∼30 Å when it switches between Ca2+-free and Ca2+-bound states. Recently, using a mutant CaM that is incapable of binding Ca2+, we confirmed the distinct binding sites for CaM in RyR1 and demonstrated that the binding location switch is due to binding of Ca2+ to CaM as opposed to effects of Ca2+ itself on the conformation of RyR1 (Huang et al., 2012). Interestingly, apo-CaM binds to RyR2 at a similar binding site to that of Ca2+-CaM on RyR1, in seeming agreement with the inhibitory effects of these two forms of CaM on their respective receptors (Huang et al., 2012).
To identify CaM-binding sequences in the primary structure of RyR, CaM overlays on RyR1- and RyR2-derived peptides were used to test the binding capacity of CaM on RyR fragments, which were expressed from RyR cDNA fragments generated from available endonuclease restriction sites (Balshaw et al., 2001; Chen and MacLennan, 1994; Guerrini et al., 1995; Menegazzi et al., 1994). A number of sequences have been reported to be potential CaM-binding sequences (Fig. 1). In RyR1, these include residues 921–1173 (Chen and MacLennan, 1994), 1975–1999 (Zhang et al., 2003a), 2063–2091 (Chen and MacLennan, 1994), 2804–2930 and 2961–3084 (Chen and MacLennan, 1994), 2937–3225 (Menegazzi et al., 1994), 3042–3057 (Guerrini et al., 1995), 3225–3662 (Balshaw et al., 2001), 3546–3655 (Menegazzi et al., 1994), 3611–3642 (Chen and MacLennan, 1994), 3614–3643 (Rodney et al., 2001; Xiong et al., 2002; Zhu et al., 2004), 3617–3634 (Guerrini et al., 1995), 4302–4430 (Balshaw et al., 2001), 4303–4328 (Chen and MacLennan, 1994), 4425–4621 (Menegazzi et al., 1994) and 4540–4557 (Guerrini et al., 1995). In RyR2, these include residues 263–614, 2724–3016, 3007–3023, 3298–3961, 3583–3603, 4480–4497 and 4548–4748 (Balshaw et al., 2001; Guerrini et al., 1995; Yamaguchi et al., 2003). The evidence supporting these hypotheses of CaM regulation is mostly based on studies of synthetic RyR peptides or RyR fragments rather than on the intact, full-length RyR. Although the use of RyR peptides helps simplify the experiments, the results gained from RyR peptide studies may not represent what occurs in the intact RyR, and thus some internal, buried RyR sequences are likely to be detected as potential CaM-binding sequences. CaM, as a soluble protein, however, can only bind to sequences exposed on the surface of RyR.
Proposed CaM-binding sites in the primary sequences of RyR1 and RyR2. Regions proposed for Ca2+-CaM binding are shown as red boxes, whereas regions proposed for both apo-CaM and Ca2+-CaM binding are shown as pink boxes. [Figure adapted from Balshaw et al. (Balshaw et al., 2001) with modification.] The two green circles indicate positions where GFP was inserted (see Materials and Methods) into the sequence of RyR2 in two of the proposed CaM-binding regions, specifically, after residues Arg3595 and Lys4269.
To further test these potential CaM-binding sequences, in this study we used site-specific GFP insertion coupled with 3D cryo-EM, to map two of the proposed CaM-binding sequences. 3D localization of inserted GFP is based on structural differences between 3D reconstructions of GFP-inserted RyR2 and no-insertion RyR2 control. The two binding sequences that we investigated were: sequence 3581–3612 in RyR2 (corresponding to sequence 3614–3643 in RyR1) and sequence 4261–4286 in RyR2 (4303–4328 in RyR1). Fig. 1 illustrates schematically the two GFP insertion sites in the primary sequence and their correlation to the reported CaM-binding sequences. The DNA sequence of GFP was inserted after Arg3595 for mapping sequence 3581–3612 (henceforth named RyR2R3595-GFP), and after Lys4269 for the sequence 4261–4286 (henceforth named RyR2K4269-GFP), respectively. Extensive experimental evidence supports the view that amino-acid residues within the sequence 3614–3643 in RyR1 and the corresponding sequence 3581–3610 in RyR2 are involved in binding CaM (Balshaw et al., 2001; Chen and MacLennan, 1994; Guerrini et al., 1995; Menegazzi et al., 1994; Rodney et al., 2001; Xiong et al., 2002; Zhang et al., 2003a; Zhu et al., 2004). Sequence 4261–4286 in RyR1 (4303–4328 in RyR2) was first detected as a CaM-binding sequence in our 125I-labeled CaM binding study (Chen and MacLennan, 1994), and later confirmed in a 35S-labeled CaM binding study (Balshaw et al., 2001). This sequence is also close to several other proposed CaM-binding sequences (Balshaw et al., 2001; Guerrini et al., 1995) (see Fig. 1). Our 3D localization of GFP in RyR2R3595-GFP and RyR2K4269-GFP demonstrated that both sequence 3581–3612 and 4261–4286 could be CaM-binding sequences, since both of them are adjacent to the CaM-binding sites determined previously (Huang et al., 2012; Samsó and Wagenknecht, 2002; Wagenknecht et al., 1997). Interestingly, the two GFP locations are also close to one another, allowing us to generate a fluorescence resonance energy transfer (FRET) probe to characterize the conformational changes around the CaM-binding site when the RyR switches between the closed and open states. Furthermore, our FRET study has also established that there are interactions between CaM-binding sequences 3581–3612 and 4261–4286 from the same subunit within the tetrameric RyR molecule. Thus, our data suggest that CaM-binding sequences 3581–3612 and 4261–4286 form an intra-subunit binding pocket for CaM.
Construction of a GFP structural marker in the CaM-binding sequences
In previous cryo-EM analyses of RyR1/RyR2–CaM interactions, the physical locations of CaM binding were determined (Huang et al., 2012; Samsó and Wagenknecht, 2002; Wagenknecht et al., 1997). To gain insight into which regions of the RyR amino-acid sequence are potentially involved in CaM binding, we used site-specific GFP insertion coupled with 3D localization of inserted GFP by cryo-EM techniques to map the CaM-binding sequences onto the 3D architecture of RyR2. Briefly, we genetically tagged the residue of interest with GFP, and identified the GFP location as an extra mass in the density map that was derived from cryo-EM. To minimize potential disruptive effects of the insertion on folding of both GFP and RyR2, two Gly-rich spacers (ten residues and nine residues) flanked the inserted GFP (Kratz et al., 1999). Most of our cryo-EM mappings using this method are consistent with other studies. For instance, 3D localization of Tyr2801 in the phosphorylation domain (Meng et al., 2007) is consistent with the docking models from two groups in which a crystal structure of RyR fragments containing the phosphorylated reside was fitted into a RyR cryo-EM structure (Sharma et al., 2012; Yuchi et al., 2012); 3D localization of Tyr846 (Wang et al., 2011) is consistent with a docking model based on a pseudo-atomic structure of the N-terminal fragment (Zhu et al., 2013); and 3D localization of Arg626 in the FK506-binding protein (FKBP)-adjoining domain by combining FRET and cryo-EM studies (Wang et al., 2011) is consistent with binding assays that implicate this region in the RyR–FKBP interaction (Girgenrath et al., 2013). However, a shortcoming of the long linkers is that they may allow GFP to move away from the target sites. In our previous mapping of Ser437 in the N-terminal region, the inserted GFP containing long linkers was located between domain 5 and 9 (Wang et al., 2007), roughly 60 Å from the insertion site that is predicted from the docking model of N-terminal ABC domains (Tung et al., 2010). The offset may come from the flexibility of linkers, from the flexibility of the loop structure of RyR2 where the Ser437 located, and from the cylindrical shape of GFP with an overall length of nearly 50 Å.
In this study, GFP mappings were done with our refined insertion protocol, in which both linkers are truncated by five or six residues, such that the entire GFP sequence, with a short Gly-rich linker (four residues) on each side, can be inserted into two of the proposed CaM-binding regions in RyR2: after Arg3595 for mapping the sequence 3581–3612, and after Lys4269 for the sequence 4261–4286. With the short linkers, we will be able to map the surface-exposed residues of RyR2 more precisely because of the reduced extendibility and flexibility from the linkers. If the GFP-labeled sequence is involved in CaM binding to RyR1, the GFP in the 3D reconstruction should be at or adjacent to one of the previously reported CaM-binding sites. The recombinant RyR2-GFP cDNAs were then expressed in HEK293 cells. The RyR2 function was characterized by measuring Ca2+ release induced by sequential additions of increasing concentrations of caffeine in HEK293 cells transfected with wild-type RyR2 (RyR2WT), RyR2R3595-GFP or RyR2K4269-GFP, using the fluorescent Ca2+ indicator dye fluo-3 AM. Fig. 2 shows that both RyR2R3595-GFP and RyR2K4269-GFP form functional Ca2+-release channel in HEK293 cells (Fig. 2B,C), with significantly reduced response to caffeine (Fig. 2H).
Functional characterization of RyR2s labeled with GFP, CFP or YFP. HEK293 cells were transfected with RyR2WT (A), RyR2R3595-GFP (B), RyR2K4269-GFP (C), RyR2R3595-CFP (D), RyR2K4269-YFP (E), or RyR2R3595-CFP/K4269-YFP (F), or co-transfected with both RyR2R3595-CFP and RyR2K4269-YFP cDNAs (G). Fluorescence intensity of fluo-3-AM-loaded transfected cells was monitored before and after the additions of various concentrations of caffeine. The numbers (under the traces) indicate caffeine concentrations. Traces shown are from representative experiments. (H) Ca2+ release in relation to cumulative caffeine concentration in transfected HEK293 cells. Data shown are means ± s.e.m. (n = 3). Except for those labeled 'NS' (not significant), all other data points of GFP-, CFP- or YFP-labeled RyR2 are significantly different from that of RyR2WT for a given caffeine concentration (P<0.05).
Two-dimensional and three-dimensional localization of R3595-GFP in RyR2
Purified RyR2R3595-GFP was imaged by cryo-EM (Fig. 3A). In the images taken without tilting the specimen, RyR2R3595-GFP showed a typical tetrameric RyR shape, which is a square with a central cross and four protruding corners (Fig. 3A). Images of untilted RyR2R3595-GFP particles were aligned and averaged to increase the signal-to-noise ratio (Fig. 3B), and compared with the averaged images of RyR2WT control (no-GFP-insertion; Fig. 3C). Four symmetrically related strong positive regions were apparent after subtracting the averaged images of the RyR2WT control from that of RyR2R3595-GFP (Fig. 3D). Statistical analysis (t-tests) showed these positive regions to be significant at more than 99.9% confidence levels. We ascribe these regions to the inserted GFP locations on RyR2R3595-GFP. Since the two-dimensional (2D) analysis described above was only done with untilted particles, all in the fourfold symmetric orientation, the localization provided XY coordinates for the GFP but the coordinate along the Z-axis (defined as parallel to the fourfold symmetry axis of RyR) was indeterminate (Fig. 3E), hence the need for 3D reconstructions.
Two-dimensional analysis of RyR2R3595-GFP and RyR2K4269-GFP. (A,F) Cryo-EM CCD image of RyR2R3595-GFP and RyR2K4269-GFP, respectively, showing protein particles embedded in a thin layer of vitreous ice. The tetrameric structure of RyR2 is well preserved, and several individual particles are marked with circles. Scale bars: 500 Å. (B,G), 2D average of RyR2R3595-GFP and RyR2K4269-GFP, respectively, determined from 321 and 300 selected individual particles with defocus value ranges between −2.8 and −3.2 µm and between −2.8 and −3.6 µm, respectively. (C,H), 2D average of RyR2WT control from 380 and 333 particles in the same defocus range as were used for B and G, respectively. (D,I) The difference map obtained by subtracting the image in C from the image in B and the image in H from G, respectively. Bright white spots represent positive differences (one spot is highlighted by an asterisk), which correspond to the four extra masses contributed by GFP insertion. The images shown in B–D and G–H represent the projection of RyR as seen from the lumen side of the sarcoplasmic reticulum, which is shown for 3D models in E and J. The asterisk highlights the location of GFP within the plane of projection. The width of frames in B–J is 544 Å.
3D reconstruction of RyR2R3595-GFP identified a square prism shape, the standard RyR2 appearance (Liu et al., 2002; Sharma et al., 1998) composed of 14 cytoplasmic domains connecting to a transmembrane region (Fig. 4A,B). Compared with the no-GFP-insertion RyR2WT control, RyR2R3595-GFP contained four major excess density regions, each located between domains 3 and 8a (highlighted with red circles in Fig. 4A,B). Subtraction of the 3D volume of RyR2WT from RyR2R3595-GFP provided a clear picture of where the excess density regions were located (Fig. 4C,D). Because these regions were within the areas detected by 2D analysis and there were no other substantial excess densities nearby, we attribute them to be the locations of inserted GFP in RyR2R3595-GFP. This location is close to the previously mapped locations of both apo-CaM- and Ca2+-CaM-binding sites on RyR1, and of apo-CaM on RyR2 (Huang et al., 2012; Samsó and Wagenknecht, 2002; Wagenknecht et al., 1997), and is therefore consistent with evidence for the RyR2 sequence 3581–3612 being involved in binding CaM.
Three-dimensional reconstruction of RyR2R3595-GFP and RyR2K4269-GFP. (A,B,E,F) Surface representations of the 3D reconstruction of RyR2R3595-GFP (A,B) and RyR2K4269-GFP (E,F), shown in a side view (A,E) and in bottom view (B,F). The side view shows the surface that would be perpendicular to the plane of the sarcoplasmic reticulum membrane, and the bottom view shows the surface parallel to the sarcoplasmic reticulum membrane, which in situ would face the sarcoplasmic reticulum lumen. Red and orange circles highlight the locations of the extra masses representing GFP. (C,D,G,H) Surface representations of the 3D reconstruction of RyR2 control shown in blue, and the difference map [subtraction of 3D volume of RyR2 control from that of RyR2R3595-GFP (C and D) or RyR2K4269-GFP (G and H)] shown in green and superimposed on the 3D reconstruction of RyR2 control.
Two-dimensional and three-dimensional localization of K4269-GFP in RyR2
We next investigated the GFP location in the RyR2K4269-GFP. Purified RyR2K4269-GFP was imaged and analyzed using identical methods to those used for RyR2R3595-GFP. Cryo-EM showed the normal, standard RyR shape (Fig. 3F). 2D analysis (Fig. 3G–I) revealed four significant, symmetrically related positive peaks in the difference map, which represented the GFP location. Comparing the 3D reconstruction of RyR2K4269-GFP with RyR2WT control, we found that the two reconstructions are similar except for the extra masses representing the inserted GFP, which were clearly present on the side of domain 3 facing domain 7 (Fig. 4E–H). This location is also close to both the previously mapped Ca2+-CaM-binding site in RyR1 and the apo-CaM-binding site in RyR2 (Huang et al., 2012; Wagenknecht et al., 1997).
FRET characterization of dynamic conformational changes around the CaM-binding site
Based on the structural information from cryo-EM, we demonstrated that both sequences, 3581–3612 and 4261–4286 in RyR2 (3614–3643 and 4303–4328 in RyR1), are adjacent to the CaM-binding sites. Fortuitously, the two GFP reporters are located in close proximity, the distance between them being ∼40 Å, a spatial arrangement that is favorable for characterizing conformational dynamics by FRET. We first replaced the GFP insertion in the RyR2R3595-GFP and RyR2K4269-GFP with a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP), respectively, to generate two single-insertion FRET probes, RyR2R3595-CFP and RyR2K4269-YFP. Subsequently, we constructed a cDNA with dual fluorescent protein insertions in the RyR2 primary sequence, RyR2R3595-CFP/K4269-YFP (Fig. 5A,B). We next carried out FRET analyses in HEK293 cells that were transfected with the cDNAs to probe for conformational changes around the CaM-binding site. Note that HEK293 cells expressing RyR2R3593-CFP, RyR2K4269-YFP, RyR2R3595-CFP/K4269-YFP, or co-expressing RyR2R3593-CFP and RyR2K4269-YFP form functional Ca2+ release channels (Fig. 2D–G). However, similar to GFP insertions, the CFP and YFP insertions also significantly suppressed the response of the RyR2 channel to caffeine (Fig. 2H).
Dynamic interaction between two CaM-binding sequences characterized by FRET analysis. (A) Structural model of the dual insertion RyR2R3595-CFP/K4269-YFP. The cyan and yellow spheres represent CFP and YFP, respectively. Each RyR2 molecule contains four FRET pairs (highlighted by green ellipses). Red curves represent a possible boundary between the four subunits. FRET signals will be detected regardless of whether two structural domains bearing R3595-CFP and K4269-YFP are contained within one RyR2 subunit (i.e. an intra-subunit interaction, top panel) or belong to two different subunits (i.e. an inter-subunit interaction, bottom panel). (B) Models of six possible hybrid RyR2 tetramers when two cDNAs encoding RyR2R3595-CFP and RyR2K4269-YFP are co-expressed. The top row shows six RyR2 tetramer structures for the situation in which two structural domains bearing R3595-CFP and K4269-YFP are contained within one RyR subunit. In this case, no FRET signal will be detected, since the distance between CFP and YFP in the two neighboring subunits is over 180 Å. The bottom row shows six RyR2 tetramer structures for the case in which the two structural domains bearing R3595-CFP and K4269-YFP belong to two different subunits. In this case, four out of six structures have at least one FRET pair (highlighted by green ellipses). Numbers between two rows are the mathematical probability ratios for the six possible structures, assuming that RyR2 tetramers formed by random assembly of the two differently labeled subunits. (C) Confocal images of a HEK293 cell expressing RyR2R3595-CFP/K4269-YFP cDNA, showing cyan and yellow fluorescence before and after photobleaching. The green ellipse demarcates the area selected for photobleaching. Scale bar: 10 µm. (D) FRET efficiency determined by photobleaching of acceptor. Data are means ± s.e.m., with the number of the cells indicated on the bars. ***P<0.001; n.s., not significant.
Fig. 5C shows the results of acceptor photobleaching experiments to measure FRET efficiency in the HEK293 cells that expressed RyR2R3595-CFP/K4269-YFP. Cyan (donor) and yellow (acceptor) fluorescence images were recorded before and after photobleaching of the acceptor and used to calculate the FRET efficiency. As shown in Fig. 5D, the average FRET efficiency in the cells that expressed RyR2R3595-CFP/K4269-YFP in the absence of any treatment was 22.1±1.1% (mean ± s.e.m., n = 33 cells). The addition of 5 mM caffeine, a pharmacological activator of RyR channel activity, significantly decreased the FRET efficiency to 8.7±0.5% (n = 30, P<0.001). The strong FRET that we observed supports the cryo-EM results that place residues R3595 and K4269 in close proximity, and the decrease in FRET efficiency upon caffeine treatment indicates that the two putative CaM-binding sequences separate from one another when the RyR channel switches from the closed to the open conformation (see model in Fig. 6).
Hypothetical model of RyR conformation changes in the CaM-binding region and translocation of CaM when the RyR channel switches from closed to open. The transparent purple mass represents the apo-CaM-binding site, and the transparent red mass is the Ca2+-CaM-binding site in RyR1. The orange mass indicates the apo-CaM-binding site in RyR2. Two structural domains bearing CaM-binding sequences 3615–3644 and 4303–4328 in RyR1 are highlighted in green. Green arrows demonstrate that the two domains move apart when the RyR channel is activated, and the purple-red arrow indicates the shift of CaM from the apo-binding site to the Ca2+ binding site that occurs in RyR1 but not in RyR2. Figure adapted from Samsó and Wagenknecht and Huang et al., with modifications (Samsó and Wagenknecht, 2002; Huang et al., 2012).
When the two cDNAs (RyR2R3595-CFP and RyR2K4269-YFP) are co-expressed in HEK293 cells, various hybrid tetrameric RyR2s could form (see models in Fig. 5B). FRET pairs will be formed only when the following two criteria are both satisfied: (1) the donor (CFP) and acceptor (YFP) are within a certain distance of each other (normally below 100 Å); (2) the two domains containing CFP and YFP must belong to two neighboring RyR2 subunits. The models in the lower panel in Fig. 5B highlight FRET pairs that occur only when the two domains form an inter-subunit interaction. No FRET will be detected in the case of an intra-subunit interaction (top panel in Fig. 5B), because the distance between CFP and YFP in the two neighboring subunits is over 180 Å, which is far beyond the distance limitation between donor and acceptor for energy transfer. Little or no significant FRET (2–3%) or caffeine-induced reduction in FRET was observed in the cells co-expressing cDNAs of RyR2R3595-CFP and RyR2K4269-YFP (Fig. 5D). These residual, non-specific FRET signals most probably arise from the diffusion of nearby RyR2R3595-CFP into the photobleached region, because FRET was measured in live cells instead of fixed cells to maintain near physiological conditions. Using a fluorescence recovery after photobleaching (FRAP) experiment, we showed that the diffusion of RyR2 in the live cells would not substantially interfere with the FRET efficiency determined by the photobleaching approach, but a 3–5% RyR2 diffusion indeed exists in live cells (Liu et al., 2010). The observation of little or no FRET in the cells co-transfected with RyR2R3595-CFP and RyR2K4269-YFP indicates that these two closely located CaM-binding sequences are from the same subunit within the homo-tetrameric RyR.
Sequences not directly involved in CaM binding
As indicated in Fig. 1, numerous regions in the sequences of RyR1 and RyR2 have been implicated in binding CaM. The physical locations of Ca2+-CaM and apo-CaM binding on the surface of RyR1 and of apo-CaM on RyR2 have previously been established by 3D cryo-EM (Huang et al., 2012; Samsó and Wagenknecht, 2002; Wagenknecht et al., 1997). For this study we focused on the two CaM-binding sequences that contain residues Arg3595 and Lys4269 (RyR2 sequence numbers) because the proposed sites that are located N-terminally to residues 3581–3610 in RyR2 (3614–3643 in RyR1) are considered unlikely on the basis of subsequent experimental findings, particularly several cryo-EM analyses of GFP-insertions and docking models of crystal structures of RyR fragments. The proposed RyR2 CaM-binding sequence 263–615 (Balshaw et al., 2001) is incompatible with the EM data of Wang et al. and the X-ray crystallography/molecular docking results from Tung et al. (Wang et al., 2007; Tung et al., 2010). The predicted CaM-binding sequence 921–1173 in RyR1 (Chen and MacLennan, 1994) is also incompatible with experimental and modeling studies on a fragment that overlaps this sequence (Zhu et al., 2013). Proposed CaM-binding sequences in the vicinity of 2724–3016 in RyR2 (Balshaw et al., 2001) and 2804–2930 in RyR1 (Chen and MacLennan, 1994) contain a phosphorylation site that, according to cryo-EM (Meng et al., 2007) and X-ray crystallography and molecular docking results (Yuchi et al., 2012) is not near the known CaM-binding locations.
One of the proposed CaM-binding sequences located N-terminally to the sequence 3614–3643 in RyR1 deserves further consideration. Zhang et al. found that a peptide corresponding to RyR1 residues 1975–1999 binds apo-CaM and that this same region was protected from tryptic digestion in the presence of apo-CaM (Zhang et al., 2003a). Several attempts have been made by our laboratory to insert GFP into the corresponding region of RyR2, but these were unsuccessful, apparently because they interfered with binding the accessory protein FKBP12.6 to RyR2, which we make use of to purify the GFP-modified RyR2. However, cryo-EM of RyR2 containing GFP insertions that lie on either side of 1975–1999 (1942–1966 in RyR2), specifically at Thr1874 and Thr2023, showing that these insertions are not in the vicinity of known CaM-binding sites (Jones et al., 2008; Zhang et al., 2003b), but further study of this region is needed to ascertain whether or not the sequence 1975–1999 is involved in direct interactions with CaM.
Sequence 4261–4286
Several proposed CaM-binding sequences are on the C-terminal side of the well-established CaM-binding sequence, 3614–3643 in RyR1 (3581–3610 in RyR2). These sequences fall within the range 4480–4748 in RyR2 and within 4302–4621 in RyR1 (Fig. 1). Previously, we used GFP insertion and cryo-EM to map residue Asp4365 in RyR2 (corresponding to Asp4413 in RyR1) to a region in domain 3 that was within a few nanometers of the apo-CaM-binding location (Liu et al., 2002). Here we have inserted GFP at Lys4269 in RyR2, which corresponds to RyR1 residue 4311, which lies within the proposed CaM-binding sequences proposed by Chen and MacLennan (4303–4328) and by Balshaw et al. (4302–4430) in RyR1 (Balshaw et al., 2001; Chen and MacLennan, 1994).
The localization of 4261–4286 was quite unambiguous. Both 2D analysis and 3D reconstruction of RyR2K4269-GFP clearly resolved the inserted GFP, which localized to the bottom of domain 3 and facing domain 7, a location that is within a few nanometers of the known apo-CaM- and Ca2+-CaM-binding locations. However, biochemical evidence supporting the involvement of 4261–4286 of RyR2 (4303–4328 of RyR1) in binding of CaM is insufficient. Only two [125I]CaM and [35S]CaM overlay experiments (Balshaw et al., 2001; Chen and MacLennan, 1994), which detected the binding of RyR1 immobilized fragments to radio-labeled CaMs, showed that 4303–4328 and 4302–4430 of RyR1 were able to bind CaM. The binding of a RyR fragment to CaM does not necessarily indicate that CaM binds to that specific sequence within the intact RyR, because RyR folding might restrict exposure of the sequence and/or its conformation. Here we have shown for an intact RyR, rather than a RyR fragment, that GFP inserted within this region is probably at least partly surface-exposed as opposed to being internal (buried). If the site is internal, the insertion would probably disrupt the structure of RyR2, at least locally, and prevent GFP localization. Additionally, the fact that the GFP in RyR2K4269-GFP is located close to the actual CaM-binding site (Huang et al., 2012; Wagenknecht et al., 1997), supports the involvement of 4261–4286 of RyR2 in CaM binding. However, strong evidence against the involvement of this sequence is the lack of an effect on CaM binding to RyR1 when a region containing residues 4274–4535 is deleted by site-directed mutagenesis (Yamaguchi et al., 2001). Also, mutations within the sequence 3581–3612 are known to eliminate CaM binding to RyR (discussed below), suggesting that the sequence 4274–4535 is not capable of independent CaM binding. It is possible that the sequence 4274–4535 is capable of binding CaM with a much lower affinity than the 3581–3612 region, but at present there is no evidence for this, or for any physiologically relevant reason for such a secondary CaM-binding site. Nevertheless, we cannot exclude this region of RyR from playing some role in CaM binding.
The interpretation of the results for GFP inserted within the sequence 3581–3612 was not straightforward because of its location in a cleft bordered by several RyR domains. GFP was found to locate between domains 3 and 8a (Fig. 4C,D), which is close to all three of the CaM-binding positions that have been determined (apo-CaM- and Ca2+-CaM-binding sites on RyR1 and apo-CaM-binding site on RyR2) (Huang et al., 2012; Samsó and Wagenknecht, 2002; Wagenknecht et al., 1997). This location is consistent with 3581–3612 of RyR2 (and by inference the homologous sequence 3615–3644 in RyR1) as an authentic CaM-binding sequence, but there was still uncertainty as to whether R3595-GFP was linked to domain 3 or domain 8a. Considering the reported locations of apo-CaM and Ca2+-CaM on RyR1 as well as apo-CaM on RyR2 (Huang et al., 2012; Samsó and Wagenknecht, 2002; Wagenknecht et al., 1997), we favor the domain 3 location for the sequence 3581–3612 because it is more compatible with both the Ca2+-CaM and apo-CaM physical binding locations. In addition, our FRET result indicates sequences 3581–3612 and 4261–4286 from the same subunit are located in close proximity in the tetrameric RyR2 molecule. This finding increases the possibility that both sequences occur on the same domain (domain 3). Future studies to determine the location of 3581–3612 more precisely could be crucial for understanding the regulatory mechanism of CaM.
Unlike sequence 4302–4328 of RyR1 (4261–4286 of RyR2), the sequence 3614–3643 of RyR1 (3581–3612 of RyR2) as a CaM-binding sequence is well supported by several types of studies, including amino-acid sequence prediction (Takeshima et al., 1989), CaM overlays (Balshaw et al., 2001; Chen and MacLennan, 1994; Guerrini et al., 1995; Menegazzi et al., 1994), point mutation and deletion mutation (Yamaguchi et al., 2001; Yamaguchi et al., 2003) as well as tryptic digestion protection (Moore et al., 1999). Sequence 3614–3643 of RyR1 (3581–3612 of RyR2) was also shown to be involved in binding both apo-CaM and Ca2+-CaM (Moore et al., 1999). We identified this sequence at the region of overlap between the apo-CaM-binding site on RyR1 and the apo-CaM-binding site on RyR2 (similar to the Ca2+-CaM-binding site on RyR1) as determined by cryo-EM (Huang et al., 2012; Samsó and Wagenknecht, 2002; Wagenknecht et al., 1997), and this is fully consistent with all available evidence that supports this sequence as being essential for CaM binding to RyRs.
Conformational changes in the CaM-binding region of RyR and translocation of CaM
Previous functional studies showed that apo-CaM activates RyR1 but inhibits RyR2, whereas Ca2+-CaM inhibits both isoforms (Balshaw et al., 2001; Buratti et al., 1995; Damiani and Margreth, 2000; Rodney et al., 2000; Tripathy et al., 1995; Yamaguchi et al., 2003). Our previous cryo-EM studies showed distinctly different binding locations of apo-CaM and Ca2+-CaM on RyR1 (Samsó and Wagenknecht, 2002; Wagenknecht et al., 1997). Interestingly, apo-CaM binds to RyR2 at a similar location to that of Ca2+-CaM on RyR1, in seeming agreement with the inhibitory effects of these two forms of CaM on their respective receptors (Fig. 6) (Huang et al., 2012). Using a mutant CaM that is incapable of binding calcium, we have determined that the mutant CaM binds to RyR1 at the apo-CaM site, regardless of the calcium concentration. The existence of the two overlapping but distinct binding sites for apo-CaM and Ca2+-CaM on RyR1 imply that the binding location switch is due to Ca2+ binding to CaM, as opposed to direct effects of Ca2+ on RyR1, and that the switch to the alternative apo-CaM site on RyR1 (but not on RyR2) is involved in the activation effect of apo-CaM on RyR1 (Huang et al., 2012).
In this study, we demonstrated by cryo-EM that two hypothesized CaM-binding sequences in RyR2, 3581–3612 and 4261–4286 (corresponding to 3614–3643 and 4302–4328 in RyR1) are both in close proximity to the previously found locations of CaM. In addition, we found that when the RyR2 channel is activated, the structural domains bearing these two sequences move apart, indicating that conformational changes associated with channel activation occur in the vicinity of receptor-bound CaM. We suggest that these conformational changes also affect CaM binding, and vice versa, which would be consistent with the known influences CaM on channel gating. Clearly, more detailed studies of conformational changes in this region of RyR are needed. RyR activation is induced either by direct interaction with DHPR (for RyR1), by a small amount of Ca2+ influx from DHPR (for RyR2) or by Ca2+ released from SR stores through nearby RyRs (RyR1 and RyR2). Global conformational changes underlie RyR activation, including changes in the clamp region (Tian et al., 2013), in the transmembrane region that result in ion channel activation (Samsó et al., 2009), and, as we show here for the first time, in the CaM-binding region of RyR1. Upon RyR activation the two fluorophore-labeled sequences move apart, a structural change that is likely to be influenced by CaM. We hypothesize that for RyR2, the presence of either apo-CaM or Ca2+-CaM inhibits the conformational change, whereas for RyR1 apo-CaM facilitates and Ca2+-CaM inhibits it, thereby accounting for the regulatory effects of CaM on the activity of RyR2 and RyR1. Additional biophysical characterization, by FRET and other techniques, of this conformational change on both RyR2 and RyR1 and in the presence of CaM and calcium should allow us to refine or reject this hypothesis. It is also possible that the conformational change we have discovered plays a role in the termination of Ca2+ release, particularly for RyR2 for which the mechanism by which Ca2+ release is terminated during excitation–contraction coupling in the heart is not understood (Cannell and Kong, 2012; Stern and Cheng, 2004).
Materials and chemicals
The detergent CHAPS, the redox reagent DTT, and the protease inhibitor leupeptin were purchased from CalBiochem (La Jolla, CA, USA). All the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). GFP-inserted mouse RyR2s were constructed, expressed in HEK293 cells and purified as described previously (Liu et al., 2002).
Construction of GFP-tagged RyR2s
The cloning and construction of the 15-kb full-length cDNA encoding the mouse RyR2 has been described previously (NCBI reference sequence NP_076357.2, GI:124430578) (Zhao et al., 1999). The cDNA encoding GFP with Gly-rich linkers (four residues) on each side and an AscI site was obtained by PCR. The AscI site was introduced into a cDNA fragment of RyR2 after a specific residue (Arg3595 or Lys4269) by overlap extension using PCR. The fragment with the AscI site was subcloned into the full-length RyR2 cDNA. The AscI–AscI fragment containing GFP and the linkers were then subcloned into the full-length RyR2 at the introduced AscI site. The sequences of all PCR fragments and the orientation of the inserted GFP cDNA were verified by DNA sequencing analysis.
Caffeine-induced Ca2+ release measurements
Free cytosolic Ca2+ concentration in transfected HEK293 cells was measured using the fluorescent Ca2+ indicator dye fluo-3 AM as described previously (Li and Chen, 2001).
Cryo-grid preparation
The expression and purification of RyR2R3595-GFP and RyR2K4269-GFP was carried out as described previously (Liu et al., 2002). Cryo-grids prepared for 2D and 3D localization of GFP-inserted RyR2s were prepared as follows. Purified GFP-inserted RyR2 (0.8 µl at a concentration about 0.2 mg/ml in 20 mM NaMOPS, pH 7.4, 1 M NaCl, 1% CHAPS, 2 mM DTT, 2 µg/ml leupeptin) was diluted with 4 µl buffer (20 mM NaMOPS, pH 7.4, 400 mM KCl, 3.0 mM EGTA, 0.5% CHAPS, 2 mM DTT, 2 µg/ml leupeptin). A 4-µl volume was applied to 300 mesh grids coated with a thin continuous carbon film suspended over a thick holey carbon film for 30 seconds. The grids were blotted with Whatman no. 1 filter paper for about 3 seconds before plunging into liquid ethane.
Cryo-electron microscopy and image processing
Cryo-EM data collection and image processing were performed in a similar manner to that described previously (Huang et al., 2012). The final 3D reconstructions of RyR2R3595-GFP (resolution 30 Å) and RyR2K4269-GFP (resolution 31 Å) were computed from 8002 and 6934 particles, respectively.
FRET measurement
For FRET analysis, the GFP insertion in the RyR2R3595-GFP and RyR2K4269-GFP were replaced by CFP and YFP, respectively. In addition to the single insertions, a dual insertion RyR2R3595-GFP/K4269-YFP was constructed as described previously (Liu et al., 2010). FRET measurements in the HEK293 cells expressing RyR2R3595-CFP/K4269-YFP, or co-expressing RyR2R3595-CFP and RyR2K4269-YFP were performed as described previously (Liu et al., 2010).
We gratefully acknowledge the 3D-EM and Advanced Light Microscopy and Image Analysis Core Facilities at the Wadsworth Center.
Z.L., T.W. and S.R.W.C. designed the research; X.H., Ying L., R.W., X.Z., Yingjie L., A.K. and Z.L. performed experiments; X.H., Yingjie L., S.R.W.C. and Z.L. analyzed data; X.H., S.R.W.C., T.W. and Z.L. wrote the manuscript.
This work was supported by the National Institutes of Health [grant numbers R01HL095541 to Z.L and R01AR040615 to T.W.]; and by the Canadian Institutes of Health Research and Heart and Stroke Foundation of Canada to S.R.W.C. Deposited in PMC for release after 12 months.
© 2013. Published by The Company of Biologists Ltd
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Protesters holding up a banner reading 'Women in Black for Peace and Justice': rally and march against the Iraq War
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Protesters holding up a banner reading 'Women in Black for Peace and Justice': rally and march against the Iraq War, March 19, 2005
From a series of images taken at a protest against the Iraq War held in New Paltz, N.Y., part of the Global Day of Action promoted by the Act Now to Stop War and End Racism Coalition (ANSWER Coalition).
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redpajama_set_name: RedPajamaCommonCrawl


Singer songwriter Elisa Korenne of New York Mills, MN writes original songs about historical people and events of the northern plains. One such figure is Rachel Calof. She traveled from Russia to North Dakota in 1894 as an immigrant homesteader and a mail order bride. Her story is a riveting and candid look at the hardships of life on the prairie.
Singer songwriter Elisa Korenne of New York Mills, Minnesota writes original songs about historical people and events of the northern plains. In 1893, northern Minnesota was experiencing a logging boom but it was a dangerous industry. Back then, health insurance was in the form of health tickets to St. Mary's Hospital in Duluth, MN. Sister Amata Mackett of the Benedictine Sisters of St. Scholastica Monastery sold these tickets to lumberjacks and quickly became know as Sister Lumberjack.
Singer-songwriter Elisa Korenne of New York Mills, Minnesota, writes songs about unusual characters and obscure events in Minnesota history.
Susan Frenier Brown was a mixed-blood Dakota Indian, and although she was surrounded by influential people who had an impact on politics in the mid and late 1800's, her own identity and story has been somewhat lost to history. Minnesota musician Elisa Korenne explores this shadowy figure in her original song "Who Was I?"
Experience a musical tribute to Billy Joel in this clip featuring vocalist Josh Groban at the annual Gershwin Prize for Popular Song. In commemoration of George and Ira Gershwin's contributions to American song and culture, the Library of Congress names an annual award to an American musican. The George and Ira Gershwin Collection is housed in the Music Division of the Library of Congress and provides a wealth of orchestrations, lyric sheets, librettos, and audio recordings.
Explore the music and dance sequences of 11 cultural groups who have settled in Santa Fe, New Mexico over the past 700 years. These diverse communities include the Native Americans, Spanish, Mexicans, Crypto-Jewish, Celtic, German, Greek, Japanese, Tibetan, Sikh and the Central Americans. All performers and narrators in these segments are of school-age.


text:
using testing::ElementsAre;

namespace blink {
namespace {

static PaintChunkProperties rootPaintChunkProperties() { return PaintChunkProperties(); }

class PaintChunkerTest : public testing::Test {
protected:
    void SetUp() override
    {
        RuntimeEnabledFeatures::setSlimmingPaintV2Enabled(true);
    }

    void TearDown() override
    {
        m_featuresBackup.restore();
    }

private:
    RuntimeEnabledFeatures::Backup m_featuresBackup;
};

TEST_F(PaintChunkerTest, Empty)
{
    Vector<PaintChunk> chunks = PaintChunker().releasePaintChunks();
    ASSERT_TRUE(chunks.isEmpty());
}

TEST_F(PaintChunkerTest, SingleNonEmptyRange)
{
    PaintChunker chunker;
    chunker.updateCurrentPaintChunkProperties(rootPaintChunkProperties());
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    Vector<PaintChunk> chunks = chunker.releasePaintChunks();

    EXPECT_THAT(chunks, ElementsAre(
        PaintChunk(0, 2, rootPaintChunkProperties())));
}

TEST_F(PaintChunkerTest, SamePropertiesTwiceCombineIntoOneChunk)
{
    PaintChunker chunker;
    chunker.updateCurrentPaintChunkProperties(rootPaintChunkProperties());
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.updateCurrentPaintChunkProperties(rootPaintChunkProperties());
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    Vector<PaintChunk> chunks = chunker.releasePaintChunks();

    EXPECT_THAT(chunks, ElementsAre(
        PaintChunk(0, 3, rootPaintChunkProperties())));
}

TEST_F(PaintChunkerTest, CanRewindDisplayItemIndex)
{
    PaintChunker chunker;
    chunker.updateCurrentPaintChunkProperties(rootPaintChunkProperties());
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.decrementDisplayItemIndex();
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    Vector<PaintChunk> chunks = chunker.releasePaintChunks();

    EXPECT_THAT(chunks, ElementsAre(
        PaintChunk(0, 2, rootPaintChunkProperties())));
}

TEST_F(PaintChunkerTest, BuildMultipleChunksWithSinglePropertyChanging)
{
    PaintChunker chunker;
    chunker.updateCurrentPaintChunkProperties(rootPaintChunkProperties());
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    PaintChunkProperties simpleTransform;
    simpleTransform.transform = TransformPaintPropertyNode::create(TransformationMatrix(0, 1, 2, 3, 4, 5), FloatPoint3D(9, 8, 7));

    chunker.updateCurrentPaintChunkProperties(simpleTransform);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    PaintChunkProperties anotherTransform;
    anotherTransform.transform = TransformPaintPropertyNode::create(TransformationMatrix(0, 1, 2, 3, 4, 5), FloatPoint3D(9, 8, 7));
    chunker.updateCurrentPaintChunkProperties(anotherTransform);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    Vector<PaintChunk> chunks = chunker.releasePaintChunks();

    EXPECT_THAT(chunks, ElementsAre(
        PaintChunk(0, 2, rootPaintChunkProperties()),
        PaintChunk(2, 3, simpleTransform),
        PaintChunk(3, 4, anotherTransform)));
}

TEST_F(PaintChunkerTest, BuildMultipleChunksWithDifferentPropertyChanges)
{
    PaintChunker chunker;
    chunker.updateCurrentPaintChunkProperties(rootPaintChunkProperties());
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    PaintChunkProperties simpleTransform;
    simpleTransform.transform = TransformPaintPropertyNode::create(TransformationMatrix(0, 0, 0, 0, 0, 0), FloatPoint3D(9, 8, 7));
    chunker.updateCurrentPaintChunkProperties(simpleTransform);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    PaintChunkProperties simpleTransformAndEffect;
    simpleTransformAndEffect.transform = simpleTransform.transform;
    simpleTransformAndEffect.effect = EffectPaintPropertyNode::create(0.5f);
    chunker.updateCurrentPaintChunkProperties(simpleTransformAndEffect);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    PaintChunkProperties simpleTransformAndEffectWithUpdatedTransform;
    simpleTransformAndEffectWithUpdatedTransform.transform = TransformPaintPropertyNode::create(TransformationMatrix(1, 1, 0, 0, 0, 0), FloatPoint3D(9, 8, 7));
    simpleTransformAndEffectWithUpdatedTransform.effect = EffectPaintPropertyNode::create(simpleTransformAndEffect.effect->opacity());
    chunker.updateCurrentPaintChunkProperties(simpleTransformAndEffectWithUpdatedTransform);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    // Test that going back to a previous chunk property still creates a new chunk.
    chunker.updateCurrentPaintChunkProperties(simpleTransformAndEffect);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    Vector<PaintChunk> chunks = chunker.releasePaintChunks();

    EXPECT_THAT(chunks, ElementsAre(
        PaintChunk(0, 1, rootPaintChunkProperties()),
        PaintChunk(1, 3, simpleTransform),
        PaintChunk(3, 5, simpleTransformAndEffect),
        PaintChunk(5, 7, simpleTransformAndEffectWithUpdatedTransform),
        PaintChunk(7, 9, simpleTransformAndEffect)));
}

TEST_F(PaintChunkerTest, BuildChunksFromNestedTransforms)
{
    // Test that "nested" transforms linearize using the following
    // sequence of transforms and display items:
    // <root xform>, <paint>, <a xform>, <paint>, <paint>, </a xform>, <paint>, </root xform>
    PaintChunker chunker;
    chunker.updateCurrentPaintChunkProperties(rootPaintChunkProperties());
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    PaintChunkProperties simpleTransform;
    simpleTransform.transform = TransformPaintPropertyNode::create(TransformationMatrix(0, 1, 2, 3, 4, 5), FloatPoint3D(9, 8, 7));
    chunker.updateCurrentPaintChunkProperties(simpleTransform);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    chunker.updateCurrentPaintChunkProperties(rootPaintChunkProperties());
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    Vector<PaintChunk> chunks = chunker.releasePaintChunks();

    EXPECT_THAT(chunks, ElementsAre(
        PaintChunk(0, 1, rootPaintChunkProperties()),
        PaintChunk(1, 3, simpleTransform),
        PaintChunk(3, 4, rootPaintChunkProperties())));
}

TEST_F(PaintChunkerTest, ChangingPropertiesWithoutItems)
{
    // Test that properties can change without display items being generated.
    PaintChunker chunker;
    chunker.updateCurrentPaintChunkProperties(rootPaintChunkProperties());
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);

    PaintChunkProperties firstTransform;
    firstTransform.transform = TransformPaintPropertyNode::create(TransformationMatrix(0, 1, 2, 3, 4, 5), FloatPoint3D(9, 8, 7));
    chunker.updateCurrentPaintChunkProperties(firstTransform);

    PaintChunkProperties secondTransform;
    secondTransform.transform = TransformPaintPropertyNode::create(TransformationMatrix(9, 8, 7, 6, 5, 4), FloatPoint3D(3, 2, 1));
    chunker.updateCurrentPaintChunkProperties(secondTransform);

    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    Vector<PaintChunk> chunks = chunker.releasePaintChunks();

    EXPECT_THAT(chunks, ElementsAre(
        PaintChunk(0, 1, rootPaintChunkProperties()),
        PaintChunk(1, 2, secondTransform)));
}

TEST_F(PaintChunkerTest, CreatesSeparateChunksWhenRequested)
{
    // Tests that the chunker creates a separate chunks for display items which
    // require it.
    PaintChunker chunker;
    chunker.updateCurrentPaintChunkProperties(rootPaintChunkProperties());
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::RequiresSeparateChunk);
    chunker.incrementDisplayItemIndex(PaintChunker::RequiresSeparateChunk);
    chunker.incrementDisplayItemIndex(PaintChunker::RequiresSeparateChunk);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::RequiresSeparateChunk);
    chunker.incrementDisplayItemIndex(PaintChunker::RequiresSeparateChunk);
    chunker.decrementDisplayItemIndex();
    chunker.decrementDisplayItemIndex();
    chunker.decrementDisplayItemIndex();
    chunker.incrementDisplayItemIndex(PaintChunker::DefaultBehavior);
    chunker.incrementDisplayItemIndex(PaintChunker::RequiresSeparateChunk);

    Vector<PaintChunk> chunks = chunker.releasePaintChunks();
    EXPECT_THAT(chunks, ElementsAre(
        PaintChunk(0, 1, rootPaintChunkProperties()),
        PaintChunk(1, 2, rootPaintChunkProperties()),
        PaintChunk(2, 3, rootPaintChunkProperties()),
        PaintChunk(3, 4, rootPaintChunkProperties()),
        PaintChunk(4, 6, rootPaintChunkProperties()),
        PaintChunk(6, 7, rootPaintChunkProperties())));
}

} // namespace
} // namespace blink

redpajama_set_name: RedPajamaGithub


By Thomas J. Donohue, President & CEO of the U.S. Chamber of Commerce. Article provided by the U.S. Chamber of Commerce. Read the original here.
Our economy is rapidly being reshaped by technology and other forces that are creating opportunity as well as disruption – and with it, insecurity for many businesses and workers.
Much of this insecurity can be attributed to two gaps that are preventing our nation from leveraging its talent and people from realizing their potential. The first is a skills gap – too many people lack the skills or credentials necessary to compete for 21st century jobs. The second is a people gap – too many businesses can't find the workers they need, when and where they need them. Closing both gaps is imperative to our competitiveness.
Businesses are already leading this national imperative. We have the ideas, influence, and incentives to drive change and create solutions.
To close the skills gap, we're working with all the stakeholders to address shortcomings in education. We're highlighting the need for high-quality childcare and early education and advocating for rigorous standards and accountability in our K-12 schools to better prepare students for college or a career.
We're stressing the importance of smart choices in post-secondary education or training so that people can get a return on their investment and earn credentials of value in the market. It's crucial that people look at learning as a lifelong endeavor – they must be entrepreneurial to stay marketable in the modern economy. We're also advancing business-led solutions, like Talent Pipeline Management™, to train workers for existing jobs.
But even as we make progress in preparing workers for 21st century jobs, we still face a growing people gap.
To help close this gap, we need to get as many people as possible off the sidelines. We must help veterans and military spouses transition to the civilian workforce. We need to make it easier and provide incentives for people to work well past age 65. And we need to tackle big social challenges, including addiction, mass incarceration, and high youth unemployment.
Closing the people gap also requires us to fix our broken immigration system. Immigrants have long been a vital part of our economy, and they can help fill gaps in our workforce. We need an effective immigration system that will respect the rule of law, respond to the needs of our economy, and reflect our nation's values.
The collective talent of our nation is what makes this country exceptional, and it's an advantage we must never cede. Yet if we are going to keep that advantage and keep the promise of opportunity to future generations of Americans, all the stakeholders must work together to solve the two-gap challenge.


text:
Auctorum (abreviado auct. o auctt.) es un término utilizado en zoología y en botánica para indicar que un nombre se está empleando con el sentido establecido por autores diferentes al original. A menudo se usa conjuntamente con nec o no para indicar la aplicación incorrecta de un nombre.

Puede usarse para indicar el número de autores. Por ejemplo, auctorum multorum (abreviado auct. mult.), que en latín significa de muchos autores, indica que muchos autores subsiguientes utilizaron el nombre en un sentido diferente al autor original.

Ejemplos
Flora Europaea indica "C. libanotis auct. mult., non L." como sinónimo de Cistus clusii, indicando con ello que varios autores emplearon erróneamente el nombre Cistus libanotis para la especie Cistus clusii utilizando este nombre de manera diferente al autor original, Carlos Linneo.

Referencias

Enlaces externos
 Auctorum en el glosario del ICZN.
 Auctorum en el glosario de Invertebrate Ireland Online.

Nomenclatura biológica
Expresiones latinas usadas en biología

redpajama_set_name: RedPajamaWikipedia


Win64/Filecoder.R is a trojan that encrypts files on fixed, removable and network drives. To decrypt files the user is requested to comply with given conditions in exchange for a password/instructions.
Win64/Filecoder.R is a trojan that encrypts files on fixed, removable and network drives.
The AES-256, RSA encryption algorithm is used.


Bill payers are finding energy suppliers both easier to deal with and more trustworthy, new research undertaken by Ipsos MORI reveals. The research shows that 74 per cent of bill payers who have switched or tried to switch their supplier in the last 12 months, found the process of switching easy, and that 79 per cent of bill payers find it easy to make a bill payment. In terms of their experience overall, the proportion of bill payers who find it easy to deal with their supplier has increased by four percentage points to 63 per cent.
The proportion of bill payers who trust their supplier to provide them with a service that meets their needs has also increased by four percentage points to 59 per cent. 59 per cent of customers trust their supplier to provide an accurate bill. The proportion of bill payers who trust their supplier to deal with complaints fairly has also increased significantly from 44 per cent to 54 per cent.
The research was commissioned by Energy UK and conducted by Ipsos MORI.
The survey was conducted between 29 August and 11 September 2014.
This represents the second wave of results, the first survey being carried out between 21 February and 10 March 2014. Findings are based on bill payers who are either solely or jointly responsible for household energy bills, and who have a mains gas or electricity supply to their home. Results are based on a total sample size of 1,420 bill payers or, for questions about switching energy supplier, on 487 bill payers that have switched energy supplier in the last 12 months.
For the questions that asked about experiences of suppliers, respondents answered the questions in terms of their electricity supplier (if they were connected to mains electricity and could name their electricity supplier). If respondents could not name their electricity supplier, but could name their gas supplier, they were asked about their gas supplier.
Both waves of results were conducted in respondent's homes using Capibus, Ipsos MORI's face-to-face weekly omnibus survey of adults aged 15+.
The results have been weighted to the known population profile of Great Britain.
The Ipsos MORI report can be read in full on the Energy UK website.


With the speed of organization today, the significance of the desktop phone can never be disregarded. Today's staff member needs an available interaction tool at any place to end up being as effective and efficient as possible. Grandstream's VoIP Phones address the requirements of individuals who utilize them, whatever their task, their place, or the time of day. However, numerous services and staff members have not benefited from the boosted abilities used by today's next-generation communication features. Grandstream IP Phones are the response for that. With an extensive range of adjustable functions in addition to a modular style, these IP Phones can assist business telephone communication of any work environment.
Grandstream Executive phone range offers an amazing customer experience for executives and High-level managers. All of the Grandstream IP Phones are double ethernet port, next generation IP phone that provides an expense-conserving service for small to large business. The SIP protocol assists to make sure that it is possible to install this IP phone can work with any SIP certified Phone Systems.
Contact Us Today. We can assist you choose the right Grandstream Phones in Abu Dhabi, Grandstream Phones in Dubai, Grandstream Phones in Umm Al Quwain, Grandstream Phones in Ras Al-Khaimah, Grandstream Phones in Sharjah, Grandstream Phones in Umm Al Quwain, Grandstream Phones in Al Ain, Grandstream Phones in Fujairah, Grandstream Phones in Ajman and Grandstream Phones in Khor Fakkan.
Each Series of Grandstream Phone offer fantastic worth to users. Grandstream office phones are ergonomically planned for natural convenience and easy to use. With different menu-driven soft essential functions, Conferencing Capabilities and simple gain access to function keys these Grandstream IP Phones are the best choices for your office telephone system. Grandstream Phones provide power, adaptability, and programmability to all user stations, optimizing efficiency and performance. Due to the fact that the whole desktop surface area is crafted for user efficiency and constantly searching for much better speed, service and efficiency, these Grandstream phones can assist your company to reach new levels.
Built-in Bluetooth for hands-complimentary operation by means of basic headsets.
Easy-to-deploy and manage with central management.


text:
Hoping to trace family in Filey
The T'oard Ship Inn in Filey.
Published: 10:16 Sunday 19 February 2017
A man researching his family tree is asking for help from Filey residents after struggling to find out more about his connections to the town.
Mick Frankland and his immediate family used to stay with his mother's relatives in the late 1940s and early 1950s.
They lived in a house just at the top of the steps going up from the cobbled landing.
There used to be an old pub called The T'oard Ship Inn and he remembers an old model ship on the outside porch of the establishment.
He said: "My mother's uncle and his brother were Filey fishermen using the cobble boats and I remember them boiling the crabs.
"I think my aunt and uncle were called Frank and Annie but I'm not sure, and it's these names in particular I am looking to trace to see where we all fit in.
"Another name springs to mind is an old fisherman called Rammy.
"He was always around so I assume he was related to me in some way. Apparently he was a bit of a character and well known in Filey.
"If there are still any family members in Filey or people who remember them I really would love to hear from them."
Filey RNLI rescue 10m fishing vessel and two crew two miles off Filey Brigg
Every Football League club ranked in order of how much money they made or lost last season
Easter holidays 2020: 8 of the best destinations to visit for warm weather in April
12 of the best restaurants in and around Whitby according to Trip Advisor reviews
These are the 'happiest' and 'unhappiest' jobs
More from Filey and Hunmanby Mercury


text:
<?php if ( ! defined('BASEPATH')) exit('No direct script access allowed');

/* Heredamos de la clase CI_Controller */
class Vendor extends CI_Controller {

	function __construct()
	{

		parent::__construct();

		/* Cargamos la base de datos */
		$this->load->database();

		/* Cargamos la libreria*/
		$this->load->library('grocery_crud');

		/* Añadimos el helper al controlador */
		$this->load->helper('url');
	}

	function index()
	{
		/*
		 * Mandamos todo lo que llegue a la funcion
		 * administracion().
		 **/
		redirect('Vendor/administracion');
	}

	/*
	 *
	 **/
	function administracion()
	{
		if (!$this->tank_auth->is_logged_in()) {
			redirect('/auth/login/');
		}

		try{

			/* Creamos el objeto */
			$crud = new grocery_CRUD();

			/* Seleccionamos el tema */
			$crud->set_theme('flexigrid');

			/* Seleccionmos el nombre de la tabla de nuestra base de datos*/
			$crud->set_table('vendor');

			/* Le asignamos un nombre */
			$crud->set_subject('vendedor');

			/* Asignamos el idioma español */
			$crud->set_language('spanish');

			/* Aqui le decimos a grocery que estos campos son obligatorios */
			$crud->required_fields(
					'VendorID',
					'Name',
					'Lastname',
					'IdentType',
					'Ident',
					'BirthDate',
					'RegisterDate',
					'Address',
					'City'
					);

			/* Aqui le indicamos que campos deseamos mostrar */
			$crud->columns(
					'VendorID',
					'Name',
					'Lastname',
					'IdentType',
					'Ident',
					'BirthDate',
					'RegisterDate',
					'Address',
					'City',
					'Phone1',
					'Phone2',
					'Mail',
					'Facebook',
					'Twitter',
					'Facebook',
					'Skype'
			);

			$crud->display_as('VendorID','ID');
			$crud->display_as('Name','Nombre');
			$crud->display_as('Lastname','Apellido');
			$crud->display_as('IdentType','Tipo Identificaci&oacuten');
			$crud->display_as('Ident','Identificaci&oacuten');
			$crud->display_as('BirthDate','Fecha Nacimiento');
			$crud->display_as('RegisterDate','Fecha Ingreso');
			$crud->display_as('Address','Direcci&oacuten');
			$crud->display_as('City','Ciudad');
			$crud->display_as('Phone1','Tel&eacutefono 1');
			$crud->display_as('Phone2','Tel&eacutefono 2');
			$crud->display_as('Phone3','Tel&eacutefono 3');

			// ver productos
			$crud->add_action('Productos',base_url() . 'assets/uploads/detalle.png','Product/admin_by_vendor');

			/* Generamos la tabla */
			$output = $crud->render();

			/* La cargamos en la vista situada en */
			$this->load->view('Vendor/administracion', $output);

		}catch(Exception $e){
			/* Si algo sale mal cachamos el error y lo mostramos */
			show_error($e->getMessage().' --- '.$e->getTraceAsString());
		}
	}
}


Premium Cabs is a leading taxi service provider in Melbourne. With extensive years of experience and excellence in the taxi transportation industry, the Melbourne taxi service strives to go above and beyond client's expectations every time. Premium Cabs has a huge assortment of taxis, yellow cabs, and cars for the convenience of clients nevertheless of their travel needs – be it for airport transfer or longer trips in Melbourne or the surrounding areas. The taxi service provider offers luxury taxis for all your airport pickup and drop-off at a standard taxi rate with no additional charge. They have a big network of professional and friendly drivers to deliver you smooth and hassle-free transportation every time.


text:
The 1968 Pontiac E-Body Grand Prix - What If?
Don KeefewriterTed Alexanderphotographer
A little-known chapter in the Grand Prix's history actually pre-dates its introduction. At the time, General Motors was developing the personal luxury E-Body platform as a competitor to the four-seat Ford Thunderbird, but was unsure early on as to which Divisions would get it. There was even a plan to use this car to resurrect the old LaSalle nameplate, which was last seen as a production car in 1940 and the subject of two revival Dream Cars from 1955.
To settle the debate, there was an internal competition between the Divisions, and GM evaluated concepts to see which Division had the best understanding of the market and best exploit the idea of a personal luxury car. Pontiac, through the GM Advanced Design Studio, submitted the XP-756 Skorpion full-scale model.
Pontiac designers were also hedging their bets, and they developed a more practical and affordable personal luxury car, one with a performance-oriented attitude. It was based on their existing short-wheelbase version of the B-Body, which was shared with the Catalina. It reached the showrooms as the 1962 Grand Prix, and for Pontiac it proved to be the right car...and the start of a lineage.
Buick got the E-Body in 1963 and named it the Riviera, while the LaSalle version never materialized. The same platform would also go on, in modified form, as the front-drive Oldsmobile Toronado and Cadillac Eldorado.
This month, Ted Alexander takes us on an intriguing flight of fancy, conjuring up what a late-second-generation E-Body Grand Prix might have looked like. Taking the basic shape and proportions of the 1966-70 Riviera, he adds a decidedly Pontiac design theme, drawing from both production and concepts of the era.
Up front, the family resemblance to the GTO is evident, though the details give this Grand Prix its own identity. The Goat-inspired Endura nose and grille mesh is 1968 correct, but there are several other details that demand some exploration.
The 1969 Pontiac Cirrus concept car (the revamped 1964 GM-X) is the inspiration for the front fender shape, which featured vertical air slots on the leading edge and fender extractors just above and below the fender crown. The Endura nose also features a chrome bumper/valance panel, which is slotted for air intake, with louvered details at the beak. The chrome extends to the lower leading edge of the fender, forming a bright band that visually flows back to the rocker-panel mouldings, which are adorned with 428 badges. The hood also features the influence of the Cirrus, with a prominent ironing-board contour, painted a semi-gloss black, and slotted eyebrow turn signals, embedded in the Endura bumper material. It adds an innovative and attractive touch.
The Riviera's roofline is replicated in this Grand Prix version but the rear quarter windows are completely different, and more closely identifiable to the LeMans and GTO than the treatment Buick used, which looked somewhat like an elongated version of the first- generation F-Car.
The rear quarters are also Pontiac influenced, featuring a lower skeg, similar to the 1967-1968 full-sized Pontiacs. The rocker trim extends to the lower rear quarters and picks up the contour of the rear bumper in the same way as it did to the front bumper. The red, inner fender liners and 5-on-5inch bolt-circle Rally I wheels shod with redline tires add a racy flair to the big E-Body.
With a car doing battle with such vehicles as the aforementioned Thunderbird, the interior would have to be attractive, innovative, and luxurious. Inside, the E-Body Grand Prix features a cockpit-style dash, a scaled-up version of what was used in the 1969 production Grand Prix, with walnut accents, full Rally gauges, and a wood wheel on a tilt and telescoping column. Four bucket seats, trimmed in glove leather, are separated by a full-length console that runs from the dash and up between the rear seats. The console houses the shifter (Hurst for manuals and Hurst Dual-Gate for automatics) and front and rear storage compartments. Like the dash, it is trimmed in walnut, with chrome accents.
Powertrain Options
With such an upscale vehicle as the E-Body Grand Prix, only Pontiac's top powertrain options would be available—no economy-minded two-barrels here. Two very special engines are custom-tailored for this special car.
The base engine would be a 428ci version of the Ram Air II. The round-port heads, hotter camshaft, and the Firebird's exhaust manifolds would give the 428 a serious jump in power. Where the production 428 was rated at 376 horsepower, we underrate this engine at an even 400 horses, with the torque at 500 lb-ft. Available with either a Muncie four-speed or a Turbo 400, it would give the performance/luxury buyer a rare manual-transmission option.
The top engine is straight from Mac McKellar's secret stash—a 428ci version of the three-valve DOHC V-8 that was on the Mar. 1968 cover of Hot Rod. Originally built for a full-size application, this engine was a technological tour de force and no doubt would have rewritten muscle car and racing history.
This version for our E-GP would feature dual four-barrel carbs on a common baseplate and McKellar's innovative fiberglass belt-drive. The cam drives on this V-8 were supposedly too large for an A-body, but the cavernous E-Body engine compartment would have easily fit them, along with streamlined exhaust manifolds.
Reportedly, this engine was capable of over 600 horsepower, so a detuned street version would have still been over the 500hp mark. For the official horsepower rating, we'll take a cue from Rolls Royce and just call it "adequate."
While the idea of a Pontiac-specific E-Body is a tantalizing notion, given the market conditions of the 1960s, it probably would not have been the right car for the time. GM upper management wisely chose Buick to receive the E-Body platform, where the elevated price could be more easily justified. Our versions would most likely have broken the bank for both the buyers and for Pontiac.
An E-Body Grand Prix would also have likely prevented the release of the real winner in the personal luxury marketplace, the 1969 G-Body Grand Prix. Still, it is fun to imagine what could have been, which of course, is the whole idea behind this series.
Please send your ideas for What If? to christopher.phillip@sorc.com


The state agency in Orihuela Costa that it helps you to find your house in the Costa Blanca.
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Our church's Bible study library is now digital...and gigantic! We are pleased to now offer you FREE access to RightNow Media, a wonderful library of thousands of video resources for all ages from churches and pastors all across the country, to help you develop and grow in His Word and your walk. Streaming directly to your computer, tablet, or mobile device, you'll find this "Netflix of Bible studies" to have relevant content for every age group or spiritual season. Parents, you'll even find that RightNow Media has a multitude of videos and resources for your kids.


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"Eli Rosenbaum: Nazi Hunter"
For a man who's devoted nearly 30 years of his life hounding the despicable men and women who've committed crimes against humanity, Eli Rosenbaum doesn't look so menacing.
Given the choice, he'd rather watch the Yankees or see a comedy than sit through another Hollywood movie about the horrors of the Holocaust. He's been living with that gruesome reality almost 24/7 ever since the U.S. Department of Justice's Office of Special Investigations hired him out of Harvard Law School in 1980.
Today the 57-year-old Long Island native is the director of the human rights and special prosecutions section, making him the Justice Department's longest-running investigator of human rights violators living in the United States.
"So my entire career is really a summer internship gone awry!" he says with a grin.
There's a kindness in his brown eyes that belies the evil he's had to face. He helped deport Boleslavs Maikovskis, a Nazi war criminal living in Mineola, and Karl Linnas, a former concentration camp commander living in Greenlawn. Because nature has finally enacted a "biological solution" to that Nazi generation, his section is now pursuing war criminals from the likes of Bosnia, Guatemala and Rwanda, who think they've found a safe haven here. His message to them: "You'll have to be looking over your shoulder for the rest of your life."
Growing up "on the south side of Old Country Road in Westbury," attending high school in East Meadow, and studying Hebrew three days a week, it's surprising how little Rosenbaum knew about the genocide of World War II until one Sunday afternoon on his family's black and white TV he saw a dramatization of the Nuremberg Trials by Peter Weiss, a German playwright. Rosenbaum couldn't have been more than 12.
"The Holocaust wasn't spoken about in my household—it was too painful for my parents," he recalls. They had both fled Germany before the war.
But Rosenbaum's father did return, wearing a U.S. Army uniform. One winter some 25 years after the war, Eli and his dad were driving through a blizzard when his father casually mentioned that he had been one of the first Americans to report on Dachau after its liberation in April 1945.
"I said, 'Well, what did you see?' I'm looking out at the road, and I didn't hear anything. Finally I look at my father, and I see that his eyes have welled with tears. His mouth is open like he wants to speak but he can't do it. He's crying…To the day he died, he never told me."
He found out for himself. Today, a father, Rosenbaum credits his wife of 25 years, Cynthia, who also has a law degree, for keeping him balanced. "She keeps me sane despite the awful stuff that I have to deal with—the subject matter of my work."
Recently Rosenbaum was in Manhattan sharing the dais at the Four Seasons with Sara Bloomfield, executive director of the U.S. Holocaust Memorial Museum in Washington, D.C., which was celebrating its 20th anniversary—and has been an invaluable resource for Rosenbaum's investigations.
The Nazis, it turns out, expecting they would win the war and rule for 1,000 years, kept meticulous records.
"We've done the best job of any law enforcement agency in the world in hunting these people down," he says. "I like to think that this effort is carried out for the victims who perished and the victims who survived."
Posted in: Anti Zog Media,Europe,Holocash,The Jewish Problem,USA


In South Carolina, all adults have the right to make decisions about their own healthcare. If there are certain treatments you would not like to receive or a person you'd like to appoint as a healthcare representative, you have the right to do so, and healthcare providers must respect your choices.
A health care power of attorney enables your representative to make almost any healthcare decision you would be able to make. These include consenting to or refusing any type of treatment or medication to reduce pain regardless of its potential for addiction, admitting or dismissing you from healthcare facilities, and taking any other necessary measures to follow your healthcare instructions and interests. These are sweeping powers, so you should take care in choosing a representative and consider limiting which powers to grant. Also, you should make sure your representative is familiar with your values.
A living will, also called a "Declaration for Desire of Natural Death" in South Carolina, allows you to provide instructions for your treatment in end-of-life circumstances. This means you can choose to withhold life-sustaining treatment if you have a terminal illness or are permanently unconscious. To qualify, a terminal illness must be incurable, irreversible, and doctors must determine that the illness would cause the patient's death if life-sustaining treatments ceased.
These can be difficult issues to think about, but planning for these issues could save your loved ones some grief and stop you from enduring an illness unnecessarily.


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Police seek driver who repeatedly nudged cyclist before running over his bike
Dark 4x4 vehicle aggressively attacked cyclist
by Sarah Barth
Sat, Sep 24, 2016 13:11
Police in York are searching for the driver of a 4x4, who repeatedly nudged the rear wheel of a bike before running over it when the cyclist got off to protest.
The car was behind a cyclist who was waiting at traffic lights, and the driver pushed against his back wheel several times.
The cyclist was waiting in the right-hand filter lane for Rawcliffe Lane when the dark-coloured car, possibly an Audi, approached on Monday, September 12th at around 5.45pm.
When the cyclist dismounted, the car ran over it, damaging the back wheel. The driver then disappeared towards Shipton Road.
No-one was injured during the attack, which took place in a busy area.
Police are appealing for witnesses or dash cam video to help their investigation, and ask the public to dial 101 and ask for PC John Armstrong or York Police.
Back in 2012 we reported how Mary Creagh, then Shadow Environment Secretary, wrote to London private hire firm Addison Lee to complain about an incident in which one of its drivers allegedly bumped her back wheel while she was waiting to cross a junction on her bike in London.
She described the incident in a letter addressed to the firm's chairman and founder John Griffin.
"I was interested to hear you a few weeks back on Radio 4, saying that your minicabs should be allowed into bus lanes," she wrote.
"Perhaps you would be interested to hear my experience of one of your drivers today? I was at the junction of Bloomsbury Square and Tottenham Court Road, which is left turn only for vehicles, with an exception to go straight ahead for cycles. The lights changed and the road was blocked by a bus. I was on the right hand side of one of your vehicles and behind a van which could not move because of the bus.
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"The lights changed back to red, the van ahead of me curled around the corner but I decided to wait at the red light rather than risk crossing the road as my sight line of any oncoming traffic was obscured. Your driver bumped into the back of my bicycle.
"When I observed that the lights had changed to red some five seconds earlier, he shouted and yelled at me.
"It was the first time I had been bumped in well over ten years. I am a slow, careful cyclist with excellent hand signals and always make eye contact with drivers.
"It was certainly an interesting experience to be barracked for obeying a red stop light when driver mythology has all cyclists down as light-jumping lunatics. Based on today's experience, your drivers' reputation for careful driving may be just as much of a myth."
Sarah Barth
After an unpromising start, having to be bribed by her parents to learn to ride without stabilisers, Sarah became rather keener on cycling in her university years, and was eventually persuaded to upgrade to proper road cycling by the prospect of a shiny red Italian bike, which she promptly destroyed by trapping a pair of knickers in the rear derailleur. Sarah writes about about cycling every weekend on road.cc.
Mungecrundle 1 hour 5 min ago
iandusud 4 hours 5 min ago


Or in the shadow of the pyramids as has happened in the past.
As for London, the Super Series Finals took place in the Broadgate and aside the noise intrusion from the football on one evening was an excellent venue and the Canary Wharf Classic is better still in terms of a venue. Queens Club for the Super Series Finals has the potential to also be an excellent venue and I noticed that tickets for the Final for that are selling very well indeed, better than last years at the same point by the look of it.
As for Manchester I agree about the cavernous nature detracting from the event, and it was almost comical seeing Shabana have to come out of the court during the semi to ask that the dish collecters from the VIP area make less noise. Having said that the whole concession area, I thought was excellent. My wife had never seen live pro squash before and thoroughly enjoyed it and is looking forward to attending again.


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module SessionsHelper
  # 登入指定的用户
  def log_in(user)
    session[:user_id] = user.id
  end

  # 在持久会话中记住用户
  def remember(user)
    user.remember
    cookies.permanent.signed[:user_id] = user.id
    cookies.permanent[:remember_token] = user.remember_token
  end

  # 如果指定用户是当前用户,返回 true
  def current_user?(user)
    user == current_user
  end

  # 返回 cookie 中记忆令牌对应的用户
  def current_user
    if (user_id = session[:user_id])
      @current_user ||= User.find_by(id: user_id)
    elsif (user_id = cookies.signed[:user_id])
      user = User.find_by(id: user_id)
      if user && user.authenticated?(:remember, cookies[:remember_token])
        log_in user
        @current_user = user
      end
    end
  end

  # 如果用户已登录,返回 true,否则返回 false
  def logged_in?
    !current_user.nil?
  end

  # 忘记持久会话
  def forget(user)
    user.forget
    cookies.delete(:user_id)
    cookies.delete(:remember_token)
  end

  # 退出当前用户
  def log_out
    forget(current_user)
    session.delete(:user_id)
    @current_user = nil
  end

  # 重定向到存储的地址或者默认地址
  def redirect_back_or(default)
    redirect_to(session[:forwarding_url] || default)
    session.delete(:forwarding_url)
  end

  # 存储后面需要使用的地址
  def store_location
    session[:forwarding_url] = request.original_url if request.get?
  end
end