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The archael small heat-shock protein (sHSP), MjHSP16.5, forms a 24-subunit oligomer with octahedral symmetry. Here, we demonstrate that the IXI motif present in the C-terminal domain is necessary for the oligomerization of MjHSP16.5. Removal increased the in vitro chaperone activity with citrate synthase as the client protein. Less predictable were the effects of the R107G substitution in MjHSP16.5 because of the differences in the oligomerization of metazoan and non-metazoan sHSPs. We present the crystal structure for MjHSP16.5 R107G and compare this with an improved (2.5 Å) crystal structure for wild-type (WT) MjHSP16.5. Although no significant structural differences were found in the crystal, using cryo-electron microscopy, we identified two 24mer species with octahedral symmetry for the WT MjHSP16.5 both at room temperature and at 60°C, all showing two major species with the same diameter of 12.4 nm. Similarly, at room temperature, there are also two kinds of 12.4 nm oligomers for R107G MjHSP16.5, but in the 60°C sample, a larger 24mer species with a diameter of 13.6 nm was observed with significant changes in the fourfold symmetry axis and dimer–dimer interface. This highly conserved arginine, therefore, contributes to the quaternary organization of non-metazoan sHSP oligomers. Potentially, the R107G substitution has functional consequences as R107G MjHSP16.5 was far superior to the WT protein in protecting βL-crystallin against heat-induced aggregation.
1. Introduction
The small heat-shock protein (sHSP) class of protein chaperone is an energy-independent class of chaperone that act as holdases when combating the potential of misfolded or damaged proteins to form aggregates. sHSPs are also assembly chaperones, associating with protein polymers, such as actin, microtubules and intermediate filaments to modulate both their oligomerization and their assemblies in cells. How such properties relate to the structure of sHSP oligomers is a key question.
HSP16.5 from Methanocaldococcus jannaschii (MjHSP16.5) was the first sHSP to be structurally characterized by crystallography [1]. The protein oligomerizes into a particle containing 24 identical subunits with octahedral symmetry. The crystal structure confirmed earlier protein structure predictions [2] regarding a central α-crystallin domain (ACD) forming a β-strand sandwich comprised eight β-strands (numbered 2–9) organized in an immunoglobulin-like fold that is flanked by the N- and C-terminal domains that dock onto hydrophobic sites on the β4/β8 edge of this β-strand sandwich at the fourfold symmetry axis of the oligomer. The β6-strand hydrogen bonds to the β2-strand of its partner subunit in the twofold axis, forming an inter-subunit composite β-sheet, a feature found in the crystal structures of other non-metazoan sHSP oligomers [1,3,4]. The β6-strand therefore contributes to the oligomerization of individual sHSPs [5–7]. The edge formed by the β2- and β7-strands is where one of the highly conserved arginine residues [8] locates: precisely, the one which when mutated in αB-crystallin (CRYAB, CTPP2, HSPB5; [9]) and αA-crystallin (CRYAA, CRYA1, HSPB4; [10]), causes cataract (R116C CRYAA and R120G CRYAB) and cardiomyopathy (R120G CRYAB).
Previous studies on the R120G mutation in CRYAB revealed an increase in oligomer size [11]. Moreover, the R120G CRYAB oligomer had lost both its temperature and pressure dependence for size, and was also compromised in its heat stability compared with wild-type (WT) CRYAB [11]. By comparing a series of substitutions at the R120 site, it was apparent that the dimer, via its intra- and inter-subunit interactions, was very important to oligomerization and particle size [11]. Furthermore, for MjHSP16.5, oligomerization and particle size can also be altered by inserting extra sequences in its N-terminal domain as seen for HSP16.5-P1N [12].
Oligomerization differs remarkably between metazoan and non-metazoan orthologues. Non-metazoan sHSPs classically form oligomers of a defined subunit number, but metazoan sHSP complexes are typically polydisperse [8,13]. In non-metazoan sHSPs, the β6-strand is integrated into its partner dimer subunit, but this does not occur in metazoan sHSPs because the loop separating the β6- and β7-strands is non-existent, and they are instead fused into one [14]. One highly conserved structural element within the sHSP family [2,8,13] that is critical to oligomerization and to sHSP polydispersity is the C-terminal domain and specifically the IXI motif, which because of its palindromic nature, allows two different binding modes into the groove of the β4/β8 edge of the ACD [15]. The position and structural function of the IXI motif was identified in the crystal structures of MjHSP16.5 [12,16] as well as in the crystal structure of wheat HSP16.9 [4]. The MjHSP16.5 IXI motif and C-terminal extension therefore links together the four and threefold units in the oligomer, and its deletion would be expected to be deleterious for oligomerization [16]. This notion is further supported by the findings that deletion of the IXI motif prevents oligomerization of both bacterial and plant sHSPs [17,18]. The mammalian sHSP, HSPB6, is naturally missing this motif and does not oligomerize, forming dimers instead [19]. For a mammalian sHSP that does possess an IXI motif, deletion of C-terminal sequences including the IXI motif in CRYAB (Q151X in CRYAB) not only prevents its self-oligomerization, but also destabilizes it, with the benefit of apparently increasing its binding to client proteins [20]. Mutating the IXI to G-X-G in either CRYAB or CRYAA did not prevent oligomerization, but did improve its function in client protein protection assays [21]. Indeed, solution nuclear magnetic resonance (NMR) studies have shown that the flexibility of the IXI motif, and the C-terminal extension, in general, is critical to its function and point mutations that alter this flexibility change the activity of the sHSP [22]. Therefore, it is clear that this motif is key to sHSP protein oligomerization, dynamics and function, and it is important to formally confirm that this key role for the IXI motif is preserved in MjHSP16.5.
In this study, we analyse the effect of deleting the IXI motif and substitute the highly conserved arginine (R107) equivalent to the disease-causing mutation in CRYAB (R120G) into MjHSP16.5. In addition, we present the crystal structure for R107G MjHSP16.5 and by cryo-electron microscopy document a temperature-dependent effect upon its quaternary structure and assess the functional consequences by in vitro chaperone assays.
2. Material and methods
(a) Cloning, mutagenesis and bacterial expression of MjHSP16.5
Methanocaldococcus jannaschii (Mj) DNA was obtained from Dr Huber (Microbiology, Regensburg, Germany). The MjHSP16.5 cDNA was generated by polymerase chain reaction (PCR) amplification using the primers 16.5FOR1: CATATGTTCGGTCGTGA CCCATTTGATTCATTATTTTG and 16.5REV1: TTATTCAATGTTGATTCCTTTCTTAATTGAGG using Pfu DNA polymerase (Fermentas, UK). PCR products were gel purified and cloned into pGEMT-Easy (Promega, UK). The cDNA was then subcloned into pET23b, using the NdeI and EcoRI sites prior to bacterial expression and purification after isopropyl-1-thio-β-d-galactopyranoside (IPTG) induction. The R107G mutation was created by PCR. The oligonucleotide pairs HSP16.5F1 and HSP16.5Rg1: AAGCTTTATTGTTCCATATATTTCTTCCTCTTCTG, and then HSP16.5Fg1: AAGCTTCCTGCAACTGTTAAGG paired with HSP16.5REV1. This strategy introduced the G107 mutation along with a distal HindIII site. After cloning both fragments into pGEMT-Easy, NdeI–HindIII, HindIII–EcoRI fragments were then ligated together into pET23b cut with NdeI and EcoRI to generate a bacterial expression vector to produce R107G MjHSP16.5. Lastly, a C-terminal truncation construct was produced, HSP16.5-G143STOP, using the primer pair MjHSP16.5Fstop1 and MjHSP16.5Rstop2 TTATTTCTTAATTGAGGATTCTGCCTTTGG. PCR fragments were then cloned into pGEMT-Easy. After blue-white selection after all the cloning steps detailed earlier, plasmids from individual clones were purified and both DNA strands sequenced, to confirm amplicon/ligation fidelity, and compared with the relevant database entry (http://srs.ebi.ac.uk/srsbin/cgi-bin/wgetz?-e+[EMBL_features-id:L77117_606]).
The expression constructs of both WT and engineered (R107G, G143X) MjHSP16.5 were transformed into BL21(DE3) pLysS Escherichia coli. Recombinant protein expression was induced using 0.5 mM IPTG for 4 h, once the bacterial culture had reached an OD600 of 0.6. Harvested pellets were resuspended in 50 mM Tris–HCl pH 8, 1 mM EDTA, 300 mM NaCl, 1 mM DTT, 0.2 mM PMSF containing complete protease inhibitor cocktail (Roche) and lysed by several freeze/thaw cycles. A supernatant fraction containing the soluble MjHSP16.5 was prepared by centrifugation of the lysate at 15 000 rpm in a JA-20 rotor (Beckman) for 30 min at 4°C after incubation for 30 min with 10 U ml−1 of benzonase nuclease (Merck Bioscience) at 4°C followed by 0.005% (w/v) polyethyleneimine for 10 min on ice.
(b) Purification of MjHSP16.5 proteins
Methanocaldococcus jannaschii is a hyperthermophile and lives in hyperthermal oceanic vents, which experience temperatures of approximately 50–95°C and NaCl concentrations of approximately 0.2–1.0 M [23]. These organisms must therefore possess unique adaptations to allow survival in such high temperatures and salinity levels [24]. With this as a background, the soluble protein fraction was incubated in a water bath at 80°C for 30 min in the presence of 1 M NaCl. Samples were then cooled on ice for 10 min and centrifuged at 48 000g for 30 min. By using this approach, almost all contaminating proteins are denatured and pelleted, and the resulting soluble fraction was 90–95% MjHSP16.5. The protein was then further purified by anion exchange chromatography and size exclusion chromatography (SEC). Purification by anion exchange chromatography used a Fractogel EMD-650S TMAE column. Peak fractions were pooled and then dialysed into 20 mM Tris–HCl pH 7.4, 100 mM NaCl in preparation for SEC.
The R107G and G143X MjHSP16.5 proteins were purified using the same purification methods as WT MjHSP16.5, by heating and centrifugation, anion exchange and SEC. Purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry as described [20]. For G143X MjHSP16.5, it was difficult to achieve more than 90 per cent. Although G143X MjHSP16.5 was heat stable, crystallization was not attempted. All pure protein samples were analysed by electron spray ionization mass spectrometry, and the measured molecular weights were within 2 kDa of the calculated molecular weight (data not shown).
(c) Size exclusion chromatography of small heat-shock proteins
To estimate the molecular size of the recombinant sHSP complexes, approximately 5 mg of purified proteins were subjected to SEC. Purified MjHSP16.5 proteins were analysed using a Superose 6 column (290 × 10 mm) at a flow rate of 0.2 ml per minute at room temperature using a Merck–Hitachi biochromatography system. The column was calibrated with the following molecular mass standards: thyroglobulin (669 kDa), apoferritin (440 kDa), β-amylase (200 kDa), bovine serum albumin (67 kDa) and carbonic anhydrase (29 kDa). The void volume of the column was determined by dextran blue (2000 kDa). The proteins were separated on the column in buffer containing either 20 mM Tris–HCl, pH 7.4 or 10 mM HEPES, pH 7.4 and 100 mM NaCl at room temperature, and the elution volume of each sample was used to estimate the molecular weight. The data were analysed using Chromeleon 6.30 software (Sunnyvale, CA, USA).
(d) Chaperone assays
Citrate synthase from porcine heart (Sigma-Aldrich, UK) was dialysed for 16 h at 4°C against 50 mM Tris–HCl pH 8.0, 2 mM EDTA and diluted to 0.49 mg ml−1. The assay mixture contained citrate synthase and MjHSP16.5 at a substrate : chaperone (4 : 1) ratio in 220 μl at a temperature of 42°C. The effectiveness of MjHSP16.5 as a chaperone at 60°C was measured using βL-crystallin as the client protein [25] and α-crystallin as a positive control. Both α-crystallin and βL-crystallin from bovine lens (Sigma-Aldrich) were dissolved in 50 mM sodium phosphate (pH 7.4), 100 mM NaCl. A chaperone-to-client ratio (1 : 1) by weight was used. Time-dependent light scattering was measured at 360 nm every 15 s over 30 min, using a Beckman DU640 spectrophotometer for both chaperone assays.
(e) Crystallization
Initial crystallization experiments were performed using an Innovadyne screenmaker and standard sitting drop trays with drop sizes of 100 and 200 nl protein solution, respectively, mixed with 100 nl reservoir solution. Small crystals were observed in a number of conditions containing 2-methyl-2,4-pentanediol (MPD) and low molecular weight polyethylene glycol. The largest, best diffracting crystals were obtained from follow-up trays with hanging drop geometry using a reservoir solution of 0.02 M sodium acetate pH 5.5, 0.02 M CalCl2 and 35 per cent MPD as precipitating agents. Crystals grew over a period of weeks to maximum dimensions of 1.0 × 0.5 × 0.5 mm.
(f) Data collection, processing and structure determination
Crystals were typically tested using the in-house Bruker MicroStar rotating anode equipped with a platinum 135 CCD detector [26]. All final diffractions were collected at the Diamond beam line I02 [27] and processed with XDS [28]. The structures of the WT and the R107G variant were solved by molecular replacement using the original MjHSP16.5 structure [1] with AMoRe [29], and refined using REFMAC [30]. All interactive modelling was performed with Coot [31]. Further details of the crystal structure determinations are summarized in table 1. Coordinates and structure factors for the R107G MhHSO16.5 have been deposited at the protein data bank (PDB ID: 4I88).
(g) Cryo-electron microscopy
The final concentrations of both R107G and WT MjHSP16.5 used for cryo-electron microscopy (cryoEM) study were 0.8 mg ml−1. The sample (3.5–4.5 µl) was applied to the GIG holey grids (LifeTrust, PR China) after the girds were treated with a glow-discharge machine (Master Plasmer). Then, the samples were blotted for 3 s with blot force three at 100 per cent humidity using the VitrobotMark IV (FEI, The Netherland). The temperature of the Vitrobot chamber was set to room temperature and 60°C, respectively, for both the WT and the R107G MjHSP16.5 proteins. The grids were fast frozen into liquid-nitrogen-cooled liquid ethane.
The frozen samples were imaged with a FEI 300 kV Titan Krios cryo-electron microscope equipped with a Gatan UltraScan4000 (model 895) 16-megapixel CCD. A nominal magnification of 96 000× corresponding to the pixel size 0.933 Å and a defocus range between −2 and −3 µm were used during data collection for the WT MjHSP16.5 both at room temperature and at 60°C and for R107G MjHSP16.5 at 60°C. For the mutant R107G at room temperature, a nominal magnification of 75 000× was used, which corresponds to the pixel size 1.196 Å. The electron dose per image was kept around 20 e− Å−2. For the R107G MjHSP16.5 at room temperature, 126 raw CCD electron micrographs were collected manually. For R107G MjHSP16.5 at 60°C and the WT MjHSP16.5 at both room temperature and at 60°C, electron micrograph exposures were made with the automatic collection package Leginon [33]. In total, 1308 raw images were collected for the R107G MJHSP16.5 at 60°C, 1160 raw images for the WT at room temperature and 2119 raw images for the WT MjHSP16.5 at 60°C. The EM maps for both WT and R107G HSP16.5 have been deposited in EMDB (EMBD IDs: EMD–2288 for the first model of WT MjHSP16.5 at room temperature (figure 4a); EMD–2289 for the second model of WT MjHSP16.5 at room temperature (figure 4b); EMD–2290 for the first model of WT MjHSP16.5 at 60°C (figure 4c); EMD–2291 for the second model of WT MjHSP16.5 at 60°C (figure 4d); EMD–2292 for the first model of R107G MjHSP16.5 at room temperature (figure 4e); EMD–2293 for the second model of R107G MjHSP16.5 at room temperature (figure 4f); EMD–2294 for the first model of R107G MjHSP16.5 at 60°C (figure 4g); EMD–2295 for the second model of R107G MjHSP16.5 at 60°C (figure 4h)).
(h) Image processing
The defocus and astigmatism of each micrograph were estimated with CTFFIND3 [34] and corrected using the 'applyctf' routine of EMAN1 [35]. Particles of R107G (14 702 and 12 870) at room temperature and at 60°C and particles of the WT MjHSP16.5 (21 001 and 38 220) at room temperature and at 60°C were picked out using a graphical processing unit-accelerated, template–based, particles selection program, Gautomatch, developed in Fei Sun's laboratory (to be published). The initial models for the earlier-mentioned four different samples were obtained by using 'startoct' in EMAN1 [35]. Two different levels of noises were added into each initial model to yield two additional models. Then for each sample, these three corresponding models were used for further competitive image alignment and model refinement by using 'multirefine' in EMAN1 [35]. During the three-dimensional reconstruction, the octahedron symmetry was imposed. The final reconstructed density map was further sharpened by application of an amplitude correction algorithm in the program BFactor [36]. CryoEM maps were segmented, displayed and fitted with atomic models, using UCSF Chimera [37]. The crystal structure of the WT MjHSP16.5 (PDB code 1SHS) was used to fit into the cryoEM maps using the UCSF Chimera package [37].
The diameters of the reconstructed HSP16.5 particles were measured using the rotation-averaged projection method. First, the reconstructed volumes were projected by the same azimuthal angle to get the corresponding projections. Second, the projections were rotation-averaged using 'proc2d' in EMAN1 [35]. Finally, the rotational-averaged projections were projected along the y-direction to obtain the respective one-dimensional profiles. The distance between the two minimal values of the one-dimensional profile was measured as the diameter of the reconstructed particle.
3. Results
(a) Characterization of wild-type, R107G and G143X MjHSP16.5
Wild-type MjHSP16.5 and the two engineered proteins R107G (the G107 substitution) and G143X MjHSP16.5 (the IXI motif deletion construct) were produced recombinantly in E. coli (figure 1a). The WT and R107G MjHSP16.5 were both purified using the modified protocol, which included a heating step (80°C for 30 min in the presence of 1M NaCl) to remove as many E. coli proteins as possible. Purification was completed by a final SEC step. Although it was previously found that at 60°C the protein tended to aggregate [38], this was not our experience (see below). R107G MjHSP16.5 was purified similarly, but the same degree of purification for G143X MjHSP16.5 was not possible and this prohibited detailed structural studies on this protein. SEC was used to analyse the oligomerization of the prepared MjHSP16.5 (figure 1b). WT and R107G MjHSP16.5 were very similar in their elution profile and consistent with a calculated oligomer of 24 subunits. Interestingly, the G143X MjHSP16.5 SEC characteristics were consistent with a dimer being the most prevalent oligomer in solution (figure 1b). WT MjHSP16.5 prepared in this way did not aggregate when heated to 80°C, the protein remaining in the supernatant fraction after sedimentation (figure 1c). SEC was also used to prepare the highly pure material for crystallization experiments (figure 1d).The in vitro chaperone activity of WT, G143X and R107G MjHSP16.5 proteins was determined using an assay based on the heat denaturation of citrate synthase (figure 1e). Results show that both WT and R107G MjHSP16.5 can protect against the heat-induced aggregation of citrate synthase, providing comparable protection (figure 1e). Interestingly, the G143X MjHSP16.5 protein provided significantly increased (approx. two times) protection (figure 1e).
(b) Crystal structures of R107G MjHSP16.5 and comparison with the wild-type structure
The R107G MjHSP16.5 crystallizes in the same rhombohedral space group with eight independent molecules per asymmetric unit as the WT protein [1]. The functional 24mer generated by crystallographic threefold axis is shown in ribbon representations in three different orientations (figure 2). Owing to the high diffraction data quality, the mutation is unambiguously confirmed even in the initial electron density map calculated with the molecular replacement solution using the WT structure. The negative electron density on all eight independent protein chains confirms the absence of side chain atoms (figure 3a). Clearly, the mutation does not cause any significant change in oligomeric state and overall structure (figure 3b).
Closer comparison between the higher resolution WT structure presented here and the R107G MjHSP16.5 shows that there are no significant changes in the main chain positions (figure 3b). The root-mean-square deviation of 6299 equivalent main chain atoms is only 0.45 Å—clearly within the accuracy of the crystal structure determination. Hence, even locally, there are no significant structural changes detectable. However, it is noteworthy that the R107 in WT MjHSP16.5 forms hydrogen bonds to the main chain carbonyl oxygen atoms of G41 and M43, and forms electrostatic interactions with the residue E98 of the partner chain (figure 3c). Although the R107G substitution does not affect the overall crystal structure of the protein in the crystal environment, the loss of these two H-bonds and the inter-molecular electrostatic interaction in the R107G MjHSP16.5 variant may very well change the dynamic behaviour in solution.
(c) Cryo-electron microscopy reconstruction of wild-type and R107G MjHSP16.5 at room temperature and at 60°C
The WT and R107G MjHSP16.5 incubated at room temperature and at 60°C were imaged and analysed by cryo-electron microscopic single particle reconstruction (see figure 4 and electronic supplementary material, figures S1 and S2). The final reconstructions of the WT MjHSP16.5 particle at room temperature converged to produce two major octahedral structures (figure 4). Although the diameters of the two structures are both 12.4 nm, the densities inside are different; one has an empty chamber (figure 4a), whereas the other is filled with very obvious central density (figure 4b). For the WT MjHSP16.5 at 60°C, there are also major octahedral structures obtained with the same diameter of 12.4 nm (figure 4c,d), similar to the room temperature particles. The final reconstructions of R107G MjHSP16.5 at room temperature were also converged to two structures with the same diameters 12.4 nm; one has an open structure around the fourfold axes (see figure 4e and electronic supplementary material, figure S1a) and the other has a closed structure at the same region (see figure 4f and electronic supplementary material, figure S1b). The chamber of both structures for R107G MjHSP16.5 at room temperature contains electron density.
The final reconstructions of R107G MjHSP16.5 at 60°C, however, revealed two significantly different structures with distinct diameters (see figure 4g,h and electronic supplementary material, figure S1c,d). The two different diameters of R107G MjHSP16.5 particles at 60°C were obvious after two-dimensional reference free classification (see the electronic supplementary material, figure S2). The smaller of the two particles is similar in size (diameter 12.4 nm) to the R107G and WT MjHSP16.5 particles at room temperature. Interestingly, the central chamber of this smaller (12.4 nm) particle for R107G MjHSP16.5 is empty in contrast to similar-sized particles at room temperature. The larger of the two, however, has a significantly increased diameter of 13.6 nm and was larger than any other structure derived in this study. These two reconstructions of the particles revealed different oligomer arrangements in the fourfold symmetry axis and the dimer–dimer interface for R107G MjHSP16.5 oligomers (see figure 5 and electronic supplementary material, figure S3). The dimer–dimer interface within the 12.4 nm diameter particle is equivalent to the arrangement in the crystal structures of WT (see the electronic supplementary material, figure S3) and R107G MjHSP16.5 (figure 5a,e). However, the distance between the dimers in the 13.6 nm particle is increased to reveal a large space at the dimer–dimer interface (figure 5d,f).
(d) The R107G mutation alters the chaperone activity of MjHSP16.5 at 60°C
The cryo-electron microscopy data suggest that the R107G substitution allows for some oligomer flexibility and therefore we considered whether this could influence the in vitro chaperone activity of MjHSP16.5. For this purpose, we used one of the earliest assays used to demonstrate the chaperone activity of sHSPs [25] because it could be conducted at 60°C and so be consistent with the cryo-electron microscopy analyses. As previously reported [25], α-crystallin very effectively prevented the heat-induced aggregation of βL-crystallin (figure 6a) when present in a 1 : 1 ratio (figure 6b). Similarly, R107G MjHSP16.5 was equally effective in suppressing the heat-induced aggregation of this artificial client protein. This is in stark contrast to the WT MjHSP16.5 (figure 6a), which appeared ineffective. Under the conditions of this assay and in the absence of an effective chaperone, βL-crystallin formed aggregates that over the course of the assay were large enough to settle under gravity, which is the reason for the variable signal towards the end of the assay. Clearly, the effect of the addition of MjHSP16.5 was quite distinct from that of either α-crystallin or R107G MjHSP16.5.
4. Discussion
(a) The role of the N- and C-terminal domains on oligomerization of MjHSP16.5
Here, we have shown that removal of the C-terminal 5 residues from MjHSP16.5 is sufficient to significantly alter its oligomerization and also its chaperone activity towards citrate synthase (figure 1e). Our data are consistent with previous reports and with a key role for this domain in the oligomerization of MjHSP16.5 [16]. Preventing oligomerization by the removal of C-terminal residues mimic data obtained for CRYAB, where the myopathy-causing mutation Q151X in CRYAB prevented subunit oligomerization, but also increased its activity in in vitro chaperone assays [20]. Similar results are presented for MjHSP16.5.
Altering the N-terminal sequences in MjHSP16.5 can also increase the particle size with subunit number increased from 24 to 48 and also the client-protein-binding properties of the protein complex [12]. Swapping the N-terminal domain of CRYAB with that from CRYAA had a similar remarkable effect upon oligomerization and upon its chaperone function [39]. Similarly, deletion of the N-terminal domain prevents the oligomerization of mammalian sHSPs, for instance, CRYAB [15], and solid-state NMR studies of CRYAB demonstrate that this domain, which includes the β2-strand, interacts with the β3-strand of a neighbouring dimer [40]. Indeed, removing both the N- and C-terminal domains of CRYAB produces a stable ACD dimer that can be crystallized [14,15]. Therefore, it is clear that though there is significant sequence variation in the N- and C-terminal sequences between members of the sHSP family, they influence greatly sHSP subunit oligomerization and also their chaperone function [41].
(b) Influence of the R107G mutation on the dimer interface and MjHSP16.5 oligomerization
Recent data suggest that the origin of metazoan sHSP particle size heterogeneity resides in the protein dimer and specifically the ACD [42]. The CRYAB ACD dimer can adopt different conformations as revealed by crystallography [14,15] and by solid-state NMR studies [40]. Mutations in the ACD that alter the dimer conformation would be expected to significantly change sHSP oligomerization, and one example of this is the disease-causing mutation R120G in CRYAB [42]. R120 in CRYAB is part of an extensive ionic interaction network that crosses the dimer interface and it influences the oligomerization properties of both CRYAB and CRYAA [43,44]. This arginine is a very highly conserved residue within the ACD domain of sHSPs [8].
As shown in this study, the introduction of glycine instead of arginine at position 107 had several interesting structural consequences for MjHSP16.5. In the first instance, the crystal structures revealed no significant difference in the electron density maps for the crystals of WT or R107G MjHSP16.5. This is likely due to the fact that in non-metazoans, the β6-strand exchanges between partner chains, which is absent in metazoan sHSPs [14,15,45], together with the crystallization conditions that constrained the oligomer packing. It was only by cryo-electron microscopy that structural differences around the dimer interface at the fourfold axis were observed and then only for MjHSP16.5 heated to 60°C. The WT MjHSP16.5 had two distinct classes represented in the samples prepared at room temperature and at 60°C, which is consistent with previous studies [38]. No changes in the dimer subunit arrangements were observable for these two different-sized particles. Indeed, the difference in these two particles was seen similarly for the R107G MjHSP16.5 at room temperature (figure 4). In both cases, the subunit compliment was unchanged (see figure 5 and electronic supplementary material, figure S3).
The dimer–dimer interface within the 12.4 nm diameter particle is equivalent to the arrangement in the crystal structures of WT (see the electronic supplementary material, figure S3) and R107G MjHSP16.5 (figure 5). The distance between the dimers in the 13.6 nm particle is increased to reveal a large space at the dimer–dimer interface. Each monomer has to adapt the conformation of its ACD loop to make appropriate interactions with the adjacent dimer. Such conformational change can be easily achieved as a result of the R107 substitution with glycine because this would abrogate the electrostatic interaction in the dimer between R107 in one monomer and E98 in an adjacent subunit to allow more flexibility in the ACD loop (figure 5), and this most likely is the reason why changes in the dimer interface can be accommodated without changing the subunit number in the assembled particle. This interpretation is consistent with the view that inter-subunit interactions are very important to oligomer size [11,42].
(c) Influence of the R107G mutation on the chaperone function of MjHSP16.5
The R107G substitution in MjHSP16.5 also has functional consequences as evidenced by the increased chaperone activity towards βL-crystallin at 60°C (figure 6). The R120G substitution in CRYAB also changed its chaperone activity, either ablating completely or reducing it for the client proteins tested [43,44,46–48]. In CRYAB, there is an extensive network of ionic interactions that determine the AP interface between dimers [15], and these are disrupted by the R120G mutation in CRYAB causing the groove formed at the dimer interface to narrow [42]. This groove is also where small peptides bind in CRYAB [42]. If it is assumed that the equivalent interface in MjHSP16.5 can also potentially bind to client, then because this interface adopts a more open state at 60°C this would offer a potential explanation of its improved chaperone function.
Thermal-induced plasticity in chaperone activity combined with oligomerization has been observed for pea HSP18.1 [49]. HSP18.1 is a monodisperse dodecamer at ambient (22°C) temperatures, but heat shock (38°C) causes higher-order oligomeric states to form [49]. The addition of unfolding client proteins induces the formation of HSP18.1-client protein complexes that are remarkably polydisperse, varying in both the number of sHSP subunits and client proteins. WT and R107G MjHSP16.5 are constrained in their oligomerization dispersity. Nevertheless, the cryo-electron microscopy data indicate that R107G MjHSP16.5 forms at least two particles of different diameter, but of constant subunit number. Others have reported that WT MjHSP16.5 is also structurally diverse for the 24mer [38,50] and our data reveal two 12.4 nm particles differing in central density. The chaperone activity of MjHSP16.5 can also be increased when oligomer dispersity is driven by N-terminal sequence changes [12,16]. In the case of MjHSP16.5-P1 [16], there was no change in either the subunit number, the dimer interface or the symmetry, rather only a change in the orientation of the C-terminal sequences. This led to the suggestion that shell size for MjHSP16.5 depends on how the N-terminal sequences influence the C-terminal sequence orientation in MjHSP16.5-P1 [16]. The observed increase in client protein affinity and chaperone activity was reasoned to be due to the exposure of the N-terminal sequences [41,51]. We document here that preventing the oligomerization of MjHSP16.5 by deleting the C-terminal five residues increases in vitro chaperone activity, which parallels data obtained for CRYAB [20]. While our data do not distinguish between the two current models of sHSP-client protein recognition and binding [51,52], the increased chaperone activity towards βL-crystallin at 60°C as a result of the R107G substitution in MjHSP16.5 illustrates how such substitutions within the ACD can also contribute to structural and functional diversity of MjHSP16.5.
Note added in proof
During the review of this manuscript, Shi et al. [53] reported cryo-electron microscope analysis for both WT and R107 MjHSP16.5. This study reported differences in the density distribution with the central cavity, which were interpreted to be due to changes in the relative position of the N-terminal domain as a result of the R107G substitution and indicating that R107G MjHSP16.5 was in an activated state. The data presented here are consistent with this interpretation, but only when the data for reconstructed particles at 60°C are considered. Moreover, our data demonstrate that the R107G substitution also facilitates the expansion of the ACD shell without the necessity of additional subunits.
We thank Dr Xiaojun Huang and Dr Gang Ji (Center for Biological Imaging, Institute of Biophysics, CAS) for their help on the cryoEM image collection. We thank Xiaoyun Pang (F.S. group) for her great assistance with lab management. This work was supported by grants to F.S. from Chinese Ministry of Science and Technology (2011CB910303) and National Natural Science Foundation of China (31021062). All the EM data were collected at the Center for Biological Imaging, Core Facility for Protein Sciences, Chinese Academy of Sciences. The financial support of the BBSRC (A.L.), the British Consulate Offices of San Francisco and Shanghai for travel funds (F.S.), Durham University Fight for Sight (R.A.Q., Project grant 1763), the BBSRC (A.L.) and the EPSRC (E.P.) are all gratefully acknowledged. We also acknowledge excellent technical support by the Diamond beam line staff. We are grateful to Ian Edwards and Nikolay Milev for technical assistance with crystallization and structure determination, and for many excellent discussions.
One contribution of 11 to a Theme Issue 'Assembly chaperones in health and disease'.
© 2013 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.


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Hanle Village, Changthang Valley – Ladakh
Hanle in Changthang region of Ladakh is one of the most beautiful, enchanting, calm and soul loosing places in India. Hanle has a lovely Hanle Monastery offering some great aerial views of whole village that is a home of about 1000 people. The views from the top of monastery are just breath-taking. Hanle also houses an Indian Astronomical Observatory which is the world's highest observatory in the world at a staggering height of 4500 Mtrs. The Hanle Observatory is operated by Indian Institute of Astrophysics, Bengaluru and currently the second highest optical observatory in the world, with an installed telescope of about 2.01 meters (6.5 feet) diameter.
Hanle Village… An Aerial View…
Hanle is about 150 KMs from Mahe and the road runs through Nyoma and then Loma, where you need to pass the bridge on right that takes you to Hanle further 50 KMs ahead of Loma. Hats off to BRO, that they have created such smooth roads in the cold desert at such high altitude and harsh conditions. As you move towards Hanle, the size of the mountains starts to decrease and you start witnessing a series of small mountains clustered together giving a feeling of a different world altogether. The journey to Nyoma – Loma and up to Hanle is extremely nice with beautiful vistas all around you and in fact it is one of those journeys which I can go ever and ever again… I had never been to such a beautiful place ever. Roads could look like freshly laid but with caution, do not cruise as there are inverted bumps left on the roads so that the water passes through them. Such bumps can damage your vehicle, so be careful with the drive :)…
Hanle is very near to the Line of Actual Control, so one need to get Inner Line Permit to visit it. You can get more details on Inner Line Permits at the link: Inner Line Permits for Leh – Ladakh
The place is actually very scenic and feels out of the world. However, in actual reality the place is extremely harsh and difficult to survive especially in winters. There is only one cemented accommodation available around the lake and the facilities like electricity, medical aid, hot running water, western toilets etc. are rare to find. The lack of oxygen makes survival difficult if your body is not properly acclimatized to its high altitude. Hence, one need to plan his/her journey extremely carefully to make it acute mountain sickness free vacation.
Hanle is located about 275 KMs from Leh town of Ladakh district.
Hanle village is one of the high altitudes villages in Ladakh at a staggering altitude close to 4300 Meters (not sure about actual)…
Season for Traveling to Hanle – Ladakh
In December to March, Hanle remains mostly frozen, extremely cold conditions with only options to stay as homestays that may not be as convenient option for you. In April types, it starts to get a bit warm and one can afford to sleep even in home stays, I did the same :)… Then, May to September it is a very pleasant season to visit Hanle and a lot of tourist visit this place now. Come October and November, the tourists starts to reside completely as cold starts to set in again and you start to again feel a bit uncomfortable at Homestays but it is still manageable.
How to Reach Hanle – Ladakh
Route from Leh to Hanle
This is the general route to Hanle that most people take to visit this ever beautiful place in Ladakh. You can reach in about 9 – 10 Hrs depending upon the number of break you take in between. The route along with distance between the subsequent places is as follows:
Leh – Karu (36 KMs) – Upshi (13 KMs) – Kumdok (19 KMs) – Kere (53 KMs) – Chumathang (34 KMs) – Mahe (22 KMs) – Nyoma (25 KMs) – Loma (22 KMs) – Hanle(51 KMs) = 275 KMs
You can cover Thicksey, Hemis and Shey monasteries while going through this route, if you haven't covered them earlier while going to Pangong Tso. This route provides the best road conditions among all other routes. So, if you are one of those who prefer relaxing vacation and do not want to rush through places then, I will suggest make a 3 day trip to Hanle via this route from Leh. Of course, I assume your body has been acclimatized properly by staying for couple of nights in Leh and spending a night or two in Nubra Valley as well before attempting to sleep in cold desert of Changthang at Hanle. If you are short on time then you can also exit towards Manali via Manali – Leh Highway via Hanle and then to Tso Moriri via this route only but you need to ensure you have enough fuel to cover about 850 KMs without any fuel pump.
Route from Pangong Tso Lake to Hanle
There exist a direct route between Hanle / Tso Moriri and Pangong Tso as well and in most general cases, you will always be doing it from direct route of Pangong Tso to Hanle because you can exit Ladakh from Tso Moriri towards Manali – Leh Highway after visiting Hanle and Tso Moriri without coming back towards Leh. Secondly, both Hanle and Tso Moriri is higher than Pangong Tso so as per acclimatization schedule you should stay near Pangong Tso before spending night at Hanle and Tso Moriri. However, all depends subject to the approval of permits for this route via Chusul. Foreigners anyhow are not at all allowed to go beyond Man – Merak Villages as permits are not issued to them for this route.
Spangmik (Pangong Tso) – Man (9 KMs) – Merak (12 KMs)– Kakstet (4 KMs) – Chusul (24 KMs) – Tsaga La (33 KMs) – Tsaga Village (20 KMs) – Loma (23 KMs) – Hanle(51 KMs) = 176 KMs
Route from Hanle to Tso Moriri
After visiting Hanle, you can opt to go towards Tso Moriri from where either you can go to Ladakh or you can exit towards Manali via Manali Leh Highway. There exist two routes to reach Tso Moriri from Hanle, one via Mahe bridge and other via Chumur. Below is the route which you can follow to reach Tso Moriri from Hanle via Mahe bridge:
Hanle – Loma (51 KMs) – Nyoma (22 KMs) – Mahe (25 KMs) – Sumdo (10 KMs) – Tso Moriri / Korzok (53 KMs) = 161 KMs
The other route goes via Chumur whose permits are hard to come by but if you are able to get them then nothing gets better and adventurous on your Ladakh journey. Below is the route which you can follow to reach Tso Moriri from Hanle via Chummur village:
Hanle – Salsal La (62 KMs) – Charchagan La (23 KMs) – Tso Moriri / Korzok (60 KMs) = 145 KMs
You need to make sure you carry fuel for about 700 – 800 KMs because you will be covering Leh – Pangong Tso – Nyoma – Loma – Hanle – Tso Moriri – Leh without any other petrol pump. And if you want to exit Ladakh through Manali – Leh Highway from Tso Moriri then also carry petrol/diesel for about 900 – 1000 KMs as the next petrol pump will be available at Tandi. Otherwise, you need to come back to Karu or Upshi, refill and move back again towards Manali – Leh Highway.
Preferred Vehicles
The higher ground clearance vehicles are preferable and 4×4 ones would definitely be helpful especially between Pangong Tso and Tso Moriri via Man – Merak – Chusul – Nyoma (Changthang) route AND Tso Moriri to Hanle via Chumur. However, for general route via Chumathang or Mahe bridge, quite a few people do it in hatchbacks and sedans as well which depends upon your skills to drive and road conditions at the time of travel.
Suggested Itinerary for Hanle – Ladakh
Generally, at least two night stay is preferable to truly witness the mesmerizing beauty and vistas at Hanle including the night sky watch. Of course, one night stay will also help you watch a star studded night sky, an opportunity to capture some galaxy in your camera. Generally I prefer like:
Day 1 | Leh – Upshi – Chumathang – Nyoma – Loma – Hanle
- Cover Landscapes eroute
- Overnight at Hanle
Day 2 | Hanle
- Cover Hanle Monastery and Hanle Observatory
- A leisure walks in the pasture lands of Hanle
- Overnight at Hanle
Day 3 | Hanle – Tso Moriri via Mahe OR Chumur (if permits allowed)
- Cover Korzok Monastery
- Overnight at Tso Moriri
Day 4 | Tso Moriri – Tso Kar – Debring – Taglang La – Rumste – Upshi – Karu – Leh
- Cover Tso Kar Lake and Taglang La (some part of Manali – Leh Highway)
- Overnight at Leh
You can also check the most common and very well balanced itinerary for whole Leh – Ladakh region covering all major tourist destination including Srinagar – Leh Highway and Manali – Leh Highway, at the below link: Most Common Itinerary for Leh – Ladakh
Accommodation Options at Hanle – Ladakh
The homestays at village Khaldo very very close to observatory. One is near the start of the village and has a landline phone. Ask people about homestay which has phone. The rooms are decent but the prices are hay-way as the owner is greedy types. There is one more homestay at the end of this village just before pasture land starts which is run by a lady named Padmadeskit Homestay (ask for this name). We stayed at this only and found it OK to stay. Do not expect too much out of a homestay. This one is more loyal to tourists as other one charge too much if he sees more tourists.
Update 2013: Padma Homestay in Hanle has been converted into a kind of decent guest house in 2013 and when I visited there in September 2013, there were 4-5 rooms there with one room having attached bath and others sharing a common bath. A big room for dining. You can read my review here in the DoW Community link: Padma (Sonam's) Guest House/Homestay, Hanle | Review
Contact Details of Padma Homestay Hanle – Ladakh
- Contact Person: Mr. Sonam Dorjay
- Attributes: Very good friend of mine, knows DoW and its vision very well, very down to earth local ready to support & help other locals of the village
- Contact Number: 09469224302, 09419834944, 08991922029,
- Email: [email protected], [email protected]
- Location: Hanle, Khaldo
Visit Umling La Pass from Hanle
Umling La pass is about 105 KMs from Hanle Village. If you have a permit to visit Hanle, then there is no extra permit required and if you are coming from Koyul side then you need to have a permit for passing Koyul as well. Refer DoW Maps for Hanle and Koyul routes: DoW – Maps of Ladakh | Nubra Valley & Changthang
The route you can take is Hanle to Photi La Pass to Photile Village (57 KMs) and then follow the route from Photile Village to Umlinga La pass for about 50 KMs at an altitude of 19300 feet. You will come across two bridges/ water crossings while going to Umlingla Pass from Photile Village. There is another route from Ukdungle village as well but it is more of a dirt track only all the way except few KMs.
Medical Facilities near Hanle – Ladakh
The nearest basic medical health center may be available at Korzok Village though I am not that sure about it. You can get basic first aid including Oxygen doses at some of the tents/camps, I guess.
Food Joints near Hanle – Ladakh
Being a remote place, you will either be eating in the home stay or at Hanle observatory guest house in which you might be staying overnight if you get the bookings.
Phone Signals at Hanle – Ladakh
BSNL phone tower has been put up in Hanle and at times the phone connectivity is present in the village. However, do not rely much on it. There is one satellite public phone installed at a house in Khaldo village in Hanle. The home with a phone also offers homestay accommodation.
Petrol Pumps near Hanle – Ladakh
Nearest Petrol Pump is at Karu (about 240 KMs) from Hanle and comes on the way to Hanle from Leh via Chumathang / Mahe Bridge. I will suggest that you top-up the fuel from Leh / Karu itself and then drive/ride up to Hanle.
ATMs near Hanle – Ladakh
You will not find any ATMs near Hanle and hence, it is always better to carry cash from Leh (the only place where you find ATM in Ladakh) itself.
It is one of the most beautiful places in the whole Trans Himalayas, I will say, to visit. Hence, if it is your may be second or third visit to Ladakh and you are keen of finding some secluded place with a different feeling and aura then this one surely is not the place you would like to exclude from your plan. I hope the above information will be useful for your upcoming Leh – Ladakh trip.
Please feel free to post any queries or doubts you might have in planning your trip to Leh – Ladakh or Hanle, in the comments section below. If you have been to Hanle before, kindly share your thoughts and other information you might feel will be worth sharing with other fellow travelers and will prove helpful to them.

subset: commoncrawl


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Location: Winther Hall 1006
WITRC Lab: (262) 472-2844
Jon Spike: (262) 472-2838
Email: firstname.lastname@example.com
As a student at the University you will use technology daily to complete assignments, write reports, gather information through the Internet, and communicate with faculty, family, and friends. It is safe to say that it is very difficult to get along in today's world of education without knowing about instructional technology and how to operate them. It is not only necessary to utilize technology as a student but as a future teacher you will be expected to have a high level of proficiency with technology. It is absolutely true that one cannot be an effective teacher in today's schools without a thorough understanding of instructional technology and their individual uses.
While it is not absolutely necessary, it is highly recommended that each student have their own computer. For those considering not owning a computer, there are public-access computer labs at UW-Whitewater. Though computer labs have generous hours you would be required to complete all of your work during these hours and many may find this inconvenient.
While it may be possible to use University computer resources while one is a resident on campus, the day will come when you will want to have a computer of your own. Because of the convenience that owning a personal computer affords, many students will elect to purchase one as soon as possible. The following link can provide you with more information: Buying a Computer.
Wisconsin Instructional Technology Resource Center (WITRC)
WITRC primarily serves College of Education & Professional Studies students and faculty. Located in Winther Hall 1006 there are several resources that WITRC makes available. Our general computing lab has approximately 20 computers and helpful staff ready to assist if you encounter a problem. The ACL Lab offers access to Promethean and SmartBoard units, a commonly used tool in current classrooms. WITRC manages two collaboration labs that include Smart Boards and video conferencing equipment available for use by students. Finally, WITRC also manages a computer lab in Williams Center 187 which contains 24 PC's and one Mac. Typical office productivity software, as well as multimedia development, graphics, and web publishing software are available on the computers managed by WITRC.
More information about WITRC's resources
General Access Labs
The McGraw and Andersen Labs are general access computing labs for use by all UW-Whitewater students. These labs are maintained by Instructional Technology Services. They offer access to general-purpose and discipline-specific software. Students can also use e mail and access the Internet from any of the lab computers.
Help Desk
The University maintains a computer Help Desk (262-472-HELP) in McGraw Hall to answer faculty and student computing questions.
ResNET provides full, round-the-clock access to the Internet and all UW-Whitewater networked computing resources, such as file servers, mainframe computers, printers, and more.
Discounted software is available to students in the University of Wisconsin system from the Wisconsin Integrated Software Catalog (WISC). Software from Adobe/Macromedia, Microsoft, and others are available for purchase with student discounts.
Among the software titles available for use in WITRC, and other general access campus computer labs are Internet Explorer, Mozilla Firefox, Apple Safari, Google Chrome, Adobe Suite, Adobe Acrobat Pro, Microsoft Office, iPhoto, iMovie, iDVD, and Inspiration.


text:
Google Steps Into Microsoft Antitrust Case
Last week, Google asked Judge Colleen Kollar-Kotelly of the U.S. District Court for the District of Columbia to extend the consent decree, reached as part of the settlement between Microsoft and the U.S. Department of Justice, to November 2009.
Google argued in court documents that although Microsoft had promised to take steps to address concerns relating...
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A figure skater's progress is measured by a series of tests, which are governed by the U.S. Figure Skating Association. EPFSC conducts test sessions throughout the year, offering Moves in the Field and Freestyle evaluations.
Most application deadlines are 7 days prior to the test date. Test sessions may be closed earlier than noted if the session is full prior to that date. If you feel there is a need for an exception, please have your coach speak with the test chair. Please see the announcements on the testing registration page for updates.
Test fees are on a graduated scale based on the level of the test with pre-preliminary tests costing $30 and increasing to $65 for a senior-level test. Home club skaters have priority on the test sessions.
- Please contact Carrie Van Beusekom, the EPFSC test chairperson, or email email@example.com for more information
Test Application
Click on the link below to register for an upcoming test session. The Eden Prairie Figure Skating Club uses Entryeeze for registration and payment.
Testing outside EPFSC
- Please check the Twin City Figure Skating Association event calendar for test sessions at area clubs. Test fees may differ.
- To test at another club, you must also notify the EPFSC test chairperson. Please email email@example.com upon registration. The hosting club will require approval from our test chair prior to test day.
Upcoming Test Schedule at EPFSC
All testing sessions are on Rink 1 at Eden Prairie Community Center. Skaters: dress and fix your hair as you would for a competition. Skaters may wear skate jackets/sweaters and gloves for a moves test, but it is best to remove them for a freestyle test.
|Testing Date
|March 7, 2017
|9am – 12:30pm
|May 2, 2017
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|August 17, 2017
|1pm – 4:30pm
|September 28, 2017
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|December 5, 2017
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|February 6, 2018
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|May 1, 2018
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|August 23, 2018
|12:30 – 4:30pm
|September 27, 2018
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|November 1, 2018
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All dates and times are subject to change. Test sessions may be cancelled if fewer than 10 test applications are submitted or judges are not available. Test applications and payments are due no later than seven (7) days before the session. Applications will be processed by seniority if more applications are received than time allows. A $25 late fee applies for applications received after the deadline. Please check this page and our calendar for any changes and/or updates.


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Formative Assessment: Weekly Summaries
In our attempt to give concrete examples of formative assessment techniques, we go back to Debra Dirksen's 2011 article "Hitting the Reset Button: Using Formative Assessment to Guide Instruction" where she discusses weekly summaries.
She discusses having students "complete longer writing assignments in which I ask them to write a weekly summary reflecting on what they've learned from class discussion, activities, and reading during the week. To prompt reflection on personal learning, I ask students to respond to the question: What did you learn personally from class discussion, activities, and readings conducted this week? For evaluation, I ask, "How do you think what was taught this week, in class and through your readings, will work in the real world?" And finally, for transformation, I ask, "How will you personally use the information?" This is an opportunity for students to engage with the material and discover what resonates with them (Taylor 2008). I can also use this information as I design future instruction"
This gets the students to really think about what they have learned that week and allows them more processing time, while giving the teacher the necessary information to assess student learning. I highlighted the three aspects of this assessment to underscore their importance: reflection, evaluation and transformation. All of these factors feed into the students getting beyond mere recall of information and being able to use that information in a different way-a key to really learning.


text:
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Philips infrared lamp gives pain relief for muscles and joints. Its 150 Watt infrared warmth penetrates deep into skin, improving blood circulation thereby giving relief for areas of 20 x 30cm. Concentric rings help to focus on specific spots.
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South African Healthcare workers face greater risk for TB, HIVVery interesting article to read on our South African Healthcare workers and how TB is affecting...


text:
This section contains 560 words
(approx. 2 pages at 400 words per page)
That was how I resurrected the past: people in their outfits, or who sat next to whom, and from there on to what we talked about, what we were like at a certain time."
-- Carrie as Narrator (chapter 1 paragraph 1)
Importance: The quote addresses how Carrie uses clothing as a touchstone in her life.
When I made something I thought of longevity; how the hours I put into it would yield me an exponential number of wearing them."
-- Carrie as Narrator (chapter 3 paragraph 29)
Importance: The quote demonstrates how Carrie values the history and quality of items that she creates. This foreshadows that she will return to Madison.
I'd want to die, too,' Mike said, and I felt a narrow gap open up between us because I wouldn't want to, couldn't see thinking in those terms at all."
-- Mike and Carrie as Narrator (chapter 12 paragraph 38)
Importance: The quote shows the depth of Mike's commitment to Carrie in contrast to the disconnect she feels.
I'd been gone two weeks...
-- Carrie as Narrator (chapter 17 paragraph 1)
This section contains 560 words
(approx. 2 pages at 400 words per page)


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Charlie Chaplin (1916)
Critic Consensus: No consensus yet.
Charlie Chaplin Photos
Movie Info
as Vagabond Police Recruit
as Missionary
as Scourge of Easy Street
as Clergyman and Policeman
as Anarchist
as Small Father and Policeman
as Ungrateful Woman
as Drug Addict
as Mission Visitor and Policeman
as Policeman
Critic Reviews for Charlie Chaplin
There are no critic reviews yet for Charlie Chaplin. Keep checking Rotten Tomatoes for updates!
Audience Reviews for Charlie Chaplin
A light and funny silent short in which Chaplin the tramp gets a job as a policeman and must put order in the London slums.
Super Reviewer
The story is interesting, not great, but it's funny enough to watch.
Super Reviewer
Classic Charlie Chaplin silent movie. Charlie is a tramp who falls in love with the church organist. He gets a job as a policeman in the South London slums, where gangs beat up policemen. Charlie becomes the hero.
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Charlie Chaplin Quotes
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Mini Rant Begin
You know kind of like the way car-holes complain about cyclists going through stop signs and the way that cyclist slow them down and keep them from ignoring the speed limit signs on the road.Mini Rant End
Still this is a great thing to see going into Shadyside. We need the infrastructure more than ever. The popularity of cycling is growing and more people are taking to their bikes as an option to their cars. The only way that people will continue to do this is if we can make it easy and convenient for them to do this. I am not talking about the hardcore fixie riding twenty something who doesn't own a car or see a reason to own a car. I'm talking about the people who just want to go out for a evening grab some food and maybe a drink or two and don't want to have to use a car, find parking, and pay for parking.
Remember these are the people who could be in that car behind you tomorrow morning.


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Sunday. The day of you could use for rest or you devote the day taking advantage of all the things on offer in the city. Today I had the appetite to do more than just lie down (although that was totally on the agenda too). Stop one was the Greek Club to take up an invitation to dine. The club is in walking distance from my home but I've never dined there. Bad mistake. The banquet (with a side order of white bait because my husband can't resist) is to die for. By the end I felt like Mr Creosote from that scene in Monty Python's Meaning of Life. I was ready to explode but in a good way. Stop two was to walk it off. Today there was only walk worth doing - Legacy Way. In a couple of weeks the 5 kilometre tunnel will be open to cars prepared to pay a toll. But today was a one-off opportunity for families of Brisbane to walk the tunnel. Brisbane over recent years has shown quite an appetite for building great big new tunnels under the city. My family and I have walked every one and today was no exception. At one point I was, apparently, metres under the dirt of Toowong Cemetery. This is not something you generally live to talk about. A walk like this gives you a whole new perspective of the city. You get a sense of just how amazing the projects are that driving through will never give you. An estimated 20,000 people took the opportunity to walk the road with no need to pay a toll. (Personally, however, I'm glad I fueled up before the walk. It's not as easy on foot as it is by car).
This is where I was going to take a photo a day in 2012 but forgot to stop. I also write something random to give you an insight into the craziness that is Susan's mind.
Sunday, May 31, 2015
May 31. Day 151. Quite the appetite for this sort of thing
Sunday. The day of you could use for rest or you devote the day taking advantage of all the things on offer in the city. Today I had the appetite to do more than just lie down (although that was totally on the agenda too). Stop one was the Greek Club to take up an invitation to dine. The club is in walking distance from my home but I've never dined there. Bad mistake. The banquet (with a side order of white bait because my husband can't resist) is to die for. By the end I felt like Mr Creosote from that scene in Monty Python's Meaning of Life. I was ready to explode but in a good way. Stop two was to walk it off. Today there was only walk worth doing - Legacy Way. In a couple of weeks the 5 kilometre tunnel will be open to cars prepared to pay a toll. But today was a one-off opportunity for families of Brisbane to walk the tunnel. Brisbane over recent years has shown quite an appetite for building great big new tunnels under the city. My family and I have walked every one and today was no exception. At one point I was, apparently, metres under the dirt of Toowong Cemetery. This is not something you generally live to talk about. A walk like this gives you a whole new perspective of the city. You get a sense of just how amazing the projects are that driving through will never give you. An estimated 20,000 people took the opportunity to walk the road with no need to pay a toll. (Personally, however, I'm glad I fueled up before the walk. It's not as easy on foot as it is by car).
Saturday, May 30, 2015
May 30. Day 150. Show a bit of backbone
Music? Yes, there was a band but there was also a fair chunk of recorded music. And as for that singing and dancing "at the same time", it didn't happen. Almost all of the singing was done by two of the ensemble members. Yep, they were great singers but it felt like cheating. That's not how it's supposed to go. So I left the theatre feeling just a little cheated. And then in what was almost Monty Pythonesque just outside the theatre I saw a group of people wrestling with a giant spine trying to
to get it on top of a car. If only I'd seen a bit more of that backbone inside the theatre.
Friday, May 29, 2015
May 29. Day 149. Making waves
I think I might have a girl crush. To be clear it has nothing to do with the fact that George Clooney has bought the rights to his book Hack Attack and intends to turn it into a film (well it might have a bit to do with that but I shall pretend otherwise). No, I have a girl crush on Nick Davies for his candor and his courage. Any man who is prepared to take on the likes of the British tabloid press and Rupert Murdock and come out on top is a bit of a hero in my book.
But it's not only that. What I loved about the British journalist's presentation at QUT Kelvin Grove tonight was his honesty, his humour and his belief in journalism and for the most part journalists.
My favourite moments from tonight's presentation were the following observations
- He's seen "stuff written on toilet walls" better than most material written by citizen journalists
- A tiny minority of "scumbag" journalists ruin it for the rest of us
- There was lots of "extracurricular bonking" going on in Fleet Street papers while they criticised others for having affairs
- The revelations of the inquiries into the British press mean that crime at the papers has dropped to about zero but for the most part it's same old, same old
- The newspapers that invade people's privacy the most have the strongest sales despite people saying they don't like privacy invaded
- Closing the News of the World was a selfish and ruthless action by Murdock
- Exposing the wrong doing of the British press made Davies feel "iffy" because it meant many hard working foot soldiers were blamed and punished because of the orders of the generals
And for now Hack Attack can sit on my bedside table alongside Game of Thrones. The level of ethical behaviour revealed in both is about the same.
Thursday, May 28, 2015
May 28. Day 148. Not for the first time
"Hetherbelle," the teacher exploded."Are you looking at those Presbyterian girls again?"
"Yes, Brother," my father replied.
"Would you like to go and join them?"
"Yes Brother," Dad said. He got the cuts for his trouble.
There's so much about that story that is a reflection of the time. Catholic schools taught by Brothers. Corporal (sometimes sadistic) punishment and, of course the way teachers addressed students. No given names back then. All surnames and apparently somewhat condescending versions of them at that.
Not these days. But fast forward a couple of generations to quite possibly the same classroom and Drama Teen being required to stack chairs as a punishment. He was taking an exceptionally long time to do. It terms out that was Eisteddfod time and there were whole dance troupes of young women warming up on the school oval before their performance in the school hall. You can't blame a boy for looking. Or perhaps you can. Drama Teen's no longer so sure staring at the girls is such an acceptable sport not now that his cousin Jess is one of those girls on the oval ... Perhaps they should bring back corporal punishment.
Wednesday, May 27, 2015
May 27. Day 147. Greek tragedy
The ancient Greeks were seriously messed up. I mean seriously some of those Greek myths were dangerously perverted in a way that if I was to write them today I might just find myself subjected to the thought police or perhaps a best selling fan fiction author with a multimillion dollar fortune. It's hard to tell. But when it comes to dangerously messed up tales, Oedipus is top of the strange tree. He accidentally kills his father and marries and has four children with his mother. On finding out the truth, he gouges his eyes out and she kills herself. It is just the sort of stuff tabloids love. It's click bait on steroids. This is probably the strongest take-home message of Brisbane writer Dan Evan's new modern interpretation of the myth Oedipus Doesn't Live Here any More which opened at the Queensland Theatre Company's Billie Brown Studio tonight. This is the type of story we should be appalled by - and we will say we are - while engaging in essentially tragedy porn and lapping up every last sordid detail. This is an intriguing work. Funny and confronting. Disgusting but charming. It will not be everyone's cup of tea (in fact I am told some people walked out of the previews). But for those who like theatre that isn't just more of the same, theatre that challenges and goes out on a limb, you could do worse than give it a go. It. certainly beat State of Origin for my idea of a way of spending a Wednesday night. And for the record, Drama Teen also loved it. But then he would. Last year, when set a drama task of creating a modern interpretation of a Greek tragedy, he decided to turn his hand to Pygmalion. In that myth, the Greek sculptor Pygmalion is so repulsed by the local prostitutes that he shuns women and instead devotes his entire attention to his work. He creates the perfect woman in stone and falls in love and as a reward for his devotion, The Gods bring his woman to life. In Drama Teen's vision, a man is so tired of meaningless Tinder-style Internet hookups that he turns his back on relationships and orders himself a blow-up doll.... He falls in love. She comes to life. Seriously messed up.
Tuesday, May 26, 2015
May 25. Day 145. Let the history books show
It is also a (not very useful) tool in the toolkit of every student I have ever met.
As such the dining room table has this week been the home to just about every book on the Cultural Revolution held by the QUT Library. They have been there so long that Rumple decided they were clearly now a permanent feature and managed to get up and get among them today. They put him to sleep. He wouldn't be the first one they've had that effect on.
While they've been there, notes have been taken, sticky notes have been attached and as far as I can tell enough information to write a PhD thesis has been collected. But actual words written, not so much.
Because you have to put off to the last minute. The books sit there as though the mere act will make words jump out and write themselves. History shows this is not the case. History shows that no student ever actual used this knowledge to change behaviour. Or perhaps it is just that Drama Teen has inherited the procrastination gene from me. Either way, I feel quietly confident, something will be submitted tomorrow. I feel equally confident there will be missed sleep as a result. Do I think, the process will improve next time? It would be rewriting history for that to happen.
Monday, May 25, 2015
May 25. Day 145. Driven to distraction
Seriously how is a person supposed to get any work done around here? For months I have been driven to distraction by the following.
1) Noise. Just about every roof in my area has been replaced as a result of the great hail storm of 2014. Looking out my window you'd swear I lived in one of those new outer suburban housing estates such is the sea of shiny new roofs. But the change has not been a silent revolution. Throwing sheets of iron from high set tin and timber homes makes A LOT of noise.
2). Workmen. They park their cars blocking the street and they often parade around with very little on. Most distracting. They also like playing FM radio very loudly. There should be rules about this.At least it wasn't Alan Jones.
And today, just to join the noise and visual "pollution" the kookaburras decided it was all a bit of a laugh. Four of them circled and swooned and joined in the general chaos out my window. Yep, most distracting.
Sunday, May 24, 2015
May 24. Day 144. Birthday boy
|Rumple added his best wishes in his usual furry way
Anyway I officially reached the age where the government sends you a Bowel Screening Kit and Australian Pensioners Insurance will have you on their books 53 days ago. Today my husband Charles caught up.
As the experienced member of the family I chose and booked the birthday activities - breakfast at the civilised time of 10am with friends at a well-recommended and quite delicious cafe and then part-sexy, part-silly circus La Soiree in the early evening. And because we are old we were home by 8.30pm.
I think it was a celebration worthy of a half century.
So raise the bat Charles you old thing. Here's to the first 50 and 50 or so more
Saturday, May 23, 2015
May 23. Day 143. Festival forgiven (previously posted as festival fury)
***UPDATE: I have been contacted by the orgainisers who have apologised and assured me that cameras ARE allowed. It was a mistake of the security company. I have been assured it has been dealt with and it won't happen again. I was invited back but due to prior commitments (it's my husband's 50th birthday) I can't go. I will return next year (with camera). I accept the apology and I shall leave the post up because it was an accurate reflection of what happened and how it made me feel ******
So instead of tasting honey puffs, I had the bitter taste of bile in my mouth. Let's be clear. This is in the terms and conditions of the Festival should you scroll down to term 24 of the fine print. **
So it is well possible that the organisers were withing their rights to prohibit me and my camera from gaining entry. This kind of thing seriously annoys me. There are rules against just about everything. And exactly how is "The Venue" - a public park - going to give me permission to do anything
But, and it is a huge but, this kind of thing is dead wrong (and almost always badly handled by security).
Here are some facts. Thousands of photos will be taken in the festival grounds this weekend. No questions. No-one will care and in fact they will be a huge promotional tool for the festival.
This is not an major international act. It is hard to see how taking photos breaches any copyright or intellectual property.
This is a festival in a public park. It is not on private property. The public should have rights too.
I live very close to Musgrave Park. I walk my dog there all the time. But at festival time big fences go up and the public can't access a public space. This situation continues for about a week.
This weekend I can't drive down one of the main streets to my local supermarket because it is closed for the festival. I accept these things without question because a festival of this type is good for the community. But I do not accept that I should be barred from taking photos. I would not profit from the photos. I take photos because I like photography and I use a Digital SLR because I like taking good photos. In fact, I have taken photos with the same model camera for the past three or four Paniyiri festivals.
My camera was around my neck because it never occurred to me to try and hide it. Like everyone else going through those gates my intention was to have a good time and take photos to prove it.
Any organisers with any brains would understand that public endorsement and photos should be embraced. Word of mouth is a much stronger motivator than advertising. Well here are the words out of my mouth. You know what you can do with your honey puffs. And, as you can see, if your intention was to stop me taking photos you failed in any case.
Friday, May 22, 2015
May 22. Day 142. I'll have what he's having
You know "that" scene in When Harry Met Sally? The one that ends with the woman in the restaurant announcing "I'll have what she's having" after Sally's exuberant display of delight? Well today my 87-year-old neighbour Margaret said quite a similar thing after being greeted by a Rumple far more enthusiastic and energetic than we've seen for a number of days. I was telling Margaret that I was about to take Rumple back to the vet for a follow up visit after this week's serious illness. She told me to
a) Tell the vet, that the medication is clearly working and
b) put in a request for a few extra doses for her.
Margaret and Rumple love each other dearly. She giggles like a little girl when he's around. It's actually a delight to watch. I know Margaret appreciates my company and the little chores I do for her but I am by no means the favourite neighbour. I may be able to buy some bread and milk but can I was my tail? Can I put a paw on the arm? Do I lick a face? Well no, not recently anyway. See Margaret and Rumple and the case for having animals in nursing homes is closed. Open and shut case. They bring so much joy. Perhaps I should hire out Rumple to be a home visitor. I might need to if he finds new and improved ways to clock up mounting vet bills ...
Thursday, May 21, 2015
May 21. Day 141. We butchered that
|Seriously, this motley crew not only solved the puzzle but did so in record time
|Jumping for joy to celebrate the win
|Enter if you dare
|They make you hand over and lock up your phones before you even start. This is no picnic in the park
Wednesday, May 20, 2015
May 20. Day 140. The fog lifts
Potter's cloak of invisibility may not have been needed if you were one of the Brisbane's sky scrapers trying to hide from view. The city was heavily veiled in a thick blanket of mist.
At home, also covered by a thick blanket, was Rumple. With him back safely where he belongs I feel like my head has emerged from the fog of anxiety. He's not yet fully himself but he's doing a much better impression of the Rumple we know and love with every hour. And I have to say his restricted diet of only poached chicken breast and rice is just about the best thing since sliced bread as far as Rumple is concerned. The medication he'll have to take for the next five days, not so much
Tuesday, May 19, 2015
May 19. Day 139. Back where he belongs
Monday, May 18, 2015
May 18. Day 38. Paw thing
I was never the kid who went down in the school playground no matter how long they had us standing perfectly still in the blazing sun at double arms distance.
Others may get squeamish at the sight of blood. I make sure I turn to get a good look at a blood test.
But not today. Today the room started swimming. I went cold and clammy. I felt woozy and I knew I was only seconds away from hitting the floor. A nearby seat saved me. It was about 7am. I'd had two hours sleep and hadn't eaten. The room was hot and stuffy and my fur baby was vomiting and about to be admitted.
But none of that really explains what happened - but it did. I love my fur friend like a child and when your child hurts, you hurt. But he is in good hands and I have to trust that. With good luck, good medicine and good vibes he might be home tomorrow afternoon. I miss him dreadfully. I'm pretty sure the feeling is mutual
Sunday, May 17, 2015
May 17. Day 137. Paw use of a Sunday
If you go down to South Bank today, you're in for a big surprise. If you go down to South Bank today you'd better go in disguise. For every dog that ever there was. Was gathered there simply because. Today's the day the puppy dogs have their walkies. Million paws walk. That's about a million reasons to drag the family out of bed and join just about every other pooch in Brisbane for the annual RSPCA fundraiser. Rumple was wearing a tie and face paint but was otherwise relying on his natural charm and good looks. We were all wearing normal street clothes. Clearly we are amateurs at the Paw Walk thing. Team Rumple probably should have been wearing matching dinosaur onsies or something. But while we were under-dressed we still enjoyed the walk and the delightful autumn weather. The occasional shower was hardly enough to rain on our parade.
Saturday, May 16, 2015
May 16. Day 136. Not happy Jan
Rumple was putting on the sad eyes and threatening to call the RSPCA over the fact that he had to have a bath. I know. That's so unfair.
And in the afternoon I joined a community action meeting at the office of Councillor Helen Abrahams. As someone who spent years in the press box reporting council, I am well aware that change does not come easy. It is not just a case of NIMBY (Not in My Back Yard) but often it seems like the BANANA (BUILD ABSOLUTELY NOTHING ANYWHERE NEAR ANYTHING) principle is at play. So it should not be surprising that residents are up in arms at plans to change traffic arrangements in my area. Yes, it is true that the "what's-in-it-for-me" motivation is at play when I say the proposal on the table is about as stupid at I've ever seen. The ability to turn into our street will be seriously restricted which will be personally massively inconvenient. So I would be voicing my opposition regardless. But the thinking is crazy. Part of the plan is to stop accidents at that intersection. That's a fine ideal but it's based on old traffic statistics. A bus stop at that intersection was moved a number of years back and there hasn't been an accident there since. So they are trying to stop accidents that don't happen. But worse, the plan is to direct turning traffic further down the street - to an intersection where there are accidents. Putting a U-Turn facility there will quite literally be another accident waiting to happen. The people at the meeting were united in their opposition and hopefully the planners will see that.
Friday, May 15, 2015
May 15. Day 135. Duck off
This afternoon the walk with Rumple took us off to the University of Queensland, a venue which Rumple finds packed with possibility. He sees every bird, lizard and turtle as a potential conquest. I'm pretty sure he thinks it is only the lead that comes between him and a duck for dinner. I'm pretty sure he is entirely wrong. Not only is he hampered by the fact that he can't swim all that well and can't fly at all but deep down he's a lover not a fighter. The only way he's going to have a feed of fowl is if someone orders it for him. I love my fur friend but not that much. The ducks, for their part, stayed just out of reach and quietly preened themselves like they were preparing themselves for a big Friday night on the town. But not this little black duck. I was home on the couch by just after dark which after this week was just about perfect.
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Just read a blog post by Tim Ferriss, the author of the 4 hour work week, and was blown away at how active his blog is! I know he gets a ton of traffic due to the fact that he has a best seller out but I was still surprised by this blog post. He had close to 600 people that retweeted this blog post and close to 200 comments. If you scroll through the post you'll see Tim actively responds to people in the comments section. In my comment I ask Tim if it's really him and not one of his many assistants. Let's see if Tim responds to my comment or allows my comment. At one point I heard or read that Tim has over 50 personal assistants.
Good example of an active blog


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WILLIAM T. CASE et al. v.
Judicial District of Litchfield
Zoning; Whether the Commission's Vote Constituted an Approval of a Zoning Application; Whether the Interpretation of the Commission's Vote Should be Resolved Based on the Common Law or on General Statutes § 8-22; Whether the Trial Court Should Defer to the Commission's Interpretation of the Legal Effect of its Vote. The plaintiffs sought approval to resubdivide their property and a special exception to allow the interior lots on the property to be served by a common driveway. After holding public hearings, two commission members voted to approve the application with conditions, and three commission members abstained. Following the vote, the five commission members unanimously agreed that the application had been denied. Thereafter, the commission published a legal notice stating that the commission, by majority vote, had denied the application. The plaintiffs appealed from the commission's denial, arguing that the vote of the commission constituted an approval of the application as a matter of law. Subsequently, the parties reached a settlement agreement, wherein the commission agreed that its vote constituted an approval of the application. The parties then filed a joint motion for judgment in accordance with their settlement agreement. In the meantime, the trial court granted the motion to intervene of several neighboring property owners. The intervenors claimed that because only two of the five members of the commission voted to approve the application, the application had not been approved by a "majority" vote as required by General Statutes § 8-22. The trial court, however, concluded that the commission approved the application under the common-law rule that provides that where a quorum of an agency is present, a majority of the votes actually cast is sufficient even though the number of votes is less than a quorum, provided there are a sufficient number of members present, whether voting or not, to constitute a quorum. The court further held that § 8-22 did not create an exception to the common-law rule because it applied only to the administrative tasks of a planning commission enumerated in the statute itself, and not to the commission's consideration of a resubdivision or special exception application. Accordingly, the court sustained the plaintiffs' appeal, ruling that the commission's vote constituted an approval. On appeal, the intervenors claim that, contrary to the trial court's conclusion, § 8-22 applies to all actions of a planning commission and requires the affirmative vote of three members of a five member commission to approve an application regardless of the number of members actually voting. Alternatively, if § 8-22 is found not to be applicable, they claim that the trial court should have deferred to the commission's own interpretation of its vote, that being that the application had been denied.