The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.
Copyright: © 2016 Appaduray et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Australian National and Medical Research Council (NHMRC) Project Grants APP1026616 (PWG, ECH) and APP1079866 (ECH, AM, PWG), Australian Research Council (ARC) Linkage, Infrastructure, Equipment and Facilities (LIEF) grant LE140100166 (PWG, ECH), and funding from the The Kids' Cancer Project (AM, NSB, PWG, ECH).
Competing interests: PWG is a Director on the Board of Novogen, a company commercialising drugs that are directed against Tpm3.1. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
The actin cytoskeleton is a dynamic meshwork of filaments that are involved in most biological processes. Precise regulation of actin filaments is crucial to maintain the integrity of these processes and involves interactions with various actin-associated and -binding proteins including the tropomyosins (Tpms) . Indeed, most non-muscle or cytoskeletal actin filaments are comprised of polymers of actin and Tpm. Tpms are α-helical coiled coil dimers that form two continuous parallel polymers along the major grooves of the actin polymer and 'bind' to actin via ionic interactions . Over 40 cytoskeletal Tpm isoforms exist in mammalian cells and define the functional capacity of actin filaments by regulating the interaction of actin filaments with actin-binding proteins and myosin motors [1, 3, 4]. Studies have indicated that the formins specify, at least in part, which Tpm isoform is incorporated into an actin filament [5, 6] which suggests that Tpm polymer formation is dependent on actin dynamics.
Distinct actin filament populations have been identified in cultured cells. There are at least four different categories of stress fibers: dorsal, ventral, transverse arcs and the perinuclear cap . In addition, two actin sub-populations have been identified at the cell cortex distinguished by very different turnover and polymerization rates . Analysis of isoform-specific Tpm dynamics associated with stress fibers has been carried out using fluorescence recovery after photobleaching (FRAP) and revealed different recovery rates. When YFP/GFP-tagged Tpms 1.7, 3.1 and 1.9 (previously Tm3, Tm5NM1 and Tm5b, respectively; ) were compared, Tpm3.1 had a higher rate of recovery on actin stress fibers . Of the four Tpm isoforms shown to be essential for stress fiber formation, 1.6 (previously Tm2), 1.7, 3.1 and 4.2 (previously Tm4), Tpm4.2 had a faster recovery than the other 3 isoforms . There is no information about the relationship between actin and Tpm dynamics in filaments of the cytoskeleton; however, recent data on the assembly of pre-myofibrils suggests that there is not an absolute relationship between actin and Tpm turnover . A secondary question is whether the placement of a tag impacts Tpm dynamics since no comparison has been carried out between N and C-terminal tagged Tpm constructs.
In this study we used FRAP to investigate the interrelationship of Tpm and actin dynamics in vitro in cultured cells and in vivo in tissues. In particular, we focused on isoform Tpm3.1 that is known to stabilize actin filaments by reducing depolymerisation  as well as recruiting myosin motors . We used fluorescent protein-tagged Tpm3.1 and actin and examined Tpm3.1 vs actin recovery in dorsal/ventral stress fibers in mouse embryo fibroblasts (MEFs) and in apical/cortical fibers in rat salivary gland acinar cells. Recovery of Tpm3.1 was determined on actin filaments perturbed with the actin-targeting drug jasplakinolide, which promotes actin filament nucleation and stabilization [14, 15]. We also investigated the impact of placing a fluorescent tag at either the N- or C-terminus of Tpm3.1 on the fidelity of its localization and recovery kinetics. Our data is compatible with a continuous dynamic exchange of Tpms occurring on actin filaments that is independent of actin filament dynamics and the location of the tag on Tpm3.1.
MEFs were isolated from day 13.5 C57Bl/6 mouse embryos and cultured as previously described . Cultured cells were maintained in Dulbecco's modified Eagle medium (DMEM) with 10% (v/v) foetal bovine serum (FBS) at 37°C, 5% CO2. For imaging experiments, MEFs at passages 1–3 were seeded into Fluorodish™ tissue culture dishes (World Precision Instruments Pty Ltd) and grown to 70–90% confluency. Cell transfections were performed using Lipofectamine 3000 reagent (Life Technologies) and plasmid DNA according to the manufacturer's instructions. Jasplakinolide (Sapphire Bioscience Pty Ltd) was added at a final concentration of 7 μM from a 1mM stock prepared in DMSO. FRAP analysis was performed within 10 s after addition of the drug.
The sequence encoding mNeonGreen (a gift from Jiwu Wang, ) was inserted at either the N- or C-terminus of human Tpm3.1, separated by a 10 amino acid linker motif (GGGGSGGGGS), and cloned into pcDNA3.1 under control of a CMV promoter (GeneArt, Invitrogen). pCAG-GFP-actin was a gift from Ryohei Yasuda (Addgene plasmid #21948) . Lifeact-RFP was a gift from Roland Wedlich-Soldner .
Transfected wildtype B6 and B6-Tpm3tm2(Δ9d)Pgun MEFs  (mice lack exon 9d of the Tpm3 gene resulting in the knockout of isoforms Tpm3.1 and Tpm3.2) were fixed in 4% PFA at RT for 30 min, permeabilized in ice cold methanol for 30 min, blocked in 2% BSA in PBS at RT for 60 min. The cells were incubated with CG3 in 2% BSA (mouse monoclonal, 1:25 ), which recognizes the 1b exon from the TPM3 gene (1 h, RT) followed by Alexa-647 conjugated donkey anti-mouse secondary antibodies (1:400 in PBS). Cells were washed 3 times with PBS and imaged using a Zeiss 880 confocal using a 63x/1.4 NA objective and sequential excitation with 488 nm and 633 nm lasers. The raw image data was deconvolved using the Airyscan processing algorithm that is included with the Zeiss Zen software package.
Protein was extracted from transfected and non-transfected MEFS in RIPA buffer and analysed by SDS-PAGE and western blotting as described previously . Protein concentration was estimated using Precision Red (Cytoskeleton, Inc). Equal amounts of protein (30 μg) were resolved on a 12% SDS-PAGE gel before electro-transfer to PVDF membranes. Non-specific binding on the blot was blocked with blocking buffer, 1% BSA in TBST (100 mM Tris-Cl, pH 7.5. 150 mM NaCl with 0.05% Tween 20). Tpm 3.1 was recognised using monoclonal ab CG3 (1:200 in blocking buffer)  and secondary antibody rabbit anti mouse Ig-conjugated horseradish peroxidase (Abcam) (1:10,000 in blocking buffer). Primary antibody was incubated overnight and secondary for 2 h with 4 x 15 min washes. Blots were developed with the Western lightening Chemiluminescence reagent (Perkin Elmer Life Sciences; Boston, MA) and exposed to x-ray film. Equal protein loading was examined by staining the protein gel blots with 0.1% (w/v) Coomassie blue R350, 20% (v/v) methanol and 10% (v/v acetic acid).
Live cell imaging was performed on a Nikon A1 inverted scanning confocal microscope fitted with a Nikon Plan Apochromat λ 60x oil immersion objective with an NA of 1.4 and an Okolab incubation chamber equilibrated to 37°C. The mNeonGreen (λem 516 nm) constructs were excited with a 488-nm laser. Transiently transfected cells with a low level of tagged Tpm3.1 expression were chosen for FRAP analysis. For time-lapse imaging, frames were acquired at 516 ms/frame at 256 pixel resolution, 300 nm per pixel, and imaged at 1 Hz. FRAP zones were bleached with a single 120 ms pulse using a 488-nm laser. 3–5 reference frames were acquired per cell, followed by a single bleach pulse, followed by image acquisition at 1 Hz for 120 s.
Animal experiments were performed in accordance with the NSW Animal Research Act (1985) and Australian National Health and Medical Research Council (NHMRC) 'Code' 8th edition (2013). All experiments were approved by the UNSW Australia Animal Care and Ethics Committee under applications 12-120B, 15-103A and 14-92A. Male Wistar rats weighing 150–225 g were obtained from the Animal Resources Centre, Perth, Australia and allowed to acclimatize for 1 week. Rats were anesthetized and salivary glands (SGs) transfected as previously described  with the following modifications: 24 μg of plasmid DNA was mixed with in vivo-jetPEI® (Polyplus Transfection) with 100 μL of 10% w/v glucose according to the manufacturer's instructions.
Rats were anesthetized and salivary glands externalized and prepared for intravital imaging as previously described . Intravital imaging was performed on a Nikon A1 inverted laser scanning confocal microscope fitted with a Plan Apochromat WI DIC N2 60x water objective with an NA of 1.27, an Okolab incubation chamber and a custom made stage insert. mNeonGreen constructs were excited with a 488 nm laser. For time-lapse imaging, frames were acquired at 477 ms/frame at 256 pixel resolution, 100 nm per pixel, and imaged at 1 Hz. FRAP zones were bleached with a single 120 ms pulse using a 488 nm laser. 3–5 reference frames were imaged per cell, followed by a single bleach pulse at a defined ROI, followed by acquisition at 1 Hz for 120 s.
Salivary glands were dissected from adult male C57Bl/6 mice, immersed in Tissue-Tek and frozen in isopentane cooled in liquid nitrogen. 10 μm sections were cut using a Cryostat (Leica CM1950) at -20°C, fixed for 30 min in 1% PFA in PBS at 4°C, treated for 5 min with MeOH at -20°C, and blocked with 5% goat serum, 5% FBS, 1% BSA in PBS for 2 h, RT. Mouse mAb 2G10 against Tpm3.1/3.2 (1:200 in blocking buffer)  was added for 12 h, RT, and then secondary antibody conjugated with AlexaFluor488 (mouse; Invitrogen) (1:750 in blocking buffer) for 1 h, RT. Images were taken with an Olympus FV1000 confocal microscope. Transfected salivary glands were excised, immersed in 50 μg/mL 10kDa dextran-Alexa647 conjugate (Invitrogen) for 10 min, placed on a glass coverslip and the surface imaged immediately as for intravital imaging.
Images were taken using NIS Elements software. Image processing and data extraction was performed using ImageJ (v1.47n). Data processing and analyses were performed using Microsoft Office Excel. All statistical p values were obtained from unpaired t-tests using Graphpad Prism 6. FRAP curves were normalized to the minimum and maximum fluorescence values using a value range of 0 (minimum fluorescence) to 1 (maximum fluorescence). Data from normalized FRAP curves were then fitted with a double exponential equation using the IgorPRO6 software complemented with the K_FRAPcalc version 9 procedure (Kota Miura, EMBL-Heidelberg, Germany) to derive half-times and mobile fractions. Fits obtained using either single or double exponential FRAP recovery model were compared using the extra sum-of-squares F test in Graphpad Prism 6 to determine whether the double exponential recovery provided a statistically significant improvement in the quality of the fit, accounting for the extra degrees of freedom in the more complex model. 95% confidence intervals on the fitted parameters were estimated using Graphpad Prism 6 using the delta method.
Radially averaged spatial recovery profiles were obtained using a custom MATLAB (Mathworks) script by computing the average intensity at increasing distances from the centre of the bleached region and normalising to the pre-bleach intensity. The spatial profile of the recovery was fitted to a Gaussian model as previously described to determine the width of the bleached region [24, 25].
N-terminal (N-Tpm3.1)- and C-terminal (C-Tpm3.1)-tagged Tpm3.1 were transfected into wild type and Tpm3.1/3.2 knockout MEFs and the tagged constructs localized predominantly to stress fibers in both cell types (Fig 1A–1D). These cells were co-stained with the CG3 antibody that detects all isoforms expressed from the TPM3 gene as well as both N-Tpm3.1 and C-Tpm3.1 (Fig 1A–1E). The CG3 antibody detects stress fibers in both untransfected (arrowheads Fig 1A) and transfected wild type MEFs (arrows Fig 1A). These stress fibers co-localize with the tagged proteins, however there are regions, particularly at the ends of stress fibers and regions of high tag density that do not co-localize with the antibody staining. This is not due to a failure of the antibody to recognise the tagged Tpm3.1 based on both Western blot results (Fig 1E) and detection of the transfected tagged proteins in Tpm3.1/3.2 knock out cells (Fig 1C and 1D). We hypothesize that there may be steric hindrance of the antibody epitope in these regions and as a result of this only the central region of stress fibers was analysed in further experiments.
Fig 1. N- and C-terminal tagged Tpm3.1 both localize to stress fibers in mouse embryo fibroblasts.
Tagged Tpm3.1 constructs were transfected into wild type and Tpm3.1/3.2 knockout mouse embryo fibroblasts (MEFs) and visualized by confocal microscopy (A and C) N-terminal tagged Tpm3.1 and (B and D) C-terminal Tpm3.1. Tpm3.1 was visualized using the CG3 antibody that recognises all isoforms from the TPM3 gene. (E) Western blot showing expression of the tagged Tpm3.1 constructs and endogenous Tpm 3.1 in primary wild type MEFs as detected by the CG3 antibody. Scale bar = 10 μm.
This result indicates that both types of tagged Tpm3.1 proteins have very similar localization patterns to each other that is surprising because there is extensive evidence that the N-termini of Tpms are crucial for Tpm function. From studies on muscle Tpms, it is known that the N-terminal residues are highly conserved [26, 27] and that acetylation of a methionine is required for normal function , regulation of actomyosin ATPase with troponin  and Tpm dimer formation . Mammalian cytoskeletal Tpms in contrast do not require acetylation to bind actin . In yeast, tagging the N-terminus of Tpm prevents acetylation of the N-terminal methionine causing the tagged protein to mis-localize  and recent studies indicate that care must be taken in interpreting functional outcomes using tagged Tpms . Nevertheless, expression of transfected N-terminally tagged Tpm3.1 shows biological activity in a cell motility assay in MEFs, but proving biological equivalence of tagged and untagged Tpm3.1 has not been established . In contrast, the C-terminus has a more variable amino acid sequence and a more flexible structure [34, 35], thus tagging the C-terminus of Tpm3.1 is expected to cause less perturbation to the normal activity of the protein, however in this experiment we observed localisation of both constructs to stress fibers.
We elected to use FRAP assay to assess the dynamics of recruitment of Tpm3.1 into actin filament bundles in relation to actin dynamics using constructs tagged at either the N- or C-termini. By photobleaching zones containing stress fibers in the interior of the cell and monitoring the fluorescence recovery we are able to characterise the kinetics of Tpm3.1 on a cell-by-cell basis (Fig 2A and 2B; S1 Table). In a typical FRAP experiment, the fluorescence will recover due to movement of unbleached molecules into the bleached zone and eventually reach a plateau. The level of the plateau provides information about the fraction of molecules that are mobile in the bleached zone (the 'mobile fraction') while the shape of the recovery provides information about the number and rates of the dynamic processes leading to recovery, for example diffusion and exchange with a cytoplasmic pool. To determine the number of recovery processes present, we fitted recovery curves from both N- and C-terminal tagged constructs to a single- and double-exponential FRAP recovery model. We found that the double exponential model provided a statistically significant improvement in the quality of the fit compared the single exponential model. The C-terminal fit R2 values were calculated at 0.9835 for the single exponential fit compared with 0.999 for the double exponential fit (p<0.0001) with the N-terminal R2 values calculated at 0.9860 and 0.9995 for the single and double exponential fits respectively (p<0.0001), suggesting there are two dominant processes contributing to the recovery. We hypothesised that there are three candidate processes potentially contributing to the recovery: (1) diffusion of unbound tropomyosin molecules in the cytoplasm, (2) exchange of tagged- for untagged-Tpm3.1 on actin filaments which are exposed to the cytoplasm and (3) relatively slower exchange of tagged- for untagged-Tpm3.1 on actin filaments located in the interior of stress fiber filament bundles which are not in direct contact with cytoplasm. To determine whether the fast recovery component observed was associated with diffusion or exchange of Tpm3.1 between actin filaments and the cytoplasm we examined in more detail the spatial profile of the recovery process. These processes will produce distinct spatial recovery profiles as illustrated schematically in S1A and S1B Fig. Recovery due to diffusion will show an increase in the width of the bleached region over time as bleached and unbleached molecules diffuse, while the width of the bleached region will remain unchanged in an exchange process [24, 25]. We computed radially averaged spatial recovery profile by averaging over the bleached regions of a number of N- and C-terminal tagged Tpm3.1 transfected cells, shown in S1C and S1D Fig. In both cases the spatial recovery profile is consistent with an exchange-based recovery. In line with this, we calculated the width of the bleached region over time in both cases by fitting to a Gaussian profile and found no increase in the width over time. We therefore concluded that diffusion does not contribute significantly to the observed recovery and the two recovery processes are associated with exchange of Tpm3.1 between cytoplasm exposed and shielded filament bundles, respectively. This conclusion is supported by visual inspection of the movies and micrographs (Fig 2A and 2B); association of tagged-Tpm3.1 with filament bundles starts almost immediately after bleaching. We hypothesise that the relatively slower recovery process is due to packing of filaments in the interior of stress fibers leading to potential steric impediments to complete exchange, independent of filament turnover.
Fig 2. N- and C-terminal tagged Tpm3.1 constructs have similar mobile fractions but dissimilar recovery rates.
(A,B) Representative images of FRAP assay in MEFs transfected with either N- or C-Tpm3.1. FRAP zones (white arrows) were bleached and cells imaged at 1 fps for 2 min. (inset A,B). Enlarged images of FRAP zones over time (s). (C,D) FRAP curves of N- or C-Tpm3.1 transfected MEFs. (E) Half-times of N- and C-Tpm3.1 recovery (see also S1 Table). Data obtained from 6 experiments, 3–15 cells per experiment. Error bars are +/- SEM. Scale bars = 10 μm.
Initial preliminary data using cells transfected with Tpm3.1-YFP were acquired for 5 min, but no stable plateau was observed. This is due to the inevitable movement of the cells during imaging (typically at 1.5 to 2 min), resulting in unbleached structures being imported into the FRAP zone with time. This is illustrated in S1 Movie, showing movement of the stress fibers through the FRAP zone after 2 min as well as the stress fiber moving in and out of the focal plane making analysis after 2 min unreliable. Therefore, to minimise motion artefacts in the FRAP recovery data, all photobleaching experiments were performed for 2 min post-bleach and fitted with a double exponential recovery model to determine the mobile fraction and recovery half-times. Although the recovery is not fully complete at 2 min, we found that the data recorded up to this point was able to sufficiently constrain the model and so obtain an accurate estimate of the mobile fraction; for N- and C- terminal tagged Tpm3.1 the spread of the 95% confidence interval on the estimated mobile fraction ranged from ±1% to ±5%.
Recovery curves for C-Tpm3.1 and N-Tpm3.1 showed identical mobile fractions of 66% in MEFs (Fig 2C and 2D), and no statistical difference between the rate of fast exchange or relative contributions of slow and fast exchange, indicating that the fast exchange process is not affected by the tag location. N-Tpm3.1, however, exhibited a significantly longer slow recovery half-time than C-Tpm3.1 (64.6±14.9s vs 40.2±3.2s, p = 0.017), indicating that this slower exchange process is inhibited by the N-terminal tag. The slower recovery half-time observed for N-Tpm3.1 (Fig 2E) is consistent with the finding that muscle Tpm with an 80 residue N-terminal fusion peptide binds with an affinity slightly greater than a non-fusion variant and many-fold greater than unacetylated Tpm . The fusion Tpm failed to be regulated by Troponin T and did not regulate the actomyosin Mg ATPase. Inserting the tag in the N-terminus could potentially alter normal Tpm regulation and binding through steric hindrance thus reducing the rate of N-Tpm3.1 incorporation into actin filaments. For these reasons we selected C-Tpm3.1 as construct of choice for subsequent cellular experiments.
To confirm these observations in vivo, we applied FRAP analysis to transfected salivary gland acinar cells in live rats using intracellular intravital microscopy. We chose the rat salivary gland as our in vivo model because of its tractability for intravital imaging and genetic manipulation . Salivary acinar cells express endogenous Tpm3.1 that is highly enriched at the apical membranes that are arranged into canaliculi (Fig 3A, white arrow). Therefore, intravital FRAP assay was carried out on the apical regions of transfected cells (Fig 3B and 3C). Both N- and C-terminally tagged proteins were localized at the apical membranes of acinar cells (Fig 3D and 3E, white arrows). FRAP recovery kinetics showed similar trends to that seen in cultured MEFs although C-Tpm3.1 has a significantly higher mobile fraction (66%) compared to N-Tpm3.1 (55%) (Fig 3F, 3G, 3I and 3J) (S2 Table). Therefore, the tagged proteins display similar but distinct activities in vitro and in vivo, in quite dissimilar actin filament structures–stress fibers vs apical filament bundles. As observed with MEFs, the half-life of recovery of C-Tpm3.1 was half of that seen with N-Tpm3.1 (Fig 3H).
Fig 3. Intracellular intravital imaging of the kinetics of N- and C-terminal tagged Tpm3.1 constructs transfected into rat salivary gland acinar cells.
(A) Confocal image of an acinus from rat submandibular salivary gland section stained with anti-Tpm3.1 (2G10) antibody. Tpm3.1 is enriched on the apical plasma membranes that form the canaliculi of acinar cells (white arrow). (B) Confocal image of a C-Tpm3.1 transfected cell (arrow) in a single acinus of a rat salivary gland in situ. Extracellular space outside the acinus was stained with 10kDa dextran Alexa 647 conjugate. (C) Illustration of the transfected acinar cell in (B), arrow shows apical membrane/canaliculi, arrowhead shows basolateral membrane. (D,E) Intravital microscopy and FRAP analysis of N- and C-Tpm3.1 constructs in live transfected rats. Numbers indicate time in sec. White arrows indicate FRAP zones on the canaliculi of rat acinar cells. (F) FRAP curves for N- and C-Tpm3.1. (G) Mobile fraction of N- and C-Tpm3.1. (H) Half-times for N- and C-Tpm3.1. (I, J) Curve fits for N- and C-Tpm3.1. 11–16 cells assayed from at least 3 animals per construct (see also S2 Table). Error bars are +/- SEM. Scale bars = 5 μm.
Comparison of recovery curves for the C-terminal tagged Tpm3.1 with Lifeact in MEFs indicates that Tpm3.1 has a high mobile fraction although not as high as Lifeact (Figs 2D and 4C). Because Lifeact engages in rapid exchange binding to actin filaments the mobile fraction is over 90% and has a very short half-time (S3 Table). However, the fact that Tpm3.1 also has a short half-time and a high mobile fraction both in MEFs and in acinar cells in vivo suggests that most of the Tpm3.1 is engaging in rapid exchange with a soluble pool. In contrast, GFP-actin shows a relatively slow recovery curve (Fig 4A and 4E) (S4 Table) that raised the possibility that Tpm3.1 may be exchanging independent of actin filament turnover.
Fig 4. The majority of actin in stress fibers is stable.
(A) Representative image and FRAP sequence of MEFs transfected with GFP-beta-actin. FRAP zone indicated by white arrow. Top panel: FRAP sequence of untreated control cells. Bottom panel: FRAP sequence after treatment with 7 μM jasplakinolide. (B) FRAP curves for GFP-actin in control and jasplakinolide treated condition. (C) FRAP curves for Lifeact-RFP in control and jasplakinolide treated condition. (D) Mobile fractions of control and drug treated GFP-actin and Lifeact-RFP (see also S3 and S4 Tables). (E) Curve fits for GFP-Actin control. (F) Curve fit for GFP-Actin treated with jasplakinolide. Data obtained from 3 separate experiments, 2–8 cells per experiment. Error bars are +/- SEM. Scale bars = 5 μm.
Wang et al.  recently showed that C-terminal tagged muscle Tpm exchange is much less sensitive to jasplakinolide than tagged actin in pre-myofibrils suggesting that Tpms can exchange independent of actin. Since our results suggest a similar conclusion we tested this in the MEF cytoskeleton by examining the cytoskeletal Tpm3.1. Our approach was to inhibit actin filament kinetics in MEFs using jasplakinolide to stabilise the actin filaments and measuring Tpm3.1 kinetics in stress fibers using FRAP analysis. Using this strategy, we specifically manipulated the kinetics of actin filaments; therefore, any recovery following photobleaching should reflect Tpm3.1 dynamics independent of actin dynamics. First, we established the conditions under which actin filaments were disrupted with drug treatment. We found that FRAP analysis of MEFs transfected with GFP-beta-actin showed very weak recovery into stress fibers (Fig 4A and 4B) and treatment with 7 μM jasplakinolide eliminated the minimal recovery of fluorescent actin into the FRAP zone (Fig 4A, inset). Although the recovery curve (Fig 4B) shows an ~50% reduction in the mobile fraction (Fig 4B, 4D, 4E and 4F) (S4 Table) this does not reflect true recovery into stress fiber bundles, but rather a recovery in fluorescence of the cytosolic G-actin pool (Fig 4A inset). Thus, treatment with jasplakinolide essentially eliminates the small amount of recovery of actin into stress fibers after photobleaching (Fig 4A).
We then investigated the kinetics of continuous diffusion as opposed to active binding of a tagged construct in our assay using Lifeact-RFP, which has extremely transient binding to actin. MEFs were transfected with Lifeact-RFP and FRAP analysis was performed on stress fiber regions in the presence and absence of 7 μM jasplakinolide. FRAP of Lifeact-RFP results in an almost instantaneous recovery in both control and drug treated conditions (Fig 4C). This is in agreement with the highly diffusive behaviour and transient binding of Lifeact constructs to its target site on actin . Interestingly, a small reduction in the mobile fraction from control to drug treated cells was observed (Fig 4D). This perhaps suggests the existence of a sub-population of Lifeact bound to actin filaments in the interior of stress fibers that cannot as readily exchange as Lifeact at the periphery of stress fibers.
Having determined that treatment with 7 μM jasplakinolide significantly impacts actin turnover on stress fiber structures, we then sought to determine the recruitment kinetics of tagged Tpm3.1. MEF cells were transfected with constructs encoding C-Tpm3.1. Regions of stress fibers were photobleached prior to addition of jasplakinolide to obtain control curves (Fig 5A, inset). Jasplakinolide was then added to cells and the same cells were immediately photobleached at a different site (Fig 5A, inset). Intriguingly, jasplakinolide had a minimal effect on tagged Tpm3.1 recruitment into stress fibers (Fig 5A, inset), where only a small but not significant reduction in the mobile fraction was observed (Fig 5B, 5C, 5D and 5E) (S5 Table). As global actin turnover is significantly inhibited in the presence of the drug (Fig 4A and 4B), we conclude that Tpm3.1 is constantly undergoing dynamic exchange on actin filaments that is independent of actin filament dynamics. This is also apparent from inspection of the images of actin and Tpm3.1 recovery in Figs 4A vs 5A. Since Tpms bind actin through weak ionic interactions [36, 37] it may be logical that Tpm3.1 on filaments undergo changes independent of actin. The small reduction in mobile fractions of tagged Tpm3.1 (Fig 5B and 5C) and Lifeact-RFP (Fig 4C and 4D) in the jasplakinolide-treated conditions perhaps suggests that jasplakinolide is inhibiting the translocation and/or polymerization of a dynamic sub-population of actin filaments that operates in stress fibers, a hypothesis which is in agreement with what has been reported for cortical actin .
Fig 5. Tpm3.1 maintains constant and rapid cycling on stress fibers in the presence of jasplakinolide.
(A) Representative image and FRAP sequence of MEFs transfected with C-Tpm3.1. FRAP zone indicated by white arrow. Top panel: FRAP sequence of untreated control cells. Bottom panel: FRAP sequence after treatment with 7 μM jasplakinolide. (B) FRAP curves of C-Tpm3.1 in control and drug-treated conditions. (C) Mobile fraction of control and drug-treated condition (see also S5 Table). (D,E) Curve fits for C-Tpm3.1 in control (D) and drug-treated condition (E). Data obtained from 3 separate experiments, 3–8 cells per experiment. Error bars are +/- SEM. Scale bars = 10 μm.
In conclusion, our results provide evidence that cytoskeletal Tpm3.1 binding to actin may be independent, at least in part, of actin dynamics. It is possible that the presence of the N- and C-terminal tags on Tpm3.1 impact the observed exchange kinetics in these experiments. It has been demonstrated previously that deletion of a single C-terminal amino acid can abolish Tpm binding to actin in yeast . However, we have shown that expression of N-terminal tagged Tpm3.1 can supress an increase in cell speed induced in Tpm3.1 KO mouse embryo fibroblasts  indicating that the presence of an N-terminal tag does not completely abolish normal Tpm3.1 function. It is also possible that the replacement kinetics we observe primarily reflects the addition of tagged Tpm3.1 to the ends of actin filaments within filament bundles; however, the similar levels of accumulation of the tagged and endogenous Tpm3.1 make this unlikely (Fig 1C). Thus, it appears that Tpm3.1 dimers located within a filament are able to break two head-to-tail overlap interactions with adjacent dimers in the polymer together with their interaction with actin in order to exchange with 'free' Tpm3.1. While each of these interactions is of low affinity [1, 2], it seems likely that a source of energy may be required to weaken these interactions and promote the exchange reaction.
S1 Fig. Spatial recovery of tagged Tpm3.1 is consistent with exchange rather than a diffusive transport process.
(A,B) Simulated spatial recovery profile for (A) diffusion and (B) exchange reaction based recovery showing (top) kymographs of fluorescent intensity and (bottom) spatial profile of recovery at selected timepoints with cartoon illustrating relationship to bleached region. Black arrows indicate change in fluorescent intensity over time. Recovery due to diffusion shows an increase in the width of the bleached zone during the recovery due to motion of bleached and unbleached molecules from the surrounding areas while recovery due to an exchange reaction shows no change in the width of the bleached zone during the recovery. (C,D) Measured radially averaged spatial recovery profiles, for (C) N-Tpm3.1 and (D) C-Tpm3.1 averaged over n = 13 and n = 9 cells, respectively. (E,F) Fitted width of recovery profile over time for (E) N-Tpm3.1 and (F) C-Tpm3.1. Error bars indicate confidence interval on fit.
S1 Movie. Movement of stress fibers within cells.
MEFs transfected with EYFP-Tpm3.1 were imaged every 1 s for 5 min. The region of the cell highlighted with the red circle was bleached at 6 s. Movie is displayed at 50 fps.
S1 Table. Half-times from double-exponential fits of N- and C-Tpm3.1 recovery in transfected MEFs.
S2 Table. Half-times from double-exponential fits of N- and C-Tpm3.1 recovery in transfected rat acinar cells.
S3 Table. Half-times from double-exponential fits of Lifeact-RFP recovery in control and drug-treated conditions.
S4 Table. Half-times from double-exponential fits of GFP-actin recovery in control and drug-treated conditions.
S5 Table. Half-times from double-exponential fits of C-Tpm3.1 recovery in control and drug-treated conditions.
We thank the UNSW BioMedical Imaging Facility under the direction of Renee Whan for help with the microscopy and the UNSW Biological Resource Centre under the direction of Stephen Danon. We also thank Kota Miura for the IgorPro FRAP curve fitting script and Michael Carnell for advice on FRAP analysis.
Conceptualization: MAA AM PWG ECH.
Data curation: NSB SCW JHS PWG ECH.
Formal analysis: MAA AM NSB CAL SCW PWG ECH.
Methodology: MAA AM CAL SCW PT JHS.
Project administration: PT PWG ECH.
Supervision: AM PT PWG ECH.
Writing – original draft: MAA.
Writing – review & editing: AM NSB SCW JHS PWG ECH.
11. Wang J, Fan Y, Dube DK, Sanger JM, Sanger JW. Jasplakinolide reduces actin and tropomyosin dynamics during myofibrillogenesis. Cytoskeleton (Hoboken, NJ). 2014;71(9):513–29. Epub 2014/08/26.
15. Holzinger A. Jasplakinolide: an actin-specific reagent that promotes actin polymerization. Methods in molecular biology (Clifton, NJ). 2009;586:71–87. Epub 2009/09/22.
16. Schevzov G, Vrhovski B, Bryce NS, Elmir S, Qiu MR, O'Neill GM, et al. Tissue-specific tropomyosin isoform composition. The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society. 2005;53(5):557–70. Epub 2005/05/06.
23. Masedunskas A, Sramkova M, Parente L, Weigert R. Intravital microscopy to image membrane trafficking in live rats. Methods in molecular biology (Clifton, NJ). 2013;931:153–67. Epub 2012/10/03.
24. Coscoy S, Waharte F, Gautreau A, Martin M, Louvard D, Mangeat P, et al. Molecular analysis of microscopic ezrin dynamics by two-photon FRAP. Proceedings of the National Academy of Sciences. 2002;99(20):12813–8.


The Bricoleur Experience with Shrine is a 2 day art and creativity workshop, the purpose of which is to impart an understanding of spontaneity, what it means to allow an interactive art piece to be born; to inspire anybody to be an artist, and to express themselves with any medium they choose.
Day One: Scavenge and glean for found materials, possibly on bicycle. Along the way, stories are told and random acts of public art created.
Day Two: Materials from day one are used to create a permanent installation (on the college of other location), such as a mosaic wall.


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Anastrepha soroana är en tvåvingeart som beskrevs av Fernandez och Juan Manuel Rodriguez 1998. Anastrepha soroana ingår i släktet Anastrepha och familjen borrflugor. Inga underarter finns listade i Catalogue of Life.

Källor

Borrflugor
soroana

redpajama_set_name: RedPajamaWikipedia


It coats domes and steeples of secret sites. It decorates the edges of the most precious books. It embellishes statutes paying tribute to figures who have left a mark on history and human imagination. Inserted into idioms, it adds substantial weight to the other words used.
Beyond its function as an ornament and regardless of era or culture, gold assumes a powerful symbolism. While it originates from the depths of the earth, it is associated with the sun and the stars. Its purity captures the light and draws the gaze to what deserves to be admired. Its ubiquity is synonymous with opulence, but a single stroke of gold suffices to evoke the richness of the soul, the spirit, and the culture.
It reflects a hidden treasure, a concrete manifestation of an intangible perfection. When handed on the top step of the podium, it attests to the medallist's superhuman nature. By adding a golden sheen to the collection's technicality, Marie Saint Pierre reawakens the tension between performance and preciousness.


Join your fellow Bobcat alumni as we take over the Xfinity Center, where the men's basketball team takes on the University of Maryland.
Register here. Your $15 registration fee includes a ticket to the game and a $15 credit for concessions at the Xfinity Center.


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Home/News/Head of Iran's Revolutionary Guards warns this is 'last day' of protests
Head of Iran's Revolutionary Guards warns this is 'last day' of protests
The head of Iran's powerful Revolutionary Guards warned protesters that Saturday would be their last day on the streets, a sign that security forces could step up their crackdown on unrest. sweeping across the country.
Iran has been hit by protests since the death of a 22-year-old Kurdish woman Mahsa Amini while in police custody last month, posing one of the boldest challenges to its leadership. clerics since the 1979 revolution.
"Don't take to the streets! Today is the last day of riots," Guards commander Hossein Salami said in some of the toughest language used during the crisis, which Iran's clerical leadership blames to their foreign enemies, including Israel and the United States. .
"This sinister plan is one cherished … in the White House and the Zionist regime," Salami said.
The much-feared Revolutionary Guards, who report directly to Supreme Leader Ayatollah Ali Khamenei, have not been deployed since the protests began on September 16. They are a elite force with a record of suppressing dissent.
VIEW | Iran protests gain strength 41 days after Mahsa Amini's death:
Iran protests gain strength 41 days after Mahsa Amini's death
More than 40 days after the death of 22-year-old Mahsa Amini in Iranian police custody, the protests continue to gain strength.
Human rights groups say at least 250 protesters have been killed and thousands arrested across Iran in protests, which have turned into a popular uprising by angry Iranians from all walks of life. society.
On Friday, video on social media showed protesters calling for the death of Khamenei and the Basij militia, which played a key role in the effort to defuse the protests.
The Intelligence Ministry and the intelligence branch of the Revolutionary Guards have accused spy agencies from the US, UK, Israel and Saudi Arabia of orchestrating the situation to destabilize the Islamic Republic.
Salami, who was speaking at the funeral of the victims of an attack claimed by the Islamic State group in Iraq and Syria (ISIS) this week, reiterated that message in a speech. directly with the protesters.
"Don't sell your honor to America and don't slap the security forces protecting you in the face," he said.
Video games can improve children's brains: study
2024 Maserati Granturismo Convertible, 2023 Honda Civic Type R: This week's top photos
Fresh rail strike called for 27 July in row over 'paltry' pay offer | Business News
Indian Premier League 2022, Punjab Kings vs Lucknow Super Giants: Mohsin Khan, Krunal Pandya Star As LSG Down PBKS after 20 innings
Best sale to shop today: Macy's, Casper, SkinStore and more
Convoy organizer Tamara Lich again granted bail
UP schools closed in THESE districts due to cold wave

redpajama_set_name: RedPajamaCommonCrawl


Thieves barge into a house from a backdoor with the intention to rob and loot money. Backdoor.ZAccess.yib here is a kind of thief in itself. It makes a PC in which it exists vulnerable and slow so that it could get in the hands of the cyber criminals. Backdoor Trojans are used by cyber thugs from a long time and this infection is a new addition to the whole family. It is contagious and spreads like in snap of a finger. Although it is not like a virus which replicates to grow, instead it downloads several exe files and items such as malware, viruses, worms, Trojans to exploit a system. It aims and targets only a PC having Windows operating system.
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Iran's chief of staff arrives in Syria, announces joint campaign against 'Zionist enemy'
By World Tribune on October 18, 2017
by WorldTribune Staff, October 18, 2017
Iran's chief of staff, during a visit to Syria on Oct. 17, pledged to increase cooperation with the regime of President Bashar Assad to confront the "Zionist enemy."
Gen. Mohammad Baqeri. / Reuters
"We arrived in Damascus in order to coordinate and cooperate to confront our common enemies: the Zionists and the terrorists," Gen. Mohammad Baqeri told Syria's state television channel.
Baqeri's visit came a day after Israeli warplanes bombed a Syrian surface-to-air missile battery.
Israeli military officials reported on Oct. 16 that the battery had fired at Israeli planes over Lebanon. The Syrian army claimed it hit an Israeli warplane after it breached its airspace at the Syria-Lebanon border.
It is "not acceptable for the Zionist regime to violate Syria anytime it wants," Baqeri said.
Baqeri arrived in Syria on Oct. 17 accompanied by several other senior Iranian military figures. Among others, he is expected to meet with Syria's defense minister.
A report in August revealed that Iran has been building a Scud missile factory in Syria near the city of Baniyas, south of Latakia.
Israeli Prime Minister Benjamin Netanyahu has said that Iran was strengthening its foothold in Syria and that Israel would "do whatever it takes" to protect its security.
announces joint campaign against 'Zionist enemy', Bashar Assad, Benjamin Netanyahu, Gen. Mohammad Baqeri, Iran's chief of staff arrives in Syria, WorldTribune.com
Iran's chief of staff arrives in Syria, announces joint campaign against 'Zionist enemy' added by World Tribune on October 18, 2017


Sigma filed a patent P2018-101132A for anti-reflection lens coating using mesoporous silica similar to Nikon's Nano Crystal Coat and Canon's "Air Sphere Coating".
This entry was posted in Sigma and tagged Patents. Bookmark the permalink. Trackbacks are closed, but you can post a comment.


The holidays and holiday related events will be here before you know it, and this dress is the perfect staple for your fall and winter wardrobe! We love the 3/4 length sleeves and flattering fit! Pair it with heeled booties and pretty jewelry. It's great for any occasion, whether it's for a romantic evening or a holiday party!


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Tag Archives: Theo Kriel
Theo Kriel's Sentence May Be Appealed By Solicitor General – Updated x2
(scroll to bottom for update) The following notification appeared in the Northern Advocate, 11 April 2010 "The Solicitor General is considering whether to appeal the 11-and-a-half year jail term handed down to a Kerikeri teenager convicted of murdering schoolgirl Liberty Templeman. Whangarei Crown prosecutor Mike Smith sent a request to Wellington asking for a possible…
April 16, 2010 in Immigration, Kerikeri, murder, New Zealand, NZ not as safe as you think, Theo Kriel, Violence on the Streets, Violent Crime, Whangarei.
Liberty Templeman's Grandmother Mother Speaks Out
Liberty Templeman's grandmother Monica Hempstead has spoken about her loss and the reasons why her family have started a petition to change the sentencing guidelines in New Zealand to make the punishment fit the crime. We talked about the petition to seek justice for Liberty a few days ago, see here: Justice for Liberty Templeman.…
April 4, 2010 in Libby Templeman, Theo Kriel.
Templeman Parents Disgusted At Kriel's Luxury 'Lodgings'
Clio Francis of the Sunday Star Times has written a feature on the treatment of teenage murderer Theo Kriel who was recently sentenced to life imprisonment for the killing of British schoolgirl Liberty Templeman in the small Northland town of Kerikeri (see posts tagged Libby Templeman) and of how he was living in a house…
April 4, 2010 in Libby Templeman, New Zealand, NZ not as safe as you think, Theo Kriel.
Justice For Liberty Rose Templeman
An petition has been started on Petitiononline.com calling for justice for Liberty Rose Templeman, the 15 year old schoolgirl who was brutally murdered and sexually assaulted in Kerikeri, New Zealand on 1 November 2008. Her attacker, a youth who was 14 at the time of her death, was sentenced to life imprisonment and must serve…
March 28, 2010 in Crime, Culture of brutality., Kerikeri, Libby Templeman, murder, New Zealand, Theo Kriel.
Templeman Parents Call For Revision of Immigration Laws, Sentence "A Mere Token"
The mother of murdered school girl Liberty Templeman has spoken about Theo Kriel the youth who killed her daughter. She has been quoted as saying the immigration laws of New Zealand should be revised: "Migrants like the Kriel family need to meet very stringent requirements before being considered" 3 News quotes her as adding: ""Send…
March 23, 2010 in Kerikeri, Theo Kriel.


Eva Stone is a teaching artist, choreographer, and founder/producer of CHOP SHOP: Bodies of Work. She received her BFA in Performance & Choreography from Arizona State University, and later earned an MA in Choreography & Choreological Studies from Trinity Laban. She's created work for Spectrum Dance Theater, Pacific Northwest Ballet/Seattle Art Museum's Sculptured Dance, Seattle International Dance Festival, Bellingham Repertory Dance, and collaboratively with Seattle Dance Project for Project Orpheus, among others, and presented work in New York, Los Angeles, London, Geneva, Montreal, and St. Petersburg. In September 2018 she launched a nationally recognized choreography program "New Voices: Choreography and Process for Young Women in Dance" at Pacific Northwest Ballet School. CHOP SHOP: Bodies of Work brings outstanding contemporary dance companies together for a unique series of performances, lectures, and master classes annually in Bellevue, WA. She is currently based in Seattle.
Lizzy Melton is a former professional dancer and current arts administrator. Lizzy earned a BA in Art History from the American University of Paris and, in addition to her work with CHOP SHOP, serves as Panel Coordinator for the City of Seattle's Office of Arts & Culture and as Company Manager for Spectrum Dance Theater. Lizzy assists with curation of the festival's artists, oversees festival marketing and outreach, leads fundraising campaigns, and provides administrative support to the festival producer and artists. The 2019 festival will mark Lizzy's 9th year supporting CHOP SHOP.
Banner Photo: Courtney Mazeika and Marlie Couto performing their duet eventually at CHOP SHOP 2018, photo by Bret Doss.


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Q: sum of rational and irrational numbers with dedekind cuts Can someone please help me to prove what is  the sum of two rational numbers (is rational obviously but why) with dedekind cuts, what is the sum of rational and an irrational and the sum of two irrational numbers also with the definition of dedekinds cuts?
I give the definition of cut dedekind
(a) Cut $A\neq \emptyset$ and $A\neq\Bbb Q$.
(b) If $x\in A$ and $y \in\Bbb Q$ and $y<x$, then $y\in A$.
(c) $A$ contains no largest number, that is if $x \in A$ then there is $y\in A$ so that $x<y$

A: I'll show that the sum of two rational cuts is rational. Suppose that $A$ and $B$ are rational cuts; then there are $a,b\in\Bbb Q$ such that $A=\{q\in\Bbb Q:q<a\}$ and $B=\{q\in\Bbb Q:q<b\}$. By definition $$A+B=\{p+q:p\in A\text{ and }q\in B\}\;;$$ I'll prove that $A+B=\{q\in\Bbb Q:q<a+b\}$, the Dedekind cut corresponding to the rational number $a+b$.

Suppose that $r\in A+B$. Then there are $p\in A$ and $q\in B$ such that $r=p+q$. Since $p\in A$, we know that $p<a$, and similarly, since $q\in B$, we know that $q<b$, so $r=p+q<a+b$. This shows that $A+B\subseteq\{q\in\Bbb Q:q<a+b\}$.
Now suppose that $r\in\Bbb Q$ and $r<a+b$. Let $d=\frac12(a+b-r)>0$, and note that $d$ is rational. Let $p=a-d$ and $q=b-d$; then $p,q\in\Bbb Q$. Moreover $p<a$ (since $d>0$), so $p\in A$, and $q<b$, so $q\in B$, and $$p+q=(a-d)+(b-d)=a+b-2d=a+b-(a+b-r)=r\;.$$ Thus, $r\in A+B$, and we've shown that $\{q\in\Bbb q:q<a+b\}\subseteq A+B$. Putting the pieces together, we conclude that $A+B=\{q\in\Bbb Q:q<a+b\}$. $\dashv$

Here I didn't have to prove directly that $A+B$ was a Dedekind cut, because I could show that it was equal to something known to be a Dedekind cut. You should try to prove that for all Dedekind cuts $A$ and $B$, $A+B=\{p+q:p\in A\text{ and }q\in B\}$ is a Dedekind cut.

*

*It's easy to show that $A+B\ne\varnothing$.

*Suppose that $r\in A+B$, $s\in\Bbb Q$, and $s\le r$; you need to show that $s\in A+B$. You know that $r=p+q$ for some $p\in A$ and $q\in B$. Let $d=\frac12(r-s)$, and consider the rational numbers $p-d$ and $q-d$.

*Suppose that $r\in A+B$; you need to show that there is an $s\in A+B$ such that $r<s$. Start by writing $r=p+q$ for some $p\in A$ and $q\in B$, and use the fact that $A$ and $B$ have no largest elements.

Once you've done that, you can show that if $A$ is a rational cut and $B$ is an irrational cut, then $A+B$ is irrational. You already know that $A+B$ is a Dedekind cut, so you just have to show that it's not a rational Dedekind cut: for each $r\in\Bbb Q$, $A+B\ne\{q\in\Bbb Q:q<r\}$. This can be done by contradiction: show that if $A+B=\{q\in\Bbb Q:q<r\}$ for some $r\in\Bbb Q$, then the cut $B$ is rational.
When both $A$ and $B$ are irrational cuts, $A+B$ can be either rational or irrational, depending on exactly which cuts $A$ and $B$ are; you can't prove any general conclusion here.

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Book Blitz: It Started With a Whisper by Shannon McCrimmon
It Started With a Whisper
by Shannon McCrimmon
Genre: YA Contemporary
Release Date: June 1st 2016
Five simple words: That's all it takes to change the course of 18-year-old Josie Graham's life in the summer of 1989.
Josie is a musical prodigy: She can sing, play guitar and is a natural on the piano. Instead of spending her last summer before
college traveling the country with her rock star father, she's made a last minute decision to spend it working at her Aunt Bernie's inn, in Ambler's Fork, North Carolina. But what could have turned her life-long passion for music into a hatred for an industry she's worked so hard to get into?
Her aunt's inn seems like the perfect place to escape, to clear her head and figure things out, but on her first day there, she almost drowns before Chic Hobbs saves her.
Chic wants nothing more in life than to leave Ambler's Fork and his sordid past behind—at least not until that day Josie Graham swims into his life. The problem is, Chic's got a secret. It's a secret he's keeping from Josie, and he's worried if it gets out, it'll ruin everything, and she'll judge him for his past mistakes the way everyone else in town has.
Josie is carrying a burden of her own. One that made her run to Ambler's Fork - away from her family and everything she's ever known.
Chic saved Josie once. Can he save her again? Or will Josie rescue him this time?
Shannon McCrimmon was born and raised in Central Florida. She attended Rollins College and earned a Master's Degree in Counseling. In 2008, she moved to the upstate of South Carolina. It was the move that inspired her to write her first novel. She currently lives in Greenville, South Carolina with her husband and toy poodle. She'd love to hear from you! Connect with her at shannonmccrimmon.com
Release Day BLITZ! The Little Antique Shop Under t...
Review: With Malice by Eileen Cook
Release Day Blitz: The Darkest Lie by Pintip Dunn
Release Day Review: Beauty and the Highland Beast ...
Blog Tour: Review & Excerpt: Beautiful by Ella Bor...
Cover Reveal & Giveaway: Lavender in Bloom by Lily...
Blog Tour: Love, or Nearest Offer by Adèle Geras
Book Blitz: It Started With a Whisper by Shannon M...


alti2d wrote: Sorry if you've already mentioned it, but where did you source the LEDs from, the 1156's in particular? Was it an easy transaction? Thanks, it's all looking good!!
It is for sale though...since we are moving to the US at the end of the year and can't take it will us.
How did you remedy that??
Thread is still holding up after like 7 years!
Too bad all the referenced posts are all broken. :( Mostly on page 1 and 2 (curtains, seafoam purge).


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The Devil and the Madonna (German: Der Teufel und die Madonna) is a 1919 German silent drama film directed by Carl Boese and starring Gertrude Welcker, Magnus Stifter and Reinhold Schünzel.

The film's sets were designed by the art director Hans Dreier.

Cast
 Magnus Stifter as Doktor Canzarro / Teufel
 Gertrude Welcker as Tochter Gesina Tradler
 Ernst Pittschau as Graf Jervis
 Emil Rameau as Meister Tradler
 Reinhold Schünzel

References

Bibliography
 Bock, Hans-Michael & Bergfelder, Tim. The Concise CineGraph. Encyclopedia of German Cinema. Berghahn Books, 2009.

External links

1919 films
Films of the Weimar Republic
German silent feature films
Films directed by Carl Boese
German black-and-white films
German drama films
1919 drama films
Silent drama films
1910s German films


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Australiaen
Australia Today
The VICE Guide To Right Now
Women Get Bored in Bed Faster Than Men
Recent studies suggest women grow disinterested in sex with a long-term sexual partner faster than men.
by Marie Boule
Photo Luis Diaz Devesa/ Getty Images
This article originally appeared on VICE Quebec.
A growing body of research suggests that keeping sex exciting is an issue for women, especially in a years-long relationship. Contrary to what the distracted boyfriend meme might have you believe, women grow disinterested in sex with a long-term sexual partner faster than men, and report lower sexual satisfaction.
The Atlantic compiled some of the recent studies on this phenomenon in a piece titled "The Bored Sex." The article mentions an English study from 2017 which shows that women have less interest in sex when they have been in together for more than a year, and women who live with their partner tend to feel less sexual desire than women who don't. Another study published in 2012 tells us that the longer a relationship lasts, the more women's sexual desire decreases, which is not the case for men. And finally, the results of a 2006 German study show that while 60 percent of women want to have "frequent" sex at the beginning of a relationship, in the four years that follow this figure drops to less than 50 percent, and after 20 years of relationship, it falls to about 20 percent. The libido of men, on the other hand, generally remains stable throughout the duration of a relationship.
"It confirms what I've seen in my patients for years," François Renaud, a sexologist and psychotherapist specializing in questions of desire, told VICE Quebec when asked about the phenomenon.
"Women have a tendency to get bored faster than men in a monogamous relationship," Renaud said. "They want a form of sexual variety, while men are often content with very little. It is often women who will lose their desire first, and sometimes even quickly enough."
Among couples who come to see him, the sexologist says that women often think that their spouse would have trouble accepting that they are "more sexual" than their partners. "To protect a little bit of the male ego and the role of the man who teaches sexuality to women, women curl up a little in a modest and conservative position," said Renaud. The couple's sex life then continues as usual, growing comfortable, redundant and predictable.
Renaud sees this as a product of how society views and understands female sexuality. "For a long time, they have been categorized as the kind that wants less sex, but, in truth, women's sexuality is repressed and women are taught to quash their sexuality, slut shaming is a good example. "
The sexologist noted that one mistake couples make is to think that frequent sexuality proof of satisfaction, as if just doing the activity was a guarantee of success in itself, and as if bad sex in heterosexual monogamous couple doesn't exist. There is also the question of the pressure that the partners feel. The idea that it's better to turn away from a partner rather than face rejection: "The first time you have sex with your partner, there is always anxiety and a fear of rejection. At some point, we become comfortable in the relationship and we do not want to live this insecurity any more. The sex therapist works with couples to try to maintain that stability and security while creating a sense of exploration with their partner."
"Often, when couples tell me about their sexuality, I tell them, 'I feel like you are masturbating in each other,' and often women will answer me: 'Absolutely.' That's what we do. We used to have an orgasm, but there is no sexual relationship."
A better idea, according to the Renaud, is to bring as much variety as possible into a relationship so that the sexual activity remains interesting. To make his point, he compares a couple's sex life to a water park: "If you always do the same slides, you end up bored."
So, happy sliding to all, and don't forget to change it up a bit.
Marie Boule is on Twitter.
This article originally appeared on VICE FRCA.


The Stevenson University athletic training staff works closely with members of the sports medicine team to provide the best injury prevention, evaluation, treatment, rehabilitation and appropriate education to all varsity student-athletes competing in intercollegiate athletics while attending Stevenson University.
The athletic training staff is housed in the newly renovated athletic training room located in the Caves Sports & Wellness Center on the Owings Mills campus. The Caves athletic training room services all 27 of the University's intercollegiate sports.


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HomeInsurance Stories: Black History Month
Insurance Stories: Black History Month
4th October 2021 5th October 2021 by HFG_user Posted in Uncategorised
As part of Black History Month we take a look at some fascinating insurance stories put together by HFG's Paul Miller (History Ambassador for the Insurance Museum) and The ACIN (African-Caribbean Insurance Network).
Covering an array of topics and a variety of influential black people, the stories show the role the insurance market played throughout their lifetimes and how reflecting on both the positives and negatives of the past can help inspire a brighter, more diverse future.
Usain Bolt is widely considered to be the greatest sprinter of all time and is a world record holder in the 100 metres, 200 metres and 4 × 100 metres relay. His achievements as a sprinter earned him the nickname "Lightning Bolt", and his awards include the IAAF World Athlete of the Year, Track & Field Athlete of the Year, BBC Overseas Sports Personality of the Year (three times) and Laureus World Sportsman of the Year (four times). When he competed at the 2012 London Olympics, a policy was taken out at Lloyd's of London insuring him against the risk of injury. In case you were wondering, the legs of the fastest man on earth were insured at Lloyd's for $200 million.
Tangy C. Morgan
Tangy C. Morgan has more than 25 years' experience in the US, Bermuda, London and Lloyd's markets. She is a senior adviser in the PRA directorate of the Bank of England and a senior advisor of Strategia Worldwide. She is also a trustee of the Ethnic Minority Foundation in the UK. Morgan was the first African-American to work at Lloyd's, a time described as 'one of the highlights of my career to date'. In her role, she was directly responsible for 42 Lloyd's syndicates, representing 50% of Lloyd's US$42 billion market, and voted on the yearly approval of all syndicates in the market. When she took this role on, she had not worked in the Lloyd's market before. Of the challenge, she said: "In the words of Robert Frost, 'two roads diverged… and I took the one less travelled by, and that has made all the difference'."
Pelé is widely regarded as the greatest footballer of all time and was, for a period, the highest paid athlete in the world. After coming to prominence in the 1958 World Cup finals, the teams he played for began taking out insurance policies on him. For example, he was insured against injury at the 1966 World Cup for £250,000. Each player had been valued based on their importance to the team and Pelé was not only the highest insured player in the Brazil team, but at the tournament too. Perhaps indicating a rise in his fame, his head alone was insured at Lloyd's for $4 million in the 1970s.
In 1998, as sports minister for Brazil, he introduced Pelé Law which aimed to modernise the country's sports industry. Article 45 of the law obliges football clubs to insure all of its players and policies must cover personal and occupational accidents. The law stipulates that the indemnity must correspond to the negotiated annual wage of each player. Pele described the legislation, which was strongly opposed by football clubs, as 'one of the most difficult goals of my life'.
Tina Turner's career has spanned five decades. She is one of the best-selling recording artists in history, having sold more than 100 million records worldwide. She was the first black artist to cover Rolling Stone. A number of insurance policies have been taken out at Lloyd's for Turner, some that support her professionally and others that have been used for publicity purposes. For example, all her tours have been insured against cancellation (her 1995 tour was covered for £3 million), and she holds a $2 million policy on her voice. She hit the headlines in the 1990s after insuring her legs at Lloyd's for $3.2 million.
Talabi Braithwaite
Talabi Braithwaite was a member of Lloyd's and is remembered in many ways as an insurance pioneer. For example, he was the first chartered insurer from Africa after becoming an associate of the ACII in 1951. That year, he returned to Nigeria to found the Law Union & Rock Insurance Company before launching T.A. Braithwaite Insurance Brokers & Co in 1958. His firm brought many new lines of business to the Nigerian market including marine insurance and life assurance. In 1960, he advised the government of the Western Region when it formed the Great Nigeria Insurance Company and, for a few years, served as president of the Insurance Institute of Nigeria. He was also the first president of the Nigerian Corporation of Insurance Brokers and held the position for 16 years, starting in 1963. Mr. Braithwaite joined Lloyd's as a member in 1969 and began underwriting for the Merrett Syndicate a year later. He sadly died in 2011.
Will Smith is one of only two hip-hop artists to receive an Oscar nomination in an acting category (Best Actor, Ali, 2001). He was recognised in 2007 at Grauman's Chinese Theater with a Hollywood Star and has starred in films that have taken more than $6 billion at the box office. Owing to his value to production companies, when he wanted to celebrate his 50th birthday in style, he was told that he must first take out a $200 million insurance policy at Lloyd's. He'd decided to bungee jump out of a helicopter from 1,000 feet into the Grand Canyon and took out a policy that covered against loss of earnings. The premium for the single event was set at $500,000.
Enjoy what you read above? Click here for the rest of the stories!
Click here to find out more about The ACIN.


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Confirmed: Aston Villa winger seals Championship loan
Villa have Jota, Trezeguet and Anwar El Ghazi and first-team football for AVFC ace would've been hard to come by
Ashley Preece
Andre Green has departed Aston Villa on a season-long loan deal, signing for Championship side Preston North End.
The 21-year-old winger was officially unveiled at Deepdale today after being told by Dean Smith to play regular football.
Green, who scored for Villa last weekend at Charlton, said: "I am happy I am here. I just spoke to the gaffer; everything is exciting and I can't wait to get started and meet the lads.
"I had a good season in the Championship last year and I am excited to be here to bring that experience I have had into this team. The lads dealt with it really well last year and hopefully I can bring some extra power to the attack of the team and we can go somewhere this season.
"The gaffer has said he wants to improve me as a player. As a winger you can see what happened to Callum [Robinson] and how much he improved over the course of last season. He wants me to come away from this season as a better player than when I have arrived and more goals is what I need to add to my game and he is confident I can do that."
Do you agree sending Andre Green out on loan?
1000+ VOTES SO FAR
Yes, he needs to play regular football
No, he could've broken into the Villa team
Aston Villa TV fixtures
Aston Villa vs Leicester City (Carabao Cup)
Tuesday, January 28 (7.45pm) - live on Sky Sports
Aston Villa vs Tottenham Hotspur
Sunday, February 16 (2pm) - live on Sky Sports
Leicester City vs Aston Villa
Monday, March 9 (8pm) - live on Sky Sports
Aston Villa vs Chelsea
Saturday, March 14 (5:30pm) - live on Sky Sports
Villa have signed fellow wide players Jota, Trezeguet and Anwar El Ghazi and first-team football for Green would have been limited.
Preston boss Alex Neil, who has snapped up the winger on a year's loan, said: "Andre played the second half of season at Aston Villa in a team that was promoted. We are very hopeful that his directness, his pace and his creativity will add to our front line and that he will score and supply goals.
Andre Green of Aston Villa
'You don't notice' Aston Villa coach reveals Liverpool and Man City message
"The experience of being involved in an event the magnitude of the Play-Offs and the Final, in particular, will help to him spur him on to have more success and hopefully that will be with Preston North End."
Green will wear his favoured the No.19 shirt at Deepdale and will be available for North End's weekend Championship opener at Millwall.
For all your latest Villa news, opinion, analysis and transfer gossip, click here


King Quilt: King quilt measures 110"x97" and has a 100% cotton shell and fill. This quilt features a deep red colored Ohio Star Star pattern with a solid red backing.
Red and White Striped King Bed Skirt: King skirt measures L 80" x W 78" with 16" drop, 100% cotton. Deck is white cotton with 3" fabric border matching the ruffle. Ruffle ratio is 1.75 x deck length on 3 sides, gathered. Front skirt panel overlaps side panel 2.5" at front corners.
Two Standard Quilted Shams: The standard shams measure 21"x27" each and are 100% cotton. Each sham features bold stripes and are hand quilted. The backs feature a 3-button closure and a 3" overlap to conceal pillow insert.
Woven Throw: This woven throw measures 50"x60" and is made using a satin weave. This throw is 100% cotton with tassels on both ends.


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Get ready for plush toys, home décor, collectibles and electronics accessories!
My Singing Monsters is a beloved, critically acclaimed game on the App Store and Google Play, with more than 70 million players worldwide. The free-to-play family-friendly title explores rich and intriguing worlds as players engage in discovery, creativity and delight by breeding and feeding Singing Monsters to build unique melodies.
My Singing Monsters is produced by mobile gaming company Big Blue Bubble. For more than 10 years, Big Blue Bubble has been creating fun, innovative, engaging and award-winning games that inspire creativity and encourage people of all ages to embrace their playful side. Big Blue Bubble's commitment has earned the studio recognition from prestigious organizations around the world, including awards from Deloitte, BAFTA and IGN for economic growth, people's choice and editor's choice, respectively. My Singing Monsters.is the studio's top-grossing flagship franchise.
Big Blue Bubble, partnering with Vancouver, Canada-based multimedia entertainment company Wind Sun Sky and The Walking Dead producer Skybound Entertainment, has licensed the My Singing Monsters gaming app property to FanWraps Inc. to produce a line of automotive, home décor, collectibles and consumer electronic accessories.
In addition, the troika has also licensed My Singing Monsters to Commonwealth Toys to produce a complete line of plush toys in all sizes for the global market.
Everyone involved is thrilled.
"With their strong fan-centric pop culture licenses, we know FanWraps will deliver quality products for our brand," says Catherine Winder, CEO of Wind Sun Sky and producer of the game's animated series. "We're also excited to bring our plush toy program to life for our fans and collectors by working with Commonwealth Toys." Catherine is known for her work at LucasFilm and on The Angry Birds Movie.
My Singing Monsters has a significant library of user-generated content with over 150 Million views on YouTube, which gives FanWraps a large audience to reach with collectibles. An automotive wrap and decal company at its inception, FanWraps has moved into the consumer electronics, home décor and collectibles market, producing a wide variety of product types customized by a licensed property and specializing in new category development.
"We've been keeping our eye on this property for a while and see incredible retail toy and collectible potential for both the game and future animated series," says Alec Kessler, Executive Vice President of Commonwealth. "This brand has an enormous following and massive fan engagement." Commonwealth Toy's portfolio includes properties such as Angry Birds, Sponge Bob, JoJo Siwa, Paw Patrol, Fingerlings, Smooshy Mushy and many more.
The first FanWraps merchandise will be introduced for the 2018 holidays while the first Commonwealth Toys characters will also roll out in time for the 2018 holidays, offered through e-commerce and at select retailers. The plush toy line will continue to build a collection of characters in a variety of sizes with plans to incorporate the musical abilities of the monsters.
These deals are brokered by Alita Friedman, CEO of Alita's Brand Bar, and licensing agency for the My Singing Monsters franchise.
Children's Retail Today publishes new articles each week about innovative, imaginative and inspiring products for children. Contact me today with your great idea!
August 29, 2018 Janet Gray Muniz	Best Kid Focused Blog Award, Big Blue Bubble, blogs for children's retailers, Children's Retail Today, Commonwealth Toys, CRT, FanWraps, Janet Gray Muniz, kids products blogs, mommy blogs, My Singing Monsters, My Singing Monsters plush toys, Sageland Creative, Skybound Entertainment, Top 40 Kids Fashion Blogs, Wind Sun Sky	1 Comment


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redpajama_set_name: RedPajamaGithub


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Q: keep getting ascii value where char is expected When a coordinate is selected, it should be replaced with a "~".  However, it's being replaced with the ascii value for the ~ instead (126). I tried a few different things, but I always get the 126 instead of the ~.  Any ideas?
Thanks for the help!
int board_is_empty(int N, int board[ROWS][COLS])
{
    int i = 0, j = 0;
    for (i = 0; i < N; i++)
    {
        for (j = 0; j < N; j++)
        {
            if (board[i][j] != '~')
            {
                return 0;
            }
        }
    }
    return 1;
}

//updates the board to replace each selected coordinate with a ~.
//returns nothing
void update_board (int board[ROWS][COLS], int row_target, int column_target)
{

    board[row_target][column_target] = '~';
}

int main(void)
{
    int game_board[ROWS][COLS] = {0};
    int rows, columns = 0;
    int players_turn = 1, target_column = -1, target_row = -1, value = 0;
    int row_selection = 0, column_selection = 0;
    int i = 0;

    initialize_game_board(game_board);
    display_board(game_board);
    generate_starting_point(game_board, &rows, &columns);

    printf ("\nPlease hit <Enter> to continue.\n");
    getchar ();

    while (board_is_empty(ROWS, game_board) != 1)
    {
        select_target (&target_row, &target_column, players_turn);
        value += game_board[target_row][target_column];
        update_board (game_board, target_row, target_column); //should cause the coordinates at target_row && target_column to be replaced with a ~
        display_board(game_board);
    }
    printf("\n%d", value);

}

A: For storing your coordinates, you are using an integer array. When you execute
board[row_target][column_target] = '~'; tilde's ascii value (126) is assigned to LHS. There is no way you can assign a character to an integer value. I think you should use some special number rather than tilde. If I were you, I would use INT_MIN or INT_MAX.

A: There isn't a difference between the character '~' and the number 126 as far as the C language is concerned, '~' == 126.
(You used "~" which i would normally use for a string, but i assume you don't actually mean that).
If you want to display a value, you have to use the correct format string. %d is for decimal integers, %c would be for characters (the variable holding the value should also be a char)

A: In C, chars are just integers. At output time they are represented as characters but internally they hold just the ASCII code of that character.
Since your board is a matrix of int's, when you assign '~' you are effectively assigning the number 126 to a position of the board. If you check that position, the expected result is to get an int equal to 126.
However, if you want to see that value as a character, you can do it by casting that number into a char:
printf("%c", value);

Take a look:
#include <stdio.h>

int main()
{
    int i = '~';
    char c = '~';

    printf("Integer: %d\n", i); /* outputs: 126 */
    printf("Char: %c\n", c); /* outputs: ~ */
    printf("Integer casted to char: %c\n", i); /* outputs: ~ */
}

That is, your value is right. You just need to get the representation you want. (If you want to be able to store the value 126 in the board and the character ~ at the same time, then you're out of luck because for C they are the same thing -you can use some other value that you know that the board isn't going to hold, like -1 or something like that).
Update:
So, if I didn't get it wrong what you're trying to do is to read numbers from a bidimensional matrix of random integers and mark each one as you go reading them.
If that is what you're trying to achieve, then your idea of using '~' to mark the read positions isn't going to work. What I meant before is that, in C, 126 and the character '~' are the exact same thing. Thus, you won't be able to differentiate those positions in which you have written a '~' character and those ones in which a random 126 is stored by chance.
If you happen to be storing positive integers in your array, then use -1 instead of '~'. That will tell you if the position has been read or not.
If you are storing any possible random integer, then there is nothing you can store in that array that you can use to mark a position as read. In this case a possible solution is to define your array like this:
typedef struct {
    int value;
    char marked;
} Position;

Position board[ROWS][COLS];

Thus, for each position you can store a value like this:
board[row][col].value = 23123;

And you can mark it as read like this:
board[row][col].marked = 'y';

Just, don't forget to mark the positions as not read (board[row][col].marked = 'n';) while you fill the matrix with random integers.

A: '~' is a character and you have declared board as a two dimensional integer array.
so when you write board[row_target][column_target] = '~';
it convert '~' it into integer i.e into its ascii value which is 126
and there for it becomes board[row_target][column_target] = 126
I will suggest make board as two dimensional character array. Hopefully it will solve your problem.
And in case if you want it as integer only then consider 126 as a special no which means '~' by declaring


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Author: raannt
Posted on May 4, 2012 June 5, 2012
Cole Mohr…THE Role Model
What is it with this kid with the April, May, June tattoo??? We are obsessed with model Cole Mohr…and not just because he is insanely good looking! We're not sure if its his boyish charm or his constant edginess which seems to be the portrayal of some Larry Clark film character. He is dark and brooding one moment and intoxicatingly handsome and classic the next. We've watched him for awhile and were intrigued when we saw him on an episode of Kell on Earth because he seemed much more down to Earth and friendly than we would have imagined, making us adore him even more! Nonetheless, this kid is here to stay…probably even long enough for us to see him make it to July, August…and even the September Issue!
Be Yourself. Be Fearless. Be Your Own Unexpected Luxury!
Posted on May 4, 2012 October 17, 2013
Out of Our Closet Today 5-4-12 – Juvenile Peace
When picking out what we're going to wear for the day…comfort usually rules. Our closet is so eclectic that we like to pull opposites together, which indicates our inner personality dictating our individual style. Alex definitely puts more thought in his outfits, but is always true to himself, whether what he is wearing is trendy or not. Neither one of us is ruled by magazine articles or runway shows, although we're obsessed with both, but we find our style happiness more in what makes us feel attractive.
Since we're constantly asked what we're wearing or where we shop, we thought it would be fun to begin highlighting one of our outfits daily. You will probably find that Alex tends to lean more towards colored pants and tighter, button down colored shirts and wingtips, while Peter sways toward the vintage, with 70's Western shirts and old concert tees. We both have a love for accessories; jewelry and watches, shoes and underwear…and luckily, we share all of it!
Out of Our Closet Today 5-4-12:
Black Sport Coat: H & M
Black Graphic Tee: We made these at a local t-shirt shop for awareness of disaster in Japan
True Religion Jeans: Saks Fifth Avenue
PEACE Bracelet(also comes in LOVE) Gift shop at The Cosmopolitan in Las Vegas
Cartier Santos Galbee Men's Watch: Cartier
Black Converse Low-Top Slides(We're addicted and have them in several colors): Converse or Nordstrom
Paulina Rubio…Boys Will Be Boys! We're In Love!
We've been HUGE fans of Paulina Rubio, "The Golden Girl" for a long time, but her new video for her single "Boys Will Be Boys" has made us fall in love with her even more! We especially love the part with the fat men in diapers! She looks so incredibly hot and badass in this video! Check it out!
Gossip Girl Rewind S05/Ep22 "Raiders of the Lost Art"
Gossip Girl raannt here! Your one and only source of the scandalous life of… who are we kidding!!! No one can do it like the original GG…and tonight's episode proved it. Let's recap.
Our first matter of business… Dan, Come!!!! On!!!! Really?? Jealous of Chuck? Sure he and Blair have some intense history but let's face it, she chose you, over him… over the glam, power, money… THE UPPER EAST SIDE… She chose you. Trust… you have nothing to worry about. And while we're at it, next time you get an offer like that – The Writers Academy in Rome – don't pass it up. (Didn't you learn anything from Lauren Conrad on The Hills???) You passed up way too many opportunities while trying to date Serena and here you go again making the same mistakes. Learn something! Although we might be a little worried too after telling her "I love you" and just getting nothing in return. Ridic!
The real Blair is back… unlike last week. The REAL Blair is back! And she is up to what she does best; scheming and taking someone down in defense of her friends and love ones, of course while using Dorota in the best possible way. I swear if we had nannies like that while growing up we wouldn't be typing this – she would be. (Unfortunately, we have no nanny…just three pups who are chewing away at our brand new boat shoes!) Ok…back to business. Blair steps into Diana's shoes – well actually, into her overly priced slutty dress – to help Nate and Chuck and Lola (Why is she still around?) to get to the bottom of finding out the true identity of Chuck's father…
Then we have dear Serena, who pretty much got sucka-punched-schemed (or whatever you want to call it) by everyone tonight. First she tries to help Diana so that she could get to the real GG. and then she tries to help her friends so that she could get to real GG and then she gets totally thrown under the bus by all… All while at a super chic party (a sex party, but still chic! Mental Note: someone needs to do this here! Our social circle is beginning to give us a headache in comparison!)
The real secret tonight was behind Door #1, Door #2 or Door #3??? Who is Chuck's real father? Actually…who are Chuck's real parents? Is he really an alien from another planet? Is he the lost child of Marilyn Monroe? Who really knows any more. This whole mother/father thing is really getting out of control. Maybe in the next last 2 episodes we'll get some answers. Until then, post below in the comments what you think would make the best story idea for Chuck's past! We'll pick our favorite and discuss it!
Gossip Girl raannt
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The Winner of RuPaul's Drag Race Season 4 Is…
All season, Sharon needles identified herself as the queen who couldn't win a pageant. Well darling, the time has come to change that title. Good thing you can now refer to yourself as RuPaul's Next Drag Superstar! Yes maam…tonight at the reunion show for RuPaul's Drag Race Season 4, spookerstar Sharon Needles, was crowned America's Next Drag Superstar! We were literally pounding our fists and feet, ecstatic that our queen took home the crown!
But the night was not left without a few other winners. All three girls in the top three represented gorgeously and we realized, while watching as they explained what the title would mean to them, that any one of them would represent the title beautifully. And how cool was it that all of their moms were their to support! We've come a long way baby!
Chad Michaels again displayed unbelievable class and style, even confronting Phi Phi about her behaviors, in a mentoring stance. The reality about Chad Michaels is simple; she is in an entirely different class, one not defined by titles or prestige. We imagine Chad having his own show in Vegas, representing the gay community with style and possibly even writing his own book. If Chad could sing…he might be the next RuPaul…but instead, we think he is defining his role as he goes.
Everyone can hate Phi Phi all they want, but she is Phierce! While she may seem a little young and too "goal focused", she's not stupid enough to realize the power of $100,000 or the title as American's Next Drag Superstar. Should she be hated because she came to win? Maybe some of her behaviors were backhanded but she owned it in the end. And let's be honest…which one of the girls would have donated the money if they had won? No…Phi Phi's not much different than some of the other girls, if not smarter. And although she could work on being kinder, she's had a tough life. She's a survivor! And in case you didn't mention what she said when asked about her father, we'll recap it for you. It was more powerful than any anti-hate message we've heard in a long time! "He does watch it and he doesn't like it. But I love me!" Powerful shit! And she looked gorgeous in the blonde, Marilyn wig! Could she be the Jennifer Hudson of RuPaul's Drag Race??? Only time will tell!
Sharon Needles…OMG!!!! We really, really thought RuPaul was going to give it to Chad. Imagine how unbelievably excited we were when Sharon's name was announced. She had her own powerful moment too when she shared that she never thought she would affect so many people, reaching out to the "gay or weird" kids, or anyone who feels different. And damn! A Ouija board head piece! Sickening! A perfect accessory to go along with her farewell "Happy Halloween everybody!" We love her so! Its kind of hard to deny…she's the Lady Gaga of drag! Well…kind of!
The rest of the show was just as incredible. RuPaul paid homage to Latrice, who incidentally won the title of Miss Congeniality. Was anyone surprised? Latrice is like the Humanitarian Ambassador of Drag. We adore her…not just because of her elegance, but because of her message. In fact, she dropped so many of these profound messages, we're wondering if she's trying to make it on the list of famous quotes. "It's alright to fall down. Get up. Look Sickening…and make them eat it!" She went on to pull out a note she received from the mother of a 10 year old girl. The message was so inspiring, displaying that drag queens are changing the world, that it emphasized the strength of Latrice's message: "Doing Me Works".
And of course there was Willam. We don't care what anyone says…we love Willam. He's real, genuine, honest, hard-working, extremely talented(just check out his YouTube videos) and gorgeous. She has sick style too! We were a little bored that he got kicked off for having his husband come to his hotel room for "conjugal visits". It almost makes you wonder if there wasn't a reason producers were looking for a reason to get Willam off the show. RuPaul was so supportive of Willam's future that it made us wonder. And for once…Willam refused to be flippant and show a gentler side. Yep…if not Sharon, Willam should have won the show! She even coined the phrase Rupologize, to which RuPaul responded "Consider that word stolen!". Willam is a genius!
The rest of the show was just as good! Jiggly looked gorgeous, but it made us sad when she cried while confronting Phi Phi, "I smell like a sucka!". Honey…you've smelled like a sucka all season…and not just because of Phi Phi! Which, by the way, made it even funnier when she kept slurping on that sucker! Did you guys miss that irony? Could someone please get the girl a Sugar Factory Couture Pop ASAP!
We can't wait for the much anticipated RuPaul's Drag Race Season 5!
And while you're waiting, check out our other RuPaul's Drag Race write-ups including interviews with the cast of Season 4, Season 3, Ongina, Shangela, Tyra or search for any of our RuPaul's Drag Race reviews in the search section!
And Please follow us on Facebook and Twitter so we can review and interview the things you love! Love you guys!
*Image Source: Zimbio
Posted on April 26, 2012 June 5, 2012
Glee Supreme Performance Award…Blaine!
Seriously…could Blaine…or Darren Criss for that matter, be any more perfect? He's gorgeous, sweet, incredibly talented and supports gays and lesbians, loving everyone. Uhhh…could we reserve you for the role of husband for our best girlfriend. And Blaine…if he wasn't already perfect, by making Kurt "supposedly" text/cheat on him, we just fell in love even more with his vulnerable heart…and those adorable dimples.
Above all else…the boy can sing! We've always loved Whitney Houston's It's Not Right But Its Ok and now we love it even more! Blaine's performance was absolutely incredible and probably the most "music video" quality performance we've seen in a few episodes. That being said, it's easy to see why this week's Glee Supreme Performance Award goes to Blaine! If you haven't seen it, we put it below to let you see what all the hypes about!
http://www.youtube.com/watch?v=W64tyo-g18A
We're In LOVE! Dance Again…Jennifer Lopez Ft. Pitbull
Have you been watching Jennifer Lopez on American Idol??? Are you one of those haters who criticizes her for not being tough enough or only saving her favorites??? Who cares…the bitch looks absolutely incredible. Obviously…dating someone younger does the body good!
We are so in love with Jennifer Lopez right now…especially her new single Dance Again Ft. Pitbull. She's all grown up now…but for us, she'll forever be Jenny From the Block!
Be Yourself. Be Fearless. Be Your Own Unexpected Luxury.
Glee…500 Tears to Graduation…Dance With Somebody
42 days left until graduation…
If you're like us, the kids of Glee have become part of your family, and just like Kurt's dad, we don't want to see them leave either. Tonight's episode, Dance With Somebody, marked the beginning of the end as the kids began to realize how numbered their days are at McKinley High. And for us…well…life will go on.
So here's what happened. The kids grieved the loss of Whitney Houston and the week was a tribute in her honor. Will tried to tie this into a learning experience, stating that the kids were grieving Whitney because they couldn't deal with the pain of their of their own goodbyes from high school. Some of the songs were amazing(Kurt's rendition of I Have Nothing was reminiscent of Whitney herself in The Bodyguard and Blaine's It's Not Right But It's Ok was ahhhh-mazing!) The rest of the songs were alright…but that's ok. Blaine hasn't been paying much attention to Kurt or giving him any compliments, and apparently they lack any kind of passion(if they can't find it does it leave much hope for the rest of us? Seriously…does it surprise anyone else they're not having sex but Brittany and Santana already have a sex tape???), so Kurt begins flirting via text with another gay teen he meets at the music shop. Ok…we could have sworn the actor playing Chandler was actually YouTube sensation Tyler Oakley…but after much Google-ing found this to be false. (But wouldn't that have been great if it had been…he's probably one of their biggest fans!) After Blaine finds the texts by snooping through Kurt's phone,(bad boy Blaine)he accuses Kurt of cheating on him. Kurt doesn't understand how this is cheating…blah, blah, blah…they end up in Emma's office and Blaine breaks down and tells Kurt he feels like he's leaving him behind. That part was actually pretty endearing and probably one of the best scenes in the show. Ok, lets see what else…Oh, yeah. Kurt's dad tells him he's going to miss him a bunch too(another great scene) and then Puck hands out all these shot glasses to the guys so they remember all of the "broments" they had together. And finally…Will wants to move the date of the wedding from December to May because he wants to make sure the Glee kids will be able to attend and perform, when in reality, we find that Will is the one who is having the most difficult time with the kids nearing graduation.
Did anyone else find it interesting that the wedding was originally set in December and Will wants to move it to May? Have you ever heard of a May-December romance defined as someone being in the spring of their life while the other is in the winter of their life? How many people watching Glee are teenagers and how many are adults, because had we watched it when we were much younger, it would have had a different impact. At one point in the show, Kurt's dad makes the comment that things change "once you go out that door" and nothing is the same. How true that is. All those things our parents and teachers told us about life changing, friends going their separate ways and life moving forward…are true. The people you know today will most likely not be the people you know 5-10 years from now.
Does that mean we become bitter and jaded and live our lives without any meaning or significance because time beats on and nobody really matters? Hell no. Life does matter. Every little second…and Glee is a reminder of that fact. And as we count down those 42 days, let's all remember what's important and treat the people in our life today as if we're on borrowed time…because like Whitney…they could be gone tomorrow!
*Photo Credits: Fox Entertainment
Who Won RuPaul's Drag Race Season 4??? Spoilers and More Spoilers!
Who won RuPaul's Drag Race Season 4??? (We'll tell you our secrets at the end!) If you watched tonight's supposed Grand Finale, you know that the secret will be kept for another week. All over the internet, threads and blogs are blowing up about people's distaste for RuPaul's possible pick of Phi Phi O'Hara as the winner of RuPaul's Drag Race Season 4. This…is about the only part of Drag Race that is similar to an authentic drag pageant; pissed off fans bitching that their girl didn't win. "It was rigged" or "It's so unfair" or "She had a snaggletooth"…which is by far the best comment. So what Phi Phi O'Hara has a snaggletooth(who even uses that word). Does that make her any less deserving to win.
The reality is that there are thousands of talented queens in the United States and all over the world who's art would warrant them a spot on the show. These three, Chad Michaels, Sharon Needles and Phi Phi O'Hara, have risen to the top. Why? Who knows, but all three are there for a very specific reason.
So here's what we heard…Wikipedia leaked on their webpage that Phi Phi had won. Then Sharon. Supposedly Phi Phi also Tweeted something about winning, but we can't find it. Most threads are saying Sharon doesn't win because the Absolut sponsor doesn't like her. Almost all of the comments on the threads and almost all of the Facebook and Twitter feeds are overwhelmingly in favor of Sharon Needles winning…and so are we!
Don't get us wrong, we love Phi Phi and Chad and believe that they deserve to be in the top three, but Sharon is just our personal favorite…and that's what it's all about. Personal favorites.
Chad is so refined and professional. We love his story about living with his mother and his long term relationship with his boyfriend. He'd be an amazing role model…maybe almost too amazing. We're not even sure Chad belongs on this show. He is the embodiment of female impersonation, dating back to the art of celebrity female illusion. He is Cher. Is it possible that Chad is too good…even for RuPaul's Drag Race?
Phi Phi O'Hara is pretty but if we're going to rate her on pretty, what about Kenya Michaels??? Gorgeous! Phi Phi's story about his father is endearing and painful, but not enough to win a show. The whole season she was referred to as a pageant queen. Could someone please inform us in what national pageant system Phi Phi holds a title? Nonetheless, she improved throughout the entire show and always came to the runway looking Phierce!
For us, Sharon is perfect because she embodies everything we already feel about ourselves. And…she's so damn stylish! Literally, Sharon is not just a drag queen…she is an artist. Her greatest scene the entire season was the look on her face after Phi Phi told her to go back to Party City. Sharon looked deeply hurt. It grabbed us. We felt it. We related. And we fell in love.(We also love all of the merchandise she has on sale at her online store Tacky Overpriced Crap; especially the Sharon Needles tattoos for $6.66)
We related because that's how we felt as kids growing up gay. Teased. Mocked. Jabbed. Hurt. Why are fans all over the country, mostly gay fans, doing the same thing to these queens, who are part of our community, by calling them weird, snaggletoothed or old? Why can't we all join together…at least in our love of drag…a fun form of entertainment? Wasn't that how Stonewall began? So back the fuck up…be nice…and remember we're all in this thing together! Races, pageants and contests have winners…and they have losers. Get over it! Maybe Sharon won…or maybe she's just the next Jennifer Hudson. Who knows…
By the way…for all you people out there that like to speculate without doing research…here's some real clues. Phi Phi and Sharon have the same PR representative, which is different from Chad Michaels. Also…Sharon has many more tour dates than either Chad or Phi Phi and hers include Absolut Tour Dates…just sayin!
Legalized Marriage Equality or a Resolution to the US National Debt???
We have a Democrat in the Presidential office…yet we still do not have national marriage equality. At the time we are writing this, the US national debt is $15,630,722,222,420.64 increasing at $3.97 Billion a day since September 28, 2007. Each citizen's share is approximately $50,000. So…as gays and lesbians…how do we vote? Do we vote for our party? Do we vote per issue? Do we vote for the person we think is most appropriate? Do we not vote at all???
The Presidential election is 6 months away, but what if we had to decide today. If you could only pick one…which would you choose?
Would You Rather: Round 7
As of today, one of the following happens…would you rather have legalized, national marriage equality OR a resolution to the US national debt??? You can only pick one!
Let us know in the comments below…and visit our other Would You Rather games in our Would You Rather section!