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Exosomes, which are 50- to 100-nm-diameter lipid vesicles, have been implicated in intercellular communication, including transmitting malignancy, and as a way for viral particles to evade detection while spreading to new cells. Previously, we demonstrated that adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. Thus, release of HSP60 from exosomes would be damaging to the surrounding cardiac myocytes. We hypothesized that 1) pathological changes in the environment, such as fever, change in pH, or ethanol consumption, would increase exosome permeability; 2) different exosome inducers would result in different exosomal protein content; 3) ethanol at "physiological" concentrations would cause exosome release; and 4) ROS production is an underlying mechanism of increased exosome production. We found the following: first, exosomes retained their protein cargo under different physiological/pathological conditions, based on Western blot analyses. Second, mass spectrometry demonstrated that the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins. Third, ethanol did not affect exosome stability but greatly increased the production of exosomes by cardiac myocytes. Fourth, ethanol- and hypoxia/reoxygenation-derived exosomes had different protein content. Finally, ROS inhibition reduced exosome production but did not completely inhibit it. In conclusion, exosomal protein content is influenced by the cell source and stimulus for exosome formation. ROS stimulate exosome production. The functions of exosomes remain to be fully elucidated.
- exosome
- proteomics
- ethanol
- valosin-containing protein
- heat shock protein 60
- alcoholic cardiomyopathy
- reactive oxygen species
studies of exosomes, which are 50- to 100-nm-diameter lipid vesicles produced by a variety of cell types, and the cardiovascular system have been very limited. Most work on exosomes has focused on cancer, and this work suggests that exosomes have a role in intercellular signaling (5, 43). Previously, we (12) found that heat shock protein (HSP)60 was released in exosomes. We (19) have also shown that extracellular (ex)HSP60 can activate the innate immune system via Toll-like receptor (TLR)4, leading to cardiac myocyte apoptosis. ExHSP60 has also been shown to contribute to myocardial injury during ischemia (25). HSP60 has been found in human and rat plasma and serum samples in studies of both cardiovascular disease and diabetes (3, 11, 24, 26, 35). We reasoned that if HSP60 was contained within the exosome, it would not be toxic. However, if HSP60 was released from the exosome, this would promote cardiac myocyte death. The stability of exosomes and their ability to retain their cargo have not been investigated. In addition, the underlying mechanisms regulating exosome formation are not well understood and have not been studied in cardiac cells. Understanding the function and fate of cardiac exosomes can provide new insights into cardiovascular disease.
We investigated the stability of exosomes under physiological and pathological conditions, including changes in temperature and pH. We hypothesized that pathological conditions, such as fever and acidosis, would increase the leakiness of exosomes and release HSP60. We also examined whether ethanol, an organic solvent, at levels seen with moderate and heavy consumption of alcoholic beverages would increase the leakiness of exosomes. To better understand the function of cardiac myocyte-derived exosomes, we studied their protein content by mass spectrometry (MS) and compared exosomes produced after two different treatments: mild hypoxia and ethanol. Finally, we hypothesized that ROS production was a common factor between hypoxia/reoxygenation and ethanol treatment leading to exosome release. The results of these experiments, which demonstrated that exosomes are quite stable, exosomal protein content varies depending on the stimulus for production, and ROS have an important role in exosome generation, are reported here.
Isolated Adult Cardiac Myocytes
Isolated adult cardiac myocytes were prepared from the hearts of 3-mo-old male Sprague-Dawley rats, as previously described (48). The animal protocol was approved by the University of California-Davis Research Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All research adhered to the American Physiological Society's "Guiding Principles in the Care and Use of Vertebrate Animals in Research." Rats were anesthetized with an intraperitoneal injection of ketamine(75 mg/kg) and xylazine (3.9 mg/kg). Before removal of the heart, a terminal event, for the preparation of isolated cardiac myocytes, deep anesthesia was confirmed by the absence of corneal and toe pinch reflexes. Adult cardiac myocytes were cultured in media containing human serum albumin. As serum samples can contain exosomes, this human serum albumin was first centrifuged at 164,000 g for 2 h to remove all exosomes, as previously described (12).
Exosome Isolation
Untreated adult cardiac myocytes in culture produce a very minimal amount of exosomes. Exosome production by cardiac myocytes was increased by hypoxia (2 h) followed by 1 h of reoxygenation, as previously described (12). In later experiments, treatment with cell culture grade ethanol, at concentrations found in humans consuming alcoholic beverages, was done for 2 h followed by 1 h of recovery, matching the timing of the hypoxia protocol, after we found that ethanol treatment greatly increased exosome production.
Two different approaches were used to purify exosomes from the media. The first was a method adhering to current good manufacturing processes that generates exosomes that can be used clinically, which was tried so that exosomes could then be used for in vivo experiments (21). Alternatively, ExoQuick (System Biosciences) was used as a second method to prepare exosomes. This approach involves less labor than the serial centrifugation we used previously (12). ExoQuick was used following the directions of the manufacturer. When exosomes were prepared from media, the media was first concentrated from 50 ml to 130 μl with Amicon Ultra filter (Millipore, Billerica, MA) with a 100,000 molecular weight cutoff before ExoQuick treatment.
Exosome Quality
As exosomes are too small to readily visualize, the quality of exosome preparations was confirmed by measuring the hydrodynamic radius with a particle sizing system (NICOMP 380 zls, PSS, Port Richey, FL). In addition, acetyl choline esterase activity, which reflects the amount of cell membrane present, was used to indirectly follow the quantity of exosomes isolated along with measurement of total protein by the BCA assay (Pierce, Rockford, IL), as previously described (12). Highly consistent preparations of exosomes were obtained with Exoquick.
Electron Microscopy
Electron microscopy was done in the core Electron Microscopy Laboratory (Department of Pathology and Laboratory Medicine, School of Medicine, University of California, Davis, CA) following the approach of Thery et al. (50). Exosomes were ultracentrifuged to generate a pellet as part of the final step of isolation. A drop of this resuspended pellet was allowed to settle on a gold-coated grid, blotted, fixed in 1% glutaraldehyde, washed for 2 min in double-distilled water, incubated in uranyl oxylate for 5 min, incubated in three separate drops of methyl cellulose with uranyl acetate with 5 min in the first two drops and 10 min in the last drop, and finally removed from methyl cellulose-uranyl acetate by slow-drag on edge on filter paper. Exosomes were visualized by standard transmission electron microscopy with a Philips CM120 microscope.
Exosome Treatments
Exosomes were placed in standard PBS for all incubations. Protein concentrations were measured by the BCA assay (Pierce). After incubations under different conditions, exosomes were isolated by centrifugation at 164,000 g for 2 h. Protein in the supernatant was concentrated in an Amicon Ultra filter (Millipore) with a 100,000 molecular weight cutoff cutoff. Exosomal (pellet) and released (supernatant) proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose, and then analyzed for HSP60 expression using our previously published methods, as detailed below.
Temperature incubations were done using precision water baths set at appropriate temperature and a 4°C refrigerator. pH was adjusted with concentrated HCl. Ethanol (100%, tissue culture grade) was added to the buffer to achieve concentrations of 21.7 and 65.1 mM. These ethanol levels correspond to legally intoxicated levels and levels found with the consumption of multiple alcoholic drinks (15). Similar concentrations have been studied using in vivo rodent models of ethanol ingestion (16).
Cell Injury
Apoptosis was assessed by quantifying DNA fragmentation with the cell death detection assay (Roche) as previously described (19). Lactate dehydrogenase (LDH) release was measured using a standard assay, as previously reported (45).
ROS Levels
ROS levels were measured by a fluorescent method with confocal microscopy. CellROX deep red (Invitrogen) was used as an indicator for cell ROS, as previously described (4). Cardiac myocytes were treated with high-dose ethanol for 2 h and changed to standard media, at which time CellROX deep red was added. Cells were incubated with CellROX deep red for 30 min, fixed with 4% paraformaldehyde in PBS, and mounted with 4′,6-diamidino-2-phenylindole for counterstaining. The intensity of CellROX fluorescence was calculated and analyzed to quantify the ROS level, as previously described (4).
Experiments of exosomal protein content were done in the University of California-Davis Proteomics Core. Samples were briefly separated using standard 10% SDS-PAGE and visualized with Instant Blue Protein Stain (Fisher Scientific). Gel pieces, containing all the proteins in the sample, were cut out and digested overnight with trypsin according to a standard protocol. LC-MS/MS analysis was performed using the standard top 15 method using a Thermo Scientific QExactive orbitrap mass spectrometer in conjunction with Paradigm MG4 HPLC (Michrom Bio Resources, Auburn, CA). Digested peptides were loaded onto a Michrom C18 trap and desalted before being separated using a Michrom 200 × 150-mm Magic C18AQ reverse-phase column. A flow rate of 2 ml/min was used. Peptides were eluted using a 60-min gradient with 2% solvent B to 35% solvent B over 40 min, 35% solvent B to 80% solvent B for 3 min, 80% solvent B for 2 min, and then a decrease from 80% to 5% solvent B in 1 min (where solvent A = 0.1% formic acid and solvent B = 100% acetonitrile). A spray voltage of 2.2 kV was used with a transfer capillary temperature of 200°C.
Data Analysis
Database searching.
Tandem mass spectra were extracted, charge state deconvoluted, and deisotoped. All MS/MS samples were analyzed using X! Tandem [version TORNADO (2010.01.01.4), The GPM; thegpm.org]. X! Tandem was set up to search the Uniprot complete rat proteome database (unknown version, 71,443 entries) plus an equal number of reverse sequences and 115 common laboratory contaminant proteins (www.gpm.org/crap/), assuming the digestion enzyme trypsin. X! Tandem was searched with a fragment ion mass tolerance of 20 ppm and a parent ion tolerance of 20 ppm. The iodoacetamide derivative of cysteine was specified in X! Tandem as a fixed modification. Deamidation of asparagine and glutamine, oxidation of methionine and tryptophan, sulphone of methionine, tryptophan oxidation to formylkynurenin of tryptophan, and acetylation of the NH2-terminus were specified in X! Tandem as variable modifications.
Criteria for protein identification.
Scaffold (version Scaffold_3.2.0, Proteome Software, Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >90.0% probability as specified by the Peptide Prophet algorithm (17a). Protein identifications were accepted if they could be established at >90.0% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (33). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Using these parameters, the false discovery rate was calculated as 3.7% on the peptide level and 0.2% on the protein level for samples searched against the rat database.
Western Blot Analysis
Western blot analysis to analyze protein expression was performed as previously described (32). Anti-HSP60 (Enzo Life Sciences, Farmingdale, NY) was used at 1:1,000, and anti-GAPDH (Fitzgerald Industries, Acton, MA) was used at 1:30,000. Antibodies to myomesin and myosin-binding protein C were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a 1:1,000 dilution. Antibodies to valosin-containing protein (VCP), tropomyosin, and α-crystallin were obtained from ABCAM (Cambridge, MA) and used at a 1:1,000 dilution. Appropriate secondary antibodies with horseradish peroxidase were used at a 1:1,000 concentration. Western blots were developed with a chemiluminescent system (Pierce), and the subsequent images were analyzed as previously described (46). We attempted to identify a loading control, which could be used in addition to Ponceau staining of the blots to confirm equivalent loading of blots. Exosomes are not cells but the products of cells subjected to different stresses, and their protein content may not be the same. GAPDH varied between ethanol-treated and hypoxia groups. Na+-K+-ATPase (Santa Cruz Biotechnology, 1:5,000), which was tested as a membrane protein, could have been more consistently present among different exosome preparations but varied among different exosome preparations and thus was not satisfactory as a loading control (data not shown). β-Actin (Millipore, 1:500), a common loading control, was not found either by MS or Western blot analysis (data not shown). Therefore, Western blot analysis was done without normalization to density of another protein, relying instead on the confirmation of equal protein loading by staining the membrane with Ponceau red before the development with antibody.
All data are expressed as means ± SE. Data were compared by ANOVA followed by the Student-Neuman-Keuls test. Normalized data were compared by ANOVA on ranks followed by a Dunnett's test. Two-way ANOVA was used to compare data on acetylcholine esterase activity. A Student's t-test was used when there were only two sets of data to be compared, such as for Western blot and ROS data. P values of <0.05 were considered to be significant.
Purified exosomes were prepared from the media of adult cardiac myocytes after brief hypoxia (2 h) and reoxygenation (1 h), as previously described (12). Exosome size was analyzed with a Nanotrak particle sizing system (NICOMP 380 zls). A representative sizing graph is shown in Fig. 1A. Exosomes were, on average, 100 nm in diameter. Electron microscopy with negative staining was used to visualize the exosomes and confirm that 50- to 100-nm membranous spheres were isolated, as shown in Fig. 1, B and C.
Exosome Integrity
As discussed above, exHSP60 causes cardiac myocyte apoptosis, and HSP60 is released from cardiac myocytes in exosomes. If HSP60 is sequestered in the exosomes, this should prevent toxicity; however, if exosomes release HSP60, it would be able to bind to TLR4 and cause apoptosis. Therefore, whether exosomes release proteins under varying conditions was assessed with three different sets of physiological/pathological conditions for as long as 18 h. A temperature range of 4–42°C had no adverse effects on exosome stability, as assessed by the release of HSP60 (Fig. 1D). The temperature of 4°C was tested not as a pathophysiological state but with regard to the issue of stability during temporary storage. Ethanol is an organic solvent, and we reasoned that consumption of ethanol might increase exosome leakiness. As shown in Fig. 1E, there was no release of HSP60 under conditions of low ethanol (21.7 mM) or high ethanol (65.1 mM), levels that correlate with legally intoxicated in most states and with the consumption of a high number of alcoholic drinks (15). Over time, the ethanol would be expected to have some evaporation, but in vivo it would decrease secondary to metabolism. Similarly, pH variation was tested, and again there was no release of HSP60 from exosomes with acidosis, which would be seen with disease states such as ischemia, sepsis, and diabetic ketoacidosis (Fig. 1F).
Previously, we (12) have found that HSP60 is predominantly associated with the exosome membrane and tightly bound to the membrane but not the transmembrane. If HSP60 is tightly bound, it might not be released by exosomes in response to changing conditions. However, GAPDH, a cytosolic protein, is also found in exosomes, as discussed below, and GAPDH might more readily be released by exosomes. As shown in Fig. 1G, after an 18-h incubation at different temperatures, neither HSP60 nor GAPDH were released from exosomes.
Clinical Grade Exosomes
Clinical grade exosomes are desirable for potential in vivo studies of exosome function. Clinical grade exosomes were isolated following the method of Lamparski et al. (21). At 18 h, almost 50% of HSP60 had been released by exosomes over a range of temperatures (Fig. 2, A and B). Similarly, ∼40% of HSP60 was released with exposure to either pH 7.4 or 7.1 (Fig. 2, C and D). Interestingly, HSP60 was present as a doublet in the protein released into the supernatant. Treatment with 21.7 and 65.1 mM ethanol did not change the release of HSP60 (Fig. 2, E and F). Further investigation showed that at as early as 2 h, half of the HSP60 had been released by exosomes regardless of treatment. With Exoquick, as shown in Fig. 1, there was little or no release of HSP60 from exosomes, but the clinical grade method, which involves multiple steps with concentrating and isolating the exosomes with high-speed centrifugation followed by filtration steps, was consistently associated with the release of HSP60 from exosomes in our hands.
Ethanol and Exosome Generation
We were surprised that ethanol did not increase exosome permeability. Even if ethanol did not effect exosome stability, it could change the production of exosomes, possibly through increased protein denaturation or other effects on the cell. Thus, we hypothesized that ethanol levels seen with alcohol consumption would increase the production of exosomes. Adult cardiac myocytes were treated with either 21.7 or 65.1 mM cell culture grade ethanol. Both treatments resulted in exosome production. The particle sizing profile for the exosomes was similar to that seen with hypoxia/reoxygenation-derived exosomes. Ethanol treatment resulted in much higher production of exosomes than hypoxia/reoxygenation. As shown in Fig. 3A, three different cardiac myocyte isolations were split, and half of the cells were treated with hypoxia/reoxygenation and half were treated with 65.1 mM ethanol. Ethanol treatment consistently generated far more exosomes than hypoxia/reoxygenation based on the protein content of the exosome preparations (P < 0.05). Furthermore, acetylcholine esterase activity in ethanol-derived exosomes tended to be higher than that in exosomes isolated after brief hypoxia/reoxygenation, but this did not reach significance (Fig. 3B). Thus, the differences in protein content were more marked than any difference in acetylcholine esterase activity, as shown in Fig. 3.
Does Exosome Generation Reflect Cardiac Myocyte Injury?
Previously, we have shown that the brief hypoxia/reoxygenation treatment used to generate exosomes does not cause LDH release, a sign of necrosis. Ethanol treatment (65.1 mM) and hypoxia/reoxygenation were each tested for evidence of cardiac myocyte necrosis (LDH release) and apoptosis (DNA fragmentation). As shown in Fig. 3, C and D, there was no increase in LDH release from cardiac myocytes with either treatment. Ethanol treatment caused a small but significant increase in DNA fragmentation, whereas hypoxia/reoxygenation had no effect on DNA fragmentation (Fig. 3, E and F). Given that the higher concentration of ethanol caused mild DNA fragmentation, the effect of low-dose ethanol (21.7 mM) on cardiac myocytes was also assessed. Low-dose ethanol caused no increase in LDH release (Fig. 4A) or DNA fragmentation (Fig. 4B).
Exosome Proteomics
To gain insight into the function of exosomes, the protein content of three different exosome preparations after ethanol treatment (65.1 mM) of cardiac myocytes was analyzed by MS. Fifty-seven proteins were identified. Of these, four proteins were clearly contaminants from cell culture, sample preparation, and protein digestion with trypsin (human serum albumin, trypsin, keratin, and human hemoglobin). Two proteins, haptoglobin and ferritin, are common serum proteins and may also have been contaminants from the cell culture media. These six proteins were excluded from analysis. As shown in Table 1, 51 different exosomal proteins were identified, ranging from membrane to cytosolic and mitochondrial proteins. Eleven proteins were only found in one of the three exosome preparations, and five proteins were identified as uncharacterized proteins. The remaining 35 proteins included tropomyosin-α1, myomesin 2 (M-band), α-crystallin B, cardiac α-actin, GAPDH, and long-chain specific acyl-CoA dehydrogenase, a mitochondrial protein. Western blot analysis was used to confirm proteins of interest. As shown in Fig. 4C, myomesin, myosin-binding protein C, VCP (also known as p97 AAA-ATPase, Cdc48, and transitional endoplasmic reticulum ATPase), and tropomyosin were all found in multiple exosome preparations, confirming the MS results. α-Crystallin was found in two of three Western blot samples examined (Fig. 4C).
We generated exosomes by two distinct treatments, which cause at most minimal harm based on LDH and DNA fragmentation. Whether these exosomes have similar protein content has a bearing on their potential function. Therefore, MS analysis of three different sets of hypoxia/reoxygenation exosomes was also done. As shown in Table 2, 33 proteins were identified in hypoxia/reoxygenation-derived exosomes, excluding serum contaminants. Hypoxia/reoxygenation-derived exosomes contained HSP27 and HSP90, neither of which was found in ethanol-derived exosomes by MS. The 2 sets of exosomes had 17 proteins in common, including HSP60, GAPDH, tropomyosin-α, myomesin, myosin-binding protein C, α-crystallin B chain, and VCP. Tables 3 and 4 show proteins only found in ethanol-derived exosomes (Table 3) or hypoxia/reoxygenation-derived exosomes (Table 4). Figure 5 shows a comparison of the expression of key proteins from the two different types of exosomes. Ethanol-derived exosomes contained more myosin-binding protein C than hypoxia/reoxygenation-derived exosomes (P < 0.05; Fig. 5, A and B). Similarly, VCP levels were higher in ethanol-generated exosomes than those released after hypoxia/reoxygenation (P < 0.01; Fig. 5, A and B). Tropomyosin levels, while fairly constant in the ethanol-treated group, varied in the hypoxia/reoxygenation-treated group and overall did not differ. HSP60 levels were somewhat higher in the ethanol-treated group, but this was not significant (Fig. 5, C and D). GAPDH levels were lower in hypoxia-treated exosomes (P < 0.05; Fig. 5, C and D).
Proteomics Control
To control for nonspecific proteins, we prepared exosomes from the media of untreated control cardiac myocytes. Such cells would produce very few exosomes, and this "exosome isolation" (blank) control was to determine what proteins were nonspecifically isolated. SDS-PAGE on this control sample showed only a single faint band, consistent with albumin (data not shown). The typical volume used for MS in the ethanol and hypoxia experiments was sent for MS analysis. As expected, human albumin was the predominant protein present followed by trypsin. LDH B chain and human keratin were both present. Finally, very small amounts of pyruvate kinase isozymes M1/M2 and cardiac α-actin were present compared with the robust amounts found in ethanol- and hypoxia/reoxygenation-derived exosomes.
Hypoxia/reoxygenation is well established as a source of ROS. We hypothesized that ROS might be a common denominator for stimulation of exosome production. Ethanol has previously been reported to increase ROS in the heart, as discussed below. CellRox red (Invitrogen) was used to stain for ROS at the end of the 2-h ethanol treatment. As shown in Fig. 6, A–C, ROS production was barely detectable in control untreated cardiac myocytes but markedly increased after ethanol treatment (P < 0.001). The mitochondria are a major source of ROS, and ethanol-generated exosomes contained a large number of mitochondrial proteins. Cells were treated with a combination of antioxidants: 10 mM N-acetyl-l-cysteine, 1 mM ascorbate, and 100 μM Trolox, based on the literature (13, 36). Treatment was begun 30 min before the initiation of ethanol treatment or hypoxia to allow equilibration. As shown in Fig. 6, D and E, antioxidant treatment significantly decreased acetylcholine esterase activity, a marker for the amount of exosomes present (Fig. 6, D and E). However, total protein/exosomes increased in groups treated with inhibitor compared with hypoxia/reoxygenation or ethanol alone, but this increase was not significant (Fig. 6F).
Although exosomes have been recognized for 15–20 yr, their actual function(s) remains elusive. Exosomes were originally described as the mechanism by which reticulocytes shed unneeded receptors and protein complexes as they matured to erythrocytes (14). One function of exosomes is thought to be the removal of denatured proteins and organelle fragments. A number of studies (20, 43) have implicated exosomes in transmitting/transferring the malignancy of cancer to other cells. Cancer cells have been found to manipulate the environment and evade immunity via exosomes (5). Exosomes released by a medulloblastoma-derived cell line promoted cell proliferation and tumor cell migration and had immune modulatory effects on lymphocytes (7). Exosomes have also been identified as allowing virus transmission from cell to cell while evading the immune system (28, 30). Thus, exosomes promote disease progression and are considered to be immunosuppressive in cancer (49). However, little is known about exosomes in the cardiovascular system.
HL-1 cells have been studied as a model of cardiac cells. Microvesicles ranging in size from 40 to 300 nm have been isolated from the media of HL-1 cells and were found to change gene expression in fibroblasts after 48 h of coincubation (52). The broad size range of these vesicles indicates a mixed population, making results difficult to interpret with respect to the specific vesicle type. Yu et al. (54) reported that neonatal cardiac myocytes released TNF-α in exosomes after prolonged hypoxia. Thus, very limited evidence supports that cardiac-derived exosomes may have important downstream signaling effects.
ExHSP60 is toxic to cardiac cells through the activation of TLR4 triggering late cardiac myocyte apoptosis (19). HSP60 is released from cardiac myocyte in exosomes, where potentially it is safely sequestered. In the present study, exosomes under a range of physiological, pathophysiological, and extreme conditions did not release HSP60. As exosomal HSP60 is tightly bound to the exosomal membrane, we tested whether GAPDH would be released from exosomes. Like HSP60, GAPDH remained within the exosome. Thus, HSP60 is stable within the exosome and not released under a range of physiological conditions.
Plasma/Serum HSP60 and Exosomes
There is a substantial literature reporting HSP60 in the plasma in various disease states (11, 24, 35). One study (41) found very high levels of plasma HSP60, 10 μg/ml or more, rivaling albumin levels in some patients. On the other hand, in a study of Italian diabetics (3), we found relatively low levels of HSP60 in serum. Similar low serum levels correlated with the development of lung problems after motor vehicle accidents (34). Most studies are done on frozen plasma or serum samples, which would rupture any exosomes. Although we have shown that HSP60 is released by cardiac myocytes via exosomes in the absence of necrosis, this does not eliminate other routes for release of HSP60 by other cell types. Plasma/serum HSP60 reflects the sum of HSP60 released by all organs and tissues. Many questions remain to be answered about the source, amount, and toxicity (or lack thereof) of plasma/serum HSP60. We and others are pursuing investigations in this area, and further work will be needed to define the role of exosomes versus other cellular mechanism in the release of HSP60 into the bloodstream. However, the results of the present study supports that HSP60 released in exosomes remains in exosomes.
Exosome Protein Content
As shown in Table 5, comparison of reported content of exosomes from other cell types shows that our exosomes shared only a handful of common proteins with previous studies. Of the 51 ethanol exosomal proteins identified, there were only a few proteins in common with previous studies, including actin, heat shock cognate 71-kDa protein, and GAPDH (2, 6, 8, 18, 29, 37, 42, 51, 53). A number of other proteins that are commonly associated with exosomes, such as HSP90, major histocompatibility complex I/II, and tetraspanins (such as CD63), were not seen in our MS analysis; however, HSP90 was found in hypoxia/reoxygenation-derived exosomes (data not shown). Although these proteins may be considered common exosome proteins, there was still a sizeable variance among proteomic studies of exosome composition (29). In addition, it should be noted that the majority of proteomic studies were taken from human cells, commonly cancer cells. These data give credence to the possibility that the composition of exosomes can vary greatly based on their origins; thus, exosomes from different cell types may serve different purposes based on their composition.
VCP is an abundant cellular protein known by several names, including p97, p97 AAA-ATPase, TER94, and CDc48 (the latter in yeast only). VCP is widely distributed throughout the cell and was originally identified for its role in ubiquitin-mediated protein degradation (1). However, results from a recent study (31) have led to an increasing list of cellular functions and processes in which VCP is involved. Besides its role in protein processing for degradation, VCP has been found to be involved in autophagy, regulation of proteins in the outer mitochondrial membrane through degradation, and endosomal sorting (17). VCP localizes to the endosomal membrane, where it binds early endosome antigen 1 (38). Inhibition of VCP or downregulation with small interfering RNA delayed trafficking of the endocytic cargo and resulted in an increase in size and clustering of the endosomes (38). The presence of VCP in cardiac myocyte-derived exosomes raises the question of whether VCP is involved in exosome formation or exosomal protein trafficking. We identified only one other study where VCP was localized in exosomes (37). VCP's role in the formation of the Golgi stacks as well as its association with the endosomal membrane certainly suggest that this protein is involved in organelle and vesicle dynamics.
Ethanol and Exosomes
An unexpected finding was that ethanol, at concentrations seen with the consumption of alcoholic beverages, greatly increased the production of exosomes by cardiac myocytes. With high-dose ethanol there was a slight increase in apoptosis, but there was none with the low dose. Neither ethanol treatment nor hypoxia/reoxygenation resulted in LDH release. There was also no evidence of apoptosis with hypoxia/reoxygenation. Ethanol has not previously been found to stimulate exosome production. The particle sizing profile for ethanol-derived exosomes compared with hypoxia/reoxygenation-derived exosomes did not differ. Ethanol-induced exosomes appeared to have increased protein content compared with hypoxia/reoxygenation-induced exosomes, based on similar acetylcholine esterase activity, which reflects the amount of membrane present, but a greatly increased exosomal protein content.
Ethanol is a hydroxyl radical scavenger leading to the production of several radicals, of which the 1-hydroxy-ethyl radical is the most abundant (80%) (39). ROS generation in the heart was detected in vivo after the ingestion of alcohol for 2 wk in a rat model (40). Others (27) have found ROS-related cardiac damage within hours of ethanol exposure, and this could be ameliorated by pretreatment with the antioxidant N-acetyl-l-cysteine. The increase in ROS would be exacerbated by a drop in mitochondrial glutathione (cytosolic levels unchanged) observed after one treatment of oral ethanol in a rodent model (16). In the present study, we found that 2 h of ethanol exposure resulted in a marked increase in ROS in cardiac myocytes. Many of the proteins in ethanol-derived exosomes were mitochondrial. We treated cardiac myocytes with a combination of potent antioxidants to reduce ROS production. Exosome quantity, based on the acetylcholine esterase assay, was decreased with both ethanol treatment and hypoxia/reoxygenation. Thus, antioxidants decreased exosome formation but did not completely inhibit it.
In the present study, we found that exosomes are quite stable under physiological as well as pathological conditions. In addition, exosome formation is greatly enhanced after brief exposure to ethanol at concentrations that would be seen after the consumption of multiple alcoholic drinks. Alcohol consumption can lead to cardiomyopathy, and some have proposed that 21–36% of dilated, nonischemic cardiomyopathy is caused by excessive ethanol consumption (10, 23). The enhanced release of exosomes after ethanol treatment raises the possibility that this increase in exosome release might have a role in the eventual development of cardiomyopathy. The increase in exosomes and their protein content after brief ethanol treatment are likely secondary to denatured proteins and cellular damage, in part from increased ROS. This is the first report, to our knowledge, identifying ethanol at levels seen in the general public as stimulating exosome production. Proteomic analysis established that the exosomes contained a wide assortment of proteins, including proteins from the mitochondria, cytosol, and plasma membrane. The protein content of ethanol- versus hypoxia/reoxygenation-derived exosomes differed, suggesting that different stimuli will result in different proteins being released in exosomes from the same cell type. Furthermore, there was a greater amount of protein present in ethanol-induced exosomes. Exosomes form in the multivesicular body, which either traffics to the lysosome or fuses with the plasma membrane, emptying its contents of exosomes into the extracellular space. The regulation of the gating of this process, lysosome versus extracellular space, remains to be understood, but given VPC's known functions, it is possible that it may have a role here, too.
Exosomes are released in the extracellular space and distributed throughout the body. The fate of exosomes remains to be elucidated. One study (43) has suggested that exosomes from cancer cells may convey malignancy to noncancerous cells. Exosomes have also been reported in the urine, and these may arise from the kidney or represent one method of clearing these vesicles. Work to date suggests that exosomes may have a role in intercellular signaling, as cancer studies would suggest (8, 9, 29, 44). Exosomes have also been suggested as a way for viruses to move between cells without being detected (47). More work is need to understand the function and fate of these small vesicles.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-077281 and HL079071 and a Merit Award from the Department of Veterans Affairs (all to A. A. Knowlton).
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: Z.A.M., K.S.K., and A.A.K. conception and design of research; Z.A.M., K.S.K., A.J.P., T.K., L.C., and K.W.F. performed experiments; Z.A.M., K.S.K., A.J.P., T.K., L.C., and A.A.K. analyzed data; Z.A.M., K.S.K., A.J.P., T.K., L.C., and A.A.K. interpreted results of experiments; Z.A.M., A.J.P., T.K., and A.A.K. prepared figures; Z.A.M. and A.A.K. edited and revised manuscript; Z.A.M., K.S.K., A.J.P., L.C., K.W.F., and A.A.K. approved final version of manuscript; A.J.P. and A.A.K. drafted manuscript.
The authors thank James Heiserman and Alice Tran for excellent technical assistance and Dr. Brett S. Phinney and Darren Weber (Proteomics Core Facility, University of California-Davis Genome Center; http://www.proteomics.ucdavis.edu/) for the help with LC-MS/MS.


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"Brilliant and comprehensive! Kivi Leroux Miller covers all the bases in this lively and insightful guide to marketing for nonprofit organizations. The breadth of her experience on both sides of the consultant-client divide shows clearly here. If you're responsible for marketing and communications and stretched to the limit and beyond, buy this book. You'll soon see how much easier your job becomes."
—Mal Warwick, author, Fundraising When Money Is Tight and How to Write Successful Fundraising Letters
"The Nonprofit Marketing Guide provides needed direction for busy nonprofit professionals who find themselves doing multiple jobs without time to focus on effective marketing and communications. This book relieves the feeling of being overwhelmed and provides strategic direction for our valuable resources of time and money."
—Allison Treppa, director of marketing and communications, Michigan Nonprofit Association
"As a tiny nonprofit with a total budget of about $100,000, we obviously have very little set aside for marketing. Kivi understands our situation and provides practical, useful, low-cost marketing advice that produces great results."
—Danielle Denhardt, board of directors, Fancy Cats Rescue Team

subset: commoncrawl


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Northwest Olympic Athletes
The Great Pacific Northwest is being represented in Sochi this year by eight athletes and one very...
2014 Olympic Winter Games
The Opening Ceremonies for the 2014 Olympic Winter Games will begin on February 7th in Sochi,...
< Fighting Suicide/Bullying In Idaho
SEATTLE (AP) - A National Park Service spokeswoman says a
Jan 17, 2012 06:15
Category: General News
Category: General News
Missing Climber Found Alive
SEATTLE (AP) - A National Park Service spokeswoman says a
66-year-old snowshoer who was found alive on Mount Rainier after
three days has been rescued. Spokeswoman Lee Taylor says it took
nine hours to bring Yong Chun Kim out of the backcountry. She says
he didn't require medical treatment and went home. Kim is an
experienced hiker who was equipped for a day trip but had no
overnight gear. He got separated from a group he was leading on the
mountain, 100 miles south of Seattle.


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In this post, I'll show you how to turn a picture frame into a tray. I'm using mine to display vintage sewing ads, needle books, sewing kits, and some beading cord. I love buying old sewing lots at thrift stores and antique shops, but I'm never sure what to do with all of the odds and ends I acquire. I love the graphics on some of these vintage sewing items–they make for a great display. I'm using this tray to hold sewing supplies and projects in progress, so it's functional and cute!
Disclosure: This post contains affiliate links, which means I may receive a percentage if you make a purchase using these links. This won't cost anything extra to you. I only link to things I like and use, and the proceeds help me to run this blog. Thank you for supporting me!
Materials used:
- Picture frame (check thrift stores and antique shops–I got mine for $1)
- Paper for background (Michaels has a great selection of single sheets that you can buy)
- Items for display
- 3m Surgical Tape
- 1/2″ Wire Nails
- Hand Drill Set with Drill Bits
- Handles, 2-Pack
Turn Your Picture Frame into a Tray
I tested out a few different backgrounds and layouts to see what looked best.
My frame was kind of old, so I had to pull up the nails holding the backing in place. Some of the nails were too warped/loose after this, so I had to replace them later on. I used pliers to pull up the nails, and I manually unscrewed the eyelets holding the string in place, so the back would lay flat.
I used the cardboard that I removed from the frame as a template to cut my paper to the right size. Then I started arranging my sewing paraphernalia on it.
Because I had a bunch of small items to display, I used surgical tape to hold them down. I use this tape to hold things in place while sewing, too. This particular tape holds things well, but also isn't hugely sticky, so you can remove it from paper/fabric with ease, just in case I decide to remove these items from the tray at some point and don't want them damaged.
As noted above, I had to replace some of the nails. I only included nails in the long sides of the frame, since I knew I wanted to add handles on the shorter sides. I didn't want to hit any nails when I drilled into the sides.
Here's what it looked like after assembly, but before adding handles.
And here's the finished product with handles. I used drawer-pull handles, but I think most handles would work. These handles are held on with screws. Since my frame was older, I didn't want to risk damaging it with an electric drill, so I pre-drilled the holes for the screws with a hand drill instead. The wood was pretty soft, so this wasn't difficult. Now I'm using it to hold sewing supplies next to my sewing machine!


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Know More About Best Reloading Press
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The Crescent Tongue and Groove Pliers are versatile utility pliers, ideal for objects of all shapes and sizes. They are forged from alloy steel and precision machined for long life and durability. The unique tooth design allows for better grip and increases torque transfer with less effort.
- Self-locking jaw opening adjustment
- Black-oxide finish
- Straight jaw design
- Long handles provide powerful leverage
- Good in quality
- Product is able to withstand wear, pressure or damage


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Fluttershy by KairiChizuruChanWatch
©2013-2017 KairiChizuruChan
This is adorable.
Aw, cute. ^^
I like the background.
I like the background.
thanks i was trying to figure out how to do png but i failed and gave up so i cropped it
Haha, sometimes .png files save as one combined level so I'd be careful, save a copy of the file as the file type your program usually uses and then save another as the .png file type. When you're saving a file below the box where you type in the name of the file there should be another box, there you can click on it and scroll through the different file types.
Hopefully that helps. ^^
Hopefully that helps. ^^
yeah i did that but sadly i got my sai from a friend so it isnt a complete english pack. so i learned full english packs do the png
Aw, that really stinks ... Maybe you could export or copy the image to another program and save a copy .png there?


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Archive for January 2013 | Monthly archive page
Always marching to the beat of his own drum, Travis Pastrana made waves yesterday in the NASCAR world with the unveiling of his car's new paint scheme. Drawing inspiration from 80′s rock, Pastrana's #60 car with Roush Fenway Racing will bring some color to the NASCAR Nationwide Series. Travis was excited to show off his
Scotty Cranmer joins HYPER Bike Co and makes the announcement with style in this new edit currently featured on www.vitalbmx.com. Congrats Scotty on the move!


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- Home
- New Orleans
- Mardi Gras Shot Glass (Set of 2)
Store Credit
You have of store credit. To use it, simply place your order and you will be able to choose store credit as the payment method when it comes time to pay for your order.
Mardi Gras Shot Glass (Set of 2)
Product Description
Mardi Gras Shot Glasses
Dine in elegance with this unique Mardi Gras Shot Glass. A sand-blasting technique leaves this creative glass with a frosted appearance and a beautiful and detailed Mardi Gras scene clearly visible. Your favorite mixed drink contrasts sharply with the white frosted glass, making the Mardi Gras design stand out.
Buy a set for yourself or for friends. These shot glasses will impress friends and family and also make a great gift!
Also appropriate gifts for fans of Carnival celebrations around the world!
Scene Includes:
- Jass and blues band
- Jass and blues band
Each glass is carefully packaged to arrive in perfect condition.
Measures 2½" Tall. Holds 2 oz.


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1545 W. Morse Avenue
Chicago, IL 60626
We host and participate in several events each year to bring our church and community together to have fun, learn and raise funds to continue our mission.
Pie Making: Whether you're an experienced baker or want to learn how to make a pie, you can join us during one of our semi annual, Saturday pie making workshops led by our talented pie maker teacher, Jill Graham. We usually make at least twelve pies to serve a good slice to each guest at the Feast the next day (but we also let you make a small pie of your own to enjoy).
Contact Jill at firstname.lastname@example.com or 847/328/5612
Pancake Breakfasts and Bake Sales: Every Spring and Fall we host a Pancake Breakfast and Bake Sale. We invite our community to come, bring their families and friends, and enjoy a wonderful breakfast of fruit or plain pancakes,sausages, orange juice, and choice of beverages. It is a wonderful time to spend together, pick up some delicious home made baked goods and support our free dinner program.
Website powered by Network Solutions®


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When you are building an empire you know that you are going to have to start off small, you can start off with a dollar store and then take over the world!! Well maybe not that quickly, there are probably a few steps in between.
In Shop Empire you will create your own SIM style set of shops, brought to you by Gamesfree-com this cute little game will really get you addicted.
You will take control of a shopping mall in each country that you play in and you must fill it with everything that the customers want. From bookshops to hair salons, men's clothing and pet stores. Make sure people also have what the need like grocery stores, pharmacies and food courts. You can even get into the entertainment business by creating cinemas and concert halls.
Use the money you earn to buy staff and add more area to your shopping mall. And everything will need to be cared for and maintained. This will include the customers, some of who might be really picky like celebrities. They will want extra attention and their high end shopping needs taken care of.
Another fun element of this game is the security, there will always be some dodgy crook that is trying to steal from you and you customers so you've got to keep them safe and feeling secure.
There is a good tutorial at the start of the game, which will teach the basics pretty quickly. The menus are easy to navigate and everything is laid out in a simple fashion.
Keeping your customers happy is the key to this game as that is how you will earn more money. You can keep your mall open 24 hours and when you have earned enough cash look at investing in new locations with even more lavish decorations to really pull in the rich clients.
Plays: 155222
Plays: 234564
Plays: 61481
Plays: 94635


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The Crafty Owl
I've had a full day today with a group of lovely ladies at my extended technique class. I'll be sharing more of those cards in the coming weeks. Meanwhile, today I'm sharing a card for this week's challenge over at CAS(E) This Sketch that also uses the current colour palette over at Fab Friday:
I was intrigued by the lovely Hannelie's sketch this week and whilst my instinct was to run a mile at the five bright colours over at Fab Friday, I suddenly had an idea that worked perfectly for me when I combined both challenges - take a look:
Here's the low-down on today's card:
Summer Seasonal Layers - I decided to use a bright, white adirondack chair diecut cut with my Seasonal Layers Thinlits for the silhouette motif - complete with cooling lemonade drink on the side for the focal point of my card. It's the perfect fit with that summery colour palette too.
Block That Colour - The five summer colours of the Fab Friday colour palette could be a challenge to use on a single card without them becoming overwhelming. The sketch though allows them to be used for big, bold blocks of colour that have impact whilst balanced by the large expanse of white space that surround them. Perfect!
Holiday Wishes - What sentiment to use on my summery card? I am soon off on my summer vacation and seriously looking forward to whiling away the hours in a chair just like this, with a drink by my side, enjoying the warmth and generally relaxing into my holiday idyll. I created a customised sentiment using the Thoughtful Banners stamp set - what better way to send someone on their summer travels?
Here is the visual summary of the key products used on today's project for you to pin for easy reference:
You can see all of my 'How To' summaries on my 'How To - Tips and Techniques' Pinterest board in case you've missed any.
I'll be back tomorrow with another project. I'm hoping for the return of warmer weather here tomorrow as otherwise I will have a couple of boisterous, stir-crazy boys on my hands as we have a friend coming over to play. Wish me luck! Until then, happy stampin'!
Stampin' Up! Supplies Used:
Tags: Seasonal Layers Thinlits, Stamping, Stampin' Up, Stampin' Up Card, Stampin' Up Card ideas, Stampin' Up Supplies, Shop online, Shop Stampin' Up online, Shop Stampin' Up! Online, Colour Blocking, Thoughtful Banners
Cards | Shop Online | Shop Stampin' Up! Online | Stamping
Monday again - doesn't a new week seem to come around so quickly? I have one James Junior on school holiday as of today, so need to be more organised than ever! Today I'm sharing a card for this week's colour challenge over at CAS Colours and Sketches:
I have been making cards for the second of this month's technique classes and these colours were perfect for another card using my Oh So Eclectic Bundle - take a look:
Here's the low-down on today's card:
Many Eclectic Layers - The Eclectic Layers Thinlits are so versatile: a number of them can be used nested, for example to cut out an outlined veined flower, or alternatively just the border edge can be used - as I've done to cut out my green-blue stamped flower here - or even just the detailed section as I've done with my other two flowers, to reveal the paper below. There really are so many different options when it comes to creating with these thinlits!
Naturally Eclectic Watercolour - I'd like to say that the lovely Berry Burst watercoloured background was all me, but I can't take the credit. Well, I can take the credit for choosing this rather lovely piece of naturally Eclectic designer series paper, but that's all! I love it when the paper does all the work!
Layering Colour - I decided to create an unorthodox 'Island Indigo on Lime Green' flower to complete my trio; whilst this might not be the most authentic of flowers, I do think the colours pair beautifully when layered together. I then added a block of indigo for my sentiment and a few glitter enamel dots to complete my card.
Enjoy your day and I'll be back tomorrow with another project. Until then, happy stampin'!
Tags: Oh So Eclectic, Eclectic Layers Thinlits, Stampin' Up, Stampin' Up Card, Stampin' Up Card ideas, Stampin' Up Supplies, Shop online, Shop Stampin' Up online, Shop Stampin' Up! Online
Today I've a quick second post - you can see my earlier post for this week's new challenge over at The Paper Players here - with my card for this week's challenge over at The Challenge, which also happens to use the sketch I used for yesterday's card from the team over at The Global Design Project:
I'm really enjoying playing along with the lovely ladies over at The Challenge again. I love those bright flowers and the striped arms of the chair worked perfectly with the sketch and inspired me to create something clean and modern - take a look:
I used my Oh So Eclectic stamp set and co-ordinating Eclectic Layers Thinlits Dies for my flowers, offsetting the layers, and combined with some stripes in black on my white card base, courtesy of the Thoughtful Banners set, which I used for my sentiment too. I added a trio of Berry Burst glittery enamel dots to finish - love these!
I really like this card and prefer it to my first GDP attempt. Funny how sometimes an inspiration photo can make all the difference to your creative direction. I'll be back tomorrow with another project. Until then, happy stampin'!
Tags: Oh So Eclectic, Eclectic Layers Thinlits, Stampin' Up, Stampin' Up Card, Stampin' Up Card ideas, Stampin' Up Supplies, Shop online, Shop Stampin' Up online, Shop Stampin' Up! Online, Thoughtful Banners
It's Sunday and time for this week's new challenge over at The Paper Players! It's my wonderful friend LeAnne's turn to host this week and she has a fun colour challenge for us to work with:
Ok, time for confessions first! I saw the colours on the badge and had such a clear idea of what I wanted to make when I was preparing my card that I pulled the colours from my ink caddy and set to work. It wasn't until I started to write this post that whilst I had used monochromatic purples, I hadn't used the three on LeAnne's swatch! I actually used Sweet Sugarplum, Rich Razzleberry and Fresh Fig for my three colours and I didn't have time to remake. So I'm hoping for forgiveness from LeAnne for choosing Fresh Fig instead of Berry Burst and that you can all use your imagination!
I took the opportunity to create a big bunch of flowers using the Beautiful Bouquet bundle - take a look:
Pick A Bunch - Like most of the team this week, I was drawn to use flowers with this monochromatic colour scheme and decide to ink up my Beautiful Bouquet stamp set and create a bunch of flowers. The thing I love most about this set is the co-ordinating framelits: the Bouquet Bunch Framelit Dies make cutting out the flower heads and stems really easy and enable you to create layered florals with ease.
A Beautiful Bouquet - In addition to the flower pieces, the stamp set also contains a great selection of sentiments in a lovely combined block and scripted font. I chose one of my favourites for my card today - the perfect greeting for a friend who means a lot that lets them know how you feel.
The Importance of White - I often talk about the importance of white on cards, usually in the context of 'white space' or clear and uncluttered space, a key feature of clean and simple cards. On today's card white plays an important role as a colour to break up the monochromatic colours and add definition to the flowers themselves.
I really like this card, even if the colours are not quite what LeAnne had in mind when she set us the challenge!
You can see all of my Product and 'How To' summaries on my 'How To - Tips and Techniques' Pinterest board in case you've missed any.
Before you reach for your ink and papers, check out what our amazing team has created to inspire you this week:
I look forward to seeing what you create with this week's colour palette. Enjoy the rest of your weekend as it will be over all too quickly; I'll be back tomorrow with another project; until then, happy stampin'!
Stampin' Up! Supplies Used:
Tags: Shop Stampin' Up! Online, Stamping Shop Stampin' Up online, Shop online, Stampin' Up, Stampin' Up Card ideas, Stampin' Up Supplies, Stampin' Up Card, The Paper Players, Beautiful Bouquet, Bouquet Bunch Framelits
Today I've a quick post as I'm playing catch-up to share a quick card that I made using this week's sketch over at The Global Design Project:
I considered all those lines and came up with this card using the new Basics (Pack 3) washi tape:
Super simple, I inset my stripes and used my top mat to frame them, then added a layered flower and sentiment using the Oh So Eclectic stamp set and co-ordinating Eclectic Layers Thinlits Dies. Washi tape is so useful for easy cards and I love that the new ones come in shades of colour across one area of the colour spectrum and are of differing widths.
I've been making lots of samples using this set in readiness for my technique class that features it next week so have lots more where this came from, including another one using the same sketch that I'll share tomorrow after my Paper Players post.
I'll be back tomorrow with my card for this week's new challenge over at The Paper Players and another project. Until then, happy stampin'!
Tags: Oh So Eclectic, Eclectic Layers Thinlits, Washi Tape, Stampin' Up, Stampin' Up Card, Stampin' Up Card ideas, Stampin' Up Supplies, Shop online, Shop Stampin' Up online, Shop Stampin' Up! Online
I'm having a busy week as the youngest of the James Juniors is on the last week of the school year this week so blogging is having to take something of a back seat whilst I get other work things in order and prepare for the long summer break! I did find time yesterday to make a card for a couple of challenges that I wanted to share with you today. Yes, another scorcher here in the UK today and I'm sharing a Christmas card! My original inspiration was this rather perplexing sketch over at CAS(E) This Sketch:
It's been swirling around in my creative subconscious for days and I was keen to play along as I love their sketches but when combined with this week's challenge over at Stamp Ink Paper I finally found my answer - take a look:
My 'Star of Light' stamp set to the rescue! This stamp set has to me one of my most-used Christmas sets last year and worth it for that fabulous swirl of trailing sparkle alone in my opinion. Some background sponging and a white glimmer star, cut using my Starlight Thinlits Dies, completes the picture. I even remembered to take a photograph of the inside to show you, so that you can see how the greeting follows on:
A card that took longer to execute than to explain, but I guess that's the beauty of the creative process! If you don't have this set, I can definitely recommend it; buy both the stamp set and co-ordinating thinlits this month and you will not only be able to get a head start on your Christmas cards, you'll get a £5 voucher to spend next month too!
I've a busy couple of days ahead but promise I'll be back soon with another project. Until then, happy stampin'!
Tags: Christmas, Starlight Thinlits Dies, Star of Light, Stampin' Up, Stampin' Up Card, Stampin' Up Card ideas, Stampin' Up Supplies, Shop online, Shop Stampin' Up online, Shop Stampin' Up! Online
Today I'm here with a post that should have gone live on Saturday but appears to have had some scheduling issues - but it's news that's too good not to share! First things first: BONUS DAYS ARE BACK! Do you remember this promotion from last year? It was so successful, Stampin' Up! have decided to repeat it this year.
For every £45.00 you spend between 1 July and 31 July (before delivery), you'll get a £4.50 Bonus Days coupon that you can use between 1 and 31 August . Since our new annual catalogue has only been out a month ago, you will probably still have some items on your wish list and this is a great opportunity to shop from it and make savings when you shop again in August with your coupons!There are some details in terms of small print that you need to take note of, as follows:
This is such a great promotion - I get to buy even more from my wishlist, then as a demonstrator I can use my coupons in August against my pre-order from the new Autumn-Winter catalogue - woohoo! For customers there is also going to be the early release of a new exclusive Christmas bundle called 'Carols Of Christmas' for sale from 1 August, so you could get started early with some and use your coupons to get a great deal!
Don't forget if you are placing an order online to also include this month's hostess code JG7PHQKR to receive a free thank you gift from me too!
Here is a sneak peak of the exclusive bundle available from 1st August - you can choose to spend your coupon on this bundle or any other items of your choice!
Can't wait until 1st August to get your hands on this great new stamp set? Well, there's more! All of us at Stampin' Up! miss Christmas so much that we decided to have another one right now with a fantastic offer for anyone out there who would like to join my team this month!
Between 1 and 31 July, every new recruit gets an Essentials Gift Pack (worth 63,50 € / £47) plus the Carols of Christmas Stamp Set (worth 24 € / £18) from the upcoming autumn/winter catalogue added to their starter kit for free! This means that new recruits will get products and business supplies worth a whopping 262,50 € / £195 for just 129 € / £99 with free shipping!
Here's what's included in the Essentials Gift Pack:
This is a great collection of basic items that along with your starter kit will enable you to hit the ground running and easily run your first event and share your love of stamping with a friend or two.
Here's a close-up of the Carols of Christmas stamp set:
There has never been a better time to join Stampin' Up!: we are at the beginning of a new quarter, so you will have a full 6 months before you need to meet your first quarterly minimum, and the run up to Christmas is a great time to build a business if you're looking to earn or boost your income doing something that you love. I am always keen to hear from people thinking about joining my team - why not give me a call to discuss what it could mean for you?
That's all from me today; happy shopping, knowing that you get a spending spree next month too!
Tags: Shop online, Shop Stampin' Up online, Stampin' Up, Stampin' Up Card ideas, Stampin' Up Supplies, Promotions, Bonus Days, Stamping, Carols Of Christmas, join my team, join Stampin' Up, Earn Extra income
Join My Team | Promotions | Shop Online | Shop Stampin' Up! Online | Stamping
It's Sunday and time for this week's new challenge over at The Paper Players! I am the hostess this week and I have a timely theme for you to work with:
We are almost at the end of the academic year here in the UK and so it's the perfect time for teacher thank you cards. It's also the start of exam results season: everything from Common Entrance Exams, to the 11 Plus, graduate degrees and next month GCSE and A Level results. Since our challenge is an international one and all countries have these things happening at different times, I thought I would broaden this week's theme to encapsulate all of these things. So whether it's a thank you for your favourite teacher or a message of congratulations, we would love to see them all in the gallery this week.
I've made my teacher thank yous already, however I know a number of children that sat GCSE exams this year and so will be requiring a few more congratulations cards than usual. I used today's challenge as an opportunity to get started! This week's sketch over at Freshly Made Sketches inspired my layout - I turned it through 90 degrees - and created a few cards using the same design; take a look:
Text Ya Later - This set was one of the first on my wish list; perfect for those tricky teenager cards, this on-trend stamp set, with it's text-speak and humorous emoticons works for children and adults alike. How many of us are in family message groups that span generations and often result in some hilarious texting faux-pas? There is so much fun to be had with these stamps, with the ability to tailor the messages to a number of different occasions. There are also some great background stamps that enable you to infill the speech bubbles with colour with ease.
Stars In Their Eyes - For the strip detail, I cut a row of stars out of coloured cardstock using one of the thinlits in the Mini Treat Bag Thinlits set, to add to the celebratory feel.
A Neat Little Trick - If you look really closely at my card, you will see that the smiley face emoticon has been punched out and added to my coloured speech bubbles. Want to know how I did it? I discovered that the single berry on the Holly Berry Builder Punch i just the right size to punch out these emoticons - handy, right?
Before I go, I also have another card that I made at the same time using the same sketch and stamp set whilst everything was out on my desk that uses this week's colours over at The Global Design Project:
Rebecca saw my congratulations cards and immediately requested a fun card for one of her friends whose birthday it is this week:
Those words just crack me up every time I read them - gotta love predictive text right? I just know this set is going to get a lot of use this year!
I look forward to seeing what you create with this week's theme. Enjoy the rest of your weekend as it will be over all too quickly; we are having the a relatively quiet day today since having had a very busy week it's the first day in over a week that the entire family will be at home together - how crazy is that?!
I'll be back tomorrow with another project; until then, happy stampin'!
Tags: Shop Stampin' Up! Online, Stamping Shop Stampin' Up online, Shop online, Stampin' Up, Stampin' Up Card ideas, Stampin' Up Supplies, Stampin' Up Card, The Paper Players, Text Ya Later, Pick A Pattern, Mini Treat Bag Thinlit Die
Before I share today's project, I just want to give you a reminder that today is the final day for the 'We <3 Hosts' promotion where you can earn an extra £30 of rewards with an order of £300 or more. If you would like to refresh your memory on the details, you can find them here. You can place your order online or alternatively feel free to call me if you'd like to place your order over the phone. It's also the final day for this month's hostess code - if you are placing an order online for between £25 and £150, don't forget to add the code B3C3DUB6 to receive an extra thank you in the post from me.
Today I'm sharing another of the cards I designed for one of my recent team training meetings - you can see one of our other makes here. This one features probably my favourite of the new in-colours, lemon lime twist:
Lovely Words For A Friend - I used the Lovely Words Thinlits for the 'friend' diecut on my card, snipping it away from the oval so that I could use it standalone. These are such great thinlits with so many ways they can be used to create interest on your cards.
CASE The Kit - The design of my card was inspired by one of the cards in the new 'Hello Friend' heat embossing product kit on page 16 of the new annual catalogue. One of the business topics we discussed at team training was how Stampin' Up!'s kits can benefit your business and our make and takes all included opportunities to practice the techniques and skills required to demonstrate with this kit effectively. There's nothing like being prepared and I'm sure my team would agree that team training is great for this kind of thing. Check out this video from the home office team that shows you the Hello Friend Kit in more detail:
Stampin' Up!'s kits are great for a crafty get-together with friends; if you're reading this and would like to arrange one with me to guide you through the fun techniques, then do get in touch.
Copper Trim - To tie in with the dip-embossed copper of my diecut sentiment, I finished my card with a length of the new copper trim. This is a really versatile trim option for cards, home decor and gifts; silky smooth, you can use it like ribbon as it ties beautiful bows, but you can also split it and unravel it and use it like net, or even stretch it and use it as a cover for small items such as chocolates (a little like a sock!)
Tags: Shop online, Shop Stampin' Up online, Shop Stampin' Up! Online, Stampin' Up, Stampin' Up Card, Stampin' Up Card ideas, Stampin' Up Supplies, In Colours, Lovely Words Thinlits, Lovely Inside and Out, Hello Friend Heat Embossing Project Kit
Cards | Host a Party | hosting | Join My Team | Shop Online | Shop Stampin' Up! Online | Stampin' Up! Events | Stamping
Today I'm sharing another of the cards from my recent technique class featuring the Happy Birthday Gorgeous bundle. The outside is a CASE (copy and share everything) of the card on page 76 of the annual catalogue:
Hip Hip Hooray - Today's card again features the 'Happy Birthday Gorgeous' stamp set, this time focusing on the balloons rather than the flowers for decoration, and also highlights the Happy Birthday Thinlit. These items are available as a bundle and whilst the thinlit is proving popular as a standalone item with many of my customers, the stamp set really is worth a look: flowers and balloons that have universal appeal and lots of great birthday sentiments, some of which also co-ordinate with the classic label punch, that I've used for the 'hip hip hooray' on my card front today. Besides, with a 10% discount when you buy both items, you are also getting even more value from your purchases - just sayin' :-)
A Happy Birthday Surprise - So you might be thinking I've lost the plot, as I mentioned above I was featuring both items in the bundle and the Happy Birthday Thinlit is no-where to be seen. Well, look what awaits when you open the card:
There it is! I made an easy pop-up mechanism so that as the recipient opens the card, they get a big, bold birthday message - how fun is that? This is so easy to do: you just need to make a couple of slits a few centimetres in from each side along the central fold and add a couple of additional scorelines in between them, then adhere the base of your diecut to the pop-out section.
A Hint Of Fancy Foil - Lastly, for a little pizzazz I added a few strips of the super-shiny Foil Frenzy cardstock with the Tangerine Tango pattern to both the outside and inside of my card. I will be using most of mine for boxes and packaging as it is super sturdy, although it's worth keeping any offcuts to use on cards as a little goes a long way.
Tags: Happy Birthday Gorgeous, Shop online, Shop Stampin' Up online, Shop Stampin' Up! Online, Stampin' Up, Stampin' Up Card, Stampin' Up Card ideas, Stampin' Up SuppliesBalloon Bouquet Punch, Happy Birthday Thinlits
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Hi I'm Joanne and live in Northampton and when I'm not looking after my husband and two beautiful children, am an avid crafter with a passion for all things Stampin' Up!
I must confess that I have never been a big rubber stamper - until I discovered Stampin' Up! that is. I love the fact that I can get such great results with co-ordinating stamps, inks, papers and punches - the possibilities are endless! Join me on my stamping journey as I share the things I love and be inspired to create amazing things yourself!
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EU's 'fanatic' Brexit negotiator threatens David Davis with 'HELL of a conversation'
THE European Parliament's Brexit negotiator Guy Verhofstadt has promised David Davis a "hell of a conversation" after the Brexiteer appeared to compare Mr Verhofstadt to Satan.
Ahead of their meeting on Tuesday, Mr Verhofstadt tweeted: "Looking forward to a hell of a conversation with David Davis tomorrow."
He also attached a link to a newspaper report in which Mr Davis said "Get thee behind me Satan!" after he was asked about the negotiator at appearance before MPs in September.
The Brexit bigwigs are meeting after it was reported that a bid to block Britain's exit from the EU is being offered financial backing by Virgin Group.
David Davis MP (left) is set for Brexit talks with negotiator Guy Verhofstadt on Tuesday
The Independent claims Richard Branson's firm has offered at least £25,000 to the anti-Brexit campaign after a memo from Blairite Alan Milburn revealed the organisation is in the final stages of development.
The anti-Brexit group has reportedly received backing from a number business and communications organisations while former Liberal Deomcrat leader Nick Clegg, keen Remainer Chuka Umunna and Bob Geldof have been in contact with the group.
The memo from Mr Milburn said: "We have been beavering away over the last few months to get a Europe campaign up and running. I'm pleased to say that substantial progress has been made.
Looking forward to a hell of a conversation with David Davis tomorrow :) https://t.co/R5r67Shm8P— Guy Verhofstadt (@GuyVerhofstadt) November 21, 2016
"I have been in discussions with an excellent potential CEO to lead the campaign.
"Virgin... are keen to help. Since we last spoke [they] have offered a further £25k, plus bigger office space, help with legal advice and a possible secondment.
"I have held discussions with Stronger In, Chuka Umunna, a new organisation called Common Ground, Bob Geldof and a number of senior politicians across the party spectrum."
The group will launch next year and plans to push through a second EU referendum - despite Prime Minister Theresa May stating that Article 50 will be triggered by next March.
Bob Geldof has reportedly been contacted by the pro-EU group
We have been beavering away over the last few months to get a Europe campaign up and running
Virgin has denied any involvement in anti-Brexit groups however.
A spokesman told The Independent: "Since the EU referendum, Virgin has not made any payments to any Brexit campaign or organisation.
"It is well known that Richard supported the Remain campaign before the referendum, and as a result people involved in all sides of the debate are interested in Virgin's views on Brexit and its impact on business.
"Virgin is interested in engaging with different organisations to understand the impact Brexit will have on the UK."
Richard Branson's Virgin Group have reportedly backed the pro-EU group
News of the pro-EU group comes as rumours that Tony Blair is preparing for a return to British politics have gained steady momentum.
Although he is not believed to have a role in the group at the moment, there are a number of Blairite former ministers involved and it be seen as a way for the former PM to creep back into British politics.


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Cookies are a favourite snack to many. It is the best snack that you can have and truly relish its every bite. There are many varieties of cookies that you may know to prepare; however, the crunchy coconut cookie is something that you must try today.
Cookies are very good for health too, as they serve many health benefits too. When you are hungry, all you need to do is to grab a cookie and have a bite and for sure your purpose will be solved.
This one's a quick delicacy for beating those hunger pangs as well.Another reason to eat cookies is that it does not contain any fat.
It is not a junk food that adds up to your calories. So, do you need any more reasons to have these delicious munchies?
Then let's start preparing the crunchy coconut cookie recipe, shall we?
Serves - 4
Preparation time - 15 minutes
Cooking time - 15 minutes
- Butter - 1 cup (unsalted)
- Brown sugar - 1 cup
- Coconut essence - 1/4th teaspoon
- Vanilla essence - 1/4th teaspoon
- Grated coconut - 1 cup
- Egg - 1
- Milk - 1/2 cup
- Maida - 1/2 cup
- Baking powder - 1/2 teaspoon
- Take a bowl and keep it on the stove and then add butter to it to melt the butter. Switch off the flame once the butter has melted.
- To this, add brown sugar and egg, and beat the mixture well.
- Then, add grated coconut, coconut essence and vanilla essence.
- Then, add a little milk and beat it again. Then add the maida flour, so that the mixture becomes a little thick. Finally add the baking powder.
- Make small, round flattened balls and place it on the cookie pan.
- Keep it in the oven at 160 degree Celcius and bake it for 15 minutes.
Serve the hot coconut cookies with hot coffee or tea or just enjoy the plain cookie.
Try this delicious cookie munchy and give us your feedback.


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With the 2nd year of my 2 year term coming to a close as your President and Chair of the Board of Director's, I would like to share some the achievements of the last year with you for awareness and acknowledgement:
On behalf of the Board of Directors, I would like to extend my sincere appreciation to the staff at the ORFA who continue to provide our members and the Board with "excellent services".
Finally, I would like to thank my fellow Board members and St. Michael's College School (my employer), for their ongoing support, especially for outgoing Board members Julie Mitchell, Justin Fidler and Past President Larry Fisher, your service to our Board and Association is much appreciated. I look forward to working with our new President, Ross Rankin and our new Board members as we continue "to be the leaders in the Recreation Facilities Profession".
Rob Tunney, RRFA
In this section


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About "Scuba Gooding Jr. " by brunosomoza and MartinRognoli
This is Scuba Gooding Jr. before having a really unfortunate accident on his leg.
Design by brunosomoza and MartinRognoli
Great style!!
Sooo great! You had me with the illustration and the title just pushed it over the awesome edge!
i want it!!
thanks for the support!
haha so good lol Love the duck floatie and fish biting his tale! Great title!
nice work!
:D awesome \m/
Very cool character
Killa illustrtion Mangs !
Haha! Very good!
Really thanks guys!
Aahah, amazing!
Great design for the Summer Challenge. Hope they print this!
Yeah I hope that too! thanks!
nice work $5!
Great Illustration! 5!
cool style, you rawk \m/
i like it
Love the colors and style!
$5 yay!! cooool :D
Hey!! thanks for such a great feedback!
i love it.
Awesome style and character!


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I'm still here! I know it's been ages since my last post, and I NEVER like to let that happen, Tomorrow we turn the calendar over to May and it will be 13 days and counting before I leave for Portland. My head is swimming with things I still need to do! I've been up at 5AM every day working on patterns, and FINALLY the early hours are paying off. I am starting to see new patterns arrive from the printer, quilts coming back from the photographer and a bedroom stacked with things that will be going with me. Oh my, the chaos before a market is something to see (:
For the next few weeks, in the middle of all the chaos, I'm going to give you peeks of what's to come. Little bits of new patterns, market preparation, and what it takes to pull off International Quilt Market. It's so much work to get there, and I'll show you some of "what's happening" at Bunny Hill!
This Sweet Dreams pillow is part of this…
…stack of pillows waiting to be packed to go.
One more and they all come tumbling down!
Stay with me, I've got lots to share! It's the events before the event and it's a bit crazy.


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...and the crowd goes wild!
her customized plan makes it easy for me.
She researched ideal platforms and approaches, and she developed a useful plan filled with practical tips and steps. Cara is a social media guru for business owners! If you want your company's social media presence to actually be part of your marketing (and client-relationship-building!) plan, you need to work with Cara.
— Terri Wangle, Spotlight Ink
She is BAD-ASS!!!!
Cara is the go to girl for all things web related.
— Dale Clark, Golf Rocks
Hiring Cara, has without a doubt, been one of the best decisions I have made for my organization.
Cara is the consummate professional, trustworthy and incredibly easy to work with. Most importantly, she executes my image and social media with the same care I would as the owner - only in a comprehensive, strategic fashion.
— Danielle Pieters, Arcadia + Haven
Cara Chace Works Magic.
I didn't know how much I needed Cara until she provided a concise, action-plan-filled review of my very new website and social media platforms.
— Diane, Totlosophy
I desperately needed a complete overhaul of my online presence.
Cara really listens and hears what is and what is not said ... her technical skills, passion for her work, and brilliant insights are evident in the results she delivers.
— Elizabeth Glick
I didn't know what I needed to focus on. I felt like I was stuck.
Fortunately, Cara Chace came along with her magic touch. Cara looked over my accounts and she gave me her "no fluff", clear-cut plan on what I needed to do and how. It's been a lifesaver with my time and sanity!
— Ashley, Salvaged Angels