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Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrPC). Here we develop a unique cell system in which epitope-tagged PrPC is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrPC, when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrPSc). Using this epitope-tagged PrPSc, we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion.
Transmissible spongiform encephalopathies or prion diseases are fatal neurodegenerative diseases of humans and animals, and are characterized by the accumulation of misfolded prion protein (PrPSc) in the brain1. Prion disease is caused by the templated misfolding of a normal host cellular prion protein (PrPC) by PrPSc, which can be derived either by infection from an exogenous prion source (for example, scrapie in sheep, bovine spongiform encephalopathy in cattle and kuru in humans), from the pathological misfolding of endogenous PrPC due to mutations in the PrP gene (for example, fatal familial insomnia) or due to rare stochastic misfolding of normal PrPC protein (for example, sporadic Creutzfeldt-Jakob disease)2. Neuropathologically, prion diseases show widespread spongiosis, neuronal loss and gliosis with strain-specific patterns of neuronal cell tropism. The spread of misfolded prion protein throughout the brain from the site of initial prion conversion appears to be a process dependent on close cell association, often occurring along well-defined synaptic tracts3. There is growing evidence that several other proteinopathies that result in neurodegeneration may follow a similar prion-like spread of protein misfolding from a localized region, making the study of this process relevant to other diseases including Alzheimer's disease and Parkinson's disease4.
Despite intense efforts, the cell biology of prion conversion and trafficking is relatively poorly defined. Cell lines that are susceptible to prion infection have been used to model the process in vitro5,6,7 but are limited because the infectious agent PrPSc is immunologically indistinguishable from the PrPC expressed on the recipient cell. This means that newly formed PrPSc generated on the recipient cell cannot be specifically identified, making the conversion process itself difficult to study directly. Previous studies have relied on prolonged culture of cell lines that stably propagate prions to clear any inocula-derived PrPSc—known to persist in cells for two weeks or more8,9—before the cell biology of newly formed PrPSc can be analysed10. Therefore, fundamental information such as the timescale of the infection process or the cellular site in which the initial PrP conversion occurs has not been established.
To study the earliest events in cellular prion infection, it is essential to distinguish the inocula-derived PrPSc species from newly formed PrPSc generated in the recipient cell. Inserting an epitope tag into the sequence of the endogenous PrPC such that the tag can be used to differentiate de novo formed PrPSc is an elegant solution to this problem; however, the generation of true infectious prions containing an epitope tag has yet to be demonstrated in cells. This is probably because the sequence sensitivity of the conversion process prevents the misfolding of most tagged PrPC molecules11. The sequence sensitivity is such that the prion seed (exogenous PrPSc) and PrPC substrate (endogenous cellular PrPC) must have homologous primary structures for efficient conversion to take place, particularly in certain key regions of the PrP molecule12,13. Hence, previous attempts to produce PrP constructs with green fluorescent protein reporter inserts or other epitope tags have not generated PrP chimerae that are capable of prion conversion14,15.
Here we report on the generation of a unique cell system in which a novel epitope-tagged PrPC is expressed in a neuroblastoma cell line in which the endogenous PrPC has been silenced by RNA interference. We show that this engineered PrPC chimera supports prion replication and results in the production of a bona fide epitope-tagged PrPSc as evidenced by both in vitro assays and mice bioassays. Using this cellular system, we have studied the earliest events in cellular prion infection and PrP misfolding; we show that prion infection of cells is extremely rapid, occurring within 1 min of prion exposure, and demonstrate that the plasma membrane is the initial site of prion conversion.
Generation of cell lines expressing epitope-tagged PrPSc
To study prion infection in cells expressing tagged PrPC constructs without any interference from the host wild-type PrPC, we produced PrP knockdown (KD) cells derived from the mouse PK1 prion-susceptible neuroblastoma cell line16, using RNA interference (RNAi) directed at the 3′ untranslated region (3′-UTR) of PrPC mRNA. This effectively silences PrP gene expression (Fig. 1a,b) and results in the complete inhibition of prion propagation (Fig. 1c). This strategy allows the expression of the mouse PrPC open reading frame from exogenous constructs lacking the 3′-UTR, and permits the expression of a panel of FLAG- and MYC-tagged PrPC chimerae. The PrP constructs were designed such that the epitope tags were inserted into regions of the murine PrPC sequence that are relatively unstructured, and therefore theoretically unlikely to interfere with prion conversion (Supplementary Fig. S1a)12,17. Wild-type PK1 cells, PrP-KD cells and cells expressing tagged PrPC constructs were exposed to Rocky Mountain Laboratory (RML) mouse prions, and their ability to support prion propagation was assayed using the Scrapie Cell Assay16. PrP constructs 22CysFLAG and 30GlyFLAG were found to reconstitute prion propagation very efficiently and contained high levels of PrPSc. However, it became apparent during the course of our study that these amino (N)-terminal FLAG tags were readily cleaved during cellular processing such that a proportion of the tagged PrPSc in the cells was not detected by the anti-FLAG antibodies, making them unsuitable for this study. Cells expressing PrPC with epitope tags placed centrally within the PrP sequence (for example, PrP-124AlaMYC, Supplementary Fig. S1a) did not support prion propagation (Fig. 1c). However, cells expressing PrP-224AlaMYC with the epitope tag inserted near the carboxy (C)-terminus were found to propagate prions robustly and reproducibly (Fig. 1c). Immunofluorescence and immunoblot analyses demonstrate that the expression levels and the cellular distribution of PrP-224AlaMYC were similar to that of wild-type PrPC in PK1 cells (Fig. 1a,b). Immunoblotting of proteinase K (PK)-digested cell lysates from RML prion-infected PrP-224AlaMYC cells demonstrates that they contain protease-resistant PrP, a characteristic feature of PrPSc18 (Supplementary Fig. S1b).
PrP-224AlaMYC cells propagate infectious tagged prions in vivo
To confirm that our infected PrP-224AlaMYC cells were generating bona fide prions, the infectious nature of the 224-AlaMYC-PrPSc was analysed in tg20 mice, a transgenic mouse line that overexpresses PrP often used in prion bioassays19. The mice were inoculated intracerebrally with extracts from RML prion-infected PrP-224AlaMYC-expressing cells. PK1 and PrP-KD cells infected in parallel with these cultures were used as positive and negative controls, respectively. Importantly, inoculation of tg20 mice with extracts from RML prion-infected PrP-KD cells (that do not propagate prions) did not result in prion disease in the mice (Fig. 1d,e and Table 1). Inoculation of tg20 mice with RML prion-infected PrP-224AlaMYC cell extracts resulted in early clinical signs of scrapie, whereas extracts from control PrP-224AlaMYC cultures that were not previously exposed to RML prions did not (Table 1). Biochemical analysis of the brains of the scrapie-affected mice showed that they contained PK-resistant PrPSc (Fig. 1d). Histological analyses revealed classical prion neuropathology with PrP deposition, neuronal loss, spongiosis and gliosis (Fig. 1e). Control animals killed in parallel with the scrapie-affected mice did not contain PrPSc or show any pathological features (Fig. 1d,e). These experiments demonstrate that our PrP-KD cells expressing PrP-224AlaMYC propagate true infectious tagged prions following RML prion exposure, and cause disease indistinguishable from that caused by RML prions propagated in wild-type PK1 cells.
Characterization of MYC-tagged PrPSc cellular distribution
To perform a detailed analysis of prion infection in cultured cells, it is necessary to distinguish newly synthesized PrPSc from the host cellular PrPC. Three established methods achieve this by utilizing the biochemical differences between native PrPC and misfolded PrPSc: These are PK digestion, guanidinium hydrochloride (GdnHCl) denaturation and formic acid extraction20,21. We compared each method in detail by immunostaining our PrP-224AlaMYC-expressing cells with anti-MYC antibodies. Both PK and formic acid treatment revealed a population of PK and formic acid-resistant MYC-tagged PrPSc in prion-infected cells (Fig. 2a,b), with a strong signal at the plasma membrane and in the perinuclear compartment (PNC), consistent with previous observations of the sub-cellular distribution of PrPSc (refs 20,22,23). In contrast, only very low levels of background immunostaining were detected in uninfected cells, indicating that PrPC had been effectively removed by these treatments. GdnHCl denaturation was less effective at removing PrPC staining (Fig. 2a,b). Similar results were obtained using ScPK1 cells that express wild-type PrPSc (ref. 20) (Supplementary Fig. S2a,b), indicating that our MYC-tagged PrPSc displays a physiologically relevant cellular localization.
The distribution of the formic acid-resistant MYC-tagged PrPSc in PrP-224AlaMYC cells was analysed in detail by dual staining of formic acid-treated cells with anti-MYC and anti-PrP antibodies; importantly, anti-MYC and anti-PrP antibody staining colocalized (Fig. 2c). Further, dual staining experiments using anti-MYC antibodies and antibodies directed against various intracellular markers show partial colocalization of these compartments with formic acid-resistant MYC-tagged PrPSc (Supplementary Fig. S3), consistent with previous reports7,20,22,24 and validating the use of formic acid treatment to reveal PrPSc in cultured cells.
MYC-tagged PrPSc is rapidly formed following prion exposure
Our novel cell system expressing MYC-tagged PrPSc combined with effective PrPC removal by formic acid or PK treatment allows de novo synthesized PrPSc to be clearly distinguished from the host RML prion inocula. This makes a detailed temporal and spatial study of the early stages of cellular prion infection feasible. Previous work in the field suggests that a 72-h exposure to prion-infected material is necessary to fully infect susceptible cells16. Therefore, we first infected PrP-224AlaMYC cells with RML prions for 3 days, and either fixed immediately or passaged in fresh media before fixation, formic acid extraction and anti-MYC staining. Maximum numbers of RML prion-infected MYC-tagged PrPSc cells were seen 6 days post infection (DPI; Fig. 3a) and stable prion propagation in ∼20% of the cells was observed up to 14 DPI (Fig. 3a). PrP-KD cells treated in the same way showed no anti-MYC immunostaining up to 6 DPI (Fig. 3a), indicating that the observed staining was dependent on PrP-224AlaMYC expression. Interestingly, a significant proportion of the PrP-224AlaMYC cells already contained MYC-tagged PrPSc after 3 days of RML prion exposure (Fig. 3a). This prompted us to examine the first 72 h of prion exposure in more detail. Figure 3b shows the proportion of infected cells at different time points of RML prion exposure and reveals that PrP-224AlaMYC cells produce PrPSc rapidly, with MYC-tagged PrPSc-positive prion-infected cells clearly detectable after 2 h of RML prion exposure. These observations suggest that cellular prion infection is faster than previously thought. Therefore, we examined the first few minutes of prion exposure in more detail. PrP-224AlaMYC cells were exposed to RML prions for very short periods before rapid washing and fixation. A significant proportion of the cells showed clearly detectable levels of formic acid-resistant MYC-tagged PrPSc after as little as 1 min of exposure to RML prions (Fig. 3c), the shortest time period technically possible to reproduce accurately within the limitations of our system. PK digestion of similar cells confirmed that protease-resistant MYC-tagged PrPSc was generated after very short periods of prion exposure (Fig. 3d).
Further experiments (Supplementary Fig. S4) confirmed that prion infection is rapid in our system. Washing the cells to remove inocula before culturing the cells in the fresh media (without inocula), demonstrated that short periods of RML prion exposure are sufficient to generate stable PrPSc propagation in our cells. Quantifying the anti-MYC label incorporated into PrP-224AlaMYC cells following RML prion exposure showed that MYC-tagged PrPSc builds up rapidly, reaching maximium levels by 2 h, which are maintained for at least 24 h. We also found that the proportion of prion-susceptible PrP-224AlaMYC cells was not increased if higher concentrations of inocula were applied.
Prion conversion occurs at the cell surface within 1 min
The steep rise in the number of prion-infected cells observed in the first 2 min following RML prion exposure indicates that this is the period when most cells first begin to synthesize PrPSc (Fig. 3c). This allowed us to study the cellular site of this initial prion conversion. Examination of cells that were fixed after 1 min of prion exposure revealed that the MYC-tagged PrPSc signal is only present at the plasma membrane. Orthogonal projections of serial confocal sections clearly showed that PrPSc is present only at the cell surface (Fig. 4a—1 min, white arrows—the z stack series is shown in Supplementary Fig. S5a). This cellular distribution is strikingly different from the more typical plasma membrane and intracellular localization observed in cells that have been exposed to RML prions for longer durations (Fig. 4a—180 min, arrows—the z stack series is shown in Supplementary Fig. S5b). Categorizing the cells into three phenotypes of different PrPSc distributions (plasma membrane, plasma membrane/diffuse intracellular and plasma membrane/PNC), and examining each phenotype at different durations following RML prion exposure revealed a rapid transition from the plasma membrane distribution seen at the earliest time point to the typical plasma membrane/PNC distribution observed later (Fig. 4b). Indeed, cells showing exclusively plasma membrane MYC-tagged PrPSc staining disappeared after 2 min of exposure to RML prions, consistent with rapid PrPSc formation, endocytosis and trafficking.
To confirm this result, we directly compared the formation and trafficking of MYC-tagged PrPSc to the endocytosis of Texas red-labelled transferrin. Transferrin endocytosis and trafficking occurs rapidly, reaching a steady-state distribution at the PNC between 8 and 16 min25 (Fig. 4c). In the same cells, PrPSc attained a steady-state distribution in nearly all cells by 4 min of exposure to prions (Fig. 4b,c). Therefore, prion protein conversion and endocytosis proceeds at rates that are similar to that of transferrin trafficking.
To determine whether PrP prion conversion can occur on the plasma membrane, we incubated pre-cooled PrP-224AlaMYC cells with RML prions at 4 °C. This temperature shift effectively abolished endocytosis in mammalian cells26. Significantly, PrPSc accumulated only on the cell surface under these conditions (Fig. 5a,b, 4 °C). Orthogonal projections of confocal sections taken through these infected cells revealed the plasma membrane distribution of MYC-tagged PrPSc (Fig. 5a—the z stack series is shown in Supplementary Fig. S6) and clearly demonstrated that endocytosis is not necessary to produce misfolded PrP.
To test this hypothesis, we used a range of pharmacological and genetic manipulations designed to inhibit the endocytic pathways that were previously implicated in prion metabolism—dynamin- and clathrin-dependent endocytosis and macropinocytosis. Dynasore was used as a direct dynamin inhibitor27 and RNAi directed at the clathrin heavy chain (CHC) was used to interfere with clathrin function. The functional efficacy of both treatments was shown by the reduced labelled transferrin uptake in the cells, a well-characterized cargo for dynamin- and clathrin-dependent endocytosis (Fig. 5b). Immunoblotting showed that RNAi treatment effectively reduced overall CHC levels (Fig. 5c). However, the transfection efficiency of the RNAi delivery varied in our cell population, with some cells showing an almost complete block in transferrin uptake and other cells maintaining residual endocytic capacity (Fig. 5b). Dynasore and RNAi directed at the CHC did not significantly affect RML prion infection or PrPSc internalization (Fig. 5b,d), and no correlation was observed between an individual cell's susceptibility to prion infection and its ability to endocytose transferrin. Macropinocytosis was targeted using the ion transport inhibitor (N-ethyl-N-isopropyl)amiloride (EIPA)28. This treatment had no effect on labelled transferrin uptake and did not affect RML prion infection in our system (Fig. 5b,d). Incubating pre-cooled PrP-224AlaMYC cells with RML prions at 4 °C confirms that blocking all endocytosis—demonstrated by the lack of transferrin uptake—does not ablate prion conversion (Fig. 5b,d).
An important role for plasma membrane PrPC expression in prion conversion was confirmed by experiments using phosphoinositide-specific phospholipase C (PIPLC) to deplete plasma membrane PrPC. This significantly reduced the generation of MYC-tagged PrPSc (Fig. 6a,b). It is interesting to note that many of the cells in the PrP-224AlaMYC cultures bind and/or internalize inocula-derived PrPSc following RML prion exposure, independent of surface PrPC expression (Fig. 6a). Further, striking examples of this phenomenon are included in Supplementary Figure S7. This suggests that other factors in addition to a simple physical association of cellular PrPC with PrPSc affect prion conversion, an observation in agreement with previously published data29,30,31.
Rapid prion conversion on the cell surface may point to a mechanism in which PrP misfolding is a stochastic event catalysed by the interaction with PrPSc in a favourable environment such as that found in lipid rafts, membrane microdomains thought to have a role in prion conversion32. We therefore tested whether reagents that disrupt lipid raft integrity would affect prion protein conversion. Two reagents that affect lipid raft function by different mechanisms—filipin33, which binds and sequesters plasma membrane cholesterol, and U18666A, which inhibits cholesterol transport to rafts34—abrogated prion conversion in our cellular system (Fig. 6c).
We report on the generation of a unique cell system in which an epitope-tagged PrPC is expressed in a prion-susceptible neuroblastoma cell line in which the host PrPC has been removed using RNAi. This engineered PrPC was shown to support prion replication resulting in the production of a true bona fide, infectious epitope-tagged PrPSc. Using this cellular system we have shown that prion infection of cells is extremely rapid, occurring within 1 min of prion exposure, and we demonstrate unequivocally that the plasma membrane is the initial site of prion conversion.
Generating an epitope-tagged PrPC chimera that is capable of prion conversion and forming an infectious tagged PrPSc is not straightforward. A C-terminal green fluorescent protein-tagged PrPC construct produced by Barmada and Harris14 failed to form PrPSc and even the insertion of a small 10-amino-acid MYC tag at ser-230 of murine PrP abrogated prion conversion when expressed in a PrPC knockout mouse background15. We designed a panel of eight different constructs based on a biophysical analysis of PrPC structure, and screened them for the ability to reconstitute prion propagation in our PrP-KD cells. In agreement with an earlier report17 PrP constructs with FLAG tags inserted near the N-terminus supported efficient prion conversion, but could not be used because this region of the protein was cleaved during the cellular processing of PrPSc. Epitope tags inserted into the central region of the PrPC molecule did not support prion propagation, consistent with previous reports on the importance of sequence conservation in this region for efficient conversion12,13. However, one of our constructs, PrP-224AlaMYC, robustly generated epitope-tagged PrPSc. Significantly, a mouse bioassay demonstrated that the PrP-224AlaMYC cells propagate genuine infectious prions. The capacity of PrP-224AlaMYC cells to produce epitope-tagged PrPSc, combined with effective protocols to remove PrPC from our cells, allowed us to examine PrP misfolding when prions initially contact and infect susceptible cells.
Our temporal analysis of the events immediately following cellular prion exposure revealed that prion infection is far faster than previously thought. Earlier studies had indicated that PrPSc/cell interactions may be rapid35, but de novo PrPSc formation was not studied directly. Here we have demonstrated in a cellular system the rapid protein misfolding and aggregation observed in cell-free experiments36. Consistent with the highly dynamic nature of prion infection of PrP-224AlaMYC cells, we observed that PrPSc had attained steady-state levels by 2 h following RML prion exposure and did not change significantly up to 24 h, indicating that PrPSc metabolism is highly dynamic and the synthesis and breakdown appear normally balanced in these cells. A detailed spatial analysis of cells shortly after prion exposure demonstrated that PrPSc forms on the plasma membrane. The plasma membrane has been suggested to be a potential site of PrP conversion7,21,22,37, which is consistent with the observed PrPSc distribution reported in different cell and in vivo systems21,24,38. Our observations that prion conversion proceeds even at 4 °C under conditions that prohibit endocytic activity in mammalian cells26 confirms that the plasma membrane alone is sufficient for the process. Supporting this idea, we have also shown that plasma membrane PrPC expression is required for efficient prion infection. PIPLC treatment effectively removed plasma membrane PrPC from PrP-224AlaMYC cells and greatly reduced the generation of PrPSc. This is consistent with previous reports showing that PIPLC treatment reduced the generation of PrPSc in chronically prion-infected PK1 cells39, and data indicating that PrPC lacking the GPI anchor (and hence not expressed on the cell surface) does not support efficient PrPSc production or stable cellular prion infection40.
The role of the plasma membrane in prion conversion was explored further by experimental treatments designed to inhibit dynamin/clathrin-dependent endocytosis and macropinocytosis, pathways that have been previously implicated in prion metabolism35,41. These manipulations had little effect on prion infection or PrPSc internalization. This suggests that either they are not involved in PrPSc internalization or that the process can occur through multiple pathways, an idea supported by the literature32. Our data also suggest that prion conversion is dependent on the presence of intact lipid rafts, specialized membrane microdomains known to be present in the plasma membrane and enriched in PrPC (refs 42,43). Supporting this idea, cofactors enriched in lipid rafts have been shown to stimulate synthetic prion conversion29,31 and reagents that disrupt raft integrity abrogate PrPSc propagation in cells44,45. Despite this, our data do not preclude a role for intracellular compartments in prion conversion, which may continue or accelerate the process following the endocytosis of PrPSc. Previous reports have implicated various cell compartments in the conversion process7,46,47. Our observation that prion infection was reduced at 4 °C may reflect a slower reaction rate due to the lower temperature as might be expected for a protein-folding-driven phenomenon, but could also be consistent with an important role for internal compartments in PrPSc propagation. As earlier studies relied on the analysis of cells already propagating prions that express intracellular PrPSc before any manipulations designed to examine the region at which misfolding takes place7,47,48, the contribution of the plasma membrane to the initial prion conversion process may have been underestimated or overlooked.
Our system has also allowed new insights into the dynamic nature of PrPSc trafficking in the cell. The ability to analyse the trafficking events immediately following prion exposure has revealed that PrPSc initially forms at the cell surface, and is rapidly endocytosed. Indeed, after 1 min of exposure to RML prions, many cells already contain intracellular PrPSc, often in a diffuse cellular distribution, possibly reflecting a transition through an endosomal compartment. After 4 min of exposure to prions, PrPSc takes up a steady-state distribution concentrated at the PNC and at the cell surface, as described in the literature20,22,23. The similarity in the appearance of these cells to cells that chronically propagate prions, and our data showing that a 2-min exposure to RML prion brain homogenate is sufficient to generate cells that continue to produce PrPSc in the absence of further prion seed, suggests that cells are stably infected by prions in a very short time period. However, membrane binding and internalization of inocula-derived PrPSc is not in itself sufficient to generate prion infection/de novo PrPSc production. This is clearly demonstrated by our novel cell system and suggests that cell factors, in addition to a simple physical interaction between PrPC and PrPSc, are required for prion formation. This idea has been postulated previously29,31 and is supported by our demonstration that increasing the concentration of inocula added to our PrP224AlaMYC cultures did not lead to an increase in the proportion of infected cells. This indicates that only a proportion of our cells contain/express the necessary susceptibility factors required for prion conversion.
In conclusion, we have presented the generation of a unique cell system producing bona fide epitope-tagged PrPSc, which was analysed using both in vitro and in vivo assays. By visualizing MYC-tagged PrPSc, we have been able to study the earliest events in cellular prion conversion. We have shown that cellular prion infection is a dynamic process that occurs within 1 min of prion exposure, and that the plasma membrane is the primary site of prion conversion. These results have important implications for our understanding of the conversion and spread of prions in vivo. Once a sufficient seed of infectious prions has been established in the brain, our data suggest that prion cellular spread is likely to proceed far more rapidly than previously thought. These data cast new light on why prion infection can be very aggressive in vivo and can affect the whole neural axis in just a few weeks.
Cell lines and DNA constructs
All cells were cultured in OptiMEM, FCS (10%) and penicllin/streptomycin. Short interfering RNA sequences targeting the 3′-UTR of PrPC were cloned into the pSUPER.retro.puro expression vector (Oligoengine) and introduced into wild-type PK1 cells by retroviral transduction49. Stable PrP knockout clones (Supplementary Fig. S1) were isolated and cultured in puromycin selection media. Tagged PrP constructs were generated by overlap extension PCR procedures using standard methods50 (primer sequences available on request), subcloned into the pLNCX-retroviral vector and then sequenced (Clontech). These were introduced into PK1-KD cells by retroviral transduction51, and stable lines expressing the tagged PrP constructs were isolated and routinely cultured in dual selection media (puromycin and G418, Invitrogen).
Cellular prion infection and PrPSc analyses
Brain homogenates were prepared from end-stage RML prion-infected CD-1 mice as a 10% homogenate in PBS16. These were diluted to 0.1% in media, and added to cells grown to 70% confluence that were seeded on poly-L-lysine-coated glass coverslips or plastic dishes. After prion incubation, cells were washed three times with warm PBS and fixed, collected for biochemical analysis or passaged in the ratio 1:8. The Scrapie Cell Assay was performed as described previously16. Cells were routinely prepared for immunofluorescence analysis by fixation in 3.7% paraformaldehyde for 12 min at 37 °C, and then washing in PBS. PrPSc was revealed specifically by a 5-min pre-treatment with 98% formic acid to remove host PrPC (ref. 20). PK digestion and GdnHCl treatment to remove PrPC and expose PrPSc were as described21.
To further characterize prion infection, PrP-224AlaMYC cells were exposed to varying concentrations of RML prions for 180 min before fixation, or incubated with 0.1% RML prions for 2 min, washed three times and fixed immediately or incubated in fresh medium lacking prions for a further 48 h before fixation. In addition, cells were pretreated with vehicle, dynasore (80 μM—added in serum-free media), EIPA (100 μM) and filipin (5 μg ml−1) for 30 min, or U18666A (1 μg ml−1) for 16 h, and then exposed to RML prions. Cells were transfected with RNAi oligos using Fugene 6 reagent according to the manufacturer's instructions. A mixture of four RNAi oligos targeting CHC (Dharmacon on target plus kit) was added at 100 nM for 30 h before the addition of RML prions. PIPLC treatment was carried out using 0.5 U ml−1 PIPLC for 2 h before the addition of RML prions39. The cells were then incubated with RML prions for 180 min in the continued presence of the treatment. Texas red-labelled transferrin (Invitrogen) was added to the media at 5 μg ml−1 for the last 10 min of the incubation. Control experiments replicating the conditions for each treatment but lacking the active reagents were carried out to check that conditions outside our treatments did not affect prion infection. No significant differences were observed. In our 4 °C experiments (Fig. 5a,b), cells were pre-cooled on ice for 30 min before the addition of RML prions made up in ice-cold media. Cells were washed three times with ice-cold PBS, fixed on ice for 10 min and then transferred to 37 °C for a further 10 min before washing with PBS and further processing.
Formic acid-treated and -untreated cells were permeabilized with cold methanol for 10 min at −20 °C and then washed with PBS, before blocking with 10% horse serum and 1% BSA in TBS. Primary antibody incubations were performed overnight at 4 °C in TBS/1% BSA. Following extensive washing in TBS, samples were incubated with AlexaFluor 488 and 543-labelled secondary antibodies diluted in TBS/1% BSA, as appropriate, for 1 h at room temperature. Cells were thoroughly washed in TBS and mounted in Antifade media (DAKO) containing 1-μg ml−1 4′,6-diamidino-2-phenylindole. The primary antibodies used in this study are detailed in Supplementary Table S1. Fluorescence images were acquired using a Zeiss LSM510 meta confocal microscope (Zeiss) with a plan-Apochromat' ×63/1.40 oil DIC objective. Control uninfected cells were processed for each experiment and LSM settings were chosen to produce minimal background staining. The same settings were used to examine all infected cells in that experiment. The extent of MYC label incorporated into the cells was determined using Volocity software (Improvision). At least 50 cells from three independent experiments were analysed per condition. Orthogonal projections were generated from z-stacks using Zeiss LSM software (Zeiss). To determine prion infection, at least 300 cells from eight randomly chosen fields were counted for each condition, and the proportion of cells showing PrPSc immunostaining after PrPC removal was calculated. All experiments were performed by two independent investigators. Blinded analysis by a third investigator was also undertaken. For cell phenotypic analyses, z-stacks encompassing the entire cell were obtained and cells were grouped into one of three categories based on the localization of MYC-tagged PrPSc: exclusively plasma membrane, plasma membrane/diffuse intracellular and plasma membrane/PNC. In each experiment, at least 50 cells were analysed for each condition.
Inoculation of tg20 mice and neuropathological analysis
Inocula were prepared from PK1, PrP-KD and PrP-224AlaMYC cell cultures exposed to prions for 72 h and sub-passaged five times 1:8 (ref. 16). Cells were harvested in sterile PBS, washed twice and then resuspended to 30 million cells per ml. Control PrP-224AlaMYC cells that were not exposed to prions were collected the same way. Inocula were stored in sterile vessels at −80 °C until use. Animals were cared for per institutional and Home Office regulations. For each of the test conditions, groups of five tg20 mice were inoculated intracerebrally with 30 μl of cell suspension. Animals were observed daily for clinical signs and culled on diagnosis of prion disease. Two animals that were not diagnosed with prion disease were culled in parallel with the diseased animals, and the remaining animals were culled 4 to 5 weeks after the last diseased animal was diagnosed. Brains were excised and examined by histopathology and western blotting52.
Cell lysates were collected for biochemical analysis in PBS, washed and resuspended in a small volume of fresh PBS52,53. The cells were lysed by three freeze/thaw cycles and benzonase (50 U ml−1, Merck) was added for 20 min at 4 °C. Protein concentrations in the lysates were determined by Bio-Rad assay (Bio-Rad). For PK treatments, the samples were diluted to 2 mg ml−1 in PBS and Triton X-100 (1%) and PK (5 or 2.5 μg ml−1) was added. Samples were digested for 30 min at 37 °C and digestion was terminated by the addition of phenylmethylsulphonyl fluoride to a 1-mM sample. Proteins were precipitated with cold methanol and resuspended in SDS sample buffer. SDS–polyacrylamide gel electrophoresis and immunoblotting were carried out as described previously52.
Data were expressed as mean plus standard error of the mean (s.e.m.), unless otherwise stated. Data were compared by two-tailed t-tests and considered significantly different when P<0.05. Degree of significance is expressed as follows: P<0.05*; P<0.01**; unless otherwise specified.
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We thank Sebastian Brandner, Jackie Linehan and Caroline Powell for help with histological analysis, Jonathon Wadsworth for advice and critical review of the manuscript, BSF for help with the animal studies, Francesca Properzi and Peter Kloehn for helpful discussions, Dave Emery (Emergen) for help with the molecular biological work and Ray Young for graphics. This work was funded by grants from the UK Department of Health, Medical Research Council and Brain Research Trust.
The authors declare no competing financial interests.
About this article
Cite this article
Goold, R., Rabbanian, S., Sutton, L. et al. Rapid cell-surface prion protein conversion revealed using a novel cell system. Nat Commun 2, 281 (2011). https://doi.org/10.1038/ncomms1282
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domain: health


text:
How to Save Bristol Bay Slideshow
June 20, 2012
1) The Wonders of Wild Salmon
According to the Monterey Bay Aquarium, wild salmon is a rarity these days, with more than half of the salmon on the global market coming from farms. Salmon farming is associated with numerous environmental and health risks, and the leading seafood sustainability organizations recommend avoiding it. This makes it all the more important to protect the wild salmon resources that we have, and Bristol Bay continues to welcome 40 million salmon to its waters each year. And if you've ever tasted farmed salmon next to wild, you already know there's just no comparison in taste.
2) Importance of Salmon to Bristol Bay
Bristol Bay revolves around salmon. The Bristol Bay salmon run generates a $2.2 billion fishing industry, creates more than 12,000 jobs each year, and provides a livelihood for more than 5,000 Alaskan natives. In contrast, the proposed Pebble Mine project predicts the creation of a mere 2,000 jobs, only half of which would remain after the mine is built.
3) The Pebble Deposit
There's no doubt that the Pebble deposit is massive, estimated to be worth $200 to $500 billion. The mass of copper, gold, and molybdenum has been on the radar of mining corporations for a while, but recently the Pebble Limited Partnership started moving plans forward to develop North America's largest open pit mine in the Bay. The catch? The Pebble deposit lies in the headwaters of the region's two most important salmon spawning streams.
4) The Risks
The risks of the proposed Pebble Mine are far-reaching. The mine would be built in a seismically active zone and require the construction of the world's largest earthen dam to contain the billions of pounds of toxic waste rock that would be generated over the lifetime of the mine. Any release of the waste or metals has the potential to negatively impact the salmon run. Scientists have found that even small traces of copper in the waters affect a salmon's ability to smell and navigate. Factor in a potential mine accident or failing of the dam and we can say goodbye to Bristol Bay salmon as we know it.
5) The EPA Assessment
On May 18 the EPA released a draft watershed assessment of Bristol Bay. The report states clearly that any mining project would have damaging effects on the salmon habitat — even without a potential mining accident. The report also opened up a 60-day public comment period during which people can weigh in on whether the EPA should prevent harmful mining activities in Bristol Bay via the Clean Water Act. Telling the EPA our opinion is an easy step we can all take to make a difference.
6) What We Can Do Now
• Speak out during the EPA's comment period, which ends July 23.
• Are you a chef or food professional? Sign this national letter to President Obama urging him to protect our wild salmon resources.
• Spread the word. Tell your family and friends. Write a letter to the editor of your local paper. Post about the comment period on your blog.
• Vote with your fork. The Bristol Bay salmon season begins at the end of June. The best way to show support for the resource is to eat Bristol Bay salmon. The survival of this fishing community depends on the choices we make as conscious consumers.

domain: news


text:
In a partnership, both parties will be willingly involved. The goal is to make the partner happy and feel at ease in the romantic relationship. The romantic bond among both associates is voluntary and mexican ladies dating a vital part of both peoples’ lives. Regardless of this, the risk of a conflicting partner is very big. During the inconsistant periods, it is advisable to leave the partner go and move on to a new person. It’s always better to remain friends than to be stuck in a dangerous cycle of anger and frustration.
A loving relationship can develop into more than one type. It might begin as being a physical or perhaps romantic connection. However , the relationship is not meant for the long-term. The partnership may be “just for the moment, ” this means it is not serious, and it doesn’t evaporate demand a commitment. This sort of relationship is perfect for someone who is busy and have the time for a long-term relationship. While a “just for now” relationship can be fun and enjoyable, it is not recommended.
A romantic romance is a meaningful exchange between two people. Within a strong partnership, you are likely to risk your life to save your companion. You can be a good match for somebody with to whom you talk about the same interests and desired goals. While a “just with respect to now” relationship may develop into a even more permanent marriage, the expectation or desire of an romantic relationship should not be the goal. Instead, the relationship could be a time to check out the world.
A loving relationship does not have to be sex or have a long-term potential. It can be based upon an emotional connection. A romantic relationship is a wonderful opportunity for both associates to develop. But you could not expect a “just with respect to now” relationship to develop in to something even more. This type of romance should be a non permanent thing. Should you be not ready for a fully commited relationship, exclusively for now is not the right option. For the time being, you can have a casual relationship with the person you like very best and find another person you can talk to.
A romantic marriage can be equally healthy and harmful. A healthy relationship can be quite a fulfilling encounter for both partners. But it really is also crucial to be realistic when it comes to your anticipations. When it comes to romantic relationships, it’s best to take into account that relationships are definitely not just about one person. Rather, they must be based on shared interests. You should also be honest together and be accessible to each other. For anyone who is not being honest with your partner with regards to your feelings, some might have an detrimental environment.
Depending on type of romantic relationship, a romantic relationship can be a dangerous affair. It’s not advisable to interact in cheating with your spouse, especially if you will be in an early stage of your romantic relationship. In case you are not sure if the partner is compatible, you might want to reconsider your romantic relationship. Regardless of the kind of relationship, there is nothing wrong with dating simply for now. And for anyone who is both completely happy in your romance, you’re still likely to be able to have a more severe one.
Intimate relationships can be quite healthy and rewarding. But they should be tempered with the simple fact of daily life. A romantic romance can last a lifetime. A “just with respect to now” romance is a short-term arrangement. Really not a long lasting commitment. The goal ought to be to be in appreciate with your partner until if you’re ready to experience a child. Then you definitely will make your romance a healthy and happy 1.
While an intimate relationship can be fun and fulfilling, it’s also never easy. There’s no such point as a ideal relationship. A person should be able to commit to a partner and respect these people. This is an essential part of a loving relationship. Yet even if you’re only in it for a while, you should not expect it to last forever. If you’re not satisfied, really far better to end the romance.
A loving relationship can last a lifetime, so it is crucial to not really anticipate too much out of your partner. It is critical to have out in the open interests, yet don’t forget to preserve a sense of independence. Having another life allows you to balance the relationships and observe after your state of mind. This is essential to ensure a happy and romantic relationship. But it’s imperative that you remember that both of you can live without each other for so long as you want.

domain: sociology


text: I've been very busy painting silk scarves this weekend. I painted some for Christmas presents and they were very successful. I was asked to create two new designs for a colleague at work. So we have 'pansies' and 'vintage rose'. Simple yet striking. Lets hope she likes them and I have a happy customer!!

domain: artistic


text:
One of my favorite places in NYC is Governors Island, a 172 acre island that is 800 yards from Manhattan (a 7 minute ferry ride!). It is the perfect place to spend a day wandering and feels like a vacation, when it is only minutes away from home (well depending on the traffic….). And even better — you can spend the night there! It is an upscale camping experience and is a memorable and truly perfect way to spend the night. Recently, I took my daughter “glamping” on Governors Island mid week and it was the highlight of our Spring.
This is Tusia on Governors Island in 2017. Since then, the green space has turned in to the property for the glamping, Collective Retreats.
Tusia on Governors in 2020. Same same, but different.
In previous years, you could take a private boat to Collective Retreats on Governors Island. But now, since they extended the hours of the public ferry, the private boat has been eliminated and you take the boat from lower Manhattan. Once you arrive, they pick you up in a golf cart to bring you onsite.
The tents are super nice — basic, but beautiful. They have AC and heat (we needed heat), outlets, nice sheets, but no bathrooms.
The people around us looked like they were on vacation for a week there! Not us — in by 5 pm, out by 7 am! LIFO (last in, first out)
Statue of Liberty with campfires and smores!
The bar and restaurant. Great and delicious food.
We played soccer……on a muddy field. But so fun. And I hate soccer.
Early morning to school (thank goodness it was no uniform day and so it made it easy to just get up and go!)
On the ferry back! We took a taxi and made it to school on time! A little bit tired, but so very happy.
Collective Retreats has glamping experiences all across the country, so if you happen to come across one, you must try it out! And if you are in NYC and want a fun overnight experience, I highly recommend Collective Retreats at Governors Island! Expect not to sleep that night — I had to use the restroom in the middle of the night and there were so many planes and even fireworks that night — but oh well, one for the books!

domain: geography


Nicole Scherzinger covers Adele’s hit ‘Rolling In The Deep’ during a private gig in Monte Carlo for Hugo Boss. How did she do guys?
The Guardian names Adele as the most Powerful Person In British Music today.
Having dominated the charts worldwide with her album, “21″ , the accolade was decided by a team of music industry experts, with a comment:
“Adele is the most powerful force in music at the moment…It is very refreshing to see something so successful done in such a sensitive way…The accolade actually goes to ‘Team Adele’ which includes the chart-topper’s manager and record label boss.”
R.Kelly always knows how to get his audience interested, from great song with catchy hooks, to heart felt ballads and classics that will be a part of RnB lovers collections forever.
Yesterday, the top songwriter performed on ‘Later With Jools Holland’ giving the audience a great set with a heart wrenching performance of ‘When A Woman Loves’ taken from his current album ‘Love Letters’. Adele also stopped by to discuss her progress, but not before expressing her love for him too.
John Legend performs at the Rock & Roots Festival in Singapore over the weekend and sings “Rolling In The Deep” by Adele!! What do you think?
Currently number one on the Billboard charts, watch Adele’s performances for her VH1 Unplugged show
Adele was on the hit show Chelsea Lately recently and spoke honestly about her latest album, ’21′, which is based on her ex-boyfriend and the breakup, she said:
I think he’s a bit bitter, because the album is doing better than anyone expected.”
Watch one of the clips of Adele’s beautiful performances as she continues with her U.S. promo tour. It’s looking like Adele could get her first U.S number one album next week!
Reports suggest that the singer’s efforts may sell as many as 275,000 to 300,000 copies by tomorrow. That figure would instantly mark her best ever week in sales!
Adele admitted the end of her “amazing, immense relationship” helped her to write a whole host of passionate songs!!
I had the most amazing, immense relationship. It didn’t last, unfortunately – it stopped being fun, but thankfully the ending wasn’t bitter. The whole thing changed me. It made me really passionate about love, life, me – pretty much everything.”
However, after the success of her debut album ‘19′, Adele was unsure how she could improve her second album, she revealed to Reveal Magazine:
Before I went into the studio I wondered how on earth I was going to beat the first album. But it was like a tap that’s difficult to turn off. The relationship I’d been in just consumed me, which is where the songs came from.
Watch the main performances from the BRIT Awards 2011 below!!
Cee Lo Green
Best British Male Solo Artist – Plan B
Best British Breakthrough Artist: Tinie Tempah
Best International Album: Arcade Fire – The Suburbs
Critics Choice: Jessie J
Best International Male Solo Artist: Cee Lo Green
Best British Single: Tinie Tempah
Best International Group: Arcade Fire
Best International Breakthrough Act: Justin Beiber
Best British Solo Female Artist: Laura Marling
Best International Female Artist: Rihanna
Best British Group: Take That
Best British Album: Mumford and Sons – Sigh No More
Watch as Adele explains and performs the story behind her track ‘Someone Like You’ live from her home!
Check out the insert below from Melanie Fiona with urban music blog The Wrap Up.
In the interview she speaks about keeping her private life private, who she’s feeling in the UK and working with Tinchy Stryder…
“Adele is one of my favorite, I really like her. I love Estelle and Amy Winehouse. I love a lot of artists that come out of the UK and of course The Beatles were from here. I’m actually doing work with Tinchy Stryder right now. I think it’s really cool that I can work with different artists and they want to work with me too.”


Fall 2020 Auditions are here!
This fall we are teaming up with the rest of the UCD a cappella community for virtual auditions. Auditionees will be required to submit a video audition via Google Forms using this link: https://tinyurl.com/y4zrlaf9
The deadline to submit will be 10/16 at 11:59 PM.
Callback emails will be sent the following week; callback auditions will be conducted over Zoom.
We can't wait to hear your lovely voices sing and beatbox for us!
How do I prepare?
You should have a 30 second solo prepared for us that shows your range, unique style, and overall ability. Expect to sing scales, vocal exercises, and answer silly questions! Most importantly, have fun!
What are the Spokes looking for?
The Spokes look for women who have a decent amount of musical knowledge (whether it's intuitive or the product of training), a range and tone that blends well with our group, and the ability to learn quickly! It also helps to have arranging, beatboxing, piano, or choreography experience.


text:
Phasing Out an Inefficient Venture Capital Tax Credit
35 Pages Posted: 14 May 2010
Date Written: May 11, 2010
In 2005, the Government of Ontario, Canada, announced the phase out of the Labour Sponsored Venture Capital Corporation (LSVCC) tax credit, which will be become effective in 2011. Some media attention has suggested this might lead to difficulty for Ontario entrepreneurs and emerging firms in raising capital. This study presents evidence from Ontario innovative healthcare firms that capital raising concerns are not related to the phasing out of the LSVCC tax credit, and this evidence is consistent with evidence of extreme underperformance of LSVCCs. However, amongst firms currently funded by LSVCCs, there is significant concern about the phase out of the tax credit, which is at least in part attributable to the terms within LSVCC shareholder agreements. Policymakers should account for firms currently funded by LSVCCs to efficiently facilitate the phase out of the tax credit.
Keywords: Entrepreneurship, Venture Capital, Tax Policy, Canada
JEL Classification: G24, G28, G32, G38, K22
Suggested Citation: Suggested Citation

domain: finance


text:
Tips to Minimize Joint Discomfort
Inflammation and discomfort in the joints can leave us feeling tired and restless throughout the
day. Using helpful methods to prevent joint inflammation is essential to reduce the chances of
discomfort. At the same time, if you suffer from joint discomfort, there are techniques to help
minimize inflammation in the surrounding areas. If you haven’t, make sure to check out these
tips to help minimize joint discomfort.
Stretch is Essential (Anytime, Anywhere)
Soreness is typically a part of any type of exercise. Unfortunately, soreness contributes to
inflammation and discomfort in the joints. If a person is new to exercise or exercising at a higher
intensity than before, this tends to lead to soreness in the muscles and joints. Although soreness
can be inevitable at times, studies have shown that stretching before and after a workout can help
reduce levels of soreness in the body. Doing stretches helps relieve tension in the muscles and
joints, which helps reduce swelling and soreness in the muscles.
Asides from exercising, your daily habits can also contribute to soreness in the body. For
example, if you tend to sit at a desk all day for work, this can lead to discomfort in the back,
shoulders, and sides. It is important to make sure to take breaks from your desks and do stretches
to help relieve discomfort in these areas of the body.
Hot and Cold Compression Treatments
If you are suffering from discomfort and inflammation in the body, recent studies have shown
that hot and cold compression therapy helps relieve the muscles. Hot compression on the
muscles helps promote circulation in the body, which is needed to reduce swelling and tension in
the muscles. On the other hand, cold compression treatments have been shown to help reduce
swelling in the joints, which helps decrease inflammation and discomfort.
Give Your Body Time to Recover
If you notice increased levels of swelling, inflammation, and discomfort in your body, it’s time
to take another look into your lifestyle. Do not hesitate to give your body the time needed to
recover and rest from workouts or high-intensity activities. Also, this is a time to recover by
methods such as massages, physical therapy, or acupuncture techniques.

domain: medical


text:
Package: Book a boat with your stay
Upgrade your stay at Suitelodges Gooilanden with your own electric sloop from 1 April to 1 October 2023*. You can pick up the sloop every day from 10:00 at Boat Rental De Otter, 4km from the park, on the Loosdrechtse Plassen. You can fully enjoy the whole day on the water; the boat should be back at the marina at 16:00.
The Pontus sloop is a spacious and modern electric tender. The sloop can accommodate 10 people and is equipped with every comfort including speakers and lounger.
*Based on availability and only final after confirmation. The supplement for the sloop is paid in advance and cannot be changed or cancelled free of charge.

domain: travel


text:
Kirin Beverage recall 900,000 cans of coffee
Japanese drinks maker Kirin Beverage has recalled around 900,000 cans of coffee.
This is due to a packaging defect that potentially changes the way the drink tastes.
The product is a limited version of the Fire brand milk coffee sold in Osaka and five other prefectures in western Japan.
An investigation was launched by Kirin Beverage after complaints from consumers that the coffee tasted different to normal.
The company found that some of the cans had a narrow crack in the metal.
Kirin Beverage says if the cans aren’t airtight and it could lead to changes in flavour or cause the drink to congeal.
Products subject to the recall have best-before dates of May, July and August 2022.
Kirin Beverage apologised for the trouble and pledged to improve its quality management.

domain: food


• Plays the STC155CENT Teton Guitars
• From New Orleans, Louisiana
“The son of Bill Frisell, and the grandson of Derek Bailey” – All About Jazz
“Like Thelonious Monk set ablaze!” – DownBeat Magazine
“Be very careful, really exciting and love surprising from the get go! – Musica Jazz, Italy
Guitarist and Composer Craig Green’s unique vision and constant exploration has spanned the last 20 years, through a wide and diverse constellation of artistic projects from jazz, avant garde, electronica and world music.
With collaboration and improvisation as the focus, Craig has scored music for stage, film, ballet and chamber ensembles in addition to a continuous output of recorded projects.
In 2016, Craig represented Teton Guitars at the TEDx Idaho Falls: Widening Circles Conference, performed for our 5th Anniversary Concert here in Idaho Falls, appeared on Teton TV, as well as numerous other Teton Guitars events and engagements.
Teton Guitars is exceptionally honored to have Craig on our roster of Teton Artists! Please visit Craig’s website for current projects and tours.


Good Man · DJ Khaled · Pusha T · Jadakiss
℗ 2017 Epic Records, a division of Sony Music Entertainment
Released on: 2017-06-23
Associated Performer: DJ Khaled feat. Pusha T & Jadakiss
Composer, Lyricist: Khaled Khaled
Off his mixtape “Hate Me Now”, presented by DJ Drama. Straight outta Harlem, Dave East doesn’t get the recognition down south like he should.
This is a throwback!
Jada adds a verse to Drizzy’s current #1 hit, “One Dance”
Eye contact, I just had a glance with her
If I dance with her, then I got a chance with her
Look her in her eye when I’m holding hands with her
I get anything I want, so who says I can’t get her?


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IOWA (KWWL) -- Iowa ranks No. 10 on the list of Most Moved from States in 2019.
According to the 2019 National Movers Study by United Van Lines, the nation’s largest household goods mover, Iowa ranks in the top 10 states that have the most residents move out.
In 2019, more residents moved out of Iowa than into the state, as 55 percent of Iowa moves were outbound.
The top outbound states (with 250 United Van Lines moves or more) for 2019 were:
- New Jersey
- New York
- North Dakota
The Midwest region continues to see more residents leaving than moving in, with nearly 57 percent of all moves within the Midwest U.S. being outbound moves. This year’s study reflects continuing trends of Americans moving to the Mountain West (56.5 percent inbound moves) and the South (54.1 percent inbound moves).
The top inbound states (with 250 United Van Lines moves or more) of 2019 were:
- South Carolina
- District of Columbia
- South Dakota
- North Carolina
- New Mexico
Read more on the study HERE.


text:
THERE are plenty of important anniversaries falling in 2017 - from historical events to celebrity birthdays and classic pop culture moments.
Here's what happened in history on October 3...
What happened in history on this day?
- 1283: Dafydd ap Gruffydd, the then Prince of Wales, becomes the first nobleman in Britain to be hung, drawn and quartered.
- 1863: Abraham Lincoln declares that the last Thursday in November will be known as Thanksgiving in America.
- 1944: German forces crush a rebellion in Warsaw, Poland after street fighting began between Soviet and Nazi troops.
- 1952: The UK tests its first atomic bomb near the Montebello Islands in Western Australia becoming the third country to have nuclear weapons.
- 1981: IRA prisoners, who have been on hunger strike in the Maze Prison end their protest
- 1990: Germany is reunited as a single country ending 45 years of division
- 1995: OJ Simpson is found not guilty of the murders of his ex-wife Nicole Simpson and Ronald Goldman in what was dubbed the 'Trial of the Century'
Which famous people were born on this day?
Does your birthday fall on October 3? If so, you're sharing with this lot:
- 1964: Clive Owen, actor
- 1968: Gwen Stefani, singer
- 1981: Zlatan Ibrahimovic, footballer
- 1987: Danny Willett, golfer
- 1988: Alicia Vikander, actress
Which other important anniversaries fall in 2017?
A number of special dates fall this year, although it's impossible to list them all.
Here's a round-up of some of the big ones in 2017...
- March, 1967: Sandie Shaw wins the Eurovision Song Contest for Britain with Puppet On A String
- May 25, 1977: Star Wars is released
- May 29, 1917: 35th US President, John F. Kennedy, was born
- June 1, 1967: The 50th anniversary of the Beatles' Sgt. Pepper's Lonely Hearts Club Band
- July 1817: The 200th anniversary of the death of author Jane Austen
- August 16, 1977: Marks 40 years since musical legend Elvis Presley died
- October 1, 1967: It is 50 years since the death of revolutionary Che Guevara
- October 14, 2007: Keeping Up With The Kardashians debuts for the first time
- October 16, 1987: Marks 30 years since Dirty Dancing was released
- October 31, 1517: Theologian Martin Luther compiled his 95 Theses
- October 31, 1887: The 125th anniversary of Sir Arthur Conan Doyle's A Study In Scarlet - his first work featuring Sherlock Holmes
- November 18, 1997: Blockbuster film Titanic was released 20 years ago (UK film premiere)
- December 3, 1967: The first human heart transplant was carried out 50 years ago

domain: history


text:
Community health is a fundamental course in the principles of personal health: nutrition, mental health, drugs, exercise, sleep and rest. Community health also explores the principles of community health sanitation, community services and public health agencies
What Will You Learn?
- The benefits of this model curriculum are to:
- To learn the fundamentals of community health
- To understand how community health practices, affect one personally as well as those around them.
- To understand how Kinesiology relates to a healthy individual lifestyle.
Organizations that contribute to community health
How communities measure disease, injury and death
Control of communicable and non-communicable diseases
How communities organize and solve health problems


text:
Three weeks from today we'll be in Ecuador!
Just a few facts about Cuenca to give you a sense of where we’ll be:
Cuenca is in the South of Ecuador, just north of the Border with Peru. The full name of the city is Santa Ana de los Cuatro ríos de Cuenca, because of the 4 rivers that run through the city. It is 8,400 feet high, and nestled in a Mountain valley. Why did we pick Cuenca? It is big enough (600 people in the metro area) to need English teachers and to have good public transportation. It has a lot of colonial architecture and is very walkable. And it has a mild climate.. described as “eternal Spring” but maybe a bit more fall-like (50-70 degrees F).
Our to-do list of details to take care of on the home front seems to be getting longer. But we’ve been very fortunate with all the big items: finding a great renter for our house, friends willing to store our car and use our piano for a year, and a teaching position (English for preschoolers) that lets us get a year visa and that will allow me to work the same hours as the kids’ school.
I’m struck by three things as we move closer to this family adventure becoming a reality.
First, it has been easier to make happen I might have imagined. Sure, we’ve been pretty deliberate about lining things up, and about saving to fund our adventure. And we’re fortunate that we have jobs that are willing to keep us employed (in Doug’s case) or give a leave of absence (in mine). And there are a lot of little details to take care of. But in the end, it isn’t as much of a stretch to make it happen as we expected.
Second: it is so different arranging to go abroad now than it was 20 years ago. That probably goes without saying. But the internet makes the logistics much easier. There are services for everything: Air B&B to line up initial housing; services that will scan your mail and let you decide what to shred, scan, or have mailed to you; banking done remotely which is essentially the same as we do now. The ability to stay connected via Skype, blog, Facebook, etc. just didn’t used to exist. I’m a bit ambivalent about this last convenience. When I’ve lived abroad before it has been the sense of immersion, of being in a totally different world and of having to make a concerted effort to connect with home that has helped define the experience. I’m interested to see how the experience is different in a more connected world. And also grateful that we’ll be able to maintain contact with family and friends.
Which brings me to the third observation. We fully expected that at least a few people that we told about our plans would think we are crazy. And maybe some have, but they haven’t said so to us. Without exception, our family, friends, work and school have been very supportive. Even those who are sad about us being so far away for so long (and we share that sadness!) are supportive of our goals. We can’t wait to share our experience with you!


A COMMUNITY bus has been hailed such a success it is extending its service.
The Yarm Community bus, which runs every two weeks to take elderly and less mobile residents shopping, has agreed to take on extra passengers in nearby Kirklevington.
Stockton Borough Council cut its annual bus subsidy of £300,000 from this year’s budget.
However some bus services have, unexpectedly, been maintained, including The Leven Valley Coaches bus that travels from Port Clarence to Wolviston and then on to Billingham, as well as the service from Stillington, serving Bishopton Avenue Stockton and Carlton.
Yarm councillor Andrew Sherris has been helping to organise a community bus.
The bus currently picks up from The Meadowings, Willey Flatts and Layfield areas and takes residents to Aldi supermarket on the outskirts of Yarm.
Cllr Sherris said: “This has proved a very popular service and we have recently introduced it to Kirklevington. Although at present only running twice a month many of our elderly residents appreciate the chance to get out and socialise as well as shop for the Thursday specials.
"It’s very important that we identify any residents in our community who are isolated and feel remote. The bus ride is a friendly and social occasion with a great driver from Stockton Community Transport who even carries bags into homes and ensures the residents are safely inside.”
“I would ask anyone who is aware of any elderly residents who may need this help or indeed any other help, to contact me.”
Coun Sherris can be contacted on 01642-786767 or firstname.lastname@example.org


Oppo F17 Pro price in Pakistan is Rs. 44,999. Oppo F17 Pro price in Pakistan & Full Specifications. Oppo official dealers and warranty providers determine the retail price for mobile phones under their official warranty.
- The price of theOppo F17 Pro in PakistanIt is Rs. 44,999
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Oppo F17 Pro Price
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Oppo’s new Oppo F17 Pro smartphone is now available. This high-end smartphone will rock your world with its impressive camera performance. The MediaTek Helio P95 is the latest chipset. Oppo F17 Pro‘s processor is paired with 8 gigabytes of RAM.
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Dual-camera selfies are possible with the front-facing camera. Expect exceptional results from the Oppo F17 Pro’s front-facing camera. It is paired with a 4000mAh Non-removable LiPo battery. Huawei may be out, but Samsung appears to have another competitor on the international market. The F17 Pro seems like a good start.
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text:
Icicle – My Heart
ARTIST NAME: Icicle
SONG TITLE: My Heart
ALBUM TITLE: Silence
Tell us your opinion on creativity and originality.
Mandatory – You always have to search, explore, experiment, and challenge yourself.
Tell us the beginning of your passion for music.
From a very young age, I needed music around and with me at all times.
Elaborate on how you make sure the quality of your sound is high.
By triple and quadruple checking if this is the very final version I’d like to hear.
But I also remember that a song or an album for that matter is like a snapshot. You must accept that this is merely a picture of a time and place.
Tell us how you get ideas to develop your songs.
I generally have a hum in my head… pretty much all the time. Sometimes I get caught by some line or lyric and I delve into it. Other times I collaborate or sit down with nothing particular in mind and just get busy.
Describe your vocal ability.
Tell us how you develop the instrumentation for this song.
Bass, drums, and vocals are what I did right away. I worked on the guitars afterward.
I spent quite a bit of time on the keyboards and sound effects. I wanted to sonically picture the beat of a heart.
State five hit songs and name the artists.
Forest – The Cure
Comfortably Numb – Pink Floyd
Driven to Tears – The Police
London Calling – The Clash
Purple Haze – The Jimi Hendrix Experience
Tell us how you entertain the audience during a live performance.
Never settle for giving less.
List the name of artists that have a similar sound to yours.
Depeche Mode, David Gahan, Arctic Monkeys, The Black Keys, Beck, Death Cab For Cutie, Folk Implosion, Kasabian, Spoon, The Soup Dragons, Talking Heads, The Killers, Blur, TV On The Radio, Franz Ferdinand, David Bowie, Queens Of The Stone Age, Charlie Winston, Modest Mouse, Dub Pistols, Kongos, Morphine, Jamiroquai, Earth Wind and Fire, Doves, Damon Albarn, Archive, Iggy Pop, Muse, The Fall, The Stone Roses, Oasis, Foals, The Chemical Brothers, Mogwai, Ratatat, Future Islands, Gorillaz, Peter Gabriel, The Heavy, Keane, Ian Brown, Kaiser Chiefs, Kaleo, Joy Division, Johnny Marr, The Kills, LCD Soundsystem, Pink Floyd, Black Rebel Motorcycle Club, Groove Armada, Thievery Corporation, Jack White, The Dead Weather, The White Stripes, The Flaming Lips, Fleet Foxes, The Fray, Of Monsters And Men, Mumford And Sons, R.E.M., The Rakes, Snow Patrol, Editors, Interpol, U2, Coldplay, Led Zeppelin, The Beatles, The Rolling Stones, Paul Simon, David Burne…
Discuss how you think your music is impacting your fans.
Motivating, comforting, stimulating, reassuring, thought-provoking, engaging… I hope!
Tell us what inspires you to sing.
This Bulgarian proverb: “He who sings thinks of no evil.”
Discuss this song.
It is a different take on another song I wrote a while back with the same title.
The premise is about losing a really close family member at a young age.
Discuss how you develop your melody.
I was just humming the lyrics over a bass line (as I often do).
Share your press release and reviews with us.
Publisher: Herd Records
Label: Herd Records
Icicle’s newest record SILENCE is one of the finest in his acclaimed catalog, taking him down yet another new sonic path five albums into his career.
This is an act whose name has been built on the progressive evolution of its own aural dynamics, innovative compositions, and stellar arrangements.
SILENCE is a bold, upfront, driving album, relying on grooves of pulsing keyboard twists, infectious guitar hooks, and the off-kilter, slinky rhythms to carry the vocals of singer/bassist Krassy Halatchev.
Lyrically, the record is demanding attention and mindfulness. The topics are broad, deep and above all – painfully honest and sincere.
These 13 songs are some of the most sensual, serendipitous, sparse recordings to come from Icicle, showcasing a new dimension in his songwriting and marking SILENCE as that crucial next sonic step forward for one of Montreal’s best artists.
Tell us how you master your songs after production.
I do everything in my studio. The same principle applies – make sure you have the right tools and put in the time it takes.
Discuss how you get your songs to digital stores.
Via distributor. There are plenty of them out there.
Discuss how to generate income through music.
There is no one or two key things here. Everything is key. Your music is your brand.
You must brand your brand just like the ones you like to do.
State your favorite radio station.
Fresh Radio and FIP.
Go into details on this song.
Losing someone dear, special and important in your life is a big blow especially when you are young… It leaves a lifetime scar.
Wishing and imagining that somehow they are still around is comforting, soothing, and strengthening. This is what this song is about.


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text:
Martial World - Chapter 91
Challenged at Spear Point
At the Ten Thousand Kill Array ashlar square –
Zhu Yan narrowed his eyes towards Lin Ming . Without saying a word, his complexion had returned to normal, but in the glint of those narrow eyes was an icy chill .
Lin Ming and Zhu Yan looked at eachother with outwards indifference . Their conflict would end in combat sooner or later, but Zhu Yan had not thought that the day would be arriving so quickly .
Lan Yunyue was quietly standing in a corner of the sword bamboo grove . She was at a complete loss looking at that incomprehensible number on the Ranking Stone . She looked at the confrontation between Zhu Yan and Lin Ming and was totally confused . How could things come to this…?
Many people in the audience were still shocked silly by the result . By now, the deacon responsible for the Ten Thousand Killing Array opened his mouth and suddenly said, “Lin Ming, congratulations for reaching rank 62 in the Ten Thousand Killing Array . According to the regulations of the Martial House, as soon as you enter into the top 80 rankings among the disciples, you have earned the qualifications to challenge any disciple of the Heavenly Abode . The winner will enter the Heavenly Abode and the loser will be eliminated and enter the Earth Hall . ”
The Seven Profound Martial House’s Heavenly Abode had exactly 72 people . These people were there because of their combat prowess, but their strength might not be ordered on the Ten Thousand Killing Array rankings .
Although the Ranking Stone rankings judged the basic strength of an individual, sometimes there existed discrepancies . For instance, specific specialties such as an assassin, who do not excel in the forward rushing fighting style of the Ten Thousand Killing Array . But in terms of killing a human, they can often surpass their ranking to defeat someone .
Therefore the Seven Profound Martial House regulations stipulated that once one entered the top 80 rankings of the Ranking Stone, they obtained the qualifications to challenge a disciple of the Heavenly Abode . Generally speaking, for this challenge, the one most often chosen was the worst ranking person in the Heavenly Abode .
Lin Ming look at the Ranking Stone . On the Ranking Stone, the names of the Heavenly Abode disciples were slightly larger than those of the Earth Hall . It was easy to see that the one worst ranked in the Heavenly Abode was a 17 year old girl named Bai Rongrong, rank 75 .
Bai Rongrong just happened to be present during this ranking war . She looked extremely pitiful as she saw Lin Ming turn his eyes on the Ranking Stone . She knew that she had no chance at all facing him . To reach rank 62 in four incense sticks and a breathe of time; even if there were two of her, she had no fancy dreams she could overcome such a monstrously evil genius .
She had to at least symbolically fight him . She just couldn’t throw in the towel and admit defeat . She had to fight him honestly and fairly . She could only hope that this abnormal fellow showed some mercy and wasn’t too ruthless…
Bai Rongrong was self-aware of her own situation; she had already resigned herself to fate .
The eyes of many of those present fell on Bai Rongrong . They all expected that this Bai Rongrong would lose . A girl was able to win sympathy from others, especially this Bai Rongrong who was always diligent in her cultivation . If the one who met such a calamity this time was a boy, chances were that most people would have the feeling of taking pleasure in other’s misfortune .
“Have you decided?” The deacon asked .
“Mmm, I’ve decided . ” Lin Ming replied . “I have a question though . Are there any restrictions on the time setting of this challenge?”
“The time is decided yourself, but preferably within a month . If your ranking falls out of the top 80, then you also lose the qualifications to challenge . ”
“I see… thank you Deacon Senior Apprentice . ”
“You’ve chosen? I need the name to report to the Heavenly Abode elder responsible for these matters . ”
“Mmm . I’ve chosen . The time is in one month . As for who…” Lin Ming turned his head . He lifted his spear and pointed it directly at a man who was staring at him with narrowed eyes, “I choose Zhu Yan!”
Everyone who heard this name froze . Especially Bai Rongrong, whose face filled with an incredulous expression of disbelief .
The entire ashlar square fell into absolute silence .
The one this Lin Ming chose was… Zhu Yan!?
Many people at first thought they heard wrong, but looking at who Lin Ming’s spear was pointed at, it was clearly directed at Zhu Yan!
Challenging someone at spear point! This was simply unconcealed provocation!
Ranked 32, Zhu Yan! This Lin Ming just wouldn’t stop astonishing everyone!
“This Lin Ming… he’s just… he’s gone insane!” Murong Zi’s lips twitched . She mumbled, “No… I’m the crazy one . If this Lin Ming is crazy, and he does crazy things and creates crazy miracles, then if he is not crazy, I am crazy . ”
Bai Jingyun was also amazed . This Lin Ming was bold! What courage! He was prepared to surpass Zhu Yan in one month!
From 62 to 32, it had to be known that the ranking was exponentially more difficult the higher one went . From 162 to 132 would not be difficult, but from 62 to 32, the hardship would increase by several times!
She had used an entire year to go from 65 to 26 .
This growth seemed high, but the truth was it was limited!
Because this year, 20 disciples who had been in front of Bai Jingyun had graduated from the Martial House . So Bai Jingyun had only actually advanced 19 ranks!
“This has become interesting . ” Ta Ku said with a wry smile . “I did not come today in vain . This Lin Ming seems to have a grudge with Zhu Yan . Otherwise, why would he aim so high?”
“Mmm, really a horrible relationship . I felt Zhu Yan’s body flash a moment ago with murderous intent, but it was instantly hidden . Lin Ming challenged Zhu Yan at spear point . That in itself was also a kind of provocation . ” Ling Sen said . Although that moment when Zhu Yan had broken out in murderous intention was not obvious, Ling Sen was able to sense it from the many years he had spent on the battlefield . He also had his Ashura martial intent, so he was particularly sensitive to murderous intent .
“Zhu Yan is a ruthless person . Although he has an aristocratic background, he is actually someone who sinks his entire heart into diligently cultivating . He is very strong, and not only that, but this kind of person has no lack of ambition . In the future he will probably join the government . Even I have kept several points of attention on him . Now this Lin Ming challenged Zhu Yan, I really want to see!” Ta Ku licked his lips in excitement . He was a subordinate of the army, and would most likely return in the future . It was better for him not to be involved in these struggles .
What would the result be after one month? No one dared to guess .
If it was before, then they would say that Lin Ming was a fool who had bitten off more than he could chew . But at present, Lin Ming had created miracle after miracle, and no one dared to look down on a single hair of his .
“Mmm? What? Lin Ming challenged Zhu Yan? And he challenged him at spear point! Is it possible that he has some enmity with Zhu Yan?” In the Eastern Palace, the Crown Prince had just gotten the news that Lin Ming had challenged Zhu Yan .
“He called out his name and also referred to him by spear point . It really is unusual . Between these two people, there should be some matters . ” Muyi also said .
“If there really is a grudge, then my chances just increased . This Zhu Yan is a cousin of my tenth brother . Zhu Yan is already joined together with my tenth brother as one . They share glory, and also defeat . I don’t know what sort of relationship exists between Lin Ming and Zhu Yan . It seems there are some oversights in my information…”
For the matters between Lin Ming and Lan Yunyue, except Lin Xiaodong, few others knew . The intelligence that the Crown Prince collected was mainly about Lin Ming’s background and parental situation, it wasn’t a completely exhaustive report .
Peng! The teacup in the Cloud Prince’s hand was crushed . He had received the sound transmitting talisman at the same time the Crown Prince had .
Naming Zhu Yan in a challenge, referring to him by spear point, this was definitely not a good sign . If there was no hatred between them, why would Lin Ming jump over so many people to find Zhu Yan?
“This Zhu Yan, he’s really making trouble for me!”
The Tenth Prince’s two eyebrows slanted upwards like scimitars and twisted together . Zhu Yan belonged to his mother’s side . About relatives from the wife or mother’s family, generally speaking, regardless if it were a prince or an emperor, they would exclude most from the mother’s side .
Since ancient times, it had been far too common for the wife’s family or mother’s family to take power . For instance, the empress or empress dowager would entrust heavy responsibilities to their family and cause their power to skyrocket . But once their influence inflated, then there would also be conspiracies and plots to control the court . There would be rebellions and plots from the women’s side to seize power .
But, this was a crucial phase for the Cloud Prince in his struggle . Anything that could give him a boost was good . Zhu Yan was a talented person, and the Cloud Prince was willing to let him work as his subordinate, but he would not take Zhu Yan as a trusted follower . Someone like Zhu Yan was simply too ambitious .
“Pass the order; I want Zhu Yan to come see me tonight!” The Tenth Prince wanted to inquire about the reason behind the feud between Zhu Yan and Lin Ming, and see whether he could convert Lin Ming from an enemy into a friend .
User rating: 4.4

domain: literature


text:
Lesley-Ann Brandt Who Plays Maze On Lucifer Tweets Using Hashtag RenewLucifer As Movement For Another Season Grows
Actress Lesley-Ann Brandt is talking about the new viral movement to see a fifth season of the Netflix series Lucifer and she's definitely joining in. Originally starting on Fox, Lucifer ran three seasons on the network before the canceled the series. Fans rallied to its aid and launched several hashtags and viral movements including #SaveLucifer.
The #SaveLucifer campaign worked and soon Netflix picked up the series for its fourth season. Now that Netflix has released Lucifer Season 4, another movement is already underway hoping to save Lucifer and bring the show back for Season 5.
Lesley-Ann Brandt plays the demon character Maze who is Lucifer's protege and protector. On Wednesday, Lesley jumped in on the action and after commenting on the new hashtag, she used it in one of her own tweets.
Lesley-Ann Brandt wrote the following.
"Yes!!! We love @netflix and We love your enthusiasm and we hope for another season so fingers crossed. #renewluciferl"
You may see the full tweet as Lesley-Ann shared it with replies below.
Fans agree with Lesley-Ann and #renewlucifer began trending on Wednesday afternoon. Many fans of the series began tweeting directly to Netflix letting them know how much they loved the fourth season and begged them not to cancel the show.
One fan was so happy for Netflix taking the show
"We can't thank netflix enough and for allowing you all to have so much creative freedom. It was truly the best season yet and we hope for many more. 😈 #RenewLucifer P.S. I hope you sing again in season 5. You were amazing!!!"
This fan isn't alone with the praise. Countless fans are online tweeting to cast members, the show's creators, and Netflix begging them to renew the season.
Lucifer is based on the Neil Gaiman stories and are about the devil being bored with hell and moving to Los Angeles. The more time Lucifer spends with a detective named Chloe, the more human he becomes.
Lucifer and Chloe fall in love but each is tortured by the implications and the show is a good study on what makes someone human and is a commentary on the power of love.
What do you think? Do you hope they #renewLucifer?