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Recent interest in understanding the spatial organization of gene expression has focused attention on nuclear structures known as speckles or interchromatin granule clusters (IGCs) revealed by immunofluorescence or electron microscopy. Staining of nuclear factors involved in pre‐mRNA splicing or, more recently, transcription, reveals 20–40 speckles per nucleus, resulting in the intriguing suggestion that speckles are nuclear sites of transcription and processing. In contrast, other investigations have observed transcription in other areas of the nucleus. In this study, we have examined the localization of active transcription as detected by uridine incorporation and recently developed RNA polymerase II antibodies, and compared this pattern with that of known splicing and polyadenylation factors. Our results indicate that in actively transcribing cells, transcription and splicing factors are dispersed throughout the nucleus with abundant sites of preferred localization. In contrast, in poorly transcribing cells, polymerase II and splicing factors localize to speckles. In nuclei inactivated for transcription by drugs or heat shock, the speckle type of co‐localization is accentuated. These observations suggest that bulk transcription and splicing occur throughout the nucleus during periods of active transcription; and that factors involved in these two processes re‐locate to minimal speckle domains during periods of inactive transcription.
In recent years, a number of studies have addressed the spatial organization of gene expression within mammalian nuclei. Key to this endeavor have been experiments localizing polymerase II‐derived nascent transcripts and the factors that transcribe, splice and polyadenylate these RNAs. A number of antibodies specific for elements of the transcription or pre‐mRNA processing machinery have been used to reveal subnuclear structures associated with pre‐mRNA (reviewed in Spector, 1993; Fakan, 1994; Moen, et al., 1995; van Driel et al., 1995). One particularly striking staining pattern is that observed using an antibody raised against the conserved splicing factor, SC35. This antibody brightly stains nuclei from a wide variety of tissue culture cells in a pattern of localized staining termed ‘speckles’ (Fu and Maniatis, 1990; Spector et al., 1991). Other splicing‐related factors were also localized to speckles (Spector, 1984, 1990; Nyman et al., 1986; Carmo‐Fonseca et al., 1992; Huang and Spector, 1992; Krause et al., 1994; for reviews, see Spector, 1993; van Driel et al., 1995). Cultured mammalian cells commonly contain 20–40 speckles, raising the intriguing possibility that RNA processing is restricted to minimal domains within mammalian nuclei. Such a hypothesis immediately suggested either that many pre‐mRNAs should be synthesized within the vicinity of speckles or that nascent pre‐mRNAs must be transported to the speckles for processing.
Experiments to address the possibility that speckles represent sites of transcription or processing of pre‐mRNA have utilized localization of either bulk or individual transcripts. Use of oligo(dT) probes to detect bulk polyadenylated RNA revealed a speckled localization (Carter et al., 1991, 1993). Several identified unique pre‐mRNAs have also been localized to speckles defined by staining with the anti‐SC35 antibody (Huang and Spector, 1991; Lawrence et al., 1993; Xing et al., 1993, 1995; reviewed in Moen et al., 1995). Speckled localization was especially prominent for microinjected RNAs or transcripts produced from transfected DNAs (Wang et al., 1991; Huang and Spector, 1996). Such data suggested that the speckled pattern reflected localized accumulation of active splicing and transcription factors.
An alternative proposal has suggested speckled nuclear structures as storage sites of splicing and transcription factors distal from the sites of bulk transcription occurring throughout the nucleoplasm and associated with chromatin (reviewed in Spector, 1993; van Driel et al., 1995). Supporting evidence for this proposal came from early electron microscopy studies in which bulk transcription was localized by short pulses of [3H]uridine incorporation. Nascent RNA was detected within the perichromatin fibrils that reside between the interchromatin granule clusters (IGCs) with little accumulation at the IGCs, or speckles (reviewed in Fakan and Puvion, 1980; Fakan, 1994; Hendzel and Bazett‐Jones, 1995). Subsequent in situ nucleotide incorporation experiments using fluorescence microscopy have confirmed this observation via the detection of transcription at many sites within the nucleus other than the speckles (Jackson et al., 1993; Wansink et al., 1993). Furthermore, multiple identified pre‐mRNAs have been localized to regions of the nucleoplasm between the speckles (Zhang et al., 1994). These results raised questions about speckles being the sites of pre‐mRNA generation and subsequent splicing. Such incorporation studies, however, are compromised by the inherent difficulty in ascribing bulk transcription to pol II‐driven pre‐mRNA synthesis (Moen et al., 1995).
Determining the function in transcription and splicing of speckles is an important issue because of the ramifications that alternate interpretations have on the spatial relationship between transcription and RNA processing within the nucleus. If transcription and processing are coupled, as studies in Drosophila indicate (Beyer et al., 1988), then it is difficult to visualize how all of the active transcription sites in the average nucleus can be effectively coupled to 20–40 speckles containing processing factors. It also becomes difficult to explain why so much transcription occurs distal to speckles when in situ incorporation experiments are performed. If transcription and splicing are spatially uncoupled within the nucleus, it is difficult to envisage how extremely large pre‐mRNAs can be made as a unit and then transported within the nucleus to speckle‐type processing centers. Thus, it is difficult to reconcile observations that indicate disperse transcription centers and localized processing centers. Given the striking visual observation that many factors involved in splicing can be found in speckles, it becomes imperative to ascertain if speckles are indeed the sites of active pre‐mRNA production and/or processing.
Recently, two antibodies directed toward RNA polymerase II were developed (Bregman et al., 1995). One of these antibodies recognizes pol II hyperphosphorylated within the C‐terminal domain (CTD) of the large subunit, whereas the other recognizes hyper‐, hypo‐ and intermediately phosphorylated forms of the CTD. In this study, we show that both pol II antibodies can recognize transcriptionally engaged polymerase molecules, indicating that both antibodies can be used to detect sites of active transcription within the nuclei of mammalian cells. Using these antibodies and other antibodies specific to RNA processing factors, we have observed that transcription and RNA processing overlap within a broad meshwork occupying the non‐nucleolar portion of the nucleus with hundreds of ‘dots’ of preferred localization overlaying the network. Observation of the meshwork was dependent on the ability to detect active transcription by bromo‐uridine triphosphate (BrU) incorporation. In cells in which BrU incorporation was abundant, BrU incorporation, the large subunit of polymerase II, and splicing and polyadenylation factors were broadly located within the nucleoplasm and did not concentrate at speckles.
In contrast, cells that poorly incorporated BrU demonstrated a strikingly different pattern in that both pol II antibodies and splicing factor antibodies co‐localized to classic speckles, suggesting a redistribution of transcription and splicing factors during periods of inactive transcription. A similar speckled localization of pol II was observed when transcription was experimentally arrested with either drugs or heat shock, supporting such a redistribution. Therefore, our data suggest that active transcription and splicing occur throughout the nucleoplasm. Furthermore, they support an alternative definition of speckles as dynamic locales within the nucleoplasm in which transcription and processing factors localize between rounds of active pre‐mRNA synthesis, perhaps for some form of recycling and reactivation. Our data also suggest that very active transcripts, such as those previously localized to speckles, may be found preferentially in, or adjacent to, speckles because they provide a local high concentration of processing factors following recycling.
Monoclonal antibodies H5 and H14 recognize transcriptionally engaged polymerase II molecules
Monoclonal antibodies reactive for phosphorylated forms of mammalian polymerase II have recently been developed (Bregman et al., 1995). One of these antibodies, mAb H5, is specific for a hyperphosphorylated form of the large subunit of pol II (IIo); a second antibody, mAb H14, recognizes various forms of the phosphorylated pol II large subunit. A population of non‐transcribing polymerase molecules that are phosphorylated on the CTD has been detected by both H5 and H14 (Bregman et al., 1995; Kim et al., 1997). However, phosphorylation of the CTD takes place concomitant with the initiation of transcription (reviewed by Dahmus, 1995); consequently, we asked if mAb H5 and H14 recognize phospho‐epitopes on actively transcribing polymerase molecules. For this purpose, we used an in situ photoaffinity labeling technique (Bartholomew et al., 1986). Briefly, nuclear run‐on transcription was performed with isolated HeLa cell nuclei in the presence of [α‐32P]CTP (labeling nucleotide), 4‐thio‐UTP (photoactive RNA–protein cross‐linking nucleotide) and 3′‐O‐methyl GTP (chain‐terminating nucleotide). Then nuclei were exposed to UV light (λmax = 310 nm) to induce covalent cross‐linking of the radiolabeled RNAs to proteins. The nuclei were extracted after digestion with RNase to trim excess RNA from the proteins, and the extract was used immediately for immunoprecipitation with mAbs H14 and H5.
The results indicated that, in the absence of the transcription inhibitor α‐amanitin, mAbs H5 and H14 immunoprecipitated three distinct [α‐32P]CTP‐radiolabeled polymerase II subunits: IIo (∼240 kDa), IIa (∼220 kDa) and IIc (140 kDa) (Figure 1A). mAb H5, which recognizes a hyperphosphorylated form(s) of polymerase II (Bregman et al., 1995), immunoprecipitated predominantly [α–32P]CTP‐radiolabeled Pol IIo. Trace [α‐32P]CTP‐radiolabeled Pol IIa was also present (Figure 1A, lane 1). mAb H14, which recognizes multiple phosphorylated forms of polymerase, immunoprecipitated [α‐32P]CTP‐radiolabeled Pol IIo as well as a significant amount of Pol IIa (Figure 1A, lane 4). In the presence of α‐amanitin, radiolabeled RNA was not detected on immunoprecipitated polymerase molecules. Thus, mAbs H5 and H14 can recognize a population of phosphorylated polymerase molecules that is transcriptionally engaged in the in situ nuclear run‐on assay.
Polymerase II localizes to many sites within the nucleoplasm
Next, we wanted to know the nuclear localization of pol II that is actively engaged in transcription. mAb H5 and mAb H14 have been shown to stain mammalian cells broadly (Bregman et al., 1995). Figure 1B and C indicates a staining of human ovarian cancer cells (OV‐MZ‐15; Möbus et al., 1994) with antibodies mAb H5 (Figure 1B) and mAb H14 (Figure 1C). Broad staining of the entire non‐nucleolar portion of the nucleus was observed, suggesting a wide distribution of pol II and, by inference, of transcription throughout the nucleoplasm. In this communication, we refer to this nucleoplasm‐wide staining as a meshwork because at higher resolution of the image, staining was not uniformly diffuse, but instead appeared as a very dense fibrogranulous structure. Overlaying the meshwork were multiple discrete sites of preferred staining distinguishable from a classic speckle staining pattern. These discrete sites were very numerous in several tested cell lines, with an average number of 100–200 per cell in a given focal plane (suggesting up to 1000 per nucleus) and were observed throughout the entire nucleoplasm (with the exception of nucleoli). Therefore, preferred sites of staining for pol II were noticeably more numerous per nucleus than the number of speckles per nucleus. Most sites detected by pol II‐specific antibodies were also smaller in appearance than classic speckles. To distinguish linguistically this latter pattern of pol II localization from that classically referred to as speckles, we have termed these multiple sites of pol II localization nuclear ‘dots’.
Polymerase II localization pattern is distinct from speckles
Speckles are frequently experimentally defined by staining with antibodies specific for splicing factors. One such factor, SC35, has been prominently localized to speckles (Fu and Maniatis, 1990; Spector et al., 1991). Figure 2A–C compares the staining pattern of cells stained with both pol II‐ and SC35‐specific antibodies. Both antibodies stained broadly throughout the nucleoplasm in a meshwork pattern. Although the meshwork was much more visible with the pol II antibody in optical sections, the two patterns of staining overlapped extensively, suggesting that a portion of the SC35 appears to be localized in the vicinity of polymerase. Superimposed on this meshwork pattern were the prominently staining speckles of SC35 (Figure 2A, green) or the nuclear dots of pol II (Figure 2B, red). A few of the speckles overlapped with the ‘dots’ of polymerase II (overlay in Figure 2C); for the most part, however, the dots of preferred pol II localization and the SC35‐defined speckles did not co‐localize. Our result suggests that the bulk of polymerase II is not localized to speckles and raises questions about models that propose speckles as the sites of active transcription and RNA processing.
Uridine incorporation and pol II overlap both in a meshwork throughout the nucleoplasm and in many preferred sites
To confirm that the pattern of staining detected with pol II‐specific antibodies represented sites of transcription, double‐label experiments were performed in which the pattern of pol II staining using antibody mAb H5 was compared with sites of UTP incorporation during an in situ transcriptional run‐on experiment (Figure 2D–I). Nascent transcripts were labeled with BrU and visually detected via the use of anti‐BrU antibodies. Human CaSki (Figure 2D–F) and OV‐MZ‐15 (Figure 2G–I) cells were used for this experiment.
For CaSki cells, two patterns of staining were observed that are represented by the two cells shown in Figure 2D–F. One pattern was seen in 60–80% of the cells and is represented by the top cell in Figure 2D. In these cells, nascent RNA as detected by confocal microscopy was localized throughout the nucleus in a pattern very similar to that of the pol II staining shown in Figure 2E (i.e. a nucleoplasm‐wide meshwork overlaid with many sites of preferred staining).
OV‐MZ‐15 cells demonstrated localization patterns of pol II and BrU incorporation similar to that of CaSki cells (Figure 2G–I). Both pol II and nucleotide incorporation occurred throughout the nucleoplasm in a complicated pattern. The merged views (Figure 2F and I) demonstrated considerable overlap of the two staining patterns (yellow) with the exclusion of nucleoli (marked as arrows). Because immunofluorescence can only provide an approximation of the actual extent of co‐localization of two antigens with immunofluorescence patterns as complex as those in Figure 2D–I, a conservative interpretation of these patterns suggests that both pol II and transcription are broadly dispersed throughout the nucleoplasm in a particulate, non‐speckled pattern.
The underlying reason for both a meshwork and ‘dotted’ distribution of both pol II and BrUTP incorporation is unclear. Given the limits in resolution of light microscopy, the meshwork pattern could represent the transcription of single, moderately transcribed genes, whereas the dots could represent either clusters of genes or individual high‐activity transcription units. Regardless of their origin, the similar appearance and location of both incorporation and polymerase suggests that both regions represent active sites of pol II‐directed transcription and that both are clearly distinguishable from speckles.
Close examination of multiple double‐labeling experiments indicated that the discrete sites of pol II and BrU staining in cells demonstrating nuclei‐wide BrU incorporation were not absolutely coincident. The size, shape or intensity in some of overlapping domains are not exactly the same. In particular, it was usually possible to observe a subfraction of pol II staining that had little associated BrU incorporation which presumably represented the subpopulation of pol II that was not actively engaged in the transcription (Kim et al., 1997). Moreover, the loci of nucleoplasmic BrU incorporation demonstrated considerable overlapping pol II staining.
In addition to H5, mAb H14, which recognizes various phosphorylated forms of the pol II large subunit, was also used in co‐localization of pol II and its transcripts. In both CaSki and OV‐MZ‐15 cells, H14 staining appeared identical to H5 pattern as a meshwork overlaid with discrete nuclear dots which overlapped with BrU staining, suggesting that different phosphorylated forms of pol II may exist in transcription domains. This result agrees with the results shown in Figure 1A that both antibodies can recognize transcriptionally engaged polymerase molecules.
Pol II localizes to speckles in cells not actively involved in transcription
A second pattern of in situ transcription was observed with CaSki, but not OV‐MZ‐15 cells, and is represented by the bottom cell in Figure 2D–F. This cell, representing 20–40% of the population, stained minimally with the anti‐BrU antibody, indicating a poor incorporation of BrU. Incorporation in these cells was restricted to nucleoli when detected with phase‐contrast microscopy (data not shown). In this second subpopulation of cells, pol II staining was concentrated into minimal domains that resembled speckles. A speckled distribution of pol II has been reported previously for MDCK cells (Bregman et al., 1995). In this latter study, speckles were preferentially seen in cells within the early G1 or late G2 phases of the cell cycle, in ∼20% of the cells within an unsynchronized cell population.
The data in Figure 2D–F indicated that the pattern of pol II localization depended on the distribution of nascent transcripts, those cells that demonstrated active, nucleoplasm‐wide transcription demonstrated nucleoplasm‐wide pol II localization, suggesting that in these cells the staining pattern of pol II‐specific antibodies represents sites of transcription of pol II transcribed genes. In contrast, the ‘speckle’ staining pattern for pol II was only observed in cells in which transcription was limited to the nucleoli, suggesting an alteration in localization of pol II, depending on the ability to detect numerous nascent transcripts. It should be noted that although mAb H5 recognizes hyperphosphorylated polymerase II, it has been shown to be capable of detecting pol II in cells arrested for transcription immediately before or after mitosis or with drug treatment (Bregman et al., 1995), indicating that the phosphorylated CTD epitope is still present under these conditions. In agreement with this interpretation, cells with and without appreciable BrU incorporation had approximately equal staining with mAb H5 (compare the two cells in Figure 2E), suggesting maintenance of the phosphorylated pol II epitope detected by this antibody in cells not actively incorporating BrU.
We interpret the lack of nucleoplasm‐wide pol II staining and BrU incorporation in the minor cell population to reflect minimal pol II‐directed transcription in these cells. Alternative interpretations, including lack of permeability to both anti‐pol II antibodies and nucleotide analogs, or inaccessibility of antigens to antibodies, seem unlikely because of the ability of pol II actively to decorate speckles in these cells as well as the ability to detect nucleotide incorporation into nucleoli in the same population.
In vitro splicing has recently been shown to be inhibited if all uridines (U) within the utilized precursor RNA were replaced with BrU, presumably due to a strict requirement for U in consensus splicing sequences (Wansink et al., 1994). To address possible problems in factor localization arising from the use of a nucleotide analog, we compared the staining patterns of BrU, pol II and SC35 in CaSki and OV‐MZ‐15 cells during in situ transcription experiments using mixtures of UTP and BrUTP in various ratios, or in the total absence of BrUTP. The BrU staining pattern remained the same when the ratio of UTP:BrUTP was varied over a 4‐fold range (data not shown), indicating minimal impact on the sites of transcription as BrUTP was added. More importantly, the pattern of pol II and SC35 staining remained the same when cells were transcribed in the absence or presence of BrUTP (data not shown). Thus, the distribution patterns of pol II and SC35 were not affected by the presence of BrU during an in situ transcription experiment.
Inactivation of transcription localizes pol II to speckles
The above experiments suggested a correlation between transcriptional state and the localization of polymerase II within the nucleoplasm. To better correlate the two, experiments were performed in which transcription was halted by either treatment with α‐amanitin or heat shock (Figures 2J–L and 3, respectively). When α‐amanitin was added to the in situ transcription of CaSki cells at a concentration sufficient to inhibit only polymerase II, BrU incorporation was severely curtailed in the entire population and bright staining was limited to nucleoli (Figure 2J). In all cells, pol II staining was highly concentrated in a minimal number of centers, to produce a pattern closely resembling that of speckles (Figure 2K and L). Thus, when pol II activity was inhibited and the synthesis of pre‐mRNA arrested, pol II staining was highly localized.
Heat shock also inhibits pre‐mRNA production. When MDCK cells were subjected to a heat shock of 45°C for 1 h, pol II distribution as monitored by staining with the mAb H5 antibody was concentrated into speckles as distinguished from the nucleoplasmic meshwork staining in cells at normal temperature (compare Figure 3B with Figure 3A). If permitted to recover at 37°C for 1 h, the speckled distribution of pol II disappeared and the normal meshwork staining pattern was restored. Recovery of pre‐heat shock distribution of pol II required normal temperature; recovery at 0°C caused maintenance of pol II staining in a speckled pattern (Figure 3C and D, respectively). As with α‐amanitin treatment, pol II distribution was throughout the nucleoplasm under conditions permitting transcription, but was more localized into a speckle‐like pattern when pre‐mRNA production was inhibited. The reversible recompartmentalization of pol II upon heat shock suggests a possible cycling of polymerase between speckles and more widely distributed sites of transcription.
To confirm that the minimal staining domains of pol II detected when transcription was arrested actually were speckles, the staining pattern of pol II and SC35 were compared in CaSki cells under conditions of in situ transcription (Figure 4). As before, pol II staining was observed in different patterns in subpopulations of cells: a broad meshwork was observed in a majority of cells with pronounced ‘speckles’ in a minority of cells (speckles marked with arrows in Figure 4A). In addition, there were cells in the population that demonstrated an intermediate pattern of pol II distribution. The speckles of pol II correlated well with the speckles of SC35 (Figure 4B). Interestingly, SC35‐detected speckles were most pronounced in those cells demonstrating pol II speckles. In those cells in which pol II was more dispersed to the meshwork and nuclear dots, the SC35 speckles were less pronounced, suggesting that SC35, like pol II, may redistribute dynamically within nuclei depending on transcription (see also Figure 6).
Polyadenylation and splicing factors overlap with sites of transcription
Similar appearance of in situ BrU incorporation and polymerase II within both a broad meshwork and dots throughout the nucleoplasm permitted us to re‐address the question of co‐localization of transcription and RNA processing with an emphasis on the non‐speckled portion of the nucleus. Figures 5 and 6 examine the localization of transcription as measured by BrU incorporation versus the localization of the polyadenylation factor CstF and the arginine–serine‐rich (SR) family of splicing factors.
CstF is a highly conserved heterotrimer involved in the polyadenylation of pol II‐transcribed pre‐mRNA. Previous studies had indicated that antibodies to CstF broadly stain nuclei (Takagaki et al., 1990). Recently, cleavage factors CstF and CPSF were also shown to be enriched in a few ‘cleavage bodies’, some of which contained newly synthesized RNA (Schul et al., 1996). When used to stain OV‐MZ‐15 cells in an in situ BrU incorporation study, antibodies to BrU and the 64 kDa subunit of CstF appeared to block each other in many cells, i.e. if a cell was significantly stained by one antibody, the intensity of the alternate staining was too weak to record. This blocking effect strongly suggested co‐localization of epitopes in nascent RNA and CstF within the nucleus. A portion of stained cells showed less blocking and permitted visualization of CstF in a broad meshwork pattern similar to the pattern of BrU incorporation (Figure 5A and B, respectively), suggesting that polyadenylation factors and polymerase II are localized in the same or adjacent regions of the nucleus. It should be noted, however, that certain ‘dots’ accentuated in the BrU incorporation stained only poorly with the anti‐CstF antibody, suggesting either a population of pol II‐directed transcripts not requiring polyadenylation, such as histone pre‐mRNA, or partial masking of one antibody for the other. Cumulatively, in addition to antibody blocking, CstF staining and BrU incorporation overlap broadly over the nuclear meshwork.
Staining with antibody 104, which detects the major members of the mammalian SR family of splicing factors (Zahler et al., 1992), also stained nuclei broadly in those cells that actively incorporated BrU (Figure 6A–C). A portion of the broad staining appeared to overlap with nascent RNA; a portion of the BrU incorporation was not associated with appreciable mAb 104 staining. Thus, the SR proteins did not seem to be as prevalently associated with pol II transcription units as did the polyadenylation factor CstF, consistent with a uniform requirement for CstF in the generation of polyadenylated RNA and a more specialized requirement for the major SR proteins family members detected by mAb 104.
As with the pol II antibodies, staining of splicing factors in those cells that actively incorporated BrU was visually different than staining in cells that poorly incorporated BrU. In these latter cells, the SR proteins detected by mAb 104 were highly localized into minimal domains of speckles. Thus, like pol II, the localization of SR proteins within the nucleoplasm was related to the ability of individual cells to demonstrate appreciable BrU incorporation.
The pattern of staining observed with the anti‐SC35 antibody was also compared with the pattern of BrU incorporation in a double‐label experiment (Figure 6D–F). Similar to the mAb 104 staining, the staining pattern of this individual SR protein varied from dispersed throughout the nucleoplasm (Figure 6E, top cell) to concentrated within minimal speckle domains (Figure 6E, bottom two cells). BrU incorporation was maximal in cells showing a dispersed SC35 pattern, and minimal in cells demonstrating a concentrated, speckle pattern (Figure 6D).
In cells demonstrating the more classical speckle pattern of SC35 staining, the limited BrU incorporation occurring did not co‐localize with the speckles (Figure 6F). Dispersed and apparent overlap of BrU incorporation and SC35 increased as the level of overall incorporation of BrU increased, suggesting that at least a portion of the SC35 is localized throughout the nucleoplasm when BrU incorporation and transcription is occurring maximally. The overlap of SC35 staining and BrU incorporation was not as pervasive as the overlap of pol II and BrU incorporation, suggesting that SC35 is not associated with all nucleoplasmic transcripts.
Localization of transcription within mammalian nuclei
Recently, it has become possible to localize RNA polymerase II in somatic nuclei at the level of light microscopy via the use of monoclonal antibodies H5 and H14 directed against the CTD of the large subunit (Bregman et al., 1995; Kim et al., 1997). Applied to several lines of cultured mammalian cells, these antibodies detected pol II throughout the nucleoplasm in a broad meshwork with several hundred sites of preferred localization. This pattern suggests that Pol II is distributed broadly in the nucleoplasm and, by inference, that pol II‐directed transcription of pre‐mRNA occurs throughout the nucleoplasm. Such an interpretation was substantiated via the use of in situ incorporation of nucleotide analogs to detect active transcription. The nuclear distribution patterns of both pol II and in situ nucleotide incorporation were highly complex, with both antigens distributed in a meshwork pattern that appeared fibrous under high magnification. Although co‐localization of complex patterns is hard to determine unambiguously via light microscopy, the patterns of pol II and BrU incorporation appeared to co‐mingle throughout the nucleoplasm, suggesting that both patterns are indicative of pol II transcription of pre‐mRNA and that such transcription is not restricted to minimal domains within mammalian nuclei.
When transcription was arrested by either drugs or heat shock, polymerase II distribution altered such that it could no longer be detected as an intensive meshwork throughout the nucleoplasm and instead appeared in minimal domains corresponding to speckles. These results suggest a dynamic situation in which polymerase II is recruited to sites of active transcription located throughout the nucleoplasm and relocates during inactive transcription to minimal domains. In this view of nuclear pre‐mRNA generation, speckles would be considered as temporary storage or perhaps recycling centers for multiple factors required for mRNA biogenesis.
Evidence that pol II could be detected in a speckled pattern has been reported previously (Bregman et al., 1995). Pol II was detected in speckles most predominantly during early G1 and late G2 stages of the cell cycle in MDCK cells, suggesting that pol II localization was concentrated in speckles during those parts of the cell cycle associated with minimal transcription. Here, we saw pol II localization to a speckle pattern in a minor population of CaSki cells during an in situ transcription experiment. This population of cells incorporated BrUTP only within nucleoli, suggesting that they were not actively engaged in pol II‐directed transcription during the labeling interval. Observation of this population of pol II localized to speckles was cell line‐specific; OV‐MZ‐15 cells, a line of human cervical carcinoma cells, for example, did not yield a speckle pattern unless transcription was deliberately blocked. Cumulatively, the observations from experiments localizing polymerase II and sites of BrU incorporation indicate that neither are localized to speckles when transcription is maximal, but that pol II can be seen in such minimal domains under natural or induced conditions when transcription is restricted.
The C‐terminal domain of polymerase II contains all known phosphorylation sites in the subunit (reviewed in Corden, 1993; Dahmus, 1995). Two pol II‐directed antibodies used in this study, H5 and H14, are directed against hyperphosphorylated or various forms of phosphorylated pol II, respectively. In experiments using immunoprecipitation of nascent in vivo RNA (Figure 1) and in situ BrU incorporation, both H5 and H14 antibodies efficiently recognize newly transcribed RNA. No significant difference was observed in their abilities to bind transcriptionally engaged polymerases, indicating the involvement of different forms of phosphorylated pol II during transcription. These results also imply that the level of phosphorylation may not be well correlated with transcriptional activity per se, although phosphorylation occurs concomitant with the initiation of transcription and elongating pol II has been shown to be hyperphosphorylated (Laybourn and Dahmus, 1990; Kang and Dahmus, 1993; Weeks et al., 1993).
Co‐localization of transcription and splicing
Elegant electron microscopy of Drosophila salivary glands has suggested that transcription and processing are coincident with 5′‐proximal introns being recognized and processed before 3′‐proximal regions of a pre‐mRNA have even been synthesized (Beyer et al., 1988). If transcription and processing are coincident, co‐localization of transcription and processing factors would be predicted. Here, we show that BrU incorporation, pol II localization and localization of one polyadenylation factor (CstF) and a family of splicing factors (the SR proteins detected by mAbs 104 and SC35) overlap throughout the nucleoplasm in a pattern of a nucleoplasmic meshwork with many sites of preferred staining that is distinguishable from speckles. This meshwork pattern of nascent RNA staining has not been the focus of previous studies because of an interest in the more visually prominent domains represented by speckles. Broad meshwork staining, however, has been noticed in studies of the distribution patterns of a number of processing factors other than SC35 (Takagaki et al., 1990; Carmo‐Fonseca et al., 1991a,b, 1992; Jenny et al., 1994; Krause et al., 1994).
Our results indicate that splicing factors are located throughout the nucleoplasm in the same area in which transcription by pol II is occurring. Like the localization of polymerase II, the localization of splicing factors was dependent upon the transcriptional activity of the cell being examined. When BrU incorporation was high, splicing factors localized throughout the nucleoplasm; when it was low, they concentrated in speckle‐like domains. Thus, both splicing and transcription factors have a dynamic localization within the nucleus and may be recruited from speckles to the position of active transcription.
Our results are also in agreement with recent biochemical studies that suggest an interaction between the transcription and processing machinery. Isolation of cDNAs that interact with the CTD of polymerase II in the yeast two‐hybrid system (Yuryev et al., 1996) identified four genes each containing a significant arginine–serine domain similar to those found in the arginine–serine family of splicing factors (Zahler et al., 1992), suggesting a direct protein–protein interaction between polymerase II and splicing factors. More directly, antibodies against polymerase II inhibit in vitro splicing and the pol II CTD peptide inhibits in vitro (Yuryev et al., 1996) and in vivo (Du and Warren, 1997) RNA splicing.
Models for nuclear organization of mRNA transcription and processing
This study examined the distribution of bulk factors. Earlier attempts to address the possibility of co‐localization of transcription and processing have used in situ hybridization techniques to detect identified individual transcripts or bulk polyadenylated RNA. Although different laboratories have observed different results, at least several abundant, actively transcribed genes have consistently been localized to speckles rather than to the inter‐speckle meshwork described in this report. Two possible explanations present themselves immediately for the discrepancy between localization of individual transcripts to speckles and the localization of bulk transcription to the region between the speckles. The first explanation, which we prefer, is that transcription units operating to produce extremely abundant pre‐mRNAs are situated near localized regions of high concentration of splicing factors—the ‘speckles’—but that most transcription units operating at lower efficiency are localized throughout the nucleoplasm. Certainly the genes that have been localized to speckles in previous studies, collagen and fibronectin, are extremely abundant RNAs in the cell types examined and might not be representative of lower copy transcription units (Xing et al., 1993, 1995). The ability of high‐activity transcription units to be found near speckles is underscored by the co‐localization of speckles and transfected plasmid DNA with transcription driven from strong promoters (Huang and Spector, 1996).
A second, and in our opinion less attractive, hypothesis is that BrU incorporation and polymerase II localization cannot be used to indicate pre‐mRNA synthesis because of the existence of a large population of polymerase II‐driven transcription to produce RNAs other than pre‐mRNAs. Early studies examining nuclear RNA indicated that the sequence complexity of nuclear RNA is not represented in mRNA (Harpold et al., 1981; Salditt‐Georgieff et al., 1981). Given the experimental limitation used in early studies, however, this non‐mRNA may in part be represented by intervening sequences (whose sizes are considerably larger than exons in vertebrates) as well as a class of RNA undergoing rapid nuclear turnover (from genes regulated at the level of nuclear RNA stabilization). In this study we document that incorporation of ribonucleotides and pol II‐localization as detected by antibodies directed against a transcriptionally competent form of pol II are not restricted to speckles, but are instead widely distributed throughout the nucleoplasm, indicating that transcription by pol II is broadly distributed. Thus, in the absence of contradictory data establishing that pol II distribution and BrU incorporation represent a bulk population of RNA other than pre‐mRNA, we interpret our results to indicate that transcription and processing occur throughout the nucleoplasm.
In addition to the broad meshwork pattern observed for most of the antibodies used in this study, we likewise observed that each also stained multiple sites of preferred localization. These were more numerous and smaller in diameter than classic speckles. For polymerase II, these ‘dots’ overlapped with BrU incorporation, indicating that they represent active sites of in situ transcription. A similar pattern of preferred staining has been noticed previously in in situ incorporation experiments (Jackson et al., 1993; Wansink et al., 1993; Iborra et al., 1996; Schul et al., 1996). The identity of these regions is unclear, but they are of an abundance to suggest that they may represent abundant transcription units. The association of these domains with the polyadenylation factor CstF underscores their likely identification as transcription units. Splicing factor antibodies only stained a subset of these locations, suggesting preferred localization of splicing factors with only a subset of pre‐mRNAs.
Overall, our results are not consistent with speckles as the preferred sites of transcription and RNA processing. Incorporating our results with those of other laboratories indicating an ability to locate individual transcription units to speckles, we suggest that transcription and splicing are coincident and localized throughout the nucleoplasm, presumably at the sites of the transcribed chromatin. In addition, we suggest that abundant transcription units, especially those driven from transfected genes, can be seen at the light microscopy level as dots or speckles of preferred staining that represent local high concentrations of both transcription and processing factors. Furthermore, we suggest a dynamic interaction between the dispersed population and the clustered population that reflects transcriptional status such that both transcriptional and processing factors can be found highly localized when transcription is minimal and highly dispersed when transcription is maximal. Combining these two suggestions indicates that speckles may be factor recycling centers which associate closely with some highly active transcription units because of the abundance of factors within the speckles.
Materials and methods
Cell culture and heat shock
Human cervical epidermoid carcinoma cells (CaSki) were grown in RPMI 1640 with 10% fetal bovine serum (FBS). HeLa and MDCK cells were grown in DMEM with 10% FBS. The human ovary cancer cell line, OV‐MZ‐15 was maintained in the same DMEM/FBS medium supplemented with 10 mM HEPES pH 7.0, 2 mM glutamine, 116 μg/ml arginine and 30 μg/ml asparagine. Heat shock was applied by incubating MDCK cells grown on coverslips at 45°C for 1 h, followed by recovery for 1 h at 37°C or 0°C as a control.
Cross‐linking of radiolabeled RNA to transcriptionally active Pol II in isolated nuclei and immunoprecipitation of labeled RNA with pol II antibodies
This procedure was adapted from an established protocol as described (Bartholomew et al., 1986). Briefly, HeLa cell nuclei (5×107/ml) were incubated in ‘run on buffer’ containing 500 mCi/ml [α‐32P]CTP (>2000 Ci/mmol, Amersham), 0.1 mM ATP, 0.1 mM 4‐thio‐UTP, 1 mM‐3′‐O‐methyl GTP, 0.12 M KCl, 5 mM Mg‐acetate, 0.05 mM EDTA, 12.5% glycerol, 0.05 mM dithiothreitol, 25 mM Tris–HCl pH 8 and 0.1 mM PMSF. The transcription reaction was carried out at 20°C for 10 min with or without α‐amanitin (10 μg/ml), after which the nuclei were exposed to UV (λmax = 310 nm) to induce covalent cross‐linking between proteins in contact with 4‐thio‐UTP‐containing radiolabeled RNA. The nuclei were subsequently digested with RNase T1 and DNase I as described (Bartholomew et al., 1986). A nuclear extract was prepared from these nuclei using ice‐cold T‐buffer (50 mM Tris–HCl pH 7.4, 250 mM NaCl, 1% Triton X‐100, 1 mM PMSF, 0.2 mM Na2VO4, 5 mM β–glycerolphosphate). Insoluble material was pelleted by centrifugation (15 000 g for 10 min), and the supernatant was used for immunoprecipitation with mAb H14 or H5 as described (Kim et al., 1997). Immunoprecipitates were washed three times in ice‐cold T‐buffer, boiled in SDS sample buffer, subjected to 6% SDS–PAGE, and processed for autoradiography.
In situ run‐on transcription
CaSki or OV‐MZ‐15 cells grown on coverslips were washed with a ‘physiological’ buffer (PB) (pH 7.4, 22 mM Na+, 130 mM K+, 1 mM Mg2+, 2.5 mM Ca2+, 31 mM Cl−, 100 mM acetate, 11 mM phosphate, 0.5 mM ATP, 1 mM DTT and 0.2 mM PMSF; Jackson et al., 1993). Samples were then slightly permeabilized with cold 0.025–0.05% Triton X‐100 in PB for 2 min. Run‐on transcription was performed by incubating cells with reaction buffer (PB containing 0.1 mM CTP, GTP and BrUTP; 2 mM MgCl2 and 4 U/ml RNasin) for 20–30 min at 30°C. Alternatively, cells were incubated with reaction buffers containing mixtures of UTP/BrUTP in a ratio of 1/3, 2/2 or 3/1, or UTP alone, respectively. For inhibition of in situ transcription, 1–2 μg/ml α‐amanitin was added to the reaction buffer during BrU incorporation. Reactions were stopped by rinsing samples with cold PB and cells were post‐permeabilized with cold 0.1–0.2% Triton X‐100 in PB for 2 min. Finally, cells were fixed in 3.2% paraformaldehyde for 20 min. The nascent RNA was detected by immunofluorescence with a commercial BrdU antibody which also recognizes BrU.
Immunofluorescence and microscopy
The following primary antibodies were used for immunofluorescence in this study: pol II large subunit antibodies, mAb H5 and mAb H14 (Bregman et al., 1995); mAb anti‐SC35 (ATCC); anti‐BrdU mAbs (Boehringer Mannheim, Seralab); mAb anti CstF 64 kDa (Takagaki et al., 1990); and mAb 104 (Zahler et al., 1992).
Cells were labeled with BrUTP and then fixed as described above. For samples without BrU labeling, cells were permeabilized for 1–2 min with 0.5% Triton X‐100 in CSK buffer (10 mM PIPES pH 7, 1 mM EGTA, 3 mM MgCl2, 20% sucrose; Fey et al., 1986), and then fixed with 3.2% paraformaldehyde in the same buffer for 20 min. Alternatively, cells (Figure 6) were fixed and then permeabilized with 0.5% Triton X‐100 in PBS. For fluorescent staining, coverslips were incubated with properly diluted antibodies at 37°C for 30 min. After washing with PBS three times, cells were incubated with diluted secondary antibodies (1:20–1:50) conjugated with FITC, TRITC or Texas Red (Jackson Immuno‐Research) for 30 min, and then washed and counterstained with DAPI solution (0.1 μg/ml) for 5 min. For double staining, fluorescent antibodies were all pre‐tested not to cross‐react with other immunoglobulins. Samples were finally mounted with glycerol/PBS (9:1) containing 0.1% p‐phenylenediamine (Sigma).
Images were obtained from fluorescent microscopy (Zeiss Axiophot; Figures 1B and C, and 3,4,5) or laser confocal microscopy (Molecular Dynamics; Figures 2 and 6). Optic sections of double‐stained samples were scanned with a Nikon laser microscope. A dual wavelength channel was used to excite FITC and TRITC/Texas Red at 488 and 568 nm respectively. Fluorescent signals of both fluorochromes were recorded simultaneously at one scan. No cross‐talk was observed. Image slides were taken from a digital camera (Polaroid, Cl 5000S) using Adobe and Powerpoint software.
We thank James L.Manley and Mark B.Roth for kindly providing antibodies, and Michael Dahmus for advice on the UV cross‐linking experiments. This work was supported by grants from the Tobacco Research Council (S.L.W.), NIH (GM38526, S.M.B.) and the American Cancer Society (S.M.B.).
- Copyright © 1997 European Molecular Biology Organization


text: 字幕表 動画を再生する 英語字幕をプリント The capital of Northern Ireland has traditionally been thought of a secondary holiday location to Dublin. However Belfast is quickly establishing itself as the place to visit for a truly authentic Irish experience. The history of Northern Ireland's capital searches back to the Bronze Ages, and during the 19th century, the city began to flourish as a naval port and ship-building center, famously being the birthplace of the Titanic. Perhaps the most famous period of Belfast's history, however, is its most tragic - a period of political conflict spanning three decades. And the city still has plenty of reminders of the troubles, in particular the striking and sometimes intimidating murals depicting various events of the times. Belfast has plenty of traditional pubs were visitors can enjoy a pint with the locals, and there are dozens of historic buildings, hotels and old streets, most of which can be explored on foot.

domain: history


text:
Hedging her bets
At first glance, Tracy Falbe looks nothing like what you’d expect from a woman running a tattoo Web site. Or a former craps dealer. Fantasy novelist, maybe. But mostly, with her calm demeanor and noticeably ink-free skin, Falbe looks more like her domesticated side—a mother of two. She is actually all of the above. After moving to Northern California from Las Vegas, where she was known as Tracy Michigan (also the pen name she used for Get Dicey, her book about playing craps), she got her degree in journalism from Chico State. She now puts her reporting skills to work creating Web sites with plenty of content, many of which tie in with her books. Her most recent venture is Destination Tattoo (http://tattoo.falbepublishing.com).
Well, tattoos are popular, so I knew there would be a general level of interest. And also, when I was younger and I really wanted to get a tattoo, I thought they were so cool and all that, but I didn’t have the money for it. Then as I got older and had the money, I didn’t really want one anymore. I see that today, compared with 10 to 15 years ago, when I was thinking about getting a tattoo, they’ve really grown in popularity. It’s interesting because there are so many different styles. There are health issues. There’s tattoo removal. It also has an artistic element. Sometimes I’ll look at a tattoo and think it’s really cool and other times I can’t even fathom why they’d want that on their body.
You also have sites on swords, dragons and casinos.
Some of my sites have a cross-commercial element with my publishing business. Years ago I used to be a craps dealer in Las Vegas. So, the first book I wrote was Get Dicey, and it was just a how-to-play-craps book. So I sell that through my Web site …. And so my little side site kind of helps me with marketing that. I’ve written a fantasy series, too, four books. So for the swords and the dragons Web sites, I find them interesting subjects to publish on, but I’m also trying to maybe catch the eye of people who might be a good target audience for my books.
What are your hopes for your business?
My business isn’t at a real big money-making level. I’m home with my kids right now, while my husband works. I have a 3-year-old and a 1-year-old. I’m trying to build in that value and hopefully a couple years down the line it will be a much more significant income. You could make $10 a month, or you could make $100,000 a month. With the wide variety of topics, I’m hoping something will just hit pay dirt.

domain: artistic


text:
Girls' Luxurious Birthday Cakes
Cakes for your Princess
The gallery shows just a few designs made by Celebration Cakes by Yvonne. Some of these have edible images. I also offer edible imaging printing of any design you wish. Depending on the chosen design the cake will be covered with a layer of fondant over either white or milk ganache, or simply a layer of buttercream. I love a new idea so please let me know your vision and I’ll make it a reality. After all, your Princess deserves the best on her special day.
Pop along to our Testimonials to see how clients have been delighted with their bespoke celebration cake.
Delivery is available throughout the Home Counties: Ascot, Bagshot, Camberley, Chobham, Crowthorne, Farnborough, Farnham, Frimley, Sandhurst, Sunningdale, Sunninghill, Virginia Waters, Wentworth, Yateley.
Please contact Celebration Cakes by Yvonne either by telephone or click the button below.

domain: design


The inventory management landscape is rapidly evolving due to automation, significantly altering retail business practices. As operations grow in complexity, there's a pressing need for more sophisticated inventory management systems that not only track but also add strategic value. Gone are the days of spreadsheet-based, haphazard order placements of the 2010s—today's retailers demand multifunctional, intelligent solutions to stay competitive.
Automated inventory management systems often outperform by eliminating biases, personal incentives and focus solely on organizational objectives.
Operational teams aim to reduce write-offs by ordering fewer low-demand items. However, when KPIs focus on sales, manual reorders might prioritize best-sellers over low-demand products, skewing inventory. Recognizing these inefficiencies, technology-driven online inventory systems emerged, replacing manual Excel and basic ERP systems. These automated systems streamline order processes, eliminating time-consuming manual calculations and enhancing efficiency.
Contemporary inventory management systems have transformed order processes by fully automating them. These systems proficiently analyze sales data across numerous SKUs, monitor fluctuating demand, and autonomously determine necessary restock quantities, seemingly fulfilling every retailer's wish. Yet, it's crucial to note that without strategic business process planning, even the most advanced software cannot guarantee a boost in profits.
Importance of inventory management system in business
In retail, financial performance hinges on the operating cycle's length, influenced by inventory turnover and business credit. A longer cycle means more funds for operations and growth. Business credit is reserved for reputable companies and relies on good vendor relations. Inventory turnover, indicating management efficiency, is calculated using stock and sales data to show time until restock, and is affected by product mix and management tools used.
So, an inventory management system software streamlines retail business processes to maintain optimal stock levels, ensuring product availability meets customer demand. These business processes are:
Replenishment of store and central warehouse inventory. Including the goods of regular demand, promotional, seasonal products (taking into account all retailers and suppliers SC restrictions);
Analysis of inventory key performance indicators;
Response to emerging problems;
Consider a retailer with 30 stores, each carrying 5,000 unique SKUs. This totals 150,000 items, each with individual demand, supply, and performance metrics. Calculating restock quantities for such a vast inventory requires meticulous analysis. This scenario underscores the necessity for an automated inventory management system. Such a system handles complex calculations and analysis, freeing managers to focus on strategic activities, KPIs, and development. When selecting or upgrading an inventory management system, prioritize the following functions:
Accurate order quantity generation for restocking each SKU.
Comprehensive demand tracking and analysis, adaptable to seasonal and sales fluctuations.
User-friendly analytics for critical inventory performance indicators.
Essential Attributes of a Robust Inventory Management System:
Advanced Automation & User-Friendly Interface: A superior inventory management system should offer high automation and a quality UI/UX. It enables creation of tailored task lists for users who can then monitor inventory levels, manage order and delivery schedules, and access customized reports on inventory status, deficits, and delayed deliveries. Moreover, it should swiftly identify and notify users of potential errors.
The system's usability and intuitive design are crucial for its overall effectiveness. It should be easily navigable, allowing users at all levels to comprehend data and operate the system with minimal learning time.
Enhancing Financial Performance An online inventory management system can bolster profitability through:
Automated tracking and restocking calculations for each SKU, with real-time inventory updates reflecting demand shifts, delivery schedules, and packaging changes.
Streamlined restocking and reshipping within the supply chain, facilitating automated order placements to suppliers, warehouses, and stores.
Tailored restocking volumes for stores and distribution centers, informed by analyses of seasonal trends, sales performance, and demand surges for promotional or perishable items.
Each product category has its nuances. An inventory management system must account for these by analyzing product-specific characteristics. For instance, in managing dry goods, the system should track:
Average daily demand
Upcoming promotions and sales
Supplier reliability and reputation
For fresh goods, an inventory management system must additionally manage:
Daily demand variations by day of the week
Targeted availability and merchandising for specific customer groups
Product expiration dates
The system should be adaptable, with customizable algorithms to suit unique business needs and objectives. For instance, when generating orders for the main warehouse, the system should factor in:
Store-specific sales demand
Promotional and sales activities per store
Seasonal demand fluctuations
Distribution Center (DC) sales
Other variables affecting orders within a delivery cycle
Advanced Order and Restocking Functions:
Order Optimization: The system refines order quantities based on cost, weight, and product count, adjusting for supplier minimums or budget constraints.
Logistics Adaptation: It splits orders to match logistical capacities, like vehicle load and fleet size.
Substitute Management: The system identifies and uses substitute products, tracking their presence in the inventory.
Supplier Diversification: It facilitates working with alternative and additional suppliers for robust supply chain management.
Managing Promotions and Seasonal Inventory:
Automated restocking post-promotions and seasonal peaks.
Detailed analysis of seasonal demand variations.
Tracking and anticipating holiday logistics delays, ensuring timely order placement.
Proactive inventory preparation for promotional periods.
Real-time monitoring and adjustment of promotional demand.
Efficient tracking of additional supplier commitments during promotions.
Balancing inventory at the end of promotional events.
Clear visual analytics for evaluating promotional sales and demand.
Performance Monitoring and Troubleshooting:
Comprehensive, real-time analytics for key metrics like stock levels, turnover, and sales.
Alerts for critical tasks needing urgent action, such as inventory adjustments for promotions.
Data Management and Transparency:
Quick generation of stock status reports and detailed order histories for responsive decision-making.
Tracking of user modifications within the system for accountability.
Advanced reporting tools for analyzing lost sales, overstock, and inventory metrics, with customizable filters.
Supplier performance reports covering reliability and order fulfillment.
Detailed inventory turnover and availability insights.
Tracking of like-for-like metrics.
Specialized reports for fresh products and promotional activities.
Audit trails for user-made changes.
Cross-Platform Accessibility and Adaptability:
Remote access from any device, anywhere.
Highly adaptable settings for bespoke business requirements, avoiding costly programming.
Seamless integration with various reporting systems.
Fast operational speed.
Round-the-clock technical support from the provider.
In summary, key considerations for selecting an inventory management system include:
Adaptive restock algorithms attuned to market and demand fluctuations.
Customizability to fit your business requirements.
Transparent, real-time analytics and KPI monitoring.
Proactive notification system for inventory-related risk management.


text:
T’was The Night Before Wembley – 12th April at London Barfly
With Frank Turner headlining a sold out Wembley Arena on 13th April – we at Xtra Mile Recordings thought what better way to celebrate then to spend the night before drinking, being merry and revelling in our incredible roster.
On the 12th April at London’s Barfly we will be getting the party started with a fantastic line-up of Xtra Mile acts that showcase the strength in our ever increasing stable. Confirmed to play are: The Loved Ones frontman Dave Hause who is currently on tour with Alkaline Trio; Devonian hard-twang roots punk rockers Crazy Arm; recent signings Jim Lockey & The Solemn Sun who will mark the release of their new album ‘Death’ out on 9th April and the undeniable talents of our very own Ben Marwood complete the bill.
In charge of keeping the night moving along without a hitch is our Master of Ceremonies - Jamie Lenman.
There will be ultra ‘special guests’ on the night too so keep your eye on twitter for all breaking news!
Selling out Wembley Arena will be a fantastic achievement for Frank and for the label at large so please come along and rejoice with us in the best way we know how.
Ticket can be purchased here:
12th April – T’was The Night Before Wembley
13th April – Frank Turner at Wembley Arena
Sign up to our mailing list & receive a free MP3 of "bones" from To Kill A King. This is the first song the band wrote together and is NOT featured on the album 'Cannibals With Cutlery' –Deluxe Edition.
Tweets by @Xtra_Mile
(c) 2014 Xtra Mile Recordings.
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text:
My mum made the most delicious but simple Christmas truffles around Christmas time and I’ve created this recipe to try and recreate those truffles she made us as kids. They are so delicious and make a lovely gift too! 🙂
They are made with mint chocolate, digestive biscuits, real double cream, white chocolate and sprinkles of choice. They are so delicious you have trouble resisting them. I used sprinkles to coat my truffles and a good quality white chocolate worked well. You could mix up the chocolate and flavours a little bit more to your liking and experiment with different toppings and chocolates.
Equipment you may need:
- a glass bowl and small pan to melt the chocolate
- a large mixing bowl for mixing up all the ingredients
- Cocktail sticks or something similiar to poke into the truffles and dip into the white chocolate.
You may enjoy these as a treat after a delicious Traditional Scottish meal. The following two courses would work really well with this recipe – my Scottish Hotch Potch soup recipe and my traditional Scottish mince and tatties. You can of course serve them up after a delicious traditional Christmas dinner too or hand them out as gifts.
Tips for making this recipe:
- I made these truffles with Green & Blacks mint chocolate but you can use a plain milk chocolate should you prefer and add in a little mint flavouring if you fancy the minty flavour that way.
- These truffles were made quite large but you could make smaller ones to fit into gift bags.
- You could place these lovely truffles into a chocolate gift box to make a really nice gift for somebody. They keep pretty well as long as they are sealed. The only fresh ingredient that might have an impact on the shelf life is the double cream.
- You can vary the sprinkles and chocolate that coat these truffles too. I have used white chocolate but you could also use milk chocolate and different sprinkles to roll them in.
- You can melt the chocolate in the microwave if you prefer. This may be easier for you as I use a pan and simmering water with a heat proof bowl on the top. Click on the link to a page I found using the microwave method.
Christmas Truffles Recipe
- 150g of mint chocolate
- 150ml of double cream
- 1/2 tsp of vanilla
- 300g of crushed digestive biscuits
- white chocolate for melting (I used two large bars about 400g)
- chocolate sprinkles & coloured sprinkles for coating the truffles (enough to cover a small plate for each)
- Heat some water in the pan and add a heat proof bowl on top of the pan to melt the chocolate in. Please be very careful and make sure the bowl is steady.
- Add all of the mint chocolate into the heat proof bowl on top of the pan and bring the pan water to a gentle simmer.
- Stir the chocolate but be very careful not to get water in the bowl of chocolate. You can also melt the chocolate in the microwave using the link in the tips above.
- Pour the double cream into the melted bowl of chocolate and sitr through until warm.
- Carefully remove the bowl of melted chocolate from the pan and pour into a large bowl. Quickly add in the digestive biscuits and vanilla and stir through until you have a nice chocolately digestive dough type consistency.
- Take a tsp of the mixture into your hand and roll into small balls. You can make larger truffles if you wish.
- Repeat until all the mixture has been turned into balls. Place them all on a tray and place in the freezer for at least an hour or over night if you wish.
- Remove the balls from the freezer and set aside.
- Lay out two plates and fill one plate with coloured sprinkles and one with chocolate sprinkles or using whatever sprinkles you choose.
- Melt the white chocolate in the same way using the pan method or the microwave and set aside.
- Quickly prick the balls with a cocktail stick and dip them into the white chocolate. Use a spoon to cover them completely in chocolate.
- Using the stick gently roll the truffle in the desired sprinkles and pop onto another tray or plate to harden.
- Repeat with all of the mixture to create all of your truffles.
If you enjoyed this recipe you may like to pin it to your favourite pinterest board using the link below:
You may also like some of our other blog recipes below:

domain: food


text:
During the summer of 2012, the FBI took down Appbucket, Applanet, and SnappzMarket, some of the largest pirate app stores at the time.
In the years that followed several people connected to the Android app sites were arrested and indicted, resulting in prison sentences for some.
Aside from the initial announcement, the cases have been handled relatively quietly by the US Government. We can report, however, that after six years all cases that we know of have now been closed.
The last remaining case was that of Gary Sharp, who was indicted in two separate cases.
The Massachusetts man, who’s now in his early thirties, worked as “super moderator” on Applanet’s servers and Facebook page, for which he received a video game as compensation. At SnappzMarket he had a more important role, dealing with finances and the administrative side of the operation.
Sharp never denied his involvement with any of these sites and pleaded guilty to conspiring to commit criminal copyright infringement in both cases. In addition, he provided the FBI with detailed background information and testified in the trial of another defendant.
Based on this cooperative stance, Sharp’s attorney requested a lower sentence, which was granted. A few weeks ago, he was sentenced to a six months prison term, which started on July 14th.
This means that, without any appeals, all pirate app store cases have now been closed.
While the enforcement efforts led to convictions in most instances, the Department of Justice isn’t publicly celebrating. Perhaps that’s due to the stories of the defendants being littered with personal problems and them being far from the hardcore criminals some might expect.
The convicts did, however, help to distribute hundreds of thousands of pirated apps. And while they haven’t made millions, the Government found that they caused substantial losses to the respective copyright holders.
For example, a defendant in Appbucket case, Mr. Blocker, earned $7,222 from his involvement with the site. However, he was held liable for over $750,000 in damages. The defendant faced years in prison, but he eventually received a sentence of two years probation earlier this year.
Two other operators of Appbucket were also sentenced to probation this year, while the founder received a prison sentence of a year and a day. Two Applanet defendants walked free, while two Snapzmarket operators previously received prison sentences of 16 and 46 months.
Most cases ended in successful convictions which will likely deter others from starting similar sites. However, as is often the case with pirate ventures, there will always be people still willing to take the risk.

domain: news


text:
EastEnders star Lacey Turner has revealed that she made a deal with Charlie Clements to leave the Square at the same time as him.
The actress, who plays Stacey Branning in the BBC One soap, admitted that when Clements's character Bradley was killed off in the live episode earlier this year, she knew it was time to go.
She told Fabulous magazine: "Charlie and I always said we'd go at the same time. For quite a while I'd been saying I was going to go and kept realising I wasn't ready. But when Charlie said he was going last year I was like, 'Right, OK, I'm going too'."
Speaking of Stacey's departure, she said: "The whole thing hints at everything she's been through in the last six years and it all comes together. I'm really happy with the way it's all worked out.
"I'd like to be more like Stacey in some ways," Turner continued. "She's so forward and says what she wants to say when she wants to say it. I'd love to be able to be more like that, but I just don't have the guts."


Cached Decoding the Internet in Global Popular Culture
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“This is the most culturally sophisticated history of the Internet yet written. We can’t make sense of what the Internet means in our lives without reading Schulte’s elegant account of what the Internet has meant at various points in the past 30 years.”—Siva Vaidhyanathan, Chair of the Department of Media Studies at The University of Virginia In the 1980s and 1990s, the internet became a major player in the global economy and a revolutionary component of everyday life for much of the United States and the world. It offered users new ways to relate to one another, to share their lives, and to spend their time—shopping, working, learning, and even taking political or social action.…
Copyright year: 2013
Publisher: New York University Press
Publication date: 3/18/2013
Size: 6.25" wide x 9.00" long x 1.00" tall


by Matt Fleming | February 9, 2016 9:26 am
In dramatic fashion, Assemblyman Rocky Chavez announced Monday he was suspending his campaign to replace the retiring Democratic Senator Barbara Boxer while at a debate for that seat.
“I think the best role I can fill for the Republican Party and moving the agenda forward … is to run for my Assembly seat,” the Oceanside Republican said during the debate’s opening comments. “I’m not going to be running for the United States Senate, and I’ll leave the field right now.”
And like that, he was gone.
Chavez had struggled to keep up financially. As of the end of 2015, Attorney General Kamala Harris, the Democratic frontrunner, had nearly $4 million in the bank. Rep. Loretta Sanchez, D-Garden Grove, was relatively close with $2.1 million.
Two former CAGOP chairmen, Duf Sundheim of Los Altos and Tom Del Beccaro of Willows, had $70,000 and $40,000, respectively.
And Chavez had $369. Just for comparison, California’s other Democratic senator, Dianne Feinstein, spent $12.1 million to retain her seat in 2012 — and a million wasn’t even close to enough for her Republican challenger.
Democrats Are Leading
Although in Chavez’s speech he said he was falling on his sword for the good of the party and the seat, the other Republicans running for the seat still face long odds, with Democrats in the state outnumbering Republicans 43.2 percent to 28 percent (23.6 percent have indicated no party preference).
According to a Field Poll last month, Harris led with 27 percent with Sanchez trailing at 15 percent. The three Republicans had a combined 13 percent (with Chavez ahead at 7 percent) and 44 percent still undecided.
And the Republicans’ chances are even murkier now in the heavily Democratic seat as California in recent years moved to a jungle primary where the top two advance to the November general election regardless of party.
Throughout the 2014 primary, voters saw minority party candidates squeak through to the general when enough candidates from the majority party split the vote, which will be Sundheim and Del Beccarro’s best chance (although either’s prospects are hurt with the other in the race as well because they’re also splitting votes).
Chavez’s recommitting to his Assembly seat does add intrigue there, however. While the seat has a 6 percent tilt towards Republicans, Chavez won re-election in 2014 with 66.9 percent against another Republican.
The two Republicans already running have a sizable fundraising advantage over Chavez. Phil Graham, the stepson of former Gov. Pete Wilson has $221,000, while Oceanside City Councilman Jerry Kern has $138,000.
In his Assembly account, Chavez has $601.
Source URL: https://calwatchdog.com/2016/02/09/chavez-drops-senate-campaign-recommits-assembly/
Copyright ©2021 CalWatchdog.com unless otherwise noted.


text:
Although many environmental psychology studies have investigated retail settings and green environments separately, the incorporation of greenery in retail areas is barely explored. By means of a 2 by 2 experimental design, we empirically studied the effect of in-store vegetation on consumer emotions and responses towards the store, considering the moderating role of the information rate of the retail setting. While introducing foliage in the store environment did not seem to evoke any feelings of excitement, in-store vegetation was found to elicit pleasure and reduce stress, albeit these effects could only be discerned in a 'complex' store interior. Given the impact of pleasure and stress on consumers' approach/avoidance responses, these findings support the potential of integrating greenery in retail store environments, especially in 'spatial dense' stores.
|Titel||Proceedings van de ES Navorsingsdag|
|Uitgeverij||Vrije Universiteit Brussel|
|Status||Published - 18 sep 2009|
|Naam||Proceedings van de ES Navorsingsdag|

domain: literature


text:
WASHINGTON (AFP) - The United States is seeking an explanation from Russia on the status of fugitive US intelligence whistleblower Edward Snowden, the White House said on Wednesday.
"We're seeking clarity from Russian authorities about Mr Snowden's status and any change in it," White House press secretary Jay Carney told reporters aboard Air Force One.
He reiterated the US position that Snowden "is neither a human rights activist nor a dissident", adding: "He has been charged with serious felonies for the unauthorized leaking of highly classified information."
"Mr Snowden should be expelled at returned to the United States," Mr Carney said.
He spoke amid conflicting reports from Moscow about whether Snowden would be allowed to leave the transit area of Moscow's Sheremetyevo Airport.
Snowden has been marooned at the airport amid an international tug of war set off by his revelations about highly secret US electronic surveillance programmes.
Russian news reports said he had been given a document allowing the former IT contractor to leave the transit area and cross the Russian border, but a lawyer who has been helping him denied that.
"He is staying here... He is here in the transit zone" at the airport, pro-Kremlin lawyer Anatoly Kucherena told reporters after meeting Snowden inside the area.

domain: politics


text:
It was approved in Curitiba City Council one new law for animal abuse. The objective of the new law was the modernization and consolidation of municipal legislation, the proposition revoked six laws that were all diluted in the new one, with this the inspection will be more agile and easier for the municipal inspectors.
“The approval of this bill is important to say that here in Curitiba we care for and respect our animals. The proposal becomes a single, integrative, utilitarian and solid law. In line with the city’s new environmental policy, the law 15,852from last year”, stressed the president of the House, Tico Kuzma (Pros).
+ Read more: Dogs and Cats also need to take care of themselves in winter; check out these tips!
The minimum amount of the fine was raised from R$200 to R$400, with the highest amount reaching R$200,000. It also expands the behaviors typified as ill-treatment: the substitute brings, for example, the express prohibition of events such as bullfights, vaquejadas and fights, “even if in a private place”, and the prohibition of collars “with blunt, cutting, piercing mechanisms”. or that generate electronic impulses or electrical discharges” in animals.
No previous seals have been dropped, but almost all have been rewritten. For example, today, in the law, it is prohibited to “physically assault or act to cause pain, suffering or harm to the animal”. In the general substitutive, the fence was changed to “harm or attack animals, whether by beating, stoning, by sharp, blunt instruments, by chemical, scalding, toxic substances, by fire or others”.
+ See also: Mexico City May (Finally) Ban Bullfights
At the beginning of the month, the president of the House of Laws, councilor Tico Kuzma received representatives of organized civil society who presented suggestions that were included in the approved text. Like the inclusion of a paragraph in article 2, to make it clear that the Municipal Guard of Curitiba also fits as a police force. In the same article, the proposition lists as mistreatment “molesting or disturbing animals kept in parks, sanctuaries, zoos or free-living”.
“One of the main changes is the expansion of what can be considered maltreatment,” continued Kuzma, who opened the discussion on the matter. One of these advances, mentioned the president of the House, is the device that deals with animal accumulators. The substitute also brings mechanisms to facilitate the identification of the offender and assigns him expenses for the recovery of the rescued animal, he pointed out.
Kuzma, like other lawmakers, stressed the importance of responsible adoption. In addition to the adoption events and the permanent campaign at the Reference Center for Animals at Risk (CRAR), the Animal Protection Network website (RPA) makes it easy to find dogs and cats waiting for a home.
“The costs paid by the violator are a novelty. The offender does have that responsibility”, supported the deputy leader of the mayor, Mauro Ignácio (União). The inspection will also benefit from the new legislation, predicts the councilor. “The House has always been attentive to this issue. [bem-estar animal]”, stressed Ignacio.
Opposition leader Professor Josete (PT) reinforced the call for adoption: “To really seek animal welfare, adopt a dog, a cat, an abandoned animal, we are collaborating with this policy”. “I think an awareness campaign is also important, before maltreatment happens, this is an aspect that needs to move forward,” she suggested.
“The legislative text reinforces the prohibition of physical or psychological punishment to encourage animals for sports competitions”, quoted Marcelo Fachinello (PSC). In addition to responsible adoption, he highlighted, in the proposal under discussion, the prohibition of collars that apply shocks (with the aim of controlling the barking of animals, for example) and the clear rule for euthanasia, when necessary and recommended by a qualified professional.
For journalist Márcio Barros (PSD), it is also necessary to invest in inspection and think about actions with the cities of Greater Curitiba, such as castration campaigns. Dalton Borba, who withdrew a substitute from his initiative, proposed in 2020, said that a “cultural change” is necessary for the citizen, to take care of animals and denounce cases of mistreatment. “It is necessary that civil society collaborate, participate, so that we have full effectiveness in this bill”, he opined.
The vote was accompanied by the director of the Fauna Research and Conservation Department of the Municipal Environment Department (SMMA), Edson Ferraz Evaristo de Paula; the president of COMUPA – Municipal Animal Protection Council, Carla Negochadle; the Vice-President of the State Council for Animal Rights (CEDA), Paulo Colnaghi; activist Laelia Tonhozi, from the SOS Bicho Movement; and the municipal guard Sandro Arioan Dias Alves, from the Animal Protection Patrol of the Municipal Guard.
Curitiba thus remains at the forefront of actions in favor of animal rights.
Complaints of mistreatment of domestic and wild animals, in addition to illegal possession, can be made to Central 156, of the Municipality of Curitiba.


- Genesis Model Type
- Equus (2011-2016)
So I was at the local shop and it turns out the passenger side light is faulty. The driver's side functioned just like you said. All off when off, halogen halo + DRL LEDs with parking lights, DRL LEDs + reg beam 3 LEDs on on, turn signals and high beam work fine too. We tried hooking up the pass side light to the driver's side cable and it was just like yesterday where only the DRL LEDs and turn signals worked. I'm not sure if there's a car lighting place that will be able to fix it.


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This is a serious philosophy book that is also an easy read. The book deals with the moral problem of whether it can be right to kill someone for the greater good: in particular, whether it is right to deliberately push a man off a bridge to stop a run-away tram, which otherwise would kill many others.
In dealing with the ethical issues that this raises, the author takes the reader through the views of Aquinus, Bentham, JS Mill, Foot, Anscombe, Singer and many other past and present philosophers. Later in the book, the author shows how neuroscience, psychology and behavioural economics are playing an increasing role in ethics.
As with all of Edmonds' books, you finish reading it feeling better informed about an important and intellectual subject. Yet he has the ability to keep the reader engaged throughout the book.

domain: library


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It’s January, and you may just now be finally getting the rest of your Christmas decorations put back into storage for next year. Holiday decorations are so magical and wonderful, and you might be feeling a little sad about your regular decor after putting everything away. But there are many ways you can decorate your home after the holidays that are still fun.
Still a winter theme
It is still winter for at least another few months, so you could actually keep some of those winter decorations out if you want. Personally, I’m all for the decorations I can leave out for half the year instead of having to go through the work of redecorating every month. My baseboard heater covers stay on all year long and match my baseboard colors, so I never have to change them. Snowmen and Pinecones are still great decorations and can make your place feel just a little festive.
Get the right pillows
One way to instantly change the feel in a room is through throw pillows. They can be in all different kinds of colors and styles, and they make your place feel more cozy. If you want to keep the feeling of joy in your home, just pick out some new pillows for your couch that seem fun and are something you can enjoy throughout the rest of the winter.
Remember, Valentine’s Day
It might feel a little too soon if you like to wait until February, but Valentine’s Day is about a month away now, and you can get out some decorations for that. Christmas is a decorations-all-over-the-house kind of holiday, and other holidays tend to just be a thing or two, but you don’t have to be that way. Go crazy with Valentine’s Day if it lifts your spirits.
Most people don’t realize how much scents affect the feel of their home. While decorating is important, the smell can make or break your whole design. Not only are candles decorative and can complete your look, they also smell wonderful and can give your room a whole new feel. You could even sneak in a pine tree smelling candle if you are feeling really nostalgic for Christmas.
One thing that can really change the look in your room is decorating your table. Get a new table runner and some kind of centerpiece that can be displayed year round. It will not only add to your current decor but will be something new and exciting you can enjoy every day.
One way to really beef up the design in your house is through artwork. You could get a large canvas, and make a piece of art yourself. It will give your room a sentimental feel and will have the exact colors you want to use, so you don’t have to worry about everything matching. Or, you could go buy some beautiful art to hang in your home. New art on the walls might be just what you need to get over the post-holiday decorating blues and start to feel happy with your decor again.
Selling a home can be a very stressful experience. If you don’t want to hire a professional to do it for you, then consider the following ideas-
The following DIY secrets can help you with your redecorating and home improvement, get great results with limited funds...
Measure the height from the floor to the top of the metal wall plate.
Follow our guide for baseboard heater cover measurement:
Any baseboard larger than 7 3/8" will be compatible with our standard cover.
Any measurement greater than 9 3/8" will fit our tall cover.
Measure from the bottom of the finned tube heating element to the top of the metal wall plate,
A measurement of 5 1/2" to 6 3/4" will fit our standard cover.
A measurement of 7 1/2" to 8 3/4" will fit our tall cover.
Measure the distance from the wall or the metal wall plate attached to the wall, to the outside of the finned tube heating element.
Any measurement of less than 3 1/8" inches from the wall will fit our
Any measurement of less than 3 1/8" inches from the wall will fit our tall cover.
EZ Snap™ Wall Widgets are used when your old or existing wall back plate has been removed or if you have to hang your new cover 1 inch or higher to bring them up to a height that will fit our installation guidelines. Just use the self-tapping screws to secure them to the top of your existing wallplate.
EZ Snap™ Floor Fidgets easily raise your new covers ¾ inch to compensate for any ¾ inch floor (wood, tile, or other) that has been installed any time since your baseboard heater was originally installed. May be used for any reason when the overall height has been shortened and the total height is less than 7-½ inches for standard height or 9-½ inches for the TALL height EZ Snap™ BaseBoard Covers. Just measure your overall desired height, subtract 1", drill a hole, preferably in a stud and attach it to the wall with the included screw.
EZ Snap™ Wall Contraptions are used when your wall back plate has been completely removed. EZ Snap™ Wall Contraptions receives your EZ Snap™ BaseBoard Cover and keeps your aluminum fin tube from sagging. These completely replace your wall back plate. To install, slide up from the bottom and make sure the top is at your desired height. Screw to the wall, then bend the front finger up to hold the fin tube in place.
Measure from wall to wall and subtract ½ inch (to allow for wiggle room when installing your new covers.) The endcaps can be pulled or slid ½ inch outward on either end to fit your existing length requirements. Choose 2 flush to wall end caps.
Measure the overall length of the existing unit with ends attached then subtract ½ inch (to allow for wiggle room when installing your new covers.) The endcaps can be pulled or slid ½ inch outward on either end to fit your existing length requirements. Choose 2 Closed or Open-end caps.
Measure from the corner to the end of the unit with ends attached then subtract ½ inch (to allow for wiggle room when installing your new covers.) The endcaps can be pulled or slid ½ inch outward on either end to fit your existing length requirements. Choose 1 Closed or Open-end & 1 flush to wall end cap.
Measure the left side from corner A to corner B (see diagram). Then subtract 3 inches for the 90 degree inside corner, then subtract another ½ inch (to allow for wiggle room when installing your new covers.) Repeat for the right side if also wall to wall. You need to subtract a total of 3½ inches from each side that is wall to wall. Choose your end caps.
Measure the left side from the corner of the wall to the end of the unit with ends caps. Then subtract 3 inches for the 90 degree inside corner, then subtract another ½ inch (to allow for wiggle room when installing your new covers.) Repeat for the right side. You need to subtract a total of 3½ inches from both left and right sides. Choose your end caps.
L-Shaped outside radiators ending in the middle of the wall:
Measure from outside corner of the wall A to the end of the radiator unit with end caps attached B, then subtract ½" (to allow for wiggle room when installing your new covers.)
Measure from outside corner of the wall to the corner of the wall, then subtract 1/2" (to allow for wiggle room when installing your new covers.)
Left leg - measure from the corner out to the end of the radiator subtract 3" for the corner and ½ (to allow for wiggle room when installing your new covers.)
Center leg - measure from corner to corner and subtract 3" for each corner a total of 6"
Right leg - measure from the corner out to the end of the radiator subtract 3" for the corner and ½" (to allow for wiggle room when installing your new covers.).
We recommend that you order covers a little longer than normal and cut them on site, as there are many opportunities for mistakes in measuring and installation. By cutting on site you can fit and cut to fit. The covers can be cut with a good quality jigsaw and a fine metal cutting blade.
Now that you’ve learned how to measure baseboard heaters,
you’re ready to order.


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Tired of battling with the pain? Put down the pills and dial up a physical therapist for relief.
If you are suffering from acute or chronic pain and have decided to go to a doctor, there are a few options that a doctor can suggest- injections, procedures, and medications.
If you are reaching physiotherapy in Surrey, you are on the safer side. It is better to call a physical therapist before going to a doctor. A physiotherapy service is a safer, more effective, and more sustainable way to get relief from pain.
In modern pain management, physiotherapy stands as a cornerstone, offering a holistic and non-invasive way to alleviate pain and restore functionality. The approach adopted by physiotherapists addresses the root cause of the problem, promoting improved quality of life and long-term relief.
In this blog article, we will discuss some strong reasons why you should consult a physiotherapist before directly going to a doctor.
Pain Conditions Treated by Physiotherapist
|Sprains and strains|
Why Physiotherapists for Pain Management?
Physiotherapists play a vital role in pain management due to their expertise in treating musculoskeletal conditions. The holistic approach adopted by them aims to promote muscle strength, posture, and flexibility.
Address the Root Cause of the Problem
Unlike medications that simply mask the problem temporarily, physiotherapists, through comprehensive assessment, address the root cause of the problem. They design personalized treatments that not only address the pain but also address the root cause. It provides relief and also promotes functional improvement, prevents recurring pain, and helps in long-term recovery.
For example :
Pain in your knees is caused by a problem in your foot. Through various massages and therapies, it will also direct work on the root cause of the problem, i.e., the foot. Physiotherapists in Surrey take a holistic approach to improving musculoskeletal health.
No Negative Side-effects
Apart from the soreness caused by exercises, physical therapy doesn’t carry any scary side effects, but medications and injections can have several side effects.
As pills and injections cause different reactions in different people, physio doesn’t carry the risk of any adverse reactions as each treatment plan is customized as per the individual, which can be adjusted through careful monitoring.
When it comes to physical therapy, physiotherapists tailor plans according to the medical history and specific needs of an individual. They make adjustments and make progress as needed, minimizing the risk of adverse outcomes.
Your physiotherapist will even watch your recovery progress and make decisions accordingly when it’s time to take the next step. Physio will also suggest when it’s the right time to do exercises at home, saving you time and money.
Promotion of Health and Wellness
Physiotherapists services focus on promoting overall health and wellness. They not only focus on pain but also ergonomics, habits, lifestyle, and posture. This approach reduces the risk of future issues and promotes long-term health.
Education on Self-management
Physiotherapists educate patients on self-management strategies. Through demonstration and deep explanation, a physiotherapist in Surrey equips individuals with the right knowledge and techniques needed to participate actively in pain management. This includes ergonomic adjustments, teaching specific exercises, posture correction, and ergonomic adjustments as per the condition of an individual.
Through self-awareness and fostering understanding, physiotherapists make informed decisions about their activities, habits, and lifestyle. This not only reduces the dependence on medications but also encourages a sustained and healthy lifestyle that aids in long-term relief.
Avoidance of Surgical Needs and Risks
Physiotherapists avoid the need for surgical intervention and its inherent risks, which is one of the compelling reasons for hiring a physiotherapist for pain management. Through a variety of non-invasive techniques, physiotherapy addresses the root cause of the problem without relying on surgical procedures. This approach prioritizes the concept of natural healing.
Provide Ergonomic Advice
Physiotherapists in Surrey offer vital ergonomic advice to the patient. Equipped with musculoskeletal advice, recognize ergonomic pain that can be triggered through daily activities. They ease muscle and joint pain by pinpointing pitfalls and refining home and workplace layouts to improve posture. In this way, physiotherapists are an indispensable source for tackling ergonomic challenges by providing a smooth journey towards health and well-being.
Provides Long-term Solution
When physiotherapists discharge a patient from their clinic, they provide a comprehensive home care plan to mitigate the symptoms. If the patient strictly follows the home care plan, they will be able to succeed in managing their long-term issues.
After thoroughly reading the article, the choice of where to go first is in your hands. If you are suffering from any kind of pain, before consuming another pill, we initially recommend you visit physiotherapists in Surrey, BC. At Khalsa Physiotherapy Clinic, our trained professionals have dedicated their lives to treating individualized pain. We have helped many patients live their lives pain-free.

domain: medical


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The popular on-demand film streaming service Kanopy is now available for free to all Cook Memorial Library card holders. Library card holders can access Kanopy and sign up to start streaming films instantly by visiting http://cityoflagrande.kanopy.com. Films can be streamed from any computer, television, mobile device or platform by downloading the Kanopy app for iOS, Android, AppleTV, Chromecast or Roku. Offering what the New York Times calls “a garden of cinematic delights,” Kanopy showcases more than 30,000 of the world’s best films, including award-winning documentaries, rare and hard-to-find titles, film festival favorites, indie and classic films, and world cinema with collections from A24, Criterion Collection, Music Box Films, Samuel Goldwyn, The Great Courses, PBS and thousands of independent filmmakers. With the motto of “thoughtful entertainment,” Kanopy provides library patrons with access to films of unique social and cultural value; films that are often difficult or impossible to access elsewhere, and programming that features diversity, with a wide array of foreign language films and films on race and current affairs. The Kanopy collection includes indie hits like “Hunt for the Wilderpeople” and “Night of the Living Dead,” classic masterpieces like “Charade,” “Sands of Iwo Jima,” and “Seven Samurai,” award-winning documentaries like the Oscar-nominated “Kedi” and “I Am Not Your Negro,” as well as the Academy Award winner for Best Picture 2018 “Moonlight.”
Patrons also have access to Kanopy Kids which is billed as thoughtful entertainment for children aged preschool and up. Parents can trust a curated selection of educational and enriching videos on Kanopy Kids with developmentally appropriate, age-based ratings from Common Sense Media, as well as additional parental controls to keep kids safe. Kanopy Kids features popular episodes of “Sesame Street” and other TV shows, hundreds of family friendly movies, and even animated children’s books like Mo Willems’ “Pigeon” and “Knuffle Bunny.” “We are thrilled to offer Kanopy’s diverse collection of films and documentaries to our library patrons,” said Kip Roberson, Cook Memorial Library director. “The subjects are far-reaching and certainly reflect the cultural interests of our patrons.” An added benefit of Kanopy is that all films in the collection come with public performance rights. “We will explore the possibility of having more movie events at the library so the community can enjoy and discuss these films together,” Roberson continued.
Cook Memorial Library patrons whose accounts are in good standing have the ability to stream up to 10 movies per month. Patrons accessing Kanopy Kids have unlimited viewing of that content. Accessing Kanopy was founded in 2008 by CEO Olivia Humphrey as an educational tool for colleges and universities. More than 5 million Kanopy users stream the most acclaimed movies and documentaries from award-winning filmmakers, and experience the best in independent, classic film, and world cinema.
For more information about this and other library programs, please contact the library at 541-962-1339.