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We are happy to introduce our latest XLProjects (XLP) member office in Santiago, Chile
Integral Chile Spa
Vitacura 2939 Oficina 2203, Las Condes 7550011,
Santiago, Chile
Tel : +56-2-2430-2400
Fax : +56-2-2430-2411
Website : https://xlprojects.net/integral-chile-s-a/
Contact : Dalia Cordova – CEO
Integral Chile S.A. is a Chilean-owned company founded in 1992, which aims to provide a comprehensive and innovative approach to the services of logistics and transportation engineering, led by a group of highly qualified professionals with extensive experience in the market.
Integral Chile S.A. offer full heavy lift and project cargo services, including oversized cargo, extra-weight cargo, and merchandise that requires specific logistics needs, including domestic transport.
Integral Chile S.A. can offer project cargo management for engineering, procurement and construction companies servicing energy, mining, chemical, petrochemical, pipeline, infrastructure and other sectors. These highly specialist shipments require individual transport planning from origin to destination.
In their activity and constant development, the company has established a significant presence in major international transport traffic both import and export from and to Chile, covering continents such as the Americas, Europe and Asia including both domestic transport and courier and general cargo. They currently have a select portfolio of clients from small businesses to large corporate groups, thanks to the constant challenge to answer their demanding needs, which has prompted Integral Chile SA to seek leadership in their market.
The current world trend for green energy and the substantial investment in clean energy technology is providing a rapid increase in business opportunities for project cargo specialists who have the required capabilities in the supply chain management of oversize plant and equipment, i.e. wind turbines.
Their Strengths Also Include:
- SICEP (Rating system of suppliers companies goods and services for mining)
We welcome Integral to the XLP team!

subset: commoncrawl


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Voltage-gated sodium channels (VGSCs) are concentrated in the depths of the postsynaptic folds at mammalian neuromuscular junctions (NMJs) where they facilitate action potential generation during neuromuscular transmission. At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, β-spectrin and ankyrin, which may help to maintain the high local density of VGSCs. Here we show in skeletal muscle, using immunofluorescence, that β-spectrin is precisely colocalized with both VGSCs and ankyrinG, the nodal isoform of ankyrin. In en face views of rat NMJs, acetylcholine receptors (AChRs), and utrophin immunolabeling are organized in distinctive linear arrays corresponding to the crests of the postsynaptic folds. In contrast, β-spectrin, VGSCs, and ankyrinG have a punctate distribution that extends laterally beyond the AChRs, consistent with a localization in the depths of the folds. Double antibody labeling shows that β-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ. Furthermore, quantification of immunofluorescence in labeled transverse sections reveals that β-spectrin is also concentrated in perijunctional regions, in parallel with an increase in labeling of VGSCs and ankyrinG, but not of dystrophin. These observations suggest that interactions with β-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.
Segregation of ion channels into distinct domains of the plasma membrane is important for the function of many cells. A major issue for neurobiology is how integral membrane proteins, such as acetylcholine receptors (AChRs)1 and voltage-gated sodium channels (VGSCs) become restricted to particular membrane domains rather than remaining freely mobile in the membrane. Association with the spectrin-based cytoskeleton represents one possible mechanism (for review see Bennett, 1990; Bennett and Gilligan, 1993). Spectrin was first characterized in red blood cells (RBCs) where anion exchanger channels in the membrane are linked through ankyrin and spectrin to the underlying actin cytoskeleton. A similar mechanism may contribute to ion channel localization in the nervous system.
One particularly important class of ion channels in excitable cells is the VGSCs, which are essential for the initiation of action potentials in most nerve and muscle cells. In the nervous system, VGSCs have been shown to be clustered at axon hillocks and initial segments (Wollner and Catterall, 1986; Angelides et al., 1988) where they are responsible for initiating action potentials, and at nodes of Ranvier in both peripheral and central neurons (Waxman and Ritchie, 1985; Kaplan et al., 1997) where they mediate saltatory conduction in myelinated axons. In skeletal muscle, electrophysiological studies using loose-patch voltage clamp recording have shown that VGSCs are nonuniformly distributed with high sodium current densities present at the neuromuscular junction (NMJ) and in the perijunctional region (Betz et al., 1984; Beam et al., 1985; Caldwell et al., 1986). These findings were subsequently confirmed by immunofluorescence studies (Angelides, 1986; Haimovich et al., 1987) and EM immunolabeling, which further revealed that VGSCs are concentrated in the depths of the postsynaptic folds and in the perisynaptic membrane, although excluded from the AChR-rich domain at the tops of the folds (Flucher and Daniels, 1989; Le Treut et al., 1990; Boudier et al., 1992). The presence of a high density of VGSCs at the NMJ is believed to lower the threshold for action potential generation (Wood and Slater, 1995) and to increase the safety factor for neuromuscular transmission (Wood and Slater, 1997).
There is considerable interest in the molecular mechanisms that account for VGSC clustering at sites of action potential generation. It has been suggested that interactions with ankyrin and spectrin may play a role in maintaining a high density of VGSCs (Srinivasan et al., 1988). Although originally described in RBCs, isoforms of both spectrin and ankyrin have been identified throughout the nervous system. Spectrin is a flexible rod-shaped protein made up of homologous α and β subunits (Bennett, 1990; Bennett and Gilligan, 1993). The β subunit contains both the actin-binding site at the NH2 terminus and the ankyrin binding site in the midregion. An isoform of β-spectrin has been shown to be present at the NMJ and in extrajunctional muscle membrane (Bloch and Morrow, 1989; Bewick et al., 1992). Bewick et al. (1992) observed that the distribution of β-spectrin was quite distinct from that of AChRs, and suggested that β-spectrin was in fact localized to the troughs of the postsynaptic folds. Spectrin is a member of a family of structurally related cytoskeleton proteins including utrophin and the muscle-specific dystrophin. Whereas the function of these two proteins is as yet unclear, their distribution in muscle fibers is well described. Utrophin is associated with AChRs at the NMJ (Ohlendiek et al., 1991; Bewick et al., 1992) and is implicated in stabilizing AChR clusters at the tops of the postsynaptic folds (Apel and Merlie, 1995). On the other hand, dystrophin, like β-spectrin, is found throughout the muscle fiber membrane and is concentrated at the NMJ where it is thought to be localized in the depths of the folds (Sealock et al., 1991; Yeadon et al., 1991; Bewick et al., 1992). This localization raises the possibility that both β-spectrin and dystrophin may have a role in localizing VGSCs to the depths of the postsynaptic folds.
Ankyrins are a family of peripheral membrane proteins that have been shown to interact with both ion channels and β-spectrin. It is now clear that there are at least three separate genes encoding the ankyrin family of proteins (for review see Lambert and Bennett, 1993). The first members of the ankyrin family described were ankyrinB, which is the major ankyrin in the nervous system, and ankyrinR, which is present in RBCs and has a restricted distribution in the nervous system. More recently, a third ankyrin, ankyrinG, has been described; it is the largest ankyrin found to date (Kordeli et al., 1995). An isoform of ankyrinG is present at the nodes of Ranvier and at the initial segment of axons in central neurons (Kordeli et al., 1990; Kordeli and Bennett, 1991; Kordeli et al., 1995; Kaplan et al., 1997), suggesting that ankyrinG mediates the interaction between VGSCs and spectrin in these regions. Using an antibody raised against RBCs, ankyrin immunoreactivity has been shown to be localized at the NMJ in the depths of the postsynaptic folds (Flucher and Daniels, 1989) suggesting that a similar interaction may account for the high density of VGSCs at the NMJ.
If ankyrinG and β-spectrin mediate VGSC clustering, then it would be expected that these three proteins would be codistributed. Whereas there is much evidence suggesting that the β-spectrin–ankyrin interaction is important for aggregating VGSCs in neurons, the in situ colocalization of these three proteins has not been explicitly demonstrated. Here we have used immunofluorescence labeling to ask whether, in skeletal muscle, β-spectrin is colocalized with both VGSCs and the isoform of ankyrin found at the nodes of Ranvier, ankyrinG. For comparison with β-spectrin, we have also examined the distribution of the related cytoskeletal proteins utrophin and dystrophin to identify whether they are colocalized with VGSCs and ankyrinG. This study is a prerequisite to developmental studies that may give insight into how distinct VGSC–cytoskeleton domains are assembled and maintained.
Materials And Methods
Soleus muscles isolated from adult female rats (∼200 g) were used throughout the experiments. For immunolabeling, the muscles were prepared in one of two ways. Whole muscles were lightly fixed in paraformaldehyde (0.5% wt/vol) for 30 min, and then washed in PBS. The muscles were then teased into small bundles of 5–15 fibers and permeabilized with Triton X-100 (1% vol/vol) in PBS for 30 min. These teased fibers, which allow NMJs to be viewed en face, were then immunolabeled as outlined below. Alternatively, muscles were prepared for cryosectioning by freezing blocks of muscle in isopentane cooled in liquid nitrogen. Transverse cryostat sections (6 μm) of the soleus muscles were cut and thaw mounted onto gelatine-coated slides. After air drying for 60 min, these slides were wrapped and stored at −40°C until required. Transverse cryosections of control and mdx mouse epitrochleoanconeous (ETA) muscles were similarly prepared.
The following primary antibodies were diluted in PBS containing 3% BSA and 0.1 M lysine as indicated. Two previously described monoclonal antibodies that recognize β-spectrin, raised against human RBC ghosts, were used. NCLSPEC2 was used in sections (1:30; Novacastra, Burlingame, CA) and RBC2/5C4 (Bewick et al., 1992) was used in teased fibers (1:2; a gift from Dr. L. Anderson, University of Newcastle upon Tyne). An affinity-purified rabbit polyclonal raised against peptides unique to the rat ankyrinG spectrin-binding domain AnkG@SpBd was used in sections at 0.01 μg/μl and in teased fibers at 0.02 μg/μl (a gift from Dr. S. Lambert, Worcester Foundation for Biomedical Research, Shrewsbury, MA) (Kordeli et al., 1995). For VGSC labeling, a rabbit polyclonal antibody AP1380 raised against a synthetic peptide EOIII, which recognizes the highly conserved segment in the intracellular III–IV loop that is present in all known vertebrate sodium channels (a gift from Dr. R. Levinson, University of Colorado School of Medicine, Denver, CO) (Dugandzija-Novakovic et al., 1995) was used at a dilution of 1:30 in sections and 1:10 for teased fibers. Control experiments where the VGSC antibody (2 μl) was preabsorbed with the peptide antigen EOIII (1 μg in 1 μl) overnight at 4°C and then applied to the tissues revealed no significant labeling above the no primary control (Fig. 1). A monoclonal antibody DY8/6C5 to a COOH-terminal epitope of dystrophin was used at 1:50 in sections and at 1:10 in teased fibers (a gift from Dr. L. Anderson) (Bewick et al., 1992). A monoclonal antibody DRP3/20C5, raised against a fusion protein containing the first 261 NH2-terminal amino acids of the utrophin sequence, was used at 1:10 in both sections and in teased fibers (a gift from Dr. L. Anderson) (also available as antibody NCLDRP2; Novacastra).
TRITC-conjugated swine anti–rabbit or rabbit anti–mouse IGs (Dako Ltd., High Wycombe, UK) were used as secondary antibodies in most experiments to recognize polyclonal and monoclonal primary antibodies, respectively. For double antibody–labeling experiments monoclonal primary antibodies were detected using a FITC-conjugated goat anti–mouse secondary (Dako Ltd.). All secondary antibodies were diluted at 1:100.
All incubations and washings were performed at room temperature except where stated and PBS was used for all washes.
Permeabilized, teased muscle fibers were incubated in primary antibodies overnight (at least 15 h) at 4°C. After allowing the fibers to warm to room temperature for 45 min, the bundles were washed for 45 min, and then incubated for 2 h in secondary antibodies. The secondary antibodies were preincubated with normal rat serum at a ratio of 2:1 for 1 h at room temperature before dilution. To enable NMJs to be identified, FITC α-bungarotoxin (BgTx) (6 × 10−7 M; Molecular Probes, Inc., Eugene, OR), which labels AChRs, was added with the secondary antibody solution. Subsequently, the fibers were washed (30 min), fixed in paraformaldehyde (1% wt/vol for 15 min), and then mounted on microscope slides in antifading fluorescence mounting medium (Vectashield; Vector Laboratories, Inc., Burlingame, CA). As a control, an identical labeling procedure was carried out with the omission of the primary antibody where diluent was applied instead.
Slide-mounted transverse sections were removed from the freezer and warmed to room temperature. Before labeling with primary antibodies, the unfixed sections were washed first with 0.1% Triton X-100 (vol/vol) for 5 min, and then washed for 15 min. The labeling procedure for sections and teased fibers was then essentially the same.
To examine the colocalization of proteins, double antibody labeling was performed with a monoclonal antibody to β-spectrin, dystrophin, or utrophin and a polyclonal antibody to either VGSCs or ankyrinG in both serial sections and in teased fiber preparations. Tissues were incubated in primary monoclonal antibodies for 6 h at 4°C, allowed to warm to room temperature, and washed as above. This was followed by incubation with FITC-conjugated goat anti–mouse for 2 h, a wash for 1 h, and then the primary polyclonal antibody was applied for 10 h at 4°C. Again the tissues were allowed to warm to room temperature, and were then washed before incubation with TRITC-conjugated swine anti–rabbit for 2 h. Finally the tissues were washed, fixed, and mounted as described above.
Double antibody–labeled transverse sections were photographed on an Optiphot-2 microscope using a ×40 oil Plan apo objective (Nikon UK Ltd., Kingston, UK). The rhodamine and fluorescein fluorescence were viewed with Nikon G-2A and B-2A filter sets, respectively. A525/20 nm bandpass emission filter (Leica UK Plc., Milkon Keynes, UK) was used to enhance the selectivity of the fluorescein images by preventing rhodamine "bleed through."
In addition, digitized images of labeled specimens were recorded using a cooled CCD camera imaging system (Astrocam, Cambridge, UK) and either a ×50 (Leitz) or a ×100 (Nikon) plan fluor oil immersion objectives on a M2B microscope (Micro Instruments Ltd., Oxford, UK) using Nikon filter blocks, G-1B (TRITC) and B-2H (FITC), with the additional narrow bandpass filter to improve fluorescein selectivity. All the digitized images were stored on optical disks for subsequent analysis using Imager 2+ software (Astrocam, Cambridge, UK) as outlined below.
To determine the localization of immunolabeling within the postsynaptic folds, double-labeled en face NMJs were also viewed with a confocal laser microscope (laser λ 488 nm for FITC; laser λ 568 nm for rhodamine) (MRC600; Bio-Rad Laboratories, Hercules, CA) using a ×60 objective (NA 1.4). NMJs were initially viewed en face. A line of interest was selected, through which a series of scans at different focal depths (0.3-μm steps) was made. From such series reconstructed transverse sections were produced. Subsequently, the entire NMJ was scanned in 10–15 steps of 0.5 μm to construct a complete en face view. The position of the transverse section was clearly identified in en face views where the fluorescence had been bleached.
Images of both fluorescein and rhodamine labeling were recorded from transverse sections of muscle fibers that were dual labeled with a primary antibody to the protein of interest and with BgTx. The presence of FITC-α-BgTx binding to AChRs was used to identify the position of the NMJ (Fig. 2), and the corresponding rhodamine image demonstrates the presence of VGSC immunolabeling around the entire muscle fiber membrane. The intensity of fluorescence was measured in each of three regions of the muscle fiber surface (Fig. 2): the NMJ (J); the perijunctional region (PJ) immediately adjacent (0–5 μm) to the NMJ; and an extrajunctional region (XJ) diametrically opposed to the NMJ. For all the images obtained the camera was operating within a range where pixel intensity and exposure time were linearly related. All intensity values were expressed as gray levels per second of exposure time. The total fluorescence intensity within each region was calculated and the value obtained was divided by the area of the region to give the mean intensity of labeling per unit area. A background value determined by applying the same procedure to sections in which the primary antibody had been omitted was subtracted to give the net labeling intensity. For each muscle fiber, the mean fluorescence intensity in J and PJ regions was normalized to that in the XJ region (assigned a value of 1). For each antibody, 10 muscle fibers in each of two soleus muscles were analyzed, and the results expressed as mean normalized fluorescence intensity ± SEM.
An important preliminary step in this quantification procedure was to establish an appropriate primary antibody dilution. If the fluorescence intensity is to be proportional to the number of protein molecules, then the primary antibodies should be used at or near saturating concentration. Using serial dilution experiments, an approximately saturating antibody concentration was determined for the primary antibodies to VGSC, ankyrinG, β-spectrin, and dystrophin. Fig. 3 shows an example of a dilution series for the NCLSPEC2 antibody to β-spectrin. Muscle sections were exposed to serial dilutions of antibody and images containing NMJs were analyzed for J and XJ labeling as outlined above (without normalizing the labeling intensity to extrajunctional levels). The mean fluorescence labeling intensity per μm2 in both J and XJ regions was plotted against antibody concentration (Fig. 3). Each point is the mean ± SEM of observations from six muscle fibers. The concentration at which the binding sites appeared saturated was used to label sections throughout the experiments. At this dilution, the greatest difference between labeling intensity in J and XJ regions of the muscle fiber can be seen.
Distribution of VGSCs and Cytoskeletal Proteins at the NMJ
β-Spectrin, a cytoskeletal protein implicated in the maintenance of VGSC clusters throughout the nervous system, is concentrated at the NMJ (Bloch and Morrow, 1989; Bewick et al., 1992). To see whether it is well placed within the NMJ to interact with VGSCs, the distribution of both VGSCs and β-spectrin was examined using confocal microscopy (Fig. 4). Dual-labeled teased muscle fibers viewed en face show that both VGSCs and β-spectrin are approximately codistributed with AChRs at the NMJ (Fig. 4). The superimposed images show clearly that labeling for both VGSCs and β-spectrin extends laterally ∼0.5 μm beyond that for AChRs. Bewick et al. (1993) reported that in en face views of NMJs a fringe of dystrophin or β-dystroglycan labeling could be seen to extend beyond AChR labeling. These authors interpreted this observed fringe of labeling as representing immunolabeling in the bottom of the postsynaptic folds.
To determine the localization of β-spectrin and VGSC immunolabeling within the postsynaptic folds, reconstructed transverse sections through the NMJ were produced. These images (Fig. 4) show that there are two discrete ion channel domains at the NMJ with AChR labeling and VGSC immunolabeling having distinctly different spatial distributions. These observations are consistent with a previous study (Flucher and Daniels, 1989) that showed, using EM immunogold labeling, that VGSCs are concentrated in the depths of the postsynaptic folds and largely excluded from the AChR-rich domain at the tops of the folds. Moreover, the pattern of β-spectrin immunolabeling is similar to that of VGSC labeling in both en face views and in reconstructed transverse sections (Fig. 4). This indicates that β-spectrin, like VGSCs, is present in the depths of the postsynaptic folds. An isoform of β-spectrin has previously been reported to be associated with AChRs in myotube cultures and at adult rat NMJs (Bloch and Morrow, 1989). Our results show that an isoform of β-spectrin is present at NMJs, which is clearly associated with VGSC-rich domains and not with AChRs, indicating that there may be multiple β-spectrin isoforms in skeletal muscle.
If ankyrinG is an intermediary in the binding of VGSCs and β-spectrin at the NMJ then its distribution would be expected to be similar to that of VGSCs. In low magnification en face views of NMJs, immunolabeling for both VGSCs and ankyrinG is concentrated at the adult NMJ and is approximately codistributed with the AChRs (Fig. 5). On closer examination however, it can be seen that labeling for ankyrinG extends laterally beyond that for AChRs, as does immunolabeling for VGSCs and β-spectrin (Fig. 4). At higher magnification, differences in the detail of the labeling patterns can be seen (Fig. 5, arrows). Whereas AChRs seem to be organized in a characteristic linear pattern corresponding to the crests of the postsynaptic folds, VGSCs and ankyrinG have a more punctate distribution with some features of the labeling pattern being quite different from that of the AChR labeling. Fig. 5 thus confirms and extends the observations of Flucher and Daniels (1989), which found that VGSCs and ankyrin are localized in the depths of the postsynaptic folds. However, we have used different antibodies, in particular the antibody used here to demonstrate ankyrin labeling is specific for the isoform of ankyrin found at the nodes of Ranvier—ankyrinG (Kordeli et al., 1995).
To test directly the hypothesis that β-spectrin is colocalized with VGSCs, teased fibers were double labeled with antibodies to VGSCs and with antibodies to the structurally related cytoskeletal proteins β-spectrin, dystrophin, and utrophin (Fig. 6). The distribution of immunolabeling of both β-spectrin and dystrophin was found to be strikingly similar to that of VGSCs (Fig 6, arrows). However, occasionally, very slight differences in the fine pattern of dystrophin and VGSC labeling could be detected. In contrast, utrophin immunolabeling was quite different to that of VGSCs, often resembling a more linear pattern similar to that associated with AChR labeling (Fig. 5). These observations suggest that the two distinct ion channel domains occupied by VGSCs and AChRs at the NMJ are matched by distinct cytoskeletal protein domains, with β-spectrin and dystrophin (but not utrophin) being colocalized with VGSCs.
Distribution of VGSCs and Cytoskeletal Proteins in the PJ Region
Electrophysiological and EM labeling studies have suggested that the local high density of VGSCs is not confined to the NMJ but extends into the perijunctional region (Caldwell et al., 1986; Flucher and Daniels, 1989; Le Treut et al., 1990). Therefore, the distribution of VGSCs and cytoskeletal proteins was examined in transverse sections of muscle that allow the entire circumference of the muscle fiber membrane to be examined. Dual labeling confirms that VGSC immunolabeling is concentrated at the NMJ (Fig. 7). As expected, VGSC immunolabeling was not uniformly distributed in the rest of the muscle fiber but appeared to be concentrated in regions close to the NMJ. Interestingly, subsequent serial sections through the same NMJ shows that this perijunctional increase in VGSC labeling is matched by increased immunolabeling for β-spectrin but not for dystrophin (Fig. 7, arrows). Utrophin immunolabeling acts as a control for the double antibody– labeling technique. Since immunolabeling for utrophin is restricted to the NMJ and to intramuscular blood vessels it can clearly be seen that there is no inappropriate cross- reaction between the antibodies used in this procedure (Fig. 7). These observations suggest that in the perijunctional region, as at the NMJ, the concentration of β-spectrin closely parallels that of VGSCs but the concentration of dystrophin does not.
If VGSC clustering is mediated via an interaction between ankyrinG and β-spectrin, then ankyrinG would also be expected to be concentrated at the NMJ and in the perijunctional region. The distribution of ankyrinG immunolabeling in the muscle fiber membrane was therefore examined and compared with that of the cytoskeletal proteins β-spectrin, utrophin, and dystrophin. Transverse sections were dual labeled with ankyrinG antibody and BgTx to allow the NMJ to be identified (Fig. 8). AnkyrinG appeared concentrated at the NMJ and in adjacent regions, compared with extrajunctional regions. Subsequent serial sections were double antibody labeled and revealed that like VGSCs, the perijunctional concentration of ankyrinG immunolabeling is mirrored by β-spectrin (Fig. 8), but not by dystrophin or utrophin (data not shown). Thus ankyrinG and β-spectrin are colocalized at the NMJ and in the perijunctional region where the concentration of ankyrinG appears to parallel that of β-spectrin.
In addition to sarcolemmal immunolabeling, Fig. 8 also reveals the presence of myoplasmic labeling by the ankyrinG antibody. The specificity of this myoplasmic labeling is confirmed by comparing Figs. 7 and 8, in which double primary antibody labeling is illustrated. In both figures the secondary antibodies used are the same. However, additional labeling of blood vessels is specific to the utrophin antibody (Fig. 7), and myoplasmic labeling is specific to the ankyrinG antibody (Fig. 8). Devarajan et al. (1996) identified a truncated isoform of ankyrinG, ankG119, in kidney cells that binds spectrin but does not associate with the plasma membrane. Northern blot analysis of the mRNA for this truncated ankyrinG isoform indicated that this isoform was also present in skeletal muscle (Devarajan et al., 1996). The immunolabeling seen in this study could correspond to this truncated ankyrinG isoform.
To assess the intensity of immunolabeling for VGSCs, ankyrinG, β-spectrin, and dystrophin regions of J, PJ, and XJ labeling were identified in transverse muscle sections (as in Fig. 2). The mean fluorescence labeling intensity per μm2 in each region was measured and normalized to that in the XJ region (Fig. 9). All the proteins studied are clearly concentrated at the NMJ. The cytoskeletal proteins ankyrinG, β-spectrin, and dystrophin are all increased three- to fourfold, whereas VGSCs are increased sixfold (Fig. 9). The increase in the cytoskeletal proteins is approximately in line with the increase in membrane at the NMJ as a result of postsynaptic folding (Wood and Slater, 1997). The greater increase in VGSCs suggests that there may actually be a higher density of channels per unit area of membrane within the folds at the NMJ than in XJ regions, as suggested previously (Caldwell et al., 1986; Flucher and Daniels, 1989). A highly significant (P < 0.001; t test) perijunctional increase of 1.5–2-fold was seen for VGSCs, ankyrinG, and β-spectrin, but not for dystrophin (Fig. 9). This analysis indicates that the concentration of β-spectrin closely parallels that of VGSCs and ankyrinG in the PJ region, but that the concentration of dystrophin does not.
Distribution of VGSC in the Absence of Dystrophin
Dystrophin and VGSCs are colocalized at the NMJ and throughout the muscle fiber membrane. Although there is no increase in dystrophin immunolabeling in the PJ region, it is still present there. To test for a possible influence of dystrophin in maintaining the high concentration of VGSCs at the NMJ, the distribution of VGSCs was examined in muscles from the mdx mouse, in which a point mutation on the X chromosome results in a profound deficiency of dystrophin (Sicinski et al., 1989). Transverse sections of ETA muscles from control and mdx mice were dual labeled with BgTx, to identify the NMJs and the VGSC antibody. Both in the presence and absence of dystrophin, VGSC immunolabeling was clearly concentrated at the NMJ and in PJ regions of mouse muscle (Fig. 10). This observation indicates that dystrophin does not play an essential role in clustering VGSCs at the NMJ in adult muscle.
The main finding of this study is that at adult rat NMJs, β-spectrin is colocalized with both VGSCs and ankyrinG. Flucher and Daniels (1989) used EM immunogold labeling to show that VGSCs are concentrated in the depths of the postsynaptic folds, whereas AChRs are restricted to the tops of the folds. We have demonstrated that these two ion channel domains can be distinguished in en face views of labeled NMJs. Confocal microscopy of immunolabeled teased fibers has shown that β-spectrin is clearly associated with VGSC-rich domains in the depths of the postsynaptic folds and not colocalized with AChRs. Furthermore, within the NMJ, the punctate pattern of labeling for VGSCs is mirrored by β-spectrin and by ankyrinG. This contrasts to the linear arrays of labeling for AChRs and utrophin, which are thought to correspond to the tops of the postsynaptic folds. The punctate labeling pattern may arise from the branching of the folds and their poor alignment with the fold openings or from the presence of VGSC microclusters within the fold membrane. In addition, we have shown that the concentration of VGSCs in perijunctional regions is paralleled by increases in the concentration of both β-spectrin and ankyrinG. The precise colocalization of β-spectrin and ankyrinG with VGSCs at the NMJ and in perijunctional regions suggests that these three proteins may interact with one another to provide a means of maintaining a high density of VGSCs at the NMJ.
The muscle cytoskeletal protein dystrophin, a member of the spectrin family, has previously been shown to have a similar distribution to that of β-spectrin at the NMJ (Bewick et al., 1992). Likewise, in this study dystrophin has a somewhat punctate distribution at the NMJ that extends beyond the AChR-labeled regions, suggestive of a localization in the depths of the postsynaptic folds. However, examination of the labeling pattern of VGSCs and dystrophin in greater detail revealed subtle differences. Furthermore, although dystrophin is concentrated at the NMJ, it is not increased in the perijunctional region. Finally, in muscles of the mdx mouse (which lacks dystrophin), VGSCs are normally distributed and concentrated at the NMJ. Together, these observations suggest that dystrophin does not play a significant role in clustering VGSCs at the NMJ.
Syntrophin, a protein closely associated with dystrophin and utrophin, contains PSD-95, DIgA, ZO-1–like (PDZ) domains that in other situations have been implicated in clustering of ion channels (Kim et al., 1995). Recently it has been reported that VGSCs copurify with syntrophin and dystrophin in skeletal and cardiac muscle (Gee et al., 1998). In the mdx mouse, which lacks dystrophin, syntrophin and other dystrophin-associated proteins are greatly reduced in abundance in the XJ region of the muscle fiber but they persist at the NMJ, presumably in association with utrophin (Matsumura et al., 1992). Although we have shown that the distribution of VGSCs in mdx mouse muscle is essentially normal, it remains possible that interactions with syntrophin represent an additional means of localizing VGSCs at the NMJ.
VGSC Concentration at the NMJ
The concentration of VGSCs in J and PJ regions is likely to be functionally important. The action potential threshold at the NMJ is lower than in extrajunctional regions (Wood and Slater, 1995) and the high safety factor for neuromuscular transmission may be attributed in part to the postsynaptic folds and the VGSCs concentrated within them (Wood and Slater, 1997). Various approaches have been used to estimate the number of VGSCs at the NMJ. Electrophysiological analyses using loose-patch voltage-clamp recording to measure sodium current density have suggested that the density of VGSCs at the NMJ was 8,000/μm2 in rat muscle (Caldwell et al., 1986), representing a 20-fold increase over XJ sodium current densities. This estimate of channel number was based on values of sodium current density and related to VGSC-specific toxin binding studies of the nodes of Ranvier. It was complicated by huge variations in sodium current density within the NMJ and by uncertainties concerning the amount of folded membrane in the patch examined. An alternative approach of EM autoradiography, using radiolabeled scorpion toxins, has subsequently suggested that there is a sevenfold increase in VGSC concentration at the NMJ compared with XJ regions (Le Treut et al., 1990), and that this may correspond to as many as 5,000 channels per μm2 in the fold membrane (Boudier et al., 1992).
In this study, we have used the intensity of fluorescent labeling as a measure of VGSC density. Whereas this approach does not lead to a measure of the absolute number of VGSCs per unit area, it is useful in providing an indication of the density of VGSC labeling within the NMJ relative to that in other parts of the same muscle fiber. Using this approach, we have found that the intensity of VGSC labeling is sixfold greater at the NMJ than in XJ regions. Since VGSCs are localized in the postsynaptic folds, the folding itself is likely to account for much of this increase. Folding results in a fivefold increase in the postsynaptic membrane area at the rat soleus NMJ (Wood and Slater, 1997). However, not all of that folded membrane is occupied by VGSCs. The boundary between the regions of high AChR and VGSC density occurs about one-third of the way into the folds (Flucher and Daniels, 1989). This suggests that the actual increase in the amount of membrane that accommodates VGSCs is about threefold, consistent with the degree of increased labeling of β-spectrin, ankyrinG, and dystrophin, which all appear to occupy the same region of the folds as VGSCs. Thus the sixfold junctional increase in VGSC labeling indicates that the junctional VGSC density per unit area is at least twice that in extrajunctional membrane and that the increase in VGSCs is clearly more than that for cytoskeletal proteins. Whereas our value for the increase in local VGSC density is lower than previous estimates, there are substantial uncertainties associated with all the methods used.
In the perijunctional region there are no folds, yet several studies have suggested that VGSCs are also concentrated in the perisynaptic region. Electrophysiological studies suggest that the perijunctional density is 5–10-fold greater than that in the extrajunctional region (Beam et al., 1985; Caldwell et al., 1986), whereas EM studies suggest that the junctional increase in VGSCs extends into the perijunctional region (Le Treut et al., 1990; Boudier et al., 1992). The observations reported here suggest that the density of VGSCs in the perijunctional region is 2.5-fold greater than in the extrajunctional region, and is thus similar to that within the postsynaptic folds at the NMJ. This increased VGSC density in the perisynaptic region may function to ensure the initial spread of depolarization away from the endplate.
VGSC Clustering and Cytoskeletal Proteins
The factors leading to the increased density of VGSCs at the NMJ are poorly understood. One possibility is that VGSCs are preferentially synthesized and inserted into the membrane in the junctional region. It is well known that the genes for AChRs and AChE are preferentially synthesized by postsynaptic myonuclei (Brenner et al., 1990; Jasmin et al., 1993; Duclert and Changeux, 1995), and recent reports indicate that this is also true for a number of other postsynaptic molecules including neural cell adhesion molecule, rapsyn, s-laminin (Moscoso et al., 1995), and utrophin (Gramolini et al., 1997; Vater et al., 1988). If there is preferential expression of VGSC genes at the NMJ, then the high concentration of VGSCs at the NMJ and in perijunctional regions might well result from a gradient of mRNA concentration with its peak at the NMJ. However, NMJ-specific gene expression is unlikely to be the entire explanation for the observations reported. It seems probable that local mechanisms must also exist to regulate the placement of ion channels at the NMJ since AChRs, which exhibit synapse-specific gene expression, are not found in perijunctional regions.
One influence on VGSC distribution and concentration at the NMJ is likely to be interaction with the underlying cytoskeleton. Here we have shown very close spatial associations between VGSCs and both β-spectrin and ankyrinG. Studies in nerve cells (Srinivasan et al., 1988; Kordeli et al., 1990; Joe and Angelides, 1992; Kaplan et al., 1997) have led to the idea that ankyrinG links VGSCs to the underlying spectrin-based cytoskeleton and that this helps to maintain the high local density of ion channels. Whereas such a general model seems plausible for the NMJ, many of the details remain uncertain. For example, the observations of this study suggest that the local density of VGSCs is increased more within the folds than that of β-spectrin and ankyrinG. Any interpretation of this depends on knowledge of the likely stoichiometric relationships between these three proteins. At present it is not clear that every VGSC must bind to one ankyrinG molecule or that one ankyrinG would have to bind to one β-spectrin molecule for VGSCs to become localized to the membrane. Since ankyrin is known to be capable of binding more than one ion channel at a time (Michaely and Bennett, 1995), it is possible that individual ankyrinG molecules would bind multiple VGSCs. Furthermore, it is not clear what fraction of β-spectrin molecules have ankyrinG bound to them or that this is the same in J, PJ, or XJ regions of the muscle fiber surface. Thus, although the observations of this study show highly suggestive parallels in the density of VGSCs, ankyrinG, and β-spectrin, the full significance of these remains uncertain.
The precise colocalization of β-spectrin with both VGSCs and ankyrinG at the NMJ suggests that interactions between these proteins play a role in the localization of VGSCs in muscle. Such a model has been proposed to account for the local high density of VGSCs at axon hillocks and initial segments, and at the nodes of Ranvier (Waxman and Ritchie, 1985; Wollner and Catterall, 1986; Srinivasan et al., 1988; Kordeli et al., 1990; Dugandzija-Novakovic et al., 1995; Kaplan et al., 1997). Our findings in skeletal muscle suggest that VGSC–ankyrinG–β-spectrin interactions may be common to all excitable cells where VGSCs are found at high density.
We are grateful to Drs. R. Levinson, S. Lambert, and L. Anderson for generous gifts of antibodies. We thank C. Young for her assistance with producing the figures and T. Booth for his assistance with confocal microscopy.
Abbreviations used in this paper
This project was supported by grants from the Muscular Dystrophy Group of Great Britain and the Myasthenia Gravis Association.
Address all correspondence to S.J. Wood's present address, Department of Physiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK. Tel.: 44-117-928-7821. Fax: 44-117-928-8923. E-mail: firstname.lastname@example.com


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Important update
It is strongly recommended to install this update when using
- The
.cycleloopdid not maintain its defined cycle time under certain circumstances (different runtimes of the cycle function)! Depending on the case, the cycle function was executed too often or too seldom than expected. With this version, the cycle time is adhered to exactly and a warning is issued if the cycle function takes to long.
- Fixes bug in
.replace_ioto replace more than 8 bits of a >1 byte long IO object (thanks Nico)
- The
prefireparameter can now also be passed to the
.reg_timereventfunction. This is analogous to


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Payline vs PayPal
Payline is a good credit card processor for most businesses. How does it compare to PayPal? Read our comparison chart below.
Payline uses an Interchange-Plus pricing model. Swiped transactions are Interchange + 0.2% + $0.10 per transaction, plus a $10 monthly fee. There is no contract and your first month is free. Payline is also known to work with high-risk merchants.
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Skip to main content
Dan Bassill, President and CEO of Tutor/Mentor Institute, LLC, will be a guest on the Nonprofit "U" radio blog talk show on Monday, August 3, 2015, from 2:00 p.m. to 2:30 p.m. Nonprofit "U" is an online forum where nonprofit stakeholders can discuss the latest developments in the sector and increase their capacity to serve their clients and build sustainable communities. Valerie F. Leonard, an expert in community and organizational development, is the host. The show will stream live from BlogTalkRadio.com/nonprofit_u. The podcast will be archived on BlogTalkRadio.com/nonprofit_u, Valeriefleonard.com/NonprofitU and Stitcher.
Monday's episode will provide an overview of how the Tutor/Mentor Institute LLC is creating tutor/mentoring hubs to "transport" low income children from poverty to careers. There will be opportunities for listeners to call in with questions and participate in live chats. Nonprofit professionals, and educators are encouraged to call in and share their stories. The live call in number is (347) 884-8121.
A place for youth to create media
World Sport Chicago is offering 50 Chicago high school student-athletes ACT tutoring, pre-college mentoring and the opportunity to receive one of 10 renewable college scholarships worth up to $5,000 annually ($20,000 total), supported by WSC and Chicago donors. Applications and more information is available at www.wscscholars.org. In order to be considered for the scholarship, all applications must be submitted online by November 16, 2011.
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Maganaris, Constantinos N., Baltzopoulos, Vasilios and Sargeant, Anthony J. (2006) Human calf muscle responses during repeated isometric plantarflexions. Journal of biomechanics, 39 (7). pp. 1249-55. ISSN 0021-9290File not available for download.
In the present study, we measured the contraction-induced shortening (dL) of individual synergistic human muscles in a repeated motor task to assess their contractile behaviour. Ultrasonography was used to obtain dL measurements in the gastrocnemius (GS) and soleus (SOL) muscles of six men performing 11 consecutive isometric plantarflexions. Contractions 1 and 11 were performed with maximal effort, and contractions 2-4, 5-7 and 8-10 were performed with efforts generating 50, 70 and 90%, respectively, of the plantarflexion moment produced in contraction 1. In contractions 5-10, the SOL muscle dL was similar (p>0.05) to that produced in contraction 1 ( approximately 6mm), indicating that the SOL muscle became fully activated at 70% of the maximum plantarflexion moment. The GS muscle dL in contractions 10 and 11 exceeded by approximately 0.5mm (p<0.05) and 1.3mm (p<0.01), respectively, that generated in contraction 1 ( approximately 10mm), despite evidence obtained by superimposed stimulation that contraction 1 was produced with full motor unit activation. The consequent paradox that the GS muscle would produce in contractions 10 and 11 a greater activation and therefore more force than its actual potential is resolved when considering the interaction between the time-dependent tensile response of tendon and the performance of muscle as dictated by the sliding filament mechanism of contraction.
Impact and Reach
Additional statistics for this dataset are available via IRStats2.


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Quarantine has got us all locked in our homes with no access to the outdoors, let alone the gym. Well, your gym bag might think it is of no use now, but Mahima Sethia, Founder of Fitness Flu doesn't think likewise. There is something in your gym bag you can use for your workout at home. No, we are not talking about the resistance band but the humble towel that everyone does have at their home. She believes Exercise and Diet are like Marriage. Exercise alone cannot help you lose weight if your nutrition is not on point. They both co-exist. It's okay if you do not have a home gym or any workout equipment, as right now it is necessary to stay at home and keep our body healthy. Health and wellness coach Mahima Sethia brings a few fun and entertaining sliding towel workouts to help you tone your body during the lockdown.
So let's get started
Inner Thigh Slide Lunges
It is an easy and effective exercise that will focus on your inner thighs and will give a nice toning to your legs and also help you improve your flexibility.
Slide Back Lunge
The classic back lunge is an easy-to-learn-exercise that helps to tone your glutes, quadriceps, hamstrings and core. The sliding lunge using a towel is a functional variation of the classic lunge exercise that enhances the level of difficulty of this exercise.
Side Sliding Pushups
Push-ups are the best exercises to tone your upper body. By using your body weight and sliding the towel, you will work on shoulder, arms and chest muscles. This is an advanced variation of classic pushups.
Leg Wiper
This is a fun towel exercise that will engage your core, legs, arms, back and help you tone full-body.
Sliding Mountain Climbers
This is a fun towel exercise that will target hips, glutes, thighs, core and will help you improve balance and coordination.
Sliding Tuck
Sliding Tuck is a very effective exercise. It strengthens multiple muscles at one time and also helps to improve the cardiovascular system. You will surely feel the burn.
Towel Hamstring Curl
It's important to train your hamstrings which contribute to functional movements such as walking. This towel workouts can help you strengthen your hamstring.
The article is authored by Mahima Sethia, CEO & Founder of Fitness Flu(registered under start-up India)


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Talk about a sick burn.
The "Real Housewives of New Jersey" crew — including Teresa Giudice, Melissa Gorga, Jackie Goldschneider, Dolores Catania, Jennifer Aydin, Margaret Josephs and friend of the cast Danielle Staub — took a trip to Steven Dann's boutique in Great Neck on Wednesday night's episode, with Josephs' belongings ending up in a Fornasetti candle worth more than $600.
Staub grabbed Josephs' red clutch and proceeded to toss the contents into the burning candle after Josephs dumped a water bottle on her head in the middle of an argument.
Dann told Giudice during the episode that the pricey piece of home decor retailed for $675 at his shop, but it is actually sold for $640, per the merchandiser. Fans can also snag it for the same price on Amazon.
"It's hand-painted in Italy!" Dann exclaimed.
Josephs, visibly embarrassed by Staub's antics, offered to reimburse Dann for the costly candle.


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From Awareness to Action: Netsuite's Full-Funnel Webinar Strategy
- On-Demand Webinar
Webinars are more than just a single event, they are a powerful mechanism for a full-funnel marketing campaign. Join Netsuite's Mark Philllips to hear how to use webinars as a driving force for pipeline acceleration and moving engaged prospects and customers across every stage of the lifecycle.
You'll learn how to:
- Attract new leads with a top-of-funnel webinar series
- Accelerate mid-funnel pipeline with webinars featuring products and case studies
- Close deals and upsell customers with hands-on demos
From Awareness to Action: Netsuite's Full-Funnel Webinar Strategy
Get ready!
Woot, thrilled to have you join us!
You will receive a confirmation email shortly.
Mark Phillips, Senior Marketing Manager, Oracle NetSuite


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South Africa's relations with Malawi's new President Lazarus Chakwera have started badly, clouded by suspicions that he helped a fugitive escape South African justice and complaints that Pretoria damaged his dignity by delaying his departure from South Africa.
By Peter Fabricius
Malawian President Lazarus Chakwera, elected in June 2020, made his first official visit to South Africa last week, meeting President Cyril Ramaphosa. It all seemed at first to be a success.
But the visit became embroiled in the controversy over the near-simultaneous flight from South Africa of the indicted Malawian TV pastor Shepherd Bushiri and his wife Mary after they had skipped bail.
Both the Malawian and South African governments have denied media speculation that the multimillionaire Bushiris — who are believed to have helped bankroll Chakwera's election campaign — escaped to Malawi on Chakwera's plane.
But Chakwera's flight home has soured relations with Pretoria anyway. On Monday, the Malawian government issued an angry statement complaining that the departure of Chakwera's aircraft from Waterkloof Air Force Base on Friday had been delayed by seven hours, mainly because South African authorities wouldn't allow the Malawian foreign minister and some other officials to board.
The Malawi government said the delay had been caused by SA's "inexplicable refusal" to allow Chakwera's advance party to board his plane at Waterkloof Air Force Base.
The statement said South African authorities had cited "unspecified security reasons" for its refusal. Pretoria had initially also refused — "for no apparent reason" — to allow the Malawians to fly the presidential plane to Johannesburg's OR Tambo International Airport to pick up the advance party. But it had eventually allowed that after several more hours of delay.
Malawi said it was "justified in its dissatisfaction with the unspecified security reasons cited for the refusal". It said Chakwera's plane had been parked at Waterkloof military airbase, a highly secure facility, throughout his visit.
The statement also complained that the passports and luggage of the advance team had been thoroughly checked by uniformed police officers and not immigration officers.
It said that all the Malawian delegation's luggage had been loaded on to Chakwera's plane by South African military personnel and at the time of the scheduled departure of the plane at 3.30pm, there had been dozens of SA security personnel on the ground, who inspected the plane, its cargo and every passenger on entry.
South Africa's efforts to get the Bushiris back to face trial may put further strain on relations with Malawi. The offences for which they have been charged are not extraditable in Malawi, it has been reported.
These "unacceptable and unsatisfactory" security procedures and the delay they caused had "breached diplomatic protocols commensurate with the dignity of President Chakwera's office and person". However, the Malawi government welcomed the SA government's statement "exculpating President Chakwera from false allegations dominating the South African media that his presidential plane was used as a conduit for trafficking two Malawians wanted by South African authorities".
But the statement complained that Pretoria had not acknowledged that its treatment of Chakwera on his departure was "improper and incongruous to the warm hospitality he received upon his arrival".
The two Malawians who the statement referred to are wealthy Malawian TV pastor Shepherd Bushiri and his wife Mary, who skipped bail of R200,000 each last week while on theft, fraud and money-laundering charges in South Africa relating to an alleged R100-million investment scam.
Apart from the unproven allegations that Chakwera helped them escape, there has also been speculation that the intense security which his delegation was subjected to on Friday arose from suspicions by South African authorities that he would be smuggled on board Chakwera's plane.
The passports of the Bushiris had been confiscated under bail conditions and South African government spokesperson Phumla Williams has publicly stated that they did not leave on Chakwera's plane or through any other official port. However, Bushiri is known to have several passports — including a diplomatic one issued by the previous Malawi government. There is now speculation that he left South Africa by road, possibly via the Beit Bridge border post between SA and Zimbabwe which is notoriously porous. Malawian information minister Gospel Kazako told ENCA News on Friday that the Bushiris had been in Malawi since Wednesday, two days before Chakwera left SA.
In contrast to Malawi's sharp criticism of Pretoria for the way it handled Chakwera's departure, there is also some criticism of him in Malawi for failing to understand diplomatic protocol.
One Malawian observer said that the president's advance party had arrived in South Africa at OR Tambo International Airport earlier last week and had been processed there by normal immigration authorities. The officials who accompanied Chakwera on his plane had been processed differently at Waterkloof Air Force Base. Then Chakwera had decided to take them all back home on his plane — possibly to save costs — which had caused bureaucratic and diplomatic complications for the South African authorities.
Daily Maverick reported on Monday that it was Malawian foreign minister Eisenhower Mkaka, a member of the advance party, who had provoked the row by causing a scene at Waterkloof Air Force Base when he was not allowed on Chakwera's plane.
South Africa's efforts to get the Bushiris back to face trial may put further strain on relations with Malawi. The offences for which they have been charged are not extraditable in Malawi, it has been reported.
But Williams said in a statement that South Africa had initiated a process to secure their extradition from Malawi "in terms of the Southern African Development Community (SADC) Protocol on Extradition and other legal instruments, to which Malawi is a signatory".
A South African official told Daily Maverick that the SADC protocol would "trump" any bilateral bar which Malawi might have on extraditing the Bushiris. Daily Maverick


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It's extremely easy to grow pretty blooms and shrubs in sunny outdoor and indoor spots, but what about the shadier areas? Thankfully there are a number of shade-loving plants available, all of which thrive without a great deal of sunlight, yet still, add colour and fragrance to our homes and gardens.
Here we have the key tips on how to grow shade plants, and the key details relating to which plants grow well in the shade and which do not.
Shade-loving plants
There are a number of shade-loving plants out there to pick from, although the following are among the most popular.
1. Astilbe
These elegant perennial plants boast an old-fashioned look and are able to thrive in both partial sunlight and shade, providing the soil is rich and moist. Their tall feathery plumes flower in the months of spring and summer, producing beautiful purple, pink, white and red flowers.
These blossoms are hugely popular as cut flowers in the home. Depending on the variant, astilbes can grow anywhere between one and six feet in height.
2. Coleus
These blooms are available in a rainbow of hues and patterns. Also known as Solenostemon scutellarioides, they're grown for their impressive foliage, rather than their flowers. When placing them in containers or vases, a great idea is to mix several shades and species with contrasting colours to create a dramatic effect.
They're often grown as annuals in cooler climates and make a great option for those looking for low maintenance plants. Many grow these plants on patios, whilst newer varieties have a much greater tolerance for full sunlight. Avoid placing them in harsh sunlight, as this will cook the plants.
3. Coral Bells
Coral bells, also known as Heuchera, feature delicate blooms, which come out in the months of late spring and early summer. This perennial's flowers are quite insignificant, however, it's dramatic foliage makes up for it. The leaves come in a range of beautiful shades, including everything from warm-orange caramel hues, to nearly black 'Obsidian' shades.
The 'Peach Flambe' is a popular variant to choose, as its young foliage begins a shade of peachy yellow, and turns a deep red over time. When fully matured, these plants reach a deep shade of plum, this usually occurs by late autumn.
Plant this picture-perfect plant in semi-shady areas or under deciduous trees. They're a great option if you wish to add a splash of colour to your home or garden.
4. Impatiens
This extremely prevalent flowering annual, also known as Impatiens wallerana, thrives in a garden with partial shade. Impatiens flowers come in a wide variety of shapes and hues, and feature singles, doubles and semi-double blooms that range from pink to orange to red.
They're easily sourced online and at your local garden centre and look great when planted in bunches in hanging baskets, garden beds, window boxes and lining walkways.
These easy to grow annuals thrive best in rich, moist soil that is able to drain well. One thing to watch out for when planting Impatiens is their susceptibility to downy mildew disease. This can, however, be avoided when they are planted in the correct environment.
5. Polka dot plant
Often referred to as the freckle face plant, botanical name: Hypoestes Phyllostachys, this impressively attractive annual, boasts an array of white, red, green and pink leaves. It is famous for its splotches and freckles, hence where the nickname comes from. This plant flourishes in rich, well-drained soil and is best suited to partial sunlight and areas boasting plenty of humidity.
When placing this plant indoors, it is a good idea to mist it on a regular basis. Simply use a spray bottle to upsurge the air moisture. The polka dot plant enhances both the indoors and outdoors, adding a fanciful touch to vases, garden beds and containers. It looks great when grown alongside coleus in pots.
Polka dot plant, impatiens and coleus are just a few of the shade-loving plants in existence, all of which thrive in darker gardens and indoor spaces, meaning you can add colour and life to areas you deemed unworthy of housing plants, blooms and shrubs.
How to shade plants
If you have decided to plant an array of shade-loving plants in your garden, yet live in a location that gets a lot of suns, you can build various areas of cover.
From floating row covers, created using a lightweight garden fabric that is safe to drape directly over plants and flowers, to support hoops, that is able to hold a cover above plants, there are many options to choose from. You may even wish to get creative and fashion something yourself.
How to grow shade plants
All plants necessitate different methods of care. Some are very easy to care for and require little water or pruning, whilst others are more high maintenance. It's important to consider how much time you have available to care for these plants and also your existing garden space.
Each plant comes with different care instructions and knowing a little more about this will help you to choose the correct shade-loving plants for your home and garden. It will also help you to ensure they thrive in the conditions you have to offer.
What are good flowering shade plants?
Some of the best flowering shade plants include:
- Bletilla
- Dead Nettle
- Caladium
- Torenia
- Foamflower
- Primulas
- Coleus
- Lungwort
- Heuchera
- Hostas
- Oakleaf hydrangeas
- Astilbe
What plants grow well in the shade?
As listed above, there are many plants in existence that thrive in shaded conditions. Before planting any one of these, do your research though. Look into their size (when fully matured), their shape, whether they are high or low maintenance, and how easy they are to obtain.
Colour should also be an important consideration, especially if you already have a certain garden theme in place.


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"Facility introduction", "Guide tour", "HighKing trail" is translated into English.
William Merrell Vories came to Japan in 1905 (Meiji 38) at the age of 24. He wanted to be an Architect but came as an English teacher. He was asked to oversee the building of the YMCA office in Omihachiman Shiga and from there went on to oversee the construction of over 1,600 houses, buildings and churches around Japan.
The Vories Rokko Cottage was built in 1934 and used by his family as their summer mountain retreat. It is a beautiful combination of Japanese and Western building styles. It is a Registered National Cultural Asset, and is one of the 100 Selected Modern Houses in Hyogo. It was purchased as a National Trust by Amenity 2000 and is open to the public. They have maintained the property beautifully and one can really experience and understand how the Vories family enjoyed spending time on Rokkosan.
In 2018, the adjacent property was purchased and in 2019 and 2020 a beautiful Mountain Forest Garden was built for visitors to walk in and enjoy the area.
It is open every Saturday, Sunday and Public Holiday from April 3rd (Sat) until November 14(Sun) from 11 am until 4pm. The Entrance Fee is ¥500. Be sure to check their website (Japanese) for more information.
It is just a nice 15 minute walk from the Mt.Rokko Guide House. Flyers and maps are available.
Detailed Access Map (Japanese)
Make sure to stop by Vories Rokko Cottage and experience a fantastic piece of history.
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In this issue: Box office news for We're the Millers, a new release from Madman, Metallica at Imax, lineups announced for Austin Film Festival and Greek Film Festival, callouts for entries from Edge and Studio, watch Pandora's Promise to meet Robert Stone, plus much more…
The Greek Film Festival is coming to town
The Delphi Bank Greek Film Festival has released it's 2013 festival dates. This year marks the twentieth anniversary and befittingly, features a "best of" program. The festival opens at Palace Cinemas Wednesday 6th November in Sydney, and Thursday 7th November in Melbourne. Following one of their most successful years yet in 2012, 2013 looks to continue this trend showcasing the best in contemporary Greek cinema for Aussie audiences. Early program announcements include Thanos Anastopoulos's The Daughter, Ilias Yannakakis' Joy and A.C.A.B. All Cats are Brilliant directed by Constantina Voulgaris.
Head to www.greekfilmfestival.com.au for more information and further program details.
We're the Millers crosses $200 million mark
With Jason Sudeikis as a drug dealer and Jennifer Aniston as a stripper all the while playing happy families, We're the Millers always had box office success written all over it. Having taken in an estimated $206 million internationally, the film has become the highest grossing R-rated comedy of all time, perhaps reflecting the general change in attitudes of the population.
It's still set to be released in Spain, France and Brazil and continues climbing on the international box office. Go see it if you haven't already, raise those revenues.
Madman gets Dragon Ball Z: Battle of the Gods
Purveyor of mostly animated goods, Madman have acquired the latest feature in the Dragon Ball Z franchise, Dragon Ball Z: Battle of the Gods. The first Dragon Ball Z movie to receive a Japanese theatrical release in 17 years, this foray into film in the series is the first to be considered as part of the official manga storyline.
When Birus, The God of Destruction awakes from a long slumber, he learns of the defeat of the galactic overlord Freeza by the hands of Son Goku. Seeking an opponent worthy of his power, Birus, along with his companion Uis, travels to the North Quadrant of the universe to challenge Goku to a battle.
Dragon Ball Z: Battle of the Gods will premiere at the 17th Japanese Film Festival, touring Australia Oct – Dec, 2013. For more information, visit www.japanesefilmfestival.net
Dragon Ball Z: Battle of the Gods will also screen in select Australian cinemas in 2014.
Metallica at Imax
For one week only, Metallica Through the Never 3D, will be at IMAX Darling Harbour, on 10 October 2013. From award-winning filmmaker Nimrod Antal, Metallica Through the Never 3D is a music-driven feature film that combines a bold narrative and spectacular live-performance footage of one of the most popular and influential rock bands in history.
Emerging young star Dane DeHaan portrays Trip, a young roadie sent on an urgent mission during Metallia's roaring live set in front of a sold out arena. But what seems like a simple assignment turns into a surreal adventure.
Get your tickets from www.imax.com.au
2013 Austin Film Festival announces lineup
2013 marks the 20th anniversary for the Austin Film Festival. They've announced their lineup for the festival with Joel and Ethan Coen's Inside Llewyn Davis as the centrepiece. Starring Oscar Isaac (in attendance), Carey Mulligan, John Goodman, Garrett Hedlund, and Justin Timberlake, the film follows a week in the life of a young folk singer as he navigates the Greenwich Village folk scene of 1961.
Additional films include: Steve McQueen's 12 YEARS A SLAVE starring Benedict Cumberbatch, Brad Pitt, Michael Fassbender and Chiwetel Ejiofor, AUGUST: OSAGE COUNTY based on the Pulitzer Prize-winning play and featuring a powerful ensemble cast including Meryl Streep and Julia Roberts; Alexander Payne's NEBRASKA starring Will Forté (in attendance) and Bruce Dern; biopic MANDELA: LONG WALK TO FREEDOM starring Idris Elba; James Franco's CHILD OF GOD; BIG SUR with Josh Lucas, Jean Marc-Barr and John Robbins in attendance; and the World Premiere of the romance drama LIGHT YEARS, starring Allison Janney and Clark Gregg. Also playing is PHILOMENA with Judi Dench and Steve Coogan; HBO'S WHOOPI GOLDBERG PRESENTS MOMS MABLEY; COLD COMES THE NIGHT starring Alice Eve and Bryan Cranston; crime drama MYSTERY ROAD; AND WINTER IN THE BLOOD with cast in attendance.
Check out the full line-up and find more information at www.austinfilmfestival.com
Young filmmakers, get your foot in the door
Content marketing agency Edge is on the hunt for talented new short film makers with a new short film competition called "The New Wave". From 9 September 2013 to 29 October 2013, the hunt is on for budding filmmakers and creatives between 18 to 25 to submit a short film centred around the theme "Play". Up for grabs is a 6 month paid internship plus $3000 cash.
Find out more from www.thenewwave.com.au
Win $10,000 with Create Studio
Studio, Foxtel's Arts and Entertainment channel returns again this year on the lookout for artistic talent to help create four new TV commercials. Give life to your ideas with your 30-second concept for a commercial that brings the Studio logo to life. In association with The Loop, this is an exciting opportunity for the next artist out there to dive into the next part of their career. Applications are now open and close at the end of October.
More information is available from www.studiotv.com.au/the-competition/
Ron Howard's Heart of the Sea begins production
From the man that brought you A Beautiful Mind, Ron Howard's newest film Heart of the Sea, has begun production in the UK. The action adventure film, based on Nathaniel Philbrick's best-selling novel follows the dramatic true journey of a whaling ship, Essex.
In the winter of 1820, the New England whaling ship Essex was assaulted by something no one could believe: a whale of mammoth size and will, and an almost human sense of vengeance. The real-life maritime disaster would inspire Melville's Moby-Dick. But that told only half the story. "Heart of the Sea" reveals the encounter's harrowing aftermath, as the ship's surviving crew is pushed to their limits and forced to do the unthinkable to stay alive. Braving storms, starvation, panic and despair, the men will call into question their deepest beliefs, from the value of their lives to the morality of their trade, as their captain searches for direction on the open sea and his first mate still seeks to bring the great whale down.
Starring Chris Hemsworth, Benjamin Walker, Cillian Murphy and Ben Whishaw, the film will be distributed by Warner Bros.
Oldboy featurette released
Hype is building as the November 27, the release date for new film Oldboy, looms before excited fans. The thriller has found a fervent online audience and has released a featurette in anticipation of the release date.
Check it out here:
Meet Oscar-nominated director of Pandora's Promise
Get the chance to meet Oscar-nominated director Robert Stone as he tours Australia promoting his new ground-breaking documentary, Pandora's Promise. Stone will be presenting Q&A's following special screenings of the film nationally. An official selection at this year's Sundance Film Festival, Pandora's Promise presents a topical and controversial argument that nuclear energy, the one technology we fear most, could save our planet from a climate catastrophe, while providing the energy needed to lift billions of people in the developing world out of poverty.
Go see him at the following:
Melbourne • Tuesday 8th October • Classic Cinema, Elsternwick
Adelaide • Wednesday 9th October • Mercury Cinema, Morphett St
Perth • Thursday 10th October • Luna Palace Cinemas, Nedlands
Canberra • Friday 11th October • The Arc Cinema, McCoy Circuit
Sydney • Saturday 12th & Sunday 13th October • Hoyts, Moore Park
Brisbane • Tuesday 14th October • Bemac Cinema, Kangaroo Point
For bookings, head to www.cinemaventures.org/pandoras-promise/
Hunger Games releases soundtrack
The soundtrack has been released for The Hunger Games: Catching Fire in a powerful, star-studded lineup featuring the likes of Coldplay, Sia and Christina Aguilera. The soundtrack will be available in stores and through all digital retailers on November 19, 2013 just before the film opens in Australia on November 21, 2013. Right now, the album can be pre-ordered via the film's official online store. Those who pre-order the soundtrack by October 27th will have an opportunity to have their names printed on an exclusive The Hunger Games: Catching Fire poster (while supplies last). All pre-orders unlock an instant download of Coldplay's "Atlas" as well.
1. "Atlas" – Coldplay
2. "Silhouettes" – Of Monsters and Men
3. "Elastic Heart" – Sia (ft. The Weeknd & Diplo)
4. "Lean" – The National
5. "We Remain" – Christina Aguilera
6. "Devil May Cry" – The Weeknd
7. "Who We Are" – Imagine Dragons
8. "Everybody Wants To Rule The World" – Lorde
9. "Gale Song" – The Lumineers
10. "Mirror" – Ellie Goulding
11. "Capital Letter" – Patti Smith
12. "Shooting Arrows At The Sky" – Santigold
13. "Place For Us" – Mikky Ekko
14. "Lights" – Phantogram
15. "Angel On Fire" – Antony and the Johnsons
Foxtel CEO confirmed for Screen Forever conference
Richard Freudenstein, the CEO and Managing Director of Australia's biggest pay tv platform Foxtel, will be the guest in a special one-on-one conversation with Bob Campbell, Managing Director of Screentime, at the Screen Forever: SPAA Conference 2013.
Richard Freudenstein was appointed CEO of Foxtel in November 2011 and has continued the work of his predecessor Kim Williams in positioning the company at the forefront of innovation in a rapidly changing media landscape. Foxtel currently has a slate of 50 local productions commissioned from 30 independent production companies, with titles as varied as Wentworth, Australia's Next Top Model, Selling Houses Australia, Paddock to the Plate, Outback Coroner and The Train.
Get more info on Screen Forever at www.screenforever.org.au


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The first medical marijuana dispensary in Harford County is set to open this week. RISE Joppa will begin serving patients on Friday, April 27.
RISE Joppa will be overseen by Green Thumb Industries (GTI), which runs dispensaries in several states. The company already operates two RISE locations in Maryland, one in Bethesda and another in Silver Spring.
"We are thrilled to join the Joppa community and look forward to serving our patients," Pete Kadens, chief executive officer of GTI, said in a statement.
"Our goal at our RISE dispensaries is to provide the very best care to our patients while offering the the most effective and quality medical marijuana available," Kadens said.
Maryland legalized medical marijuana in 2013 and the first dispensary in Maryland opened in December 2017. Currently there are 46 licensed dispensaries across the state.
Medical use of marijuana has been legalized in 29 states, the District of Columbia, Puerto Rico and Guam, according to the National Conference of State Legislatures.
The process to obtain cannabis legally in Maryland entails registering with the Maryland Medical Cannabis Commission and obtaining certification from a medical provider registered with the state commission.
Conditions for which people may be prescribed cannabis include cachexia, anorexia, wasting syndrome, severe pain, severe nausea, seizures, severe or persistent muscle spasms, glaucoma, post-traumatic stress disorder and chronic pain.
Of the opening of RISE Joppa, Kadens said: "This is a great day for the patients of Maryland and the state's medical cannabis program."
Operators of the dispensary say they hope to help curb opioid use in Harford County.
"We are so honored to be able to bring relief to more patients in Maryland who are suffering and can benefit from medical cannabis," GTI's Maryland Market President Andy Grossman said in a statement.
"Harford County has been especially hard hit by the opioid epidemic," Grossman added, "and GTI is hopeful that by offering an alternative treatment for chronic pain and other conditions we can have a positive impact on this issue which is so important to families and the community."


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tvOS SDK Release Notes for tvOS 9.0
The tvOS SDK leverages many of the same frameworks and technologies that you are already familiar with when developing for iOS. However, please note that all libraries and frameworks that you use in your tvOS apps must be built for tvOS, including any 3rd-party libraries. Do not link your tvOS app against frameworks or libraries that are not built with tvOS. Attempting to do so will result in a build failure. Furthermore, bitcode is now required for all tvOS apps. All apps and frameworks in the app bundle need to include bitcode.
tvOS SDK 9.0 provides support for developing tvOS apps. It is packaged with a complete set of Xcode tools, compilers, and frameworks for creating apps for tvOS. These tools include the Xcode IDE and the Instruments analysis tool, among many others.
This version of tvOS is intended for installation only on devices registered with the Apple Developer Program. Attempting to install this version of tvOS in an unauthorized manner could put your device in an unusable state.
Bug Reporting
For issues not mentioned in the Notes and Known Issues section, please file bugs through the Apple Developer website (https://developer.apple.com/bug-reporting/). Additionally, you may discuss these issues and tvOS SDK 9.0 in the Apple Developer Forums: https://forums.developer.apple.com/community/pre-release/tvos-beta.
Notes and Known Issues
For tvOS Metal development, it is now required that tvOS specific feature set enum is used. Specifically:
Parallax Images
Please regenerate any LCR images from source material using the latest SDK. There have been several improvements to performance, size, and accuracy of LCR images.
Known Issue
In the Simulator, if the remote sleeps, when it wakes back up motion control will no longer work.
Workaround: Restart the app.
On Demand Resources
Known Issues
When using the beginAccessingResourcesWithCompletionHandler: method, any resource that has already downloaded will report a progress of 0.0 even though it is available locally.
Initial On Demand Resource asset packs are not currently downloaded during install-time. They can be fetched at first runtime.
Tap To Setup requires iOS 9.1 or later.
StoreKit (In-App purchases) do not work in the Simulator.
Copyright © 2018 Apple Inc. All rights reserved. Terms of Use | Privacy Policy | Updated: 2015-10-21


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Read Online or Download A Forgotten Sector: The Training of Ancillary Staff in Hospitals PDF
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I wanted to keep a log for myself of what has been happening with my health. I want to encourage society to stop this insane bs of comparing weight. And I want to give someone insight on my treatment therapy in hopes of helping him or her.
Category: Multiple Sclerosis
This is my journey with multiple sclerosis, a rare autoimmune disease. From details on my attacks to tests to medication insight, I hope my story helps others.
Who makes these decisions? Who finger-points which people "survive," which benefit?
My point is this: Facebook can blow up with stupid viral videos of old not-even-good movie clips with the words "Bye Felicia" and deep debates on the color of a dress, and people can bitch about our government as those running constantly say bigoted, racist remarks while still being handed a microphone . . . but practically nothing is being done to actually help anymore.
I had a flare-up which (to me) is basically a reminder that you have MS, a sign that your body is still trying to hurt itself.
It has been three years since I was diagnosed with Multiple Sclerosis. In the beginning, people and articles said, "Things would make more sense soon." This filled me with more anxiety. What "things"? When was "soon"? It seemed to be a riddled threat, one I could not figure out… until now. I realize I was looking at it all wrong. The articles and people meant things — the ticking time bomb — had already gone off and that I would get answers as to why soon, now.
It's strange because sometimes when I talk about MS in person, I feel furthest from people. Yet, here — with the light of my computer screen — I feel like I have someone with me. I'm not judged. I'm not told I'm wrong or right… or how I should act or what to think. I'm not told anything. I'm just given a space. A space to think. And I believe when one can delve into their own thoughts — be allowed to fully think — that person is never alone.
I had my multiple sclerosis second opinion appointment a few days ago. And I finally got the answers I've been wanting.
I took to the internet for the first time to research multiple sclerosis. So it is here, on my blog, I have a stress reliever, a different form of a scrapbook, a way to record major information (mainly MS details). I also truly want to share my research with someone newly diagnosed… or someone who knows a person newly diagnosed. Information can be overwhelming, but hopefully, it will be more brief and not as scary here.
For me, this month simply meant two things: doctors… doctors… an all-important MRI exam… and yep, more doctors. Maybe by the end of November it would mean one more thing: a diagnosis. …Maybe.
Soon, James and I will be married. And instead of relishing in our wedding plans, I'm concentrating on November. November means doctors, and tests, and possibly answers.
James told me recently he doesn't necessarily believe people are given more than they can handle. Instead, he thinks people are given what they and their support group can handle. I'm beginning to believe this.


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MAYVILLE – It's Presidential Primary day today in New York State, and registered Republicans throughout the state will be heading to the polls to decide which candidate they want to see represent their party in November. In Chautauqua County, all 54 polling sites will be opening at noon and staying open until 9 p.m.
Polling will be at the same location as regular voting sites, although the County Board of Elections says that poll sites for all voters in the village of Fredonia, Charlotte and three election districts on the east side of Jamestown have been changed for 2012.
In Jamestown Ward 1 Districts 2, 3 & 4 will now be voting at the IBEW Local 106 Hall at 322 James Ave. – across from the JCC Parking lot. Also, Charlotte voters will now be voting at the Sinclairville Fire Station and Fredonia village voters will now be voting at Wheelock School.
The four Republican Candidates vying for voters endorsements are front-runner Mitt Romney, former Pennsylvania U.S. Senator Rick Santorum, former house speaker Newt Gingrich, and Texas Congressman Ron Paul. No other political parties have contested primary elections and New York State has a closed primary system, which limits Tuesday's voting to Republicans.
Voters can determine their current affiliation or registration status and find primary night results by visiting www.votechautauqua.com. Voters may also contact the Board of Elections directly by calling 753-4580, 363-4580 or 661-7580.