text:
An Alpha-Catulin Homologue Controls Neuromuscular Function through Localization of the Dystrophin Complex and BK Channels in
The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.
Published in the journal:
. PLoS Genet 6(8): e32767. doi:10.1371/journal.pgen.1001077
Category: Research Article
doi: 10.1371/journal.pgen.1001077
The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.
Precise control of membrane excitability, largely determined by ion channels, is of utmost importance for neuronal and muscle function. The regulation of ion channel localization, density and gating properties thus provides an effective way to control the excitability within these cells [1]. Indeed, the localization and gating properties of ion channels are often regulated or modified by cytoskeletal and signaling proteins, or auxiliary ion channel subunits expressed in a cell-type specific manner [2]. Potassium channels are critical in determining the excitability of cells, because potassium ions are dominant charge carriers at the cell resting potential. Among potassium channels, the large conductance, voltage- and calcium-dependent potassium BK channels (also called SLO-1 or Maxi-K) are uniquely gated by coincident calcium signaling and membrane depolarization [3], [4]. This feature of BK channels provides a crucial negative feedback mechanism for calcium-induced functions, and plays an important role in determining the duration of action potentials [3]. BK channels are widely expressed in a variety of cell types and are implicated in many physiological processes, including the regulation of blood pressure [5], neuroendocrine signaling [6], smooth muscle tone [7], and neural network excitability [8], [9].
Mounting evidence indicates that BK channels can interact with a variety of proteins that modulate channel function, or control membrane trafficking. For example, the Drosophila BK channel, dSLO, interacts with SLO binding protein (slob), which in turn modulates the channel gating properties [10]. Similarly, mammalian BK channels associate with auxiliary beta subunits that influence channel activation time course and voltage-dependence [11]. In yeast two hybrid screens, the cytoplasmic C-terminal tail of mammalian BK channels has been shown to interact with several proteins, including cytoskeletal elements, such as actin-binding proteins [12], [13] and a microtubule-associated protein [14]. These cytoskeletal proteins are partially co-localized with BK channels, and appear to increase cell surface expression of BK channels in cultured cells [12], [13]. However, it remains to be determined whether these proteins have any role in controlling the localization of BK channels to specific areas of the plasma membrane in vivo. Robust activation of BK channels requires higher intracellular calcium concentrations (>10 µM), which only occur in the immediate vicinity of calcium-permeable channels [4]. Hence, the localization of BK channels to specific areas (i.e. calcium nanodomains) where calcium-permeable ion channels are located is physiologically important for BK channel activation.
In C. elegans, loss-of-function mutations in slo-1 partially compensate for the synaptic release defects of C. elegans syntaxin (unc-64) mutants [15] and lead to altered alcohol sensitivity [16]. Recent studies in C. elegans have also implicated SLO-1 in muscle function [17]. slo-1 mutants display an exaggerated anterior body angle, referred to as the head-bending phenotype that is shared by mutants that are defective in the C. elegans dystrophin complex [18]–[20]. Recent evidence that the C. elegans dystrophin complex interacts with SLO-1 channels via SLO-1 interacting protein, ISLO-1, explains this phenotypic overlap [21]. However, C. elegans dystrophin complex mutants do not appear to alter the biophysical properties of BK channels per se [17]. Similarly, ISLO-1 does not modify SLO-1 channel properties [21]. Rather, ISLO-1 tethers SLO-1 near the dense bodies of muscle membranes, where L-type calcium channels (EGL-19) are localized [21]. Consequently, defects in the dystrophin complex or ISLO-1 cause a large reduction in SLO-1 protein levels in muscle membrane, which in turn causes muscle hyper-excitability leading to enhanced intracellular calcium levels. This perturbation of calcium homeostasis has been postulated to be one of the first steps in the degenerative muscle pathogenesis associated with disruption of the dystrophin complex [22].
In this study, we performed a forward genetic screen to identify additional genes responsible for SLO-1 localization and function in C. elegans. We identified ctn-1, an orthologue of α-catulin, as a novel gene that controls SLO-1 localization and function in muscles and neurons. Our analysis showed that ctn-1 uses different strategies to localize SLO-1 in these two cell types. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 near L-type calcium channels via ISLO-1. In neurons, CTN-1 localizes SLO-1 independent of the dystrophin complex.
A genetic screen for suppressor mutants of gain-of-function slo-1 identifies genes that interact with the dystrophin gene
Loss-of-function slo-1 mutants exhibit a jerky locomotion and head bending phenotype [15]. By contrast, gain-of-function slo-1 mutants exhibit sluggish movement combined with low muscle tone [16]. When slo-1(gf) mutant animals are mechanically stimulated, they fail to make a normal forward movement, and tend to curl ventrally (Video S1). To identify genes that regulate slo-1 function, we performed a forward genetic screen to isolate mutants that suppress the phenotypes of the slo-1(ky399) gain-of-function mutant. Based on a previous genetic study [21], suppressor genes were expected to encode slo-1, components of the dystrophin complex, as well as novel proteins that control neuronal or muscular function of SLO-1. As expected, several loss-of-function alleles of slo-1 were isolated. In addition to these intragenic suppressors, several mutants could be segregated away from slo-1(gf) (Figure 1A and Video S1) and exhibited the head bending phenotype. Genetic mapping and complementation testing determined that these extragenic suppressors include dyb-1 and stn-1 which encode two homologous components of the dystrophin complex, dystrobrevin and syntrophin respectively. Additionally we isolated cim6 and eg1167 suppressors that represent novel genes. Compared to slo-1(ky399) and cim6;slo-1(ky399) mutants, eg1167;slo-1(ky399) mutants exhibited a profound improvement in the locomotion speed (Figure 1A).
It was previously observed that slo-1(gf) mutants retain significantly more eggs than wild-type animals due to low activity of the egg-laying muscles [16]. We found that suppressor mutants abolish an egg laying defect of slo-1(gf) mutants and retain eggs in uteri at levels similar to wild-type animals (Figure 1B).
ctn-1 encodes an α-catulin orthologue that has homology to α-catenin and vinculin
To understand the role of novel genes in slo-1 function, we pursued the identification of genes that mapped to chromosomal locations neither previously implicated in BK channel function, nor encoding known components of the dystrophin complex. Two mutations, cim6 and eg1167, both mapped to the left side of chromosome I and failed to complement each other for head bending, suggesting that these two mutations represent alleles of the same gene. Our quantitative analysis for locomotion and egg laying phenotypes showed that the locomotion speed of eg1167;slo-1(gf) was higher than that of cim6;slo-1(gf) whereas egg laying was comparable in both strains (Figure 1A and 1B). We further mapped eg1167 to a 250 kb interval and rescued the phenotype of eg1167 by generating transgenic animals with the fosmid WRM0621cC01 (Figure S1). Next, we rescued the head bending phenotype of eg1167 with a transgene consisting of the ctn-1 gene (Y23H5A.5) and approximately 4 kb upstream of the translation initiation codon (Figure 2A and 2B). The same transgene caused eg1167;slo-1(gf) double mutants to revert to the slo-1(gf) phenotype, displaying sluggish movement and retention of late-staged eggs in uteri (Figure 2C and 2D). These results indicate that a genetic defect in ctn-1 is responsible for suppression of the slo-1(gf) phenotypes.
The ctn-1 gene is orthologous to mammalian α-catulin (39.4% identity to human α-catulin), and is named on the basis of sequence similarity to both α-catenin and vinculin (Figure 2A) [23]. Vinculin and α-catenin are membrane-associated cytoskeletal proteins found in focal adhesion plaques and cadherens junctions. In C. elegans, vinculin (DEB-1) is localized to the dense bodies of body wall muscle and is essential for attachment of actin thin filaments to the sarcolemma [24], whereas α-catenin (HMP-1) is localized to hypodermal adherens junctions and is essential for proper enclosure and elongation of the embryo [25]. Based on its homology to vinculin/α-catenin and the localization of mammalian α-catulin [26], CTN-1 is likely to interact with other cytoskeletal proteins, which may in turn affect SLO-1 function. Additionally, the ctn-1 gene encodes a predicted coiled-coil domain. Such a coiled-coil domain mediates the interaction between dystrophin and dystrobrevin [27], two components of the dystrophin complex, although we do not know if the coiled-coil domain of CTN-1 is important for the interaction with these proteins (Figure 2A).
We determined nucleotide sequence of the predicted exons and exon-intron boundaries of the ctn-1 gene in eg1167 and cim6. The mutation sites found in both alleles create translation-termination codons (R144>STOP in eg1167, Q521>STOP in cim6) (Figure 2A). eg1167 exhibits complete suppression of slo-1(gf) phenotypes (see below) and is hence considered as a severe loss-of-function or null allele. All subsequent experiments were carried out with eg1167, unless mentioned otherwise. Although both eg1167 and cim6 mutants alone exhibit the head-bending phenotype, they differ with respect to suppression of slo-1(gf) phenotypes. Whereas ctn-1(eg1167) suppresses all aspects of the slo-1(gf) phenotype, ctn-1(cim6) completely suppresses the egg-laying defect of slo-1(gf) (Figure 1B), but not the locomotory defect (Figure 1A). These results suggest that the C-terminal third of CTN-1 is required for normal egg laying and head bending, but is not necessary to mediate the locomotion speed defect of slo-1(gf) mutants.
To elucidate the function of CTN-1, we examined the expression pattern of the ctn-1 gene using a ctn-1 promoter-tagged GFP reporter (Figure 2E–2H). We observed GFP fluorescence in body wall muscles, pharyngeal muscle, egg-laying muscle and enteric muscle of transgenic animals as well as in most, if not all, neurons of the nerve ring and ventral nerve cord.
CTN-1 has two distinct functions in neurons and muscles
Based on the ctn-1 expression pattern and the phenotypic differences between eg1167 and cim6, we investigated whether the head-bending phenotype and the suppression of sluggish movement of slo-1(gf) mutants are separable by expressing ctn-1 minigenes under the control of either muscle- or neuron-specific promoters in ctn-1 and ctn-1;slo-1(gf) mutant animals. Muscle, but not neuronal, expression of ctn-1 rescued the head-bending phenotype of the ctn-1 mutant (Figure 2B and Figure S1C). These results are consistent with previous reports that the head-bending phenotype is due to perturbations in muscle function [17]–[19]. Furthermore, muscle expression of ctn-1 in ctn-1;slo-1(gf) mutants resulted in egg retention to the level observed in slo-1(gf) mutants, whereas neuronal expression of ctn-1 did not alter the number of eggs retained in the uteri of ctn-1;slo-(gf) mutants (Figure 2C). Conversely, neuronal expression of ctn-1 in ctn-1;slo-1(gf) mutants reverted the seemingly normal locomotion of ctn-1;slo-1(gf) to the sluggish, uncoordinated locomotion of the slo-1(gf) mutant, whereas muscle expression of ctn-1 did not (Figure 2D). These results indicate that the sluggish, uncoordinated locomotory phenotype of slo-1(gf) mutants comes from presynaptic depression, but not from direct suppression of muscle excitability. Together with the allele specific phenotypic differences indicating different regions of CTN-1 are required for normal locomotory speed and head bending, these results suggest that CTN-1 uses two distinct mechanisms for mediating SLO-1 function in muscle and neurons by interacting with different sets of genes.
CTN-1 controls the integrity of the dystrophin complex and the localization of SLO-1 in muscle
Most, if not all, of the mutants that exhibit the head bending phenotype have a defect in either a component of the dystrophin complex or proteins that interact with the dystrophin complex [17]–[19]. The dystrophin complex is localized near muscle dense bodies [21]. Because ctn-1 mutants exhibit the head bending phenotype, we determined the subcellular localization of CTN-1 using a GFP-tagged CTN-1 transgene, which rescues the head bending phenotype (data not shown). GFP::CTN-1 exhibited a punctate expression pattern that resembled that of the dense bodies (Figure 3A). To further define the localization of CTN-1, we stained GFP-tagged CTN-1 transgenic animals with GFP antibodies and vinculin/DEB-1 antibodies that recognize the attachment plaque and dense bodies. CTN-1::GFP is localized in close proximity to, or partially colocalized with, vinculin/DEB-1 in dense bodies, but not in the attachment plaques, indicating that CTN-1 is localized near dense bodies (Figure 3A). This expression pattern of CTN-1, along with the head bending phenotype of ctn-1 mutants, prompted us to examine whether the ctn-1 mutation disrupts the integrity of the dystrophin complex. We compared the expression pattern of a component of the dystrophin complex, SGCA-1 (an α-sarcoglycan homolog) in wild-type, dys-1, slo-1 and ctn-1 animals using a GFP-tagged SGCA-1 that rescues the head bending phenotype of sgca-1 mutants [21] (Figure 3B). GFP::SGCA-1 exhibited a punctate expression pattern in the muscle membrane of wild-type and slo-1 mutant animals. By contrast, GFP puncta were greatly diminished in dys-1 and ctn-1 mutants. These results indicate that ctn-1 is critical for maintaining the dystrophin complex near the dense bodies.
We previously demonstrated that ISLO-1 interacts with STN-1 through a PDZ domain-mediated interaction, thereby linking SLO-1 to the dystrophin complex [21]. Because we failed to observe a component of the dystrophin complex in the muscle membrane of ctn-1 mutants, we examined mCherry-tagged ISLO-1 in the muscle membrane of wild-type and ctn-1 mutant animals. The punctate mCherry::ISLO-1 fluorescence was observed in wild-type muscle membranes, but was greatly reduced in ctn-1 mutant (Figure 3C). These results further strengthen the notion that CTN-1 is required for maintaining the integrity of the dystrophin complex.
Based on the genetic interaction between ctn-1 and slo-1, and the observation that the integrity of the dystrophin complex and ISLO-1 localization are disrupted in ctn-1 mutants, we hypothesized that CTN-1 regulates the localization of SLO-1 in muscle. To test this hypothesis, we examined the localization of GFP-tagged SLO-1 in muscles of wild-type, dys-1, and ctn-1 animals (Figure 4A and 4B). The punctate SLO-1::GFP expression pattern in the muscle membrane of wild-type animals was greatly diminished in the muscles of either dys-1 or ctn-1 mutant. Interestingly, the protein levels of SLO-1::GFP were not significantly different in wild-type, dys-1 and ctn-1 animals (Figure S2B), indicating that mislocalized SLO-1 does not necessarily undergo degradation. The mislocalization of SLO-1 in dys-1 mutants is consistent with the requirement of the dystrophin complex for ISLO-1 localization [21]. These results further indicate that CTN-1 stabilizes or maintains the punctate muscle expression of SLO-1::GFP in a dystrophin complex-dependent manner.
CTN-1 regulates presynaptic release by controlling the localization of SLO-1
In mammals, BK channels are found in neuronal somata, dendrites and presynaptic terminals [28], [29]. An immunoelectron microscopy study indicates that BK channels are not homogeneously distributed in neurons, but are clustered, presumably near calcium channels [30]. We addressed whether SLO-1 is evenly distributed or clustered in C. elegans neurons by examining SLO-1::GFP. Wild-type animals displayed patches of fluorescence along the ventral nerve cord or near cell bodies under high magnification (Figure 4C and 4D, Figure S2). Tissue-specific rescue experiments demonstrated that ctn-1 mediates SLO-1 function in neurons independent of the dystrophin complex (Figure 2D). Therefore, we compared neuronal SLO-1::GFP expression in dys-1 and ctn-1 mutant animals. The clustered GFP expression observed along the ventral cord of both wild-type and dys-1 mutant animals contrasted with the uniform GFP localization in ctn-1 mutants (Figure 4C and 4D). These results indicate that ctn-1 mutation disrupts the neuron-specific clustering of SLO-1::GFP independent of the dystrophin complex.
SLO-1 contributes to the repolarization of the synaptic terminal following neuronal stimulation, thereby terminating neurotransmitter release. Consequently loss-of-function slo-1 mutants are hypersensitive to the paralyzing effects of aldicarb, an acetylcholinesterase inhibitor, a phenotype indicative of enhanced acetylcholine release. Consistent with this interpretation, electrophysiological recordings from neuromuscular junctions of slo-1 loss-of-function mutants exhibit prolonged evoked synaptic responses [15], [16]. If CTN-1 regulates SLO-1 localization in motor neurons and thus slo-1 function, we would expect ctn-1 mutants to exhibit similar pharmacological and synaptic changes. Indeed, we found that ctn-1 mutants were hypersensitive to aldicarb compared to wild-type animals (Figure S3). To confirm this observation directly, we measured synaptic responses from the neuromuscular junctions of dissected wild-type and ctn-1 mutant animals engineered to express channelrhodopsin-2 in motor neurons [31] (Figure 5). Evoked synaptic responses were elicited by blue light activation of channelrhodopsin-2 and recorded from voltage-clamped post-synaptic body wall muscle cells. Consistent with our pharmacological data and localization results, recordings from ctn-1 showed prolonged evoked synaptic responses similar to those of slo-1(lf) mutants (Figure 5B and 5C). Furthermore, muscular expression of ctn-1 in ctn-1 mutant animals rescued the head-bending phenotype (Figure 2B), but did not rescue prolonged evoked synaptic responses (Figure S3B). These data strongly suggest that altered synaptic responses of ctn-1 mutants result from a neuronal defect.
In contrast to the slo-1(lf) mutants, evoked responses of slo-1(gf) mutants were short-lived (Figure 5C), and the charge integral, a measure of total ion flux during the evoked response, was significantly reduced (Figure 5E). Our genetic analyses demonstrated that the ctn-1 mutation suppresses the sluggish locomotory phenotype of slo-1(gf) mutants and disrupts SLO-1 localization (Figure 1A, Figure 4C and 4D). If this is due to loss of neuronal SLO-1(gf) channels, the ctn-1 mutation should suppress the evoked response defects of slo-1(gf). Consistent with this prediction, the decay time of the ctn-1;slo-1(gf) double mutants (t1/2 = 6.61±0.53 ms) was significantly longer than slo-1(gf) (t1/2 = 3.23±0.21 ms) (Figure 5D), and the charge integral was restored to wild-type levels (Figure 5E). Interestingly, ctn-1 mutants did not convert the decay time of slo-1(gf) evoke responses to that of slo-1(lf), indicating that residual SLO-1 function may be mediated by dispersed SLO-1 channels.
In a genetic screen to identify novel regulators of SLO-1, we found two alleles of ctn-1, a gene which encodes an α-catulin orthologue. CTN-1 mediates normal bending of the anterior body through SLO-1 localization near the dense bodies of body wall muscles. CTN-1 also maintains normal locomotory speed through SLO-1 localization within neurons. Based on our data, we propose a model for ctn-1 function in localizing SLO-1 (Figure 6). In muscles, CTN-1 interacts with the dystrophin complex. It is also possible that CTN-1 may influence the stability of another protein that directly interacts with the dystrophin complex. Loss of CTN-1 function disrupts the integrity of the dystrophin complex, thus compromising ISLO-1 and SLO-1 localization near muscle dense bodies, where L-type calcium channels are present. Disruption of SLO-1 localization is expected to uncouple local calcium increases from SLO-1-dependent outward-rectifying currents, resulting in muscle hyper-excitation. Previous studies have shown that the head bending phenotype, shared among mutants that have a defect in the dystrophin complex or its associated proteins, results from muscle hyperexcitability [17]–[19], [32]. Our data further show that this head-bending phenotype does not result from a synaptic transmission defect, but from a muscle excitation and contraction defect. In neurons, SLO-1 localization is not mediated through the dystrophin complex, suggesting that CTN-1 interacts with other proteins to localize SLO-1 to specific neuronal domains.
Why does CTN-1 use two distinct mechanisms to localize SLO-1 to subcellular regions of muscles and neurons? BK channels are functionally coupled with several different calcium channels (including voltage-gated L-type and P/Q-type calcium channels and IP3 receptors) that are localized in different subcellular regions [30], [33], [34]. Although it has not been determined whether all of these calcium channels are functionally coupled with SLO-1 in C. elegans, these calcium channels are distributed in different regions of neurons. For example, the L-type calcium channel (EGL-19) is mainly expressed in the cell body and the P/Q type calcium channel (UNC-2) is concentrated at the presynaptic terminals [35], [36]. A distinct set of proteins is perhaps required for SLO-1 channel localization near different calcium channels.
How CTN-1 interacts with the dystrophin complex in muscle remains to be determined. It has been suggested that mammalian α-catulin interacts with the hydrophobic C-terminus of dystrophin resulting from alternative splicing [37]. However, the C. elegans dys-1 gene does not encode a hydrophobic C-terminus. Thus, CTN-1 may interact with a different domain of dystrophin, or with another component of the dystrophin complex. In this regard, it is noteworthy that both mammalian dystrophin and C. elegans DYS-1 have multiple spectrin repeat domains, and that the N-terminal region of vinculin which exhibits homology to that of α-catulin (Figure S1) is known to bind the spectrin repeat domain of α-actinin [38]. By extension, we speculate that the N-terminal region of CTN-1 may bind the spectrin repeat domain of DYS-1 directly. Alternatively, the coiled-coil domain of dystrophin, which is known to interact with the coiled-coil domain of dystrobrevin [27], may potentially bind the coiled-coil domain of CTN-1. Interestingly, CTN-1 exhibits high homology to vinculin in both the N-terminal and C-terminal regions (Figure S1B). The C-terminal region of vinculin interacts with cytoskeletal molecules or regulators (F-actin, inositol phospholipids and paxillin) in focal adhesion and adherens junctions [39]. Because the C-terminal region of CTN-1 is also necessary for normal head bending, we speculate that this C-terminal region may be important for tethering the dystrophin complex to other cytoskeletal proteins.
In mammalian striated muscle, dystrophin is enriched in costameres [40] which are analogous to C. elegans dense bodies. A costamere is a subsarcolemmal protein assembly that connects Z-disks to the sarcolemma, and is considered to be a muscle-specific elaboration of the focal adhesion in which integrin and vinculin are abundant. Compromised costameres have been postulated to be an underlying cause of several different myopathies [40]. It was recently shown that ankyrin-B and -G recruit the dystrophin complex to costameres [41]. Based on overall high homology of ctn-1 to vinculin and α-catenin, we speculate that CTN-1 similarly interacts with cytoskeletal proteins in the dense bodies, and links the dystrophin complex to the dense bodies.
Another intriguing conclusion from our data is that loss of CTN-1 does not completely abolish SLO-1 function. Complete abolishment of SLO-1 function in ctn-1 mutant should alter the decay time for evoked synaptic responses of ctn-1;slo-1(gf) to the same degree as slo-1(lf) mutants, rather than to that of wild-type animals (Figure 5D). Mutants including slo-1(gf), that have defects in neural activation or membrane depolarization, are reported to cause str-2, a candidate odorant receptor gene, to be expressed in both AWC olfactory neurons whereas wild-type animals express str-2 in only one of the AWC pair [42]. We find that ctn-1 mutation does not suppress the misexpression of str-2 in both AWC neurons in slo-1(gf) mutants, suggesting that ctn-1 mutations do not completely abolish SLO-1 function (unpublished observations, HK). It is thus likely that the defect in SLO-1 localization in ctn-1 mutants makes it less responsive to local calcium nanodomains found at presynaptic terminals and dense bodies, but still able to respond to depolarization-induced global calcium increases, albeit at a lower level.
In conclusion, we have identified ctn-1, a gene encoding the C. elegans homolog of α-catulin, and demonstrated that CTN-1 mediates SLO-1 localization in muscles and neurons by dystrophin complex-dependent and -independent mechanisms, respectively. How SLO-1 is localized to certain neuronal domains will require further screening of slo-1(gf) suppressor mutants. Given that proteins affecting components of the dystrophin complex are likely to contribute to the pathogenesis of muscular dystrophy, α-catulin is a candidate causal gene for a form of muscular dystrophy in humans.
Materials and Methods
Strains and genetics
The genotypes of animals used in this study are: N2 (wild-type), CB4856, dys-1(eg33) I, stn-1(tm795) I, ctn-1(eg1167) I, ctn-1(cim6) I, slo-1(eg142) V, slo-1(ky399gf) V and sgca-1(tm1232) X. The following transgenes were used in this study: cimIs1[slo-1a::GFP, rol-6(d)] [21], cimIs5[mCherry::islo-1, ofm-1::GFP] [21], zxIs6[unc-17::chop-2(H134R)-yfp; lin-15(+)] [31], cimIs6[GFP::sgca-1, rol-6(d)], cimEx5[ctn-1, ofm-1::GFP], cimIs7[GFP::ctn-1, rol-6(d)], cimEx6[Pmyo-3ctn-1, Pmyo-3GFP, ofm-1::GFP] and cimEx7[PH20ctn-1, PH20GFP,ofm-1::GFP].
Genetic screen for suppressor mutants of slo-1(ky399)
Gain-of-function slo-1(ky399) mutants were mutagenized by exposure to 50 mM EMS (ethane methyl sulfonate) for 4 h [43]. Suppressors that suppress or ameliorate the sluggish locomotory phenotype of slo-1(ky399gf) mutants were selected from F2 progeny of the mutagenized animals. We screened approximately 5,000 haploid genome size for suppressor mutants and identified a total of 17 suppressor mutants. Genetic analysis of these suppressor mutants indicates that three of these have a second mutation in the slo-1 gene. In addition, we found that eight have mutations in genes causing head-bending phenotype (2 alleles of dyb-1, 3 alleles of stn-1 and 2 alleles of ctn-1). The remaining six mutants do not exhibit distinct locomotory phenotypes when segregated from slo-1(gf).
Genetic mapping and cloning
For genetic mapping, slo-1(ky399) mutants were outcrossed 12 times to the CB4856 strain. The resulting strain was used for SNP (single nucleotide polymorphism) mapping [44]. Alternatively, we used CB4856 as a mapping strain when mapping is based on the head-bending phenotype. For transgenic rescue, fosmid clones purchased from Gene services Inc. (Cambridge, UK) were injected into the gonad of ctn-1 mutant at 2 ng/µl along with ofm-1::GFP marker (30 ng/µl). Once we rescued the head bending phenotype of ctn-1 with a single fosmid, we rescued ctn-1 mutant with a genomic DNA fragment encompassing the entire coding sequence of ctn-1 and approximately 4 kb upstream of the putative translation site.
To verify the predicted coding sequence of ctn-1, we first performed BLAST search analysis using the genomic sequences of C. briggsae and C. remanei. This analysis suggested that the first and 12th exons are longer than predicted in WormBase (WS208), and that an additional exon (10th exon) is present. Second, we sequenced C. elegans ORF ctn-1 clone (9349620) and confirmed the 10th and the 12th exon sequences. Third, we performed sequence analysis of the DNA fragment obtained from RT-PCR with a primer set (SL1 and an internal primer) and identified the trans-splicing site which is 29 bp upstream of the newly-defined translation initiation site. Our analysis indicates that ctn-1 encodes a predicted protein with 784 amino acids (Figure S1B).
Constructs and transformation
The ctn-1 genomic DNA (approximately 4 kb upstream of the promoter and the entire coding sequence) was amplified by the expand long template PCR system (Roche Applied Science) and used directly for rescue. For PH20ctn-1 and Pmyo-3ctn-1 constructs, the neuron-specific H20 [45] or muscle-specific myo-3 promoter sequences were fused to the translation initiation site of the ctn-1 genomic DNA in frame by the overlapping extension PCR (Roche). For localization of CTN-1, we inserted the GFP sequence to the translation initiation site of ctn-1 cDNA, and then the ctn-1 promoter sequence was inserted before the GFP sequence. The resulting construct rescued the head-bending phenotype of ctn-1 mutants and was used for generating integrated transgenic animals. For GFP::sgca-1 construct, the GFP sequence was inserted in-frame right after the signal sequence of sgca-1 open-reading frame as described previously [21]. Transgenic strains were made as described [46] by injecting DNA constructs (2–10 ng/µl) along with a co-injection marker DNA (pRF4(rol-6(d)) or ofm-1::GFP) into the gonad of hermaphrodite animals at 100 ng/µl. We obtained at least 3 independent transgenic lines for rescue, and found that all lines show similar results.
Measurement of locomotory speed
To remove bacteria attached to animals, approximately fifteen age-matched (30 hr after L4 stage) hermaphrodite animals for each genotype were placed on a NGM (nematode growth medium) agar plate without bacteria for 15 min. The animals were then placed inside one of two copper rings embedded in a NGM plate. We found that age of agar plate influences the speed of animals, probably because the surface tension resulting from the liquid surrounding animals slows down movement. We used approximately one week-old plates for our assay, and compared with the speed of wild-type control animals. Video frames from two different genotypes were simultaneously acquired with a dissecting microscope equipped with Go-3 digital camera (QImaging) for 2 min with a 500 ms interval and 20 ms exposure. We measured the average speed of animals by using Track Objects from ImagePro Plus (Media Cybernetics).
Measurement of the number of eggs
The activity of egg laying muscle was measured indirectly by counting eggs retained in uteri. Single age-matched (30 hrs post-L4) animals (total 15 for each genotype) were placed in each well of a 96 well plate that contains 1% alkaline hypochlorite solution. The eggshells protect embryos from dissolution by alkaline hypochlorite. After 15 min incubation, the remaining eggs were counted in each well.
Body curvature analysis
Body curvature analysis was previously described [47]. A single animal was transferred to an agar plate and its movement was recorded at 20 frames per second. We limited image acquisition within 15 to 60 seconds after transfer, because the head bending phenotype is prominent when animals are stimulated to move forward rapidly. A custom-written software automatically recognizes the animal and assigns thirteen points spaced equally from the tip of nose to the tail along the midline of the body, and produces the pixel coordinates of thirteen points. First supplementary angles were calculated from the coordinates of the first three points with MATLAB software. First angle data were obtained when the head swing of an animal reached the maximal extension to the dorsoventral side.
Western blot analysis
Mixed stage worms were washed and collected in M9 buffer. Equal volume of 2× Laemmli sample buffer was added to the worm pellets. The resulting worm suspension was heated at 90 °C for 10 min, centrifuged at 20,000 g for 10 min, and then immediately loaded on 7.5% SDS-PAGE gel. The Western blot analysis was performed using anti-GFP antibody (Clontech, JL-8) and anti-α-tubulin antibody (Developmental hybridoma bank, AA4.3).
Microscopy imaging
Fixation and immunostaining procedures are previously described [21]. Fluorescence images were observed under a Zeiss Axio Observer microscope with 40× objective (water-immersion, NA: 1.2) or an Olympus Fluoview 300 confocal microscope with a 60× objective (oil-immersion, NA: 1.4) or 100× objective (oil-immersion, NA: 1.4). We typically observed more than 50 animals for each genotype. Images for quantification were acquired under an identical exposure time, gains and pinhole diameter. The intensity of puncta from acquired images was analyzed using linescan (Metamorph, Molecular Devices) and presented as values obtained by subtracting background levels from the peak grey levels of puncta.
Electrophysiological recordings
Electrophysiological methods were as previously described [48]. Briefly, animals raised on 80 µM retinal plates, were immobilized with cyanoacrylic glue and a lateral cuticle incision was made to expose the ventral medial body wall muscles. Muscle recordings were made in the whole-cell voltage-clamp configuration (holding potential −60 mV) using an EPC-10 patch-clamp amplifier and digitized at 2.9 kHz. The extracellular solution consisted of (in mM): NaCl 150; KCl 5; CaCl2 5; MgCl2 4, glucose 10; sucrose 5; HEPES 15 (pH 7.3, ∼340mOsm). The patch pipette was filled with (in mM): KCl 120; KOH 20; MgCl2 4; (N-tris[Hydroxymethyl] methyl-2-aminoethane-sulfonic acid) 5; CaCl2 0.25; Na2ATP 4; sucrose 36; EGTA 5 (pH 7.2, ∼315mOsm). All of the animals carry a transgene (zxIs6) that expresses channelrhodopsin-2 under the control of the cholinergic motor neuron (unc-17)-specific promoter. Evoked currents were recorded in a body-wall muscle after eliciting neurotransmitter release by a 10 ms illumination using a 470 nm LED (Thor labs) triggered with a TTL pulse from the EPC10 pulse generator [31]. Evoked post-synaptic responses were acquired using Pulse software (HEKA) run on a Dell computer. Subsequent analysis and graphing was performed using Pulsefit (HEKA), Mini analysis (Synaptosoft Inc) and Igor Pro (Wavemetrics). The data were analyzed with one-way ANOVA followed by Dunnett's multiple comparison.
Supporting Information
1. LaiHC
2006 The distribution and targeting of neuronal voltage-gated ion channels. Nat Rev Neurosci 7 548 562
2. LevitanIB
2006 Signaling protein complexes associated with neuronal ion channels. Nat Neurosci 9 305 310
3. SalkoffL
2006 High-conductance potassium channels of the SLO family. Nat Rev Neurosci 7 921 931
4. FaklerB
2008 Control of K(Ca) channels by calcium nano/microdomains. Neuron 59 873 881
5. BrennerR
2000 Vasoregulation by the beta1 subunit of the calcium-activated potassium channel. Nature 407 870 876
6. LovellPV
2001 Pituitary control of BK potassium channel function and intrinsic firing properties of adrenal chromaffin cells. J Neurosci 21 3429 3442
7. WernerME
2005 Erectile dysfunction in mice lacking the large-conductance calcium-activated potassium (BK) channel. J Physiol 567 545 556
8. DuW
2005 Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder. Nat Genet 37 733 738
9. ShrutiS
2008 A seizure-induced gain-of-function in BK channels is associated with elevated firing activity in neocortical pyramidal neurons. Neurobiol Dis 30 323 330
10. SchopperleWM
1998 Slob, a novel protein that interacts with the Slowpoke calcium-dependent potassium channel. Neuron 20 565 573
11. LuR
2006 MaxiK channel partners: physiological impact. J Physiol 570 65 72
12. TianL
2006 A noncanonical SH3 domain binding motif links BK channels to the actin cytoskeleton via the SH3 adapter cortactin. FASEB J 20 2588 2590
13. KimEY
2007 Interactions with filamin A stimulate surface expression of large-conductance Ca2+-activated K+ channels in the absence of direct actin binding. Mol Pharmacol 72 622 630
14. ParkSM
2004 Direct interaction between BKCa potassium channel and microtubule-associated protein 1A. FEBS Lett 570 143 148
15. WangZW
2001 SLO-1 potassium channels control quantal content of neurotransmitter release at the C. elegans neuromuscular junction. Neuron 32 867 881
16. DaviesAG
2003 A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell 115 655 666
17. Carre-PierratM
2006 The SLO-1 BK channel of Caenorhabditis elegans is critical for muscle function and is involved in dystrophin-dependent muscle dystrophy. J Mol Biol 358 387 395
18. KimH
2004 SNF-6 is an acetylcholine transporter interacting with the dystrophin complex in Caenorhabditis elegans. Nature 430 891 896
19. BessouC
1998 Mutations in the Caenorhabditis elegans dystrophin-like gene dys-1 lead to hyperactivity and suggest a link with cholinergic transmission. Neurogenetics 2 61 72
20. GieselerK
1999 Dystrobrevin- and dystrophin-like mutants display similar phenotypes in the nematode Caenorhabditis elegans. Neurogenetics 2 87 90
21. KimH
2009 The dystrophin complex controls bk channel localization and muscle activity in Caenorhabditis elegans. PLoS Genet 5 e1000780 doi:10.1371/journal.pgen.1000780
22. AldertonJM
2000 Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes. J Biol Chem 275 9452 9460
23. JanssensB
van RoyF
1999 Human alpha-catulin, a novel alpha-catenin-like molecule with conserved genomic structure, but deviating alternative splicing. Biochim Biophys Acta 1447 341 347
24. BarsteadRJ
1989 The basal component of the nematode dense-body is vinculin. J Biol Chem 264 10177 10185
25. CostaM
1998 A putative catenin-cadherin system mediates morphogenesis of the Caenorhabditis elegans embryo. J Cell Biol 141 297 308
26. WiesnerC
2008 Alpha-catulin, a Rho signalling component, can regulate NF-kappaB through binding to IKK-beta, and confers resistance to apoptosis. Oncogene 27 2159 2169
27. Sadoulet-PuccioHM
1997 Dystrobrevin and dystrophin: an interaction through coiled-coil motifs. Proc Natl Acad Sci U S A 94 12413 12418
28. HuH
2001 Presynaptic Ca2+-activated K+ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release. J Neurosci 21 9585 9597
29. SailerCA
2006 Immunolocalization of BK channels in hippocampal pyramidal neurons. Eur J Neurosci 24 442 454
30. KaufmannWA
2009 Large-conductance calcium-activated potassium channels in purkinje cell plasma membranes are clustered at sites of hypolemmal microdomains. J Comp Neurol 515 215 230
31. LiewaldJF
2008 Optogenetic analysis of synaptic function. Nat Methods 5 895 902
32. GieselerK
2001 Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans. J Mol Biol 307 107 117
33. EdgertonJR
2003 Distinct contributions of small and large conductance Ca2+-activated K+ channels to rat Purkinje neuron function. J Physiol 548 53 69
34. PrakriyaM
1999 BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. J Neurophysiol 81 2267 2278
35. GrachevaEO
2008 Direct interactions between C. elegans RAB-3 and Rim provide a mechanism to target vesicles to the presynaptic density. Neurosci Lett 444 137 142
36. SahekiY
2009 Presynaptic CaV2 calcium channel traffic requires CALF-1 and the alpha(2)delta subunit UNC-36. Nat Neurosci 12 1257 1265
37. BiggarWD
2002 Duchenne muscular dystrophy: current knowledge, treatment, and future prospects. Clin Orthop Relat Res 88 106
38. BoisPR
2005 Structural dynamics of alpha-actinin-vinculin interactions. Mol Cell Biol 25 6112 6122
39. ZieglerWH
2006 The structure and regulation of vinculin. Trends Cell Biol 16 453 460
40. ErvastiJM
2003 Costameres: the Achilles' heel of Herculean muscle. J Biol Chem 278 13591 13594
41. AyalonG
2008 An ankyrin-based mechanism for functional organization of dystrophin and dystroglycan. Cell 135 1189 1200
42. TroemelER
1999 Lateral signaling mediated by axon contact and calcium entry regulates asymmetric odorant receptor expression in C. elegans. Cell 99 387 398
43. BrennerS
1974 The genetics of Caenorhabditis elegans. Genetics 77 71 94
44. WicksSR
2001 Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nat Genet 28 160 164
45. ShioiG
2001 Mutations affecting nerve attachment of Caenorhabditis elegans. Genetics 157 1611 1622
46. MelloCC
1991 Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. Embo J 10 3959 3970
47. Pierce-ShimomuraJT
2008 Genetic analysis of crawling and swimming locomotory patterns in C. elegans. Proc Natl Acad Sci U S A 105 20982 20987
48. RichmondJE
2006 Electrophysiological recordings from the neuromuscular junction of C. elegans. WormBook 1 8
ŠtítkyGenetika Reprodukční medicína
Článek vyšel v časopise
PLOS Genetics
2010 Číslo 8
Nejčtenější v tomto čísle
Tomuto tématu se dále věnují…
- Common Inherited Variation in Mitochondrial Genes Is Not Enriched for Associations with Type 2 Diabetes or Related Glycemic Traits
- Disease-Associated Mutations That Alter the RNA Structural Ensemble
- Transcriptional Regulation by CHIP/LDB Complexes
- Genetic Analysis of Baker's Yeast Msh4-Msh5 Reveals a Threshold Crossover Level for Meiotic Viability
Zvyšte si kvalifikaci online z pohodlí domova
Všechny kurzy


text:
Tough first quarter for collective investments
The local collective investment schemes industry reported a 3.2% drop in assets under management for the first quarter of this year as a result of difficult market conditions and subdued net inflows on the back of bruised investor sentiment.
According to industry statistics for the quarter and year ended March 2018, released this week by the Association for Savings and Investment South Africa (Asisa), assets under management in unit trust funds and exchange traded funds dropped to R2.18 trillion over the first quarter of this year from R2.25 trillion at the end of December 2017.
Sunette Mulder, senior policy adviser at Asisa, says not only did the JSE All Share Index report a drop of 6% in the first quarter, contributing to lower assets under management, but the industry attracted quarterly net inflows of only R3 billion for the quarter.
"The collective investments industry last reported net inflows in the single digits in the third quarter of 2014 when net inflows came in at only R2 billion.
"This was the same quarter that marked the start of the African Bank saga, which unnerved investors.
"It is probably no coincidence that investors were spooked in the first quarter of this year as the Steinhoff debacle continued to unfold."
Mulder says despite the anaemic net inflows for the first quarter of this year, the industry attracted healthy net inflows of R107bn for the 12 months to the end of March 2018.
She says that year-on-year the local collective investments industry has delivered a steady growth in assets. At the end of March assets stood at R2.18 trillion, compared with R2.07 trillion at the end of the first quarter of 2017.
In the quarter to the end of March, net outflows from South African Interest Bearing Variable Term portfolios were R4.4bn and those from money market portfolios were R9bn.
SA Multi Asset High Equity portfolios, on the other hand, attracted strong net inflows of R9.7bn.
SA General Equity portfolios were also popular with investors, attracting the second-highest net inflows for the quarter of R4.4bn.
Mulder says these patterns indicate that resilient investors in all likelihood used the market turmoil as a buying opportunity.
At the end of March, investors had a choice of 1 584 funds, an increase of 62 from 2017.

subset: commoncrawl


text:
JACKSON — Surgeons at the University of Mississippi Medical Center recently transplanted a pancreas and kidney into a 49-year-old man, marking the first such event for the state and final piece of UMMC's abdominal transplant line.
"Our goal has been to build a complete and high-functioning abdominal transplant program," said Dr. Christopher Anderson, UMMC associate professor of transplant surgery and division chief of transplant and hepatobiliary surgery. "The pancreas transplant program represents that last piece of the abdominal program and is the culmination of a lot of hard work from our transplant team and the entire institution. University Transplant can now serve the state's kidney, liver and pancreas needs while keeping patients close to home."
The patient has suffered from type 1 diabetes since childhood and subsequently developed kidney failure. He is in good condition yesterday and is expected to return home this week or early next.
Dr. Mark Earl, assistant professor of transplant surgery, performed the operation, assisted by Anderson and Dr. Andrew Gaugler, a chief surgery resident.
During a 24-hour span that included the milestone pancreas operation, UMMC teams transplanted five organs into four Mississippians. Other patients received a heart, kidney and liver.
"It's remarkable how far organ transplant — at this very institution — has come, from Dr. James Hardy transplanting the first human lung just over 50 years ago to these recent outstanding achievements," said Dr. Marc Mitchell, professor and chair of the Department of Surgery.
In addition to Gaugler, two general surgery residents, a third-year medical student, a critical-care fellow and a student nurse anesthetist observed or participated in the operation.
The transplant program began administrative and logistical preparations about a year ago for the first pancreas transplant. In late September, University Transplant received pancreas approval from the United Network for Organ Sharing, the nation's non-profit oversight organization for transplantation.
… we'd like to ask for your support. More people are reading the Mississippi Business Journal than ever before, but advertising revenues for all conventional media are falling fast. Unlike many, we do not use a pay wall, because we want to continue providing Mississippi's most comprehensive business news each and every day. But that takes time, money and hard work. We do it because it is important to us … and equally important to you, if you value the flow of trustworthy news and information which have always kept America strong and free for more than 200 years.
If those who read our content will help fund it, we can continue to bring you the very best in news and information. Please consider joining us as a valued member, or if you prefer, make a one-time contribution.Click for more info


text:
It is a testament to the quality of St Andrews' nightlife that we require any sort of transportation (beyond mere walking) to get us home after a night out. Even the most remote student flat is reachable within a half hour by foot. Despite this, however, a heady night out often leaves us in such a state that some kinetic assistance is required, frequently in the form of a taxi. More understandably, students who struggle to cart their groceries home or who dislike the idea of walking in the rain may also opt for a cab ride as they undertake the lengthy sojourn from Tesco to David Russell Apartments.
Despite the constant sight of students piling into cabs following an evening at the Vic or throughout a particularly windy afternoon, few people would admit to enjoying taxi rides. No one likes attempting to issue directions to their house in the Badlands, scrambling for change to pay the overpriced fare, or struggling to reconcile the driver's taste in music with their own. The litany of reasons to dislike cabs goes on. As a result, four St Andreans have taken it upon themselves to offer us an alternative: rickshaws.
Fourth-year student James Webb, inspired by his time spent driving a rickshaw in India, united with third- year student Charlie Buchanan-Smith and fellow fourth years Abraham McCauley and Simon Pepper to establish Chariots of Hire, a local business like no other. Based upon prior rickshaw escapades and a proclivity for cycling, the foursome launched their brand in the first semester of the 2015-2016 school year. Meticulous research led the team to Aberdeen, where they purchased three state-of-the-art rickshaws, equipped with WiFi and speakers. The goal being to support one of the main tenets of the business: to entertain. As Pepper explained: "Taxi rides are a run-of-the-mill occurrence. We offer people a more holistic experience."
Considering their entrepreneurial spirits, one may expect the business owners to be students of management or economics. Instead they study courses as varied as chemistry, French, geology and biology. They explained to The Saint that this diversity aids them in the social aspect of their business. The ability to chat with guests about such an extensive list of subjects further improves the rickshawing affair. Pepper recalls conversing in French with a pair of customers one evening, surely making the ride home one of their more memorable experiences using private transportation.
In their debut appearance, Chariots of Hire stationed their rickshaws at the Old Course for GIG's glamorous For Your Eyes Only casino night. The black tie-clad guests were intrigued by the prospect of such a unique form of transport and gamely clamoured into the vehicles. Cycling in its own right is no easy task, but combining it with a heavy rickshaw and poor weather conditions necessitates a certain amount of fitness that the four boys possess in spades. The team returned to rickshaw again at Masquerade Ball and later at the Kate Kennedy Club's annual toga party, in both instances earning rave reviews from their customers and ensuring their viability in the personal transportation industry.
Another notable feature of Chariots of Hire is their fare – or rather, the lack thereof. Rather than billing clients, the boys allow their customers to choose their rate of pay based on the quality of the ride. By allowing people to pay however much or little they want, the service is guaranteed to be top-notch as the boys seek to deliver a genuine experience.
With their wealth of life experience and assortment of interests, it is no wonder that the foursome is so keen to provide a positive, social experience for its customers. The rickshaws also allow customers to play music on request (McCauley fondly reminisces about one ride spent listening to Sorry by Justin Bieber on repeat), as the free WiFi permits their clients to use YouTube or Spotify en route. Most importantly, the customer's safety is ensured should a potential emergency situation arise. Having undergone council-sanctioned training to obtain their street trader's license in accordance to Scottish law, all four members of the startup are able to adequately cater to any needs their guests may have, large and small.
The rickshaws themselves, built in Germany and outfitted to every modern convenience, are impressive specimens in their own right. Far from the rickety contraptions that one might picture, the vehicles rival cars in their convenience factor. With the aid of indicators, lights, brakes and seat-belts, the rickshaws can obey all the expected rules of traffic as they soar to speeds of 40 miles per hour. Such rules were kept in mind during a Vic-sponsored rickshaw race from the Vic to Janettas Gelateria, which saw the rickshaws pushed to their limit as they skilfully navigated their way along South Street. Formally inspected and council approved, these eco-friendly vehicles are maintained to the highest standards and kept in a secure location when not in use.
Chariots of Hire seeks not only to offer an alternative method of transportation, but also to revolutionise the local travel industry. McCauley says he hopes to offer tours of St Andrews, aided by the knowledge accumulated during his four years as a student here spent traversing the town. This way, tourists and residents alike can witness the hidden gems that they may previously have overlooked, all from the comfort of a rickshaw and with the aid of their knowledgeable drivers. The group's humanitarian ethos is demonstrated in its involvement with Sitara, one of the many student-run charity fashion shows on campus. Chariots of Hire has offered the free use of a rickshaw to be used for the event's photobooth.
Although their primary business during the school year comes from students, the boys hope to increase their potential clientele during the summer golf season in St Andrews. Chariots of Hire has been in talks with The New Inn regarding a potential partnership, with the goal of contributing a unique brand of tour to a market suffused with foot traffic. Having spent years learning the ins and outs of our town, the drivers now possess enough talent and street smarts to take the road less travelled and to present St Andrews not only as a golfing hotspot, but as our home.
St Andrews is filled with the ambitious and the business-minded, as evidenced by the sheer amount of charity events, student blogs and budding organisations that we see advertised on Facebook each day. However, Chariots of Hire stands out amongst this crowd for its intrepid premise and creative payment model. The team describes their startup as an opportunity to demonstrate that while internships and grad schemes may be beneficial, we all have the potential to start our careers earlier and on our own time. As equal partners and their own bosses, the four boys set their own hours and work for as long as they desire.
Webb, McCauley, Pepper and Buchanan-Smith all come from different walks of life, but neither their passion for the peculiar, nor their passion for Chariots of Hire can be denied. It may be in early stages yet, but their business does appear to be gaining traction as they begin the process of obtaining a logo and arranging events on a more regular basis. The beginning of the new year and the promise of second semester's full social calendar have provided the momentum they will need to foster their growing business, taking it from a niche market to a mainstream resource.
Even if one prefers walking or taxiing home after a night out in town, the idea of taking a rickshaw, even just once, should not be so quickly dismissed. It could be the ride of your life.
To learn more about Chariots of Hire, check out their Facebook page.


text:
The former leader of Newcastle City Council is to help decide how £2bn in taxpayers' cash is distributed across the country.
Lord Shipley, a Liberal Democrat peer who led the authority from 2006 to 2010, will join Tory peer Lord Heseltine to oversee Local Growth Deals, a flagship Government scheme to create jobs in the private sector.
But the appointment could mean he finds himself thrust into a row between local authorities in the North East, which are at loggerheads over plans for a new combined authority.
Seven local councils in the region had drawn up plans to merge some of their functions and create a new body, following in the footsteps of councils in the Greater Manchester area which have similar arrangements. The move was prompted partly by Government warnings that the authorities would be far more likely to receive funding if they worked together.
But Sunderland Council has now written to the Government asking it to delay the plans, while South Tyneside is believed to have given only qualified approval.
Lord Shipley was appointed by Nick Clegg, the Deputy Prime Minster, and Greg Clark, the Minister for the Cities. Mr Clark is also the Minister who has been pushing North East councils to form a joint authority.
Along with Lord Heseltine, he will advise Ministers in the merits of bids for funding drawn up Local Enterprise Partnerships. These are agencies run jointly by councils and the business community.
The cash will come from a £2bn Local Growth Fund created by the Government to support the creation of private sector jobs.
The fund is designed to be a "single pot" of money for replacing an array of different schemes - including "city deals" - which areas applied to in the past.
Local Enterprise Partnerships have been told they must demonstrate how their plans will use local authority assets and creating a long-term strategy to put growth at the heart of everything councils do.
Lord Heseltine and Lord Shipley will advise ministers on individual proposals brought forward by the LEPs, and will challenge the LEPs themselves to prod uce the best possible proposals.
Lord Shipley said: "Following on from the success of the City Deals, I am delighted to be involved in the next exciting chapter of the Government's decentralisation agenda."
Mr Clegg said: "This is another step forward in handing more power from Whitehall to local people. I am delighted that Lord Heseltine and Lord Shipley have agreed to bring their expertise and enthusiasm to bear on Local Growth Deals."
And Mr Clark said: "As we work towards the completion of the second phase of City Deals, these local Growth Deals will take the Government vision for local growth to the next level, and the huge experience of Lord Heseltine and Lord Shipley will be vital to that.
"As the economy turns the corner, Local Growth Deals will ensure not only that every part of the country can take advantage of the recovery, but has the opportunity to be right there in the driving seat."


text:
Omega oil blends are an excellent source of monounsaturated and polyunsaturated fatty acids (Omega-3 & 6 respectively). These healthy fats have been seen to exert anti-inflammatory effects on our body, resulting in better recovery after training, reduced muscle and joint inflammation, and reduced irritation to the lining of our arteries supporting heart health!
Add 5ml to your meals and/or protein shake 3x a day!
Try Optimum Health Omega Oil Blend for an ideal ratio of Omega 3, 6 & 9 fatty acids!


text:
Hegemony is not only an exceptional addition to the Samael discography alongside albums such as "Passage" and "Eternal" the album will also mark the band's 30th anniversary!
The band states: The creative process that leads to the completion of « Hegemony » took over 4 years. Throughout that time we lived with and saw each song growing into what they finally became.
« Hegemony » is an album that acknowledges the world we're living in, and emphasized on the effort to take control of that world. It's all about change and evolution. It's a strong statement, a testament of our time and a call for revolution."
The album will be out on October 13th via Napalm records.
Orders: https://shop.napalmrecords.com/samael
- Hegemony
- Samael
- Angel of Wrath
- Rite of Renewal
- Red Planet
- Black Supremacy
- Murder or Suicide
- This World
- Against All Enemies
- Land of the Living
- Dictate of Transparency
- Helter Skelter
- Storm of Fire (Bonus Track)
Support your favorite magazine by donation to cover some webhosting expenses - that will be more than appreciated!


text:
This month, I have several looming deadlines. To help me de-stress, I stitched up two mini Wholecloth quilted Mandalas. Some might think this an odd choice for stress relief, but I really enjoy the Zen and meditative nature of these mandala designs.
Ok--enough procrastinating for me...back to tackling my quilting deadlines!
The first mini mandala took a little over 2 hours, but almost half of that time was spent ripping out the background stitching. Initially, I used the same Medium Butter thread from the center mandala design and went with my comfort zone of Zig Zag Meander for the background. Almost a 3rd of the way through quilting the background I ran out of bobbin thread. While initially frustrating, this proved to be a good thing as it gave me time to reevaluate and realize that the background quilting was all wrong for this design! So I spent almost 45 minutes ripping out all the stitching. Again, this gave me time to reevaluate what was needed in the background and come up with a much better design solution involving a contrasting thread color and a much better motif that just radiates out from the mandala.
|Celestial Mandala, Finishes 12" x 12"
I had so much fun creating this mini mandala that the next morning, I set out to quilt a companion quilt in under one hour using a different set of thread colors. As I was using the same basic design, I found more efficient pathways.
|Ocean Waves Mandala, Finishes 12" x 12"
Technically, this took a little over one hour to quilt, but I took a few extra minutes to record a quick Free Motion Quilting video demo as I stitched out the background flame quilting-so you too could experience some Zen quilting!!
|Zen Quilting
Aurifil 50-weight threads were used for all the quilting:
- Celestial Mandala: Medium Butter (2130) and Storm at Sea Variegated (4655)
- Ocean Waves: Bright Orange (1133) and Creme de Menthe Variegated (4662)
I love how the variegated threads add wonderful contrast, color & texture to the background.
|Aurifil Adds AMAZING Contrast, Color & Texture!!


text:
Brendan Gleeson on Netflix
This is a regularly updated list with movies, series and documentaries with Brendan Gleeson on Netflix. The best rated item with Brendan Gleeson on Netflix is "Stonehearst Asylum" and appeared on screen in 2014.
About Brendan Gleeson
Brendan Gleeson is an Irish actor. His best-known films include the Harry Potter films, Braveheart, Gangs of New York, 28 Days Later, and the role of Michael Collins in The Treaty. He won an Emmy award in 2009 for his portrayal of Winston Churchill in the film Into the Storm. Gleeson was born in Dublin, Ireland and has described himself as having been an avid reader as a child. After leaving school for a short while, he worked for two years in an office with a health board. He then graduated from University College Dublin and went on to Drama School in London at the Royal Academy of Dramatic Art (RADA). Just after completing the 3 Year Acting Drama Course at RADA, Gleeson built a very respectable CV at the Royal National Theatre, Shakespeare's Globe Theatre, and the Royal Shakespeare Company in Stratford-upon-Avon. Gleeson became known for his tremedous stage presence, particularly while performing in such works of William Shakespeare as King Lear, Richard III, and Hamlet. Gleeson started his film career at the age of 34. He first came to prominence in Ireland for his role as Michael Collins in The Treaty a television film broadcast on RTÉ One, and for which he won a Jacob's Award in 1992. Gleeson has subsequently acted in more than 60 films including Braveheart, I Went Down, Michael Collins, Gangs of New York, Cold Mountain, 28 Days Later, Troy, Kingdom of Heaven, Lake Placid, Artificial Intelligence: AI and The Village. He won critical acclaim for his performance as Irish gangster Martin Cahill in John Boorman's 1998 film The General. Gleeson lives in Malahide, County Dublin. He is a talented fiddle player, with an interest in Irish folklore. He can be found from time to time playing at traditional music seisiúins in Hughes' pub in Chancery Street in Dublin 7. His brother, Barry Gleeson, is a well-known singer in An Góilín traditional singers' club, and his son Domhnall is an actor. Gleeson is known for his love of football and recounts tales of attempting to get a clear radio reception so he could listen to how his team, Aston Villa, were faring on a Saturday.
Below you find an overview of all movies and series with Brendan Gleeson on Netflix. We found 35 titles with Brendan Gleeson on Netflix.
Titles with Brendan Gleeson on Netflix
Stonehearst Asylum
The Grand Seduction
The Smurfs 2
Mission: Impossible II
The Secret of Kells
The Raven
The General
Assassin's creed
Edge of Tomorrow
Kingdom of Heaven
The Tiger's Tail
In the Heart of the Sea
Harry Potter and the Order of the Phoenix
Harry Potter and the Deathly Hallows: Part I
The Cup
A.I. Artificial Intelligence
Harry Potter and the Goblet of Fire
Trespass Against Us
Live By Night
Trespass Against Us
The Tailor of Panama
Safe House
Perrier's Bounty
28 Days Later
Lake Placid
Dark Blue
Alone in Berlin
Song of the Sea
Paddington 2
Suffragette (Italian Version)
The Ballad of Buster Scruggs


text:
The air conditioned fitness studio benefits from state of the art Life Fitness equipment, with an excellent mix of both cardiovascular and resistance equipment.
Our Cardiovascular Equipment will enable you to reach your individual goals, with our multiple programmes we're sure to have the workout for you.
Our state-of-the-art Life Fitness equipment has built in TV, FM radio and Real-time Heart Rate Monitoring all using the LCD touchscreen.
- 4 x Treadmills
- 2 x Elliptical Trainers
- 2 x Recumbant Bikes
- 1 x Upright Cycle
- 2 x Concept2 Rowers
Resistance Equipment
Our extensive Resistance Equipment offers you all you need for a full body workout. State-of-the-art machines allow you to reach your goals and beyond.
Below is a list of our Resistance Equipment:-
- Ad/Abductor Machine
- Pec Dec/Rear Fly
- Back Extension
- Ab Machine
- Lat Pull Down
- Chest Press
- Shoulder Press
- Seated Leg Extension
- Seated Leg Curl
- Seated Leg Press
- Cable Column


text:
Friday, November 6, 2020
Registration begins at 7:45 a.m.
Breakfast begins at 8:30 a.m.
Once a year, the Association of Fundraising Professionals Central Florida Chapter (AFP) is proud to
recognize and honor many heroes who make dreams come true through a community-wide event called National
Philanthropy Day. This year's Label
National Philanthropy Day Awards will take
place on Friday, November 6, 2020 at the prestigious
Ritz-Carlton Orlando, Grande Lakes.
National Philanthropy Day acknowledges the entire spectrum of services that the non-profit and civic
and service sectors provide, as well as the profound impact that philanthropy has on the fabric of
society. This annual event is also a fundraiser for the Central Florida Chapter of the AFP to provide
scholarships and educational opportunities to further individual members' professional development.


text:
Gossip, Deception and Custom Essay Service
This is but one of the most difficult tasks faced by most pupils. Only the best essay creating agency since you can not be in a position isn't needed by you will not necessarily imply. On-line article writing services give excellent newspapers .
There's no more easy way to receive your academic documents completed. Far as you'd like custom article creating low rates, make certain you're not falling operate. There are a lot of gains about the choice to obtain custom essay newspapers with all the struggle that leads to overdue essays and poor excellent.
Be sure your composition is exceptional. Essays create a great deal of issues for several college students.
You may even incorporate your personal thoughts, science news, proverbs and I'd like to reveal your essay! It truly is by no means ever straightforward to compose an essay from scratch each day or 2. With these supplies from the web, picking out top essay writing organizations can be not as simple as it may possibly show cheap essay writer up.
An editor gives you a rating to your paper. You've got to realize the quality of your informative article will subject an entire ton, without a writer is likely to deliver top-notch materials for peanuts. He'll start looking the important information for each and they understand that the issue.
The rising amount of essay producing services will be overwhelming. Ordinarily, best article writing services present a group of editors and writers that are on paper any form of academic missions expert. Our crew of professional custom-made article asked where can we find professional essay authors.
One of the best causes of the pupils to have essays from composing service that is online is they want to procure results. There certainly really are a range of reasons why pupils buy an essay online. In the modern day https://essayhelp-now.com/ they are lucky to get composition producing companies available online and writing help.
The author will even have to comprehend that which books are employed from the class and also everything the class material is. You will have to earn your choice in the way,, but although there's a great deal of creating essays companies all over the Web . It's very easy to go into the internet and decide on the web site which gives the professional hints of article writing although not simply the a variety of composition services but.
Additionally, lots of pupils are involved that some body can notice they will have used this service. Since you see, you will find a great deal of factors to purchase expert assistance by means of your custom writing. When you take a look at that time that you have to possess, companies is merely a indication of great expert essay http://www.bio.fsu.edu/courses/pcb4673/answering_essay_questions.html.
RESULT-ORIENTED CUSTOM ESSAY AND THE MOST EFFECTIVE support entire, our support is a writing service which puts its ESL clients over and beforehand the rest . Both services have positive aspects and pitfalls. On account of a single particular of those finest writers group, our clients're delighted with every and every composing we deliver.
You may even restrain the process of compose essays . Each mission has been made particularly for every buyer. Thus after you order a customized essay service in the you can be certain it will be furnished by an expert with astounding expertise and ideal grasp of the topic on your issue and all of your requirements.
If your refund petition is all about the characteristic of work done the company is going to go through the assignment that is completed. Our approach is straightforward and forthright as we don't ever wish to gratify our clients into finding out the facets of producing so forth and in order. It is operate on a sliding-scale that's dependant on a few important aspects.
It is also feasible to opt for the au thor besides being the capacity to customize the sequence. Together with the progress in education degrees, the documents might be more complicated. In experienced authors may face issues, not equipped to present their critical quality's assignment.
Whenever you conduct into the following informative article assist, you should take a peek at an individual's purchase price's caliber and selling cost. Moreover, you're getting to need a newspaper urgently that you set is processed that you need to be certain that the buy price. As soon as a order is completed, it's sent to the quality assurance section.


text:
Canadian Cannabis Clinics set to open in Thunder Bay, Ontario
Canadian Cannabis Clinics, a medical marijuana clinic will open in Thunder Bay, Ont., Thursday.
The company has been expanding across the country and trying to set up in areas they consider under serviced, said Ronan Levy, one of the executive directors.
"Everywhere in Canada you have a largely aging population and with people getting older, those are the people who often find the most benefit from medical cannabis because they tend to have the most ailments that seem to benefit from medical cannabis and Thunder Bay fits those parameters as well," Levy said.
The new facility on Arthur Street in Thunder Bay is the 20th clinic to be opened by the company.


text:
Disjoint disks
For college and university level advanced Mathematics
Post Reply
First unread post • 12 posts
Re: Disjoint disks
What if we have a disk centered at $(0,0)$ of radius $r$, and another one centered at $(\epsilon,2r+\epsilon)$ for sufficiently small $\epsilon$, and so on? Then we can have a disk with centre at every possible real distance along the $x$ axis. This is what I meant previously.
"Everything should be made as simple as possible, but not simpler." - Albert Einstein
Re: Disjoint disks
sufficiently small মানে বুঝলাম না। যাই হোক, যদি মনে হয় আসলেই real axis এ সবগুলারে বসানো যাবে, বসানো হোক, who cares ...
Have you read the Forum Rules and Guidelines?
Have you read the Forum Rules and Guidelines?
Post Reply


text:
A powerful core value you can add to your life is "give your family your best and everyone else the rest."
The people you love and that love you deserve your best.
Entrepreneurs and go-getters are always saying what they do is for their family but is it really?
How can something you do benefit your family is you are not present both mentally and physically to enjoy/experience with them?
Are you giving your family your best?
In this "Get Your Mind Right Podcast" episode, Ryan Stewman talks about why and how you need to always give your family your best.
Give Your Family Your Best
iTunes – https://apple.co/2pLTdNr
YouTube – https://bit.ly/2GfVw21
Libsyn – https://bit.ly/2pK07Ds
Set a reminder with your alarm to listen to it daily as soon as you get out of bed. When you get out of bed, let me in your head.


text:
|Home Obituaries / Condolences Staff Contacts / Maps
Lucille K. (Carlson) Robley
Jan. 17, 1922 - Apr. 1, 2019
Sutton resident Lucille K. (Carlson) Robley, 97, passed away Monday, April 1, 2019, at the Sutton Community Home in Sutton, NE.
Funeral services will be conducted at 2:00 p.m., Saturday, April 6, 2019, at Hope Reformed Church in Sutton, NE with Rev. Scott Henry officiating. Interment will follow at the Sutton Cemetery. Visitation will be Friday, from 1 p.m. 8 p.m., with the family greeting friends from 5 p.m. 7 p.m., at Sutton Memorial Chapel in Sutton, NE.
Memorial Contributions may be directed to Hope Reformed Church in Sutton, NE.
Lucille was born January 17, 1922, to Adam D. and Henrietta F. (Griess) Bauer in Arriba, CO. They later moved to a farm by Lushton where she and her siblings were raised. She went to country school, then to Sutton High School and graduated with the class of 1940. She married Everett Carlson on April 8, 1944, in Lincoln, NE. They lived in Lincoln and Omaha where they were both employed at the Glenn L. Martin Bomber Plant. She was a Rosie the Riveter and helped assemble the B-29 Superfortress planes.
They later bought a farm southwest of Sutton where their three children were raised. She started working at the Sutton Community Home in Sutton when it first opened in 1964 and worked until she retired in 1995.
Everett passed away from injuries sustained in a car accident on icy roads on November 22, 1978. She married Duayne W. Robley on October 27, 1981. She lived in her home till shortly before her death.
Lucille loved the Lord, her children, her grandchildren, and her great-grandchildren--the latest being born just one week before she passed away. She was also expecting another great-grandchild in July, and her first great-great-grandchild in May. She also had a love for all animals.
She leaves to mourn her passing her husband, Duayne at the Vets Home in Kearney, NE; son, Dennis (Charlotte) Carlson of Hastings; daughters, Connie (Rollie) Johnson and JoAnn (Mark) Brown both of Sutton; 7 grandchildren; 20 great-grandchildren; and a host of nieces, nephews and friends.
She was preceded in death by her husband, Everett; her parents, Adam and Henrietta; her brother, Harold; and her sisters, Shirley White and Eleanor Heinbigner.
We are confident, I say, and willing rather to be absent from the body, and to be present with the Lord II Corinthians 5:8.
Leave Your Condolences
|Copyright ©2020 Prairie Services, LLC d/b/a Sutton Memorial Chapel/McLaughlin Funeral Chapel. All Rights Reserved. Web Design by TheBluePig


text:
August 2020 S M T W T F S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Rank Me (Pretty Please!)
Have A Look Around
- Who - About me
- What - About this blog
- See - Cool pics that I love
- Watch - Fave videos
- Visit - Guest book
- Notice - Legal crap
Recent Posts
Types Of Rants
Tag! You're It
- advice
- American Idol
- baby
- blog
- blogging
- books
- breastfeeding
- cartoon
- celebrities
- child
- children
- Christmas
- comments
- Dancing With the Stars
- death
- diapers
- drinking
- economy
- entertainment
- family
- father
- Florida
- friends
- funny
- gifts
- going back to work
- growing up
- hockey
- holidays
- Hollywood
- hope
- humour
- husband
- joke
- Jon and Kate Plus 8
- kids
- learning
- lessons
- letting go
- life
- list
- marriage
- memories
- milestones
- mother
- motherhood
- musings
- Nadya Suleman
- nursing
- observations
- Olympics
- opinions
- parenthood
- parenting
- pictures
- playing
- poem
- post
- pregnancy
- random
- reality shows
- recession
- reflections
- retail
- sharing
- shopping
- social media
- So You Think You Can Dance
- television
- thoughts
- vacation
- work
- YouTube
What Are You Looking For?
Tag Archives: vacation
On our Florida vacation, we decided that our Boo-Boo was a little young for Disneyworld. For a place that iconic, I want to take him when he is old enough to actually remember the experience. But, how can you not … Continue reading
Posted in family Tagged child, Florida, memories, milestones, parenting, pictures, reminiscing, Sea World, vacation 6 Comments
Didn't You Get The Memo?
When I took my son to the beach for the first time, I wanted to take some shots of his footprints in the sand. You see, I'm quite the artiste. Not. Lol. Unfortunately, there were two things conspiring against me and … Continue reading
Posted in family Tagged child, family, Florida, growing up, letting go, memories, milestones, motherhood, parenting, vacation 4 Comments
Vive La Difference!
When you go on vacation, you not only want to relax and chill, but you also want to see and experience things that you wouldn't get back home. These things don't necessarily have to be big or even positive. But, … Continue reading
Ah, The Memories!
Finally! Here are some vacation pics that I would like to share with you. More pics to come!
Posted in family Tagged child, Florida, growing up, life, memories, milestones, pictures, reflections, sharing, shopping, vacation 4 Comments
Love Is In The Air
After many headaches (passports, packing, car rental, etc.), we finally had our family vacation in Orlando. In fact, it was our first vacation since we got married. For shame! I worried about how my Boo-Boo was going to be on … Continue reading
Posted in family Tagged children, growing up, life, memories, musings, observations, parenthood, thoughts, vacation 1 Comment
Home Sweet Home
Wah! It's over! We're back finally. While it was fun, it certainly is good to be home. I hope to blog about our first family vacation soon. I'm just in the midst of unpacking, cleaning, and catching up. Whew! So … Continue reading
Posted in family Tagged blog, children, economy, growing up, husband, learning, life, milestones, parenthood, thoughts, vacation 2 Comments
Leaving On A Jet Plane
Finally! We are flying out of here tomorrow! After much planning, headache, expense, and last-minute change of venue (Orlando instead of Naples), we will be on our way. As I have mentioned before, this will be our first family vacation. … Continue reading
Posted in family Tagged blog, child, growing up, husband, life, milestones, parenthood, thoughts, vacation 1 Comment


text:
It is not allowed for men to wear silk cloths. Is it permissible to use/own silk bed-throws?. There are men's cloths known as Andi Cotton Punjabi which is quite popular in some areas. In Andi cotton, 5% to 20% silk is mixed. Is such cloth is permissible to wear?
There is a difference of opinion regarding this. The hanafi Madhab permits clothing in which there is less than 50% silk.
In the Hanafi school, it is permitted without dislike to wear items of clothing in which there is less than 50% silk content. [Nahlawi, al-Hadhr wa'l Ibaha]
But scholars at IslamQA regard all kinds of clothing that have pure silk mixture to be haram except in the following cases and if the mixture is larger than "four fingers' width" of silk.
The scholars allowed men to wear silk in certain exceptional circumstances where there is shar'i evidence to support this, such as Muslim combatants in war who are allowed to wear silk in order to show off and annoy the enemy, which is a kind of psychological warfare. People who are sick may also be permitted to wear silk to relieve their suffering, as Anas reported that the Prophet (peace and blessings of Allaah be upon him) allowed 'Abd al-Rahmaan ibn 'Awf and Ibn al-Zubayr (may Allaah be pleased with them both) to wear silk because of a skin irritation that they suffered from. (Reported by al-Bukhaari, 10/295, and Muslim, 3/1646).
The scholars also allowed men to wear garments containing four fingers' width of silk, because of the hadeeth of 'Umar ibn al-Khattaab (may Allaah be pleased with him) who said that the Prophet (peace and blessings of Allaah be upon him) forbade the wearing of silk except for an area the width of two fingers, or three or four." (Reported by Muslim, 3/1644).
Now, it's up to you to choose between these two different views. You should also keep in mind that the prophet (peace and blessings of Allaah be upon him) said:
"Whoever wears silk in this world will not wear it in the Hereafter." (al-Bukhaari, 5832).
Just a remainder, all kinds of silk are halal for women.
Wearing Silk is Haram no matter how much percent. But if you have a disease on your skin and only silk can help, so its allowed.
Salaam, and welcome to Islam.SE. We expects answers here to be substantial and backed up by Islamic sources. Please consider improving your answer, and also have a look at How do I write good answers – infatuated Feb 24 '15 at 9:26
Assalamu alaikum, I was shown a silk blend sherwani for my wedding in Lahore. The shop keeper told me provided it isnt majority silk it is allowed; and that making a halal living was as much a concern for him as it was for us. I asked around and that was the answer I was getting ie a material is only considered to be 'silk' when that is the majority. It is established that small amounts of silk can be uesed (up to four fingers length), so threads and baring and patches are fine. I don't know if sleeping in bedsheets counts as wearing it.
1Salam and welcome on Islam SE. Please take your time and take our tour, check our help center and visit Islam Meta to learn more about this site. I also recommend you to read How to Answer to learn what makes a good answer. – Medi1Saif♦ May 30 '16 at 10:28


text:
Will Bots Replace Human Agents?
It's certainly a topic that captures the imagination. Will my job be replaced by a robot? For some, the introduction of AI into the workplace is the first step in the rise of Skynet. However, in reality, the human touch is still required in most white-collar roles.
In this article from the Financial Times, they spend time with a Customer Support Officer who utilizes AI to generate automated responses to customer queries. When asked whether he feels his job is under threat, he says: "I wouldn't say it's put my job at risk — it's made my job better." With bots handling the more repetitive inquiries typically received in a contact center, human agents are freed to offer an empathetic service to customers who require more complex support.
The future of customer service is humans and bots working side-by-side to deliver the best experience. Which brings us neatly on to the release of our latest guide...
The Definitive Guide to Customer Service in the Era of Messaging for the Airline Industry
This is the first Conversocial guide that I've contributed to since becoming CEO and I couldn't be happier with how it turned out! While it's aimed at the airline industry, it's packed with great takeaways that are relevant for any brand delivering customer service via messaging channels.
One particular theme that I think will become more widely discussed over the coming months is the need for brands to become uni-channel rather than omni-channel. Being omni-channel means you're likely a jack of all trades (as opposed to a master of one), particularly when it comes to customer service. If you spread your teams and their capabilities too thin, how can you deliver the best service to your customers?
Instead, you should be focussing on fewer channels and going deeper. For example, low-cost airline Volaris reduced agent response time by 43% and average handling time by 29% by making Messenger their key customer service channel and deflecting away from email and phone.
National Customer Service Week
Next week, October 7th-12th, is officially National Customer Service Week. While I appreciate it may not be the hottest date on everyone's calendar, I think it's as good a time as any to think about your own customer service. According to a report by Gartner, 64% of people say Customer Experience is more important than price when making a purchase. Customer service is integral to delivering that experience and consumers now expect to have conversations with brands in their preferred channel - messaging. If you do one thing next week, take 5 minutes to consider how you can future proof your contact center with the use of messaging and automation.
That's it for another Conversation Round-Up. As always, I'd love to hear from you. You can carry on the conversation @IdoHacohen.


text:
24" x 43" Country Blue & Navy U.S. HAND WOVEN Small Area Rag Rug
Fabric Model: TRG-SG24/43-695
- Size: 24" x 43"
- Fiber Content: Cotton, polyester, rayon.
- Care Instructions: Machine wash gentle cycle; line dry. Use "Color Catcher" by Shout to prevent discoloration.
- Description: Throw rug in shades of country blue, navy and ivory. Texture & density created by selvage of upholstery fabric. Approximately 1/2" thick. Hand-woven in Iowa.
- Uses: Suitable for bathroom rug, kitchen rug, entry rug, living room rug, bedroom rug & door mat rug.


text:
Leaf Designs Bright Green Flora Table Mats - Set of 6
Beautifully designed multicoloured flora & fauna fabric table mats in a set of 6 by Leaf Designs . Enjoy the spring season & the meal with these colourful mats! Uplift the beauty and utility quotient of your dining table while keeping it away from the stains, with the help of these table mats. With a variety of designs, shapes, and colours, these table mats will bring out large difference to your dining experience that will never go unnoticed. Care guide:Gentle hand wash in cold water Disclaimer: Product colour may vary slightly due to photographic lighting sources or your monitor settings.
Additional Information
|Poly Duck
|Poly Duck
|Weight (In KG)
FAQ Question
The Product will be dispatched within 2-3 working days from the date of placing your order.
We are proud to offer a 100% Satisfaction Guarantee 48 Hours Risk Free Return Policy.
We are confident you will be happy with your purchase. If for any reason the product does not meet your expectations, you may return it in its original condition with all original packing materials within 02 calendar days from the shipment date for an exchange or full refund of your purchase price, excluding shipping costs.
Please inspect your order immediately upon arrival. Do not dispose of the box or any packaging materials until you have inspected your order and are satisfied with the product you have received.


text:
_Social Media
To achieve a pole position in a competitive environment, many companies offer services provided at the cucomter's location, for the customer or even in cooperation with the customer. "Service sells" is the subject - that's where service marketing starts. That opens up a variety of possibilities, like customer mailings, by the employee on-location or by efficient public relations. Especially PR marketing has recently gained massively both acceptance and relevance, which can be traced back to the many new channels of communication - especially in the area of social media. How to use these new channels efficiently? That's a task for the specialists in service marketing.
The whole lot of new communication possibilities provided by channels like Twitter and Facebook, weblogs and bulletin boards all sound very attractive, but also confusing at times. It doesn't always seem to be clear what impacts they have, who's actually using then and how to adopt them for the means of service marketing. One thing comes clear though: The classic advertising does not work very well in the area of service marketing.
We're taking you one step further. Entrust us with your social media activity - be it Twitter, Facebook, weblogs or bulleting boards.
Try our introductory offer:
Starting with inventory-taking: What activtity is mentioned online yet, where do you or does your product already appear, maybe even without your cognition? We investigate that for you.
- Seach bulletin boards, user groups and communities for certain phrases (e.g. your company name or product), continuous documentation (PDF files) in a predefined interval, key aspect analysis regarding the importance of online entries, summary as fully written report, starting at 39,00 Euros* hourly rate.
(E.g. daily search for a predefined phrase in the web, on news siites and appointed bulleting boards, complete documentation, balanced analysis with fully written report once a week, 399,00* Euros)


text: Canada's new foreign buyer tax in British Columbia could lead to a price drop of 10 percent by the end of next year in Vancouver, reports The Globe and Mail (30 August 2016). House sales in Vancouver could also drop fall 3 percent this year, and potentially decline more than 22 percent next year as well. According to Juwai Data, Chinese searches for houses worth more than $1 million plunged 55 percent in July 2016 because of 'very dramatic and abundant' coverage on this foreign buyer tax. Juwai Data also showed Chinese buyers turning away from Vancouver to look at more affordable cities elsewhere in Canada. "The shift to other cities has actually been going on for months, with buyer demand momentum shifting to other cities with similar appeal but lower entry prices," said Matthew Moore, President of the Americas for Juwai.com. [This article is available on subscription only]


text:
Hard to imagine a salt and pepper pot could excite much interest. After all, you get them for around €3.50 from Ikea. If you have something more than €50 million spare, though, you could try and purchase the Saliera…
- Fabulous golden salt cellar from the mid-16th century
- Originally created by Benvenuto Cellini for King Francis I of France
- Once thought lost after an art theft
- Entered Habsburg hands as a gift to Archduke Ferdinand II
- See also: Kunsthistorisches Museum tickets & visitor info | Best art exhibits
The Cellini salt cellar
The Saliera occupies pride of place in the Kunstkammer Chamber of Wonders at Vienna's Kunsthistorisches Museum (better known as the KHM).
The Kunstkammer bursts with amazing items and sits proudly in my top 10 places to visit. But the Saliera towers over everything. Ostensibly, it's a piece of tableware to hold salt and pepper. And two receptacles (a ship and temple) do indeed serve that purpose.
But it's so much more than that.
Taking a pinch of salt or pepper from the Saliera is an act of submission, an acknowledgement that you are mere dirt on the shoe of the owner's prestige and power.
(Salt cellar (Saliera) Benvenuto Cellini 1540-1543, Paris, gold, enamel, ebony, ivory 26.3 cm x 28.5 cm x 21.5 cm © Kunsthistorisches Museum Wien)
Inside the gallery it calls home, the Saliera rests on its own in a free-standing glass cabinet, spotlights creating a pool of light in a low-lit room. Two figures rest on its surface, legs intertwined. A man ("Sea") holds a trident and sits back almost lasciviously, six-pack to the fore. Opposite him, a woman ("Land") reclines with one hand pinching a breast.
Elsewhere, a few glass display cases hold other French treasures. For example, some 16th-century cameos, a pendant of gold and enamel, or 16th-century painted enamel tableware from Limoges. These are just window dressing in the presence of the Saliera.
Gold is the piece's dominant theme, urged into place by the hands of Benvenuto Cellini, an Italian goldsmith, sculptor and all-round creative genius. Few of his works have survived, but the Saliera is one of them.
(Ferdinand II of Tyrol, one-time owner of the Saliera
Image courtesy of the Rijksmuseum)
Cellini completed his salt cellar in 1543 as a commission for Francis I, King of France. A later king, Charles IX, made a gift of it to Archduke Ferdinand II of Tyrol, architect of the enormous Schloss Ambros collection that later passed into the ownership of the KHM.
The Saliera's story does not end there, though. The salt cellar fell victim to an opportunistic theft in 2003. Fortunately, a contrite thief led police to its whereabouts just under three years later.
How to get to the Saliera
Once you've found the KHM, enter the main entrance hall with its glorious dome; the stairs to the left take you up into the Kunstkammer.
Once inside the Kunstkammer, go straight on through the galleries until you can't go any further and have to turn left or right. On the left is Gallery 24 (Saal XXIV) and inside is the Saliera.
Address: Burgring 7, 1010 Vienna


text:
Happy New Year, everybody.
We hope you got the best out of your holiday time. We sure did and are now eager to get back to work. We are starting this year with a few changes to our curriculum and would like to inform you about them.
Our in-house classes would be held during the 4-day week, as opposed to 5-day week before, with the identical courses spread out to around once in 5 weeks, as opposed to 4. For example, you can take our Microsoft Word course in Kelowna tomorrow, January 6, or on Tuesday, February 17. This is done to ensure that we are able to balance corporate and individual clients more effectively, as well as never cancel any class, regardless of the number of students registered.
Our schedule is currently set up until March, to give you the possibility to plan ahead. As always, you can book any course through our website or phone (250-861-8324).
We also continue to successfully provide tailored courses to dozens of corporate clients in BC, Alberta, and the world (remotely). Anything from our scheduled courses and more could be redesigned for you specific needs. Give us a call for more information.
Finally, we are still an authorized exam-testing centre, for those who are looking to get Microsoft Office Specialist certification; and sometimes our lab is available for rent, for those who are looking for a place to hold a meeting or a small conference. Contact us to get more information on both.


text:
The Single Most Lucrative Form Of Content?
There's a tragic, unnecessary mistake a lot of entrepreneurs and even large companies make in their marketing.
It's how they distribute their content efforts… and it's all kinds of wrong. 🙂
Press play and watch as I give you the whole shpiel, and give you the better alternative.
One that helps you make MUCH more money, just by shifting your efforts a tiny bit.
Let's go!
PS. If you're reading this on June 15, 2016 or earlier… there's JUST enough time to get in on the Copywriting Hotline before it defaults to waiting list only!