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Formation of the neuromuscular junction (NMJ) requires agrin, a factor released from motoneurons, and MuSK, a transmembrane tyrosine kinase that is activated by agrin. However, how signal is transduced from agrin to MuSK remains unclear. Here we report that low-density lipoprotein receptor (LDLR)-related protein (LRP) 4 (LRP4) functions as a co-receptor of agrin. LRP4 is specifically expressed in myotubes and is concentrated at the NMJ. The extracellular domain of LRP4 interacts with neuronal, but not muscle, agrin. Expression of LRP4 enables agrin binding activity and MuSK signaling in cells that otherwise does not respond to agrin. Suppression of LRP4 expression attenuates agrin binding activity, agrin-induced MuSK tyrosine phosphorylation and AChR clustering in muscle cells. LRP4 also interacts with MuSK in a manner that is stimulated by agrin. Finally, we showed that LRP4 becomes tyrosine-phosphorylated in agrin-stimulated muscle cells. These observations identify LRP4 as a functional co-receptor of agrin that is necessary for agrin-induced MuSK signaling and AChR clustering.
The NMJ is a cholinergic synapse that conveys signals from motor neurons to muscle cells. It has served as an informative model of synaptogenesis because of its simplicity and accessibility (Sanes and Lichtman, 1999; Sanes and Lichtman, 2001). NMJ formation requires communication between presynaptic motor neurons and postsynaptic muscle fibers (Brandon et al., 2003; Fu et al., 2008; Li et al., 2008; Sanes and Lichtman, 2001). Agrin is a main nerve-derived organizer of postsynaptic differentiation at the NMJ (McMahan, 1990). Mice lacking agrin displayed severe deficits in NMJ formation (Gautam et al., 1996). Introduction of agrin into denervated muscles elicits formation of postsynaptic apparatus (Bezakova et al., 2001; Gesemann et al., 1995; Herbst and Burden, 2000; Jones et al., 1997). MuSK is a component of the agrin receptor complex (DeChiara et al., 1996; Jennings et al., 1993; Lin et al., 2001; Valenzuela et al., 1995; Yang et al., 2001). MuSK mutant mice do not form the NMJ and prepattern of aneuronal AChR-rich sites in the central region (Kim and Burden, 2008; Lin et al., 2001; Yang et al., 2001). Moreover, agrin induces rapid tyrosine phosphorylation of MuSK in cultured muscle cells (Glass et al., 1996a). Agrin is no longer able to induce AChR clusters in MuSK−/− myotubes and the agrin sensitivity can be restored by the introduction of wild-type MuSK (Glass et al., 1996a; Zhou et al., 1999). These observations provide evidence that agrin functions by stimulating MuSK. A fundamental gap in our understanding of agrin/MuSK signaling, despite of its essential role for NMJ formation and maintenance (Hesser et al., 2006; Kim and Burden, 2008), is poor understanding of how signals are transmitted from agrin to MuSK because the two proteins do not interact directly (Glass et al., 1996a). A hypothetical molecule MASC (myotube associated specificity component) was proposed to serve as a binding partner for agrin to transduce signals to MuSK (Glass et al., 1996a). Despite extensive studies, the identity of this co-receptor of agrin remains unclear.
LRP4 [or MEGF7 for multiple epidermal growth factor (EGF)-like domain 7] is a member of the LDLR family, and contains a large extracellular N-terminal region that possesses multiple EGF repeats and LDLR repeats, a transmembrane domain and a short C-terminal region without an identifiable catalytic motif (Johnson et al., 2005; Lu et al., 2007; Tian et al., 2006; Yamaguchi et al., 2006). It was identified by a motif trap screen of genes encoding proteins with multiple EGF domains (Nakayama et al., 1998). Remarkably, LRP4 is required for NMJ formation as well as the development of the limb, lung, kidney, and ectodermal organs (Johnson et al., 2005; Simon-Chazottes et al., 2006; Weatherbee et al., 2006). Mice lacking LRP4 die at birth with deficits that resemble the phenotype observed in MuSK mutant mice (Weatherbee et al., 2006). The phenotypic similarity suggested that LRP4 plays a role in MuSK signaling (Weatherbee et al., 2006). However, molecular mechanisms of how LRP4 regulates NMJ formation remain unclear.
Here we provide evidences that LRP4 functions as a co-receptor of agrin. LRP4 is expressed specifically in myotubes and concentrated at the NMJ. The extracellular domain of LRP4 binds to neuronal, but not muscle, agrin. LRP4 also interacts with MuSK in a manner enhanced by agrin. Suppression of LRP4 expression attenuated agrin binding activity, agrin-induced MuSK tyrosine phosphorylation and AChR clustering in muscle cells. LRP4 expression, on the other hand, reconstitutes agrin binding and MuSK activation by agrin in cells that would otherwise not respond to agrin. These observations identify LRP4 as a key component of the receptor complex of agrin.
Because neuronal agrin binds only to myotubes, but not myoblasts (Glass et al., 1996b), we characterized the expression of LRP4 in developing myotubes. C2C12 myoblasts were switched fusion medium to induce muscle differentiation. Under these conditions, myotubes began to form 48 hr after medium switch (Luo et al., 2002; Luo et al., 2003; Si et al., 1996). Developing myotubes were collected and LRP4 expressed analyzed by immunoblotting with anti-LRP4 antibody. As shown in Figure 1A, LRP4 was barely detectable in myoblasts, but its expression gradually increased as myotubes matured. As control, expression of MuSK was examined in same preparations, whose expression was known to be regulated by muscle differentiation (Glass et al., 1996b; Ip et al., 2000; Valenzuela et al., 1995) (Figure 1A). These results indicate that LRP4, like MuSK, is expressed in well differentiated myotubes, but not myoblasts. Next, we investigated LRP4 distribution in vivo by staining muscle sections with anti-LRP4 antibody. The immunoreactivity of LRP4, as well as MuSK, showed a pattern of labeling similar to that of rhodamine-conjugated α-bungarotoxin (R-BTX) that labels AChRs (Figures 1B), suggesting that LRP4, like MuSK, is enriched at the NMJ. This notion was supported by results from immunoblot analysis of LRP4 expression of muscles. Hemi-diaphragms were divided into three regions: the central, narrow region, where NMJs are enriched, as synaptic region; the region close to ligaments to the ribs as non-synaptic region; and the middle in between. The AChR was enriched in the synaptic, but not non-synaptic, region (Figure 1C). In agreement with results of immunostaining, LRP4 was readily detectable in the synaptic region where AChRs were enriched. However, little, if any, LRP4 was found in the non-synaptic region. Together these results demonstrated that LRP4 is specifically expression in myotubes and is enriched at the NMJ, suggesting a role of LRP4 in NMJ formation.
Next, we determined whether agrin binds to LRP4. A secreted form of neuronal agrin, Flag-nAgrin, was generated that comprised the C-terminus of neuronal agrin fused with the Flag epitope, and a secreted form of LRP4 (i.e., LRP4N–Myc), which consisted of the LRP4 extracellular domain tagged by the Myc epitope. Flag-nAgrin was immobilized on beads and incubated with LRP4N-Myc. As shown in Figure 2A, LRP4N–Myc was precipitated with neuronal agrin, suggesting that the two proteins interact in solution. In contrast, Flag-nAgrin did not precipitate MuSKect–Myc, which consisted of the extracellular region of the kinase (Figure 2B), in agreement with previous findings that agrin and MuSK do not directly bind to each other (Glass et al., 1996a). Moreover, Flag-nAgrin did not interact with LRP6N-Myc, which comprised Myc-tagged extracellular domain of LRP6, a homologous member of the LRP family whose extracellular structural organization resembles that of LRP4 (Figure 2C). As control, LRP6N-Myc was able to interact with Wnt-1-HA when the two proteins were incubated together (Figure 2D), indicating proper folding and specific binding of LRP6N-Myc. Furthermore, LRP4N-Myc did not co-precipitate with Wnt-1-HA (Figure 2E), suggesting that the two proteins do not interact. These results demonstrate that agrin binds specifically to the extracellular domain of LRP4, but not that of MuSK, or LRP6 and on the other hand, Wnt-1 interacts with LRP6, but not LRP4. In support of this notion, the extracellular domain of LRP4 was able to neutralize neuronal agrin and thus prevented it from stimulating MuSK tyrosine phosphorylation and AChR clustering (Figure S1).
To determine whether the interaction is direct, we produced recombinant agrins, nAgrin-AP and mAgrin-AP, which contained the C-terminal region of neuronal and muscle agrin, respectively. They were fused with the heat-insensitive human placental isozyme of alkaline phosphatase (AP) (Flanagan and Cheng, 2000) (Figure 3A). The activity of the AP recombinant proteins was tested in AChR cluster assays. As shown in Figure 3B, nAgrin-AP was able to stimulate AChR clustering in C2C12 myotubes, indicating proper folding of the recombinant neuronal agrin protein. In contrast, mAgrin-AP or AP alone had little effect on AChR clustering. Next, we characterized the binding activity of the AP proteins to muscle cells by in-cell assays as described in Experimental Procedures. AP binding to myoblasts or myotubes was minimal (Figure 3C). mAgrin-AP binding to myoblasts was higher than that of AP alone, presumably because myoblasts express alpha-dystroglycan to which muscle agrin is known to interact (Bowe et al., 1994; Campanelli et al., 1996; Campanelli et al., 1994; Gee et al., 1994; Gesemann et al., 1996; Hopf and Hoch, 1996; Sugiyama et al., 1994). The mAgrin-AP binding to myotubes was higher in comparison with that in myoblasts because alpha-dystroglycan expression was increased during muscle differentiation. nAgrin-AP binding to myoblasts was similar to that of mAgrin-AP (Figure 3C). However, nAgrin-AP binding was significantly higher in myotubes than in myoblasts (Figure 3C), in agreement with earlier reports (Glass et al., 1996a) and the LRP4 expression pattern in developing muscle cells (Figure 1). These results demonstrate differential ability of recombinant muscle and neuronal agrins in binding to myotubes. Having established that nAgrin-AP was able to bind to myotubes and stimulate AChR clustering, we next characterized the interaction between LRP4 and nAgrin-AP. LRPN4-Myc was purified and immobilized on plates and incubated with purified nAgrin-AP. After wash, the AP activity bound to immobilized LRPN4-Myc was assayed by a modified ELISA (enzyme-linked immunosorbent assay). In comparison with control (AP alone), there was a significant increase in AP activity when nAgrin-AP were incubated with LRP4N-Myc (Figure 3D), suggesting direct interaction between the two proteins, i.e., independent of a third protein. Quantitatively, the interaction between neuronal agrin and LRP4 was dose-dependent, saturable, and of high affinity (Kd values of 0.5 ± 0.053 nM) (Figure 3E and 3F). This affinity is comparable to that (0.1–0.5 nM) of LRP6 for Dkk1 and Dkk2 (Bafico et al., 2001; Mao et al., 2001; Semenov et al., 2001). In contrast, muscle agrin, which lacks four and eight amino acid inserts at the Y and Z sites, respectively (Figure 3A) and is 1000 times less potent than neuronal agrin in stimulating AChR clusters (Gesemann et al., 1995; Reist et al., 1992), did not appear to bind to LRP4 (Figure 3D). The binding of LRP4N-Myc to muscle agrin was minimal even at high concentrations (Figure 3E). Together, these results suggest LRP4 binds specifically to neuronal agrin with high affinity. These results indicate that LRP4 binds to neuronal, but not muscle, agrin in a manner that is concentration-dependent, saturable and of high affinity.
To determine whether agrin binds to LRP4 in vivo, we expressed exogenous LRP4 in C2C12 myoblasts that, unlike myotubes, do not bind neuronal agrin (Glass et al., 1996a) (Figure 3C). Myoblasts were transfected with full length LRP4 or the empty vector (as control). Intact transfected myoblasts were incubated with AP alone, nAgrin-AP or mAgrin-AP. The AP activity bound to cell surface was measured in situ after heat inactivation of endogenous AP. As shown in Figure 4A and 4B, when incubated with AP alone, control and LRP4-transfected myoblasts show no difference in AP activity. However, nAgrin-AP binding was significantly higher to LRP4-transfected myoblasts in comparison with control, indicating that LRP4 enables myoblasts to interact with neuronal agrin. By contrast, transfection of MuSK had no consistent effect on binding to nAgrin-AP, in agreement with earlier observations that agrin does not bind to MuSK (Glass et al., 1996a) (Figure 2B). In addition to myoblasts, HEK293 cells were able to bind to nAgrin-AP after LRP4 transfection (Figure 4C).
The in situ binding activity generated by transfected LRP4 had the following characters. First, it was dose-dependent. Increase in LRP4 expression in transfected HEK293 cells led to higher nAgrin-AP binding activity (Figure 4C). Probably due to rate-limiting surface integration of overexpressed LRP4, nAgrin-AP binding was not further increased in cells transfected with 2 µg of DNA. Earlier studies have reported that overexpressed LRP4 is retained in the endoplasmic reticulum (Lu et al., 2007; Obermoeller-McCormick et al., 2001). Notice that the blot reveals total, but not surface, LRP4 (Figure 4C). Second, LRP4 binding was specific for neuronal agrin because the amount of mAgrin-AP bound to transfected myoblasts and HEK293 cells was minimal, and not concentration-dependent (Figure 4A–4C). Notice that mAgrin-AP, like nAgrin-AP, also contained the AP and the Myc and His tags. Inability of mAgrin-AP to bind to LRP4-transfected cells indicate that binding to LRP4 does not involve the AP or tags. Third, the binding activity was LRP4 specific. Expression of LRP5, another member of the LRP family (Herz and Bock, 2002), did not increase agrin binding in transfected cells (Figure 4D). Last, nAgrin-AP binding was similar between cells transfected with LRP4 alone and those co-transfected with LRP4 and MuSK (Figure 4A and 4B), indicating that the neuronal agrin binding activity is mainly contributed by LRP4 although LRP4 and MuSK could interact in muscle cells (see below). Taken together, these results demonstrate the ability of LRP4 to reconstitute agrin binding in cells that otherwise do not interact with agrin.
Next, we determined if LRP4 was able to reconstitute MuSK signaling in cells that do not respond to agrin. MuSK is a receptor tyrosine kinase whose activation has been shown to be upstream of all known agrin signaling cascades (Fuhrer et al., 1997; Glass et al., 1997; Glass et al., 1996a; Herbst and Burden, 2000; Luo et al., 2002; Strochlic et al., 2005; Wang et al., 2008; Zhou et al., 1999). Therefore we first examined whether LRP4 expression enables MuSK activation by agrin in HEK293 cells that do not express LRP4 (Figure 4E). Flag-MuSK was transfected into HEK293 cells with or without LRP4 and transfected cells were stimulated with neuronal agrin. As shown in Figure 4E and 4G, agrin was unable to elicit MuSK tyrosine phosphorylation in HEK293 cells transfected with MuSK alone. Intriguingly, LRP4 co-expression enabled agrin to activate MuSK, indicating that LRP4 could be an agrin receptor able to stimulate MuSK. Basal tyrosine phosphorylation of MuSK, i.e., in the absence of agrin, was increased by LRP4, which could suggest a role of LRP4 in MuSK auto-activation, presumably by its direct interaction with the kinase (see below). Agrin-induced AChR clustering requires the intracellular tyrosine kinase Abl (Finn et al., 2003). To further investigate the role of LRP4, we examined Abl activation by anti-phospho-Abl antibody in cells co-expressing LRP4 and MuSK. As shown in Figure 4F and 4G, active Abl was barely detectable in cells transfected with Myc-MuSK alone, regardless of agrin stimulation. In contrast, agrin elicited a significant increase in phospho-Abl in cells co-expressing LRP4 and Myc-MuSK. Together, these results indicate that LRP4 expression enables binding activity for neuronal agrin, MuSK activation, and initiation of intracellular signaling in cells that otherwise do not respond to agrin.
We next determined if LRP4 is necessary for agrin/MuSK signaling by a loss-of-function approach. To this end, we generated several microRNA constructs of LRP4. As shown in Figure 5A, miLRP4-1062 was most potent in inhibiting LRP4 expression. First, we determined if repression of LRP4 affects agrin binding to intact muscle cells. C2C12 myoblasts were transfected with miLRP4-1062 or the control miRNA that encoded scramble sequence, and resulting myotubes were incubated with AP, mAgrin-AP or nAgrin-AP and assayed for AP activity by in-cell staining. In comparison with control miRNA, miLRP4-1062 did not appear to alter binding activity of AP and mAgrin-AP to myotubes (Figure 5B and 5C). However, myotubes transfected with miLRP4-1062 had lower levels of nAgrin-AP staining in comparison with those transfected with the control vector (Figure 5B and 5C), indicating a necessary role of endogenous LRP4 for neuronal agrin binding. Second, we tested whether LRP4 is required for agrin to stimulate tyrosine phosphorylation of MuSK. MuSK was precipitated from myotubes transfected with control miRNA or miLRP4-1062 and assayed for tyrosine phosphorylation. Expression of miRNA constructs was indicated by the presence of GFP that was encoded by the parental vector. As shown in Figure 5D, transfection of miLRP4-1062 reduced expression of endogenous LRP4, but not MuSK or β-actin. Remarkably, agrin-induced MuSK tyrosine phosphorylation was attenuated in myotubes transfected with miLRP4-1062 in comparison with control miRNA (Figure 5E). These results suggest that MuSK activation is impaired when LRP4 levels were reduced.
Finally, we investigated whether LRP4 is necessary for agrin-induced AChR clustering. Myoblasts were transfected with control miRNA or miLRP4-1062, or miRNA constructs against MuSK and LRP5 that reduced expression of MuSK and LRP5, respectively (data not shown). Transfected myotubes were stimulated without or with agrin and AChR clusters in GFP-expressing myotubes scored as described previously (Zhang et al., 2007). Expression of these miRNA constructs did not appear to alter basal AChR clusters. However, the number of agrin-induced AChR clusters was reduced in myotubes transfected with miLRP4-1062 (Figure 5F), suggesting a necessary role of LRP4 in agrin-induced clustering. Similar reduction was observed in myotubes expressing miMuSK-1161, as expected. Transfection with miLRP5-1490, however, had no effect on agrin-induced AChR clustering, in agreement with the observation that LRP5 does not bind to neuronal agrin (Figure 4D).
In a working model, LRP4 serves as a co-receptor that binds to agrin and, together with MuSK, stimulates AChR clustering. To examine the relationship among agrin, LRP4 and MuSK, we determined whether LRP4 interacts with MuSK and if so, if the interaction is regulated by agrin. Secreted Flag-MuSKect, which comprised the entire extracellular region of MuSK fused with the Flag epitope, was incubated with LRP4N–Myc in the absence or presence of agrin. Flag-MuSKect alone was able to co-precipitate with LRP4N–Myc (Figure 6A and 6B), indicative of direct binding between the extracellular domains of MuSK and LRP4. Quantitatively, the interaction between MuSK and LRP4 was dose-dependent and saturable, and of high affinity (Kd values of 0.45 ± 0.041 nM, Figure 6C and 6D). Interestingly, the amount of LRP4 co-precipitated with Flag-MuSKect was increased by agrin (Figure 6A and 6B). In contrast, as control, LRP6N-Myc failed to co-precipitate with Flag-MuSKect regardless of the presence or absence of agrin (Figure 6E). These observations suggest that LRP4 and MuSK form a complex in the absence of the ligand agrin; however, agrin, via binding to LRP4, enhances the LRP4-MuSK interaction. To test this hypothesis further, we examined if full length MuSK and LRP4 interact with each other in cells. LRP4 and Flag-MuSK were co-transfected into HEK293 cells. MuSK was precipitated from cell lysates by a Flag antibody and the resulting immunocomplex was analyzed for LRP4. As shown in Figure 6F, LRP4 co-precipitated with MuSK in transfected cells, in support of the notion that the two proteins interact in transfected cells. Moreover, the LRP4-MuSK association was detectable in mouse muscle homogenates (Figure 6G), suggesting in vivo interaction of the two proteins.
To further investigator the role of LRP4 in agrin signaling, we examined whether the LRP4-MuSK interaction in muscle cells is regulated by neuronal agrin. C2C12 myotubes were treated without or with agrin for 1 hr. Myotubes were subjected to immunoprecipitation with anti-LRP4 antibody and resulting precipitates were probed for MuSK. As shown in Figure 7A, MuSK co-precipitated with LRP4 from cells in the absence of agrin, suggesting basal interaction of the two proteins and in agreement with in vitro binding results (Figure 6A–D). The co-precipitation was increased in agrin-stimulated myotubes (Figure 7A and 7B). These observations indicate that LRP4 and MuSK form a complex in a manner that is up-regulated by agrin.
LRP4 has a large intracellular domain containing six tyrosine residues. Recent evidence indicates that LRP4, immunopurified from the brain, could be phosphorylated on serine residues presumably by CaMK II (Tian et al., 2006). Other members of the LRP family, LRP5 and LRP6, become phosphorylated upon activation of the Wnt canonical pathway (Ding et al., 2008). Unlike LRP5 and LRP6, LPR4 has a NPXY motif in the intracellular region that may be phosphorylated by a tyrosine kinase (Herz and Bock, 2002). Having demonstrated that LRP4 interacts with MuSK and the interaction is enhanced by agrin, we determined whether LRP4 itself becomes phosphorylated on tyrosine residues. C2C12 myotubes were stimulated with neuronal agrin for 1 hr and lysates were subjected immunoprecipitation of LRP4 and MuSK, respectively. Resulting precipitates were probed with the anti-phospho-tyrosine antibody 4G10. As shown in Figure 7C and 7D, LRP4 as well as MuSK became tyrosine-phosphorylated in agrin-stimulated myotubes. This result suggests a role of LRP4 in agrin signaling.
Major findings of this paper are as follows. First, LRP4 is specifically expressed in myotubes, but not myoblasts and is concentrated at the NMJ (Figure 1). Second, it is both necessary and sufficient to bind to agrin and to activate MuSK signaling that leads to AChR clustering. Using three different assays (in solution, on solid phase, and in cells), we demonstrate that neuronal agrin was able to interact directly with the extracellular region of LRP4 (Figure 2, Figure 3, and Figure 4). The binding activity of LRP4 was specific because 1) LRP4 binding to muscle agrin was minimal; 2) the binding is concentration-dependent and of high affinity with a sub-nanomolar Kd; and 3) neuronal agrin did not bind to LRP5 or LRP6, two other members of the LRP family that are highly homologous to LRP4. Third, expression of LRP4 enabled binding activity for neuronal agrin and MuSK signaling in cells that otherwise did not respond to agrin (Figure 4). Fourth, suppression of LRP4 expression attenuated agrin binding activity and agrin-induced MuSK phosphorylation and AChR clustering in muscle cells (Figure 5). Fifth, LRP4 could interact with MuSK in a manner that is increased by agrin (Figure 6 and Figure 7). Finally, LRP4 became tyrosine-phosphorylated in muscle cells in response to agrin stimulation (Figure 7). These observations indicate that LRP4 can bind to agrin and transmit signals to MuSK, suggesting that it may serve as a functional receptor for agrin. Based on these observations, we propose a working hypothesis where LRP4 interacts with MuSK at basal levels in the absence of the ligand. Upon agrin stimulation, the interaction was increased to activate MuSK and subsequent downstream signal cascades for AChR clustering (Figure 7E).
Despite the essential role of MuSK in NMJ formation, mechanisms of how it is activated and how it acts to control NMJ formation remain elusive. Recent studies have shed light on intracellular pathways downstream of MuSK. They are thought to involve the adapter protein Dok-7 (Okada et al., 2006), and several enzymes including Src-family kinase (Ferns et al., 1996; Mittaud et al., 2001; Mohamed et al., 2001; Qu and Huganir, 1994; Wallace, 1991), Abl (Finn et al., 2003), casein kinase 2 (Cheusova et al., 2006), geranylgeranyl transferase I (GGT) (Luo et al., 2003), GTPases of the Rho family (Weston et al., 2003; Weston et al., 2000), and Pak1, a serine/threonine kinase that is activated by Rho GTPases (Luo et al., 2002). Although agrin is known to activate MuSK, the two proteins, however, do not interact directly. The MASC co-receptor was hypothesized that has to be myotubes specific and is able to transmit signal from agrin to MuSK (Glass et al., 1996a). Remarkably, LRP4 is a protein specifically expressed in myotubes, not in myoblasts (Figure 1), fulfilling a requirement of MASC. Second, LRP4 is able to reconstitute agrin binding and MuSK signaling in cells that otherwise do not respond to agrin (Figure 4). Third, LRP4 is required for agrin binding and induced MuSK signaling and AChR clustering in muscle cells (Figure 5). Fourth, genetic studies have demonstrated that phenotypes of LRP4 mutant mice are similar to those in MuSK mutant (Weatherbee et al., 2006). LRP4 mutants die at birth with defects in both pre- and post-synaptic differentiation and in particular, the rapsyn-dependent scaffold fails to assemble in LRP4 mutants. These results provide strong evidence that LRP4 satisfies essential criteria of serving a functional co-receptor of agrin.
The identification of LRP4 as a co-receptor for agrin could provide insight into mechanisms of how agrin stimulation leads to AChR clustering. First, bridging agrin and MuSK, LRP4 could transmit signal to MuSK and thus activate intracellular cascades that have been identified, leading to AChR clustering. Second, LRP4 may regulate MuSK activity. MuSK and LRP4 co-precipitate in vitro and in muscle cells in the absence of agrin (Figure 6 and Figure 7), and tyrosine phosphorylation of MuSK is increased in cells co-expressing LRP4 (Figures 4E and 4G). These observations may suggest that LRP4 promotes MuSK auto-activation, presumably by regulating MuSK dimerization. Exactly how LRP4 regulates MuSK function and the stoichiometry of the LRP4-MuSK interaction warrant further investigation. Third and alternatively, LRP4 itself may function as a signal transducer. The juxtamembrane cytoplasmic region of LRP4 contains a NPXY motif. This motif in LDLR, LRP1 and LRP2 has been shown to serve as a docking site for cytoplasmic adaptor proteins through a phosphotyrosine binding (PTB) domain (Herz and Bock, 2002). Intriguingly, LRP4 becomes tyrosine phosphorylated upon agrin stimulation (Figure 7C and 7D). It would be interesting to investigate whether tyrosine phosphorylation of LRP4 is necessary for agrin signaling and AChR clustering and whether phosphorylated LRP4 binds to PTB domain-containing proteins. One such protein is Dok7, which is essential for NMJ formation (Okada et al., 2006).
Wnt signaling is implicated in synapse formation (Ciani and Salinas, 2005). Wnt-7a released from granule cells induces axon and growth cone remodeling in mossy fibers (Hall et al., 2000). In C. elegans, Wnt signaling positions NMJs by inhibiting synaptogenesis (Klassen and Shen, 2007). NMJ formation in Drosophila requires Wnt signaling (Mathew et al., 2005; Packard et al., 2002). However, it remains unclear whether Wnt signaling regulates mammalian NMJ formation. Wnt ligands act by binding to the receptor complex of Frizzled and LRP5/6 (Cadigan and Liu, 2006; He et al., 2004; Malbon and Wang, 2006; Schulte and Bryja, 2007). Subsequently, signal is believed to be transmitted to the adapter protein Dishevelled (Dvl), which interacts with Frizzled, to initiate intracellular canonical and non-canonical pathways. Intriguingly, MuSK, like Frizzled, interacts with both a LRP protein (i.e., LRP4) and Dvl (Luo et al., 2002). In addition, MuSK contains an extracellular CRD domain that is highly homologous to that in Frizzled that interacts with Wnt (Glass et al., 1996a; Valenzuela et al., 1995). Moreover, a number of Wnt signaling molecules including APC and β-catenin have been implicated in MuSK cascades (Li et al., 2008; Wang et al., 2003; Zhang et al., 2007). These observations raise a question whether the agrin-LRP4-MuSK signaling is regulated by a Wnt ligand that may interact with LRP4 and/or MuSK. We showed that LRP4 does not bind Wnt-1 (Figure 2E). This, however, does not exclude possible involvement of one of the 18 other Wnt proteins in mouse (Clevers, 2006)(The Wnt Homepage: www.stanford.edu/~rnusse/wntwindow). On the other hand, the "Wnt signaling" molecules (including Dvl, APC, and β-catenin) may simply function in a manner independent of Wnt signaling in mammalian NMJ formation.
It is of interest to note that the phenotypes of MuSK and LRP4 mutant mice are more severe than those of agrin mutant. In LRP4 or MuSK mutants, but not agrin mutants, AChR clusters are absent when clusters begin to assemble at E13.5 and the rapsyn-dependent scaffold fails to assemble (Lin et al., 2001; Weatherbee et al., 2006). These observations could suggest the existence of a signaling pathway that requires MuSK and/or LRP4, but not agrin. This pathway may regulate the formation of aneuronal AChR clusters prior to the arrival of motoneuron terminals or assembly of rapsyn-dependent scaffold. It may be regulated by a ligand that could interact with MuSK and/or LRP4. In light of the above discussion, such ligand may be a Wnt protein.
Agrin is expressed in the brain (Cohen et al., 1997; Mann and Kroger, 1996; O'Connor et al., 1994). Suppression of its expression impairs dendritic development and synapse formation in cultured hippocampal neurons (Bose et al., 2000; Ferreira, 1999). Agrin-deficient neurons appear to be resistant to excitotoxic injury and agrin heterozygous mice are less sensitive to kainic acid-induced seizure and mortality (Hilgenberg et al., 2002). Agrin is thought to bind to the a3 subunit of Na+/K+-ATPase in neurons and thus regulates their function (Hilgenberg et al., 2006). LRP4 expression is enriched in the brain and could interact with postsynaptic scaffold proteins including PSD-95 and SAP97 (Lu et al., 2007; Tian et al., 2006; Weatherbee et al., 2006). The identification of LRP4 as a co-receptor of neuronal agrin may shed light on molecular mechanisms of how agrin and LRP4 work in the brain.
Taq DNA polymerase, T4 DNA ligase, and restriction enzymes were purchased from Promega. Horseradish peroxidase conjugated goat anti-mouse and goat anti-rabbit antibodies and enhanced chemifluoresent (ECL) reagents for Western blotting were from Amersham. Rhodamine-αBTX (R-BTX) was from Molecular Probes. Oligonucleotides were synthesized by Operon Biotechnologies. Unless otherwise specified, all chemicals were from Sigma-Aldrich. Antibodies were purchased from Sigma (Flag M2, F3165); Torrey Pines Biolabs (GFP, TP401); Upstate Biotechnology (4G10, 05–1050); Cell Signaling (Phospho-c-Abl, 2861); Novus (β-actin, NB600-501). Rabbit anti-MuSK antibodies were described previously (Luo et al., 2002). Rabbit anti-LRP4 antibody was described previously (Lu et al., 2007). Rat anti-AChR α-subunit (mAb35) antibody was a gift from Dr. Richard Rotundo. Rat anti-AChR β-subunit (mAb124) antibody was a gift from Dr. Jon Lindstrom.
Original agrin constructs were gifts from Zach Hall. Agrin-AP constructs were generated by fusing neuronal and muscle agrin (aa 1145–1940) (Ferns et al., 1993) with AP in pAPtag-5. To generate Flag-MuSK, the MuSK DNA was generated by PCR and subcloned in EcoRI/XbaI sites in pFlag-CMV1 downstream of an artificial signal peptide sequence and a Flag epitope. LRP4-Myc was generated by subcloning the full length LRP4 DNA into NheI and HindIII sites in pcDNA3.1-MycHis (Invitrogen) with 3 Alanines insert after amino acid 1746. LRP4N-Myc was generated by subcloning LRP4 extracellular domain DNA into NheI and NotI sites in pcDNA3.1-MycHis. LRP5 DNA was amplified with pCMV-Sports6-LRP5 (Open Biosystems) as template and subcloned into XbaI and NotI sites in pcDNA3.1-MycHis to generate LRP5-Myc. LRP4-, LRP5- and MuSK-miRNA constructs were generated using the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit (Invitrogene, K4936-00). Oligonucleotide sequences for miRNA constructs were: for mi-MuSK-1161 5'-TGCTG TAACA CAGCA GAGCC TCAGC AGTTT TGGCC ACTGA CTGAC TGCTG AGGCT GCTGT GTTA-3' (sense), 5'- CTGTA ACACA GCAGC CTCAG CAGTC AGTCA GTGGC CAAAA CTGCT GAGGC TCTGC TGTGT TAC -3' (antisense); for mi-LRP5-1490 5'- TGCTG ATCAC AGGGT GCAAC ACAAT GGTTT TGGCC ACTGA CTGAC CATTG TGTCA CCCTG TGAT -3' (sense), 5'- CCTGA TCACA GGGTG ACACA ATGGT CAGTC AGTGG CCAAA ACCAT TGTGT TGCAC CCTGT GATC -3' (antisense); for mi-LRP4-1062: 5'-TGCTG TTAAC ATTGC AGTTC TCCTC AGTTT TGGCC ACTGA CTGAC TGAGG AGATG CAATG TTAA-3' (sense), 5'-CCTGT TAACA TTGCA TCTCC TCAGT CAGTC AGTGG CCAAA ACTGA GGAGA ACTGC AATGT TAAC-3' (antisense), for mi-LRP4-2603: 5'-TGCTG AATAC ATGTA CCCGC CCATG GGTTT TGGCC ACTGA CTGAC CCATG GGCGT ACATG TATT-3'(sense), 5'-CCTGA ATACA TGTAC GCCCA TGGGT CAGTC AGTGG CCAAA ACCCA TGGGC GGGTA CATGT ATTC-3' (antisense), for mi-LRP4-5355: 5'-GCTGT AGCAC AGCTG ATTAT ACACG GTTTT GGCCA CTGAC TGACC GTGTA TACAG CTGTG CTA-3'(sense), 5'-CCTGT AGCAC AGCTG TATAC ACGGT CAGTC AGTGG CCAAA ACCGT GTATA ATCAG CTGTG CTAC-3' (antisense). The authenticity of all constructs was verified by DNA sequencing. The following constructs were described previously: MuSK-AP (Wang et al., 2008); pcDNA-LRP4 (Lu et al., 2007); Wnt1-HA(Zhang et al., 2007); and Wnt1-Myc, LRP6-N-Myc, and mfz8CRD-IgG (Tamai et al., 2000).
HEK293 cells and mouse C2C12 muscle cells were maintained and transfected as previously described (Zhang et al., 2007). In some experiments, myotubes were transfected with lipofectamine 2000 (Invitrogen, 11668-019). The cells were incubated with a mixture of DNA, lipofectamine and serum-free medium for 8 hr before being switched to the fusion medium. The DNA: lipofectamine ratio in the mixture was 1 µg: 2µl. The optimal volume of the mixture for 24-well dishes was 200 µl per well with 2 µg plasmid DNA.
To produce recombinant proteins, HEK293 were transfected with respective plasmids. Twenty-four hours after transfection, cells were switched to Dulbecco's Modified Eagle Medium supplemented with reduced concentration (0.05%) of fetal bovine serum, and secreted proteins were harvested 24 hr later. nAgrin-AP, mAgrin-AP, or MuSK-AP recombinant proteins, which contained 6-His-tags that were encoded by pAPtag-5, were purified by affinity chromatography using TALON Resins (BD Biosciences).
Flag-nAgrin was immobilized to protein A Sepharose beads (that were preabsorbed with anti-Flag antibody), which were incubated with 1 ml (0.5 nM) of LRP4N-Myc, MuSKect-Myc, or LRP6N-Myc condition medium, and Flag-nAgrin-bound proteins were isolated by bead precipitation and resolved by SDS-PAGE and visualized by immunoblot with anti-Myc antibody. In some experiments, LRP4N-Myc, LRP6N-Myc or MuSKect-Myc was incubated with Wnt-1-HA immobilized on beads. LRP4 and LRP6 that were co-precipitated with Wnt-1 were analyzed by immunoblot with anti-Myc antibody.
Maxi-Sorp Immuno Plates (Nunc) were coated with purified LRP4-Myc at 4°C overnight, and then incubated with 1% BSA in PBS to block non-specific binding. Coated wells were incubated with purified AP fusion proteins and the AP activity was measured using pNPP as substrate.
Live C2C12 myoblasts or myotubes in 15-mm dishes were incubated at room temperature for 90 min with 500 µl of 5 nM nAgrin-AP, mAgrin-AP or AP. Cells were washed three times with the HABH buffer (0.5 mg/ml bovine serum albumin, 0.1% NaN3, 20 mM HEPES, pH 7.0 in Hank's balanced salt solution) and fixed in 60% acetone, 3% formaldehyde in 20 mM HEPES (pH 7.0) for 15 sec. Fixed cells were washed once in 20 mM HEPES (pH 7.0), 150 mM NaCl, incubated at 65°C for 100 min to inactivate endogenous AP, washed again in the AP buffer (0.1 M Tris-HCl, pH 9.4/0.1 M NaCl/5 mM MgCl2) and stained at room temperature overnight with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) (165 µg/ml)/nitroblue tetrazolium (NBT) (330 µg/ml) in the AP buffer. Digital photographs of stained cells were analyzed by using the NIH ImageJ software. In some experiments, Agrin-AP-bound cells were lyzed in the lysis buffer (1% Triton-X100, 10 mM Tris, pH 8.0). After the inactivation of the endogenous AP, lysates were assayed for AP activity using p-nitrophenyl phosphate (pNPP) as substrate.
These assays were performed as previously described (Luo et al., 2002; Zhang et al., 2007; Zhu et al., 2008). Unless otherwise indicated, the final concentration of recombinant neuronal agrin was 1 nM to stimulate muscle cells. Band intensity of immunoblot was analyzed by using the ImageJ software.
Data of multiple groups was analyzed by ANOVA, followed by a student-Newman-Keuls test. Two-tailed Student's t test was used to compare data between two groups. Differences were considered significant at P < 0.05. Values and error bars in figures denote mean ± SEM.
The authors wish to thank Dr. Zach Hall for original constructs of agrin and MuSK, Dr. Xi He for Wnt, LRP6 and Frizzled constructs, and Dr. Jon Lindstrom and Dr. Rick Rotundo for anti-AChR antibodies. We are grateful to members of the Mei and Xiong laboratories for discussion. This work was support in part by grants from NIH (L.M. and W.C.X) and Muscular Dystrophy Association (L.M.).
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LCA Tejas: An Indian Fighter – With Foreign Help
May 19/16: Indian Air Force (IAF) chief Air Chief Marshal Arup Raha became the first chief to fly the indigenous HAL Light Combat Aircraft (LCA) Tejas fighter in a short sortie on Tuesday. Raha's flight came during a visit to meet team LCA in Bengalaru which involved the inauguration of the Tejas' painting hanger. The only other top IAF officer to have flown the LCA was Deputy chief of Air Staff Air Marshal SBP Sinha, in September 2014.
India's Light Combat Aircraft program is meant to boost its aviation industry, but it must also solve a pressing military problem. The IAF's fighter strength has been declining as the MiG-21s that form the bulk of its fleet are lost in crashes, or retired due to age and wear. Most of India's other Cold War vintage aircraft face similar problems.
In response, some MiG-21s have been modernized to MiG-21 'Bison' configuration, and other current fighter types are undergoing modernization programs of their own. The IAF's hope is that they can maintain an adequate force until the multi-billion dollar 126+ plane MMRCA competition delivers replacements, and more SU-30MKIs arrive from HAL. Which still leaves India without an affordable fighter solution. MMRCA can replace some of India's mid-range fighters, but what about the MiG-21s? The MiG-21 Bison program adds years of life to those airframes, but even so, they're likely to be gone by 2020.
That's why India's own Tejas Light Combat Aircraft (LCA) project is so important to the IAF's future prospects. It's also why India's rigid domestic-only policies are gradually being relaxed, in order to field an operational and competitive aircraft. Even with that help, the program's delays are a growing problem for the IAF. Meanwhile, the west's near-abandonment of the global lightweight fighter market opens a global opportunity, if India can seize it with a compelling and timely product.
LCA Tejas: India's Lightweight Fighter
Radar Love: Weapons & Fire Control
Engines & Alternatives
In the Navy… Naval LCA
LCA Tejas: Program, Prospects, and Future
The Program
Industrial Team
Tejas Prospects: Think Globally, Begin Locally
Contracts and Key Events
2006 – 2008
Appendix A: DID Analysis & Op/Ed (2006)
Appendix B: The Kaveri Saga – Keystone, or Killer?
\Additional Readings & Sources
Background: LCA Tejas
Background: Ancillary Technologies & Weapons
Background: Tejas Mk.II Technologies
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subset: commoncrawl


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Most people enjoy the modern technologies and conveniences of today — "smart" phones, tablets, cable and satellite TV among them — but there are those who choose to live off the grid in the unspoiled wilderness, where dangers like mudslides, falling trees and bears are all part of life. "Mountain Men" profiles some of them. Eustace Conway, who has lived at the western edge of the Blue Ridge Mountains in North Carolina for more than 25 years, teaches interns about the old ways of living with nature. Tom Oar needs an entire year to prepare for the 7-month-long winter on Montana's Yaak River. In Alaska, Marty Meierotto must gather enough wood to survive — in complete isolation — winters that can have temperatures drop to 60 degrees below zero. It's not easy, but for these mountaineers, it's life as they know it.
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Fixed PriceBudget: $200
Start DateJanuary 15, 2014
I am looking for a UNITY 3D Expert for a long term project
developing multiple apps .
i am basically looking for someone to provide / build 1 or 2 full games (can be based on existing / purchesed templates) , and Reskin them for me multiple times: changing the name , logo, images , assets & the background , but maintaining the same logic .
I will provide the concepts and the 3d assets , and i need you to provide all the rest and publish the games to the different stores on my account . i
I will pay per app / copy .
basically i am talking about 2-3 new duplications every a week .
the job requires the ability to to provide all the app assets
(main 3d models are provided by me) , and publish the app to IOS/ANDROID
Is this something you can /want to do ?
About the Client
Tel Aviv 08:07 AM
Jobs Posted
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Total Spent
23 Hires, 0 Active


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Investment options
Suited to your investment needs
Building an investment portfolio can help you create a more secure and sustainable financial future and give you the freedom to live life how you want to, not how your bank balance dictates. A Commonwealth Financial Planner can give you professional advice about which investment option, or mix of investments, best suits your individual needs. When you're ready to start investing, consider scheduling a no-obligation consultation – and we'll cover the cost of your first appointment.
Term deposits
If you prefer the security of investing for a specified period of time and knowing what your investment will be worth at the end, a term deposit may be right for you.
Commonwealth Direct Investment Account
If you have a large lump sum that you'd like to earn interest on while you plan your next investment move, you may want to consider opening a Commonwealth Direct Investment Account.
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If you're looking for access to a variety of sophisticated investment types, or asset classes, managed funds may be right for you. They're an easy way to diversify your portfolio, and your money is managed by professionals.
Investment Growth Bond
Combining the benefits of a managed fund with the security of a life insurance policy in a simple, tax-effective investment vehicle, the Investment Growth Bond offers a range of investment options.
Protected loan
Commonwealth Bank offers a fixed-term Protected Loan that allows you to borrow money to build a share portfolio for yourself or your self-managed super fund (SMSF). The Protected Loan also gives you the flexibility you need to create a geared portfolio tailored to your investment strategy.
- Important information
The information contained on this web page is of a factual nature only and is not intended to constitute financial product advice. It has been prepared by Commonwealth Financial Planning Limited without considering your individual objectives, financial situation or needs. You should consider its appropriateness in light of your circumstances and consider seeking professional advice relevant to your individual needs before making a decision based on this information. Commonwealth Financial Planners are Representatives or Authorised Representatives of Commonwealth Financial Planning Limited ABN 65 003 900 169, AFSL 231139, a wholly owned but non-guaranteed subsidiary of Commonwealth Bank of Australia ABN 48 123 123 124.


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Executive Director, Marketing Services (1660)
- Employer
- Hearst Magazines
- Location
- New York
- Posted
- Nov 27, 2012
- Closes
- Dec 27, 2012
- Contact
- Hearst Recruiter
- Category
- Publishing
- Duration
- Full Time
The Company:
Hearst Digital Media is high performing team responsible for delivering digital excellence within the organization. Be the marketing voice and strategic vision —spearheading advertising-led digital marketing solutions across the entire portfolio of women's brands. Inspire the combined talents of the marketing, design, ad product, and research teams to create innovative solutions for advertising partners.
The Role:
- Lead and empower a large cross-disciplinary team: marketing, design, ad product, research
- Work with senior management to define strategy and deliver against the business objectives for women's group
- Create solutions within a diverse portfolio of sites across different categories (food, home, beauty, fashion) and platforms (mobile, social, video)
- Partner across departments and divisions (magazines, business development, editorial)
- Be a tireless innovator, cheerleader, and digital advocate
- Mentor and empower a team of smart, enthusiastic marketers
- Create and establish communication of our positioning in the marketplace (presentations, materials, research)
- Supervise the development and progress of key marketing initiatives to ensure the strategy/purpose is achieved
- Ability to identify products and platform that drive scalable results via internal and external partners
The Impact:
You are crucial to the growth of the business—shaping the strategy, message, and ad product for Hearst Digital Media. Your vision will continue to elevate Hearst as an industry innovator, capitalizing on new technologies, products, and platforms to develop revenue-generating marketing solutions. Your knowledge and passion will inform and inspire the sales and marketing teams as well as clients.
The Requirements:
- Bachelor's Degree in related field
- 10+ years experience with at least 3 years in a management position
- Exceptional digital knowledge across platforms (desktop, tablet, mobile, social)
- Advertiser-driven media marketing experience
- Strong Powerpoint skills; knowledge of comScore, Omniture, @plan; Photoshop and Salesforce a plus
- Curiosity and intelligence are required; patience and a good sense of humor are preferred
The Benefits:
- Work with and learn from best-in-class teams; our employees rock!
- Fast paced and challenging projects
- Diverse and forward-thinking leadership
- Competitive salary, benefits and training
- App Lab
- State-of-the-art building
- In-house digital studio
- Two art galleries
- Corporate gym that puts Ritz Carlton's to shame (9,000 sq. ft.)
- Company café that will satisfy the most discerning NYC foodies (350 seats)


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WE wish to report on our experience supervising second-year psychiatric residents who spend six months in adult outpatient work. The setting of our study is the Adult Outpatient Clinic in the Department of Psychiatry, University of Rochester (NY) Medical Center.
The Adult Psychiatric Outpatient Clinic was established for those in the community and surrounding counties who cannot afford private care, and affords diagnostic evaluations and therapeutic services of many types. It receives referrals from a variety of sources, and about 25% of the patients are self-referred. A detailed study of our clinic operation has been recently reported.1
Over the years we have evolved various procedures for evaluating the patients referred to us in an attempt to be more flexible and responsive and offer professional help as quickly as possible. At the present time we employ a triage method of a brief "screening" interview of every new patient by
Thank you for submitting a comment on this article. It will be reviewed by JAMA Psychiatry editors. You will be notified when your comment has been published. Comments should not exceed 500 words of text and 10 references.
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Indicate all relevant conflicts of interest of each author below, including all relevant financial interests, activities, and relationships within the past 3 years including, but not limited to, employment, affiliation, grants or funding, consultancies, honoraria or payment, speakers' bureaus, stock ownership or options, expert testimony, royalties, donation of medical equipment, or patents planned, pending, or issued. If all authors have none, check "No potential conflicts or relevant financial interests" in the box below. Please also indicate any funding received in support of this work. The information will be posted with your response.
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Madison's Richard Davis named an NEA Jazz Master
Richard Davis, a bassist and a professor of music at the University of Wisconsin — Madison, has been named one of the 2014 National Endowment for the Arts Jazz Masters, one of the highest honors for an American jazz musician.
His three 2014 fellow masters are among the nation's most elite jazz figures: Keith Jarrett, one of the leading improvising pianists ever; Anthony Braxton, a pioneering avant-garde saxophonist; and Jamey Aebersold, whose Play-A-Long series of educational recordings has been used by countless musicians. »Read Full Article(2)
Summerfest adds 80-foot Ferris wheel
What's old is new again at Summerfest.
The Ferris wheel is back. Not the Ferris wheel that once sat on the Midway but a new one soaring 80 feet into the sky and set up on the north end of Henry W. Maier Festival Park. »Read Full Article
Pete's Pops, a cart selling frozen treats, launching in June
A cart called Pete's Pops, peddling frozen-fruit confections in flavors like strawberry basil, will debut in Milwaukee within a couple of weeks, with a second expected in Wauwatosa around the Fourth of July. »Read Full Blog Post(1)
Miranda Lambert; Lucy Hale; Matisyahu; the Orwells; Paul Weller; Bob Mould; Hamilton Leithauser
Matisyahu »Read Full Article
Delayed Glendale Hofbrauhaus not likely to open in 2014
On Tap Today: Local documentary premiere 'Can You Hear Us Now?' at Oriental Theatre
Tragically, teens affected by violence in Milwaukee and beyond is an issue that isn't going away any time soon. Boys & Girls Clubs of Greater Milwaukee recognizes that, and now the new documentary "Can You Hear Us Now?" gives voice to area teens who have struggled with trauma, with gang violence, with the loss of loved ones who have been shot and killed.
A year in the making, the 40-minute documentary will premiere at 6:30 p.m. tonight at Landmark's Oriental Theatre, 2230 N. Farwell Ave. Admission is free; register for a ticket at eventbrite.com. »Read Full Blog Post


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The Planetary Society is proud to honor those in our community who help to promote the future of space exploration through two very special awards, The Cosmos Award for Outstanding Public Presentation of Science and The Thomas O. Paine Memorial Award for the Advancement of Human Exploration of Mars.
The Thomas O. Paine Memorial Award
The Thomas O. Paine Memorial Award for the Advancement of Human Exploration of Mars
2010 Honoree Dr. Stephen Hawking
Through his books, television series and public lectures, Dr. Stephen Hawking, our 2010 Cosmos Award honoree, has engaged the public for over 20 years in some of the most profound questions of our existence – from the origins of the universe to the nature of black holes.
The 2007 Cosmos Award for Outstanding Public Presentation of Science was presented October 19, 2007 in New York City to Paula Apsel, Senior Executive Producer of the Emmy Award-winning series NOVA
Filmmaker James Cameron was awarded the first Cosmos Award in recognition of his IMAX films that have allowed audiences to join true-life adventures with real explorers that are as exciting as any fictional plot that Hollywood could devise.
Beyond The Horizon, There's More To Explore!
Become a member of The Planetary Society and together we will create the future of space exploration.
Essential Advocacy
Our Advocacy Program provides each Society member a voice in the process.
Funding is critical. The more we have, the more effective we can be, translating into more missions, more science, and more exploration.
Space in Images
Pretty pictures and
awe-inspiring science.
Join The Planetary Society
Let's explore the cosmos together!


text:
How to design a killer catalogue
Boost retail sales with letterbox media.
New prepaid mobile offers from Coles and Kogan
Optus and Vodafone launch new prepaid mobile plans.
Chocoholics buy their treats from supermarkets
Chocolate bar sales in convenience good, but room to improve.
Two sports drinks or more a day for 1.5 million Australians
Powerade and Gatorade still ahead of the pack.
Coles catches bigger share of deli spend
The supermarket chain has been making steady gains.
Consumers to spend $42.1B in pre-Christmas sales
Retailers are expected to see signs of improved consumer confidence when an estimated $42.1 billion is predicted to go through retail tills before Christmas, according to Roy Morgan Research.
Business confidence shows signs of economic recovery
Roy Morgan Research's business confidence survey in July indicated there are tentative signs the Australian economy is recovering.
The hardware rival
Hardware giant Bunnings has long been a key player in the home improvement space but since Woolworths-owned Masters Home Improvement entered the market it has changed things up. Or has it?
Consumers prefer Aussie made
Following recent news of News Limited getting behind local retailers in Australia, a similar shop local campaign has been launched by Australian Made.
Smartphone, app shopping least favourite choice
According to a study compared to a recent, similar survey in the UK, Australians are about half as likely to use an app or a smartphone for online purchases.
ARA partners with Roy Morgan to industry insight
Australian Retailers Association has partnered with Roy Morgan Research to gain better knowledge and insight to retailers operating the sector.
Customers satisfied with The Good Guys
The Good Guys has increased its customer satisfaction ahead of Betta Electrical and Dick Smith in the latest customer satisfaction survey for electrical stores.
1-10 11-12


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Geno Smith Preferred To Work Through Pain
The more prudent course of action, especially so early in the preseason, might have been to give Geno Smith a day or two to rest a sore ankle.
But the rookie quarterback didn't want to take a day off, and the New York Jets didn't stand in his way.
According to Manish Mehta of the New York Daily News, Smith ?looked much slower in his drop-backs? and ?jogged with a noticeable limp,? in his first work after spraining his right ankle against the Lions Saturday. Offensive coordinator Marty Mornhinweg repeatedly told him ?don?t limp,? throughout the season, but Smith wanted to get through it.
?Those guys are constantly putting their bodies on the line,? Smith said of his teammates. ?So a little injury such as an ankle shouldn?t keep me out of practice. It?s important to show those guys that no matter what happens, I?m going to always come out there and give it my best.?
Read more on Pro Football Talk.


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Alan's Auto Recycling Inc telephone number is (605) 388-0550.
Telefax: No fax number available
Website: No web address available
Biz owner? Moderate reviews or edit listing.
Search entire category for Truck Repairing & Service
Truck Repairing and Service, in Rapid City, refers to repairing services offered at repair shops to fix worn out and dysfunctioning truck parts. Services may include repair of engine, brake, clutch, batteries, electronic systems, exhaust systems, heating and air conditioning systems, transmission service, radiator repair, fixing damaged or broken body parts, oiling and lubricating parts, etc. Repair and maintenance services keep trucks in good condition.
Governor Cuomo Launches $5 Billion Clean Energy Fund To Grow New York's Clean Energy Economy
Clean Energy Fund will advance solar, wind, energy efficiency and other clean tech industries to spur economic development and reduce harmful emissions (New York, NY) Governor Andrew M. Cuomo today announced the New York State Public Service Commission's approval of a 10-year, $5 billion Clean Energy Fund to accelerate the growth of New York's clean energy economy, address climate..


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When a person has hardwood floors they need to keep them clean and steaming hardwood floors will help get off the dirt and the grim. Steaming hardwood floors is easier to do then most people would think. The steamer can be filled just with water or a gentle cleaning solution can be added.
While the steamer heats up sweet up any loose dirt. When the steamer is ready start at one end of the room and move to the other. Use a back and forth motion for cleaning when the streamer is pushed forward stream will be released. When the steamer is pulled back the dirt from the floor will be removed. This should be done on the floor until it is clean. If the floor is wet it can be gently dried with a clean cloth.
Steaming a hardwood floor will help remove stuck on dirt and grime. Within a matter of minutes a person can enjoy a clean floor once again.
Welcome to www.eggmag.co.uk


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Senior Biztalk Developer needed
This project received 9 bids from talented freelancers with an average bid price of $77 AUD / hour.Get free quotes for a project like this
Skills Required
Project Budgetmin $50 AUD / hour
Total Bids9
Project Description
Our client is a leading technology consulting and solutions company who is in need of several Senior BizTalk Developers for a big project starting up in Perth.
The successful candidate will be able to hit the ground running, and be able to meet every deadline that's presented.
Experience required:
- BizTalk 2010 Enterprise Experience
- 5+ Years Development and Architecture using BizTalk
- SQL Server 2008 R2 and 2012 Experience
- SAP Integration Experience and Knowledge
The contract will initially cover a period of 3 months, with the possibility of extension included.
Contact me for further details on (03) 9017 0779.
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- Times Online


text:
Erie hospital has new website
Saint Vincent Hospital has switched its online home to Allegheny Health Network's new website, www.ahn.org.
People who type the Erie hospital's former web addresses will automatically be redirected to Saint Vincent's home page on the AHN website. The site includes pages for all eight of the network's hospitals.
"A singular website that unites all of our facilities is a natural extension of our branding," said AHN spokesman Dan Laurent.
It took about a year to create the site, which is based on the award-winning site for the former West Penn Allegheny Health System.
"The website launched (Monday), though we had a soft launch of the website a few weeks ago to get people used to seeing ahn.org," said Julie DeMarco, Allegheny Health Network's project leader for the website's development.
Saint Vincent patients will still be able to locate a primary care or specialty physician, find out when a particular office is open, and learn about specific procedures and treatments.
"If they search for a physician, they will get all of the ones in the Allegheny Health Network," DeMarco said. "They then can use the filtering tools to narrow their choices."
Patients can now request a physician appointment through the site, DeMarco said. The site also offers links for AHN services not offered at Saint Vincent, including transplants.
Westfield (N.Y.) Memorial Hospital, which was part of Saint Vincent Health System before it joined AHN, also has its own page on the new site.
Saint Vincent's Twitter account is also now part of AHN, though the hospital still has a separate Facebook page.
DAVID BRUCE can be reached at 870-1736 or by e-mail. Follow him on Twitter at twitter.com/ETNbruce.


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Interest Level
Grades K - 2
Reading Level
Grade level Equivalent: 4.3
Lexile® Measure: AD700L
Guided Reading: O
- Informational
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Elizabeth Cady Stanton stood up and fought for what she believed in. From an early age, she knew that women were not given rights equal to men. But rather than accept her lesser status, Elizabeth went to college and later gathered other like-minded women to challenge the right to vote. This inspiring story is about an extraordinary woman who changed America forever because she wouldn't take no for an answer
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Context Evidence-based medical education requires rigorous studies appraising educational efficacy.
Objectives To assess trends over time in methods used to evaluate undergraduate medical education interventions and to identify whether participation of medical education departments or centers is associated with more rigorous methods.
Data Sources The PubMed, Cochrane Controlled Trials Registry, Campbell Collaboration, and ERIC databases (January 1966–March 2007) were searched using terms equivalent to students, medical and education, medical crossed with all relevant study designs.
Study Selection We selected publications in all languages from every fifth year, plus the most recent 12 months, that evaluated an educational intervention for undergraduate medical students. Four hundred seventy-two publications met criteria for review.
Data Extraction Data were abstracted on number of participants; types of comparison groups; whether outcomes assessed were objective, subjective, and/or validated; timing of outcome assessments; funding; and participation of medical education departments and centers. Ten percent of publications were independently abstracted by 2 authors to assess validity of the data abstraction.
Results The annual number of publications increased over time from 1 (1969-1970) to 147 (2006-2007). In the most recent year, there was a mean of 145 medical student participants; 9 (6%) recruited participants from multiple institutions; 80 (54%) used comparison groups; 37 (25%) used randomized control groups; 91 (62%) had objective outcomes; 23 (16%) had validated outcomes; 35 (24%) assessed an outcome more than 1 month later; 21 (14%) estimated statistical power; and 66 (45%) reported funding. In 2006-2007, medical education department or center participation, reported in 46 (31%) of the recent publications, was associated only with enrolling more medical student participants (P = .04); for all studies from 1969 to 2007, it was associated only with measuring an objective outcome (P = .048). Between 1969 and 2007, the percentage of publications reporting statistical power and funding increased; percentages did not change for other study features.
Conclusions The annual number of published studies of undergraduate medical education interventions demonstrating methodological rigor has been increasing. However, considerable opportunities for improvement remain.
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UFC on FOX 5: Raphael Assuncao vs Mike Easton
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post #2
Thread Starter
Gabba Gabba Hey
Join Date: Sep 2008
Location: Pensacola, FL
Posts: 19,934
Round 1 - Referee Steve Newport in charge of this bantamweight matchup. Easton bouncing on the outside, and he misses a spinning kick. Assuncao with a snapping low kick, and he's patient from the outside to start. Easton flurries inside. Assuncao moves out of the way and throws up a high kick. Counter straight for Assuncao, who looks content to let his opponent come to him. Assuncao moving his head and looking for angles, but Easton's non-stop movement making that hard. Halfway mark. Assuncao misses on a high kick. Easton starting to reach with his punches, and Assuncao is timing them well. Final minute. Close round. It's Easton moving forward, but Assuncao is having success on the feet. Assuncao shoots in, but Easton defends well. Assuncao stays tight in a clinch. Reset in the final 10 seconds. MMAjunkie.com leans toward Easton, 10-9, but Assuncao had moments and seemed to claim some late momentum.
Round 2 - Assuncao whiffs on a high kick, as does Easton. Assuncao still content to let Easton come to him. Assuncao misses a kick but comes back with a slick backfist that scores. Leg kicks traded. Low kick from Assuncao, but he eats a counter. Assuncao starting to walk forward a bit, and Easton looks a little out of sorts in terms of rhythm. Easton complains of an eyepoke, but Newport didn't see anything, so the fight carries on. Halfway mark, and big exchanges. Assuncao into the clinch, and he lands a knee to the cup. Assuncao didn't think it was low, but Easton gets a break. Easton aggressive again on the restart. He's definitely moving forward, but Assuncao seems to be more effective in his efforts. Crowd wants the pace to quicken. Assuncao kicks the face and the leg. Easton shoots in, but Assuncao sprawls and pulls easily away in the final minute. Assuncao misses a spinning backfist and stays upright when Easton pushes in. Another close round, but MMAjunkie.com leans toward Assuncao, 10-9.
Round 3 - Easton again the one moving forward, but he's unable to really land anything. Kick to the body for Assuncao, who follows again with a left. Assuncao switches to southpaw and lands two punches. He's managing the distance well and staying away from opponent's attacks while launching his own. Assuncao shoots in and looks for a takedown. He gets Easton off-balance but can't take top position. Easton scrambles away, though Assuncao chases and shoots in again. Halfway mark, and Assuncao tries again for a takedown that won't come. Crowd again implores action, but Assuncao is using smart tactics as he stay away from the big shots. Assuncao pushes in again to the clinch but lets it go as the final minute opens. Easton kicks low but eats a counter. Assuncao a little off-balance, and Easton wants a takedown, but the Brazilian pulls away.Round ends just as it played out all along. Close again, but MMAjunkie.com leans toward Assuncao, 10-9, and awards him the fight 29-28. Raphael Assuncao def. Mike Easton via unanimous decision (29-28, 30-27, 30-27).
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Utah Valley Wolverines at Houston Baptist Huskies Basketball
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You may secretly -- or not so secretly -- steer clear of collaborative projects, but Stephanie Buckhanon Crowder highlights four key benefits.
Higher Education Career Advice
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Anxious about landing an academic job after graduation, Shantá R. Robinson ultimately discovered that true "How I Made It" stories involve uncertainties, mishaps and mistakes.
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August 26, 2016
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Once Roberto Abadie came to the realization that he wouldn't get tenure, he began to see the upside.
August 24, 2016
Hillary Clinton's comment at the Democratic convention suggests we might be entering an era in which women can see ourselves represented in many diverse champions, writes Judith S. White.
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Much of the conversation about career exploration focuses on the importance of identifying our skills, but we often don't take the time to think about our core values and how they connect to our skills, argues Laura N. Schram.
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None of those games are really suitable for 5 or 8 year olds though are they.Originally Posted by Sovieto
"as amusing as your comment it its not entirely true. most 360s dont break and even when they do, in most situations it doesnt break multiple times"
I know this dont apply to all 360s but my brother had to ship his back twice... but he think it may have been fudged in the shipping truck on the return.
None of those games are really suitable for 5 or 8 year olds though are they.Originally Posted by Sovieto
Originally Posted by PIZ
The gamestops/eb games/etc by me have them on display as well as the Target's/Walmarts/Etc however usually they aren't hooked up. But atleast at a store like gamestop or something it's relatively small so if it isn't hooked up you have a chance of someone working there helping you out and hooking it up for you to test it out. Also the nice part about those stores is the emplyees tend to be gamers where as a lot of the time(not all the time though) the employees at big department stores aren't that into gaming, it's just a paycheck(nothing wrong with that, i've been in that boat before too). So even if the Wii at gamestop can't be hooked up you generally can speak to someone who knows the system and the games.
I am fast. To give you a reference point, I'm somewhere between a snake and a mongoose. And a panther.
~Argue for your limitations,and they're yours...
-Feel free to add my wii and send me a message and i'll add you back
-Mario Strikers Charged: 442495 403215
-MoH: H2 Persona- DexterMorgan114
uhh that wasnt the point :/Originally Posted by raisinghelen
Everything in this signature except for this text was made by Wiired, he is my creator and almighty.
well yeah it was because we are trying to see which console is suitable for this mans kids who are 5 and 8
Mr wii man is here
mario strikers fc code: 232037 285654
wii fc code : 5367 7688 3777 9378
pm me if u wanna add
AHHHHH Hitler had stormtroopers
Then what was the point? Its supposed to be a thread helping decide what console would be suitable for 5 and 8 year old kids, not one extoiling why a console is good due to the amount of mature games it has. Besides if you are including unreleased 360/PS3 games in their 'good game list' then you could also include the upcoming Wii games too, like Mario Galaxy for a start.Originally Posted by Sovieto
It would be more helpful to list the 360 or PS3 games that would actually be good for young children. The only decent reason ive read so far is that the 360 is similar to the original Xbox so the controls wouldnt be much of a change.


text: As Senate Leaves For Recess, House Keeps Working On A Border Bill By Tamara Keith • Aug 1, 2014 ShareTwitter Facebook Google+ Email Listen Listening... / Originally published on August 1, 2014 6:14 am Copyright 2014 NPR. To see more, visit http://www.npr.org/. Related Programs: Morning Edition on WKNOMorning Edition on WKNO HD-2View the discussion thread.


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Home > Sunglasses > Tom Ford > ANOUSHKA (FT0371 - 20B)
Anoushka FT 0371 is available in 3 additional attractive styles
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Size and availability
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These sunglasses are the perfect accessory on the beach, on the way to the office or in the city. There is only one slight drawback: all the envious looks you will attract. The FT0371 is available in the Edel-Optics online shop in other styles from Tom Ford collections 2015 and 2016.
This frame was specially designed for genuine power women. Graceful design and expressiveness are interspersed with classical chic. Eyewear is in. Full rim is extremely popular, because you feel that you get more eyeglass for your money. It is not just about their resilience, but also about the visibility of material and design, which are of the highest quality. The butterfly look is characterized by the thick full frame. The classic shape has an enduring popularity and is always en vogue. Stars like Marilyn Monroe made the look famous. This eye-catching shape is particularly suitable for faces with broad cheeks. The butterfly shape attracts everyone's attention. Due to its extravagance, it is absolutely perfect for confident personalities. The look is particularly suitable for somewhat shorter faces because the special shape makes the faces appear elongated and, thus, more slender. Plastic frames like these combine durability and are comfortable to wear. The FT0371 is a very snug fit on the ears and nose. Grey suits every style and every outfit. It looks good with almost every skin tone and is more unobtrusive than black. Eyewear in grey is your perfect everyday companion. Grey lenses are a classic in sunglasses. Their high degree of neutrality with regard to the field of view, makes them particularly suitable for everyday use. Functionally, you are, of course, on the safe side, too. With 100% UV eye protection, the sun can immediately start shining.
If this pair is your favourite, you can order them without hesitation. We have them in stock and can send them off immediately at a super affordable Edel-Optics price. If you buy at Edel-Optics you get the best price, because "in the sale" is our standard.
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Environmental Management Companies serving Ghana
Current filtering criteriaYou can remove a filter by clicking on the x
Category: Environmental Management ×
Subcategory: Environmental Management ×
Employees number: 11-100 ×
Industry: Energy - Climate Change ×
Serving: Africa ×
Serving: Ghana ×
ICT International
based in Armidale, AUSTRALIA
ICT International has been designing, manufacturing and providing monitoring solutions for soil, plant and environmental research since 1982. The success of our instruments stems from our worldwide experience in the application of quantitative technologies to soil moisture monitoring, plant water use and environmental studies.Our systems are at the cutting edge of ...
ifu Hamburg GmbH
based in Hamburg, GERMANY
ifu Hamburg GmbH (Institute for Environmental Informatics Hamburg Ltd.) is a software development and consulting company helping companies to achieve sustainable production and strengthen their competitiveness. We deliver software solutions that support corporate environmental tasks and activities such as Material Flow Management, Resource Efficiency, Life Cycle ...
Macdonald & Company - Environmental & Energy
based in London, UNITED KINGDOM
Macdonald & Company Environmental & Energy is a specialist team of qualified recruiters sourcing Environmental & Energy professionals globally. Our team covers the vast range of professional disciplines across both sectors and sources jobs for individuals at all levels. We have offices in the UAE, United Kingdom, Hong Kong, and South Africa, enabling us to offer global ...
CSRware, Inc.
based in San Francisco, CALIFORNIA (USA)
CSRware offers Conflict Minerals Management, Supply Chain and Energy & Sustainability software with EH&S integration. Meet CMRT, SEC reporting or other regulatory deadlines quickly, easily and accurately. To do your job well, you need accurate information at the right time. Unfortunately, that information lives in an ever-increasing mess of fragmented spreadsheets, ...
Certification Europe
based in Dublin, IRELAND
Certification Europe is an accredited certification body which provides International Organization for Standardization (ISO) management system certification and inspection services to organisations globally. ISO Standards provide a recognised framework to achieve best practice management. Certification Europe can certify your organisation to Quality (ISO 9001), ...


text:
street rods
Here are some keywords which we've determined lie within the same circle of relevance as street rods.
rods, street, cars, muscle cars, hot, classic cars, hot rods, hot rod, ford, custom, classic, sale, news, parts, trucks, rod parts, rod, rat rods, old, kits
1. auto auto-roundup.com
2. round auto-roundup.com
Enter a domain name or a keyword.
# a b c d e f g h i j k l m n o p q r s t u v w x y z


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Now its $ 126 and sinking . Oh, what a feeling !
Put the bottle down Dead-Ender. Yeah it's up 4x! Great drunken post. Now back to the assembly line lunchtime is over.
You have it all wrong.Government Motors is in that range.A company with lots of money & good managemaent without unions blood suckers price out over $100.Do your research doofus.


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a incredibly large and humungous dick/penis
"ok, now get on your knees and suck my donkey!"
"my donkey is so big that it could eat your donkey!"
"my donkey is so big that it could eat your donkey!"
de M "Smoke'M" Crackaz 25 août 2006
Something you yell out after you trick someone. Kind of like "made you look" but its cooler.
For example, if you're playing basketball and you throw a fake pass and the guy defending you turns and looks for the ball, you might yell DONKEY.
de HRadam 27 novembre 2005
A big swinging dick. Sourced from "hung like a donkey" or "donkey dick."
My friend Karen loves donkey!
de Robert MacKenzie 29 août 2005
Here comes Jesus riding on a donkey. Hosanna, hosanna. Hosanna to the King!
de Steven 3 mars 2004
When used as an adverb donkey implies sexual gusto.
She went donkey on that monkey.
de Elroy Fitz 1 février 2004
An assignment (ass)
I have to go work on my donkey.
de Kiomo 29 octobre 2003
A term of identification used for anoyone who can closely imitate the stunned demeanor that donkey kong takes when he receives a serious blow to the head. This shortened form is more easily said in conversation than "donkey kong". D.K. is just too cool to be considered for such a person. Also, it carries connotations of Shrek's sidekick.
Josh Good is donkey. Call him that when you see him on Lycoming Campus and elsewhere.
de shorky 17 février 2005


text: Why Lily Allen isn't Lily Cooper anymore Tue-21-Jan-14 08:40 Share this Tell Us What You Think WTF 0 LOL 0 FAIL 0 OMG 0 HOT 0 CUTE 0 ] By Jay-Jay FeeneySinger LILY ALLEN has made a comeback using her maiden name even though she calls herself Lily Rose Cooper in real life because her record company bosses forced her to.She wanted to relaunch herself using her married name but her label insisted she had to keep using the name she was most known by. You May Also Like Grounded Lilly Allen honeymoons in Paris Is Lily Allen's marriage in trouble? Lilly Allen face of U.K.'s Elle August cover Lily Allen & Azealia Banks continue their Twitter Feud Become a fan of The Edge on Facebook and on Twitter .


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Hello Dear Readers, My promises to update this blog regularly… OK, I'm not even going to insult your intelligence. In my defence (or defense depending on where you are reading this) I have been writing… I'm also going to appear at a few events during the upcoming weeks (nice change of subject there). The first is an author Chat arranged by the Women's National Book Association- Atlanta Chapter! A bit about this organisation: The Women's National Book Association was established in … →
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BRIGHTON — City Council received the city's annual report during its July 1 board meeting and approved it unanimously following City Manager Manuel Esquibel's presentation.
"This is just a quick little snapshot of what's gone on in the past year," he said. "This is the fifth time we've done it in this manner, and so it's been received by our citizens very well."
The annual report contains a list of the city's accomplishments in 2013, along with demographic and economic statistics, information about the city's sales tax revenue and the city's revenues and expenses. The report also gives information on how the city's essential services were utilized during the year.
The Brighton Police Department received 622 more calls for service in 2013, bringing the total number of service calls to 34,195. Although there was an increase in calls, the department had 351 fewer arrests — bringing the total number of arrests to 2,365 — and 761 fewer summons issued — bringing the total number of summons issued to 5,225. The courts processed 5,510 citations in 2013, 457 fewer citations than were issued in 2012.
The water department pumped a total of 1.077 billion gallons of water last year, 0.228 billion gallons fewer than the previous year. Throughout 2013, residents in the city used an average 4.44 million gallons of water each day, about 0.9 million gallons less than in 2012. The city was able to treat about 24 million gallons of water more than it used in 2012, bringing the total amount of water treated in 2013 to 755 million.
The Recreation Center and Eagle View Adult Center served more people in 2013. The recreation center had 19,770 more admissions in 2013, bringing the total number of admissions to 174,553, and had 140 more program participants than the 10,380 it served in 2012. Eagle View Adult Center had 1,748 more participants than the 154,783 it served in 2012. The Parks and Recreation department had 399 fewer participants in its service programs, down from 11,070 participants in 2012.
The Publics works department was able to resurface 0.3 miles of street in 2013 and slurry sealed 3.7 miles of road, a 0.7-mile increase in repairs made over the previous year. The cemetery saw 10 more interments, up to 138 from 128 in 2012.
The city collected $19,330,483 in sales tax last year, which was included in its total revenues of 58,821,000. The city's expenditures were $51,66300.
Copies of the city's annual report can be found at city hall or on the city's website www.brightonco.gov.
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text: Hi there ! A customer is planning to use our cluster suite (most probably with 2 nodes cluster running RHEL5 cluster suite). The fencing mechanism will be some rsaII cards. Right now, the rsaII cards are on a another network than the one used for heartbeat. The problem is, it is dedicated to an admin team wich does not want the cluster suite solution to have access to this specific network. The customer does not know yet if he will accept to create a new network and put the rsaII cards (and only them) on it. Would it be a problem as far as the cluster suite and the support coverage is concerned if those devices were available on the same network as the one used for heatbeat ? I would be tempted to say yes to both, because i dont know how one node could fence the other in case there was a communication problem between the 2 node on the heartbeat. The following article seems to states it can be done (even without any quorum disk): http://kbase.redhat.com/faq/docs/DOC-16712 The article is still unverified though. Is it ok to give the customer a green flag using a two node cluster (without quorum disk) where heartbeat and fencing use the same network ? -- Pierre Amadio <pamadio redhat com> Technical Account Manager mobile: +33 685 774 477 Red Hat France SARL, 1 rue du Général Leclerc, 92047 Paris La Défense Cedex, France. Siret n° 421 199 464 00064


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Shadey's Rugged Run
April 27, 2013
Shadey's Rugged Run is a 3.5 mile obstacle/mud run designed to challenge participants as they run through mud, fire, water, walls and much more! Proceeds benefit the Lancaster Bible College Athletic Department and Penn State Hershey Children's Miracle Network. 7175608200
Saturday, April 27th at Lancaster Bible College
First wave starts at 9:00am
Last wave starts at 2:30pm
www.shadeysruggedrun.com to sign up


text:
What is a qui tam lawsuit?
Qui tam allows a private individual to file a lawsuit in the name of the U.S. Government, charging fraud by government contractors and other entities who receive or use government funds, and share in the recovery of any monies received (as much as 30% of the total). Successful qui tam suits help stop dishonest conduct and prevent similar conduct from happening in the future.Initially enacted during the Civil War, the False Claims Act is also known as the "Lincoln Act," the Whistleblower's Act, or (more recently) the qui tam statute. This is a simplified version of the original Latin phrase "qui tam pro domino rege quam pro seipse," which translates to "he who sues for the king as for himself." The law initially targeted dishonest suppliers during the war because the government could not investigate and prosecute the fraud. This extends into today, since our federal government is enormous and is unable to look at every small detail of taxpayer funds.
Since 1986, more than 4,000 qui tam suits have been filed after the federal false claims act was strengthened to make it easier and more rewarding for private citizens to sue. The government has recovered billions as a result of these suits, and hundreds of millions have been paid out to whistleblowers.
LaBovick Law Group – Civil Justice Prosecutors
Filing A Qui Tam Lawsuit
A private citizen, often known as a relator or a whistleblower, can file qui tam claim by filing under the False Claims Act in the United States District Court and must be served on the government. The defendant must NOT be served until the courts orders this to be done. It must be filed under seal, and must be accompanied by a comprehensive memorandum, not filed in court, but served on the government, detailing the facts of the complaint, together with copies of all relevant documents.It is not permissible for the attorney or the relator/whistleblower to discuss the case or to disclose its existence to anyone, including the defendant and the media. If they do so it could jeopardize the case and affect the government's ability to investigate the allegations in secret. Failure to follow these unique statutory requirements can result in a dismissal of the action.
After the complaint is filed under seal, and the memo and documents are served on the government, it has 60 days to intervene or decline to intervene, move for an extension of time to determine whether to intervene, seek dismissal of the action, or settle the case. Usually, the government will request numerous extensions of the 60-day initial investigatory period, since, 60 days is an unrealistically short period of time for the government to complete an investigation.
Upon completion of its investigation, the government has the option to take over the case. Regardless of whether the government intervenes, the relator/whistleblower (if he or she followed the proper procedures and is not precluded from recovery) is still entitled to a share of the recovery, and may pursue the case himself or herself on behalf of the government.The government allows a relator to receive a part of the proceeds if funds are recovered from the defendant. The relator/whistleblower is entitled to at least 15 percent, but no more than 25 percent, of what the government recovers if the government joins the case. If the relator/whistleblower proceeds with a lawsuit against a defendant after the government decides not to be involved in the lawsuit, the relator/whistleblower is entitled to at least 25 percent, but not more than 30 percent, of the money the government recovers which includes damages for the false bills, tripled, plus civil penalties of from $5,000 to $10,000 per false claim. To recover this reward, the relator/whistleblower must follow the statutory requirements. Simply providing basic information to the government does not entitle a whistleblower to recover under the False Claims Act. To state a cause of action under qui tam, a relator may allege that defendant either:
Knowingly presented or caused to be presented, to an officer or employee of the United States government a false or fraudulent claim for payment or approval.
Knowingly, made, used, or caused to be made or used, a false record or statement to get a false or fraudulent claim paid by the government.
Conspired to defraud the government by getting a false or fraudulent claim allowed or paid.
Most of the state false claims acts are based on the federal False Claims Act, and allow qui tam whistleblowers to bring claims on behalf of the state. However, there are important differences between these state false claims acts, and potential whistleblowers must pay careful attention to the specific requirements of each statute.The following 28 states have enacted their own False Claims Acts:
California — Colorado — Connecticut — Delaware — Florida — Georgia — Hawaii
Illinois — Indiana — Louisiana — Maryland — Massachusetts — Michigan — Minnesota
Montana — Nevada — New Hampshire — New Jersey — New Mexico — New York — North Carolina
Oklahoma — Oregon — Rhode Island — Tennessee — Texas — Virginia — WisconsinIt is important that a whistleblower seek the assistance of an attorney that is experienced and knowledgeable on the federal and state statutes. In some instances a fraud scheme can impact the federal government and multi-state governments. Many states enacted a False Claims Act after Congress gave financial incentives to states with false claims acts that are modeled after the federal False Claims Act. These financial incentives have helped states recover billions of dollars in new found revenue for their states. It is highly likely that more states will enact a False Claims Act to maximize the revenue for their state. There are three municipalities with their own false claims acts: Chicago, New York, and the District of Columbia. Do you have any questions about the False Claims Act, Tax Fraud or Qui Tam? Call us today for a free case evaluation. We are available 24 hours a day/7 days a week to discuss your personal matter at 561-655-1777 or toll-free at 866-522-6842.