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A major question in cell biology is how molecular specificity is achieved by different growth factor receptors that activate apparently identical signaling events. For the neurotrophin family, a distinguishing feature is the ability to maintain a prolonged duration of signal transduction. However, the mechanisms by which neurotrophin receptors assemble such a sustained signaling complex are not understood. Here we report that an unusual ankyrin‐rich transmembrane protein (ARMS+kidins220) is closely associated with Trk receptor tyrosine kinases, and not the EGF receptor. This association requires interactions between transmembrane domains of Trk and ARMS. ARMS is rapidly tyrosine phosphorylated after binding of neurotrophins to Trk receptors and provides a docking site for the CrkL–C3G complex, resulting in Rap1‐dependent sustained ERK activation. Accordingly, disruption of Trk–ARMS or the ARMS–CrkL interaction with dominant‐negative ARMS mutants, or treatment with small interference RNA against ARMS substantially reduce neurotrophin‐elicited signaling to ERK, but without any effect upon Ras or Akt activation. These findings suggest that ARMS acts as a major and neuronal‐specific platform for prolonged MAP kinase signaling by neurotrophins.
Growth factor signaling is characterized by a common set of events, including receptor dimerization, protein phosphorylation, recruitment of adaptor proteins and stimulation of downstream signaling cascades. Many signal transduction events are shared between different cellular receptor systems, raising the issue of how molecular specificity is achieved to give a unique biological response (Chao, 1992). This is particularly relevant to neurotrophin signaling, which is responsible for a wide variety of neuronal functions including survival, differentiation, axonal and dendritic growth, cell death, neurotransmitter secretion and neuronal activity (Bibel and Barde, 2000; Poo, 2001; Chao, 2003; Huang and Reichardt, 2003).
The actions of neurotrophins depend upon two different transmembrane receptor types, the Trk receptor tyrosine kinase and the p75 neurotrophin receptor that belongs to the TNF receptor family (Huang and Reichardt, 2003). Nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and NT‐4 all recognize the p75 receptor, but NGF binds preferentially to TrkA, BDNF and NT‐4 to TrkB, and NT‐3 to TrkC receptors. Trk and p75 receptors can engage distinct signaling modules when expressed independent of each other (Carter et al, 1996; Yoon et al, 1998). In the nervous system where both receptors are frequently co‐expressed, however, p75 receptor also acts as a co‐receptor for Trk to increase neurotrophin's binding affinity and to enhance Trk specificity for cognate ligands (Bibel and Barde, 2000; Chao, 2003).
What are the critical signaling events that emanate from Trk receptors? After neurotrophin binding, activated Trk receptors recruit adaptor proteins, such as Shc and FRS‐2, and effectors, such as phosphatidylinositol 3‐kinase (PI3‐kinase) and phospholipase‐C‐γ (PLC‐γ) (Chao, 2003; Huang and Reichardt, 2003). The key docking sites on the Trk receptor are tyrosine residue 490 in the juxtamembrane region and tyrosine residue 785 at the end of the cytoplasmic domain, which binds to PLC‐γ. Activation of PI3‐kinase and MAP kinase activities are both mediated by Shc protein binding to the Trk receptor at tyrosine 490 (Huang and Reichardt, 2003).
Despite our understanding of neurotrophin signaling, many of these pathways, such as ERK, Akt, PLC, PKC and Ras, are not unique to neurotrophin signaling. Each signaling component is universally used in many different contexts and by other growth factors and cytokines. This problem is accentuated by the findings that, in addition to cell differentiation, neurotrophins have effects upon many aspects of neuronal activity that result in synaptic responses that are extremely rapid (Blum et al, 2002) or that can be long lasting (Sherwood and Lo, 1999).
A distinguishing feature of neurotrophin signaling has been the sustained activation of MAP kinase activity (Marshall, 1995). Continuous ERK activity occurs for hours after NGF treatment of PC12 cells, whereas EGF treatment provides a transient activation (Qui and Green, 1992; Cowley et al, 1994). The small G protein Rap1 accounts at least in part for the ability of neurotrophins to signal for long time periods, by activating the B‐Raf and the MEK/ERK pathway (York et al, 1998). Recent studies further implicate a critical role of the Rap1–B‐Raf pathway in retrograde endosomal signaling (Kao et al, 2001; Wu et al, 2001; Stork, 2003).
A still unanswered question is how prolonged signaling through the Rap1/ERK pathway is connected to the Trk receptor. A resolution to this issue would provide an insight into how neurotrophins are able to signal in a manner that selectively triggers a sustained MAP kinase response, as well as explain how trophic responses unique to neurotrophins are generated. Here we report that a novel transmembrane protein substrate of Trk receptors, Ankyrin‐Rich Membrane Spanning (ARMS) protein, is directly involved in these downstream signaling events for neurotrophins.
Kinetics of neurotrophin signal transduction
The ARMS gene was originally identified as an interacting protein for the p75 neurotrophin receptor (Kong et al, 2001), as well as a target for protein kinase D, Kidins220, or a kinase D‐interacting substrate of 220 kDa (Iglesias et al, 2000). ARMS is a highly conserved 220 kDa protein possessing 11 ankryin repeats, four closely spaced transmembrane domains and a large C‐terminal cytoplasmic tail. The ARMS protein is tyrosine phosphorylated by Trk receptors in response to NGF and BDNF (Kong et al, 2001), but it lacks the usual binding motifs associated with tyrosine kinase substrates, such as pleckstrin or SH2 domains. Since propagation of Trk signaling is associated with rapid tyrosine phosphorylation, we assessed how rapidly the ARMS protein is phosphorylated compared to more well‐known Trk substrates, such as Shc and PLC‐γ.
In PC12 cells treated with NGF for brief intervals, tyrosine phosphorylation of ARMS occurred simultaneously with the detection of phosphorylated TrkA receptors (Figure 1A). The phosphorylation of ARMS also appeared at the same time as Shc and PLC‐γ, indicating that ARMS was among the first proteins to be activated by NGF. This rapid phosphorylation was also observed in primary cultures of cortical neurons treated with BDNF (Figure 1B and Supplementary Figure S1). By contrast, other downstream events, such as activation of ERK and Akt, proceeded after the phosphorylation of ARMS, Shc and PLC‐γ (Figure 1A), consistent with their known roles in the Trk signal transduction cascade (Huang and Reichardt, 2003).
Transmembrane interaction of ARMS with Trk receptors
The rapid phosphorylation of ARMS by Trk suggests that, like Shc and PLC‐γ, ARMS may be directly associated with Trk receptors. To test whether an interaction exists between these two proteins, TrkA, TrkB and TrkC receptors were coexpressed with FLAG‐tagged ARMS protein in HEK293 cells. The associations were assessed by co‐immunoprecipitation. Indeed, all the three Trk receptors associate with ARMS (Figure 2A). In contrast, ARMS did not interact with the EGF receptor (Figure 2B), consistent with our previous finding that EGF does not stimulate ARMS tyrosine phosphorylation (Kong et al, 2001).
To verify that the Trk–ARMS interaction also occurs in vivo, detergent lysates of E18 cortical neurons were immunoprecipitated with an antiserum raised against Trk, followed by Western blotting with anti‐ARMS antibody. As shown in Figure 2C, stable complex formation between TrkB and ARMS was clearly detected. Furthermore, punctate staining of ARMS protein was found to co‐localize significantly with TrkB in E18 cortical neurons (Figure 2D), thus supporting the immunoprecipitation analysis of an endogenous Trk–ARMS interaction.
To identify the domains responsible for the interaction between ARMS and the Trk receptor, a series of truncations were made in the Trk receptor and the ARMS protein (Figure 3). Each truncated mutant was tested in cotransfection and immunoprecipitation experiments. Trk receptors contain two extracellular IgG domains responsible for ligand binding, a single transmembrane sequence and a consensus tyrosine kinase domain in the intracellular domain. Notably, neither the cytoplasmic domain nor the extracellular domains of Trk were sufficient to maintain an interaction with ARMS (Figure 3B; Supplementary Figure S2), suggesting that transmembrane or nearby sequences of Trk were responsible. Indeed, this interpretation was supported by an analysis of a chimeric receptor of TrkA that contained the transmembrane domain of the EGF receptor flanked by the extracellular and intracellular domains of TrkA (Figure 3A). Unlike other Trk mutants that were tested, TrkA‐TM EGFR did not interact with ARMS in immunoprecipitation experiments (Figure 3A and B).
The domain of ARMS necessary for Trk association was also determined. A large deletion of the N‐terminal ankyrin repeats as well as a deletion of the C‐terminal tail of ARMS did not interfere with its association with the Trk receptor (Figure 3C and D; Supplementary Figure S3). Consistent with the Trk mapping analysis (Figure 3A and B), the transmembrane segments of ARMS (i.e. ARMS3 and ARMS4.2) were found to interact with Trk receptors and can do so without the majority of intracellular sequences (Supplementary Figure S3). These results were corroborated by testing whether the ARMS transmembrane domains alone exerted any effect upon Trk association with ARMS. We therefore expressed the fourth transmembrane segment of ARMS (ARMS4.2) in the presence of full‐length epitope‐tagged ARMS and TrkA receptors in HEK293 cells. The association of FLAG‐tagged ARMS and Trk was assessed as a function of increasing levels of ARMS4.2. A noticeable decrease in the association of ARMS and TrkA was observed in the presence of ARMS4.2 protein (Figure 3E). Taken together, we conclude that the transmembrane domains are major sites of interaction between ARMS and Trk, which can be disrupted by expressing the membrane‐spanning segments of ARMS.
ARMS‐dependent neurotrophin signaling
The interaction between Trk receptors and ARMS requires transmembrane domains of each protein, suggesting that the association is constitutive and that ARMS is uniquely positioned to coordinate rapid Trk signal transduction via its multiple protein interaction motifs. We therefore sought to characterize the role of ARMS in neurotrophin action. Since ectopic expression of the transmembrane domains of ARMS (ARMS4.2) disengages endogenous TrkA–ARMS complexes (Figure 3E), the effect of ARMS4.2 on NGF signaling was first examined. PC12 cells transfected with full‐length ARMS or ARMS4.2 were compared for neuritogenic responsiveness to NGF. While the majority of control (GFP‐transfected) PC12 cells displayed pronounced neurite outgrowth after 2 days of NGF treatment, expression of wild‐type ARMS augmented NGF‐induced neuronal differentiation (Figure 4A). Significantly, PC12 transfectants expressing the ARMS4.2 mutant protein displayed a 50% decrease in neurite‐bearing population (Figure 4A) after comparable duration of NGF treatment. These data suggest that ARMS functionally interacts with TrkA and that disruption of this interaction results in aborted NGF‐dependent signal transduction critical for neurite outgrowth.
Neuronal differentiation of PC12 cells is closely associated with sustained MAP kinase activation elicited by NGF, whereas mitogenic growth factors, such as EGF, induce a relatively transient activation of MAP kinase (Marshall, 1995). To assess if ARMS modulates these signaling events, MAP kinase activation in ARMS4.2‐transfected PC12 cells was monitored by immunostaining with phospho‐MAP kinase (p‐MAPK)‐specific antibodies. As shown in Figure 4B, PC12 cells expressing FLAG‐ARMS or FLAG‐ARMS4.2 were stimulated with NGF for 5 or 40 min, two time points that can discriminate between transient and sustained MAP kinase activation (Qui and Green, 1992; York et al, 1998). The total numbers of p‐MAPK‐positive cells expressing GFP, FLAG‐ARMS, or FLAG‐ARMS4.2 were similar after NGF treatment for 5 min (Figure 4B). However, PC12 cells transfected with FLAG‐ARMS4.2 construct showed a significant reduction in the number of p‐MAPK‐positive cells at the later time point (Figure 4B). Importantly, ARMS4.2 mutant did not perturb the transient MAP kinase activation by EGF, consistent with earlier data that EGF treatment normally evokes a transient MAP kinase cascade in PC12 cells (Qiu and Green, 1991; Qui and Green, 1992). The results suggest that ARMS is a specific substrate for Trk receptor signaling (Figures 2 and 3; Kong et al, 2001). In addition, the lack of an effect of ARMS4.2 on transient MAP kinase activation evoked by either NGF or EGF suggests that the previously identified pathway for Ras‐dependent MAP kinase activation via FRS2 (Kouhara et al, 1997; Stork, 2003) is not affected by ARMS4.2 expression.
Since in vitro binding studies indicated that TrkB also interacts with ARMS (Figure 2A), the role of ARMS in BDNF signaling was evaluated in cortical neurons, which express endogenous TrkB receptor and ARMS (Barbacid, 1994; Kong et al, 2001). Accordingly, transfected cortical neurons were treated with BDNF (50 ng/ml) for 40 min (Figure 4D, panels d–l) and the proportion of p‐MAPK‐positive cells was determined (Figure 4C). The results indicated a clear decrease in the number of p‐MAPK‐positive neurons after ARMS4.2 expression (Figures 4C and D, panels g–i), when compared to control cultures (Figures 4C and D, panels d–f). Interestingly, we found no effect of ARMS4.2 on BDNF‐dependent activation of Akt (Figure 4D, panels j–l), suggesting that functional TrkB–ARMS interaction is specifically required for modulating the kinetics of MAP kinase activation. These observations lend support to the hypothesis that ARMS is involved in the sustained activation of MAP kinase mediated by Trk receptors upon neurotrophin treatment.
NGF induces complex formation between Trk, ARMS and CrkL
The ability of neurotrophins to activate the prolonged MAP cascade requires the integration of Ras‐ and Rap1‐dependent pathways. In PC12 cells, Rap1 is responsible for the sustained phase of MAP kinase activation via B‐Raf, whereas Ras activity is associated with the earliest initial response (Vossler et al, 1997; York et al, 1998). Our findings with the ARMS4.2 mutant suggest a specific role for ARMS in maintaining late‐phase MAP kinase activation (Figure 4). Since the Crk adaptor protein family is involved in neurotrophin‐dependent differentiation and signaling to Rap1 (Teng et al, 1995, 1996; York et al, 1998; Feller, 2001), we asked whether NGF regulates the interactions between Trk and Crk proteins.
To address this question, Crk proteins and TrkA were co‐immunoprecipitated in PC12nnr cells (Green et al, 1986), which stably expressed either exogenous wild‐type or mutant TrkA receptors (ΔShc) that do not interact with the adaptor protein FRS2 (Kouhara et al, 1997). An inducible interaction was clearly detectable with wild‐type Trk and a Trk receptor mutant (ΔShc) missing the consensus Shc‐binding site ENPQY (Figure 5A). It had been proposed that association of Crk proteins with Trk was facilitated by FRS2 (Meakin et al, 1999; Kao et al, 2001). However, the interaction of Crk with Trk receptors did not depend upon the Shc site or FRS2 (Figure 5A). Therefore, the association of Crk with TrkA did not require Shc phosphorylation or FRS2 activation.
In endogenous co‐immunoprecipitation experiments, we also found that CrkL is specifically associated with ARMS and TrkA in an NGF‐inducible manner in PC12 cells (Figure 5B). This interaction increased minutes after NGF treatment. Consistent with the mapping results from transfected cells, these findings suggest that ARMS is involved in the association between TrkA and CrkL. This complex was observed constitutively, but increased with NGF treatment (Figure 5B).
Tyrosine phosphorylation events may change the specificity of Crk binding, leading to differential association with downstream proteins. This may be manifested by a change in the binding specificities of Crk to different signaling proteins, such as C3G or Abl (Feller, 2001). To evaluate this possibility, we carried out a time course of association of CrkL, activated TrkA and the guanine nucleotide exchange factor (GEF) C3G, in response to NGF in PC12 cells. After NGF treatment, there was a noticeable increase in the amount of endogenous C3G and TrkA associated with CrkL over a time period of 3 h (Figure 5C). This increase was specific to C3G, since Abl, another interactor of CrkL, did not show a corresponding change. The increased association of C3G with CrkL paralleled the activation of the TrkA receptor. NGF, therefore, induced an association of CrkL with C3G, which can lead to increases in Rap1 and MAPK activities associated with prolonged responses by neurotrophins. Taken together with the association of ARMS and CrkL, these results indicate that a complex of proteins forms downstream of the Trk receptor, which is enhanced upon neurotrophin treatment.
ARMS may therefore serve as a molecular scaffold for Trk‐dependent signaling via CrkL. If so, ARMS overexpression would be expected to enhance complex formation between CrkL and TrkA. To address this possibility, we carried out co‐immunoprecipitation analysis of FLAG‐tagged CrkL with TrkA in the presence or absence of exogenous ARMS. HEK293 cells harbor a low level of endogenous ARMS protein that can facilitate TrkA–CrkL binding. As shown in Figure 5D, complex formation between TrkA and CrkL was augmented by additional ARMS protein expression, suggesting that one function of ARMS is to link activated Trk to CrkL. Consistent with the role of Crk adaptors in Rap1 signaling (Feller, 2001), the Rap1 GEF C3G was also found to complex with CrkL in this assay (Figure 5D). These results suggest that endogenous ARMS–CrkL and CrkL–C3G complexes occur constitutively, but also that these associations can be significantly augmented by NGF stimulation.
Specificity of ARMS–CrkL interaction
Analysis of the ARMS protein sequence reveals a region rich in polyprolines (Pro) in the cytoplasmic domain (Figure 6A). In particular, P1081PLP, P1089PRPP and P1131QHP on ARMS conform to the consensus SH3‐binding site for the Crk family of adaptor proteins, which modulate Rap1 activity via C3G (Feller, 2001). To determine which of these polyproline sequences of ARMS might be involved in binding to Crk family members, we carried out in vitro GST‐binding experiments with the polyproline segment of ARMS. Utilizing cellular lysates from HEK293 cells that express endogenous c‐Crk II and the related CrkL, a specific interaction of CrkL, but not c‐Crk II, with GST–ARMS7.2 was detected (Figure 6B). No binding was detected with the GST–ARMS7.3 or GST–ARMS7.6 proteins. Binding of CrkL was therefore localized to the second of these three polyproline sequences of ARMS (P1089PRPP). To confirm these results, a corresponding GST–ARMS7.2 fusion protein lacking the P1089PRPP sequence (ARMS 7.2ΔP) was next tested for CrkL binding. No interaction of GST–ARMS7.2ΔP proteins with CrkL was observed (Figure 6B). These findings verify that there is direct binding of ARMS and CrkL, and identify the requirement of the P1089PRPP sequence for this interaction.
ARMS mediates Rap1‐dependent MAP kinase activation
The binding interactions between ARMS and CrkL suggest that downstream signaling to Rap1 may be directly affected by disrupting this complex. ARMS interacts with Trk receptors via transmembrane domains, but with CrkL via its polyproline region (Figures 2, 3 and 6B). Therefore, ARMS mutants with a deleted P1089PRPP polyproline sequence (ARMSΔP) might be expected to disengage Trk signaling to CrkL. Given that activation of Rap1 is necessary for prolonged MAP kinase activity in PC12 cells (York et al, 1998), we considered whether expression of ARMSΔP specifically affects Rap1 and MAP kinase activation by neurotrophins.
Toward this end, we generated stable PC12 cell clones expressing FLAG‐ARMS or FLAG‐ARMSΔP (Figure 6C). The state of Rap1 activity in these cells was evaluated with a GST protein fused to the Rap1‐binding domain of RalGDS (GST‐RalGDS RBD), which specifically binds only to the active, GTP‐bound form of Rap1 (Hermann et al, 1996). Cells were treated with NGF for 5 and 40 min and cell lysates were subjected to GST‐RalGDS RBD pull‐down assays. Consistent with the above model, NGF‐dependent Rap1 activation was detected in control as well as FLAG‐ARMS‐expressing PC12 cells, but is completely abolished in cells expressing the ARMSΔP protein (Figure 6D). In contrast, we observed no differences in the amount of active Ras between PC12 cells that express FLAG‐ARMS and FLAG‐ARMSΔP (Figure 6D). These results therefore verify the specificity of the ARMSΔP mutant on NGF‐dependent Rap1 signaling.
Next, we sought to correlate the effects of ARMSΔP on Rap1 activation to downstream MAP kinase events. In contrast to wild‐type ARMS‐expressing cells, expression of ARMSΔP resulted in a marked decrease in p‐MAPK protein at longer but not short time points (Figure 6E). Consistent with the NGF role in maintaining MAP kinase activity (York et al, 1998), ARMSΔP did not affect the initial activation of MAP kinase, but the longer duration of this activity. Interestingly, this was not accompanied by a decrease in pAkt by immunoblot analysis (Figure 6E), a result that was previously observed by staining primary cultures treated with the ARMS deletion mutant 4.2 (Figure 4D). Similar to the previous experiments using ARMS4.2, NGF‐dependent neurite outgrowth in PC12 cells is also significantly reduced by ARMSΔP expression (Figure 6F). Finally, transient expression of ARMSΔP in cultured cortical neurons was found to perturb sustained MAPK activation by BDNF (Figure 6G), suggesting a conservation of ARMS function among the neurotrophins.
Loss of function of ARMS
The function of ARMS in mediating prolonged MAPK responses has been gleaned largely from transfection of mutant constructs into cells. As another means of investigating the effects of ARMS in neurotrophin signaling, we attempted to decrease the expression of the endogenous ARMS proteins in PC12 cells. Small interference RNA (siRNA) was generated against the rat ARMS cDNA sequence. ARMS expression was specifically reduced by at least 80% in cells transfected with siRNA (Figure 7). This reduction impaired the ability of the PC12 cells to activate Rap1 (Figure 7A) and the sustained MAPK pathway (40‐min treatment; Figure 7B), but without appreciable effect on the Ras activation or on early MAP kinase activation (5‐min treatment) in response to NGF. By contrast, Akt activation was not affected at any time point (Figure 7B), consistent with the specificity of ARMS signaling to the prolonged MAP kinase pathway (Figures 4D and 6E). The lack of effect upon Akt separates this signaling pathway from the prolonged MAPK pathway influenced by ARMS. Therefore, the siRNA experiments indicated that levels of ARMS are instrumental in the sustained Rap1‐dependent MAPK activation elicited by NGF. Finally, the observations that ARMS siRNA abolished both early and delayed Rap1 activation but only interfered with late‐phase MAP kinase activity are consistent with a bimodal mechanism of Ras and Rap1 in early‐ and late‐phase MAP kinase induction (York et al, 1998).
Many signaling pathways are not unique to neurotrophins but are utilized by other growth factors and cytokines in different cell contexts. Here we have found that the ARMS protein serves as an important downstream platform for the Trk neurotrophin receptor in neurons. There are several unique features of the ARMS protein. First, it is closely associated with Trk receptors through interactions between transmembrane domains. Second, ARMS becomes tyrosine phosphorylated by neurotrophins for many hours and days (Kong et al, 2001), consistent with the proposed role of maintaining neurotrophin signaling. Third, although ARMS lacks conventional motifs such as SH2, SH3 and PH domains, it contains a C‐terminal PDZ‐binding motif (SIL), a SAM domain, 11 ankyrin repeats and a polyproline region. In this study, we have found that the P1089RPPP sequence in the polyproline region of ARMS is responsible for sustaining MAP kinase activity and that this polyproline sequence interacts specifically with CrkL. Our data support a model in which the interactions of ARMS and CrkL lead to activation of the small G protein Rap1 that accounts for the ability of neurotrophins to signal for sustained duration.
ARMS‐specific neurotrophin signaling
This report has identified a functional interaction between ARMS and CrkL in maintaining late‐phase ERK activity via the Rap1 GTPase. The ARMS–CrkL interaction is specific, as the related c‐CrkII proein does not bind to ARMS (Figure 6B). Consistent with nonoverlapping functions for CrkL and c‐Crk II in Trk signaling, inactivation of the CrkL gene resulted in neural crest defects (Guris et al, 2001), similar to defects when the neurotrophins and Trk genes are inactivated (Huang and Reichardt, 2003).
Although MAP kinase activities are influenced by multiple neurotrophin‐elicited signal transduction mechanisms (Kouhara et al, 1997; Qian et al, 1998; Atwal et al, 2000), our findings identify a unique pathway that accounts for the prolonged MAP kinase activity. This conclusion is supported by Trk receptor mutant knockin experiments, in which the Shc‐binding site in the TrkB receptor was mutated by changing the consensus tyrosine residue to phenylalanine (Minichiello et al, 1998). This mutation resulted in a complete lack of association of Shc with TrkB. When BDNF was administered to cortical neurons isolated from TrkBShc/Shc mutant mice, MAP kinase activation was reduced, but not eliminated completely (Minichiello et al, 1998). Likewise, TrkCShc/Shc mutation also yielded a similar pattern of response to NT‐3 (Postigo et al, 2002). In the latter study, MAP kinase activity still persisted after 60 min of neurotrophin treatment, indicating that sustained MAP kinase activation does not rely solely upon Shc binding.
Our findings with the ARMS protein suggest a new mechanism to account for the in vitro and in vivo findings that have been published. Association of ARMS with Trk receptors generates a scaffold, by which the CrkL–C3G–Rap1 proteins are assembled. Prolonged MAP kinase activation is facilitated by the formation of this complex. Disruption of the Trk–ARMS–CrkL interaction with two different mutant ARMS proteins (ARMS4.2 and ARMSΔP) perturbs this pathway, but still allows transient MAP kinase signaling events to occur.
Role of tyrosine phosphorylation
Since ARMS consists of multiple consensus motifs for serine/threonine and tyrosine kinases (Iglesias et al, 2000), an outstanding issue is the role of tyrosine phosphorylation in modulating the ARMS–CrkL interaction. The available data indicate that the constitutive interactions between Trk and ARMS and between ARMS and CrkL are critical for the initiation of Rap1‐dependent signaling (Figures 4 and 6). One potential mechanism is that a Trk–ARMS–CrkL complex changes its recruitment for C3G upon NGF‐induced phosphorylation. This is supported by the increase in C3G recruitment with CrkL (Figure 5C). We therefore propose that a preformed Trk–ARMS–CrkL complex (via the CrkL SH3 domain) facilitates CrkL SH2 domain binding to tyrosine‐phosphorylated ARMS upon neurotrophin stimulation, thereby releasing the CrkL SH3 domain for recruitment of C3G. Previous studies indicated that conformational changes occur intramolecularly in Crk proteins that modify the binding specificities of Crk to other signaling proteins (Escalente et al, 1999; Kurokawa et al, 2001; Ting et al, 2001). Resolution of this issue will require the future identification and mutational analysis of ARMS phosphorylation sites.
Implications of ARMS signaling
The rapid and sustained kinetics of ARMS tyrosine phosphorylation by Trk is reminiscent of other receptor systems that utilize scaffolds for signal transduction. This includes the insulin receptor, which mediates the bulk of its signaling through IRS proteins (Shpakov and Pertseva, 2000) and by T‐cell antigen receptors that use LAT, a transmembrane protein which serves as a scaffold for a variety of SH2 domain‐containing proteins. The specificity of ligand–receptor signaling appears to involve the coupling of each receptor to unique multidomain adaptors.
Presynaptic and postsynaptic functions are dramatically influenced by neurotrophins (Poo, 2001). It is attractive to consider that neurons have evolved distinctive mechanisms to regulate synaptic function over different time courses. The recent finding that BDNF can exert synaptic responses over millisecond time intervals (Blum et al, 2002), coupled with the ability of neurotrophins to produce physiological effects of extended duration, suggests that there are specialized mechanisms for neurotrophin receptors. This is further supported by recent observations that AMPA receptor distribution is determined by the opposing actions of Ras and Rap (Zhu et al, 2002), which can display specific consequences upon LTP (Morozov et al, 2003). Due to the close proximity of the ARMS protein with Trk receptors and its time course of phosphorylation, it is tempting to speculate that ARMS may serve as a bridge between neurotrophins and changes in short‐term and long‐term synaptic strength, in addition to the process of neuronal differentiation.
Materials and methods
NGF was obtained from Harlan (Indianapolis, IN) and BDNF and EGF were from Peprotech (Rocky Hill, NJ). The following antibodies were used: 4G10, CrkL were obtained from Upstate Biotech (Lake Placid, NY), Trk (C‐14 and B‐3), PLC‐γ, EGFR, Crk II, C3G and Rap1 were from Santa Cruz Biotechnology (Santa Cruz, CA), p‐MAPK, p‐Akt, MAPK and Akt were from Cell Signaling Technology (Beverly, MA), anti‐FLAG was from Sigma (St Louis, MO), polyclonal ARMS antibody has been previously described (Kong et al, 2001). Anti‐TrkA antiserum (RTA) that recognizes the extracellular domain of TrkA was generously provided by L Reichardt (UCSF).
Constructs of different ARMS domains were amplified by PCR using the rat ARMS cDNA (Kong et al, 2001) as a template. Deletion of amino acids P1089–P1093 in ARMS was performed by site‐directed mutagenesis to generate ARMSΔP. Deletion constructs of ARMS were cloned in pFLAG‐CMV1 or pFLAG‐CMV2 vectors, or in pGEX6P to generate recombinant proteins in Escherichia coli. The TrkAΔJM construct was made in pCMV5, by ligating PCR‐generated cDNA fragments of rat TrkA (Yano et al, 2000). All PCR fragments were verified by DNA sequencing.
DNA transfections, preparation of cell lysates and immunoblotting
Plasmid DNA was transiently transfected into HEK293 cells (2 × 106 cells/plate) using the calcium phosphate method. PC12 cells and cortical neurons were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. For PC12 cell immunoprecipitations, 1–2 × 107 cells were used. Western blotting was performed according to Arévalo et al (2000).
Preparation of GST fusion proteins and in vitro binding assays
ARMS7, ARMS7.2 (amino acids P1057–G1110), ARMS7.2ΔP (ARMS 7.2 with amino acids P1089–P1093 deleted), ARMS7.3 (amino acids P1057–G1088) and ARMS‐7.6 (amino acids P1132–L1151) were amplified by PCR and subcloned in pGEX6P‐1 vector. GST–ARMS proteins were purified according to Yano et al (2001).
Stably transfected PC12 cells
PC12 cells were cotransfected with 20 μg of pFLAG‐ARMS or pFLAG‐ARMSP and 2 μg of pCDNA3 with Lipofectamine 2000. To obtain stable transfectants, cells were treated with 500 μg/ml G418. After 15–20 days of G418 selection, resistant clones were subjected to Western blot analysis using FLAG antibodies to verify ARMS or ARMSΔP protein expression. Positive clones were maintained in 200 μg/ml G418 thereafter.
siRNA against ARMS
ARMS siRNA was obtained using the Silencer™ siRNA Cocktail Kit (RNase III) (Ambion). As a template for the in vitro transcription, a fragment of 400 bp corresponding to the nucleotides 3952–4357 of the ARMS rat sequence was subcloned in pcDNA3 in both orientations. PC12 cells in 24‐well plates were transfected with ARMS siRNA (0.5 μg/well) or mock transfected using Lipofectamine 2000 in two consecutive days. After 24 h, cells were starved overnight and treated with NGF (100 ng/ml) for different time points before immunoblotting.
Preparation and crosslinking of cortical neuron lysates
Cortical neurons were obtained from E17–19 rats and cultured in Neurobasal medium (Gibco) supplemented with B‐27 and 0.4 mM glutamine in poly‐l‐lysine‐coated plates. For co‐immunoprecipitation, primary cultured neurons DIV 8–11 were rinsed with PBS and proteins were crosslinked with 1 mM dithiobis‐succinimidylpropionate (DSP, Pierce Biotechnology, Rockford, IL) in PBS for 30 min. Immunoprecipitated complexes were boiled for 5 min to cleave the crosslinked proteins, and resolved by SDS–PAGE and Western blotting with antibodies against Trk and ARMS.
Ras and Rap1 activation assays
PC12 cells and stable PC12 transformants expressing FLAG‐ARMS or FLAG‐ARMSΔP were used for these experiments. Recombinant GST‐RalGDS Rap‐binding domain (RBD) and GST‐Raf‐Ras‐binding domain were produced in bacteria as described above. Pull‐down assays were used to detect endogenous levels of GTP‐bound Rap1 or Ras proteins (Wu et al, 2001).
Supplementary data
Supplementary data are available at The EMBO Journal Online.
Supplementary Information
Supplementary data [emboj7600253-sup-0001.pdf]
We thank Belén Domínguez, Pilar Pérez, Dionisio Martin‐Zanca, Barbara Hempstead and the members of the lab for comments and support, Louis Reichardt for generously providing RTA antibody, and Lani Mutstacchi for primary cultures. This work was supported by the NIH (NS21072 and HD023315) and by a Postdoctoral Fellowship from the Spanish Education Ministry (JCA).
- Copyright © 2004 European Molecular Biology Organization


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By Francisco Chinesta, Elias Cueto
This ebook teams the most advances in fabric forming, contemplating assorted strategies, either traditional and non-conventional. It focuses
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Additional info for Advances in material forming : Esaform 10 Years on
Example text
The increasing computation power allows better analyzing dislocation densities associated to different mechanisms, it allows for instance to study the induced microstructure during not only monotonic strain path but also two-stage strain paths[71]. The current evolution of multiscale models concerns the modelling of multiple phase materials (while single phase models were initially developed). Body centred cubic (bcc) and face centred cubic (fcc) material were first investigated because of their rather continuous behaviour.
It allows to describe complex hardening behaviours induced by strainpath changes. The model is described by 13 material parameters and depends on four state variables: P , S , X , R . The variable P is a second order-tensor that depicts the polarity of the persistent dislocation structures (PDS) and S is a fourth-order tensor that describes the directional strength of the PDS's. The scalar R represents the isotropic 136 Advances in Material Forming – Esaform 10 years on hardening due to the randomly distributed dislocations and the second-order tensor X is the back stress.
Proceedings of ICOTOM 6, vol. 1. The Iron and Steel Institute of Japan, Tokyo (1981) 347–355 8. : Slip geometry in partially constrained deformation. Acta Metall. 30 (1982) 695 9. : On the equivalence of the relaxed Taylor theory and the Bishop–Hill theory for partially constrained plastic deformation of crystals. Mater. Sci. Eng. 55 (1982) 69–77 10. : Generalisation of the relaxed constraints models for the prediction of deformation textures. Revue de Métallurgie-CIT/Science et Génie des matériaux (1997) 1483-1494 11.

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With crime levels rising by 70% in the last three years within León city in Mexico, OptiBus who operates the cities only Bus Rapid Transit (BRT) system needed an optimal solution for keeping their 350,000 daily ridership safe when using their transport network. Through deploying an InfiNet Wireless solution they have been able to overhaul their network and transmit CCTV images in real-time from their 53 bus stops across the city via an increased network speed of 8Mbps per subscriber point…
With crime levels rising by 70% in the last three years within León city in Mexico, OptiBus who operates the cities only Bus Rapid Transit (BRT) system needed an optimal solution for keeping their 350,000 daily ridership safe when using their transport network. Through deploying an InfiNet Wireless solution they have been able to overhaul their network and transmit CCTV images in real-time from their 53 bus stops across the city via an increased network speed of 8Mbps per subscriber point. The new system has also allowed them to bring their overall monitoring system completely online, increasing efficiencies and safety.
OptiBus Director, Fernando Hernandez Garcia explains why they needed to update their wireless canopy system, "We previously had an outdated wireless canopy system in place, but it only offered a maximum through-put of 4Mpbs, and was causing the network to become increasingly saturated, with real time video becoming impossible, negating the purpose of CCTV. We were also unable to collect the data from the cameras remotely, meaning that extra staff had to be employed to perform this task manually at each station. If a crime was committed, there would also be a significant delay in getting the video evidence due to the distances of the stations and cameras. We needed to upgrade our system to something that provided real-time coverage and also improved data collection and our overall data monitoring systems."
OptiBus looked at all wireless solution suppliers assessing sustainability, reliability and price point, but through working with Tec45 – an approved partner for InfiNet Wireless products, they decided to carry out a trial with the InfiNet equipment. The first trial was for one base station and 10 InfiMAN Point-to-Multipoint (PMP) subscribers. The test was extremely successful, achieving 8Mbps per subscriber- enough to transmit video in real-time, as opposed to their previous canopy system's capabilities of 160kbps per subscriber. With a favorable outcome from the trial OptiBus decided to go ahead and extend the project to five base stations, with three Point-to-Point (PTP) InfiLINK systems and 55 InfiMAN PMP subscribers.
OptiBus has seen a significant improvement since the overhaul. One of the biggest achievements from the project is the ability to support cameras at bus stations with a network that has bandwidth capacity to transmit images in real time, and enabling staff to now have the added capability to be able to monitor and collect data and passenger billing information remotely. With one monitoring centre in the OptiBus head office, staff are now able to collate data from 53 connected bus stops across five separate base stations. The new technology has also allowed OptiBus to bring its avoidance alarm, telephone usage, report collection system, video security and overall system monitoring completely online – providing a much more efficient and reliable network.
Fernando Hernandez Garcia, OptiBus Director concluded, "Before we started using the InfiNet Wireless network, we couldn't see real-time video and it was practically impossible to monitor security in the city. Today, the InfiNet Wireless equipment helps us to collect billing information and utilise IP telephony and station alarms. Now, we can monitor security cameras in real-time which are linked to security agencies across the city and we work together to provide a safe and secure city for citizens."
Tec45 provided total support to OptiBus during the process of implementing the changes – putting together the proposal, engineering the process and overseeing the project, integrating OptiBus' systems and continue to provide after-care, post-sale services and technical support.
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On 10 June 2015, Phyathai 2 hospital signed a Direct Billing agreement with China's most established and most internationally awarded premium health insurance service provider in order to provide a cashless, convenient & claims-free experience to MSH members.
Dr.Anantasak Apairatana, Hospital excecutive director, says " it is our privilege to partner with MSH CHINA, a leading high-end health insurance services provider in China. The MOU signed today will allow us to work more closely together to reach the goal of providing convenient and claims-free experience to our valued patients as well as MSH members"
Dr Lily Sun, Senior Operations Director of MSH CHINA says "MSH invested significant time and resources in carefully assessing the medical capabilities available in Asia. We are very happy to sign an agreement with world class Phyathai 2 hospital to accept our MSH member cards for direct billing access to their facility. The last thing we want our members to be worried about is whether they have enough cash for the medical treatment. Our partnership today effectively resolves this concern for our members."
Phyathai 2 Hospital has been a leading medical provider in Thailand, located right in the heart of Bangk0k city and just 4 minutes walk from Sanam Pao BTS station. We offer a full range of health care services by physicians who are board certified specialists and trained in the top medical schools from Thailand, USA and Europe. The hospital was accredited by HA (Hospital Accreditation), Thailand Energy Saving Awards 2014 and JCI (Joint Commission International) – the gold standard in international healthcare provision. This ensures our commitment to providing the highest quality standard of healthcare in the comfortable and reassuring environment.
MSH is a world leader in the design and management of international healthcare solutions with members across more than 194 countries. We provide round-the-clock services via 4 regional headquarters in Paris, Calgary, Dubai & Shanghai. We have a multi-cultural team of over 60 nationalities speaking more than 40 languages. Our extensive global medical network partners over 860,000 accredited providers around the world. In Asia, our medical network covers Thailand, Singapore, Philippines, Indonesia, Malaysia, Cambodia, India, Vietnam, Japan, South Korea, Taiwan, HK and China.


text:
R&D Internship M/W
April 14, 2018
Working in an English-speaking and fast-paced environment, you will assist the R&D team charged with the responsibility for development of sub-systems of the CorWave LVAD, including requirements, design and testing.
Your tasks will cover different aspects of our cross-disciplinary R&D program ranging from design engineering to biological interfaces, use of software tools and materials selection. It will include:
- Design and simulation using the latest CAE tools
- Test set-up, automation and analysis using tools such as Labview and Matlab
- Review of literature and other source materials to determine industry best practices
- Development of requirements, specifications, test protocols and reports to document your work
A few projects you might work on:
- Design of surgical components and tools, optimization of actuator and pump head design
- Coordination of pre-clinical and clinical experiments
- Improvement and maintenance of automated hydrodynamic, blood interface and durability bench tests
- Research and development of new testing methods leveraging basic physics and fundamental research
- Implementation of a control and monitoring system (electronics and software) for implantable blood pumps
The candidate must have direct experience in the development of medical medical devices.
- Studying engineering with a major in one of the following areas: mechanical, electronics or biomedical engineer
- Previous working experience (internship, other) in the industry is preferred
The successful candidate will be highly motivated, hard-working, autonomous, team oriented, and driven by a positive attitude. Flexibility to work in a start-up environment, requiring lean, efficient use of resources are required for success in this position.
Offer and Application
- Starting date: Q2/Q3 2018
- Salary will be competitive and commensurate with experience and qualifications
- Location : Clichy-Levallois / Metro line 13, train L
Please send your CV and letter of introduction to: firstname.lastname@example.com, "Reference R&D Internship"


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Beam Oceana 400 satellite terminal is specifically designed to operate with the Inmarsat "FleetPhone" in maritime applications, allowing access to reliable voice and data communications services at any time. The Oceana 400 has an RJ11/POTS interface which allows you to connect up to five phones (corded or cordless) or integrate a PABX system.It can support cable runs of up to 600m (2000 feet), with in-built intelligence to support standard ring, busy and dial tones.The Oceana 400 terminal is supplied with the ISD710 Maritime Antenna (Active) to provide a complete integrated solution that is ready for use at all times. The Oceana 400 is IP53 rated making the terminal suitable for onboard solutions.
The "FleetPhone" service provides low cost voice services allowing phone calls to be charged to the vessel or a prepay card account, Data transmits simple text emails with small attachments, and SMS messages can be sent and received.The Oceana 400 is ideal for those working or sailing outside of cellular coverage.
The terminal is wall mountable and comes with a wall mounting plate or desk mountable. The USB data access allows configuration of the terminal and quick connection of a laptop or computer to have data connection, providing access to the laptop or computer to satellite connectivity.
Product Features
Oceana 400 Satellite Terminal
ISD710 Maritime Antenna (Active)
110-240V AC Plug Pack 15v DC Out
10-32V DC Power Cable
USB Cable
2 x screws for wall mounting
User Manual
Quick Start Guide
Technical Specifications Physical Specifications •8.0 x 7.5 x 2.1 (inches) •1.28lbs Connectors/Interfaces •POTS/RJ11: RJ11/2-wire, 5REN @ 600m, Adjustable dial, ring, busy tone configured frequency and adaptive impedance. •Inmarsat Antenna: TNC-Female •GPS Antenna: SMA-Female •10-32V DC: 4-way microFit (AC/DC adapter, or DC lead) •Configuration/Data USB Mini B Environmental Specifications •Operating Range: -4°F to +131°F •Storage: -22°F to +176°F •Humidity: <=75% RH Tx Operating Frequency •1626.5 - 1660.5 MHz Rx Operating Frequency •1518 – 1565 MHz (Inmarsat) •1565.19 – 1585.65 MHz (GPS) RF Output Impedance •50 Ohms Carrier frequencies •Channel 0 = 1626.525 MHz •Channel 169 = 1660.475 MHz TX Modulation GMSK RX Modulation •OQPSK
All prices are in USD.


text:
Magnetic Content Combo
"Unless a company is producing a steady stream of (quality) content, the audience it earns will begin to decay."
Hubspot State of Inbound 2015 Report
"Companies that blog generate 67% more leads per month than those who do not blog"
Hubspot State of Inbound 2015 Report
Creating content is hard.
Creating high-quality content that engages your audience and meets your firm's goals is even harder. Consistent content creation… ugh… a perpetual struggle. And, where on earth can you find the time to blog, write a newsletter or that long form piece you know Google loves but keep putting off?
Struggling with content creation doesn't mean you must abandon your content marketing efforts.
There's a way out.
Magnetic Content Combo is for you if:
- Your blog was last updated during Hillary Clinton's run for the White House
- You're struggling to feed your list with quality content (the content beast has a rapacious appetite you know J)
- You and your strained team are spending more and more time writing content which keeps you away from your core duties
- You blog erratically when inspiration strikes or you can find the time to write
- Your newsletter is sent out once in a while
- You don't know how to stand out content wise in the vast sea of sameness
What this package includes:
- 4 Optimized posts
- 4 teaser emails
- 2 Newsletters
- Blog templates
Bonuses (Pick one)
- Site content audit & report (worth $2000)
- Special report (worth $2000)
- Long form piece (worth $1700)
And these are the results you'll get:
- More productivity since you'll be freed up to focus on your core duties.
- An increase in organic traffic
- More social shares as people rave about your delicious content
- List growth as more people sign up because they're attracted by the high-quality content
- A more engaged audience that comments more and responds to your email marketing campaigns
- Higher conversions when you sell your products/services since you'll be a trusted source whose content your crowd delights in
Investment: $1999
Ready to start? Email me now


text:
When you search for a 'Plumber near me', what do you hope to find? You're probably looking for a local plumber who will provide fast service and stand behind their work. At ENCO, we've been providing residential & commercial plumbing services in DFW for more than 30 years—and our team is ready to take your call right now.
If you need help with your plumbing, DFW has no shortage of companies looking to earn your business. But when there's an emergency, you can't afford to wait for regular business hours—or rely on a company without the manpower to fit you into their schedule. Our experienced plumber service teams are always on-call to address your plumbing issues. Whether that's a leaking pipe, a clogged sewer line, or a broken water heater, you deserve a plumber in DFW that can be on-site as soon as possible. We look forward to becoming the company you call any time, day or night, to fix your plumbing problems.
Gas water heater repair and maintenance is a common request for units that are reaching the end of their useful lifespan (about 8-10 years). If repair isn't cost-effective, you may need a gas water heater replacement. We can help with the selection process as well as performing the gas water heater installation in your home or business. Delivery is free of charge, and we'll haul off your old unit at no cost as well.
Are you tired of running out of hot water? Tankless water heater installation provides a constant, unlimited supply of hot water with no need to store 50 gallons in a traditional heater that could leak or burst. We are happy to walk you through the benefits of this newer technology so you can decide if it's the right choice. These systems are more complex than traditional heaters, so our team also provides tankless water heater mainuenance to keep your new unit in great condition year after year.
No matter what system you have in place, natural gas, electric, propane, or tankless, our team can provide water heater repair services 24/7 to remove mineral buildup, fix leaks, and extend the lifespan of your unit. We provide water heater installation services throughout the DFW area for homes and businesses as well. If you are interested in energy-efficient, worry-free water heater replacement, we can upgrade your existing system with a solution that makes sense.
Water stains, and suspiciously high water bills can all spell hidden trouble with your plumbing. If you are noticing any signs that you might have a water leak, it's smart to get it checked out. Left uncorrected, a leak can cause significant property damage. Plumbing leaks can occur in any area of your home. Did you know that even small water leaks can cost you thousands of dollars in high water bills and property damage? Shifting soil in North Texas is a common cause of pipe failures, which is why our slab leak detection and yard leak detection services are in high demand. Early intervention to identify and fix leaks can help curtail the cost of foundation repairs, flooring replacement, landscape renovation, and more.
The most important thing to do if you suspect a gas leak is to have the gas shut off immediately! Once that's done, we can come out and find where the leak is occurring. A gas leak can occur with either a traditional or tankless water heater. Maintenance is key to preventing problems with old, worn connections. If you are getting a new tankless water heater installed, by a professional it is critical for ensuring there are no gas leaks on your system.
Slab leaks from hot or cold water pipes can threaten the foundation of an entire building—and repairs can be costly. Don't add to that expense by hiring a plumber who will rip up all your floors looking for the leak. Our team has the equipment to locate and fix leaks while minimizing damage to your property. If the issue is related to your hot water system or a severe leak has damaged your water heater, repair or even water heater replacement may be needed. When you purchase a new water heater with ENCO, we provide free delivery, free disposal of your own unit, and expert water heater installation to help minimize the risk of future leaks.
Hydro jet drain cleaning is an alternative to the older power snake or "roto-rooter" approach. It uses a high-pressure stream of water to remove blockages in your sewer line (including those caused by roots and debris). The results from our hydro jetter service tend to last longer than traditional methods for clearing drain lines. Maintenance may only be needed once every few years instead of annually.
Is your sewer line clogged? Unfortunately, the city may not take responsibility for fixing this issue. And the last thing you want to deal with is sewage backing up into your property causing a foul odor and a serious health hazard. Our team works quickly to clear obstructions and get rid of roots in sewer line systems on your property. We use video equipment to accurately identify leaks or other areas of damage so that we can perform sewer line repair right away.


text:
This week on Rhythm City
Suffo gives Mzi an ultimatum over Pearl, Mapula is blamed for Melusi's actions, and Kop learns his house is not in his name. Rhythm City, weekdays at 7PM.
Suffo gives Mzi an ultimatum over Pearl. Mapula gets blamed for Melusi's actions. Kop learns his house is not in his name.
David tries to recruit Suffo to bring down Pearl and Mzi's relationship. Fats refuses to hand the title deed over to Kop. Mapula's trials aren't over yet, but Rene stands up for her.
Watch last week's episodes now on e On Demand.
David learns that Mzi doesn't know about the Pearl-Suffo engagement. Puleng plays hard ball with Suffo. Mapula hides her suspension from school.
David seems to be all loving and understanding when he suggests the Genaros and Ndlovus meet for dinner. But what are his real plans? Kop has a new idea for getting the house.
Dumi confronts Melusi and Mapula plans to run away. Suffo calls David's bluff, and Kop secures the house.
Watch Rhythm City, weekdays at 7PM. Follow the show on Twitter and like the Facebook page.


text:
|March, 2009
|Tracey et al.
|Deep vein thrombosis prevention
|January, 2009
|Weintraub et al.
|Stimulation Device for Treating Osteoarthritis
|November, 2008
|Gill et al.
|December, 2009
|Gastrointestinal stimulator device for digestive and eating disorders
|November, 2008
|Supply circuit for implantable medical devices
|August, 2007
|Device for the irradiation of the ciliary body of the eye
|April, 2006
|Pallikaris et al.
|November, 2006
|Kaplan et al.
|Enhancing human ventricular contractility, perfusion, and rhythm by enhancing laminar coordination of ventricular myocardial repolarization by the His-Purkinje system
|March, 2004
|Craig et al.
|March, 2010
|Altman et al.
|October, 2008
|Dong et al.
[0001] The subject invention relates generally to implantable cardioverter/defibrillators and, more particularly, to detection of cardiac arrhythmias with subcutaneous implantable cardioverter/defibrillators.
[0002] Defibrillation/cardioversion is a technique employed to counter arrhythmic heart conditions including some tachycardias or fast heart rhythms originating in the atria and/or ventricles. Typically, electrodes are employed to stimulate the heart with electrical impulses or shocks, of a magnitude substantially greater than pulses used in cardiac pacing. A variety of shock waveforms are used for both defibrillation and pacing, including truncated exponentially decaying monophasic and biphasic waveform pulses as well as pulses where the waveform maintains a relatively constant current over the duration of delivery to the myocardium.
[0003] Defibrillation/cardioversion systems include body implantable electrodes that are connected to a hermetically sealed container housing the electronics, battery supply and capacitors. The entire system is referred to as an implantable cardioverter/defibrillators (ICD). The electrodes used in ICDs can be in the form of patches applied directly to epicardium, or, more commonly, the electrodes are located on the distal regions of small cylindrical insulated catheters that typically enter the subclavian venous system, pass through the superior vena cava and, into one or more endocardial areas of the heart. Such electrode systems are called intravascular or transvenous electrodes. U.S. Pat. Nos. 4,603,705, 4,693,253, 4,944,300, 5,105,810, the disclosures of which are all incorporated herein by reference, disclose intravascular or transvenous electrodes, employed either alone, in combination with other intravascular or transvenous electrodes, or in combination with an epicardial patch or subcutaneous electrodes. Compliant epicardial defibrillator electrodes are disclosed in U.S. Pat. Nos. 4,567,900 and 5,618,287, the disclosures of which are incorporated herein by reference. A sensing epicardial electrode configuration is disclosed in U.S. Pat. No. 5,476,503, the disclosure of which is incorporated herein by reference.
[0004] In addition to epicardial and transvenous electrodes, subcutaneous electrode systems have also been developed. For example, U.S. Pat. Nos. 5,342,407 and 5,603,732, the disclosures of which are incorporated herein by reference, teach the use of a pulse monitor/generator surgically implanted into the abdomen and subcutaneous electrodes implanted in the thorax. This system is far more complicated to use than current ICD systems using transvenous lead systems together with an active can electrode and therefore it has no practical use. It has in fact never been used because of the surgical difficulty of applying such a device (3 incisions), the impractical abdominal location of the generator and the electrically poor sensing and defibrillation aspects of such a system.
[0005] Recent efforts to improve the efficiency of ICDs have led manufacturers to produce ICDs that are small enough to be implanted in the infraclavicular pectoral region, a site allowing access to the subclavian venous system. In addition, advances in circuit design have enabled the housing of the ICD to form a subcutaneous electrode. Some examples of ICDs in which the housing of the ICD serves as an optional additional electrode are described in U.S. Pat. Nos. 5,133,353, 5,261,400, 5,620,477, and 5,658,321 the disclosures of which are incorporated herein by reference.
[0006] ICDs are now an established therapy for the management of life threatening cardiac rhythm disorders, primarily ventricular fibrillation (VF) and also ventricular tachycardia (VT). ICDs are very effective at treating VF and VT, but traditional ICD implantation still requires significant surgery and surgical skill, especially regarding lead insertion into the venous system and lead positioning in the heart.
[0007] As ICD therapy becomes more prophylactic in nature and used in progressively less ill individuals, including children, the requirement of ICD therapy to use intravenous catheters and transvenous leads is a major impediment to very long term management as most individuals will develop complications related to lead system malfunction, fracture or infection sometime in the 5-10 year time frame, often earlier. In addition, chronic transvenous lead systems, their removal and reimplantation, can damage major cardiovascular venous systems and the tricuspid valve, as well as result in life threatening perforations of the great vessels and heart. Consequently, use of transvenous lead systems, despite their many known advantages, are not without their chronic patient management limitations in those with life expectancies of >5 years. The problem of lead complications is even greater in children where body growth can substantially alter transvenous lead function and lead to additional cardiovascular problems and revisions. Moreover, transvenous ICD systems also increase cost and require specialized interventional rooms and equipment as well as special skill for insertion. These systems are typically implanted by cardiac electrophysiologists who have had a great deal of extra training.
[0008] In addition to the background related to ICD therapy, the present invention requires a brief understanding of a related therapy, the automatic external defibrillator (AED). AEDs employ the use of cutaneous patch electrodes, rather than implantable lead systems, to effect defibrillation under the direction of a bystander user who treats the patient suffering from VF with a portable device containing the necessary electronics and power supply that allows defibrillation. AEDs can be nearly as effective as an ICD for defibrillation if applied to the victim of ventricular fibrillation promptly, i.e., within 2 to 3 minutes of the onset of the ventricular fibrillation. AEDs, unlike ICDs, only make shock/no shock decisions, as they are relieved of the burden of needing to deliver complicated tiered therapeutic responses that the ICD encumbers as a consequence of detecting and treating a multitude of rhythm problems with a multitude of therapies. AEDs either shock for a life-threatening event or they do not shock. ICDs, on the other hand, are designed for a variety of interventions and use a different technological approach for arrhythmia detection, redetection, assessment of effectiveness and therapy.
[0009] AED therapy has great appeal as a tool for diminishing the risk of death in public venues such as in airplanes. However, an AED must be used by another individual, not the person suffering from the potential fatal rhythm. It is more of a public health tool than a patient-specific tool like an ICD. Because >75% of cardiac arrests occur in the home, and over half occur in the bedroom, patients at risk ID of cardiac arrest are often alone or asleep and can not be helped in time with an AED. Moreover, its success depends to a reasonable degree on an acceptable level of skill and calm by the bystander user.
[0010] What is needed therefore for life-threatening arrhythmias, especially for children and for prophylactic long term use for adults at risk of cardiac arrest, is a novel combination of the two forms of therapy which would provide prompt and near-certain defibrillation, like an ICD, but without the long-term adverse sequelae of a transvenous lead system while simultaneously harboring most of the simpler diagnostic and therapeutic technological approaches of an AED. What is also needed is a cardioverter/defibrillator that is of simple design and can be comfortably implanted in a patient for many years.
[0011] Further, an ICD is needed that can detect various types of cardiac arrhythmias to provide a patient with adequate therapy according to the type of cardiac arrhythmia experienced by the patient. Although ICDs have multiple secondary functions, they primarily have two key functions: detection and therapy of life threatening cardiac arrhythmias. ICDs constantly monitor a patient's cardiac activity and analyze the cardiac activity to determine the appropriate therapy that should be delivered to the patient. The type of therapies available to be delivered to the patient include pacing, which can be single or dual chamber, therapy to correct slow heart rates or bradycardia called anti-bradycardia pacing, therapy to correct slow and moderately fast ventricular tachycardia (VT) and sometimes atrial tachyarrhythmias, called anti-tachycardia pacing (ATP), and therapy to correct ventricular fibrillation (VF) or high energy shocks. Thus, given the various types of therapies available, it is very important to classify the type of cardiac arrhythmias appropriately.
[0012] Detection schemes of cardiac arrhythmias are characterized using two indexes of performance: sensitivity and specificity. Sensitivity generally refers to the ability of the detection scheme or algorithm to accurately detect an abnormal heart rhythm for which the physician desires the device to treat. Specificity generally refers to the ability of the detection scheme or algorithm to not treat rhythms that the physician determines the device should not treat, such as sinus tachycardia. Sensitivity values for VT/VF are typically between 90% to 98%. There is constantly a tradeoff between the ability to detect an abnormal rhythm and the desire to prevent treatment of a normal rhythm. The higher the sensitivity, the more likely the detection algorithm will result in an inappropriate therapy of a normal rhythm and thus, lower specificity. The higher the specificity, the less likely the device will be able to detect rhythms that should be treated and thus, the lower the sensitivity. Despite a desire to limit false positive interventions, specificity values are typically only between 70% to 90% in order to not miss therapy of a life-threatening disorder. In practice, there is a constant tension between sensitivity and specificity.
[0013] Factors influencing detection algorithm performance include hardware performance, lead electrode placement, electrode design, electrode shape, inter-electrode spacing and the analytical capabilities and design of the detection scheme itself. Because ICDs are battery powered and, therefore, an unlimited supply of power is not available to perform complex and power intensive analytical functions, the energy required to perform the analytical functions is also a factor influencing detection scheme performance.
[0014] Another factor that influences the sensitivity and specificity performance of a detection algorithm is the clinical balance between false negatives and false positives for each arrhythmia that the detection algorithm is required to detect. The more arrhythmias required to detect, the more overlap and complexity in the algorithm. For example, one might require a very high sensitivity for fast VT/VF, given its lethality, and therefore accept a lower specificity compared to the relatively benign arrhythmia of atrial fibrillation (AF).
[0015] A false negative therapeutic decision results from a detection scheme that calls a bona fide treatable event "normal". But, depending upon the arrhythmia, some false negatives are more tolerable than others. On the other end of the spectrum is a false positive therapeutic decision that results from a detection algorithm that calls a "normal" event an "abnormal" event, thus inappropriately indicating that therapy should be delivered. False positives for therapy of VT are more clinically acceptable than false positives for rhythms like AF where the former is immediately life threatening and the later is seldom so.
[0016] A false negative can result in a missed arrhythmia and can lead to death, and a false positive may result in an inappropriate shock that will be uncomfortable, but not life threatening. Therefore, it is understandable that detection algorithms are skewed to result in false positives and reduce the occurrence of false negatives to zero for rhythms like fast VT but are more conservative for rhythms like AF where lower sensitivity is more clinically acceptable. Performance of typical detection schemes in current devices result in approximately 15% to 45% false positives for life threatening disorders like VF and fast VT.
[0017] Typically, ICDs primarily use a rate-based classification scheme. That is, the intervals between successive heart beats are measured and, depending on their values, they are classified as slow, normal, or fast. Slow heart beats are treated with pacing (i.e., anti-bradycardia pacing), when the rate reaches a critically low level, according to a physician's direction via the programming of the device. Programmable parameters for slow heart rates include primarily rate and hysteresis. Normal heart rates are left alone, where normal is usually defined in the range of 40-100 bpm. Fast heart beats are often further classified into three zones with various therapies ascribed to each zone. For example, the lowest zone may have a series of anti-tachycardia pacing (ATP) therapies manuevers programmed for rhythms like monomorphic VT that may fall in this lowest tachycardia rate zone. The next higher zone may integrate a limited number of ATP attempts with a moderate energy shock therapy of approximately between 5-10 Joules for that zone. Finally, the highest zone may have a scheme programmed to deliver the highest output shock energy for rhythms falling in this zone having VF or VT with the fastest and therefore most life threatening rates.
[0018] Additional rate-based qualifiers have been used to improve the specificity of detection schemes. Examples of such qualifiers include parameters like sudden onset to eliminate false positive detection of sinus tachycardia and heart rate interval stability to eliminate false positive detection of rapid AF for VT. Sudden onset refers to tachyarrhythmias that are a result of a precipitous increase in heart rate as opposed to sinus tachycardia where the higher rates tend to come on gradually. The term stability in detection algorithms usually, but not always, refers to the coupling interval between heart beats as a way to distinguish the characteristically irregular interval-to-interval time sequence of AF from more regular cardiac rhythms like sinus tachycardia or monomorphic ventricular tachycardia where the interval to interval time sequence is more regular and rarely varies by more than 30 ms unlike AF where variation by 40 ms or more is the rule. Although typically faster in rate, VF does, like AF, exhibit interval-to-interval instability. It can be distinguished from AF in a detection algorithm mostly by its typically much faster rate but also by overlaying other measures in addition to rate and interval-to-interval stability that result from an examination of the electrocardiographic QRS features.
[0019] The term stability, when used in detection algorithms, can have another meaning and refer to electrocardiogram (ECG) QRS signal stability, i.e., the ability of the QRS to have identical or at least very similar signal characteristics on a beat-to-beat basis. In one example of stability, one can examine QRS duration or width, QRS amplitude, QRS slew rate (rate of change of the voltage signal), QRS signal template matching, and/or QRS signal frequency content as an indicator of beat-to-beat stability. VF, for example, under this example of stability would be highly unstable as the QRS is constantly changing in all of the ways indicated above in this paragraph. Confirming the presence of VF, regardless of rate, would aid dramatically in applying a shock/no-shock decision-making process.
[0020] Recent developments in QRS morphology measurements involve a scheme in which certain areas within a cardiac QRS complex are measured and compared against a normal set as defined by the physician at the time of implant or as defined automatically by the device during preset follow-up measurement intervals.
[0021] There are other issues that enter into the ability of ICDs to detect abnormal heart rhythms depending on the cardiac information that is actually presented to the device's sensing electrodes (which may represent only part of the complete set of cardiac information at any point in time), the ICD energy available to analyze the information, the time required or allowed to make the analysis, and the ability or lack thereof for the device to modify itself over time with respect to changes in the patient's underlying cardiac disease process or even such shorter duration events as changes in body positioning.
[0022] With respect to limitations in cardiac information available to the device's sensing electrodes, current devices use a first electrode at the tip deep inside the right ventricle as one half of an electrode sensing pair. The second electrode of the pair can be a shocking coil also in the right ventricle and in close proximity to the first electrode, a second cylindrical electrode approximately 0.5 to 2 cm away from the first tip electrode, or the metallic housing of the device itself. Because the first electrode is in close proximity to cardiac tissue, it is more sensitive to electric fields present in the tissue closest to it and less sensitive to the entire electric field generated by all of the cardiac tissue. This contrast to a subcutaneous ICD sensing pair that has the advantage of a true far-field cardiac signal.
[0023] With respect to energy limitations of current devices, the ability to process and analyze cardiac signals depends on the energy available to the devices. Current devices are limited on the amount of energy available for analyzing signals because the devices must reserve a significant portion of the energy for pacing and shocking over the course of several years. For example, one measure of a device's performance is its ability to filter out unwanted signals, this is called common mode rejection ratio (CMRR). The higher the CMRR of a device, the better able it is to reject signals common to both electrodes. Current devices have a CMRR of about 50 db. Energy is required to achieve higher CMRR values. Another aspect of the analysis requiring energy is the use of a microprocessor with appropriate software inside the device to analyze cardiac information. Continuous running of a microprocessor in an implantable device would result in a rapid battery drain with unacceptably short battery longevity. As a result, current devices reserve a portion of hardware dedicated to crude analysis of the underlying rhythm on a continuous basis and only activate the microprocessor when further analysis is needed. Once activated, the microprocessor, along with its software programming, further analyze the cardiac information, decide on a course of action and, when finished, go back to inactive mode.
[0024] For a subcutaneous only ICD, these issues of arrhythmia detection sensitivity and specificity, false positives and false negatives, battery depletion, and algorithm design require significantly different considerations and unique hardware considerations and algorithm design compared to typical ICDs with 1-2 sensing leads in immediate contact with the myocardium.
[0025] What is then needed is an implantable cardioverter/defibrillator that has different sensing capabilities to ensure that an ICD using only subcutaneous shocking and sensing electrodes performs at acceptable levels as well as has the ability to adapt itself to changes over time.
[0026] According to one aspect of the invention, an apparatus and method are provided for detecting cardiac arrhythmias with a subcutaneous implantable cardioverter/defibrillator. The method includes the steps of sensing cardiac signal information from far field electrodes implanted in a patient, amplifying the sensed cardiac signal information, filtering the amplified cardiac signal information, measuring parameters such as rate, amplitude, QRS pulse width, cardiac QRS slew rate, QRS signal frequency content, QRS morphology, and stability measures of these parameters, and processing and integrating the parameters of the cardiac signal information to determine if the cardioverter/defibrillator needs to initiate therapeutic action.
[0027] For a better understanding of the invention, reference is now made to the drawings where like numerals represent similar objects throughout the figures where:
[0033] A flow diagram of an algorithm for detecting various types of cardiac arrhythmias is shown in
[0034] One feature of S-ICD and US-ICD devices that facilitates cardiac rhythm monitoring is the size and location of the lead. Far field cardiac signals are more easily detected by use of larger electrodes. The S-ICD and US-ICD devices typically use electrode size much larger than that used in standard ICD electrode systems for sensing VT/VF and thus are more appropriate for far field ECG sensing. In addition, the location in the 3
[0035] In addition, S-ICD and US-ICD devices typically provide an electrode that is inward facing toward the heart to facilitate improved arrhythmia detection and rhythm monitoring. This design of the electrode compensates for not being directly in contact with the heart, like the sensing electrodes of the common transvenous ICD. The compensating features of this type of lead electrode for the S-ICD allow better rhythm detection by facing the myocardium and simultaneously avoiding or minimizing exposure to surface artifacts and noise that may be easily detected by subcutaneous sensing electrodes.
[0036] In addition, the S-ICD and US-ICD devices have two or more electrodes that provide a far-field view of cardiac electrical activity that includes the ability to record the P-wave of the electrocardiogram as well as the QRS. In one embodiment of the present invention, the detection algorithm can detect the onset of AF by referencing to the P-wave recorded during normal sinus rhythm and monitoring for its change in rate, morphology, amplitude and frequency content as well as its timing and polarity relationship to the QRS. For example, a well-defined P-wave that abruptly disappears and is replaced by a low-amplitude, variable morphology signal would be a strong indication of the absence of sinus rhythm and the onset of AF. In an alternative embodiment of the detection algorithm, the ventricular detection rate could be monitored for stability of the R-R coupling interval. In the examination of the R-R interval sequence, AF can be recognized by providing irregularly spaced R-R intervals due to variable conduction of atrial beats to the ventricles through the atrioventricular node. A R-R interval plot during AF appears "cloudlike" in appearance when many R-R intervals are plotted over time and compared to sinus rhythm or other supraventricular arrhythmias. Moreover, a distinguishing feature of AF when compared to ventricular fibrillation is that the QRS morphology is similar on a beat-by-beat basis during AF despite the irregularity in the R-R coupling interval. In yet another embodiment, AF may be detected by seeking to compare the timing and amplitude relationship of the detected P-wave of the electrocardiogram to the detected QRS (R-wave) of the electrocardiogram. Normal sinus rhythm has a fixed relationship that can be placed into a template-matching algorithm that can be used as a reference point should the relationship change.
[0037] In other aspects of the AF detection process, one may include alternative electrodes that may be brought to bear in the S-ICD or US-ICD systems either by placing them in the detection algorithm circuitry through a programming maneuver or by manually adding such additional electrode systems to the S-ICD or US-ICD at the time of implant or at the time of follow-up evaluation. One may also use electrodes for the detection of AF that may or may not also be used for the detection of ventricular arrhythmias, given the different anatomic locations of the atria and ventricles with respect to the S-ICD or US-ICD housing and surgical implant sites.
[0038] Once AF is detected, therapy can be withheld since AF is relatively benign or the arrhythmia can be treated by delivery of a synchronized shock using energy levels up to the maximum output of the device therapy for terminating AF or for terminating other supraventricular arrhythmias. Synchronization can be conducted by detecting the onset of the QRS signal and delivering the atrial cardioversion shock during the synchronized time period. The S-ICD or US-ICD electrode system can be used to treat both atrial and ventricular arrhythmias not only with shock therapy but also with pacing therapy. In a further embodiment of the treatment of AF or other atrial arrhythmias, one may be able to use different electrode systems than what is used to treat ventricular arrhythmias. Another embodiment would be to allow for different types of therapies (amplitude, waveform, capacitance, etc.) for atrial arrhythmias compared to ventricular arrhythmias.
[0039] An algorithm for detecting various types of cardiac arrhythmias with an S-ICD or a US-ICD will be described below with respect to
[0040] It is also known that while S-ICD and US-ICD devices are able to pace, the devices are not intended for long term pacing. Typically, the S-ICD provides short term pacing in the order of a few hours as an emergency therapy so that the patient can consult with a physician or other care-giver. As a result, some of the energy that would be used for pacing is available for better processing and sophisticated analysis of the sensed cardiac signals. Accordingly, the S-ICD has energy available to achieve CMRR values of approximately 70 db to 100 db, in one example.
[0041] As noted above, S-ICDs use signals gathered from far field electrodes as opposed to electrodes that are in contact with the heart of a patient. As a result, a more sophisticated set of features can be used to make a device with higher specificity performance characteristics. A variety of detection schemes are possible with both the information available by the electrode placement as well as with the processing power available.
[0042] In addition to the use of the sense circuitry for detection of atrial and ventricular information, sense circuitry can check for the presence or the absence of respiration. The respiration rate and or minute volume can be detected by monitoring the impedance across the thorax using subthreshold currents delivered across the active can and the high voltage subcutaneous lead electrode (or the 2 can electrodes in one US-ICD embodiment) and monitoring the frequency in undulation in the waveform that results from the undulations of transthoracic impedance during the respiratory cycle. If there is no undulation, then the patent is not respiring and this lack of respiration can be used to confirm the QRS findings of cardiac arrest. The impedance measurement using the S-ICD and US-ICD electrodes can also be used to extract stroke volume, ejection fraction or cardiac output measures providing a hemodynamic sensor input that can be combined with rate, amplitude, QRS pulse width, cardiac QRS slew rate, QRS signal frequency content, QRS morphology, and stability measures of these parameters to further refine the shock/no-shock decision-making process.
[0043] The activity level of the patient can also be detected using a miniature 1-D, 2-D or 3-D accelerometer. Piezoelectric crystals can also be used as a sensor to determine the activity level of the patient. Activity level of the patient can be another input parameter used by the detection algorithm to further refine the shock/no-shock decision-making process.
[0044] Even the patient's position can be detected by using a miniature mercury switch that is actuated when the patient is horizontal. The patient's position can be used as another input parameter to the detection algorithm to further refine the shock/no-shock decision-making process.
[0045] Referring now to
[0046] Electrocardiogram (ECG)
[0047] In an embodiment, from the narrow-band filter
[0048] From QRS detector
[0049] In an embodiment, after processing by R-R interval detector
[0050] In an embodiment, from A/D converter
[0051] In one embodiment of the detection algorithm, rate is measured on a continuous basis. Other parameters may have registers associated for fine tuning by a programmer and/or determined periodically or continually on a beat-by-beat basis during normal sinus rhythm (NSR) and used to enhance the detection algorithms effectiveness by compensating for the relatively slow-changing effects many of the input parameters may have due to body positional changes, substrate changes and drug effects.
[0052] For example, short-term or long-term NSR averages of QRS width and R-wave amplitude would help compute the variability of these parameters with greater insensitivity to cardiac substrate changes.
[0053] It is also envisaged that many if not all of these parameters will be stored on a beat-by-beat basis in a circular buffer, allowing more sophisticated algorithms to scan back in time and take into account the past behavior of the system.
[0054] In addition, to conserve power consumption, a predetermined threshold will be used to enable the advanced analysis when multiple input variables are evaluated. Several parameters such as rate, cardiac QRS pulse width, cardiac QRS slew rate, and the various measures of signal stability are, in one embodiment, measured on a continuous basis. In another embodiment, the non-rate measures may be turned on only when the rate exceeds a predetermined limit. The range of this predetermined limit could be 50-240 bpm. In one embodiment, the limit for activation of the non-rate detection measures would be 140 bpm. In another embodiment, the predetermined threshold that allows advanced analysis may be a measure other than rate, for example, QRS width or some other measure. In any case, each of these parameters has associated registers for fine tuning or modifying the parameters on either a periodic basis or a more dynamic basis such as that associated with body positional changes.
[0056] In one embodiment of the detection algorithm, rate is used as a first criterion in the detection algorithm. As shown in
[0057] The parameter integration block
[0059] In another embodiment, the decision to shock/no-shock can be performed with as few as one parameter. For example a single scalar value can be set using QRS width. Beats with a QRS width wider than a threshold are classified as ventricular tachycardia. The threshold maximum be fixed or may be adjusted based on NSR computed measures or other calculations to account for long-term cardiac substrate changes.
[0060] In another embodiment, a single parameter, such as rate, can be monitored such that once it reaches a threshold, the detection algorithm can then analyze other parameters to determine the shock/no-shock decision, thus, conserving battery power.
[0061] In another embodiment, the decision to shock/no-shock can be performed with three parameters. For example, in three dimensions, one axis represents QRS stability, another axis represents QRS slew-rate and the third axis represents rate. The decision boundary can then be represented as a surface, which can be a simple plane or a more complex 3-dimensional surface. The decision boundary surface can be computed analytically or empirically to determine the shock/no-shock decision. Such a surface could also be updated and changed according to a continuous or semi-continuous or periodic measurement of reference values for that patient.
[0062] While a 2-dimensional embodiment of the detection algorithm is shown, other embodiments are possible such as an n-dimensional embodiment that employs n different input parameters.
[0063] Artificial neural networks can also be used to determine the shock/no-shock decision boundary by emulating the observed properties of biological nervous systems and drawing on the analogies of adaptive biological learning. The principal feature of artificial neural networks is a structure of highly interconnected processing elements that are analogous to neurons and are tied together with weighted connections that are analogous to synapses. Learning involves the changes that occur to the synaptic connections between neurons.
[0064] Neural networks are well suited to process n-dimensional input parameters and can be trained to produce a shock/no-shock decision often with imprecise or noisy data. Many different neural structures are possible. For example, one structure comprises a multilayer perceptron that is trained with a backpropagation of error algorithm, although numerous other structures and training algorithms could also be effective.
[0065] Numerous characteristics and advantages of the invention covered by this document have been set forth in the foregoing description. It will be understood, however, that this disclosure is, in many aspects, only illustrative. Changes may be made in details, particularly in matters of shape, size and arrangement of parts without exceeding the scope of the invention. The invention's scope is defined, of course, in the language in which the appended claims are expressed.


text:
"When I came in for an interview, I found the Principal and all the staff I met to be very kind and welcoming. After touring the school, I took very warmly to the fact that the A Level classes were small, meaning that lessons were very personal and you didn't have to shout to have your voice heard. I came to DLD with terrible grades in my A levels and no motivation for further education. However, after the excellent quality of teaching that I received, it motivated me to study and find ambition."
"DLD is really just a large family, there are various different types of people that you can engage with, from all parts of the world, so conversation never gets boring. The location is also perfect, right in the centre of London."
Erkan is going to study Middle Eastern Studies at SOAS, University of London.
A level courses:
Government and Politics A
Sociology B
Turkish B


text:
Sheryl Watkins T.C.R.G – Principal Teacher
Sheryl has been involved in Irish dancing for more than 35 years. Having started classes at the age of eight, Sheryl has enjoyed a successful and enjoyable competitive Irish dancing career both as a dancer and as a teacher. A five-time Queensland solo championship runner-up and Queensland Irish Association & Mid Year Feis Title winner, Sheryl has won many state figure dancing titles and is an Australian figure dance champion. She was also ranked in the Top 5 nationally for solos. In 1986, she was a recall finalist in the World Irish Dancing Championships, which were held in Limerick in Ireland, finishing in the Top 20.
Sheryl was also invited to perform with the internationally-acclaimed Irish group "The Chieftains" during their Brisbane tour in 1987 and took the stage at QPAC with the famous musicians. In 1989, "The Chieftains" invited her to join their Australian tour but the arrival of a baby daughter only weeks before the tour meant Sheryl was unable to accept the offer.
After retiring from competitive dance, Sheryl set her sights on teaching and was appointed principal teacher at the Queensland Irish Association, where she schooled almost 70 students from children to adults. Under her instruction, the QIA Dancers collected many solo and figure dancing titles including an Australian figure dancing championship title in 1991.
After two years at the QIA, Sheryl established her own dance school – the WATKINS ACADEMY OF IRISH DANCE – with classes operating in Ipswich and Brisbane's western suburbs. Since its inception in 1992, the WATKINS ACADEMY OF IRISH DANCE has produced many solo and figure dance champions and its dancers have been placed in the Top 10 in the world.
Sheryl Watkins is a qualified Irish dance teacher, having passed the comprehensive TCRG (Teachers) Exam. Registered with the An Coimisiun Le Rinci Gaelacha (Irish Dancing Commission) in Dublin and the Australian Irish Dancing Association.
Lorin Grey T.C.R.G
Lorin put on her first pair of Irish dancing shoes in 1992 in Mount Isa. Lorin quickly showed a passion and a gift for Irish dance and competed in her first State and National Championships in 1995. Since then, Lorin has enjoyed a wonderful career as a dancer having won five State solo titles in 2004, 2005, 2007, 2008 and 2009.
In 2008, Lorin realised a long-time goal by winning the Australian Senior Ladies Solo Title at the National Championships in Hobart. Lorin went on to win a prestigious World Championships Solo Medal in 2009, placing 18th overall. Lorin had previously also held a World recall medal. Lorin also competed in team dancing and her career highlights boast many State Titles and a National Title in 2007 in the Senior Mixed 4-Hand Own Choreography Jig event. Her most recent team achievement was finishing Runner-Up National Champions in the hotly-contested Senior Figure Dance event at the Australian Championships held in Wollongong in 2010. Now a qualified Irish dance teacher, having passed the tough TCRG exam, Lorin is enjoying the new phase in her Irish dancing career as a teacher.


text: Posted on May 2, 2012 by Emily Cotton Posted in Uncategorized . This is so random, but I really enjoy Lauren O Lauren, this girl is crazy! enjoy! Em More from my siteIn my Ipod – Throw Backs '90's baby'Friday Funny –Recipe of the day – Caribbean Curry CrabRandom – Missing Jamaica'Beautiful Mess' – Taren inspiredRandom – Usain Jokes


text:
My interactions with Japanese business professionals were fairly limited in number, but significant, especially in a class I took called Business Project. In this class, Adidas Japan came in and presented us with a marketing related problem that they are currently facing. We were tasked with finding the best way to measure Net Promoter Score (NPS), and where we could measure it best. We formed groups to solve this problem, and in mid-October and early December we went to Adidas Japan's headquarters to present our research and solutions.
During my two visits to Adidas Japan's HQ, I had several opportunities to network with current employees and Rikkyo alumni at Adidas. The employees were more than willing to talk about what it's like to work in Japan and their experiences with Adidas Japan. I also met several senior executives and mid-level managers that were happy to talk about their career paths, and what they liked or disliked about working in Japan.
I learned a lot about searching for jobs from Japanese students. Looking for a job at a Japanese company in Japan is very different from the United States. Internships differ between the two countries as they usually last one or two days in Japan versus two or three months in the United States. These one day internships are unpaid. Students do most of their network through these internships and career fairs. In Japan, looking for a job once you graduate is called "Job Hunting" as they typically take time off of school to schedule a lot of interviews, do as many one day internships as possible, and go to a lot of career fairs. Japanese workers rarely change companies. As far as networking goes, reach out to your professors and counselors to find out about career fairs and potential job opportunities. Several of the professors at Rikkyo teach part-time and work at various firms. Most networking techniques that work in the U.S. also work in Japan, so put them to use and be persistent.


text:
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