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28 Feb 2014:
The PLOS Computational Biology Staff
Correction: Mathematical Modeling of Heterogeneous Electrophysiological Responses in Human
Electrical activity plays a pivotal role in glucose-stimulated insulin secretion from pancreatic -cells. Recent findings have shown that the electrophysiological characteristics of human -cells differ from their rodent counterparts. We show that the electrophysiological responses in human -cells to a range of ion channels antagonists are heterogeneous. In some cells, inhibition of small-conductance potassium currents has no effect on action potential firing, while it increases the firing frequency dramatically in other cells. Sodium channel block can sometimes reduce action potential amplitude, sometimes abolish electrical activity, and in some cells even change spiking electrical activity to rapid bursting. We show that, in contrast to L-type -channels, P/Q-type -currents are not necessary for action potential generation, and, surprisingly, a P/Q-type -channel antagonist even accelerates action potential firing. By including SK-channels and dynamics in a previous mathematical model of electrical activity in human -cells, we investigate the heterogeneous and nonintuitive electrophysiological responses to ion channel antagonists, and use our findings to obtain insight in previously published insulin secretion measurements. Using our model we also study paracrine signals, and simulate slow oscillations by adding a glycolytic oscillatory component to the electrophysiological model. The heterogenous electrophysiological responses in human -cells must be taken into account for a deeper understanding of the mechanisms underlying insulin secretion in health and disease, and as shown here, the interdisciplinary combination of experiments and modeling increases our understanding of human -cell physiology.
Author Summary
Insulin is a glucose-lowering hormone secreted from the pancreatic -cells in response to raised plasma glucose levels, and it is now well-established that defective insulin secretion plays a pivotal role in the development of diabetes. The -cells are electrically active, and use electrical activity to transduce an increase in glucose metabolism to calcium influx, which triggers insulin release. Experimental and theoretical studies on -cells from rodents have provided valuable insight in their electrophysiology. However, human -cells differ from their rodent counterparts in several aspects including their electrophysiological characteristics. We show that the electrophysiological responses in human -cells to a range of experimental manipulations are heterogeneous. We extend a previous mathematical model of electrical activity in human -cells to investigate such heterogeneous and nonintuitive electrophysiological responses, and use our findings to obtain insight in previously published insulin secretion measurements. By adding a glycolytic component to the electrophysiological model, we show that oscillations in glucose metabolism might underlie slow oscillations in electrical activity, calcium levels and insulin secretion observed experimentally. We conclude that the interdisciplinary combination of experiments and modeling increases our understanding of human -cell physiology and provides new insight in -cell heterogeneity.
Citation: Riz M, Braun M, Pedersen MG (2014) Mathematical Modeling of Heterogeneous Electrophysiological Responses in Human β-Cells. PLoS Comput Biol 10(1): e1003389. doi:10.1371/journal.pcbi.1003389
Editor: Bard Ermentrout, University of Pittsburgh, United States of America
Received: July 17, 2013; Accepted: October 22, 2013; Published: January 2, 2014
Copyright: © 2014 Riz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: MGP was partly supported by the Lundbeck Foundation, and the EU via a Marie Curie Intra-European Fellowship. The work in Padova was supported by a grant from Sanofi AG Frankfurt, Germany. MB was supported by the CIHR (MOP-106435) and the CFI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and have the following conflicts: The work in Padua was partially supported by a research grant from Sanofi.
Glucose-stimulated insulin secretion from human pancreatic -cells relies on the same major signaling cascade as their rodent counterparts, with electrical activity playing a pivotal role. Following metabolism of the sugar, ATP-sensitive potassium channels (K(ATP)-channels) close in response to the elevated ATP/ADP-ratio, which triggers action potential firing and -influx through voltage-gated calcium channels. The resulting increase in intracellular calcium leads to insulin release by -dependent exocytosis [1]–[4]. However, the electrophysiological properties of human and rodent -cells show important differences, e.g., with respect to their palette of expressed -channels and the role of -channels, which contribute to electrical activity in human but not in rodent -cells [1], [3].
Mathematical modeling has played important roles in studying the dynamics of electrical activity in rodent -cells [5], [6], and could plausibly aid in understanding the electrophysiological responses in human -cells, and how they might differ from rodent cells. Recently, the first model of electrical activity in human -cells [7] was constructed from careful biophysical characterizations of ion channels in human -cells, mainly from Braun et al. [3]. The model [7] included -channels, three types of -channels, an unspecified leak-current, and several -channels: delayed rectifier (Kv) -channels, large-conductance (BK) -sensitive -channels, human ether-a-go-go (HERG) -channels as well as K(ATP)-channels. Recently evidence for small conductance (SK) -sensitive -channels in human -cells was published [4], [8], a current not included in the mathematical model [7].
The model [7] was shown to reproduce, depending on parameter values, spiking or rapid bursting electrical activity, which could be modified in accordance with a series of experiments by simulating pharmacological interventions such as ion channel blocking. These experiments were in general straightforward to interpret, also without a model. For example, the facts that blocking depolarizing - or -currents slowed or abolished electrical activity [3] are as one would expect.
Here, we extend the previous model for human -cells [7] by including SK-channels and dynamics, and show that the model now has reached a level of maturity that allows us to get insight in less intuitive experimental findings. We find experimentally that SK-channels in some cells play an important role in controlling electrical activity, while they have virtually no effect in other cells. Using the extended version of the model, we show that this difference can be explained by differences in the excitability of the cells. Moreover we find that SK-channels can substitute for HERG-channels in controlling rapid bursting. We also show that blocking -channels in some cells can transform spiking behavior into rapid bursting, in contrast to the usual effect of -channel blockers, which in general reduce or abolish spiking behavior [3], [9], [10]. Using our model we suggest that this happens in cells with a large -current and that BK-channels play a prominent role. In addition, we suggest that SK-channels might underlie the surprising result that blocking depolarizing P/Q-type -channels enhances electrical activity, in contrast to the effect of L- or T-type -channel antagonists, which reduce excitability and electrical activity [3]. Our model is then used to investigate paracrine effects of -aminobutyric acid (GABA) and muscarinic signaling on electrical activity. Finally, we show experimentally slow oscillations in electrical activity that might underlie pulsatile insulin secretion from human pancreatic islets, and by adding an oscillatory glycolytic component [11] to the electrophysiological model, we simulate such slow bursting patterns.
To investigate a series of experimental observations, we have extended our previous model of electric activity in human -cells [7] by including several additional components of human -cell physiology, as described in the following, and in greater details in the Methods section. The mathematical modeling was carefully based on experimental data, as was the development of the core electrophysiological part modeled previously [7]. The extended model includes small conductance -activated -channels (SK-channels), which are expressed in human -cells [4], [8]. The size of the SK-current was estimated from experimental measures [8]. We made a special effort to carefully model the submembrane dynamics of , since SK-channels are controlled by the submembrane concentration (), which reacts rapidly to each action potential so that activation of SK-channels might influence the generation and shape of action potentials during spiking electrical activity. In order to study paracrine signalling, our extended model also includes currents due to -aminobutyric acid (GABA) activation of GABAA receptors, which are ligand-gated channels operating in human -cells [12]. Finally, a glycolytic oscillator [11] has been added to the model to account for slow oscillations in ATP levels in human -cells [13], [14], which have been suggested to underlie slow patterns of electrical activity, oscillations, and pulsatile insulin release.
Summarizing, the new version of the model now includes components from glucose metabolism, additional electrophysiological components (SK-channels and GABAA receptors), and dynamics, leading to a global model of human -cell physiology, which, importantly, is based as far as possible on published data from human -cells.
SK channels
When stimulated by glucose, human -cells show electrical activity [1], [3]. Human -cells express SK-channels [4], [8], which might participate in controlling electrical activity. To study the role of SK-channels in human -cells, we included SK-channels and dynamics in our previous model [7]. The new model with standard parameters produces spiking electrical activity (Fig. 1A), which is virtually unaffected by setting the SK-conductance nS/pF simulating SK-channels block. This model prediction was confirmed by our experimental data, and was also observed in at least one cell by Jacobson et al. [8]. Fig. 1B shows an example of spiking electrical activity in a human -cell stimulated by 6 mM glucose, where addition of the SK1-3 channel blocker UCL 1684 (0.2 µM) did not affect the spiking pattern. Unchanged or marginal effects on electrical activity were also seen with a specific SK4 channel antagonist, TRAM-34 (1 µM, Fig. 1C). However, in some cells TRAM-34 application increased the action potential dramatically (Fig. 1D) in agreement with observations with the SK-channel antagonist apamin [8]. Note that before SK-channel block, the cell in Fig. 1D was almost quiescent, and fired action potentials very infrequently and randomly. This increase in spike frequency can be simulated by a stochastic version of the model. By including noise in the K(ATP) current, an otherwise silent cell produces infrequent action potentials evoked by random perturbations (Fig. 1E). When the SK-conductance is set to 0 nS/pF, the cell starts rapid action potential firing driven by the underlying deterministic dynamics. The model analysis indicates that this mechanism only works if the cell is very near the threshold for electrical activity in the absence of the SK-channel antagonist. Düfer et al. [15] suggested a similar, important role for SK4 channels in promoting electrical activity in murine -cells at subthreshold glucose concentrations. Summarizing, cell-to-cell heterogeneity can explain the differences seen in the electrophysiological responses to SK-channel antagonists.
Note the differences in time-scales. A: Simulation, with default parameters, showing no effect of SK-channels block ( nS/pF during the period indicated by the gray bar). B: Experimental recording of spiking electrical activity in the same human -cell before (left) and during (right) application of the SK1-3 channel antagonist UCL-1684 (0.2 µM). C: Experimental recording of spiking electrical activity in the same human -cell before (left) and during (right) application of the SK4 channel antagonist TRAM-34 (1 µM), which had little effect on the action potential frequency in this cell. D: Experimental recording of spiking electrical activity in the same human -cell before (left) and during (right) application of the SK4 channel antagonist TRAM-34 (1 µM), which accelerated the action potential frequency in this cell. E: Stochastic simulation reproducing the dramatic effect of SK-channels block ( nS/pF during the period indicated by the gray bar). Other parameters took default values, except nS/pF.
In addition to spiking electrical activity, human -cells often show rapid bursting, where clusters of a few action potentials (active phases) are separated by hyperpolarized silent phases [1], [4], [9], [10], [16] (Fig. 2A). The extended model presented here can also reproduce this behavior (Fig. 2B) as could the previous version of the model [7], where the alternations between silent and active phases were controlled by HERG-channels. In contrast, in the present version of the model the rapid burst pattern (Fig. 2B, upper trace) can be controlled by SK-channels, which in turn are regulated by and ultimately by bulk cytosolic levels (). The simulated cytosolic concentration shows the characteristic sawtooth pattern (Fig. 2B, lower trace) of a slow variable underlying bursting [17], [18]. Thus, as in the pioneering model by Chay and Keizer [19], increases during the active phase and activates SK-channels, which eventually repolarize the cell. During the silent phase decreases and SK-channels close, allowing another cycle to occur.
A: Experimental recording of rapid bursting in a human -cell. B: Simulation of bursting driven by via SK-channels. Default parameters except nS/pF, nS/pF, mV.
Blocking voltage-dependent -channels in human -cells showing spiking electrical activity with tetrodotoxin (TTX) typically reduces the action potential amplitude by ∼10 mV, and broadens its duration [3], [9], [10] (Fig. 3A). The previous version of the model [7] could reproduce these results, though the reduction in peak voltage was slightly less than observed experimentally. The inclusion of SK-channels in the model leads to a greater reduction in the spike amplitude (Fig. 3B, upper trace) when -channels are blocked. This improvement is because of a mechanism where the slower upstroke in the presence of -channel blockers allows submembrane to build up earlier and to higher concentrations (Fig. 3B, lower trace), and consequently to activate more SK-channels, which in turn leads to an earlier repolarization reducing the action potential amplitude. In other experiments (Fig. 3C) [16], TTX application suppresses action potential firing. In agreement, simulated spiking electrical activity can be suppressed by TTX application if the cell is less excitable because of, for example, smaller -currents (Fig. 3D, upper, black trace). Before TTX application, the simulated cell had less hyperpolarized inter-spike membrane potential ( mV; Fig. 3D) compared to the simulation with default parameters ( mV, Fig. 3B). This finding is in accordance with experimental recordings (compare Fig. 3A and 3C). The cessation of action potential firing leads to a reduction in simulated (Fig. 3D, lower, black trace). The model predicts that spiking, electrical activity can continue in presence of TTX even in less excitable cells, e.g., with lower depolarizing -currents, if the hyperpolarizing K(ATP)-current is sufficiently small (Fig. 3D, upper, gray trace). In this case, is nearly unchanged (Fig. 3D, lower, gray trace). Hence, it is the relative sizes of the depolarizing and hyperpolarizing currents that determine whether TTX application silences the cell or allows the cell to remain in a region where action potential firing continues. The model thus predicts that in some cells, which stop firing action potentials in the presence of TTX, increased glucose concentrations or sulfonylureas (K(ATP)-channel antagonists) could reintroduce spiking electrical activity.
A: TTX caused a reduction in action potential amplitude in this human -cell. B: Simulation with default parameters showing V (upper trace, left axis) and (lower trace, right axis), reproducing the data in panel A. C: TTX abolished action potential firing in this human -cell. D: Simulations of V and with default parameters except nS/pF. With default K(ATP)-channel conductance nS/pF, the simulation reproduces the data in panel C (black traces). When nS/pF, the model shows continued firing with -channel block (gray traces). E: TTX changed spiking into rapid bursting electrical activity in this human -cell. F: Simulation showing V (upper), (middle), and (lower), reproducing the data in panel E. Parameters took default values, except nS/pF, ms, nS/pF, nS/pF, nS/pF, and mV. The extracellular glucose concentration was 6 mM in all experiments. Each couple of experimental traces (panels A, C and E) is from the same human -cell before (left) and during (right) application of TTX. In the simulations, the -channel conductance was set to 0 nS/pF during the period indicated by the gray bars.
More surprisingly, TTX application can change spiking electrical activity to rapid bursting in some cells (Fig. 3E). This behavior can also be captured by the model (Fig. 3F). To simulate this behavior it was necessary to increase the size of the -current. Without TTX, the big -current leads to large action potentials, which activate sufficient BK-current to send the membrane potential back to the hyperpolarized state, allowing a new action potential to form. With -channels blocked, there is insufficient depolarizing current to allow full action potentials to develop. In consequence, less BK-current is activated (Fig. 3F, lower trace), and the membrane potential enters a regime with more complex dynamics where smaller spikes appear in clusters from a plateau of ∼−40 mV. The change to bursting activity leads to a notable increase in simulated (Fig. 3F, middle trace).
High-voltage activated L- and P/Q-type -currents are believed to be directly involved in exocytosis of secretory granules in human -cells [1], [3], [4], [20], [21]. Blocking L-type -channels suppresses electrical activity [3], which is reproduced by the model (Fig. 4A) [7], and the lack of electrical activity is likely the main reason for the complete absence of glucose stimulated insulin secretion in the presence of L-type -channel blockers [3]. Thus, L-type -channels participate in the upstroke of action potentials and increases excitability of human -cells.
A: Spiking electrical activity is suppressed by L-type -channel block in the model with default parameters, and nS/pF during the period indicated by the gray bar. B: Spiking electrical activity is accelerated by the application of -agatoxin IVA in human -cells. Recordings from the same human -cell in 6 mM extracellular glucose before (left) and during (right) application of 200 nM -agatoxin IVA. C: Model simulation with default parameters of the membrane potential during spiking electrical activity under control conditions and after blockage of P/Q-type -channels ( nS/pF during the period indicated by the gray bar). D: In the model, the peak submembrane -concentration is lower when P/Q-type channels are blocked. E: The reduced activate less SK-current when P/Q-type channels are blocked.
In contrast, and surprisingly, application of the P/Q-type -channel antagonist -agatoxin IVA does not block or slow down electrical activity, but leads to an increased spike frequency (Fig. 4B). Electrical activity continues also in our model simulations of P/Q-type channel block with slightly increased spike frequency (Fig. 4C). Reduced entry leads to lower peak concentrations in the submembrane space (; Fig. 4D). As a consequence, less hyperpolarizing SK-current is activated (Fig. 4E), which leads to an increase in spike frequency (Fig. 4C). Hence, the reduction in excitability caused by blockage of the P/Q-type -current can be overruled by the competing increase in excitability due to the smaller SK-current. Experimentally, -agatoxin IVA application reduced the action potential amplitude slightly in 3 of 4 cells (by 2.0–4.3 mV), a finding that was quantitatively reproduced by the model, although the reduction was larger (∼7.5 mV in Fig. 4C). A direct conclusion from Fig. 4B is that the P/Q-type -current is not needed for the action potential upstroke, unlike the L-type current, probably because of the fact that P/Q-type channels activate at higher membrane potentials than L-type channels. The fact that electrical activity persists with P/Q-type -channels blocked, albeit with lower peak , could underlie the finding that -agatoxin IVA only partly inhibits insulin secretion [3].
Paracrine effects on electrical activity
The neurotransmitter -aminobutyric acid (GABA) is secreted from pancreatic -cells, and has been shown to stimulate electrical activity in human -cells [12]. In human -cells, GABA activates GABAA receptors, which are ligand-gated channels, thus creating an additional current. Notably, the reversal potential in human -cells is less negative than in many neurons, and positive compared to the -cell resting potential, which means that currents, such as the GABAA receptor current, stimulate action potential firing in -cells. Hence, GABA is a excitatory transmitter in -cells, in contrast to its usual inhibitory role in neurons. We simulate the addition of GABA by raising the GABAA receptor conductance. In a silent model cell with a rather large K(ATP)-conductance, simulated GABA application leads to a single action potential whereafter the membrane potential settles at ∼−45 mV (Fig. 5A), in close correspondence with the experimental results [12]. In an active cell, the simulation of activation of GABAA receptors leads to a minor depolarization and increased action potential firing (Fig. 5B), as found experimentally [12].
A: Simulation of application of 100 µM GABA to a silent cell (reproducing Fig. 7A in [12]). Default parameters except nS/pF. GABA application was simulated by setting to 0.1 nS/pF during the period indicated by the gray bar. B: Simulation of application of 10 µM GABA to an active cell (reproducing Fig. 7B in [12]). GABA application was simulated by setting to 0.020 nS/pF during the period indicated by the gray bar. Other parameters took default values. C: Experimental recording of spiking electrical activity in the same human -cell before (left) and during (right) application of carbachol (20 µM). D: Simulation of accelerated action potential firing due to carbachol application. Default parameters except nS/pF. Carbachol application was simulated by increasing to 0.030 nS/pF during the period indicated by the gray bar.
Another neurotransmitter, acetylcholine, might also play a paracrine role in human pancreatic islets, where it is released from -cells, and activates muscarinic receptors in -cells [22]. Muscarinic receptor activation by acetylcholine triggers a voltage-insensitive -current in mouse pancreatic beta-cells [23], and similarly, the muscarinic agonist carbachol activates nonselective leak channels (NALCN) in the MIN6 -cell line [24]. Based on these findings, it was speculated that muscarinic activation of NACLN currents in human -cells might participate in the positive effect of acetylcholine and carbachol on insulin secretion [4]. Experimentally, we found that carbachol (20 µM) accelerates action potential firing (Fig. 5C). We tested the hypothesis of a central role of leak current activation by increasing the leak conductance in the model to simulate carbachol application, which caused accelerated action potential firing. The simulation thus reproduced the experimental data, and lends support to the hypothesis that carbachol and acetylcholine can accelerate action potential firing via muscarinic receptor-dependent stimulation of NALCN currents [4].
Slow oscillations
We finally use our model to address the origin of slow rhythmic patterns of electrical activity in human -cells (Fig. 6A) [4], [25], which likely underlie slow oscillations in intracellular [26], [27] and pulsatile insulin release [28], [29]. Based on accumulating evidence obtained in rodent islets [5], [30], we have previously speculated that oscillations in metabolism could drive these patterns [7]. In support of this hypothesis, oscillations in ATP levels with a period of 3–5 minutes have been observed in human -cells [13], [14]. By adding a glycolytic component [11], which can oscillate due to positive feedback on the central enzyme phosphofructokinase (PFK), our model can indeed simulate such periodic modulation of the electrical pattern, where action potential firing is interrupted by long silent, hyperpolarized periods, which drives slow oscillations (Fig. 6).
A: Experimental recording of slow oscillations in action potential firing in a human -cell exposed to 10 mM glucose. B–D: Simulation of slow bursting driven by glycolytic oscillations with glucose concentration mM and default parameters, except nS/pF, nS/pF, nS/pF. Oscillations in glycolysis create pulses of FBP (B), which via ATP production modulates K(ATP) channels in a periodic fashion (C). The rhythmic changes in K(ATP) conductance drives slow patterns of electrical activity (D), which causes oscillations in the intracellular concentration (E).
Human -cells show complex and heterogeneous electrophysiological responses to ion channel antagonists. It can therefore sometimes be difficult to reach clear conclusions regarding the participation of certain ion channels in the various phases of electrical activity, in particular since some of the electrophysiological responses are nonintuitive as shown here. A deeper understanding of the role of ion channels in electrical activity and insulin secretion could have important clinical benefits, since it might help in the development of new anti-diabetic drugs.
We have here shown how mathematical modeling can help in interpreting various electrophysiological responses, and in particular, to study the effect of competing effects and cell heterogeneity. The role of SK-channels in human -cells is still not clear. We (Fig. 1) and others [8] have found heterogeneous electrophysiological responses to SK-channel antagonists. Our model suggests that these differences can be caused by underlying variations in cell excitability: Less excitable -cells that produce action potentials evoked mostly by stochastic channel dynamics show a clear increase in action potential frequency when SK-channels are blocked (Fig. 1DE). In contrast, spiking electrical activity in very active cells is driven by the deterministic dynamics caused by ion channel interactions, and is nearly unchanged by SK-channel blockers (Fig. 1A–C). We showed also that rapid bursting activity can be driven by and SK-channels (Fig. 2), which could add a complementary mechanism to HERG-channel dynamics [7] for the control of rapid bursting.
The wide range of responses to TTX could be accounted for by a single model but with different parameters, i.e., differences in the relative size of the various currents. A peculiar finding is the qualitative change from spiking to rapid bursting seen in some cells (Fig. 3E). We suggest that this happens in human -cells with large -currents. The blockage of this depolarizing current reduces the amplitude of the action potentials, and as a consequence, the size of the hyperpolarizing BK-current. Under the right conditions, the combination of these competing events allows the membrane potential to enter a bursting regime controlled by SK- and/or HERG-channels (Fig. 3F). Interestingly, it has been found that TTX reduces insulin secretion evoked by 6 mM glucose greatly, but at glucose levels of 10–20 mM, the effect of TTX on secretion is smaller [3], [9], [10]. Based on our simulations showing that less excitable cells cease to fire in the presence of TTX (Fig. 3D, black traces), but that lower can reintroduce spiking activity (Fig. 3D, gray traces), we suggest that at low, near-threshold glucose levels TTX abolishes electrical activity in many cells, which reduces the and consequently insulin secretion greatly (Fig. 3D, black traces). At higher glucose concentrations, -cells have lower K(ATP)-conductance and in some of the cells that stop firing in low glucose concentration the effect of TTX on electrical activity and is smaller (Fig. 3D, gray traces). Hence, more -cells remain active in the presence of TTX at high than at low glucose levels. Consequently, insulin secretion is more robust to TTX at higher glucose concentrations.
Similarly, insulin release is more affected by the P/Q-type -channel blocker -agatoxin IVA at 6 mM (−71%) than at 20 mM (−31%) glucose [3]. This is in contrast to L-type -channel antagonists, which abolish insulin secretion at both high (15–20 mM) and low (6 mM) glucose concentrations [1], [3], [10]. These results concerning L-type -channel block are easily explained by the fact that L-type channel activity is necessary for action potential generation [3] (Fig. 4A). In contrast, we showed that electrical activity in human -cells not only persists, but is accelerated by -agatoxin IVA (Fig. 4B). The counter-intuitive finding of increased excitability and electrical activity when the depolarizing P/Q-type -current is blocked by -agatoxin IVA can be accounted for by an even greater reduction in the hyperpolarizing SK-current due to reduced -influx and consequently lower .
Our mathematical modeling confirmed that GABA released from human -cells can have a role as a positive feedback messenger. GABA application has been shown to depolarize both silent and active human -cells [12], which was reproduced here. A detailed characterization of GABAA receptor currents would refine the analysis presented here.
Data from mouse -cells [23] and the MIN-6 -cell line [24] suggest that muscaric agonists such as carbachol and acetylcholine stimulate insulin secretion partly by activating NACLN currents. Using our model we could translate this finding to the human scenario, thus testing the hypothesis that this mechanism is also operating in human -cells [4]. Our simulations confirmed that increased leak currents can underlie the change in electrical activity found experimentally (Fig. 5C). The incretin hormone glucagon-like peptide 1 (GLP-1) has also been shown to act partly via activation of leak channels [31], a mechanism which might be involved in activating otherwise silent -cells [7], [32], [33]. These results suggest that leak currents could play important roles in controlling electrical activity in -cells, and potentially be pharmacological targets. Further studies are clearly needed to investigate these questions.
We were also able to simulate slow rhythmic electrical activity patterns by adding an oscillatory glycolytic component to the model. To date, there is to our knowledge no evidence of oscillations in glycolytic variables in human (or rodent) -cells or islets, but ATP levels have been found to fluctuate rhythmically also in human -cells [13], [14], supporting the idea of metabolism having a pacemaker role. In agreement, data from rodent -cells show accumulating evidence for oscillations in metabolism playing an important role in controlling pulsatile insulin secretion [5], [30]. It will be interesting to see if these findings in rodents are applicable to human -cells.
Regarding the model development, the inclusion of SK-channels in the model provided insight that was not within reach with the previous version of the model [7]. Besides the direct investigation of the role of SK-channels, the acceleration in action potential firing seen with P/Q-type channel blockers (Fig. 4) can not be reproduced by the older version of the model without SK-channels [7]. Moreover, considering the effect of TTX on spike amplitude, a better correspondence between experiments and simulations was found with SK-channels included in the model. To model SK-channel activation accurately, we made a special effort to describe carefully. Submembrane responds rapidly to an action potential, while integrates many action potentials. The rapid submembrane dynamics has important consequences for the study of the role of SK-channels in spiking electrical activity, e.g., it was crucial for explaining the larger effect of TTX on spike amplitude in this version of the model. Most models of electrical activity in rodent -cells do not include a submembrane compartment, but these models were typically built to explain the slow bursting patterns seen in rodent islets with a period of tens of seconds. For these long time scales, the rapid dynamics in the submembrane compartment is not important. In contrast, the situation is different in human -cells with their faster dynamics.
We build on the previously published Hodgkin-Huxley type model for human -cells [7], which was mainly based on the results of Braun et al. [3], who carefully assured that investigated human islet cells were -cells. We include SK-channels in the model. Since these channels are -sensitive and located at some distance from -channels [34] we also model -dynamics in a submembrane layer controlling SK-channel activity.
The membrane potential (measured in mV) develops in time (measured in ms) according to(1)All currents (measured in pA/pF), except the SK-current and the GABAA receptor mediated current , are modeled as in [7]. Expressions and parameters are given below. For the stochastic simulation in Fig. 1E, we included "conductance noise" [35] in the K(ATP) current by multiplying by a stochastic factor , where is a standard Gaussian white-noise process with zero mean and mean square , see also [36]–[38].
SK-channels are assumed to activate instantaneously in response to elevations at the plasma membrane but away from channels [34], and are modeled as [39](2)In human -cells, flash-released triggered a ∼10 pA current at a holding current of mV, presumably through SK-channels [8]. Assuming that SK-channels were nearly saturated by , the maximal SK-conductance is estimated to be nS/pF. Here, = 10 pF is the capacitance of the plasma membrane [3].
In Eq. 2, is the submembrane concentration (; measured in µM), which is described by a single compartment model [21](3)where is the ratio of free-to-total , µmol/pA/ms changes current to flux, and and are the volumes of the submembrane compartment and the bulk cytosol, respectively. describes the flux of from the submembrane compartment to the bulk cytosol, is the flux through plasma membrane -ATPases, and represents flux through the - exchanger. Cytosolic (; measured in µM) follows(4)where describes SERCA pump-dependent sequestration of into the endoplasmic reticulum (ER), and is a leak flux from the ER to the cytosol. Expressions and parameters for the fluxes are taken from [40].
The submembrane compartment volume is estimated based on the considerations of Klingauf and Neher [41], who found that a shell model (in contrast to a domain model) describes submembrane satisfactorily when the shell-depth is chosen correctly. The dynamics between channels can be estimated from a shell model at a depth of ∼23% of the distance to a -channel. In mouse -cells the interchannel distance has been estimated to be nm [42]. Moreover, SK-channels are located nm from channels [34].
Based on these considerations, we modeled the submembrane space controlling SK-channels as a shell of depth nm. The radius of a human -cell is µm, which gives cell volume (), shell volume () and internal surface area () of the shell, of(5)The flux-constant can then be calculated as [43](6)where is a typical length scale. We set to 1 µm, which together with the diffusion constant for , [41], [44], gives .
In human -cells, GABA activates GABAA receptors, which are ligand-gated channels. We model the current carried by GABAA receptor as a passive current with the expression(7)where is the GABAA receptor conductance, and mV is the chloride reversal potential [4]. We estimate from the findings that 1 mM GABA evokes a current of pA/pF (but with substantial cell-to-cell variation) at a holding potential of −70 mV [12], which yields a conductance of nS/pF. To simulate the changes in firing patterns evoked by lower GABA concentrations (10 or 100 µM) [12], we take into consideration the does-response curve [45] for the subunits, which are the most highly expressed subunits in human -cells [12]. At 10 µM the GABA-evoked current is -fold smaller compared to 1 mM GABA, and we set nS/pF. At 100 µM, the reduction is about 2-fold compared to 1 mM. We used nS/pF to simulate application of 100 µM GABA.
To investigate slow electrical patterns (Fig. 6) we added a glycolytic component [11], which drives ATP levels and K(ATP) channel activity. The glycolytic subsystem can oscillate due to positive feedback on the enzyme phosphofructokinase (PFK) from its product fructose-1,6-bisphosphate (FBP). The glycolytic equations are(8)(9)(10)where is the rate of glucokinase, which phosphorylates glucose to glucose-6-phosphate (G6P). G6P is assumed to be in equilibrium with fructose-6-phosphate (F6P), the substrate for PFK, and is the sum of G6P and F6P. is the rate of PFK producing FBP, which is subsequently removed by fructose-bisphosphate aldolase (FBA), which produces glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone-phosphate (DHAP) with rate . DHAP and G3P are assumed to be in equilibrium, and indicates their sum. Finally, G3P serves as substrate for glyceraldehyde-3-phosphate dehydrogenase (GAPDH with rate ), which via the lower part of glycolysis eventually stimulates mitochondrial ATP production. We introduce a phenomenological variable that mimics ATP levels, and is model by(11)The K(ATP) conductance depends inversely on , and is modeled as(12)Expressions and parameters are given below.
Simulations were done in XPPAUT [46] with the cvode solver, except the stochastic simulation in Fig. 1E, which was performed with the implicit backward Euler method. Computer code can be found as supplementary material, or downloaded from http://www.dei.unipd.it/ pedersen.
Human pancreatic islets were obtained with ethical approval and clinical consent from non-diabetic organ donors. All studies were approved by the Human Research Ethics Board at the University of Alberta. The islets were dispersed into single cells by incubation in free buffer and plated onto 35 mm plastic Petri dishes. The cells were incubated in RPMI 1640 culture medium containing 7.5 mM glucose for >24 h prior to the experiments. Patch-pipettes were pulled from borosilicate glass to a tip resistance of 6–9 MΩ when filled with intracellular solution. The membrane potential was measured in the perforated-patch whole-cell configuration, using an EPC-10 amplifier and Patchmaster software (HEKA, Lambrecht, Germany). The cells were constantly perifused with heated bath solution during the experiment to maintain a temperature of C. The extracellular solution consisted of (in mM) 140 NaCl, 3.6 KCl, 0.5 , 1.5 , 10 HEPES, 0.5 , 5 and 6 glucose (pH was adjusted to 7.4 with NaOH). The pipette solution contained (in mM) 76 , 10 KCl, 10 NaCl, 1 , 5 HEPES (pH 7.35 with KOH) and 0.24 mg/ml amphotericin B. -cells were identified by immunostaining (18 out of 28 cells) or by size when immunostaining was not possible (cell capacitance >6 pF, [3]). Tetrodotoxin (TTX) and -agatoxin IVA were purchased from Alomone Labs (Jerusalem, Israel), UCL-1684 was obtained from R&D Systems (Minneapolis, MN), TRAM-34 from Sigma-Aldrich (Oakville, ON, Canada). Figures with experimental responses to ion channel antagonists (Figs. 1, 3, 4 and 5) show recordings from the same cell before (ctrl) and after application of the blocker.
Model equations and parameters
For completeness, we report all expressions and parameters of the mathematical model here. For details, please refer to the Modeling section above and the previous article [7].
The main variables, membrane potential, , submembrane , , and cytosolic , , are described by(13)(14)(15)
The currents are(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)where(27)and activation variables (and similarly inactivations variabels, , where denotes the type of current) follow(28)where (respectively ) is the time-constant of activation (respectively inactivation for ), and (respectively ) is the steady-state voltage-dependent activation (respectively inactivation) of the current. The steady-state activation (and inactivation) functions are described with Boltzmann functions,(29)except(30)for -dependent inactivation of L-type channels. The time-constant for activation of Kv-channes is assumed to be voltage-dependent [3], [7],(31) -fluxes are [40](32)(33)(34)Glycolysis was modeled by [11](35)(36)(37)which controls the electrophysiological subsystem via the "ATP-mimetic" and K(ATP)-channels, as described by(38)(39)Here(40)(41)(42)(43)where(44)(45)(46)and(47)Default parameters are given in Table 1.
This work was initiated while MGP was affiliated with Lund University Diabetes Centre (LUDC), Malmö, Sweden. MR thanks LUDC for hospitality during her stay in Malmö. Tragically and unexpectedly Dr. Matthias Braun passed away far too young while this work was in press. We will remember him for his pleasant personality and as an outstanding islet electrophysiologist. Without his superb scientific skills the modelling presented here would not have been possible.
Author Contributions
Conceived and designed the experiments: MR MB MGP. Performed the experiments: MR MB MGP. Analyzed the data: MR MB MGP. Wrote the paper: MGP. Wrote the computer code: MR MGP. Revised manuscript: MR MB MGP. Approved final version of manuscript: MR MB MGP.
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text: Neil DeGrasse Tyson Calls Out Prometheus For Its Sketchy ScienceAndrew Liszewski6/09/12 2:00pmFiled to: SciencetwitterMoviesSpaceNeil deGrasse TysonPrometheustweet265EditInvite manuallyPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink Most science fiction fans are happy to shrug off technical inaccuracies while enjoying a movie. But not if you're the director of the Hayden Planetarium and a world-renowned astrophysicist. Which is why Neil DeGrasse Tyson couldn't help but question one of Prometheus' technical gaffes.In the film they travel 35 light years into space, which Charlize Theron's character comments as being about half a billion miles from Earth. But that estimate's a little off. Like, way off. As Tyson points out, half a billion miles from Earth would only put you just past Jupiter. In reality, a single light year equates to 5.88 trillion miles, not billion. So 35 light years would have put the crew some 205.8 trillion miles away from the Earth. Well past Jupiter, and well outside of our solar system. So thanks Neil for keeping an eye out for bad science in movies. The world's a more well-informed place as long as you're keeping watch. [Twitter - Neil DeGrasse Tyson via Geekosystem] Reply265 repliesLeave a reply

subset: commoncrawl


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The iOS version is said to be a port of the PS3 game, although we've yet to see just how it looks. Upon its release, Wizardry: Labyrinth of Lost Souls will be playable on iPhone 4s, iPads and 4th generation iPod Touches.
Boot Hill Heroes July 24 • 9:00am PDT/12:00pm EDT
Tales from the Borderlands July 31 • 9:00am PDT/12:00pm EDT
Breath of Fire IV Continues! Every Monday • 5:00pm PDT/8:00pm EDT
Grandia II Continues! Every Wednesday • 2:00pm PDT/5:00pm EDT


text:
By doing some quick math, it becomes clear that by speaking just one language, organizations are only reaching about a quarter of online users. To expand reach and engage with a wider audience, global businesses must adopt multilingual communications. Otherwise, they risk abandoning potential customers who will instead turn to competitors who communicate with them on a local level.
Mar 02, 2004
The Wall Street Journal Online at WSJ.com has announced an enhanced version of In Today's Paper, which is designed to make it easy for online subscribers to "flip through" the site, particularly print readers accustomed to the print layout. In Today's Paper is one page on the site where all of the articles from that day's print Journal can be found online; the page features all print headlines and the section front articles feature summaries from the online editors.
Other online-only features include: Scoops and updated news every day; Journal archives; Personalized tracking of industries; Email articles to colleagues and friends; Interactive graphics and articles; More columns and discussions from Journal writers; Global edition with full content of Asian and European Journals every day; Research on nearly 30,000 companies worldwide; and Personal finance tools in the newly redesigned Personal Journal section. Prior to the enhancements, the In Today's Paper section showed a glossary of just the section front articles (A1, B1, C1, W1), but nothing inside the paper was available in that section. Now, the enhanced In Today's Paper section, provides a glossary showing every headline listing of major articles organized by section of the Journal paper in one place.


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*Kit includes: 6-ft of 1/2, 20-ft of 3/8, 8-ft of 1/4 diameter, expandable chrome covering plus 6-chrome zip ties and black and clear shrink tubing*Use what the professionals use to cover cables, hoses and wires quickly and inexpensively*Expands easily to fit over ends for smooth installation and long lasting protection*Dynamic chrome covering adds a bright, shiny appearance*Fits snugly for a clean, custom look*More than enough product to do your entire motorcycle*Can be used to customize any motorcycle
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Cycle House Chrome Set of 2 caps for front fork tubes for Suzuki M109R
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30 June 2016
Gavi Board reappoints WHO's Flavia Bustreo as vice-chair
Board also confirms appointment of four new members and two alternates.
26 May 2016
Gavi welcomes price trend for pentavalent vaccine
Latest price information published by UNICEF.
Gavi welcomes contribution of US$ 76 million from Government of Japan
New multi-year commitment will help Vaccine Alliance partners towards goal of immunising 300m children by 2020.
23 May 2016
World Humanitarian Summit - Gavi's Fragility and Immunisation Policy
Gavi to review its response to humanitarian emergencies and fragile settings in 2016.
22 April 2016
Gavi tackling environmental challenges head on
Commitment to seek improvements in sustainability.
17 March 2016
Gavi welcomes new record low price for pneumococcal vaccine
GSK commitment is latest step towards improving access to PCV but challenges remain
11 February 2016
Japan commits US$ 18.5 million to support immunisation in Ebola-affected region
Pledge from G7 President will help revitalise immunisation services and strengthen health systems.
05 February 2016
Study shows near elimination of Hib disease in Kilifi region of Kenya after introduction of vaccine
Welcome Trust and Gavi funded research also shows Hib booster not needed for long-term protection.
03 December 2015
Gavi Board thanks Dagfinn Høybråten for inspirational leadership
Gavi Board Chair steps down after five years of unparalleled achievement.
26 November 2015
Organisation of Islamic Cooperation approves Gavi membership to key health committee
Vaccine Alliance support already helping to immunise millions of children in OIC member states.
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December 3, 2013
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These Bird Ocean poems are examples of Bird poems about Ocean. These are the best examples of Bird Ocean poems written by international PoetrySoup poets
If you don't find the poem you want here, try our incredible, super duper, all-knowing, advanced poem search engine.
The atmosphere rings with the bell like calls
of the plover flock, long before they are spotted.
The flight herringbones a grey fedora sky.
Markings of white and coal black weave,
wing-stitched, a blanket maker's dream.
Sigh makers they close on the beach
at high tide, the horizon shivers the
sand blanches. These ravishing scavengers
light on the tattered edge of wet to dry,
dawdling with the dead.
Plovers are diminutive scroungers, one-legged
dancers, hopping to the pull of tide, dining on
crab-eggs in black-tie and feathered tails, their
gray skull caps lined with a black brow.
Sparrow-small birds dress to the nines.
A feast for the birds, fall crisps, crab moltings,
go on for endless miles. September is beginning
and soon winged ones will fly to sunny shores.
The cold Atlantic will moan for the loss of music,
the unstitched sky will part. The avian choir is off
to the mud flats of Carolina.
First Published Eunoia Review January 2015
Copyright © Debbie Guzzi | Year Posted 2015
Say something nice, and a short little while
She will respond with kind words and a smile.
True to her nature, she always will say
Something so nice that I cannot repay.
Bright and unique, every contest that's new
Challenges, helps, and expands us anew.
Don't know that form? Then it's time to begin;
Do a good job and it's sure of a win.
Trying a form that you can't understand?
Andrea's knowledge is always at hand.
Gifted in phrasing and rhyming for sure;
Words she has written in minds will endure.
Poems of flow'rs and a sweet little beach
Dance in the dreams of this sweet little peach!
Sijos of seahorses dance in the sea;
Starfish and dolphins keep good company.
Kyrielles of yellow burst forth like sunflowers:
Rubaiyats of robins in Spring's gentle showers.
Sonnets of sparrows and songbirds fly high
Twined with the cumulus clouds in the sky.
Sunsets of lavender scatter their light;
Sapphics of stars wink their way thro' the night.
Footle sized fireflies twinkle till dawn,
Making a fairyland right on her lawn;
Then in the morning, she wakes and recalls
Visions of dreams still contained in her walls.
Out of her window, or deep in her mind,
She will not rest till the poem she finds.
Thus, she has poems by beauty inspired;
Mixed with her skill, that is why she's admired.
{Just too late for the contest. Bummer!}
Copyright © Isaiah Zerbst | Year Posted 2015
Look! Splashing fat fish.
Circling gull hovers low,
eyeing breakfast dish
For Picture Perfect Haiku #2
Contest by SKAT A
Copyright © Darlene Gifford | Year Posted 2015
Life Reflects
bird sat on a perch
ocean reflects upon clouds
rain fills a great void
Haiku, contest entry
Copyright © Robert Lindley | Year Posted 2014
Lonely sea birds cry out
Young fish washed up on shore
No hunger tonight. Full
Copyright © Pam Deremer | Year Posted 2015
Scrambling across the sky
Oh, just to know
How the sun bird flies
With my brain in tow
Growing in my mind
Fluttering on a wing
Flight from shoreline
Oh, how he sings
Lift on high old one
Perch upon thy crag
Closest to the sun
Waving your feathered flag
How we so enjoy
Your screech of ahoy!
Copyright © ANDREA TRAVIS | Year Posted 2015
Cormorant and I
Share tempestuous moment
Wide eyed ocean watch
Safely removed from spindrift
Waves, spume and spray batter shore.
Copyright © Delice Arleen Skelly | Year Posted 2015
Written March 25, 2013
Any minute now
The sun will rise again
A ship sets sail for the ocean
Another's coming in
I don't quite know
Where I should even begin
The day breathes a sigh
That makes me wonder why
The rain falls only in the den
Where we sit so patiently and wait
The same couch from our first date
Maybe this is just our fate
What house is this I'm in
The paint is wearing thin
The sound of stories told
Has got me growing old
Just you and me my darling
And all the red starlings
Any minute now
The night will come again
We'll set sail for the ocean
Watch others coming in
If I ventured in the slipstream
Would I find you in my dreams
So be still my love
Don't fear this life you're in
Things will change this time around
I'll let the light shine in
I'm just waiting
For this life to begin
Copyright © Brandon Carter | Year Posted 2013
seagull rides the waves
waiting for a chance to strike
a helpless victim
Copyright © Marty King | Year Posted 2014
on powder white sand
watching the birds relaxing
just digging the scene
Copyright © Marty King | Year Posted 2015
Keep on singing, bird
I cannot see you
as I sit enclosed
by the four walls of my room
But I do not need to see you
with my eyes
Your song goes to the centre
of my being
making me ever aware of
the kinship of my spirit
with yours
I do not hear you anymore
Perhaps you have flown away
but the vibrations
that your song has set up
within my soul
will go on
like the never-ending waves
of the ocean
Thank you, bird
Copyright © john beharry | Year Posted 2013
On the bobbing pipers run
Across the sandy beach, such fun
All together off they race
Always with the quickest pace
Piping for their unseen food they go
That's just within their reach below
Flying in a flock unfurled and fast
Swooping down as water passed
Scurrying on tiny legs they speed
Off they race to catch their feed
A moving ornament delight
A Dazzling picture when in flight
Copyright © John Squires | Year Posted 2015
Night Time By The Ocean
Night-time is beautiful
When it's so calm and cool
When full moon's in the sky
And white sea gulls they cry
Everything seems filled with mystery
As full moon it seems to touch the sea
It a lovely scene
All feels so serene
Such a show it mesmerizes me
Something about the night
It gives me much delight
The silence everywhere
The stillness darkness shares
As the water slaps against the sand
How the ocean darkness seems so grand
Lovely place to be
Here beside the sea
As I sit here watching from dry land
24 May 2016
Pedro's Nine-Liner--- Created by Peter Duggan 11/22/15
Stanzas two for style book to look nice on page..
Rhyme is aaccccddc
Meter is 6,6,6,6,9,9,5,5,9
Subject optional
Copyright © peter duggan | Year Posted 2016
Missing that Shinning Sea
Often starring at ocean waves,
just blocks from its sea gulls and sandy shores.
Daily visits never grew old; the water was always cold.
And the gulls fly low, enjoying their favorite spots.
Arrested with fascination of seals resting on solid rocks,
campers and outdoorsmen alike, parked by the sea side
Residing in the sunset district and sleeping by the sound of ocean tides,
A young family was often captured, viewing beauty from bay windows.
They engulfed the cool breezes and observed the rolling fog;
walking along its windy shores, building castles in the sand
Neither rooster crows nor cricket sounds, or even lightning bugs;
sweet sleep danced to the music of fog horns, as they sounded out faithfully;
Ships from Far East ports, passing slowly through the golden gate;
delivering huge containers of merchandize, made mostly in China
Just a short walk from the Pacific is that great and gorgeous park.
Strolling along beaten paths, or laughing and flying kites,
a father and his two kids played with sheer delight;
and embraced the breezes, mostly calm and cool
06082016 cj PS
Copyright © curtis johnson | Year Posted 2016
On wings of whisper seagulls rise
Like an unhurried, gracious fleet
Which sails upon a sea of sighs
And takes my dreams away with it.
The clouds and waves anoint the sky
With drops of myrrh and hues of blue
Whose colors blend and tower high,
While masts glide free and out of view.
How soft and languid is the roll
Of vessels tethered to the pier!
I wonder if they hear the call
Of friends afar and fellows near.
They roll with jealousy and grief
While many others, far away,
Might float over a glowing reef
Or laze upon a peaceful bay.
But here, where seagulls roam above,
The sad and silent fellows weep
For fear their unrequited love
May die forgotten in the deep.
Find my poems and published poetry volumes at www.eton-langford.com
Copyright © Eton Langford | Year Posted 2016
Hi old companion from primitive dark
When we look at the moon in delight
You roll your eyes in curious quirk
Why don't we wait and love dark night
Like you do whether in sun or in rain
For you know beauty sparkles best then
When amidst intense stories of night
You do have your lovely dancing height
Look there the moon is splashing on
The dream waters of the Mediterranean
Now you will perhaps disembark
For your primary pleasure plunder
I am keeping awake to watch the spark
The art of hunting in green grandeur
July 20, 2016
Copyright © Probir Gupta | Year Posted 2016
A wise bird, wiser than the west
It blend beak of voice and a beacon afar
Deftly blending clouds with feathers
Spread over wicked continents
Which is shifting opinions as in tectonic plates
Continent of men, clouds of winged meanings
Drifting oceans, Dithering wings
They meet in swarms and grooves
In a season of stealth and drones
No one wants an yellow messenger
That whispers to the wind, vividly
It could tilt and slope, incline and invade
The valley of vultures and other dreaded signs
No one want an yellow messenger
That could see beyond the tropic clouds
That could foretell the origin of nemesis
That could groove vision in your timid eyes
That bridges light unto your iris canals
No one want an yellow messenger
That carves blissful voices in your yellow belles
Bile is bile, vile is vile, primate is primate and so is the Continental drift
Copyright © Gokul Alex | Year Posted 2015


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From the customer's point of view Groupon is a great thing with almost astonishing surprises and deals daily. But retailers are struggling with practically giving their services away, often to their very own customer base.
Read Whole Story
Get top stories and blog posts emailed to me each day. Newsletters may offer personalized content or advertisements. Learn more.


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Under DDoS Attack?
Need Immediate DDoS Mitigation Support?
A Verisign DDoS expert will contact you shortly
or Call 1‑703‑948‑4299
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The Verisign Labs team brings a tremendous breadth of expertise and talent.
As Principal Research Scientist for advanced computing research, Dow Summers is currently exploring ways for Verisign to take advantage of fast-path packet processing techniques, improve small-packet networking performance of virtualized servers and investigate hardware-based deep-packet inspection. Dow joined Verisign in 1999 and was initially focused on transitioning our DNS services to 64-bit computing. Among other significant contributions, Dow also pioneered our passive real-time monitoring system for DNS and our other high-availability services.
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Posted by Paul
Trust, competence and experience are 3 very important qualities that are required when you need an attorney and Phillip Tatlow provided me with more than enough of all three.
My case was lengthily, as well as complicated and he never once hesitated at spending the time and resources necessary to bring it to a positive conclusion. When I first approached Phil about my case he was more than forthright about my issues and he did not hesitate to develop a plan that allowed us to reach a successful conclusion.
My experience with Mr.Tatlow has been nothing but positive and I would highly recommend him if you need an attorney. I'm confident that you'll be glad that you consulted with him. He is an excellent attorney and more importantly a good man.
Posted by James
When I approached Phil about my job-related disability situation, I had very little hope that I had a case against the insurance company that my employer had hired. Within a couple of hours Phil had assured me that I did have a case and a very good chance of collecting funds due me.
Over a period of years, because the insurance company initially denied my claim, Phil time and time again provided extraordinary professionalism, determination, experience and expertise which led to my being awarded the funds we were initially requesting, plus having his fees covered by the insurance company. Phil has fantastic communication skills which made it easy for me to understand the various steps he was taking and which gave me tremendous confidence that I had chosen the right attorney to represent me.
Posted by Chris
Phil is an exceptionally talented lawyer. I trust him and his firm absolutely to handle all of my legal matters.
Posted by Greg
Mr. Tatlow represented me after an auto accident caused by a careless driver 15 years ago. As my medical bills piled up I knew I needed expert help and thank God for Mr. Tatlow. He did a great job for me and my family. I have since referred several friends to Mr. Tatlow for a variety of legal needs and we all agree that he is outstanding.
Posted by William
Phil represented me in an ERISA case. After denying me three times by my company for the short term disability, the insurence company denied me long term disability benefits because of the denial for short term. For a little over a year I contacted at least 9 attorneys who all said my case was not winable and wouldn't be profitable for them. They didn't even check it out. I found Phil by chance and he said my case was a difficult one and I may not win, but he took on the case. After a couple of years dealing with what I call inept attorneys and insurance executives, the judge found in our favor. We even had to go to Federal Court and after a few questions that Phil handled ever so brilliantly we won again. I call Phil my Clark Kent. He is so mild mannered, but when push comes to shove, the cape comes out and he is almost indestructable. Phil will always have first shot in any legal matter I may have.
Posted by Butch
Mr. Tatlow represented my wife and I in what I considered to be a very complex personal injury case. We found him to be caring, committed and compassionate while exhibiting genuine concern for my physical well-being as a result of my injury. His knowledge of the laws pertaining to our specific case proved to be very extensive which served to instill confidence from the onset. It was obvious that the opposing attorneys respected Mr. Tatlow's integrity and abilities which I feel ultimately helped us gain a favorable settlement in the case. A most memorable moment of the case for me turned out to be the masterful way Mr. Tatlow deposed a less than honest insurance "hired gun" Physician. Justice was served when the Physician was exposed to be unscrupulous through Mr. Tatlow's skillful questioning. I would certainly recommend Mr. Tatlow to anyone in need of a stellar attorney.
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Phil Tatlow has the knowledge, skills, and desire necessary to successfully navigate the legal channels for his clients. I personally have used his legal services several times in the past few years and would not hesitate to recommend him to family and friends.
Posted by Bob
Great attorney that is personal and willing to fight for your rights and stand up to insurance companies and big corporations. He took my case when know one else believed in me and helped me win a good recovery.


text:
my last article was perfect for today's news...why didn't SA publish it? 0 commentsOct 6, 2009 8:44 AM
when i look around at some of the articles being published on SA, it makes me wonder why they aren't publishing mine? some guy wrote an article the other day that the US has only 10 years of natural gas reserves, and SA published it.
but my last article, "the big picture", which talks about the problems of the US dollar as a result of foreign oil addiction doesn't get published. yet this morning, we have news from the British Independent newspaper, that the middle eastern oil producers are working with chinese, japanese, and european countries to begin trading oil in a basket of currencies and gold as opposed to the U.S. dollar. now, the saudis and others denied this report, yet the dollar is trading down (big surprise) and oil is trading up. and so is gold...both mentioned in my last article.
meanwhile, there were two intentionally leaked items over the weekend. one in the NY Times saying that iran was much further ahead in the nuclear bomb works, and one in a London paper saying israeli intelligence determined russian physicists were working with iran on the bomb, and that is why netanyahu visited russia recently. combine these leaks with obama's pause in afghanistan, and it is becoming clearer that the U.S. is considering striking in iran sooner rather than later. i mean, if the US could go to war in iraq for no reason whatsoever, why wouldn't the US go to war in iran if they have, or will soon have, a nuclear weapon? and why not do it while oil inventories are at 20 year highs and the world is still in recession. of course the big issue (other than all the innocent people that will die...), is, can the U.S. prevent iran from pluggin up the straits of hormuz and bottling up oil deliveries?
what a mess!! all because the U.S. is too ignorant to adopt natural gas transportation and is completely reliant on oil to drive its economy and on devaluing the dollar to pay off its tremendous debt. what a ridiculous way to run a country.Back To Michael Fitzsimmons' Instablog HomePage »
Instablogs are blogs which are instantly set up and networked within the Seeking Alpha community. Instablog posts are not selected, edited or screened by Seeking Alpha editors, in contrast to contributors' articles.Full index of posts »
$KMI rally could bode well for Bostco JV with $TLP Feb 17, 2016
Buffet's move into $KMI convinces me he will buy $PSX lock-stock-n-barrel. I predicted this back in 2012: http://seekingalpha.com/a/n0sf Feb 16, 2016
$SU &COSWF - Suncor responds: http://yhoo.it/1m0dRFN Check out my article on this takeover battle: http://seekingalpha.com/a/27qom Dec 3, 2015
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- Our Impact
Report Finds the Value of Business Leadership in Early Learning
Thu, July 28, 2016
It's elementary: Investing in early education benefits everyone.More»
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It's elementary: Investing in early education benefits everyone.More»
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In partnership with Project NEXT, the School District and Beyoncé, volunteers helped raise awareness about the pivotal role music plays in helping children to learn.More»
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For this Alexander there are {FIRSTCOUNT} different names on Yasni.
In the ranking of the most common first names Alexander is on position {NAMERANK}.
Male first name (English, German, Dutch): Alexander, the (foreign) men defensive end, protector of men, Ancient Greek (Greek mythology); alexein fend protect =; recognized = the man, known by Alexander the Great, king of Macedonia ( 356-323 BC). so far contributed eight popes named Alexander


text:
Classic-style rooms with free Wi-Fi are offered at Il Ramingo B&B. All rooms have shared bathrooms. Guests can make... 추가 정보 »
Centro storico is a B&B located in an ancient palace located in the middle of the historical centre inside the... 추가 정보 »
Lying just a few steps from the walled city center and only 700from the railway station, the guest house 'Sogni... 추가 정보 »
B&B Domus Clotilde is a recently renoveted guesthouse in the rustic style. The property offers free city maps and... 추가 정보 »
Villa Sunrise is dated from 1930 and we kept the appearance of the house: Windows, floor, doors. We offer a rich... 추가 정보 »
Gallo Pappa B&B was created with the goal of making guests feel welcome in a very quiet, comfortable space, with... 추가 정보 »
The b&b has been recently eco renovated using local natural materials (terracotta tiles, "Matraia" stones, chestnut... 추가 정보 »
This apartment is delicious, nice and functional. A careful renovation which was completed in October 2012 has... 추가 정보 »
Servizi: Accoglienza. Ogni ospite verrà accolto con la massima cortesia e disponibilità. Sarà offerto un cocktail di... 추가 정보 »
Livio, with many work experiences also abroad, and Jessica, with a Bachelor in Science of Tourism and a year of... 추가 정보 »
Viacarrara16lucca is a charming and characteristic apartment located in a nice building with a gorgeous view of the... 추가 정보 »
Tropical B&B is a nice guest house at 10 minutes walking from the town, near to the bus stops where buses pass... 추가 정보 »
For the Tuscan holiday of your dreams! Nestling among the gentle hills of Tuscany and within easy reach of the dreamy... 추가 정보 »
Canx policy 72hrs. Free car parking available. Taxes are included. We do not serve any breakfast. Baby until 3... 추가 정보 »
Lucca in Villa, an art nouveau villa restored preserving the epoch features, is nearby the wonderful historical... 추가 정보 »
'Alla Dolce vita ' is placed in the very heart of historic center of Lucca, just a few steps away from the roman... 추가 정보 »


text:
Another Baby Cake
I am much happiers with this baby but the bc flowers were melting all over the place! It was too hot in the house! But anyway, much happier with the baby's face especially!
Comments (3)
CarolAnn on
Cakerunner and her daughter died April 07 in an auto accident. They walk with Jesus now. I miss you Becky!!
Login To Leave A Comment


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PSN Accounts
Edit Page Last Edit: 4 years 6 months ago
I have a Question about the PSN Accounts.
My question is can you have One PSN account on two or more ps vita memory cards and not have to do factory reset to switch the memory cards?
Moving this to Ask Your PlayStation Vita Question...
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This item from Carol's Collectibles is now in the Shop Backroom
More from Carol's Collectibles
Dolly Dingle Paper Dolls
This is a very nice paper doll book with thirty 1978 reprints of Grace Drayton's Dolly Dingles. This edition is a selection of dolls that was originally published in the March, April and May , 1913, and January-December, 1917, issues of Pictorial Review. There are so many cute Dolls---a Santa page, many dollies and pets, furniture, costumes---so much in these pages!
It was published in the USA by Dover Publications and copyrighted by the Margaret Woodbury Strong Museum, a toy museum.
The pages are printed on hard stock paper and have beautiful color. Some of the dolls included in this volume are Dolly Dingle of Dingle Dell, In the Land of the Cherry Blossom, and Visits.....to many countries.
This book has not been used and is in great condition. It has 30 dolls, as stated above, and 100 clothes. There are 32 pages.
We are happy to combine shipping when possible. CA residents please expect to pay 8.25% sales tax.
If you were the pending buyer of this item, go to My Account to view, track and check payment for this item.
Carol's Collectibles
Carol Campbell, On Request, CA
Just a little taste of unusual.
We have some good summer values and would consider reasonable offers. . . . . . interesting books and paper dolls.


text:
Liverpool Message Board
*Asked to account for Hodgson's appointment in July, chairman Martin Broughton explained: "He was really focused on, 'I believe, and I've got a track record that demonstrates it, that I can take the existing players, get much better performances out of them and buy constructively to build for a better future.'" On both counts, Hodgson has failed. His inherited players have regressed and his signings, without exception, have flopped. Paul Konchesky's only use is an embodiment of Liverpool's present plight and the mistake of appointing a mid-table manager for one of English football's biggest jobs.
* Notice how much better Liverpool were after the introduction of additional firepower? Tactically inept is a default charge to be issued by disgruntled and accountable armchair pundits but pairing Steven Gerrard with Fernando Torres is pointless unless there is a player in the midfield capable of providing quality service. Criticising Gerrard and Torres has become a popular pastime but they are being wasted in Hodgson's otherwise-guileless team.
* Joe Cole's transfer to the centre lasted almost as long as the predictions that he would be the signing of the summer. The left of midfield may not be his preferred position, but there are no excuses for his inability to cross a ball over the head of Phil Jagielka for the entire ninety minutes. Value for money because he was signed on a free transfer? In wages alone, Cole has earned more than a million pounds since moving to the north-west. On this form, Cole could be the first 'Bosman' since Winston Bogarde to be declared a waste of money.
*In retrospect, the mistake that was made was the implication that Rodriguez occasionally produces better. He is the definition of a nothing player.
* But while the red side of Stanley Park is looking backwards, the blue side can start peering upwards. The impression remains that they remain two or three players short of a Champions League outfit, with the 'two or three' determined by Louis Saha's state of fitness. Otherwise, Everton are a very decent outfit. John W Henry could have been excused for wondering if he had bought the wrong club of Liverpool. With £20m - money that, unfortunately, Bill Kenwright doesn't have for David Moyes' disposal - they would be there or thereabouts with the elite.
* Too good to go down? Along with a vote of confidence, there are no more dreaded words in football. In their current situation, Liverpool are not too good to go down. They are too bad to stay up. Unless something drastic changes at Liverpool, and Hodgson's refusal to change personnel at Goodison Park suggests that drastic change is not in his make-up, the unthinkable really could occur. Liverpool are in a relegation fight.
Just to say these are NOT my views but taken from another article...some of which I think is a fair assesment..Sort: Newest | Oldest | Most Replied Expand all replies
I cant see Roy recovering from this, he just doesnt look like he has it in him to get the players fired up. Blackburn at Anfield then Bolton away have never looked so difficult and I can think of nothing worse then that clown Allardyce gobbing off in the papers all week! He will probably declare his interest in the Liverpool job this week!
We can definitely get out of this mess but the manager needs to change, he just has to. O'Neill and Dalglish look the most likely options but I seriously believe that we could get Mourihno at the end of the season!
- 1 Reply to Hobsey
The only job Mourinho would leave Real Madrid for is either the Man U job, or being the national coach of Portugal, if he DID come to Liverpool he'd probably see it as a "stop-gap" till either of the other roles become vacant..
their team is not better how it works is better- a clueless manager is in charge at LFC...
i think you need to get a life
- 1 Reply to Gary S
End of the day, how many games has Hodgson got to save himself? next 3 games 2 games or more?? Jamie Redknapp was right that its partly to do with Benitez with the present make up of the squad, Alonso,Mascherano, Crouch go, he wasted near 30 million on Aquliani and Dossena, fact of the matter is, if the team had players like, Sneijder, Van De Vaart, and Higuain, the prospects would have been greater, (just wanted to add that in.. I know its irrelevant to what needs to happen NOW...)
I reckon the best man at the moment for the Liverpool job, some may not agree with me here, is MARCELO LIPPI, he's out of a job I think, he's got a proven calibre (one poor tournament doesn't make a manager) he needs to learn english, (but if Ancelotti can do it, so can Lippi!) Mourinho won't leave Madrid for no other job, aside from Man U possibly, Hiddink won't leave Turkey, so I reckon, Lippi could be the man..
Woys biggest fan ! Woys biggest fan ! Oct 18, 2010 09:47 Flag
Mourinho could possibly take us from the top 4 to a title but when has he ever taken a team from the bottom to the top ?
He has always walked into top jobs where they were contenders before he arrived & with the help of God knows how many millions gave them that final push to get them over the line .
In managerial terms hes considered a great salesman (Selling his public image) & a bit of a fraud .
Kenny Dalglish on the other hand has proven himself in that department getting Blackburn promoted , keeping them up & then winning the league .
Just look at the 2 comments from the respective coaches.. One is graceful, honest and even confident he has players to match Liverpool's quality AND that they could go on to play for "big" clubs. Then you have Roy in denial, stating domination and it was the best team performance under his management!! I would have said the win turned draw on opening day vs Arsenal down a man for 45+ minutes was the best performance, downhill ever since... If today was good, I guess 8-0 @ Chelsea will be acceptable and positive.. Roberto Dimatteo, Ian Holloway, and Owen Coyle look like geniuses at the moment..
To be honest, rather than investing in players I'd prefer if Purslow took profits and NESV threw in, and we paid Jose Mourinho 40 million dollars, or pounds just to get us into the top 6..
David Moyes: "The smile's not come off my face. It's been a long time. I'm really disappointed I've not been able to win more derbies. We play against a really good football club but it's not been because of a lack of effort. In the past we've probably lacked the quality to match Liverpool. I don't think we do now. We've got players of real quality who could play for the big clubs. Whoever scored the first goal was going to be really important and I think we always looked more likely in the first 20-25 minutes. I was delighted to get in front because both teams have not been scoring that freely."
Roy Hodgson: "We suffered at the hands of an early onslaught which you invariably do at Goodison but towards the end of the first half we started to even things out. From what I saw I thought we dominated the second half totally. I thought the shape of the team was good, the quality of our passing and movement was good. We didn't score goals and Everton did but I refuse to accept that we were in any way outplayed or any way inferior. I watched the performance and the second half was as good as I saw a Liverpool team play under my management that is for sure."
Woys biggest fan ! Woys biggest fan ! Oct 18, 2010 07:36 Flag
You need to get yer eye on the ball .
Our situation is we are bottom 3 with zero team spirit .
Spurs don't compare or matter .
A Yahoo! User Oct 18, 2010 06:56 Flag
I agree with the first two, especially second as that leads on to the third with which I don't agree with. For Cole to be effective in a 433 system we need a strong midfield and this is where Hodgson shot himself in the foot.
The rest I don't agree with, especially with Everton being better than us. They are not.
I don't think we'll go down, as Hodgson will be fired well before that happens :)
- 1 Reply to A Yahoo! User
Fair point, that I don't think the owners would allow it to continue for long especially with the money they've just put in, at the moment Liverpool would be lucky to get a draw against blackburn next week...its gone beyond a joke..


text:
The reason we chose this motel was because it was close to I-10 and La Mesilla. At check-in the clerk said, "I am giving you one of the new rooms". Not knowing what he ment, we assumed it had been recently renovated. If this was a new room, can't imagine what the old ones were like. Big hole in the carpet right inside the door. Furniture average but bed was a pillow-top and good. Halls were in need of being vacuumed. Especially around the elevator. TV reception terrible. Staff friendly and breakfast average. Price, above average for a comfort inn but not for the area.
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text:
SOURCE: WaPo
tags: museums, Holocaust, anniversaries, United States Holocaust Memorial Museum
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Holocaust museum hits 20th anniversaryBreaking News
tags: museums, Holocaust, anniversaries, United States Holocaust Memorial Museum
Today marks the 20-year anniversary of the opening of the United States Holocaust Memorial Museum. Called "a living memorial to the Holocaust," and hailed as groundbreaking, its unsparing narrative–populated with artifacts, oral and video histories, and conversations with survivors –has attracted nearly 35 million visitors. Two days of formal anniversary observances kick off next week with a National Tribute Dinner April 28 and a National Tribute to Holocaust Survivors and World War II Veterans April 29 featuring former President Bill Clinton, and Founding Chairman Elie Wiesel....
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- Historian James Harris says Russian archives show we've misunderstood Stalin
- The Invisible Labor of Women's Studies
- Lincoln University historian mourns decision to abolish the history major
- Hamilton College conservative historian questions diversity requirement
- Historians on Donald Trump: A Huge Hit on Facebook


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Whitehall in Franklin County, Ohio — The American Midwest (Great Lakes)
Norton Field
Erected 2000 by Ohio Bicentennial Commission, The Longaberger Company, City of Whitehall, Holy Spirit Catholic Church, and The Ohio Historical Society. (Marker Number 31-25.)
Marker series. This marker is included in the Ohio Historical Society / The Ohio History Connection marker series.
Location. 39° 58.503′ N, 82° 52.989′ W. Marker is in Whitehall, Ohio, in Franklin County. Marker is on Broad Street, on the right when traveling east. Click for map. Marker is about 500 feet east of intersection with Yearling Road, and about 200 feet NE of Holy Spirit Catholic Church. Marker is in this post office area: Columbus OH 43213, United States of America.
Other nearby markers. At least 8 other markers are within 3 miles of this marker, measured as the crow flies. Columbus Depot (approx. 0.6 miles away); Original Port Columbus Airport Terminal (approx. one mile away); Forest Lawn Veterans Memorial A Brief History of Eastmoor / Eastmoor Polo Field (approx. 2 miles away); Bexley World War II Memorial (approx. 2.9 miles away); Bexley (approx. 2.9 miles away); Camp Bushnell (approx. 2.9 miles away); Veterans of Foreign Wars Memorial (approx. 2.9 miles away).
Categories. • 20th Century • Air & Space • Heroes • Military • Notable Persons • Patriots & Patriotism • War, World I • War, World II •
Credits. This page originally submitted on , by William Fischer, Jr. of Fort Scott, Kansas. This page has been viewed 1,607 times since then. Photos: 1, 2, 3, 4, 5. submitted on , by William Fischer, Jr. of Fort Scott, Kansas. • Bill Pfingsten was the editor who published this page. This page was last revised on June 16, 2016.