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Previous electrophysiological and anatomical studies of Ascaris suum motor neurons demonstrated a strong correlation between functional interactions and the presence of anatomically defined synapses. However, one example of a physiologically robust synaptic connection was encountered for which no anatomical evidence of direct chemical synapses was found. This involved synaptic transmission from an identified excitatory motor neuron to its inhibitory partner. In this study, pressure injection of horseradish peroxidase or nickel lysine into inhibitory motor neurons revealed numerous spines projecting from the main neuronal process toward the neuromuscular surface that then branched and extended fine, longitudinal processes up to 130 microm in length. Subsequent examination of nickel lysine-injected spines by electron microscopy revealed numerous chemical synapses, including for the first time direct inputs from the excitatory neuron. However, the numbers of synapses from this motor neuron were very small relative to inputs from other identified cells. Thus, direct synapses are unlikely to explain the robust nature of this physiological interaction.

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Current-clamp studies of cultured leech Retzius cells revealed inward rectification in the form of slow voltage sags in response to membrane hyperpolarization. Sag responses were eliminated in Na(+)-free saline and blocked by Cs+, but not Ba2+. Voltage clamp experiments revealed a Cs(+)-sensitive inward current activated by hyperpolarization negative to -70 mV. Cs+ decreased the frequency of spontaneous impulses in Retzius cells of intact ganglia. Plateau potentials were evoked in Retzius cells following block of Ca2+ influx with Ni2+ and suppression of K+ currents with internal tetraethylammonium. Plateau potentials continued to be expressed with Li+ as the charge carrier, but were eliminated when Na+ was replaced with N-methyl-D-glucamine. A persistent Na+ current with similar pharmacology that activated positive to -40 mV and reached its peak amplitude near -5 mV was identified in voltage-clamp experiments. Inactivation of the persistent Na+ current was slow and incomplete. The current was revealed by slow voltage ramps and persisted for the duration of 5-s voltage steps. Persistent Na+ current may underlie Na(+)-dependent bursting recorded in neurons of intact ganglia exposed to Ca2(+)-channel blockers.

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Leech neurons exposed to salines containing inorganic Ca(2+)-channel blockers generate rhythmic bursts of impulses. According to an earlier model, these blockers unmask persistent Na+ currents that generate plateau-like depolarizations, each triggering a burst of impulses. The resulting increase in intracellular Na+ activates an outward Na+/K+ pump current that contributes to burst termination. We tested this model by examining systematically the effects of six transition metal ions (Co2+, Ni2+, Mn2+, Cd2+, La3+, and Zn2+) on the electrical activity of neurons in isolated leech ganglia. Each ion induced bursting activity, but the amplitude, form, and persistence of bursting differed with the ion used and its concentration relative to Ca2+. All ions tested suppressed chemical synaptic transmission between identified motor neurons, consistent with block of voltage-dependent Ca2+ currents in these cells. In addition, a strong correlation between suppression of synaptic transmission and burst amplitudes was obtained. Finally, burst duration was increased and the rate of repolarization decreased in reduced K+ saline, as expected for pump-dependent repolarization. These results provide further support for the hypothesis that a novel form of oscillatory electrical activity driven by persistent Na+ currents and the Na+/K+ pump occurs in leech ganglia exposed to Ca(2+)-channel blockers.

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1. Individual leech Retzius (Rz) cells were removed from mid-body ganglia and plated in cell culture on concanavalin A or polylysine. Experiments on the majority of cells were performed after 6-11 days in culture. Isolated Rz cells were superfused with normal leech saline (NS), cobalt saline (Ca2+ replaced with Co2+), or one of a variety of other modified salines. 2. Prolonged plateau potentials (PPs) with durations ranging from several seconds to nearly 2 min were evoked in isolated Rz cells in response to 1-s depolarizing current pulses delivered under discontinuous current clamp. Some PPs terminated spontaneously while others were terminated with hyperpolarizing current pulses. PPs were associated with a dramatic increase in the input conductance of the neuron. The PP decayed slightly over time, and this decay was accompanied by a small decrease in the input conductance. 3. PP duration was enhanced by penetrating cells with electrodes containing tetraethylammonium (TEA) and by bathing cells in Co2+ saline, but PPs were evoked also in NS and using electrodes without TEA. The effects of TEA and Co2+ saline suggest that voltage-dependent and especially calcium-dependent outward currents normally suppress plateau formation. 4. PPs occurred most reliably in neurons with extensive neurite sprouting. Isolated somata with few or no neurites usually failed to express PP, although there were several exceptions to this trend. 5. PPs persisted when Ca2+ was replaced with either of the calcium channel blockers Co2+, Ni2+, or Mn2+, when 200 microM Cd2+ was added to normal saline, or when Na+ was replaced with Li+. In contrast, PPs were eliminated rapidly when Na+ was replaced with N-methyl-D-glucamine. 6. Isolated Rz cells also expressed repetitive PPs either spontaneously or in response to injection of sustained depolarizing current. Spontaneous repetitive PPs were suppressed by hyperpolarizing current. Repetitive PPs in isolated Rz cells are similar in many respects to the bursting electrical activity induced by Co2+ saline in Rz and other neurons in intact ganglia. 7. The ionic dependence and prolonged duration of PPs suggest that these responses are generated by a persistent voltage-dependent Na+ current. A quantitative computer simulation of PPs was achieved using a depolarization-activated Na+ conductance with very slow inactivation. Repetitive PPs were simulated by addition of a slow outward current in the form of an electrogenic pump.

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A complete understanding of animal behavior at the cellular level requires detailed information on the intrinsic biophysical properties of neurons, muscles, and the synaptic connections they make. In the past 10 to 15 years, electrophysiological studies of leech neurons have revealed a diverse array of voltage-gated ionic conductances distinguished by their pharmacological sensitivity to classic ion channel blockers. Voltage-clamp studies have provided new information about the kinetics and voltage-dependence of Na+ conductances, several K+ currents, including IA, IK and IK(Ca.), and high- and low-voltage-gated Ca2+ conductances. These studies showed that the action potentials of most leech neurons result from the usual sequence of permeability changes to Na+, K+, and Ca2+ ions. They also added insight as to the role played by particular combinations of conductances in providing individual neurons with electrical properties appropriate for the particular information they encode. Evidence is accumulating on the modulatory actions fo endogenous neurotransmitters such as FMRFamide, serotonin, and octopamine on motor behaviors in the animal. Parallel studies suggest that changes in behavior can be explained, at least in part, by the alteration of firing patterns of selected neurons and muscles resulting from modulation of multiple ion conductances. This makes the leech exceptionally attractive for neuroethological studies because it is one of the simplest organisms in which the methods of psychology and neurobiology can be combined. Information gathered from this animal will therefore increase our understanding regarding general principles underlying the cellular basis of behavior.

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1. The heartbeat central pattern-generating network of the medicinal leech contains elemental neural oscillators, comprising reciprocally inhibitory pairs of segmental heart interneurons, that use graded as well as spike-mediated synaptic transmission. We are in the process of developing a general computer model of this pattern generator. Our modeling goal is to explore the interaction of membrane currents and synaptic transmission that promote oscillation in heart interneurons. As a first step toward this goal, we have developed a computer model of graded synaptic transmission between reciprocally inhibitory heart interneurons. Previously gathered voltage-clamp data of presynaptic Ca2+ currents and simultaneous postsynaptic currents and potentials (5 mM external [Ca2+]) were used as the bases of the model. 2. We assumed that presynaptic Ca2+ current was composed of distinct fast (ICaF) and slow (Icas) components because there are two distinct time courses of inactivation for this current. We fitted standard Hodgkin-Huxley equations (Eq. 1 and 2, APPENDIX) to these components using first-order activation and inactivation kinetics. 3. Graded synaptic transfer in the model is based on calculation of a dimensionless variable [P]. A portion of both IcaF and ICaS determined by a factor A contributes to [P], and a removal factor B decreases [P] (Eq. 4, APPENDIX). [P] can be roughly equated to the [Ca2+] in an unspecified volume that is effective in causing transmitter release. Transmitter release, and thus postsynaptic conductance, is related to [P]3 (Eq. 3, APPENDIX). 4. We adapted our model to voltage-clamp data gathered at physiological external [Ca2+] (2.0 mM) and tested it for shorter presynaptic voltage steps. Presynaptic Ca2+ currents and synaptic transfer were well simulated under all conditions. 5. The graded synaptic transfer model could be used in a network simulation to reproduce the oscillatory activity of a reciprocally inhibitory pair of heart interneurons. Because synaptic transmission in the model is an explicit function of presynaptic Ca2+ current, the model should prove useful to explore the interaction between membrane currents and synaptic transmission that promote and modulate oscillation in reciprocally inhibitory heart interneurons.

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In the previous paper we showed that serotonin had several effects on the electrical properties of swim-gating neurons (cells 204) of the leech. These included membrane potential depolarization, induction of a sag voltage response, and enhancement of rebound responses. Here we investigate the ionic basis of these changes by comparing responses of cell 204 to injected current pulses in experimental salines containing modified concentrations of Na+, K+, Ca2+, or Cl-. Our data indicate that serotonin modulates multiple conductances in cell 204. However, most effects of serotonin can be explained by enhancement of two Na(+)-dependent conductances, a Cs(+)-sensitive cation conductance (gh) and a persistent Na+ conductance (gNaS). Both conductances contribute to the resting potential depolarization and increased amplitude of postinhibitory rebound responses induced by serotonin. In addition, enhanced gh underlies a sag potential elicited by hyperpolarizing current pulses in serotonin-treated cells. Hyperpolarizing rebound responses following depolarizing current pulses are composed of Na(+)-dependent and Na(+)-independent components, both of which are enhanced by serotonin. Activation of an electrogenic Na+/K+ pump may underlie the prolonged Na(+)-dependent component. The Na(+)-independent component decays within 1 s and may be produced by a voltage- or Ca(2+)-activated K+ conductance.

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The effects of serotonin on the electrical properties of swim-gating neurons (cell 204) were examined in leech (Hirudo medicinalis) nerve cords. Exposure to serotonin decreased the threshold current required to elicit swim episodes by prolonged depolarization of an individual cell 204 in isolated nerve cords. This effect was correlated with a more rapid depolarization and an increased impulse frequency of cell 204 in the first second of stimulation. In normal leech saline, brief depolarizing current pulses (1 s) injected into cell 204 failed to elicit swim episodes. Following exposure to serotonin, however, identical pulses consistently evoked swim episodes. Thus, serotonin appears to transform cell 204 from a gating to a trigger cell. Serotonin had little effect on the steady-state current-voltage relation of cell 204. However, serotonin altered the membrane potential trajectories in response to injected current pulses and increased the amplitude of rebound responses occurring at the offset of current pulses. These changes suggest that serotonin modulates one or more voltage dependent conductances in cell 204, resulting in a more rapid depolarization and greater firing rate in response to injected currents. Thus, modulation of intrinsic ionic conductances in cell 204 may account in part for the increased probability of swimming behavior induced by serotonin in intact leeches.

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Triple-therapy (low-dose cyclosporine-azathioprine-prednisone) immunosuppression regimen was compared with double-therapy (cyclosporine-prednisone) in 91 consecutive nonrandomized adult cadaveric renal transplant recipients. Both groups were comparable with respect to ethnic diversity, prior transplants, and diabetes. The majority of patients with delayed function (ATN) were maintained on triple therapy, and the use of antilymphocyte agents was more common in the triple-therapy group (52% vs. 7%; P = 0.0001). Triple-therapy patients experienced increased acute rejection episodes (1.4 vs. 0.8 per patient, P = 0.03), required more courses of additional steroid pulse therapy (4.3 vs. 1.6 per patient; P = 0.001), and developed serious infections more frequently (37% vs. 15%; P = 0.05), especially CMV infections (17% vs. 0; P = 0.03), compared with double-therapy patients. However, the increased overall infection rate and CMV infection rate were observed only in those patients who received antilymphocyte agents compared with those who did not (46% vs. 21%; P = 0.02 for all infections, 26% vs. 4%; P = 0.006 for CMV). Additional steroid pulse therapy was associated with increased CMV infections (24% vs. 0; P = 0.03) but not with overall infections. One-year allograft and patient survival were equivalent in both groups. Exclusion of ATN patients from analysis did not alter the findings. This experience confirms the overall efficacy of triple-therapy immunosuppression in renal transplant recipients but suggests that triple therapy may be associated with more acute rejection episodes, greater immunosuppression requirements, and a resultant increase in infections, especially CMV.

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1. Leech ganglia were superfused with salines in which Ca2+ was replaced with equimolar concentrations of Co2+, Ni2+, or Mn2+. These salines elicited rhythmic membrane potential oscillations with cycle periods ranging from 8 to 25 s in all neurons examined within the ventral nerve cord. 2. Rhythmic activity consisted of a rapid depolarization to a prolonged (3-6 s) plateau level, followed by a rapid repolarization. Each depolarization elicited a burst of action potentials. Peak-to-trough amplitudes of the plateau depolarizations were up to 40 mV in some cells. The plateau depolarizations were separated by slowly depolarizing ramp potentials. 3. Oscillations in all neurons were synchronized (in phase) both within individual ganglia and between ganglia linked by connective nerves. Rhythmic activity in isolated ganglia persisted after the interposed connective nerves were cut. 4. The occurrence of oscillatory activity was strongly correlated with the block of chemical synaptic transmission. 5. Electrotonic interactions persisted during oscillatory activity and may be one mechanism by which oscillations are synchronized. 6. The phase of rhythmic impulse bursts monitored with extracellular electrodes could be reset by electrical stimulation of connective nerves but not by injection of current pulses into individual neurons. Phase reset appeared to occur within one cycle and to a fixed phase point (plateau termination). 7. Oscillatory activity was eliminated by 75-100% reductions of [Na+]o (Na+ replaced with N-methyl-D-glucamine). Smaller reductions of Na+ (by 25-50%) increased the cycle period of oscillations. 8. The Na(+)-K+ pump inhibitors ouabain and strophanthidin disrupted oscillations. Cells were depolarized by approximately 20 mV and fired tonically. After the initial washout of the inhibitors, cells repolarized and became quiescent. After several minutes of continued washing, oscillatory activity resumed. 9. A conceptual model is proposed to explain the mechanisms underlying oscillatory activity induced by Ca2+ channel blockers. According to this model, depolarizing plateaus are generated by a noninactivating Na+ conductance. Na+ influx during the plateau leads to an increase in [Na+]i, which activates an electrogenic Na(+)-K+ pump that contributes to plateau termination. 10. A quantitative computer simulation incorporating six types of currents (capacity, outward rectifying potassium, inward rectifying potassium, sodium, leakage, and an electrogenic sodium pump) demonstrates the plausibility of the conceptual model. 11. These data suggest that a novel Na(+)-based mechanism for membrane potential oscillation is revealed by blockade of Ca2+ channels in leech ganglia.

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Synaptic transmission between reciprocally inhibitory heart interneurons (HN cells) of the medicinal leech was examined in the absence of Na-mediated action potentials. Under voltage clamp, depolarizing steps from a holding potential of -60 mV elicited 2 kinetically distinct components of inward current in the presynaptic HN cell: an early transient current that inactivates within 200 msec and a persistent current that only partially decays over several seconds. Both currents begin to activate near -60 mV. Steady-state inactivation occurs over the voltage range between -70 and -45 mV and is completely removed by 1-2-sec hyperpolarizing voltage steps to -80 mV. The inward currents are carried by Ca2+, Ba2+, or Sr2+ ions, but not by Co2+, Mn2+, or Ni2+. These same inward currents underlie the burst-generating plateau potentials previously described in HN cells (Arbas and Calabrese, 1987a,b). With a presynaptic holding potential of -60 mV, the threshold for transmitter release is near -45 mV. Postsynaptic currents in the contralateral HN cell have a reversal potential near -60 mV. The largest postsynaptic currents (300-400 pA) exhibit an initial peak response that is followed by a more slowly decaying component. The persistent component of Ca2+ current in the presynaptic neuron is strongly correlated with the prolonged component of the postsynaptic current, while the transient presynaptic Ca2+ current appears to correspond to the early peak of postsynaptic current. These data are consistent with the hypothesis that voltage-dependent calcium currents contribute to the oscillatory capability of reciprocally inhibitory HN cells by (1) generating the plateau potential that drives the burst of action potentials and (2) underlying the release of inhibitory transmitter onto the contralateral cell.

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Fifty-one consecutive vascular access procedures were randomized to either the Medtronic plasma TFE or Gore-Tex polytetrafluoroethylene (PTFE) conduits in patients requiring immediate dialysis from December 1989 to April 1990. There were 49 forearm loop fistulas and 2 upper arm grafts. Fifty of these fistulas were cannulated within 48 h of placement to avoid use of subclavian venous catheters for hemodialysis. Complications related to the early cannulation of these fistulas included 2 hematomas in the plasma TFE group, and 3 hematomas in the Gore-Tex group (p = 1.00). Two patients with Gore-Tex grafts were systemically heparinized prior to hematoma formation after thrombectomy of their accesses. There were no adverse sequelae in these 5 patients, and none of the hematomas interfered with further dialytic therapy. One patient in the plasma TFE group and 3 patients in the Gore-Tex group developed cellulitis within the first month of placement (p = 0.65). All were treated with intravenous vancomycin with resolution of the erythema. None of the plasma TFE and 3 of the Gore-Tex fistulas thrombosed within 30 days of placement (p = 0.22). All were salvaged by thrombectomy. Both the plasma TFE and Gore-Tex vascular conduits may be used after surgical placement for early dialytic therapy and are associated with minimal early complications. The early use of these fistulas may eliminate the need for subclavian venous cannulation in most patients with renal failure, thus diminishing the incidence of subclavian venous stenosis and thrombosis. Further observation of these grafts will be necessary to determine the effect of immediate cannulation on their long-term performance for hemodialysis.

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The ability of ventral inhibitory motor neurons of the nematode Ascaris to generate slow depolarizing potentials was investigated using intracellular recording and current injection. In quiescent cells, regenerative depolarizations with peak amplitudes of approximately 20 mV and durations of several 100 ms were evoked in response to brief depolarizing current pulses. Repetitive slow potentials were produced in response to sustained depolarizing currents in a threshold-dependent manner. Repetitive slow potentials also occurred spontaneously, exhibiting cycle periods of about 700 ms. The ability of inhibitory motor neurons to generate slow potentials was blocked by addition of Co++, Cd++, or other Ca-channel blockers to the saline but not by TTX or substitution of Na+ with Tris. The amplitude and duration of slow potentials were increased in the presence of Ba++, Sr++, and TEA. Spontaneous slow potentials exhibited characteristics expected of intrinsically generated oscillations, including frequency modulation by injection of prolonged offset currents, phase resetting by brief current pulses, and suppression by strong hyperpolarization. Slow potentials appear to be generated in the ventral nerve cord processes and/or cell body of the motor neuron, and they produce rhythmic inhibitory postsynaptic potentials in ventral muscle cells. Slow potentials may therefore contribute to locomotory or other motor behaviors of the animal.

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Heart interneurons (HN cells) in isolated ganglia of the medicinal leech were voltage-clamped with single microelectrodes. Hyperpolarizing voltage steps elicited a slow inward current (Ih), which underlies the characteristic depolarizing response of HN cells to injection of prolonged hyperpolarizing current pulses (Arbas and Calabrese, 1987a). The conductance underlying Ih begins to activate near -mV and is fully activated between -70 and -80 mV. The activation kinetics of Ih are slow and voltage dependent. The activation time constant (tau h) ranges from approximately 2 sec at -60 mV to near 700 msec at -100 mV. Ih persists in low Ca2+ (0.1 mM), 5 mM Mn2+ saline and exhibits a reversal potential of -21 +/- 5 mV. The reversal potential is shifted by altering [Na+]o or [K+]o but is unaffected by changes in [Cl-]o. Ih is blocked by extracellular Cs+ (1-5 mM) but not Ba2+ (5 mM) or TEA (25 mM). Low concentrations of Cs+ (100-200 microM) cause a partial block that exhibits strong voltage dependence. Temperature changes were also shown to affect Ih. Both the rate of activation and the steady-state amplitude of Ih are enhanced by temperature increases. HN cells are interconnected by inhibitory chemical synapses, and their normal electrical activity consists of bursts of action potentials separated by periods of inhibition. During the inhibitory phase of rhythmic bursting activity, HN cells hyperpolarize to a voltage range where Ih is activated. Block of Ih with extracellular Cs+ (4 mM) disrupted the normal bursting activity of HN cells. These results are consistent with the hypothesis that Ih contributes to escape from inhibitory inputs during normal bursting activity.

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The nematode nervous system is distinguished by the small number and morphological simplicity of its neurons. Recently, the shapes and synaptic interactions of each of the 302 neurons in the small free-living nematode, Caenorhabditis elegans, have been determined from reconstructions of serial sections by electron microscopy. Comparable anatomical studies of the large parasitic nematode Ascaris have concentrated on the dorsal and ventral nerve cords where reconstructions of motor neurons by light microscopy led to the identification of seven distinct types of motor neurons, each corresponding to a homologous cell type in C. elegans. In this study the shapes of the 13 neurons with cell bodies in the retrovesicular ganglion (RVG) of Ascaris suum were reconstructed from light micrographs of serial sections. In other preparations the morphology of RVG neurons was observed in whole mounts after the cells were impaled with microelectrodes and injected with the fluorescent dye Lucifer yellow. The intracellular electrodes also permitted electrical recordings and revealed that one type of cell, the AVF-like interneuron, expresses spontaneous repetitive plateau potentials. Comparisons of neuronal morphologies in the retrovesicular ganglia of Ascaris and C. elegans suggest that each neuron in Ascaris can be assigned a corresponding homolog in C. elegans. These data provide further evidence for a remarkable conservation of neuronal morphology in nematodes despite large differences in size and habitat.

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We observed ectopic soft tissue calcification affecting seven patients following orthotopic liver transplantation. The cause of such calcification is unknown, but potential pathogenetic factors include hyperparathyroidism, calcium administered during and following surgery, renal failure, acid-base changes and citrate in fresh frozen plasma. To investigate some of the mechanisms underlying ectopic calcification following liver transplantation, we determined preoperative levels of ionized serum calcium, phosphate, magnesium, parathyroid hormone (midmolecule assay) and 1,25-(OH)2 vitamin D in 20 patients who underwent 24 liver transplants. In addition, these parameters were measured weekly in 15 patients during the first month after liver transplantation. Preoperatively, 5 of the 20 patients had elevated serum levels of parathyroid hormone, and 9 others had low levels of 1,25-(OH)2 vitamin D. After liver transplantation, ectopic calcification was found in seven patients (47%). The organs affected in order of frequency were lungs, liver graft, colon, vascular walls, kidneys, adrenal glands and gastric mucosa. One patient with ectopic calcification of both lungs had markedly restricted pulmonary function as well as radiologic evidence of osteopenia and pathologic fractures of three vertebrae. Postoperatively, increased parathyroid hormone levels were found in all patients who developed soft tissue calcification. Parathyroid hormone levels peaked during the second week after transplantation and were higher at all times compared to subjects without calcification. Five of the seven patients with ectopic calcification had associated renal failure. Individuals who developed calcification had received significantly more fresh frozen plasma, red blood cells and elemental calcium postoperatively, but showed no difference in serum levels of calcium, magnesium, vitamin D, total plasma CO2 or phosphate levels when compared to patients without calcification.(ABSTRACT TRUNCATED AT 250 WORDS)

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