Facilitated sequence assembly using densely labeled optical DNA barcodes:A combinatorial auction approach

<div><p>The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard “foreign” contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.</p></div

Capsaicin- resistant arterial baroreceptors

BACKGROUND: Aortic baroreceptors (BRs) comprise a class of cranial afferents arising from major arteries closest to the heart whose axons form the aortic depressor nerve. BRs are mechanoreceptors that are largely devoted to cardiovascular autonomic reflexes. Such cranial afferents have either lightly myelinated (A-type) or non-myelinated (C-type) axons and share remarkable cellular similarities to spinal primary afferent neurons. Our goal was to test whether vanilloid receptor (TRPV1) agonists, capsaicin (CAP) and resiniferatoxin (RTX), altered the pressure-discharge properties of peripheral aortic BRs. RESULTS: Periaxonal application of 1 μM CAP decreased the amplitude of the C-wave in the compound action potential conducting at <1 m/sec along the aortic depressor nerve. 10 μM CAP eliminated the C-wave while leaving intact the A-wave conducting in the A-δ range (<12 m/sec). These whole nerve results suggest that TRPV1 receptors are expressed along the axons of C- but not A-conducting BR axons. In an aortic arch – aortic nerve preparation, intralumenal perfusion with 1 μM CAP had no effect on the pressure-discharge relations of regularly discharging, single fiber BRs (A-type) – including the pressure threshold, sensitivity, frequency at threshold, or maximum discharge frequency (n = 8, p > 0.50) but completely inhibited discharge of an irregularly discharging BR (C-type). CAP at high concentrations (10–100 μM) depressed BR sensitivity in regularly discharging BRs, an effect attributed to non-specific actions. RTX (≤ 10 μM) did not affect the discharge properties of regularly discharging BRs (n = 7, p > 0.18). A CAP-sensitive BR had significantly lower discharge regularity expressed as the coefficient of variation than the CAP-resistant fibers (p < 0.002). CONCLUSION: We conclude that functional TRPV1 channels are present in C-type but not A-type (A-δ) myelinated aortic arch BRs. CAP has nonspecific inhibitory actions that are unlikely to be related to TRV1 binding since such effects were absent with the highly specific TRPV1 agonist RTX. Thus, CAP must be used with caution at very high concentrations

Postnatal sulfur dioxide exposure reversibly alters parasympathetic regulation of heart rate

Perinatal sulfur dioxide exposure disrupts parasympathetic regulation of cardiovascular activity. Here, we examine the relative risks of prenatal versus postnatal exposure to the air pollutant and the reversibility of the cardiovascular effects. Two groups of animals were used for this study. For prenatal exposure, pregnant Sprague-Dawley dams were exposed to 5 parts per million sulfur dioxide for 1 hour daily throughout gestation and with their pups after birth to medical-grade air through 6 days postnatal. For postnatal exposure, dams were exposed to air, and after delivery along with their pups to 5 parts per million sulfur dioxide through postnatal day 6. ECGs were recorded from pups on postnatal day 5 to examine changes in heart rate. Whole-cell patch-clamp electrophysiology was used to examine changes in neurotransmission to cardiac vagal neurons in the nucleus ambiguus on sulfur dioxide exposure. Postnatal sulfur dioxide exposure diminished glutamatergic neurotransmission to cardiac vagal neurons by 40.9% and increased heart rate, whereas prenatal exposure altered neither of these properties. When postnatal exposure concluded on postnatal day 5, excitatory neurotransmission remained decreased through day 6 and returned to basal levels by day 7. ECGs showed that heart rate remained elevated through day 6 and recovered by day 7. On activation of the parasympathetic diving reflex, the response was significantly blunted by postnatal sulfur dioxide exposure through day 7 but recovered by day 8. Postnatal, but not prenatal, exposure to sulfur dioxide can disrupt parasympathetic regulation of cardiovascular activity. Neonates can recover from these effects within 2 to 3 days of discontinued exposure. © 2013 American Heart Association, Inc

Postnatal sulfur dioxide exposure reversibly alters parasympathetic regulation of heart rate

Perinatal sulfur dioxide exposure disrupts parasympathetic regulation of cardiovascular activity. Here, we examine the relative risks of prenatal versus postnatal exposure to the air pollutant and the reversibility of the cardiovascular effects. Two groups of animals were used for this study. For prenatal exposure, pregnant Sprague-Dawley dams were exposed to 5 parts per million sulfur dioxide for 1 hour daily throughout gestation and with their pups after birth to medical-grade air through 6 days postnatal. For postnatal exposure, dams were exposed to air, and after delivery along with their pups to 5 parts per million sulfur dioxide through postnatal day 6. ECGs were recorded from pups on postnatal day 5 to examine changes in heart rate. Whole-cell patch-clamp electrophysiology was used to examine changes in neurotransmission to cardiac vagal neurons in the nucleus ambiguus on sulfur dioxide exposure. Postnatal sulfur dioxide exposure diminished glutamatergic neurotransmission to cardiac vagal neurons by 40.9% and increased heart rate, whereas prenatal exposure altered neither of these properties. When postnatal exposure concluded on postnatal day 5, excitatory neurotransmission remained decreased through day 6 and returned to basal levels by day 7. ECGs showed that heart rate remained elevated through day 6 and recovered by day 7. On activation of the parasympathetic diving reflex, the response was significantly blunted by postnatal sulfur dioxide exposure through day 7 but recovered by day 8. Postnatal, but not prenatal, exposure to sulfur dioxide can disrupt parasympathetic regulation of cardiovascular activity. Neonates can recover from these effects within 2 to 3 days of discontinued exposure. © 2013 American Heart Association, Inc

Identification and dissociation of cardiovascular neurons from the medulla for patch clamp analysis

This study describes a preparation that will enable us to study, using voltage clamp techniques, ionic currents from dissociated cardiovascular neurons that have retained their anatomical and functional identity of the intact animal. To identify dispersed preganglionic cardiac motorneurons various fluorescent dyes (rhodamine, fluorogold, microspheres, bizbenzimide and dextrans) were examined to determine which can be absorbed by preganglionic cardiac motorneuron nerve terminals (without surgical penetration of cardiac tissue), transported retrogradely to their soma in the medulla and retained during dissociation of the neurons. Rhodamine fulfilled these criteria. Dissociated preganglionic cardiac motorneurons had resting membrane potentials of -52.4 ± 3 mV and input resistances of 236 ± 71 MΩ (mean ± S.E.M., n = 10). Depolarizing voltage steps to -50 mV or above evoked a tetrodotoxin (TTX) sensitive inward sodium current followed by a biphasic outward current. © 1991

Perinatal sulfur dioxide exposure alters brainstem parasympathetic control of heart rate

AimsSulfur dioxide (SO2) is an air pollutant that impedes neonatal development and induces adverse cardiorespiratory health effects, including tachycardia. Here, an animal model was developed that enabled characterization of (i) in vivo alterations in heart rate and (ii) altered activity in brainstem neurons that control heart rate after perinatal SO 2 exposure.Methods and resultsPregnant Sprague-Dawley dams and their pups were exposed to 5 parts per million SO2 for 1 h daily throughout gestation and 6 days postnatal. Electrocardiograms were recorded from pups at 5 days postnatal to examine changes in basal and diving reflex-evoked changes in heart rate following perinatal SO2 exposure. In vitro studies employed whole-cell patch-clamp electrophysiology to examine changes in neurotransmission to cardiac vagal neurons within the nucleus ambiguus upon SO2 exposure using a preparation that maintains fictive inspiratory activity recorded from the hypoglossal rootlet. Perinatal SO2 exposure increased heart rate and blunted the parasympathetic-mediated diving reflex-evoked changes in heart rate. Neither spontaneous nor inspiratory-related inhibitory GABAergic or glycinergic neurotransmission to cardiac vagal neurons was altered by SO2 exposure. However, excitatory glutamatergic neurotransmission was decreased by 51.2% upon SO2 exposure. This diminished excitatory neurotransmission was tetrodotoxin-sensitive, indicating SO2 exposure impaired the activity of preceding glutamatergic neurons that synapse upon cardiac vagal neurons.ConclusionsDiminished glutamatergic, but unaltered inhibitory neurotransmission to cardiac vagal neurons provides a mechanism for the observed SO2-induced elevated heart rate via an impairment of brainstem cardioinhibitory parasympathetic activity to the heart. © 2013 Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2013

Perinatal sulfur dioxide exposure alters brainstem parasympathetic control of heart rate

AimsSulfur dioxide (SO2) is an air pollutant that impedes neonatal development and induces adverse cardiorespiratory health effects, including tachycardia. Here, an animal model was developed that enabled characterization of (i) in vivo alterations in heart rate and (ii) altered activity in brainstem neurons that control heart rate after perinatal SO 2 exposure.Methods and resultsPregnant Sprague-Dawley dams and their pups were exposed to 5 parts per million SO2 for 1 h daily throughout gestation and 6 days postnatal. Electrocardiograms were recorded from pups at 5 days postnatal to examine changes in basal and diving reflex-evoked changes in heart rate following perinatal SO2 exposure. In vitro studies employed whole-cell patch-clamp electrophysiology to examine changes in neurotransmission to cardiac vagal neurons within the nucleus ambiguus upon SO2 exposure using a preparation that maintains fictive inspiratory activity recorded from the hypoglossal rootlet. Perinatal SO2 exposure increased heart rate and blunted the parasympathetic-mediated diving reflex-evoked changes in heart rate. Neither spontaneous nor inspiratory-related inhibitory GABAergic or glycinergic neurotransmission to cardiac vagal neurons was altered by SO2 exposure. However, excitatory glutamatergic neurotransmission was decreased by 51.2% upon SO2 exposure. This diminished excitatory neurotransmission was tetrodotoxin-sensitive, indicating SO2 exposure impaired the activity of preceding glutamatergic neurons that synapse upon cardiac vagal neurons.ConclusionsDiminished glutamatergic, but unaltered inhibitory neurotransmission to cardiac vagal neurons provides a mechanism for the observed SO2-induced elevated heart rate via an impairment of brainstem cardioinhibitory parasympathetic activity to the heart. © 2013 Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2013

Characterization of calcium currents in aortic baroreceptor neurons

1. Calcium currents in identified rat aortic baroreceptors were characterized with the perforated patch whole-cell voltage-clamp technique. Aortic baroreceptors were distinguished from other neurons by the presence of a fluorescent tracer that was previously applied to the aortic depressor nerve. The diversity of calcium currents in unidentified neurons dissociated from neonatal rat nodose ganglia were also examined. 2. A population of aortic baroreceptors (63%, 7 of 11) possessed a low-threshold, also referred to as a T-type, calcium current. This current was typically \u3c100 pA in 2 mM Ca [72.7 ± 20.9 (SE) pA, n = 7], had a rapid activation and inactivation, and inactivated completely at conditioning voltages positive to -50 mV. 3. All aortic baroreceptors possessed high-threshold calcium currents that were activated at voltages positive to -30 mV, with typical maximum amplitudes of 600-1,000 pA (826 ± 79 pA, n = 11). 4. The high-threshold current inactivated with three exponential rates of decay of τ = 10.7 ± 2.2 ms, 138 ± 14.6 ms, and a third τ \u3e 3 s. It was not possible to separate the kinetic components of inactivation with conditioning voltages (voltage-dependent inactivation), activation thresholds, deactivation kinetics, or calcium- channel antagonists. 5. The voltage-dependent inactivation of high-threshold calcium currents began at voltages positive to -70 mV and became steeply voltage dependent between -60 and -10 mV. Unexpectedly, the three decay constants were present after all conditioning voltages. There were no conditioning voltages that excluded any component. 6. ω-Conotoxin (GVIA) blocked ~70% of the high-threshold current and inhibited all kinetic components of inactivation equally. ω-Conotoxin (1 μM) inhibited the magnitude of calcium current activated after each conditioning voltage without any change in the voltage dependence of inactivation. The dihydropyridine antagonist nimodipine (2 μM) had little or no effect. 7. Analysis of tail currents indicated that high-threshold calcium currents activate at voltages positive to -30 mV. ω-Conotoxin inhibited the tail currents but did not alter the threshold or range of voltages over which activation occurred. 8. High-threshold tail currents deactivated with a single exponential rate after different test voltages and varying duration. 9. In conclusion, the calcium currents in aortic baroreceptors have been characterized. A population of aortic baroreceptors possess a low-threshold (T-type) calcium current. All aortic baroreceptors possess high-threshold calcium currents that inactivate with three rates of decay. The entire high- threshold calcium current profile activated at voltages positive to -30 mV, inactivated after conditioning voltages positive to -70 mV, and deactivated with a single rate of decay, was insensitive to nimodipine, and was blocked (70%) by ω-conotoxin