Rate Of Origin Firing Research Articles

The TRESLIN-MTBP complex couples completion of DNA replication with S/G2 transition

SummaryIt has been proposed that ATR kinase senses the completion of DNA replication to initiate the S/G2 transition. In contrast to this model, we show here that the TRESLIN-MTBP complex prevents a premature entry into G2 from early S-phase independently of ATR/CHK1 kinases. TRESLIN-MTBP acts transiently at pre-replication complexes (preRCs) to initiate origin firing and is released after the subsequent recruitment of CDC45. This dynamic behavior of TRESLIN-MTBP implements a monitoring system that checks the activation of replication forks and senses the rate of origin firing to prevent the entry into G2. This system detects the decline in the number of origins of replication that naturally occurs in very late S, which is the signature that cells use to determine the completion of DNA replication and permit the S/G2 transition. Our work introduces TRESLIN-MTBP as a key player in cell-cycle control independent of canonical checkpoints.

Classification of naturally evoked compound action potentials in peripheral nerve spatiotemporal recordings

Peripheral neural signals have the potential to provide the necessary motor, sensory or autonomic information for robust control in many neuroprosthetic and neuromodulation applications. However, developing methods to recover information encoded in these signals is a significant challenge. We introduce the idea of using spatiotemporal signatures extracted from multi-contact nerve cuff electrode recordings to classify naturally evoked compound action potentials (CAP). 9 Long-Evan rats were implanted with a 56-channel nerve cuff on the sciatic nerve. Afferent activity was selectively evoked in the different fascicles of the sciatic nerve (tibial, peroneal, sural) using mechano-sensory stimuli. Spatiotemporal signatures of recorded CAPs were used to train three different classifiers. Performance was measured based on the classification accuracy, F1-score, and the ability to reconstruct original firing rates of neural pathways. The mean classification accuracies, for a 3-class problem, for the best performing classifier was 0.686 ± 0.126 and corresponding mean F1-score was 0.605 ± 0.212. The mean Pearson correlation coefficients between the original firing rates and estimated firing rates found for the best classifier was 0.728 ± 0.276. The proposed method demonstrates the possibility of classifying individual naturally evoked CAPs in peripheral neural signals recorded from extraneural electrodes, allowing for more precise control signals in neuroprosthetic applications.

Nitric Oxide Orchestrates a Power-Law Modulation of Sympathetic Firing Behaviors in Neonatal Rat Spinal Cords.

Nitric oxide (NO) is a diffusible gas and has multifarious effects on both pre- and postsynaptic events. As a consequence of complex excitatory and inhibitory integrations, NO effects on neuronal activities are heterogeneous. Using in vitro preparations of neonatal rats that retain the splanchnic sympathetic nerves and the thoracic spinal cord as an experimental model, we report here that either enhancement or attenuation of NO production in the neonatal rat spinal cords could increase, decrease, or not change the spontaneous firing behaviors recorded from splanchnic sympathetic single fibers. To elucidate the mathematical features of NO-mediated heterogeneous responses, the ratios of changes in firing were plotted against their original firing rates. In log-log plots, a linear data distribution demonstrated that NO-mediated heterogeneity in sympathetic firing responses was well described by a power function. Selective antagonists were applied to test if glycinergic, GABAergic, glutamatergic, and cholinergic neurotransmission in the spinal cord are involved in NO-mediated power-law firing modulations (plFM). NO-mediated plFM diminished in the presence of mecamylamine (an open-channel blocker of nicotinic cholinergic receptors), indicating that endogenous nicotinic receptor activities were essential for plFM. Applications of strychnine (a glycine receptor blocker), gabazine (a GABAA receptor blocker), or kynurenate (a broad-spectrum ionotropic glutamate receptor blocker) also caused plFM. However, strychnine- or kynurenate-induced plFM was diminished by L-NAME (an NO synthase inhibitor) pretreatments, indicating that the involvements of glycine or ionotropic glutamate receptor activities in plFM were secondary to NO signaling. To recapitulate the arithmetic natures of the plFM, the plFM were simulated by firing changes in two components: a step increment and a fractional reduction of their basal firing activities. Ionotropic glutamate receptor activities were found to participate in plFM by both components. In contrast, GABAA receptor activities are involved in the component of fractional reduction only. These findings suggest that NO orchestrates a repertoire of excitatory and inhibitory neurotransmissions, incurs a shunting effect on postsynaptic membrane properties, and thus, alters sympathetic firing in a manner of plFM. We propose that the plFM mediated by NO forms a basic scheme of differential controls for heterogeneous sympathetic regulation of visceral functions.

Analysis of Fission Yeast Single DNA Molecules on the Megabase Scale Using DNA Combing.

DNA combing enables the quantitative analysis of DNA replication, DNA recombination, DNA-protein interaction, and DNA methylation along genomic single DNA molecules at 1kb resolution. However, DNA combing has been restricted to short 200-500kb long DNA fragments, which introduces significant bias in data analysis. An improved DNA combing methodology that allows to routinely image Mb-scale single DNA molecules and occasionally up to full-length fission yeast chromosomes is presented in this chapter. DNA combing of Mb-scale single DNA molecules can be applied to accurately measure the dynamic properties of DNA replication such as the rate of origin firing, replication fork velocity, fork directionality and the frequency of fork blockage. In addition, Mb-scale single DNA molecules enable the quantitative analysis of complex genomic rearrangements including gross chromosomal translocations, repetitive DNA sequences, large deletions, and duplications, which are difficult to investigate with deep sequencing strategies.

Use of spatiotemporal templates for pathway discrimination in peripheral nerve recordings: a simulation study

Objective. Extraction of information from the peripheral nervous system can provide control signals in neuroprosthetic applications. However, the ability to selectively record from different pathways within peripheral nerves is limited. We investigated the integration of spatial and temporal information for pathway discrimination in peripheral nerves using measurements from a multi-contact nerve cuff electrode. Approach. Spatiotemporal templates were established for different neural pathways of interest, and used to obtain tailored matched filters for each of these pathways. Simulated measurements of compound action potentials propagating through the nerve in different test cases were used to evaluate classification accuracy, percentage of missed spikes, and ability to reconstruct the original firing rates of the neural pathways. Main results. The mean Pearson correlation coefficients between the original firing rates and estimated firing rates over all tests cases was found to be 0.832 ± 0.161, 0.421 ± 0.145, 0.481 ± 0.340 for our algorithm, Bayesian spatial filters, and velocity selective recordings respectively. Significance. The proposed method shows that the spatiotemporal templates were able to provide more robust spike detection and reliable pathway discrimination than these existing algorithms.

The spatial and temporal organization of origin firing during the S-phase of fission yeast.

Eukaryotes duplicate their genomes using multiple replication origins, but the organization of origin firing along chromosomes and during S-phase is not well understood. Using fission yeast, we report the first genome-wide analysis of the spatial and temporal organization of replication origin firing, analyzed using single DNA molecules that can approach the full length of chromosomes. At S-phase onset, origins fire randomly and sparsely throughout the chromosomes. Later in S-phase, clusters of fired origins appear embedded in the sparser regions, which form the basis of nuclear replication foci. The formation of clusters requires proper histone methylation and acetylation, and their locations are not inherited between cell cycles. The rate of origin firing increases gradually, peaking just before mid S-phase. Toward the end of S-phase, nearly all the available origins within the unreplicated regions are fired, contributing to the timely completion of genome replication. We propose that the majority of origins do not fire as a part of a deterministic program. Instead, origin firing, both individually and as clusters, should be viewed as being mostly stochastic.

Regulation of DNA Replication within the Immunoglobulin Heavy-Chain Locus During B Cell Commitment

The temporal order of replication of mammalian chromosomes appears to be linked to their functional organization, but the process that establishes and modifies this order during cell differentiation remains largely unknown. Here, we studied how the replication of the Igh locus initiates, progresses, and terminates in bone marrow pro-B cells undergoing B cell commitment. We show that many aspects of DNA replication can be quantitatively explained by a mechanism involving the stochastic firing of origins (across the S phase and the Igh locus) and extensive variations in their firing rate (along the locus). The firing rate of origins shows a high degree of coordination across Igh domains that span tens to hundreds of kilobases, a phenomenon not observed in simple eukaryotes. Differences in domain sizes and firing rates determine the temporal order of replication. During B cell commitment, the expression of the B-cell-specific factor Pax5 sharply alters the temporal order of replication by modifying the rate of origin firing within various Igh domains (particularly those containing Pax5 binding sites). We propose that, within the Igh CH-3′RR domain, Pax5 is responsible for both establishing and maintaining high rates of origin firing, mostly by controlling events downstream of the assembly of pre-replication complexes.

Do replication forks control late origin firing in Saccharomyces cerevisiae?

Recent studies of eukaryotic DNA replication timing profiles suggest that the time-dependent rate of origin firing, I(t), has a universal shape, which ensures a reproducible replication completion time. However, measurements of I(t) are based on population averages, which may bias the shape of the I(t) because of imperfect cell synchrony and cell-to-cell variability. Here, we measure the population-averaged I(t) profile from synchronized Saccharomyces cerevisiae cells using DNA combing and we extract the single-cell I(t) profile using numerical deconvolution. The single cell I(t) and the population-averaged I(t) extracted from DNA combing and replication timing profiles are similar, indicating a genome scale invariance of the replication process, and excluding cell-to-cell variability in replication time as an explanation for the shape of I(t). The single cell I(t) correlates with fork density in wild-type cells, which is specifically loosened in late S phase in the clb5Δ mutant. A previously proposed numerical model that reproduces the wild-type I(t) profile, could also describe the clb5Δ mutant I(t) once modified to incorporate the decline in CDK activity and the looser dependency of initiation on fork density in the absence of Clb5p. Overall, these results suggest that the replication forks emanating from early fired origins facilitate origin firing in later-replicating regions.

Ganglionic transmission in a vasomotor pathway studied in vivo

Intracellular recordings were made in vivo from 40 spontaneously active cells in the third lumbar sympathetic ganglion of urethane-anaesthetized rats. In 38/40 cells ongoing action potentials showed strong cardiac rhythmicity (93.4 +/- 1.9% modulation) indicating high barosensitivity and probable muscle vasoconstrictor (MVC) function. Subthreshold excitatory postsynaptic potentials (EPSPs) showed the same pattern. The 38 barosensitive neurons fired action potentials at 2.9 +/- 0.3 Hz. All action potentials were triggered by EPSPs, most of which were unitary events. Calculations indicated that <5% of action potentials were triggered by summation of otherwise subthreshold EPSPs. 'Dominant' synaptic inputs with a high safety factor were identified, confirming previous work. These were active in 24/38 cells and accounted for 32% of all action potentials; other ('secondary') inputs drove the remainder. Inputs (21 dominant, 19 secondary) attributed to single preganglionic neurons fired at 1.38 +/- 0.16 Hz. An average of two to three preganglionic neurons were estimated to drive each ganglion cell's action potentials. When cells were held hyperpolarized to block spiking, a range of spontaneous EPSP amplitudes was revealed. Threshold equivalent was defined as the membrane potential value that was exceeded by spontaneous EPSPs at the same frequency as the cell's original firing rate. In 10/12 cells examined, a continuum of EPSP amplitudes overlapped threshold equivalent. Small changes in cell excitability could therefore raise or lower the percentage of preganglionic inputs triggering action potentials. The results indicate that vasoconstrictor ganglion cells in vivo mostly behave not as 1:1 relays, but as continuously variable gates.

Instability of motor unit firing rates during prolonged isometric contractions in human masseter

The firing patterns of up to 4 concurrently active masseter motor units were studied with intramuscular electrodes during a continuous isometric contraction of 15 min duration, in which the subject maintained the mean firing rate of one selected unit at 10 Hz. With this paradigm the net excitation (i.e. mean firing rate) of one unit in the muscle was controlled. This served as the reference for the functional state of other active units during the prolonged contraction. With the mean firing rate of one unit in the muscle fixed, 58% of other active units showed a slow, statistically-significant change in mean firing rate over the 15 min. The initial firing rate of the units did not influence the change in rate. The original firing rate hierarchy, which in short-term contractions reflects the recruitment order, was altered during the prolonged contraction. The explanation for these differential changes in motoneuron net excitation is not clear; they could be intrinsic to the motoneurons or perhaps mediated by reflex pathways. The selective facilitation or suppression of some motor units with continuous activation means that the original size-structured combination of motor units can be modified during a prolonged contraction.

Are Noradrenaline Excitations Artefacts ?

IT has frequently been reported that noradrenaline applied by microiontophoresis depresses firing in neurones of the central nervous system1–11. Excitatory responses have also been found, however, both in cells which are, and in cells which are not, depressed by noradrenaline12–19. In the former case, depression is followed by a delayed excitation. In all cases excitatory responses are delayed and often reach a maximum after the end of the application, after which the cell does not recover its original firing rate for 2–3 min. Furthermore, the excitant effects of noradrenaline are stereospecific for l-noradrenaline and can be blocked by the common adrenergic blocking agents12–14,18 whereas the depressant responses are not stereospecific and cannot usually be blocked by these agents2,5, 8,12–14. It has therefore been suggested that noradrenaline is an excitatory transmitter in the brain, and that the depressant effects represent a non-specific effect on cellular membranes or processes14.