Holliday junction resolvase in Schizosaccharomyces pombe has identical endonuclease activity to the CCE1 homologue YDC2

A novel Holliday junction resolving activity has been identified in fractionated cell extracts of the fission yeast Schizosaccharomyces pombe . The enzyme catalyses endonucleolytic cleavage of Holliday junction-containing chi DNA and synthetic four-way DNA junctions. The activity cuts with high specificity a synthetic four-way junction containing a 12 bp core of homologous sequences but has no activity on another four-way junction (with a fixed crossover point), a three-way junction, linear duplex DNA or duplex DNA containing six mismatched nucleotides in the centre. The major cleavage sites map as single nicks in the vicinity of the crossover point, 3' of a thymidine residue. These data indicate that the activity has a strong DNA structure selectivity as well as a limited sequence preference; features similar to the Holliday junction resolving enzymes RuvC of Escherichia coli and the mitochondrial CCE1 (cruciform-cuttingenzyme 1) of Saccharomyces cerevisiae. A putative homologue of CCE1 in S.pombe (YDC2_SCHPO) has been identified through a search of the sequence database. The open reading frame of this gene has been cloned and the encoded protein, YDC2, expressed in E.coli . The purified recombinant YDC2 exhibits Holliday junction resolvase activity and is, therefore, a functional S.pombe homologue of CCE1. The resolvase YDC2 shows the same substrate specificity and produces identical cleavage sites as the activity obtained from S. pombe cells. Both YDC2 and the cellular activity cleave Holliday junctions in both orientations to give nicks that can be ligated in vitro. The partially purified Holliday junction resolving enzyme in fission yeast is biochemically indistinguishable from recombinant YDC2 and appears to be the same protein

Design and validation of a virtual player for studying interpersonal coordination in the mirror game

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.The mirror game has been recently proposed as a simple, yet powerful paradigm for studying interpersonal interactions. It has been suggested that a virtual partner able to play the game with human subjects can be an effective tool to affect the underlying neural processes needed to establish the necessary connections between the players, and also to provide new clinical interventions for rehabilitation of patients suffering from social disorders. Inspired by the motor processes of the central nervous system (CNS) and the musculoskeletal system in the human body, in this paper we develop a novel interactive cognitive architecture based on nonlinear control theory to drive a virtual player (VP) to play the mirror game with a human player (HP) in different configurations. Specifically, we consider two cases: the former where the VP acts as leader and the latter where it acts as follower. The crucial problem is to design a feedback control architecture capable of imitating and following or leading a human player in a joint action task. Movement of the end-effector of the VP is modeled by means of a feedback controlled Haken-Kelso-Bunz (HKB) oscillator, which is coupled with the observed motion of the HP measured in real time. To this aim, two types of control algorithms (adaptive control and optimal control) are used and implemented on the HKB model so that the VP can generate a human-like motion while satisfying certain kinematic constraints. A proof of convergence of the control algorithms is presented in the paper together with an extensive numerical and experimental validation of their effectiveness. A comparison with other existing designs is also discussed, showing the flexibility and the advantages of our control-based approach.This work was funded by the European Project AlterEgo FP7 ICT 2.9 - Cognitive Sciences and Robotics, Grant Number 600610

Dynamical systems analysis of spike-adding mechanisms in transient bursts

Transient bursting behaviour of excitable cells, such as neurons, is a common feature observed experimentally, but theoretically, it is not well understood. We analyse a five-dimensional simplified model of after-depolarisation that exhibits transient bursting behaviour when perturbed with a short current injection. Using one-parameter continuation of the perturbed orbit segment formulated as a well-posed boundary value problem, we show that the spike-adding mechanism is a canard-like transition that has a different character from known mechanisms for periodic burst solutions. The biophysical basis of the model gives a natural time-scale separation, which allows us to explain the spike-adding mechanism using geometric singular perturbation theory, but it does not involve actual bifurcations as for periodic bursts. We show that unstable sheets of the critical manifold, formed by saddle equilibria of the system that only exist in a singular limit, are responsible for the spike-adding transition; the transition is organised by the slow flow on the critical manifold near folds of this manifold. Our analysis shows that the orbit segment during the spike-adding transition includes a fast transition between two unstable sheets of the slow manifold that are of saddle type. We also discuss a different parameter regime where the presence of additional saddle equilibria of the full system alters the spike-adding mechanism

Mathematical modeling of gonadotropin-releasing hormone signaling.

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Gonadotropin-releasing hormone (GnRH) acts via G-protein coupled receptors on pituitary gonadotropes to control of reproduction. These are Gq-coupled receptors that mediate acute effects of GnRH on the exocytotic secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as the chronic regulation of their synthesis. GnRH is secreted in short pulses and GnRH effects on its target cells are dependent upon the dynamics of these pulses. Here we overview GnRH receptors and their signaling network, placing emphasis on pulsatile signaling, and how mechanistic mathematical models and an information theoretic approach have helped further this field.This work was funded Project Grants from MRC (93447) and the BBSRC (J014699). KTA and MV gratefully acknowledge the financial support of the EPSRC via grant EP/N014391/1 and an MRC Biomedical Informatics Fellowship (MR/K021826/1), respectively

Control of clustered action potential firing in a mathematical model of entorhinal cortex stellate cells.

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.The entorhinal cortex is a crucial component of our memory and spatial navigation systems and is one of the first areas to be affected in dementias featuring tau pathology, such as Alzheimer's disease and frontotemporal dementia. Electrophysiological recordings from principle cells of medial entorhinal cortex (layer II stellate cells, mEC-SCs) demonstrate a number of key identifying properties including subthreshold oscillations in the theta (4-12 Hz) range and clustered action potential firing. These single cell properties are correlated with network activity such as grid firing and coupling between theta and gamma rhythms, suggesting they are important for spatial memory. As such, experimental models of dementia have revealed disruption of organised dorsoventral gradients in clustered action potential firing. To better understand the mechanisms underpinning these different dynamics, we study a conductance based model of mEC-SCs. We demonstrate that the model, driven by extrinsic noise, can capture quantitative differences in clustered action potential firing patterns recorded from experimental models of tau pathology and healthy animals. The differential equation formulation of our model allows us to perform numerical bifurcation analyses in order to uncover the dynamic mechanisms underlying these patterns. We show that clustered dynamics can be understood as subcritical Hopf/homoclinic bursting in a fast-slow system where the slow sub-system is governed by activation of the persistent sodium current and inactivation of the slow A-type potassium current. In the full system, we demonstrate that clustered firing arises via flip bifurcations as conductance parameters are varied. Our model analyses confirm the experimentally suggested hypothesis that the breakdown of clustered dynamics in disease occurs via increases in AHP conductance.The contribution of MG, KTR and JB was generously supported by a Wellcome Trust Institutional Strategic Support Award (WT105618MA). MG and KT gratefully acknowledge the financial support of the EPSRC via grant EP/N014391/1. LT’s doctoral studentship is supported by the Alzheimer’s Society in partnership with the Garfield Weston Foundation (grant reference 231). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

ruvA Mutants that resolve Holliday junctions but do not reverse replication forks

RuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination

Critical currents in vicinal YBa2_2Cu3_3O7−δ_{7-\delta} films

Most measurements of critical current densities in YBa$_2$Cu$_3$O$_{7-\delta}$ thin films to date have been performed on films where the \textit{c}-axis is grown normal to the film surface. With such films, the analysis of the dependence of $j_c$ on the magnetic field angle is complex. The effects of extrinsic contributions to the angular field dependence of $j_c$, such as the measurement geometry and disposition of pinning centres, are convoluted with those intrinsically due to the anisotropy of the material. As a consequence of this, it is difficult to distinguish between proposed FLL structure models on the basis of angular critical current density measurements on \textit{c}-axis films. Films grown on mis-cut (vicinal) substrates have a reduced measurement symmetry and thus provide a greater insight into the critical current anisotropy. In this paper previous descriptions of the magnetic field angle dependence of $j_c$ in YBa$_2$Cu$_3$O$_{7-\delta}$ are reviewed. Measurements on YBa$_2$Cu$_3$O$_{7-\delta}$ thin films grown on a range of vicinal substrates are presented and the results interpreted in terms of the structure and dimensionality of the FLL in YBa$_2$Cu$_3$O$_{7-\delta}$. There is strong evidence for a transition in the structure of the flux line lattice depending on magnetic field magnitude, orientation and temperature. As a consequence, a simple scaling law can not, by itself, describe the observed critical current anisotropy in YBa$_2$Cu$_3$O$_{7-\delta}$. The experimentally obtained $j_c(\theta)$ behaviour of YBCO is successfully described in terms of a kinked vortex structure for fields applied near parallel to the \textit{a-b} planes.Comment: 10 pages, 12 figures, Submitted to PR

Information Transfer via Gonadotropin-Releasing Hormone Receptors to ERK and NFAT: Sensing GnRH and Sensing Dynamics

This is the final version of the article. Available from Oxford University Press via the DOI in this record.Information theoretic approaches can be used to quantify information transfer via cell signaling networks. In this study, we do so for gonadotropin-releasing hormone (GnRH) activation of extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells (NFAT) in large numbers of individual fixed LβT2 and HeLa cells. Information transfer, measured by mutual information between GnRH and ERK or NFAT, was <1 bit (despite 3-bit system inputs). It was increased by sensing both ERK and NFAT, but the increase was <50%. In live cells, information transfer via GnRH receptors to NFAT was also <1 bit and was increased by consideration of response trajectory, but the increase was <10%. GnRH secretion is pulsatile, so we explored information gained by sensing a second pulse, developing a model of GnRH signaling to NFAT with variability introduced by allowing effectors to fluctuate. Simulations revealed that when cell–cell variability reflects rapidly fluctuating effector levels, additional information is gained by sensing two GnRH pulses, but where it is due to slowly fluctuating effectors, responses in one pulse are predictive of those in another, so little information is gained from sensing both. Wet laboratory experiments revealed that the latter scenario holds true for GnRH signaling; within the timescale of our experiments (1 to 2 hours), cell–cell variability in the NFAT pathway remains relatively constant, so trajectories are reproducible from pulse to pulse. Accordingly, joint sensing, sensing of response trajectories, and sensing of repeated pulses can all increase information transfer via GnRH receptors, but in each case the increase is small.This work was supported by Biochemical and Biophysical Science Research Council Grant BBSRC BB/J014699/1 (to C.A.M. and K.T.-A.). M.V. acknowledges the support of the Medical Research Council (a strategic skills development fellowship in biomedical informatics) and the Engineering and Physical Sciences Research Council via Grant EP/N014391/1

Information Transfer in Gonadotropin-releasing Hormone (GnRH) Signaling: extracellular signal-regulated kinase (ERK)-mediated feedback loops control hormone sensing

The computation model used in the study of GnRH signalling which was used to generate the data appearing in this paper is in ORE at http://hdl.handle.net/10871/27844Cell signaling pathways are noisy communication channels, and statistical measures derived from information theory can be used to quantify the information they transfer. Here we use single cell signaling measures to calculate mutual information as a measure of information transfer via gonadotropin-releasing hormone (GnRH) receptors (GnRHR) to extracellular signal-regulated kinase (ERK) or nuclear factor of activated T-cells (NFAT). This revealed mutual information values <1 bit, implying that individual GnRH-responsive cells cannot unambiguously differentiate even two equally probable input concentrations. Addressing possible mechanisms for mitigation of information loss, we focused on the ERK pathway and developed a stochastic activation model incorporating negative feedback and constitutive activity. Model simulations revealed interplay between fast (min) and slow (min-h) negative feedback loops with maximal information transfer at intermediate feedback levels. Consistent with this, experiments revealed that reducing negative feedback (by expressing catalytically inactive ERK2) and increasing negative feedback (by Egr1-driven expression of dual-specificity phosphatase 5 (DUSP5)) both reduced information transfer from GnRHR to ERK. It was also reduced by blocking protein synthesis (to prevent GnRH from increasing DUSP expression) but did not differ for different GnRHRs that do or do not undergo rapid homologous desensitization. Thus, the first statistical measures of information transfer via these receptors reveals that individual cells are unreliable sensors of GnRH concentration and that this reliability is maximal at intermediate levels of ERK-mediated negative feedback but is not influenced by receptor desensitization.This work was supported by a Biochemical and Biophysical Science Research Council award (BBSRC BB/J014699/1; to C. A. M. and K. T.-A.)

Information Transfer in Gonadotropin-Releasing Hormone (GnRH) Signaling:Extracellular Signal-Regulated Kinase (ERK)-Mediated Feedback Loops Control Hormone Sensing

Cell signaling pathways are noisy communication channels, and statistical measures derived from information theory can be used to quantify the information they transfer. Here we use single cell signaling measures to calculate mutual information as a measure of information transfer via gonadotropin-releasing hormone (GnRH) receptors (GnRHR) to extracellular signal-regulated kinase (ERK) or nuclear factor of activated T-cells (NFAT). This revealed mutual information values <1 bit, implying that individual GnRH-responsive cells cannot unambiguously differentiate even two equally probable input concentrations. Addressing possible mechanisms for mitigation of information loss, we focused on the ERK pathway and developed a stochastic activation model incorporating negative feedback and constitutive activity. Model simulations revealed interplay between fast (min) and slow (min-h) negative feedback loops with maximal information transfer at intermediate feedback levels. Consistent with this, experiments revealed that reducing negative feedback (by expressing catalytically inactive ERK2) and increasing negative feedback (by Egr1-driven expression of dual-specificity phosphatase 5 (DUSP5)) both reduced information transfer from GnRHR to ERK. It was also reduced by blocking protein synthesis (to prevent GnRH from increasing DUSP expression) but did not differ for different GnRHRs that do or do not undergo rapid homologous desensitization. Thus, the first statistical measures of information transfer via these receptors reveals that individual cells are unreliable sensors of GnRH concentration and that this reliability is maximal at intermediate levels of ERK-mediated negative feedback but is not influenced by receptor desensitization