Stochastic line integrals and stream functions as metrics of irreversibility and heat transfer.

Stochastic line integrals are presented as a useful metric for quantitatively characterizing irreversibility and detailed balance violation in noise-driven dynamical systems. A particular realization is the stochastic area, recently studied in coupled electrical circuits. Here we provide a general framework for understanding properties of stochastic line integrals and clarify their implementation for experiments and simulations. For two-dimensional systems, stochastic line integrals can be expressed in terms of a stream function, the sign of which determines the orientation of nonequilibrium steady-state probability currents. Theoretical results are supported by numerical studies of an overdamped two-dimensional mass-spring system driven out of equilibrium. Additionally, the stream function permits analytical understanding of the scaling dependence of stochastic area growth rate on key parameters such as the noise strength for both linear and nonlinear springs.

Repulsive expansion dynamics in colony growth and gene expression.

Spatial expansion of a population of cells can arise from growth of microorganisms, plant cells, and mammalian cells. It underlies normal or dysfunctional tissue development, and it can be exploited as the foundation for programming spatial patterns. This expansion is often driven by continuous growth and division of cells within a colony, which in turn pushes the peripheral cells outward. This process generates a repulsion velocity field at each location within the colony. Here we show that this process can be approximated as coarse-grained repulsive-expansion kinetics. This framework enables accurate and efficient simulation of growth and gene expression dynamics in radially symmetric colonies with homogenous z-directional distribution. It is robust even if cells are not spherical and vary in size. The simplicity of the resulting mathematical framework also greatly facilitates generation of mechanistic insights.

Experimental metrics for detection of detailed balance violation.

We report on the measurement of detailed balance violation in a coupled, noise-driven linear electronic circuit consisting of two nominally identical RC elements that are coupled via a variable capacitance. The state variables are the time-dependent voltages across each of the two primary capacitors, and the system is driven by independent noise sources in series with each of the resistances. From the recorded time histories of these two voltages, we quantify violations of detailed balance by three methods: (1) explicit construction of the probability current density, (2) constructing the time-dependent stochastic area, and (3) constructing statistical fluctuation loops. In comparing the three methods, we find that the stochastic area is relatively simple to implement and computationally inexpensive and provides a highly sensitive means for detecting violations of detailed balance.

Fluctuation loops in noise-driven linear dynamical systems.

Understanding the spatiotemporal structure of most probable fluctuation pathways to rarely occurring states is a central problem in the study of noise-driven, nonequilibrium dynamical systems. When the underlying system does not possess detailed balance, the optimal fluctuation pathway to a particular state and relaxation pathway from that state may combine to form a looplike structure in the system phase space called a fluctuation loop. Here, fluctuation loops are studied in a linear circuit model consisting of coupled RC elements, where each element is driven by its own independent noise source. Using a stochastic Hamiltonian approach, we determine the optimal fluctuation pathways, and analytically construct corresponding fluctuation loops. To quantitatively characterize fluctuation loops, we study the time-dependent area tensor that is swept out by individual stochastic trajectories in the system phase space. At long times, the area tensor scales linearly with time, with a coefficient that precisely vanishes when the system satisfies detailed balance.

Linear population allocation by bistable switches in response to transient stimulation.

Many cellular decision processes, including proliferation, differentiation, and phenotypic switching, are controlled by bistable signaling networks. In response to transient or intermediate input signals, these networks allocate a population fraction to each of two distinct states (e.g. OFF and ON). While extensive studies have been carried out to analyze various bistable networks, they are primarily focused on responses of bistable networks to sustained input signals. In this work, we investigate the response characteristics of bistable networks to transient signals, using both theoretical analysis and numerical simulation. We find that bistable systems exhibit a common property: for input signals with short durations, the fraction of switching cells increases linearly with the signal duration, allowing the population to integrate transient signals to tune its response. We propose that this allocation algorithm can be an optimal response strategy for certain cellular decisions in which excessive switching results in lower population fitness.

Steering most probable escape paths by varying relative noise intensities.

We demonstrate the possibility to systematically steer the most probable escape paths (MPEPs) by adjusting relative noise intensities in dynamical systems that exhibit noise-induced escape from a metastable point via a saddle point. With the use of a geometric minimum action approach, an asymptotic theory is developed that is broadly applicable to fast-slow systems and shows the important role played by the nullcline associated with the fast variable in locating the MPEPs. A two-dimensional quadratic system is presented which permits analytical determination of both the MPEPs and associated action values. Analytical predictions agree with computed MPEPs, and both are numerically confirmed by constructing prehistory distributions directly from the underlying stochastic differential equation.

Aggregation according to classical kinetics: from nucleation to coarsening.

A previous paper [Y. Farjoun and J. C. Neu, Phys. Rev. E 78, 051402 (2008)] presents a simple kinetic model of the initial creation transient, starting from pure monomer. During this transient the majority of clusters are created, and the distribution of cluster sizes that emerges from it is predicted to be discontinuous at the largest cluster size. It is well known that the further evolution according to the Lifshitz-Slyozov model of coarsening preserves this discontinuity. The result is at odds with the original proposal of Lifshitz and Slyozov, that the physical late-stage coarsening distribution is the smooth one. The current paper presents an analytic-numerical solution of the Lifshitz-Slyozov equations, starting from the discontinuous creation distribution. Of course, this analysis selects the discontinuous late-stage coarsening distribution, but there is much more. It resolves the intermediate stages between the creation transient and late-state coarsening and provides specific scales of time and cluster size that characterize the onset of coarsening.

Myxobacteria gliding motility requires cytoskeleton rotation powered by proton motive force.

Myxococcus xanthus is a Gram-negative bacterium that glides over surfaces without the aid of flagella. Two motility systems are used for locomotion: social-motility, powered by the retraction of type IV pili, and adventurous (A)-motility, powered by unknown mechanism(s). We have shown that AgmU, an A-motility protein, is part of a multiprotein complex that spans the inner membrane and periplasm of M. xanthus. In this paper, we present evidence that periplasmic AgmU decorates a looped continuous helix that rotates clockwise as cells glide forward, reversing its rotation when cells reverse polarity. Inhibitor studies showed that the AgmU helix rotation is driven by proton motive force (PMF) and depends on actin-like MreB cytoskeletal filaments. The AgmU motility complex was found to interact with MotAB homologs. Our data are consistent with a mechanochemical model in which PMF-driven motors, similar to bacterial flagella stator complexes, run along an endless looped helical track, driving rotation of the track; deformation of the cell surface by the AgmU-associated proteins creates pressure waves in the slime, pushing cells forward.

Motor-substrate interactions in mycoplasma motility explains non-Arrhenius temperature dependence.

Mycoplasmas exhibit a novel, substrate-dependent gliding motility that is driven by approximately 400 "leg" proteins. The legs interact with the substrate and transmit the forces generated by an assembly of ATPase motors. The velocity of the cell increases linearly by nearly 10-fold over a narrow temperature range of 10-40 degrees C. This corresponds to an Arrhenius factor that decreases from approximately 45 k(B)T at 10 degrees C to approximately 10 k(B)T at 40 degrees C. On the other hand, load-velocity curves at different temperatures extrapolate to nearly the same stall force, suggesting a temperature-insensitive force-generation mechanism near stall. In this article, we propose a leg-substrate interaction mechanism that explains the intriguing temperature sensitivity of this motility. The large Arrhenius factor at low temperature comes about from the addition of many smaller energy barriers arising from many substrate-binding sites at the distal end of the leg protein. The Arrhenius dependence attenuates at high temperature due to two factors: 1), the reduced effective multiplicity of energy barriers intrinsic to the multiple-site binding mechanism; and 2), the temperature-sensitive weakly facilitated leg release that curtails the power stroke. The model suggests an explanation for the similar steep, sub-Arrhenius temperature-velocity curves observed in many molecular motors, such as kinesin and myosin, wherein the temperature behavior is dominated not by the catalytic biochemistry, but by the motor-substrate interaction.

Activating function of needle electrodes in anisotropic tissue.

We present an analytical solution for the electrical potential and activating function (AF) established by cylindrical needle electrodes in anisotropic tissue. We compare this activating function to (1) AF computed assuming line-source electrodes and (2) AF computed using a finite element program. The results show that when the fiber is two needle diameters away from the electrodes, the maximum of the AF for needle electrodes is 1.43-times larger than for line-source electrodes, which results in lower thresholds for stimulation and electroporation. Therefore, for fibers that are close to the stimulating electrodes, one would benefit from using the formula that accounts for the electrodes' geometry.

Exhaustion of nucleation in a closed system.

We determine the distribution of cluster sizes that emerges from an initial phase of homogeneous aggregation with conserved total particle density. The physical ingredients behind the predictions are essentially classical: Supercritical nuclei are created at the Zeldovich rate, and before the depletion of monomers is significant, the characteristic cluster size is so large that the clusters undergo diffusion-limited growth. Mathematically, the distribution of cluster sizes satisfies an advection partial differential equation (PDE) in "size space." During this creation phase, clusters are nucleated and then grow much larger than the critical size, so nucleation of supercritical clusters at the Zeldovich rate is represented by an effective boundary condition at zero size. The advection PDE subject to the effective boundary condition constitutes a "creation signaling problem" for the evolving distribution of cluster sizes during the creation era. Dominant balance arguments applied to the advection signaling problem show that the characteristic time and cluster size of the creation era are exponentially large in the initial free-energy barrier against nucleation, G*. Specifically, the characteristic time is proportional to e(2/5)G*/kBT} and the characteristic number of monomers in a cluster is proportional to e(3/5)G*/kBT. The exponentially large characteristic time and cluster size give a posteriori validation of the mathematical signaling problem. In a short note, Marchenko [JETP Lett. 64, 66 (1996)] obtained these exponentials and the numerical prefactors 2/5 and 3/5. Our work adds the actual solution of the kinetic model implied by these scalings, and the basis for connection to subsequent stages of the aggregation process after the creation era.

Singular perturbation analysis of the pore creation transient.

Electroporation, in which electric pulses create transient pores in the cell membrane, is an important technique for drug and DNA delivery. Electroporation kinetics is mathematically described by an advection-diffusion boundary value problem. This study uses singular perturbation to derive a reduced description of the pore creation transient in the form of a single integrodifferential equation for the transmembrane voltage Vt. The number of pores and the distribution of their radii are computed from Vt. The analysis contains two nonstandard features: the use of the voltage deviation to peel away the strong exponential dependence of pore creation upon the transmembrane potential, and the autonomous approximation of the pore evolution. Comparing the predictions of the reduced equation with the simulations of the original problem demonstrates that this analysis allows one to predict with good accuracy the number and distribution of pores as a function of the electric pulse strength.

Accordion waves in Myxococcus xanthus.

Myxococcus xanthus are Gram-negative bacteria that glide on solid surfaces, periodically reversing their direction of movement. When starved, M. xanthus cells organize their movements into waves of cell density that sweep over the colony surface. These waves are unique: Although they appear to interpenetrate, they actually reflect off one another when they collide, so that each wave crest oscillates back and forth with no net displacement. Because the waves reflect the coordinated back and forth oscillations of the individual bacteria, we call them "accordion" waves. The spatial oscillations of individuals are a manifestation of an internal biochemical oscillator, probably involving the Frz chemosensory system. These internal "clocks," each of which is quite variable, are synchronized by collisions between individual cells using a contact-mediated signal-transduction system. The result of collision signaling is that the collective spatial behavior is much less variable than the individual oscillators. In this work, we present experimental observations in which individual cells marked with GFP can be followed in groups of unlabeled cells in monolayer cultures. These data, together with an agent-based computational model demonstrate that the only properties required to explain the ripple patterns are an asymmetric biochemical limit cycle that controls direction reversals and asymmetric contact-induced signaling between cells: Head-to-head signaling is stronger than head-to-tail signaling. Together, the experimental and computational data provide new insights into how populations of interacting oscillators can synchronize and organize spatially to produce morphogenetic patterns that may have parallels in higher organisms.

Developmental waves in myxobacteria: A distinctive pattern formation mechanism.

In early stages of their development, starving myxobacteria organize their motion to produce a periodic pattern of traveling cell density waves. These waves arise from coordination of individual cell reversals by contact signaling when they collide. Unlike waves generated by reaction-diffusion instabilities, which annihilate on collision, myxobacteria waves appear to pass through one another unaffected. Here we analyze a mathematical model of these waves developed earlier [Proc. Natl. Acad. Sci. USA 98, 14 913 (2001)]]. The mechanisms which generate and maintain the density waves are clearly revealed by tracing the reversal loci of individual cells. An evolution equation of reversal point density is derived in the weak-signaling limit. Linear stability analysis determines parameters favorable for the development of the waves. Numerical solutions demonstrate the stability of the fully developed nonlinear waves.

Induction of RNA interference in dendritic cells.

Dendritic cells (DC) reside at the center of the immunological universe, possessing the ability both to stimulate and inhibit various types of responses. Tolerogenic/regulatory DC with therapeutic properties can be generated through various means of manipulations in vitro and in vivo. Here we describe several attractive strategies for manipulation of DC using the novel technique of RNA interference (RNAi). Additionally, we overview some of our data regarding yet undescribed characteristics of RNAi in DC such as specific transfection strategies, persistence of gene silencing, and multi-gene silencing. The advantages of using RNAi for DC genetic manipulation gives rise to the promise of generating tailor-made DC that can be used effectively to treat a variety of immunologically mediated diseases.

Characterization of an inducible vancomycin resistance system in Streptomyces coelicolor reveals a novel gene (vanK) required for drug resistance.

Vancomycin is the front-line therapy for treating problematic infections caused by methicillin-resistant Staphylococcus aureus (MRSA), and the spread of vancomycin resistance is an acute problem. Vancomycin blocks cross-linking between peptidoglycan intermediates by binding to the D-Ala-D-Ala termini of bacterial cell wall precursors, which are the substrate of transglycosylase/transpeptidase. We have characterized a cluster of seven genes (vanSRJKHAX) in Streptomyces coelicolor that confers inducible, high-level vancomycin resistance. vanHAX are orthologous to genes found in vancomycin-resistant enterococci that encode enzymes predicted to reprogramme peptidoglycan biosynthesis such that cell wall precursors terminate in D-Ala-D-Lac rather than D-Ala-D-Ala. vanR and vanS encode a two-component signal transduction system that mediates transcriptional induction of the seven van genes. vanJ and vanK are novel genes that have no counterpart in previously characterized vancomycin resistance clusters from pathogens. VanK is a member of the Fem family of enzymes that add the cross-bridge amino acids to the stem pentapeptide of cell wall precursors, and vanK is essential for vancomycin resistance. The van genes are organized into four transcription units, vanRS, vanJ, vanK and vanHAX, and these transcripts are induced by vancomycin in a vanR-dependent manner. To develop a sensitive bioassay for inducers of the vancomycin resistance system, the promoter of vanJ was fused to a reporter gene conferring resistance to kanamycin. All the inducers identified were glycopeptide antibiotics, but teicoplanin, a membrane-anchored glycopeptide, failed to act as an inducer. Analysis of mutants defective in the vanRS and cseBC cell envelope signal transduction systems revealed significant cross-talk between the two pathways.

Model of creation and evolution of stable electropores for DNA delivery.

Electroporation, in which electric pulses create transient pores in the cell membrane, is becoming an important technique for gene therapy. To enable entry of supercoiled DNA into cells, the pores should have sufficiently large radii (>10 nm), remain open long enough for the DNA chain to enter the cell (milliseconds), and should not cause membrane rupture. This study presents a model that can predict such macropores. The distinctive features of this model are the coupling of individual pores through membrane tension and the electrical force on the pores, which is applicable to pores of any size. The model is used to explore the process of pore creation and evolution and to determine the number and size of pores as a function of the pulse magnitude and duration. Next, our electroporation model is combined with a heuristic model of DNA uptake and used to predict the dependence of DNA uptake on pulsing parameters. Finally, the model is used to examine the mechanism of a two-pulse protocol, which was proposed specifically for gene delivery. The comparison between experimental results and the model suggests that this model is well-suited for the investigation of electroporation-mediated DNA delivery.

Electrical energy required to form large conducting pores.

This study computes the contribution of the externally induced transmembrane potential to the energy of large, highly conductive pores. This work was undertaken because the pore energy formulas existing in the literature predict qualitatively different behavior of large pores: the original formula proposed by Abidor et al. in 1979 implies that the electrical force expanding the pore increases linearly with pore radius, while later extensions of this formula imply that this force decreases to zero for large pores. Starting from the Maxwell stress tensors, our study derives the formula for the mechanical work required to deform a dielectric body in an ionic solution with steady-state electric current. This formula is related to a boundary value problem (BVP) governing electric potentials and fields in a proximity of a pore. Computer simulations yield estimates of the electrical energy for pores of two different shapes: cylindrical and toroidal. In both cases, the energy increases linearly for pore radii above approximately 20 nm, implying that the electrical force expanding the pore asymptotes to a constant value for large pores. This result is different from either of the two energy formulas mentioned above. Our study traces the source of this disagreement to approximations made by previous studies, which are suitable only for small pores. Therefore, this study provides a better understanding of the energy of large pores, which is needed for designing pulsing protocols for DNA delivery.

Modeling postshock evolution of large electropores.

The Smoluchowski equation (SE), which describes the evolution of pores created by electric shocks, cannot be applied to modeling large and long-lived pores for two reasons: (1) it does not predict pores of radius above 20 nm without also predicting membrane rupture; (2) it does not predict postshock growth of pores. This study proposes a model in which pores are coupled by membrane tension, resulting in a nonlinear generalization of SE. The predictions of the model are explored using examples of homogeneous (all pore radii r are equal) and heterogeneous (0

Protein interactions and membrane geometry.

The difficulty in growing crystals for x-ray diffraction analysis has hindered the determination of membrane protein structures. However, this is changing with the advent of a new method for growing high quality membrane protein crystals from the lipidic cubic phase. Although successful, the mechanism underlying this method has remained unclear. Here, we present a theoretical analysis of the process. We show that it is energetically favorable for proteins embedded in the highly curved cubic phase to cluster together in flattened regions of the membrane. This stabilizes the lamellar phase, permitting its outgrowth from the cubic phase. A kinetic barrier-crossing model is developed to determine the free energy barrier to crystallization from the time-dependent growth of protein clusters. Determining the values of key parameters provides both a rational basis for optimizing the experimental procedure for membrane proteins that have not yet been crystallized and insight into the analogous cubic to lamellar transitions in cells. We also discuss the implications of this mechanism for protein sorting at the exit sites of the Golgi and endoplasmic reticulum and the general stabilization of membrane structures.