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ABSTRACT: Many signaling circuits face a fundamental tradeoff between accelerating their response speed while maintaining final levels below a cytotoxic threshold. Here, we describe a transcriptional circuitry that dynamically converts signaling inputs into faster rates without amplifying final equilibrium levels. Using time-lapse microscopy, we find that transcriptional activators accelerate human cytomegalovirus (CMV) gene expression in single cells without amplifying steady-state expression levels, and this acceleration generates a significant replication advantage. We map the accelerator to a highly self-cooperative transcriptional negative-feedback loop (Hill coefficient ∼7) generated by homomultimerization of the virus's essential transactivator protein IE2 at nuclear PML bodies. Eliminating the IE2-accelerator circuit reduces transcriptional strength through mislocalization of incoming viral genomes away from PML bodies and carries a heavy fitness cost. In general, accelerators may provide a mechanism for signal-transduction circuits to respond quickly to external signals without increasing steady-state levels of potentially cytotoxic molecules.
Cell 12/2012; 151(7):1569-80. · 32.40 Impact Factor
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ABSTRACT: Gene expression occurs either as an episodic process, characterized by pulsatile bursts, or as a constitutive process, characterized by a Poisson-like accumulation of gene products. It is not clear which mode of gene expression (constitutive versus bursty) predominates across a genome or how transcriptional dynamics are influenced by genomic position and promoter sequence. Here, we use time-lapse fluorescence microscopy to analyze 8,000 individual human genomic loci and find that at virtually all loci, episodic bursting-as opposed to constitutive expression-is the predominant mode of expression. Quantitative analysis of the expression dynamics at these 8,000 loci indicates that both the frequency and size of the transcriptional bursts varies equally across the human genome, independent of promoter sequence. Strikingly, weaker expression loci modulate burst frequency to increase activity, whereas stronger expression loci modulate burst size to increase activity. Transcriptional activators such as trichostatin A (TSA) and tumor necrosis factor α (TNF) only modulate burst size and frequency along a constrained trend line governed by the promoter. In summary, transcriptional bursting dominates across the human genome, both burst frequency and burst size vary by chromosomal location, and transcriptional activators alter burst frequency and burst size, depending on the expression level of the locus.
Proceedings of the National Academy of Sciences 10/2012; 109(43):17454-9. · 9.68 Impact Factor
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ABSTRACT: Neural chips, which are capable of simultaneous multisite neural recording and stimulation, have been used to detect and modulate neural activity for almost thirty years. As neural interfaces, neural chips provide dynamic functional information for neural decoding and neural control. By improving sensitivity and spatial resolution, nano-scale electrodes may revolutionize neural detection and modulation at cellular and molecular levels as nano-neuron interfaces. We developed a carbon-nanofiber neural chip with lithographically defined arrays of vertically aligned carbon nanofiber electrodes and demonstrated its capability of both stimulating and monitoring electrophysiological signals from brain tissues in vitro and monitoring dynamic information of neuroplasticity. This novel nano-neuron interface may potentially serve as a precise, informative, biocompatible, and dual-mode neural interface for monitoring of both neuroelectrical and neurochemical activity at the single-cell level and even inside the cell. FROM THE CLINICAL EDITOR: The authors demonstrate the utility of a neural chip with lithographically defined arrays of vertically aligned carbon nanofiber electrodes. The new device can be used to stimulate and/or monitor signals from brain tissue in vitro and for monitoring dynamic information of neuroplasticity both intracellularly and at the single cell level including neuroelectrical and neurochemical activities.
Nanomedicine: nanotechnology, biology, and medicine 03/2012; 8(4):419-23. · 5.44 Impact Factor
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ABSTRACT: Compartmentalization is essential in the organization of biological systems, playing a fundamental role in modulating biochemical activity. An appreciation of the impact that biological compartments have on chemical reactions and an understanding of the physical and chemical phenomena that affect their assembly and function have inspired the development of synthetic compartments. Organic compartments assembled from amphiphilic molecules or derived from biological materials, have formed the basis of initial work in the field. However, inorganic and hybrid organic-inorganic compartments that capitalize on the optical and catalytic properties of metal and semiconductor materials are emerging. Methods for arraying these microcompartment and nanocompartment materials in higher order systems promise to enable the scaling and integration of these technologies for industrial and commercial applications.
Current opinion in biotechnology 02/2012; 23(4):522-8. · 7.82 Impact Factor
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ABSTRACT: Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion.
Nucleic Acids Research 12/2011; 40(8):3763-74. · 8.03 Impact Factor
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ABSTRACT: A key factor to the implementation of devices with vertically aligned carbon nanofibers (VACNFs) is fundamental understanding of how to control fluctuations in the growth direction of the fibers. Here we demonstrate synthesis of VACNF on transparent and insulating substrates by continuous direct current (DC) plasma for realization of cellular interface suitable for transmission optical microscopy. To maintain continuous glow discharge above the substrate, a metal grid electrode layer (Cr) was deposited over silica with windows of exposed silica ranging in size from 200 μm to 1 mm. This electrode geometry allows for synthesis of VACNFs even within an insulating window. This observation and the observed trends in the alignment of nanofibers in the vicinity of grid electrodes have indicated that the alignment does not correspond to the direction of the electric field at the substrate level, contrary to previously proposed alignment mechanism. Computational modeling of the plasma with this grid cathode geometry has shown that nanofiber alignment trends follow calculated ion flux direction rather than electrical field. The new proposed alignment mechanism is that ion sputtering of the carbon film on a catalyst particle defines the growth direction of the nanofibers. With this development, fiber growth direction can be better manipulated through changes in ionic flux direction, opening the possibility for growth of nanofibers on substrates with unique geometries.
ACS Applied Materials & Interfaces 08/2011; 3(9):3501-7. · 4.53 Impact Factor
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ABSTRACT: Controlling the movement of nanoscale objects is a significant goal of nanotechnology. Dewetting-induced ejection of nanodroplets could provide another means of achieving that goal. Molecular dynamics simulations were used to investigate the dewetting-induced ejection of nanoscale liquid copper nanostructures that were deposited on a graphitic substrate. Nanostructures in the shape of a circle, square, equilateral, and isosceles triangle dewet and form nanodroplets that are ejected from the substrate with a velocity that depends on the initial shape and temperature. The dependence of the ejected velocity on shape is ascribed to the temporal asymmetry of the mass coalescence during the droplet formation; the dependence on temperature is ascribed to changes in the density and viscosity. The results suggest that dewetting induced by nanosecond laser pulses could be used to control the velocity of ejected nanodroplets.
ACS Nano 08/2011; 5(9):7130-6. · 10.77 Impact Factor
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ABSTRACT: A better understanding of how confinement, crowding and reduced dimensionality modulate reactivity and reaction dynamics will aid in the rational and systematic discovery of functionality in complex biological systems. Artificial microfabricated and nanofabricated structures have helped elucidate the effects of nanoscale spatial confinement and segregation on biological behavior, particularly when integrated with microfluidics, through precise control in both space and time of diffusible signals and binding interactions. Examples of nanostructured interfaces for synthetic biology include the development of cell-like compartments for encapsulating biochemical reactions, nanostructured environments for fundamental studies of diffusion, molecular transport and biochemical reaction kinetics, and regulation of biomolecular interactions as functions of microfabricated and nanofabricated topological constraints.
Current opinion in biotechnology 05/2011; 22(4):516-26. · 7.82 Impact Factor
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ABSTRACT: The ability to synthesize free-standing, individual carbon nanofibres (CNFs) aligned perpendicularly to a substrate has enabled fabrication of a large array of devices with nanoscale functional elements, including electron field emission sources, electrochemical probes, neural interface arrays, scanning probes, gene delivery arrays and many others. This was made possible by development of a catalytic plasma process, with DC bias directing the alignment of nanofibres. Successful implementation of prototypical devices has uncovered numerous challenges in the integration of this synthesis process as one of the steps in device fabrication. This paper is dedicated to these engineering and fundamental difficulties that hinder further device development. Relatively high temperature for catalytic synthesis, electrical conductivity of the substrate to maintain DC discharge and other difficulties place restrictions on substrate material. Balancing non-catalytic carbon film deposition and substrate etching, non-uniformity of plasma due to growth of the high aspect ratio structures, plasma instabilities and other factors lead to challenges in controlling the plasma. Ultimately, controlling the atomistic processes at the catalyst nanoparticle (NP) and the behaviour of the NP is the central challenge of plasma nanosynthesis of vertically aligned CNFs.
Journal of Physics D Applied Physics 04/2011; 44(17):174008. · 2.54 Impact Factor
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ABSTRACT: The inherently small system sizes involved imply that, in the absence of large applied fields designed to overwhelm them, fluctuations will play a major role in determining the response and functionality of nanoscale systems. Theoretical advances over the past two decades have provided fresh insight into fluctuations and their role at the nanoscale, even in the presence of arbitrarily large applied external fields. In contrast to traditional engineered systems, Nature's approach to nanotechnology is to embrace and to exploit fluctuations and noise to create adaptable, persistent, optimized functional architectures. We describe some of the mechanisms by which Nature exploits noise, with the goal of applying these lessons to engineered physical and chemical nanosystems. In particular, we emphasize the critical role of the tails of distributions of properties in both physical and biological nanosystems and their impact on system behavior.
ACS Nano 04/2011; 5(4):2425-32. · 10.77 Impact Factor
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ABSTRACT: Thin-film dewetting can be exploited to self-assemble and organize nanoparticles. Crucial to this effort is the understanding of the nanoscale liquid phase dynamics, and molecular dynamics simulations (MD) provide a powerful tool in this respect. In this paper we demonstrate that MD simulations utilizing a Lennard-Jones (LJ) interface potential can be effectively used to study various wetting regimes of nanoscale Cu disks on graphite. It was found that both the dewetting velocity and the equilibrium contact angle increase with a decrease in the Cu-C potential, and that the retraction velocities obtained are characteristic of dewetting phenomena governed by inertial and capillary forces. This phenomena leads to a change in morphology, from disks to nanodroplets, which, in turn, when using the most accurate LJ potential, jump off the graphitic substrate with a velocity on the order of 140 m/s. This ejection velocity is consistent with the previous experimental observation that nanoscale Au triangles deposited on graphite or glass jump when exposed to a pulsed laser above the melting threshold. Interestingly, the Cu ejection velocity decreases when the liquid Cu disks are deposited on a suspended graphene membrane. Finally, a Rayleigh-Plateau-like instability, which leads to the breakup of a pseudo-one-dimensional liquid Cu nanowire in nanodroplets, is revealed when the MD simulations are performed using different LJ interface potentials.
Physical Review E 04/2011; 83(4 Pt 1):041603. · 2.26 Impact Factor
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ABSTRACT: Cells offer natural examples of highly efficient networks of nanomachines. Accordingly, both intracellular and intercellular communication mechanisms in nature are looked to as a source of inspiration and instruction for engineered nanocommunication. Harnessing biological functionality in this manner requires an interdisciplinary approach that integrates systems biology, synthetic biology, and nanofabrication. Here, we present a model system that exemplifies the synergism between these realms of research. We propose a synthetic gene network for operation in a nanofabricated cell mimic array that propagates a biomolecular signal over long distances using the phenomenon of stochastic resonance. Our system consists of a bacterial quorum sensing signal molecule, a bistable genetic switch triggered by this signal, and an array of nanofabricated cell mimic wells that contain the genetic system. An optimal level of noise in the system helps to propagate a time-varying AHL signal over long distances through the array of mimics. This noise level is determined both by the system volume and by the parameters of the genetic network. Our proposed genetically driven stochastic resonance system serves as a testbed for exploring the potential harnessing of gene expression noise to aid in the transmission of a time-varying molecular signal.
Nano communication networks. 03/2011; 2(1):39-49.
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ABSTRACT: The behavior of a 50 nm Cu–Ni alloy film on Si in a process of reactive solid-state dewetting is presented. The films were annealed at a range of temperatures (300–700 °C) in 1% H2 99% N2 reducing atmosphere. The resulting alloy and silicide particles formed by film dewetting and film reaction with the substrate were distinguished by selective wet etching and examined by scanning electron microscopy and spectroscopy. After potassium hydroxide etch, regions that etch slower than silicon substrate have distribution statistics similar to the alloy and silicide particles prior to their removal, indicating strong coupling between mass transport across the interface and along the surface.
Applied Physics Letters 12/2010; 97(25):253101-253101-3. · 3.84 Impact Factor
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ABSTRACT: Analysis of noise in gene expression has proven a powerful approach for analyzing gene regulatory architecture. To probe the regulatory mechanisms controlling expression of HIV-1, we analyze noise in gene-expression from HIV-1's long terminal repeat (LTR) promoter at different HIV-1 integration sites across the human genome. Flow cytometry analysis of GFP expression from the HIV-1 LTR shows high variability (noise) at each integration site. Notably, the measured noise levels are inconsistent with constitutive gene expression models. Instead, quantification of expression noise indicates that HIV-1 gene expression occurs through randomly timed bursts of activity from the LTR and that each burst generates an average of 2-10 mRNA transcripts before the promoter returns to an inactive state. These data indicate that transcriptional bursting can generate high variability in HIV-1 early gene products, which may critically influence the viral fate-decision between active replication and proviral latency.
Biophysical Journal 04/2010; 98(8):L32-4. · 3.65 Impact Factor
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ABSTRACT: Anode geometry can significantly affect the electrochemical synthesis of conductive polymers. Here, the effects of anode dimensions on the electropolymerization of pyrrole are investigated. Band microelectrodes were prepared with widths ranging from 2 to 500 mum. The anode dimension has a significant effect on the resulting thickness of polymer film. The electropolymerization process deviates significantly from that predicted by simple mass transfer considerations when electrode dimensions are less than approximately 20 mum. Polymer film thickness is thinner than expected when electrode dimensions become less than approximately 10 mum. A simple mathematical model was derived to explain the observed effects of anode dimensions on the polymerization process. Simulation results confirm that diffusive loss of reaction intermediates accounts for the observed experimental trends. The described simulation facilitates understanding of the electropolymerization processes and approaches to the controlled deposition of polypyrrole, particularly at the submicron scale, for microelectromechanical systems and biomedical applications.
Journal of Applied Physics 07/2009; 105(12):124312. · 2.17 Impact Factor
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ABSTRACT: We report a strategy for immobilizing dsDNA (double-stranded DNA) onto vertically aligned carbon nanofibers and subsequently releasing this dsDNA following penetration and residence of these high aspect ratio structures within cells. Gold-coated nanofiber arrays were modified with self-assembled monolayers (SAM) to which reporter dsDNA was covalently and end-specifically bound with or without a cleavable linker. The DNA-modified nanofiber arrays were then used to impale, and thereby transfect, Chinese hamster lung epithelial cells. This mechanical approach enables the transport of bound ligands directly into the cell nucleus and consequently bypasses extracellular and cytosolic degradation. Statistically significant differences were observed between the expression levels from immobilized and releasable DNA, and these are discussed in relation to the distinct accessibility and mode of action of glutathione, an intracellular reducing agent responsible for releasing the bound dsDNA. These results prove for the first time that an end-specifically and covalently SAM-bound DNA can be expressed in cells. They further demonstrate how the choice of immobilization and release methods can impact expression of nanoparticle delivered DNA.
Nanotechnology 05/2009; 20(14):145304. · 3.98 Impact Factor
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ABSTRACT: We demonstrate the resolution and characteristics of a nanolithography process utilizing electron beam induced deposition (EBID) of W(CO)(6) and C(10)H(8) to define the imaging and masking layers. Lines and dot matrices were defined/written with various electron beam doses onto both polymethylmethacrylate (PMMA) coated silicon substrates (PMMA-Si) and bare silicon substrates (Si). The selectivity of the W(CO)(x) for the PMMA dry development process (no measurable etching) and the silicon ( approximately 18:1) reactive ion etch was very good. C(10)H(8) directly patterned on Si also provided good selectivity for the silicon etch process, 21:1. The pattern transfer of the EBID material patterns into the silicon had high fidelity. The resolution scaled with exposure dose and was correlated with the EBID broadening/scattering via a Monte Carlo simulation. Using the bi-layer approach, imaging layers on PMMA-Si, a silicon nanowire resolution of 13.5 nm and linewidth of 24.5 nm are demonstrated. Furthermore, using the single-layer approach, EBID directly on Si, a silicon nanowire resolution of 33 nm is demonstrated.
Nanotechnology 12/2008; 19(50):505302. · 3.98 Impact Factor
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ABSTRACT: We report the effective and site-specific binding of long double stranded (ds)DNA to high aspect ratio carbon nanofiber arrays. The carbon nanofibers were first coated with a thin gold layer to provide anchorage for two controllable binding methods. One method was based on the direct binding of thiol end-labeled dsDNA. The second and enhanced method used amine end-labeled dsDNA bound with crosslinkers to a carboxyl-terminated self-assembled monolayer. The bound dsDNA was first visualized with a fluorescent, dsDNA-intercalating dye. The specific binding onto the carbon nanofiber was verified by a high resolution detection method using scanning electron microscopy in combination with the binding of neutravidin-coated fluorescent microspheres to the immobilized and biotinylated dsDNA. Functional activity of thiol end-labeled dsDNA on gold-coated nanofiber arrays was verified with a transcriptional assay, whereby Chinese hamster lung cells (V79) were impaled upon the DNA-modified nanofibers and scored for transgene expression of the tethered template. Thiol end-labeled dsDNA demonstrated significantly higher expression levels than nanofibers prepared with control dsDNA that lacked a gold-binding end-label. Employing these site-specific and robust techniques of immobilization of dsDNA onto nanodevices can be of advantage for the study of DNA/protein interactions and for gene delivery applications.
Nanotechnology 10/2008; 19(43):435301. · 3.98 Impact Factor
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ABSTRACT: Stochastic fluctuations (or "noise") in the single-cell populations of molecular species are shaped by the structure and biokinetic rates of the underlying gene circuit. The structure of the noise is summarized by its autocorrelation function. In this article, we introduce the noise regulatory vector as a generalized framework for making inferences concerning the structure and biokinetic rates of a gene circuit from its noise autocorrelation function. Although most previous studies have focused primarily on the magnitude component of the noise (given by the zero-lag autocorrelation function), our approach also considers the correlation component, which encodes additional information concerning the circuit. Theoretical analyses and simulations of various gene circuits show that the noise regulatory vector is characteristic of the composition of the circuit. Although a particular noise regulatory vector does not map uniquely to a single underlying circuit, it does suggest possible candidate circuits, while excluding others, thereby demonstrating the probative value of noise in gene circuit analysis.
Proceedings of the National Academy of Sciences 09/2008; 105(31):10809-14. · 9.68 Impact Factor
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ABSTRACT: Thin nickel films were patterned into various shapes and treated with a series of laser pulses. The edges and vertices of the patterned shapes act as programable instabilities, which enable directed assembly via dewetting when the laser energy density is above the melting threshold. The pattern formations were monitored as a function of laser pulse and the retraction process was attributed liquid dewetting and a subsequent resolidification. The calculated retraction velocity (83 m / s ) and liquid lifetime (12.3 ns ) were consistent with the measured nickel retraction distances. The vertices of the shapes had an initially larger retraction velocity which was attributed to an additional in-plane curvature.
Applied Physics Letters 07/2008; · 3.84 Impact Factor
