The Experts below are selected from a list of 249 Experts worldwide ranked by ideXlab platform
Jorge E Contreras - One of the best experts on this subject based on the ideXlab platform.
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a novel voltage clamp dye uptake assay reveals saturable transport of molecules through calhm1 and connexin channels
The Journal of General Physiology, 2020Co-Authors: Pablo S Gaete, Mauricio A Lillo, William Lopez, Yu Liu, Andrew L Harris, Wenjuan Jiang, Yun Luo, Jorge E ContrerasAbstract:Large-pore channels permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, their mechanisms of Molecular permeation and selectivity remain mostly unexplored. Combining fluorescence microscopy and electrophysiology, we developed a novel technique that allows kinetic analysis of Molecular permeation through connexin and CALHM1 channels in Xenopus oocytes rendered translucent. Using this methodology, we found that (1) Molecular Flux through these channels saturates at low micromolar concentrations, (2) kinetic parameters of Molecular transport are sensitive to modulators of channel gating, (3) Molecular transport and ionic currents can be differentially affected by mutation and gating, and (4) N-terminal regions of these channels control transport kinetics and permselectivity. Our methodology allows analysis of how human disease-causing mutations affect kinetic properties and permselectivity of Molecular signaling and enables the study of Molecular mechanisms, including selectivity and saturability, of Molecular transport in other large-pore channels.
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a novel voltage clamp dye uptake assay reveals saturable transport of molecules through calhm1 and connexin channels
bioRxiv, 2020Co-Authors: Pablo S Gaete, Mauricio A Lillo, William Lopez, Yu Liu, Andrew L Harris, Jorge E ContrerasAbstract:ABSTRACT Channels that are permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, mechanisms of Molecular permeation and selectivity of these channels remain mostly unexplored due to the lack of quantitative methodologies. To address this need, we developed a novel two-electrode voltage clamp (TEVC)/dye uptake assay to examine the kinetics of Molecular permeation of channels formed by human connexins (hCx), and the calcium homeostasis modulator (hCALHM1). We expressed hCx26, hCx30, and hCALHM1 individually in Xenopus laevis oocytes. To quantify the uptake of small Molecular dyes through these channels, we developed a protocol that renders oocytes translucent – thereby amenable to optical detection techniques – without affecting the functional properties of the expressed channels. To control membrane potential and to determine functional channel expression accurately, dye uptake was evaluated in conjunction with TEVC. Using this methodology, we found that: (1) CALHM1 and Cx30 hemichannels display saturable transport of molecules that could be described by Michaelis-Menten kinetics, with apparent KM and Vmax; (2) Kinetic parameters for Molecular transport through CALHM1 are sensitive to voltage and extracellular calcium; (3) Significant transport of molecules occurs through CALHM1 when there are little or no ionic currents through the channels; (4) Cx mutations in the N-terminal region significantly affect kinetics of transport and permselectivity. Our results reveal that Molecular Flux through these channels has a rate-limiting step, that the kinetic parameters of Molecular transport are sensitive to modulators of channel gating and that Molecular transport and ionic currents can be differentially affected. Our methodology allows the analysis of how human mutations causing diseases affect kinetic properties and permselectivity of Molecular signaling and enables the study of Molecular mechanisms, including selectivity and saturability, associated with Molecular transport in large-pore channels.
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A novel voltage clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels
2020Co-Authors: Pablo S Gaete, Mauricio A Lillo, William Lopez, Yu Liu, Andrew L Harris, Jorge E ContrerasAbstract:ABSTRACT Channels that are permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, mechanisms of Molecular permeation and selectivity of these channels remain mostly unexplored due to the lack of quantitative methodologies. To address this need, we developed a novel two-electrode voltage clamp (TEVC)/dye uptake assay to examine the kinetics of Molecular permeation of channels formed by human connexins (hCx), and the calcium homeostasis modulator (hCALHM1). We expressed hCx26, hCx30, and hCALHM1 individually in Xenopus laevis oocytes. To quantify the uptake of small Molecular dyes through these channels, we developed a protocol that renders oocytes translucent – thereby amenable to optical detection techniques – without affecting the functional properties of the expressed channels. To control membrane potential and to determine functional channel expression accurately, dye uptake was evaluated in conjunction with TEVC. Using this methodology, we found that: (1) CALHM1 and Cx30 hemichannels display saturable transport of molecules that could be described by Michaelis-Menten kinetics, with apparent KM and Vmax; (2) Kinetic parameters for Molecular transport through CALHM1 are sensitive to voltage and extracellular calcium; (3) Significant transport of molecules occurs through CALHM1 when there are little or no ionic currents through the channels; (4) Cx mutations in the N-terminal region significantly affect kinetics of transport and permselectivity. Our results reveal that Molecular Flux through these channels has a rate-limiting step, that the kinetic parameters of Molecular transport are sensitive to modulators of channel gating and that Molecular transport and ionic currents can be differentially affected. Our methodology allows the analysis of how human mutations causing diseases affect kinetic properties and permselectivity of Molecular signaling and enables the study of Molecular mechanisms, including selectivity and saturability, associated with Molecular transport in large-pore channels.
Rahul Sarpeshkar - One of the best experts on this subject based on the ideXlab platform.
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fast and precise emulation of stochastic biochemical reaction networks with amplified thermal noise in silicon chips
IEEE Transactions on Biomedical Circuits and Systems, 2018Co-Authors: Jaewook Kim, Sung Sik Woo, Rahul SarpeshkarAbstract:The analysis and simulation of complex interacting biochemical reaction pathways in cells is important in all of systems biology and medicine. Yet, the dynamics of even a modest number of noisy or stochastic coupled biochemical reactions is extremely time consuming to simulate. In large part, this is because of the expensive cost of random number and Poisson process generation and the presence of stiff, coupled, nonlinear differential equations. Here, we demonstrate that we can amplify inherent thermal noise in chips to emulate randomness physically, thus alleviating these costs significantly. Concurrently, Molecular Flux in thermodynamic biochemical reactions maps to thermodynamic electronic current in a transistor such that stiff nonlinear biochemical differential equations are emulated exactly in compact, digitally programmable, highly parallel analog “cytomorphic” transistor circuits. For even small-scale systems involving just 80 stochastic reactions, our 0.35-μm BiCMOS chips yield a 311× speedup in the simulation time of Gillespie's stochastic algorithm over COPASI, a fast biochemical-reaction software simulator that is widely used in computational biology; they yield a 15 500× speedup over equivalent MATLAB stochastic simulations. The chip emulation results are consistent with these software simulations over a large range of signal-to-noise ratios. Most importantly, our physical emulation of Poisson chemical dynamics does not involve any inherently sequential processes and updates such that, unlike prior exact simulation approaches, they are parallelizable, asynchronous, and enable even more speedup for larger-size networks.
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Synthetic Biology: A Unifying View and Review Using Analog Circuits
IEEE Transactions on Biomedical Circuits and Systems, 2015Co-Authors: Rahul SarpeshkarAbstract:We review the field of synthetic biology from an analog circuits and analog computation perspective, focusing on circuits that have been built in living cells. This perspective is well suited to pictorially, symbolically, and quantitatively representing the nonlinear, dynamic, and stochastic (noisy) ordinary and partial differential equations that rigorously describe the Molecular circuits of synthetic biology. This perspective enables us to construct a canonical analog circuit schematic that helps unify and review the operation of many fundamental circuits that have been built in synthetic biology at the DNA, RNA, protein, and small-molecule levels over nearly two decades. We review 17 circuits in the literature as particular examples of feedforward and feedback analog circuits that arise from special topological cases of the canonical analog circuit schematic. Digital circuit operation of these circuits represents a special case of saturated analog circuit behavior and is automatically incorporated as well. Many issues that have prevented synthetic biology from scaling are naturally represented in analog circuit schematics. Furthermore, the deep similarity between the Boltzmann thermodynamic equations that describe noisy electronic current flow in subthreshold transistors and noisy Molecular Flux in biochemical reactions has helped map analog circuit motifs in electronics to analog circuit motifs in cells and vice versa via a ‘cytomorphic’ approach. Thus, a body of knowledge in analog electronic circuit design, analysis, simulation, and implementation may also be useful in the robust and efficient design of Molecular circuits in synthetic biology, helping it to scale to more complex circuits in the future.
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Analog synthetic biology
Philosophical Transactions of the Royal Society A, 2014Co-Authors: Rahul SarpeshkarAbstract:We analyse the pros and cons of analog versus digital computation in living cells. Our analysis is based on fundamental laws of noise in gene and protein expression, which set limits on the energy, time, space, Molecular count and part-count resources needed to compute at a given level of precision. We conclude that analog computation is significantly more efficient in its use of resources than deterministic digital computation even at relatively high levels of precision in the cell. Based on this analysis, we conclude that synthetic biology must use analog, collective analog, probabilistic and hybrid analog–digital computational approaches; otherwise, even relatively simple synthetic computations in cells such as addition will exceed energy and Molecular-count budgets. We present schematics for efficiently representing analog DNA–protein computation in cells. Analog electronic flow in subthreshold transistors and analog Molecular Flux in chemical reactions obey Boltzmann exponential laws of thermodynamics and are described by astoundingly similar logarithmic electrochemical potentials. Therefore, cytomorphic circuits can help to map circuit designs between electronic and biochemical domains. We review recent work that uses positive-feedback linearization circuits to architect wide-dynamic-range logarithmic analog computation in Escherichia coli using three transcription factors, nearly two orders of magnitude more efficient in parts than prior digital implementations.
Pablo S Gaete - One of the best experts on this subject based on the ideXlab platform.
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a novel voltage clamp dye uptake assay reveals saturable transport of molecules through calhm1 and connexin channels
The Journal of General Physiology, 2020Co-Authors: Pablo S Gaete, Mauricio A Lillo, William Lopez, Yu Liu, Andrew L Harris, Wenjuan Jiang, Yun Luo, Jorge E ContrerasAbstract:Large-pore channels permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, their mechanisms of Molecular permeation and selectivity remain mostly unexplored. Combining fluorescence microscopy and electrophysiology, we developed a novel technique that allows kinetic analysis of Molecular permeation through connexin and CALHM1 channels in Xenopus oocytes rendered translucent. Using this methodology, we found that (1) Molecular Flux through these channels saturates at low micromolar concentrations, (2) kinetic parameters of Molecular transport are sensitive to modulators of channel gating, (3) Molecular transport and ionic currents can be differentially affected by mutation and gating, and (4) N-terminal regions of these channels control transport kinetics and permselectivity. Our methodology allows analysis of how human disease-causing mutations affect kinetic properties and permselectivity of Molecular signaling and enables the study of Molecular mechanisms, including selectivity and saturability, of Molecular transport in other large-pore channels.
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a novel voltage clamp dye uptake assay reveals saturable transport of molecules through calhm1 and connexin channels
bioRxiv, 2020Co-Authors: Pablo S Gaete, Mauricio A Lillo, William Lopez, Yu Liu, Andrew L Harris, Jorge E ContrerasAbstract:ABSTRACT Channels that are permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, mechanisms of Molecular permeation and selectivity of these channels remain mostly unexplored due to the lack of quantitative methodologies. To address this need, we developed a novel two-electrode voltage clamp (TEVC)/dye uptake assay to examine the kinetics of Molecular permeation of channels formed by human connexins (hCx), and the calcium homeostasis modulator (hCALHM1). We expressed hCx26, hCx30, and hCALHM1 individually in Xenopus laevis oocytes. To quantify the uptake of small Molecular dyes through these channels, we developed a protocol that renders oocytes translucent – thereby amenable to optical detection techniques – without affecting the functional properties of the expressed channels. To control membrane potential and to determine functional channel expression accurately, dye uptake was evaluated in conjunction with TEVC. Using this methodology, we found that: (1) CALHM1 and Cx30 hemichannels display saturable transport of molecules that could be described by Michaelis-Menten kinetics, with apparent KM and Vmax; (2) Kinetic parameters for Molecular transport through CALHM1 are sensitive to voltage and extracellular calcium; (3) Significant transport of molecules occurs through CALHM1 when there are little or no ionic currents through the channels; (4) Cx mutations in the N-terminal region significantly affect kinetics of transport and permselectivity. Our results reveal that Molecular Flux through these channels has a rate-limiting step, that the kinetic parameters of Molecular transport are sensitive to modulators of channel gating and that Molecular transport and ionic currents can be differentially affected. Our methodology allows the analysis of how human mutations causing diseases affect kinetic properties and permselectivity of Molecular signaling and enables the study of Molecular mechanisms, including selectivity and saturability, associated with Molecular transport in large-pore channels.
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A novel voltage clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels
2020Co-Authors: Pablo S Gaete, Mauricio A Lillo, William Lopez, Yu Liu, Andrew L Harris, Jorge E ContrerasAbstract:ABSTRACT Channels that are permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, mechanisms of Molecular permeation and selectivity of these channels remain mostly unexplored due to the lack of quantitative methodologies. To address this need, we developed a novel two-electrode voltage clamp (TEVC)/dye uptake assay to examine the kinetics of Molecular permeation of channels formed by human connexins (hCx), and the calcium homeostasis modulator (hCALHM1). We expressed hCx26, hCx30, and hCALHM1 individually in Xenopus laevis oocytes. To quantify the uptake of small Molecular dyes through these channels, we developed a protocol that renders oocytes translucent – thereby amenable to optical detection techniques – without affecting the functional properties of the expressed channels. To control membrane potential and to determine functional channel expression accurately, dye uptake was evaluated in conjunction with TEVC. Using this methodology, we found that: (1) CALHM1 and Cx30 hemichannels display saturable transport of molecules that could be described by Michaelis-Menten kinetics, with apparent KM and Vmax; (2) Kinetic parameters for Molecular transport through CALHM1 are sensitive to voltage and extracellular calcium; (3) Significant transport of molecules occurs through CALHM1 when there are little or no ionic currents through the channels; (4) Cx mutations in the N-terminal region significantly affect kinetics of transport and permselectivity. Our results reveal that Molecular Flux through these channels has a rate-limiting step, that the kinetic parameters of Molecular transport are sensitive to modulators of channel gating and that Molecular transport and ionic currents can be differentially affected. Our methodology allows the analysis of how human mutations causing diseases affect kinetic properties and permselectivity of Molecular signaling and enables the study of Molecular mechanisms, including selectivity and saturability, associated with Molecular transport in large-pore channels.
Yuzhong Qian - One of the best experts on this subject based on the ideXlab platform.
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simulation of supersonic Molecular beam injection fueling into h mode plasmas on east using bout
Physics of Plasmas, 2020Co-Authors: Yuzhong Qian, Tian Xia, Y Q Huang, Z Sun, G Z Zuo, X C Meng, Mingguang Huang, Bin Cao, Y M WangAbstract:Transport dynamic 2D simulations for supersonic Molecular beam injection (SMBI) fueling into H-mode deuterium plasmas on the Experimental Advanced Superconducting Tokamak (EAST) is first simulated using a seven-field two-fluid model in the BOUT++ framework. The SMB is assumed to be injected into plasma from the midplane at the low field side with a fixed width and constant Molecular Flux. The different densities and injection velocities of SMBI are investigated within the upper single-null geometry of EAST. The simulations indicate that the SMBI has a self-shielding effect on molecules' inward transport into the plasma, and the deposition of SMBI leads to a large increase in plasma density and decrease in plasma temperature. There is a velocity threshold for SMB penetrating the pedestal and depositing at the top of density pedestal. However, the deposition point would be back toward the plasma boundary after SMB arriving at the deepest penetration position. Comparing the different Molecular injection velocities and densities, the outcomes demonstrate that the depth of deposition is closely related to the injection velocity rather than the Molecular density. The simulated results show good agreement with the EAST experiments by comparing the electron density profiles obtained by simulation and experiment, respectively. These results will be helpful for guiding future experiments, as well as the design of the SMBI system.
Y M Wang - One of the best experts on this subject based on the ideXlab platform.
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simulation of supersonic Molecular beam injection fueling into h mode plasmas on east using bout
Physics of Plasmas, 2020Co-Authors: Yuzhong Qian, Tian Xia, Y Q Huang, Z Sun, G Z Zuo, X C Meng, Mingguang Huang, Bin Cao, Y M WangAbstract:Transport dynamic 2D simulations for supersonic Molecular beam injection (SMBI) fueling into H-mode deuterium plasmas on the Experimental Advanced Superconducting Tokamak (EAST) is first simulated using a seven-field two-fluid model in the BOUT++ framework. The SMB is assumed to be injected into plasma from the midplane at the low field side with a fixed width and constant Molecular Flux. The different densities and injection velocities of SMBI are investigated within the upper single-null geometry of EAST. The simulations indicate that the SMBI has a self-shielding effect on molecules' inward transport into the plasma, and the deposition of SMBI leads to a large increase in plasma density and decrease in plasma temperature. There is a velocity threshold for SMB penetrating the pedestal and depositing at the top of density pedestal. However, the deposition point would be back toward the plasma boundary after SMB arriving at the deepest penetration position. Comparing the different Molecular injection velocities and densities, the outcomes demonstrate that the depth of deposition is closely related to the injection velocity rather than the Molecular density. The simulated results show good agreement with the EAST experiments by comparing the electron density profiles obtained by simulation and experiment, respectively. These results will be helpful for guiding future experiments, as well as the design of the SMBI system.