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Darel J Hunting - One of the best experts on this subject based on the ideXlab platform.
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loss of cellular transformation efficiency induced by dna irradiation with low energy 10 ev electrons
Journal of Physical Chemistry B, 2014Co-Authors: Saloua Kouass Sahbani, L Sanche, Pierre Cloutier, A D Bass, Darel J HuntingAbstract:Low energy electrons (LEEs) of energies less than 20 eV are generated in large quantities by ionizing radiation in Biological Matter. While LEEs are known to induce single (SSBs) and double strand breaks (DSBs) in DNA, their ability to inactivate cells by inducing nonreparable lethal damage has not yet been demonstrated. Here we observe the effect of LEEs on the functionality of DNA, by measuring the efficiency of transforming Escherichia coli with a [pGEM-3Zf (−)] plasmid irradiated with 10 eV electrons. Highly ordered DNA films were prepared on pyrolitic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap2+). The uniformity of these films permits the inactivation of approximately 50% of the plasmids compared to <10% using previous methods, which is sufficient for the subsequent determination of their functionality. Upon LEE irradiation, the fraction of functional plasmids decreased exponentially with increasing electron fluence, while LEE-induced isolated base damage, frank DSB, and n...
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loss of cellular transformation efficiency induced by dna irradiation with low energy 10 ev electrons
The Journal of Physical Chemistry, 2014Co-Authors: Saloua Kouass Sahbani, L Sanche, Pierre Cloutier, A D Bass, Darel J HuntingAbstract:Low energy electrons (LEEs) of energies less than 20 eV are generated in large quantities by ionizing radiation in Biological Matter. While LEEs are known to induce single (SSBs) and double strand breaks (DSBs) in DNA, their ability to inactivate cells by inducing nonreparable lethal damage has not yet been demonstrated. Here we observe the effect of LEEs on the functionality of DNA, by measuring the efficiency of transforming Escherichia coli with a [pGEM-3Zf (−)] plasmid irradiated with 10 eV electrons. Highly ordered DNA films were prepared on pyrolitic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap²⁺). The uniformity of these films permits the inactivation of approximately 50% of the plasmids compared to <10% using previous methods, which is sufficient for the subsequent determination of their functionality. Upon LEE irradiation, the fraction of functional plasmids decreased exponentially with increasing electron fluence, while LEE-induced isolated base damage, frank DSB, and non DSB-cluster damage increased linearly with fluence. While DSBs can be toxic, their levels were too low to explain the loss of plasmid functionality observed upon LEE irradiation. Similarly, non-DSB cluster damage, revealed by transforming cluster damage into DSBs by digestion with repair enzymes, also occurred relatively infrequently. The exact nature of the lethal damage remains unknown, but it is probably a form of compact cluster damage in which the lesions are too close to be revealed by purified repair enzymes. In addition, this damage is either not repaired or is misrepaired by E. coli, since it results in plasmid inactivation, when they contain an average of three lesions. Comparison with previous results from a similar experiment performed with γ-irradiated plasmids indicates that the type of clustered DNA lesions, created directly on cellular DNA by LEEs, may be more difficult to repair than those produced by other species from radiolysis.
Boris Rubinsky - One of the best experts on this subject based on the ideXlab platform.
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mass transfer into Biological Matter using isochoric freezing
Cryobiology, 2021Co-Authors: Boris RubinskyAbstract:This paper is a theoretical study of a protocol for transport of high concentrations of cryoprotectants into Biological Matter, using isochoric freezing. Unlike isobaric freezing, where the entire system freezes at temperatures lower than the freezing temperature, in isochoric freezing a substantial portion of the system remains unfrozen at temperatures below freezing. In isochoric freezing cryopreservation, the system is designed in such a way that the Biological Matter remains unfrozen and surrounded by an unfrozen solution. The protocol in this study involves the freezing of an isochoric systems along the "liquidus line" at which water and ice are in thermodynamic equilibrium. Rejection of solutes by ice increases the concentration of the solutes in the unfrozen solution surrounding the unfrozen Biological Matter, leading, thereby, to transport of increasingly higher concentrations of cryoprotectants into the Biological Matter, as the temperature of the system is lowered and the toxicity of the cryoprotectants is reduced.
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phase change interface stability during isochoric solidification of an aqueous solution
Applied Physics Letters, 2020Co-Authors: Matthew J Powellpalm, Gideon Ukpai, Yuanheng Zhao, Cristina Bilbaosainz, Liubiao Chen, Junjie Wang, Boris RubinskyAbstract:The stability of solid–liquid interfaces during solidification is a physical phenomenon of fundamental interest with a wide range of practical applications, including the freezing of Biological Matter for medical and agricultural purposes. Much of the classical research in this field treats solidification in isobaric (constant-pressure) systems in which the phase transition typically occurs under constant atmospheric pressure. Recent research has found, however, that freezing in isochoric (constant-volume) systems in which the solidifying material is confined within a high-strength constant-volume chamber held at subfreezing temperatures gives rise to many atypical physical phenomena, and understanding of the solid–liquid interface behavior under isochoric conditions is currently lacking. In this work, we study the stability and propagation of the solid–liquid interface during isochoric freezing of aqueous solutions. Using a mathematical model of heat and mass transfer during solidification coupled with multiple criteria for predicting the emergence of interfacial instabilities based on temperature and concentration gradients in the phase transition region, we find that isochoric freezing significantly stabilizes the solid–liquid interface relative to isobaric freezing, suggesting the potential for extended growth of planar, non-dendritic interfaces.
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a three dimensional model for analysis and control of phase change phenomena during 3d printing of Biological tissue
Bioprinting, 2020Co-Authors: Gideon Ukpai, Boris RubinskyAbstract:Abstract Three-dimensional (3D) bioprinting is one of the fastest advancing and most promising techniques for tissue engineering. However, despite numerous developments in 3D bioprinting, issues of size scalability remain. Two primary factors limiting the scale of printable objects are the structural properties of the bioinks and the unsustainable time it takes to bioprint large structures. Freezing has been proposed as a possible solution to both, improving the mechanical properties of the soft aqueous hydrogel bioinks and reducing the adverse effects of cell metabolism with the lowered temperature. In addition, it eases the longer-term cryopreservation of the larger tissue constructs as they can be frozen element by element at faster (more preferable) and uniform cooling rates during printing rather than whole after 3D bioprinting. However, for freezing to be a feasible 3D bioprinting technique, there is a need to better understand the effect of phase change during printing on Biological Matter. This can be done by rigorous experimentation or alternatively, using mathematical tools and models. Though, most techniques of 3D printing are inherently thermal processes, limited thermal analysis has been done on extrusion-based (fused deposition modeling) printing processes to characterize thermal effects. This paper introduces a model for analysis of the thermal phase change process during the 3D bioprinting of Biological Matter at low temperature. The model was developed and experimentally validated for an extrusion-based printing process of aqueous Biological material. It was used to investigate the conditions under which a constant freezing rate could be achieved during multilayer printing and the size limitations in conduction dependent freezing from a cold surface during printing. Overall, this model could be a useful tool for the design and control of the 3D printing protocols.
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thermodynamic theory and experimental validation of a multiphase isochoric freezing process
Journal of Biomechanical Engineering-transactions of The Asme, 2019Co-Authors: Matthew J Powellpalm, Justin Aruda, Boris RubinskyAbstract:Freezing of the aqueous solutions that comprise Biological materials, such as isotonic physiological saline, results in the formation of ice crystals and the generation of a hypertonic solution, both of which prove deleterious to Biological Matter. The field of modern cryopreservation, or preservation of Biological Matter at sub-freezing temperatures, emerged from the 1948 discovery that certain chemical additives such as glycerol, known as cryoprotectants, can protect cells from freeze-related damage by depressing the freezing point of water in solution. This gave rise to a slew of important medical applications, from the preservation of sperm and blood cells to the recent preservation of an entire liver, and current cryopreservation protocols thus rely heavily on the use of additive cryoprotectants. However, high concentrations of cryoprotectants themselves prove toxic to cells, and thus there is an ongoing effort to minimize cryoprotectant usage whilst maintaining protection from ice-related damage. Herein we conceive from first principles a new, purely thermodynamic method to eliminate ice formation and hypertonicity during the freezing of a physiological solution: multiphase isochoric freezing. We develop a comprehensive thermodynamic model to predict the equilibrium behaviors of multiphase isochoric systems of arbitrary composition and validate these concepts experimentally in a simple device with no moving parts, providing a baseline from which to design tailored cryopreservation protocols using the multiphase isochoric technique.
Mark C Leake - One of the best experts on this subject based on the ideXlab platform.
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single molecule techniques in biophysics a review of the progress in methods and applications
Reports on Progress in Physics, 2018Co-Authors: Helen Miller, Zhaokun Zhou, Jack W Shepherd, Adam J M Wollman, Mark C LeakeAbstract:Single-molecule biophysics has transformed our understanding of biology, but also of the physics of life. More exotic than simple soft Matter, bioMatter lives far from thermal equilibrium, covering multiple lengths from the nanoscale of single molecules to up to several orders of magnitude higher in cells, tissues and organisms. Biomolecules are often characterized by underlying instability: multiple metastable free energy states exist, separated by levels of just a few multiples of the thermal energy scale k B T, where k B is the Boltzmann constant and T absolute temperature, implying complex inter-conversion kinetics in the relatively hot, wet environment of active Biological Matter. A key benefit of single-molecule biophysics techniques is their ability to probe heterogeneity of free energy states across a molecular population, too challenging in general for conventional ensemble average approaches. Parallel developments in experimental and computational techniques have catalysed the birth of multiplexed, correlative techniques to tackle previously intractable Biological questions. Experimentally, progress has been driven by improvements in sensitivity and speed of detectors, and the stability and efficiency of light sources, probes and microfluidics. We discuss the motivation and requirements for these recent experiments, including the underpinning mathematics. These methods are broadly divided into tools which detect molecules and those which manipulate them. For the former we discuss the progress of super-resolution microscopy, transformative for addressing many longstanding questions in the life sciences, and for the latter we include progress in 'force spectroscopy' techniques that mechanically perturb molecules. We also consider in silico progress of single-molecule computational physics, and how simulation and experimentation may be drawn together to give a more complete understanding. Increasingly, combinatorial techniques are now used, including correlative atomic force microscopy and fluorescence imaging, to probe questions closer to native physiological behaviour. We identify the trade-offs, limitations and applications of these techniques, and discuss exciting new directions.
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single molecule techniques in biophysics a review of the progress in methods and applications
arXiv: Biological Physics, 2017Co-Authors: Helen Miller, Zhaokun Zhou, Jack W Shepherd, Adam J M Wollman, Mark C LeakeAbstract:Single-molecule biophysics has transformed our understanding of the fundamental molecular processes involved in living Biological systems, but also of the fascinating physics of life. Far more exotic than a collection of exemplars of soft Matter behaviour, active Biological Matter lives far from thermal equilibrium, and typically covers multiple length scales from the nanometre level of single molecules up several orders of magnitude to longer length scales in emergent structures of cells, tissues and organisms. Biological molecules are often characterized by an underlying instability, in that multiple metastable free energy states exist which are separated by energy levels of typically just a few multiples of the thermal energy scale of kBT, where kB is the Boltzmann constant and T the absolute temperature, implying complex, dynamic inter-conversion kinetics across this bumpy free energy landscape in the relatively hot, wet environment of real, living Biological Matter. The key utility of single-molecule biophysics lies in its ability to probe the underlying heterogeneity of free energy states across a population of molecules, which in general is too challenging for conventional ensemble level approaches which measure mean average properties. Parallel developments in both experimental and theoretical techniques have been key to the latest insights and are enabling the development of highly-multiplexed, correlative techniques to tackle previously intractable Biological problems. Experimentally, technological developments in the sensitivity and speed of biomolecular detectors, the stability and efficiency of light sources, probes and microfluidics, have enabled and driven the study of heterogeneous behaviours both in vitro and in vivo that were previously undetectable by ensemble methods...
Saloua Kouass Sahbani - One of the best experts on this subject based on the ideXlab platform.
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loss of cellular transformation efficiency induced by dna irradiation with low energy 10 ev electrons
Journal of Physical Chemistry B, 2014Co-Authors: Saloua Kouass Sahbani, L Sanche, Pierre Cloutier, A D Bass, Darel J HuntingAbstract:Low energy electrons (LEEs) of energies less than 20 eV are generated in large quantities by ionizing radiation in Biological Matter. While LEEs are known to induce single (SSBs) and double strand breaks (DSBs) in DNA, their ability to inactivate cells by inducing nonreparable lethal damage has not yet been demonstrated. Here we observe the effect of LEEs on the functionality of DNA, by measuring the efficiency of transforming Escherichia coli with a [pGEM-3Zf (−)] plasmid irradiated with 10 eV electrons. Highly ordered DNA films were prepared on pyrolitic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap2+). The uniformity of these films permits the inactivation of approximately 50% of the plasmids compared to <10% using previous methods, which is sufficient for the subsequent determination of their functionality. Upon LEE irradiation, the fraction of functional plasmids decreased exponentially with increasing electron fluence, while LEE-induced isolated base damage, frank DSB, and n...
-
loss of cellular transformation efficiency induced by dna irradiation with low energy 10 ev electrons
The Journal of Physical Chemistry, 2014Co-Authors: Saloua Kouass Sahbani, L Sanche, Pierre Cloutier, A D Bass, Darel J HuntingAbstract:Low energy electrons (LEEs) of energies less than 20 eV are generated in large quantities by ionizing radiation in Biological Matter. While LEEs are known to induce single (SSBs) and double strand breaks (DSBs) in DNA, their ability to inactivate cells by inducing nonreparable lethal damage has not yet been demonstrated. Here we observe the effect of LEEs on the functionality of DNA, by measuring the efficiency of transforming Escherichia coli with a [pGEM-3Zf (−)] plasmid irradiated with 10 eV electrons. Highly ordered DNA films were prepared on pyrolitic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap²⁺). The uniformity of these films permits the inactivation of approximately 50% of the plasmids compared to <10% using previous methods, which is sufficient for the subsequent determination of their functionality. Upon LEE irradiation, the fraction of functional plasmids decreased exponentially with increasing electron fluence, while LEE-induced isolated base damage, frank DSB, and non DSB-cluster damage increased linearly with fluence. While DSBs can be toxic, their levels were too low to explain the loss of plasmid functionality observed upon LEE irradiation. Similarly, non-DSB cluster damage, revealed by transforming cluster damage into DSBs by digestion with repair enzymes, also occurred relatively infrequently. The exact nature of the lethal damage remains unknown, but it is probably a form of compact cluster damage in which the lesions are too close to be revealed by purified repair enzymes. In addition, this damage is either not repaired or is misrepaired by E. coli, since it results in plasmid inactivation, when they contain an average of three lesions. Comparison with previous results from a similar experiment performed with γ-irradiated plasmids indicates that the type of clustered DNA lesions, created directly on cellular DNA by LEEs, may be more difficult to repair than those produced by other species from radiolysis.
Thomas C T Michaels - One of the best experts on this subject based on the ideXlab platform.
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reaction rate theory for supramolecular kinetics application to protein aggregation
Molecular Physics, 2018Co-Authors: Thomas C T Michaels, Samo Curk, Peter G Bolhuis, Lucie X Liu, Anđela Saric, Tuomas P. J. KnowlesAbstract:Probing reaction mechanisms of supramolecular processes in soft and Biological Matter, such as protein aggregation, is inherently challenging. This is because these processes involve multiple molec...
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reaction rate theory for supramolecular kinetics application to protein aggregation
Molecular Physics, 2018Co-Authors: Thomas C T Michaels, Samo Curk, Anđela Šarić, Peter G Bolhuis, Tuomas P. J. KnowlesAbstract:Probing reaction mechanisms of supramolecular processes in soft and Biological Matter, such as protein aggregation, is inherently challenging. This is because these processes involve multiple molecular mechanisms that are associated with the rearrangement of large numbers of weak bonds, resulting in complex free energy landscapes with many kinetic barriers. Reaction rate measurements at different temperatures can offer unprecedented insights into the underlying molecular mechanisms. However, to be able to interpret such measurements, a key challenge is to establish which properties of the complex free energy landscapes are probed by the reaction rate. Here, we present a reaction rate theory for supramolecular kinetics based on Kramers theory of diffusive reactions over multiple kinetic barriers. We find that reaction rates for protein aggregation are of the Arrhenius-Eyring type and that the associated activation energies probe only one relevant barrier along the respective free energy landscapes. We apply this advancement to interpret, in experiments and in coarse-grained computer simulations, reaction rates of amyloid aggregation in terms of molecular mechanisms and associated thermodynamic signatures. These results suggest a practical extension of the concept of rate-determining steps for complex supramolecular processes and establish a general platform for probing the underlying energy landscape using kinetic measurements.
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reaction rate theory for supramolecular kinetics application to protein aggregation
arXiv: Biological Physics, 2018Co-Authors: Thomas C T Michaels, Samo Curk, Anđela Šarić, Peter G Bolhuis, Tuomas P. J. KnowlesAbstract:Probing the reaction mechanisms of supramolecular processes in soft- and Biological Matter, such as protein aggregation, is inherently challenging. These processes emerge from the simultaneous action of multiple molecular mechanisms, each of which is associated with the rearrangement of a large number of weak bonds, resulting in a complex free energy landscape with many kinetic barriers. Reaction rate measurements of supramolecular processes at different temperatures can offer unprecedented insights into the underlying molecular mechanisms and their thermodynamic properties. However, to be able to interpret such measurements in terms of the underlying microscopic mechanisms, a key challenge is to establish which properties of the complex free energy landscapes are probed by the reaction rate. Here, we present a reaction rate theory for supramolecular kinetics based on Kramers rate theory for diffusive reactions over multiple kinetic barriers, and apply the results to protein aggregation. Using this framework and Monte Carlo simulations, we show that reaction rates for protein aggregation are of the Arrhenius-Eyring type and that the associated activation energies probe only one relevant barrier along the respective free energy landscapes. We apply this advancement to interpret, both in experiments and in coarse-grained computer simulations, reaction rate measurements of amyloid aggregation kinetics in terms of the underlying molecular mechanisms and associated thermodynamic signatures. Our results establish a general platform for probing the mechanisms and energetics of supramolecular phenomena in soft- and Biological Matter using the framework of chemical kinetics.