Electric field bridging-effect in electrified microfibrils' scaffolds.

Introduction: The use of biocompatible scaffolds combined with the implantation of neural stem cells, is increasingly being investigated to promote the regeneration of damaged neural tissue, for instance, after a Spinal Cord Injury (SCI). In particular, aligned Polylactic Acid (PLA) microfibrils' scaffolds are capable of supporting cells, promoting their survival and guiding their differentiation in neural lineage to repair the lesion. Despite its biocompatible nature, PLA is an electrically insulating material and thus it could be detrimental for increasingly common scaffolds' electric functionalization, aimed at accelerating the cellular processes. In this context, the European RISEUP project aims to combine high intense microseconds pulses and DC stimulation with neurogenesis, supported by a PLA microfibrils' scaffold. Methods: In this paper a numerical study on the effect of microfibrils' scaffolds on the E-field distribution, in planar interdigitated electrodes, is presented. Realistic microfibrils' 3D CAD models have been built to carry out a numerical dosimetry study, through Comsol Multiphysics software. Results: Under a voltage of 10 V, microfibrils redistribute the E-field values focalizing the field streamlines in the spaces between the fibers, allowing the field to pass and reach maximum values up to 100 kV/m and values comparable with the bare electrodes' device (without fibers). Discussion: Globally the median E-field inside the scaffolded electrodes is the 90% of the nominal field, allowing an adequate cells' exposure.

Effects of Environmental and Electric Perturbations on the pKa of Thioredoxin Cysteine 35: A Computational Study.

Here we present a theoretical-computational study dealing with the evaluation of the pKa of the Cysteine residues in Thioredoxin (TRX) and in its complex with the Thioredoxin-interacting protein (TXNIP). The free energy differences between the anionic and neutral form of the Cysteine 32 and 35 have been evaluated by means of the Perturbed Matrix Method with classical perturbations due to both the environment and an exogenous electric field as provided by Molecular Dynamics (MD) simulations. The evaluation of the free energies allowed us to show that the effect of the perturbing terms is to lower the pKa of Cysteine 32 and Cysteine 35 with respect to the free amino-acid. On the other hand, in the complex TRX-TXNIP, our data show an enhanced stabilization of the neutral reduced form of Cys 35. These results suggest that external electric stimuli higher than 0.02 V/nm can modulate the Cysteine pKa, which can be connected to the tight regulation of the TRX acting as an antioxidant agent.

A rationale for non-linear responses to strong electric fields in molecular dynamics simulations.

Many approaches for calculation of the field-dependent electric properties of water solutions rely on the Onsager and Kirkwood theories of polar dielectrics. Such basic theories implicitly consider the electric field intensity to fulfill the so-called 'weak field conditions', i.e. to produce a linear response in the system. In this work we made use of molecular dynamics simulations to investigate possible non-linear effects induced by high intensity electric fields, specifically continuous wave bursts with nanosecond duration, comparing them with the ones predicted by the theory. We found that field intensities above 0.15 V nm-1 produce remarkable nonlinear responses in the whole 100 MHz-100 GHz frequency window considered, with the onset of higher order polarization signals, which are the clear fingerprint of harmonic distorsions. That non-linear response turned out to depend on the considered frequency. We finally show that MD outcomes are consistent with a modelization based on an extended formulation of the Langevin function including a frequency-dependent parameter.

Electric-driven membrane poration: A rationale for water role in the kinetics of pore formation.

Electroporation is a well-established technique used to stimulate cells, enhancing membrane permeability by inducing reversible membrane pores. In the absence of experimental observation of the dynamics of pore creation, molecular dynamics studies provide the molecular-level evidence that the electric field promotes pore formation. Although single steps in the pore formation process are well assessed, a kinetic model representing the mathematical description of the electroporation process, is lacking. In the present work we studied the basis of the pore formation process, providing a rationale for the definition of a first-order kinetic scheme. Here, authors propose a three-state kinetic model for the process based on the assessed mechanism of water defects intruding at the water/lipid interface, when applying electric field intensities at the edge of the linear regime. The methodology proposed is based on the use of two robust biophysical quantities analyzed for the water molecules intruding at the water/lipid interface: (i) number of hydrogen bonds; (ii) number of contacts. The final model, sustained by a robust statistical sampling, provides kinetic constants for the transitions from the intact bilayer state to the hydrophobic pore state.

Nanosecond pulsed electric signals can affect electrostatic environment of proteins below the threshold of conformational effects: The case study of SOD1 with a molecular simulation study.

Electric fields can be a powerful tool to interact with enzymes or proteins, with an intriguing perspective to allow protein manipulation. Recently, researchers have focused the interest on intracellular enzyme modifications triggered by the application of nanosecond pulsed electric fields. These findings were also supported by theoretical predictions from molecular dynamics simulations focussing on significant variations in protein secondary structures. In this work, a theoretical study utilizing molecular dynamics simulations is proposed to explore effects of electric fields of high intensity and very short nanosecond duration applied to the superoxide dismutase (Cu/Zn-SOD or SOD-1), an important enzyme involved in the cellular antioxidant defence mechanism. The effects of 100-nanosecond pulsed electric fields, with intensities ranging from 108 to 7x108 V/m, on a single SOD1 enzyme are presented. We demonstrated that the lowest intensity of 108 V/m, although not inducing structural changes, can produce electrostatic modifications on the reaction centre of the enzyme, as apparent from the dipolar response and the electric field distribution of the protein active site. Electric pulses above 5x108 V/m produced a fast transition between the folded and a partially denatured state, as inferred by the secondary structures analysis. Finally, for the highest field intensity used (7x108 V/m), a not reversible transition toward an unfolded state was observed.

Tubulin response to intense nanosecond-scale electric field in molecular dynamics simulation.

Intense pulsed electric fields are known to act at the cell membrane level and are already being exploited in biomedical and biotechnological applications. However, it is not clear if electric pulses within biomedically-attainable parameters could directly influence intra-cellular components such as cytoskeletal proteins. If so, a molecular mechanism of action could be uncovered for therapeutic applications of such electric fields. To help clarify this question, we first identified that a tubulin heterodimer is a natural biological target for intense electric fields due to its exceptional electric properties and crucial roles played in cell division. Using molecular dynamics simulations, we then demonstrated that an intense - yet experimentally attainable - electric field of nanosecond duration can affect the bß-tubulin's C-terminus conformations and also influence local electrostatic properties at the GTPase as well as the binding sites of major tubulin drugs site. Our results suggest that intense nanosecond electric pulses could be used for physical modulation of microtubule dynamics. Since a nanosecond pulsed electric field can penetrate the tissues and cellular membranes due to its broadband spectrum, our results are also potentially significant for the development of new therapeutic protocols.

Controlling ionic conductivity through transprotein electropores in human aquaporin 4: a non-equilibrium molecular-dynamics study.

Electroporation is a matter of intensive ongoing research interest, and a much-neglected topic in trans-membrane proteins, particularly in view of such promising potential applications in medicine and biotechnology. In particular, selected such novel and exciting applications are predicated on controlling ionic conductivity through electro-pores. Here, we scrutinise the mechanisms of ions' electric conductivity, by means of structural rearrangements, through quasi-stable electro-pores through human-AQP4 as a well-representative prototype of trans-membrane ionic conduction, achieving exquisite control over ionic permeability manipulated by the application of intense static electric fields.

Transprotein-Electropore Characterization: A Molecular Dynamics Investigation on Human AQP4.

Electroporation characterization is a topic of intensive interest probed by extensive ongoing research efforts. Usually, these studies are carried out on lipid-bilayer electroporation. Surprisingly, the possibility of water-channel electropore formation across transmembrane proteins themselves, particularly in view of such a promising application, has not yet been elucidated. The present work examines the geometrical and kinetic aspects of electropores and their stability in such a protein milieux (as opposed through the phospholipid membranes) in depth, by means of scrutiny of such a process in human-AQP4 as a well-representative prototype. The residues forming the electropore's walls, organized in loops, reveal the formation mechanism by their dipole alignment and translational response in response to applied axial electric fields in nonequilibrium molecular dynamics simulation. The magnitude of sustaining electric fields (keeping a stable electropore open) were determined. This suggests that transmembrane proteins could play a central role in electroporation applications, e.g., in medicine and biotechnology.

A wide-band bio-chip for real-time optical detection of bioelectromagnetic interactions with cells.

The analytical and numerical design, implementation, and experimental validation of a new grounded closed coplanar waveguide for wide-band electromagnetic exposures of cells and their optical detection in real-time is reported. The realized device fulfills high-quality requirements for novel bioelectromagnetic experiments, involving elevated temporal and spatial resolutions. Excellent performances in terms of matching bandwidth (less than -10 dB up to at least 3 GHz), emission (below 1 × 10-6 W/m2) and efficiency (around 1) have been obtained as revealed by both numerical simulations and experimental measurements. A low spatial electric field inhomogeneity (coefficient of variation of around 10 %) has been achieved within the cell solutions filling the polydimethylsiloxane reservoir of the conceived device. This original bio-chip based on the grounded closed coplanar waveguide concept opens new possibilities for the development of controlled experiments combining electromagnetic exposures and sophisticated imaging using optical spectroscopic techniques.

Human aquaporin 4 gating dynamics under axially oriented electric-field impulses: A non-equilibrium molecular-dynamics study.

Human aquaporin 4 has been studied using non-equilibrium molecular dynamics simulations in the absence and presence of pulses of external electric fields. The pulses were 100 ns in duration and 0.005-0.015 V/Å in intensity acting along the pores' axes. Water diffusivity and the dipolar response of various residues of interest within the pores have been studied. Results show relatively little change in levels of water permeability per se within aquaporin channels during axially oriented field impulses, although care must be taken with regard to statistical certainty. However, the spatial variation of water permeability vis-à-vis electric-field intensity within the milieu of the channels, as revealed by heterogeneity in diffusivity-map gradients, indicates the possibility of somewhat enhanced diffusivity, owing to several residues being affected substantially by external fields, particularly for HIS 201 and 95 and ILE 93. This has the effect of increasing slightly intra-pore water diffusivity in the "pore-mouths" locale, albeit rendering it more spatially uniform overall vis-à-vis zero-field conditions (via manipulation of the selectivity filter).

Human Aquaporin 4 Gating Dynamics under Perpendicularly-Oriented Electric-Field Impulses: A Molecular Dynamics Study.

Human aquaporin 4 has been studied using molecular dynamics (MD) simulations in the absence and presence of pulses of external static electric fields. The pulses were 10 ns in duration and 0.012-0.065 V/Å in intensity acting along both directions perpendicular to the pores. Water permeability and the dipolar response of all residues of interest (including the selectivity filter) within the pores have been studied. Results showed decreased levels of water osmotic permeability within aquaporin channels during orthogonally-oriented field impulses, although care must be taken with regard to statistical certainty. This can be explained observing enhanced "dipolar flipping" of certain key residues, especially serine 211, histidine 201, arginine 216, histidine 95 and cysteine 178. These residues are placed at the extracellular end of the pore (serine 211, histidine 201, and arginine 216) and at the cytoplasm end (histidine 95 and cysteine 178), with the key role in gating mechanism, hence influencing water permeability.

In silico characterization of protein partial molecular volumes and hydration shells.

In this paper we present a computational approach, based on NVT molecular dynamics trajectories, that allows the direct evaluation of the protein partial molecular volume. The results obtained for five different globular proteins demonstrate the accuracy of this computational procedure in reproducing protein partial molecular volumes, providing quantitative characterization of the hydration shell in terms of the protein excluded volume, hydration shell ellipsoidal volume and related solvent density. Remarkably, our data indicate for the hydration shell a ≈10% solvent density increase with respect to the liquid water bulk density, in excellent agreement with the available experimental data.

Water response to intense electric fields: A molecular dynamics study.

This paper investigated polarization properties of water molecules in close proximity to an ionic charge in the presence of external electric fields by using an approach based on simulations at the atomic level. We chose sodium and chloride ions in water as examples of dilute ionic solutions and used molecular dynamics simulations to systematically investigate the influence of an external static electric field on structural, dipolar, and polarization properties of water near charged ions. Results showed that a threshold electric field higher than 10(8) V/m is needed to affect water polarization and increase mean dipole moment of water molecules close to the ion. A similar threshold holds for water permittivity profiles, although a field 10× higher is needed to ensure that water permittivity is almost constant independently of the position close to the ion. Electric fields of such intensities can greatly enhance polarizability of water in hydration shells around ions.

Communication: influence of nanosecond-pulsed electric fields on water and its subsequent relaxation: dipolar effects and debunking memory.

Water has many intriguing and anomalous physical properties that have puzzled and titillated the scientific community for centuries, perhaps none more so than the proposition that water may retain some (permanent) "memory" of conditions (e.g., dilution) or electric fields to which it has been subject. Here, we have performed non-equilibrium molecular dynamics simulations of liquid water in external electric-field nanosecond pulses, at 260-310 K, and gauged significant non-thermal field effects in terms of dipolar response. Response of both system- and individual-dipoles has been investigated, and autocorrelation functions of both show more significant effects in stronger fields, with more sluggish relaxation. Crucially, we show that once the field is removed, the dipoles relax, exhibiting no memory or permanent dipolar alignment. We also quantify the time scales for system dynamical-dipolar properties to revert to zero-field equilibrium behaviour.

Human aquaporin 4 gating dynamics under and after nanosecond-scale static and alternating electric-field impulses: a molecular dynamics study of field effects and relaxation.

Water self-diffusion and the dipolar response of the selectivity filter within human aquaporin 4 have been studied using molecular dynamics (MD) simulations in the absence and presence of pulses of external static and alternating electric fields. The pulses were approximately 50 and 100 ns in duration and 0.0065 V/Å in (r.m.s.) intensity and were either static or else 2.45 or 100 GHz in frequency and applied both along and perpendicular to the channels. In addition, the relaxation of the aquaporin, water self-diffusion and gating dynamics following cessation of the impulses was studied. In previous work it was determined that switches in the dihedral angle of the selectivity filter led to boosting of water permeation events within the channels, in the presence of identical external static and alternating electric fields, although applied continuously. Here the application of field impulses (and subsequently, upon removal) has shown that it is the dipolar orientation of the histidine-201 residue in the selectivity filter which governs the dihedral angle, and hence influences water self-diffusion; this constitutes an appropriate order parameter. The dipolar response of this residue to the applied field leads to the adoption of four distinct states, which we modelled as time-homogeneous Markov jump processes, and may be distinguished in the potential of mean force (PMF) as a function of the dipolar orientation of histidine-201. The observations of enhanced "dipolar flipping" of H201 serve to explain increased levels of water self-diffusion within aquaporin channels during, and immediately following, field impulses, although the level of statistical certainty here is lower. Given the appreciable size of the energy barriers evident in PMFs computed directly from deterministic MD (whether in the absence or presence of external fields), metadynamics calculations were undertaken to explore the free-energy landscape of histidine-201 orientation with greater accuracy and precision. These indicate that electric fields do alter the free-energy profile of the H201 side-chain orientation, wherein a perturbation of the symmetric bimodal state evident in the zero-field case is observed. These effects are dependent on the field intensities.

Effect of high exogenous electric pulses on protein conformation: myoglobin as a case study.

Protein folding and unfolding under the effect of exogenous perturbations remains a topic of great interest, further enhanced by recent technological developments in the field of signal generation that allow the use of intense ultrashort electric pulses to directly interact at microscopic level with biological matter. In this paper, we show results from molecular dynamics (MD) simulations of a single myoglobin molecule in water exposed to pulsed and static electric fields, ranging from 10(8) to 10(9) V/m, compared to data with unexposed conditions. We have found that the highest intensity (10(9) V/m) produced a fast transition (occurring within a few hundreds of picoseconds) between folded and unfolded states, as inferred by secondary structures and geometrical analysis. Fields of 10(8) V/m, on the contrary, produced no significant denaturation, although a relevant effect on the protein dipolar behavior was detected.

Modeling of chemical reactions in micelle: water-mediated keto-enol interconversion as a case study.

The effect of a zwitterionic micelle environment on the efficiency of the keto-enol interconversion of 2-phenylacetylthiophene has been investigated by means of a joint application of experimental and theoretical/computational approaches. Results have revealed a reduction of the reaction rate constant if compared with bulk water essentially because of the different solvation conditions experienced by the reactant species, including water molecules, in the micelle environment. The slight inhibiting effect due to the application of a static electric field has also been theoretically investigated and presented.