Dynamic Personality of Proteins and Effect of the Molecular Environment.

Protein dynamics display distinct traits that are linked to their specific biological function. However, the interplay between intrinsic dynamics and the molecular environment on protein stability remains poorly understood. In this study, we investigate, by incoherent neutron scattering, the subnanosecond time scale dynamics of three model proteins: the mesophilic lysozyme, the thermophilic thermolysin, and the intrinsically disordered ß-casein. Moreover, we address the influence of water, glycerol, and glucose, which create progressively more viscous matrices around the protein surface. By comparing the protein thermal fluctuations, we find that the internal dynamics of thermolysin are less affected by the environment compared to lysozyme and ß-casein. We ascribe this behavior to the protein dynamic personality, i.e., to the stiffer dynamics of the thermophilic protein that contrasts the influence of the environment. Remarkably, lysozyme and thermolysin in all molecular environments reach a critical common flexibility when approaching the calorimetric melting temperature.

Piezoelectric Yield of Single Electrospun Poly(acrylonitrile) Ultrafine Fibers Studied by Piezoresponse Force Microscopy and Numerical Simulations.

Quantitative converse piezoelectric coefficient (d33) mapping of polymer ultrafine fibers of poly(acrylonitrile) (PAN), as well as of poly(vinylidene fluoride) (PVDF) as a reference material, obtained by rotating electrospinning, was carried out by piezoresponse force microscopy in the constant-excitation frequency-modulation mode (CE-FM-PFM). PFM mapping of single fibers reveals their piezoelectric activity and provides information on its distribution along the fiber length. Uniform behavior is typically observed on a length scale of a few micrometers. In some cases, variations with sinusoidal dependence along the fiber are reported, compatibly with a possible twisting around the fiber axis. The observed features of the piezoelectric yield have motivated numerical simulations of the surface displacement in a piezoelectric ultrafine fiber concerned by the electric field generated by biasing of the PFM probe. Uniform alignment of the piezoelectric axis along the fiber would comply with the uniform but strongly variable values observed, and sinusoidal variations were occasionally found on the fibers laying on the conductive substrate. Furthermore, in the latter case, numerical simulations show that the piezoelectric tensor's shear terms should be carefully considered in estimations since they may provide a remarkably different contribution to the overall deformation profile.

Electron FLASH radiotherapy in vivo studies. A systematic review.

FLASH-radiotherapy delivers a radiation beam a thousand times faster compared to conventional radiotherapy, reducing radiation damage in healthy tissues with an equivalent tumor response. Although not completely understood, this radiobiological phenomenon has been proved in several animal models with a spectrum of all kinds of particles currently used in contemporary radiotherapy, especially electrons. However, all the research teams have performed FLASH preclinical studies using industrial linear accelerator or LINAC commonly employed in conventional radiotherapy and modified for the delivery of ultra-high-dose-rate (UHDRs). Unfortunately, the delivering and measuring of UHDR beams have been proved not to be completely reliable with such devices. Concerns arise regarding the accuracy of beam monitoring and dosimetry systems. Additionally, this LINAC totally lacks an integrated and dedicated Treatment Planning System (TPS) able to evaluate the internal dose distribution in the case of in vivo experiments. Finally, these devices cannot modify dose-time parameters of the beam relevant to the flash effect, such as average dose rate; dose per pulse; and instantaneous dose rate. This aspect also precludes the exploration of the quantitative relationship with biological phenomena. The dependence on these parameters need to be further investigated. A promising advancement is represented by a new generation of electron LINAC that has successfully overcome some of these technological challenges. In this review, we aim to provide a comprehensive summary of the existing literature on in vivo experiments using electron FLASH radiotherapy and explore the promising clinical perspectives associated with this technology.

3D Printed Piezoelectric BaTiO3/Polyhydroxybutyrate Nanocomposite Scaffolds for Bone Tissue Engineering.

Bone defects are a significant health problem worldwide. Novel treatment approaches in the tissue engineering field rely on the use of biomaterial scaffolds to stimulate and guide the regeneration of damaged tissue that cannot repair or regrow spontaneously. This work aimed at developing and characterizing new piezoelectric scaffolds to provide electric bio-signals naturally present in bone and vascular tissues. Mixing and extrusion were used to obtain nanocomposites made of polyhydroxybutyrate (PHB) as a matrix and barium titanate (BaTiO3) nanoparticles as a filler, at BaTiO3/PHB compositions of 5/95, 10/90, 15/85 and 20/80 (w/w%). The morphological, thermal, mechanical and piezoelectric properties of the nanocomposites were studied. Scanning electron microscopy analysis showed good nanoparticle dispersion within the polymer matrix. Considerable increases in the Young's modulus, compressive strength and the piezoelectric coefficient d31 were observed with increasing BaTiO3 content, with d31 = 37 pm/V in 20/80 (w/w%) BaTiO3/PHB. 3D printing was used to produce porous cubic-shaped scaffolds using a 90° lay-down pattern, with pore size ranging in 0.60-0.77 mm and good mechanical stability. Biodegradation tests conducted for 8 weeks in saline solution at 37 °C showed low mass loss (∼4%) for 3D printed scaffolds. The results obtained in terms of piezoelectric, mechanical and chemical properties of the nanocomposite provide a new promising strategy for vascularized bone tissue engineering.

Ultrathin Ambipolar Polyelectrolyte Capacitors Prepared via Layer-by-Layer Assembling.

Miniaturized solid state capacitors leveraging migration of unipolar ions in a single polyelectrolyte layer sandwiched between metal electrodes, namely, polyelectrolyte capacitors (PECs), have been recently reported with areal capacitance up to 100-200 nF mm-2 . Nonetheless, application of PECs in consumer and industrial electronics has been hindered so far by their small operational frequency range, up to a few kHz, due to the resistive behavior (phase angle >-45°) of PECs in the range kHz-to-MHz. Here, it is reported on multilayer polyelectrolyte capacitors (mPECs) that leverage as dielectric an ambipolar nanometer-thick (down to 10 nm) stack of anionic and cationic polyelectrolytes assembled layer-by-layer between metal electrodes to eliminate the resistive behavior at frequencies from kHz to MHz. This significantly extends the operational range of mPECs over PECs. mPECs with areal capacitance as high as 25 nF mm-2 at 20 Hz and full capacitive behavior from 100 mHz to 10 MHz are demonstrated using different assembling conditions and anionic/cationic polyelectrolyte pairs. The mPECs reliably operate over time for >300 million cycles, at different biasing voltages up to 3 V, and temperatures up to 80 °C, showing a reversible capacitive behavior without significant hysteresis. Application of mPECs in flexible electronics, also operating at high frequency, is envisaged.

Synthesis and Characterization of Core-Double-Shell-Structured PVDF-grafted-BaTiO3/P(VDF-co-HFP) Nanocomposite Films.

Core-double-shell-structured nanocomposite films consisting of polyvinylidene fluoride-grafted-barium titanate (PVDF-g-BT) incorporated into a P(VDF-co-hexafluoropropylene (HFP)) copolymer matrix were produced via a solution mixing method for energy storage applications. The resulting films were thoroughly investigated via spectroscopic, thermal, and morphological analyses. Thermogravimetric data provided an enhancement of the thermal stability, while differential scanning calorimetry indicated an increase in the crystallinity of the films after the addition of PVDF-g-BT. Moreover, broadband dielectric spectroscopy revealed three dielectric processes, namely, glass-rubber relaxation (αa), relaxation associated with the polymer crystalline phase (αc), and slower relaxation in the nanocomposites resulting from the accumulation of charge on the interface between the PVDF-g-BT filler and the P(VDF-co-HFP) matrix. The dependence of the dielectric constant from the composition was analyzed, and we found that the highest permittivity enhancement was obtained by the highest concentration filler added to the largest concentration of P(VDF-co-HFP). Mechanical analysis revealed an improvement in Young's modulus for all nanocomposites versus pristine P(VDF-co-HFP), confirming the uniformity of the distribution of the PVDF-g-BT nanocomposite with a strong interaction with the copolymer matrix, as also evidenced via scanning electron microscopy. The suggested system is promising for use in high-energy-density storage devices as supercapacitors.

Dielectric Characterization of Core-Shell Structured Poly(vinylidene fluoride)-grafted-BaTiO3 Nanocomposites.

Dielectric properties of poly(vinylidene fluoride)-grafted-BaTiO3 (PVDF-g-BT) core-shell structured nanocomposites obtained from Reversible Addition Fragmentation chain Transfer (RAFT) polymerization of VDF were investigated by Broadband Dielectric Spectroscopy (BDS). The dielectric constant increased along with the BT content, about +50% by addition of 15 vol% of BT, which was around 40% more than expected from predictions using the usual dielectric modeling methods for composite materials, to be ascribed to the effect of the interfacial core-shell structure. The known dielectric relaxations for PVDF were observed for the neat polymer as well as for its nanocomposites, not affected by the presence of nanoparticles. A relaxation process at higher temperatures was found, due to interfacial polarization at the amorphous-crystalline interface, due to the high crystallinity of materials produced by RAFT. Isochronal BDS spectra were exploited to detect the primary relaxation of the amorphous fraction. Thermal analysis demonstrated a very broad endotherm at temperatures much lower than the usual melting peaks, possibly due to the ungrafted fraction of the polymer that is more easily removable by repeated washing of the pristine material with acetone.

Local Piezoelectric Response of Polymer/Ceramic Nanocomposite Fibers.

Effective converse piezoelectric coefficient (d33,eff) mapping of poly(vinylidene fluoride) (PVDF) nanofibers with ceramic BaTiO3 nanoparticle inclusions obtained by electrospinning was carried out by piezoresponse force microscopy (PFM) in a peculiar dynamic mode, namely constant-excitation frequency-modulation (CE-FM), particularly suitable for the analysis of compliant materials. Mapping of single nanocomposite fibers was carried out to demonstrate the ability of CE-FM-PFM to investigate the nanostructure of semicrystalline polymers well above their glass transition temperature, such as PVDF, by revealing the distribution of piezoelectric activity of the nanofiber, as well as of the embedded nanoparticles employed. A decreased piezoelectric activity at the nanoparticle site compared to the polymeric fiber was found. This evidence can be rationalized in terms of a tradeoff between the dielectric constants and piezoelectric coefficients of the component materials, as well as on the mutual orientation of polar axes.

A new calculation method for the free electron fraction of an ionization chamber in the ultra-high-dose-per-pulse regimen.

The free electron fraction is the fraction of electrons, produced inside the cavity of an ionization chamber after irradiation, which does not bind to gas molecules and thereby reaches the electrode as free electrons. It is a fundamental quantity to describe the recombination processes of an ionization chamber, as it generates a gap of positive charges compared to negative ones, which certainly will not undergo recombination. The free electron fraction depends on the specific chamber geometry, the polarizing applied voltage and the gas thermodynamic properties. Therefore, it is necessary to evaluate such fraction in an accurate and easy way for any measurement condition. In this paper, a simple and direct method for evaluating the free electron fraction of ionization chambers is proposed. We first model the capture process of the electrons produced inside an ionization chamber after the beam pulse; then we present a method to evaluate the free electron fraction based on simple measurements of collected charge, by varying the applied voltage. Finally, the results obtained using an Advanced Markus chamber irradiated with a Flash Radiotherapy dedicated research Linac (ElectronFlash) to estimate the free electron fraction are presented. The proposed method allows the use of a conventional ionization chamber for measurements in ultra-high-dose-per-pulse (UHDP) conditions, up to values of dose-per-pulse at which the perturbation of the electric field due to the generated charge can be considered negligible.

A new solution for UHDP and UHDR (Flash) measurements: Theory and conceptual design of ALLS chamber.

Ultra-High dose-per-pulse regimens (UHDP), necessary to trigger the "FLASH" effect, still pose serious challenges to dosimetry. Dosimetry plays a crucial role, both to significantly improve the accuracy of the radiobiological experiments necessary to fully understand the mechanisms underlying the effect and its dependencies on the beam parameters, and to be able to translate such effect into clinical practice. The standard ionization chamber in UHDP region is significantly affected by the effects of the electric field generated by the enormous density of charges produced by the dose pulse. This work describes the theory and the conceptual design of a gas chamber (the ALLS chamber) which overcomes the above-mentioned problems.

Specific Interactions and Environment Flexibility Tune Protein Stability under Extreme Crowding.

Macromolecular crowding influences protein mobility and stability in vivo. A precise description of the crowding effect on protein thermal stability requires the estimate of the combined effects of excluded volume, specific protein-environment interactions, as well as the thermal response of the crowders. Here, we explore an ideal model system, the lysozyme protein in powder state, to dissect the factors controlling the melting of the protein under extreme crowding. By deploying state-of-the art molecular simulations, supported by calorimetric experiments, we assess the role of the environment flexibility and of intermolecular electrostatic interactions. In particular, we show that the temperature-dependent flexibility of the macromolecular crowders, along with specific interactions, significantly alleviates the stabilizing contributions of the static volume effect.

The Dynamics of Hydrated Proteins Are the Same as Those of Highly Asymmetric Mixtures of Two Glass-Formers.

Customarily, the studies of dynamics of hydrated proteins are focused on the fast hydration water ν-relaxation, the slow structural α-relaxation responsible for a single glass transition, and the protein dynamic transition (PDT). Guided by the analogy with the dynamics of highly asymmetric mixtures of molecular glass-formers, we explore the possibility that the dynamics of hydrated proteins are richer than presently known. By providing neutron scattering, dielectric relaxation, calorimetry, and deuteron NMR data in two hydrated globular proteins, myoglobin and BSA, and the fibrous elastin, we show the presence of two structural α-relaxations, α1 and α2, and the hydration water ν-relaxation, all coupled together with interconnecting properties. There are two glass transition temperatures T g α1and T g α2 corresponding to vitrification of the α1 and α2 processes. Relaxation time τα2(T) of the α2-relaxation changes its Arrhenius temperature dependence to super-Arrhenius on crossing T g α1 from below. The ν-relaxation responds to the two vitrifications by changing the T-dependence of its relaxation time τν(T) on crossing consecutively T g α2 and T g α1. It generates the PDT at T d where τν(T d) matches about five times the experimental instrument timescale τexp, provided that T d > T g α1. This condition is satisfied by the hydrated globular proteins considered in this paper, and the ν-relaxation is in the liquid state with τν(T) having the super-Arrhenius temperature dependence. However, if T d < T g α1, the ν-relaxation fails to generate the PDT because it is in the glassy state and τν(T) has Arrhenius T-dependence, as in the case of hydrated elastin. Overall, the dynamics of hydrated proteins are the same as the dynamics of highly asymmetric mixtures of glass-formers. The results from this study have expanded the knowledge of the dynamic processes and their properties in hydrated proteins, and impact on research in this area is expected.

Isochronal Superposition of the Structural α-Relaxation and Invariance of Its Relation to the ß-Relaxation to Changes of Thermodynamic Conditions in Methyl m-Toluate.

The dielectric spectra of methyl m-toluate (MMT) in supercooled liquid and glassy states were measured over wide ranges of temperature T at ambient and elevated pressures P. We found that the frequency dispersion of the loss peak contributed by the structural α-relaxation is invariant to changes of P and T, while keeping the loss peak frequency fα(T,P) constant. This isochronal superposition property of the α-relaxation holds for different choices of fα(T,P). The invariant frequency dispersions for the same fα(T,P) are also indicated by the fractional exponent ßKWW in the Fourier transform of the Kohlrausch-Williams-Watts (KWW) function. Similarly, the fragility m index of MMT keeps approximately constant on varying pressure, largely different from H-bonded glass formers. The secondary ß-relaxation at a frequency higher than fα(T,P) is found to shift to lower frequencies by elevating pressure in concert with the α-relaxation. The ratio τα(T,P)/τß(T,P) is approximately unchanged to variations of T and P while keeping τα(T,P) constant. These properties observed in MMT offer experimental evidence of the dynamic correlation between α- and ß-relaxations in pure small-molecule glass-formers.

Coincident Correlation between Vibrational Dynamics and Primary Relaxation of Polymers with Strong or Weak Johari-Goldstein Relaxation.

The correlation between the vibrational dynamics, as sensed by the Debye-Waller factor, and the primary relaxation in the presence of secondary Johari-Goldstein (JG) relaxation, has been investigated through molecular dynamics simulations. Two melts of polymer chains with different bond length, resulting in rather different strength of the JG relaxation are studied. We focus on the bond-orientation correlation function, exhibiting higher JG sensitivity with respect to alternatives provided by torsional autocorrelation function and intermediate scattering function. We find that, even if changing the bond length alters both the strength and the relaxation time of the JG relaxation, it leaves unaffected the correlation between the vibrational dynamics and the primary relaxation. The finding is in harmony with previous studies reporting that numerical models not showing secondary relaxations exhibit striking agreement with experimental data of polymers also where the presence of JG relaxation is known.

Isochronal superposition and density scaling of the α-relaxation from pico- to millisecond.

The relaxation dynamics in two van der Waals bonded liquids and one hydrogen-bonding molecular liquid are studied as a function of pressure and temperature by incoherent neutron scattering using simultaneous dielectric spectroscopy. The dynamics are studied in a range of alpha relaxation times from pico- to milliseconds, primarily in the equilibrium liquid state. In this range, we find that isochronal superposition and density scaling work not only for the two van der Waals liquids but also for the hydrogen-bonding liquid, though the density scaling exponent is much smaller for the latter. Density scaling and isochronal superposition are seen to break down for intra-molecular dynamics when it is separated in time from the alpha relaxation, in close agreement with previous observations from molecular dynamics simulations.

Direct Experimental Characterization of Contributions from Self-Motion of Hydrogen and from Interatomic Motion of Heavy Atoms to Protein Anharmonicity.

One fundamental challenge in biophysics is to understand the connection between protein dynamics and its function. Part of the difficulty arises from the fact that proteins often present local atomic motions and collective dynamics on the same time scales, and challenge the experimental identification and quantification of different dynamic modes. Here, by taking lyophilized proteins as the example, we combined deuteration technique and neutron scattering to separate and characterize the self-motion of hydrogen and the collective interatomic motion of heavy atoms (C, O, N) in proteins on the pico-to-nanosecond time scales. We found that hydrogen atoms present an instrument-resolution-dependent onset for anharmonic motions, which can be ascribed to the thermal activation of local side-group motions. However, the protein heavy atoms exhibit an instrument-resolution-independent anharmonicity around 200 K, which results from unfreezing of the relaxation of the protein structures on the laboratory equilibrium time (100-1000 s), softening of the entire bio-macromolecules.

High-pressure cell for simultaneous dielectric and neutron spectroscopy.

In this article, we report on the design, manufacture, and testing of a high-pressure cell for simultaneous dielectric and neutron spectroscopy. This cell is a unique tool for studying dynamics on different time scales, from kilo- to picoseconds, covering universal features such as the α relaxation and fast vibrations at the same time. The cell, constructed in cylindrical geometry, is made of a high-strength aluminum alloy and operates up to 500 MPa in a temperature range between roughly 2 and 320 K. In order to measure the scattered neutron intensity and the sample capacitance simultaneously, a cylindrical capacitor is positioned within the bore of the high-pressure container. The capacitor consists of two concentric electrodes separated by insulating spacers. The performance of this setup has been successfully verified by collecting simultaneous dielectric and neutron spectroscopy data on dipropylene glycol, using both backscattering and time-of-flight instruments. We have carried out the experiments at different combinations of temperature and pressure in both the supercooled liquid and glassy state.

Critical structural fluctuations of proteins upon thermal unfolding challenge the Lindemann criterion.

Internal subnanosecond timescale motions are key for the function of proteins, and are coupled to the surrounding solvent environment. These fast fluctuations guide protein conformational changes, yet their role for protein stability, and for unfolding, remains elusive. Here, in analogy with the Lindemann criterion for the melting of solids, we demonstrate a common scaling of structural fluctuations of lysozyme protein embedded in different environments as the thermal unfolding transition is approached. By combining elastic incoherent neutron scattering and advanced molecular simulations, we show that, although different solvents modify the protein melting temperature, a unique dynamical regime is attained in proximity of thermal unfolding in all solvents that we tested. This solvation shell-independent dynamical regime arises from an equivalent sampling of the energy landscape at the respective melting temperatures. Thus, we propose that a threshold for the conformational entropy provided by structural fluctuations of proteins exists, beyond which thermal unfolding is triggered.

Direct Evidence of Relaxation Anisotropy Resolved by High Pressure in a Rigid and Planar Glass Former.

Rigid molecular glass-formers with no internal degrees of freedom nonetheless have a single secondary ß-relaxation. For a rigid and planar molecule, 1-methylindole (1MID), although a secondary relaxation is resolved at ambient pressure, its properties do not conform to the rules established for rigid molecules reported in early studies. By applying pressure to the dielectric spectra of 1MID, we find the single secondary relaxation splits into two. The slower one is pressure sensitive showing connections to the α-relaxation as observed in other rigid molecules, while the faster one is almost pressure insensitive and dominate the loss at ambient pressure. The two secondary relaxations, identified to associate with the out-of-plane and in-plane rotations of the rigid and planar 1MID, are resolved and observed for the first time by increasing density via elevating pressure.