Fluctuation Theorems for Heat Exchanges between Passive and Active Baths.

In addition to providing general constraints on probability distributions, fluctuation theorems allow us to infer essential information on the role played by temperature in heat exchange phenomena. In this numerical study, we measure the temperature of an out-of-equilibrium active bath using a fluctuation theorem that relates the fluctuations in the heat exchanged between two baths to their temperatures. Our setup consists of a single particle moving between two wells of a quartic potential accommodating two different baths. The heat exchanged between the two baths is monitored according to two definitions: as the kinetic energy carried by the particle whenever it jumps from one well to the other and as the work performed by the particle on one of the two baths when immersed in it. First, we consider two equilibrium baths at two different temperatures and verify that a fluctuation theorem featuring the baths temperatures holds for both heat definitions. Then, we introduce an additional Gaussian coloured noise in one of the baths, so as to make it effectively an active (out-of-equilibrium) bath. We find that a fluctuation theorem is still satisfied with both heat definitions. Interestingly, in this case the temperature obtained through the fluctuation theorem for the active bath corresponds to the kinetic temperature when considering the first heat definition, while it is larger with the second one. We interpret these results by looking at the particle jump phenomenology.

Dual mechanisms contribute to enhanced voltage dependence of an electric fish potassium channel.

The voltage dependence of different voltage-gated potassium channels, described by the voltage at which half of the channels are open (V1/2), varies over a range of 80 mV and is influenced by factors such as the number of positive gating charges and the identity of the hydrophobic amino acids in the channel's voltage sensor (S4). Here we explore by experimental manipulations and molecular dynamics simulation the contributions of two derived features of an electric fish potassium channel (Kv1.7a) that is among the most voltage-sensitive Shaker family potassium channels known. These are a patch of four contiguous negatively charged glutamates in the S3-S4 extracellular loop and a glutamate in the S3b helix. We find that these negative charges affect V1/2 by separate, complementary mechanisms. In the closed state, the S3-S4 linker negative patch reduces the membrane surface charge biasing the channel to enter the open state while, upon opening, the negative amino acid in the S3b helix faces the second (R2) gating charge of the voltage sensor electrostatically biasing the channel to remain in the open state. This work highlights two evolutionary novelties that illustrate the potential influence of negatively charged amino acids in extracellular loops and adjacent helices to voltage dependence.

Work Fluctuations for a Harmonically Confined Active Ornstein-Uhlenbeck Particle.

We study the active work fluctuations of an active Ornstein-Uhlenbeck particle in the presence of a confining harmonic potential. We tackle the problem analytically both for stationary and generic uncorrelated initial states. Our results show that harmonic confinement can induce singularities in the active work rate function, with linear stretches at large positive and negative active work, at sufficiently large active and harmonic force constants. These singularities originate from big jumps in the displacement and in the active force, occurring at the initial or ending points of trajectories and marking the relevance of boundary terms in this problem.

Dynamics of Motility-Induced Clusters: Coarsening beyond Ostwald Ripening.

We study the dynamics of clusters of active Brownian disks generated by motility-induced phase separation, by applying an algorithm that we devised to track cluster trajectories. We identify an aggregation mechanism that goes beyond Ostwald ripening but also yields a dynamic exponent characterizing the cluster growth z=3, in the timescales explored numerically. Clusters of mass M self-propel with enhanced diffusivity D∼Pe^{2}/sqrt[M]. Their fast motion drives aggregation into large fractal structures, which are patchworks of diverse hexatic orders, and coexist with regular, orientationally uniform, smaller ones. To bring out the impact of activity, we perform a comparative study of a passive system that evidences major differences with the active case.

The DTX Protein Family: An Emerging Set of E3 Ubiquitin Ligases in Cancer.

Until recently, Deltex (DTX) proteins have been considered putative E3 ligases, based on the presence of an E3 RING domain in their protein coding sequence. The human DTX family includes DTX1, DTX2, DTX3, DTX3L and DTX4. Despite the fact that our knowledge of this class of E3-ubiquitin ligases is still at an early stage, our understanding of their role in oncogenesis is beginning to unfold. In fact, recently published studies allow us to define specific biological scenarios and further consolidate evidence-based working hypotheses. According to the current evidence, all DTX family members are involved in the regulation of Notch signaling, suggesting a phylogenetically conserved role in the regulation of this pathway. Indeed, additional evidence reveals a wider involvement of these proteins in other signaling complexes and cancer-promoting mechanisms beyond NOTCH signaling. DTX3, in particular, had been known to express two isoform variants (DTX3a and DTX3b). The recent identification and cloning of a third isoform variant in cancer (DTX3c), and its specific involvement in EphB4 degradation in cancer cells, sheds further light on this group of proteins and their specific role in cancer. Herein, we review the cumulative knowledge of this family of E3 Ubiquitin ligases with a specific focus on the potential oncogenic role of DTX isoforms in light of the rapidly expanding findings regarding this protein family's cellular targets and regulated signaling pathways. Furthermore, using a comparative and bioinformatic approach, we here disclose a new putative motif of a member of this family which may help in understanding the biological and contextual differences between the members of these proteins.

Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IRA Signal in Malignant Mesothelioma.

EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein-protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as "DTX3c"), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway.

Detecting Liquid-Liquid Phase Separations Using Molecular Dynamics Simulations and Spectral Clustering.

A stringent test of the accuracy of empirical force fields is reproducing the phase diagram of bulk phases and mixtures. Exploring the phase diagram of mixtures requires the detection of phase boundaries and critical points. In contrast to most solid-liquid transitions, in which a global order parameter (average density) can be used to discriminate between two phases, some demixing transitions entail relatively subtle changes in the local environment of each molecule. In such cases, finite sampling errors and finite-size effects make the identification of trends in local order parameters extremely challenging. Here we analyze one such example, namely a methanol/hexane mixture, and compute several local and global structural properties. We simulate the system at various temperatures and study the structural changes associated with demixing. We show that despite a seemingly continuous transformation between mixed and demixed states, the topological properties of the H-bond network change abruptly as the system crosses the demixing line. In particular, by using spectral clustering, we show that the distribution of cluster sizes develops a fat tail (as expected from percolation theory) in the vicinity of the critical point. We illustrate a simple criterion to identify this behavior, which results from the emergence of large system-spanning clusters from a collection of aggregates. We further tested the spectral clustering analysis on a Lennard-Jones system as a standard example of a system with no H-bonds, and also, in this case, we were able to detect the demixing transition.

Nonequilibrium Thermodynamics of DNA Nanopore Unzipping.

Using theory and simulations, we carried out a first systematic characterization of DNA unzipping via nanopore translocation. Starting from partially unzipped states, we found three dynamical regimes depending on the applied force f: (i) heterogeneous DNA retraction and rezipping (f<17 pN), (ii) normal (17 pN60 pN) drift-diffusive behavior. We show that the normal drift-diffusion regime can be effectively modeled as a one-dimensional stochastic process in a tilted periodic potential. We use the theory of stochastic processes to recover the potential from nonequilibrium unzipping trajectories and show that it corresponds to the free-energy landscape for single-base-pair unzipping. Applying this general approach to other single-molecule systems with periodic potentials ought to yield detailed free-energy landscapes from out-of-equilibrium trajectories.

Molecular states and spin crossover of hemin studied by DNA origami enabled single-molecule surface-enhanced Raman scattering.

The study of biologically relevant molecules and their interaction with external stimuli on a single molecular scale is of high importance due to the availability of distributed rather than averaged information. Surface enhanced Raman scattering (SERS) provides direct chemical information, but is rather challenging on the single molecule (SM) level, where it is often assumed to require a direct contact of analyte molecules with the metal surface. Here, we detect and investigate the molecular states of single hemin by SM-SERS. A DNA aptamer based G-quadruplex mediated recognition of hemin directs its placement in the SERS hot-spot of a DNA Origami Nanofork Antenna (DONA). The configuration of the DONA structure allows the molecule to be trapped at the plasmonic hot-spot preferentially in no-contact configuration with the metal surface. Owing to high field enhancement at the plasmonic hot spot, the detection of a single folded G-quadruplex becomes possible. For the first time, we present a systematic study by SM-SERS where most hemin molecule adopt a high spin and oxidation state (III) that showed state crossover to low spin upon strong-field-ligand binding. The present study therefore, provides a platform for studying biologically relevant molecules and their properties at SM sensitivity along with demonstrating a conceptual advancement towards successful monitoring of single molecular chemical interaction using DNA aptamers.

Environment-Dependent Stability and Mechanical Properties of DNA Origami Six-Helix Bundles with Different Crossover Spacings.

The internal design of DNA nanostructures defines how they behave in different environmental conditions, such as endonuclease-rich or low-Mg2+ solutions. Notably, the inter-helical crossovers that form the core of such DNA objects have a major impact on their mechanical properties and stability. Importantly, crossover design can be used to optimize DNA nanostructures for target applications, especially when developing them for biomedical environments. To elucidate this, two otherwise identical DNA origami designs are presented that have a different number of staple crossovers between neighboring helices, spaced at 42- and 21- basepair (bp) intervals, respectively. The behavior of these structures is then compared in various buffer conditions, as well as when they are exposed to enzymatic digestion by DNase I. The results show that an increased number of crossovers significantly improves the nuclease resistance of the DNA origami by making it less accessible to digestion enzymes but simultaneously lowers its stability under Mg2+ -free conditions by reducing the malleability of the structures. Therefore, these results represent an important step toward rational, application-specific DNA nanostructure design.

Dataset of numerical simulations for aeroelastic control of an aero engine compressor cascade using plasma actuators.

The dataset presented here regard the analysis reported in the research article entitled "Comparison of different plasma actuation strategies for aeroelastic control on a linear compressor cascade" De Giorgi et al. (2021) [1]. These data are related to the Computational Fluid Dynamics (CFD) assessment of different plasma actuation strategies for the aeroelastic control of an aero engine compressor cascade in subsonic flow conditions. The authors evaluated the accuracy of numerical computations using experimental results. Here, both experimental and raw data of the CFD simulations are presented.

Probing ion channel functional architecture and domain recombination compatibility by massively parallel domain insertion profiling.

Protein domains are the basic units of protein structure and function. Comparative analysis of genomes and proteomes showed that domain recombination is a main driver of multidomain protein functional diversification and some of the constraining genomic mechanisms are known. Much less is known about biophysical mechanisms that determine whether protein domains can be combined into viable protein folds. Here, we use massively parallel insertional mutagenesis to determine compatibility of over 300,000 domain recombination variants of the Inward Rectifier K+ channel Kir2.1 with channel surface expression. Our data suggest that genomic and biophysical mechanisms acted in concert to favor gain of large, structured domain at protein termini during ion channel evolution. We use machine learning to build a quantitative biophysical model of domain compatibility in Kir2.1 that allows us to derive rudimentary rules for designing domain insertion variants that fold and traffic to the cell surface. Positional Kir2.1 responses to motif insertion clusters into distinct groups that correspond to contiguous structural regions of the channel with distinct biophysical properties tuned towards providing either folding stability or gating transitions. This suggests that insertional profiling is a high-throughput method to annotate function of ion channel structural regions.

Binding Sites and the Mechanism of Action of Propofol and a Photoreactive Analogue in Prokaryotic Voltage-Gated Sodium Channels.

Propofol, one of the most commonly used intravenous general anesthetics, modulates neuronal function by interacting with ion channels. The mechanisms that link propofol binding to the modulation of distinct ion channel states, however, are not understood. To tackle this problem, we investigated the prokaryotic ancestors of eukaryotic voltage-gated Na+ channels (Navs) using unbiased photoaffinity labeling (PAL) with a diazirine derivative of propofol (AziPm), electrophysiological methods, and mutagenesis. AziPm inhibits Nav function in a manner that is indistinguishable from that of the parent compound by promoting activation-coupled inactivation. In several replicates (8/9) involving NaChBac and NavMs, we found adducts at residues located at the C-terminal end of the S4 voltage sensor, the S4-S5 linker, and the N-terminal end of the S5 segment. However, the non-inactivating mutant NaChBac-T220A yielded adducts that were different from those found in the wild-type counterpart, which suggested state-dependent changes at the binding site. Then, using molecular dynamics simulations to further elucidate the structural basis of Nav modulation by propofol, we show that the S4 voltage sensors and the S4-S5 linkers shape two distinct propofol binding sites in a conformation-dependent manner. Supporting the PAL and MD simulation results, we also found that Ala mutations of a subset of adducted residues have distinct effects on gating modulation of NaChBac and NavMs by propofol. The results of this study provide direct insights into the structural basis of the mechanism through which propofol binding promotes activation-coupled inactivation to inhibit Nav channel function.

Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4-S5 linker in activation and slow inactivation.

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.

A Versatile DNA Origami-Based Plasmonic Nanoantenna for Label-Free Single-Molecule Surface-Enhanced Raman Spectroscopy.

DNA origami technology allows for the precise nanoscale assembly of chemical entities that give rise to sophisticated functional materials. We have created a versatile DNA origami nanofork antenna (DONA) by assembling Au or Ag nanoparticle dimers with different gap sizes down to 1.17 nm, enabling signal enhancements in surface-enhanced Raman scattering (SERS) of up to 1011. This allows for single-molecule SERS measurements, which can even be performed with larger gap sizes to accommodate differently sized molecules, at various excitation wavelengths. A general scheme is presented to place single analyte molecules into the SERS hot spots using the DNA origami structure exploiting covalent and noncovalent coupling schemes. By using Au and Ag dimers, single-molecule SERS measurements of three dyes and cytochrome c and horseradish peroxidase proteins are demonstrated even under nonresonant excitation conditions, thus providing long photostability during time-series measurement and enabling optical monitoring of single molecules.

Particle-based Ising model.

We characterize equilibrium properties and relaxation dynamics of a two-dimensional lattice containing, at each site, two particles connected by a double-well potential (dumbbell). Dumbbells are oriented in the orthogonal direction with respect to the lattice plane and interact with each other through a Lennard-Jones potential truncated at the nearest neighbor distance. We show that the system's equilibrium properties are accurately described by a two-dimensional Ising model with an appropriate coupling constant. Moreover, we characterize the coarsening kinetics by calculating the cluster size as a function of time and compare the results with Monte Carlo simulations based on Glauber or reactive dynamics rate constants.

RNA Pore Translocation with Static and Periodic Forces: Effect of Secondary and Tertiary Elements on Process Activation and Duration.

We use MD simulations to study the pore translocation properties of a pseudoknotted viral RNA. We consider the 71-nucleotide-long xrRNA from the Zika virus and establish how it responds when driven through a narrow pore by static or periodic forces applied to either of the two termini. Unlike the case of fluctuating homopolymers, the onset of translocation is significantly delayed with respect to the application of static driving forces. Because of the peculiar xrRNA architecture, activation times can differ by orders of magnitude at the two ends. Instead, translocation duration is much smaller than activation times and occurs on time scales comparable at the two ends. Periodic forces amplify significantly the differences at the two ends, for both activation times and translocation duration. Finally, we use a waiting-times analysis to examine the systematic slowing downs in xrRNA translocations and associate them to the hindrance of specific secondary and tertiary elements of xrRNA. The findings provide a useful reference to interpret and design future theoretical and experimental studies of RNA translocation.

Effective temperatures in inhomogeneous passive and active bidimensional Brownian particle systems.

We study the stationary dynamics of an active interacting Brownian particle system. We measure the violations of the fluctuation dissipation theorem, and the corresponding effective temperature, in a locally resolved way. Quite naturally, in the homogeneous phases the diffusive properties and effective temperature are also homogeneous. Instead, in the inhomogeneous phases (close to equilibrium and within the MIPS sector) the particles can be separated in two groups with different diffusion properties and effective temperatures. Notably, at fixed activity strength the effective temperatures in the two phases remain distinct and approximately constant within the MIPS region, with values corresponding to the ones of the whole system at the boundaries of this sector of the phase diagram. We complement the study of the globally averaged properties with the theoretical and numerical characterization of the fluctuation distributions of the single-particle diffusion, linear response, and effective temperature in the homogeneous and inhomogeneous phases. We also distinguish the behavior of the (time-delayed) effective temperature from the (instantaneous) kinetic temperature, showing that the former is independent of the friction coefficient.

Directional translocation resistance of Zika xrRNA.

xrRNAs from flaviviruses survive in host cells because of their exceptional dichotomic response to the unfolding action of different enzymes. They can be unwound, and hence copied, by replicases, and yet can resist degradation by exonucleases. How the same stretch of xrRNA can encode such diverse responses is an open question. Here, by using atomistic models and translocation simulations, we uncover an elaborate and directional mechanism for how stress propagates when the two xrRNA ends, [Formula: see text] and [Formula: see text], are driven through a pore. Pulling the [Formula: see text] end, as done by replicases, elicits a progressive unfolding; pulling the [Formula: see text] end, as done by exonucleases, triggers a counterintuitive molecular tightening. Thus, in what appears to be a remarkable instance of intra-molecular tensegrity, the very pulling of the [Formula: see text] end is what boosts resistance to translocation and consequently to degradation. The uncovered mechanistic principle might be co-opted to design molecular meta-materials.

Polyamine blockade and binding energetics in the MthK potassium channel.

Polyamines such as spermidine and spermine are found in nearly all cells, at concentrations ranging up to 0.5 mM. These cations are endogenous regulators of cellular K+ efflux, binding tightly in the pores of inwardly rectifying K+ (Kir) channels in a voltage-dependent manner. Although the voltage dependence of Kir channel polyamine blockade is thought to arise at least partially from the energetically coupled movements of polyamine and K+ ions through the pore, the nature of physical interactions between these molecules is unclear. Here we analyze the polyamine-blocking mechanism in the model K+ channel MthK, using a combination of electrophysiology and computation. Spermidine (SPD3+) and spermine (SPM4+) each blocked current through MthK channels in a voltage-dependent manner, and blockade by these polyamines was described by a three-state kinetic scheme over a wide range of polyamine concentrations. In the context of the scheme, both SPD3+ and SPM4+ access a blocking site with similar effective gating valences (0.84 ± 0.03 e0 for SPD3+ and 0.99 ± 0.04 e0 for SPM4+), whereas SPM4+ binds in the blocked state with an ∼20-fold higher affinity than SPD3+ (Kd = 28.1 ± 3.1 µM for SPD3+ and 1.28 ± 0.20 µM for SPM4+), consistent with a free energy difference of 1.8 kcal/mol. Molecular simulations of the MthK pore in complex with either SPD3+ or SPM4+ are consistent with the leading amine interacting with the hydroxyl groups of T59, at the selectivity filter threshold, with access to this site governed by outward movement of K+ ions. These coupled movements can account for a large fraction of the voltage dependence of blockade. In contrast, differences in binding energetics between SPD3+ and SPM4+ may arise from distinct electrostatic interactions between the polyamines and carboxylate oxygens on the side chains of E92 and E96, located in the pore-lining helix.