The dimerized pentraxin-like domain of the adhesion G protein-coupled receptor 112 (ADGRG4) suggests function in sensing mechanical forces.

Adhesion G protein-coupled receptors (aGPCRs) feature large extracellular regions with modular domains that often resemble protein classes of various function. The pentraxin (PTX) domain, which is predicted by sequence homology within the extracellular region of four different aGPCR members, is well known to form pentamers and other oligomers. Oligomerization of GPCRs is frequently reported and mainly driven by interactions of the seven-transmembrane region and N or C termini. While the functional importance of dimers is well-established for some class C GPCRs, relatively little is known about aGPCR multimerization. Here, we showcase the example of ADGRG4, an orphan aGPCR that possesses a PTX-like domain at its very N-terminal tip, followed by an extremely long stalk containing serine-threonine repeats. Using X-ray crystallography and biophysical methods, we determined the structure of this unusual PTX-like domain and provide experimental evidence for a homodimer equilibrium of this domain which is Ca2+-independent and driven by intermolecular contacts that differ vastly from the known soluble PTXs. The formation of this dimer seems to be conserved in mammalian ADGRG4 indicating functional relevance. Our data alongside of theoretical considerations lead to the hypothesis that ADGRG4 acts as an in vivo sensor for shear forces in enterochromaffin and Paneth cells of the small intestine.

Bridging two insect flight modes in evolution, physiology and robophysics.

Since taking flight, insects have undergone repeated evolutionary transitions between two seemingly distinct flight modes1-3. Some insects neurally activate their muscles synchronously with each wingstroke. However, many insects have achieved wingbeat frequencies beyond the speed limit of typical neuromuscular systems by evolving flight muscles that are asynchronous with neural activation and activate in response to mechanical stretch2-8. These modes reflect the two fundamental ways of generating rhythmic movement: time-periodic forcing versus emergent oscillations from self-excitation8-10. How repeated evolutionary transitions have occurred and what governs the switching between these distinct modes remain unknown. Here we find that, despite widespread asynchronous actuation in insects across the phylogeny3,6, asynchrony probably evolved only once at the order level, with many reversions to the ancestral, synchronous mode. A synchronous moth species, evolved from an asynchronous ancestor, still preserves the stretch-activated muscle physiology. Numerical and robophysical analyses of a unified biophysical framework reveal that rather than a dichotomy, these two modes are two regimes of the same dynamics. Insects can transition between flight modes across a bridge in physiological parameter space. Finally, we integrate these two actuation modes into an insect-scale robot11-13 that enables transitions between modes and unlocks a new self-excited wingstroke strategy for engineered flight. Together, this framework accounts for repeated transitions in insect flight evolution and shows how flight modes can flip with changes in physiological parameters.

Motif-pattern dependence of biomolecular phase separation driven by specific interactions.

Eukaryotic cells partition a wide variety of important materials and processes into biomolecular condensates-phase-separated droplets that lack a membrane. In addition to nonspecific electrostatic or hydrophobic interactions, phase separation also depends on specific binding motifs that link together constituent molecules. Nevertheless, few rules have been established for how these ubiquitous specific, saturating, motif-motif interactions drive phase separation. By integrating Monte Carlo simulations of lattice-polymers with mean-field theory, we show that the sequence of heterotypic binding motifs strongly affects a polymer's ability to phase separate, influencing both phase boundaries and condensate properties (e.g. viscosity and polymer diffusion). We find that sequences with large blocks of single motifs typically form more inter-polymer bonds, which promotes phase separation. Notably, the sequence of binding motifs influences phase separation primarily by determining the conformational entropy of self-bonding by single polymers. This contrasts with systems where the molecular architecture primarily affects the energy of the dense phase, providing a new entropy-based mechanism for the biological control of phase separation.

Reversible and irreversible mitochondrial swelling: Effects of variable mitochondrial activity.

An extended biophysical model was obtained by upgrading the previously reported one (Khmelinskii and Makarov, 2021). The upgraded model accommodates variations of solute transport rates through the inner mitochondrial membrane (IMM) within the mitochondrial population, described by a Gaussian distribution. However, the model may be used for any functional form of the distribution. The dynamics of system parameters as predicted by the current model differed from that predicted by the previous model in the same initial conditions (Khmelinskii and Makarov, 2021). The amount of change varied from one parameter to the other, remaining in the 1-38% range. The upgraded model fitted the available experimental data with a better accuracy (R = 0.993) compared to the previous model (R = 0.978) using the same experimental data (Khmelinskii and Makarov, 2021). The fitting procedure also estimated the Gaussian distribution parameters. The new model requires much larger computational resources, but given its higher accuracy, it may be used for better analysis of experimental data and for better prediction of MS dynamics in different initial conditions. Note that activities of individual mitochondria in mitochondrial populations should vary within biological tissues. Thus, the currently upgraded model is a better tool for biological and bio-medical applications. We believe that this model is much better adapted to the analysis of MS dynamics in vivo.

Mass Spectrometric and Bio-Computational Binding Strength Analysis of Multiply Charged RNAse S Gas-Phase Complexes Obtained by Electrospray Ionization from Varying In-Solution Equilibrium Conditions.

We investigated the influence of a solvent's composition on the stability of desorbed and multiply charged RNAse S ions by analyzing the non-covalent complex's gas-phase dissociation processes. RNAse S was dissolved in electrospray ionization-compatible buffers with either increasing organic co-solvent content or different pHs. The direct transition of all the ions and the evaporation of the solvent from all the in-solution components of RNAse S under the respective in-solution conditions by electrospray ionization was followed by a collision-induced dissociation of the surviving non-covalent RNAse S complex ions. Both types of changes of solvent conditions yielded in mass spectrometrically observable differences of the in-solution complexation equilibria. Through quantitative analysis of the dissociation products, i.e., from normalized ion abundances of RNAse S, S-protein, and S-peptide, the apparent kinetic and apparent thermodynamic gas-phase complex properties were deduced. From the experimental data, it is concluded that the stability of RNAse S in the gas phase is independent of its in-solution equilibrium but is sensitive to the complexes' gas-phase charge states. Bio-computational in-silico studies showed that after desolvation and ionization by electrospray, the remaining binding forces kept the S-peptide and S-protein together in the gas phase predominantly by polar interactions, which indirectly stabilized the in-bulk solution predominating non-polar intermolecular interactions. As polar interactions are sensitive to in-solution protonation, bio-computational results provide an explanation of quantitative experimental data with single amino acid residue resolution.

Recent Advances in Understanding the Role of Cartilage Lubrication in Osteoarthritis.

The remarkable lubrication properties of normal articular cartilage play an essential role in daily life, providing almost frictionless movements of joints. Alterations of cartilage surface or degradation of biomacromolecules within synovial fluid increase the wear and tear of the cartilage and hence determining the onset of the most common joint disease, osteoarthritis (OA). The irreversible and progressive degradation of articular cartilage is the hallmark of OA. Considering the absence of effective options to treat OA, the mechanosensitivity of chondrocytes has captured attention. As the only embedded cells in cartilage, the metabolism of chondrocytes is essential in maintaining homeostasis of cartilage, which triggers motivations to understand what is behind the low friction of cartilage and develop biolubrication-based strategies to postpone or even possibly heal OA. This review firstly focuses on the mechanism of cartilage lubrication, particularly on boundary lubrication. Then the mechanotransduction (especially shear stress) of chondrocytes is discussed. The following summarizes the recent development of cartilage-inspired biolubricants to highlight the correlation between cartilage lubrication and OA. One might expect that the restoration of cartilage lubrication at the early stage of OA could potentially promote the regeneration of cartilage and reverse its pathology to cure OA.

Visualizing bleb mass dynamics in single cells using quantitative phase microscopy.

Understanding biological responses to directed energy (DE) is critical to ensure the safety of personnel within the Department of Defense. At the Air Force Research Laboratory, we have developed or adapted advanced optical imaging systems that quantify biophysical responses to DE. One notable cellular response to DE exposure is the formation of blebs, or semi-spherical protrusions of the plasma membrane in living cells. In this work, we demonstrate the capacity of quantitative phase imaging (QPI) to both visualize and quantify the formation of membrane blebs following DE exposure. QPI is an interferometric imaging tool that uses optical path length as a label-free contrast mechanism and is sensitive to the non-aqueous mass density, or dry mass, of living cells. Blebs from both CHO-K1 and U937 cells were generated after exposure to a series of 600 ns, 21.2 kV/cm electric pulses. These blebs were visualized in real time, and their dry mass relative to the rest of the cell body was quantified as a function of time. It is our hope that this system will lead to an improved understanding of both DE-induced and apoptotic blebbing.

Understanding cytoskeletal avalanches using mechanical stability analysis.

Eukaryotic cells are mechanically supported by a polymer network called the cytoskeleton, which consumes chemical energy to dynamically remodel its structure. Recent experiments in vivo have revealed that this remodeling occasionally happens through anomalously large displacements, reminiscent of earthquakes or avalanches. These cytoskeletal avalanches might indicate that the cytoskeleton's structural response to a changing cellular environment is highly sensitive, and they are therefore of significant biological interest. However, the physics underlying "cytoquakes" is poorly understood. Here, we use agent-based simulations of cytoskeletal self-organization to study fluctuations in the network's mechanical energy. We robustly observe non-Gaussian statistics and asymmetrically large rates of energy release compared to accumulation in a minimal cytoskeletal model. The large events of energy release are found to correlate with large, collective displacements of the cytoskeletal filaments. We also find that the changes in the localization of tension and the projections of the network motion onto the vibrational normal modes are asymmetrically distributed for energy release and accumulation. These results imply an avalanche-like process of slow energy storage punctuated by fast, large events of energy release involving a collective network rearrangement. We further show that mechanical instability precedes cytoquake occurrence through a machine-learning model that dynamically forecasts cytoquakes using the vibrational spectrum as input. Our results provide a connection between the cytoquake phenomenon and the network's mechanical energy and can help guide future investigations of the cytoskeleton's structural susceptibility.

Configurational fingerprints of multicellular living systems.

Cells cooperate as groups to achieve structure and function at the tissue level, during which specific material characteristics emerge. Analogous to phase transitions in classical physics, transformations in the material characteristics of multicellular assemblies are essential for a variety of vital processes including morphogenesis, wound healing, and cancer. In this work, we develop configurational fingerprints of particulate and multicellular assemblies and extract volumetric and shear order parameters based on this fingerprint to quantify the system disorder. Theoretically, these two parameters form a complete and unique pair of signatures for the structural disorder of a multicellular system. The evolution of these two order parameters offers a robust and experimentally accessible way to map the phase transitions in expanding cell monolayers and during embryogenesis and invasion of epithelial spheroids.

On the electrical passivity of astrocyte potassium conductance.

Predominant expression of leak-type K+ channels provides astrocytes a high membrane permeability to K+ ions and a hyperpolarized membrane potential that are crucial for astrocyte function in brain homeostasis. In functionally mature astrocytes, the expression of leak K+ channels creates a unique membrane K+ conductance that lacks voltage-dependent rectification. Accordingly, the conductance is named ohmic or passive K+ conductance. Several inwardly rectifying and two-pore domain K+ channels have been investigated for their contributions to passive conductance. Meanwhile, gap junctional coupling has been postulated to underlie the passive behavior of membrane conductance. It is now clear that the intrinsic properties of K+ channels and gap junctional coupling can each act alone or together to bring about a passive behavior of astrocyte conductance. Additionally, while the passive conductance can generally be viewed as a K+ conductance, the actual representation of this conductance is a combined expression of multiple known and unknown K+ channels, which has been further modified by the intricate morphology of individual astrocytes and syncytial gap junctional coupling. The expression of the inwardly rectifying K+ channels explains the inward-going component of passive conductance disobeying Goldman-Hodgkin-Katz constant field outward rectification. However, the K+ channels encoding the outward-going passive currents remain to be determined in the future. Here, we review our current understanding of ion channels and biophysical mechanisms engaged in the passive astrocyte K+ conductance, propose new studies to resolve this long-standing puzzle in astrocyte physiology, and discuss the functional implication(s) of passive behavior of K+ conductance on astrocyte physiology.

Liquid-Liquid Phase Separation in Biology: Specific Stoichiometric Molecular Interactions vs Promiscuous Interactions Mediated by Disordered Sequences.

Extensive studies in the past few years have shown that nonmembrane bound organelles are likely assembled via liquid-liquid phase separation (LLPS), a process that is driven by multivalent protein-protein and/or protein-nucleic acid interactions. Both stoichiometric molecular interactions and intrinsically disordered region (IDR)-driven interactions can promote the assembly of membraneless organelles, and the field is currently dominated by IDR-driven biological condensate formation. Here we discuss recent studies that demonstrate the importance of specific biomolecular interactions for functions of diverse physiological condensates. We suggest that phase separation based on combinations of specific interactions and promiscuous IDR-driven interactions is likely a general feature of biological condensation under physiological conditions.

Identification and Characterization of Specific Protein-Lipid Interactions Using Molecular Simulation.

Interactions with lipids can dramatically shape and define the activity of membrane proteins. Here, we describe tools that allow the identification of these interactions using molecular dynamics simulation. Additionally, we provide the details of how to use different methods to probe the affinity of these interactions.

Vimentin intermediate filaments stabilize dynamic microtubules by direct interactions.

The cytoskeleton determines cell mechanics and lies at the heart of important cellular functions. Growing evidence suggests that the manifold tasks of the cytoskeleton rely on the interactions between its filamentous components-actin filaments, intermediate filaments, and microtubules. However, the nature of these interactions and their impact on cytoskeletal dynamics are largely unknown. Here, we show in a reconstituted in vitro system that vimentin intermediate filaments stabilize microtubules against depolymerization and support microtubule rescue. To understand these stabilizing effects, we directly measure the interaction forces between individual microtubules and vimentin filaments. Combined with numerical simulations, our observations provide detailed insight into the physical nature of the interactions and how they affect microtubule dynamics. Thus, we describe an additional, direct mechanism by which cells establish the fundamental cross talk of cytoskeletal components alongside linker proteins. Moreover, we suggest a strategy to estimate the binding energy of tubulin dimers within the microtubule lattice.

Volume measurement and biophysical characterization of mounds in epithelial monolayers after intracellular bacterial infection.

Mechanical forces are important in (patho)physiological processes, including how host epithelial cells interact with intracellular bacterial pathogens. As these pathogens disseminate within host epithelial monolayers, large mounds of infected cells are formed due to the forceful action of surrounding uninfected cells, limiting bacterial spread across the basal cell monolayer. Here, we present a protocol for mound volume measurement and biophysical characterization of mound formation. Modifications to this protocol may be necessary for studying different host cell types or pathogenic organisms. For complete details on the use and execution of this protocol, please refer to Bastounis et al. (2021).

Protein phase separation and its role in chromatin organization and diseases.

In the physical sciences, solid, liquid, and gas are the most familiar phase states, whose essence is their existence reflecting the different spatial distribution of molecular components. The biological molecules in the living cell also have differences in spatial distribution. The molecules organized in the form of membrane-bound organelles are well recognized. However, the biomolecules organized in membraneless compartments called biomolecular condensates remain elusive. The liquid-liquid phase separation (LLPS), as a new emerging scientific breakthrough, describes the biomolecules assembled in special distribution and appeared as membraneless condensates in the form of a new "phase" compared with the surrounding liquid milieu. LLPS provides an important theoretical basis for explaining the composition of biological molecules and related biological reactions. Mounting evidence has emerged recently that phase-separated condensates participate in various biological activities. This article reviews the occurrence of LLPS and underlying regulatory mechanisms for understanding how multivalent molecules drive phase transitions to form the biomolecular condensates. And, it also summarizes recent major progress in elucidating the roles of LLPS in chromatin organization and provides clues for the development of new innovative therapeutic strategies for related diseases.

Magnetoencephalography: physics, techniques, and applications in the basic and clinical neurosciences.

Magnetoencephalography (MEG) is a technique used to measure the magnetic fields generated from neuronal activity in the brain. MEG has a high temporal resolution on the order of milliseconds and provides a more direct measure of brain activity when compared with hemodynamic-based neuroimaging methods such as magnetic resonance imaging and positron emission tomography. The current review focuses on basic features of MEG such as the instrumentation and the physics that are integral to the signals that can be measured, and the principles of source localization techniques, particularly the physics of beamforming and the techniques that are used to localize the signal of interest. In addition, we review several metrics that can be used to assess functional coupling in MEG and describe the advantages and disadvantages of each approach. Lastly, we discuss the current and future applications of MEG.

Biophysical interaction between self-assembled branched DNA nanostructures with bovine serum albumin and bovine liver catalase.

Branched DNA (bDNA) nanostructures have emerged as self-assembled biomaterials and are being considered for biomedical applications. Herein, we report the biophysical interaction between self-assembled bDNA nanostructure with circulating protein bovine serum albumin (BSA) and cellular enzyme bovine liver catalase (BLC). The binding between bDNA and BSA or BLC was confirmed through the decrease in fluorescence spectra. The Stern-Volmer data supports for non-covalent bonding with ~1 binding site in case of BSA and BLC thus advocating a static binding. Furthermore, FTIR and ITC study confirmed the binding of bDNAs with proteins through hydrogen bonding and van der Waals interaction. The negative free energy observed in ITC represent spontaneous reaction for BLC-bDNA interaction. The biophysical interaction between bDNA nanostructures and proteins was also supported by DLS and zeta potential measurement. With an increase in bDNA concentrations up to 100 nM, no significant change in absorbance and CD spectra was observed for both BLC and BSA which suggests structural stability and unaffected secondary conformation of proteins in presence of bDNA. Furthermore, the catalytic activity of BLC was unaltered in presence of bDNAscr even with increasing the incubation period from 1 h to 24 h. Interestingly, the time-dependent decrease in activity of BLC was protected by bDNAmix. The thermal melting study suggests a higher Tm value for proteins in presence of bDNAmix which demonstrates that interaction with bDNAmix increases the thermal stability of proteins. Collectively these data suggest that self-assembled DNA nanostructure may bind to BSA for facilitating circulation in plasma or binding to intracellular proteins like BLC for stabilization, however the secondary conformation of protein or catalytic activity of enzyme is unaltered in presence of bDNA nanostructure. Thus, the newly established genomic sequence-driven self-assembled DNA nanostructure can be explored for in vitro or in vivo experimental work in recent future.

A generic approach to study the kinetics of liquid-liquid phase separation under near-native conditions.

Understanding the kinetics, thermodynamics, and molecular mechanisms of liquid-liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.

Physical fitness is associated with better technical performance in adolescent alpine ski racers after controlling for practice time: A retrospective regression analysis.

In an effort to evaluate the mechanisms underpinning performance in alpine ski racing, researchers have focused on the predictive validity of measures derived from fitness assessments. However, a limitation of this literature is the absence of practice time, since prolonged training may naturally develop specific fitness capacities, making some tests of physical ability less predictive of performance. We examine the relationship between fitness tests, practice, and performance using linear regressions with fitness test data, practice history data, and performance results from adolescent alpine ski racers attending professional development academies in the United States (N = 82). Only aerobic capacity (i.e. 20 m shuttle run) was significantly associated with more practice time. After controlling for practice hours, 5.5-6.5% of variance in ski performance was significantly explained by assessments of lower body power (i.e. standing long jump, triple jump), anaerobic capacity (i.e. 60 s box jump), and upper body strength/endurance (i.e. push-ups). Findings highlight the important role of anaerobic power on alpine ski racing performance, which may be developed outside of regular practice, possibly through weight training or physical maturation. The small variance explained by physical/physiological measures suggests that superior ski performance is likely a product of various skills and characteristics (e.g. technical, tactical, perceptual-cognitive, psychosocial).

Green Synthesis of Copper Oxide Nanoparticles using Cucumis Sativus (Cucumber) Extracts and their Bio-Physical and Biochemical Characterization for Cosmetic and Dermatologic Applications.

BACKGROUND & OBJECTIVE: Nanoparticles are used in cosmetic and dermatologic products, due to better skin penetration properties. Incorporation of natural products exhibiting medicinal properties in nano-preparations could significantly improve the efficacy of these products and improve the quality of life without the side effects of synthetic formulations. METHODS: We here report the green synthesis of Copper Oxide nanoparticles, using Cucumber extract, and their detailed bio-physical and bio-chemical characterization. RESULTS: These Copper Oxide-Cucumber nanoparticles exhibit significant anti-bacterial and anti-fungal properties, Ultra Violet-radiation protection ability and reactive-oxygen species inhibition properties. Importantly, these nanoparticles do not exhibit significant cellular toxicity and, when incorporated in skin cream, exhibit skin rejuvenating properties. CONCLUSION: Our findings have implications for nanoparticle-based cosmetics and dermatologic applications.