Protein arginine methyltransferases (PRMTs) catalyze arginine methylation, a key post-translational modification (PTM) regulating chromatin organization, RNA metabolism, and signaling. Recent studies reveal that PRMT-mediated methylation also modulates liquid-liquid phase separation (LLPS), which organizes membraneless condensates controlling transcription, stress response, and genome stability. Dysregulated PRMT activity disrupts condensate dynamics, contributing to cancer and neurodegenerative diseases. In cancer, PRMT1, PRMT5, and PRMT6 promote tumor progression via methylation-dependent condensates that enhance oncogenic transcription and stress resistance. In the nervous system, PRMT1, PRMT4, PRMT5, PRMT6, and PRMT8 regulate LLPS of proteins, linking aberrant methylation to ALS and Huntington’s disease. This review highlights PRMTs as key modulators of phase separation and potential therapeutic targets in both oncology and neurodegeneration.
In the quantitative characterization of experimental systems the observation of a time-independent response can signify the attainment of thermodynamic equilibrium. However, there are also situations where the time-independence of experimental response merely reflects the attainment of a kinetic steady state. Although quantitative expressions derived on the basis of kinetic steady-state attainment retain validity for a system at thermodynamic equilibrium, those based on attainment of thermodynamic equilibrium do not apply to systems merely reflecting the attainment of a kinetic steady state. This survey of experimental procedures encountered during research endeavours over the past six decades has revealed many situations where the source of the time-independent instrumental response was correctly identified, but also a number where the responses were misinterpreted.
The article presents a study on the disruption of the bacterial membrane by celandine (CME) and dandelion (TOE) extracts containing bioactive compounds responsible for their antibacterial activity. The in vitro microbiological tests showed antibacterial effect both on Gram-positive (Staphylococcus aureus and Streptococcus pyogenes) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) pathogenic bacteria species. However, in the case of celandine herb extract, the effect was stronger, especially for Gram-positive bacteria. For a deeper understanding of the mechanism of antibacterial action, both studied extracts were subjected to biophysical studies on artificial bacterial lipid membranes, modeled with the Langmuir monolayers technique. The monolayer investigations were performed using two approaches: (i) monitoring the penetration of extracts injected into the aqueous subphase beneath a compressed lipid film, and (ii) comparing surface pressure-area isotherms recorded on a subphase with and without addition of extracts. These experiments confirmed the activity of TOE and CME at the membrane level, demonstrating the penetration of extract components into the membranes and their fluidizing effect. AFM analysis of the domain structures of the films transferred onto solid supports (Langmuir-Blodgett) revealed differences in film topography induced by the extracts and are in a good agreement with the results of biological tests and analytical analyses of the tested extracts. These findings confirm that the stronger antibacterial effect of CME, associated with a higher content of bioactive compounds, exerts a more destructive effect on the bacterial lipid membranes compared to TOE.
Advances in artificial intelligence, particularly in deep learning, are transforming the field of protein structure prediction. AlphaFold has emerged as a benchmark tool due to its remarkable ability to model protein folding based solely on amino acid sequences. Nevertheless, despite these impressive achievements, several prediction failures still occur. In this study, we focus on two multidomain proteins, LicT from Bacillus subtilis and P1 from the Rice Yellow Mottle Virus, that have long resisted structural characterization. Both proteins exhibit inter-domain flexibility, dimerization, and dynamic behavior in response to phosphorylation (for LicT) or zinc binding (for P1). Here, we screen the performances of AlphaFold versions against these two proteins endowed with a complex conformational landscape and show the present impossibility to correctly predict all domains simultaneously.
A quick, noninvasive method based on 31P NMR spectroscopy to measure the pH of samples containing phosphate buffer is presented. By taking advantage of all four phosphate species populated at acidic, mildly acidic, mildly alkaline and alkaline pH, the method is applicable over a wide range of pH values, from 1 to 13. This tool is most precise at pH values near the pKa values, namely from 5.8 to 8.0 and 10.6 to 13.0. To facilitate its use, a web application is presented that calculates the pH value directly from phosphate 31P chemical shift (ppm) when sodium or potassium is the cation: ( https://rmni.iqf.csic.es/software/31phnmr/ ). In addition, the potential of this approach to monitor reactions which generate or consume H+ is illustrated by following the hydrolysis of GTP catalyzed by Ras-like protein in brain 1a (Rab1a), an essential protein linked to Parkinson’s disease and tuberculosis. The intrinsic GTPase rate of Rab1a is found to be 3.3 ± 0.8·10− 3 min at 37 °C, which places Rab1a amoung the Rabs with slow GTPase rates and the longer lived “active” GTP-bound states.
Bitter taste type 2 G protein-coupled receptors (TAS2R GPCRs) detect bitter substances on the tongue, but they are also prevalent in tissues throughout the human body that play a role in cancer and other diseases, making them targets for pharmacological research. However, structural information on G protein (GP)-bound TAS2R complexes resolved by cryogenic electron microscopy (cryo-EM) is available for only 4 of the 25 TAS2Rs. Herein, we show that AlphaFold (AF) predictions of GP-bound TAS2R14 and TAS2R46 agree well with cryo-EM structure determinations, thus suggesting that the AF2 predicted structures for all 25 TAS2Rs could be used as starting points for drug development. Although useful starting points, we show that Molecular Dynamics (MD) studies are needed to refine the AF2 structures to get sufficient accuracy for drug design. We find that the MD simulations agree well with the cryo-EM structures at 85 K, but significant changes occur in the MD structures at 310 K that arise from thermal fluctuations frozen-out in the cryo-EM structure, making them far less pronounced at 85 K. We conclude that for TAS2Rs for which cryo-EM structures are not available, AF2 predicted structures followed by MD at 310 K provide a basis for TAS2R drug discovery.
N-acylmelatonins (NAMs), a class of fatty acid amides featuring melatonin as the polar head group, demonstrate significant biomedical potential. In this study, a homologous series of saturated NAMs (n = 9–18) were synthesized and fully characterized via FTIR, NMR and HRMS. The biophysical self-assembly behaviour was assessed by powder X-ray diffraction and fluorescence spectroscopy. The d-spacing determined from PXRD increases linearly with an increment of 0.90 Å per CH₂ group, suggesting that the NAMs adopt a tilted bilayer structure. Fluorescence spectroscopy was employed to measure the critical micellar concentrations (CMCs) of NAMs by monitoring the spectral changes of 8-anilinonaphthalene-1-sulfonate (ANS). Fluorescence emission and lifetime measurements of NAMs show a red-shift ( 10 nm) in emission maxima and a 2.4-fold emission intensity enhancement due to the hydrophobic acyl chain. Antioxidant properties, determined by DPPH radical scavenging assays, increased with concentration, showcasing potent activity. Notably, NAMs exhibited enhanced antimicrobial efficacy against clinically relevant bacterial and fungal strains, with minimum inhibitory concentrations comparable to standard drugs. In vitro anticancer screening revealed significant cytotoxicity against multiple human cancer cell lines, particularly with medium-chain NAMs. These findings highlight the broad-spectrum therapeutic potential of NAMs, driven by variable acyl chain length, self-assembly characteristics and strong biological applications.
Geobacter bacteria produce multiheme c-type cytochrome nanowires that are involved in long-range extracellular electron transfer. The ability of these protein nanowires to conduct electrical current makes them promising candidates for electronic devices, offering several functional and sustainable advantages over traditional materials. Therefore, this study focused on synthesizing hybrid protein fibers that mimic the natural Geobacter extracellular nanowires. To achieve this, a mutated PpcA triheme protein variant was used as the building block, with thiol-ene coupling employed to bind the protein molecules. This engineered PpcA variant (PpcAK9CK22C) maintained a structure similar to that of the native protein. Thermal denaturation studies revealed a two-state process, with a melting temperature of 62 ± 1 °C and an enthalpy change of 61 ± 2 kcal/mol. The new protein nanowires showed a lower heme group content than the precursor protein and displayed distinct secondary structure features, with a slight reduction in helical content and an increase in β-sheet and unordered structures. Their thermal stability also differed, as it could not be described by the same model applied to the PpcA variant. Despite these differences, the nanowires retained their ability to undergo redox cycling. Morphologically, they consisted of linear single-protein filaments extending over 300 nm in length.
The green alga Chlamydomonas reinhardtii (CR) has been explored as the live component of biohybrid microrobots due to its intrinsic motility. However, cell performance depends on physiological state, which evolves with culture age. Using optical tweezers, we quantify the rotational dynamics of individual CR cells across lag, exponential, stationary, and death phases. We reveal a clear performance trajectory: rotation speed and corresponding hydrodynamic torque peak during the mid-exponential phase and decrease as cultures mature. Additionally, counterclockwise rotation consistently generates higher hydrodynamic torque than clockwise rotation. These findings provide quantitative benchmarks linking CR’s motility characteristics to growth phase, offering guidelines for designing living micromachines and establishing rotational dynamics as a sensitive proxy for cellular metabolic health.
We investigated the interaction of liquid crystal droplets, formed by a phase separating system of elongated virus particles and polymer with rod-like impurities. The virus particles were Pf4, which when mixed with sodium alginate as polymer, phase separated into an isotropic phase in coexistence with a nematic phase of the virus particles. The nematic phase appeared in the form of spindle-shaped liquid crystal droplets, which are called tactoids. We studied the interaction of this mixture with micrometer sized rods, which were either bacteria (Bacillus subtilis or Escherichia coli) or inert colloidal particles (made of SU8 photoresist polymer or silica). Confocal laser scanning microscopy was used to examine the mixtures and four different dominant configurations were found, classified as no attachment, partial attachment of the rod to the tactoid, a “sandwich” state, where two tactoids interact with the same rod, and lastly encapsulation of the rods by the tactoids. We categorized the results in terms of surface properties and rod geometry (size and aspect ratio). We further investigated the system through numerical calculation in a simplified two-dimensional model. Finally, we addressed the role of confining the experimental system from three dimensions to a quasi two-dimensional setup, a relevant scenario in bacteria biofilms.
Viruses are complex supramolecular assemblies that propagate their genetic material from cell to cell, thereby relying on host cell mechanisms. Employing a combination of passive and active strategies, they efficiently package, transport and release nucleic acids. While structural and biochemical techniques offer insights into certain, static aspects of the viral life cycle, recent advancements in biophysical approaches now allow for direct measurement of their inherent dynamic activities in the research field commonly referred to as physical virology. One of these methods is optical tweezers, enabling the precise measurement of force and position at the single-molecule level over time. Over the past decades, the ability to optically trap beads and to manipulate biomolecules has revolutionised medical and biophysical research. In this paper, we provide a comprehensive analysis of optical tweezers, exploring its integration with imaging modalities and review its diverse applications in the study of viruses and viral components. In particular we focus on studies that use optical tweezers to study virus-cell interactions, genome packaging using molecular motors and co-assembly of viral assembly proteins with their nucleic acid.
Interleukin-6 (IL-6) is an aggregation-prone cytokine whose recombinant production from Escherichia coli inclusion bodies remains challenging because of inefficient refolding. Here, we present a limited batch-mode process-analytical study comparing two practical buffer-exchange operations, “fast” and “slow”, during batch dilution refolding of IL-6. Using SDS-PAGE, size-exclusion chromatography (SEC), dynamic light scattering (DLS), and far-UV circular dichroism (CD) spectroscopy, we examined dimerization and aggregation under acidic and guanidine denaturation workflows. The data suggest that IL-6 forms predominantly non-covalent dimers and that batch buffer-exchange operation and timescale are associated with aggregation outcome. Because the acidic and guanidine workflows differed in starting solvent composition, residual denaturant, and final protein concentration, the results should be interpreted as comparisons of complete workflow conditions rather than isolated denaturant effects. Overall, the combined use of SEC, DLS, and far-UV CD provides a practical framework for monitoring IL-6 refolding outcomes, while mechanistic assignments remain beyond the scope of this study.
A novel constitutive material model for the collagenous tissue of bridging veins is presented in this article. The walls of blood vessels are structurally reinforced by the collagen fibers, which are interconnected with an elastin matrix. Collagen fibers are oriented according to a specific probability distribution, with a greater abundance of fibers along the longitudinal axis. It is possible to analyze the collective mechanical behavior of collagen fibers using statistical mechanics techniques. The article describes how to derive the macroscopic behavior of collagenous tissue using the formalism of the canonical ensemble of statistical mechanics. One of the innovations of this formulation is the incorporation of a damage variable that increases significantly under excessive stretching. This formalism enables the derivation of a constitutive material model that is thermodynamically consistent, in which entropy increases irreversibly with the level of damage. In the model, an increase in damage results in a permanent increase in entropy. Furthermore, for a given level of damage, an increase in strain induces organization in the fibers, which is consistent with the behavior of an entropically elastic material. In addition, the thermodynamic properties of the model account for specific empirical observations, such as the slight contraction that occurs in a blood vessel when it is heated. The model’s mechanical suitability was assessed by subjecting it to uniaxial tensile tests on bridging veins. The findings provide strong evidence that the proposed constitutive model accurately reproduces the stress–strain curves ( R^2_adj> 0.99 ).
Host–guest interactions between cyclodextrins and small molecules are fundamental in drug delivery, supramolecular chemistry, and molecular sensing. In this study, we applied Flow-Induced Dispersion Analysis (FIDA) to investigate the binding of a tryptophan derivative with α-, β-, and γ-cyclodextrins. For this purpose, L-tryptophan (trp) was fluorescently labelled with fluorescein (Fl-trp) to serve as a detectable indicator, enabling both direct binding and competitive displacement assays. Direct FIDA measurements revealed preferential binding of Fl-trp to β-cyclodextrin, with an apparent association constant K 470 M− 1, while α- and γ-cyclodextrins showed weaker interactions, in agreement with literature. In competitive displacement experiments, the addition of unlabelled trp that binds to cyclodextrins effectively shifts the equilibria to the displacement of Fl-trp from the cyclodextrin cavities. The titration with [trp]tot allows to indirectly monitor the complex dissociation via changes in Rₕ. These results demonstrate that FIDA can be successfully applied to quantify small-molecule host–guest interactions, including weak or transient complexes, by tracking relative Rₕ changes. This approach provides a versatile, quantitative complement to conventional techniques such as NMR, ITC, or fluorescence titrations, and can be extended to other small-molecule binding systems in complex matrices.
Spheroids are of great interest in the study of cancer as they can partially mimic the tumour microenvironment, thus allowing to investigate several aspects of cell – microenvironment interactions in healthy and diseased conditions, including those pertaining to mechanobiology. Atomic Force Microscopy (AFM) is a versatile tool for studying biological samples and their mechanobiological properties. In AFM, the tip shape and dimensions determine the contact geometry between the tip and the sample and the length scales at which the mechanical properties are probed. Given the complex multiscale structure of spheroids, the choice of tip geometry and size would allow, in principle, to dissect the mechanical response of the overall system into the contributions of the constituents, from the single cell level to the cellular aggregate. In this work, we studied the mechanical properties of spheroids derived from four cell lines (A549, NHLF, HT-29, and CCD-18Co cells). Our studies revealed that using different contact geometries in the fitting procedure results in significantly different Young’s modulus values, highlighting the multiscale response of these complex cellular systems and the importance of a precise experimental design and choice of the AFM probe for the nanomechanical measurements. We observed that the location of F-actin filaments is correlated with the rigidity of the spheroids.
Quantitative metabolic profiling of bacterial communities provides biophysical insight into cellular respiration and energy transduction under controlled environmental conditions. The oxygen consumption rate (OCR) represents a direct, integrative readout of respiratory activity and has emerged as a sensitive phenotypic parameter for characterizing microbial physiology across different metabolic states. Here, we present an experimental protocol for measuring bacterial respiration using the Seahorse XFe96 Analyzer (Agilent), a microplate-based platform that enables high temporal resolution measurements of oxygen flux. Although originally developed for eukaryotic cell cultures, we adapted this technology to bacterial systems, allowing the simultaneous analysis of multiple strains in a medium-throughput format. The 96-well configuration, combined with sequential injection of up to four compounds, including metabolic substrates, stressors, and alternative electron acceptors, enables real-time perturbation of respiration and monitoring of dynamic OCR responses. To investigate bacterial respiration under physiologically and environmentally relevant oxygen tensions, OCR measurements were additionally performed under hypoxic conditions (5
Amyloid assembly is governed by a balance between intrinsic sequence determinants and environmental cues that modulate nucleation. The RIP homotypic interaction motif (RHIM) of receptor-interacting protein kinase 3 (RIPK3) provides a tractable model to dissect these principles. In solution, amyloid formation strictly requires the conserved VQVG RHIM core tetrad; a deliberately core-disrupted variant (VQVG→AAAA) fails to assemble under aggregation permissive conditions. Here, we use Thioflavin-T fluorescence assays and NMR spectroscopy to show that negatively charged lipidic interfaces unlock a latent amyloidogenic potential in this mutant. These results delineate two separable dimensions of amyloidogenesis, namely a sequence-encoded propensity and an environmental component that can catalyze nucleation by templating molecular proximity and orientation.
